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. 2023 Sep 20;24(18):14341.
doi: 10.3390/ijms241814341.

Effects of Dibutylphthalate and Steroid Hormone Mixture on Human Prostate Cells

Aldo Mileo  1 , Teresa Chianese  1 , Gianluca Fasciolo  1 , Paola Venditti  1 , Anna Capaldo  1 , Luigi Rosati  1   2 , Maria De Falco  1   3
Affiliations

Effects of Dibutylphthalate and Steroid Hormone Mixture on Human Prostate Cells

Aldo Mileo et al. Int J Mol Sci. .

Abstract

Phthalates are a family of aromatic chemical compounds mainly used as plasticizers. Among phthalates, di-n-butyl phthalate (DBP) is a low-molecular-weight phthalate used as a component of many cosmetic products, such as nail polish, and other perfumed personal care products. DBP has toxic effects on reproductive health, inducing testicular damage and developmental malformations. Inside the male reproductive system, the prostate gland reacts to both male and female sex steroids. For this reason, it represents an important target of endocrine-disrupting chemicals (EDCs), compounds that are able to affect the estrogen and androgen signaling pathways, thus interfering with prostate homeostasis and inducing several prostate pathologies. The aim of this project was to investigate the effects of DBP, alone and in combination with testosterone (T), 17β-estradiol (E2), and both, on the normal PNT1A human prostate cell-derived cell line, to mimic environmental contamination. We showed that DBP and all of the tested mixtures increase cell viability through activation of both estrogen receptor α (ERα) and androgen receptor (AR). DBP modulated steroid receptor levels in a nonmonotonic way, and differently to endogenous hormones. In addition, DBP translocated ERα to the nucleus over different durations and for a more prolonged time than E2, altering the normal responsiveness of prostate cells. However, DBP alone seemed not to influence AR localization, but AR was continuously and persistently activated when DBP was used in combination. Our results show that DBP alone, and in mixture, alters redox homeostasis in prostate cells, leading to a greater increase in cell oxidative susceptibility. In addition, we also demonstrate that DBP increases the migratory potential of PNT1A cells. In conclusion, our findings demonstrate that DBP, alone and in mixtures with endogenous steroid hormones, acts as an EDC, resulting in an altered prostate cell physiology and making these cells more prone to cancer transformation.

