David R. Geter

Dose-Response Modeling of Early Molecular and Cellular Key Events in the CAR-Mediated Hepatocarcinogenesis Pathway

Low-dose extrapolation and dose-related transitions are paramount in the ongoing debate regarding the quantification of cancer risks for nongenotoxic carcinogens. Phenobarbital (PB) is a prototypical nongenotoxic carcinogen that activates the constitutive androstane receptor (CAR) resulting in rodent liver tumors. In this study, male and female CD-1 mice administered dietary PB at 0, 0.15, 1.5, 15, 75, or 150 mg/kg-day for 2 or 7 days to characterize multiple apical and molecular endpoints below, at (∼75 mg/kg-day), and above the carcinogenic dose level of PB and examine these responses using benchmark dose modeling. Linear toxicokinetics were observed for all doses. Increased liver weight, hepatocellular hypertrophy, and mitotic figures were seen at 75 and 150 mg/kg-day. CAR activation, based on Cyp2b qPCR and pentoxyresorufin dealkylase activity, occurred at doses ≥ 1.5 mg/kg-day. The no-observable transcriptional effect level for global gene expression was 15 mg/kg-day. At 2 days, several xenobiotic metabolism and cell protective pathways were activated at lower doses and to a greater degree in females. However, hepatocellular proliferation, quantified by bromodeoxyuridine immunohistochemistry, was the most sensitive indicator of PB exposure with female mice more sensitive than males, contrary to sex-specific differences in sensitivity to hepatocarcinogenesis. Taken together, the identification of low-dose cellular and molecular transitions in the subtumorigenic dose range aids the understanding of early key events in CAR-mediated hepatocarcinogenesis.

Mode of action and human relevance analysis for nuclear receptor-mediated liver toxicity: A case study with phenobarbital as a model constitutive androstane receptor (CAR) activator

Abstract The constitutive androstane receptor (CAR) and pregnane X receptor (PXR) are important nuclear receptors involved in the regulation of cellular responses from exposure to many xenobiotics and various physiological processes. Phenobarbital (PB) is a non-genotoxic indirect CAR activator, which induces cytochrome P450 (CYP) and other xenobiotic metabolizing enzymes and is known to produce liver foci/tumors in mice and rats. From literature data, a mode of action (MOA) for PB-induced rodent liver tumor formation was developed. A MOA for PXR activators was not established owing to a lack of suitable data. The key events in the PB-induced liver tumor MOA comprise activation of CAR followed by altered gene expression specific to CAR activation, increased cell proliferation, formation of altered hepatic foci and ultimately the development of liver tumors. Associative events in the MOA include altered epigenetic changes, induction of hepatic CYP2B enzymes, liver hypertrophy and decreased apoptosis; with inhibition of gap junctional intercellular communication being an associative event or modulating factor. The MOA was evaluated using the modified Bradford Hill criteria for causality and other possible MOAs were excluded. While PB produces liver tumors in rodents, important species differences were identified including a lack of cell proliferation in cultured human hepatocytes. The MOA for PB-induced rodent liver tumor formation was considered to be qualitatively not plausible for humans. This conclusion is supported by data from a number of epidemiological studies conducted in human populations chronically exposed to PB in which there is no clear evidence for increased liver tumor risk.

Simultaneous quantitation of testosterone, estradiol, ethinyl estradiol, and 11-ketotestosterone in fathead minnow fish plasma by liquid chromatography/positive atmospheric pressure photoionization tandem mass spectrometry

