Increased levels of chromosomal aberrations and DNA damage in a group of workers exposed to formaldehyde.

Formaldehyde (FA) is a commonly used chemical in anatomy and pathology laboratories as a tissue preservative and fixative. Because of its sensitising properties, irritating effects and cancer implication, FA accounts probably for the most important chemical-exposure hazard concerning this professional group. Evidence for genotoxic effects and carcinogenic properties in humans is insufficient and conflicting, particularly in regard to the ability of inhaled FA to induce toxicity on other cells besides first contact tissues, such as buccal and nasal cells. To evaluate the effects of exposure to FA in human peripheral blood lymphocytes, a group of 84 anatomy pathology laboratory workers exposed occupationally to FA and 87 control subjects were tested for chromosomal aberrations (CAs) and DNA damage (comet assay). The level of exposure to FA in the workplace air was evaluated. The association between genotoxicity biomarkers and polymorphic genes of xenobiotic-metabolising and DNA repair enzymes were also assessed. The estimated mean level of FA exposure was 0.38±0.03 ppm. All cytogenetic endpoints assessed by CAs test and comet assay % tail DNA (%TDNA) were significantly higher in FA-exposed workers compared with controls. Regarding the effect of susceptibility biomarkers, results suggest that polymorphisms in CYP2E1 and GSTP1 metabolic genes, as well as, XRCC1 and PARP1 polymorphic genes involved in DNA repair pathways are associated with higher genetic damage in FA-exposed subjects. Data obtained in this study show a potential health risk situation of anatomy pathology laboratory workers exposed to FA (0.38 ppm). Implementation of security and hygiene measures may be crucial to decrease risk. The obtained information may also provide new important data to be used by health care programs and by governmental agencies responsible for occupational health and safety.

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.

The role of CCNH Val270Ala (rs2230641) and other nucleotide excision repair polymorphisms in individual susceptibility to well-differentiated thyroid cancer.

Well-differentiated thyroid cancer (DTC) is the most common form of thyroid cancer (TC); however, with the exception of radiation exposure, its etiology remains largely unknown. Several single nucleotide polymorphisms (SNPs) have previously been implicated in DTC risk. Nucleotide excision repair (NER) polymorphisms, despite having been associated with cancer risk at other locations, have received little attention in the context of thyroid carcinogenesis. In order to evaluate the role of NER pathway SNPs in DTC susceptibility, we performed a case-control study in 106 Caucasian Portuguese DTC patients and 212 matched controls. rs2230641 (CCNH), rs2972388 (CDK7), rs1805329 (RAD23B), rs3212986 (ERCC1), rs1800067 (ERCC4), rs17655, rs2227869 (ERCC5), rs4253211 and rs2228529 (ERCC6) were genotyped using TaqMan® methodology, while conventional PCR-RFLP was employed for rs2228000 and rs2228001 (XPC). When considering all DTC cases, only rs2230641 (CCNH) was associated with DTC risk; a consistent increase in overall DTC risk was observed for both the heterozygous genotype (OR=1.89, 95% CI=1.14-3.14) and the variant allele carriers (OR=1.79, 95% CI=1.09-2.93). Histological stratification analysis confirmed an identical effect on follicular TC (OR=2.72, 95% CI=1.19-6.22, for heterozygous; OR=2.44, 95% CI=1.07­5.55, for variant allele carriers). Considering papillary TC, the rs2228001 (XPC) variant genotype was associated with increased risk (OR=2.33, 95% CI=1.05-5.16), while a protective effect was observed for rs2227869 (ERCC5) (OR=0.26, 95% CI=0.08­0.90, for heterozygous; OR=0.25, 95% CI=0.07-0.86, for variant allele carriers). No further significant results were observed. Our results suggest that NER polymorphisms such as rs2230641 (CCNH) and, possibly, rs2227869 (ERCC5) and rs2228001 (XPC), may influence DTC susceptibility. However, larger studies are required to confirm these results.

Lack of relevance of HIF-1α polymorphisms in breast cancer in a Portuguese population.

We aimed to study the prevalence of Hypoxia inducible factor-1α (HIF-1α) P582S and A588T polymorphisms in a Portuguese breast cancer population and its effect on the transcriptional activity of HIF-1α in MCF7 breast adenocarcinoma cells. We performed a polymerase chain Reaction--restriction fragment length polymorphism (PCR-RFLP)-based genotyping of a Portuguese breast cancer population for two HIF-1α polymorphisms. Furthermore, by site-directed mutagenesis, we generated a variant form of HIF-1α and compared its transcriptional activity with the wild-type HIF-1α in MCF7 cells. There were no significant differences in genotypic frequencies for P582S and A588T between breast cancer patients and controls, nor between the transcriptional activity of the 582S mutant and the wild-type HIF-1α. In conclusion, there is no association between HIF-1α polymorphisms and incidence of breast cancer in the Portuguese examined population. Furthermore, the P582S mutation does not affect transcriptional activity of HIF-1α in breast cancer cells, contrary to previous findings in other cell lines, suggesting that the impact of this mutation is cell-type specific.

