Inhibitory effect of CDA-II, a urinary preparation, on aflatoxin B(1)-induced oxidative stress and DNA damage in primary cultured rat hepatocytes.

The effects of CDA-II (cell differentiation agent II; a urinary preparation) on both aflatoxin B(1) (AFB(1))-induced cell injury and DNA damage were investigated using cultured rat hepatocytes. CDA-II was able to suppress both the lipid peroxidation and lactate dehydrogenase leakage induced by AFB(1). Glutathione (GSH) depletion by AFB(1) was replenished by CDA-II treatment. Under these experimental conditions, CDA-II enhanced the activity of GSH peroxidase, but not GSH S-transferase. By evaluation of unscheduled DNA synthesis, CDA-II reduced AFB(1)-induced DNA damage in hepatocyte cultures. These findings suggest that CDA-II can inhibit cytotoxicity of AFB(1) through enhancing the activity of GSH peroxidase and preventing GSH depletion.

Synergistic effects of nicotine on arecoline-induced cytotoxicity in human buccal mucosal fibroblasts.

Areca quid chewing has been linked to oral submucous fibrosis and oral cancer. Arecoline, a major areca nut alkaloid, is considered to be the most important etiologic factor in the areca nut. In order to elucidate the pathobiological effects of arecoline, cytotoxicity assays, cellular glutathione S-transferase (GST) activity and lipid peroxidation assay were employed to investigate cultured human buccal mucosal fibroblasts. To date, there is a large proportion of areca quid chewers who are also smokers. Furthermore, nicotine, the major product of cigarette smoking, was added to test how it modulated the cytotoxicity of arecoline. At a concentration higher than 50 microg/ml, arecoline was shown to be cytotoxic to human buccal fibroblasts in a dose-dependent manner by the alamar blue dye colorimetric assay (P<0.05). In addition, arecoline significantly decreased GST activity in a dose-dependent manner (P<0.05). At concentrations of 100 microg/ml and 400 microg/ml, arecoline reduced GST activity about 21% and 46%, respectively, during a 24 h incubation period. However, arecoline at any test dose did not increase lipid peroxidation in the present human buccal fibroblast test system. The addition of extracellular nicotine acted synergistically on the arecoline-induced cytotoxicity. Arecoline at a concentration of 50 microg/ml caused about 30% of cell death over the 24 h incubation period. However, 2.5 mM nicotine enhanced the cytotoxic response and caused about 50% of cell death on 50 microg/ml arecoline-induced cytotoxicity. Taken together, arecoline may render human buccal mucosal fibroblasts more vulnerable to other reactive agents in cigarettes via GST reduction. The compounds of tobacco products may act synergistically in the pathogenesis of oral mucosal lesions in areca quid chewers. The data presented here may partly explain why patients who combined the habits of areca quid chewing and cigarette smoking are at greater risk of contracting oral cancer.

Disulfide bonds stabilize JC virus capsid-like structure by protecting calcium ions from chelation.

To investigate the role of disulfide bonds in the capsid structure, a recombinant JC virus-like particle (VLP) was used. The major capsid protein, VP1, of the JC virus was expressed in yeast cells. The yeast-expressed VP1 was self-assembled into a VLP. Disulfide bonds were found in the VLP which caused dimeric and trimeric VP1 linkages as demonstrated by non-reducing SDS-PAGE. The VLP remained intact when disulfide bonds were reduced by dithiothreitol. The VLP without disulfide bonds could be disassembled into capsomeres by EGTA alone, but those with disulfide bonds could not be disassembled by EGTA. Capsomeres were reassembled into VLPs in the presence of calcium ions. Capsomeres formed irregular aggregations instead of VLPs when treated with diamide to reconstitute the disulfide bonds. These results indicate that disulfide bonds play an important role in maintaining the integrity of the JC VLP by protecting calcium ions from chelation.

Effect of diallyl sulfide and diallyl disulfide, the active principles of garlic, on the aflatoxin B(1)-induced DNA damage in primary rat hepatocytes.

