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Rhus vernicifluaStokes (RVS) exert cardiovascular protective activity by promoting blood circulation, but its active ingredients and underlying mechanism have yet to be identified. This study investigated the vascular effects of RVS, focusing on vasoconstriction and smooth muscle Ca2+signaling. RVS heartwood extract attenuated contraction of aortic rings induced by the vasoconstrictors serotonin and phenylephrine, and inhibited the Ca2+signaling evoked by serotonin in vascular smooth muscle cells. Subsequent activity-guided fractionation identified fisetin as an active constituent exerting a Ca2+inhibitory effect. Fisetin could inhibit major Ca2+mobilization pathways including extracellular Ca2+influx mediated by the L-type voltage-gated Ca2+channel, Ca2+release from the intracellular store and store-operated Ca2+entry. In accordance with Ca2+inhibitory effect, fisetin attenuated vasoconstriction by serotonin and phenylephrine. These results suggest that the anticontractile effect, which is presumably mediated by inhibition of Ca2+signaling, may contribute to the improvement of blood circulation by RVS.

Metallothionein (MT) is a cysteine-rich protein that binds to and is inducible by heavy metals such as cadmium and zinc. However, the precise mechanism of MT induction by other metals remains unclear. In the present study, we investigated the mechanism of MT induction by manganese, focusing on the involvement of cytokine production. Administration of MnCl(2) to mice resulted in the induction of MT dose-dependently in the liver with little accumulation of manganese. Speciation analysis of metals in the liver cytosol showed that the major metal bound to the induced MT was zinc. Administration of MnCl(2) caused an increase in mRNA levels of interleukin-6 (IL-6) in the liver as well as an increase in serum levels of IL-6 but not those of other inflammatory cytokines. Subsequently, serum levels of serum amyloid A (SAA), an acute-phase protein induced by IL-6, increased with a peak at 24 h. However, no increase in serum alanine aminotransferase activity was observed, suggesting that manganese enhanced the production of IL-6 and SAA without causing liver injury. In response to IL-6, the expression of a zinc transporter, ZIP14, was enhanced in the liver, possibly contributing to the synthesis of hepatic zinc-MT. In IL-6-null mice, the induction of hepatic MT by treatment with MnCl(2) was completely suppressed to the control level. These results suggest that manganese is a unique metal that induces the synthesis of hepatic MT completely depending on the production of IL-6 without accompanying liver injury.

Plasmid pSV2MT‐I encoding mouse metallothionein‐I (MT‐I) designed to be expressed under the control of an SV40 promoter was introduced into human HeLa S3 cells. Several transformants (HeLa/MTH) carrying multi‐copies of mouse MT‐I cDNA in their genomes were isolated. These transformants produced 4 to 20‐fold larger amounts of MT than their parent cells. The MT levels in HeLa/MTH were well correlated with the extent of resistance to cadmium, but not with that to cis‐platinum (cis‐DDP) in vitro.To study the role of MT in resistance to cis‐DDP in vivo, nude mice were inoculated subcutaneously with two independent HeLa/MTH clones. MT levels in these tumors were about 3‐fold higher than those in the parental cells. The growth of tumors derived from either HeLa/MTH clone was not inhibited in the presence of 15 μmol/kg of cis‐DDP, which completely inhibited the growth of tumors derived from the parental HeLa cells. These data strongly suggest that the elevated level of MT confers resistance to cis‐DDP in vivobut not in vitro.Thus, the results of this study indicate that in vitrodeterminations of the influence of MT on cis‐DDP resistance may underestimate its importance in in vivosituations.

The effect of preinduction of metallonthionein (MT) by bismuth subnitrate (BSN) on the adverse effects and antitumor activity of γ-ray irradiation was investigated in mice. Preinduction of MT by oral administration of BSN significantly reduced the lethal effects and bone marrow injury caused by total body irradiation with γ-rays. A significant increase in the MT concentration in bone marrow was observed in mice treated with BSN. In tumor-bearing mice, pretreatment with BSN did not compromise the antitumor activity of γ-ray irradiation although bone marrow injury was remarkably suppressed. These results suggest that BSN pretreatment is an effective method for protection against side-effects in radiotherapy.

