The use of biomarkers to study the effects of the mixture of diuron and hexazinone on small and large O. niloticus

Pesticides have made possible a safer and plentiful supply of food; however, the ultimate sink for many of these contaminants is the aquatic environment. We analyzed the commercial mixture Velpar K® WG, which is composed of the pesticides diuron (46.8% m/m) and hexazinone (13.2% m/m), as well as inert ingredients (40.0% m/m). The present study aimed to evaluate the effects of the herbicide mixture on Oreochromis niloticus of different sizes. To this end, we analyzed biomarkers in small and large O. niloticus exposed to a mixture of herbicides at 125, 250 and 500 ug L-1 for 72h. EROD increased activity in small fish exposed to the herbicide mixture at 250 and 500 ug L-1. The GST activity and levels of the antioxidant enzymes GPx and CAT remained the same in the treated fish, compared with the control. The level of the antioxidant enzyme SOD measured in the fish gills was changed in animals exposed to the herbicide mixture at 250 ug L-1. MDA analysis did not show lipid peroxidation. The comet assay evidenced widespread DNA damage, but the micronucleus test did not show mutagenicity. Hepatosomatic (HSI) analysis did not indicate any alterations in liver morphology. The biomarkers response in the fish depended on the size of the individuals

Evaluation of cytotoxicity, mutagenicity and genotoxicity of the herbicides tebuthiuron and trifluralin and its effects on expression of cellular stress responses genes

Os herbicidas são destinados ao controle das ervas daninhas e seu uso torna o fornecimento de alimentos abundante e livre de pragas, porém a exposição ocupacional e ambiental a esses compostos pode trazer riscos à saúde. O Brasil é o maior consumidor de praguicidas desde 2008, e os herbicidas correspondem por 45% do volume dessas substâncias. O tebutiurom e a trifluralina são herbicidas muito utilizados em culturas de cana-de-açúcar, e apesar de serem descritos como seletivos em seu mecanismo de ação, seus efeitos em organismos não alvo, como células humanas, são pouco conhecidos. O objetivo do trabalho foi avaliar os efeitos dos herbicidas tebutiuron e trifluralina em organismos não alvo. Para isso, utilizou-se a linhagem celular HepG2, e as concentrações testadas dos herbicidas foram de 1 a 100 ?mol/L e o tempo de exposição variou de 4 h à 14 dias de acordo com o ensaio. Utilizou-se também as linhagens TA98 TA100, TA97a e TA1535 da bactéria S typhimurium. Neste caso, as concentrações testadas dos herbicidas variaram de 0,1 à 5000 ?g/placa e o tempo de exposição foi de 66 h. As análises indicaram que o tebutiurom não apresenta potencial citotóxico, genotóxico, ou mutagênico nas condições testadas, evidenciando sua seletividade. Testes com a trifluralina, no entanto, mostraram que HepG2 apresentaram uma diminuição da capacidade em formar clones quando expostas à 100 ?mol/L por 14 dias, e uma redução na densidade celular quando expostas à 50 e 100 ?mol/L por 24, 48 e 72h. Tais efeitos ocorreram devido a uma diminuição da viabilidade celular, observada em 50 e 100 ?mol/L pelo ensaio MTT, e devido a um bloqueio no ciclo celular na fase S, evidenciado em 100 ?mol/L, ambos nos tempos de 24, 48 e 72h. O tipo de morte celular inicialmente observada foi a apoptose, através da marcação por anexina V em 100 ?mol/L após 48 e 72 h de exposição, e através da condensação e fragmentação nuclear em 100 ?mol/L após 24 e 48 h de exposição. Em 72 h, observou-se também necrose em 100 ?mol/L, por meio dos testes anexina V/PI e liberação de LDH. A morte celular pode estar relacionada à diminuição do potencial de membrana mitocondrial, observada em 50 e 100 ?mol/L após 24, 48 e 72h, e a um aumento na produção de espécies reativas, efeito observado em células expostas à 100 ?mol/L de trifluralina por 24 e 48 h. No entanto, observou-se que a via de resposta ao estresse oxidativo Keap1/Nrf2-ARE não foi ativada no tempo analisado de 24 h. Além disso, os testes de cometa e micronúcleo não indicaram potencial da trifluralina em provocar danos no material genético de HepG2. Complementarmente, o teste de Ames em linhagens de S typhimurium também não evidenciaram potencial mutagênico do herbicida. As análises com a trifluralina mostraram que o herbicida, apesar de não induzir genotoxicidade e mutagenicidade, possui potencial citotóxico em HepG2, indicando que pode afetar organismos não alvo, como células humanasHerbicides are used to control weeds in agriculture. The use of these chemicals makes possible an abundant and pest free supply of food. However, occupational and environmental exposure to these compounds can lead to health risks. Brazil is the largest consumer of pesticides since 2008, and herbicides match for 45% of the volume of these substances. The tebutiurom and trifluralin herbicides are widely used in sugarcane crops, and although they are described as selective in its mechanism of action, effects on non-target organisms, such as human cells, are are poorly known. The aim of this work was to evaluate the effects of tebuthiuron and trifluralin herbicides on non-target organisms. To this end, we used the cell line HepG2, and herbicides were tested at concentrations of 1 to 100 ?M and the exposure time ranged from 4 h to 14 days in accordance with the assay. We also used the strains TA100 TA98, TA97a and TA1535 of the bacterium S. typhimurium. In this case, the herbicide concentrations tested ranged from 0.1 to 5000 ?g / plate and the exposure time was 66 h. Analyzes indicated that the tebutiurom has no cytotoxic, genotoxic or mutagenic potential at tested conditions, highlighting its selectivity. Tests with the Trifluralin, however, showed that HepG2 had a decreased ability in forming clones when exposed to 100 ?M for 14 days, and a reduction in cell density when exposed to 50 and 100 ?M of the herbicide for 24, 48 and 72h. These effects occurred due a decrease in cell viability observed at 50 and 100 ?M by MTT assay, and due to a block in the cell cycle into S phase, as evidenced in 100 ?M, both at 24, 48 and 72h. The type of cell death detected first was apoptosis. It was observed by staining with annexin V in cells exposed to 100 ?M of trifluralin during 48 and 72 h, and through the nuclear condensation and fragmentation in exposure with 100 ?M during 24 and 48 h of exposure. At 72 h, necrosis was also observed in 100 ?M through the annexin V / PI and LDH assays. Cell death occurrence may be associated with decreased mitochondrial membrane potential observed in 50 and 100 ?M after 24, 48 and 72h of exposure, and also may be associated with incresed production of reactive species, observed in cells exposed to 100 ?M of trifluralin for 24 and 48 h. However, it was observed that the oxidative stress response pathway Keap1 / Nrf2-ARE was not activated within 24 hours. Furthermore, comet assay and micronucleus test indicated no potential of trifluralin in causing DNA damage of HepG2. In addition, the Ames test using S. typhimurium strains also showed no mutagenic potential of the herbicide. The analyzes showed that the trifluralin, despite not induce genotoxicity and mutagenicity, have cytotoxic potential in HepG2, indicating that can affect non-target organisms, such as human cells