The objective of this study was to investigate the effect of the active principles in garlic-- diallyl sulfide (DAS) and diallyl disulfide (DADS)--on aflatoxin B(1) (AFB(1))-induced DNA damage in primary rat hepatocytes. Primary rat hepatocytes, induced with DNA damage using 10 microM AFB(1) were used as an experimental model. According to the results of LDH leakage, 0.5 and 2 mM of DAS or 0.5 and 1 mM of DADS significantly increased the viability of hepatocytes compared with the AFB(1) controls after 4, 8 and 24 h treatment (P<0.05). According to the results of unscheduled DNA synthesis (UDS) test, 0.5 and 2 mM of DAS or 0.5 and 1 mM of DADS could significantly decrease the DNA damage induced by AFB(1) (P<0.05). Furthermore, 0.5 and 2 mM DAS or 0.5 and 1 mM DADS could increase the glutathione S-transferase (GST) and glutathione peroxidase (GPx) activities as compared with the AFB(1) controls after 24 h treatment (P<0.05). Results of immunoblot analysis of cytosolic GST isoenzyme indicate that the levels of GST isoform Ya, Yb2 and Yc were markedly increased after treatment with 0.5 and 2 mM DAS or 0.5 and 1 mM DADS compared with the AFB(1) control. These results indicate that 0.5 and 2 mM DAS or 0.5 and 1 mM DADS might protect hepatocytes from AFB(1)-induced DNA damage via increasing the activities of GST and GPx.

Cytotoxicity and arecoline mechanisms in human gingival fibroblasts in vitro.

Betel nut chewing, like cigarette smoking, is a popular oral habit which impinges on the daily lives of a population of approximately 200 million. People who chew betel nuts have a higher prevalence of periodontal diseases than those who do not. Many of the undesirable effects of betel nuts have been attributed to arecoline, a major component of the particular alkaloid in betel nuts. In this in vitro study, we have focused on the effects of arecoline and the role it could play in periodontal breakdown via its direct effects on human gingival fibroblasts. Human gingival fibroblasts were derived from three healthy individuals undergoing crown-lengthening procedures. We found that arecoline is cytotoxic to human gingival fibroblasts at a concentration higher than 50 micrograms/ml by depleting intracellular thiols and inhibiting mitochondrial activity (P < 0.05). In addition, the cells displayed a marked arrest at G2/M phase in a dose-dependent manner. Repeated and long-term exposure to arecoline could impair the gingival fibroblast functions. As they are cytotoxic, the use of betel nut products in conjunction with periodontal therapy may interfere with optimal healing and/or lead to further periodontal breakdown.

Dietary fat and garlic oil independently regulate hepatic cytochrome p(450) 2B1 and the placental form of glutathione S-transferase expression in rats.

The individual and combined effects of dietary fat and garlic oil on two drug-metabolizing enzymes, cytochrome P(450) 2B1 and the placental form of glutathione (GSH) S-transferase (PGST), in rat liver were examined in this study. Rats were fed a low corn oil, high corn oil or high fish oil diet and received various amount of garlic oil (0, 30, 80, 200 mg/kg body) orally three times per week for 6 wk. The fat energy in the low and high fat diets accounted for 11.6 and 45.7% of total energy, respectively. Final body weights did not differ among the three dietary fat groups and were not affected by garlic oil treatment. The fatty acid profile in hepatic phospholipids revealed higher eicosapentaenoic acid [20:5(n-3)] and docosahexaenoic acid [22:6(n-3)] levels in the fish oil-fed group than in the low and high corn oil-fed groups (P < 0.05). In contrast, the corn oil-fed groups had greater hepatic phospholipid arachidonic acid [20:4(n-6)] levels (P < 0.05). Both dietary fat and garlic oil significantly affected hepatic cytochrome 7-pentoxyresorufin O-dealkylase (PROD) activity and GST activity toward ethacrynic acid. Rats fed the high fish oil diet had 85 and 51% higher PROD activity compared with those fed the low or the high corn oil diet, respectively (P < 0.05). The GST activity in the high fish oil and the high corn oil groups was 33 and 18% higher than that in the low corn oil group (P < 0.05), respectively, and the GST activity in rats fed the high fish oil diet was higher than in those fed the high corn oil diet (P < 0.05). Garlic oil dose-dependently increased GST activity. No interaction between dietary fat and garlic oil on PROD or GST activity was noted. Northern and Western blot analysis revealed that dietary fish oil increased both cytochrome P(450) 2B1 and PGST mRNA and protein levels. Cytochrome P(450) 2B1 and PGST mRNA and protein levels were also dose-dependently increased by garlic oil treatment. The effects of garlic oil and dietary fat on P(450) 2B1 and PGST mRNA and protein expression were independent. These results indicate that dietary fat and garlic oil independently modulate P(450) 2B1 and PGST expression at transcriptional and/or post-transcriptional stages.

