Rock paintings and microorganisms: a new insight on Escoural cave

European cave art is of tremendous importance to understand the cultural traditions of the Upper Palaeolithic (35 000 – 10 000 BP) populations. Indeed, Prehistoric communities performed numerous cave paintings all over Western Europe. Understanding these artworks should provide a better knowledge of these early cultural aspects. Although numerous studies have been carried out to analyse the materials used by those communities, nothing has been done on the techniques’ palette of Escoural Cave’s representations. The present work aims at providing the very first data about the techniques and materials used by the Prehistoric to perform the cave paintings of Escoural (Alentejo, Portugal), and the microorganisms possibly endangering this unique parietal art. In situ observations coupled with an extensive micro-sampling and micro-destructive analyses allowed to characterize the coloured material and the way they were applied on the walls of the cave. Both red and black pigments present major composition’s disparities among the different paintings and drawings, supporting a more complex occupations’ chronology than what was earlier thought. The Palaeolithic paintings have suffered deterioration from environmental conditions and include chemical, mechanical and aesthetic alterations, possibly as a result of fungal activity. The standard techniques for biological assessments used in these contexts provided important insights on the diversity of the microbial population, though they have accuracy limitations. To understand the extent and viability of the existing microbiota, DNA quantification and biomarkers analyses, such as desidrogenase activity were performed and correlated with ergosterol amounts; RESUMO: A Arte Rupestre Europeia é de grande importância para compreender as tradições culturais da população do Paleolítico Superior (35 000 - 10 000 BP). De fato, as comunidades Pré-históricas realizaram inúmeras pinturas rupestres em toda a Europa Ocidental, sendo crucial compreender estas obras de forma a proporcionar um melhor conhecimento destes ancestrais aspectos culturais. Embora vários estudos tenham sido realizados para analisar os materiais utilizados por estas comunidades, nada foi efetuado sobre a técnica de execução das representações presentes na Gruta do Escoural. O presente trabalho visa fornecer os primeiros dados sobre as técnicas e materiais utilizados na Pré-História para executar as pinturas rupestres de Escoural (Alentejo, Portugal) bem como caracterização dos microorganismos possivelmente associados aos danos deste bem único. Observações in situ, juntamente com uma extensa micro-amostragem e análises micro-destrutivas permitiu caracterizar os pigmentos utilizados e a forma como eles foram aplicados nas paredes da caverna. Tanto os pigmentos vermelhos como os pretos apresentam composição distinta nas diversas pinturas e desenhos aí representados, apoiando a presença de diferentes ocupações contrariamente ao que se pensava até então. As pinturas Paleolíticas têm sofrido deterioração, devido às condições ambientais, nomeadamente alterações químicas, mecânicas e, possivelmente como resultado da atividade fúngica. As técnicas usualmente utilizadas para a avaliação de contaminação biológica fornecem informação importante sobre a diversidade da população microbiana, embora apresentem algumas limitações. Para entender a extensão e a viabilidade da microbiota existente, a quantificação de DNA e análise de biomarcadores como actividade de desidrogenases foram realizadas e correlacionadas com o conteúdo em ergosterol.

Introduction of an automated system for the diagnosis and quantification of hepatitis B and hepatitis C viruses.

Hepatitis B virus (HBV) and Hepatitis C virus (HCV) infections pose major public health problems because of their prevalence worldwide. Consequently, screening for these infections is an important part of routine laboratory activity. Serological and molecular markers are key elements in diagnosis, prognosis and treatment monitoring for HBV and HCV infections. Today, automated chemiluminescence immunoassay (CLIA) analyzers are widely used for virological diagnosis, particularly in high-volume clinical laboratories. Molecular biology techniques are routinely used to detect and quantify viral genomes as well as to analyze their sequence; in order to determine their genotype and detect resistance to antiviral drugs. Real-time PCR, which provides high sensitivity and a broad dynamic range, has gradually replaced other signal and target amplification technologies for the quantification and detection of nucleic acid. The next-generation DNA sequencing techniques are still restricted to research laboratories.The serological and molecular marker methods available for HBV and HCV are discussed in this article, along with their utility and limitations for use in Chronic Hepatitis B (CHB) diagnosis and monitoring.

