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In vitro investigation of the molecular mechanisms of hepatotoxicity
18
Workshop 4. In Vitro Cell Models for Investigating Molecular Mechanism of Toxicity
GSTM1 enzyme. We have examined the smoking-induced expression of CYP1A 1 by measuring ethoxyresorufin O-deethylase activity and by immunohistochemistry in human lungs from patients both with and without the GSTM1 enzyme. In contrast to the previous in-vitro study, no association was found between the GSTM1 genotype and CYP1 A 1 inducibility in vivo. Our findings do not therefore support the hypothesis that the GSTM1 enzyme participates in the inhibition of smoking-induced stimulation of CYP1A1 transcription. [1] Vaury,C. etal., CancerRes., 55, 5520-5523, 1995.
Keywords: metabolic enzymes; genetic susceptibility ~
POLYMORPHISMS IN XENOGENES AND SUSCEPTIBILITY TO OBSTRUCTIVE LUNG DISEASE
Christopher A.D. Smith *, Ann Cantlay, David J. Harrison.
University of Edinburgh, Department of Pathology, Edinburgh, UK The various lineages of cells in the lung are subject to oxidative stress in many ways but the primary sources of dangerous environmental agents are cigarette smoke, occupational exposure to solvents and both particulate and gaseous airborne pollutants. All of these potential hazards contain factors (xenobiotics and oxidants) that may (i) have direct cytotoxic effects, (ii) promote primary genotoxic events and (iii) generate ROIs, all of which can induce severe macromolecular, cellular and tissue damage leading to lung dysfunction, obstruction and cancer. We decided to study the molecular genetics of xenobiotic metabolising enzymes in relation to obstructive lung disease related to oxidant stress. Using PCR based genotyping assays for polymorphic xenogenes we have shown that the high inducible form of CYP1A1 and the GSTM1 null (mutant) genotypes are associated with increased risk of developing COPD and emphysema. No association was seen with CYP2D6 null or NAT-2 genotype. A genetic polymorphism which produces slow microsomal epoxide hydrolase activity (mEPHX) is highly penetrant in obstructive lung diseases, suggesting that highly reactive epoxides may be major contributing intermediates in pathogenesis. Interestingly, no association between any of the polymorphic xenogenes studied and the development of lung cancer or asthma was noted. The slow (mutant) form of mEPHX is four and six-fold more common in cohorts of patients suffering from COPD and emphysema respectively. These results imply that xenogenes, in particular mEPHX, play a key detoxification role in the human lung.
Keywords: emphysema; lung; mEPHX; smoking; polymorphisms; xenogenes
W4. In Vitro Cell Models for Investigating Molecular Mechanism of Toxicity 6-~IN VITROINVESTIGATION OF THE MOLECULAR MECHANISMS OF HEPATOTOXICITY J.V. Castell, M.J. G6mez-Lech6n. Unidad de Hepatologia Experi-
mental Centre de lnvestigacidn, Hospital U. La Fe, Valencia, Spain Substances capable of causing hepatocyte damage are known as hepatotoxins. They are classified according to whether they exert their effects in all individuals in a dose-dependent manner (intrinsic hepatotoxins) or appear only in some individuals (idiosyncratic hepatotoxins). Intrinsic hepatotoxins may act directly on cellular systems (active) or after biotransformation by hepatocytes (latent), while idiosincratic toxicity may be the consequence of an abnormal pathway for drug metabolism which may have a geno- or phenotypic basis (metabolic idiosincrasy) or be mediated by the immune system (allergic hepatitis). Biotransformation reactions can lead to
the production of more reactive (toxic) metabolites. In the course of reactions catalyzed by P450 production, drug-derived reactive metabolites and/or active oxygen species may result. Some intermediates are potent electrophiles able to react with nucleophiles and covalently bind to macromolecules (proteins, DNA) or carboncentered radicals capable of initiating radical-chain reactions (lipid peroxidation). There are also examples that illustrate the ability of drug conjugates to react with proteins. Many hepatotoxins act indirectly by altering the energetic balance of cells, either by dramatically increasing the energy demand, reducing ATP production or by dissipating NAD(P)H in futile cycles. This is accompanied by alterations of ion homeostasis which generally precedes irreversible stages of cell destruction via necrosis or apoptosis. Against these potential hazards, hepatocytes have specific defence mechanisms (enzymes, GSH, and repair mechanisms). Ultimately, the balance between bioactivation, detoxification and defence mechanisms is what determines whether a compound will or will not elicit a toxic effect. Allergic drug hepatitis constitutes another mechanisms for hepatocyte injury (necrosis and/or cholestasis) that involves the participation of T-cells and/or antibodies directed against drug epitopes, or autoantigens. Cultures of hepatocytes, including those of human origin, have advantages when it comes to examining toxicity mechanisms of xenobiotics as they allow researchers a more flexible definition of experimental conditions and access to easily monitoring critical cell end-points.
