Toxicological Evaluation of Flumequine in Pubertal Male Rats After Oral Administration for Six Weeks.

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.

Evaluation of hepatotoxicity of chemicals using hepatic progenitor and hepatocyte-like cells derived from mouse embryonic stem cells: effect of chemicals on ESC-derived hepatocyte differentiation.

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.

Antimutagenic and anticarcinogenic effect of methanol extracts of Petasites japonicus Maxim leaves.

The methanol extract from the leaves of Petasites japonicus Maxim (PJ) was studied for its (anti-)mutagenic effect with the SOS chromotest and reverse mutation assay. The (anti-)carcinogenic effects were evaluated by the cytotoxicity on human cancer line cells and by the function and the expression of gap junctions in rat liver epithelial cell. PJ extracts significantly decreased spontaneous beta-galactosidase activity and beta-galactosidase activity induced by a mutagen, ICR, in Salmonella (S.) typhimurium TA 1535/pSK 1002. All doses of the extract (0.08-100 mg/plate) decreased the reversion frequency induced by benzo (alpha)pyrene (BaP) in S. typhimurium TA 98. It decreased not only the spontaneous reversion frequency but also that induced by BaP in S. typhimurium TA 100. PJ extract showed greater cytotoxic effects on human stomach, colon and uterus cancer cells than on other cancer cell types and normal rat liver epithelial cells. Dye transfers though gap junctions were significantly increased by PJ extracts at concentrations greater than 200 microg/mL and the inhibition of dye transfer by 12-O-tetradecanoylphorobol-13-acetate (TPA) was obstructed in all concentrations of PJ. PJ significantly increased the numbers of gap junction protein connexin 43, and increased the protein expression decreased by TPA in a dose-dependent manner. Based on these findings, PJ is suggested to contain antimutagenic and anticarcionogenic compounds.

Antimutagenic and Anticarcinogenic Effect of Methanol Extracts of Sweetpotato (Ipomea batata) Leaves.

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 ß-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 ß-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.

Evaluation of embryotoxic potential of olaquindox and vitamin a in micromass culture and in rats.

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.

Time-dependent changes in lead and delta-aminolevulinic acid after subchronic lead exposure in rats.

The time-dependent changes in lead (Pb) concentrations in major tissues, serum and urine, and the Pb biomarker delta-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.

Gene expression profiles and pathways in skin inflammation induced by three different sensitizers and an irritant.

It is often difficult to discriminate between chemically induced skin irritation and sensitization due to their similar clinical, pathological, and immunological responses. More information than that currently available from local lymph node assays (LLNAs), such as data from gene expression and pathway analysis, can provide more insightful data than the assay itself for distinguishing skin sensitization from skin irritation. This study investigated the gene expression profiles and pathways in ear skins of mice topically exposed daily for three consecutive days to the known strong contact sensitizer 1-chloro-2,4-dinitrobenzene, the skin contact sensitizer 2-phenyl-4-ethoxymethylene-5-oxazolone, the skin or respiratory sensitizer toluene 2,4-diisocyanate, or to the non-sensitizing irritant croton oil. All the sensitizers induced histological changes in ear tissues similar to those induced by the croton oil. In gene expression microarrays, sensitizers up-regulated 193 genes and down-regulated 61 genes in ear skin following chemical exposure. 13 genes whose expression was affected by more than two-fold by all three of the sensitizers, but not by the irritant, were selected by microarray analysis. Microarray and real-time RT-PCR analyses revealed that, of these genes, the allergic inflammation-related genes Oasl2 and Zbp1 were up-regulated in skin inflammation by the sensitizers. In gene expression pathway analysis of all the sensitizers and the croton oil, the top functions of the 48 genes were related to cytokine and cytokine receptors interactions, and only two genes (Cxcl9 and Cxcl10) were specific to skin sensitizer-induced skin inflammation. Thus, although contact sensitizer-induced skin inflammation is similar to irritant-induced responses in terms of histological changes and gene expression profiles, the regulation of allergic inflammation-related gene transcripts, such as those of Oasl2 and Zbp1 or Cxcl9 and Cxcl10, could help to discriminate skin sensitization from chemically induced skin inflammation.

