To identify gene expression that can be closely involved in chemical-induced hepatocellular hypertrophy, the hepatic gene expression profile was assessed by cDNA microarray analysis in male F344 rats fed for 3 days, 4 weeks, and 13 weeks a diet containing a hepatocellular hypertrophy inducer, either phenobarbital (500 ppm), clofibrate (2,500 ppm), or piperonyl butoxide (20,000 ppm). The results showed that, in all treatment groups, the increased expressional rate of the Grin2c gene, which encodes the N-methyl-D-aspartate receptor 2C subunit (NR2C), was the highest among those of all the genes tested, as compared with the corresponding gene expression in rats fed a normal diet. Moreover, real-time RT-PCR analysis showed that the expression levels of the Grin2c gene in rats fed with each chemical clearly increased in a chemical treatment period-dependent fashion, and that the increased rate was closely correlated with the grade of hypertrophy of hepatocytes rather than with the increased rate in liver weight. These results suggest the possibility that chemical-induced NR2C expression relates to the development of hepatocellular hypertrophy.

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To date, biological risk assessment studies of chemicals that induce DNA lesions have been primarily based on the action of DNA polymerases during replication. However, DNA lesions interfere not only with replication but also with transcription. Therefore, detecting the damaging effects of DNA lesions during transcription might be important for estimating the safety of chemical mutagens and carcinogens. However, methods to address these effects have not been developed. Here, we report a simple, non-isotopic method for determining the toxicity of chemical agents by visualizing transcription in a mammalian cell system. The method is based on the measurement of the incorporation of bromouridine (as the uridine analogue) into the nascent RNA during RNA synthesis inhibition (RSI) induced by the stalling of RNA polymerases at DNA lesions on the transcribed DNA strand, which triggers transcription-coupled nucleotide excision repair (TC-NER). When we tested chemical agents (camptothecin, etoposide, 4-nitroquinoline-1-oxide, mitomycin C, methyl methanesulfonate, and cisplatin) in HeLa cells by the method, RSI indicative of genomic toxicity was observed in the nucleoli of the tested cells. This procedure provides the following advantages: 1) it uses common, affordable mammalian cells (HeLa cells, WI38VA13 cells, human dermal fibroblasts, or Chinese hamster ovary cells) rather than genetically modified microorganisms; 2) it can be completed within approximately 8 hr after the cells are prepared because RNA polymerase responses during TC-NER are faster than other DNA damage responses (replication, recombination, and apoptosis); and 3) it is safe because it uses non-radioactive bromouridine and antibodies to detect RNA synthesis on undamaged transcribed DNA strands.

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Stir bar sorptive extraction is a technique used for extracting target substances from various aqueous matrixes such as environmental water, food, and biological samples. This type of extraction is carried out by rotating a coated stir bar is rotated in the sample solution. In particular, Twister bar is a commercial stir bar that is coated with polydimethylsiloxane (PDMS) and used to perform sorptive extraction. In this study, we developed a method for simultaneous detection of amphetamine, methamphetamine, 3,4-methylenedioxyamphetamine, 3,4-methylenedioxymethamphetamine, and a Δ⁹-tetrahydrocannabiniol (THC) metabolite in human urine. For extracting the target analytes, the Twister bar was simply stirred in the sample in the presence of a derivatizing agent. Using this technique, phenethylamines and the acidic THC metabolite can be simultaneously extracted from human urine. This method also enables the extraction of trace amounts of these substances with good reproducibility and high selectivity. The proposed method offers many advantages over other extraction-based approaches and is therefore well suited for screening psychoactive substances in urine specimens.

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The aim of this study was to investigate the protective effects of olive leaf extract (OLE) on genotoxicity and oxidative damage in cultured human blood cells treated with permethrin (PM) in the presence of a rat liver S9 mix containing cytochrome P 450 enzymes. Anti-genotoxic activities of OLE were studied using sister chromatid exchange (SCE) and chromosome aberration (CA) tests and furthermore total antioxidant capacity (TAC) and total oxidative status (TOS) were examined to determine the oxidative damage. Our results clearly revealed that treatment with PM (200 mg/l) alone increased SCE and CA rates and TOS level, decreased TAC level in cultured human blood cells. The OLE alone at the all tested doses did not induce any significant changes in the genotoxicity endpoint. However OLE leads to increases of plasma TAC level in vitro. OLE starts showing this positive effect at 100 mg/l. The combined treatment showed significant improvements in cytogenetic and biochemical parameters tested. Moreover, this improvement was more pronounced in the group received the high dose of the OLE. It could be concluded that the ethanol extract of OLE induced its genoprotective effect via the increase in the antioxidant capacity, inhibition of oxidative stress and scavenging of free radicals.

