Marta Pingarilho

Genotoxic effects of occupational exposure to lead and influence of polymorphisms in genes involved in lead toxicokinetics and in DNA repair

Lead is still widely used in many industrial processes and is very persistent in the environment. Although toxic effects caused by occupational exposure to lead have been extensively studied, there are still conflicting results regarding its genotoxicity. In a previous pilot study we observed some genotoxic effects in a population of lead exposed workers. Thus, we extended our study analysing a larger population, increasing the number of genotoxicity endpoints, and including a set of 20 genetic polymorphisms related to lead toxicokinetics and DNA repair as susceptibility biomarkers. Our population comprised 148 workers from two Portuguese factories and 107 controls. The parameters analysed were: blood lead levels (BLL) and δ-aminolevulinic acid dehydratase (ALAD) activity as exposure biomarkers, and T-cell receptor (TCR) mutation assay, micronucleus (MN) test, comet assay and OGG1-modified comet assay as genotoxicity biomarkers. Lead exposed workers showed markedly higher BLL and lower ALAD activity than the controls, and significant increases of TCR mutation frequency (TCR-Mf), MN rate and DNA damage. Oxidative damage did not experience any significant alteration in the exposed population. Besides, significant influence was observed for VDR rs1544410 polymorphism on BLL; APE1 rs1130409 and LIG4 rs1805388 polymorphisms on TCR-Mf; MUTYH rs3219489, XRCC4 rs28360135 and LIG4 rs1805388 polymorphisms on comet assay parameter; and OGG1 rs1052133 and XRCC4 rs28360135 polymorphisms on oxidative damage. Our results showed genotoxic effects related to occupational lead exposure to levels under the Portuguese regulation limit of 70 μg/dl. Moreover, a significant influence of polymorphisms in genes involved in DNA repair on genotoxicity biomarkers was observed.

Genotoxic effects of doxorubicin in cultured human lymphocytes with different glutathione S-transferase genotypes.

Doxorubicin (Dox) is a widely used drug in oncology with a broad spectrum of interactions with various cellular components; therefore, it is likely to act through different mechanisms. In clinical practice there is inter-individual variability in cytotoxic drug response and in the occurrence of adverse reactions. Glutathione S-transferases (GSTM1, GSTT1 and GSTP1) are thought to be involved in the detoxification of endogenous and exogenous genotoxicants. The aim of this work is the assessment of a possible influence of polymorphisms in GSTs on the levels of genetic damage induced in vitro by Dox in cultured human lymphocytes. For this purpose, whole blood cultures from individuals with different genotypes for GSTM1, GSTT1 and GSTP1 were exposed to Dox and the cytokinesis-blocked micronucleus (CBMN) assay was used as the endpoint for chromosomal damage in the lymphocytes. Genotyping of GSTM1 and GSTT1 was carried out by multiplex PCR and the GSTP1-Ile105Val polymorphism was determined by PCR/RFLP. The total number of micronuclei present in 1000 binucleated cells and the frequency of micronucleated binucleated lymphocytes in the different individuals were analyzed considering the GSTM1, GSTT1 and GSTP1 genotypes. The results obtained suggest that GSTM1 and GSTT1 deletion polymorphisms do not modify significantly the genotoxic potential of Dox. However, the GSTP1 Ile105Val polymorphism was associated with an increase of micronucleated binucleated cells induced by Dox. Lymphocytes from homozygous individuals for the variant form (Val/Val) presented a significant increase in micronucleated binucleated cells (approximately 1.5-fold; p<0.05) when compared with individuals with at least one wild-type allele. These results suggest a possible role for GSTP1 on the modulation of the genotoxicity induced by Dox, which may be considered in cancer therapy.

Mechanistic insights into the cytotoxicity and genotoxicity induced by glycidamide in human mammary cells

Acrylamide (AA) is a well-known industrial chemical classified as a probable human carcinogen. Benign and malignant tumours at different sites, including the mammary gland, have been reported in rodents exposed to AA. This xenobiotic is also formed in many carbohydrate-rich foods prepared at high temperatures. For this reason, AA is an issue of concern in terms of human cancer risk. The epoxide glycidamide (GA) is thought to be the ultimate genotoxic AA metabolite. Despite extensive experimental and epidemiological data focused on AA-induced breast cancer, there is still lack of information on the deleterious effects induced by GA in mammary cells. The work reported here addresses the characterisation and modulation of cytotoxicity, generation of reactive oxygen species, formation of micronuclei (MN) and quantification of specific GA-DNA adducts in human MCF10A epithelial cells exposed to GA. The results show that GA significantly induces MN, impairs cell proliferation kinetics and decreases cell viability at high concentrations by mechanisms not involving oxidative stress. KU55933, an inhibitor of ataxia telangiectasia mutated kinase, enhanced the cytotoxicity of GA (P < 0.05), supporting a role of this enzyme in regulating the repair of GA-induced DNA lesions. Moreover, even at low GA levels, N7-GA-Gua adducts were generated in a linear dose-response manner in MCF10A cells. These results confirm that human mammary cells are susceptible to GA toxicity and reinforce the need for additional studies to clarify the potential correlation between dietary AA exposure and breast cancer risk in human populations.

