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Ibbotson S.H.,University of Dundee | Dawe R.S.,University of Dundee | Dinkova-Kostova A.T.,University of Dundee | Weidlich S.,University of Dundee | And 5 more authors.
British Journal of Dermatology | Year: 2012

Summary Background There is marked interpatient variation in responses to psoralen-ultraviolet A (PUVA) photochemotherapy. Identification of molecular biomarkers of PUVA sensitivity may facilitate treatment predictability. The glutathione S-transferases (GSTs) influence cutaneous defence against UV radiation-induced oxidative stress and are therefore candidate biomarkers of PUVA sensitivity. Several human GSTs, including GSTM1 and GSTT1, are polymorphic, and null polymorphisms have been associated with increased UVB erythemal sensitivity and skin cancer risk. PUVA also increases skin cancer risk. Objectives To investigate the effect of GST genotype on PUVA sensitivity. Methods We investigated GST genotype in patients starting PUVA (n = 111) and the effects of 8-methoxypsoralen (8-MOP) on antioxidant response element (ARE)-regulated gene expression in mammalian cells. Results Lower minimal phototoxic doses (MPD) (P = 0·022) and higher serum 8-MOP concentrations (P = 0·052) were seen in GSTM1-null allele homozygotes compared with patients with one or two active alleles. In a subset of patients with psoriasis (n = 50), the GSTM1 genotype was not associated with PUVA outcomes, although MPD [hazard ratio (HR) 1·37; 95% confidence interval (CI) for HR 1·15-1·64] and GSTT1-null (HR 2·39; 95% CI for HR 1·31-4·35) and GSTP1b (HR 1·96; 95% CI for HR 1·10-3·51) genotypes were associated with clearance of psoriasis in this patient group. Exposure of mammalian cells to 8-MOP induced gene expression via the ARE, a regulatory sequence in promoters of cytoprotective genes including GSTs, suggesting that these genes may be implicated in 8-MOP metabolism. Conclusion The polymorphic human GSTs are associated with PUVA sensitivity. Further studies are required to examine the clinical relevance of these preliminary findings. © 2011 British Association of Dermatologists.

Deeni Y.Y.,University of Dundee | Deeni Y.Y.,University of Abertay Dundee | Ibbotson S.H.,University of Dundee | Woods J.A.,University of Dundee | And 3 more authors.
PLoS ONE | Year: 2013

Background:There are unpredictable inter-individual differences in sensitivity to psoralen-UVA (PUVA) photochemotherapy, used to treat skin diseases including psoriasis. Psoralens are metabolised by cytochrome P450 enzymes (P450), and we hypothesised that variability in cutaneous P450 expression may influence PUVA sensitivity. We previously showed that P450 CYP1B1 was abundantly expressed in human skin and regulated by PUVA, and described marked inter-individual differences in cutaneous CYP1B1 expression.Objectives:We investigated whether CYP1B1 made a significant contribution to 8-methoxypsoralen (8-MOP) metabolism, and whether individuality in CYP1B1 activity influenced PUVA sensitivity.Methods:We used E. coli membranes co-expressing various P450s and cytochrome P450 reductase (CPR) to study 8-MOP metabolism and cytotoxicity assays in CYP1B1-expressing mammalian cells to assess PUVA sensitivity.Results:We showed that P450s CYP1A1, CYP1A2, CYP1B1, CYP2A6 and CYP2E1 influence 8-MOP metabolism. As CYP1B1 is the most abundant P450 in human skin, we further demonstrated that: (i) CYP1B1 interacts with 8-MOP (ii) metabolism of the CYP1B1 substrates 7-ethoxyresorufin and 17-β-estradiol showed concentration-dependent inhibition by 8-MOP and (iii) inhibition of 7-ethoxyresorufin metabolism by 8-MOP was influenced by CYP1B1 genotype. The influence of CYP1B1 on PUVA cytotoxicity was further investigated in a Chinese hamster ovary cell line, stably expressing CYP1B1 and CPR, which was more sensitive to PUVA than control cells, suggesting that CYP1B1 metabolises 8-MOP to a more phototoxic metabolite(s).Conclusion:Our data therefore suggest that CYP1B1 significantly contributes to cutaneous 8-MOP metabolism, and that individuality in CYP1B1 expression may influence PUVA sensitivity. © 2013 Deeni et al.

Singh R.,University of Leicester | Arlt V.M.,Institute of Cancer Research | Henderson C.J.,Cancer Research UK Molecular Pharmacology Unit | Phillips D.H.,Institute of Cancer Research | And 2 more authors.
Journal of Chromatography B: Analytical Technologies in the Biomedical and Life Sciences | Year: 2010

