E.A. Hassoun

In vitro and in vivo generation of reactive oxygen species, DNA damage and lactate dehydrogenase leakage by selected pesticides

Reactive oxygen species may be involved in the toxicity of various pesticides and we have, therefore, examined the in vivo effects of structurally dissimilar polyhalogenated cyclic hydrocarbons (PCH), such as endrin and chlordane, chlorinated acetamide herbicides (CAH), such as alachlor, and organophosphate pesticides (OPS), such as chlorpyrifos and fenthion, on the production of hepatic and brain lipid peroxidation and DNA-single strand breaks (SSB), two indices of oxidative stress and oxidative tissue damage. The selected pesticides were administered p.o. to female Sprague-Dawley rats in two 0.25 LD50 doses at 0 h and 21 h and killed at 24 h. In a parallel set of experiments, we have determined the in vitro effects of these pesticides on the DNA-SSB and enhanced lactate dehydrogenase leakage (LDH) from neuroactive PC-12 cells in culture. In vitro production of reactive oxygen species by these pesticides was also assessed by determining the enhanced chemiluminescence responses of hepatic and brain homogenates. Following treatment of rats with endrin, chlordane, alachlor, chlorpyrifos and fenthion, increases of 2.8-, 3.0-, 4.2-, 4.3- and 4.8-fold were observed in hepatic lipid peroxidation, respectively, while at these same doses, increases in lipid peroxidation of 2.4-, 2.1-, 3.6-, 4.6- and 5.3-fold, respectively, were observed in brain homogenates. Increases of 4.4-, 3.9-, 1.6-, 3.0- and 3.5-fold were observed in hepatic DNA-SSB following treatment of the rats with endrin, chlordane, alachlor, chlorpyrifos and fenthion, respectively, while at these same doses, increases of 1.9-, 1.7-, 2.2-, 1.4-, 1.4-fold, respectively, were observed in brain nuclear DNA-SSB. Following in vitro incubation of hepatic and brain tissues with 1 nmol/ml of each of the five pesticides, maximum increases in chemiluminescence occurred within 4-7 min of incubation and persisted for over 10 min. Increases of 3.0-, 2.7-, 3.6-, 4.9- and 4.4-fold were observed in chemiluminescence following in vitro incubation of the liver homogenates with endrin, chlordane, alachlor, chlorpyrifos and fenthion, respectively, while increases of 1.7-, 1.8-, 2.0-, 3.4- and 3.7-fold, respectively, were observed in the brain homogenates. Increases of 2.2-, 2.3-, 2.9-, 2.9- and 3.4-fold were observed in the chemiluminescence responses in the liver homogenates of the animals treated with endrin, chlordane, alachlor, chlorpyrifos and fenthion, respectively, while increases of 1.8-, 2.0-, 3.2-, 2.9- and 2.4-fold, respectively, were observed in the brain homogenates. Cultured neuroactive PC-12 cells were incubated with the pesticides and the release of the enzyme lactate dehydrogenase (LDH) into the media as an indicator of cellular damage and cytotoxicity was examined. Maximal release of LDH from cultured PC-12 cells was observed at 100 nM concentrations of the pesticides. Increases of 2.3-, 2.5-, 2.8-, 3.1 and 3.4-fold were observed in LDH leakage following incubation of the PC-12 cells with endrin, chlordane, alachlor, chlorpyrifos and fenthion, respectively. Following incubation of the cultured PC-12 cells with 100 nM concentrations of these same pesticides, increases in DNA-SSB of 2.5-, 2.2-, 2.1-, 2.4- and 2.5-fold, respectively, were observed. The results clearly demonstrate that these different classes of pesticides induce production of reactive oxygen species and oxidative tissue damage which may contribute to the toxic manifestations of these xenobiotics. Reactive oxygen species may serve as common mediators of programmed cell death (apoptosis) in response to many toxicants and pathological conditions.

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.

