4-Methylthio-3-butenyl isothiocyanate (MTBITC) induced apoptotic cell death and G2/M cell cycle arrest via ROS production in human esophageal epithelial cancer cells.

To investigate the chemopreventive mechanisms of 4-Methylthio-3-butenyl isothiocyanate (MTBITC), we analyzed cell viability, cell cycle distribution, and expression levels for cell cycle and apoptosis-related proteins in MTBITC-treated malignant esophageal KYSE510 cells, with and without the reactive oxygen species (ROS) scavenger N-acethyl-L-Cysteine (NAC). MTBITC dose-dependently reduced cell viability and Bcl2 protein expression, while it induced cleavages of caspase-3, caspase-9, and PARP-1, suggesting that reduced cell viability occurred through the mitochondrial apoptotic pathway in KYSE510 cells. In cell cycle distribution analysis, MTBITC (20-40 µM) induced cell cycle arrest at G2/M phase. Furthermore, MTBITC induced Chk1 and Akt phosphorylations and decreased p27 protein expression. Both apoptotic- and cell cycle-related changes induced by MTBITC treatment were abolished by NAC. These results suggest that MTBITC has chemopreventive potential for esophageal carcinogenesis by elimination of cancer cells via induction of mitochondrial apoptotic cell death, G2/M cell cycle arrest, and ROS production.

Regulation of NRF2, AP-1 and NF-κB by cigarette smoke exposure in three-dimensional human bronchial epithelial cells.

Cigarette smoke (CS) is a complex mixture of chemicals and interacts with various physiological processes. We previously reported that nuclear factor erythroid 2-related factor 2 (NRF2) was the most sensitive transcription factor to aqueous CS extract (AqCSE) exposure in monolayer cultured human bronchial epithelial cell lines. Recently, in vitro three-dimensional (3D) culture models have been used to supplement pharmacological and toxicological assessments. Bronchial epithelium models in particular are useful for the evaluation of substances that directly contact the respiratory tract, such as CS. In the present study, we used 3D-cultured human bronchial epithelial cells (HBECs) to assess activation of transcription factors and relevant gene expression in response to AqCSE, primarily focusing on NRF2 and nuclear factor-kappa B (NF-κB) pathways. The 3D-cultured HBECs exposed to AqCSE showed expression of NRF2 and its nuclear translocation in addition to upregulation of genes related to oxidative stress. Our results suggest that the NRF2 pathway was the dominant pathway when 3D-cultured HBECs were exposed to AqCSE at a low dose, supporting our previous findings that NRF2 was the most sensitive transcription factor in response to AqCSE. Expression and nuclear translocation of NF-κB were not increased, although proinflammatory genes were upregulated. However, another inflammation-related transcription factor, activation protein 1, was induced by AqCSE. Gene classification analysis suggested that induction of the inflammatory response by AqCSE was dependent on NRF2 and activation protein 1 rather than NF-κB.

4-Methylthio-3-butenyl isothiocyanate mediates nuclear factor (erythroid-derived 2)-like 2 activation by regulating reactive oxygen species production in human esophageal epithelial cells.

4-Methylthio-3-butenyl isothiocyanate (MTBITC) extracted from daikon (Raphanus sativus), which shows antimutagenicity, may have applications as an effective chemopreventive agent in several cancers; however, few reports have described the associated mechanisms. We investigated whether MTBITC induced cytoprotective genes, including phase II enzymes, in Het-1A human esophageal epithelial cells. HMOX1, NQO1, and GCLC mRNA levels and nuclear factor (erythroid-derived 2)-like 2 (Nrf2) protein levels were increased in Het-1A cells treated with 10 µM MTBITC. Reactive oxygen species (ROS) tended to increase when Het-1A cells were treated with MTBITC, and the increases in ROS and Nrf2 expression in the cells treated with MTBITC were completely abolished by treatment with N-acetyl-l-cysteine. We also examined the relationships between Nrf2 activation and mitogen-activated protein kinase (MAPK) signaling by western blot analysis. MTBITC induced extracellular signal-regulated kinase, c-Jun N-terminal kinase, and p38 phosphorylation in Het-1A cells; however, MTBITC did not affect the relationship between Nrf2 activation and MAPK responses. In the present study, we found that MTBITC induced Nrf2 activation and cytoprotective genes via ROS production in Het-1A cells. These results suggest that MTBITC may have the potential for preventing esophageal carcinogenesis through modification of carcinogen metabolism by phase II enzyme induction via ROS production.

