Risk assessment of substances used in food supplements: the example of the botanical Gymnema sylvestre.

Botanicals and preparations derived from these are among the substances frequently added to foods and food supplements, yet the safety of many botanicals has not been systematically assessed. In the context of the EU-FORA fellowship programme, the fellow performed an assessment on the safety of the botanical Gymnema sylvestre, in accordance with EFSA's guidance on the assessment of safety of botanicals. Although preparations of G. sylvestre are marketed as food supplements, they may appeal to people who are suffering from metabolic syndrome and/or diabetes mellitus. A scientific literature search was carried out using PubMed/MEDLINE and EMBASE electronic databases. Experience was gained by the fellow in systematic data extraction from scientific publications, structuring of the data and evaluating toxicological key parameters, outcomes of clinical significance, pharmacokinetic and pharmacodynamic interactions, uncertainties and methodological shortcomings of studies. Limited evidence from toxicological in vivo studies and human clinical studies suggested lack of relevant adverse effects of this botanical. However, human studies provided some indications that certain Gymnema extracts may enhance the glucose-lowering effects of certain antidiabetic drugs. Considering the uncertainties for the composition of different Gymnema preparations, potential herb-drug interactions and the indications of glucose lowering or hypoglycaemic effects, the use of Gymnema-based food supplements in combination with authorised antidiabetic drugs may be associated with risks. The procedures learned for the safety evaluation of Gymnema may be similarly applied by the fellow for the risk assessment of other substances with nutritional or physiological effect added to foods and food supplements. Furthermore, apart from learning by conducting exercises in risk assessment, the fellow was able to develop other skills (e.g. communication skills), diversify his competencies and expand his network of scientific connections for future collaborations in the field of nutritional risk assessment.

Mixture effects of azole fungicides on the adrenal gland in a broad dose range.

Consumers are exposed to low concentrations of a variety of pesticide residues in or on food. Some of them might interfere with the endocrine system. While each individual active substance has been extensively tested for toxicity and safety, potential combination effects possibly resulting from combined exposure to different pesticides have seldomly been tested so far, especially in vivo. Since the adrenal gland is a key endocrine organ, we investigated if and how substances of a group of fungicides presumed to interfere with the biosynthesis of steroid hormones affect this organ when applied individually and in combination in a broad dose range. A 28-day feeding study was conducted in Wistar rats by using three (tri)azole fungicides considered to potentially affect the endocrine system (cyproconazole, epoxiconazole and prochloraz) individually at five dose levels, ranging from 0.9ppm to 2400ppm, and in combination at three dose levels. The parameters analysed included classical toxicology (pathology, histopathology, clinical chemistry) and molecular toxicology endpoints (gene expression arrays and quantitative real time PCR e.g. of Star, HSD3ß, Cyp11a1, Cyp11b1, Cyp11b2, Cyp 21, ApoE), as well as hormone analysis. A dose-dependent decrease in the adrenal gland weight of rats treated with epoxiconazole alone, which was accompanied by an atrophy of the adrenal gland as well as by an increase in the serum cholesterol level and which only became statistically significant at the top dose levels, was observed. These effects were attenuated in the combination experiments, although the same epoxiconazole concentration was used.

Assessment of three approaches for regulatory decision making on pesticides with endocrine disrupting properties.

