In this study, a microfluidic culture device and related evaluation methods were developed using deep learning to construct a rapid assessment platform for peripheral neuropathy caused by typical anticancer drugs. Primary rodent dorsal root ganglia were cultured in a microfluidic culture device that separated the cell body and neurites, and morphological changes in the neuritis were analyzed using immunofluorescence imaging. Successful culture of separated neurites in the microfluidic device for more than 1 month indicated that this test process, including culture, drug stimulation, and fluorescence observation, results in a viable outcome. In addition, cultured samples were treated with several anticancer drugs known to cause peripheral neurotoxicity (i.e., vincristine, oxaliplatin, and paclitaxel), and morphological changes in the neuritis were analyzed using deep learning for image analysis. After training, artificial intelligence (AI) could identify morphological changes in the neurites caused by each compound and precisely predict toxicity, even at low concentrations. For the test compounds, AI could also precisely detect neurotoxicity based on neurite images, even at low concentrations. Our results suggest that this microfluidic culture system is useful for in vitro toxicity assessment.

Hyperuricemia, an elevated urate concentration in blood, is the precursor state of gout and is associated with metabolic syndrome and chronic kidney disease. The increasing prevalence of hyperuricemia in recent years is thought to be due to environmental factors, particularly diet. Here, we investigated whether and how fructose induces an increase in plasma urate concentration using a rat model. Oral administration of fructose increased plasma urate concentration within 15–30 min, without an increase in urate excretion into urine or gastrointestinal tract, and this action was evident at a dose of 7.5 g/kg b.w. On the other hand, the same dose of glucose did not elicit a hyperuricemic effect at all, and thus the effect is thought to be a specific property of fructose. The absence of an increase in urinary or gastrointestinal excretion suggested that the increase did not result from an increase in urate production. Instead, a decrease in urate clearance could have a potential to explain the increase in plasma urate concentration. In conclusion, the present study characterized the time course and dose response of a fructose-induced increase in plasma urate concentration using a rat model.

The pharmaceutical concentrations and antimicrobial activity in the Ayase River were investigated. The water sources of the Ayase River are agricultural wastewater and effluent from septic tanks during the irrigation season and effluent from a sewage treatment plant and septic tanks during the non-irrigation season. Acetaminophen, bezafibrate, carbamazepine, clarithromycin, and diphenhydramine levels were in the range of 1.9–495 ng/L. During the irrigation season, pharmaceutical concentrations were higher downstream than upstream due to the inflow of agricultural wastewater in upstream areas. However, during the non-irrigation season, since most of the water sources were effluent from a sewage treatment plant and septic tanks, pharmaceutical concentrations were extremely high from upstream to downstream. The pharmaceutical concentrations during the non-irrigation season were at the same levels as those downstream of the Arakawa River, which was polluted by the effluent from Japan’s largest sewage treatment plant. This extremely high pharmaceutical concentration in the non-irrigated season was found only in the Ayase River but not in the nearby Nakagawa and Furutone rivers, which are both urban water sources. The maximum concentration of clarithromycin in the Ayase River was higher than the predicted no-effect concentration for green algae, indicating that the water from the Ayase River may affect aquatic organisms. Antimicrobial activity analysis in the Ayase River using hypersusceptible Escherichia coli lacking the TolC component of the multidrug efflux system revealed that the river water had antimicrobial activity, suggesting potential risks, such as bactericidal action and development of antibiotic resistance in river water.