The effect of selenite coadministration on the toxicity and antitumor activity of repeated treatment with high doses ofcis-diamminedichloroplatinum (cis-DDP) was examined in mice. Sodium selenite was injected s.c. into separate abdominal sites of mice together withcis-DDP at a molar ratio of 1:3.5 (selenite tocis-DDP) on day 0. The same amount of selenite was given daily for 4 subsequent days (days 1–4). This fixed administration schedule was repeated weekly for a total of 7 weeks. Under the experimental conditions used, the lethal toxicity, renal toxicity [indicated by an increase in blood urea nitrogen (BUN) and plasma creatinine levels], hepatic toxicity (indicated by an increase in plasma GPT and GOT activity), and myelotoxicity (indicated by a decrease in the numbers of leukocytes and platelets) observed in mice given repeated doses ofcis-DDP alone (15 or 25 µmol/kg, s.c.) were significantly depressed by the coadministration of sodium selenite. Treatment withcis-DDP alone (15,20, or 25µmol/kg, s.c.) resulted in some dose-dependent prolongation of the life span of mice transplanted either s.c. with colon adenocarcinoma 38 (colon 38) or i.p. with P388 leukemia (P388) but did not completely depress the tumor growth, and the animals died of either progressive disease orcis-DDP-induced toxicity. However, following the coadministration of 7.1 µmol/kg selenite with 25 µmol/kgcis-DDP, all of the mice transplanted either s.c. with colon 38 or i.p. with P388 survived for as long as 4 months after the end of the treatment and showed no evidence of malignancy. These results indicate that selenite coadministration enables the use of increasing doses ofcis-DDP and, consequently, enhances the antitumor effect ofcis-DDP by depressing its side effects.

The effects of bismuth nitrate pretreatment on the toxicity and antitumor activity ofcis-diamminedichloroplatinum (cisplatin, CDDP) were examined in nude mice that had been inoculated with human bladder-tumor tissue. Pretreatment with bismuth nitrate depressed the renal toxicity of CDDP without compromising its activity against a transplantable human bladder tumor. Renal metallothionein (MT) and bismuth (Bi) levels in nude mice were markedly increased by Bi preadministration, but no significant MT induction was observed in inoculated human bladder-tumor tissue in which only a trace amount of Bi was incorporated. Furthermore, it was confirmed that tumor platinum (Pt) concentrations in CDDP-treated mice were not affected by Bi pretreatment. Thus, the administration of Bi compounds prior to chemotherapy with CDDP may provide an effective mode of treatment for advanced bladder tumors.

Abstract: To elucidate the protective role of metallothionein (MT) in the prevention of cisplatinum (cis-DDP) toxicity, we investigated the lethal and renal toxicities caused by cis-DDP in MT-null transgenic mice in comparison with wild-type control mice, and examined the effect of pretreatment with bismuth nitrate or zinc sulfate on the cis-DDP nephrotoxicity. The MT-null mice were of mixed 129 Ola and C57BL/6 genetic background. Since no differences in cis-DDP nephrotoxicity were observed between these strains, C57BL/6J mice were used as the wild-type control. The basal MT levels in the kidneys were negligible in the MT-null mice and 2.93 ± 0.77 μg/g tissue in the C57BL/6J mice. In terms of both the lethal and renal toxicities of cis-DDP, MT-null mice were far more sensitive than C57BL/6J mice. Preinduction of renal MT synthesis by administration of bismuth nitrate or zinc sulfate protected C57BL/6J mice from cis-DDP nephrotoxicity. In the case of MT-null mice, however, renal MT could not be induced by pretreatment with these metal compounds, and renal toxicity of cis-DDP was not prevented by this pretreatment. These results suggest that MT is an important factor with the potential to suppress the development of cis-DDP toxicity.