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The mechanism of toxicity of structurally diverse environmental toxicants including heavy metals and polyhalogenated and polycyclic hydrocarbons may involve a common cascade of events which entails an oxidative stress and production of reactive oxygen species. We have determined the comparative effects of single 0.01, 0.10 and 0.50 LD(50) doses of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), endrin, naphthalene and sodium dichromate (chromium VI) on lipid peroxidation, DNA fragmentation and enhanced production of superoxide anion (cytochrome c reduction) in liver and brain tissues of C57BL/6NTac mice. The effects of a single acute oral 0.50 LD(50) dose of these xenobiotics on hepatic and brain lipid peroxidation were investigated at 0, 12, 24, 48, and 96 h after treatment, while the effects of 0.10 LD(50) and 0.01 LD(50) doses of these xenobiotics were at 0, 24, 48, 72, and 96 h after treatment. Dose- and time-dependent effects were observed with all four xenobiotics. At a 0.50 LD(50) dose of TCDD, endrin, naphthalene and chromium VI, maximum increases in cytochrome c reduction (superoxide anion production) of approximately 5.7-, 5.4-, 5.3- and 4.1-fold, respectively, were observed in hepatic tissues. TCDD showed an increasing effect through 96 h. Endrin and naphthalene demonstrated a maximum effect at 12-24 h, while chromium VI exhibited a maximum effect at 48 h. With respect to lipid peroxidation, at a 0.50 LD(50) dose both endrin and chromium VI induced the maximum effect at 48 h of treatment, while naphthalene demonstrated the maximum effect after 24 h of treatment. TCDD demonstrated a continued effect through 96 h of treatment. At a 0.50 LD(50) dose TCDD, endrin, naphthalene and chromium VI produced maximum increases in hepatic lipid peroxidation of approximately 3.5-, 3.1-, 3.7- and 3.3-fold in hepatic tissues, respectively. Similar results were obtained in hepatic and brain DNA fragmentation at 0.50 LD(50) doses. Lesser effects were observed with 0.10 and 0.01 LD(50) doses of these xenobiotics as compared to the 0.50 LD(50) dose. The results clearly demonstrate that these diverse xenobiotics induce dose- and time-dependent oxidative stress and tissue damage in the liver and brain tissues of mice.

Helicobacter pylori has been identified in the pathogenesis of chronic active gastritis and peptic ulcer disease and is epidemiologically linked to gastric cancer and lymphoma. Our previous studies have demonstrated enhanced production of reactive oxygen species (ROS) in cultured gastric adenocarcinoma cells (ATCC CRL/1739) in association with H. pylori. Recently, we have isolated and cultured normal human gastric mucosal cells (GMC) from H. pylori-negative endoscopic biopsies. The integrity of these mucosal cells was determined by periodic acid-Schiff staining. We assessed the effects of various H. pylori strains including 60190 (a 87-kDa cytotoxin producing strain), ATCC 43504, and 60190-v1 (in which the cytotoxin gene has been disrupted) on the primary culture of human gastric mucosal cells. The induction of ROS and DNA fragmentation in the mucosal cells in association with these H. pylori strains were assessed by cytochrome c reduction (an index of superoxide anion production), hydroxyl radical production, and DNA fragmentation. Following incubation of the mucosal cells with 1:0.5 and 1:1 ratios of H. pylori strain 60190, approximately 6.2- and 9.9-fold increases were observed in cytochrome c reduction, respectively, as compared to mucosal cells in the absence of H. pylori, demonstrating the production of superoxide anion. The detection of hydroxyl radicals based on the formation of 2,3-dihydroxybenzoic acid and 2,5-dihydroxybenzoic acid was determined by using a high-performance liquid chromatograph equipped with an electrochemical detector. Approximately 3.5- and 7.7-fold increases in hydroxyl radical production were observed following incubation of the mucosal cells with 1:0.5 and 1:1 ratios of H. pylori, respectively. Approximately 3.6- and 4.5-fold increases in DNA fragmentation were observed in gastric mucosal cells following incubation with 1:0.5 and 1:1 ratios of H. pylori, respectively. The effects of culture supernatant preparations from H. pylori strains 60190 and 60190-v1 on the enhanced production of ROS and increased DNA fragmentation in mucosal cells were also investigated. Culture supernatant preparations, the prime source of the 87-kDa cytotoxin, from both H. pylori strains 60190 and 60190-v1 were extracted under identical conditions to determine the role of 87-kDa cytotoxin on the enhanced production of ROS and DNA fragmentation. The cytotoxin rich-H. pylori strain 60190 induced greater production of ROS and DNA fragmentation in mucosal cells as compared to the supernatant preparation from H. pylori strain 60190-v1, in which the cytotoxin gene has been disrupted. This study demonstrates that H. pylori induces enhanced production of ROS and DNA damage in association with human gastric mucosal cells and that the 87-kDa cytotoxin protein plays a prime role in the induction of oxidative stress and DNA damage.

To understand the bioavailability and mechanistic pathways of cytoprotection by IH636 grape seed proanthocyanidin extract (GSPE, commercially known as ActiVin) a series of in vitro and in vivo studies were conducted. Comparative protective abilities of GSPE, and vitamins C and E, singly and in combination, were assessed against smokeless tobacco extract (STE)-induced oxidative stress, DNA fragmentation and apoptotic cell death in a primary culture of normal human oral keratinocytes. GSPE protected against STE-induced oxidative stress, DNA damage and apoptotic cell death, and provided better protection as compared to vitamins C and E, singly and in combination. The bioavailability and protective ability of GSPE were examined against acetaminophen (AP)-induced hepato- and nephrotoxicity, amiodarone (AM)-induced lung toxicity, doxorubicin (DX)-induced cardiotoxicity and dimethylnitrosamine (DM)-induced spleenotoxicity in mice. GSPE-fed animals were compared with GSPE-untreated mice to evaluate the protective ability of GSPE against these structurally diverse drugs/chemicals. Serum chemistry changes, histopathology and DNA damage were evaluated. Results indicate that GSPE preexposure prior to the drugs/chemicals such as AP, AM, DX or DM treatment, provided near complete protection in terms of serum chemistry changes and inhibition of both forms of cell death, e.g., apoptosis and necrosis. DNA damage in various tissues triggered by these agents was significantly reduced in GSPE-fed animals. Histopathological examination of multiple target organs provided similar data. The results suggest that GSPE exposure is bioavailable and provides significant multiorgan protection against structurally diverse drug- and chemical-induced toxic assaults. Further, these studies exhibited a series of mechanistic information including free radical scavenging ability, anti-endonucleolytic activity, cytochrome P450 2E1 inhibitory activity, anti-necrotic, anti-apoptotic and anti-carcinogenic activities, modulatory effects on antioxidative and apoptotic regulatory genes such as Bcl2, c-myc and p53, which may be responsible for the novel chemoprotective properties exhibited by GSPE.

Grape seed proanthocyanidins have been reported to possess a broad spectrum of pharmacological and medicinal properties against oxidative stress. We have demonstrated that IH636 proanthocyanidin extract (GSPE) provides excellent protection against free radicals in both in vitro and in vivo models. GSPE had significantly better free radical scavenging ability than vitamins C, E and beta-carotene and demonstrated significant cytotoxicity towards human breast, lung and gastric adenocarcinoma cells, while enhancing the growth and viability of normal cells. GSPE protected against tobacco-induced apoptotic cell death in human oral keratinocytes and provided protection against cancer chemotherapeutic drug-induced cytotoxicity in human liver cells by modulating cell cycle/apoptosis regulatory genes such as bcl2, p53 and c-myc. Recently, the bioavailability and mechanistic pathways of cytoprotection by GSPE were examined on acetaminophen-induced hepatotoxicity and nephrotoxicity, amiodarone-induced pulmonary toxicity, doxorubicin-induced cardiotoxicity, DMN-induced immunotoxicity and MOCAP-induced neurotoxicity in mice. Serum chemistry changes, integrity of genomic DNA and histopathology were assessed. GSPE pre-exposure provided near complete protection in terms of serum chemistry changes and DNA damage, as well as abolished apoptotic and necrotic cell death in all tissues. Histopathological examination reconfirmed these findings. GSPE demonstrated concentration-/dose-dependent inhibitory effects on the drug metabolizing enzyme cytochrome P450 2E1, and this may be a major pathway for the anti-toxic potential exerted by GSPE. Furthermore, GSPE treatment significantly decreased TNFalpha-induced adherence of T-cells to HUVEC by inhibiting VCAM-1 expression. These results demonstrate that GSPE is highly bioavailable and may serve as a potential therapeutic tool in protecting multiple target organs from structurally diverse drug- and chemical-induced toxicity.

The oral use of chewing tobacco has greatly increased in recent years, and this usage is associated with cancers of the mouth, lip, nasal cavities, esophagus and gut. Oral cancer accounts for 3% of all cancers in U.S.A. and is the seventh most common cancer. Previous studies in our laboratory have demonstrated the protective abilities of a novel IH636 grape seed proanthocyanidin extract (GSPE) against reactive oxygen species both in vitro and in vivo models, and provided significantly better protection as compared to vitamins C, E and beta-carotene. In the recent past, we have demonstrated smokeless tobacco (STE)-induced oxidative stress, apoptotic cell death in a primary culture of normal human oral keratinocytes (NHOK), and have compared the protective abilities of vitamins C and E, singly and in combination, and GSPE in this pathobiology [Free Rad. Biol. Med., 26, 992-1000 (1999)]. In the present study, we have assessed the protective role of vitamins C and E, and GSPE against STE-induced modulation of intracellular oxidized states in NHOK cells as demonstrated by laser scanning confocal microscopy. Approximately 11%, 26%, 28% and 50% protection were observed following incubation with vitamin C, vitamin E, a combination of vitamins C plus E, and GSPE, respectively. DNA fragmentation was assessed as an index of oxidative DNA damage and similar results were observed. Furthermore, the cellular viability and functional roles of Bcl-2, p53 and c-myc genes were assessed in STE-induced oxidative stress in NHOK cells. NHOK cells were treated with STE (0-200 micrograms/ml) for 24 h and changes in the expression of Bcl-2, p53 and c-myc genes were measured by reverse transcriptase-polymerase chain reaction (RT-PCR), and the protective effect of GSPE was assessed. Approximately a 2.0-fold increase in p53 gene expression was observed following incubation of the oral keratinocytes with 100 micrograms/ml of STE, beyond which the expression of p53 decreased, confirming increased apoptotic cell death with a higher concentration of STE as reported earlier. GSPE significantly modulated STE-induced changes in p53. The expression of antiapoptotic Bcl-2 gene decreased with STE treatment and the expression of Bcl-2 gene increased significantly following preincubation with GSPE. No significant change in the expression of transcription factor c-myc gene responsible for cell cycle growth was observed following incubation with STE and/or GSPE. Thus, c-myc may not be involved in STE-induced cytotoxicity towards NHOK cells. These results suggest that antioxidant protection of STE-induced cellular injury is associated with alterations in Bcl-2 and p53 expression.

