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Orally administered Cd is predominantly distributed to the intestine, and the majority of this mucosal Cd is bound to metallothionein (MT). MT attenuates heavy metal-induced cytotoxicity by sequestering these metals and lowering their intracellular concentrations. In addition, MT acts as an extracellular transporter of orally administered Cd to the kidney. Because of its low molecular weight, the Cd-MT complex is freely filtered at the glomerulus, and the filtered Cd-MT is then incorporated into renal proximal tubular cells. Megalin, a multiligand endocytic receptor (also known as low-density lipoprotein receptor-related protein 2 or Lrp2), acts as the receptor for Cd-MT in a renal proximal tubular cell model. Here, we used the soluble form of 39-kDa receptor-associated protein (sRAP; also known as Lrpap1), a ligand of megalin, to inhibit megalin function, and then analyzed the effect of megalin loss on Cd-MT distribution and Cd-MT-induced nephrotoxicity in an animal model. Administration of sRAP to mice caused acute loss of megalin function by removing megalin in the brush border membrane. The pre-injection of sRAP decreased renal Cd content and decreased Cd-MT-induced kidney damage. Our results demonstrate that sRAP reduces Cd-MT-induced kidney toxicity in vivo.

The Gln-Val-Val-Ala-Gly sequence, which occurs frequently in several natural thiol proteinase inhibitors, and derivatives were synthesized by conventional solution methods and their effect on thiol proteinases were examined. The studies led us to the conclusion that certain of these peptides exhibited a weak inhibitory effect on the thiol proteinase, papain. One of them, Z-Gln-Val-Val-Ala-Gly-OMe, showed a protective effect on papain from natural thiol proteinase inhibitor-induced inactivation. The relationship between structure and activity of these derivatives was studied and certain conclusions were derived on possible mode of action of these inhibitors. From these studies, it was concluded that Z-Gln-Val-Val-OMe was the smallest peptide to exhibit some effect on papain.

Mouse MT-I (metallothionein-I) transcription is regulated by MTF-1 (metal-response-element-binding transcription factor-1) which is recruited to the promoter in response to zinc. Cr(VI) [chromium(VI)] pretreatment blocks zinc-activation of the endogenous MT-I gene and attenuates zinc-activation of MT-I-promoter-driven luciferase reporter genes in transient transfection assays. Chromatin immunoprecipitation assays revealed that Cr(VI) only modestly reduces recruitment of MTF-1 to the MT-I promoter in response to zinc, but drastically reduces the recruitment of RNA polymerase II. These results suggest that Cr(VI) inhibits the ability of MTF-1 to transactivate this gene in response to zinc. Zinc has recently been shown to induce the formation of a co-activator complex containing MTF-1 and the histone acetyltransferase p300 which plays an essential role in the activation of MT-I transcription. In the present study, co-immunoprecipitation assays demonstrated that Cr(VI) pretreatment blocks the zinc-induced formation of this co-activator complex. Thus Cr(VI) inhibits mouse MT-I gene expression in response to zinc by interfering with the ability of MTF-1 to form a co-activator complex containing p300 and recruiting RNA polymerase II to the promoter.

The genomic DNA of crucian carp (Carassius cuvieri) metallothionein-II (ccMT-II), with its upstream region, was obtained. The sequence analysis of its upstream region revealed several putative cis-acting elements including seven metal regulatory elements (MREs), three activator protein 1 (AP1), two glucocorticoid response elements (GREs), etc. The seven MREs locate into two clusters, a distal cluster with four MREs within -800/-600bp from the translation start site and a proximal cluster with three MREs close to TATA box. In transient luciferase gene expression assays, both of the distal and proximal cluster MREs have significantly shown synergistic effects in the transcription of ccMT-II gene; the proximal cluster of MREs serves as the major elements in metal inducing activity; Zn(2+) and Cd(2+) served as much stronger inducers than Cu(2+) shown in ccMT-II expression. The two GRE homologous sequences in ccMT-II promoter showed not to be inductive in either HepG2 or HEK293.

Structures of 1,8-(ArZ)2C14H6O2 and 9-(MeO)-1,8-(ArZ)2C14H7 (Z = S, Se) are determined by X-ray crystallographic analysis. Five C─Z- - -O- - -Z─C atoms of the compounds align linearly, which are analyzed by the extended hypervalent 5c–6e model, based on QC calculations. CT of the 5c–6e occurs as the σ*(C─Z) ← n p (O)→ σ*(Z─C) direction.

Zinc is an essential and multifunctional element for all cells. Metallothionein (MT) is a low molecular weight, cystein-rich, metal-binding protein that is involved in zinc homeostasis. During the acute-phase reaction, hepatic MT production is induced and hepatic zinc accumulation is stimulated. We previously reported that MT-I and II-deficient (MT-null) mice are highly sensitive to the lethal effects of lipopolysaccharide (LPS) plus D-galactosamine (GalN). The sensitization may relate to attenuatoin of α1-acid glycoprotein (AGP) expression in MT-null mice. In the present study, we hypothesized that MT-induced hepatic zinc accumulation promotes AGP expression and prevents LPS/GalN-induced lethality. To determine whether zinc reduces LPS/GalN toxicity, zinc was administered to mice. Simultaneous administration of zinc and LPS/GalN showed no effect on the lethality of LPS/GalN in mice. Zinc administration at 3 hr prior to LPS/GalN challenge reduced LPS/GalN-induced death. However, zinc pre-administration at 24 hr before LPS/GalN challenge did not reduce LPS/GalN-induced death. The expression of AGP mRNA was elevated at 3 hr after zinc administration, 24-hr pretreatment was ineffective. The protective effect of zinc was observed in both wild-type and MT-null mice. These results show that the protective effects of zinc were not caused by MT induction, but by AGP expression. We suggest that MT-induced hepatic zinc accumulation may promote AGP expression and thus prevent LPS/GalN-induced lethality.

