Diquat causes mouse testis injury through inducing heme oxygenase-1-mediated ferroptosis in spermatogonia.

Diquat dibromide (DQ) is a globally used herbicide in agriculture, and its overuse poses an important public health issue, including male reproductive toxicity in mammals. However, the effects and molecular mechanisms of DQ on testes are limited. In vivo experiments, mice were intraperitoneally injected with 8 or 10 mg/kg/ day of DQ for 28 days. It has been found that heme oxygenase-1 (HO-1) mediates DQ-induced ferroptosis in mouse spermatogonia, thereby damaging testicular development and spermatogenesis. Histopathologically, we found that DQ exposure caused seminiferous tubule disorders, reduced germ cells, and increased sperm malformation, in mice. Reactive oxygen species (ROS) staining of frozen section and transmission electron microscopy (TEM) displayed DQ promoted ROS generation and mitochondrial morphology alterations in mouse testes, suggesting that DQ treatment induced testicular oxidative stress. Subsequent RNA-sequencing further showed that DQ treatment might trigger ferroptosis pathway, attributed to disturbed glutathione metabolism and iron homeostasis in spermatogonia cells in vitro. Consistently, results of western blotting, measurements of MDA and ferrous iron, and ROS staining confirmed that DQ increased oxidative stress and lipid peroxidation, and accelerated ferrous iron accumulation both in vitro and in vivo. Moreover, inhibition of ferroptosis by deferoxamine (DFO) markedly ameliorated DQ-induced cell death and dysfunction. By RNA-sequencing, we found that the expression of HO-1 was significantly upregulated in DQ-treated spermatogonia, while ZnPP (a specific inhibitor of HO-1) blocked spermatogonia ferroptosis by balancing intracellular iron homeostasis. In mice, administration of the ferroptosis inhibitor ferrostatin-1 effectively restored the increase of HO-1 levels in the spermatogonia, prevented spermatogonia death, and alleviated the spermatogenesis disorders induced by DQ. Overall, these findings suggest that HO-1 mediates DQ-induced spermatogonia ferroptosis in mouse testes, and targeting HO-1 may be an effective protective strategy against male reproductive disorders induced by pesticides in agriculture.

High expression of 3-hydroxy-3-methylglutaryl-CoA synthase 2 (HMGCS2) associated with Diquat-induced damage.

Diquat (DQ) is a commonly used bipyridine herbicide known for its toxic properties and adverse effects on individuals. However, the mechanism underlying DQ-induced damage remain elusive. Our research aimed to uncover the regulatory network involved in DQ-induced damage. We analyzed publicly accessible gene expression patterns and performed research using a DQ-induced damage animal model. The GSE153959 dataset from the Gene Expression Omnibus collection and the animal model of DQ-induced kidney injury were used to identify differentially expressed genes (DEGs). Pathways including the regulation of DNA-templated transcription in response to stress, RNA polymerase II transcription regulator complex and transcription coregulatory activity were shown to be enriched in 21 DEGs. We used least absolute shrinkage and selection operator (LASSO) regression analysis to find possible diagnostic biomarkers for DQ-induced damage. Then, we used an HK-2 cell model to confirm these results. Additionally, we confirmed that 3-hydroxy-3-methylglutaryl-CoA synthase 2 (HMGCS2) was the major gene associated with DQ-induced damage using multi-omics screening. The sample validation strongly suggested that HMGCS2 has promise as a diagnostic marker and may provide new targets for therapy in the context of DQ-induced damage.

Mitochondria-associated endoplasmic reticulum membranes involve in oxidative stress-induced intestinal barrier injury and mitochondrial dysfunction under diquat exposing.

Many factors induced by environmental toxicants have made oxidative stress a risk factor for the intestinal barrier injury and growth restriction, which is serious health threat for human and livestock and induces significant economic loss. It is well-known that diquat-induced oxidative stress is implicated in the intestinal barrier injury. Although some studies have shown that mitochondria are the primary target organelle of diquat, the underlying mechanism remains incompletely understood. Recently, mitochondria-associated endoplasmic reticulum membranes (MAMs) have aroused increasing concerns among scholars, which participate in mitochondrial dynamics and signal transduction. In this study, we investigated whether MAMs involved in intestinal barrier injury and mitochondrial dysfunction induced by diquat-induced oxidative stress in piglets and porcine intestinal epithelial cells (IPEC-J2 cells). The results showed that diquat induced growth restriction and impaired intestinal barrier. The mitochondrial reactive oxygen species (ROS) was increased and mitochondrial membrane potential was decreased following diquat exposure. The ultrastructure of mitochondria and MAMs was also disturbed. Meanwhile, diquat upregulated endoplasmic reticulum stress marker protein and activated PERK pathway. Furthermore, loosening MAMs alleviated intestinal barrier injury, decrease of antioxidant enzyme activity and mitochondrial dysfunction induced by diquat in IPEC-J2 cells, while tightening MAMs exacerbated diquat-induced mitochondrial dysfunction. These results suggested that MAMs may be associated with the intestinal barrier injury and mitochondrial dysfunction induced by diquat in the jejunum of piglets.