Chromium (VI) is a widely used industrial chemical, extensively used in paints, metal finishes, steel including stainless steel manufacturing, alloy cast irons, chrome, and wood treatment. On the contrary, chromium (III) salts such as chromium polynicotinate, chromium chloride and chromium picolinate, are used as micronutrients and nutritional supplements, and have been demonstrated to exhibit a significant number of health benefits in rodents and humans. However, the cause for the hexavalent chromium to induce cytotoxicity is not entirely understood. A series of in vitro and in vivo studies have demonstrated that chromium (VI) induces an oxidative stress through enhanced production of reactive oxygen species (ROS) leading to genomic DNA damage and oxidative deterioration of lipids and proteins. A cascade of cellular events occur following chromium (VI)-induced oxidative stress including enhanced production of superoxide anion and hydroxyl radicals, increased lipid peroxidation and genomic DNA fragmentation, modulation of intracellular oxidized states, activation of protein kinase C, apoptotic cell death and altered gene expression. In this paper, we have demonstrated concentration- and time-dependent effects of sodium dichromate (chromium (VI) or Cr (VI)) on enhanced production of superoxide anion and hydroxyl radicals, changes in intracellular oxidized states as determined by laser scanning confocal microscopy, DNA fragmentation and apoptotic cell death (by flow cytometry) in human peripheral blood mononuclear cells. These results were compared with the concentration-dependent effects of chromium (VI) on chronic myelogenous leukemic K562 cells and J774A.1 murine macrophage cells. Chromium (VI)-induced enhanced production of ROS, as well as oxidative tissue and DNA damage were observed in these cells. More pronounced effect was observed on chronic myelogenous leukemic K562 cells and J774A.1 murine macrophage cells. Furthermore, we have assessed the effect of a single oral LD50 dose of chromium (VI) on female C57BL/6Ntac and p53-deficient C57BL/6TSG p53 mice on enhanced production of superoxide anion, lipid peroxidation and DNA fragmentation in the hepatic and brain tissues. Chromium (VI)-induced more pronounced oxidative damage in p53 deficient mice. This in vivo study highlighted that apoptotic regulatory protein p53 may play a major role in chromium (VI)-induced oxidative stress and toxicity. Taken together, oxidative stress and oxidative tissue damage, and a cascade of cellular events including modulation of apoptotic regulatory gene p53 are involved in chromium (VI)-induced toxicity and carcinogenesis.

Naphthalene is a bicyclic aromatic compound that is widely used in various domestic and commercial applications. Previous studies in our laboratory have demonstrated enhanced production of reactive oxygen species, lipid peroxidation and DNA fragmentation in both in vitro and in vivo models following treatment with naphthalene. Melatonin (N-acetyl-5-methoxytryptamine), an indole hormone, is the chief secretory product of the pineal gland and is an efficient free radical scavenger and antioxidant, both in vitro and in vivo. In this study, we have investigated the ability of 1 mM melatonin to protect against naphthalene-induced oxidative stress and DNA damage in cultured macrophage J774A.1 cells. No significant changes were observed when these macrophage cells were treated with 100 microM naphthalene. Approximately 2.0-, 4.2- and 4.4-fold increases in cytochrome c reduction were observed at 200, 400 and 500 mM concentrations of naphthalene, demonstrating the increased production of superoxide anion. At 24 h, lipid peroxidation increased by approximately 1.4-, 2.1- and 2.2-fold following treatment of these cells with 200, 400 and 500 mM concentrations of naphthalene, respectively, while 1.6-, 2.8- and 2.8-fold increases in DNA fragmentation were observed at these same concentrations. Two hour pretreatment of these cultured cells with 1 mM melatonin provided approximately 26-44% decreases in lipid peroxidation, superoxide anion production and DNA fragmentation in cells treated with 400 and 500 microM naphthalene. Cellular viability decreased significantly when cells were incubated with concentrations of naphthalene greater than 100 microM, while preincubation with melatonin significantly increased the cellular viability. These results demonstrate that naphthalene may induce toxic manifestations by enhanced production of reactive oxygen free radicals, resulting in lipid peroxidation and DNA damage, while preincubation with melatonin significantly suppressed cytoxicity in J774A.1 macrophage cells.

In this study the induction of oxidative stress in the hepatic and brain tissues of rats after subchronic exposure to various mixtures of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and two of its congeners, namely 2,3,4,7,8-pentachlorodibenzofuran (PeCDF) and 3,3',4,4',5-pentachlorobiphenyl (PCB 126) was investigated. Four mixtures of TCDD and its congeners, corresponding to 10, 22, 46 and 100 ng of toxic equivalence (TEQ) kg(-1) day(-1), were administered to groups of rats for 13 weeks. The animals were sacrificed at the end of the exposure period and the biomarkers of oxidative stress, including the production of superoxide anion, lipid peroxidation and DNA single-strand breaks (SSBs), were determined in the hepatic and brain tissues. All mixtures caused dose-dependent increases in the production of superoxide anion, lipid peroxidation and DNA SSBs in both tissues, with significantly higher damage in the hepatic compared with the brain tissues. The 22 ng TEQ dose level (TEQ = 22) contains TCDD, PeCDF and PCB 126 at levels that correspond to 7.3, 14.5 and 73.3 ng kg(-1) day(-1), respectively, and it produced effects that correspond to ca. 50% of the maximal production of superoxide anion, lipid peroxidation and DNA SSBs in the hepatic and brain tissues of those animals. Relative to the doses that are required to produce 50% of the maximal production of the biomarkers of oxidative stress by the individual congeners in hepatic and brain tissues of rats, the concentrations of the congeners in TEQ = 22 did result in significant interactivity, probably in the form of additive effects in the hepatic but not in brain tissues., (Copyright 2001 John Wiley & Sons, Ltd.)

Grape seed proanthocyanidins are known to possess a broad spectrum of pharmacological, medicinal and therapeutic properties. Previous studies in our laboratories have demonstrated the various protective abilities of a novel IH636 grape seed proanthocyanidin extract (GSPE) against various pathologic conditions. However no extensive safety studies have been conducted on grape seed proanthocyanidins to date. This study demonstrates the acute and chronic safety studies on GSPE. Acute oral toxicity, dermal toxicity, dermal irritation and eye irritation studies have been conducted. The LD50 of GSPE was found to be greater than 5000 mg/kg when administered once orally via gastric intubation to fasted male and female albino rats. The LD50 of GSPE was found to be greater than 2000 mg/kg when administered once for 24 hr to the clipped, intact skin of male and female albino rats. In addition, 2000 mg/kg was found to be the no-observed-effect level (NOEL) for systemic toxicity under the conditions of the study. In a dermal irritation study, GSPE received a descriptive rating classification of moderately irritating. Extensive chronic studies were also conducted. We have assessed the effects of chronic administration of 100 mg GSPE/kg/day for twelve months and its effect on seven vital target organs, namely, brain, heart, intestine, kidney, liver, lung and spleen, and on serum chemistry changes in male B6C3F1 mice. Furthermore, the dose-dependent chronic effects of GSPE in female B6C3F1 mice were evaluated. Mice were fed 0, 100, 250 or 500 mg GSPE/kg/day for six months and the effects of GSPE exposure were examined on brain, duodenum, heart, kidney, liver, lung, pancreas and spleen, and on serum chemistry changes in female mice. These acute studies demonstrated that GSPE is safe and did not cause any detrimental effects in vivo under the conditions investigated in this study.

In order to assess a possible role of the natural glutathione defense system in the pathogenesis of rheumatoid arthritis (RA), serum reduced glutathione levels (GSH), glutathione reductase (GSR), glutathione S-transferase (GST), glutathione peroxidase (GSH-Px) and alkaline phosphatase (ALP) activities, lipid peroxidation (MDA content) and indexes of inflammation were evaluated in 58 rheumatic patients. Rheumatoid athritis was associated with significant depletion (ca. 50%) in GSH levels compared with normal control subjects. Serum levels of the detoxifying enzymes GSR and GSH-Px decreased by ca. 50% and 45%, respectively, whereas a threefold increase in the activity of GST was observed. A 1.2-fold increase in ALP was observed in patients with RA. These effects were accompanied by a 3.1-fold increase in serum MDA content. The MDA content was higher in RA patients who were seropositive for rheumatoid factor as well as positive for C-reactive proteins. The erythrocyte sedimentation rate for all patients with RA was approximately 13.8-fold higher than for the control group, and was higher among RA patients who were positive for C-reactive proteins and exhibited seropositivity for rheumatoid factor. Patients with RA receiving gold therapy exhibited significantly lower MDA levels whereas all other factors that were measured were not effected. The results support a hypothesis that defense mechanisms against reactive oxygen species are impaired in RA., (Copyright 2001 John Wiley & Sons, Ltd.)

Chromium and cadmium are widely used industrial chemicals. The toxicities associated with both metal ions are well known. However, less information is available concerning the mechanisms of toxicity. The results of in vitro and in vivo studies demonstrate that both cations induce an oxidative stress that results in oxidative deterioration of biological macromolecules. However, different mechanisms are involved in the production of oxidative stress by chromium and cadmium. Chromium undergoes redox cycling, while cadmium depletes glutathione and protein-bound sulfhydryl groups, resulting in enhanced production of reactive oxygen species such as superoxide ion, hydroxyl radicals, and hydrogen peroxide. These reactive oxygen species result in increased lipid peroxidation, enhanced excretion of urinary lipid metabolites, modulation of intracellular oxidized states, DNA damage, membrane damage, altered gene expression, and apoptosis. Enhanced production of nuclear factor-kappaB and activation of protein kinase C occur. Furthermore, the p53 tumor suppressor gene is involved in the cascade of events associated with the toxicities of these cations. In summary, the results clearly indicate that although different mechanisms lead to the production of reactive oxygen species by chromium and cadmium, similar subsequent mechanisms and types of oxidative tissue damage are involved in the overall toxicities.