Optimisation of a quantitative polymerase chain reaction-based strategy for the detection and quantification of human herpesvirus 6 DNA in patients undergoing allogeneic haematopoietic stem cell transplantation

Human herpesvirus 6 (HHV-6) may cause severe complications after haematopoietic stem cell transplantation (HSCT). Monitoring this virus and providing precise, rapid and early diagnosis of related clinical diseases, constitute essential measures to improve outcomes. A prospective survey on the incidence and clinical features of HHV-6 infections after HSCT has not yet been conducted in Brazilian patients and the impact of this infection on HSCT outcome remains unclear. A rapid test based on real-time quantitative polymerase chain reaction (qPCR) has been optimised to screen and quantify clinical samples for HHV-6. The detection step was based on reaction with TaqMan® hydrolysis probes. A set of previously described primers and probes have been tested to evaluate efficiency, sensitivity and reproducibility. The target efficiency range was 91.4% with linearity ranging from 10-106 copies/reaction and a limit of detection of five copies/reaction or 250 copies/mL of plasma. The qPCR assay developed in the present study was simple, rapid and sensitive, allowing the detection of a wide range of HHV-6 loads. In conclusion, this test may be useful as a practical tool to help elucidate the clinical relevance of HHV-6 infection and reactivation in different scenarios and to determine the need for surveillance.

Quantification of mitochondrial DNA mutation load

Mitochondrial DNA (mtDNA) mutations are an important cause of genetic disease and have been proposed to play a role in the ageing process. Quantification of total mtDNA mutation load in ageing tissues is difficult as mutational events are rare in a background of wild-type molecules, and detection of individual mutated molecules is beyond the sensitivity of most sequencing based techniques. The methods currently most commonly used to document the incidence of mtDNA point mutations in ageing include post-PCR cloning, single-molecule PCR and the random mutation capture assay. The mtDNA mutation load obtained by these different techniques varies by orders of magnitude, but direct comparison of the three techniques on the same ageing human tissue has not been performed. We assess the procedures and practicalities involved in each of these three assays and discuss the results obtained by investigation of mutation loads in colonic mucosal biopsies from ten human subjects.

Quantification of mitochondrial DNA mutation load

Mitochondrial DNA (mtDNA) mutations are an important cause of genetic disease and have been proposed to play a role in the ageing process. Quantification of total mtDNA mutation load in ageing tissues is difficult as mutational events are rare in a background of wild-type molecules, and detection of individual mutated molecules is beyond the sensitivity of most sequencing based techniques. The methods currently most commonly used to document the incidence of mtDNA point mutations in ageing include post-PCR cloning, single-molecule PCR and the random mutation capture assay. The mtDNA mutation load obtained by these different techniques varies by orders of magnitude, but direct comparison of the three techniques on the same ageing human tissue has not been performed. We assess the procedures and practicalities involved in each of these three assays and discuss the results obtained by investigation of mutation loads in colonic mucosal biopsies from ten human subjects.

Ultrasensitive Simultaneous Quantification of 1,N(2)-Etheno-2 `-deoxyguanosine and 1,N(2-)Propano-2 `-deoxyguanosine in DNA by an Online Liquid Chromatography-Electrospray Tandem Mass Spectrometry Assay