Chromium-induced excretion of urinary lipid metabolites, DNA damage, nitric oxide production, and generation of reactive oxygen species in Sprague-Dawley rats

Chromium and its salts induce cytotoxicity and mutagenesis, and vitamin E has been reported to attenuate chromate-induced cytotoxicity. These observations suggest that chromium produces reactive oxygen species which may mediate many of the untoward effects of chromium. We have therefore examined and compared the effects of Cr(III) (chromium chloride hexahydrate) and Cr(VI) (sodium dichromate) following single oral doses (0.50 LD50) on the production of reactive oxygen species by peritoneal macrophages, and hepatic mitochondria and microsomes in rats. The effects of Cr(III) and Cr(VI) on hepatic mitochondrial and microsomal lipid peroxidation and enhanced excretion of urinary lipid metabolites as well as the incidence of hepatic nuclear DNA damage and nitric oxide (NO) production were also examined. Increases in lipid peroxidation of 1.8- and 2.2-fold occurred in hepatic mitochondria and microsomes, respectively, 48 hr after the oral administration of 25 mg Cr(VI)/kg, while increases of 1.2- and 1.4-fold, respectively, were observed after 895 mg Cr(III)/kg. The urinary excretion of malondialdehyde (MDA), formaldehyde (FA), acetaldehyde (ACT) and acetone (ACON) were determined at 0-96 hr after Cr administration. Between 48 and 72 hr post-treatment, maximal excretion of the four urinary lipid metabolites was observed with increases of 1.5- to 5.4-fold in Cr(VI) treated rats. Peritoneal macrophages from Cr(VI) treated animals 48 hr after treatment resulted in 1.4- and 3.6-fold increases in chemiluminescence and iodonitrotetrazolium reduction, indicating enhanced production of superoxide anion, while macrophages from Cr(III) treated animals showed negligible increases. Increases in DNA single strand breaks of 1.7-fold and 1.5-fold were observed following administration of Cr(VI) and Cr(III), respectively, at 48 hr post-treatment. Enhanced production of NO by peritoneal exudate cells (primarily macrophages) was monitored following Cr(VI) administration at both 24 and 48 hr post-treatment with enhanced production of NO being observed at both timepoints. The results indicate that both Cr(VI) and Cr(III) induce an oxidative stress at equitoxic doses, while Cr(VI) induces greater oxidative stress in rats as compared with Cr(III) treated animals.

Protective effects of antioxidants against endrin-induced hepatic lipid peroxidation, DNA damage, and excretion of urinary lipid metabolites

Oxidative stress is believed to play a pivotal role in endrin-induced hepatic and neurologic toxicity. Therefore, the effects of the antioxidants vitamin E succinate and ellagic acid have been examined on hepatic lipid peroxidation, DNA single-strand breaks (SSB), and the urinary excretion of lipid metabolites following an acute oral dose of 4.5 mg endrin/kg. Groups of rats were pretreated with 100 mg/kg vitamin E succinate for 3 d followed by 40 mg/kg on day 4, or 6.0 mg ellagic acid/kg for 3 d p.o. followed by 3.0 mg/kg on day 4 or the vehicle. Endrin was administered p.o. on day 4 2 hr after treatment with the antioxidant. All animals were killed 24 h after endrin administration. Vitamin E succinate pretreatment decreased the endrin-induced increase in hepatic mitochondrial and microsomal lipid peroxidation by approximately 60% and 40%, respectively. Ellagic acid pretreatment reduced the endrin-induced increased in mitochondrial and microsomal lipid peroxidation by approximately 76 and 79%, respectively. Both vitamin E succinate and ellagic acid alone produced small but nonsignificant decreases in hepatic mitochondrial and microsomal lipid peroxidation. A 3.3-fold increase in the incidence of hepatic nuclear DNA single-strand breaks was observed 24 h after endrin administration. Pretreatment of rats with vitamin E succinate, vitamin E, and ellagic acid decreased endrin-induced DNA-SSB by approximately 47%, 22%, and 21%, respectively. Pretreatment of rats with vitamin E succinate decreased the endrin-induced increase in the urinary excretion of malondialdehyde, acetaldehyde, formaldehyde, and acetone by approximately 68, 65, 70, and 55%, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Adriamycin-induced hepatic and myocardial lipid peroxidation and DNA damage, and enhanced excretion of urinary lipid metabolites in rats

