Estimation of Apple Intake for the Exposure Assessment of Residual Chemicals Using Korea National Health and Nutrition Examination Survey Database.

The aims of this study were to develop strategies and algorithms of calculating food commodity intake suitable for exposure assessment of residual chemicals by using the food intake database of Korea National Health and Nutrition Examination Survey (KNHANES). In this study, apples and their processed food products were chosen as a model food for accurate calculation of food commodity intakes uthrough the recently developed Korea food commodity intake calculation (KFCIC) software. The average daily intakes of total apples in Korea Health Statistics were 29.60 g in 2008, 32.40 g in 2009, 34.30 g in 2010, 28.10 g in 2011, and 24.60 g in 2012. The average daily intakes of apples by KFCIC software was 2.65 g higher than that by Korea Health Statistics. The food intake data in Korea Health Statistics might have less reflected the intake of apples from mixed and processed foods than KFCIC software has. These results can affect outcome of risk assessment for residual chemicals in foods. Therefore, the accurate estimation of the average daily intake of food commodities is very important, and more data for food intakes and recipes have to be applied to improve the quality of data. Nevertheless, this study can contribute to the predictive estimation of exposure to possible residual chemicals and subsequent analysis for their potential risks.

Contributing effect of various washing procedures and additives on the decline pattern of diethofencarb in crown daisy, a model of leafy vegetables.

The effects of various washing procedures, including stagnant, running, and stagnant and running tap water, and the use of washing solutions and additives, namely NaCl (1% and 2%), vinegar (2%, 5%, and 10%), detergent (0.5% and 1%), and charcoal (1% and 2%), on the reduction rate of diethofencarb were estimated in field-incurred crown daisy, a model of leafy vegetables, grown under greenhouses located in 3 different areas (Gwangju, Naju, and Muan). The original Quick, Easy, Cheap, Effective, Rugged, and Safe "QuEChERS" method was modified for extraction and liquid chromatography-tandem mass spectrometry (LC/MS/MS) was used for analysis. The recovery of diethofencarb in unwashed and washed samples was satisfactory and ranged between 84.28% and 115.32% with relative standard deviations (RSDs) of <6%. The residual levels decreased following washing with stagnant, running, and stagnant+running tap water (i.e., decline in levels increased from 65.08% to 85.02%, 69.99 to 86.79, and 74.75 to 88.96, respectively). The percentage of decline increased and ranged from 77.46% to 91.19% following washing with various solutions. Application of 1% detergent was found to be the most effective washing method for reducing the residues in crown daisy. Additionally, washing with stagnant and running tap water or even stagnant water for 5 min might reduce the residue levels substantially, making the prepared food safe for human consumption.

Detection of pyridaben residue levels in hot pepper fruit and leaves by liquid chromatography-tandem mass spectrometry: effect of household processes.

Following quick, easy, cheap, effective, rugged and safe (QuEChERS) and LC/MS/MS analysis, pyridaben residual levels were determined in unprocessed and processed hot pepper fruit and leaves. The linearities were satisfactory with determination coefficients (R(2)) in excess of 0.995 in processed and unprocessed pepper fruit and leaves. Recoveries at various concentrations were 79.9-105.1% with relative standard deviations ≤15%. The limits of quantitation of 0.003-0.012 mg/kg were very low compared with the maximum residue limits (2-5 mg/kg) set by the Ministry of Food and Drug Safety, Republic of Korea. The effects of various household processes, including washing, blanching, frying and drying under different conditions (water volume, blanching time and temperature) on residual concentrations were evaluated. Both washing and blanching (in combination with high water volume and time factor) significantly reduced residue levels in hot pepper fruit and leaves compared with other processes. In sum, the developed method was satisfactory and could be used to accurately detect residues in unprocessed and processed pepper fruit and leaves. It is recommended that pepper fruit/leaves be blanched after washing before being consumed to protect consumers from the negative health effects of detected pesticide residues.

Bisphenol A exerts estrogenic effects by modulating CDK1/2 and p38 MAP kinase activity.

