Neonatal Persistent Exposure to 6‐Propyl‐2‐thiouracil, a Thyroid‐Disrupting Chemical, Differentially Modulates Expression of Hepatic Catalase and C/EBP‐β in Adult Rats

Abstract

Persistent exposure of rats to 6-propyl-2-thiouracil (PTU) from birth resulted in decreases in plasma thyroid hormone (TH) levels and hepatic expression of catalase and CCAAT enhancer binding protein β (C/EBP-β). Catalase promoter region (-185 to +52) that contains binding sites for C/EBP-β showed an augmentation in the methylation level along with a change in methylation pattern of CpG islands in response to PTU treatment. PTU withdrawal on 30 days of birth restored TH levels and C/EBP-β to control rats in adulthood. Although catalase expression was restored to some extent in adult rats in response to PTU withdrawal, a permanent change in its promoter CpG methylation pattern was recorded. The results suggest that downregulation of adult hepatic catalase gene in response to persistent neonatal PTU exposure may not solely be attributed to thyroid-disrupting properties of PTU. It is possible that besides thyroid-disrupting behavior, PTU may impair expression of hepatic catalase by altering methylation pattern of its promoter.

Neonatal Exposure to 6-n-Propyl-Thiouracil, an Anti-Thyroid Drug, Alters Expression of Hepatic DNA Methyltransferases, Methyl CpG-Binding Proteins, Gadd45a, p53, and PCNA in Adult Male Rats

“…Western blotting of liver homogenates (20–25 μg) was performed by incubating the PVDF membrane with rabbit polyclon al anti-G3PDH (1: 1,000), anti-DNMT1 (1: 500), anti-DNMT3a (1: 500), anti-DNMT3b (1: 500), anti-MBD4 (1: 500), anti-MeCP2 (1: 500), and Gadd45a (1: 500) followed by HRP-conjugated anti-rabbit goat IgG (G3PDH – 1: 7,500, and DNMT1, DNMT3a, DNMT3b, MBD4, MeCP2, and Gadd45α – 1: 5,000) and expressed as previously described [4]. …”

“…Chemicals, biochemicals, molecular biology reagents, kits used in the present study, animal maintenance, care, Western blotting, and qPCR have been mentioned earlier in detail [4]. Rabbit polyclonal anti-glyceraldehyde-3 phosphate dehydrogenase (G3PDH), DNMT1, DNMT3a, DNMT3b, MBD4, MeCP2, Gadd45a, p53, PCNA, biotin goat anti-rabbit IgG, and horseradish peroxidase (HRP)-conjugated anti-rabbit goat IgG were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA), whereas streptavidin-HRP conjugate was purchased from Genei, India.…”

“…Tissue preparation, total RNA isolation, cDNA synthesis, and real-time PCR analysis with gene-specific primer using SYBR Green PCR master mix have been described earlier [4]. The primer sequences are presented in Table 1.…”

“…Liver sections were incubated with rabbit polyclonal primary antibodies against DNMT1 (1: 30), DNMT3a (1: 25), DNMT3b (1: 25), MBD4 (1: 25), MeCP2 (1: 25), Gadd45α (1: 25), PCNA (1: 25), and p53 (1: 50) followed by washing, incubating with biotinylated goat anti-rabbit IgG (1: 200) and streptavidin-HRP conjugate (1: 200), and visualized by incubating with 0.5% (w/v) 3,3-diaminobenzidine (DAB) followed by methyl green counter staining [4]. …”

“…It has been reported that the interruption of actions of thyroid hormones by anti-thyroid drugs such as methimazole or PTU during the foetal and neonatal period of vertebrates results in impairment of hepatic gene expression in adulthood [3, 4]. …”