An in vitro developmental neurotoxicity screening assay for retinoic acid-induced neuronal differentiation using the human NT2/D1 cell line.

Traditional approaches (e.g., neurobehavior, neuropathology) can detect alterations in apical endpoints indicative of developmental neurotoxicity (DNT). However, there is an increasing desire to understand mode-of-action (MOA) for DNT effects; thus, this short communication describes initial work on a neuronal differentiation assay. Basically, our laboratory used the human NT2/D1 cell line to develop an assay to evaluate toxicants for effects on all-trans retinoic acid (RA)-induced neuronal differentiation. Based on literature reports, we selected a neuronal protein, neuronal class III ß-tubulin (ß3-tubulin), as a marker of differentiation. For this assay, cultured RA-treated NT2 cells were trypsinized to individual cells, methanol fixed, and labeled with a ß3-tubulin specific monoclonal antibody (TUJ1). Characterization studies using 100,000 cells/sample showed that NT2 cells had appreciable expression of ß3-tubulin starting around day 7 of the differentiation process with a peak expression noted around day 12. Methylmercury, 22(R)-hydroxycholesterol, N-(4-hydroxyphenol)retinamide (4HPR), and 9-cis retinoic acid were selected as initial test compounds. Of these, only 9-cis RA, which is known to affect the RA pathway, was positive for specific impacts on differentiation. These results demonstrate the feasibility of using a flow cytometry method targeting specific cellular biomarkers for evaluating effects on neuronal differentiation. Additional assays are needed to detect compounds targeting other (non-RA) neuronal differentiation pathways. Ultimately, a battery of in vitro assays would be needed to evaluate the potential MOAs involved in altered neuronal differentiation.

Characterization of nuclear receptor-mediated murine hepatocarcinogenesis of the herbicide pronamide and its human relevance.

The key events responsible for mouse liver tumors induced by a pesticide (viz., pronamide) were investigated in a series of studies employing molecular, biochemical, cellular, and apical endpoints. Based on these studies, it was demonstrated that the liver tumors were mediated by a mode of action (MoA) involving nuclear receptors (NRs) through the following key events: (1) CAR and PPAR-α receptor activation, (2) increased hepatocellular proliferation, eventually leading to (3) hepatocellular tumors. Specifically, gene expression analysis indicated robust, simultaneous coactivation of the CAR and PPAR-α NRs, as indicated by the induction of hepatic Cyp2b10 and Cyp4a10 transcripts, in response to dietary administration of pronamide to mice. The presence of hepatocellular hypertrophy and peroxisome proliferation was indicative of the activation of these two NRs at carcinogenic dose levels. Demonstrated induction of Cyp2b10 gene and protein, however, was not accompanied by enhancement of the corresponding enzyme activity (7-pentoxyresorufin-O-dealkylase (PROD)), suggesting that pronamide administration resulted in mechanism-based (suicide) inhibition of the enzyme in vivo. This was confirmed with an in vitro assay for suicide inhibition, where pronamide and/or its metabolites irreversibly inhibited Cyp2b10-mediated PROD activity. Analysis of hepatocellular proliferation via BrdU incorporation indicated a clear dose- and duration-related induction of S-phase DNA synthesis only in animals treated at and above the carcinogenic dose level. The available MoA data were evaluated for weight-of-evidence based upon the Bradford Hill criteria, followed by a human relevance framework. The conclusion from this evaluation is that pronamide-induced mouse liver tumors occur via an NR-mediated MoA involving CAR and PPAR-α activation and this MoA is not relevant to humans based on qualitative/quantitative differences between mice and humans.

Evaluation of potential endocrine activity of 2,4-dichlorophenoxyacetic acid using in vitro assays.

The herbicide 2,4-dichlorophenoxyacetic acid (2,4-D) was evaluated in five in vitro screening assays to assess the potential for interaction with the androgen, estrogen and steroidogenesis pathways in the endocrine system. The assays were conducted to meet the requirements of the in vitro component of Tier 1 of the United States Environmental Protection Agency's Endocrine Disruptor Screening Program (EDSP), and included assays for estrogen receptor (ER) binding (rat uterine cytosol ER binding assay), ER-mediated transcriptional activation (HeLa-9903-ERα transactivation assay), androgen receptor (AR) binding (rat prostate cytosol AR binding assay), aromatase enzymatic activity inhibition (recombinant human CYP19 aromatase inhibition assay), and interference with steroidogenesis (H295R steroidogenesis assay). Results from these five assays demonstrated that 2,4-D does not have the potential to interact in vitro with the estrogen, androgen, or steroidogenesis pathways. These in vitro data are consistent with a corresponding lack of endocrine effects observed in apical in vivo animal studies, and thus provide important supporting data valuable in a comprehensive weight of evidence evaluation indicating a low potential of 2,4-D to interact with the endocrine system.