Human gene icb-1 was initially described as a gene with increased expression in endometrial tumor cells differentiated in vitro by culture on a reconstituted basement membrane. We provide evidence for a more general involvement of icb-1 gene function in cellular differentiation processes. We report the up-regulation of icb-1 transcript levels in HL-60 promyelocytic leukemia cells during their in vitro differentiation induced by all- trans retinoic acid, vitamin D 3 or DMSO. Increased icb-1 mRNA levels could be observed both in monocytic and granulocytic differentiation. We also report the identification of the novel icb-1 splice variants icb-1β and γ , and the spleen-specific isoform icb-1δ . Expression of icb-1 can be used as a novel marker for in vitro differentiation processes of HL-60 cells.

Human choriocarcinoma cell lines have been used as placental models for the study of endocrine function, including aromatase (CYP19) activity and the secretion of human CG (hCG). In the present study, we investigated the effects of trialkyltin compounds, which are suspected endocrine disrupters, on aromatase activity and hCG secretion in human choriocarcinoma JAR, JEG-3, and BeWo cells. Protein synthesis as measured by 35 S-methionine incorporation in all cell lines was markedly decreased by treatment with both tributyltin (TBT) and triphenyltin (TPT) at concentrations above 3 10 7 M, due to cytotoxicity. In JAR cells, 35 S-methionine uptake was decreased by 50% at 3 10 7 M of TBT. At a TPT concentration of 1 10 7 M, protein synthesis in JAR cells was not affected, whereas JEG-3 and BeWo cells demonstrated slightly decreases. In all cell lines, both TBT and TPT increased levels of hCG secretion and aromatase activity in a dose- and timedependent fashion following exposure to nontoxic concentration ranges. In addition, these trialkyltin compounds enhanced 8-bromo-cAMP-induced hCG secretion and aromatase activity in JAR cells. TBT caused dose-related increases in steady-state mRNA levels of both hCG and CYP19 in JAR cells following 24- or 48-h exposure to nontoxic concentrations of TBT. However, these mRNA changes in JAR cells were not comparable to the changes in both hCG secretion and aromatase activity. These results indicate that the observed trialkyltin-induced alterations in human choriocarcinoma cells are due to other mechanism in addition to a regulation of hCG and CYP19 mRNA levels. Our studies suggest that trialkyltin compounds are potent stimulators of human placental hCG production and aromatase activity in vitro; and the placenta represents a potential target organ for trialkyltin compounds, whose endocrine-disrupting effects might be the result of local changes in hCG and estrogen concentrations in pregnant women. (J Clin Endocrinol Metab 87: 2830 –2837, 2002)

Metallothionein (MT) is a low-molecular-mass, cysteine-rich metal binding protein thought to be involved in the detoxification of heavy metals and scavenging of free radicals. MT is directly induced not only by heavy metals, but also by hormones and cytokines. The present study, which uses mice with genetic deletions of the MT proteins (MT−/- mice), was designed to evaluate the effects of MT on the expression of pro-inflammatory cytokines in macrophages. We found that the production of tumour necrosis factor (TNF) induced by lipopolysaccharide (LPS) in peritoneal macrophages is up-regulated by MT via the modulation of nuclear factor κB (NF-κB) activity. This conclusion is supported by the following observations: (1) LPS stimulated the secretion of less TNF activity from MT−/- peritoneal exudate macrophages (PEMs) than from wild-type controls (MT+/+ mice) without a difference in the pattern of kinetics; (2) LPS-stimulated expression of TNF-α mRNA was decreased in MT−/- PEMs; (3) LPS-stimulated activation of NF-κB was decreased in MT−/- PEMs; and (4) production of TNF in PEMs of MT−/- mice after LPS treatment in vivo was decreased (compared with MT+/+ PEMs). Expression of other inflammatory cytokines, interleukin (IL)-1α and IL-6 mRNA, which were modulated by NF-κB, were also down-regulated in MT−/- PEMs. Thus MT plays a key role in the LPS-induced activation of PEMs via the modulation of NF-κB activity.

Macrophage colony stimulating factor (M-CSF) plays an important role in the proliferation and differentiation of mononuclear phagocytes. The present study investigates the effect of zinc on M-CSF expression in MC3T3-E1 and L929 cells. Zinc dose-dependently increased M-CSF mRNA levels. The time-course of zinc-induced M-CSF mRNA expression peaked at 6 h. Stability studies of mRNA using actinomycin D revealed that zinc does not affect M-CSF mRNA stability. We examined the function of the M-CSF gene promoter using a luciferase reporter assay. A construct containing the -467/+39 region of the promoter was upregulated by zinc. In the presence of cycloheximide, zinc did not induce a greater increase in the M-CSF mRNA than cycloheximide alone. To confirm the effect of MT on M-CSF mRNA expression, mouse lung fibroblasts (MLFs) were prepared from MT+/+ and MT-/- mice. Zinc induced an increase in the expression of M-CSF in MT+/+ MLFs, but this response was not evident in MT-/- MLFs. Moreover, overexpression of MT upregulated M-CSF mRNA expression as well as M-CSF secretion. Our findings suggest that MT expression mediates zinc regulation of M-CSF gene expression at the transcriptional level.

Scalarane-type sesterterpenes, PHC-1–PHC-7, which have been isolated from a marine sponge, increased hemoglobin production in human chronic myelogenous leukemia cell line K562 at the concentration of 0.1–5 μg/ml. PHC-1, the major constituent, induced the expression of glycophorin A and the enucleation for K562 cells. These sesterterpenes were found to induce erythroid differentiation in K562 cells. In addition, PHC-1 induced G1 arrest of the cell cycle of K562 cells.