An environmental pollutant, 9,10-phenanthrenequinone, activates human TRPA1 via critical cysteines 621 and 665.

Transient receptor potential ankyrin 1 (TRPA1) is activated by noxious cold, mechanical stimulation, and irritant chemicals. In our recent study, 9, 10-phenanthrenequinone (9,10-PQ) is the most potent irritant for activation of NRF2 among 1395 cigarette smoke components and it may be, therefore, important to find its additional targets. Here, we show that 9,10-PQ functions as an activator of TRPA1 in human embryonic kidney (HEK) cells expressing human wild-type TRPA1 (HEK-wTRPA1) and human alveolar A549 (A549) cells. Application of 9,10-PQ at 0.1-10 µmol/L induced a concentration-dependent Ca2+ response as well as inward currents at -50 mV in HEK-wTRPA1 cells. The current response was blocked by TRPA1 antagonists, HC-030031 (HC) and A-967079. To test whether 9,10-PQ affects the cysteine residues of TRPA1, we expressed mutant TRPA1 channels in HEK cells (HEK-muTRPA1) in which six different cysteine residues were replaced with serine. Among them, a mutation of cysteine 621 (C621S) abolished the 9,10-PQ-induced Ca2+ and current responses. The channel activity induced by 9,10-PQ was also abolished in excised inside-out patches isolated from HEK-muTRPA1 cells with the C621S substitution. Although a mutation of cysteine 665 (C665S) reduced the 9,10-PQ-induced response, channel sensitization by pretreatment with Cu2+ plus 1,10-phenanthroline and by internal dialysis of 3 µmol/L Ca2+ restored the response. However, a double mutant with C621S and C665S substitutions had little response to 9,10-PQ, even when sensitized by Ca2+ dialysis. In A549 cells, 9,10-PQ induced an HC-sensitive Ca2+ response. Our findings demonstrate that 9,10-PQ activation of human TRA1 is dependent on cysteine residues 621 and 665.

Toxic effects of 4-methylthio-3-butenyl isothiocyanate (Raphasatin) in the rat urinary bladder without genotoxicity.

We recently reported that 4-methylthio-3-butenyl isothiocyanate (MTBITC) exerts chemopreventive effects on the rat esophageal carcinogenesis model at a low dose of 80 ppm in a diet. In contrast, some isothiocyanates (ITCs) have been reported to cause toxic effects, promotion activity, and/or carcinogenic potential in the urinary bladder of rats. In the present study, we investigated whether MTBITC had toxic effects in the urinary bladder similar to other ITCs, such as phenethyl ITC (PEITC). First, to examine the early toxicity of MTBITC, rats were fed a diet supplemented with 100, 300 or 1000 ppm MTBITC for 14 days. Treatment with 1000 ppm MTBITC caused increased organ weights and histopathological changes in the urinary bladder, producing lesions similar to those of 1000 ppm PEITC. In contrast, rats treated with 100 or 300 ppm MTBITC showed no signs of toxicity. Additionally, we performed in vivo genotoxicity studies to clarify whether MTBITC may exhibit a carcinogenic potential through a genotoxic mechanism in rats. Rats were treated with MTBITC for 3 days at doses of 10, 30 or 90 mg kg-1 body weight by gavage, and comet assays in the urinary bladder and micronucleus assays in the bone marrow were performed. No genotoxic changes were observed after treatment with MTBITC at all doses. Overall, these results suggested that the effects of MTBITC in the rat urinary bladder are less than those of PEITC, but that MTBITC could have toxic effects through a nongenotoxic mechanism in the urinary bladder of rats at high doses. Copyright © 2016 John Wiley & Sons, Ltd.

Lack of in vivo mutagenicity of 1,2-dichloropropane and dichloromethane in the livers of gpt delta rats administered singly or in combination.

