Mode of action analysis for fluxapyroxad-induced rat liver tumour formation: evidence for activation of the constitutive androstane receptor and assessment of human relevance.

The fungicide fluxapyroxad (BAS 700 F) has been shown to significantly increase the incidence of liver tumours in male Wistar rats at dietary levels of 1500 and 3000 ppm and in female rats at a dietary level of 3000 ppm via a non-genotoxic mechanism. In order to elucidate the mode of action (MOA) for fluxapyroxad-induced rat liver tumour formation a series of in vivo and in vitro investigative studies were undertaken. The treatment of male and female Wistar rats with diets containing 0 (control), 50, 250, 1500 and 3000 ppm fluxapyroxad for 1, 3, 7 and 14 days resulted in a dose-dependent increases in relative weight at 1500 and 3000 ppm from day 3 onwards in both sexes, with an increase in relative liver weight being also observed in male rats given 250 ppm fluxapyroxad for 14 days.

Evaluation of the human hazard of the liver and lung tumors in mice treated with permethrin based on mode of action.

The non-genotoxic synthetic pyrethroid insecticide permethrin produced hepatocellular adenomas and bronchiolo-alveolar adenomas in female CD-1 mice, but not in male CD-1 mice or in female or male Wistar rats. Studies were performed to evaluate possible modes of action (MOAs) for permethrin-induced female CD-1 mouse liver and lung tumor formation. The MOA for liver tumor formation by permethrin involves activation of the peroxisome proliferator-activated receptor alpha (PPARα), increased hepatocellular proliferation, development of altered hepatic foci, and ultimately liver tumors.

Club Cells Are the Primary Target for Permethrin-Induced Mouse Lung Tumor Formation.

Permethrin has been shown to increase lung adenomas in female CD-1 mice, but not in male mice or Wistar rats. The proposed mode of action (MOA) for permethrin-induced female mouse lung tumor formation involves morphological changes in Club cells; increased Club cell proliferation; increased Club cell hyperplasia, and lung tumor formation. In this study, the treatment of female CD-1 mice with tumorigenic doses (2500 and 5000 ppm) of permethrin, but not with a nontumorigenic dose (20 ppm), for 14 and/or 28 days increased Club cell replicative DNA synthesis.

Critical evaluation of the human relevance of the mode of action for rodent liver tumor formation by activators of the constitutive androstane receptor (CAR).

Many nongenotoxic chemicals have been shown to produce liver tumors in mice and/or rats by a mode of action (MOA) involving activation of the constitutive androstane receptor (CAR). Studies with phenobarbital (PB) and other compounds have identified the key events for this MOA: CAR activation; increased hepatocellular proliferation; altered foci formation; and ultimately the development of adenomas/carcinomas. In terms of human relevance, the pivotal species difference is that CAR activators are mitogenic agents in mouse and rat hepatocytes, but they do not stimulate increased hepatocellular proliferation in humans.

Differential lymphatic versus portal vein uptake of the synthetic pyrethroids deltamethrin and cis-permethrin in rats.

The objective of this study was to obtain data on pathways of absorption of the synthetic pyrethroids deltamethrin (DLM) and cis-permethrin (CPM) following oral administration to rats. Adult male Sprague-Dawley rats with cannulated mesenteric lymph ducts and hepatic portal veins were given single doses of either 5 mg/kg DLM or 60 mg/kg CPM via the duodenum and lymph and portal blood samples collected for up to 300 min. The pyrethroid dosing vehicles (5 mL/kg body weight) were either corn oil or glycerol formal.

Kinetics of metabolism of deltamethrin and - and -permethrin . Studies using rat and human liver microsomes, isolated rat hepatocytes and rat liver cytosol.

The kinetics of metabolism of deltamethrin (DLM) and - and -permethrin (CPM and TPM) was studied in male Sprague-Dawley rat and human liver microsomes. DLM metabolism kinetics was also studied in isolated rat hepatocytes, liver microsomes and cytosol. Apparent intrinsic clearance (CL) values for the metabolism of DLM, CPM and TPM by cytochrome P450 (CYP) and carboxylesterase (CES) enzymes in rat and human liver microsomes decreased with increasing microsomal protein concentration.

