Seung Jun Kwack

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.

Acrylamide induces cell death in neural progenitor cells and impairs hippocampal neurogenesis.

Acrylamide (ACR) is a well-known neurotoxin in mammalian species that causes neuropathy characterized by ataxia and skeletal muscle weakness. Therefore, ACR-mediated axon damage in the central and peripheral nervous systems is considered to be central-peripheral axonopathy. However, the molecular mechanisms underlying ACRs toxicity to neural progenitor cells are unknown. This study investigated the adverse effects of ACR on mouse multipotent neural progenitor cells and adult hippocampal neurogenesis. ACR significantly reduced the proliferation of neural progenitor cells, and high ACR concentrations induced apoptotic and necrotic cell death. We found that elevated intracellular levels of reactive oxygen species were involved in ACR-mediated cytotoxicity. Interestingly, the administration of ACR to young mice resulted in a significant decrease in the number of newly generated cells in the dentate gyrus of the hippocampus, suggesting an impairment of adult neurogenesis. These results suggest that ACRs deleterious effects on the central nervous system are due to the death of neural progenitor cells and impaired adult neurogenesis.

Neurotoxic Effects of Alcohol and Acetaldehyde During Embryonic Development

Alcohol drinking during pregnancy results in abnormal fetal development, including fetal alcohol syndrome (FAS) in humans and experimental animals. FAS is characterized by two major effects, including central nervous system (CNS) dysfunction and multiple anomalies recognizable mainly as a typical face. However, the mechanisms of alcohol-induced embryotoxicity have not been clearly demonstrated. The aim of the present study was to investigate the possible mechanisms underlying ethanol-induced FAS in the developing embryo. First, ethanol-induced developmental abnormalities were investigated in vitro. Postimplantation embryos at gestation day (GD) 9.5 were cultured for 48 h and observed for morphological changes. Ethanol-mediated changes in proteins regulated apoptosis (p53 and bcl-2), antioxidant (vitamin E and catalase) activities, generation of reactive oxygen species (ROS), and oxidative DNA damage shown as 8-hydroxy-2′-deoxyguanosine (8-OHdG) were measured in embryonic midbrain cells. Alcohol or acetaldehyde significantly induced cytotoxicity in cultured rat embryonic midbrain cells. The levels of p53, bcl-2, and 8-OHdG were concomitantly changed by alcohol and acetaldehyde treatment in midbrain cells. Injured cells induced by ROS were increased by alcohol or acetaldehyde treatment in midbrain cells. Cotreatment with alcohol or acetaldehyde and catalase decreased cytotoxicity in midbrain cells. In postimplantation embryo culture, alcohol or acetaldehyde-treated embryos showed retardation of embryonic growth and development in a concentration-dependent manner. These results indicate that alcohol and its metabolite acetaldehyde induce fetal developmental abnormalities by disrupting cellular differentiation and growth. Data demonstrate that some antioxidants can partially protect against the alcohol-induced embryonic developmental toxicity.

Pharmacokinetic disposition and tissue distribution of bisphenol A in rats after intravenous administration