Recent EU legislation has introduced endocrine disrupting properties as a hazard-based "cut-off" criterion for the approval of active substances as pesticides and biocides. Currently, no specific science-based approach for the assessment of substances with endocrine disrupting properties has been agreed upon, although this new legislation provides interim criteria based on classification and labelling. Different proposals for decision making on potential endocrine disrupting properties in human health risk assessment have been developed by the German Federal Institute for Risk Assessment (BfR) and other regulatory bodies. All these frameworks, although differing with regard to hazard characterisation, include a toxicological assessment of adversity of the effects, the evaluation of underlying modes/mechanisms of action in animals and considerations concerning the relevance of effects to humans. Three options for regulatory decision making were tested upon 39 pesticides for their applicability and to analyze their potential impact on the regulatory status of active substances that are currently approved for use in Europe: Option 1, based purely on hazard identification (adversity, mode of action, and the plausibility that both are related); Option 2, based on hazard identification and additional elements of hazard characterisation (severity and potency); Option 3, based on the interim criteria laid down in the recent EU pesticides legislation. Additionally, the data analysed in this study were used to address the questions, which parts of the endocrine system were affected, which studies were the most sensitive and whether no observed adverse effect levels were observed for substance with ED properties. The results of this exercise represent preliminary categorisations and must not be used as a basis for definitive regulatory decisions. They demonstrate that a combination of criteria for hazard identification with additional criteria of hazard characterisation allows prioritising and differentiating between substances with regard to their regulatory concern. It is proposed to integrate these elements into a decision matrix to be used within a weight of evidence approach for the toxicological categorisation of relevant endocrine disruptors and to consider all parts of the endocrine system for regulatory decision making on endocrine disruption.

Meeting report: international workshop on endocrine disruptors: exposure and potential impact on consumers health.

The French Agency for Food, Environmental and Occupational Health and Safety (Anses) hosted a two-day workshop on Endocrine Disruptors: Exposure and Potential Impact on Consumers Health, bringing together participants from international organizations, academia, research institutes and from German, Swedish, Danish and French governmental agencies. The main objective of the workshop was to share knowledge and experiences on endocrine disruptors (ED) exposure and potential impact on consumers' health, to identify current risk assessment practices and knowledge gaps and issue recommendations on research needs and future collaboration. The following topics were reviewed: (1) Definition of ED, (2) endpoints to be considered for Risk assessment (RA) of ED, (3) non-monotonic dose response curves, (4) studies to be considered for RA (regulatory versus academic studies), (5) point of departure and uncertainty factors, (6) exposure assessment, (7) regulatory issues related to ED. The opinions expressed during this workshop reflect day-to-day experiences from scientists, regulators, researchers, and others from many different countries in the fields of risk assessment, and were regarded by the attendees as an important basis for further discussions. Accordingly, the participants underlined the need for more exchange in the future to share experiences and improve the methodology related to risk assessment for endocrine disrupters.

Assessment strategies and decision criteria for pesticides with endocrine disrupting properties relevant to humans.

There is growing concern that environmental substances with a potential to modulate the hormonal system may have harmful effects on human health. Consequently, a new EU regulation names endocrine disrupting properties as one of the cut-off criteria for the approval of plant protection products, although it currently fails to provide specific science-based measures for the assessment of substances with such properties. Since specific measures are to be presented by the European Commission in 2013 the development of assessment and decision criteria is a key challenge concerning the implementation of this new EU regulation. Proposals of such decision criteria for substances with potential endocrine disrupting properties in human health risk assessment were developed by the German Federal Institute for Risk Assessment (BfR) and discussed at an expert workshop in November 2009. Under consideration of the requirements laid down within the new plant protection product legislation and the scientific discussions during the workshop, a conceptual framework on evaluation of substances for endocrine disrupting properties in a regulatory context is presented in this paper. Central aspects of the framework include assessment of adversity of effects, establishment of a mode/mechanism of action in animals, considerations concerning the relevance of effects to humans and two options for a regulatory decision.

Synergistic acceleration of thyroid hormone degradation by phenobarbital and the PPAR alpha agonist WY14643 in rat hepatocytes.

