Calbindin-D9k as a sensitive molecular biomarker for evaluating the synergistic impact of estrogenic chemicals on GH3 rat pituitary cells

VO, THUY T.B.,AN, BEUM-SOO,YANG, HYUN,JUNG, EUI-MAN,HWANG, INHO,JEUNG, EUI-BAE Spandidos Publications 2012 INTERNATIONAL JOURNAL OF MOLECULAR MEDICINE Vol.30 No.5

<P>Various endocrine-disrupting chemicals (EDCs) such as bisphenol A (BPA), alkylphenols [4-nonylphenol (NP) and 4-tert octylphenol (OP)] and isobutylparaben (IBP) are a constant concern due to their widespread distribution. It has been reported that some combinations of hormone-disrupting chemicals are much more powerful than any of the chemicals alone. In this study, we measured the expression of an estrogenic biomarker gene, calbindin-D9k (CaBP-9k), and progesterone receptor (PR) to evaluate the individual or combined estrogenic activity of BPA, NP, OP and IBP in GH3 rat pituitary cells. Most doses of the individual compounds and all the doses of the combined chemicals significantly increased CaBP-9k and PR mRNA and protein expression compared to the vehicle (except for PR expression after treatment with OP and NP at 10-7 M). Of note, high doses (10-6 and 10-5 M) of the EDC combinations increased the translational and transcriptional levels of CaBP-9k by 1.3- to 2.4-fold compared to each individual equivalent concentrations of EDCs. To determine whether the increased CaBP-9k gene expression was induced via intracellular estrogen receptor (ER), we blocked ER signaling using fulvestrant, an ER antagonist. The results showed that fulvestrant significantly reversed the CaBP-9k and PR upregulation following treatment with individual EDCs or their combinations. Taken together, we conclude that combinations of BPA, NP, OP and IBP in GH3 rat pituitary cells have synergistic estrogenic activities mediated by ER signaling. In addition, the expression of the CaBP-9k gene may be used as a biomarker to assess the synergistic effects of EDCs in vitro.</P>

An evaluation of estrogenic activity of parabens using uterine calbindin-d9k gene in an immature rat model.

Vo, Thuy T B,Jeung, Eui-Bae Academic Press 2009 TOXICOLOGICAL SCIENCES Vol.112 No.1

<P>In the present study, calbindin-D9k (CaBP-9k), a potent biomarker for screening estrogen-like environmental chemicals in vivo and in vitro, was adopted to examine the potential estrogen-like property of the following parabens: propyl-, isopropyl-, butyl-, and isobutylparaben. Immature female rats were administered for 3 days from postnatal day 14 to 16 with 17alpha-ethinylestradiol (EE, 1 mg/kg body weight [BW]/day) or parabens (62.5, 250, and 1000 mg/kg BW/day). In uterotrophic assays, significantly increased uterus weights were detected in the EE-treated group and in the groups treated with the highest dose of isopropyl-, butyl-, and isobutylparaben. In addition, these parabens induced uterine CaBP-9k messenger RNA (mRNA) and protein levels, whereas cotreatment of parabens and fulvestrant, a pure estrogen receptor (ER) antagonist, completely reversed the paraben-induced gene expression and increased uterine weights. To investigate the ER-mediated mechanism(s) by which parabens exert their effects, the expression level of ER-alpha and progesterone receptor (PR) was analyzed. Exposure to EE or parabens caused a dramatic decrease in expression of both ER-alpha mRNA and protein levels, whereas cotreatment with fulvestrant reversed these effects. These data showed the difference of CaBP-9k and ER-alpha expression, suggesting that CaBP-9k may not express via ER-alpha pathway. In the effect of parabens on CaBP-9k expression through PR mediation, a significantly increased expression of uterine PR gene, a well-known ER-regulating gene, at both transcriptional and translational levels was indicated in the highest dose of isopropyl- and butylparaben. These parabens-induced PR gene expression was completely blocked by fulvestrant. This result indicates that CaBP-9k expression may involve with PR mediates in the estrogenic effect of paraben in immature rat uteri. Taken together, parabens exhibited an estrogen-like property in vivo, which may be mediated by a PR and/or ER-alpha signaling pathway. In addition, our results expanded the current understanding of the potential adverse effects of parabens associated with their estrogen-like activities. Further investigation is needed to elucidate in greater detail the adverse effects of parabens in humans and wildlife.</P>

Differential Effects of Flutamide and Di-(2-ethylhexyl) phthalate on Male Reproductive Organs in a Rat Model

VO, Thuy T. B.,JUNG, Eui-Man,DANG, Vu Hoang,JUNG, Kikyung,BAEK, Jounghee,CHOI, Kyung-Chul,JEUNG, Eui-Bae Society for Reproduction and Development 2009 Journal of Reproduction and Development Vol.55 No.4

