N-hexane Inhalation During Pregnancy Alters Ovary Development And DNA Methylation In Rat Offspring

N-hexane is a kind of solvent that widely used in food making, medicine production, and the organics synthesis. As it is volatile at the room temperature, the inhalation is the most frequent way in the occupational exposure. N-hexane is previously considered to be a low or micro-poisonous chemical so as to be widely used in various industries, the "Poisonous Apple" incident in2008had drawn public attentions, however, recent studies have shown that n-hexane and its metabolites had serious effects on female endocrine and reproductive system. In addition, The fundamental dates of toxicology had indicated that n-hexane exposure during gestation might induce offspring dysplasia and no gene mutations induced by were observed. As the above, we had established a model of pregnancy n-hexane exposure in order to detect the role of DNA methylation in the ovary development and ovarian granulosa cell functions in the adult offspring.Objective:To detect the impact on the development of reproductive system in the female offspring after gestational n-hexane exposure;another purpose was to investigate the methylation status of genome promoter, differential gene ontology, and methylation patterns in the ovarian granulosa cells of F1female rats after n-hexane exposure during pregnancy. And to explore the relationship between promoter methylation status, mRNA levels of the hormone synthesis genes, and the hormond levels in the ovarian granulosa cells of F1female rats after n-hexane exposure during pregnancy.Methods:1. Clean grade Wistar rats, whose body weights ranged from210-230g for females and300-320g for males, were caged. Each group contained5pregnant animals. F0pregnant rats inhaled n-hexane on gestational days1-20(4h per day) at doses of0,100,500,2500, and12500ppm. In the F1rats the sex ratio at birth, body weight growth, vaginal opening time, and estrous cycle were observed. Tissue sections were cut from each rat’s ovary with a rotary microtome, and then consecutively stained with hematoxylin and eosin (H&E). After these, the proportion of each stage follicles was calculated and pathologic changes were detected. The ovarian granulosa cells of the female F1generation was isolated and cultured for4h, then the progesterone and estradiol in the supernatant of the culture solution were measured using Chemiluminescent enzyme immunoassay (CLEIA)2. Genome DNA (gDNA) of ovarian granulosa cells in F1generation females was extracted using DNeasy Blood&Tissue Kit, The gDNA of each sample was sonicated and immunoprecipitated with5-mC antibody. Later the immunoprecipitated gDNA was hybridized with Nimblegen385Kpromoter Plus CpG Island Arrays. The differential methylation genes were defined as Peak Score>2compared with control. Several methods were used to analyze the differential genes, including the Gene Ontology Consortium tools, the DAVID Functional Annotation Clustering Tool, hierarchical clustering, and KEGG pathway analysis.3. The differential methylation peak (compared with control) in the promoter of hormone synthesis genes were determined using Signalmap software, afterwards the methylation status were verified by MS-HRM method. The mRNA levels of corresponding genes were determined using RT-PCR. Results:1. The live births of12500ppm group were significantly fewer than any other group, the male/female ratio showed a downtrend as the exposure dose increased. There were no differences between the control and n-hexane exposure group in body weight growth, however, n-hexane exposure group seemed to show a slower increase of body weights after PD36. There were also no differences between the control and n-hexane exposure group in virginal opening time. Compared with the control, the preoestrus durations were significantly longer in100,500, and2500ppm groups; the estrus durations of500and2500ppm groups were longer than that of the control. The diestrus duration and of12500ppm groups were shorter than the control. The atretic follicles proportion in12500ppm was lower than the control. Estradiol levels of2,500ppm and12,500ppm group were significantly lower than that of the control. Compared with the control, progesterone levels in100ppm and500ppm exposure group were significantly higher than that of the control, but progesterone levels in12,500ppm were significantly lower.2. The number of shared demethylated genes was higher than that of methylated genes, and the differentially methylated genes were enriched in cell death and apoptosis, cell growth and hormone regulation. The methylation profiles of the offspring from the500ppm and control groups were different from those of the2,500and12,500ppm groups. Furthermore, the methylation status of genes in the PI3K-Akt and NF-kappa B signaling pathways was changed after n-hexane exposure. The Cyp11a1, Cyp17a1, Hsd3b1, Cyp1a1, and Srd5a1promoters were hypermethylated in the n-hexane-exposed groups.3. promoter methylation of the genes related with hormone secretion in the ovarian granulosa cells of rat offspring:(1) Star, Cyp11a1, Cyp17a1, and Hsd3b genes showed differential promoter methylation of after n-hexane exposure. The specific as follows: In500ppm group, compared with control promoter methylation degrees of Star, Cyp11a1and Cyp17a1were lower, meanwhile those of Hsd3b showed no difference. In2500ppm group, compared with control, promoter methylation degrees of Cyp11a1and Cyp17a1were higher, meanwhile those of Star and Hsd3b showed no difference. In12500ppm group, compared with control, promoter methylation degrees of all the four genes were higher than those of the control.(2) Using the MS-HRM, the methylation degrees in local region of the Star gene promoter were as below:12500ppm>2500ppm> control>500ppm>100ppm; the methylation degrees in local region of the Cyp11al gene promoter were as below:25%>12500ppm>2500ppm> control>500ppm和100ppm; The methylation degrees in local region of the Cyp17al gene promoter were as below:12500and2500ppm> control>500and100ppm; the methylation degrees in local region of the Hsd3b gene promoter were as below:12500ppm>2500ppm and Control>100and500ppm. In total, results of MS-HRM were consistent with those of the MeDIP-CHIP.(3) Star, Cyp11a1, Cyp17a1, and Hsd3b showed differential mRNA levels after n-hexane exposure. The specific as follows:in Star and Cypllalgene expression, compared with control, the mRNA levels of12500ppm group were significantly lower, but those of500ppm group were significantly higher; in cyp17al gene expression, the mRNA levels of2500and12500ppm group were significantly lower than those of the control; in Hsd3b gene expression, the mRNA levels of12500ppm group were significantly lower than those of the control.Conclusions:1. N-hexane exposure during pregnancy had obvious embryo toxicity, and interfered with ovarian developments and functions in adult F1female offspring.2. N-hexane exposure during pregnancy could alter the methylation status of the promoter in the offspring ovarian granulosa cells, especially high doses of n-hexane exposure (2500and12500ppm) significant changed gene promoter methylation of cell apoptosis and hormone synthesis genes.3. After n-hexane exposure in gestation, the relationships between sex hormone secretion ability, the promoter methylation and mRNA levels alteration of genes Star, Cyp11a1, Cyp17al and Hsd3b in F1ovarian granulosa cells might exist.