Liver Tumors In Rodents Research Articles

Translational Insights into Molecular Mechanisms of Chemical Hepatocarcinogenesis for Improved Human Risk Assessment

Background: Hepatocellular carcinoma (HCC) is a prevalent liver cancer with major risk factors being hepatitis viral infections, alcohol, non-alcoholic fatty liver disease, and aflatoxin exposure. Both genotoxic and non-genotoxic agents can induce HCC through mechanisms involving DNA damage, oxidative stress, chronic inflammation, and disrupted signaling pathways like MAPK/ERK, PI3K/AKT, WNT/β-catenin and PPARα. While rodent assays are utilized to detect potential chemical hepatocarcinogens, species differences in pathways like PPARα and CAR/PXR activation impact human risk assessment. Purpose: This analysis provides an updated, critical examination of species concordance in mechanisms of hepatic carcinogenesis to inform human safety assessment of rodent liver tumor findings. Main Body: Rodent assays including 2-year bioassays, transgenic models, and short-term studies detect liver tumors through lifetime exposure or early biomarkers. However, rodent-specific PPARα and CAR/PXR activation, along with human risk factors like hepatitis, highlight key interspecies differences. Determining mode of action relevance requires evaluating mechanistic validity and pivotal key events leading to tumors across species. Non-genotoxic compounds eliciting rodent liver tumors can activate PPARα, CAR/PXR, and other pathways triggering increased cell replication; but downstream signaling may differ in human liver. Understanding applicability of these mechanisms in humans as well as incorporating human risk factors into experimental models is critical for accurate risk assessment. Conclusion: In summary, elucidating conserved versus divergent molecular mechanisms of hepatic carcinogenesis between rodents and humans is essential for appropriately interpreting rodent findings and safeguarding human health through science-based risk assessment frameworks and regulatory decision-making processes around potential chemical hazards.

Risk assessment of N-nitrosamines in food.

EFSA was asked for a scientific opinion on the risks to public health related to the presence of N-nitrosamines (N-NAs) in food. The risk assessment was confined to those 10 carcinogenic N-NAs occurring in food (TCNAs), i.e. NDMA, NMEA, NDEA, NDPA, NDBA, NMA, NSAR, NMOR, NPIP and NPYR. N-NAs are genotoxic and induce liver tumours in rodents. The in vivo data available to derive potency factors are limited, and therefore, equal potency of TCNAs was assumed. The lower confidence limit of the benchmark dose at 10% (BMDL10) was 10μg/kg body weight (bw) perday, derived from the incidence of rat liver tumours (benign and malignant) induced by NDEA and used in a margin of exposure (MOE) approach. Analytical results on the occurrence of N-NAs were extracted from the EFSA occurrence database (n=2,817) and the literature (n=4,003). Occurrence data were available for five food categories across TCNAs. Dietary exposure was assessed for two scenarios, excluding (scenario 1) and including (scenario 2) cooked unprocessed meat and fish. TCNAs exposure ranged from 0 to 208.9ng/kg bw perday across surveys, age groups and scenarios. 'Meat and meat products' is the main food category contributing to TCNA exposure. MOEs ranged from 3,337 to 48 at the P95 exposure excluding some infant surveys with P95 exposure equal to zero. Two major uncertainties were (i) the high number of left censored data and (ii) the lack of data on important food categories. The CONTAM Panel concluded that the MOE for TCNAs at the P95 exposure is highly likely (98-100% certain) to be less than 10,000 for all age groups, which raises a health concern.

Saroglitazar suppresses the hepatocellular carcinoma induced by intraperitoneal injection of diethylnitrosamine in C57BL/6 mice fed on choline deficient, l-amino acid- defined, high-fat diet

