In vivo effects of ascorbate and glutathione on the uptake of chromium, formation of chromium(V), chromiumDNA binding and 8-hydroxy-2'-deoxyguanosine in liver and kidney of Osteogenic Disorder Shionogi rats following treatment with chromium(VI)
Jeu-Ming P. Yuann1,
Ke Jian Liu2,4,
Joshua W. Hamilton1,3 and
Karen E. Wetterhahn1,
1 Department of Chemistry, Dartmouth College,
2 Department of Radiology and
3 Department of Pharmacology and Toxicology, Dartmouth Medical School, Hanover, NH 03755-3564, USA
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Abstract
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Several previous in vitro studies have indicated that ascorbate and glutathione are the major reductants of Cr(VI) in cells. In order to evaluate the in vivo effects of ascorbate and glutathione on Cr(VI)-induced carcinogenesis, Cr uptake and the formation of Cr(V), CrDNA adducts and 8-hydroxy-2'-deoxyguanosine (8-OH-dG) were measured in the liver and kidney of Osteogenic Disorder Shionogi (ODS) rats that lack the ability to synthesize ascorbate. Despite a 10-fold difference in tissue ascorbate levels among different dietary ascorbate groups, the Cr(V) signal intensity, Cr uptake and total CrDNA binding were not affected in either organ. Treatment of ODS rats with Cr(VI) (10 mg/kg) had no substantial effect on the levels of ascorbate and glutathione in these tissues. The levels of Cr(V) and CrDNA binding were ~2-fold higher in the liver than in the kidney, although the levels of total Cr uptake were similar in both tissues. Cr uptake levels were significantly lower in the liver and kidney of ODS rats treated with high levels of ascorbate and a high dose of Cr(VI) (40 mg/kg), suggesting a detoxifying role played by plasma ascorbate. Similarly, modulation of glutathione levels by N-acetyl-L-cysteine, L-buthionine-S,R-sulfoximine or phorone in these animals by up to 2-fold had little or no consistent effect on Cr uptake, CrDNA binding, Cr(V) levels or 8-OH-dG formation in either organ. One possible explanation is that reduction of ascorbate and glutathione concentration to <10 and 50%, respectively, of normal in these two organs still provides threshold levels of these two reductants that are in excess of what is needed for significant reductive activation of Cr(VI). Alternatively, it is possible that ascorbate and glutathione do not play a major role in the formation of Cr(V), CrDNA binding or 8-OH-dG and that other cellular reductants, such as cysteine or other amino acids, might be more important reductants of Cr(VI) in vivo.
Abbreviations: BHT, butylated hydroxytoluene; BSO, L-buthionine-S,R-sulfoximine; DETAPAC, diethylenetriamine pentaaetic acid; dG, 2'-deoxyguanosine; DTNB, 5,5'-dithiobis-(2-nitrobenzoic acid); NAC, N-acetyl-L-cysteine; NPSH, non-protein sulfhydryl; 8-OH-dG, 8-hydroxy-2'-deoxyguanosine; ODS, osteogenic disorder Shionogi; PBS, phosphate-buffered saline; ROS, reactive oxygen species; TCA, trichloroacetic acid.
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Introduction
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Certain Cr(VI) compounds have been shown to be human carcinogens (1). Cr(VI) can also cause DNA damage and toxic effects in both humans and animals (2). Epidemiological evidence has documented a high risk of cancer of the lung, larynx and nasal cavities in workers in industries producing chromate (3,4). However, Cr(VI) itself is relatively unreactive toward DNA in vitro in the absence of a chemical or enzymatic reductant (5). According to the uptakereduction model of Cr(VI) carcinogenicity (6,7), Cr(VI) (as chromate CrO42) under physiological conditions crosses the cell membrane through the anion transport system (8) and is then rapidly reduced to lower oxidation states, i.e. Cr(V), Cr(IV) and Cr(III), by a variety of intracellular components. Cr(VI) can also react with intracellular reductants to generate unstable radical species, e.g. hydroxyl radical (HO.) (9), thiyl radicals (RS.) (10), ascorbate radical (Asc.) (11) and carbon-based radicals (R.) (12). Any of these Cr or radical species may potentially target DNA and cause various types of damage. Among the possible reductants in cells, L-ascorbate (vitamin C) and glutathione are two of the non-enzymatic reductants that have been postulated to play important roles in the reduction of Cr(VI) (6). Ascorbate was attributed to the reducing capacity in lung lavage fluids of adult rats (13). On the other hand, in rats pretreated with diethylmaleate, an electrophilic glutathione depleting agent, the injected Cr(VI) was excreted primarily as Cr(VI), suggesting the importance of the reducing power of glutathione in rats (14). However, ascorbate reduces Cr(VI) in vitro at a faster rate than does glutathione under normal physiological conditions (15). Furthermore, recent studies have shown that ascorbate, but not glutathione, is the principal reductant of Cr(VI) in vitro in ultrafiltrates from rat liver, lung and kidney (16,17). Nevertheless, the role of ascorbate in the in vivo reduction of Cr(VI) is not known and the effect of glutathione levels on the in vivo reduction of Cr(VI) at different levels of ascorbate has also not been established.
