Analysis of EPA's endocrine screening battery and recommendations for further review.

EPA's Endocrine Disruptor Screening Program Tier 1 battery consists of eleven assays intended to identify the potential of a chemical to interact with the estrogen, androgen, thyroid, or steroidogenesis systems. We have collected control data from a subset of test order recipients from the first round of screening. The analysis undertaken herein demonstrates that the EPA should review all testing methods prior to issuing further test orders. Given the frequency with which certain performance criteria were violated, a primary focus of that review should consider adjustments to these standards to better reflect biological variability. A second focus should be to provide detailed, assay-specific direction on when results should be discarded; no clear guidance exists on the degree to which assays need to be re-run for failing to meet performance criteria. A third focus should be to identify permissible differences in study design and execution that have a large influence on endpoint variance. Experimental guidelines could then be re-defined such that endpoint variances are reduced and performance criteria are violated less frequently. It must be emphasized that because we were restricted to a subset (approximately half) of the control data, our analyses serve only as examples to underscore the importance of a detailed, rigorous, and comprehensive evaluation of the performance of the battery.

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.

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.

Relevance weighting of tier 1 endocrine screening endpoints by rank order.

Weight of evidence (WoE) approaches are recommended for interpreting various toxicological data, but few systematic and transparent procedures exist. A hypothesis-based WoE framework was recently published focusing on the U.S. EPA's Tier 1 Endocrine Screening Battery (ESB) as an example. The framework recommends weighting each experimental endpoint according to its relevance for deciding eight hypotheses addressed by the ESB. Here we present detailed rationale for weighting the ESB endpoints according to three rank ordered categories and an interpretive process for using the rankings to reach WoE determinations. Rank 1 was assigned to in vivo endpoints that characterize the fundamental physiological actions for androgen, estrogen, and thyroid activities. Rank 1 endpoints are specific and sensitive for the hypothesis, interpretable without ancillary data, and rarely confounded by artifacts or nonspecific activity. Rank 2 endpoints are specific and interpretable for the hypothesis but less informative than Rank 1, often due to oversensitivity, inclusion of narrowly context-dependent components of the hormonal system (e.g., in vitro endpoints), or confounding by nonspecific activity. Rank 3 endpoints are relevant for the hypothesis but only corroborative of Ranks 1 and 2 endpoints. Rank 3 includes many apical in vivo endpoints that can be affected by systemic toxicity and nonhormonal activity. Although these relevance weight rankings (WREL ) necessarily involve professional judgment, their a priori derivation enhances transparency and renders WoE determinations amenable to methodological scrutiny according to basic scientific premises, characteristics that cannot be assured by processes in which the rationale for decisions is provided post hoc.

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.

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 × 10(8) 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.

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 assay's specificity at this time.

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.

Oncogene expression profiles in K6/ODC mouse skin and papillomas following a chronic exposure to monomethylarsonous acid.

We have previously observed that a chronic drinking water exposure to monomethylarsonous acid [MMA(III)], a cellular metabolite of inorganic arsenic, increases tumor frequency in the skin of keratin VI/ornithine decarboxylase (K6/ODC) transgenic mice. To characterize gene expression profiles predictive of MMA(III) exposure and mode of action of carcinogenesis, skin and papilloma RNA was isolated from K6/ODC mice administered 0, 10, 50, and 100 ppm MMA(III) in their drinking water for 26 weeks. Following RNA processing, the resulting cRNA samples were hybridized to Affymetrix Mouse Genome 430A 2.0 GeneChips(R). Micoarray data were normalized using MAS 5.0 software, and statistically significant genes were determined using a regularized t-test. Significant changes in bZIP transcription factors, MAP kinase signaling, chromatin remodeling, and lipid metabolism gene transcripts were observed following MMA(III) exposure as determined using the Database for Annotation, Visualization and Integrated Discovery 2.1 (DAVID) (Dennis et al., Genome Biol 2003;4(5):P3). MMA(III) also caused dose-dependent changes in multiple Rho guanine nucleotide triphosphatase (GTPase) and cell cycle related genes as determined by linear regression analyses. Observed increases in transcript abundance of Fosl1, Myc, and Rac1 oncogenes in mouse skin support previous reports on the inducibility of these oncogenes in response to arsenic and support the relevance of these genomic changes in skin tumor induction in the K6/ODC mouse model.

