The Brazilian founder mutation TP53 p.R337H is uncommon in Portuguese women diagnosed with breast cancer.

Since the first studies reporting the TP53 p.R337H mutation as founder mutation in Southern and Southeastern Brazil, there has been controversy on its origin. Preliminary analysis of a small subset of Brazilian mutation carriers revealed that the haplotype incided on a Caucasian background. The vast majority of carriers identified today reside in Brazil or, if identified in other countries, are Brazilian immigrants. To our knowledge, the only two exceptions of carriers without a recognizable link with Brazil are two European families, from Portugal and Germany. Haplotype analysis in the Portuguese family revealed the same haplotype identified in Brazilian individuals, but in the German family, a distinct haplotype was found. Knowing that a significant proportion of women with breast cancer (BC) in Southern Brazil are p.R337H carriers, we analyzed p.R337H in a Portuguese cohort of women diagnosed with this disease. Median age at diagnosis among the first 573 patients tested was 60 years and 100 (17.4%) patients had been diagnosed at or under the age of 45 years. Mutation screening failed to identify the mutation in the 573 patients tested. These results are in contrast with the mutation frequency observed in a study including 815 BC-affected women from Brazil, in which carrier frequencies of 12.1 and 5.1% in pre- and postmenopausal women were observed, respectively. These findings suggest that the Brazilian founder mutation p.R337H, the most frequent germline TP53 mutation reported to date, is not a common germline alteration in Portuguese women diagnosed with BC.

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.

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) genes, either alone or in combination.

Genotoxic damage in hospital workers exposed to ionizing radiation and metabolic gene polymorphisms.

Of all workers exposed globally to synthetic sources of radiation, medical personnel represent the largest group, but receive relatively low doses. Accidental or therapeutic acute radiation exposure of humans was observed to induce various forms of cytogenetic damage, including the possibility of increasing the incidence of micronuclei (MN) and chromosomal aberrations (CA). The aim of this study was to assess occupationally induced chromosomal damage in a large population of hospital workers exposed to low doses of ionizing radiation (IR). The cytokinesis-block MN and comet assays were used to examine peripheral blood lymphocytes (PBL) of 31 exposed workers to IR and 33 control subjects corresponding in gender, age, and smoking. Glutathione S-transferases (GSTM1, GSTT1, and GSTP1) are postulated to be involved in the detoxification of endogenous and exogenous genotoxicants. The association between these biomarkers and polymorphic genes of xenobiotic metabolizing enzymes was thus also assessed. MN frequency was significantly higher in the exposed subjects compared controls. Comet assay results showed a significant increase of tail length in workers exposed to IR. Data obtained suggest that GSTM1, GSTT1, and GSTP1 polymorphism do not modify significantly the genotoxic potential of IR. Therefore, the exposed medical personnel need to carefully apply radiation protection procedures and minimize, as low as possible, IR exposure to avoid possible genotoxic effects.

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.

The role of common variants of non-homologous end-joining repair genes XRCC4, LIG4 and Ku80 in thyroid cancer risk.

Variations, such as single nucleotide polymorphisms (SNPs) in DNA damage repair genes have been pointed out as possible factors to cancer predisposition. Ionizing radiation (IR) induces DNA double strand breaks (DSBs) and is the main recognized risk factor for thyroid cancer. However, most of the patients do not show chronic contact with IR and the other factors have non-concordant data. Thus, thyroid cancer could be due to gene variations in association with certain exogenous factors. One of the pathways that repair DSBs is DNA non-homologous end-joining (NHEJ) that comprises several polymorphic genes. We intend to study the role of polymorphic variants in XRCC4, LIG4 and Ku80 genes, since there is scarcity of data on the role of these genes in thyroid cancer susceptibility. We carried out a hospital-based case-control study in a Caucasian Portuguese population (109 patients and 217 controls) to estimate the potential role of the XRCC4 (N298S and T134I), LIG4 (T9I) and Ku80 (Ex21-238Gright curved arrow A, Ex21+338Tright curved arrow C, Ex21-352Cright curved arrow A, Ex21+466Aright curved arrow G) polymorphisms in the individual susceptibility for this disease. The results here reported do not associate these polymorphisms with susceptibility for non-familial thyroid cancer. However, when the data were analyzed according to the type of tumour, significant results for Ku80 Ex21-238Gright curved arrow A and Ex21+466Aright curved arrow G were found for papillary tumours (adjusted OR = 2.281; 95% CI =; 1.063-4.894; P=0.034). Taken together these results suggest that some of these variants in NHEJ genes can contribute to thyroid cancer susceptibility. However, further studies with a larger sample size will be needed to support our results.

