The Toxicologist - Society of Toxicology

1676 TWO BIOMARKERS FOR EVALUATION OF
ORGANOPHOSPHATE (OP) POISONING IN HUMANS.
G. H. DeOliveira 1 , G. L. Emerick 1 , K. A. Belaz 2 , M. Gonçalves 1 and R. V.
Oliveira 2 . 1 Natural Actives Principles and Toxicology, UNESP Sao Paulo State
University, Araraquara, Sao Paulo, Brazil and 2 Department of Chemistry, University
Federal of Sao Carlos, Sao Carlos, Sao Paulo, Brazil.
World Health Organization recognizes that the activity of acetylcholinesterase
(AChE) is a useful preventative biomarker to overexposure to OP. On the other
hand, in spite of the neuropathy target esterase (NTE) had been identified by
Johnson (1969) as a target of neuropathy induced OP, we did not have knowledge
of the normal value of it activity into human lymphocyte. Therefore, this work
aimed to establish reference value for normal activity of AChE in erythrocytes and
NTE in lymphocytes (LNTE) in blood of donors of the hemocenter of Araraquara,
SP Brazil. Besides all the requirements that a blood donor must have, we applied a
questionnaire prior to a screening and know more about some habits of each volunteer.
In a total of 45 volunteers we conducted the following key questions: Is
there any industry near your home? Do you smoke? Do you have difficulty remembering
things? Are you taking any medicine? Then the venous blood was collected
in Vacutainer tubes with sodium heparin. We made the separation of lymphocytes
from 2.5 ml of blood using the Histopaque-1077 and for the separation of erythrocytes
we centrifuged 0.5 ml of blood at 2500 rpm for 5 min. The values obtained
for AChE and LNTE were 6.82 ± 1.29 μmol/min/g proteins and 7.02 ± 1.85
μmol/min/g proteins, respectively. It was made a separation between men and
women and 77.8% of the volunteers were men with values of 6.8 ± 1.2
μmol/min/g protein for AChE and 7.3 ± 1.78 μmol/min/g protein for LNTE. The
women had values of 6.23 ± 1.01 μmol/min/g proteins for AChE and 6.64 ± 1.89
μmol/min/g proteins for LNTE. We conclude that these data may be useful in future
work aimed at delineating the activity of NTE as a biomarker of effect for agricultural
workers from exposure to neuropathic OP. Human Ethics Committee,
Resolution: 31/2009. Financial support: FAPESP 2009/51048-8.
1677 GLOBAL GENE EXPRESSION CHANGES BY HUMAN
UROEPITHELIAL CELLS EXPOSED TO LOW-LEVEL
MONOMETHYLARSONOUS ACID.
X. Zheng, P. Novak, S. M. Wnek, M. K. Medeirous, V. Chyan and J. A.
Gandolfi. Pharmacology/Toxicology, University of Arizona, Tucson, AZ.
Global gene expression effects of a 3 month exposure of low concentrations of
monomethylarsonous acid [MMA(III), a metabolite of arsenate] to human uroepithelial
(UROtsa) cells was investigated. To identify the molecular targets of
MMA(III) in human cells, total RNA was isolated from UROtsa cells exposed for
0-3 months to 50 nM MMA(III) and then an Affymetrix Human gene array (28K
genes) was used to carry out the cDNA microarray assay. Small, progressive changes
are seen with some genes over at 1-2 month exposure to MMA(III) followed by a
much larger change between 2-3 month of exposure. Using a log2 cut-off, there are
168 up-regulated genes and 442 down-regulated genes in response to 3 month exposure.
The gene changes at 3 month of exposure are coincident with these cells
having an increase in proliferation, DNA damage, and eventual capabilities to
grown in soft agar and form tumors in immuno-compromised mice. Six up-regulated
genes and six down-regulated genes were selected to validate the array data
and used as “marker” genes related to cellular and molecular functions: TM-
PRSS11A and KRT7 for cell cycle, CXCR4 and TGFBI for cell proliferation,
PDGFRA and IRF1for cell differentiation, CTNND2 and ICAM1 for cell adhesion,
CRYAB and DUSP1 for oxidative stress, and ERCC2 and DCLRE1C for
DNA repair. In general, these genes demonstrated a time-dependent change starting
at 1 month exposure to 50 nM MMA(III) with greater alterations seen at the 3month
time point. More detailed chronological assessment of the global gene alterations
along with their correlation to phenotypic changes should provide clues to
the underlying the mechanisms of human diseases related to chronic arsenic exposure.
(NIEHS 04940 and 06694).
1678 METABOLIC PROFILING OF SERUM FROM RATS
DOSED WITH ACETAMINOPHEN AND CARBON
TETRACHLORIDE.
R. D. Beger, L. Pence, L. K. Schnackenberg, J. Sun, T. Schmitt, X. Yang, J.
Greenhaw, S. Bhattacharyya, W. Salminen and D. L. Mendrick. Division of
Systems Biology, NCTR, U.S. FDA, Jefferson, AR.