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This study examined the neurobehavioral changes and alteration in gene expression in the brains of metallothionein (MT)-I/II null mice exposed to low-levels of mercury vapor (Hg⁰) during postnatal development. MT-I/II null and wild-type mice were repeatedly exposed to Hg⁰ at 0.030 mg/m³ (range: 0.023-0.043 mg/m³), which was similar to the current threshold value (TLV), for 6 hr per day until the 20th day postpartum. The behavioral effects were evaluated with locomotor activity in the open field (OPF), learning ability in the passive avoidance response (PA) and spatial learning ability in the Morris water maze (MM) at 12 weeks of age. Hg⁰-exposed MT-I/II null mice showed a significant decrease in total locomotor activity in females, though learning ability and spatial learning ability were not affected. Immediately after Hg⁰ exposure, mercury concentrations in the brain did not exceed 0.5 µg/g in any animals. Hg⁰ exposure resulted in significant alterations in gene expression in the brains of both strains using DNA microarray analysis. The number of altered genes in MT-I/II null mice was higher than that in wild-type mice and calcium-calmodulin kinase II (Camk2a) involved in learning and memory in down-regulated genes was detected. These results provide useful information to elucidate the development of behavioral toxicity following low-level exposure to Hg⁰.

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The antioxidant efficacy of heated garlic juice (HGJ) in liver was evaluated with that of ascorbic acid (AA) in rats exposed to acute dose of cadmium (4 mg kg^-1 bd. wt). The rats were either given HGJ (100 mg kg^-1 bd. wt) orally, daily for 4 weeks or AA (100 mg kg^-1 bd. wt) orally, daily for 4 weeks or both or cadmium (4 mg kg^-1 bd. wt) intraperitoneally for 3 days. Another group of rats was given cadmium (4 mg kg^-1 bd. wt) intraperitoneally for 3 days after pretreatment with either HGJ (100 mg kg^-1 bd. wt) or ascorbic acid (100 mg kg^-1 bd. wt) for 4 weeks and the liver excised. The results obtained show that AA and HGJ significantly reduced the level of liver malondialdehyde (MDA) induced by cadmium compared to control (p < 0.05) but AA tends to be more potent when compared with HGJ. The presence of either HGJ or AA also significantly reduced the levels of ROS in the presence of cadmium (Cd). The presence of either AA or HGJ pre-treatment produced significant increase in liver SOD and Catalase activities when compared with rats treated with Cd alone. There was no significant reduction in the activities of these enzymes in the presence of cadmium compared to control. Western blot shows that the expressions of Nrf2 and NQO1 in the liver were significantly increase by 3 and 1.7-fold respectively in the AA pretreated mice when compared with Cd. However no significant changes were seen in HGJ pretreated rats. The expression of HO-1 was not significantly increase in the AA pretreated rats. The results show that though both ascorbic acid and HGJ are efficient in preventing Cd-induced damage in the rat liver, ascorbic acid appeared to be a more powerful antioxidant than heated garlic juice in preventing cadmium-induced oxidative damage in liver and its action may be mediated in parts via Nrf2-keap1 pathway.

