Recent evidence for toxic effects of NH3 exposure on lung injury: Protective effects of L-selenomethionine.

Ammonia (NH3) is a common air pollutant, which poses a serious threat to farm animals. L-selenomethionine is organic selenium (Se), which can inhibit intracellular ROS generation, block ROS-dependent autophagy, promote mitochondrial energy metabolism, and enhance the body's immunity. Lung, as an important organ of the respiratory system, is highly susceptible to the toxic effects of NH3. However, there were few studies on the mechanism of toxic effects of NH3 on lung tissues. The aim of this study was to investigate the effect of NH3 on the lungs in pigs and the alleviating effect of L-selenomethionine. Twenty-four Large White*Duroc*Min pigs were randomly assigned to 4 groups: control group, NH3 group, Se group, and NH3 +Se group. The results showed that exposure to NH3 caused damage and inflammation in lung tissues and significantly increased blood NH3 concentration. NH3 induced changes of oxidative stress indexes (GSH, GSH-Px, SOD, MDA, Keap1, Nrf2, and HO-1) and expressions of energy metabolism related genes (HK1, HK2, PFK, PK, LDHA, and HIF-1α). Ultrastructure showed that mitochondrial damage and autophagosome increased significantly, and the expression levels of autophagy related genes (Beclin1, ATG5, ATG7, ATG10, and p62) changed. However, the addition of L-selenomethionine alleviated the above changes, but there was still a significant difference compared with the control group (P < 0.05). This finding can provide a new evidence for mitigation of NH3 toxicity.

Intestinal barrier dysfunction induced by ammonia exposure in pigs in vivo and in vitro: The protective role of L-selenomethionine.

Ammonia has been reported to have a variety of toxicity to aquatic animals, farm animals and humans. However, its potential toxicity on the intestines remains unknown. L-selenomethionine is one of the important organic selenium sources. However, the mitigating effect of L-selenomethionine on ammonia exposure toxicity is still lacking. Therefore, in this study, the mechanism of toxic action of ammonia on intestinal tract and the detoxification effect of L-selenomethionine were examined. We evaluated the intestinal toxicity of ammonia and the alleviating effect of L-selenomethionine in an in vivo model, and then verified it in vitro model by a variety of cutting-edge experimental techniques. Our results showed that ammonia exposure causes oxidative stress, necroptosis, Th1/Th2 imbalance and inflammation in the intestinal tissue and the intestinal cells, and L-selenomethionine had a significant mitigation effect on the changes of these indexes induced by ammonia. In conclusion, ammonia exposure caused oxidative stress and Th1/Th2 imbalance in the porcine small intestine and IPEC-J2 cells, and that excessive ROS accumulation-mediated necroptosis targeted inflammatory responses, resulting in the destruction of tight connections of intestinal cells, thereby causing intestinal barrier dysfunction. L-selenomethionine could effectively reduce the intestinal injury caused by ammonia exposure and antagonize the toxic effect of ammonia.

Protective role of selenium on ammonia-mediated nephrotoxicity via PI3K/AKT/mTOR pathway: Crosstalk between autophagy and cytokine release.

Ammonia (NH3) is a hazardous substance to human and animal health. Selenium (Se) is an essential micronutrient with multiple health benefits. The present study aimed to verify whether and how Se supplementation has a protective role against NH3 mediated-nephrotoxicity in pigs. A Se-NH3 interaction model was established in pigs and the kidney samples were collected after a 30-day treatment period. The results showed that NH3 exposure inhibited the PI3K/AKT/mTOR pathway and enhanced the secretion of inflammatory cytokines to induce autophagy and inflammation. Se can regulate the PI3K/AKT/mTOR pathway and attenuate the secretion of inflammatory cytokines altered by NH3 to reduce autophagy and inflammation. In addition, Se co-treatment inhibited ROS production, elevated the activities of antioxidant systems, and increased the expression of 13 selenoproteins in pig kidneys caused by NH3 exposure. These results implied that L-selenomethionine can moderate NH3-induced nephrotoxicity in pigs. Our study gives new ideas for the specific mechanism of NH3 nephrotoxicity and provides a reference for comparative medicine and clinical medication.

