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<strong>Journal</strong> <strong>of</strong> <strong>Cell</strong> <strong>and</strong><br />
Molecular Biology<br />
• Sulfabenzamide promotes autophagy through p53/ DRAM pathway<br />
MGP polymorphisms in ischemic s<strong>tr</strong>oke<br />
<s<strong>tr</strong>ong>Volume</s<strong>tr</strong>ong> <s<strong>tr</strong>ong>10</s<strong>tr</strong>ong> · No 1· June 2012<br />
http://<s<strong>tr</strong>ong>jcmb</s<strong>tr</strong>ong>.<s<strong>tr</strong>ong>halic</s<strong>tr</strong>ong>.<s<strong>tr</strong>ong>edu</s<strong>tr</strong>ong>.<strong>tr</strong><br />
Survivin -625G/C polymorphism in non-small cell lung cancer
<strong>Journal</strong> <strong>of</strong> <strong>Cell</strong> <strong>and</strong><br />
Molecular Biology<br />
<s<strong>tr</strong>ong>Volume</s<strong>tr</strong>ong> <s<strong>tr</strong>ong>10</s<strong>tr</strong>ong> · Number 1<br />
June 2012<br />
İstanbul-TURKEY
Haliç University<br />
Faculty <strong>of</strong> Arts <strong>and</strong> Sciences<br />
<strong>Journal</strong> <strong>of</strong> <strong>Cell</strong> <strong>and</strong> Molecular Biology<br />
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<strong>Journal</strong> <strong>of</strong> <strong>Cell</strong> <strong>and</strong> Molecular Biology<br />
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Kemal BÜYÜKGÜZEL, Zonguldak, Turkey<br />
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Ayla ÇELİK, Mersin, Turkey<br />
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Zihni DEMİRBAĞ, Trabzon, Turkey<br />
Gizem DİNLER DOĞANAY, İstanbul, Turkey<br />
Mustafa DJAMGÖZ, London, UK<br />
Aglika EDREVA, S<strong>of</strong>ia, Bulgaria<br />
Ünal EGELİ, Bursa, Turkey<br />
Anne FRARY, İzmir, Turkey<br />
H<strong>and</strong>e GÜRER ORHAN, İzmir, Turkey<br />
Nermin GÖZÜKIRMIZI, İstanbul, Turkey<br />
Ferruh ÖZCAN, İstanbul, Turkey<br />
Asım KADIOĞLU, Trabzon, Turkey<br />
Maria V. KALEVITCH, Pennsylvania, USA<br />
Nevin Gül KARAGÜLER, İstanbul, Turkey<br />
Valentine KEFELİ, Pennsylvania, USA<br />
Meral KENCE, Ankara, Turkey<br />
Fatma Neşe KÖK, İstanbul, Turkey<br />
Uğur ÖZBEK, İstanbul, Turkey<br />
Ayşe ÖZDEMİR, İstanbul, Turkey<br />
Pınar SAİP, Istanbul, TURKEY<br />
Sevtap SAVAŞ, Toronto, Canada<br />
Müge TÜRET SAYAR, İstanbul, Turkey<br />
İsmail TÜRKAN, İzmir, Turkey<br />
Mehmet TOPAKTAŞ, Adana, Turkey<br />
Meral ÜNAL, İstanbul, Turkey<br />
İlhan YAYLIM ERALTAN, İstanbul, Turkey<br />
Selma YILMAZER, İstanbul, Turkey<br />
Ziya ZİYLAN, İstanbul, Turkey
<strong>Journal</strong> <strong>of</strong> <strong>Cell</strong> <strong>and</strong> Molecular Biology<br />
CONTENTS<br />
<s<strong>tr</strong>ong>Volume</s<strong>tr</strong>ong> <s<strong>tr</strong>ong>10</s<strong>tr</strong>ong> · Number 1 · June 2012<br />
Review Article<br />
Production <strong>and</strong> indus<strong>tr</strong>ial applications <strong>of</strong> laccase enzyme<br />
M. IMRAN, M.J. ASAD, S.H. HADRI, S. MEHMOOD<br />
Research Articles<br />
Isolation <strong>and</strong> biochemical identification <strong>of</strong> Escherichia coli from<br />
wastewater effluents <strong>of</strong> food <strong>and</strong> beverage indus<strong>tr</strong>y<br />
T. FARASAT, Z. BILAL, F. YUNUS<br />
Investigation <strong>of</strong> the MGP promoter <strong>and</strong> exon 4 polymorphisms in<br />
patients with ischemic s<strong>tr</strong>oke in the Ukrainian population<br />
A.V. ATAMAN, V.Y. GARBUSOVA, Y.A. ATAMAN, O.I. MATLAJ,<br />
O.A. OBUCHOVA<br />
Investigation <strong>of</strong> the association <strong>of</strong> survivin gene -625G/C polymorphism<br />
in non-small cell lung cancer<br />
Survivin geni -625G/C polimorfizminin Küçük Hücreli Dışı Akciğer<br />
Kanseri ile ilişkisinin araştırılması<br />
E. AYNACI, E. COŞKUNPINAR, A. EREN, O. KUM, Y. M. OLTULU, N.<br />
AKKAYA, A. TURNA, İ. YAYLIM, P. YILDIZ<br />
Effects <strong>of</strong> prenatal <strong>and</strong> neonatal exposure to lead on white blood cells in<br />
Swiss mice<br />
R. SHARMA, K. PANWAR, S. MOGRA<br />
Sulfabenzamide promotes autophagic cell death in T-47D breast cancer<br />
cells through p53/ DRAM pathway<br />
R. MOHAMMADPOUR, S. SAFARIAN, S. FARAHNAK, S.<br />
HASHEMINASL, N. SHEIBANI<br />
Media optimization for amylase production in solid state fermentation<br />
<strong>of</strong> wheat bran by fungal s<strong>tr</strong>ains<br />
M. IRFAN, M. NADEEM, Q. SYED<br />
Guidelines for Authors<br />
Front cover image: “DNA s<strong>tr</strong><strong>and</strong>s on abs<strong>tr</strong>act”<br />
Shutterstock image ID: 704<s<strong>tr</strong>ong>10</s<strong>tr</strong>ong>25<br />
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<strong>Journal</strong> <strong>of</strong> <strong>Cell</strong> <strong>and</strong> Molecular Biology <s<strong>tr</strong>ong>10</s<strong>tr</strong>ong>(1): 1-11, 2012 Review Article 1<br />
Haliç University, Printed in Turkey.<br />
http://<s<strong>tr</strong>ong>jcmb</s<strong>tr</strong>ong>.<s<strong>tr</strong>ong>halic</s<strong>tr</strong>ong>.<s<strong>tr</strong>ong>edu</s<strong>tr</strong>ong>.<strong>tr</strong><br />
Production <strong>and</strong> indus<strong>tr</strong>ial applications <strong>of</strong> laccase enzyme<br />
Muhammad IMRAN *1,2 , Muhammad J. ASAD 1 , Saqib H. HADRI 1 <strong>and</strong> Sajid<br />
MEHMOOD 2<br />
1 Department <strong>of</strong> Biochemis<strong>tr</strong>y, Pir Mehr Ali Shah Arid Agriculture University, Rawalpindi, Pakistan<br />
2 Department <strong>of</strong> Biochemis<strong>tr</strong>y <strong>and</strong> Biotechnology, University <strong>of</strong> Gujrat, Pakistan<br />
(* author for correspondence; mirzaimran42@gmail.com)<br />
Received: 22 April 2011; Accepted: 15 May 2012<br />
Abs<strong>tr</strong>act<br />
Laccase is an enzyme that has potential ability <strong>of</strong> oxidation. It belongs to those enzymes, which have innate<br />
properties <strong>of</strong> reactive radical production, <strong>and</strong> its utilization in many fields has been ignored because <strong>of</strong> its<br />
unavailability in the commercial field. There are diverse sources <strong>of</strong> laccase producing organisms like<br />
bacteria, fungi <strong>and</strong> plants. Textile, pulp <strong>and</strong> paper indus<strong>tr</strong>ies discharge a huge quantity <strong>of</strong> waste in the<br />
environment, <strong>and</strong> the disposal <strong>of</strong> this waste is a big problem. To solve this problem, work has done to<br />
discover such an enzyme, which can detoxify these wastes <strong>and</strong> is not harmful to the environment. Laccases<br />
use oxygen <strong>and</strong> produce water as by product. They can degrade a range <strong>of</strong> compounds including phenolic <strong>and</strong><br />
non-phenolic compounds. They also have ability to detoxify a range <strong>of</strong> environmental pollutants. Their<br />
property to act on a range <strong>of</strong> subs<strong>tr</strong>ates <strong>and</strong> also to detoxify a range <strong>of</strong> pollutants have made them to be<br />
usable for several purposes in many indus<strong>tr</strong>ies including paper, pulp, textile <strong>and</strong> pe<strong>tr</strong>ochemical indus<strong>tr</strong>ies.<br />
Keywords: Laccase, solid state fermentation, oxidation, enzyme, fungi.<br />
Lakkaz enziminin üretimi ve endüs<strong>tr</strong>iyel uygulamaları<br />
Özet<br />
Lakkaz, potansiyel oksidasyon yeteneği olan bir enzimdir. Reaktif radikal üretim özelliği olan enzimlere<br />
dahildir ve birçok al<strong>and</strong>aki kullanımı, ticari al<strong>and</strong>a uygun olmaması nedeniyle göz ardı edilmektedir. Bakteri,<br />
mantar ve bitki gibi lakkaz üreten çeşitli organizma kaynakları vardır. Tekstil, kağıt hamuru ve kağıt<br />
endüs<strong>tr</strong>isi çevreye büyük miktarda atık salmaktadır ve bu atıkların uzaklaştırılması büyük bir problemdir. Bu<br />
sorunu çözmek üzere, atıkları detoksifiye eden ve çevreye zararlı olmayan bir enzim keşfetmek için<br />
çalışmalar yapılmıştır. Bu enzim oksijen kullanır ve yan ürün olarak su üretir. Lakkaz, fenolik ve fenolik<br />
olamayan bileşikleri içeren bir dizi bileşiği parçalayabilir. Ayrıca, bir dizi çevresel kirleticiyi detoksifiye<br />
etme yeteneği vardır. Çeşitli subs<strong>tr</strong>atlar üzerine etki etme ve ayrıca bir dizi kirliliği detoksifiye etme özelliği,<br />
bu enzimleri çeşitli amaçlarla tekstil, kâğıt hamuru, kâğıt ve pe<strong>tr</strong>okimya endüs<strong>tr</strong>isini kapsayan birçok<br />
endüs<strong>tr</strong>ide kullanılabilir kılmaktadır.<br />
Anahtar kelimeler: Lakkaz, katı hal fermentasyonu, oksidasyon, enzim, mantarlar.<br />
In<strong>tr</strong>oduction<br />
Laccase was first discovered in the sap <strong>of</strong> the<br />
Japanese lacquer <strong>tr</strong>ee Rhus vernicifera, <strong>and</strong> its<br />
characteristic as a metal containing oxidase was<br />
discovered by Ber<strong>tr</strong><strong>and</strong> in 1985 (Giardina et al.,<br />
20<s<strong>tr</strong>ong>10</s<strong>tr</strong>ong>). Since then, laccases have also been found in<br />
various basidiomycetous <strong>and</strong> ascomycetous fungi<br />
<strong>and</strong> thus far fungal laccases have accounted for the<br />
most important group <strong>of</strong> multicopper oxidases<br />
(MCOs) with respect to number <strong>and</strong> extent <strong>of</strong><br />
characterization (Giardina et al., 20<s<strong>tr</strong>ong>10</s<strong>tr</strong>ong>).<br />
The large quantity <strong>of</strong> laccases have been widely<br />
reported inside white-rot fungi. A number <strong>of</strong>
2Muhammad IMRAN et al.<br />
laccase genes have been isolated <strong>and</strong> distinguished<br />
for this purpose (Mayer <strong>and</strong> Staples, 2002). The<br />
improvement in laccase appearance, characterized<br />
by an increase in protein <strong>and</strong> mRNA level, was<br />
illus<strong>tr</strong>ated with Picnoprus cinnabarinus, Pleurotus<br />
sajor caju <strong>and</strong> Trametes versicolor (Eggert et al.<br />
1996, Solden <strong>and</strong> Dobson 2001, Collins <strong>and</strong><br />
Dobson 1997).<br />
A number <strong>of</strong> species <strong>of</strong> genus Pleurotus have<br />
been explained as manufacturers <strong>of</strong> laccase<br />
(Leonowicz et al. 2001). We freshly reported that a<br />
s<strong>tr</strong>ain <strong>of</strong> P. pulmonarius produce laccase as the<br />
main ligninolytic enzymes while cultured on wheat<br />
bran solid state medium (Souza et al. 2002). In the<br />
current study, numerous phenolic <strong>and</strong> aromatic<br />
compounds s<strong>tr</strong>ucturally related to lignin were<br />
calculated for their capability to arouse laccase<br />
production by P. pulmonarius. (Solden <strong>and</strong><br />
Dobson, 2001).<br />
P. pulmonarius was pr<strong>of</strong>icient <strong>of</strong> mounting on a<br />
wide variety <strong>of</strong> phenolic <strong>and</strong> aromatic compounds.<br />
Laccase production by P. pulmonarius could be<br />
considerably improved by including an equimolar<br />
combination <strong>of</strong> ferulic acid <strong>and</strong> vanillin as inducer.<br />
The cons<strong>tr</strong>uction <strong>of</strong> different laccase is<strong>of</strong>orm in<br />
reply to phenolics implicates a possible task <strong>of</strong> this<br />
enzyme in the detoxification processes (Souza et<br />
al., 2002)<br />
Numerous white-rot fungi, counting Trametes<br />
versicolor, make ex<strong>tr</strong>a cellular copper-containing<br />
phenol oxidases (E C 1.<s<strong>tr</strong>ong>10</s<strong>tr</strong>ong>.3.2), named laccases<br />
(Birhanli <strong>and</strong> Yesilada, 2006). The two major likely<br />
natural functions at<strong>tr</strong>ibuted to fungal laccases are,<br />
first, their participation in lignin degradation,<br />
mutually with supplementary ligninolytic enzymes<br />
such as peroxidases, <strong>and</strong> second, their function in<br />
fungal virulence as key cause in pathogenesis in<br />
opposition to plant hosts (Gianfreda et al., 1999).<br />
As well, laccases display in vivo other functions<br />
that are the foundation <strong>of</strong> several indus<strong>tr</strong>ial<br />
applications. For instance, in Aspergillus nidulans,<br />
laccases take action on pigment development in<br />
fungal spores (Smith et al., 1997). A number <strong>of</strong><br />
fungi also ooze laccases to take away either<br />
potentially toxic phenols released through lignin<br />
degradation or toxins formed by others organisms.<br />
As a result, the enzyme has probable applications in<br />
the textile indus<strong>tr</strong>ies, dye, as well as for the<br />
degradation <strong>of</strong> a variety <strong>of</strong> xenobiotics, which are<br />
recognized as ecological pollutants (Rama et al.<br />
1998, Jolivalt et al. 1999, Mougin et al., 2000).<br />
Laccase-producing fungi have also been<br />
reported to be helpful apparatus for xenobiotic<br />
removal in liquid effluents as well as in soil<br />
bioremediation (Gianfreda et al. 1999, Jolivalt et al.<br />
2000). Our outcomes demons<strong>tr</strong>ate that the resulting<br />
alteration products themselves are likely to<br />
encourage biological effects moreover on<br />
degrading or non-target organisms. So, an entire<br />
characterization <strong>of</strong> these compounds is essential for<br />
an entire assessment <strong>of</strong> the remediation processes<br />
(Souza et al., 2002).<br />
Laccase represents a family <strong>of</strong> coppercontaining<br />
polyphenol oxidases (PPO) & are<br />
usually called multicopper oxidases (MCO)<br />
(Birhanli <strong>and</strong> Yesilada, 2006; Arora <strong>and</strong> Sharma,<br />
20<s<strong>tr</strong>ong>10</s<strong>tr</strong>ong>; Giardina et al., 20<s<strong>tr</strong>ong>10</s<strong>tr</strong>ong>). Laccases catalyze the<br />
oxidation <strong>of</strong> various substituted phenolic<br />
compounds by using molecular oxygen as the<br />
elec<strong>tr</strong>on acceptor (Sharma et al., 2007). These<br />
enzymes have less subs<strong>tr</strong>ate specificity <strong>and</strong> have<br />
the ability to degrade a range <strong>of</strong> xenobiotics<br />
including indus<strong>tr</strong>ial colored wastewaters (Souza et<br />
al., 2006).<br />
Laccases exhibit broad subs<strong>tr</strong>ate range, which<br />
varies from one laccase to another. Although it is<br />
known to be diphenol oxidase, monophenols like 2,<br />
6-dimethoxy phenol or guaiacol are better<br />
subs<strong>tr</strong>ates than phenols (e.g., catechol or<br />
hydroquinone) (Baldrian, 2006; Arora <strong>and</strong> Sharma,<br />
20<s<strong>tr</strong>ong>10</s<strong>tr</strong>ong>).<br />
Laccases catalyze monoelec<strong>tr</strong>onic oxidation <strong>of</strong><br />
molecules to corresponding reactive radicals with<br />
the help <strong>of</strong> four copper atoms, which form the main<br />
catalytic core <strong>of</strong> the laccase, accompanied with the<br />
diminution <strong>of</strong> oxygen to water molecules <strong>and</strong><br />
simultaneous oxidation <strong>of</strong> subs<strong>tr</strong>ate to produce<br />
radicals (Arora <strong>and</strong> Sharma, 20<s<strong>tr</strong>ong>10</s<strong>tr</strong>ong>). All subs<strong>tr</strong>ates<br />
cannot be directly oxidized by laccases, either<br />
because <strong>of</strong> their large size which res<strong>tr</strong>icts their<br />
pene<strong>tr</strong>ation into the enzyme active site or because<br />
<strong>of</strong> their particular high redox potential. To<br />
overcome this hindrance, suitable chemical<br />
mediators are used which are oxidized by the<br />
laccase <strong>and</strong> their oxidized forms are then able to<br />
interact with high redox potential subs<strong>tr</strong>ate targets<br />
(Arora <strong>and</strong> Sharma, 20<s<strong>tr</strong>ong>10</s<strong>tr</strong>ong>).<br />
In fungi, laccases carry out a variety <strong>of</strong><br />
physiological roles including morphogenesis,<br />
fungal plant pathogen/host interaction, s<strong>tr</strong>ess<br />
defense, <strong>and</strong> lignin degradation (Gianfreda et al.,<br />
1999; Giardina et al., 20<s<strong>tr</strong>ong>10</s<strong>tr</strong>ong>). Laccases have been<br />
found in nearly all woodrotting fungi analyzed so<br />
far (Heinzkill <strong>and</strong> Messner, 1997; Giardina et al.,<br />
20<s<strong>tr</strong>ong>10</s<strong>tr</strong>ong>) <strong>and</strong> are almost ubiquitary enzymes as they<br />
have been isolated from plants, from some kinds <strong>of</strong><br />
bacteria, <strong>and</strong> from insects too (Enguita et al., 2003;<br />
Sharma et al., 2007; Giardina et al., 20<s<strong>tr</strong>ong>10</s<strong>tr</strong>ong>).
Laccase has many applications in other fields,<br />
like medical diagnosis, pharmaceutical indus<strong>tr</strong>y.<br />
Laccase has also applications in the agriculture area<br />
by clearing herbicides, pesticides <strong>and</strong> some<br />
explosives in soil. It is also used in the preparation<br />
<strong>of</strong> some important drugs, like anticancer drugs, <strong>and</strong><br />
added in some cosmetics to r<s<strong>tr</strong>ong>edu</s<strong>tr</strong>ong>ce their toxicity.<br />
Laccase also has the ability to form polymers <strong>of</strong><br />
value able importance (Couto <strong>and</strong> Herrera, 2006).<br />
Solid state fermentation (SSF) is a technique in<br />
which fungi are grown on solid subs<strong>tr</strong>ate or<br />
subs<strong>tr</strong>ate moistened with a low quantity <strong>of</strong> mineral<br />
salt solution <strong>and</strong> it has a great potential to produce<br />
enzyme especially where the fermented raw<br />
materials are used as a source <strong>of</strong> nu<strong>tr</strong>ients for the<br />
fungi. The enzymes produced by this method have<br />
several applications in several fields including food<br />
<strong>and</strong> fermentation indus<strong>tr</strong>y. These enzymes are also<br />
used to prepare several bioactive compounds. SSF<br />
system is much better than the submerged system<br />
because a number <strong>of</strong> reasons. The benefits <strong>of</strong> SSF<br />
over SMF include the high production <strong>of</strong> the<br />
enzyme, <strong>and</strong> fewer effluent generation. Moreover,<br />
comparably simple equipment is required for SSF<br />
(P<strong>and</strong>ey, 1994).<br />
Neurospora is a genus <strong>of</strong> kingdom fungi that<br />
has become a popular experimental model<br />
organism (Davis et al., 2002). Laccases have<br />
copper atoms at their catalytic sites <strong>and</strong> are<br />
oxidative enzymes (EC 1.<s<strong>tr</strong>ong>10</s<strong>tr</strong>ong>.3.2) which are widely<br />
found in many species <strong>of</strong> fungi, where they are<br />
involved in lignin degradation, in higher plants<br />
where they are involved in biosynthesis <strong>of</strong> lignin<br />
(Mayer <strong>and</strong> Staples, 2002; Sharma <strong>and</strong> Kuhad,<br />
2008), in bacteria (Claus, 2003; Liers et al., 2007),<br />
<strong>and</strong> in insects (Litthauer et al., 2007). Some species<br />
<strong>of</strong> fungi <strong>and</strong> insects produce laccases as<br />
in<strong>tr</strong>acellular proteins, but most <strong>of</strong> the laccases are<br />
produced as ex<strong>tr</strong>acellular proteins by all other types<br />
<strong>of</strong> producers (Arora <strong>and</strong> Sharma, 20<s<strong>tr</strong>ong>10</s<strong>tr</strong>ong>).<br />
Laccase production in various organisms<br />
Production <strong>of</strong> laccase in fungi<br />
Laccase production occurs in various fungi over a<br />
wide range <strong>of</strong> taxa. Fungi from the deuteromycetes,<br />
ascomycetes (Aisemberg et al., 1989) as well as<br />
basidiomycetes are the known producers <strong>of</strong> laccase<br />
(Sadhasivam et al., 2008). Among them,<br />
basidiomycetes are considered efficient laccase<br />
producers, especially white rot fungi (Revankar <strong>and</strong><br />
lele, 2006; Sadhasivam et al., 2008). Laccase<br />
production has not been reported in lower fungi,<br />
Production <strong>and</strong> indus<strong>tr</strong>ial applications <strong>of</strong> laccase 3<br />
i.e., Zygomycetes <strong>and</strong> Chy<strong>tr</strong>idiomycetes. However,<br />
these groups have not yet been studied in detail<br />
(Arora <strong>and</strong> Sharma, 20<s<strong>tr</strong>ong>10</s<strong>tr</strong>ong>).<br />
Trametes versicolor, Chaetomium<br />
thermophilum <strong>and</strong> Pleurotus eryngii are well<br />
known producers <strong>of</strong> laccase. It has been reported<br />
that some Trichoderma species, including T.<br />
harzianum has the ability to produce polyphenol<br />
oxidases (Kiiskinen et al., 2004; Sadhasivam et al.,<br />
2008).<br />
Laccase has been produced by many species <strong>of</strong><br />
s<strong>of</strong>t, white rot fungi, geophilous saprophytic fungi.<br />
Laccase has also been produced by many edible<br />
mushrooms including the oyster mushroom<br />
Pleurotus os<strong>tr</strong>eatus, the rice mushroom Lentinula<br />
edodes <strong>and</strong> champignon Agaricus bisporus. Other<br />
laccase producers <strong>of</strong> wood-rotting fungi include T.<br />
hirsuta (C. hirsutus), T. villosa, T. gallica, Cerrena<br />
maxima, Lentinus tigrinus, T. ochracea, Pleurotus<br />
eryngii, Trametes (Coriolus) versicolor,<br />
Coriolopsis polyzona, etc. (Morozova et al., 2007).<br />
In fungal physiology, laccases are involved in plant<br />
pathogenesis, pigmentation, detoxification, lignin<br />
degradation (Sadhasivam et al., 2008) <strong>and</strong> also in<br />
development <strong>of</strong> morphogenesis <strong>of</strong> fungi (Baldrian,<br />
2006; Morozova et al., 2007).<br />
Laccases <strong>of</strong> wood-colonizing basidiomycetes<br />
(white rot fungi) have been thoroughly studied (not<br />
least also with respect to laccase-mediator<br />
interaction), <strong>and</strong> many <strong>of</strong> them purified <strong>and</strong><br />
characterized on the protein <strong>and</strong> gene level (Liers et<br />
al., 2007).<br />
Mishra et al. (2008) have used cyanobacterial<br />
biomass <strong>of</strong> water bloom, groundnut shell (GNS)<br />
<strong>and</strong> dye effluent as culture medium for the<br />
production <strong>of</strong> laccase by Coriolus versicolor. They<br />
found the laccase production to be <s<strong>tr</strong>ong>10</s<strong>tr</strong>ong>.15±2.21<br />
U/ml in the medium having groundnut shell <strong>and</strong><br />
cyanobacterial bloom in a ratio <strong>of</strong> 9:1 (dry weight<br />
basis) at initial pH 5.0 <strong>and</strong> 28±2 o C temperature.<br />
Half life <strong>of</strong> enzyme was 74 min at 60 o C. Kinetic<br />
analysis <strong>of</strong> laccase with ABTS were also<br />
determined, Km <strong>and</strong> Vmax were found to be 0.29mM<br />
<strong>and</strong> 9.49mol/min respectively. Azide <strong>and</strong><br />
hydroxylamine exerted significant inhibition on<br />
production <strong>of</strong> thermostable laccase.<br />
It is reported that Phanerochaete chrysosporium<br />
NCIM 1197 also secretes ex<strong>tr</strong>acellular laccase.<br />
They also studied effect <strong>of</strong> several inducers on the<br />
production <strong>of</strong> laccase. Among several inducers<br />
tested copper sulphate has the greatest tendency to<br />
enhance the produce <strong>of</strong> laccase. Laccase production<br />
increased 3.5 fold in the presence <strong>of</strong> copper sulfate
4Muhammad IMRAN et al.<br />
as compared to con<strong>tr</strong>ol. Laccase production under<br />
SSF, batch fermentation in a laboratory scale<br />
bioreactor <strong>and</strong> static liquid culture was also<br />
compared. The maximum production <strong>of</strong> laccase<br />
was achieved after five days <strong>and</strong> it was found to be<br />
48.89±1.82 U/L, 30.21±1.66 <strong>and</strong> 22.56±1.22 U/L,<br />
respectively (Gnanamani et al., 2006).<br />
The white-rot fungus Trametes pubescens MB<br />
89 is a source <strong>of</strong> the laccase production at indus<strong>tr</strong>ial<br />
level. Ex<strong>tr</strong>acellular laccase formation is<br />
considerably enhanced by the addition <strong>of</strong> Cu (II) in<br />
the low quantities in the simple glucose medium.<br />
When using glucose, a typically repressing<br />
subs<strong>tr</strong>ate, as the main carbon source, significant<br />
laccase formation by T. pubescens only started<br />
when glucose was completely consumed from the<br />
culture medium. In addition, the ni<strong>tr</strong>ogen source<br />
employed had an important effect on laccase<br />
synthesis. When using an optimized medium<br />
containing glucose (40 g/L), peptone from meat (<s<strong>tr</strong>ong>10</s<strong>tr</strong>ong><br />
g/L), <strong>and</strong> MgSO4.7H2O <strong>and</strong> stimulating enzyme<br />
formation by the addition <strong>of</strong> 2.0 mM Cu, maximal<br />
laccase activities obtained in a batch cultivation<br />
were approximately 330 U ml – l (Galhaup et al.,<br />
2002).<br />
Production <strong>of</strong> laccase in plants<br />
Laccases are a diverse group <strong>of</strong> multi-copper<br />
proteins with broad subs<strong>tr</strong>ate specificity, originally<br />
discovered in the exudates <strong>of</strong> Rhus vernicifera, the<br />
Japanese lacquer <strong>tr</strong>ee <strong>and</strong> subsequently<br />
demons<strong>tr</strong>ated as a fungal enzyme as well (Sharma<br />
<strong>and</strong> Kuhad, 2008). The plants in which the laccase<br />
enzyme has been detected include lacquer, mango,<br />
mung bean, peach, pine, prune, <strong>and</strong> sycamore<br />
(Arora <strong>and</strong> Sharma, 20<s<strong>tr</strong>ong>10</s<strong>tr</strong>ong>). Techniques are also<br />
being developed to express laccase in the crop<br />
plants. Recently, laccase has been expressed in the<br />
embryo <strong>of</strong> maize (Zea mays) seeds (Bailey et al.,<br />
2004; Arora <strong>and</strong> Sharma, 20<s<strong>tr</strong>ong>10</s<strong>tr</strong>ong>).<br />
Laccase is envolved in polymerization <strong>of</strong> lignin<br />
units; p coumaryl, coniferyl, sinapyl alcohols <strong>and</strong> in<br />
the synthesis <strong>of</strong> lignin in the plants (Morozova et<br />
al., 2007). If the comparison between plant <strong>and</strong><br />
fungal laccases is taken up, the former takes part in<br />
radical-based polymerization <strong>of</strong> lignin (Ranocha et<br />
al., 2002; Arora <strong>and</strong> Sharma, 20<s<strong>tr</strong>ong>10</s<strong>tr</strong>ong>), whereas fungal<br />
laccase con<strong>tr</strong>ibutes to lignin biodegradation due to<br />
which it has gained considerable significance in<br />
green technology (Arora <strong>and</strong> Sharma, 20<s<strong>tr</strong>ong>10</s<strong>tr</strong>ong>).<br />
Production <strong>of</strong> laccase in bacteria<br />
Laccase in bacteria is present in<strong>tr</strong>acellularly <strong>and</strong> as<br />
periplasmic protoplast (Claus, 2003; Arora <strong>and</strong><br />
Sharma, 20<s<strong>tr</strong>ong>10</s<strong>tr</strong>ong>). The first bacterial laccase was<br />
found in the plant root associated bacterium,<br />
Azospirillum lip<strong>of</strong>erum (Givaudan et al., 1993;<br />
Sharma et al., 2007; Sharma <strong>and</strong> Kuhad, 2008),<br />
where it was shown to be involved in melanin<br />
formation (Faure et al., 1994; Sharma <strong>and</strong> Kuhad,<br />
2008). Laccase has been discovered in a number <strong>of</strong><br />
bacteria including Bacillus subtilis, Bordetella<br />
compes<strong>tr</strong>is, Caulobacter crescentus, Escherichia<br />
coli, Mycobacterium tuberculosum, Pseudomonas<br />
syringae, Pseudomonas aeruginosa, <strong>and</strong> Yersinia<br />
pestis (Alex<strong>and</strong>re <strong>and</strong> Zhulin, 2000; Enguita et al.,<br />
2003; Arora <strong>and</strong> Sharma, 20<s<strong>tr</strong>ong>10</s<strong>tr</strong>ong>). Recently,<br />
Steno<strong>tr</strong>ophomonas maltophilia s<strong>tr</strong>ain was found to<br />
be laccase producing, which was used to degrade<br />
synthetic dyes (Galai et al., 2008; Arora <strong>and</strong><br />
Sharma, 20<s<strong>tr</strong>ong>10</s<strong>tr</strong>ong>).<br />
Laccase containing six putative copper binding<br />
sites were discovered in marine bacterium<br />
Marinomonas mediterranea, but no functional role<br />
was assigned to this enzyme (Amat et al., 2001;<br />
Sharma <strong>and</strong> Kuhad, 2008). Some <strong>of</strong> the reported<br />
laccases have the ability to perform the activity at<br />
very crucial conditions like in the presence <strong>of</strong> high<br />
conc. <strong>of</strong> Cl - 1 <strong>and</strong> Cu +2 <strong>and</strong> even at neu<strong>tr</strong>al pH<br />
values. The enzyme produced by Sinorhizobium<br />
meliloti is a one <strong>of</strong> the examples <strong>of</strong> such enzymes<br />
<strong>and</strong> is a protein having two subunits with pI 6.2 <strong>and</strong><br />
the molecular weight <strong>of</strong> the subunits is 45 kDa each<br />
(Morozova et al., 2007), whereas laccase produced<br />
by Pseudomonas putida is also an example <strong>of</strong> such<br />
enzyme <strong>and</strong> is a single subunit 59 kDa protein<br />
which works well at pH 7.0 (Morozova et al.,<br />
2007). Both enzymes can oxidize syringaldazine.<br />
Niladevi et al. (2009) used response surface<br />
methodology for the optimization <strong>of</strong> different<br />
nu<strong>tr</strong>itional <strong>and</strong> physical parameters for the<br />
production <strong>of</strong> laccase by the filamentous bacteria<br />
S<strong>tr</strong>eptomyces psammoticus MTCC 7334 in<br />
submerged fermentation. Incubation temperature,<br />
incubation period, agitationrate, concen<strong>tr</strong>ations <strong>of</strong><br />
yeast ex<strong>tr</strong>act, MgSO4.7H2O, <strong>and</strong> <strong>tr</strong>ace elements<br />
were found to influence laccase production<br />
significantly.<br />
A new laccase gene (cotA) was cloned from<br />
Bacillus licheniformis <strong>and</strong> expressed in Escherichia<br />
coli. The recombinant protein CotA was purified<br />
<strong>and</strong> showed spec<strong>tr</strong>oscopic properties typical for<br />
blue multi-copper oxidases. The enzyme has a<br />
molecular weight <strong>of</strong> ~65kDa <strong>and</strong> demons<strong>tr</strong>ates<br />
activity towards canonical laccase subs<strong>tr</strong>ates 2, 2’azino-bis<br />
(3-ethylnenzothiazoline-6sulphonic acid)<br />
(ABTS), syringaldazine (SGZ) <strong>and</strong> 2, 6-
dimethoxyphenol (2, 6-DMP). Kinetic constants Km<br />
<strong>and</strong> kcat for ABTS were <strong>of</strong> 6.5±0.2 µM <strong>and</strong> 83s -1 ,<br />
for SGZ <strong>of</strong> 4.3+0.2 µM <strong>and</strong> <s<strong>tr</strong>ong>10</s<strong>tr</strong>ong>0s -1 , <strong>and</strong> for 2, 6-<br />
DMP <strong>of</strong> 56.7+1.0 µM <strong>and</strong> 28 s -1 . Highest oxidizing<br />
activities towards ABTS were obtained at 85 o C<br />
(Koschorreck et al., 2009).<br />
Production <strong>of</strong> laccase in insects<br />
The laccase enzyme has also been characterized in<br />
different insects, e.g., Bombyx, Calliphora,<br />
Diploptera, Drosophila, Lucilia, M<strong>and</strong>uca, Musca,<br />
Orycetes, Papilio, Phormia, Rhodnius,<br />
Sarcophaga, Schistocerca, <strong>and</strong> Tenebrio (Arora<br />
<strong>and</strong> Sharma, 20<s<strong>tr</strong>ong>10</s<strong>tr</strong>ong>).<br />
In insects, laccases have been suggested to be<br />
active in cuticle sclerotization (Dittmer et al., 2004;<br />
Sharma <strong>and</strong> Kuhad, 2008). Recently, two is<strong>of</strong>orms<br />
<strong>of</strong> laccase 2 gene have been found to catalyse<br />
larval, pupal, <strong>and</strong> adult cuticle tanning in Tribolium<br />
castaneum (Arakane et al., 2005; Sharma <strong>and</strong><br />
Kuhad, 2008)<br />
Applications <strong>of</strong> laccase<br />
Laccases have many biotechnological applications<br />
because <strong>of</strong> their oxidation ability towards a broad<br />
range <strong>of</strong> phenolic <strong>and</strong> non-phenolic compounds<br />
(Figure 1) (Mohammadian et al., 20<s<strong>tr</strong>ong>10</s<strong>tr</strong>ong>).<br />
Other applications <strong>of</strong> laccase include the<br />
cleaning the indus<strong>tr</strong>ial effluents, mostly from<br />
indus<strong>tr</strong>ies like paper indus<strong>tr</strong>y, pulp, textile &<br />
pe<strong>tr</strong>ochemical indus<strong>tr</strong>ies. Laccase are also used in<br />
the medical diagnostics <strong>and</strong> for cleaning herbicides,<br />
pesticides <strong>and</strong> some explosives in soil. Laccase has<br />
many applications in agricultural, medicinal <strong>and</strong><br />
indus<strong>tr</strong>ial areas (Arora <strong>and</strong> Sharma, 20<s<strong>tr</strong>ong>10</s<strong>tr</strong>ong>).<br />
Laccases are also to clean the water in many<br />
purification systems. It has also applications in<br />
medical side to prepare certain drugs like anticancer<br />
drugs <strong>and</strong> it is added in cosmetics to<br />
minimize their toxic effects. Laccase has the<br />
enormous ability to remove xenobiotic substances<br />
<strong>and</strong> produce polymeric products <strong>and</strong> that is why<br />
they are being used for many bioremediation<br />
purposes (Couto <strong>and</strong> Herrera, 2006).<br />
Now researchers are working on enzymatic<br />
synthesis <strong>of</strong> organic compounds, laccase-based<br />
biooxidation, <strong>and</strong> bio<strong>tr</strong>ansformation <strong>and</strong> biosensor<br />
development. The yield <strong>of</strong> laccase can be increased<br />
by optimizing different cultural conditions (Arora<br />
<strong>and</strong> Sharma, 20<s<strong>tr</strong>ong>10</s<strong>tr</strong>ong>).<br />
Production <strong>and</strong> indus<strong>tr</strong>ial applications <strong>of</strong> laccase 5<br />
Figure 1. Scheme <strong>of</strong> applications <strong>of</strong> laccase<br />