Sensing of mitochondrial DNA by ZBP1 promotes RIPK3-mediated necroptosis and ferroptosis in response to diquat poisoning.

Diquat (DQ) poisoning is a severe medical condition associated with life-threatening implications and multiorgan dysfunction. Despite its clinical significance, the precise underlying mechanism remains inadequately understood. This study elucidates that DQ induces instability in the mitochondrial genome of endothelial cells, resulting in the accumulation of Z-form DNA. This process activates Z-DNA binding protein 1 (ZBP1), which then interacts with receptor-interacting protein kinase 3 (RIPK3), ultimately leading to RIPK3-dependent necroptotic and ferroptotic signaling cascades. Specific deletion of either Zbp1 or Ripk3 in endothelial cells simultaneously inhibits both necroptosis and ferroptosis. This dual inhibition significantly reduces organ damage and lowers mortality rate. Notably, our investigation reveals that RIPK3 has a dual role. It not only phosphorylates MLKL to induce necroptosis but also phosphorylates FSP1 to inhibit its enzymatic activity, promoting ferroptosis. The study further shows that deletion of mixed lineage kinase domain-like (Mlkl) and the augmentation of ferroptosis suppressor protein 1 (FSP1)-dependent non-canonical vitamin K cycling can provide partial protection against DQ-induced organ damage. Combining Mlkl deletion with vitamin K treatment demonstrates a heightened efficacy in ameliorating multiorgan damage and lethality induced by DQ. Taken together, this study identifies ZBP1 as a crucial sensor for DQ-induced mitochondrial Z-form DNA, initiating RIPK3-dependent necroptosis and ferroptosis. These findings suggest that targeting the ZBP1/RIPK3-dependent necroptotic and ferroptotic pathways could be a promising approach for drug interventions aimed at mitigating the adverse consequences of DQ poisoning.

Supplementation with grape seed extract, onion peel extract, or rosemary extract in the diet alleviates growth inhibition, liver damage, and oxidative stress induced by diquat in Lohmann chicks.

The present study aimed to assess the impact of grape seed extract (GSE), onion peel extract (OPE), and rosemary extract (ROE) on Diquat-induced growth restriction and oxidative stress in Lohmann chicks. A total of 200 chicks were randomly assigned to 5 diets: the positive control (PC) group, the negative control (NC) group, GSE group, OPE group, and ROE group. During the first 7 d of trial, compared with NC and PC groups, the GSE group enhanced average daily feed intake (ADFI). From day 8-21, diquat injection resulted in reduced growth performance, increased platelet volume distribution width (PWD), malondialdehyde (MDA) concentration, and activities of alanine aminotransferase (ALT) in chick serum; it also decreased total protein (TP), albumin (ALB), globulin (GLB) concentration, activities of superoxide dismutase (SOD) and glutathione S-transferase (GST) in chick serum; furthermore, it increased MDA concentration while decreasing GST activities in liver. The NC group exhibited lower average daily gain (ADG) than other groups. Compared with NC group, GSE group reduced ALT activities, MDA levels, and red cell distribution width (RDW), and PDW concentration; it also increased SOD, GST activities. The ROE group lowered ALT activities and MDA concentration. The OPE group decreased ALT activities, and MDA levels, RDW, and PDW concentration, and increased SOD activities of chicks. These results suggest that supplementing antioxidants in diets alleviated oxidative stress in chicks challenged by improving antioxidant capacity and liver function.

Dietary curcumin supplementation alleviates diquat-induced oxidative stress in the liver of broilers.

