Rhee Da Lee

COMPARATIVE EVALUATION OF ALKYLPHENOLIC COMPOUNDS ON ESTROGENIC ACTIVITY IN VITRO AND IN VIVO

This study was undertaken to compare the sensitivity of screening test methods and to investigate the structure-activity relationships of the estrogenic activity of alkylphenolic compounds (APs) using in vitro and in vivo assays. Two in vitro systems, MCF-7 cell proliferation (E-screen assay) and competitive binding assay to estrogen receptor (ER), were selected to evaluate the estrogenic effects. Uterotrophic assay and Calbindin-D (CaBP9K 9K) mRNA expression were also examined in ovariectomized Sprague-Dawley female rats. A series of APs with various alkyl groups were examined, namely, 4-propylphenol, 4-butylphenol, 4- t -butylphenol, 4-pentylphenol, 4-nonylphenol, 4-octylphenol, 4- t -octylphenol, and 4-phenylphenol, and 17 g -estradiol (E2) was used as a positive control. In the E-screen assay, E2 was found to induce maximum proliferation of MCF-7 cells at 1 n M . Among the APs, 4- t -octylphenol and 4-nonylphenol were found to be considerably more potent than any other compound and estrogenic effects were detectable at 1 and 10 µ M , respectively. 4- t -Octylphenol and 4-nonylphenol inhibited the binding of E2 to the ER of MCF-7 cells in a competitive ER binding assay. The uterotrophic effects to APs (10, 50, 200, and 400 mg/kg/d) were compared to E2 (1 µg/kg) in ovariectomized rats after treatment for 3 d. 4-Nonylphenol, 4- t -octylphenol, and 4-phenylphenol produced dose-dependent increases in the uterine weights of ovariectomized rats. In the CaBP-9K mRNA expression test, CaBP-9K mRNA levels were detected in the uteri of ovariectomized rats treated with 4-pentylphenol (400 mg/kg), 4-nonylphenol, 4-phenylphenol (200 and 400 mg/kg), and 4- t -octylphenol (50 mg/kg and above), respectively. In the dot blot assay, CaBP-9K mRNA levels were significantly increased in rats exposed to 4- t -octylphenol (200 and 400 mg/kg), 4-pentylphenol, 4-nonylphenol, and 4-phenylphenol (400 mg/kg), respectively. Among the APs, compounds with bulky alkyl groups or higher carbon numbers possessed higher estrogenic capacity. In addition, the pattern of CaBP9K expression correlated with that of the 3-d uterotrophic assay. Therefore, our results suggest that the CaBP-9K gene might be used as a potential biomarker for the screening of endocrine disruptors.

Multigeneration Reproductive and Developmental Toxicity Study of bar Gene Inserted into Genetically Modified Potato on Rats

Each specific protein has an individual gene encoding it, and a foreign gene introduced to a plant can be used to synthesize a new protein. The identification of potential reproductive and developmental toxicity from novel proteins produced by genetically modified (GM) crops is a difficult task. A science-based risk assessment is needed in order to use GM crops as a conventional foodstuff. In this study, the specific characteristics of GM food and low-level chronic exposure were examined using a five-generation animal study. In each generation, rats were fed a solid pellet containing 5% GM potato and non-GM potato for 10 wk prior to mating in order to assess the potential reproductive and developmental toxic effects. In the multigeneration animal study, there were no GM potato-related changes in body weight, food consumption, reproductive performance, and organ weight. Polymerase chain reaction (PCR) was carried out using extracted genomic DNA to examine the possibility of gene persistence in the organ tissues after a long-term exposure to low levels of GM feed. In each generation, the gene responsible for bar was not found in any of the reproductive organs of the GM potato-treated male and female rats, and the litter-related indexes did not show any genetically modified organism (GMO)-related changes. The results suggest that genetically modified crops have no adverse effects on the multigeneration reproductive-developmental ability.Each specific protein has an individual gene encoding it, and a foreign gene introduced to a plant can be used to synthesize a new protein. The identification of potential reproductive and developmental toxicity from novel proteins produced by genetically modified (GM) crops is a difficult task. A science-based risk assessment is needed in order to use GM crops as a conventional foodstuff. In this study, the specific characteristics of GM food and low-level chronic exposure were examined using a five-generation animal study. In each generation, rats were fed a solid pellet containing 5% GM potato and non-GM potato for 10 wk prior to mating in order to assess the potential reproductive and developmental toxic effects. In the multigeneration animal study, there were no GM potato-related changes in body weight, food consumption, reproductive performance, and organ weight. Polymerase chain reaction (PCR) was carried out using extracted genomic DNA to examine the possibility of gene persistence in the organ tissues after a long-term exposure to low levels of GM feed. In each generation, the gene responsible for bar was not found in any of the reproductive organs of the GM potato-treated male and female rats, and the litter-related indexes did not show any genetically modified organism (GMO)-related changes. The results suggest that genetically modified crops have no adverse effects on the multigeneration reproductive–developmental ability.

