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On September 27, 2012, an explosion from hydrofluoric acid occurred in Gumi city of Gyeongbuk province, Republic of Korea, exposing livestock animals nearby to Hydrofluoric acid (HF). This study aimed at evaluating the HF exposure among cattle raised near the accident site by determining the fluoride ion (F(−1)) levels and other biochemical parameters in the animals’ urine and serum. The study groups included 90 cattle raised on farms near the accident site and, as controls, 21 cattle raised on a farm more than 100 km away from the accident site. Urine and blood serum samples were taken from 10% to 20% of the cattle on each farm that were present 17 days after the accident. The F(−1) concentrations in the samples were analysed by the fluoride-ion-selective electrode method or a biochemistry analyser. The mean F(−1) levels in the cattle serum samples (expressed as mg/L) were 0.23 (100 m), 0.15 (500 m), 0.23 (800 m), 0.11 (900 m), 0.07 (1.2 km), 0.16 (1.5 km), and 0.10 in the control group. The mean F(−1) levels in the cattle urine samples (expressed as F(−1) mg/g creatinine) were 27.8 (100 m), 24.4 (500 m), 11.1 (800 m), 16.3 (900 m), 3.02 (1.2 km), 9.16 (1.5 km), and 3.58 in the control group. The mean ± SD concentrations of calcium ions in serum (expressed as mg/dL) were 9.72 ± 0.41 (100 m), 9.54 ± 0.57 (500 m), 8.31 ± 0.44 (800 m), 9.06 ± 0.40 (900 m), 8.36 ± 0.89 (1.2 km), 9.13 ± 0.98 (1.5 km), and 10.48 ± 1.43 in the control group. The serum and urine F(−1) levels in cattle exposed to HF decreased with the distance from the accident site, suggesting that the relative F(−1) levels in urine after normalization through concentration of urinary creatinine could be a more reliable biomarker for HF exposure in cattle than the urine F(−1) level alone.

To develop a new simple and simultaneous purification method for mycotoxins in feeds and grains, magnetic nanoparticles (MNPs) conjugated with monoclonal antibodies (mAbs) against mycotoxins were used to separate aflatoxin B1 (AFB1), zearalenone (ZEA) and deoxynivalenol (DON). For a single spike of each mycotoxin into the buffer solution (16% MeOH in PBS), mean recoveries were 93.1–95.0% for AFB1 (5–20 ng/mL spiked), 87.2–96.0% for ZEA (125–500 ng/mL spiked) and 75.2–96.9% for DON (250–1,000 ng/mL spiked) by HPLC and ELISA. Recoveries of AFB1 (20 ng/mL) and ZEA (500 ng/mL) simultaneously spiked into the buffer solution were 87.0 and 99.8%, respectively. Recovery rates of AFB1/DON and DON/ZEA spiked simultaneously were 86.2%/76.6% and 92.0%/86.7%, respectively, at concentrations of 20 ng/mL AFB1, 500 ng/mL ZEA, and 1,000 ng/mL DON. Recoveries using the novel mAb–MNP conjugated system in a buffer solution simultaneously spiked with AFB1, ZEA and DON were 82.5, 94.6 and 73.4%, respectively. Recoveries of DON in animal feed were 107.7–132.5% at concentrations of 250–1,000 ng/g spiked in feed. The immunoaffinity chromatography (IAC) clean-up method was compared with the purification method using novel mAb–MNP. After fortification of animal feed with AFB1 (5, 10 and 20 ng/g feed) and ZEA (125, 250 and 500 ng/g feed), AFB1 and ZEA were purified using both the methods. In the case of the novel mAb-MNP conjugated system, mean recoveries for AFB1 were 89.4, 73.1 and 88.3% at concentrations of 5, 10 and 20 ng/g feed, respectively. For ZEA, mean recoveries were 86.7, 85.9 and 79.1% at concentrations of 125, 250 and 500 ng/g, respectively. For IAC purification, recoveries were 42.9–45.1% for AFB1 and 96.8–103.2% for ZEA. In conclusion, the present purification method using monoclonal antibodies conjugated to MNPs can be used for simple and simultaneous purification of mycotoxins from feed and maize.

Background Although previous in vivo studies explored urinary microRNA (miRNA), there is no agreement on nephrotoxicity-specific miRNA biomarkers. Objectives In this study, we assessed whether urinary miRNAs could be employed as biomarkers for nephrotoxicity. Methods For this, literature-based candidate miRNAs were identified by reviewing the previous studies. Female Sprague-Dawley rats received subcutaneous injections of a single dose or repeated doses (3 consecutive days) of gentamicin (GEN; 137 or 412 mg/kg). The expression of miRNAs was analyzed by real-time reverse transcription-polymerase chain reaction in 16 h pooled urine from GEN-treated rats. Results GEN-induced acute kidney injury was confirmed by the presence of tubular necrosis. We identified let-7g-5p, miR-21-3p, 26b-3p, 192-5p, and 378a-3p significantly upregulated in the urine of GEN-treated rats with the appearance of the necrosis in proximal tubules. Specifically, miR-26-3p, 192-5p, and 378a-3p with highly expressed levels in urine of rats with GEN-induced acute tubular injury were considered to have sensitivities comparable to clinical biomarkers, such as blood urea nitrogen, serum creatinine, and urinary kidney injury molecule protein. Conclusions These results indicated the potential involvement of urinary miRNAs in chemical-induced nephrotoxicity, suggesting that certain miRNAs could serve as biomarkers for acute nephrotoxicity.

Biofluid-based biomarkers provide an efficient tool for hazard identification of chemicals. Here, we explored the potential of microRNAs (miRNAs) as biomarkers for hepatotoxicity of chemicals by linking in vitro to in vivo animal models. A search of the literature identified candidate circulating miRNA biomarkers of chemical-induced hepatotoxicity. The expression of candidate miRNAs (miR-122, miR-151a, miR-192, miR-193a, miR-194, miR-21, miR-29c), was determined by real-time reverse transcription-polymerase chain reaction in in vivo acute liver injury induced by acetaminophen, and then were further compared with those of in vitro cell assays. Candidate miRNAs, except miR-29c, were significantly or biologically upregulated by acetaminophen, at a dose that caused acute liver injury as confirmed by hepatocellular necrosis. Except miR-122 and miR-193a, other miRNAs elevated in in vivo models were confirmed by in vitro models using HepG2 cells, whereas they failed by in vitro models using human primary hepatocytes. These findings indicate that certain miRNAs may still have the potential of toxicological biomarkers in linking in vitro to in vivo hepatotoxicity.

Background Although previous in vivo studies explored urinary microRNA (miRNA), there is no agreement on nephrotoxicity-specific miRNA biomarkers. Objectives In this study, we assessed whether urinary miRNAs could be employed as biomarkers for nephrotoxicity. Methods For this, literature-based candidate miRNAs were identified by reviewing the previous studies. Female Sprague-Dawley rats received subcutaneous injections of a single dose or repeated doses (3 consecutive days) of gentamicin (GEN; 137 or 412 mg/kg). The expression of miRNAs was analyzed by real-time reverse transcription-polymerase chain reaction in 16 h pooled urine from GEN-treated rats. Results GEN-induced acute kidney injury was confirmed by the presence of tubular necrosis. We identified let-7g-5p, miR-21-3p, 26b-3p, 192-5p, and 378a-3p significantly upregulated in the urine of GEN-treated rats with the appearance of the necrosis in proximal tubules. Specifically, miR-26-3p, 192-5p, and 378a-3p with highly expressed levels in urine of rats with GEN-induced acute tubular injury were considered to have sensitivities comparable to clinical biomarkers, such as blood urea nitrogen, serum creatinine, and urinary kidney injury molecule protein. Conclusions These results indicated the potential involvement of urinary miRNAs in chemical-induced nephrotoxicity, suggesting that certain miRNAs could serve as biomarkers for acute nephrotoxicity.