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 mug/mL in Krebs buffered media (KRH), KRH + 4% bovine serum albumin (BSA), and KRH + 25 muM 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 mug/ml. In KRH media, CPF-generated ROS was observed at 4 and 5 h at 500 and 1000 mug/mL, and at 1 to 5 h at 5000 mug/mL CPF. In KRH + 4% BSA, ROS was seen only at 5 h in 5000 mug/mL CPF. Trolox significantly reduced CPF- and paraquat-induced ROS. Calculated CPF-albumin binding at 1, 10, and 100 mug/mL CPF in 4% BSA was 96%, 75%, and 15%. These data show CPF at >/=500 mug/mL induced ROS in PC12 cells, but the addition of the antioxidant Trolox and 4% BSA dramatically reduced ROS levels.

Molecular biomarkers of oxidative stress associated with bromate carcinogenicity.

Potassium bromate (KBrO3) is a chemical oxidizing agent found in drinking water as a disinfection byproduct of surface water ozonation. Chronic exposures to KBrO3 cause renal cell tumors in rats, hamsters and mice and thyroid and testicular mesothelial tumors in rats. Experimental evidence indicates that bromate mediates toxicological effects via the induction of oxidative stress. To investigate the contribution of oxidative stress in KBrO3-induced cancer, male F344 rats were administered KBrO3 in their drinking water at multiple concentrations for 2-100 weeks. Gene expression analyses were performed on kidney, thyroid and mesothelial cell RNA. Families of mRNA transcripts differentially expressed with respect to bromate treatment included multiple cancer, cell death, ion transport and oxidative stress genes. Multiple glutathione metabolism genes were up-regulated in kidney following carcinogenic (400 mg/L) but not non-carcinogenic (20 mg/L) bromate exposures. 8-Oxodeoxyguanosine glycosylase (Ogg1) mRNA was up-regulated in response to bromate treatment in kidney but not thyroid. A dramatic decrease in global gene expression changes was observed following 1mg/L compared to 20 mg/L bromate exposures. In a separate study oxygen-18 (18O) labeled KBrO3 was administered to male rats by oral gavage and tissues were analyzed for 18O deposition. Tissue enrichment of 18O was observed at 5 and 24 h post-KBr18O3 exposure with the highest enrichment occurring in the liver followed by the kidney, thyroid and testes. The kidney dose response observed was biphasic showing similar statistical increases in 18O deposition between 0.25 and 50 mg/L (equivalent dose) KBr18O3 followed by a much greater increase above 50 mg/L. These results suggest that carcinogenic doses of potassium bromate require attainment of a threshold at which oxidation of tissues occurs and that gene expression profiles may be predictive of these physiological changes in renal homeostasis.

Tribromomethane exposure and dietary folate deficiency in the formation of aberrant crypt foci in the colons of F344/N rats.

Folate and folic acid are forms of the B vitamin that are involved in the synthesis, repair, and functioning of DNA and are required for the production and maintenance of cells. Low levels of folate have been associated with several forms of cancer, including colon cancer. Aberrant crypt foci (ACF), identified as putative precursor lesions in the development of colon cancer, have been induced by the drinking water disinfection by-product, tribromomethane (TBM). To investigate whether ACF induced by TBM could be promoted by a diet devoid of dietary folate, male F344/N rats were exposed to 500 mg/l of TBM in drinking water and fed either a normal or no folate diet (NFD) for 26 weeks. At the conclusion of the study, colons were excised and examined for ACF. Rats exposed to TBM and fed a NFD, evident by significantly reduced serum folate concentrations and elevated serum homocysteine levels, had significant increases of ACF when compared to rats exposed to TBM and fed a normal diet. This study highlights the important role that diet, especially folate intake, represents in protecting the colon against TBM-induced ACF.

Identification of chemical modulators of the constitutive activated receptor (CAR) in a gene expression compendium.

