The Gut Microbiome and Xenobiotics: Identifying Knowledge Gaps.

There is an increasing awareness that the gut microbiome plays a critical role in human health and disease, but mechanistic insights are often lacking. In June 2018, the Health and Environmental Sciences Institute (HESI) held a workshop, "The Gut Microbiome: Markers of Human Health, Drug Efficacy and Xenobiotic Toxicity" (https://hesiglobal.org/event/the-gut-microbiome-workshop) to identify data gaps in determining how gut microbiome alterations may affect human health. Speakers and stakeholders from academia, government, and industry addressed multiple topics including the current science on the gut microbiome, endogenous and exogenous metabolites, biomarkers, and model systems. The workshop presentations and breakout group discussions formed the basis for identifying data gaps and research needs. Two critical issues that emerged were defining the microbial composition and function related to health and developing standards for models, methods and analysis in order to increase the ability to compare and replicate studies. A series of key recommendations were formulated to focus efforts to further understand host-microbiome interactions and the consequences of exposure to xenobiotics as well as identifying biomarkers of microbiome-associated disease and toxicity.

From the Cover: Genomic Effects of Androstenedione and Sex-Specific Liver Cancer Susceptibility in Mice.

Current strategies for predicting carcinogenic mode of action for nongenotoxic chemicals are based on identification of early key events in toxicity pathways. The goal of this study was to evaluate short-term key event indicators resulting from exposure to androstenedione (A4), an androgen receptor agonist and known liver carcinogen in mice. Liver cancer is more prevalent in men compared with women, but androgen-related pathways underlying this sex difference have not been clearly identified. Short-term hepatic effects of A4 were compared with reference agonists of the estrogen receptor (ethinyl estradiol, EE) and glucocorticoid receptor (prednisone, PRED). Male B6C3F1 mice were exposed for 7 or 28 days to A4, EE, or PRED. EE increased and PRED suppressed hepatocyte proliferation, while A4 had no detectable effects. In a microarray analysis, EE and PRED altered >3000 and >670 genes, respectively, in a dose-dependent manner, whereas A4 did not significantly alter any genes. Gene expression was subsequently examined in archival liver samples from male and female B6C3F1 mice exposed to A4 for 90 days. A4 altered more genes in females than males and did not alter expression of genes linked to activation of the mitogenic xenobiotic receptors AhR, CAR, and PPARα in either sex. A gene expression biomarker was used to show that in female mice, the high dose of A4 activated the growth hormone-regulated transcription factor STAT5b, which controls sexually dimorphic gene expression in the liver. These findings suggest that A4 induces subtle age-related effects on STAT5b signaling that may contribute to the higher risk of liver cancer in males compared with females.

Editor's Highlight: Mechanistic Toxicity Tests Based on an Adverse Outcome Pathway Network for Hepatic Steatosis.

Risk assessors use liver endpoints in rodent toxicology studies to assess the safety of chemical exposures. Yet, rodent endpoints may not accurately reflect human responses. For this reason and others, human-based invitro models are being developed and anchored to adverse outcome pathways to better predict adverse human health outcomes. Here, a networked adverse outcome pathway-guided selection of biology-based assays for lipid uptake, lipid efflux, fatty acid oxidation, and lipid accumulation were developed. These assays were evaluated in a metabolically competent human hepatocyte cell model (HepaRG) exposed to compounds known to cause steatosis (amiodarone, cyclosporine A, and T0901317) or activate lipid metabolism pathways (troglitazone, Wyeth-14,643, and 22(R)-hydroxycholesterol). All of the chemicals activated at least one assay, however, only T0901317 and cyclosporin A dose-dependently increased lipid accumulation. T0901317 and cyclosporin A increased fatty acid uptake, decreased lipid efflux (inferred from apolipoprotein B100 levels), and increased fatty acid synthase protein levels. Using this biologically-based evaluation of key events regulating hepatic lipid levels, we demonstrated dysregulation of compensatory pathways that normally balance hepatic lipid levels. This approach may provide biological plausibility and data needed to increase confidence in linking invitro-based measurements to chemical effects on adverse human health outcomes.

