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This study describes the maternal-fetal disposition of bisphenol A and its distribution into the placenta and amniotic fluid after iv injection (2 mg/kg) to pregnant Sprague-Dawley rats. Bisphenol A was distributed extensively to the placenta and fetus, with their respective AUC values 4.4- and 2.2-fold greater than AUC for the maternal serum. In contrast, the distribution of bisphenol A into the amniotic fluid was low, with the mean amniotic fluid-to-maternal serum AUC ratio of 0.2. The decay curves of bisphenol A in the placenta, fetus, and amniotic fluid paralleled that of the maternal serum during the terminal elimination phase. A five-compartment open model consisting of the maternal central, maternal peripheral, placental, fetal, and amniotic fluid compartments was used to describe the disposition of bisphenol A in pregnant rats, with the elimination occurring from the maternal central and fetal compartments. Based on this model, bisphenol A delivered to the placenta was transferred primarily to the fetus [kpf/(kpf + kpc + kpa) = 65.4 %], with the remaining fraction transported to the maternal central (33.2%) and amniotic fluid (1.4%) compartments. Bisphenol A was eliminated from the amniotic fluid by the fetal (63.9%) and placental (36.1%) routes. On the other hand, bisphenol A was eliminated from the fetus primarily by the placental route back to mother [kfp/(kfp + kfa + kfo) = 100%], with the amniotic route playing an insignificant role in fetal elimination. The percent contribution of the fetal elimination to the total elimination in the maternal-fetal unit was 0.0% [CLfoAUCfetus/(CLcoAUCmaternal serum + CLfoAUCfetus)]. The pharmacokinetic model used in this study provides insights into the routes of elimination of bisphenol A in the maternal-fetal rat upon maternal administration.

The effects of pH and temperature on benzo[a]pyrene(BaP)-DNA, -protein, and -lipid adducts were compared in rat hepatocytes treated with [3H]-BaP. In primary rat hepatocytes, BaP-DNA, -protein, and -lipid adducts were formed in a concentration-dependent manner (0.31-2.80 mmol BaP), and the formation of BaP-lipid adducts, specifically triglyceride (TG) adducts, correlated well with that of DNA or protein adducts. As incubation time increased (1-24 h), the formation of DNA or protein adducts was also elevated, while BaP-TG adducts decreased after 3 h of reaction. pH had no effect on the formation of [3H]-BaP-DNA, and -protein adducts, and the TG adducts decreased with increasing pH (3.4-11.4). With an increase in incubation temperature (30-45 degrees C), on the other hand, the adduct formation was increased with increasing temperature up to 40 degrees C and then decreased sharply. These results suggest that the physicochemical properties of BaP-DNA, -protein, and -TG adducts might be important to consider when cells or humans who have been exposed to BaP are monitored for biomarkers.

Bepotastine salts (nicotinate and salicylate) were investigated for their physicochemical properties to develop novel salt forms of bepotastine, bioequivalent to the bepotastine besilate-loaded tablet (Talion). These bepotastine salts of either nicotinate- or salicylate-loaded tablets were prepared by conventional wet granulation method, and dissolution profiles and pharmacokinetics in beagle dogs were compared to those of Talion. A novel bepotastine nicotinate has a higher solubility at varying pH levels (1.2, 4, or 6.8) than salicylate-loaded or besilate-loaded salt. In addition, those bepostastine salt forms (nicotinate and salicylate) are stable in heat, light, and water. Further, the novel nicotinate- and salicylate-loaded tablets showed similar dissolution rates to Talion in several selected dissolution media and were bioequivalent to Talion in beagle dogs in terms of area under the concentration-time curve (AUC) and maximum observed concentration (Cmax). A pharmacokinetic study performed in beagle dogs demonstrated that test and reference products were found to be bioequivalent in terms of safety, efficacy, and pharmacokinetic properties. These results suggest that bepostastine nicotinate and salicylate formulations are considered applicable candidates and are well tolerated versus the conventional bepostastine besilate formulation.