// Mingzhu Fang 1, 2, 3, 4 , Pamela A. Ohman Strickland 1, 3, 4 , Hwan-Goo Kang 5 and Helmut Zarbl 1, 2, 3, 4 1 School of Public Health, Rutgers, The State University of New Jersey, Piscataway, NJ, USA 2 Environmental and Occupational Health Sciences Institute, Rutgers, The State University of New Jersey, Piscataway, NJ, USA 3 NIEHS Center for Environmental Exposures and Disease, Rutgers, The State University of New Jersey, Piscataway, NJ, USA 4 Cancer Institute of New Jersey, Rutgers, The State University of New Jersey, Piscataway, NJ, USA 5 Veterinary Drugs & Biologics Division, Animal and Plant Quarantine Agency, Gimcheon-si, Gyeongsangbuk-do, Republic of Korea Correspondence to: Mingzhu Fang, email: fang@eohsi.rutgers.edu Keywords: genetic susceptibility, circadian rhythm, DNA damage response and repair, Period 2, SIRT1 Received: May 24, 2016 Accepted: February 09, 2017 Published: February 24, 2017 ABSTRACT We previously demonstrated that chemopreventive methylselenocysteine (MSC) prevents N -Nitroso- N -methylurea (NMU)-induced mammary carcinogenesis in the susceptible Fischer 344 (F344) rats by enhancing NAD + -dependent SIRT1 activity, restoring circadian expression of Period 2 (Per2) and circadian controlled genes. Here, we show that compared to the genetically resistant Copenhagen (COP) rat strain, mammary glands of the F344 rats have a 4-hour phase delay in circadian expression of Per2 . Consequently, F344 rats failed to increase SIRT1 activity and circadian expression of Per2 and DDRR genes after exposure to NMU. Exposure of COP rats to NMU had the opposite effect, enhancing SIRT1 activity, increasing circadian expression of Per2 and DDRR genes. Significantly, SIRT1 activity and circadian expression of Per2 and DDRR genes in NMU-treated F344 rats on a chemopreventive regimen of MSC approximated those in NMU-treated COP rats. These results indicated that COP rats have an increased capacity to maintain NAD + -dependent SIRT1 activity under genotoxic stress. This contention was supported by increased stability of the period and phase of circadian locomotor activity in COP vs F344 rats exposed to changing light conditions. The increased sensitivity and rapid response of COP to changing light were correlated with the enhanced circadian response of this strain to carcinogen. Disturbance of circadian rhythm by jet lag also disrupted circadian expression of Per2 and DDRR genes, and accelerated mammary tumorigenesis in rodent models. These results suggested that uncoupling of DDRR responses from circadian control by environmental stresses and endogenous factors increases susceptibility to mammary carcinogenesis, possibly by inducing a promutagenic state.

Acetylcholinesterase (AChE) activity level can be used as a diagnostic marker for anticholinesterase pesticide poisoning. In this study, we aimed to establish a baseline level of normal brain AChE activity in wild birds. AChE activity was measured in the brains of 87dead wild birds (26 species). The level of AChE activity ranged from 6.40 to 15.9 µmol/min/g of brain tissue in normal wild birds. However, the brain tissue AChE activity level in wild birds exposed to organophosphate (OP) pesticide was 48.0%-96.3% of that in the normal birds. These results may serve as reference values to facilitate routine diagnosis and monitoring of OP-poisoned wild birds.

With the increased use of cell therapy in the veterinary sector, there is a growing demand for the development of cell-based medicinal products and the determination of their safety. Currently, the Korean Animal and Plant Quarantine Agency has established a guideline for evaluating the safety of cell-based medicinal products for animal use. The guideline includes items related to definition, classification, management, manufacturing procedure and quality control (standard and test method), stability testing, toxicity testing, pharmacological testing, and performance of clinical trials. In addition, testing protocols related to safety assessment of animal cell-based products such as chromosome karyotyping, tumorigenicity testing, confirmatory testing of biodistribution and kinetics, and target animal safety testing are described in detail. Moreover, because cell-based medicinal products are novel therapies, deviations from traditional designs may be justified in order to obtain relevant safety information on the treatment. Additionally, this guideline can be amended on the basis of new scientific findings.

We previously reported that dietary methylselenocysteine (MSC) inhibits N-methyl-N-nitrosourea (NMU)-induced mammary tumorigenesis by resetting circadian gene expression disrupted by the carcinogen at the early stage of tumorigenesis. To investigate the underlying mechanism, we developed a circadian reporter system comprised of human mammary epithelial cells with a luciferase reporter driven by the promoter of human PERIOD 2 (PER2), a core circadian gene. In this in vitro model, NMU disrupted cellular circadian rhythm in a pattern similar to that observed with SIRT1-specific inhibitors; in contrast, MSC restored the circadian rhythms disrupted by NMU and protected against SIRT1 inhibitors. Moreover, NMU inhibited intracellular NAD+/NADH ratio and reduced NAD+-dependent SIRT1 activity in a dose-dependent manner, while MSC restored NAD+/NADH and SIRT1 activity in the NMU-treated cells, indicating that the NAD+-SIRT1 pathway was targeted by NMU and MSC. In rat mammary tissue, a carcinogenic dose of NMU also disrupted NAD+/NADH oscillations and decreased SIRT1 activity; dietary MSC restored NAD+/NADH oscillations and increased SIRT1 activity in the mammary glands of NMU-treated rats. MSC-induced SIRT1 activity was correlated with decreased acetylation of BMAL1 and increased acetylation of histone 3 lysine 9 at the Per2 promoter E-Box in mammary tissue. Changes in SIRT1 activity were temporally correlated with loss or restoration of rhythmic Per2 mRNA expression in NMU-treated or MSC-rescued rat mammary glands, respectively. Together with our previous findings, these results suggest that enhancement of NAD+-dependent SIRT1 activity contributes to the chemopreventive efficacy of MSC by restoring epigenetic regulation of circadian gene expression at early stages of mammary tumorigenesis.

To elucidate the effect on the H19 gene methylation of sperm and organs in offspring by chlorpyrifos-methyl (CPM) exposure during organogenesis period, CPM was administered at doses of 4 (CPM4), 20 (CPM20), and 100 (CPM100) mg/kg bw/day from 7 days post coitum (d.p.c.) to 17 d.p.c. after mating CAST/Ei (♂) and B6 (♀). Anogenital distance (AGD) was measured at postnatal day (PND) 21. Clinical signs, body weights, feed and water consumption, organs weights, serum hormone values, and H19 methylation level of organ and sperm were measured at PND63. Body weights were significantly lower than control until PND6. AGD was significantly decreased in the CPM100 group in males and increased in the CPM20 group in females. The absolute weights of the thymus and epididymis were significantly increased for males in all of CPM treatment groups. In the CPM20 group, absolute weights of liver, kidney, heart, lung, spleen, prostate gland, and testes were significantly increased. Testosterone concentrations in serum were significantly increased by CPM treatment in males. H19 methylation level of liver and thymus showed decreased pattern in a dose-dependent manner in males. The levels of H19 methylation in sperm were 73.76 ± 7.16% (Control), 57.84 ± 12.94% (CPM4), 64.24 ± 3.79% (CPM20), and 64.24 ± 3.79% (CPM100). Conclusively, CPM exposure during organogenesis period can disrupt H19 methylation in sperm, liver, and thymus and disturb the early development of offspring. © 2015 Wiley Periodicals, Inc. Environ Toxicol 30: 1355–1363, 2015.