In the present work, for the first time, a rapid and sensitive liquid chromatography/positive atmospheric pressure photoionization tandem mass spectrometry (LC/APPI-MS/MS) method has been developed and validated for the simultaneous quantitation of testosterone, estradiol, ethinyl estradiol, and 11-ketotestosterone in fathead minnow fish plasma using no more than 10 microL of plasma. Compounds present in plasma were directly derivatized with dansyl chloride and 25 microL of the derivatized mixture was injected into the LC/APPI-MS/MS system. The gradient chromatographic elution was achieved on an Agilent Zorbax SB-C18 analytical column (2.1 mm x 50 mm, 1.8 microm particle size) with mobile phases consisting of acetonitrile, water and acetic acid. The flow rate was 0.5 to 0.7 mL/min and the total run time was 11.5 min. The lower limits of quantitation for testosterone, estradiol, ethinyl estradiol, and 11-ketotestosterone and were 1, 1, 1, and 2.5 ng/mL, respectively. Intra-batch precision was less than 19.4% and inter-batch precision was less than 11.7% for all four analytes. Accuracy was within 83.5-115.4% of nominal concentrations. This method is used for quantitation of sex steroid levels in fathead minnow tested in endocrine disruptor screening experiments.

Quantitation of glutathione by liquid chromatography/positive electrospray ionization tandem mass spectrometry.

Glutathione (GSH) is a tripeptide composed of glutamate, cysteine, and glycine. It is present in practically all cells and has several important roles, such as preventing the oxidation of the sulfhydryl groups of proteins within a cell. Evidence for GSH deficiency or depletion has been found in a variety of diseases and toxicity-related studies, including diabetes and induction of oxidative stress to form reactive oxygen species which cause DNA, lipid, and protein oxidations. A simple, selective, and sensitive analytical method for measuring low levels of GSH in biological fluids would therefore be desirable to conduct GSH deficiency or depletion-related mechanistic toxicity studies. Here a method for both low- and high-level quantitation of GSH from cultured cells and rat liver tissues via liquid chromatography/positive electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS) has been developed. The lower limit of quantitation (LOQ) of the method was 5 ng/mL. The method is linear over a wide dynamic concentration range of 5.0 to 5000.0 ng/mL, with a correlation coefficient R2 > 0.99. The intra-day assay precision relative standard deviation (RSD) values for all quality control (QC) samples were < or =16.31%, with accuracy values ranging from 94.13 to 97.80%. The inter-day assay precision RSD values for all QC samples were < or =15.94%, with accuracy values ranging from 94.51 to 100.29%. With this method, low levels of GSH from diethyl maleate (DEM)-treated mouse lymphoma cells, and GSH in rat liver tissues, were quantified.

An integrated approach for prospectively investigating a mode-of-action for rodent liver effects

Registration of new plant protection products (e.g., herbicide, insecticide, or fungicide) requires comprehensive mammalian toxicity evaluation including carcinogenicity studies in two species. The outcome of the carcinogenicity testing has a significant bearing on the overall human health risk assessment of the substance and, consequently, approved uses for different crops across geographies. In order to understand the relevance of a specific tumor finding to human health, a systematic, transparent, and hypothesis-driven mode of action (MoA) investigation is, appropriately, an expectation by the regulatory agencies. Here, we describe a novel approach of prospectively generating the MoA data by implementing additional end points to the standard guideline toxicity studies with sulfoxaflor, a molecule in development. This proactive MoA approach results in a more robust integration of molecular with apical end points while minimizing animal use. Sulfoxaflor, a molecule targeting sap-feeding insects, induced liver effects (increased liver weight due to hepatocellular hypertrophy) in an initial palatability probe study for selecting doses for subsequent repeat-dose dietary studies. This finding triggered the inclusion of dose-response investigations of the potential key events for rodent liver carcinogenesis, concurrent with the hazard assessment studies. As predicted, sulfoxaflor induced liver tumors in rats and mice in the bioassays. The MoA data available by the time of the carcinogenicity finding supported the conclusion that the carcinogenic potential of sulfoxaflor was due to CAR/PXR nuclear receptor activation with subsequent hepatocellular proliferation. This MoA was not considered to be relevant to humans as sulfoxaflor is unlikely to induce hepatocellular proliferation in humans and therefore would not be a human liver carcinogen.