This study purposed to investigate the alleviating effect of dietary curcumin supplementation on oxidative stress in the liver of broilers induced by diquat. One-day-old Cobb broilers (400) were selected and randomly divided into 5 groups, with 8 replicates and 10 broilers per replicate. The control group and the diquat group were fed the basal diet, while the curcumin supplementation groups were fed the basal diet supplemented with different amounts of curcumin (50, 100, and 150 mg/kg). On d 21 of the test, 1 broiler was randomly selected from each replicate and intraperitoneally injected with 20 mg/mL of diquat solution at a dose of 1 mL/kg BW or equivalent physiological saline (for the control group). After 48 h of feeding, the selected broilers were slaughtered for analysis. The results show that diquat treatment reduced the antioxidant capacity of the liver, caused oxidative stress, and affected its lipid metabolism. However, diet supplementation using curcumin completely or partially reversed the effect of diquat on the liver of broilers. The blood alanine aminotransferase activity, total bilirubin and total protein levels, and liver Caspase-3 mRNA abundance in broilers were lower or significantly lower in the curcumin supplementation group than in the diquat group (P < 0.05). The curcumin supplementation groups had significantly higher total antioxidant capacity activity but significantly lower malondialdehyde in the liver of broilers than the diquat group (P < 0.05). The blood triglyceride level of broilers was lower or significantly lower in the curcumin supplementation groups than in the diquat group (P < 0.05). The activities of cetyl coenzyme A carboxylase in the liver were significantly lower in the 150 mg/kg curcumin supplementation groups than in the DQ group (P < 0.05). In conclusion, dietary curcumin supplementation could ameliorate the effects of diquat-induced oxidative stress on the antioxidant capacity, tissue morphology, and lipid metabolism of the liver of broilers, thus protecting the liver. The recommended dosage for broiler diets is 100 to 150 mg/kg curcumin.

[Experimental study on the toxicokinetics and gastrointestinal damage in rats poisoned with acute diquat poisoning at different exposure doses].

OBJECTIVE: To observe the toxicokinetic parameters, absorption characteristics and pathomorphological damage in different parts of the gastrointestinal tract of rats poisoned with different doses of diquat (DQ). METHODS: Ninety-six healthy male Wistar rats were randomly divided into a control group (six rats) and low (115.5 mg/kg), medium (231.0 mg/kg) and high (346.5 mg/kg) dose DQ poisoning groups (thirty rats in each dose group), and then the poisoning groups were randomly divided into 5 subgroups according to the time after exposure (15 minutes and 1, 3, 12, 36 hours; six rats in each subgroup). All rats in the exposure groups were given a single dose of DQ by gavage. Rats in the control group was given the same amount of saline by gavage. The general condition of the rats was recorded. Blood was collected from the inner canthus of the eye at 3 time points in each subgroup, and rats were sacrificed after the third blood collection to obtain gastrointestinal specimens. DQ concentrations in plasma and tissues were determined by ultra-high performance liquid chromatography and mass spectrometry (UPHLC-MS), and the toxic concentration-time curves were plotted to calculate the toxicokinetic parameters; the morphological structure of the intestine was observed under light microscopy, and the villi height and crypt depth were determined and the ratio (V/C) was calculated. RESULTS: DQ was detected in the plasma of the rats in the low, medium and high dose groups 5 minutes after exposure. The time to maximum plasma concentration (Tmax) was (0.85±0.22), (0.75±0.25) and (0.25±0.00) hours, respectively. The trend of plasma DQ concentration over time was similar in the three dose groups, but the plasma DQ concentration increased again at 36 hours in the high dose group. In terms of DQ concentration in gastrointestinal tissues, the highest concentrations of DQ were found in the stomach and small intestine from 15 minutes to 1 hour and in the colon at 3 hours. By 36 hours after poisoning, the concentrations of DQ in all parts of the stomach and intestine in the low and medium dose groups had decreased to lower levels. Gastrointestinal tissue (except jejunum) DQ concentrations in the high dose group tended to increase from 12 hours. Higher doses of DQ were still detectable [gastric, duodenal, ileal and colonic DQ concentrations of 6 400.0 (1 232.5), 4 889.0 (6 070.5), 10 300.0 (3 565.0) and 1 835.0 (202.5) mg/kg respectively]. Light microscopic observation of morphological and histopathological changes in the intestine shows that acute damage to the stomach, duodenum and jejunum of rats was observed 15 minutes after each dose of DQ, pathological lesions were observed in the ileum and colon 1 hour after exposure, the most severe gastrointestinal injury occurred at 12 hours, significant reduction in villi height, significant increase in crypt depth and lowest V/C ratio in all segments of the small intestine, damage begins to diminish by 36-hour post-intoxication. At the same time, morphological and histopathological damage to the intestine of rats at all time points increased significantly with increasing doses of the toxin. CONCLUSIONS: The absorption of DQ in the digestive tract is rapid, and all segments of the gastrointestinal tract may absorb DQ. The toxicokinetics of DQ-tainted rats at different times and doses have different characteristics. In terms of timing, gastrointestinal damage was seen at 15 minutes after DQ, and began to diminish at 36 hours. In terms of dose, Tmax was advanced with the increase of dose and the peak time was shorter. The damage to the digestive system of DQ is closely related to the dose and retention time of the poison exposure.

Pterostilbene Confers Protection against Diquat-Induced Intestinal Damage with Potential Regulation of Redox Status and Ferroptosis in Broiler Chickens.