Histone acetyltransferases (HATs) are separated into two types. Type A HATs act on nucleosomal histones and thus primarily function in transcriptional regulation, while cytoplasmic HATs (type B) are known as enzymes that modify free histones before their assembly into chromatin, and may also function outside the nucleus. N-alpha-acetyltransferase 60 (NAA60) is the most recently discovered type B HATs, which are also known as N-terminal acetyltransferases (NATs) and are found only in multicellular eukaryotes. NAA60 localizes to the Golgi complex and possesses a unique ability to catalyze the acetylation of membrane-anchored proteins at the N-terminus and free histones at the lysine side chains, the biological significance of which remains unclear. To investigate the cellular functions of NAA60 and its relation to other cytoplasmic HATs, Hat1, we generated NAA60- or HAT1-deficient cells and NAA60/HAT1-double deficient cells using a chicken B lymphocyte leukemia DT40 cell line. Although NAA60-deficient cells did not show any impairment in cell growth and showed a slight sensitivity to DNA damage agents, NAA60/HAT1-double deficient cells exhibited an additive increase in sensitivity to methyl methanesulfonate (MMS) and 4-nitroquinoline 1-oxide (4-NQO) when compared to HAT1-deficient cells, which were previously reported to be moderately sensitive to these agents. These results predict that each type B HATs might contribute differently in regulation of repair of chemical induced DNA lesions.

This study aimed to evaluate the genotoxicity of 2-[2-(acetylamino)-4-[bis(2-hydroxyethyl)amino]-5-methoxyphenyl]-5-amino-7-bromo-4-chloro-2H-benzotriazole (PBTA-6) in fish. Adult zebrafish (Danio rerio) were exposed to PBTA-6 solution of 0.3 to 10 mg/L for 96 hr. Genotoxicity was evaluated using alkaline single-cell gel electrophoresis (comet assay) and micronucleus (MN) tests on blood and gills. Although PBTA-6 showed no positive response to MN induction, it caused a concentration-dependent increase in DNA damage. Fish exposed to PBTA-6 (10 mg/L) for 96 hr followed by a 96 hr recovery period in tap water were also examined; DNA damage decreased to the level before treatment within 96 hr. To compare the cytochrome P450 (CYP) activity in different tissues, CYP1A (ethoxyresorufin-O-deethylase; EROD and methoxyresorufin-O-demethylase; MROD) and CYP2B (penthoxyresorufin-O-depentylase; PROD and benzyloxyresorufin-O-debenzylase; BROD) activities were analyzed in liver and gill microsomes. The results showed that PBTA-6 markedly induced PROD activity in gills and caused DNA damage, but the results show that this damage could be reverted if the exposed fish are returned to tap water.

The effects of six cooked food-derived heterocyclic carcinogenic amines (HCAs), including 2-amino-9H-pyrido[2,3-b]indole acetate (AαC) and its 3-methyl derivative (MeAαC), on cytochrome P450 3A (CYP3A) induction were examined using a human hepatoblastoma cell-derived HPL-A3 cell line, previously established for a luciferase-reporter gene assay for the detection of a CYP3A4 inducer. AαC and MeAαC, but not the four other HCAs, enhanced luciferase activity. Quantitative RT-PCR further confirmed the significant induction of CYP3A4 mRNA by AαC and MeAαC. Since CYP3A4 gene expression is primarily regulated by the pregnane X receptor (PXR) and occasionally by other receptors, such as the aryl hydrocarbon receptor and vitamin D receptor, the effects of siRNAs for these regulator genes on AαC- and MeAαC-mediated increases in luciferase activity were investigated further. PXR siRNA, but not other siRNAs, significantly reduced AαC- and MeAαC-induced luciferase activities. These results demonstrate for the first time that AαC and MeAαC, among the six HCAs tested, increase CYP3A4 mRNA via PXR activation in human hepatoblastoma-derived HPL-A3 cells.

