Research for Cytomegalovirus Mutations Associated With Resistance to Antivirals in Kidney Transplant Receptors.

Cytomegalovirus (CMV) mutations associated with antiviral resistance have become a major problem related to high mortality in kidney transplant patients. The aim of the study was to investigate mutations in the CMV genes UL97 and UL54 associated with antiviral resistance. A retrospective observational cohort study was carried out at Hospital Ophir Loyola (HOL), a reference in Kidney Transplantation. A total of 81 patients who underwent kidney transplantation were followed up between 2016 and 2018 were monitored for CMV viral load by performing qPCR. Sanger sequencing was performed on 66 patients. All CMV-positive kidney transplant recipients were included. Mutations were observed in 15 samples (22.72%) from patients. Most cases involved UL97 mutations. Mutation in UL54 without mutation in UL97 was detected in only 2 cases. Resistance mutations in UL97 were identified, such as M460V, L595S, H520Q, two co-mutations D465R + Del524 and A594P + D413A and a 3 codon deletion (del598-601). The search for mutations in the CMV genes identified mutations that confer resistance to conventional antivirals, such as ganciclovir and cidofovir, used in the treatment of these patients. Confirmation of the association with increased CMV viral load in transplanted patients, due to mutation in resistance genes, requires phenotypic analysis for confirmation purposes. These were the first findings in patients in northern Brazil that we know of.

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

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.

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.

Detection of Anisakidae larvae parasitizing Plagioscion squamosissimus and Pellona castelnaeana in the State of Pará, Brazil / 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 Pará, 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 (L3) 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.

Cinco espécimes de Plagioscion squamosissimus do rio Xingu e dez espécimes de Pellona castelnaeana da Ilha do Mosqueiro, ambos no Estado do Pará, Brasil, foram examinados para investigar a presença de nematodas Anisakidae, devido ao seu potencial zoonótico. Também foram determinados seus índices de parasitismo e sítios de infestação. Esse é o primeiro registro de Anisakidae parasitando Pellona castelnaeana. Foram encontradas 484 larvas de Anisakidae de terceiro estágio (L3), sendo 42 em P. squamosissimus e 442 em P. castelnaeana. Os índices parasitários de anisaquídeos coletados de P. squamosissimus compreenderam prevalência (P) de 100%, intensidade de infecção média (IM) de 8.4, amplitude de infecção (AI) de 1-13, abundância média (AM) de 8,4 e local de infecção (LI) na cavidade abdominal. P. castelnaeana apresentou P = 100%, IM = 44,2, AI = 10-114, AM = 44,2 e LI = cavidade abdominal, ceco e estômago. O ciclo de vida desses nematodas pode ser completo na bacia Amazônica, já que os dois hospedeiros fazem parte da dieta de cetáceos da região, que atuam como hospedeiros finais. Esses achados têm consequência importante na epidemiologia de anisaquíases, devendo haver mais atenção em relação à proteção humana contra esse problema de saúde pública.

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.

Investigation into the cytotoxicity and mutagenicity of the Marajó Archipelago waters using Plagioscion squamosissimus (Perciformes: Sciaenidae) as a bioindicator.

Maintaining water quality within tolerable limits is a basic need of the riverside communities in the Amazon. Using endemic aquatic organisms as biological models is useful for monitoring the environment. In this study, potential cytotoxic and genotoxic damages in Plagioscion squamosissimus (commonly known as silver croaker) from the Marajó Archipelago were evaluated using a flow cytometry assay and a survey of micronuclei (MN) frequency as well as other nuclear abnormalities (NA). P. squamosissimus specimens were collected at four locations in the Marajó Archipelago. Blood samples from these fish were used in the flow cytometry assay and piscine micronucleus test, and the resulting data were analyzed using analysis of variance (ANOVA). We did not observe a difference in the erythrocyte cell cycle distribution among the samples (P=0.9992), which suggests the absence of cytotoxic agent-induced apoptosis. The piscine micronucleus test exhibited differences in the samples from São Sebastião da Boa Vista (SSBV), and those from Anajás produced the highest mutagenicity indices. The MN frequencies were low for all groups, but the groups exhibited significantly different frequencies (P=0.0033). Reniform nuclei, nuclei with extensions, and lobed nuclei were combined and considered NA. The frequency differences for these NA were significant among sampling sites (P <0.0001). This report is the first to use flow cytometry in fish to evaluate cytotoxic agent-induced apoptosis. The micronucleus test results indicate the presence of pollutants that can change the genetic material of the fish studied. We also demonstrate that the Amazonian fish P. squamosissimus is important not only as a comestible species but also as an adequate model for biomonitoring in aquatic environments.

Description of the karyotype of Geophagus cf. proximus (Perciformes, Cichlidae, Geophaginae) / Descrição do cariótipo de Geophagus cf. proximus (Perciformes, Cichlidae, Geophaginae)

This study described the karyotype of Geophagus cf. proximus. Specimens were collected in Água Preta Lake, Parque Ambiental de Belém, Pará State, Brazil. The karyotype were 2n = 48 chromosomes (FN = 60: 12M/SM+36ST/A) and no sexual chromosome differentiation. C-banding showed centromeric staining in all chromosomes. The first chromosome pair, besides centromeric coloration, presented a totally heterochromatic long arm. The nucleolus organizer regions (NORs) were studied by means of AgNO3. NORs were located at the short arm of the second chromosome pair.

Este estudo descreve o cariótipo de Geophagus cf. proximus. Espécimes foram coletados no lago Água Preta, Parque Ambiental de Belém, Estado do Pará, Brasil. O cariótipo obtido apresentou 2n = 48 cromossomos (NF = 60: 12M/SM+36ST/A), sem diferenciação de cromossomos sexuais. O bandeamento C mostrou marcações centroméricas em todos os cromossomos. O primeiro par cromossômico, além da coloração centromérica, apresentou o braço longo totalmente heterocromático. As regiões organizadoras do nucléolo (RONs) foram estudadas por meio da coloração de AgNO3. As RONs foram encontradas no braço curto do segundo par de cromossomos.

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.