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

The kinetics of metabolism of deltamethrin (DLM) and cis- and trans-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 (CLint) 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. However, when apparent CLint values were corrected for nonspecific binding to allow calculation of unbound (i.e., corrected) CLint values, the unbound values did not vary greatly with microsomal protein concentration. Unbound CLint values for metabolism of 0.05-1 µM DLM in rat liver microsomes (CYP and CES enzymes) and cytosol (CES enzymes) were not significantly different from rates of DLM metabolism in isolated rat hepatocytes. This study demonstrates that the nonspecific binding of these highly lipophilic compounds needs to be taken into account in order to obtain accurate estimates of rates of in vitro metabolism of these pyrethroids. While DLM is rapidly metabolised in vitro, the hepatocyte membrane does not appear to represent a barrier to the absorption and hence subsequent hepatic metabolism of this pyrethroid.

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. Treatment with 100 and 300 mg/kg/day PBO and 500 ppm NaPB increased relative liver weight which was associated with hepatocyte hypertrophy, with hepatocyte replicative DNA synthesis (RDS) being increased after 7 days treatment. The treatment of CD-1 mice with 30-300 mg/kg/day PBO for 14 days resulted in significant dose-dependent increases in hepatic microsomal cytochrome P450 (CYP) content and 7-pentoxyresorufin O-depentylase (PROD) activity and in hepatic Cyp2b10 mRNA levels. In contrast, PBO produced a biphasic effect on markers of activation of the peroxisome proliferator-activated receptor alpha (PPARα), with small increases in microsomal lauric acid 12-hydroxylase activity and hepatic Cyp4a10 mRNA levels being observed in mice given 100 mg/kg/day with PBO, with either no increase or a significant inhibition being observed in mice given 300 mg/kg/day PBO. The hepatic effects of PBO in male CD-1 mice were generally similar to those produced by NaPB and were reversible after the cessation of treatment for 28 days. Studies were also performed in male C57BL/6J (wild type) mice and in hepatic CAR and pregnane X receptor (PXR) knockout mice (CAR KO/PXR KO mice), where in the CAR KO/PXR KO mice PBO had little effect on markers of CAR activation, but produced some increases in markers of PPARα activation. The treatment of male CD-1 mouse hepatocytes for 4 days with 5-50 µM PBO, 10-1000 µM NaPB and 25 ng/mL epidermal growth factor (EGF) resulted in significant increases in hepatocyte RDS. While treatment of hepatocytes from one male and one female human donor with 5-500 µM PBO and 10-1000 µM NaPB for 4 days had no effect on hepatocyte RDS, treatment with EGF resulted in significant increases in RDS in both human hepatocyte preparations. In summary, PBO is predominantly a hepatic CAR activator at carcinogenic dose levels in CD-1 mice, with activation of hepatic CAR resulting in a suppression of the effect of PBO on hepatic PPARα. A robust MOA for PBO-induced mouse liver tumour formation has been established, this MOA being similar to that previously identified for NaPB and some other rodent liver CAR activators. Based on the lack of effect of PBO on RDS in human hepatocytes, it is considered that the MOA for PBO-induced mouse liver tumour formation is qualitatively not plausible for humans.

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 1.11.1.8).2. An assay was developed for rat thyroid gland microsomal TPO activity, employing L-tyrosine as the physiological substrate, with analysis of the formation of the 3-iodo-L-tyrosine (3MIT) metabolite by ultra-performance liquid chromatography-mass spectrometry-mass spectrometry.3. Formation of 3MIT was linear with respect to both rat thyroid gland microsomal protein concentration and incubation time, whereas only small quantities of 3,5-diodo-L-tyrosine were formed.4. Studies were performed with nine known TPO inhibitors. The most potent inhibitors were 3-amino-1,2,4-triazole, ethylene thiourea, methimazole and 6-propyl-2-thiouracil which had IC50 values (i.e. concentration to produce a 50% inhibition of enzyme activity) of 0.059, 0.791, 1.07 and 1.96 µM, respectively, whereas the least potent inhibitor was sodium perchlorate which had an IC50 value of 13,800 µM.5. For five inhibitors, where literature data were available, the observed IC50 values obtained in this study employing rat thyroid gland microsomes and L-tyrosine as substrate were similar to those previously reported using the spectrophotometric guaiacol oxidation assay.

