Minoru Higashimoto

Cadmium induces apoptosis partly via caspase-9 activation in HL-60 cells

Cadmium (Cd), a potent immunotoxic metal, induces apoptosis both in vitro and in vivo. However, the mode of action remains unclear. We previously reported that Cd-induced apoptosis was partly dependent on mitochondria. In the present study, we investigated the involvement of caspase-9, which is the apex caspase in the mitochondoria-dependent apoptosis pathway, in Cd-induced apoptosis in human promyelocytic leukemia HL-60 cells. A specific inhibitor of caspase-9, Z-LEHD-FMK, partly inhibited DNA fragmentation induced by Cd treatment in HL-60 cells. Moreover, treatment of HL-60 cells with Cd resulted in the appearance of Cytochrome c (Cyt c), a potent activator of caspase-9, in the cytosol at 3 h, which closely paralleled the activation of caspase-9. Caspase-9 is an initiator caspase that is a potent activator of downstream effector caspases such as caspase-3. Caspase-3 activation was subsequent to the Cyt c release at 6 h. DNA fragmentation, an index of induction of apoptosis, also appeared 6 h after Cd treatment. The effects were more pronounced at 9 h after Cd addition. A broad-specificity inhibitor of caspases, Z-Asp-CH(2)-DCB, inhibited caspase-3 activation and DNA fragmentation induced by Cd in a dose-dependent fashion. The results suggest that Cd-induced apoptosis is partly caused by caspase-9 activation triggered by Cyt c.

Specific induction of metallothionein synthesis by mitochondrial oxidative stress

Metallothionein (MT), a sulfhydryl-rich protein, may be increased by administration of a variety of agents, including metals, cytokines and oxidative stress agents. Mitochondria are a major source of reactive oxygen species, but antioxidant systems against mitochondrial free radicals are not fully understood. In this study, we examined the induction of MT synthesis by administration of mitochondrial-specific reactive oxygen generators such as antimycin A (AA), an electron transfer inhibitor, and 2,4-dinitrophenol (DNP), an uncoupling agent. Subcutaneous administration of AA to mice significantly increased the hepatic MT concentration in a dose- and time-dependent manner. AA slightly elevated glutathione peroxidase (GSHPx) activity, but the rate of increase in GSHPx (1.3-fold) was smaller than that in MT (11.8-fold). Other antioxidants such as catalase, manganese-superoxide dismutase (Mn-SOD), copper/zinc-superoxide dismutase (Cu/Zn-SOD) and GSHPx were not activated by AA treatment. Moreover, administration of DNP induced the synthesis of MT in the liver. Although DNP slightly elevated Mn-SOD activity, the rate of increase in Mn-SOD (1.3-fold) was smaller than that in MT (3.7-fold). Other antioxidants such as catalase, Cu/Zn-SOD and GSHPx were not activated by DNP treatment. These data suggest that MT plays a major role in protection against oxidative stress induced in mitochondria.

Mercuric chloride induces apoptosis via a mitochondrial-dependent pathway in human leukemia cells.

Mercurial compounds modulate immunologic functions by inducing cytotoxicity. Although mercury chloride (HgCl(2)) is known to induce apoptosis in various immune system cells, the mechanism of the induction of apoptosis is poorly understood. In this study, we examined the activation of caspase-3, an important cysteine aspartic protease, during HgCl(2)-induced apoptosis in a human leukemia cell line (HL-60 cells). Both DNA fragmentation, a characteristic of apoptotic cells, and proteolysis of poly(ADP-ribose) polymerase (PARP), a substrate of caspase-3, occurred at 6 h after HgCl(2) treatment in HL-60 cells. These results suggest that the activation of caspase-3 was involved in HgCl(2)-induced apoptosis. The release of cytochrome c (Cyt c) from mitochondria into the cytosol, which is an initiator of the activation of caspase cascades, was also observed in HgCl(2)-treated HL-60 cells. Moreover, the release of Cyt c from mitochondria was observed in HgCl(2)-treated mitochondria isolated from mice liver, and this was followed by mitochondrial permeability transition (PT). The PT was inhibited by cyclosporin A (CsA), a potent inhibitor of PT. CsA also suppressed the occurrence of DNA fragmentation induced by HgCl(2) treatment in HL-60 cells. Taken together, these findings indicate that HgCl(2) is a potent inducer of apoptosis via Cyt c release from the mitochondria in HL-60 cells.

Antioxidant property of metallothionein in fasted mice.

