Production of hydrogen peroxide and methionine sulfoxide by epigallocatechin gallate and antioxidants.

Epigallocatechin gallate (EGCG) induced apoptosis-associated characteristics in human oral tumor cell lines more efficiently than ascorbates, gallic acid, vitamin K, flavonoids or steroidal saponins. Since catalase partially inhibited the cytotoxic activity of EGCG, the possible involvement of hydrogen peroxide (H2O2) in cell death induction was investigated, using TCPO chemiluminescence method. Production of H2O2 by EGCG, sodium ascorbate, gallic acid or catechin reached a maximum level within 30 minutes, and was increased up to a plateau level above pH 8. Under optimal conditions, 1 mM EGCG was converted to 1 mM H2O2. At neutral pH, EGCG produced the highest amount of H2O2, followed by gallic acid, sodium ascorbate and catechin. EGCG produced methionine sulfoxide from methionine in the culture medium, while the methionine oxidation by EGCG was significantly reduced in the presence of serum. ESR spectroscopy showed that EGCG, gallic acid and sodium ascorbate, but not catechin, produced radicals under alkaline condition and that all these compounds scavenged superoxide anion, produced by hypoxanthine-xanthine oxidase reaction. EGCG also effectively scavenged the ascorbate and gallate radicals, more efficiently than other compounds. These data suggest that the apoptosis induction by EGCG may be mediated by H2O2 produced in the culture medium.

Radical intensity and carcinogenic activity of benz[c]acridines.

Among 13 benz[c]acridines, six 7-methyl-substituted compounds (7-methylbenz[c]acridine, 7,9-dimethylbenz[c]acridine, 7,10-dimethylbenz[c]acridine, 7,11-dimethylbenz[c]acridine, 7,9,10-trimethylbenz[c]acridine, 7,9,11-trimethylbenz[c]acridine) were carcinogenic, while the other seven compounds (benz[c] acridine, 8-methylbenz[c]acridine, 9-methylbenz[c]acridine, 10-methylbenz[c]acridine, 11-methylbenz[c]acridine, 5,7-dimethylbenz[c]acridine, cis-5,6-dihydroxy-5,6-dihydrobenz[c]acridine) were inactive. Using both McLachlan-Hückel molecular orbital (McLachlan-HMO) and HMO methods, all the carcinogenic compounds were shown to have the elevated pi-spin density at 12th nitrogen atom of their molecules, as compared with noncarcinogenic compounds. Electron spin resonance (ESR) spectroscopy, however, revealed that both carcinogenic and noncarcinogenic compounds produced no detectable amounts of radical. This is in contrast to ascorbates, gallates and benzo[a]phenothiazines, which induced apoptosis by radical mediated mechanism(s). Amino acid analysis demonstrated that methionine oxidation is not involved in the induction of carcinogenic activity by benz[c]acridines.

Effect of Trolox, a synthetic analog of alpha-tocopherol, on cytotoxicity induced by UV irradiation and antioxidants.

The addition of Trolox, a synthetic analog of alpha-tocopherol, significantly reduced the cytotoxicity induced by UV irradiation and antioxidants, such as ascorbate, gallate and caffeate. Ascorbate and gallate, but not UV irradiation, stimulated the oxidation of methionine to methionine sulfoxide in the culture medium, possibly because of their prooxidant actions. Trolox slightly, but significantly reduced the methionine oxidation. On the other hand, alpha-tocopherol showed a much lower protective effect against ascorbate and gallate-induced cytotoxicity, and failed to reduce the methionine oxidation induced by these agents. ESR spectroscopy showed that both Trolox and alpha-tocopherol did not significantly change the radical intensity of ascorbate and gallate. The present study suggests that the antioxidative efficacy of Trolox surpasses that of alpha-tocopherol.

Effect of methionine depletion on growth and apoptosis in various tumor cell lines.

Sodium ascorbate, sodium 5,6-benzylidene-L-ascorbate (SBA), gallic acid and caffeic acid induced apoptotic cell death in human myelogenous leukemic cell lines, and stimulated oxidation of methionine into methionine sulfoxide in the culture medium. When various tumor cell lines were cultured in methionine-free medium, their growth was nearly terminated at G1 phase of the cell cycle, producing much smaller number of apoptotic cells. Addition of methionine sulfoxide to the methionine-free medium did not stimulate the apoptosis induction. These data suggest that induction of apoptosis by ascorbates, gallate or by caffeate cannot be simply explained by methionine oxidation.

Copper, but not iron, enhances apoptosis-inducing activity of antioxidants.

