| In recent years,with the rapid development of industry and agriculture,a lot of chemicals which hazardous to environment and human health were released into the natural environment.Plenty of evidence suggests that many chemical contaminants in environment are interfered with the normal physiological activities of wild animals and humans.Recently,a marked decline in amphibian populations has been observed globally.Although climate change and habitat destruction have been invoked as potential contributing factors,evidence is mounting that pesticides may be primarily responsible for many of these catastrophic declines.Previous studies have stressed that the tadpoles can be used as reference organisms in assessing the risk of lethal and sublethal effects induced by several emerging pollutants,including agrochemicals.The adverse.effects of pesticides on amphibians,especially larvae,should therefore be investigated in water ecological risk assessments.The class of chemicals known as the"organophosphate insecticides"(OPs)is one of most widely used pesticides after the prohibition of organochlorine.OPs used massively and repeatedly because of their high toxic and moderate persistence,were widely used worldwide.OPs account for 70%of all pesticides used in the world.Trichlorfon,or O,O-dimethyl(1-hydroxy 2,2,2-trichloroethyl)phosphonate,is a selective organophosphate insecticide that is widely used in aquaculture and agriculture as a treatment for various parasitic infestations.Trichlorfon has been widely used in the aquaculture.However,the trichlorfon-polluted waters fromthe fish and agriculture industries are potential threats to non-targeted species,such as fish,crabs,and shrimps,Therefore,the expansion of aquaculture worldwide has generated concerns about its impact on both public and environmental health.Trichlorfon is able to phosphorylate serine hydrolase at the esterasic active site,the primary action mode of which is via inhibition of acetylcholinesterase(AChE)in the myconeutral junction of skeletal muscle and of the cholinergic synapses of nervous tissue.However,studies have shown that the effects of trichlorfon were not restricted to AChE inhibition;it was also capable of inducing oxidative stress and altering the antioxidant defense system of organisms.However,there is a lack of experimental data on the effects of sublethal trichlorfon on amphibians,as available studies focus on toxic effects.Metabolic changes were observed in aquatic animals exposed to trichlorfon,which created widespread disturbances of enzymatic activities and oxidative metabolism in general physiological processes.The developmental toxicity and genotoxic effects of some pesticides on amphibians very rarely cause lethality,the risk assessment of trichlorfon as a long-term pollutant in aquatic ecosystems remains to be comprehensively investigated in a specific species.However,the molecular mechanism of the metabolic pathways involved in xenobiotic detoxification induced by trichlorfon is still unclear.The results will be helpful in gaining valuable information on the use and standardized management of OPs,water environment management and provide important scientific basis.The unique characteristics of amphibians render them as an indicator species for changes in the environment;for example,they have highly permeable skin and they reproduce and grow through critical hormone-regulated developmental stages in aquatic environments.Because amphibians spend the first phase of their life cycle in aquatic environments,their highly permeable skin makes them particularly sensitive to contaminants.Furthermore,the low mobility and high diversity in their reproductive modes render them vulnerable to environmental changes.The liver is prone to oxidative stress when there is excessive production of reactive oxygen species,which will be reduced to superoxide anions.These redox reactions occur principally in the liver,which is critical in the detoxification of xenobiotics.The Chinese brown frog(Rana chensinensis)is a unique amphibian species that is widely distributed in northern China.The larval stage is highly sensitive to aquatic chemical pollution in the environment,making it vital in aquatic environment monitoring and a convenient native species in which to research ecotoxicology in local amphibians.OPs are also capable of altering an organism's antioxidant defense system,inducing oxidative stress both in vitro and in vivo.OPs insecticides create widespread disturbances in general physiological processes,such as enzymatic activities,oxidative metabolism,oxygen consumption,and osmoregulation.Oxidative stress occurs when the critical balance between oxidants and antioxidants is disrupted due to the depletion of antioxidants or excessive accumulation of the reactive oxygen species(ROS),or both,leading to damage.The antioxidant defense system includes enzymes such as superoxide dismutase(SOD),glutathione peroxidase(GPx),catalase(CAT).glutathioneS-transferase(GST)and other low molecular weight scavengers such as glutathione(GSH).SOD is the first enzyme to respond to O,?and offers the greatest response to oxidative stress.Superoxide dismutase(EC 1.15.1.1),such as the cytosolic Cu,Zn-SOD or the mitochondrial Mn-SOD,metabolizes superoxide anion into a less reactive species,molecular oxygen and hydrogen peroxide(H2O2).Catalase(EC 1.11.1.6,CAT)is an antioxidant enzyme protecting organisms