Free radicals have been implicated in over a hundred disease conditions in humans, including arthritis, hemorrhagic shock, atherosclerosis, advancing age, ischemia and reperfusion injury of many organs, Alzheimer and Parkinson's disease, gastrointestinal dysfunctions, tumor promotion and carcinogenesis, and AIDS. Antioxidants are potent scavengers of free radicals and serve as inhibitors of neoplastic processes. A large number of synthetic and natural antioxidants have been demonstrated to induce beneficial effects on human health and disease prevention. However, the structure-activity relationship, bioavailability and therapeutic efficacy of the antioxidants differ extensively. Oligomeric proanthocyanidins, naturally occurring antioxidants widely available in fruits, vegetables, nuts, seeds, flowers and bark, have been reported to possess a broad spectrum of biological, pharmacological and therapeutic activities against free radicals and oxidative stress. We have assessed the concentration- or dose-dependent free radical scavenging ability of a novel IH636 grape seed proanthocyanidin extract (GSPE) both in vitro and in vivo models, and compared the free radical scavenging ability of GSPE with vitamins C, E and beta-carotene. These experiments demonstrated that GSPE is highly bioavailable and provides significantly greater protection against free radicals and free radical-induced lipid peroxidation and DNA damage than vitamins C, E and beta-carotene. GSPE was also shown to demonstrate cytotoxicity towards human breast, lung and gastric adenocarcinoma cells, while enhancing the growth and viability of normal human gastric mucosal cells. The comparative protective effects of GSPE, vitamins C and E were examined on tobacco-induced oxidative stress and apoptotic cell death in human oral keratinocytes. Oxidative tissue damage was determined by lipid peroxidation and DNA fragmentation, while apoptotic cell death was assessed by flow cytometry. GSPE provided significantly better protection as compared to vitamins C and E, singly and in combination. GSPE also demonstrated excellent protection against acetaminophen overdose-induced liver and kidney damage by regulating bcl-X(L) gene, DNA damage and presumably by reducing oxidative stress. GSPE demonstrated excellent protection against myocardial ischemia-reperfusion injury and myocardial infarction in rats. GSPE was also shown to upregulate bcl(2) gene and downregulate the oncogene c-myc. Topical application of GSPE enhances sun protection factor in human volunteers, as well as supplementation of GSPE ameliorates chronic pancreatitis in humans. These results demonstrate that GSPE provides excellent protection against oxidative stress and free radical-mediated tissue injury.

The abilities of single doses of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) to induce oxidative stress in hepatic and some extra-hepatic tissues of animals, are well documented. In this study we have investigated the induction of oxidative stress in hepatic and brain tissues of rats after subchronic (13 weeks) exposure to TCDD and two of its congeners, namely 2,3,4,7,8-pentachlorodibenzofuran (PeCDF) and 3,3',4,4',5-pentachlorobiphenyl (PCB126). TCDD, PeCDF and PCB126 were administered daily to groups of rats at various doses, for 13 weeks, and biomarkers of oxidative stress, including the production of superoxide anion, lipid peroxidation and DNA-single strand breaks (SSBs), were determined in the hepatic and brain tissues at the end of the exposure period. The three congeners caused dose-dependent increases in the production of superoxide anion, lipid proxidation and DNA-SSBs, with maximal effects achieved at doses ranging between 10-100, 20-92, and 300-550 ng/kg per day for TCDD, PeCDF and PCB126, respectively. The doses that produce 50% of maximal responses by each of the xenobiotics in the hepatic and brain tissues were found to be within the ranges of 7-34, 13-32, and 137-400 ng/kg per day for TCDD, PeCDF and PCB126, respectively. The results of the study suggest that subchronic exposures to TCDD, PeCDF and PCB126 induce significant oxidative damage in the hepatic and brain tissues of rats, with more damage observed in the brain as compared to the hepatic tissues. Also, as inducers of oxidative stress in the hepatic and brain tissues, TCDD is the most potent among the three congeners and PCB126 being the least potent.

It has been postulated that tumor suppressor genes are involved in the cascade of events leading to the toxicity of diverse xenobiotics. Therefore, we have assessed the comparative effects of 0.01, 0.10, and 0.50 median lethal doses (LD(50)) of 2,3,7, 8-tetrachlorodibenzo-p-dioxin (TCDD), endrin, naphthalene, and sodium dichromate (VI) [Cr(VI)] on lipid peroxidation, DNA fragmentation, and enhanced production of superoxide anion (cytochrome c reduction) in liver and brain tissues of p53-deficient and standard C57BL/6NTac mice to determine the role of p53 gene in the toxic manifestations produced by these diverse xenobiotics. In general, p53-deficient mice are more susceptible to all four xenobiotics than C57BL/6NTac mice, with dose-dependent effects being observed. Specifically, at a 0.50 LD(50) dose, naphthalene and Cr(VI) induced the greatest toxicity in the liver tissue of mice, and naphthalene and endrin exhibited the greatest effect in the brain tissue. At this dose, TCDD, endrin, naphthalene, and Cr(VI) induced 2.3- to 3.7-fold higher increases in hepatic lipid peroxidation and 1.8- to 3.0-fold higher increases in brain lipid peroxidation in p53-deficient mice than in C57BL/6NTac mice. At a 0. 10 LD(50) dose, TCDD, endrin, naphthalene, and Cr(VI) induced 1.3- to 1.8-fold higher increases in hepatic lipid peroxidation and 1.4- to 1.9-fold higher increases in brain lipid peroxidation in p53-deficient mice than in C57BL/6NTac mice. Similar results were observed with respect to DNA fragmentation and cytochrome c reduction (superoxide anion production). For example, at the 0.10 LD(50) dose, the four xenobiotics induced increases of 1.6- to 3. 0-fold and 1.5- to 2.1-fold in brain and liver DNA fragmentation, respectively, and increases of 1.5- to 2.3-fold and 1.4- to 2.5-fold in brain and liver cytochrome c reduction (superoxide anion production), respectively, in p53-deficient mice compared with control C57BL/6NTac mice. These results suggest that the p53 tumor suppressor gene may play a role in the toxicity of structurally diverse xenobiotics.

Sodium dichromate [Cr(VI)] and cadmium chloride [Cd(II)] are both cytotoxic and mutagenic. This study examined the toxic and apoptotic potentials of these two cations on three cell types in vitro, namely, human chronic myelogenous leukemic (CML) K562 cells, promyelocytic leukemic HL-60 cells, and normal human peripheral blood mononuclear cells. The cells were incubated with 0-100 microM concentrations of the two cations for 0, 24, or 48 hours at 37 degrees C. Both Cr(VI) and Cd(II) induced changes in intracellular oxidized states of cells, which were detected using laser scanning confocal microscopy. Cell cycle modulation and apoptosis of the K562 cells by Cr(VI) and Cd(II) were determined by flow cytometry. Significant decreases in the G2/M phase were observed in the Cr(VI) and Cd(II) treated CML cells compared with untreated cells. At 12.5 microM, Cr(VI) induced greater apoptosis in K562 cells as compared with Cd(II). In the K562 cells, 2.2- and 3.0-fold increases in DNA fragmentation were observed following incubation with 12.5 and 25 microM Cr(VI), respectively, and 1.2- and 1.7-fold increases in DNA fragmentation were observed with Cd(II). Furthermore, approximately 2.7- and 4.9-fold increases in cytochrome c reduction were observed following incubation with 12.5 and 25 microM Cr(VI), respectively, and 1.6- and 3.3-fold increases in cytochrome c reduction were observed with Cd(II), demonstrating enhanced production of superoxide anion. Approximately 3.1 to 6-fold increases in hydroxyl radical production were observed following incubation of the K562 cells with these cations at 12.5 and 25 microM concentrations. These results in K562 cells were compared with promyelocytic leukemic HL-60 cells and normal human peripheral blood mononuclear cells. More pronounced effects were observed on K562 and HL-60 cells, and much lesser effects were observed on normal human peripheral blood mononuclear cells. The results demonstrate that both cations are toxic, producing oxidative tissue damage and apoptosis. Furthermore, more drastic effects were observed on K562 and HL-60 cells as compared with normal human peripheral blood mononuclear cells.

Chromium and cadmium are widely used industrial chemicals. The toxicities associated with both metal ions are well known. However, less information is available concerning the mechanisms of toxicity. The results of in vitro and in vivo studies demonstrate that both cations induce an oxidative stress that results in oxidative deterioration of biological macromolecules. However, different mechanisms are involved in the production of the oxidative stress by chromium and cadmium. Chromium undergoes redox cycling, while cadmium depletes glutathione and protein-bound sulfhydryl groups, resulting in enhanced production of reactive oxygen species such as superoxide ion, hydroxyl radicals, and hydrogen peroxide. These reactive oxygen species result in increased lipid peroxidation, enhanced excretion of urinary lipid metabolites, modulation of intracellular oxidized states, DNA damage, membrane damage, altered gene expression, and apoptosis. Enhanced production of nuclear factor-kappaB and activation of protein kinase C occur. Furthermore, the p53 tumor suppressor gene is involved in the cascade of events associated with the toxicities of these cations. In summary, the results clearly indicate that although different mechanisms lead to the production of reactive oxygen species by chromium and cadmium, similar subsequent mechanisms and types of oxidative tissue damage are involved in the overall toxicities.

