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Biomedical subjects

M D Burke

Publications and source records attributed to M D Burke.

At least 37 records · Page 2Linked to original sources
PubMed1996

Xenobiotic metabolism in rat, dog, and human precision-cut liver slices, freshly isolated hepatocytes, and vitrified precision-cut liver slices.

Human, rat, and dog phase I and phase II xenobiotic metabolism in precision-cut liver slices and freshly isolated hepatocytes was compared using a range of substrates. Carbamazepine (50 microM) and styrene (2 mM) were used as probes to study the maintenance of cytochrome P450 and epoxide hydrolase-mediated metabolism in male Sprague-Dawley rat, precision-cut liver slices and hepatocytes. Carbamazepine metabolism in both models resulted in the formation of the bioactive 10,11-epoxide (KM = 766 microM and Vmax = 2.5 pmol/min/mg protein in precision-cut slices). Epoxide formation was higher (2.4-fold) in hepatocytes than slices. Styrene was deactivated to styrene diol at a higher rate in hepatocytes (9.7-fold) than slices. The lower rate of metabolism in slices compared with hepatocytes confirms our previous observations using testosterone, 7-ethoxycoumarin, 1-chloro-2,4-dinitrobenzene and 2-(5'-chloro-2'-phosphoryloxyphenyl)-6-chloro-4-(3H)-quinazolinone in the rat. Testosterone 6 beta-hydroxylation in human liver slices was similar to cultured hepatocytes, but lower than in freshly isolated hepatocytes. 7-Ethoxycoumarin O-deethylation was higher in freshly isolated human hepatocytes, as was the ratio of glucuronide to 7-hydroxycoumarin. Testosterone hydroxylations, 7-ethoxycoumarin O-deethylation, and 1-chloro-2,4-dinitrobenzene conjugation were also lower in male beagle dog slices, compared with freshly isolated hepatocytes. Attempts at long-term preservation of dog liver slices using vitrification and storage for up to 9 days at -196 degrees C resulted in the retention of phase I and phase II metabolism, although conjugation was lower than in freshly prepared slices. Xenobiotic metabolism in short-term incubations is consistently lower in dog and rat precision-cut slices than in freshly isolated hepatocytes; whereas, in humans, this quantitative difference is partly hidden by the large interindividual variation.

Adolescent↗
PubMed1995

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.

Adult↗
PubMed1995

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.

Aged↗
PubMed1995

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.

Antimalarials↗
PubMed1995

The immunohistochemical localization of drug-metabolizing enzymes in prostate cancer.

The major groups of enzymes involved in activating and detoxifying therapeutic drugs, not least several anti-cancer drugs, include the cytochromes P450 (P450s), epoxide hydrolase, and glutathione S-transferases (GSTs). The expression of these enzymes in malignant tumours is one possible mechanism of anti-cancer drug resistance. This study has investigated the presence, cellular localization, and distribution of drug-metabolizing enzymes in prostate cancer. The P450 subfamilies CYP1A, CYP2C, and CYP3A were present in 63, 25, and 61 per cent of tumours, respectively. Epoxide hydrolase was identified in 96 per cent of tumours. GST-alpha and GST-mu were expressed in 29 and 41 per cent of tumours, respectively, while there was no immunoreactivity for the pi form of GST. The absence of GST-pi in prostate cancer contrasts with the frequent expression of GST-pi observed in other types of malignant tumour. In non-neoplastic prostatic epithelium, there was expression of CYP1A, CYP2C, epoxide hydrolase, and the different forms of GST, while there was no apparent immunoreactivity for CYP3A.

Aged↗
PubMed1995

Human placental cytochrome P450 and quinone reductase enzyme induction in relation to maternal smoking.

Components of cigarette smoke such as cadmium and polycyclic aromatic hydrocarbons have been shown to induce quinone reductase (QR) activity in placental explants. This study examines the relationship of maternal smoking habit and maternal plasma cotinine concentration with the activities in vitro of both QR and the cytochrome P450 (CYP1A) marker ethoxyresorufin O-deethylase (EROD) in placental tissue. Maternal plasma samples were taken at Week 34 of gestation, and placental tissues were obtained at term. Plasma cotinine concentrations were determined by high-performance liquid chromatography. Trophoblast cytosolic QR and microsomal EROD activities were measured by resazurin reduction and ethoxyresorufin O-dealkylation respectively. QR activity was inhibited 70% by a mixture of dicoumarol (1 microM) and rutin (20 microM). Plasma cotinine concentrations correlated significantly (P < 0.001) with both declared smoking rate (r = 0.67, N = 37) and placental EROD activity (r = 0.63, N = 36), but not with QR activity, whether measured as total QR activity or specifically as either DT-diaphorase or carbonyl reductase. It is concluded that smoking up to 40 cigarettes per day induces EROD but does not affect QR activity in the placenta at term.

