Streptozotocin (STZ), a toxic glucose analogue for pancreatic β cells, has been demonstrated as a treatment option for insulinoma. Glucose-dependent insulinotropic polypeptide (GIP), an incretin hormone, is secreted by K cells in the duodenum in response to food intake and acts on pancreatic β cells, leading to increased secretion of insulin. We previously reported that GIP gene expression in the liver is modified in a diabetic setting. However, the role of GIP in the liver has not been fully elucidated; we aimed to discover its effects on type 1 diabetes by focusing on GIP protein expression in a type 1 diabetes rat model following STZ administration. In this study, we assessed whether glucose and lipid metabolism affected GIP expression in the liver following STZ-induced diabetes. Diabetes was induced by intraperitoneal injection with 70 mg/kg STZ. We expected that blood glucose levels would be higher because of STZ treatment as a result of reduced insulin secretion in pancreatic β cells. Interestingly, GIP was expressed only in the liver of STZ-treated rats; however, blood glucose levels were not elevated. On the other hand, blood triglyceride and cholesterol levels were higher in STZ-treated rats with hepatic GIP protein expression than in control rats. These findings indicated that GIP protein expression in the liver possibly has hypoglycemic action, which may ameliorate the damage of pancreatic β cells induced by STZ treatment, rather than affect glycolipid metabolism.

Cadmium (Cd) is an environmental contaminant known to exert toxic effects on various tissues. Metallothionein (MT) acts as a protective protein with high affinity for Cd. However, among the four isoforms of MT, the physiologic function of MT-III in the liver of mice exposed to Cd chronically has not been determined. In the present study, we examined the susceptibility of MT-III null mice to hepatotoxicity by exposure to Cd for 67 weeks. Cd exposure reduced the body weight of wild-type mice but not MT-III null mice. MT-I/II null mice were also exposed to Cd; as expected, they died at 18 weeks of exposure. Long-term exposure to Cd exhibited mild hepatotoxicity to wild-type mice, and the effects of MT-III on hepatotoxicity were not extensive. Long-term exposure to Cd increased mRNA levels of MT-I and MT-II in the livers of wild-type mice and MT-III null mice. These results suggest that long-term exposure to Cd may contribute similar sensitivity to the livers of MT-III null mice as that of wild-type mice because expression of MT-I and MT-II was induced in the liver of both types of mice.

Cadmium (Cd) is a toxic heavy metal that is particularly damaging to proximal tubular cells of the kidney. Cd-induced renal toxicity is associated with perturbation of the ubiquitin proteasome system. Our previous study demonstrated that Cd increased gene expression of ubiquitin-coding genes, UBB, UBC, and UBA80. Notably, knockdown of the polyubiquitin gene UBB by siRNA transfection significantly decreased ubiquitinated protein levels that had been increased by Cd treatment. The present study showed that in contrast to UBB, knockdown of the monoubiquitin gene UBA80 did not diminish the Cd-induced protein ubiquitination in HK-2 cells. Taken together, our results suggest that polyubiquitin rather than monoubiquitin is preferably engaged in Cd-induced accumulation of ubquitinated proteins in HK-2 cells.

Cadmium (Cd) is a non-essential toxic metal widely distributed throughout the environment. Cd is reported to be toxic to the fetus, so we aimed to investigate changes in gene expression in the liver of fetal mice exposed to Cd during gestation. We exposed pregnant mice to Cd (5 mg/kg) and collected fetal livers to perform DNA microarray analysis. The expression of 1,669 genes was found to be increased more than 2.0-fold, while that of 194 genes was decreased less than 0.5-fold in fetal livers following Cd exposure during gestation. We categorized the Cd-changed genes in terms of cell cycle and cell proliferation, apoptosis, cell growth and differentiation, cellular defense, metabolism, transport, transcription, signal transduction, metal homeostasis, and ubiquitin proteasome system. These results provide useful information about fetal toxicity following gestational Cd exposure.

To reduce the false positives in in vitro mammalian genotoxicity tests for chromosomal damage, which is caused by severe cytotoxicity, OECD test guidelines have adopted new cytotoxicity indices, and we developed a retrospective evaluation workflow that can be used to identify false positives that are ultimately deemed negative when the new indices are used. Overall, 14 chemicals were estimated to have a negative result. The aim of the present study was to validate the strategy used for the identification of false positives via re-evaluation of these 14 chemicals using the in vitro micronucleus test to evaluate the clastogenicity and aneugenicity. As a result, 11 chemicals became negatives, whereas the other three chemicals remained positives. In particular, chemicals of high priority for re-evaluation are more likely to become negative. Therefore, we conclude that our developed strategy is useful to find false positives that have specifically shown chromosomal aberrations at doses inducing strong cytotoxicity. These chemicals can be correctly evaluated as negatives using the new cytotoxicity indices.

