Retrospective application of ICH M7 to anti-hypertensive drugs in Brazil: Risk assessment of potentially mutagenic impurities.

Potentially mutagenic impurities are likely to be formed in any drug substance, since their synthesis requires reactive intermediates which may also react with DNA. The ICH M7 guideline, which defines how to risk assess and control mutagenic impurities, was first published in 2014 and is not to be applied retrospectively; however, some impurities have been found above the permitted limits in drug products which were already on the market. This study assessed the implications of applying ICH M7 retrospectively to anti-hypertensive drugs marketed in Brazil by performing a risk assessment and establishing control strategies. The manufacturing processes of 15 drug substances were evaluated and 262 impurities were identified, from which 21% were classified as potentially mutagenic. Most of the impurities were identified below ICH M7 acceptable limits, except for impurities described in a pharmacopoeial monograph. Compendial specifications are defined based on scientific evidence and play an important role in setting quality and safety standards for pharmaceuticals, however there are opportunities for further alignment with ICH guidelines, aiming for a holistic assessment of the impurities profile to ensure the safety of medicines.

Classifying micropollutants by environmental risk in groundwater using screening analysis associated to a hybrid multicriteria method combining (Q)SAR tools, fuzzy AHP and ELECTRE.

This study aimed to evaluate and monitor pesticides in groundwater of the Serra Geral aquifer, located in the Paraná Basin 3 (southern Brazil), using Liquid Chromatography coupled with a Quadrupole-Time-of-Flight Mass Spectrometer (LC-QTOF MS). A total of 117 samples, collected in three different moments, were analyzed over 36 months. Groundwater samples from 35 wells and four surface water points were monitored in each sampling campaign. A pesticide screening methodology was proposed with the tentative identification of 1607 pesticides and pesticide metabolites. The application of the proposed methodology resulted in the verification of 29 pesticides and pesticide metabolites, 7 as confirmed analytes and 22 as suspect compounds. (Q)SAR in silico predictions and GUS index calculation provided data on the potential environmental risk of the identified compounds, with eight endpoints considered. After in silico predictions, an alternative hybrid multicriteria method was applied, combining the weighting of endpoints of fuzzy AHP and micropollutants classification by environmental risk using ELECTRE. The fuzzy AHP results indicated the greatest importance of mutagenicity among the eight evaluated indicators, while the scarce influence of the physicochemical properties on the environmental risk suggested their exclusion from the model. Accordingly, the ELECTRE results highlighted the prevalence of thiamethoxam and carbendazim as the most dangerous for the environment. The application of the proposed method enabled the selection of the compounds that must be monitored, considering mutagenicity and toxicity predictions for environmental risk analysis.

In vitro genotoxic and mutagenic effects of water samples from Sapucaia and Esteio streams (Brazil) under the influence of different anthropogenic activities.

Pollution of aquatic ecosystems is associated with the discharge of mainly industrial and urban effluents, which may cause damage to public health. This study aims to evaluate the cytotoxic, genotoxic, and mutagenic potential of surface water samples under the influence of different anthropogenic effluents in a human-derived liver cell line (HepG2). Samples were collected in Esteio and Sapucaia streams (Rio Grande do Sul; Brazil), which flow into the Sinos River, a source of water supply for more than one million people. Physicochemical and microbiological analyses were performed as well as an analysis of inorganic elements using the PIXE technique (Particle-Induced X-Ray Emission). The presence of pharmaceutical compounds and caffeine was evaluated by gas chromatography coupled to mass spectrometry. The cytotoxicity, genotoxicity, and mutagenicity of the samples were evaluated in HepG2 cells by cell viability assays, alkaline Comet Assay and Cytokinesis-block micronucleus (CBMN) assay. We verified alterations in the physicochemical and microbiological parameters and detected caffeine, diethyltoluamide, and different inorganic elements that corresponded to elements from domestic and industrial effluents and agricultural runoff. Although the samples in the concentration used were not cytotoxic, water samples from all sites induced DNA damage. However, it is difficult to attribute these damages to a specific substance since the factors are a complex mixture of different compounds. Despite this, it is observed that both urban and industrial contributions had a similar effect in the cells evaluated. Such results demonstrate the need to perform biomonitoring of surface waters under anthropogenic influence, especially those that flow into rivers that are a source of public supply water. We also highlight the need for research into emerging pollutants in these aquatic environments.