Reactive oxygen species (ROS) are implicated in the pathogenesis of chemically induced gastric mucosal injury. We have investigated the effects of ethanol, hydrochloric acid (HCl), and sodium hydroxide (NaOH) on: (1) enhanced production of ROS including superoxide anion and hydroxyl radicals, (2) modulation of intracellular oxidized states by laser scanning confocal microscopy, and (3) DNA fragmentation, indices of oxidative tissue, and DNA damage in a primary culture of normal human gastric mucosal cells (GC), which were isolated and cultured from Helicobacter pylori-negative endoscopic biopsies from human subjects. The induction of ROS and DNA damage in these cells following exposure to ethanol (15%), HCl (150 mM) and NaOH (150 mM) were assessed by cytochrome c reduction (superoxide anion production), HPLC detection for enhanced production of hydroxyl radicals, changes in intracellular oxidized states by laser scanning confocal microscopy, and DNA damage by quantitating DNA fragmentation. Furthermore, the protective ability of bismuth subsalicylate (BSS) was assessed at concentrations of 25, 50, and 100 mg/liter. Incubation of GC with ethanol, HCI, and NaOH increased superoxide anion production by approximately 8.0-, 6.1-and 7.1-fold and increased hydroxyl radical production by 13.3-, 9.6-, and 8.9-fold, respectively, compared to the untreated gastric cells. Incubation of GC with ethanol, HCl, and NaOH increased DNA fragmentation by approximately 6.7-, 4.3-, and 4.8-fold, respectively. Approximately 20.3-, 17.5-, and 13.1-fold increases in fluorescence intensities were observed following incubation of gastric cells with ethanol, HCl, and NaOH, respectively, demonstrating dramatic changes in the intracellular oxidized states of GC following exposure to these necrotizing agents. Preincubation of GC with 25, 50, and 100 mg/liter of BSS decreased ethanol-induced increases in intracellular oxidized states in these cells by 36%, 56%, and 66%, respectively, demonstrating a concentration-dependent protective ability by BSS. Similar results were observed with respect to BSS in terms of superoxide anion and hydroxyl radical production, and DNA damage. The present study demonstrates that ethanol, HCl, and NaOH induce oxidative stress and DNA damage in GC and that BSS can significantly attenuate gastric injury by scavenging these ROS.

Reactive oxygen species (ROS) are implicated in the pathogenesis of stress-induced gastrointestinal mucosal injury. In the present study, we have investigated the effects of acute and chronic stress on the enhanced production of ROS including superoxide anion [SA; as determined by cytochrome c reduction (CCR)] and hydroxyl radicals (OH), and correlated the enhanced production of these free radicals with increased lipid peroxidation, membrane microviscosity and DNA fragmentation, indices of oxidative tissue damage, in the gastric and intestinal mucosa of female Sprague-Dawley rats. Furthermore, the protective ability of bismuth subsalicylate (BSS) against the gastrointestinal mucosal injury induced by acute and chronic stress was determined. Acute stress was induced for a period of 90 min, while chronic stress was induced for 15 min/day for 15 consecutive days. Half of the animals exposed to acute stress were pretreated orally with 15 mg BSS/kg 30 min prior to the exposure to acute stress. Similarly, half of the animals exposed to water-immersion restraint chronic stress were pretreated orally with 7.5 mg BSS/kg/day for 15 consecutive days 30 min prior to the exposure to chronic stress. Acute stress produced greater injury to both gastric and intestinal mucosa as compared to chronic stress. Acute stress increased CCR and OH production by 10.0- and 14.3-fold, respectively, in the gastric mucosa, and 10.4- and 17.0-fold, respectively, in the intestinal mucosa. Pretreatment with BSS prevented the acute stress-induced increase in CCR and OH production. Acute stress increased lipid peroxidation, DNA fragmentation and membrane microviscosity by 3.6-, 4.0- and 11.6-fold, respectively, in gastric mucosa, and 4.1-, 5.0- and 16.2-fold, respectively, in intestinal mucosa. BSS decreased acute stress-induced lipid peroxidation, DNA fragmentation and membrane microviscosity by approximately 26, 35 and 30%, respectively, in gastric mucosa, and by 20, 36 and 30%, respectively, in the intestinal mucosa. Chronic stress increased CCR and OH production by 4.8- and 6.3-fold, respectively, in gastric mucosa, and 4.6- and 6.9-fold, respectively, in intestinal mucosa. Chronic stress increased lipid peroxidation and DNA fragmentation by 2.9- and 3.3-fold, respectively, in gastric mucosa, and 3.3- and 4.2-fold, respectively, in intestinal mucosa. BSS decreased chronic stress-induced lipid peroxidation, DNA fragmentation and membrane microviscosity by approximately 41, 44 and 45%, respectively, in gastric mucosa, and by 39, 52 and 51%, respectively, in the intestinal mucosa. Daily administration of BSS provided greater protection against chronic stress-induced oxidative gastrointestinal injury as compared to the acute stress. These results demonstrate that both acute and chronic stress can induce gastrointestinal mucosal injury through enhanced production of ROS, and that BSS can significantly protect against gastrointestinal mucosal injury.

Grape seed proanthocyanidins are natural antioxidants which possess a broad spectrum of chemoprotective properties against free radicals and oxidative stress. In this study, we have assessed the cytotoxicity of a novel IH636 grape seed proanthocyanidin extract (GSPE) against MCF-7 human breast cancer cells, A-427 human lung cancer cells, CRL-1739 human gastric adenocarcinoma cells and K562 chronic myelogenous leukemic cells at 25 and 50 mg/lit concentrations for 0-72 h using cytomorphology and MTT cytotoxicity assay. In addition, we compared the effects on normal human gastric mucosal cells and normal J774A.1 murine macrophage cells with the effects on the cancer cell lines. Concentration- and time-dependent cytotoxic effects of GSPE were observed on the MCF-7 breast cancer, A-427 lung cancer and gastric adenocarcinoma cells. Following incubation of the MCF-7 cells with 25 mg/lit of the GSPE approximately 6.5, 30 and 43% inhibitions in cell growth were observed at 24, 48 and 72 h of incubation, respectively, while incubation of the MCF-7 cells with 50 mg/lit of the GSPE resulted in 11, 35 and 47% inhibition in cell growth at these same points, respectively. Similar results were observed in the A-427 and gastric adenocarcinoma cells. GSPE exhibited no cytotoxicity toward the neoplastic K562 myelogenous leukemic cells. However, GSPE enhanced the growth and viability of the normal human gastric mucosal cells and J774A.1 murine macrophage cells. These data demonstrate that GSPE exhibited cytotoxicity towards some cancer cells, while enhancing the growth and viability of the normal cells which were examined.

We have investigated the effects of a smokeless tobacco extract (STE) on lipid peroxidation, cytochrome c reduction, DNA fragmentation and apoptotic cell death in normal human oral keratinocyte cells, and assessed the protective abilities of selected antioxidants. The cells, isolated and cultured from human oral tissues, were treated with STE (0-300 microl;g/ml) for 24 h. Superoxide anion production was determined by cytochrome c reductase. Oxidative tissue damage was determined by lipid peroxidation and DNA fragmentation, whereas apoptotic cell death was assessed by flow cytometry. STE-induced fragmentation of genomic DNA was also determined by gel electrophoresis. The comparative protective abilities of vitamin C (75 microM), vitamin E (75 microM), a combination of vitamins C & E (75 microM each), and a novel grape seed proanthocyanidin (IH636) extract (GSPE) (100 microg/ml) against STE induced oxidative stress and tissue damage were also determined. Following treatment of the cells with 300 microg STE/ml 1.5-7.6-fold increases in lipid peroxidation, cytochrome c reduction and DNA fragmentation were observed. The addition of the antioxidants to cells treated with STE provided 10-54% decreases in these parameters. Approximately 9, 29, and 35% increases in apoptotic cell death were observed following treatment with 100, 200, and 300 microg STE/ml, respectively, and 51-85% decreases in apoptotic cell death were observed with the antioxidants. The results demonstrate that STE produces oxidative tissue damage and apoptosis, which can be attenuated by antioxidants including vitamin C, vitamin E, a combination of vitamins C plus E and GSPE. GSPE exhibited better protection against STE than vitamins C and E, singly and in combination.

Radiation generates a variety of free radicals during the exposure of biological tissues through radiolysis of water. These free radicals are highly reactive and cause oxidative damage to biological molecules. This study examined the protective ability of aspirin against radiation induced oxidative stress. The study assessed the protective effect of aspirin (0.05 mM, 0.10 mM, 0.50 mM) on the generation of free radicals during exposure of J774A.1 macrophage cells to radiation (13.25 cGy). Approximately a 2.2-fold increase in superoxide anion formation as determined by cytochrome c reduction was observed following exposure of the cells to radiation for 20 one second exposures. Preincubation with aspirin exhibited a dose dependent decrease in free radical production as assessed by chemiluminescence and cytochrome c reduction. Aspirin also produced a concentration dependent reduction in radiation induced DNA damage in the cells. The data indicate that radiation of these cells results in production of reactive oxygen species and DNA damage, and aspirin can decrease these effects in a concentration dependent manner.

Naphthalene is a bicyclic aromatic compound that is widely used in various domestic and commercial applications including lavatory scent disks, soil fumigants and moth balls. Little information is available regarding the mechanism of naphthalene toxicity. We have assessed the oral, low dose (0.05 LD50) chronic effects of naphthalene (110 mg/kg/day p.o. in corn oil) for 120 consecutive days on lipid peroxidation and DNA fragmentation in the liver and brain tissues of female Sprague-Dawley rats. The animals were sacrificed on 0, 15, 30, 45, 60, 75, 90, 105 and 120 days of treatment. Maximum increases in hepatic and brain lipid peroxidation and DNA fragmentation were observed between 90 and 105 days of treatment. Following administration of naphthalene for 90 days, approximately 1.4- and 1.3-fold increases in lipid peroxidation were observed in the hepatic and brain tissues, respectively, while under the same conditions and time points 1.9- and 2.5-fold increases in hepatic and brain DNA fragmentation were observed, respectively. These results demonstrate that low dose chronic administration of naphthalene induces an oxidative stress resulting in tissue damaging effects that may contribute to the toxicity and carcinogenicity of naphthalene.