Exocyclic DNA adducts produced by exogenous and endogenous compounds are emerging as potential tools to study a variety of human diseases and air pollution exposure. A highly sensitive method involving online reverse-phase high performance liquid chromatography with electrospray tandem mass spectrometry detection in the multiple reaction monitoring mode and employing stable isotope-labeled internal standards was developed for the simultaneous quantification of 1,N(2)-etheno-2`-deoxyguanosine (1,N(2)-epsilon dGuo) and 1,N(2)-propano-2`-deoxyguanosine (1,N(2)-propanodGuo) in DNA. This methodology permits direct online quantification of 2`-deoxyguanosine and ca. 500 amol of adducts in 100 mu g of hydrolyzed DNA M the same analysis. Using the newly developed technique, accurate determinations of 1,N(2)-etheno-2`-deoxyguanosine and 1,N2-propano-2`-deoxyguanosine levels in DNA extracts of human cultured cells (4.01 +/- 0.32 1,N(2)-epsilon dGuo/10(8) dGuo and 3.43 +/- 0.33 1,N(2)-propanodGuo/10(8) dGuo) and rat tissue (liver, 2.47 +/- 0.61 1,N(2)-epsilon dGuo/10(8) dGuo and 4.61 +/- 0.69 1,N(2)-propanodGuo/108 dGuo; brain, 2.96 +/- 1.43,N(2)-epsilon dGuo/10(8) dGuo and 5.66 +/- 3.70 1,N(2)-propanoclGuo/10(8) dGuo; and lung, 0,87 +/- 0.34 1,N(2)-edGuo/ 10(8) dGuo and 2.25 +/- 1.72 1,N(2)-propanodGuo/10(8) dGuo) were performed. The method described herein can be used to study the biological significance of exocyclic DNA adducts through the quantification of different adducts in humans and experimental an with pathological conditions and after air pollution exposure.

Quantification of global DNA methylation with infrared fluorescence in liver and muscle tissues of differentially fed boars

Methylation of cytosine residues at CpG sites is involved in various biological processes to control gene regulation and gene expression. Global DNA methylation is changed in different tumors and in cloned animals. Global DNA methylation can be accurately quantified by dot blot analysis with infrared (IR) fluorophores. Methylated lambda DNA was used as model DNA to develop and validate an immunochemical assay with IR fluorescence detection. Two different IR fluorophores were used, one to detect 5-methylcytosine and another to account for DNA loading. A sensitive infrared detection method was established which is suitable for accurate and reproducible quantification of global DNA methylation across a wide dynamic range. This method was subsequently employed to quantify global DNA methylation in liver and in muscle tissues of boars which have received either a control diet or a methyl supplemented diet in an ongoing study. A significant difference in global DNA methylation is indicated in muscle but not in liver tissue between the two groups of boars.

Simultaneous A8344G heteroplasmy and mitochondrial DNA copy number quantification in Myoclonus Epilepsy and Ragged-Red Fibers (MERRF) syndrome by a multiplex Molecular Beacon based real-time fluorescence PCR

The association of a particular mitochondrial DNA (mtDNA) mutation with different clinical phenotypes is a well-known feature of mitochondrial diseases. A simple genotype–phenotype correlation has not been found between mutation load and disease expression. Tissue and intercellular mosaicism as well as mtDNA copy number are thought to be responsible for the different clinical phenotypes. As disease expression of mitochondrial tRNA mutations is mostly in postmitotic tissues, studies to elucidate disease mechanisms need to be performed on patient material. Heteroplasmy quantitation and copy number estimation using small patient biopsy samples has not been reported before, mainly due to technical restrictions. In order to resolve this problem, we have developed a robust assay that utilizes Molecular Beacons to accurately quantify heteroplasmy levels and determine mtDNA copy number in small samples carrying the A8344G tRNALys mutation. It provides the methodological basis to investigate the role of heteroplasmy and mtDNA copy number in determining the clinical phenotypes.

