3-methylcholanthrene-induced monooxygenase (O-deethylation) activity of human lymphocytes.

With a direct fluorescence assay, the levels of mixed-function oxidase activity were determined in mitogen-activated human lymphocytes. The O-deethylation of ethoxyresorufin to resorufin was used to quantitate mixed-function oxidase activity. Ethoxyresorufin O-deethylase activity was low to nondetectable in noninduced, mitogen-activated cells, but it was readily detected in 3-methylcholanthrene, mitogen-activated lymphocytes. The activity was: (a) dependent on assay time and number of lymphocytes; (b) dependent on the presence of reduced nicotinamide adenine dinucleotide phosphate; (c) stable to freezing at -80 degrees for at least 2 weeks; (d) reproducibly detected in duplicate samples of blood from one individual when cultured and assayed at the same time; but (e) quite variable in samples of blood from one individual at different times. Since in hepatic and pulmonary tissue of model animal systems ethoxyresorufin is a specific substrate for cytochrome P-448-associated monooxygenases, the use of this chemical could proffer an assay that specifically measures human cytochrome P-448-associated activity.

Tumor-specific expression of cytochrome P450 CYP1B1.

Cytochrome P450 CYP1B1 is a recently cloned dioxin-inducible form of the cytochrome P450 family of xenobiotic metabolizing enzymes. An antibody raised against a peptide specific for CYP1B1 was found to recognize CYP1B1 expressed in human lymphoblastoid cells but not to recognize other forms of cytochrome P450, particularly CYP1A1 and CYP1A2. Using this antibody, the cellular distribution and localization of CYP1B1 were investigated by immunohistochemistry in a range of malignant tumors and corresponding normal tissues. CYP1B1 was found to be expressed at a high frequency in a wide range of human cancers of different histogenetic types, including cancers of the breast, colon, lung, esophagus, skin, lymph node, brain, and testis. There was no detectable immunostaining for CYP1B1 in normal tissues. These results provide the basis for the development of novel methods of cancer diagnosis based on the identification of CYP1B1 in tumor cells and the development of anticancer drugs that are selectively activated in tumors by CYP1B1.

Cytochrome P450 CYP3A5 in the human anterior pituitary gland.

The cytochromes P450 are a key group of enzymes involved in the metabolism of xenobiotics and several biologically active endogenous compounds. The expression of CYP3A5 has been identified by reverse transcriptase-polymerase chain reaction in human pituitary gland and shown by immunohistochemistry to be localized to growth hormone containing cells of the anterior pituitary gland. This is the first direct identification of an individual P450 subfamily in the pituitary gland and the presence of CYP3A in the pituitary gland may play a role in regulating growth hormone secretion.

The influence of culture medium composition on drug metabolising enzyme activities of the human liver derived Hep G2 cell line.

When grown in the standard Dulbecco's medium the human liver derived Hep G2 hepatoma cell line shows only 10-20% of the cytochrome P-450-dependent mixed function oxidase (MFO) activity of freshly isolated human adult hepatocytes. However, the MFO activities and, to a lesser extent, the activities of UDP-glucuronyltransferase and glutathione-S-transferase can be increased by altering the composition of the growth medium. Modified Earle's medium was more effective in this respect than Williams' E medium and increased the O-dealkylations of ethoxyresorufin, benzyloxyresorufin and pentoxyresorufin 50-, 30- and 10-fold, respectively.

Studies on the maintenance of cytochromes P-450 and b5, monooxygenases and cytochrome reductases in primary cultures of rat hepatocytes.

The cytochrome P-450 content of rat hepatocytes declined rapidly over 72 h in culture, due primarily to denaturation to cytochrome P-420. Six different media were investigated for their ability to conserve cytochrome P-450 during culture, and the most successful was a modified Earle's medium. After 72 h culture in this medium, cytochromes P-450 and b5, NADH-cytochrome b5- and NADPH-cytochrome c-reductases were maintained at 40, 100, 35 and 52% of fresh cell values, respectively. Cytochrome P-450 showed differential functional stability during culture with ethoxyresorufin O-deethylation being more stable than either pentoxyphenoxazone O-depentylation or biphenyl 4-hydroxylation. Monooxygenase than did cytochrome P-450 content. This discrepancy was not explained by loss of flavin nucleotides, FMN or FAD.