(Morozova et al., 2007)<br />
Applications <strong>of</strong> laccase in food indus<strong>tr</strong>ies<br />
Wine stabilization<br />
Laccase is used to improve the quality <strong>of</strong> drinks<br />
<strong>and</strong> for the stabilization <strong>of</strong> certain perishable<br />
products containing plant oils (Morozova et al.,<br />
2007). In food indus<strong>tr</strong>y, wine stabilization is the<br />
main application <strong>of</strong> laccase (Duran <strong>and</strong> Esposito,<br />
2000; Rosana et al., 2002).<br />
Polyphenols have undesirable effects on wine<br />
production <strong>and</strong> on its organoleptic characteristics,<br />
so their removal from the wine is very necessary<br />
(Rosana et al., 2002). Many innovative <strong>tr</strong>eatments,<br />
such as enzyme inhibitors, complexing agents, <strong>and</strong><br />
sulfate compounds, have been proposed for the<br />
removal <strong>of</strong> phenolics responsible for discoloration,<br />
haze, <strong>and</strong> flavor changes but the possibility <strong>of</strong> using<br />
enzymatic laccase <strong>tr</strong>eatments as a specific <strong>and</strong> mild<br />
technology for stabilizing beverages against<br />
discoloration <strong>and</strong> clouding represents an at<strong>tr</strong>active<br />
alternative (Cantarelli et al., 1989; Arora <strong>and</strong><br />
Sharma, 20<s<strong>tr</strong>ong>10</s<strong>tr</strong>ong>). Since such an enzyme is not yet<br />
allowed as a food additive, the use <strong>of</strong> immobilized<br />
laccase might be a suitable method to overcome<br />
such legal barriers as in this form it may be<br />
classified as technological aid. So laccase could<br />
find application in preparation <strong>of</strong> must, wine <strong>and</strong> in<br />
fruit juice stabilization (Minussi et al., 2002; Arora<br />
<strong>and</strong> Sharma, 20<s<strong>tr</strong>ong>10</s<strong>tr</strong>ong>).<br />
Baking indus<strong>tr</strong>y<br />
In the bread-making process laccases affix bread<br />
<strong>and</strong>/or dough-enhancement additives to the bread<br />
dough, these results in improved freshness <strong>of</strong> the<br />
bread texture, flavour <strong>and</strong> the improved<br />
machinability (Minussi et al., 2002).<br />
Laccase is also one <strong>of</strong> the enzymes used in the<br />
baking indus<strong>tr</strong>y. Laccase enzyme is added in the
6Muhammad IMRAN et al.<br />
baking process which results in the oxidizing effect,<br />
<strong>and</strong> also improves the s<strong>tr</strong>ength <strong>of</strong> s<strong>tr</strong>uctures in<br />
dough <strong>and</strong>/or baked products. Laccase imparts<br />
many characteristics to the baked products<br />
including an improved crumb s<strong>tr</strong>ucture, increased<br />
s<strong>of</strong>tness <strong>and</strong> volume. A flour <strong>of</strong> poor quality can be<br />
also used in this process using laccase enzyme<br />
(Minussi et al., 2002).<br />
Applications <strong>of</strong> laccase in textile indus<strong>tr</strong>y<br />
Synthetic dyes are widely used in such indus<strong>tr</strong>ies as<br />
textile, leather, cosmetics, food <strong>and</strong> paper printing<br />
(Forgacsa et al., 2004). Reactive dyes are coloured<br />
molecules used to dye cellulose fibres (Tavares et<br />
al., 2009). These dyes result in the production <strong>of</strong><br />
large amounts <strong>of</strong> high-colored wastewater. A<br />
special problem is found in the application <strong>of</strong><br />
synthetic dyes that they are resistant to<br />
biodegradation (Wesenberg et al., 2003, Moilanen<br />
et al., 20<s<strong>tr</strong>ong>10</s<strong>tr</strong>ong>).<br />
Normally, from <s<strong>tr</strong>ong>10</s<strong>tr</strong>ong> to 50% <strong>of</strong> the initial dye<br />
load will be present in the dyebath effluent, giving<br />
rise to a highly coloured effluent (V<strong>and</strong>evivere et<br />
al., 1998; Moilanen et al., 20<s<strong>tr</strong>ong>10</s<strong>tr</strong>ong>). Therefore, the<br />
<strong>tr</strong>eatment <strong>of</strong> indus<strong>tr</strong>ial effluents containing<br />
aromatic compounds is necessary prior to final<br />
discharge to the environment (Khlifia et al., 20<s<strong>tr</strong>ong>10</s<strong>tr</strong>ong>).<br />
Nowadays, environmental regulations in most<br />
coun<strong>tr</strong>ies require that wastewater must be<br />
decolorized before its discharge (Moilanen et al.,<br />
20<s<strong>tr</strong>ong>10</s<strong>tr</strong>ong>) to r<s<strong>tr</strong>ong>edu</s<strong>tr</strong>ong>ce environmental problems related to<br />
the effluent (Tavares et al., 2009). A wide range <strong>of</strong><br />
physicochemical methods has been developed for<br />
the degradation <strong>of</strong> dye-containing wastewaters<br />
(V<strong>and</strong>evivere et al., 1998; Tavares et al., 2009).<br />
Wastewaters from textile dying process are usually<br />
<strong>tr</strong>eated by physical or chemical processes, which<br />
include physical–chemical processes elec<strong>tr</strong>okinetic<br />
coagulation, elec<strong>tr</strong>ochemical des<strong>tr</strong>uction,<br />
irradiation, precipitation, ozonation, or the Katox<br />
method that involves the use <strong>of</strong> active carbon <strong>and</strong><br />
the mixture <strong>of</strong> certain gases (air) (Banat et al.,<br />
1996, Khlifia et al., 20<s<strong>tr</strong>ong>10</s<strong>tr</strong>ong>, Tavares et al., 2009).<br />
However, due to the chemical nature, molecular<br />
size <strong>and</strong> s<strong>tr</strong>ucture <strong>of</strong> the reactive dyes these<br />
classical processes can cause a problem in the<br />
environment <strong>and</strong> better <strong>tr</strong>eatments can be obtained<br />
using bioprocesses (Tavares et al., 2009). Recently,<br />
enzymatic <strong>tr</strong>eatments have at<strong>tr</strong>acted much interest<br />
in the decolourization/degradation <strong>of</strong> textile dyes in<br />
wastewater as an alternative s<strong>tr</strong>ategy to<br />
conventional chemical <strong>and</strong> physical <strong>tr</strong>eatments,<br />
which present serious limitations (Cristovao et al.,<br />
2008, Tavares et al., 2009).<br />
Five indigenous fungi P. os<strong>tr</strong>eatus IBL-02, P.<br />
chrysosporium IBL-03, Coriolus versicolor IBL-<br />
04, G. lucidum IBL-05 <strong>and</strong> S. commune IBL-06<br />
were screened for decolorization <strong>of</strong> four vat dyes,<br />
Cibanon red 2B-MD, Cibanon golden-yellow PK-<br />
MD, Cibanon blue GFJ-MD <strong>and</strong> Indanthrene direct<br />
black RBS. The screening experiment was run for<br />
<s<strong>tr</strong>ong>10</s<strong>tr</strong>ong> days with 0.01% dye solutions prepared in<br />
alkaline Kirk’s basal nu<strong>tr</strong>ient medium in <strong>tr</strong>iplicate<br />
(250 ml flasks). Every 48 h samples were read on<br />
their respective wavelengths to determine the<br />
percent decolorization. It was observed that C.<br />
versicolor IBL-04 could effectively decolorized all<br />
the four vat dyes at varying incubation times but<br />
best results were shown on Cibanon blue GFJ-MD<br />
(90.7%) after 7 days, followed by golden yellow<br />
(88%), Indanthrene direct black (79.7%) <strong>and</strong><br />
Cibanon red (74%). P. chrysosporium also showed<br />
good decolorization potential on Cibanon blue<br />
(87%), followed by Cibanon golden-yellow<br />
(74.8%), Red (71%), <strong>and</strong> Indanthrene direct black<br />
(54.6%) (Asghar et al., 2008).<br />
Decolourization <strong>and</strong> detoxification <strong>of</strong> a textile<br />
indus<strong>tr</strong>y effluent by laccase from Trametes <strong>tr</strong>ogii in<br />
the presence <strong>and</strong> the absence <strong>of</strong> laccase mediators<br />
had been investigated. It was found that laccase<br />
alone was not able to decolourize the effluent<br />
efficiently even at the highest enzyme<br />
concen<strong>tr</strong>ation tested: less than <s<strong>tr</strong>ong>10</s<strong>tr</strong>ong>% decolourization<br />
was obtained with 9 U/mL reaction mixtures. To<br />
enhance effluent decolourization, several potential<br />
laccase mediators were tested at concen<strong>tr</strong>ations<br />
ranging from 0 to 1mM. Most potential mediators<br />
enhanced decolourization <strong>of</strong> the effluent, with 1hydroxybenzo<strong>tr</strong>iazol<br />
(HBT) being the most<br />
effective (Khlifia et al., 20<s<strong>tr</strong>ong>10</s<strong>tr</strong>ong>).<br />
Moilanen et al. (20<s<strong>tr</strong>ong>10</s<strong>tr</strong>ong>) used the crude laccases<br />
from the white-rot fungi Cerrena unicolor <strong>and</strong><br />
Trametes hirsuta for their ability to decolorize<br />
simulated textile dye baths. The dyes used were<br />
Remazol Brilliant Blue R (RBBR) (<s<strong>tr</strong>ong>10</s<strong>tr</strong>ong>0 mg/L),<br />
Congo red (12.5 mg/L), Lanaset Grey (75 mg/L)<br />
<strong>and</strong> Poly R-478 (50 mg/L). They assessed the effect<br />
<strong>of</strong> redox mediators on dye decolorization by<br />
laccases. The result was that C. unicolor laccase<br />
was able to decolorize all the dyes tested. It was<br />
especially effective towards Congo red <strong>and</strong> RBBR<br />
with 91 <strong>and</strong> 80% <strong>of</strong> color removal in 19.5 h despite<br />
the fact that simulated textile dye baths were used.<br />
Applications in pharmaceutical indus<strong>tr</strong>y<br />
Laccases have been used for the synthesis <strong>of</strong><br />
several products <strong>of</strong> pharmaceutical indus<strong>tr</strong>y (Arora<br />
<strong>and</strong> Sharma, 20<s<strong>tr</strong>ong>10</s<strong>tr</strong>ong>). The first chemical <strong>of</strong> the
pharmaceutical importance that has been prepared<br />
using laccase enzyme is actinocin that has been<br />
prepared from 4-methyl-3-hydroxyanthranilic acid.<br />
This compound has anticancer capability <strong>and</strong> works<br />
by blocking the <strong>tr</strong>anscription <strong>of</strong> DNA from the<br />
tumor cell (Burton, 2003).<br />
Another example <strong>of</strong> the anticancer drugs is<br />
Vinblastine, which is useful for the <strong>tr</strong>eatment <strong>of</strong><br />
leukemia. The plant Catharanthus roseus naturally<br />
produces vinblastine. This plant produces small<br />
amount <strong>of</strong> this compound. Katarantine <strong>and</strong><br />
vindoline are the precursors <strong>of</strong> this<br />
pharmaceutically important compound. These<br />
precursors are produced in higher quantities <strong>and</strong> are<br />
easy to purifiy. Laccase is used to convert these<br />
precursors into vinblastine. A 40% conversion <strong>of</strong><br />
these precursors into the final product has been<br />
obtained using laccase (Yaropolov et al., 1994).<br />
The use <strong>of</strong> laccase in such conversion reactions has<br />
made the preparation <strong>of</strong> several important<br />
compounds with useful properties, like antibiotics,<br />
possible (Pilz et al., 2003).<br />
Catechins have the antioxidant ability <strong>and</strong><br />
Laccases can oxidize catechins. These catechins<br />
consist <strong>of</strong> small units <strong>of</strong> tannins <strong>and</strong> these are<br />
important antioxidants found in tea, herbs <strong>and</strong><br />
vegetables. Catechins have the tendency to hunt<br />
free radicals <strong>and</strong> their property makes them useful<br />
in preventing several diseases including cancer,<br />
inflammatory <strong>and</strong> cardiovascular diseases. The<br />
catechins have less antioxidant ability; this property<br />
can be increased by using laccase <strong>and</strong> has resulted<br />
in the conversion <strong>of</strong> catechins in several products<br />
with enhanced antioxidant capability (Kurisawa et<br />
al., 2003).<br />
Laccase has applications in the synthesis <strong>of</strong><br />
hormone derivatives. In<strong>tr</strong>a et al. (2005) <strong>and</strong> Nico<strong>tr</strong>a<br />
et al. (2004) have reported that laccase has the<br />
ability to seperate innovative dimeric derivatives <strong>of</strong><br />
the β-es<strong>tr</strong>adiol <strong>and</strong> <strong>of</strong> the phytoalexin resvera<strong>tr</strong>ol.<br />
Isoeugenol oxidation coniferyl alcohol <strong>and</strong> totarol<br />
gave new dimeric derivatives (Ncanana et al.,<br />
2007) <strong>and</strong> a mixture <strong>of</strong> dimeric <strong>and</strong> te<strong>tr</strong>americ<br />
derivatives (Shiba et al., 2000) respectively,<br />
whereas the oxidation <strong>of</strong> substituted imidazole has<br />
resulted in the production <strong>of</strong> even more complex<br />
subs<strong>tr</strong>ances. These new formed imidazoles or<br />
oligomerization products (2–4) can be used for<br />
pharmacological purposes (Kurisawa et al., 2003).<br />
Aromatic <strong>and</strong> aliphatic amines can be converted<br />
into 3-(3, 4-dihydroxyphenyl)-propionic acid using<br />
laccase based oxidation. The derivatives have the<br />
antiviral natural activity <strong>and</strong> can be used for<br />
Production <strong>and</strong> indus<strong>tr</strong>ial applications <strong>of</strong> laccase 7<br />
pharmaceutical purposes (Ncanana et al., 2007).<br />
Conclusion<br />
Laccases are produced by various sources like<br />
fungi, bacteria <strong>and</strong> insects. They have many<br />
indus<strong>tr</strong>ial applications because <strong>of</strong> their innate<br />
ability <strong>of</strong> oxidation <strong>of</strong> a broad range <strong>of</strong> phenolic<br />
<strong>and</strong> non-phenolic compounds. Laccase is utilized in<br />
drink indus<strong>tr</strong>y to improve the quality <strong>of</strong> drinks <strong>and</strong><br />
for stabilization <strong>of</strong> some perishable products having<br />
plant oils. Laccases have the potential for the<br />
synthesis <strong>of</strong> several useful drugs in pharmaceutical<br />
indus<strong>tr</strong>y because <strong>of</strong> their high value <strong>of</strong> oxidation<br />
potential. Laccases have also <strong>tr</strong>emendous ability <strong>of</strong><br />
oxidation <strong>of</strong> harmful <strong>and</strong> indus<strong>tr</strong>ial products <strong>and</strong><br />
belongs to those enzymes, which have instinctive<br />
properties <strong>of</strong> immediate radical production. Laccase<br />
enzyme has the property to act on a range <strong>of</strong><br />
subs<strong>tr</strong>ates <strong>and</strong> to detoxify a range <strong>of</strong> pollutants,<br />
which have made them to be useful in many<br />
indus<strong>tr</strong>ies including paper, pulp, textile <strong>and</strong><br />
pe<strong>tr</strong>ochemical indus<strong>tr</strong>ies.<br />
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textile wet-processing indus<strong>tr</strong>y: review <strong>of</strong><br />
emerging technologies. J Chem Technol<br />
Biotechnol. 72: 289–302, 1998.<br />
Wesenberg D, Kyriakides I, Agathos N. White-rot<br />
fungi <strong>and</strong> their enzymes for the <strong>tr</strong>eatment <strong>of</strong><br />
indus<strong>tr</strong>ial dye effluents. Biotechnol Adv. 22:<br />
161–187, 2003.<br />
Yaropolov AI, Skorobogatko OV, Vartanov SS,<br />
Varfolomeyev SD. Laccase: Properties,<br />
catalytic mechanism <strong>and</strong> applicability. Appl<br />
Biochem Biotechnol. 49: 257–280, 1994.<br />
Production <strong>and</strong> indus<strong>tr</strong>ial applications <strong>of</strong> laccase 11
<strong>Journal</strong> <strong>of</strong> <strong>Cell</strong> <strong>and</strong> Molecular Biology <s<strong>tr</strong>ong>10</s<strong>tr</strong>ong>(1):13-18, 2012 Research Article 13<br />
Haliç University, Printed in Turkey.<br />
http://<s<strong>tr</strong>ong>jcmb</s<strong>tr</strong>ong>.<s<strong>tr</strong>ong>halic</s<strong>tr</strong>ong>.<s<strong>tr</strong>ong>edu</s<strong>tr</strong>ong>.<strong>tr</strong><br />
Isolation <strong>and</strong> biochemical identification <strong>of</strong> Escherichia coli from<br />
wastewater effluents <strong>of</strong> food <strong>and</strong> beverage indus<strong>tr</strong>y<br />
Tasnim FARASAT*, Zubia BILAL, Fakhar-un-Nisa YUNUS<br />
Department <strong>of</strong> Zoology, Lahore College for Women University, Lahore, Pakistan.<br />
(* author for correspondence; tasnimfarasat@hotmail.com)<br />
Received: 20 May 2011; Accepted: 11 May 2012<br />
Abs<strong>tr</strong>act<br />
The aim <strong>of</strong> this study was the isolation <strong>and</strong> biochemical identification <strong>of</strong> E. coli from indus<strong>tr</strong>ial wastewater<br />
effluents. Sixty samples were collected from different sources in Lahore. The results revealed that E.coli was<br />
found in higher concen<strong>tr</strong>ation in wastewater <strong>of</strong> food <strong>and</strong> beverage indus<strong>tr</strong>ies. Wastewater is an important<br />
reservoir for E.coli <strong>and</strong> presented significant acute toxicity if released into the receiving water body without<br />
being adequately <strong>tr</strong>eated. Results revealed the presence <strong>of</strong> both gram negative <strong>and</strong> positive bacteria. There<br />
was nonsignificant variation among all the samples <strong>of</strong> wastewater. The highest concen<strong>tr</strong>ation <strong>of</strong> E.coli was<br />
observed in wastewater <strong>of</strong> food indus<strong>tr</strong>y Site A (Rettigon road) <strong>and</strong> beverage indus<strong>tr</strong>y Site F (Wahdat road).<br />
Biochemical <strong>and</strong> serological tests confirmed the presence <strong>of</strong> E.coli.<br />
Keywords: E .coli, wastewater, food indus<strong>tr</strong>y, beverage indus<strong>tr</strong>y, effluent.<br />
Yiyecek ve içecek endüs<strong>tr</strong>isi atıksu deşarjlarından Escherichia coli eldesi ve biyokimyasal<br />
tanımlanması<br />
Özet<br />
Bu çalışmanın amacı endüs<strong>tr</strong>iyel atıksu deşarjlarından E. coli eldesi ve biyokimyasal tanımlanmasıdır.<br />
Lahor’da farklı kaynaklardan 60 örnek topl<strong>and</strong>ı. Sonuçlar yiyecek ve içecek endüs<strong>tr</strong>isi atıksularında daha<br />
fazla konsan<strong>tr</strong>asyonda E. coli bulunduğunu gösterdi. Atıksu önemli bir E.coli deposudur ve yeterli olarak<br />
muamele edilmeden alıcı su kaynağına salınırsa ileri derecede toksik olabilir. Sonuçlar hem gram pozitif,<br />
hem de gram negatif bakteri varlığını gösterdi. En yüksek E. coli konsan<strong>tr</strong>asyonu A bölgesi (Rettigon yolu)<br />
yiyecek endüs<strong>tr</strong>isinin ve F bölgesi (Wahdat yolu) içecek endüs<strong>tr</strong>isinin atıksularında gözlemlendi. Biyolojik<br />
ve serolojik testler E. coli varlığını doğruladı.<br />
Anahtar kelimeler: E.coli, atıksu, yiyecek endüs<strong>tr</strong>isi, içecek endüs<strong>tr</strong>isi, atıksu deşarjı.<br />
In<strong>tr</strong>oduction<br />
Indus<strong>tr</strong>ial waste is the most common source <strong>of</strong><br />
water pollution in the present day (Ogedengbe <strong>and</strong><br />
Akinbile, 2004) <strong>and</strong> it increases yearly due to the<br />
fact that indus<strong>tr</strong>ies are increasing as most coun<strong>tr</strong>ies<br />
are getting indus<strong>tr</strong>ialized. Indus<strong>tr</strong>ies produce wastes<br />
which are peculiar in terms <strong>of</strong> type, volume <strong>and</strong><br />
frequency depending on the type <strong>of</strong> indus<strong>tr</strong>y <strong>and</strong><br />
population that uses the product (Odumosu, 1992).<br />
Water <strong>and</strong> wastewater management constitutes a<br />
practical problem for the food <strong>and</strong> beverage<br />
indus<strong>tr</strong>y. In spite <strong>of</strong> significant improvement over<br />
the last 20 years, water consumption <strong>and</strong> disposal<br />
remain critical from environmental <strong>and</strong> economic<br />
st<strong>and</strong>point (Fillaudeau et al., 2005).<br />
A food processing indus<strong>tr</strong>y is involved with the<br />
total environment from the farm to the customer.<br />
Water is absolutely necessary for many steps in the<br />
food processing indus<strong>tr</strong>y. At present, there is no<br />
economical substitute <strong>of</strong> water. Consequently water<br />
conservation <strong>and</strong> water reuse are necessary. By
14 Tasnim FARASAT et al.<br />
practicing conservation <strong>and</strong> reuse, the amount <strong>of</strong><br />
liquid waste <strong>and</strong> pollution potential from the food<br />
processing is r<s<strong>tr</strong>ong>edu</s<strong>tr</strong>ong>ced (Mercer, 1964).<br />
On a global scale, contamination <strong>of</strong> drinking<br />
water by pathogenic bacteria causes the most<br />
significant health risk to humans, <strong>and</strong> there have<br />
been countless numbers <strong>of</strong> disease outbreaks <strong>and</strong><br />
poisonings resulting from exposure to un<strong>tr</strong>eated or<br />
poorly <strong>tr</strong>eated drinking water. However, significant<br />
risks to human health may also result from<br />
exposure to toxic contaminants that are <strong>of</strong>ten<br />
globally ubiquitous in waters from which drinking<br />
water is derived. The presence <strong>of</strong> E. coli is a<br />
definite indication <strong>of</strong> fecal contamination (WHO,<br />
2004). Some E. coli s<strong>tr</strong>ains can cause a wide variety<br />
<strong>of</strong> intestinal <strong>and</strong> ex<strong>tr</strong>a-intestinal diseases, such as<br />
diarrhea, urinary <strong>tr</strong>act infections, septicemia, <strong>and</strong><br />
neonatal meningitis (Orskov <strong>and</strong> Orskov, 1992).<br />
The magnitude <strong>of</strong> the problem <strong>of</strong> bacterial<br />
contamination deserves more elaborative studies<br />
from the point <strong>of</strong> production <strong>of</strong> waste effluents to<br />
the point <strong>of</strong> consumption at all intermediary levels.<br />
The aim <strong>of</strong> the present research was isolation <strong>and</strong><br />
biochemical identification <strong>of</strong> E.coli from indus<strong>tr</strong>ial<br />
effluents <strong>of</strong> food <strong>and</strong> beverage indus<strong>tr</strong>ies in Lahore.<br />
Material <strong>and</strong> methods<br />
Sample collection<br />
Sampling was completed in two successive months<br />
from March to April for the microbial assessment<br />
<strong>of</strong> waste effluents from food <strong>and</strong> beverage<br />
indus<strong>tr</strong>ies. Total <strong>of</strong> sixty samples were collected. In<br />
March, effluents <strong>of</strong> food indus<strong>tr</strong>ies were collected<br />
from indus<strong>tr</strong>ies near Rettigon road, Township<br />
indus<strong>tr</strong>ial area, Township indus<strong>tr</strong>ial estate. In April,<br />
effluents <strong>of</strong> beverage indus<strong>tr</strong>ies were collected from<br />
indus<strong>tr</strong>ies near Multan road <strong>and</strong> Wahdat road. Data<br />
from each sample was collected <strong>and</strong> recorded in the<br />
data book. Samples were collected in hermetically<br />
sealed, sterilized falcon tubes <strong>and</strong> were kept at 4ºC<br />
until analysis.<br />
Sample processing<br />
The technique described by Theodor Escherich,<br />
1885 was used for isolation <strong>of</strong> E.coli (Escherich,<br />
1885). To prevent contamination, the area was<br />
swabbed with 70% ethanol prior to opening any<br />
sample container. Samples (0.5 ml) were taken in<br />
<s<strong>tr</strong>ong>10</s<strong>tr</strong>ong> ml LB (Luria Bertani) broth medium in test<br />
tube, <strong>and</strong> vortexed for one minute <strong>and</strong> left for thirty<br />
minutes at room temperature. Then supernatant<br />
(1ml) was taken from this test tube <strong>and</strong> a 2-fold<br />
serial dilution was prepared (Reddy, 2007). After<br />
this, 500 ml from the final dilution tube was<br />
spreaded on the pe<strong>tr</strong>i dishes (Pyrex) <strong>of</strong> MacConkey<br />
medium <strong>and</strong> LB medium. Pe<strong>tr</strong>i dishes were kept in<br />
the incubator for 24 hours at 37ºC (Hajna <strong>and</strong><br />
Perry, 1939). After 24 hours, plates were studied<br />
for the colonies <strong>of</strong> microbes grown on the media.<br />
Microorganisms grown on MacConkey agar are<br />
capable <strong>of</strong> metabolizing lactose which produces<br />
acid by-products that lower the pH <strong>of</strong> the media<br />
which causes the neu<strong>tr</strong>al red indicator to turn red,<br />
<strong>and</strong> if sufficient acid is produced, a zone <strong>of</strong><br />
precipitated bile develops around the colony<br />
(Koneman, 2005). Different biochemical tests<br />
(Werkman, 1930; O'Meara, 1931; Vaughn et al.,<br />
1939; Silva et al., 1980) were performed for the<br />
identification <strong>of</strong> E. coli in the waste effluents <strong>of</strong><br />
food <strong>and</strong> beverage indus<strong>tr</strong>y (Table 1).<br />
Serological tests<br />
Commercial latex kits are available for O157, O26,<br />
<strong>and</strong> H7 s<strong>tr</strong>ains <strong>of</strong> E. coli. O157 antiserum has been<br />
shown to cross-react with other organisms<br />
including E. hermanii (frequently found in foods)<br />
(Hopkins <strong>and</strong> Hilton, 2000; Law, 2000). Tests<br />
incorporated positive <strong>and</strong> negative con<strong>tr</strong>ol<br />
organisms <strong>and</strong> con<strong>tr</strong>ol latex. Test was performed by<br />
a slide agglutination test using somatic (O) or<br />
flagella (H) antisera. Some pathogenic bacteria<br />
were nonmotile.<br />
Results<br />
E. coli was cultured on LB medium <strong>and</strong><br />
MacConkey medium for morphological<br />
characterization. After 24 hrs, two types <strong>of</strong> colonies<br />
were isolated under microscopic examination. All<br />
the isolated colonies were pink on MacConkey<br />
medium, while creamy yellow on LB medium.<br />
E. coli was observed in highest concen<strong>tr</strong>ation<br />
from wastewater samples <strong>of</strong> indus<strong>tr</strong>y (Site A)<br />
whereas in wastewater samples <strong>of</strong> indus<strong>tr</strong>y (Site B)<br />
six samples indicated the presence <strong>of</strong> E. coli which<br />
was confirmed by biochemical <strong>and</strong> serological test.<br />
Four samples were <strong>of</strong> gram positive bacteria which<br />
may be Bacillus subtilus or Bacillus thuringiensis.<br />
In indus<strong>tr</strong>ial effluent (Site C) eight samples were <strong>of</strong><br />
gram negative while two samples were <strong>of</strong> gram<br />
positive bacteria. It was observed that waste<br />
effluents <strong>of</strong> food indus<strong>tr</strong>y (Site A) revealed greater<br />
percentage <strong>of</strong> gram negative bacteria.<br />
In wastewater samples <strong>of</strong> indus<strong>tr</strong>y (Site D) five<br />
were gram negative, while five were gram positive<br />
bacteria. Six samples in indus<strong>tr</strong>ial effluent (Site E)
Samples<br />
Sources<br />
Indus<strong>tr</strong>ial<br />
effluent (A)<br />
Indus<strong>tr</strong>ial<br />
effluent(B)<br />
Indus<strong>tr</strong>ial<br />
effluent(C)<br />
Indus<strong>tr</strong>ial<br />
effluent(D)<br />
Indus<strong>tr</strong>ial<br />
effluent(E)<br />
Indus<strong>tr</strong>ial<br />
effluent(F)<br />
Identification <strong>of</strong> E. coli from wastewater 15<br />
Table 1. Biochemical identification <strong>of</strong> E. coli in indus<strong>tr</strong>ial wastewaters<br />
Indole<br />
Test<br />
Spot<br />
Indole<br />
Test<br />
Kovacs<br />
Indole<br />
Test<br />
Methyl Red<br />
Test<br />
Voges<br />
Proskeur<br />
Test<br />
Ammonium<br />
acetate Test<br />
Simmon's Test<br />
Ammonium<br />
Ci<strong>tr</strong>ate Test<br />
+ + + + - + -<br />
+ + + + - + -<br />
+ + + + - + -<br />
+ + + + - + -<br />
+ + + + - + -<br />
+ + + + - + -<br />
were gram negative <strong>and</strong> four were gram positive<br />
bacteria. In the wastewater samples <strong>of</strong> beverage<br />
indus<strong>tr</strong>y (Site F) all samples were <strong>of</strong> gram negative<br />
bacteria (Figure 1 <strong>and</strong> 2).<br />
The average value <strong>of</strong> gram negative bacteria in<br />
wastewater <strong>of</strong> food indus<strong>tr</strong>y (Site A) was 5.<s<strong>tr</strong>ong>10</s<strong>tr</strong>ong> ±<br />
0.34. The average value <strong>of</strong> gram negative bacteria<br />
in wastewater <strong>of</strong> food indus<strong>tr</strong>y (Site B) was 4.66 ±<br />
0.66 while in wastewater <strong>of</strong> food indus<strong>tr</strong>y (Site C)<br />
was 4.50 ± 0.50. The average value <strong>of</strong> gram<br />
negative bacteria in wastewater <strong>of</strong> beverage<br />
indus<strong>tr</strong>y (Site D) was 5.0 ± 0.70, whereas in the<br />
wastewater <strong>of</strong> beverage indus<strong>tr</strong>y (Site E) was 3.8 ±<br />
0.60 <strong>and</strong> in the wastewater <strong>of</strong> beverage indus<strong>tr</strong>y<br />
(Site F) was 4.0 ± 0.33. Student’s t-test revealed a<br />
non significant difference (P >0.05) between gram<br />
positive <strong>and</strong> gram negative bacteria.<br />
Figure 1. Percentage <strong>of</strong> gram negative bacteria in all indus<strong>tr</strong>ial wastewater samples. Food Indus<strong>tr</strong>y A:<br />
Rettigon road; Food Indus<strong>tr</strong>y B: Township indus<strong>tr</strong>ial area; Food Indus<strong>tr</strong>y C: Township indus<strong>tr</strong>ial estate;<br />
Beverage indus<strong>tr</strong>y D: Multan Road; Beverage indus<strong>tr</strong>y E: Multan road; Beverage indus<strong>tr</strong>y F: Wahadat road
16 Tasnim FARASAT et al.<br />
Figure 2. Number <strong>of</strong> E.coli colonies (mean ±SE) in wastewater samples <strong>of</strong> indus<strong>tr</strong>ies. Sites A, B, C: Food<br />
indus<strong>tr</strong>ies at Rettigon road, Township indus<strong>tr</strong>ial area, Township indus<strong>tr</strong>ial Estate Sites D, E, F: Beverage<br />
indus<strong>tr</strong>ies at Multan Road <strong>and</strong> Wahadat road.<br />
Discussion<br />
The bacterium E. coli is one <strong>of</strong> the best <strong>and</strong> most<br />
thoroughly studied free-living organisms. It is also<br />
a remarkably diverse species because some E.coli<br />
s<strong>tr</strong>ains live as harmless commensals in animal<br />
intestines. E. coli is a widely used indicator <strong>of</strong> fecal<br />
contamination in water bodies. External contact <strong>and</strong><br />
subsequent ingestion <strong>of</strong> bacteria from fecal<br />
contamination can cause de<strong>tr</strong>imental health effects<br />
(Money et al., 2009).<br />
Stomach cramps, nausea <strong>and</strong> vomiting are the<br />
symptoms caused by E. coli, however serious<br />
complications can also occur. Water samples were<br />
the only nonfecal samples that tested positive for E.<br />
coli. Water has been implicated in human outbreaks<br />
<strong>and</strong> the studies revealed that water may be an<br />
important source <strong>of</strong> 0157:H7 on farms (Karmali,<br />
1989).<br />
The present research work was conducted to<br />
isolate E. coli from food <strong>and</strong> beverage indus<strong>tr</strong>ial<br />
effluents. Effluents are good primary reservoir for<br />
E. coli. Sixty different samples from food <strong>and</strong><br />
beverage indus<strong>tr</strong>ies were processed for the isolation<br />
<strong>of</strong> E. coli. The food <strong>and</strong> beverage indus<strong>tr</strong>ies uses<br />
large volume <strong>of</strong> water as it is suitable, clean, <strong>and</strong> a<br />
quite inexpensive resource, both as a constituent <strong>of</strong><br />
many products, <strong>and</strong> for other production<br />
requirements. Microbial growth in drinks due to<br />
contaminated water supplies or sugar syrups can<br />
cause discoloration, <strong>of</strong>f flavors <strong>and</strong> shortened shelflife,<br />
as well as increasing the risk <strong>of</strong> infection to<br />
consumers (Noronha et al., 2002).<br />
However, selective media are universally used<br />
in water monitoring <strong>and</strong> were employed in the<br />
United States Environmental Protection Agency<br />
epidemiological investigations, suggesting that<br />
culturable fecal indicator counts are valid predictors<br />
<strong>of</strong> disease risk (Sinton et al., 1994). Sewage can<br />
serve as a vehicle for entering into human <strong>and</strong><br />
nonhuman hosts either by direct contact or through<br />
contamination <strong>of</strong> drinking water supplies (Boczek<br />
et al., 2007).<br />
The results revealed that the highest percentage<br />
<strong>of</strong> E. coli was observed in the waste effluents <strong>of</strong><br />
indus<strong>tr</strong>ies (A <strong>and</strong> F). ANOVA showed non<br />
significant (P > 0.05) variation. Student’s t-test also<br />
revealed non significant difference between gram<br />
positive <strong>and</strong> gram negative bacteria.<br />
According to Boczek <strong>and</strong> colleagues (2007) the<br />
occurrence <strong>of</strong> clonal group in wastewater<br />
demons<strong>tr</strong>ates a potential mode for the dissemination<br />
<strong>of</strong> this clonal group in the environment, with<br />
possible secondary <strong>tr</strong>ansmission to human or<br />
animal hosts. Chalmers <strong>and</strong> colleagues (2000)<br />
demons<strong>tr</strong>ated that the effluent had a significant<br />
pollution potential, mainly due to the low pH <strong>and</strong><br />
high concen<strong>tr</strong>ation <strong>of</strong> E. coli. The results also
demons<strong>tr</strong>ated that the wastewater presented<br />
significant acute toxicity, <strong>and</strong> could cause diseases<br />
if released into the receiving body without being<br />
adequately <strong>tr</strong>eated. This represents a dangerous<br />