Characterization of Hepatitis B virus (HBV) genotypes in patients from Rondonia, Brazil

Background: Hepatitis B virus (HBV) can be classified into nine genotypes (A-I) defined by sequence divergence of more than 8% based on the complete genome. This study aims to identify the genotypic distribution of HBV in 40 HBsAg-positive patients from Rondonia, Brazil. A fragment of 1306 bp partially comprising surface and polymerase overlapping genes was amplified by PCR. Amplified DNA was purified and sequenced. Amplified DNA was purified and sequenced on an ABI PRISM (R) 377 Automatic Sequencer (Applied Biosystems, Foster City, CA, USA). The obtained sequences were aligned with reference sequences obtained from the GenBank using Clustal X software and then edited with Se-Al software. Phylogenetic analyses were conducted by the Markov Chain Monte Carlo (MCMC) approach using BEAST v.1.5.3. Results: The subgenotypes distribution was A1 (37.1%), D3 (22.8%), F2a (20.0%), D4 (17.1%) and D2 (2.8%). Conclusions: These results for the first HBV genotypic characterization in Rondonia state are consistent with other studies in Brazil, showing the presence of several HBV genotypes that reflects the mixed origin of the population, involving descendants from Native Americans, Europeans, and Africans.

Sequence-specific electrochemical detection of Alicyclobacillus acidoterrestris DNA using electroconductive polymer-modified fluorine tin oxide electrodes

This study outlines the quantification of low levels of Alicyclobacillus acidoterrestris in pure cultures, since this bacterium is not inactivated by pasteurization and may remain in industrialized foods and beverages. Electroconductive polymer-modified fluorine tin oxide (FTO) electrodes and multiple nanoparticle labels were used for biosensing. The detection of A. acidoterrestris in pure cultures was performed by reverse transcription polymerase chain reaction (RT-PCR) and the sensitivity was further increased by asymmetric nested RT-PCR using electrochemical detection for quantification of the amplicon. The quantification of nested RT-PCR products by Ag/Au-based electrochemical detection was able to detect 2 colony forming units per mL (CFU mL(-1)) of spores in pure culture and low detection and quantification limits (7.07 and 23.6 nM, respectively) were obtained for the target A. acidoterrestris on the electrochemical detection bioassay.

Real-time Polymerase Chain Reaction Quantification of Porphyromonas gingivalis and Tannerella forsythia in Primary Endodontic Infections

Introduction: Porphyromonas gingivalis and Tannerella forsythia are anaerobic bacteria commonly involved in root canal infections. Although previous investigations have assessed these species by strictly qualitative approaches, accurate determination of their cell levels by a sensitive quantitative technique may contribute with additional information regarding relevance in pain of endodontic origin. Method: The root canal levels of P gingivalis, T forsythia, and total bacteria were investigated by a quantitative polymerase chain reaction (PCR) assay based on unique copy molecular markers. A total of 32 symptomatic (n = 14) and asymptomatic (n = 18) cases of endodontic infections were analyzed. Root canal samples were collected; genomic DNA was extracted and submitted to SYBR Green I real-time PCR targeting the rgpB (P gingivalis), bspA (T forsythia), and rpoB (total bacteria) single copy genes. Results: Overall, R gingivalis, T forsythia, and the coexistence of both species were encountered in 28%, 66%, and 22% of the subjects, respectively. P gingivalis and T forsythia levels ranged from 5.65 x 10(-6) to 1.20 x 10(-2) and from 5.76 x 10(-6) to 1.35 x 10(-1). T forsythia was highly prevalent and numerous in the study groups, whereas P gingivalis was moderately frequent and less abundant, displaying 19-fold lower average levels than the former. Conclusions: The endodontic levels of P gingivalis and T forsythia, individually or in conjunction, did not display significant associations with the manifestation of pain of endodontic origin. (J Endod 2009,35:1518-1524)

Quantification of Cyclobutane Pyrimidine Dimers Induced by UVB Radiation in Conidia of the Fungi Aspergillus fumigatus, Aspergillus nidulans, Metarhizium acridum and Metarhizium robertsii

