Mutagenicity of a novel 2-phenylbenzotriazole (non-chlorinated 2-phenylbenzotriazole-9) in mice.

Dinitrophenylazo dyes can form 2-phenylbenzotriazoles (PBTAs) in the textile dyeing process upon the addition of chemical reducing agents. Some dinitrophenylazo dyes, as well as their respective reduced (non-chlorinated) and chlorinated PBTAs, are now found in rivers owing to wastewater from textile plants. This study aimed to investigate the genotoxicity of a new PBTA derived from C.I. Disperse Violet 93 azo dye, namely non-Cl PBTA-9. Primary DNA damage in the blood, liver, and colon cells, micronucleated cells in the bone marrow, and gene expression (NAT2, CYP1A1, TRP53, and CDKN1A) in liver cells were observed in mice, at acute oral exposure (gavage) doses of 5, 50, and 500 µg/kg body weight (b.w.). The non-chlorinated PBTA-9 caused DNA damage in the blood and liver (at 500 µg/kg b.w.) and in colon cells (at 5, 50, and 500 µg/kg), and increased the frequency of micronucleated cells in the bone marrow (at 5 and 50 µg/kg). No histological alterations or gene expression changes were observed. In conclusion, in vivo exposure to non-chlorinated PBTA-9 induced genetic damage in various rodent tissues, corroborating results previously obtained from the Ames test. Because this compound has been detected in rivers, exposure to humans and biota is a major concern.

Effect of doxorubicin on cardiac lipid metabolism-related transcriptome and the protective activity of Alda-1.

The use of doxorubicin (DOX) as an antineoplastic drug is compromised by its cardiotoxicity risk. Although several mechanisms have been proposed for DOX-induced cardiac dysfunction, there is still increased interest in assessing its effects. Likewise, it is important to find protocols that can prevent or minimize the side effects of DOX without hindering its antitumor activity. Thus, this study was designed to investigate the molecular mechanisms underlying DOX cardiotoxicity, with a special focus on cardiac energy metabolism and the ability of Alda-1 (ALDH2 agonist) to prevent DOX-induced cardiac alterations. We explored the effects of DOX on the histological morphology of the myocardium, on lipid profile, and on the expression of genes related to fatty acid metabolism, in the presence and absence of Alda-1 (8 mg/kg body weight; b.wt.). Two DOX treatment protocols were used: a single dose of DOX (4 mg/kg b.wt.); four doses of DOX (4 mg/kg b.wt.), one dose/week, for 4 weeks. Treatment with DOX caused a progressive injury in the cardiac tissue and an increase in the blood total cholesterol, high-density lipoproteins, very low-density lipoproteins and triglyceride, as well as an up-regulation of FABP4 (DOX and DOX + Alda-1 groups) and Slc27a2 (in DOX-treated animals). Alda-1 administration promoted reduction in the severity of the histopathological injuries (after single dose of DOX) and Slc27a2 overexpression was restored. In conclusion, the study revealed novel insights regarding the development of DOX-mediated cardiomyopathy, indicating a relationship between DOX exposure and FABP4 and Slc27a2 overexpression, and confirmed the cardioprotective effect of Alda-1.

The HUman MicroNucleus project on eXfoLiated buccal cells (HUMN(XL)): the role of life-style, host factors, occupational exposures, health status, and assay protocol.

The human buccal micronucleus cytome assay (BMCyt) is one of the most widely used techniques to measure genetic damage in human population studies. Reducing protocol variability, assessing the role of confounders, and estimating a range of reference values are research priorities that will be addressed by the HUMN(XL) collaborative study. The HUMN(XL) project evaluates the impact of host factors, occupation, life-style, disease status, and protocol features on the occurrence of MN in exfoliated buccal cells. In addition, the study will provide a range of reference values for all cytome endpoints. A database of 5424 subjects with buccal MN values obtained from 30 laboratories worldwide was compiled and analyzed to investigate the influence of several conditions affecting MN frequency. Random effects models were mostly used to investigate MN predictors. The estimated spontaneous MN frequency was 0.74‰ (95% CI 0.52-1.05). Only staining among technical features influenced MN frequency, with an abnormal increase for non-DNA-specific stains. No effect of gender was evident, while the trend for age was highly significant (p<0.001). Most occupational exposures and a diagnosis of cancer significantly increased MN and other endpoints frequencies. MN frequency increased in heavy smoking (≥40cig/day, FR=1.37; 95% CI 1.03-.82) and decreased with daily fruit consumption (FR=0.68; 95% CI 0.50-0.91). The results of the HUMN(XL) project identified priorities for validation studies, increased the basic knowledge of the assay, and contributed to the creation of a laboratory network which in perspective may allow the evaluation of disease risk associated with MN frequency.