Ammonia exposure causes the disruption of the solute carrier family gene network in pigs.

Ammonia is the main harmful gas in livestock houses. However, the toxic mechanism of ammonia is still unclear. Therefore, we examined the effects of ammonia exposure on different tissues of fattening pigs by histological analysis and transcriptome techniques in this study. The results showed that there were varying degrees of pathological changes in liver, kidney, hypothalamus, jejunum, lungs, spleen, heart and trachea of fattening pigs under ammonia exposure. Notably, the extent of damage in liver, kidney, jejunum, lungs, hypothalamus and trachea was more severe than that in heart and spleen. Transcriptome results showed that ammonia exposure caused changes in 349, 335, 340, 229, 120, 578, 407 and 115 differentially expressed genes in liver, kidney, spleen, lung, trachea, hypothalamus, jejunum and heart, respectively. Interestingly, the changes in solute vector (SLC) family genes were found in all 8 tissues, and the verified gene results (SLC11A1, SLC17A7, SLC17A6, SLC6A4, SLC22A7, SLC25A3, SLC28A3, SLC7A2, SLC6A6, SLC38A5, SLC22A12, SLC34A1, SLC26A1, SLC26A6, SLC27A5, SLC22A8 and SLC44A4) were consistent with qRT-PCR results. In conclusion, ammonia exposure can cause pathological changes in many tissues and organs of fattening pigs and changes in the SCL family gene network. Importantly, the SCL family is involved in the toxic mechanism of ammonia. Our findings will provide a new insight for better assessing the mechanism of ammonia toxicity.

Novel insights into cytochrome P450 enzyme and solute carrier families in cadmium-induced liver injury of pigs.

Cadmium (Cd) is a typical pollutant and carcinogen in environment. Exposure assessment of contaminants is an important component of occupational and environmental epidemiological studies. Early studies of Cd have focused on aquatic animals, chickens and rats. However, toxicological evaluation of Cd in pigs has not been reported. Therefore, twelve pigs were randomly divided into two groups (n = 6): the control group and the Cd group (Cd content: 15 ± 0.242 mg/kg feed) in this study, the experimental period was 30 d, and the toxic effects of Cd on the liver of weanling piglets were examined by antioxidant function, liver function, Cd content, histological examination and transcriptomics. The results showed that the changes of antioxidant function, liver function and Cd content were significant in the liver. Transcriptional profiling results showed that 399 differentially expressed genes (DEGs) were significantly up-regulated while 369 DEGs were remarkably down-regulated in Cd group, and which were concentrated in three ontologies: molecular function, cellular component and biological processes. Interestingly, significant changes in some genes of the cytochrome P450 enzyme (CYP450) and solute carrier (SLC) families have been observed and were consistent with qRT-PCR results. In conclusion, Cd could cause liver injury in weanling piglets and change the transcriptomic characteristics of liver. CYP450 and SLC families play an indispensable role in Cd-mediated hepatotoxicity. Importantly, changes in mRNA levels of CYP2B22, CYP7A1, CYP8B1, SLC26A8, SLC11A1, SLC27A2 and SLC22A7 induced by Cd have been reported for the first time. Our findings will provide a new insight for better assessing the mechanism of Cd toxicity to the liver.

Selenomethionine protects against ammonia-induced apoptosis through inhibition of endoplasmic reticulum stress in pig kidneys.

Ammonia (NH3) emission is a common threat to farm animals. Selenium (Se) is known for its antioxidant property and can resist several stressors affecting farm animals. The aims of this study were (Ⅰ) to determine how excess NH3 exert nephrotoxic effects in pigs and (Ⅱ) to investigate whether selenomethionine has an alleviative effect on NH3 toxicity. Two diets supplemented with different doses of Se (0.22 mg/kg or 0.50 mg/kg) and two concentrations of NH3 (< 5 mg/m3 or 89.8 mg/m3) were used in a 2 × 2 factorial design trial for a period of 30 days. The results showed that NH3 exposure caused apoptosis and increased the number of apoptotic cells in pig kidneys. Further, the activities of antioxidant enzymes were decreased, and the transcriptional and translational levels of endoplasmic reticulum stress-related genes, Bcl-2 and Caspase family members were increased under NH3 exposure. In addition, Wnt/ß-catenin signaling pathway was suppressed after NH3 treatment. Dietary supplement with selenomethionine appears to offer protection against NH3-induced kidney injury in pigs and the pathologic changes above were alleviated. Our findings provide additional insight into the mechanism of NH3 toxicity in pigs while elucidating the role of Se as a potential antidote against NH3 poisoning.

