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Induction of Oxidative Stress by Chronic Administration of Sodium Dichromate [Chromium VI] and Cadmium Chloride [Cadmium II] to Rats
Free Radical Biology & Medicine, Vol. 22, No. 3, pp. 471–478, 1997 Copyright q 1996 Elsevier Science Inc. Printed in the USA. All rights reserved 0891-5849/97 $17.00 / .00
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Original Contribution INDUCTION OF OXIDATIVE STRESS BY CHRONIC ADMINISTRATION OF SODIUM DICHROMATE [CHROMIUM VI] AND CADMIUM CHLORIDE [CADMIUM II] TO RATS D. Bagchi,* P. J. Vuchetich,* M. Bagchi,* E. A. Hassoun,† M. X. Tran,* L. Tang,* and S. J. Stohs* *School of Pharmacy and Allied Health Professions, Creighton University, Omaha, NE 68178, USA; and †College of Pharmacy, University of Toledo, Toledo, OH 43606, USA (Received 29 January 1996; Revised 30 April 1996; Accepted 20 June 1996)
Abstract—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. Copyright q 1996 Elsevier Science Inc. Keywords—Chromium, Cadmium, Oxidative stress, Lipid peroxidation, DNA single strand breaks, Liver, Brain, Malondialdehyde, Formaldehyde, Acetaldehyde, Acetone, Free radicals
icity, reproductive toxicity, genotoxicity, carcinogenicity, and environmental toxicity of chromium among industrially exposed chromium workers engaged in chrome plating, leather tanning, and stainless steel industries. This transition metal can exist in various oxidation states ranging from 02 to /6, but states 3 (III) and 6 (VI) are the most predominant oxidation states that are commonly encountered. Investigations in our laboratories have shown that at equitoxic doses (0.50 LD50) Cr(VI) more effectively induces formation of reactive oxygen species and causes oxidative tissue and DNA damage as compared to Cr(III).3 The chromate ion [CrO4]02, the dominant form
INTRODUCTION
Chromium is a widely used industrial chemical, finding uses in steel, alloy, cast irons, chrome, paints, metal finishes, and wood treatment. Chromium occurs in the workplace primarily in the valence forms Cr(III) and Cr(VI). Chromium is widely known to cause allergic dermatitis as well as toxic and carcinogenic effects in humans and animals.1,2 Von Burg and Liu2 have summarized the acute toxicity, chronic toxicity, neurotoxAddress correspondence to: Dr. S. J. Stohs, School of Pharmacy and Allied Health Professions, Creighton University, 2500 California Plaza, Omaha, NE 68178. 471
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of Cr(VI) in neutral aqueous solutions, can readily cross cellular membranes via nonspecific anion carriers, while Cr(III) is poorly transported across membranes.4 The differences in membrane transport may explain the differences in the abilities of these two forms of chromium to induce the formation of reactive oxygen species and produce oxidative tissue damaging effects. It has been demonstrated that the Cr(V) complexes that are produced in the reduction of Cr(VI) by cellular biological reductants react with hydrogen peroxide to generate hydroxyl radicals, which in turn, act as initiators of the primary events in Cr(VI) cytotoxicity.5 Hydrogen peroxide suppresses Cr(V) and enhances the formation of hydroxyl radicals through a Cr(V)-catalyzed Haber-Weiss–like reaction. Jones et al.6 have provided evidence that indicates that hydroxyl radicals are generated from a Cr(V) intermediate that is responsible for causing DNA strand breaks. Chronic oral administration of sodium dichromate [Cr(VI)] at a dose as low as 10 mg/kg/d (0.20 LD50) for a period of up to 90 d induces oxidative tissue and DNA damage, and the maximal effects were observed at approximately 45 d of treatment.7 Cadmium is an abundant, nonessential element that is widely used in electroplating and galvanizing, as a color pigment in paints, and in batteries. Soluble cadmium salts accumulate and result in toxicity to liver, brain, kidneys, lungs, heart, testes, and the central nervous system.1 The mechanisms responsible for the toxicity of cadmium are not entirely clear. It has been shown to elevate lipid peroxidation in tissues soon after exposure.8 Fariss9 has shown that free radical scavengers and antioxidants are useful in protecting against cadmium toxicity. Studies in our laboratories have demonstrated that a single oral 0.50 LD50 dose of cadmium chloride [Cd(II), 44 mg/kg] to rats results in increased hepatic mitochondrial and microsomal lipid peroxidation, hepatic glutathione depletion, and enhanced excretion of urinary lipid metabolites.10 In the present study, the low-dose chronic effects of orally administered sodium dichromate [Cr(VI), 2.5 mg/kg/d] and cadmium chloride [Cd(II), 4.4 mg/kg/d] on excretion of urinary lipid metabolites, hepatic and brain mitochondrial and microsomal lipid peroxidation, and hepatic and brain nuclear DNA single-strand breaks were monitored in rats for a period of 120 d. These concentrations of Cr(VI) and Cd(II) salts correspond to approximately 0.05 LD50 daily doses. MATERIALS AND METHODS
Chemicals Sodium dichromate [Cr(VI)] and cadmium chloride [Cd(II)] were obtained from Aldrich, Milwaukee, WI.
