Challenges and opportunities for overcoming dog use in agrochemical evaluation and registration.

Progress in developing new tools, assays, and approaches to assess human hazard and health risk provides an opportunity to re-evaluate the necessity of dog studies for the safety evaluation of agrochemicals. A workshop was held where partic­ipants discussed the strengths and limitations of past use of dogs for pesticide evaluations and registrations. Opportunities were identified to support alternative approaches to answer human safety questions without performing the required 90-day dog study. Development of a decision tree for determining when the dog study might not be necessary to inform pesticide safety and risk assessment was proposed. Such a process will require global regulatory authority participation to lead to its acceptance. The identification of unique effects in dogs that are not identified in rodents will need further evaluation and determination of their relevance to humans. The establishment of in vitro and in silico approaches that can provide critical data on relative species sensitivity and human relevance will be an important tool to advance the decision process. Promising novel tools including in vitro comparative metabolism studies, in silico models, and high-throughput assays able to identify metabolites and mechanisms of action leading to development of adverse outcome pathways will need further development. To replace or eliminate the 90-day dog study, a collaborative, multidisciplinary, international effort that transcends organi­zations and regulatory agencies will be needed in order to develop guidance on when the study would not be necessary for human safety and risk assessment.

Bridging Sex-Specific Differences in the CAR-Mediated Hepatocarcinogenesis of Nitrapyrin Using Molecular and Apical Endpoints.

Nitrapyrin, a nitrification inhibitor, produces liver tumors in B6C3F1 mice. In a 2-year oncogenicity study, increased incidence of mice with hepatocellular tumors was observed following exposure to 125 (females only) or 250 mg/kg/day (males and females) nitrapyrin in the diet. Previous data was generated in male mice to support a mode-of-action (MoA) characterized by constitutive androstane receptor (CAR) nuclear receptor (NR) activation, increased hepatocellular proliferation, and subsequent hepatocellular foci and tumor formation. Uncertainty as to the relevance of this MoA for females remained given the increased sensitivity to tumor formation in female mice. A targeted MoA study was conducted to evaluate CAR activation and hepatic responses in female mice treated with the female carcinogenic dose of nitrapyrin for 4 days. Nitrapyrin induced a treatment-related increase in hepatocellular hypertrophy and hepatocellular proliferation. Nitrapyrin also induced a dose-related increase in the Cyp2b10/CAR-associated transcript and liver weights. Nitrapyrin-induced liver weights and Cyp2b10 gene expression for both males and females were compared to data generated from three other established CAR activators; methyl isobutyl ketone, phenobarbital, and sulfoxaflor. The response observed in female mice following exposure to nitrapyrin was within range of the degree of change observed in mice following exposure to tumorigenic doses of other CAR activators. Consistent with the liver MoA in male mice, these data support a CAR-mediated mode of action for nitrapyrin-induced liver tumors in female mice, with the understanding that a focused approach minimizing animal use can bridge male and female datasets when sex-specific carcinogenic differences are observed.

A rat subchronic study transcriptional point of departure estimates a carcinogenicity study apical point of departure.

Considerations of human relevance and animal use are driving research to identify new approaches to inform risk assessment of chemicals and replace guideline-based rodent carcinogenicity tests. Here, the hypothesis was tested across four agrochemicals that 1) a rat 90-day transcriptome-based BEPOD is protective of a rat carcinogenicity study and 2) a subchronic liver or kidney BEPOD would approximate a cancer bioassay apical POD derived from other organs and a rat subchronic BEPOD would approximate a mouse cancer bioassay apical POD. Using RNA sequencing and BMDExpress software, liver and/or kidney BEPOD values were generated in male rats exposed for 90 days to either Triclopyr Acid, Pronamide, Sulfoxaflor, or Fenpicoxamid. BEPOD values were compared to benchmark dose-derived apical POD values generated from rat 90-day and rodent carcinogenicity studies. Across all four agrochemicals, findings showed that a rat 90-day study BEPOD approximated the most sensitive apical POD (within 10-fold) generated from the 90-day rat study and long-term rodent carcinogenicity studies. This study supports the conclusion that a subchronic transcriptome-based BEPOD could be utilized to estimate an apical POD within a risk-based approach of chronic toxicity and carcinogenicity agrochemical assessment, abrogating the need for time- and resource-intensive rodent carcinogenicity studies and minimizing animal testing.