Conidia are responsible for reproduction, dispersal, environmental persistence and host infection of many fungal species. One of the main environmental factors that can kill and/or damage conidia is solar UV radiation. Cyclobutane pyrimidine dimers (CPD) are the major DNA photoproducts induced by UVB. We examined the conidial germination kinetics and the occurrence of CPD in DNA of conidia exposed to different doses of UVB radiation. Conidia of Aspergillus fumigatus, Aspergillus nidulans and Metarhizium acridum were exposed to UVB doses of 0.9, 1.8, 3.6 and 5.4 kJ m-2. CPD were quantified using T4 endonuclease V and alkaline agarose gel electrophoresis. Most of the doses were sublethal for all three species. Exposures to UVB delayed conidial germination and the delays were directly related both to UVB doses and CPD frequencies. The frequencies of dimers also were linear and directly proportional to the UVB doses, but the CPD yields differed among species. We also evaluated the impact of conidial pigmentation on germination and CPD induction on Metarhizium robertsii. The frequency of dimers in an albino mutant was approximately 10 times higher than of its green wild-type parent strain after exposure to a sublethal dose (1.8 kJ m-2) of UVB radiation.

Quantification of Listeria monocytogenes in minimally processed leafy vegetables using a combined method based on enrichment and 16S rRNA real-time PCR

Modern lifestyle markedly changed eating habits worldwide, with an increasing demand for ready-to-eat foods, such as minimally processed fruits and leafy greens. Packaging and storage conditions of those products may favor the growth of psychrotrophic bacteria, including the pathogen Listeria monocytogenes. In this work, minimally processed leafy vegetables samples (n = 162) from retail market from Ribeirao Preto, Sao Paulo, Brazil, were tested for the presence or absence of Listeria spp. by the immunoassay Listeria Rapid Test, Oxoid. Two L. monocytogenes positive and six artificially contaminated samples of minimally processed leafy vegetables were evaluated by the Most Probable Number (MPN) with detection by classical culture method and also culture method combined with real-time PCR (RTi-PCR) for 16S rRNA genes of L monocytogenes. Positive MPN enrichment tubes were analyzed by RTi-PCR with primers specific for L. monocytogenes using the commercial preparation ABSOLUTET (TM) QPCR SYBR (R) Green Mix (ABgene, UK). Real-time PCR assay presented good exclusivity and inclusivity results and no statistical significant difference was found in comparison with the conventional culture method (p < 0.05). Moreover, RTi-PCR was fist and easy to perform, with MPN results obtained in ca. 48 h for RTi-PCR in comparison to 7 days for conventional method. (C) 2009 Elsevier Ltd. All rights reserved.

GP5+/6+ SYBR Green methodology for simultaneous screening and quantification of human papillomavirus

Background: Detection and quantification of human papillomavirus (HPV) may help in predicting the evolution of HPV infection and progression of associated lesions. Objectives: We propose a novel protocol using consensus primers GP5+/6+ in a SYBR Green quantitative real-time (Q-RT) polymerase chain reaction (PCR). The strategy permits screening for HPV infection and viral load quantification simultaneously. Study design: DNA from 153 archived cervical samples, previously tested for HPV detection by GP5+/6+ PCR and typed by EIA-RLB (enzyme immunoassay-reverse line blot) or sequence analysis, was analysed using SYBR Green Q-RT PCR. Melting temperature assay (T(m)) and cycle threshold (C(t)) were used to evaluate HPV positivity and viral load. The T(m) in the range of 77-82 degrees C was considered to be positive for HPV-DNA. HPV results generated through GP5+/6+ conventional PCR were considered the gold standard against which sensitivity and specificity of our assay were measured. Results: Out of 104 HPV positive samples, 100 (96.2%) were also determined as positive by SYBR Green Q-RT PCR; of the 49 HPV-negative samples, all were determined as negative. There was an excellent positivity agreement (K = 0.94) between the SYBR Green Q-RT and the previous methods employed. The specificity and sensitivity were 100% and 96.2%, respectively. Comparison of SYBR Green Q-RT and TaqMan oligo-probe technologies gave an excellent concordance (pc = 0.95) which validated the proposed strategy. Conclusions: We propose a sensitive and easy-to-perform technique for HPV screening and viral load quantification simultaneously. (C) 2009 Elsevier B.V. All rights reserved.

