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On December 11, 2019, California's Developmental and Reproductive Toxicant Identification Committee (DARTIC) met to consider the addition of cannabis smoke and ΔWhile the HID included both human and animal data, this set of three reviews will highlight the animal-derived data pertaining to somatic development (Part I), neurodevelopmental effects (Part II), and proposed neurodevelopmental mechanisms of action (Part III).Endogenous cannabinoids (eCBs) and their receptors serve many critical functions in normal development. ΔIn reaching its decision to list cannabis and Δ

This review summarizes the most common potential pathways of neurodevelopmental toxicity due to perinatal exposure to Δ

Many nitrosamines are potent carcinogens, with more than 30 listed under California’s Proposition 65. Recently, nitrosamine contamination of commonly used drugs for treatment of hypertension, heartburn, and type 2 diabetes has prompted numerous Food and Drug Administration (FDA) recalls in the US. These contaminants include the carcinogens NDMA (N-nitrosodimethylamine) and NDEA (N-nitrosodiethylamine) and the animal tumorigen NMBA (N-nitroso-N-methyl-4-aminobutyric acid). NMBA and NDEA are metabolically and/or structurally related to NDMA, an N-nitrosomethyl-n-alkylamine (NMA), and 12 other carcinogenic NMAs. These nitrosamines exhibit common genotoxic and tumorigenic activities, with shared target tumor sites amongst chemicals and within a given laboratory animal species. We use the drug valsartan as a case study to estimate the additional cancer risks associated with NDMA and NDEA contamination, based on nitrosamine levels reported by the US FDA, cancer potencies developed by California’s Proposition 65 program and the US Environmental Protection Agency (EPA), and specific exposure scenarios. These estimates suggest that nitrosamine contamination in drugs that are used long-term can increase cancer risks and pose a serious concern to public health.

We developed an integrated, modular approach to predicting chemical toxicity relying on in vitro assay data, linkage of molecular targets to disease categories, and software for ranking chemical activity and examining structural features (chemotypes). We evaluate our approach in a proof-of-concept exercise to identify and prioritize chemicals of potential carcinogenicity concern. We identified 137 cancer pathway-related assays from a subset of U.S. EPA's ToxCast platforms. We mapped these assays to key characteristics of carcinogens and found they collectively assess 5 of 10 characteristics. We ranked all 1061 chemicals screened in Phases I and II of ToxCast by their activity in the selected cancer pathway-related assays using Toxicological Prioritization Index software. More chemicals used as biologically active agents (eg, pharmaceuticals) ranked in the upper 50% versus lower 50%. Twenty-three chemotypes are enriched in the top 5% (n = 54) of chemicals; these features may be important for their activity in cancer pathway-related assays. The biological coverage of the ToxCast assays related to cancer pathways is limited and short-term assays may not capture the biology of some key characteristics. Metabolism is also minimal in the assays. The ability of our approach to identify chemicals with cancer hazard is limited with the current input data, but we expect that our approach can be applied with future iterations of ToxCast and other data for improved chemical prioritization and characterization. The novel approach and proof-of-concept exercise described here for ranking chemicals for potential carcinogenicity concern is modular, adaptable, and amenable to evolving data streams.

Research on disease causation often attempts to isolate the effects of individual factors, including individual genes or environmental factors. This reductionist approach has generated many discoveries, but misses important interactive and cumulative effects that may help explain the broad range of variability in disease occurrence observed across studies and individuals. A disease rarely results from a single factor, and instead results from a broader combination of factors, characterized here as intrinsic (I) and extrinsic (E) factors. Intrinsic vulnerability or resilience emanates from a variety of both fixed and shifting biological factors including genetic traits, while extrinsic factors comprise all biologically-relevant external stressors encountered across the lifespan. The I×E concept incorporates the multi-factorial and dynamic nature of health and disease and provides a unified, conceptual basis for integrating results from multiple areas of research, including genomics, G×E, developmental origins of health and disease, and the exposome. We describe the utility of the I×E concept to better understand and characterize the cumulative impact of multiple extrinsic and intrinsic factors on individual and population health. New research methods increasingly facilitate the measurement of multifactorial and interactive effects in epidemiological and toxicological studies. Tiered or indicator-based approaches can guide the selection of potentially relevant I and E factors for study and quantification, and exposomics methods may eventually produce results that can be used to generate a response function over the life course. Quantitative data on I×E interactive effects should generate a better understanding of the variability in human response to environmental factors. The proposed I×E concept highlights the role for broader study design in order to identify extrinsic and intrinsic factors amenable to interventions at the individual and population levels in order to enhance resilience, reduce vulnerability and improve health.