Bacillus coagulans is a lactic acid, spore-forming gram-positive bacteria that has the potential of probiotic benefits. To evaluate the toxicological profiles of Bacillus coagulans strain SANK70258 (active element of LACRIS^TM-S), a 90-day oral gavage dose study was conducted in rats. The microbe was administered by oral gavage to 6-week old Crl:CD(SD) rats (10 males and 10 females/group) for 90 days at dose levels of 0, 500, 1000, and 2000 mg/kg/day. According to the results, no deaths occurred in either males or females, and no treatment-related changes were observed in any clinical sign including a detailed observation with functional observational battery (FOB), functional test, motor activity, body weight, food consumption, ophthalmoscopy, urinalysis, hematology, blood chemistry, organ weight, necropsy or histopathology. In conclusion, Bacillus coagulans strain SANK70258 had no subchronic toxicity in rats and the no-observed-adverse-effect-level (NOAEL) was judged to be greater than 2000 mg (about 1 × 10¹² CFU)/kg/day. The microbe is harmless and can be used as a probiotic.

Most living beings including human beings have endogenous 24-hr rhythms, called circadian rhythms. The most important factor to alter the circadian rhythms is light. Therefore, this biological rhythms can easily disrupt by exposure to light at night (LAN). In modern society, rotating shift work with night work (night shift work) is essential working arrangement and induces circadian disruption to workers by LAN exposure. Epidemiologic studies indicate that a long-term biological rhythm disruption induced by a long-term LAN exposure causes many serious health disorders including immune dysfunction. Cd also dysregulates the immune system. Main exposure source of Cd is tobacco smoking and food intake in living environment. Further, in work places with handling Cd, occupational environment also be an exposure source with high concentration of Cd. In this report, we paid attention to and examined the combined effect of Cd and LAN on immune system. Mice were kept under the light/dark shift condition and/or injected CdCl2 (0.33 or 1.0 mg/kg) twice a week for 4 weeks followed by sacrificing 7 days after the last injection. The mitogen activity induced by phytohemagglutinin was markedly reduced by shift condition; further, this reduced mitogen activity was completely inhibited by additional Cd treatment. This result indicates a possibility of enhancement of immune dysfunction by Cd and LAN combination. Our result suggests that Cd exposure when being the circadian disruption had potential for more inhibition of immune function.

Acute liver disease may develop due to various causes and occur by different mechanisms. Carbon tetrachloride (CCl₄), a well-known hepatic toxicant, was selected as a model of alkylating agents that do not induce glutathione depletion. Our previous study indicated CCl₄-induced hepatotoxicity was decreased by pretreatment with the Japanese herbal medicine “Juzen-taiho-to” (JTX), suggesting that prophylaxis with JTX protects mice from CCl₄-induced acute hepatic toxicity. In contrast, bromobenzene (BB) is a known glutathione-depleting agent. Although BB-induced hepatotoxicity also promotes lipid peroxidation, the mechanism of hepatic injury is different from that of CCl₄. Hence, in this study, we investigated whether pretreatment with JTX ameliorated BB-induced hepatotoxicity. Mice injected with BB showed increased plasma levels of hepatic injury markers (alanine aminotransferase and aspartate aminotransferase) in addition to hepatic lipid peroxidation. Pretreatment with JTX decreased BB-induced plasma levels of hepatic injury markers. BB-induced hepatotoxicity is mainly caused by oxidative stress. JTX pretreatment also decreased BB-induced lipid peroxidation. Our results suggest that JTX has the potential to protect against BB-induced hepatotoxicity and modulate oxidative stress.

Ocular instillation toxicity studies (OITSs) are one of general toxicity studies. Yet, OITSs have a unique characteristic that the test article is directly administered as eye drops to the target organ. Compared with general toxicity studies aiming systemic exposure, the study design of OITSs is somewhat distinctive in selecting test species, dosing formulation, administration volume/frequency and ocular examinations. After the administration of eye drops, the exposure level is high in the ocular surface, whereas the bioavailability in the eye balls, especially in the posterior segment, is low. In contrast to the general toxicity studies aiming systemic exposure, the absolute systemic exposure level in OITSs is generally low, while the systemic bioavailability is relatively high. These pharmacokinetic features determine the profiles of local and systemic toxicities in OITSs. Systemic toxicities are more often found in animals of relatively small body size, and are in most cases related with pharmacological actions. Current progress in ophthalmologic imaging technologies enables advanced safety evaluation using imaging biomarkers. Bioanalysis detecting drug levels present in blood in trace amount leads to a detailed safety assessment of systemic toxicity and yields accurate safety margins. Recognizing the peculiar characteristics of OITSs, toxicologists need to propose an appropriate study design and strategy of safety evaluation. Further discussion may be awaited on rationales for testing both sexes, and for conducting separated toxicity studies to evaluate systemic toxicity. This mini-review provides insight regarding current status and points to consider of OITSs.

