| Formaldehyde (FA) is named as ants aldehyde. Its molecular formula is CH3CHO. Formaldehyde contains unsaturated carbonyl, with strong activity of oxidization, and its chemical properties is active. Formaldehyde is widespread in human living environment, where it is form a variety of health-related products, such as indoor decoration materials, printing ink, textile fibers, cosmetics, pesticides, chemical cleaners, disinfectant, preservative, and the combustion of various fuels, containing formaldehyde at varying degrees. In the past 20 years, as the large-scale development of china in building decoration and construction, the degree of exposure to formaldehyde is higher than the past. It is reported that urea-formaldehyde resin in the indoor decoration materials can slowly release low concentration of formaldehyde for a few years even decades, and lead to a potential hazard of human health.In vitro experiments, the results have shown that formaldehyde is a genotoxic carcinogen, and can induce the DNA double-strands in peripheral blood lymphocytes, human leukemia cells (HL-60), alveolar macrophages and testicular cells breaking down. And as increasing of concentrations of formaldehyde, cells present a cross-linking effect of DNA-DNA or DNA-protein. Epidemiological studies also confirm that long-term exposure to low concentrations of formaldehyde can lead to inflammation of eye and respiratory tract, bronchial asthma, neurobehavioral abnormalities, and increase the risk of nasopharyngeal or lung cancer. For female, formaldehyde can also increase the risk of infertility, stillbirth and teratism. At present, the biological mechanisms of female reproductive toxicity which is induced by formaldehyde are yet unclear. Especially, under the low-dose exposure conditions, it is necessary to clarify the toxicological mechanism of formaldehyde at the cellular and molecular level. Ovaries play an important role in reproduction of female animals, oogonia in ovarians can pass fallopian tube, enter uterus and wait for fertilization in formation of a mature oocyte by mitosis and meiosis. If female animals of sexual maturity are exposed to environment containing formaldehyde, development and maturation or fertilization of oocyte are disturbed. As a result, it will lead to miscarriage, stillbirth and other serious consequences.In our study, Sprague-Dawley (SD) female rats were chosen as experimental animals treated with different concentrations (low to high) of formaldehyde. Serum, ovarian and uterus tissue, oocytes and granulosa cells were collected. The characteristics of cell and molecular toxicity in ovaries or uterus of female rats induced by formaldehyde are elaborated from the changes of function of ovarian reserve, ovarian tissue and cell morphology, expression of apoptosis related genes, etc. Further, it is expected to provide a reference for the detailed mechanisms of female reproductive toxicity induced by formaldehyde.Part I Toxicological effects of formaldehyde on ovarian function and tissue structure of female rats(?) Effects of formaldehyde on function of ovarian reserve in female SD rats Objective:To explore the effects of subacute exposure to formaldehyde on ovarian reserve function in female rats.Methods:Forty female Sprague-Dawley (SD) rats were randomly divided into a control group and three formaldehyde groups. The rats in formaldehyde groups were intraperitoneally treated with formaldehyde at 0.2,2.0 and 20.0 mg/kg.d for 14 days, respectively. Estradiol (E2) was detected by chemiluminescent, luteinizing hormone (LH), follicle stimulating hormone (FSH) and Inhibin B were detected by radioimmunoassay or enzyme-linked immunoadsordent assay (ELISA). Ovaries or body weights of female rats were measured and organic coefficient of ovaries was counted.Results:Compared with the control group, the levels of serum estradiol (E2) and Inhibin B in the groups treated with formaldehyde decreased significantly (P<0.05). The level of serum FSH increased and organic coefficient of ovaries decreased significantly (P<0.05), while the levels of LH did not change obviously (P>0.05).Conclusions:The injury to ovarian reserve function in female rats is resulted from formaldehyde exposure.(?) The pathological effects of Formaldehyde on micro-or ultra-structure of tisue and cells of ovarian in female SD ratsObjective:To study the effects of formaldehyde on ovarian histopathology structure, oocyte and granulosa cell micro-or ultra-structure of female rats.Methods:Healthy female rats were injected intraperitoneally with different doses of formaldehyde (20.0,2.0,0.2mg/kg), once a day for 14 days. These rats were killed at the end of treatment with formaldehyde. Morphological changes of ovary cells were observed by light microscopy and the ultrastructure of oocyte and granulosa cells was observed by electron microscope. The apoptosis of oocyte and granulosa cells was determined by terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) staining.Results:Formaldehyde induced pathological structural changes in ovarian tissue of female rats, mainly as mild atrophy of ovarian cortex, structural disorder, interstitial