Coumarin is a naturally occurring sweet-smelling benzopyrone that may be extracted from plants or synthesized for commercial uses. Its uses include as a flavoring agent, fragrance enhancer, and odor-masking additive. We reviewed and evaluated the scientific evidence on the carcinogenicity of coumarin, integrating information from carcinogenicity studies in animals with mechanistic and other relevant data, including data from toxicogenomic, genotoxicity, and metabolism studies, and studies of human variability of a key enzyme, CYP2A6. Increases in tumors were observed in multiple studies in rats and mice in multiple tissues. Our functional pathway analysis identified several common cancer-related biological processes/pathways affected by coumarin in rat liver following in vivo exposure and in human primary hepatocytes exposed in vitro. When coumarin 7-hydroxylation by CYP2A6 is compromised, this can lead to a shift in metabolism to the 3,4-epoxidation pathway and increased generation of electrophilic metabolites. Mechanistic data align with 3 key characteristics of carcinogens, namely formation of electrophilic metabolites, genotoxicity, and induction of oxidative stress. Considerations of metabolism, human variability in CYP2A6 activity, and coumarin hepatotoxicity in susceptible individuals provide additional support for carcinogenicity concern. Our analysis illustrates the importance of integrating information on human variability in the cancer hazard identification process.

US EPA's Toxicity Forecaster (ToxCastTM) is a tool with potential use in evaluating safer consumer products, conducting chemical alternatives analyses, prioritizing chemicals for exposure monitoring, and ultimately performing screening-level risk assessments. As a case study exploring a potential use of ToxCast, we evaluated ToxCast results for ortho-phthalates focused on the well-established toxicological endpoints of some members of this class. We compared molecular perturbations measured in ToxCast assays with the known apical toxicity endpoints of o-phthalates reported in the open literature to broadly reflect on the predictive capability of the high-throughput screening (HTS) assays. We grouped the ToxCast assays into defined sets to examine o-phthalate activity and potency. This study revealed several links between key molecular events assayed in vitro and chemical-specific hazard traits. In general, parent o-phthalates are more active than their monoester metabolites. The medium-chain length o-phthalate group is also more active than other o-phthalate groups, as supported by Toxicological Priority Index ranking and statistical methods. Some HTS assay results correlated with in vivo findings, but others did not. For example, there was a notable lack of assay activity to explain the known male reproductive toxicity of these compounds. Ultimately, HTS data resources such as ToxCast may inform us of sensitive upstream toxicity endpoints and may assist in the rapid identification of environmental chemical hazards for screening and prioritization. However, this case study shows that the absence of positive results in ToxCast in vitro assays cannot be interpreted as absence of related in vivo toxicity, and limited biological coverage by the assays remains a concern.

Our objective was to test the hypothesis that self-assembling peptide hydrogel scaffolds provide cues that enhance the chondrogenic differentiation of bone marrow stromal cells (BMSCs). BMSCs were encapsulated within two unique peptide hydrogel sequences, and chondrogenesis was compared with that in agarose hydrogels. BMSCs in all three hydrogels underwent transforming growth factor-beta1-mediated chondrogenesis as demonstrated by comparable gene expression and biosynthesis of extracellular matrix molecules. Expression of an osteogenic marker was unchanged, and an adipogenic marker was suppressed by transforming growth factor-beta1 in all hydrogels. Cell proliferation occurred only in the peptide hydrogels, not in agarose, resulting in higher glycosaminoglycan content and more spatially uniform proteoglycan and collagen type II deposition. The G1-positive aggrecan produced in peptide hydrogels was predominantly the full-length species, whereas that in agarose was predominantly the aggrecanase product G1-NITEGE. Unique cell morphologies were observed for BMSCs in each peptide hydrogel sequence, with extensive cell-cell contact present for both, whereas BMSCs in agarose remained rounded over 21 days in culture. Differences in cell morphology within the two peptide scaffolds may be related to sequence-specific cell adhesion. Taken together, this study demonstrates that self-assembling peptide hydrogels enhance chondrogenesis compared with agarose as shown by extracellular matrix production, DNA content, and aggrecan molecular structure.

