Carlos Alberto Machado da Rocha

Studies of micronuclei and other nuclear abnormalities in red blood cells of Colossoma macropomum exposed to methylmercury

The frequencies of micronuclei (MN) and morphological nuclear abnormalities (NA) in erythrocytes in the peripheral blood of tambaqui (Colossoma macropomum), treated with 2 mg.L−1 methylmercury (MeHg), were analyzed. Two groups (nine specimens in each) were exposed to MeHg for different periods (group A - 24 h; group B - 120 h). A third group served as negative control (group C, untreated; n = 9). Although, when compared to the control group there were no significant differences in MN frequency in the treated groups, for NA, the differences between the frequencies of group B (treated for 120 h) and the control group were extremely significant (p < 0.02), thus demonstrating the potentially adverse effects of MeHg on C. macropomum erythrocytes after prolonged exposure.

The Micronucleus Assay in Fish Species as an Important Tool for Xenobiotic Exposure Risk Assessment—A Brief Review and an Example Using Neotropical Fish Exposed To Methylmercury

Micronucleus (MN) assay has been extensively used in the evaluation of DNA damage. Mutagenesis and genotoxicity studies employed this methodology to evaluate possible genotoxic risk due to exposition to hazardous xenobiotics in different organisms, including aquatic sentinel organisms. MN assay in such species is sensitive, fast, and an important biomarker of mutagenic exposure in the environment. The use of bioassays, considering the toxic effects of isolated or combined contaminants, is also important since the environmental variants are minimized. The aim of this study is to gather and evaluate published data on the use of fish MN assay in biomonitoring and genotoxicity assays. In addition, we show the results of both micronuclei and other nuclear abnormalities in erythrocytes from Colossoma macropomum, exposed to methylmercury. Specimens (n = 9) were subjected to 2 mg/L of methylmercury, with an equal control group. Chi-square test was performed to compare the frequencies of nuclear abnormalities between control and treatment groups. The contingence table of χ2 test showed a significant increase of altered cells in the exposed group. Our results support the importance of MN test as an effective indicator for genotoxicity in fishes, which can be used with exposition bioindicators of human populations exposed to chemical pollutants of consuming water.

Parasitic Helminths of the Freshwater Neotropical Fish Hoplias malabaricus (Characiformes, Erythrinidae) from South America Basins

This study gathered the largest amount of evidence available concerning the presence of helminths in the Hoplias malabaricus species (Trahira, traíra in Portuguese) from limnic ecosystems in South America. At least 18 species of helminths belonging to 15 genera, 14 families, 10 orders, and 8 classes were found. The greatest diversity of parasitic species was observed among the Digeneans and acanthocephalus. Throughout the text, helminths are presented in four sections corresponding to their phyla: Platyhelminthes, Acanthocephala, Nematoda, and Annelida. The different niches occupied by the Trahira in the heteroxenic helminth cycles are also analyzed, verifying whether this fish acts as an intermediate or a definitive host.This study gathered the largest amount of evidence available concerning the presence of helminths in the Hoplias malabaricus species (Trahira, traira in Portuguese) from limnic ecosystems in South America. At least 18 species of helminths belonging to 15 genera, 14 families, 10 orders, and 8 classes were found. The greatest diversity of parasitic species was observed among the Digeneans and acanthocephalus. Throughout the text, helminths are presented in four sections corresponding to their phyla: Platyhelminthes, Acanthocephala, Nematoda, and Annelida. The different niches occupied by the Trahira in the heteroxenic helminth cycles are also analyzed, verifying whether this fish acts as an intermediate or a definitive host.

Protective effect of prolactin against methylmercury-induced mutagenicity and cytotoxicity on human lymphocytes.

Mercury exhibits cytotoxic and mutagenic properties as a result of its effect on tubulin. This toxicity mechanism is related to the production of free radicals that can cause DNA damage. Methylmercury (MeHg) is one of the most toxic of the mercury compounds. It accumulates in the aquatic food chain, eventually reaching the human diet. Several studies have demonstrated that prolactin (PRL) may be differently affected by inorganic and organic mercury based on interference with various neurotransmitters involved in the regulation of PRL secretion. This study evaluated the cytoprotective effect of PRL on human lymphocytes exposed to MeHg in vitro, including observation of the kinetics of HL-60 cells (an acute myeloid leukemia lineage) treated with MeHg and PRL at different concentrations, with both treatments with the individual compounds and combined treatments. All treatments with MeHg produced a significant increase in the frequency of chromatid gaps, however, no significant difference was observed in the chromosomal breaks with any treatment. A dose-dependent increase in the mitotic index was observed for treatments with PRL, which also acts as a co-mitogenic factor, regulating proliferation by modulating the expression of genes that are essential for cell cycle progression and cytoskeleton organization. These properties contribute to the protective action of PRL against the cytotoxic and mutagenic effects of MeHg.