Addition of either CuCl or CuCl2 significantly enhanced sodium ascorbate or sodium 5,6-benzylidene-L-ascorbate (SBA)-induced cytotoxicity and internucleosomal DNA cleavage in human promyelocytic leukemic HL-60 cells. On the other hand, the addition of either FeCl2 or FeCl3 inhibited the cytotoxic activity of ascorbate. These effects were observed even if the cells were exposed for only 20 minutes to metals and ascorbates. Copper also stimulated the gallate or caffeate-induced apoptotic cell death, whereas iron was inhibitory. Both copper and iron enhanced the radical intensity of ascorbates, but slightly reduced the radical intensity of gallate and caffeate, suggesting that radical intensity is not the sole determinant of apoptosis induction. Metals did not significantly change the methionine oxidation stimulated by ascorbate, gallate or caffeate. Methionine oxidation may not be indispensable for antioxidant-induced apoptosis.

Methionine oxidation and apoptosis induction by ascorbate, gallate and hydrogen peroxide.

Addition of either ascorbate, gallate, caffeate or hydrogen peroxide to the culture medium stimulated the oxidation of free methionine to methionine sulfoxide, regardless of the presence or absence of the cells. In methionine-free medium, growth of human myelogenous leukemic cell lines was nearly stopped by G1 arrest, without induction of internucleosomal DNA cleavage. Methionine sulfoxide, a major oxidation product of methionine, was neither growth-promoting nor cytotoxic. On the other hand, methional, a deamination and decarboxylation product of methionine, was highly cytotoxic. The present study suggests that the apoptosis induction by these antioxidants and oxidant cannot simply be explained by methionine oxidation or depletion.

[Distribution of free amino acids and related compounds in ocular fluids of rat--I. Aging and inherited cataracts].

The distribution of free amino acids and their related compounds has been determined in the aqueous humors of Wistar strain and Ihara cataract f-strain (ICR) aging rats. Taurine was the most abundant amino acid in aqueous humors except in ICR rats of 16 and 70 weeks. It was supposed that the increase of serine and glutamine, and the decrease of aspartate, proline and glycine in aqueous humors were related to aging. There were interesting changes in amino acids related to opacity of lens, concentration of taurine was lower than that of Wistar rats in ICR rats of 4, 16, and 70 weeks, alanine and citrulline increased in Wistar rats and decreased in ICR rats, and histidine increased in ICR rats with aging. The changes in free amino acids in aqueous humors were the greatest in ICR rats, and these data will provide useful clues for the formation of cataract and the transportation of amino acids.

[Quantitative estimation of the relationship between amino acids and cataracts in rat lens].

The concentration of free amino acids and their related compounds has been determined in the lenses of ICR (f) strain rat and in the Wistar strain rat's lenses which were cultured with diethyl maleate. It was supposed that the decrease of cystathionine and the increase of serine in lenses of ICR with aging were related with development of senile cataracts. The increase of cystathionine in lenses cultured were suggested that synthesis of taurine is done by cystathionine pathway. Quantitative changes of amino acids were higher than normal of glutamine, glycine and aspartate in lenses cultured. It was supposed that the changes were the flow in lens from medium for synthesis of glutathione and glucose.

Purification and properties of steroid sulfatase from human placenta.

Steroid sulfatase was purified approximately 170-fold from normal human placental microsomes and properties of the enzyme were investigated. The major steps in the purification procedure included solubilization with Triton X-100, column chromatofocusing, and hydrophobic interaction chromatography on phenylsepharose CL-4B. The purified sulfatase showed a molecular weight of 500-600 kDa on HPLC gel filtration, whereas the enzyme migrated as a molecular mass of 73 kDa on sodium dodecyl sulfate polyacrylamide gel electrophoresis. The isoelectric point of steroid sulfatase was estimated to be 6.7 by isoelectric focusing in polyacrylamide gel in the presence of 2% Triton X-100. The addition of phosphatidylcholine did not enhance the enzyme activity in the placental microsomes obtained from two patients with placental sulfatase deficiency (PSD) after solubilization and chromatofocusing. This result indicates that PSD is the result of a defect in the enzyme rather than a defect in the membrane-enzyme structure. Amino acid analysis revealed that the purified human placental sulfatase did not contain cysteine residue. The Km and Vmax values of the steroid sulfatase for dehydroepiandrosterone sulfate (DHA-S) were 7.8 microM and 0.56 nmol/min, while those for estrone sulfate (E1-S) were 50.6 microM and 0.33 nmol/min, respectively. The results of the kinetic study suggest the substrate specificity of the purified enzyme, but further studies should be done with different substrates and inhibitors.