Tipping the Balance: Hepatotoxicity and the 4 Apical Key Events of Hepatic Steatosis.

Hepatic steatosis is a condition were fat accumulates in the liver and it is associated with extra-hepatic diseases related to metabolic syndrome and systemic energy metabolism. If not reversed, steatosis can progress to steatohepatitis and irreversible stages of liver disease including fibrosis, cirrhosis, hepatocellular carcinoma, and death. From a public health standpoint, identifying chemical exposures that may be factors in steatosis etiology are important for preventing hepatotoxicity and liver disease progression. It is therefore important to identify the biological events that are key for steatosis pathology mediated by chemical exposure. In this review, we give a current overview of the complex biological cascades that can disrupt lipid homeostasis in hepatocytes in the context of 4 apical key events central to hepatic lipid retention: hepatic fatty acid (FA) uptake,de novoFA and lipid synthesis, FA oxidation, and lipid efflux. Our goal is to review these key cellular events and visually summarize them using a network for application in pathway-based toxicity testing. This effort provides a foundation to improve next-generation chemical screening efforts that may be used to prevent and ultimately reverse the growing incidence of fatty liver disease in our population.

Dose and Effect Thresholds for Early Key Events in a PPARα-Mediated Mode of Action.

Current strategies for predicting adverse health outcomes of environmental chemicals are centered on early key events in toxicity pathways. However, quantitative relationships between early molecular changes in a given pathway and later health effects are often poorly defined. The goal of this study was to evaluate short-term key event indicators using qualitative and quantitative methods in an established pathway of mouse liver tumorigenesis mediated by peroxisome proliferator-activated receptor alpha (PPARα). Male B6C3F1 mice were exposed for 7 days to di (2-ethylhexyl) phthalate (DEHP), di-n-octyl phthalate (DNOP), and n-butyl benzyl phthalate (BBP), which vary in PPARα activity and liver tumorigenicity. Each phthalate increased expression of select PPARα target genes at 7 days, while only DEHP significantly increased liver cell proliferation labeling index (LI). Transcriptional benchmark dose (BMDT) estimates for dose-related genomic markers stratified phthalates according to hypothetical tumorigenic potencies, unlike BMDs for non-genomic endpoints (relative liver weights or proliferation). The 7-day BMDT values for Acot1 as a surrogate measure for PPARα activation were 29, 370, and 676 mg/kg/day for DEHP, DNOP, and BBP, respectively, distinguishing DEHP (liver tumor BMD of 35 mg/kg/day) from non-tumorigenic DNOP and BBP. Effect thresholds were generated using linear regression of DEHP effects at 7 days and 2-year tumor incidence values to anchor early response molecular indicators and a later phenotypic outcome. Thresholds varied widely by marker, from 2-fold (Pdk4 and proliferation LI) to 30-fold (Acot1) induction to reach hypothetical tumorigenic expression levels. These findings highlight key issues in defining thresholds for biological adversity based on molecular changes.

Adverse Outcome Pathways-Organizing Toxicological Information to Improve Decision Making.

The number of chemicals for which environmental regulatory decisions are required far exceeds the current capacity for toxicity testing. High-throughput screening commonly used for drug discovery has the potential to increase this capacity. The adverse outcome pathway (AOP) concept has emerged as a framework for connecting high-throughput toxicity testing (HTT) and other results to potential impacts on human and wildlife populations. As a result of international efforts, the AOP development process is now well-defined and efforts are underway to broaden the participation through outreach and training. One key principle is that AOPs represent the chemical-agnostic portions of pathways to increase the generalizability of their application from early key events to overt toxicity. The closely related mode of action framework extends the AOP as needed when evaluating the potential risk of a specific chemical. This in turn enables integrated approaches to testing and assessment (IATA), which incorporate results of assays at various levels of biologic organization such as in silico; HTT; chemical-specific aspects including absorption, distribution, metabolism, and excretion (ADME); and an AOP describing the biologic basis of toxicity. Thus, it is envisaged that provision of limited information regarding both the AOP for critical effects and the ADME for any chemical associated with any adverse outcome would allow for the development of IATA and permit more detailed AOP and ADME research, where higher precision is needed based on the decision context.