Ammonia exposure induces oxidative stress and inflammation by destroying the microtubule structures and the balance of solute carriers in the trachea of pigs.

Ammonia (NH3) is the most alkaline gaseous compound in the atmosphere and the primary gas pollutant in the piggery. It can cause irritation and damage to the airway after inhalation. However, the effects and toxicity mechanism of NH3 on the trachea are still unclear. In order to evaluate the toxic effects of NH3 inhalation on pig trachea, the changes of oxidative stress parameters (SOD, GSH, GSH-Px, and MDA), tissue structure and transcriptome in the trachea of pigs were examined after 30 days of exposure to NH3. Our results showed SOD, GSH-Px and GSH in the trachea in the NH3-treatment group were significantly decreased (P < 0.05) compared with the control group, on the contrary, MDA content was significantly higher (P < 0.05). The analysis of differentially expressed genes (DEGs) showed that 2542 DEGs (1109 up-regulated DEGs and 1433 down-regulated DEGs) were significantly changed under NH3 exposure, including many DEGs associated with inflammation, oxidative stress, microtubule activity and SLC family, and the qRT-PCR verification results of these DEGs were consistent with the transcriptome results. The results indicated that NH3 exposure could break down the mucosal barrier of the respiratory tract, induce oxidative stress and inflammation, reduce the activity of microtubules and disrupt the balance of SLC transporters. In this study, transcriptome analysis was used for the first time to explore the toxic mechanism of NH3 on pig trachea, providing new insights for better assessing the toxicity mechanism of NH3, as well as references for comparative medicine.

H2S exposure-induced oxidative stress promotes LPS-mediated hepatocyte autophagy through the PI3K/AKT/TOR pathway.

Hydrogen sulfide (H2S), a common air pollutant and toxic gas, is detrimental to organisms and the environment. Exposure to highly concentrated H2S can induce oxidative stress and autophagy. However, the mechanism underlying the liver damage caused by H2S has not been identified. Lipopolysaccharide (LPS), the key component of endotoxin, can induce oxidative stress and autophagy. For this experiment, we used one-day-old chickens as model organisms to evaluate the effects of H2S combined with LPS on oxidative stress and autophagy. The four groups (control group, LPS group, H2S group and H2S-LPS group) were observed by electron microscopy, detected by oxidative stress kit, analyzed by quantitative real-time quantitative PCR, and analyzed by Western blot. We found that the activities of antioxidant enzymes (superoxide dismutase, antioxidant glutathione, catalase, and glutathione peroxidase) decreased in the H2S group compared to those in the control group; however, malondialdehyde levels in the H2S group increased. Molecular-level studies showed that the expression of genes associated with the PI3K/ AKT/ TOR pathways in the H2S group decreased, whereas the expression of other autophagy-related genes (Beclin1, ATG5 and the ratio of LC3-II/ LC3-I) increased compared to that in the control group. These findings suggest that H2S caused oxidative stress and induced autophagy through the PI3K/ AKT/ TOR pathway in chicken liver cells. Additionally, exposure to H2S aggravated LPS-induced oxidative stress and autophagy injury. Capsule: Aerial exposure to H2S can cause oxidative stress in chicken livers and induce autophagy through the PI3K/AKT/TOR pathway, and can aggravate LPS-induced oxidative stress and autophagy.

Ammonia exposure causes the imbalance of the gut-brain axis by altering gene networks associated with oxidative metabolism, inflammation and apoptosis.