Several links exist between the two mechanisms of neuronal degeneration (i.e., oxygen radical production and mitochondrial damage) proposed to have a role in Parkinson's disease. Indeed, mitochondria are critical targets for the toxic injury induced by oxygen radicals, and experimental evidence suggests that mitochondrial damage may cause an increased generation of oxygen radicals. A potentially important link between these two mechanisms of neurodegeneration is glutathione. Because of the scavenging activity of glutathione against accumulation of oxygen radicals, its decrease in the brains of parkinsonian patients has been interpreted as a sign of oxidative stress; however, this change may also result from or lead to mitochondrial damage. It is conceivable therefore that regardless of whether oxidative stress or mitochondrial damage represents the initial insult, these toxic mechanisms may both contribute to neuronal degeneration via changes in glutathione levels.

In this study we assessed the two forms of monoamine oxidase (MAO) in the caudate, putamen, and substantia nigra of young (4-year-old), intermediate-aged (11-year-old), and aged (20-year-old) squirrel monkeys and in the striata of young (2-month-old) and older (10-month-old) C57Bl/6 mice. MAO A and B activities were determined by measuring the rate of oxidation of the specific substrates phenethylamine and serotonin. In squirrel monkey, the vast majority of MAO activity was MAO B with activity of this isoform 10 times greater than of MAO A, while in mice the activity of the two forms was approximately equivalent. Although mice demonstrated nearly twofold selective increases in striatal MAO B between 2 and 10 months of age, neither MAO B nor A showed statistically significant changes with age in squirrel monkeys. These results document the marked differences between nonhuman primates and rodents with respect to the relative activities and the effects of age on MAO A and B, and indicate that increased MAO is not an inevitable feature of aging.

The present study examined neurochemical, morphological and functional markers of the nigrostriatal dopamine system in young, intermediate-aged and old squirrel monkeys. Striking reductions in motoric activity were observed with advancing age. significant age-related loss of dopamine occurred in the substantia nigra (70%) and the putamen (30%) but not in the caudate. There was a strong correlation between the reductions in motoric activity and the loss of putamen dopamine. However, nigrostriatal dopamine loss did not appear to be the consequence of age-related loss of dopaminergic nigral neurons since the number of tyrosine immunoreactive cells was not significantly different among the three age groups. These results suggest that the aging squirrel monkey demonstrates the age-related loss of nigrostriatal dopamine thought to occur in humans and identify this non-human primate as a useful model to further investigate the underlying mechanism(s) and functional consequences of age-related decline of the nigrostriatal dopamine system. In addition, the selective loss of dopamine in the putamen but not the caudate parallels the regional vulnerability observed in Parkinson's disease, an age-related neurodegenerative disorder, raising the possibility of a relationship between normal aging and the development of this disease. Finally, because the number of tyrosine hydroxylase (TH) positive cells remains constant with age, these results raise the possibility that therapeutic strategies aimed at increasing dopamine concentrations may benefit elderly individuals.

Antimycin A, KCN, and 1-methyl-4-phenylpyridinium ion (MPP+) all produced a marked depletion of cellular GSH levels in freshly isolated hepatocytes. This effect was consistently observed before the onset of cytotoxicity and seemed to be correlated with the loss of cellular ATP induced by these mitochondrial poisons. Concentrations of GSSG remained unchanged both intracellularly and extracellularly, indicating that oxidation was not involved in the events leading to GSH depletion. Approximately 40% of the decrease of intracellular GSH was accounted for by efflux of this tripeptide, assessed by increased formation of cysteinyl-glutathione when hepatocytes were incubated in the presence of 0.2 m m cystine. Therefore, an overall loss of glutathione was observed during incubations with all three inhibitors of mitochondrial function. Addition of 10 m m fructose to the incubation media substantially protected against GSH depletion caused by antimycin A, KCN, and MPP+. These results indicate that energy-dependent mechanisms are involved in the maintenance of intracellular GSH levels, and suggest that GSH depletion may be a general phenomenon associated with impairment of mitochondrial function.

