Study On Reproductive And Embryonic Developmental Toxicity And Mechanism Of Formaldehyde And Benzene In Decorated House Indoor Air

Objective:To monitor the pollution status and trends of formaldehyde, homologues of benzene, and TVOCs in decorated houses indoor air, to investigate the menstruation condition and cyophoric process and outcome of the newly-married female who live in the new decorated house, and to explore the female reproductive damage by indoor air pollution in new decorated house.Methods:The reproduction function of 323 newly-married female who lives in the new decorated house and 72 newly-married female who don't lives in the new decorated house were investigated, and To monitor the pollution status and trends of formaldehyde, homologues of benzene, and TVOCs of in their house indoor air.1. The menstruation condition and cyophoric process and outcome of the 395 newly-married female and their risk factors were investigated by using the questionnaire designed, and persistent observation for 1.5-3 years.2. The concentration of indoor air's formaldehyde, benzene, toluene, xylene and TVOCs was monitored in the different time points at living new house,3rd month and 6th month. The methods of monitoring pollutants were according to the "Indoor air quality standard". 3. The informant was divided into exposed and non-exposed according to that if their indoor air pollutant's concentration is beyond the "Indoor air quality standard", and comparing the incidence of irregular menstruation and anomalous pregnancy outcome between exposed and non-exposed4. The risk factors of women's menstruation and cyophoric process and outcome were analyzed by Logistic regression analysis.Results1. The decoration material mainly including man-made board, all kinds of paint, glue,adhesive, compound floor board, and so on could emit air pollutant.2. The concentration of indoor air formaldehyde of 57.89% decorated house exceed the "Indoor air quality standard" at living new house, at 3rd month and 6th month., the exceeding rate is 50.15% and 25.39% respectively, and there were significant differences between the three time points (P＜0.05). The decorated house that concentration of indoor air benzene, toluene and xylene at living new house exceeded "indoor air quality standard" were 41.18%, 58.82% and 51.70% respectively, The exceeding rate of benzene, toluene and xylene 3 and 6 months after living new house was lower than that at living new house (P＜0.05). The exceeding rate of benzene, toluene and xylene at the sixth month after living new house dropped to 5.57%,21.98% and 24.15% respectively. The exceeding rate homologues of benzene in decorated house indoor air was lower than that of formaldehyde (P＜0.05). The decorated house that concentration of indoor air TVOCs at living new house exceeded "indoor air quality standard" was 60.06%, after 3 and 6 months, the concentration of TVOCs and exceeding rate of decorated house were both lower than that at living new house(P<0.05). At the sixth months, there still was 29.93% decorated house TVOCs concentration exceeding "indoor air quality standard".3. The incidence of abnormal menstruation (including menstrual cycle,menstrual period irregular,menstrual bleeding abnormal,painful menstruation) of exposed women were higher than that of non-exposure group(P<0.05). The spontaneous abortion incidence in exposed group was higher than that in non-exposed group(P<0.05), RR was 2.35,95% CI was 1.09-5.05. The pregnant complication incidence in exposed group was higher than that in non-exposed group(P<0.05), RR was 1.86,95% CI was 1.22-2.84.8.87% babies had birth defect, which was more than that of non-exposed group(P<0.05), RR was 2.43,95% CI was 1.06-5.58.4. The results by logistic regression analysis showed that the main factors affecting women's reproductive functions and menstruation are indoor air's formaldehyde, benzene, toluene, xylene and TVOCs, poor ventilation, staying home long each day. In addition, drinking was a major risk factor too.conclusions1. Indoor air of decorated house is heavily polluted by formaldehyde, benzene, toluene, xylene and TVOCs. But as time goes on, the pollutant concentration fall gradually,2. Indoor air pollution in decorated house will damage female reproductive function and menstruation, cause to menstruation abnormal, high spontaneous abortion incidence and birth-defect. Objective To study embryonic developmental toxicity of formaldehyde and benzene in vitro and to explore the interaction type of formaldehyde and benzene.Methods1. The whole embryo culture system in vitro was adopted. The 8.5-day embedded mouse embryos were co-cultured with immediately centrifugal serum (ICS) of rat containing different concentration formaldehyde