Cell cycle was disturbed in the MNNG-induced initiation stage during in vitro two-stage transformation of Balb/3T3 cells.

In vitro two-stage transformation, an important method for the screening of carcinogens, is a valuable approach for the mechanistic study of multi-stage carcinogenesis. However, very little is known about the molecular and cellular mechanisms, particularly in terms of cell cycle control during in vitro two-stage transformation. We improved the in vitro two-stage transformation method using N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) as an initiator and cadmium as a promoter, and reconfirmed the promotional effect of cadmium (Fang et al., 2001a). To determine the alterations of cell cycle control in the MNNG-induced initiation stage during transformation, we examined the effects of MNNG on Balb/3T3 A31 cell growth, and determined the alterations of the protein and/or mRNA levels of cyclins B1, D1, E, and G, PCNA, GADD45, p27, and wild-type p53. After 4 hour treatment of MNNG, populations of G2/M phase distribution and apoptotic fraction and the cyclin G mRNA level increased, while the cyclin B1 mRNA level decreased in a concentration-dependent manner. Wild-type p53, p27, and GADD45 protein levels also increased as a function of MNNG concentrations. However, cyclin D1, cyclin E, and PCNA expressions remained unchanged. During the initiation stage, PCNA protein expression decreased on the first day after MNNG-treatment, then increased gradually during the following 6 days, and further increased on the first day after cadmium treatment. Although wild-type p53 and p27 protein expressions also showed temporary retardation on the first day after MNNG-treatment, the expressions increased gradually during the following 6 days, but decreased rapidly by the cadmium treatment. These results indicated that during the initiation stage, MNNG induced G2/M arrest and apoptosis with increased expressions of wild-type p53, p27, and GADD45 proteins; and down-regulated mRNA level of cyclin B1 and up-regulated mRNA level of cyclin G. In addition, although a few of the G2/M-arrested cells proliferated gradually, most cells continued to be suppressed and inactivated by the over-expressions of wild-type p53 and p27 until the cadmium treatment.

The analgesic efficacy of bee venom acupuncture for knee osteoarthritis: a comparative study with needle acupuncture.

The aim of this investigation was to determine whether bee venom (BV) administered directly into an acupoint was a clinically effective and safe method for relieving the pain of patients with knee osteoarthritis (OA) as compared to traditional needle acupuncture. We evaluated the efficacy of BV acupuncture using both pain relief scores and computerized infrared thermography (IRT) following 4 weeks of BV acupuncture treatment. We observed that a significantly higher proportion of subjects receiving BV acupuncture reported substantial pain relief as compared with those receiving traditional needle acupuncture therapy. Furthermore, the IRT score was significantly improved and paralleled the level of pain relief.

Effects of nonylphenol, bisphenol A, and their mixture on the viviparous swordtail fish (Xiphophorus helleri).

A number of fish species have been used for studies on endocrine disrupting chemicals (EDCs). However, despite the widespread use of oviparous fish, relatively little attention has been given to viviparous species. This study investigated the effects of EDCs in a viviparous fish and examined the possible usefulness of the fish as an alternative model for the studies on EDCs. Swordtails (Xiphophorus helleri) were exposed to nonylphenol (NP), bisphenol A (BPA), and their mixture. Both short-term (3-d) and relatively long-term (60-d) exposures were carried out using adult male and 30-d-old juvenile fish, respectively. Following the short-term exposure, both NP and BPA caused vitellogenin mRNA expression. Flow cytometric analysis and terminal deoxynucleotidyl transferase assay on the testes of treated fish indicated reproductive damage. Histopathological analysis found degenerative and necrotic cells in seminiferous tubules following the exposure to 100 ppb NP. The testes with lesions were also associated with highly suppressed spermatogenesis. Following the long-term exposure, both NP and BPA exposures significantly affected the growth of swordtails. In all cases, the results showed that the mixture was always more potent than a single chemical and that swordtail fish can be a useful model for the study of endocrine disruptors.

Bee venom pretreatment has both an antinociceptive and anti-inflammatory effect on carrageenan-induced inflammation.

Although the injection of bee venom (BV) has been reported to evoke tonic pain and hyperalgesia, there is conflicting evidence in the literature indicating that BV can also exert an anti-inflammatory and antinociceptive effects on inflammation. In this regard, BV has been traditionally used in Oriental medicine to relieve pain and to treat chronic inflammatory diseases such as rheumatoid arthritis. The present study was designed to test the hypothesis that BV induces acute nociception under normal conditions, but that it can serve as a potent anti-inflammatory and antinociceptive agent in a localized inflammatory state. The experiments were designed to evaluate the effect of BV pretreatment on carrageenan (CR)-induced acute paw edema and thermal hyperalgesia. In addition, spinal cord Fos expression induced by peripheral inflammation was quantitatively analyzed. In normal animals subcutaneous BV injection into the hindlimb was found to slightly increase Fos expression in the spinal cord without producing detectable nociceptive behaviors or hyperalgesia. In contrast pretreatment with BV (0.8 mg/kg) 30 min prior to CR injection suppressed both the paw edema and thermal hyperalgesia evoked by CR. In addition, there was a positive correlation between the percent change in paw volume and the expression of Fos positive neurons in the spinal cord. These results indicate that BV pretreatment has both antinociceptive and anti-inflammatory effects in CR-induced inflammatory pain. These data also suggest that BV administration may be useful in the treatment of the pain and edema associated with chronic inflammatory diseases.