Anti-inflammatory effects of glycosaminoglycan (GAG) derived from cricket (Gryllus bimaculatus, Gb) were investigated in a complete Freund's adjuvant (CFA)-treated chronic arthritic rat model. This GAG produced a significant anti-edema effect as evidenced by inhibition of C-reactive protein (CRP) and rheumatoid factor, and interfered with atherogenesis by reducing proinflammatory cytokine levels of (1) vascular endothelial growth factor (VEGF) production in human umbilical vein endothelial cells (HUVEC), (2) interleukin-6, (3) prostaglandin E2-stimulated lipopolysaccharide in RAW 264.7 cells, and (4) tumor necrosis factor (TNF)-α production in normal splenocytes, in a dose-dependent manner. This GAG was also found to induce nitric oxide (NO) production in HUVEC cells and elevated endothelial nitric oxide synthase (eNOS) activity levels. Histological findings demonstrated the fifth lumbar vertebrae (LV) dorsal root ganglion, which was linked to the paw treated with Gb GAG, was repaired against CFA-induced cartilage destruction. Further, combined indomethacin (5 mg/kg)-Gb GAG (10 mg/kg) inhibited more effectively CFA-induced paw edema at 3 h and 2 or 3 d after treatment to levels comparable to only the anti-inflammatory drug indomethacin. Ultraviolet (UV)-irritated skin inflammation also downregulated nuclear factor κB (NFκB) activity in transfected HaCaT cells. Data suggest that the anti-inflammatory effects of GAG obtained from cricket (Gb) may be useful for treatment of inflammatory diseases including chronic arthritis.

In order to investigate the potential application of blood biomarkers as surrogate indicators of carcinogen-adduct formation in target-specific tissues, temporal formation of benzo[a]pyrene (BaP)-associated DNA adducts, protein adducts, or lipid damage in target tissues such as lung, liver, and kidney was compared with globin adduct formation or plasma lipid damage in blood after continuous intraperitoneal (ip) injection of [(3)H]BaP into female ICR mice for 7 d. Following treatment with [(3)H]BaP, formation of [(3)H]BaP-DNA or -protein adducts in lung, liver, and kidney increased linearly, and persisted thereafter. This finding was similar to the observed effects on globin adduct formation and plasma lipid damage in blood. The lungs contained a higher level of DNA adducts than liver or kidneys during the treatment period. Further, the rate of cumulative adduct formation in lung was markedly greater than that in liver. Treatment with a single dose of [(3)H]BaP indicated that BaP-globin adduct formation and BaP-lipid damage in blood reached a peak 48 h after treatment. Overall, globin adduct formation and lipid damage in blood were significantly correlated with DNA adduct formation in the target tissues. These data suggest that peripheral blood biomarkers, such as BaP-globin adduct formation or BaP-lipid damage, may be useful for prediction of target tissue-specific DNA adduct formation, and for risk assessment after exposure.

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

This study was designed to investigate the possible protective effects of antioxidants (vitamin E, beta-carotene, vitamin C, and red ginseng) on lipid peroxidation in smokers (> or = 20 cigarettes/day). Male student smokers were given antioxidant supplements for 4 wk. Smokers had significantly higher plasma levels of total cholesterol, triacylglycerols, and malondialdehyde (MDA) than nonsmokers. No corresponding significant differences in lipid profiles were found between smokers and nonsmokers. Smokers had significantly lower baseline concentrations of plasma vitamin C, beta-carotene, and alpha-tocopherol. After antioxidant (200 IU vitamin E, 9 mg beta-carotene, 500 mg vitamin C, or 1.8 g red ginseng) supplementation for 4 wk, smokers had significantly higher concentrations of plasma antioxidants. After 4 wk of antioxidant supplementation with betacarotene, high-density lipoprotein (HDL) cholesterol concentrations in smokers were significantly increased. Overall, plasma MDA concentrations gradually decreased after antioxidant supplementation over the 4-wk period. Moreover, a significant reduction in plasma MDA concentrations was observed after vitamin E supplementation. The results of our study support the hypothesis that lipid peroxidation concentrations are inversely correlated with plasma antioxidant concentrations. Our data suggest that smokers have insufficient concentrations of antioxidant vitamins in plasma and that supplementation with antioxidants might protect smokers from oxidative damage.

A rapid and sensitive HPLC method with fluorescence detection has been developed for the micro-sample analysis of bisphenol A in rat serum. Compounds were separated on a Spherisorb C18 analytical column with acetonitrile-0.1% triethylamine in deionized water, 54:46 (v/v), as mobile phase. Bisphenol A was isolated by one-step extraction with dichloromethane. The calibration plot was linear (r=0.999) over the concentration range 1–1000 ng mL−1. The limits of detection and quantitation were 0.2 ng mL−1 and 1 ng mL−1, respectively, for 100-μL serum samples. The assay was successfully used for a pharmacokinetic study after intravenous administration of a low dose (0.1 mg kg−1) of bisphenol A to rats.

