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. 2012 Jul;33(4):563-576.
doi: 10.1016/j.reprotox.2012.02.004. Epub 2012 Mar 5.

Perfluorooctanoic acid effects on ovaries mediate its inhibition of peripubertal mammary gland development in Balb/c and C57Bl/6 mice

Yong Zhao  1 , Ying S Tan  2 , Mark J Strynar  3 , Gloria Perez  4 , Sandra Z Haslam  5 , Chengfeng Yang  6
Affiliations

Perfluorooctanoic acid effects on ovaries mediate its inhibition of peripubertal mammary gland development in Balb/c and C57Bl/6 mice

Yong Zhao et al. Reprod Toxicol. 2012 Jul.

Abstract

Exposure to perfluorooctanoic acid (PFOA), a synthetic perfluorinated compound and an agonist of peroxisome proliferator-activated receptor α (PPARα), causes stunted mouse mammary gland development in various developmental stages. However, the underlying mechanisms remain poorly understood. We found that peripubertal PFOA exposure significantly inhibited mammary gland growth in both Balb/c and C57Bl/6 wild type mice, but not in C57Bl/6 PPARα knockout mice, and Balb/c mice were more sensitive to PFOA inhibition. PFOA caused (1) delayed or absence of vaginal opening and lack of estrous cycling during the experimental period; (2) decreases in ovarian steroid hormonal synthetic enzyme levels; and (3) reduced expression of estrogen- or progesterone-induced mammary growth factors. Supplementation with exogenous estrogen and/or progesterone reversed the PFOA inhibitory effect on mammary gland. These results indicate that PFOA effects on ovaries mediate its inhibition of mammary gland development in Balb/c and C57Bl/6 mice and that PPARα expression is a contributing factor.

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Conflict of interest statement

Conflict of interest: the authors declare that there are no conflicts of interest.