No mutations found in exon 2 of gene p16CDKN2A during rat tongue carcinogenesis induced by 4-nitroquinoline-1-oxide.

The medium-term tongue carcinogenesis assay is a useful model for studying oral squamous cell carcinomas phase by phase. The present study aimed to investigate mutations in exon 2 of gene p16CDKN2A during rat tongue carcinogenesis induced by 4-nitroquinoline 1-oxide (4NQO) using direct DNA-sequencing method. A total of 30 male Wistar rats were treated with 4-nitroquinoline 1-oxide (4NQO) in drinking water for 4, 12, and 20 weeks at 50 ppm dose. Ten animals were used as negative control. No histopathological changes in tongue epithelia were observed among controls or in the group treated for 4 weeks with 4NQO. Following 12-week treatment, hyperplasia and epithelial dysplasia were found in mild and moderate forms. At 20 weeks, the tongue presented moderate and/or severe oral dysplasia and squamous cell carcinoma, with squamous cell carcinoma in the majority of animals. No mutations were found in any experimental period evaluated that corresponded to normal oral mucosa, hyperplasia, dysplasia and squamous cell carcinomas. Taken together, our results suggest that p16CDKN2A mutations in exon 2 are not involved in the multistep tongue carcinogenesis of Wistar rats induced by 4NQO.

Effect of lycopene on doxorubicin-induced cardiotoxicity: an echocardiographic, histological and morphometrical assessment.

Doxorubicin is an excellent chemotherapeutic agent utilized for several types of cancer but the irreversible doxorubicin-induced cardiac damage is the major limitation for its use. Oxidative stress seems to be associated with some phase of the toxicity mechanism process. To determine if lycopene protects against doxorubicin-induced cardiotoxicity, male Wistar rats were randomly assigned either to control, lycopene, doxorubicin or doxorubicin + lycopene groups. They received corn oil (control, doxorubicin) or lycopene (5 mg/kg body weight a day) (lycopene, doxorubicin + lycopene) by gavage for a 7-week period. They also received saline (control, lycopene) or doxorubicin (4 mg/kg) (doxorubicin, doxorubin + lycopene) intraperitoneally by week 3, 4, 5 and 6. Animals underwent echocardiogram and were killed for tissue analyses by week 7. Mean lycopene levels (nmol/kg) in liver were higher in the doxorubicin + lycopene group (5822.59) than in the lycopene group (2496.73), but no differences in lycopene were found in heart or plasma of these two groups. Lycopene did not prevent left ventricular systolic dysfunction induced by doxorubicin. However, morphologic examination revealed that doxorubicin-induced myocyte damage was significantly suppressed in rats treated with lycopene. Doxorubicin treatment was followed by increase of myocardium interstitial collagen volume fraction. Our results show that: (i) doxorubicin-induced cardiotoxicity was confirmed by echocardiogram and morphological evaluations; (ii) lycopene absorption was confirmed by its levels in heart, liver and plasma; (iii) lycopene supplementation provided myocyte protection without preventing interstitial collagen accumulation increase; (iv) doxorubicin-induced cardiac dysfunction was not prevented by lycopene supplementation; and (v) lycopene depletion was not observed in plasma and tissues from animals treated with doxorubicin.

Influence of endogenous and synthetic female sex hormones on human blood cells in vitro studied with comet assay.