Oxidative stress causes damage to macromolecules, including proteins, DNA, and lipid, and has been recognized as a crucial driver of the onset and progression of several intestinal disorders. Pterostilbene, one of the natural antioxidants, has attracted considerable attention owing to its multiple biological activities. In the present study, we established an oxidative stress model in broiler chickens via injection with diquat to investigate whether pterostilbene could attenuate diquat-induced intestinal damage and reveal the underlying mechanisms. We found that diquat-induced decreases in the activities of superoxide dismutase and glutathione peroxidase and the level of reduced glutathione and the increase in hydrogen peroxide content in plasma and jejunum were significantly alleviated by pterostilbene (P < 0.05). Pterostilbene supplementation also decreased intestinal permeability and jejunal apoptosis rate, improved jejunal villus height and the ratio of villus height to crypt depth, and promoted the transcription and translation of jejunal tight junction proteins occludin and zona occludens 1 in diquat-challenged broilers (P < 0.05). Furthermore, pterostilbene reversed diquat-induced mitochondrial injury in the jejunum, as indicated by the decreased reactive oxygen species level and elevated activities of superoxide dismutase 2 and mitochondrial respiratory complexes (P < 0.05). Importantly, administering pterostilbene maintained iron homeostasis, inhibited lipid peroxidation, and regulated the expression of the markers of ferroptosis in the jejunum of diquat-exposed broilers (P < 0.05). The nuclear factor erythroid 2-related factor 2 signaling pathway in the jejunum of diquat-exposed broilers was also activated by pterostilbene (P < 0.05). In conclusion, our study provides evidence that pterostilbene alleviates diquat-induced intestinal mucosa injury and barrier dysfunction by strengthening antioxidant capacity and regulating ferroptosis of broiler chickens.

Effects of dietary supplementation with chlorogenic acid on growth performance, antioxidant capacity, and hepatic inflammation in broiler chickens subjected to diquat-induced oxidative stress.

This study was conducted to investigate the protective effects of chlorogenic acid (CGA) on broilers subjected to (DQ)-induced oxidative stress. In experiment 1, one hundred and ninety-two male one-day-old Ross 308 broiler chicks were distributed into 4 groups and fed a basal diet supplemented with 0, 250, 500, or 1,000 mg/kg CGA for 21 d. In experiment 2, an equivalent number of male one-day-old chicks were allocated to 4 treatments for a 21-d trial: 1) Control group, normal birds fed a basal diet; 2) DQ group, DQ-challenged birds fed a basal diet; and 3) and 4) CGA-treated groups: DQ-challenged birds fed a basal diet supplemented with 500 or 1,000 mg/kg CGA. The intraperitoneal DQ challenge was performed at 20 d. In experiment 1, CGA administration linearly increased 21-d body weight, and weight gain and feed intake during 1 to 21 d (P < 0.05). CGA linearly and/or quadratically increased total antioxidant capacity, catalase, superoxide dismutase, and glutathione peroxidase activities, elevated glutathione level, and reduced malondialdehyde accumulation in serum, liver, and/or jejunum (P < 0.05). In experiment 2, compared with the control group, DQ challenge reduced body weight ratio (P < 0.05), which was reversed by CGA administration (P < 0.05). DQ challenge increased serum total protein level, aspartate aminotransferase activity, and total bilirubin concentration (P < 0.05), which were normalized when supplementing 500 mg/kg and/or 1,000 mg/kg CGA (P < 0.05). DQ administration elevated hepatic interleukin-1ß, tumor necrosis factor-α, and interleukin-6 levels (P < 0.05), and the values of interleukin-1ß were normalized to control values when supplementing CGA (P < 0.05). DQ injection decreased serum superoxide dismutase activity, hepatic catalase activity, and serum and hepatic glutathione level, but increased malondialdehyde concentration in serum and liver (P < 0.05), and the values of these parameters (except hepatic catalase activity) were reversed by 500 and/or 1,000 mg/kg CGA. The results suggested that CGA could improve growth performance, alleviate oxidative stress, and ameliorate hepatic inflammation in DQ-challenged broilers.

Effects of L-theanine on intestinal morphology, barrier function, and MAPK signaling pathways in diquat-challenged piglets.