Integration of novel approaches demonstrates simultaneous metabolic inactivation and CAR-mediated hepatocarcinogenesis of a nitrification inhibitor.

Nitrapyrin, a nitrification inhibitor, produces liver tumors in mice at high doses. Several experiments were performed to investigate molecular, cellular, and apical endpoints to define the key events leading to the tumor formation. These data support a mode-of-action (MoA) characterized by constitutive androstane receptor (CAR) nuclear receptor activation, increased hepatocellular proliferation leading to hepatocellular foci and tumor formation. Specifically, nitrapyrin induced a dose-related increase in the Cyp2b10/CAR-associated transcript and protein. Interestingly, the corresponding enzyme activity (7-pentoxyresorufin-O-dealkylase (PROD) was not enhanced due to nitrapyrin-mediated suicide inhibition of PROD activity. Nitrapyrin exposure elicited a clear dose-responsive increase in hepatocellular proliferation in wild-type mice, but not in CAR knock-out mice, informing that CAR activation is an obligatory key event in this test material-induced hepatocarcinogenesis. Furthermore, nitrapyrin exposure induced a clear, concentration-responsive increase in cell proliferation in mouse, but not human, hepatocytes in vitro. Evaluation of the data from repeat dose and MoA studies by the Bradford Hill criteria and a Human Relevance Framework (HRF) suggested that nitrapyrin-induced mouse liver tumors are not relevant to human health risk assessment because of qualitative differences between these two species.

Dose-response analysis of epigenetic, metabolic, and apical endpoints after short-term exposure to experimental hepatotoxicants.

Identification of sensitive and novel biomarkers or endpoints associated with toxicity and carcinogenesis is of a high priority. There is increasing interest in the incorporation of epigenetic and metabolic biomarkers to complement apical data; however, a number of questions, including the tissue specificity, dose-response patterns, early detection of those endpoints, and the added value need to be addressed. In this study, we investigated the dose-response relationship between apical, epigenetic, and metabolomics endpoints following short-term exposure to experimental hepatotoxicants, clofibrate (CF) and phenobarbital (PB). Male F344 rats were exposed to PB (0, 5, 25, and 100 mg/kg/day) or CF (0, 10, 50, and 250 mg/kg/day) for seven days. Exposure to PB or CF resulted in dose-dependent increases in relative liver weights, hepatocellular hypertrophy and proliferation, and increases in Cyp2b1 and Cyp4a1 transcripts. These changes were associated with altered histone modifications within the regulatory units of cytochrome genes, LINE-1 DNA hypomethylation, and altered microRNA profiles. Metabolomics data indicated alterations in the metabolism of bile acids. This study provides the first comprehensive analysis of the apical, epigenetic and metabolic alterations, and suggests that the latter two occur within or near the dose response curve of apical endpoint alterations following exposure to experimental hepatotoxicants.

Predicting the future: opportunities and challenges for the chemical industry to apply 21st-century toxicity testing.

Interest in applying 21st-century toxicity testing tools for safety assessment of industrial chemicals is growing. Whereas conventional toxicology uses mainly animal-based, descriptive methods, a paradigm shift is emerging in which computational approaches, systems biology, high-throughput in vitro toxicity assays, and high-throughput exposure assessments are beginning to be applied to mechanism-based risk assessments in a time- and resource-efficient fashion. Here we describe recent advances in predictive safety assessment, with a focus on their strategic application to meet the changing demands of the chemical industry and its stakeholders. The opportunities to apply these new approaches is extensive and include screening of new chemicals, informing the design of safer and more sustainable chemical alternatives, filling information gaps on data-poor chemicals already in commerce, strengthening read-across methodology for categories of chemicals sharing similar modes of action, and optimizing the design of reduced-risk product formulations. Finally, we discuss how these predictive approaches dovetail with in vivo integrated testing strategies within repeated-dose regulatory toxicity studies, which are in line with 3Rs principles to refine, reduce, and replace animal testing. Strategic application of these tools is the foundation for informed and efficient safety assessment testing strategies that can be applied at all stages of the product-development process.

Concurrent evaluation of general, immune, and genetic toxicity endpoints as part of an integrated testing strategy.