Expression of cytochrome P450 CYP1B1 in breast cancer.

The expression of CYP1B1 has been identified in breast cancer using the reverse transcriptase-polymerase chain reaction and immunoblotting. CYP1B1 mRNA was expressed in the majority of breast tumours and immunoblotting of breast tumours identified a single protein band of molecular weight 60 kDa corresponding to the predicted molecular weight of human CYP1B1. This is the first study to identify CYP1B1 expression in a tumour where it may represent a previously unknown pathway for the metabolism of oestradiol and chemotherapeutic drugs.

Creatine kinase isoenzymes. Predictive value in the early diagnosis of acute myocardial infarction.

This is a prospective study of the value of the creatine kinase (CK) isoenzyme determination in the early diagnosis of acute myocardial infarction. The presence or absence of the MB isoenzyme was correlated with electrocardiogram and standard enzymes. The frequency of falsely positive and falsely negative results for CK-MB, electrocardiogram and each standard enzyme was calculated and, using the elements of conditional probability theory, their predictive values for the diagnosis of acute myocardial infarction were determined. Results indicate that CK-MB combines the best attributes of the electrocardiogram and standard enzyme tests: detectable MB isoenzyme activity by acrylamide slab electrophoresis has a predictive value for the diagnosis if acute myocardial infarction comparable to that of a positive electrocardiogram; absence of MB isoenzyme activity, in the 24 hour period following the onset of symptoms, excludes the diagnosis of acute myocardial infarction with a probability equivalent to that provided by normal standard enzyme results.

Involvement of human cytochromes P450 (CYP) in the reductive metabolism of AQ4N, a hypoxia activated anthraquinone di-N-oxide prodrug.

PURPOSE: To establish the role of the human cytochromes P450 (CYPs) in the reductive metabolism of the novel anthraquinone di-N-oxide prodrug AQ4N. METHODS AND MATERIALS: Metabolism of AQ4N was conducted in a panel of 17 human phenotyped liver microsomes. AQ4N and metabolites were detected by reverse phase isocratic HPLC. CYP inhibitors and Spearman rank correlation were used to determine the significance of AQ4N metabolism versus specific CYP activity and/or expression. RESULTS: Anaerobic metabolism of AQ4N to the 2-electron reduction product, AQM, and the 4-electron reduced tertiary amine, AQ4, occurred in all 17 human liver microsome preparations. The range (+/- SE) for total AQ4N turnover was 14.26 +/- 1.43 nmol/incubate (highest) to 3.65 +/- 1.05 nmol/incubate (lowest). Metabolism was not detected in the absence of NADPH or microsomes. In aerobic incubates, AQM was less than 4% of anaerobic values whereas AQ4 was undetectable. CYP-mediated metabolism of AQ4N was inhibited completely by ketoconazole (KET) and carbon monoxide (CO), two global inhibitors of CYP-mediated metabolism. AQ4N metabolism correlated significantly with probes for CYP 3A, specifically benzoxylresorufin O-dealkylation [r(s) = 0.70,p <0.01] and tamoxifen N-demethylation (r(s) = 0.85, p < 0.01), but not with probes for CYPs 2C, 2D, and 1A. CYP 3A involvement was confirmed by the use of the CYP 3A specific inhibitor, triacetyloleandomycin (TAO), which repressed the formation of AQM to 13% of the uninhibited value and abolished completely the formation of AQ4. Alpha-naphthoflavone (ANF), an inhibitor of CYP 2C and 1A, had no significant effect on AQ4N metabolism. CONCLUSIONS: These data suggest that the human CYP 3A enzymes can contribute to the reductive metabolism of AQ4N. CYP 3A enzymes are highly expressed in a broad spectrum of human cancers. The results show that AQ4N requires anaerobic conditions for CYP 3A-mediated reduction and hence this subfamily of enzymes is likely to selectively activate AQ4N in hypoxic tumors.

Enzyme histochemical demonstration of NADH dehydrogenase on resin-embedded tissue.