MPTP (1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine) is converted by monoamine oxidase B to its putative toxic metabolite MPP+ (1-methyl-4-phenylpyridinium ion) via MPDP+ (1-methyl-4-phenyl-2,3-dihydropyridinium ion). Both the parent compound and these two major metabolites were toxic to isolated rat hepatocytes with MPDP+ being the most toxic and MPP+ the least effective. MPP+ produced a slight increase in lipid peroxidation above control levels in hepatocytes, while both MPTP and MPDP+ showed antioxidant effects. The latter two compounds also protected against chemically and nonchemically induced lipid peroxidation in rat liver microsomes. MPDP+ was effective at much lower concentrations than MPTP. MPDP+ was also markedly more efficient when NADPH was used to induce microsomal lipid peroxidation. Lipid peroxidation as a consequence of oxygen radical generation is therefore unlikely to be involved in MPTP toxicity in vitro and the rationale of using chain-breaking antioxidants as protective agents in vivo needs a more careful evaluation.

1-methyl-4-phenylpyridine (MPP+) is the putative toxic metabolite of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and is structurally similar to the herbicide paraquat (PQ++). We have therefore compared the effects of MPP+ and PQ++ on a well characterized experimental model, namely isolated rat hepatocytes. PQ++ generates reactive oxygen species within cells by redox cycling and its toxicity to hepatocytes was potentiated by pretreatment with 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU), an inhibitor of glutathione reductase. In BCNU-treated cells, PQ++ caused GSH depletion, lipid peroxidation and cell death. These cytotoxic effects were prevented by the antioxidant N,N'-diphenyl-p-phenylenediamine (DPPD) and the iron-chelating agent desferrioxamine. MPP+ also caused GSH depletion in BCNU-treated hepatocytes but its cytotoxicity was not markedly affected by BCNU, nor was it accompanied by significant lipid peroxidation. DPPD and desferrioxamine also failed to prevent MPP+-induced cell death. We conclude that the production of active oxygen species is likely to play a major role in PQ++ cytotoxicity, while MPP+-induced cell damage may involve additional, more important toxic mechanisms.

Summary The loss of viability of isolated rat hepatocytes exposed to either 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) or its toxic metabolite 1-methyl-4-phenylpyridinium ion (MPP + ) was prevented by addition of fructose to the incubation medium. This protection was dependent on fructose concentration, being complete at 10 mM. Addition of fructose dramatically delayed MPTP- and MPP + -induced depletion of ATP and was accompanied by a significant accumulation of lactate, indicating the occurrence of enhanced glycolytic production of ATP. Glucose was much less effective against MPTP and MPP + toxicity, probably because it is a relatively poor substrate for glycolysis in liver cells. We conclude that depletion of ATP is a critical event in MPTP cytotoxicity in our in vitro model system, and that the use of alternative sources of ATP production may represent an important protective device against the effects of this toxic agent.

1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and its oxidized metabolite 1-methyl-4-phenylpyridine (MPP+) produce a rapid depletion of intracellular ATP in isolated rat hepatocytes. This effect was dose-dependent and was consistently observed before the onset of toxicity. The monoamine oxidase inhibitor pargyline provided significant protection against MPTP-induced cell death and ATP loss, but had no effect with MPP+. Thus, ATP depletion may play a critical role in MPTP toxicity, possibly via the metabolic production of MPP+.

The role of active oxygen species and lipid peroxidation in the toxic effects of diquat, paraquat and other bipyridyl herbicides remains controversial. In vitro studies have shown that these compounds are potent generators of active oxygen species by redox cycling and that they stimulate lipid peroxidation. In vivo studies have failed, however, to show clear evidence of lipid peroxidation resulting from toxic exposures to these compounds. We have directly compared the abilities of three bipyridyl herbicides, diquat (DQ), paraquat (PQ) and benzyl viologen (BV), to generate superoxide anion radical (O2-.) in rat liver microsomes and H2O2 in hepatocytes and correlated this with their relative toxicities to a compromised isolated hepatocyte system. DQ was the most potent generator of O2-. and H2O2, being slightly more potent than BV and much better than PQ. This ability of the bipyridyls to generate active oxygen was positively correlated with the ability to induce toxicity in hepatocytes pretreated with 1,3-bis-(2-chloroethyl)-1-nitrosourea (BCNU) to inhibit their glutathione reductase activity, i.e. DQ greater than BV greater than PQ. DQ caused a rapid depletion of cellular GSH and a concomitant increase in GSSG in this system. Toxicity, measured as loss of plasma membrane integrity, was pronounced after only 30-60 min of incubation and was accompanied by a significant increase in lipid peroxidation. The onset of lipid peroxidation could not be separated temporally from the expression of toxicity. However, the total inhibition of lipid peroxidation by the antioxidants Trolox C, promethazine and N,N'-diphenyl-p-phenylenediamine only delayed toxicity, indicating that, even though lipid peroxidation may play some role in enhancing bipyridyl herbicide toxicity, it is not essential for the toxicity to manifest itself.

