Preliminary evaluation of DNA damage related with the smoking habit measured by the comet assay in whole blood cells

Artículo de publicación ISIThe alkaline single-cell gel electrophoresis (SCGE) assay, also called the comet assay, is a rapid and simple method for the detection of DNA damage in individual cells. The objective of this study was to establish if the alkaline SCGE assay in whole blood cells gives similar results as the same method in isolated lymphocytes, because whole blood cells are simpler and more economical to use, specifically in human genotoxic biomonitoring. To validate the method, we first used mouse blood cells, because mouse is one of the most commonly used animals in genetic toxicology testing. Groups of seven CF1 male mice were given i.p. injections of relatively low doses of methyl methanesulfonate (25 mg/kg body weight), a direct acting genotoxic agent, or cyclophosphamide (50 mg/kg body weight), which requires metabolic activation. Three, 6, 8, 12, 16, 20, and 65 hours after treatment, 5 ML of blood were collected from each animal and were processed for the alkaline SCGE assay. On the basis of an analysis of tail moment, the results showed that this assay can detect DNA damage induced by both kinds of alkylating mutagens. We then did a preliminary study to assess the status of DNA damage in a young (19 to 23 years old) healthy population of male smokers (n = 6) and nonsmokers (n = 6) using the comet assay in whole blood cells. A significant difference was observed between the two groups, showing that the method is able to detect DNA damage in the smoking group despite the short time that the volunteers had actually been smoking

Prevalence of TPMT and ITPA gene polymorphisms and effect on mercaptopurine dosage in Chilean children with acute lymphoblastic leukemia

Artículo de publicación ISIBackground: Mercaptopurine (6-MP) plays a pivotal role in treatment of childhood acute lymphoblastic leukemia (ALL); however, interindividual variability in toxicity of this drug due to genetic polymorphism in 6-MP metabolizing enzymes has been described. We determined the prevalence of the major genetic polymorphisms in 6-MP metabolizing enzymes in Chilean children with ALL. Methods: 103 Chilean pediatric patients with a confirmed diagnosis of ALL were enrolled. DNA was isolated from whole blood and genetic polymorphism in thiopurine S-methyltransferase (TPMT) and inosine triphosphate pyrophosphatase (ITPA) coding genes were detected by polymorphism chain reaction-restriction fragment length (PCR-RFLP) assay. Results: The total frequency of variant TPMT alleles was 8%. TPMT*2, TPMT*3A and TPMT*3B alleles were found in 0%, 7%, and 1% of patients, respectively. For ITPA, the frequency of P32T allele was 3%. We did not observe any homozygous variant for TPMT and ITPA alleles. We also analyzed a subgroup of 40 patients who completed the maintenance phase of ALL treatment, and we found that patients carrying a TPMT gene variant allele required a significantly lower median cumulative dosage and median daily dosage of 6-MP than patients carrying wild type alleles. Conclusion: TMPT genotyping appears an important tool to further optimize 6-MP treatment design in Chilean patients with ALL.This work was supported by grants from Fundación Nuestros Hijos (JM and MJF) and FONDECYT 1120809 (MJF). We are indebted to and pleased to acknowledge Drs. Mary V. Relling and Cristine Crews for TPMT activity measurement