In vivo comet assays are often used to evaluate genotoxicity. The advantage of an in vivo comet assay is that it can be applied to a variety of organs. In general, the liver and stomach are used in an in vivo comet assay. However, there have also been some reports on the in vivo comet assay of the thyroid gland, which is a common target organ for carcinogens in rodents. Ethyl methanesulfonate (EMS) is listed as a compound likely to be the positive control compound of choice in an in vivo comet assay, since it is known to cause DNA damage in a variety of tissues, including the liver, stomach, kidneys, and bone marrow. In contrast, there have been no reports on in vivo comet assays of the thyroid gland in rats treated with EMS. In the present study, we examined whether EMS also exhibits DNA damage in the in vivo comet assay of the thyroid gland in rats. EMS was administered orally at a dose of 200 mg/kg. The results showed a significant increase in mean % tail DNA in the thyroid gland, the extent of which was similar to that in the liver. Since histopathological examination of the thyroid gland revealed no histological findings, the increase in mean % tail DNA was most likely due to DNA damage rather than tissue damage. These results suggest that EMS can be used as a positive control compound in the in vivo comet assay of the thyroid gland in rats.

Deubiquitinases are an important regulator of ubiquitin-mediated signaling pathways because of their ability to cleave the isopeptide bond that connects the ubiquitin chain to the target protein. We previously reported that the expression of many proteins involved in methylmercury toxicity is regulated by the ubiquitin-proteasome system. In this study, double-stranded siRNAs against mRNAs of approximately 60 deubiquitinases were transfected into human embryonic kidney (HEK) 293 cells, and ubiquitin-specific protease 54 (USP54) was identified as a deubiquitinase that increases the sensitivity of HEK293 cells to methylmercury by RNA interference.

Polymethoxyflavones in which all hydroxyl groups are capped by methyl residues show high membrane permeability, metabolic resistance, and diverse biological activities such as cell growth inhibitory effects responsible for their anti-cancer activities. They are expected to be as biotherapeutic drugs for maintaining human health via contributing prevention and treatment of serious illnesses such as cancers, neurodegenerative diseases, inflammatory bowel diseases, lipid metabolism disorders, and so on. Human monoblastic leukemia U937 cell line has been used as an excellent in vitro cell model system for studying macrophage differentiation induced by various cell differentiation inducers such as all-trans retinoic acid (ATRA). In this study, we investigated the influences of two typical polymethoxyflavones, such as nobiletin and tangeretin, on the ATRA-induced superoxide anion (O₂^-)-generating ability of U937 cells. Nobiletin and tangeretin suppressed cell proliferation of U937 cells in a dose-dependent manner. At a concentration of 10 μM, tangeretin drastically brought about down-regulation of the ATRA-induced O₂^--generating ability (to ~15% of ATRA-treated cells) although nobiletin showed moderate inhibitory effects on the ATRA-induced O₂^--generating ability (to ~65% of ATRA-treated cells). Quantitative RT-PCR and immunoblotting revealed that tangeretin down-regulates the ATRA-induced O₂^--generating ability via suppressing gene exprerssion levels of gp91-phox (mRNA: to ~75%, protein: to ~70% of ATRA-treated cells) and p47-phox (mRNA: to ~75%, protein: to ~40% of ATRA-treated cells) genes. These findings demonstrated that tangeretin shows not only the inhibitory effects on cell growth but also the strong suppressing effects on the ATRA-induced O₂^--generating ability of U937 cells.

In recent years, paraoxybenzoic acid esters (parabens) have been used in pharmaceuticals, cosmetics, and food additives. Parabens have been reported to have a weak estrogenic effect in in vitro test systems, and it is presumed that the longer the alkyl chain of the paraben, the greater its endocrine-disrupting and reproductive function effects. However, the effects of parabens on human health are still unclear. In this study, we evaluated the effects of six parabens (methyl p-hydroxybenzoate [MP], ethyl p-hydroxybenzoate [EP], propyl p-hydroxybenzoate [PP], isopropyl p-hydroxybenzoate [IPP], butyl p-hydroxybenzoate [BP], and isobutyl p-hydroxybenzoate [IBP]) on the reproductive function of the model organism Caenorhabditis elegans. We used 25, 50, and 100 µg/mL solutions of parabens in 0.1% dimethyl sulfoxide (DMSO). Bioassays (growth and maturation effect tests and reproduction effect tests) were performed on L1 larvae of wild-type C. elegans. In the growth effects test, all parabens were observed to have no effect. In the maturation effects test, there was a significant decrease in maturity at each concentration of five of the six parabens, with the exception being MP. In the reproduction effects test, a significant decrease in the number of lifetime offspring was observed at each concentration of five of the six parabens, with the exception being EP. This decrease was remarkable with PP, which has been reported to adversely affect reproductive function in rats. It is necessary to continue to focus on the estrogen-like action of parabens, including PP, and perform genetic analyses, such as RNA sequencing.