Recently, adipose tissue-derived mesenchymal stem cells (MSCs) were reported to be a valuable cell source for generating induced pluripotent stem (iPS) cells because adipose tissue is readily obtainable and possesses high quantities of MSCs. To date, little research has been conducted to characterize the efficiency of iPS induction in adipose tissue. Human iPS cells were generated from adipose tissue-derived MSCs (AT-MSCs) and stromal cells (AT-SCs) via a retroviral delivery system. The reprogramming efficiency of AT-MSCs and AT-SCs were 0.008 and 0.014%, respectively, in terms of the expression of the TRA-1-60 antibody and 0.023 and 0.041%, respectively, regarding alkaline phosphatase staining. This differential reprogramming capability resulted from different levels of viral infectivity between the two cell types. Viral infectivity using pMXs-IRES-GFP was notably low in AT-MSCs and the quantities of OSKM (OCT4, SOX2, KLF4 and c-MYC) in AT-MSCs were 22.5–31.6% of AT-SCs. In conclusion, the amount of transgenes leads to a higher reprogramming efficiency in AT-SCs compared with AT-MSCs. The present study shows that the induction efficiency of pluripotent cells from somatic or adult stem cells could depend on the viral susceptibility of the target cells. Therefore, non-MSCs could be a more efficient source for reprogramming by retrovirus-mediated iPS induction.

The circadian rhythm is a fundamental physiological process present in all organisms that regulates biological processes ranging from gene expression to sleep behavior. In vertebrates, circadian rhythm is controlled by a molecular oscillator that functions in both the suprachiasmatic nucleus (SCN; central pacemaker) and individual cells comprising most peripheral tissues. More importantly, disruption of circadian rhythm by exposure to light-at-night, environmental stressors and/or toxicants is associated with increased risk of chronic diseases and aging. The ability to identify agents that can disrupt central and/or peripheral biological clocks, and agents that can prevent or mitigate the effects of circadian disruption, has significant implications for prevention of chronic diseases. Although rodent models can be used to identify exposures and agents that induce or prevent/mitigate circadian disruption, these experiments require large numbers of animals. In vivo studies also require significant resources and infrastructure, and require researchers to work all night. Thus, there is an urgent need for a cell-type appropriate in vitro system to screen for environmental circadian disruptors and enhancers in cell types from different organs and disease states. We constructed a vector that drives transcription of the destabilized luciferase in eukaryotic cells under the control of the human PERIOD 2 gene promoter. This circadian reporter construct was stably transfected into human mammary epithelial cells, and circadian responsive reporter cells were selected to develop the in vitro bioluminescence assay. Here, we present a detailed protocol to establish and validate the assay. We further provide details for proof of concept experiments demonstrating the ability of our in vitro assay to recapitulate the in vivo effects of various chemicals on the cellular biological clock. The results indicate that the assay can be adapted to a variety of cell types to screen for both environmental disruptors and chemopreventive enhancers of circadian clocks.

The circadian rhythm is a fundamental physiological process present in all organisms that regulates biological processes ranging from gene expression to sleep behavior. In vertebrates, circadian rhythm is controlled by a molecular oscillator that functions in both the suprachiasmatic nucleus (SCN; central pacemaker) and individual cells comprising most peripheral tissues. More importantly, disruption of circadian rhythm by exposure to light-at-night, environmental stressors and/or toxicants is associated with increased risk of chronic diseases and aging. The ability to identify agents that can disrupt central and/or peripheral biological clocks, and agents that can prevent or mitigate the effects of circadian disruption, has significant implications for prevention of chronic diseases. Although rodent models can be used to identify exposures and agents that induce or prevent/mitigate circadian disruption, these experiments require large numbers of animals. In vivo studies also require significant resources and infrastructure, and require researchers to work all night. Thus, there is an urgent need for a cell-type appropriate in vitro system to screen for environmental circadian disruptors and enhancers in cell types from different organs and disease states. We constructed a vector that drives transcription of the destabilized luciferase in eukaryotic cells under the control of the human PERIOD 2 gene promoter. This circadian reporter construct was stably transfected into human mammary epithelial cells, and circadian responsive reporter cells were selected to develop the in vitro bioluminescence assay. Here, we present a detailed protocol to establish and validate the assay. We further provide details for proof of concept experiments demonstrating the ability of our in vitro assay to recapitulate the in vivo effects of various chemicals on the cellular biological clock. The results indicate that the assay can be adapted to a variety of cell types to screen for both environmental disruptors and chemopreventive enhancers of circadian clocks.

Introduction Veterinarians use flumequine (FLU) widely but its toxicological effects are still unclear. Material and Methods FLU doses of 53, 200, or 750 mg/kg were administered orally for six weeks to pubertal male rats for evaluation of their toxicity. Results Weight gain was poorer after seven days of exposure to FLU 750, but relative weights of the brain, adrenal and thyroid glands, and testes were notably higher. Haematological and lipid profile parameters, cardiac markers, and inorganic phosphate significantly increased in the FLU 750 group. Blood glucose, oestradiol and serum concentrations of immunoglobulins G (IgG) and E (IgE) significantly decreased after treatment. The levels of interleukins 10 (IL-10) and 6 (IL-6) fell significantly in the FLU 200 and FLU 750 groups. Cytochrome P450, family 1, subfamily A, polypeptide 1 (CYP1A1) and cyclooxygenase-2 (Cox-2) expression amplified after treatment. Serum levels of free triiodothyronine (fT3) and free thyroxine (fT4) reduced in the FLU 200 and FLU 750 groups without changes in total T3 or T4 level. All doses of FLU significantly depressed concentrations of thyroid-stimulating hormone (TSH) and testosterone. Histopathology of thyroid glands from rats treated with FLU 750 showed degeneration and depletion of thyroid follicular epithelial cells. Expression of 8-hydroxydeoxyguanosine (8-OHdG) was increased in a dose-dependent manner in the brain, but decreased in the testes. Expression of CYP1A1 increased in the adrenal and pituitary glands. Conclusion The results of this study suggest that the toxicity of FLU in rats is an effect of its disruptive influence on the pituitary-thyroid hormonal system and on the dysfunction of the immune system.

Partially reprogrammed cells [preinduced pluripotent stem cells (pre-iPSCs)] commonly stall at epigenetic barriers, and this is one of the major failures in the reprogramming process. These cells can be converted to the fully reprogrammed state by reducing epigenetic blocks. In this study, we established three iPSC lines and two pre-iPSC lines induced by the doxycycline (dox)-inducible lentiviral system. In the pre-iPSC lines maintained under dox treatment (dox(+)), a small portion of embryonic stem cell (ESC)-like colonies spontaneously emerged after dox withdrawal (dox(-)), and major differentiation into fibroblast-like cells occurred. The spontaneous conversions based on the number of stage-specific embryonic antigen-1-positive (SSEA-1(+)) colonies were 0.006 ± 0.004% [mean ± standard deviation (SD)] for the #89-7D line and 0.016 ± 0.004% for the #102-2D line. The SSEA-1(+) colonies did not express the Nanog protein. However, the colonies showed characteristics typical of fully reprogrammed iPSCs after further expansion. To determine whether spontaneous conversion could be improved by epigenetic modification, we applied four small molecules-valproic acid (VPA), sodium butyrate (SB), trichostatin (TSA), and 5-aza-2'-deoxycytidine (5-Aza)-in both pre-iPSC lines. SB was the most effective molecule in enhancing the number of SSEA-1(+) colonies (32- to 39-fold) at day 5 of dox(-) treatment. In addition, the expression of pluripotent genes (sox2 and nanog) was increased by SB. After exposure to SB, we found that the expression of four reprogramming factors and cell cycle-related genes was relatively increased compared to their expression following dox(+) of pre-iPSCs. These changes caused by SB might play an important role in the spontaneous conversion of partially reprogrammed cells.

is Table S1. presenting a list of primer sets and Table S2. presenting antibody information. (DOCX 31 kb)

contains supplementary materials and methods presenting TRA-1-60 livestaining and Annexin V staining; Figure S1. showing the immunocytochemical phenotype stage on hepatic differentiation; Figure S2. showing the YM155 effect on stage I; Figure S3. showing expression of apoptosis-related genes (BIRC5 and BAX) during the whole hepatic differentiation of QIA7; Figure S4. showing Annexin V staining with QIA7, QIA7-iHeps (7Â days of differentiation) and hAT-SCs after YM155 treatment for 24Â h; Figure S5. showing the livestaining of TRA-1-60 at stage II (day 7 of differentiation) after YM155 treatment for 24Â h; and Figure S6. showing the histological analysis of teratomas derived from QIA7 and QIA7-iHeps without pretreatment of YM155. (DOCX 11505 kb)

