Tamoxifen induces short-term cumulative DNA damage and liver tumors in rats: promotion by phenobarbital.

Tamoxifen administered in the diet (420 ppm) to Wistar rats (TOX:P) for only 3 months caused cumulative hepatic DNA damage as assessed by 32P-postlabeling, consistent with the proposal that tamoxifen is a genotoxic carcinogen in this species. Promotion of tumor development with phenobarbital after discontinuation of dietary tamoxifen resulted in the formation of liver carcinomas after 9 months. At 12 and 20 months in this study, the majority of these rats had liver carcinomas. Rats treated with tamoxifen for 3 months but not promoted with phenobarbital also developed liver tumors over a longer period of time. These tumors were predominantly adenomas, with one carcinoma, and occurred at a lower incidence than the tumors produced by promotion with phenobarbital. Rats treated with phenobarbital alone did not develop tumors after 20 months. Tamoxifen-induced DNA adducts were relatively persistent, with only a 38% decrease 3 months after tamoxifen treatment had been discontinued. This demonstrates that, in a susceptible species (the rat), tamoxifen can cause initiation of liver cancer after only 3 months exposure. It is proposed that the persistence of such DNA adducts may account for the ability of phenobarbital to promote a high incidence of liver carcinoma, even after discontinuation of tamoxifen treatment. These data are relevant to the concern for women given prophylactic tamoxifen for long periods in that even if there is a relatively small amount of cumulative tamoxifen-induced liver DNA damage, liver tumors could be promoted by other agents, even after the cessation of tamoxifen treatment.

The effect of N-methylprotoporphyrin and succinyl-acetone on the regulation of heme biosynthesis in chicken hepatocytes in culture.

The essential features of hepatic protoporphyria, namely inhibition of ferrochelatase, accumulation of protoporphyrin and stimulation of 5-aminolevulinic acid synthase (ALA-S) were all obtained by treating chicken hepatocytes in culture with small doses of N-methylprotoporphyrin. Both N-methylprotoporphyrin and succinyl-acetone, another inhibitor of heme biosynthesis, stimulated ALA-S when given on their own and also enhanced the stimulation of ALA-S caused by phenobarbital.

Two pathways of iron-catalyzed oxidation of bilirubin: effect of desferrioxamine and trolox, and comparison with microsomal oxidation.

The bilirubin-degrading activity of liver microsomes from rats induced with 3-methylcholanthrene has been shown to be markedly stimulated by addition of 3,3',4,4',5,5'-hexabromobiphenyl, a polyhalogenated chemical which resembles in size and shape the most effective inducers of cytochrome P450IA1, but lacks the structural features necessary for it to be metabolised. The degradation of bilirubin by this microsomal system has been compared to oxidation by a chemical model system involving H2O2 and Fe-EDTA (ethylenediaminetetraacetic acid). In both systems bilirubin disappearance was accompanied by bleaching. However, when either desferrioxamine or Trolox were present in the chemical model system, the rate of bilirubin oxidation was greatly enhanced and, at the same time, bilirubin was largely or entirely converted to biliverdin, a pathway of oxidation which proceeds by dehydrogenation. In the presence of desferrioxamine, biliverdin was also further oxidised to an unidentified red pigment.

Hemozoin stability and dormant induction of heme oxygenase in hemozoin-fed human monocytes.