In vitro culture of primary hepatocytes for drug screening purposes remains a challenge as cells rapidly lose their function in conventional culture conditions. Thin oxygen permeable membranes have shown, through direct oxygenation, beneficial effects for long-term culture and cellular function with freshly isolated hepatocytes. However, culture of cryopreserved hepatocytes, a standard for the industry, has shown limits due to high cellular damage, leading to low cellular function. In addition, high sorption of drug screening compounds on PDMS oxygen permeable membranes has rendered evaluation of different molecules, aimed at the improvement of the culture of those cells difficult. Here, culture of cryopreserved hepatocytes was performed on PMP membranes, known to exhibit exceptionally low sorption characteristics. A mixture of anti-apoptotic and anti-inflammatory compounds to improve cell viability during adhesion was tested and evaluation in terms of cellular damage and drug metabolism was performed after 24 hr and 72 hr. Components of the mixture were shown to have beneficial effect on Reactive Oxygen Species production after 6 hr of adhesion as well as on mitochondrial activity and LDH release after 24 hr. Effects in improving recovery of albumin and drug metabolism, could be efficiently measured after 72 hr as a result of the use of PMP. The presented results demonstrate the compatibility of PMP oxygen-permeable membrane-based culture with cryopreserved hepatocytes for efficient drug screening.

Toluene had been designated as a priority assessment chemical substance under the Japanese Chemical Substances Control Law (CSCL), and as a result of prioritization, a detailed human health hazard assessment was conducted under Assessment II. We evaluated its general, reproductive, and developmental toxicities, as well as its genotoxicity and carcinogenicity, based on the hazard information provided by domestic and international risk assessment organizations, and the following hazard assessment values for oral and inhalation exposure are proposed. The hazard assessment value of 0.223 mg/kg/day for oral exposure was calculated from a no-observed-adverse-effect level (NOAEL) of 312 mg/kg/day (equal to an average daily dose of 223 mg/kg/day) based on liver and kidney weight increases in a 13-week oral toxicity study in rats by using an uncertainty factor (UF) of 1,000 (interspecies variation: 10, intraspecies variation: 10, and short test period: 10). The hazard assessment value of 0.1 ppm (0.383 mg/m³) for inhalation exposure was calculated from a NOAEL of 45 ppm (equal to a continuous exposure level of 10.7 ppm) based on toxic effects on the central nervous system found in epidemiological investigations of occupational exposure by using a UF of 100 (intraspecies variation: 10 and severe effect: 10).

Skin is the primary tissue exposed to ambient air pollution because it acts as an interface between the body and the surrounding atmosphere. We previously reported that particulate matter 2.5 (PM2.5) induced oxidative stress and subsequent chemokine release in the human epidermis, followed by neutrophil chemotaxis. We identified in this study that the leaf extracts from Camellia sinensis and Argania spinosa showed high radical scavenging activity as evaluated by 2,2-diphenyl-1-(2,4,6-trinitrophenyl)-hydrazinyl and 2,2-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid assays. PM2.5 exposure induced lipid peroxidation, IL-8 release and neutrophil migration in human 3-dimensional cultured epidermis. Pretreatment with leaf extracts from Camellia sinensis or Argania spinosa significantly suppressed the above harmful effects elicited by PM2.5. Taken together, both extracts can protect the epidermis from PM2.5 exposure. Camellia sinensis and Argania spinosa extracts could be added to a novel cosmetic that protects skin from air pollution.

