The present investigation was undertaken to determine the distribution of 1,2-dichloropropane (DCP) in the blood, liver, kidney, lung, and abdominal fat of rats after oral administration. Male rats were orally administered 62 or 125 mg/kg body weight doses of DCP dissolved in corn oil by gavage, and the concentrations in the blood and tissues were measured. The DCP concentration in the abdominal fat was much greater than in the blood and other tissues. Twenty-four-hr after oral administration, DCP could still be detected in the blood and abdominal fat in the 62-mg/kg group, and in the blood, liver, kidney, lung, and abdominal fat in the 125-mg/kg group. Our results are valuable data pertaining to the pharmacokinetics of DCP and to human health risk assessment of oral exposure to DCP.
We developed a modified local lymph node assay based on ATP (LLNA:DA), termed the Two-Stage LLNA:DA, to further reduce the animal numbers in the identification of sensitizers. In the Two-Stage LLNA:DA procedure, 13 chemicals ranging from non-sensitizers to extreme sensitizers were selected. The first stage used reduced LLNA:DA (rLLNA:DA) to screen out sensitive chemicals. The second stage used LLNA:DA based on OECD 442 (A) to classify those potential sensitizers screened out in the first stage. In the first stage, the SIs of the methyl methacrylate, salicylic acid, methyl salicylate, ethyl salicylate, isopropanol and propanediol were below 1.8 and need not to be tested in the second step. Others continued to be tested by LLNA:DA. In the second stage, sodium lauryl sulphate and xylene were classified as weak sensitizers. a-hexyl cinnamic aldehyde and eugenol were moderate sensitizers. Benzalkonium chloride and glyoxal were strong sensitizers, and phthalic anhydride was an extreme sensitizer. The 9/9, 11/12, 10/11, and 8/13 (positive or negative only) categories of the Two-Stage LLNA:DA were consistent with those from the other methods (LLNA, LLNA:DA, GPMT/BT and HMT/HPTA), suggesting that Two-Stage LLNA:DA have a high coincidence rate with reported data. In conclusion, The Two-Stage LLNA:DA is in line with the “3R” rules, and can be a modification of LLNA:DA but needs more study.
Certain interindividual differences affecting the efficacy of drug treatment and adverse drug reactions are caused by genetic variants, and their phenotypic effects differ among ethnic groups. In this study, we used whole exome sequencing (WES) systematically to identify germline mutations that influence the activities of drug-metabolizing enzymes, as well as that of a transporter. We analyzed DNA isolated from blood samples from 2,042 Japanese patients with diverse cancers. We identified sequence variants of CYP2B6 (rs3745274), CYP2C9 (rs1057910), CYP2C19 (rs4986893), CYP2C19 (rs4244285), TPMT (rs1142345), NAT2 (rs1799930), NAT2 (rs1799931), UGT1A1 (rs4148323), COMT (rs4680), ABCB1 (rs1045642), and CDA (rs60369023). Wider application of WES will help to determine the effects of mutations on the activities of proteins encoded by drug response genes, and the information gained will accelerate the development of personalized therapies for patients with cancer. Moreover, this knowledge may provide clues for preventing cancer before the onset of symptoms.
Previously, we showed that phototoxicity assessments in Sprague-Dawley (SD) rats can detect phototoxic potential to the same degree as those in guinea pigs. In this study, we examined whether phototoxicity assessments can be incorporated into general toxicology studies, using SD rats. Three phototoxic compounds were tested. Acridine and 8-methoxypsoralen (8-MOP) were transdermally administered, and 8-MOP and lomefloxacin were orally administered. The animals were allocated to three groups for each compound: single-dose, repeated-dose, and repeated-dose plus toxicokinetics (TK). The single-dose group was irradiated with UV-A and UV-B after a single administration of the drug. The repeated-dose and TK groups were irradiated after 8 days of repeated administration of the drug. Blood samples were also collected from the TK group on days 1 and 7 after administration. The phototoxic compounds resulted in skin reactions in all the groups, with no difference in the degree of skin reaction among the three groups. In the TK measurements, all of the phototoxic compounds were detected in the plasma samples, and the irradiation timing was close to the Tmax. These results indicate that phototoxic potential could be evaluated in the TK group, and phototoxicity assessments could be incorporated into general toxicology studies. This reduces the number of studies and animals required, thus shortening the research and development period, and supporting the 3Rs principle of animal experiments. The study also provides information regarding appropriate irradiation timings, differences between the sexes, and dose-response, in turn enabling the phototoxic risk of the compounds to be clearly evaluated.
