Effect Of Low Dose BPA Prenatal Exposure On Morphologic Changes During Long Bone Development

Objective:Bisphenol A(BPA)is one of the most widely used Endocrine disrupting compounds in the world.BPA can be used to synthesize polycarbonate plastics and epoxy resins,which can cause reproductive dysfunction,change in fat metabolism,and damage to brain function and increase susceptibility to prostate and breast cancer.Previous studies have shown that bone tissue may be particularly sensitive to low-dose BPA during development.Therefore,the effect of low-dose BPA exposure on offspring development is of great significance.Endochondral ossification occurs rapidly in long bones during embryonic and early postnatal period.There are few reports about the effect of BPA on endochondral ossification,and the mechanism is not very clear.The formation and degradation of extracellular matrix is the key to skeletal development.MMP-9 and MMP-13 have the ability to cut extracellular matrix components in bone tissue.The effects of BPA on the expression of MMP-9and MMP-13 in bone tissue has not been reported.Our group previously found that low-dose BPA exposure in drinking water during gestation and lactation could decreased growth plate height and delayed the formation of secondary ossification centers in adolescent offspring.To further explore the effects of low-dose BPA exposure during gestation and lactation on the development of long bones during embryonic and lactation,a Wistar rat model of low-dose BPA exposure during gestation and lactation was established to investigate the formation of primary ossification center and secondary ossification center during long bone development.The changes in several key periods of formation,and the expression changes of MMP-9 and MMP-13 in it,provide clues to the study of the mechanism of the effects of low-dose BPA exposure during pregnancy and lactation on the growth of offspring long bones and the prevention and treatment of BPA bone toxicity in accordance with.Methods: The male and female Wistar rats were caged together,and the pregnant rats were individually fed.On the 0th day of pregnancy,pregnant rats were randomly divided into Control group,0.01 mg/LBPA group,and 0.1 mg/LBPA group.Pregnant rats were exposed to BPA from drinking water from day 0 to weaning(day 21 afterdelivery).Newborn,2W and 3W pups were taken and their body weight,body length,tail length,tibia and femur length were measured.The left tibia was fixed and embedded to prepare paraffin sections for HE staining,Van Gesion / Alixin double staining,Von Kossa staining,and immunohistochemical staining.The whole skeleton of newborn rats was stained with Alixin blue / Alizarin Red S.Results: 1.Effects of low-dose BPA exposure during pregnancy on body weight,body length,tail length,tibia and femur length and mineralization of newborn pups:Compared with the control group,the weight of newborn pups in each BPA group decreased,except for 0.01 Except for the newborn female rats in the mg/L BPA group,the differences were statistically significant(P<0.05).The body length(nose tip-tail tip)of the newborn male rats in the 0.1mg / L group was significantly lower than that in the control group,and the difference was statistically significant(P<0.05).The tail length of the newborn male rats in the 0.1 mg/L group and the newborn female rats in the 0.01 mg/L group were significantly lower than those in the control group(P<0.01).In the BPA group,the skeleton of newborn pups is shorter than that of the control group,and the tail is shortened.The bone length of the long bones of the forelimbs and hind limbs is shorter than that of the control group.Ossification point,except for the newborn male rats in the 0.1 mg/L BPA group,the newborn rats in the other BPA groups did not develop.The length of the tibia and femur in the BPA group decreased compared with the control group,and the length of the tibia and femur of the newborn male rats in the 0.1 mg / L group and the newborn female rats in the 0.01 mg/L group were significantly shorter than those in the control group.The difference was statistically significant(P<0.05).The length of the tibial and femoral mineralized areas of the newborn male rats in the 0.1mg/LBPA group was significantly shorter than that in the control group(P<0.05).The other BPA groups were also reduced compared to the control group,but the difference was not significant.In the control group,the trabeculae of the newborn pups were thin and long.In the BPA group,the shape of the trabeculae was irregular,sparse,broken,thick and short,and some of them were flaky or punctate.Mineralization has occurred around the cells and terminal hypertrophic chondrocytes,and mineralization has hardly occurred in theBPA group or only around terminal mast chondrocytes.Except for the femur of newborn male rats in the 0.1mg / LBPA group,the bone volume fraction(BV / TV)of the BPA group was lower than that of the control group,and the tibia of male rats was significantly different.The