BackgroundSaroglitazar is a novel PPAR-α/γ agonist with predominant PPAR-α activity. In various preclinical models, saroglitazar has been shown to prevent & reverse symptoms of NASH. In view of these observations, and the fact that NASH is a progressive disease leading to HCC, we hypothesized that saroglitazar may prevent the development of HCC in rodents.MethodsHCC was induced in C57BL/6 mice by a single intraperitoneal injection of 25 mg/kg diethylnitrosamine (DEN) at the age of 4 weeks and then feeding the animal a choline-deficient, L-amino acid- defined, high-fat diet (CDAHFD) for the entire study duration. Eight weeks after initiation of CDAHFD, saroglitazar (1 and 3 mg/kg) treatment was started and continued for another 27 weeks.ResultsSaroglitazar treatment significantly reduced the liver injury markers (serum ALT and AST), reversed hepatic steatosis and decreased the levels of pro-inflammatory cytokines like TNF-α in liver. It also resulted in a marked increase in serum adiponectin and osteopontin levels. All disease control animals showed hepatic tumors, which was absent in saroglitazar (3 mg/kg)- treatment group indicating 100% prevention of hepatic tumorigenesis. This is the first study demonstrating a potent PPARα agonist causing suppression of liver tumors in rodents, perhaps due to a strong anti-NASH activity of Saroglitazar that overrides its rodent-specific peroxisome proliferation activity.ConclusionThe data reveals potential of saroglitazar for chemoprevention of hepatocellular carcinoma in patients with NAFLD/NASH.

Development of a strategy to identify and evaluate direct and indirect activators of constitutive androstane receptor in rats.

Constitutive androstane receptor (CAR) is a nuclear receptor that plays a key role in drug metabolism and disposition and in the development of liver tumors in rodents. CAR is activated by ligands and indirect activators, which do not bind to the receptor but activate it through cellular signaling. In this study, we sought to identify direct and indirect activators of rat CAR (rCAR). Assessment of the influence of mutations on the transcriptional activity of rCAR identified a mutant termed rCAR-3A-G354Q that displays low constitutive activity and high ligand responsiveness. Reporter assays using the mutant were performed with compounds that increased the mRNA levels of Cyp2b1, a CAR target gene, in rat primary hepatocytes. Several compounds activated rCAR-3A-G354Q and were implicated as rCAR ligands. Since indirect CAR activators are considered to display little species differences, we then determined CYP2B6 mRNA levels in human hepatocyte-like HepaRG cells after treatment with compounds that increased Cyp2b1 mRNA levels in rat hepatocytes but did not activate rCAR-3A-G354Q. The results demonstrated six compounds as possible rCAR indirect activators. Taken together, the combined measurement of Cyp2b1 mRNA levels in rat primary hepatocytes and rCAR-3A-G354Q activation in reporter assays can be useful for evaluating rCAR activation by chemicals.

Chronic Treatment With WY-14643 Induces Tumorigenesis and Triglyceride Accumulation in Mouse Livers.

Peroxisome proliferator-activated receptor α (PPARα) is closely related to lipid metabolism and various liver diseases. Previous study has shown that chronic treatment with PPARα agonist WY-14643 can induce liver tumors in rodents, but the implications of this process on lipid metabolism in the liver remain unclear. Thus, this study aimed to explore the influences of chronic treatment with WY-14643 on the liver and hepatic lipid metabolism. Wild-type C57BL/6 mice were treated with WY-14643 (100 mg/kg/week, i.p.) or corn oil, and liver and serum samples were collected for testing after 42 weeks of WY-14643 treatment. The results showed that hepatomegaly, liver tumors with mild liver injury, and hepatocyte proliferation were induced in mice treated with WY-14643. The mRNA and protein expression levels of PPARα downstream targets acyl-CoA oxidase 1 and cytochrome P450 4A were significantly upregulated in the WY-14643-treated group. Lipidomic analysis revealed that chronic treatment with WY-14643 disturbed lipid homeostasis, especially triglycerides (TGs), which were significantly elevated after WY-14643 treatment. Moreover, TG homeostasis-related genes were significantly increased in the WY-14643-treated group. In conclusion, these findings demonstrated that hepatomegaly and liver tumors induced by chronic treatment with WY-14643 in mice are accompanied by hepatocyte proliferation and TG accumulation. SIGNIFICANCE STATEMENT: The present study clearly demonstrated that sustained peroxisome proliferator-activated receptor α (PPARα) activation by chronic treatment with WY-14643 induces hepatomegaly and liver tumors with triglyceride accumulation by regulating lipid homeostasis-related genes in mice. These findings may help to clarify the influences of sustained PPARα activation on liver lipid homeostasis and provide data for the clinically rational use of drugs that can activate PPARα.

