Arsenite and cadmium promote the development of mammary tumors

Abstract

Previous studies demonstrate that the heavy metal cadmium and the metalloid arsenite activate estrogen receptor-alpha in breast cancer cells by forming a high-affinity complex with the ligand-binding domain of the receptor and that environmentally relevant doses of cadmium have estrogen-like activity in vivo. The present study showed that in estrogen-receptor positive cells, arsenite and cadmium increased the global expression of estrogen-responsive genes and that an environmentally relevant dose of arsenite also had estrogen-like activity in vivo. Similar to estrogens, exposure of ovariectomized animals to arsenite induced the expression of the progesterone receptor, GREB1, and c-fos in the mammary gland and the expression of complement C3, c-fos, and cyclin D1 in the uterus and the increase was blocked by the antiestrogen ICI-182,780. When virgin female animals were fed a diet, that mimics exposure to either arsenite or cadmium, and challenged with the chemical carcinogen dimethylbenzanthracene, there was an increase in the incidence of mammary tumors and a decrease in the time to tumor onset, but no difference in the total number of tumors, tumor multiplicity, or total tumor volume. Together with published results, these data showed that environmentally relevant amounts of arsenite and cadmium had estrogen-like activity in vivo and promoted mammary tumorigenesis.

Figure 1.

Gene expression profile in MCF-7 cells treated with estradiol, arsenite, or cadmium. MCF-7 cells were treated for 24 h with estradiol (E2; 1 nM), arsenite (As; 1 µM), or cadmium (Cd; 1 µM) in the absence or presence of the ERα antagonist ICI-182,780 (ICI; 500 nM). (A) Two-dimensional clustering of genes responsive to treatment with estradiol, arsenite, or cadmium in absence and presence of ICI-182,780. 2,167 genes were selected that showed significant difference, i.e. more than 2-fold with (P < 0.05), in three or more treatments. (B) Venn diagram comparing the number of significantly up-regulated and down-regulated genes in cells treated with estradiol, arsenite, or cadmium. (n = 4 for treatments in the absence of ICI-182,780; n = 3 for treatments in the presence of ICI-182,780).

Figure 2.

Gene expression profiles of selected ERa-responsive genes. MCF-7 cells were treated with estradiol, arsenite, or cadmium in the absence or presence of ICI-182,780 as described in Figure 1. (A) Two-dimensional clustering of 108 ER-responsive genes. (B) Heatmap of the 17 genes with the greatest fold change.

Figure 3.

Effects of arenite on the expression of ERα regulated genes in the mammary gland. Virgin female Sprague-Dawley rats were ovariectomized at 28 days of age and allowed to recover for 2 to 3 weeks prior to treatment for 4 days with ethinyl estradiol (EE; 50 µg/kg bw/day by oral gavage) or arsenite (As; 5 µg/kg bw/day by i.p.) in the presence and absence of the antiestrogen ICI-182,780 (ICI; 500 μg/kg bw/day by s.c.). The third mammary gland was removed, RNA was extracted, and mRNA was measured by qRT-PCR. Data are presented as relative expression of progesterone receptor (PgR; A), GREB-1 (B), and c-fos (C) mRNA normalized to GAPDH mRNA (RQ). For each gene, separate comparisons and T-tests were conducted for ethinyl estradiol (EE) to control (C), arsenite (As) to C, and arsenite plus the antiestrogen ICI-182,780 (As+ICI) to As. (mean ± SD; n = 13–15 animals/group; *P = 0.05; **P = 0.005).

Figure 4.

Effects of arsenite on the expression of ERα regulated genes in the uterus. Virgin female Sprague-Dawley rats were ovariectomized at 28 days of age and allowed to recover for 2 to 3 weeks prior to treatment for 4 days with ethinyl estradiol (EE; 50 µg/kg bw/day by oral gavage) or arsenite (5 µg/kg bw/day by i.p.) in the presence and absence of the antiestrogen ICI-182,780 (ICI; 500 μg/kg bw/day by s.c.). The uterus was removed, RNA was extracted, and mRNA was measured by qRT-PCR. Data are presented as relative expression of complement C3 (C3; A), c-fos (B), and cyclin D1 (C) mRNA normalized to GAPDH mRNA (RQ). For each gene, separate comparisons and T-tests were conducted for ethinyl estradiol (EE) to control (C), arsenite (As) to C, and arsenite plus the antiestrogen ICI-182,780 (As+ICI) to As. (mean ± SD; n = 13–15 animals/group; *P = 0.05; **P = 0.005; ***P = 0.0005).

Figure 5.

Effects of arsenite and cadmium on mammary tumorigenesis. Virgin female Sprague-Dawley rats at 25 days of age were placed on the control diet or the control diet containing 0.2 mg/kg diet of arsenite or cadmium to mimic dietary exposure the metalloid and metal (approximately 10 μg/kg bw/day). At 49 days of age, the animals were challenged with DMBA (50 mg/kg bw by oral gavage). Tumor development was monitored beginning 11 weeks after treatment with the carcinogen. (A) tumor incidence in animals fed the metalloid or metal containing diet and challenged with DMBA (closed symbols; n = 20 animals/group) and in animals fed the metalloid or metal containing diet but not challenged with DMBA (open symbols; n = 10 animals/group); (B) time to tumor onset in animals fed the arsenite containing diet and treated with DMBA (n = 20 animals/group); (C) time to tumor onset in animals fed the cadmium containing diet and treated with DMBA (n = 20 animals/group); (D) total tumor volume (n = 20 animals/group); (E) total number of tumors (n = 20 animals/group); (F) tumor multiplicity (n = 20 animals/group).

Figure 6.

Effects of arsenite and cadmium on body weight. Virgin female Sprague-Dawley rats at 25 days of age were placed on the control diet or the control diet containing 0.2 mg/kg diet of arsenite or cadmium to mimic dietary exposure the metalloid and metal (approximately 10 μg/kg bw/day). At 49 days of age, the animals were either challenged or not with DMBA (50 mg/kg bw by oral gavage) as described above. Body weight was monitored beginning 11 weeks after treatment with the carcinogen. (A) Animals fed the arsenite or cadmium containing diets and challenged with DMBA (mean ± SEM; n = 19–20); (B) Animals fed the arsenite or cadmium containing diets (mean ± SEM; n = 9–10).