We describe a method for enzyme histochemical demonstration of NADH dehydrogenase in cold (4 degrees C)-processed resin-embedded tissue. The effects on NADH dehydrogenase activity of processing tissue through a variety of dehydrating agents and embedding in three different acrylic resins were evaluated. The optimal procedure to maintain NADH dehydrogenase activity used a short (3-hr) fixation in 1% paraformaldehyde solution, followed by dehydration in acetone and embedding in glycol methacrylate resin. Embedding of tissue in resin combined preservation and accurate localization of NADH dehydrogenase activity with good tissue morphology. Blocks of the resin-embedded tissue could be stored at room temperature for at least 6 months without loss of NADH dehydrogenase activity.

Enzyme histochemistry on freeze-dried, resin-embedded tissue.

We have developed a method for histochemical demonstration of a wide range of enzymes in freeze-dried, resin-embedded tissue. Freeze-dried tissue specimens were embedded without fixation at low temperature (4 degrees C or -20 degrees C) in glycol methacrylate resin or LR Gold resin. Enzyme activity was optimally preserved by embedding the freeze-dried tissue in glycol methacrylate resin. All enzymes studied (oxidoreductases, esterases, peptidases, and phosphatases), except for glucose-6-phosphatase, were readily demonstrated. The enzymes displayed high activity and were accurately localized without diffusion when tissue sections were incubated in aqueous media, addition of colloid stabilizers to the incubating media not being required. Freeze-drying combined with low-temperature resin embedding permits the demonstration of a wide range of enzymes with accurate enzyme localization, high enzyme activity, and excellent tissue morphology.

Localization of microsomal epoxide hydrolase in normal and neoplastic human kidney.

Microsomal epoxide hydrolase is a xenobiotic metabolizing enzyme that catalyzes the conversion of toxic and carcinogenic epoxides to less toxic dihydrodiols. The cellular localization and distribution of microsomal epoxide hydrolase were investigated for the first time in normal and neoplastic human kidney. Light microscopic immunohistochemical studies using an alkaline phosphatase-anti-alkaline phosphatase technique showed that in normal kidney there was a wide distribution of epoxide hydrolase immunoreactivity. The main localization of epoxide hydrolase immunoreactivity was to the proximal and distal tubule epithelial cells. Strong epoxide hydrolase immunoreactivity was also identified in epithelium of the collecting ducts. In addition, epoxide hydrolase immunoreactivity was present in vascular endothelial cells, including endothelial cells lining glomerular capillaries. Epoxide hydrolase immunoreactivity was identified in all the renal tumors, and in each tumor immunoreactivity for epoxide hydrolase was localized to tumor cells. Immunoblotting of both normal kidney and tumor microsomes confirmed the presence of a single protein band of molecular weight 49 KD corresponding to the molecular weight of human hepatic microsomal epoxide hydrolase.

Alkoxyresorufin O-dealkylases: association with the murine Ah locus.

The hepatic microsomal dealkylation of a series of alkoxyresorufins and the oxidation of phenoxazone to resorufin were investigated in C57BL/6 and DBA/2 mice of both sexes. In both strains of mice and in both sexes the dealkylation rate decreased with increasing length of the alkyl chain. With all alkoxyresorufins the dealkylation rates were higher in the C57BL mice than the DBA mice, whereas the rate of phenoxazone hydroxylation was higher in the latter. In the C57BL mice, and to a lesser extent in the DBA mice, females were more efficient in dealkylating the resorufin ethers. Treatment with 3-methylcholanthrene (3MC) enhanced the rates of dealkylation of all alkoxyresorufins in the C57BL mice but not in the DBA mice, the extent of stimulation being highest for the propoxy- and butoxyresorufins and least for pentoxy-, heptoxy- and benzyloxyresorufins. The same treatment had no effect on the oxidation of phenoxazone in either strain of mice. It is concluded that the dealkylation of alkoxyresorufins, not the oxidation of phenoxazone, is associated with the murine Ah locus.

Decreased hepatic microsomal cytochrome P450 due to indomethacin: protective roles of 16,16-dimethylprostaglandin F2 alpha and inducing agents.