Impact of Histotripsy on Development of Intrahepatic Metastases in a Rodent Liver Tumor Model.

Simple SummaryHistotripsy is a novel technique that mechanically disrupts tumors, through precisely controlled acoustic cavitation. There is insufficient evidence regarding the effects of histotripsy on the risk of recurrence and metastases, following tumor debulking. The aim of this study is to evaluate the effect of partial histotripsy tumor ablation (~50–75% tumor volume targeted) on untargeted tumor progression, survival outcomes, risk of metastases and immune infiltration, in an orthotopic, immunocompetent, metastatic rodent hepatocellular carcinoma (HCC) model. Even with partial ablation, complete local tumor regression was observed in 81% of treatment rats, with no recurrence or metastasis. In contrast, 100% of the untreated control animals showed local tumor progression and intrahepatic metastases. Histotripsy-treated animals had statistically significant improved survival outcomes compared to controls (p-value < 0.0001). Histotripsy-treated animals had increased immune infiltration, as compared to controls, which may have contributed to the eventual regression of the untargeted tumor region in partial histotripsy-treated tumors.Histotripsy has been used for tumor ablation, through controlled, non-invasive acoustic cavitation. This is the first study to evaluate the impact of partial histotripsy ablation on immune infiltration, survival outcomes, and metastasis development, in an in vivo orthotopic, immunocompetent rat HCC model (McA-RH7777). At 7–9 days post-tumor inoculation, the tumor grew to 5–10 mm, and ~50–75% tumor volume was treated by ultrasound-guided histotripsy, by delivering 1–2 cycle histotripsy pulses at 100 Hz PRF (focal peak negative pressure P– >30 MPa), using a custom 1 MHz transducer. Complete local tumor regression was observed on MRI in 9/11 histotripsy-treated rats, with no local recurrence or metastasis up to the 12-week study end point, and only a <1 mm residual scar tissue observed on histology. In comparison, 100% of untreated control animals demonstrated local tumor progression, developed intrahepatic metastases, and were euthanized at 1–3 weeks. Survival outcomes in histotripsy-treated animals were significantly improved compared to controls (p-value < 0.0001). There was evidence of potentially epithelial-to-mesenchymal transition (EMT) in control tumor and tissue healing in histotripsy-treated tumors. At 2- and 7-days post-histotripsy, increased immune infiltration of CD11b+, CD8+ and NK cells was observed, as compared to controls, which may have contributed to the eventual regression of the untargeted tumor region in histotripsy-treated tumors.

Metronidazole enhances steatosis-related early-stage hepatocarcinogenesis in high fat diet-fed rats through DNA double-strand breaks and modulation of autophagy.

Nonalcoholic fatty liver disease is a hepatic disorder with deposition of fat droplets and has a high risk of progression to steatosis-related hepatitis and irreversible hepatic cancer. Metronidazole (MNZ) is an antiprotozoal and antimicrobial agent widely used to treat patients infected with anaerobic bacteria and intestinal parasites; however, MNZ has also been shown to induce liver tumors in rodents. To investigate the effects of MNZ on steatosis-related early-stage hepatocarcinogenesis, male rats treated with N-nitrosodiethylamine following 2/3 hepatectomy at week 3 were received a control basal diet, high fat diet (HFD), or HFD containing 0.5% MNZ. The HFD induced obesity and steatosis in the liver, accompanied by altered expression of Pparg and Fasn, genes related to lipid metabolism. MNZ increased nuclear translocation of lipid metabolism-related transcription factor peroxisome proliferator-activated receptor gamma in hepatocytes, together with altered liver expression of lipid metabolism genes (Srebf1, Srebf2, Pnpla2). Furthermore, MNZ significantly increased the number of preneoplastic liver foci, accompanied by DNA double-strand breaks and late-stage autophagy inhibition, as reflected by increased levels of γ-H2AX, LC3, and p62. Therefore, MNZ could induce steatosis-related hepatocarcinogenesis by inducing DNA double-strand breaks and modulating autophagy in HFD-fed rats.