1,2-Dichloropropane (1,2-DCP) and dichloromethane (DCM) are possible causative agents associated with the development of cholangiocarcinoma in employees working in printing plant in Osaka, Japan. However, few reports have demonstrated an association between these agents and cholangiocarcinoma in rodent carcinogenicity studies. Moreover, the combined effects of these compounds have not been fully elucidated. In the present study, we evaluated the in vivo mutagenicity of 1,2-DCP and DCM, alone or combined, in the livers of gpt delta rats. Six-week-old male F344 gpt delta rats were treated with 1,2-DCP, DCM or 1,2-DCP + DCM by oral administration for 4 weeks at the dose (200 mg kg-1 body weight 1,2-DCP and 500 mg kg-1 body weight DCM) used in the carcinogenesis study performed by the National Toxicology Program. In vivo mutagenicity was analyzed by gpt mutation/Spi- assays in the livers of rats. In addition, gene and protein expression of CYP2E1 and GSTT1, the major enzymes responsible for the genotoxic effects of 1,2-DCP and DCM, were analyzed by quantitative polymerase chain reaction and western blotting. Gpt and Spi- mutation frequencies were not increased by 1,2-DCP and/or DCM in any group. Additionally, there were no significant changes in the gene and protein expression of CYP2E1 and GSTT1 in any group. These results indicated that 1,2-DCP, DCM and 1,2-DCP + DCM had no significant impact on mutagenicity in the livers of gpt delta rats under our experimental conditions. Copyright © 2016 John Wiley & Sons, Ltd.

4-Methylthio-3-butenyl isothiocyanate (raphasatin) exerts chemopreventive effects against esophageal carcinogenesis in rats.

To examine the effects of 4-methylthio-3-butenyl isothiocyanate on esophageal carcinogenesis, male 6-week-old F344 rats were subcutaneously injected with 0.5 mg/kg body weight N-nitrosomethylbenzylamine three times per week for 5 weeks and fed a diet supplemented with 80 ppm 4-methylthio-3-butenyl isothiocyanate, equivalent to 6.05 mg/kg body weight/day for the initiation stage, 4.03 mg/kg body weight/day for the promotion stage, or 4.79 mg/kg body weight/day for all stages. Although the incidence of lesions was not affected by 4-methylthio-3-butenyl isothiocyanate treatment, the multiplicity of squamous cell papilloma in the esophagus was significantly decreased in rats in the 4-methylthio-3-butenyl isothiocyanate initiation stage group (1.13 ± 0.74), 4-methylthio-3-butenyl isothiocyanate promotion stage group (1.47 ± 0.99), and 4-methylthio-3-butenyl isothiocyanate all stage group (1.47 ± 1.13) as compared with rats treated with N-nitrosomethylbenzylamine alone (3.00 ± 1.46). Immunohistochemical analysis revealed that 4-methylthio-3-butenyl isothiocyanate induced apoptosis, suppressed cell proliferation, and increased p21 expression when administered in the promotion phase. These modifying effects were not observed in the rats treated with 4-methylthio-3-butenyl isothiocyanate alone. Our results indicated that 4-methylthio-3-butenyl isothiocyanate may exert chemopreventive effects against N-nitrosomethylbenzylamine-induced esophageal carcinogenesis in rats.

Activation of transcription factors in human bronchial epithelial cells exposed to aqueous extracts of mainstream cigarette smoke in vitro.

This study aimed to identify the most sensitive transcription factor activated by cigarette smoke extract (CSE) and to explore cigarette smoke components that have high biological activities in a cell-base assay. Previously, we found evidence that implicated 10 different transcription factors as having a high biological activity to CSE in vitro, based on the results of a comprehensive gene expression profile. For this study, luciferase reporter assays for each transcription factor were developed in two types of human bronchial epithelial cells: NCI-H292 and BEAS-2B cells. The results demonstrated that the nuclear factor erythroid 2-related factor 2 (NRF2)/anti-oxidant response element (ARE) pathway was the most sensitive in response to CSE. Consistently, hemo oxygenase-1 (HO-1), a downstream target gene of NRF2, was effectively up-regulated in BEAS-2B cells exposed to CSE. Moreover, among 1395 cigarette smoke components, naphthoquinones including 9,10-phenaotrenquinone, quinones, benzenediols and α, ß-unsaturated carbonyls, were identified as major smoke components that contribute to activating the NRF2/ARE pathway, as indicated by the ARE-reporter assay in BEAS-2B cells. Taken together, NRF2 appears to be a key molecule in the CSE-induced cellular response, and the employed methodology is helpful for the analysis of molecular and cellular effects by CSE.

Early Detection of Genotoxic Urinary Bladder Carcinogens by Immunohistochemistry for γ-H2AX.

