The Toxicologist - Society of Toxicology

1671 CYTOTOXICITY COMPARISON OF DIFFERENT
ORGAN TOXICANTS IN ORGAN SPECIFIC CELL LINES.
Z. Lin, R. Swiss, Y. Will and M. Pletcher. Compound Safety Prediction, Pfizer
Global Research & Development, Groton, CT.
Attrition of drugs for safety liabilities in preclinical development as well as in late
stage clinical trials continues to be a challenge for the pharmaceutical industry for
financial and patient welfare reasons. Hepato, cardio and nephrotoxicity remain the
primary forms of drug-induced injury leading to compound termination. As a result,
in vitro cellular screening paradigms are being deployed as common practice in
the early stages of drug discovery as a means to predict these end organ toxicities
and prevent high-risk compounds from reaching in vivo evaluations. In this study
we investigated if using a hepatic, cardiac and kidney-derived cell line could accurately
predict specific organ toxicities. We tested 140 cardiotoxicants, 250 hepatotoxicants,
and 60 nephrotoxicants in H9c2 (embryonic myocardium), HepG2 (hepatocellular
carcinoma) and NRK-E52 (kidney proximal tubule) cell for their
cytotoxicity . The experiments were designed and carried out in such way that all
cells were seeded in the same density and exposed to the same diluted drugs at 11
different doses for 24 hours. Cell viability assays (ATP depletion) were carried out
in triplicate using a Promega Cell viability assay kit and IC50 values were calculated
for each drug x cell line treatment. The data showed that the majority of compounds,
regardless of their different organ toxicities, had similar cytotoxic effects in
all three cell lines. Only a very small percentage of compounds showed differential
response in the cell lines and that differential response did not show a correlation
between the known in vivo organ toxicity and the tissue of origin of the cell line.
Our results suggest that different cell lines have relatively equal value in assessing
general cytotoxicity and that organ toxicity cannot be accurately predicted using
such a simple approach. Organ toxicity potentially results from compound accumulation
in a particular tissue, metabolism and off target expression.
1672 STORE-OPERATED CALCIUM CHANNELS PLAY A
SIGNIFICANT ROLE IN ROS-INDUCED NECROTIC
CELL DEATH.
F. M. Ramirez, R. Xie, S. S. Lau and T. J. Monks. Southwest Environmental
Health Sciences Center, Department of Pharmacology & Toxicology, College of
Pharmacy, University of Arizona, Tucson, AZ.
Oxidative stress is associated with many pathological conditions, including renal
disease. The generation of reactive oxygen species (ROS) leads to DNA strand
breaks, which activate the DNA repair enzyme poly (ADP-ribose) polymerase
(PARP)-1. Hyper-activation of PARP-1 leads to NAD and ATP depletion and to
increases in intracellular calcium concentrations (iCa2+), all of which contribute to
necrotic cell death. The relationship between iCa2+ and oxidative stress induced
was determined in renal proximal tubule epithelial cells (HK-2) exposed to the potent
nephrotoxicant 2,3,5-tris-(glutathion-S-yl)hydroquinone (TGHQ). Inhibitors
of calcium channels, such as the inner mitochondrial membrane calcium uniporter
(MCU), the endoplasmic reticulum inositol 1,4,5-triphosphate receptor (IP3R),
and store-operated calcium channels (SOCs) in the plasma membrane were used to
pre-treat and co-treat HK-2 cells in the presence and absence of TGHQ in order to
determine their role in cell death. Ru360 (10μM), a MCU inhibitor, and TMB-8
(100μM), an IP3R inhibitor, had no effect on TGHQ-induced cell death. In contrast,
2-APB (100μM), a SOCs inhibitor, maintained cell viability and the mitochondrial
membrane potential to ~60% of controls. Overall, the data reveal that although
the MCU and the IP3R do not appear to play a direct role in
TGHQ-induced cell death, SOCs have a significant role in response to oxidative
stress. We are currently investigating the relationship between PARP-1 and SOCs
in TGHQ-induced cell death via direct measurement of NAD and calcium imaging.
(P30ES006694)
1673 PROTECTION OF HYDROQUINONE-INDUCED
APOPTOSIS BY FAU IS MEDIATED BY NQO1 IN W7.2
MOUSE THYMOMA CELLS.
S. H. Inayat-Hussain 1 , L. E. Siew 1 , M. K. Chan 1 , N. F. Rajab 1 , D. Ross 2 and G.
T. Williams 3 . 1 Environmental Health Program, Universiti Kebangsaan Malaysia,
Kuala Lumpur, Malaysia, 2 School of Pharmacy, University of Colorado at Denver,
Denver, CO and 3 Institute for Science and Technology in Medicine and School of Life
Sciences, Keele University, Keele, United Kingdom.