Mode of action analysis for pesticide-induced rodent liver tumours involving activation of the constitutive androstane receptor: relevance to human cancer risk.

A number of non-genotoxic chemicals, including some pesticides, have been shown to increase the incidence of liver tumours in rats and/or mice. Frameworks for analysing the modes of action (MOAs) by which chemicals produce liver tumours in rodents and the relevance of such tumour data for human risk assessment have now been established. One common MOA for rodent liver tumour formation by non-genotoxic chemicals involves activation of the constitutive androstane receptor (CAR). Key and associative events for a CAR-activation MOA include receptor activation, liver hypertrophy, induction of cytochrome P450 enzyme activities, increased replicative DNA synthesis, altered hepatic foci and liver tumours. While some effects of rodent CAR activators can be observed in human liver, a major species difference is that, unlike rodents, CAR activators do not increase replicative DNA synthesis in human hepatocytes. The CAR-activation MOA for rodent liver tumour formation is thus not plausible for humans, and hence such compounds do not pose a hepatocarcinogenic hazard for humans.

Evaluation of the metabolism and hepatotoxicity of xenobiotics utilizing precision-cut slices.

1. Precision-cut liver slices are a valuable in vitro model system to study the metabolism and toxicity of xenobiotics. Liver slices retain tissue architecture so that all cell types are present and intercellular communication between the various cell types is retained. 2. Precision-cut liver slices from humans and other species have been used to study pathways of phase I (e.g. cytochrome P450-dependent biotransformations) and II (e.g. conjugation with D-glucuronic acid, sulphate and glutathione) metabolism of a wide range of xenobiotics. 3. Liver slices can also be employed to investigate the induction and inhibition of xenobiotic metabolizing enzymes and to obtain kinetic data on the rates of metabolism of xenobiotics. 4. Precision-cut liver slices from humans and other species have been used to study the toxicity of a wide variety of xenobiotics. Toxicity can be assessed by various techniques including gene expression, morphological examination and a wide range of biochemical endpoints. 5. Precision-cut liver slices can be utilized to examine species differences in hepatic xenobiotic metabolism and xenobiotic-induced toxicity, thus permitting comparisons between animal species and humans.

In vitro assays for induction of drug metabolism.

Hepatic microsomal cytochrome P450 (CYP) forms have a major role in the metabolism of drugs and other chemicals. Primary hepatocyte cultures from humans and experimental animals are a valuable in vitro system for studying the effects of chemicals on CYP forms. This chapter describes methods to evaluate CYP form induction in human and rat hepatocytes cultured in a 96-well plate format. The use of a 96-well plate format permits studies to be performed with relatively small numbers of hepatocytes and obviates the need to harvest cells and prepare subcellular fractions prior to the assay of enzyme activities. The induction of CYP1A and CYP3A forms in human and rat hepatocytes can be determined by measurement of 7-ethoxyresorufin O-deethylase and testosterone 6beta-hydroxylase activities, respectively, whereas 7-benzyloxy-4-trifluoromethylcoumarin (BFC) O-debenzylase can be employed to assess both CYP1A and CYP2B form induction in rat hepatocytes. An assay for determining the protein content of hepatocytes cultured in a 96-well plate format is also described.

Transcriptome analysis provides new insights into liver changes induced in the rat upon dietary administration of the food additives butylated hydroxytoluene, curcumin, propyl gallate and thiabendazole.

Transcriptomics was performed to gain insight into mechanisms of food additives butylated hydroxytoluene (BHT), curcumin (CC), propyl gallate (PG), and thiabendazole (TB), additives for which interactions in the liver can not be excluded. Additives were administered in diets for 28 days to Sprague-Dawley rats and cDNA microarray experiments were performed on hepatic RNA. BHT induced changes in the expression of 10 genes, including phase I (CYP2B1/2; CYP3A9; CYP2C6) and phase II metabolism (GST mu2). The CYP2B1/2 and GST expression findings were confirmed by real time RT-PCR, western blotting, and increased GST activity towards DCNB. CC altered the expression of 12 genes. Three out of these were related to peroxisomes (phytanoyl-CoA dioxygenase, enoyl-CoA hydratase; CYP4A3). Increased cyanide insensitive palmitoyl-CoA oxidation was observed, suggesting that CC is a weak peroxisome proliferator. TB changed the expression of 12 genes, including CYP1A2. In line, CYP1A2 protein expression was increased. The expression level of five genes, associated with p53 was found to change upon TB treatment, including p53 itself, GADD45alpha, DN-7, protein kinase C beta and serum albumin. These array experiments led to the novel finding that TB is capable of inducing p53 at the protein level, at least at the highest dose levels employed above the current NOAEL. The expression of eight genes changed upon PG administration. This study shows the value of gene expression profiling in food toxicology in terms of generating novel hypotheses on the mechanisms of action of food additives in relation to pathology.

