Embryo Toxicity Of Mice Exposure To Combined Benzene And Formaldehyde At Phases Of Follicular And Embryonic Implantation

BackgroundBezene(BZ) or Formaldehyde(FA),as the important industrial solvent,widely existed in building materials or decoration materials.With the development of people's living standard,it is becoming more and more popular to have house decorated before living in.Thus,the two chemicals have become main indoor air pollutants after decoration.Most of people's time was spent in the house,so the hazard to human health from indoor air pollutantion has drawn extensive attentions. The previous epidemiology studies showed that exposure to BZ or FA alone could obviously affect the menstrual cycles and reproductive function.Animal experiments showed that exposure to BZ or FA could lead to maternal toxicity and developmental disabilities of embryos.Though BZ and FA are commonly found in combination in the environment of newly-decorated rooms,there were few reports on their combined effects.In addition,the embryo toxicity from BZ and FA may be different after exposure at different reproductive stages.Thus,this study was desiged to examine the embryo toxicity of mice after exposed to benzene and formaldehyde at phases of follicular and embryonic implantation,using a factorial design.ObjectiveTo investigate embryo toxicity of mice and compare the differences between the follicular phase and the implantation phase after combined exposure to benzene and formaldehyde;To study the mechanism of embryo toxicity from the perspective of DNA-protein crosslink.Methods1.Animal models:embryo toxicity of exposure to combined benzene and formaldehyde at the phases of follicular and embryonic implantation in mice(1) Experimental design:4×4 factorial experimental design was used in the present study.The two factors,benzene and formaldehyde,were involved and each had four levels:0,0.875,8.75 and 87.5 mg/kg BZ and 0,7.875,15.75 and 31.5 mg/kg FA.The same volume of olive oil and/or distilled water was used to establish a baseline point of reference and used as diluent in preparation of test chemicals.(2) Exposure design:â‘ Exposure time:Following an acclimatization period of one week,each experimental female animal was injected intraperitoneally at the phases of follicle and implantation.The follicular phase for toxicant exposure was at three days before mating while the implantation phase for toxicant exposure was on the pregnant day of 4-6.â‘¡Exposure way:The chemicals were injected by intreperitoneal exposure.The amount of chemicals daily injected was determined by the body weight.(3) Superovulation technique:Following an acclimatization period of one week, each experimental female mouse was intraperitoneally injected with 10 U pregnant mare serum(PMSG),and then 10 U human chorionic gonadotrophin(HCG) at interval 48h to equal estrous cycle and to promote ovulation.Later,female mice were mated with mature males(1:1).Embolus in the vaginal smear was examined in the next morning.Successful mating was determined by the presence of embolus and was designated as day 1 of gestation(GD₁).No pregnant mice will be dropped after mating in continuous three days.(4) Endpoint events:Physical condition of each mouse was assessed and change in maternal body weight was recorded daily.All animals were sacrificed by cervical dislocation at GD₉ The livers,spleen,kidneys,ovaries and uterus were removed and weighted.The implanted conceptuses were counted and weighted. (5) Statistical treatment:Spss13.0 statistical software was used for statistical treatment.Variances were evaluated by Homogeneity-of-variance test first.If variances were equal,statistical treatment were performed with factorial analysis of variance(ANOVA),followed by LSD's post hoc tests.If variances were unequal, statistical differences were evaluated by nonparametric Kruskal-Wallis test followed by Mann-Whitney U-test.Statistical significances were determined at level ofα=0.05.2.DNA-protein crosslink experimentsThe free DNA and crosslinked DNA were separated from tissue cell according to KCL-SDS method.Fluorescent dyes Hoechst33258 was added into sample,and keep the final concentration for 30minutes in the dark place.F-4500 fluorescence decompn spectrophotometric instrument was used for measuring fluorescence value at 350nm excited light and 450nm emission light.The standard curve between DNA concentration and fluorescence value was built.Then,DNA-protein crosslink was computed by formulaη=A/(A+B).Spss13.0 statistical software was used for statistical treatment.Variances were evaluated by Homogeneity-of-variance test first.If variances were equal,statistical treatment were performed with factorial analysis of variance(ANOVA);If variances were unequal,statistical treatment were performed with rank test.Statistical significances were determined at level ofα=0.05.3.Association between ovarian damage and embryo implantationSpss13.0 was used to analyze the effect of embryo toxicity or ovarian toxicity from combine BZ and FA,or the correlation coefficients association between ovarian damage and embryo implantation.Statistical significances were determined at level ofα=0.05.Results1.Animal models:embryo toxicity of exposure to combined benzene and formaldehyde at the phases of follicular and embryonic implantation in mice(1) In the follicular phase:BZ did not have obvious embryo toxicity;FA resulted in reduced embryo implantation(P=0.02) and lowered embryo weight (P=0.00);The weight of ovarian and uterus decreased significantly(P=0.00 and P=0.05,respectively);BZ and FA had significant interaction on reduced ovarian weight(F=2.17,P=0.02 ).â‘¡In the implantation phases:BZ had no significant effect on the embryo implantation,the weight of total and average embryos,the weight of ovarian and uterus,and so did FA;The spleen weight and kidney weight changed greatly in BZ or FA groups(P＜0.05).â‘¢Combined effects:Compared with FA, embryo toxicity was weaken by exposure to mixture of low or median BZ dose and high FA dose in follicular phase;Compared with individual FA,embryo toxicity was weaken by exposure to mixture of median or high BZ and FA dose.So certain dosage of BZ and FA had interaction on embryo implantation.2.DNA-protein crosslink experiment(1) BZ had no effect on DPC in the follicular phase(P=0.136),while FA had significant effect on DPC(P=0.000).Compared with control,low dosage was greatly different(Z=-3.930,P=0.000),so was the median dosage(Z=-4.846,P=0.000) or the high dosage(Z=-4.544,P=0.000).(2) BZ or FA had no effect on DPC in the implantation phase(P=0.721, P=0.127)(3) The DPC in the follicular phase was greater than that in the implantation phase.BZ and FA had remarkable interaction on DPC(F=39.87,P=0.000)Conclusion1.FA resulted in obvious embryo toxicity,which was greater than BZ in the follicular phase.BZ or FA exposure in the follicular phase was more sensitive than that in the implantation phase.2.Ovarian damage was more obvious in the follicular phase than that in the implantation phase,which may result in the reduced embryo implantation.3.BZ and FA had certain interaction on the embryo toxicity in the determined dosage.4.There was a significant correlation between ovarian damage and number of embryo implantation,which indicated that maybe the ovarian damage resulted in the reduced embryo implantation.