Enhanced oxidative stress with a gradient between plasma and muscle interstitial fluid in patients with end stage renal failure on hemodialysis.

OBJECTIVE: The concentration of 8-iso-prostaglandin-F2 alpha (8-iso-PGF2 alpha in biological fluids has been considered as the most reliable biochemical index of the lipid peroxidation and oxidative stress in patients with several pathological conditions including end stage renal failure. However, there is no reference regarding the influence of Hemodialysis (HD) on the values of 8-iso-PGF2 alpha in the muscle Interstitial Fluid (IF) of patients with end stage renal failure. The aim of our study was to determine 8-iso-PGF2 alpha concentration in the IF during hemodialysis and the gradient between plasma and IF in patients with end stage renal failure. DESIGN: In this study, two microdialysis probes were inserted into the vastus lateralis muscle of the right leg of six male patients with end stage renal failure who were on hemodialysis, and in six healthy males (controls). The samples of IF (12 dialysate fluids) were collected after an equilibration of 30 min: a) during the 1st hour preceding hemodialysis (group CRF0), b) during the 1st, 2nd, 3rd and 4th hour while on hemodialysis (groups CRF1, CRF2, CRF3 and CRF4) and c) during the 1st hour following hemodialysis (group CRF5). At the end of the above periods and simultaneously, blood samples were drawn from the arteriovenous fistula. In the controls, the IF samples (twelve dialysate fluids) were collected during a period of one hour and the blood samples at the end of this period. The levels of 8-iso-PGF2 alpha were measured with an enzyme-immunoassay method. Statistical evaluation was carried out with the statistical program NCSS 2000 and the ANOVA test. RESULTS: Plasma and IF levels of 8-iso-PGF2 alpha in the patients were significantly higher than in controls at base line. During hemodialysis, the 8-iso-PGF2 alpha rose progressively both in plasma and IF but remained higher in plasma than in IF. CONCLUSIONS: Lipid peroxidation is higher in patients on hemodialysis than in controls but it is lower in the IF compared to plasma. The mechanism for this gradient is speculative.

Aberrant p16 promoter methylation among Greek lung cancer patients and smokers: correlation with smoking.

Genetic and environmental factors (dietary and smoking) influence lung cancer epidemiology and induce epigenetic modifications that should be assessed in individual populations. We analyzed p16 methylation among Greek non-small cell lung carcinoma patients and smokers using two-stage methylation-specific polymerase chain reaction. One hundred and fifty specimens from cancerous and adjacent non-cancerous tissue, bronchial washings and sputum from patients and 48 specimens, mostly sputum, from disease-free smokers were included. p16 methylation was very frequent in biopsies (82.85%) and bronchial washings (non-small cell lung carcinoma, 80.35%; small cell lung carcinoma, 16.66%) from patients, but also in adjacent non-cancerous tissue (45.71%). Concordance of p16 methylation and positivity by cytological examination was 51.78%. Methylation was also observed in sputum from asymptomatic cytology-negative smokers (22.5%) and chronic obstructive pulmonary disease patients (three of eight). Among disease-free individuals, methylation correlated only with heavy smoking (>50 pack-years, P<0.001) and differed among male and female disease-free smokers. In summary, p16 methylation is very frequent among non-small cell lung carcinoma patients, and correlates with heavy cigarette consumption only in disease-free smokers.

Non-CpG cytosine methylation of p53 exon 5 in non-small cell lung carcinoma.

Non-CpG methylation of cytosine residues, a mechanism associated with regulation of gene expression, has not been investigated in human cancer until now. Analysis of the p53 exon 5 mutation spectrum in mutation databases for lung cancer reveals frequent GC>AT transitions, several of which occur at non-CpG sequences. To investigate the involvement of cytosine methylation in this mutagenesis process, we analyzed the methylation profile of p53 exon 5, in lung carcinoma. In this report, we present evidence that extensive clustered non-CpG methylation is observed in three regions of this exon, namely the sequences spanning codons 156-159, 175-179 and the 3' splice site, as well as in scattered CpA sequences. This methylation pattern was verified using direct methylation sequencing, and a two-stage methylation-specific PCR assay (MSP), designed for the detection of methylation in a GC rich region (oligo C sequence, of codons 175-179) of exon 5. The results from this MSP assay reveal that DNA from cancerous specimens was more heavily methylated in non-CpG cytosines, compared to that from non-cancerous lung tissue of cancer patients (14/19 cancerous and 6/19 non-cancerous, respectively). DNA isolated from human leucocytes and some non-cancerous specimens (2/19) was free of non-CpG methylation. Careful analysis of the mutations reported in p53 mutation databases also provides corroborating evidence that the high incidence of GC>AT mutations in the p53 gene, observed in lung cancer, might also be related to non-CpG methylation, as well as to the overall increase of methylation sites in this locus.

Intramuscular administration of very high dose of alpha-tocopherol protects liver from severe ischemia/reperfusion injury.

The effect of intramuscular administration of high (30 mg/kg body weight for 3 days) or very high (300 mg/kg body weight for 3 days) doses of a-tocopherol to Wistar rats subjected to total severe warm hepatic ischemia and reperfusion was investigated. After a 60-minute period of total hepatic ischemia and 120 minutes of reperfusion, animals were killed, and liver samples were taken for determination of malondialdehyde (MDA) and histological examinations. Blood samples were also taken for assay of serum a-tocopherol, alanine transaminase (ALT), aspartate transaminase (AST), and lactic dehydrogenase (LDH). Additional animals were followed for a 7-day survival rate determination. Results showed that ischemia and reperfusion decreased the survival rate to 10%, whereas the levels of AST, ALT, and LDH in serum were increased compared with levels in animals that were sham operated. The MDA concentrations in liver were also increased, from 1.142 to 1.567 nmoles/g, whereas the levels of a-tocopherol in serum were decreased from 10.20 to 1.80 mmol/L. Pretreatment with a-tocopherol increased the viability to 50% and 70%, for the high and very high doses, respectively, and decreased the levels of AST, ALT, and LDH in serum. It also decreased the MDA concentrations in liver to 0.975 and 0.774 nmoles/g for the high and very high doses of a-tocopherol, respectively, whereas it increased the level of a-tocopherol in serum to 11.25 and 13.02 mmol/L for the high and very high doses, respectively. Histological examinations showed protection of the liver parenchyma in the animals treated with a-tocopherol.