The Journal of Toxicological Sciences Vol. 40(2015) No. 1
February

Prenatal diesel exhaust (DE) exposure is associated with detrimental health effects in offspring. Although previous reports suggest that DE exposure affects the brain of offspring in the developmental period, the molecular events associated with the health effects have largely remained unclear. We hypothesized that the DNA methylation state would be disrupted by prenatal DE exposure. In the present study, the authors examined the genome-wide DNA methylation state of the gene promoter and bioinformatically analyzed the obtained data to identify the molecular events related to disrupted DNA methylation. Pregnant C57BL/6J mice were exposed to DE (DEP; 0.1 mg/m³) in an inhalation chamber on gestational days 0-16. Brains were collected from 1-day-old and 21-day-old offspring. The genomewide DNA methylation state of the brain genome was analyzed by methylation-specific DNA immunoprecipitation and subsequent promoter tiling array analysis. The genes in which the DNA methylation level was affected by prenatal DE exposure were bioinformatically categorized using Gene Ontology (GO). Differentially methylated DNA regions were detected in all chromosomes in brains collected from both 1-day-old and 21-day-old offspring. Altered DNA methylation was observed independently of the presence of CpG island. Bioinformatic interpretation using GO terms showed that differentially methylated genes with CpG islands in their promoter were commonly enriched in neuronal differentiation and neurogenesis. The results suggest that prenatal DE exposure causes genome-wide disruption of DNA methylation in the brain. Disrupted DNA methylation would disturb neuronal development in the developmental period and may be associated with health and disease in later life.

Acid-electrolyzed water (AEW) is commonly used as a disinfectant in the agricultural and medical fields. Although several studies have been conducted to examine its toxicity in vitro and in vivo, the cytotoxic mechanism of AEW has never been verified. The purpose of the present study was to elucidate the underlying mechanism by which AEW exerts its in vitro cytotoxic effect. Mouse fibroblasts treated with AEW experienced dilution rate-dependent cytotoxic effects in the 100% confluent phase as well as in the mitotic phase. The levels of intracellular reactive oxygen species (ROS) increased significantly in fully-confluent cells treated with undiluted and four times diluted AEW. In both of these treatments, cytotoxicity was also observed. It is thus concluded that the in vitro cytotoxicity of AEW is attributable to increased intracellular ROS. Additionally, the ROS responsible for these effects appears to be, at least in part, hydroxyl radical because the increase in intracellular ROS was attenuated by post-treatment with dimethyl sulfoxide, a hydroxyl radical scavenger, and with the antioxidant polyphenol, proanthocyanidin.

Recent evidence shows that tumor microenvironment containing heterogeneous cells may be involved in cancer initiation, growth and tumor cell response to anticancer therapy. Chemotherapy was designed to make toxic impact on malicious cells in organisms, however, the means to protect healthy cells against chemical toxicity are still unsuccessful. As known, the majority of tumor surrounding cells are cancer-associated adipocytes which influence cancer development, progression and treatment. Targeting the components of tumor microenvironment in combination with conventional cancer treatment may become an effective cancer therapy strategy. However, little is known about adipocyte death mechanisms during combined chemo- and targeted therapy. The importance of c-Jun-NH₂-terminal kinase (JNK) signaling in tumor development and treatment has been demonstrated using various in vitro and in vivo cancer models. The aim of this study was to ascertain adipocyte viability during simultaneous stress kinase JNK inhibition and exposure to one of the most commonly used anticancer drugs cis-diamminedichloroplatinum II (cisplatin). Our model involved adipocyte-like cells (ADC) which were obtained during in vitro differentiation of adult rabbit muscle-derived stem cells. Cisplatin induced apoptotic cell death. During 24-hr cisplatin treatment gradual, strong and prolonged increase of both JNK and its target protein c-Jun phosphorylation was found in ADC. Pre-treatment of cells with SP600125 decreased cisplatin-induced activation of c-Jun and promoted apoptosis. Upregulation of proapoptotic Bax and downregulation of antiapoptotic Bcl-2 proteins were found to be regulated in JNK-dependent manner. Thus, the results prove the antiapoptotic role of activated JNK in adipocyte-like cells treated with cisplatin.

