Toxic Effects And Mechanisms Of Norgestrel On The Digestive Gland Of Clam Mactra Veneriformis

Progestin,a type of steroid hormone,was widely used in human contraception,assisted reproduction,cancer treatment,and regulation of animal reproduction and growth.Due to incomplete treatment by sewage treatment plants and direct discharge of wastes from agricultural or livestock activities,they enter the aquatic environment and eventually enter the ocean.A few to tens of thousands ng/L of progestins have been detected in water discharged from livestock farms,surface water runoff,and marine ecosystems.However,a few ng/L progestin can have toxic effects on aquatic organisms.Currently,the toxicological research of progestins on aquatic organisms is mainly focused on fish,while the toxicological research on invertebrates with important ecological functions in aquatic ecosystems is urgently needed.In this study,the buried clam Mactra veneriformis was exposed to a typical progestin,norgestrel(NGT)for 21 days.The bioaccumulation of NGT,histological structure analysis,and transcriptomic and metabonomic analysis were performed in the digestive gland.And the toxic effects and the mechanisms of NGT on the digestive gland of Mactra veneriformis was elucidated.The results are showed as follows.1.Chemical methods were used to study the bioaccumulation of NGT in the digestive gland of M.veneriformis after 21 days of exposure to NGT(0,10,and 1000ng/L).The bioaccumulation of NGT in 10 ng/L NGT group and 1000 ng/L NGT group was 2.97 ± 0.14 and 202.77 ± 5.80 μg/kg(wet weight),respectively,and the biological concentration factor(BCF)was 386 and 268,respectively.This indicated that the digestive gland of this clam can effectively bioaccumulate NGT.2.The effects of NGT on histological structure of digestive gland of M.veneriformis were analyzed using paraffin section and electron microscope section.The microstructure showed that NGT caused swelling of the epithelial cells and blurring of the basement membrane.The ultrastructure showed that NGT caused damage to mitochondria and endoplasmic reticulum,which influenced protein synthesis and transportation,and caused cell dysfunction.These damaged structures may be related to oxidative stress.3.Transcriptomic analysis was conducted to identify the molecular toxicological effects and mechanisms of NGT on the digestive gland of this clam.Compared with the control group,309(202 upregulated,107 downregulated)and 340(156 upregulated,184 downregulated)differentially expressed genes were identified in 10 ng/L and 1000ng/L group,respectively.KEGG pathway analysis showed that differentially expressed genes were significantly enriched in Wnt signaling pathway,steroid hormone metabolism,antioxidant defense,amino acid metabolism,and other pathways.These results showed that NGT mainly interfered with detoxification metabolism,antioxidant defense,carbohydrate and amino acid metabolism,steroid hormone metabolism,and immune function in M.veneriformis.4.Metabonomic analysis of the digestive gland was conducted to reveal the effect of NGT on the metabolites in the clam.Compared with the control group,46metabolites(6 upregulated,40 downregulated)and 54 metabolites(18 upregulated,32downregulated)with significant differences in content were identified in the 10 ng/L and 1000 ng/L group,respectively.The different metabolites mainly involved in biological processes such as carnitine,carbohydrates,amino acids,and steroid hormones.KEGG pathway analysis showed that different metabolites were significantly enriched in pathways such as ABC transporters,amino acid metabolism,and carbohydrate metabolism,which suggested that NGT led to enhanced detoxification metabolism,impaired protein synthesis,and disordered energy metabolism in M.veneriformis.All the results indicated that the digestive glands of M.veneriformis can efficiently bioaccumulate NGT.NGT mainly interfered with the detoxification system,antioxidant defense,carbohydrate and amino acid metabolism,and steroid hormone metabolism of M.veneriformis,resulting in the damage to the digestive gland.This study enriches the toxicological research of progestin on the invertebrates and provides scientific data for the ecological risk assessment of progestin.