Recently, it was reported that in vitro CrDNA binding resulted in DNA polymerase arrest at specific sites, suggesting that this may be responsible for the inhibition of DNA synthesis by Cr (18). Treatment of rat lung cytosol with L-ascorbate oxidase, which specifically eliminated ascorbate, prior to incubation with Cr(VI) and DNA completely inhibited in vitro CrDNA binding (17). Depletion of glutathione with L-buthionine-S,R-sulfoximine (BSO), an inhibitor of glutathione synthase, had little or no effect on in vivo CrDNA binding in Cr(VI)-treated SpragueDawley rats (19).
Cr(V) has been implicated as the intermediate species responsible for direct CrDNA adduct formation in vitro and in vivo (20,21). Cr(VI) has been shown to react with ascorbate to generate Cr(V) and carbon-based radicals that caused CrDNA binding and DNA strand breaks, respectively, in vitro using calf thymus DNA or pBR322 plasmid DNA (22). Reaction of pBR322 DNA with Cr(VI) in the presence of various thiols led to the formation of Cr(V) and CrDNA adducts and the levels of Cr bound to DNA correlated with the levels and stability of the Cr(V) species formed (20). In 14-day-old chick embryos treated with Cr(VI) in vivo, a correlation was observed between CrDNA binding, formation of DNAprotein crosslinks and Cr(V) formation in the liver. In contrast, in chick embryo red blood cells, Cr(VI) caused DNA single-strand breaks, whereas Cr(V) and DNAprotein crosslinks were not observed (21,23,24). Cr(V) has also been detected by low frequency EPR in live mice following i.v. injection of Cr(VI) (25). In cultured cells, Cr(V) has been implicated in the formation of DNA strand breaks. Suppression of Cr(V) formation by the metal chelator o-phenanthroline has been shown to result in a decrease in chromate-induced DNA breaks and/or alkali-labile sites in Chinese hamster V-79 cells (26). In addition, hydrogen peroxide-resistant Chinese hamster ovary cells were found to have less Cr(V) and simultaneously fewer DNA strand breaks than those in the parental cells (27). Pretreatment of V-79 cells with ascorbate decreased Cr(VI)-induced alkaline-labile sites and Cr(V), but increased DNAprotein crosslinks (28).
Cr(VI) has been shown to react with H2O2 to generate HO. and Cr(V) species in vitro (9). Both Cr(VI) and Cr(V) species, but not Cr(III), are capable of oxidizing 2'-deoxyguanosine (dG) bases of calf thymus DNA to generate 8-hydroxy-2'-deoxyguanosine (8-OH-dG) (29). 8-OH-dG is therefore a useful marker for Cr(VI) generation of HO. and other reactive oxygen species (ROS). In addition, formation of 8-OH-dG has been shown to cause mispairing with adenine during DNA replication, giving rise to G:C
T:A transversion mutations (30,31). 8-OH-dG has been implicated in mutational events responsible for carcinogenesis and is also believed to play an important role in the natural process of aging (32). Ascorbate has been shown to increase the levels of 8-OH-dG in vitro (33,34). Cr(VI) treatment had no effect on the levels of 8-OH-dG in the liver and kidney of normal SpragueDawley rats (19). However, the effect of Cr(VI) treatment on the formation of 8-OH-dG in vivo at different levels of ascorbate and glutathione has not been addressed.
Most animals, including normal rats, have the ability to synthesize L-ascorbate from D-glucose via the D-glucuronic acid pathway (35), in which the last step, the conversion of L-gulono-
-lactone into L-ascorbic acid is catalyzed by L-gulonolactone oxidase (36). For some animals, including primates and guinea pigs, this enzyme is missing and regular dietary supplementation of ascorbate is required. The Osteogenic Disorder Shionogi (ODS) rat is a mutant of the Wistar strain that also lacks L-gulonolactone oxidase. Addition of ascorbate at 300 p.p.m. or greater in their drinking water prevents any sign of ascorbate deficiency, but dietary levels below this can lead to serious ascorbate deficiencies (37). Rats were able to grow and reproduce normally when the diet was supplemented with
300 p.p.m. ascorbate (38). Therefore, ODS rats provide an ideal model in which the levels of ascorbate in tissue can be modulated. The purpose of this study was to investigate the roles of ascorbate and glutathione in the reduction of Cr(VI) in vivo and subsequent DNA damage by measuring the effects of dietary ascorbate and glutathione modulation on the levels of Cr uptake, Cr(V) formation, total CrDNA binding and 8-OH-dG formation in livers and kidneys of ODS rats.
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Materials and methods
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Chemicals
All chemicals were reagent grade or better and used without further purification. L-Ascorbate (sodium salt), 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB), glutathione, L-ascorbate oxidase (from Cucurbita spp.), ribonuclease A (RNase A, from bovine pancreas), butylated hydroxytoluene (BHT), dG, nuclease P1 (from Penicillium citrinum), N-acetyl-L-cysteine (NAC), BSO, acivicin (
-amino-3-chloro-4,5-dihydro-5-isoxazole-acetic acid) and alkaline phosphatase I (type VII-S, from bovine intestinal mucosa) were purchased from Sigma Chemical Co. (St Louis, MO). Phorone was purchased from Aldrich Chemical Co. (Milwaukee, WI). RNase A was prepared in ribonuclease buffer (10 mM Tris, 15 mM NaCl, pH 7.4) to make a final concentration of 10 mg/ml. 8-OH-dG was purchased from Cayman Chemical Co. (Ann Arbor, MI). Proteinase K were purchased from Gibco BRL (Gaithersburg, MD). All enzymes were stored at 20°C.