Fluvoxamine is one of the typical selective serotonin-reuptake inhibitors. While its combined use with QT-prolonging drugs has been contraindicated because of the increase in plasma concentrations of such drugs, information is still limited whether fluvoxamine by itself may directly prolong the QT interval. We examined electropharmacological effects of fluvoxamine together with its pharmacokinetic profile by using the halothane-anesthetized dogs (n = 4). Fluvoxamine was intravenously administered in three escalating doses of 0.1, 1 and 10 mg/kg over 10 min with a pause of 20 min between the doses. The low dose provided therapeutic plasma drug concentration, whereas the middle and high doses attained approximately 10 and 100 times of the therapeutic ones, respectively. Supra-therapeutic concentration of fluvoxamine exerted the negative chronotropic, inotropic and hypotensive effects; and suppressed the atrioventricular nodal and intraventricular conductions, indicating inhibitory actions on Ca²⁺ and Na⁺ channels, whereas it delayed the repolarization in a reverse use-dependent manner, reflecting characteristics of rapidly activating delayed rectifier K⁺ current channel-blocking property. Fluvoxamine prolonged the terminal repolarization phase at 100 times higher concentration than the therapeutic, indicating its proarrhythmic potential. Thus, fluvoxamine by itself has potential to directly induce long QT syndrome at supra-therapeutic concentrations.

Nonylphenol ethoxylate (NPE) is a non-ionic surfactant, that is degraded to short-chain NPE and 4-nonylphenol (NP) by bacteria in the environment. NP, one of the most common environmental endocrine disruptors, exhibits weak estrogen-like activity. In this study, we investigated whether oral administration of NP (at 0.5 and 5 mg/kg doses) affects spatial learning and memory, general activity, emotionality, and fear-motivated learning and memory in male and female Sprague-Dawley (SD) rats. SD rats of both sexes were evaluated using a battery of behavioral tests, including an appetite-motivated maze test (MAZE test) that was used to assess spatial learning and memory. In the MAZE test, the time required to reach the reward in male rats treated with 0.5 mg/kg NP group and female rats administered 5 mg/kg NP was significantly longer than that for control animals of the corresponding sex. In other behavioral tests, no significant differences were observed between the control group and either of the NP-treated groups of male rats. In female rats, inner and ambulation values for animals administered 0.5 mg/kg NP were significantly higher than those measured in control animals in open-field test, while the latency in the group treated with 5 mg/kg NP was significantly shorter compared to the control group in step-through passive avoidance test. This study indicates that oral administration of a low-dose of NP slightly impairs spatial learning and memory performance in male and female rats, and alters emotionality and fear-motivated learning and memory in female rats only.

Inorganic phosphate (Pi) plays crucial roles in several biological processes and signaling pathways. Pi uptake is regulated by sodium-dependent phosphate (Na/Pi) transporters (NPTs). Moreover, Pi is used as a food additive in food items such as sausages, crackers, dairy products, and beverages. However, the high serum concentration of phosphate (> 5.5 mg/dL) can cause adverse renal effects, cardiovascular effects including vascular or valvular calcification, and stimulate bone resorption. In addition, Pi can also alter vital cellular signaling, related to cell growth and cap-dependent protein translation. Moreover, intake of dietary Pi, whether high (1.0%) or low (0.1%), affects organs in developing mice, and is related to tumorigenesis in mice. The recommended dietary allowance (RDA) of Pi is the daily dietary intake required to maintain levels above the lower limit of the range of normal serum Pi concentration (2.7 mg/dL) for most individuals (97-98%). Thus, adequate intake of Pi (RDA; 700 mg/day) and maintenance of normal Pi concentration (2.7-4.5 mg/dL) are important for health and prevention of diseases caused by inadequate Pi intake.