Human monocytes avidly ingest malarial pigment, hemozoin. Phagocytosed hemozoin persists in the monocyte for a long time and modifies important monocyte functions. Stability of phagocytosed hemozoin may depend on modifications of the hemozoin heme moiety or reduced ability to express heme-inducible heme oxygenase. We show here that the spectral characteristics of alkali-solubilized hemozoin were identical to those of authentic heme, although hemozoin was solubilized by alkali much more slowly than authentic heme. Alkali-solubilized hemozoin was a substrate of microsomal rat heme oxygenase and bilirubin reductase, with bilirubin as the main final product. Hemozoin feeding to human monocytes did not induce heme oxygenase, but authentic heme and alkali-solubilized hemozoin supplemented to hemozoin-fed monocytes induced heme oxygenase and were degraded normally. Lysosomes isolated from hemozoin-fed monocytes released only traces of heme while lysosomes from erythrocyte-fed monocytes liberated considerable quantities of heme. Lack of heme release from hemozoin did not depend on proteolysis-resistant, heme-binding proteins, since lysosomal proteases fully degraded hemozoin-associated proteins but did not solubilize hemozoin. In conclusion, our data indicate that lack of induction of HO1 is due to the intrinsic structural characteristics of hemozoin and not to hemozoin-mediated impairment of the mechanism of HO1 induction.

The mechanism of the suicidal, reductive inactivation of microsomal cytochrome P-450 by carbon tetrachloride.

1. Stoichiometric losses of microsomal haem and cytochrome P-450 were observed when carbon tetrachloride (CCl4) was incubated anaerobically with rat liver microsomes using NADPH or sodium dithionite as a reducing agent. A rapid destruction of haem was also observed during the non-enzymatic reductive incubation of CCl4 with soluble haem preparations (methaemalbumin) in presence of sodium dithionite. The results indicate that haem is both the site and the target of the suicidal activation of CCl4 by cytochrome P-450. 2. When an additional, fluorimetric assay for haem determination was used, an equimolar loss of protoporphyrin IX fluorescence was also observed in both the enzymatic and non-enzymatic system, indicating that the haem moiety of cytochrome P-450 has undergone a structural change, involving either loss or labilization of the porphyrin tetrapyrrolic structure. In both systems the loss of porphyrin was prevented by carbon monoxide (CO). 3. A dichlorocarbene-cytochrome P-450 ligand complex is partially responsible for the difference spectrum obtained on addition of CCl4 to anaerobically reduced rat liver microsomes. A molar extinction coefficient for this complex has been calculated. The carbene trapping agent 2,3-dimethyl-2-butene (DMB) strongly inhibited (greater than 95%) the formation of this spectrum but did not modify the loss of haem in reduced CCl4-supplemented microsomal incubations. The results suggest that dichlorocarbene (:CCl2) is not significantly involved in CCl4-dependent haem destruction. 4. Pretreatment of rats with different microsomal enzyme inducers was responsible for similar but not identical patterns of :CCl2 and CO formation and haem loss during incubation of CCl4 with reduced microsomes. This indicates a critical role of CCl4 metabolism in the suicidal destruction of cytochrome P-450 haem and suggests that the apoprotein of cytochrome P-450 is capable of modulating not only the metabolism of CCl4 to :CCl2 but also the hydrolysis of :CCl2 to CO. 5. Inactivation of cytochrome P-450 by CCl4 with reduced microsomes from Aroclor-pretreated rats was saturable and followed pseudo first-order kinetics. This provides further evidence to conclude that CCl4 activation is a suicidal process where the reactive metabolite(s) formed bind to haem, we predict, in a one to one stoichiometry. 6. The partition ratio between loss of cytochrome P-450 haem and CCl4 metabolism by liver microsomes from Aroclor pretreated rats has been investigated using limiting concentrations of CCl4. It was calculated that approximately 26 molecules of CCl4 had to be metabolised to achieve the loss of one molecule of haem.

Induction of CYP2B1 and 3A1, and associated monoxygenase activities by tamoxifen and certain analogues in the livers of female rats and mice.