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Testicular toxicity of chemical substances has been generally assessed by sperm properties and histology. However, the methods can provide only a few information of the mechanism of the toxicity. The aim of this study is to show a method that can evaluate an overview of testicular toxic mechanisms using a tissue-specific microarray and classification of genes using Medical Subject Headings (MeSH). Male ICR mice (6 weeks old) were treated with doxorubicin hydrochloride (0, 0.1, 0.3 mg/kg/time, three times per week) by subcutaneous injection for 6 weeks (until 11 weeks old). Six weeks after the final administration, tissue and blood samples were obtained. Testes were subjected to gene expression analysis using quantitative RT-PCR and cDNA microarray (testis2). To interpret the microarray data, genes were classified using MeSH related to the functions of testis and sperm. Doxorubicin (both 0.1 and 0.3 mg/kg group) induced a decrease in sperm normal morphology and mortality, daily sperm production, and the number of Sertoli cells in the seminiferous tubules. Quantitative RT-PCR and microarray analysis showed dysregulation of mRNA expression levels of genes related to Sertoli cells, germ cells and spermatogenesis. Analysis of microarray data showed a significant enrichment of a total of ten MeSH categories including Spermatogenesis, Sertoli cells, Germ cells and Male infertility. This article concluded that analysis using testicular specific microarray combined with MeSH showed a more comprehensive overview of testicular toxic mechanisms than existing methods; i.e., examination of sperm properties and the histological examinations.

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Embryonic stem cells (ES cells), pluripotent cells derived from the inner cell mass of blastocysts, differentiate in vitro into a variety of cell types representing all three germ layers. They therefore constitute one of the most promising in vitro tools for developmental toxicology. To assess the developmental toxicity of chemicals using ES cells easily, identification of effective marker genes is a high priority. We report here altered gene expression during ES cell differentiation into myocardiac and neural cells on treatment with some embryotoxic and non-embryotoxic chemicals. Decreases in several undifferentiated markers such as Oct3/4 and Nanog, and elevated expression of genes associated with heart development or the central nervous system, respectively, were found on microarray analysis. Under differentiation of ES cells into myocardic cells, 107 genes were substantially up-regulated. Decrease in the expression of 13 genes of these (Hand1, Pim2, Tbx20, Myl4, Myl7, Hbb-bh1, Hba-a1, Col1a2, Hba-x, Cmya1, Pitx2, Smyd1 and Adam19) was observed specifically by embryotoxic chemicals. Of the 107 genes up-regulated under differentiation into neurons, 22 genes (Map2, Cpe, Marcks, Ptbp2, Sox11, Tubb2b, Vim, Arx, Emx2, Pax6, Basp1, Ddr1, Ndn, Sfrp, Ttc3, Ubqln2, Six3, Dcx, L1cam, Reln, Wnt1 and Nnat) showed reduced expression specifically by embryotoxic chemicals. Almost all gene sets identified in this study are known to be indispensable for differentiation and development of heart and brain tissues, and thus may serve in early detection or prediction of embryotoxicity of chemicals in vitro.

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The toxicity of proguanil was studied on reproductive activities in male rats using in vivo and in vitro methods. Groups of ten to twelve-week-old rats were administered proguanil (2.9 mg/kg body weight) daily for 5 days and 6 weeks respectively. Thereafter, body and reproductive organ weights were taken, sperm parameters were analyzed, while the histology of the testis and epididymis were carried out. Also, serum levels of testosterone, luteinizing hormone and follicle stimulating hormone were determined. Sertoli cells obtained from sixteen to eighteen day-old-rats were cultured and treated with 0.3 μM to 10 μM of proguanil for 5 days after which Sertoli cell viability and nuclei integrity were determined. Also, the genetic expressions of transferrin and Glial cell line-derived neurotrophic factor (G.D.N.F.) were assessed. The data obtained were analyzed using the analysis of variance (ANOVA) with Students-Newman-Keuls multiple comparison test. The results showed duration dependent significant decrease in body and organ weights and also in sperm parameters. Serum testosterone level was significantly decreased while luteinizing hormone and follicle stimulating hormone showed no significant change. Duration dependent degeneration was also observed in the testis and epididymis of treated rats. These changes were restored in recovery experiment. Viability and DNA integrity of treated Sertoli cells reduced in a dose and duration dependent manner. However, the genetic expressions of transferrin and Glial cell line-derived neurotropic factor were normal. These results show that proguanil could induce infertility in rats which may act by distorting the blood-testis barrier formed by the Sertoli cells.