Indomethacin administration to rats caused a dose-dependent decrease in hepatic microsomal cytochrome P450, aminopyrine N-demethylase, ethoxyresorufin O-de-ethylase and benzyloxyresorufin O-debenzylase, accompanied by selective alterations in microsomal sodium dodecylsulphate polyacrylamide gel electrophoretograms. High doses (greater than or equal to 8.5 mg/kg) caused the disappearance of certain of the SDS-PAGE proteins tentatively identified as being different forms of cyt. P450, together with either increases, decreases or no change in some of the non-cyt. P450 proteins in the electrophoretogram. Concomitant administration of 16,16-dimethylprostaglandin F2 alpha gave dose-dependent protection against the deleterious effects of indomethacin on the enzymic and electrophoretic parameters of cyt. P450, but did not prevent the changes due to indomethacin in the non-cyt. P450 proteins on the electrophoretogram. In contrast, prior phenobarbitone or 3-methylcholanthrene induction prevented the effects of indomethacin on both cyt. P450 and the other microsomal proteins. Concomitant administration of SKF-525A exacerbated the effects of indomethacin on cyt. P450 and the other proteins. Indomethacin coadministration with 3-methylcholanthrene resulted in the major 3MC-induced putative cyt. P450 apoprotein having a lower mol. wt than usual. Conversely, indomethacin did not prevent the induction by SKF-525A of a different putative cyt. P450 apoprotein, despite causing decreases in cyt. P450 as determined spectrophotometrically and enzymologically. The results indicate that indomethacin rather than one of its metabolites is responsible for the decrease in cyt. P450 and that the mechanisms of protection by prostaglandin and inducing agents are, respectively, different.

Are the decreases in hepatic cytochrome P-450 and other drug-metabolising enzymes caused by indomethacin in vivo mediated by intestinal bacterial endotoxins? 16,16-Dimethylprostaglandin F2 alpha prevents decreases in hepatic drug-metabolising enzymes due to exogenous endotoxin.

Administration of either indomethacin (8.5 mg/kg) or E. coli endotoxin (3.5 mg/kg) to rats caused significant decreases in a variety of drug-metabolising enzyme activities. Either agent markedly decreased biphenyl 4-hydroxylase by 72-80% and caused lesser decreases (21-64%) in cyt. P-450, aminopyrine N-demethylase, ethoxyresorufin O-deethylase (EROD), benzyloxyphenoxazone O-debenzylase (BPOD), cyt. b5, NADPH-cyt. c reductase, NADH-cyt. b5 reductase, epoxide hydrolase (EH) and glucuronyl transferase (GT). The decreases in GT (21-22%) were significantly less than in cyt. P-450 (45-57%). Sulphotransferase was not affected by either indomethacin or endotoxin. The overall pattern of relative decreases in the different enzymes was similar for either indomethacin or endotoxin. Four activities, however, were affected to a significantly greater extent by indomethacin than by endotoxin at 2-6 mg/kg: EROD, BPOD, cyt. b5 and EH. Additionally, hepatic glutathione was decreased by indomethacin but not by endotoxin. Indomethacin or endotoxin caused similar but not identical decreases in selected protein bands in the "cyt. P-450 region" of microsomal SDS-polyacrylamide gel electrophoretograms. Concomitant administration of 16,16-dimethylprostaglandin F2 alpha afforded significant (50-100%) protection against all the above-mentioned effects of indomethacin or endotoxin. The effects of indomethacin on cyt. P-450 were lessened by concomitant administration of a mixture of neomycin, polymyxin B and bacitracin. Throughout the study there was a close correlation between the extent of decrease in hepatic cyt. P-450 and the degree of intestinal ulceration caused by indomethacin. It was concluded that bacterial endotoxins liberated into the portal blood as a result of indomethacin-induced ulceration of the small intestine probably only partially mediated the effects of indomethacin on hepatic drug-metabolising enzymes. The protection afforded by 16,16-dimethylprostaglandin F2 alpha could have been due to both the prevention of ulceration and to a direct cytoprotective effect on the liver.

Denaturation of cytochrome P-450 by indomethacin and other non-steroidal anti-inflammatory drugs: evidence for a surfactant mechanism and a selective effect of a p-chlorophenyl moiety.