Evaluation of Genotoxic Effects of Xenobiotics in Fishes Using Comet Assay—A Review

Comet assay is a useful tool on the evaluation of DNA damage, and it can estimate the genetic risk followed by xenobiotic exposures. This technique has been extensively used to detect DNA lesions in a large number of tissues because its application requires viable cells that are not necessarily mitotically active. It has been recognized that the use of aquatic organisms as sentinels is of fundamental importance on environmental biomonitoring studies, and the DNA strand breaks in these organisms are fast and sensible indicators of pollutant exposure. Additionally, these bioassays detect single or associated toxic effects of these contaminants and reduce the influence of different environmental variability. The current literature presents a large number of in vitro and in vivo analyses using Comet Assay with fishes from Brazilian fauna; however, the use of endemic species is preferential considering the importance of standardization of the methodology that is employed to detect the DNA damage.

Mutagenic and histopathological effects of hexavalent chromium in tadpoles of Lithobates catesbeianus (Shaw, 1802) (Anura, Ranidae)

The potential mutagenic and histopathological effects of the hexavalent chromium were investigated in Lithobates catesbeianus tadpoles. These larvae (GS 25-31) were exposed to three nominal concentrations of potassium dichromate (4, 12, and 36 mg L-1) and 5 mg L-1 of Cyclophosphamide as a positive control (PC), for 24 h. A negative control (NC) was also added to the experiment. Our results showed that, in general, the micronuclei (MN) were less frequent than the erythrocyte nuclear abnormalities (ENA); there was a significant difference in the frequency of MN between the NC and all treated groups (p < 0.05) in a concentration-dependent curve, in addition the PC did not differ from the chromium treatments. Also, only PC and the group treated with potassium dichromate at 36 mg L-1 showed significantly higher frequencies of ENA than NC (p < 0.05). Chromium treatments promoted cell retention in the Sub-G1 phase and a decrease of cells in the S and G2/M phases indicating inhibition of the cell cycle. All treatments with chromium led to liver and kidney histopathological lesions, especially with 36 mg L-1 (greater number of lesions). In conclusion, hexavalent chromium was mutagenic to L. catesbeianus tadpoles and its toxic effects also resulted in anti-mitotic activity, besides inducing histopathological alterations in liver and kidney. Amphibians have been proven to be useful bioindicators, and we suggest that tadpoles of different species can be used to represent the environmental impacts in aquatic ecosystems.

In vitro assessment of cytotoxic, genotoxic and mutagenic effects of antimalarial drugs artemisinin and artemether in human lymphocytes

Abstract Artemisinin is a substance extracted from the Chinese plant Artemisia annua L. widely used in natural medicine for the treatment of various diseases. Artemether is a substance synthesized from artemisinin, and both drugs are commonly administered in the treatment of malaria. Although considered effective antimalarial drugs, very little is known about the genotoxic, cytotoxic and mutagenic effects of these drugs. Therefore, in the present study, we evaluated the genotoxic, mutagenic and cytotoxic effects of artemisinin (12.5, 25 and 50 µg/mL) and artemether (7.46; 14.92 and 29.84 µg/mL) in cultured human lymphocytes using the comet assay, the micronucleus test and the cytotoxicity assay for detection of necrosis and apoptosis by acridine orange/ethidium bromide staining. Our results showed a significant increase (p < 0.05) in the rate of DNA damage measured by comet assay and in the micronucleus frequency after treatment with both drugs. It was also observed that only artemisinin induced a statistically significant increase (p < 0.05) in the number of lymphocytes with death by necrosis 48 h after treatment. The results demonstrated that these two drugs induce mutagenic, genotoxic and cytotoxic effects in cultured human lymphocytes. Our data indicate the need for caution in the use of such drugs, since genotoxic/mutagenic effects may increase the risk of carcinogenesis.