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.

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.

Analysis of the interaction of phytoestrogens and synthetic chemicals: an in vitro/in vivo comparison.

In the evaluation of chemical mixture toxicity, it is desirable to develop an evaluation paradigm which incorporates some critical attributes of real world exposures, particularly low dose levels, larger numbers of chemicals, and chemicals from synthetic and natural sources. This study evaluated the impact of low level exposure to a mixture of six synthetic chemicals (SC) under conditions of co-exposure to various levels of plant-derived phytoestrogen (PE) compounds. Estrogenic activity was evaluated using an in vitro human estrogen receptor (ER) transcriptional activation assay and an in vivo immature rat uterotrophic assay. Initially, dose-response curves were characterized for each of the six SCs (methoxyclor, o,p-DDT, octylphenol, bisphenol A, beta-hexachlorocyclohexane, 2,3-bis(4-hydroxyphenyl)-propionitrile) in each of the assays. The six SCs were then combined at equipotent ratios and tested at 5-6 dose levels spanning from very low, sub-threshold levels, to a dose in which every chemical in the mixture was at its individual estrogenic response threshold. The SC mixtures also were tested in the absence or presence of 5-6 different levels of PEs, for a total of 36 (in vitro) or 25 (in vivo) treatment groups. Both in vitro and in vivo, low concentrations of the SC mixture failed to increase estrogenic responses relative to those induced by PEs alone. However, significant increases in response occurred when each chemical in the SC mixture was near or above its individual response threshold. In vitro, interactions between high-doses of SCs and PEs were greater than additive, whereas mixtures of SCs in the absence of PEs interacted in a less than additive fashion. In vivo, the SC and PE mixture responses were consistent with additivity. These data illustrate a novel approach for incorporating key attributes of real world exposures in chemical mixture toxicity assessments, and suggest that chemical mixture toxicity is likely to be of concern only when the mixture components are near or above their individual response thresholds. However, these data suggest that extrapolation from in vitro assays to in vivo mixture effects should be approached with caution.

Gene expression dose-response of liver with a genotoxic and nongenotoxic carcinogen.

Tumorigenic mechanisms due to chemical exposure are broadly classified as either genotoxic or nongenotoxic. Genotoxic mechanisms are generally well defined; however nongenotoxic modes of tumorgenesis are less straightforward. This study was undertaken to help elucidate dose-response changes in gene expression (transcriptome) in the liver of rats in response to administration of known genotoxic or nongenotoxic liver carcinogens. Male Big Blue Fischer 344 rats were treated for 28-days with 0, 0.1, 0.3, 1.0, or 3.0 mg/kg/day of the genotoxin 2-acetylaminofluorene (AAF) or 0, 10, 30, 60, or 100 mg/kg/day of the nongenotoxin phenobarbital (PB). Transcriptome analysis was performed using the relatively focused Clontech Rat Toxicology II microarray (465 genes) and hybridized with 32P-labeled cDNA target. The analysis indicated that after 28 days of treatment, AAF altered the expression of 14 genes (9 up- and 5 down-regulated) and PB altered the expression of 18 genes (10 up- and 8 down-regulated). Of the limited genes whose expression was altered by AAF and PB, four were altered in common, two up-regulated, and two down-regulated. Several of the genes that show modulation of transcriptional activity following AAF and PB treatment display an atypical dose-response relationship such that the expression at the higher doses tended to be similar to that of control. This high-dose effect could potentially be caused by adaptation, toxicity, or tissue remodeling. These results suggest that the transcriptional response of the cells to higher doses of a toxic agent is likely to be different from that of a low-dose exposure.

A comparison of in vitro and in vivo EDSTAC test battery results for detecting antiandrogenic activity.