Effect of Pyrethrins on cytochrome P450 forms in cultured rat and human hepatocytes.

High doses of Pyrethrins produce liver and thyroid gland tumours in rats by modes of action involving the induction of hepatic xenobiotic metabolising enzymes. The aim of this study was to compare the effects of Pyrethrins with those of the rat liver and thyroid tumour promoter sodium Phenobarbital on some cytochrome P450 (CYP) forms in cultured rat and human hepatocytes. The treatment of female Sprague-Dawley rat and human (both male and female) hepatocytes for 72 h with 0-1000 microM Pyrethrins and 0-1000 microM Phenobarbital did not result in any marked cytotoxicity. In rat hepatocytes both Pyrethrins and Phenobarbital produced an induction of 7-benzyloxy-4-trifluoromethylcoumarin O-debenzylase activity (a CYP1A/2B form marker) and CYP2B1 and CYP2B1/2 mRNA levels. Pyrethrins and Phenobarbital also induced CYP3A-dependent testosterone 6beta-hydroxylase activity in rat hepatocytes. In human hepatocytes Pyrethrins and Phenobarbital induced both testosterone 6beta-hydroxylase activity and CYP3A4 mRNA levels and also increased CYP2B6 mRNA levels. The effects of Pyrethrins and Phenobarbital were concentration-dependent and exhibited a threshold. These results demonstrate that the effects of Pyrethrins on CYP forms in cultured rat and human hepatocytes are qualitatively similar to those of Phenobarbital. Pyrethrins induce CYP2B and CYP3A forms in cultured rat hepatocytes and can induce CYP3A and CYP2B forms in human hepatocytes. While CYP form induction by Pyrethrins, Phenobarbital and related compounds can be associated with liver and thyroid gland tumour formation in rodents, epidemiological data for Phenobarbital suggests that such effects do not occur in humans.

A mode of action for induction of liver tumors by Pyrethrins in the rat.

High doses of Pyrethrins produce liver tumors in female rats. To elucidate the mode of action for tumor formation, the hepatic effects of Pyrethrins have been investigated. Male Sprague-Dawley CD rats were fed diets containing 0 (control) and 8000 ppm Pyrethrins and female rats' diets containing 0, 100, 3000 and 8000 ppm Pyrethrins for periods of 7, 14 and 42 days and 42 days followed by 42 days of reversal. As a positive control, rats were also fed diets containing 1200-1558 ppm sodium Phenobarbital (NaPB) for 7 and 14 days. The treatment of male rats with 8000 ppm Pyrethrins, female rats with 3000 and 8000 ppm Pyrethrins and both sexes with NaPB resulted in increased liver weights, which were associated with hepatocyte hypertrophy. Hepatocyte replicative DNA synthesis was also increased by treatment with Pyrethrins and NaPB. The treatment of male and female rats with Pyrethrins and NaPB produced significant increases in hepatic microsomal cytochrome P450 (CYP) content and a marked induction of CYP2B-dependent 7-pentoxyresorufin O-depentylase and testosterone 16beta-hydroxylase activities. Significant increases were also observed in CYP3A-dependent testosterone 6beta-hydroxylase activity. The hepatic effects of Pyrethrins were dose-dependent in female rats with 100 ppm being a no effect level and on cessation of treatment were reversible in both sexes. This study demonstrates that Pyrethrins are mitogenic CYP2B form inducers in rat liver. The mode of action for Pyrethrins-induced rat liver tumor formation appears to be similar to that of NaPB and some other non-genotoxic CYP2B inducers of hepatic xenobiotic metabolism.

Effect of thiabendazole on some rat hepatic xenobiotic metabolising enzymes.