Effect of diterpenoid kaurenoic acid on genotoxicity and cell cycle progression in gastric cancer cell lines

The goal of our study was to evaluate the effect of kaurenoic acid, obtained from copaiba oil resin, in gastric cancer (GC) and a normal mucosa of stomach (MNP01) cell lines. The compound was tested at concentrations of 2.5, 5, 10, 30 and 60μg/mL. Comet and micronucleus assays were used to access its potential genotoxicity in vitro. Moreover, we evaluated the effect of kaurenoic acid in cell cycle progression and in the transcription of genes involved in the control of the cell cycle: MYC, CCND1, BCL2, CASP3, ATM, CHK2 and TP53. Kaurenoic acid induced an increase on cell DNA damage or micronucleus frequencies on GC cell lines in a dose-dependent manner. The GC and MNP01 cell lines entering DNA synthesis and mitosis decreased significantly with kaurenoic acid treatment, and had an increased growth phase compared with non-treated cells. The treatment induced apoptosis (or necrosis) even at a concentration of 2.5μg/mL in relation to non-treated cells. GC cell lines presented reduced MYC, CCND1, BCL2 and CASP3 transcription while ATM, CHK2 and TP53 increased in transcription in relation to non-treated cells, especially at a concentration above 10μg/mL. The gene transcription in the MNP01 (non-treated non-cancer cell line) was designated as a calibrator for all the GC cell lines. In conclusion, our results showed that kaurenoic acid obtained from Copaifera induces DNA damage and increases the micronuclei frequency in a dose-dependent manner in GC cells, with a significant genotoxicity observed above the concentration of 5μg/mL. Moreover, this compound seems to be able to induce cell cycle arrest and apoptosis in GC cells.

Detecção de larvas de Anisakidae parasitando Plagioscion squamosissimus e Pellona castelnaeana no Estado do Pará, Brasil

Five specimens of Plagioscion squamosissimus from Xingu River and ten specimens of Pellona castelnaeana from Mosqueiro Island, both in the State of Para, Brazil, were examined to investigate the presence of anisakid nematodes, due to their zoonotic potential. Their parasitism indices and sites of infection were also determined. This is the first record of Anisakidae parasitizing Pellona castelnaeana . Four-hundred and eighty-four third-stage larvae (L 3 ) of Anisakidae were found, of which 42 were found in P. squamosissimus and 442 in P. castelnaeana . The parasitism indices of the anisakid collected from the P. squamosissimus comprised prevalence (P) of 100%, mean infection intensity (MI) of 8.4, range of infection (RI) of 1-13, mean abundance (MA) of 8.4, and infection site (IS) in the abdominal cavity. P. castelnaeana showed P = 100%, MI = 44.2, RI = 10-114, MA = 44.2, and IS = abdominal cavity, cecum and stomach. The life cycle of these nematodes can be completed in the Amazon basin, since the two hosts fish are part of the cetacean diet of the region, which participate as final hosts. These findings have an important consequence on epidemiology of anisakiasis, so attention should be extended to human protection against this public health risk.

Perfil dos exames citopatológicos do colo do útero realizados na Casa da Mulher, Estado do Pará, Brasil

Cervical cancer usually has silent and slow progression, remaining to be among the most incident cancers in Brazil. Its screening is performed by Pap test which besides detects cancer previously, allows the research of infection agents. A retrospective analytical study about the results of Pap test was conducted in Casa da Mulher, Pará State, Brazil from September 2012 to August 2013 with data from books of prevention of cervical cancer (PCC). During that period, 2,202 PCC tests were carried out at Casa da Mulher, most of them (52.36%) from women aged of 40 to 60 years old. The age group in the sample showed a weak positive correlation (r = 0.1429) with the frequency of microbiological findings and weak negative correlation (r = -0.2571) with the frequency of cytological changes. Microbiological findings were identified in three species: Gardnerella vaginalis (23.48%), Candida sp. (12.44%) and Trichomonas vaginalis (0.68%). The prevalence of cytological abnormalities in tests was 5.72% and did not differ significantly (p = 0.7546) from the reported ones in other publications. Atypia cells of undetermined significance corresponded to 2.679%, below the range estimated by Brazilian National Cancer Institute. The total proportion of potentially malignant neoplastic lesions (high-grade squamous intra epithelial lesion and carcinoma) was 1.09%, and it is most frequently between the ages of 30 to 39 years old. The increase in PCC examinations in the female population needs to be achieved and health promotion should be done by intersectoral partnerships, community participation and collective responsibility for the quality of life.