public health risk, which needs future evaluation<br />
<strong>and</strong> con<strong>tr</strong>ol. Culture-independent analysis in<br />
various environmental samples has been used to<br />
catalog this species <strong>and</strong> also to assess the impact <strong>of</strong><br />
human activity <strong>and</strong> interactions with microbes on<br />
natural microbial communities.<br />
According to Barreto-Rodrigues <strong>and</strong> colleagues<br />
(2008), the objective <strong>of</strong> the work was to<br />
characterize the effluent originating from a<br />
Brazilian TNT production indus<strong>tr</strong>y. Analyses were<br />
performed using physical, chemical, spec<strong>tr</strong>oscopic<br />
<strong>and</strong> ecotoxicological assays, which demons<strong>tr</strong>ated<br />
that the effluent had a significant pollution<br />
potential, mainly due to the low pH <strong>and</strong> high<br />
concen<strong>tr</strong>ation <strong>of</strong> TNT (156 ± <s<strong>tr</strong>ong>10</s<strong>tr</strong>ong> mg L −1 ). The<br />
results also demons<strong>tr</strong>ated that the effluent causes<br />
significant acute toxicity, <strong>and</strong> could cause countless<br />
damages if released into the rivers without being<br />
properly <strong>tr</strong>eated. The observed pollution potential<br />
justifies studies to evaluate <strong>tr</strong>eatment technologies<br />
or recover the residue generated in the TNT<br />
indus<strong>tr</strong>y. From a total <strong>of</strong> 149 E. coli s<strong>tr</strong>ains, 87 E.<br />
coli s<strong>tr</strong>ains were from raw wastewater <strong>and</strong> 62<br />
s<strong>tr</strong>ains from <strong>tr</strong>eated wastewater by stabilization<br />
ponds. Within these s<strong>tr</strong>ains two <strong>and</strong> four positive<br />
serological reaction to E. coli 0157 were found for<br />
raw <strong>and</strong> <strong>tr</strong>eated wastewater, respectively.<br />
In the same direction, Muller <strong>and</strong> his colleagues<br />
(2001) carried out a study on E.coli 0157:H7 s<strong>tr</strong>ains<br />
in water sources in South Africa <strong>and</strong> they did not<br />
find any evidence <strong>of</strong> EHEC 0157 while virulence<br />
factors present in the 96% <strong>of</strong> analyzed samples<br />
(196), however 8 isolates from 8 samples<br />
demons<strong>tr</strong>ated the presence <strong>of</strong> Stx1 <strong>and</strong> Stx2.<br />
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<strong>Journal</strong> <strong>of</strong> <strong>Cell</strong> <strong>and</strong> Molecular Biology <s<strong>tr</strong>ong>10</s<strong>tr</strong>ong>(1):19-26, 2012 Research Article 19<br />
Haliç University, Printed in Turkey.<br />
http://<s<strong>tr</strong>ong>jcmb</s<strong>tr</strong>ong>.<s<strong>tr</strong>ong>halic</s<strong>tr</strong>ong>.<s<strong>tr</strong>ong>edu</s<strong>tr</strong>ong>.<strong>tr</strong><br />
Investigation <strong>of</strong> the MGP promoter <strong>and</strong> exon 4 polymorphisms in<br />
patients with ischemic s<strong>tr</strong>oke in the Ukrainian population<br />
Alex<strong>and</strong>er V. ATAMAN *1, Victoria Y. GARBUSOVA 2 , Yuri A. ATAMAN 3 , Olga I.<br />
MATLAJ 4 , Olga A. OBUCHOVA 1<br />
1 Sumy State University, Department <strong>of</strong> Physiology, Pathophysiology <strong>and</strong> Medical Biology, Sumy, Ukraine<br />
2 Sumy State University, Scientific Laboratory <strong>of</strong> Molecular Genetic Research, Sumy, Ukraine<br />
3 Sumy State University, Department <strong>of</strong> Internal Medicine, Sumy, Ukraine<br />
4<br />
Sumy Clinical Hospital No.5, Sumy, Ukraine<br />
(*author for correspondence; ataman_av@mail.ru )<br />
Received: 13 February 2012; Accepted: 18 May 2012<br />
Abs<strong>tr</strong>act<br />
Ma<strong>tr</strong>ix γ-carboxyglutamic acid protein (MGP) is a vitamin K-dependent protein playing a pivotal role in<br />
preventing arterial calcification. In the present study, we aimed to investigate the relation between three<br />
single nucleotide polymorphisms <strong>of</strong> MGP gene <strong>and</strong> ischemic s<strong>tr</strong>oke (IS) in the Ukrainian population. 170 IS<br />
patients <strong>and</strong> 124 healthy con<strong>tr</strong>ols were recruited to the study. MGP SNPs were examined by PCR-RFLP<br />
methodology. The dis<strong>tr</strong>ibution <strong>of</strong> homozygous carriers <strong>of</strong> the major allelic variant, <strong>and</strong> heterozygous <strong>and</strong><br />
homozygous minor allele variants <strong>of</strong> the T-138C MGP promoter polymorphism (rs1800802) in patients with<br />
IS was 61.2%, 31.2% <strong>and</strong> 7.6%, respectively. The corresponding dis<strong>tr</strong>ibutions <strong>of</strong> the variants in the con<strong>tr</strong>ol<br />
group were 59.7%, 35.6%, 4.8%. With regard to the G-7A promoter polymorphism (rs1800801), the<br />
respective dis<strong>tr</strong>ibutions were 35.9%, 48.8% <strong>and</strong> 15.3%, compared to 43.5%, 50% <strong>and</strong> 6.5% in the con<strong>tr</strong>ol<br />
group. Finally, the respective dis<strong>tr</strong>ibutions according to the Thr83Ala exon 4 polymorphism (rs4236) were<br />
39.4%, 48.8% <strong>and</strong> 11.8%, compared to 34.7%, 53.2% <strong>and</strong> 12.1% in the con<strong>tr</strong>ol group. Using logistic<br />
regression analysis, it was estimated that A/A genotype (G-7A polymorphism) was significantly (P=0.016)<br />
associated with IS (OR=2.943; 95% CI: 1.218–7.<s<strong>tr</strong>ong>10</s<strong>tr</strong>ong>9) in the Ukrainian population. A-allele homozygotes <strong>of</strong><br />
female sex had a risk <strong>of</strong> IS more than 7 times higher compared with carriers <strong>of</strong> G/G genotype.<br />
Keywords: Ma<strong>tr</strong>ix Gla protein, single nucleotide polymorphism, ischemic s<strong>tr</strong>oke, arterial calcification,<br />
Ukrainian population.<br />
Ukrayna popülasyonunda iskemik inme hastalarında MGP promotör ve ekzon 4<br />
polimorfizminin araştırılması<br />
Özet<br />
Ma<strong>tr</strong>is γ–karboksiglutamik asit proteini (MGP) vitamin-K bağımlı protein olup arteriyal kalsitleşmeyi<br />
önlemede önemli rol oynar. Bu çalışmada, MGP geninin üç tek nükleotit polimorfizmi (TNP) ile Ukrayna<br />
popülasyonunda iskemik inme (İİ) arasındaki ilişkiyi araştırmayı hedefledik. Çalışmaya 170 İİ hastası ve 124<br />
sağlıklı kon<strong>tr</strong>ol katıldı. MGP TNP’leri PCR-RFLP metodolojisi ile test edildi. İH hastalarında T-138C MGP<br />
promoter polimorfizminin (rs1800802) majör alel varyantının homozigot taşıyıcılarının ve heterozigot ve<br />
homozigot minör alel varyantlarının dağılımları sırası ile, %61.2, %31.2 ve %7.6’dır. Kon<strong>tr</strong>ol grubunda ilgili<br />
varyant dağılımları %59.7, %35.6 ve %4.8’dir. G-7A promoter polimorfizminde (rs1800801) ise ilgili<br />
dağılımlar %43.5, %50 ve %6.5 olan kon<strong>tr</strong>ol grubu ile karşılaştırıldığında %35.9, %48.8 ve %15.3’dir. Son<br />
olarak, Thr83Ala ekzon 4 polimorfizmine (rs4236) göre dağılımlar %34.7, %53.2 ve %12.1 olan kon<strong>tr</strong>ol<br />
grubu ile karşılaştırıldığında %39.4, %48.8 ve %11.8’dir. Lojistik regresyon analizi kullanarak, Ukrayna<br />
popülasyonunda İİ ile A/A genotipinin (G-7A polimorfizmi) anlamlı (P=0.016) olarak ilişkili olduğu<br />
(OR=2.943; 95% CI: 1.218–7.<s<strong>tr</strong>ong>10</s<strong>tr</strong>ong>9) tahmin edilmiştir.<br />
Anahtar kelimeler: Ma<strong>tr</strong>is Gla protein, tek nükleotid polimorfizmi, iskemik inme, arteriyal kalsifikasyon,<br />
Ukrayna popülasyonu.
20 Alex<strong>and</strong>er V. ATAMAN et al.<br />
In<strong>tr</strong>oduction<br />
Ischemic s<strong>tr</strong>oke (IS) is, in many instances, the<br />
consequence <strong>of</strong> a thrombus forming on a ruptured<br />
atherosclerotic plaque. Ex<strong>tr</strong>acellular ma<strong>tr</strong>ix<br />
calcification is considered to be a novel marker <strong>of</strong><br />
atherosclerosis <strong>and</strong> related to both coronary artery<br />
<strong>and</strong> cerebrovascular disease. It has been shown that<br />
arterial calcification in major vessel beds is<br />
associated with vascular brain disease (Bos et al.,<br />
2011).<br />
Recent studies suggest that in addition to<br />
modifiable risk factors, such as hypertension,<br />
hyperlipidemia, <strong>and</strong> cigarette smoking, there is a<br />
s<strong>tr</strong>ong genetic component to the development <strong>of</strong><br />
arterial calcification. For instance, the heritability<br />
<strong>of</strong> the presence <strong>of</strong> coronary artery calcification has<br />
been estimated to be up to 50% (Post et al., 2007).<br />
Key genes known to be involved in the<br />
regulation <strong>of</strong> the complex process <strong>of</strong> ectopic s<strong>of</strong>t<br />
tissue mineralization are those acting as<br />
calcification inhibitors such as ma<strong>tr</strong>ix γcarboxyglutamic<br />
acid protein (MGP), osteocalcin<br />
(BGP), osteoprotegerin (Opg), <strong>and</strong> fetuin (Abedin<br />
et al., 2004; Doherty et al., 2004; Giachelli, 2004;<br />
Guzman, 2007; Weissen-Plenz et al., 2008).<br />
Among those, MGP, a vitamin K-dependent<br />
protein, is widely accepted as playing a pivotal role<br />
in preventing local mineralization <strong>of</strong> the vascular<br />
wall (Luo et al., 1997; Schurgers et al., 2005;<br />
Proudfoot <strong>and</strong> Shanahan, 2006). It has been shown<br />
that the anticalcifying activity <strong>of</strong> MGP depends<br />
upon the γ-carboxylation <strong>of</strong> specific glutamic acid<br />
(Glu) residues in MGP. This vitamin K-dependent<br />
reaction yields γ-carboxyglutamic acid (Gla)<br />
residues, which are then able to bind calcium<br />
(Murshed et al., 2004).<br />
The human MGP gene is located on<br />
chromosome 12p (Cancela et al., 1990). Among the<br />
large number <strong>of</strong> identified MGP single nucleotide<br />
polymorphisms (SNPs) eight are under the most<br />
intensive investigation: two SNPs are located in<br />
exons, <strong>and</strong> six in the ups<strong>tr</strong>eam region <strong>of</strong> the MGP<br />
gene. In vi<strong>tr</strong>o studies suggest that SNPs in MGP are<br />
associated with altered promoter activity<br />
(Herrmann et al., 2000; Farzaneh-Far et al., 2001;<br />
Kobayashi et al., 2004). In addition, there is some<br />
evidence that MGP SNPs are associated with<br />
arterial calcification (Herrmann et al., 2000;<br />
Brancaccio et al., 2005; Crosier et al., 2009),<br />
although these results are not consistent (Kobayashi<br />
et al., 2004; Taylor et al., 2005).<br />
There are a large number <strong>of</strong> studies in which the<br />
association <strong>of</strong> varies gene polymorphisms with IS<br />
has been investigated (Kubo, 2008; Debette <strong>and</strong><br />
Seshadri, 2009; Matarin et al., 2009; Wang et al.,<br />
2009; Low et al., 2011), but only in one <strong>of</strong> them the<br />
MGP SNPs were a subject <strong>of</strong> interest (del Rio-<br />
Espinola et al., 20<s<strong>tr</strong>ong>10</s<strong>tr</strong>ong>).<br />
The purpose <strong>of</strong> the present study was to<br />
investigate the association <strong>of</strong> three MGP SNPs (T-<br />
138C, G-7A, Thr83Ala) with IS in the Ukrainian<br />
population.<br />
Materials <strong>and</strong> methods<br />
Study groups<br />
The study recruited 170 IS patients (57,6% men<br />
<strong>and</strong> 42,4% women) 40 to 85 years <strong>of</strong> age (mean age<br />
[± SE] 64,7±0,7) admitted to Sumy Clinical<br />
Hospital No.5. A final diagnosis <strong>of</strong> IS was<br />
established on the basis <strong>of</strong> clinical, computed<br />
tomography <strong>and</strong> magnetic resonance imaging<br />
examinations. Each case <strong>of</strong> IS was assessed<br />
according to TOAST criteria (Adams et al., 1993).<br />
The patients with IS <strong>of</strong> cardioembolic origin <strong>and</strong><br />
undetermined etiology were excluded from the<br />
study group. The con<strong>tr</strong>ol group consisted <strong>of</strong> 124<br />
clinically healthy individuals with the absence <strong>of</strong><br />
cardio- <strong>and</strong> cerebrovascular pathologies, as<br />
confirmed by medical history, ECG, <strong>and</strong><br />
measurement <strong>of</strong> arterial pressure <strong>and</strong> biochemical<br />
data. The study had been previously approved by<br />
the Ethic Committee <strong>of</strong> the Medical Institute <strong>of</strong><br />
Sumy State University. Appropriate informed<br />
consent was obtained from all patients <strong>and</strong> con<strong>tr</strong>ol<br />
subjects. The participants were unrelated Ukrainian<br />
people from the northeastern region <strong>of</strong> Ukraine.<br />
Blood sampling for genotyping was performed<br />
under sterile conditions into 2.7 ml tubes (S-<br />
Monovette [Sarstedt, Germany]) containing EDTA<br />
potassium salt as an anticoagulant, samples were<br />
frozen <strong>and</strong> stored at -20ºC.<br />
Genotyping <strong>of</strong> SNPs<br />
DNA for genotyping was ex<strong>tr</strong>acted from the venous<br />
blood using commercially available kits (Isogene<br />
Lab Ltd, Russia) according to the manufacturer’s<br />
protocol. To identify MGP SNPs the polymerase<br />
chain reaction (PCR) with subsequent res<strong>tr</strong>iction<br />
fragment length polymorphism (RFLP) analysis<br />
was performed as previously described (Garbuzova<br />
et al., 2012). Briefly, specific regions <strong>of</strong> the MGP<br />
gene were amplified using pairs <strong>of</strong> specific primers.
For T-138C polymorphism (rs1800802) they<br />
were (F) 5`-<br />
AAGCATACGАТGGCCAAAACTTCTGCA-3`<br />
<strong>and</strong> (R) 5`-<br />
GAACTAGCAТТGGAACTTTTCCCAACC-3`;<br />
for G-7A polymorphism (rs1800801): (F) 5`-<br />
CTAGTTCAGTGCCAACCCTTCCCCACC-3`<br />
<strong>and</strong> (R) 5`-<br />
TAGCAGCAGTAGGGAGAGAGGCTCCCA-3`;<br />
for Thr83Ala polymorphism (rs4236): (F) 5`-<br />
TCAATAGGGAAGCCTGTGATG-3` <strong>and</strong> (R) 5`-<br />
AGGGGGATACAAAATCAGGTG -3`. PCR<br />
products were digested using res<strong>tr</strong>iction enzymes:<br />
BseNI (for T-138C), NcoI (for G-7A), <strong>and</strong> Eco477<br />
(for Thr83Ala). The res<strong>tr</strong>iction fragments were<br />
separated by elec<strong>tr</strong>ophoresis <strong>and</strong> analysed on an<br />
ethidium bromide-stained 2.5% agarose gel<br />
visualized using ul<strong>tr</strong>aviolet <strong>tr</strong>ansillumination.<br />
Statistical analysis<br />
Using the Pearson χ 2 test, allelic frequencies in<br />
healthy con<strong>tr</strong>ols <strong>and</strong> IS patients were found to be in<br />
Hardy-Weinberg equilibrium. Statistical analysis<br />
was performed to assess the independent main <strong>and</strong><br />
Genotype Con<strong>tr</strong>ol group<br />
(n=124)<br />
MGP polymorphisms in ischemic s<strong>tr</strong>oke 21<br />
joint effects <strong>of</strong> all analyzed SNPs. To detect the<br />
s<strong>tr</strong>ongest main effect <strong>of</strong> three MGP SNPs the<br />
logistic regression method was applied by using<br />
SPSS 17.0. A comparison <strong>of</strong> variables between the<br />
IS subgroups was performed using ANOVA.<br />
Differences were considered statistically significant<br />
with a P-value < 0.05.<br />
Results<br />
Genotypes <strong>of</strong> three studied MGP polymorphisms<br />
are summarized in Table 1. As shown, major allele<br />
homozygous <strong>and</strong> heterozygous, <strong>and</strong> minor allele<br />
homozygous T-138C polymorphisms <strong>of</strong> the MGP<br />
promoter were detected in 61.2%, 31.2% <strong>and</strong> 7.6%<br />
<strong>of</strong> the IS group, respectively (con<strong>tr</strong>ol group: 59.7,<br />
35.5% <strong>and</strong> 4.8%). Analysis <strong>of</strong> the G-7A promoter<br />
polymorphism yielded respective figures <strong>of</strong> 35.9%,<br />
48.8% <strong>and</strong> 15.3% (con<strong>tr</strong>ol group: 43.5%, 50% <strong>and</strong><br />
6.5%). The dis<strong>tr</strong>ibution <strong>of</strong> genotypes when<br />
analyzing Thr83Ala polymorphism (exon 4) was<br />
39.4%, 48.8% <strong>and</strong> 11.8% in IS group (con<strong>tr</strong>ol<br />
group: 34.7%, 53.2% <strong>and</strong> 12.1).<br />
Table 1. Genotypes <strong>of</strong> MGP polymorphisms in patients with ischemic s<strong>tr</strong>oke (IS) <strong>and</strong> con<strong>tr</strong>ol subjects. Data<br />
presented as n (%). A – major allele; a – minor allele<br />
Promoter T-138C Promoter G-7A Exon 4 Thr83Ala<br />
IS group<br />
(n=170)<br />
Con<strong>tr</strong>ol group<br />
(n=124)<br />
IS group<br />
(n=170)<br />
Con<strong>tr</strong>ol group<br />
(n=124)<br />
IS group<br />
(n=170)<br />
AA 74 (59.7) <s<strong>tr</strong>ong>10</s<strong>tr</strong>ong>4 (61.2) 54 (43.5) 61 (35.9) 43 (34.7) 67 (39.4)<br />
Aa 44 (35.5) 53 (31.2) 62 (50.0) 83 (48.8) 66 (53.2) 83 (48.8)<br />
aa 6 (4.8) 13 (7.6) 8 (6.5) 26 (15.3) 15 (12.1) 20 (11.8)<br />
The differences in the dis<strong>tr</strong>ibution <strong>of</strong> allelic<br />
variants between the con<strong>tr</strong>ol <strong>and</strong> IS groups were<br />
close to the level <strong>of</strong> statistical significance only for<br />
the G-7A promoter polymorphism (P=0,051). In<br />
women, but not in men, the differences between G-<br />
7A genotypes frequency in IS <strong>and</strong> con<strong>tr</strong>ols were<br />
significant as shown in Table 2.<br />
Using logistic regression analysis (Table 3), it<br />
was estimated that A/A genotype (G-7A<br />
polymorphism) was significantly (P=0.016)<br />
associated with IS (OR=2.943; 95% CI, 1.218 –<br />
7.<s<strong>tr</strong>ong>10</s<strong>tr</strong>ong>9). Respective analysis for male <strong>and</strong> female<br />
subjects is presented in Table 4. Women who were<br />
minor A-allele homozygotes had a risk <strong>of</strong> IS more<br />
then 7 times higher compared with female carriers<br />
<strong>of</strong> G/G genotype.<br />
Some clinical characteristics <strong>of</strong> IS patients with<br />
various MGP genotypes are presented in Table 5.<br />
There were no differences in the studied parameters<br />
between major allele homozygotes, heterozygotes,<br />
<strong>and</strong> minor allele homozygotes for all three<br />
polymorphisms (with the exception <strong>of</strong> sex<br />
dis<strong>tr</strong>ibution for G-7A polymorphism).
22 Alex<strong>and</strong>er V. ATAMAN et al.<br />
Table 2. Genotypes <strong>of</strong> G-7A MGP promoter polymorphism in female <strong>and</strong> male patients with ischemic<br />
s<strong>tr</strong>oke (IS) <strong>and</strong> con<strong>tr</strong>ol subjects. Data presented as n (%).<br />
Table 3. Results <strong>of</strong> logistic regression analysis <strong>of</strong> association between MGP polymorphisms <strong>and</strong> ischemic<br />
s<strong>tr</strong>oke.Homozygotes by major allele were considered as a reference group. SE – st<strong>and</strong>ard error, OR – odds<br />
ratio, CI – confidential interval<br />
SNP Genotype<br />
Women Men<br />
Genotype Con<strong>tr</strong>ol IS Con<strong>tr</strong>ol IS<br />
Coefficient <strong>of</strong><br />
regression<br />
SE<br />
Wald<br />
statistic<br />
Pvalue<br />
OR<br />
%95 CI<br />
Lower<br />
%95 CI<br />
Upper<br />
Promoter T/C -0.186 0.258 0.521 0.470 0.830 0.500 1.377<br />
T-138C C/C 0.382 0.526 0.527 0.468 1.465 0.522 4.<s<strong>tr</strong>ong>10</s<strong>tr</strong>ong>7<br />
Promoter G/A 0.193 0.253 0.584 0.445 1.213 0.739 1.991<br />
G-7A A/A 1.079 0.450 5.752 0.016 2.943 1.218 7.<s<strong>tr</strong>ong>10</s<strong>tr</strong>ong>9<br />
Exon 4 Thr/Ala -0.235 0.259 0.824 0.364 0.790 0.476 1.313<br />
Thr83Ala Ala/Ala -0.265 0.402 0.435 0.5<s<strong>tr</strong>ong>10</s<strong>tr</strong>ong> 0.767 0.349 1.687<br />
Discussion<br />
Arterial calcification is an abnormal process that<br />
can greatly increase morbidity <strong>and</strong> mortality (Lehto<br />
et al., 1996). MGP is considered one <strong>of</strong> the most<br />
relevant physiological inhibitors <strong>of</strong> s<strong>of</strong>t tissue<br />
mineralization known today. In mice, targeted<br />
deletion <strong>of</strong> the MGP gene causes extensive<br />
calcification <strong>of</strong> the elastic lamellae <strong>of</strong> the<br />
abdominal aorta (Luo et al., 1997). Extensive<br />
vascular calcification is also induced when γcarboxylation<br />
<strong>of</strong> MGP is inhibited using the<br />
vitamin K-antagonist, warfarin (Price et al., 1998).<br />
In the present study, we explored association<br />
between genetic variation in the MGP gene <strong>and</strong> the<br />
risk <strong>of</strong> IS development. Analysing MGP SNPs, we<br />
found the G-7A promoter polymorphism to be<br />
associated with IS in Ukrainian population. We did<br />
not revealed statistically significant relation<br />
between the other two studied polymorphisms (T-<br />
138C, Thr83Ala) <strong>and</strong> IS.<br />
Published data on the MGP SNPs association<br />
with MGP serum concen<strong>tr</strong>ation <strong>and</strong> artery<br />
calcification, <strong>and</strong> the consequences <strong>of</strong><br />
G/G 18 (40.0) 21 (29.2) 36 (45.6) 40 (40.8)<br />
G/A 25 (55.6) 34 (47.2) 37 (46.8) 49 (50.0)<br />
A/A 2 (4.4) 17 (23.6) 6 (7.6) 9 (9.2)<br />
Total 45 72 79 98<br />
P-value 0.022 0.798<br />
atherosclerosis (myocardial infarction in<br />
particularly) are con<strong>tr</strong>adictory.<br />
Farzaneh et al. (2001) did not find any<br />
relationship between the G-7A polymorphism <strong>and</strong><br />
serum MGP level in healthy persons (Netherl<strong>and</strong>s),<br />
but did detect the significant association <strong>of</strong> T-138C<br />
polymorphism with above-mentioned parameter.<br />
The highest level <strong>of</strong> serum MGP was revealed in<br />
the C/C homozygotes <strong>and</strong> the lowest one – in T/T<br />
homozygotes.<br />
In con<strong>tr</strong>ast to the above study, Crosier et al.<br />
(2009) found no association <strong>of</strong> the T-138C<br />
polymorphism with serum MGP concen<strong>tr</strong>ation, but<br />
they showed a significant relationship between the<br />
other two polymorphisms (G-7A, Tht83Ala) <strong>and</strong><br />
serum MGP levels in the healthy men <strong>and</strong> women<br />
(USA). In minor allele homozygotes, the serum<br />
MGP concen<strong>tr</strong>ation was the lowest, in major allele<br />
homozygotes the highest, in heterozygotes the<br />
intermediate values were registered.<br />
In the same study, it was shown that all three<br />
MGP SNPs (T-138C, G-7A, Thr83Ala) are related<br />
to the coronary artery calcification (CAC) in men,<br />
but not in women (Crosier et al., 2009).
MGP polymorphisms in ischemic s<strong>tr</strong>oke 23<br />
Table 4. Logistic regression analysis <strong>of</strong> association between G-7A MGP promoter polymorphism <strong>and</strong><br />
ischemic s<strong>tr</strong>oke in male <strong>and</strong> female subjects. OR – odds ratio, CI – confidential interval<br />
Sex Allele OR (CI) P-value<br />
Women A/A vs. G/G 7.286 (1.479-35.895) 0.015<br />
G/A vs. G/G 1.166 (0.516-2.632) 0.712<br />
Men A/A vs. G/G 1.350 (0.437-4.166) 0.602<br />
G/A vs. G/G 1.192 (0.641-2.217) 0.579<br />
Table 5. Clinical characteristics <strong>of</strong> ischemic s<strong>tr</strong>oke patients with respect to genotypes. Data are mean ± SE.<br />
A/A A/a a/a P<br />
T-138C polymorphism<br />
n <s<strong>tr</strong>ong>10</s<strong>tr</strong>ong>4 53 13<br />
Age, years 65.4±0.92 63.0±1.39 64.7±2.13 0.288<br />
Gender, M/F 58/46 35/18 5/8 0.162*<br />
BMI (M), kg/m 2 27.8±0.56 27.4±0.64 28.1±1.22 0.862<br />
BMI (F), kg/m 2 29.1±0.74 29.4±0.93 29.0±1.17 0.789<br />
Systolic BP, mmHg 168±2.9 165±3.6 168±9.8 0.780<br />
Diastolic BP, mmHg 96±1.7 94±1.8 93±3.8 0.628<br />
Fasting glucose, mmol/L 5.9±0.15 5.9±0.2 6.1±0.53 0.916<br />
G-7A polymorphism<br />
n 61 83 26<br />
Age, years 63.0±1.15 65.3±1.04 66.8±2.09 0.164<br />
Gender, M/F 40/21 49/34 9/17 0.026*<br />
BMI (M), kg/m 2 27.2±0.45 27.9±0.68 28.6±1.53 0.536<br />
BMI (F), kg/m 2 28.3±0.8 29.9±0.86 28.2±1.13 0.315<br />
Systolic BP, mmHg 167±3.7 167±3.3 167±5.2 0.996<br />
Diastolic BP, mmHg 97±2.0 94±1.8 97±2.4 0.593<br />
Fasting glucose, mmol/L 5.8±0.18 6.0±0.17 6.2±0.35 0.481<br />
Thr83Ala polymorphism<br />
n 67 83 20<br />
Age, years 65.1±1.2 64.4±1.0 64.7±2.1 0.912<br />
Gender, M/F 44/23 45/38 9/11 0.176*<br />
BMI (M), kg/m 2 27.6±0.47 27.6±0.67 28.3±1.9 0.891<br />
BMI (F), kg/m 2 29.4±0.84 28.6±0.78 29.9±1.53 0.668<br />
Systolic BP, mmHg 163±3.7 171±3.0 163±6.6 0.252<br />
Diastolic BP, mmHg 95±1.8 96±1.7 95±4.4 0.812<br />
Fasting glucose, mmol/L 5.9±0.2 6.0±0.17 5.9±0.3 0.859
24 Alex<strong>and</strong>er V. ATAMAN et al.<br />
In some studies, MGP polymorphisms were<br />
also shown to be associated with arterial<br />
calcification <strong>and</strong> myocardial infarction (MI)<br />
(Herrmann et al., 2000; Brancaccio et al., 2005),<br />
while in others (Kobayashi et al., 2004; Taylor et<br />
al., 2005) no association between MGP SNPs <strong>and</strong><br />
cardiovascular events was found. Moreover, in the<br />
studies in which such associations were reported,<br />
the relationship between the type <strong>of</strong> MGP<br />
polymorphism <strong>and</strong> arterial calcification was<br />
different. For example, in the AXA study, the<br />
minor alleles -7A <strong>and</strong> 83Ala were associated with<br />
increased femoral artery calcification (Herrmann et<br />
al., 2000), while in the above-mentioned study by<br />
Crosier et al. (2009), the same alleles were linked<br />
to a decreased level <strong>of</strong> CAC.<br />
It should be noted that the majority <strong>of</strong> studies<br />
cited here was devoted to the relation <strong>of</strong> MGP to<br />
CAC <strong>and</strong> MI. As to cerebral artery atherosclerosis<br />
<strong>and</strong> its severe events such as IS, the role <strong>of</strong> arterial<br />
calcification in this disease <strong>and</strong> the association <strong>of</strong><br />
MGP with cerebrovascular pathology were the<br />
subject <strong>of</strong> investigation <strong>and</strong> discussion only in a<br />
few publications. In particular, Bos et al. (2011)<br />
established a close relationship between<br />
calcification in the various vessel beds outside the<br />
brain <strong>and</strong> imaging markers <strong>of</strong> vascular brain<br />
disease. Calcification in each vessel bed was<br />
shown to be associated with the presence <strong>of</strong><br />
cerebral infarcts <strong>and</strong> with larger volume <strong>of</strong> white<br />
matter lesions (WMLs). The most prominent<br />
associations were found between the in<strong>tr</strong>acranial<br />
carotid calcification <strong>and</strong> WML volume <strong>and</strong><br />
between the ex<strong>tr</strong>acranial carotid calcification <strong>and</strong><br />
infarcts.<br />
Acar et al. (2012) studied a relationship <strong>of</strong><br />
serum MGP levels to the development <strong>of</strong><br />
in<strong>tr</strong>acerebral hemorrhages (ICH) <strong>and</strong> found that in<br />
patients with ICH, serum MGP concen<strong>tr</strong>ation was<br />
much lower than in con<strong>tr</strong>ol group. Moreover, in the<br />
non-survivors, the serum MGP levels were<br />
statistically significantly lower in comparison to the<br />
survivors. According to the authors, measurement<br />
<strong>of</strong> this parameter may be <strong>of</strong> value to estimate<br />
mortality.<br />
At present, there are only a few publications<br />
concerning relation <strong>of</strong> the MGP SNPs to<br />
cerebrovascular disease. Analysing 236<br />
polymorphisms, del Rio-Espinola et al. (20<s<strong>tr</strong>ong>10</s<strong>tr</strong>ong>)<br />
showed that only two <strong>of</strong> them (G-7A <strong>of</strong> MGP <strong>and</strong><br />
T-1C <strong>of</strong> CD40) were related to the brain vessel<br />
reocclusion after fibrinolysis in IS patients. In our<br />
study, it was shown that the G-7A polymorphism <strong>of</strong><br />
MGP was associated with IS. In our previous<br />
investigation (Harbusova et al., 2011), this variant<br />
<strong>of</strong> the MGP promoter polymorphism was found to<br />
be in association with the acute coronary syndrome<br />
(ACS). Minor allele homozygotes (A/A) had<br />
significantly higher risk <strong>of</strong> ACS as well as IS. This<br />
could means that there are some common<br />
mechanisms <strong>of</strong> pathogenesis in both ACS <strong>and</strong> IS<br />
concerning to MGP. Those may be atherosclerosis,<br />
arterial calcification, <strong>and</strong> thrombosis.<br />
The relation <strong>of</strong> MGP to blood vessels<br />
calcification is well known (see above). With<br />
respect to coagulation <strong>and</strong> thrombi formation, it can<br />
be suggested that MGP is somehow connected with<br />
these processes (Krueger et al., 2009). Such an<br />
assumption is based on the fact that MGP belongs<br />
to vitamin K-dependent proteins, a large number <strong>of</strong><br />
which are procoagulants (prothrombin, factor V,<br />
etc) <strong>and</strong> can influence blood clotting <strong>and</strong> thrombi<br />
formation in the coronary <strong>and</strong> cerebral arteries. In<br />
some papers (Wallin et al., 2008), an antagonistic<br />
relationship between calcification <strong>and</strong> coagulation<br />
is discussed. Therefore, MGP can be considered as<br />
a connecting link between these two processes.<br />
Certainly, this assumption requires experimental as<br />
well as clinical pro<strong>of</strong>s, <strong>and</strong> research in this<br />
direction should be continued.<br />
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<strong>Journal</strong> <strong>of</strong> <strong>Cell</strong> <strong>and</strong> Molecular Biology <s<strong>tr</strong>ong>10</s<strong>tr</strong>ong>(1):27-32, 2012 Research Article 27<br />
Haliç University, Printed in Turkey.<br />
http://<s<strong>tr</strong>ong>jcmb</s<strong>tr</strong>ong>.<s<strong>tr</strong>ong>halic</s<strong>tr</strong>ong>.<s<strong>tr</strong>ong>edu</s<strong>tr</strong>ong>.<strong>tr</strong><br />
<br />
Survivin geni -625G/C polimorfizminin Küçük Hücreli Dışı Akciğer<br />
Kanseri ile ilişkisinin araştırılması<br />
Engin AYNACI 1 , Ender COŞKUNPINAR 2 , Ayşe EREN 2 , Onur KUM 1 , Yasemin<br />
MÜŞTERİ OLTULU 2 , Nergiz AKKAYA 2 , Akif TURNA 3 , İlhan YAYLIM 2 , Pınar<br />
YILDIZ *1 .<br />
1 Yedikule Chest Diseases <strong>and</strong> Thoracic Surgery Training Hospital, Istanbul, Turkey<br />
2 Department <strong>of</strong> Molecular Medicine, Institute <strong>of</strong> Experimental Medicine, Istanbul University, Istanbul,<br />
Turkey<br />
3 Cerrahpaşa Medicine Faculty, Department <strong>of</strong> Thoracic Surgery, Istanbul University, Istanbul, Turkey<br />
(*author for correspondence; pinary70@yahoo.com)<br />
Received: 14 February 2012; Accepted: 21 May 2012<br />
Özet<br />
Akciğer kanseri tüm kanser türleri arasında görülme sıklığı olarak ikinci sırada, kanser sebepli ölümler<br />
arasında ise ilk sırada gelmektedir. Survivin geni 17q25 kromozomal bölgesinde lokalizedir ve 142 amino<br />
asitten oluşan bir protein kodlar. Survivin (BIRC5) apoptozu düzenleyen önemli bir protein ailesi olan<br />
apoptoz proteinlerinin inhibitörü (IAPs) olarak ilk bulunan inhibitörlerden biridir ve özellikle kanser<br />
hücrelerinde ifadesi gerçekleşir. Survivin genindeki polimorfizm survivin üretimi ve aktivitesine etki<br />
edebilir, bu nedenle akciğer kanserine hassasiyet sağlar. Survivin genindeki aşırı ifadenin çok çeşitli<br />
maligniteleri içeren kanser türlerinde hastalık gelişimi, nüksü ve prognozu ile ilişkili olduğu bilinmektedir.<br />
Bu çalışmada bir Türk popülasyonunda survivin geni promotör bölgesi üzerinde bulunan -625G/C gen<br />
polimorfizmi ile küçük hücreli dışı akciğer kanseri arasında, hastalığın gelişimi ile ilgili olası ilişkilerin<br />
araştırılması amaçl<strong>and</strong>ı. Çalışmaya 146 hasta, 98 kon<strong>tr</strong>ol olgu dahil edildi. Yöntem olarak PCR-RFLP tekniği<br />
kullanıldı. Sonuç olarak survivin -625G/C genotip dağılımları incelendiğinde hasta ve kon<strong>tr</strong>ol grupları<br />
arasında istatistiksel olarak anlamlı fark olmadığı tespit edilmiştir.<br />
Anahtar kelimeler: Küçük hücreli dışı akciğer kanseri, survivin, gen polimorfizmi, PCR-RFLP, biyobelirteç.<br />
Investigation <strong>of</strong> the association <strong>of</strong> survivin gene -625G/C polymorphism in non-small cell<br />
lung cancer<br />
Abs<strong>tr</strong>act<br />
Lung cancer is the second most common cancer type diagnosed <strong>and</strong> first in cancer related deaths among all<br />
cancers worldwide. The survivin gene is located on human chromosome 17q25, encoding a protein consisting<br />
<strong>of</strong> 142 amino acids. Survivin is one <strong>of</strong> the first reported inhibitors <strong>of</strong> apoptosis proteins, which is an<br />
important family <strong>of</strong> proteins that regulate apoptosis. Survivin gene polymorphism may affect the survivin<br />
production <strong>and</strong> activity, thus providing sensitivity for the development <strong>of</strong> lung cancer. The overexpression <strong>of</strong><br />
survivin gene was found to be associated with disease development, recurrence <strong>and</strong> prognosis in various<br />
malignancies, including cancers. In this study the demons<strong>tr</strong>ation <strong>of</strong> the prognosis related associations<br />
between the -625G/C gene polymorphism located on the survivin promoter region <strong>and</strong> non small cell lung<br />
cancer in a Turkish population was aimed. 146 patients <strong>and</strong> 98 con<strong>tr</strong>ol subjects included to the study. PCR-<br />
RFLP technique was used as the method. According to survivin -625G/C genotype dis<strong>tr</strong>ibution analysis, no<br />
statistically significant difference between patients <strong>and</strong> con<strong>tr</strong>ols were found.<br />
Keywords: Non small cell lung cancer, survivin, gene polymorphism, PCR-RFLP, biomarker.