The effect of thiabendazole (TB) on some rat hepatic xenobiotic metabolising enzymes has been investigated. Male Sprague-Dawley rats were fed control diet or diets containing 102-5188 ppm TB for 28 days. As a positive control for induction of hepatic xenobiotic metabolism, rats were also fed diets containing 1457 and 10,155 ppm butylated hydroxytoluene (BHT). Treatment with TB and BHT resulted in dose-dependent increases in relative liver weight. TB was found to be a mixed inducer of cytochrome P450 (CYP) forms in the CYP1A and CYP2B subfamilies. The administration of high doses of TB resulted in the induction of 7-ethoxyresorufin O-deethylase and 7-pentoxyresorufin O-depentylase activities, CYP1A1, CYP1A2, CYP2B1 and CYP2B1/2 mRNA levels and CYP1A2 and CYP2B1/2 apoprotein levels. In contrast, BHT was a CYP2B form inducer, increasing 7-pentoxyresorufin O-depentylase activity, CYP2B1 and CYP2B1/2 mRNA levels and CYP2B1/2 apoprotein levels. Both TB and BHT induced GSH S-transferase activities towards a range of substrates. In addition, TB and BHT markedly induced GSTP1 mRNA levels, but had only a small effect on GSTT1 mRNA levels. In summary, these results demonstrate that TB induces both phase I and II xenobiotic metabolising enzymes in rat liver.

Metabolism of nicotine and induction of CYP1A forms in precision-cut rat liver and lung slices.

The aim of this study was to investigate xenobiotic metabolism and induction of cytochrome P450 (CYP) forms in precision-cut rat liver and lung slices, employing nicotine as a model compound. Freshly cut rat liver and lung slices metabolised nicotine to the major metabolite cotinine. Observed Km values for cotinine formation in liver and lung slices were 323 and 41.7 microM, respectively, with corresponding V(max) values of 47.2 and 3.21 pmol/min/mg protein, respectively. Rat liver and lung slices were cultured for 48 h with Aroclor 1254, benzo(a)pyrene, nicotine and cotinine. Both Aroclor 1254 and benzo(a)pyrene produced a marked induction of CYP1A-dependent 7-ethoxyresorufin O-deethylase activity in both liver and lung slices. However, while nicotine induced 7-ethoxyresorufin O-deethylase activity in lung slices, but not in liver slices, cotinine did not induce enzyme activity in either liver or lung slices. Overall, while higher rates of nicotine metabolism were observed in rat liver slices, nicotine-induced CYP1A form induction was observed in lung slices. These results demonstrate the usefulness of precision-cut tissue slices for studying tissue differences in xenobiotic metabolism and CYP form induction.

Studies on the metabolism of the thiofurans furfuryl mercaptan and 2-methyl-3-furanthiol in rat liver.

The metabolism of two thiofurans, namely furfuryl mercaptan (FM) and 2-methyl-3-furanthiol (MTF), to their corresponding methyl sulphide and methyl sulphoxide derivatives has been studied in male Sprague-Dawley rat hepatocytes and liver microsomes. Rat hepatocytes converted FM to furfuryl methyl sulphoxide (FMSO) and MTF to 2-methyl-3-(methylthio)furan sulphoxide (MMFSO). Liver microsomes catalysed the NADPH-dependent metabolism of furfuryl methyl sulphide (FMS) to FMSO and 2-methyl-3-(methylthio)furan sulphide (MMFS) to MMFSO. FMS and MMFS metabolism to their thiofuran methyl sulphoxide derivatives was induced by the treatment of rats with Aroclor 1254 and inhibited in liver microsomes treated with 1-aminobenzotriazole. The NADPH-dependent metabolism of FM to FMSO and MTF to MMFSO in liver microsomes was observed in the presence of S-adenosylmethionine. In summary, both thiofurans can be metabolised in rat liver to their thiofuran methyl sulphide derivatives which can be subsequently S-oxidised to form thiofuran methyl sulphoxides. FM and MTF appear to be substrates for rat hepatic microsomal thiol methyltransferase and the S-oxidation of FMS and MMFS appears to be primarily catalysed by cytochrome P450 forms.

Comparative studies on the cytochrome p450-associated metabolism and interaction potential of selegiline between human liver-derived in vitro systems.