Ammonia is an acknowledged environment pollutant in atmosphere with irritating smell. Previous studies have shown that excessive ammonia has toxic effects on farm animals and humans. However, the detail toxicity mechanism of ammonia to pigs is still unknown so far. In order to clarify the mechanism of ammonia toxicity, we established a porcine exogenous ammonia poisoning model and assessed the effects of ammonia on the gut-brain axis by transcriptome sequencing, histological observation and chemical analysis. Our results showed that after 30 d of ammonia exposure, 578 differentially expressed genes (DEGs) and 407 DEGs were obtained in the hypothalamus and jejunum, respectively. These DEGs were enriched into Gene Ontology terms associated with inflammation, oxidative metabolism, apoptosis, and the highly expressed genes among these DEGs were verified by real-time quantitative PCR. The content of glutathione and the activities of glutathione peroxidase and superoxide dismutase were significantly decreased, while malondialdehyde content was increased after ammonia exposure. Corticotropin releasing factor, substance P, 5-hydroxytryptamine and ghrelin contents in serum elevated significantly. Furthermore, pathologic observation in the ammonia group revealed infiltration of lymphocytes in the hypothalamus and significant decrease of jejunal epithelial cells. Our results indicated that ammonia exposure mediated changes in transcriptional profiles, pathological damage, oxidative stress and brain-gut peptide of the pig jejunum and hypothalamus, and induced the imbalance of the brain-gut axis through the "oxidative stress-inflammation-apoptosis" interaction network. Our study not only provides a new perspective for the toxicity assessment of ammonia, but also enriches the toxicology mechanism of ammonia.

Oxidative stress, inflammation, and glycometabolism disorder-induced erythrocyte hemolysis in selenium-deficient exudative diathesis broilers.

Selenium (Se) deficiency causes injury of diversified tissues and cells, including livers, hearts, skeletal muscles, and erythrocytes. The aim of the present study is to explore the molecular mechanism of erythrocyte hemolysis due to Se deficiency in broilers. One hundred and eighty broilers (male/female, 1 day old) were randomly divided into two groups and fed with either a normal Se content diet (C group, 0.2 mg Se/kg) or a Se-deficient diet (ED group, 0.008 mg Se/kg) for 45 days. During the trial period of 15-30 days, biological properties such as osmotic fragility, fluidity, phospholipid components of cell membrane, adenosine triphosphatase activities, and antioxidant function of erythrocytes in broilers were examined. Moreover, the messenger RNA (mRNA) expressions of genes associated with inflammation, glycometabolism, and avian uncoupling protein (avUCP) were detected. We found that compared with the C group, hemolysis rate, degree of polarization, and microviscosity of erythrocytes were increased in broilers of the ED group. The composition of erythrocyte membrane lipids was changed. Meanwhile, the antioxidant function of erythrocytes was weakened and mRNA levels of inflammatory genes were stimulated by Se deficiency (p < 0.05). In addition, mRNA expressions of rate-limiting enzymes in glycometabolism were effected and avUCP mRNA level was downregulated (p < 0.05) in the ED group. It has been concluded from the results that oxidative stress, inflammatory response, and glycometabolism disorder lead to erythrocyte hemolysis by changing the structure and function of erythrocyte membrane in ED broilers suffered from Se deficiency.

Impaired immune function and structural integrity in the gills of common carp (Cyprinus carpio L.) caused by chlorpyrifos exposure: Through oxidative stress and apoptosis.

As one of the mucosal lymphatic tissues, the gill is an important immune organ in fish. Water environmental pollutants enter fish body through the gill. Therefore, the gill is the initial site where pollutants produce toxic effects in water. Chlorpyrifos (CPF), a broad-spectrum organophosphate insecticide, is widely used for agricultural pests and causes river pollution. In the present study, we investigated histopathological effect, oxidative stress indexes (SOD, GSH, T-AOC, and MDA), and apoptosis-related genes (P53, PUMA, Bax, Bcl-2, Apaf-1, Caspase-9, and Caspase-3) in the gills of common carp exposed to CPF. The results indicated that CPF exposure decreased SOD, T-AOC, and GSH; increased MDA; decreased Bcl-2 mRNA expression; and increased P53, PUMA, Bax, Apaf-1, Caspase-9, and Caspase-3 mRNA expressions in common carp gills. Our results proved that CPF exposure caused oxidative stress and apoptosis in common carp gills; CPF exposure destroyed the structural integrity and affected the immune function through oxidative stress and apoptosis in common carp gills. These will provide evidence for the toxic effects of water environmental pollutants on immune function and structural integrity in fish gills.

