Chemopreventive effect and lack of genotoxicity and mutagenicity of the exopolysaccharide botryosphaeran on human lymphocytes.

Carbohydrate biopolymers of fungal-origin are an important natural resource in the search for new bioagents with therapeutic and nutraceutical potential. In this study the mutagenic, genotoxic, antigenotoxic and antioxidant properties of the fungal exopolysaccharide botryosphaeran, a (1→3)(1→6)-ß-D-glucan, from Botryosphaeria rhodina MAMB-05, was evaluated. The mutagenicity was assessed at five concentrations in Salmonella typhimurium by the Ames test. Normal and tumor (Jurkat cells) human T lymphocyte cultures were used to evaluate the genotoxicity and antigenotoxicity (Comet assay) of botryosphaeran alone and in combination with the mutagen methyl methanesulfonate (MMS). The ability of botryosphaeran to reduce the production of reactive oxygen and nitrogen species (RONS) generated by hydrogen peroxide was assessed using the CM-H2DCFDA probe in lymphocyte cultures under different treatment times. None of the evaluated botryosphaeran concentrations were mutagenic in bacteria, nor induced genotoxicity in normal and tumor lymphocytes. Botryosphaeran protected lymphocyte DNA against damage caused by MMS under simultaneous treatment and post-treatment conditions. However, botryosphaeran was not able to reduce the RONS generated by H2O2. Besides the absence of genotoxicity, botryosphaeran exerted a protective effect on human lymphocytes against genotoxic damage caused by MMS. These results are important in the validation of botryosphaeran as a therapeutic agent targeting health promotion.

Cytotoxic and genotoxic effects of high concentrations of the immunosuppressive drugs cyclosporine and tacrolimus in MRC-5 cells.

Immunosuppressive drugs are used to suppress immune system activity in transplant patients and reduce the risk of organ rejection. The present study evaluated the potential cytotoxic, genotoxic and mutagenic of the immunosuppressive drugs cyclosporine (CsA) and tacrolimus (FK-506) on normal human fibroblasts (MRC-5 cells). Based on plasma concentrations of the immunosuppressive drugs, which were obtained from the records of kidney transplant patients at the Kidney Institute of Londrina, Brazil, 11 concentrations of each immunosuppressive were chosen to evaluate cell viability using the MTT assay. From these results, CsA and FK-506 concentrations of 135, 300, 675, and 1520 ng/ml and 8, 16, 24, and 32 ng/ml, respectively, were evaluated using (i) the comet assay, (ii) the nuclear division index (NDI), (iii) the micronucleus test (CBMN) and (iv) cell proliferation curves generated by quantifying cell numbers and protein levels. In this study, 1520 to 3420 ng/ml CsA decreased cell viability after 48 h of exposure. Genotoxic effects were observed only with a concentration of 1520 ng/ml after 3h of exposure and with concentrations of 675 and 1520 ng/ml after 24h of exposure. Mutagenic effects were observed only for the concentration of 1520 ng/ml. FK-506 decreased cell viability after 72 h of exposure for concentrations up to 20 ng/ml; genotoxic effects were observed with concentrations up to 8 ng/ml for both treatment times (3 and 24h) and mutagenic effects were observed with concentrations of 24 and 32 ng/ml after 24h of treatment. The cell proliferation curves demonstrated the absence of cytostatic effects of these drugs, and these data were confirmed by the NDI analysis. Our results suggest that concentrations lower than 300 ng/ml of CsA and 16 ng/ml of FK-506 are safe for use, as they did not induce genotoxic and mutagenic damage or affect MRC-5 cell viability and proliferation.

Base excision repair genes XRCC1 and APEX1 and the risk for prostate cancer.