Cadmium-induced alterations of connexin expression in the promotion stage of in vitro two-stage transformation.

During the multistage carcinogenesis, functions of several key genes involved in the cell cycle control and cell-cell communication can be damaged. Gap junction intercellular communication (GJIC) is known to transfer small, water-soluble molecules through intercellular channels composed of proteins called connexins (Cxs). Therefore, aberrant expression of Cx may be one of the critical factors for the clonal expansion of initiated cells during the two-stage transformation. We already improved the classical in vitro two-stage transformation method using N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) as an initiator and cadmium as a promoter on Balb/3T3 A31 cells, and reconfirmed the promotional effect of cadmium with this method (Fang, M.Z., Cho, M.H., Lee, H.W., 2001. Improvement of in vitro two-stage transformation assay and detection of the promotional effect of cadmium, Toxicol. In Vitro (in press). In this study, precise roles of Cd on Cx expression in normal Balb/3T3 A31 and during the promotion stage of the in vitro two-stage transformation were elucidated. For this purpose, the Cx43, Cx32 and Cx26 protein levels, Cx43 and Cx26 mRNA levels and the cellular distribution location of Cx43 protein were determined. Normal Balb/3T3 cells expressed Cx43 and Cx32, but not Cx26. After a short-term treatment of cadmium on normal cells, phosphorylation of Cx43 protein increased and Cx32 protein level decreased. However, during the promotion stage of the in vitro two-stage transformation, transformed cells treated with cadmium for long periods expressed Cx43 and Cx32 highly, similar to the level of normal Balb/3T3 cells, compared to the nontransformed cells. Moreover, Cx43 of the transformed cells was distributed mostly in the perinuclear region rather than the intercellular membrane. These data suggest that cadmium may inhibit the GJIC by increasing the phosphorylation of Cx43 and decreasing the expression of Cx32 in the normal Balb/3T3 A31 cells. Our results also suggest that these changes are not associated with the cell transformation; transformed cells may reexpress Cx43 and Cx32 similar to the normal cells, though Cx43 protein is distributed aberrantly during the transformation process. Further studies are needed to clarify the relationship between transformation and posttranslational modification of the Cx proteins.

Effect of bee venom and its melittin on apical transporters of renal proximal tubule cells.

Renal failure by bee venom may be related to a malfunction of renal transporters. However, the effects of bee venom on apical membrane transporters of renal proximal tubular cells are not yet known. The aim of this study was to examine the effects of dried bee venom of Apis mellifera and its melittin on apical transporter activity of primary cultured rabbit kidney proximal tubule cells. Bee venom (1 microg/ml) decreased the cell viability and increased lactate dehydrogenase activity over 30-min treatments. Its effect was blocked by mepacrine or AACOCF(3) (10(-6) M; phospholipase A(2) inhibitors). However, there was no effect on cell viability at a concentration of 0.01 microg/ml of bee venom. Thus, we investigated the effect of bee venom (1 microg/ml) on the activity of renal transporters at 30 min. Bee venom inhibited alpha-methyl-D-glucopyranoside, Pi, and Na(+) uptakes, but increased Ca(2+) uptake. These effects of bee venom were blocked by mepacrine or AACOCF(3) (10(-6) M), and bee venom-induced stimulation of Ca(2+) uptake was also blocked by methoxyverapamil and nifedipine (L-type calcium channel blockers). In addition, bee venom increased [(3)H]-arachidonic acid release by 216 % of that of control. In all experiments, bee venom melittin (0.5 microg/ml) had an identical effect to that of bee venom itself. In conclusion, bee venom inhibited, in part, alpha-MG, Pi, and Na(+) uptakes through its melittin which increased Ca(2+) uptake and arachidonic acid release in primary cultured rabbit renal proximal tubule cells.

Enhancement of biliary excretion of aflatoxin B(1) and suppression of hepatic ornithine decarboxylase activity by 2-(allylthio)pyrazine in rats.

