Role of reactive metabolites in drug-induced hepatotoxicity.

Drugs are generally converted to biologically inactive forms and eliminated from the body, principally by hepatic metabolism. However, certain drugs undergo biotransformation to metabolites that can interfere with cellular functions through their intrinsic chemical reactivity towards glutathione, leading to thiol depletion, and functionally critical macromolecules, resulting in reversible modification, irreversible adduct formation, and irreversible loss of activity. There is now a great deal of evidence which shows that reactive metabolites are formed from drugs known to cause hepatotoxicity, such as acetaminophen, tamoxifen, isoniazid, and amodiaquine. The main theme of this article is to review the evidence for chemically reactive metabolites being initiating factors for the multiple downstream biological events culminating in toxicity. The major objectives are to understand those idiosyncratic hepatotoxicities thought to be caused by chemically reactive metabolites and to define the role of toxic metabolites.

The Exeter Contemporary flanged cemented acetabular component in primary total hip arthroplasty.

AIMS: We report on the outcome of the Exeter Contemporary flanged cemented all-polyethylene acetabular component with a mean follow-up of 12 years (10 to 13.9). This study reviewed 203 hips in 194 patients. 129 hips in 122 patients are still in situ; 66 hips in 64 patients were in patients who died before ten years, and eight hips (eight patients) were revised. Clinical outcome scores were available for 108 hips (104 patients) and radiographs for 103 hips (100 patients). PATIENTS AND METHODS: A retrospective review was undertaken of a consecutive series of 203 routine primary cemented total hip arthroplasties (THA) in 194 patients. RESULTS: There were no acetabular component revisions for aseptic loosening. Acetabular revision was undertaken in eight hips. In four hips revision was necessitated by periprosthetic femoral fractures, in two hips by recurrent dislocation, in one hip for infection and in one hip for unexplained ongoing pain. Oxford and Harris hip scores demonstrated significant clinical improvement (all p < 0.001). Radiolucent lines were present in 37 (36%) of the 103 acetabular components available for radiological evaluation. In 27 of these, the line was confined to zone 1. No component had migrated. CONCLUSION: Kaplan-Meier survivorship, with revision for aseptic loosening as the endpoint, was 100% at 12.5 years and for all causes was 97.8% (95% confidence interval 95.6 to 100) when 40 components remained at risk. The Exeter Contemporary flanged cemented acetabular component demonstrates excellent survivorship at 12.5 years. TAKE HOME MESSAGE: The Exeter Contemporary flanged cemented acetabular component has excellent clinical outcomes and survivorship when used with the Exeter stem in total hip arthroplasty.

Efficient preparations of the beta-glucuronides of dihydroartemisinin and structural confirmation of the human glucuronide metabolite.

New and greatly improved preparations of the 12alpha,1'beta- (5) and 12beta,1'beta- (6) glucuronides of dihydroartemisinin (DHA, 2) are reported using anomeric hydroxy and imidate glucuronate intermediates. Comparison of the synthetic and natural materials shows that the human metabolite of DHA is the 12alpha-epimer 5.

Mechanism-based design of parasite-targeted artemisinin derivatives: synthesis and antimalarial activity of benzylamino and alkylamino ether analogues of artemisinin.

Several artemisinin derivatives linked to benzylamino and alkylamino groups were synthesized in order to enhance accumulation within the malaria parasite. The in vitro antimalarial activity was assessed against the chloroquine sensitive HB3 strain and the chloroquine resistant K1 strain of Plasmodium falciparum. In general the incorporation of amino functionality enhances the activity relative to artemisinin. The most potent analogue in the series was compound 6 which was severalfold more active than artemisinin against both strains of P. falciparum used in the study.

Novel, potent, semisynthetic antimalarial carba analogues of the first-generation 1,2,4-trioxane artemether.

Ten novel, second-generation, fluorinated ether and ester analogues of the potent first-generation analogues artemether (4a) and arteether (4b) have been designed and synthesized. All of the compounds demonstrate high antimalarial potency in vitro against the chloroquine-sensitive HB3 and -resistant K1 strains of Plasmodium falciparum. The most potent derivative 8 was 15 times more potent than artemisinin (2) against the HB3 strain of P. falciparum. In vivo, versus Plasmodium berghei in the mouse, selected derivatives were generally less potent than dihydroartemisinin with ED(50) values of between 5 and 8 mg/kg. On the basis of the products obtained from the in vitro biomimetic Fe(II)-mediated decomposition of 8, the radical mediator of biological activity of this series may be different from that of the parent drug, artemisinin (2).