The role of extracellular Ca2+ in toxic liver injury was examined in two in vitro models of the rat liver, the isolated perfused liver and isolated hepatocytes. The toxins t-butylhydroperoxide and carbon tetrachloride, as well as the Ca2+ ionophore, heptafluorodimethyloctanedione (FOD), were employed to induce cellular injury and death. Lipid peroxidation was also measured as the formation of thiobarbituric acid-reactive products. Cell death was measured by the release of lactate dehydrogenase in the perfused liver model and by the uptake of trypan blue in isolated hepatocytes. Toxin-induced cellular injury and death occurred in both in vitro models in the presence and absence of extracellular Ca2+, indicating that an influx of Ca2+ was not essential for toxic liver injury. The degree of toxicity seen in the perfused liver model was independent of increases or decreases in the total calcium concentration present in the liver tissue, providing further evidence that cell death is not dictated solely by changes in cellular calcium content. Isolated hepatocytes differed from the perfused liver, however, undergoing more lipid peroxidation and toxin-induced cell death when incubated in the absence of extracellular Ca2+ than in its presence. While suggesting that extracellular Ca2+ concentrations may have some influence on the expression of toxicity, these results demonstrate the nonessential role extracellular Ca2+ plays in the events leading to toxic liver injury.

In order to clarify the role of oxidative processes in cytotoxicity we have studied the metabolism and toxicity of 2-methyl-1,4-naphthoquinone (menadione) and its 2,3 dimethyl (DMNQ) and 2,3 diethyl (DENQ) analogs in isolated rat hepatocytes. The two analogs, unlike menadione, cannot alkylate nucleophiles directly and were considerably less toxic than menadione. This decreased toxicity was consistent with the inability of DMNQ and DENQ to alkylate but we also found them to undergo lower rates of redox cycling in hepatocytes and a higher ratio of two electron as opposed to one electron reduction relative to menadione. Thus, facile analysis of the respective roles of alkylation and oxidation in cytotoxicity was not possible using these compounds. In hepatocytes pretreated with bischloroethyl-nitrosourea (BCNU) to inhibit glutathione reductase, all three naphthoquinones caused a potentiation of reduced glutathione (GSH) removal/ oxidized glutathione (GSSG) generation and cytotoxicity relative to that observed in control cells. These data show that inhibition of hepatocyte glutathione reductase by BCNU results in enhanced naphthoquinone-induced oxidative challenge and subsequent cellular toxicity. That DMNQ and DENQ are cytotoxic, albeit at high concentrations, and that this cytotoxicity is potentiated by BCNU pretreatment suggest that oxidative processes alone can be a determinant of cytotoxicity.

Incubation of isolated hepatocytes in the presence of either the parkinsonian-inducing compound 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) or its putative toxic metabolite 1-methyl-4-phenylpyridinium ion (MPP + ) led to a depletion of intracellular reduced glutathione (GSH), which was mostly recovered as glutathione disulfide (GSSG). However, both MPTP- and MPP + -induced glutathione perturbances were relatively unaffected by the prior inhibition of glutathione reductase with 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU), suggesting that intracellular oxidation was not the major mechaism involved in the GSH loss. Inclusion of cystine in the incubation mixtures revealed a timedependent formation of cysteinyl glutathione (CySSG), indicating that an increased efflux was mostly responsible for the MPTP- and MPP + -induced GSH depletion. Therefore, the measurement of GSSG, which is apparently formed extracellularly, was not associated with oxidative stress.