Energy balance is maintained by controlling both energy intake and energy expenditure. Thyroid hormones play a crucial role in regulating energy expenditure. Their levels are adjusted by a tight feedback-controlled regulation of thyroid hormone production/incretion and by their hepatic metabolism. Thyroid hormone degradation has previously been shown to be enhanced by treatment with phenobarbital or other antiepileptic drugs due to a CAR-dependent induction of phase II enzymes of xenobiotic metabolism. We have recently shown, that PPAR alpha agonists synergize with phenobarbital to induce another prototypical CAR target gene, CYP2B1. Therefore, it was tested whether a PPAR alpha agonist could enhance the phenobarbital-dependent acceleration of thyroid hormone elimination. In primary cultures of rat hepatocytes the apparent half-life of T3 was reduced after induction with a combination of phenobarbital and the PPAR alpha agonist WY14643 to a larger extent than after induction with either compound alone. The synergistic reduction of the half-life could be attributed to a synergistic induction of CAR and the CAR target genes that code for enzymes and transporters involved in the hepatic elimination of T3, such as OATP1A1, OATP1A3, UGT1A3 and UGT1A10. The PPAR alpha-dependent CAR induction and the subsequent induction of T3-eliminating enzymes might be of physiological significance for the fasting-induced reduction in energy expenditure by fatty acids as natural PPAR alpha ligands. The synergism of the PPAR alpha agonist WY14643 and phenobarbital in inducing thyroid hormone breakdown might serve as a paradigm for the synergistic disruption of endocrine control by other combinations of xenobiotics.

Influence of antimonite, selenite, and mercury on the toxicity of arsenite in primary rat hepatocytes.

The long-term toxicity of arsenic (As) as a result of exposure to contaminated drinking water might be modified by coinciding exposures to elements like selenium, antimony, or mercury. In this study the influence of tetravalent selenite, trivalent antimonite, and divalent mercury was investigated in vitro using cultured primary rat hepatocytes. The cell vitality was assessed in the 3-[4,5-dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide] (MTT), assay with concurrent exposures of the cells to up to 50 microM sodium arsenite(III) and a potential modifier [50 microM sodium(IV) selenite, 10 microM antimony(III) chloride, 25 microM mercuric(II) chloride], which indicated an additive increase in the combined cytotoxicity. Sodium arsenite was tested for genotoxicity in the micronucleus test in a concentration range of 0.25 up to 7.5 microM. In this range, the MTT conversion was at least 80%, indicating high cell viability. Adose-dependent induction of micronuclei was observed. The lowest concentration causing a significantly elevated frequency of micronuclei was 1 microM As (p < 0.05). A significant influence (i.e., reduction of the combined genotoxicity as a result of the presence of a potential modifier) was only observed for 10 and 25 microM antimony chloride (p < 0.05, Fisher's exact test). The metabolic methylation of arsenite was not affected by concurrent incubation with any of the potential modifiers.

Low-level self-tolerance to arsenite in human HepG2 cells is associated with a depressed induction of micronuclei.

Arsenic (As) is one of the most important global environmental toxicants. There is evidence that humans may develop tolerance to As's toxicity. For instance, it is known that uptake of small amounts of As leads to an acquisition of elevated resistance to the element's acute toxicity. Moreover, it was suggested that As-exposed native Andean females of Atacameño ethnicity may have acquired resistance to skin cancer. It is not known how such adaptation could be mechanistically conferred. In this context, the biological selection and cloning of human cells tolerant to As provides a valuable approach to investigate this question. By the means of a 12 weeks culture with increasing doses of As, three different As-resistant clones of the human hepatoma cell line HepG2 were selected. These three clones were similarly and roughly two-fold resistant to the acute toxicity of arsenite (50% reduction of neutral red (NR) uptake at 65 microM versus 115 microM; HepG2 control versus clones HepG2 K1, HepG2 K11 and HepG2 K14, respectively). Moreover, in the cytokinesis-block micronucleus test, these clones showed a significantly reduced induction of micronuclei (MNi) indicating elevated resistance to As genotoxicity as well (e.g. mean MNi rates at a concentration of 25 microM arsenite: 28.5 (control) versus 21.6 (HepG2 K1), 18 (HepG2 K11), and 16 (HepG2 K14), respectively, each P<0.05). The tolerance was neither associated with mRNA induction of putatively As-extruding membrane transporters multidrug resistance-associated protein 1 (MRP1), 2, or 3 nor to mRNA induction of the ubiquitously expressed mammalian ABC half-transporter UMAT (ABCB6). Changes in the metabolic methylation of As could not be detected. There were no differences in the cellular levels of GSH when comparing the clones and the parental line. Taken together the data showed that low-level tolerance to As-mediated cytotoxicity in human HepG2 cells was associated with enhanced resistance to As-induced DNA damage as well.

