RESUMO
Acrylamide (AA) is a probable human carcinogen generated in carbohydrate-rich foodstuffs upon heating. Glycidamide (GA), formed via epoxidation, presumably mediated by cytochrome P450 2E1, is considered to be the active metabolite that plays a central role in the genotoxicity of AA. The aim of this work was to evaluate the cytogenetic damage induced by AA and GA in cultured human lymphocytes by use of the sister chromatid exchange (SCE) assay. Furthermore, this report addresses the role of individual genetic polymorphisms in key genes involved in detoxification and DNA-repair pathways (BER, NER, HRR and NHEJ) on the induction of SCE by GA. While AA induced the number of SCE/metaphase only slightly, especially for the highest concentration tested (2000µM), GA markedly induced SCEs in a concentration-dependent manner up to concentrations of 750µM, leading to an increase in SCEs of up to about 10-fold compared with controls. By combining DNA damage in GA-treated lymphocytes and data on polymorphisms, associations between the induction of SCEs with GSTP1 (Ile105Val) and GSTA2 (Glu210Ala) genotypes are suggested.
Assuntos
Acrilamida/toxicidade , Dano ao DNA , Reparo do DNA , Compostos de Epóxi/toxicidade , Mutagênicos/toxicidade , Polimorfismo Genético , Troca de Cromátide Irmã , Glutationa S-Transferase pi/genética , Glutationa Transferase/genética , Humanos , Inativação Metabólica/genética , Isoenzimas/genética , Linfócitos/efeitos dos fármacosRESUMO
Acrylamide (AA) is a suspected human carcinogen found to be generated during the heating of carbohydrate-rich foodstuffs. AA exhibits 'Michael-type' reactivity towards reduced glutathione (GSH), resulting in vivo in the urinary excretion of mercapturic acid conjugates. GSH is a key factor for mammalian cell homeostasis, with diverse functions that include, among others, the conjugation of electrophilic compounds and the detoxification of products generated by oxidative stress. Therefore, studies focusing on the modulation of GSH are of great importance for the understanding of the mechanisms of AA-induced toxicity. This report addresses this issue by analyzing cytotoxicity (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) reduction assay) and clastogenicity (chromosomal aberrations) as endpoints in V79 cells after exposure to AA. The experiments described herein include the evaluation of the effect of buthionine sulfoximine (BSO), an effective inhibitor of GSH synthesis, GSH-monoethyl ester (GSH-EE), a compound that is taken up by cells and intracellularly hydrolysed to GSH, and also GSH exogenously added to culture medium. Pre-treatment with BSO increased the cytotoxicity and the frequency of aberrant cells excluding gaps (ACEG) induced by AA. While pre-treatment with GSH-EE did not modify the cytotoxicity or the frequency of ACEG induced by AA, co-treatment with AA and GSH decreased both parameters, rendering the cells less prone to the toxic effects of AA. In vitro studies in a cell-free system, using monochlorobimane (MCB), a fluorescent probe for GSH, were also performed in order to evaluate the role of AA in GSH depletion. The results show that spontaneous conjugation of AA with GSH in the extracellular medium is involved in the protection given by GSH. In summary, these results reinforce the role of GSH in the modulation of the cytotoxic and clastogenic effects induced by AA, which may be relevant in an in vivo exposure scenario.
Assuntos
Acrilamida/toxicidade , Aberrações Cromossômicas/induzido quimicamente , Glutationa/metabolismo , Animais , Antioxidantes/toxicidade , Butionina Sulfoximina , Contagem de Células , Linhagem Celular , Células Cultivadas , Cricetinae , Dano ao DNA/efeitos dos fármacos , Dano ao DNA/genética , HumanosRESUMO
Acrylamide (AA) is a suspected human carcinogen generated in carbohydrate-rich foodstuffs upon heating. Glycidamide (GA), formed via epoxidation, presumably mediated by cytochrome P450 2E1, is thought to be the active metabolite playing a central role in AA genotoxicity. In this work we investigated DNA damage induced by AA and GA in mammalian cells, using V79 Chinese hamster cells. For this purpose, we evaluated two cytogenetic end points, chromosomal aberrations (CAs) and sister chromatid exchanges (SCEs), as well as the levels of specific GA-DNA adducts, namely, N7-(2-carbamoyl-2-hydroxyethyl)guanine (N7-GA-Gua) and N3-(2-carbamoyl-2-hydroxyethyl)adenine (N3-GA-Ade) using high-performance liquid chromatography coupled with tandem mass spectrometry. GA was more cytotoxic and clastogenic than AA. Both AA and GA induced CAs (breaks and gaps) and decreased the mitotic index. GA induced SCEs in a dose-responsive manner; with AA, SCEs were increased at only the highest dose tested (2mM). A linear dose-response relationship was observed between the GA concentration and the levels of N7-GA-Gua. This adduct was detected for concentrations as low as 1 microM GA. N3-GA-Ade was also detected, but only at very high GA concentrations (>or= 250 microM). There was a very strong correlation between the levels of N7-GA-Gua in the GA- and AA-treated cells and the extent of SCE induction. Such correlation was not apparent for CAs. These data suggest that the induction of SCEs by AA is associated with the metabolism of AA to GA and subsequent formation of depurinating DNA adducts; however, other mechanisms must be involved in the induction of CAs.
Assuntos
Acrilamida/toxicidade , Adenina/análogos & derivados , Aberrações Cromossômicas/induzido quimicamente , Adutos de DNA/metabolismo , Compostos de Epóxi/toxicidade , Guanina/análogos & derivados , Mutagênicos/toxicidade , Troca de Cromátide Irmã/efeitos dos fármacos , Adenina/metabolismo , Animais , Linhagem Celular , Cricetinae , Cricetulus , Relação Dose-Resposta a Droga , Guanina/metabolismo , Estrutura MolecularRESUMO
PURPOSE: Within the EU RENEB project, seven laboratories have taken part in training and harmonisation activities to strengthen triage gamma-H2AX-based radiation exposure assessment. This has culminated in a second triage biodosimetry exercise. MATERIALS AND METHODS: Whole blood and separated lymphocyte samples were homogenously irradiated with 60Co gamma rays at 0.5, 2.5 (blind samples), 0 and 2 Gy (reference samples). Following post-exposure incubations of 4 and 24 h, 16 samples were shipped on ice packs to each partner. The samples were stained and scored for gamma-H2AX foci, using manual and/or automated fluorescence microscope scoring strategies. Dose estimates were obtained and used to assign triage categories to the samples. RESULTS: Average dose estimates across all the laboratories correlated well with true doses. The most accurate assignment of triage category was achieved by manual scoring of the 4-h blood and lymphocyte samples. Only three samples out of a total of 46 were miscategorized in a way that could have adversely effected the clinical management of a radiation casualty. CONCLUSIONS: This inter-comparison exercise has demonstrated that following a recent acute radiation exposure, the gamma-H2AX assay could be a useful triage tool that can be successfully applied across a network of laboratories.