SUMMARY1. Single smooth muscle cells obtained by enzymic dispersion of the longitudinal muscle layer of guinea-pig ileum were used for recording membrane currents under whole-cell voltage clamp in response to carbachol (100 /bM, unless otherwise stated) or histamine (100 jam) applied extracellularly.2. At a holding potential of 0 mV, a transient outward current was evoked by carbachol and histamine. Responses to the two agonists were very similar in size and time course to the current response to caffeine (10 mM). The response to carbachol was virtually absent in the presence of histamine, and vice versa. Caffeine was without effect in the presence of either of these agonists. Inclusion of EGTA (10 or 20 mM) in the pipette abolished the responses to carbachol, histamine and caffeine. Thus, the outward current responses were considered to represent opening of Ca2+_ activated K+ channels in response to a massive release of Ca2+ from the same stores by these three agents.3. An inward current was evoked by carbachol and histamine, but not by caffeine at a holding potential of -40 mV, which was considered to represent opening of cationic channels. The carbachol-induced inward current was much longer in duration and larger in size than the histamine-induced inward current.4. Inclusion of GDPpJS (2 mm) in the pipette abolished the inward and outward current responses to histamine, but inhibited only part of those to carbachol.5. When the holding potential was held at 0 mV with inclusion of GTPyS (0'1-1 mM) in the pipette, spontaneous transient outward currents appeared immediately after break-through but disappeared a few minutes later. Under these conditions, caffeine (10 mM) was almost without effect, suggesting that GTPyS had released Ca2+ stores. When the holding potential was held at -40 mV and GTPyS (0-1 or 0-2 mm) was present in the pipette, an inward current developed a few minutes after break-through. During the GTPyS-induced inward current, application of carbachol or histamine produced no further inward current. However, when 0-01 mmGTPyS was included in the pipette solution, carbachol-and histamine-induced inward currents were potentiated.6. Pretreated with 2-5 gug/ml pertussis toxin (PTX) did not change noticeably the MS 9646 106 S. KOMORI, M. KA WAI, T. TAKEWAKI AND H. OHASHI outward current responses to carbachol and histamine, but abolished or markedly reduced the inward current responses.7. The results suggest that stimulation of muscarinic receptor or histamine receptor caused release of Ca2+ from storage sites and activation of cationic channels, and that regardless of the receptor type, calcium store release may be mediated via a PTX-insensitive G-protein, while the cation channels are activated via another G-protein which is sensitive to PTX.

In single cells isolated from guinea-pig ileal smooth muscle, held under voltage clamp at -40 mV or -50 mV by patch pipette in the whole-cell recording mode, carbachol (CCh) evoked an oscillatory inward cationic current. The frequency of current oscillations increased with increasing CCh concentration. CCh-evoked current oscillations were followed very closely by oscillations in intracellular free Ca2+ estimated from the Indo-1 signal, and were abolished by inclusion of EGTA in the pipette solution. Ryanodine and heparin, but not nifedipine, blocked the generation of current oscillations. CCh-evoked current oscillations were abolished upon withdrawal of extracellular calcium and restored upon its reintroduction. Inclusion of GTP[gamma S] in the pipette solution caused the generation of an oscillatory inward current, which was blocked by ryanodine. The present results are consistent with the hypothesis that CCh-evoked cationic current is gated by activation of a G protein and is steeply dependent on [Ca2+]i, fluctuations in the release of Ca2+ from stores during carbachol's action produce oscillations in [Ca2+]i which cause similar oscillations in the cationic current.

No abstract

To clarify whether enzymatically modified isoquercitrin (EMIQ) or melatonin (MLT) supplementation reduces oxidative stress-mediated hepatocellular tumor-promoting effect of oxfendazole (OX), a benzimidazole anthelmintic, male rats were administered a single intraperitoneal injection of N-diethylnitrosamine (DEN) and were fed a diet containing OX (500 ppm) for 10 weeks with or without EMIQ (2,000 ppm) or MLT (100 ppm) in the drinking water after DEN initiation. One week after the commencement of the administration of OX, rats were subjected to two-thirds of partial hepatectomy. The number of GST-P-positive foci promoted by OX was significantly inhibited by the combined antioxidant EMIQ or MLT administration, and the area of GST-P-positive foci was inhibited by the administration of MLT. Real-time RT-PCR analysis revealed decreases in mRNA expression levels of cytochrome P450, family 2, subfamily b, polypeptide 2 (Cyp2b2) and malic enzyme 1 (Me1) in the DEN-OX-EMIQ and DEN-OX-MLT groups and decreases in mRNA expression levels of Cyp1a1 and aldo-keto reductase family 7, member A3 (Akr7a3) in the DEN-OX-MLT group compared to those in the DEN-OX group. In in vitro ROS production assay, inhibited production of NADPH-dependent ROS was observed by the treatment with EMIQ or MLT. These results suggest that coadministration of EMIQ or MLT suppresses the hepatocellular tumor-promoting activity of OX in rats through the decrease in ROS production by the activation of CYPs.