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Ethylene glycol monomethyl ether (EGME) induces testicular lesion in rats and human.  To investigate miRNAs expression in EGME testicular lesion, miRNA array assay and real-time RT-PCR analysis were conducted by using testis in rats treated with 50 and 2,000 mg/kg EGME for 6 and 24 hr. The expression of corresponding target gene for miRNAs was also examined. At 50 mg/kg, there were no changes in the gene expression and histopathological examination. At 2,000 mg/kg, slight decrease of phacytene spermatocytes with cell shrinkage and nucleus pyknosis at 6 hr and remarkable decrease (or cell death) of phacytene spermatocytes with Sertoli cell vacuolation at 24 hr were observed. After 24 hr, miR-449a and miR-92a decreased obviously and, miR-320, miR-134 and miR-188 increased, while only miR-760-5p increased after 6 hr. Above these miRNAs are reported to have an important role for spermatogenesis. The gene expression of Bcl-2, target for miR-449a, increased and therefore it is considered anti-apoptotic reaction has started in this stage. The expression of high mobility group AT-hook 2 (target of miR-92a) which regulates histone structure, was increased. Furthermore, histone deacethylase 4, targets for miR-320, was also affected. Above prohibiting apoptosis or activating epigenetic genes might be protective reaction to spermatocytes death under the miRNAs regulation in EGME testicular lesion.

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The purpose of our study was to examine the altered gene expression associated with nongenotoxic chemical-mediated liver hypertrophy and successive liver tumor promotion. Five-week-old male rats were fed a basal diet or a diet containing phenobarbital (PB) or clofibrate (CF) for 3 days, 4 weeks, and 13 weeks. Hepatic expression profiling of cell growth- and stress-related genes, as well as those involved in xenobiotic metabolism, was performed by DNA microarray and/or real time quantitative reverse transcription-polymerase chain reaction. The induction of liver hypertrophy and hepatic cytochrome P450 (CYP) isoforms (CYP2B1/2B2 for PB and CYP4A1 for CF) by PB and CF were clearly observed at all the treatment periods examined. Genes encoding DNA damage-inducible 45 (GADD45) family proteins, in particular GADD45g (GADD45 gamma) were down-regulated by treatment with either PB or CF for 4 and 13 weeks. The chemical-mediated development of liver hypertrophy, induction of hepatic CYPs, and suppression of hepatic GADD45g gene at week 13 disappeared at 4 weeks following cessation of the chemical treatment. Additionally, DNA microarray data indicated that cell cycle-related genes such as cyclins CCNB1 and CCNA2 and cyclin-dependent kinase inhibitor CDKN3 were also down-regulated by treatment with either PB or CF at 13 weeks. Since GADD45 functions as a chemical and radiation stress sensor by interacting with cyclins and cyclin-dependent kinase inhibitors, the decrease in the gene expression of GADD45g mRNA observed in this study, may be associated with nongenotoxic chemical-induced tumor promotion of hepatocarcinogenesis rather than liver hypertrophy. 

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The Nanoculture plate (NCP) is a recently developed plate which essentially consists of a textured surface with specific characteristics that induce spheroid formation: microfabrications with a micro-square pattern on the culture surface. The NCP can be used to generate uniform adhesive spheroids of cancer cell lines using conventional techniques without the need of any animal compounds. In this study, we assessed the performance of human hepatoma cell line HepG2 cells cultured with an NCP to evaluate the effects of the NCP on their hepatocyte-specific functions. The NCP facilitated the formation of three-dimensional (3D) HepG2 cell architecture. HepG2 cells cultured with an NCP exhibited enhanced mRNA expression levels of albumin and cytochrome P450 (CYP) enzymes compared to those cultured with a two-dimensional (2D) conventional plate. The expression levels of two specific liver-enriched transcription factors, hepatocyte nuclear factor 4α (HNF4α) and CCAAT/enhancer binding protein α (C/EBPα), were higher in HepG2 cells grown with the NCP than those in HepG2 cells grown with conventional plates before albumin and CYP enzymes expression levels were increased. The inducibility of CYP1A2 and CYP3A4 mRNA following exposure to inducers in HepG2 cells cultured with an NCP was comparable to that in HepG2 cells cultured with conventional plates, while the expression levels of CYP1A2 and CYP3A4 mRNA following exposure to inducers were higher when using an NCP than when using conventional plates. These results suggest that the use of an NCP enhances the hepatocyte-specific functions of HepG2 cells, such as drug-metabolizing enzyme expression, making the NCP/HepG2 system a useful tool for evaluating drug metabolism in vitro.