The comet assay has been conducted with numerous cell lines to assess in vitro genotoxicity. In order to use the comet assay as part of an in vitro test for evaluating genotoxicity, however, there are cell-specific factors that need to be better understood. In this present study we have evaluated some factors that may impact upon the DNA damage detected in whole blood (WB) cells and lymphocytes (ILs). Experiments were conducted comparing responses of both cells, and investigating the effects of the female hormonal cycle, and oral contraceptive (OC) use on DNA damage detection in the in vitro comet assay, at three sampling time. No significant differences were detected in the basal levels of DNA damage detected in ILs and WB cells from women OC users and non-users and from men. Basal DNA damage in ILs was unaffected by gender and stage of the menstrual cycle or the stage of the treatment schedule. Our results also indicated that the H2O2 induces DNA damage in human lymphocytes independently of gender, low-dose OC use and hormonal fluctuation. However, data showed that in 3rd sampling of menstrual cycle, lymphocytes were more resistant to H2O2-induced DNA damage than those from OC users and men.

Genomic instability in non-neoplastic oral mucosa cells can predict risk during 4-nitroquinoline 1-oxide-induced rat tongue carcinogenesis.

4-Nitroquinoline 1-oxide (4NQO)-induced rat tongue carcinogenesis is a useful model for studying oral squamous cell carcinoma. The aim of this study was to investigate the level of DNA damage induced by 4NQO in oral mucosa cells by the single cell gel (comet) assay. Male Wistar rats were distributed into three groups of 10 animals each and treated with 50 ppm 4NQO solution by drinking water for 4, 12 or 20 weeks. Ten animals were used as negative control. Statistically significant increase of DNA damage was observed in non-neoplastic oral cells at four weeks of 4NQO administration when compared with control (P < 0.05). The level of DNA damage was directly associated with the severity of histological changes. The results suggest that histologically normal tissue is able to harbor genetically unstable cells contributing to the initiation of oral carcinogenesis. Genomic instability appears to be associated with the risk and progression of oral cancer.

Anesthesia of fish with benzocaine does not interfere with comet assay results.

Fish blood erythrocytes are frequently used as sentinels in biomonitoring studies. Usually, fish blood is collected by painful cardiac or caudal vein punctures. Previous anesthesia could decrease animal suffering but it is not known at present whether anesthesia can cause confounding effects. Therefore, using the alkaline single cell gel (SCG)/comet assay with blood erythrocytes of the cichlid fish Nile tilapia, we tested for a possible modulation of induced DNA damage (methyl methanesulfonate; MMS) by the anesthetic benzocaine administered by bath exposure (80mg/l for approximately 10min). Furthermore, benzocaine (80-600mg/l) was tested for its genotoxic potential on fish erythrocytes in vitro and for potential interactions with two known genotoxins (MMS and hydrogen peroxide). Our results did neither indicate a significant increase in the amount of DNA damage (even after a 48h follow-up), nor indicated interactions with MMS-induced DNA damage when fish were exposed to benzocaine in vivo. There was also no increase in DNA damage after in vitro exposure of fish erythrocytes to benzocaine. Clear concentration-related effects were observed for the two genotoxins in vitro, which were not significantly altered by the presence of benzocaine. These results suggest that anesthesia of fish does not confound comet assay results and the use of blood samples from anesthetized fish can be recommended with regard to animal welfare.

A deteccao de substancias cancerígenas em bioensaios alternativos de média-duracao / The detection of chemical carcinogen in alternative medium-term biossays

Atualmente, o método standard para identificacao de substancias cancerígenas é teste de longa-duracao, con roedores. No Brasil nao há condicoes para a sua execucao, devido á inexistencia de especialistas e de infra-estructura adecuada. En consequencia, o país é dependente dos conhecimentos gerados no exterior sobre o risco que determinados compostos químicos impoem ás populacoes e ao meio ambiente. Além disto, as desvantagens operacionais de teste delonga-duiracao levaram ao desenvolvimento de bioensaios alternativos, mais convenientes. O Instituto Brasileiro de Meio Ambiente(IBAMA) adotou oficialmente o bioensaio de média-duracao para múltiplos orgaos, no rato, como fonte de evidencia do potencial cancerígeno de agentes químicos. Con isto, procura atender a demanda de substancias que precisam ser testadas no país, antes de colocadas no mercado e, ao mesmo tempo, aum,entar o know-how nacional em carcinogénese química e avaliacao de risco cancerígeno