Alpha-class glutathione S-transferases involved in the detoxification of aflatoxin B1 in ducklings.

The objective of this study was to identify the key glutathione S-transferase (GST) isozymes involved in the detoxification of Aflatoxin B1 (AFB1) in ducks' primary hepatocytes. The full-length cDNA encoding the 10 GST isozymes (GST, GST3, GSTM3, MGST1, MGST2, MGST3, GSTK1, GSTT1, GSTO1 and GSTZ1) were isolated/synthesized from ducks' liver and cloned into the pcDNA3.1(+) vector. The results showed that pcDNA3.1(+)-GSTs plasmids were successfully transferred into the ducks' primary hepatocytes and the mRNA of the 10 GST isozymes were overexpressed by 1.9-3274.7 times. Compared to the control, 75 µg/L (IC30) or 150 µg/L (IC50) AFB1 treatment reduced the cell viability by 30.0-50.0% and increased the LDH activity by 19.8-58.2% in the ducks' primary hepatocytes. Notably, the AFB1-induced changes in cell viability and LDH activity were mitigated by overexpression of GST and GST3. Compared to the cells treated with AFB1, exo-AFB1-8,9-epoxide (AFBO)-GSH, as the major detoxified product of AFB1, was increased in the cells overexpression of GST and GST3. Moreover, the sequences, phylogenetic and domain analysis revealed that the GST and GST3 were orthologous to Meleagris gallopavo GSTA3 and GSTA4. In conclusion, this study found that the ducks' GST and GST3 were orthologous to Meleagris gallopavo GSTA3 and GSTA4, which were involved in the detoxification of AFB1 in ducks' primary hepatocytes.

T-2 toxin-induced intestinal damage with dysregulation of metabolism, redox homeostasis, inflammation, and apoptosis in chicks.

T-2 toxin is a worldwide problem for feed and food safety, leading to livestock and human health risks. The objective of this study was to explore the mechanism of T-2 toxin-induced small intestine injury in broilers by integrating the advanced microbiomic, metabolomic and transcriptomic technologies. Four groups of 1-day-old male broilers (n = 4 cages/group, 6 birds/cage) were fed a control diet and control diet supplemented with T-2 toxin at 1.0, 3.0, and 6.0 mg/kg, respectively, for 2 weeks. Compared with the control, dietary T-2 toxin reduced feed intake, body weight gain, feed conversion ratio, and the apparent metabolic rates and induced histopathological lesions in the small intestine to varying degrees by different doses. Furthermore, the T-2 toxin decreased the activities of glutathione peroxidase, thioredoxin reductase and total antioxidant capacity but increased the concentrations of protein carbonyl and malondialdehyde in the duodenum in a dose-dependent manner. Moreover, the integrated microbiomic, metabolomic and transcriptomic analysis results revealed that the microbes, metabolites, and transcripts were primarily involved in the regulation of nucleotide and glycerophospholipid metabolism, redox homeostasis, inflammation, and apoptosis were related to the T-2 toxin-induced intestinal damage. In summary, the present study systematically elucidated the intestinal toxic mechanisms of T-2 toxin, which provides novel ideas to develop a detoxification strategy for T-2 toxin in animals.

Invited review: Remediation strategies for mycotoxin control in feed.

Mycotoxins are secondary metabolites of different species of fungi. Aflatoxin B1 (AFB1), deoxynivalenol (DON), zearalenone (ZEN) and fumonisin B1 (FB1) are the main mycotoxins contaminating animal feedstuffs. These mycotoxins can primarily induce hepatotoxicity, immunotoxicity, neurotoxicity and nephrotoxicity, consequently cause adverse effects on the health and performance of animals. Therefore, physical, chemical, biological and nutritional regulation approaches have been developed as primary strategies for the decontamination and detoxification of these mycotoxins in the feed industry. Meanwhile, each of these techniques has its drawbacks, including inefficient, costly, or impractically applied on large scale. This review summarized the advantages and disadvantages of the different remediation strategies, as well as updates of the research progress of these strategies for AFB1, DON, ZEN and FB1 control in the feed industry.

Selenium mitigated aflatoxin B1-induced cardiotoxicity with potential regulation of 4 selenoproteins and ferroptosis signaling in chicks.