This study aimed to explore the protective effects of L-theanine supplementation on the diquat-challenged weaned piglets. A total of 160 weaned piglets were randomly divided into 4 groups using a 2 × 2 two-factor design, there were 4 replicates per group and 10 pigs per replicate. Piglets were fed diets (with 1000 mg/kg L-theanine addition or not), then challenged with diquat or saline on day 7. 21 days after challenge, two pigs from each replicate were selected for sample collection. Results showed that supplement with 1000 mg/kg L-theanine down-regulated the diarrhea rate, serum D-lactate level, tumor necrosis factor-α, and phosphorylation of extracellular regulated protein kinases (ERK1/2), p38 mitogen-activated protein kinase (MAPK), and c-Jun N-terminal kinase (JNK) signaling in pigs without diquat challenge (p < 0.05). While for diquat-challenged piglets, L-theanine addition increased average daily gain, jejunum villus height, and interferon-γ level (p < 0.05). Meanwhile, L-theanine addition decreased the diarrhea rates and mortality, serum D-lactate level, and phosphorylation of ERK and JNK in diquat-challenged pigs (p < 0.05). These results demonstrate that L-theanine pretreatment could alleviate diquat-induced oxidative stress and improve intestinal barrier function in diquat-challenged weaned piglets, which can be attributed to suppression of MAPK phosphorylation signaling pathways.

Hydroxytyrosol attenuates diquat-induced oxidative stress by activating Nrf2 pathway and modulating colonic microbiota in mice.

This study was conducted to investigate the antioxidant effects of hydroxytyrosol (HT) administration in diquat (DQ)-challenged mice. The results showed that HT treatment markedly alleviated DQ-induced oxidative stress, which was indicated by the enhanced total antioxidant capacity (T-AOC), increased activities of superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), and catalase and decreased malondialdehyde (MDA) concentration in serum. Additionally, HT increased the mRNA expression levels of NF-E2-related factor 2 (Nrf2) and its downstream genes, including NADPH quinone oxidoreductase 1 (NQO1) and catalase (CAT) in the small intestine of DQ-challenged mice. 16S rRNA gene sequencing results showed that HT treatment increased the relative abundance of Firmicutes and Lactobacillus and decreased the relative abundance of Bacteroidetes. Interestingly, Pearson correlation analysis showed that there were strong association between colonic Firmicutes, Lactobacillus, and Bacteroidetes and the activities of serum antioxidant enzymes. Meanwhile, HT significantly enhanced the colonic butyrate concentration in DQ-challenged mice. Additionally, HT treatment decreased the serum metabolites involving in glycerophospholipid metabolism, pentose, and glucuronate interconversions, which were associated with alleviated oxidative stress. These results indicate that oral administration of 100 mg/kg body weight HT alleviates oxidative stress in DQ-challenged mice, which may involve Nrf2 signaling pathways via modulation of colonic microbiota.

Experimental study on the toxicokinetics and gastrointestinal damage in rats poisoned with acute diquat poisoning at different exposure doses / 中华危重病急救医学

OBJECTIVE@#To observe the toxicokinetic parameters, absorption characteristics and pathomorphological damage in different parts of the gastrointestinal tract of rats poisoned with different doses of diquat (DQ).@*METHODS@#Ninety-six healthy male Wistar rats were randomly divided into a control group (six rats) and low (115.5 mg/kg), medium (231.0 mg/kg) and high (346.5 mg/kg) dose DQ poisoning groups (thirty rats in each dose group), and then the poisoning groups were randomly divided into 5 subgroups according to the time after exposure (15 minutes and 1, 3, 12, 36 hours; six rats in each subgroup). All rats in the exposure groups were given a single dose of DQ by gavage. Rats in the control group was given the same amount of saline by gavage. The general condition of the rats was recorded. Blood was collected from the inner canthus of the eye at 3 time points in each subgroup, and rats were sacrificed after the third blood collection to obtain gastrointestinal specimens. DQ concentrations in plasma and tissues were determined by ultra-high performance liquid chromatography and mass spectrometry (UPHLC-MS), and the toxic concentration-time curves were plotted to calculate the toxicokinetic parameters; the morphological structure of the intestine was observed under light microscopy, and the villi height and crypt depth were determined and the ratio (V/C) was calculated.@*RESULTS@#DQ was detected in the plasma of the rats in the low, medium and high dose groups 5 minutes after exposure. The time to maximum plasma concentration (Tmax) was (0.85±0.22), (0.75±0.25) and (0.25±0.00) hours, respectively. The trend of plasma DQ concentration over time was similar in the three dose groups, but the plasma DQ concentration increased again at 36 hours in the high dose group. In terms of DQ concentration in gastrointestinal tissues, the highest concentrations of DQ were found in the stomach and small intestine from 15 minutes to 1 hour and in the colon at 3 hours. By 36 hours after poisoning, the concentrations of DQ in all parts of the stomach and intestine in the low and medium dose groups had decreased to lower levels. Gastrointestinal tissue (except jejunum) DQ concentrations in the high dose group tended to increase from 12 hours. Higher doses of DQ were still detectable [gastric, duodenal, ileal and colonic DQ concentrations of 6 400.0 (1 232.5), 4 889.0 (6 070.5), 10 300.0 (3 565.0) and 1 835.0 (202.5) mg/kg respectively]. Light microscopic observation of morphological and histopathological changes in the intestine shows that acute damage to the stomach, duodenum and jejunum of rats was observed 15 minutes after each dose of DQ, pathological lesions were observed in the ileum and colon 1 hour after exposure, the most severe gastrointestinal injury occurred at 12 hours, significant reduction in villi height, significant increase in crypt depth and lowest V/C ratio in all segments of the small intestine, damage begins to diminish by 36-hour post-intoxication. At the same time, morphological and histopathological damage to the intestine of rats at all time points increased significantly with increasing doses of the toxin.@*CONCLUSIONS@#The absorption of DQ in the digestive tract is rapid, and all segments of the gastrointestinal tract may absorb DQ. The toxicokinetics of DQ-tainted rats at different times and doses have different characteristics. In terms of timing, gastrointestinal damage was seen at 15 minutes after DQ, and began to diminish at 36 hours. In terms of dose, Tmax was advanced with the increase of dose and the peak time was shorter. The damage to the digestive system of DQ is closely related to the dose and retention time of the poison exposure.

