Instituto de Salud y Ambiente del Litoral (ISAL, UNL-CONICET), Facultad de Bioquímica y Ciencias Biológicas, Universidad Nacional del Litoral, Santa Fe, Argentina; Cátedra de Patología Humana, Facultad de Bioquímica y Ciencias Biológicas, Universidad Nacional del Litoral, Santa Fe, Argentina.
Instituto de Salud y Ambiente del Litoral (ISAL, UNL-CONICET), Facultad de Bioquímica y Ciencias Biológicas, Universidad Nacional del Litoral, Santa Fe, Argentina.
Environ Res. 2020 Dec;191:110185. doi: 10.1016/j.envres.2020.110185. Epub 2020 Sep 16.
The plastic monomer and plasticizer bisphenol A (BPA), and the UV-filter benzophenone-3 (BP3) have been shown to have estrogenic activities that could alter mammary gland development. Our aim was to analyze whether BPA or BP3 direct exposure affects the functional differentiation of the mammary gland using an in vitro model. Mammary organoids were obtained and isolated from 8 week-old virgin female C57BL/6 mice and were differentiated on Matrigel with medium containing lactogenic hormones and exposed to: a) vehicle (0.01% ethanol); b) 1 × 10 M or 1 × 10 M BPA; or c) 1 × 10 M, 1 × 10 M or 1 × 10 M BP3 for 72 h. The mRNA and protein expression of estrogen receptor alpha (ESR1) and progesterone receptor (PR) were assessed. In addition, mRNA levels of PR-B isoform, glucocorticoid receptor (GR), prolactin receptor (PRLR) and Stat5a, and protein expression of pStat5a/b were evaluated at 72 h. The mRNA and protein expression of milk proteins and their DNA methylation status were also analyzed. Although mRNA level of PRLR and GR was similar between treatments, mRNA expression of ESR1, total PR, PR-B and Stat5a was increased in organoids exposed to 1 × 10 M BPA and 1 × 10 M BP3. Total PR expression was also increased with 1 × 10 M BPA. Nuclear ESR1 and PR expression was observed in all treated organoids; whereas nuclear pStat5a/b alveolar cells was observed only in organoids exposed to 1 × 10 M BPA and 1 × 10 M BP3. The beta-casein mRNA level was increased in both BPA concentrations and 1 × 10 M BP3, which was associated with hypomethylation of its promoter. The beta-casein protein expression was only increased with 1 × 10 M BPA or 1 × 10 M BP3. In contrast, BPA exposure decreased alpha-lactalbumin mRNA expression and increased DNA methylation level in different methylation-sensitive sites of the gene. Also, 1 × 10 M BPA decreased alpha-lactalbumin protein expression. Our results demonstrate that BPA or BP3 exposure alters milk protein synthesis and its transcriptional regulation during mammary gland differentiation in vitro.
塑料单体和增塑剂双酚 A（BPA）以及紫外线滤光剂二苯甲酮-3（BP3）已被证明具有雌激素活性，可改变乳腺发育。我们的目的是使用体外模型分析 BPA 或 BP3 的直接暴露是否会影响乳腺的功能分化。从 8 周龄处女 C57BL/6 小鼠中获得和分离乳腺器官，并在含有泌乳激素的 Matrigel 上分化，并暴露于：a）载体（0.01%乙醇）；b）1×10^-7^ M 或 1×10^-6^ M BPA；或 c）1×10^-7^ M、1×10^-6^ M 或 1×10^-6^ M BP3 72 小时。评估雌激素受体 alpha（ESR1）和孕激素受体（PR）的 mRNA 和蛋白表达。此外，在 72 小时时评估 PR-B 同工型、糖皮质激素受体（GR）、催乳素受体（PRLR）和 Stat5a 的 mRNA 水平以及磷酸化 Stat5a/b 的蛋白表达。还分析了乳蛋白的 mRNA 和蛋白表达及其 DNA 甲基化状态。尽管处理之间的 PRLR 和 GR mRNA 水平相似，但在暴露于 1×10^-7^ M BPA 和 1×10^-6^ M BP3 的器官中，ESR1、总 PR、PR-B 和 Stat5a 的 mRNA 表达增加。总 PR 表达也随着 1×10^-7^ M BPA 而增加。所有处理的器官均观察到核 ESR1 和 PR 表达；而仅在暴露于 1×10^-7^ M BPA 和 1×10^-6^ M BP3 的器官中观察到核磷酸化 Stat5a/b 肺泡细胞。两种 BPA 浓度和 1×10^-6^ M BP3 均增加了β-酪蛋白的 mRNA 水平，这与其启动子的低甲基化有关。仅在暴露于 1×10^-7^ M BPA 或 1×10^-6^ M BP3 时，β-酪蛋白蛋白表达增加。相比之下，BPA 暴露降低了α-乳白蛋白的 mRNA 表达并增加了基因中不同甲基化敏感位点的 DNA 甲基化水平。此外，1×10^-7^ M BPA 降低了α-乳白蛋白的蛋白表达。我们的结果表明，BPA 或 BP3 暴露会改变体外乳腺分化过程中乳蛋白的合成及其转录调控。
