Induction of hepatic metallothionein synthesis by endoplasmic reticulum stress in mice.

Metallothionein (MT) is a small sulfhydryl-rich protein whose levels are elevated by various inducers of organelle stresses, such as nuclear stress (cisplatin), mitochondrial stress (antimycin A, 2,4-dinitrophenol) and lysosomal stress (paraquat). Although abnormal folding of protein in the endoplasmic reticulum (ER) causes ER stress, induction of MT synthesis by ER stress has never been investigated. In this study, we examined the induction of MT by an inducer of ER stress, tunicamycin (Tun), which induces ER stress by inhibiting N-linked glycosylation of protein in the ER. Administration of Tun (0.5-1.5 mg/kg, sc) increased hepatic MT levels in C57BL/6J mice (3.1-fold). The maximal increase in hepatic MT was observed 48-96 h after the administration of Tun (1.0 mg/kg). Expressions of MT-I, II and glucose-regulated protein 78 (Bip/GRP78), which is a molecular chaperone induced by ER stress, mRNA were also detected by administration of Tun. Thapsigargin (Thap), a generator of ER stress by inhibiting ER Ca(2+)-ATPase, also increased both hepatic MT levels and expression of MT-I and -II mRNA. The level of expression of Bip/GRP78 mRNA induced by Tun administration in MT-null mice was greater than that in wild-type mice. Taken together, these findings suggest that inhibitors of ER are potent inducers of MT.

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.

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.

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.

Inhibitory effects of tea extracts on the mutagenicity of 1-methyl-1, 2,3,4-tetrahydro-beta-carboline-3-carboxylic acid on treatment 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 major mutagen precursor in soy sauce on treatment with nitrite and ethanol, was strongly decreased by the addition of hot water extracts of green, black and oolong teas in the reaction mixture when it was treated with 50mM nitrite at pH3.0, 37 degrees C for 60min in the presence of 7.5% ethanol. The mutagenicity-decreasing activity of the teas was scarcely decreased by washing the teas with chloroform and benzene and was partly decreased by butanol and ethyl acetate. Typical polyphenols such as catechins were shown to have the antimutagenicity dose dependently. The antimutagenicity and the reducing power of tea extracts gave a positive good correlation. The results suggest that the mutagenicity of MTCCA on treatment with nitrite in the presence of ethanol may be decreased by the mixed fractions of lyophilic components such as polyphenols, which have high reducing power such as catechins and the other compounds which have little reducing power including the derivatives of the catechins and so on. Although the antimutagenicity of teas and catechins was also considerably effective when they were added after the nitrosation, that of black tea and some catechins was less effective.

Inhibitory effects of citrus fruits on the mutagenicity of 1-methyl-1,2,3,4-tetrahydro-beta-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.

Mutagenicity of 1-methyl-1,2,3,4-tetrahydro-beta-carboline-3-carboxylic acid treated with nitrite in the presence of alcohols.

The mutagenicity of a product produced from 1-methyl-1,2,3,4-tetrahydro-beta-carboline-3-carboxylic acid (MTCCA), which is a component in soy sauce, after treatment with 50 mM nitrite at pH 3, 37 degrees C, for 60 min in the presence of 7.5% ethanol was much higher than that in the absence of ethanol during the nitrite treatment. The enhancement of the mutagenicity of nitrite-treated MTCCA by ethanol required simultaneous treatment of MTCCA with nitrite and ethanol. The mutagenicity of MTCCA treated with nitrite in the presence and absence of ethanol was detected in the same fractions on HPLC and was highest for Salmonella typhimurium strain YG1029 possessing elevated O-acetyltransferase activity among the several Salmonella test strains, suggesting that the same mutagen belonging to aromatic compounds was produced both in the presence and absence of ethanol. Methanol, n-propanol and isopropanol as well as ethanol were also observed to have an augmenting effect. However, the sugars glucose and sucrose had no effect. When MTCCA was treated with nitrite in the presence of commercial alcoholic beverages equivalent to 1.25-10% ethanol, Japanese 'sake' and 'shochu' were demonstrated to have a highly augmenting effect and beer, wine, whisky and brandy to have a mildly augmenting effect.

Mutagenicity of soy sauce treated with nitrite in the presence of ethanol or alcoholic beverages.

The mutagenicity induced by soy sauce after reaction with 50 mM nitrite at pH 3, 37 degrees C, for 60 min in the presence of 1.25-10% ethanol was reduced in proportion to the ethanol concentration. The mutagenicity of soy sauce treated with nitrite was also reduced in the presence of commercial alcoholic beverages, Japanese sake, wine, 'shochu', whiskey and brandy, but not beer, in proportion to the concentration. The mutagenicity of nitrite-treated tyramine, which is a major precursor of a mutagen in soy sauce treated with nitrite, was strongly reduced in the presence of ethanol, n-propanol or isopropanol and more strongly reduced in the presence of methanol, but was increased twofold in the presence of the sugars glucose or sucrose. The reduction of the mutagenicity of nitrite-treated tyramine required simultaneous treatment of tyramine with ethanol and nitrite. The mutagenicity of tyramine treated with nitrite was clearly reduced in the presence of shochu and whiskey, similarly to ethanol. Analysis by high-performance liquid chromatography revealed that the reduction of the mutagenicity of nitrite-treated tyramine in the presence of ethanol resulted from the reduced production of mutagenic 3-diazotyramine from tyramine.

