Obesity and type 2 diabetes mellitus have become worldwide epidemics. Evidence indicates that glucose-dependent insulinotropic polypeptide (GIP) secreted by the intestines may partially underlie these conditions, considering that GIP levels are associated with lipid deposition and fat mass expansion. However, recent studies have found that GIP is also present in other tissues, such as the liver. Notably, one study discovered through microarray analyses of livers from obese diabetic rats that the transcriptional modulation of GIP also occurred in the liver. Otsuka Long-Evans Tokushima Fatty (OLETF) rats were chosen for this experiment because previous studies have shown that thiamine (vitamin B1) could successfully decrease the tendency of the animal toward obesity and mitigate the complications of diabetes. Here, the rats were randomly assigned to either the control (non-supplemented) or thiamine-supplemented (2 g thiamine/L in drinking water) groups. For this investigation, unlike that for young rats, OLETF rats were chosen for the experimental period at 93 weeks of age. Ageing is also a risk factor for diabetes and its complications. In this study, hepatic GIP expression was analysed using western blotting, suggesting that GIP was present in the livers of both obese diabetic OLETF rats and obese diabetic rats that received ongoing thiamine supplementation. Results showed that hepatic GIP expression had occurred and that liver-derived GIP may exist. Moreover, results showed that ongoing thiamine supplementation modified the hepatic GIP expression and prevented additional weight gain and complications arising from obesity and diabetes.

The effects of N^G-Nitro-_L-arginine-methyl ester (_L-NAME), an inhibitor of nitric oxide (NO) synthase, on decidual cell proliferation were examined to investigate possible NO depletion-mediated mechanisms of reproductive toxicity. Mid-to-late pregnant rats were injected intraperitoneally with _L-NAME (100 mg/kg body weight) and 5-bromo-2′-deoxyuridine (BrdU, 100 mg/kg body weight) at 12 hr and 1 hr before sacrifice, respectively, on day 13.5, 17.5, or 21.5, and the BrdU-positive proliferating decidual cells were detected. In control rats, proliferating decidual cells were observed sporadically on days 13.5 and 17.5; however, fewer such cells were observed at day 21.5. _L-NAME reduced the number of proliferating decidual cells by approximately half at days 13.5 and 17.5 but not at day 21.5. These results suggested that in mid-to-late pregnant rats, decidual cell proliferation was maintained by a mechanism involving NO signaling, and NO depletion reduced it only to a limited extent. The absent effect at day 21.5 may be due to the low incidence of proliferating decidual cells or the changing role of NO near the term of pregnancy.

Transdermal sensitization by Glupearl 19S, which is a hydrolyzed wheat protein with a molecular weight of tens of thousands and present in a facial soap, has been reported. Intact skin is considered to function as a barrier; thus, a substance with molecular weight as high as that of Glupearl 19S cannot penetrate to the skin and cause sensitization. We studied whether moderate- or high-molecular-weight materials could penetrate into the skin by using intact and injured Yucatan micropig skin in vitro. Fluorescein isothiocyanate (FITC)-labeled dextran (MW 4000, FD4) and ovalbumin (FITC-OVA) were applied as 1% aqueous solutions with or without 1% sodium dodecyl sulfate (SDS) as a surfactant. FD4 penetrated in the stratum corneum of intact and delipidized skin, especially when combined with SDS. It also penetrated into the dermis of stripped skin (the stratum corneum was removed by tape stripping). FD4 was observed in some scratches of the skin and diffused into the epidermis. FITC-OVA was partially observed in the stratum corneum of intact and delipidized skin. In the case of stripped skin, FITC-OVA did not penetrate in the viable epidermis or dermis because it could not pass through tight junctions even if they were open. FITC-OVA was observed in every scratch 2 min after the application and did not diffuse into the surrounding area as FD4 did. It is considered that FITC-OVA penetrates skin defects and remains at the site, which increases the possibility of sensitization by Langerhans cells.

3-Methylpentane, isooctane, and isononane are acyclic branched saturated hydrocarbons with carbon numbers C6, C8, and C9, respectively. To assess human risk, we conducted a combined repeated-dose and reproductive/developmental oral toxicity studies in rats. [Organization for Economic Co-operation and Development (OECD) Test Guideline 422]. Each hydrocarbon was administered by gavage to rats at three doses (plus a control group). All three chemicals targeted the liver and kidney. An increase in liver weight without hepatic injury was observed as the adaptive response to the chemical treatments. Males treated by each chemical showed α2u-globulin nephropathy, which is a rat-specific finding that bears no human relevance. Reproduction/developmental toxicity parameters showed no treatment-related effects in parents or offspring at any dose for the three chemicals, except for the retardation of offspring bodyweight development which may be a secondary effect of a maternal systemic condition or direct effect on offspring in isononane. No observed adverse effect levels (NOAELs) of repeated toxicity in either sex were determined to be 300 mg/kg/day for 3-methylpentane, 100 mg/kg/day for isooctane, and 250 mg/kg/day for isononane. NOAELs of reproductive/developmental toxicity in parents and offspring were determined to be 1000 mg/kg/day for 3-methylpentane, and 300 mg/kg/day for isooctane. For isononane, NOAELs were determined to be 1000 mg/kg/day for reproduction, and 250 mg/kg/day for offspring development. These results provide new toxicological information and support the category assessment of published reports that evaluate the acyclic branched saturated hydrocarbons as low-toxicity substances.