Treatment of ODS rats
The ODS rats were initially purchased from Shionogi Research Laboratory (Shiga, Japan) and bred under license at the Veterans Administration Research Center Hospital (White River Junction, VT). The ascorbate-free (basal) diets were purchased from Zeigler Bros. (Gardners, PA). Six to eight-week-old male ODS rats were treated with various levels (50 and 800 p.p.m.) of ascorbic acid or sodium ascorbate in their drinking water for 23 weeks before the experiments. Sodium dichromate (Na2Cr2O7·2H2O, 10 or 40 mg/kg) was injected into the rats i.p. After killing, liver and kidney were immediately excised, rinsed in ice-cold phosphate-buffered saline (PBS) (136.9 mM NaCl, 2.682 mM KCl, 10.14 mM Na2HPO4, 1.764 mM KH2PO4), blotted and stored at 70°C until analysis.
Modulation of glutathione levels in rats
Treatment of rats with NAC was carried out according to Gandy et al. (39). Phorone was administered as 250 mg/ml 0.9% sesame oil/kg, i.p. 4 h before Cr(VI) (39).
Due to its low solubility, BSO in aqueous solution was prepared according to Monroe and Eaton (40). In order to avoid precipitation of BSO in solution, BSO was administrated as 4.0 mmol/5 ml PBS/kg i.p. while the BSO solution was still warm, 5 h before Cr(VI) (41).
Ascorbate assay
Ascorbate was assayed by the method of Berger et al. (42) with minor modifications. Ascorbate was analyzed using a Hewlett Packard 1050 HPLC system connected with an LC-4C ECD (electrochemical detection) controller (Bioanalytical Systems, West Layfayette, IN). The mobile phase was composed of 49 mM sodium acetate, 0.024% n-octylamine and 1 mM EDTA, adjusted to pH 4.0 with HPLC grade o-phosphoric acid and continuously purged with helium, administered at a flow rate of 0.6 ml/min using a Rainin C-18 column (4.6 mm i.d.x150 mm) with a spherical packing diameter of 5 µm.
Due to the instability of ascorbate, all the steps from tissue homogenization to the injection of samples on the HPLC were done within 2 h. Approximately 0.1 g of tissue was homogenized in 10 vol (w/v) of trichloroacetic acid (TCA)/diethylenetriamine pentaaetic acid (DETAPAC) buffer (5% TCA treated with AG 50W-X8 for 24 h, 1 mM DETAPAC, deaerated by Ar for 2 h before use) using a Wheaton glass homogenizer with Teflon pestle at a speed of 5000 r.p.m. for ~10 s. The homogenate was centrifuged at 12 400 g and 4°C for 10 min. After centrifugation, the supernatant was then filtered with an LC13 PVDF 0.2 µm acrodisc (Gelman Sciences, Ann Arbor, MI). The filtrate was then diluted 10- and 50-fold (v/v) with TCA/DETAPAC buffer for low and high ascorbate samples, respectively, before analysis. Sodium ascorbate of known concentrations (6380 pmol/100 µl) was used as a standard for quantitation of ascorbate levels of samples. Because the extinction coefficient of ascorbate varies from 7500 to 20 400/M/cm depending on pH (43), the ascorbate standard was quantified in 1 mM HCl solution. Under these conditions, >95% of the ascorbate is in the acidic form (pKa1 = 4.25) (44), which has an established extinction coefficient of 10 000/M/cm at
= 243 nm (45).
Non-protein sulfhydryl (NPSH) assay
Approximately 0.1 g of tissue was used in the determination of glutathione levels, expressed as NPSH (46), in the liver and kidney of ODS rats. In rat liver and kidney, the NPSH pool is principally composed of glutathione (47). Tissues were homogenized in 10 vol (w/v) of ice-cold TCA/DETAPAC buffer and centrifuged as described above. Samples were kept on ice until being assayed. Freshly made DTNB in water (500 µl of 1 mM) was added to 100 µl of supernatant or freshly prepared glutathione standard (0100 nmol/100 µl) followed by 2 ml of 5% sodium citrate. The pH range of these solutions was 5.505.75. After 5 min, the absorbance at 412 nm was determined using a Perkin Elmer Lambda 2 UV/VIS spectrometer. The extinction coefficient of DTNBglutathione product at 412 nm was 1.41 ± 0.08x104/M/cm, comparable with the literature value of 1.42x104/M/cm (48).
Preparation of tissue ultrafiltrates
Approximately 0.2 g of accurately weighed tissue was homogenized as described above in 10 vol (w/v) of ice-cold and deaerated Tris/DETAPAC buffer (100 mM TrisHCl, chelated with AG 50W-X8, 1 mM DETAPAC, pH 7.0 at 37°C). The homogenate was then deaerated with a mild stream of Ar for ~30 s and centrifuged at 12 000 g and 4°C for 15 min. The supernatant was then transferred into the sample reservoir of a Centricon 30 (30 000 mol. wt cut-off) ultrafiltration unit (Amicon, Beverley, MA) and was then centrifuged at 5000 g and 4°C for 5 h in a Sorvall SA 600 rotor (Du Pont Instruments). The ultrafiltrates were diluted 2-fold (v/v) (final dilution 20-fold w/v) with Tris/DETAPAC buffer, stored on ice and used within 2 h.