Evaluation of Embryo-larval Toxicity of the Antibiotic Ceftriaxone in the Fish Danio rerio (Zebrafish)

Antibiotics comprise a class of pharmaceutical products that have been generating increasing global concern due to their detection in environmental matrices. Cephalosporins constitute the largest group of antibiotics employed for human and animal treatment in most countries. Ceftriaxone belongs to the class of third-generation cephalosporins and can reach the aquatic environment. Studies in the literature have shown the toxic effect of ceftriaxone on zebrafish at high concentrations, and on cyanobacteria at environmental concentrations. The aim of this study was to evaluate the effects of ceftriaxone (0.05 mg/L, 0.5 mg/L, 5.0 mg/L, 50 mg/L and 100 mg/L) on the development of embryos and larvae of zebrafish. The Fish Embryotoxicity Test (FET) was performed according to OECD protocol n. 236. The results showed that the tested concentrations of ceftriaxone did not cause lethal or sublethal effects on zebrafish embryos and larvae. However, considering data in the literature, added to the fact that cephalosporin-class antibiotics are widely used, the use and disposal of ceftriaxone cannot be neglected

Biochemical and genotoxic effects of a commercial formulation of the herbicide tebuthiuron in Oreochromis niloticus of different sizes

Pesticides are serious contaminants because they are designed to eliminate pests, but they also affect non-target species. The present study aimed to evaluate the biochemical and genetic effects of the herbicide tebuthiuron in Oreochromis niloticus of different sizes. Thus, we analyzed biomarkers in small and large O. niloticus specimens exposed to 62.5, 125 and 250 mg L-1 of tebuthiuron for 72 hours. Fish exposed to 250 mg L-1 had high mortality rates; therefore, the data could not be used. The results showed an increase in EROD activity in fish exposed to 125 mg L-1, but no GST alteration. Antioxidant enzymes GPx and CAT were altered only in the liver of treated fish compared to the control group: CAT decreased in large fish, and GPx increased in small fish. The MDA analysis did not evidence lipid peroxidation. High DNA damage in exposed small fish (not in large fish) was observed using comet assay, but a micronucleus test did not show mutagenicity. Moreover, a comparison between control groups with specimens of different sizes revealed that small fish are more susceptible than large fish to the tebuthiuron effects, since increased comet scores was observed only for smaller fish.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP