Ezdihar A. Hassoun

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.

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.

The role of antioxidant enzymes in tcdd-induced oxidative stress in various brain regions of rats after subchronic exposure

The induction of oxidative stress by TCDD in various brain regions of rats has been investigated after subchronic exposure. TCDD was administered by gavage to female Sprague-Dawley rats at daily doses of 0, 10, 22, and 46 ng/kg for 13 weeks. The brains were dissected to cerebral cortex (Cc), hippocampus (H), cerebellum (C), and brain stem (Bs); the production of superoxide anion (SA) and lipid peroxides and the activities of the antioxidant enzymes superoxide dismutase (SOD), catalase, and glutathione peroxidase (GSH-Px) were determined in those regions. TCDD caused dose-dependent increases in the production of SA and lipid peroxidation in Cc and H and those were associated with dose-dependent suppressions of SOD. While a TCDD dose of 10 ng/kg/d resulted in significant increases in catalase and GSH-Px activities in Cc and H, doses of 22 and 46 ng/kg/d resulted in dose-dependent suppressions of these two enzymes in the same regions. In the C and Bs, TCDD treatment did not result in significant production of SA and lipid peroxidation but it resulted in dose-dependent increases in the activities of various antioxidant enzymes. These results suggest that Cc and H are vulnerable to TCDD-induced oxidative stress after subchronic exposure, and that C and Bs are protected against that effect.

The Effects of Ellagic Acid and Vitamin E Succinate on Antioxidant Enzymes Activities and Glutathione Levels in Different Brain Regions of Rats After Subchronic Exposure to TCDD

Ellagic acid (EA) and vitamin E succinate (VES) were previously shown to protect against 2, 3, 7, 8-tetrachlorodibenzo-p-dioxin (TCDD)-induced reactive oxygen species (ROS) overproduction in certain brain regions of rats after subchronic exposure. The current study was designed to assess the modulation of antioxidant enzyme activities and glutathione (GSH) levels as protective measures for VES and EA against TCDD-induced ROS overproduction in four regions of rat brain. TCDD was administered to groups of rats at a daily dose of 46 ng/kg for 90 d. EA and VES were administered to some other groups of rats either alone or simultaneously with TCDD, every other day for 90 d. At the end of the treatment period, animals were sacrificed and brain regions were dissected, including cerebral cortex (Cc), hippocampus (H), cerebellum (C), and brainstem (Bs), for assay of superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GSH-Px) activities, as well as GSH levels. While treatment of rats with VES alone or in combination with TCDD resulted in significant increases in SOD and CAT activities in different brain regions, treatment with EA resulted in a significant rise in total GSH levels and GSH-Px activity in those regions. Results may suggest antioxidant modulation by VES and EA as a mechanism for the previously observed protection by these compounds against TCDD-induced ROS overproduction in brain. Data also indicate there are two different pathways in the protection provided by the two antioxidants.

The induction of oxidative stress and cellular death by the drinking water disinfection by-products, dichloroacetate and trichloroacetate in J774.A1 cells.

The in vitro toxicity of the drinking water disinfection by products dichloroacetate (DCA) and trichloroacetate (TCA) were studied using the J774A.1 macrophage cell line. DCA and TCA were added to cell cultures at concentrations ranging between 8-32 mM and incubated for 24, 36 and 60 h. DCA and TCA effects on cellular viability, lactate dehydrogenase (LDH) release and superoxide anion (SA) production by the cells, as well as superoxide dismutase (SOD) activities of the cells were determined. DCA and TCA caused time- and concentration-dependent increases in cellular death, in LDH release and production of SA by the cells. The compounds also caused modulations in SOD activities of the cells, with increases observed at the lower concentrations and/or shorter periods of incubations and suppression with the higher concentrations and/or longer periods of incubation. The results of the study indicate that DCA and TCA induce macrophage activation and that the activation is associated with cellular toxicity. Also, DCA and TCA are found to be equitoxic to J774.A1 cells.

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 (O−2), 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 O−2 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.

Dichloroacetate- and trichloroacetate-induced phagocytic activation and production of oxidative stress in the hepatic tissues of mice after acute exposure.