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Auranofin, a disease-modifying gold compound, has been empirically applying to the management of rheumatoid arthritis. We investigated a protective effect of auranofin against hepatic injury induced by cocaine. Cocaine (75 mg/kg) markedly increased serum alanine amino transferase (ALT) (4,130 IU/l) and aspartate amino transferase (AST) (1,730 IU/l) activities at 16 hr after treatment, and induced hepatic necrosis surrounding central veins in mice. Concurrently, overexpression of heme oxygenase-1 (HO-1), a rate-limiting enzyme for heme degradation and an oxidative stress marker, was identified at the edges of cocaine-mediated necrotic area. Auranofin (10 mg/ml, i.p.) significantly induced hepatic HO-1 protein in mice from 12 hr after treatment. Interestingly, pretreatment with auranofin resulted in the prevention of the increase of serum ALT and AST activities in a dose-dependent manner. On the other hand, although cocaine increased tumor necrosis factor α (TNFα) gene expression in mouse livers, cocaine-induced liver injury was observed in TNFα deficient mice as well as wild-type mice. Auranofin-inducded HO-1 gene expression was observed in human primary hepatocytes as well as mouse primary hepatocytes. The present findings suggest that auranofin is effective in preventing cocaine-induced hepatic injury, and HO-1 may contribute to protect aginst chemically-induced cytotoxicity.

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Methicillin-resistant Staphylococcus aureus (MRSA) along with other resistant bacteria have become a significant social and clinical problem. Therefore, there is an urgent need to develop bioactive compounds from natural products as alternatives to the very few antibiotics that remain effective. Recently, the efflux mechanism has been identified as the main contributor to antibiotic resistance in bacteria. This study therefore aimed to evaluate tetrandrine (TET), an efflux pump inhibitor (EPI), as a potential antibiotic against MRSA. We investigated the antimicrobial activity of TET against 17 MRSA strains, of which 3 selected strains were studied in further detail using a time-kill assay. When these bacterial strains (1 × 10⁶ colony-forming units (cfu)/ml) were incubated with TET in a time-kill assay, log-scale bactericidal activity was observed, which lasted for 24 hr. In addition, TET exhibits a synergistic effect when combined with the multi-drug resistance (MDR)-efflux pump substrate ethidium bromide (EtBr). Structure-function studies of the antibiotic activity of TET in combination with EtBr may lead to the discovery of more effective efflux pump inhibitors.

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Serum alkaline phosphatase (ALP) activity is frequently measured in toxicity studies. Itoh et al. (2002) reported that a commercially available polyacrylamide-gel (PAG) disk electrophoresis kit used in humans (AlkPhor System, Jokoh Co., Ltd., Tokyo, Japan) for identifying serum ALP isoenzymes was useful for veterinary clinicopathological diagnosis in mongrel dogs. In the present study, based on the report of Itoh et al. (2002), we tried to expand the application range of this kit to laboratory beagle dogs which are commonly used in toxicity studies. In order to identify the origin of each ALP isoenzyme, tissue ALP extracts from the liver, bone and small intestine and serum samples were treated with neuraminidase, anti-small intestinal ALP antibody, ALP inhibitor levamisole and/or wheat germ agglutinin (WGA). The main serum ALP isoenzymes in 5-month-old intact beagle dogs were bone-derived (bone and atypical ALP: corresponding to human variant bone ALP) and they tended to decrease with age. However, liver-derived ALP isoenzyme greatly increased in the serum of cholestasis model dogs. The cholestasis model dogs also had a large molecular ALP detected in the resolving gel. This ALP could be originated from intestinal ALP or corticosteroid-induced ALP (CALP), because the activity remained even after levamisole inhibition. CALP was observed in intact laboratory beagle dogs with individual differences. These results suggest that the present method is a useful tool for detecting serum ALP isoenzymes in laboratory beagle dogs and concomitant levamisole inhibition with another gel is applicable for the evaluation of organ toxicity.