against hydrogen peroxide(H2O2),which is a reactive oxygen species(ROS)generated in many situations of stress.H2O2 is decomposed to water and molecular oxygen by the peroxisomal and mitochondrial catalase.Glutathione-S-transferase(EC 2.5.1.18,GST),a family of enzymes with a key role in the general biotransformation of xenobiotics and endogenous substances.GST is involved in the detoxification of xenobiotics and highly reactive electrophilic components can be removed before they covalently bind to tissue nucleophilic compounds,which would lead to toxic effects.There were sufficient active antioxidant enzymes to remove reactive oxygen species(ROS),and preventing the increase in lipid peroxidation in organisms when the duration concentration levels were lower than the critical level.Otherwise,oxidative stress occurs when the critical balance between oxidants and antioxidants is disrupted due to the depletion of antioxidants or excessive accumulation of the reactive oxygen species(ROS),or both,leading to damage.The repair of the level of steroid hormone could also inhibit oxidative stress reaction.The development of molecular biology technologies has provided a better understanding of the molecular mechanisms in non-model organisms under toxicological responses.RNA sequencing(RNA-seq)is a new method for both mapping and quantifying the transcriptome;it has clear advantages over the existing approaches and improves our understanding of the extent and complexity of the eukaryotic transcriptome including non-model organisms that lack completely sequenced genomes.RNA-seq has been recently applied in some amphibian species,including Odorrana margaretae and R.chensinensis,but the use of the high-throughput RNA-seq technology for studying environmental stresses is still limited.In this study,we investigated the effects of low doses of trichlorfon on R.chensinensis tadpoles after exposure.Tadpoles were exposed to 0.01,0.1 and 1.0 mg/L trichlorfon for 2 and 4 weeks.Survival,growth,development and mortality were monitored regularly over the course of exposure.Low doses of trichlorfon induced DNA damage in the erythrocytes of this species after exposure for 4 weeks.Finally,the increases in the number of leukocytes induced by trichlorfon were analyzed.Conventional agarose gel electrophoresis was used to visualize the DNA from liver tissues and revealed smears and laddering.The activities of superoxide dismutase(SOD),catalase(CAT),glutathione S-transferase(GST)and the content of malondialdehyde(MDA)in hepatic tissue were examined to evaluate the effects of oxidative stress and lipid peroxidation.The histopathological alternations to the liver were observed through light and transmission electron microscopy(TEM).To explore the interference of trichlorfon in gene transcription,the differentially expressed genes in the liver of R.chensinensis exposed to trichlorfon were characterized using the RNA-seq platform in the current study.Notably,relative expressions of metabolism-related genes,including both up-and down-regulated genes,were also validated by qPCR.1.Evaluation of toxic effects of low doses of trichlorfon on Rana tadpoles.Effects of trichlorfon on growth were evaluated by measuring the weight and the length of tadpoles.Reductions in gro"wth and disruptions to the development of tadpoles were observed in trichlorfon treatments.The results showed that trichlorfon caused a concentration-related reduction in the survival rate of the tadpoles after 4 weeks of exposure.All treated groups exhibited deformities at the end of the experiment.The most frequent deformities of exposed tadpoles were axial flexures,skeletal malformations and lateral kinks.There was a concentration-and time-dependent increase in the rate of malformations.Our results suggest that Rana tadpoles are sensitive to trichlorfon concentrations likely to occur in their aquatic habitats.The tadpoles treated with trichlorfon showed increased rates of deformities,slowed development and delayed metamorphosis.The results are consistent with those of other studies showing adverse effects in various amphibians.These results confirm that exposure exerts an effect on the thyroid axis by decreasing levels of thyroid hormone(T3)in amphibians.2.Evaluation of genotoxic effects of low doses of trichlorfon on Rana chensinensis tadpoles.MN analysis is widely used as a bioassay of genotoxicity to detect small chromosomal fragments produced by clastogens or vagrant chromosomes produced by aneugens.The frequency of MN in the erythrocytes varied as a dependent function of time and concentration.A significant increase in the frequency of MN was observed at 4 weeks of treatment for all tested concentrations of trichlorfon compared to negative controls.An increase in the frequency of other nuclear abnormalities was observed only in tadpoles exposed to 1.0 mg/L trichlorfon.In addition,a concentration-and duration-dependent increase in the frequency of leukocytes was observed at all concentrations.An approximately two-fold increase in the frequency of leukocytes was noted at 0.1 and 1.0 mg/L,indicating the genotoxic effects of trichlorfon.This result suggested that morphological nuclear abnormalities are indicators of genetic damage.The increase in the number of leukocytes induced by trichlorfon indicates that more leukocytes were pumped from the haematopoietic tissue into the peripheral blood to defend against the damage