Prostate cancer is the second cause of cancer death in Brazilian men. One of the relevant phenomena to the inherited susceptibility is the presence of allelic variants in genes involved with the DNA repair pathway. The aim of this study was to analyze the frequencies of prevalent, heterozygous and rare genotypes of the base excision repair genes APEX1 and XRCC1 in a case-control study and relate the genotypes with tumoral aggressiveness. DNA from peripheral blood of 172 patients and 172 controls were analyzed by RFLP-PCR method. The polymorphisms were also evaluated in relation to clinical and pathological parameters. The OR (Odds Ratio) and confidence interval (CI = 95%) were used in the association study and the Chi-square and ANOVA tests for the evaluation of histopathological parameters. The rare genotypes frequencies of the gene APEX1 increased the risk for the development of prostate cancer (OR = 1.68 95% CI 1.10-2.58). No association was found for the gene XRCC1 (OR = 0.82 95% CI 0.53-1.27). The combined analysis for both genes did not show association with this neoplasia (OR = 1.27 95% CI 0.79-20.5). The relationship of XRCC1 and APEX1 genotypes with cancer aggressiveness through the correlation with histopathological parameters, did not find any association. Our results suggest that the polymorphism in the gene APEX1 may be indicated as a potential marker for prostate cancer risk.

Mutagenicity of Mouriri pusa Gardner and Mouriri elliptica Martius.

Mouriri pusa Gardner and Mouriri elliptica Martius are fruit-bearing plants of the Melastomataceae family, popularly known in Brazil as puçá-preto or jaboticaba-do-cerrado, and they are used in folk medicine for the treatment of gastric ulcers. In this study, we employ the Ames test to assess the mutagenicity of compounds obtained from the leaves of these species. The methanol extract of the M. pusa was mutagenic to the Salmonella typhimurium strains TA98, TA97a and TA100, with or without metabolic activation. The methanol extract of M. elliptica induced mutagenic activity in TA98 when metabolized with S9 fraction and TA97a with and without S9, but with lower mutagenicity index (MI) and potencies values than those for M. pusa. Enriched fractions of flavonoids and tannins of M. pusa were also evaluated and they demonstrated positive mutagenicity. The highest values of MI and potency were obtained with the flavonoid fraction, which contains large amounts of quercetin, quercetin glycosides and myricetin. These compounds are probably related to the mutagenicity observed in the Ames test. The dichloromethane extract was not mutagenic in any of the test conditions employed.

Genotoxicity and antigenotoxicity of cashew (Anacardium occidentale L.) in V79 cells.

The use of plants for the treatment of diseases continues to rise although there are few studies providing proof of these effects. One of these plants is the Anacardium occidentale, popularly known as the cashew. The present study evaluated the possible genotoxic and protective activities of cashew stem bark methanolic extract, in vitro, using methyl methanesulfonate (MMS) as a positive control, to compare possible mechanisms of DNA damage induction in the Comet assay. The antigenotoxicity protocols used were pre, simultaneous and post-treatment in relation to MMS. In genotoxicity and antigenotoxicity assessments, besides MMS, PBS was used as the negative control and three concentrations of the A. occidentale extract (500 microg/mL, 1000 microg/mL and 2000 microg/mL) were used on Chinese hamster lung fibroblasts (V79 cells). The Comet assay revealed that the two lowest concentrations tested presented no genotoxic activity, whereas the highest presented genotoxicity. All of the concentrations showed protective activity in simultaneous and post-treatment in relation to MMS. Further studies are required to identify the substances that comprise the extract and more clearly comprehend the antigenotoxic mechanism detected in this study.

Genotoxic and mutagenic effects of diesel oil water soluble fraction on a neotropical fish species.

Numerous spills and leakages involving petroleum and its derivatives have recently occurred in Brazilian rivers. Considering the lack of information regarding the genotoxic response of neotropical fish to these events and the predominance of information regarding saltwater fish, which offers no genuine comparisons, the present work aimed to evaluate the genotoxicity and mutagenicity of the diesel water soluble fraction (DWSF) on the neotropical fish Prochilodus lineatus under acute (6, 24 and 96h) and subchronic (15 days) exposures, using the comet (SCGE) and micronucleus assays. The results indicated genotoxic and mutagenic damage in erythrocytes of P. lineatus exposed to DWSF. Comet scores for fish exposed to DWSF in all experimental periods were significantly higher than the respective negative control groups (fish exposed to clean water for the same period). The relative frequencies of micronucleated erythrocytes for P. lineatus exposed to DWSF under acute and subchronic treatment were also significantly higher than their respective negative controls. Taken together these results showed that acute and subchronic exposures to DWSF produce mutagenic and genotoxic effects on the blood cells of P. lineatus and that the combination of comet and micronucleus assays proved to be both suitable and useful in the evaluation of the genotoxicity of diesel oil due to their complementary action.