The heterocyclic aromatic amine, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) is formed by the grilled cooking of certain foods such as meats, poultry and fish. PhIP has been shown to induce tumours in the colon, prostate and mammary glands of rats and is regarded as a potential human dietary carcinogen. PhIP is metabolically activated via cytochrome P450 mediated oxidation to an N-hydroxylamino-PhIP intermediate that is subsequently converted to an ester by N-acetyltransferases or sulfotransferases and undergoes heterolytic cleavage to produce a PhIP-nitrenium ion, which reacts with DNA to form the N-(deoxyguanosin-8-yl)-2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP-C8-dG) adduct. Thus far, the detection and quantification of PhIP-DNA adducts has relied to a large extent on 32P-postlabelling methodologies. In order to expand the array of available techniques for the detection and improved quantification of PhIP-C8-dG adducts in DNA we have developed an online column-switching liquid chromatography (LC)-electrospray ionization (ESI)-tandem mass spectrometry (MS/MS) selected reaction monitoring (SRM) method incorporating an isotopically [13C10]-labelled PhIP-C8-dG internal standard for the analysis of DNA enzymatically hydrolysed to 2'-deoxynucleosides. A dose-dependent increase was observed for PhIP-C8-dG adducts when salmon testis DNA was reacted with N-acetoxy-PhIP. Analysis of DNA samples isolated from colon tissue of mice treated by oral gavage daily for 5 days with 50mg/kg body weight of PhIP resulted in the detection of an average level of 14.8±3.7 PhIP-C8-dG adducts per 106 2'-deoxynucleosides. The method required 50μg of hydrolysed animal DNA on column and the limit of detection for PhIP-C8-dG was 2.5fmol (1.5 PhIP-C8-dG adducts per 108 2'-deoxynucleosides). In summary, the LC-ESI-MS/MS SRM method provides for the rapid automation of the sample clean up and a reduction in matrix components that would otherwise interfere with the mass spectrometric analysis, with sufficient sensitivity and precision to analyse DNA adducts in animals exposed to PhIP. © 2010 Elsevier B.V.

Kostrzewa-Nowak D.,University Of Szczecin | Bieg B.,Maritime University Of Szczecin | Paine M.J.I.,Cancer Research UK Molecular Pharmacology Unit | Wolf C.R.,Cancer Research UK Molecular Pharmacology Unit | Tarasiuk J.,University Of Szczecin
Anti-Cancer Drugs | Year: 2012

The aim of this study was to examine the role of structural factors of antitumour anthraquinone derivatives and analogues in the ability to undergo bioreductive activation by NADPH cytochrome P450 reductase (CPR) and determine the impact of this activation on increasing the activity especially with regard to multidrug resistant (MDR) tumour cells. It was found that at a high NADPH concentration (500 μmol/l), the anthracenedione agent ametantrone, with an unmodified quinone structure, was susceptible to CPR-dependent reductive activation. In contrast, it was shown that compounds with modified quinone grouping (benzoperimidine BP1, anthrapyridone CO1 and pyrazolopyrimidoacridine PPAC2) did not undergo reductive activation by CPR. This suggests that the presence of a modified quinone function is the structural factor excluding reductive activation of antitumour anthraquinone derivatives and analogues by CPR. In the second part of the work, the ability of antitumour anthraquinone derivatives and analogues to inhibit the growth of the human promyelocytic, sensitive leukaemia HL60 cell line as well as its MDR sublines exhibiting two different phenotypes of MDR related to the overexpression of P-glycoprotein (HL60/VINC) or MRP1 (HL60/DOX) was studied in the presence of exogenously added CPR. A significant increase in the activity of ametantrone with an unmodified quinone structure after its reductive conversion by CPR was observed against HL60 as well as HL60/VINC and HL60/DOX cells, whereas in the case of quinone-modified compounds (BP1, CO1 and PPAC2), the presence of the activation system had no effect on their activity against the sensitive and MDR tumour cells examined. © 2012 Lippincott Williams & Wilkins, Inc.

McLaughlin L.A.,Cancer Research UK Molecular Pharmacology Unit | Ronseaux S.,Cancer Research UK Molecular Pharmacology Unit | Finn R.D.,Cancer Research UK Molecular Pharmacology Unit | Henderson C.J.,Cancer Research UK Molecular Pharmacology Unit | Wolf C.R.,Cancer Research UK Molecular Pharmacology Unit
Molecular Pharmacology | Year: 2010

We demonstrated recently that cytochrome b5 plays an important in vivo role in hepatic cytochrome P450 (P450) function [J Biol Chem 283:31385-31393, 2008]. We have now generated a model in which cytochrome b 5 has been deleted in all tissues [cytochrome b5 complete null (BCN)], which surprisingly results in a viable mouse despite the putative in vivo roles of this protein in lipid and steroid hormone metabolism and the reduction of methemoglobin. In contrast to the liver-specific deletion, complete deletion of cytochrome b5 leads to a neonatal increase in the expression of many hepatic P450s at both the protein and mRNA level. In extrahepatic tissues, some changes in P450 expression were also observed that were isoformdependent. In vitro cytochrome P450 activities in liver, kidney, lung, and small intestine of BCN mice were determined for a range of model substrates and probe drugs; a profound reduction in the metabolism of some substrates, particularly in lung, kidney, and small intestine, was observed. In vivo, the metabolism of metoprolol was significantly altered in BCN mice, in contrast to the previous finding in the liver-specific cytochrome b5 deletion, suggesting that extrahepatic cytochrome b5 plays a significant role in its disposition. Testicular Cyp17 hydroxylase and lyase activities were also significantly reduced by cytochrome b5 deletion, leading to significantly lower levels of testicular testosterone. The BCN mouse provides an additional model system with which to further investigate the functions of cytochrome b5, particularly in extrahepatic tissues. Copyright © 2010 The American Society for Pharmacology and Experimental Therapeutics.

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