The use of mode of action information in risk assessment: quantitative key events/dose-response framework for modeling the dose-response for key events.

The HESI RISK21 project formed the Dose-Response/Mode-of-Action Subteam to develop strategies for using all available data (in vitro, in vivo, and in silico) to advance the next-generation of chemical risk assessments. A goal of the Subteam is to enhance the existing Mode of Action/Human Relevance Framework and Key Events/Dose Response Framework (KEDRF) to make the best use of quantitative dose-response and timing information for Key Events (KEs). The resulting Quantitative Key Events/Dose-Response Framework (Q-KEDRF) provides a structured quantitative approach for systematic examination of the dose-response and timing of KEs resulting from a dose of a bioactive agent that causes a potential adverse outcome. Two concepts are described as aids to increasing the understanding of mode of action-Associative Events and Modulating Factors. These concepts are illustrated in two case studies; 1) cholinesterase inhibition by the pesticide chlorpyrifos, which illustrates the necessity of considering quantitative dose-response information when assessing the effect of a Modulating Factor, that is, enzyme polymorphisms in humans, and 2) estrogen-induced uterotrophic responses in rodents, which demonstrate how quantitative dose-response modeling for KE, the understanding of temporal relationships between KEs and a counterfactual examination of hypothesized KEs can determine whether they are Associative Events or true KEs.

N-acetyltransferase 2 activity and folate levels.

AIMS: To determine whether increased N-acetyltransferase (NAT) activity might have a toxic effect during development and an influence on folate levels since previous work has shown that only low levels of exogenous NAT can be achieved in constitutionally transgenic mice (Cao et al. 2005). MAIN METHODS: A human NAT1 tet-inducible construct was used that would not be expressed until the inducer was delivered. Human NAT1 cDNA was cloned into pTRE2 and injected into mouse oocytes. Two transgenic lines were crossed to mouse line TgN(rtTahCMV)4Uh containing the CMV promoted "tet(on)". Measurements of red blood cell folate levels in inbred strains of mice were performed. KEY FINDINGS: Only low levels of human NAT1 could be achieved in kidney (highly responsive in other studies) whether the inducer, doxycycline, was given by gavage or in drinking water. An inverse correlation of folate levels with Nat2 enzyme activity was found. SIGNIFICANCE: Since increasing NAT1 activity decreases folate in at least one tissue, the detrimental effect of expression of human NAT1 in combination with endogenous mouse Nat2 may be a consequence of increased catabolism of folate.

An N-ethyl-N-nitrosourea-induced mutation in N-acetyltransferase 1 in mice.

Genetic variation in human N-acetyltransferases (NAT) has been implicated in susceptibility to aromatic amine and hydrazine carcinogens and therapeutic drugs. There are mouse models for variability of human NAT1; however mice with genetic differences in Nat1 (corresponding to human NAT2), have not been available. N-Ethyl-N-nitrosourea (ENU) mutagenesis was used to create genetic variation in Nat1. Among a number of mutations identified, a base-pair change substituting threonine for isoleucine at position 95 was recovered and studied. Molecular models suggested that this substitution would alter substrate binding. Analysis of hepatic Nat1 activity with the selective substrate isoniazid showed that there was a significant reduction in enzymatic activity in the homozygous mutants compared to the parental strain.

Confirmation of the role of N-acetyltransferase 2 in teratogen-induced cleft palate using transgenics and knockouts.

Previous work on Dilantin- and hydrocortisone-induced cleft palate and cleft lip with or without cleft palate using congenics for the N-acetyltransferase loci (Nat1 and Nat2 are closely linked) and recombinant inbred lines implicated the Nat1,2 region in susceptibility to teratogen-induced orofacial clefting. Since Nat1 does not differ between the two strains, Nat2 appeared to be responsible. We have now tested this conclusion using transgenics and knockouts. Transgenics for human NAT1 (equivalent to mouse Nat2) and knockouts for Nat2 were tested for susceptibility to Dilantin, hydrocortisone, and 6-aminonicotinamide-induced orofacial clefting. We found that Nat2 greatly influences teratogen-induced orofacial clefting on the A/J background but not on the C57BL/6J background. The magnitude and direction of the effects depended on which teratogen was used. The Nat2 knockout did not make C57BL/6J susceptible or A/J (already with very low activity) more susceptible but significantly decreased sporadic clefting in the A/J strain. We conclude that only the A/J strain, with several loci affecting orofacial clefting, is influenced by Nat2.