All other chemicals used in this study were obtained from Sigma Chemical Co., St. Louis, MO, and were of analytical grade or of the highest grade available. Animals and treatment Female Sprague–Dawley rats, weighing 160 to 180 g, were purchased from Sasco, Inc. (Omaha, NE). The animal research protocol for this project was approved by the Creighton University Animal Research Committee (ARC#0156). All animals were housed two per cage and allowed to acclimate to the environment for 4–5 d prior to experimental use. The animals were allowed free access to tap water and food (Purina Rodent Lab Chow #5001). The rats were maintained at a temperature of 217C with lighting from 0600 to 1800 h, daily. Chromium (VI)-treated groups of animals received a daily dose of 2.5 mg sodium dichromate/kg orally in water for 120 d. Cadmium (II)-treated groups of animals received a daily dose of 4.4 mg cadmium chloride/kg orally in water for 120 d. The control animals received the vehicle. Animals were placed in metabolism cages for urine collection on days 0, 15, 30, 45, 60, 75, 90, 105, and 120 d of treatment as described below. Other animals were killed by decapitation at these same time points. Urine collection Rats were placed in metabolism cages (Nalgene Co., Rochester, NY) for urine collection for 6 h. During urine collection, the animals were allowed free access to tap water but received no food. Animals were allowed food and water ad lib between urine collection periods. The urine-collecting vessels were positioned over styrofoam containers filled with dry ice, which permitted the collection of urine in the frozen state over the 6.0-h time periods. The samples were stored at 0707C until analyzed.11 All samples were analyzed within 1 week of collection. Identification and quantitation of urinary lipid metabolites The lipid excretion products in rats were identified by using a Hewlett-Packard GC system coupled to a Finnigan Incos 50 quadrupole mass spectrometer as previously described by Shara et al.11 2,4-Dinitrophenylhydrazine was used as the derivatizing agent in the identification of the urinary metabolites by GC-MS. 2,4-Dinitrophenylhydrazine derivatives of urinary lipid metabolites were quantitated by HPLC at 330 nm using an acetonitrile:water (49:51, v/v) mobile phase at a flow rate of 1 ml/min.11 The HPLC system consisted of a
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model 510 Waters pump (Milford, MA), a Waters model U6K loop injector, a Waters mBondapak C18 (10 ˚ , 30 cm 1 3.9 mm inner dimm particle size, 125 A ameter) column fitted with a Rainin RP-18 5-mm ODGU precolumn cartridge and a model 484 Waters tunable absorbance detector. The recorder chart speed was set at 0.25 cm/min. Details of the calibration curves for each of the synthetic hydrazones and their extraction efficiencies have been described by Shara et al.11 The data are expressed as nmol/kg/6 h. Lipid peroxidation Lipid peroxidation was determined on liver and brain mitochondria and microsomes from control and treated rats according to the method of Buege and Aust,12 based on the formation of thiobarbituric acid reactive substances (TBARS). Malondialdehyde was used as the standard and prepared according to the method of Largilliere and Melancon.13 A molar extinction coefficient of 1.56 1 105 M01 cm01 was used. DNA single-strand breaks DNA damage in hepatic nuclei was measured as single-strand breaks (SSB) by the alkaline elution method as previously described by us.14 DNA content was measured microfluorimetrically with 3,5-diaminobenzoic acid dihydrochloride as the complexing agent, with activation and emission wavelengths of 436 and 521 nm, respectively. The elution constant (k), which is used as a measure of DNA damage, was calculated from the formula k Å 02.30 1 slope of the plot of percent DNA remaining on the filter vs. volume of elute. Statistical analysis Data for each group were subjected to analysis of variance (ANOVA). Scheffe’s S method was used as the post hoc test. Significance between pair of mean values was also determined by Student’s t-test in some cases. The data are expressed as the mean { standard deviation (SD) of four to six animals. The level of statistical significance employed in all cases was p õ .05. RESULTS
The chronic effects of sodium dichromate [Cr(VI)] and cadmium chloride [Cd(II)] on lipid peroxidation in hepatic and brain mitochondria and microsomes are summarized in Figs. 1 and 2. Lipid peroxidation studies, based on the formation of thiobarbituric acid reactive substances (TBARS), were conducted on days 0, 15, 30, 45, 60, 75, 90, and 120 of treatment. An age-
Fig. 1. Female Sprague – Dawley rats were treated orally with either 2.5 mg sodium dichromate [Cr(VI)]/kg/d or 4.4 mg cadmium chloride [Cd(II)]/kg/d for 120 days, and lipid peroxidation was determined as TBARS content (nmol/mg protein) in the hepatic and brain mitochondrial and microsomal membranes after 0, 15, 30, 45, 60, 75, or 120 d of treatment. The data represent the increased lipid peroxidation of: (A) hepatic mitochondria; (B) hepatic microsomes. Each value represents the mean { S.D. of four to six animals. Values with nonidentical superscripts are significantly different (p õ .05).
dependent increase in lipid peroxidation was observed over the 120 d of this study in control animals. Lipid peroxidation increased 46 and 26% in hepatic mitochondria (Fig. 1A) and microsomes (Fig. 1B), respectively, in control animals between days 0 and 120. In these same control animals, approximately 34 and 36% increases in brain mitochondrial (Fig. 2A) and microsomal (Fig. 2B) lipid peroxidation were observed between days 0 and 120. Maximum increases in hepatic and brain mitochondrial and microsomal lipid peroxidation occurred at approximately 60–75 d of treatment (Fig. 1 and 2) with both Cr(VI) and Cd(II), and no significant increases were observed beyond 75 d of treatment. Approximately 1.8- and 2.1-fold increases in TBARS content
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a 0.05 LD50 daily dose of Cd(II) as compared to this same dose of Cr(VI). DNA single-strand breaks (SSB) are another index of oxidative stress and oxidative DNA damage. The effects of chronic Cr(VI) and Cd(II) administration on liver and brain nuclear DNA single-strand breaks (SSB) are shown in Fig. 3. DNA-SSB were measured on days 0, 15, 30, 45, 60, 75, and 120 of treatment. In control animals, approximately 70 and 103% increases in DNA-SSB were observed in the hepatic and brain nuclei, respectively, between days 0 and 120. The maximum increases in DNA-SSB in the hepatic and brain nuclei relative to control values were observed between 30 and 45 d of treatment with both Cr(VI) and Cd(II) (Fig. 3). Maximum increases of 2.4fold and 3.8-fold were observed in the hepatic nuclear
Fig. 2. Female Sprague – Dawley rats were treated orally with either 2.5 mg sodium dichromate [Cr(VI)]/kg/d or 4.4 mg cadmium chloride [Cd(II)]/kg/d for 120 days, and lipid peroxidation was determined as TBARS content (nmol/mg protein) in the hepatic and brain mitochondrial and microsomal membranes after 0, 15, 30, 45, 60, 75, or 120 d of treatment. The data represent the increased lipid peroxidation of: (A) brain mitochondria; and (B) brain microsomes. Each value represents the mean { S.D. of four to six animals. Values with nonidentical superscripts are significantly different (p õ .05).
were observed in the hepatic mitochondria following treatment of the rats for 75 d with Cr(VI) and Cd(II), respectively, as compared to control animals (Fig. 1A), while increases of approximately 1.7-fold were observed in the hepatic microsomes at this time point following chronic treatment of the animals with both salts (Fig. 1B). Maximum increases in TBARS content of 2.0-fold and 2.4-fold were observed in the brain mitochondria following treatment with Cr(VI) and Cd(II) ions (Fig. 2A), respectively, as compared to control animals, while under the same conditions maximum increases of 1.9-fold and 2.2-fold were observed in brain microsomal lipid peroxidation following daily treatment of rats with Cr(VI) and Cd(II) salts, respectively (Fig. 2B). Thus, modestly greater increases in liver and brain lipid peroxidation were observed in response to
Fig. 3. Female Sprague–Dawley rats were treated orally with either 2.5 mg sodium dichromate [Cr(VI)/kg/d] of 4.4 mg cadmium chloride [Cd(II)/kg/d] for 120 d, and nuclear DNA single-strand breaks were determined by the alkaline elution technique after 0, 15, 30, 45, 60, 75, or 120 d of treatment. The data represent the incidence of increased DNA-SSB of: (A) hepatic nuclei; and (B) brain nuclei. Each value represents the mean { SD of four to six animals. Values with nonidentical superscripts are significantly different (p õ .05).
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DNA-SSB following treatment of the animals with Cr(VI) and Cd(II) ions (Fig. 3A), respectively, as compared to control animals, while under the same treatment conditions approximately 1.3-fold and 2.0-fold increases in brain nuclear DNA-SSB were observed following treatment of the animals with Cr(VI) and Cd(II) salts, respectively (Fig. 3B). The effects of daily Cr(VI) (2.5 mg/kg/day) and Cd(II) (4.4 mg/kg/day) administration on the excretion of the urinary lipid metabolites malondialdehyde, formaldehyde, acetaldehyde, and acetone during 120 d of treatment are presented in Fig. 4, respectively. These metabolites provide another index of lipid peroxidation.11 Urine samples were collected for 6 h on days 0, 15, 30, 45, 60, 75, 90, 105, and 120 of treatment, and were analyzed on these same days. In control animals, gradual increases in the excretion of the four metabolites occurred with time. Approximately 111, 34, 162, and 65% increases in urinary excretion of malondialdehyde, formaldehyde, acetaldehyde, and acetone, respectively, were observed between days 0 and 120 (Fig.