Bisphenol A (BPA) is considered to be an endocrine disruptor, but the mechanisms by which it disrupts endocrine functions are poorly understood. Here, we have shown that BPA binds both estrogen receptor (ER)-α and ER-beta (ER-ß) using a fluorescence polarization competitive binding assay. In addition, we found that BPA induced cell proliferation by modulating cell cycle-related genes in the MCF-7 human mammary cancer cell line. Moreover, using a BG1 luciferase ER transactivation assay, we found that BPA has estrogenic activity. Modulating the MAPK pathway by using an ERK inhibitor (PD98059) or a JNK inhibitor (SP600125) had no effect on the ability of BPA to induce estrogenic activity. However, the antiestrogen, ICI 182,780, and the p38 inhibitor, PD 169316 successfully blocked BPA-induced estrogenic activity. Our findings suggest that BPA mimics ER-dependent estrogenic activity by targeting proteins that regulate the cell cycle and p38 MAPK.

Evaluation of in vitro screening system for estrogenicity: comparison of stably transfected human estrogen receptor-α transcriptional activation (OECD TG455) assay and estrogen receptor (ER) binding assay.

The estrogenic activity of industrial chemicals, di(2-ethylhexyl) phthalate (DEHP), di(n-butyl) phthalate (DBP), benzylbutyl phthalate (BBP), diethyl phthalate (DEP), tetrabromobisphenol A (TBBPA), bisphenol A (BPA), and nonylphenol (NP), was compared using OECD test guideline 455(TG455), stably transfected transcriptional activation (STTA) and estrogen receptor (ER) binding assays. The estrogenic activity of BBP, BPA and NP were approximately 180,000-fold (PC(50), 4.32 x 10(-6 )M), 5,000-fold (PC(50), 1.26 x 10(-7) M) and 120,000-fold (PC(50), 2.92 x 10(-6 )M) less than 17ß-estradiol (PC(50), 2.43 x 10(-11)M), whereas DEHP, DBP and DEP did not show any estrogenicity activity in the STTA assay. Moreover, binding affinities to human ERα of BBP, BPA, and NP were approximately 200,000-fold (IC(50), 4.91 x 10(-4) M), 8000-fold (IC(50), 1.92 x 10(-5) M) and 1400-fold (IC(50), 3.34 x 10(-6) M) less than 17ß-estradiol (IC(50), 2.45 x 10(-9) M) in competitive human ERα binding assay. The relative potencies of STTA assay were very similar to ER binding, E-screen, and Yeast screening assays. Therefore, our results suggested that OECD test guideline TG455 may be useful as a screening test for potential endocrine disruptors.

Estrogenic Activity of Persistent Organic Pollutants and Parabens Based on the Stably Transfected Human Estrogen Receptor-α Transcriptional Activation Assay (OECD TG 455).

Screening of estrogenic activity on dichloro diphenyl trichloroethane (DDT), dichloro diphenyl dichloro ethylene (DDE), dieldrin, heptachlor, aldrin, chlordane, lindane, polybrominated diphenyl ethers (PBDE) and parabens was compared using Organization for Economic Cooperation and Development (OECD) test guideline 455 (TG455). The estrogenic activity of DDT was 58,000-fold (PC50, 1.67 × 10(-6) M) less than 17ß-estradiol(E2) (PC50, 2.88 × 10(-11) M) but DDE, dieldrin, heptachlor, aldrin, chlordane, lindane and PBDE did not show any estrogenic activity in this assay system. In the case of paraben compounds, the rank of relative transcriptional activation (logRTA) was butyl paraben -1.63752 (PC50, 1.25 × 10(-7)M) > isobutyl paraben -2.34008 (PC50, 6.3 × 10(-7)M) > ethyl paraben -2.64016 (PC50, 1.26 × 10(-6) M) > isopropyl paraben -2.73993 (PC50, 1.58 × 10(-6)M) > propyl paraben -2.84164 (PC50, 2.0 × 10(-6) M). Our data suggest that OECD test guideline TG455 may be useful as a screening tool for potential endocrine disruptors.

Identification of post-generation effect of 3,4-methylenedioxymethamphetamine on the mouse brain by large-scale gene expression analysis.