Cr(VI) reductase assay
The assay for Cr(VI) reductase was followed as previously described (16) with minor modifications. Ultrafiltrate (0.9 ml) was mixed with either 50 µl Tris/DETAPAC buffer or L-ascorbate oxidase (100 U/ml in Tris/DETAPAC buffer) in a 1 ml quartz cuvette and incubated at 37°C for 4 min in a temperature controlled sample compartment of a Lambda 2 spectrophotometer (Perkin Elmer). Na2Cr2O7 (50 µl of 1.25 mM) in Tris/DETAPAC buffer was then added to the cuvette to give a final Cr(VI) concentration of 0.125 mM. After ~0.5 min, the absorbance decay was monitored at 372 nm for 3 min. The reduction rate constant of Cr(VI) in both liver and kidney ultrafiltrates was represented by the slope of the linear phase natural log of Cr(VI) absorbance at 372 nm versus time.
Cr uptake
Analysis of Cr uptake in livers and kidneys of ODS rats was carried out according to the previously described protocol (49).
EPR measurement of Cr(V)
A Bruker ER-220D X-band EPR spectrometer was used for measurement of Cr(V) in tissues at liquid nitrogen temperature (77 K). Liver and kidney tissues were collected from the animals immediately following sacrifice and were made into a cylindrical column (0.30 g) using a 1 ml syringe. The samples were stored at 77 K until analysis and transferred directly into a finger Dewar filled with liquid nitrogen for measurement in the EPR cavity at 77 K. Instrument conditions were: microwave frequency, 9.5 GHz; microwave power, 6.4 mW; modulation amplitude, 4.0 G; scan time, 50 s; time constant, 320 ms; scan range, 100 G. The tissue Cr(V) concentration was calculated from the standard curve determined by using known concentrations of the Cr(V) standard K3CrO8 (50) versus the corresponding EPR peak areas.
CrDNA binding
DNA isolation was performed by following the protocol described previously (51).
8-OH-dG assay
DNA samples used in the analysis of 8-OH-dG levels in the liver and kidney of ODS rats were obtained according to the pronase/ethanol method developed by Adachi et al. (34). DNA digestion was carried out as described previously with minor modifications (34). Before DNA digestion, the isolated DNA pellet was covered with a thin layer of 95% ethanol and 0.01 BHT of ~50 µl. Caution was taken that the DNA pellet was never exposed to air. An aliquot of 100 µl deaerated SSC solution (5 mM sodium citrate, 20 mM sodium chloride, pH 6.5) was added to each DNA sample in the same 15 ml Corning centrifuge tube. The DNA sample was then heated in a 95°C water bath for 3 min and cooled down on ice immediately. Nuclease P1 (5 U dissolved in 200 mM acetate, 100 mM ZnCl2, pH 4.8) was added to the heat-denatured DNA sample and this DNA sample was then left in a 45°C water bath for 30 min. Alkaline phosphatase (2 U dissolved in 500 mM Tris, pH 7.4) was added and the DNA sample was returned to a water bath at 37°C. After 60 min, the DNA sample was filtered through a Gelman LC 13 PVDF 0.2 µm acrodisc. The filtered DNA sample of 10 µl was then immediately injected into the HPLC apparatus for analysis.
HPLC analysis of both dG and 8-OH-dG was performed on a Hewlett Packard 1050 HPLC system. The mobile phase was an isocratic eluent of 10 mM NaH2PO4 in 8% methanol, pH 5.5, and continuously purged with helium at a flow rate of 1 ml/min using a Rainin C-18 column (4.6 mm i.d.x250 mm) with a 5 µm spherical packing diameter (Woburn, MA). Detection of dG was performed with a diode-array detector monitored at 254 nm. The dG concentration in the digested DNA sample was calculated from an authentic dG standard curve, as determined according to a dG extinction coefficient of 1.30x104/M/cm in 100 mM phosphate buffer, pH 7.0, at a wavelength of 254 nm (52). The concentration of 8-OH-dG was determined with a Coulochem II electrochemical detector from ESA (Chelmsford, MA). The 8-OH-dG in the digested DNA sample was determined from an 8-OH-dG standard curve of authentic 8-OH-dG, as determined according to an 8-OH-dG extinction coefficient of 1.03x104/M/cm in water at a wavelength of 293 nm (33). Note that the standard samples for dG and 8-OH-dG were prepared separately in order to minimize the background 8-OH-dG due to oxidation of dG. Also, authentic dG and 8-OH-dG samples were analyzed before and after all the samples to obtain averaged peak areas of the dG and 8-OH-dG standards.
Statistical analysis
One-way analysis of variance (ANOVA) was performed when comparing significant differences in more than three groups. When statistically significant differences were indicated, unpaired Student's t-test was used. The results were expressed as means ± standard deviations (SD) of replicate independent samples. The level set for statistical significance was P < 0.05.
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Results
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Because liver and kidney have been shown to be the organs with the highest Cr accumulation in treated rats (53), they were used as target organs for studying Cr(VI)-induced damage in ODS rats. In ODS rats maintained on an ascorbate-free diet for 2 weeks, hemorrhaging around the eyes and nose was noticed in all rats. Moreover, because of ascorbate deficiency, rats given an ascorbate-free diet cannot walk. However, only a few rats maintained on the 50 p.p.m. ascorbate diet had these symptoms (37). Therefore, for humane reasons, the lowest ascorbate treatment of ODS rats was set at 50 p.p.m. for all subsequent studies.