Evaluation of the cytotoxic and mutagenic potential of Rosmarinus officinalis L. essential oil through the Allium cepa Leach bioassay

The species Rosmarinus officinalisL. (rosemary) is an herb from the Lamiaceae family, widely used in cooking as a food preservative, seasoning or condiment. It also stands out for its therapeutic properties, mainly presenting antioxidant, antibacterial and antitumor activity. The aim of this study was to evaluate the cytotoxic and mutagenic activity of R. officinalis L. essential oil through the Allium cepabioassay. This test constitutes an excellent plant model routinely used due to its sensitivity, low cost and good correlation with test systems in mammals. The defined concentrations for carrying out the test were 750, 243, 81 and 27 µg mL-1. Five bulbs were used, 4 roots of each, measuring approximately 2 cm, and they were analyzed on two slides. All assays were performed at least in triplicate and compared to the negative control. The statistical test of analysis of variance (ANOVA with a fixed factor) was used, followed by Tukey's multiple comparisons test, for p< 0.05. For this purpose, the GraphPad Prism program (version 6.0) was used. The results showed a cytotoxic and mutagenic effect for all concentrations used of the essential oil of R. officinalisL. However, it is important to conduct further research using other genotoxicological tests with different endpoints and at different concentrations, in order to clarify the interaction of the essential oil of the species R. officinalisL. with the genetic material of the cell and its possible mechanism of action.(AU)

Cytotoxicity and mutagenicity of the waters of the Marrecas River (Paraná, Brazil) to bullfrogs (Lithobates catesbeianus).

Animals have a long history of assessing ecosystem responses to environmental disturbances, and amphibians stand out for presenting themselves as good animal model and bioindicators of environmental quality. The main purpose of the present work was to investigate the cellular effects of contamination of waters of the Marrecas River, located in the southwest of the state of Paraná, Brazil. Therefore, the objective of the present study was to investigate and monitor the cytotoxic and mutagenic effect, with bullfrog tadpoles, and to discuss these effects with land use along this hydrographic basin. Mutagenic effects were determined by micronucleus assay, and cytotoxicity by other nuclear changes, such as segmented cells, binucleated cells, cells with buds and reniform cells. Water samples were obtained at nine sites along the Marrecas River, covering areas with rural and urban hydrological contribution. For each site, four samples were collected, along the years 2017 and 2018, encompassing the four seasons (summer, autumn, winter, and spring). The results showed mutagenic and cytotoxic effect in four sampling sites, and only cytotoxic effect in other four sites. These effects may be due, possibly, to the use of different agrochemicals across the hydrographic basin region, which have predominant hydrological contributions from crops. Data of this study indicate the presence of cytotoxic and mutagenic contaminants in the waters of the Marrecas River, which can generate environmental problems on the river fauna/flora, and can also affect the local population health.

Evaluation of toxicity and mutagenicity of a synthetic effluent containing azo dye after Advanced Oxidation Process treatment.

Approximately 20% industrial water pollution comes from textile dyeing process, with Azo dyes being a major problem in this scenario and requiring new forms of efficient treatment. Effluent treatments using the Advanced Oxidation Processes (AOP) are justified by the potential of application in the dyed effluent treatments once they can change the Azo dye chemical structure. Thus, this study aimed to evaluate the toxicity and mutagenic capacity of a synthetic effluent containing Amido Black 10B (AB10B) azo dye before treatment with AOP, named Gross Synthetic Effluent (GSE), and after the AOP, named Treated Synthetic Effluent (TSE). Daphnia magna and Allium cepa tests were used to evaluate acute toxicity effects and chromosomal mutagenesis, respectively. The Salmonella/microsome assay was performed to evaluate gene mutations. In silico assays were also performed aiming to identify the mutagenic and carcinogenic potential of the degradation byproducts of AB10B. There was 100% immobility to D. magna after 24 h and 48 h of treatments with TSE, showing EC50 values around 5%, whereas GSE did not show acute toxicity. However, GSE induced chromosomal mutations in A. cepa test. Both GSE and TSE were not able to induce gene mutations in S. typhimurium strains. These effects can be associated with two byproducts generated with the cleavage of the azo bonds of AB10B, 4-nitroaniline and -2-7-triamino-8-hydroxy-3-6-naphthalinedisulfate (TAHNDS). In conclusion, AOP is an efficient method to reduce the mutagenicity of synthetic effluent containing AB10B and additional methods should be applied aiming to reduce the toxicity.

