Obesity Induces Elevated Oxidative Stress in Uteri of Reproductive Age Mice

Abstract

Abstract Obesity is an independent risk factor for endometrial cancer. We hypothesize that obesity changes endometrial physiology, increasing cellular vulnerability to cancer development. We previously found widespread increases in cytochrome P450 (CYP) expression in the uteri of diet-induced obese mice. CYP enzymatic activity is associated with oxidative stress and altered intracellular estrogen metabolism, which increases the potential for oncogenesis. To assess oxidative stress and related DNA damage in uterine tissue, we measured protein carbonyl (PC) and 8-oxo-2’-deoxyguanosine (8-OHdG) levels, respectively, in obese and lean mice. Post-pubertal C57BL/6 female mice were fed high-fat chow (HF; 45% fat; n=20) or normal chow (NC; 18% calories from fat; n=10) for either 10 or 22 weeks. The mice underwent insulin tolerance testing and MRI for body composition. Vaginal smears were analyzed over 10 days for estrous cycling (n=5 per group). PC and 8-OHdG levels were quantified from uterine tissue lysates and DNA fractions using colorimetric ELISAs and analyzed by 2-tailed t-tests. The HF mice had 64% higher body fat than NC mice after 10 weeks of diet (24±2% vs. 15±1%; p<0.003) and 176% higher body fat after 22 weeks of diet (35±2% vs. 13±2%; p<0.0001). Additionally, compared to NC mice, insulin levels in HF mice were 1.6-fold higher after 10 weeks (0.89±0.05 vs. 0.55±0.03 ng/mL; p<0.0008) and 2.8-fold higher after 22 weeks (3.56±0.49 vs. 1.28±0.21 ng/mL; p<0.005). Estrous cycle analysis showed obesity-related disruptions by 22 weeks, with HF mice spending 30±3% of their time in the proestrus phase versus 18±2% in NC mice (p<0.03). After 10 weeks of diet, HF and NC mice had similar levels of PC (p=NS). By 22 weeks of diet, however, the HF mice had a 2.3-fold increase in PC compared to the NC mice (1.11±0.2 vs. 0.49±0.09 nmol/mg; p<0.02). While HF and NC mice had similar levels of 8-OHdG for each diet group (p=NS, respectively), 8-OHdG levels increased with age between 10 and 22 weeks of diet in both HF (7.51±1.25 vs. 12.41±1.87 ng/mL; p<0.03) and NC (6.87±1.28 vs. 15.16±2.88 ng/mL; p<0.01) mice. We show that the uteri of reproductive age mice are altered by chronic diet-induced obesity and hyperinsulinemia. Uterine oxidative stress was higher with increased duration and severity of obesity, and may also be affected by the disrupted HPO axis. In contrast, obesity did not appear to promote DNA damage, suggesting that DNA repair mechanisms remain intact under conditions of increased oxidative stress. The age-dependent increase in 8-OHdG levels demonstrates an expected accumulation of DNA damage over time, which reinforces C57BL/6 mice as an adequate model for human endometrial disease. In sum, this study suggests that chronic high-fat consumption and long-term obesity increase oxidative stress over time and may contribute to the development of a pre-cancerous phenotype in human endometrium.