Polymorphisms in base excision repair genes and thyroid cancer risk.

Thyroid cancer (TC) is the most frequent endocrine malignancy, accounting however for only 1-2% of all human cancers, and the best-established risk factor for TC is radiation exposure, particularly during childhood. Since the BER pathway seems to play an important role in the repair of DNA damage induced by IR and other genotoxicants, we carried out a hospital-based case-control study in order to evaluate the potential modifying role of 6 BER polymorphisms on the individual susceptibility to non-familial TC in 109 TC patients receiving iodine-131, and 217 controls matched for age (± 2 years), gender and ethnicity. Our results do not reveal a significant involvement of XRCC1 Arg194Trp and Arg399Gln, OGG1 Ser326Cys, APEX1 Asp148Glu, MUTYH Gln335His and PARP1 Val762Ala polymorphisms on the individual susceptibility towards TC, mostly in agreement with the limited available evidence. By histological stratification analysis, we observed that the association between the presence of heterozygosity in the MUTYH Gln335His polymorphism and TC risk almost reached significance for the papillary subtype of TC. This was the first time that the putative association between this polymorphism and TC susceptibility was evaluated. However, since the sample size was modest, the possibility of a type I error should not be excluded and this result should, therefore, be interpreted with caution. More in depth studies involving larger populations should be pursued in order to further clarify the potential usefulness of the MUTYH Gln335His genotype as a predictive biomarker of susceptibility to TC and the role of the remaining BER polymorphisms on TC susceptibility.

Preparation of organometallic ruthenium-arene-diaminotriazine complexes as binding agents to DNA.

The reactions of two diaminotriazine ligands 2,4-diamino-6-(2-pyridyl)-1,3,5-triazine (2-pydaT) and 6-phenyl-2,4-diamino-1,3,5-triazine (PhdaT) with ruthenium-arene precursors led to a new family of ruthenium(II) compounds that were spectroscopically characterized. Four of the complexes were cationic, with the general formula [(η(6)-arene)Ru(κ(2)-N,N-2-pydaT)Cl]X (X=BF(4), TsO; arene=p-cymene: 1·BF(4), 1·TsO; arene=benzene: 2·BF(4), 2·TsO). The neutral cyclometalated complex [(η(6)-p-cymene)Ru(κ(2)-C,N-PhdaT*)Cl] (3) was also isolated. The structures of complexes 2·BF(4) and 3·H(2)O were determined by X-ray diffraction. Complex 1·BF(4) underwent a partial reversible-aquation process in water. UV/Vis and NMR spectroscopic measurements showed that the reaction was hindered by the addition of NaCl and was pH-controlled in acidic solution. At pH 7.0 (sodium cacodylate) Ru-Cl complex 1·BF(4) was the only species present in solution, even at low ionic strength. However, in alkaline medium (KOH), complex 1·BF(4) underwent basic hydrolysis to afford a Ru-OH complex (5). Fluorimetric studies revealed that the interaction of complex 1·BF(4) with DNA was not straightforward; instead, its main features were closely linked to ionic strength and to the [DNA]/complex ratio. The bifunctional complex 1·BF(4) was capable of interacting concurrently through both its p-cymene and 2-pydaT groups. Cytotoxicity and genotoxicity studies showed that, contrary to the expected behavior, the complex species was biologically inactive; the formation of a Ru-OH complex could be responsible for such behavior.

SNPs/pools: a methodology for the identification of relevant SNPs in breast cancer epidemiology.

The identification of allelic variants of human genes is of great importance when assessing genetic susceptibility. The emerging role of genetic polymorphisms in association studies has created the need for high throughput genotyping methodologies that allow a more rapid identification of relevant polymorphisms related to individual cancer risk enabling the extension to large-scale association studies. DNA pooling methodology may be of great importance considering the cost, time and labor that are involved in large-scale genotyping analysis carried out on individual samples. Alternatively, when using pooled samples which are made up of DNA from many individuals treated as a single sample, these factors are decrease drastically. In this way, the use of DNA pooling methodology, as a pre-selection tool, allows the identification of the most relevant polymorphisms to be studied, facilitating the estimation of the allelic frequency of each SNP in different populations. The present study initially aimed to validate the DNA pooling approach for the identification of genetic polymorphisms potentially associated with individual cancer risk generating pools with known allelic frequencies and using studies ongoing in the laboratory. Finally, our main aim was to test the accuracy of the pooled DNA analysis comparing the results of the allelic frequencies determined using pooled samples with the allelic frequency previously estimated by individual genotyping and previously published. In order to analyze the possibility of establishing differences between populations, we created DNA pools using a Portuguese control population, a breast cancer population and a Xavante Indian population characterized by a total absence of breast cancer cases. The pools were firstly created with known allelic frequencies, previously determined by individual genotyping, and, latter, randomly incremented in sample size to 200 patients and controls. Our results showed that the DNA pooling approach was a useful tool for the analysis of allelic distribution in the different populations studied. Ιn conclusion, our results showed that this methodology can be applied as an effective approach to identify SNPs of importance in genetic susceptibility to disease which may be used in association studies conducted subsequently by individual genotyping.