and benzene for 48 hours. HE staining and light microscope was used to observe the effects of single or combinative formaldehyde and benzene on growth and development of embryo, morphodifferentiation of tissue and organ.2. Embryonic limb bud and midbrain cells at 12-day gestational age were separated. Micromass culture was used to investigate the effects of benzene and formaldehyde on differentiation and proliferation of limb bud and midbrain cells.3. Fetal mouse limb bud at 12-day gestational age was cultured in a homeothermia rotating apparatus to observe the influence of formaldehyde and benzene on fetal mouse limb bud development and differentiation.Results1. Embryonic development in blank group and solvent group are essentially coincident between cultured for 48hr in vitro and 10.5d gestational age in vivo(P>0.05).2. Benzene had obvious toxic effects on development of embryos in a dose-dependent manner. In 1000μmol/L group, the embryonic growth and develop- ment, and morphodifferentiantion of tissue and organs were suppressed, abnormal embryos was observed, and also fetal death was viewed. Formaldehyde could affect growth and development of embryos in mouse on organogenesis, and also have evident dose-dependent manner, and it could induce malformation including araphia and paramorphia of body position, chest and heart, or fetal death.3. The morphodifferentiation of embryonic tissue and organs was no influenced in (10+3)μmol/L formaldehyde & benzene group. With the increasing formaldehyde & benzene concentration, the indexes of growth and morphodifferentiation were lower than that of control(P<0.05), diameter of saccus vitellinus, crown-rump length, head length and number of arthromera were obviously decreased. The abnormal embryo rate and death rate were much higher than that of control. The major appearances including cephalosome, eyes, ear, arcus branchialis and body position were abnormal, limb bud small. It showed in microscope that benzene and formaldehyde could cause disordered structure and irregular arrangement of splanchnic wall VYS endodermis cells. There were cell polarity missed, degeneration and engorgement, and there was vacuole in endochylema.4. Benzene and formaldehyde could cause embryonic death. Death rate of embryo was 11.1% in 27μmol/L formaldehyde, and the rate was 33.3% and 44.4% respectively in 27μmol/L formaldehyde combined with 10μmol/L and 100μmol/L benzene. Abnormal embryo rate of embryo was 11.1% in 9μmol/L formaldehyde, and the rate was 44.4% and 88.9% in 9μmol/L formaldehyde combined with 10μmol/L and 100μmol/L benzene. It showed that the intensity of the combination toxic action and teratogenic action on embryo was much stronger than the action of the mix of the two agents'solitary effect. The results illustrated that the two agents exhibited synergistic toxic effect.5. Benzene and formaldehyde could inhibite the proliferation and differentiation of embryonic limb bud and midbrain cells in a dose-dependent manner in vitro. The cell differentiative inhibition of formaldehyde and benzene was exceeded cell proliferative inhibition. The IDC50 and IPC50 of benzene for limb bud cells were 89.93μmol/L and 396.77μmol/L respectively. The IDC50 and IPC50 of benzene for midbrain cells were 116.29μmol/L and 831.46μmol/L respectively. The IDC50 and IPC50 of formaldehyde for limb bud cells were 16.46μmol/L and 26.92μmol/L respectively. The IDC50 and IPC50 of formaldehyde for midbrain cells were 116.29μmol/L and 831.46μmol/L respectively.6. The development and differentiation of fetal mouse fore limb buds were inhibited by benzene and formaldehyde. With the increasing of benzene and formaldehyde concentration, cartilagineus development and differentiation of limb was low, the Neubert score was decreased in a dose-dependent manner.Conclusions1. Benzene and formaldehyde cause embryo toxicity and teratogenicity in vitro. They could suppressed the development and differentiation of limb bud organ and inhibited proliferation and differentiation of embryo limb bud cells and midbrain cells.2. Benzene and formaldehyde could damage structure and function of embryo VYS, induce degeneration and death of embryo cells.3. The interaction of benzene and formaldehyde on mouse embryo was appeared to be synergistic effect. Objective To study the reproductive and embryonic developmental toxicity of benzene and formaldehyde for mouse, and to explore the interaction type and their toxic mechanism. To provide experimental and theoretical evidence for the further evaluating the reproductive and embryonic developmental toxicity of benzene and formaldehyde.Method 54 male and 108 female Kunming mice were randomly divided into 9 groups respectively,6 male and 12 female mice in each