dilatation and congestion of small blood vessels and fibrous tissue proliferation, etc. And the damage in the rat ovarian increased with dose of formaldehyde. The ultra-structure of oocyte and granulosa cells in ovarian of female rats was also observed by electron microscope after formaldehyde treatment, especially in feature of nuclear membrane shrinkage, chromatin margination, and nucleoli smaller or dissappear, etc. The changes of ultra-structure in ovaries of rats increased with dose of formaldehyde. The experiment of terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling suggested also that apoptosis of oocyte and granulosa cells was induced by formaldehyde in middle and high doses groups, and apoptosis rate of oocyte and granulosa cells was different significantly between groups.Conclusions:Formaldehyde has adverse effects on ovarian histological structure, and micro-or ultra-structure in female rats and can lead to apoptosis of oocyte and granulosa cells. Part II Effect of formaldehyde on expressions of Fas apoptosis pathway-related genes of ovary tissues in female ratsObjective:To explore the mechanism of formaldehyde inducing ovarian toxicity in female rats by observing effect of formaldehyde on the expression of Fas and caspase-8,and the activity of caspase-3 and caspase-8 of ovary tissues in female rats.Methods:Forty female Sprague-Dawley(SD) rats were randomly divided into a control group and three formaldehyde groups at the level of different dose. The rats in three formaldehyde groups were intraperitoneally injected with different doses of formaldehyde (20.0,2.0 and 0.2 mg/kg), once a day for 14 days. At the end of treatment with formaldehyde, all rats were sacrificed and their ovaries were collected for detecting the expression of Fas and caspase-8 mRNA by reverse transcription polymerase chain reaction (RT-PCR), the expression of Fas protein by method of western-blot, and the activities of caspase-8 and caspase-3 by spectro-photometric method.Results:Compared with the control group, the expression of Fas mRNA and its protein and caspase-8 mRNA and the activity of caspase-8 and caspase-3 of ovary tissues in the rats significantly increased with the dose of formaldehyde (P<0.05).Conclusion:The increase of Fas gene expression and the activity of caspase-8 and caspase-3 may be the important causes of apoptosis in ovarian of female rats induced by formaldehyde. Part?Interference effect of formaldehyde on expression of mitochondria-dependent apoptosis related genes in ovary or uterus tissues of female ratsObjective:To explore the interference effect of formaldehyde on expression of voltage-dependent anion channel(VDAC1), adenine nucleotide translocator(ANT1), or 8-hydroxyguanine glycosylase (hOGG1) mRNA in ovarian or uterus of female rats.Methods:Twenty female Sprague-Dawley(SD) rats were randomly divided into a control group and three formaldehyde intraperitoneally injected groups at the dose of 0.4,4.0,40.0 mg/kg.d, respectively for 5 days. At the end of formaldehyde treatment, all rats were sacrificed and their ovaries and uterus were collected for detecting the expression of VDAC1, ANT1, or hOGGl mRNA by method of reverse transcription quantitive polymerase chain reaction (RT-qPCR).Results:The expression of VDAC1 and ANT1 mRNA increased significantly in ovarian and uterus, compared with the control group, but the change of expressional level of VDAC1 and ANT1 in uterus was more sensitive than that in ovaries in low, mediate, and high dose groups(P<0.05). However, the mRNA expression of hOGG1 in ovaries was similar to uterus. The level of expression was not significantly different in low dose group, but expression of hOGG1 mRNA decreased significantly in mediate and high dose groups(P<0.05).Conclusion:Under formaldehyde treatment condition, the expression of VDAC1 and ANT1 mRNA increased significantly and the expression of oxidative damage repair gene hOGGl mRNA decreased significantly in ovarian or uterus. It was associated closely with mitochondria damage and further activation of apoptosis procedure.In the sum, under different concentration of formaldehyde exposure conditions, formaldehyde can cause damage to function of ovarian reserve, especially the decrease of serum concentration of estradiol (E2), inhibin B, but follicle stimulating hormone (FSH) levels increased significantly. Meanwhile, pathological change in ovarian tissue or oocytes and granulosa cells of female rats is also induced by formaldehyde, showing features of ovarian cortical atrophy, structural disorder, fibrous tissue hyperplasia, and oocytes or granulosa cells show features of shrinkage of nuclear membrane, swelling of mitochondria, and shrink or disappear of nucleoli, etc. At the molecular level, the results also suggest that formaldehyde can induce abnormal expression of Fas or mitochondria apoptosis pathway related gene in ovarian or uterus tissue of female rats, such as Fas, caspase-8, caspase-3, VDAC1, ANT1, and hOGG1, etc. It is associated closely with activation of Fas or mitochondria pathway of apoptosis procedure. |
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