Effects of Cr(VI) on ascorbate levels in vivo
Tissue ascorbate levels of ODS rats treated with different concentrations of ascorbate in their drinking water for ~2 weeks were measured by HPLC-ECD. Ascorbate levels in both liver and kidney increased in proportion to the increase in concentration of ascorbate in the drinking water (Figure 1
). In ODS rats treated with high dietary ascorbate (800 p.p.m.), the liver and kidney ascorbate levels were comparable with those of normal SpragueDawley rats (16). At the lowest dietary ascorbate (50 p.p.m.), the tissue ascorbate level was ~10-fold lower in both liver and kidney. The kidney ascorbate concentration was ~1.5-fold lower than in liver regardless of the other treatment conditions. A 1 h Cr(VI) treatment had no effect on tissue ascorbate levels in either liver or kidney following i.p. injection (10 mg/kg sodium dichromate) (Figure 1
). However, a transient decrease (P = 0.006) in liver ascorbate was observed 15 min after administration of Cr(VI) at 800 p.p.m. dietary ascorbate (Figure 2
). A similar trend was observed in kidney (P = 0.0578). Ascorbate levels returned to control levels in both liver and kidney by 0.5 h after treatment and remained constant over 2 h (Figure 2
).

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Fig. 1. Ascorbate concentration in the liver and kidney of ODS rats maintained at various dietary levels of ascorbate following treatment with Cr(VI). Rats were given sodium ascorbate at 50 or 800 p.p.m. in drinking water for 2 weeks and were then killed 1 h after a single i.p. injection of either sodium dichromate (10 mg/kg, 67 µmol Cr/kg) (solid bars) or saline (open bars). Ascorbate levels in (A) liver and (B) kidney were analyzed by HPLC-ECD as described in Materials and methods. Data represent the means ± SD of determinations from individual animals (n = 1116 rats/group).
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Effect of Cr(VI) and glutathione modulating agents on glutathione levels in vivo
The effects of various glutathione modulating agents on the levels of glutathione and ascorbate were measured in the liver and kidney of ODS rats treated with Cr(VI) and different dietary levels of ascorbate. Dietary ascorbate had no effect on the levels of glutathione (measured as NPSH) in the liver and kidney (Figure 3
). Glutathione levels were ~2-fold higher in liver than in kidney except in those rats treated with NAC. Glutathione levels were decreased in liver to ~50% of control upon NAC treatment, while no effect was observed in kidney. Glutathione levels in the liver and kidney were all significantly decreased by BSO or phorone treatment in all ODS rats, independent of ascorbate or Cr(VI) treatment (Figure 3
). BSO and phorone had approximately the same relative effects on depletion of glutathione in the liver and kidney.
Ascorbate levels in liver and kidney were measured in ODS rats treated with Cr(VI) for 1 h with/without the glutathione modulating agents, NAC, BSO or phorone (Figure 4
). Ascorbate levels were slightly elevated in the liver of ODS rats at 800 p.p.m. dietary ascorbate and treated with Cr(VI) for 1 h following treatment with either BSO or phorone.
Effect of ascorbate on the reduction of Cr(VI) by tissue ultrafiltrates in vitro
Ascorbate had previously been shown to be the major in vitro reductant in liver, kidney and lung ultrafiltrates of SpragueDawley rats since L-ascorbate oxidase, which specifically depletes ascorbate, was shown to decrease the level of Cr(VI) reductase activity in ultrafiltrates by 8095% (16,17). In vitro Cr(VI) reductase activity was examined in the absence or presence of L-ascorbate oxidase pretreatment in liver and kidney ultrafiltrates of ODS rats treated with high and low dietary ascorbate. As shown in Figure 5
, the Cr(VI) reduction rate constant was approximately four times higher in liver and kidney ultrafiltrates of ODS rats at high dietary ascorbate (800 p.p.m.) than those at low dietary ascorbate (50 p.p.m.). Cr(VI) reductase activity was ~2-fold higher in the liver than in the kidney in both groups of rats. When ultrafiltrates were treated with L-ascorbate oxidase to deplete ascorbate, ~80% of the Cr(VI) reductase activity was lost in the ultrafiltrates of ODS rats maintained at 800 p.p.m. ascorbate. However, in rats maintained on low (50 p.p.m.) ascorbate, no significant effect of L-ascorbate oxidase was observed in liver or kidney ultrafiltrates (Figure 5
).

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Fig. 5. Effect of dietary ascorbate on in vitro Cr(VI) reductase activity in liver and kidney ultrafiltrates of ODS rats. Rats were given sodium ascorbate at 50 or 800 p.p.m. in drinking water for 2 weeks. Ultrafiltrates of (A) liver or (B) kidney were prepared as described in Materials and methods and were pretreated with either Tris buffer (open bars) or L-ascorbate oxidase in Tris buffer (solid bars). Sodium dichromate was added to ultrafiltrates and absorbance at 372 nm was monitored for 3 min. Data represent the means ± SD of determinations from individual animals (n = 3 rats/group). *Significantly different from control, P < 0.05.