DNA damage induced by acrylamide: roe of genetic polymorphisms in DNA damage levels

RESUMO:Em 1994 a acrilamida (AA) foi classificada pela IARC como um provável cancerígeno para o homem. Para além da utilização de AA em numerosas aplicações industriais, a AA está também presente numa grande variedade de alimentos ricos em amido e processados a temperaturas elevadas. Esta exposição através da ingestão de produtos alimentares despoletou elevadas preocupações ao nível do risco para a saúde pública e poderá implicar um risco adicional para o aparecimento de cancro. A glicidamida (GA), o metabolito epóxido formado a partir da oxidação da AA provavelmente através do citocromo P450 2E1, é considerada por vários estudos, o principal responsável pela carcinogenicidade da AA. Actualmente existe uma escassez de resultados relativamente aos mecanismos de genotoxicidade da AA e GA em células de mamífero. Por este motivo, o objectivo deste estudo centra-se na avaliação das consequências genéticas da exposição à AA e GA, recorrendo-se para tal ao uso de células de mamífero como modelo. Tendo como base este objectivo avaliou-se a citotoxicidade da AA e GA, através do ensaio do MTT, e realizaram-se dois testes citogenéticos, o teste das aberrações cromossómicas (CAs) e o teste da troca de cromátides irmãs (SCEs), de modo a avaliar as lesões de DNA induzidas por estes compostos em células de hamster Chinês V79. Os resultados globalmente mostraram que a GA é mais citotóxica e clastogénica do que a AA. No âmbito deste trabalho, foi também efectuada a quantificação de aductos específicos de DNA, nomeadamente N7-(2-carbamoil-2-hidroxietil)guanina (N7-GA-Gua) e N3-(2-carbamoil-2-hidroxietil)adenina (N3-GA-Ade). Os resultados obtidos permitem afirmar que os níveis de N7-GA-Gua e a concentração de GA apresentam uma relação linear dose-resposta. Foi também identificada uma óptima correlação entre os níveis de N7-GA-Gua e a frequência de troca de cromátides irmãs. Adicionalmente, e de forma a compreender os mecanismos de toxicidade da AA, estudaram-se os mecanismos dependentes da modulação do glutationo reduzido (GSH), nomeadamente da butionina sulfoximina (BSO), um inibidor da síntese de GSH, do GSH-monoetil estér (GSH-EE), um composto permeável nas células e que é intra-celularmente hidrolisado a GSH e ainda do GSH adicionado exogenamente ao meio de cultura, em células V79. Os resultados obtidos reforçaram o papel da modulação do GSH nos efeitos de citotoxicidade e clastogenicidade da AA. Para além dos estudos efetuados com células V79, procedeu-se também à determinação da frequência de SCEs, à quantificação de aductos específicos de DNA, bem como ao ensaio do cometa alcalino em amostras de dadores saudáveis expostos à AA e GA. Tanto os resultados obtidos através do ensaio das SCE, como pela quantificação de aductos específicos de DNA, ambos efectuados em linfócitos estimulados, originaram resultados comparáveis aos obtidos anteriormente para as células V79, reforçando a ideia de que a GA é bastante mais genotóxica do que a AA. Por outro lado, os resultados obtidos pelo ensaio do cometa para exposição à AA e GA mostraram que apenas esta última aumenta o nível das lesões de DNA. Outro objectivo deste trabalho, foi a identificação de possíveis associações existentes entre as lesões de DNA, quantificadas através do ensaio das SCEs e do cometa, e biomarcadores de susceptibilidade, tendo em conta os polimorfismos genéticos individuais envolvidos na destoxificação e nas vias de reparação do DNA (BER, NER, HRR e NHEJ) em linfócitos expostos à GA. Tal permitiu identificar associações entre os níveis de lesão de DNA determinados através do ensaio das SCEs, e os polimorfismos genéticos estudados, apontando para uma possível associação entre o GSTP1 (Ile105Val) e GSTA2 (Glu210Ala) e a frequência de SCEs. Por outro lado, os resultados obtidos através do ensaio do cometa sugerem uma associação entre as lesões de DNA e polimorfismos da via BER (MUTYH Gln335His e XRCC1 Gln39Arg) e da via NER (XPC Ala499val e Lys939Gln), considerando