28 Engin AYNACI et al.<br />
Giriş<br />
Akciğer kanseri tüm dünyada kansere bağlı<br />
ölümlerin önde gelen sebebi olarak bilinmektedir.<br />
Bununla birlikte özellikle Amerika’da kansere bağlı<br />
ölüm oranları arasında akciğer kanseri sıklığı<br />
gitgide azalmakta, fakat Çin gibi sigara tüketiminin<br />
özellikle son 20 yılda arttığı bazı ülkelerde akciğer<br />
kanseri sebepli ölüm oranının arttığı<br />
gözlenmektedir. Amerika’da 2008 yılında 215.020<br />
yeni vaka belirlenirken, 161.840 kişinin bu hastalık<br />
sebebiyle öldüğü, kayıtlarda yer almaktadır.<br />
Akciğer kanserinin küçük hücreli (KHAK) ve<br />
küçük hücreli dışı (KHDAK) olmak üzere iki tipi<br />
vardır. Son 60 yıldır akciğer kanserinde hastalık<br />
gelişiminin kalıtsal bir temele oturduğu<br />
belirtilmektedir. (Julian et al., 2008).<br />
Programlanmış hücre ölümü olarak bilinen apoptoz,<br />
önemli bir hücre büyüme kon<strong>tr</strong>ol mekanizmasıdır<br />
(Yuan-Hung et al., 2009; Thompson, 1995).<br />
Survivin (BIRC5 olarak da bilinir) apoptozu<br />
düzenleyen önemli bir protein ailesi olan apoptoz<br />
proteinlerinin inhibitörü (IAP) olarak ilk bulunan<br />
inhibitörlerden biridir ve özellikle kanser<br />
hücrelerinde ifadesi gerçekleşir (Reed,1997).<br />
Survivin terminal tetikleyici kaspaz-3 ve kaspaz-9<br />
aktivitesini inhibe ederek her iki apoptoz yolunun<br />
baskılanmasını bloke eder (Nicholson <strong>and</strong><br />
Thornberry, 1997). Ayrıca survivin apoptotik<br />
uyarıcıyla indüklenen interlökin (IL-3), Fas<br />
(CD95), Bax, tümör nekroz faktörü α, kaspazlar ve<br />
antikanser ilaçlarınının etkisini yok eder (Chan et<br />
al., 2009; Yun-Hong et al., 2004). Mitozda<br />
düzenleyici rol oynadığı da çeşitli yayınlarda (Chan<br />
et al., 2009) bildirilmiş olan survivin geni 17q25<br />
kromozomal bölgesinde lokalizedir ve 142 amino<br />
asitten oluşan bir protein kodlar (Chiou et al., 2003;<br />
Deveraux et al., 1997; Uren et al., 1998).<br />
Survivin ayrıca mikrotübül dinamiklerinin<br />
düzenlenmesinde de önemli rol oynar (Li et al.,<br />
1998; Li <strong>and</strong> Altieri, 1999; Altieri, 2006; Giodini et<br />
al., 2002). Survivin geni promotör bölgesindeki<br />
polimorfizmler genin <strong>tr</strong>anskripsiyonuna etki ettiği<br />
için gen aktivitesini ve ekspresyonunu değiştirerek<br />
akciğer kanserine yatkınlık sağlayabilir (Jin Sung et<br />
al., 2008).<br />
Survivin hücre döngüsünde G2/M fazında bol<br />
miktarda eksprese olur ve G1 fazında hızlı<br />
regülasyon sergiler (Li et al., 1998). Bu durum<br />
<strong>tr</strong>anskripsiyonel basamakta kon<strong>tr</strong>ol edilir ve hücre<br />
döngüsüne bağlı elementler (CDE) ve hücre<br />
döngüsü homoloji bölgeleri (CHR) survivin<br />
promotörünün proksimal bölgesinde lokalize olur<br />
(Masayuki et al., 2000; Li <strong>and</strong> Altieri, 1999).<br />
Survivin geni ekspresyon düzeylerindeki artışın<br />
bazı hastalıklar için prognostik belirteç olabileceği<br />
düşünülmektedir (Chun-Hua et al., 20<s<strong>tr</strong>ong>10</s<strong>tr</strong>ong>). Survivin<br />
genellikle embriyonik dokularda ifade olur ve<br />
görülen homozigot mutasyonların erken<br />
embriyonik dönemde ölümle sonuçlanması bu gen<br />
ailesinin hücre gelişimi, farklılaşması ve homeostaz<br />
sürecinde çok önemli rol oynadığını göstermektedir<br />
(Chan et al., 2009).<br />
Çeşitli tek-nükleotit polimorfizmleri survivin<br />
gen bölgesi promotöründe tespit edilmiştir.<br />
Bunlardan en çok bilineni ve literatürde en fazla<br />
çalışması yapılmış olan CDE/CHR reseptör<br />
bağlayıcı bölgede lokalize olan -31G/C gen<br />
polimorfizmidir. Survivin geninin promotör<br />
bölgesindeki bu mutasyon sonucu hücre<br />
döngüsünden bağımsız olarak genin<br />
<strong>tr</strong>anskripsiyonu ve bunun sonucunda da aşırı ifadesi<br />
görülür (Xu et al., 2004).<br />
Bu çalışmada KHDAK hastalarında PCR-RFLP<br />
tekniği kullanılarak survivin geni promotör<br />
bölgesindekiki -625G/C (rs8073069)<br />
polimorfizminin bir Türk popülasyonunda KHDAK<br />
hastalığına yatkınlığı araştırılmıştır.<br />
Akciğer kanseri ile ilgili olarak eldeki verilerin<br />
doğru olarak kullanılması ve buna ek olarak<br />
hastalık oluşumu ya da gelişiminin anlaşılmasına<br />
yönelik belirteçlerin ve genetik mekanizmaların<br />
anlaşılması özellikle hastalığın erken tanısı ve<br />
tedavi sürecinde bu hastalar için anlamlı olacaktır.<br />
Materyal ve metod<br />
Örneklerin tanımı<br />
Çalışma ile ilgili olarak öncelikle İstanbul<br />
Üniversitesi İstanbul Tıp Fakültesi Etik<br />
Değerlendirme Komisyonu’ndan 09.06.20<s<strong>tr</strong>ong>10</s<strong>tr</strong>ong> tarih<br />
ve 20<s<strong>tr</strong>ong>10</s<strong>tr</strong>ong>/228-36 dosya numarası ile etik kurul onayı<br />
alındı. Çalışmaya Yedikule Göğüs Hastalıkları ve<br />
Göğüs Cerrahisi Eğitim ve Araştırma Hastanesi 3.<br />
Klinikte ve İstanbul Üniversitesi Cerrahpaşa Tıp<br />
Fakültesi Göğüs Cerrahisi Kliniğinde tanı konulan<br />
toplam 146 KHDAK olgusu ile yine aynı<br />
kliniklerde tetkik edilen ve kronik hastalık veya<br />
malignite bulgusu saptanmayan 98 sağlıklı kon<strong>tr</strong>ol<br />
olgusu alındı. Çalışmaya girmeyi kabul edenlere<br />
gönüllü olur imzaladıktan sonra 1 adet EDTA’lı<br />
tüpe <s<strong>tr</strong>ong>10</s<strong>tr</strong>ong> ml kanları alınarak soğuk zincirle<br />
laboratuvara ulaştırıldı.<br />
DNA izolasyonu<br />
Gönüllülerden alınan kanlardan High Pure PCR<br />
Template Preparation Kit (Roche, Manheim)<br />
protokolüne uygun olarak genomik DNA
izolasyonu yapıldı ve daha sonra Nano Drop<br />
Spek<strong>tr</strong><strong>of</strong>otome<strong>tr</strong>e kullanılarak, elde edilen<br />
DNA’ların konsan<strong>tr</strong>asyonları ölçüldü. DNA’lar<br />
konsan<strong>tr</strong>asyonları <s<strong>tr</strong>ong>10</s<strong>tr</strong>ong>0 ng/μl olacak şekilde<br />
seyreltildi.<br />
PCR<br />
Survivin geni promotör bölgesindeki -625G/C<br />
polimorfizmine özgü primerler (Tablo 1) dizayn<br />
edildi. PCR, total hacim 25 µl ve <s<strong>tr</strong>ong>10</s<strong>tr</strong>ong>X PCR Buffer<br />
(MBI Fermentas), 1mM MgCl2, 0.2 mM dNTP,<br />
<br />
KHDAK’de survivin polimorfizmi 29<br />
0.375 mM her bir primer, <s<strong>tr</strong>ong>10</s<strong>tr</strong>ong>0 ng genomik DNA ve<br />
1 U Taq DNA polimeraz (MBI Fermentas) olacak<br />
şekilde dizayn edildi. Amplifikasyon şartları,<br />
95°C’de <s<strong>tr</strong>ong>10</s<strong>tr</strong>ong> dakika ilk denatürasyondan sonra<br />
95°C’de 45 saniye, 72°C’de 60 saniye 5 döngü,<br />
takiben 94°C’de 45 saniye, 60°C’de 45 saniye ve<br />
72°C’de 60 saniye 30 döngü, uzama aşamasında da<br />
<s<strong>tr</strong>ong>10</s<strong>tr</strong>ong> dakika 72°C’de olacak şekilde düzenlendi.<br />
Optimum amplifikasyon şartları sağlanarak PCR<br />
ürünleri %2 lik agaroz jel elek<strong>tr</strong><strong>of</strong>orezinde<br />
yürütüldü.<br />
Tablo 1. Survivin geni promotör -625 G/C bölgesi primerleri (FP: İleri Primer, RP: Geri Primer)<br />
Polimorfizm Primer Dizileri PCR Ürün boyu<br />
-625C/G<br />
(rs8073069)<br />
Res<strong>tr</strong>iksiyon analizi<br />
FP: 5’-TGTTCATTTGTCCTTCATGCGC-3’<br />
RP: 5’-CCAGCCTAGGCAACAAGAGCAA-3’<br />
Amplifiye olan PCR ürünleri BstUI res<strong>tr</strong>iksiyon<br />
enzimi ile uygun tamponu içeren karışım<br />
hazırl<strong>and</strong>ıktan sonra 37°C de 4 saat inkübe edildi.<br />
Kesim ürünleri % 3’lük agaroz jelde <s<strong>tr</strong>ong>10</s<strong>tr</strong>ong>0 volt<br />
elek<strong>tr</strong>ik altında, 20 dakika yürütüldükten sonra UV<br />
altında incelenerek genotipler tespit edildi.<br />
İstatistiksel analiz<br />
AJJC tarafından yayınlanmış olan evrelendirme<br />
sistemine göre tümör evrelendirmesi yapılan hasta<br />
olguları ve kon<strong>tr</strong>ollere ait veriler SPSS 15.0<br />
programına yüklendi ve kategorik verilerin<br />
karşılaştırılmasında ki-kare testi ve parame<strong>tr</strong>ik ttesti<br />
kullanıldı.<br />
Şekil 1. Survivin geni -625 G/C bölgesi PCR<br />
görüntüsü<br />
Sonuçlar<br />
125 bç<br />
BstUI kesimi sonrası<br />
ürün boyları<br />
CC: 125<br />
CG: 125/<s<strong>tr</strong>ong>10</s<strong>tr</strong>ong>4/21<br />
GG: <s<strong>tr</strong>ong>10</s<strong>tr</strong>ong>4/21<br />
Çalışmaya 146 KHDAK hastası ve 98 kon<strong>tr</strong>ol olgu<br />
dahil edildi. Hastaları yaş ortalaması 60,41 yaş<br />
(±9,71), kon<strong>tr</strong>ol olgularının yaş ortalaması 55,23<br />
yaş (±8,8.) di. Çalışmaya dahil edilen hastalardan<br />
132’si erkek (%90,4), 14’ü kadın (%9,6), kon<strong>tr</strong>ol<br />
grubu olgularının ise 56’sı erkek (%57,1), 42’si<br />
kadındı (%42,9). Hasta ve kon<strong>tr</strong>ol grubuna ait<br />
genotip ve alel dağılımları Tablo 2’de verilmiştir.<br />
PCR sonucu elde edilen bant boyu 125 baz çifti<br />
büyüklüğünde (Şekil 1); BstUI res<strong>tr</strong>iksiyon enzimi<br />
kesimi sonucu elde edilen bant büyüklükleri ise<br />
125, <s<strong>tr</strong>ong>10</s<strong>tr</strong>ong>4 ve 21 baz çifti olarak görüntülendi (Şekil<br />
2).<br />
Şekil 2. BstUI kesimi %3’lük<br />
jel elek<strong>tr</strong><strong>of</strong>orezi görüntüsü. M: 50 bp DNA markörü
30 Engin AYNACI et al.<br />
<br />
Tablo 2. Hasta ve kon<strong>tr</strong>ol grubuna ait genotip ve alel dağılımları<br />
Genotipler ve Alel<br />
dağılımları<br />
Kon<strong>tr</strong>ol<br />
Grubu<br />
N=98<br />
Hasta<br />
Grubu<br />
N=146<br />
-625G/C N % N %<br />
GG 56 57.1 72 49.3<br />
GC 32 32.7 57 39.1<br />
CC <s<strong>tr</strong>ong>10</s<strong>tr</strong>ong> <s<strong>tr</strong>ong>10</s<strong>tr</strong>ong>.2 17 11.6<br />
Aleller<br />
P değeri χ2<br />
0.484 1.45<br />
tamamlamış<br />
yapmış<br />
yapmış<br />
G<br />
C<br />
144<br />
52<br />
56.06<br />
43.94<br />
201<br />
91<br />
58.59<br />
41.41<br />
0.27 1.21<br />
Tartışma<br />
düzeyindeki artışın -31G/C polimorfizmi ile ilişkili<br />
olduğunu ve bu artışın hem mRNA düzeyinde hem<br />
de protein düzeyinde meydana geldiğini<br />
Kanser oluşumunda apoptoz mekanizmasındaki<br />
bozukluklar önemli rol oynamaktadır. Apoptoz,<br />
farklı inhibe ve aktive edici ajanlar tarafından<br />
kon<strong>tr</strong>ol altında tutulan önemli bir olaydır. Kanserde<br />
apoptozun çeşitli anti-apoptotik proteinler<br />
tarafından inhibisyonu söz konusudur.<br />
Survivin, hücre döngüsünün düzenlenmesinde<br />
temel rol oynayan başlıca anti-apoptotik faktördür.<br />
Ayrıca survivinin Bcl-2 ve diğer IAPlerin aksine<br />
farklılaşmasını normal dokularda<br />
anlatımı olmayan ancak çeşitli kanser tiplerinde<br />
ifade edilen bir protein olduğu bilinmektedir. Bu<br />
durum survivin genindeki anormal ifadenin<br />
<strong>tr</strong>anskripsiyonel regülasyon bozukluğuna sebep<br />
olduğunun açık bir göstergesidir. Apoptozu inhibe<br />
eden diğer proteinlerde de bulunan BIR<br />
(“Baculovirus IAP Repeat”) bölgesi ile kaspazlara<br />
bağlanarak etkisini göstermektedir. Dai ve ark.<br />
20<s<strong>tr</strong>ong>10</s<strong>tr</strong>ong> yılında yaptıkları çalışmada survivin geni<br />
promotor bölgesindeki polimorfizmlerin<br />
KHDAK’de gen modifikasyonuna neden<br />
olabileceğini ileri sürmüşlerdir.<br />
Jang ve ark. tarafından yapılan çalışmada -31 G<br />
alelinin -31 C aleline göre önemli derecede düşük<br />
<strong>tr</strong>anskripsiyonel aktiveye sahip olduğu ve bu<br />
durumun -31G/C polimorfizminden etkilenerek<br />
ortaya çıktığı ve buna bağlı olarak -31G/C<br />
polimorfizminin akciğer kanserine yatkınlıkta<br />
önemli bir rolü olduğu belirtilmektedir. (Jang et al.,<br />
2008). Xu ve ark. kanser hücre hatları ile<br />
oldukları bir çalışmada survivin gen ifadesi<br />
.<br />
bildirmişlerdir.<br />
Klinik perspektiften bakıldığında kişisel<br />
paternlerle klinik özelliklerin öngörülmesinde<br />
hastaların genetik parmak izinde survivin geni<br />
ifadesi düzeylerinde ve genetik varyantlarda oluşan<br />
değişikliklerin olası tedaviye yanıtta erken bir<br />
belirteç olabileceğini söylemek mümkündür.<br />
Örneğin plevral efüzyondaki yüksek survivin<br />
düzeylerinin kötü prognoz göstergesi olduğu Lan<br />
ve ark. 20<s<strong>tr</strong>ong>10</s<strong>tr</strong>ong> yılındaki yayınında gösterilmiştir.<br />
Yang ve ark 2009 yılında özefagus kanserli<br />
hastalarda yaptıkları bir çalışmada C alleline sahip<br />
olmanın hastalık riskini 1.4 kat arttırdığını<br />
söylemektedirler. Özefagus kanseri hastalarında<br />
survivin -625G/C promotor polimorfizminin p53<br />
düzeyine bağlı olan survivin yüksek ifadesi<br />
olasılığını artırdığı düşünülmektedir.<br />
Sonuç olarak bir Türk popülasyonu üzerinde<br />
olduğumuz bu çalışmada survivin geni -625<br />
G/C bölgesi (rs8073069) polimorfizminin küçük<br />
hücreli dışı akciğer kanseri hastalığına yatkınlık<br />
sağladığına dair herhangi bir bulgu elde edilemedi.<br />
Ancak olgu sayısının artırılması ve polimorfik<br />
bölgenin özellikle <strong>tr</strong>anskripsiyonun aktivitesine etki<br />
eden promotör bölgesinde olmasından dolayı<br />
yapılabilecek ekspresyon çalışmaları ile özellikle<br />
tanı öncesi ve sonrası gen anlatım ifadesine bağlı<br />
değişikliklerin öngörülmesine yardımcı olabilecek<br />
daha anlamlı sonuçlara ulaşılabileceği<br />
kanaatindeyiz.
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Chen, Xiao-Jun Zhou, İnfluence <strong>of</strong> survivin <strong>and</strong><br />
caspase-3 on cell apoptosis <strong>and</strong> prognosis in<br />
gas<strong>tr</strong>ic carcinoma. World J Gas<strong>tr</strong>oenterol.<br />
<s<strong>tr</strong>ong>10</s<strong>tr</strong>ong>(13):1984-1988, 2004
<strong>Journal</strong> <strong>of</strong> <strong>Cell</strong> <strong>and</strong> Molecular Biology <s<strong>tr</strong>ong>10</s<strong>tr</strong>ong>(1):33-40, 2012 Research Article 33<br />
Haliç University, Printed in Turkey.<br />
http://<s<strong>tr</strong>ong>jcmb</s<strong>tr</strong>ong>.<s<strong>tr</strong>ong>halic</s<strong>tr</strong>ong>.<s<strong>tr</strong>ong>edu</s<strong>tr</strong>ong>.<strong>tr</strong><br />
Effects <strong>of</strong> prenatal <strong>and</strong> neonatal exposure to lead on white blood<br />
cells in Swiss mice<br />
Ragini SHARMA*, Khushbu PANWAR, Sheetal MOGRA<br />
Environmental <strong>and</strong> Developmental Toxicology Research Lab, Department <strong>of</strong> Zoology, M. L. S. University,<br />
Udaipur- 313001 Rajasthan, India<br />
(* author for correspondence; taurasragini@yahoo.com)<br />
Received: 27 August 2011; Accepted: 25 May 2012<br />
Abs<strong>tr</strong>act<br />
Lead exposure is one <strong>of</strong> the major environmental issues for children <strong>and</strong> women <strong>of</strong> child bearing age. It<br />
crosses the placental barrier <strong>and</strong> its greater intestinal absorption in fetus results in developmental defects.<br />
Lead, as one <strong>of</strong> the environmental pollutants, can threat the lives <strong>of</strong> animals <strong>and</strong> human beings in many ways;<br />
especially during developing stages. The present study was carried out to study the alterations in different<br />
types <strong>of</strong> white blood cells (WBC) due to chronic lead acetate toxicity in neonates, which passes from adult<br />
pregnant female during gestation <strong>and</strong> lactation. Lead acetate was administered orally at 8, 16, 32 mg /kg/BW<br />
to pregnant Swiss mice from <s<strong>tr</strong>ong>10</s<strong>tr</strong>ong>th day <strong>of</strong> gestation to 21th day <strong>of</strong> lactation. Hematopathological <strong>and</strong><br />
numerical alterations in the WBCs were examined in the neonates after birth at postnatal days 1, 7, 14 <strong>and</strong><br />
21. Blood smears examined illus<strong>tr</strong>ate that lead induces disturbances in the development <strong>of</strong> different types <strong>of</strong><br />
WBCs during postnatal development <strong>and</strong> lead to an abrupt neu<strong>tr</strong>ophilic degeneration, immature cells,<br />
abnormal neu<strong>tr</strong>ophils, reactive <strong>and</strong> plasmacytoid lymphocytes. The results <strong>of</strong> the present study emphasize<br />
that prenatal lead exposure is ex<strong>tr</strong>emely dangerous to developing fetus.<br />
Keywords: Lead acetate, Swiss albino mice, prenatal, neonatal, white blood cells.<br />
Swiss farelerde prenatal ve yenidoğan kurşun maruziyetinin beyaz kan hücreleri üzerine<br />
etkileri<br />
Özet<br />
Kurşun maruziyeti çocuklar ve çocuk doğurma çağındaki kadınlar için majör çevresel konulardan birisidir.<br />
Plasental bariyeri geçer ve fetusta barsaklardan emilimi, gelişimsel defektlerle sonuçlanır. Çevresel<br />
kirliliklerden biri olan kurşun, birçok yönden özellikle gelişim çağı boyunca insan ve hayvan hayatını tehdit<br />
etmektedir. Bu çalışma, yetişkin hamile dişilerden gebelik ve laktasyon süresince yenidoğanlara geçen<br />
kurşun asetatın yarattığı kronik toksisite sebebiyle lökositlerin farklı tiplerindeki değişimleri incelemek için<br />
yapılmıştır. Kurşun asetat, hamile Swiss farelere gebeliğin <s<strong>tr</strong>ong>10</s<strong>tr</strong>ong>. gününden laktasyonun 21. gününe kadar ağız<br />
yoluyla 8, 16, 32 mg /kg//BW şeklinde uygulanmıştır. Yenidoğanlarda doğumdan sonraki 1, 7, 14 ve 21.<br />
günlerde beyaz kan hücrelerindeki hematopatolojik ve sayısal değişiklikler incelenmiştir. Kurşunun doğum<br />
sonrası gelişim sırasında lökositlerin farklı tiplerinin gelişimindeki bozuklukları indüklediğini ve ani<br />
nö<strong>tr</strong><strong>of</strong>ilik olgunlaşmamış dejenerasyona, hücreler, anormal nö<strong>tr</strong><strong>of</strong>illere, reaktif ve plazmasitoid lenfositlere<br />
neden olduğunu göstermek için kan yaymaları incelenmiştir. Bu çalışmanın sonuçları prenatal kurşun<br />
maruziyetinin gelişen fetus için son derece tehlikeli olduğunu vurgulamaktadır.<br />
Anahtar kelimeler: Kurşun asetat, Swiss albino fare, prenatal, yenidoğan, lökosit.
