High post-pubertal parental ethanol consumption on development, inflammation, and protein and gene expression in the breast and ovary of ethanol-naive female offspring

Abstract

Background: The parental influence on the offspring starts before conception, therefore, the mother and the father lifestyle are decisive in the development, in the phenotypic alterations and in the genesis of diseases in the offspring. Our group reported that parental alcohol exposure, on preconception, impairs the development of female and male offspring, with the mechanisms less evident in females. The increase of early ethanol uses among young, which could harm their offspring, continuous in the public policy debate. Thus, we will evaluate whether the high postpubertal parental ethanol consumption impairs on development, oxidative stress, inflammation and global and gene protein expression of the ovary and breast, and what is the maternal, paternal or both contributions. Our results will contribute to the awareness of the use of early ethanol for future generations and, in female offspring, will help in a translational way in preventive methods and care. Material and methods: 40 couples of rats of the UChB variety, voluntary consumers of high amounts of ethanol (> 2g / kg / day), will be divided into four groups (n = 10 couples / group): control (C), fed with chow and water; ethanol (E), fed with feed, water and free access to ethanol by males and females; mothers exposed to ethanol (ME), fed feed and water, with access to ethanol being exclusive to females, and fathers exposed to ethanol (PE), fed feed and water, with access to ethanol being exclusive to males. Animals with access to ethanol will receive a bottle of 10% ethanol on the postnatal day (PND) 65 to 80, with withdrawal after this period. Only animals with ethanol intake > 2g / kg / day will continue in the experiment. Animals will be mating on PND 100. After pregnancy, the dams will be monitored. At the birth of the offspring, the female offspring will be monitored, being divided into four phases for analysis: juvenile (DPN 30), puberty (DPN 50), adult (DPN 150) and late adult (DPN 300), with 10 females / phase / group. The ovaries and breasts will be collected, weighed and stored. Uterine weight and retroperitoneal, visceral and ovarian fat will be recorded. For each phase analyzed, five rats will have the right breast collected for total assembly. To verify ovarian and breast function and development, ER-±, RP and IGFR-1 will be analyzed by immunohistochemistry and Western blot. Cell proliferation and apoptosis will be evaluated by Ki-67 and TUNEL, respectively. The ovarian concentration of reproductive hormones (17-² estradiol and progesterone) and pro-inflammatory mediators (IL-1², IL-6 and TNF-±) in the ovary and breast will be analyzed by immunoassays. For oxidative stress, the activities of superoxide dismutase, reduced glutathione, catalase and lipid hydroperoxide will be evaluated. A phase will be chosen for the evaluation of protein expression by mass spectrometry (LC-Ms/Ms). The genes of interest for mammary gene expression evaluation by RT-qPCR will be selected from the results obtained by proteomics.