Integrated testing strategies involve the assessment of multiple endpoints within a single toxicity study and represent an important approach for reducing animal use and streamlining testing. The present study evaluated the ability to combine general, immune, and genetic toxicity endpoints into a single study. Specifically, this study evaluated the impact of sheep red blood cell (SRBC) immunization, as part of the T-cell dependent antibody response (TDAR) assay, on organ weights, micronuclei (MN) formation (bone marrow and peripheral blood), and the Comet assay response in the liver of female F344/DuCrl rats treated with cyclophosphamide (CP) a known immunosuppressive chemical and genotoxicant. For the TDAR assay, treatment with CP resulted in a dose-dependent decrease in the antibody response with a suppression of greater than 95% at the high dose. Injection with SRBC had no impact on evaluated organ weights, histopathology, hematology, and clinical chemistry parameters. Analysis of MN formation in bone marrow and peripheral blood revealed a dose-dependent increase in response to CP treatment. Injection with SRBC had no impact on the level of MN in control animals and did not alter the dose response of CP. There was a slight increase in liver DNA damage in response to CP as measured by the Comet assay; however, injection with SRBCs did not alter this endpoint. Overall these data provide strong support for the concurrent assessment of general, immune, and genetic toxicology endpoints within a single study as part of an integrated testing strategy approach.

Propiconazole-enhanced hepatic cell proliferation is associated with dysregulation of the cholesterol biosynthesis pathway leading to activation of Erk1/2 through Ras farnesylation.

Propiconazole is a mouse hepatotumorigenic fungicide designed to inhibit CYP51, a key enzyme in the biosynthesis of ergosterol in fungi and is widely used in agriculture to prevent fungal growth. Metabolomic studies in mice revealed that propiconazole increased levels of hepatic cholesterol metabolites and bile acids, and transcriptomic studies revealed that genes within the cholesterol biosynthesis, cholesterol metabolism and bile acid biosyntheses pathways were up-regulated. Hepatic cell proliferation was also increased by propiconazole. AML12 immortalized hepatocytes were used to study propiconazole's effects on cell proliferation focusing on the dysregulation of cholesterol biosynthesis and resulting effects on Ras farnesylation and Erk1/2 activation as a primary pathway. Mevalonate, a key intermediate in the cholesterol biosynthesis pathway, increases cell proliferation in several cancer cell lines and tumors in vivo and serves as the precursor for isoprenoids (e.g. farnesyl pyrophosphate) which are crucial in the farnesylation of the Ras protein by farnesyl transferase. Farnesylation targets Ras to the cell membrane where it is involved in signal transduction, including the mitogen-activated protein kinase (MAPK) pathway. In our studies, mevalonic acid lactone (MVAL), a source of mevalonic acid, increased cell proliferation in AML12 cells which was reduced by farnesyl transferase inhibitors (L-744,832 or manumycin) or simvastatin, an HMG-CoA reductase inhibitor, indicating that this cell system responded to alterations in the cholesterol biosynthesis pathway. Cell proliferation in AML12 cells was increased by propiconazole which was reversed by co-incubation with L-744,832 or simvastatin. Increasing concentrations of exogenous cholesterol muted the proliferative effects of propiconazole and the inhibitory effects of L-733,832, results ascribed to reduced stimulation of the endogenous cholesterol biosynthesis pathway. Western blot analysis of subcellular fractions from control, MVAL or propiconazole-treated cells revealed increased Ras protein in the cytoplasmic fraction of L-744,832-treated cells, while propiconazole or MVAL reversed these effects. Western blot analysis indicated that phosphorylation of Erk1/2, a protein downstream of Ras, was increased by propiconazole. These data indicate that propiconazole increases cell proliferation by increasing the levels of cholesterol biosynthesis intermediates presumably through a negative feedback mechanism within the pathway, a result of CYP51 inhibition. This feedback mechanism increases Erk1/2 signaling through mevalonate-mediated Ras activation. These results provide an explanation for the observed effects of propiconazole on hepatic cholesterol pathways and on the increased hepatic cell proliferation induced by propiconazole in mice.

The hepatocarcinogenic conazoles: cyproconazole, epoxiconazole, and propiconazole induce a common set of toxicological and transcriptional responses.

