Differences in MCF-7 response to endocrine disruptors in waste, superficial, and treated water from Southern Brazil.

The aim of this study was to evaluate the effect of possible endocrine disruptors in surface and wastewater using a cell proliferation assay in an estrogen-responsive cell line (MCF-7). This study was conducted in the Sinos River (Brazil). The residual water was collected from a Pilot Treatment Plant (using Typha domingensis) and surface waters of the Luis Rau stream, the Sinos River, and the Water Treatment Station (WTS). After exposures (24-120 h), a Sulforhodamine B assay was performed to determine the proliferation rate. The higher increase in proliferation rate was observed with the Luiz Rau stream and the sewage treated by macrophytes in a flotation filter. The results from WTS water remained with a proliferation rate similar to the negative control at all times, suggesting that the conventional treatment is partially effective for the withdrawal of endocrine-disrupting agents. The study demonstrated the efficiency of the MCF-7 line in assessing endocrine disruption caused by wastewater and surface water samples. Our results indicate that conventional water treatment can partially remove the polluting load of endocrine disruptors, minimizing their environmental and public health impacts. Besides, it demonstrates the need to expand sanitary services to improve the population's quality of life.

Permitted daily exposure from preclinical studies of Ginkgo biloba L. dry extract

Abstract Resolution 658/2022 of the Brazilian Regulatory Agency requires the determination of the permitted daily exposure (PDE) of pharmaceutical agents. Ginkgo biloba L. is used therapeutically to treat memory deficits and other brain diseases. However, published results indicate that more studies are needed to confirm the safety of Ginkgo biloba. This study aimed to evaluate the dry extract of Ginkgo biloba L. leaves PDE as an ingredient in an oral pharmaceutical product in preclinical studies using mice. Acute oral toxicity and repeated dose experiments were performed based on OECD guidelines, as well as genotoxicity tests. The results indicate that Ginkgo biloba L. has low acute toxicity, no liver toxicity, and does not alter blood glucose levels. No changes in weight gain were observed, but food intake decreased in males during the first week of treatment at the highest dose. Hematological parameters were not altered in males, whereas females presented lower leukocyte and lymphocyte counts and higher neutrophil counts at the highest dose. The lipid profile was not altered in males, whereas total cholesterol was increased in females. The estimated PDE was 0.1 mg/day and, when related to the maximum residual concentration, indicates that the cleaning process used is safe and does not require reassessment.

Cytotoxicity evaluation of haloperidol, clozapine and a new molecule with antipsychotic potential, PT-31, in NIH-3T3 cells

Abstract Schizophrenia is an illness that affects 26 million people worldwide. However, conventional antipsychotics present side effects and toxicity, highlighting the need for new antipsychotics. We aimed to evaluate the cytotoxicity of haloperidol (HAL), clozapine (CLO), and a new molecule with antipsychotic potential, PT-31, in NIH-3T3 cells. The neutral red uptake assay and the MTT assay were performed to evaluate cell viability and mitochondrial activity, morphological changes were assessed, and intracellular reactive oxygen species (ROS) detection was performed. HAL and CLO (0.1 µM) showed a decrease in cell viability in the neutral red uptake assay and in the MTT assay. In addition, cell detachment, content decrease, rounding and cell death were also observed at 0.1 µM for both antipsychotics. An increase in ROS was observed for HAL (0.001, 0.01 and 1 µM) and CLO (0.01 and 1 µM). PT-31 did not alter cell viability in any of the assays, although it increased ROS at 0.01 and 1 µM. HAL and CLO present cytotoxicity at 0.1 µM, possibly through apoptosis and necrosis. In contrast, PT-31 does not present cytotoxicity to NIH-3T3 cells. Further studies must be performed for a better understanding of these mechanisms and the potential risk of conventional antipsychotics

Study of the acute and repeated dose 28-day oral toxicity in mice treated with PT-31, a molecule with a potential antipsychotic profile.