Effects of organosulfur compounds from garlic oil on the antioxidation system in rat liver and red blood cells.

The modulation of garlic oil (GO) and three allyl compounds, diallyl sulfide (DAS), diallyl disulfide (DADS) and diallyl trisulfide (DATS), on the antioxidation system in rat livers and red blood cells was examined. Rats were orally administered GO (200 mg/kg body weight), DAS (20, 80 mg/kg body weight), DADS (80 mg/kg body weight) or DATS (70 mg/kg body weight) three times a week for 6 weeks. Control rats received corn oil (2 ml/kg body weight) alone. GO, DADS and DATS treatment significantly increased the glutathione (GSH) content (48-84%) in red blood cells (P < 0.05). DATS displayed a greater enhancement than GO and DADS (P < 0.05). Hemolysis induced by tert-butyl hydroperoxide was not suppressed by GO or allyl compound treatment although higher GSH content was evident. Hepatic GSH was not influenced by garlic components. In rat livers, DADS and DATS significantly increased the activity of GSH reductase (46 and 54%, respectively) and of GSH S-transferase (GST) (63 and 103%, respectively), but decreased the GSH peroxidase activity (27 and 28%, respectively). In contrast, GSH reductase and GST activities in the DAS group, either 20 or 80 mg/kg body weight, were similar to the control group. A decrease of GSH peroxidase activity was observed in rats dosed with 80 mg/kg body weight (P < 0.05). An increase in GST activity and a decrease in GSH peroxidase activities were also noted in GO-treated rats (P < 0.05). In red blood cells, three GSH-related antioxidant enzyme activities were not affected by garlic oil and its organosulfur components. Immunoblot assay showed that, accompanying the increase in hepatic GST activity, GO, DADS, DAS (80 mg/kg body weight) and DATS increased the expression of GST Ya, Yb1 and Yc proteins. Results indicate that GO and three allyl compounds play a differential role in modulation of the GSH-related antioxidant system in rat livers and red blood cells.

Adverse effects of arecoline and nicotine on human periodontal ligament fibroblasts in vitro.

BACKGROUND, AIMS: The habit of betel nut chewing impinges on the daily lives of approximately 200 million people. Betel quid chewers have a higher prevalence of periodontal diseases than non-chewers. This study examined the pathobiological effects of arecoline, a major component of the betel nut alkaloids, on human periodontal ligament fibroblasts (PDLF) in vitro. METHOD: Cell viability, proliferation, protein synthesis, and cellular thiol levels were used to investigate the effects of human PDLF exposed to arecoline levels of 0 to 200 microg/ml. In addition, nicotine was added to test how it modulated the effects of arecoline. RESULTS: Arecoline significantly inhibited cell proliferation in a dose-dependent manner. At concentrations of 10 and 30 microg/ml, arecoline suppressed the growth of PDLF by 20% and 50% (p < 0.05), respectively. Arecoline also decreased protein synthesis in a dose-dependent manner during a 24-h culture period. A 100 microg/ ml concentration level of arecoline significantly inhibited protein synthesis to only 50% of that in the untreated control (p < 0.05). Moreover, arecoline significantly depleted intracellular thiols in a dose-dependent manner. At concentrations of 25 microg/ml and 100 microg/ml, arecoline depleted about 18% and 56% of thiols (p < 0.05), respectively. This suggests that arecoline itself might augment the destruction of periodontium associated with betel nut use. Furthermore, the addition of nicotine acted with a synergistic effect on the arecoline-induced cytotoxicity. At a concentration of 60 microg/ml, arecoline suppressed the growth of PDLF by about 33% and 5 mM nicotine enhanced the arecoline-induced cytotoxic response to cause about 66% cell death. CONCLUSION: During thiol depletion, arecoline may render human PDLF more vulnerable to reactive agents within cigarettes. Taken together, people who combine habits of betel nut chewing with cigarette smoking could be more susceptible to periodontium damage than betel nut chewing alone.