DNA constructs designed to produce short hairpin, interfering RNAs in transgenic mice sometimes show early lethality and an interferon response.

Arylamine N-acetyltransferase (NAT) genes were targeted for inhibition using short hairpin RNA (shRNA) using two different RNA polymerase III promoters. Constructs were developed for NAT1 and NAT2, the endogenous mouse genes, and for human NAT1. There were fetal and neonatal deaths with these constructs, perhaps due in part to an interferon response as reflected in increases in oligoadenylate synthetase I mRNA levels. Seven out of 8 founders with the U6 promoter generated offspring but only 2 gave positive offspring. Out of 15 founders for H1 promoted constructs, only 4 had positive offspring. When transgenic lines were successfully established, the expression of the targeted genes was variable between animals and was not generally inhibitory.

Hepatic gene expression and lipid homeostasis in C57BL/6 mice exposed to hydrazine or acetylhydrazine.

Hydrazine (HD) and acetylhydrazine (AcHD) are metabolites of the antituberculosis drug isoniazid (INH) that have been implicated in INH-induced liver damage. The hepatotoxicity of AcHD and HD were compared in adult male C57Bl/6J mice by evaluating hepatic histopathology, plasma biochemistry, and hepatic gene expression. By all measures, HD had significantly greater effects than AcHD. There was no evidence of liver damage following exposure to AcHD (300 mg/kg, po). However, HD at this dose caused marked hepatic necrosis, macrovesicular degeneration, and steatosis. Lipid accumulation was initiated 2 h after HD exposure, with hepatic macrovesicular degeneration evident after 4 h, and severe necrosis by 36 h. Gene expression profiles were compared 24 h following 100 mg/kg po of HD or AcHD. HD changed the hepatic expression of more genes than AcHD, particularly lipid synthesis, transport, and metabolism genes that may be involved in steatosis. Hepatic expression of genes regulated by peroxisome proliferator activated receptors (PPAR) and sterol regulatory element binding protein (SREBP) transcription factors was increased only by HD. The hepatotoxicty and hepatic gene expression profile of HD, but not AcHD, indicate that exposure to HD initiates a process whereby the production and intracellular transport of hepatic lipids is favored over the removal of fatty acids and their metabolites.

The effects of genetic variation in N-acetyltransferases on 4-aminobiphenyl genotoxicity in mouse liver.

Inbred, congenic and transgenic strains of mice were characterized for acetylation of p-aminobenzoic (PABA) and the carcinogen 4-aminobiphenyl (4ABP). C57Bl/6 mice have the NAT2*8 allele, A/J mice have NAT2*9 and congenic B6.A mice have NAT2*9 on the C57Bl/6 background. The first transgenic strain with human NAT1, the functional equivalent of murine NAT2, was also tested. The murine NAT2*9 allele correlated with a slow phenotype measured with the murine NAT2 selective substrate PABA. The two strains having this allele also had a lower capacity to acetylate 4ABP. A line with five copies of the human NAT1 transgene was bred for at least five generations with either C57Bl/6 or A/J mice. There was no significant change in PABA NAT activity on the C57Bl/6 background but a 2.5-fold increase was seen in hNAT1:A/J compared with A/J. The effect of variation in NATs on 4ABP genotoxicity was assessed in these strains. Twenty-four hours after exposure to a single oral dose of 120 mg 4ABP/kg, hepatic 4ABP-DNA adducts were detected by immunofluoresence in all strains. Nuclear fluorescence intensities (mean+/-S.D.) were 41.1+/-3.6 for C57Bl/6, 37.9+/-1.11 for A/J and 36.3+/-2.44 for B6.A. There was no correlation between murine NAT2 alleles and 4ABP-DNA adduct levels. Similar results were seen with the transgenic strains. The data indicate that the range of variation present in these strains of mice was insufficient to alter susceptibility to 4ABP genotoxicity. The impact of these relatively modest differences in the acetylation of the activation of 4ABP may be masked by other competing biotransformation reactions since 4ABP is a substrate for both NAT1 and NAT2. Mouse models with variation in both isoforms are needed to adequately assess the role of variation in NATs in susceptibility to 4ABP genotoxicity.

