ABSTRACT:Human carbonyl reductase (CBR) activity accounts for a significant fraction of the metabolism of endogenous and xenobiotic carbonyl compounds. It is possible that genetic polymorphisms in CBR1 and CBR3 are key for the wide interindividual variability in the disposition of CBR drug substrates. We pinpointed a single nucleotide polymorphism in CBR3 (CBR3 V244M) that encodes for a V244 to M244 change. Blacks showed a higher frequency of the M244 allele (q ‫؍‬ 0.51, n ‫؍‬ 49) than did whites (q ‫؍‬ 0.31, n ‫؍‬ 70; p ‫؍‬ 0.003). In addition, DNA variation panels from 10 ethnic groups presented a wide range of CBR3 V244M genotype distributions. Kinetic experiments with the recombinant CBR3 protein variants and menadione revealed that CBR3 M244 has significantly higher V max than does CBR3 V244 (V max CBR3 M244 ‫؍‬ 40.6 ؎ 1.3 mol/min ⅐ mg versus V max CBR3 V244 ‫؍‬ 19.6 ؎ 2.0 mol/min ⅐ mg, p ‫؍‬ 0.002). In contrast, both isoforms presented similar K m values (K m CBR3 M244 ‫؍‬ 22.9 ؎ 2.9 M versus K m CBR3 V244 ‫؍‬ 24.6 ؎ 3.2 M, p ‫؍‬ 0.43). Assays with NADP(H) demonstrated a higher V maxNADP(H) (1.6-fold) and increased catalytic efficiency (V maxNADP(H) /K mNADP(H) ) for CBR3 M244 compared with CBR3 V244 (p ‫؍‬ 0.013). Comparative three-dimensional analyses based on the structure of the homologous porcine carbonyl reductase suggested that the V244M substitution is positioned in a region critical for interactions with the NADP(H) cofactor. These studies demonstrate that the common CBR3 V244M polymorphism encodes for CBR3 isoforms with distinctive enzymatic properties.

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ABSTRACT:Human carbonyl reductase 1 (CBR1) metabolizes endogenous and xenobiotic substrates such as the fever mediator, prostaglandin E2 (PGE 2 ), and the anticancer anthracycline drug, daunorubicin. We screened 33 CBR1 full-length cDNA samples from white and black liver donors and performed database analyses to identify genetic determinants of CBR1 activity. We pinpointed a single nucleotide polymorphism on CBR1 (CBR1 V88I) that encodes for a valine-toisoleucine substitution for further characterization. Human carbonyl reductase 1 (CBR1) is a monomeric cytosolic enzyme that catalyzes the two-electron reduction of biologically and pharmacologically active substrates by using NADPH as cofactor . For example, CBR1 converts the potent fever mediator prostaglandin E 2 (PGE 2 ) into the less active metabolite PGF 2␣ . Recent studies have shown that the transcription of CBR1 in lung and liver is down-regulated to suppress the catabolism of PGE 2 during the course of the febrile response (Ivanov et al., 2003;Ivanov and Romanovsky, 2004). CBR1 also plays a predominant role in the metabolism of the anticancer anthracyclines doxorubicin and daunorubicin by catalyzing the formation of their corresponding C-13 alcohol metabolites (doxorubicinol and daunorubicinol). Anthracycline C-13 alcohol metabolites circulate in plasma, have half-lives similar to those of the parent compounds, and are devoid of significant tumor cell-killing activity. The use of anthracyclines for cancer treatment is hampered by the development of clinical cardiotoxicity in some patients (Wouters et al., 2005). The pathogenesis of anthracycline-related cardiotoxicity is complex and appears to be mediated by a combination of oxidative stress and intracardiac metabolic perturbations induced by C-13 anthracycline alcohol metabolites (Minotti et al., 2004). In human myocardium, cytosolic carbonyl reductases (CBRs), and aldo/keto reductases catalyze the two-electron reduction of the anthracycline side chain C-13 carbonyl group to form cardiotoxic alcohol metabolites (e.g., doxorubicinol, daunorubicinol). CBR activity is the main source of quinone detoxification in humans, and biochemical studies showed that aldo/keto reductases have 7-to 18-fold lower catalytic efficiencies (V max /K m ) for the reduction of anthracycline substrates than CBRs (Wermuth et al., 1986;Ohara et al., 1995;Rosemond and Walsh, 2004;Covarrubias et al., 2006). The key role of CBR1 during the pathogenesis of anthracycline-related cardiotoxicity has been established in biochemical studies and in experiments with murine models (Mordente et al., 2001). Mice overexpressing human CBR1 in heart showed high intracardiac levels of doxorubicinol and increased signs of myocardial damage after doxorubicin administration . Conversely, mice with a