Adriamycin produces clinically useful responses in a variety of human cancers including lymphomas, leukemias, and solid tumors. However, the toxicity of adriamycin has limited its usefulness. Iron-catalyzed free radical reactions as the peroxidation of membrane lipids, inactivation of critical enzymes, and the inhibition of DNA, RNA and protein synthesis in heart, liver and kidney have been implicated in the toxicity of adriamycin. In order to further assess the role of oxidative stress in the toxicity of adriamycin, the effects of adriamycin were examined on the urinary excretion of lipid metabolites at 0, 6, 12, 24, 48 and 72 h post-treatment, and on myocardial and hepatic lipid peroxidation and nuclear DNA single strand breaks at 24 h post-treatment following single oral and intravenous (i.v.) doses of 10 mg/kg adriamycin. Urinary malondialdehyde (MDA), formaldehyde (FA), acetaldehyde (ACT) and acetone (ACON) excretion was significantly increased at all time points examined. Following the oral administration of adriamycin, maximum excretion of MDA, FA, ACT and ACON of 6.2-, 2.7-, 3.7- and 2.2-fold relative to control values, respectively, occurred 24 h after treatment. However, following the i.v. administration of adriamycin, greatest increases in excretion of MDA, FA and ACT reaching 6.9-, 3.3- and 6.3-fold relative to control values, respectively, were observed 6 h after treatment, while the greatest increase in ACON excretion of 4.2-fold relative to control values occurred 12 h post-treatment. Following oral and i.v. administration of adriamycin, significant increases were observed in myocardial and hepatic lipid peroxidation in mitochondrial and microsomal membranes, and myocardial and hepatic nuclei DNA single strand breaks 24 h after treatment. The results indicate that adriamycin administration induces myocardial and hepatic lipid peroxidation which may be responsible for enhanced excretion of urinary lipid metabolites as a result of membrane damage, and also induces enhanced DNA damage. These effects may be due to adriamycin-induced production of reactive oxygen species.

Production of reactive oxygen species by peritoneal macrophages and hepatic mitochondria and microsomes from endrin-treated rats

Recent studies have shown that the administration of endrin to rodents induces lipid peroxidation in various tissues and decreases glutathione content. These results suggest that endrin produces reactive oxygen species and/or free radicals. We have therefore examined the effect of endrin (4.5 mg/kg) on the production of reactive oxygen species by peritoneal macrophages and hepatic mitochondria and microsomes in rats. The effects of endrin on hepatic mitochondrial and microsomal lipid peroxidation and membrane fluidity as well as the incidence of hepatic nuclear DNA damage were also examined. Twenty-four hours after endrin administration, significant increases in the production of chemiluminescence by the three tissue fractions were observed. Furthermore, peritoneal macrophages from endrin-treated animals resulted in 3.0- and 2.8-fold increases in cytochrome c and iodonitrotetrazolium (INT) reduction, indicating enhanced production of superoxide anion. Endrin administration also resulted in significant increases in lipid peroxidation of mitochondrial and microsomal membranes as well as decreases in the fluidity of these two membranous fractions. A significant increase in hepatic nuclear DNA single-strand breaks also occurred in response to endrin administration. The results indicate that macrophage, mitochondria, and microsomes produce reactive oxygen species following endrin administration, and these reactive oxygen species may contribute to the toxic manifestations of endrin.