DNA double-strand breaks (DSBs) induced by exposure to genotoxic agents are known to cause genome instability and cancer development. To evaluate the applicability of γ-H2AX, a sensitive marker of DSBs, in the early detection of genotoxicity and carcinogenicity of chemicals using animal models, we examined γ-H2AX expression in urinary bladders of rats. Six-week-old male F344 rats were orally treated for 4 weeks with a total of 12 chemicals divided into 4 categories based on genotoxicity and carcinogenicity in the urinary bladder. Animals were sacrificed at the end of administration or after 2 weeks of recovery, and immunohistochemistry for γ-H2AX was performed. At week 4, γ-H2AX expression in bladder epithelial cells was significantly increased by all 4 genotoxic bladder carcinogens as compared with the controls, whereas the 3 chemicals that were genotoxic but not carcinogenic in the bladders did not cause upregulation of γ-H2AX. After the recovery period, γ-H2AX expression was markedly reduced in all groups but remained significantly elevated in rats treated with 3 of the 4 genotoxic bladder carcinogens. Although slight increases in γ-H2AX expression were induced by a weak bladder carcinogen with equivocal genotoxicity (phenethyl isothiocyanate) and 2 nongenotoxic bladder carcinogens (melamine and uracil) at week 4, these differences were not significant and were thought to be associated with activated proliferation by urothelial hyperplasia, as demonstrated by increased Ki67-positive cells. These results suggested that γ-H2AX may be a potential biomarker for the early detection of genotoxic bladder carcinogens.

Multiple episodic evolution events in V1R receptor genes of East-African cichlids.

Fish use olfaction to detect a variety of nonvolatile chemical signals, and thus, this sense is key to survival and communication. However, the contribution of the olfactory sense to social-especially reproductive-interactions in cichlids is still controversial. To obtain insights into this issue, we investigated the genes encoding V1Rs-possible candidates for reproductive pheromone receptors-among East-African cichlids. Interestingly, we found an excess of nonsynonymous over synonymous substitutions in four of six V1R genes in multiple cichlid lineages. First, we found that highly dimorphic V1R2 allele groups were shared among the cichlids inhabiting all East-African Great Lakes emerged through the episodic accumulation of the nonsynonymous substitutions prior to the radiation of the Lake Tanganyika species flock. We further detected such episodic events in V1R1 of the tribe Tropheini, and in V1R3 and V1R6 of the tribe Trematocarini. The excess of nonsynonymous substitutions in these examples were indicated as dN/dS > 1, which were all statistically significant by Fisher's exact test. Furthermore, we speculate that the amino acid changes in these episodic events are likely functional switch because they occurred in the putative ligand-binding pocket. Our finding of the occurrence of multiple episodic events and the unexpected gene diversity in one unique gene family is suggestive of the contribution of the V1R to the species diversification and the social interaction in cichlids.

Rapid and local autoregulation of cerebrovascular blood flow: a deep-brain imaging study in the mouse.

The brain obtains energy by keeping the cerebral blood flow constant against unexpected changes in systemic blood pressure. Although this homeostatic mechanism is widely known as cerebrovascular autoregulation, it is not understood how widely and how robustly it works in the brain. Using a needle-like objective lens designed for deep-tissue imaging, we quantified the degree of autoregulation in the mouse hippocampus with single-capillary resolution. On average, hippocampal blood flow exhibited autoregulation over a comparatively broad range of arterial blood pressure and did not significantly respond to pressure changes induced by the pharmacological activation of autonomic nervous system receptors, whereas peripheral tissues showed linear blood flow changes. At the level of individual capillaries, however, about 40% of hippocampal capillaries did not undergo rapid autoregulation. This heterogeneity suggests the presence of a local baroreflex system to implement cerebral autoregulation.

Novel multiwavelength microscopic scanner for mouse imaging.

Real-time in vivo imaging of molecular targets at (sub)cellular resolution is essential in better understanding complex biology. Confocal microscopy and multiphoton microscopy have been used in the past to achieve this goal, but their true capabilities have often been limited by bulky optics and difficult experimental set-ups requiring exteriorized organs. We describe here the development and validation of a unique near-infrared laser scanning microscope system that uses novel optics with a millimeter footprint. Optimized for use in the far red and near-infrared ranges, the system allows an imaging depth that extends up to 500 microm from a 1.3-mm-diameter stick objective, which is up to 2 cm in length. We show exceptionally high spatial, temporal, and multiwavelength resolutions of the system and show that it can be applied to virtually any internal organ through a keyhole surgical access. We demonstrate that, when combined with novel far red imaging probes, it is possible to image the cellular details of many organs and disease processes. The new optics, coupled with the use of near-infrared probes, should prove immensely valuable for in vivo cancer imaging.