Neonatal ontogeny of murine arylamine N-acetyltransferases: implications for arylamine genotoxicity.

Age-related changes in the expression of xenobiotic biotransformation enzymes can result in differences in the rates of chemical activation and detoxification, affecting responses to the therapeutic and/or toxic effects of chemicals. Despite recognition that children and adults may exhibit differences in susceptibility to chemicals, information about when in development specific biotransformation enzymes are expressed is incomplete. N-acetyltransferases (NATs) are phase II enzymes that catalyze the acetylation of arylamine and hydrazine carcinogens and therapeutic drugs. The postnatal expression of NAT1 and NAT2 was investigated in C57Bl/6 mice. Hepatic NAT1 and NAT2 messenger RNAs (mRNAs) increased with age from neonatal day (ND) 4 to adult in a nonlinear fashion. The presence of functional proteins was confirmed by measuring NAT activities with the isoform selective substrates p-aminobenzoic acid and isoniazid, as well as the carcinogens 2-aminofluorene and 4-aminobiphenyl (4ABP). Neonatal liver was able to acetylate all of the substrates, with activities increasing with age. Protein expression of CYP1A2, another enzyme involved in the biotransformation of arylamines, showed a similar pattern. The genotoxicity of 4ABP was assessed by determining hepatic 4ABP-DNA adducts. There was an age-dependent increase in 4ABP-DNA adducts during the neonatal period. Thus, developmental increases in expression of NAT1 and NAT2 genes in neonates are associated with less 4ABP genotoxicity. The age-related pattern of expression of biotransformation enzymes in mice is consistent with human data for NATs and suggests that this may play a role in developmental differences in arylamine toxicity.

Prenatal expression of N-acetyltransferases in C57Bl/6 mice.

Exposure to carcinogens such as 4-aminobiphenyl (4ABP), found in tobacco smoke and other combustion products, results in the formation of detectable levels of 4ABP-hemoglobin adducts in cord blood and 4ABP-DNA adducts in conceptal tissue. The presence of these adducts requires that the parent compound undergo biotransformation. When exposure occurs in utero, the maternal, placental and conceptal tissues are all possible sites for the formation of DNA-reactive products. One step in the activation of 4ABP is catalyzed by N-acetyltransferases (NAT). The expression of NAT was evaluated in gestational day (GD) 10-18 conceptal tissues from C57Bl/6 mice. There was a quantitative increase in NAT1 and NAT2 mRNAs with increasing gestational age that was also reflected in age-related changes in functional protein measured as 4ABP-NAT activity. The ability to acetylate 4ABP increased from GD10 to 18 and was lower in conceptal tissue than in adult liver. The potential toxicologic significance of prenatal NAT expression was assessed by formation of 4ABP-DNA adducts. At GD 15 and 18, 4ABP-DNA adducts were detected by immunohistochemistry 24 h following a single oral dose of 120 mg 4ABP/kg. Based on nuclear fluorescence, conceptual 4ABP-DNA adducts were present at similar levels at GD15 and 18. Levels of 4ABP-DNA adducts were significantly higher in maternal liver compared with the conceptus. Results from this study show that both NAT genes were expressed prenatally and that functional enzymes were present. These data support the possible in situ generation of reactive products by the conceptus. The relative contributions of maternal activation of 4ABP and that by the conceptus remain to be determined.

Measuring the activity of arylamine N-acetyltransferase (NAT).

This unit describes methods for measuring the activity of arylamine N-acetyltransferases (NAT). Genetic polymorphisms in NAT 1 and NAT 2 have been associated with susceptibility to aromatic amines carcinogens and effects of therapeutic drugs. Evaluation of the activities associated with substrates of NATs is helpful in elucidating the contribution of these enzymes to the pharmacologic and toxicologic effects of arylamines and hydrazines.