Metallothionein (MT) is a low-molecular-weight and sulfur-rich protein that is induced by not only heavy metals but also physiological stresses such as fasting and restraint stresses. Although MT plays a role as a radical scavenger and a regulator of metabolism of metals, the biological function of MT induced by fasting stress has not been elucidated. In this study, we investigated the antioxidative role of MT in fasted mice. In fasted mice, the lipid peroxidation level of the liver was elevated by 24-h fasting stress, and pre-induction of hepatic MT by Zn diminished hepatic lipid peroxidation. Although 24-h fasting stress induced MT synthesis in the liver, other antioxidants such as catalase, manganese-superoxide dismutase (Mn-SOD), and glutathione peroxidase (GSHPx) were not activated in the liver. Moreover, the hepatic MT level was still elevated by fasting stress after seven cycles of repetition of alternate fasting and feeding every 24 h, but the activities of catalase, Mn-SOD and GSHPx were unchanged. These results indicate that MT induced by fasting stress plays partly as an antioxidant.

Tissue-dependent preventive effect of metallothionein against DNA damage in dyslipidemic mice under repeated stresses of fasting or restraint.

AIMS To investigate the effect of repeated stress on DNA damage in seven organs of dyslipidemic mice, and the preventive role of metallothionein (MT). MAIN METHODS Female adult 129/Sv wild-type and MT-null mice fed high-fat diet (HFD) were repeatedly subjected to mild stress of fasting or restraint in weeks 2 to 4 of 4-week study period. Serum cholesterol level, DNA damage in the liver, pancreas, spleen, bone marrow, kidney, lung and gastric mucosa, and other parameters were determined. KEY FINDINGS Body weights were increased in both types of mice fed HFD compared to those fed standard diet (STD), and further increased by 12 h-fasting, while they were markedly decreased by 1-3 h-restraint. Fasting accelerated accumulation of fat in the liver, and increase in serum cholesterol of both types of mice fed HFD. Feeding of HFD increased DNA damage in the pancreas, spleen and bone marrow of both types of mice, compared with those fed STD. In the wild-type mice fed HFD, 24 h-fasting increased DNA damage in the liver and spleen, while restraint increased the damage in the liver, pancreas, spleen and bone marrow. DNA damage in the cells of organs was markedly increased in the MT-null mice. Specifically, damage in the liver, pancreas, spleen and bone marrow was greatly increased with the intensity of stress increased, and the damage was much greater in the restraint mice than in the fasting mice. SIGNIFICANCE MT plays a tissue-dependent preventive role against DNA damage in various murine organs induced by repeated stress.

Inhibitory effects of citrus fruits on the mutagenicity of 1-methyl-1,2,3,4-tetrahydro-β-carboline-3-carboxylic acid treated with nitrite in the presence of ethanol

It has been shown that the mutagenicity of 1-methyl-1,2,3,4-tetrahydro-beta-carboline-3-carboxylic acid (MTCCA), a mutagen precursor in soy sauce treated with nitrite, was strongly increased when it was treated with nitrite in the presence of alcohols. We found that the mutagenicity of MTCCA treated with 50 mM nitrite at pH 3, 37 degrees C for 60 min in the presence of 7.5% ethanol was reduced by the addition of citrus fruits sudachi (Citrus sudachi), lemon (C. limon) and yuzu (C. junos), to the reaction mixture. The mutagenicity-reducing activity per weight of flavedos (outer colored portions of peel) of the citrus fruits was considerably higher than that of the juices. The juices of the other citrus fruits commercially available in Japan also had mutagenicity-reducing activity against the nitrite-treated MTCCA. Among the many components of citrus fruits, dietary fibers lignin and pectin showed strong antimutagenic activity in the reaction mixture, suggesting that the mixed fractions of these components including lignin, pectin, D-limonene, naringin, hesperidin, ascorbic acid and citric acid reduce the mutagenicity of MTCCA in the reaction mixture containing nitrite and ethanol.

Property of metallothionein as a Zn pool differs depending on the induced condition of metallothionein.