Metallothionein (MT) can modulate transcriptional activity in vitro. We examined whether the absence of MT affects gene expression in vivo. We compared the hepatic RNA profiles of wild-type and MT-null neonatal mice using improved differential display. The hepatic MT level was maximal during neonatal development. We identified five cDNA fragments that were expressed in MT-null mice at different levels from those in wild-type mice. Two were fragments of MT-I and mutant MT-I cDNA. The sequences of the other cDNA fragments were identical to those of contrapsin, transketolase, and vanin-3. The latter two were up-regulated, whereas contrapsin was down-regulated in neonatal MT-null mice. These mRNA levels were remarkably different between the two strains of neonatal mice. Further characterization of the regulated mRNA identified here will determine whether or not they are primary or secondary effects of an MT deficiency.

The biological roles of metallothionein (MT) in connection with defense systcins have been previously proposed. Some compounds in which toxicity is reduced by pre-synthesis of MT (e.g. some heavy metals and radical-producing compounds) can induce MT synthesis. Inflammation-producing compounds induce MT synthesis specific to the liver, rcduccd hy preadministration of glucocorticoid, and accompanied by the elevation of fibrinogen and ceruloplasmin in the plasma. MT inducing factor(s) for culturing hepatoma cells was detected in the serum of LPS-treated mice, and the presence of glucocorticoid at the physiological level was necessary for its MT-inducing activih. MT synthesis hy serum MT-inducing factor in cultured cells, and that by n-hexane administration in the liver were inhibited by the addition or preadministration of anti interleukin (IL)-6 antibody. In IL-6-transgenic micc. a high concentration of MT was observed specifically in the liver. These results show that MT synthesis by inflammation-p...

Hepatoprotective activities of zinc and sakuraso-saponin against toxicity of carbon tetrachloride were investigated in metallothionein (MT)-deficient mice. Pretreatment of control 129/Sv mice with zinc or sakuraso-saponin blocked carbon tetrachloride-induced elevation of plasma transaminase activities. Quantitatively equivalent protection against carbon tetrachloride-induced hepatic damage was also observed in MT-deficient mice. Zinc and sakuraso-saponin caused elevation of hepatic MT levels in control 129/Sv mice, whereas hepatic MT was undetectable in MT-deficient mice. To examine the possibility that sakuraso-saponin-induced hepatoprotection is mediated by endogenous zinc, the hepatic concentration of zinc was analyzed. Hepatic zinc concentration in MT-deficient mice was not changed by the treatment of sakuraso-saponin. Injection of sakuraso-saponin caused a decrease of activity of aniline hydroxylation. The suppression of cytochrome P450 appears to be a mechanism by which sakuraso-saponin protects mice from the hepatotoxic effects of carbon tetrachloride. These findings indicate that the hepatoprotective activity of zinc or sakuraso-saponin is not dependent on their MT-inducing activity.

We investigated the effects of deferoxamine on the differentiation of embryonal carcinoma F9 cells. Deferoxamine, a widely used therapeutic agent for thalassemia and iron overload, was found to induce F9 cell differentiation and to have some unique characteristics compared with other chelators, hinokitiol and dithizone, which were previously reported to induce differentiation of these cells. This hydrophilic agent induced reversible differentiation as did sodium butyrate, whereas other chelators did not. However, morphological features of the cells after deferoxamine-induced differentiation were similar to those of cells incubated with the other chelators. The differentiation-inducing activity of deferoxamine was abolished by preincubation with Fe3+ ions, similarly to the other chelators examined. Moreover, cell proliferation was inhibited by treatment with this agent, and the numbers of cells in the colonies were reduced by apoptosis. Based on these results, we conclude that deferoxamine induces differentiation and apoptosis of F9 cells via chelation of extracellular and/or intracellular Fe3+ ions.

Metallothionein-inducing activities of 11 saponins were investigated in mice. Of the saponins investigated, echinoside A and sakuraso-saponin were highly effective. Sakuraso-saponin showed dose-dependent and time-dependent induction of hepatic metallothionein. The isoforms of the induced metallothionein in the liver were determined to be metallothionein 1 and 2. Induction of metallothionein was observed specifically in the liver and heart. Echinoside A showed similar effects to sakuraso-saponin except that no induction was observed in the heart. Pretreatment of mice with these saponins blocked CCl4-induced hepatic injury, such as the elevation of plasma transaminase activity and centrilobular necrosis in the liver. CCl4-induced elevation of lipid peroxide level in the liver was also blocked by injection of sakuraso-saponin. The hepatoprotective activities of the saponins found in this study may have been due to their MT-inducing activity. © 1997 by John Wiley & Sons, Ltd.

Exposure to mild stress by chemicals and radiation causes DNA damage and leads to acquired stress resistance. Although the linear no-threshold (LNT) model of safety assessment assumes risk from any dose, evidence from radiological research demonstrates a conflicting hormetic phenomenon known as the hormesis effect. However, the mechanisms underlying radiation hormesis have not yet been clarified, and little is known about the effects of low doses of chemical carcinogens. We analyzed the efficacy of pretreatment with low doses of the alkylating agent N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) on the subsequent induction of cell transformation and gastric ulceration by high-dose MNNG. We used an in vitro Balb/3T3 A31-1-1 cell transformation test and monitored the formation of gastric ulcers in 5-week-old male ICR mice that were administered MNNG in drinking water. The treatment concentrations of MNNG were determined by the cell survival rate and past reports. For low-dose in vitro and in vivo experiments, MNNG was used at 0.028 μM, and 2.8 μg/mL, respectively. The frequency of cell transformation induced by 10 μm MNNG was decreased by low-dose MNNG pretreatment to levels similar to that of spontaneous transformation. In addition, reactive oxygen species (ROS) and mutation frequencies induced by 10 μm MNNG were decreased by low-dose MNNG pretreatment. Importantly, low-dose MNNG pretreatment had no effect on cell proliferation. In vivo studies showed that the number of gastric ulcers induced by 1 mg/mL MNNG decreased after low-dose MNNG pretreatment. These data indicate that low-dose pretreatment with carcinogens may play a beneficial role in the prevention of chemical toxicity under specified conditions.