Cotinine↗
PubMed1995

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.

Adult↗
PubMed1995

Drug toxicity mechanisms in human hepatoma HepG2 cells: cyclosporin A and tamoxifen.

1. Mechanisms of drug toxicity operating in human HepG2 hepatoma cells have been assessed using cyclosporin A (CsA) and tamoxifen as examples. 2. Either 150 microM CsA or 50 microM tamoxifen caused approximately 50% loss of HepG2 cell viability. alpha-Tocopherol (32 microM) almost completely prevented cell death due to either CsA or tamoxifen. Tamoxifen stimulated malondialdehyde formation. The toxicity of CsA but not tamoxifen was increased by the glutathione synthesis inhibitor, buthionine-S,R-sulphoximine, and decreased by the glutathione precursor, L-cysteine. Thus, while both CsA and tamoxifen toxicities involved lipid peroxidation, reduced glutathione (or sulphydryl groups) protected against CsA but not tamoxifen. 3. CsA was metabolized to M1 and/or M17 in HepG2 cells. The effects of the cytochrome P450 inhibitors, ketoconazole and metyrapone, indicated that P450 played a role in the toxicity of CsA but not tamoxifen. The effects of superoxide dismutase and cytochrome c indicated that tamoxifen toxicity involved superoxide formation. 4. These results show that several oxidative mechanisms of drug toxicity operate in HepG2 cells.

Animals↗
PubMed1995

Expression of xenobiotic metabolizing enzymes in tumours of the urinary bladder.

The cytochromes P450, epoxide hydrolase and glutathione S-transferases are several of the major groups of enzymes involved in the metabolism of xenobiotics and these enzymes may have a role in influencing the response of tumours to anti-cancer drugs. In this study the cell specific expression of individual xenobiotic metabolizing enzymes has been investigated using immunohistochemistry in primary transitional cell tumours of the urinary bladder. The cytochromes P450 CYP1A, CYP2C and CYP3A, were present in 68, 28 and 68% of tumours respectively and the expression of CYP1A correlated with bladder tumour grade (P = 0.03). Epoxide hydrolase was identified in 84% of tumours while the alpha, mu and pi forms of glutathione S-transferase were expressed in 56, 72 and 52% of tumours respectively. In normal bladder epoxide hydrolase and glutathione S-transferase pi were the main enzymes expressed while there was no expression of CYP2C.

Adult↗
PubMed1995

Clinical laboratory consultation.

The future of healthcare delivery systems will depend on their ability to provide the best care at the least cost. In the clinical laboratory, this translates to ensuring that decreased test use means appropriate use. The latter is unlikely to occur unless clinical laboratories provide practicing physicians with advice on test strategy and interpretation of results. Effective clinical laboratory consultation requires an identification with clinical problems. Important components of the identification process are an appreciation of the probabilistic rather than deterministic nature of the relation of clinical and laboratory data to disease and the inevitable tradeoff between false negatives and false positives that clinical circumstances demand. Institution of consultation services requires that the clinical laboratory adopt a proactive approach to advocating test strategy and interpreting results. Consultation on test strategy is likely to be facilitated by further developments in practice guidelines and by advances in informatics and telemedicine.

Chemistry, Clinical↗
PubMed1995

Quantitative differences in phase I and II metabolism between rat precision-cut liver slices and isolated hepatocytes.

Testosterone (250 microM), 7-ethoxycoumarin (25 microM), and 1-chloro-2,4-dinitrobenzene (CDNB, 50 microM) were used as substrates to compare phase I and II metabolism in rat precision-cut liver slices and rat hepatocytes. Overall clearance to metabolites was significantly greater in hepatocytes for testosterone (1.9- to 16.9-fold), 7-ethoxycoumarin (O-deethylation, 14.8-fold; glucuronidation, 3.1-fold), and CDNB (8.7-fold). The same metabolites for each of the substrates were detected in slices and hepatocytes. However, the ratio of sulfate to glucuronide conjugation was higher in slices, in line with the markedly slower rate of formation of 7-hydroxycoumarin. The slower rate of CDNB conjugation could not be explained by differences in the intracellular concentration of glutathione. These data suggest that metabolism in precision-cut slices is limited by diffusion of the substrate. This was illustrated by the use of 2-(5'-chloro-2'-phosphoryloxyphenyl)-6-chloro-4-(3H)-quinazolinone as a fluorescent probe to investigate diffusion in slices and hepatocytes.