Evidence of mutagenic and lethal effects of herbicides on Amazonian frogs / Evidências de efeitos mutagênicos e letais de herbicidas em sapos da Amazônia

Amphibians are excellent bioindicators because they are sensitive to chemical pollution and can indicate ecosystem changes due to the presence of or exposure to chemical compounds. Here we report evidence of the impact of herbicides, including glyphosate, on amphibians in a locality in the central Brazilian Amazon and compare it with data from other biomes in Brazil. We observed malformations in three species of Leptodactylus and local extinctions of Scinax ruber and Rhinella marina from reproductive sites close to an area where herbicides had been applied. The observations in the Amazon are similar to reports from Brazils Atlantic forest on morphological anomalies and mortality in amphibians exposed to herbicides. We warn of the threat of expanding crops for the production of biofuels in the Amazon due to their cultivation being associated with agrochemicals, including glyphosate, posing a threat to the biodiversity of the Amazon biome.

Os anfíbios são ótimos bioindicadores porque são sensíveis à poluição química, indicando alterações ecossistêmicas pela presença de ou pela exposição a compostos químicos. Aqui nós relatamos evidências do impacto de herbicidas, incluindo glifosato, em anfíbios em uma localidade na Amazônia central brasileira e as comparamos com dados de outros biomas no Brasil. Nós observamos malformações em três espécies de Leptodactylus e extinção local de Scinax ruber e Rhinella marina em sítios reprodutivos próximos a uma área onde herbicidas haviam sido aplicados. Os resultados observados na Amazônia são semelhantes aos observados na Mata Atlântica do Brasil, com alterações morfológicas e mortalidade em anfíbios expostos a herbicidas. Alertamos para a ameaça de expansão de culturas para a produção de biocombustíveis na Amazônia, devido ao fato de o cultivo estar associado a agroquímicos, incluindo o glifosato, representando uma ameaça à biodiversidade do bioma Amazônia.

Evaluation of effect of hazardous contaminants in areas for the abstraction of drinking water.

The lower portion of Taquari River is influenced by compounds from anthropic activities causing concern about the drinking water supplied to cities in the region. The study objective was to investigate the presence of contaminants at drinking water abstraction sites, defining the mutagenic effects of these stressors as an ecosystem quality parameter and its possible effects on human health. Geographic Information System techniques were used to investigate sources of contamination and it was found that agricultural activities predominated with a few medium and high potential pollutant agricultural activities, besides a soil area that was contaminated and undergoing an intervention process. Mutagenic effects were evaluated by Salmonella/microsome assay using TA98, TA97a, TA100, YG1041 and YG1042 strains in the presence and absence of metabolic activation (S9). Mutagenesis found in organic sediment extracts and surface water samples showed the prevalence of direct-acting mutagens at the drinking water abstraction sites. Taquari (Ta032, the sampling points were named according to the initial letters of the river (Ta), followed by the number of kilometers from the mouth) showed the highest mutagenic potency in sediment, while Ta063, at Bom Retiro do Sul, presented it in the water sample. In the Triunfo region (Ta011) there were significant responses in sediment and in water samples. The samples at General Câmara (Ta006) showed the least presence of contaminants. The Allium cepa test applied to sediments in natura showed significant micronucleus induction in Ta032 in accordance with the Salmonella/microssome assay. The test performed on Danio rerio embryos (FET) in the in natura water samples did not present significant responses. Chemical analyses of polycyclic aromatic hydrocarbons and metals already identified as chemical markers in the area indicated a small contribution to the mutagenic potency, calling attention to the fact that other direct-acting pollutants may be present at the drinking water abstraction sites.

Occurrence of 3-nitrobenzanthrone and other powerful mutagenic polycyclic aromatic compounds in living organisms: polychaetes.