Mesothelioma is a highly malignant tumor with a poor prognosis and limited treatment options. Although cisplatin (CDDP) is an effective anticancer drug, its response rate is only 20%. Therefore, discovery of biomarkers is desirable to distinguish the CDDP-susceptible versus resistant cases. To this end, differential proteome analysis was performed to distinguish between mesothelioma cells of different CDDP susceptibilities, and this revealed that expression of annexin A4 (ANXA4) protein was higher in CDDP-resistant cells than in CDDP-susceptible cells. Furthermore, ANXA4 expression levels were higher in human clinical malignant mesothelioma tissues than in benign mesothelioma and normal mesothelial tissues. Finally, increased susceptibility was observed following gene knockdown of ANXA4 in mesothelioma cells, whereas the opposite effect was observed following transfection of an ANXA4 plasmid. These results suggest that ANXA4 has a regulatory function related to the cisplatin susceptibility of mesothelioma cells and that it could be a biomarker for CDDP susceptibility in pathological diagnoses.

A new procedure for one-carbon homologation of carbonyl compounds is described. The method is based on the rearrangement of β-oxido carbenoid which is generated via the ligand exchange reaction of the sulfinyl group of α-chloro β-hydroxy sulfoxide with tert-butyllithium. Addition of the carbanion of aryl 1-chloroalkyl sulfoxides to carbonyl compounds gave the adducts in good yields. The β-oxido carbenoid rearrangement of the adducts gave one-carbon homologated carbonyl compounds having an α-alkyl substituent. A similar reaction of the adducts derived from carbonyl compounds with chloromethyl p-tolyl sulfoxide yielded a procedure for a methylene insertion. The stereochemistry of the β-oxido carbenoid rearrangement is also discussed.

The concentration of plasma angiotensinogen increases upon induction of inflammation. Studies were carried out using serum samples collected from mice and rats after injection of lipopolysaccharide (LPS) to determine whether interleukin-6 (IL-6) is a mediator responsible for the inflammation-induced increase of angiotensinogen synthesis in liver cells. Serum collected from mice or rats 2 and 4 hr after injection of LPS contained a factor that stimulated [35S]methionine incorporation into angiotensinogen newly synthesized by rat hepatoma H4IIEC3 (H4) cells. Assay of IL-6 using an IL-6-dependent murine hybridoma, MH60.BSF2 cells, showed the presence of IL-6-like activity in sera of mice or rats 2 and 4 hr after injection of LPS. Anti-mouse IL-6 monoclonal antibody completely inhibited not only the IL-6-like activity present in LPS-treated mouse serum but also the ability of the serum to stimulate angiotensinogen synthesis of H4 cells. These results suggest that increased synthesis of angiotensinogen in the liver after induction of inflammation is mediated by IL-6, a cytokine important in immune reactions and the hepatic acute-phase response.

Metallothionein (MT) is a small, cysteine-rich protein active in zinc homeostasis, cadmium detoxification, and protection against reactive oxygen species. Mouse MT-I gene transcription is regulated by metal response element-binding transcription factor-1 (MTF-1), which is recruited to the promoter by zinc. We examined alterations in the chromatin structure of the MT-I promoter associated with enhanced transcriptional activation. MTF-1 proved essential for zinc-induced epigenetic changes in the MT-I promoter. Chromatin immunoprecipitation assays demonstrated that zinc treatment rapidly decreased Lys⁴-trimethylated and Lys⁹-acetylated histone H3 in the promoter and decreased total histone H3 but not histone H3.3. Micrococcal nuclease sensitivity of the MT-I promoter was increased by zinc. Thus, the chromatin structure in the promoter may be locally disrupted by zinc-induced nucleosome removal. Without MTF-1 these changes were not observed, and an MTF-1 deletion mutant recruited to the MT-I promoter by zinc that did not recruit the coactivator p300 or activate MT-I transcription did not affect histone H3 in the MT-I promoter in response to zinc. Interleukin-6, which induces MT-I transcription independently of MTF-1, did not reduce histone H3 levels in the promoter. Rapid disruption of nucleosome structure at the MT-I promoter is mediated by zinc-responsive recruitment of an active MTF-1-coactivator complex.

The identification of cytokine adjuvants capable of inducing an efficient mucosal immune response against viral pathogens has been long anticipated. Here, we attempted to identify the potential of tumor necrosis factor superfamily (TNFS) cytokines to function as mucosal vaccine adjuvants. Sixteen different TNFS cytokines were used to screen mucosal vaccine adjuvants, after which their immune responses were compared. Among the TNFS cytokines, intranasal immunization with OVA plus APRIL, TL1A, and TNF-alpha exhibited stronger immune response than those immunized with OVA alone. TL1A induced the strongest immune response and augmented OVA-specific IgG and IgA responses in serum and mucosal compartments, respectively. The OVA-specific immune response of TL1A was characterized by high levels of serum IgG1 and increased production of IL-4 and IL-5 from splenocytes of immunized mice, suggesting that TL1A might induce Th2-type responses. These findings indicate that TL1A has the most potential as a mucosal adjuvant among the TNFS cytokines.