There are both advantages and disadvantages in the application of electromagnetic fields (EMF) for health: the former is deep brain stimulation for neurodegenerative disease in medicine and the latter is the possible association with a tumor. In this study, we examined the effect of EMF (50 Hz; 100 μT) on rat neural stem cells in vitro. During handling with culture cells, there are two phases of the state of neural stem cells isolated from rat brains; one phase is a cellular suspension in the medium (no anchorage), and another phase is anchorage to the bottom of the culture dishes. The effect of EMF on neural stem cells in vitro was dependent on these cellular phases. Upon anchorages, the cultured neural stem cells migrated along the radial axis, and exposure of these migratory neural stem cells to EMF (50 Hz; 100 μT) facilitates the migration and incorporation of BrdU 1.3~1.4 folds. However, these effects of EMF were not seen once the cellular suspension (no anchorage) was exposed. Even when the neural stem cells fully migrated, there were no effects of EMF on the retinoic acid-induced differentiation. Thus, there is a cell phase sensitive to EMF in the cultured neural stem cells.

2-Mercaptobenzimidazole (MBI) and its methyl derivatives 4-methyl-MBI (4-MeMBI), 5-methyl-MBI (5-MeMBI), and 4(or 5)-methyl-MBI (4(5)-MeMBI) are widely applied industrial agents with substantial thyrotoxicity and hepatotoxicity detected in rats in vivo. Here, we examined the in vitro cytotoxicity of MBI and its derivates in cultured SIRC rabbit corneal cells. SIRC cells were cultured in the presence of the test chemicals for 72 hr, and cell viability was determined by estimating the number of cells using a crystal violet staining assay. The median lethal concentration (LC₅₀) was calculated for each of the chemicals. The methyl derivatives showed higher cytotoxicity than MBI, which is in contrast to previous in vivo findings demonstrating higher thyrotoxicity and hepatotoxicity of MBI compared to its derivates. According to the LC₅₀ values, the ranking of the tested agents in terms of cytotoxicity was 5-MeMBI (761.5 µM) ≥ 4-MeMBI (796.3 µM) ≥ 4(5)-MeMBI (822.9 µM) > MBI (1002.9 µM). The present results suggest that the lower thyrotoxicity and hepatotoxicity of methyl derivatives of MBI is related to their faster detoxification in vivo, because SIRC cells are considered to have lower drug-metabolizing activity than hepatic cells.

We previously reported that continuous exposure to α-glycosyl isoquercitrin (AGIQ) from the fetal stage to adulthood facilitates fear-extinction learning in rats. The present study investigated the combined effect of continuous exposure to AGIQ with voluntary exercise or environmental enrichment on learning and behaviors in rats. For this purpose, maternal Long-Evans rats were either untreated or treated with 0.5% AGIQ in basal diet from gestational day 6 to day 21 post-delivery. Offspring in both groups were weaned on postnatal day 21 and reared thereafter either in a standard cage, a wheel cage or an environmental enrichment cage until the end of the experiment with or without exposure to AGIQ. Fear memory, locomotor activity and anxiety-like behavior in open field test, spatial memory and nonspatial memory were assessed in adulthood. Environmental enrichment without AGIQ exposure, as well as AGIQ exposure in standard cage, showed a tendency for facilitation of fear-extinction learning. However, exposure to AGIQ and environmental enrichment did not act synergistically. Voluntary exercise only decreased the total distance traveled in the open field test in the condition with or without AGIQ exposure, suggesting induction of anxiety-like behavior. Body weight from lactation period to adulthood, body and brain weights at the end of the experiment did not change by exposure to AGIQ under any cage condition. Therefore, there was no beneficial or detrimental effect of voluntary exercise and environment enrichment on the outcome of behavior or general conditions by continuous AGIQ exposure from the fetal stage.

Nucleolin (NCL) is an abundant DNA-, RNA-, and protein-binding protein that is expressed ubiquitously in eukaryotic cells, implicating it in many cellular functions such as gene silencing, senescence, cytokinesis, and proliferation. NCL is also involved in angiogenesis events including vascular endothelial cell migration and proliferation. We previously found that lead, an environmental pollutant with vascular toxicity, inhibits the repair process of injured vascular endothelium via suppression of cell proliferation as a result of a lower proliferative response of cells to endogenous fibroblast growth factor-2. The present study investigated the expression of NCL in proliferating bovine aortic vascular endothelial cells and the role of NCL in proliferation of the cells in the presence or absence of lead. We found that the expression of NCL protein was independent of cell density. Both siRNA-mediated NCL knockdown and an anti-NCL-neutralizing antibody inhibited the proliferation of vascular endothelial cells without non-specific cell damage, indicating that NCL was involved in endothelial cell proliferation. However, lead significantly inhibited the proliferation of vascular endothelial cells without changing the expression level of NCL. Therefore, NCL may positively regulate spontaneous proliferation, but is not involved in the inhibition of proliferation by lead in vascular endothelial cells.