Rats were administered zearalenone (ZEA) via gavage at dosages of 0, 1, 5, and 30 mg/kg for 36 days. On treatment day 8, inactivated porcine parvovirus vaccine (Vac) was injected intraperitoneally. Antibody production against porcine parvovirus was then measured as a function of ZEA treatment. Compared to the vaccine alone, ZEA treatment, with or without Vac, decreased the serum level of IgG. The level of IgM decreased in all ZEA groups at day 22, but the decrease was sustained only in the medium-dose ZEA group at day 36. The level of IgA was unchanged in the Vac only and ZEA groups at day 22, but was decreased in the 5 mg/kg ZEA plus Vac group compared to the Vac only group at day 36. The level of IgE was decreased by all doses of ZEA at day 22, but was unaffected in ZEA plus Vac groups compared to the Vac only group. The levels of IL-1 in the thymus and spleen; INF-γ in serum; IL-2, IL-6, and IL-10 in the thymus; and IL-10 and IFN-γ in the spleen decreased after ZEA administration. Furthermore, the levels of IL-1β in the spleen and mesenteric lymph node, IL-1β in the thymus, IL-2 in the thymus and spleen, IL-6 in the thymus, IL-10 and IFN-γ in the spleen, and GM-CSF and TNF-α in the thymus decreased after vaccination in rats exposed to ZEA. In conclusion, these results suggest that ZEA exposure via drinking water can cause an immunosuppressive effect by decreasing immunoglobulins in serum and cytokines in lymphoid organs.

The aim of this study was to identify whether chlorpyrifos methyl (CPM) exposure during pregnancy leads to changes in the methylation patterns of H19 gene. CPM 4, 20, 100 mg/kg bw/day was administered to 4 pregnant mice per group between 7 and 12 days post coitum (d.p.c.). Pregnant mice were killed at 13 d.p.c. The genomic methylation in primordial germ cells (PGCs) and fetal organs (the liver, intestine, and placenta) was examined. Four polymorphism sites in the H19 alleles of maternal (C57BL/6J) and paternal (CAST/Ei) alleles were identified at nucleotide position 1407, 1485, 1566, and 1654. The methylation patterns of 17 CpG sites were analyzed. The methylation level in male and female PGCs was not altered by CPM treatment in the maternal allele H19. The methylation level of the paternal H19 allele was altered in only male PGCs in response to the CPM treatment. The methylation level at a binding site for the transcriptional regulator CTCF2 was higher than that at the CTCF1 binding site in all CPM-treated groups. In the placenta, the aggregate methylation level of H19 was 56.89%in control group. But, those levels were ranged from 47.7% to 49.89% after treatment with increasing doses of CPM. H19 gene from the liver and intestine of 13 d.p.c. fetuses treated with CPM was hypomethylated as compared with controls, although H19 mRNA expression was unaltered. In the placenta, H19 expression was slightly increased in the CPM-treated group, although not significantly. IGF2 expression levels were not significantly changed in the placenta. In conclusion, CPM exposure during pregnancy alters the methylation status of the H19 gene in PGCs and embryonic tissues. We infer that these alterations are likely related to changes in DNA demethylase activity.

To clarify the role of stem cells in hepatocarcino- genesis, the expression of epithelial cell adhesion molecule (EpCAM) and proliferating cell nuclear antigen (PCNA) was investigated in mouse hepatic tumors and embryonic cell lineages. Ten ICR mice were treated with diethylnitrosamine (DEN) at 14 days of age and sacrificed at 36 weeks subsequent to DEN treatment to obtain the hepatic tumors. Mouse embry- onic stem cells, hepatic progenitor cells and hepatocyte‑like cells, representing 0, 22 and 40 days of differentiation, respec- tively, were treated in vitro with DEN at four doses (0, 1, 5 and 15 mM; G1, G2, G3 and G4, respectively) for 24 h and RNA was isolated. A total of 71 hepatic tumors were obtained from the DEN-treated mice. EpCAM expression was increased mainly in hepatic tumor cells, although it was also detected in the surrounding visually normal cells. Double staining showed that EpCAM and PCNA were co-expressed in numerous tumor cells. In vitro, EpCAM expression was significantly different for G4 at day 0 (P

Genomic analysis in the local lymph node assays (LLNAs) is useful for assessing skin sensitization of chemicals and providing insights into mechanisms of sensitization. In this study, we collected 1406 genes from previous microarray findings, validated changes in their expression by RT-PCR analysis in local lymph nodes draining skin exposed to different sensitizers, and interpreted their biological function through pathway-based genomic analysis, in which 468 genes were identified as being in the KEGG pathway database. The top-ranked functions (P

In vitro prediction of hepatotoxicity can enhance the performance of non-clinical animal testing for identifying chemical hazards. In this study, we assessed high-content analysis (HCA) using multi-parameter cell-based assays as an in vitro hepatotoxicity testing model using various hepatotoxicants and human hepatocytes such as HepG2 cells and human primary hepatocytes (hPHs). Both hepatocyte types were exposed separately to multiple doses of ten hepatotoxicants associated with liver injury whose mechanisms of action have been described. HCA data were obtained using fluorescence probes for nuclear size (Hoechst), mitochondrial membrane potential (TMRM), cytosolic free calcium (Fluo-4AM), and lipid peroxidation (BODIPY). Cellular alterations were observed in response to all hepatotoxicants tested. The most sensitive parameter was TMRM, with high sensitivity at a low dose, next was BODIPY, followed by Fluo-4AM. HCA data from HepG2 cells and hPHs were generally concordant, although some inconsistencies were noted. Both hepatocyte types showed mild or severe mitochondrial impairment and lipid peroxidation in response to several hepatotoxicants. The results demonstrate that the application of HCA to in vitro hepatotoxicity testing enables more efficient hazard identification, and further, they suggest that certain parameters could serve as sensitive endpoints for predicting the hepatotoxic potential of chemical compounds.

The toxicities of phenanthrene (PH) and pyrene (PY) are less than benzo (a) pyrene (BaP) , but both compounds are found in higher concentrations in the air, feed, and food. Most PAHs are metabolized to hydroxylated compounds by the hepatic cytochrome P450 monooxigenases system. Metabolites are excreted into urine and feces. We determined concentrations of PH, PY and BaP in muscle and hydroxylated metabolites, 3-OH-PH, 1-OH-PY, and 3-OH-BaP, respectively, in urine from dairy cattle (n = 24) . We also evaluated the relationship between parent compounds in muscle and their metabolites in urine. Concentrations of PH and PY in muscle ranged from 0.7~4.8 ng/g (1.8 ± 1.7) and 0.4~4.1 ng/g (1.2 ± 1.2) , respectively. Concentrations of 3-OH-PH and 1-OH-PY in urine ranged from 0.1~5.9 ng/ml (2.9 ± 3.7) and 0.5~3.6 ng/ml (1.9 ± 2.3) , respectively. Correlation coefficient for PY concentration in muscle versus 1-OH-PY in urine was 0.657 and for PH concentration in muscle versus 3-OH-PH in urine was 0.579.Coefficient determination for PY and PH concentrations in muscle was 0.886 and for 1-OH-PY and 3-OHPH in urine was 0.834. This study suggests that 1-OH-PY and 3-OH-PH could be used as biomarkers for PAHs exposure in dairy cattle.