34Ragini SHARMA et al.<br />
In<strong>tr</strong>oduction<br />
Lead has been recognized as a biological toxicant<br />
<strong>and</strong> different doses have been used to study leadinduced<br />
alterations Prenatal exposure to lead<br />
produces toxic effects in the human fetus, including<br />
increased risk <strong>of</strong> preterm delivery, low birth<br />
weight, <strong>and</strong> impaired mental development; because<br />
during the period <strong>of</strong> early organogenesis the onset<br />
<strong>of</strong> greatest susceptibility to teratogenesis occurs<br />
(Falcon et al., 2003). This highly sensitive or<br />
critical period is the time during which a small dose<br />
<strong>of</strong> a teratogen produces high percentage <strong>of</strong> fetuses<br />
that exhibit malformations <strong>of</strong> the organ in question<br />
(Wilson, 1973; Desesso et al., 1996).<br />
Pregnancy <strong>and</strong> breastfeeding can cause a state<br />
<strong>of</strong> physiological s<strong>tr</strong>ess that increases bone turnover<br />
<strong>of</strong> lead. Lead stored in the bone moves into the<br />
blood, increasing the mother’s blood lead level <strong>and</strong><br />
passing to the fetus, affecting fetal development.<br />
Lead is tightly bound to red blood cells, enhancing<br />
<strong>tr</strong>ansfer from maternal circulation through the<br />
placenta to the fetus. Fetus is more sensitive to lead<br />
because the fetal blood-brain barrier is more<br />
permeable. The toxic effects <strong>of</strong> lead on blood<br />
indices are well known.<br />
Lead potentially induces oxidative s<strong>tr</strong>ess <strong>and</strong><br />
evidence is accumulating to support the role <strong>of</strong><br />
oxidative s<strong>tr</strong>ess in the pathophysiology <strong>of</strong> lead<br />
toxicity. Lead is capable <strong>of</strong> inducing oxidative<br />
damage to brain, heart, kidneys, <strong>and</strong> reproductive<br />
organs. The mechanisms for lead-induced oxidative<br />
s<strong>tr</strong>ess include the effects <strong>of</strong> lead on membranes,<br />
DNA, <strong>and</strong> antioxidant defense systems <strong>of</strong> cells<br />
(Ahamed <strong>and</strong> Siddiqui, 2007). Lead interferes with<br />
a variety <strong>of</strong> body processes <strong>and</strong> is toxic to the body<br />
systems including cardiovascular, reproductive,<br />
hematopoietic, gas<strong>tr</strong>ointestinal <strong>and</strong> nervous systems<br />
(Kosnett, 2006), renal functions (Patocka <strong>and</strong><br />
Cerny, 2003) <strong>and</strong> release <strong>of</strong> glutamate (Xu et al.,<br />
2006). It affects the hematological system even at<br />
concen<strong>tr</strong>ations below <s<strong>tr</strong>ong>10</s<strong>tr</strong>ong>μg/dl (ATSDR, 2005).<br />
Many reports are available regarding lead<br />
toxicity <strong>and</strong> its deleterious effects in various<br />
species <strong>of</strong> animals <strong>and</strong> there has been lot <strong>of</strong> work<br />
carried out on pharmacokinetics <strong>and</strong> genotoxicity<br />
but very few researchers <strong>tr</strong>ied to correlate<br />
haematopathological alterations <strong>of</strong> lead acetate in<br />
different white blood cells at different dose levels<br />
in laboratory animals, especially in mice.<br />
Therefore the current study was performed to<br />
clarify the lead induced hematological changes,<br />
especially those related to white blood cells, during<br />
gestational <strong>and</strong> lactational exposure to lead in<br />
Swiss mice.<br />
Materials <strong>and</strong> methods<br />
Sexually mature r<strong>and</strong>om bred Swiss mice with the<br />
age <strong>of</strong> 5-6 weeks, weighing 25-30 gm was used for<br />
this study. During the entire experimental period,<br />
the animals were fed on a st<strong>and</strong>ard diet <strong>and</strong> water<br />
ad libitum. Mice were kept in the ratio <strong>of</strong> 1:4 males<br />
<strong>and</strong> females, respectively, <strong>and</strong> females showing<br />
vaginal plugs were separated in the con<strong>tr</strong>ol <strong>and</strong> lead<br />
<strong>tr</strong>eated group. Lead acetate solution was prepared<br />
by dissolving 4gm lead acetate in 12ml distilled<br />
water. Pregnant Swiss mice were given lead acetate<br />
at a concen<strong>tr</strong>ation <strong>of</strong> 8, 16 <strong>and</strong> 32 mg (266.66,<br />
533.33, <strong>and</strong> <s<strong>tr</strong>ong>10</s<strong>tr</strong>ong>66.66 mg/kg/bodyweight) from <s<strong>tr</strong>ong>10</s<strong>tr</strong>ong> th<br />
day <strong>of</strong> gestation to 21 st day <strong>of</strong> lactation. Blood<br />
samples were obtained from the tail <strong>of</strong> pups from<br />
each litter at days 1,7,14 <strong>and</strong> 21 day after birth. The<br />
tip <strong>of</strong> the tail was cleaned with spirit before being<br />
cut with a sharp blade <strong>and</strong> was not squeezed to<br />
avoid dilution <strong>of</strong> blood by tissue fluid.<br />
Blood cells were studied in smears prepared by<br />
spreading a drop <strong>of</strong> blood thinly over a clean <strong>and</strong><br />
sterilized microscopic slide with the help <strong>of</strong> another<br />
slide moved over the first at the angle <strong>of</strong> 45ᵒ after<br />
discarding first drop <strong>of</strong> blood. These blood films<br />
were air-dried <strong>and</strong> fixed in absolute methanol for<br />
15 minutes by dipping the film briefly in a Coplin<br />
jar containing absolute methanol. After fixation the<br />
slides were removed <strong>and</strong> air-dried. Afterward blood<br />
smears were stained with freshly made Giemsa<br />
stain diluted with water buffered to pH 6.8 or 7.0<br />
(1:9) stain <strong>and</strong> buffer respectively. The slides were<br />
washed by briefly dipping the slide in <strong>and</strong> out <strong>of</strong> a<br />
Coplin jar <strong>of</strong> buffered water <strong>and</strong> air dried again for<br />
taking observations. The erythrocytes appear pink<br />
to purple, whereas leukocytes turned blue black in<br />
color. All the experimental work was approved by<br />
the Institutional Animal Ethics Committee.<br />
No./CS/Res/07/759.<br />
Group 1- Con<strong>tr</strong>ol (distilled water only).<br />
Group 2- Exposure to 8 mg lead acetate (266.66<br />
mg/kg BW) from <s<strong>tr</strong>ong>10</s<strong>tr</strong>ong>th day <strong>of</strong> gestation up to 21st<br />
day <strong>of</strong> lactation.<br />
Group 3- Exposure to 16 mg lead acetate<br />
(533.33 mg/kg BW) from <s<strong>tr</strong>ong>10</s<strong>tr</strong>ong>th day <strong>of</strong> gestation up<br />
to 21st day <strong>of</strong> lactation.<br />
Group 4- Exposure to 32 mg lead acetate<br />
(<s<strong>tr</strong>ong>10</s<strong>tr</strong>ong>66.66 mg/kg BW) from <s<strong>tr</strong>ong>10</s<strong>tr</strong>ong>th day <strong>of</strong> gestation up<br />
to 21st day <strong>of</strong> lactation.<br />
The statistical analysis was performed following<br />
t-test for the comparison <strong>of</strong> data between different<br />
experimental groups. The data was calculated using
prism s<strong>of</strong>tware to calculate the p values. <s<strong>tr</strong>ong>10</s<strong>tr</strong>ong>0 WBC<br />
from each group were counted at different weeks,<br />
different cell types were identified <strong>and</strong> % ratio was<br />
calculated. For numerical observation highest dose<br />
level was selected.<br />
Results<br />
In the con<strong>tr</strong>ol group all the WBCs showed normal<br />
appearance. The neu<strong>tr</strong>ophils in con<strong>tr</strong>ol group were<br />
examined by a very characteristic nucleus with<br />
condensed chromatin. It is divided into 3-5 lobes<br />
(Fig.1A, 1, 2, 5 <strong>and</strong> 6) at birth which was observed<br />
with an increase by 5 to 6 lobes (Fig.1B, 1 <strong>and</strong> 3) at<br />
the termination <strong>of</strong> lactation, connected by thin<br />
s<strong>tr</strong><strong>and</strong>s <strong>of</strong> chromatin. Lymphocytes were round or<br />
ovoid at the time <strong>of</strong> birth (Fig. 1A, 3 <strong>and</strong> 4) but<br />
further on they were found notched or slightly<br />
indented (Fig. 1B, 5 <strong>and</strong> 6). The chromatin was<br />
generally diffusely dense. Ordinarily, nucleoli were<br />
not visible. A perinuclear clear zone surrounding<br />
the nucleus was visible after first week <strong>of</strong> lactation<br />
in some cells. The cytoplasm stained light blue <strong>and</strong><br />
ranges from sparse to moderately abundant in<br />
amount. The monocyte in con<strong>tr</strong>ol group were round<br />
with smooth margins, the nucleus was oval,<br />
indented <strong>and</strong> slightly folded (Fig.1B, 4). The<br />
chromatin material was moderately clumped <strong>and</strong><br />
relatively less dense compared to that <strong>of</strong><br />
neu<strong>tr</strong>ophils or lymphocytes. There was no visible<br />
nucleolus with abundant cytoplasm.<br />
The adminis<strong>tr</strong>ation <strong>of</strong> lead acetate altered the<br />
appearance <strong>and</strong> caused s<strong>tr</strong>uctural changes. The<br />
following hematological observations were taken<br />
during postnatal period from birth till the<br />
termination <strong>of</strong> the lactation period upon exposure<br />
<strong>of</strong> different doses <strong>of</strong> lead acetate:<br />
1. At the time <strong>of</strong> birth (PND1)<br />
Abnormal neu<strong>tr</strong>ophils: In lead <strong>tr</strong>eated groups<br />
the neu<strong>tr</strong>ophils showed s<strong>tr</strong>uctural abnormalities in<br />
their nucleus including improper segmentation <strong>and</strong><br />
lesser condensation <strong>of</strong> nucleus. At a lower dose the<br />
chromatin material was condensed, all the lobes<br />
were interconnected with each other <strong>and</strong> form a<br />
nodule like s<strong>tr</strong>ucture at one side (Fig.1C, 1).<br />
Degeneration: In lead <strong>tr</strong>eated group most <strong>of</strong> the<br />
neu<strong>tr</strong>ophils appeared in degenerating state in which<br />
the chromatin material was very less condensed,<br />
fused <strong>and</strong> there was no sign <strong>of</strong> clear lobulization<br />
<strong>and</strong> segmentation (Fig. 1C, 2).<br />
Immature cells: In lead <strong>tr</strong>eated group the<br />
number <strong>of</strong> immature cells was increased (Fig. 1C,<br />
3).<br />
Prenatal <strong>and</strong> neonatal exposure to lead 35 <br />
Ring shaped: In lead <strong>tr</strong>eated groups, some<br />
neu<strong>tr</strong>ophils showed abnormal ring like appearance<br />
<strong>and</strong> diffuse chromatin material, with unclear<br />
cytoplasm. In 32 mg lead <strong>tr</strong>eated group<br />
vacuolization in chromatin material was also<br />
observed ((Fig. 1C, 4).).<br />
Lymphocyte: Reactive (Fig. 1C: 5 <strong>and</strong> 6) <strong>and</strong><br />
cleaved (Fig. C, 5) types <strong>of</strong> lymphocytes were<br />
observed in lead <strong>tr</strong>eated groups.<br />
Monocytes: At postnatal day 1 we cannot<br />
identify any s<strong>tr</strong>uctural change in shape <strong>and</strong> size <strong>of</strong><br />
monocyte as observed on postnatal day 21.<br />
2. During first <strong>and</strong> second week <strong>of</strong> postnatal period<br />
(PND7&14)<br />
The following observations were taken at first to<br />
second week after birth:<br />
Degenerated neu<strong>tr</strong>ophils: In lead <strong>tr</strong>eated group<br />
overall numbers <strong>of</strong> neu<strong>tr</strong>ophils were increased<br />
particularly with degenerated neu<strong>tr</strong>ophils, however,<br />
their number was less than postnatal day 1. In 16<br />
mg lead group on postnatal day 7 the nuclear<br />
material <strong>of</strong> neu<strong>tr</strong>ophil was less condensed <strong>and</strong><br />
nucleus was divided into 2-3 unequal lobes. The<br />
cytoplasm <strong>of</strong> neu<strong>tr</strong>ophil appeared colorless. At the<br />
dose <strong>of</strong> 32mg lead at postnatal day 7, this severity<br />
<strong>of</strong> degeneration was very much increased so that<br />
the lobes were broken into many small fragments.<br />
No sign <strong>of</strong> lobulization <strong>and</strong> appropriate<br />
segmentation <strong>of</strong> neu<strong>tr</strong>ophils were found (Fig. 1D,<br />
1).<br />
Ring shaped neu<strong>tr</strong>ophils: In con<strong>tr</strong>ast to<br />
postnatal day 1, ring like nucleus was not observed<br />
in lead <strong>tr</strong>eated group at postnatal day 7.<br />
Different types <strong>of</strong> neu<strong>tr</strong>ophils: At higher dose<br />
32 mg lead <strong>tr</strong>eated groups apoptotic or necrotic<br />
neu<strong>tr</strong>ophils were more prominent. These<br />
neu<strong>tr</strong>ophils were characterized by 3-4 separate <strong>and</strong><br />
equal lobes with less condensed chromatin <strong>and</strong><br />
diffuse cytoplasmic region (Fig. 1D, 2).<br />
Immature cells: Review <strong>of</strong> the lead <strong>tr</strong>eated<br />
smear revealed that most <strong>of</strong> the leukocytes were<br />
myelocytes, b<strong>and</strong>s, myeloblast <strong>and</strong> other immature<br />
<strong>and</strong> unidentified white blood cells with left shift in<br />
leucocytes. A left shift is an increase in the number<br />
<strong>of</strong> b<strong>and</strong> neu<strong>tr</strong>ophils <strong>and</strong> other immature cell <strong>of</strong> the<br />
granulocytic lineage in the peripheral blood (Fig.<br />
1D, 3).<br />
Various lymphocytes: Adminis<strong>tr</strong>ation <strong>of</strong> lead<br />
acetate produced great variation in lymphocyte<br />
s<strong>tr</strong>ucturally as well as numerically. Various types <strong>of</strong><br />
lymphocytes such as plasmacytoid, reactive, oval,<br />
irregular, binucleated <strong>and</strong> cleaved lymphocytes<br />
were identified, whereas only reactive <strong>and</strong> cleaved
36Ragini SHARMA et al.<br />
lymphocytes were seen in postnatal day 1,<br />
exclusively in lead <strong>tr</strong>eated group.<br />
Lead <strong>tr</strong>eated group with 16 mg lead acetate<br />
produced large lymphocytes <strong>and</strong> most <strong>of</strong> the<br />
lymphocytes were having irregular; clumpy <strong>and</strong><br />
smudgy chromatin material with very dense<br />
nucleus (Fig. 1D, 4). The cytoplasm appeared<br />
completely absent as the nucleus reached its largest<br />
size <strong>and</strong> covered all the cytoplasmic area. Overall,<br />
number <strong>of</strong> lymphocytes decreased in most <strong>of</strong> the<br />
groups. At higher dose (32 mg lead) the<br />
plasmacytoid lymphocytes (eccen<strong>tr</strong>ic nucleus <strong>and</strong><br />
intensely blue / basophilic cytoplasm) (Fig. 1D, 6)<br />
<strong>and</strong> reactive lymphocytes were observed. Reactive<br />
lymphocyte was characterized by relatively very<br />
large, irregular but flattened nucleus with fine<br />
chromatin <strong>and</strong> agranular light blue stained<br />
cytoplasm (Fig. 1D, 5).<br />
3. At the end <strong>of</strong> lactation period (PND21)<br />
Abnormal nuclear segmentation: It includes<br />
abnormal segmentation <strong>of</strong> nucleus, in which the<br />
nuclear lobes were connected with each other. It<br />
gave abnormal appearance <strong>of</strong> nucleus <strong>and</strong><br />
chromatin condensation in most <strong>of</strong> the neu<strong>tr</strong>ophils<br />
(Fig. 1E, 1).<br />
Degeneration: In lower doses <strong>of</strong> lead diffuse<br />
appearance <strong>of</strong> chromatin material was observed in<br />
neu<strong>tr</strong>ophils <strong>and</strong> the lobes were fused with each<br />
other as any segmentation was not observed,<br />
whereas in higher lead <strong>tr</strong>eated group the neu<strong>tr</strong>ophils<br />
presented fragmented chromatin material <strong>and</strong> very<br />
less condensation <strong>of</strong> nucleus which finally leads to<br />
cell lysis (Fig. 1E, 2). The nuclear arrangement was<br />
distorted, as appear that all the lobes were<br />
intermingled with each other <strong>and</strong> in some cases<br />
form a nodule at one side known as sessile nodule<br />
appeared like hypersegmentation (Fig. 1E, 3).<br />
Immature cells: In lead <strong>tr</strong>eated group the<br />
numbers <strong>of</strong> immature cells were increased. A left<br />
shift i.e. presence <strong>of</strong> immature neu<strong>tr</strong>ophils, b<strong>and</strong>s,<br />
metamyelocytes, myelocytes <strong>and</strong> other unidentified<br />
immature cells were observed (Fig. 1F, 1 to 6).<br />
Lymphocytes: As the dose level increased the<br />
number <strong>of</strong> lymphocytes decreased. In higher dose<br />
lead <strong>tr</strong>eated group the lymphocyte appeared large in<br />
size with higher volume <strong>of</strong> cytoplasm. The shape <strong>of</strong><br />
the nucleus also vary from round to elliptical in<br />
s<strong>tr</strong>ucture, termed as reactive lymphocyte (Fig. 1E,<br />
4). Some lymphocytes <strong>tr</strong>ansformed into<br />
plasmocytoid lymphocyte in which the lymphocyte<br />
contains basophilic cytoplasm <strong>and</strong> eccen<strong>tr</strong>ic<br />
nucleus (Fig. 1E, 5).<br />
Monocytes: In lead <strong>tr</strong>eated groups the shape <strong>and</strong><br />
s<strong>tr</strong>ucture <strong>of</strong> the monocyte were modified <strong>and</strong> the<br />
shape <strong>of</strong> the nucleus was also altered from the<br />
normal reniform (kidney shaped) nucleus. The<br />
indentation <strong>of</strong> the nucleus became larger <strong>and</strong><br />
deeper from periphery to center. At higher dose<br />
level intensity <strong>of</strong> the indentation was increased so<br />
that the normal range <strong>of</strong> nucleo-cytoplasmic ratio<br />
was disturbed (Fig. 1E, 6). Numerical changes in<br />
different types <strong>of</strong> WBC <strong>and</strong> percent variations in<br />
different types are incorporated in Table 1 <strong>and</strong> 2<br />
respectively. In present investigation, after<br />
evaluating all the cell types, we can conclude that<br />
lead acetate at PND 1 <strong>and</strong> 14 caused significant<br />
increase in number <strong>of</strong> neu<strong>tr</strong>ophils <strong>and</strong> decrease in<br />
lymphocytes, while there was no significant<br />
difference in the number <strong>of</strong> neu<strong>tr</strong>ophils <strong>and</strong><br />
lymphocytes at PND 7 <strong>and</strong> 21.<br />
Table 1. Various types <strong>of</strong> WBCs at different postnatal days <strong>tr</strong>eated with lead acetate.<br />
Groups Neu<strong>tr</strong>ophils Lymphocytes Monocytes<br />
Con<strong>tr</strong>ol at PND 1 59.25±1.70 38.5±1.29 2.25±1.70<br />
Lead acetate at PND1 66.00±2.16** 28.25±2.06** 5.75±1.70*<br />
Con<strong>tr</strong>ol at PND 7 57.75±2.21 41.75±2.21 0.75±0.95<br />
Lead acetate at PND7 61.75±3.5 37.5±2.88 0.75±0.95<br />
Con<strong>tr</strong>ol at PND 14 55.25±3.40 44.5±3.<s<strong>tr</strong>ong>10</s<strong>tr</strong>ong> 0.25±0.5<br />
Lead acetate at PND14 61.25±1.70** 37.75±1.70** 1.00±0.81<br />
Con<strong>tr</strong>ol at PND 21 47.75±2.5 47.75±2.21 4.5±2.38<br />
Lead acetate at PND21 52.25±4.57 44.00±2.26 3.75±2.75<br />
Values were expressed as means ± S.D.; 4 animals /group;*=p
Prenatal <strong>and</strong> neonatal exposure to lead 37 <br />
Table 2. Percent variation in different types <strong>of</strong> WBCs in lead <strong>tr</strong>eated groups<br />
Lead acetate at PND1<br />
Lead acetate at PND7<br />
Lead acetate at PND14<br />
Lead acetate at PND21<br />
Neu<strong>tr</strong>ophils Lymphocytes Monocytes<br />
Normal 12.3%<br />
Degenerated 12.1%<br />
Ring shaped 8.2%<br />
Immature 4.1%<br />
Abnormal <s<strong>tr</strong>ong>10</s<strong>tr</strong>ong>.4%<br />
Normal 12.6%<br />
Degenerated 8.4%<br />
Abnormal 16.8%<br />
Immature <s<strong>tr</strong>ong>10</s<strong>tr</strong>ong>.8%<br />
Ring shaped 6%<br />
Normal 19.2%<br />
Degenerated 19%<br />
Abnormal 9.6%<br />
Immature 6.4%<br />
Apoptotic 6.4%<br />
Normal 6.18%<br />
Degenerated18.5%<br />
Ring shaped 1.5%<br />
Abnormal 17%<br />
Immature 7.4%<br />
Normal 13%<br />
Reactive 17.33%<br />
Cleaved 8.6%<br />
Normal 12.2%<br />
Plasmacytoid 7.4%<br />
Reactive 9.8%<br />
Binucleated 2.4%<br />
Large 4.2%<br />
Normal 4%<br />
Plasmacytoid 4%<br />
Reactive 12.3%<br />
Binucleated 8.2%<br />
Large 4%<br />
Irregular 2%<br />
Oval 2%<br />
Normal <s<strong>tr</strong>ong>10</s<strong>tr</strong>ong>%<br />
Abnormal 16.4%<br />
Plasmacytoid1.4%<br />
Reactive 4.4%<br />
Large 5.8%<br />
Irregular 5.86%<br />
Normal 2.0%<br />
Abnormal 3.7%<br />
Abnormal 0.75%<br />
Abnormal 1%<br />
Normal 2%<br />
Abnormal 1.7%<br />
Figure 1. A: Peripheral blood smear <strong>of</strong> con<strong>tr</strong>ol group showing neu<strong>tr</strong>ophil (1-2), lymphocytes (3-4), at the time <strong>of</strong> birth,<br />
neu<strong>tr</strong>ophils (5-6) during second <strong>and</strong> third week <strong>of</strong> lactation. B: Con<strong>tr</strong>ol group showing neu<strong>tr</strong>ophil (1), lymphocyte (2),<br />
during second <strong>and</strong> third week <strong>of</strong> lactation, <strong>and</strong>, neu<strong>tr</strong>ophil (3), lymphocytes (4-5) <strong>and</strong> monocyte (6) at the termination <strong>of</strong><br />
lactation. C: Peripheral blood smear <strong>of</strong> lead <strong>tr</strong>eated group showing abnormal neu<strong>tr</strong>ophil (1), degenerated neu<strong>tr</strong>ophil (2),<br />
immature cell (3), ring like neu<strong>tr</strong>ophil (4), 5 – cleaved (upper WBC) (5) <strong>and</strong> reactive (lower WBC) lymphocyte (5 <strong>and</strong> 6)<br />
at the time <strong>of</strong> birth. D: Lead <strong>tr</strong>eated group showing degenerated neu<strong>tr</strong>ophil (1), necrotic (2), immature cell (3), large<br />
lymphocyte (4), reactive (5) <strong>and</strong> plasmacytoid lymphocyte (6) During first <strong>and</strong> second week <strong>of</strong> postnatal period. E: Lead<br />
<strong>tr</strong>eated group showing - abnormal neu<strong>tr</strong>ophil (1), degenerated neu<strong>tr</strong>ophil (2), hypersegmented neu<strong>tr</strong>ophil (3), reactive<br />
lymphocyte (4), plasmacytoid lymphocyte (5) <strong>and</strong> reactive monocyte (6) at the termination <strong>of</strong> lactation. F: Lead <strong>tr</strong>eated<br />
group showing different immature cells at the termination <strong>of</strong> lactation (1- 6). (All Giemsa stain, 450x).
38Ragini SHARMA et al.<br />
Discussion<br />
Changes in leukocyte parameters are <strong>of</strong>ten one <strong>of</strong><br />
the hallmarks <strong>of</strong> infection. These include changes<br />
in number <strong>and</strong> in cellular morphology. Review <strong>of</strong><br />
the peripheral blood smear can provide significant<br />
insight into the possible presence <strong>of</strong> infection. Early<br />
changes during infection may include an increase in<br />
the number <strong>of</strong> b<strong>and</strong>s, even before the development<br />
<strong>of</strong> leukocytosis. A great shift to immaturity (left<br />
shift) may occur when infection is severe, with<br />
metamylocytes or even earlier forms present on the<br />
peripheral blood smear. There are many evidences<br />
<strong>of</strong> studies conducted on adults <strong>and</strong> RBC concerning<br />
lead toxicity, but very few reports are available<br />
regarding haematopathological alterations <strong>of</strong> lead<br />
acetate in different white blood cells. Significant<br />
decrease in RBC count, hematocrit (Hct) <strong>and</strong><br />
hemoglobin (Hb) were seen in rats <strong>and</strong> human with<br />
high blood lead levels. (Alexa et al., 2002; Noori et<br />
al., 2003; Othman et al., 2004; Toplan et al., 2004)<br />
In our study the con<strong>tr</strong>ol groups showed all the<br />
leukocytes in normal appearance. Still some altered<br />
types <strong>of</strong> WBCs were also observed. The<br />
adminis<strong>tr</strong>ation <strong>of</strong> lead acetate alters the s<strong>tr</strong>ucture<br />
<strong>and</strong> number <strong>of</strong> WBCs. The nuclear arrangement<br />
was also distorted. In lead <strong>tr</strong>eated groups the shape<br />
<strong>and</strong> s<strong>tr</strong>ucture <strong>of</strong> the monocyte was also altered with<br />
reniform (kidney shaped) nucleus. At higher dose<br />
level this intensity <strong>of</strong> indentation was increased so<br />
that the normal range <strong>of</strong> nucleo-cytoplasmic ratio is<br />
disturbed <strong>and</strong> appeared as reactive monocytes. Our<br />
findings are also in support <strong>of</strong> DeNicola et al.<br />
(1991) with the evidence <strong>of</strong> reactive monocytes<br />
enclosing the cytoplasm became more intensely<br />
basophilic <strong>and</strong> vacuolated. This usually indicates a<br />
chronic inflammatory process or may be seen with<br />
hemoplasmas in the cat.<br />
Toxicity in neu<strong>tr</strong>ophils is defined by the<br />
presence <strong>of</strong> Döhle bodies (small, basophilic<br />
aggregates <strong>of</strong> RNA in the cytoplasm), diffuse<br />
cytoplasmic basophilia etc. In our study each lead<br />
<strong>tr</strong>eated group in neonatal period, represents<br />
increased number <strong>of</strong> degenerated neu<strong>tr</strong>ophils<br />
particularly at birth. In the 16 mg lead exposed<br />
group, during first week <strong>of</strong> lactation, the nuclear<br />
material <strong>of</strong> cell was less condensed <strong>and</strong> nucleus<br />
was divided into 2-3 unequal lobes with colorless<br />
cytoplasm. At higher dose <strong>of</strong> 32 mg lead, this<br />
severity <strong>of</strong> degeneration was very much increased<br />
with many small fragments <strong>of</strong> nuclear material <strong>and</strong><br />
no sign <strong>of</strong> lobulization <strong>and</strong> appropriate<br />
segmentation <strong>of</strong> neu<strong>tr</strong>ophils were observed.<br />
In a study performed on young dogs,<br />
development <strong>of</strong> anemia, leukocytosis,<br />
monocytopenia, polychromato-philia, glycosuria,<br />
increased serum urobilinogen, <strong>and</strong> hematuria has<br />
been reported (Zook, 1972). Lead suppresses bone<br />
marrow hematopoiesis, probably through its<br />
interaction with the enteric iron absorption (Klader,<br />
1779; Chnielnika, 1994). In some reports,<br />
leukocytosis has been at<strong>tr</strong>ibuted to the lead-induced<br />
inflammation (Yagminas et al., 1990).<br />
Hogan <strong>and</strong> Adams, (1979) reported a threefold<br />
increase in neu<strong>tr</strong>ophil <strong>and</strong> monocyte count along<br />
with severe leukocytosis in the young rats that were<br />
exposed to lead. The present investigation revealed<br />
that adminis<strong>tr</strong>ation <strong>of</strong> lead acetate alters the<br />
appearance <strong>and</strong> cause s<strong>tr</strong>uctural changes. The<br />
nuclear arrangement was distorted with<br />
intermingled lobes <strong>and</strong> in some cases formed a<br />
sessile nodule.<br />
Con<strong>tr</strong>oversies exist about monocytes; since in<br />
some studies lead-induced monocytopenia<br />
(Xintaras, 1992) <strong>and</strong> in others significant increases<br />
in monocyte count have been reported (Yagminas<br />
et al., 1990). The reason for such difference is<br />
probably due to the extent <strong>of</strong> lead-induced<br />
inflammation.<br />
Mugahi et al. (2003) investigated additional<br />
hematotoxic effects <strong>of</strong> lead on the erythroid cell<br />
lineage <strong>and</strong> leukocytes following long-term<br />
exposure in rats. Wahab et al. (20<s<strong>tr</strong>ong>10</s<strong>tr</strong>ong>) showed that<br />
lead caused a significant decrease in hematocrit,<br />
RBC, WBC, hemoglobin concen<strong>tr</strong>ation, mean<br />
corpuscular hemoglobin, mean corpuscular<br />
hemoglobin concen<strong>tr</strong>ation <strong>and</strong> lymphocyte <strong>and</strong><br />
monocyte count; <strong>and</strong> significant increase in<br />
neu<strong>tr</strong>ophil count. The results <strong>of</strong> the present study<br />
are also parallel to the above findings. In lead<br />
exposed pups there was significant increase in the<br />
number <strong>of</strong> neu<strong>tr</strong>ophils at different weeks after birth,<br />
but decrease in the number <strong>of</strong> lymphocytes. The<br />
shortened life span <strong>of</strong> erythrocytes is due to<br />
increased fragility <strong>of</strong> the blood cell membrane <strong>and</strong><br />
r<s<strong>tr</strong>ong>edu</s<strong>tr</strong>ong>ced hemoglobin production is due to decreased<br />
levels <strong>of</strong> enzymes involved in hemesynthesis<br />
(Guidotti et al., 2008). It has long been known that<br />
hematopoiesis <strong>and</strong> heme synthesis affected by lead<br />
poisoning (Doull et al., 1980).<br />
In our study reactive <strong>and</strong> cleaved type <strong>of</strong><br />
lymphocyte were observed at the time <strong>of</strong> birth in<br />
lead <strong>tr</strong>eated groups which were reinstated by<br />
increased number <strong>of</strong> plasmacytoid, reactive, large,<br />
oval, irregular, binucleated <strong>and</strong> cleaved<br />
lymphocytes in further days <strong>of</strong> lactation. In the<br />
current investigation at higher dose (32 mg) we
found apoptotic or necrotic neu<strong>tr</strong>ophils were more<br />
prominent in the first <strong>and</strong> second week <strong>of</strong> lactation.<br />
These neu<strong>tr</strong>ophils were characterized by 3-4<br />
separate <strong>and</strong> equal lobes with less condensed<br />
chromatin <strong>and</strong> diffuse cytoplasmic region.<br />
Lead <strong>tr</strong>eated group at the termination <strong>of</strong><br />
lactation, include abnormal nuclear segmentation,<br />
giving abnormal appearance <strong>of</strong> nucleus <strong>and</strong><br />
chromatin condensation in most <strong>of</strong> the neu<strong>tr</strong>ophils<br />
<strong>and</strong> forming a ring like nucleus in some<br />
neu<strong>tr</strong>ophils. Villagra et al., (1997) also postulates<br />
that lead exposure doubles total <strong>and</strong> segmented<br />
neu<strong>tr</strong>ophils in both es<strong>tr</strong>ogens <strong>tr</strong>eated <strong>and</strong> un<strong>tr</strong>eated<br />
rats but causes a three-fold increase in b<strong>and</strong><br />
neu<strong>tr</strong>ophils in animals without es<strong>tr</strong>ogen <strong>tr</strong>eatment,<br />
but not in animals <strong>tr</strong>eated with es<strong>tr</strong>ogen. With a<br />
disappearance <strong>of</strong> non-degranulated eosinophils, the<br />
decrease in non-degranulated eosinophils was<br />
under the effect <strong>of</strong> lead exposure. He also<br />
demons<strong>tr</strong>ates that prepubertal rat exposure to lead<br />
affects blood neu<strong>tr</strong>ophil <strong>and</strong> eosinophil leukocyte<br />
levels <strong>and</strong> induces eosinophil degranulation.<br />
Vyskocil et al., (1991) discovered the effect <strong>of</strong><br />
lead on b<strong>and</strong> neu<strong>tr</strong>ophils reveals an increased<br />
neu<strong>tr</strong>ophilopoiesis rather than release from<br />
in<strong>tr</strong>avascularly sequestered forms in lead-exposed<br />
animals.<br />
In lead <strong>tr</strong>eated group from birth till the<br />
termination <strong>of</strong> lactation, the number <strong>of</strong> immature<br />
cells was increased. There was asynchrony <strong>of</strong><br />
maturation between nucleus <strong>and</strong> cytoplasm. During<br />
normal granulocytopoiesis the lengthening <strong>and</strong><br />
pinching <strong>of</strong> the nucleus were coordinated with<br />
progressive condensation <strong>of</strong> the chromatin with<br />
accelerated maturation nuclear division may be skip<br />
<strong>and</strong> cells retain immature features, because toxic<br />
changes <strong>of</strong> lead accompanies a left shift i.e.<br />
presence <strong>of</strong> immature neu<strong>tr</strong>ophils, b<strong>and</strong>s,<br />
metamyelocytes, myelocytes <strong>and</strong> other unidentified<br />
immature cells. White Blood <strong>Cell</strong>s generally<br />
increase as compared to the con<strong>tr</strong>ol level. The<br />
increase in WBC count indicates the activation <strong>of</strong><br />
defense mechanism <strong>and</strong> immune system <strong>of</strong> gasoline<br />
workers (Whitby, 1980). These findings are also in<br />
confirmations, with our results.<br />
In conclusion, lead exposure leads to various<br />
hematological disorders in white blood cells<br />
including neu<strong>tr</strong>ophilic degeneration, immature<br />
cells, abnormal neu<strong>tr</strong>ophils, reactive <strong>and</strong><br />
plasmacytoid lymphocyte, reactive monocyte etc.<br />
The present study indicates that after adminis<strong>tr</strong>ation<br />
<strong>of</strong> 266.66, 533.33 <strong>and</strong> <s<strong>tr</strong>ong>10</s<strong>tr</strong>ong>66.66 mg/kg/body weight<br />
doses <strong>of</strong> lead acetate WBCs show s<strong>tr</strong>uctural<br />
abnormalities in their nucleus <strong>and</strong> cytoplasm<br />
Prenatal <strong>and</strong> neonatal exposure to lead 39 <br />
including improper segmentation <strong>and</strong> lesser<br />
condensation <strong>of</strong> nucleus. Lead causes fluctuations<br />
in the number <strong>of</strong> various cell types at different<br />
stages <strong>of</strong> postnatal development. The exposure to<br />
lead possesses the potentials to induce hazardous<br />
biological effects during pre <strong>and</strong> postnatal<br />
development in Swiss mice.<br />
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<strong>Journal</strong> <strong>of</strong> <strong>Cell</strong> <strong>and</strong> Molecular Biology <s<strong>tr</strong>ong>10</s<strong>tr</strong>ong>(1): 41-54, 2012 Research Article 41<br />
Haliç University, Printed in Turkey.<br />
http://<s<strong>tr</strong>ong>jcmb</s<strong>tr</strong>ong>.<s<strong>tr</strong>ong>halic</s<strong>tr</strong>ong>.<s<strong>tr</strong>ong>edu</s<strong>tr</strong>ong>.<strong>tr</strong><br />
Sulfabenzamide promotes autophagic cell death in T-47D breast<br />
cancer cells through p53/ DRAM pathway<br />
Raziye MOHAMMADPOUR 1 , Shahrokh SAFARIAN *1 , Soroor FARAHNAK 1 , Sana<br />
HASHEMINASL 1 , Nader SHEIBANI 2<br />
1<br />
School <strong>of</strong> Biology, College <strong>of</strong> Science, University <strong>of</strong> Tehran, Tehran, Iran<br />
2<br />
Department <strong>of</strong> Ophthalmology <strong>and</strong> Visual Sciences, School <strong>of</strong> Medicine <strong>and</strong> Public Health, University <strong>of</strong><br />
Wisconsin, Madison, USA<br />
(* author for correspondence; safarian@ibb.ut.ac.ir )<br />
Received: 26 March 2012; Accepted: 29 May 2012<br />
Abs<strong>tr</strong>act<br />
Sulfonamides exhibit their antitumor effects through multiple mechanisms including inhibition <strong>of</strong> membrane<br />
bound carbonic anhydrases, prevention <strong>of</strong> microtubule assembly, cell cycle arrest, <strong>and</strong> inhibition <strong>of</strong> angiogenesis.<br />
Here, sulfabenzamide’s mechanisms <strong>of</strong> action on T-47D breast cancer cells were determined. <strong>Cell</strong>s incubated with<br />
sulfabenzamide exhibited negligible levels <strong>of</strong> apoptosis, necrosis <strong>and</strong> cell cycle arrest when compared to un<strong>tr</strong>eated<br />
cells. These results were confirmed by morphological examinations, DNA fragmentation assays, flow cytome<strong>tr</strong>ic<br />
<strong>and</strong> real time RT-PCR analysis. Surprisingly, despite negligible detection <strong>of</strong> DNA fragmentation, a considerable<br />
increase in caspase-3 activity was observed in cells incubated with sulfabenzamide. The increased expression ratio<br />
<strong>of</strong> DFF-45/DFF-40 indicated that caspase-3-related DNA fragmentation was blocked <strong>and</strong> apoptosis symptoms<br />
could not be seen. However, the effects <strong>of</strong> caspase-3 for PARP1 <strong>and</strong> DNA-PK deactivation resulted in autophagy<br />
induction. The overexpression <strong>of</strong> critical genes involved in autophagy, including ATG5, p53 <strong>and</strong> DRAM, indicated<br />
that in T-47D cells sulfabenzamide-induced antiproliferative effect was mainly exerted through induction <strong>of</strong><br />
autophagy. Furthermore, downregulation <strong>of</strong> AKT1 <strong>and</strong> AKT2 as well as over expression <strong>of</strong> PTEN resulted in<br />
attenuation <strong>of</strong> AKT/mTOR survival pathway showing that death autophagy should be occurred in sulfabenzamide<br />
<strong>tr</strong>eatment.<br />
Keywords: Sulfabenzamide, breast cancer, autophagy, apoptosis, p53.<br />
Sülfobenzamid, T-47D meme kanseri hücrelerinde p53/DRAM yolağı aracılığıyla ot<strong>of</strong>ajik hücre ölümünü<br />
teşvik eder<br />
Sülfonamidler, membrana bağlı karbonik anhidraz inhibisyonunu, mikrotubül toplanmasının engellenmesini, hücre<br />
siklusunun durdurulmasını ve anjiyogenez inhibisyonunu içeren çoklu mekanizmalarla antitümör etkilerini<br />
göstermektedirler. Burada, T-47D meme kanser hücreleri üzerinde sülfobenzamid mekanizmasının etkisi<br />
belirlenmiştir. Sülfobenzamid ile inkübe edilen hücreler yapılmamış uygulama hücrelerle karşılaştırıldıklarında<br />
önemsenmeyecek seviyede apoptoz, nekroz ve hücre siklusunun durmasını ortaya koymuştur. Bu sonuçlar<br />
morfolojik incelemelerle, DNA fragmantasyon analizleriyle, flow sitome<strong>tr</strong>ik ve gerçek zamanlı RT-PCR<br />
analizleriyle doğrulanmıştır. Şaşırtıcı bir şekilde, DNA fragmantasyonunun ihmal edilebilecek tespitine rağmen,<br />
sülfobenzamidle edilmiş inkübe hücrelerde kaspaz 3 aktivitesinde dikkate değer artış bir gözlenmiştir. DFF-45/<br />
DFF-40’ artmış ın ekspresyon oranı, kaspaz 3 ile ilişkili DNA fragmantasyonunun durdurulduğunu ve apoptoz<br />
belirtilerinin görülemeyeceğini işaret etmektedir. Bununla birlikte PARP1 ve DNA-PK deaktivasyonu için kaspaz<br />
3’ün etkileri ot<strong>of</strong>aji indüklenmesiyle sonuçlanmaktadır. ATG5, p53 ve DRAM gibi ot<strong>of</strong>ajide yer alan kritik<br />
genlerin aşırı ekspresyonu T-47D sülfobenzamid-indüklenmiş hücrelerinde antiproliferatif etkinin çoğunlukla<br />
ot<strong>of</strong>aji indüksiyonu aracılığıyla uygul<strong>and</strong>ığını belirtmektedir. Ayrıca, PTEN aşırı ekspresyonu gibi AKT1 ve<br />
AKT’’nin azalarak düzenlenmesi, ot<strong>of</strong>aji ölümünün sülfobenzamid uygulamasıyla meydana geldiğini gösteren<br />
sağ AKT1/mTOR kalım yolağının etkisinin azalmasıyla sonuçlanmaktadır.<br />
Anahtar kelimeler: sülfobenzamid, meme kanseri, ot<strong>of</strong>aji, apoptoz, p53.
