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Chem. Res. Toxicol. 2005, 18, 24-27
Glutathione Depletion Enhances the Formation of Endogenous Cyclic DNA Adducts Derived from t-4-Hydroxy-2-nonenal in Rat Liver Fung-Lung Chung,* Despina Komninou, Lei Zhang, Raghu Nath, Jishen Pan, Shantu Amin, and John Richie American Health Foundation Cancer Center, Institute for Cancer Prevention, Valhalla, New York 10595 Received September 28, 2004
Earlier, we detected the cyclic adducts of deoxyguanosine (dG) derived from t-4-hydroxy-2nonenal (HNE), a long chain R,β-unsaturated aldehyde (enal) product from oxidation of ω-6 polyunsaturated fatty acids, in tissue DNA of rats and humans as endogenous DNA damage. Recent evidence implicates the cyclic HNE adducts in human liver carcinogenesis. Because glutathione (GSH) protects against oxidative stress, we undertook a study to examine the effect of GSH depletion on the HNE-derived cyclic adducts in vivo. Four F344 rats were administered L-buthionine-(S,R)-sulfoximine (BSO), a potent inhibitor of GSH biosynthesis, at 10 mM in drinking water for 2 weeks. Rats in the control group were given water only. Livers were harvested, and each liver was divided into portions for GSH and DNA adduct analyses. The BSO treatment depleted hepatic GSH by 77%; the GSH levels were reduced from 6.3 ( 0.3 in the control rats to 1.5 ( 0.1 µmol/g tissues in the treated group. The formation of HNE-dG adducts, analyzed by an HPLC-based 32P-postlabeling assay, was increased by 4-fold, from 6.2 ( 2.2 nmol/mol dG in liver DNA of control rats to 28.5 ( 16.1 nmol/mol dG in the rats treated with BSO (p < 0.05). The formation of 8-oxodG in liver DNA was also increased as a result of BSO treatment, although the increase was not statistically significant. These results further support the endogenous origin of HNE-dG adducts and, more importantly, indicate a critical role that GSH plays in protecting against in vivo formation of the promutagenic cyclic DNA adducts derived from HNE.
Introduction Glutathione (GSH)1 is an endogenous thiol that plays an important role as an antioxidant in regulating cellular redox status (1, 2). Depleting GSH in tissues often results in an increase in oxidative stress as measured with products of reactive oxygen radicals and the induced DNA damage. These results indicate that GSH protects tissue DNA against damage caused by oxidation. Alternatively, GSH also functions as a nucleophile capable of scavenging DNA reactive intermediates, such as activated carcinogens, by a conjugation reaction commonly mediated by GSH transferases (3). Therefore, GSH is believed to have a dual role in alleviating the damage to cellular DNA and proteins. The cellular GSH is tightly regulated, and as a result, its levels are not easily modulated (4). However, L-buthionine-(S,R)-sulfoxime (BSO) is an inhibitor of γ-glutamylcysteine synthetase in GSH biosynthesis, and the treatment with BSO efficiently depletes GSH in tissues (4, 5). Therefore, the treatment with BSO provides an approach to investigate the role of GSH in various biological and physiological functions. * To whom correspondence should be addressed. Present address: Department of Oncology, Lombardi Comprehensive Cancer Center, Georgetown University Medical Center, 3800 Reservoir Road NW, Washington, DC 20057. Tel: 202-687-3021. E-mail: flc6@ georgetown.edu. 1Abbreviations: GSH, glutathione; Acr, acrolein; Cro, crotonaldehyde; PUFAs, polyunsaturated fatty acids; BSO, L-buthionine-(S,R)sulfoximine; dG, deoxyguanosine; HNE, t-4-hydroxy-2-nonenal; PdG, 1,N2-propanodeoxyguanosine.
Cyclic 1,N2-propanodeoxyguanosine (PdG) adducts are ubiquitous DNA lesions that have been detected in tissues of untreated rodents and humans (6, 7). The cyclic PdG adducts that have been detected in vivo include the earlier ones from acrolein (Acr) and crotonaldehyde (Cro) and a more recent one from the long-chain t-4-hydroxy2-nonenal (HNE), which are detected as four structure isomers (8-11). Studies have shown that enals derived from oxidation of polyunsaturated fatty acids (PUFAs) are a major source for the formation of PdG adducts (12, 13). We have previously shown that Acr- and Crodeoxyguanosine (dG) adducts primarily come from ω-3 fatty acids, whereas HNE-dG adducts are exclusive products of oxidized ω-6 PUFAs (14). Figure 1 shows the structures of HNE-dG adduct isomers that have been detected in vivo. The site-specific mutagenesis studies have shown that the Acr-dG adduct isomers that are predominantly detected in vivo are much less genotoxic; they do not cause mispairing in double-stranded DNA because of efficient repair and accurate translesion synthesis (1517). On the contrary, Hussain et al. reported that HNE induces G to T mutations predominantly at the third base of codon 249 of the p53 gene of human lymphoblastoids: a mutation that coincides with the mutation hotspot in human cancer, particularly in human liver cancer (18). The same mutation also occurs in the liver DNA of Wilson’s disease patients who have a higher risk of liver cancer, believed to be caused by the elevated lipid
10.1021/tx049728+ CCC: $30.25 © 2005 American Chemical Society Published on Web 12/24/2004
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Figure 1. Structures of four diastereomers of HNE-dG adducts detected in vivo.
peroxidation in that tissue as a result of abnormal copper accumulation (19). Furthermore, studies have shown that HNE preferentially binds to the third base of codon 249 of human p53 gene with the formation of HNE-dG adducts (20). Recently, Fernandes et al. showed that HNE-dG adducts induce primarily G to T mutations in a stereoselective manner in mammalian cells (21). Taken together, these results suggest a possible role of HNEdG adducts in the mutation at the hotspot of codon 249 in the human p53 gene. We reported earlier that levels of PdG adducts of Acr and Cro in liver DNA increased by 2-20-fold, respectively, in rats treated with BSO (22). These results are explained by the depletion of hepatic GSH, which serves as an antioxidant and a scavenger of enals. However, the effects of GSH depletion on HNE-dG adducts in vivo have not been studied. The purpose of this study is to investigate the role of GSH in the formation of HNEdG adducts in liver DNA of F344 rats.