There is growing evidence of transgenerational effects of a single exposure to chemicals, whose mechanism is implicated to be epigenetic. However, it is largely unknown whether psychiatric diseases such as ADHD or autism caused by environmental chemicals might be transmitted. Rotenone (3 mg/kg), a dopaminergic toxin was orally exposed to Wistar male pups at 5-day old. Their spontaneous motor activity was higher 1.3 fold than that of control rats at 11 weeks of age. At 26 weeks of age, the hyperactive rat (F0) was mated with Wistar female rats. We established the two strains of such mating and found the spontaneous motor activity of the offspring (F1) were much higher 1.5~2.0 fold than those of both control offspring and the parents. Thus, in this study I show the rat hyperactivity caused by neonatal rotenone lesions was transmitted to next generation, indicating the de novo inheritance.

Using a rat dextran sulfate sodium (DSS)-colitis model, we elucidated that the expression of miRNAs in colorectal tissues, plasma, and feces, particularly miR-31a-5p, miR-181b-5p, and miR-223-3p, could be used as noninvasive biomarkers to evaluate the reversibility of the model. We further investigated whether changes in miRNA levels were reproducible in chronic DSS-induced colitis in rats. Male SD rats were administered 5% DSS in drinking water for two cycles. Cycle 1 consisted of a 7-d dosing period and 14-d recovery period, followed by Cycle 2 consisting of a 5-d dosing period and 7-d recovery period. In-life parameters and the disease activity index (DAI) were respectively examined or calculated daily. Colon length and pathological changes were assessed postmortem in Cycle 2. A panel of nine miRNAs was also measured in colorectal tissues, plasma, and feces using digital PCR. The changes in DAI score and colon length were evident in Cycle 2. Erosive and inflammatory changes were observed in the colon and rectum following DSS treatment. At the end of the off-dose period of Cycle 2, the histological changes in the rectum worsened, while the colon changes showed recovery. The expression patterns of all miRNAs were almost the same in Cycle 2 when compared to those in a previous study (Kodama et al., 2021). Fecal miR-223-3p could be also a useful non-invasive indicator to evaluate the reversibility in chronic DSS-induced colitis in rats.

Neonicotinoids are potent agonists of nicotinic acetylcholine receptors that exert insecticidal effects by causing abnormal excitation of the nervous system. Neonicotinoids and their metabolites effect in mammals, including humans, have become a concern. In the present study, we evaluated the effects of chronic exposure of two neonicotinoids, acetamiprid (ACE) and clothianidin (CTD), on Caenorhabditis elegans. We used 1, 10, 100, and 1000 µM solutions of nicotine, ACE, and CTD dissolved in 1% dimethyl sulfoxide (DMSO). Bioassays and motility tests, which are neurotoxicity assessments, were performed on the L1‒L2 larvae of wild-type C. elegans. To evaluate the effect of exposure over multiple generations and the correlation between concentrations and generations, the same study was conducted on the second and third generations of the exposed group. The bioassay results showed concentration-dependent adverse effects: body length, maturity rate, and lifetime number of pups decreased for both ACE and CTD for the first generation. In a multi-generation study, the effect intensified with the progression of generations, and the toxicity of both ACE and CTD was cumulative. This effect was more pronounced in breeding studies. The motility test results showed concentration-dependent adverse effects, such as a decrease in the number of behaviors for both ACE and CTD in both tests for the first generation. In a multi-generation study, the effect intensified with the progression of generations, and this effect was more pronounced with ACE exposure. Thus, the chronic exposure to ACE and CTD may cause cross-generational adverse effects, especially on C. elegans reproduction and motion.