The potential estrogenic activities of bisphenol-A were investigated in vitro (E-screen and estrogen receptor competitive binding bioassays) and in vivo (uterotrophic assay). Uterotrophic responses were evaluated using mature ovariectomized Sprague-Dawley female rats treated subcutaneously with bisphenol A (1, 5, 10, 50, and 100 mg/kg/day), E2 (0.3 microgram/kg), and DES (0.3 microgram/kg) for 3 consecutive days. In a MCF-7 cell proliferation assay, E2 and DES used as positive estrogens induced maximum proliferation of MCF-7 cells at 1.0 nM, whereas BPA slightly induced MCF-7 cell proliferation at a higher level of 0.1 microM and maximum proliferation at 10 microM. In a competitive binding assay, E2 and DES showed inhibition of 17 beta-[3H]estradiol binding to the rat uterus ER with an IC50 of 1.0 nM and 0.5 nM, respectively. However, BPA had an IC50 of 5 microM, which was approximately 5,000 or 10,000-fold greater than the IC50 of E2 and DES. In uterotrophic assays, uterus (wet and blotted) and vagina weights were significantly increased at the dose of BPA 100 mg/kg/day in OVX Sprague-Dawley rats. These studies demonstrate that BPA exhibits weak estrogenic activity in all experimental systems, and thus its migration from epoxy resins or polycarbonate products should be controlled not to exceed a safety levels for humans.

In this study, we demonstrated the in vitro and in vivo formation of carcinogen-lipid adduct and its correlation with DNA or protein adducts. The lipids from serum or hepatocyte membranes of Sprague-Dawley rats, human serum and standard major lipids were in vitro reacted with benzo[a]pyrene (B[a]P) and B[a]P metabolites. 7, 8-Dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene(BPDE-I), an ultimate carcinogenic form of B[a]P, was covalently bound to triglyceride (TG). BPDE-I-TG adducts isolated by thin-layer chromatography (TLC) were further detected by high-performance liquid chromatography. TGs, including triolein, tripalmitin and tristearin, showed positive reactions with BPDE-I. However, cholesterol, phospholipids (phosphatidylcholine, phosphatidyl-ethanolamine, phosphatidyl-inositol and sphingomyelin) and non-esterified fatty acids (palmitic acid, oleic acid, linoleic acid and stearic acid) did not react with BPDE-I. In addition, other B[a]P metabolites (B[a]P-phenols and -diols) did not react with TG. TG appeared to be the most reactive lipid yet studied with respect to its ability to form an adduct with BPDE-I. There was a clear-cut dose-related formation of [1,3-(3)H]BPDE-I-lipid adducts in vitro between TG and [1,3-(3)H]BPDE-I. In an animal study, BPDE-I-TG was also formed in the serum of rats orally treated with B[a]P (25 mg/rat). Also, obvious correlations between [(3)H]B[a]P related-biomolecule adducts (DNA or protein) or lipid damage and the BPDE-I-TG adducts were obtained in various tissues of mice i.p. treated with [(3)H]B[a]P. These data suggest that TG can form an adduct with BPDE-I, as do other macromolecules (DNA, RNA and protein). Therefore, a carcinogen-lipid adduct would be a useful biomarker for chemical carcinogenesis research and cancer risk assessment.

The correlation between the steady-state level of inducible nitric oxide synthase (iNOS) mRNA and skin tumors induced following treatment with 12-o-tetradecanoylphorbol-13-acetate (TPA) was investigated in transgenic TG-AC mice, which carry the v-Ha-ras oncogene fused to the promoter of the mouse embryonic alpha-like, zeta-globin gene. In animals treated with TPA (2.5 microg x 2/week, for 2 weeks), the increase of iNOS mRNA was locally confined only to the regions of papillomas, but not to the skin tissues surrounding the papillomas. However, the tissues surrounding the papillomas showed only a minor increase in the steady-state level of iNOS mRNA. These data suggest that iNOS gene expressions may underlie tumorigenesis during TPA promotion in TG-AC mice.

The chemopreventive effects of antioxidants (vitamin E, beta-carotene, vitamin C and red ginseng) on oxidative DNA and protein (globin) damages were comparatively investigated in the peripheral blood of smokers (or = 20 cigarettes/day). Smokers showed a lower baseline level of plasma micronutrients (vitamin C and beta-carotene) (P0.01) and higher baseline level of oxidative DNA or protein damage than non-smokers (N = 5; P0.05). During daily supplementation of antioxidants (200 IU vitamin of E, 9 mg of beta-carotene, 500 mg of vitamin C, or 1.8 g of red ginseng) for 4 weeks, smokers plasma antioxidant concentrations increased linearly, while their mean levels of 8-hydroxydeoxyguanosine (8-OHdG) and carbonyl contents decreased compared with those in smokers supplemented with a placebo (P0.05). Levels of urinary and plasma cotinine remained steady in smokers regardless of supplementation with antioxidants. 8-OHdG and carbonyl content decreased in a time-dependent manner (as the total intake dose increased) after supplementation with vitamin E (8-OHdG, 33.8%; carbonyl content, 43.6%) or red ginseng (8-OHdG, 31.7%; carbonyl content, 21.3%). These preliminary data suggest that supplementation with antioxidants might protect smokers from oxidative damages and could reduce cancer risk or other diseases caused by free radicals associated with smoking.

