Sex steroid hormones regulate constitutive expression of Cyp2e1 in female mouse liver

Abstract

CYP2E1 is of paramount toxicological significance because it metabolically activates a large number of low-molecular-weight toxicants and carcinogens. In this context, factors that interfere with Cyp2e1 regulation may critically affect xenobiotic toxicity and carcinogenicity. The aim of this study was to investigate the role of female steroid hormones in the regulation of CYP2E1, as estrogens and progesterone are the bases of contraceptives and hormonal replacement therapy in menopausal women. Interestingly, a fluctuation in the hepatic expression pattern of Cyp2e1 was revealed in the different phases of the estrous cycle of female mice, with higher Cyp2e1 expression at estrus (E) and lower at methestrus (ME), highly correlated with that in plasma gonadal hormone levels. Depletion of sex steroids by ovariectomy repressed Cyp2e1 expression to levels similar to those detected in males and cyclic females at ME. Hormonal supplementation brought Cyp2e1 expression back to levels detected at E. The role of progesterone appeared to be more prominent than that of 17β-estradiol. Progesterone-induced Cyp2e1 upregulation could be attributed to inactivation of the insulin/PI3K/Akt/FOXO1 signaling pathway. Tamoxifen, an anti-estrogen, repressed Cyp2e1 expression potentially via activation of the PI3K/Akt/FOXO1 and GH/STAT5b-linked pathways. The sex steroid hormone-related changes in hepatic Cyp2e1 expression were highly correlated with those observed in Hnf-1α, β-catenin, and Srebp-1c. In conclusion, female steroid hormones are clearly involved in the regulation of CYP2E1, thus affecting the metabolism of a plethora of toxicants and carcinogenic agents, conditions that may trigger several pathologies or exacerbate the outcomes of various pathophysiological states.

Keywords: 17β-estradiol; Cyp2e1; estrous cycle; mice; progesterone.

Fig. 1.

Differentiation in Cyp2e1, Hnf-1α, β-catenin, and Srebp-1c expression patterns within the different phases of the estrous cycle. A: fluctuations in Cyp2e1 expression within the distinct phases of the estrous cycle: proestrus (PE), estrus (E), methestrus (ME), diestrus (DE). All comparisons took place with basal Cyp2e1 mRNA levels at E. Values are expressed as means ± SE (n = 10). B–D: fluctuations in Hnf-1α, β-catenin, and Srebp-1c expression within the distinct phases of the estrous cycle and correlation of these changes with those in relative Cyp2e1 mRNA levels. CYP2E1-dependent p-nitrophenol hydroxylase (PNP) activity. *P < 0.05, **P < 0.01, ***P < 0.001. CYP2E1 Western blot images contain 1 sample per treatment. Each lane in the capture represents Cyp2e1 protein amount in the microsomes of 1 mouse.

Fig. 2.

Sex differentiation in hepatic Cyp2e1 expression pattern. A: Cyp2e1 mRNA, protein and activity level in cyclic females at E in ovariectomized (OV) and male mice. Comparisons took place with basal Cyp2e1 mRNA levels at E. CYP2E1-dependent PNP activity. B: effect of hormonal replacement with 17β-estradiol (E₂) and/or progesterone on Cyp2e1 expression. Comparisons took place with basal Cyp2e1 mRNA levels in OV mice. Values are expressed as means ± SE (n = 10). CYP2E1 Western blot images contain 1 sample per treatment. Each lane in the capture represents CYP2E1 protein amount in the microsomes of 1 mouse. C: Correlation of changes in relative Cyp2e1 mRNA levels with plasma progesterone and E₂ levels. **P < 0.01, ***P < 0.001.

Fig. 3.

Assessment of impact of hormonal replacement on Cyp2e1 expression. Effect of hormonal replacement with E₂ and/or progesterone on Hnf-1α (A), β-catenin (B), and Srebp-1c (C) expression and correlation of changes in relative Hnf-1α, β-catenin, and Srebp-1c mRNA levels with those in relative Cyp2e1 mRNA levels. All comparisons took place with basal mRNA levels in OV mice. Values are expressed as means ± SE (n = 10). **P < 0.01, ***P < 0.001.

Fig. 4.

Effect of hormonal replacement with E₂ and/or progesterone on Pgc-1α mRNA expression. Comparison took place with basal Pgc-1α mRNA levels in OV mice. ***P < 0.001.

Fig. 5.

Effect of hormonal replacement with E₂ and/or progesterone on CYP2E1 expression in humanized CYP2E1 mice. CYP2E1 mRNA and activity levels are expressed as means ± SE (n = 10). Comparisons took place with basal CYP2E1 mRNA or PNP activity levels in OV mice, respectively. **P < 0.01. CYP2E1 Western blot images contain 1 sample per treatment. Each lane in the capture represents CYP2E1 protein amount in the microsomes of 1 transgenic mouse.

Fig. 6.

Assessment of impact of female steroid hormones on PI3K/Akt/FOXO1 signaling pathway. Western blot analysis of Akt and FOXO1 phosphorylation (p) in hepatic nuclear, cytosolic, and total (t) cellular proteins of male mice, cyclic females at E, and OV mice that received placebo or hormonal replacement using pellets containing estradiol and/or progesterone. Western blot images contain 1 sample per treatment, which is representative of 3 samples per group tested. Each lane in the capture represents total or phosphorylated Akt or FOXO1 in total cellular, nuclear, or cytosolic proteins of 1 mouse, respectively.

Fig. 7.

Effect of hormonal replacement with E₂ and/or progesterone on eNos (A) and iNos (B) mRNA expression. Values are expressed as means ± SE (n = 10). All comparisons took place with basal mRNA levels in OV mice. *P < 0.05, **P < 0.01, ***P < 0.001.

Fig. 8.

Assessment of impact of female steroid hormones and tamoxifen on GH/STAT5b signaling pathway. Western blot analysis of STAT5b phosphorylation in hepatic nuclear and cytosolic proteins of male mice, cyclic females at E, treated with normal saline or tamoxifen, and OV mice that received placebo or hormonal replacement using pellets containing estradiol and/or progesterone. Western blot images contain 1 sample per treatment, which is representative of 3 samples per group tested. Each lane represents total or phosphorylated STAT5b in nuclear or cytosolic proteins of 1 mouse.

Fig. 9.

Assessment of effect of tamoxifen on Cyp2e1 expression and PI3K/Akt/FOXO1 activation. A: effect of tamoxifen on Cyp2e1, Hnf-1α, β-catenin, and Pgc-1α mRNA expression. Values are expressed as means ± SE (n = 10). **P < 0.01, ***P < 0.001. B: assessment of tamoxifen-induced effect on Akt, FOXO1, and JNK activation by Western blot. Western blot images contain 1 sample per treatment, which is representative of 3 samples per group tested. Each lane in the capture represents total or phosphorylated Akt, FOXO1, or JNK in total cellular proteins of 1 mouse.

Fig. 10.

Regulation of CYP2E1 in the liver. Hormonal, cytokine-, and adrenoceptor (AR)-linked pathways involved in CYP2E1 regulation; β- and α-cells are located in pancreas. Stimulation of D₂-receptors and α₂-ARs, which are expressed on pancreatic β-cell membranes, restricts glucose-stimulated insulin release. The opposite is true following β₂-AR stimulation (18, 53). Dashed lines depict inhibition.