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The beneficial effects of Acanthopanax divaricatus var. albeofructus (ADA) extracts have been assessed by mutagenic and anti-mutagenic activities by Ames test. Mutation of Salmonella typhimurium strains TA 98, TA 100, TA1535, TA1537, and Escherichia coli WP2 uvr A was assayed in duplicates by the procedure of Maron and Ames in the presence or absence of S9 mix. As a result, ADA extracts were not mutagenic for S. typhimurium strains TA 98, TA 100, TA1535, TA1537, and E. coli by the Ames assay. Anti-mutagenic activity was assayed by the Ames mutagenicity assay using histidine mutant of S. typhimurium strains TA 98 and TA 100, using the plate-incorporation method. 2-Aminoanthrancene (2-AA), 2-(2-furyl)-3-(5-nitro-2-furyl) acrylamide (AF-2), and sodium azide (NaN₃) were used as the mutagens. ADA extracts showed a strong anti-mutagenic activity against 2-AA-induced mutagenesis which requires liver-metabolizing enzymes, and the same extract exhibited inhibitory effects on AF-2 and NaN₃-induced mutagenesis in the absence of liver-metabolizing enzymes. The data indicate that ADA extracts contain anti-mutagenic activities against typical mutagens. The anti-mutagenic property of ADA provides additional health supplemental value to the other claimed therapeutic properties of the plant.

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Acetaminophen (APAP) is one of the most commonly used drugs worldwide to reduce fever, particularly in children. It is generally considered to be a safe drug. However, a number of studies have shown that regular use of APAP increases the risk of developing allergic diseases. Nonetheless, no animal models have been used to investigate these findings. Therefore, we aimed to create an animal model of APAP-induced pruritus in mice. APAP (0.25% and 0.5%) was administered via drinking water daily from infancy, and a suboptimal concentration of 2,4,6-trinitrochlorobenzene (TNCB) was applied repeatedly to each ear three times a week for 7 weeks to evoke chronic allergic contact dermatitis. Neither 0.25% nor 0.5% APAP was overtly hepatotoxic after 73 days of daily administration. Repeated challenge with TNCB evoked increase in the number of scratching bouts compared to day 1. This increase in the number of scratching bouts was significant in 0.25% and 0.5% APAP groups but not in the group treated with TNCB alone. Daily administration of 0.5% APAP significantly increased in the number of scratching bouts compared to TNCB alone on day 29. This animal model will be useful for investigating the mechanism underlying the increased risk of development of eczema caused by regular APAP use and for examining safer and more effective therapy with APAP.

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To investigate the effects of chlorella on methylmercury (MeHg) transfer to the fetus during pregnancy, female C57BL/6N mice (aged 10 weeks) were housed for 7 to 8 weeks, from 4 weeks before mating to birth, with diets containing 0% or 10% chlorella powder (CP) and MeHg-containing drinking water (2 µg Hg/ml). The consumption volume of the MeHg-containing water was limited to 15 ml/mouse/week throughout the experiment. Distilled water and a basal diet (0% CP) was given to control mice. Except for the mating period, during the 5^th week, mice were housed individually until parturition. Two neonates were randomly selected from each mother mouse within 24 hr after parturition for Hg analysis of the blood, brain, liver, and kidneys. Mother mice were sacrificed on the same day as neonates to obtain tissue samples for Hg analysis. The blood and brain Hg levels of both neonates and mothers in the CP diet group were significantly lower than those in the basal diet group. Although the hepatic and renal Hg levels were not significant in mothers between the two dietary groups, in neonates, the CP diet group showed significantly lower Hg levels in these tissues than the basal diet group. The results obtained here revealed that continuous CP intake suppressed MeHg transfer to the fetus, in addition to effective suppressing MeHg accumulation in brains of the mothers.

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The aim of this study was to elucidate whether dietary zinc-deficiency and its recovery play a role in controlling autonomic thermoregulation. We investigated the effects of dietary zinc-deficiency and its recovery on autonomic thermoregulation by measuring the rectal temperature, an index of deep body temperature.  The weaned male Sprague Dawley rats were randomly divided into the dietary zinc-deficient diet (0.6 mg zinc/kg diet) group and the control diet (35.2 mg zinc/kg diet) group, and were fed for 4 weeks.  In the recovery period, the rats of two groups were fed with the control diet for 3 weeks.  The rectal temperature was significantly decreased throughout the period of zinc-deficiency and the hypothermic responses during the experimental period were recovered to the control group levels at least within 1 week in the recovery process from dietary zinc-deficiency. These results suggest that the deep temperature in rats was clearly associated with the dietary zinc intake levels.

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