The androgen receptor (AR) transactivation, binding, and Hershberger assays are being developed for large-scale screening of chemicals for endocrine activity. The goal of this study was to evaluate the correlation between in vitro and in vivo antiandrogenicity assays using a variety of compounds (p,p'-DDE, flutamide (FLUT), spironolactone, procymidone, RU486, methoxychlor (MXC), benzo(a)pyrene (BAP), and selected metabolites). For the AR transactivation assay, AR(+) LNCaP prostate carcinoma cells were transfected with an inducible luciferase reporter construct (pGudLuc7ARE) and exposed for 24 h to test materials (< or = 10 microM) in the presence and absence of 1 nM of the AR agonist R-1881. Each of these materials, including the hydroxlated metabolites of BAP and MXC, produced significant antiandrogenic activity in vitro as evidenced by their inhibition of the response to R-1881. Similarly, in vitro AR binding experiments using the recombinant ligand-binding domain (LBD) of the human AR and fluorescence polarization (FP) methodology yielded IC50s comparable to that of testosterone for RU486 and 9-OH-BAP. Other parent compounds and metabolites exhibited lesser binding affinity. In vivo antiandrogenic activity was evaluated with the Hershberger assay, wherein castrated male CD rats were dosed by gavage for 10 days with (mg/kg per day): MXC (10, 50, 100, and 200), BAP (1, 10, 50, and 100), RU486 (1, 5, 10, and 25), and FLUT (10) in the presence of 0.4 mg/kg per day (sc) of testosterone propionate (TP). Neither BAP nor MXC produced significant decreases in accessory sex tissue (AST) weights relative to TP control. However, 200 MXC resulted in a significant decrease in body weight and 100 BAP significantly increased absolute and relative liver weights. RU486 (25) produced significant decreases in ventral prostate, seminal vesicle, and Cowper's gland weights without affecting body weight. FLUT (10) decreased all AST weights measured. The antiandrogenic activities of the remaining materials (p,p'-DDE, spironolactone, and procymidone) have been demonstrated in previous Hershberger assays. These data indicate the importance of including in vivo results in assessing the endocrine activity of test materials and further stress the importance of a weight of evidence approach in assessing endocrine activity of test materials.

Profiles of gene expression changes in L5178Y mouse lymphoma cells treated with methyl methanesulfonate and sodium chloride.

Treatment of cells with genotoxic chemicals is expected to set into motion a series of events including gene expression changes to cope with the damage. We have investigated gene expression changes in L5178Y TK(+/-) mouse lymphoma cells in culture following treatment with methyl methanesulfonate (MMS), a direct acting genotoxin, and sodium chloride (NaCl), which induces mutations in these cells through indirect mechanisms at high concentrations. The mouse lymphoma cells were treated for 4 or 24 h and the cells were harvested for RNA isolation at the end of the treatment. Analysis of the transcriptome was performed using Clontech Mouse 1.2K cDNA microarrays (1185 genes) and hybridized using 32P-labeled cDNA. The microwell methodology was used to quantify the mutagenic response. Of the genes examined, MMS altered the expression (1.5-fold or more) of only five (four at 4 h and one after 24 h treatment). NaCl altered two genes after 4 h treatment, but after 24 h it altered 19 genes (13 down- and six up-regulated). Both compounds altered the expression of several genes associated with apoptosis and NaCl altered genes involved in DNA damage/response and GTP-related proteins. This, along with other data, indicates that the widely used L5178Y TK(+/-) mouse lymphoma cells in culture are relatively recalcitrant in terms of modulating gene expression to deal with genotoxic insult.

Identification of transcriptome profiles for the DNA-damaging agents bleomycin and hydrogen peroxide in L5178Y mouse lymphoma cells.

It is believed that some aspects of genotoxicity are associated with changes in the transcription levels of certain genes, especially those involved in DNA repair and cell cycle control. Additionally, it is hypothesized that chemicals sharing a common mode of genotoxicity should exhibit similar changes in gene expression. We have evaluated these hypotheses by analyzing transcriptome profiles of mouse lymphoma L5178Y/TK(+/-) cells treated with bleomycin and hydrogen peroxide, two mutagens that produce genotoxicity by generating reactive free radicals. The cells were treated for 4 hr and RNA was isolated at the end of the treatment and after a 20 hr recovery. Transcriptome analyses were performed using the Clontech Mouse 1.2K cDNA microarray (1,185 genes) and hybridization with a (32)[P]-labeled probe. Of the genes examined, each mutagen altered the expression (1.5-fold or greater) of only two genes after the 4 hr treatment. In cells allowed to recover for 20 hr after treatment, bleomycin and hydrogen peroxide altered the expression of 8 and 5 genes, respectively. Many of the altered genes have some association with apoptosis. Of these genes, three (the genes encoding granzyme A, integrin beta 7, and 45 kDa calcium-binding protein precursor) were in common between chemical treatments. The expression of DNA repair and cell cycle controlling genes present on the array was not affected by the treatments. These results show that bleomycin and hydrogen peroxide both have unique and commonly regulated genes that have the potential to serve as biomarkers of exposure to agents causing DNA damage by free radical mechanisms.