The aim of this project was to determine the cytotoxic components from the venom of king cobra, Ophiophagus hannah. Venom was purified by a combination of gel-filtration, ion-exchange and reversed-phase chromatographic steps. The biochemical properties of the cytotoxic component were consistent with those of l -amino acid oxidase. The molecular weight of the enzyme was estimated to be 150 000 by gel filtration and 70 000 under the denaturing conditions of SDS-PAGE, indicating a dimer. It has an isoelectric point of 4.5 and is a glycoprotein. The N-terminal sequence of l -amino acid oxidase from the king cobra venom was determined to be SVINLEESFQEPEYE. The cytotoxicity of l -amino acid oxidase was observed in stomach cancer, murine melanoma, fibrosarcoma, colorectal cancer and Chinese hamster ovary cell lines. Cytotoxicity resulted in the loss of ability in attachment and inhibition of cell proliferation. The cytotoxic protein decreased the level of cell proliferation by 74% according to [3H]thymidine uptake assay. The mechanism of enzyme action may be related to the inhibition of thymidine incorporation and an interaction with DNA.

α-Linolenic acid (ALA), a carboxylic acid with 18 carbons and three cis double bonds, is an essential fatty acid needed for human health and can be acquired via regular dietary intake of foods that contain ALA or dietary supplementation of foods high in ALA, for example flaxseed. ALA has been reported to have cardiovascular-protective, anti-cancer, neuro-protective, anti-osteoporotic, anti-inflammatory, and antioxidative effects. ALA is the precursor of longer chain omega-3 fatty acids, eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), but its beneficial effects on risk factors for cardiovascular diseases are still inconclusive. The recommended intake of ALA for cardiovascular health is reported to be 1.1-2.2g/day. Although there are limited toxicological data for ALA, no serious adverse effects have been reported. The evidence on an increased prostate cancer risk in association with dietary ALA is not conclusive. Based on the limited data currently available, it may be concluded that ALA may be beneficial as a nutraceutical/pharmaceutical candidate and is safe for use as a food ingredient.

New potential chemotherapeutic strategies are required to overcome multidrug resistance (MDR) in cancer. This study investigated the anticancer effect of a novel anthracene derivative MHY412 on doxorubicin-resistant human breast cancer (MCF-7/Adr) cells. We measured cell viability and the expression of apoptosis-related genes; in addition, the antitumor activity of MHY412 was confirmed using an in vivo tumor xenograft model. MHY412 significantly inhibited the proliferation of MCF-7/Adr and MCF-7 cells in a concentration-dependent manner. Notably, the half-maximal inhibitory concentration (IC50) values of MHY412 in MCF-7/Adr (0.15 µM) and MCF-7 (0.26 µM) cells were lower than those of doxorubicin (MCF-7/Adr, 13.6 µM and MCF-7, 1.26 µM) after treatment for 48 h. MHY412 at low concentrations induced S phase arrest, but at high concentrations, the number of MCF-7/Adr cells in the sub-G1 phase significantly increased. MHY412-induced sub-G1 phase arrest was associated with inhibition of cyclin, cyclin-dependent kinase 2 (CDK2) and p21 expression in MCF-7/Adr cells. MHY412 markedly reduced P-glycoprotein (P-gp) expression and increased apoptotic cell death in MCF-7/Adr cells. Cleavage of poly-ADP ribose polymerase, reduced Bcl-2 expression, and increased in cytochrome c release in MCF-7/Adr cells confirmed the above results. In addition, MHY412 markedly inhibited tumor growth in a tumor xenograft model of MCF-7/Adr cells. Our data suggest that MHY412 exerts antitumor effects by selectively modulating the genes related to cell cycle arrest and apoptosis. In particular, MHY412 is a new candidate agent for the treatment of Bcl-2 overexpressed doxorubicin-resistant human breast cancer.