Breast cancer risk and common single nucleotide polymorphisms in homologous recombination DNA repair pathway genes XRCC2, XRCC3, NBS1 and RAD51.

The possible role for DNA repair deficiencies in cancer development, namely in breast cancer has been the subject of increasing interest since it has been reported that breast cancer patients might be deficient in the repair of DNA damage. Exposure to ionizing radiation has been pointed out as a risk factor for breast cancer, and the type of DNA lesions induced by this carcinogen can be repaired by homologous recombination DNA repair (HRR) pathway. To evaluate the potential modifying role of some single nucleotide polymorphisms (SNP) in HRR involved genes on the individual susceptibility to breast cancer we carried out a hospital based case-control study in a Caucasian Portuguese population (289 histological confirmed breast cancer patients and 548 control individuals). We genotyped 4 SNPs in 4 different HRR pathway genes, XRCC2 (Ex3+442G>A, R188H, rs3218536), XRCC3 (Ex8-5C>T, T241M, rs861539), NBS1 (Ex5-32C>G, E185Q, rs1805794) and RAD51 5'UTR (Ex1-59G>T, rs1801321), tagging 41 SNPs in these genes. The frequency of the different polymorphisms in the Portuguese control population is similar to the ones reported for other Caucasian populations, and the deviation of the Hardy-Weinberg equilibrium was only observed for the XRCC2 (Ex3+442G>A, R188H, rs3218536) polymorphism in the control population. The results obtained, after logistic regression analysis, did not reveal a major role of these polymorphisms on breast cancer susceptibility. However, when the population was stratified according to breast feeding (women that breast fed and women that never breast fed) it is observed, in women that never breast fed, that the heterozygous individuals for the XRCC2 (Ex3+442G>A, R188H, rs3218536) polymorphism have a decreased risk for breast cancer [adjusted OR=0.45; 95% CI=0.22-0.92] (P=0.03). Additionally, after stratification according to menopausal status, our results suggest that post-menopausal women carrying at least one variant allele for the XRCC3 (Ex8-5C>T, T241M, rs861539) polymorphism have a lower risk for breast cancer [adjusted OR=0.67; 95% CI, 0.47-0.94] (P=0.03). Most of the studies suggest that breastfeeding may be responsible for 2/3 of the estimate reduction of breast cancer. The longer the duration of breastfeeding the lower the potential risk associated with breast cancer. Therefore, in our study the potential protective role of the variant allele of XRCC2 (Ex3+442G>A, R188H, rs3218536), in never breast fed women, might be related with a more efficient DNA repair activity.

Association of polymorphisms in genes of the homologous recombination DNA repair pathway and thyroid cancer risk.

BACKGROUND: Ionizing radiation exposure has been pointed out as a risk factor for thyroid cancer. The double-strand breaks induced by this carcinogen are usually repaired by homologous recombination repair pathway, a pathway that includes several polymorphic genes. Since there is a scarcity of data about the involvement of these gene polymorphisms in thyroid cancer susceptibility, we carried out a case-control study in a Caucasian Portuguese population. METHODS: We genotyped 109 patients and 217 controls for the XRCC3 T241M, XRCC2 R188H, NBS1 E185Q, and RAD51 Ex1-59G>T polymorphisms to evaluate their potential main effects on risk for this pathology. RESULTS: The results obtained showed that for the RAD51 Ex1-59G>T polymorphism, the homozigosity for the variant allele was associated with an almost significant increase of the odds ratio (OR) (adjusted OR = 1.9; confidence interval 95%: 1.0-3.5; p = 0.057). Additionaly, when the XRCC3 T241M data were analyzed concerning the presence of at least one wild-type allele, we observed that individuals homozygous for the variant allele had a higher risk for thyroid cancer (adjusted OR = 2.0; confidence interval 95%: 1.1-3.6; p = 0.026). When the data were analyzed according to the number of RAD51 Ex1-59G>T and XRCC3 T241M variant alleles, the coexistence of three or more variant alleles in either gene was associated to a significant higher risk (three variant alleles: adjusted OR = 2.9, p = 0.036; four variant alleles: adjusted OR = 8.0, p = 0.006). CONCLUSIONS: Since XRCC3 is involved in the assembly and stabilization of RAD51 protein multimers at double-strand break sites, we cannot exclude that the interaction of both polymorphisms can lead to a decreased DNA repair capacity and consequently increased risk for thyroid cancer.

The role of GSTA2 polymorphisms and haplotypes in breast cancer susceptibility: a case-control study in the Portuguese population.