This study examined the dose-linearity pharmacokinetics of bisphenol A, a U.S. Environmental Protection Agency (EPA) classified endocrine disruptor, in rats following iv administration. Upon iv injection of 0.2, 0.5, 1, or 2 mg/kg, serum levels of bisphenol A declined biexponentially, with mean initial distribution and elimination half-life ranges of 4?8.2 min and 38.6?62.2 min, respectively. There were no significant alterations in the systemic clearance rate (mean range 90.1?123.6 ml/min/kg) and the steady-state volume of distribution (mean range 4.6?6.0 L/kg) as a function of the administered dose. In addition, the area under the serum concentration?time curve linearly rose as the dose was increased. In a second study, bisphenol A was given by simultaneous iv bolus injection plus infusion to steady state, and levels were measured in serum and various organs. When expressed in concentration terms (e.g., amount accumulated per gram organ weight), bisphenol A was found predominantly in the lung, followed by kidneys, thyroid, stomach, heart, spleen, testes, liver, and brain. Ratios of the organ to serum bisphenol A concentrations exceeded unity for all the organs examined (ratio range 2.0? 5.8) except for brain (ratio 0.75). Given the high systemic clearance and short elimination half-life, bisphenol A is unlikely to accumulate significantly in the rat.This study examined the dose-linearity pharmacokinetics of bisphenol A, a U.S. Environmental Protection Agency (EPA) classified endocrine disruptor, in rats following iv administration. Upon iv injection of 0.2, 0.5, 1, or 2 mg/kg, serum levels of bisphenol A declined biexponentially, with mean initial distribution and elimination half-life ranges of 4-8.2 min and 38.6-62.2 min, respectively. There were no significant alterations in the systemic clearance rate (mean range 90.1-123.6 ml/min/kg) and the steady-state volume of distribution (mean range 4.6-6.0 L/kg) as a function of the administered dose. In addition, the area under the serum concentration-time curve linearly rose as the dose was increased. In a second study, bisphenol A was given by simultaneous iv bolus injection plus infusion to steady state, and levels were measured in serum and various organs. When expressed in concentration terms (e.g., amount accumulated per gram organ weight), bisphenol A was found predominantly in the lung, followed by kidneys, thyroid, stomach, heart, spleen, testes, liver, and brain. Ratios of the organ to serum bisphenol A concentrations exceeded unity for all the organs examined (ratio range 2.0-5.8) except for brain (ratio 0.75). Given the high systemic clearance and short elimination half-life, bisphenol A is unlikely to accumulate significantly in the rat.

Potential Risk of Bisphenol a Migration From Polycarbonate Containers After Heating, Boiling, and Microwaving

The migration levels of bisphenol A (BPA) were analyzed in food samples by high-performance liquid chromatography (HPLC) from polycarbonate (PC) bottles subjected to simulated use by heating with microwave, heating in a boiling water bath, or filling them with boiling hot water (100°C). Migration testing performed in PC bottles filled with steamed rice or hot cooked pork, standing at room temperature, or heated in a boiling water bath (100°C) showed that BPA was not detected at the limit of detection (LOD) of 1 μg/L (ppb). In contrast, heating by microwaving to 100°C for 9 min increased BPA migration levels from 6 to 18 ppb and from 5 to 15 ppb for steamed rice or for cooked pork, respectively. In addition, 3 different PC bottles were tested by filling them with boiling hot water (100°C) and leaving them to stand at room temperature for up to 3 h. The mean BPA levels from the bottles increased in a time-dependent manner, with the range of not detected (ND) to 2.5 ppb after 60 min. However, none of the PC bottles released BPA at levels that exceed the recently established specific migration limits (SML) of 600 ppb established by European Union and Korea Food and Drug Administration (KFDA). Data suggest that the use of PC plastic bottles in our daily life is considered safe in Korea.

Di(2-ethylhexyl) Phthalate Induces Apoptosis Through Peroxisome Proliferators-Activated Receptor-Gamma and ERK 1/2 Activation in Testis of Sprague-Dawley Rats

Di(2-ethylhexyl) phthalate (DEHP) is a well-known hepatic and reproductive toxicant whose toxicity may be mediated by peroxisome proliferators-activated receptor (PPAR). This study examined the effects of DEHP on the expression of PPAR-regulated genes involved in testicular cells apoptosis. Sprague-Dawley male rats were treated orally with 250, 500, or 750 mg/kg/d DEHP for 28 d, while control rats were given corn oil. The levels of cell cycle regulators (pRb, cyclins, CDKs, and p21) and apoptosis-related proteins were analyzed by Western blot analysis. The role of PPAR-gamma (PPAR-γ), class B scavenger receptor type 1 (SR-B1), and ERK1/2 was further studied to examine the signaling pathway for DEHP-induced apoptosis. Results showed that the levels of pRB, cyclin D, CDK2, cyclin E, and CDK4 were significantly lower in rats given 500 and 750 mg/kg/d DEHP, while levels of p21 were significantly higher in rat testes. Dose-dependent increases in PPAR-γ and RXRα proteins were observed in testes after DEHP exposure, while there was a significant decrease in RXRγ protein levels. In addition to PPAR-γ, DEHP also significantly increased SR-B1 mRNA and phosphorylated ERK1/2 protein levels. Furthermore, DEHP treatment induced pro-caspase-3 and cleavage of its substrate protein, poly(ADP-ribose) polymerase (PARP), in a dose-dependent manner. Data suggest that DEHP exposure may induce the expression of apoptosis-related genes in testes through induction of PPAR-γ and activation of the ERK1/2 pathway.