Transcriptomic analyses of livers from mice exposed to 1,4-dioxane for up to 90 days to assess potential mode(s) of action underlying liver tumor development

1,4-Dioxane is a volatile organic compound with industrial and commercial applications as a solvent and in the manufacture of other chemicals. 1,4-Dioxane has been demonstrated to induce liver tumors in chronic rodent bioassays conducted at very high doses. The available evidence for 1,4-dioxane-induced liver tumors in rodents aligns with a threshold-dependent mode of action (MOA), with the underlying mechanism being less clear in the mouse than in rats. To gain a better understanding of the underlying molecular mechanisms related to liver tumor development in mice orally exposed to 1,4-dioxane, transcriptomics analysis was conducted on liver tissue collected from a 90-day drinking water study in female B6D2F1/Crl mice (Lafranconi et al., 2020). Using tissue samples from female mice exposed to 1,4-dioxane in the drinking water at concentrations of 0, 40, 200, 600, 2,000 or 6,000 ppm for 7, 28, and 90 days, transcriptomic analyses demonstrate minimal treatment effects on global gene expression at concentrations below 600 ppm. At higher concentrations, genes involved in phase II metabolism and mitotic cell cycle checkpoints were significantly upregulated. There was an overall lack of enrichment of genes related to DNA damage response. The increase in mitotic signaling is most prevalent in the livers of mice exposed to 1,4-dioxane at the highest concentrations for 90 days. This finding aligns with phenotypic changes reported by Lafranconi et al. (2020) after 90-days of exposure to 6,000 ppm 1,4-dioxane in the same tissues. The transcriptomics analysis further supports overarching study findings demonstrating a non-mutagenic, threshold-based, mitogenic MOA for 1,4-dioxane-induced liver tumors.

Application of humanized mice to toxicology studies: Evaluation of the human relevance of the mode of action for rodent liver tumor formation by activators of the constitutive androstane receptor (CAR).

The constitutive androstane receptor (CAR)-mediated mode of action (MOA) for phenobarbital (PB)-induced rodent liver tumor formation has been established, with increased hepatocyte proliferation, which is a key event in tumor formation. Previous studies have demonstrated that PB and other CAR-activators stimulate proliferation in cultured rodent hepatocytes, but not in cultured human hepatocytes. However, in the genetically humanized CAR and pregnane X receptor (PXR) mouse (hCAR/hPXR mouse, downstream genes are still mouse), PB increased hepatocyte proliferation and tumor production in vivo. In contrast to the hCAR/hPXR mouse, studies with chimeric mice with human hepatocytes (PXB-mouse, both receptor and downstream genes are human) demonstrated that PB did not increase human hepatocyte proliferation in vivo. PB increased hepatocyte proliferation in a chimeric mouse model with rat hepatocytes, indicating that the lack of human hepatocyte proliferation is not due to any functional defect in the chimeric mouse liver environment. Gene expression analysis demonstrated that the downstream genes of CAR/PXR activation were similar in hCAR/hPXR and CD-1 mice, but differed from those observed in chimeric mice with human hepatocytes. These findings strongly support the conclusion that the MOA for CAR-mediated rodent liver tumor formation is qualitatively implausible for humans. Indeed, epidemiological studies have found no causal link between PB and human liver tumors. There are many similarities with respect to hepatic effects and species differences between rodent CAR and peroxisome proliferator-activated receptor α activators. Based on our research, the chimeric mouse with human hepatocytes (PXB-mouse) is reliable for human cancer risk assessment of test chemicals.