Simultaneous determination of diquat and its two primary metabolites in rat plasma by ultraperformance liquid chromatography-tandem mass spectrometry and its application to the toxicokinetic study.

PURPOSE: This study aimed to develop and validate an ultraperformance liquid chromatography-tandem mass spectrometry to simultaneously determine diquat (DQ) and its two primary metabolites in rat plasma and its application to the toxicokinetic study. METHOD: The chromatographic separation of DQ and its two primary metabolites was performed with hydrophilic interaction chromatography column by adding formic acid and ammonium acetate in mobile phase in stepwise elution mode. DQ and its two primary metabolites were detected by liquid chromatography-tandem mass spectrometry in positive mode. RESULTS: The lower limit of quantification ranging from 0.3 to 3.0 ng/mL for DQ and its two primary metabolites was achieved by using only 50 µL of rat plasma. The maximum concentration (Cmax) was 977 ng/mL, half-life (t1/2) was 13.1 h, and area under the plasma concentration-time curve (AUC0-t) was 2770 h*ng/mL for DQ, Cmax was 47.1 ng/mL, t1/2 was 25.1 h, and AUC0-t was 180 h·ng/mL for diquat monopyridone (DQ-M) and Cmax was 246 ng/mL, t1/2 was 8.2 h, and AUC0-t was 2430 h·ng/mL for diquat dipyridone (DQ-D), respectively. CONCLUSIONS: The validated method was shown to be suitable for simultaneous determination of diquat and its two primary metabolites in rat plasma. This study is the first to study the toxicokinetics of DQ and its two primary metabolites.

Protective effect of biogenic selenium nanoparticles against diquat-induced acute toxicity via regulation of gut microbiota and its metabolites.

Selenium nanoparticles (SeNPs) with unique biological properties have been suggested as a safer and more effective platform for delivering of Selenium for biological needs. In this study, we investigated the association between gut microbiota altered by SeNPs supplementation and its metabolites under oxidative stress conditions through 16S rDNA gene sequencing analysis and untargeted metabolomics. The results showed that dietary supplementation with SeNPs attenuated diquat-induced acute toxicity in mice, as demonstrated by lower levels of inflammatory effector cells, and biochemical markers in serum such as aspartate aminotransferase (AST), alanine aminotransferase (ALT), blood urea nitrogen (BUN) and lactate dehydrogenase (LDH). SeNPs also reversed the perturbed gut microbiota composition induced by diquat, decreased the Firmicutes/Bacteroidetes ratio, and increased the abundance of beneficial bacteria such as Akkermansia, Muribaculaceae, Bacteroides and Parabacteroides. Untargeted fecal metabolomics showed that SeNPs can regulate the production of steroids and steroid derivatives, organonitrogen compounds, pyridines and derivatives and other metabolites. Microbiome-metabolome correlation analysis suggested that Parabacteroides was the key bacteria for the SeNPs intervention, which might upregulate the levels of metabolites such as trimethaphan, emedastine, berberine, desoxycortone, tetrahydrocortisone. This study demonstrated that dietary SeNPs supplementation can extensively modulate the gut microbiota and its metabolism, thereby alleviating diquat-induced acute toxicity.

Metabolomics analysis of the potential toxicological mechanisms of diquat dibromide herbicide in adult zebrafish (Danio rerio) liver.

