Metabolic Activation Of Pyrrolizidine Alkaloids In Placenta And Fetal Liver Caused Developmental Toxicity In Fetal Rats

Background:Pyrrolizidine alkaloids(PAs)are a type of defense constituents with similar chemical structure and wildly present in plants.PAs were attracting people’s attention due to their severe hepatoxicity and wide range of sources.3%of flowering plants contained toxic PAs,and 660 kinds of PAs and their N-oxide metabolites have been found in more than 6000 plants in worldwide.Research showed that unsaturated PAs exhibited notable hepatoxicity after metabolic activation mainly by cytochrome P450(CYP)3A to form electrophilic intermediates(pyrrolic esters),which attack a variety of important intracellular nucleophilic substances(i.e.DNA and protein),lead to apoptosis of hepatocytes,hepatic megalocytosis,hepatic veno-occlusive disease and so on.Studies have demonstrated that prenatal PAs exposure to high doses of monocrotaline(MCT)during the middle and late pregnancy can cause developmental toxicity.However,the metabolic activation of PAs in uterine was still unclear.Objective:Using retrorsine(RTS)and MCT as the representative of the PAs,the purpose of this research was to reveal the effects of PAs on fetus during pregnancy,understand the metabolic profile of PAs in maternal-placental-fetal unit(including maternal liver,placenta and fetal liver),and explore the developmental toxicity and metabolic injury caused by PAs in fetus.Methods:Microsomes of maternal liver,neonatal liver,fetal liver and placenta were prepared by employment of normal pregnant Wistar rats.Wistar pregnant rats were randomly divided into five groups,namely control group,RTS5 group,RTS 10 group,RTS20 group and MCT20 group(n=10-12).From day 9 of gestation day(GD9)to GD20,the pregnant rats were gavaged with 5 mg/kg,10 mg/kg,20mg/kg RTS and 20 mg/kg MCT per day respectvely,the control group were given the same volume of solvent.We recorded the daily weight of pregnant rats during this period,and calculated the weight grow rate of pregnant rats.At GD20,the pregnant rats were anesthetized by ether anesthesia,and the fetal liver and placenta were collected and weighed.Hematoxylin-eosin(HE)staining was used to observe the morphological changes of maternal liver,fetal liver and placenta.Western blot was used to detect the CYP3A expression of maternal and fetal liver of normal,RTS20 and MCT20 group rats;And the serum content of RTS and MCT,the pyrrole-protein adducts(PPAs)of liver and placenta,GSH conjugates with RTS or MCT were detected by LC/MS/MS,Realtime PCR was used to detect the mRNA expression of CYP3 A isoforms in maternal liver,fetal liver and neonatal liver,recombinant CYP3A4 and CYP3A7 protein was used to detect their activity to RTS.Results:① the expreesion of CYP3A and the activity of normal maternal,fetal liver and placental:the results showed that quantity of CYP3A protein in fetal liver was significantly lower than that in maternal liver(P<0.01),and expression of CYP3A in male fetus was slightly higher than that of female.Compared with maternal liver,expression of CYP3A protein in placenta was comparative less,especially for placenta from female pups(P<0.01).Placenta CYP3A expression from male fetuses exceeded that in females(P<0.05).Michaelis-Menten kinetic analysis found that maternal liver microsomal activity for RTS and MCT were higher than that of fetal liver;Maternal hepatic CLint for RTS and MCT,which were calculated by Vmax/Km,were 21.9-and 8.6-fold higher than that of female fetal liver,and were 10.8-and 5.2-fold higher than that of male fetal liver,respectively;②the basic situation of maternal and fetal rats:Body weight grow rate of pregnant rats,weight of dam,fetus,fetal liver and placenta on GD20,which exposed to 10mg/kg,20 mg/kg RTS or 20mg/kg MCT,were lower than that of control group(P<0.01).③Maternal and fetal liver and placenta morphology:HE results showed that the maternal and fetal liver and placenta have different extent of tissue damage due to exposure to different doses of RTS or MCT,especially for 20 mg/kg RTS and MCT.Female and male pups in 20 mg/kg RTS or MCT group exhibited increased number of parenchyma cells and loss of hepatocytes,especially for female,increase of vacuolar cells was seen even in 10 mg/kg RTS group.Compared with the control group,RTS and MCT exposure caused morphology disorder in placenta tissue,especially for 20 mg/kg RTS and MCT.Glycogen cells in junctional zones were necrosis and accompanied with cysts and congestion,cells of labyrinthine zone were disorganized and reduced.Placentas of both genders showed similar morphological changes;④ The content of PAs in serum and PPAs in differrent tissue:In all PA-treatment groups,serum concentrations of RTS and MCT in fetal serum were parallel to that in maternal serum,and there was no difference in serum PAs concentration between different genders of pups;The PPAs content in the maternal liver was higer than that in placenta and fetal liver;the content of PPAs in RTS20 and MCT20 group of female fetal liver was higher than that of male(P<0.05),especially for MCT20 group(P<0.01);⑤ The effect of PAs on the expression of CYP3A in fetal liver:prenatal exposure to 20 mg/kg RTS or MCT induced the expression of CYP3A in femal pup liver(P<0.01),while inhibited that in male(P<0.05);⑥The CYP3A mRNA expreesions in maternal,fetal and neonatal liver were different,CYP3A1/23 was predominant in the matanal liver,and the fetal liver was only detected the expression of CYP3A18 and 3A62,but the expression was very low,while CYP3A1/23,3A2,3A18 and 3A62 were detected in the neonatal liver,and the CYP3A2 was higher than that in maternal liver;and the catalic activity towards RTS of neonatal liver microsome was higher than that in maternal and fetal liver;⑦ the activity of CYP3A7 to RTS was higher than that of CYP3A4.Conclusion:Our study indicated that RTS and MCT were capable to permeate placenta barrier and then enter fetal circulation.Local metabolic activation of PAs in fetal liver was gender-dependent,which probably due to gender-specific induction of CYP3A and P-glycoprotein,and may be responsible for PA-induced fetal hepatic injury,especially for female pups.The activity of CYP3A7 to RTS was higher than that of CYP3A4,which might cause the difference in metabolic injury between mother and fetus.