No androgenic/anti-androgenic effects of bisphenol-A in Hershberger assay using immature castrated rats.

Several studies have demonstrated that bisphenol A (BPA) exhibited weak estrogenic activity in the 3-day uterotrophic assay using ovariectomized (OVX) and immature rats (Toxicol. Lett. 115 (2000) 231; Regul. Toxicol. Pharmacol. 32 (2000) 118; J. Toxicol. Sci. 26 (2001) 111) and BPA also possessed anti-androgenic activity in in vitro yeast based assays (J. Endocrinol. 158 (1998) 327). To investigate anti-androgenic effects of BPA. a rodent Hershberger assay was carried out using immature Sprague-Dawley male rats. An androgen agonist, testosterone (0.4 mg/kg per day), was administered for 7 consecutive days by subcutaneous (s.c.) injection as a positive control. Additionally, a pure androgen antagonist, flutamide (1, 5. 10 mg/kg per day. oral) was co-administered with testosterone (0.4 mg/kg per day s.c.). BPA was also administered orally with or without testosterone (0.4 mg/kg per day, s.c.) for 7 consecutive days. In the testosterone treated groups, glans penis, seminal vesicles, ventral prostate, and levator ani plus bulbocavernosus muscles (LABC) weights were significantly increased compared with control. However. flulamide dose-dependently inhibited the testosterone-induced re-growth of seminal vesicles, ventral prostate, and LABC, with a significant decrease at flutamide 1.0 mg/kg and above (P<0.05). Serum LH levels were also significantly increased (5 mg/kg and above, P<0.05), but no changes in serum testosterone levels. In contrast, BPA had no effects on the re-growth of seminal vesicles, ventral prostate and LABC induced by testosterone, and no significant differences were observed in serum LH and testosterone levels. In summary, the Hershberger assay could be a sensitive method for detecting androgenic or anti-androgenic chemicals, but BPA did not exhibit any androgenic or anti-androgenic activities in Hershberger assay.

DIFFERENTIAL GENE PROFILES IN DEVELOPING EMBRYO AND FETUS AFTER IN UTERO EXPOSURE TO ETHANOL

Alcohol consumption during pregnancy results in morphological abnormalities in the fetuses of humans and experimental animals, and is referred to as fetal alcohol syndrome (FAS). However, the molecular mechanism underlying FAS has not been completely elucidated. The aim of the present study was to investigate the potential molecular mechanisms of ethanol-induced FAS in the developing embryo and fetus. cDNA microarray analysis was used to screen for altered gene profiles. Ethanol at a teratogenic dosage (3.8 g/kg, twice a day) was administered intraperitoneally to pregnant C57Bl/6J mice from gestation day (GD) 6 to 8. Morphologic observations showed excessive malformations of the craniofacial regions (reduction of the face, the absence of eyes, nose, jaw, and mandible, underdevelopment of vibrissae areas, cleft lip, and palate) in ethanol-exposed embryos (GD 10) and fetusus (GD 15). cDNA microarray analysis showed alterations in several gene profiles, including the “palate, lung, and nasal epithelium clone (plunc),” “neurofilament,” and “pale ear.” Of these genes, the expressions of plunc were confirmed by reverse-transcription polymerase chain reaction (RT-PCR) and whole-mount in situ hybridization. The plunc was highly expressed in the craniofacial region, specifically in upper airways and nasopharyngeal epithelium. RT-PCR analysis revealed that normal plunc mRNA expression levels were present in GD 15 fetuses, but not in GD 10 embryos. Interestingly, ethanol significantly downregulated the plunc expression in GD 15 fetuses. Our results suggest that ethanol-induced FAS is due in part to the downregulation of plunc expression in the fetus, and this gene may be a candidate biological marker for FAS.

Inhibitory effect of peroxisome proliferator-activated receptor gamma agonist on ochratoxin A-induced cytotoxicity and activation of transcription factors in cultured rat embryonic midbrain cells.