The nuclear receptor family member constitutive activated receptor (CAR) is activated by structurally diverse drugs and environmentally-relevant chemicals leading to transcriptional regulation of genes involved in xenobiotic metabolism and transport. Chronic activation of CAR increases liver cancer incidence in rodents, whereas suppression of CAR can lead to steatosis and insulin insensitivity. Here, analytical methods were developed to screen for chemical treatments in a gene expression compendium that lead to alteration of CAR activity. A gene expression biomarker signature of 83 CAR-dependent genes was identified using microarray profiles from the livers of wild-type and CAR-null mice after exposure to three structurally-diverse CAR activators (CITCO, phenobarbital, TCPOBOP). A rank-based algorithm (Running Fisher's algorithm (p-value ≤ 10(-4))) was used to evaluate the similarity between the CAR biomarker signature and a test set of 28 and 32 comparisons positive or negative, respectively, for CAR activation; the test resulted in a balanced accuracy of 97%. The biomarker signature was used to identify chemicals that activate or suppress CAR in an annotated mouse liver/primary hepatocyte gene expression database of ~1850 comparisons. CAR was activated by 1) activators of the aryl hydrocarbon receptor (AhR) in wild-type but not AhR-null mice, 2) pregnane X receptor (PXR) activators in wild-type and to lesser extents in PXR-null mice, and 3) activators of PPARα in wild-type and PPARα-null mice. CAR was consistently activated by five conazole fungicides and four perfluorinated compounds. Comparison of effects in wild-type and CAR-null mice showed that the fungicide propiconazole increased liver weight and hepatocyte proliferation in a CAR-dependent manner, whereas the perfluorinated compound perfluorooctanoic acid (PFOA) increased these endpoints in a CAR-independent manner. A number of compounds suppressed CAR coincident with increases in markers of inflammation including acetaminophen, concanavalin A, lipopolysaccharide, and 300 nm silica particles. In conclusion, we have shown that a CAR biomarker signature coupled with a rank-based similarity method accurately predicts CAR activation. This analytical approach, when applied to a gene expression compendium, increased the universe of known chemicals that directly or indirectly activate CAR, highlighting the promiscuous nature of CAR activation and signaling through activation of other xenobiotic-activated receptors.

The induction of aberrant crypt foci (ACF) in the colons of rats by trihalomethanes administered in the drinking water.

Bromodichloromethane (BDCM) and bromoform (TBM) have been demonstrated to be colon carcinogens in male and female F344/N rats following administration by corn oil gavage. Our chronic bioassay of BDCM administered in the drinking water failed to demonstrate colon cancer in male F344/N rats. In the present study we addressed the capability of trihalomethanes (THMs) administered in drinking water to induce aberrant crypt foci (ACF), early putative preneoplastic lesions, in the colons of male F344/N rats and B6C3F(1) mice. BDCM was tested in the A/J mouse strain. Rats and B6C3F(1) mice were exposed to isomolar concentrations of the THMs [0.5 g/l chloroform (TCM), 0.7 g/l BDCM, 0.9 g/l dibromochloromethane (DBCM), or 1.1 g/l (TBM)] for 13 weeks. A/J mice were exposed to 0.5 g/l BDCM in the drinking water for 13 and 30 weeks. Deionized water and 0.25% Alkamuls EL-620 were the negative and vehicle controls. ACF incidence (percent) and number (ACF/colon) for the rat were: combined controls, 0; AOM, 100%, 27.17+/-6.28 (P<0.01); TCM, 16.7%, 0.17+/-0.17; BDCM, 83.3%, 1.50+/-0.56 (P<0.01); DBCM, 50%, 1.17+/-0.65 (P<0.01); TBM, 66.7%, 1.17+/-0.40 (P<0.01). THM-induced ACF primarily occurred in the rectal segment of the colon (92%). No ACF were observed in the colons of B6C3F(1) mice following 13 weeks of THM treatment or in the colons of A/J mice following 13 and 30 weeks of BDCM exposure. These studies demonstrate that brominated THMs administered in the drinking water significantly induced preneoplastic ACF in the colon of rats.

Analysis of in vivo and in vitro DNA strand breaks from trihalomethane exposure.