To estimate the exposure of pharmacists to drugs during tablet crushing, we collected room dust in four pharmacies and a hospital and analyzed the concentrations of the drug ingredient. The ingredient concentrations in the room dust were detected in the range of 15–18000 µg/m³, and the median concentration was 320 µg/m³. The amount of exposure to pharmacists was estimated between 0.8–960 µg/crush, with a median of 17 µg/crush, when the respiratory volume of the pharmacist was 8 L/min. These maximum and median values were more than 10 times higher than those during the previously reported powder preparations, demonstrating that the working environment for pharmacists who crushed tablets posed more health hazards. As countermeasures, working on a bench with dust remover reduced the exposure by 99.0% compared to that on a normal bench, and wearing a medical mask reduced the exposure by 97%. The combined reduction rate of both measures was calculated to be over 99.9%. Moreover, we compared the estimated exposure by the crusher with the rotatory blade and that with two rotatory mortars and found that the estimated exposure using the latter was much less (lower than 1/1000) than that with the former. Thus, the above measures can be used to reduce the exposure of pharmacists to drugs during tablet crushing.

Kaempferol is a kind of natural flavonoid in many edible plants and reportedly has various physiological effects. In the present study, we conducted the genotoxicity (in vitro and in vivo) and 13-week subchronic toxicity studies of a new product, a kaempferol aglycone-rich food produced from enzyme-treated horseradish leaves, to evaluate its safety. In the bacterial reverse mutation test, the kaempferol aglycone-rich product showed positive results in some Salmonella typhimurium strains in the presence or absence of metabolic activation as well as other flavonoids. However, it did not increase micronucleated polychromatic erythrocytes taken from male Sprague−Dawley (SD) rats administered orally by gavage up to 4000 mg/kg for 2 consecutive days. In the 13-week subchronic toxicity study in SD rats, the kaempferol aglycone-rich product was orally administered by gavage once daily to SD rats for 13 weeks (91 days) at a dose of 500, 1000, or 2000 mg/kg/day. No toxic changes were observed at up to 2000 mg/kg/day. In conclusion, these findings indicated that the kaempferol aglycone-rich product was not genotoxic in vivo. The no-observed-adverse-effect level for both male and female rats was 2000 mg/kg/day, the highest dose tested, in the 13-week subchronic toxicity study in rats, suggesting it is safe for use as a food.

The inevitable use of mercury (Hg) and the regular release of its waste into the ecosystem through artisanal and small-scale gold mining (ASGM) activities particularly, in developing countries such as Ghana require constant monitoring and evaluation of Hg contamination and its potential toxicities particularly, in samples such as food. This study evaluated the potential human health risks associated with total mercury (THg) and methylmercury (MeHg) levels of mercury-contaminated cassavas from farms in selected ASGM communities around Obuasi, Ghana. The THg and MeHg levels were evaluated using the direct Hg analyser, MA-3000 while the human health risk assessment was done using the USEPA risk assessment model. The estimated average daily intake for ingestion (eAvDI_(ing)) (mg/kgbw/day) and the hazard quotient (HQ) for THg levels of the samples were above the USEPA reference values of 3 x 10⁻⁴ and 1, respectively. This means that residents ingest more Hg through consumption of cassava, hence long-term repeated exposures to the cassavas may be associated with detrimental human health effects in future. MeHg levels may not cause any human health effects due to eAvDI_(ing) and HQ below 1 x 10⁻⁴ and 1, respectively. However, constant releases of mercury waste and subsequent bioaccumulation along the food chain can cause MeHg levels to increase with time above the USEPA acceptable daily intake. Such levels may be detrimental to human health. Therefore, there is the need for regular and strict monitoring of ASGM activities within the studied communities and other communities involved in ASGM to protect human health and preserve ecosystem integrity.