Comparison of the Hepatic Effects of Phenobarbital in Chimeric Mice Containing Either Rat or Human Hepatocytes With Humanized Constitutive Androstane Receptor and Pregnane X Receptor Mice.

Using a chimeric mouse humanized liver model, we provided evidence that human hepatocytes are refractory to the mitogenic effects of rodent constitutive androstane receptor (CAR) activators. To evaluate the functional reliability of this model, the present study examined mitogenic responses to phenobarbital (PB) in chimeric mice transplanted with rat hepatocytes, because rats are responsive to CAR activators. Treatment with 1000 ppm PB for 7 days significantly increased replicative DNA synthesis (RDS) in rat hepatocytes of the chimeric mice, demonstrating that the transplanted hepatocyte model is functionally reliable for cell proliferation analysis.

Metabolism of bifenthrin, β-cyfluthrin, λ-cyhalothrin, cyphenothrin and esfenvalerate by rat and human cytochrome P450 and carboxylesterase enzymes.

The metabolism of bifenthrin (BIF), β-cyfluthrin (CYFL), λ-cyhalothrin (CYHA), cyphenothrin (CYPH) and esfenvalerate (ESF) was studied in liver microsomes, liver cytosol and plasma from male Sprague-Dawley rats aged 90, 21 and 15 days and from adult humans. Pyrethroid metabolism was also studied with some human expressed cytochrome P450 (CYP) and carboxylesterase (CES) enzymes. All five pyrethroids were metabolised by adult (90 day old) rat hepatic microsomal CYP and CES enzymes and by cytosolic CES enzymes.

Physiologically Based Pharmacokinetic Modeling in Risk Assessment: Case Study With Pyrethroids.

The assessment of potentially sensitive populations is an important application of risk assessment. To address the concern for age-related sensitivity to pyrethroid insecticides, life-stage physiologically based pharmacokinetic (PBPK) modeling supported by in vitro to in vivo extrapolation was conducted to predict age-dependent changes in target tissue exposure to 8 pyrethroids. The purpose of this age-dependent dosimetry was to calculate a Data-derived Extrapolation Factor (DDEF) to address age-related pharmacokinetic differences for pyrethroids in humans.

Piperonyl butoxide: Mode of action analysis for mouse liver tumour formation and human relevance.

In a 79 week bioassay the pesticide synergist piperonyl butoxide (PBO) was shown to significantly increase the incidence of hepatocellular adenoma (but not hepatocellular carcinoma) in male CD-1 mice at dietary levels of 100 and 300 mg/kg/day PBO and in female mice at a dietary level of 300 mg/kg/day. As PBO is not a genotoxic agent, a series of investigative studies were undertaken to elucidate the mode of action (MOA) for PBO-induced mouse liver tumour formation. Male CD-1 mice were fed diets to provide intakes of 0 (control), 30, 100 and 300 mg/kg/day PBO and for purposes of comparison 500 ppm sodium phenobarbital (NaPB), a known constitutive androstane receptor (CAR) activator, for 7 and 14 days.

An Evaluation of the Human Relevance of the Liver Tumors Observed in Female Mice Treated With Permethrin Based on Mode of Action.

In 2-year studies, the nongenotoxic pyrethroid insecticide permethrin produced hepatocellular tumors in CD-1 mice but not in Wistar rats. Recently, we demonstrated that the mode of action (MOA) for mouse liver tumor formation by permethrin involves activation of the peroxisome proliferator-activated receptor alpha (PPARα), resulting in a mitogenic effect. In the present study, the effects of permethrin and 2 major permethrin metabolites, namely 3-phenoxybenzoic acid and trans-dichlorochrysanthemic acid, on cytochrome P450 mRNA levels and cell proliferation (determined as replicative DNA synthesis) were evaluated in cultured CD-1 mouse, Wistar rat, and human hepatocytes.