Previous studies suggest long-term feeding of tamoxifen (Z-1-[4-(2-dimethylamino-ethoxy)phenyl]1,2-diphenyl-1-butane) to rats gives rise to liver tumours, while mice are resistant. The effects of tamoxifen on cytochrome P450 isoenzymes and associated monoxygenase activities in the livers of female Fischer rats and C57Bl/6 and DBA/2 mice have been compared. Total microsomal cytochrome P450 was not induced in the livers of rats given tamoxifen (45 mg/kg daily for 4 days) and was in fact significantly reduced after 3 days treatment. In contrast, there was a 30-60-fold increase in the metabolism of benzyloxy- and pentoxyresorufins to resorufin. Ethoxyresorufin O-deethylase was induced only 2.5-fold. The regio- and stereo-specific hydroxylation of testosterone following tamoxifen pretreatment of rats showed a general time- and dose-dependent induction. 6 beta- and 16 alpha-hydroxylation of testosterone together with oxidation to androstenedione were increased 2-3-fold while 2 beta-hydroxylation was induced only marginally, suggesting that tamoxifen produces a mixed pattern of induction with a significant phenobarbitone-like component. No induction of the 2 beta- or 6 beta-hydroxylation pathway occurred in either mouse strain. In rats, immunoblotting experiments with polyclonal antibodies raised against CYP2B1 or 3A1 showed that tamoxifen pretreatment resulted in 2-3-fold increases in both CYP2B1, 2B2 and 3A1 proteins, relative to controls. Immunohistochemistry of rat liver sections showed a centrilobular localization of these induced proteins. Similar patterns of induction as measured by immunoblotting experiments and testosterone hydroxylation were seen following the administration of structurally related analogues, toremifene and droloxifene (3-hydroxytamoxifen), thought to be non-carcinogenic in the rat. No induction of these monooxygenase activities was seen in C57Bl/6 mice and only small increases in benzyloxy and pentoxyresorufin metabolism were in DBA/2 mice. It is suggested that the induction of cytochrome P450-dependent activities by tamoxifen may result in accelerated liver metabolism of this drug with important implications for the disposition of tamoxifen in vivo and also for its metabolic conversion to genotoxic metabolite(s). The difference in inducibility of cytochrome P450-dependent monooxygenase activities between rats and mice offers a plausible and testable hypothesis that the difference in tamoxifen metabolism between the two species may contribute to their carcinogenic response to tamoxifen.

Formation of cobalt protoporphyrin by chicken hepatocytes in culture. Relationship to decrease of 5-aminolaevulinate synthase caused by cobalt.

Cobalt protoporphyrin generated from 5-amino[4-14C]laevulinate by homogenates or primary cultures of chick embryo liver exposed to CoCl2 was found to be radioactivity unextractable by acid/acetone, when extra protein was added. The activity of ferrochelatase was required for formation of cobalt protoporphyrin since inhibition of ferrochelatase with 3,5-diethoxycarbonyl-1,4-dihydrocollidine (in the presence of cycloheximide) inhibited formation of cobalt protoporphyrin and resulted in accumulation of protoporphyrin. Cobalt protoporphyrin was detected spectrophotometrically in hepatocyte cultures exposed to the combination of 2-allyl-2-isopropylacetamide and CoCl2: (1) as the pyridine haemochrome of the protein pellet remaining after acid-acetone extraction of the cells, or (2) as the material extracted from the protein pellet with acetic acid-pyridine-chloroform. The amount of cobalt protoporphyrin increased with increasing CoCl2 concentration as cellular haem declined. The decrease in haem was about equal to the amount of cobalt protoporphyrin that accumulated. 2-Allyl-2-isopropylacetamide and polychlorinated biphenyls were both powerful inducers of 5-aminolaevulinate synthase. The former led to protoporphyrin accumulation, whereas with the latter, uroporphyrin accumulated, probably due to a concomitant decrease in activity of uroporphyrinogen decarboxylase. The decrease in activity of 5-aminolaevulinate synthase produced by administration of CoCl2 was greater after treatment with 2-allyl-2-isopropylacetamide than after treatment with allylisopropylacetamide and 3,4,3',4'-tetrachlorobiphenyl. We conclude: (a) that cobalt protoporphyrin is readily formed in cultured hepatocytes, and (b) that its formation accounts for the action of cobalt on 5-aminolaevulinate synthase.

Species differences in the covalent binding of [14C]tamoxifen to liver microsomes and the forms of cytochrome P450 involved.