Selegiline was used as a model compound in a project aimed at comparing, evaluating, and integrating different in vitro approaches for the prediction of cytochrome p450 (p450)-catalyzed hepatic drug metabolism in humans (EUROCYP). Metabolic predictions were generated using homology modeling, cDNA-expressed p450 enzymes, human liver microsomes, primary cultured human hepatocytes, and precision-cut human liver slices. All of the in vitro systems correctly indicated the formation of two dealkylated metabolites, desmethylselegiline and methamphetamine. The metabolic instability of selegiline was demonstrated by all of the in vitro systems studied. Estimates of clearance varied from 16 l/h to 223 l/h. With the exception of one approach, all systems underpredicted the in vivo clearance in humans (236 l/h). Despite this, all approaches successfully classified selegiline as a high clearance compound. Homology modeling suggested the participation of CYP2B6 in the demethylation of selegiline and of CYP2D6 in the depropargylation of the drug. Studies with recombinant expressed enzymes and with human hepatic microsomal fraction supported the involvement of CYP2B6 but not of CYP2D6. These techniques also suggested the involvement of CYP1A2, CYP2C8, and CYP2C19 in the biotransformation of selegiline. In vitro, CYP2B6 was the most active form of p450 involved in selegiline metabolism. Metabolism by several enzymes operating in parallel implies a low interaction potential for the drug. None of the techniques alone was able to predict all aspects of the metabolic and kinetic behavior of selegiline in vivo. However, when used as an integrated package, all significant characteristics were predictable.

Induction of cytochrome P450 enzymes in cultured precision-cut human liver slices.

Precision-cut human liver slices obtained from 11 donors were cultured for 72 h in a defined medium (serum free Williams' medium E) supplemented with 0.1 microM insulin and 0.1 microM dexamethasone (DEX). Liver slices were treated with 50 microM concentrations of beta -naphthoflavone (BNF), lansoprazole, rifampicin (RIF), DEX and methylclofenapate and 500 microM sodium phenobarbital (NaPB). The relative apoprotein levels of 12 cytochrome P450 (P450) enzymes were determined in liver slice microsomes using a panel of antipeptide antibodies. Treatment with BNF significantly induced mean levels of CYP1A2 apoprotein to 160% of levels in 72-h control (no test compound) human liver slice microsomes. NaPB significantly induced levels of CYP3A4 apoprotein to 255% of control and RIF significantly induced levels of CYP2C19 and CYP3A4 apoproteins to 265 and 330% of control, respectively. In addition, treatment with RIF increased levels of CYP2A6 apoprotein to 205% of control, and treatment with both NaPB and RIF increased levels of CYP2B6 apoprotein to 370 and 615% of control, respectively. However, these increases were not statistically significant, owing to a variable response between liver slice preparations from different subjects, this being apparent for all inducible P450s. In contrast, none of the compounds examined significantly increased levels of CYP2C8, CYP2C9, CYP2D6, CYP2E1, and CYP4A11 apoproteins. Levels of CYP1A1 apoprotein were not detected in any liver slice sample, either before or after treatment with the model inducers. Overall, these results demonstrate the utility of cultured human liver slices for assessing the effects of chemicals on P450 enzymes.

Dose-related effects of the peroxisome proliferator methylclofenapate in rat liver.

Male Sprague-Dawley rats were fed diets containing 0 (control) and 2.5-750 ppm of the peroxisome proliferator methylclofenapate (MCP) for 1, 4 and 13 weeks. In other studies MCP has been shown to produce liver tumors at dietary levels of 50 and 250, but not 10 ppm. MCP treatment produced increases in relative liver weight and activities of peroxisomal and microsomal fatty acid oxidising enzymes at all time points at doses as low as 10 and 2.5 ppm, respectively. Replicative DNA synthesis was studied by implanting osmotic pumps containing 5-bromo-2'-deoxyuridine during study weeks 0-1, 3-4 and 12-13. Hepatocyte labelling index values were significantly increased by treatment with 10-750 ppm MCP for 1 week and 150-750 ppm MCP for 13 weeks. Treatment with 50-750 ppm MCP for 13 weeks increased hepatic peroxisome proliferator-activated receptor alpha and transforming growth factor-ß1 gene expression to 150-165 and 150-170% of control, respectively. These results demonstrate that while low doses of MCP produce sustained hepatomegaly and peroxisome proliferation in rat liver, higher doses are required to produce a sustained stimulation of replicative DNA synthesis.