Synthesis, antimalarial activity, biomimetic iron(II) chemistry, and in vivo metabolism of novel, potent C-10-phenoxy derivatives of dihydroartemisinin.

The combination of TMSOTf and AgClO(4) promotes the efficient C-10-phenoxylation of dihydroartemisinin (3) in good chemical yield and excellent stereoselectivity. All of the new phenoxy derivatives have potent in vitro antimalarial activity. On the basis of the excellent yield and stereoselectivity obtained for the p-trifluoromethyl derivative 7b, this compound and the parent phenyl-substituted derivative 5b were selected for in vivo biological evaluation against Plasmodium berghei in the mouse model and for metabolism studies in rats. Compound 7b demonstrated excellent in vivo antimalarial potency with an ED(50) of 2.12 mg/kg (cf. artemether = 6 mg/kg) versus P. berghei. Furthermore, from preliminary metabolism studies, this compound was not metabolized to dihydroartemisinin; suggesting it should have a longer half-life and potentially lower toxicity than the first-generation derivatives artemether and arteether. From biomimetic Fe(II)-catalyzed decomposition studies and ESR spectroscopy, the mechanism of action of these new lead antimalarials is proposed to involve the formation of both primary and secondary C-centered cytotoxic radicals which presumably react with vital parasite thiol-containing cellular macromolecules.

Assessment of the effect of malaria infection on hepatic clearance of dihydroartemisinin using rat liver perfusions and microsomes.

1. The clearance of dihydroartemisinin (DHA) in control and malaria-infected (MI) rats was investigated using the isolated perfused rat liver (IPRL) model and hepatic microsomal studies. 2. In the recirculating IPRL, clearance of DHA was reduced from a mean (s.d.) of 8.2+/-1.8 ml min(-1) in controls (n=8) to 6.0+/-1.0 ml min(-1) in MI (n=8; P<0.01). Clearance in control livers was similar to the perfusion flow rate, suggesting a high hepatic extraction ratio for DHA. 3. Single-pass IPRL studies in controls (n=8) showed that DHA bioavailability at 1.3, 8 and 38 microm was 0.026+/-0.020, 0.043+/-0.025 and 0.14+/-0.06, respectively (P<0.001 for 8 microM vs 38 microM). In MI livers (n=5), DHA bioavailability at 8 and 38 microM was 0.18+/-0.07 and 0.40+/-0.08, respectively (P=0.002). Bioavailability was higher in the MI group than in controls (P=0.01 at 8 microM and P<0.001 at 38 microM). DHA-glucuronide was the sole biliary metabolite. 4. Hepatic microsomal studies of DHA-glucuronide formation showed a significantly lower Vmax but no significant change in Km, in MI compared to control livers (n=6). Intrinsic metabolic clearance (Vmax/Km) was higher in control than in MI livers (5.2+/-1.3 and 2.5+/-1.4 microl min(-1) mg(-1), respectively; P=0.006). 5. These studies demonstrate that DHA has a high, concentration-dependent hepatic extraction ratio that is reduced by 20-30% in the P. berghei rodent malaria model. The impaired hepatic clearance of DHA in MI is attributable to a reduction in intrinsic metabolic clearance.

Drug-protein conjugates--XVI. Studies of sorbinil metabolism: formation of 2-hydroxysorbinil and unstable protein conjugates.

