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In this study, we investigated the neurobehavioral alterations and modifications of gene expression in the brains of female mice exposed to low-level mercury vapor and/or methylmercury during postnatal development. The mice were exposed to low-level mercury vapor at a mean concentration of 0.094 mg/m3and supplied with tap water containing 5 ppm methylmercury from postnatal day 11 to 12 weeks of age. Behavioral analyses were performed at 17 weeks of age. Total locomotor activity in the open field test and the retention trial performance in the passive avoidance test were significantly reduced in the combined exposure group compared with those in the control group. The differences in locomotor activity and performance in the retention trial at 17 weeks were no longer detected at 45 weeks. These results suggest that the effect of aging on the behavioral abnormalities resulting from postnatal exposure to mercury complexes are not significant. In the microarray analysis of brains in the combined exposure group, the gene expression levels of Ano2and Sgk1were decreased. Real-time RT-PCR analysis confirmed these changes caused by combined mercury exposure, showing significant downregulation of Ano2and Sgk1in the cerebrum. These genes play key roles in the brain as a calcium-activated chloride channel and as a kinase that responds to cellular stress, respectively. Our findings provide insight into the neurobehavioral changes caused by combined mercury exposure.

The generation and collection of cigarette smoke (CS) is a prerequisite for any toxicology study on smoking, especially an in vitroCS exposure study. In this study, the effects on blood and vascular function were tested with two widely used CS preparations to compare the biological effects of CS with respect to the CS preparation used. CS was prepared in the form of total particulate matter (TPM), which is CS trapped in a Cambridge filter pad, and cigarette smoke extract (CSE), which is CS trapped in phosphate-buffered saline. TPM potentiated platelet reactivity to thrombin and thus increased aggregation at a concentration of 25~100 μg/mL, whereas 2.5~10% CSE decreased platelet aggregation by thrombin. Both TPM and CSE inhibited vascular contraction by phenylephrine at 50~100 μg/mL and 10%, respectively. TPM inhibited acetylcholine-induced vasorelaxation at 10~100 μg/mL, but CSE exhibited a minimal effect on relaxation at the concentration that affects vasoconstriction. Neither TPM nor CSE induced hemolysis of erythrocytes or influenced plasma coagulation, as assessed by prothrombin time (PT) and activated partial thromboplastin time (aPTT). Taken together, CS affects platelet activity and deteriorates vasomotor functions in vitro.However, the effect on blood and blood vessels may vary depending on the CS preparation. Therefore, the results of experiments conducted with CS preparations should be interpreted with caution.

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.

Chronic exposure to cadmium (Cd) is known to adversely affect renal function. Our previous studies indicated that Cd induces p53-dependent apoptosis by inhibiting gene expression of the ubiquitin-conjugating enzyme (Ube) 2d family in both human and rat proximal tubular cells. In this study, the effects of Cd on protein expression of p53 and apoptotic signals in the kidney and liver of mice exposed to Cd for 12 months were examined, as well as the effects of Cd on p53 protein levels and gene expression of the Ube2d family in various cell lines. Results showed that in the kidney of mice exposed to 300 ppm Cd for 12 months, there was overaccumulation of p53 proteins in addition to the induction of apoptosis, which was triggered specifically in the proximal tubules. Interestingly, the site of apoptosis was the same as that of p53 accumulation in the proximal tubules. In the liver of mice chronically exposed to Cd, gene expression of the Ube2d family tended to be slightly decreased, together with slight apoptosis without the accumulation of p53 protein. In rat small intestine epithelial (IEC-6) cells, Cd decreased not only the p53 protein level but also gene expression of Ube2d1, Ube2d2and Ube2d4. In human brain microvascular endothelial cells (HBMECs), Cd did not suppress gene expression of the Ube2d family, but increased the p53 protein level. In human brain astrocytes (HBASTs), Cd only increased gene expression of UBE2D3. These results suggest that Cd-induced apoptosis through p53 protein is associated with renal toxicity but not hepatic toxicity, and the modification of p53 protein by Cd may vary depending on cell type.

The integration of knowledge concerning the regulation of metallothionein (MT) with research on its proposed functions is necessary to clarify how MT affects cellular processes. MT expression is induced/enhanced in various tissues by a number of physiological mediators through several response elements in the MT gene promoter. The cellular accumulation of MT depends on the availability of cellular zinc derived from the diet. MT modulates: 1) the binding and exchange/ transport of heavy metals such as zinc, cadmium, or copper under physiological conditions and cytoprotection from their toxicities, and 2) the release of gaseous mediators such as hydroxyl radicals or nitric oxide. In addition, MT reportedly affects a number of cellular processes, such as gene expression, apoptosis, proliferation, and differentiation. Given the genetic approach, the apparently healthy status of MT-deficient mice argues against an essential biological role for MT; however, the molecule may be critical in cells/tissues/organs in times of stress, since MT expression is also evoked/enhanced by various stresses. In particular, because metallothionein (MT) is induced by inflammatory stress, its roles in inflammation are implied. Also, MT expression in the lung can be enhanced by inflammatory stimuli, suggesting that its expression correlates with inflammatory lung diseases. In this paper, we review the role of MT of various inflammatory conditions in the lung.