Glutamine utilization in activated lymphocytes from rats receiving endotoxin.

BACKGROUND: A beneficial effect of supplemental glutamine for lymphocyte function in patients under metabolic stress has been suggested. Nevertheless, it is not clear how glutamine is used by lymphocytes when under stress. This time course study investigated the effect of endotoxin-induced stress on in vitro glutamine utilization and glutamine-dependent proliferation of activated lymphocytes. METHODS: Metabolic stress was modeled by intraperitoneal (ip) administration of endotoxin (5 mg/kg body wt) to rats. Control animals were injected with sterile saline. Cervical lymph node lymphocytes collected from animals 6, 12, 24, and 48 h following injection were activated with concanavalin A. Proliferation of these activated lymphocytes in the presence of 0.1-2 mM glutamine was determined. The glutamine utilization rate and glutaminase activity in the activated lymphocytes were also determined. RESULTS: The proliferation rate of lymphocytes was not affected by ip administration of endotoxin 6 h following the insult, however, 12, 24, and 48 h following the insult, the maximal response was suppressed (P < 0.05). In addition, at 12, 24, and 48 h, the concentration of glutamine for the maximal response of lymphocytes was lower than that for the control group (P < 0.05). Throughout the investigation period, both the glutamine utilization rate and glutaminase activity in the activated lymphocytes were decreased time-dependently. CONCLUSION: The present study demonstrates that glutamine utilization by lymphocytes under a mitogenic challenge in vitro is significantly decreased in the late period after endotoxin injection. This is at least partly due to decreased glutaminase activity and is associated with decreased proliferation rate of mitogen-activated lymphocytes.

Root canal sealers cause cytotoxicity and oxidative damage in hepatocytes.

Elutable substances and degradation products from root canal sealers may gain access to periodontal tissue in many ways. The aim of this study was to investigate damage and oxidative stress caused by treatment with root canal sealers in primary cultured rat hepatocytes. Four root canal sealers were used: Canals, Sealapex, AH 26, and AH Plus. Toxicity was detected by assessing lactate dehydrogenase (LDH) leakage, and oxidative stress was determined by analysis of glutathione (GSH) levels. One-way analysis of variance was performed on the data. As the concentrations of sealer were increased, LDH leakage increased (p < 0.01). Increased GSH depletion accompanied LDH leakage increase. GSH levels were reduced to zero at a concentration of 0.05% for Canals and 0.10% for AH 26. These results confirmed the hypothesis that root canal sealers can cause cytotoxicity in hepatocytes. The Canals and AH 26 sealers can also cause oxidative stress in hepatocytes.

Prostaglandin E2 is involved in the increase of cytochrome P-450 2B1 expression by alpha-tocopheryl succinate in primary rat hepatocytes in the presence of phenobarbital.

The modulation of cytochrome P-450 2B1 expression by alpha-tocopheryl succinate and whether prostaglandin E2 is involved in this modulation in primary rat hepatocytes in the presence of phenobarbital were investigated. A primary rat hepatocyte culture model that faithfully reproduces the phenobarbital response observed in vivo was used. Intracellular alpha-tocopherol content was dose dependently increased by alpha-tocopheryl succinate incubation. Hepatocytes were demonstrated to have prostaglandin E2-synthesizing capability. alpha-Tocopheryl succinate inhibited prostaglandin E2 synthesis by hepatocytes and increased cytochrome P-450 2B1 expression in the presence of phenobarbital; however, it had little effect on intracellular cAMP level. To mimic the exogenous source of prostaglandin E2 from nonparenchymal cells, various concentrations of prostaglandin E2 were added to the cell culture. High doses of exogenous prostaglandin E2 (100 and 1,000 nM) inhibited the cytochrome P-450 2B1 expression in the presence of phenobarbital compared with low doses (1 and 10 nM); however, the presence of high doses of prostaglandin E2 had no effect on intracellular cAMP level. Forskolin significantly increased intracellular cAMP level and inhibited cytochrome P-450 2B1 expression in the presence of phenobarbital. The results of this study indicate that alpha-tocopheryl succinate increases cytochrome P-450 2B1 expression via its inhibition of prostaglandin E2 synthesis in the presence of phenobarbital; however, changes in intracellular cAMP level are not related to cytochrome P-450 2B1 expression.