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.

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.

Epidemic dispersion of HIV and HCV in a population of co-infected Romanian injecting drug users

Co-infections with HIV and HCV are very frequent among people who inject drugs (PWID). However, very few studies comparatively reconstructed the transmission patterns of both viruses in the same population. We have recruited 117 co-infected PWID during a recent HIV outbreak in Romania. Phylogenetic analyses were performed on HIV and HCV sequences in order to characterize and compare transmission dynamics of the two viruses. Three large HIV clusters (2 subtype F1 and one CRF14_BG) and thirteen smaller HCV transmission networks (genotypes 1a, 1b, 3a, 4a and 4d) were identified. Eighty (65%) patients were both in HIV and HCV transmission chains and 70 of those shared the same HIV and HCV cluster with at least one other patient. Molecular clock analysis indicated that all identified HIV clusters originated around 2006, while the origin of the different HCV clusters ranged between 1980 (genotype 1b) and 2011 (genotypes 3a and 4d). HCV infection preceded HIV infection in 80.3% of cases. Coincidental transmission of HIV and HCV was estimated to be rather low (19.65%) and associated with an outbreak among PWID during detention in the same penitentiary. This study has reconstructed and compared the dispersion of these two viruses in a PWID population.

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.

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.

Abstract 4199: Genetic variation in the in vitro genotoxic response to glycidamide and gene expression of DNA repair genes

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Acrylamide (AA) is a suspected human carcinogen generated in carbohydrate-rich foodstuff upon heating. Glycidamide (GA), formed via epoxidation presumably mediated by cytochrome P450 2E1, is thought to be the active metabolite that plays a central role in AA genotoxicity. The high levels of AA that are orally consumed could be an additional factor for global cancer risk. However, cancer risk estimation in population is still problematic and the mechanisms of DNA repair triggered by GA are still poorly understood. The aim of this work was to evaluate the role of DNA repair genes in the genotoxicity induced by GA in human cells, using complementary methodologies. For this purpose, the effect of GA (250 µM) in cultured human lymphocytes from healthy non-smoker individuals (n=13) was assessed using the sensitive sister-chromatid exchange assay (SCE, 46 h in vitro exposure). GA was found to be genotoxic, with a large inter-individual variation. Therefore we studied the possible influence of individual genetic polymorphisms of key genes codifying for DNA repair enzymes related to the BER (XRCC1, OGG1, PARP1, PARP4, APEX1 and MUTYH), NER (RAD23B, ERCC1, ERCC2, ERCC4, ERCC5, ERCC6 and XPC) and HRR (RAD51, NBS, XRCC3 and XRCC2) pathways. No relevant associations between SCE/cell levels and the individual genetic polymorphisms were found. However, the only individual with the ERCC1 Q504K polymorphism revealed a significant increase in GA-induced levels of SCE/cell (p<0.03). We further studied the effect of the same concentration of GA in peripheral white blood cells from healthy individuals (n=25) using the comet assay (CA, 1 hour in vitro incubation). GA also showed to be genotoxic. The % DNA in Tail for all individuals was analysed considering the abovementioned polymorphisms. In this assay, the MUTYH H335Q was found to influence the repair capacity of GA, being the variant genotype associated with an increased damage in the comet assay (6.1± 1.7 heterozygous versus 11.9 ± 1.8 homozygous variant; p<0.001). Finally, a different strategy focusing on the role of DNA repair enzymes upon GA treatment was used. The non-malignant mammary cell line MCF10A was exposed to GA (10 µM) for 24 h and gene expression was studied using RT2 ProfilerTM PCR Array: Human DNA Damage Signalling Pathway by SABiosciencesTM. Concerning gene expression results, GA at this lower concentration level did not influence the expression of the genes considered when compared to respective non-treated controls. Further studies should be conducted in order to test other exposure periods and use other human cell types. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4199. doi:10.1158/1538-7445.AM2011-4199