In this work we report the occurrence of powerful mutagenic 3-nitrobenzanthrone (3-NBA), in addition to 18 polycyclic aromatic hydrocarbons (PAHs), 6 oxygenated PAHs and 27 nitrated PAHs in polychaete worms. Benzanthrone (BA), another important mutagenic polycyclic aromatic compound (PAC) also was detected in the samples. Polychaete annelids have great ecological relevance, being widely distributed in different environmental conditions, from intertidal zones up to seven thousand feet deep areas. They are abundantly found in both contaminated and uncontaminated areas and, therefore, used as indicators of the pollution status of a given area. As we know, so far, most of these PACs has not been previously reported in living organisms before. The 3-NBA concentrations determined in this study were within 0.11-5.18 µg g-1. Other relevant PACs such as PAHs, quinones and nitro-PAHs were found in maximum concentrations at 0.013 µg g-1 (coronene) to 11.1 µg g-1 (benzo[k]fluoranthene), 0.823 µg g-1 (9,10-phenenthrenequinone) to 12.1 µg g-1 (1,4-benzoquinone) and 0.434 (1-nitronaphthalene) µg g-1 to 19.2 µg g-1 (6-nitrobenzo[a]pyrene), respectively. Principal component analysis (PCA), ternary correlations and diagnostic ratios were employed in order to propose probable sources for PACs. Although statistical analysis preliminarily has indicated both pyrogenic and petrogenic contributions, petrogenic sources were predominant reflecting the impacts of petroleum exploration and intensive traffic of boats in the study area.

Mutagenicity as a parameter in surface water monitoring programs-opportunity for water quality improvement.

Effect-based analyses are being recognized as excellent tools to a comprehensive and reliable water quality evaluation to complement physical and chemical parameters. The Salmonella/microsome mutagenicity test was introduced in the São Paulo State water quality-monitoring program in 1999 and waters from 104 sites used to the production of drinking water were analyzed. Samples were tested after organic extraction, using the microsuspension version of the Salmonella/microsome assay with strains TA98 and TA100 with and without S9-mammalian metabolic system. Of the 1720 water samples analyzed in 20 years, 20% were positive; TA98 was the most sensitive strain, detecting alone 99%. Results were presented in hazard categories to facilitate water managers' understanding and general public communication. Hot spots of mutagenicity were identified, and pollution sources investigated. A flow scheme with instructions of how to proceed in case of mutagenic samples was developed and implemented in the monitoring program. Enforcement actions were taken to reduce exposure of humans and aquatic biota to mutagenic compounds. The results presented provide scientific basis for the incorporation of the Salmonella/microsome assay in a regulatory framework, and to guide water-quality managers. The inclusion of a mutagenicity assay using standardized conditions proved to be an opportunity to improve the quality of water, and the strategy presented here could be applied by any environmental agency around the world. Environ. Mol. Mutagen. 61:200-211, 2020. © 2019 Wiley Periodicals, Inc.

Evidence of mutagenic and lethal effects of herbicides on Amazonian frogs / Evidências de efeitos mutagênicos e letais de herbicidas em sapos da Amazônia

Amphibians are excellent bioindicators because they are sensitive to chemical pollution and can indicate ecosystem changes due to the presence of or exposure to chemical compounds. Here we report evidence of the impact of herbicides, including glyphosate, on amphibians in a locality in the central Brazilian Amazon and compare it with data from other biomes in Brazil. We observed malformations in three species of Leptodactylus and local extinctions of Scinax ruber and Rhinella marina from reproductive sites close to an area where herbicides had been applied. The observations in the Amazon are similar to reports from Brazils Atlantic forest on morphological anomalies and mortality in amphibians exposed to herbicides. We warn of the threat of expanding crops for the production of biofuels in the Amazon due to their cultivation being associated with agrochemicals, including glyphosate, posing a threat to the biodiversity of the Amazon biome.(AU)

Os anfíbios são ótimos bioindicadores porque são sensíveis à poluição química, indicando alterações ecossistêmicas pela presença de ou pela exposição a compostos químicos. Aqui nós relatamos evidências do impacto de herbicidas, incluindo glifosato, em anfíbios em uma localidade na Amazônia central brasileira e as comparamos com dados de outros biomas no Brasil. Nós observamos malformações em três espécies de Leptodactylus e extinção local de Scinax ruber e Rhinella marina em sítios reprodutivos próximos a uma área onde herbicidas haviam sido aplicados. Os resultados observados na Amazônia são semelhantes aos observados na Mata Atlântica do Brasil, com alterações morfológicas e mortalidade em anfíbios expostos a herbicidas. Alertamos para a ameaça de expansão de culturas para a produção de biocombustíveis na Amazônia, devido ao fato de o cultivo estar associado a agroquímicos, incluindo o glifosato, representando uma ameaça à biodiversidade do bioma Amazônia.(AU)

Sclerotinia Sclerotiorum (White Mold): Cytotoxic, Mutagenic, and Antimalarial Effects In Vivo and In Vitro.