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4A–D). Increases occurred in the excretion of these metabolites at each time point. The effects of daily Cr(VI) and Cd(II) administration on the urinary excretion of malondialdehyde are presented in Fig. 4A. A maximum increase in malondialdehyde excretion was observed at approximately 75 d of treatment with both Cr(VI) and Cd(II) relative to control values. Increases of 2.1-, and 1.8-fold occurred in the urinary excretion of malondialdehyde after 75 d of Cr(VI) and Cd(II) treatment, as compared to control animals. Maximum formaldehyde excretion occurred between 60–75 d of daily treatment for both Cr(VI) and Cd(II) relative to control values (Fig. 4B). Urine levels of formaldehyde in Cr(VI) and Cd(II)-treated animals increased by approximately 1.8- and 1.5-fold on day 75 of treatment, respectively, relative to control animals. The maximum increases in acetaldehyde excretion during daily treatment with Cr(VI) and Cd(II) occurred at approximately days 60 and 75, respectively, relative
Fig. 4. Female Sprague–Dawley rats were treated orally with either 2.5 mg sodium dichromate [Cr(VI)/kg/d] or 4.4 mg cadmium chloride [Cd(II)/kg/d] for 120 days, and urine samples were collected for 6.0 h over dry ice after 0, 15, 30, 45, 60, 75, 90, 105, or 120 days of treatment. The data represent the excretion of: (A) malondialdehyde; (B) formaldehyde; (C) acetaldehyde; and (D) acetone. Each value represents the mean { SD of four to six animals. Values with nonidentical superscripts are significantly different (p õ .05).
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to control values (Fig. 4C). In chronic Cr(VI)-treated animals the urinary excretion of acetaldehyde increased by approximately 2.1- and 1.9-fold on treatment day 75, respectively, relative to control animals. Maximum increases in acetone excretion during treatment with Cr(VI) and Cd(II) were observed at approximately 60 and 45 d of the study, respectively, relative to control values (Fig. 4D). Increases of approximately 2.1- and 1.5-fold occurred in acetone excretion on day 60 with Cr(VI) and Cd(II) treatment, respectively (Fig. 4D), relative to control animals. DISCUSSION
Because most toxicities associated with Cr(VI) and Cd(II) involve low-dose chronic exposure, the current studies were conducted using a daily oral dose of 2.5 mg/kg for Cr(VI) and 4.4 mg/kg of Cd(II) for up to 120 d. Each daily dose represents approximately a 0.05 LD50 value in rats. The production of tissue damage under these conditions was assessed by measuring hepatic and brain mitochondrial and microsomal lipid peroxidation, hepatic, and brain DNA single-strand breaks (SSB), and the urinary excretion of lipid metabolites over the 120-d study. No previous studies have examined the effects of chronic, low-dose daily administration of Cr(VI) or Cd(II), and previous investigations have assessed oxidative tissue damage primarily in liver. The results clearly demonstrate that a low-dose daily administration of the two cations results in time-dependent increases in hepatic (Fig. 1) and brain (Fig. 2) microsomal, and mitochondrial lipid peroxidation, hepatic, and brain nuclear DNA-SSB (Fig. 3), and enhanced urinary excretion of lipid metabolites (Fig. 4). Greater increases in lipid peroxidation were observed in both liver and brain by Cd(II) as compared to Cr(VI), while maximum increases in lipid peroxidation in liver and brain mitochondria and microsomes in response to both Cr(VI) and Cd(II) occurred between 60–75 d of treatment. In previous studies, a dose of 10 mg/kg Cr(VI) resulted in maximum increases in hepatic mitochondrial and microsomal lipid peroxidation following 45–60 d of treatment,7 and the maximum increase in hepatic DNA-SSB was observed after 30 d of treatment.7 Furthermore, in these studies maximum enhanced urinary excretion of malondialdehyde and acetone was observed at the 45-d time point, while the maximum increases in excretion of acetaldehyde and formaldehyde were observed at approximately 30 and 60 d of treatment, respectively.7 In the present study, significant increases in lipid peroxidation and DNA damage were observed after 15 d of treatment with both Cr(VI) and Cd(II) with max-
imum increases occurring between 30–45 d of treatment. Thus, dose and time-dependent increases in these parameters occur. Significant increases in the urinary excretion of the four lipid metabolites were observed in most cases after 30 d of treatment with the two cations. Thus, the results clearly indicate that low-dose daily exposure results in cumulative effects which ultimately plateau after sufficient exposure has occurred. The data also confirm that exposure to Cr(VI) and Cd(II) results in oxidative tissue-damaging effects. Reactive oxygen species are implicated in the toxicity of Cr(VI). The in vitro and in vivo effects of oxygen scavengers, glutathione, vitamin E, and vitamin C on Cr(VI)-induced injuries including DNA damage, lipid peroxidation, enzyme inhibition, cytotoxicity, and mutagenesis were extensively reviewed by Sugiyama et al.15,16 Vitamin E dramatically decreases Cr(VI)-induced cytotoxicity, lipid peroxidation, and DNA damage, suggesting the involvement of reactive oxygen species and/or free radicals in these processes. Furthermore, evidence indicates that reactive oxygen species are produced from Cr(VI) via the decomposition of the intermediary Cr(V) (O2)4 03 ion, resulting in DNA damage.17–19 The toxicity of cadmium [Cd(II)] may also involve reactive oxygen species. Manca et al.20 examined lipid peroxidation in liver, kidneys, brains, lungs, heart, and testes of rats given cadmium chloride intraperitoneally. The animals received from 25–1250 mg Cd (II)/kg, and were sacrificed 24-h posttreatment. Greatest increases in lipid peroxidation were demonstrated in lungs and brain as well as liver, based on the formation of thiobarbituric acid reactive substances (TBARS). These results indicate that lipid peroxidation is an early and sensitive consequence of Cd(II) exposure. Previous studies in our laboratory have shown that a single acute oral dose of either Cr(VI) (25 mg/kg) or Cd(II) (44 mg/kg) results in the production of reactive oxygen species and oxidative tissue damage within 24 h after treatment.3,10 The doses that were used are the approximate 0.50 LD50 values for Cr(VI) and Cd(II). Both Cr(VI) and Cd(II) resulted in significant increases in the urinary excretion of malondialdehyde, formaldehyde, acetaldehyde, and acetone, with maximum excretion occurring between 48 and 72 h posttreatment. Both cations resulted in significant increases in lipid peroxidation in hepatic mitochondria and microsomes as well as hepatic nuclear DNA-SSB.3,10 Furthermore, peritoneal macrophages from Cr(VI)-treated rats resulted in significant increases in chemiluminescence and iodonitrotetrazolium reduction, indicating enhanced production of superoxide anions.3 As previously noted, reactive oxygen species are produced from Cr(VI) via the decomposition of the in-