Limb bud (LB) and central nerve system (CNS) cells were prepared from 12.5 day old pregnant female Crj:CD (SD) rats and treated with olaquindox and vitamin A. Cytotoxicity and inhibition on differentiation were measured in each cell. Three doses of olaquindox (4, 21 and 100 mgkg) , and 0.2 and 75 mg/kg of vitamin A were administered to pregnant rat for 11 days from 6(th) to 16(th) of pregnancy. IC50 values of olaquindox for proliferation and differentiation in CNS cell were 22.74 and 28.32 μg/ml and 79.34 and 23.29 μg/ml in LB cell and those values of vitamin A were 8.13 and 5.94 μg/ml in CNS cell and 0.81 and 0.05 μg/ml in LB cell, respectively. Mean body weights of pregnant rats were decreased at high dose of olaquindox (110 mg/kg) but relative ovary weight, number of corpus lutea, and number of implantation were not changed. Resorption and dead fetus were increased at high dose of olaquindox, and relative ovary weight, the number of corpus lutea and implantation, and sex ratio of male to female were not significantly changed in all dose of olaquindox. Mean fetal and placenta weights were significantly (p < 0.01) decreased in rats of high group. Seven fetuses out of 103 showed external anomaly like bent tail, and 10 out of 114 fetuses showed visceral anomalies at high group. The ossification of sternebrae and metacarpals were significantly (p < 0.01) increased by low and middle dose of olaquindox but it was significantly (p < 0.01) prohibited by high dose of olaquindox. In rats treated with vitamin A, the resorption and dead fetus were increased by high dose. Mean fetal weights were significantly (p < 0.01) increased by low dose but significantly (p < 0.01) decreased by high dose. Thirty four fetuses out of 52 showed external anomaly; bent tail (1) , cranioarchschisis (14) , exencephaly (14) , dome shaped head (22) , anophthalmia (15) , brcahynathia (10) and others (19) . Forty five fetuses out of 52 showed soft tissue anomaly; cleft palate (42/52) and anophthalmia (22/52) by high dose of vitamin A. Sixty one fetuses out of 61 (85.2%) showed skull anomaly; defect of frontal, partial and occipital bone (21/61) , defect of palatine bone (52/61) and others (50/61) . In summary, we support that vitamin A is strong teratogen based on our micromass and in vivo data, and olaquindox has a weak teratogenic potential in LB cell but not in CNS cell. We provide the in vivo evidence that a high dose of olaquindox could have weak embryotoxic potential in rats.

The time-dependent changes in cadmium (Cd) concentration were studied in Female Sprague-Dawley (SD) rats during and after Cd exposure via drinking water (10 and 50 ppm) for 30 days. The cadmium concentration in muscle, liver, kidney, blood plasma, and urine, and the metallothionein concentration in blood plasma were determined every 10 days during exposure and every 7 days after exposure for 3 weeks. The muscle Cd concentration did not change during, and neither after, exposure. The liver Cd concentration increased from 1.4 to 3.3 (at 10 ppm) and from 6.1 to 10.1 folds (at 50 ppm) during exposure and remained higher than those of controls in both groups even during post-exposure period. The kidney Cd concentrations were 2.3 to 5.1 (at 10 ppm) and 4.9-14.0 folds (at 50 ppm) higher than those of controls during exposure and also remained elevated during the post-exposure period. Plasma Cd concentrations were not significantly different from those of controls in both groups. Urine Cd concentrations were more than 2 folds (at 10 ppm) and 6.5 to 12.6 folds (at 50 ppm) higher than those of controls but rapidly decreased over the 7 days of withdrawal. Blood plasma metallothionein concentrations were more than 2.4 folds (at 10 ppm) and 3.1 to 7.4 folds (at 50 ppm) , and they remained elevated till 7 days (10 ppm) and 14 days (at 50 ppm) after exposure. Our data support that Cd in urine could be a useful biomarker during Cd exposure period and metallothionein in blood plasma could be as a supportive biological marker for during and post Cd exposure.

There has been some concern that certain non-sensitizing irritants may yield false positive results in the murine local lymph node assay (LLNA). This study compared gene expression profiles in lymph nodes draining skin following exposure to sensitizers and irritants, to identify gene transcripts that could distinguish sensitizers from irritants. After treating CBA/N mouse ears for 3 days with the sensitizers 1-chloro-2,4-dinitrobenzene, 2-phenyl-4-ethoxymethylene-5-oxazolone, or toluene-2,4-diisocyanate or the non-sensitizing irritants croton oil or nonanoic acid, auricular lymph nodes and ear tissues were excised. Sensitizer-induced changes in parameters such as ear thickness, lymph node weight, and cell count also occurred in irritant-treated mouse tissues. However, gene transcripts such as Ifi27, Il12rb1, Ifng, and Zbp1, which are related to T-cell activation, were shown by gene expression microarrays and real-time RT-PCR analyses to be up-regulated in auricular lymph nodes by sensitizers exclusively. These findings suggest that gene expression analysis may enable distinction between sensitizing chemicals and non-sensitizing irritants.

Deoxynivalenol (DON) is a common food borne mycotoxin and occurs predominantly in grains such as wheat, barley, oats, etc. DON induces systemic health problems such as loss of appetite, emesis and diarrhea in both human and farm animals. Reliable diagnostic parameters for DON intoxication are needed to prevent deep health impact. In order to establish useful diagnostic parameters, we investigated clinical signs, hematological values, serum biochemical values, gross-, histo- and toxico-pathological findings in B6C3F1 male mice after oral administration of DON (0.83, 2.5 and 7.5 mg/kg) for 8 days. Body weight gain was significantly decreased at the highest dose of DON. Anorexia, ataxia, for crudness and lack of vigor were observed at the highest dose DON group. In hematological values, the numbers of WBC and platelets and hemoglobin content were reduced with decreased neutrophil and monocytes by 7.5 mg/kg DON. Prothrombin time (PT) and activated partial thromboplastin time (aPTT) were prolonged in a dose-dependent manner and the content of fibrinogen was elevated at high dose of DON. Of serum biochemical values, total protein, globulin, BUN, cholesterol and test-osterone were reduced but total bilirubin and albumin/globulin ratio increased. The enzyme activity of alkaline phosphatase was decreased while that of alanine aminotransferase was elevated. Relative organ weights of thymus, seminal vesicle/prostate and testes were dose-dependently reduced but those of liver and left adrenal gland increased with dose dependency. As for pathological findings, atrophy of thymus, seminal vesicle/prostate and testes and submucosal edema and ulceration in stomach and depletion of lymphocytes in thymus cortex were observed. In conclusion, these clinical, hematological, blood biochemical and patholgical parameters obtained in the present studies can be used for diagnosis of DON-mycotoxicosis, especially, low WBC, platelets, protein, BUN and testosterone and delayed prothrombin time can be available as for reliable diagnostic parameters.

Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous environmental contaminants present in air and food. Among PAHs, benzo(a)pyrene(BaP), phenanthrene (PH) and pyrene (PY) are considered to be important for their toxicity or abundance. To investigate the changes of biomarkers after PAH exposure, rats were treated with BaP (150 microg/kg) alone or with PH (4,300 microg/kg) and PY (2,700 microg/kg) (BPP group) by oral gavage once per day for 30 days. 7-ethoxyresorufin-O-deethylase activity in liver microsomal fraction was increased in only BaP groups. The highest concentration (34.5 ng/g) of BaP, was found in muscle of rats treated with BaP alone at 20 days of treatment; it was 23.6 ng/g in BPP treated rats at 30 days of treatment. The highest PH concentration was 47.1 ng/g in muscle and 118.8 ng/g in fat, and for PY it was 29.7 ng/g in muscle and 219.9 ng/g in fat, in BPP groups. In urine, 114-161 ng/ml 3-OH-PH was found, while PH was 41-69 ng/ml during treatment. 201-263 ng/ml 1-OH-PY was found, while PH was 9-17 ng/ml in urine. The level of PY, PH and their metabolites in urine was rapidly decreased after withdrawal of treatment. This study suggest that 1-OH-PY in urine is a sensitive biomarker for PAHs; it was the most highly detected marker among the three PAHs and their metabolites evaluated during the exposure period and for 14 days after withdrawal.