Plasticizers or plastic materials such as phthalates, bisphenol-A (BPA), and styrene are widely used in the plastic industry and are suspected endocrine-disrupting chemicals (EDC). Although plastic materials such as polypropylene (PP) and polyethylene terephthalate (PET) are not EDC and are considered to be safe, their potential properties as EDC have not been fully investigated. In this study, plastic samples eluted from plastic food containers (PP or PET) were investigated in Sprague-Dawley rats using Hershberger and uterotrophic assays. In the Hershberger assay, 6-wk-old castrated male rats were orally treated for 10 consecutive days with plastic effluent at 3 different doses (5 ml/kg) or vehicle control (corn oil, 1 ml/100 g) to determine the presence of both anti-androgenic and androgenic effects. Testosterone (0.4 mg/ml/kg) was subcutaneously administered for androgenic evaluation as a positive control, whereas testosterone (0.4 mg/ml/kg) and flutamide (3 mg/kg/day) were administered to a positive control group for anti-androgenic evaluation. The presence of any anti-androgenic or androgenic activities of plastic effluent was not detected. Sex accessory tissues such as ventral prostate or seminal vesicle showed no significant differences in weight between treated and control groups. For the uterotrophic assay, immature female rats were treated with plastic effluent at three different doses (5 ml/kg), with vehicle control (corn oil, 1 ml/100 g), or with ethinyl estradiol (3 μg/kg/d) for 3 d. There were no significant differences between test and control groups in vagina or uterine weight. Data suggest that effluents from plastic food containers do not appear to produce significant adverse effects according to Hershberger and uterotrophic assays.

Exposure to the wide variety of chemicals used for manufacturing consumer products commonly occurs daily and the consequences to health are beneficial. However, some of these products are hazardous and exert deleterious effects on humans and the ecosystem. To protect consumers from exposure to hazardous chemicals, appropriate risk management systems are needed. Developed countries such as the United States and Canada have developed their own risk management systems for regulating hazardous agents. However, the risk management systems prepared by developed countries may not be readily applicable to developing or underdeveloped countries because of certain economic, political, cultural, or social factors in each country. In general, a risk management framework includes evaluation components of risk assessment, risk confrontation, risk intervention, risk communication, and risk management, but these may differ in specifics. The European Commission (EC) requires a socioeconomic analysis for formulating restrictions suggested by the European Chemicals Agency (ECHA). The EC has an early warning system for safety management termed the Rapid Alert System (RAPEX). Korea, Australia, and Japan also developed integrated network systems for risk management of consumer products. Monitoring entails the collection of information and evaluation. The risk assessment process includes scientific evaluation of potential adverse health effects. Risk communication tasks are to (1) identify stakeholders, (2) develop stakeholder analysis, (3) assess stakeholder acceptability, (4) consult with stakeholders, (5) inform stakeholders about their options, (6) evaluate control options, and (7) monitor changing issues. The risk management process involves weighing policy options and selecting regulatory options. The decision-making step is related to the determination of governmental or voluntary actions. This review examines the critical points of risk management system in Korea to effectively control hazardous agents for human safety and compliance with global harmonization.

d-Limonene, a major constituent of citrus oils, is a monoterpene widely used as a flavor/fragrance additive in cosmetics, foods, and industrial solvents as it possesses a pleasant lemon-like odor. d-Limonene has been designated as a chemical with low toxicity based upon lethal dose (LD50) and repeated-dose toxicity studies when administered orally to animals. However, skin irritation or sensitizing potential was reported following widespread use of this agent in various consumer products. In experimental animals and humans, oxidation products or metabolites of d-limonene were shown to act as skin irritants. Carcinogenic effects have also been observed in male rats, but the mode of action (MOA) is considered irrelevant for humans as the protein α(2u)-globulin responsible for this effect in rodents is absent in humans. Thus, the liver was identified as a critical target organ following oral administration of d-limonene. Other than the adverse dermal effects noted in humans, other notable toxic effects of d-limonene have not been reported. The reference dose (RfD), the no-observed-adverse-effect level (NOAEL), and the systemic exposure dose (SED) were determined and found to be 2.5 mg/kg/d, 250 mg/kg//d, and 1.48 mg/kg/d, respectively. Consequently, the margin of exposure (MOE = NOAEL/SED) of 169 was derived based upon the data, and the hazard index (HI = SED/RfD) for d-limonene is 0.592. Taking into consideration conservative estimation, d-limonene appears to exert no serious risk for human exposure. Based on adverse effects and risk assessments, d-limonene may be regarded as a safe ingredient. However, the potential occurrence of skin irritation necessitates regulation of this chemical as an ingredient in cosmetics. In conclusion, the use of d-limonene in cosmetics is safe under the current regulatory guidelines for cosmetics.

