The Toxicologist - Society of Toxicology

1577 AN IN VITRO MODEL SYSTEM FOR ASSESSING THE
EFFECTS OF OIL DISPERSANTS IN HUMAN
HEPATOCYTES.
O. Bandele, M. Santillo and P. Wiesenfeld. Toxicology, Office of Applied Research
and Safety Assessment, Center for Food Safety and Applied Nutrition, U.S. FDA,
Laurel, MD.
Because of the unprecedented amount of oil dispersants utilized during the 2010
Deepwater Horizon oil spill, understanding the toxicity of these chemicals is crucial.
These dispersants may accumulate in aquatic species, be passed down the food
chain, where if consumed in significant quantities may have a negative affect on
human health. The majority of ingested chemicals, drugs, pollutants, and toxins are
metabolized by the liver. We therefore examined the effects of several oil dispersants
on a human liver cell line, HepG2/C3A, which serves as an in vitro cell model. We
employed spectrofluorimetry-based endpoint assays to assess the toxicity of 10-500
ppm EC9527A, EC9500A, and ZI-400 in HepG2/C3A cells. Significant increases
in cell death were observed after exposing cells to EC9527A and EC9500A for a
maximum of 72 hr (LC50 ~325 ppm), whereas ZI-400 was moderately cytotoxic
(LC50 > 400 ppm). Endpoint assays that monitored reactive oxygen species
demonstrated that all dispersants caused a dose-dependent accumulation of reactive
oxygen species in HepG2/C3A cells. We also observed that EC9500A and ZI-400
disrupted the mitochondrial membrane electrical potential, monitored by the uptake
of rhodamine 123. In addition, EC9527A may inhibit P-glycoprotein activity.
Our initial findings suggest that the dispersants examined in this study may act
through different mechanisms. These results also demonstrate an effective in vitro
approach to identify and evaluate dispersants and related chemicals that may be potential
hepatotoxins.
1578 CHARACTERIZATION OF IONIC LIQUID
COMPOUNDS FOR TOXICOLOGICAL EVALUATION.
J. W. Algaier 1 , C. C. Pearson 1 , A. D. Ammenhauser 1 , Q. Lawrence 1 , J. L.
Cookinham 1 , L. G. Siemann 1 , R. K. Harris 1 , B. Jayaram 2 and C. S. Smith 2 .
1 Midwest Research Institute, Kansas City, MO and 2 NIH, NIEHS, Research Triangle
Park, NC.
Ionic liquid (IL) compounds are a new class of chemicals replacing regular organic
solvents and are used in diverse reactions under ‘green chemistry’. The structural
flexibility of IL’s and the varied array of available anions have made it possible to
tune solvents for a specific reaction. However, little is known about the toxicity of
these compounds and for this reason four ionic liquids have been selected for toxicological
evaluation by the NIEHS, National Toxicology Program (NTP), which
are 1-Butyl-3-methyl-imidazolium chloride, 1-Butyl-1-methyl-pyrrolidinium chloride,
1-ethyl-3-methylimidazolium chloride, and N-Butyl-pyridinium chloride.
For this work, identity and impurity determination used several methods including
direct infusion liquid chromatography/mass spectrometry (LC/MS), where parent
and daughter ion scan spectra confirmed identity. The infrared (FT-IR) and nuclear
magnetic resonance (NMR) spectra are consistent with the literature structures.
Because of the hydrophobic nature of ionic liquid compounds, special handling
procedures using inert argon atmosphere were required, especially to determine the
water content by Karl Fischer, which averaged 0.36%. The volatile content by
weight loss on drying was attempted, but only successful with one compound at
0.75% since a constant weight loss was not achieved. Chloride content was quantitated
using ion chromatography and elemental analysis for carbon, hydrogen, nitrogen
and chloride was performed; results from both methods matched theoretical
values within +/- 0.5%. The HPLC purity profile analysis using an isocratic acetonitrile-ammonium
acetate mobile phase with a cyano column and ultraviolet
(UV) and evaporative light scattering detection (ELSD) confirmed no observed impurities
under these conditions. The results confirmed the identity of each ionic liquid
compound with purity values greater than 99%.
1579 ANALYSIS OF ACUTE AND CHRONIC EFFECTS OF
HETEROCYCLIC AROMATIC AMINES IN
DIFFERENTIATED HUMAN HEPATOMA HEPARG CELLS.
J. Dumont 1, 2 , R. Josse 1, 2 , C. Lambert 1, 2 , S. Anthérieu 1, 2 , C. Guguen-
Guillouzo 1, 2 , A. Guillouzo 1, 2 and M. Robin 1, 2 . 1 UMR991, INSERM, Rennes,
France and 2 University of Rennes 1, Rennes, France. Sponsor: M. Pallardy.