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.

To clarify the physiological role(s) of metallothionein (MT) in carcinogenesis, we studied the susceptibility of MT-null mice to chemically mediated carcinogenesis in the 7,12-dimethylbenz[a]anthracene (DMBA)/12-O-tetradecanoylphorbol-13-acetate (TPA)-induced two-stage carcinogenesis model. The MT-null mice were subjected to a single topical application of DMBA (50 or 100 micro g/mouse) and, 1 week later, to promotion with TPA (10 micro g/mouse) twice a week for 20 weeks. At week 21, nearly all of the MT-null mice developed tumors in the skin, in contrast to only 10-40% of wild-type mice. No tumors were observed in MT-null or wild-type mice that were administered TPA alone. By using the PCR-restriction fragment length polymorphism and PCR-single strand conformation polymorphism methods, we found a transversion of A182 to T in codon 61 of c-Ha-ras in the papilloma tissue of MT-null mice and wild-type mice but failed to find any mutations in the c-Ki-ras and c-N-ras genes. In two-stage skin carcinogenesis induction by DMBA/TPA, p53 and p21(WAF1/Cip1) expression levels were found to be increased in MT-null mice compared with wild-type mice. As to an earlier change at the promotion stage triggered by TPA application, MT-null mice were found to have both hyperplasia of the epithelium and a marked degree of inflammation in the basal layer, indicating that the induced as well as endogenous MT acted as a protective factor against tumorigenesis. In conclusion, the present study has demonstrated that MT has antitumorigenic potential in both the initiation and promotion stages of the two-stage chemical skin carcinogenesis model.

Poly(ADP-ribose) polymerase is a 113-kDa nuclear enzyme that binds to both damaged DNA and to RNA associated with actively transcribed regions of chromatin. Binding of poly(ADP-ribose) polymerase to DNA lesions activates it, catalyzing the covalent addition of multiple ADP-ribose polymers to the enzyme (automodification). During apoptosis, poly(ADP-ribose) polymerase is cleaved by caspase-3, resulting in the formation of an N-terminal 24-kDa fragment, containing the DNA binding domain, and a C-terminal 89-kDa catalytic fragment. The functional relevance of this cleavage is not well understood. We therefore prepared a recombinant 24-kDa poly(ADP-ribose) polymerase fragment and investigated the role of this fragment in DNA repair and transcription. The 24-kDa fragment retained its binding affinity for both DNA breaks and RNA. In an in vitrocell-free DNA repair assay, this fragment inhibited rejoining of DNA breaks and suppressed ADP-ribose polymer formation by competing with poly(ADP-ribose) polymerase in binding to DNA breaks. With regard to transcription, it has recently been demonstrated that binding of poly(ADP-ribose) polymerase to transcribed RNA reduces the rate of transcript elongation and that automodification of poly(ADP-ribose) polymerase bound to DNA breaks results in up-regulation of transcription. We tested the 24-kDa fragment for its ability to suppress transcript elongation, and we found that it competed against the up-regulation of transcription mediated by full-length poly(ADP-ribose) polymerase. The ability of the 24-kDa fragment to inhibit DNA repair, ADP-ribose polymer formation, and damage-dependent up-regulation of transcription may contribute to the apoptotic shift from cell survival to cell death mode.

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.

To investigate the relationship between cadmium (Cd) toxicity, intestinal absorption, and its distribution to various tissues in rats treated orally with minimum amounts of Cd, 14 female rats per dose group per time point were given diets consisting of 28% purified diet and 72% ordinary rice containing Cd-polluted rice (0.02, 0.04, 0.12, or 1.01 ppm of Cd) or CdCl2(5.08, 19.8, or 40.0 ppm of Cd) for up to 8 months. At 1, 4, and 8 months after the commencement of Cd treatment, seven rats per group were euthanized for pathological examinations to determine the Cd concentrations in the liver and kidneys and metallothionein (MT) in the liver, kidneys, intestinal mucosa, serum, and urine. One week before each period of 1, 4, and 8 months, the remaining seven rats in each group were administered a single dosage of 109Cd, a tracer, to match the amounts of the designated Cd doses (about 1.2 to 2400 μg/kg body wt). They were euthanized 5 days later to determine the distribution of Cd to various tissues. No Cd-related toxic changes were observed. The concentrations of Cd in the liver and kidneys at any time point and MT in the liver, kidney, serum, and urine at 4 and 8 months increased dose-dependently, whereas MT in the intestinal mucosa did not alter markedly at any time point. The distribution rates of Cd to the liver increased dose-dependently (40% at lower doses to 60% at higher doses), whereas those to the kidney decreased dose-dependently (20% at lower doses to 10% at higher doses). The Cd retention rates 5 days after 109Cd administration (amounts of Cd in various tissues/amounts of Cd administered) ranged from 0.2 to 1.0% at any time point. These results suggest that the distribution of Cd to the liver and kidneys after the oral administration vary depending on the dosage levels of Cd. The difference of the distribution pattern of Cd to the liver and kidney is probably due to the difference in the form of the absorbed Cd, i.e., free ion or Cd–MT complex, although not closely related to the MT in the intestinal mucosa.

