Oxidative mechanisms in the toxicity of chromium and cadmium ions.

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.

Production of superoxide anion, lipid peroxidation and DNA damage in the hepatic and brain tissues of rats after subchronic exposure to mixtures of TCDD and its congeners.

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.

Oxidative mechanisms in the toxicity of chromium and cadmium ions.

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.

Oxidative stress in female B6C3F1 mice following acute and subchronic exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD).

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is a highly persistent trace environmental contaminant and is one of the most potent toxicants known to man. Hassoun et al. (1998, Toxicol. Sci. 42, 23-27) reported an increase in the production of reactive oxygen species (ROS) in the brain of female B6C3F1 mice following subchronic exposure to TCDD at doses as low as 0.45 ng/kg/day. In the present study, oxidative stress was characterized in liver, spleen, lung, and kidney following subchronic (0.15-150 ng/kg; 5 days/week for 13 weeks, po) or acute exposure (0.001-100 microg/kg, po) to TCDD in order to investigate the interaction between tissue concentration and time for production of ROS. Seven days following acute administration of TCDD, mice were sacrificed; they demonstrated increases in liver superoxide anion production (SOAP) and thiobarbituric acid reactive substances (TBARS) at doses of 10 and 100 microg/kg, associated with hepatic TCDD concentrations of 55 and 321 ng/g, respectively. Liver obtained from mice following subchronic TCDD exposure demonstrated an increase in SOAP and TBARS above controls at doses of 150 ng/kg/day with liver TCDD concentration of only 12 ng/g. Interestingly, glutathione (GSH) levels in lung and kidney following sub-chronic TCDD exposure were decreased at the low dose of 0.15 ng/kg/day. This effect disappeared at higher TCDD doses. The data suggest that higher tissue TCDD concentrations are required to elicit oxidative stress following acute dosing than with subchronic TCDD exposure. Therefore, the mechanism of ROS production following TCDD exposure does not appear to be solely dependent upon the concentration of TCDD within the tissue. In addition, very low doses of TCDD that result in tissue concentrations similar to the background levels found in the human population produced an effect on an oxidative stress endogenous defense system. The role of this effect in TCDD-mediated toxicity is not known and warrants further investigation.

The relative abilities of TCDD and its congeners to induce oxidative stress in the hepatic and brain tissues of rats after subchronic exposure.

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.

Ricin-induced toxicity in the macrophage J744A.1 cells: the role of TNF-alpha and the modulation effects of TNF-alpha polyclonal antibody.

Ricin is a natural toxin of the castor beans (Ricinus communus). We studied the time- and concentration-dependent effects of ricin on the release of TNF-alpha and lactate dehydrogenase (LDH), as well as the modulation of the ricin-induced effects by TNF-alpha antibody in the J774A.1 cells. When added at concentrations ranging from 0 to 1000 ng/mL, ricin caused concentration-dependent increases in the release of TNF-alpha after incubation for 12 to 24 hours. Concentration-dependent increases in the leakage of LDH were also observed after incubation of the cells with those concentrations of ricin for 24 to 48 hours. Addition of 5 units/mL of rabbit anti-mouse TNF-alpha polyclonal antibody (TNF-alpha antibody) 2 hours prior to the addition of ricin resulted in a decrease in the ricin-induced toxicity, indicated by the release of LDH by the cells. However, when added at concentrations higher than 5 units/mL, the antibody resulted in either no effect or an increase in the ricin-induced LDH leakage. These results suggest that secretion of TNF-alpha by the macrophages in response to ricin plays a significant role in the toxicity of ricin and that TNF-alpha antibody can antagonize the effects of ricin in this cell line when added at relatively low concentrations.

Time- and concentration-dependent production of superoxide anion, nitric oxide, DNA damage and cellular death by ricin in the J774A.1 macrophage cells.

The time- and concentration-dependent effects of ricin on some biomarkers of cellular toxicity, including production of superoxide anion (O2-), nitric oxide (NO), and DNA single strand breaks (SSB), as well as cellular death, have been examined in the J774A.1 macrophage cell cultures. Various concentrations of ricin have been added to various cell cultures, and the cells were incubated for 12, 24, 36, and 48 hours. Following 12 hour incubation, ricin did not cause significant increases in any of those biomarkers. However, time- and concentration-dependent increases were observed in the induction of all the biomarkers after incubation for 24-48 hours. Approximately twofold increases in the production of O2- were observed after incubation with 1 and 10 ng/mL of ricin for 24 and 36-48 hours, respectively. The concentrations of ricin that caused approximately twofold increases in the rate of DNA-SSB are 10 and 1-10 ng/mL after 24 and 36-48 hours incubation, respectively. Approximately twofold increases in NO production were only observed after incubation of the cultures with 1-10 ng/mL of ricin for 36-48 hours. Fifty percent reductions in cellular viability were also observed with ricin concentrations of 10-100, 10, and 1-10 ng/mL, after incubation for 24, 36, and 48 hours, respectively.

Subchronic effects of smokeless tobacco extract (STE) on hepatic lipid peroxidation, DNA damage and excretion of urinary metabolites in rats.

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.

Induction of oxidative stress in brain tissues of mice after subchronic exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin.

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.

Modulation of TCDD-induced fetotoxicity and oxidative stress in embryonic and placental tissues of C57BL/6J mice by vitamin E succinate and ellagic acid.

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.

Induction of oxidative stress by chronic administration of sodium dichromate [chromium VI] and cadmium chloride [cadmium II] to rats.

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.

TCDD endrin and lindane induced increases in lipid metabolites in maternal sera and amniotic fluids of pregnant C57BL/6J and DBA/2J mice.

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.

Cadmium-induced production of superoxide anion and nitric oxide, DNA single strand breaks and lactate dehydrogenase leakage in J774A.1 cell cultures.

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.

TCDD, endrin and lindane induced oxidative stress in fetal and placental tissues of C57BL/6J and DBA/2J mice.

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.

Cadmium-induced excretion of urinary lipid metabolites, DNA damage, glutathione depletion, and hepatic lipid peroxidation in Sprague-Dawley rats.

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.

Role of iron in ricin-induced lipid peroxidation and superoxide production.

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.

Comparative teratological studies on TCDD, endrin and lindane in C57BL/6J and DBA/2J mice.

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.

Potential chemoprotectant activity of mechanism-based glycosidase inhibitors against ricin toxicity in Chinese hamster ovary and macrophage J774A.1 cell cultures.

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.

Potential chemoprotectant activity of 3'-azido-3'-deoxythymidine (AZT) and 2',3'-dideoxycytidine (DDC) against ricin toxicity in Chinese hamster ovary and macrophage J774A.1 cell cultures.

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.

Naphthalene-induced oxidative stress in rats and the protective effects of vitamin E succinate.

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.