Comparative studies on the effects of sodium phenobarbital and two other constitutive androstane receptor (CAR) activators on induction of cytochrome P450 enzymes and replicative DNA synthesis in cultured hepatocytes from wild type and CAR knockout rats.

Nongenotoxic chemicals can produce liver tumours in rats and mice by a mitogenic mode of action involving activation of the constitutive androstane receptor (CAR). The aim of this study was to evaluate the usefulness of cultured hepatocytes from normal (wild type; WT) and CAR knockout (KO) rats to screen compounds as potential activators of rat CAR and to validate this test system. Cultured hepatocytes from male Sprague-Dawley WT and CAR KO rats were treated with either 100 and 1000 μM sodium phenobarbital (NaPB), 3-100 μM fluquinconazole (FQZ), or 3-300 μM 3-(difluoromethyl)-1-methyl-N-(3´,4´,6-trifluoro[1,1´-biphenyl]-2-yl)-1H-pyrazole-4-carboxamide (TI1) for 96 h.

Analysis of β-catenin gene mutations and gene expression in liver tumours of C57BL/10J mice produced by chronic administration of sodium phenobarbital.

In this study liver tumours produced in male and female mice of the low spontaneous liver tumour incidence C57BL/10J strain treated for 99 weeks with 1000 ppm in the diet with the model constitutive androstane receptor (CAR) activator sodium phenobarbital (NaPB) were analysed for β-catenin mutations by Western immunoblotting and DNA/RNA analysis. Some gene array analysis was also performed to identify genes involved in CAR activation and in β-catenin and Hras gene mutations. Analysis of 8 male and 2 female NaPB-induced liver tumour samples (comprising 2 adenomas, 6 carcinomas and 2 samples containing separate adenomas and carcinomas) revealed truncated β-catenin forms in just 4 male liver tumour samples, with the presence of the truncated β-catenin forms being confirmed by β-catenin exon 1-3 mutation analysis.

Development and Application of a Life-Stage Physiologically Based Pharmacokinetic (PBPK) Model to the Assessment of Internal Dose of Pyrethroids in Humans.

To address concerns around age-related sensitivity to pyrethroids, a life-stage physiologically based pharmacokinetic (PBPK) model, supported by in vitro to in vivo extrapolation (IVIVE) was developed. The model was used to predict age-dependent changes in target tissue exposure of 8 pyrethroids; deltamethrin (DLM), cis-permethrin (CPM), trans-permethrin, esfenvalerate, cyphenothrin, cyhalothrin, cyfluthrin, and bifenthrin. A single model structure was used based on previous work in the rat.

Metazachlor: Mode of action analysis for rat liver tumour formation and human relevance.

In a 2-year study the herbicide metazachlor (BAS 479H) was shown to significantly increase the incidence of liver tumours in female Wistar rats at a dietary level of 8000 ppm. As metazachlor is not a genotoxic agent, a series of in vivo and in vitro investigative studies were undertaken to elucidate the mode of action (MOA) for metazachlor-induced female rat liver tumour formation. Male and female Wistar rats were given diets containing 0 (control), 200 and 8000 ppm metazachlor for 3, 7, 14 and 28 days.

An assay for screening xenobiotics for inhibition of rat thyroid gland peroxidase activity.

1. A number of chemicals have been shown to produce disruption of the thyroid gland, resulting in reduced thyroid hormone synthesis, by a mechanism involving inhibition of thyroid peroxidase (TPO) activity (EC 1.11.

Evaluation of Age-Related Pyrethroid Pharmacokinetic Differences in Rats: Physiologically-Based Pharmacokinetic Model Development Using In Vitro Data and In Vitro to In Vivo Extrapolation.

An in vitro to in vivo (IVIVE) extrapolation based-physiologically based pharmacokinetic (PBPK) modeling approach was demonstrated to understand age-related differences in kinetics and how they potentially affect age-related differences in acute neurotoxic effects of pyrethroids. To describe the age-dependent changes in pyrethroid kinetics, it was critical to incorporate age-dependent changes in metabolism into the model. As such, in vitro metabolism data were collected for 3 selected pyrethroids, deltamethrin (DLM), cis-permethrin, and trans-permethrin, using liver microsomes and cytosol, and plasma prepared from immature and adult rats.

