Effect of genotype and methylation of CYP2D6 on smoking behaviour.

OBJECTIVE: Cigarette smoking is one of the most influential environmental factors affecting the DNA methylation patterns. The addiction-causing substance of tobacco smoke, nicotine, has also shown the potential to alter DNA methylation patterns. However, genetics has a strong influence on DNA methylation patterns, which in turn may affect an individual's smoking behaviour. MATERIALS AND METHODS: We studied eight functional gene variants of one of the most important drug-metabolizing enzymes, CYP2D6, in relation to smoking behaviour in our well-characterized study population consisting of 1230 Whites of Russian origin. In addition, potential associations between methylation levels in a CpG island in the CYP2D6 gene and sex, age, different smoking-related phenotypes and CYP2D6 genotypes were studied. RESULTS: Both age and sex were found to be associated with the methylation level of the CYP2D6 gene. The CYP2D6 methylation pattern also showed high genotype dependence; compared with the extensive metabolizer genotype, the poor metabolizer genotype occurred notably more frequently with higher methylation status (odds ratio 5.05, 95% confidence interval 2.14-11.90). Moreover, higher methylation levels were found to be related inversely to heavier smoking (odds ratio 0.56, 95% confidence interval 0.35-0.91). We also found associations between the CYP2D6 genotype and smoking habits; the poor metabolizer genotype tended to decrease the risk of becoming a heavy smoker compared with the extensive metabolizers, whereas the ultrarapid metabolism-related genotypes tended to increase the risk. CONCLUSION: The CYP2D6-related metabolic capacity seems to be related to cigarette consumption both through genetic and through epigenetic mechanisms.

Molecular crosstalk between a chemical and a biological stressor and consequences on disease manifestation in rainbow trout.

The aim of the present study was to examine the molecular and organism reaction of rainbow trout, Oncorhynchus mykiss, to the combined impact of two environmental stressors. The two stressors were the myxozoan parasite, Tetracapsuloides bryosalmonae, which is the etiological agent of proliferative kidney disease (PKD) and a natural stressor to salmonid populations, and 17ß-estradiol (E2) as prototype of estrogen-active chemical stressors in the aquatic environment. Both stressors, the parasite and estrogenic contaminants, co-exist in Swiss rivers and are discussed as factors contributing to the decline of Swiss brown trout populations over the last decades. Using a microarray approach contrasting parasite-infected and non-infected rainbow trout at low or high estrogen levels, it was observed that molecular response patterns under joint exposure differed from those to the single stressors. More specifically, three major response patterns were present: (i) expression responses of gene transcripts to one stressor are weakened by the presence of the second stressor; (ii) expression responses of gene transcripts to one stressor are enhanced by the presence of the second stressor; (iii) expression responses of gene transcripts at joint treatment are dominated by one of the two stressors. Organism-level responses to concurrent E2 and parasite treatment - assessed through measuring parasite loads in the fish host and cumulative mortalities of trout - were dominated by the pathogen, with no modulating influence of E2. The findings reveal function- and level-specific responses of rainbow trout to stressor combinations, which are only partly predictable from the response to the single stressors.

The hypermethylation of the O6-methylguanine-DNA methyltransferase gene promoter in gliomas--correlation with array comparative genome hybridization results and IDH1 mutation.

The use of molecular markers in the diagnostics of gliomas aids histopathological diagnosis and allows their further classification into clinically significant subgroups. The aim of this study was to characterize the methylation pattern of the O(6) -methylguanine-DNA methyltransferase (MGMT) promoter, gene copy number aberrations, and isocitrate dehydrogenase I (IDH1) mutation in gliomas. We studied 51 gliomas (15 oligodendrogliomas, 18 oligoastrocytomas, 3 astrocytomas, and 15 glioblastomas) by pyrosequencing, array comparative genome hybridization (CGH), and immunohistochemistry. MGMT hypermethylation was observed in 100% of oligoastrocytomas, 93% of oligodendrogliomas, and 47% of glioblastomas. The most frequently altered chromosomal regions were deletions of 1p31.1/21.1-22.2 and 19q13.3qter in oligodendroglial tumors, and losses of 9p21.3, 10q25.3qter, and 10q26.13-26.2 in glioblastomas. Deletions on 9p and 10q, and gain of 7p were associated with the unmethylated MGMT phenotype, whereas deletion of 19q and oligodendroglial morphology was associated with MGMT hypermethylation. IDH1 mutation showed positive correlation with MGMT hypermethylation and loss of 1p/19q. Our results suggest that MGMT promoter methylation, analyzed by pyrosequencing, is a frequent event in oligodendroglial tumors, and it correlates with IDH1 mutation and 19q loss in gliomas. Pyrosequencing proved a good method for assessing the degree of MGMT methylation in formalin-fixed paraffin-embedded glioma samples. However, further studies are needed to confirm a clinically relevant cut-off point for MGMT methylation in gliomas.