We investigated the potential toxicity of gum ghatti, which is added to food for emulsifying, thickening, and stabilizing, after 4 weeks of repeated oral administration at a dose of 8000 mg/kg/day to male and female SD rats. Although food consumption was significantly reduced in males in the gum ghatti group compared with those in the distilled water group from Day 18 onwards, the change was minor, there was no pathological evidence of digestive tract abnormalities, and there were no significant changes in body weight; therefore, the change in food consumption was judged to be of no toxicological significance. Hematology and blood biochemistry revealed statistically significant differences in some parameters between the gum ghatti group and the distilled water group. These changes were all within the normal range of physiological variation and therefore were not considered to represent the effects of gum ghatti. In addition, general signs, body weight, and pathology showed no changes in either sex attributable to gum ghatti. Thus, all changes observed were of no toxicological significance and within the normal range of physiological variation, suggesting gum ghatti has no toxic effects in rats.

Artisanal and Small Scale Gold Mining (ASGM) contributes as the biggest source of mercury (Hg) emission in Southeast Asian countries including Indonesia. Rice paddies are the most impacted agriculture area caused by Hg emission from ASGM sewage water. It may pose to health risk effect to humans due to the consumption of the rice as the staple food. This study was aimed to evaluate the Hg accumulation in rice and health effect to residents in Lebaksitu ASGM area by analysis of total mercury (THg) and methylmercury (MeHg) in hair. Two villages were selected in this study, Hg hotspot village (Lebak-1) and low Hg exposure village (Lebak-2). The THg concentration in rice ranged from 9.1-115 µg/kg with an average of 32.2 µg/kg. MeHg concentration in rice constituted 14.7-81.8% of the THg. Rice in Lebak-1 had higher THg and MeHg concentrations than those in Lebak-2. The mean THg and MeHg concentration in hair were 3.2 mg/kg and 1.78 mg/kg, respectively. Residents in Lebak-1 had significantly higher THg and MeHg in hair than those collected from Lebak-2. The MeHg ratio to THg in hair varied widely ranged from 15.68-92.43%. There was a significant correlation between high intake of MeHg from rice and the accumulation of MeHg in the hair. It was concluded that rice is the potential source of MeHg exposure to humans through daily consumption in rice consumer countries.

2-Azahypoxanthine (AHX) and imidazole-4-carboxamide (ICA) are fairy-ring causing compounds from a mushroom-forming fungus, Lepista sordida, and 2-aza-8-oxohypoxanthine (AOH) is a metabolite of AHX in plants. However, the safety of AOH had not yet been elucidated. In this study, we focused on AOH and performed safety evaluations of the compound using in vitro and human patch tests for cosmetic applications. In the Ames test, AOH was not mutagenic to any of the test bacterial strains (> 5000 μg/plate). In vitro skin irritation and skin sensitization studies using reconstructed human epidermis and peptides that contained lysine and cysteine showed that AOH was not a skin irritant (cell viability > 50%) and did not exhibit skin sensitization. This compound also did not exhibit cytotoxicity under ultraviolet- or sham-irradiation in the alternative phototoxicity test using BALB/c 3T3 cells (mean photo effect < 0.1) and no skin reaction was observed in the patch test on human skin. Thus, we concluded that AOH is safe as a cosmetic ingredient. This is the first study in which safety evaluation tests were performed on AOH.

Since phytoestrogens (e.g. biochanin A, coumestrol, daidzein, genistein and glycitein) are structurally similar to estrogen, they mimic estrogen function. Phytoestrogens consist in certain edible plants, especially in soybeans. Among them, interestingly, equol that has the greatest estrogenic activity is generated from daidzein by gut microbes. Therefore, the relationship between phytoestrogens and gut (including intestinal cells and bacteria) is being watched with strong interest. In this paper, we revealed that all-trans retinoic acid (RA) dramatically enhances cytotoxicity of several phytoestrogens against U937 cells. While β-estradiol and phytoestrogens tested showed no effect on the viability of U937 cells in the absence of RA, 10 μM coumestrol, (±)-equol and genistein brought about remarkably reduced viability of U937 cells at 24 h (to ~15%, ~7% and ~35%, respectively) in the presence of 1 μM RA. In particular, the cytotoxicity of (±)-equol was drastically enhanced in the presence of RA. Moreover, very interestingly, (±)-equol showed no effect on the viability of human peripheral neutrophils even in the presence of RA. As is well known, human monoblastic leukemia U937 cells have been used as an in vitro model for macrophage that exists in intestine and plays significant roles to maintain intestinal homeostasis. These data suggest that equol not only can serve as an effective modifier in therapy for leukemia in combination with RA but also may affect the maintenance of intestinal homeostasis.