<P>Endocrine disruptors (EDs) with androgenic and anti-androgenic effects may alter reproductive function by binding to androgenic receptors (AR) and inducing or modulating AR-dependent responses in the male reproductive system. However, the molecular mechanism(s) underlying these events remains unclear. In the present study, pregnant Sprague Dawley (SD) rats were treated with testosterone propionate (TP), flutamide (Flu) and di-(2-ethylhexyl) phthalate (DEHP) from gestation days (GD) 11 to 21. Interestingly, maternal exposure to Flu or DEHP caused fluctuations in the neonatal levels of serum testosterone (T) and luteinizing hormone (LH). Serum testosterone and LH were upregulated by Flu, but these hormones were down-regulated by DEHP. The anogenital distances (AGD) of male newborns were determined at post-neonatal days (PND) 1, 21 and 63. Male rats treated prenatally with DEHP (100 mg/kg mother's body weight) or Flu showed an AGD shorter than that of control rats. At PND 63, sperm concentration, viability and motility were reduced in the maternal DEHP and Flu-treated groups. The numbers of seminiferous tubules were reduced in the Flu and DEHP-treated offspring when compared with the vehicle- and TP-treated groups, and the tubules of the testes at PND 63 were disrupted by a high dose of Flu. In addition, we found differential gene expression patterns by microarray analysis following ED exposure, particularly in sex determination-related genes. Although Flu and DEHP are considered to be identical with regard to their anti-androgenic effects, their effects on developing male reproductive organs were distinct, suggesting that Flu competes with endogenous T, while DEHP influences a different step in androgenesis.</P>

Construction of an Agroinfectious Clone of a Korean Isolate of Sweet Potato Symptomless Virus 1 and Comparison of Its Infectivity According to Agrobacterium tumefaciens Strains in Nicotiana benthamiana

Phuong T. Ho(Phuong T. Ho ),Hee-Seong Byun(Hee-Seong Byun),Thuy T. B. Vo(Thuy T. B. Vo ),Aamir Lal(Aamir Lal ),Sukchan Lee(Sukchan Lee),Eui-Joon Kil(Eui-Joon Kil) 한국식물병리학회 2023 Plant Pathology Journal Vol.39 No.3

Sweet potato symptomless virus 1 (SPSMV-1) is a single-stranded circular DNA virus, belonging to the genus Mastrevirus (family Geminiviridae) that was first identified on sweet potato plants in South Korea in 2012. Although SPSMV-1 does not induce distinct symptoms in sweet potato plants, its co-infection with different sweet potato viruses is highly prevalent, and thus threatens sweet potato production in South Ko-rea. In this study, the complete genome sequence of a Korean isolate of SPSMV-1 was obtained by Sanger sequencing of polymerase chain reaction (PCR) am-plicons from sweet potato plants collected in the field (Suwon). An infectious clone of SPSMV-1 (1.1-mer) was constructed, cloned into the plant expression vec-tor pCAMBIA1303, and agro-inoculated into Nicotiana benthamiana using three Agrobacterium tumefaciens strains (GV3101, LBA4404, and EHA105). Although no visual differences were observed between the mock and infected groups, SPSMV-1 accumulation was detected in the roots, stems, and newly produced leaves through PCR. The A. tumefaciens strain LBA4404 was the most effective at transferring the SPSMV-1 genome to N. benthamiana. We confirmed the viral replication in N. benthamiana samples through strand-specific amplifi-cation using virion-sense- and complementary-sense-specific primer sets.

Dietary Calcium and 1,25-Dihydroxyvitamin D₃ Regulate Transcription of Calcium Transporter Genes in Calbindin-D9k Knockout Mice

KO, Sang-Hwan,LEE, Geun-Shik,VO, Thuy T. B.,JUNG, Eui-Man,CHOI, Kyung-Chul,CHEUNG, Ki-Wha,KIM, Jae Wha,PARK, Jong-Gil,OH, Goo Taeg,JEUNG, Eui-Bae Society for Reproduction and Development 2009 Journal of Reproduction and Development Vol.55 No.2

<P>The effect(s) of oral calcium and vitamin D<SUB>3</SUB> were examined on the expression of duodenal and renal active calcium transport genes, i.e., calbindin-D9k (<I>CaBP-9k</I>) and calbindin-D28k (<I>CaBP-28k</I>), transient receptor potential cation channels (<I>TRPV5</I> and <I>TRPV6</I>), Na<SUP>+</SUP>/Ca<SUP>2+</SUP> exchanger 1 (<I>NCX1</I>) and plasma membrane calcium ATPase 1b (<I>PMCA1b</I>), in <I>CaBP-9k</I> KO mice. Wild-type (WT) and KO mice were provided with calcium and vitamin D<SUB>3</SUB>-deficient diets for 10 weeks. The deficient diet significantly decreased body weights compared with the normal diet groups. The serum calcium concentration of the WT mice was decreased by the deficient diet but was unchanged in the KO mice. The deficient diet significantly increased duodenal transcription of <I>CaBP-9k</I> and <I>TRPV6</I> in the WT mice, but no alteration was observed in the KO mice. In the kidney, the deficient diet significantly increased renal transcripts of <I>CaBP-9k</I>, <I>TRPV6</I>, <I>PMCA1b</I>, <I>CaBP-28k</I> and <I>TRPV5</I> in the WT mice but did not alter calcium-relating genes in the KO mice. Two potential mediators of calcium-processing genes, vitamin D receptor (VDR) and parathyroid hormone receptor (PTHR), have been suggested to be useful for elucidating these differential regulations in the calcium-related genes of the KO mice. Expression of VDR was not significantly affected by diet or the KO mutation. Renal <I>PTHR</I> mRNA levels were reduced by the diet, and reduced expression was also seen in the KO mice given the normal diet. Taken together, these results suggest that the active calcium transporting genes in KO mice may have resistance to the deficiency diet of calcium and vitamin D<SUB>3</SUB>.</P>