Breast cancer 1 (BRCA1)-deficient embryos develop normally but are more susceptible to ethanol-initiated DNA damage and embryopathies ☆

摘要

Highlights ? Heterozygous (+/?) brca1 conditional knockout (cKO) embryos develop normally. ? +/? brca1 cKO embryos have 28% less BRCA1 protein than wild-type (WT) littermates. ? Ethanol-exposed BRCA1 - deficient mice have more oxidatively damaged DNA than WTs. ? Ethanol-exposed BRCA1 cKO embryos exhibit more embryopathies than WT littermates. ? BRCA1 protects the embryo from ethanol-enhanced oxidative stress—a novel role. Abstract The breast cancer 1 ( brca1 ) gene is associated with breast and ovarian cancers, and heterozygous (+/?) brca1 knockout progeny develop normally, suggesting a negligible developmental impact. However, our results show BRCA1 plays a broader biological role in protecting the embryo from oxidative stress. Sox2-promoted Cre-expressing hemizygous males were mated with floxed brca1 females, and gestational day 8 +/? brca1 conditional knockout embryos with a 28% reduction in protein expression were exposed in culture to the reactive oxygen species (ROS)-initiating drug ethanol (EtOH). Untreated +/? brca1 -deficient embryos developed normally, but when exposed to EtOH exhibited increased levels of oxidatively damaged DNA, measured as 8-oxo-2'-deoxyguanosine, γH2AX, which is a marker of DNA double strand breaks that can result from 8-oxo-2'-deoxyguanosine, formation, and embryopathies at EtOH concentrations that did not affect their brca1 -normal littermates. These results reveal that even modest BRCA1 deficiencies render the embryo more susceptible to drug-enhanced ROS formation, and corroborate a role for DNA oxidation in the mechanism of EtOH teratogenesis. Graphical abstract Figure options Download full-size image Download as PowerPoint slide Abbreviations 8-oxo-2′-deoxyguanosine , 8-oxodGuo ; BER , Base excision repair ; brca1 , Breast cancer 1 ; csb , Cockayne Syndrome B ; dGuo , deoxyguanosine ; DSB , double strand break ; ELISA , enzyme linked immunosorbent assay ; EtOH , ethanol ; FASD , fetal alcohol spectrum disorder ; GD , gestational day ; MDMA , methylenedioxymethamphetamine ; METH , methamphetamine ; NCC , neural crest cell ; NOX , NADPH oxidases ; Nrf2 , nuclear factor erythroid 2-related factor 2 ; ogg1 , oxoguanine glycosylase 1 ; ROS , reactive oxygen species ; γH2AX , phosphorylation of histone H2AX Keywords Breast cancer 1 (BRCA1) ; Oxidatively damaged DNA ; DNA repair ; Ethanol ; Embryo culture ; Embryopathies prs.rt("abs_end"); 1. Introduction Brca1 is a DNA repair gene commonly associated with certain familial breast and ovarian cancers [1] . Studies in knockout models have shown that mice lacking both copies of the brca1 gene have poor genomic integrity and are not viable [2] . The morphological basis has been characterized by rapid disorganized proliferation of neuroepithelium and increased cell death [3] . However, progeny missing only one copy of the brca1 gene appear to develop normally [3] , suggesting that minor BRCA1 deficiencies have a negligible developmental impact. A number of population-based studies have investigated the developmental consequences of brca1 mutations on sex differences, miscarriage rates and prevalence of mutation carriers as well as double mutants. While it has been suggested in small population studies that there are differences in birth rates and sex ratios [4] and [5] , this has not been confirmed in larger population studies [6] and [7] . Clinically, there have been no confirmed cases of a viable individual with two mutated copies of the brca1 gene [8] , although screening of lymphocyte DNA from patients in Scotland with breast or ovarian cancer revealed a woman who lacked the wild-type allele for brca1 , and was the offspring of two carriers of the brca1 mutation [9] . However, the finding that this individual carried two copies of the mutated gene was likely the result of a technical error in the screening method [10] . Taken together, population studies on brca1 mutations and embryonic development suggest that double mutants are not viable, while heterozygous carriers of the mutation develop normally but are more susceptible to developing cancer later in life [7] and [11] . The BRCA1 protein contains a ring domain at the N-terminal primarily associated with E3 ubiquitin ligase activity and a C-terminal responsible for tumor suppression and recruitment of DNA repair enzymes [12] . When evaluating the two domains of the BRCA1 protein, it was found that the C-terminal is not essential for development. However, mice lacking the C-terminal were at significant risk of developing tumors as early as 1 month after birth in a tissue- and time-dependent manner [13] . This finding suggests that the multiple functions of BRCA1 in DNA repair are not required for normal embryonic and fetal development at least with respect to gross morphology. Evaluation of a number of other DNA repair genes in mouse models has similarly shown that progeny deficient in DNA repair exhibit normal gross morphology, but are more susceptible to developmental anoma