Inhibitors of mdr1-dependent transport activity delay accumulation of the mdr1 substrate rhodamine 123 in primary rat hepatocyte cultures.

P-glycoproteins (P-gps) encoded by mdr1 (multidrug resistance) genes mediate extrusion of numerous lipophilic xeno- and endobiotics through the plasma membrane. Rhodamine 123 (Rh123), a fluorescent dye which is accumulated by mitochondria, is a mdr1 substrate and a well-established tool to study mdr1 transport activity. Inhibitors of mdr1-dependent transport such as verapamil or cyclosporin A have been found to decrease Rh123 efflux from mdr1-expressing cells. Mdr1b gene expression increases with time in primary rat hepatocyte culture. In hepatocytes cultured for 4 days and expressing high levels of P-gp, intracellular Rh123 accumulation was enhanced in the presence of mdr1 inhibitors (cyclosporin A, 8 and 80 microM, verapamil, 8 and 80 microM, or triton X-100, 8 microM). Surprisingly, in hepatocytes expressing low levels of P-gp (after 1 day of culture), time-dependent Rh123 accumulation was not enhanced, but delayed by cyclosporin A, verapamil or triton X-100. In these cells orthovanadate (50 microM), an inhibitor of P-glycoprotein ATPase activity, suppressed Rh123 accumulation, while tetraethylammonium (200 microM), an organic cation transporter (OCT) substrate, had no effect. The paradoxical delay in Rh123 accumulation by verapamil and cyclosporin A occurred eventhough these compounds decreased dye extrusion from Rh123 pre-loaded cells. These observations suggest that a hitherto unknown mechanism which is sensitive to modulators of mdr1-activity contributes to Rh123 uptake or accumulation in primary rat hepatocytes.

Expression and functional activity of P-glycoprotein in cultured hepatocytes from Oncorhynchus mykiss.

P-glycoproteins encoded by multidrug resistance 1 (mdr1) genes are ATP-dependent transporters located in the plasma membrane that mediate the extrusion of hydrophobic compounds from the cell. Using cultured isolated rainbow trout hepatocytes, we characterized an mdr1-like transport mechanism of the teleost liver. Immunoblots with the monoclonal antibody C219, which recognizes a conserved epitope of P-glycoproteins, revealed the presence of immunoreactive protein(s) of 165 kDa in trout liver and cultured hepatocytes. In trout liver sections, the immunohistochemistry with C219 stained bile canalicular structures. Compounds known to interfere with mdr1-dependent transport (verapamil, vinblastine, doxorubicin, cyclosporin A, and vanadate) all increased the accumulation of rhodamine 123 by hepatocytes. Verapamil, vinblastine, and cyclosporin A decreased the efflux of rhodamine 123 from hepatocytes preloaded with rhodamine 123. By contrast, the substrate of the canalicular cation transporter tetraethylammonium and the inhibitor of the multidrug resistance-associated protein MK571 had no effect on rhodamine 123 transport. The results demonstrate the presence of an mdr1-like transport system in the teleost liver and suggest its function in biliary excretion.

Repression of phenobarbital-dependent CYP2B1 mRNA induction by reactive oxygen species in primary rat hepatocyte cultures.

