Higher sensitivity of female cells to ethanol: methylation of DNA lowers Cyp2e1, generating more ROS.

BACKGROUND: Cells taken from mouse embryos before sex differentiation respond to insults according to their chromosomal sex, a difference traceable to differential methylation. We evaluated the mechanism for this difference in the controlled situation of their response to ethanol. METHODS: We evaluated the expression of mRNA for alcohol dehydrogenase (ADH), aldehyde dehyrogenases (ALDH), and a cytochrome P450 isoenzyme (Cyp2e1) in male and female mice, comparing the expressions to toxicity under several experimental conditions evaluating redox and other states. RESULTS: Females are more sensitive to ethanol. Disulfiram, which inhibits alcohol dehydrogenase (ADH), increases cell death in males, eliminating the sex dimorphism. The expressions ADH Class 1 to 4 and ALDH Class 1 and 2 do not differ by sex. However, females express approximately 8X more message for Cyp2e1, an enzyme in the non-canonical pathway. Female cells produce approximately 15% more ROS (reactive oxygen species) than male cells, but male cells contain approximately double the concentration of GSH, a ROS scavenger. Scavenging ROS with N-acetyl cysteine reduces cell death and eliminates sex dimorphism. Finally, since many of the differences in gene expression derive from methylation of DNA, we exposed cells to the methyltransferase inhibitor 5-aza- 2-deoxycytidine; blocking methylation eliminates both the difference in expression of Cyp2e1 and cell death. CONCLUSION: We conclude that the sex-differential cell death caused by ethanol derives from sex dimorphic methylation of Cyp2e1 gene, resulting in generation of more ROS.

What cell death does in development.

Cell death is prominent in gametogenesis and shapes and sculpts embryos. In non-mammalian embryos one sees little or no cell death prior to the maternal-zygotic transition, but, in mammalian embryos, characteristic deaths of one or two cells occur at the end of compaction and are apparently necessary for the separation of the trophoblast from the inner cell mass. Considerable sculpting of the embryo occurs by cell deaths during organogenesis, and appropriate cell numbers, especially in the CNS and in the immune system, are generated by massive overproduction of cells and selection of a few, with death of the rest. The timing, identity, and genetic control of specific cells that die have been well documented in Caenorhabditis, but in other embryos the stochastic nature of the deaths limit our ability to do more than identify the regions in which cells will die. Complete disruption of the cell death machinery can be lethal, but many mutations of the regulatory machinery yield only modest or no phenotypes, indicating substantial redundancy and compensation of regulatory mechanisms. Most of the deaths are apoptotic and are identified by techniques used to recognize apoptosis, but techniques identifying lysosomes (whether in dying or involuting cells or in the phagocytes that invade the tissue) also reveal patterns of cell death. Aberrant cell deaths that produce known phenotypes are typically localized, indicating that the mechanism of activating a programmed death in a specific region, rather than the mechanism of death, is aberrant. These results lead us to conclude that we need to know much more about the conversations among cells that lead cells to commit suicide.

Sex-dependent regulation of cytochrome P450 family members Cyp1a1, Cyp2e1, and Cyp7b1 by methylation of DNA.

Sexual differences are only partially attributable to hormones. Cultured male or female cells, even from embryos before sexual differentiation, differ in gene expression and sensitivity to toxins, and these differences persist in isolated primary cells. Male and female cells from Swiss Webster CWF mice manifest sex-distinct patterns of DNA methylation for X-ist and for cytochrome P450 (CYP; family members 1a1, 2e1m, and 7b1. Dnmt3l is differentially expressed but not differentially methylated, and Gapdh is neither differentially methylated nor expressed. CYP family genes differ in expression in whole tissue homogenates and cell cultures, with female Cyp expression 2- to 355-fold higher and Dnmt3l 12- to 32-fold higher in males. DNA methylation in the promoters of these genes is sex dimorphic; reducing methylation differences reduces to 1- to 6-fold differences in the expression of these genes. Stress or estradiol alters both methylation and gene expression. We conclude that different methylation patterns partially explain the sex-based differences in expression of CYP family members and X-ist, which potentially leads to inborn differences between males and females and their different responses to chronic and acute changes. Sex-differential methylation may have medical effects.