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termediary Cr(V)(O2)4 03 ion, and are believed to result in tissue damaging effects.17–19 The mechanism whereby Cd(II) induces an oxidative stress is not entirely clear. Cd(II) does not induce production of reactive oxygen species through a Haber-Weiss–like redox cycling mechanism as is characteristic of chromium, iron, copper, and vanadium.1 However, Cd(II) does cause depletion of glutathione and protein-bond sulfhydryls that can result in the production of an oxidative stress with subsequent oxidative tissue damage. This mechanism for the production of an oxidative stress is characteristic of other metal ions including mercury, nickel and lead.1 The sources of the oxidative lipid metabolites that are detected in the urine (Fig. 4) are not entirely clear. The increases in these products may occur via a variety of mechanisms including free radical-induced cellular injury and enhanced lipid peroxidation and/or b-oxidation. Radiolabeled malondialdehyde administered to rats is extensively metabolized to acetate and carbon dioxide.21 Thus, the urinary excretion of acetaldehyde may arise as an intermediate product in the breakdown of malondialdehyde that is formed due to lipid peroxidation. The enhanced formation of acetone in response to disease states such as diabetes as a consequence of increased b-oxidation is well known.22 Rat liver microsomes metabolize glycerol to formaldehyde,23,24 with the glycerol being a product of the metabolism of triglycerides by adipose tissues that possess the glycerol activating enzyme glycerol kinase. Liver and brown adipose tissues are known to have high glycerol kinase levels. Several other possible sources of formaldehyde include the breakdown of malondialdehyde to acetaldehyde or acetate and a one carbon fragment and/or cleavage of a one carbon fragment from the acetoacetic acid with the formation of acetone. In summary, the current results in conjunction with previous studies indicate that the daily low dose administration of sodium dichromate [Cr(VI)] and cadmium chloride [Cd(II)] to rats results in the formation of reactive oxygen species, enhanced hepatic and brain lipid peroxidation and nuclear DNA damage, and increased excretion of urinary lipid metabolites. At the doses that were employed (approximately 0.05 LD50), Cd(II) resulted in greater increases in hepatic and brain lipid peroxidation as well as DNA-SSB while Cr(VI) produced greater increases in the urinary excretion of lipid metabolites. These results may be due to differences in mechanisms as well as toxicokinetics. Good correlations appear to exist between the peroxidation of membrane lipids and DNA-SSB with the enhanced excretion of the urinary lipid metabolites. These ef-
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fects may contribute to the toxicity and carcinogenicity induced by hexavalent chromium and bivalent cadmium. Acknowledgements — This study was supported in part by a grant from the Air Force Office of Scientific Research (#94-1-0048). The authors thank Ms. LuAnn Schwery for technical assistance.
REFERENCES 1. Stohs, S. J.; Bagchi, D. Oxidative mechanisms in the toxicity of metal ions. Free Radic. Biol. Med. 18:321–336; 1995. 2. Von Burg, R.; Liu, D. Chromium and hexavalent chromium. J. Appl. Toxicol. 13:225–230; 1993. 3. Bagchi, D.; Hassoun, E. A.; Bagchi, M.; Stohs, S. J. Chromiuminduced excretion of urinary lipid metabolites, DNA damage, nitric oxide production, and generation of reactive oxygen species in Sprague–Dawley rats. Comp. Biochem. Physiol. 110C:177–187; 1995. 4. Danielsson, B. R. G.; Hassoun, E.; Dencker, L. Embryo toxicity of chromium: Distribution in pregnent mice and effects in embryonic cells in vitro. Arch. Toxicol. 51:233–245; 1982. 5. Kawanishi, S.; Inoue, S.; Sano, S. Mechanism of DNA cleavage induced by sodium chromate (VI) in the presence of hydrogen peroxide. J. Biol. Chem. 261:5952–5958; 1986. 6. Jones, P.; Kortenkamp, A.; O’Brien, P. , et al. Evidence for the generation of hydroxyl radicals from a chromium (V) intermediate isolated from the reaction of chromate with glutathione. Arch. Biochem. Biophys. 286:652–655; 1991. 7. Bagchi, D.; Hassoun, E. A.; Bagchi, M.; Muldoon, D. F.; Stohs, S. J. Oxidative stress induced by chronic administration of sodium dichromate [Cr(VI)] to rats. Comp. Biochem. Physiol. 110C:281–287; 1995. 8. Muller, L. Consequences of cadmium toxicity in rat hepatocytes: Mitochondrial dysfunction and lipid peroxidation. Toxicology 40:285–292; 1986. 9. Fariss, M. W. Cadmium toxicity: Unique cytoprotective properties of alpha tocopheryl succinate in hepatocytes. Toxicology 69:63–77; 1991. 10. Bagchi, D.; Bagchi, M.; Hassoun, E. A.; Stohs, S. J. Cadmiuminduced excretion of urinary lipid metabolites, DNA damage, glutathione depletion, and hepatic lipid peroxidation in Sprague– Dawley rats. Biol. Trace Elem. Res. 53:143–154; 1996. 11. Shara, M. A.; Dickson, P. H.; Bagchi, D.; Stohs, S. J. Excretion of formaldehyde, malondialdehyde, acetaldehyde and acetone in the urine of rats in response to oxidative stress. J. Chromatogr. 576:221–233; 1992. 12. Buege, J. A.; Aust, S. D. Microsomal lipid peroxidation. Methods Enzymol 52:302–310; 1978. 13. Largilliere, C.; Melancon, S. B. Free malondialdehyde determination in human plasma by high performance liquid chromatography. Anal. Biochem. 170:123–126; 1988. 14. Bagchi, M.; Hassoun, E. A.; Bagchi, D.; Stohs, S. J. Endrininduced increases in hepatic lipid peroxidation, membrane microviscosity and DNA damage in rats. Arch. Environ. Contam. Toxicol. 23:1–5; 1992. 15. Sugiyama, M. Effects of vitamins on chromium(VI)-induced damage. Environ. Health Perspect. 92:63–70; 1992. 16. Sugiyama, M. Role of physiological antioxidants in chromium (VI)-induced cellular injury. Free Radic. Biol. Med. 12:397–407; 1992. 17. Kawanishi, S.; Inoue, S.; Sano, S. Mechanism of DNA cleavage induced by sodium dichromate (VI) in the presence of hydrogen peroxide. J. Biol. Chem. 261:5952–5958; 1986. 18. Shi, X.; Dalal, N. S. Chromium (V) and hydroxyl radical formation during the glutathione reductase-catalyzed reduction of chromium (VI). Biochem. Biophys. Res. Commun. 163:627–634; 1989. 19. Shi, X.; Dalal, N. S. On the hydroxyl radical formation in the
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reaction between hydrogen peroxide and biologically generated chromium (V) species. Arch. Biochem. Biophys. 277:342–350; 1990. 20. Manca, D.; Ricard, A. C.; Trottier, B.; Chevalier, G. Studies on lipid peroxidation in rat tissues following administration of low and moderate doses of cadmium chloride. Toxicology 67:303– 323; 1991. 21. Dhanakoti, S. N.; Draper, H. H. Response of urinary malondialdehyde to factors that stimulate lipid peroxidation in vivo. Lipids 22:643–646; 1991.
22. Foster, D. W. Diabetes mellitus. In: Braunwald, E.; Isselbacher, K. J.; Petersdorf, R. G.; Wilson, J. D.; Marten, J. B.; Fauci, A. S., eds. Harrison’s principles of internal medicine. 11th ed. New York: McGraw-Hill; 1987:1778–1796. 23. Winters, D. K.; Clejan, L. A.; Cederbaum, A. I. Oxidation of glycerol to formaldehyde by rat liver microsomes. Biochem. Biophys. Res. Commun. 153:612–617; 1988. 24. Clejan, L. A.; Cederbaum, A. I. Stimulation by paraquat of microsomal and cytochrome P-450-dependent oxidation of glycerol to formaldehyde. Biochem. J. 295:781–786; 1993.