2-(Allylthio)pyrazine (2-AP), a synthetic pyrazine derivative with an allylsulfur moiety, has protective effects against chemically-induced hepatic toxicity. Previous studies have shown that 2-AP significantly reduces the formation of preneoplastic foci in rats exposed to aflatoxin B(1) (AFB(1)). The present study was designed to determine whether 2-AP could increase the biliary excretion of metabolites of AFB(1) in rats treated with this carcinogen and whether the agent could alter the activity of ornithine decarboxylase (ODC), which is considered to be associated with tumor promotion. Rats were pretreated with 2-AP (p.o.) at a daily dose of 50 mg/kg for 5 consecutive days. AFB(1) (5 mg/kg) was administered intraperitoneally 2 h after the last dose of 2-AP. Amounts of principal AFB(1) metabolites, AFB(1)-glutathione and a glucuronide conjugate secreted in bile juice was increased by 56 and 50%, respectively, after the 2-AP treatment. Levels of radiolabelled AFB(1) covalently bound to calf thymus DNA catalyzed by microsomes obtained from 2-AP-treated rats (10 and 50 mg/kg, for 5 days) were reduced by 47 to 66%. ODC activity in AFB(1)-treated rats was determined by the three-step medium-term hepatocarcinogenesis assay. Rats were treated with 2-AP at the daily doses of 10, 25 and 50 mg/kg for 16 consecutive days. During this period, four repeated doses of AFB(1) (1.0 mg/kg) were given to the animals. Rats were then subjected to two-third partial hepatectomy, followed by administration of phenobarbital. 2-AP inhibited AFB(1)-induced ODC activity by 40 to 66%, as determined at the 44th day. Inhibition of AFB(1)-induced ODC activity by 2-AP in conjunction with acceleration of AFB(1) elimination through metabolic conjugation may contribute to its chemopreventive effects against this carcinogen.

Thromboxane A2 synthase inhibition and thromboxane A2 receptor blockade by 2-[(4-cyanophenyl)amino]-3-chloro-1,4-naphthalenedione (NQ-Y15) in rat platelets.

The effects of 2-[(4-acetylphenyl)amino]-3-chloro-1,4-naphthalenedione (NQ-Y15), a synthetic 1,4-naphthoquinone derivative, on platelet activity and its mechanism of action were investigated. NQ-Y15 caused a concentration-dependent inhibition of the aggregation induced by thrombin, collagen, arachidonic acid (AA), and A23187. The IC50 values of NQ-Y15 on thrombin (0.1 U/mL)-, collagen (10 microg/mL)-, AA (50 microM)-, and A23187 (2 microM)-induced aggregation were 36.2 +/- 1.5, 6.7 +/- 0.7, 35.4 +/- 1.7, and 93.1 +/- 1.4 microM, respectively. NQ-Y15 also inhibited thrombin-, collagen-, AA-, and A23187-stimulated serotonin secretion in a concentration-dependent manner. However, a high concentration (100 microM) of NQ-Y15 showed no significant inhibitory effect on ADP-induced primary aggregation, which is independent of thromboxane A2 (TXA2) production in rat platelets. In fura-2-loaded platelets, the elevation of intracellular free calcium concentration stimulated by AA, thrombin, and 4-bromo-A23187 was inhibited by NQ-Y15 in a concentration-dependent manner. The formation of TXA2 caused by AA, thrombin, and collagen was inhibited significantly by NQ-Y15. NQ-Y15 inhibited TXA2 synthase in intact rat platelets, since this agent reduced the conversion of prostaglandin (PG) H2 to TXA2. Similarly, NQ-Y15 selectively inhibited the TXA2 synthase activity in human platelet microsomes, whereas it had no effect on activity of phospholipase A2, cyclooxygenase, and PGI2 synthase in vitro. NQ-Y15 inhibited platelet aggregation induced by the endoperoxide analogue U46619 in human platelets, indicating TXA2 receptor antagonism, possibly of a competitive nature. These results suggest that the antiplatelet effect of NQ-Y15 is due to a combination of TXA2 synthase inhibition with TXA2 receptor blockade, and that it may be useful as an antithrombotic agent.

Effect of T-2 toxin on lipid peroxidation in rats: elevation of conjugated diene formation.

A single dose of T-2 toxin (3.6 mg/kg, oral) enhanced conjugated diene formation in rat liver, spleen, kidney, thymus and bone marrow, implying that lipid peroxidation was stimulated. Lipid peroxidation showed apparent specificity in each organ as time elapsed (1-6 h). In liver and kidney maximum stimulation (+74% and +72%, respectively) was noted at 1 h after T-2 treatment. In spleen and thymus, maximum values were observed at 3 h (+95% and +32%, respectively). In bone marrow, a continuous elevation was noted which reached a maximum at 6 h (+112%). Results obtained from serum transaminase assay and histological examination suggested that T-2 toxin exhibited low hepatotoxicity even when the rat received a dose close to the oral LD50.