Xenobiotic-metabolizing cytochrome P-450 (P-450) enzymes not only play a pivotal role in elimination of foreign compounds but also contribute to generation of toxic intermediates, including reactive oxygen species, that may elicit cellular damage if produced excessively. Expression of several xenobiotic-metabolizing P-450 enzymes is induced by phenobarbital (PB). Pronounced induction is observed for the rat CYP2B1 isoform. A primary rat hepatocyte culture system was used to investigate whether reactive oxygen species might modulate PB-dependent CYP2B1 induction. In cells cultivated for 3 days with 1.5 mM PB, substantial CYP2B1 mRNA induction was observed (100%). Addition of H(2)O(2) or of the catalase inhibitor 3-amino-1,2,4-triazole (AT) to the medium repressed induction to approximately 30% (at 1 mM H(2)O(2) and 2 mM AT, respectively). Accordingly, treatment of hepatocytes with PB and the glutathione precursor N-acetylcysteine (NAC) led to enhanced PB-dependent induction (to over 1000% at 10 mM NAC). In primary hepatocyte cultures transfected with a CYP2B1 promoter-luciferase construct containing approximately 2.7 kilobase pairs of the native CYP2B1 promoter sequence, PB-dependent reporter gene activation was repressed by AT and stimulated by N-acetylcysteine. Furthermore, a 263-base pair CYP2B1 promoter fragment encompassing the phenobarbital-responsive enhancer module conferred suppression of PB-dependent luciferase expression by AT and activation by NAC in a heterologous SV40-promoter construct. In summary, these data demonstrate a regulatory mechanism that is dependent on the cellular redox status, which modulates CYP2B1 mRNA induction by PB on the transcriptional level, thus representing a feedback mechanism preventing further P-450-dependent production of reactive oxygen intermediates under oxidative stress.

Induction of cytochrome P450 2B1 by pyrethroids in primary rat hepatocyte cultures.

Numerous xenobiotics are capable of inducing their own metabolism and by enzyme induction can also lead to enhanced biotransformation of other xenobiotics. In this project, we examined the influence of pyrethroids (permethrin, cypermethrin, and fenvalerate) on the expression and activity of the phenobarbital (PB)-inducible cytochrome P450 2B1 isoform (CYP2B1) in primary rat hepatocyte cultures. Incubation of hepatocyte cultures with pyrethroids resulted in a marked CYP2B1 induction. Among the tested pyrethroids, permethrin elicited the most pronounced induction of CYP2B1 mRNA, which exceeded maximal induction achieved by PB at concentrations approximately 10-fold higher. Furthermore, permethrin induced CYP3A1 mRNA expression, while the expression of the CYP1A1 isoform, which in vivo is not responsive to PB treatment, was not significantly affected by pyrethroids. Permethrin-dependent enhancement of CYP2B1 and CYP3A1 mRNA expression was repressed by the hepatotrophic cytokine epidermal growth factor, which is known to also inhibit PB-dependent induction of CYP2B1. Several metabolites of permethrin formed by hepatocytes (3-(2',2'-dichlorovinyl)-2,2-dimethylcyclopropanecarboxylic acid, 3-phenoxybenzyl alcohol, and 3-phenoxybenzoic acid) were ineffective in inducing CYP2B1 mRNA. Furthermore, permethrin stimulated the expression of the luciferase reporter gene under control of the CYP2B1 promoter (comprising the PB-responsive enhancer module) in transiently transfected primary hepatocyte cultures. Thus, permethrin-stimulated gene expression occurred on the transcriptional level. Taken together, these results indicate that the pyrethroid permethrin is a PB-like inducer. Due to its superior potency in induction, permethrin appears as a useful substance for mechanistic studies to elucidate the mechanism of enzyme induction by phenobarbital.

Physiological oxygen tensions modulate expression of the mdr1b multidrug-resistance gene in primary rat hepatocyte cultures.

P-Glycoprotein transporters encoded by mdr1 (multidrug resistance) genes mediate extrusion of an array of lipophilic xenobiotics from the cell. In rat liver, mdr transcripts have been shown to be expressed mainly in hepatocytes of the periportal region. Since gradients in oxygen tension (pO(2)) may contribute towards zonated gene expression, the influence of arterial and venous pO(2) on mRNA expression of the mdr1b isoform was examined in primary rat hepatocytes cultured for up to 3 days. Maximal mdr1b mRNA levels (100%) were observed under arterial pO(2) after 72 h, whereas less than half-maximal mRNA levels (40%) were attained under venous pO(2). Accordingly, expression of mdr protein and extrusion of the mdr1 substrate rhodamine 123 were maximal under arterial pO(2) and reduced under venous pO(2). Oxygen-dependent modulation of mdr1b mRNA expression was prevented by actinomycin D, indicating transcriptional regulation. Inhibition of haem synthesis by 25 microM CoCl(2) blocked mdr1b mRNA expression under both oxygen tensions, whereas 80 microM desferrioxamine abolished modulation by O(2). Haem (10 microM) increased mdr1b mRNA levels under arterial and venous pO(2). In hepatocytes treated with 50 microM H(2)O(2), mdr1b mRNA expression was elevated by about 1.6-fold at venous pO(2) and 1.5-fold at arterial pO(2). These results support the conclusion that haem proteins are crucial for modulation of mdr1b mRNA expression by O(2) in hepatocyte cultures and that reactive oxygen species may participate in O(2)-dependent signal transduction. Furthermore, the present study suggests that oxygen might be a critical modulator for zonated secretion of mdr1 substrates into the bile.