Human carbonyl reductase 1 (CBR1) metabolizes a variety of substrates, including the anticancer doxorubicin and the antipsychotic haloperidol. The transcriptional regulation of CBR1 has been largely unexplored. Therefore, we first investigated the promoter activities of progressive gene-reporter constructs encompassing up to 2.4 kilobases upstream of the translation start site of CBR1. Next, we investigated whether CBR1 mRNA levels were altered in cells incubated with prototypical receptor activators (e.g., dexamethasone and rifampicin). CBR1 mRNA levels were significantly induced (5-fold) by the ligand of the aryl hydrocarbon receptor (AHR) ␤-naphthoflavone. DNA sequence analysis revealed two xenobiotic response elements ( Ϫ122 XRE and Ϫ5783 XRE) with potential regulatory functions. CBR1 promoter constructs lacking the Ϫ122 XRE showed diminished (9-fold) promoter activity in AHR-proficient cells incubated with ␤-naphthoflavone. Fusion of Ϫ5783 XRE to the Ϫ2485 CBR1 reporter construct enhanced its promoter activity after incubations with ␤-naphthoflavone by 5-fold. Furthermore, we tested whether the potent AHR ligand 2,3,7,8-tetrachlorodibenzo-pdioxin (TCDD) induced Cbr1 expression in Ahr ϩ/Ϫ and Ahr Ϫ/Ϫ mice. TCDD induced hepatic Cbr1 mRNA (TCDD, 2-fold) and Cbr1 protein levels (TCDD, 2-fold) in Ahr ϩ/Ϫ mice compared with vehicle-injected controls. In contrast, no significant Cbr1 mRNA and Cbr1 protein induction was detected in livers from Ahr Ϫ/Ϫ mice treated with TCDD. These studies provide the first insights on the functional characteristics of the human CBR1 gene promoter. Our data indicate that the AHR pathway contributes to the transcriptional regulation of CBR1.

The understanding of the cytochrome P450 3A SNP in antiretroviral therapy is important, because it is highly inducible, extremely polymorphic and metabolizes many of the drugs that are key components of highly active antiretroviral therapy regimens. This enzyme is prolific and promiscuous towards drug and xenobiotic substrate selection and it is also unpredictable among individuals, having a 5- to 20-fold variability in its ability to contribute to drug clearance. The importance of human CYP3A pharmacogenetics is also gaining attention in other established areas of pharmacotherapy as it may contribute to the goal of predicting efficacy and/or toxicity, specifically with the discovery of null allele CYP3A4*20. This review summarizes the current understanding, implications of genetic variation in the CYP3A enzymes, the central role of CYP3A in linking human genetics, the pharmacokinetics and resulting pharmacodynamic responses to certain antiretroviral drugs, and their eventual place in applied clinical pharmacotherapy.