The metabolism of sorbinil [+)6-fluoro-spiro (chroman-4, 4'-imidazolidine)-2',5' dione), an aldose reductase inhibitor associated with immunological adverse reactions, was studied in vivo and in vitro with particular reference to the formation of protein conjugates of 2-hydroxysorbinil and their further metabolism. [8-3H]Sorbinil was rapidly and extensively metabolized in the rat. 2-Hydroxysorbinil (2HSB) and a phenolic primary alcohol (2,4-imidazolidinedione 5-(2-hydroxyethyl)-5-(5-fluoro-2-hydroxyphenyl); IHFH) were its principal urinary metabolites; over 0-24 hr, they represented 17.0 +/- 0.7% (mean +/- SD, N = 4) and 7.1 +/- 0.7% of the dose, respectively. [3H]2HSB isolated from urine and re-administered was converted to IHFH. Chronic dosing with sorbinil (150 mg/kg x 5) induced 2-hydroxylation of the drug, the 0-24 hr urinary excretion of 2HSB increasing from 17.0 +/- 0.7% to 24.7 +/- 3.4% of the dose (P less than 0.05 by Students' paired t-test). The biotransformation of 2HSB to IHFH was rationalized in terms of an open-chain aldehyde intermediate. Since aldehydes form both stable and unstable protein adducts, 2HSB was potentially a pro-reactive metabolite and initiator of the hypersensitivity reaction associated with sorbinil. However, [3H]2HSB was neither metabolized by human liver microsomes nor underwent irreversible binding to the microsomal protein. Nevertheless, the mild reductant sodium cyanoborohydride, although without effect on microsomal binding of [3H]2HSB, enhanced binding to human serum albumin. Formation of unstable Schiff base adducts was indicated.

Drug-protein conjugates--V. Sex-linked differences in the metabolism and irreversible binding of 17 alpha-ethinylestradiol in the rat.

Sex-linked differences in the disposition, biotransformation, excretion and irreversible binding of [6, 7-3H]17 alpha-ethinylestradiol [( 3H]EE2) in Wistar rats have been observed. Three hours after i.v. administration of [3H]EE2 (5 micrograms/kg) the livers of males contained twice as much 3H-labelled material as those of females. The biliary metabolites were largely glucuronides in both sexes, but males also excreted arylsulphates. The principal metabolites liberated from biliary conjugates by enzymes were 2-hydroxyEE2 and 2-methoxyEE2 in females and males, respectively. Biliary elimination of 3H over 3 hr was slightly greater in males (P less than 0.05). Radiolabelled material was irreversibly bound to hepatic microsomal and soluble protein. The material bound to microsomes represented 0.24 +/- 0.07% (mean +/- S.D.) of the dose in males and 0.56 +/- 0.10% in females (P less than 0.001). Oxygenation of the steroid D-ring was not indicated, and 2-hydroxyEE2 appears to be the precursor of the reactive metabolite. The metabolic basis of the sex-linked difference in irreversible binding is discussed.

Drug-protein conjugates--VIII. The metabolic fate of the dinitrophenyl hapten conjugated to albumin.

Dinitrofluorobenzene has been used as a model chemically reactive metabolite to investigate factors which determine the fate of drug-protein conjugates formed in vivo. The disposition of homologous and heterologous albumin conjugated with 3H-dinitrophenyl groups (3H-DNP) has been investigated in the male Wistar rat. After intravenous administration, conjugates were cleared from plasma, predominantly through the liver, and the hapten was excreted into bile and urine as the novel amino acid derivative N2-acetyl-N6-DNP-lysine. It is assumed that the product arises from lysosomal hydrolysis of the conjugate, followed by N-acetylation, since its biliary excretion was significantly reduced in animals pretreated with suramin, an inhibitor of lysosomal proteolysis. The clearance of DNP-albumin conjugates was dependent upon the degree of conjugation; conjugates with an epitope density of greater than 20 had a short (ca. 1 hr) half-life. In a second study, the disposition of DNP-autologous protein conjugates was monitored in the rabbit over 21 days. The plasma concentration-time profile of the serum conjugates indicated that clearance was dependent upon non-immune mechanisms and that intravenous administration of DNP-serum protein conjugates did not elicit an anti-DNP response.

Oxidative dehalogenation of 2-fluoro-17 alpha-ethynyloestradiol in vivo. A distal structure-metabolism relationship of 17 alpha-ethynylation.