Nicardipine hydrochloride (NIC), a dihydropyridine calcium-channel blocking agent, has been widely used for the treatment of hypertension. Especially, nicardipine hydrochloride injection is used as first-line therapy for emergency treatment of abnormally high blood pressure. Although NIC has an attractive pharmacological profile, one of the dose-limiting factors of NIC is severe peripheral vascular injury after intravenous injection. The goal of this study was to better understand and thereby reduce NIC-mediated vascular injury. Here, we investigated the mechanism of NIC-induced vascular injury using human dermal microvascular endothelial cells (HMVECs). NIC decreased cell viability and increased percent of dead cells in a dose-dependent manner (10-30 μg/mL). Although cell membrane injury was not significant over 9 hr exposure, significant changes of cell morphology and increases in vacuoles in HMVECs were observed within 30 min of NIC exposure (30 μg/mL). Autophagosome labeling with monodansylcadaverine revealed increased autophagosomes in the NIC-treated cells, whereas caspase 3/7 activity was not increased in the NIC-treated cells (30 μg/mL). Additionally, NIC-induced reduction of cell viability was inhibited by 3-methyladenine, an inhibitor of autophagosome formation. These findings suggest that NIC causes severe peripheral venous irritation via induction of autophagic cell death and that inhibition of autophagy could contribute to the reduction of NIC-induced vascular injury.

Genotoxicity is the most commonly used endpoint to predict the carcinogenicity of chemicals. The International Conference on Harmonization (ICH) M7 Guideline on Assessment and Control of DNA Reactive (Mutagenic) Impurities in Pharmaceuticals to Limit Potential Carcinogenic Risk offers guidance on (quantitative) structure-activity relationship ((Q)SAR) methodologies that predict the outcome of bacterial mutagenicity assay for actual and potential impurities. We examined the effectiveness of the (Q)SAR approach with the combination of DEREK NEXUS as an expert rule-based system and ADMEWorks as a statistics-based system for the prediction of not only mutagenic potential in the Ames test, but also genotoxic potential in mutagenicity and clastogenicity tests, using a data set of 342 chemicals extracted from the literature. The prediction of mutagenic potential or genotoxic potential by DEREK NEXUS or ADMEWorks showed high values of sensitivity and concordance, while prediction by the combination of DEREK NEXUS and ADMEWorks (battery system) showed the highest values of sensitivity and concordance among the three methods, but the lowest value of specificity. The number of false negatives was reduced with the battery system. We also separately predicted the mutagenic potential and genotoxic potential of 41 cosmetic ingredients listed in the International Nomenclature of Cosmetic Ingredients (INCI) among the 342 chemicals. Although specificity was low with the battery system, sensitivity and concordance were high. These results suggest that the battery system consisting of DEREK NEXUS and ADMEWorks is useful for prediction of genotoxic potential of chemicals, including cosmetic ingredients.

In view of the known sensitivity of the developing central nervous system to pollutants, we sought to assess the effects of exposure to uranium (U) — a heavy metal naturally present in the environment — on the behavior of young rats and the impact of oxidative stress on their hippocampus. Pups drank U (in the form of uranyl nitrate) at doses of 10 or 40 mg.L^-1 for 10 weeks from birth. Control rats drank mineral water. Locomotor activity in an open field and practice effects on a rotarod device decreased in rats exposed to 10 mg.L^-1 (respectively, -19.4% and -51.4%) or 40 mg.L^-1 (respectively, -19.3% and -55.9%) in compared with control rats. Anxiety (+37%) and depressive-like behavior (-50.8%) were altered by U exposure only at 40 mg.L^-1. Lipid peroxidation (+35%) and protein carbonyl concentration (+137%) increased significantly after exposure to U at 40 mg.L^-1. A significant increase in superoxide dismutase (SOD, +122.5%) and glutathione peroxidase (GPx, +13.6%) activities was also observed in the hippocampus of rats exposed to 40 mg.L^-1. These results demonstrate that exposure to U since birth alters some behaviors and modifies antioxidant status.