Inhibition of tert-butyl hydroperoxide-induced cell membrane bleb formation by alpha-tocopherol and glutathione.

Multiple bleb formation on cell membrane is common during cell death. The effects of alpha-tocopherol and glutathione (GSH) on tert-butyl hydroperoxide (TBH)-induced membrane changes in rat hepatocytes were studied. Over 60 min of exposure, TBH (0.5-2.0 mM) caused a dose-dependent membrane blebbing. Cells pretreated with buthionine sulfoximine, a GSH synthesis inhibitor, had significantly greater blebbing and lactate dehydrogenase (LDH) leakage under 0.5 mM TBH treatment as compared to cells without pretreatment. However, the protective effect of GSH disappeared when the TBH concentration was increased to 2.0 mM. In the presence of alpha-tocopheryl succinate (TS) pretreatment, it was noted that bleb formation, expressed as the percentage of cells bearing blebs, the average bleb size, or the onset of blebbing, was partially suppressed even when TBH concentration was 2.0 mM. TBH-induced thiobarbituric acid reactive substances and LDH leakage were completely abolished by TS pretreatment. Accompanying bleb formation, membrane-insoluble actin was noted to decrease by immunoblot assay. The decrease in actin was also suppressed by TS. These results indicated that intracellular GSH and alpha-tocopherol status are important to the TBH-induced cell membrane abnormality. Furthermore, TS plays a defensive role against blebbing when GSH is exhausted by TBH.

Comparison of the effect of fish oil and corn oil on chemical-induced hepatic enzyme-altered foci in rats.

The effects of fish oil and corn oil diets on diethylnitrosamine initiation/phenobarbital promotion of hepatic enzyme-altered foci in female Sprague-Dawley rats were investigated. Groups of 12 rats were initiated with diethylnitrosamine (15 mg/kg) at 24 h of age. After weaning, they received diets containing either 13.5% fish oil plus 1. 5% corn oil or 15% corn oil for 24 weeks. Rats fed fish oil had significantly greater liver weight, relative liver weight, spleen weight, and relative spleen weight than rats fed corn oil (p < 0.05). Hepatic phospholipid fatty-acid profile was significantly affected by the type of dietary lipid. The rats fed fish oil had significantly greater hepatic phospholipid 20:5 and 22:6 than rats fed corn oil; in contrast, the rats fed corn oil had significantly greater hepatic phospholipid 18:2 and 20:4 than rats fed fish oil (p < 0.05). Rats fed fish oil had significantly lower hepatic vitamin E and PGE(2) content but significantly greater hepatic lipid peroxidation than rats fed corn oil (p < 0.05). The hepatic levels of antioxidant enzymes (GSH reductase and GST) were significantly greater in rats fed fish oil than in rats fed corn oil (p < 0.05). Except for PGST-positive foci (foci area/tissue area), all the other foci parameters (GGT-positive foci area/tissue area, GGT-positive foci no./cm(2), GGT-positive foci no./cm(3), PGST-positive foci no. /cm(2), and PGST-positive foci no./cm(3)) measured in the fish oil group were 10-30% of those in the corn oil group (p < 0.05). Analyses of Pearson correlation coefficient revealed a positive correlation between hepatic GGT- or PGST-positive foci number (no. /cm(2)) and PGE(2) content (r = 0.66, P = 0.01; r = 0.56, P = 0.02, respectively) but a negative correlation between GGT- and PGST-positive foci (no./cm(2)) and lipid peroxidation (r = -0.8, P = 0.0006; r = -0.58, P = 0.01, respectively), GSH/(GSH + GSSG) ratio (r = -0.61, P = 0.05; r = -0.4, P = 0.14, respectively), GSH reductase (r = -0.75, P = 0.002; r = -0.53, P = 0.02, respectively), and GST activities (r = -0.65, P = 0.01; r = -0.44, P = 0.07, respectively). Similar correlation between foci number (no./cm(3)) and PGE(2), lipid peroxidation, GSH/(GSH + GSSG) ratio, GSH reductase, and GST activities were obtained. The results of this study show that dietary fish oil significantly inhibited hepatic enzyme-altered foci formation compared with corn oil in rats. These results suggest that the possible mechanisms involved in this process are the stimulation of hepatic detoxification system, changes in membrane composition, inhibition of PGE(2) synthesis, the enhancement of GSH-related antioxidant capacity, and the enhancement of lipid peroxidation by fish oil.