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Original Contribution INDUCTION OF OXIDATIVE STRESS BY CHRONIC ADMINISTRATION OF SODIUM DICHROMATE [CHROMIUM VI] AND CADMIUM CHLORIDE [CADMIUM II] TO RATS D. Bagchi,* P. J. Vuchetich,* M. Bagchi,* E. A. Hassoun,† M. X. Tran,* L. Tang,* and S. J. Stohs* *School of Pharmacy and Allied Health Professions, Creighton University, Omaha, NE 68178, USA; and †College of Pharmacy, University of Toledo, Toledo, OH 43606, USA (Received 29 January 1996; Revised 30 April 1996; Accepted 20 June 1996)
Abstract—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. Copyright q 1996 Elsevier Science Inc. Keywords—Chromium, Cadmium, Oxidative stress, Lipid peroxidation, DNA single strand breaks, Liver, Brain, Malondialdehyde, Formaldehyde, Acetaldehyde, Acetone, Free radicals
icity, reproductive toxicity, genotoxicity, carcinogenicity, and environmental toxicity of chromium among industrially exposed chromium workers engaged in chrome plating, leather tanning, and stainless steel industries. This transition metal can exist in various oxidation states ranging from 02 to /6, but states 3 (III) and 6 (VI) are the most predominant oxidation states that are commonly encountered. Investigations in our laboratories have shown that at equitoxic doses (0.50 LD50) Cr(VI) more effectively induces formation of reactive oxygen species and causes oxidative tissue and DNA damage as compared to Cr(III).3 The chromate ion [CrO4]02, the dominant form
INTRODUCTION
Chromium is a widely used industrial chemical, finding uses in steel, alloy, cast irons, chrome, paints, metal finishes, and wood treatment. Chromium occurs in the workplace primarily in the valence forms Cr(III) and Cr(VI). Chromium is widely known to cause allergic dermatitis as well as toxic and carcinogenic effects in humans and animals.1,2 Von Burg and Liu2 have summarized the acute toxicity, chronic toxicity, neurotoxAddress correspondence to: Dr. S. J. Stohs, School of Pharmacy and Allied Health Professions, Creighton University, 2500 California Plaza, Omaha, NE 68178. 471
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of Cr(VI) in neutral aqueous solutions, can readily cross cellular membranes via nonspecific anion carriers, while Cr(III) is poorly transported across membranes.4 The differences in membrane transport may explain the differences in the abilities of these two forms of chromium to induce the formation of reactive oxygen species and produce oxidative tissue damaging effects. It has been demonstrated that the Cr(V) complexes that are produced in the reduction of Cr(VI) by cellular biological reductants react with hydrogen peroxide to generate hydroxyl radicals, which in turn, act as initiators of the primary events in Cr(VI) cytotoxicity.5 Hydrogen peroxide suppresses Cr(V) and enhances the formation of hydroxyl radicals through a Cr(V)-catalyzed Haber-Weiss–like reaction. Jones et al.6 have provided evidence that indicates that hydroxyl radicals are generated from a Cr(V) intermediate that is responsible for causing DNA strand breaks. Chronic oral administration of sodium dichromate [Cr(VI)] at a dose as low as 10 mg/kg/d (0.20 LD50) for a period of up to 90 d induces oxidative tissue and DNA damage, and the maximal effects were observed at approximately 45 d of treatment.7 Cadmium is an abundant, nonessential element that is widely used in electroplating and galvanizing, as a color pigment in paints, and in batteries. Soluble cadmium salts accumulate and result in toxicity to liver, brain, kidneys, lungs, heart, testes, and the central nervous system.1 The mechanisms responsible for the toxicity of cadmium are not entirely clear. It has been shown to elevate lipid peroxidation in tissues soon after exposure.8 Fariss9 has shown that free radical scavengers and antioxidants are useful in protecting against cadmium toxicity. Studies in our laboratories have demonstrated that a single oral 0.50 LD50 dose of cadmium chloride [Cd(II), 44 mg/kg] to rats results in increased hepatic mitochondrial and microsomal lipid peroxidation, hepatic glutathione depletion, and enhanced excretion of urinary lipid metabolites.10 In the present study, the low-dose chronic effects of orally administered sodium dichromate [Cr(VI), 2.5 mg/kg/d] and cadmium chloride [Cd(II), 4.4 mg/kg/d] on excretion of urinary lipid metabolites, hepatic and brain mitochondrial and microsomal lipid peroxidation, and hepatic and brain nuclear DNA single-strand breaks were monitored in rats for a period of 120 d. These concentrations of Cr(VI) and Cd(II) salts correspond to approximately 0.05 LD50 daily doses. MATERIALS AND METHODS
Chemicals Sodium dichromate [Cr(VI)] and cadmium chloride [Cd(II)] were obtained from Aldrich, Milwaukee, WI.
All other chemicals used in this study were obtained from Sigma Chemical Co., St. Louis, MO, and were of analytical grade or of the highest grade available. Animals and treatment Female Sprague–Dawley rats, weighing 160 to 180 g, were purchased from Sasco, Inc. (Omaha, NE). The animal research protocol for this project was approved by the Creighton University Animal Research Committee (ARC#0156). All animals were housed two per cage and allowed to acclimate to the environment for 4–5 d prior to experimental use. The animals were allowed free access to tap water and food (Purina Rodent Lab Chow #5001). The rats were maintained at a temperature of 217C with lighting from 0600 to 1800 h, daily. Chromium (VI)-treated groups of animals received a daily dose of 2.5 mg sodium dichromate/kg orally in water for 120 d. Cadmium (II)-treated groups of animals received a daily dose of 4.4 mg cadmium chloride/kg orally in water for 120 d. The control animals received the vehicle. Animals were placed in metabolism cages for urine collection on days 0, 15, 30, 45, 60, 75, 90, 105, and 120 d of treatment as described below. Other animals were killed by decapitation at these same time points. Urine collection Rats were placed in metabolism cages (Nalgene Co., Rochester, NY) for urine collection for 6 h. During urine collection, the animals were allowed free access to tap water but received no food. Animals were allowed food and water ad lib between urine collection periods. The urine-collecting vessels were positioned over styrofoam containers filled with dry ice, which permitted the collection of urine in the frozen state over the 6.0-h time periods. The samples were stored at 0707C until analyzed.11 All samples were analyzed within 1 week of collection. Identification and quantitation of urinary lipid metabolites The lipid excretion products in rats were identified by using a Hewlett-Packard GC system coupled to a Finnigan Incos 50 quadrupole mass spectrometer as previously described by Shara et al.11 2,4-Dinitrophenylhydrazine was used as the derivatizing agent in the identification of the urinary metabolites by GC-MS. 2,4-Dinitrophenylhydrazine derivatives of urinary lipid metabolites were quantitated by HPLC at 330 nm using an acetonitrile:water (49:51, v/v) mobile phase at a flow rate of 1 ml/min.11 The HPLC system consisted of a