The aim of the present study was to explore the underlying mechanism of selenium (Se)-mediated detoxification of aflatoxin B1 (AFB1)-induced cardiotoxicity in chicks. A Se-deficient, corn-soybean meal-basal diet (36 µg Se/kg, BD) and three test diets (BD+1.0 mg AFB1/kg, 0.3 mg Se/kg, or 1.0 mg AFB1/kg+0.3 mg Se/kg) were used in a 3-wk 2 × 2 factorial design trial (n = 30 chicks/group). Dietary AFB1 led to induced (P < 0.05) serum creatine kinase and creatine kinase MB isoenzyme activities and heart histopathologic lesions. However, Se deficiency aggravated most of these alterations induced by AFB1. Moreover, mRNA levels of two ferroptosis activators (solute carrier family 11 Member 2 and transferrin) were upregulated (P < 0.05) in the AFB1-treated groups. Additionally, Se deficiency reduced (P < 0.05) glutathione peroxidase (GPX) 3 and thioredoxin reductase 3 mRNA and GPX activity but increased (P < 0.05) selenoprotein M and selenophosphate synthetase 2 mRNA in the heart in AFB1-administered groups. The in vitro study showed that Se alleviated (P < 0.05) AFB1-reduced cell viability and induced (P < 0.05) ROS and ferroptosis in H9C2 cardiac cells. It also downregulated (P < 0.05) two ferroptosis activators (long-chain acyl-CoA synthetase 4 and solute carrier family 11 Member 2) in the AFB1-treated groups in the H9C2 cells. In conclusion, this study illustrated that Se alleviates AFB1-induced cardiotoxicity and cardiomyocyte damage potentially related to the regulation of redox status, 4 selenoproteins, and ferroptosis-related signaling.

Mitigation of Aflatoxin B1 Hepatoxicity by Dietary Hedyotis diffusa Is Associated with Activation of NRF2/ARE Signaling in Chicks.

The objective of this study was to explore the mechanism of Hedyotis diffusa (HD) in mediating the detoxification of aflatoxin B1 (AFB1)-induced hepatic injury in chicks. A total of 144 one-day-old male broilers (Cobb 500) were randomly assigned to four treatment groups (n = 6 cages/diet, 6 chicks/cage). After three days of acclimation, the broilers were fed either a control diet (Control), Control plus 0.5 mg/kg of AFB1, or Control plus 0.5 mg/kg AFB1 with 500 or 1000 mg/kg HD for two weeks. Both serum and liver were collected at the end of the feeding trial for biochemistry, histology, and NF-E2-related nuclear factor 2 (NRF2)/antioxidant response element (ARE) signaling analysis. Compared with Control, the AFB1 treatment caused liver injury and decreased (p < 0.05) body weight gain, feed intake, feed conversion ratio, and serum albumin and total protein by 6.2-20.7%. AFB1 also induced swelling, necrosis, and severe vacuolar degeneration in chicks' livers. Notably, HD supplementation at 500 and 1000 mg/kg mitigated (p < 0.05) the alterations induced by AFB1. HD supplementation alleviated (p < 0.05) AFB1-induced impairment in hepatic glutathione peroxidase activity, protein carbonyl, and exo-AFB1-8,9-epoxide (AFBO)-DNA concentrations by 57.7-100% and increased (p < 0.05) the activities of superoxide dismutase and catalase by 23.1-40.9% more than those of AFB1 treatment alone. Furthermore, HD supplementation at the two doses upregulated (p < 0.05) NRF2, NAD(P)H: quinone oxidoreductase-1, heme oxygenase-1, glutathione cysteine ligase catalytic subunit, and glutathione-S transferase A2 and A3 in livers relative to the AFB1 group by 0.99-3.4-fold. Overall, dietary supplementation of HD at a high dose displayed better protection effects against aflatoxicosis. In conclusion, a dietary HD supplementation at 500 and 1000 mg/kg protected broilers from AFB1-induced hepatotoxicity, potentially due to the activation of NRF2/ARE signaling in the chicks.

Dietary Se deficiency dysregulates metabolic and cell death signaling in aggravating the AFB1 hepatotoxicity of chicks.