Schizophrenia is a psychiatric disorder that affects 1% of the world population and is treated with antipsychotics, which may induce important biochemical and hematological alterations. Since it is necessary to verify the safety of new molecules with antipsychotic potential, the present study aimed to evaluate the oral toxicity of PT-31, a putative α2-adrenoreceptor agonist, after acute (2000 mg/kg) and repeated doses (28 days) gavage treatment, in three different doses: minimum effective dose in animal models (10 mg/kg), twice the dose (20 mg/kg), and four times the dose (40 mg/kg), as recommended by the OECD guidelines. Balb/C female adult mice were used, and biochemical, hematological, and histopathological analyses were performed. PT-31 10 and 20 mg/kg did not cause biochemical alterations related to hepatic and renal toxicity, and neither altered glycemic and lipid profiles. The preclinical dose of PT-31 also did not promote mice histopathological changes in the liver, kidney, and brain. In the hematimetric parameters, PT-31 only increased HGB at 20 mg/kg, and MCH and MCHC at 40 mg/kg. However, all the tested doses of PT-31 showed platelet increase, which must be better investigated. Therefore, further studies are needed to investigate the safety of PT-31 as a potential antipsychotic drug.

Cytotoxic and genotoxic effects induced by associated commercial glyphosate and 2,4-D formulations using the Allium cepa bioassay.

Studies assessing the toxicity of glyphosate and 2,4-dichlorophenoxyacetic acid mixture are scarce. The aim of this study was to evaluate the cytotoxicity and genotoxicity of the mixture of these herbicides using Allium cepa. Roots were exposed to glyphosate (1.56 and 11.66 mg mL-1), 2,4-D (0.28 and 17.5 mg mL-1) and mixture for 24 h, based on the average concentration applied in the field and the acute reference dose (ARfD) established in Brazil. Both isolated and associated herbicides induced a significative decrease in mitotic index (MI) (P < 0.0001) in all tested concentrations. Regarding the genotoxicity results, 2,4-D and the mixture showed, at concentrations applied in the field, a significative increase of chromosomal anomalies (CA) index compared to control (P < 0.0001) and glyphosate (P = 0.024 and P = 0.0002, respectively). All tested groups from the ARfD showed a significative difference compared to the control group (P < 0.0001), as well as glyphosate and 2,4-D isolated compared to the mixture (P = 0.0005 and P < 0.0001, respectively). The most observed CA were apoptotic bodies, giant cells, and nuclear erosions. We emphasize the need for further studies assessing the toxicity of these herbicides' mixture due to the distinct effects caused in different organisms.

Repeated dose of meloxicam induces genotoxicity and histopathological changes in cardiac tissue of mice.

Meloxicam is the non-steroidal anti-inflammatory drug most used in small animals; however, studies on genotoxicity, oxidative stress, and histopathologic alterations in cardiac tissue are limited, especially at therapeutical doses used in these animals. This study evaluated the toxic effects caused by the treatment involving repeated low at higher doses of meloxicam in mice, by genotoxicity, oxidative stress, and histopathological parameters. Mice (CF1, male) received, by gavage, meloxicam at the therapeutic dose indicated for small animals (0.1 mg/kg) and at higher doses (0.5 and 1 mg/kg) for 28 days. Later, they were euthanized for blood and organ analysis. Oxidative stress was analyzed by the plasma ferric reduction capacity (FRAP) and catalase, and genotoxicity, by the comet assay and the micronucleus test. Heart, liver, lung, and kidney tissues were analyzed by the histology, and stomach and duodenum were analyzed with a magnifying glass. The relative weight of organs did not present significant alterations. However, congestion of duodenum vessels was observed at the three tested doses and caused hyperemia of stomach mucosa at 1 mg/kg. In the heart histology there was a reduction in the number of cardiomyocytes, accompanied by an increase in cell diameter (possible cell hypertrophy) dose-dependent. The highest tested dose of meloxicam also increased the DNA damage index, without alterations in the micronucleus test. Meloxicam did not affect the catalase activity but increased the FRAP (1 mg/kg). Meloxicam at the dose prescribed for small animals could potentially cause cardiac histopathologic alterations and genotoxic effects.

Potential use of distinct biomarkers (trace metals, micronuclei, and nuclear abnormalities) in a heterogeneous sample of birds in southern Brazil.

The analysis of metal concentrations in bird feathers and genotoxicity tests are tools used to evaluate anthropogenic impacts on ecosystems. We investigated the response of birds, used as bioindicators, to disturbances observed in three areas with distinctive environmental characteristics (natural, agricultural, and urban) in southern Brazil. For this purpose, we quantified metals (Mn, Cu, Cr, and Zn) in feathers and determined the number of micronuclei (MN) and other nuclear abnormalities (NA) in 108 birds from 25 species and 17 families captured in the study area. No significant differences was found in the metal concentrations and the number of MN and NA between the sampling areas. Zn and Cu concentrations were significantly higher in insectivorous than those in omnivorous birds. The Zn concentration was significantly different between some species, and the Cu concentration was significantly higher in juveniles than that in adults. The best generalized linear models showed that omnivorous birds had more MN and NA and that juveniles and birds with better body condition index had increased NA numbers. This study demonstrates that the analyzed variables contribute in different ways to the result of each biomarker, mainly due to particular ecological and physiological characteristics of each species. We conclude that wild birds have the potential to be used as environmental bioindicators in the study area, but future studies should focus on one or a few species whose ecological and physiological habits are well known.