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model 510 Waters pump (Milford, MA), a Waters model U6K loop injector, a Waters mBondapak C18 (10 ˚ , 30 cm 1 3.9 mm inner dimm particle size, 125 A ameter) column fitted with a Rainin RP-18 5-mm ODGU precolumn cartridge and a model 484 Waters tunable absorbance detector. The recorder chart speed was set at 0.25 cm/min. Details of the calibration curves for each of the synthetic hydrazones and their extraction efficiencies have been described by Shara et al.11 The data are expressed as nmol/kg/6 h. Lipid peroxidation Lipid peroxidation was determined on liver and brain mitochondria and microsomes from control and treated rats according to the method of Buege and Aust,12 based on the formation of thiobarbituric acid reactive substances (TBARS). Malondialdehyde was used as the standard and prepared according to the method of Largilliere and Melancon.13 A molar extinction coefficient of 1.56 1 105 M01 cm01 was used. DNA single-strand breaks DNA damage in hepatic nuclei was measured as single-strand breaks (SSB) by the alkaline elution method as previously described by us.14 DNA content was measured microfluorimetrically with 3,5-diaminobenzoic acid dihydrochloride as the complexing agent, with activation and emission wavelengths of 436 and 521 nm, respectively. The elution constant (k), which is used as a measure of DNA damage, was calculated from the formula k Å 02.30 1 slope of the plot of percent DNA remaining on the filter vs. volume of elute. Statistical analysis Data for each group were subjected to analysis of variance (ANOVA). Scheffe’s S method was used as the post hoc test. Significance between pair of mean values was also determined by Student’s t-test in some cases. The data are expressed as the mean { standard deviation (SD) of four to six animals. The level of statistical significance employed in all cases was p õ .05. RESULTS
The chronic effects of sodium dichromate [Cr(VI)] and cadmium chloride [Cd(II)] on lipid peroxidation in hepatic and brain mitochondria and microsomes are summarized in Figs. 1 and 2. Lipid peroxidation studies, based on the formation of thiobarbituric acid reactive substances (TBARS), were conducted on days 0, 15, 30, 45, 60, 75, 90, and 120 of treatment. An age-
Fig. 1. Female Sprague – Dawley rats were treated orally with either 2.5 mg sodium dichromate [Cr(VI)]/kg/d or 4.4 mg cadmium chloride [Cd(II)]/kg/d for 120 days, and lipid peroxidation was determined as TBARS content (nmol/mg protein) in the hepatic and brain mitochondrial and microsomal membranes after 0, 15, 30, 45, 60, 75, or 120 d of treatment. The data represent the increased lipid peroxidation of: (A) hepatic mitochondria; (B) hepatic microsomes. Each value represents the mean { S.D. of four to six animals. Values with nonidentical superscripts are significantly different (p õ .05).
dependent increase in lipid peroxidation was observed over the 120 d of this study in control animals. Lipid peroxidation increased 46 and 26% in hepatic mitochondria (Fig. 1A) and microsomes (Fig. 1B), respectively, in control animals between days 0 and 120. In these same control animals, approximately 34 and 36% increases in brain mitochondrial (Fig. 2A) and microsomal (Fig. 2B) lipid peroxidation were observed between days 0 and 120. Maximum increases in hepatic and brain mitochondrial and microsomal lipid peroxidation occurred at approximately 60–75 d of treatment (Fig. 1 and 2) with both Cr(VI) and Cd(II), and no significant increases were observed beyond 75 d of treatment. Approximately 1.8- and 2.1-fold increases in TBARS content
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a 0.05 LD50 daily dose of Cd(II) as compared to this same dose of Cr(VI). DNA single-strand breaks (SSB) are another index of oxidative stress and oxidative DNA damage. The effects of chronic Cr(VI) and Cd(II) administration on liver and brain nuclear DNA single-strand breaks (SSB) are shown in Fig. 3. DNA-SSB were measured on days 0, 15, 30, 45, 60, 75, and 120 of treatment. In control animals, approximately 70 and 103% increases in DNA-SSB were observed in the hepatic and brain nuclei, respectively, between days 0 and 120. The maximum increases in DNA-SSB in the hepatic and brain nuclei relative to control values were observed between 30 and 45 d of treatment with both Cr(VI) and Cd(II) (Fig. 3). Maximum increases of 2.4fold and 3.8-fold were observed in the hepatic nuclear
Fig. 2. Female Sprague – Dawley rats were treated orally with either 2.5 mg sodium dichromate [Cr(VI)]/kg/d or 4.4 mg cadmium chloride [Cd(II)]/kg/d for 120 days, and lipid peroxidation was determined as TBARS content (nmol/mg protein) in the hepatic and brain mitochondrial and microsomal membranes after 0, 15, 30, 45, 60, 75, or 120 d of treatment. The data represent the increased lipid peroxidation of: (A) brain mitochondria; and (B) brain microsomes. Each value represents the mean { S.D. of four to six animals. Values with nonidentical superscripts are significantly different (p õ .05).
were observed in the hepatic mitochondria following treatment of the rats for 75 d with Cr(VI) and Cd(II), respectively, as compared to control animals (Fig. 1A), while increases of approximately 1.7-fold were observed in the hepatic microsomes at this time point following chronic treatment of the animals with both salts (Fig. 1B). Maximum increases in TBARS content of 2.0-fold and 2.4-fold were observed in the brain mitochondria following treatment with Cr(VI) and Cd(II) ions (Fig. 2A), respectively, as compared to control animals, while under the same conditions maximum increases of 1.9-fold and 2.2-fold were observed in brain microsomal lipid peroxidation following daily treatment of rats with Cr(VI) and Cd(II) salts, respectively (Fig. 2B). Thus, modestly greater increases in liver and brain lipid peroxidation were observed in response to
Fig. 3. Female Sprague–Dawley rats were treated orally with either 2.5 mg sodium dichromate [Cr(VI)/kg/d] of 4.4 mg cadmium chloride [Cd(II)/kg/d] for 120 d, and nuclear DNA single-strand breaks were determined by the alkaline elution technique after 0, 15, 30, 45, 60, 75, or 120 d of treatment. The data represent the incidence of increased DNA-SSB of: (A) hepatic nuclei; and (B) brain nuclei. Each value represents the mean { SD of four to six animals. Values with nonidentical superscripts are significantly different (p õ .05).
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DNA-SSB following treatment of the animals with Cr(VI) and Cd(II) ions (Fig. 3A), respectively, as compared to control animals, while under the same treatment conditions approximately 1.3-fold and 2.0-fold increases in brain nuclear DNA-SSB were observed following treatment of the animals with Cr(VI) and Cd(II) salts, respectively (Fig. 3B). The effects of daily Cr(VI) (2.5 mg/kg/day) and Cd(II) (4.4 mg/kg/day) administration on the excretion of the urinary lipid metabolites malondialdehyde, formaldehyde, acetaldehyde, and acetone during 120 d of treatment are presented in Fig. 4, respectively. These metabolites provide another index of lipid peroxidation.11 Urine samples were collected for 6 h on days 0, 15, 30, 45, 60, 75, 90, 105, and 120 of treatment, and were analyzed on these same days. In control animals, gradual increases in the excretion of the four metabolites occurred with time. Approximately 111, 34, 162, and 65% increases in urinary excretion of malondialdehyde, formaldehyde, acetaldehyde, and acetone, respectively, were observed between days 0 and 120 (Fig.