Metabolic activation to catechols and their oxidation products is variously considered to contribute to the genotoxic, cytotoxic, transforming and tumour-promoting activities of exogenous steroidal oestrogens. 2-Fluoro-17 alpha-ethynyloestradiol (2-FEE2) was synthesized as a prototype of pharmacologically active derivatives of 17 beta-oestradiol which are resistant to metabolic activation in vivo. It possessed high affinity for the rat uterine oestrogen receptor and was oestrogenic in rats. Biliary metabolites of [6,7-3H]2-FEE2 (0.73 mumol/kg, 157 micrograms/kg, i.v.) in female rats were characterized: 87% of the radiolabel was excreted, principally as 2-FEE2 glucuronide, over 6 hr. Although 2-fluoro-17 beta-oestradiol is not metabolized to C-2 oxygenated products in vivo, 2-FEE2 underwent rapid and appreciable oxidative defluorination. 2-Hydroxy-17 alpha-ethynyloestradiol and 2-methoxy-17 alpha-ethynyloestradiol represented, respectively, 8% and 13% of the dose. Fluorination nevertheless restricted C-2 oxygenation to ca. 28% of that which 17 alpha-ethynyloestradiol undergoes in female rats. C-4 oxygenation of 2-FEE2, resulting in catechol formation, occurred but to a lesser extent (ca. 12% of dose). None of the major and identified minor biliary metabolites was a product of metabolic activation at the ethynyl function. A mechanistic rationalization of the long range enhancement by 17 alpha-ethynylation of oxidative defluorination at C-2 is presented.

Drug-protein conjugates--XIV. Mechanisms of formation of protein-arylating intermediates from amodiaquine, a myelotoxin and hepatotoxin in man.

The enzymic and non-enzymic formation of protein-arylating intermediates from amodiaquine (AQ,7-chloro-4-(3'-diethylamino-4'-hydroxyanilino) quinoline), an anti-malarial associated with agranulocytosis and liver damage in man, was studied in vitro. [14C]AQ in phosphate buffer, pH 7.4, under air was autoxidized to a reactive derivative(s) which possessed characteristics indicative of a semiquinone/quinone imine: reduction by NADPH and ascorbic acid, conjugation with thiols and irreversible binding to microsomal and soluble proteins. Cysteinyl SH groups were major sites of arylation. Radiolabelled material irreversibly bound to HSA after 24 hr and to human liver microsomes after 4 hr represented 26.5 +/- 1.8% and 31.4 +/- 0.6% (means +/- SD, N = 3) of incubated [14C]AQ (10 microM), respectively. The quinone imine of AQ(AQQI) was synthesized, and displayed the same oxidative and electrophilic reactions as the product(s) of AQ's autoxidation. A water-soluble product formed in buffered solutions of AQ and N-acetylcysteine was identified as an AQ mercapturate by comparison with an adduct prepared from synthetic AQQI. Irreversible binding of [14C]AQ was inhibited by a radical scavenger; this indicated that the semiquinone imine contributed to the binding. Although AQ was extensively de-ethylated by human liver microsomes, oxidation by cytochrome P-450 did not appear to be principally responsible for its activation and irreversible binding in microsomal incubations. AQ was oxidized to protein-arylating intermediates by horseradish peroxidase. It also formed reactive derivatives, possibly N-chloro compounds, in chlorine solutions. These findings indicated that AQ can give rise to chemically reactive species by at least three distinct mechanisms, viz. autoxidation in neutral solution under air, peroxidase-catalyzed oxidation and N-chlorination. Formation of such species in liver and myeloid cells might be responsible for the adverse reactions associated with AQ.

The metabolism of 2- and 4-fluoro-17 beta-oestradiol in the rat and its implications for oestrogen carcinogenesis.

2-Fluoro-[6,7-3H]17 beta-oestradiol([3H]2-FE2) and 4-fluoro-[6,7-3H]17 beta-oestradiol([3H]4-FE2) were synthesized by the fluorination and reduction of [3H]oestrone and purified by HPLC. [3H]2-FE2 and [3H]4-FE2 (72.5 micrograms/kg; 0.25 mumol/kg) were administered i.v. to anaesthetized female and male Wistar rats (N = 4) with biliary cannulae. Bile was collected for 6 hr. Female rats administered [3H]2-FE2 excreted 85% of the dose into bile over 6 hr whilst male rats excreted 77%. After the administration of [3H]4-FE2, female and male rats excreted 72 and 83% of dose into bile over 6 hr, respectively. The biliary metabolites were glucuronides in all cases. The principal metabolite of [3H]2-FE2 liberated from biliary conjugates by beta-glucuronidase was 2-fluoroestrone in both female rats (64% of dose) and male rats (57%). No 2-hydroxylated, i.e. oxidatively defluorinated, metabolites were detected in either sex. In contrast, 2-hydroxylation of [3H]4-FE2 did occur, but only in female rats: 2-hydroxy-4-fluoro-oestrone (22%) and 2-methoxy-4-fluoroestrone (17%) were identified as biliary aglycones. However, the major metabolite was 4-fluoroestrone (4FE1; 38%). In male rats, 4-FE1 and 4-fluoro D-ring-oxygenated products were the principal biliary aglycones. The differences in metabolism between the two fluoro analogues and oestradiol are discussed with particular reference to the possible involvement of 2- and 4-hydroxy (catechol) oestrogens in oestrogen toxicity.