Background Multidrug resistance is a major problem in the treatment of breast cancer, and a number of studies have attempted to find an efficient strategy with which to overcome it. In this study, we investigate the synergistic anticancer effects of resveratrol (RSV) and doxorubicin (Dox) against human breast cancer cell lines. Methods The synergistic effects of RSV on chemosensitivity were examined in Dox-resistant breast cancer (MCF-7/adr) and MDA-MB-231 cells. In vivo experiments were performed using a nude mouse xenograft model to investigate the combined sensitization effect of RSV and Dox. Results and conclusion RSV markedly enhanced Dox-induced cytotoxicity in MCF-7/adr and MDA-MB-231 cells. Treatment with a combination of RSV and Dox significantly increased the cellular accumulation of Dox by down-regulating the expression levels of ATP-binding cassette (ABC) transporter genes, MDR1, and MRP1. Further in vivo experiments in the xenograft model revealed that treatment with a combination of RSV and Dox significantly inhibited tumor volume by 60%, relative to the control group. General significance These results suggest that treatment with a combination of RSV and Dox would be a helpful strategy for increasing the efficacy of Dox by promoting an intracellular accumulation of Dox and decreasing multi-drug resistance in human breast cancer cells.

DNA adduct formation assay (DAFA) was carried out to compare dose responses with the Ames test and chromosomal aberration test using aflatoxin B1 (AFB1) and benzo[a]pyrene (BaP). In the bacterial mutation test, AFB1 and BaP (0-1 microgram/plate) were all positive in TA97a and TA100 with dose-related revertants. However, the slopes of the dose-response curves were gradual (slope 0.55-3.73, r = .84-.98). In the chromosome aberration test, a significant increase in the percentage of chromosomal aberrations was obtained from male ICR mouse spleen cells treated with AFB1 and BaP, but a dose-related increase was insensitive (slope 0.09-0.23, r = .75-.78). The incidence of chromosomally aberrant spleen cells treated with BaP was significantly increased compared with AFB1. DAFA was performed in vitro with [3H]-AFB1 and [3H]BaP. These two carcinogens were able to induce genotoxicity and showed good dose-related increases in terms of DNA adduct formation (slope 0.78-1.28, r = 1.00). Coefficients of variation (CV) for the slope of each dose-response curve were much lower in DAFA in vitro (CV 15.09- 18.34%) than those in any other test (CV 19.69-99.33%, Ames test; 18.89-44.58%, chromosome aberration test). Furthermore, DAFA in vivo was performed to investigate organotropic DNA adduct formation and persistence in Sprague-Dawley rats ip or orally treated with AFB1 and BaP. DNA adducts were monitored for 48-96 h by enzyme-linked immunosorbent assay (ELISA) using corresponding monoclonal antibodies, 6A10 and 8E11. DAFA in vivo demonstrated that the liver and kidney might be the probable target organs for AFB1 with the highest formation and persistence of DNA adducts and the lung and liver for BaP regardless of the route of administration. The results suggest that DAFA in vitro could be useful for detecting genotoxic compounds, and DAFA in vivo should also be considered as a good alternative method for the screening of organ-specific chemical carcinogens.

A metabolomics approach using proton nuclear magnetic resonance (NMR) was applied to investigate metabolic alterations following adriamycin (ADR) treatment for gastric adenocarcinoma. After BALB/c-nu/nu mice were implanted with human gastric adenocarcinoma, ADR (1 or 3 mg kg−1 per day) was intraperitoneally administered for 5 days. Urine was collected on days 2 and 5 and analyzed by NMR. The levels of trimethylamine oxide (TMAO, ×0.3), hippurate (×0.3) and taurine (×0.6) decreased significantly (P

Sirtuins (SIRTs), NAD+-dependent class III histone deacetylases (HDACs), play an important role in the regulation of cell division, survival and senescence. Although a number of effective SIRT inhibitors have been developed, little is known about the specific mechanisms of their anticancer activity. In this study, we investigated the anticancer effects of sirtinol, a SIRT inhibitor, on MCF-7 human breast cancer cells. Apoptotic and autophagic cell death were measured. Sirtinol significantly inhibited the proliferation of MCF-7 cells in a concentration-dependent manner. The IC50 values of sirtinol were 48.6 µM (24 h) and 43.5 µM (48 h) in MCF-7 cells. As expected, sirtinol significantly increased the acetylation of p53, which has been reported to be a target of SIRT1/2. Flow cyto-metry analysis revealed that sirtinol significantly increased the G1 phase of the cell cycle. The upregulation of Bax, downregulation of Bcl-2 and cytochrome c release into the cytoplasm, which are considered as mechanisms of apoptotic cell death, were observed in the MCF-7 cells treated with sirtinol. The annexin V-FITC assay was used to confirm sirtinol-induced apoptotic cell death. Furthermore, the expression of LC3-II, an autophagy-related molecule, was significantly increased in MCF-7 cells after sirtinol treatment. Autophagic cell death was confirmed by acridine orange and monodansylcadaverine (MDC) staining. Of note, pre-treatment with 3-methyladenine (3-MA) increased the sirtinol-induced MCF-7 cell cytotoxicity, which is associated with blocking autophagic cell death and increasing apoptotic cell death. Based on our results, the downregulation of SIRT1/2 expression may play an important role in the regulation of breast cancer cell death; thus, SIRT1/2 may be a novel molecular target for cancer therapy and these findings may provide a molecular basis for targeting SIRT1/2 in future cancer therapy.

