Effects Of Genetic Variations Of Human Cytochrome P450 Oxidoreductase On The Metabolic Activation Of Anticancer Prodrugs And Environmental Toxins

POR(EC 1.6.2.4) is a flavoprotein localized in the endoplasmic reticulum and the outer membrane of the nuclear envelope in eukaryotic cells.It is the only flavoprotein that mediates electron transfer from NADPH to CYP enzymes(Poter et al.,1991). Disruption of POR gene in knockout mice results in early embryonic lethality(Shen et al.,2002;Otto et al.,2003),while liver-specfic knockout of POR results in a profound disruption of hepatic drug metabolism,but these mice are morphologically and reproductively normal.POR has the ability to reduce a host of exogenous electron accptors,including cytochrome c,potassium ferricyanide,and 2,6-dichloroindophenol(DCIP),as well as the therapeutically important compounds such as mitomycin c and the benzotriazine SR4233,and environmental toxin,paraquat.The gene encoding human POR is quite genetically polymorphic.Since six naturally occurring human POR variants with single amino acid changes(Y181D, A287P,R457H,V492E,C569Y,and V608F) have been identified in 2004(Arlt et al., 2004;Fluck et al.,2004),a total of 40 have been reported till now(Miller et al.,2008; Hart et al.,2007;Dhir et al.,2007).However,those previous work about the POR variants mainly focused on their relation to the P450 oxidoreductase deficiency,this new disorder of steroidogenesis.Seldom study has done about the influence of POR genetic polymorphisms on the metabolic activation of drugs and environmental toxins. In the present work,we invested the potential role of POR genetic variations in the metabolic activation of anticancer drugs and environmental toxins.First,we extended our study to investigate the effect of a total of 28 reported missense POR variants on the MMC metabolic activation.Then,we also established a double transfected Flp-In^TM CHO cell system,which co-expressing wild-type human CYPs with wild-type human POR or selected variants POR,to investigate the influence of those POR genetic variations on supporting the CYP2B6 in metabolic activation of Cyclophosphamide (CPA) and the CYP2A13 in mebabolic activation of Aflatoxin B₁(AFB₁).1.Generation of stable transfectant Flp-In^TMCHO cells expressing human POR and MMC-induced cytotoxicity.In the present work,the recombinant plasmids containing the cDNAs of 28 POR variants were successfully constructed,and stable transfected to the Flp-In^TMCHO cells. The Flp-In^TM CHO cells stable expressing wild-type and variant POR were selected and determined by western blot.Later the cells were treated with MMC to examine the effect of genetic variations of human POR on the metabolic activation of MMC.The MMC-induced cell death in the most variants cells was not different from the wild-type POR cells.However,the viability was significantly increased in the cells expressing the R316W,H628P and V631I variants of POR.For the Y459H and L565P variant cells, the cell viability was the same as that of the negative control cells(transfected with vector alone).The stable expressing cells generated in the present work may further be applied in the study of other POR involved compounds activation.2.CPA-induced cytotoxicity in Flp-In^TM CHO cells co-expressing human CYP2B6 and wild-type or variant PORThe Flp-In^TM CHO cells expressing human CYP2B6 were generated by random transfection first,and monoclone with the highest expression level of CYP2B6 was selected and determined by activity assay.The wild-type or variant POR cDNA was induced into the Flp-In^TM site of the cells stable expressing CYP2B6.Later those cells co-expressing CYP2B6 and wild-type or variant POR were used to examine the effect of genetic variation of human POR on the supporting of CYP2B6 in the metabolic activation of CPA.As shown in the results,cells co-expressing the M263V and C569Y variants responded to the CPA treatment in a manner similar to that of the wild-type POR cells.In contrast,cells co-expressing the other five huan POR variants were more resistant to CPA-induced cytotoxicity than the cells co-expressing wild-type POR.In particular,the response to CPA in the cells expressing the Y181D and L565P variants was nearly the same to that in the cells expressing CYP2B6 alone.The result also indicated that the effect of different POR variations may be substrate dependant.3.Aflatoxin B₁-induced cytotoxicity in Flp-In^TM CHO cells co-expressing human CYP2A13 and wild-type or variant PORFlp-In^TM CHO cells co-expressing human CYP2A13 and wild-type or variant POR were generated and used to examine the effect of genetic variation of human POR on the supporting of CYP2A13 in the metabolic activation of Aflatoxin B₁.As shown in the results,cells co-expressing the M263V and A503V variants responded to the Aflatoxin B₁ treatment in a manner similar to that of the wild-type POR cells.In contrast,cells co-expressing the other three human POR variants were more resistant to Aflatoxin B₁-induced cytotoxicity than the cells co-expressing wild-type POR.In particular,the response to Aflatoxin B₁ in the cells expressing the Y459H variant was very similar to that in the cells expressing CYP2A13 alone.Comparing with the results of last part,the results indicated that the POR variants may have different effects on supporting different CYPs with different substrates as well.There is no easy way to predict the effect of all the POR variants on all the bioactivation of substrates.The co-expressing cell system established in the present work may be further applied in the studies of other CYPs or substrates,and also could be used to examine the effect of CYP genetic variations.Our results suggested that the genetic variations of POR may serve as a biomarker to predict the POR-involved metabolic activation of anticancer drugs and environmental toxins.Our work does a good attempt to set up a database about the influence of POR genetic polymorphisms for the clinical use.