Structure-dependent genotoxic potencies of selected pyrrolizidine alkaloids in metabolically competent HepG2 cells

1,2-unsaturated pyrrolizidine alkaloids (PAs) are natural plant constituents comprising more than 600 different structures. A major source of human exposure is thought to be cross-contamination of food, feed and phytomedicines with PA plants. In humans, laboratory and farm animals, certain PAs exert pronounced liver toxicity and can induce malignant liver tumors in rodents. Here, we investigated the cytotoxicity and genotoxicity of eleven PAs belonging to different structural classes. Although all PAs were negative in the fluctuation Ames test in Salmonella, they were cytotoxic and induced micronuclei in human HepG2 hepatoblastoma cells over-expressing human cytochrome P450 3A4. Lasiocarpine and cyclic diesters except monocrotaline were the most potent congeners both in cytotoxicity and micronucleus assays with concentrations below 3 μM inducing a doubling in micronuclei counts. Other open di-esters and all monoesters exhibited weaker or much weaker geno- and cytotoxicity. The findings were in agreement with recently suggested interim Relative Potency (iREP) factors with the exceptions of europine and monocrotaline. A more detailed micronuclei analysis at low concentrations of lasiocarpine, retrorsine or senecionine indicated that pronounced hypolinearity of the concentration–response curves was evident for retrorsine and senecionine but not for lasiocarpine. Our findings show that the genotoxic and cytotoxic potencies of PAs in a human hepatic cell line vary in a structure-dependent manner. Both the low potency of monoesters and the shape of prototype concentration–response relationships warrant a substance- and structure-specific approach in the risk assessment of PAs.

Quantitation of DNA reactive pyrrolic metabolites of senecionine – A carcinogenic pyrrolizidine alkaloid by LC/MS/MS analysis

Pyrrolizidine alkaloids (PAs) are carcinogenic phytochemicals, inducing liver tumors in experimental rodents. We previously determined that (±)-6,7-dihydro-7-hydroxy-1-hydroxymethyl-5H-pyrrolizine (DHP), 7-glutathione-DHP, 7-cysteine-DHP, 7-N-acetylcysteine-DHP, and 1-CHO-DHP are DNA reactive pyrrolic metabolites potentially associated with PA-induced liver tumor initiation. In this study, we developed an LC/MS/MS multiple reaction monitoring (MRM) mode method to identify and quantify these metabolites formed from the metabolism of senecionine, a carcinogenic PA, by mouse, rat, and human liver microsomes, and primary rat hepatocytes. Together with the chemically prepared standards of these metabolites, this represents an accurate and convenient LC/MS/MS analytical method for quantifying these five reactive pyrrolic metabolites in biological systems.

Iron-Oxide Nanocluster Labeling of Clostridium novyi-NT Spores for MR Imaging–Monitored Locoregional Delivery to Liver Tumors in Rat and Rabbit Models

PurposeTo label Clostridium novyi-NT spores (C. novyi-NT) with iron oxide nanoclusters and track distribution of bacteria during magnetic resonance (MR) imaging-monitored locoregional delivery to liver tumors using intratumoral injection or intra-arterial transcatheter infusion. Materials and MethodsVegetative state C. novyi-NT were labeled with iron oxide particles followed by induction of sporulation. Labeling was confirmed with fluorescence microscopy and transmission electron microscopy (TEM). T2 and T2* relaxation times for magnetic clusters and magnetic microspheres were determined using 7T and 1.5T MR imaging scanners. In vitro assays compared labeled bacteria viability and oncolytic potential to unlabeled controls. Labeled spores were either directly injected into N1-S1 rodent liver tumors (n = 24) or selectively infused via the hepatic artery in rabbits with VX2 liver tumors (n = 3). Hematoxylin-eosin, Prussian blue, and gram staining were performed. Statistical comparison methods included paired t-test and ANOVA. ResultsBoth fluorescence microscopy and TEM studies confirmed presence of iron oxide labels within the bacterial spores. Phantom studies demonstrated that the synthesized nanoclusters produce R2 relaxivities comparable to clinical agents. Labeling had no significant impact on overall growth or oncolytic properties (P >.05). Tumor signal-to-noise ratio (SNR) decreased significantly following intratumoral injection and intra-arterial infusion of labeled spores (P <.05). Prussian blue and gram staining confirmed spore delivery. ConclusionsC. novyi-NT spores can be internally labeled with iron oxide nanoparticles to visualize distribution with MR imaging during locoregional bacteriolytic therapy involving direct injection or intra-arterial transcatheter infusion.