The bioactivation of amodiaquine by human polymorphonuclear leucocytes in vitro: chemical mechanisms and the effects of fluorine substitution.

Amodiaquine, a 4-aminoquinoline antimalarial, has been associated with hepatitis and agranulocytosis in humans. Drug hypersensitivity reactions, especially agranulocytosis, have been attributed to reactive intermediates generated by the oxidants discharged from stimulated polymorphonuclear leucocytes (PMN). The metabolism of amodiaquine to both stable and chemically reactive metabolites by human PMN has been investigated in vitro. Incubation of [14C]-amodiaquine with PMN resulted in irreversible binding of radiolabel to protein and depletion of intracellular reduced glutathione, which were enhanced by phorbol myristate acetate (PMA), a PMN activator. Two metabolites were identified: the C-5' glutathione adduct of amodiaquine, derived from both endogenous and exogenous glutathione, and 4-amino-7-chloroquinoline, which was presumed to be formed by hydrolysis of amodiaquine quinoneimine. Desethylamodiaquine, the major plasma metabolite of amodiaquine in humans, also underwent bioactivation to a chemically reactive species in the presence of PMA-stimulated PMN. Substitution of the 4'-hydroxyl group in amodiaquine with fluorine significantly reduced irreversible binding to protein and abolished depletion of intracellular glutathione in the presence of PMA. These findings indicate that the bioactivation of amodiaquine by PMN is associated with the formation of a quinoneimine intermediate. Such a reactive metabolite, if produced in PMN or bone marrow in vivo, may be responsible for the drug's myelotoxicity.

Drug-protein conjugates--X. The role of protein conjugation in the disposition of dinitrofluorobenzene.

The metabolism and irreversible protein binding of 2,4-[3,5-3H]dinitrofluorobenzene (3H-DNFB), a model chemically reactive compound, were studied in the rat. 3H-DNFB given intravenously (5 micrograms, 5 mg or 25 mg per kg) to anaesthetized cannulated rats was rapidly metabolized via the mercapturic acid pathway. The metabolites were extensively eliminated in bile and urine: predominantly as the glutathione conjugate and mercapturate in bile, and as the mercapturate in urine. Only ca. 3-10% of the doses remained in the liver, kidneys, spleen, heart and lungs at 3 hr. Dinitrophenyl mercapturate was the principal urinary metabolite in conscious rats dosed i.v. (5 mg or 25 mg per kg). Only 15-25% of the radiolabelled material in liver and kidney at 3 hr was irreversibly bound to protein, but 45-99% of that in the other organs and 49-88% in plasma was irreversibly bound. Preliminary evidence for the metabolism of 3H-DNFB (5 mg/kg and 25 mg/kg doses) to N2-acetyl-N6-DNP-lysine, a novel conjugate and metabolite of dinitrophenylated proteins in vivo, is presented.

In vitro metabolism of dexamethasone (DEX) in human liver and kidney: the involvement of CYP3A4 and CYP17 (17,20 LYASE) and molecular modelling studies.