We investigated if access to a running wheel after in utero exposure to benzyl butyl phthalate (BBP) ameliorates the toxicological effects of BBP exposure. Our purpose was to determine if post-birth exercise after prenatal BBP exposure could reverse alterations in mammary gland development. Twenty-five female pups were exposed to 500 mg/kg of BBP on days 9-15 in utero and analyzed for mammary gland development and morphology. Mice either had access to a running wheel beginning at 8 weeks of age or were in a cage with a “locked” wheel to prevent running activity. Whole mount staining showed delayed mammary gland morphology development, regardless of wheel exposure in the treated groups. Additional histology staining revealed BBP exposed mice that were not allowed exercise, had larger ducts with multiple cell layers containing proliferative cells suggesting a favorable environment for tumor growth. In addition, there was a significant increase in progesterone status in the mouse mammary gland at 20 weeks but not 10 weeks, regardless of wheel exposure. BBP exposure led to abnormal mammary gland development in female mice and access to a running wheel helped ameliorate some, but not all, of the harmful effects due to BBP exposure at either 10 weeks or 20 weeks of age. Our results are significant because they indicate that exercise can reverse most of the BBP-initiated alterations in the mouse mammary gland, and physical activity has a positive impact on most developmental parameters in the mouse mammary gland.

The discarded internal organs of the Japanese giant scallop (Patinopecten yessoensis) are abundant resources rich in n-3 polyunsaturated fatty acids (PUFA), such as eicosapentaenoic acid and docosahexaenoic acid. However, they have not been utilized due to contamination with toxic substances such as cadmium (Cd) and the occurrence of diarrhetic shellfish toxins (DST). We have successfully prepared a high-quality scallop oil (SCO) from its internal organs with negligible contamination with Cd and DST. The scallop internal organs were obtained from two different scallop processing areas, Mutsu and Uchiura bays, Japan, and referred to as SCO-M and SCO-U, respectively. To evaluate the safeties of SCO-M and SCO-U as food ingredients and n-3 PUFA supplements, repeated 28-day and 13-week dose oral toxicity studies in rats were conducted. Rats were fed diets containing 1% and 5% of SCO-M and SCO-U, respectively, in the repeated 28-day dose oral toxicity study and 5% SCO-M or SCO-U in the repeated 13-week dose oral toxicity study (limit test). No adverse toxicological effects were observed when rats were fed diets containing SCO-M and/or SCO-U at up to 5% for 28 days and 13 weeks. These results suggest that SCO-M and SCO-U are safe products in terms of subacute toxicity under these experimental conditions.

Oxidative stress is involved in the development of many neurological diseases. The interactions between catecholamines and copper or iron generate reactive oxygen species that lead to oxidative DNA damage in vitro. Furthermore, catechol structure is essential for DNA damage. Here, we clarified the effect of aminoethyl side chains on DNA damage. Endogenous catecholamines (dopamine, noradrenaline, and adrenaline) were more effective than other catechols (catechol, 4-ethylcatechol, and 3,4-dihydroxybenzylamine) in strand break and base oxidation of calf thymus DNA. The presence of copper caused more DNA damage than iron. Furthermore, adrenaline oxidized to adrenochrome more rapidly by copper than iron. Leukoadrenochrome, an oxidation intermediate formed by the intramolecular cyclization of aminoethyl side chains, rapidly increased the formation of 8-hydroxy-2′-deoxyguanosine compared with adrenalin. These results show the effect of aminoethyl side chains in catecholamine-induced oxidative DNA damage. This mechanism may partly show the vulnerability of catecholaminergic neurons against oxidative stress.

Recently, a “pack cooking” method, which uses heat-resistant plastic bags, has attracted attention for being simple and safe for emergencies such as natural disasters. One serving is filled in a pack. It uses less kitchenware and clean water, so “pack cooking” will particularly be a powerful tool for soup runs. However, its risk for food poisoning has not been given attention. It is important to consider the safety of “pack-cooked” meals, because medical shortage is as much a problem as food shortage during natural disasters. We conducted a bacteriological evaluation of curry and rice as typical “pack-cooked” meals, assuming that they were stored at room temperature for a long time. “Pack-cooked” meal samples were stored at 25°C and 4°C for 24 hr, and their homogenates were used as sample for bacteriological analysis. Obvious standard plate count bacteria were found in “pack-cooked” curry, which was stored at 25°C for 24 hr, whereas those stored at 4°C for 24 hr did not. Furthermore, there was a sample that was stored at 25°C for 24 hr in which coliform bacteria were detected. Since 25°C is equivalent to room temperature, our results demonstrate that the “pack-cooked” curry stored at room temperature for 24 hr could cause food poisoning. While “pack cooking” is a useful method, it is important to understand that prolonged storage at room temperature of “pack-cooked” meals should be avoided.