Glutathione-S-transferases (GSTs) are a super-family of phase II metabolizing enzymes that catalyse the detoxification of a large range of endogenous and exogenous toxic compounds, playing an important role in protecting cells against damage, through glutathione conjugation with electrophilic substances. Polymorphic variation in these enzymes that affect its activity seems to be related to individual susceptibility to various human diseases, including cancer. Of the GST super-family, the alpha class GSTs have commonly been described as one of the most versatile class, since it is responsible for detoxification of compounds such as bilirubin, bile acids and penicillin, thyroid and steroid hormones, allowing its solubilization and storage in the liver. Among the alpha class, GSTA1 and GSTA2 isoforms are the most widely expressed in human tissues. Additionally, these enzymes can catalyse conjugation of the nitrogen mustard group of alkylating anticancer drugs, some heterocyclic amines and alpha,beta-unsaturated aldehydes. Since some risk factors for increased breast cancer risk could be related to high production of reactive oxygen species during the metabolism of estrogens by catechol estrogens, or to the exposure to genotoxic compounds, and some of these toxic compounds are usually metabolized by GSTA2, we carried out a hospital based case-control study in a Caucasian Portuguese population (291 breast cancer patients without familiar history of breast cancer and 547 controls matched for age, sex and ethnicity) in order to evaluate the potential modifying role of three non-synonymous polymorphisms in the GSTA2 gene (P110S Ex 5+56C>T;, rs2234951; S112T Ex5+63G>C, rs2180314 and E210A Ex7+83A>C, rs6577) on the individual susceptibility to breast cancer. Our data show that the studied polymorphisms are in strong linkage disequilibrium, but no association was observed between individual GSTA2 polymorphisms and haplotypes and individual susceptibility to breast cancer.

Cytotoxicity and chromosomal aberrations induced by acrylamide in V79 cells: role of glutathione modulators.

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.

Genetic effects and biotoxicity monitoring of occupational styrene exposure.

Styrene is a commercially important chemical widely used in the manufacture of synthetic rubber, resins, polyesters, and plastics. The highest levels of human exposure to styrene occurs in occupational settings, especially during the production of reinforced plastic products, which involve manual lay-up or spray-up operations. In these settings, absorption of styrene occurs mainly through inhalation and, to a minor extent, via skin contact. A variety of biological markers (biomarkers) have been developed for genotoxic agents. Three types of biomarkers are identified: biomarkers of exposure, of effect and of susceptibility. Biomarkers of exposure measure the chemical itself or its metabolites in body fluids. Biomarkers of effect measure indicators of damage by the exposure. Biomarkers of effect are generally pre-clinical indicators of abnormalities and the most frequently used in genotoxicity assessment are sister chromatid exchanges (SCEs), chromosomal aberrations (CAs) and micronuclei (MN). More recently, the use of the single cell gel electrophoresis assay (SCGE), or comet assay has been proposed as a useful biomarker for early effects. The third type is a biomarker of susceptibility, which indicates that the individual is vulnerable to the effect of a xenobiotic or to the effects of a group of such compounds. This type of biomarkers are related with individual genetic polymorphisms that could lead to different capacities to activate, detoxify or repair DNA lesions arising from exposure to chemical carcinogens. Styrene metabolism involves cytochrome P450 enzymes (CYP)-mediated that by oxidation convert styrene to its reactive metabolite styrene-7,8-oxide (SO) which is capable of binding covalently with macromolecules and is considered to be directly responsible for the genotoxic effects of styrene. SO, is mainly hydrolysed to styrene glycol by the microsomal epoxide hydrolase (mEH), and subsequently oxidized by alcohol and aldehyde dehydrogenases to the main urinary metabolites, mandelic acid (MA) and phenylglyoxylic acid (PGA) (major pathway). MA and PGA represent more than 95% of the urinary metabolites of styrene. Further transformation of MA and PGA, via transamination of alpha-keto- and alpha-hydroxyacids into the corresponding amino acid, leads to phenylglycine. Evidence of the carcinogenicity of styrene has been reported in studies with mice. Epidemiologic evidence does not suggest a causal association between styrene and any forms of cancer in humans. However, the possibility of a small elevation of risk for one or more cancers cannot be ruled out. The International Agency for Research on Cancer (IARC) has designated styrene as possibly carcinogenic to humans (group 2B). Concern about the potential carcinogenicity of styrene stems largely from the ability of its metabolite, SO to bind covalently to DNA and to its activity in a variety of genotoxicity test systems. SO has been classified by IARC in group 2A, probably carcinogenic to humans. Styrene exposure has been reported to cause an increase in DNA and haemoglobin adducts and in the frequency of CAs; there is less evidence for an association between styrene exposure and the frequency of SCEs. This article thoroughly reviews all available published data on the genetic effects of styrene and the biotoxicity markers of exposure monitoring.

Cytogenetic damage induced by acrylamide and glycidamide in mammalian cells: correlation with specific glycidamide-DNA adducts.

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.