Although diquat is a widely used water-soluble herbicide in the world, its sublethal adverse effects to fish have not been well characterised. In this study, histopathological examination and biochemical assays were applied to assess hepatotoxicity and combined with gas chromatography-mass spectrometry (GC-MS)-based metabolomics analysis to reveal overall metabolic mechanisms in the liver of zebrafish (Danio rerio) after diquat exposure at concentrations of 0.34 and 1.69 mg·L-1 for 21 days. Results indicated that 1.69 mg·L-1 diquat exposure caused cellular vacuolisation and degeneration with nuclear abnormality and led to the disturbance of antioxidative system and dysfunction in the liver. No evident pathological injury was detected, and changes in liver biochemistry were not obvious in the fish exposed to 0.34 mg·L-1 diquat. Multivariate statistical analysis revealed differences between profiles obtained by GC-MS spectrometry from control and two treatment groups. A total of 17 and 22 metabolites belonging to different classes were identified following exposure to 0.34 and 1.69 mg·L-1 diquat, respectively. The metabolic changes in the liver of zebrafish are mainly manifested as inhibition of energy metabolism, disorders of amino acid metabolism and reduction of antioxidant capacity caused by 1.69 mg·L-1 diquat exposure. The energy metabolism of zebrafish exposed to 0.34 mg·L-1 diquat was more inclined to rely on anaerobic glycolysis than that of normal zebrafish, and interference effects on lipid metabolism were observed. The metabolomics approach provided an innovative perspective to explore possible hepatic damages on fish induced by diquat as a basis for further research.

Effects of Curcumin on Mitochondrial Function, Endoplasmic Reticulum Stress, and Mitochondria-Associated Endoplasmic Reticulum Membranes in the Jejunum of Oxidative Stress Piglets.

Mitochondria-associated endoplasmic reticulum (ER) membranes (MAMs) are not only critical for the communication between two organelles but also crucial for cellular processes such as energy metabolism, calcium signaling, and mitochondrial dynamics. The effects of curcumin on jejunal mitochondria, ER, and MAMs in piglets under diquat-induced oxidative stress were assessed. Twenty-four piglets (35 days old, weaned at 21 days, 9.54 ± 0.28 kg, six piglets per group) were used in the study: (1) control group; (2) control + curcumin group; (3) diquat group; and (4) diquat + curcumin group. Curcumin was mixed with the basic diet at 200 mg/kg and fed to piglets. Piglets were administered intraperitoneally of 0.9% saline solution or diquat at 10 mg/kg body weight on the first day. Compared with the diquat group, curcumin improved jejunal morphology and barrier function. Meanwhile, curcumin improved mitochondrial function and ultrastructure, alleviated endoplasmic reticulum stress (ERS), and inhibited apoptosis induced by diquat. Moreover, curcumin prevented excessive MAM formation and alleviated MAM disorder. In conclusion, dietary curcumin ameliorated jejunal damage and mitochondrial dysfunction, attenuated ERS, and alleviated MAM disorder in oxidative stress piglets induced by diquat.

Ellagic Acid Alleviates Diquat-Induced Jejunum Oxidative Stress in C57BL/6 Mice through Activating Nrf2 Mediated Signaling Pathway.

Ellagic acid (EA) is the main constituent found in pomegranate rind, which has anti-inflammatory and antioxidant effects. However, whether EA can alleviate diquat-induced oxidative stress is still unknown. Here, the effects and mechanisms of EA on jejunum oxidative stress induced by diquat was investigated. Oxidative stress was induced in mice by administrating diquat (25 mg/kg body weight) followed by treatment with 100 mg/kg body weight EA for 5 days. Results showed that oral administration of EA significantly ameliorated diquat-induced weight loss and oxidative stress (p < 0.05) evidenced by reduced ROS production in the jejunum. Furthermore, EA up-regulated the mRNA expression of the antioxidant enzymes (Nrf2, GPX1 and HO-1) when mice were challenged with diquat, compared with the diquat group (p < 0.05). Importantly, pharmacological inhibition of Nrf2 by ML385 counteracted the EA-mediated alleviation of jejunum oxidative stress, as evidence by body weight and ROS production. Also, immunohistochemistry staining confirmed the markedly decreased jejunal Nrf2 expression. The up-regulated effect on NQO1 and HO-1 mRNA expression induced by EA was diminished in mice treated with ML385 (p < 0.05). Together, our results demonstrated that therapeutic and preventative EA treatment was effective in reducing weight loss and oxidative stress induced by diquat through the Nrf2 mediated signaling pathway.

Characteristics of circRNAs expression profiles in the piglets intestine induced by oxidative stress.