Autoantibody against oxidized low-density lipoproteins may be enhanced by cigarette smoking.

A total of 59 healthy male subjects (32 smokers and 27 nonsmokers) who had no reported systemic disease and did not take alcohol and vitamin supplementation were included. The levels of autoantibody to oxidized low-density lipoproteins (ox-LDL) in smokers and age-matched nonsmokers were compared. The plasma levels of antioxidants that can affect the formation of ox-LDL were also measured, and correlation analyses between anti ox-LDL IgG and plasma antioxidants, controlling for age and body mass index (BMI), were performed. Plasma alpha-tocopherol and uric acid concentrations of nonsmokers (2.78+/-1.09 microg/mg total lipid and 6.96+/-1.69 mg/dl, respectively) were significantly higher than those of smokers (1.68+/-0.48 microg/mg total lipid and 6.15+/-1.14 mg/dl, respectively) (P<0.05). Although plasma ascorbate and retinol levels were not significantly different between smokers and nonsmokers, smokers older than 45 years old had significantly lower plasma ascorbate levels (0.32+/-0.17 mg/dl) than age-matched nonsmokers (0. 53+/-0.14 mg/dl) (P=0.036). Higher level of plasma anti ox-LDL IgG was noted in the group of smokers compared with nonsmokers (515+/-409 mU/ml vs. 407+/-268 mU/ml, respectively) under the statistic method of Chi-Square test (P=0.049). A significant negative correlation was found between plasma anti ox-LDL IgG and alpha-tocopherol in the combined population as well as in the smoker group (r=-0.26, p=0.047; r=-0.48, p=0.006; respectively). However, there was no correlation between plasma anti ox-LDL IgG and the levels of other antioxidants. These results suggest that reduced concentrations of alpha-tocopherol are associated with cigarette smoking. The significantly negative correlation between plasma anti ox-LDL IgG and alpha-tocopherol in the entire study population as well as in the smoker group suggests that plasma alpha-tocopherol may be partially effective if not totally at protecting LDL from oxidative damage caused by cigarette smoking and dietary supplementation with alpha-tocopherol may provide a protective effect against LDL oxidation, especially in smokers.

Lactate dehydrogenase leakage of hepatocytes with AH26 and AH Plus sealer treatments.

Numerous root canals filling materials are available in the field of dentistry, based on various formulas that contain a variety of different and partly mutagenic components, such as epoxy resin sealers, Ca(OH)2-based materials, and zinc oxide-eugenol cements. AH Plus root canal sealer will not release formaldehyde according to the manufacturer, although AH26 does. The purpose of this study was to analyze the leakage of lactate dehydrogenase (LDH) from rat hepatocytes after treatment with AH26 and AH Plus root canal sealers in vitro. Hepatocytes from male Sprague-Dawley rats were used to test the cytotoxicity of AH26 and AH Plus. The root canal sealers were mixed and then dissolved in the dimethyl sulfoxide to final concentrations of 0.01%, 0.04%, and 0.1% (wt/vol), with a dimethyl sulfoxide concentration of < 0.05%. Dosage-dependent and time-dependent lactate dehydrogenase leakage values were measured and tested by one-way ANOVA. The results showed that both AH26 and AH Plus are toxic to rat hepatocytes. At a low concentration, AH26 had a higher toxicity than AH Plus to rat hepatocytes.

Suppression of altered hepatic foci development by a high fish oil diet compared with a high corn oil diet in rats.