Acute irradiation stimulates oxidative stress and DNA damage responses. However, it is unknown whether chronic irradiation (IR) at a low dose rate causes similar responses, and epidemiological studies of radiation-exposed people with low doses have reported effects on cardiovascular diseases. Therefore, we investigated the cellular effects under low dose rate of IR in human vascular endothelial cells as a model for cardiovascular diseases. We demonstrated that a low dose rate of IR induces phosphorylation of p38MAPK and STAT1, which is related to cGAS, and increases p21, a cellular senescence-regulatory factor. A low dose rate of IR also causes a remarkable formation of micronuclei in human vascular endothelial cells. DIA proteome analysis in human vascular endothelial cells indicated an increase in oxidative stress- and inflammation-related protein levels, and a decrease in protein levels related to the repression of micronuclei formation following exposure to low dose rate of IR. These results suggest that a low dose rate of IR might induce oxidative stress and micronuclei formation, which could activate the cGAS pathway and subsequently lead to cellular senescence.

There are several subtypes of gastric cancer, such as diffuse-type gastric cancer (GC) and intestinal-type GC. Diffuse-type GC is known to be more malignant than intestinal-type GC, showing high metastasis, recurrence and anti-cancer drug resistance. The malignant phenotype of diffuse-type GC includes cancer stem cell (CSC)-like features and epithelial-mesenchymal transition (EMT). By analyzing the molecular network in these tumors, it is possible to reveal the mechanisms of anti-cancer drug resistance, therapeutic targets and drug safety. Upon the analyses of the molecular network in diffuse- and intestinal-type GC, a regulatory network for RNA virus infection was obtained. This study aims to reveal the relationship between cancer and RNA virus infection in detail. RNA virus infection-related molecules and cancer-related molecules were analyzed using network analysis tools, such as Ingenuity Pathway Analysis (IPA), and molecular networks related to RNA virus infection mechanisms. Regulator effect analysis revealed the involvement of RNA virus infection network in diffuse-type GC. c-Jun N-terminal kinase (JNK) and BCL2 like 11 (BCL2L11) in the Coronavirus Pathogenesis Pathway were activated. In conclusion, this research suggested the relationship between the mechanisms of RNA virus infection and diffuse-type GC. This study may be useful for virus infection control and cancer drug discovery by clarifying the relationship between the mechanism of RNA virus infection and cancer.

4,4'-Oxybis(benzenesulfonohydrazide) (OBSH) is a blowing agent widely used in the manufacture of porous plastics and rubber. OBSH was notified as an additive in the Japanese positive list system for food utensils, containers and packaging. The in vitro mutagenicity of OBSH was shown extensively in bacterial reverse mutation assays, a DNA repair test, and a chromosomal aberration test. Few studies exist on in vivo genotoxic evaluation on OBSH apart from an in vivo micronuclei test. To clarify in vivo mutagenicity, we conducted a transgenic rodent gene mutation (TGR) assay (OECD TG 488). We dosed male Muta^TM Mouse with OBSH by oral gavage at 0 (negative control), 25, 50, and 100 mg/kg/day for 28 consecutive days, and evaluated mutant frequencies (MFs) of lacZ in the liver and glandular stomach (5 mice/group). We observed two deaths and a reduction in body weight at 100 mg/kg/day. Although we exposed Muta^TM Mouse to OBSH orally for 28 days up to the maximum tolerated dose, we did not detect in vivo mutagenicity in the liver and glandular stomach. In contrast, in the positive control we detected significantly increased MFs. The results of this study suggest that OBSH is not mutagenic in vivo.

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[Fundam. Toxicol. Sci., 4, 57-75 (2017) ]
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Error:
These subjects continued to consume increasing doses of IMD.


Correction:
These subjects stopped to consume increasing doses of IMD.