Effects of Gestational Exposure to Decabromodiphenyl Ether on Reproductive Parameters, Thyroid Hormone Levels, and Neuronal Development in Sprague-Dawley Rats Offspring

Polybrominated diphenyl ethers (PBDE) are a class of brominated flame retardants that are recognized as global environmental contaminants with potential adverse effects on human health. This study examined the effects of prenatal exposure to PBDE on reproductive organs, neuronal development, and levels of thyroid hormones. Pregnant rats were exposed to the vehicle or deca-bromodiphenyl ether (BDE) (BDE-209; 5, 40, or 320 mg/kg body weight/d) during gestation days (GD) 6–18. There was a significant decrease in body weight gain in F1 male offspring exposed to high-dose (320 mg/kg) BDE-209. Significant increases in thyroid weight and a decrease in adrenal weight were observed in high-dose BDE-209. Thyroxine (T4) concentrations were significantly lower in F1 female offspring exposed to BDE-209 at postnatal day (PND) 42. This reduction was more pronounced in the group exposed to higher doses. A low dose (5 mg/kg) of BDE-209 significantly reduced serum estradiol concentration in female offspring but did not affect testosterone levels in males. There was no significant effect on hippocampal neurogenesis in BDE-209 treatment groups. In conclusion, there was no apparent association between thyroid hormone concentrations and low birth weight in F1 rats after gestational exposure to BDE-209.

Comparative Toxicological Evaluation of Phthalate Diesters and Metabolites in Sprague-Dawley Male Rats for Risk Assessment

In order to comparatively assess the systemic toxicity and sperm parameters, nine phthalate diesters, including di(2-ethylhexyl) phthalate (DEHP), di(n-butyl) phthalate (DBP), di-n-octyl phthalate (DnOP), diethyl phthalate (DEP), butylbenzyl phthalate (BBP), dimethyl phthalate (DMP), di-isodecyl phthalate (DIDP), diundecyl phthalate (DUP), and di-isononyl phthalate (DINP), and five phthalate monoesters, including mono(2-ethylhexyl) phthalate (MEHP), monobutyl phthalate (MBuP), monobenzyl phthalate (MBeP), monoethyl phthalate (MEP), monomethyl phthalate (MMP), and phthalic acid (PA) were administered orally to Sprague-Dawley male rats at 250 (phthalate monoesters and PA) or 500 mg/kg body weight (bw)/d (phthalate diesters) for 4 wk. Liver weights were significantly increased in g roups treated with DEHP, DBP, BBP, DIDP, DINP, MEHP, and MBuP compared to the control. Testes weights were significantly reduced only in DEHP, DBP, and MEHP-treated groups compared to the control. Significant decreases in red blood cell (RBC) and hematocrit (Ht) levels were observed in DEHP-treated rats, whereas significant increases in mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), and platelet (PLT) levels were found in the DEHP-treated group. Hemoglobin (Hb) level was reduced only in the DMP group. Similar to effects on testis and epididymal weights, DEHP and MEHP significantly reduced sperm numbers and motility. In particular, DnOP, DBP, BBP, MEP, MBuP, DUP, DINP, and MBeP significantly lowered the sperm counts and sperm motility of epididymal sperm, detected by a change in the sperm motion parameters. The strongest to the weakest adverse effects for sperm motility were as follows: DEHP > DBP > DnOP > DUP > DIDP > BBP among diesters and MBuP > MEP > MEHP among monoesters, respectively. These results suggest that the adverse effects of phthalate esters (PEs) on sperm parameters in male rats are greater with phthalate diesters than monoesters, which may be useful for the risk assessment of phthalates.