Naphthalene is a bicyclic aromatic compound that is widely used in various domestic and commercial applications including lavatory scent disks, soil fumigants and moth balls. However, little information is available regarding the mechanism of naphthalene toxicity. We have assessed the concentration-dependent in vitro effects of naphthalene on increased lipid peroxidation, cytochrome c reduction, hydroxyl radical production, modulation of intracellular oxidized states by laser scanning confocal microscopy, and DNA fragmentation in cultured macrophage J774A.1 cells. The cells were incubated with 0-500 microM concentrations of naphthalene for 0, 12 and 24 h at 37 degrees C. Concentration- and time-dependent changes were observed. No significant changes were observed with concentrations of naphthalene up to 100 microM. At 24 h, lipid peroxidation increased by 1.8-, 2.4- and 2.9-fold at 200, 300 and 500 microM concentrations of naphthalene. Approximately 2.0-, 3.1- and 4.6-fold increases in cytochrome c reduction were observed at 200, 300 and 500 microM concentrations of naphthalene, respectively, at this time point demonstrating the production of superoxide anion, while under the same conditions approximately 2.4-, 3.2- and 4.9-fold increases in hydroxyl radical production were observed, respectively. Following incubation of these cells with 200 and 500 microM concentrations of naphthalene 2.3- and 4.7-fold increases in fluorescence intensity were observed, respectively, as compared to the untreated cells. At 24 h, approximately 1.8-, 2.3- and 3.0-fold increases in DNA fragmentation were observed following incubation with 200, 300 and 500 microM concentrations of naphthalene, respectively. Naphthalene also produced concentration- dependent decreases in cell viability. At the 12 h time point, significant changes were observed only with 300 and 500 microM concentrations of naphthalene. These results demonstrate that naphthalene may induce toxic manifestations by enhanced production of oxygen free radicals, resulting in lipid peroxidation and DNA damage.

The oral use of moist smokeless tobacco products (snuff) is causally associated with cancer of the mouth, lip, nasal cavities, esophagus and gut. The mechanism by which smokeless tobacco constituents produce genetic and tissue damage is not known. Recent studies in our laboratories have shown that an aqueous extract of smokeless tobacco (STE) activates macrophages with the resultant production of reactive oxygen species (ROS), including nitric oxide. Furthermore, the administration of acute doses of STE (125-500 mg/kg) to rats induces dose dependent increases in mitochondrial and microsomal lipid peroxidation, enhances DNA single strand breaks, and significantly increases the urinary excretion of the lipid metabolites malondialdehyde, formaldehyde, acetaldehyde and acetone. Since the use of tobacco is a chronic process, the effects of an aqueous extract of STE in rats following low dose exposure were examined. Female Sprague-Dawley rats were treated orally with 25 mg STE/kg every other day for 105 days. The effects of subchronic treatment of STE on hepatic microsomal and mitochondrial lipid peroxidation and the incidence of hepatic nuclear DNA damage were assessed. Lipid peroxidation increased 1.4- to 3.3-fold in hepatic mitochondria and microsome with STE treatment between 0 and 105 days with respect to control animals while hepatic DNA single strand breaks increased up to 3.4-fold. Maximum increases in lipid peroxidation and DNA single strand breaks occurred between 75 and 90 days of treatment. Urinary excretion of the four lipid metabolites malondialdehyde, formaldehyde, acetaldehyde and acetone was monitored by high pressure liquid chromatography (HPLC) with maximum increases being observed between 60 and 75 days of treatment. The results clearly indicate that low dose subchronic administration of STE induces an oxidative stress resulting in tissue damaging effects which may contribute to the toxicity and carcinogenicity of STE.

1. The comparative protective abilities of a grape seed proanthocyanidin extract (GSPE) (25-100 mg/kg), vitamin C (100 mg/kg), vitamin E succinate (VES) (100 mg/kg) and beta-carotene (50 mg/kg) on 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced lipid peroxidation and DNA fragmentation in the hepatic and brain tissues, as well as production of reactive oxygen species by peritoneal macrophages, were assessed. 2. Treatment of mice with GSPE (100 mg/kg), vitamin C, VES and beta-carotene decreased TPA-induced production of reactive oxygen species, as evidenced by decreases in the chemiluminescence response in peritoneal macrophages by approximately 70%, 18%, 47% and 16%, respectively, and cytochrome c reduction by approximately 65%, 15%, 37% and 19%, respectively, compared with controls. 3. GSPE, vitamin C, VES and beta-carotene decreased TPA-induced DNA fragmentation by approximately 47%, 10%, 30% and 11%, respectively, in the hepatic tissues, and 50%, 14%, 31% and 11%, respectively, in the brain tissues, at the doses that were used. Similar results were observed with respect to lipid peroxidation in hepatic mitochondria and microsomes and in brain homogenates. 4. GSPE exhibited a dose-dependent inhibition of TPA-induced lipid peroxidation and DNA fragmentation in liver and brain, as well as a dose-dependent inhibition of TPA-induced reactive oxygen species production in peritoneal macrophages. 5. GSPE and other antioxidants provided significant protection against TPA-induced oxidative damage, with GSPE providing better protection than did other antioxidants at the doses that were employed.

The ability of single doses of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) to induce oxidative stress in hepatic and some extrahepatic tissues of animals is well documented. However, no previous study has examined the ability of TCDD to induce oxidative stress and tissue damage in brain in vivo. In this study the ability of TCDD to induce oxidative stress in brain tissues of mice was studied after subchronic exposures. Groups of female B6C3F1 mice were treated orally with TCDD (0, 0.45, 1.5, 15, and 150 ng/kg/day) for 13 weeks, 5 days/week. The animals were euthanized 3 days after the last treatment and brain tissues were collected. Biomarkers of oxidative stress including production of superoxide anion, lipid peroxidation, and DNA-single-strand breaks (SSB) were determined. TCDD treatment resulted in significant and dose-dependent increases in the production of superoxide anion as assessed by reduction of cytochrome c. Significant increases were also observed in lipid peroxidation and DNA-SSB in those tissues, as assessed by the presence of thiobarbituric acid-reactive substances and the alkaline elution technique, respectively. These results clearly indicate that subchronic exposure to low doses of TCDD can induce oxidative tissue damage in brain tissues which may at least in part play a role in the effects of TCDD on the central nervous system., (Copyright 1998 Academic Press.)

1. The comparative protective abilities of zinc L-methionine, zinc DL-methionine, zinc sulfate, zinc gluconate, L-methionine, DL-methionine, and vitamin E succinate (VES) on 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced lipid peroxidation, DNA fragmentation, and glutathione depletion in the hepatic and brain tissues, and production of reactive oxygen species by peritoneal macrophages were assessed. In addition, mice were fed a zinc-deficient diet for 5 weeks, and treated with TPA and/or zinc L-methionine or zinc DL-methionine, and similar studies were conducted. 2. The zinc-deficient diet induced oxidative stress in the hepatic and brain tissues as well as in the peritoneal macrophages as evidenced by significantly enhanced lipid peroxidation. DNA fragmentation, glutathione depletion, and production of reactive oxygen species. 3. Treatment of mice with zinc L-methionine, zinc DL-methionine, and VES decreased TPA-induced reactive oxygen species production as evidenced by significant decreases in chemiluminescence in peritoneal macrophages by approximately 45%, 31%, and 47%, respectively, and cytochrome c reduction by approximately 54%, 35%, and 41%, respectively, as compared with control values. Similar results were observed with liver and brain lipid peroxidation, DNA fragmentation, and glutathione depletion. 4. Zinc salts and antioxidants provided significant protection against TPA-induced oxidative damage. Zinc L-methionine provided the best protection.

Oxygen free radicals are implicated in the pathogenesis of stress and food/alcohol-induced gastrointestinal injury. We have investigated the effects of restraint stress, spicy food diet, high-fat diet and 40% ethanol on the enhanced production of reactive oxygen species, including superoxide anion and hydroxyl radicals, and on DNA fragmentation, lipid peroxidation and membrane microviscosity (indices of oxidative tissue damage) in gastric and intestinal mucosa of Sprague-Dawley rats. Furthermore, the protective ability of bismuth subsalicylate (BSS; 15 mg kg(-1) was determined against the gastrointestinal mucosal injury induced by these stressors. Animals on the high-fat diet consumed 31% more food as compared to other animals. Animals on the spicy food diet consumed ca. 23% more water as compared to control animals, and the high-fat diet animals consumed 17% less water. Restraint stress provided greater injury to both gastric and intestinal mucosa as compared to other stressors. Restraint stress, spicy food diet, high-fat diet and ethanol increased superoxide anion production by 10.0-, 4.3-, 5.7- and 4.8-fold, respectively, in the gastric mucosa, and by 10.4-, 5.3-, 7.0- and 5.5-fold in the intestinal mucosa. Exposure to restraint stress, spicy food diet, high-fat diet and 40% ethanol also increased hydroxyl radical production by ca. 14.3-, 4.5-, 3.5- and 4.8-fold, respectively, in the gastric mucosa, and by 17.0-, 4.8-, 3.5- and 4.7-fold in the intestinal mucosa. Bismuth subsalicylate administration to the animals provided significant protection against superoxide anion and hydroxyl radical production. Restraint stress, spicy food diet, high-fat diet and ethanol increased lipid peroxidation by 3.6-, 2.4-, 2.6- and 2.0-fold, respectively, in the gastric mucosa, and by 4.1-, 3.5-, 3.6- and 2.7-fold in intestinal mucosa. Administration of BSS decreased restraint stress, spicy food diet, high-fat diet and ethanol-induced gastric mucosal lipid peroxidation by ca. 26%, 36%, 45% and 18%, and intestinal mucosa lipid peroxidation by 20%, 21%, 46% and 42%, respectively. Approximately 4.0-, 2.0-, 2.4- and 2.0-fold increases in DNA fragmentation were observed in the gastric mucosa of rats exposed to restraint stress, spicy food diet, high-fat diet and 40% ethanol, respectively, and similar increases in the intestinal mucosa. These same four stressors increased membrane microviscosity by 11.6-, 6.1-, 7.3- and 5.4-fold, respectively, in the gastric mucosa, and by 16.2-, 7.9-, 9.5- and 7.8-fold in the intestinal mucosa. Bismuth subsalicylate exerted significant protection against DNA damage and changes in membrane microviscosity induced by the four stressors. Excellent correlations existed between the production of reactive oxygen species and the tissue damaging effects in both gastric and intestinal mucosa. In summary, the results demonstrate that physical and chemical stressors can induce gastrointestinal oxidative stress and mucosal injury through enhanced production of reactive oxygen species, and that BSS can significantly attenuate gastrointestinal injury by scavenging these reactive oxygen species.

