João Pereira de Lima

Induction of sister chromatid exchange by acrylamide and glycidamide in human lymphocytes: role of polymorphisms in detoxification and DNA-repair genes in the genotoxicity of glycidamide.

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.

Genotoxic effect of exposure to metal(loid)s. A molecular epidemiology survey of populations living and working in Panasqueira mine area, Portugal

Previous studies investigating the exposure to metal(loid)s of populations living in the Panasqueira mine area of central Portugal found a higher internal dose of elements such as arsenic, chromium, lead, manganese, molybdenum and zinc in exposed individuals. The aims of the present study were to evaluate the extent of genotoxic damage caused by environmental and occupational exposure in individuals previously tested for metal(loid) levels in different biological matrices, and the possible modulating role of genetic polymorphisms involved in metabolism and DNA repair. T-cell receptor mutation assay, comet assay, micronucleus (MN) test and chromosomal aberrations (CA) were performed in a group of 122 subjects working in the Panasqueira mine or living in the same region. The modifying effect of polymorphisms in GSTA2, GSTM1, GSTP1, GSTT1, XRCC1, APEX1, MPG, MUTYH, OGG1, PARP1, PARP4, ERCC1, ERCC4, and ERCC5 genes was investigated. Significant increases in the frequency of all biomarkers investigated were found in exposed groups, however those environmentally exposed were generally higher. Significant influences of polymorphisms were observed for GSTM1 deletion and OGG1 rs1052133 on CA frequencies, APEX1 rs1130409 on DNA damage, ERCC1 rs3212986 on DNA damage and CA frequency, and ERCC4 rs1800067 on MN and CA frequencies. Our results show that the metal(loid) contamination in the Panasqueira mine area induced genotoxic damage both in individuals working in the mine or living in the area. The observed effects are closely associated to the internal exposure dose, and are more evident in susceptible genotypes. The urgent intervention of authorities is required to protect exposed populations.

Genetic Polymorphisms in Detoxification and DNA Repair Genes and Susceptibility to Glycidamide-Induced DNA Damage

Acrylamide (AA) is a probable human carcinogen formed in carbohydrate-rich foodstuffs upon heating. Glycidamide (GA), the AA metabolite formed by epoxidation, is considered the ultimate genotoxic agent. In this study, the in vitro genotoxic potential of AA and GA in human whole blood leukocytes was compared using the alkaline comet assay. Although AA did not induce significant DNA damage in the concentrations tested (up to 1000 μM), GA markedly increased the percentage of tail DNA at concentrations ≥250 μM. Further, this study addressed the role of genetic polymorphisms in key genes involved in metabolism and DNA repair pathways (BER, NER, HRR, and NHEJ) on GA-induced genotoxicity assessed by the alkaline comet assay. The results obtained 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.

DNA repair genes polymorphisms and genetic susceptibility to Philadelphia-negative myeloproliferative neoplasms in a Portuguese population: The role of base excision repair genes polymorphisms

The role of base excision repair (BER) genes in Philadelphia-negative (PN)-myeloproliferative neoplasms (MPNs) susceptibility was evaluated by genotyping eight polymorphisms [apurinic/apyrimidinic endodeoxyribonuclease 1, mutY DNA glycosylase, earlier mutY homolog (E. coli) (MUTYH), 8-oxoguanine DNA glycosylase 1, poly (ADP-ribose) polymerase (PARP) 1, PARP4 and X-ray repair cross-complementing 1 (XRCC1)] in a case-control study involving 133 Caucasian Portuguese patients. The results did not reveal a correlation between individual BER polymorphisms and PN-MPNs when considered as a whole. However, stratification for essential thrombocythaemia revealed i) borderline effect/tendency to increased risk when carrying at least one variant allele for XRCC1_399 single-nucleotide polymorphism (SNP); ii) decreased risk for Janus kinase 2-positive patients carrying at least one variant allele for XRCC1_399 SNP; and iii) decreased risk in females carrying at least one variant allele for MUTYH SNP. Combination of alleles demonstrated an increased risk to PN-MPNs for one specific haplogroup. These findings may provide evidence for gene variants in susceptibility to MPNs. Indeed, common variants in DNA repair genes may hamper the capacity to repair DNA, thus increasing cancer susceptibility.

Glycidamide genotoxicity modulated by Caspases genes polymorphisms

Acrylamide (AA) is amongst acknowledged carcinogenic dietary factors. Its DNA-reactive metabolite is glycidamide (GA). The present study intended to correlate the role of key polymorphic genes of apoptosis (CASP7, CASP8, CASP9, CASP10, LTA and TNFRSF1B) with biomarkers of effect of DNA damage, namely the sister chromatid exchange assay (SCE) and the comet assay in whole blood cells exposed to GA. The aim was to assess as a proof of concept the role that pro-apoptotic effector proteins might have in the yields of genotoxic effects when those effector proteins are coded by polymorphic genes. Whole blood from a small group of volunteers was exposed to GA to assess DNA damage and the volunteers were genotyped for polymorphic genes related to apoptosis pathways. A relation between the induction of SCE and several variants of the polymorphism CASP8 rs1035142 G>T was observed. Also, a relation between the % tail DNA and the CASP10 I522L polymorphism was found. Furthermore, associations between % tail DNA and several SNP-SNP interactions of CASP8 and CASP10 were found. A possible correlation between DNA damage and the genetic susceptibility, bestowed by polymorphic genes in the apoptosis inducing pathways was verified.