A unique medicinal mushroom Antrodia cinnamomea has been used for centuries to treat various human diseases. Recent studies revealed its potent pharmacological effects including anti-cancer, anti-inflammation, anti-oxidant, anti-diabetic, neuroprotection and hepatoprotection. The present study was aimed to investigate the toxicological effects of A. cinnamomea health food product “Leader Antrodia cinnamomea Capsule (LACC)” by measuring its genotoxic, oral toxic and teratotoxic effects in vitro and in vivo. Result of Ames test with 5 strains of Salmonella typhimurium shows no sign of increase in the numbers of revertant colonies upon exposure to LACC. Treatment of Chinese Hamster Ovary cells (CHO-K1) with LACC did not affect increase in the frequency of chromosomal aberration in vitro. In addition, treatment with LACC did not affect the proportions of immature to total erythrocytes and the number of micronuclei in the immature erythrocytes of ICR mice. Moreover, acute oral toxicity (14-days single-dose) or prolonged oral toxicity (28- and 90-days repeated oral dose) tests with rats showed that there were no observable adverse effects were found. Furthermore, teratological studies with LACC (500-2500 mg/kg/day) for 20 days, shows no observable segment II reproductive and developmental toxic evidences in pregnant SD rats and their fetus. These toxicological assessments strongly support the safety efficacy of LACC for human consumption.

The oral toxicity of phorate oxon (PHO), with emphasis on gender- and age-related effects, was characterized in the Sprague-Dawley rat. The oral LD₅₀ (95% fiducial limits) for PHO in corn oil was 0.88 (0.79, 1.04) mg/kg in males and 0.55 (0.46, 0.63) mg/kg in females with a probit slope of 15. Females had higher baseline blood cholinesterase titers, but males were significantly more tolerant. Younger rats generally had lower absolute cholinesterase blood titers. However as PHO challenges increased, baseline-normalized cholinesterase inhibition was independent of age and gender. Butyrylcholinesterase (BChE) and especially acetylcholinesterase (AChE) in brains of younger females were affected more than that in either males or older females. In summary, while female rats, especially older females, had higher titers relative to males, female rats were more susceptible in terms of absolute cholinesterase inhibition and 24‑hr lethality data, but the differences were not observed when titers were normalized to baseline levels.

Female C57BL mice were exposed to a low level of mercury vapor (Hg⁰, 0.096 mg/m³) and was given the solution containing 5-ppm methylmercury (MeHg) during the growth period to examine the influence on the neurobehavioral function after birth. Exposure period was 4 weeks at 3 to 7 weeks of age. At 10 weeks of age, three behavioral tests were conducted; open field (OPF) test, passive avoidance response (PA) test, and eight-arm radial maze (RM) test. To evaluate the influence of aging, additional behavioral tests were performed at 79 weeks of age. With respect to the results of the three behavioral tests conducted at 10 to 14 weeks of age, there were no significant differences between the Hg⁰/MeHg/Hg⁰+MeHg and control groups. Furthermore, there were also no significant differences between each exposure and control group on behavioral tests performed at 79 to 83 weeks of age after the completion of mercury exposure. The concentration of mercury in the brain after the completion of exposure was the highest in the Hg⁰+MeHg group, followed by the MeHg and Hg⁰ groups. The values in the Hg⁰+MeHg and MeHg groups were ≤ 3.0 μg/g. The value in the Hg⁰ group was ≤ 1.0 μg/g. There were no differences in the brain concentration of mercury after 1 year between the Hg⁰/MeHg and control groups. However, in the Hg⁰+MeHg group, it was significantly higher than in the control group, suggesting that the disappearance of mercury in the brain is delayed in comparison with the exposure to Hg⁰ or MeHg alone. These results showed that there was no influence of low-level Hg⁰+MeHg exposure during the growth period on neurobehavioral manifestation. However, the disappearance of mercury in the brain was delayed in comparison with the exposure to Hg⁰ or MeHg alone.

In hospitals and pharmacies, many kinds of pharmaceutical tablets are frequently crushed to powder in order to facilitate administration. However, there is concern about the influence of this process on the health of pharmacy workers. In this study, we conducted model experiments to estimate the potential exposure of pharmacy workers to pharmaceutical particles during tablet crushing and transfer of powder. Tablets were crushed in a tablet mill. Particulates released into the air during and after milling and transfer to a mortar were counted with a dust counter, and collected on the filter of an air sampler. Amounts of pharmaceutical active ingredients collected on the filter were determined by high-performance liquid chromatography (HPLC). During tablet crushing, particulates were released into the air. We found that the particle concentration in air was highest during transfer of the powder from the tablet mill to the mortar. The amount of active ingredient collected on the filter of the air sampler was significantly higher in the case of Loxonin, as compared with Neurovitan. Although conditions under which tablets are crushed are likely to vary greatly in practice, our results and calculations at least indicate that unmasked workers might routinely inhale microgram levels of active ingredients during tablet crushing and transfer of the resulting powder. Our results should be helpful in designing appropriate protective measures and in developing professional guidelines to minimize occupational exposure of pharmacy workers to drugs.

