The Toxicologist - Society of Toxicology

1170 GENOTOXICITY STUDIES ON REFERENCE
SMOKELESS TOBACCO PRODUCTS USING
GREENSCREEN HC GADD45A-GFP ASSAY.
J. H. Lauterbach 1 , L. Birrell 2, 1 , P. A. Cahill 2 , C. Jagger 2 and N. Billinton 2 .
1
Lauterbach & Associates, LLC, Macon, GA and 2 Gentronix Ltd., Manchester,
United Kingdom.
Some health experts have recommended that smokers, who refuse to quit or refuse
to use nicotine replacement therapies, switch to low TSNA smokeless tobacco
products (STP). US-style moist snuff is the most popular of the STP, but has attracted
much criticism from those against STP use on account of toxicological and
addiction concerns. Use of standard in vitro assays (e.g., Ames) to assess STP toxicity
was of limited utility in distinguishing product types and brands within a type
(Rickert et al., Regul. Toxicol. Pharmacol. 2009 53:121-33). This study sought to
assess the genotoxicity of DMSO extracts of the Reference Smokeless Tobacco
products (1S1, 2S1, loose-leaf chewing tobacco; 1S2, dry snuff; 1S3, 2S3, moist
snuff) in the increasingly widely used in vitro mammalian GreenScreen HC assay
(GADD45a-GFP reporter hosted in TK6 cells). Extracts of tobacco products are
inherently autofluorescent and so GreenScreen HC data were collected by flow cytometry
to circumvent fluorescence interference. Genotoxicity data generated in
this study for the five standard samples were not consistent with the levels of TSNA
reported in these products (e.g., at 3h-S9 potency by LEC showed low TSNA STP
had higher genotoxicity than high TSNA STP). However, the genotoxicity may
stem from other sources as found in detailed GC-MS analyses of the same products.
The GreenScreen HC assay appears to have utility in distinguishing between STP
at the biological level.
1171 HISTOPATHOLOGY AND NEUROTOXICITY
SCREENING IN F344 RATS IN A 90-DAY INHALATION
EXPOSURE TO AEROSOLIZED SYNTHETIC JET FUEL.
B. A. Wong 1 , C. U. Parkinson 1 , M. F. Struve 2 , G. A. Willson 3 , D. J. Wagner 4 ,
D. R. Mattie 5 and D. E. Dodd 1 . 1 The Hamner Institutes for Health Sciences,
Research Triangle Park, NC, 2 North Carolina State University, Raleigh, NC,
3
Experimental Pathology Laboratories, Research Triangle Park, NC, 4 Naval Health
Research Center, EHEL, Wright-Patterson AFB, OH and 5 AFRL/711 HPW/RHPB,
Wright-Patterson AFB, OH.
Development of synthetic fuels is being pursued to augment or replace petroleumderived
JP-8 jet fuel for the US military. One such product is a jet fuel produced by
the Fischer-Tropsch (FT) synthetic process from natural gas. During operations
and maintenance, personnel may be exposed to jet fuel vapors and aerosols. To assess
potential toxicity, a study was conducted with F344 rats exposed by whole
body inhalation to 0, 200, 700, and 2000 mg/m3 of an aerosol/vapor mixture of
FT jet fuel for 6 hours per day, 5 d/wk for 90 days. Towards the end of the exposure
period, rats underwent neurotoxicity (motor activity and functional observational
battery (FOB)) screening. After the last exposure, animals were euthanized and selected
tissues examined for histopathology. For rats exposed to 2000 mg/m3 of FT
jet fuel, there was an overall reduction in motor activity in males, and a reduction of
initial exploratory activity in female rats. During FOB evaluation, female rats exposed
to FT jet fuel at 2000 mg/m3 had a reduced number of rears. There was no
other behavioral neurotoxicity observed in FT jet fuel exposed male or female rats.
Animals exposed to 2000 mg/m3 of jet fuel showed somewhat lower body weights.
Histopathology changes were observed in the kidneys of male rats, and in the nose
and lungs of males and females. In the kidneys, males in the high exposure group
showed an accumulation of hyaline droplets in the proximal convoluted tubular
cells. Nasal effects included olfactory epithelial degeneration in the 2000 mg/m3
males and females. Minimal to mild multifocal areas of inflammatory cell infiltration
were seen in the lungs for males and females at 2000 mg/m3. This information
is being used to help develop occupational exposure limits for synthetic jet fuels.
1172 STUDIES ON THE GENOTOXICITY OF 2, 5-
DIMETHYLFURAN, A POTENTIAL BIOFUEL.
M. Fromowitz, J. Shuga, A. Yip Wlassowsky, L. Zhang and M. T. Smith. School
of Public Health, University of California Berkeley, Berkeley, CA.
2,5- Dimethylfuran (2, 5-DMF) is currently being explored as an alternative biofuel
to ethanol, because of its high energy density, and its stability for liquid transport.