Animals↗
PubMed1994

Cytochrome P450 specificities of alkoxyresorufin O-dealkylation in human and rat liver.

The O-dealkylations of ethoxyresorufin and pentoxyresorufin are widely used activity probes for measuring the cytochrome P450 forms, CYP1A1 and CYP2B1, respectively, and their induction by xenobiotics, but there is confusion in the literature about which P450 forms are detected in human and rat liver microsomes by these and homologous alkoxyresorufins. High performance liquid chromatographic analysis confirmed that O-dealkylation to resorufin was the sole or predominant route of metabolism for both short-chain and long-chain alkoxyresorufins and benzyloxyresorufin by rat liver microsomes. The purified 3-methylcholanthrene (3MC)-induced rat P450 forms, CYP1A1 and CYP1A2, and a possible variant form, CYP1A1*, showed substrate selectivities for propoxyresorufin, methoxyresorufin and ethoxyresorufin, respectively. Purified phenobarbitone (PB)-induced CYP2B1 was selective for benzyloxyresorufin and pentoxyresorufin. Purified constitutive CYP2C6 was much less active than CYP2B1 or the CYP1A forms but showed distinctive selectivity for benzyloxy-, propoxy- and butoxyresorufin. CYP1A2 and CYP2C6 metabolised n-propoxy- and n-butoxyresorufin much more rapidly (8-23-fold) than iso-propoxy- and iso-butoxyresorufin, whereas CYP1A1 and CYP2B1 showed only small differences (2-5-fold) between the n- and iso-homologues and CYP1A1* and CYP2B2 did not discriminate between them. The results show that ratios between different alkoxyresorufin O-dealkylation (AROD) activities can be more useful than absolute values of single activities for identifying P450 forms. Anti-P450 antibody and furafylline inhibition of rat liver microsomal AROD confirmed that ethoxyresorufin was a selective probe for CYP1A1 in 3MC-induced and isosafrole (ISF)-induced microsomes and that pentoxy- and benzyloxyresorufins both selectively measured CYP2B1 in PB-induced and ISF-induced microsomes. Ethoxyresorufin was not a selective probe for CYP1A in liver microsomes from untreated or PB-induced rats, however, where it was metabolised mainly by CYP2C6 and CYP2B1, respectively. Pentoxyresorufin and benzyloxyresorufin were metabolised by several different P450 forms in non-induced rat liver microsomes but mainly by the CYP1A subfamily in 3MC-induced microsomes and by CYP2B1 in PB- and ISF-induced microsomes. Although with purified rat P450s methoxyresorufin appeared not effectively to discriminate CYP1A2 from CYP1A1, CYP1A1* or CYP2C6, furafylline inhibition indicated that methoxyresorufin was a selective measure of CYP1A2 in uninduced and 3MC-induced rat liver microsomes but not in ISF- or PB-induced microsomes. In human liver microsomes, antibody inhibition and furafylline inhibition showed that ethoxyresorufin and methoxyresorufin were metabolised mainly by CYP1A2, whilst benzyloxyresorufin metabolism was due mainly to the CYP3A subfamily but also involved CYP1A2 and CYP2A6.(ABSTRACT TRUNCATED AT 400 WORDS)

Adult↗
PubMed1994

Evidence for a new cytochrome P450 form induced by 3-methylcholanthrene in rats.

Evidence is presented for a new 3-methylcholanthrene (3MC)-induced form of cytochrome P450, P450MCX, in rat liver microsomes. P450MCX was co-purified with CYP1A1 from 3MC-treated male Sprague-Dawley rats but was resolved by gel electrophoresis. The M(r) of P450MCX (56,700) was intermediate between CYP1A1 (57,000) and CYP1A2 (54,800). Monoclonal antibodies showed that P450MCX was immunorelated to both CYP1A1 and CYP1A2 but not to CYP2B1, CYP2C6 or CYP3A1. Immunoreactive P450MCX was not detectable in liver microsomes from untreated rats but was highly induced by 3MC and Aroclor 1254, although not induced by isosafrole. The N-terminal amino acid sequence of P450MCX, obtained from an electroblotted sample resolved on SDS-PAGE, did not match any known cytochrome P450 or other protein. P450MCX may be a new member of the CYP1 family.

Amino Acid Sequence↗