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4A–D). Increases occurred in the excretion of these metabolites at each time point. The effects of daily Cr(VI) and Cd(II) administration on the urinary excretion of malondialdehyde are presented in Fig. 4A. A maximum increase in malondialdehyde excretion was observed at approximately 75 d of treatment with both Cr(VI) and Cd(II) relative to control values. Increases of 2.1-, and 1.8-fold occurred in the urinary excretion of malondialdehyde after 75 d of Cr(VI) and Cd(II) treatment, as compared to control animals. Maximum formaldehyde excretion occurred between 60–75 d of daily treatment for both Cr(VI) and Cd(II) relative to control values (Fig. 4B). Urine levels of formaldehyde in Cr(VI) and Cd(II)-treated animals increased by approximately 1.8- and 1.5-fold on day 75 of treatment, respectively, relative to control animals. The maximum increases in acetaldehyde excretion during daily treatment with Cr(VI) and Cd(II) occurred at approximately days 60 and 75, respectively, relative
Fig. 4. Female Sprague–Dawley rats were treated orally with either 2.5 mg sodium dichromate [Cr(VI)/kg/d] or 4.4 mg cadmium chloride [Cd(II)/kg/d] for 120 days, and urine samples were collected for 6.0 h over dry ice after 0, 15, 30, 45, 60, 75, 90, 105, or 120 days of treatment. The data represent the excretion of: (A) malondialdehyde; (B) formaldehyde; (C) acetaldehyde; and (D) acetone. Each value represents the mean { SD of four to six animals. Values with nonidentical superscripts are significantly different (p õ .05).
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to control values (Fig. 4C). In chronic Cr(VI)-treated animals the urinary excretion of acetaldehyde increased by approximately 2.1- and 1.9-fold on treatment day 75, respectively, relative to control animals. Maximum increases in acetone excretion during treatment with Cr(VI) and Cd(II) were observed at approximately 60 and 45 d of the study, respectively, relative to control values (Fig. 4D). Increases of approximately 2.1- and 1.5-fold occurred in acetone excretion on day 60 with Cr(VI) and Cd(II) treatment, respectively (Fig. 4D), relative to control animals. DISCUSSION
Because most toxicities associated with Cr(VI) and Cd(II) involve low-dose chronic exposure, the current studies were conducted using a daily oral dose of 2.5 mg/kg for Cr(VI) and 4.4 mg/kg of Cd(II) for up to 120 d. Each daily dose represents approximately a 0.05 LD50 value in rats. The production of tissue damage under these conditions was assessed by measuring hepatic and brain mitochondrial and microsomal lipid peroxidation, hepatic, and brain DNA single-strand breaks (SSB), and the urinary excretion of lipid metabolites over the 120-d study. No previous studies have examined the effects of chronic, low-dose daily administration of Cr(VI) or Cd(II), and previous investigations have assessed oxidative tissue damage primarily in liver. The results clearly demonstrate that a low-dose daily administration of the two cations results in time-dependent increases in hepatic (Fig. 1) and brain (Fig. 2) microsomal, and mitochondrial lipid peroxidation, hepatic, and brain nuclear DNA-SSB (Fig. 3), and enhanced urinary excretion of lipid metabolites (Fig. 4). Greater increases in lipid peroxidation were observed in both liver and brain by Cd(II) as compared to Cr(VI), while maximum increases in lipid peroxidation in liver and brain mitochondria and microsomes in response to both Cr(VI) and Cd(II) occurred between 60–75 d of treatment. In previous studies, a dose of 10 mg/kg Cr(VI) resulted in maximum increases in hepatic mitochondrial and microsomal lipid peroxidation following 45–60 d of treatment,7 and the maximum increase in hepatic DNA-SSB was observed after 30 d of treatment.7 Furthermore, in these studies maximum enhanced urinary excretion of malondialdehyde and acetone was observed at the 45-d time point, while the maximum increases in excretion of acetaldehyde and formaldehyde were observed at approximately 30 and 60 d of treatment, respectively.7 In the present study, significant increases in lipid peroxidation and DNA damage were observed after 15 d of treatment with both Cr(VI) and Cd(II) with max-
imum increases occurring between 30–45 d of treatment. Thus, dose and time-dependent increases in these parameters occur. Significant increases in the urinary excretion of the four lipid metabolites were observed in most cases after 30 d of treatment with the two cations. Thus, the results clearly indicate that low-dose daily exposure results in cumulative effects which ultimately plateau after sufficient exposure has occurred. The data also confirm that exposure to Cr(VI) and Cd(II) results in oxidative tissue-damaging effects. Reactive oxygen species are implicated in the toxicity of Cr(VI). The in vitro and in vivo effects of oxygen scavengers, glutathione, vitamin E, and vitamin C on Cr(VI)-induced injuries including DNA damage, lipid peroxidation, enzyme inhibition, cytotoxicity, and mutagenesis were extensively reviewed by Sugiyama et al.15,16 Vitamin E dramatically decreases Cr(VI)-induced cytotoxicity, lipid peroxidation, and DNA damage, suggesting the involvement of reactive oxygen species and/or free radicals in these processes. Furthermore, evidence indicates that reactive oxygen species are produced from Cr(VI) via the decomposition of the intermediary Cr(V) (O2)4 03 ion, resulting in DNA damage.17–19 The toxicity of cadmium [Cd(II)] may also involve reactive oxygen species. Manca et al.20 examined lipid peroxidation in liver, kidneys, brains, lungs, heart, and testes of rats given cadmium chloride intraperitoneally. The animals received from 25–1250 mg Cd (II)/kg, and were sacrificed 24-h posttreatment. Greatest increases in lipid peroxidation were demonstrated in lungs and brain as well as liver, based on the formation of thiobarbituric acid reactive substances (TBARS). These results indicate that lipid peroxidation is an early and sensitive consequence of Cd(II) exposure. Previous studies in our laboratory have shown that a single acute oral dose of either Cr(VI) (25 mg/kg) or Cd(II) (44 mg/kg) results in the production of reactive oxygen species and oxidative tissue damage within 24 h after treatment.3,10 The doses that were used are the approximate 0.50 LD50 values for Cr(VI) and Cd(II). Both Cr(VI) and Cd(II) resulted in significant increases in the urinary excretion of malondialdehyde, formaldehyde, acetaldehyde, and acetone, with maximum excretion occurring between 48 and 72 h posttreatment. Both cations resulted in significant increases in lipid peroxidation in hepatic mitochondria and microsomes as well as hepatic nuclear DNA-SSB.3,10 Furthermore, peritoneal macrophages from Cr(VI)-treated rats resulted in significant increases in chemiluminescence and iodonitrotetrazolium reduction, indicating enhanced production of superoxide anions.3 As previously noted, reactive oxygen species are produced from Cr(VI) via the decomposition of the in-
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termediary Cr(V)(O2)4 03 ion, and are believed to result in tissue damaging effects.17–19 The mechanism whereby Cd(II) induces an oxidative stress is not entirely clear. Cd(II) does not induce production of reactive oxygen species through a Haber-Weiss–like redox cycling mechanism as is characteristic of chromium, iron, copper, and vanadium.1 However, Cd(II) does cause depletion of glutathione and protein-bond sulfhydryls that can result in the production of an oxidative stress with subsequent oxidative tissue damage. This mechanism for the production of an oxidative stress is characteristic of other metal ions including mercury, nickel and lead.1 The sources of the oxidative lipid metabolites that are detected in the urine (Fig. 4) are not entirely clear. The increases in these products may occur via a variety of mechanisms including free radical-induced cellular injury and enhanced lipid peroxidation and/or b-oxidation. Radiolabeled malondialdehyde administered to rats is extensively metabolized to acetate and carbon dioxide.21 Thus, the urinary excretion of acetaldehyde may arise as an intermediate product in the breakdown of malondialdehyde that is formed due to lipid peroxidation. The enhanced formation of acetone in response to disease states such as diabetes as a consequence of increased b-oxidation is well known.22 Rat liver microsomes metabolize glycerol to formaldehyde,23,24 with the glycerol being a product of the metabolism of triglycerides by adipose tissues that possess the glycerol activating enzyme glycerol kinase. Liver and brown adipose tissues are known to have high glycerol kinase levels. Several other possible sources of formaldehyde include the breakdown of malondialdehyde to acetaldehyde or acetate and a one carbon fragment and/or cleavage of a one carbon fragment from the acetoacetic acid with the formation of acetone. In summary, the current results in conjunction with previous studies indicate that the daily low dose administration of sodium dichromate [Cr(VI)] and cadmium chloride [Cd(II)] to rats results in the formation of reactive oxygen species, enhanced hepatic and brain lipid peroxidation and nuclear DNA damage, and increased excretion of urinary lipid metabolites. At the doses that were employed (approximately 0.05 LD50), Cd(II) resulted in greater increases in hepatic and brain lipid peroxidation as well as DNA-SSB while Cr(VI) produced greater increases in the urinary excretion of lipid metabolites. These results may be due to differences in mechanisms as well as toxicokinetics. Good correlations appear to exist between the peroxidation of membrane lipids and DNA-SSB with the enhanced excretion of the urinary lipid metabolites. These ef-
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fects may contribute to the toxicity and carcinogenicity induced by hexavalent chromium and bivalent cadmium. Acknowledgements — This study was supported in part by a grant from the Air Force Office of Scientific Research (#94-1-0048). The authors thank Ms. LuAnn Schwery for technical assistance.