The distribution of metal and metalloid species in each of the cell compartments is termed as “metallome”. It is important to elucidate the molecular mechanism underlying the beneficial or toxic effects exerted by a given metal or metalloid on human health. Therefore, we developed a method to measure intracellular metal ion concentration (particularly, intracellular calcium ion) in fission yeast. We evaluated the effects of nitric acid (HNO3), zymolyase, and westase treatment on cytolysis in fission yeast. Moreover, we evaluated the changes in the intracellular calcium ion concentration in fission yeast in response to treatment with/without micafungin. The fission yeast undergoes lysis when treated with 60% HNO3,which is simpler and cheaper compared to the other treatments. Additionally, the intracellular calcium ion concentration in 60% HNO3-treated fission yeast was determined by inductively coupled plasma atomic emission spectrometry. This study yields significant information pertaining to measurement of the intracellular calcium ion concentration in fission yeast, which is useful for elucidating the physiological or pathological functions of calcium ion in the biological systems. This study is the first step to obtain perspective view on the effect of the metallome in biological systems.
Amphotericin B (AmpB) nephrotoxicity was used to assess the utility of drug‑induced kidney injury (DIKI) biomarkers in an exploratory study in male cynomolgus monkeys. All animals had quantifiable levels of AmpB in plasma on days 1 and 4. There were no clinical signs of AmpB‑induced toxicity in this study. The gold standard method used to confirm AmpB‑induced DIKI was anatomic pathology which revealed microscopic lesions with varying grades of severity. Immunolocalization of alpha‑1 microglobulin (α‑1M), kidney injury molecule 1 (KIM‑1), osteopontin (OPN) and neutrophil gelatinase‑associated lipocalin (NGAL) proteins was evaluated in formalin‑fixed, paraffin‑embedded monkey kidney tissue sections. AmpB related immunoreactivities were identified in distinct nephron segments of treated monkeys including α‑1M in damaged proximal tubule epithelium, KIM‑1 in damaged medullary tubule epithelium, OPN mostly in the infiltrating cells of cortical tubule interstitium, and NGAL in the granular and cellular cast in dilatated cortical tubules. Variations in α‑1M, KIM‑1, OPN and NGAL immunolocalization appear as promising DIKI protein biomarkers when monitoring for AmpB‑induced corticomedullary tubule injury in male cynomolgus monkeys.
This study was designed to identify early-response genes of chemical-induced renal carcinogenicity for the prediction of chemical carcinogenicity in rats. We conducted a 28-day repeated-dose test in male Crl:CD (SD) rats with 12 carcinogens and 10 non-carcinogens as the training dataset, and five carcinogens and five non-carcinogens as the validation dataset. Renal gene expression profiles were analyzed by using a microarray. Fifteen candidate genes were selected from the gene expression profiles of the training dataset as genes that showed specific expression in response to carcinogens. To assess the prediction performance of the candidate genes for renal carcinogenicity, a prediction formula was developed on the basis of the gene expression data. When this formula was applied to the training dataset to check its predictive performance, all of the carcinogens and non-carcinogens were predicted correctly; the prediction formula was then applied to the validation dataset, and five carcinogens and four non-carcinogens were correctly predicted. However, 4-Hydroxy-m-phenylenediammonium dichloride (AMIDOL), a known non-renal carcinogen, was judged as positive. Therefore, the accuracy of the prediction formula for renal carcinogenicity was 100% for the training dataset and 90% for the validation dataset. Among the predictive genes, Hamp and Ranbp1 are known to be important for cell growth and cell cycle regulation, which are important events in carcinogenesis. Given our current limited knowledge of the genes responsible for renal carcinogenesis, the identification of candidate genes of chemical-induced renal carcinogenicity by use of this gene expression-based prediction method represents a promising advance in renal carcinogen identification.