Humans are continuously exposed to low doses of various food and environmental
contaminants. Prediction of their toxicity and risk assessment for human health
represent currently a major challenge. We have compared the effects of acute and
repeat exposure to two common heterocyclic aromatic amines (HAA), 2-Amino-1methyl-6-phenylimidazo[4,5-b]pyridine
(PhIP) and 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline
(MeIQx), singly or in equimolar mixture, in differentiated
human hepatoma HepaRG cells using various experimental approaches, including
the comet assay to assess DNA damage and transcriptomic technologies. After a 24
h-exposure, PhIP and MeIQx, singly and in mixture, exerted differential effects on
apoptosis, oxidative stress, DNA and cytochrome P450 (CYP) activities. Only
PhIP caused DNA damage. PhIP was also a stronger inducer of CYP1A1 and
CYP1B1 expression and activity than MeIQx. In contrast, only MeIQx significantly
induced CYP1A2 activity. The combination of PhIP with MeIQx caused an
oxidative stress and showed synergistic effects on apoptosis but inhibitory effects on
DNA damage. In addition, we analyzed gene expression profiles after either a single
24 h- or a repeat 28 day-exposure to PhIP or MeIQx. The most responsive genes to
both HAA were downstream targets of the arylhydrocarbon receptor: CYP1A1 and
CYP1A2 after both time points and CYP1B1 and ALDH3A1 after 28 days. Several
other genes, mainly related to cell growth, apoptosis and cancer, exhibited to a
lesser extent both time-dependent and compound-specific expression changes. Our
findings highlight the need to investigate both acute and chronic effects of contaminants,
singly and in mixture, as well as their interactions when assessing risk for
human health and demonstrate the unique properties of HepaRG cells as a metabolically
competent cell line for chemical toxicity testing. (Dumont et al. TAAP.
2010, 245:256 and Dumont et al. TAAP in press. Financial support: ANR, contract
06SEST17).
1580 THE MAXIMUM CUMULATIVE RATIO (MCR): A TOOL
FOR ASSESSING THE NEED FOR CUMULATIVE RISK
ASSESSMENTS.
X. Han and P. S. Price. Toxicology & Environmental Research & Consulting, The
Dow Chemical Company, Midland, MI.
Cumulative risk assessments are resource intensive. Maximum cumulative ratio
(MCR) is a tool that identifies chemicals where cumulative risk assessments are
most necessary. The MCR is the ratio of the total risk of a group of chemicals to the
largest single-chemical risk. Smaller values of MCR imply less need for risk assessments.
Two case studies of the approach are presented. Methods: Biomonitoring
data were taken from NHANES, for serum levels of 26 dioxins, furans, and PCBs
(D/F/Ps) and urine levels of 6 phytoestrogens and 3 estrogenic phenols (PEs). The
cumulative toxicity of the levels of the chemicals was evaluated using toxicity equivalency
factors (TEFs). WHO and Haws et al. TEFs were used for D/F/Ps and TEFs
for PEs were developed from published toxicity data. MCR values were determined
and plotted against individuals’ total body burden. Simulation models were used to
investigate the impact of uncertainty in correlations, uncertainty in the TEFs, and
adding more chemicals to the assessment. Results: MCR values for individuals were
found to range from 1-5 for D/F/Ps and 1-3.5 for PEs. Mean values for PEs (1.7)
were lower than D/F/Ps (3.3). Values decreased with higher body burdens for PEs
and for D/F/Ps when TEFs from Haws et al. were used but increased slightly when
the WHO TEFs were used for the D/F/Ps. Simulations show that increasing the
number of chemicals had a moderate effect and increasing inter-chemical correlations
increased values by ~1.5. Conclusions: In both cases the majority of toxicity
came from a small fraction of chemicals, the D/F/Ps were dominated by 4-5 of the
26 chemicals while the PEs were dominated by 1-2 of the 9 chemicals. The MCR
values decreased with total risk for PEs but not for D/F/Ps. These findings suggest
that there is more need for a cumulative risk assessment for D/F/Ps than for PEs
and demonstrate the ability of the MCR to differentiate between mixtures that do,
and do not, warrant cumulative risk assessments.
1581 EVALUATION OF THE POTENTIAL IMPACT OF
INHIBITION OF TRICHLOROETHYLENE
METABOLISM IN THE LIVER ON EXTRAHEPATIC
TOXICITY.
C. R. Eklund, M. V. Evans and J. Simmons. NHEERL, U.S. EPA, Research
Triangle Park, NC.
The interaction between trichloroethylene (TCE) and chloroform (CHCl3) is less
than additive, with co-exposure to TCE and CHCl3 resulting in less hepatic and
renal toxicity than observed with CHCl3 alone. Vapor uptake data demonstrate
that co-exposure to CHCl3 decreases the rate of disappearance of TCE from the
vapor uptake chamber. Physiologically-based pharmacokinetic (PBPK) modeling of
vapor uptake data for TCE alone, CHCl3 alone, and binary mixtures of TCE and
CHCl3 indicate that competitive inhibition best describes the effect of CHCl3 on
TCE metabolism. We have previously determined that the acute neurotoxic effects
of TCE are predicted on the basis of the momentary concentration of TCE in either
the blood or the brain. Therefore, the purpose of the present study was to understand
the impact of decreased TCE metabolism in the presence of CHCl3 on the
concentration of TCE in the blood. The vapor-uptake, five-compartment (blood,
liver, fat, slowly and rapidly perfused) PBPK model was used to estimate the concentration
of TCE in blood at the end of a 10 min, 1 hr or 8 hr exposure to initial
starting concentrations of either 500 ppm TCE alone, 500 ppm TCE in combination
with 500 ppm CHCl3, 1000 ppm TCE alone and 1000 ppm TCE in combination
with 1000 pm CHCl3. At both 500 and 1000 ppm TCE, concurrent exposure
to CHCl3 resulted in small increases in blood TCE concentrations at all three
SOT 2011 ANNUAL MEETING 339