Involvement of Peroxisome Proliferator-Activated Receptor-Alpha in Liver Tumor Production by Permethrin in the Female Mouse.

The nongenotoxic pyrethroid insecticide permethrin produced hepatocellular tumors in CD-1 mice but not in Wistar rats. Recently, based on findings of a Pathology Working Group involving an expert panel of pathologists, it was concluded that permethrin increased liver tumors at 2500 and 5000 ppm in female mice, but no treatment-related tumorigenic response occurred in male mice at dose levels examined in the 2-year bioassay. To evaluate a possible mode of action (MOA) for the permethrin female CD-1 mouse hepatocellular tumors, a number of investigative studies were conducted.

Human relevance of rodent liver tumour formation by constitutive androstane receptor (CAR) activators.

A large number of nongenotoxic chemicals have been shown to increase the incidence of liver tumours in rats and/or mice by a mode of action (MOA) involving activation of the constitutive androstane receptor (CAR). Studies with the model CAR activator phenobarbital (PB) and its sodium salt (sodium phenobarbital; NaPB) have demonstrated that the key and associative events for rat and mouse liver tumour formation include CAR activation, increased hepatocyte replicative DNA synthesis (RDS), induction of cytochrome P450 CYP2B subfamily enzymes, liver hypertrophy, increased altered hepatic foci and hepatocellular adenomas/carcinomas. The key species difference between the rat and mouse compared to humans, is that human hepatocytes are refractory to the mitogenic effects of PB/NaPB and other CAR activators.

Metabolism of deltamethrin and cis- and trans-permethrin by human expressed cytochrome P450 and carboxylesterase enzymes.

The metabolism of the pyrethroids deltamethrin (DLM), cis-permethrin (CPM) and trans-permethrin (TPM) was studied in human expressed cytochrome P450 (CYP) and carboxylesterase (CES) enzymes. DLM, CPM and TPM were metabolised by human CYP2B6 and CYP2C19, with the highest apparent intrinsic clearance (CL) values for pyrethroid metabolism being observed with CYP2C19. Other CYP enzymes contributing to the metabolism of one or more of the three pyrethroids were CYP1A2, CYP2C8, CYP2C9*1, CYP2D6*1, CYP3A4 and CYP3A5.

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.

Metabolism of deltamethrin and cis- and trans-permethrin by rat and human liver microsomes, liver cytosol and plasma preparations.

The metabolism of deltamethrin (DLM), cis-permethrin (CPM) and trans-permethrin (TPM) was studied in liver microsomes, liver cytosol and plasma from male Sprague-Dawley rats aged 15, 21 and 90 days and from adult humans. DLM and CPM were metabolised by rat hepatic microsomal cytochrome P450 (CYP) enzymes and to a lesser extent by microsomal and cytosolic carboxylesterase (CES) enzymes, whereas TPM was metabolised to a greater extent by CES enzymes. In human liver, DLM and TPM were mainly metabolised by CES enzymes, whereas CPM was metabolised by CYP and CES enzymes.

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.

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

The hepatic and thyroid gland effects of the constitutive androstane receptor (CAR) activator sodium phenobarbital (NaPB) and the pregnane X receptor (PXR) activator pregnenolone-16α-carbonitrile (PCN) were examined in male Sprague-Dawley wild-type (WT) and knockout (KO) rats lacking both hepatic CAR and PXR receptors (CAR KO/PXR KO rats). The treatment of WT rats for 7 d with 500 ppm NaPB in the diet and 100 mg/kg/d PCN by gavage resulted in increased relative liver weight, hepatocyte hypertrophy, increased hepatocyte replicative DNA synthesis (RDS) and induction of cytochrome P450 CYP2B and CYP3A subfamily enzymes. NaPB and PCN also induced thyroid gland follicular cell RDS and hepatic microsomal UDP-glucuronosyltransferase activity towards thyroxine as substrate.