Sickle Cell Trait, Malaria and Sensorineural Hearing Loss–A Case-Control Study from São Tomé and Príncipe

Background: Hearing loss is a problem with higher incidence in South Asia, Asia Pacific and sub-Saharan Africa. In these countries there is also associated history of anemia and malaria. Objective: This study aims to identify a putative role of Beta globin mutation - sickle cell trait and HL in Sao Tome and Principe population. Methods: A retrospective case-control study of a convenience sample was collected during Otolaryngologist Humanitarian Missions in Sao Tome and Principe. Control group includes individuals with normal hearing in both ears, and the case group has participants presenting bilateral or unilateral HL. It was evaluated the potential risk factors and sickle cell trait with HL, as well self-report of malaria infection, consanguinity, familial history of HL. The HbS gene point mutation (Glu6Val) was determined by PCR-RFLP. Results: Our results showed a statistical significance between HL - oral language and self-report of HL. Taken altogether, our data did not reveal association between sickle cell trait and HL. However, a statistical association between HL and self-report of malaria was found. Conclusion: No association between sickle cell trait and the high prevalence of HL was found. Self-report of Malaria was found as a risk factor for the development of HL in Sao Tome and Principe population. The multifactorial profile of HL shall not exclude the relevance of other etiologic factors than Malaria to justify the high prevalence of HL in Sao Tome and Principe and further investigation must be applied.

Abstract 82: Gene expression induced by acrylamide and glycidamide in mammalian cells.

Abstract Acrylamide (AA) has been classified as a probable human carcinogen by IARC. AA is generated in carbohydrate-rich foodstuff upon heating and its discovery in a variety of fried foods has raised public health concerns since oral consumption of AA could be an additional risk factor for cancer. Glycidamide (GA), formed via epoxidation, presumably by cytochrome P450 2E1, is considered the ultimate genotoxic agent playing a central role in AA carcinogenesis. Previous studies done by our group reported a strong correlation between GA-induction of Sister Chromatid Exchanges (SCE) and GA-DNA adducts, suggesting the possible involvement of Base Excision Repair (BER) and Homologous Recombination Repair (HRR) pathways in AA/GA induced DNA damage. Presently there is a lot of information concerning the mechanisms of genotoxicity of AA and GA in mammalian cells, however this is not the case for the role of AA and GA in gene expression. In view of this, we studied gene expression in non-malignant mammary cell line (MCF10A) and in isolated human lymphocytes using a high-throughput technique, the RT2 Profiler PCR arrays: Human DNA Damage Signaling Pathway. In our experimental protocol we exposed MCF10A cells to AA and GA (10 μM) for 1, 2 and 24 h periods. Moreover, to discern the possible differences of cultured cell lines and isolated lymphocytes, we exposed isolated lymphocytes from two healthy donors to GA (10 μM) for a period of 2 h. A value of p ≤0.05, and a fold change greater than 1.1 and lower than -1.1, were considered to be significant in gene expression dysregulation. Concerning MCF10A cell line, the results obtained in 24h exposure showed that neither AA nor GA influenced the expression of the genes considered when compared to respective non-treated controls. However, for 1 h exposure it was observed that of the 84 studied genes, only GADD45A was up-regulated and GTSE1 was down-regulated for both compounds. On the other hand, MRE11A was only up-regulated in GA exposure. In what respect to 2 h exposure, of all the genes studied, 7 were significantly up-regulated (DMC1, GADD45G, MPG, RAD9A, XRCC3, XPA and XRCC6) and 2 down-regulated (GTF2H1 and GTSE1) for AA exposure and 16 were up-regulated (AIFM1, ATRX, DMC1, GADD45G, MNAT1, MPG, PMS2L3, RAD18, RAD51, RAD51L1, RAD9A, TREX1, XPA, XPC, XRCC3 and XRCC6) and 1 down-regulated (GTSE1) for GA exposure. The results obtained regarding the study done with isolated lymphocytes exposed to GA (10 μM) for a 2 h period showed that GA up-regulated 1 gene for each donor, RAD51 for donor 1 and UNG for donor 2. However, when individuals were pooled, no statistical significance results were observed, what may be related to the importance of inter-individual variability in gene expression. In fact, several Single Nucleotide Polymorphisms (SNPs) can modulate the level of DNA damage induced by AA and GA, and for that reason, this variability should be taking into account. The overall results obtained from the genes identified in this methodology are in general associated with DNA repair, specifically with BER and HRR, but also with apoptosis. In the future, Western blots should be performed in order to analyze the protein levels associated with representative genes identified and further studies should be conducted in human lymphocytes concerning other exposure periods, to account for variations in reaction between cell lines and isolated human lymphocytes. Moreover the number of donors should be increased to ascertain the possible effect of inter-individual variations in our study. Citation Format: Marta Pingarilho, João P. Lima, Célia Martins, José Rueff, Jorge F. Gaspar. Gene expression induced by acrylamide and glycidamide in mammalian cells. [abstract]. In: Proceedings of the AACR Special Conference on Post-GWAS Horizons in Molecular Epidemiology: Digging Deeper into the Environment; 2012 Nov 11-14; Hollywood, FL. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2012;21(11 Suppl):Abstract nr 82.