group.8 groups' male mice respectively breathed in benzene vapor(5,10,15)mg/m3, formaldehyde vapor(3,6,9)mg/m3 and formaldehyde & benzene (3+6) mg/m3 and (6+9) mg/m3 at 1 st to 7th day,2hr/day. Control group's male mice breathed in air of cabinet at 1st to 7th day,2hr/day.8 groups' female mice also respectively breathed in benzene vapor(5,10,15)mg/m3, formaldehyde vapor(3, 6,9)mg/m3 and formaldehyde & benzene (3+6) mg/m3 and (6+9) mg/m3 at 28th to 34th day,2hr/day. control group's Female mice also breathed in air of cabinet at 28th to 34th day,2hr/day. At 35th day, Same group's famle and male mice were inter-mateed to form pregnant mice. To observe interbreeding rate and pregnant rate. The growing and development situation of male mice were observated during 1st to 35th day. All mice were killed after inter-mateed, and testicular organ coefficient was calculated. The sperm mortality and malformation rate were observed in all mice epididymis. In each group, one testis of every mouse were used to doing conventional pathology section, HE staining to observe the testicular tissue morphous changes, and anothers were homogenised and centrifuged, the concentration of NO, T-AOC and Ca2+, the activity of ATPase were detected in the supernatant using spectrophotometric method.Every experimental group's pregnant mice respectively breathed in benzene vapor(5,10,15)mg/m3, formaldehyde vapor(3,6,9)mg/m3 and formaldehyde & benzene (3+6) mg/m3 and (6+9) mg/m3 at pregnancy 7th to 15th day,2hr/day, and control group's pregnant mice breathed in air of cabinet at pregnancy 7th to 15th day,2hr/day. The pregnant mice were sacrificed at 18th day of pregnancy and the blood was gathered using to detect estradiol and progesterone by enzyme immunoassay. The mouse embryos were isolated. The growth development of placenta and embryos were measured, the absorbed and dead embryos were counted, the external appearances and internal organ malformations were observed. The organ coefficient of heart, liver, spleen, kidney and ovary in the pregnant mice was calculated. The ovary and placenta of all pregnant mouse were used to doing conventional pathology section, HE staining to observe the ovarian and placental tissue morphous changes. The expression levels of estrogen receptors in placental tissue was measured by immunohistochemical method and to quantitate using image analysis.Results1. During experiment, there were no change of the hair and activity of mice in treatment groups and control group, also no acute poisoning and death in each group. The interbreeding rate was 100% in each group. The pregnant rate and abortion rate in high-concentration formaldehyde or(and) benzene and were higher than that of control(P<0.05). Comparing to same concentration formaldehyde or benzene, the pregnant rate and abortion rate were higher in joint group.2. Middle and high concentration benzene or(and) formaldehyde could decreased the growth of male mice and pregnant mice body weight(P<0.05), and there was a certain degree of dose-effect relationship. The growth rate of pregnant mice body weight in joint groups was lower than that of in same concentration benzene or formaldehyde, the difference was statistical significance (P<0.01). The joint toxic effect of benzene and formaldehyde on body weight was synergism.3. The testicular organ coefficient was decreased with the increasing of concentration of benzene or(and) formaldehyde, and the testicular organ coefficient in treatment groups was lower than that of in control group (P<0.01), there was statistical significance except the low-dose benzene or formaldehyde group. And the inhibition of benzene and formaldehyde for it also was dose-effect relationship. The synergistic toxic effect of benzene and formaldehyde could been found in testicular organ coefficient. There were dead sperm and the sperm shape abnormality in the control and the treatment groups. In the control, the percentages of dead sperm, malformed sperm and testis cell apoptosis were 4.55%,5.86% and 4.95% respectively. And there was higher in the treatment groups compared with the control group(P<0.01). The larger the dose of benzene or(and) formaldehyde were, the higher the percentages of dead sperm and malformed sperm were, and the synergistic toxic effect of benzene and formaldehyde could been detected in this. The main type of sperm malformation were big head sperm, small head sperm, double head sperm, banana head sperm, crook head sperm, short tail sperm and fold tail sperm etc.4. In air control, the seminiferous tubules had regular morphology and clear border; and spermatocyte, primary spermatocyte, secondary spermatocyte arranged densely form outer to inner, and their shape and structure were clear in convoluted