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Effect of ascorbate and glutathione on Cr uptake in vivo
The effect of ascorbate levels on the rate and level of Cr uptake was examined. Following i.p. injection, Cr was taken up rapidly in both the liver and kidney of ODS rats maintained at either 50 or 800 p.p.m. dietary ascorbate (Figure 6
). Cr levels were half maximal or greater in both tissues as early as 15 min after Cr(VI) administration and the level of Cr remained constant between 30 min and 2 h following treatment (Figure 6
). The levels of tissue Cr were comparable in both liver and kidney. A significantly lower uptake of Cr was observed in rats at 800 as compared with 50 p.p.m. dietary ascorbate in both liver and kidney, but only at the 15 min time point (Figure 6
). Cr uptake in ODS rats treated with different dietary levels of ascorbate and with various glutathione modulating agents was unchanged in either tissue after treatment with 10 mg/kg Cr(VI) for 1 h (data not shown). In contrast, both liver and kidney exhibited a significantly higher Cr uptake in rats at 50 than at 800 p.p.m. dietary ascorbate at a higher Cr(VI) dose (40 mg/kg) (Figure 7
), suggesting a protective effect of plasma ascorbate against the toxicity of Cr(VI).
Effect of ascorbate and glutathione on Cr(V) formation in vivo
Cr(V) formation was measured in liver and kidney of ODS rats maintained at various levels of ascorbate following i.p. injection of Cr(VI) (10 mg/kg). Cr(V) species with g = 1.985 (
H = 14.5 and 12.5 G in liver and kidney, respectively) were observed in both the liver and kidney, which was superimposed on the endogenous radical signal (g = 2.004,
H = 14 G) observed in both liver and kidney (Figure 8
). The Cr(V) signal reached maximum intensity 30 min after Cr(VI) administration in the liver, whereas in kidney the Cr(V) signal was maximal at 15 min (Figure 9
). The decay rate of the Cr(V) signal intensity appeared to be faster in the liver than in the kidney. Two hours after Cr(VI) treatment, ~30 and 70% of the maximal Cr(V) signal intensity remained in the liver and kidney, respectively. Cr(V) EPR signal intensity was 2- to 4-fold higher in the liver than in the kidney of ODS rats regardless of the levels of ascorbate treatment. At 15 min after injection of Cr(VI), the steady-state concentration of Cr(V) species was estimated to be 3 and 1% of the total tissue Cr in the liver and kidney, respectively. The liver Cr(V) EPR signal was transiently but significantly higher (P = 0.0116) at low (50 p.p.m.) than at high dietary ascorbate (800 p.p.m.) at 30 min. These results suggest that, as with Cr uptake, a higher ascorbate level appeared to decrease the initial rate of Cr(V) formation in the liver but had little overall effect on Cr(V) formation or decay in liver and kidney.

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Fig. 8. Representative EPR spectra from the liver and kidney of ODS rats maintained at 50 p.p.m. dietary ascorbate following treatment with Cr(VI). (A) Liver and (B) kidney was removed 30 min after i.p. injection of sodium dichromate (10 mg/kg). Tissue Cr(V) was measured at 77 K in a finger Dewar filled with liquid nitrogen using a standard X-band EPR spectrometer.
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There were no consistent effects of glutathione modulation on formation of Cr(V) in liver and kidney. For example, Cr(V) signal was increased by phorone but not by BSO in liver and by BSO but not phorone in kidney, despite the fact that both agents depleted glutathione to a similar extent in both tissues. This occurred at both the low and high levels of dietary ascorbate (Figure 10
). There was also no effect of NAC on Cr(V) in either tissue at low and high ascorbate levels, although NAC decreased glutathione levels by ~50% in liver (data not shown).
Effect of ascorbate and glutathione on CrDNA binding in vivo
There were appreciable levels of CrDNA binding in both liver and kidney at 15 min after administration of Cr(VI), which increased over a 2 h period in both tissues (Figure 11
). CrDNA binding was ~2-fold higher in liver than in kidney. Decreased ascorbate levels do not appear to significantly affect the levels of CrDNA binding in either the liver or kidney under these conditions. CrDNA binding was significantly increased (~2-fold) in the liver and kidney of ODS rats treated with NAC at both low and high dietary ascorbate (Figure 12
). Neither BSO nor phorone had any effect on CrDNA binding under the same conditions (data not shown).
Effects of Cr(VI), ascorbate and glutathione on 8-OH-dG formation in vivo
ODS rats maintained at different levels of dietary ascorbate (50 and 800 p.p.m.) were treated with BSO or/and Cr(VI) (10 mg/kg) and 8-OH-dG levels were determined in liver and kidney. 8-OH-dG levels were very similar (~12x105 8-OH-dG/dG) in both liver and kidney and neither Cr nor ascorbate had any effect on the endogenous levels of 8-OH-dG (data not shown). Different levels of glutathione modulated by BSO also had no effect on the levels of 8-OH-dG.