The objective of this study was to use isobaric tags for relative and absolute quantitation (iTRAQ) proteomic technology to systematically analyze the hepatotoxic mechanism of aflatoxin B1 (AFB1) and its prevention by Se in broilers. Four groups of day-old broilers were allocated into a 2 × 2 factorial design trial that fed a Se-deficient based diet (BD) or the BD + 1.0 mg AFB1/kg, 0.3 mg Se/kg, or 1.0 mg AFB1/kg plus 0.3 mg Se/kg for 3 wk. Dietary AFB1 increased serum ALT and decreased total protein and albumin concentrations, and induced hepatic histopathological lesions in Se adequate groups. Notably, Se deficiency exacerbated these AFB1-induced changes. Furthermore, Se deficiency reduced hepatic glutathione peroxidase but increased thioredoxin reductase and glutathione S-transferase activities and 8-hydroxydeoxyguanosine concentration in AFB1 administrated groups. Moreover, AFB1 dysregulated 261 co-differentially expressed proteins (DEPs) in both Se adequate and deficiency diets, and Se deficiency dysregulated 64 DEPs in AFB1 administrated diets. These DEPs are mainly related to phase I and II metabolizing enzymes, heat shock proteins, DNA repair, fatty acid metabolism and apoptosis. The in vitro study has verified that aldo-keto reductase family1, member10 plays an important role in AFB1-induced hepatotoxicity and Se-mediated detoxification of AFB1 in a chicken leghorn male hepatoma cells. Conclusively, this study has analyzed the hepatic proteome response to dietary AFB1 and Se, and thus shed new light on the mechanisms of hepatotoxicity of AFB1 and its detoxification by Se in broilers.

Yeast culture promotes the production of aged laying hens by improving intestinal digestive enzyme activities and the intestinal health status.

Yeast culture (YC) positively affects the performance of laying hens. The purpose of the present study was to explore the underlying mechanism for the YC-mediated performance improvement. Sixty 67-week-old Hy-Line Brown laying hens were randomly allocated into 2 experimental groups with 5 replicates of 6 birds each. One group was fed a control diet, whereas the other received the control diet supplemented with YC at 3.0 g/kg; treatment lasted for 8 wk. The results showed that dietary YC supplementation increased (P < 0.05) the total egg weight (11.2-13.6%) and egg-laying rate (13.0-13.5%) but decreased (P < 0.05) the feed/egg ratio by 9.3 to 11.0% during weeks 5 to 6 and 7 to 8 compared with the control. However, egg quality, including eggshell strength, eggshell thickness, egg weight, albumen height, egg yolk color, and Haugh unit, was not affected (P > 0.05) by YC supplementation. Furthermore, dietary YC supplementation increased (P < 0.05) chymotrypsin and ɑ-amylase activities by 54.8 to 62.5% in the duodenal chyme and reduced (P < 0.05) plasma endotoxin by 44.1%. YC dietary supplementation also upregulated (P < 0.05) the mRNA levels of intestinal barrier-related genes (occludin and claudin 1) and antimicrobial peptides genes (ß-defensin 1 and 7 and cathelicidin 1 and 3) in the duodenum or jejunum compared with the control. In conclusion, dietary YC supplementation improved the performance of aged laying hens, potentially through the upregulation of intestinal digestive enzyme activities and intestinal health-related gene expression.

The response of glandular gastric transcriptome to T-2 toxin in chicks.

This study was conducted to determine the effect of T-2 toxin on the transcriptome of the glandular stomach in chicks using RNA-sequencing (RNA-Seq). Four groups of 1-day-old Cobb male broilers (n = 4 cages/group, 6 chicks/cage) were fed a corn-soybean-based diet (control) and control supplemented with T-2 toxin at 1.0, 3.0, and 6.0 mg/kg, respectively, for 2 weeks. The histological results showed that dietary supplementation of T-2 toxin at 3.0 and 6.0 mg/kg induced glandular gastric injury including serious inflammation, increased inflammatory cells, mucosal edema, and necrosis and desquamation of the epithelial cells in the glandular stomach of chicks. RNA-Seq analysis revealed that there were 671, 1393, and 1394 genes displayed ≥2 (P < 0.05) differential expression in the dietary supplemental T-2 toxin at 1.0, 3.0, and 6.0 mg/kg, respectively, compared with the control group. Notably, 204 differently expressed genes had shared similar changes among these three doses of T-2 toxin. GO and KEGG pathway analysis results showed that many genes involved in oxidation-reduction process, inflammation, wound healing/bleeding, and apoptosis/carcinogenesis were affected by T-2 toxin exposure. In conclusion, this study systematically elucidated toxic mechanisms of T-2 toxin on the glandular stomach, which might provide novel ideas to prevent adverse effects of T-2 toxin in chicks.