Species differences in the NADPH-dependent covalent binding of [14C]tamoxifen to liver microsomes have been studied using preparations from humans, female F344 rats and DBA/2 mice. Protein binding has been used as an index of metabolic activation and as a surrogate for DNA binding in order to establish which forms of cytochrome P450 are responsible for genotoxicity. A panel of 12 human liver microsomes has been characterized and immunoquantified for nine cytochrome P450 isoenzymes. Binding of tamoxifen (45 microM) (25 +/- 2.5 pmol/15 min/mg protein, mean +/- SE) correlated (P < 0.05) with CYP3A4 and CYP2B6 content. Covalent binding of [14C]tamoxifen to microsomal preparations from human breast tumour tissue could also be detected but at levels 7-fold lower than in liver. The covalent binding of tamoxifen to mice, rat or human liver microsomal preparations increased with increasing substrate concentration. Covalent binding of [14C]tamoxifen (45 microM) in rats was 3.8-fold and mice 17-fold higher than in human liver microsomal preparations. In mice, the apparent Km (9.6 +/- 1.9 microM) was very much lower than for rats (119 +/- 41 microM). Pretreatment of female rats with phenobarbitone or dexamethasone resulted in a 4- to 5-fold increase in [14C]tamoxifen binding, relative to controls, consistent with the involvement of CYP2B1 and CYP3A1 in the metabolic activation. It cannot be distinguished at present if the same reactive metabolites are involved in protein and DNA binding. The greater potential of mouse liver microsomes to activate tamoxifen, relative to rats, does not reflect DNA damage or hepatocarcinogenicity seen following dosing with tamoxifen in vivo. It is concluded that covalent binding of tamoxifen to protein in vitro cannot be directly related to the carcinogenic potential of this compound. However, in the three species investigated, results suggest that the rat is a better model than the mouse for human liver microsomal activation of tamoxifen both with respect to kinetic parameters and the pattern of metabolic products.

Role of cytochrome P450 1A2 in bilirubin degradation Studies in Cyp1a2 (-/-) mutant mice.

In congenital jaundice, which is due to defects of bilirubin gluruconidation, bilirubin is degraded by an alternative pathway into unidentified products. Previously, it was shown that plasma bilirubin levels can be decreased in rats with this defect by inducers of CYP1A enzymes. Here, liver microsomes from rats or mice treated with beta-naphthoflavone (BNF) or 3-methylcholanthrene (3 MC) had increased activity for bilirubin degradation. The activity was further stimulated by addition of the coplanar molecule 3,4,3',4'-tetrachlorobiphenyl (TCB). There was more stimulation of bilirubin degradation by TCB in microsomes from BNF-treated rats than in microsomes from BNF-treated mice. CYP1A1 to CYP1A2 ratios were greater in rats treated with BNF. In Cyp1a2 (-/-) mutant mice, 3-MC treatment did not increase the rate of bilirubin degradation, but TCB increased this degradation severalfold. Between SWR and C57BL/6 inbred mouse strains that have a 2-fold difference in hepatic constitutive CYP1A2 levels, there was also a 2-fold difference in bilirubin degradation; TCB did not stimulate in either strain. We conclude that CYP1A2 is responsible for microsomal bilirubin degradation in the absence of TCB. TCB was required for bilirubin degradation by CYP1A1. Manipulation of CYP1A2 may be of therapeutic benefit in patients with these diseases of bilirubin conjugation.

Metabolic activation of halothane to neoantigens in C57Bl/10 mice: immunochemical studies.