Bioavailability and mammary excretion of bisphenol A in Sprague-Dawley rats

This study reports the absolute oral bioavailability and mammary excretion of bisphenol A in rats. The oral bioavailability was determined after administration of relatively low iv (0.1 mg/kg) and oral (10 mg/kg) doses of bisphenol A to rats. After iv injection, serum levels of bisphenol A declined biexponentially, with the mean initial distribution and terminal elimination half-lives being 6.1 - 1.3 min and 52.5 - 2.4 min, respectively. The systemic clearance (Cl s ) and the steady-state volume of distribution ( V ss ) averaged 107.9 - 28.7 ml/min/kg and 5.6 - 2.4 L/kg, respectively. Upon oral administration, the maximum serum concentration ( C max ) and the time to reach the maximum concentration ( T max ) were 14.7 - 10.9 ng/ml and 0.2 - 0.2 h, respectively. The apparent terminal elimination half-life of bisphenol A (21.3 - 7.4 h) after oral administration was significantly longer than that after iv injection, indicating the flip-flop of the absorption and elimination rates. The absolute oral bioavailability of bisphenol A was low (5.3 - 2.1%). To determine the extent of mammary excretion, bisphenol A was given by simultaneous iv bolus injection plus infusion to steady state at low, medium, and high doses. The steady-state serum levels of bisphenol A were linearly increased with higher dosing rates. The systemic clearance (mean range, 119.2-154.1 ml/min/kg) remained unaltered over the dosing rate studied. The levels of bisphenol A in milk exceeded those in serum, with the steady-state milk to serum concentration ratio being 2.4-2.7.

Risk Assessment of Bisphenol a Migrated from Canned Foods in Korea

Exposure and risk assessment of bisphenol A (BPA) was conducted on consumption of canned foods in Korean adults. Sixty-one canned food items with different brands purchased from retail outlets in markets were analyzed for BPA concentration by high-performance liquid chromatography (HPLC) coupled with fluorescence detection. Limits of detection (LOD) were 3 μg/kg for solid and 2 μg/kg for liquid foods. BPA was detected from 7 groups of food items, such as tuna (n = 8), fish (n = 11), fruits (n = 9), vegetables (n = 12), meats (n = 13), coffee (n = 5), and tea (n = 3) in the range from not detected (ND) to 136.14 μg/kg. Mean concentrations of BPA were 3.1 μg/kg (ND−21.5 μg/kg) for vegetables, 8.3 μg/kg (ND−14.26) for tea, 8.6 μg/kg (ND−54.56 μg/kg) for fruits, 24.49 μg/kg (ND−98.30 μg/kg) for meats, 39.78 μg/kg (ND−125.25 μg/kg) for fish, 43.7 μg/kg (ND−116.88 μg/kg) for tuna, and 45.51 μg/kg (ND−136.14 μg/kg) for coffee, in the order of magnitude. Based on daily dietary intake of canned food items and concentrations of BPA, human exposure level to BPA was estimated to be 1.509 μg/kg body weight (bw)/d, well below the tolerable daily intake (TDI) or reference dose (RfD) of 50 μg/kg, bw/d set by the European Commission, U.S.EPA, and South Korea, . Therefore, the potential risk for BPA contamination due to consumption of each canned food items was calculated to be (1.509 μg/kg bw/d)/(50 μg/kg bw/d) = 0.03, which is the hazard index [HI = exposure level/(RfD or TDI)]. Evidence indicates that the levels of BPA levels in canned foods are not likely to constitute a safety concern for consumers in Korea.