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.

Abstract: The effect of pretreatment with metallothionein (MT)-inducing metals (Zn, Cu, Bi, Co, Cd or Hg) on paraquat (PQ) toxicity was investigated in mice. PQ lethality was remarkably reduced by pretreatment with the above MT-inducing metals. The protective effect of pretreatment with these metals on PQ lethality was significantly correlated with MT levels in the lung, a target tissue of PQ toxicity, in mice administered MT-inducing metals, but not with MT in the liver or kidney. The increase in pulmonary lipid peroxidation in mice treated with PQ was significantly inhibited by Zn pretreatment. Zn was the most effective of the MT-inducing metals used in this experiment in protecting mice against PQ lethality. Of those monitored, the only pulmonary free radical scavenging factor increased by Zn pretreatment was MT. Other free radical scavenging factors (activities of Superoxide dismutase, glutathione peroxidase and catalase, and concentration of non-protein thiols level) were not influenced by Zn treatment. These results indicate that the induction of pulmonary MT protects against the lethality and lung toxicity of PQ. Pulmonary MT may scavenge free radicals produced by PQ, thereby protecting against lethal pulmonary toxicity.

We examined the role of metallothionein in the chemosensitivity of transplanted tumors in mice. The antitumor activities of cisplatin, adriamycin, bleomycin, peplomycin, cyclophosphamide, and melphalan were significantly suppressed when the concentration of metallothionein in the tumor was increased to only twice the control level. On the other hand, the antitumor activities of mitomycin C, 5-fluorouracil, and vinblastine were hardly affected by increases in the concentration of metallothionein in the tumors in mice. Moreover, all the antitumor drugs examined increased the concentration of metallothionein in transplanted tumors to a level that was high enough to suppress the antitumor activity of these drugs. These observations suggest that treatment of patients with certain antitumor drugs might result in the resistance of their tumors to multiple drugs.

The Michael addition reaction of a series of alkyl vinyl sulfoxides (CH2=CH-SO-R; R = CH3 (MVSO), C2H5 (EVSO), t-C4H9 (BVSO)) with poly(vinyl alcohol) (PVA) was carried out after investigating the model reaction with isopropyl alcohol. The addition reaction was found to proceed in the presence of sodium hydroxide as catalyst. This sulfinylation of PVA proceeded more easily with MVSO and EVSO than with BVSO, and a polymer containing up to 60 mol% sulfoxide unit was obtained with MVSO and EVSO. The solution viscosity as well as the solubility behavior of the modified PVA' having various types and contents of sulfoxide group were also examined.

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.

The protective effects of metallothionein (MT) preinduction by bismuth subnitrate (BSN) against the renal toxicity of cis-diamminedichloroplatinum (cis-DDP), the cardiotoxicity of adriamycin (ADR), and the lethal and bone marrow toxicities of these drugs were observed in mice receiving cis-DDP and ADR simultaneously. Preinduction of MT biosynthesis by BSN, which is currently used as an antidiarrheal drug, did not affect the antitumor activities of the two drugs, suggesting that the beneficial effects of the preinduction of MT biosynthesis by BSN may be applicable for therapy involving cis-DDP and ADR, either alone or in combination.

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In mammalian cells, mRNA transcription is initiated with the aid of transcription initiation factors. Of these, TFIIH has also been shown to play an essential role in nucleotide excision repair (NER), which is a versatile biochemical pathway that corrects a broad range of DNA damage. Since the dual role of TFIIH is conserved among eukaryotes, including yeast and mammalian cells, the sharing of TFIIH between NER and RNA transcription initiation might provide some survival advantage. However, the functional relationship between NER and RNA transcription initiation through TFIIH is not yet understood. We have developed an optimized cell-free assay which allows us to analyze NER and RNA transcription under identical conditions. In this assay, NER did not compete with RNA transcription, probably because the extracts contained sufficient amounts of TFIIH to support both processes. Thus, NER can be considered functionally independent of RNA transcription initiation despite the fact that both processes use the same factor.