C57Bl/10 mice were given halothane (10 mmol/kg, intraperitoneally) and microsomal proteins were analysed for the presence of trifluoroacetylated (TFA) neoantigens by SDS-gel electrophoresis followed by immunoblotting using a polyclonal anti-TFA antibody. In microsomal preparations from liver, lung and olfactory tissues, a 54 kDa TFA adduct was detectable 1 h after dosing. After 3-48 h, multiple bands were detected in liver (45-100 kDa) and in the lung (26-57 kDa) and in one experiment in which [14C]halothane was given, several immunoreactive bands from liver microsomes were shown to contain a covalently bound metabolite of the drug. In olfactory tissue, initially (1 h), a major band of 54 kDa and a less prominent component of about 50 kDa were seen. The number of bands increased at later times but the additional bands were far fewer than in liver. The rate of decay of the 54 kDa adduct was also measured in both liver and olfactory microsomes and found to be compatible with the reported turnover of total liver cytochrome P-450. 24 h after treating mice with halothane (10 mmol/kg), no TFA neoantigens could be detected on the outer cell surface of isolated viable hepatocytes when analysed by fluorescence activated flow cytometry. In contrast, non-viable cells, or those fixed in acetone were all positive. Using immunohistochemistry, TFA neoantigens were demonstrated in the centrilobular area of the liver, the non-ciliated bronchiolar epithelial (Clara) cells of the lung, proximal tubular cells of the kidney and the respiratory and olfactory epithelium of nasal tissues.

Effects of 2-[1-(ethoxyimino)propyl]-3-hydroxy-5-(2,4,6-trimethylphenyl) cyclohex-2-enone on hepatic haem biosynthesis: species differences in hepatic porphyria.

2-[1-(Ethoxyimino)propyl]-3-hydroxy-5-(2,4,6-trimethylphenyl) cyclohex-2-enone (ETC) is a novel alkyl ketone herbicide. Continuous administration of ETC to mice for 28 days resulted in marked liver enlargement and severe intrahepatic cholestasis. These effects have been shown to result directly from a rapid and marked accumulation of porphyrin in the liver. The porphyrin which accumulates in the liver has been identified as protoporphyrin IX and dose response and time course studies confirm prior inhibition of mitochondrial ferrochelatase as the causal lesion. ETC was a very potent porphyrinogenic compound in mice, with a no-effect level for a single oral dose of 1 mg/kg. Rats and hamsters were insensitive to this type of hepatotoxicity following single oral doses of up to 750 mg/kg or following repeated, and indeed prolonged administration. The sensitivity of different species to ETC-induced porphyria correlated with the effect of ETC on hepatic ferrochelatase activity. The inhibition of ferrochelatase activity and the hepatic porphyria in mice were both found to be readily reversible upon withdrawal of ETC.

The glutathione-linked metabolism of 2-allyl-2-isopropy-lacetamide in rats. Further evidence for the formation of a reactive metabolite.

2-Allyl-2-isopropylacetamide (AIA) causes a depletion of liver glutathione in rats only if the animals have been pretreated with phenobarbitone. Phenobarbitone stimulates the excretion in bile of a component derived from AIA and glutathione which is apparently not the same as the conjugate formed by reaction of the two components in simple solutions. The significance of these findings are considered in relation to the suggestion that AIA is metabolised to an epoxide by the microsomal enzyme system; in addition several differences between AIA and the non-porphyrogenic compound, acrylamide, are discussed.

A difference between two strains of rats in their liver non-haem iron content and in their response to the porphyrogenic effect of hexachlorobenzene.

Female Agus rats developed hepatic porphyria at a much faster rate than female Porton-Wistar rats when fed a diet containing 0.01% of hexachlorobenzene (HCB). They also showed a greater inhibition of liver uroporphyrinogen decarboxylase [EC 4.1.1.37] activity and a marked stimulation of 5-aminolaevulinate synthetase [EC 2.3.1.37]. The difference between the two strains could not be correlated with differences in the liver concentrations of HCB. However, control Agus rats were found to possess significantly higher levels of total non-haem iron in their livers than the Porton animals. This was particularly apparent after 24 h of starvation and is further evidence for the involvement of iron in the pathogenesis of HCB-induced porphyria. The posterior lobes of the livers from the Agus rats given HCB became porphyric more slowly than the remainder with less severe inhibition of uroporphyrinogen decarboxylase. In contrast to their increased susceptibility to HCB, the Agus rats were less susceptible to another prophyrogenic agent, 3,5-diethoxycarbonyl-1,4-dihydrocollidine.