REFERENCES 1. Stohs, S. J.; Bagchi, D. Oxidative mechanisms in the toxicity of metal ions. Free Radic. Biol. Med. 18:321–336; 1995. 2. Von Burg, R.; Liu, D. Chromium and hexavalent chromium. J. Appl. Toxicol. 13:225–230; 1993. 3. Bagchi, D.; Hassoun, E. A.; Bagchi, M.; Stohs, S. J. Chromiuminduced excretion of urinary lipid metabolites, DNA damage, nitric oxide production, and generation of reactive oxygen species in Sprague–Dawley rats. Comp. Biochem. Physiol. 110C:177–187; 1995. 4. Danielsson, B. R. G.; Hassoun, E.; Dencker, L. Embryo toxicity of chromium: Distribution in pregnent mice and effects in embryonic cells in vitro. Arch. Toxicol. 51:233–245; 1982. 5. Kawanishi, S.; Inoue, S.; Sano, S. Mechanism of DNA cleavage induced by sodium chromate (VI) in the presence of hydrogen peroxide. J. Biol. Chem. 261:5952–5958; 1986. 6. Jones, P.; Kortenkamp, A.; O’Brien, P. , et al. Evidence for the generation of hydroxyl radicals from a chromium (V) intermediate isolated from the reaction of chromate with glutathione. Arch. Biochem. Biophys. 286:652–655; 1991. 7. Bagchi, D.; Hassoun, E. A.; Bagchi, M.; Muldoon, D. F.; Stohs, S. J. Oxidative stress induced by chronic administration of sodium dichromate [Cr(VI)] to rats. Comp. Biochem. Physiol. 110C:281–287; 1995. 8. Muller, L. Consequences of cadmium toxicity in rat hepatocytes: Mitochondrial dysfunction and lipid peroxidation. Toxicology 40:285–292; 1986. 9. Fariss, M. W. Cadmium toxicity: Unique cytoprotective properties of alpha tocopheryl succinate in hepatocytes. Toxicology 69:63–77; 1991. 10. Bagchi, D.; Bagchi, M.; Hassoun, E. A.; Stohs, S. J. Cadmiuminduced excretion of urinary lipid metabolites, DNA damage, glutathione depletion, and hepatic lipid peroxidation in Sprague– Dawley rats. Biol. Trace Elem. Res. 53:143–154; 1996. 11. Shara, M. A.; Dickson, P. H.; Bagchi, D.; Stohs, S. J. Excretion of formaldehyde, malondialdehyde, acetaldehyde and acetone in the urine of rats in response to oxidative stress. J. Chromatogr. 576:221–233; 1992. 12. Buege, J. A.; Aust, S. D. Microsomal lipid peroxidation. Methods Enzymol 52:302–310; 1978. 13. Largilliere, C.; Melancon, S. B. Free malondialdehyde determination in human plasma by high performance liquid chromatography. Anal. Biochem. 170:123–126; 1988. 14. Bagchi, M.; Hassoun, E. A.; Bagchi, D.; Stohs, S. J. Endrininduced increases in hepatic lipid peroxidation, membrane microviscosity and DNA damage in rats. Arch. Environ. Contam. Toxicol. 23:1–5; 1992. 15. Sugiyama, M. Effects of vitamins on chromium(VI)-induced damage. Environ. Health Perspect. 92:63–70; 1992. 16. Sugiyama, M. Role of physiological antioxidants in chromium (VI)-induced cellular injury. Free Radic. Biol. Med. 12:397–407; 1992. 17. Kawanishi, S.; Inoue, S.; Sano, S. Mechanism of DNA cleavage induced by sodium dichromate (VI) in the presence of hydrogen peroxide. J. Biol. Chem. 261:5952–5958; 1986. 18. Shi, X.; Dalal, N. S. Chromium (V) and hydroxyl radical formation during the glutathione reductase-catalyzed reduction of chromium (VI). Biochem. Biophys. Res. Commun. 163:627–634; 1989. 19. Shi, X.; Dalal, N. S. On the hydroxyl radical formation in the
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reaction between hydrogen peroxide and biologically generated chromium (V) species. Arch. Biochem. Biophys. 277:342–350; 1990. 20. Manca, D.; Ricard, A. C.; Trottier, B.; Chevalier, G. Studies on lipid peroxidation in rat tissues following administration of low and moderate doses of cadmium chloride. Toxicology 67:303– 323; 1991. 21. Dhanakoti, S. N.; Draper, H. H. Response of urinary malondialdehyde to factors that stimulate lipid peroxidation in vivo. Lipids 22:643–646; 1991.
22. Foster, D. W. Diabetes mellitus. In: Braunwald, E.; Isselbacher, K. J.; Petersdorf, R. G.; Wilson, J. D.; Marten, J. B.; Fauci, A. S., eds. Harrison’s principles of internal medicine. 11th ed. New York: McGraw-Hill; 1987:1778–1796. 23. Winters, D. K.; Clejan, L. A.; Cederbaum, A. I. Oxidation of glycerol to formaldehyde by rat liver microsomes. Biochem. Biophys. Res. Commun. 153:612–617; 1988. 24. Clejan, L. A.; Cederbaum, A. I. Stimulation by paraquat of microsomal and cytochrome P-450-dependent oxidation of glycerol to formaldehyde. Biochem. J. 295:781–786; 1993.
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