Dexamethasone (DEX) has previously been shown to be extensively metabolised to 6-hydroxylated and side-chain cleaved metabolites in human liver in vitro. CYP3A4 is responsible for 6alpha- and 6beta-hydroxylation of DEX and CYP17 is thought to mediate side-chain cleavage to generate 9alphafluoro-androsta-1,4-diene-11beta-hydroxy-16alpha-methyl-3,17-dione (9alphaF-A). Although 9alphaF-A has not previously been isolated as a metabolite in its unhydroxylated form in human liver incubations, it is formed as an intermediate metabolite, which is subsequently rapidly hydroxylated to OH-9alphaF-A. A main part of this study has been to conclusively show that DEX undergoes extensive side-chain cleavage to form 9alphaF-A in human kidney fractions, which is in contrast to profiles obtained for DEX metabolism in parallel human liver microsomal incubations where 6-hydroxylation is the predominant pathway. Furthermore, molecular models of CYP3A4 and CYP17 (17,20 lyase) have been used to model the enzyme fits of DEX. From these modelling studies it has been shown that DEX complements both putative enzyme active sites in orientations likely to lead to the formation of the metabolites identified in vitro. We have also been able to rationalise the preferential formation of the 6betaOH-DEX isomer.

Obstacles to the prediction of estrogenicity from chemical structure: assay-mediated metabolic transformation and the apparent promiscuous nature of the estrogen receptor.

Information on structure-activity relationships (SAR) and pathways of metabolic activation would facilitate the preliminary screening of chemicals for estrogenic potential. Published crystallographic studies of the estrogen receptor (ER) imply an essential role of the two hydroxyl groups on estradiol (17beta-E(2)) for its binding to ER. The influence of these hydroxyl groups on ER binding and estrogenicity was evaluated by the study of 17beta-E(2) with one or both of these hydroxyl groups removed (17beta-desoxyestradiol and 3, 17beta-bisdesoxyestradiol, respectively). 6-Hydroxytetralin (17beta-E(2) with its C- and D-rings removed) and other synthetic estrogens were also studied. The estrogenicity assays comprised a yeast ER-mediated transcription assay, mammalian cell transcription assays incorporating either ER alpha or ER beta, and the immature rat uterotrophic assay. With the exception of 6-hydroxytetralin in the uterotrophic assay, all the chemicals were active in all the assays. Hydroxylation of the two desoxy compounds to estradiol was shown to occur in immature female rats, but metabolism was not implicated in the responses observed in the ER-binding and yeast systems. It is concluded that the 3-hydroxyl and 17beta-hydroxyl groups of 17beta-E(2) are not absolute requirements for estrogenicity. It would therefore be of value to the derivation of SAR for estrogenicity were the crystal structure of the bisdesoxy-E(2)/ER complex to be evaluated.

The sexually differentiated metabolism of [6,7-3H]17 beta-oestradiol in rats: male-specific 15 alpha- and male-selective 16 alpha-hydroxylation and female-selective catechol formation.

The oxygenated-metabolite profiles of exogenous 17 beta-oestradiol (E2) in adult male and female Wistar rats have been characterized and major sex-dependent biotransformations observed which correlate with the regioselectivities of known sexually differentiated hepatic P450. [6,7-3H]E2 (27 micrograms/kg) was given i.v. The metabolites of E2 were rapidly and extensively excreted in bile (46 and 78% of the dose over 1 and 6 h, respectively). Female rats metabolized E2 by one major pathway: oxidation to oestrone (E1) followed by C-2 hydroxylation and O-methylation; the principal aglycones (0-1 h bile collections) were E1 (14%), 2-hydroxyE1 (2-OHE1) (42%) and 2-methoxyE1 (24%). Male rats metabolized E2 principally by two parallel composite pathways of E1 hydroxylation which yielded a complex mixture of mono- and di-oxygenated compounds: 15 alpha-OHE1 (33%), 2,15 alpha-diOHE1 (7%), and 2-methoxy-15 alpha OHE1 (14%); 16 alpha-OHE1 (13%), 2,16 alpha-diOHE1 (4%) and 2-methoxy-16 alpha-OHE1 (2%). 15 alpha-Hydroxylation was unique to males. The balance of aromatic and alkyl hydroxylation in males was dose-dependent: at 3 mg/kg, 15 alpha-hydroxylation was decreased approx. 50% in favour of 2-hydroxylation whilst 16 alpha-hydroxylation was largely unaffected. The male-specific 15 alpha-hydroxylation and male-predominant 16 alpha-hydroxylation of E1 derived from E2 in vivo may be ascribable to the male-specific isoforms P450IIC13 and P450IIC11, respectively.