The hyperglycemia and hyperoxidation that characterize diabetes lead to reduced vitamin C (L-ascorbic acid, AA) levels in diabetic humans and animals. We examined the possibility that diabetes-induced low plasma AA levels impair AA distribution to various tissues and that these changes are closely related to the development of diabetic complications. AA levels were markedly decreased in the plasma and increased in the adrenals of mice with streptozotocin (STZ)-induced diabetes. Consistently with these results, in [1-(14)C]AA accumulation assays, the efficiency of [1-(14)C]AA accumulation was significantly higher in the adrenals (which had the greatest ability to accumulate [1-(14)C]AA) of diabetic mice than in those of controls. Expression of sodium-dependent vitamin C transporter (SVCT)-2, a transporter of AA, was upregulated in diabetic adrenals. Furthermore, increased AA incorporation into the diabetic adrenals by SVCT-2 led to increased plasma norepinephrine, triglyceride and free fatty acid levels in mice with STZ-induced diabetes. Therefore, oversupplementation with AA could be deleterious in diabetic patients, because overexpression of adrenal SVCT-2 in diabetes could lead to excessive AA uptake, thus enhancing norepinephrine production and exacerbating some diabetic complications. Interestingly, however, treatment with AA dose-dependently abolished the increased expression of adrenal SVCT-2 and normalized the abovementioned plasma parameters in diabetic mice. These results suggest SVCT-2-mediated increases in AA uptake by the adrenals followed by excessive production of plasma norepinephrine may play a pivotal role in the development of diabetic complications.

Metal-responsive transcription factor-1 (MTF-1), which is involved in sensing heavy metal load, induces the transcription of several protective genes. The mouse Mtf-1 gene is essential, and Mtf-1(-/-) embryos die from liver degeneration. We showed that DNA synthesis induced in hepatocytes by epidermal growth factor (EGF) was delayed by inhibition of MTF-1. To inhibit MTF-1 activity, MTFDeltaC, a C-terminal deletion mutant of MTF-1, was expressed by infection with the virus Ad5MTFDeltaC. Lactate dehydrogenase (LDH) release and/or caspase-3/7 activation was not observed under our experimental conditions. The inhibitory effect of MTFDeltaC on EGF-dependent DNA synthesis in hepatocytes was not eliminated by zinc addition. EGF-dependent extracellular signal-related kinase (ERK) phosphorylation, an essential reaction for EGF-dependent DNA synthesis, was decreased in MTF-1-inhibited hepatocytes. Moreover, decrease of ERK phosphorylation was observed by using siRNA in MTF-1-downregulated hepatocytes. These results indicate that MTF-1 is particularly important for proper hepatocyte proliferation. This is the first report to suggest the function of MTF-1 in the ERK pathway.

Organotin compounds, such as tributyltin (TBT) and triphenyltin (TPT), are typical environmental contaminants and suspected endocrine-disrupting chemicals because they cause masculinization in female mollusks. However, it remains unclear whether organotin compounds also cause crucial toxicities in human sexual development and reproductive functions. We investigated the effects of 17 tin compounds on the catalytic activity and mRNA expression of 17beta-hydroxysteroid dehydrogenase type I (17beta-HSD I) in human choriocarcinoma JAr cells. At nontoxic concentrations, both trialkyltins with propyl, butyl or cyclohexyl substituents on the tin atom and triphenyltin (TPT) enhanced 17beta-HSD I mRNA transcription and enzyme activity in a dose-dependent fashion. Although tetraalkyltin compounds such as tetrabutyltin and tributylvinyltin also increased the mRNA expression and enzyme activity of 17beta-HSD I, the concentrations necessary for activation were >30-100 times greater than those for trialkyltins. Inorganic tin had no effect on the catalytic activity and mRNA expression of 17beta-HSD I. Interestingly, diphenyltin and monophenyltin, which are metabolites of TPT, enhanced 17beta-HSD I activity with a concomitant increase in mRNA expression, whereas dibutyltin and monobutyltin, which are metabolites of tributyltin, enhanced 17beta-HSD I activity without a concomitant increase in mRNA expression. These results suggest that organotin compounds are potent stimulators of 17beta-estradiol biosynthesis to enhance 17beta-HSD I activity in the human placenta in vitro; the placenta represents a potential target organ for these compounds, whose endocrine-disrupting effects might be the result of local changes in 17beta-estradiol concentrations in pregnant women.

Retinoid X receptor (RXR) is a nuclear receptor that plays important and multiple roles in mammalian development and homeostasis. We previously reported that, in human choriocarcinoma cells, tributyltin chloride and triphenyltin hydroxide, which are typical environmental contaminants and cause masculinization in female mollusks, are potent stimulators of human chorionic gonadotropin production and aromatase activity, which play key endocrine functions in maintaining pregnancy and fetal development. However, the molecular mechanism through which these compounds stimulate these endocrine functions remains unclear. Our current study shows that trialkyltin compounds, including tributyltin chloride and triphenyltin hydroxide, function as RXR agonists. Trialkyltins directly bind to the ligand-binding domain of RXR with high affinity and function as transcriptional activators. Unlike the natural RXR ligand, 9-cis-retinoic acid, the activity of trialkyltins is RXR specific and does not activate the retinoic acid receptor pathway. In addition, trialkyltins activate RXR to stimulate the expression of a luciferase reporter gene containing the human placental promoter I.1 sequence of aromatase, suggesting that trialkyltins stimulate human placental endocrine functions through RXR-dependent signaling pathways. Therefore, our results suggest that activation of RXR may be a novel mechanism by which trialkyltins alter human endocrine functions.

Fluorometric detection of O2-• is performed based on desulfonylation of 3 to the corresponding fluoresceins 4 through nucleophilic substitution, and this fluorescing process is quite specific toward O2-• over H2O2, t-BuOOH, NaOCl, 1O2, HO•, NO•, and ONOO-. Furthermore, effects of glutathione, cytochrome P450 reductase/NADPH, and diaphorase/NADH are relatively small on the fluorescing process of probe 3 with X = Y = F, which is useful to detect O2-• released from neutrophils stimulated by phorbol myristate acetate with satisfactory sensitivity.