This work aimed includes performing the sclerotia chemical profile and evaluates their biological effects on mutagenesis, oxidative stress, cancer, and malaria. A chemical profile was determined by ultraperformance liquid chromatography mass spectrometry (UHPLC-HRMS) analysis dereplicating norditerpenoid dilactone, sclerolide, and other compounds. The GI50 values to cancer cells (19.8 to 277.6 µg/mL) were higher than normal (16.05 µg/mL), meaning high cytotoxicity. Regarding the oxidative stress, the results showed that the all AcOET fraction concentrations tested on IMR90 noncancer cell increased reactive oxygen species (ROS) production in more intense way (by fivefold) than in tested cancer cells. The in vivo study showed an increase of the following biomarkers (by 296.00%): % DNA in comet tail in peripheral blood and liver cells; micronucleated erythrocytes and colon cells and lipid serum peroxidation. These results indicate the sclerotia as genotoxic and mutagenic agent and its contamination may lead to fungal toxic effects with a risk to human health.

Assessment of surface water using Allium cepa test and histological analysis in Rhamdia quelen.

This study aimed to assess the cytotoxic, genotoxic, and mutagenic potentials of water samples collected in the Alegre River Basin, located in a predominantly rural area with no sewage treatment facilities in the Espírito Santo State, Brazil, using Allium cepa test. Also, gills and liver of Rhamdia quelen, a common fish species of the region, were histologically analyzed. A semi-quantitative analysis was performed and a histopathological alterations index (HAI) was determined. Our findings indicated that the waters of this river basin were cytotoxic (mitotic index reduction) and/or genotoxic (chromosomal abnormalities induction). Mutagenicity (micronuclei induction) was not observed for any water sample. The values for HAI showed that the waters caused moderate histological alterations in R. quelen. Liver was more sensitive than gills. It is necessary to implement a sewage treatment system and raise awareness on inappropriate management and disposal of agrochemicals in order to allow the recovery of Alegre River.

Assessment of sediment mutagenicity in areas under the influence of a contaminated site undergoing a remediation process.

Soil contamination enters aquatic ecosystems affecting sediment quality. The region studied is the Taquari River, Brazil, close to a site contaminated by wood preservatives, with a runoff route into the river. The first stage of the remediation process (In this article, the terms intervention and remediation have been used with slightly different meanings. We consider intervention to be the first phase of the remediation process, which aims to remove active sources) was an intervention to remove the main active sources. The Salmonella/microsome assay and polycyclic aromatic hydrocarbons (PAHs) were used to assess sediment quality in organic extracts during different intervention phases. The strains used were TA98, TA97a, and TA100 with and without S9mix (±S9). The results indicated the presence of pro-mutagens at site Ta010 (closest to the contaminated site) in all samplings, and the highest result occurred before intervention for TA100 + S9 (1,672 ± 215.9 rev/g). These values decreased during (83 ± 23.6 rev/g) and after this process (403 ± 105.9 rev/g), although the PAHs concentrations increased. Samples from this site presented PAHs with a carcinogenic potential during the assessed periods. After intervention, Ta006 (4 km downstream from Ta010) showed the most significant mutagenesis for TA100 + S9 (764 ± 230.2 rev/g) and, although the total PAHs values were lower, the species considered carcinogenic had higher concentrations. Mutagenesis predicted values of PAHs confirmed that carcinogenic species were predominantly detected by TA100, and the other PAHs by TA97a strains. Marked contaminant release to the river was observed, mainly in Ta010 at different periods. Mutagenicity and PAHs values in an internal stream, upstream from Ta010, showed a dispersion route of these agents. Thus, contamination in Ta010 and possible contribution to Ta006, after intervention, provides a warning regarding environmental quality in the region. Environ. Mol. Mutagen. 59:625-638, 2018. © 2018 Wiley Periodicals, Inc.

Characterization of imatinib mesylate formulations distributed in South American countries: Determination of genotoxic impurities by UHPLC-MS/MS and dissolution profile.