The ability of vitamin E succinate and ellagic acid to modulate 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-induced developmental toxicity and oxidative damage in embryonic/fetal and placental tissues was studied in C57BL/6J mice. Vitamin E succinate (100 mg/kg per day) and ellagic acid(6 mg/kg per day) were administered by gavage to groups of pregnant mice on days 10, 11 and 12 of gestation and 40 mg vitamin E succinate/kg or 3 mg ellagic acid/kg on day 13 of gestation. A number of animals from the vitamin E succinate and ellagic acid treated groups also received 30 microg TCDD/kg on day 12 of gestation, 2 h prior to vitamin E succinate or ellagic acid treatment. Groups of treated animals were terminated on day 14 of gestation, and the biomarkers of oxidative stress, including superoxide anion production and the induction of lipid peroxidation and DNA-single strand breaks (SSB), were determined in whole embryonic and placental tissues homogenates. Groups of treated animals were also killed on day 18 of gestation for investigation of the fetotoxic and teratogenic effects as well as effects on the placentae. Vitamin E succinate and ellagic acid significantly decreased TCDD-induced fetal growth retardation fetal death and placental weight reduction, with no significant ameliorating effects on TCDD-induced malformations including cleft palate and hydronephrosis. Vitamin E succinate treatment resulted in decreases of 77-88%, 70-87%, and 21-47% in the production of superoxide anion, lipid peroxidation and DNA-SSB, respectively, in embryonic and placental tissues, while ellagic acid caused 47-98%, 79-93%, and 37-53% decreases, respectively, in these parameters. These results indicate that TCDD-induced fetal death and fetal and placental weight reductions in C57BL/6J mice may be due to oxidative damage induced by TCDD, and ellagic acid and vitamin E succinate provide protection against those effects. Ellagic acid provided better protection than vitamin E succinate against TCDD-induced fetal growth retardation and increases in lipid peroxidation in embryonic and placental tissues.

Oxygen free radicals (OFR) are implicated in the pathogenesis of stress, chemically induced gastric lesions, and gastrointestinal injury. The concentration-dependent scavenging abilities of bismuth subsalicylate (SBS), colloidal bismuth subcitrate (CBS), and selected OFR scavengers, including superoxide dismutase (SOD), catalase, mannitol, and allopurinol were examined against biochemically or chemically generated superoxide anion, hydroxyl radical, and hypochlorite radical plus hypochlorous acid based on a chemiluminescence assay. Furthermore, both gastric (GM) and intestinal mucosa (IM) were individually exposed in vitro to these free radical generating systems, and the concentration-dependent protective abilities of SBS and CBS against lipid peroxidation (LP) were compared with selected OFR scavengers. In addition, 24-hr fasted rats were orally treated with the necrotizing agents 0.6 M HCl, 0.2 M NaOH, 80% ethanol, and aspirin (200 mg/kg). The extent of tissue injury in the GM and IM was determined by assessing LP, DNA fragmentation, and membrane microviscosity. Dose- and time-dependent in vivo protective abilities of CBS (100 mg/kg) and SBS (15 mg/kg) were also assessed. Following incubations with superoxide anion and hydroxyl radical generating systems in the presence of 125 mg SBS/liter, approximately 47% and 61% inhibitions were observed in the chemiluminescence response, respectively, while 48% and 46% inhibitions were observed with 125 mg CBS/liter. SBS and CBS exerted similar abilities towards hypochlorite radical plus hypochlorous acid. Approx. 3.1- and 3.7-fold increases in LP were observed in the GM and IM of rats following oral administration of 0.6 M HCl. Pretreatment of the rats with SBS and CBS decreased 0.6 M HCl-induced LP in the GM by approx. 39% and 27%, respectively, with similar decreases in LP in the IM. SBS exhibited better protective abilities towards 0.6 M HCl and 0.2 m NaOH-induced GM and IM injury as compared to CBS. SBS and CBS provided similar protection towards 80% ethanol-induced gastric injury, while CBS exerted a superior protective ability towards aspirin-induced gastric injury. The results demonstrate that both SBS and CBS can scavenge reactive oxygen species and prevent tissue damage produced by OFR.

The concentration-dependent effects of chromium picolinate and chromium nicotinate were assessed on the enhanced production of reactive oxygen species including superoxide anion and hydroxyl radicals, and lipid peroxidation and DNA fragmentation in cultured macrophage J774A.1 cells. The macrophage cells were incubated with 0-50 micrograms/ml [corrected] concentrations of these chromium (III) salts for 0 and 24 hrs at 37 degrees C. Concentration-dependent effects were observed. Lipid peroxidation increased by 1.3-1.5-fold following treatment of these cells with chromium picolinate while at these same concentrations of chromium nicotinate approximately 1.2-1.8-fold increases in lipid peroxidation were observed. Increases of 1.0-1.5-fold occurred in the production of superoxide anion as determined by cytochrome c reduction following treatment with chromium picolinate while with these same concentrations and conditions only 1.1-1.2-fold increases in cytochrome c reduction were observed following treatment with chromium nicotinate. Approximately 1.2-1.5-fold increases in hydroxyl radical production were observed following treatment of these macrophage cells with increasing concentrations of chromium picolinate and chromium nicotinate. Incubation of the cells with 30-50 micrograms/ml concentrations of chromium picolinate produced 1.2-1.6 fold increases in DNA fragmentation, while under these same conditions with chromium nicotinate 1.2-1.3-fold increases in DNA fragmentation occurred. No significant loss in cell viability was observed with either chromium salt. These results demonstrate that incubation of macrophage J774A.1 cells with these chromium salts induces low levels of oxidative stress as demonstrated by the biochemical assay techniques employed in this study.

This review describes the interrelationship between two important biological factors, intracellular calcium overloading and oxygen-derived free radicals, which play a crucial role in the pathogenesis of myocardial ischemic reperfusion injury. Free radicals are generated during the reperfusion of ischemic myocardium, and polyunsaturated fatty acids in the membrane phospholipids are the likely targets of the free radical attack. On the other hand, activation of phospholipases can provoke the breakdown of membrane phospholipids which results in the activation of arachidonate cascade leading to the generation of prostaglandins, and oxygen free radicals can be produced during the interconversion of the prostaglandins. In conclusion, it has been emphasized that the two seemingly different causative factors of reperfusion injury, intracellular calcium overloading and free radical generation are, in fact, highly interrelated.

Various pesticides and transition metals induce oxidative deterioration of biological macromolecules. Protein kinase C (PKC) may mediate these effects. However, no information is available regarding whether these xenobiotics can modulate PKC which is a critical event signaling the increase in endothelial permeability and cell proliferation. Female Sprague-Dawley rats were treated p.o. with two 0.25 LD50 doses of selected pesticides and transition metal salts at 0 and 21 h, and killed at 24 h. PKC activities were measured in liver and brain tissues. Cultured PC-12 cells were incubated for 24 h with 50, 100 or 200 nM concentrations of these pesticides, while 0.20, 0.40 or 0.60 microM concentrations of cadmium chloride (Cd(II)) and sodium dichromate (Cr(VI)) salts were employed. PKC activations were observed in the hepatic and brain cytosol fractions by all xenobiotics. Approximately 1.4- to 2.0-fold and 1.6- to 3.5-fold increases in PKC activity in the hepatic and brain cytosol fractions were observed, respectively. In the hepatic tissues, the greatest increases in activities were observed with TCDD, chlorpyrifos, endrin and Cd(II), while chlorpyrifos and fenthion exerted the greatest increases in the brain tissues. In cultured PC-12 cells, the greatest activation of PKC was observed primarily with 100-nM concentrations of the pesticides. The maximum effects were induced by chlorpyrifos, fenthion, Cd(II) and Cr(VI) salt. The results clearly indicate that pesticides as well as Cd(II) and Cr(VI) salts can modulate a vital component of the cell signaling pathway, namely PKC activity. PKC may be a target of free radicals and oxidative stress, leading to altered cell proliferation and differentiation.

Proanthocyanidins, a group of polyphenolic bioflavonoids, have been reported to exhibit a wide range of biological, pharmacological and chemoprotective properties against oxygen free radicals. We have assessed the concentration-dependent oxygen free radical scavenging abilities of a grape seed proanthocyanidin extract (GSPE), vitamin C and vitamin E succinate (VES) as well as superoxide dismutase, catalase and mannitol against biochemically generated superoxide anion and hydroxyl radical using a chemiluminescence assay and cytochrome c reduction. A concentration-dependent inhibition was demonstrated by GSPE. At a 100 mg/l concentration, GSPE exhibited 78-81% inhibition of superoxide anion and hydroxyl radical. Under similar conditions, vitamin C inhibited these two oxygen free radicals by approximately 12-19%, while VES inhibited the two radicals by 36-44%. The combination of superoxide dismutase and catalase inhibited superoxide anion by approximately 83%, while mannitol resulted in an 87% inhibition of hydroxyl radical. The results demonstrate that GSPE is a more potent scavenger of oxygen free radicals as compared to vitamin C and VES.

Recent studies have demonstrated that both chromium (VI) and cadmium (II) induce an oxidative stress, as determined by increased hepatic lipid peroxidation, hepatic glutathione depletion, hepatic nuclear DNA damage, and excretion of urinary lipid metabolites. However, whether chronic exposure to low levels of Cr(VI) and Cd(II) will produce an oxidative stress is not shown. The effects of oral, low (0.05 LD50) doses of sodium dichromate [Cr(VI); 2.5 mg/kg/d] and cadmium chloride [Cd(II); 4.4 mg/kg/d] in water on hepatic and brain mitochondrial and microsomal lipid peroxidation, excretion of urinary lipid metabolites including malondialdehyde, formaldehyde, acetaldehyde and acetone, and hepatic nuclear DNA-single strand breaks (SSB) were examined in female Sprague-Dawley rats over a period of 120 d. The animals were treated daily using an intragastric feeding needle. Maximum increases in hepatic and brain lipid peroxidation were observed between 60 and 75 d of treatment with both cations. Following Cr(VI) administration for 75 d, maximum increases in the urinary excretion of malondialdehyde, formaldehyde, acetaldehyde, and acetone were 2.1-, 1.8-, 2.1-, and 2.1-fold, respectively, while under the same conditions involving Cd(II) administration approximately 1.8-, 1.5-, 1.9-, and 1.5-fold increases were observed, respectively, as compared to control values. Following administration of Cr(VI) and Cd(II) for 75 d, approximately 2.4- and 3.8-fold increases in hepatic nuclear DNA-SSB were observed, respectively, while approximately 1.3- and 2.0-fold increases in brain nuclear DNA-SSB were observed, respectively. The results clearly indicate that low dose chronic administration of sodium dichromate and cadmium chloride induces an oxidative stress resulting in tissue damaging effects that may contribute to the toxicity and carcinogenicity of these two cations.