Melamine-induced renal toxicity is associated with crystal formation in the kidney following exposure to melamine and cyanuric acid. However, metabolomic profiling of intact kidney tissue after chronic intake of melamine and cyanuric acid (M + CA) mixtures has rarely been studied. The present study investigated the melamine-induced renal toxicity by determining metabolites in the kidney through [(1)H]nuclear magnetic resonance. Melamine (63 mg/kg) and cyanuric acid (6.3 mg/kg) were co-administered to rats via oral gavage for 30 days. The mixture of M + CA (63/6.3 mg/kg) induced nephrotoxicity, as determined by increased blood urea nitrogen (BUN) and creatinine levels. The kidney weights were significantly increased in the animals treated with M + CA (63/6.3 mg/kg). The histological analysis revealed epithelial degeneration and necrotic cell death in the proximal and distal tubules. Furthermore, various metabolites were altered in both renal medullar and cortical tissues. In the medullar tissues, asparagine, choline, creatinine, cysteine, ethanolamine, glucose, isoleucine, glutamine, and myo-inositol levels were elevated, but glucitol, phenylalanine, tyrosine, and sn-glycero-3-levels were reduced. In the cortex, ethanolamine, hypoxanthine, isoleucine and o-phosphoethanolamine levels were increased, whereas formate, glucose, glutathione, threonine, and myo-inositol levels were decreased, suggesting the M + CA-induced renal cell injury. These data suggest that a mixture of M + CA-induced metabolites may be useful biomarkers for the detection of kidney injury.

A metabolomics approach using proton nuclear magnetic resonance (NMR) was applied to investigate metabolic alterations following adriamycin (ADR) treatment for gastric adenocarcinoma. After BALB/c-nu/nu mice were implanted with human gastric adenocarcinoma, ADR (1 or 3 mg kg(-1) per day) was intraperitoneally administered for 5 days. Urine was collected on days 2 and 5 and analyzed by NMR. The levels of trimethylamine oxide (TMAO, ×0.3), hippurate (×0.3) and taurine (×0.6) decreased significantly (P 0.05), whereas the levels of 3-indoxylsulfate (×12.6), trigonelline (×1.5), citrate (×2.5), trimethylamine (TMA, ×2.0) and 2-oxoglutarate (×2.3) increased significantly (P 0.05) in the tumor model. After ADR treatment, TMAO, hippuarte and taurine were increased significantly on day 5 compared with those of the tumor model. The levels of 2-oxoglutarate, 3-indoxylsulfate, trigonelline, TMA and citrate, which increased in the tumor model, significantly decreased to those of normal control by ADR treatment. Furthermore, the ratio between TMA and TMAO was dramatically altered in both tumor and ADR-treated groups. Overall, metabolites such as TMAO, TMA, 3-indoxylsulfate, hippurate, trigonelline, citrate and 2-oxoglutarate related to the tricarboxylic acid (TCA) cycle might be considered as therapeutic targets to potentiate the efficacy of ADR. Thus, these results suggest that the metabolomics analysis of tumor response to ADR treatment may be applicable for demonstrating the efficacy of anticancer agent, ADR and treatment adaptation.

4-Hydroxy-2-nonenal (4-HNE) is a major end product of lipid peroxidation of membrane n-6 polyunsaturated fatty acids, which are found in food products. In order to examine the toxicity attributed to 4-HNE, a subacute toxicity study was conducted in Sprague-Dawley (SD) rats. For this study, 4 groups of 10 male and 10 female rats were administered by gavage either 0 (control), 0.5, 2.5, or 12.5 mg 4-HNE/kg body weight/d for 28 d, and then sacrificed for blood and tissue sampling. No significant changes in body weight or clinical signs were observed, but biochemical analysis showed significant alterations in hepatotoxicity biomarkers, such as levels of serum albumin and total bilirubin, and aspartate aminotransferase (AST) activity, and in nephrotoxicity biomarkers, such as levels of blood urea nitrogen (BUN) and creatinine and activity of alkaline phosphatase (ALP), and urinary creatinine and protein levels at 0.5 mg/kg/d. In addition, significant increases in kidney and brain weights and a significant decrease in small intestine weight were noted at 12.5 mg/kg/d. Histologic examinations of kidneys showed hyaline droplets or accumulation of hyaline bodies in renal tubules and degeneration of tubular epithelium cells. These results demonstrate that oral daily exposure to 4-HNE for 28 d produced hepatotoxicity and nephrotoxicity. The no-observed-adverse-effect level (NOAEL) for 4-HNE was calculated to be