Environmental electrophiles readily interact with reactive sulfur species (RSS), resulting in decline of cellular RSS levels accompanied by formation of their sulfur adducts. In the present study, we examined the effects of combined environmental electrophiles on consumption of persulfide and on cytotoxicity in HepG2 cells. A convenient assay with SSP4, a fluorometric probe for detection of per/polysulfides, indicated that each environmental electrophile caused compound-dependent consumption of persulfide. Consumption of persulfide by combined exposure to methylmercury (MeHg) and cadmium (Cd) was greater than that of the single exposure. Consistent with this finding, combined exposure to environmental electrophiles (MeHg, Cd, and 1,4-naphthoquinone) exacerbated concentration-dependent cellular toxicity in HepG2 cells compare to single exposure to each compound. As humans are exposed to environmental electrophiles daily, more attention should be paid to the study of combined exposure to environmental electrophiles, which can modulate cellular levels of RSS and disrupt redox homeostasis.

Drug-induced liver injury (DILI) is one of the serious and frequent drug-related adverse events and is classified into intrinsic and idiosyncratic types. Almost all of DILI caused in humans is known to be idiosyncratic type. The estimation of the potential risk for a drug candidate to induce idiosyncratic DILI is important to facilitate the development of new drugs, however, the estimation is difficult from the results of non-clinical toxicity studies using animals. We have previously reported the in vitro combination assay of mitochondrial function and apoptosis using human primary hepatocytes as a useful model for estimation of the risk of idiosyncratic DILI. In this study, to improve the in vitro assay for estimation of the risk of idiosyncratic DILI, we evaluate the usefulness of HepaRG cells for the mitochondrial function assay. We measured the oxygen consumption rate (OCR) as an endpoint of mitochondrial function in HepaRG cells treated with some compounds causing idiosyncratic DILI (troglitazone, leflunomide, ranitidine and diclofenac) and others known not to cause idiosyncratic DILI (acetaminophen and ethanol) and compared the results in HepaRG cells and with those for human primary hepatocytes as previously reported. As the results, HepaRG cells showed comparable or even higher sensitivity for detecting mitochondrial dysfunction than human primary hepatocytes, in all tested compounds. Taking into account these results and many other useful properties of the cells, HepaRG cells are considered to be much more suitable for this mitochondrial function assay than the human primary hepatocytes.

Maltosyltrehalose syrup (TG4 syrup) is an enzymatically derived starch hydrolysate consisting of only glucose. The main components are maltotetraose (G4) and maltosyltrehalose (TG4). G4 is a common component of essentially all starch hydrolysates, and consists of only four glucose molecules with α-1,4 linkages. TG4 also consists of four glucose units, but the terminal glycosidic bond at the reducing end of the glucose chain is inverted, which results in the final bond being α-1,1. The two terminal glucose units form a trehalose disaccharide molecule, attached to a maltose molecule. The presence of the trehalose moiety in the TG4 molecule results in unique functional and technical properties from what is found in other α-1,4 linked oligosaccharides. This paper presents results of standardized toxicity studies of TG4 syrup, including an in vitro bacterial mutagenicity test, a 90-day oral feeding study in rats, and a 90-day oral toxicity (gavage) study in rats. Treatment with TG4 syrup resulted in no microbial mutagenic activity or growth inhibition in either Salmonella typhimurium or Escherichia coli, even at the maximum dose of 5,000 μg/plate. The NOAEL in the 90-day oral feeding study was calculated as 10% of TG4 syrup in the diet, which was equal to 6,818 and 7,464 mg/kg/day (dwb) in male and female rats, respectively. The 90-day oral gavage toxicity study had a NOAEL of 5,000 mg/kg/day (dwb) in male and female rats. Taken together these data showed that TG4 syrup was safe for use in these studies, suggesting it is safe for consumption by humans.

Obesity is caused by a chronic positive energy balance, which not only increases the amount of lipid in adipose tissue, but also, in non-adipose tissue. Excessive accumulation of lipids in tissues may lead to cell dysfunction or cell death, a phenomenon known as lipotoxicity. The aim of this study was to investigate the effects of high-fat diet (HFD) feeding on the sciatic nerves of both male and female mice. HFD feeding induced increased mRNA levels of Bax and Bcl2 in HFD group of males only, although the accumulation of fatty acids in the sciatic nerves was induced by HFD feeding in the both sexes. To determine whether estrogen was involved in the inhibitory effects of HFD feeding-induced increased expression of apoptosis-related genes, ovariectomized (OVX) females were fed a normal diet (ND) or HFD with or without daily ethinylestradiol treatment for 1 week. In OVX mice, the mRNA levels of Bax and Bcl2 in HFD group were higher than in ND group. In contrast, in OVX mice treated with ethinylestradiol, there was no significant between ND and HFD groups. In conclusion, HFD induced apoptosis in the sciatic nerves of males, but not females, and estrogen had inhibitory effects on HFD-induced apoptosis in the sciatic nerves of females. Long-term feeding studies are needed to investigate the pathological effects on sciatic nerves of mice fed HFD as pathological findings were not observed under our study condition.