The Pig-a gene is well-known to encode a key enzyme essential in the biosynthesis of glycosylphosphatidylinositol (GPI), which attaches CD molecules (Cluster differentiation), such as CD55 and CD59, to red blood cells (RBCs) membranes. In this study, the blood was marked with the special antigen CD45-PE (Phycoerythrin) to separate the erythrocytes from the leukocytes, then the Pig-a mutant frequency (MF) of RBCs could be investigated without pivotal antigen CD59-FITC (Fluorescein isothiocyanate), and the optimal ENU concentration was determined to be 100 mg/kg/day. The optimal cell number to count and the stability of specimens were determined. At last, the in vivo Pig-a gene mutation assay was utilized to detect the potential genotoxicity of Cis-Dichlorodiammineplatinum (DP), Procarbazine Hydrochloride (PH), and Triptolide (TP). The results indicated that the Pig-a gene mutation obviously occurred in the PH treatment group. In the DP treatment group, an irregular shape with a slightly serrated border in erythrocytes was observed in the blood smear. However, no obvious Pig-a gene mutations were detected in the DP treatment and TP treatment groups under the experimental conditions. In conclusion, this primary in vivo Pig-a gene mutation assay will provide an easy protocol to use in screening potential genotoxic compounds.

The Bhas 42 cell transformation assay is a sensitive and effective mutation assay, and it has been widely used to detect and evaluate potential carcinogenic risks, including initiation assay and promotion assay. In this transformation assay, the number of transformed foci is the only criterion. However, it is nonquantitative because the shape is irregular. Nevertheless, a high-throughput Bhas 42 cell transformation assay was developed, which includes the usage of hydrogen peroxide in 96-well plates. In this way, transformed foci are quantitatively analysed in proportion to the optical density value (OD value). In this study, the Bhas 42 cell transformation assay was established using the criterion of the OD value. As a result, 3-methylcholanthrene (3-MCA) presented strong tumour initiation activity, whereas the 12-O-tetradecanoylphorbol-13-acetate (TPA) showed strong tumour promotion activity. Lowdaphne Stringbush Flower and Leaf (LSFL, also named as Wikstroemia chamaedaphne (Bunge) Meissn) and Rhubarb (Rheum palmatum L.) showed strong tumour promotion activity, but the tumour promotion activity of Lilac Daphne Flower Bud (LDFB, also named as Daphne genkwa Siebold & Zucc) was equivocal. Surprisingly, the OD value displayed a strong correlation with the number of transformed foci. In conclusion, the high-throughput Bhas 42 cell transformation assay was feasible.

Carbon tetrachloride (CCl₄) is a well-known hepatotoxic chemical. Exposure to CCl₄ produces free radicals, which induce oxidative stress and cause hepatic injury. We demonstrated previously that pretreatment with zinc (Zn), which induces metallothionein (MT) expression, prevents CCl₄-induced lethal toxicity in a dose-dependent manner. While MT has been suggested as a possible hepatoprotective protein, its mechanism of protection remains unknown. In the current study, we evaluated the protective mechanism of MT, an endogenous scavenger of free radicals, against CCl₄-induced toxicity through subcutaneous administration of 50 mg/kg Zn (as ZnSO4) once daily for three consecutive days, prior to a single intraperitoneal injection of 4 g/kg CCl₄ in male ddY mice. Our results showed that Zn pretreatment significantly decreased aspartate aminotransferase and total cholesterol levels, 6-hr after CCl₄ injection, as well as lipid peroxidation. Moreover, CCl₄-induced hepatic calcium level was downregulated by pretreatment with Zn while Zn-induced MT expression decreased by more than 500 μg/g liver (43%) in the Zn + CCl₄-treated group, implying that MT was consumed by CCl₄-induced free radicals. These findings suggest that prophylaxis with Zn protects mice from CCl₄-induced acute hepatotoxicity, presumably by inducing the expression of free radical-scavenging MT.

Perfluoroalkyl acids (PFAAs) have been widely used since 1950s. The long chained-PFAAs, such as perfluorooctanoic acid (PFOA) are persistent and bio-accumulative, and are detected in humans. PFOA, which is a peroxisome proliferator activated receptor (PPAR) α agonist, has been suggested to be a carcinogen in epidemiological and animal studies. In some studies PFOA is shown to be non-mutagenic in Ames and micronucleus tests, but in other studies it caused oxidative DNA damage and micronucleus formation. However, there has been no report that has examined whether PFOA-induced genotoxicity is mediated by PPARα. In order to relate genotoxicity of PFAAs to PPARα, we conducted two kinds of comet assays (cellular and acellular), a micronucleus (MN) test, and a TK mutation assay with and without PPARαantagonists by using human lymphoblastoid cells. PFAAs at 125-1000 μg/mL showed positive responses in the cellular comet assay but not in the MN test and TK mutation assay. A PPARα antagonist GW6471 (2 μg/mL) only partly reduced PFOA-induced DNA damage (in the cellular comet assay), but abolished PFOA-induced intracellular ROS formation. PFAAs with 8-12 carbons also showed positive responses in the acellular comet assay where there is no cellular function such as PPAR. Therefore, PFOA-induced DNA damage was partly related to the oxidative stress via PPARα, without manifestation of chromosome aberration and point mutation in this cell line.