length of the tibial and femoral callus of newborn pups in the BPA group was lower than that of the control group,and the length of the femoral callus of newborn male rats in the BPA group was significantly lower than that of the control group,the difference was statistically significant(P<0.05);The groups were thinner,and the femur of newborn male pups in the 0.1 mg / L and 0.01 mg/LBPA groups was significantly different from the control group(P<0.05).In each BPA group,the morphology of chondrocytes in the tibia and femur growth plates of male and female offspring changed,the chondrocyte column in the proliferative zone became shorter,the number of flat cells decreased,and the number of round cells increased.The normal stacking of cells is no longer apparent;mast chondrocytes become smaller.2.The effects of low-dose BPA exposure during pregnancy on body weight,body length,tail length,tibia and femur length and mineralization of 2W pups:all indicators of pups in the 2W BPA group have a downward trend compared with the control group,0.01 mg / The tibia length of male offspring of LBPA group decreased significantly(P<0.05).In the 2W BPA group,the trabeculae of tibia secondary ossification were mostly primary trabeculae,and there were more cartilage islands than the control group.The measurement results showed that the secondary ossification centers of newborn pups had a downward trend,and the SOC area of male pups in the BPA group decreased most significantly(P<0.05).In the 2W BPA group,the anterior hypertrophic area of the tibia of male offspring was significantly thickened(P<0.05),the total growth plate and the hypertrophic area were thickened,and the proliferation area became shorter,but the difference was not significant.At2 W,the morphology of chondrocytes in the tibial growth plate of male offspring in the BPA group was similar to that in newborn rats.The chondrocyte column in the proliferative zone became shorter,the number of flat cells decreased,and the number of abnormal cell shapes(rounded)increased.The normal stacking of cells is no longer apparent.The cells became smaller and the intercellular density increased;the cells inthe hypertrophic region also became smaller than the control group,the number of irregular cells increased,and the cell size was uneven;the transition from the proliferation zone to the hypertrophic zone was no longer obvious.3.Effects of low-dose BPA exposure during pregnancy on body weight,body length,tail length,tibia and femur length,and mineralization in 3W pups: There was no difference in the parameters of 3W BPA pups.Taking the right tibia and femur of the 3W pups in each group,the length measurement results showed that there was no significant difference in the length of the tibia and femur of the pups in each group compared with the control group.At 3W,the primary spongy bone of the tibia of male offspring in the BPA group was significantly shorter than that in the control group,but there was no significant difference in the females.The area of secondary ossification centers of 3W male pups was most significantly reduced(P<0.05),and the area of secondary ossification centers of female pups was not significantly different.At 3W,the morphological changes of chondrocytes in the tibial growth plate of male offspring in the BPA group were similar to those in the newborn and at 2W.The length measurement of the growth plate area showed that compared with the control group,the anterior hypertrophic area of the tibia of male pups in the BPA group was significantly thickened at 3W(P<0.05),and the hypertrophic area became thinner.The 0.01 mg / LBPA group was different from the control group.Significant(P<0.05).The total growth plate thickened and the proliferation area became shorter,but the difference was not significant.4.Effect of low-dose BPA exposure during pregnancy on the expression of marker proteins in long bones of young rats: The expression of MMP-13 in the tibial secondary ossification center of male young rats in the newborn BPA group was lower than that in the control group,while MMP-The expression of13 was higher than that of the control group;the expression of MMP-13 in the tibial growth plate of male offspring of 2W and 3W BPA groups was also significantly higher than that of the control group(P<0.05).The expression of MMP-9 protein in the tibia of male offspring of the newborn BPA group was significantly lower than that in the control group(P<0.05),while the expression of MMP-9 protein in the tibia of male offspring in the BPA group was significantly higher than that in the controlgroup at 2W and 3W,of which 0.1mg / The difference in group L was statistically significant(P<0.05).Conclusion: 1.Low-dose BPA exposure during pregnancy and lactation can affect the development of long bone growth plates,POC,and SOC in the offspring during embryonic and lactation.2.Low-dose BPA exposure during gestation can change the expression of MMP-9 and MMP-13 in long bones of newborn,2W and 3W daughter rats.