Monitoring of cytochrome P-450 1A activity by determination of the urinary pattern of caffeine metabolites in Wistar and hyperbilirubinemic Gunn rats.

Various studies suggest that induction of cytochrome P-450 1A (CYP1A) might be a valuable therapeutic modality for reducing the hyperbilirubinemia of infants with Crigler-Najjar syndrome type I (CNS-I), a severe form of congenital jaundice. To evaluate inducers of CYP1A as possible tools in the treatment of hyperbilirubinemia, a novel assay was established, based on the analysis of the urinary pattern of caffeine metabolites in rats. Wistar rats received [1-Me-(14)C]-caffeine (10 mg/kg i.p.), before and 48h after administration of the potent CYP1A inducer 5,6-benzoflavone (BNF) (80 mg/kg, i.p.). A substantial increase in the fractions of the terminal caffeine metabolites 1-methyluric acid (1-U), 1-methylxanthine (1-X), and a concomitant decrease in the caffeine demethylation product 1,7-dimethylxanthine (1,7-X) was observed after application of BNF. The ratio of the caffeine metabolites (1-U+1-X)/1,7-X may serve as an index of CYP1A activity in rats in vivo. Hyperbilirubinemic, homozygous (jj) Gunn rats are an accepted model for human CNS-I. In male jj Gunn rats treated with BNF or with indole-3-carbinol (I3C, 80 mg/kg, oral gavage), the inducing effect of BNF and 13C on CYP1A activity was confirmed by the urinary pattern of caffeine metabolites, and was parallelled by a decrease in plasma bilirubin levels. These data demonstrate the usefulness of the established caffeine assay for the evaluation of inducers of CYP1A as tools for reducing hyperbilirubinemia and further confirm the potential value of I3C in the treatment of CNS-I.

Metabolism of 4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) in primary cultures of rat alveolar type II cells.

The tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) induces primarily lung tumors, which are assumed to derive from malignant transformation of alveolar type II (AII) cells within the lung. To elicit its carcinogenic effects, NNK requires metabolic activation by cytochrome P-450 (CYP)-mediated alpha-hydroxylation. Therefore, in this study the metabolism of NNK and expression of the NNK-activating CYP isoform CYP2B1 were investigated in primary cultures of rat AII cells. Although basal expression of CYP2B1 decreased in a time-dependent manner during culture of AII cells, substantial CYP2B1 protein expression was observed in AII cell cultures after the first 24 h. When AII cells were incubated with 0. 05 microM [5-(3)H]NNK, N-oxidation of NNK, which is thought to represent a detoxification pathway, was predominant (42%). alpha-Hydroxylated metabolites resulting from metabolic activation of NNK amounted to 35% of all detected metabolites. However, the proportion of alpha-hydroxylated metabolites decreased to 17% of all detected metabolites when AII cells were incubated with a 100-fold higher concentration of NNK (5 microM). In summary, this study indicates a remarkable activity of cultured AII cells to metabolize NNK, leading to substantial metabolic activation of NNK, which was more pronounced in incubations at low NNK concentration. Because exposure to NNK via cigarette smoking is thought to lead to very low plasma NNK concentrations (1-15 pM), these data suggest that metabolic activation of NNK in cigarette smokers might occur to a larger extent than would be expected according to previous metabolic studies performed with high (micromolar) NNK concentrations.

Contribution of mdr1b-type P-glycoprotein to okadaic acid resistance in rat pituitary GH3 cells.