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Discussion
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Cr(VI) treatment had little or no effect on the overall levels of ascorbate and glutathione in either the liver or kidney of ODS rats treated with a non-overtly toxic dose of sodium dichromate (10 mg/kg). These results are consistent with our previous findings in normal SpragueDawley rats that Cr(VI) had no effect on ascorbate and glutathione levels in livers or kidneys 90 min after treatment with 20 mg/kg sodium dichromate (16). Glutathione levels also remained constant up to 2 h after Cr(VI) treatment in both the liver and kidney of ODS rats. Cr(VI) treatment was also shown to increase the liver ascorbate levels in ODS rats treated with either BSO or phorone at the high ascorbate level. The increased levels of ascorbate are most likely due to the effects of these glutathione depleting agents, BSO and phorone, rather than Cr(VI) alone. Phorone has been shown to increase the levels of ascorbate in subcellular fractions of rat liver (54) and it was reported that phorone can increase the levels of ascorbate up to 3-fold in normal SpragueDawley rats (16). Since ODS rats lack the ability to synthesize ascorbate de novo, the effect of phorone in increasing ascorbate levels does not involve its biosynthesis per se. Only when both phorone and Cr(VI) were injected together were the levels of ascorbate increased.
It is interesting to note that the levels of glutathione (measured as NPSH) in the liver of ODS rats treated with both high and low ascorbate were not enhanced by NAC treatment as expected, but decreased to ~50% of control levels. In normal SpragueDawley rats, liver glutathione levels were increased up to 126% of control levels after rats were treated under the same conditions (39). Moreover, NAC has been shown to increase the levels of cysteine, but not glutathione, in Hepa-1 wild-type cells (55). This difference indicates that ODS rats are somehow physiologically different from normal rats. As expected, the glutathione levels of ODS rats treated with BSO or phorone were decreased and Cr(VI) had no effect on the levels of glutathione.
The levels of Cr taken up in the liver and kidney of ODS rats treated with Cr(VI) were about the same and the glutathione modulating agents had no effect on the levels of Cr in each organ. When ODS rats were injected with a higher, toxic dose of Cr(VI) (40 mg/kg), a lower uptake was observed in both liver and kidney of ODS rats maintained at high levels of ascorbate (800 p.p.m.). Ascorbate has been shown to be the most effective aqueous phase antioxidant in human blood plasma (56). In ODS rats, plasma ascorbate levels are proportional to the hepatic values, regardless of the maintained level of ascorbate (15800 p.p.m.) (57). Thus, ascorbate appears to play an antioxidant role in the plasma by reducing Cr(VI) to non-toxic Cr(III) species, which has a far lower ability to enter cells and can circulate in blood plasma only (58,59). Therefore, the lower initial Cr(V) in the liver and lower Cr uptake in both organs of rats treated at 800 p.p.m. ascorbate are most likely due to the reduction of Cr(VI) by plasma ascorbate. Nevertheless, at the lower, non-overtly toxic dose of Cr(VI), this difference was not observed.
The levels of in vivo CrDNA binding were ~2.0x103 and 6.0x104 Cr/nucleotide for liver and kidney, respectively, for ODS rats treated with a non-overtly toxic dose of Cr(VI) at both the high and low ascorbate levels. All of these results are very comparable with the levels of CrDNA binding in normal SpragueDawley rats reported previously (19), suggesting that ascorbate has no obvious effect on the level of CrDNA binding.
The Cr(V) signal (g = 1.985) observed in the liver and kidney of ODS rats treated with Cr(VI) was also detected in other in vivo systems with similar g values. Ueno et al. (60) have shown that treatment of male albino mice with potassium dichromate (1040 mg/kg) resulted in three in vivo Cr(V) species (g = 1.992, 1.984 and 1.974) in the livers of these mice. Cr(V) EPR species were also detected as early as 15 min after i.p. injection of Cr(VI) and persisted for at least 12 h. Cr(V) (g = 1.980,
H = 2.5) was also detected by low frequency EPR in live mice injected i.v. with Cr(VI) (25). All of these results suggest the importance of Cr(V) species formation during the course of Cr(VI) metabolism in vivo.
In the present study, the levels of Cr(V) were closely correlated with the levels of CrDNA binding in both the liver and kidney of ODS rats. The consistently higher levels of Cr(V) and CrDNA binding in the liver relative to the kidney possibly reflect the different reductant environments of these two organs in ODS rats, since the levels of Cr uptake in both organs were similar. It was previously suggested that both the reduction of Cr(VI) and stabilization of the Cr(V) product (by coordination to protein ligands) is required for CrDNA binding (61). Recently, it was suggested that Cr(V) formed through intracellular reduction of Cr(VI) can undergo ligand exchange and bind to the phosphate group moiety present in both DNA and deoxyribonucleotides (62). In the in vitro reaction between ascorbate and Cr(VI) in the presence of calf thymus DNA, the highest levels of Cr bound to DNA were observed for reaction conditions that produce the most Cr(V) species, suggesting a role for Cr(V) in the formation of CrDNA adducts (12,22). Nevertheless, in the present study, modulation of either ascorbate or glutathione had no simultaneous effect on both Cr(V) and CrDNA binding, suggesting that formation of Cr(V) and CrDNA adducts in vivo may not necessarily be due to the same reductant.