Effects of low corn oil, high corn oil, and high fish oil diets on altered hepatic foci development in female Sprague-Dawley rats were investigated. Rats assigned to Groups 1-4 were initiated with saline as the control and those assigned to Groups 5-7 were initiated with diethylnitrosamine (DEN 15 mg/kg) at 24 hours of age. After weaning, all rats, except those in Group 1, received 500 ppm phenobarbital (PB) in their diet as tumor promoter for three months. Altered hepatic foci development was significantly lower in DEN-initiated rats fed the high fish oil + PB diet than in DEN-initiated rats fed the high corn oil + PB diets. Liver weight and relative liver weight were significantly greater in rats fed the high fish oil + PB diet than in rats fed the other diets, and hepatic biotransformation/detoxification enzyme activities were greater in rats fed the fish oil + PB diets than in rats fed the other diets. These results suggest that the effect of a high fish oil diet on altered hepatic foci may occur through regulation of hepatic biotransformation/detoxification enzyme activities, leading to alteration in the tumor-promoting action of PB. Dietary lipid significantly affected the hepatic phospholipid fatty acid composition of rats. n-3 polyunsaturated fatty acids were incorporated into membrane phospholipid at the expense of n-6 polyunsaturated fatty acids. A high fish oil diet caused greater oxidative stress in rats, as measured by plasma vitamin E level, red blood cell glutathione status, liver lipid peroxidation, and hepatic glutathione reductase activity. Pearson's correlation analysis indicated that the foci number was negatively correlated to the liver thiobarbituric acid-reactive substance and 7-pentoxyresorufin O-dealkylase activity, and the foci area was negatively correlated to the liver thiobarbituric acid-reactive substance activity (p < 0.05) in rats of groups that developed foci. These results suggest that the type of dietary lipid is the more important determinant for gamma-glutamyl transpeptidase-positive foci development than the amount of dietary lipid when rats consumed approximately the same amount of calories in all the dietary groups, and the underlying mechanisms may be partially ascribed to the antioxidant/oxidation status and biotransformation/detoxification system of rats.

Lack of effect of dietary alpha-tocopherol on chemically induced hepatocarcinogenesis in rats.

We investigated the effects of alpha-tocopherol on diethylnitrosamine (DEN) initiation-phenobarbital (PB) promotion of hepatic foci in female Sprague-Dawley rats. Groups of eight rats were initiated with DEN (15 mg/kg) at 24 hours of age. After weaning, they received diets containing 500 ppm PB and various concentrations of alpha-tocopherol, deficient (0 ppm), adequate (100 ppm), and supplemented (5,000 ppm), for 24 weeks. Rats fed alpha-tocopherol-supplemented diets had significantly greater hepatic alpha-tocopherol levels than those fed alpha-tocopherol-deficient or -adequate diets (p < 0.05). Liver lipid peroxidation (measured as thiobarbituric acid-reactive substances) was significantly greater in rats fed alpha-tocopherol-deficient diets than in those fed alpha-tocopherol-adequate or -supplemented diets (p < 0.05). The dietary alpha-tocopherol level had no significant effect on the ratios of reduced glutathione (GSH) to oxidized GSH or reduced GSH to total GSH in the liver or on the plasma prostaglandin E2 concentration or on the activities of hepatic cytosolic and particulate protein kinase C. Rats fed alpha-tocopherol-adequate or -supplemented diets had significantly greater hepatic glutathione S-transferase, GSH reductase, and GSH peroxidase activities than those fed alpha-tocopherol-deficient diets (p < 0.05). The dietary alpha-tocopherol level did not significantly affect the formation of hepatic gamma-glutamyl transpeptidase- and placental glutathione S-transferase-positive foci. These results suggest that alpha-tocopherol does not influence hepatic foci formation and that reactive oxygen species may not be the underlying mechanism of hepatic foci formation in this DEN initiation-PB promotion model of hepatocarcinogenesis.

Cytopathologic effects of arecoline on human gingival fibroblasts in vitro.

