Study Of Reproductive And Developmental Toxicity On Male Mice Caused By Fetal Exposure To Diphenylmethane Diisocyanate

Diphenylmethane diisocyanate (MD1) has been widely used in making polyurethane products in Asia, Europe and North America; it is the most manufactured and employed of all isocyanates in the world. Scientists have been concerned about its health threat to people who have access to it ever since. Existing studies showed that MDI entering body mostly via respiratory tract can induce occupational asthma, and cause damage to immune system as well as respiratory system; moreover, it has genotoxicity . Our previous studies indicated that MDI has reproductive toxicity, for reasons of causing weight loss in testes of mice, exerting significant influences on activities of some testicular enzymes that are related with sperm producing and male fertility, and on levels of sex-related hormones such as testosterone, luteotrophic hormone and follicle stimulating hormone. Relevant research by Gamer AO, Buschmann J et al. showed that high dose MDI aerosol of 12mg/m3 inhaled from gestational day 6 through 15 by pregnant Wistar rats can cause maternal toxicity and developmental toxicity, while 3mg/m³ was considered having no embryotoxicity. The present study is to further explore potential effects of MDI exposure in pregnancy on the development of reproductive, endocrine and other related systems of male mouse offspring, and their functions in maturity.Objective:Exposing male KM mice at fetus period to MDI by maternal administration, observing and measuring the development of their reproductive, endocrine and other related systems, and their reproductive system functions in maturity, to further explore potential endocrine disrupting effects of MDI, its reproductive and developmental toxicities and the possible mechanisms, to provide study on MDI of its endocrine disrupting effects and reproductive and developmental toxicities with theories and experimental evidences. Methods:Sixteen pregnant SP mice from Laboratory Animal Center of Shandong University (licence No.: SCXK (LU) 20030004) weighing 45-60g were randomly divided into four equal groups according to LD₅₀ (oral) of MDI for mouse being 2200mg/kg, i.e. One control group (maize oil was given) and three MDI groups (275mg/kg, 137.5mg/kg, 68.75mg/kg MDI dissolved in maize oil were given respectively). The intragastrical administration started from gestational day 14 and ended before delivery, once every day. Male pups were separated from their dams on postnatal day 21 and performed eye-pulling surgery to obtain blood samples at postnatal day 56, and then sacrificed by vertebra dislocating. General conditions, bodyweight changes and delivery times of the dams were observed and recorded. Numbers, sex ratios, bodyweight changes, survival rates, eyes opening ages, preputial separation ages and bodyweights of the cubs in each litter were specifically observed and recorded after their birth. Thymus glands, spleens, livers, and testes, epididymides and kidneys of both sides were detached and weighed to calculate relative organ weights respectively after their sacrifice. Two testes from each group were randomly selected and used for making paraffin sections. Other testes were temporarily stored at -80℃for further detecting activities of SDH, ACP, AKP, NOS, ATPase and SOD, contents of proteins (used for calculation), NO and MDA, and levels of testosterone in testis. Epididymides of left side were used for determination of sperm motility, and those of the other side preserved at -20℃for sperm count and calculating sperm deformity ratio. The obtained blood was centrifuged to attain sera which were thereafter collected and stored at -20℃for further detection of T, LH and FSH levels.Results:1. Effects of MDI on general conditions and deliveries of pregnant mice No evident toxic symptoms were observed in any MDI group, no evident pathological changes in internal organs were observed neither when dissection was performed, the general conditions of pregnant mcie kept well, the delivery times of pregnant mice were basically normal, with no statistical differences between groups (P>0.05). The bodyweight increases of pregnant mice in MDI groups were lower than in the control group during administration, and showed a declining tendency with the increase of MDI concentration, but the differences were not statistically significant (P>0.05). There were no statistical differences between any groups in offspring numbers of a litter and sex ratio of a group (P>0.05).2. Effects of MDI on general growth conditions of male mouse offspringThere was no abnormality but developmental retardation was observed in the MDI treated groups, no evident pathological changes in internal organs were observed when dissection was performed. The survival rates of the offspring before postnatal day 21 were not statistically different between any two groups (P>0.05). The eyes opening ages of the offspring in MDI groups were delayed comparing with those in the control group. The average bodyweights of the offspring in MDI groups were all lower than those in the control group (P<0.01) persistently. The relative weights of spleen in MDI groups were significantly higher than those in the contriol group (P< 0.01).3. Effects of MDI on development of genitals and sperm parameters of male mouse offspringThere was no difference between MDI groups and the maize oil group in preputial separation ages of male offspring (P>0.05), but the bodyweights when prepuces fully separated in MDI groups were lower than those in the maize oil group (P<0.01). The weights and relative weights of testis in 275mg/kg MDI group were significantly lower than in the other three groups (P<0.01 or P<0.05). Sperm motilities showed a declining tendency with the increase of MDI concentration, sperm deformity ratios were all lower in MDI groups than in the control group, but there were no statistical differences between MDI treated groups and the control group in sperm motility, sperm count and deformity ratio (P>0.05). 4. Effects of MDI on activities of some testicular enzymes of male mouse offspringActivities of SDH, ATPase and ACP showed declining tendencies with the increase of MDI concentration, and there were differences between MDI groups and the control group (P<0.01 or P<0.05). Activities of AKP in MDI groups were all lower than those in the control goup, showing a rising tendency with the increase of MDI concentration, significant differences only existed between 68.75mg/kg MDI group or 137.5mg/kg MDI group and the control group (P<0.01, P<0.05). Activities of total NOS and c-NOS were all lower in MDI groups than in the control group (P<0.01 or P<0.05), but activities of i-NOS between MDI groups and the control group were not different (P>0.05) in spite of the rising tendency with the increase of MDI concentration. NO contents of testis in MDI groups were all lower than those in the control group, but not significantly (P>0.05). Activities of SOD in 137.5mg/kg and 275mg/kg MDI groups were lower than those in 68.75mg/kg MDI group and the control group (P<0.01 or P<0.05). The contents of MDA in MDI groups were lower than those in the control group, but the differences were not statistically significant (P >0.05).5. Effects of MDI on testis tissue structures of male mouse offspringWith the increase of MDI concentration, the walls of seminiferous tubules in MDI groups became thinner gradually, layers of cells lessened, the arrangement disordered, but the basic structures were integral, with mature sperms observed in the tubules.6. Effects of MDI on sex-related hormones of male mouse offspringThere were no statistical differences in serum FSH and T levels between MDI groups and the control group (P>0.05). Serum LH levels showed a rising tendency with the increase of MDI concentration, but only between 275mg/kg MDI group and the control group, the difference was statistically significant (P<0.01). Testis T levels showed a rising tendency with the increase of MDI concentration, but not significantly (P>0.05). Conclusion:MDI has not been proved to have endocrine disrupting effects; MDI does have embryotoxicity, intrauterine exposure to MDI at fetus period can influence the growth and development of mouse offspring, and the developmental retardation of testis tissues can influence activities of many important testicular enzymes and secretions of sex-related hormones, finally leading to possible functional disorders of testis and declination of reproductive capacity.