In order to characterize human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) sheets as a model for detecting drug-induced conduction disturbance, we examined their electrophysiological and electropharmacological properties by using the multi-electrode array system with a programmed electrical stimulation protocol. At pre-drug control, the conduction speed, effective refractory period and field potential duration were 0.14 ± 0.01 m/sec, 453 ± 10 msec and 361 ± 9 msec, respectively at a cycle length of 1,000 msec (n = 18). Shortening the pacing cycle length from 1,000 to 600 msec decreased the conduction speed and field potential duration, but prolonged the effective refractory period. Disopyramide, lidocaine and flecainide decreased the conduction speed but prolonged the effective refractory period and field potential duration, whereas the reverse was true for verapamil. Thus, conduction properties of the cell sheet may largely depend on the extent of Na+ channel availability as is the case in the human ventricle. Importantly, there was no relationship between the conduction delay and 1st spike amplitude reduction after the treatment of Na+ channel blockers. These findings may provide crucial guide on future application of this new technology for early phase safety pharmacological screening of new chemical entities.
Epidemiological studies suggest that lung cancer, which is a major cause of cancer death, has a critical association with cigarette smoking. Tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) in cigarette smoke is a major risk factor for carcinogenesis. However, the mechanisms by which NNK promotes cancer development have not been fully elucidated. Growing evidence suggests that lung cancer originates from cancer stem cells (CSCs), which are a minor population of lung cancer cells. In the present study, we investigated the effects of NNK on the CSCs in A549 human lung cancer cells using flow cytometry with aldehyde dehydrogenase (ALDH), a functional marker of CSCs. We found that NNK increased the proportion of ALDH-positive cells in a dose-dependent manner. A Wnt inhibitor PNU74654 reduced NNK-induced expression levels of Wnt target gene Dkk1 and increase in ALDH-positive cells. We next examined the signaling pathway that mediates the NNK-induced increase in ALDH-positive cells via Wnt signaling. DCF assay revealed that NNK induced reactive oxygen species (ROS) production. The ROS scavenger N-acetylcysteine (NAC) inhibited the NNK-induced Wnt activation and increase in ALDH-positive cells. These data suggest that NNK-induced ROS activate the Wnt signaling pathway in A549 cells. These findings would provide new insights into the role of NNK in the lung CSCs.
The ATP assay is a highly sensitive and versatile method for measuring cytotoxicity. However, the correlation between the cell viability results obtained using the ATP assay and those obtained using direct cell counting has not been widely reported. Therefore, to evaluate the reliability and limitations of the ATP assay, we compared the results of ATP assay with those of automatic cell counter, which can measure the number and diameter of cells directly, by using 24 compounds and repeating individual experiments thrice. The correlation between the data was low for 7 of the 24 compounds (r2 < 0.8, at least 2 out of 3 experiments). These were the top 7 of the 11 compounds that induced cell hypertrophy. These 7 compounds were also observed to increase the area of mitochondria. However, the last 4 of the 11 compounds increased the cell size but did not increase the mitochondrial area. For the remaining 13 compounds, which had no effect on cell size, a good correlation was observed between the results of the two methods (r2 > 0.8, at least 2 out of 3 experiments), and the cell size was effectively the same as that of the controls. We concluded that the poor correlation between the two methods was attributable to an increase in the content of intracellular ATP because of the chemically induced cell and mitochondrial hypertrophy. We showed that the ATP assay is unsuitable for assessing the cytotoxicity of compounds that induce cell hypertrophy with increase in the mitochondrial area and ATP content.