Heavy metal-induced transcriptional activation of the genes coding for metallothionein (MT) is mediated by a cis-acting DNA element, the metal-responsive element (MRE). MRE-binding transcription factor-1 (MTF-1) is a highly conserved heavy metal-induced transcriptional activator. MTF-1 also activates transcription in response to oxidative stress and regulates the expression of several cytoprotective factor genes, including MT, gamma-glutamylcysteine synthetase, and Cu/Zn-superoxide dismutase. It is thus thought that MTF-1 plays a role in cellular stress response. The physiological role of MTF-1 remains unclear because of the lack of MTF-1-specific activators and/or inhibitors. To obtain an MTF-1-specific inhibitor, we constructed an MTFDeltaC (amino acids 1-317), a C-terminal deletion mutant of MTF-1. MTFDeltaC could bind MRE and competed with MTF-1 for MTF-MRE complex formation. Transient expression of MTFDeltaC in HepG2 cells reduced MRE-driven gene expression, demonstrating that MTFDeltaC is dominant to MTF-1. HepG2 cells stably expressing MTFDeltaC showed increased susceptibility to the cytotoxic effects of tert-butyl hydroperoxide (tBH). Furthermore, we constructed Ad5MTFDeltaC, a recombinant adenovirus that expresses MTFDeltaC. Infection with the virus induced MTFDeltaC expression and increased susceptibility to the cytotoxic effects of tBH. These results indicate that MTF-1 participates in controlling the cellular redox state.

Sodium-dependent vitamin C transporter (SVCT) 2 facilitates reduced ascorbic acid (AA) transport in MC3T3-E1 osteoblasts. Our previous studies suggested that Zn-induced osteoblast differentiation and Ca 2+ -, PO 4 3− -stimulated osteopontin (OPN) expression might result from their up-regulation effect on SVCT2 expression and AA uptake. Here, we investigated the role of SVCT2 on osteoblast differentiation by using SVCT2-overexpressing cells. Two clones of SVCT2-introduced cells overexpressed SVCT2 mRNA by 2.8- and 3.1-fold those of control cells, which resulted in obvious increase of AA uptake by 2.1- and 2.4-fold in V max with no change in K m . Alkaline phosphatase activity, hydroxyproline content significantly increased in SVCT2-overexpressing cells, and the induction of OPN mRNA was through up-regulation of OPN promoter activity by SVCT2 overexpression. Moreover, SVCT2-overexpressing cells exhibited more ability to promote mineralization and increase calcium deposition under the stimulation of 5 mM β-glycerophosphate. These findings indicate that SVCT2 stimulates osteoblast differentiation and mineralization.

Zinc is an essential trace element that increases osteoblast numbers and bone formation. However, the mechanisms involved in the Zn-induced differentiation of osteoblasts are poorly understood. We examined the roles of l -ascorbic acid (AA) and its transporter, sodium-dependent vitamin C transporter (SVCT) 2, in the Zn-induced expression of osteoblastic differentiation markers. Zinc time- and dose-dependently induced SVCT2 mRNA expression in the absence or presence of AA. Western blotting and kinetic assays showed that Zn increased functional SVCT2 protein levels and AA transport. In the presence of AA, 50 μM Zn enhanced mRNA expression of the osteoblastic differentiation markers alkaline phosphatase, α1(I) procollagen, osteopontin (OPN), and osteocalcin (OCN) by 3.9-, 3.8-, 3.3-, and 3.5-fold, respectively; in the absence of AA, the Zn-induced increase was 2.8-, 2.5-, 1.3-, and 1.1-fold, respectively. These findings suggest that AA and SVCT2 mediate Zn-induced OPN and OCN expression and partly regulate Zn-induced osteoblastic differentiation.

Suppressors of cytokine signaling (SOCS) proteins possess common structures, a SOCS box at the C-terminus and a SH2 domain at their center. These suppressors are inducible in response to cytokines and act as negative regulators of cytokine signaling. The ASB proteins also contain the SOCS box and the ankyrin repeat sequence at the N-terminus, but do not have the SH2 domain. Although Socs genes are directly induced by several cytokines, no Asb gene inducers have been identified. In this study, we screened the specific genes expressed in the course of differentiation of HL-60 cells, and demonstrated that ASB-2, one of the ASB proteins, was rapidly induced by all-trans retinoic acid (ATRA). Typical retinoid receptors (RARs) or retinoid X receptors (RXRs) binding element (RARE/RXRE) were presented in the promoter of the Asb-2 gene. We showed that RARα, one of the RARs, binds to the RARE/RXRE in the Asb-2 promoter. In addition, we demonstrated by luciferase reporter assay that this element was a functional RARE/RXRE. These findings indicate that ASB-2 is directly induced by ATRA and may act as a significant regulator, underlying such physiological processes as cell differentiation.

Mice treated with lipopolysaccharide (LPS)/D-galactosamine (GalN) selectively develop hepatic failure. The acute-phase protein alpha(1)-acid glycoprotein (AGP) has been demonstrated to protect mice from LPS/GalN-induced lethality. Metallothionein (MT), which is a low-molecular weight, cysteine-rich, metal-binding protein, is also induced in the acute-phase reaction. However, the specific function of MT in acute-phase response remain to be elucidated. We showed that MT-null mice were more sensitive to LPS/GalN-induced lethality than wild-type mice. The increase in vital mediator levels, TNF-alpha and NO were of similar levels in wild-type and MT-null mice. A remarkable increase in plasma platelet-activating factor levels was not observed in our experimental conditions. On the other hands, the mRNA level of AGP in the response to LPS/GalN was decreased in MT-null mice compared to wild-type mice. These results indicated that MT may have the potential to prevent LPS/GalN-induced lethality, at least through the attenuation of AGP induction.