The role of CYP forms in the metabolism and metabolic activation of HCFCs and other halocarbons.

The use of hydrochlorofluorocarbons (HCFCs) such as HCFC-123 (2,2-dichloro-1,1,1-trifluoroethane) and HCFC-141b (1,1-dichloro-1-fluoroethane) is becoming widespread as replacements for the ozone depleting chlorofluorocarbons. Hepatic activation of HCFC-123 or the unsaturated perchloroethylene through oxidative pathways leads to the formation of the electrophiles trifluoroacetyl chloride or trichloroacetyl chloride, respectively. These can react with epsilon-NH(2) functions of lysine in proteins and give rise to neoantigens. In the case of HCFC-123, this reaction is catalysed primarily by CYP2E1 and to a much lesser extent by the constitutive CYP2C19, CYP2B6 and CYP2C8. For perchloroethylene, the extent of activation is less and the reaction is catalysed primarily by the CYP2B family. While acute hepatotoxicity has been seen in humans exposed to HCFC-123 or halothane, little short- or long-term toxicity in rodents is observed. No immunological related toxicity of perchloroethylene has been reported in exposed humans. Long-term exposure of rats can lead to renal tubule carcinomas and in mice, hepatocellular carcinomas. These toxic reactions do not appear to be directly related to the formation of the putative trichloroacetyl chloride intermediate.

Selective inactivation of rat and bovine olfactory cytochrome P450 by three haloethanes.

The effects of halothane, 1,1-dichloro-2,2,2-trifluoroethane (HCFC-123) and 1,1-dichloro-1-fluoroethane (HCFC-141b) on the P450 system in olfactory and hepatic microsomes of bovine and rat have been investigated. In the in vitro experiments, all three compounds decreased olfactory CYP-dependent activities in microsomes from both species, especially under anaerobic conditions, halothane showing the greatest effect. Hepatic activities were not affected. A selective olfactory CYP depletion was also observed in vivo after treatment with halothane, but not with HCFC-123 or HCFC-141b. A loss of olfactory ethoxycoumarin-O-deethylase activity was also found both in vitro and in vivo experiments, suggesting that a CYP2A isoform may be the main target of inactivation. The present results therefore suggest that CYP2A, the major isoform expressed in the olfactory tissue of mammals, may be particularly prone to catalyze the reductive metabolism of halothane both in anaerobic and aerobic conditions.

Neoantigen formation and clastogenic action of HCFC-123 and perchloroethylene in human MCL-5 cells.

In this study, the metabolic activation of 2,2-dichloro-1,1,1-trifluoroethane (hydrochlorofluorocarbons-123, HCFC-123), halothane or 1,1-dichloro-1-fluoroethane (HCFC-141b) was compared to that of perchloroethylene, using lymphoblastoma derived cell lines expressing human CYP1A1, CYP1A2, CYP2E1, CYP2A6 and CYP3A4 (MCL-5 cells). A dose dependent increase in micronucleus formation was detected over a nominal concentration range of 0.05-2 mM for HCFC-123 and halothane, but this was not seen with HCFC-141b. No dose response for HCFC-123 was seen in a control cHo1 cell line not expressing this cytochrome P450's. Cell lines expressing individual human cytochrome P-450 (CYP) forms were also used to define the enzymes responsible for the clastogenic events and to investigate the formation of immunoreactive protein by microsomal fractions. It was shown that CYP2E1 or CYP2B6 catalysed the clastogenic response, but CYP2D6, CYP3A4, CYP1A2 or CYP1A1 all appeared to be inactive. The formation of neoantigenic trifluoroacetylated protein adducts by microsomal mixtures incubated with HCFC-123 and NADPH was catalysed primarily by CYP2E1 and to a lesser extent by CYP2C19, whereas, only trace levels of immunoreactive protein were seen with microsomes expressing CYP2B6 or CYP2C8. With perchloroethylene as a substrate, the extent of activation was low in comparison with HCFC-123, as judged by the absence of micronuclei formation in the MCL-5 cell line and the weak immunoreactivity of proteins following Western blotting. CYP1A2, CYP2B6 and CYP2C8 appeared to be responsible for perchloroethylene immunoreactivity and in contrast to the findings with the HCFC's, no activation of perchloroethylene by CYP2E1 could be detected. These results show that even though both saturated and unsaturated halocarbons can result in neoantigen formation, there is a marked difference in the specificity of the CYP enzymes involved in their metabolic activation.