The metabolism of 2,4-dibromo [6,7-3H]17 beta-oestradiol in the rat: ring-A dibromination blocks male-specific 15 alpha-hydroxylation and catechol formation.

The metabolism in the rat of 2,4-dibromo-17 beta-oestradiol (2,4-DBE2), a compound of potential use for tumour imaging and assessment, has been studied. 2,4-DB[6,7-3H]E2 was synthesized by bromination of [6,7-3H]E2 with N-bromosuccinimide, and administered (40 micrograms/kg, i.v.) to anaesthetized male and female rats. Metabolites were rapidly and extensively excreted in bile (60 and 82% of the dose over 1 and 6 h, respectively). No unchanged compound was excreted. 2,4-DBE2 was almost entirely oxidized to 2,4-DB-oestrone; which was largely eliminated as its glucuronide but partly (approx. 30%) metabolized to 2,4-DB-16 alpha-hhydroxyoesterone and, to a minor extent, 2,4-DB-oestriol. No products of either oxidative or reductive debromination were detected. Neither of the two oxidative transformations of 2,4-DBE2 in the rat, in contrast with those of exogenous E2, was sex-selective, and 2,4-DB-oestrone underwent less extensive hydroxylation than oestrone formed from E2. In female rats, the substituents selectively redirected the principal site of hydroxylation from C-2 to C-16, whereas in males they had no significant effect on the existing 16 alpha-hydroxylation but did block the major pathway, 15 alpha-hydroxylation. Thus the sexual differentiation of E2 oxidative metabolism was abolished by direct blockage causing metabolic switching to a latent reaction in the female rat and long-range inhibition of the vicinal hydroxylation in the male rat.

Cortisol metabolism by human liver in vitro--I. Metabolite identification and inter-individual variability.

The measurement of urinary 6 beta-hydroxycortisol (6 beta-OHF) has been widely used as a non-invasive clinical test to detect cytochrome P450 induction. Although only a minor biotransformation, 6 beta-OHF formation represents a sensitive target for many P450-inducing drugs and environmental chemicals in man. There is good evidence that an isozyme of the P450IIIA subfamily is predominantly responsible for 6 beta-hydroxylase activity and therefore it has been suggested that urinary 6 beta-OHF is a marker of the induction of P450IIIA. The basis of the present study was that in order to realistically assign to 6 beta-OHF the status of a P450IIIA marker we should characterize all the metabolites of cortisol produced by human liver and assess inter-liver variability. Incubations at 37 degrees C for 2 h contained [3H]cortisol (0.1 microCi, 1 or 50 microM), MgCl2 (10 mM), microsomal or cytosolic protein (3 mg), an NADPH-regenerating system and 1/15 M phosphate buffer (pH 7.4) to give a final volume of 0.5 ml. Extraction with ethyl acetate (2 x 2 ml) was followed by radiometric HPLC analysis. Metabolites were identified by co-chromatography with authentic standards and mass spectrometry (electron impact and chemical ionization). All the microsomal incubations (n = 6 livers) produced 6 alpha-hydroxycortisol (6 alpha-OHF), 6 beta-OHF, 20 beta-dihydroxycortisol, 20 beta-dihydroxycortisone, cortisone, and 3 alpha, 5 beta-tetrahydrocortisone (3 alpha, 5 beta-THE), while five produced 6 beta-hydroxycortisone and four produced 3 alpha, 5 beta-tetrahydrocortisol (3 alpha, 5 beta-THF). The cytosolic incubations gave a much simpler metabolic profile, with 3 alpha, 5 beta-THF the major metabolite and 3 alpha, 5 beta-THE a minor metabolite. There was considerable inter-individual variability in metabolite profiles from microsomal incubations. 6 beta-OHF varied from 2.8 to 31.7%. Major metabolites were cortisone and 3 alpha, 5 beta-THE. Inter-liver variability was less for cytosolic incubations, the major metabolite always being 3 alpha, 5 beta-THF. In conclusion we have rigorously identified the hepatic metabolites of cortisol formed in vitro. The highly complex and variable hepatic metabolism of cortisol clearly limits the use of urinary 6 beta-OHF excretion as a marker of baseline P450IIIA activity in man.