os genes isoladamente ou combinados. Estes estudos contribuem para um melhor entendimento da genotoxicidade e carcinogenicidade da AA e GA em células de mamífero, bem como da variabilidade da susceptibilidade individual na destoxificação e reparação de lesões de DNA provocadas pela exposição a estes xenobióticos alimentares. ----------- ABSTRACT:Acrylamide (AA) has been classified as a probable human carcinogen by IARC. Besides being used in numerous industrial applications, AA is also present in a variety of starchy cooked foods. This AA exposure scenario raised concerns about risk in human health and suggests that the oral consumption of AA is an additional risk factor for cancer. A considerable number of findings strongly suggest that the reactive metabolite glycidamide (GA), an epoxide generated presumably by cytochrome P450 2E1, plays a central role in AA carcinogenesis. Until now there are a scarcity of results concerning the mechanisms of genotoxicity of AA and GA in mammalian cells. In view of that, the study described in this thesis aims to unveil the genetic consequences of AA and GA exposure using mammalian cells as a model system. With this aim we evaluated the cytotoxicity of AA and GA using the MTT assay and subsequently performed two cytogenetic end-points: chromosomal aberrations (CAs) and sister chromatid exchanges (SCEs), in order to evaluate DNA damage induced by these compounds in V79 Chinese hamster cell line. The results showed that GA was more cytotoxic and clastogenic than AA. Within the scope of this thesis the quantification of specific DNA adducts were also performed, namely N7-(2-carbamoyl-2-hydroxyethyl)guanine (N7-GA-Gua) and N3-(2-carbamoyl-2-hydroxyethyl)adenine (N3-GA-Ade). Interestingly, the GA concentration and the levels of N7-GA-Gua presented a linear dose-response relationship. Further, a very good correlation between the levels of N7-GA-Gua and the extent of SCEs were observed. In order to understand the mechanisms of AA-induced toxicity, the modulation of reduced glutathione (GSH)-dependent mechanisms were studied, namely the evaluation of the effect of buthionine sulfoximine (BSO), an effective inhibitor of GSH synthesis, of GSH-monoethyl ester (GSH-EE), a cell permeable compound that is intracellularly hydrolysed to GSH and also of GSH endogenously added to culture medium,z in V79 cell line. The overall results reinforced the role of GSH in the modulation of the cytotoxic and clastogenic effects induced by AA.Complementary to the studies performed in V79 cells, SCEs, specific DNA-adducts and alkaline comet assay in lymphocytes from healthy donors exposed to AA and GA were also evaluated. Both, the frequency of SCE and the quantification of specific GA DNA adducts, produced comparable results with those obtained in V79 cell line, reinforcing the idea that GA is far more genotoxic than AA. Further, the DNA damaging potential of AA and GA in whole blood leukocytes evaluated by the alkaline comet assay, showed that GA, but not AA, increases DNA damage. Additionally, this study aimed to identify associations between DNA damage and biomarkers of susceptibility, concerning individual genetic polymorphisms involved in detoxification and DNA repair pathways (BER, NER, HRR and NHEJ) on the GA-induced genotoxicity assessed by the SCE assay and by the alkaline comet assay. The extent of DNA damage determined by the levels of SCEs induced by GA seems to be modulated by GSTP1 (Ile105Val) and GSTA2 (Glu210Ala) genotypes. Moreover, the results obtained from the comet assay suggested associations between DNA damage and polymorphisms of BER (MUTYH Gln335His and XRCC1 Gln399Arg) and NER (XPC Ala499Val and Lys939Gln) genes, either alone or in combination. The overall results from this study contribute to a better understanding of the genotoxicity and carcinogenicity of AA and GA in mammalian cells, as well as the knowledge about the variability in individual susceptibility involved in detoxification and repair of DNA damage due to these dietary xenobiotics.