CYP2C9 genotype modifies activity of the renin-angiotensin-aldosterone system in hypertensive men.

BACKGROUND: Two variants of the CYP2C9 gene, CYP2C9*2 and CYP2C9*3, have been indicated to have impaired enzyme function, and thus suspected to reduce the formation of the active metabolite of losartan. Cytochrome P450 (CYP) enzymes are also involved in eicosanoid biosynthesis and regulation of blood pressure (BP) and sodium homeostasis. METHODS: We studied the impact of these variants on BP response to losartan and three other antihypertensive drugs and on baseline indicators of the activity of the renin-angiotensin-aldosterone system. The participants were 217 moderately hypertensive Finnish men that participated in the double-blind, cross-over, placebo-controlled GENRES Study. RESULTS: BP responses to losartan did not differ between CYP2C9*2 or CYP2C9*3 allele carriers and CYP2C9*1*1 patients. A suggestive finding of less pronounced ambulatory BP response to losartan in CYP2C9*1*3 patients with low-normal kidney function was made. At baseline of the GENRES Study, CYP2C9*1*3 patients had significantly lower plasma renin activity and aldosterone levels than CYP2C9*1*1 patients (both P values 0.004). In a replication study in patients with treatment-resistant hypertension, men with CYP2C9*3 allele also had lower plasma renin activity (P = 0.03) and aldosterone levels (P = 0.18). In addition, these men had attenuated renin and aldosterone responses in captopril challenge test (P = 0.29 and 0.006, respectively). CONCLUSION: The CYP2C9*3 allele was associated with lower activity of the renin-angiotensin-aldosterone system in hypertensive men, which may reflect a more efficient sodium reabsorption capacity. CYP2C9*2 and CYP2C9*3 alleles do not influence the antihypertensive effect of losartan in men with essential hypertension and normal kidney function.

Involvement of TRPC3 channels in calcium oscillations mediated by OX(1) orexin receptors.

Oscillations of intracellular Ca2+ provide a novel mechanism for sustained activation of cellular processes. Receptor-activated oscillations are mainly thought to occur through rhythmic IP3-dependent store discharge. However, as shown here in HEK293 cells 1 nM orexin-A (Ox-A) acting at OX1 receptors (OX1R) triggered oscillatory Ca2+ responses, requiring external Ca2+. These responses were attenuated by interference with TRPC3 channel (but not TRPC1/4) function using dominant negative constructs, elevated Mg2+ (a blocker of many TRP channels) or inhibition of phospholipase A2. These treatments did not affect Ca2+ oscillations elicited by high concentrations of Ox-A (100 nM) in the absence of external Ca2+. OX1R are thus able to activate TRPC(3)-channel-dependent oscillatory responses independently of store discharge.

Orexin-A-induced Ca2+ entry: evidence for involvement of trpc channels and protein kinase C regulation.

The orexins are peptide transmitters/hormones, which exert stimulatory actions in many types of cells via the G-protein-coupled OX(1) and OX(2) receptors. Our previous results have suggested that low (subnanomolar) concentrations of orexin-A activate Ca(2+) entry, whereas higher concentrations activate phospholipase C, Ca(2+) release, and capacitative Ca(2+) entry. As shown here, the Ca(2+) response to subnanomolar orexin-A concentrations was blocked by activation of protein kinase C by using different approaches (12-O-tetradecanoylphorbol acetate, dioctanoylglycerol, and diacylglycerol kinase inhibition) and protein phosphatase inhibition by calyculin A. The Ca(2+) response to subnanomolar orexin-A concentrations was also blocked by Mg(2+), dextromethorphan, and tetraethylammonium. These treatments neither affected the response to high concentrations of orexin-A nor the thapsigargin-stimulated capacitative entry. The capacitative entry was instead strongly suppressed by SKF96365. An inward membrane current activated by subnanomolar concentrations of orexin-A and the currents activated upon transient expression of trpc3 channels were also sensitive to Mg(2+), dextromethorphan, and tetraethylammonium. Responses to subnanomolar concentrations of orexin-A (Ca(2+) elevation, inward current, and membrane depolarization) were voltage-dependent with a loss of the response around -15 mV. By using reverse transcription-PCR, mRNA for the trpc1-4 channel isoforms were detected in the CHO-hOX1-C1 cells. The expression of truncated TRPC channel isoforms, in particular trpc1 and trpc3, reduced the response to subnanomolar concentrations of orexin-A but did not affect the response to higher concentrations of orexin-A. The results suggest that activation of the OX(1) receptor leads to opening of a Ca(2+)-permeable channel, involving trpc1 and -3, which is controlled by protein kinase C.