A nuclear localization signal (NLS) has been detected in several nuclear proteins. Classical NLS-mediated nuclear pore targeting is performed by using the cytosolic factors, importin alpha and importin beta, whereas nuclear translocation requires the small GTPase, Ran. In the present study, we demonstrated that nuclear localization of metallothionein (MT) differs from that of classical NLS-mediated substrates. In digitonin-permeabilized BALB/c3T3 cells, biotinylated MT was localized in the nucleus in the presence of ATP and erythrocyte cytosol in the same manner as for SV40 large T NLS-conjugated allophycocyanin (APC-NLS). Under ATP-free conditions, nuclear rim-binding was observed in both transport substrates. Rim-binding of labeled MT was competitively inhibited by the addition of an excess amount of unlabeled MT. Different elution profiles were observed for the localization-promoting activities of MT in the cytosol compared to those of APC-NLS. Furthermore, nuclear localization of MT was determined to be a wheat germ agglutinin-insensitive, GTPgammaS-sensitive, and anti-Ran antibody-sensitive process. Green fluorescent protein-metallothionein (GFP-MT) fusion protein was also localized in the nucleus in the stable transformant of CHL-IU cells. These results strongly suggest that the targeting by MT of the nuclear pore is mediated by cytosolic factor(s) other than importins and that MT requires Ran for its nuclear localization.

Based on previous findings that liver zinc and metallothionein (MT) levels increase after tumor transplantation, zinc metabolism in tumor-bearing mice was studied to clarify the role of zinc–MT in host defense systems. Zinc in the hepatic cytosolic MT fraction did not increase in tumor-bearing mice fed a zinc-deficient diet, suggesting that dietary zinc is necessary for apo-MT induction in the liver after tumor transplantation and is then incorporated into the apo-MT. When 65 ZnCl 2 was intravenously injected, liver 65 Zn levels in the tumor-bearing mice were higher than those in control mice for 72 h after the injection. Pancreatic and blood 65 Zn levels in tumor-bearing mice were lower than those in controls for 24 h (pancreas) and 6 h (blood) after the injection. These findings indicate that the hepatic zinc response via MT induction influences zinc metabolism in the body after tumor transplantation. Moreover, 65 Zn uptake in the liver of MT-deficient tumor-bearing mice was lower than that in control tumor-bearing mice 1 h after injection. 65 Zn uptake in the tumor and blood 65 Zn levels in the MT-deficient tumor-bearing mice were higher than those in the control tumor-bearing mice. Tumor weight increased more in MT-deficient mice than in control mice. The formation of zinc–MT in the liver of tumor-bearing mice might decrease blood zinc availability for tumors and other tissues, such as the pancreas.

We investigated the effect of diphenylthiocarbazone (dithizone) and its structurally related compounds on the differentiation and apoptosis of two human myeloid leukemia cell lines. Dithizone caused a time- and concentration-dependent induction of differentiation in both the promyelocytic leukemia cell line HL-60 cells and the myeloblastic leukemia cell line ML-1 cells, as measured by nitroblue tetrazolium (NBT) reducing activity. Morphological changes and esterase activities confirmed that this differentiation took place. The induction of differentiation required the addition of dithizone to the culture medium for at least 12 h. The differentiation inducing activity was inhibited by the preincubation of dithizone with various metal ions such as Pb2+, Zn2+, Cu2+ and Mn2+ ions, but not with Fe3+ and Mg2+ ions. In addition, the DNA extracted from dithizone-treated HL-60 cells showed a typical ladder pattern characteristic of apoptosis in agarose gel electrophoresis. A quantitative analysis of DNA fragmentation revealed that this apoptosis was concentration- and time-dependent in both the HL-60 and ML-1 cells. Dithizone-induced apoptosis was also inhibited by preincubation with Mn2+ ions, but not with Mg2+ ions. These results indicate that dithizone induces both differentiation and apoptosis in HL-60 and ML-1 cells through a unique mechanism including metal chelation.

It has been demonstrated that ascorbic acid is present in human gastric juice at a very high level even in the fasting state. In this study, we confirmed such a physiological event in pylorus-ligated rats and investigated the mechanism for gastric ascorbic acid secretion using the rat in vivo perfusion method. Gastric juice from fasting rats after a 4-hr ligation contained a 6-fold higher level of ascorbic acid than that in sera, indicating the presence of its active transport system across the gastric wall. To identify an endogenous mediator, four kinds of gastric stimulants were compared for their secretory abilities. Although all four secretagogues used stimulated acid secretion to comparable levels, ascorbic acid secretion was stimulated only by carbamylcholine chloride (carbachol). Carbachol-stimulated ascorbic acid secretion was abolished by atropine pretreatment, showing that it is mediated via a muscarinic cholinergic receptor. This cholinergic stimulation was also demonstrated in a rat mutant that genetically lacks the ability to synthesize ascorbic acid in the liver, similar to humans. In addition, a potent inhibitor of the gastric proton pump, which is a final regulatory component in the mechanism of acid secretion, caused no inhibition of ascorbic acid secretion in Osteogenic Disorder Shionogi (ODS) rats. These results are the first evidence indicating that ascorbic acid, the reduced form of vitamin C, is actively and independently secreted into the gastric juice under regulation of a cholinergic receptor system.

We propose a novel alternative approach, an advanced method for recently developed strategies, for identifying differentially expressed genes. Firstly, double-stranded cDNAs were digested using Sau 3AI and the 3′-end restriction fragments of the cDNA were ligated to a double-stranded adapter. Next, the restriction fragments were directly amplified using several combinations of adapter-specific primers and FITC-labeled oligo dT primers. The selected cDNA fragments were displayed on a polyacrylamide gel. Neither nested PCR nor purification of 3′-end fragments are necessary. We examined the validity of this approach by evaluating gene expression changes during granulocytic differentiation of HL-60 cells. This method can theoretically detect almost all gene expression changes more rapidly and through simpler manipulations than by any other approach.

T-kininogen is known to be an acute-phase reactant as well as a kininogen in rat plasma. Three kinds of cytokines, interleukin-1, tumor necrosis factor and interferon, were assayed for their abilities to stimulate hepatic production of T-kininogen. Of these cytokines, interferon was able to stimulate hepatic production of T-kininogen, but few effects were observed for interleukin-1 and tumor necrosis factor. In addition, the stimulatory effect of interferon was inhibited by tumor necrosis factor. Our data suggest that interferon is a candidate for the leukocyte-derived factor mediating the acute-phase response of T-kininogen.