I--~-~
CELL SIGNALLING ALTERATIONS AS END-POINT FOR IN VITRO NEUROTOXICITY TESTING
Anna D. Rossi 1,2 Luigi Manzo * 2, Sten Orrenius I, Pierluigi Nicotera 3. i Institute of Environmental Medicine, Division
of Toxicology, Stockholm, Sweden; 2 Toxicology Unit, University of Pavia and Clinica del Lavoro Medical Centre, Pavia, Italy; 3Laboratory of Molecular Toxicology, University of Konstanz, Germany Low level exposure to environmental contaminants can result in biological changes that, in many cases, are difficult to detect in the whole animal or in humans presenting only sub-clinical manifestations. Recent evidence suggests that the development of delayed disorders in the CNS following single or chronic exposures to toxic chemicals may originate from alterations in cell signalling systems. We have previously shown that exposure of PC12 cells to concentrations of inorganic mercury in the range of environmental exposure levels can promote calcium channel alterations that in turn result in disturbances of the cell differentiation program. In cerebellar granule cells we found instead that the altered calcium responses to depolarisation following exposure to 0.1-1 /zM inorganic mercury caused cytoskeletal alterations and finally apoptosis. Cerebellar granule cells express glutamate receptors sensitive to NMDA. We tested the effect of mercury on NMDA-stimulated Ca 2+ response and delayed cytotoxicity. When cells were stimulated with NMDA and subsequently treated with inorganic mercury the latter promoted sustained elevation of the intracellular free Ca 2+. Under these condition, for example in the presence of 0.1-0.3/zM mercury, concentrations of NMDA which are not normally cytotoxic in this system (i.e. 50/zM) become toxic. These data indicate that (i) mercury levels that are within the range of environmental exposure affect normal signal responses to neurotransmitters (ii) such alterations may lead to inappropriate activation of neural cell apoptosis. It is concluded that appropriate in vitro model systems can reveal subtle neurotoxic changes such as those induced by the exposure to only marginally elevated concentrations of environmental chemicals. Supported by Grants EV5V-CT940508 and ENV4-CT96-0173 from the European Commission.
Keywords: mercury; neurotoxicity; in vitro assays; signalling
Workshop 4. In Vitro Cell Models for Investigating Molecular Mechanism of Toxicity
GSTM1 enzyme. We have examined the smoking-induced expression of CYP1A 1 by measuring ethoxyresorufin O-deethylase activity and by immunohistochemistry in human lungs from patients both with and without the GSTM1 enzyme. In contrast to the previous in-vitro study, no association was found between the GSTM1 genotype and CYP1 A 1 inducibility in vivo. Our findings do not therefore support the hypothesis that the GSTM1 enzyme participates in the inhibition of smoking-induced stimulation of CYP1A1 transcription. [1] Vaury,C. etal., CancerRes., 55, 5520-5523, 1995.
Keywords: metabolic enzymes; genetic susceptibility ~
POLYMORPHISMS IN XENOGENES AND SUSCEPTIBILITY TO OBSTRUCTIVE LUNG DISEASE
Christopher A.D. Smith *, Ann Cantlay, David J. Harrison.
University of Edinburgh, Department of Pathology, Edinburgh, UK The various lineages of cells in the lung are subject to oxidative stress in many ways but the primary sources of dangerous environmental agents are cigarette smoke, occupational exposure to solvents and both particulate and gaseous airborne pollutants. All of these potential hazards contain factors (xenobiotics and oxidants) that may (i) have direct cytotoxic effects, (ii) promote primary genotoxic events and (iii) generate ROIs, all of which can induce severe macromolecular, cellular and tissue damage leading to lung dysfunction, obstruction and cancer. We decided to study the molecular genetics of xenobiotic metabolising enzymes in relation to obstructive lung disease related to oxidant stress. Using PCR based genotyping assays for polymorphic xenogenes we have shown that the high inducible form of CYP1A1 and the GSTM1 null (mutant) genotypes are associated with increased risk of developing COPD and emphysema. No association was seen with CYP2D6 null or NAT-2 genotype. A genetic polymorphism which produces slow microsomal epoxide hydrolase activity (mEPHX) is highly penetrant in obstructive lung diseases, suggesting that highly reactive epoxides may be major contributing intermediates in pathogenesis. Interestingly, no association between any of the polymorphic xenogenes studied and the development of lung cancer or asthma was noted. The slow (mutant) form of mEPHX is four and six-fold more common in cohorts of patients suffering from COPD and emphysema respectively. These results imply that xenogenes, in particular mEPHX, play a key detoxification role in the human lung.
