A H Gibbs

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.

Liver production of N-alkylated porphyrins caused in mice by treatment with substituted dihydropyridines: Evidence that the alkyl group on the pyrrole nitrogen atom originates from the drug

The porphyrogenic drug, 3,Sdiethoxycarbonyl1,4-dihydro-2,4,6_trimethylpyridine causes a marked inhibition of the enzyme protohaem ferro-lyase (EC 4.99.1 .l) in the liver of rats, mice and chick embryos [l-4] an effect which is thought to be responsible for the very pronounced accumulation of protoporphyrin seen in this type of experimental porphyria. We have isolated a potent inhibitor of protohaem ferro-lyase from the liver of mice made porphyric by treatment with this drug [5,6] and have identified the inhibitor as N-methyl protoporphyrin [7]. Inhibition of protohaem ferro-lyase has also been obtained both in vivo and in vitro with synthetic N-alkylated porphyrins [7-91, and the size of the alkyl group present on the pyrrole nitrogen atom has been shown to be important for the inhibitory effect, N-ethylmesoporphyrin being less active than N-methylmesoporphyrin [8]. Isotopic experiments have suggested that the N-methylated protoporphyrin produced by treatment with 3,5-diethoxycarbonyl-1,4-dihydro-2,4,6-trimethylpyridine originates from liver haem [5,6,10], but the source of the methyl group bound onto the pyrrole nitrogen atom has not yet been determined. The following findings have raised the possibility that the methyl group may originate from the 4-methyl substituent of the drug: under relatively mild chemical conditions certain dihydropyridines lose their 4-alkyl substituent on oxidation and that this alkyl group can be donated to suitable nucleophiles [ 111. A series of dihydropyridine analogues have been compared for their ability to inhibit liver protohaem ferro-lyase

Conversion of liver haem into N-substituted porphyrins or green pigments: Nature of the substituent at the pyrrole nitrogen atom

Drugs can promote the conversion of liver haem into modeled porphyrins (or green pigments) of two distinct classes. Pigments of the first class are obtained by treatment with unsaturated drugs containing at least one allyl, vinyl or ethynyl side chain. These drugs are metabolized by cytochrome P450 into reactive derivatives, which then become bound onto the porphyrin nucleus of the haem moiety of the cytochrome, converting it into modified porphyrins (reviewed in [I ,2]). The nature of the drug metabolite responsible is still controversial: epoxides have been suggested ([2] and references therein), but there is also a proposal [33 specifically excluding epoxides and emphasizing other structural features of the metabolite, such as the presence of an ‘activated’ carbony1 grouping. Green pigments of this class do not inhibit liver protohaem ferro-lyase [4] (the enzyme which converts protoporphyrin to haem) and for this reason none of the unsaturated drugs responsible will cause a very marked increase in liver protoporphyrin in v&o. In contrast 3,5-diethoxycarbonyl-1,4-d~ydrocollidine, griseofulvin and isogriseofulvin produce liver accumulation of a second type of green pigment with strong inhibitory properties towards protohaem ferrolyase in vitro [4-61. Marked inhibition of the liver enzyme is also observed in vivo after treatment with this second group of drugs [7,8] and as a consequence, the liver concentration of protoporphyrin increases markedly giving rise to the biochemical picture of hepatic protoporphyria. The ‘inhibitory’ pigment also appears to originate from turnover of liver haem [4],

Inactivation of cytochrome P-450 and production of N-alkylated porphyrins caused in isolated hepatocytes by substituted dihydropyridines Structural requirements for loss of haem and alkylation of the pyrrole nitrogen atom

Drugs with unsaturated side chains and certain dihydropyridines both convert liver haem into Nalkylated porphyrins, but the underlying mechanisms differ in the two cases. With unsaturated drugs, for example 2-allyl-2-isopropylacetamide (AIA), a monooxygenated derivative of the drug becomes bound [l] onto one of the pyrrole nitrogen atoms [2-41 of liver haem; whereas with 3,5diethoxycarbonyl-1,4-dihydro-2,4,6-trimethylpyridine (DDC) only the 4-methyl substituent of the drug is transferred onto the pyrrole nitrogen atom [5-71 and the product, N-methyl protoporphyrin, is a powerful inhibitor of ferrochelatase (EC 4.99.1.1) [8,9]. There is strong evidence that the N-alkylated porphyrins produced by unsaturated compounds all originate from the haem of cytochrome P-450 [ 10-121, but it is less clear from which pool of hepatic haem N-methyl protoporphyrin originates after treatment with DDC; and the detailed mechanism of the transmethylation reaction involved has not yet been elucidated. The aim of the experiments described in this paper has been to clarify wether N-methyl protoporphyrin originates from the haem of cytochrome P450 and whether exogenous haem can be utilized

Structural Isomerism and chirality of N-monosubstituted protoporphyrins: Possible relevance to binding of N-methyl protoporphyrin by ferrochelatase and to orientation of haem in cytochrome P450

A modified porphyrin or green pigment which is a powerful inhibitor of ferrochelatase (protohaem ferrolyase, EC 4.99.1.1) has been isolated from the liver of mice treated with 3,5-diethoxycarbonyl-1,4-dihydrocoUidine (DDC) [1] and the inhibitory porphyrin has been identified as N-methyl protoporphyrin [2-4]. Interaction of the inhibitory porphyrin with the active centre of ferrochelatase is important for inhibition of the enzyme [5,6]. Authentic N-methylated meso and protoporphyrin have been synthesized and shown to be powerful inhibitors of the enzyme in vivo and in vitro [2,6,7]. Synthetic N-methyl protoporphyrin could be separated by high-performance liquid chromatography (HPLC) into 2 separate fractions possessing different inhibitory activity and possibly representing different structural isomers, where different pyrrole nitrogens had been methylated [2]. Here, we provide evidence that the two HPLC fractions contain in fact different structural isomers of N-methyl protoporphyrin. The different inhibitory activity of these two isomeric fractions is now confirmed and extended to their respective zinc chelates. In the isomers with less inhibitory activity, where the propionic acid substituted rings of protoporphyrin are N-methylated, the spatial arrangement and distance between the two propionic acid sideehains may be significantly altered. We also wish to report that the naturally occurring N-methyl protoporphyrin and the N-alkylated protoporphyrins produced in vivo from degradation of the haem of cytochrome P450 during metabolism of unsaturated chemicals are all optically active and appear to possess the same absolute configuration.

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 P450s. 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 HCFCs, 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.

Role of Inducible Cytochrome P450 in the Liver Toxicity of Polyhalogenated Aromatic Compounds

Both major inducible forms of liver cytochrome P450 can interact with polyhalogenated biphenyls (PBCS) of the appropriate configuration to produce oxidizing species, as judged by the degradation of bilirubin, a readily oxidizable compound, by isolated microsomes in vitro. Planar PCBs are active with cytochrome P450 IA1, while non-planar di-orthosubstituted compounds are effective with cytochrome P450 IIB1.Evidence for uroporphyrinogen oxidation under similar conditions has also been obtained with 3-methylcholanthrene-induced rat liver microsomes, where the stimulatory effect of a planar biphenyl, though not as great and consistent as with bilirubin, could also be demonstrated. The nature of the oxidizing species produced by the microsomes is still uncertain, but bilirubin undergoing enzymic oxidation shows similar spectral changes as in a chemical oxidation system. we suggest that a PCB of the appropriate steric configuration may first induce the relevant cytochrome in vivo, then become bound to the active site of the induced cytochrome and lead to production of oxidative species. These may then attack not only bilirubin, but also other key targets in the cell, including hexahydroporphyrins (porphyrinogens) and DNA bases.