seminiferous tubule; The convoluted seminiferous tubule was situated in the cernter, and it contained large numbers of mature and normal sperm cells. The testicular histopathology in all treatment groups showed:the seminiferous tubules had irregular morphology and unclear border, spermatocyte, primary spermatocyte and secondary spermatocyte didn't arrange closely form outer to inner, moreover their shape and structure were damaged in convoluted seminiferous tubule. The convoluted seminiferous tubule was deviated form the center, even it was lack, only parts of seminiferous tubules contained some mature sperm cells.5. With the increases of the exposure concentration of formaldehyde or(and) benzene, T-AOC, ATPase activity in all treatment groups were decreased(P<0.01), but the contents of NO and Ca2+ in all treatment groups were increased(P<0.01). moreover also there was dose-effect relationship. Formaldehyde and benzene had a significant synergistic effect on T-AOC, ATPase activity, contents of NO and Ca2+.6. Comparing to air control, the organ coefficient of heart, spleen and kidney of the pregnant mice was no significant difference between all groups (P>0.05), but the organ coefficient of liver and ovary in the middle and high concentration formaldehyde or(and) benzene groups were lower than that of air control(P<0.05). The organ coefficient of liver and ovary in joint groups were lower than that of the same concentration benzene or formaldehyde group (P<0.05). Formaldehyde and benzene had a significant synergistic toxic effect on the organ coefficient of liver and ovary.7. The rate of the absorbed and dead embryo and deformans embryo were no statistical significance between low-concentration formaldehyde or benzene group and air control (P>0.05). Middl and high concentration formaldehyde or benzene and joint groups could result in the increasing of the rate of the absorbed and dead embryo and embryos malformations(P<0.05). And middle and high concentration formaldehyde or(and) benzene and groups could inhibit the development of embryo(P<0.05), moreover, it could induce increasing the rate of embryo malformations(P<0.05). Formaldehyde and benzene had a significant synergistic toxic effect on the embryonic development in vivo.8. The development of every grade ovarian follicle was normal in air control. The atret-ovarian follicle was increased in exposure to formaldehyde or benzene, but the constitution every grade ovarian follicle had on apparente abnormal in formaldehyde or benzene groups.9. The high-concentration formaldehyde or(and) benzene could inhibit the development of placenta(P<0.05) and result in placental weight descent (P＜0.05). The constitution of placenta was normal, and exist a large of glycogen in glyco-cell of trophoderm in the air control. In high-concentration formaldehyde or(and) benzene group, the trophoblastic cell changed smaller, and its arrangement was conglobation, a bulk of trophoblastic cell terminal line was dim, cytoplasm was eryth-staining, karyopycnosis, the glyco-cell in trophoderm reduced or vanished, the lacouna of blood sinus was narrowing and its number could decreased. A few cell of giant cell layer desquamated, the euchromatin of many cell was condense, and a few cell had nuclear inclusion.10. The level of estrogen were no statistical significance among all groups (P>0.05). Compared with the control group, the blood-serum progesterone in all benzene or(and) formaldehyde groups was increase and had in a dose-dependent manner(P<0.01). There was statistical significance comparing with the all all benzene or(and) formaldehyde groups(P<0.05). The expression positive rate of ERa of placental villus in middle and high-dose all benzene or(and) formaldehyde groups was higher than that of in air control(P<0.01). But the expression positive rate of ERβof placental villus was on statistical significance among all groups (P>0.05).Conclusions1. Formaldehyde and benzene can inhibite the growth and development of mice, and damage mice reproductive cells, result in sperm malformation or death. Formaldehyde and benzene have synergistic toxic effect on reproductive system in male mouse.2. Formaldehyde and benzene may have the embryonic developmental toxicity and induce mouse embryonic teratogenicity in vivo.3. The mechanism of the embryonic developmental toxicity and teratogenicity of formaldehyde and benzene for mouse embryo may be to damage the placental constitution, to inhibit the progesterone secretion, and to alter the placental ERa expression. The mechanism of reproductive harm of formaldehyde and benzene for male mice may be to damage antioxygen ability, to inhibit ATPase activity and to influence NO metabolism and Ca2+ disbalance in generative cell, and so on.