Keywords: androgens; di-n-butyl phthalate (DBP); endocrine-disrupting chemicals (EDCs); estrogens; phthalates; prostate gland; steroid receptors.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
MTT assay after 24 h of treatment with testosterone (T) (A), 17β-estradiol (E2) (B), and dibutylphthalate (DBP) (C). Cell proliferation was induced by E2, T, and DBP at 10−6, 10−8, and 10−9 M, respectively. When used in combination, all of the mixtures stimulated cell proliferation, with DBP + T showing the greatest effect. DBP + E2 + T showed a less proliferative effect than DBP (D). When ICI182,780 10−5 M was used, the effects were reverted (E). Flutamide 10−7 M decreased cell viability in all of the experimental classes, except for E2 and, to a lesser extent, DBP + E2 (F) (Dunnet’s test: * p < 0.05, *** p < 0.001, **** p < 0.0001).
Figure 2
Figure 2
Western blot analysis: quantification of ERα and AR after 30 min, 2 h, and 4 h of exposure to T, E2, and DBP, alone and in mixture. Details provided in the text (A). The graphs represent the optical density (OD) ratio of ERα, and AR is normalized to the OD of GAPDH (B) (Dunnett test: *** p < 0.001).
Figure 3
Figure 3
Localization of ERα after 30 min, 2 h, and 4 h of exposure to DBP 10−9 M. DBP showed an estrogen-like behavior, inducing ERα cytoplasm–nucleus translocation after 2 h and 4 h. Alexa Fluor 488 (green) and nuclear staining (Höechst-blue) were analyzed by immunofluorescence. Scale bar: 10 µm.
Figure 4
Figure 4
Localization of ERα after 30 min, 2 h, and 4 h of exposure to E2 10−6 M. E2 induced cytoplasm–nuclear ERα translocation after 30 min and 2 h. Alexa Fluor 488 (green) and nuclear staining (Höechst-blue) were analyzed by immunofluorescence. Scale bar: 10 µm.
Figure 5
Figure 5
Localization of ERα after 30 min, 2 h, and 4 h of exposure to DBP + E2. The mixture induced cytoplasm–nuclear ERα translocation in the same way as E2. Alexa Fluor 488 (green) and nuclear staining (Höechst-blue) were analyzed by immunofluorescence. Scale bar: 10 μm.
Figure 6
Figure 6
Localization of ERα after 30 min, 2 h, and 4 h of exposure to DBP + T. The mixture did not translocate ERα to the nucleus in any of the treatments. Alexa Fluor 488 (green) and nuclear staining (Höechst-blue) were analyzed by immunofluorescence. Scale bar: 10 μm.
Figure 7
Figure 7
Localization of ERα after 30 min, 2 h, and 4 h of exposure to T + E2. The mixture induced cytoplasm–nuclear ERα translocation after 2 h and 4 h. Alexa Fluor 488 (green) and nuclear staining (Höechst-blue) were analyzed by immunofluorescence. Scale bar: 10 μm.
Figure 8
Figure 8
Localization of ERα after 30 min, 2 h, and 4 h of exposure to DBP + T + E2. The mixture induced cytoplasm–nuclear ERα translocation after 2 h and 4 h. Alexa Fluor 488 (green) and nuclear staining (Höechst-blue) were analyzed by immunofluorescence. Scale bar: 10 μm.
Figure 9
Figure 9
Localization of AR after 30 min, 2 h, and 4 h of exposure to DBP 10−9 M. DBP did not translocate AR to the nucleus in any of the treatments. Alexa Fluor 594 (red) and nuclear staining (Höechst-blue) were analyzed by immunofluorescence. Scale bar: 10 μm.
Figure 10
Figure 10
Localization of AR after 30 min, 2 h, and 4 h of exposure to T 10−8 M. T translocated AR to the nucleus after 30 min and 4 h of the treatments, exhibiting a cyclic behavior inside the cell. Alexa Fluor 594 (red) and nuclear staining (Höechst-blue) were analyzed by immunofluorescence. Scale bar: 10 μm.
Figure 11
Figure 11
Localization of AR after 30 min, 2 h, and 4 h of exposure to DBP + T. The mixture translocated AR to the nucleus in the same way as T. Alexa Fluor 594 (red) and nuclear staining (Höechst-blue) were analyzed by immunofluorescence. Scale bar: 10 μm.
Figure 12
Figure 12
Localization of AR after 30 min, 2 h, and 4 h of exposure to DBP + E2. AR translocation did not occur, but AR is in the perinuclear area in all experimental times. Alexa Fluor 594 (red) and nuclear staining (Höechst-blue) were analyzed by immunofluorescence. Scale bar: 10 μm.
Figure 13
Figure 13
Localization of AR after 30 min, 2 h, and 4 h of exposure to T + E2. The mixture induced AR translocation in all time treatments. Alexa Fluor 594 (red) and nuclear staining (Höechst-blue) were analyzed by immunofluorescence. Scale bar: 10 μm.
Figure 14
Figure 14
Localization of AR after 30 min, 2 h, and 4 h of exposure to DBP + T + E2. The mixture induced AR translocation in all time treatments. Alexa Fluor 594 (red) and nuclear staining (Höechst-blue) were analyzed by immunofluorescence. Scale bar: 10 μm.
Figure 15
Figure 15
Effect of treatments on PNT1A cells redox homeostasis. Lipid hydroperoxides (A), susceptibility to oxidative stress (B), total ROS (C), and cell soluble antioxidant capacity (D) (Dunnett test: * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001).
Figure 16
Figure 16
Wound healing assay performed after 24 h of exposure to T, E2, and DBP alone and in mixture. There was an increase in cell migration in all experimental classes except for the mixture T + E2 (A). After ICI 182,780 treatment, DBP and E2 alone did not affect cell migration (B). After flutamide treatment, except for the mixture DBP + T, all the experimental classes were able to increase cell migration, with a peak after E2 and T + E2 treatments (C). The assay was performed using July stage, which allowed us to follow the scratch in time lapse. Average rate of healing was obtained as the ratio between space and time. Details are in the text (Dunnett test, * p < 0.05; **** p < 0.0001).
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