caused by environmental pollution.3.Evaluation of DNA damage and apoptosis effects of low doses of trichlorfon on Rana chensinensis tadpoles.SCGE assays are widely used in toxicology,environmental monitoring,and other fields to detect DNA damage and have become valuable as a biomarker to monitor amphibians in contaminated areas.Tadpoles exposed to various concentrations of trichlorfon exhibited significantly higher levels of DNA damage than did the corresponding controls,and the DNA damage was concentration-and time-dependent:trichlorfon induced higher levels of DNA damage in the cells as the concentration in the culture media increased.The cytotoxic effect of trichlorfon was further examined by evaluating apoptosis-associated DNA fragmentation in hepatic tissues.Trichlorfon induced a weak ladder-like fragmentation pattern.Untreated cells showed intact DNA,while DNA ladders gradually formed with increasing doses of trichlorfon and increasing exposure time.These results are consistent with the data acquired through SCGE.In summary,the pesticide trichlorfon may induce DNA damage and apoptosis in two ways:(1)via oxidative stress(for instance,excessive ROS),which may promote DNA damage and apoptosis;and(2)via the mitochondrial pathway.4.Oxidative stress in the frog,Rana chensinensis,when exposed to low doses of trichlorfon.The activities of superoxide dismutase(SOD),catalase(CAT),glutathione S-transferase(GST)and the content of malondialdehyde(MDA)in hepatic tissue were examined to evaluate the effects of oxidative stress and lipid peroxidation.The results showed that SOD and CAT activities were increased in the livers of frogs exposed to various concentrations of trichlorfon.GST activity showed no significant changes at any concentration after 2 weeks of exposure,whereas there was an initial increase after exposure to 0.1 mg/L of trichlorfon at 4 weeks.The content of MDA revealed a significant decrease after exposure.These results suggest that low doses of trichlorfon could induce oxidative stress,lipid peroxidation and hepatic lesions in frogs,which shows that even lower,non-lethal doses of trichlorfon are potentially toxic to amphibians.The increased activities in SOD and CAT in this study could be related to the increased oxidative stress caused by trichlorfon exposures.In the oxidative station,the SOD-CAT system converted the oxidative stress to less reactive species.The increase of SOD activity indicated an increase in O2-production,which supports the hypothesis that a compensatory up-regulation of antioxidants occurs after the initial generation of ROS.The enhancement in CAT activity was attributed to an increase in the concentration of its substrate,which is the major metabolite in SOD catalysis.In the present study,no sensitive response of GST was observed in the livers of frogs exposed to trichlorfon for 2 weeks,which might indicate that this enzyme is not involved in the metabolism of trichlorfon or that GST is not a very sensitive endpoint as a response at this time point.Increased GST in tissues should indicate the development of a defensive mechanism to counteract the effects of trichlorfon,which reflects the possibility of a more efficient protection against pesticide toxicity.GST-mediated conjugation may be an important mechanism for detoxifying peroxidized lipid breakdown products.Reduced or unchanged MDA contents may reflect the protective effects of the antioxidant system in R.chensinensis,as observed at different times.5.Pathological analysis in the frog,Rana chensinensis,when exposed to low doses of trichlorfon.The histopathological alternations to the liver were observed through light and transmission electron microscopy(TEM).Histopathological and ultrastructural studies showed that trichlorfon induced hyalinization,vacuolation,nucleus necrosis,and cellular swelling in hepatocytes.The histocytopathological alterations suggest that the treated frogs faced a metabolic crisis caused by serious tissue damage,which resulted in the alteration of metabolism caused by oxidative stress and a disorder of lipid metabolism.These biochemical events might mediate a number of redox-dependent processes and alterations in mitochondrial function,which in turn may have triggered the activation of specific cascades.The dilation of mitochondria,endoplasmic reticulum stress and nuclei damage observed in the hepatocyte ultrastructure in the groups treated with trichlorfon may activate the regulation of the intrinsic pathways of apoptosis,which can be induced by environmental stress or toxicity.6.Transcriptome analysis of Rana chensinensis liver under trichlorfon stress.To explore the interference of trichlorfon in gene transcription,the differentially expressed genes in the liver of R.chensinensis exposed to trichlorfon were characterized using the RNA-seq platform in the current study.A search of all unigenes against non-redundant protein sequence(Nr),non-redundant nucleotide(Nt),Swiss-Prot,Kyoto Encyclopaedia of Genes and Genomes(KEGG),Clusters of Orthologous Groups(COG)and Gene Ontology(GO)databases resulted in 22,888,21,719,20,934,16,923,7,375 and 15,631 annotations,respectively,and provided a total of 27,781 annotated unigenes.Among the annotated unigenes,16,923 were mapped to 257 signalling pathways.The hits from the COG prediction were functionally classified into 24 categories.After parsing the GO annotations output,15,631(28.3%)GO terms