BACKGROUND: Epidemiological studies have linked the consumption of chlorinated surface waters to an increased risk of two major causes of human mortality, colorectal and bladder cancer. Trihalomethanes (THMs) are by-products formed when chlorine is used to disinfect drinking water. The purpose of this study was to examine the ability of the THMs, trichloromethane (TCM), bromodichloromethane (BDCM), dibromochloromethane (DBCM), and tribromomethane (TBM), to induce DNA strand breaks (SB) in (1) CCRF-CEM human lymphoblastic leukemia cells, (2) primary rat hepatocytes (PRH) exposed in vitro, and (3) rats exposed by gavage or drinking water. METHODS: DNA SB were measured by the DNA alkaline unwinding assay (DAUA). CCRF-CEM cells were exposed to individual THMs for 2 hr. Half of the cells were immediately analyzed for DNA SB and half were transferred into fresh culture medium and incubated for an additional 22 hr before testing for DNA SB. PRH were exposed to individual THMs for 4 hr then assayed for DNA SB. F344/N rats were exposed to individual THMs for 4 hr, 2 weeks, and to BDCM for 5 wk then tested for DNA SB. RESULTS: CCRF-CEM cells exposed to 5- or 10-mM brominated THMs for 2 hr produced DNA SB. The order of activity was TBM>DBCM>BDCM; TCM was inactive. Following a 22-hr recovery period, all groups had fewer SB except 10-mM DBCM and 1-mM TBM. CCRF-CEM cells were found to be positive for the GSTT1-1 gene, however no activity was detected. No DNA SB, unassociated with cytotoxicity, were observed in PRH or F344/N rats exposed to individual THMs. CONCLUSION: CCRF-CEM cells exposed to the brominated THMs at 5 or 10 mM for 2 hr showed a significant increase in DNA SB when compared to control cells. Additionally, CCRF-CEM cells exposed to DBCM and TBM appeared to have compromised DNA repair capacity as demonstrated by an increased amount of DNA SB at 22 hr following exposure. CCRF-CEM cells were found to be positive for the GSTT1-1 gene, however no activity was detected. No DNA SB were observed in PRH or F344/N rats exposed to individual THMs.

The effects of a high animal fat diet on the induction of aberrant crypt foci in the colons of male F344/N rats exposed to trihalomethanes in the drinking water.

Aberrant crypt foci (ACF), identified as putative precursor lesions in the development of colon cancer, were induced by brominated trihalomethanes (THMs) administered in the drinking water of rats. To investigate whether ACF induced by THMs could be promoted by a diet high in saturated animal fat, male F344/N rats were exposed to 0.5, 0.7, 0.9 or 1.1 g/l of trichloromethane (TCM), bromodichloromethane (BDCM), dibromochloromethane (DBCM) and tribromomethane (TBM), respectively, in drinking water. All animals were fed a Purina 5001 diet with half receiving the normal 4.5% fat feed and half receiving feed supplemented with 19% animal fat. After 26 weeks of treatment, colons were excised and examined for ACF. No difference in ACF was noted between animals fed a normal or high fat diet and exposed to TCM, BDCM or DBCM. However, animals exposed to TBM and fed a high fat diet showed a significant and near two-fold increase in ACF when compared to TBM exposed animals fed a normal diet.

Vehicle and mode of administration effects on the induction of aberrant crypt foci in the colons of male F344/N rats exposed to bromodichloromethane.

Bromodichloromethane (BDCM) and tribromomethane given by corn oil gavage were previously found to induce neoplasia in the large intestine of rats. Our chronic bioassay of BDCM administered in drinking water failed to produce colon neoplasia in male F344/N rats. We recently reported that BDCM induces aberrant crypt foci (ACF), putative precursor lesions in the development of colon cancer, when included in the drinking water of male rats. To investigate whether ACF induced by BDCM could be promoted by corn oil (CO), male F344/N rats were exposed to 0.7 g BDCM/L in drinking water or 50 mg BDCM/kg body weight by oral gavage in CO. Animals exposed to drinking water, CO, or 15 mg/kg azoxymethane (AOM) (ip) constituted the negative, vehicle, and positive controls. After 26 wk, colons were examined for ACF. A significant decrease in water consumption was observed in both the positive controls and BDCM-treated animals; however, no difference was noted in final body weight. The administration of CO to AOM-exposed animals produced a significant increase in total ACF when compared to AOM alone. BDCM produced a significant increase in ACF when compared to control, but no difference was noticed between BDCM exposure by oral CO gavage and control. Additionally, no difference was noted between BDCM exposure by drinking water and by oral CO gavage. This study demonstrates that the formation of ACF is independent of the route of BDCM exposure (drinking water vs. oral corn oil gavage), with both routes producing similar ACF values of 1.33 +/- 0.49 and 1.5 +/- 0.51 ACF/colon.

MX [3-chloro-4-(dichloromethyl)-5-hydroxy-2[5H]-furanone], a drinking-water carcinogen, does not induce mutations in the liver of cII transgenic medaka (Oryzias latipes).