Okadaic acid as well as other, structurally different, inhibitors of serine/threonine phosphatases 1 and 2A induce apoptosis in pituitary GH3 cells. Incubation with stepwise raised concentrations of okadaic acid resulted in the isolation of cells that were increasingly less sensitive to the cytotoxic effect of this agent. After about 18 months cells were selected that survived at 300 nM okadaic acid, which is about 30 times the initially lethal concentration. This study revealed that a major pharmacokinetic mechanism underlying cell survival was the development of a P-glycoprotein-mediated multidrug resistance (MDR) phenotype. The increase in mRNA levels of the mdr1b P-glycoprotein isoform correlated with the extent of drug resistance. Functional assays revealed that increasing drug resistance was paralleled by a decreased accumulation of rhodamine 123, a fluorescent dye which is a substrate of mdr1-mediated efflux activity. Resistance could be abolished by structurally different chemosensitizers of P-glycoprotein function like verapamil and reserpine but not by the leukotriene receptor antagonist MK571 which is a modulator of the multidrug resistance-associated protein (MRP). Okadaic acid resistance included cross-resistance to other cytotoxic agents that are substrates of mdr1-type P-glycoproteins, like doxorubicin and actinomycin D, but not to non-substrates of mdr1, e.g. cytosine arabinoside. Thus, functional as well as biochemical features support the conclusion that okadaic acid is a substrate of the mdr1-mediated efflux activity in rat pituitary GH3 cells. Maintenance of resistance after withdrawal of okadaic acid as well as metaphase spreads of 100 nM okadaic acid-resistant cells suggested a stable MDR genotype without indications for the occurrence of extrachromosomal amplifications, e.g. double minute chromosomes.

Mimicry in primary rat hepatocyte cultures of the in vivo perivenous induction by phenobarbital of cytochrome P-450 2B1 mRNA: role of epidermal growth factor and perivenous oxygen tension.

Treatment of male rats with phenobarbital (PB) results in a perivenous and mid-zonal pattern of cytochrome P-450 (CYP)2B1 mRNA expression within the liver acinus. The mechanism of this zonated induction is still poorly understood. In this study sinusoidal gradients of oxygen and epidermal growth factor (EGF) besides those of the pituitary-dependent hormones growth hormone (GH), thyroxine (T4), and triiodothyronine (T3) were considered to be possible determinants for the zonated induction of the CYP2B1 gene in liver. Moreover, heme proteins seem to play a key role in oxygen sensing. Therefore, the influence of arterial (16% O2) and venous (8% O2) oxygen tension (pO2), and of the heme synthesis inhibitors CoCl2 and desferrioxamine (DSF) on PB-dependent CYP2B1 mRNA induction as well as the repression by EGF and, for comparison, by GH, T4, and T3, of the induction under arterial and venous pO2 were investigated in primary rat hepatocytes. Within 3 days, phenobarbital induced CYP2B1 mRNA to maximal levels under arterial pO2 and to about 40% of maximal levels under venous pO2. CoCl2 annihilated induction by PB under both oxygen tensions, whereas desferrioxamine and heme abolished the positive modulation by O2, suggesting that heme is a necessary component for O2 sensing. EGF suppressed CYP2B1 mRNA induction by PB only under arterial but not under venous pO2, whereas GH, T4, and T3 inhibited induction under both arterial and venous pO2. Thus, in hepatocyte cultures, an O2 gradient in conjunction with EGF mimicked the perivenous induction by PB of the CYP2B1 gene observed in the liver in vivo.

High-performance liquid chromatographic method for simultaneous determination of [1-methyl-14C]caffeine and its eight major metabolites in rat urine.