Validation of urinary sphingolipid metabolites as biomarker of effect for fumonisins exposure in Kenyan children

Context: Fumonisins (FNs), a group of mycotoxins produced mainly by Fusarium species, are ubiquitous food contaminants, especially for maize. Fumonisin B1 (FB1) caused severe toxicities in farm animals, induced kidney and liver tumours in rodents and is associated with many human adverse health effects, including oesophageal cancer. International Agency for Research on Cancer (IARC) categorizes FB1 as a possible human carcinogen (Group 2B). Inhibition of ceramide synthesis and disruption of sphingolipids metabolism are well studied as the major mechanisms of FB1-induced toxicity. Increases in sphinganine (Sa) and decrease in sphingosine (So) levels and their ratio are validated biomarkers of FB1 effects.Methods: In this study, we measured urinary levels of Sa, So and Sa/So in 284 children aged 1–14 years who consume maize as a staple diet. Exfoliated cells from urine were processed and sphingolipids quantified by High Pressure Liquid Chromatography.Results and conclusions: Sa and So were detectable in 95.07% and 98.94% of samples, respectively. Creatinine adjusted mean levels and standard deviation of Sa, So and Sa/So ratio were 1.23 ± 2.18, 4.99 ± 8.3 and 0.296 ± 0.587 nM. These results further confirmed the findings in studies with human adults, i.e. urinary Sa, So levels and Sa/So ratio are good biomarkers to assess FNs exposure in children.

Cross-species comparison of CAR-mediated procarcinogenic key events in a 3D liver microtissue model.

Characterisation of the mode of action (MOA) of constitutive androstane receptor (CAR)-mediated rodent liver tumours involves measurement 5 key events including activation of the CAR receptor, altered gene expression, hepatocellular proliferation, clonal expansion and increased hepatocellular adenomas/carcinomas. To test whether or not liver 3D microtissues (LiMTs) recapitulate CAR- mediated procarcinogenic key events in response to the prototypical CAR activator phenobarbital (PB) we performed hepatocyte proliferation (LI%) analysis in rat and human LiMTs using a microTMA technology in conjunction with integrated transcriptomics (microarray) and proteomics analysis. The rationale for this approach was that LiMTs containing parenchymal and non-parenchymal cells (NPCs) are more physiologically representative of liver and thus would generate data more relevant to the in vivo situation. Rat and human LiMTs were treated with PB over a range of concentrations (500 uM - 2000 uM) and times (24 h - 96 h) in a dose-response/time-course analysis. There was a dose-dependent induction of LI% in rat LiMTs, however there was little or no effect of PB on LI% in human LiMTs. ATP levels in the rat and human LiMTs were similar to control in all of the PB treatments. There was also a dose- and time-dependent PB-mediated RNA induction of CAR regulated genes CYP2B6/Cyp2b2, CYP3A7/Cyp3a9 and UGT1A6/Ugt1a6 in human and rat LiMTs, respectively. These CAR regulated genes were also upregulated at the protein level. Ingenuity pathways analysis (IPA) indicated that there was a significant (Z score >2.0;-log p value >) activation of CAR by PB in both human and rat LiMTs. These results indicate that human and rat LiMTs showed the expected responses at the level of PB-induced hepatocyte proliferation and enzyme induction with rat LiMTs showing significant dose-dependent effects while human LiMTs showed no proliferation response but did show dose-dependent enzyme induction at the RNA and protein levels. In conclusion LiMTs serve as a model to provide mechanistic data for 3 of the 5 key events considered necessary to establish a CAR-mediated MOA for liver tumourigenesis and thus can potentially reduce the use of animals when compiling mechanistic data packages.

Mode of action and human relevance of rodent liver tumours produced by nongenotoxic CAR and PPARα activators

Mode of action and human relevance of rodent liver tumours produced by nongenotoxic CAR and PPARα activators

Minimum datasets to establish a CAR-mediated mode of action for rodent liver tumors