Polybrominated diphenyl ethers (PBDEs), commonly used flame retardants, have been reported as potential endocrine disruptor and neurodevelopmental toxicants, thus giving rise to the public health concern. The goal of this study was to investigate the relationship between umbilical cord blood, maternal blood, and breast milk concentrations of PBDEs in South Korean. We assessed PBDE levels in paired samples of umbilical cord blood, maternal blood, and breast milk. The levels of seven PBDE congeners were measured in 21 paired samples collected from the Cheil Woman’s Hospital (Seoul, Korea) in 2008. We also measured thyroid hormones levels in maternal and cord blood to assess the association between PBDEs exposure and thyroid hormone levels. However, there was no correlation between serum thyroxin (T4) and total PBDEs concentrations. The total PBDEs concentrations in the umbilical cord blood, maternal blood, and breast milk were 10.7 ± 5.1 ng g −1 lipid, 7.7 ± 4.2 ng g −1 lipid, and 3.0 ± 1.8 ng g −1 lipid, respectively. The ranges of total PBDE concentrations observed were 2.28–30.94 ng g −1 lipid in umbilical cord blood, 1.8–17.66 ng g −1 lipid in maternal blood, and 1.08–8.66 ng g −1 lipid in breast milk. BDE-47 (45–73% of total PBDEs) was observed to be present dominantly in all samples, followed by BDE-153. A strong correlation was found for major BDE-congeners between breast milk and cord blood or maternal blood and cord blood samples. The measurement of PBDEs concentrations in maternal blood or breast milk may help to determine the concentration of PBDEs in infant.

Publisher Summary Many chemicals, including endocrine disruptors, are known to produce various toxicities such as developmental or reproductive toxicity, carcinogenicity, mutagenicity, immunotoxicity and neurotoxicity; this chapter discusses mutagenicity and carcinogenicity. Mechanisms of carcinogenesis due to exposure to endocrine disruptors may be associated with free radical generation and biomolecule (e.g., DNA, protein, lipid) damage after the dysregulation of hormones such as estrogen, testosterone, thyroid and insulin. Each subtype of ovarian carcinoma has different clinical characteristics, biological behaviors and responses to chemotherapy. Endometriosis is an estrogen-dependent disease characterized by the presence of endometrial glands and stroma outside the uterine cavity. It is a common gynecological disorder as well as a major cause of infertility. It also addresses vaginal cancer, testicular cancer and prostate cancer. The fact that epigenetic mechanisms may play a role in endocrine disruption helps to explain the transgenerational effects of chemicals. The epigenetic mechanism involves the alteration of DNA in the germ line that appears to transmit transgenerational adult-onset disease, including spermatogenic defects, prostate disease, kidney disease and cancer. Mechanisms of carcinogenesis due to exposure to endocrine disruptors may be associated with free radical generation and biomolecule (e.g., DNA, protein, lipid) damage after the dysregulation of hormones such as estrogen, testosterone, thyroid and insulin.

The purpose of this study was to determine the effects of di(n-butyl) phthalate (DBP) administration on male reproductive organ development in F1 Sprague-Dawley rats following in utero exposure. During gestation days (GD) 10-19, pregnant rats were administered daily, orally, DBP at 250, 500, or 700 mg/kg or flutamide (1, 12.5, or 25 mg/kg/d) as a positive control. The male offspring were sacrificed at 31 d of age. DBP and flutamide dose-dependently significantly increased the incidence of hypospadias and cryptorchidism in F1 male offspring. The weights of testes and accessory sex organs (epididymides, seminal vesicles, ventral prostate, levator ani plus bulbocavernosus muscles (LABC), and Cowper's glands) were significantly reduced in DBP-treated animals. Furthermore, cauda agenesis of epididymides and ventral prostate atrophy were observed in high-dose 700-mg/kg DBP males. Anogenital distance (AGD) and levels of dihydrotestosterone (DHT) and testosterone were significantly decreased in the DBP (700 mg/kg/d)-treated groups. In particular, the expression of androgen receptor (AR) and 5α-reductase type 2 in the proximal penis was markedly depressed following administration of DBP (700 mg/kg/d) or flutamide (25 mg/kg/d). The expression of sonic hedgehog (Shh) in the urethral epithelium of the proximal penis was significantly less in the DBP (700 mg/kg/d)- or flutamide (25 mg/kg/d)-treated groups. In addition, DBP dose-dependently significantly increased the expression of estrogen receptor (ER α) in the undescended testis. Data demonstrated that in utero exposure to DBP produced several abnormal responses in male reproductive organs, and these effects may be due to disruption of the stage-specific expression of genes related to androgen-dependent organs development.