Epoxidized fatty acids are generated during food processing and detected in various lipid- and oil-containing foods. Although compounds with an epoxide structure have high reactivity and there is increasing concern about their potential toxic effects such as genotoxicity and cellular damage, information regarding the toxicity of epoxy fatty acids is largely unknown. Therefore, we conducted three in vitro genotoxicity studies (bacterial reverse mutation assay, in vitro micronucleus test, and p53R2-dependent luciferase reporter gene assay) and HepG2 cytotoxicity assays for epoxyoleic acid (EOA) and diepoxylinoleic acid (DELA). The in vitro genotoxicity results of EOA and DELA were uniformly negative. In the cytotoxicity assay, EOA and DELA induced weak cytotoxicity at high concentrations, but the effect was similar to those of oleic and linoleic acids at low concentrations. Considering the existing toxicity information on epoxidized soybean oil, which is a similar compound, there might be little concern concerning the health effects of epoxy fatty acids at low concentrations.

The accumulation of free fatty acids induces lipotoxicity in neural cells. Estrogen, 17β-estradiol (E2) protects against the damage of cells in various organs and tissues. However, the role of E2 on lipotoxicity in neural cells remains unclear. In this study, we investigated the effects of E2 on stearic acid (saturated fatty acid)- and oleic acid (unsaturated fatty acid)-induced cytotoxicity in retinoic acid-induced mouse neuroblastoma Neuro-2a differentiated into neural cells. Cell viability was evaluated by lactate dehydrogenase release from Neuro-2a neural cells. Stearic acid and oleic acids suppressed the cell viability in a dose-dependent manner. E2 prevented oleic acid-induced cytotoxicity but had no effect on stearic acid-induced cytotoxicity. ERα-selective agonist prevented cytotoxicity in Neuro-2a neural cells. In contrast, ERβ-selective agonist slightly significantly enhanced the cytotoxicity in the presence of oleic acid. Oleic acid, but not stearic acid, increased the mRNA level of p62/Sqstm1. E2 treatment statistically significantly, but slightly, enhanced the stearic acid-induced Bax expression. In contrast, E2 and ERα-selective agonist inhibited the oleic acid-induced the p62/Sqstm1 expression. Our results suggested that fatty acids induced cytotoxicity in Neuro-2a neural cells, and estrogen prevented the oleic acid-induced cytotoxicity via ERα, but not ERβ. Further studies are needed to understand the role of ERβ in neuron injury under normal conditions.

The purpose of this study was to profile cytokine storms (cytokine release syndrome) in the LPS-induced disseminated intravascular coagulation (DIC)-cynomolgus monkey model by measuring changes in 22 cytokines using Luminex. In this study, increases were noted in 20 cytokines, excluding IL-4 and IL-17A. Specifically, IL-6, IL-8, G-CSF and TNF-α, pro-inflammatory cytokines, and IL-10, an anti-inflammatory cytokine, as well as MCP-1, markedly increased by 10,000 pg/mL or more. In addition to the marked increases in the pro-inflammatory cytokines IL-6 and G-CSF, the concentrations of IL-5, IL-18, IFN-γ, VEGF and IL-15 increased continuously. Also, in addition to the marked increases in the pro-inflammatory cytokine IL-8 as well as in MCP-1, the concentrations of IL-1ra, IL-2, IL-1β, IL-12/23 (p40), GM-CSF and TGF-α gradually decreased after initially increasing. On the other hand, in addition to the marked increases in the pro-inflammatory cytokine TNF-α and anti-inflammatory cytokine IL-10, MIP-1β and MIP-1α transiently increased and then rapidly disappeared from serum. IL-13 increased at 6 hr after administration only. Since the behavior of cytokines in this monkey model was similar to those noted in DIC in humans, this model will be useful for evaluating the efficacy of anti-DIC drugs. In addition, this model will also be useful for assessing the risk of cytokine storm development, which is a serious adverse effect of certain types of antibody drugs and CAR-T cell-based therapies.

5-Fluorocytosine (5-FC) is an antimycotic and teratogenic compound. Oral administration of 5-FC to pregnant rats on gestation days (GD) 9 and 13 was shown to induce thoracolumbar supernumerary ribs (TSR, 14th rib) and abnormal digits, respectively, in fetuses. This study investigated the effects of 5-FC on homeobox genes, which control the anterior-posterior-axis. 5-FC (75 mg/kg) was administered orally on GD9 and GD13, and tissues collected from cranial and caudal regions of TSR sites were analyzed. Following 5-FC administration on GD9, the levels of expression of Hoxa10, which determine the position of the thoracic and lumbar vertebrae, were decreased at GD13. Analysis of hindlimbs 6 hours after administration on GD13 showed decreases in expression of Hoxa11, Hoxd12, and Hoxd13, the Hox genes responsible for limb formation from the proximal to distal, and from the anterior to posterior directions. The present findings showed that altered expression of Hox genes contributes to 5-FC teratogenicity.