Chlorpyrifos-methyl (CPM) suppressed androgenic activity in Hershberger assay using castrated rats. Acute oral lowest-observed-adverse-effect-level (LOAEL) and no-observed-adverse-effect-level (NOAEL) was evaluated as 12 and 0.1 mg/kg bw, respectively, based on its major effect of cholinesterase inhibition. Also, repeated oral NOAEL was 0.1 mg/kg bw/day based on adrenal damage in rats. We investigated one-generation reproductive toxicity of CPM focusing on endocrine-disrupting effects by the administration of 1, 10 and 100 mg/kg bw/day CPM to mature SD rats (F0) through pre-mating, mating, gestation and lactation period and to their offspring (F1) until 13 weeks age via gavage. A group treated with corn oil served as vehicle control. In F0 rats, the most affected organs were adrenal glands as increased in weight at all doses of CPM in males and at 10 and 100 mg/kg CPM in females and adrenal vacuolation at CPM 10 and 100 mg/kg. The relative and absolute ovaries and the absolute seminal vesicle weights were decreased but the weights of liver, spleen or kidneys were increased at 100 mg/kg CPM. Parameters representing reproductive performances as mating ratio, gestation length and delivery index were not affected, except for decreased fertility index and numbers of implantation and born pups and a higher male sex ratio of pups at CPM 100 mg/kg. F1 pups exposed to CPM 100 mg/kg in utero and via maternal milk showed lower body weight with changes of absolute or relative weights of brain, ovary, liver, spleen and epididymis and decreased absolute not relative anogenital distance at weanling time. The time of vaginal patency and preputial separation and estrous cycling pattern of F1 rats were not impacted by CPM. After further 10 weeks oral administration until 13 weeks old, adrenal glands, brain, liver, spleen or kidneys tended to be increased, while thyroid gland, testes and ventral prostate of F1 male rats were decreased at CPM 10 or 100 mg/kg. Histopathologically, necrosis or vacuolation of thyroid follicular epithelial cells and adrenal cortical cells were observed at all doses of CPM. Serum levels of estradiol, testosterone, T4 and T3 were significantly lower while TSH and cholesterol were higher in both F1 female and male rats treated with CPM though dose-responsiveness was not clear in F1 females. Decreased sperm were counted in F1 rats at CPM 100 mg/kg. As a whole, LOAEL and NOAEL was evaluated as 10 and 1 mg/kg bw, respectively, based on decreased estradiol and T4 and increased TSH in serum of F1 male rats, and when considering histopathological alteration of adrenal and thyroid glands, LOAEL assumed to be lower than 1 mg/kg bw. This study elucidates that CPM exhibit weak reproductive toxicity in F0 rats exposed at adulthood and negligible effects in F1 offspring exposed in utero and via lactation at weanling, but induce anti-androgenic effect and hypothyroidism after long term exposure from in utero through sexual maturation of F1 rats.

We evaluated the estrogenic and androgenic activity of butylated hydroxyanisole (BHA) using immature rat uterotrophic assay and Hershberger assay. To investigate (anti-) estrogenicity, BHA alone or with 17beta-estradiol was administered to 20-days-old immature female rats for three consecutive days. Absolute and relative uterine weights were significantly decreased by BHA (50, 100, 250, 500 mg/kg) alone and 17beta-estradiol-stimulated weights of uterine and vagina were also decreased by BHA (500 mg/kg), while uterine epithelial cell height was not affected. In Hershberger assay, BHA alone or with testosterone propionate (TP) was administered daily to 51-days-old castrated male rats for 10 days. BHA alone or with testosterone propionate (TP) caused no significant effect on androgen-dependent accessory sex organ weights; seminal vesicle/coagulative glands, glans penis, Cowper's gland, ventral prostate gland and levator ani plus bulbocarvernosus muscle. Although, the relative weight of ventral prostate gland was increased by the co-treatment of BHA 250 mg/kg with TP 0.4 mg/kg compared to that of TP alone, the relative and absolute weights of other androgen-dependent organs and absolute and formalin-fixed ventral prostate gland weight showed no changes. Our studies suggest that BHA have anti-estrogenic activity for the decrease of uterine weight in immature female rat but have negligible effect on the androgenic activity in castrated male rats.

Chlorpyrifos-methyl (CPM), an organophosphate insecticide, widely used for grain storage and agriculture, has been suspected as endocrine disrupter by a few in vitro studies. This study was performed to investigate the (anti-) estrogenicity and (anti-) androgenicity of CPM in vivo using immature rat uterotrophic assay and rat Hershberger assay. CPM with or without 17beta-estradiol were administered to 20 days old female rats to investigate its (anti-) estrogenic activity. Uterine and vaginal weight, uterine epithelial cell height were not affected by the treatment of CPM (2, 10, 50, 250 mg/kg). CPM 250 mg/kg potentiated relative vagina weight in 17beta-estradiol treated immature female rats without any changing of uterine weight. Relative liver weight was increased with decrease of body weight by CPM 250 mg/kg treatment. Uterine cell proliferation tested with bromodeoxyuridine labeling index was not observed in CPM treated rats. CPM with or without testosterone propionate were administered to castrated rat of 51 days old for 10 days to investigate the (anti-)androgenic activity,. The weight of relative and absolute androgen-dependent accessory sex organs; seminal vesicle with coagulating glands (SV/CG), ventral prostate gland (VP), glans penis (GP), levator ani plus bulbocarvernosus muscle (LABC) and Cowper's gland (CG,) were unchanged by the treatment of CPM alone. While CPM induced the increase of relative adrenal gland weight, CPM 50mg/kg decreased the weights of CV/CG, VP, CG and LABC without change of GP without changing of GP when it was treated with TP. In conclusion, CPM dose not show estrogenic and anti-estrogenic activity in immature female rats, but it represents anti-androgenic activity by inhibition of the TP-stimulated increase of the weight of accessory sex organs.

In the present study, we differentiated hepatocyte-like cells (HLCs) from human adipose tissue-derived mesenchymal stem cells (AT-MSCs). The hepatic differentiation was confirmed by increases in hepatic proteins or genes, the cytochrome P450 (CYP) activities, albumin secretion, and glycogen storage. To determine the developmental toxic effect of arsanilic acid (Ars) and acetaminophen (AAP) on the hepatic development, the differentiating cells were treated with the test chemicals (below IC12.5) from day 4 to day 13. The enzymatic activities of lactate dehydrogenase (LDH), alanine aminotransferase (ALT), and aspartate aminotransferase (AST) did not significantly differ in response to Ars treatment. AAP treatment increased the activities of all enzymes in a dose-dependent manner, significantly at concentrations of 2.5 and 5 mM of AAP. On the expressions of hepatic genes for Ars, the expressions were significantly inhibited by more than 0.5 mM for Albumin (ALB), but only 2.5 mM for α-feto protein (AFP). In the AAP-treated group, the expressions of ALB and AFP were significantly decreased at the concentrations exceeding 0.625 mM. The activities of CYP3A4 were not changed by both treatments. The activities of CYP1A2 were increased by AAP, whereas it was decreased by Ars treatment. In conclusion, AAP could cause serious adverse effects during the hepatic development as compared to Ars.