Dichoroacetate (DCA) and trichloroacetate (TCA) are by‐products formed during chlorination of the drinking water and were found to be hepatotoxic and hepatocarcinogenic in rodents. In this study, the abilities of the compounds to induce oxidative stress and phagocytic activation have been studied in B6C3F1 mice. Groups of mice were administered 300 mg/kg of either DCA or TCA, p.o, and were sacrificed after 6 or 12 h. Peritoneal lavage cells (PLCs) were isolated and assayed for superoxide anion (SA) production, and hepatic tissues were assayed for the production of SA, lipid peroxidation (LP), and DNA‐single strand breaks (SSBs). TCA resulted in significant production of SA in the PLCs, and in the production of SA, LP, and DNA‐SSBs in the hepatic tissues, 12 h after dosing, as compared with the control. DCA administration, on the other hand, resulted in significant increases in the productions of LP and DNA‐SSBs in the hepatic tissues at both time points, and in SA production in PLCs and hepatic tissues, 6 h after dosing. However, DCA‐induced increases in SA production in PLC and hepatic tissues declined at the 12‐h time point, reaching control level in the hepatic tissues. These results may implicate the contribution of phagocytic activation to the induction of oxidative stress in the hepatic tissues and also the role of SA production in the induction of LP and/or DNA damage in those tissues, in response to the compounds. The results also suggest studying the involvement of these mechanisms in the long‐term hepatotoxicity/hepatocarcinogencity of the compounds.

Ricin-induced toxicity in the macrophage J744A.1 cells: The role of TNF-? and the modulation effects of TNF-? 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‐α and lactate dehydrogenase (LDH), as well as the modulation of the ricin‐induced effects by TNF‐α 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‐α 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‐α 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‐α by the macrophages in response to ricin plays a significant role in the toxicity of ricin and that TNF‐α antibody can antagonize the effects of ricin in this cell line when added at relatively low concentrations.

Dichloroacetate- and Trichloroacetate-Induced Modulation of Superoxide Dismutase, Catalase, and Glutathione Peroxidase Activities and Glutathione Level in the livers of Mice after Subacute and Subchronic exposure

Dichloroacetate (DCA) and trichloroacetate (TCA) were previously found to induce various levels of oxidative stress in the hepatic tissues of mice after subacute and subchronic exposures. The cells are known to have several protective mechanisms against production of oxidative stress by different xenobiotics. To assess the roles of the antioxidant enzymes and glutathione (GSH) in DCA- and TCA-induced oxidative stress, groups of B6C3F1 mice were administered either DCA or TCA at doses of 7.7, 77, 154, and 410 mg kg−1 day−1, by gavage for 4 weeks (4-W) and 13 weeks (13-W), and superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GSH-Px) activities, as well as GSH were determined in the hepatic tissues. DCA at doses ranging between 7.7–410, and 7.7–77 mg kg−1 day−1, given for 4-W and 13-W, respectively, resulted in either suppression or no change in SOD, CAT, and GSH-Px activities, but doses of 154–410 mg DCA kg−1 day−1 administered for 13-W were found to result in a significant induction of the three enzyme activities. TCA administration on the other hand, resulted in increases in the SOD and CAT activities, but caused suppression of GSH-Px activity in both the periods. Except for the DCA doses of 77–154 mg kg−1 day−1 administered for 13-W that resulted in a significant reduction in the GSH levels, all other DCA as well as TCA treatments produced no changes in GSH. Since these enzymes are involved in the detoxification of the reactive oxygen species (ROS), superoxide anion (SA), and H2O2, it is concluded that SA is the main contributor to DCA-induced oxidative stress, while both ROS contribute to that of TCA. The increase in the enzyme activities associated with 154–410 mg DCA kg1− day−1 in the 13-W period suggest their role as protective mechanisms contributing to the survival of cells modified in response to those treatments.

Protective effects of lazaroid U74389F (16-desmethyl tirilazad) on endrin-induced lipid peroxidation and DNA damage in brain and liver and regional distribution of catalase activity in rat brain

Endrin, a poly-halogenated cyclic hydrocarbon, induces hepatic lipid peroxidation, modulates calcium homeostasis, decreases membrane fluidity, and increases nuclear DNA damage. Little information is available on the neurotoxicity of endrin. The effects of endrin on lipid peroxidation, DNA damage, and regional distribution of catalase activity were assessed in rat brain and liver 24 h following an acute oral dose of 4.5 mg endrin/kg. Lipid peroxidation associated with whole brain mitochondria increased 2.4-fold, whereas microsomal lipid peroxidation increased 2.8-fold following endrin administration. Lipid peroxidation also increased 2.0-fold both in hepatic mitochondria and microsomes. Catalase activity decreased 24% in the hypothalamus, 23% in the cortex, 38% in the cerebellum, and 11% in the brain stem in response to endrin. A 4.3-fold increase in brain nuclear DNA-single strand breaks (SSB) was observed in endrin-treated rats. Pretreatment of rats intraperitoneally with the lazaroid U74389F (16-desmethyl tirilazad) (10 mg/kg in two doses) attenuated the biochemical consequences of endrin-induced oxidative stress. The administration of U74389F in citrate buffer (pH 3.8) provided better protection than administering the lazaroid in corn oil, decreasing endrin-induced lipid peroxidation by 50-80% and DNA-SSB by approximately 72% in liver and 85% in brain, while ameliorating the suppressed catalase activity. The data suggest an involvement of an oxidative stress in the neurotoxicity and hepatotoxicity induced by endrin, which can be attenuated by the lazaroid U74389F.