Chlorpyrifos induced oxidative stress to promote apoptosis and autophagy through the regulation of miR-19a-AMPK axis in common carp.

Chlorpyrifos (CPF) has become a mainly pollution in water environment. Micro-RNAs (miRNAs) play an important part in the development of apoptosis and autophagy. However, the potential mechanism of CPF induced kidney toxicity and the roles of miRNAs are still unclear. To explore the underlying mechanism, the kidney of common carp exposed to different concentrations of CPF for 40 days was used as a research object. We found that CPF could damage the ultrastructure and function of kidney; and also caused antioxidant system disorder. CPF inhibited the mRNA level of miR-19a which improved AMP-activated protein kinase (AMPK). Furthermore, the detection of apoptosis and autophagy relative genes showed that the expressions of TSC complex subunit 2 (TSC2), light chain 3 (LC3), Dynein, tumor protein 53 (p53), Bcl-2 associated X protein (Bax), caspase-3 and caspase-9 were enhanced and the expressions of nechanistic target of rapamycin (mTOR), Ras homolog mTORC1 binding (Rheb) and B-cell lymphoma (Bcl-2) were reduced in dose-dependent way. Taken together, we conclude that CPF causes oxidative stress and miR-19a-AMPK axis disorder, thereby promotes apoptosis and autophagy in common carp kidney. Our study will provide theoretical basis for toxicology research and environmental protection of CPF.

The co-expression of circRNA and mRNA in the thymuses of chickens exposed to ammonia.

Ammonia (NH3) is one of major air pollutants in intensive poultry houses, affecting chicken health. Circular RNA (circRNA) is a novel type of RNA that can regulate gene expression and be associated with various biological activities. However, the changes of circRNA caused by excess NH3 in chickens have not been investigated. We found differentially expressed genes and morphological changes in the thymuses of chickens exposed to NH3 on day 42. We used a combination of RNA deep sequencing, qRT-PCR, and bioinformatic analysis to explore regulatory mechanism of circRNA and mRNA. Transcriptional profiling results showed that 5 circRNA genes and 100 mRNA genes were significantly dyregulated by high NH3. The results from GO items showed that immune response and the regulation of cytokine production were involved in the mechanisms of chickens exposed to NH3. Co-expression analysis found that circRNA-mRNA network was correlated with oxidative stress and inflammation. NH3 exposure decreased mRNA expression of antioxidant-related genes (GPx and GST4) and increased the mRNA expression of inflammation-related genes (IL-1ß, IL-6, IL-8, and iNOS) in chicken thymuses. Histopathologic analysis demonstrated that NH3 caused inflammatory injury in chicken thymuses. In conclusion, the co-expression of circRNA and mRNA took part in chicken thymus inflammatory injury caused by NH3. Our study further enriches the mechanism of NH3 toxicity on chickens, which may be valuable for human and animal health protection.

Identification of signal pathways for immunotoxicity in the spleen of common carp exposed to chlorpyrifos.

Chlorpyrifos (CPF) is an environmental pollutant due to its high toxicity to aquatic animals. Because CPF was detected in aquatic environments in many countries, it has been widely concerned by researchers. Although the immunotoxicity of CPF to fish had been reported, the immunotoxicity mechanism is still not clear. Recently, transcriptome analysis has become a major method to study the toxic mechanism of pollutants in environmental toxicology. However, the immunotoxicity identification of CPF on fish had not been reported by transcriptome analysis. In the present study, we examined the effects of CPF on organismal system in the spleen of common carp by transcriptome analysis. We have successfully constructed a database of transcriptome analysis of carp spleens under exposure to CPF and found 773 differentially expressed genes (DEGs) (including 498 up-regulated DEGs and 275 down-regulated DEGs) and 4 branches (containing 33 known KEGG pathways). Some genes associated with the 4 pathways (Complement and coagulation cascades, PPAR signaling pathway, Fat digestion and absorption, and Collecting duct acid secretion) contained in organismal system were validated by quantitative real-time PCR and showed significant improvement compared with the control group. Our results indicated that exposure to CPF caused a change in the signal pathways of organismal system in carp spleens. The present study provides new insights into the immunotoxicity mechanism and risk assessment of CPF, as well as references for comparative medicine.