A Novel Modified Hydrated Sodium Calcium Aluminosilicate (HSCAS) Adsorbent Can Effectively Reduce T-2 Toxin-Induced Toxicity in Growth Performance, Nutrient Digestibility, Serum Biochemistry, and Small Intestinal Morphology in Chicks.

The objective of this study was to evaluate the ability of a modified hydrated sodium calcium aluminosilicate (HSCAS) adsorbent to reduce the toxicity of T-2 toxin in broilers. Ninety-six one-day-old male broilers were randomly allocated into four experimental groups with four replicates of six birds each. The four groups, 1-4, received a basal diet (BD), a BD plus 6.0 mg/kg T-2 toxin, a BD plus 6.0 mg/kg T-2 toxin with 0.05% modified HSCAS adsorbent, and a BD plus 0.05% modified HSCAS adsorbent, respectively, for two weeks. Growth performance, nutrient digestibility, serum biochemistry, and small intestinal histopathology were analyzed. Compared to the control group, dietary supplementation of T-2 toxin decreased (p < 0.05) body weight gain, feed intake, and the feed conversion ratio by 11.4%-31.8% during the whole experiment. It also decreased (p < 0.05) the apparent metabolic rates of crude protein, calcium, and total phosphorus by 14.9%-16.1%. The alterations induced by T-2 toxin were mitigated (p < 0.05) by the supplementation of the modified HSCAS adsorbent. Meanwhile, dietary modified HSCAS adsorbent supplementation prevented (p < 0.05) increased serum aspartate aminotransferase by T-2 toxin at d 14. It also prevented (p < 0.05) T-2 toxin-induced morphological changes and damage in the duodenum, jejunum, and ileum of broilers. However, dietary supplementation of the modified HSCAS adsorbent alone did not affect (p > 0.05) any of these variables. In conclusion, these findings indicate that the modified HSCAS adsorbent could be used against T-2 toxin-induced toxicity in growth performance, nutrient digestibility, and hepatic and small intestinal injuries in chicks.

Proanthocyanidins Alleviates AflatoxinB1-Induced Oxidative Stress and Apoptosis through Mitochondrial Pathway in the Bursa of Fabricius of Broilers.

Aflatoxin B1 (AFB1) is a serious threat to the poultry industry. Proanthocyanidins (PCs) demonstrates a broad range of biological, pharmacological, therapeutic, and chemoprotective properties. The aim of this study was to investigate the ameliorative effects of PCs against AFB1-induced histopathology, oxidative stress, and apoptosis via the mitochondrial pathway in the bursa of Fabricius (BF) of broilers. One hundred forty-four one-day old Cobb chicks were randomly assigned into four treatment groups of six replicates (6 birds each replicate) for 28 days. Groups were fed on the following four diets; (1) Basal diet without addition of PCs or AFB1 (Control); (2) basal diet supplemented with 1 mg/kg AFB1 from contaminated corn (AFB1); (3) basal diet supplemented with 250 mg/kg PCs (PCs); and (4) basal diet supplemented with 1 mg/kg AFB1 + 250 mg/kg PCs (AFB1+ PCs). The present study results showed that antioxidant enzymes activities of total superoxide dismutase (T-SOD), catalase (CAT), glutathione peroxidase (GSH-Px), and glutathione S-transferase (GST) in AFB1 treated group were (p < 0.05) decreased, whereas malondialdehyde (MDA) contents were significantly increased in comparison with the control group. Furthermore, we found that dietary PCs treatment ameliorated AFB1-induced oxidative stress in the BF through inhibiting the accumulation of MDA content and enhancing the antioxidant enzymes activities (T-SOD, CAT, GSH-Px, and GST). Similarly, PCs markedly enhanced messenger RNA (mRNA) expression of antioxidant genes (SOD, CAT, GPx1, and GST) in comparison with AFB1 group. Moreover, histological results showed that PCs alleviated AFB1-induced apoptotic cells in the BF of broilers. In addition, both mRNA and protein expression results manifested that mitochondrial-apoptosis-associated genes (Bax, caspase-9, caspase-3, and p53 and cytochrome c) showed up-regulation, while (Bcl-2) showed down-regulation in AFB1 fed group. The supplementation of PCs to AFB1 diet significantly reversed the mRNA and protein expression of these apoptosis-associated genes, as compared to the AFB1 group. Our results demonstrated that PCs ameliorated AFB1-induced oxidative stress by modulating the antioxidant defense system and apoptosis in the BF through mitochondrial pathway in broilers.