Conazoles are fungicides used as agricultural pesticides and pharmaceutical products. We investigated whether a common core of toxicological and transcriptional responses underlies the observed carcinogenic effects of three conazoles: cyproconazole, epoxiconazole, and propiconazole. In studies where mice were fed diets of these conazoles for 30 days, we found a common set of toxicological effects altered by these conazoles: hepatomegaly, hepatocellular hypertrophy, decreased serum cholesterol, decreased hepatic levels of all-trans-retinoic acid, and increased hepatic cell proliferation. Microarray-based transcriptional analysis revealed 330 significantly altered probe sets common to these conazoles, many of which showed strong dose responses for cytochrome P450, glutathione S-transferase, and oxidative stress genes. More detailed analyses identified a subset of 80 altered genes common to the three conazoles that were associated with cancer. Pathways associated with these genes included xenobiotic metabolism, oxidative stress, cell signaling, and cell proliferation. A common TGFα-centric pathway was identified within the 80-gene set, which, in combination with the toxicological and other transcriptomic findings, provides a more refined toxicity profile for these carcinogenic conazoles.

Propiconazole increases reactive oxygen species levels in mouse hepatic cells in culture and in mouse liver by a cytochrome P450 enzyme mediated process.

Propiconazole induces hepatocellular carcinomas and hepatocellular adenomas in mice and promotes liver tumors in rats. Transcriptional, proteomic, metabolomic and biochemical studies of hepatic tissues from mice treated with propiconazole under the conditions of the chronic bioassay indicated that propiconazole induced oxidative stress. Here we sought to identify the source of the reactive oxygen species (ROS) induced by propiconazole using both AML12 immortalized mouse hepatocytes in culture and liver tissues from mice. We also sought to further characterize the nature and effects of ROS formation induced by propiconazole treatment in mouse liver. ROS was induced in AML12 cells by propiconazole as measured by fluorescence detection and its formation was ameliorated by N-acetylcysteine. Propiconazole induced glutathione-S-transferase (GSTα) protein levels and increased the levels of thiobarbituric acid reactive substances (TBARS) in AML12 cells. The TBARS levels were decreased by diphenylene iodonium chloride (DPIC), a cytochrome P450 (CYP) reductase inhibitor revealing the role of CYPs in ROS generation. It has been previously reported that Cyp2b and Cyp3a proteins were induced in mouse liver by propiconazole and that Cyp2b and Cyp3a proteins undergo uncoupling of their CYP catalytic cycle releasing ROS. Therefore, salicylic acid hydroxylation was used as probe for ROS formation using microsomes from mice treated with propiconazole. These studies showed that levels of 2,3-dihydroxybenzoic acid (an ROS derived metabolite) were decreased by ketoconazole, melatonin and DPIC. In vivo, propiconazole increased hepatic malondialdehyde levels and GSTα protein levels and had no effect on hepatic catalase or superoxide dismutase activities. Based on these observations we conclude that propiconazole induces ROS in mouse liver by increasing CYP protein levels leading to increased ROS levels. Our data also suggest that propiconazole induces the hydroxyl radical as a major ROS form.

Phosphorylation of CAP-G is required for its chromosomal DNA localization during mitosis.

Condensin is a 5 subunit complex that plays an important role in the structure of chromosomes during mitosis. It is known that phosphorylation of condensin subunits by cdc2/cyclin B at the beginning of mitosis is important for condensin activity, but the sites of these phosphorylation events have not been identified nor has their role in regulating condensin function. Here we identify two threonine residues in the CAP-G subunit of condensin, threonines 308 and 332, that are targets of cdc2/cyclin B phosphorylation. Mutation of these threonines to alanines results in defects in CAP-G localization with chromosomes during mitosis. These results are the first to identify phosphorylation sites within the condensin complex that regulate condensin localization with chromosomal DNA.

PRC1 associates with the hsp70i promoter and interacts with HSF2 during mitosis.