Endrin-induced increases in hepatic lipid peroxidation, membrane microviscosity, and DNA damage in rats

Endrin is a polyhalogenated cyclic hydrocarbon pesticide which produces hepatic and neurologic toxicity. Previous studies have indicated that endrin induces hepatic lipid peroxidation. In order to further assess the possible role of lipid peroxidation in the toxicity of endrin, the dose- and time-dependent effects of endrin on hepatic lipid peroxidation, membrane microviscosity and DNA damage in rats were examined. Rats were treated with 0, 3.0, 4.5, or 6.0 mg endrin/kg as a single oral dose in corn oil, and the animals were killed 0, 12, 24, 48, or 72 h post-treatment. Dose-dependent increases in hepatic mitochondrial and microsomal lipid peroxidation and microviscosity as well as nuclear DNA single strand breaks were observed as early as 12 h post-treatment. Maximum increases in these three parameters occurred 24 h after endrin administration at all three doses. While the incidence in DNA damage decreased with time after 24 h, the incidence of lipid peroxidation and microviscosity of microsomal and mitochondrial membranes remained relatively constant. Dose- and time-dependent increases in liver and spleen weight/body weight ratios with decreases in thymus weight/body weight ratios were observed. The data indicate that endrin administration induces hepatic lipid peroxidation which may be responsible for the increased membrane microviscosity as a result of membrane damage as well as enhanced DNA damage.

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.

Comparative studies on lipid peroxidation and DNA-single strand breaks induced by lindane, DDT, chlordane and endrin in rats

1. A variety of structurally dissimilar polyhalogenated cyclic hydrocarbons produce similar toxic effects. The molecular mechanisms involved in the production of these toxic manifestations is not known. 2. We have proposed that reactive oxygen species may be involved, and have therefore examined the time-dependent effects of lindane (30 mg/kg), DDT (40 mg/kg), chlordane (120 mg/kg), and endrin (4.5 mg/kg) on the production of hepatic mitochondrial and microsomal lipid peroxidation and DNA single strand breaks, two indices of oxidative stress. 3. All four xenobiotics resulted in significant increases in hepatic lipid peroxidation and DNA damage. Earliest (6 hr) increases in both lipid peroxidation and DNA damage were observed following lindane administration. Time-dependent increases in both parameters were observed following endrin administration. 4. Maximum increases in DNA single strand breaks of 2.8- and 2.5-fold were observed 12 hr after DDT and chlordane administration, respectively, while a 4.4-fold increase was observed 24 hr after endrin administration. 5. The results demonstrate that the four structurally dissimilar polyhalogenated hydrocarbons produce oxidative tissue damage which may contribute to the toxic manifestations of these xenobiotics, and exhibit different toxicokinetic properties.

Oxidative stress induced by chronic administration of sodium dichromate [Cr(VI)] to rats

Chromium occurs in the workplace primarily in the valence forms Cr(III) and Cr(VI). Recent studies have demonstrated that sodium dichromate [Cr(VI)] induces greater oxidative stress as compared with Cr(III), as indicated by the production of reactive oxygen species by peritoneal macrophages and hepatic mitochondria and microsomes, and enhanced excretion of urinary lipid metabolites and hepatic DNA-single strand breaks (SSB) following acute oral administration of Cr(III) and Cr(VI). We have therefore examined the chronic effects of sodium dichromate dihydrate [Cr(VI); 10 mg (33.56 mumol)/kg/day] on hepatic mitochondrial and microsomal lipid peroxidation, enhanced excretion of urinary lipid metabolites including malondialdehyde (MDA), formaldehyde (FA), acetaldehyde (ACT), acetone (ACON) and propionaldehyde (PROP), and hepatic DNA damage over a period of 90 days. The maximal increases in hepatic lipid peroxidation and DNA damage were observed at approximately 45 days of treatment. Maximum increases in the urinary excretion of MDA, FA, ACT, ACON and PROP were 3.2-, 2.6-, 4.1-, 3.3- and 2.1-fold, respectively, while a 5.2-fold increase in DNA-SSB was observed. The results clearly indicate that chronic sodium dichromate administration induces oxidative stress resulting in tissue damaging effects which may contribute to the toxicity and carcinogenicity of hexavalent chromium.