The tridecapeptide corresponding to the sequence 165-177 of T-kininogen (tryptic peptide) (I) containing Gln-Val-Val-Ala-Gly sequence was synthesized by a conventional solution method and its effect on thiol proteinase was examined. Although Z-Gln-Val-Val-Ala-Gly-OMe showed inhibitory activity towards papain and protective activity against T-kininogen-induced inhibition of papain, the tryptic peptide obtained did not exhibit any effect on thiol proteinase. In order to study the relationship between structure and effect on thiol proteinase, several compounds (II-VI) modified with various groups at the N-terminus of the Gln-Val-Val-Ala-Gly sequence were synthesized. Peptides V and VI, which have an aromatic ring at the N-terminus, exhibited weak inhibitory and significant protective activities. A small peptide such as Gln-Val-Val-Ala-Gly might be better able to approach papain than the tryptic peptide (I), and the aromatic ring associated with Gln-Val-Val-Ala-Gly at the N-terminus apparently strengthened the binding ability of the peptide to papain.

The Gln-Val-Val-Ala-Gly sequence occurs frequently in several thiol proteinase inhibitors, suggesting that this conserved sequence may be one of the reactive sites in the inhibitors. In order to study the effects of the sequence on thiol proteinases, we synthesized H-Gln-Val-Val-Ala-Gly-OH (VI) and its related peptide derivatives by the conventional solution method and examined their effects on the enzymatic activity of papain. We found that Z-Gln-Val-Val-Ala-Gly-OMe (IV) not only inhibits papain but also protects it from T-kininogen, which is one of thiol proteinase inhibitors. Apparently this peptide (IV) binds with some part of papain in competition with T-kininogen.

Thiol proteinase inhibitors isolated from various tissues have a fairly conservative common amino acid sequence, Gln-Val-Val-Ala-Gly. We synthesized various kinds of Gln-Val-Val-Ala-Gly derivatives by the solution method. Z-Gln-Val-Val-Ala-Ala-pNA, Z-Gln-Val-Val-Ala-pNA and Z-Gln-Val-Val-pNA weakly inhibited the amidolytic activity of papain toward Bz-Arg-βNA and exhibited a stronger protective activity than corresponding Z-peptide methyl ester against thiol proteinase inhibitor (such as T-kininogen)-induced inhibition of papain.Suc-Gln-Val-Val-Ala-Ala-pNA exhibited more potent inhibitory activity toward papain than Z-Gln-Val-Val-Ala-Ala-pNA, but it did not show any protective effect. The circular dichroism spectra of the pNA derivatives and papain suggested that the pNA moiety of the peptide participated in binding with some part of the enzyme other than the catalytic site.

Plasma levels of angiotensinogen were significantly increased in rats following treatment with lipopolysaccharide (LPS). However, no such acute-phase response in the plasma angiotensinogen level was induced in hypophysectomized rats. Implantation of peritoneal exudate cells (PEC), which had been collected from rats treated with i.p. injection of LPS and glycogen, into the peritoneal cavities of normal rats also caused elevation of plasma angiotensinogen levels. No significant changes in plasma levels of corticosterone were observed in rats receiving either LPS or LPS-stimulated PEC. These results indicate that the acute-phase response of angiotensinogen is mediated by leukocytes-derived cytokine, and glucocorticoid is essential for its stimulatory effect on the hepatic production of angiotensinogen.

Angiotensinogen has been identified as one of the acute-phase reactants. In vitro studies were carried out using the Reuber H35 hepatoma cell line to identify the species of cytokines contributing to the increased synthesis of angiotensinogen in the liver. Angiotensinogen secretion by H35 cells was maximally increased 4-fold by the addition of 10(-7) M dexamethasone. Under this condition, angiotensinogen secretion was further stimulated by B cell stimulatory factor 2/interleukin-6 (IL-6, 50 U/ml), but not by interleukin-1 or interferon-alpha. In the absence of glucocorticoid, IL-6 did not affect angiotensinogen secretion by H35 cells, indicating that the presence of glucocorticoid is required for the stimulatory activity of IL-6. These results suggest that IL-6 is a mediator responsible for the increased synthesis of angiotensinogen in the liver during acute inflammation.

Studies were carried out in order to characterize the kininogen in rat urine. Rat urine contained a component which was cross-reactive with antibody to rat plasma T-kininogen. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of rat urine revealed a single antigenic band corresponding to the molecular weight of plasma T-kininogen. Induction of acute inflammation in rats by an injection of lipopolysaccharide caused an increase in the urinary excretion of immunoreactive T-kininogen in parallel with an elevation of plasma T-kininogen. Kininogen partially purified from rat urine by affinity chromatography using S-carboxymethylated papain-agarose liberated only T-kinin upon trypsinization, but not upon treatment with rat glandular kallikreins. From these results, we conclude that T-kininogen is the major kininogen present in rat urine.

Acute phase responses of plasma angiotensinogen and kininogen were studied in rats. Plasma angiotensinogen levels increased about 3-fold during the first 8 hr, and returned to normal at 48 hr, following the induction of acute inflammation by lipopolysaccharide (LPS). Plasma kininogen reached maximum levels at 48 hr following LPS administration. In adrenalectomized rats, plasma angiotensinogen levels decreased significantly, and the administration of LPS did not elevate plasma angiotensinogen levels. In contrast, plasma kininogen levels were increased by adrenalectomy, as well as by sham-operation. Dexamethasone significantly increased plasma angiotensinogen levels in adrenalectomized rats as well as in normal rats, but aldosterone did not. Plasma kininogen levels of normal rats were not changed by the administration of dexamethasone or aldosterone. From these results, it was concluded that the acute phase response of plasma angiotensinogen is mediated by glucocorticoid, but that of plasma kininogen is not.