Hydroquinone (HQ) is a major metabolite of benzene. However, the role of fau in
HQ-induced apoptosis is still poorly defined. fau (Finkel-Biskis-Reilly murine sarcoma
virus (FBR-MuSV)-associated ubiquitously expressed gene) was identified
using functional expression cloning as a potential tumor suppressor gene. In this
360 SOT 2011 ANNUAL MEETING
study, downregulation of fau by the antisense orientation of fau (rfau) in W7.2 cells
was investigated whether it offered any protection on HQ induced DNA damage,
reactive oxygen species (ROS) and apoptosis. Our data showed that HQ induced
apoptosis via DNA damage and ROS was abrogated using a stable clone of W7.2
rfau cells. Further investigation revealed that there was an elevation of NAD(P)H:
quinone oxidoreductase 1 (NQO1) protein in W7.2 rfau cells. Importantly, inhibition
of NQO1 enzymatic activity using MAC220, a specific inhibitor and knockdown
of NQO1 by siRNA rendered the cells to be susceptible to HQ induced toxicity.
Taken together, this study suggests that cytoprotection of HQ toxicity by
downregulation of fau is mediated by NQO1 and there is a possible role for fau in
the manifestation of benzene toxicity.
1674 AFLATOXIN PREVALENCE AND HEPATOCELLULAR
CARCINOMA IN GHANA: A REGIONAL ANALYSIS.
N. J. Mitchell 1 , P. E. Jolly 2 , C. M. Jolly 3 , A. Marroquin-Cardona 1 and T. D.
Phillips 1 . 1 College of Veterinary Medicine, Texas A&M University, College Station,
TX, 2 Department of Epidemiology, School of Public Health, University of Alabama at
Birmingham, Birmingham, AL and 3 Department of Agricultural Economics and
Rural Sociology, Auburn University, Auburn, AL.
Cancer incidence is rising in Africa with approximately 650,000 people developing
cancer annually with an 80% mortality rate. Hepatocellular carcinoma (HCC) is
one of the most common malignancies in Ghanaian men and women. Mortality
among Ghanaian patients with HCC accounts for 21.15% and 10.9% of all cancer
related deaths respectivley. Multiple factors play a role in the etiology of HCC; one
major risk factor is chronic exposure to low levels of aflatoxins (AFs) from the diet.
It is well established that the predominant congener, aflatoxin B 1 (AFB 1 ), is one of
the most potent naturally-occurring hepatocarcinogens in humans. A well-defined
biomarker of exposure, aflatoxin M 1 (AFM 1 ), has been correlated with an elevated
risk of HCC in several human populations. In this study, urinary AFM 1 biomarkers
from 324 Ghanaians (representing the Ashanti, Greater Accra and Central regions)
were analyzed for future possible correlations with cancer prevalence and dietary intake
of AFs. Biomarkers were extracted from urine using AflaTest® immunoaffinty
columns and measured using HPLC analysis with fluorescence detection. AFM 1
was detected in 43.4% of the urines with values ranging from 233.2 pg/dl to
818,769 pg/dl urine. The calculated median value was 3,313.4 pg/dl urine.
Differences were noted in the percent mortality rates from HCC in each region.
Specific values were as follows; Ashanti 25.18%, Greater Accra 43.55%, and
Central 9.36%. AFs exposure and HCC incidence is an ongoing public health
problem in Ghana, especially in the Ashanti and Greater Accra regions where HCC
mortality rate is the highest. Determination of the prevalence of AFs in different regions
of Ghana is an important tool to assess the predominant sources of exposure,
as well as those populations that are most at risk for HCC due to AFs.
1675 CYTOCHROME P4501A PROMOTER
POLYMORPHISMS AND ACTIVITY IN NATURAL
POPULATIONS ADAPTED TO CHRONIC POLLUTION.
L. Williams 1 and M. Oleksiak 2 . 1 Environmental and Molecular Toxicology, North
Carolina State University, Raleigh, NC and 2 University of Miami, Miami, FL.
Cytochrome P4501A (CYP1A) has been shown to be refractory to prototypic inducers
in populations of the estuarine minnow, Fundulus heteroclitus, adapted to
chronic anthropogenic pollution. Two single nucleotide polymorphisms (SNPs) in
the promoter and first intron of the CYP1A promoter were previously found to be
under selection, indicating that natural selection was acting on the CYP1A promoter
or loci linked to these SNPs. In order to understand the role of the CYP1A
promoter and these selectively important SNPs, the promoter and first intron and
exon were sequenced in multiple individuals in one polluted (New Bedford
Harbor) population resistant to PCBs and other anthropogenic contaminants and
two reference populations north and south of the polluted site. The CYP1A promoter
was extremely variable (an average of 21% of the promoter nucleotides varied
among all populations) and there were no fixed differences between populations.
The inducibility of the promoter was also explored; the promoter was
induced by a prototypic PAH, 3-methylcholanthrene, in a dose-dependent manner.
When promoters from multiple individuals per population were tested, the CYP1A
promoter was significantly induced in the polluted New Bedford Harbor population
as compared to both reference populations. This is in contrast to CYP1A expression
in vivo which is refractory to induction in New Bedford Harbor individuals.
Overall, the variation in the promoter does not explain the variation in in vitro
promoter expression in all tested populations. These results indicate that the underlying
mechanism for the in vivo transcriptional phenotype may lay further upstream
or downstream of the CYP1A promoter region which was sequenced
(1600bp), or involve proteins which were not available in the cell line (top minnow)
in which the transfection assays were completed.