Methods for investigating the Mode of Action (MoA) for rodent liver tumors via constitutive androstane receptor (CAR) activation are outlined here, based on current scientific knowledge about CAR and feedback from regulatory agencies globally. The key events (i.e., CAR activation, altered gene expression, cell proliferation, altered foci and increased adenomas/carcinomas) can be demonstrated by measuring a combination of key events and associative events that are markers for the key events. For crop protection products, a primary dataset typically should include a short-term study in the species/strain that showed the tumor response at dose levels that bracket the tumorigenic and non-tumorigenic dose levels. The dataset may vary depending on the species and the test compound. As examples, Case Studies with nitrapyrin (in mice) and metofluthrin (in rats) are described. Based on qualitative differences between the species, the key events leading to tumors in mice or rats by this MoA are not operative in humans. In the future, newer approaches such as a CAR biomarker signature approach and/or in vitro CAR3 reporter assays for mouse, rat and human CAR may eventually be used to demonstrate a CAR MoA is operative, without the need for extensive additional studies in laboratory animals.

Comparison of the hepatic and thyroid gland effects of sodium phenobarbital in wild type and constitutive androstane receptor (CAR) knockout rats and pregnenolone-16α-carbonitrile in wild type and pregnane X receptor (PXR) knockout rats

A number of chemicals produce liver and thyroid gland tumours in rodents by nongenotoxic modes of action (MOAs). In this study the hepatic and thyroid gland effects of the constitutive androstane receptor (CAR) activator sodium phenobarbital (NaPB) were examined in male Sprague-Dawley wild type (WT) rats and in CAR knockout (CAR KO) rats and the effects of the pregnane X receptor (PXR) activator pregnenolone-16α-carbonitrile (PCN) were examined in WT and PXR knockout (PXR KO) rats. Rats were either fed diets containing 0 (control) or 500 ppm NaPB or were dosed with 0 (control) or 100 mg/kg/day PCN orally for 7 days. The treatment of WT rats with NaPB and PCN for 7 days resulted in increased relative liver weight, increased hepatocyte replicative DNA synthesis (RDS) and the induction of cytochrome P450 CYP2B and CYP3A subfamily enzyme, mRNA and protein levels. In marked contrast, the treatment of CAR KO rats with NaPB and PXR KO rats with PCN did not result in any increases in liver weight and induction of CYP2B and CYP3A enzymes. The treatment of CAR KO rats with NaPB had no effect on hepatocyte RDS, while PCN produced only a small increase in hepatocyte RDS in PXR KO rats. Treatment with NaPB had no effect on thyroid gland weight in WT and CAR KO rats, whereas treatment with PCN resulted in an increase in relative thyroid gland weight in WT, but not in PXR KO, rats. Thyroid gland follicular cell RDS was increased by the treatment of WT rats with NaPB and PCN, with NaPB also producing a small increase in thyroid gland follicular cell RDS in CAR KO rats. Overall, the present study with CAR KO rats demonstrates that a functional CAR is required for NaPB-mediated increases in liver weight, stimulation of hepatocyte RDS and induction of hepatic CYP enzymes. The studies with PXR KO rats demonstrate that a functional PXR is required for PCN-mediated increases in liver weight and induction of hepatic CYP enzymes; with induction of hepatocyte RDS also being largely mediated through PXR. The hepatic effects of NaPB in CAR KO rats and of PCN in PXR KO rats are in agreement with those observed in other recent literature studies. These results suggest that CAR KO and PXR KO rats are useful experimental models for liver MOA studies with rodent CAR and PXR activators and may also be useful for thyroid gland MOA studies.

Case examples of an evaluation of the human relevance of the pyrethroids/pyrethrins-induced liver tumours in rodents based on the mode of action.