The compound 3,4-methylenedioxymethamphetamine (MDMA or ecstasy) is a synthetic, psychoactive drug chemically similar to the stimulant methamphetamine and the hallucinogen mescaline. Accumulated data has revealed potential toxic effects associated with MDMA on brain serotonin and dopamine neurons in animal models. However, the relevance of these adverse effects on prenatal exposure to this drug remains unknown. In this study, we demonstrated that prenatal (F0) exposure to MDMA caused permanent large-scale transcriptional changes in the brains of the offspring (F1), especially in the cerebral cortex, by gene expression profiling analysis. The expression analysis of the brain of F1 pups, after maternal ingestion of MDMA (20 mg/kg MDMA), revealed significant transcriptional changes in both male and female pups. Supervised analysis resulted in the identification of 804 outlier genes in males and 1784 outlier genes in females as MDMA-associated genes in the F1 generation. Most of the functional categories of genes, among the outlier genes, were intracellular signaling pathways, including the MAPK signaling pathway, Wnt signaling pathway, and neuroactive ligand-receptor interaction pathway. Although these genes were affected by MDMA exposure in utero, their association with brain dysfunction requires further investigation. The results of this study suggest that prenatal MDMA exposure may affect the developing brain.

ATG5 expression induced by MDMA (ecstasy), interferes with neuronal differentiation of neuroblastoma cells.

The amphetamine derivative 3, 4-methylenedioxymethamphetamine (MDMA) has become a popular recreational drug, and has also been shown to cause serotonergic neurotoxicity. This report shows that MDMA impairs brain development in a whole mouse embryo culture. The results of quantitative real-time PCR analysis showed that autophagy-related protein 5 (Atg5) expression is elevated in mouse embryo and neuroblastoma cells after MDMA treatment. This elevated Atg5 expression interferes with the neuronal differentiation of neuroblastoma cells such as SH-SY5Y and PC12 cells. Thus, our results suggest that the use of MDMA during pregnancy may impair neuronal development via an induction of Atg5 expression.

Transcriptomic configuration of mouse brain induced by adolescent exposure to 3,4-methylenedioxymethamphetamine.

The amphetamine derivative (+/-)-3,4-methylenedioxymethamphetamine (MDMA or ecstasy) is a synthetic amphetamine analogue used recreationally to obtain an enhanced affiliative emotional response. MDMA is a potent monoaminergic neurotoxin with the potential to damage brain serotonin and/or dopamine neurons. As the majority of MDMA users are young adults, the risk that users may expose the fetus to MDMA is a concern. However, the majority of studies on MDMA have investigated the effects on adult animals. Here, we investigated whether long-term exposure to MDMA, especially in adolescence, could induce comprehensive transcriptional changes in mouse brain. Transcriptomic analysis of mouse brain regions demonstrated significant gene expression changes in the cerebral cortex. Supervised analysis identified 1028 genes that were chronically dysregulated by long-term exposure to MDMA in adolescent mice. Functional categories most represented by this MDMA characteristic signature are intracellular molecular signaling pathways of neurotoxicity, such as, the MAPK signaling pathway, the Wnt signaling pathway, neuroactive ligand-receptor interaction, long-term potentiation, and the long-term depression signaling pathway. Although these resultant large-scale molecular changes remain to be studied associated with functional brain damage caused by MDMA, our observations delineate the possible neurotoxic effects of MDMA on brain function, and have therapeutic implications concerning neuro-pathological conditions associated with MDMA abuse.

Cytotoxicity of 5-fluorouracil: Effect on endothelial differentiation via cell cycle inhibition in mouse embryonic stem cells.

Embryonic stem cells (ESCs) are known to characteristics for pluripotency and self-renewal, but the precise mechanisms of ES-derived cells to specific toxicants have not been determined. Here, we evaluated the cytotoxicity of 5-fluorouracil (5-FU) and see its effect on cell viability, proliferation, and differentiation in mouse ESC-derived endothelial differentiation. Mouse ESCs were exposed to 5-FU (10 microM) and combined with probucol (50 microM) for 24h, which is an antagonist of 5-FU. Changes in gene expression as a result of 5-FU exposure in mouse ESC-derived endothelial precursor cells (ES-EPCs) were assessed using an oligonucleotide microarray (AB1700). The expression of Oct-4 was decreased during the differentiation of mouse ESCs into endothelial cells; otherwise, the expression of PECAM was increased. Mouse ES-EPCs were shown to have a decrease in viability (49.8%) and PECAM expression, and induce G1/S phase (31.1%/60.6%) when compared with/without treatment of 5-FU. Expression of cell cycle-related proteins was increased in endothelial precursor cells exposed to 5-FU without probucol treatment. From theses results suggest that 5-FU inhibit endothelial differentiation as well as inducing the G1/S phase arrest. We propose that mouse ES-EPCs might be a useful tool for screening the cytotoxicity of compounds in endothelial cells.