The tumor-promoting effects of oxfendazole (OX), a benzimidazole anthelmintic, were investigated using a medium-term rat hepatocarcinogenesis model. Six-week-old male F344 rats received an intraperitoneal injection of N-diethylnitrosamine (DEN) and were given a powdered diet containing 0 or 500 ppm OX for 6 weeks from 2 weeks after DEN treatment. All animals were subjected to two-thirds partial hepatectomy 1 week after OX treatment. The numbers and areas of glutathione S-transferase placental form (GST-P)-positive foci were significantly increased in the livers of rats treated with OX, with concomitantly increased cell proliferation, compared with those in the livers of the DEN alone group. Quantitative real-time RT-PCR analysis revealed that OX induced not only mRNA expression of phase I enzymes Cyp1a1, Cyp1a2, but also Nrf2-regulated phase II enzymes such as Gpx2, Nqo1, Yc2, Akr7a3 and Gstm1, presumably due to an adaptive response against OX-induced oxidative stress. Reactive oxygen species production increased in microsomes isolated from the livers of OX-treated rats. Furthermore, OX enhanced oxidative DNA damage (as assessed by 8-hydroxydeoxyguanosine; 8-OHdG) and lipid peroxidation (as assessed by thiobarbituric acid-reactive substances; TBARS). These results suggest that administration of OX at a high dose and for a long term enhances oxidative stress responses, which may contribute to its tumor-promoting potential in rats.

No abstract

Piperonyl butoxide (PBO) is a pesticide synergist used with pyrethroids as a domestic insecticide, and it acts as a non-genotoxic hepatocarcinogen in rats and mice. To clarify whether oxidative stress is involved in the liver tumor-promoting effect of PBO in mice, male mice were subjected to two-thirds partial hepatectomy, followed by N-diethylnitrosamine (DEN) treatment, and given a diet containing 0.6% PBO for 25 weeks. The incidences of cytokeratin (CK) 8/18-positive foci, adenomas, and carcinomas significantly increased in the DEN + PBO group compared with the DEN-alone group. The PCNA-positive ratio significantly increased in non-tumor hepatocytes, CK8/18-positive foci and adenomas in the DEN + PBO group compared with the DEN-alone group. PBO increased reactive oxygen species (ROS) production in microsomes but did not change oxidative DNA damage as assessed by 8-hydroxydeoxyguanosine (8-OHdG). In real-time RT-PCR, PBO upregulated the expression of genes related to metabolism, such as Cytochrome P450 1a1, 2a5, and 2b10, and metabolic stress, such as Por and Nqo1, but downregulated Egfr and Ogg1. PBO also increased early response genes downstream of mitogen-activated protein kinase (MAPK), such as c-Myc that is induced by excessive ROS production, and G1/S transition-related genes, such as E2f1 and Ccnd1. Thus, PBO can generate ROS via the metabolic pathway without any induction of oxidative DNA damage, activate cell growth, increase c-Myc- and E2F1-related pathways, and act as a liver tumor promoter of DEN-induced hepatocarcinogenesis in mice.

ABSTRACT. In order to clarify whether cytokeratin (CK) 8/18 is a useful immunohistochemical marker for hepatocellular proliferative lesions in mice, partially hepatectomized male ICR mice were given 0.6% piperonyl butoxide (PBO) for 8 (Experiment I) or 25 weeks (Experiment II) after N-diethylnitrosamine (DEN) initiation treatment, and the livers were subjected to histological examinations on hematoxylin and eosin (HE) stained sections, CK8/18 immunohistochemistry and gamma-glutamyl transpeptidase (GGT) histochemistry. In Experiment I, the multiplicity of hepatocellular foci in paraffin-embedded sections which were observed in HE-stained sections and positive for CK8/18 was 10.17 and 18.50, respectively, while that of hepatocellular foci in frozen sections which were observed in HEstained sections and positive/negative for GGT was 6.17 and 8.17, respectively. In Experiment II, the total multiplicity of hepatocellular foci in paraffin-embedded sections which were observed in HE-stained sections and positive/negative for CK8/18 was 4.47 and 23.17, respectively, while that of hepatocellular foci in frozen sections which were observed in HE-stained sections and positive/negative for GGT was 2.50 and 3.50, respectively. Most of the hepatocellular adenomas and carcinomas observed in HE-stained sections were positive for CK8/18, but some of the adenomas were negative for CK8/18. These findings indicate that more hepatocellular proliferative lesions can be detected in CK8/18 immunohistochemistry in addition to those observed in HE-stained sections, and suggest that CK8/ 18 may become a useful immunohistochemical marker for detecting hepatocellular proliferative lesions in mice. The rat liver medium-term bioassay system first established by Ito et al. [14] has been repeatedly used for the detection of carcinogenic or tumor promoting potential of chemical substances and has a great advantage due to reproducibility and reliability for generation of data within 8 weeks [8]. For assessment of promoting effects of the test chemicals, glutathione S-transferase placental form (GST-P)-positive foci are used as the primary endpoint. Moreover, since production of GST-P positive foci has been closely correlated with the actual tumor yields [11,21], they are regarded as the reliable preneoplastic lesion in rats. However, it is generally recognized that this immunohistochemical marker is not reactive for liver preneoplastic and neoplastic lesions of mice. It has been shown that gammaglutamyl transpeptidase (GGT) play a role in multistage hepatocarcinogenesis and the enhanced expression of this enzyme is observed in preneoplastic altered hepatocellular foci, hepatocellular adenomas, and hepatocellular carcinomas in rats and mice [13]. Therefore, GGT is used as a histochemical marker for these proliferative lesions in mice. However, this histochemical staining of GGT is not suitable for routine histopathological examinations, because frozen sections are necessary for this staining. In addition, there is a disadvantage that almost all th...