Structural improvement of higher education in environmental toxicology in Latin America and Europe.

Industrial development has resulted in an increased release of chemicals and other agents into the environment, resulting in damage to the environment as well as increasing the risk of adverse effects on human health. Environmental toxicology (ET) is the discipline responsible for assessing the risks to human health and the environment from the effects of new chemicals and those already present in the environment. The development of human resources in toxicology is therefore a priority in both Latin America (LA) and the European Union (EU), although LA professionals are more involved in risk evaluation than in risk assessment compared to their EU colleagues. A solid background in general toxicology will enable those interested in environmental issues to tackle local problems. Moreover, the increasing globalization of markets and, therefore, of the necessary regulations, requires harmonisation of postgraduate programmes to ensure that risk assessment and management related to the environment are dealt with uniformly and by highly qualified scientists. The Inaugural Meeting of the ALFA-OMET Toxicology', a 2-year programme supported by the European Commission, offered the opportunity to discuss a number of these issues. The present status of existing ET courses in the EU and LA and the corresponding professional profiles in the two regions were examined, and a harmonized academic curriculum for a postgraduate professional profiles in the two regions were examined, and a harmonized academic curriculum for a postgraduate course in environmental toxicology was developed. Finally, a course programme for toxicology and a specialization in environmental toxicology designed by a panel of experts was discussed, and its relevance as a model for other specialisation programmes was analysed. Exercises such as those performed by ALFA-OMET may be useful not only in promoting discussion for the implementation of national and international professional registers in LA, but also in encouraging the same, ongoing process in the EU.

N-Dealkylation of chlorimipramine and chlorpromazine by rat liver microsomal cytochrome P450 isoenzymes.

The role of different cytochrome P450 isozymes (CYP) in the N-demethylation of chlorimipramine and chlorpromazine has been investigated in liver microsomes from rats by studying the effects of multiple subchronic doses of chlorimipramine, chlorpromazine, phenobarbital and beta-naphthoflavone on the N-demethylation of ethylmorphine, mono-N-demethyl-chlorimipramine and chlorpromazine and on the hydroxylation of aniline. With control microsomes, CYP-dependent metabolism of chlorimipramine and chlorpromazine (100 nmol; 30 min incubation) resulted in the formation of predominantly chlorimipramine (46.5 +/- 4.9 nmol) whereas chlorpromazine (14.1 +/- 0.9 nmol) accounted for only part of the overall metabolism of chlorpromazine. Multiple doses of chlorimipramine increased the capacity of microsomes to N-demethylate ethylmorphine (9.8 +/- 0.73 and 6.08 +/- 0.06 nmol min(-1) (mg protein)(-1) for chlorimipramine-treated and control rats, respectively) as well as itself (4.65 +/- 0.25 and 3.10 +/- 0.33 nmol min(-1) (mg protein)(-1), respectively). Multiple doses of chlorpromazine induced aniline-hydroxylase activity (1.11 +/- 0.16 and 0.94 +/- 0.06 nmol min(-1) (mg protein)(-1) for chlorimipramine and control microsomes, respectively) but the capacity to N-demethylate itself was unchanged. Phenobarbital treatment induced ethylmorphine N-demethylation activity, but did not affect N-demethylation activity, towards chlorimipramine and chlorpromazine. In control microsomes the N-demethylation capacity of chlorimipramine or chlorpromazine (0.160 +/- 0.025 and 0.015 +/- 0.003 nmol min(-1) (mg protein)(-1), respectively) was one order of magnitude lower than that of chlorimipramine or chlorpromazine. The capacity to N-demethylate either chlorimipramine or chlorpromazine was increased by treatment with either phenobarbital or beta-naphthoflavone. In control microsomes, sulphaphenazole markedly inhibited both chlorimipramine-N-mono- and di-N-demethylation, whereas quinidine markedly inhibited the rate of formation of chlorpromazine. The CYP2C and CYP2D subfamilies seem to be involved in the mono N-demethylation of chlorimipramine and chlorpromazine, respectively. Moreover the CYP1A and CYP2B subfamilies might participate in the N-demethylation of either chlorimipramine or chlorpromazine. This could have important implications in the clinical use of chlorimipramine and chlorpromazine in view of the genetic polymorphism of CYP2C and CYP2D isozymes in man.