1. The concentration-dependent scavenging abilities of zinc DL-methionine, zinc sulfate, zinc gluconate, zinc picolinate and selected free radical scavengers, including superoxide dismutase (SOD), catalase, mannitol, allopurinol and DL-methionine, were examined against biochemically generated superoxide anion, hydroxyl radical and hypochlorite radical plus hypochlorous acid, by chemiluminescence and cytochrome c reduction. 2. Zinc methionine was the most effective of the zinc compounds that were tested. Following incubations with superoxide anion, hydroxyl radical, and hypochlorite radical-generating systems, in the presence of 50 microM zinc DL-methionine approximately 38%, 47% and 28% inhibition in reactive oxygen species generation was observed, respectively, compared to control groups. 3. The protective abilities of various zinc salts, as well as selected free radical scavengers and antioxidants were also assessed on phorbol ester (TPA)-induced lactate dehydrogenase (LDH) release from cultured PC-12 cells. Preincubation showed better protection than coincubation. Approximately 45% and 50% inhibition in TPA-induced LDH leakage was observed following preincubation with 50 microM zinc DL-methionine and 50 microM vitamin E succinate, respectively. Zinc DL-methionine exhibited better protection against LDH leakage than any other zinc salt tested. 4. The results indicate that zinc DL-methionine can attenuate the biochemical consequences of oxygen free radicals, and is comparable to other well-known antioxidants and free radical scavengers in the in vitro system that was employed.

TCDD, endrin and lindane induce an oxidative stress and enhance lipid peroxidation in fetal and placental tissues of mice. The levels of the products resulting from altered lipid metabolism, including malondialdehyde (MDA), formaldehyde (FA), acetaldehyde (ACT) and acetone (ACON) have been determined in maternal sera and amniotic fluids of pregnant C57BL/6J and DBA/2J mice after oral administration of single fetotoxic doses of TCDD, endrin and lindane on day 12 of gestation, using high pressure liquid chromatography (HPLC). Under these conditions, TCDD given at a dose of 30 micrograms/kg body weight to C57BL/6J mice produced 2.5-3.9 and 1.7-4.0 fold increases in the levels of the four metabolites in maternal sera and the amniotic fluids, respectively. TCDD given to DBA/2J mice at a dose of 60 micrograms/kg produced 1.5-1.7 and 1.7-2.2 fold increases in the levels of these metabolites in maternal sera and the amniotic fluids, respectively. Endrin, when given at a dose of 4.5 mg/kg body weight to either mouse strain, produced increases of 1.9-3.1 and 1.7-3.2-fold in the levels of the four metabolites in maternal sera and the amniotic fluids, respectively, in the C57BL/6J mice and increases of 1.4-1.6 and 1.2-1.5 fold in the levels of these metabolites in maternal sera and the amniotic fluids, respectively, in the DBA/2J mice. Lindane given at a dose of 30 mg/kg body weight to C57BL/6J mice produced 1.5-2.0 and 1.3-1.7-fold increases in the four metabolites in maternal serum and amniotic fluids, respectively, while administration of this same dose to DBa/2J mice produced 1.2-1.5 and 1.1-1.5 fold increases, respectively. Increases in the levels of lipid metabolites occur in maternal serum and amniotic fluid as a result of enhanced lipid peroxidation in response to TCDD, endrin and lindane. Lipid peroxidation may participate in the fetotoxic effects of these xenobiotics and these effects are observed regardless of the Ah-responsiveness of the mice, although higher levels of the metabolites are produced by TCDD in Ah-responsive mice.

The involvement of reactive oxygen species in the toxicity of cadmium (Cd) has been proposed. We have, therefore, examined the effects of this cation on the production of superoxide anion and nitric oxide and DNA single strand breaks in J774A.1 macrophage cells in culture as well as the effects on lactate dehydrogenase (LDH) leakage and cell viability. Following a 48-h incubation, over 2-fold increases in superoxide anion and nitric oxide (NO) production were observed at a Cd concentration of approximately 0.60 microM, while a 50% decrease in viability was observed at this concentration. LDH leakage paralleled the superoxide anion and nitric oxide production. Concentration-dependent increases in DNA single strand breaks (SSB) were observed after incubation with Cd with a maximum increase occurring at a concentration of approximately 0.40 microM. The results indicate that Cd is toxic to the J774A.1 cell line, and support the hypothesis that the toxicity may be due at least in part to an oxidative stress induced by the production of reactive oxygen species following exposure to this cation.

The abilities of TCDD, endrin and lindane to induce oxidative stress in fetal and placental tissues have been studied after the oral administration of these xenobiotics to pregnant C57BL/6J and DBA/2J mice. Production of superoxide anion, lipid peroxidation and DNA-single strand breaks (SSB) was determined in live fetal and placental tissues 48 hr after administration of single teratogenic doses of the compounds on day 12 of gestation. Oxidative stress and its biomarkers were also determined in livers of day 18 fetuses after administration on day 12 of gestation. TCDD given at doses of 30 and 60 micrograms/kg body weight to the C57BL/6J and DBA/2J mice, respectively, produced increases of 1.3-2.7-fold in superoxide anion production, 1.6-1.9-fold in lipid peroxidation and 2.1-4.4-fold in DNA-SSB. Endrin, given at a dose of 4.5 mg/kg body weight to C57BL/6J and DBA/2J mice, produced increases of 1.3-2.8-fold in superoxide production, 1.4-1.8-fold in lipid peroxidation and 1.4-4.7-fold in DNA-SSB. Lindane when given at a dose of 30 mg/kg body weight to C57BL/6J and DBA/2J mice produced increases of 1.6-3.0-fold in superoxide production, 1.3-2.1-fold in lipid peroxidation and 1.4-5.0-fold in DNA-SSB. The results suggest that superoxide production, lipid peroxidation and DNA-SSB in fetal and placental tissues may participate in the fetotoxic effects of TCDD and other polyhalogenated cyclic hydrocarbons, and that TCDD-induced oxidative damage in fetal and placental tissues is mediated at least in part by the Ah-receptor. The results also indicate that TCDD as an inducer of oxidative tissue damage in the embryos and placentas is approximately 150 and 1000 times more potent than endrin and lindane, respectively, in C57BL/6J mice, and 75 and 500 times more potent than endrin and lindane, respectively, in the DBA/2J mouse strain.

The chloroacetamide insecticide alachlor, polyhalogenated cyclic hydrocarbons endrin and chlordane and the organophosphate pesticides chlorpyrifos and fenthion induce oxidative tissue damaging effects including lipid peroxidation and nuclear DNA-single strand breaks. The mechanism involved in the induction of oxidative stress by these xenobiotics is unknown. No information is available regarding whether these pesticides can induce the expression of heat shock (stress) protein (Hsp) genes as a common protective mechanism against tissue damage. The pesticides were administered p.o. individually to female Sprague-Dawley rats in two 0.25 LD50 doses at 0 h and 21 h. The animals were killed at 24 h, and liver, brain, heart and lung tissues were removed to examine the induction of Hsps by Western and Northern blot analysis. In a separate series of experiments, cultured neuroactive PC-12 cells were treated 24 h with 50, 100 or 200 nM concentrations of these pesticides. Alachlor, endrin, chlorpyrifos and fenthion induced Hsp89 alpha and Hsp89 beta in hepatic and brain tissues, as well as in cultured PC-12 cells. Chlordane induced some expression of Hsp89 alpha but not Hsp89 beta in the hepatic and brain tissues of treated rats. Some expression of Hsp89 beta was observed in lung tissues of endrin and alachlor treated animals. These findings were substantiated by Western blot analysis using Hsp90 antibody. Except chlordane all these pesticides induced enhanced synthesis of Hsp90 in cultured PC-12 cells. The results indicate striking tissue differences in the patterns of the Hsps induced by the pesticides which were used, and demonstrate that these pesticides can induce the expression of Hsp89 alpha and Hsp89 beta genes in various target organs of rats. The results support the hypothesis that these genes may be mechanistically involved in protecting tissues against oxidative stress induced by structurally diverse pesticides.

Reactive oxygen species (ROS) and Helicobacter pylori have been identified as pathogenic factors in several gastrointestinal disorders. Since little information is available regarding the mechanistic pathways of H. pylori-induced gastric injury, the potential role of ROS was investigated. The induction of ROS in gastric cells (GC) by H. pylori was assessed using chemiluminescence, cytochrome c reduction, nitrobluetetrazolium (NBT) reduction and lactate dehydrogenase (LDH) leakage. The dose-dependent protective abilities of selected ROS scavengers on LDH leakage were determined. Following incubation of GC with three strains of H. pylori (1:1), approximately 5.7-8.0 and 3.8-4.3 fold increases were observed in cytochrome c and NBT reduction, respectively, demonstrating production of ROS. Enhanced chemiluminescence responses of 2.1- and 3.7-fold were observed following incubation of GC with H. pylori (ATCC 43504) at ratios of 1:1 and 1:10, respectively. Approximately 2.2- and 3.5-fold increases in LDH leakage were observed at GC:H. pylori (ATCC 43504) ratios of 1:1 and 1:10, respectively. Substantial inhibition of LDH leakage from GC in the presence of H. pylori was observed following co-incubations with selected ROS scavengers with cimetidine serving as the best chemoprotectant. The antioxidants and H2-receptor antagonists had no effect on growth of H. pylori cells. This study demonstrates that H. pylori induces enhanced production of ROS in GC, and enhances membrane damage.

Recent studies have described lipid peroxidation to be an early and sensitive consequence of cadmium exposure, and free radical scavengers and antioxidants have been reported to attenuate cadmium-induced toxicity. These observations suggest that cadmium produces reactive oxygen species that may mediate many of the untoward effects of cadmium. Therefore, the effects of cadmium (II) chloride on reactive oxygen species production were examined following a single oral exposure (0.50 LD50) by assessing hepatic mitochondrial and microsomal lipid peroxidation, glutathione content in the liver, excretion of urinary lipid metabolites, and the incidence of hepatic nuclear DNA damage. Increases in lipid peroxidation of 4.0- and 4.2-fold occurred in hepatic mitochondria and microsomes, respectively, 48 h after the oral administration of 44 mg cadmium (II) chloride/kg, while a 65% decrease in glutathione content was observed in the liver. The urinary excretion of malondialdehyde (MDA), formaldehyde (FA), acetaldehyde (ACT), and acetone (ACON) were determined at 0-96 h after Cd administration. Between 48 and 72 h posttreatment maximal excretion of the four urinary lipid metabolites was observed with increases of 2.2- to 3.6-fold in cadmium (II) chloride-treated rats. Increases in DNA single-strand breaks of 1.7-fold were observed 48 h after administration of cadmium. These results support the hypothesis that cadmium induces production of reactive oxygen species, which may contribute to the tissue-damaging effects of this metal ion.