Simultaneous quantitation of testosterone and estradiol in human cell line (H295R) by liquid chromatography/positive atmospheric pressure photoionization tandem mass spectrometry

The possible interaction of environmental contaminants with the endocrine system has been an environmental concern since the early 1990s. To examine these interactions test guidelines have been introduced by regulatory agencies to screen for possible endocrine active compounds. One of these guidelines is the EPAs OPPTS 890.1550 [Steroidogenesis (Human Cell Line-H295R)]. This guideline requires the quantification of two major biomarkers (testosterone and estradiol) in various biological test systems. Traditional quantitation methodologies such as Radioimmunoassay (RIA) and Enzyme-linked Immunosorbent Assay (ELISA) have been used to quantify low levels of steroids. However, those methodologies have drawbacks such as the radioactive safety, antibody availability, separate assay for each biomarker, and lack of selectivity. In the current study, a rapid and sensitive liquid chromatography/positive atmospheric pressure photoionization tandem mass spectrometry method (LC/APPI-MS/MS) has been developed and validated for the simultaneous quantitation of testosterone and estradiol in the H295R cell line. Briefly, the media from cultured cells was extracted with dichloromethane (CH(2)Cl(2)) containing internal standards of both testosterone-d(3) and estradiol-(13)C(3); then, the extracted organic layer was concentrated down to dryness. The final residue was derivatized with dansyl chloride solution, and directly analyzed by LC/APPI-MS/MS. The calibration curves, with concentration ranging from 10 to 2500 pg/mL, were linear with coefficient >0.99. The lower limits of quantitation for both testosterone and estradiol were 10 pg/mL. This method was successfully validated to support requirements of the current EPA Steroidogenesis guideline. This type of method may also provide value for rapid and precise measurements of these two hormones in other in vitro or in vivo test systems.

Evaluation of the amphibian metamorphosis assay: Exposure to the goitrogen methimazole and the endogenous thyroid hormone L‐thyroxine

The U.S. Environmental Protection Agency (U.S. EPA) has included an amphibian metamorphosis assay (AMA) to detect thyroid active chemicals in Tier 1 testing of their endocrine screening program. To understand the variability, specificity, and reliability of the key endpoints of this assay, two exposure studies with Xenopus laevis tadpoles were conducted with two known thyroid-active compounds, namely, methimazole or L-thyroxine, for a total of 21 d. In addition, various increased-flow-rate treatments were included in the exposures to evaluate the effects of physical stress on metamorphic development. The endpoints examined in the exposures were wet weight, snout-vent length, hind-limb length, developmental stage, and thyroid and gonadal histopathology. As expected, the results indicated that both methimazole and L-thyroxine were thyroid active in the AMA, hind-limb length and thyroid histopathology being the most sensitive endpoints of thyroid activity. Tadpoles that were exposed to the various physical stressors in these experiments showed no signs of altered metamorphic development, and exposure to the thyroid-active compounds had no effect on the developing gonad of X. laevis. Taken together, these results support the use of the AMA as a Tier 1 endocrine screen for detection of potential thyroid pathway activity; however, the lack of a true negative response (no-effect) during the validation process prevents a full evaluation of this assays specificity at this time.

Investigations of Oxidative Stress, Antioxidant Response, and Protein Binding in Chlorpyrifos Exposed Rat Neuronal PC12 Cells