A typical DNA-damaging agent 4-nitroquinoline 1-oxide (4NQO) is known as an experimental oral carcinogen. Although 4NQO was initially characterized as a UV-mimetic agent, it shows more complex effects inducing production of various covalent adducts, oxidative damage and DNA single strand break in DNA. To understand roles of histone acetyltransferase GCN5, which protects cells against UV-irradiation, on repair of 4NQO-induced DNA damage, we studied the sensitivity of chicken homozygous DT40 mutants, ΔGCN5, against 4NQO. After 4NQO treatment, the viability of ΔGCN5 was appreciably reduced (to ~25% at 6 hr) as compared to that of wild type DT40. Semiquantitative RT-PCR showed that transcription of DNA polymerase η (POLH) gene whose deficiency is responsible for a variant form of xeroderma pigmentosum was drastically down-regulated in ΔGCN5 (to ~25%). However, overexpression of POLH could not rescue ΔGCN5 from the enhanced sensitivity to 4NQO, unlike UV-irradiation. Our data suggested that GCN5 participates in control of the sensitivity against 4NQO, and the molecular mechanisms of GCN5-mediated repair of the 4NQO-induced DNA lesions are highly complex.

Quetiapine fumarate (QF) is a widely used antipsychotic agent for the first-line treatment of schizophrenia, with good tolerability and compliance in humans and no observed adverse effects against liver. Taking advantages of zebrafish, which can be utilized in rapid drug screening and acute toxicity assessment, our study determined a certain hepatotoxicity of QF for the first time, indicating a potential safety hazard of this commonly used drug to humans.

Methyl cinnamate (MC) and essential oils containing MC possess beneficial antimicrobial, antifungal, and insecticidal effects, among others. Such effects are related to the biocidal action of MC. The antioxidant activity of MC has also been reported elsewhere. It has been suggested that MC may be cytotoxic to cells exposed to oxidative stress. To test this possibility, the effect of MC on rat thymocytes was examined while the cells were subjected to oxidative stress induced by hydrogen peroxide (H₂O₂). Flow cytometric techniques with appropriate fluorescent probes were used for quantification. MC increased cell vulnerability to oxidative stress via acceleration of the cell death process and/or potentiation of oxidative stress. The use of MC is widespread because of its beneficial actions, and thus further attention should be paid to whether MC is effective under oxidative stress.

Cancer cells can develop resistance to anti-cancer agents. Although some mechanisms have been suggested for this resistance to treatments, further detailed research is required. Historically, sesquiterpene lactones (SLs) have been shown to exhibit toxicity in humans and animals due to their chemical nature. Among the SLs identified to date, (–)-xanthatin, which was originally obtained in an extract from Xanthium strumarium, is reportedly less toxic to animals. Furthermore, accumulating evidence suggests that some SLs can kill cancer cells. Therefore, we have focused on (–)-xanthatin and established a method for the chemical synthesis of SLs in order to obtain a pure form. Although we showed that (–)-xanthatin exerts anti-proliferative effects on highly aggressive (poorly differentiated) human MDA-MB-231 breast cancer cells via a mechanism involving the induction of GADD45γ, a tumor suppressor gene, other molecular target(s) of the molecule have not yet been identified. In the present study, we employed chemically synthesized pure (–)-xanthatin to investigate the targets involved in (–)-xanthatin-mediated cell death. The results obtained revealed marked increases in FosB, the expression of which is suggested to be down-regulated in poorly differentiated breast cancers, and the stimulated expression of FosB as well as cell death by (–)-xanthatin was abrogated by N-acetyl-L-cysteine (a ROS-scavenging agent). The possible participation of FosB in (–)-xanthatin-evoked cell death is discussed.

Organic-inorganic hybrid molecules, which have organic structure and metal atoms, can exhibit various biological activities that are distinctly different from their components. Consequently, organic-inorganic hybrid molecules are considered an effective tool to analyze biological systems. Herein, we investigated the cytotoxicity of zinc, copper, and rhodium complexes, with either 1,10-phenanthroline or 2,9-dimethyl-1,10-phenanthroline as a common ligand, in cultured vascular endothelial cells. The copper complexes, that is, dichloro(1,10-phenanthroline) copper and dichloro(2,9-dimethyl-1,10-phenanthroline) copper, exhibited high cytotoxicity accompanied by considerable accumulation inside the cells. Potassium tetrachloro(1,10-phenanthroline) rhodate also exhibited cytotoxicity and considerable accumulation. Thus, it was found that the cytotoxicity of organic-inorganic hybrid molecules to vascular endothelial cells depends on the interaction between the intramolecular metal and ligand, which facilitates their uptake by the cells.

Animal models can help determine the etiology of neurodegenerative diseases such as Parkinson’s disease. Here, we conducted transcriptome analysis of the rotenone model of Parkinson’s disease in rats. Exposure of 9-week-old Wistar rats to rotenone at 3 mg/kg/day for 14 days reduced spontaneous motor activity to 49% of that of control rats. Immunohistochemical analysis revealed increased expression of major histocompatibility complex (MHC) molecules in the substantia nigra of rotenonetreated rats, which was deduced by DNA array analysis. Further, gene set enrichment analysis of the transcriptome extracted the presence of a cytokine network, which included TNF-α. The expression of these proteins tended to be reduced at developed states of the disease. Thus, our analyses of the rotenone rat model still provides new insights into the etiology of Parkinson’s disease.