Detection of the exercise-associated changes in serum proteins by two-dimensional electrophoresis under non-denaturing conditions.

The changes in serum proteins caused by physical exercise (10-km run) were examined using two-dimensional electrophoresis under non-denaturing conditions. Two specific proteins were found to increase remarkably in amount. Both proteins had a slightly higher molecular mass than albumin, which suggests that they are albumin-bound with large amounts of sugars or lipids. However, these proteins were not adsorbed on an anti-albumin affinity column, and they were not stained by either periodic acid-Schiff base or Sudan Black. The molecular mass was determined to be 25,000 by sodium dodecyl sulphate polyacrylamide gel electrophoresis. The changes of the specific proteins in competitors in a triathlon race were also examined.

Major pathway for putrescine synthesis induced by 1 alpha,25-dihydroxyvitamin D3 in chick duodenum.

We have reported that a single injection of 1 alpha,25-dihydroxyvitamin D3 into vitamin D-deficient chicks produces a marked accumulation of putrescine in the duodenum by an interconversion pathway. In the present study, we examined the effect of N1,N2-bis(2,3-butadienyl)-1,4-butanediamine, a specific irreversible inhibitor of polyamine oxidase, on the duodenal putrescine synthesis induced by 1 alpha,25-dihydroxyvitamin D3. Addition of N1,N2-bis(2,3-butadienyl)-1,4-butanediamine to an assay mixture completely inhibited the activity of duodenal polyamine oxidase in vitro. Prior administration of N1,N2-bis(2,3-butadienyl)-1,4-butanediamine to chicks completely blocked the 1 alpha,25-dihydroxyvitamin D3-induced increase in duodenal accumulation of putrescine in vivo. The increase of the duodenal accumulation of putrescine by 1 alpha,25-dihydroxyvitamin D3 in vitamin D-deficient chicks coincided quantitatively with the amount of N1-acetylspermidine synthesized from spermidine after the injection of the vitamin into the chicks pretreated with the inhibitor of polyamine oxidase. These results clearly indicate that spermidine N1-acetyltransferase plays a preferential role in the increase in duodenal putrescine synthesis by 1 alpha,25-dihydroxyvitamin D3. The rapidly proliferating and maturing epithelium of small intestines will provide a good model for investigating the role of the interconversion of polyamine metabolism in cell growth and differentiation.

Spermidine N1-acetyltransferase has a larger role than ornithine decarboxylase in 1 alpha,25-dihydroxyvitamin D3-induced putrescine synthesis.

We have reported that a single injection of 1 alpha,25-dihydroxyvitamin D3 (1 alpha,25(OH)2D3), the active form of vitamin D3, into vitamin D-deficient chicks produces a marked increase in the formation of duodenal putrescine by two pathways, one from ornithine and one from spermidine (Shinki, T., Takahashi, N., Kadofuku, T., Sato, T., and Suda, T. (1985) J. Biol. Chem. 260, 2185-2190). In this work, the conversion of [3H]ornithine into [3H]putrescine catalyzed by ornithine decarboxylase was compared with the conversion of [14C]spermidine into [14C]putrescine catalyzed by spermidine N1-acetyltransferase and polyamine oxidase. Using the in situ duodenal loop method in the presence or absence of alpha-difluoromethylornithine, we evaluated the relative contributions of these two pathways in the 1 alpha,25(OH)2D3-induced duodenal synthesis of putrescine. Prior administration of alpha-difluoromethylornithine inhibited neither the 1 alpha,25(OH)2D3-induced increase in duodenal spermidine N1-acetyltransferase activity nor the vitamin-induced enhancement of the duodenal putrescine content, although it completely suppressed the duodenal ornithine decarboxylase activity induced by 1 alpha,25(OH)2D3. The duodenal content of spermidine decreased time-dependently after injection of 1 alpha,25(OH)2D3. The increase of duodenal putrescine by 1 alpha,25(OH)2D3 coincided quantitatively with the amount of putrescine synthesized from spermidine but not from ornithine after injection of the vitamin. These unexpected results clearly indicate that spermidine N1-acetyltransferase has a larger role than ornithine decarboxylase in the increase of duodenal putrescine synthesis induced by 1 alpha,25(OH)2D3. The polyamine metabolism reported here may be related to the characteristics of intestinal epithelial cells such as the short lifetime (90-108 h) and typical gradient of differentiation from the crypt to villus regions.

Detection of the changes in cellular proteins in regenerating rat liver by high-resolution two-dimensional electrophoresis.