Measurement of the bone length is not used routinely in toxicity studies but used to examine growth and bone toxicity. To investigate the utility of measuring the bone length in toxicity studies and to identify the appropriate site, we evaluated femur, tibia, humerus, and sternum, in a 14-day repeated dose oral toxicity study of Dexamethasone (DEX), which is known to cause growth retardation and osteoporosis, in young, periadolescent, and adult rats. To observe the effect of decreased food consumption, we also evaluated the changes in each diet-restricted group in which the food intake restricted to levels corresponding to that consumed by the DEX-treated periadolescent and adult rats. Significant decreases of the bone length at all the measured sites and histopathological findings in growth plates and/or trabecular bone were observed in the DEX-treated young and periadolescent rats. Significant decreases of the femoral length and decreased trabecular bone were observed in the DEX-treated adult rats. No histopathological changes were observed in any of the diet-restricted groups, while decreases of the femoral length, similar to that in the DEX-treated adult rats were observed in the diet-restricted adult rats. The results suggested that measurement of the bone length in femur, tibia, humerus, and sternum was useful in young and periadolescent rats, and measurement of the femoral length was useful in adult rats. Moreover, our results showed that the decreases in the femoral length in the DEX-treated adult rats were not only related to the DEX-treatment, but were also influenced by the decreased food consumption.

Arsenic is an environmental pollutant and is a possible risk factor for vascular diseases such as atherosclerosis. Vascular proteoglycans (PGs) are key molecules in the initiation and progression of atherosclerosis. We previously demonstrated that arsenite, but not arsenate, decreases the synthesis of both heparan sulfate proteoglycans (HSPGs) and chondroitin/dermatan sulfate proteoglycans (CS/DSPGs) in cultured vascular endothelial cells. In the present study, we aimed to identify the PG molecules whose expression is decreased by arsenite, using a culture system of bovine aortic endothelial cells. The results indicate that a 24-hr treatment of arsenite significantly decreases the mRNA levels of a large HSPG perlecan, small HSPGs—syndecan-1, -2 and -3—, and a small CS/DSPG biglycan in vascular endothelial cells without nonspecific cell damage; the expression of syndecan-4 mRNA was unaffected by arsenite. The decreased expression of perlecan, syndecan-1 and biglycan genes began after 3 hr of arsenite treatment. However, arsenate did not change the mRNA expression levels of perlecan and biglycan in the cells. These results suggest that the inhibition of synthesis by arsenite occurs in particular types of proteoglycans, i.e. perlecan, syndecan-1, -2, -3, and biglycan in vascular endothelial cells.

In the inhalation field, lipids such as egg phosphatidylcholine (PC), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) and dipalmitoylphosphatidylcholine (DPPC), are considered to be generally recognized as safe (GRAS), comprising materials that are endogenous to the lungs and locally present in large quantities. Indeed, PC, DSPC and DPPC may be used to form liposomes which are known to promote an increase in drug retention time and reduce the toxicity of drugs after administration. Unfortunately, published literature guidance about the safety evaluation of these lipids as pharmaceutical excipients for use in inhaled products and about application for marketing authorization, is very limited. The purpose of this article is to review the potential toxicity of DSPC for pulmonary administration. Given the use of air and vehicle controls in a range of inhalation toxicology studies as well as negative genotoxicity and also reproductive toxicity results, it is thought that the use of DSPC is shown to be safe for pulmonary administration.

One of the risk factors for drug-induced liver injury (DILI) is the diabetic state. Our previous investigation showed that liver injury after repeated oral dosing with allyl alcohol and carbamazepine was enhanced more in the Spontaneously Diabetic Torii-Lepr^fa (SDT fatty) rats than in the Sprague-Dawley (SD) rats. It was caused by lower hepatic detoxification due to depleted hepatic glutathione synthesis. This is because simvastatin, frequently used in diabetic patients, shows a positive high reaction in a GSH adduct assay in vitro although the GSH adduction is considered not to be a major metabolic pathway of simvastatin. Therefore, in the present study, effects of simvastatin-treatment on the liver were compared between the Sprague-Dawley (SD) rats and SDT fatty rats in order to obtain additional information to estimate the potential risk of DILI in the diabetic state. There were no effects with simvastatin on the liver in the SD or SDT fatty rats after 13-week oral dosing of simvastatin. These results indicate that simvastatin does not have potential to induce liver injury in diabetic state and are consistent with the reports for the clinical use of simvastatin in the diabetic patients.