Predicting drug-induced liver injury is still a big challenge for the research and development of pharmaceuticals. Primary human hepatocytes (PHH) are the gold standard cell sources for examining drug metabolism, drug-drug interaction (DDI) and hepatotoxicity in humans in vitro. However, their supply does not meet the demand of laboratories sufficiently, and only a limited number of lots of PHH are commercially available. Recently, human iPS cell-derived hepatocytes have been reported and are anticipated to be used as an alternative to PHH. However, drug metabolizing activities of these human iPS-derived hepatocytes have not been well characterized and quantitative target values for PHH alternatives remain unknown. In this study, we collected 179 data sheets of commercially available PHH lots from 4 vendors to clarify the characteristics of PHH in drug metabolism and DDI. We identified the most frequently observed value ranges (MFRs) of the activities of major drug metabolizing enzymes (CYP3A4/5, CYP1A2, CYP2C9, CYP2C19, CYP2D6, CYP2E1, UGT, and SULT) and drug transporters. Moreover, we also identified MFRs of fold changes of CYP inductions by each typical inducer both in the mRNA levels and the enzyme activity levels of CYP1A2, CYP2B6, and CYP3A. We suggest that these MFRs are the first milestone to develop and evaluate human iPS cell-derived hepatocytes as alternatives to PHH in pharmaceutical research for assessing absorption, distribution, metabolism, excretion, and toxicity.

According to International Conference on Harmonization guidelines, each drug in development for administration to women of child-bearing potential must be tested for possible developmental toxicities using at least two species (a rodent and non-rodent). With the high cost and slow pace of embryonic-fetal toxicity testing in mammals, both the zebrafish embryonic toxicity test (ZET) and mouse embryonic stem cell test (mEST) have been shown to be useful to assess developmental toxicity of various chemical compounds, including human pharmaceutical drugs, in a high-throughput manner. However, comparative study of the sensitivity and specificity of these methods using the same set of human pharmaceutical drugs is scarce. In this study, we assessed developmental toxicity tests of 39 chemical compounds, including human pharmaceutical drugs, in both the ZET and mEST. The accuracy, sensitivity, and specificity of the ZET were 69%, 59%, and 82%, respectively. The accuracy, sensitivity, and specificity of the mEST were 64%, 50%, and 82%, respectively. As a result, both the ZET and mEST showed acceptable accuracies compared with rat embryo-fetal toxicity study and Food and Drug Administration pregnancy categories. By comparing the results between the ZET and mEST, we identified different types of true positives and true negatives. Thus, complementary tests using both the ZET and mEST may better predict the developmental toxicity of human pharmaceuticals.

We have previously identified Akr1 as one factor in the reduction of methylmercury toxicity in yeast, and reported that Akr1’s palmitoyl transferase activity is necessary for reducing methylmercury toxicity. Palmitoylation transferases are highly conserved from yeast to humans. As such, we prepared SH-SY5Y neuroblastoma cells capable of overexpressing HIP14, a human homolog of Akr1, and investigated the cell’s sensitivity to methylmercury. Our results showed that, compared with the control, the cells were resistant to methylmercury. Thus, we believe the palmitoyl transferase activity of HIP14 in humans can play an important role in the reduction of methylmercury toxicity.

The in vivo nanotoxicity of nanoparticles is drawing increased attention as concerns grow over the biosafety of nanotechnology. TiO₂ nanoparticles are coated to decrease the potential of harmful effects due to their photoactivity. Rutile-type alumina-coated titanium dioxide nanoparticles (Al₂O₃-TiO₂-NPs) are frequently used in cosmetics to improve their dispersion stability. We herein discuss the effects of Al₂O₃-TiO₂-NPs exposure during pregnancy on mouse spermatogenesis. Pregnant mice were injected five times, once each with 0.1 mL of sequentially diluted concentrations of a Al₂O₃-TiO₂-NPs suspension (1, 10, 100 or 1,000 μg/mL) and received doses of 0.5, 5, 50 and 500 μg, respectively. Prior to injection, the size distribution of the Al₂O₃-TiO₂-NPs was analyzed by dynamic light scattering (DLS) measurement. The average diameter was increased in dose-dependent manner from an average of 153.8 nm to 654.6 nm. The offspring testes were examined at 12 weeks postpartum. The agglomerates in the testicular sections were small (< 200 nm). They were confirmed by the characteristic peaks of the Ti and Al elements on field emission-scanning electron microscope/energy dispersive X-ray spectroscopy (FE-SEM/EDS). Low cellular adhesion and degenerated Sertoli cells were observed in the seminiferous epithelium of all of the Al₂O₃-TiO₂-NP recipient groups by a histological analysis. The detrimental function of the Sertoli cells resulted in the formation of abnormal spermatozoa. The results suggested that Al₂O₃-TiO₂-NPs that transferred from the mother’s body affected spermatogenesis in the offspring.

To investigate the adaptive response of astrocytes to an environmental chemical, methylmercury (MeHg), we herein examined transcription factors activated in rat primary astrocytes treated with 10 μM MeHg for 6 hr using the Combo Protein/DNA Array. The activities of 38 transcription factors increased by 5-fold or greater in response to MeHg. The paired box family of transcription factors were strongly activated by MeHg exposure, which are considered to be activated downstream of NGF and BDNF upregulation induced by MeHg. Nrf-2 (an antioxidant response element) was also activated, which has been reported to act on MeHg detoxification.