Risk assessment of ciprofloxacin, flavomycin, olaquindox and colistin sulfate based on microbiological impact on human gut biota.

Trace levels of veterinary antibiotics that reside in livestock products may disturb the balance of human intestinal microbiota and impair the colonized barrier function, which is critical to protect against the invasion or overgrowth of exogenous pathogens. We investigated the colonization barrier disruption effect of ciprofloxacin, flavomycin, olaquindox and colistin sulfate by the minimum inhibitory concentration (MIC) assay in pure culture of human gut bacteria and evaluated the no-observed-effect-concentration (NOEC) and acceptable daily intake (ADI) based on the microbiological impact. MICs of the antibiotics were tested for total 100 isolates composed of 10 isolates from each of 10 predominant genera of human faeces that were freshly collected from healthy women at 1x10(5) and 1x10(9) colony-forming units (CFU)/ml. MIC assay was also conducted with 10 ATCC standard bacteria species of human fecal microbiota for the comparison with freshly isolated human fecal mirobiota. The most susceptible bacteria were Escherichia coli for ciprofloxacin and colistin sulfate, Fusobacterium spp. for flavomycin and Eubacterium spp. for olaquindox. MIC values were lower at 1x10(5) than at 1x10(9)CFU/ml. The susceptibility of feacal microbiota freshly isolated from healthy women tended to be similar with those of ATCC standard bactera. NOEC (microg/ml) and ADI (microg/kg BW/day) were evaluated as 0.008 and 0.15 for ciprofloxacin, 0.25 and 1 for flavomycin, 0.125 and 3 for olaquindox and 1.0 and 7 for colistin sulfate, respectively.

Plasma haptoglobin and immunoglobulins as diagnostic indicators of deoxynivalenol intoxication.

This study aimed to discover potential biomarkers for dioxynivalenol (DON) intoxication. B6C3F1 male mice were orally exposed to 0.83, 2.5 and 7.5 mg/kg body weight (bw) DON for 8 days and the differential protein expressions in their blood plasma were determined by SELDI - Time-of-Flight/Mass Spectrometry (TOF/MS) and the immunoglobulins (Igs) G, A, M and E in the serum were investigated. 11.7 kDa protein was significantly highly expressed according to DON administration and this protein was purified by employing a methyl ceramic HyperD F column with using optimization buffer for adsorption and desorption. The purified protein was identified as a haptoglobin precursor by peptide mapping with using LC/Q-TOF/MS and MALDI-TOF/MS and this was confirmed by western blotting and ELISA. IgG and IgM in serum were decreased in a dose-dependent manner and IgA was decreased at 7.5 mg/kg bw DON administration, but the IgE level was not changed. To compare the expressions of haptoglobin and the Igs patterns between aflatoxin B1 (AFB1), zearalenone (ZEA) and DON intoxications, rats were orally administered with AFB1 1.0, ZEA 240 and DON 7.5 mg/kg bw for 8 days. Haptoglobin was increased only at DON 7.5 mg/kg bw, while it was slightly decreased at ZEA 240 mg/kg bw and it was not detected at all at AFB1 1.0 mg/kg bw. IgG and IgA were decreased by DON, but IgG, IgA, IgM and IgE were all increased by AFB1. No changes were observed by ZEA administration. These results show that plasma haptoglobin could be a diagnostic biomarker for DON intoxication when this is combined with examining the serum Igs.

Intracellular expression of cytokines and granzyme B in auricular lymph nodes draining skin exposed to irritants and sensitizers.