A selective and sensitive reversed-phase liquid chromatographic method was developed for the simultaneous analysis of [1-Me-14C]caffeine and its eight major radiolabelled metabolites in rat urine. The separation of the complex mixture of caffeine metabolites was achieved by gradient elution with a dual solvent system using an endcapped C18 reversed-phase column, which in contrast to commonly used C18 reversed-phase columns also allows the separation of the two isomers of 6-amino-5-(N-formylmethylamino)-1,3-dimethyluracil (1,3,7-DAU), a caffeine metabolite of quantitative importance predominantly occurring in rat. As caffeine is metabolised primarily by members of the cytochrome P450 1A (CYP1A) subfamiliy, determination of the pattern of caffeine metabolites in rat urine enables analysis of activities of this important enzyme subfamily in vivo. Since CYP1A is suggested to be involved in the detoxification of bilirubin, the assay may be applied to search for untoxic inducers of CYP1A which might be of pharmacological interest in the treatment of hyperbilirubinaemia.

Reactive oxygen species participate in mdr1b mRNA and P-glycoprotein overexpression in primary rat hepatocyte cultures.

P-glycoproteins encoded by multidrug resistance type 1 (mdr1) genes mediate ATP-dependent efflux of numerous lipophilic xenobiotics, including several anticancer drugs, from cells. Overexpression of mdr1-type transporters in tumour cells contributes to a multidrug resistance phenotype. Several factors shown to induce mdr1 overexpression (UV irradiation, epidermal growth factor, tumour necrosis factor alpha, doxorubicin) have been associated with the generation of reactive oxygen species (ROS). In the present study, primary rat hepatocyte cultures that exhibit time-dependent overexpression of the mdr1b gene were used as a model system to investigate whether ROS might participate in the regulation of intrinsic mdr1b overexpression. Addition of H2O2 to the culture medium resulted in a significant increase in mdrlb mRNA and P-glycoprotein after 3 days of culture, with maximal (approximately 2-fold) induction being observed with 0.5-1 mM H2O2. Furthermore, H2O2 led to activation of poly(ADP-ribose) polymerase, a nuclear enzyme activated by DNA strand breaks, indicating that ROS reached the nuclear compartment. Thus, extracellularly applied H2O2 elicited intracellular effects. Treatment of rat hepatocytes with the catalase inhibitor 3-amino-1,2,4-triazole (2-4 mM for 72 h or 10 mM for 1 h following the hepatocyte attachment period) also led to an up-regulation of mdrlb mRNA and P-glycoprotein expression. Conversely, antioxidants (1 mM ascorbate, 10 mM mannitol, 2% dimethyl sulphoxide, 10 mM N-acetylcysteine) markedly suppressed intrinsic mdr1b mRNA and P-glycoprotein overexpression. Intracellular steady-state levels of the mdrl substrate rhodamine 123, determined as parameter of mdr1-type transport activity, indicated that mdr1-dependent efflux was increased in hepatocytes pretreated with H2O2 or aminotriazole and decreased in antioxidant-treated cells. The induction of mdr1b mRNA and of functionally active mdr1-type P-glycoproteins by elevation in intracellular ROS levels and the repression of intrinsic mdrlb mRNA and P-glycoprotein overexpression by antioxidant compounds support the conclusion that the expression of the mdr1b P-glycoprotein is regulated in a redox-sensitive manner.

Molecular cDNA cloning and tissue distribution of mRNA encoding a novel ATP-binding cassette (ABC) half-transporter.

The majority of proteins belonging to the ATP-binding cassette (ABC) superfamily catalyzes translocation of substrates across biological membranes. Employing a reverse transcription-PCR approach with degenerate primers, we have identified a full-length cDNA from rat hepatocytes encoding a novel ABC transporter termed umat (ubiquitously expressed mammalian ABC half-transporter). The deduced sequence of 836 amino acids comprises an N-terminal membrane anchor domain and a single conserved C-terminal nucleotide binding fold, specifying umat as an ABC half-transporter. While the first 250 amino acid positions are highly divergent from other ABC transporters, clusters of conserved residues are evident along the rest of the protein. The greatest sequence similarity was observed with the fission yeast heavy metal tolerance protein hmt1 (44.5% identity in a 626-amino-acid overlap). Umat mRNA, expressed in all tissues analyzed, was most abundant in testis. Substantial umat mRNA expression in cultured primary rat hepatocytes suggests that hepatocyte cultures should represent an adequate model for investigation of umat function and regulation.