The biotransformation of the tertiary amine 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) was studied in isolated rat hepatocytes, in order to assess the relative contributions of the metabolic reactions previously described in studies with subcellular preparations. The oxidative pathway which produces the toxic metabolite 1-methyl-4-phenylpyridinium ion (MPP+) via the generation of the 2,3-dihydropyridinium derivative, MPDP+, accounted for approximately 90% of MPTP metabolism by hepatocytes. Mitochondrial monoamine oxidase type B (MAO B) was specifically responsible for the initial step of this conversion. In the endoplasmic reticulum, cytochrome P-450 catalyzed the demethylation of MPTP to form PTP, while the flavin-containing monooxygenase (FMO) was responsible for the generation of MPTP N-oxide. No other metabolite was detected in our hepatocyte incubations under any of the experimental conditions used. After pretreatment with the specific MAO B inhibitor, deprenyl, the rates of production of the two microsomal metabolites were enhanced, but the overall rate of MPTP conversion decreased by more than 60%. On the other hand, no significant difference in the rate of MPTP metabolism was found after the inhibition of cytochrome P-450 by SKF 525-A or with the use of methimazole, a competitive substrate for FMO. The SKF 525-A and methimazole treatments selectively inhibited the formation of PTP and MPTP N-oxide, respectively, but had no significant effect on the rate and extent of MPTP toxicity. MPP+ was the only metabolite which accumulated within the cell compartment under all the experimental conditions.(ABSTRACT TRUNCATED AT 250 WORDS)

Oxidative stress and covalent binding have been proposed as possible mechanisms involved in the cytotoxic effects of the parkinsonism-causing compound 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). However, the toxicity induced by MPTP in isolated rat hepatocytes seems to be relatively independent of oxygen radical-induced oxidative stress. Here we demonstrate that MPTP cytotoxicity is not potentiated by pretreatment with 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU), an inhibitor of glutathione reductase, nor prevented by the antioxidant N,N′-diphenyl-p-pphenylenediamine (DPPD) or the iron-chelating agent desferrioxamine. Moreover, preincubation of hepatocytes with diethylmaleate to lower the level of intracellular reduced glutathione (to 20% of the initial value) did not affect either the rate or extent of MPTP cytotoxicity. Thus, nucleophilic soluble thiols do not seem to play a protective role against MPTP-induced cell damage, in contrast to what one would have expected if covalent protein binding and oxidative stress were involved as toxic mechanisms. On the other hand, MPTP cytotoxicity was potentiated by pretreatment of hepatocytes with cytochrome P-450 inhibitors (e.g., SKF 525A and metyrapone) and a more rapid depletion of ATP was observed in these experimental conditions. We conclude that mitochondrial damage and subsequent ATP depletion are likely to play a critical role in the toxicity of MPTP to isolated hepatocytes and that the metabolism of MPTP via the cytochrome P-450 monooxygenase system can be considered to be a detoxifying pathway.

The glutathione peroxidase (GSH-Px)-like reduction of H2O2 by the selenoorganic compound 2-phenyl-1,2-benzoisoselenazol-3(H)-one (PZ51: Ebselen) was studied using glutathione (GSH) and the therapeutic agent N-acetylcysteine (NAC) to provide reducing equivalents. In a purely chemical system containing H2O2 and in an enzymatic system of glucose/glucose oxidase-generated H2O2 Ebselen alone did not reduce H2O2. Ebselen in combination with either GSH (1 mM) or NAC (1 mM) was capable of reducing H2O2 in both systems. In these non-cellular systems GSH was a more effective source of reducing equivalents than NAC. The GSH-Px-like activity of Ebselen was further investigated in a cellular system. The redox-cycling bipyridylium compound diquat generates active oxygen species, depletes intracellular glutathione, and is cytotoxic in isolated hepatocytes pretreated with the glutathione reductase inhibitor 1,3-bis(Z-chloro-ethyl)-1-nitrosourea (BCNU). Ebselen alone did not ameliorate diquat cytotoxicity, but in combination with either GSH (1 mM) or NAC (1 mM) it produced a significant delay in diquat-induced cytotoxicity. Further additions of either GSH (0.5 mM) or NAC (0.5 mM) at 30 min intervals provided significantly more protection against diquat-induced cytotoxicity and intracellular GSH depletion than the single 1 mM addition. Thus, the combination of Ebselen and NAC may provide an effective antidote in cases of overexposure to bipyridylium herbicides, such as diquat and paraquat.