To elucidate the effect on the H19 gene methylation of sperm and organs in offspring by chlorpyrifos-methyl (CPM) exposure during organogenesis period, CPM was administered at doses of 4 (CPM4), 20 (CPM20), and 100 (CPM100) mg/kg bw/day from 7 days post coitum (d.p.c.) to 17 d.p.c. after mating CAST/Ei (♂) and B6 (♀). Anogenital distance (AGD) was measured at postnatal day (PND) 21. Clinical signs, body weights, feed and water consumption, organs weights, serum hormone values, and H19 methylation level of organ and sperm were measured at PND63. Body weights were significantly lower than control until PND6. AGD was significantly decreased in the CPM100 group in males and increased in the CPM20 group in females. The absolute weights of the thymus and epididymis were significantly increased for males in all of CPM treatment groups. In the CPM20 group, absolute weights of liver, kidney, heart, lung, spleen, prostate gland, and testes were significantly increased. Testosterone concentrations in serum were significantly increased by CPM treatment in males. H19 methylation level of liver and thymus showed decreased pattern in a dose-dependent manner in males. The levels of H19 methylation in sperm were 73.76 ± 7.16% (Control), 57.84 ± 12.94% (CPM4), 64.24 ± 3.79% (CPM20), and 64.24 ± 3.79% (CPM100). Conclusively, CPM exposure during organogenesis period can disrupt H19 methylation in sperm, liver, and thymus and disturb the early development of offspring.

This study was performed to assess the neurotoxic effects of methylmercury, arsanilic acid and danofloxacin by quantification of neural-specific proteins in vitro. Quantitation of the protein markers during 14 days of differentiation indicated that the mouse ESCs were completely differentiated into neural cells by Day 8. The cells were treated with non-cytotoxic concentrations of three chemicals during differentiation. Low levels of exposure to methylmercury decreased the expression of GABAA-R and Nestin during the differentiating stage, and Nestin during the differentiated stage. In contrast, GFAP, Tuj1, and MAP2 expression was affected only by relatively high doses during both stages. Arsanilic acid affected the levels of GABAA-R and GFAP during the differentiated stage while the changes of Nestin and Tuj1 were greater during the differentiating stage. For the neural markers (except Nestin) expressed during both stages, danofloxacin affected protein levels at lower concentrations in the differentiated stage than the differentiating stage. Acetylcholinesterase activity was inhibited by relatively low concentrations of methylmercury and arsanilic acid during the differentiating stage while this activity was inhibited only by more than 40 µM of danofloxacin in the differentiated stage. Our results provide useful information about the different toxicities of chemicals and the impact on neural development.

In this study, we developed a novel tool for purifying two mycotoxins, aflatoxin B1 (AFB1) and zearalenone (ZEN), in feed. This system utilized monoclonal antibodies (mAbs) against AFB1 and ZEN, and magnetic nanoparticles (MNPs). Among ten MNPs with different diameters and functional groups, a 100-nm diameter MNP (fMA) conjugated to an amine group (-NH2) was found to be optimum for coupling with mAbs. The optimal mAb concentrations for coupling to the fMA along with mycotoxin purification capacities of the fMA-mAb conjugates (fMA-AFB1 and fMA-ZEN) were determined. A comparison of mean recovery rates (from corn and product X feed) between the fMA-mAb conjugates and immunoaffinity columns (IAC-AFB1 and IAC-ZEN) showed that the rate for fMA-AFB1 (90~92% and 81~88%) was higher (p > 0.05) than that of IAC-AFB1 (81~84% and 72~78%) for AFB1 (5, 10, 15 ng/mL), and the rate for fMA-ZEN (99~100% and 92~94%) was significantly higher (p < 0.01) than that of IAC-ZEN (86~88% and 81~88%) for ZEN (10, 25, 50 ng/mL) except at a concentration of 10 ng/mL, demonstrating the remarkable purification efficiency of the novel fMA-mAb method. Additionally, mycotoxin purification was much faster using our novel method (approx. 5 min) than the IAC-based technique (> 30 min). This study suggests that the novel purification system we developed would be a useful tool for monitoring and regulating mycotoxin contamination in feed, and replace IAC methods.

Mice were exposed to deoxynivalenol (DON) via drinking water at a concentration of 2 mg/L for 36 days. On day 8 of treatment, inactivated porcine parvovirus vaccine (PPV) was injected intraperitoneally. The relative and absolute weight of the spleen was significantly decreased in the DON-treated group (DON). Antibody titers to parvovirus in serum were 47.9 ± 2.4 in the vaccination group (Vac), but 15.2 ± 6.5 in the group treated with DON and vaccine (DON + Vac). The IgA and IgG was not different in the DON, Vac an,d DON + Vac groups. IgM was significantly lower only in the DON + Vac group. However IgE was significantly increased in the Vac and DON + Vac group, but no change was observed between the Vac and DON + Vac groups. The concentrations of IL-2, IL-4, GM-CSF, MCP-1 and Rantes in serum, and IL-1α in mesenteric lymph node and MIP-1β in spleen were significantly increased by DON treatment compared to control. The concentrations of IL-2, IL-5, IL-6, IL-9, IL-12, IL-13 and Rantes in thymus, of IL-2 in spleen, and of IL-1α, IL-1β, IL-3, IL-5, IL-10, IL-17, G-CSF, GM-CSF and MCP-1 in mesenteric lymph nodes were significantly decreased in mice compared to those in the Vac group, while concentrations of IL-1α, IL-2, IL-9, IL-13,G-CSF, GM-CSF, IFN-γ, MCP-1, MIP-1α and TNF-α were significantly increased in serum compared to the Vac group. In conclusion, the results presented here indicate that exposure to DON at 2.0 mg/L via drinking water can disrupt the immune response in vaccinated mice by modulating cytokines and chemokines involved in their immune response to infectious disease.

The aim of this study was to identify whether chlorpyrifos methyl (CPM) exposure during pregnancy leads to changes in the methylation patterns of H19 gene. CPM 4, 20, 100 mg/kg bw/day was administered to 4 pregnant mice per group between 7 and 12 days post coitum (d.p.c.). Pregnant mice were killed at 13 d.p.c. The genomic methylation in primordial germ cells (PGCs) and fetal organs (the liver, intestine, and placenta) was examined. Four polymorphism sites in the H19 alleles of maternal (C57BL/6J) and paternal (CAST/Ei) alleles were identified at nucleotide position 1407, 1485, 1566, and 1654. The methylation patterns of 17 CpG sites were analyzed. The methylation level in male and female PGCs was not altered by CPM treatment in the maternal allele H19. The methylation level of the paternal H19 allele was altered in only male PGCs in response to the CPM treatment. The methylation level at a binding site for the transcriptional regulator CTCF2 was higher than that at the CTCF1 binding site in all CPM-treated groups. In the placenta, the aggregate methylation level of H19 was 56.89%in control group. But, those levels were ranged from 47.7% to 49.89% after treatment with increasing doses of CPM. H19 gene from the liver and intestine of 13 d.p.c. fetuses treated with CPM was hypomethylated as compared with controls, although H19 mRNA expression was unaltered. In the placenta, H19 expression was slightly increased in the CPM-treated group, although not significantly. IGF2 expression levels were not significantly changed in the placenta. In conclusion, CPM exposure during pregnancy alters the methylation status of the H19 gene in PGCs and embryonic tissues. We infer that these alterations are likely related to changes in DNA demethylase activity.