Arecoline, a major betel nut alkaloid, has been detected in saliva obtained during betel nut chewing in concentrations up to 140 micrograms/ml, corresponding to 0.9 mM. Arecoline in the millimolar concentration range might participate in the initiation and/or progression of periodontal disease during the long-term effects of betel nut chewing. In this study, cell growth, cell proliferation, assessment of cytoplasmic enzyme lactate dehydrogenase (LDH) and collagen synthesis were used to investigate the effects of human gingival fibroblasts exposed to arecoline levels of 0-200 micrograms/ml. Control culture exhibited a normal monolayer of long spindle-shaped fibroblast morphology. Arecoline-treated human gingival fibroblasts showed a more rounded appearance and detached at the higher concentrations. At concentrations higher than 75 micrograms/ml, many cells had detached from the surface of the petri dish and numerous floating cells could be seen under the inverted microscope. At a concentrations higher than 25 micrograms/ml, arecoline inhibited cell growth, proliferation and collagen synthesis and increased LDH leakage in a dose-dependent manner (P < 0.05). These results indicate that arecoline is a cytotoxic agent to human gingival fibroblasts. Repeated and long-term exposure to arecoline could impair gingival fibroblast function. Betel quid chewers might be more susceptible to destruction of the periodontium and less responsive to a regeneration procedures during periodontal therapy.

Affinity purification of Schistosoma japonicum glutathione-S-transferase and its site-directed mutants with glutathione affinity chromatography and immobilized metal affinity chromatography.

A C-terminally polyhistidine-tagged protein of Schistosoma japonicum glutathione-S-transferase, named as SjGST/His, and its Cys85-->Ser, Cys138-->Ser, and Cys178-->Ser site-directed mutants were prepared and highly expressed in Escherichia coli. Both immobilized metal affinity chromatography (IMAC) and glutathione (GSH) affinity chromatography were used to purify these four enzymes. All of them were purified with equal efficiency by Ni2+-chelated nitrilotriacetic acid agarose gel, but not by GSH Sepharose 4B gel. The protein amounts of wild-type and Cys85-->Ser enzymes purified by the latter gel were three to seven-fold greater than those of the other two enzymes purified by the same gel, while their specific activities were two-fold lower, presumably because of the occurrence of noncovalent aggregation. Both purification methods yielded highly pure enzymes, while there were minor amounts of inter- and intra-disulfide forms in the IMAC purified enzymes except for the Cys85-->Ser mutant. Addition of dithiothreitol to GSH-affinity purified enzymes shifted all of their mass spectra of matrix-assisted laser desorption/ionization-time of flight mass spectrometry toward low molecular-mass regions, while addition of GSH to IMAC purified enzymes shifted the spectra toward high molecular-mass regions. The shift values of wild-type enzyme were larger than those of the three mutants, indicating that the Cys85, Cys138, and Cys178 residues were S-thiolated by GSH during the GSH-affinity purification. This result was confirmed by isoelectric focusing. These findings suggest that IMAC is more efficient than the conventional GSH-affinity system for the purification of SjGST/His enzyme, especially for its mutants and fusion proteins.

Effect of garlic active principle, diallyl disulfide, on cell viability, lipid peroxidation, glutathione concentration and its related enzyme activities in primary rat hepatocytes.

This study investigated the effects of various concentrations and incubation time intervals of diallyl disulfide (DADS), the active principle of garlic, on: 1. cell viability, 2. lipid peroxidation, and 3. glutathione (GSH) concentration and its related enzyme activities of rat hepatocytes. According to the results of LDH leakage and microscopic examination, 0.5 and 1 mM DADS did not significantly affect the viability of hepatocytes. However, significant decrease in cell viability according to increased LDH leakage and significant changes in morphology of hepatocytes were observed at 2 mM DADS (P < 0.05). Lipid peroxidation was also detected when the hepatocytes were treated with 2 mM DADS. At 0.5 mM DADS, a higher GSH content was found in the hepatocytes although not at a statistically significant level. 0.5 and 1 mM DADS has little effect on the activities of glutathione-S-transferase (GST) and glutathione peroxidase (GPx); however a significant decrease in GST, GPx and glutathione reductase (GRd) activities was observed at 2 mM DADS. Once the media of 2 mM DADS was replaced with fresh medium at 24 hr treatment, the activities of GST, GRd and GPx were recovered, although they were still lower than the control values.