CYP1A1 and GSTP1 polymorphisms in an oral cancer case-control study.

CYP1A1 and GSTP1 polymorphisms have been associated with a higher risk to develop several cancers, including oral squamous cell carcinoma (OSCC), which is closely related to tobacco and alcohol consumption. Both genes code for enzymes that have an important role in activating or detoxifying carcinogenic elements found in tobacco and other compounds, and polymorphic variants of these genes may result in alterations of the enzymatic activity. The CYP1A1 gene codes for the enzyme aryl hydrocarbon hydroxylase, which is responsible for the metabolism of polycyclic aromatic hydrocarbons. The investigated polymorphism, Ile/Val, seems to increase the activity of the enzyme in homozygous individuals, leading to an accumulation of carcinogens. The Ile/Val polymorphism occurs because of an A->G transition at exon 7, resulting in the CYP1A1*2B allele. The GSTP1*B variant shows an A->G transition at exon 5, changing the amino acid Ile to Val, with a reduced catalytic activity of the enzyme. Due to this reduction, the carriers of mutant alleles lost the capability to metabolize carcinogens, which could be responsible for a higher susceptibility to cancer. We conducted a case-control study in a group of 72 cases with newly diagnosed OSCC and 60 healthy controls matched for age, gender, smoking habits, and ethnicity. We used PCR methods to identify the allelic variants CYP1A1*2B and GSTP1*B. The data obtained showed no statistically significant association of allelic or genotypic variants of CYP1A1*2B (OR = 1.06; 95% CI = 0.49-2.29) and GSTP1*B (OR = 1.40; 95% CI = 0.70-2.79) with OSCC.

CYP1A1 and GSTP1 polymorphisms in an oral cancer case-control study

CYP1A1 and GSTP1 polymorphisms have been associated with a higher risk to develop several cancers, including oral squamous cell carcinoma (OSCC), which is closely related to tobacco and alcohol consumption. Both genes code for enzymes that have an important role in activating or detoxifying carcinogenic elements found in tobacco and other compounds, and polymorphic variants of these genes may result in alterations of the enzymatic activity. The CYP1A1 gene codes for the enzyme aryl hydrocarbon hydroxylase, which is responsible for the metabolism of polycyclic aromatic hydrocarbons. The investigated polymorphism, Ile/Val, seems to increase the activity of the enzyme in homozygous individuals, leading to an accumulation of carcinogens. The Ile/Val polymorphism occurs because of an A->G transition at exon 7, resulting in the CYP1A1*2B allele. The GSTP1*B variant shows an A->G transition at exon 5, changing the amino acid Ile to Val, with a reduced catalytic activity of the enzyme. Due to this reduction, the carriers of mutant alleles lost the capability to metabolize carcinogens, which could be responsible for a higher susceptibility to cancer. We conducted a case-control study in a group of 72 cases with newly diagnosed OSCC and 60 healthy controls matched for age, gender, smoking habits, and ethnicity. We used PCR methods to identify the allelic variants CYP1A1*2B and GSTP1*B. The data obtained showed no statistically significant association of allelic or genotypic variants of CYP1A1*2B (OR = 1.06; 95 percent CI = 0.49-2.29) and GSTP1*B (OR = 1.40; 95 percent CI = 0.70-2.79) with OSCC.

Shiitake (Lentinula edodes (Berkeley) Pegler) extracts as a modulator of micronuclei induced in HEp-2 cells.

Shiitake (Lentinula edodes (Berkeley) Pegler) is one of the most consumed mushrooms, for both therapeutic purposes and as food, therefore, the study of its biological properties is of great interest for producers and consumers. Aqueous extracts of the shiitake mushroom (L. edodes (Berkeley) Pegler) were evaluated by the micronucleus test (MN) in HEp-2 cells in vitro, to analyze their possible mutagenic and antimutagenic activities. None of the three extract concentrations tested (0.5, 1.0 and 1.5mg/mL) presented mutagenicity at any of the preparation temperatures (4 degrees C, 22+/-2 degrees C and 60 degrees C). In the antimutagenicity evaluation, all extract concentrations at all preparation temperatures presented a strong protective activity for the HEp-2 cells in response to the alkylating agent methyl methanesulfonate (MMS) in the different treatment protocols: pre-treatment, simultaneous treatment and post-treatment. The extracts prepared at 22+/-2 degrees C presented the lowest frequencies of MN in the evaluations of mutagenicity and antimutagenicity, indicating these as the best option for potential therapeutic use.