Ricin has been shown to induce oxidative stress in the livers of mice in vivo. These studies examined ricin-induced hepatic microsomal lipid peroxidation in mice, and the modulation thereof by iron and desferrioxamine. In addition, the studies investigated the production of superoxide anion by microsomes, mitochondria, and macrophages. Ricin (25 micrograms/kg, in vivo) increased microsomal lipid peroxidation by approximately 1.8-fold relative to control animals. This effect was abrogated by adding desferrioxamine to the microsomes. Fe2+ increased lipid peroxidation approximately 15-fold and 5-fold when added to microsomes from control and ricin-treated animals, respectively. Adding ricin to microsomes from control animals, however, decreased lipid peroxidation in a concentration-dependent manner. Desferrioxamine decreased lipid peroxidation by 47% and 64% in the absence and presence of ricin (5 micrograms/ml), respectively. Ricin, added to mitochondria from untreated animals decreased lipid peroxidation by 26% and 17% in the presence and absence of Fe2+, respectively. The administration of ricin (5 and 25 micrograms/kg) to mice increased microsomal, mitochondrial and macrophage superoxide anion production, in a dose-dependent fashion. The results suggest that iron mediated production of superoxide anion may be involved in the process of oxidative stress induced by ricin.

The possible role of reaction oxygen species in the toxicity of smokeless tobacco was explored. In order to determine possible sources of reactive oxygen species in response to smokeless tobacco, rat peritoneal macrophages (3 x 10(6)/ml) and hepatic mitochondria and microsomes (1 mg protein/ml) from untreated female Sprague-Dawley rats were incubated with an aqueous smokeless tobacco extract (STE) (200 micro g/ml). STE resulted in rapid increases in chemiluminescence with maximum increases occurring at approximately 6 min for the macrophages and 8 min for mitochondria and microsomes. Maximum increases in chemiluminescence of 1.4-, 3.2-, and 3.1-fold relative to control values occurred for macrophages, mitochondria, and microsomes, respectively. Hepatic mitochondria and microsomes (1 mg protein/ml) from female Sprague-Dawley rats were incubated at 37 degrees C for 60 min in the presence of 0-500 micro g/ml STE. Potential tissue damage was measured as lipid peroxidation, and dose-dependent increases of 1.1-2.4-fold occurred in mitochondria and microsomes. Pre-incubation with various oxygen free radical scavengers including superoxide dismutase (SOD) (100 micro g/ml), catalase (100 micro g/ml), SOD + catalase (100 micro g/ml each), mannitol (1.25 mmol/ml), and allopurinol (100 micro g/ml) inhibited STE (200 micro g/ml) induced lipid peroxidation by 15% to 70%. Previous studies in our laboratories strongly suggest that STE induces the production of oxygen free radicals which cause tissue-damaging effects. We therefore examined the cytotoxicity of STE by incubating cultured human oral epidermal carcinoma (KB) cells with STE, and assessing the release of the enzyme lactate dehydrogenase (LDH) into the media as an indicator of cellular membrane damage. The amount of LDH released by STE was both concentration- and time-dependent. The results demonstrate that oral cells, peritoneal macrophages, and hepatic mitochondria and microsomes produce reactive oxygen species following in vitro incubation with an aqueous extract of smokeless tobacco. Tissue damage in response to STE may occur as the result of reactive oxygen species production.

The teratogenic effects of endrin and lindane have been determined and compared to those induced by TCDD in the fetuses of C57BL/6J and DBA/2J mice after the administration of single oral doses to pregnant mice on day 12 of gestation. TCDD produced dose-dependent decreases in fetal weight, fetal thymic weight and placental weight, and dose-dependent increases in fetolethality, cleft palate formation and hydronephrosis at doses of 10-30 and 30-60 micrograms/kg body weight in C57BL/6J and DBA/2J mice, respectively. No maternal death was observed at the given doses in both strains of mice. Endrin (4.5 and 6 mg/kg body weight) and lindane (30 and 45 mg/kg body weight) produced significant decreases in fetal weight and placental weight in C57BL/6J and DBA/2J mice, and dose-dependent decreases in fetal thymic weight in C57BL/6J mice but not DBA/2J mice. Endrin and lindane caused 0-25 and 14-25% maternal deaths, respectively, at the above mentioned doses. Neither cleft plate nor hydronephrosis were induced by endrin or lindane in the two strains of mice. The results support the hypothesis that TCDD-induced cleft plate and hydronephrosis involve mechanisms that are Ah (aryl hydrocarbon) receptor mediated. However, other fetotoxic effects induced by TCDD, and the fetotoxic effects induced by endrin and lindane may involve additional unknown mechanisms that are not related to the Ah-receptor.

The abilities of the triacetylated galacto- and gluco-derivatives of 2-deoxy-2-fluoro-D-pyranosyl fluoride as well as alpha- and beta-N-bromoacetyl-D-galactopyranosylamine to inhibit the cytotoxicity of ricin in vitro in macrophage J774A.1 and Chinese hamster ovary (CHO) cell lines were determined. Leakage of lactate dehydrogenase (LDH) and aspartate aminotransferase (AST) from the cells into the culture media were used as indicators of ricin cytotoxicity. The potential chemoprotectants were used in concentrations ranging from 10(-8) to 10(-4) g ml-1. Of the four potential mechanism-based, site-specific glycosidase inhibitors that were tested, 3,4,6-tri-O-acetyl-2-deoxy-2-fluoro- beta-D-glucopyranosyl fluoride exhibited the greatest chemoprotectant activity. The ricin-induced LDH release was inhibited in a concentration-dependent manner by this compound, with the LDH leakage returning to control values in the presence of the highest concentration of this chemoprotectant in both cell cultures when given 4 h prior to ricin. This compound exhibited a small but significant inhibition of AST release from both cell cultures when given simultaneously with ricin. 3,4,6-Tri-O-acetyl-2-deoxy-2-fluoro-beta-D-galactopyranosyl fluoride exhibited a small but significant chemoprotective effect only at the highest concentration in both cell cultures when given simultaneously with ricin. Both the alpha- and beta-isomers of N-bromoacetyl-D-galactopyranosylamine exhibited activity against ricin toxicity in the CHO cell line, with the beta-isomer exhibiting greatest activity. The beta-isomer exhibited greater cytotoxicity in the absence of ricin, as demonstrated by the release of both enzymes from the cultured CHO cells. Further studies will be required to assess the utility of these compounds as chemoprotectants against ricin toxicity in vivo.

The protein toxin ricin is one of the most toxic substances known. No specific chemoprotective agents are available against ricin or similar protein toxins. Previous investigations have suggested that deoxynucleoside analogs may be effective in decreasing the toxicity of ricin. We have therefore examined the effects of 3'-azido-3'-deoxythymidine (AZT) and 2',3'-dideoxycytidine (DDC) in decreasing the cytotoxicity of ricin in J774A.1 macrophage and Chinese hamster ovary (CHO) cells in culture by assessing the release of lactate dehydrogenase (LDH) and aspartate aminotransferase (AST) from the cells, as well as decreased cell viability. The results clearly indicate that DDC provided partial protection against ricin toxicity in both cell culture systems based on the leakage of LDH and AST. Concentration-dependent effects between 10(-10) and 10(-4) g ml-1 were produced. Under similar conditions, AZT had no effect on the toxicity of ricin in these two cell culture systems. When assessing cell viability, DDC almost doubled the viability of both CHO and J774A.1 cells at a concentration of 10(-4) g ml-1 in the presence of ricin. The results demonstrate that DDC but not AZT exhibits a chemoprotective effect against ricin toxicity in the two cell culture systems employed.

Quinone metabolites of naphthalene (NAP) are known to produce lipid peroxidation. However, the ability of naphthalene to induce oxidative stress in experimental animals has not been extensively investigated. Furthermore, the effects of vitamin E succinate [(+)-alpha-tocopherol acid succinate; VES] on naphthalene-induced oxidative stress and tissue damage were assessed. Female Sprague-Dawley rats were treated with a single oral dose of 1100 mg naphthalene/kg (0.50 LD50) in corn oil. Vitamin E succinate-treated rats received 100 mg VES/kg/day orally for 3 d before naphthalene treatment, and 40 mg VES/kg/d after NAP administration. Hepatic and brain tissues and urine samples were collected 0, 12, 24, 48, and 72 h after NAP treatment. Naphthalene treatment resulted in a 2.1-fold increase in lipid peroxidation in liver and brain mitochondria at the 24-h time point. Increases in hepatic and brain mitochondrial lipid peroxidation in VES plus NAP-treated rats were 39-46% less than NAP treated rats at 24 h. DNA-single strand breaks increased 3.0-fold in hepatic tissues in NAP treated rats, and increased only 1.6-fold in VES protected rats at the 24-h time point. Glutathione (GSH) decreased by 83 and 49% in hepatic and brain tissues, respectively, in NAP-treated rats at the 24-h time point, while GSH content in VES plus NAP-treated rats decreased 47 and 21% in hepatic and brain tissues, respectively, at this same time point. Microsomal membrane fluidity, a measurement of membrane damage, increased 1.9- and 1.7-fold in liver and brain tissues, respectively, in NAP-treated rats, and only 1.3- and 1.2-fold in NAP plus VES-treated rats at the 24-h time point. The urinary excretion of malondialdehyde (MDA), formaldehyde (FA), acetaldehyde (ACT), and acetone (ACON) was determined at 0-96 h after NAP administration. Between 12-24 h after NAP administration maximal excretion of the four urinary lipid metabolites was observed, with increases of 4.5-, 2.7-, 2.3-, and 2.8-fold for MDA, FA, ACT, and ACON, respectively, at the 24-h time point. VES reduced the NAP-induced excretion of these urinary metabolites by 28-49% 24 h after NAP administration. These results support the hypothesis that NAP induces oxidative stress and tissue damage, and that vitamin E succinate provides significant protection.