Pharmacogenomic studies of the anticancer and immunosuppressive thiopurines mercaptopurine and azathioprine.

AIMS
To examine the allelic variation of three enzymes involved in 6-mercaptopurine/azathioprine (6-MP/AZA) metabolism and evaluate the in?uence of these polymorphisms on toxicity, haematological parameters and metabolite levels in patients with acute lymphoblastic leukaemia (ALL) or in?ammatory bowel disease (IBD).
METHODS
Clinical data and blood samples were collected from 19 ALL paediatric patients and 35 IBD patients who were receiving 6-MP/AZA therapy. All patients were screened for seven genetic polymorphisms in three enzymes involved in mercaptopurine metabolism [xanthine oxidase, inosine triphosphatase (C94?A and IVS2+21A?C) and thiopurine methyltransferase]. Erythrocyte and plasma metabolite concentrations were also determined. The associations between the various genotypes and myelotoxicity, haematological parameters and metabolite concentrations were determined.
RESULTS
Thiopurine methyltransferase variant alleles were associated with a preferential metabolism away from 6-methylmercaptopurine nucleotides (P = 0.008 in ALL patients,P = 0.038 in IBD patients) favouring 6-thioguanine nucleotides (6-TGNs) (P = 0.021 in ALL patients). Interestingly, carriers of inosine triphosphatase IVS2+21A?C variants among ALL and IBD patients had signi?cantly higher concentrations of the active cytotoxic metabolites, 6-TGNs (P = 0.008 in ALL patients,P = 0.047 in IBD patients). The study con?rmed the association of thiopurine methyltransferase heterozygosity with leucopenia and neutropenia in ALL patients and reported a signi?cant association between inosine triphosphatase IVS2+21A?C variants with thrombocytopenia (P = 0.012).
CONCLUSIONS
Pharmacogenetic polymorphisms in the 6-MP pathway may help identify patients at risk for associated toxicities and may serve as a guide for dose
individualization.

Characterization of SodC, a periplasmic superoxide dismutase from Burkholderia cenocepacia

Burkholderia cenocepacia is a gram-negative, non-spore-forming bacillus and a member of the Burkholderia cepacia complex. B. cenocepacia can survive intracellularly in phagocytic cells and can produce at least one superoxide dismutase (SOD). The inability of O2- to cross the cytoplasmic membrane, coupled with the periplasmic location of Cu,ZnSODs, suggests that periplasmic SODs protect bacteria from superoxide that has an exogenous origin (for example, when cells are faced with reactive oxygen intermediates generated by host cells in response to infection). In this study, we identified the sodC gene encoding a Cu,ZnSOD in B. cenocepacia and demonstrated that a sodC null mutant was not sensitive to a H2O2, 3-morpholinosydnonimine, or paraquat challenge but was killed by exogenous superoxide generated by the xanthine/xanthine oxidase method. The sodC mutant also exhibited a growth defect in liquid medium compared to the parental strain, which could be complemented in trans. The mutant was killed more rapidly than the parental strain was killed in murine macrophage-like cell line RAW 264.7, but killing was eliminated when macrophages were treated with an NADPH oxidase inhibitor. We also confirmed that SodC is periplasmic and identified the metal cofactor. B. cenocepacia SodC was resistant to inhibition by H2O2 and was unusually resistant to KCN for a Cu,ZnSOD. Together, these observations establish that B. cenocepacia produces a periplasmic Cu,ZnSOD that protects this bacterium from exogenously generated O2- and contributes to intracellular survival of this bacterium in macrophages.

Impaired activities of antioxidant enzymes elicit endothelial dysfunction in spontaneous hypertensive rats despite enhanced vascular nitric oxide generation

Objective: Enhanced oxidative stress is involved in mediating the endothelial dysfunction associated with hypertension. The aim of this study was to investigate the relative contributions of pro-oxidant and anti-oxidant enzymes to the pathogenesis of endothelial dysfunction in genetic hypertension. Methods: Dilator responses to endothelium-dependent and endothelium-independent agents such as acetylcholine (ACh) and sodium nitroprusside were measured in the thoracic aortas of 28-week-old spontaneously hypertensive rats (SHR) and their matched normotensive counterparts, Wistar Kyoto rats (WKY). The activity and expression (mRNA and protein levels) of endothelial nitric oxide synthase (eNOS), p22-phox, a membrane-bound component of NAD(P)H oxidase, and antioxidant enzymes, namely, superoxide dismutases (CuZn- and Mn-SOD), catalase and glutathione peroxidase (GPx), were also investigated in aortic rings. Results: Relaxant responses to ACh were attenuated in phenylephrine-precontracted SHR aortic rings, despite a 2-fold increase in eNOS expression and activity. Although the activity and/or expression of SODs, NAD(P)H oxidase (p22-phox) and GPx were elevated in SHR aorta, catalase activity and expression remained unchanged compared to WKY. Pretreatment of SHR aortic rings with the inhibitor of xanthine oxidase, allopurinol, and the inhibitor of cyclooxygenase, indomethacin, significantly potentiated ACh-induced relaxation. Pretreatment of SHR rings with catalase and Tiron, a superoxide anion (O) scavenger, increased the relaxant responses to the levels observed in WKY rings whereas pyrogallol, a O -generator, abolished relaxant responses to ACh. Conclusion: These data demonstrate that dysregulation of several enzymes, resulting in oxidative stress, contributes to the pathogenesis of endothelial dysfunction in SHR and indicate that the antioxidant enzyme catalase is of particular importance in the reversal of this defect. © 2003 European Society of Cardiology. Published by Elsevier B.V. All rights reserved.

Involvement of Nox2 NADPH oxidase in adverse cardiac remodeling after myocardial infarction.

Oxidative stress plays an important role in the development of cardiac remodeling after myocardial infarction (MI), but the sources of oxidative stress remain unclear. We investigated the role of Nox2-containing reduced nicotinamide-adenine dinucleotide phosphate oxidase in the development of cardiac remodeling after MI. Adult Nox2(-/-) and matched wild-type (WT) mice were subjected to coronary artery ligation and studied 4 weeks later. Infarct size after MI was similar in Nox2(-/-) and WT mice. Nox2(-/-) mice exhibited significantly less left ventricular (LV) cavity dilatation and dysfunction after MI than WT mice (eg, echocardiographic LV end-diastolic volume: 75.7+/-5.8 versus 112.4+/-12.3 microL; ejection fraction: 41.6+/-3.7 versus 32.9+/-3.2%; both P