The effects of 15-deoxy- j 12,14 -prostaglandin J 2 (15-deoxy PGJ 2 ) on ochratoxin A (OTA)-induced neurotoxicity and on the activation of transcription factors activator protein-1 (AP-1) and nuclear factor-kappa B (NF- s B) were investigated in cultured rat embryonic midbrain cells. Twelve-day rat embryo midbrain cells were cultured for 48 h. OTA (0.5 or 1 µg/ml) and/or 15-deoxyPGJ 2 (0.5 µ M ) were then added for 48 h. Cell number and neurite outgrowth were determined to assess the neurotoxicity of OTA. AP-1 and NF- s B activation was determined by gel mobility shift assay after 3 h of exposure to OTA and/or 15-deoxy PGJ 2 . OTA caused concentration-dependent reductions in neurite outgrowth and cell number, and induced AP-1 and NF- s B activation. Cotreatment with 15-deoxy PGJ 2 (0.5 µ M ) blocked OTA-induced decrease in neurite outgrowth and cell number and inhibited AP-1 and NF- s B activation. 15-Deoxy PGJ 2 (0.5 µ M ) caused the expression of peroxisome proliferator-activated receptor-gamma (PPAR- n ) in the cells. Results show that 15-deoxy PGJ 2 blocked OTA-induced neurotoxicity by inhibiting AP-1 and NF- s B activation in cultured rat embryonic midbrain cells.

Assessment of estrogenic and androgenic activities of tetramethrin in vitro and in vivo assays.

Tetramethrin, a synthetic pyrethroid insecticide, is used globally for agriculture, and thus potential environmental exposure to tetramethrin is a concern. Environmental chemicals that are hormonally active (particularly estrogen or androgen) may adversely affect the reproductive and endocrine systems. However, little is known about the estrogenic and androgenic activities of tetramethrin. In this study, uterine CaBP-9k gene expression assay and a uterotrophic assay were conducted for estrogenic activity assessment of tetramethrin, and a Hershberger assay was conducted for androgenic activity. Estrogen receptor (ERα and ERβ) protein levels were also measured in tetramethrin-treated rat uteri. Northern blot analysis showed reduction in uterine CaBP-9k mRNA levels in response to tetramethrin, as well as when rats were given both tetramethrin and 17β-estradiol (E2). In the uterotrophic assay using 18-d-old female Sprague-Dawley rats, subcutaneous treatment with tetramethrin (5 to 800 mg/kg/day) for 3 d led to a statistically significant decrease in absolute and relative uterine wet weights at all doses tested. Moreover, tetramethrin blocked the effect of E2 on uterine weights. In addition, tetramethrin reduced absolute and relative vaginal wet weights, and also inhibited the increases of vaginal weights produced by E2. Tetramethrin showed no androgenic on antiandrogenic activities in the Hershberger assay. These results suggest that tetramethrin might exert endocrine-disrupting effects on female rats through antiestrogenic action.

EFFECTS OF OCHRATOXIN A ON CYTOTOXICITY AND CELL DIFFERENTIATION IN CULTURED RAT EMBRYONIC CELLS

In the present study, the effects of ochratoxin A (OTA) on cytotoxicity, cell differentiation, and other cell functions in the embryonic midbrain cells, which are dopaminergic, were compared to those in the limb bud cells, which are nondopaminergic, to assess the selectivity of OTA central action. Twelve-day rat embryo midbrain and limb bud cells were cultured in Dulbeccos modified Eagles medium nutrient and Hams F12 (1:1) mixture containing 10% Nuserum for 96 h in the presence of various concentrations of OTA. OTA significantly reduced the levels of protein, DNA and glutathione, and [3H]thymidine incorporation into DNA in both embryonic midbrain and limb bud cells in a similar concentration-dependent manner. The IC50 values for cytotoxicity measured by neutral red uptake were 1.10 µ M in the midbrain cells and 1.05 µ M in the limb bud cells. The IC50 values of cell differentiation were 1.10 µ M in the midbrain cells and 1.0 µM in the limb bud cells. The addition of exogenous glutathione (32.5 µ M) did not change the OTA-induced fall in protein and DNA levels, or the IC50 values of cytotoxicity and differentiation in the midbrain and limb bud cells. Data show that OTA does not appear to exert a selective toxic dopaminergic cell action and that OTA-induced cytotoxicity and inhibition of cell differentiation were not prevented by exogenous glutathione.