Indomethacin added to rat liver microsomes in vitro resulted in the denaturation of cytochrome P-450 to cytochrome P-420. This was NADPH independent, appeared to be non-enzyme mediated, did not involve free radicals or lipid peroxidation and was prevented by glycerol, butylated hydroxytoluene or SKF-525A. Indomethacin in vitro also caused a loss of cytochrome b5, NADH-cytochrome b5 reductase, NADPH-cytochrome c reductase and epoxide hydrolase activities, but an activation of UDP-glucuronyltransferase. Amongst a total of 22 non-steroidal anti-inflammatory drugs and derivatives there was a highly significant correlation between the extent of their denaturation of cytochrome P-450 and their surfactant potency. The results suggest that the denaturation of cytochrome P-450 by certain non-steroidal anti-inflammatory drugs was due to a detergent-like, membrane-perturbing action of the drugs and that in most cases the denaturation also involved a specific effect of a p-chlorophenyl moiety of the drug.

Hydroxylation of the antimalarial drug 58C80 by CYP2C9 in human liver microsomes: comparison with mephenytoin and tolbutamide hydroxylations.

58C80 [2-(4-t-butylcyclohexyl)-3-hydroxy-1,4-naphthoquinone] is an experimental naphthoquinone antimalarial drug which undergoes extensive alkyl hydroxylation to a single t-butylhydroxy metabolite in man in vivo and also in human liver microsomes, where this is catalysed primarily by a 54 kDa CYP2C9 form of cytochrome P450, P450hB20-27. Microsomal 58C80 hydroxylation (58OH) activity showed a marked inter-individual variation in a bank of 39 individual human livers but did not correlate with the immunoquantified levels of either of two microsomal proteins (54 and 50 kDa, respectively) recognised by a polyclonal antibody against CYP2C9 (P450hB20-27). Neither 58OH activity nor the concentrations of the CYP2C9-immunorelated proteins showed any relationship with the individuals' age, sex, cigarette smoking habit, alcohol consumption or clinical drug treatment, including long term antiepileptic therapy with phenobarbitone or phenytoin. 58OH activity did not correlate with either TBOH (tolbutamide hydroxylation) or MPOH (S-mephenytoin 4'-hydroxylation) activities, while 58C80 inhibited both TBOH and MPOH in human liver microsomes non-competitively (Ki = 30 and 175 microM for TBOH and MPOH, respectively). 58C80 could be a useful model substrate for measuring human CYP2C activity in vitro.

Cytochrome P450 2C9 is responsible for hydroxylation of the naphthoquinone antimalarial drug 58C80 in human liver.

2-(4-t-Butylcyclohexyl)-3-hydroxy-1,4-naphthoquinone (58C80) is an experimental naphthoquinone antimalarial drug which undergoes extensive alky hydroxylation in man. By means of purification, N-terminal amino acid sequencing and inhibition by antibodies and sulfaphenazole, we have identified the form of cytochrome P450 primarily responsible for 58C80 hydroxylation in human liver, P450hB20-27, to be a member of the P450 2C9 subfamily. P450hB20-27 is a low-spin haemoprotein with molecular mass 54 kDa. 58C80 hydroxylation in human liver microsomes was dependent on either NADPH or NADH, with the activity supported by NADH being 35% of that supported by NADPH. With purified P450hB20-27 cytochrome b5 stimulated the NADH-dependent activity 8-fold but inhibited the NADPH-dependent activity by 30%. 58C80 is a novel substrate structure for human P450 2C and these results significantly broaden the range of drugs which have been directly shown (i.e. using a purified enzyme as opposed to expressed cDNA) to be metabolized by human P450 2C forms that are incontrovertibly expressed in human liver in vivo.

The effects of cigarette smoke compared to 3-methylcholanthrene and phenobarbitone on alkoxyresorufin metabolism by lung and liver microsomes from rats.