Keywords: emphysema; lung; mEPHX; smoking; polymorphisms; xenogenes
W4. In Vitro Cell Models for Investigating Molecular Mechanism of Toxicity 6-~IN VITROINVESTIGATION OF THE MOLECULAR MECHANISMS OF HEPATOTOXICITY J.V. Castell, M.J. G6mez-Lech6n. Unidad de Hepatologia Experi-
mental Centre de lnvestigacidn, Hospital U. La Fe, Valencia, Spain Substances capable of causing hepatocyte damage are known as hepatotoxins. They are classified according to whether they exert their effects in all individuals in a dose-dependent manner (intrinsic hepatotoxins) or appear only in some individuals (idiosyncratic hepatotoxins). Intrinsic hepatotoxins may act directly on cellular systems (active) or after biotransformation by hepatocytes (latent), while idiosincratic toxicity may be the consequence of an abnormal pathway for drug metabolism which may have a geno- or phenotypic basis (metabolic idiosincrasy) or be mediated by the immune system (allergic hepatitis). Biotransformation reactions can lead to
the production of more reactive (toxic) metabolites. In the course of reactions catalyzed by P450 production, drug-derived reactive metabolites and/or active oxygen species may result. Some intermediates are potent electrophiles able to react with nucleophiles and covalently bind to macromolecules (proteins, DNA) or carboncentered radicals capable of initiating radical-chain reactions (lipid peroxidation). There are also examples that illustrate the ability of drug conjugates to react with proteins. Many hepatotoxins act indirectly by altering the energetic balance of cells, either by dramatically increasing the energy demand, reducing ATP production or by dissipating NAD(P)H in futile cycles. This is accompanied by alterations of ion homeostasis which generally precedes irreversible stages of cell destruction via necrosis or apoptosis. Against these potential hazards, hepatocytes have specific defence mechanisms (enzymes, GSH, and repair mechanisms). Ultimately, the balance between bioactivation, detoxification and defence mechanisms is what determines whether a compound will or will not elicit a toxic effect. Allergic drug hepatitis constitutes another mechanisms for hepatocyte injury (necrosis and/or cholestasis) that involves the participation of T-cells and/or antibodies directed against drug epitopes, or autoantigens. Cultures of hepatocytes, including those of human origin, have advantages when it comes to examining toxicity mechanisms of xenobiotics as they allow researchers a more flexible definition of experimental conditions and access to easily monitoring critical cell end-points.
I--~-~
CELL SIGNALLING ALTERATIONS AS END-POINT FOR IN VITRO NEUROTOXICITY TESTING
Anna D. Rossi 1,2 Luigi Manzo * 2, Sten Orrenius I, Pierluigi Nicotera 3. i Institute of Environmental Medicine, Division
of Toxicology, Stockholm, Sweden; 2 Toxicology Unit, University of Pavia and Clinica del Lavoro Medical Centre, Pavia, Italy; 3Laboratory of Molecular Toxicology, University of Konstanz, Germany Low level exposure to environmental contaminants can result in biological changes that, in many cases, are difficult to detect in the whole animal or in humans presenting only sub-clinical manifestations. Recent evidence suggests that the development of delayed disorders in the CNS following single or chronic exposures to toxic chemicals may originate from alterations in cell signalling systems. We have previously shown that exposure of PC12 cells to concentrations of inorganic mercury in the range of environmental exposure levels can promote calcium channel alterations that in turn result in disturbances of the cell differentiation program. In cerebellar granule cells we found instead that the altered calcium responses to depolarisation following exposure to 0.1-1 /zM inorganic mercury caused cytoskeletal alterations and finally apoptosis. Cerebellar granule cells express glutamate receptors sensitive to NMDA. We tested the effect of mercury on NMDA-stimulated Ca 2+ response and delayed cytotoxicity. When cells were stimulated with NMDA and subsequently treated with inorganic mercury the latter promoted sustained elevation of the intracellular free Ca 2+. Under these condition, for example in the presence of 0.1-0.3/zM mercury, concentrations of NMDA which are not normally cytotoxic in this system (i.e. 50/zM) become toxic. These data indicate that (i) mercury levels that are within the range of environmental exposure affect normal signal responses to neurotransmitters (ii) such alterations may lead to inappropriate activation of neural cell apoptosis. It is concluded that appropriate in vitro model systems can reveal subtle neurotoxic changes such as those induced by the exposure to only marginally elevated concentrations of environmental chemicals. Supported by Grants EV5V-CT940508 and ENV4-CT96-0173 from the European Commission.
Keywords: mercury; neurotoxicity; in vitro assays; signalling