Study On Systemic And Reproductive Toxicity Of Acetochlor In Male Mice

Acetochlor is one of the three most abundantly used herbicides in China which is a pre-emergence herbicide belonging to the chloroacetanilide family.Its chemical properties are stable,not easy to volatilize and photolysis under natural conditions.Acetochlor is extensively used in the world,it has been classified as“likely to be carcinogenic to humans”and suspected endocrine disruptors by the USEPA and EUEPA.There are many manufacturers of acetochlor and occupational exposures to acetochlor in China,but there is still no standardized concentration limit for acetochlor in the workplace.Accordingly,there was growing concern for the risk of acetochlor to human health and environmental impact.The toxic effects and the possible mechanisms are still belongs to the research hotspot for scholars.However,the current research of acetochlor mainly focuses on the evaluation of environmental behaviors,the detection of environmental residues and decomposition processes by related technologies,the toxicity studies are mostly concentrated on non-mammalian animals.Reports of the effects on the male reproductive system in mice are rare.In this study,C57BL/6 male mice and GC-1spermatogonia were used to analyze the systemic and reproductive toxicity of acetochlor in vivo and in vitro.GC-1 spermatogonia were used to tested the verification of reproductive toxicity and the possible toxicological mechanisms.The results of this study will provide a new theoretical basis for the formulation of acetochlor related standards and the protection of acetochlor occupational contact population.Objectives:To investigate the systemic and reproductive toxicity of acetochlor in male mice and explore the relationship between acetochlor induced oxidative stress,endocrine dysfunction and reproductive cell degenerative lesions and apoptosis.Methods:SPF C57BL/6 male mice were randomly divided into four groups:Control,250 mg/kg,500 mg/kg,and 1000 mg/kg,with 10 mice in each group.Gavage once a day and the control group was given corn oil for 30 days.The body weight of the mice was monitored and recorded every week.After the end of the 30th day,the mice were sacrificed.The blood was taken to test the blood biochemical and testosterone levels.The organ/body ratios of each mice were analyzed,the testicular tissue was subjected to oxidative stress measurements and pathological sections.The spermatozoa were taken from the epididymis to examine the sperm deformity rate.CCK-8 Cell Counting Kit was used to test the viability of GC-1 spermatogonia after different concentrations of acetochlor exposed for 12 h.LDH release assay was used to test the toxic effect of acetochlor on cells.SOD,GSH,and MDA were used to detect the oxidative stress response in GC-1 spermatogonia.AnnexinV-FITC/PI double staining method was used to detect the apoptosis.Western blot was used to detect the expression of proteins JNK/SAPK,p38,p44/42?ERK1/2?,p53,Bax,Bcl-2,caspase3 and caspase-9 to investigate the possible mechanism of apoptosis induced by acetochlor.Results:1.The systemic toxicity of acetochlor-treated C57BL/6 male miceThe growth rate of body weight obviously slowed as the concentration of acetochlor increased.The organ/body ratios of heart?liver?spleen?kidney and epididymis were statistically ascendant as the increases of acetochlor concentration.Compared to the control group,the levels of BUN and CREA were significantly reduced in three dose groups compared with control;the levels of CHO were significantly increased in both middle-and high-dose groups;the levels of ALT?AST and TG were significantly elevated in high-dose groups and TP?ALB?and GLU level were no significantly difference.2.The reproductive toxicity of acetochlor in C57BL/6 male miceThe serum testosterone levels in mice exposed to acetochlor increased significantly;the level of SOD decreased and the level of MDA increased.The level of GSH decreased overall but showed no trend change.The pathology of testis was progressive with a degenerative lesions:the number of spermatozoa decreased,the arrangement of spermatogenic tubules was disordered,and vacuolization was significantly altered;the sperm aberration ratio of acetochlor exposed male mice was increased.3.Effect of acetochlor on GC-1 spgsThe cell viability of acetochlor-treated GC-1 spermatogonia were decreased,the IC50 of acetochlor to GC-1spgs was 4.80×10^-4M;cytotoxicity overall showed an upward trend,the cytotoxicity of 10^-3M group was about 4 fold change than the control;The level of SOD and GSH decreased gradually,and the level of MDA increased significantly;The rate of cell apoptosis increased with the increase of the acetochlor concentration.4.Acetochlor may mediate apoptotic pathways affecting GC-1 spgs through ERK-p53-Bcl-2 cascadeWestern blotting was used to detect the expression of related pathway proteins in acetochlor-treated GC-1 spgs.Actin as an internal reference.Acetochlor can significantly reduce the expression of p44/42?ERK1/2?in GC-1 spgs,increase the expression of pro-apoptotic protein Bax,decrease the expression of anti-apoptotic protein Bcl-2,and activate downstream apoptosis cascade,caspase3 and caspase9protein expression increased.In addition,there was a dose-response increase in the expression of p53 protein,but there was no significant change in the expression of JNK/SAPK and p38 in each group.In summary,acetochlor may regulate the ERK-p53-Bcl-2 cascade response through oxidative stress and mediate apoptotic pathways affecting GC-1 spgs cell survival.Conclusion:1.Acetochlor has a systemic toxity effect on C57BL/6 mice.2.Acetochlor has reproductive system toxicity to C57BL/6 mice and ICR mice.3.Acetochlor has obvious cytotoxic effects on GC-1 spgs.Acetochlor may mediate apoptotic pathways affecting GC-1 spgs through ERK-p53-Bcl-2 cascade.