Mutagenicity assays with Salmonella have shown that 3-chloro-4-(dichloromethyl)-5-hydroxy-2[5H]-furanone (MX), a drinking-water disinfection by-product, is a potent mutagen, accounting for about one-third of the mutagenic potency/potential of chlorinated drinking water. The ability of MX to induce mutations was investigated in the liver of medaka (Oryzias latipes), a small fish model, utilizing the cII transgenic medaka strain that allows detection of in vivo mutations. Methylazoxymethanol acetate (MAMAc), a carcinogen in medaka, served as a positive control. Fish were exposed to MX at 0, 1, 10, or 30 mg/L for 96 h, whereas the MAMAc exposures were for 2 h at 0, 0.1, 1, or 10 mg/L. Both exposures were conducted under static water conditions and with fasted medaka. Following exposure, fish were returned to regular culture conditions to allow mutation expression for 15 or 40 d for MX or for 15 or 32 d for MAMAc. Mutations were not induced in medaka exposed to MX for 96 h. However, a concentration- and time-dependent increase in mutations was observed from the livers of fish exposed to 1 and 10 mg/L MAMAc. In conclusion, mutation induction was not observed in the livers of cII medaka exposed to MX for 96 h; however, studies are planned to examine mutation induction in the gills and skin to explore the possibility that MX-induced DNA damage occurs primarily in the tissues of initial contact.

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.

Measurement of deoxyinosine adduct: Can it be a reliable tool to assess oxidative or nitrosative DNA damage?

Adenosine deaminases (ADA) are key enzymes that deaminate adenosine (A) or deoxyadenosine (dA) and produce inosine or deoxyinosine (dI), respectively. While ADA only deaminates free dA, reactive nitrogen species (RNS) or reactive oxygen species (ROS) deaminate adenine base on the DNA and leave dI, which is a pre-mutagenic lesion. Therefore, dI adduct in the genomic DNA has been considered a biomarker of DNA damage caused by RNS or by ROS. In the presented study, genomic DNA was isolated from frozen calf thymus in low or room temperature, with or without an addition of antioxidant. The number of dI in the DNA was measured using liquid chromatography-tandem mass spectrometry. While low temperature (LT) work-up with an addition of antioxidant in reagents helped to prevent artifactual formation of oxidative DNA lesions in the calf thymus DNA (CTD), it also significantly inhibited activities of proteinase, which in turn resulted in significant ADA contamination in the final DNA samples. ADA remained in LT-CTD completely deaminated most dA when the DNA was subjected to enzymatic hydrolysis to single nucleosides. The ADA contamination in the DNA was significantly reduced when DNA was isolated from pre-isolated nuclear fraction rather than from entire tissue homogenates. However, enzymes used for DNA hydrolysis were confirmed to contain significant amounts of ADA. Therefore, these enzymes would increase deamination of dA during DNA hydrolysis. Artifactual dI production by contaminated ADA was dramatically reduced by an addition of EHNA (erythro-9-(2-hydroxy-3-nonyl)adenine), which is a potent inhibitor of ADA. However, time- and temperature-dependent dI production from dA in phosphate buffer solution was observed. More importantly, TEMPO, an antioxidant commonly used to prevent DNA oxidation, was found to deaminate dA independent to ADA. Overall, these findings indicate that assay methods measuring dI or other dA DNA adducts in genomic DNA should be carefully validated to minimize artificial errors caused by dA deamination. Recommendations to overcome those technical challenges were discussed in this presentation.

Early alterations in protein and gene expression in rat kidney following bromate exposure.

Bromate, a common disinfectant byproduct of drinking water ozonation, has been linked to human and animal renal toxicity, including renal cell carcinomas in multiple animal species. Here, we evaluate changes in protein and gene expression through two-dimensional difference gel electrophoresis (2D-DIGE) and Affymetrix arrays to identify potential modes of action involved in potassium bromate carcinogenicity. Male rats were exposed to potassium bromate in drinking water at concentrations of 0, 1, 20 and 400 ppm for two weeks. Differential expression of glycolytic proteins including enolase 1 (Eno1), triosephosphate isomerase 1 (Tpi1) and glyceraldehyde-3-phosphate dehydrogenase (Gapdh) suggests that bromate toxicity is associated with changes in energy consumption and utilization in renal cells involving up-regulation of glycolytic processes that may be the result of altered mitochondrial function. Several alterations in glycolysis and mitochondrial gene transcripts were also observed to be consistent with this mode of action. These studies provide insight into early events in renal cell physiology altered by bromate exposure.