Hepatotoxicity is a major reason that drugs are withdrawn post-market or have a
black box warning label. Current clinical chemistry biomarkers of liver injury are
not always adequate in terms of specificity; therefore new specific biomarkers of
liver injury are needed. Metabolic profiling methods were used to find and evaluate
biomarkers of liver toxicity with two classic hepatotoxins. Male Sprague-Dawley
rats were dosed with acetaminophen (APAP) or carbon tetrachloride (CCl4) and
blood samples were collected at 6 hours, 24 hours, and 3 days after dosing. Rats
were dosed with either a single dose of 0 (0.5% methylcellulose), 100 or 1250
mg/kg APAP or given 0 (corn oil), 50 or 2000 mg/kg CCl4 for 3 consecutive days.
Rats were sacrificed at 6 and 24 hours and 3 days and tissues collected for
histopathology. Nuclear magnetic resonance, gas chromatography coupled to mass
spectrometry and ultra performance liquid chromatography coupled to mass spectrometry
were used to profile bile acids and to discover new biomarkers of hepatotoxicity.
Histopathology showed minimal to moderate centrilobular necrosis at 24
hours after high doses of APAP. For CCl4, histopathology showed mild to marked
centrilobular necrosis at 24 hours after high dosing and minimal to mild centilobular
necrosis at 3 days after dosing. Serum levels of cholic acid and glycocholic acid
were significantly higher at 24 hours after a high dose of APAP or CCl4.
Taurocholic acid was increased at 6 and 24 hours after a high dose of CCl4.
Metabolic profiling of blood samples from rats dosed separately with APAP or CCl4
was successful in finding potential biomarkers of liver toxicity. Several bile acids
were elevated before traditional clinical chemistry markers of liver injury. More liver
toxicity studies are needed to determine whether these potential biomarkers are
more specific for liver injury than current clinical chemistry biomarkers.
1679 CHARACTERIZATION OF PEPTIDE ADDUCTS OF
REACTIVE NAPHTHALENE METABOLITES IN THE
URINE OF MALE MICE BY MASS SPECTROMETRY.
N. Pham 2, 1 , A. Buckpitt 1, 2 , D. Morin 1 and W. T. Jewell 2 . 1 Veterinary Medicine,
Molecular Biosciences, University of California Davis, Davis, CA and 2 Pharmacology
and Toxicology, University of California Davis, Davis, CA.
Naphthalene, a volatile polycyclic aromatic hydrocarbon generated during combustion,
is metabolized to reactive metabolites which bind covalently to proteins in
vivo and in vitro. In general, covalent binding levels correlate with the species and
tissue selective toxicity associated with naphthalene. The substantial differences in
susceptibility to naphthalene toxicity in different rodent species combined with
widespread human exposure supports the need to understand whether this compound
poses a risk for humans. Although covalent adducts with hemoglobin and
albumin have been used as surrogates for examining the generation of reactive
metabolites, their measurements have not lead to a clearly established relationship
between adduct levels and toxicity. The hypothesis being tested in these studies is
that the formation of critical adducts in target tissues can be followed by measuring
downstream products, specifically adducted peptides, in the urine. To establish
methodology for measuring adducted peptides in the urine, 24 hr urine samples
were obtained from mice treated with 50/50 mixtures of 2H/1H-naphthalene either
IP or by inhalation. Separation of water soluble metabolites and adducted peptides
by reverse phase HPLC followed by HR-MS revealed the presence of numerous
putative peptide adducts with the expected isotopic ratios and molecular masses
which varied from 600 to 900Da. Partial sequences obtained for some of these entities
and differences in chromatographic/mass spectral characteristics with known
water soluble metabolites of naphthalene further confirmed the presence of urinary
peptide adducts. The urine remains a relatively unexplored biofluid for investigation
of protein/peptide adducts that could act as markers of metabolic processes key
to the mechanisms of toxicity of numerous chemicals which undergo metabolic activation.
Characterization and identification of these potential candidates is ongoing.
Supported by NIEHS 04311 and 04699.
1680 INSECTICIDE RESIDUES AND THEIR SELECTED
BIOMARKERS IN PRODUCE: FIELD STUDIES USING
MALATHION AND FENPROPATHRIN IN
STRAWBERRIES.
L. Chen 1 , Z. Chen 1, 2 , Y. Liu 1 , T. Lopez 1, 2 , G. Sankaran 1, 2 , H. Vega 1 and R. I.
Krieger 1, 2 . 1 Personal Chemical Exposure Program, Department of Entomology,
University of California, Riverside, CA and 2 Environmental Toxicology Graduate
Program, University of California, Riverside, CA.
Trace levels of pesticides used in conventional or organic crop protection and degradation
products may occur in produce. Low level human dietary and non-occupational
urine biomonitoring studies may be confounded by preformed pesticide biomarkers
used to infer potential human pesticide exposure. This research
investigated the formation of potential urine biomarkers in strawberries under normal
field conditions in Santa Maria, CA. Malathion and fenpropathrin insecticides
were sprayed (lb/A) in August, 2010. Residue and biomarker analysis using
SOT 2011 ANNUAL MEETING 361