ABSTRACT Chlorpyrifos (CPF) is a widely used organophosphate insecticide. In addition to its known properties of cholinesterase inhibition, the production of reactive oxygen species (ROS) has been suggested as a possible toxic mechanism. To investigate CPF-generated ROS, rat neuronal PC12 cells were exposed to CPF concentrations of 0 to 5000 μg/mL in Krebs buffered media (KRH), KRH + 4% bovine serum albumin (BSA), and KRH + 25 μM of the antioxidant Trolox for 0 to 5 h. Paraquat served as a positive control for ROS. The fluorescent probe 2,7-dichlorodihydro-fluorescein and the MTS assay were used to measure ROS and cytotoxicity, respectively. Examinations into CPF-albumin binding were also conducted. CPF was not strongly cytotoxic to PC12 cells, causing only mild cytotoxicity at 5000 μg/ml. In KRH media, CPF-generated ROS was observed at 4 and 5 h at 500 and 1000 μg/mL, and at 1 to 5 h at 5000 μg/mL CPF. In KRH + 4% BSA, ROS was seen only at 5 h in 5000 μg/mL CPF. Trolox significantly reduced CPF- and paraquat-induced ROS. Calculated CPF-albumin binding at 1, 10, and 100 μg/mL CPF in 4% BSA was 96%, 75%, and 15%. These data show CPF at ≥500 μg/mL induced ROS in PC12 cells, but the addition of the antioxidant Trolox and 4% BSA dramatically reduced ROS levels.

Frequency and Spectrum of lacI Mutations in the Liver of Big Blue Mice Following the Administration of Genotoxic Carcinogens Singly and in Series

Transgenic mouse models offer a unique opportunity to study in vivo mutagenicity in any tissue of interest. In this study, the authors have determined the liver mutant frequency (MF) and mutational spectra (MS) of 12 week-old male Big Blue B6C3F1 transgenic mice exposed to the genotoxic carcinogens benzo[a]pyrene (B[a]P; 250 mg/kg/day), N-nitrosodimethylamine (NDMA; 7 mg/kg/day), or N-ethyl-1-nitrosourea (ENU; 50 mg/kg/day) singly (3 daily oral doses) or in series (B[a]P on day 1, NDMA on day 2, and ENU on day 3). All genotoxic agents, alone or in series, increased MF in the liver (three-to sixfold). MS analyses of liver DNA revealed a high percentage of G:C → A:T transitions in the control (88%) and the NDMA (64%) groups. In contrast, B[a]P, ENU, and the series treatment induced a high percentage (≥50%) of transversions. Significantly, 46% (19 out of 41) of the mutations in the series treatment group occurred at CpG dinucleotides, compared to less than 22% in the other treatment groups. The MS from the series exposure was most similar to B[a]P with a high percentage of transversion mutations occurring at guanine nucleotides (36%). These preliminary data suggest that genotoxic carcinogens, when exposed in series, produce a unique MS profile characterized not only by shifts in mutation class but also sequence context.

Genetic damage, but limited evidence of oxidative stress markers in diethyl maleate-induced glutathione depleted mouse lymphoma L5178Y (TK+/-) cell cultures

Depletion of glutathione (GSH) in cells exposed to certain xenobiotics has been proposed to result in oxidative stress, which could lead to damage of cellular macromolecules such as proteins, lipids, and DNA. Diethyl maleate (DEM) is known to conjugate with GSH and rapidly lower cellular GSH levels. The objective of this study was to investigate the influence of DEM-induced GSH depletion on various genotoxicity and gene expression end points in mouse lymphoma L5178Y (TK+/-) cell cultures. Cells were exposed to DEM for 4 h at concentrations of 0, 6.7, 13.5, 26.9, 53.8, 107.6, 215.3, and 430.6 µg/mL (0.039–2.5 mM). Genotoxicity was evaluated by examining the induction of in vitro micronuclei (20 h post-treatment) and DNA strand breaks as measured by comet (immediately following treatment), and correlating these observations to cellular GSH levels. In the current study, GSH was decreased more than 50% at the lowest test concentration (6.7 µg/mL) and more than 95% at ≥ 107.6 µg/mL. A significant increase in micronuclei and DNA strand breaks was observed at concentrations of ≥ 26.9 µg/mL. Gene expression of seven apoptosis and oxidative-stress related genes showed significant alterations in only three genes only at the highest test concentration. Quantifiable levels of 8-OH-dG (≥ 2 adducts per 1 × 108 NT) were not detected at any treatment concentration. These results demonstrate an association between DEM-induced genotoxicity and GSH depletion in mouse lymphoma L5178Y (TK+/-) cells, but not with other oxidative markers.