Effects of Atrazine and Chlorpyrifos on Autophagy-Related Genes in the Brain of Common Carp: Health-Risk Assessments.

This study assessed the impacts of atrazine (ATR), chlorpyrifos (CPF), and a combined ATR/CPF exposure on the brain of common carp (Cyprinus carpio L.). The carp were sampled after a 40-days exposure to CPF and ATR, individually or in combination, followed by a 40-days recovery period to measure autophagy and antioxidant activity. The results indicate that the anti-superoxide anion and anti-hydroxy radical activities decreased upon exposure to ATR, CPF, and the ATR/CPF combination but increased after a subsequent 40-days recovery period. Quantitative real-time PCR and Western blot analyses revealed that the mRNA and protein levels of LC3B and dynein in common carp decreased significantly after exposure to ATR and CPF alone or in combination. Moreover, the mRNA and protein levels of beclin1 gene decreased significantly only in the 116 and 11.3 µg/L treatment groups. However, the mRNA and protein levels of all tested genes increased significantly after a 40-days recovery. Transmission electron microscope demonstrated the occurrence of autolysosomes in the recovery groups but not in the exposure groups. These results suggest that exposure to ATR, CPF, or their combination promotes oxidative stress and autophagic responses in the brain of common carp.

Acute and subchronic toxic effects of atrazine and chlorpyrifos on common carp (Cyprinus carpio L.): Immunotoxicity assessments.

Atrazine (ATR) and chlorpyrifos (CPF) are widely used pesticides in agricultural practices throughout world. It has resulted in a series of toxicological and environmental problems, such as impacts on many non-target aquatic species, including fish. The spleen and head kidney in the bony fish are the major hematopoietic organs, and play a crucial part in immune responses. This study evaluated the subchronic effects of ATR and CPF on the mRNA and protein levels of HSP60, HSP70 and HSP90 in the immune organs of common carp and compared the acute and subchronic effects of ATR and CPF on the swimming speed (SS) of common carp. The results of acute toxicity tests showed that the 96 h-LC50 of ATR and CPF for common carp was determined to be 2.142 and 0.582 mg/L, respectively. Meanwhile, acute and subacute toxicity of ATR and CPF in common carp resulted in hypoactivity. We also found that the mRNA and protein levels of HSP60, HSP70 and HSP90 genes were induced in the spleen and head kidney of common carp exposed to ATR and CPF in the subchronic toxicity test. Our results indicate that ATR and CPF are highly toxic to common carp, and hypoactivity in common carp by acute and subchronic toxicity of ATR and CPF may provide a useful tool for assessing the toxicity of triazine herbicide and organophosphorous pesticides to aquatic organisms. In addition, the results from the subchronic toxicity test exhibited that increasing concentration of ATR and CPF in the environment causes considerable stress for common carp, suggesting that ATR and CPF exposure cause immunotoxicity to common carp.

Effects of atrazine and chlorpyrifos on oxidative stress-induced autophagy in the immune organs of common carp (Cyprinus carpio L.).

Atrazine (ATR) and chlorpyrifos (CPF) are the most common agrochemical in the freshwater ecosystems of the world. This study assessed the effects of ATR (4.28, 42.8 and 428 µg/L), CPF (1.16, 11.6 and 116 µg/L) and combined ATR/CPF (1.13, 11.3 and 113 µg/L) on common carp head kidneys and spleens following 40 d exposure and 40 d recovery treatments. Nitric oxide (NO) content, activities of anti hydroxyl radical (AHR), anti superoxide anion (ASA), peroxidase (POD) and inducible nitric oxide synthase (iNOS), and the mRNA levels of the autophagy genes (LC3-II, dynein, TOR) were determined. The results indicate that the antioxidant enzyme (AHR, ASA, POD and iNOS) activities and NO content in the head kidney and spleen of the common carp increased significantly after a 40 d exposure to ATR and CPF alone or in combination. The mRNA levels of LC3-II and dynein in common carp increased significantly after exposure to ATR and CPF alone, or in combination. Moreover, the mRNA levels of LC3-II and dynein decreased significantly after a 40-d recovery. However, the mRNA levels of TOR gene for all decreased significantly at the end of the exposure and the recovery. To our knowledge, this is the first study to report the oxidative stress-induced autophagic effects in the common carp by exposure to ATR, CPF and the ATR/CPF combination. The information presented in the present study may be helpful to understanding the mechanisms of autophagy induced by ATR, CPF and the ATR/CPF combination in fish.