Δ⁹-Tetrahydrocannabinol (Δ⁹-THC), an active ingredient of marijuana, evokes a number of biological effects including anti-cancer and anti-estrogenic actions. We and others have so far focused on and investigated the latter action. We recently reported that Δ⁹-THC up-regulates the expression of estrogen receptor β (ERβ, ESR2), resulting in the abrogation of 17β-estradiol (E2)-mediated ERα signaling (Takeda et al., Chem. Res. Toxicol., 26, 1073-1079, 2013). This finding may shed light on the possible endocrine-disrupting mechanism(s) employed by cannabinoids including Δ⁹-THC. Although previous studies have suggested that HU-210, a synthetic analog of Δ⁹-THC, evokes a set of endocrine alterations closely related to those of Δ⁹-THC, none have examined the effects of cannabinoids with a focus on the expression of ERβ, a “suppressive” molecule for ERα-mediated signaling. Thus, we herein determined whether HU-210 is also an endocrine modifier similar to Δ⁹-THC using ERα-positive MCF-7 cells in which the expression of ERβ is maintained at very low levels. The results of the present study revealed that HU-210, despite having a similar structure to Δ⁹-THC, did not modulate E2/ERα signaling or induce ERβ.

In recent years, human induced pluripotent stem cell-derived cardiomyocytes (hiPSCMs) and human embryonic stem cell-derived cardiomyocytes (hES-CMs) have been widely used to develop cardiotoxicity assessment systems. To accurately evaluate the arrhythmogenic potential of drugs, we tested three types of hiPS-CMs (iCell Cardiomyocytes, Cellartis hiPS-CM, and Cor.4U) and a type of hES-CMs (Cellartis Pure hES-CM) using impedance technology. All CMs were cultured as confluent monolayers and their beating activity was analyzed with an impedance-based real-time monitoring instrument, the xCELLigence RTCA Cardio System. Measurement of impedance provided information about the CM beating rate and impedance amplitude between negative and positive peaks. Although all CMs except iCell CMs showed similar beating rates, their amplitudes were different. In addition, the patterns of contraction and relaxation were notably different between iCell CMs and the other CMs. The hiPSCMs and hES-CMs were treated with two arrhythmogenic drugs, sotalol and moxifloxacin, for 24 hr; the results showed that the drugs induced arrhythmic beating patterns on all CMs, although the response was varied. Thus, a more suitable type of CM remains to be found for optimal evaluation of the arrhythmogenic potential of drugs.

Structured regularization is a mathematical technique which incorporates prior structural knowledge among variables into regression analysis to make a sparse estimation reflecting relationships among them. Abundance of structural information in biology, such as pathways formed by genes, transcripts, and proteins, especially suits well its application. Previously, we reported on the first application of latent group Lasso, a group-based regularization method, in toxicogenomics, with genes regulated by the same transcription factor treated as a group. We revealed that it achieved good predictive performances comparable to Lasso and allowed us to discuss mechanisms behind liver weight gain in rats based on selected groups. Latent group Lasso, however, does not lead to a sparse estimation, due to large group sizes in our analytical setting. In this study, we applied graph-based regularization methods, generalized fused Lasso and graph Lasso, for the same data, with regulatory networks formed by transcription factors and their downstream genes as a graph. These methods are expected to make a sparser estimation since they select variables based on edges. Comparisons showed that graph Lasso generated an accurate, biologically relevant and sparse model that could not have been possible with latent group Lasso and generalized fused Lasso.

Endocrine disruptors are ubiquitous in nature. Indeed, some pesticides are not only insect killers, they also function as “endocrine disruptors” in humans and animals; therefore, concern regarding the possible health risks of pesticides for humans is growing. However, few studies have investigated the adverse effects induced by pesticides. One study previously suggested that fipronil reduced the levels of 17β-estradiol (E2), a natural ligand for estrogen receptor α (ESR1, ERα), in female Wistar rat plasma. In the present study, we focused on three relatively novel insecticides: fipronil (a phenyl pyrazole), acetamiprid (a neonicotinoid), and imidacloprid (a neonicotinoid). The effects of these 3 insecticides on the expression of ERα as well as E2/ERα-mediated signaling were examined in an ERα-positive MCF-7 human breast cancer cell line. The results obtained showed that fipronil selectively down-regulated the expression of ERα, and its regulated gene, CDC2, and also that PES1, an upstream signaling molecule for the regulation of ERα, was suppressed by the insecticide. We discussed the potential of fipronil as an antiestrogen.