Grape Seed Proanthocyanidin Extract Alleviates AflatoxinB1-Induced Immunotoxicity and Oxidative Stress via Modulation of NF-κB and Nrf2 Signaling Pathways in Broilers.

Aflatoxin B1 (AFB1) is a widely spread mycotoxin contaminates food and feed, causing severe oxidative stress damages and immunotoxicity. Grape seed proanthocyanidin (GSPE), a natural antioxidant with wide range of pharmacological and medicinal properties. The goal of the present study was to investigate the protective effects of GSPE against AFB1-induced immunotoxicity and oxidative stress via NF-κB and Nrf2 signaling pathways in broiler chickens. For the experiment, 240 one-day old Cobb chicks were allocated into four dietary treatment groups of six replicates (10 birds per replicate): 1. Basal diet (control); 2. Basal diet + AFB1 1mg/kg contaminated corn (AFB1); 3. Basal diet + GSPE 250 mg/kg (GSPE); 4. Basal diet + AFB1 1 mg/kg + GSPE 250 mg/kg (AFB1 + GSPE). The results showed that GSPE significantly decreased serum inflammatory cytokines TNF-α, IFN-γ, IL-1ß, IL-10, and IL-6 induced by AFB1. Similarly, GSPE + AFB1 treated group revealed a significant decrease in mRNA expressions of pro-inflammatory cytokines (TNF-α, IFN-γ, IL-1ß, and IL-6) in the splenic tissue compared to the AFB1 treatment group. In addition, western blotting results manifested that GSPE treatment normalized the phosphorylation of nuclear factor kappa B (p65) and the degradation of IκBα protein induced by AFB1. Furthermore, GSPE enhanced the antioxidant defense system through activating the nuclear factor-erythroid-2-related factor (Nrf2) signaling pathway. The mRNA and protein expression level of Nrf2 and its down streaming associated genes were noted up-regulated by the addition of GSPE, and down-regulated in the AFB1 group. Taken together, GSPE alleviates AFB1-induced immunotoxicity and oxidative damage by inhibiting the NF-κB and activating the Nrf2 signaling pathways in broiler chickens. Conclusively, our results suggest that GSPE could be considered as a potential natural agent for the prevention of AFB1-induced immunotoxicity and oxidative damage.

Correction: Ameliorative Effects of Grape Seed Proanthocyanidin Extract on Growth Performance, Immune Function, Antioxidant Capacity, Biochemical Constituents, Liver Histopathology and Aflatoxin Residues in Broilers Exposed to Aflatoxin B1. Toxins 2017, 9, 371.

The authors wish to make the following correction to their paper [...].

Dietary Silymarin Supplementation Alleviates Zearalenone-Induced Hepatotoxicity and Reproductive Toxicity in Rats.

Background: Zearalenone (ZEN) can cause serious defects in development and reproduction in humans and animals. Silymarin shows antioxidant and estrogenic effects. Objective: This study was conducted to determine if silymarin can antagonize ZEN-induced hepatic and reproductive toxicities. Methods: Thirty-five 21-d-old female Sprague-Dawley rats (n = 7/diet) were fed a control diet (Ctrl) or Ctrl plus 20 mg ZEN/kg or Ctrl plus 20 mg ZEN/kg with 100, 200, or 500 mg silymarin/kg for 6 wk. Serum, livers, ovaries, and uterus were collected at week 6 for biochemistry, hormone, and redox status and selected gene and protein assays. Results: The consumption of ZEN decreased (P < 0.05) the final body weight by 17.9%, induced liver injury, increased (P < 0.05) aspartate aminotransferase and alkaline phosphatase activities, and decreased (P < 0.05) total protein and albumin concentrations in serum by 16.7-40.6%. ZEN also caused reproductive toxicity, including decreased (P < 0.05) 17ß-estradiol and increased (P < 0.05) follicle-stimulating hormone concentrations in serum by 12.7-46.3% and induced histopathologic alterations in the liver, ovaries, and uterus. Interestingly, these alterations induced by ZEN were alleviated (P < 0.05) by silymarin supplementation at 100, 200, and 500 mg/kg. Moreover, silymarin supplementation at the 3 doses mitigated (P < 0.05) ZEN-induced impairment in hepatic glutathione peroxidase activity, total antioxidant capacity, and malondialdehyde concentration by 17.6-100%. Meanwhile, silymarin supplementation at all doses upregulated (P < 0.05) phospho-ribosomal protein S6 kinase 1 (p-RPS6KB1) and 3ß-hydroxysteroid dehydrogenase (HSD3B) by 43.0-121% but downregulated (P < 0.05) AMP-activated protein kinase (AMPK) and 3α-hydroxysteroid dehydrogenase (HSD3A) in the liver relative to the ZEN group by 11.2-40.6%. In addition, silymarin supplementation at all doses elevated (P < 0.05) HSD3B by 1.8- to 2.5-fold and decreased (P < 0.05) estrogen receptor 1 (ESR1), ATP binding cassette (ABC) c1, and Abcc5 in ovaries and the uterus by 10.7-63.2%. Conclusion: Dietary silymarin supplementation at 100, 200, and 500 mg/kg protected rats from ZEN-induced hepatotoxicity and reproductive toxicity, potentially through improvement in the antioxidant capacity and regulation in the genes related to protein synthesis, ZEN metabolism, hormone synthesis, and ABC transporters in the tissues.