Mitosis is a series of events leading to division of a cell by the process known as cytokinesis. Protein regulating cytokinesis 1 (PRC1) is a CDK substrate that associates with the mitotic spindle and functions in microtubule bundling. Previous studies revealed that loss of PRC1 is associated with chromosomal mis-segregation and atypical chromosome alignment. HSF2 is a DNA binding protein that we previously showed bookmarks the hsp70i gene during mitosis, an epigenetic mechanism which allows the hsp70i gene to re-establish transcriptional competence early in G1. Another study demonstrated that HSF2-/- mouse embryonic fibroblasts (MEFs) exhibit increased numbers of multinucleated cells vs. wild-type MEFs. This suggests that HSF2 is important for proper cytokinesis, but the mechanism was unknown. Here we report the existence of a direct interaction between HSF2 and PRC1. HSF2 and PRC1 associate during mitosis and co-localize during this phase of the cell cycle. PRC1 does not interact with the related protein HSF1, indicating the specificity of the HSF2-PRC1 interaction. Intriguingly, PRC1 is associated with the hsp70i promoter during mitosis. These results provide a potential mechanistic basis for the defective cytokinesis phenotype exhibited by HSF2-/- cells, as well as suggest a potential role for PRC1 in HSF2-mediated gene bookmarking.

Interaction of HSF1 and HSF2 with the Hspa1b promoter in mouse epididymal spermatozoa.

The Hspa1b gene is one of the first genes expressed after fertilization, with expression observed in the male pronucleus as early as the one-cell stage of embryogenesis. This expression can occur in the absence of stress and is initiated during the minor zygotic genome activation. There is a significant reduction in the number of embryos developing to the blastocyte stage when HSPA1B levels are depleted, which supports the importance of this protein for embryonic viability. However, the mechanism responsible for allowing expression of Hspa1b during the minor zygotic genome activation (ZGA) is unknown. In this report, we investigated the role of HSF1 and HSF2 in bookmarking Hspa1b during late spermatogenesis. Western blot results show that both HSF1 and HSF2 are present in epididymal spermatozoa, and immunofluorescence analysis revealed that some of the HSF1 and HSF2 proteins in these cells overlap the 4',6'-diamidino-2-phenylindole-stained DNA region. Results from chromatin immunoprecipitation assays showed that HSF1, HSF2, and SP1 are bound to the Hspa1b promoter in epididymal spermatozoa. Furthermore, we observed an increase in HSF2 binding to the Hspa1b promoter in late spermatids versus early spermatids, suggesting a likely period during spermatogenesis when transcription factor binding could occur. These results support a model in which the binding of HSF1, HSF2, and SP1 to the promoter of Hspa1b would allow the rapid formation of a transcription-competent state during the minor ZGA, thereby allowing Hspa1b expression.

HSF1-TPR interaction facilitates export of stress-induced HSP70 mRNA.

Stress conditions inhibit mRNA export, but mRNAs encoding heat shock proteins continue to be efficiently exported from the nucleus during stress. How HSP mRNAs bypass this stress-associated export inhibition was not known. Here, we show that HSF1, the transcription factor that binds HSP promoters after stress to induce their transcription, interacts with the nuclear pore-associating TPR protein in a stress-responsive manner. TPR is brought into proximity of the HSP70 promoter after stress and preferentially associates with mRNAs transcribed from this promoter. Disruption of the HSF1-TPR interaction inhibits the export of mRNAs expressed from the HSP70 promoter, both endogenous HSP70 mRNA and a luciferase reporter mRNA. These results suggest that HSP mRNA export escapes stress inhibition via HSF1-mediated recruitment of the nuclear pore-associating protein TPR to HSP genes, thereby functionally connecting the first and last nuclear steps of the gene expression pathway, transcription and mRNA export.

Identification of Xenopus heat shock transcription factor-2: conserved role of sumoylation in regulating deoxyribonucleic acid-binding activity of heat shock transcription factor-2 proteins.

Heat shock transcription factor (Hsf)-1 and Hsf2 are members of the heat shock factor (HSF) protein family involved in heat shock protein (hsp) gene regulation, a regulation that is critical for the ability of cells to survive exposure to stress conditions. Although the role of Hsf1 in binding and activating transcription of hsp gene promoters in response to cell stress is well established, how Hsf2 enhances stress-induced hsp expression is not understood. To gain an insight into the critical conserved features of the regulation and function of Hsf2, we have identified and characterized the Hsf2 protein from Xenopus laevis. We found that, similar to its human counterpart, Xenopus Hsf2 is sumoylated at lysine 82 and that, as it does in human Hsf2, the modification event of the small ubiquitin-related modifier 1 functions to increase the deoxyribonucleic acid-binding activity of this transcription factor in Xenopus. These results indicate that sumoylation is an evolutionarily conserved modification of Hsf2 proteins, supporting the position of this modification as a critical regulator of Hsf2 function.