In the view of animal welfare considerations, we investigated the suitability of modifying the rat liver micronucleus test with partial hepatectomy to include administration of an analgesic drug to minimize pain and distress as much as possible. The effects of the analgesic, buprenorphine, on the genotoxicity evaluation of structural chromosome aberration inducers (cyclophosphamide, diethylnitrosamine and 1,2-dimethylhydrazine) and numerical chromosome aberration inducers (colchicine and carbendazim) were examined. The genotoxicants were given orally to 8-week-old male F344 rats a day before or after partial hepatectomy and hepatocytes were isolated 4 days after the partial hepatectomy. Buprenorphine was injected subcutaneously twice a day with at least a 6-hr interval for 2 days from just after partial hepatectomy. As results, buprenorphine caused neither change in clinical signs (except for one animal death) nor increase in the incidence of micronucleated hepatocytes of vehicle treated animals. In the case of concomitant treatment of buprenorphine and a genotoxicant, one out of 8 animals died in each group given buprenorphine with cyclophosphamide, carbendazim or colchicine (lower dose level only). Slight changes in clinical signs were noted in the group given buprenorphine with cyclophosphamide or carbendazim. A statistically significant increase in the incidence of micronucleated hepatocytes was obtained in concomitant treatment of buprenorphine and genotoxicant compared with genotoxicant alone for 1,2-dimethylhydrazine, colchicine and carbendazim. It is concluded that use of buprenorphine as an analgesic drug to minimize pain and distress for rats that are given partial hepatectomy is not appropriate under the present experimental conditions, because it could enhance the general toxicity and genotoxicity of the test chemical.

Spiroquinazolinone compounds have been considered as a new series of potent apoptosis-inducing agents. In this study, anti-proliferative and apoptotic effects of the derivatives from the spiroquinazolinone family were investigated in the human chronic myeloid leukemia K562 cells. The K562 cells were treated with various concentrations of the spiroquinazolinone (10-300 µM) for 3 days and cell viability was determined by MTT growth inhibition assay. 4t-QTC was more active among these compounds with IC50 of 50 ± 3.6 µM and was selected for further studies. Apoptosis, as the mechanism of cell death was investigated morphologically by acridine orange/ethidium bromide (AO/EtBr) double staining, cell surface expression assay of phosphatidyl serine by Annexin V/PI technique, as well as the formation of DNA ladder. The K562 cells underwent apoptosis upon a single dose (at IC50 value) of the 4t-QTC compound, and over-expressed caspase-3 expression by more than 1.7-fold, following a 72 hr treatment. Furthermore, RT-PCR and Western blot analysis revealed that treatment of the K562 cells with 4t-QTC down-regulates and up-regulates the expression of Bcl-2 (anti-apoptotic) and Bax (pro-apoptotic), respectively. Based on the present data, it seems that these compounds from the spiroquinazolinone family are good candidates for further evaluation as an effective chemotherapeutic family acting through induction of apoptosis in chronic myeloid leukemia.

Aspirin has been reported to regulate lipid metabolism. However, the mechanism underlying the regulation is not clear. We presently investigated aspirin’s promotion of AMP-activated protein kinase (AMPK) pathway activation in human hepatoma HepG2 cells by examining AMPK expression, the promotion of AMPK activation. Then we investigated the influence of aspirin-promoted AMPK signaling on fatty acid oxidation in HepG2 cells. The results demonstrated that aspirin treatment did not regulate the expression of AMPK and its downstream target, Acetyl-Coenzyme A Carboxylase (ACC), but activated the AMPK signaling pathway by promoting the phosphorylation of AMPK and ACC. And, interestingly, the promotion by aspirin is dependent of cellular esterase, which catalyzes aspirin to salicylate. Moreover, the activated AMPK signaling promoted the fatty acid oxidation, by promoting expression of Carnitine palmitoyltransferase I (CPT1) and Medium-Chain Acyl-CoA Dehydrogenase (MCAD) in both mRNA and protein levels. Thus, we confirmed in this study that aspirin promoted lipid oxidation by upregulating the AMPK signaling pathway.