Rodent carcinogenicity studies are useful for screening for human carcinogens but they are not perfect. Some modes of action (MOAs) lead to cancers in both experimental rodents and humans, but others that lead to cancers in rodents do not do so in humans. Therefore, analysing the MOAs by which chemicals produce tumours in rodents and determining the relevance of such tumour data for human risk are critical. Recently, experimental data were obtained as case examples of an evaluation of the human relevance of pyrethroid (metofluthrin and momfluorothrin)- and pyrethrins-induced liver tumours in rats based on MOA. The MOA analysis, based on the International Programme on Chemical Safety (IPCS) framework, concluded that experimental data strongly support that the postulated MOA for metofluthrin-, momfluorothrin- and pyrethrins-produced rat hepatocellular tumours is mediated by constitutive androstane receptor (CAR) activation. Since metofluthrin and momfluorothrin are close structural analogues, reproducible outcomes for both chemicals provide confidence in the MOA findings. Furthermore, cultured human hepatocyte studies and humanized chimeric mouse liver studies demonstrated species difference between human hepatocytes (refractory to the mitogenic effects of these compounds) and rat hepatocytes (sensitive to their mitogenic effects). These data strongly support the hypothesis that the CAR-mediated MOA for liver tumorigenesis is of low carcinogenic risk for humans. In this research, in addition to cultured human hepatocyte studies, the usefulness of the humanized chimeric liver mouse models was clearly demonstrated. These data substantially influenced decisions in regulatory toxicology. In this review I comprehensively discuss the human relevance of the CAR-mediated MOA for rodent liver tumorigenesis based on published information, including our recent molecular research on CAR-mediated MOA.

Comparison of the effects of sodium phenobarbital in wild type and humanized constitutive androstane receptor (CAR)/pregnane X receptor (PXR) mice and in cultured mouse, rat and human hepatocytes

Phenobarbital (PB), a constitutive androstane receptor (CAR) activator, produces liver tumours in rodents by a mitogenic mode of action involving CAR activation. In this study, the hepatic effects of sodium phenobarbital (NaPB) were compared in male C57BL/6J wild type (WT) mice and in humanized mice, where both the mouse CAR and pregnane X receptor (PXR) have been replaced by their human counterparts (hCAR/hPXR mice). Investigations were also performed in cultured male C57BL/6J and CD-1 mouse, male Sprague-Dawley rat and male and female human hepatocytes. The treatment of WT and hCAR/hPXR mice with 186–984 ppm NaPB in the diet for 7 days resulted in increased relative liver weight, hypertrophy and induction of cytochrome P450 (CYP) enzyme activities. Treatment with NaPB also produced dose-dependent increases in hepatocyte replicative DNA synthesis (RDS), with the effect being more marked in WT than in hCAR/hPXR mice. While the treatment of cultured C57BL/6J and CD-1 mouse, Sprague-Dawley rat and human hepatocytes with 100 and/or 1000 μM NaPB for 4 days induced CYP enzyme activities, increased RDS was only observed in mouse and rat hepatocytes. However, as a positive control, epidermal growth factor increased RDS in hepatocytes from all three species. In summary, although human hepatocytes are refractory to the mitogenic effects of NaPB, treatment with NaPB induced RDS in vivo in hCAR/hPXR mice, which is presumably due to the human CAR and PXR receptors operating in a mouse hepatocyte regulatory environment. As the response of the hCAR/hPXR mouse to the CAR activator NaPB differs markedly from that of human hepatocytes, the hCAR/hPXR mouse is thus not a suitable animal model for studies on the hepatic effects of nongenotoxic rodent CAR activators.

Biphasic hCAR Inhibition-Activation by Two Aminoazo Liver Carcinogens

Detailed dose-response data recently archived by the National Center for Biotechnology Information (NCBI) identified 853 human CAR (hCAR) agonists by quantitative high-throughput screening (qHTS) assays applied to >9,000 chemicals tested at ≥14 concentrations using n = 3–48 replicates. By re-examining NCBI data on 746 agonists with replicate data sets each satisfying additional quality criteria, ∼95% had average values of agonist-specific Hill-model slopes estimated by NCBI that exceed 1 (i.e., exhibited an overall sublinear low-dose dose-response), and two unambiguously biphasic hCAR inhibitor-agonists were identified, 4-aminoazobenzene (n = 37) and ortho-aminoazotoluene (n = 3), both of which also cause rodent liver tumors. Although evidently rare among hCAR agonists, such biphasic responses add to evidence that nuclear receptors can exhibit complex patterns of low-dose response, consistent with previous observations and theoretical predictions for endpoints governed by ultrasensitive molecular switches. The pronounced biphasic hCAR response pattern observed for 4-aminoazobenzene is particularly noteworthy insofar as it was identified with statistical power that exceeds that of most if not all other receptor-mediated biphasic cellular responses to any single-chemical exposure reported to date.