We previously reported the skin sensitizing potential of 41 different chemicals using a modified local lymph node assay with an elicitation phase (LLNA:DAE). Of these, the measured skin sensitizing potential for 39 was consistent with the reported results from a murine local lymph node assay (LLNA). Furthermore, the increase in the weight of the left ear lymph node per 1% test dose of each chemical (as measured using the LLNA:DAE method) seemed to reflect the class of skin sensitization potency as evaluated by LLNA. However, this relationship has never been quantitatively investigated. In the present study, we sought to determine the connection between the results of the LLNA:DAE method, in terms of lymph node weight, and the skin sensitization potency as indicated by the EC3 value. We evaluated the concentration of each chemical needed to increase the weight of the left ear lymph node by 2 mg, 1 mg, 0.5 mg, and 0.25 mg during the elicitation phase using the LLNA:DAE method. These concentrations were then assessed in terms of how they directly compared to the LLNA EC3 value as well as how they matched the range of the EC3 value (under 1%, 1-10% and over 10%). Finally, clear quantitative relationships between the EC values at 1 mg and the EC3 values were observed using various statistical regression models. These results indicate that results obtained using the LLNA:DAE method can potentially be used to speculate the EC3 range found using LLNA method for these 29 kinds of skin sensitizers.

Acotiamide hydrochloride hydrate (acotiamide-HH) is the first approved drug in the world for the treatment of patients with functional dyspepsia in Japan. A statistically significant increase in the incidence of endometrial adenocarcinoma was found in a 104-week carcinogenicity study in rats, in a non-dose-dependent manner, and it was considered that further evaluation was required to clarify this issue. Therefore, we performed a uterotrophic bioassay using immature female rats, which is mentioned in the Organization for Economic Co-operation and Development (OECD) Guideline 440, to evaluate the effect of acotiamide-HH on estrogen, which is one of the most important mechanisms causing increase in the incidence of endometrial adenocarcinoma. The positive control substance selected was 17α-ethinyl estradiol (EE). While EE caused a dose-dependent increase in uterine weight, no increase in uterine weight, histopathological changes, or endometrial proliferation activity were observed in the acotiamide-HH treatment groups at doses of up to 1000 mg/kg. Based on this result, we concluded that acotiamide-HH has no potential risk to cause imbalance of the sex hormone environment in female rats.

Chronic infection with the hepatitis C virus (HCV) frequently induces steatosis, which is characterized by the accumulation of lipid droplets (LDs) in hepatocytes. Steatosis is a significant risk factor for liver cancer. The HCV structural protein core is distributed on the surface of the endoplasmic reticulum (ER) and in LDs, thereby increasing LD levels. In this work, we attempt to elucidate the effect of the core protein on LD generation using yeast cells. We found that the core localized to the cytosolic surface of the ER in yeast and is able to increase LD levels when overexpressed from an inducible GAL1 promoter for 3 hr. The effect of the core was conserved among three different HCV serotypes: 1b, 2a and 3a. While the ER stress inducer tunicamycin both elicited an unfolded stress response (UPR) and increased LD levels, the core did not induce the UPR. The RNA viral genome changes rapidly due to its high mutation rate in order to replicate under a variety of circumstances. Our observations suggest a functional analogy between core function in hepatocytes and in yeast cells and thus might be applicable to the screening of small molecules that impair the core-ER interaction.

Picrorhiza kurroa is a well-known ayurvedic or herbal medicine which is used very commonly in the treatment of various diseases. Therefore, we studied the oral toxicity of Picrorhiza kurroa rhizome extract in rats. A single high dose of the extract at 2000 mg/kg body weight was tested on Wistar rats. Mortality/viability and clinical signs were recorded on test day 0 (prior to administration), 7, 14 and at death. All animals appeared normal from day one to throughout the experimental procedure. Picrorhiza kurroa rhizome extract is non-toxic to rats and helped in weight gain with LD₅₀ > 2000 mg/kg body weight. Oral administration of Picrorhiza kurroa is not connected with any toxicologically significant effects and the data could provide satisfactory preclinical evidence of safety to launch a clinical trial on a standardized formulation of the plant extracts.

Any devices used in toxicity studies should be validated. In the present study, the physiological conditions of rats implanted with a new micro-infusion pump, iPRECIO^®, were examined to evaluate its availability for toxicity studies. Five or six animals/group of 6-week-old male CD(SD) rats received either sham surgery or the implantation surgery for either the iPRECIO^® (iP) pump or a standard osmotic infusion pump (OSM) under the back skin. This was followed by 4 weeks (experiment I) or 13 weeks (experiment II) of observation. During the observation period, the iP- and OSM-animals received a continuous infusion of 2.0 or 2.5 μL/hr of saline via the external jugular vein. In experiment I, standard hematologic and blood chemical parameters used in toxicity studies were measured at weeks 1, 2, and 4. The iP-animals showed no abnormal changes in any parameters at any point when compared with the OSM-/SHAM-animals. In experiment II (only iP- and SHAM-animals), necropsy and histopathological examination were performed at weeks 1, 2, 4, and 13. The histopathological examination revealed foreign material-induced inflammatory changes in the dorsal subcutaneous tissue (implantation site) of the iP-animals, including the infiltration of polymorphonuclear or mononuclear cells, edema, granulation and fibrous capsule formation. However, the abnormalities were limited to the implantation site. These results suggest that the implantation of iPRECIO^® exerted no significant impact on the physiological condition of the rats. Therefore, we concluded that iPRECIO^® is applicable for toxicity studies.