Time-dependent porphyric response in mice subchronically exposed to arsenic.

1. A time-course study was carried out in mice subchronically exposed to As III (as sodium arsenite) or As V (as sodium arsenate), via drinking water, relating the pattern of urinary porphyrin excretion to the renal and hepatic enzyme activities of porphobilinogen deaminase (PBGD), uroporphyrinogen III synthetase (URO III-S), uroporphyrinogen decarboxylase (URO-D) and coproporphyrinogen oxidase (COPRO-O), as well as to the hepatic porphyrin accumulation in the treated animals. 2. A time-dependent, wave-like porphyric response was found in mice exposed to As V, and the increases seen in total urinary porphyrins (at 3 weeks of exposure) corresponded to an increased activity of PBGD and Uro III-S in liver. 3. Significant decreases in renal URO-D and hepatic and renal COPRO-O activities were found in treated mice; these inhibitions were more pronounced in animals exposed to As III. 4. The combination of these enzymic effects may explain the time-dependent porphyric response of mice subchronically exposed to As. Finally, the relative magnitudes of URO-D and COPRO-O inhibitions may determine the pattern of porphyrin concentration observed in urine and tissues. 5. The decrease in renal URO-D activity may help to explain the inversion in the coproporphyrin/uroporphyrin ratio previously reported in humans chronically exposed to As; however, there were differences between the urinary porphyrin profiles found in both species. The possible reasons for the similarities and differences are briefly discussed.

Altered urinary porphyrin excretion in a human population chronically exposed to arsenic in Mexico.

1. A detailed study of the urinary excretion pattern of porphyrins in humans chronically exposed to As via drinking water was performed using high performance liquid chromatography (HPLC) 2. Thirty-six individuals (15 men and 21 women) were selected from a town which had 0.400 mg L-1 of As in drinking water. The control group consisted of thirty-one individuals (13 men and 18 women) whose As concentration in drinking water was 0.020 mg L-1. 3. The major abnormalities in the urinary porphyrin excretion pattern observed in arsenic-exposed individuals were: (a) significant reductions in coproporphyrin III excretion resulting in decreases in the COPRO III/COPRO I ratio, and (b) significant increases in uroporphyrin excretion. Both alterations were responsible for the decrease in the COPRO/URO ratio. 4. No porphyrinogenic response was found in individuals with urinary As concentrations below 1,000 micrograms of As g-1 of creatinine. However, as arsenic concentrations exceeded this value, the excretion of porphyrins (except coproporphyrin III) increased proportionally. 5. The prevalence of clinical signs of arsenicism showed a direct relationship to both As concentration in urine and time-weighted exposure to As. A direct relationship between time-weighted exposure and alterations in urinary porphyrin excretion ratios was also observed. 6. The alterations found are compatible with a lower uroporphyrinogen decarboxylase activity in arsenic-exposed individuals. However, the similarities in the urinary porphyrin excretion pattern between As-exposed individuals and Dubin-Johnson syndrome patients suggest that impairments in the excretion of coproporphyrin isomers may also contribute to the pattern observed.