Lactational coumestrol exposure increases ovarian apoptosis in adult rats.

This study is the first to examine the increased apoptosis in the adult rat ovary after lactational exposure to coumestrol (COU), a potent phytoestrogen. Lactating dams were gavaged at doses of 0.01, 0.1, 1, and 10 mg/kg COU during the lactation period and the reproductive effects of female pups were investigated in young adults. Rats were sacrificed at postnatal days (PND) 81-84. Ovarian weights were reduced significantly at 0.1 and 1.0 mg/kg COU. The reduction in the ovarian weight occurred in parallel with an increase in the apoptosis at PND 135-140. A marked dose-dependent increase in the expressions of active caspase-3 and -7 was observed in ovarian granulosa cells. Immunostaining for active caspase-3 and the TUNEL staining of apoptotic cells were also increased in ovaries exposed to COU in a dose-dependent manner. These results suggest new sights into the effect of lactational exposure to COU on the female reproductive health.

Differentiation of endothelial cells derived from mouse embryoid bodies: a possible in vitro vasculogenesis model.

Mouse embryonic stem cells (mES cells), which are pluripotent and self-renewal cells, are derived from the inner cell mass of mouse blastocysts. The objective of this study was to construct more efficient mES cell-derived embryoid bodies (EBs) for use as a vasculogenesis model and as an in vitro vascular toxicity testing model. EBs were formed for 3 days using hanging drop cultures and plated on gelatin-coated plates in endothelial growth medium-2 (EGM-2) to promote vascular development. The differentiation of mES cell-derived EBs was confirmed by reverse transcription-polymerase chain reaction (RT-PCR), immunocytochemistry, and flow cytometry within 7 days after plating EBs. The mRNA and protein expressions of vascular endothelial growth factor receptors-2 (FLK-1), platelet endothelial cell adhesion molecule (PECAM), and vascular endothelial-cadherin (VE-cadherin) were observed in differentiated mES cells. When placed in matrigel, mES cell-derived endothelial like cells formed networks similar to vascular structures. mES cells were also exposed to 5-fluorouracil (5-FU), a strong inhibitor of vessel formation, and its cytotoxicity was determined using MTT assays. The inhibitory concentrations (IC50) of 5-FU for mES cells and C166 cells were 0.72 microM and 1.04 microM, respectively. These results demonstrate that mES cells can be used to study vasculogenesis and for cytotoxicity screening.

Chronic 3,4-methylenedioxymethamphetamine treatment suppresses cell proliferation in the adult mouse dentate gyrus.

We investigated the influence of chronic 3,4-methylenedioxymethamphetamine (MDMA) treatment on cell proliferation in the adult dentate gyrus. Mice were orally treated with MDMA (1.25 mg/kg-40 mg/kg) or saline for 30 days. To label dividing cells, mice were given 5-bromo-2'-deoxyuridine (BrdU) for 4 days from the day after the last administration of MDMA, and their brains were examined 24 h later. MDMA dose-dependently induced a decrease in the number of BrdU-positive cells in the male and female dentate gyrus. Our results suggest that chronic exposure to MDMA suppresses cell proliferation in the dentate gyrus.

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.

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.

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 (ERalpha and ERbeta) 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 17beta-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.

Potential estrogenic and antiandrogenic effects of permethrin in rats.

Many environmental chemicals including pesticides have been reported to possess hormonal activities, and thus are classified as endocrine disruptors. Permethrin, a synthetic pyrethroid insecticide, is used worldwide, which provides potential environmental exposure. However, relatively few studies have reported on hormonal activities, particularly estrogenic and androgenic activities of permethrin, and the results of these studies are in some respects contradictory. Therefore, this study investigated the potential estrogenic and androgenic activities of permethrin in vitro and in vivo. We conducted an uterine Calbindin-D9k (CaBP-9k) gene expression assay and an uterotrophic assay for estrogenic activity, and a Hershberger assay for androgenic activity. The CaBP-9k gene, one of the intracellular calcium binding proteins, is estrogen-responsive in the uterus. The rat uterotrophic and Hershberger assays are generally used as in vivo short-term screening assays for detecting the estrogenic and androgenic activities of chemicals, although these assays are still being validated by the Organization for Economic Cooperation and Development (OECD). Northern blot analysis showed the induction of uterine CaBP-9k mRNA level in response to permethrin as well as co-administration of permethrin with E2. In the uterotrophic assay using 18-day-old female rats, subcutaneous treatments with permethrin (10 to 800 mg/kg) for three days increased relative uterine wet weights, and E2-induced uterine weights. These effects were statistically significant at 800 and 200 mg/kg, respectively. Moreover, permethrin-induced uterine weights were inhibited by the co-administration of ICI 182,780, an antiestrogen. In the Hershberger assay, the administration of permethrin orally to testosterone propionate-treated castrated male rats led to statistically significant reductions in androgen-dependent sex accessory tissue (ventral prostate, seminal vesicles, levator ani and bulbocavernosus muscles, Cowper's gland and glans penis) weights at all doses tested (10, 50 and 100 mg/kg). These results suggest that permethrin might have estrogen-like effects on female rats, but antiandrogen-like effects on males.