Assessment of DNA damage by extracts and fractions of Strychnos pseudoquina, a Brazilian medicinal plant with antiulcerogenic activity.

Strychnos pseudoquina St. Hil. is a native plant of the Brazilian Savannah, used in popular medicine to treat a number of conditions. Since it contains large quantities of alkaloids with proven antiulcer activity, we tested the genotoxic potential of crude extracts and fractions containing alkaloids and flavonoids from the leaves of this plant, on Salmonella typhimurium and performed the micronucleus test on peripheral blood cells of mice treated in vivo. The results showed that the methanol extract of the leaves of S. pseudoquina is mutagenic to the TA98 (-S9) and TA100 (+S9, -S9) strains of Salmonella. The dichloromethane extract was not mutagenic to any of the tested strains. Fractions enriched with alkaloids or flavonoids were not mutagenic. In vivo tests were done on the crude methanol extract in albino Swiss mice, which were treated, by gavage, with three different doses of the extract. The highest dose tested (1800 mg/kgb.w.) induced micronuclei after acute treatment, confirming the mutagenic potential of the methanol extract of the leaves of S. pseudoquina. In high doses, constituents of S. pseudoquina compounds act on DNA, causing breaks and giving rise to micronuclei in the blood cells of treated animals.

Assessment of the ability of Imazaquin herbicide to induce chromosomal aberrations in vitro in cultured Chinese hamster ovary cells and micronuclei in vivo in mice.

The agricultural chemicals marketed to increase food production may not only combat pests and weeds but also present toxic properties and cause genetic damage to the fauna and flora. The Imazaquin herbicide (Scepter 70 DG-Cyanamid) has been widely used in soybean fields in Paraná (Brazil), but information on its genotoxicity is scarce. Thus, in vivo and in vitro studies were carried out to assess the possible clastogenic effect of this herbicide on eukaryote cells. In the in vitro studies, the Chinese hamster ovarian cell lines CHO-K1 (wild) and CHO xrs-5 (mutant) were treated at the three phases of the cell cycle (G1, S and G2) for chromosome aberration (CA) analysis. The in vivo assessment was carried out by the micronucleus test (MN) on Swiss mice (Mus musculus) bone marrow cells. The herbicide did not induce a significant increase in the CA frequency in any of the treatments. No statistically significant differences were observed in the MN frequencies among the groups treated with the herbicide and the negative control. From the test system used in this study, we can conclude that the Imazaquin herbicide did not act as a clastogenic agent either in vitro or in vivo.

Genotoxicity and antigenotoxicity assessment of shiitake (Lentinula edodes (Berkeley) Pegler) using the Comet assay

The mushroom shiitake (Lentinula edodes (Berkeley) Pegler) is been widely consumed in many countries, including Brazil, because of its pleasant flavor and reports of its therapeutic properties, although there is little available information on the genotoxicity and/or antigenotoxicity of this mushroom. We used the Comet assay and HEp-2 cells to evaluate the in vitro genotoxic and antigenotoxic activity of aqueous extracts of shiitake prepared in three different concentrations (0.5, 1.0 and 1.5 mg/mL) and three different temperatures (4, 22 and 60 ºC), using methyl methanesulfonate (MMS) as a positive control and untreated cells as a negative control. Two concentrations (1.0 and 1.5 mg/mL) of extract prepared at 4 ºC and all of the concentrations prepared at 22 ± 2 and 60 ºC showed moderate genotoxic activity. To test the protective effect of the three concentrations of the extracts against the genotoxicity induced by methyl methanesulfonate, three protocols were used: pre-treatment, simultaneous-treatment and post-treatment. Treatments were repeated for all combinations of preparation temperature and concentration. Two extracts (22 ± 2 ºC 1.0 mg/mL (simultaneous-treatment) and 4 ºC 0.5 mg/mL (post-treatment)) showed antigenotoxic activity.