There are very few peer reviewed, published toxicological studies on 2,5-
DMF and hence it is difficult to assess its potential risks as a biofuel. Published
Ames tests were negative but a clastogenicity study found that a 3 hour exposure to
8mM of 2,5-DMF induced significant increases in chromosome breaks and exchanges
in cultured Chinese hamster ovary cells (Cancer Letters 13: 89-95, 1981).
Preliminary studies using a murine in vitro erythroid micronucleus system (Proc.
Natl Acad. Sci. USA 104:8737–8742, 2007) established in our laboratory also suggest
that 2,5-DMF, is clastogenic. Lineage negative hematopoietic progenitor cells
were isolated through immunomagnetic separation from the bone marrow of 6-8
week old black C57BL/6J mice. The cells were cultured in an IMDM based
medium containing 5U/ml human recombinant erythropoietin to induce erythropoiesis.
The cells were treated with 1mM 2, 5- DMF with or without human S9
metabolism activation 24 hours after seeding. After one hour of exposure the cells
were washed and fed with media devoid of erythropoietin. The cells were harvested
and prepared on slides at 48 hours. The slides were stained with acridine orange,
blinded, and examined by fluorescence microscope. Over 2000 polychromatic erythrocytes
on each slide were scored for the presence of micronuclei. Micronuclei
frequencies were tabulated and statistical significance was determined by t test. A
significant increase in micronucleus frequency, compared to vehicle controls, was
induced by a 1mM, 1-hour exposure of 2,5-DMF, with and without human liver
S9 fraction metabolism. Further studies on the potential genotoxic effects of 2,5-
DMF are underway.
1173 REPEAT DOSE ORAL AND REPRODUCTIVE TOXICITY
OF THE CHLORINATED FLAME RETARDANT
DECHLORANE PLUS (DP).
W. J. Brock 1 , R. Schroeder 2 , C. MacKnight 2 , J. VanSteenhouse 2 and J. Nyberg 3 .
1
Brock Scientific Consulting, Montgomery Village, MD, 2 MPI Research, Mattawan,
MI and 3 Occidental Chemical Corporation, Wichita, KS.
DP was introduced as a flame retardant as an alternative to brominated flame retardants
but the published data are limited. Rats were treated with doses of 750, 1500
or 5000 mg/kg by gavage. Controls were treated with corn oil. For the subchronic
toxicity phase, animals were observed for clinical signs of toxicity including FOB
changes, body weight and food consumption changes and for effects on clinical and
anatomic pathology. No effects were observed from clinical signs, body weights,
food consumption, neurobehavioral and FOB evaluations. In addition, no effects
were observed on clinical pathology parameters, and no organ weight effects were
observed. In the reproductive toxicity phase, no effects were noted on clinical signs
of toxicity, body weights or food consumption. Estrous cyclicity, reproduction and
fertility indices, parturition (gestation length, litter size), pup body weights, sex ratios
and clinical findings through LD4 were unaffected. No effects were noted on
GD 20 uterine implantation data, fetal body weights or fetal sex ratios. No fetal external
and visceral malformations or variations were observed. Mortalities occurred
across all dose groups, including controls. Microscopic evidence of gavage-related
errors consisted of adhesions, inflammation and fibrosis in the thoracic and pleural
cavities with evidence of esophageal perforations noted in some animals.
Microscopic findings associated with an antigenic stimulus, immune response
and/or a physiological stress response secondary to the presence of test article material
in the thoracic cavity were observed. These findings were not considered to be
test article related as they were not dose dependent and were observed only in animals
with evidence of suspected gavage injury. Viscosity measurements of the DP
suspensions in corn oil suggested that the high viscosity of the suspensions most
likely contributed to the mis-dosing and mortalities observed in this study. The
NOAEL was 5000 mg/kg.
1174 EMODIN INHIBITED ATP BINDING TO
TOPOISOMERASE II TO INDUCE DNA DOUBLE-
STRAND BREAKS.
Y. Li, Y. Luan and J. Ren. Shanghai Institute of Materia Medica, Chinese Academy
of Science, Shanghai, China.
Emodin has been widely used as a component of laxatives, whereas it can lead to
genotoxicity. However, the mechanism underlining its genotoxicity is not entirely
clear. We applied different assays to investigate the mechanisms by which emodin
induces genotoxicity. Without metabolic activation, emodin at 80 μg/mL was
mildly genotoxic as indicated in thymidine kinase (TK) gene mutation assay and
micronucleus (MN) test. But in the neutral comet assay and the detection of γ-
H2AX, emodin at 80 μg/mL induced DNA double-strand breaks (DSBs).
Moreover, results obtained from inhibitions of kDNA decatenation and relaxation
of supercoiled pBR322 induced by topoisomerase II (Topo II) showed that emodin
inhibited Topo II activity. Further, using both aclarubicin, a Topo II catalytic inhibitor,
and HL-60/MX2 cells deficient in Topo II, we showed that emodin-triggered
DSBs s were in a Topo II –dependent manner. However, emodin did not intercalate
into DNA. In contrast, emodin interacted with Topo II by competing
with ATP for binding to the ATPase domain of human Topo II α and inhibited
ATP hydrolysis. Taken together, these results suggested that emodin inhibited ATP
binding to Topo II to induce DSBs.
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