Although cardiac activity is known to differ between species in many respects, most evaluations of the cardiac effects of low-frequency electric and magnetic fields, which have a stimulant effect on electrically activated cells, have been performed in non-human experimental animals and cells, and the effects in humans have been assessed using theoretical models. In recent years, it has been verified that human cardiomyocytes differentiated from human induced pluripotent stem cells (hiPS-CM) are useful for evaluating human responses to various cardioactive compounds. In this study, we applied hiPSCMs for the first time to evaluate the human cardiac effects of power-frequency magnetic fields (MFs). After preparation of hiPS-CMs, we subjected a hiPS-CM monolayer formed on a multi-electrode array to short-term exposure to a 50 Hz MF at 400 mT with recording of the extracellular field potentials. The field potential duration of the hiPS-CMs did not differ significantly pre- and post-exposure, indicating that under these conditions, exposure to a 50 Hz MF at 400 mT does not affect the electrical activity of hiPSCMs.
SUMOylation is widely reported as a cytoprotective response against cell stress induced by ischemia and reperfusion. However, it is still unclear what other cell stressors trigger protein SUMOylation, what mechanisms enhance and maintain the enhanced SUMOylation, and if it is a general protective mediator against other cytotoxic stresses, toxic heavy metal cadmium for instance. Here, we show increased levels of SUMOylation and decreased levels of the deSUMOylation enzyme SENP3 in PC12 cells exposed to CdCl2. In addition, SENP3 knockdown reduced cadmium-induced apoptosis and cell death in PC12 cells, while SENP3 overexpression enhanced cell death. These results suggest that SENP3 is an important regulator of the cell response to cadmium stress. Our finding is consistent with previous reports of SENP3 and SUMOylation in ischemia, and implies that the regulation of SENP3 levels and subsequent changes in SUMOylation could be a general cytoprotective mechanism.
N,N-Dimethylformamide (DMF), a solvent commonly used in factories, can induce liver toxicities, including hepatitis, fibrosis, cirrhosis and hepatoma. It is well known that the gut microbial community plays a role in the metabolism of many toxic substance and in liver regeneration. However, the effect of DMF on rat gut microbial community is poorly understood. The gut microbiotas in control rats and rats exposed to DMF were characterized by high-throughput sequencing of the bacterial 16S rRNA gene.The levels of biochemical parameters in the serum of rats, including cholesterol, bile acid, alanine aminotransferase (ALT), and aspartate aminotransferase (AST), were evaluated. The weight was lower in the DMF exposure group than in the control group. DMF exposure led to changes in gut microbiotas that were reflected in a decreased abundance of Prevotellaceae, Lactobacillaceae, and increased abundance of S24-7, Baceroidaceae, Rikenellaceae and Peptostreptococcaceae. Compared with control group, the cholesterol level was substantially reduced in the DMF exposure group (p < 0.05), while the concentration of bile acid was significantly increased in the DMF exposure group (p < 0.05). The present data established that the gut microbiotasy were changed after DMF exposure, and it revealed the relationship between DMF and gut microbiotas for the first time.
The aim of the present study is to investigate whether or not bromobenzene (BB) toxicity varies with circadian periodicity. Seven-week-old male ICR mice were injected with 900 mg/kg (5.73 mmol/kg) BB intraperitoneally at 4 different time points of a day (zeitgeber time [ZT]: ZT0, ZT6, ZT12, and ZT18). Mortality was then monitored for 7 days after injection. Interestingly, mice were sensitive to BB acute toxicity at ZT6 while tolerant at ZT18. Moreover, in mice that were given a non-lethal dose of BB (540 mg (3.44 mmol)/kg), levels of alanine aminotransferase and aspartate aminotransferase, used as markers of hepatic injury, markedly increased in response to injection at ZT6, but did not increase significantly in response to injection at ZT18. In contrast, the markers of renal injury (creatinine and blood urea nitrogen), showed no significant difference in response to the two injection times. To further investigate this extreme circadian variation, we examined hepatic and renal lipid peroxidation levels, and conducted histopathological studies. Similar to our observation with alanine aminotransferase and creatinine, hepatic lipid peroxidation and histopathological changes were more pronounced than renal changes, and showed circadian variation. Our present investigation demonstrated that BB-induced mortality had clear circadian variation, and suggested that hepatic injury was one of the important factors for determination of this variation.