BACKGROUD: Oxidative stress (OS) can affect the expression of key genes and destroy the intestinal structure. However, it is unclear how OS regulates the expression of circular RNAs (circRNAs), microRNAs (miRNAs) and mRNAs. OBJECTIVE: The aim of this study was to examine the expression of circRNAs, miRNAs and mRNAs exposed to OS. METHODS: Piglets were exposed to diquat (DQ), a herbicide, and the activity of antioxidant enzymes and the morphology of the intestine were investigated. We utilized whole transcriptome sequencing to examine the global expression of circRNAs, miRNAs and mRNAs in the jejunum. RESULTS: Compared to controls, 751 circRNAs, 731 miRNAs and 164 mRNAs were differentially expressed in diquat-treated piglets. Kyoto Encyclopedia of Genes and Genomes pathway analysis indicated that oxidative phosphorylation, RNA degradation and ubiquitin-mediated proteolysis were closely associated with OS. CONCLUSIONS: Our results indicated that diquat-induced OS alters the intestinal structure, resulting in the differential expression of circRNAs, miRNAs and mRNAs in the jejunum of piglets. Meanwhile, OS weakened the enzyme antioxidant system in serum of piglets. Our results provide a foundation for further studies on the mechanisms involved in the response to OS in the jejunum.

Chaperon-mediated autophagy can regulate diquat-induced apoptosis by inhibiting α-synuclein accumulation cooperatively with macroautophagy.

α-Synuclein, which is associated with Parkinson's disease, is cleared by the ubiquitin-proteasome system and autophagy lysosome system. Chaperon-mediated autophagy (CMA) and macroautophagy are major subtypes of autophagy and play a critical role in pesticide-induced α-synucleinopathy. In this study, we explored the role of CMA in diquat (DQ)-induced α-synucleinopathy and characterized the relationship between CMA and macroautophagy in the clearance of pathologic α-synuclein for the prevention of DQ neurotoxicity. DQ was cytotoxic to SH-SY5Y cells in a concentration-dependent manner, as shown by decreased cell viability and increased cytotoxicity. DQ treatment was also found to induce autophagy such as CMA and macroautophagy by monitoring the expression of Lamp2A and microtubule-associated protein 1A/1B light chain 3B (LC3-II) respectively. Following DQ treatment, SH-SY5Y cells were found to have induced phosphorylated and detergent-insoluble α-synuclein deposits, and MG132, a proteasome inhibitor, effectively potentiated both CMA and macroautophagy for preventing α-synuclein aggregation. Interestingly, CMA impairment by Lamp2A-knock down decreased the LC3II expression compared to in DQ-treated cells transfected with control siRNA. In Lamp2-knock down cells, pathologic α-synuclein was increased 12 h after DQ treatment, but there was no change observed at 24 h. In DQ-treated cells, macroautophagy by 3-methyladenine and bafilomycin inhibition increased Lamp2A expression, indicating an increase in CMA activity. In addition, CMA modulation affected apoptosis, and inhibiting lysosome activity by NH4Cl increased apoptosis in DQ-treated cells. An increase in autophagy was confirmed to compensate for the decrease in lysosome activity. Pretreatment with z-VAD-fmk, a pan-caspase inhibitor, significantly enhanced the macroautophagy response of DQ-exposed cells without alterations in Lamp2A expression. Our results suggest that CMA can regulate DQ-induced α-synucleinopathy cooperatively with macroautophagy, and crosstalk between macroautophagy and CMA plays an important role in DQ-induced cytotoxicity. Taken together, autophagy modulation may be a useful treatment strategy in pesticide-induced neurodegenerative disorders through preventing α-synucleinopathy.

Vitamin D3 Protects Mice from Diquat-Induced Oxidative Stress through the NF-κB/Nrf2/HO-1 Signaling Pathway.

Vitamin D3, as an indispensable and fat-soluble micronutrient, plays an important role in the health of humans and animals. At present, studies are focusing on the calcium absorption and immunoregulation function of vitamin D3; this study was aimed at exploring the antioxidative stress ability of vitamin D3 on diquat-induced intestinal dysfunction of ICR mice and the underlying mechanism. The results showed that oral gavage of vitamin D3 daily significantly improved the body weight gain and immune organ index and significantly reverted the abnormal changes of ALT, AST, SOD, GSH-Px, T-AOC, and MDA in the serum and jejunum induced by diquat. The addition of vitamin D3 also significantly reduced the concentration of DAO, D-LA, and certain proinflammatory cytokines in serum. Moreover, vitamin D3 improved the pathological morphology of the duodenum, jejunum, colon, liver, and kidney tissues, and it also largely attenuated the degree of inflammatory infiltration of macrophages and cell apoptotic index of jejunal epithelial tissue induced by diquat. The results demonstrated that vitamin D3 significantly recovered the intestinal barrier injury by enhancing the expression of mucins and tight junction proteins in the jejunum. In addition, the results indicated that vitamin D3 could significantly reduce the phosphorylation level of NF-κB (p65) and enhance the expression of Nrf2 and HO-1 in the jejunum compared with the diquat-induced group. This study suggested that oral administration of vitamin D3 can protect mice against oxidative damage by inhibiting the phosphorylation level of NF-κB (p65) and activating Nrf2-related signaling pathways.