Imatinib mesylate (IM) is an anti-neoplasic drug used for the treatment of cancer. Recent new guidelines specify daily doses and concentration limits for genotoxic impurities (GTIs) in pharmaceutical final products. Therefore, in this work an analytical method using UHPLC-MS/MS was developed, validated and applied to characterize IM tablets for two GTIs: N-(2-methyl-5-aminophenyl)-4-(3-pyridyl)-2-pyrimidine amine (Imp. 1), and N-[4-methyl-3-(4-methyl-3-yl-pyrimidin-2-ylamino)-phenyl]-4- chloromethyl benzamide (Imp. 2), simultaneously. Additionally, dissolution data of IM tablets were compared using a methodology recommended by the US Food and Drug Administration. The UHPLC method utilized an Acquity BEH C18 (150 × 2.1 mm, 1.7 µm) maintained at 40°C. The mobile phase consisted of ammonium formate 0.063% (phase A) and acetonitrile plus 0.05% formic acid (phase B) in gradient elution. A sensitive method for determination of previously mentioned GTIs in IM tablets was successfully developed and applied. Overall, the formulations analyzed in this work showed low levels of Imp. 1 and Imp. 2. However, the sample named D1 showed very high levels of Imp. 1 and failed to meet the requirements established by the US Food and Drug Administration for dissolution data. Periodic verification of GTIs in pharmaceutical formulations is important to minimize safety risks, so analytical methods to determine it need be available and implemented in routine analysis.

Biomass burning particles in the Brazilian Amazon region: Mutagenic effects of nitro and oxy-PAHs and assessment of health risks.

Emissions from burning of biomass in the Amazon region have adverse effects on the environment and human health. Herein, particulate matter (PM) emitted from biomass burning in the Amazon region during two different periods, namely intense and moderate, was investigated. This study focused on: i) organic characterization of nitro- and oxy-polycyclic aromatic hydrocarbons (PAHs); ii) assessment of the excess lifetime cancer risk (LCR); and iii) assessment of the in vitro mutagenic effects of extractable organic matter (EOM). Further, we compared the sensitivity of two mutagenicity tests: Salmonella/microsome test and cytokinesis-block micronucleus (CBMN) with human lung cells. Among the nitro-PAHs, 2-nitrofluoranthene, 7-nitrobenz[a]anthracene, 1-nitropyrene, and 3-nitrofluoranthene showed the highest concentrations, while among oxy-PAHs, 2-metylanthraquinone, benz[a]anthracene-7,12-dione, and 9,10-anthraquinone were the most abundant. The LCR calculated for nitro-PAH exposure during intense biomass burning period showed a major contribution of 6-nitrochrysene to human carcinogenic risk. The EOM from intense period was more mutagenic than that from moderate period for both TA98 and YG1041 Salmonella strains. The number of revertants for YG1041 was 5-50% higher than that for TA98, and the most intense responses were obtained in the absence of metabolic activation, suggesting that nitroaromatic compounds with direct-acting frameshift mutagenic activity are contributing to the DNA damage. Treatment of cells with non-cytotoxic doses of EOM resulted in an increase in micronuclei frequencies. The minimal effective dose showed that Salmonella/microsome test was considerably more sensitive in comparison with CBMN mainly for the intense burning period samples. This was the first study to assess the mutagenicity of EOM associated with PM collected in the Amazon region using Salmonella/microsome test. The presence of compounds with mutagenic effects, particularly nitro- and oxy-PAHs, and LCR values in the range of 10-5 indicate that the population is potentially exposed to an increased risk of DNA damage, mutation, and cancer.

The micronucleus test for the oral mucosa: global trends and new questions.

This study reviews global trends in the publication of papers on the micronucleus test of the exfoliated cells of the oral mucosa in mammals as an approach for environmental biomonitoring. This test has been widely used due to its precision for the detection of chromosome damage. Our temporal analysis showed a significant increase (p < 0.05) in the publication of papers on the oral mucosa over the past 33 years. Brazil was the country that published most papers (24% of the total), followed by India (16%), the USA (10%), Mexico (7%), and Turkey (6%). A further 30 countries contributed the other 37% of the papers. Overall, 99% of the micronucleus studies involved human subjects, and only 1% involved other mammals. As many wild mammals are subject to the same environmental pressures as humans, in particular chemical pollution, it seems likely that many species are equally susceptible to genotoxicogical damage. We emphasize the importance of applying this technique to the analysis of the oral mucosa of wild mammals, as well as the evaluation of its effectiveness, as observed in humans, and the expansion of the available approaches to the monitoring of environmental quality.