Evaluation of transgenerational effects caused by metals as environmental pollutants in Daphnia magna.

The present study aimed to evaluate the acute and chronic toxicity of environmentally relevant concentrations of metals (Mn, Al, Fe, and Pb) in Daphnia magna and the generational transposition of reproductive and morphological damages. The effective concentration for 10% of the organisms from each metal was obtained by the acute toxicity test (96 hours); then, another five concentrations lower than this one were defined for the chronic experimentation (21 days), in which the number of neonates generated by each individual was checked daily. At the end of the exposition, the lengths and number of morphological damages were recorded in each adult daphnid. During this, the molt generated on the 14th and 21st days were collected and cultivated for posterior evaluation of the same parameters. Alterations in the reproductive performance were observed in the organisms exposed to manganese and aluminum (4.0 and 0.5 mg L-1, respectively). Organisms exposed to aluminum (0.05 mg L-1) and iron (0.27 mg L-1) showed a reduction in body length. It is also noteworthy that the molt of these adults and their respective offspring also presented reproductive alterations, especially the molt from the 14th day of lead exposure (0.02 mg L-1) and the 21st day of manganese exposure (4.0 mg L-1). Such effects allow us to conclude that environments polluted by metals can reduce the ability of the species to maintain themselves in the ecosystem. In addition, there is a need to increase the control and monitoring of metals, such as aluminum, which present risks even in low concentrations.

Impact of Anthropogenic Activities on Water Quality of The Paranhana River, Southern Brazil

Abstract: The Paranhana River, located in Southern Brazil, is one of the major tributaries of the Sinos River basin and receives mainly industrial and domestic effluents. In the present study, water physicochemical and microbiological analyses, condition factor, micronucleus test, gill histopathology and metal bioaccumulation in the muscle of the native fish Bryconamericus iheringii collected at two sites (S1 and S2) of the Paranhana River under different degrees of anthropogenic pressures were assessed in four sampling campaigns. Data from water quality parameters, condition factor, mucous cells proliferation in fish gills and bioaccumulation of chromium and manganese in muscle evidenced higher impacts at S2, whereas a higher genotoxic potential was observed at S1. Gill histopathological alterations were found in fish captured at both sampling sites. Temporal variations in all biomarkers analyzed and bioaccumulation of manganese and nickel were observed at S1, whereas only variations in condition factor, gill alterations and bioaccumulation of manganese and aluminum were found at S2. Our study evidences that S1 is under minor anthropogenic impacts and that the high urbanization at S2 reflects in a poor water quality. Nonetheless, the human consumption of fish from the Paranhana River should be avoided given the high concentrations of cadmium, chromium and lead.

In situ monitoring of the Sinos River, southern Brazil: water quality parameters, biomarkers, and metal bioaccumulation in fish.

The Sinos River is an important water supply in Southern Brazil and receives industrial, agricultural, and domestic effluents which may affect aquatic biota. Water physicochemical and microbiological analyses, biomarker responses (scaled mass index (SMI), gill histopathology, and micronucleus and nuclear abnormality (MN and NA) frequencies), and metal bioaccumulation in muscle were assessed in the fish species Bryconamericus iheringii (Characidae) captured at three sampling sites (S1, S2, and S3) in four sampling periods. The mean values of five parameters (total phosphorus, thermotolerant coliforms, aluminum, iron, and lead) exceeded the limits established by the Brazilian legislation at the three sampling sites. Although physicochemical analysis indicated higher impacts at S3, in some samples, significantly higher MN frequencies and bioaccumulation of manganese in fish muscle were observed at S1, whereas low SMI and higher concentrations of aluminum and zinc in fish muscle were found at S2. Histopathological alterations in gills were observed in fish collected at the three sampling sites; however, no spatial differences were observed, indicating similar environmental conditions with respect to this biomarker. Moreover, temporal variation of biomarker responses and metal bioaccumulation were found at all sampling sites. Furthermore, the consumption of fish from the Sinos River should be avoided given the concentrations of chromium (all samples), cadmium, and lead in fish muscle above the threshold for safe human consumption.