ACMA (9-amino-6-chloro-2-methoxy acridine) forms three complexes in the presence of DNA.

The interaction of ACMA (9-amino-6-chloro-2-methoxy acridine) (D) with DNA (P) has been studied by absorbance, fluorescence, circular dichroism, spectrophotometry, viscometry and unwinding electrophoresis. A T-jump kinetic study has also been undertaken. The experimental data show that, totally unlike other drugs, ACMA is able to form with DNA three complexes (PD(I), PD(II), PD(III)) that differ from each other by the characteristics and extent of the binding process. The main features of PD(I) fulfil the classical intercalation pattern and the formation/dissociation kinetics have been elucidated by T-jump techniques. PD(II) and PD(III) are also intercalated species but, in addition to the dye units lodged between base pairs, they also bear dye molecules externally bound, more in PD(III) relative to PD(II). A reaction mechanism is put forward here. Comparison between absorbance, fluorescence and kinetic experiments has enabled us to determine the binding constants of the three complexes, namely (6.5 ± 1.1) × 10(4) M(-1) (PD(I)), (5.5 ± 1.5) × 10(4) M(-1) (PD(II)) and (5.7 ± 0.03) × 10(4) M(-1) (PD(III)). The Comet assay reveals that the ACMA binding to DNA brings about genotoxic properties. The mutagenic potential studied by the Ames test reveals that ACMA can produce frameshift and transversion/transition mutations. ACMA also is able to produce base-pair substitution in the presence of S9 mix. Moreover, the MTT assays have revealed cytotoxicity. The biological effects observed have been rationalized in light of these features.

8,15-epoxylabdane and norlabdane diterpenoids from Eragrostis viscosa.

Four labdanes with a 8alpha,15-epoxy ring (8alpha,15-epoxylabdan-16beta-oic acid; 8alpha,15-epoxy-16-norlabdan-13-one; 8alpha,15-epoxy-16-norlabdane; and 16-acetoxy-8alpha,15-epoxylabdane) and the known compound ambreinolide were isolated from the hexane extract of the aerial parts of the grass Eragrostis viscosa. The structures of all compounds were established based on spectroscopic data and the X-ray analysis of 8alpha,15-epoxy-16-norlabdan-13-one. The hexane extract presented moderate activity against the snail Biomphalaria glabrata. 8alpha,15-Epoxylabdan-16beta-oic acid showed no mutagenic activity for doses up to 1000 microg/plate and no significant clastogenic activity for doses up to 100 microg/ml.

Association of common variants in mismatch repair genes and breast cancer susceptibility: a multigene study.

BACKGROUND: MMR is responsible for the repair of base-base mismatches and insertion/deletion loops. Besides this, MMR is also associated with an anti-recombination function, suppressing homologous recombination. Losses of heterozygosity and/or microsatellite instability have been detected in a large number of skin samples from breast cancer patients, suggesting a potential role of MMR in breast cancer susceptibility. METHODS: We carried out a hospital-based case-control study in a Caucasian Portuguese population (287 cases and 547 controls) to estimate the susceptibility to non-familial breast cancer associated with some polymorphisms in mismatch repair genes (MSH3, MSH4, MSH6, MLH1, MLH3, PMS1 and MUTYH). RESULTS: Using unconditional logistic regression we found that MLH3 (L844P, G>A) polymorphism GA (Leu/Pro) and AA (Pro/Pro) genotypes were associated with a decreased risk: OR = 0.65 (0.45-0.95) (p = 0.03) and OR = 0.62 (0.41-0.94) (p = 0.03), respectively.Analysis of two-way SNP interaction effects on breast cancer revealed two potential associations to breast cancer susceptibility: MSH3 Ala1045Thr/MSH6 Gly39Glu - AA/TC [OR = 0.43 (0.21-0.83), p = 0.01] associated with a decreased risk; and MSH4 Ala97Thr/MLH3 Leu844Pro - AG/AA [OR = 2.35 (1.23-4.49), p = 0.01], GG/AA [OR = 2.11 (1.12-3,98), p = 0.02], and GG/AG [adjusted OR = 1.88 (1.12-3.15), p = 0.02] all associated with an increased risk for breast cancer. CONCLUSION: It is possible that some of these common variants in MMR genes contribute significantly to breast cancer susceptibility. However, further studies with a large sample size will be needed to support our results.