Mechanism of antifertility in male rats treated with 3-monochloro-1,2-propanediol (3-MCPD).

3-Monochloro-1,2-propanediol (3-MCPD) is a food contaminant that is often found in foods containing acid-hydrolyzed (AH) protein, like seasonings and savory food products. The purpose of the present study was to investigate the effects of 3-MCPD on male fertility, sperm, and hormonal levels and its antifertility mechanism. In vivo male fertility testing was performed to observe the adverse effects of 3-MCPD on the functioning of the male reproductive system and pregnancy outcome. 3-MCPD (0.01-5 mg/kg) was administered daily by gavage to Sprague-Dawley (SD) male rats for 4 wk. At the end of the pretreatment period, male rats were mated overnight with untreated females. Males successfully inducing pregnancy were sacrificed to assess sperm parameters, reproductive organ histopathology, and spermatogenesis. The resulting pregnant females were sacrificed on 20 of gestation to evaluate pregnancy outcome. The paternal administration of 3-MCPD (5 mg/kg) was found to result in adverse effects on male fertility and pregnancy outcome without inducing remarkable histopathological changes in testes and epididymides. Additionally, 3-MCPD (5 mg/kg) significantly reduced sperm motility, copulation, fertility indices, and the number of live fetuses showed steep dose-response curves. 3-MCPD did not affect spermatogenesis or induce hormonal changes in the blood and testes of male rats. An in vitro hormone assay using primary isolated Leydig cells showed no significant changes in related hormone levels after 3-MCPD treatment. To evaluate the effects of 3-MCPD on apoptotic induction and H+-ATPase levels in the testis and epididymis, 10 or 100 mg/kg of 3-MCPD was administered by gavage to male rats and testes and epididymides were examined at 3, 6, 12, and 24 h later. Apoptosis was not detected in the testes of animals treated with 100 mg/kg 3-MCPD. However, the level of H+-ATPase in the cauda epididymis was reduced by 3-MCPD treatment. These results indicate that 3-MCPD induced a spermatotoxic effect, which was mediated by reduced H+-ATPase expression in the cauda epididymis, and suggest that an altered pH level in the cauda epididymis might lead to a disruption of sperm maturation and the acquisition of motility.

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.

Proliferation and hepatic differentiation of adult-derived progenitor cells.

Hepatic progenitor cells, capable of maturing into hepatocytes and biliary cells, are hypothesized to be involved in all forms of liver regeneration and may prove clinically useful at reconstituting damaged livers. A murine hepatic progenitor cell population from young adult liver tissue has been isolated and characterized to establish a model for the development of liver cell therapies and for analysis of immune responses after transplantation. Hepatic progenitor cells were isolated from 3- to 6-week-old C57BL/6 mice using modifications of a two-stage liver perfusion technique followed by low speed centrifugation. Cellular analysis by phase contrast, fluorescent and confocal microscopy demonstrated that the hepatic progenitors (1) formed ex vivo colonies with a morphological appearance similar to committed hepatocytic progenitors isolated from embryonic mice and rats; (2) they are smaller than mature hepatocytes; (3) in culture they demonstrated peak expression of an oval cell marker at day 14, whereas albumin expression continued to increase beyond day 21 of culture, and (4) a subset of the progenitors phenotypically differentiated into mature hepatocytes or biliary cells. The unique antigenic profile of these hepatic progenitor cells and their ability to differentiate suggests that purification of the cells should allow for their potential use in transplantation.