42 Raziye MOHAMMADPOUR et al.<br />
In<strong>tr</strong>oduction<br />
Sulfonamides are synthetic antibacterial agents<br />
with diverse pharmacological effects including<br />
antibacterial, antiviral, antidiabetic, antithyroid, <strong>and</strong><br />
diuretic. Their antibacterial effects are con<strong>tr</strong>ibuted<br />
to the interfering with enzyme activities responsible<br />
for folic acid synthesis by competing for para<br />
aminobenzoic acid. These drugs are selectively<br />
toxic for prokaryotes (Owa et al., 1999; Fukuoka et<br />
al., 2001; Yokoi et al., 2002; Supuran 2003). Two<br />
novel sulfonamides, E7070 <strong>and</strong> E70<s<strong>tr</strong>ong>10</s<strong>tr</strong>ong>, are potently<br />
effective against cancer cells via inhibition <strong>of</strong><br />
tubulin polymerization <strong>and</strong> proliferation. The<br />
ma<strong>tr</strong>ix metalloprotease (MMP) inhibitory effects <strong>of</strong><br />
sulfonamides have been evaluated for <strong>tr</strong>eatment <strong>of</strong><br />
arthritis <strong>and</strong> cancer (Fukuoka et al., 2001; Ozawa et<br />
al., 2001; Supuran et al., 2003; Mohan et al., 2006).<br />
Sulfabenzamide, 4-Amino-N-benzoyl-benzenesulfonamide,<br />
is a sulfonamide derivative used for<br />
<strong>tr</strong>eatment <strong>of</strong> specific vaginal infections in<br />
combination with sulfathiazole <strong>and</strong> sulfacetamide<br />
(Valley <strong>and</strong> Balmer, 1999).<br />
Knowledge regarding alterations in signaling<br />
pathways <strong>and</strong> the type <strong>of</strong> cell death induced by<br />
chemotherapeutic drugs is the first <strong>and</strong> most<br />
important step in design <strong>of</strong> effective <strong>tr</strong>eatments.<br />
Furthermore, manipulation <strong>of</strong> autophagy has the<br />
potential to improve anticancer therapeutics.<br />
Studies have shown that autophagy protects cancer<br />
cells against antitumor effects <strong>of</strong> some drugs by<br />
blocking the apoptotic pathway <strong>and</strong> maintaining<br />
ATP levels. In con<strong>tr</strong>ast, other cancer cells undergo<br />
autophagic cell death (ACD or type II programmed<br />
cell death, PCDII) after anticancer therapies<br />
(Kondo et al., 2005; Kondo <strong>and</strong> Kondo, 2006).<br />
Various anticancer drugs that activate ACD in<br />
breast cancer cells have been reported including<br />
vitamin D analog, EB<s<strong>tr</strong>ong>10</s<strong>tr</strong>ong>89, Tamoxifen <strong>and</strong> other<br />
anties<strong>tr</strong>ogen agents (Hoyer-Hansen et al., 2005).<br />
Tamoxifen induced autophagic pathway occurs<br />
through down regulation <strong>of</strong> AKT activity<br />
(Yokoyama et al., 2009). The 3'-methoxylated<br />
analogue isocannflavin B (IsoB) exhibits an<br />
inhibitory effect on T-47D cell proliferation, which<br />
is accompanied by the appearance <strong>of</strong> an intense<br />
in<strong>tr</strong>acytoplasmic vacuolization <strong>of</strong> autophagic origin<br />
(Brunelli et al., 2009).<br />
Here, we choose sulfabenzamide for assessing<br />
its antitumor activity in T-47D breast cancer cell<br />
line. Our main objective was to determine whether<br />
this drug can be used as an antitumor drug in<br />
medicine. From this point <strong>of</strong> view, we could<br />
ascertain that there is a correlation between the<br />
expression level <strong>of</strong> some critical genes <strong>and</strong><br />
induction <strong>of</strong> death autophagy in T-47D cells.<br />
Materials <strong>and</strong> methods<br />
Reagents<br />
Culture medium, RPMI 1640, <strong>and</strong> fetal bovine<br />
serum were from Gibco (Engl<strong>and</strong>); penicillin<br />
s<strong>tr</strong>eptomycin solution, DNA laddering kit,<br />
Annexin-V-FLOUS Staining Kit, Propidium<br />
Iodide (PI) kit, caspase-3 fluorome<strong>tr</strong>ic<br />
immunosorbent enzyme assay kit, 4',6- Diamidino -<br />
2-phenylindole (DAPI) kit were all acquired from<br />
Roche (Germany); MTT was from Sigma<br />
(Engl<strong>and</strong>); sulfabenzamide <strong>and</strong> doxorubicin were<br />
from Sina Darou (Iran) <strong>and</strong> Ebewe Pharma<br />
(Aus<strong>tr</strong>ia), respectively. QuantiFast SYBR Green<br />
PCR master mix <strong>and</strong> RNeasy plus Mini kit were<br />
provided from Qiagen (USA). RevertAidTM M-<br />
MuLV reverse <strong>tr</strong>anscriptase <strong>and</strong> r<strong>and</strong>om hexamer<br />
were purchased from Fermentas (Germany).<br />
<strong>Cell</strong> culture<br />
Epithelial tumor cell line, T-47D, stemmed from<br />
human ductal breast tissue, was provided from<br />
National <strong>Cell</strong> Bank <strong>of</strong> Pasteur Institute (Tehran,<br />
IRAN; ATCC number HTB-133). <strong>Cell</strong>s were<br />
maintained in RPMI 1640 medium supplemented<br />
with heat-inactivated (35 min, 56°C) fetal bovine<br />
serum (<s<strong>tr</strong>ong>10</s<strong>tr</strong>ong>% v/v) <strong>and</strong> penicillin s<strong>tr</strong>eptomycin<br />
solution (1% v/v) <strong>and</strong> incubated in humidified<br />
condition; 95% air <strong>and</strong> 5% CO2 at 37°C.<br />
Drug preparation <strong>and</strong> <strong>tr</strong>eatments<br />
Regarding the obtained results from MTT assays,<br />
LC50 for sodium sulfabenzamide <strong>and</strong> doxorubicin<br />
after 48 h were estimated at <s<strong>tr</strong>ong>10</s<strong>tr</strong>ong>.8 <strong>and</strong> 0.337×<s<strong>tr</strong>ong>10</s<strong>tr</strong>ong> -3<br />
mM, respectively. After reaching confluency (~<br />
80%), cells were incubated with freshly prepared<br />
drugs at the LC50 concen<strong>tr</strong>ations, harvested by<br />
<strong>tr</strong>ypsin-EDTA, washed three times by phosphatebuffered<br />
saline, <strong>and</strong> stored at -70°C.<br />
Cytotoxicity/Viability assay<br />
In brief, <s<strong>tr</strong>ong>10</s<strong>tr</strong>ong> 4 cells/well were seeded in a 96 well<br />
culture plate <strong>and</strong> incubated with different<br />
concen<strong>tr</strong>ations <strong>of</strong> drugs for 24, 48 <strong>and</strong> 72 h. MTT<br />
was then added to the wells (4 mg/ml or <s<strong>tr</strong>ong>10</s<strong>tr</strong>ong>0<br />
µg/well) <strong>and</strong> the produced formazan was<br />
systematically assessed using Elisa micro plate
eader at the wavelength <strong>of</strong> 570 nm. The percent <strong>of</strong><br />
cell viability related to each drug concen<strong>tr</strong>ation was<br />
estimated in relation to the un<strong>tr</strong>eated sample. All<br />
assays were done at least three times unless stated<br />
otherwise.<br />
Apoptosis quantification<br />
After washing <s<strong>tr</strong>ong>10</s<strong>tr</strong>ong> 6 cells with PBS, cell pellets were<br />
re-suspended in <s<strong>tr</strong>ong>10</s<strong>tr</strong>ong>0 µl <strong>of</strong> ready to use Annexin/PI<br />
buffer (20 µl <strong>of</strong> each Annexin <strong>and</strong> PI buffer in 1 ml<br />
incubation buffer) for <s<strong>tr</strong>ong>10</s<strong>tr</strong>ong>-15 min at 25˚C. Samples<br />
were then diluted in 500 µl <strong>of</strong> incubation buffer <strong>and</strong><br />
analyzed by flow cytome<strong>tr</strong>y (Partech Pass, USA)<br />
using FloMax s<strong>of</strong>tware.<br />
<strong>Cell</strong> cycle analysis<br />
5×<s<strong>tr</strong>ong>10</s<strong>tr</strong>ong> 5 drug <strong>tr</strong>eated cells were incubated with DAPI<br />
solution (<s<strong>tr</strong>ong>10</s<strong>tr</strong>ong> µg/ml <strong>and</strong> 6% Triton X-<s<strong>tr</strong>ong>10</s<strong>tr</strong>ong>0 in PBS)<br />
for 30 min in the dark at 4ºC. Using a flow<br />
cytometer fluorescent emission <strong>of</strong> applied indicator<br />
was detected (excitation <strong>and</strong> emission wavelength<br />
<strong>of</strong> 359 nm <strong>and</strong> 461 nm, respectively) <strong>and</strong> the<br />
analysis was performed using FloMax s<strong>of</strong>tware.<br />
Morphological studies <strong>of</strong> the apoptotic cells<br />
<strong>Cell</strong>s were cultured on cover slips coated with Poly<br />
L-lysine <strong>and</strong> exposed to drugs for 48 h. Following<br />
staining with Annexin V-FITC (20 µg/ml) <strong>and</strong> PI<br />
(20 µg/ml) in the dark for <s<strong>tr</strong>ong>10</s<strong>tr</strong>ong>-15 min, samples were<br />
examined using a fluorescent microscope (Carl<br />
Zeiss-Germany) using 450-500 nm excitation <strong>and</strong><br />
515-565 nm emission filters.<br />
Measurement <strong>of</strong> caspase-3 activity<br />
Following drug <strong>tr</strong>eatments, cells were harvested<br />
<strong>and</strong> incubated in lysis buffer on ice for 1 minute.<br />
After cen<strong>tr</strong>ifugation, sample supernatants were used<br />
for caspase-3 activity measurements using AC-<br />
DEVED-AFC fluorescent subs<strong>tr</strong>ate as<br />
recommended by the supplier. The concen<strong>tr</strong>ation <strong>of</strong><br />
enzyme-released AFC was estimated using<br />
fluorospec<strong>tr</strong>ophotometer (HITACHI model MPF4-<br />
Japan) at 400 nm excitation <strong>and</strong> 505 nm emission<br />
wavelengths.<br />
DNA laddering assay<br />
2×<s<strong>tr</strong>ong>10</s<strong>tr</strong>ong> 6 drug <strong>tr</strong>eated cells were lysed with an equal<br />
volume <strong>of</strong> binding/lysis buffer for <s<strong>tr</strong>ong>10</s<strong>tr</strong>ong> minutes at 15-<br />
25 º C. The obtained ex<strong>tr</strong>act was processed as<br />
recommended by the supplier. Elec<strong>tr</strong>ophoresis <strong>of</strong><br />
the samples in 1% agarose gel at 75 volt for 90<br />
minutes revealed DNA cleavage pattern <strong>of</strong> cells<br />
Sulfabenzamide promotes autophagic cell death 43<br />
relative to positive con<strong>tr</strong>ol (DNA ex<strong>tr</strong>acted<br />
prepared from U937 cells incubated 3h with 4 µM<br />
camptothecin).<br />
Preparation <strong>of</strong> total RNA, cDNA synthesis <strong>and</strong> real<br />
time RT-PCR<br />
Total RNA was purified using the RNeasy Qiagen<br />
kit according to the manufacturer’s<br />
recommendation. First s<strong>tr</strong><strong>and</strong> cDNA was generated<br />
using RevertAidTM M-MuLV reverse <strong>tr</strong>anscriptase<br />
<strong>and</strong> 5µg <strong>of</strong> RNA with r<strong>and</strong>om hexamer primers.<br />
Real time quantitative RT-PCR was performed<br />
using the QuantiFast SYBR Green PCR Master<br />
Mix under the following program: 95˚C for 5 min<br />
followed by 40 cycles (95˚C for <s<strong>tr</strong>ong>10</s<strong>tr</strong>ong> sec, annealing<br />
for 25 sec <strong>and</strong> extension at 72˚C for 30 sec).<br />
Analysis was done using Corbett rotor-gene 6000<br />
s<strong>of</strong>tware based on the comparative Ct method (or<br />
ΔΔCt method). The relative amount <strong>of</strong> target<br />
materials was quantified compared to the reference<br />
gene (GAPDH). Primers were prepared by TAG<br />
(Copenhagen, Denmark) <strong>and</strong> were used to amplify<br />
specific regions <strong>of</strong> cDNA as listed in Table1.<br />
Statistical analysis<br />
For all methods statistical analysis were performed<br />
by the SPSS version 16 <strong>and</strong> Excel 2007 s<strong>of</strong>twares.<br />
Statistical analysis for MTT assay, flow cytome<strong>tr</strong>y,<br />
caspase-3 activity were performed by one way<br />
ANOVA <strong>and</strong> real time RT-PCR methods were<br />
carried out by t-test. All results are presented as<br />
mean ± st<strong>and</strong>ard deviation (p< 0.05 was considered<br />
statistically significant).<br />
Results<br />
Sulfabenzamide inhibits the proliferation <strong>of</strong> T-47D<br />
cells<br />
The MTT assay was used to evaluate the viability<br />
<strong>of</strong> T-47D cells incubated with different<br />
concen<strong>tr</strong>ations <strong>of</strong> sulfabenzamide (0.0-20 mM) or<br />
doxorubicin (0.0-0.6 µM) after 24, 48 <strong>and</strong> 72 h<br />
(chemical s<strong>tr</strong>uctures are shown in Figure 1A). We<br />
checked toxic effects <strong>of</strong> doxorubicin on T-47D<br />
since it had been reported that its anticancer effects<br />
on different cell types exerts through distinct<br />
cellular processes (apoptosis or cell cycle arrest).<br />
Thus, it could be utilized as a con<strong>tr</strong>ol in our<br />
experiments. The 50% growth inhibition (LC50)<br />
concen<strong>tr</strong>ation for sulfabenzamide <strong>and</strong> doxorubicin<br />
after 48 h, were calculated as <s<strong>tr</strong>ong>10</s<strong>tr</strong>ong>.8 mM <strong>and</strong> 0.33<br />
µM, respectively, <strong>and</strong> utilized in the following<br />
experiments (Figure 1B).
44 Raziye MOHAMMADPOUR et al.<br />
Table 1. List <strong>of</strong> primers. Forward <strong>and</strong> reverse primer pairs for PTEN gene were designed to amplify a region which could<br />
not anneal to PTEN pseudogene. Primer for p53 was designed for the mutant form present in T-47D cells.<br />
Gene Accession number primers<br />
F: CCAGGTGGTCTCCTCTGACTTCAACAG<br />
PCR product(bp)<br />
GAPDH NC_000012.11<br />
R: AGGGTCTCTCTCTTCTTCCTCTTGTGCTCT<br />
F: GTGAGATATGGTTTGAATATGAAGGC<br />
218<br />
ATG5 NC_000006.11<br />
R: CTCTTAAAATGTACTGTGATGTTCCAA<br />
F: GGAGAGGAGCCATTTATTGAAACT<br />
122<br />
beclin1 NC_000017.<s<strong>tr</strong>ong>10</s<strong>tr</strong>ong><br />
R: AGAGTGAAGCTGTTGGCACTTTCTG<br />
F: CTTGGATTGGTGGGATGTTTC<br />
<s<strong>tr</strong>ong>10</s<strong>tr</strong>ong>4<br />
DRAM NC_000012.11<br />
R: GATGATGGACTGTAGGAGCGTGT<br />
F: CCAGATGGAAAGACGTTTTTGTG<br />
135<br />
AKT1 NC_000014.8<br />
R: GAGAACAAACTGGATGAAATAAA<br />
F: CTGCGGAAGGAAGTCATCATTGC<br />
<s<strong>tr</strong>ong>10</s<strong>tr</strong>ong>6<br />
AKT2 NC_000019.9<br />
R: CGGTCGTGGGTCTGGAAGGCATAC<br />
F: CAAACTTTTTCAGAGGGGATCG<br />
125<br />
caspase-3 NC_000004.11<br />
R: GCATACTGTTTCAGCATGGCAC<br />
F: AAGAAGCTGAGCGAGTGTC<br />
261<br />
bax NC_000019.9<br />
R: GGCCCCAGTTGAAGTTGC<br />
F: ATGGAACTAACTATGTTGGACTATG<br />
157<br />
cyclinB1 NC_000005.9<br />
R: AGTATATGACAGGTAATGTTGTAGAGT<br />
F: AGGGGGAAACACCAGAATCAAGTG<br />
138<br />
bcl-2 NC_000018.9<br />
R: CCCAGAGAAAGAAGAGGAGTTATAA<br />
F: GGTCTTGTGGACAGTAGTTTGCC<br />
113<br />
AIF NC_000023.<s<strong>tr</strong>ong>10</s<strong>tr</strong>ong><br />
R: TCTCACTCTCTGATCGGATACCA<br />
F:CCTGTGCAGCTGTGGGTTGATTT<br />
115<br />
p53 NC_000017.<s<strong>tr</strong>ong>10</s<strong>tr</strong>ong><br />
R: AGGAGGGGCCAGACCATCGCTAT<br />
F: TTGGAGTCCCGATTTCAGAG<br />
150<br />
DFF40 NC_000001.<s<strong>tr</strong>ong>10</s<strong>tr</strong>ong><br />
R: CTGTCGAAGTAGCTGCCATTG<br />
F:TTCTGTGTCTACCTTCCAATACTA<br />
194<br />
DFF45 NC_000001.<s<strong>tr</strong>ong>10</s<strong>tr</strong>ong><br />
R:CTGTCTGTTTCATCTACATCAAAG<br />
F: TAACATTAGTCTGGATGGTGTAGA<br />
127<br />
PARP1 NC_000001.<s<strong>tr</strong>ong>10</s<strong>tr</strong>ong><br />
R: TTACCTGAGCAATATCATAGACAAT<br />
F: TGGCATTACAGACATCTTTAGTTT<br />
113<br />
DNA-PK NC_000008.<s<strong>tr</strong>ong>10</s<strong>tr</strong>ong><br />
R: ACTTTAGGATTTCTTCTCTACATTCA<br />
F: TGGCTAAGTGAAGATGACAATCATG<br />
111<br />
PTEN NC_0000<s<strong>tr</strong>ong>10</s<strong>tr</strong>ong>.<s<strong>tr</strong>ong>10</s<strong>tr</strong>ong><br />
R: GCACATATCATTACACCAGTTCGT<br />
81
Sulfabenzamide promotes autophagic cell death 45<br />
Figure 1. A) Chemical s<strong>tr</strong>ucture <strong>of</strong> sulfabenzamide <strong>and</strong> doxorubicin. B) Viability curve <strong>of</strong> sodium<br />
sulfabenzamide <strong>and</strong> doxorubicin <strong>tr</strong>eated T-47D cells. Percent viability <strong>of</strong> cells incubated with sodium<br />
sulfabenzamide <strong>and</strong> doxorubicin was calculated relative to the related un<strong>tr</strong>eated con<strong>tr</strong>ols after 24, 48 <strong>and</strong> 72<br />
h. Each point relates to the mean value <strong>of</strong> at least three independent experiments. The related correlation<br />
coefficient (r 2 ) was adjusted until the best fit for the selected mathematical function was used to interpolate<br />
the experimental points.<br />
T-47D cells do not exhibit DNA fragmentation <strong>and</strong><br />
apoptotic morphology in the presence <strong>of</strong><br />
sulfabenzamide or doxorubicin<br />
Unlike DNA fragmentation patterns observed in<br />
DNA ex<strong>tr</strong>acted from U937 cells incubated with<br />
camptothecin (as a positive con<strong>tr</strong>ol <strong>of</strong> DNA<br />
laddering kit), the gel elec<strong>tr</strong>ophoresis <strong>of</strong> DNA<br />
prepared from cells incubated with sulfabenzamide<br />
(<s<strong>tr</strong>ong>10</s<strong>tr</strong>ong>.8 mM) or doxorubicin (0.33 µM) showed no<br />
DNA ladder or smear pattern confirming lack <strong>of</strong><br />
apoptosis or necrosis in these cells (Figure 2A).<br />
Morphological analysis <strong>of</strong> sulfabenzamide <strong>and</strong><br />
doxorubicin <strong>tr</strong>eated cells, double stained with<br />
Annexin-FITC <strong>and</strong> PI, evaluated by fluorescent<br />
microscopy <strong>and</strong> confirmed the results <strong>of</strong> DNA<br />
laddering analysis. There were few cells having<br />
morphological characteristics <strong>of</strong> apoptotic <strong>and</strong><br />
necrotic cells (Figures 2B, 2C). Early (young)<br />
apoptotic cells have rounded shape <strong>and</strong> shiny green<br />
membrane because PI cannot pene<strong>tr</strong>ate into the<br />
cells <strong>and</strong> Annexin-FITC binds to the externally<br />
membrane-exposed phosphatidylserines (Figures<br />
2B, 2C). Late apoptotic <strong>and</strong> necrotic cells have<br />
membrane permeability for PI so their nuclei are<br />
stained red (Figure 2C). The main difference<br />
between necrotic <strong>and</strong> late apoptotic cells is the<br />
potency <strong>of</strong> late apoptotic cells for simultaneous<br />
staining <strong>of</strong> nuclei <strong>and</strong> membrane-exposed<br />
phosphatidylserines with PI <strong>and</strong> Annexin-FITC,<br />
respectively. Membrane blebbing, which is a<br />
common feature <strong>of</strong> apoptotic cells was seen in<br />
Figure 2B. Evidently, healthy cells cannot be seen<br />
under fluorescent microscope since they were not<br />
stained with either <strong>of</strong> the fluorescent dyes (Figure<br />
2E).
46 Raziye MOHAMMADPOUR et al.<br />
Figure 2. A) DNA laddering analysis. 1-3 µg DNA prepared from 2×<s<strong>tr</strong>ong>10</s<strong>tr</strong>ong> 6 cells was resolved by<br />
elec<strong>tr</strong>ophoresis in a 1% agarose gel. DNA fragmentation was observed only in positive con<strong>tr</strong>ol (camptothecin<br />
<strong>tr</strong>eated) cells, but it was not detected in con<strong>tr</strong>ol or cells incubated with doxorubicin or sodium<br />
sulfabenzamide. B) Observation <strong>of</strong> the morphology <strong>of</strong> early apoptotic cells using fluorescent microscopy<br />
following double staining with Annexin V-FITC <strong>and</strong> PI. Morphological characteristics <strong>of</strong> early apoptotic<br />
cells (rounded green shiny cells showing membrane blebbing) in sulfabenzamide <strong>tr</strong>eated cells. C)<br />
Observation <strong>of</strong> the morphology <strong>of</strong> late apoptotic <strong>and</strong> necrotic cells using fluorescent microscopy following<br />
double staining with Annexin V-FITC <strong>and</strong> PI. Morphological characteristics <strong>of</strong> late apoptotic (flattened green<br />
shiny cells showing red dense nuclei) <strong>and</strong> necrotic cells (red dense spheres lacking green shiny membrane) in<br />
sulfabenzamide <strong>tr</strong>eated cells. Similar results were observed for doxorubicin (not shown). Living cells due to<br />
lack <strong>of</strong> staining with dyes are not detectable in fluorescent visual field (E) but are visible using phase con<strong>tr</strong>ast<br />
microscopy (D).
Sulfabenzamide promotes autophagic cell death 47<br />
Figure 3. Caspase-3 activity was increased in cells incubated with sulfabenzamide or doxorubicin. Enzyme<br />
activity in the con<strong>tr</strong>ol, sodium sulfabenzamide, or doxorubicin <strong>tr</strong>eated cells were 1.308±0.115, 2.07±0.08,<br />
<strong>and</strong> 2.496±0.11 nM.h -1 , respectively. St<strong>and</strong>ard curve based on emission (Y axis) <strong>of</strong> different concen<strong>tr</strong>ation <strong>of</strong><br />
free AFC (nM) is plotted (inset). Diagram <strong>of</strong> free AFC is plotted in 400 nm excitation <strong>and</strong> 505 nm emission<br />
wavelengths.<br />
Table 2. Numerical results <strong>of</strong> flow cytome<strong>tr</strong>y analysis. Results are the mean value ± SD for at least three<br />
replicated experiments. Each column named with Qi which includes data related to the quadrant that are Q1<br />
(PI + <strong>and</strong> Annexin V-FITC - ) or Q1+Q2 (Q2 is the region for PI + <strong>and</strong> Annexin V-FITC + ) indicated percent value<br />
<strong>of</strong> necrotic cells, <strong>and</strong> columns Q4 (PI - <strong>and</strong> Annexin V-FITC + ) or Q2+Q4 show percent values <strong>of</strong> apoptotic cells<br />
(see text). Column Q3 (PI - <strong>and</strong> Annexin V-FITC - ) indicates percent value <strong>of</strong> normal cells. Column <strong>of</strong> G1, S<br />
<strong>and</strong> G2/M represent the percent value <strong>of</strong> the cells placed in each related phase <strong>of</strong> cell cycle. NC, Dox <strong>and</strong> SU<br />
are abbreviations for Negative Con<strong>tr</strong>ol, Doxorubicin <strong>and</strong> Sulfabenzamide, respectively.<br />
Treated<br />
cells<br />
Q3 Q1 Q2 Q4 Q1+Q2 Q2+Q4 G1<br />
S G2<br />
NC 98.94±0.72 0.90±0.46 0.05±0.04 0.35±0.09 0.96±0.50 0.40±0.11 67.06±5.79 16.39±4.27 16.54±3.20<br />
DOX 97.60±1.12 1.33±0.68 0.015±0.02 1.05±0.90 1.34±0.67 1.06±0.90 30.58±1.14 40.55±4.65 28.86±3.51<br />
SU 98.58±0.56 0.27±0.19 0.09±0.08 0.86±0.57 0.37±0.1 0.96±0.54 48.80±4.28 27.47±4.39 22.70±3.79
48 Raziye MOHAMMADPOUR et al.<br />
Caspase-3 activity was increased in the<br />
sulfabenzamide <strong>and</strong> doxorubicin <strong>tr</strong>eated cells<br />
Using caspase-3 specific subs<strong>tr</strong>ate, subsequent<br />
releasing <strong>of</strong> the fluorescent product (AFC) was<br />
measured <strong>and</strong> average enzymatic velocity was<br />
calculated (three independent experiments) as<br />
16.6±1.42, 26.2±1.3 <strong>and</strong> 38.3±0.85 (∆F.h -1 , ∆F<br />
means fluorescent intensity alteration) for un<strong>tr</strong>eated<br />
cell, sulfabenzamide or doxorubicin <strong>tr</strong>eated cells,<br />
respectively. Using the st<strong>and</strong>ard curve <strong>of</strong> free AFC,<br />
enzymatic activity was calculated as 1.308±0.115,<br />
2.07± 0.08 <strong>and</strong> 2.496± 0.11nM.h -1 , respectively<br />
(Figure 3).<br />
Comparing with un<strong>tr</strong>eated cells, caspase-3<br />
activity was increased in drug <strong>tr</strong>eated samples.<br />
Elevated activity <strong>of</strong> caspase-3, which is a sign <strong>of</strong><br />
apoptosis induction, is in con<strong>tr</strong>ast with the DNA<br />
laddering results <strong>and</strong> is further discussed below.<br />
Sulfabenzamide did not induce apoptosis but<br />
induced a minimal shift from G1 to S <strong>and</strong> G2/M<br />
phases <strong>of</strong> the cell cycle<br />
Using flow cytome<strong>tr</strong>ic analysis <strong>and</strong> Annexin-FITC<br />
<strong>and</strong> PI staining, the incidence <strong>of</strong> apoptosis <strong>and</strong><br />
necrosis in un<strong>tr</strong>eated, sulfabenzamide, or<br />
doxorubicin <strong>tr</strong>eated cells were quantified (Figure<br />
4A <strong>and</strong> Table 2).<br />
Congruent with graph interpretation methods<br />
applied in most publications, the sum <strong>of</strong> cell<br />
populations in regions Q2 (PI + <strong>and</strong> Annexin V-<br />
FITC + ) <strong>and</strong> Q4 (PI - <strong>and</strong> Annexin V-FITC + ) were<br />
considered as early <strong>and</strong> late apoptotic cells (Hsu et<br />
al., 2006; Tyagi et al., 2006; Dowejko et al., 2009;<br />
LaPensee et al., 2009). In addition, regions Q1 (PI +<br />
<strong>and</strong> Annexin V-FITC - ) <strong>and</strong> Q3 (PI - <strong>and</strong> Annexin V-<br />
FITC - ) indicated necrotic <strong>and</strong> unscathed<br />
populations, respectively. In some publications, cell<br />
percentages located in Q1 <strong>and</strong> Q2 (Q2 is the region<br />
for PI + <strong>and</strong> Annexin V-FITC + ) quarters are<br />
considered as necrotic cells (Davis et al., 2000). In<br />
these studies, Q4 quarter (PI - <strong>and</strong> Annexin V-<br />
FITC + ) represented the percentage <strong>of</strong> apoptotic<br />
cells. Therefore, in Table 2 determination <strong>of</strong><br />
necrotic cells was performed separately via Q1, as<br />
well as Q1+Q2, <strong>and</strong> the estimation for apoptotic<br />
cells was carried out as Q2+Q4 as well as Q4, in<br />
order to indicate that the low percentages <strong>of</strong><br />
apoptotic <strong>and</strong> necrotic cells observed was not<br />
influenced by the applied analytical methods.<br />
Flow cytome<strong>tr</strong>y is useful for calculating the<br />
percentages <strong>of</strong> cells existing in various stages <strong>of</strong> the<br />
cell cycle including G1, S <strong>and</strong> G2/M. To make a<br />
practical use <strong>of</strong> this technique, cells were stained<br />
with DAPI, which enters the nucleus <strong>and</strong> binds to<br />
DNA <strong>and</strong> emanates fluorescent emission.<br />
Although, no significant change in the normal<br />
pattern <strong>of</strong> cell dis<strong>tr</strong>ibution throughout the cell cycle<br />
was observed for sulfabenzamide <strong>tr</strong>eated cells (18%<br />
shift from G1 to S <strong>and</strong> G2/M) a considerable<br />
<strong>tr</strong>ansition (37%) was detected from G1 to S (main<br />
<strong>tr</strong>ansition) <strong>and</strong> G2/M in cells incubated with<br />
doxorubicin as positive con<strong>tr</strong>ol (Figure 4B <strong>and</strong><br />
Table 2).<br />
Alterations in expression <strong>of</strong> proapoptotic,<br />
prosurvival <strong>and</strong> autophagic genes in<br />
sulfabenzamide <strong>and</strong> doxorubicin <strong>tr</strong>eated cells<br />
The changes in expression level <strong>of</strong> apoptotic, cell<br />
survival <strong>and</strong> autophagic genes were evaluated using<br />
real time RT-PCR. With respect to the results<br />
shown in Figure 5 as well as its insets it can be seen<br />
that in sulfabenzamide <strong>tr</strong>eatments some apoptotic<br />
genes (DFF-45 <strong>and</strong> DNA-PK ) were over expressed<br />
while some others were down regulated (PARP1,<br />
Bax, Bcl-2 <strong>and</strong> AIF) or retained their expression<br />
level in a constant condition (DFF-40 <strong>and</strong> caspase-<br />
3). Moreover, some critical genes which are<br />
important in cell survival pathway were also down-<br />
regulated (AKT1 <strong>and</strong> AKT2) or over expressed<br />
(PTEN). Alterations in gene expression were<br />
evaluated for some autophagic genes such as<br />
ATG5, p53 <strong>and</strong> DRAM indicating higher amounts<br />
<strong>of</strong> the related <strong>tr</strong>anscripts in drug <strong>tr</strong>eated cells<br />
relative to the un<strong>tr</strong>eated ones. In doxorubicin<br />
<strong>tr</strong>eated cells some apoptotic genes were focused<br />
<strong>and</strong> their alterations including over expression <strong>of</strong><br />
caspase-3, DNA-PK, DFF-45 <strong>and</strong> Bax; down<br />
regulation <strong>of</strong> DFF-40 <strong>and</strong> constant expression <strong>of</strong><br />
AIF <strong>and</strong> PARP1 were evaluated (Figure 5).
Sulfabenzamide promotes autophagic cell death 49<br />
Figure 4. A) Two dimensional plots <strong>of</strong> Annexin V-FITC against PI related to the flow cytome<strong>tr</strong>ic<br />
experiments. Two dimensional diagrams from flow cytome<strong>tr</strong>ic studies showed that the percentage <strong>of</strong><br />
apoptotic cells (cells located in the Q4 area or total cells in Q2 + Q4) <strong>and</strong> necrosis (cell located in Q1 or in<br />
Q1+Q2) do not show dramatic differences compared with con<strong>tr</strong>ol cells. B) Effects <strong>of</strong> sodium sulfabenzamide<br />
<strong>and</strong> doxorubicin on the cell cycle dis<strong>tr</strong>ibution. FL4-A indicates the area under the registered elec<strong>tr</strong>ical signal<br />
<strong>of</strong> each stained cell. The curves from left to right relate to G1, S, G2/M phases <strong>of</strong> the cell cycle in con<strong>tr</strong>ol,<br />
doxorubicin or sodium sulfabenzamide <strong>tr</strong>eated samples.