Cr(V) was significantly increased by phorone but not by BSO in liver and by BSO but not by phorone in kidney, whereas NAC had no effect on Cr(V) in either tissue. An inverse relationship between glutathione levels and in vivo Cr(V) has been reported previously (63,64). In Chinese hamster V-79 cells, the depletion of glutathione by BSO caused an increase in cellular levels of Cr(V) species without affecting the overall level of Cr(III) (63). Similarly, BSO had no effect on the formation of Cr(V) in the liver of mice treated with Cr(VI), although the systematic toxicity caused by Cr(VI) was decreased by BSO treatment (60). In the present study, it was shown that ascorbate had no effect on the formation of Cr(V) species generated in ODS rats treated with Cr(VI). Therefore, it is most likely that the formation of Cr(V) species in vivo involves the reaction between Cr(VI) and glutathione.
In those ODS rats treated with NAC and Cr(VI) at either high or low ascorbate, CrDNA binding levels were increased significantly in both liver and kidney. It is unlikely that the increase in CrDNA binding in liver is due to the effects of glutathione, since glutathione levels were decreased by NAC and acivicin treatment. Also, BSO and phorone had no effect on CrDNA binding. It is more likely that the increase in CrDNA binding was due to the higher levels of cysteine, since NAC acts as a cysteine precursor for glutathione synthesis. Cysteine and tyrosine exhibited the highest activity for formation of complexes with DNA and Cr in vitro, whereas glutathione showed only ~1015% of the activity of cysteine (65). Furthermore, the extent of protein complexing to DNA by Cr(III) in vitro also depends upon the content of cysteine and histidine in the proteins (65). Amino acids complexed to DNA were purified from chromate-exposed cells and were shown to be free amino acids that had reacted with Cr and DNA in the cell (66). Nevertheless, the increase in CrDNA binding in the liver and kidney of NAC-treated ODS rats was only ~2-fold. This is perhaps due to the fact that amino acids and glutathione crosslinked by Cr to DNA are estimated to account for only ~50% of the total Cr bound to DNA (66).
Cr(VI) treatment and the levels of ascorbate or glutathione had no effect on the levels of 8-OH-dG in vivo, suggesting that the reaction between Cr(VI) and H2O2 in vivo may not be a significant pathway for Cr-induced DNA damage. This could be due to the fact that the H2O2 concentration is <0.1 µM in cells (67) and that there are significant mechanisms in vivo for scavenging H2O2, HO. and other ROS.
Our results suggest that plasma ascorbate may play a significant role as an extracellular detoxifying agent of Cr(VI) in ODS rats, presumably by reducing extracellular Cr(VI) to the non-toxic and poorly absorbed Cr(III). Conversely, one interpretation of the unchanged levels of Cr(V), CrDNA binding and 8-OH-dG in the liver and kidney of ODS rats treated with different dietary levels of ascorbate is that ascorbate is not the principal intracellular reductant of Cr(VI) in these tissues. This conclusion would appear to contradict several previous findings obtained in vitro (16,17), which suggested that ascorbate plays a major role in the reduction of Cr(VI) and subsequent Cr-induced DNA damage. These apparently conflicting results may reflect differences between in vitro model systems and the more complex in vivo physiological conditions of a whole animal, under which more than one competing pathway for the reduction of Cr(VI) may be operating simultaneously. An alternative possibility is that, even at 50 p.p.m. dietary ascorbate, which is the lowest dose we could use without crippling the animals and which provides only one tenth of the normal tissue ascorbate level, there is still enough ascorbate in ODS rat organs to fully reduce Cr(VI) and cause DNA damage in vivo. This is a difficult issue to resolve in vivo. It is not possible to completely deplete ascorbate even in the organs of ODS rats treated with a 0 p.p.m. (ascorbate-free) diet, owing to the fact that glutathione-dependent dehydroascorbate reductase has recently been shown to have a wide, likely ubiquitous, tissue distribution, suggesting its importance in maintaining adequate tissue levels of functionally active ascorbate (68,69). Furthermore, glutathione was shown to be able to non-enzymatically reduce dehydroascorbate to produce ascorbate (70). Therefore, it may not be possible to completely deplete in vivo ascorbate in the organs of ODS rats, at least in part due to these endogenous physiological processes.
Modulation of glutathione levels by 2-fold or greater also did not have a major effect on any of the parameters measured in our study. It is difficult to modulate glutathione levels beyond this range in vivo and even a 50% depletion may be insufficient to see an effect since this would still be expected to provide glutathione levels in large excess over Cr(VI) (mM versus µM concentrations, respectively). Future studies should also consider other possible thiol reductants in vivo, such as cysteine (20,71), which have been shown to have high reactivities towards reducing Cr(VI). However, it is not possible to completely deplete these important small molecular reductants in vivo to fully test their roles in Cr(VI) metabolism in vivo, since they are critical for maintenance of redox status and other important physiological functions. On the other hand, if these reductants are still in excess even under the extreme conditions of these experiments, our results suggest that more modest physiological fluctuations in glutathione and ascorbate levels have little or no effect on overall Cr(VI) metabolism or DNA damage in vivo.
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Acknowledgments
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We would like to thank Mr William (Jay) Bement for his technical assistance. This project was funded by PHS grant ES07167 awarded by the National Cancer Institute, DHHS (K.E.W.) and by an American Cancer Society Institutional Research Grant (IN157K) from the Norris Cotton Cancer Center (K.J.L.). This paper is dedicated to the late Dr Karen E.Wetterhahn (19481997).
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Notes
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4 To whom correspondence should be addressed. Email: jim.liu{at}dartmouth.edu 
5
Deceased. 
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Received October 6, 1998;
revised February 25, 1999;
accepted March 17, 1999.