Atrazine and chlorpyrifos exposure induces liver autophagic response in common carp.

Under normal conditions, autophagy occurs at basal levels but can be induced rapidly in response to stress conditions and extracellular signals. Increasing experimental evidence indicates that the expression of autophagy-related genes play very important roles in toxicology. Atrazine (ATR) and chlorpyrifos (CPF) are the most common agrochemical in the freshwater ecosystems of the world. This study assessed the effects of ATR, CPF and combined ATR/CPF exposure on the liver of common carp. Carp were sampled after a 40-d exposure to ATR and CPF, individually or in combination, followed by a 40-d recovery to measure the mRNA and protein levels of autophagy-related genes in the liver. In addition, we also investigated the change in ultrastructure in the liver. The results revealed that the mRNA and protein levels of microtubule-associated protein 1 light chain 3 B (LC3B) and dynein were significantly induced in the treated groups compared to the solvent control group. Transmission electron microscope assays indicated that autolysosomes were observed in the exposure and recovery groups. These results indicated that ATR and CPF could induce autophagy in carp liver. To the best of our knowledge, this is the first report to study the autophagy effects caused by sub-chronic exposure to ATR, CPF and the ATR/CPF combination in common carp. The information presented in the present study may provide new insights into the mechanisms used by fish to adapt to stressful environments.

Assessment of pesticide residues and gene expression in common carp exposed to atrazine and chlorpyrifos: Health risk assessments.

This study assessed the impacts of atrazine (ATR), chlorpyrifos (CPF) and combined ATR/CPF exposure on the kidney of common carp (Cyprinus carpio L.). The carp were sampled after a 40-d exposure to CPF and ATR, individually or in combination, followed by a 40-d recovery to measure the expression levels of heat shock proteins genes (HSP60, HSP70 and HSP90) and pesticide residues in the kidney tissue. The results revealed that the mRNA and protein levels of HSP60, HSP70 and HSP90 were induced in the kidney of common carp by ATR, CPF, and ATR/CPF mixture. The accumulated amounts of ATR, CPF, and their metabolites in the kidney tissues exhibited dose-dependency. These results exhibited that increasing concentration of ATR and CPF in the environment causes considerable stress for common carp, suggesting that the expression levels of HSP60, HSP70 and HSP90 may act as potential biomarkers for assessing the environmental ATR and CPF risk for carp.

Effects of atrazine and chlorpyrifos on DNA methylation in the liver, kidney and gill of the common carp (Cyprinus carpio L.).

Pesticide exposure has repeatedly been associated with cancers, although the molecular mechanisms behind this association are largely undetermined. Abnormal DNA methylation plays a key role in the process of some disease. However, little was known about the effect of pesticides on DNA methylation in the common carp. In this study, we investigated the mRNA levels of DNA methyltransferases (DNMTs) and methyl-CpG-binding protein DNA-binding domain protein 2 (MBD2) as well as the DNA methylation levels in the liver, kidney and gill of the common carp (Cyprinus carpio L.) after 40-d exposure to atrazine (ATR) and chlorpyrifos (CPF) alone or in combination, and a 40-d recovery period. Juvenile common carp were exposed to various concentrations of ATR (at concentrations of 4.28, 42.8 and 428µg/L), CPF (1.16, 11.6 and 116µg/L), and an ATR/CPF mixture (at concentrations of 1.13, 11.3 and 113µg/L). The results revealed that the levels of genomic DNA methylation decreased in all tissues after 40d of exposure to ATR and CPF either individually or in combination. Moreover, the mRNA expression of DNMTs was down-regulated in all treatment groups. In contrast, the mRNA expression of MBD2 was up-regulated. These results demonstrated that long-term exposure to ATR, CPF and ATR/CPF mixtures could disrupt genomic DNA. It might imply that DNA methylation is involved in the toxicity caused by ATR and CPF in the common carp.