Less Carcinogenic Chlorinated Estrogens Applicable to Hormone Replacement Therapy

Abstract

Human estrogens prescribed for hormone replacement therapy (HRT) are known to be potent carcinogens. To find safer estrogens, several chlorinated estrogens were synthesized and their carcinogenic potential were determined. A pellet containing either 2-chloro-17β-estradiol (2-ClE₂) or 4-chloro-17β-estradiol (4-ClE₂) was implanted subcutaneously for 52 weeks into August Copenhagen Irish (ACI) rats, a preferred animal model for human breast cancer. 17β-Estradiol (E₂) frequently induced mammary tumors while both 2-ClE₂ and 4-ClE₂ did not. Their 17α-ethinyl forms, thought to be orally active estrogens, were also synthesized. Neither 2-chloro-17α-ethinylestradiol (2-ClEE₂) nor 4-chloro-17α-ethinylestradiol (4-ClEE₂) induced tumors. The less carcinogenic effects were supported by histological examination of mammary glands of ACI rats treated with the chlorinated estrogens. A chlorine atom positioned at the 2- or 4-position of E₂ may prevent the metabolic activation, resulting in reducing the carcinogenicity. 2-ClE₂ and 4-ClE₂ administered subcutaneously and 2-ClEE₂ and 4-ClEE₂ given orally to ovariectomized rats all showed uterotrophic potency, albeit slightly weaker than that of E₂. Our results indicate that less carcinogenic chlorinated estrogens retaining estrogenic potential could be safer alternatives to the carcinogenic estrogens now in use for HRT.

Keywords: DNA damage; chlorination; estrogen; hormone replacement therapy; mammary tumor; uterotrophic activity.

Figure 1

Proposed oxidative mechanism of chlorinated estrogens.

Figure 2

Structures of E₂ and EE₂ and their chlorinated compounds.

Figure 3

Cumulative incidence of mammary tumors in chlorinated estrogen-treated rats. Development of mammary tumors in ACI rats implanted with placebo (n = 5), E₂ [1.25 mg (n = 5), 2.5 mg (n = 10)], 2-ClE₂ [5.0 mg (n = 6)], or 4-ClE₂ [5.0 mg (n = 6)] pellets was monitored once a week for 52 weeks.

Figure 4

Morphological examination of mammary glands of chlorinated estrogen-treated rats. Mammary glands were collected from ACI rats at the end of the experiments. Mammary tissue of ACI rats implanted with placebo (A), E₂ (2.5 mg) (B), 2-ClE₂ (5.0 mg) (C), 4-ClE₂ (5.0 mg) (D), 2-ClEE₂ (5.0 mg) (E), or 4-ClEE₂ (5.0 mg) (F) and stained with hematoxylin (magnification 10×).

Figure 5

Uterotrophic potential of chlorinated estrogens on OVX-rats. (A) OVX-rats (4 rats/group) were treated subcutaneously for 3 days with 2-ClE₂ (3.4 or 34 μg/rat/day) or 4-ClE₂ (3.4 or 34 μg/rat/day). The rats treated with E₂ (3.0 μg/rat/day) were used as positive controls. The negative control rats received vehicle only. (B) OVX-rats (4 rats/group) were treated orally for 3 days with 2-ClEE₂ (18 or 54 μg/rat/day) or 4-ClE₂ (18 or 54 μg/rat/day). The rats treated with EE₂ (16.5 μg/rat/day) were used as positive controls. On day 4, uterine wet-weight/bw ratios were calculated and compared to that obtained for OVX-rats that received vehicle, as described in Materials and Methods. Statistical analysis (one-way ANOVA with Tukey’s post hoc test) was performed for multiple comparisons to evaluate differences; *, p < 0.05 (control vs. 2-ClEE₂ (54 μg/rat)); ****, p < 0.0001 (control vs. E₂ (3.0 μg/rat), 2-ClE₂ (34 μg/rat), 4-ClE₂ (34 μg/rat), EE₂ (16.5 μg/rat), or 4-ClE₂ (54 μg/rat)).