were finally obtained,with 52.18%for biological process,34.83%for cellular components and 12.99%for molecular functions.The three main GO categories were classified into 53 subcategories.A total of 16,923(30.64%)unigenes were mapped to 257 KEGG signalling pathways,including metabolic pathways,endocytosis,MAPK signalling pathway,and PPAR signalling pathway.7.Identification of DEGs induced by trichlorfon stress.To identify genes that displayed significant changes in expression during trichlorfon stress,DEGs were analysed by comparing the treated and control libraries.In all,the abundance of 3,329 filtered unigenes of varying overall length showed evidence of differential transcription.A higher number of genes were down-regulated than up-regulated in response to trichlorfon stress.From the data sets,a total of 1444 up-regulated and 1885 down-regulated DEGs were detected after exposure to trichlorfon for 4 weeks.The functional annotations of the unigenes according to the COG,GO and KEGG databases provided ample numbers of candidate genes and more information about their biological features in R.chensinensis under trichlorfon stress.Therein,unigene 22540,CL307.Contig2,and CL1105.Contigl were classified into metabolic pathways,whereas CL3031.Contig2,CL307.Contig2,and CL1105.Contigl were classified as the metabolism of xenobiotics and drugs.Additionally,antioxidant genes including endothelial nitric oxide synthase(eNOS)and glutathione S-transferase(GST)were also identified in this transcriptome database.All of this information suggests that metabolism and antioxidant defence systems should be activated to protect amphibians against pathogen infections under trichlorfon stress.8.Validation of RNA-seq profiles by qPCR.To test and verify the credibility of the RNA-seq results,qPCR experiments were performed for validation.Four candidate genes involved in metabolic pathways and metabolism of xenobiotics,which included both induced and repressed genes,were selected to perform qPCR and be compared with the RNA-seq data at the gene transcript level.The results showed that the expression patterns for the four DEGs corroborated with the RNA-seq-based regulation analysis.These results also further confirmed the reliability of the RNA-seq data and the accuracy of the Trinity assembly.Altered eNOS expression in the liver is an important factor in the progression of liver fibrogenesis to stress.The increased CL307.contig2(CYP2C)expression levels in the liver,which could lead to enhanced trichlorfon metabolism.Meanwhile,there is strong evidence suggesting that the activation of CYP2C epoxygenase in endothelial cells is an essential step in the NO-independent vasodilatation of several vascular beds.The decreased levels of CYP3A may hinder the metabolism of trichlorfon,resulting in the accumulation of the compound and its toxicity.Down-regulation of CL3031.contig2(glutathione S-transferase kappa 1)gene expression highlighted the fact that this enzyme probably plays divergent physiological roles during trichlorfon detoxification in R.chensinensis.Different xenobiotics might affect transcription factors and then further affect the transcript of GST expression and the incomplete degradation of xenobiotics absorbed by the organisms could possible explanations for these phenomena.The results indicated that these DEGs are implicated in response to trichlorfon stress.Furthermore,the bioinformatics analysis of the transcriptome in this study generated a large number of novel genes,which will be of great value for understanding the toxicity mechanisms of trichlorfon in non-target species.Taken together,this study demonstrates that low doses of trichlorfon are developmentally toxic in R.chensinensis tadpoles,with consequences including increased mortality,delayed developmental progress,prolonged metamorphosis time and increased incidences of morphological alterations.Additionally,low doses of trichlorfon can induce DNA damage in the erythrocytes and hepatic cells of amphibian larvae.Trichlorfon-induced DNA damage showed a dose-response relationship.Moreover,smeared apoptosis was revealed by a DNA fragmentation assay.Repeated trichlorfon exposure to amphibians at low doses caused the highlighted antioxidant response involving a different modulation of SOD,CAT,GST and MDA.The SOD activity was the-most sensitive parameter.In addition,trichlorfon could-induce serious histocytopathological damage,which then resulted in the alteration of the metabolism of the organism.We have determined the transcriptomic response of R.chensinensis under trichlorfon stress with RNA-seq to examine the liver transcriptome profile and identify DEGs.Metabolic pathways-related transcripts were detected in this dataset.All these results suggest that the wild frog populations may be affected by pesticides in agroecosystems,which has profound implications in light of the amphibian decline.The data obtained in this research can serve as a fundamental reference for evaluating the potential negative ecological effects of organophosphate insecticides in amphibians,which is helpful for the long-term conservation of amphibian species.Furthermore,the bioinformatics analysis of the transcriptome in this study generated a large number of novel genes,which will be of great value for understanding the toxicity mechanisms of trichlorfon in non-target species. |
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