Organotin compounds such as triphenyltin (TPT), which are common environmental pollutants, had been widely used as antifouling agents for ship bottoms. Although toxic effects of organotins through nuclear receptors such as retinoid X receptor (RXR) and peroxisome proliferator-activated receptor (PPAR) γ have been well demonstrated, other mechanisms underlying organotin-induced toxicity have hardly been reported. In the present study, we focused on the transcription factor GA-binding protein (GABP), which regulates the expression of various housekeeping genes, as a novel target of TPT toxicity. We investigated the change of GABPα subunit protein expression induced by TPT. Although 100-500 nM concentration of TPT was not found to affect the total protein expression of GABPα, TPT significantly decreased nuclear translocation of GABPα in human embryonic kidney (HEK) 293T cells. In addition, TPT increased intracellular reactive oxygen species (ROS) levels. Both inhibition of GABPα nuclear translocation and the increase in ROS levels were observed in menadione (an ROS inducer)-treated HEK293T cells. Our results indicate that TPT causes inhibition of GABPα nuclear translocation, which may be triggered by ROS production. This might have serious implications in cellular physiology, thereby affecting cell survival.

D-allulose is one of the rare sugars with almost zero calories and several health benefits. Previous studies have reported the safety of D-allulose in normal, overweight/obese, and diabetic humans. However, one study reported significant increases in T-Cho and LDL-C after 12 weeks of D-allulose intake; this report was not a randomized controlled trial and these changes were considered to be due to seasonal variations. We, therefore, conducted a randomized, double-blind, placebo-controlled trial in 90 subjects with high LDL-C levels for 48 weeks to clarify the influence of long-term D-allulose consumption on cholesterol metabolism and efficacy. Subjects were randomly divided into 3 groups: high-dose D-allulose (15 g D-allulose/day), low-dose D-allulose (5 g D-allulose/day), and placebo group (0 g D-allulose/day); each subject consumed a daily test beverage for 48 weeks. Clinical examinations were performed every eight weeks, beginning from initial consumption until week 52. No significant increases in T-Cho and LDL-C between test groups were observed, and 48 weeks of D-allulose consumption did not change risk factors for atherosclerotic cardiovascular disease. Furthermore, no clinical problems were recognized for other parameters. Additionally, significant improvements in hepatic enzyme activities, fatty liver score, and glucose metabolism after long-term D-allulose consumption were observed. The results from our study revealed that 1) D-allulose consumption is considered safe for long-term intake up to a year, and 2) D-allulose may be effective for improving hepatic functions and glucose metabolism.

Cytochrome P450s (CYPs) are involved in the metabolism of various drugs, and may generate toxic metabolites or intermediates that result in drug-induced liver injury (DILI). Consequently, inducers of CYPs may promote DILI. In a draft test guideline, the Organisation for Economic Co-operation and Development (OECD) recommends measurement of the metabolic activity of CYP as an index for assessing CYP-inducing activity. However, change of mRNA level has also been used as a simple parameter to evaluate CYP induction. In this study, therefore, we examined the usefulness and limitations of mRNA expression measurement for evaluation of the induction of CYP1A2, CYP2B6, and CYP3A4 by omeprazole, phenobarbital, and rifampicin (RIF), respectively, in HepaRG cells, a well-established cell line derived from human hepatocellular carcinoma. The results of mRNA measurement correlated well with the results of metabolic activity measurement in the lower concentration ranges for all inducers, even though we observed significant decreases in albumin and urea secretion in the presence of 10 µM RIF, reflecting its known hepatotoxicity. Our results indicate that mRNA measurements and metabolic activity measurements in HepaRG cells generally give comparable results for fold-induction of CYPs.

Azoxystrobin is a broad-spectrum fungicide having a wide usage. However, the toxic effect of azoxystrobin in humans is not reported. In Japan, azoxystrobin was detected at a five-fold higher concentration than the normal upper limit (2.5 mg/kg) in a shipment of an Australian barley used in different food products. Thus, there is a chance of azoxystrobin exposure through food to humans, and hence it is imperative to study the toxic effects of this compound. In this study, the toxic effect of azoxystrobin was evaluated to predict its adverse effects on human. Azoxystrobin at 3-30 µM (approximately 1.2-12.1 mg/L) raised the intracellular Zn²⁺ concentration of rat thymic lymphocytes. This increase was due to an influx of extracellular Zn²⁺ and a release of intracellular Zn²⁺. Azoxystrobin partially inhibited the temperature-dependent Zn²⁺ influx, thus jeopardizing the cellular Zn²⁺ homeostasis. Because Zn²⁺ is an important intracellular messenger in lymphocytes, this altered Zn²⁺ homeostasis might lead to adverse effects if the blood concentration of azoxystrobin reaches 3 µM or more in humans. However, by extrapolating the azoxystrobin pharmacokinetics data of rats to human, it can be predicted that such high blood concentration may be unlikely in humans.