Carbon-nanotube yarn (CNT-Y) made from high-purity, highly crystalized, double-walled carbon nanotubes is an advanced material with excellent electrical conductivity and flexibility; hence, it could potentially be used as a novel electrode for biopotential measurements. To our knowledge, the present study is the first in which CNT-Y electrodes were used to conduct electrocardiography (ECG) and electroencephalography (EEG) on experimental animals. All procedures and biopotential measurements were performed under isoflurane anesthesia. The CNT-Y electrodes were attached to the animals by creating a single interrupting suture on the skin. The lead II electrode configuration was used for ECG recording, i.e., the positive, negative, and body-earth electrodes were placed on the left apex of the auricular surface, the interscapular region, and the cervical region, respectively. The bipolar lead was used for EEG recording, with the exploring and reference electrodes on the bregma and base of the right auricular surface, respectively. Using CNT-Y electrodes, we obtained a clear ECG waveform from rats and a guinea pig; the QRS amplitude was ~1.4 mV. In rats, we obtained an EEG waveform with an amplitude of ~150 µV; the peak frequency was 0.8 Hz and the range was ~3 Hz according to power spectral density analysis. In the guinea pig, we obtained an EEG waveform with an amplitude of ~500 µV; the first peak was 0.1 Hz, the second peak was 1 Hz, and the range was ~3 Hz. These results show that CNT-Y could be used in toxicology studies to easily and inexpensively obtain high-resolution biological signals.

The present study was conducted to survey the influence of canola oil (CAN) ingestion on the steroid hormone production in stroke-prone spontaneously hypertensive rats (SHRSP). Male SHRSP were fed a diet containing 10 wt/wt% soybean oil (SOY, the control) or CAN as the sole dietary fat for 8 weeks. Plasma concentration of luteinizing hormone (LH) was similar in the 2 dietary groups. However, the plasma testosterone level in the CAN group, 1.36 ± 0.271 ng/mL, was lower than in the SOY group, 2.79 ± 0.514 ng/mL (p < 0.05, unpaired t-test; n = 10), and plasma concentration of aldosterone in the CAN group, 345 ± 79.6 pg/mL, was higher than in the SOY group, 159 ± 33.7 pg/mL (p < 0.05, unpaired t-test; n = 10). In the testis, the expressions of mRNA for StAR, CYP11A1, CYP17, 3βHSD and 17βHSD and the amounts of the corresponding proteins were significantly decreased. However, in the adrenal gland, the expressions of mRNA for StAR, CYP11A1, 3βHSD and CYP11B1 in the CAN group were not different from those in the SOY group, but the expression of mRNA and the amount of the corresponding protein for CYP11B2 were increased significantly in the CAN group. These findings are indicative of a peripheral, testicular toxicity of CAN. The decreased testosterone and the concomitantly increased aldosterone may play a role in the aggravation by CAN of the genetic diseases (i.e., metabolic syndrome-like complications) in male SHRSP.

Methylmercury (MeHg) reacts with nucleophilic sulfur species to form sulfur adducts, such as the low-toxic metabolite bismethylmercury sulfide [(MeHg)₂S]. We found that protein-bound persulfides interact with MeHg to form (MeHg)₂S and identified glutathione S-transferase pi 1 as a S-sulfhydrated protein involved in (MeHg)₂S formation. Although glyceraldehyde 3-phosphate dehydrogenase (GAPDH), a house-keeping protein abundantly expressed in various tissues, has been reported to undergo S-sulfhydration in the presence of sulfur donors or cystathionine γ-lyase, the biological significance of this post-translational modification is poorly understood. In this study, we investigated the possible interaction between GAPDH and MeHg to form (MeHg)₂S. High-performance liquid chromatography/atomic absorption spectrophotometry revealed that (MeHg)₂S was formed during the reaction of MeHg with a model of cysteine persulfide and GAPDH following incubation of the protein with NaHS. After reacting with NaHS, GAPDH C152S and C156S mutants transformed MeHg into (MeHg)₂S, whereas formation of the sulfur adduct was not observed for the C247S mutant, suggesting that Cys247 is critical for conversion of MeHg to (MeHg)₂S. These results suggest that the sulfane sulfur on Cys247 of GAPDH plays a protective role in reducing MeHg toxicity.