In human liver, the two-electron reduction of quinone compounds, such as menadione is catalyzed by cytosolic carbonyl reductase (CBR) and NAD(P)H:quinone oxidoreductase (NQO1) activities. We assessed the relative contributions of CBR and NQO1 activities to the total menadione reducing capacity in liver cytosols from black (n=31) and white donors (n=63). Maximal menadione reductase activities did not differ between black (13.0+/-5.0 nmol/min mg), and white donors (11.4+/-6.6 nmol/min mg; p=0.208). In addition, both groups presented similar levels of CBR activities (CBR(blacks)=10.9+/-4.1 nmol/min mg) versus CBR(whites)=10.5+/-5.8 nmol/min mg; p=0.708). In contrast, blacks showed higher NQO1 activities (two-fold) than whites (NQO1(blacks)=2.1+/-3.0 nmol/min mg versus NQO1(whites)=0.9+/-1.6 nmol/min mg, p<0.01). To further explore this disparity, we tested whether NQO1 activity was associated with the common NQO1(*)2 genetic polymorphism by using paired DNA samples for genotyping. Cytosolic NQO1 activities differed significantly by NQO1 genotype status in whites (NQO1(whites[NQO1*1/*1])=1.3+/-1.7 nmol/min mg versus NQO1(whites[NQO1*1/*2+NQO1*2/*2])=0.5+/-0.7 nmol/min mg, p<0.01), but not in blacks (NQO1(blacks[NQO1*1/*1])=2.6+/-3.4 nmol/min mg versus NQO1(blacks[NQO1*1/*2])=1.1+/-1.2 nmol/min mg, p=0.134). Our findings pinpoint the presence of significant interethnic differences in polymorphic hepatic NQO1 activity.

Modulation of calcium channels plays an important role in many cellular processes. Previous studies have shown that the L-type Ca 2؉ channels in Drosophila larval muscles are modulated via a cAMP-protein kinase A (PKA)-mediated pathway. This raises questions on the identity of the steps prior to cAMP, particularly the endogenous signal that may initiate this modulatory cascade. We now present data suggesting the possible role of a neuropeptide, pituitary adenylyl cyclase-activating polypeptide (PACAP), in this modulation. Mutations in the amnesiac (amn) gene, which encodes a polypeptide homologous to human PACAP-38, reduced the L-type current in larval muscles. Conditional expression of a wild-type copy of the amn gene rescued the current from this reduction. Bath application of human PACAP-38 also rescued the current. PACAP-38 did not rescue the mutant current in the presence of PACAP-6 -38, an antagonist at type-I PACAP receptor. 2 ,5 -dideoxyadenosine, an inhibitor of adenylyl cyclase, prevented PACAP-38 from rescuing the amn current. In addition, 2 ,5 -dideoxyadenosine reduced the wild-type current to the level seen in amn, whereas it failed to further reduce the current observed in amn muscles. H-89, an inhibitor of PKA, suppressed the effect of PACAP-38 on the current. The above data suggest that PACAP, the type-I PACAP receptors, and adenylyl cyclase play a role in the modulation of L-type Ca 2؉ channels via cAMP-PKA pathway. The data also provide support for functional homology between human PACAP-38 and the amn gene product in Drosophila.

Intermittent fasting dietary restriction (IF-DR) is recently reported to be an effective intervention to retard age associated disease load and to promote healthy aging. Since sustaining long term caloric restriction regimen is not practically feasible in humans, so use of alternate approach such as late onset short term IF-DR regimen which is reported to trigger similar biological pathways is gaining scientific interest. The current study was designed to investigate the effect of IF-DR regimen implemented for 12 weeks in middle age rats on their motor coordination skills and protein and DNA damage in different brain regions. Further, the effect of IF-DR regimen was also studied on expression of energy regulators, cell survival pathways and synaptic plasticity marker proteins. Our data demonstrate that there was an improvement in motor coordination and learning response with decline in protein oxidative damage and recovery in expression of energy regulating neuropeptides. We further observed significant downregulation in nuclear factor kappa B (NF-κB) and cytochrome c (Cyt c) levels and moderate upregulation of mortalin and synaptophysin expression. The present data may provide an insight on how a modest level of short term IF-DR, imposed in middle age, can slow down or prevent the age-associated impairment of brain functions and promote healthy aging by involving multiple regulatory pathways aimed at maintaining energy homeostasis.