Mutagenic and genotoxic effects and metal contaminations in fish of the Amambai River, Upper Paraná River, Brazil.

The present study evaluated mutagenic and genotoxic effects and metal accumulation in the liver and musculature of Pterygoplichthys ambrosetti and Prochilodus lineatus in the Amambai River, a tributary of the Upper Paraná River in Brazil. We also evaluated the potential for these fish species as environmental bioindicators and performed risk assessment for conservation purposes. We found that P. ambrosetti had a higher frequency of micronuclei compared to P. lineatus (p < 0.0001). There were no significant differences between species in other erythrocyte nuclear abnormalities (ENA) (p > 0.05). For both species, the liver contained a higher concentration of metals (Cd, Pb, Cr, Cu, Fe, Zn, and Ni) than the musculature did (p < 0.0001). Of the two species, P. ambrosetti was more suitable to evaluate mutagenic and genotoxic effects and metal accumulation in the liver and musculature, likely due to its resident behavior; hence, we highlight the potential of this species for use as an environmental bioindicator. The concentrations of non-essential metals observed in the fish confirm conditions of environmental stress in the Amambai River, possibly related to the discharge of pollutants and exacerbated by lack of native vegetation cover along the watercourse.

Effect of green juice and their bioactive compounds on genotoxicity induced by alkylating agents in mice.

Kale juice (Brassica oleracea L. var. acephala D.C.) is a reliable source of dietary carotenoids and typically contains the highest concentrations of lutein (LT) and beta-carotene (BC) among green leafy vegetables. As a result of their antioxidant properties, dietary carotenoids are postulated to decrease the risk of disease occurrence, particularly certain cancers. The present study aimed to (1) examine the genotoxic and antigenotoxic activity of natural and commercially available juices derived from Brassica oleracea and (2) assess influence of LT or BC against DNA damage induced by alkylating agents such as methyl methanesulfonate (MS) or cyclophosphamide (CP) in vivo in mice. Male Swiss mice were divided into groups of 6 animals, which were treated with water, natural, or commercial Brassica oleraceae juices (kale), LT, BC, MMS, or CP. After treatment, DNA damage was determined in peripheral blood lymphocytes using the comet assay. Results demonstrated that none of the Brassica oleraceae juices or carotenoids produced genotoxic effects. In all examined cell types, kale juices or carotenoids inhibited DNA damage induced by MMS or CP administered either pre- or posttreatment by 50 and 20%, respectively. Under our experimental conditions, kale leaf juices alone exerted no marked genotoxic or clastogenic effects. However, a significant decrease in DNA damage induced by MMS or CP was noted. This effect was most pronounced in groups that received juices, rather than carotenoids, suggesting that the synergy among constituents present in the food matrix may be more beneficial than the action of single compounds. Data suggest that the antigenotoxic properties of kale juices may be of therapeutic importance.

The genotoxicity and cytotoxicity of tannery effluent in bullfrog (Lithobates catesbeianus).

Some of the most polluting activities occur in bovine skin processing. Tannery generates effluents containing high concentrations of heavy metals and organic compounds. The phases composing the leather production process generate a large volume of tannery effluents that are often discarded in aquatic environments without any previous treatment. However, the effect these xenobiotics have on adult representatives belonging to the class Amphibia remains unknown. Thus, the aim of the present study is to assess the geno- and cytotoxic effects of tannery effluent on adult male bullfrogs (Lithobates castesbeianus) exposed to it. Accordingly, the animals were divided into the following groups: negative control (tannery effluent-free water), positive control (cyclophosphamide), and effluent (water added with 5% tannery effluent). The animals were euthanized for blood collection, and erythrocyte analyses were conducted after 35 and 90 days of exposure. The micronuclei (MN) frequency and the frequency of other nuclear abnormalities in each of the animals in the experimental groups were assessed in 2000 erythrocytes. According to the present results, the exposure to tannery effluents increased MN frequency as well as other nuclear abnormalities (i.e., lobed nuclei, binucleated cell, kidney-shaped nuclei, notched nuclei, and apoptotic cell) in the erythrocytes of animals in the effluent group and in the positive control group after 35 and 90 exposure days. Thus, the current study corroborated the hypothesis that the tannery effluent has aneugenic and clastogenic potential in adult male bullfrogs (L. castesbeianus). The present study is the first to report such effect.