Mutagenicity and antimutagenicity of extracts of three spices and a medicinal plant in Thailand.

Three kinds of spices (caraway, coriander and black pepper seeds) and a medicinal plant called 'tong tak' in Thai (Baliospermum axillar, a species of the spurge family) were fractionated into hot water, methanol and hexane extracts. These extracts were not mutagenic for Salmonella typhimurium strains TA98 and TA100 by the Ames assay. However, when the extracts were treated with nitrite, samples of the water and methanol extracts were mutagenic for strain TA100 without metabolic activation. The mutagenicity of the nitrite-treated methanol and hot water extracts of black pepper was highest (8380 and 22,200 His+ per 0.1 g of spice powder, respectively), and that of the nitrite-treated hot water extracts of caraway and tong tak was moderate. The hot water extracts were examined for their antimutagenic activity against mutagenicity induced by various carcinogens by the Ames assay, using the preincubation technique. The tested samples (equivalent to 1-2 mg of spice powder) reduced the mutagenicity induced by 2.7 nmole (397 ng) of N-methyl-N'-nitro-N-nitrosoguanidine by more than 84%, and that induced by dimethylnitrosamine (1.48 mg) or ICR-170 (10 ng) by 30-60%. However, they did not inhibit the mutagenic activity of 1-nitropyrene, 3-nitrofluoranthene, AF-2, methyl methanesulfonate, N-ethyl-N'-nitro-N-nitrosoguanidine, 2-aminoanthracene, 2-acetylaminofluorene, benzo[a]pyrene or IQ.

Mutagenicity of nitrite-treated soy sauce in Chinese hamster V79 cells.

Mutagenicity of 4 kinds of Japanese soy sauce and tyramine, which is a precursor of a mutagen (3-diazotyramine) and present in soy sauce, after nitrite treatment under a yellow lamp was studied in Chinese hamster V79 cells with 6-thioguanine (TG) as a selective marker. The frequencies of mutation caused by nitrite-treated samples of soy sauce increased in the V79 cells in a dose-dependent manner. Four kinds of nitrite-treated soy sauce induced 139 to 473 TG-resistant mutants per 10(5) clonable cells per milliliter of soy sauce equivalent in the cells. Nitrite-treated tyramine was also mutagenic for the cells; it induced 8.6, 13.3, and 18.3 TG-resistant mutants per 10(5) clonable cells at concentrations of 20 micrograms, 56 micrograms, and 112 micrograms, respectively.

Augmenting effect of a nonmutagenic fraction in soy sauce on mutagenicity of 3-diazotyramine produced in the nitrite-treated sauce.

When 25 kinds of Japanese soy sauce at a concentration of 5% were incubated with 50mM sodium nitrite (pH 2) at 37 degrees for 1 hr, the reaction mixtures induced 34-834 (average 368 +/- 228) revertants per microliter of soy sauce equivalent in Salmonella typhimurium strain TA100 in the absence of S9 mix. The mutagen(s) formed was very unstable under natural daylight and a fluorescent lamp but quite stable under a yellow lamp as well as in the dark. In addition to the known precursors, i.e., tyramine and 1-methyl-1,2,3,4-tetrahydro-beta-carboline-3-carboxylic acid, 1-methyl-1,2,3,4-tetrahydro-beta-carboline, which caused weak mutagenesis, was found in the soy sauce. However, the sum of the activities of the three mutagen-precursors after nitrite treatment accounted for only a part of the mutagenicity of nitrite-treated soy sauce. There was in the soy sauce a factor which increased ninefold the mutagenicity of nitrite-treated tyramine, 3-diazotyramine. Therefore, tyramine was considered the principal precursor of the mutagen produced in the nitrite-treated soy sauce. These three precursors together with the mutagenicity augmentation accounted for all the mutagenicity of nitrite-treated sauce. The mutagenicity-augmenting factor in the soy sauce was nonmutagenic before and after nitrite treatment and was stable to heat and light irradiation.

Ultrastructure of the radial components of the mouse optic nerve and its changes during Wallerian degeneration.

Radial components of the optic nerve of the mouse were studied by using thin sections and freeze-fracture replicas. The investigations were performed on normal optic nerves, as well as on those undergoing Wallerian degeneration following eyeball enucleation. Normal radial components in thin sections were observed as a series of light lines composed of small electron-lucent dots situated in the interperiod lines across the myelin sheath. They are frequently found in those parts of the myelin sheath lying near the outer and inner processes of the oligodendroglia. Radial components in freeze-fracture replica were observed as a parallel array of many ridges composed of a row of particles. The particles of radial components located in the deeper part of the myelin sheath lose their linear arrangement and fall into disorder in a relatively early post-operative period. The parallel array of rows of particles located closely beneath the outer processes of the oligodendroglia remained intact for a long period, even in a markedly distorted myelin sheath. The present observations suggest that the radial components are resistant against the disintegration of the myelin lamellae during Wallerian degeneration.