Efficacy of 2-hydroxy-4-methylthiobutanoic acid compared to DL-Methionine on growth performance, carcass traits, feather growth, and redox status of Cherry Valley ducks.

The objective of this study was to compare the bio-efficacy of 2-hydroxy-4-methylthiobutanoic acid (DL-HMTBA) with that of DL-methionine (DLM) as sources of methionine in terms of the growth performance, carcass traits, feather growth, and redox statuses of Cherry Valley ducks. Six hundred and thirty male ducks were randomly allotted to 9 dietary treatment groups with 7 replicates of 10 birds each. The first group received a basal diet (BD) without methionine addition that was deficient in the total number of sulfur amino acids. In Groups 2 to 5 and Groups 6 to 9, the BD was supplemented with 4 increasing doses of methionine as either DLM or DL-HMTBA. The trial was run from ages 1 to 42 d. Dietary supplementation with DLM and DL-HMTBA improved body weight gain and feed intake as well as weights of carcasses, breast meat, and feathers compared with the BD. No significant difference was observed between the 2 methionine sources on growth performance, carcass traits, and feather growth. Concentrations of some redox markers in the pectoralis major muscle were improved by addition of methionine to the BD. However, a significant difference was observed between DLM and DL-HMTBA in this respect, as the supplementation of DL-HMTBA significantly increased the total antioxidant capacity, the activities of glutathione peroxidase, and the concentration of reduced glutathione in the pectoralis major muscle, compared with DLM. No significant difference between methionine sources was found with regard to the concentrations of oxidized glutathione and malondialdehyde in the pectoralis major muscle. Both DLM and DL-HMTBA increased malondialdehyde concentrations in the pectoralis major muscle compared with the BD. In conclusion, these results indicated that DLM and DL-HMTBA have equal biological value for the growth performance, carcass traits, and feather growth of Cherry Valley duck. Moreover, the improved antioxidant capacity observed with DL-HMTBA makes this a better candidate than DLM for lowering the oxidation process in the meat during post-mortem storage and thereby contributes to a better duck meat quality.

Prenatal exposure to zearalenone disrupts reproductive potential and development via hormone-related genes in male rats.

The present study investigated the reproductive and developmental toxicity of male offspring induced by prenatal ZEN exposure and explored the possible mechanism. 64 pregnant rats were allocated into four groups and fed with ZEN contaminated (0, 5, 10 and 20 mg/kg) diet during the whole gestation period. The results showed that, F1 male foetal viability was not affected while newborn bodyweight (BW) was significantly decreased after prenatal exposure to ZEN. Decreased BW was found on postnatal day (PND) 21 but not on PND 63 in ZEN exposed male rats. Moreover, adult testis weight increased with seminiferous tubules atrophy as well as decreased spermatocytes and mature sperms (35% and 31%) in ZEN-treated rats. Meanwhile, circulating levels of luteinizing hormone and testosterone decreased while estradiol increased in ZEN-treated rats. These impairments concurred with down-regulations of 3ß-HSD and StAR in both mRNA and protein levels in weaned and adult testis. Furthermore, gene and protein expressions of GnRHr and Esr1 were inhibited in the ZEN-treated foetal brain. These results suggested that prenatal ZEN exposure disrupted the system regulating the reproductive hormones and testis development through hormone related genes, which may result in a reproductive dysfunction in adult male offspring.