The murine local lymph node assay (LLNA) has been extensively utilized to evaluate sensitizing chemicals. However, there have been some concerns that its use to discriminate between classes of chemicals is minimal. It is thus desirable to identify better or alternative immune endpoints with in LLNA itself. Here, we evaluated the protein and/or mRNA levels of cytokines and granzyme B (GzmB), a cytotoxic lymphocyte product, to discriminate between sensitizers and irritants and to characterize the chemical sensitizers when used as supplemental indicators in LLNA endpoints. For this, CBA/N mice were topically treated daily with a well-known chemical sensitizer such as a strong contact sensitizer 1-chloro-2,4-dinitrobenzene (DNCB), a skin contact sensitizer 2-phenyl-4-ethoxymethylene-5-oxazolone (OXA), and a skin or respiratory sensitizer toluene 2,4-diisocyanate (TDI), and the non-sensitizing irritants, croton oil (CRO) and nonanoic acid (NA), for 3 consecutive days. The protein and/or mRNA levels in auricular lymph nodes draining the ear skin were then analyzed by real-time RT-PCR and immunoassay. The sensitizers, but not the irritants, evoked pronounced interleukin (IL)-2, IL-3 and IL-4 or interferon (IFN)-gamma. Significantly, different sensitizers evoked different cytokine patterns of IL-4 and IFN-gamma, as DNCB strongly up-regulated both IFN-gamma and IL-4, OXA up-regulated IFN-gamma strongly but IL-4 weakly, and TDI up-regulated IL-4 strongly but IFN-gamma weakly. The sensitizers also strongly up-regulated GzmB mRNA, while the irritants had a much weaker effect. Thus, these cytokines and GzmB mRNA may be useful as additional endpoints for discriminating between irritants and sensitizers or contact and respiratory sensitizers in the LLNA.

Analysis of differential gene expression in auricular lymph nodes draining skin exposed to sensitizers and irritants.

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.

Changes of biomarkers with oral exposure to benzo(a)pyrene, phenanthrene and pyrene in rats.

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.

Effect of chlorpyrifos-methyl on steroid and thyroid hormones in rat F0- and F1-generations.

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.

Evaluation of estrogenic and androgenic activity of butylated hydroxyanisole in immature female and castrated 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.

Rapid bioassay for the determination of dioxins and dioxin-like PCDFs and PCBs in meat and animal feeds.

Over the past several years, the numerous contamination incidents have raised concerns over the presence of halogenated aromatic hydrocarbons, such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), and related chemicals in foods and feeds. Here we applied a sensitive recombinant mouse hepatoma cell (H1L1.1c2) bioassay for the determination of dioxins and dioxin-like polychlorinated dibenzofurans (PCDFs) and biphenyls (PCBs) in meat and animal feeds. These cells responded to TCDD-like chemicals with dose-dependent induction of firefly luciferase activity, and the minimal detection limit of TCDD in the cell was 16 fg. Induction equivalency factors determined for pure TCDD-like polychlorinated dibenzo-p-dioxins (PCDDs), PCDFs, and PCBs in the bioassay were well-correlated with the World Health Organization's toxic equivalency factors. To determine the applicability of the bioassay system to detect those compounds presence in meat and feed samples, cell bioassays for 17 TCDD-like PCDDs and PCDFs congeners-spiked lipid extracted from beef or animal feed were performed. Mean recoveries of TCDD-like chlorinated PCDDs and PCDFs congeners from spiked beef or feed fat ranged from 61.2 to 122.3%. Within-laboratory coefficients of variation for analysis as index of precision were lower than 5.2%, and the calculated limits of detection and quantitation were 0.33 and 1 pg toxicity equivalency quantity (TEQ)/0.5 g fat, respectively. Correlation between bioassay- and high-resolution gas chromatography-mass spectrometry (HR-GC-MS)-determined TEQs for 10 meat samples was 0.85, with 1.2 times higher in bioassay than HR-GC-MS. The correlation between bioassay- and HR-GC-MS-determined TEQs in 10 animal feed products was 0.81, with 2.1 times higher in bioassay than HR-GC-MS. Overall, these results demonstrated that the recombinant cell bioassay can be used for the rapid detection and quantitation of PCDDs and dioxin-like PCDFs and PCBs in meats and animal feeds.