50 Raziye MOHAMMADPOUR et al.<br />
Figure 5. Quantitative real time RT-PCR analysis histograms. Real time RT-PCR for the selected genes for<br />
sulfabenzamide (A) <strong>and</strong> Doxorubicin (B) <strong>tr</strong>eated T-47D cells were determined as described in Methods. The<br />
relative amount <strong>of</strong> target material was quantified compared to the reference gene using the comparative Ct<br />
(ΔΔCt) method. The statistical significant differences are indicated with * <strong>and</strong> ** for 0.01
independent <strong>of</strong> changes in the related mRNA level<br />
(Figures 3 <strong>and</strong> 5). The increased activity <strong>of</strong><br />
caspase-3 was negated via overexpression <strong>of</strong> DFF-<br />
45. DFF-45 is the natural inhibitor <strong>of</strong> DFF-40<br />
(CAD) (Liu et al., 1997). In addition, the increased<br />
expression <strong>of</strong> DFF-45, along with a modest<br />
increase (for sulfabenzamide) <strong>and</strong> a significant<br />
decrease (for doxorubicin) in DFF-40 expression<br />
(Figure 5), indicated that the increased activity <strong>of</strong><br />
caspase-3 could be blocked by the increased<br />
expression ratio <strong>of</strong> DFF-45/DFF-40. This r<s<strong>tr</strong>ong>edu</s<strong>tr</strong>ong>ces<br />
the level <strong>of</strong> active DFF-40 to <strong>tr</strong>igger DNA<br />
fragmentation <strong>and</strong> appearance <strong>of</strong> apoptotic<br />
symptoms. Furthermore, it has been reported that<br />
caspases are activated during autophagy in dying<br />
cells <strong>and</strong> are suppressed for apoptosis induction<br />
(Martin et al., 2004; Yu et al., 2004). Therefore, it<br />
can be d<s<strong>tr</strong>ong>edu</s<strong>tr</strong>ong>ced that during autophagy, the effects<br />
<strong>of</strong> activated caspase-3 on their downs<strong>tr</strong>eam<br />
subs<strong>tr</strong>ates (like DFF-40) should be suppressed by<br />
special factors (e.g. DFF-45 in T-47D cells) only in<br />
those cellular routes which are involved in the<br />
appearance <strong>of</strong> apoptosis symptoms (e.g. DNA<br />
fragmentation).<br />
<strong>Cell</strong> cycle arrest, an important cellular target<br />
affected by sulfabenzamide <strong>and</strong> doxorubicin, was<br />
analyzed using flow cytome<strong>tr</strong>y. Incubation <strong>of</strong> T-<br />
47D cells with 0.33 µM doxorubicin resulted in a<br />
significant accumulation <strong>of</strong> cells in S phase, <strong>and</strong> to<br />
a lesser extent in G2/M phase <strong>of</strong> the cell cycle<br />
(Figure 4B <strong>and</strong> Table 2). Thus, doxorubicin exerts<br />
its antiproliferative action mainly through cell cycle<br />
arrest. Induced mitotic catas<strong>tr</strong>ophe following<br />
increased activation <strong>of</strong> cyclinB1/Cdc2 may occur<br />
while cells are delayed, particularly in G2 phase <strong>of</strong><br />
the cell cycle (Lindqvist et al., 2007). The induced<br />
G2/M arrest along with down regulation <strong>of</strong><br />
cyclinB1 expression confirmed that anticancer<br />
activity <strong>of</strong> doxorubicin is not via mitotic<br />
catas<strong>tr</strong>ophe (Figure 5 <strong>and</strong> Table 2). In con<strong>tr</strong>ast to<br />
doxorubicin, minimal cell cycle arrest in S <strong>and</strong><br />
G2/M phases (totally 18%) was observed in T-47D<br />
cells incubated with <s<strong>tr</strong>ong>10</s<strong>tr</strong>ong>.8 mM sulfabenzamide<br />
(Figure 4B <strong>and</strong> Table2). Thus, cell cycle arrest<br />
could not be mainly responsible for a 50%<br />
r<s<strong>tr</strong>ong>edu</s<strong>tr</strong>ong>ction in cell viability in the presence <strong>of</strong><br />
sulfabenzamide.<br />
As we know, when apoptosis is blocked or<br />
delayed autophagy <strong>tr</strong>iggered <strong>and</strong> vice versa. These<br />
possibilities are consistent with our findings<br />
regarding lack <strong>of</strong> apoptosis in drug <strong>tr</strong>eated cells <strong>and</strong><br />
induction <strong>of</strong> autophagy. The induced<br />
overexpression <strong>of</strong> ATG5 supported that autophagy<br />
Sulfabenzamide promotes autophagic cell death 51<br />
<strong>tr</strong>iggered in the presence <strong>of</strong> sulfabenzamide (Figure<br />
5). This could be probably occurred through the<br />
increase in Bax activity working on mitochondrial<br />
membrane to result in activation <strong>of</strong> caspase-3 for<br />
PARP1 <strong>and</strong> DNA-PK deactivation <strong>and</strong> autophagy<br />
induction. It has been reported that induction <strong>of</strong><br />
autophagy by PUMA (the p53-inducible BH3-only<br />
protein) depends on Bax/Bak <strong>and</strong> can be<br />
reproduced by overexpression <strong>of</strong> Bax (Yee et al.,<br />
2009). Here, in doxorubicin <strong>tr</strong>eatment, increase in<br />
Bax activity could be occurred in parallel with the<br />
increment <strong>of</strong> Bax <strong>tr</strong>anscripts affecting on the cells<br />
for caspase-3 activation <strong>and</strong> changing the cell's<br />
destiny toward autophagy (Figure 5). This notion<br />
could be also supplied in sulfabenzamide <strong>tr</strong>eatment<br />
aside from the mild decrease in Bax expression<br />
because activation <strong>of</strong> the existed Bax molecules in<br />
the cells could be happened for caspase-3 activation<br />
<strong>and</strong> autophagy induction (Figure 5). It has been<br />
also reported that proteolytic cleavage <strong>of</strong> PARP1,<br />
performed by caspase-3, produces specific<br />
proteolytic cleavage fragments which are involved<br />
in the cell’s decision to change its fate from<br />
apoptosis toward autophagy (Munoz-Gamez et al.,<br />
2009; Chaitanya et al., 20<s<strong>tr</strong>ong>10</s<strong>tr</strong>ong>). Induction <strong>of</strong><br />
autophagic cell death is dependent on DNA-PK<br />
inhibition (Daido et al., 2005). Thus, the increased<br />
activity <strong>of</strong> caspase-3 could finally deactivate<br />
PARP1 (has a decreased <strong>and</strong> constant expression<br />
level in sulfabenzamide <strong>and</strong> doxorubicin<br />
<strong>tr</strong>eatments, respectively) <strong>and</strong> DNA-PK (has an<br />
increased <strong>and</strong> invariable expression level in<br />
sulfabenzamide <strong>and</strong> doxorubicin <strong>tr</strong>eatments,<br />
respectively) until apoptosis was blocked <strong>and</strong><br />
autophagy induced (Figures 3 <strong>and</strong> 5).<br />
Despite the existence <strong>of</strong> some con<strong>tr</strong>oversies<br />
regarding the possible role <strong>of</strong> autophagy in tumor<br />
progression by promoting cell survival, autophagy<br />
can exist as a backup mechanism promoting<br />
cellular death when other mortality mechanisms are<br />
not functional. Hyperactivation <strong>of</strong> autophagy above<br />
the threshold point leads to unlimited self-eating <strong>of</strong><br />
the cells causing autophagy or type II programmed<br />
cell death (Hoyer-Hansen et al., 2005; Maiuri et al.,<br />
20<s<strong>tr</strong>ong>10</s<strong>tr</strong>ong>). Based on our data, downregulation <strong>of</strong> AKT1<br />
<strong>and</strong> AKT2 as well as upregulation <strong>of</strong> PTEN in<br />
sulfabenzamide <strong>tr</strong>eated cells indicated that cell<br />
survival pathways were slowed down (Figure 5). In<br />
addition, down regulation <strong>of</strong> bcl-2 was happened<br />
along with the induction <strong>of</strong> autophagy (Figure 5). It<br />
has been reported that targeted silencing <strong>of</strong> bcl-2<br />
expression (an anti-autophagic gene) in human<br />
breast cancer cells with RNA-interference has
52 Raziye MOHAMMADPOUR et al.<br />
promoted autophagic cell death <strong>and</strong> thus presents a<br />
therapeutic potential (Akar et al., 2008).<br />
p53 is involved in decreasing cell survival<br />
potency through inactivation <strong>of</strong> AKT/mTOR<br />
pathways, <strong>and</strong> stimulation <strong>of</strong> autophagy via<br />
<strong>tr</strong>ansactivation <strong>of</strong> DRAM (Maiuri et al., 2007).<br />
Thus, the observed increased expression level <strong>of</strong><br />
DRAM <strong>and</strong> p53 genes support our conclusion that<br />
the repression <strong>of</strong> AKT/mTOR survival pathway<br />
(via p53 overexpression) <strong>and</strong> autophagy induction<br />
(via increased DRAM <strong>tr</strong>anscripts) are responsible<br />
for r<s<strong>tr</strong>ong>edu</s<strong>tr</strong>ong>ced viability <strong>of</strong> T-47D cells <strong>and</strong> induction<br />
<strong>of</strong> death inducing autophagy in the presence <strong>of</strong><br />
sulfabenzamide (Figure 5). T -47D cells contain<br />
only a single copy <strong>of</strong> the p53 missense mutation<br />
(Schafer et al., 2000). It has been reported by<br />
various studies that mutant p53 may lose its natural<br />
antitumor activity (Lim et al., 2009). Interestingly,<br />
in the presence <strong>of</strong> sulfabenzamide the antitumor<br />
activities <strong>of</strong> mutant form <strong>of</strong> p53 should return to the<br />
normal activities <strong>of</strong> the wild type form to induce<br />
autophagic cell death. This is very similar to the<br />
mechanism <strong>of</strong> action for some antitumor drugs<br />
reactivating mutant p53 to kill cancerous cells<br />
(Lambert et al., 2009).<br />
Evidently, checking <strong>of</strong> the protein expression<br />
levels using other supplementary methods such as<br />
western blotting could provide us better documents<br />
to support the presented real time RT-PCR data.<br />
But, in our work, we found that the registered<br />
alterations for the level <strong>of</strong> RNA <strong>tr</strong>anscripts were in<br />
a good consistence with the expected cellular<br />
behaviors when the proteins' expression levels or<br />
their activities were theoretically going to become<br />
changed in parallel with the RNA levels in the<br />
cells. Therefore, regardless <strong>of</strong> some exceptions,<br />
evaluating RNA <strong>tr</strong>anscripts could provide us an<br />
adequate image illus<strong>tr</strong>ating the changes in the<br />
proteins’ expression levels in the cells.<br />
Conclusions<br />
In summary, we showed that cell cycle arrest (<strong>and</strong><br />
possibly autophagy) may play a role in action <strong>of</strong><br />
doxorubicin on T-47D cells. However, the<br />
con<strong>tr</strong>ibution <strong>of</strong> apoptosis <strong>and</strong> cell cycle arrest<br />
antiproliferative effect <strong>of</strong> sulfabenzamide on T-47D<br />
cells is minimal. These observations are in con<strong>tr</strong>ast<br />
to many reports in which the mechanism <strong>of</strong> action<br />
<strong>of</strong> sulfonamide derivatives on cancer cells<br />
at<strong>tr</strong>ibuted to the conventional processes <strong>of</strong><br />
apoptosis <strong>and</strong> cell cycle arrest. We believe that<br />
induction <strong>of</strong> autophagic cell death in T-47D cells is<br />
<strong>tr</strong>iggered through p53/DRAM pathway (occurred<br />
along with decreasing <strong>of</strong> Akt/mTOR pathway) <strong>and</strong><br />
this is a reasonable cellular axis to justify our<br />
results.<br />
Abbreviations<br />
AKT: v-akt murine thymoma viral oncogene<br />
homolog, mTOR: Mechanistic Target Of<br />
Rapamycin, PTEN: Phosphatase <strong>and</strong> Tensin<br />
homolog, DRAM: Damage Regulated Autophagy<br />
Modulator, ATG5:Autophagy related gene 5,<br />
Beclin1: Bcl2 Interacting protein 1, PARP1: Poly<br />
ADP-Ribose Polymerase 1, DFF-40/CAD: DNA<br />
Fragmentation Factor 40/ Caspase-Activated<br />
DNase, Bax: Bcl2-Associated X protein, Bcl-2: B-<br />
<strong>Cell</strong> Lymphoma 2, AIF: Apoptosis Inducing<br />
Factor, DFF-45/iCAD: DNA Fragmentation<br />
Factor 45/inhibitor <strong>of</strong> Caspase-Activated DNase,<br />
Cdc2: <strong>Cell</strong> Division Cycle protein 2, ARF: ADP<br />
Ribosylation Factor, GAPDH: Glyceraldehyde-3-<br />
Phosphate Dehydrogenase, ACD: Autophagic <strong>Cell</strong><br />
Death, PCDII: type II Programmed <strong>Cell</strong> Death,<br />
RPMI: Roswell Park Memorial Institute.<br />
Conflict <strong>of</strong> interest<br />
The authors declare that they have no competing<br />
interest.<br />
Authors' con<strong>tr</strong>ibutions<br />
SS designed the study <strong>and</strong> experiments, analyzed<br />
<strong>and</strong> interpreted data <strong>and</strong> also prepared the<br />
manuscript. RM carried out the experiments <strong>and</strong><br />
participated in data analysis as well as writing the<br />
initial draft <strong>of</strong> the manuscript. SH <strong>and</strong> SF<br />
participated in performing the experiments. NS<br />
con<strong>tr</strong>ibuted on giving scientific comments <strong>and</strong> also<br />
carried out final editing <strong>of</strong> the manuscript.<br />
Acknowledgements<br />
Iran National Science Foundation (INSF) <strong>and</strong><br />
Research Council <strong>of</strong> University <strong>of</strong> Tehran have<br />
been gratefully appreciated by the authors because<br />
<strong>of</strong> their foundational supports for this work.<br />
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<strong>Journal</strong> <strong>of</strong> <strong>Cell</strong> <strong>and</strong> Molecular Biology <s<strong>tr</strong>ong>10</s<strong>tr</strong>ong>(1): 55-64, 2012 Research Article 55<br />
Haliç University, Printed in Turkey.<br />
http://<s<strong>tr</strong>ong>jcmb</s<strong>tr</strong>ong>.<s<strong>tr</strong>ong>halic</s<strong>tr</strong>ong>.<s<strong>tr</strong>ong>edu</s<strong>tr</strong>ong>.<strong>tr</strong><br />
Media optimization for amylase production in solid state<br />
fermentation <strong>of</strong> wheat bran by fungal s<strong>tr</strong>ains<br />
Muhammad IRFAN*, Muhammad NADEEM, Quratualain SYED<br />
Food & Biotechnology Research Center (FBRC), Pakistan Council <strong>of</strong> Scientific & Indus<strong>tr</strong>ial Research<br />
(PCSIR) Laboratories Complex, Ferozpure Road Lahore, Pakistan.<br />
(* author for correspondence; mirfanashraf@yahoo.com)<br />
Received: 02 April 2012; Accepted: 30 May 2012<br />
Abs<strong>tr</strong>act<br />
The present study is concerned with the optimization <strong>of</strong> environmental <strong>and</strong> cultural conditions for the<br />
production <strong>of</strong> α-amylase from wheat bran in solid state fermentation by locally isolated s<strong>tr</strong>ains <strong>of</strong> Aspergillus<br />
niger- ML-17 <strong>and</strong> Rhizopus oligosporus-ML-<s<strong>tr</strong>ong>10</s<strong>tr</strong>ong> . The whole fermentation process was carried out in 250 ml<br />
Erlenmeyer flask. Different parameters were optimized for each s<strong>tr</strong>ain to obtain maximum enzyme yield. For<br />
Aspergillus niger-ML-17, incubation period <strong>of</strong> 96 h, initial diluent pH <strong>of</strong> 5.0, 30°C incubation temperature,<br />
inoculum size <strong>of</strong> 5% were found to be optimum for the production <strong>of</strong> α-amylase. Amylase production was<br />
enhanced from 2.3 ± 0.014 IU to 4.4 ± 0.042 IU by supplementing the fermentation media with maltose<br />
(0.25%), yeast ex<strong>tr</strong>act (0.25%), NaNO3 (0.25%), MgSO4 (0.2%), NaCl (0.5%), Tween-80 (0.001%) <strong>and</strong><br />
Asparginine (0.0001%) . In case <strong>of</strong> Rhizopus oligosporus-ML-<s<strong>tr</strong>ong>10</s<strong>tr</strong>ong>, the optimized cultural conditions for the<br />
production <strong>of</strong> 2.5 ±0.023 IU were inoculum size <strong>of</strong> <s<strong>tr</strong>ong>10</s<strong>tr</strong>ong>%, initial pH <strong>of</strong> the diluent 6.0, incubation temperature<br />
<strong>of</strong> 35 o C for 96h. Further addition <strong>of</strong> maltose (25%), yeast ex<strong>tr</strong>act (0.25%), NH4NO3 (0.25%), MgSO4 (0.2%),<br />
NaCl (0.75%), Tween soluble starch (0.001%) <strong>and</strong> asparginine (0.0001%) to the medium significantly<br />
enhance the enzyme production up to 3.2 ± 0.027 IU.<br />
Keywords: Amylase, wheat bran, A.niger, R.oligosporus, solid state fermentation<br />
Buğday kepeği katı hal fermentasyonunda mantar suşlarıyla amilaz üretimi için besiyeri<br />
optimizasyonu<br />
Özet<br />
Bu çalışma yerel olarak izole edilen Aspergillus niger- ML-17 ve Rhizopus oligosporus-ML-<s<strong>tr</strong>ong>10</s<strong>tr</strong>ong> suşları<br />
tarafından buğday kepeği katı hal fermentasyonunda amilaz üretimi için çevre ve kültür koşullarının<br />
optimizasyonuyla ilgilidir. Tüm fermentasyon süreci 250 ml erlende yapılmıştır. Maksimum enzim ürünü<br />
elde etmek amacıyla her suş bir için farklı parame<strong>tr</strong>eler optimize edilmiştir. Aspergillus niger- ML-17 için 96<br />
saat inkübasyon süresi, çözücünün başlangıç pH’sı olarak 5.0, 30°C inkübasyon sıcaklığı, % 5 ekim<br />
büyüklüğü α- amilaz üretimi için optimum bulunmuştur. Amilaz üretimi fermentasyon ortamına maltoz<br />
(%0,25), maya özütü (% 0,25), NaNO3 (% 0,25), MgSO4 (% 0,2), NaCl (% 0,5), Tween-80 (% 0,001) ve<br />
asparajin (% 0,0001) eklenmesiyle 2,3 ± 0,014 IU’ dan 4,4 ± 0,042 IU’ ya artmıştır. Rhizopus oligosporus-<br />
ML-<s<strong>tr</strong>ong>10</s<strong>tr</strong>ong> için ise; 2,5 ± 0,023 IU üretim için optimize edilen kültür koşulları %<s<strong>tr</strong>ong>10</s<strong>tr</strong>ong> ekim büyüklüğü, çözücünün<br />
başlangıç pH’sı 5,0, inkübasyon sıcaklığı 35°C, 96 saat olacak şekildedir. Besiyerine maltoz (25%), maya<br />
özütü (0.25%), NH4NO3 (0.25%), MgSO4 (0.2%), NaCl (0.75%), Tween çözünür nişasta (0.001%) ve<br />
Asparginine (0.0001%) eklenmesi enzim üretimini önemli ölçüde artırarak 3.2 ± 0.027 IU seviyesine<br />
ulaştırmıştır.<br />
Anahtar kelimeler: Amilaz, buğday kepeği, A.niger, R.oligosporus, katı hal fermentasyonu
56 Muhammad IRFAN et al.<br />
In<strong>tr</strong>oduction<br />
Amylases are a group <strong>of</strong> hydrolases that can<br />
specifically cleave the O-glycosidic bonds in<br />
starch. Two important groups <strong>of</strong> amylases are<br />
glucoamylase <strong>and</strong> α-amylase. Glucoamylase (exo-<br />
1,4-α-D-glucan glucanohydrolase, E.C. 3.2.1.3)<br />
hydrolyzes single glucose units from the nonr<s<strong>tr</strong>ong>edu</s<strong>tr</strong>ong>cing<br />
ends <strong>of</strong> amylose <strong>and</strong> amylopectin in a<br />
stepwise manner (Anto et al., 2006). Whereas αamylases<br />
(endo-1,4-α-D-glucan glucohydrolase,<br />
E.C. 3.2.1.1) are ex<strong>tr</strong>acellular enzymes that<br />
r<strong>and</strong>omly cleave the 1,4-α-D-glucosidic linkages<br />
between adjacent glucose units inside the linear<br />
amylose chain (Anto et al., 2006, Cas<strong>tr</strong>o et al.,<br />
20<s<strong>tr</strong>ong>10</s<strong>tr</strong>ong>, P<strong>and</strong>ey et al., 2005).<br />
Alpha-amylases are widely dis<strong>tr</strong>ibuted in nature<br />
<strong>and</strong> can be derived from various sources such as<br />
plants, animals <strong>and</strong> microorganisms (P<strong>and</strong>ey et al.,<br />
2005, Reddy et al., 2003 3-4). However, fungal <strong>and</strong><br />
bacterial amylases have predominant applications<br />
in the indus<strong>tr</strong>ial sector. Major advantage <strong>of</strong> using<br />
fungi for the production <strong>of</strong> amylases is the<br />
economical bulk production capacity <strong>and</strong> ease <strong>of</strong><br />
manipulation. Many species <strong>of</strong> Aspergillus <strong>and</strong><br />
Rhizopus are used as a source <strong>of</strong> fungal α-amylase<br />
(P<strong>and</strong>ey et al., 2005).<br />
Usually amylase production from fungi has<br />
been carried out using well defined chemical media<br />
by submerged fermentation (SMF) <strong>and</strong> solid state<br />
fermentation (SSF) in recent times (Mir<strong>and</strong>a et al.,<br />
1999). The economics <strong>of</strong> enzyme production using<br />
inexpensive raw materials can make an indus<strong>tr</strong>ial<br />
enzyme process competitive (Couto <strong>and</strong> Sanroman,<br />
2006).<br />
For the microbial α-amylase production, two<br />
types <strong>of</strong> fermentation methods are mainly used i.e.<br />
submerged <strong>and</strong> solid state (Norouzian et al., 2006).<br />
Submerged fermentation (SmF) is comparatively<br />
advanced <strong>and</strong> commercially important enzymes are<br />
<strong>tr</strong>aditionally produced by this method (Hashemi et<br />
al., 20<s<strong>tr</strong>ong>10</s<strong>tr</strong>ong>). Whereas, solid state fermentation (SSF)<br />
is an old technology <strong>and</strong> has been used since 2600<br />
BC. However, in recent year SSF has emerged as a<br />
well developed biotechnological tool for the<br />
production <strong>of</strong> enzymes (Bhatnagar et al., 20<s<strong>tr</strong>ong>10</s<strong>tr</strong>ong>).<br />
Nowadays, spec<strong>tr</strong>um <strong>of</strong> applications <strong>of</strong> αamylase<br />
is also extending in many other areas such<br />
as analytical chemis<strong>tr</strong>y, clinical <strong>and</strong> medicinal<br />
diagnosis e.g. diagnosis <strong>of</strong> acute inflammation <strong>of</strong><br />
pancreas, macroamylasemia, perforated pelvic ulcer<br />
<strong>and</strong> mumps (Anto et al., 2006, Nimkar et al., 20<s<strong>tr</strong>ong>10</s<strong>tr</strong>ong>,<br />
Chimata et al., 20<s<strong>tr</strong>ong>10</s<strong>tr</strong>ong>, Muralikrishna et al., 2005).In<br />
this study we reported here the process<br />
optimization for amylase production from two<br />
fungal species i.e. Aspergillus niger-ML-17 <strong>and</strong><br />
Rhizopus oligosporus-ML-<s<strong>tr</strong>ong>10</s<strong>tr</strong>ong> using wheat bran as a<br />
solid subs<strong>tr</strong>ate fermentation.<br />
Materials <strong>and</strong> Methods<br />
Subs<strong>tr</strong>ate<br />
Wheat bran was procured from local market <strong>of</strong><br />
Lahore city <strong>and</strong> was used as a subs<strong>tr</strong>ate for amylase<br />
production in solid state fermentation.<br />
Microorganism <strong>and</strong> culture maintenance<br />
Fungal s<strong>tr</strong>ain <strong>of</strong> Aspergillus niger-ML-17 <strong>and</strong><br />
Rhizopus oligosporus-ML-<s<strong>tr</strong>ong>10</s<strong>tr</strong>ong> was obtained from<br />
the Microbiology Laboratory <strong>of</strong> Food <strong>and</strong><br />
Biotechnology Research Center (FBRC), PCSIR<br />
Laboratories Complex, Lahore. The culture was<br />
maintained on Potato-Dex<strong>tr</strong>ose-Agar (PDA) slants.<br />
The slants were grown at 30°C for 5 days <strong>and</strong><br />
stored at 4°C.<br />
Inoculum preparation<br />
<s<strong>tr</strong>ong>10</s<strong>tr</strong>ong> mL <strong>of</strong> sterilized distilled water was added to a<br />
sporulated 5 days old PDA slant culture. An<br />
inoculum needle was used to dislodge the spore<br />
clusters under sterilized conditions <strong>and</strong> then it was<br />
shaken thoroughly to prepare homogenized spore<br />
suspension.<br />
Solid-state fermentation<br />
<s<strong>tr</strong>ong>10</s<strong>tr</strong>ong> g wheat bran amended with <s<strong>tr</strong>ong>10</s<strong>tr</strong>ong> mL <strong>of</strong> mineral<br />
salt solution containing (g/L) KH2PO4 <s<strong>tr</strong>ong>10</s<strong>tr</strong>ong>, MgSO4 2,<br />
NaCl 2 <strong>and</strong> MnSO4 0.5 was taken in 250 mL cotton<br />
plugged Erlenmeyer flask, mixed homogenously<br />
<strong>and</strong> sterilized at 121°C for 15 min in an autoclave.<br />
Thereafter, the flask material was cooled at room<br />
temperature <strong>and</strong> inoculated with 1 mL spore<br />
suspensions <strong>of</strong> Aspergillus niger-ML-17 <strong>and</strong><br />
Rhizopus oligosporus-ML-<s<strong>tr</strong>ong>10</s<strong>tr</strong>ong>. The flasks were then<br />
incubated at 30°C for 5 days.<br />
Optimization <strong>of</strong> cultural <strong>and</strong> nu<strong>tr</strong>itional parameters<br />
Various process parameters were optimized for<br />
maximal enzyme production such as fermentation<br />
period (24-168 h), incubation temperature (20-<br />
40°C), initial pH (4-8), inoculum size (2, 5, 7, <s<strong>tr</strong>ong>10</s<strong>tr</strong>ong>,<br />
13, 15, 17%). Experiments were also performed to<br />
evaluate the influence <strong>of</strong> different carbon sources<br />
(maltose, glucose, galactose, lactose, sucrose,<br />
arabinose, cellulose, soluble starch) <strong>and</strong> ni<strong>tr</strong>ogen<br />
sources (yeast ex<strong>tr</strong>act, peptone, <strong>tr</strong>yptone, urea,
ammonium ci<strong>tr</strong>ate, ammonium phosphate,<br />
NH4NO3, (NH4)2SO4, NH4Cl <strong>and</strong> NaNO3) different<br />
metal salts (MnCl2, ZnCl2, CaCl2, MgSO4, FeSO4),<br />
NaCl concen<strong>tr</strong>ation (0.25-2.0%), surfactants<br />
(Tween-80, Triton-X-<s<strong>tr</strong>ong>10</s<strong>tr</strong>ong>0, sodium dodecyl<br />
sulphate, sodium lauryl sulphate) <strong>and</strong> different<br />
amino acids (asparginine, aspartic acid, proline,<br />
cystine, arginine ) on α-amylase production under<br />
the optimized fermentation conditions.<br />
Recovery <strong>of</strong> enzyme<br />
After the specified incubation period (in each case),<br />
50 mL <strong>of</strong> distilled water was added in each flask<br />
containing fermented mash <strong>and</strong> placed on a shaker<br />
at 200 rpm for 60 min. Afterward, the mixture was<br />
filtered <strong>and</strong> cen<strong>tr</strong>ifuged at 8,000 rpm for 15 min at<br />
4°C to remove the fungal spores <strong>and</strong> unwanted<br />
particles. The clear supernatant thus obtained after<br />
cen<strong>tr</strong>ifugation was used as a source <strong>of</strong> crude<br />
enzyme.<br />
Alpha amylase activity<br />
The activity <strong>of</strong> α-amylase was assayed as described<br />
earlier (Irfan et al., 2011) by incubating 0.5 mL <strong>of</strong><br />
the diluted enzyme with 0.5 mL soluble starch (0.5<br />
%, w/v) prepared in 0.1 M sodium Phosphate<br />
buffer, pH= 7. After incubation at 60 ºC for <s<strong>tr</strong>ong>10</s<strong>tr</strong>ong><br />
minutes the reaction was stopped <strong>and</strong> the r<s<strong>tr</strong>ong>edu</s<strong>tr</strong>ong>cing<br />
sugars released were assayed colorime<strong>tr</strong>ically by<br />
the addition <strong>of</strong> 1 mL <strong>of</strong> 3-5-dini<strong>tr</strong>osalicylic acid<br />
reagent. One enzyme activity unit (U) was defined<br />
as the amount <strong>of</strong> enzyme releasing 1 µmol <strong>of</strong><br />
maltose from the subs<strong>tr</strong>ate in 1 minute at 60 ºC.<br />
Statistical analysis<br />
All the data was statistically (SD) analyzed by<br />
using Micros<strong>of</strong>t excel computer programme.<br />
Results<br />
Amylase production in solid state fermentation 57<br />
The most widely used enzyme in the indus<strong>tr</strong>y for<br />
starch hydrolysis is α-amylase. These enzymes<br />
account for 65 % <strong>of</strong> enzyme market in the world<br />
(Muralikrishna et al., 2005). For the production <strong>of</strong><br />
commercially important enzymes, selection <strong>of</strong> a<br />
particular s<strong>tr</strong>ain remains a tedious task. Amylolytic<br />
enzymes are commonly produced by filamentous<br />
fungi preferably from species <strong>of</strong> Aspergillus <strong>and</strong><br />
Rhizopus (P<strong>and</strong>ey et al., 2005).<br />
Time course study for the production <strong>of</strong> αamylase<br />
Figure 1 depicts the time course study (24-120 h)<br />
for the production <strong>of</strong> α-amylase by Aspergillus<br />
niger-ML-17 <strong>and</strong> Rhizopus oligosporus-ML-<s<strong>tr</strong>ong>10</s<strong>tr</strong>ong><br />
using solid state fermentation. Optimum<br />
fermentation period <strong>of</strong> 96 h was best for both<br />
Aspergillus niger-ML-17 (4.7 ±0.14 IU) <strong>and</strong><br />
Rhizopus oligosporus-ML-<s<strong>tr</strong>ong>10</s<strong>tr</strong>ong> (2.7 ±0.08 IU) in<br />
solid state fermentation. Further increase in the<br />
incubation period decreased the enzyme secretion.<br />
Therefore, incubation time <strong>of</strong> 96 h was selected as<br />
optimum time for the production <strong>of</strong> α-amylase in<br />
the subsequent experimental work. Maximum<br />
accumulation <strong>of</strong> α-amylase occurs during stationary<br />
phase. Further increase in incubation period<br />
decreased the production <strong>of</strong> α-amylase. It might be<br />
due to the deficiency <strong>of</strong> nu<strong>tr</strong>ients, accumulation <strong>of</strong><br />
toxic substances <strong>and</strong> proteolysis <strong>of</strong> α-amylase as<br />
interpreted by many workers (Chamber et al., 1999,<br />
Shafique et al., 2009). Abu et al., (2005) also<br />
reported maximum α-amylase production by<br />
Aspergillus niger after an incubation period <strong>of</strong> 96 h.<br />
Figure 1. Time course study <strong>of</strong> amylase production (Error bars represent the SD among replicates).
58 Muhammad IRFAN et al.<br />
Effect <strong>of</strong> initial pH <strong>of</strong> diluent on α-amylase<br />
production<br />
The effect <strong>of</strong> different initial pH (4-6.5) <strong>of</strong> the<br />
diluent on α-amylase production by Aspergillus<br />
niger-ML-17 <strong>and</strong> Rhizopus oligosporus-ML-<s<strong>tr</strong>ong>10</s<strong>tr</strong>ong><br />
using SSF is shown in figure 2. Enzyme production<br />
was maximum (3.7±0.015 IU) when initial pH <strong>of</strong><br />
the diluent was adjusted at 5.0 using s<strong>tr</strong>ains <strong>of</strong><br />
A.niger-ML-17 in SSF. When s<strong>tr</strong>ain <strong>of</strong><br />
R.ologosporus –ML-<s<strong>tr</strong>ong>10</s<strong>tr</strong>ong> was employed for amylase<br />
production, it gave better yield (2.7 ± 0.016 IU) at<br />
6.0 initial pH <strong>of</strong> the diluent. Further increase in the<br />
initial pH <strong>of</strong> the diluent resulted decreased in the<br />
enzyme activity. It is due to the fact that fungal<br />
growth was optimum at this pH <strong>and</strong> hence the<br />
enzyme production. Initial pH <strong>of</strong> the medium is<br />
known to affect the synthesis <strong>and</strong> secretion <strong>of</strong> αamylase.<br />
Alpha amylase production by microbial<br />
s<strong>tr</strong>ains s<strong>tr</strong>ongly depends on the ex<strong>tr</strong>acellular pH as<br />
it influences many enzymatic reactions as well as<br />
the <strong>tr</strong>ansport <strong>of</strong> various components across the cell<br />
membrane (Ellaiah et al., 2002). In con<strong>tr</strong>ast to the<br />
present findings, Alva et al. (2007) achieved the<br />
optimal α-amylase production at initial pH 5.8 by<br />
Aspergillus sp. Conversely, this might be because<br />
the requirement <strong>of</strong> slightly acidic pH for optimum<br />
growth <strong>of</strong> fungi (Liu et al., 2008, Sun et al., 2009).<br />
Figure 2. Effect <strong>of</strong> initial pH <strong>of</strong> diluents on amylase production (Error bars represent the SD among<br />
replicates)<br />
Effect <strong>of</strong> temperature on the production <strong>of</strong> αamylase<br />
Figure 3 showed the effect <strong>of</strong> varying incubation<br />
temperature (20-40ºC) on the production <strong>of</strong> αamylase<br />
by Aspergillus niger-ML-17 <strong>and</strong><br />
R.oligosporus-ML-<s<strong>tr</strong>ong>10</s<strong>tr</strong>ong> using SSF. Maximal enzyme<br />
production (3.5 ±0.034 IU) was obtained in<br />
fermentation flask that was incubated at 30ºC after<br />
the conidial inoculation <strong>of</strong> A.niger-ML-17. On the<br />
other h<strong>and</strong> R.oligosporus-ML-<s<strong>tr</strong>ong>10</s<strong>tr</strong>ong> gave better<br />
enzyme production (2.5 ±0.023 IU) at 35 o C. All the<br />
other flasks that were incubated on temperatures<br />
other than these gave comparatively less production<br />
<strong>of</strong> α-amylase. Alpha amylase production by fungi is<br />
related to the growth which sequentially depends<br />
upon the incubation temperature. Many other<br />
researchers have also reported 30°C as optimum<br />
temperature for the fungal growth <strong>and</strong> enzyme<br />
production. This is because the enzyme production<br />
is growth associated <strong>and</strong> 30°C is optimum<br />
temperature for fungi <strong>and</strong> subsequently α-amylase<br />
production (Shafique et al., 2009, Dakhmouche et<br />
al., 2006).
Amylase production in solid state fermentation 59<br />
Figure 3. Effect <strong>of</strong> incubation temperature on amylase production (Error bars represent the SD among<br />
replicates).<br />
Effect <strong>of</strong> inoculum size on the production <strong>of</strong> αamylase<br />
Effect <strong>of</strong> inoculum size was checked by varying the<br />
concen<strong>tr</strong>ation <strong>of</strong> spores <strong>of</strong> A.niger-ML-17 <strong>and</strong><br />
R.oligosporus-ML-<s<strong>tr</strong>ong>10</s<strong>tr</strong>ong> in solid state fermentation <strong>of</strong><br />
wheat bran. Optimum inoculum size <strong>of</strong> 5% (3.5<br />
±0.014 IU) <strong>and</strong> <s<strong>tr</strong>ong>10</s<strong>tr</strong>ong>% (2.9 ±0.011 IU) <strong>of</strong> A.niger-<br />
ML-17 <strong>and</strong> R.oligosporus-ML-<s<strong>tr</strong>ong>10</s<strong>tr</strong>ong> gave highest<br />
yield <strong>of</strong> enzyme production respectively as shown<br />
in figure 4. By increasing the size <strong>of</strong> inoculum<br />
resulted in decreased enzyme production. Increased<br />
inoculum size resulted in increases moisture level<br />
which ultimately decreased the fungal growth <strong>and</strong><br />
enzyme production (Sharma et al., 1996). Ammar<br />
<strong>and</strong> El-Safey (2003) obtained highest yield <strong>of</strong><br />
amylase production from A. flavus var columnaris<br />
using inoculum size <strong>of</strong> 0.0637 x <s<strong>tr</strong>ong>10</s<strong>tr</strong>ong> 6 /ml -1 . In<br />
another study 5 x <s<strong>tr</strong>ong>10</s<strong>tr</strong>ong> 6 spores per flask gave<br />
maximum enzyme production by Aspergillus niger<br />
ATCC 16404 (Dakhmouche et al., 2006). Zambare<br />
(20<s<strong>tr</strong>ong>10</s<strong>tr</strong>ong>) obtained highest enzyme production (1672-<br />
1693 U/gdfs) at inoculum level <strong>of</strong> 5-8% (v/w) in<br />
solid state fermentation using s<strong>tr</strong>ain <strong>of</strong> A.oryzae.<br />
Figure 4. Effect <strong>of</strong> inoculum size on amylase production (Error bars represent the SD among replicates).<br />
Effect <strong>of</strong> different carbon sources on the<br />
production <strong>of</strong> α-amylase<br />
Different carbon sources i.e. soluble starch,<br />
glucose, galactose, lactose, arabinose, cellulose,<br />
maltose <strong>and</strong> sucrose were also evaluated for the<br />
production <strong>of</strong> α-amylase (Figure 5). A.niger-ML-<br />
17 better used the maltose as a carbon source <strong>and</strong><br />
improved enzyme production (4.4 ± 0.042 IU) as<br />
compared to con<strong>tr</strong>ol while soluble starch (3.2 ±<br />
0.027 IU) was found best carbon source for<br />
amylase production in case <strong>of</strong> R.oligosporus-ML-<br />
<s<strong>tr</strong>ong>10</s<strong>tr</strong>ong>. All the other tested carbon sources gave<br />
comparatively less enzyme production which is<br />
supplemented to the fermentation medium at<br />
concen<strong>tr</strong>ation <strong>of</strong> 0.25% (w/v). Therefore, soluble<br />
starch <strong>and</strong> maltose was optimized as a carbon<br />
source in the further experimental work<br />
R.oligosporus-ML-<s<strong>tr</strong>ong>10</s<strong>tr</strong>ong> <strong>and</strong> A.niger-ML-17<br />
respectively. Many other workers also reported<br />
starch as the best carbon source for the production<br />
<strong>of</strong> alpha amylase (Gigras et al., 2002, Dharani<br />
2004). It is because α-amylase is an ex<strong>tr</strong>acellular<br />
enzyme <strong>and</strong> its production is increased by its<br />
subs<strong>tr</strong>ate (Chimata et al., 20<s<strong>tr</strong>ong>10</s<strong>tr</strong>ong>; Varalakshmi et al.,<br />
2009).