The changes in cellular proteins in regenerating rat liver after partial hepatectomy were examined by high-resolution two-dimensional electrophoresis. The cellular proteins in regenerating rat livers were separated into two fractions (soluble and insoluble protein fractions) and the proteins in each fraction were analysed by means of two-dimensional electrophoresis. A rapid increase in three proteins and a rapid decrease in two proteins were detected after partial hepatectomy. The changes in these proteins were in parallel with the regeneration rate of liver, suggesting a close relationship with the proliferation of liver after partial hepatectomy.

Induction of spermidine N1-acetyltransferase by 1 alpha,25-dihydroxyvitamin D3 as an early common event in the target tissues of vitamin D.

We have reported that the duodenal ornithine decarboxylase activity and the tissue content of putrescine increase markedly after a single intravenous injection of 1 alpha,25-dihydroxyvitamin D3 (1 alpha,25(OH)2D3) into vitamin D-deficient chicks (Shinki, T., Takahashi, N., Miyaura, C., Samejima, K., Nishii, Y., and Suda, T. (1981) Biochem. J. 195, 685-690). In the present study, we examined in the same experimental system the effect of 1 alpha,25(OH)2D3 on the activity of duodenal spermidine N1-acetyltransferase, a rate-limiting enzyme catalyzing the conversion from spermidine to putrescine. The duodenal spermidine N1-acetyltransferase activity began to increase 30 min after a single intravenous injection of 625 ng of 1 alpha,25(OH)2D3 and attained a maximum in 2 h. As little as 1.25 ng of 1 alpha,25(OH)2D3 induced a small but significant increase in the spermidine N1-acetyltransferase activity, and the maximal response was obtained by 125 ng of the vitamin. The dose levels of 1 alpha,25(OH)2D3 required to induce duodenal spermidine N1-acetyltransferase activity were only one-tenth as much as those required to induce ornithine decarboxylase activity. The spermidine N1-acetyltransferase activity was induced commonly in the target tissues of vitamin D, whereas ornithine decarboxylase activity occurred only in intestine. The 1 alpha,25(OH)2D3-induced spermidine N1-acetyltransferase activity was greatly inhibited by prior administration of actinomycin D or cycloheximide. When 30,000 X g intestinal supernatants were incubated with [14C]spermidine, [14C]putrescine was formed. These results clearly indicate that the induction of spermidine N1-acetyltransferase activity is the 1 alpha,25(OH)2D3-induced earliest de novo synthesis in several proteins induced by the vitamin reported to date and that the 1 alpha,25(OH)2D3-induced duodenal synthesis of putrescine occurs by the pathways from both ornithine and spermidine.

Detection of changes in rabbit serum proteins after partial hepatectomy by means of two-dimensional electrophoresis under non-denaturing conditions.

The changes in rabbit serum proteins after partial hepatectomy were examined by means of two-dimensional electrophoresis utilizing isoelectric focusing in a 4% polyacrylamide gel in the first dimension and a 4-30% pore gradient polyacrylamide gel in the second dimension. A rapid increase in seven proteins was observed after partial hepatectomy and a rapid decrease in two proteins. Major serum proteins, including albumin, immunoglobulin G, immunoglobulin M and alpha 2-macroglobulin, did not change. The time course of the changes was examined using a densitometer; the maxima of the changes were observed on day 3 after partial hepatectomy.

Increase of transferrin receptors in regenerating rat liver cells after partial hepatectomy.

The change of transferrin receptors in regenerating rat liver cells after partial hepatectomy was demonstrated. The binding of 125I-labeled transferrin to the cells began to increase after 24 h of partial hepatectomy and reached about double that of the non-operated normal rat liver cells. A Scatchard analysis of binding parameters showed 1.62 X 10(5) (Ka: 1.04 X 10(7) M-1) in normal cells and 3.36 X 10(5) (Ka: 1.23 X 10(7) M-1) in regenerating cells. This reflected on increase of transferrin receptor number and little change occurred in the binding affinity between transferrin and its surface receptor.

Detection of the changes in protein distribution in rat serum after partial hepatectomy using two-dimensional electrophoresis under non-denaturing conditions.

The changes in rat serum protein distribution after partial hepatectomy were examined using two-dimensional electrophoresis, utilizing isoelectric focusing in polyacrylamide gel in the first dimension and pore gradient polyacrylamide gel electrophoresis in the second dimension. Drastic decreases in amount of protein were observed at more than twenty spot positions, and drastic increases in amount or newly appeared proteins were observed at eight spot positions. The amounts of albumin, immunoglobulin M and alpha 2-macroglobulin did not change relatively after hepatectomy. The time course of the changes was examined using a densitometer, and it was observed that almost all the serum proteins changed drastically at 24 h after hepatectomy.