The rates of metabolism of phenoxazone and a homologous series of its ethers (alkoxyresorufins) by liver and lung microsomes of rats exposed to cigarette smoke were compared with the metabolism in rats pretreated with 3-methylcholanthrene (3MC) or phenobarbitone (PB). The rate of resorufin production was dependent on the length of the ether side chain. Liver and lung microsomes from control rats differed in their activity profiles (rate of resorufin production plotted against side-chain length), showing highest activity with ethoxy- and benzyloxyresorufin respectively. 3MC and PB selectively induced hepatic microsomal resorufin production with only certain of the substrates and the two agents differed in their selectivity, inducing most greatly with ethoxy- and benzyloxyresorufin respectively. Pulmonary microsomal resorufin production was induced by 3MC with a substrate selectivity similar to that shown for liver, but PB suppressed pulmonary metabolism with all the substrates. A single, short exposure to cigarette smoke induced ethoxyresorufin O-deethylase activity transiently in liver and lung microsomes. Three consecutive daily short exposures to cigarette smoke caused a weak 3MC-like induction of liver microsomal alkoxyresorufin metabolism, but the effect on lung microsomes was like weak 3MC and PB inductions combined. It is concluded that cigarette smoke induces selected cytochrome P-450-linked alkoxyresorufin O-dealkylase activities to a similar extent in both lung and liver and that the effects of cigarette smoke are characteristic of both 3MC-type and non-3MC-type inducers.

Mixed function oxidase and UDP-glucuronyltransferase activities in the human Hep G2 hepatoma cell line.

In cultured human hepatoma cells phenolphthalein glucuronidation was increased 3-fold by 2 mM phenobarbitone (PB) in the culture medium but not by 25 microM benz(a)anthracene (BA), while 1-naphthol glucuronidation was not increased by either PB or BA. Ethoxyresorufin O-deethylation (EROD) was increased 15-fold by BA but not by PB, while the O-dealkylations of pentoxyresorufin (PROD) and benzyloxyresorufin (BROD) were increased by either PB or BA. The BROD activity increased by BA was sensitive to inhibition by alpha-naphthoflavone whereas that induced by PB was not. This suggests induction of different cytochrome P-450 isoenzymes. Control Hep G2 cells had similar glucuronide conjugation and cytochrome reductase activities to freshly isolated human adult hepatocytes, but had lower O-dealkylation and elevated microsomal epoxide hydrolase activities.

Ethoxy-, pentoxy- and benzyloxyphenoxazones and homologues: a series of substrates to distinguish between different induced cytochromes P-450.

The individual members of a homologous series of phenoxazone ethers related to ethoxyresorufin were O-dealkylated, and the parent compound phenoxazone was ring-hydroxylated, each at different rates with hepatic microsomes of untreated rats. A structure-activity relationship (SAR) was plotted, relating the rate of O-dealkylation to the length and type of the ether side-chain. Phenobarbitone (PB), 3-methylcholanthrene (MC), Aroclor 1254 (ARO), isosafrole (ISO) and SKF-525A each induced preferentially the O-dealkylation of different members of the homologous series, resulting in the appearance of 5 different SAR plots, which characterized and differentiated between the 5 different inducers. beta-Napthoflavone (BNF) had a similar effect to MC, whereas pregnenolone 16 alpha-carbonitrile treatment caused no large change in the metabolism of any of the substrates tested. For characterizing the effects of the different inducers it was largely sufficient to compare the O-dealkylations of just 4 of the ethers: methoxy-, ethoxy-, pentoxy- and benzyloxyphenoxazone. Very high degrees of induction were seen. MC and ARO each induced preferentially the O-dealkylation of ethoxyphenoxazone (51- and 61-fold respectively). PB and SKF-525A each induced preferentially the O-dealkylation of pentoxyphenoxazone (283- and 324-fold respectively). ISO induced preferentially the O-dealkylation of benzyloxyphenoxazone (43-fold). For any particular induced type of microsomes the substrate with the fastest metabolism was not necessarily the substrate whose metabolism was induced the most, so that in order to characterize each of the 5 different inducers (PB, MC/BNF, ARO, ISO, SKF) it was necessary to compare both the degrees of induction and the specific activities of the reactions. Experiments with purified cyt. P-450 isozymes showed that ethoxyphenoxazone and pentoxyphenoxazone were highly selective substrates for the major isozymes induced by MC and PB respectively, whilst benzyloxyphenoxazone was a good substrate for both isozymes. Experiments using the organic inhibitors metyrapone and alpha-naphthoflavone and inhibitory antibodies against individual cyt. P-450 isozymes indicated that similar substrate selectivities occurred with the monooxygenase system in the microsomal membrane. It is suggested that the use of some or all of these homologous phenoxazone ethers will provide both a simple routine test for the characterization of several types of inducing agents and a powerful tool for investigating the biochemical basis for cyt. P-450 isozyme substrate selectivity.