60 Muhammad IRFAN et al.<br />
Figure 5. Effect <strong>of</strong> different carbon sources on amylase production (Error bars represent the SD among<br />
replicates).<br />
production by employing s<strong>tr</strong>ains <strong>of</strong> A.niger-ML-17<br />
Effect <strong>of</strong> different ni<strong>tr</strong>ogen sources on the<br />
<strong>and</strong> R.oligosporus-ML-<s<strong>tr</strong>ong>10</s<strong>tr</strong>ong>. Thus, yeast ex<strong>tr</strong>act was<br />
production <strong>of</strong> α-amylase<br />
used as an optimized organic ni<strong>tr</strong>ogen sources in<br />
Figure 6 depicts the effect <strong>of</strong> ni<strong>tr</strong>ogen sources this further research. Hashemi et al., (20<s<strong>tr</strong>ong>10</s<strong>tr</strong>ong>) also<br />
(peptone, urea, <strong>tr</strong>yptone, casein, skimmed milk <strong>and</strong> obtained maximum α-amylase production (140<br />
yeast ex<strong>tr</strong>act NH4SO4, NH4NO3, NaNO3, U/g) with NH4NO3 but at a level <strong>of</strong> 1% (w/v).<br />
ammonium ci<strong>tr</strong>ate & ammonium phosphate) on the Amylase production is enhanced with the addition<br />
production <strong>of</strong> α-amylase by Aspergillus niger-ML- <strong>of</strong> organic ni<strong>tr</strong>ogen sources (Hamilton et al., 1999,<br />
17 <strong>and</strong> R.oligosporus-ML-<s<strong>tr</strong>ong>10</s<strong>tr</strong>ong> using SSF. Among all Hayashida et al., 1998 28,29). Many workers (Anto<br />
the selected inorganic ni<strong>tr</strong>ogen sources maximum et al., 2006, Pederson <strong>and</strong> Neilson 2000, Oshoma et<br />
amylase production (3.9 ±0.1IU) was obtained, al., 20<s<strong>tr</strong>ong>10</s<strong>tr</strong>ong>, Valaparla 20<s<strong>tr</strong>ong>10</s<strong>tr</strong>ong>) reported that yeast<br />
when NaNO3 (0.25%, w/v) was used in medium ex<strong>tr</strong>act as an organic ni<strong>tr</strong>ogen source produces<br />
inoculated with spores <strong>of</strong> A.niger-ML-17 while the maximum amylase production. Pederson <strong>and</strong><br />
s<strong>tr</strong>ain R.oligosporus-ML-<s<strong>tr</strong>ong>10</s<strong>tr</strong>ong> best utilized (2.6 ±0.08 Neilson (2000) optimized (NH4)2SO4 for maximum<br />
IU) NH4NO3 at concen<strong>tr</strong>ation <strong>of</strong> 0.25% for amylase amylase productivity by A. oryzae.<br />
production. In case <strong>of</strong> organic ni<strong>tr</strong>ogen sources<br />
yeast ex<strong>tr</strong>act was found better for amylase<br />
Figure 6. Effect <strong>of</strong> ni<strong>tr</strong>ogen sources on amylase production (Error bars represent the SD among replicates).<br />
Effect <strong>of</strong> different metal salts on amylase<br />
production in SSF<br />
Different metal salts (MnCl2, ZnCl2, CaCl2, MgSO4<br />
<strong>and</strong> FeSO4) were added to the fermentation<br />
medium to enhance the amylase yield by A.niger-<br />
ML-17 <strong>and</strong> R.oligosporus-ML-<s<strong>tr</strong>ong>10</s<strong>tr</strong>ong> in solid state<br />
fermentation. Results (Fig. 7) revealed that addition<br />
<strong>of</strong> MgSO4 at 0.1% concen<strong>tr</strong>ation to the medium<br />
favored the enzyme production by both tested<br />
fungi. Of all the metal salts FeSO4 lowers the<br />
enzyme production as compared to con<strong>tr</strong>ol.<br />
Rameshkumar <strong>and</strong> Sivasudha (2011) reported that<br />
supplementation <strong>of</strong> 0.1% CaCl2 as a mineral source<br />
to the medium effectively increased the amylase<br />
production by B.subtilis in solid state fermentation.<br />
According to Negi <strong>and</strong> Banerjee (20<s<strong>tr</strong>ong>10</s<strong>tr</strong>ong>) addition <strong>of</strong><br />
HgCl2 to the medium increases the amylase<br />
production up to 2.44 folds. Most <strong>of</strong> the study<br />
indicated that the enzyme did not require a specific<br />
ion for their proper functioning (Reyed 2007).<br />
Sanghvi et al., (2011) produced amylase from<br />
Chrysosporium asperatum in submerged<br />
fermentation <strong>and</strong> reported that supplementation <strong>of</strong><br />
FeCl3 in the medium as a mineral source slightly<br />
increased the amylase production.
Amylase production in solid state fermentation 61<br />
Figure 7. Effect <strong>of</strong> different metal salts on amylase production (Error bars represent the SD among<br />
replicates).<br />
Effect <strong>of</strong> different concen<strong>tr</strong>ation <strong>of</strong> NaCl on<br />
amylase production in SSF<br />
In this experiment, different concen<strong>tr</strong>ations <strong>of</strong> NaCl<br />
(0.25-2.0 %) were employed to check the effect <strong>of</strong><br />
different concen<strong>tr</strong>ation <strong>of</strong> NaCl on amylase<br />
production by A.niger-ML-17 <strong>and</strong> R.oligosporus-<br />
ML-<s<strong>tr</strong>ong>10</s<strong>tr</strong>ong>. Results indicated (Fig. 8) that A.niger-ML-<br />
17 showed maximum (3.6 ±0.17 IU) enzyme<br />
production at 0.5% concen<strong>tr</strong>ation <strong>of</strong> NaCl in the<br />
medium while R.oligosporus-ML-<s<strong>tr</strong>ong>10</s<strong>tr</strong>ong> gave<br />
maximum titer <strong>of</strong> amylase (2.5± 0.13 IU) at 0.75%<br />
concen<strong>tr</strong>ation <strong>of</strong> NaCl. By increasing the NaCl<br />
concen<strong>tr</strong>ation beyond this decline in enzyme<br />
production was observed. Kokab et al., (2003)<br />
produced amylase from Bacillus subtilis in solid<br />
state fermentation having medium containing 1.5%<br />
concen<strong>tr</strong>ation <strong>of</strong> NaCl. Patel et al., (2005) purified<br />
<strong>and</strong> characterized the amylase enzyme from<br />
A.oryzae in SSF having medium with 0.1%<br />
concen<strong>tr</strong>ation <strong>of</strong> NaCl.<br />
Figure 8. Effect <strong>of</strong> different concen<strong>tr</strong>ations <strong>of</strong> NaCl on amylase production (Error bars represent the SD<br />
among replicates).<br />
Effect <strong>of</strong> surfactants on amylase production<br />
Figure 9 illus<strong>tr</strong>ated the effect <strong>of</strong> different<br />
surfactants on amylase production by A.niger –ML-<br />
17 <strong>and</strong> R.oligosporus-ML-<s<strong>tr</strong>ong>10</s<strong>tr</strong>ong>. Result showed that<br />
addition <strong>of</strong> Tween 80 (0.05 v/w) to the medium is<br />
effective in enzyme production by both A.niger-<br />
ML-17 (3.7 IU) <strong>and</strong> R.oligosporus-ML-<s<strong>tr</strong>ong>10</s<strong>tr</strong>ong> (2.5 IU)<br />
in SSF. SDS <strong>and</strong> SLS had no s<strong>tr</strong>ong influence on<br />
enzyme production in solid state fermentation by<br />
tested fungi. Negi <strong>and</strong> Banerjee (20<s<strong>tr</strong>ong>10</s<strong>tr</strong>ong>) reported<br />
that Triton-X-<s<strong>tr</strong>ong>10</s<strong>tr</strong>ong>0 <strong>and</strong> sodium laurate sulphate are<br />
the stimulator for enzyme production but sodium<br />
laurate sulphate increased amylase production by<br />
1.28 folds. Gupta et al. (2008) also reported that<br />
different surfactants like Tween-80, Triton X-<s<strong>tr</strong>ong>10</s<strong>tr</strong>ong>0<br />
<strong>and</strong> SDS had s<strong>tr</strong>ong influence on amylase<br />
production in submerged fermentation using<br />
Aspergillus niger. Vu et al. (2011) stated that use <strong>of</strong><br />
Tween-80 as a surfactant effectively enhances the<br />
cellulose production in solid state fermentation.<br />
Production <strong>of</strong> hydrolytic enzymes can be enhanced<br />
by the surfactants <strong>and</strong> fatty acids (Singh et al.,<br />
1991). When surfactants are added, they enhance<br />
the microbial growth in SSF by promoting the<br />
pene<strong>tr</strong>ation <strong>of</strong> water into the solid subs<strong>tr</strong>ate ma<strong>tr</strong>ix,<br />
leading in an increase <strong>of</strong> surface area (Asgher et al.,<br />
2006).
62 Muhammad IRFAN et al.<br />
Figure 9. Effect <strong>of</strong> different surfactants on amylase production (Error bars represents the SD among<br />
replicates).<br />
Effect <strong>of</strong> different amino acids on amylase<br />
production in SSF<br />
To check the effect <strong>of</strong> different amino acids on<br />
amylase production, various amino acids like<br />
asparaginine, aspartic acid, praline, cysteine <strong>and</strong><br />
arginine were evaluated by A.niger –ML-17<strong>and</strong><br />
R.oligosporus-ML-<s<strong>tr</strong>ong>10</s<strong>tr</strong>ong> in SSF. Results (Fig. <s<strong>tr</strong>ong>10</s<strong>tr</strong>ong>)<br />
revealed that supplementation <strong>of</strong> asparginine with<br />
concen<strong>tr</strong>ation <strong>of</strong> 0.0001% favored enzyme<br />
production in solid state fermentation. Rest <strong>of</strong> the<br />
amino acids showed no significant effect on<br />
enzyme production.<br />
Sidkey et al. (20<s<strong>tr</strong>ong>10</s<strong>tr</strong>ong>), isolated different s<strong>tr</strong>ains<br />
from natural environment <strong>and</strong> screened for amylase<br />
production. Among various screened s<strong>tr</strong>ains<br />
Aspergillus flavus was found to be potent amylase<br />
producer when supplementations <strong>of</strong> methionine as<br />
an amino acid cotent to the medium. Some workers<br />
(Moustafa, 2002; Sidkey et al., 1996) reported that<br />
acidic amino acids like glutamic <strong>and</strong> aspartic acids<br />
are the best inducers for amylase production.<br />
Figure <s<strong>tr</strong>ong>10</s<strong>tr</strong>ong>. Effect <strong>of</strong> different amino acids on amylase production (Error bars represent the SD among<br />
replicates).<br />
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Reddy AS, Jharat R, Byrne N. Purification <strong>and</strong><br />
properties <strong>of</strong> amylase from A review. J Food<br />
Biochem. 21:281-302, 2003.<br />
Reyed RM. Biosynthesis <strong>and</strong> properties <strong>of</strong><br />
ex<strong>tr</strong>acellular amylase by encapsulation<br />
Bifidoba<strong>tr</strong>ium bifidum in batch culture.<br />
Aus<strong>tr</strong>alian J Basic Appl Sci. 1: 7-14, 2007.<br />
Sanghvi GV, Rina DK, Kishore SR. Isolation,<br />
Optimization, <strong>and</strong> Partial Purification <strong>of</strong><br />
Amylase from Chrysosporium asperatum by<br />
Submerged Fermentation. J Microb Biotechnol.<br />
21(5):470–476, 2011.<br />
Shafique S, Bajwa R, Shafique S. Screening <strong>of</strong><br />
Aspergillus niger <strong>and</strong> A. flavus s<strong>tr</strong>ains for ex<strong>tr</strong>a<br />
cellular α-amylase activity. Pak J Bot.<br />
41(2):897-905, 2009.<br />
Sharma DK, Tiwari M, Behere BK. Solid state<br />
fermentation <strong>of</strong> new subs<strong>tr</strong>ates for production <strong>of</strong><br />
cellulase <strong>and</strong> other biopolymer hydrolyzing<br />
enzymes. Appl Biochem Biotechnol. 15:495-<br />
500, 1996.<br />
Sidkey NM, Abo-Shadi MA, Al-Mu<strong>tr</strong>afy AM,<br />
Sefergy F, Al-Reheily N. Screening <strong>of</strong><br />
Microorganisms Isolated from some Enviro-<br />
Agro-Indus<strong>tr</strong>ial Wastes in Saudi Arabia for<br />
Amylase Product. J American Sci. 6(<s<strong>tr</strong>ong>10</s<strong>tr</strong>ong>):926-<br />
939, 20<s<strong>tr</strong>ong>10</s<strong>tr</strong>ong>.<br />
Sidkey NM, Shash SM, Ammar MS. Regulation <strong>of</strong><br />
α-amylase biosynthesis by Aspergillus sp. ,S-7<br />
attaching the Nile Hyacinth homogenate<br />
produced under laboratory scale fermentation<br />
conditions. Al-Azhar Bullutin Sci. 7(1):437-488,<br />
1996.<br />
Singh A, Abidi AB, Darmwal NS, Agrawal AK.<br />
Influence <strong>of</strong> nu<strong>tr</strong>itional factors <strong>of</strong> cellulase<br />
production from natural lignocellulosic residues<br />
by Aspergillus niger. Agri Biol Res. 7:19-27,<br />
1991.<br />
Sun H, Ge X, Wang L, Zhao P, Peng M.<br />
Microbial production <strong>of</strong> raw starch digesting<br />
enzymes. Afr J Biotechnol. 8 (9):1734-1739,<br />
2009.<br />
Varalakshmi KN, Kumudini BS, N<strong>and</strong>ini BN,<br />
Solomon J, Suhas R, Mahesh B, Kavitha AP.<br />
Production <strong>and</strong> characterization <strong>of</strong> α-amylase<br />
from Aspergillus niger JGI 24 isolated in<br />
Bangalore. Polish J Microbiol. 58: 29-36,<br />
2009.<br />
Valaparla VK. Purification <strong>and</strong> properties <strong>of</strong> a<br />
thermostable α-amylase by Acremonium<br />
Sporosulcatum. Int J Biotechnol Biochem.<br />
6(1):25–34, 20<s<strong>tr</strong>ong>10</s<strong>tr</strong>ong>.<br />
Vu VH, Pham TA, Kim K. Improvement <strong>of</strong> fungal<br />
cellulase production by mutation <strong>and</strong><br />
optimization <strong>of</strong> solid state fermentation.<br />
Mycobiology. 39(1): 20-25, 2011.<br />
Zambare V. Solid state fermentation <strong>of</strong> Aspergillus<br />
oryzae for Glucoamylase Production on Agro<br />
residues. Int J Life Sci. 4:16-25, 20<s<strong>tr</strong>ong>10</s<strong>tr</strong>ong>.
<strong>Journal</strong> <strong>of</strong> <strong>Cell</strong> <strong>and</strong> Molecular Biology - GUIDELINES for AUTHORS<br />
General<br />
<strong>Journal</strong> <strong>of</strong> <strong>Cell</strong> <strong>and</strong> Molecular Biology<br />
(J<strong>Cell</strong>MolBiol) is an international journal which<br />
covers original works in the field <strong>of</strong> cell biology,<br />
molecular biology, genetics, microbiology,<br />
neurobiology, bioinformatics <strong>and</strong> related topics.<br />
The <strong>of</strong>ficial language <strong>of</strong> the journal is English,<br />
however manuscripts in Turkish are accepted as<br />
well.<br />
Conditions for publication<br />
This journal publishes research articles, review<br />
articles, short communications, book/s<strong>of</strong>tware<br />
reviews, case reports <strong>and</strong> letters to the editor.<br />
Research articles: Only original con<strong>tr</strong>ibutions will<br />
be accepted which have not been published<br />
previously. Manuscripts should not exceed 15<br />
papers <strong>of</strong> printed text, including tables, figures <strong>and</strong><br />
references<br />
Review articles: Reviews <strong>of</strong> recent developments in<br />
a research field <strong>and</strong> ideas will be accepted.<br />
Manuscripts should not exceed 15 papers <strong>of</strong> printed<br />
text. Illus<strong>tr</strong>ations are encouraged.<br />
Short communications: These include small-scale<br />
investigations or innovative methods, techniques,<br />
clinical <strong>tr</strong>ials <strong>and</strong> epidemiological studies. It should<br />
not exceed 3 pages.<br />
Letters to editor: These include opinions, news <strong>and</strong><br />
suggestions. Letters should not exceed 2 papers <strong>of</strong><br />
printed text.<br />
Case Reports: These include individual<br />
observations based on small numbers <strong>of</strong> subjects.<br />
This type <strong>of</strong> research cannot indicate causality but<br />
may indicate areas for further research.<br />
REVISED<br />
December 2011<br />
Manuscripts should be submitted by e-mail to:<br />
<strong>Journal</strong> <strong>of</strong> <strong>Cell</strong> <strong>and</strong> Molecular Biology<br />
Haliç Üniversitesi<br />
Fen Edebiyat Fakültesi<br />
Moleküler Biyoloji ve Genetik Bölümü<br />
Sıracevizler Cad. No:29<br />
Bomonti-Şişli 34381, İstanbul-TÜRKİYE<br />
Tel: +90 212 343 08 87, Fax: +90 212 231 06 31<br />
E-Mail: <s<strong>tr</strong>ong>jcmb</s<strong>tr</strong>ong>@<s<strong>tr</strong>ong>halic</s<strong>tr</strong>ong>.<s<strong>tr</strong>ong>edu</s<strong>tr</strong>ong>.<strong>tr</strong><br />
65<br />
Book/s<strong>of</strong>tware reviews: Short but concise<br />
description <strong>of</strong> the book/s<strong>of</strong>tware, not exceeding a<br />
page. Book/s<strong>of</strong>tware reviews are not peer reviewed.<br />
Presentation<br />
Papers should be typed clearly, double-spaced with<br />
3 cm wide margins.<br />
Manuscripts should be prepared using Word<br />
Processor.<br />
Cover Letter: You may briefly explain your work<br />
<strong>and</strong> its con<strong>tr</strong>ibution to present knowledge.<br />
Title Page: The first page <strong>of</strong> your manuscript<br />
should be a title page containing the type <strong>of</strong> paper;<br />
the title; all authors' full names, <strong>and</strong> affiliations;<br />
<strong>and</strong> the corresponding author's contact address<br />
(including phone <strong>and</strong> fax numbers) <strong>and</strong> e-mail<br />
address. The title should be as short as possible, but<br />
should give adequate information regarding the<br />
contents. Authors should also state a running title<br />
<strong>of</strong> no more than 50 characters including spaces.<br />
All pages must be numbered.<br />
<s<strong>tr</strong>ong>Full</s<strong>tr</strong>ong> Paper<br />
The full paper should be divided into the following<br />
parts in the order indicated:
66<br />
Abs<strong>tr</strong>act: A brief, informative abs<strong>tr</strong>act, not<br />
exceeding 200 words, should be provided in<br />
English <strong>and</strong> in Turkish. For authors who are not<br />
native Turkish speakers, J<strong>Cell</strong>MolBiol can provide<br />
the Turkish abs<strong>tr</strong>act.<br />
Keywords: Immediately following the abs<strong>tr</strong>act,<br />
authors should provide 5 keywords or phrases that<br />
reflect the content <strong>of</strong> the article.<br />
In<strong>tr</strong>oduction should include theory, hypotheses,<br />
prior work<br />
Material <strong>and</strong> methods may include subheadings<br />
Results: If the study consists <strong>of</strong> different parts,<br />
subheadings in this section should be consistent<br />
with subheadings in the methods.<br />
Discussion<br />
Acknowledgements should precede the list <strong>of</strong><br />
references<br />
References: Papers cited in the manuscript should<br />
be listed in alphabetical order according to the first<br />
author's surname.<br />
Tables <strong>and</strong> Figures<br />
• Tables <strong>and</strong> figures should both be embedded<br />
within the text in their appropriate positions <strong>and</strong> be<br />
submitted separately.<br />
• Elec<strong>tr</strong>onically submitted figures are preferred in<br />
*.jpg or *.tiff (min. 300 dpi) formats. Bar scales<br />
should be drawn directly on the figures when<br />
necessary. Figure legends should not be included in<br />
the *jpg or *tif files.<br />
• Each table should be accompanied by a short<br />
ins<strong>tr</strong>uctive title line plus an explanatory caption at<br />
the top. Indicate footnotes within tables by<br />
superscript letters <strong>and</strong> type footnotes below the<br />
table.<br />
• All the tables <strong>and</strong> figures must be referred to<br />
within the text.<br />
Units, Abbreviations <strong>and</strong> Scientific Names<br />
• Only SI units should be used. Current<br />
abbreviations can be used without explanation,<br />
others must be explained.<br />
• All acronyms/abbreviations must be explained<br />
in parenthesis after their first occurrence. If many<br />
unfamiliar acronyms/abbreviations are used, please<br />
REVISED<br />
December 2011<br />
compile them in an "Abbreviations" section at the<br />
end <strong>of</strong> the paper.<br />
• Latin expressions should be typed in italics.<br />
Referencing<br />
• In the text, citations with two authors should<br />
take the form: Smith <strong>and</strong> Robinson,1990. If several<br />
papers are cited by the same author in the same<br />
year, they should be lettered in sequence (1990a),<br />
(1990b), etc. When papers are by more then two<br />
authors they should be cited as Smith et al.,1990. In<br />
cases where more than one reference is written for<br />
the same sentence, they should appear in ascending<br />
publication order, e.g. (Jones et al., 2005; Smith et<br />
al., 2007; Brown et al., 2009).<br />
• In the list, references must be placed in<br />
alphabetical order. The following models for the<br />
reference list cover all situations. The punctuation<br />
given must be exactly followed. The journal titles<br />
should be abbreviated appropriately.<br />
Redford IR. Evidence for a general relationship<br />
between the induced level <strong>of</strong> DNA double<br />
s<strong>tr</strong><strong>and</strong> breakage <strong>and</strong> cell killing after Xirradiation<br />
<strong>of</strong> mammalian cells. Int J Radiat<br />
Biol. 49: 611- 620, 1986.<br />
Tccioli CE, Cottlieb TM, Blund T. Product <strong>of</strong> the<br />
XRCCS gene <strong>and</strong> its role in DNA repair <strong>and</strong><br />
V(D)J recombination. Science. 265: 1442-1445,<br />
1994<br />
Ohlrogge JB. Biochemis<strong>tr</strong>y <strong>of</strong> plant acyl carrier<br />
proteins. The Biochemis<strong>tr</strong>y <strong>of</strong> Plants: A<br />
Comprehensive Treatise. Stumpf PK <strong>and</strong> Conn<br />
EE (Ed). Academic Press, New York. 137-157,<br />
1987.<br />
Brown LA. How to cope with your supervisor. PhD<br />
Thesis. University <strong>of</strong> New Orleans, 2005.<br />
Web document with no author: Leafy seadragons<br />
<strong>and</strong> weedy seadragons 2001. Re<strong>tr</strong>ieved<br />
November 13, 2002, from http:// www.<br />
windspeed.net.au/jenny/seadragons/<br />
Web document with author: Dawson J, Smith L,<br />
Deubert K. Referencing, not plagiarism.<br />
Re<strong>tr</strong>ieved October 31, 2002 from http:<br />
//study<strong>tr</strong>ekk.lis.curtin.<s<strong>tr</strong>ong>edu</s<strong>tr</strong>ong>.au/<br />
• Only papers published or in press should be<br />
cited in the literature list. Unpublished results,<br />
including submitted manuscripts <strong>and</strong> those in<br />
preparation, should be indicated as unpublished<br />
data in the text.
Submission Policies <strong>and</strong> Authorship<br />
Upon submission <strong>of</strong> a manuscript, it is accepted<br />
that all co-authors have approved the contents <strong>of</strong><br />
the manuscript <strong>and</strong> its submission by the<br />
corresponding author, <strong>and</strong> that the corresponding<br />
author is authorized to represent all co-authors in<br />
pre-publication discussions with J<strong>Cell</strong>MolBiol.<br />
The corresponding author is responsible for<br />
ensuring that all the con<strong>tr</strong>ibutors to the relevant<br />
work are listed as authors <strong>and</strong> that all authors have<br />
aggreed to the manuscript’s content <strong>and</strong> its<br />
submission to the J<strong>Cell</strong>MolBiol. In case the <strong>Journal</strong><br />
happens to be aware <strong>of</strong> an authorship dispute,<br />
authorship must be approved in writing by all <strong>of</strong> the<br />
parties.<br />
Cost<br />
There are no submission fees or page charges.<br />
Criteria for the Selection <strong>of</strong> Manuscripts<br />
Manuscripts should meet the following criteria: The<br />
study conducted is material is original <strong>and</strong> ethical,<br />
the writing is clear; the study methods are<br />
appropriate, the data are valid, the conclusions are<br />
reasonable <strong>and</strong> supported by the data; the<br />
information is important; <strong>and</strong> the topic is<br />
interesting to our readership.<br />
Editorial Processes<br />
Researchers may request informal feedback from<br />
the editors in a particular manuscript. The<br />
presubmission process aids in the submission<br />
decision for authors.<br />
When J<strong>Cell</strong>MolBiol receives a manuscript, the<br />
Editor-in-Chief will first decide whether the<br />
manuscript meets the formal criteria specified with<br />
“Guidelines for Authors” <strong>and</strong> whether it fits within<br />
the scope <strong>of</strong> the <strong>Journal</strong>. In case <strong>of</strong> doubt on the<br />
basis <strong>of</strong> initial review, the Editor-in-Chief will<br />
consult other members <strong>of</strong> the Editorial Board.<br />
Manuscripts that are found suitable for peer review<br />
will be assigned to two expert reviewers. Reviewers<br />
may either be Editorial/Advisory Board members<br />
or external experts selected by the Editorial Board.<br />
The corresponding author is notified by e-mail<br />
when the editor decides to send a paper for review.<br />
The reviewers will have up to three weeks to<br />
review the submitted article. After peer review, the<br />
editor will contact the author. If the author is<br />
required to submit a revised version, the revised<br />
version has to be submitted by the author within<br />
REVISED<br />
December 2011<br />
67<br />
two weeks. Otherwise, the manuscript will be<br />
removed from the manuscript submission queue<br />
<strong>and</strong> will be considered rejected.<br />
In cases where the referees have requested welldefined<br />
changes to the manuscript, editors may<br />
request a revised manuscript that addresses to<br />
referees’ concerns. The revised version is sent back<br />
to the original referees for re-review. In cases<br />
where the referees’ concerns are more wideranging,<br />
editors may reject the manuscript. The<br />
revised manuscript should be accompanied by a<br />
cover letter that includes a point-by-point response<br />
to referees’ comments <strong>and</strong> an explanation <strong>of</strong> how<br />
the manuscript has been changed.<br />
As a matter <strong>of</strong> policy, we do not suppress referees’<br />
reports, any comments directed to authors are<br />
<strong>tr</strong>ansmitted regardless <strong>of</strong> what we may think <strong>of</strong> the<br />
content. On rare occasions, we may edit a report to<br />
remove <strong>of</strong>fensive language or comments to reveal<br />
confidentiality.<br />
The final decision to accept or reject a manuscript<br />
will be made by the Editor-in-Chief. If it becomes<br />
apparent that there are serious problems with the<br />
scientific content or with violations <strong>of</strong> our<br />
publishing policies, the Editor-in-Chief also<br />
reserves the right to reject a paper even after it has<br />
been accepted.<br />
After acceptance, the Editor-in-Chief may make<br />
further changes to the text <strong>and</strong> figures so that the<br />
manuscript is readable <strong>and</strong> clear. Page pro<strong>of</strong>s will<br />
be sent to the corresponding author via email for<br />
checking before publication. Corresponding authors<br />
are sent pro<strong>of</strong>s <strong>and</strong> are welcome to discuss<br />
proposed changes with the Editor-in-Chief, but<br />
J<strong>Cell</strong>MolBiol reserves the right to make the final<br />
decision about the style. Corrected pro<strong>of</strong>s should be<br />
sent back within three days <strong>of</strong> receipt, otherwise the<br />
Editor-in-Chief reserves the rights to correct the<br />
pro<strong>of</strong>s himself <strong>and</strong> to send the material for<br />
publication. In cases where the authors do not<br />
submit the appropriately signed Publication<br />
Agreement Form, the manuscript is drawn from<br />
publication process even if it is accepted.<br />
Appeals<br />
Authors have the right to ask the Editor-in-Chief to<br />
reconsider a rejection decision, which is considered<br />
an appeal. Decisions are reversed only if the Editor<br />
is convinced that the original decision was a serious<br />
mistake. If an appeal merits further consideration,<br />
the Editor may send the author’s response or the<br />
revised paper to one or more referees, or Editor
68<br />
may ask one referee to comment on the concerns<br />
raised by another referee.<br />
Advance Online Publication<br />
J<strong>Cell</strong>MolBiol provides Advance Online Publication<br />
<strong>of</strong> articles, which benefit authors with an earlier<br />
publication date <strong>and</strong> allows the readers’ access to<br />
accepted papers several weeks before they appear<br />
in print<br />
Ethical Issues<br />
For manuscripts reporting experiments on live<br />
vertebrates or higher invertebrates, authors must<br />
declare that the study was approved by the<br />
institutional ethics committee. Papers describing<br />
investigations on human subjects must include a<br />
statement that informed consent was obtained from<br />
all subjects.<br />
Plagiarism<br />
If portions <strong>of</strong> the manuscript have already been<br />
published by the author on other journals or<br />
websites, J<strong>Cell</strong>MolBiol Editorial Board needs to<br />
know which portions <strong>of</strong> the manuscript have been<br />
previously published <strong>and</strong> where. The author should<br />
include a note in the cover letter indicating which<br />
portions have been published elsewhere.<br />
In case <strong>of</strong> any suspicion on scientific misconduct or<br />
dishonesty in research, J<strong>Cell</strong>MolBiol reserves the<br />
right to forward any submitted manuscript to an<br />
appropriate authority for investigation.<br />
Copyright Notice<br />
It is the responsibility <strong>of</strong> the submitting author to<br />
ensure that the authorship <strong>of</strong> the paper reflects the<br />
con<strong>tr</strong>ibutions <strong>of</strong> the authors to the work described,<br />
<strong>and</strong> that all listed authors have agreed to the<br />
submission <strong>of</strong> the manuscript in its current form.<br />
Conditions <strong>of</strong> publication in J<strong>Cell</strong>MolBiol are that<br />
the paper has not already been published elsewhere;<br />
that it is not currently being considered for<br />
publication else-where; all persons designated as<br />
authors should qualify for authorship, <strong>and</strong> all those<br />
who qualify should be listed. If accepted, Haliç<br />
University <strong>and</strong> J<strong>Cell</strong>MolBiol have the exclusive<br />
license to publish.<br />
J<strong>Cell</strong>MolBiol is freely available to individuals <strong>and</strong><br />
institutions. Copies <strong>of</strong> this <strong>Journal</strong> <strong>and</strong> articles in<br />
this journal may be dis<strong>tr</strong>ibuted for research or for<br />
<s<strong>tr</strong>ong>edu</s<strong>tr</strong>ong>cational purposes free <strong>of</strong> charge. However,<br />
REVISED<br />
December 2011<br />
commercial use <strong>of</strong> articles contained herein is<br />
prohibited without the written consent <strong>of</strong> the<br />
Editor-in-Chief.<br />
Publication Agreement<br />
The corresponing author is required to assign the<br />
Publication Agreement Form in order to publish the<br />
submitted manuscript in J<strong>Cell</strong>MolBiol.
<strong>Journal</strong> <strong>of</strong> <strong>Cell</strong> <strong>and</strong><br />
<s<strong>tr</strong>ong>Volume</s<strong>tr</strong>ong> <s<strong>tr</strong>ong>10</s<strong>tr</strong>ong> · No 1 · June 2012<br />
Review Article<br />
Molecular Biology<br />
Production <strong>and</strong> indus<strong>tr</strong>ial applications <strong>of</strong> laccase enzyme<br />
M. IMRAN, M.J. ASAD, S.H. HADRI, S. MEHMOOD<br />
Research Articles<br />
Isolation <strong>and</strong> biochemical identification <strong>of</strong> Escherichia coli from wastewater effluents <strong>of</strong> food<br />
<strong>and</strong> beverage indus<strong>tr</strong>y<br />
T. FARASAT, Z. BILAL, F. YUNUS<br />
Investigation <strong>of</strong> the MGP promoter <strong>and</strong> exon 4 polymorphisms in patients with ischemic<br />
s<strong>tr</strong>oke in the Ukrainian population<br />
A.V. ATAMAN, V.Y. GARBUSOVA, Y.A. ATAMAN, O.I. MATLAJ, O.A. OBUCHOVA<br />
Investigation <strong>of</strong> the association <strong>of</strong> survivin gene -625G/C polymorphism in non-small cell<br />
lung cancer<br />
E. AYNACI, E. COŞKUNPINAR, A. EREN, O. KUM, Y. M. OLTULU, N. AKKAYA, A.<br />
TURNA, İ. YAYLIM, P. YILDIZ<br />
Effects <strong>of</strong> prenatal <strong>and</strong> neonatal exposure to lead on white blood cells in Swiss mice<br />
R. SHARMA, K. PANWAR, S. MOGRA<br />
Sulfabenzamide promotes autophagic cell death in T-47D breast cancer cells through p53/<br />
DRAM pathway<br />
R. MOHAMMADPOUR, S. SAFARIAN, S. FARAHNAK, S. HASHEMINASL, N. SHEIBANI<br />
Media optimization for amylase production in solid state fermentation <strong>of</strong> wheat bran by<br />
fungal s<strong>tr</strong>ains<br />
M. IRFAN, M. NADEEM, Q. SYED<br />
Guidelines for Authors<br />
1<br />
13<br />
19<br />
27<br />
33<br />
41<br />
55<br />
65