Embryonic stem cell testing is an alternative model system to assess drug and chemical toxicities because of its similar developmental characteristics with in vivo embryogenesis and organogenesis. This study evaluated the toxicity of chemicals at specific developmental stages of mouse embryonic stem cell (ESC)-derived hepatic differentiation; hepatic progenitor cells (HPCs), and hepatocyte-like cells (HCs). The toxic effects of carbon tetrachloride (CCl(4)), 5-fluorouracil (5-FU), and arsanilic acid (Ars) were evaluated by measuring the expressions of Cytokeratin (CK18) and GATA binding protein 4 (GATA-4) and the activities of aspartate transaminase (AST), lactate dehydrogenase (LDH), and alkaline phosphatase (ALP) during the hepatic differentiation process. Non-toxic doses of three chemicals at a range of 25 to 500 μM for CCl(4), 12.5 to 800 nM for 5-FU and 6.25 to 400 mM for Ars were treated. In the CCl(4)-treated group, significant decreases (P 0.05) of the marker expression were observed by more than 300 μM from day 10 in CK18 and by more than 400 μM of CCl(4) from day 22 in GATA-4, respectively. However, both markers were decreased (P 0.01) by treatments of all doses at day 40. In the 5-FU-treated group, the expressions of two proteins were not affected by any of the doses at day 10 and 22, whereas the GATA-4 expression was decreased (P 0.05) by more than 400 nM of 5-FU at days 28 and 40. In the Ars-treated group, the CK18 expression was inhibited (P 0.05) by more than 100 mM of Ars at day 22 but showed a tendency to recover. Although the GATA-4 was inhibited by all doses at day 22, the inhibition of GATA-4 recovered at days 28 and 40. ALP activities of three chemicals were significantly increased (P 0.05) by a dose-dependent manner. The activities of AST and LDH were prone to be increased by more than 300 μM of CCl(4,) but not affected by all doses of 5-FU except for 800 nM of 5-FU in AST activities. In the Ars, the enzyme activities were significantly increased (P 0.05) by more than 50 μM of Ars in AST and more than 6.25 μM of Ars in LDH. The present results indicate that CCl(4) has a more toxic effect on HCs, whereas Ars is more toxic to HPCs. Additionally, in vitro alternative testing using ESC-derived HPCs and HCs could provide useful information on chemical toxicity during the hepatic differentiation process and could be a useful model system for assessing chemical hepatotoxicity.

Through the present study, we produced a monoclonal antibody against aflatoxin B1 (AFB1) using AFB1- carboxymethoxylamine BSA conjugates. One clone showing high binding ability was selected and it was applied to develop a direct competitive ELISA system. The epitope densities of AFB1-CMO against BSA and KLH were about 1 : 6 and 1 : 545, respectively. The monoclonal antibody (mAb) from cloned hybridoma cell was the IgG1 subclass with λ-type light chains. The IC50s of the monoclonal antibody developed for AFB1, AFB2, AFG1 and AFG2 were 4.36, 7.22, 6.61 and 29.41 ng/ml, respectively, based on the AFB1-KLH coated ELISA system and 15.28, 26.62, 32.75 and 56.67 ng/ml, respectively, based on the mAb coated ELISA. Cross-relativities of mAb to AFB1 for AFB2, AFG1 and AFG2 were 60.47, 65.97 and 14.83% in the AFB1-KLH coated ELISA, and 59.41, 46.66 and 26.97% in the mAb coated ELISA, respectively. Quantitative calculations for AFB1 from the AFB1-Ab ELISA and AFB1-Ag ELISA ranged from 0.25 to 25 ng/ml (R(2) > 0.99) and from 1 to 100 ng/ml (R(2) > 0.99), respectively. The intra- and inter-assay precision CVs were < 10% in both ELISA assay, representing good reproducibility of developed assay. Recoveries ranged from 79.18 to 91.27%, CVs ranged from 3.21 to 7.97% after spiking AFB1 at concentrations ranging from 5 to 50 ng/ml and following by extraction with 70% methanol solution in the Ab-coated ELISA. In conclusion, we produced a group specific mAb against aflatoxins and developed two direct competitive ELISAs for the detection of AFB1 in feeds based on a monoclonal antibody developed.

Genomic analysis in the local lymph node assays (LLNAs) is useful for assessing skin sensitization of chemicals and providing insights into mechanisms of sensitization. In this study, we collected 1406 genes from previous microarray findings, validated changes in their expression by RT-PCR analysis in local lymph nodes draining skin exposed to different sensitizers, and interpreted their biological function through pathway-based genomic analysis, in which 468 genes were identified as being in the KEGG pathway database. The top-ranked functions (P 0.01) identified as being affected by the sensitizers were associated with aspects of cell growth, such as DNA replication, cell cycle regulation and pyrimidine metabolism. All the sensitizers tested (DNCB, OXA and TDI) induced significant up-regulation of Psme4, which is associated with DNA replication; Tfdp1, which is related to cell cycle regulation; and Dut, which is involved in pyrimidine metabolism. Specific changes were also shown in functional categories related to the immune response, including cytokines and their receptors. Genes identified in these functional categories, such as Ccl21c, Cxcl9, Cxcl10, Ifng and Il12rb1, were found to have functional relevance. These findings may enhance our understanding and assessment of chemical sensitizers, and enable us to distinguish sensitizers from irritants and to classify chemicals as contact sensitizers.

The present study was conducted to investigate the antimutagenic potential of the methanolic extract from the leaves of sweet potato (Ipomea batatas, IB) with the SOS chromotest (umu test) and Salmonella typhimurium TA 98 and TA 100. The anticarcinogenic effects were also studied by calculation of the IC50 on human cancer cell lines and investigating the function of gap junction in rat liver epithelial cells. The IB extract inhibited dose-dependently the β-galactosidase activity induced spontaneously at concentration of more than 200 mg/ml in S. typhimurium TA 1535/pSK 1002, and decreased significantly (p < 0.01) the β-galactosidase activities induced by mutagen 6-chloro-9-[3- (2-chloroethylamino) proylamino]-2-methoxyacridine dihydrochloride (ICR) at dose of more than 0.4 mg/0.1 ml. The IB extract showed no effect on the spontaneous reversions of S. typhimurium TA 98 and 100 but benzo (α) pyrene (BaP) -stimulated reversions were decreased dose-dependently (p < 0.01) at the concentration of more than 100 mg/ml. The IC50 value of stomach cancer cells was lower than that of normal rat liver epithelial cells, but the values of colon and uterine cancer cell lines were similar to those of normal rat liver epithelial cells. The transfer of dye through gap junctions was not affected by treatment of the IB extracts at any concentration during treatment periods. The simultaneously treatment of IB extract and 12-O-tetradecanoylphorbol-13-acetate (TPA) effectively prevented the inhibition of dye transfer induced by TPA 1 hour after treatment at all exposed concentrations. The number of gap junctions was significantly (p < 0.01) increased by the treatment with IB extract at concentrations of more than 40 μg/ml. The inhibition of the expression of gap junction proteins by TPA (0.01 μg/ml) was recovered dose dependently by the simultaneous treatment of IB extracts. Our data suggest that Ipomea batatas has antimutagenic and anticarcionogenic activity in vitro.

The time-dependent changes in lead (Pb) concentrations in major tissues, serum and urine, and the Pb biomarker δ-aminolevulinic acid (ALA) concentration in urine were studied in rats after sub-chronic Pb exposure. Female Sprague-Dawley (SD) rats were exposed to Pb in drinking water at concentrations of 100 ppm and 1000 ppm for 30 days. The Pb concentration in muscle, liver, kidney, plasma and urine, and the ALA concentration in urine were determined during exposure and every 7 days after exposure for 3 weeks. The muscle Pb concentration did not change post exposure. The liver Pb concentration increased 2.2 to 2.8 times (100 ppm group) and 3.9 to 7.4 times (1000 ppm group) during exposure, then decreased rapidly. Kidney Pb concentrations were 8.0 to 14.3 times (100 ppm group) and 13.8 to 28.5 times (1000 ppm group) higher than controls during exposure and decreased for 1 to 2 weeks post exposure. Plasma Pb concentrations were 1.2 to 3.3 times (100 ppm group) and 2.9 to 5.8 times (1000 ppm group) higher than control concentrations during exposure and decreased time-dependently in the 1000-ppm group after exposure. Urine Pb concentrations were 8.5 to 10.7 times (100 ppm group) and 30.4 to 51.1 times (1000 ppm group) higher than control concentrations during exposure and rapidly decreased after exposure, though concentrations remained up to 4 times higher than controls in the 1000 ppm exposure group. Urine ALA concentrations increased 1.7 to 2.6 and 7.1 to 32.7 times during exposure in the 100 ppm and 1000 ppm groups respectively, and remained elevated for 21 days post exposure. Our data support that urine Pb concentration is a useful marker for acute Pb exposure or post exposure. Urine ALA may be a predicator of biological response to Pb exposure.