Metallothionein (MT) is a low-molecular-weight, cysteine-rich, heavy metal-binding protein with several putative physiological functions as a radical scavenger and a regulator of metabolism of Zn. Although the induction of hepatic MT by a wide range of stressors is a well-known phenomenon, little is known about the role of MT in stressful situations. Since MT regulates Zn metabolism, we investigated the differences between affinities of MT for Zn in various stressful conditions in this study. Divalent cadmium ions are capable of displacement of Zn from MT in vitro. Therefore, we assayed the binding of Zn to MT induced by various stimuli using in vivo administration of Cd. MT was induced by paraquat (PQ), a reactive oxygen generator, fasting stress and restraint stress. Apo-MT induced by them bound to Zn in vivo. Zn, which bound to MT induced by PQ, was displaced by the administration of Cd. However, Zn that had bound to MT induced by fasting stress and restraint stress was not displaced by Cd. Moreover, we assessed the in vivo affinity of Zn to MT induced by fasting stress under the condition of subchronic Cd exposure. Cd was administered to mice by subcutaneously implanted Alzet osmotic minipumps, which released constant amounts of Cd over a 14-day period. After 4 days, mice were fasted for 24 h and hepatic MT was examined. Interestingly, it was found that Zn had displaced Cd bound to MT. These findings indicate that the affinity of MT for Zn differs depending on the stimulus by which MT was induced. This is the first report on differences in the properties of MT depending on the stimulus used to induce MT.

Different responses of metallothionein and leptin induced in the mouse by fasting stress.

Changes in metallothionein (MT) and leptin under fasting stress were studied. MT content in the liver of mice increased markedly during continuous fasting periods up to 66 h. Hepatic content of MT increased significantly in mice during the first three cycles of alternate daily fasting-feeding, and then the rate of increase gradually decreased with repetition of this cycle. At the end of 10 cycles, the hepatic MT content was still greater in stressed mice than in the control, although the rate of increase decreased. On the other hand, the plasma concentration of leptin decreased dramatically during continuous fasting. The plasma leptin level recovered to the basal level at the end of 10 cycles of fasting-feeding. These data indicate that MT induction was strongly increased, but leptin was scarcely induced under the continuous fasting stress, and that rates of the changes in MT and leptin levels were gradually reduced under the repeated fasting stress, which may result from an inborn tolerance. Fasting presumably causes translocation of zinc from intracellular to extracellular space, from which it is taken into target organs. The increased zinc-bound MT under the continuous fasting may partly result from the need to maintain zinc and protect tissues against oxidative damage.

Enhancement of MT synthesis by leptin in fasted mice.

It is known that metallothionein (MT) synthesis occurs in the liver in various stressful situations such as immobilization and fasting. However, the mechanism of MT synthesis in stressful situations is not fully understood. In this study, we examined the involvement of leptin, the obese gene product, in MT synthesis induced by fasting stress. Despite an increase in hepatic MT levels induced by 24-hr fasting in wild-type mice, both wild-type and MT-null mice showed decreases in plasma leptin levels after 24 hr of fasting. Hepatic MT levels increased to levels comparable with that in control mice in ICR, C3H, 129Sv and Balb/c mice fasted for 24 hr, and plasma leptin levels decreased significantly. Repetition of fasting and feeding in turn every 24 hr caused a gradual decrease in hepatic MT levels after the fasting period. In contrast, the reduced plasma leptin levels increased after the fasting period with repetition of fasting-feeding cycles. The findings indicate that there is an adaptation to starvation. On the other hand, subcutaneous leptin infusion in fasted mice via an osmotic pump resulted in increases in hepatic MT levels compared to the levels in vehicle-treated mice after 24 hr of fasting. Only leptin infusion had no effect on hepatic MT levels. These results suggest that MT synthesis in fasting stress is not correlated with decrease in plasma leptin levels but that leptin itself is a potent inducer of MT in a fasting situation.

Early changes induced by environmental stresses in insulin sensitivity-related genes.

Although adipocytes have been shown to secrete various adipocytokines, the factors that influence them are not clearly understood. We investigated whether repeated exposure to mild stresses affects the expression of adipocytokines in white adipose tissue (WAT). Male ICR mice were exposed to repeated restraint and tail-pinch (10 min/day) for 2 weeks. The exposure to restraint and tail-pinch stresses did not cause any change in body or liver weight, decreased the weight of WAT, and resulted in a slight increase in plasma concentration of corticosterone, indicating that mild stress did not result in any changes in blood glucose and insulin. Enhanced gene expression of adiponectin, which is associated with increased insulin sensitivity, and resistin and tumor necrosis factor-α (TNF-α), which are associated with decreased insulin sensitivity, was observed in the WAT of stressed mice. These results indicate that WAT is sensitive to mild stress, which may cause changes in the expression of adipocytokines, both improving and decreasing insulin sensitivity even during the normal stage of glucose metabolism. Thus, stress may be a factor that modulates insulin sensitivity.