Figures

Fig. 1
Fig. 1. Effect of peripubertal PFOA exposure on body weight in Balb/c, C57Bl/6 wild type and C57Bl/6 PPARα knockout mice
Three-week old female Balb/c (A), C57Bl/6 wild type (B) or PPARα knockout (C) mice were treated with vehicle control (deionized H2O) or PFOA (2.5mg/kg for Balb/c mice, 7.5mg/kg for C57Bl/6 mice) for 4 weeks. Body weight was monitored daily during PFOA dosing period. Mouse body weight is presented as mean ± standard deviation (n=5). * p<0.05 compared to vehicle control-treated mice.
Fig. 2
Fig. 2. Peripubertal PFOA exposure inhibits mammary gland development in Balb/c and C57Bl/6 wild type mice but not in C57Bl/6 PPARα knockout mice
Three-week old female Balb/c, C57Bl/6 wild type or PPARα knockout mice were treated with vehicle control (deionized H2O) or PFOA (2.5mg/kg for Balb/c mice, 7.5mg/kg for C57Bl/6 mice) for 4 weeks. Mice were sacrificed 24 h after the last treatment and mammary gland whole mounts were prepared as described in Materials and Methods. Representative photomicrographs of mammary gland whole mounts from vehicle control- and PFOA -treated mice. Note the reduced ductal length (dotted lines) and reduced number of TEB (arrow heads) in PFOA-treated wild type Balb/c and C57BL/6 mammary glands
Fig. 3
Fig. 3. Plasma PFOA levels in vehicle control- and PFOA-treated mice
Three-week old female Balb/c, C57Bl/6 wild type and PPARα knockout mice were treated with vehicle control or indicated doses of PFOA for 4 weeks and sacrificed 24 h after the last treatment. Mouse plasma samples were collected for analyzing PFOA levels as described in Materials and Methods. Plasma samples of Balb/c mice treated with 1 or 5 mg/kg of PFOA; of C57Bl/6 wild type mice treated with 1, 5, or 10 mg/kg of PFOA; and of C57Bl/6 PPARα−/− mice treated with 5 mg/kg of PFOA were from previous studies (refs 12,13). All mice were treated the same way as those in current study and plasma PFOA levels were determined in the same laboratory under the same conditions. Mouse plasma PFOA levels are presented as mean ± standard deviation (n=5). PFOA levels in vehicle control-treated mice are lower than 0.01 μg/ml. a p<0.05, compared with vehicle control group; b p<0.05, compared with 1 mg/kg of PFOA-treated Balb/c or C57Bl/6 mice; c p<0.05, compared with 2.5 mg/kg of PFOA-treated Balb/c mice; d p<0.05, compared with 5 mg/kg of PFOA-treated Balb/c mice; e p<0.05, compared with 5 mg/kg of PFOA-treated C57Bl/6 wild type mice; f p<0.05, compared with 5 mg/kg of PFOA-treated C57Bl/6 PPARα knockout (PPARα−/−) mice; g p<0.05, compared with 7.5 or 10 mg/kg of PFOA-treated C57Bl/6 wild type mice; h p<0.05, compared with 7.5 mg/kg of PFOA-treated C57Bl/6 PPARα knockout (PPARα−/−) mice.
Fig. 4
Fig. 4. Relative expression levels of kidney organic anionic transporters (OATs) in wild type Balb/c, C57Bl/6 and C57BL/6 PPARα knockout mice
Balb/c and C57Bl/6 mice were sacrificed 24 h after the last vehicle control or PFOA treatment. RNA samples were prepared from frozen kidneys and used for quantitative RT-PCR analysis of OAT expression levels as described in Materials and Methods. The OAT levels were expressed relative to Balb/c mice (mean ± standard deviation, n=3). * p<0.05 compared to Balb/c mice; # p<0.05 compared to Balb/c and C57Bl/6 wild type mice.
Fig. 5
Fig. 5. PFOA treatment decreases ovarian protein levels of steroid hormone synthetic enzymes in Balb/c and C57Bl/6 wild type mice but not in C57Bl/6 PPARα knockout mice
A. Effect of PFOA treatment on ovarian protein levels of steroid hormone synthetic enzymes. Balb/c, C57Bl/6 wild type and PPARα knockout mice were sacrificed 24 h after the last vehicle control or PFOA treatment. Protein samples prepared from frozen ovaries were used for Western blot analysis as described in Materials and Methods. Representative Western blots of protein levels of StAR, CYP11A1, HSD3β1, HSD17β1, aromatase and PPARα in vehicle control- or PFOA-treated ovaries. GAPDH served as a protein loading control. B. Western blot analysis of PPARα protein levels in mouse ovaries. Representative Western blots of protein levels of PPARα in vehicle control- or PFOA-treated ovaries. GAPDH served as a protein loading control. C. Quantification of Western blot analysis for the effect of PFOA treatment on ovarian protein levels of steroid hormone synthetic enzymes in Balb/c and C57Bl/6 wild type and PPARα knockout mice (mean ± standard deviation, n=3). * p< 0.05, compared with Vehicle Control-treated group in each mouse strain.
Fig. 6
Fig. 6. PFOA treatment reduces mammary gland protein levels of Areg, HGF, ERα, EGFR and PCNA in Balb/c and C57Bl/6 wild type mice but not in C57Bl/6 PPARα knockout mice
A. Effect of PFOA treatment on mammary gland protein levels of Areg, HGF, ERα, EGFR and PCNA. Balb/c, C57Bl/6 wild type and PPARα knockout mice were sacrificed 24 h after the last vehicle control or PFOA treatment. Protein samples were prepared from frozen mammary glands and used for Western blot analysis as described in Materials and Methods. Representative Western blots of protein levels of Areg, HGF, ERα, EGFR, PCNA and PPARα in vehicle control- or PFOA-treated mammary glands. GAPDH served as a protein loading control. B. Quantification of Western blot analysis for the effect of PFOA treatment on mammary gland protein levels of Areg, HGF, ERα, EGFR and PCNA in Balb/c and C57Bl/6 wild type and PPARα knockout mice (mean ± standard deviation, n=3). * p< 0.05, compared with Vehicle Control-treated group in each mouse strain.
Fig. 7
Fig. 7. Effect of PFOA treatment on the expression of Areg and ERα in Balb/c and C57Bl/6 wild type mammary gland luminal epithelial cells (LECs)
Mice were sacrificed 24 h after the last vehicle control or PFOA treatment and mammary glands were processed for dual immunofluorescent staining of Areg and ERα as described in Materials and Methods. A. and B. Representative images of immunoflourescence staining of Areg (green) and ERα (red) in mammary gland sections from vehicle control- or PFOA-treated Balb/c (A) or C57Bl/6 wild type (B) mice. Nuclei were counterstained with DAPI (blue). C. Quantitation of Areg and/or ERα positive mammary LECs in vehicle control- or PFOA-treated mice. Data (mean ± standard deviation, n=3) are presented as percent of total LECs. Blue bar = ERα positive cells; red bar = Areg positive cells; and yellow bar = Areg and ERα double positive cells. a, b. c p<0.05, compared with vehicle control-treated mice.
Fig. 8
Fig. 8. Supplement of exogenous estradiol (E), progesterone (P) or E+P overcomes PFOA mammary gland inhibition in Balb/c and C57Bl/6 mice
Three-week old female Balb/c or C57Bl/6 wild type mice were treated with vehicle control or PFOA for 1 week, then the mice were treated with vehicle control plus hormone C (vehicle) (A) or PFOA plus C (vehicle), E, P or E+P (B) for another week as described in Materials and Methods. Mice were sacrificed 24 h after the last PFOA/hormones treatment and mammary gland whole mounts were prepared as described in Materials and Methods. Representative photomicrographs of mammary gland whole mounts from vehicle control-, PFOA-, or PFOA plus hormone-treated mice. Note the reduced ductal length (dotted lines) and reduced number of TEB (arrow heads) in PFOA-treated wild type Balb/c and C57BL/6 mammary glands (B 7dC) and the increased ductal length and numbers of TEBs in 7dE, 7dP and 7dE+P supplemented PFOA-treated glands.
Fig. 9
Fig. 9. Effect of supplement of exogenous hormones on mammary gland protein levels of Areg, HGF, ERα, EGFR and PCNA in vehicle control- and PFOA-treated mice
A. Effect of supplement of exogenous hormones on mammary gland protein levels of Areg, HGF, ERα, EGFR and PCNA. Balb/c and C57Bl/6 Wild type mice were sacrificed 24 h after the last PFOA+hormone treatments. Representative Western Blots from protein samples prepared from frozen mammary glands as described in Materials and Methods. GAPDH served as a protein loading control. B. Quantification of Western blot analysis for the effect of supplement of exogenous hormones on mammary gland protein levels of Areg, HGF, ERα, EGFR and PCNA in vehicle control- and PFOA-treated mice (mean ± standard deviation, n=3). * p<0.05 compared with vehicle control + 7dC in each mouse strain; # p<0.05 compared with PFOA + 7dC in each mouse strain.
Fig. 10
Fig. 10. Effect of supplement of exogenous estradiol (E) on the expression of Areg and ERα in PFOA-treated Balb/c and C57Bl/6 wild type LECs
Mice were sacrificed 24 h after the last PFOA/hormones treatment and mammary glands were processed for dual immunofluorescent staining of Areg and ERα as described in Materials and Methods. Representative images of immunoflourescence staining of Areg (green) and ERα (red) in mammary gland sections from Balb/c (A) or C57Bl/6 wild type (B) mice. Nuclei were counterstained with DAPI (blue).
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