Effects of butylated hydroxyanisole on the development and functions of reproductive system in rats.

Butylated hydroxyanisol (BHA) is a widely used antioxidant for long preservation of food products, cosmetics and pharmaceuticals. Although BHA is generally recognized as safe, it is classified as a suspected endocrine-disrupting compound. We investigated the effects of BHA on reproductive function and development by the treatment of mature male and female SD rats (F0) through pre-gestation, gestation and lactation period and of their offspring (F1) until 13 weeks old via gavage with BHA 0 (corn oil, vehicle control), 10, 100 and 500 mg/kg bw/day. Organ weights of liver, adrenal gland and thyroid gland of F0 rats were increased by BHA 500 mg/kg but those of spleen and ventral prostate were decreased without significant difference in terminal body weight. Reduced serum testosterone and thyroxine (T4) were observed with dose-dependent manner in F0 male rats. Mating rate was decreased and cohabitation duration for conception was longer without differences in the number, motility and morphology of sperm by BHA 500 mg/kg. Body weight of F1 offspring was significantly decreased with change of relative weight of liver and brain by BHA 500 mg/kg at PND21. Sexual maturation indicated by vaginal opening and preputial separation was delayed by BHA 500 mg/kg. The weights of liver and adrenal gland were increased while those of spleen, vagina, testes and ventral prostate were decreased in F1 rats exposed to BHA 100 or 500 mg/kg for 13 weeks. Also, BHA 500 mg/kg reduced the velocity of sperm motion and number with smaller-sized sperm head in F1 male rats and slightly shortened estrous cycle length with higher frequency of estrus and lower frequency of diestrus stages in F1 female rats. Lower serum T4 and testosterone contents with higher serum cholesterol levels were also observed by BHA 500 mg/kg. Increased follicular cell height, and exfoliated and vacuolated follicular epithelial cells were observed in thyroids of F1 female and males rats exposed to BHA 500 mg/kg. This study elucidates that high dose of BHA induce weak dysfunction and underdevelopment of reproductive system of male and female rats with the change of T4 and testosterone levels, sex organ weights and sexual maturation and histological lesions of thyroid gland.

Chlropyrifos-methyl shows anti-androgenic activity without estrogenic activity in 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.

Changes of urinary and blood porphyrin profiles by exposure to PCBs, lead or diazinon in rats.

Porphyrin profiles in excreta or blood have been useful biomarkers for monitoring exposure of hazardous xenobiotics to human or animals. We evaluated and compared the changes in urinary and blood copro-, uro-, and protoporphyrins during and after the exposure of Aroclor1254 (PCBs), lead (Pb) or diazinon to rats. PCBs (10, 50 and 100 mg/kg bw), Pb (62.5, 250 and 1,000 ppm) and diazinon (10, 30 and 90 mg/k bw) were administered to rats via gavage (PCBs and diazinon) or via drinking water (Pb) daily for 5 w. Urine and blood were collected weekly for the 5 w of exposure and for 5 w after withdrawal. Three urinary porphyrins and blood protoporphyrin increased gradually with PCB dose in a time dependent manner and remained elevated for 5 w after withdrawal. Urinary porphyrins were increased rapidly by Pb and then returned to normal immediately after Pb withdrawal while blood protoporphyrin remained high but gradualy decreased during the 5 w after withdrawal. Diazinon did not affect blood and urinary porphyrins as did PCBs and Pb, but just induced a weak increase of urinary coproporphyrin. Urinary and blood porphyrin profiles can be used as biomarkers for exposure assessment to PCBs and Pb. The normal ranges of blood porphyrins in cattle, pigs, chickens and rats were also established for use as biomarkers.