Methods and Materials Chemicals. BSO, GSH, 8-oxodG, micrococcal nuclease, apyrase, alkaline phosphatase, and nuclease P1 were purchased from Sigma Aldrich Co. (St. Louis, MO). Spleen phosphodiesterase was obtained from Worthington Biochemical (Lakewood, NJ), and [γ-32P]ATP and T4 polynucleotide kinase were from Amersham (Piscataway, NJ). HNE was synthesized by a published method (23). HNE-dG adduct standards were prepared according to a previously published method (10). All other reagents, unless otherwise stated, were purchased from SigmaAldrich Co. and Fisher Chemical (Fair Lawn, NJ). Animal Treatment. Eight 3 month old male F344 rats were obtained from Charles River Labs (Kingston, NY). Animals were housed in the Research Animal Facility and maintained under standard conditions (25 °C, 12 h light and 12 h dark cycle) and fed ad libitum a modified AIN-76A diet (5% corn oil; Dyets, Bethlehem, PA). After 1 week of acclimatization, the animals were divided into two groups (four rats/group). In the treated group, the rats were given tap water ad libitum containing 10 mM BSO for 2 weeks, and in the control group, the rats were given water without BSO. The water consumption was measured, and freshly prepared BSO solutions were provided three times a week. Body weights were recorded on days 0, 2, 4, 11, and 14. On the 14th day, the rats were sacrificed by CO2 euthanasia, and the livers were removed and minced. A portion of fresh liver was immediately processed for GSH determination, and the remaining tissue was frozen at -80 °C until DNA isolation for adduct assays. GSH Analysis. Fresh liver minces were homogenized in 5% metaphosphoric acid (10%, w/v). After centrifugation at 14000g
Chem. Res. Toxicol., Vol. 18, No. 1, 2005 25 for 2 min, the acid soluble fractions were removed, and total GSH and GSSG were measured by the method of Tietze (24) modified for use with 96 well plates (25). Briefly, 50 µL of tissue extract diluted 10-fold in 100 mM NaHP2O4/5 mM EDTA buffer at pH 7.5 was added to 50 µL of GSH oxidoreductase (5 units/ 50 µL) and 50 µL of 2.5 mM 5,5′-dithiobis(2-nitrobenzoic acid). The reaction was then initiated by the addition of 50 µL of 1.2 mM NADPH in buffer. The rate of color change at 410 nm, which is proportional to the amount of total GSH in the samples, was monitored in a Dynatech MR5000 plate reader (Denkendorf, Germany). HNE-dG Adducts Analysis. A portion of the liver was used for DNA isolation by a modified Marmur’s method (26). The purity of DNA was determined by the ratio of 260 nm/280 nm (>1.8). Liver DNA (80-100 µg, 2 µg/µL in NaCl/citrate buffer) was hydrolyzed by incubating with micrococcal nuclease and spleen phosphodiesterase to 3′-monophosphates, and HNE-dG adducts were analyzed by a previously described 32P-postlabeling/HPLC method (27). Briefly, the DNA hydrolysate was applied to a preconditioned C18 solid phase extraction column (Varian C18 Bond Elut, 3 mL/500 mg) and was eluted with 3 mL of 50 mM ammonium formate (pH 7.0), followed by 3 mL of 10% MeOH in ammonium formate. The fraction containing the HNE-dG 3′-monophosphates was eluted with 6 mL of 50% MeOH, dried, and enriched with nuclease P1 to convert normal nucleotides to nucleosides, followed by labeling with [γ-32P]ATP at 37 °C for 40 min and then treatment with apyrase for 20 min at 37 °C. The labeled 3′,5′-bisphosphates of HNE-dG were separated by TLC on a prewashed polyethyleimine cellulose sheet (Machery Nagel, Duren, Germany) in 2.25 M NaH2PO4 (pH 3.5) for 16-18 h. The radioactive adduct spot, identified by comparison to the simultaneously labeled 3′-monophosphates of HNE-dG standard, was excised and extracted with 1.5 mL of 2-propanol:ammonia mixture by shaking in a water bath for 12 min at 37 °C. The extract was filtered through an Acrodisc syringe filter to a 7 mL glass vial, and the filtrate was evaporated to dryness in a speedvac. The resulting residue was reconstituted in 750 µL of H2O. After the residue was spiked with the synthetic 3′,5′-bisphosphates of HNE-dG as UV markers, the mixture was purified on a reversed phase HPLC column, followed by an ion pair column (details in ref 27). The collected fraction containing 3′,5′-bisphosphates of HNE-dG was again dried in a speedvac and reconstituted in 500 µL of water. Half of the purified fraction containing the adducts was analyzed on a reversed phase HPLC system with dual detection of UV and radioflow detectors. The identities were first established by comigration with the UV standards. For final confirmation, the other half of the adduct fraction was either hydrolyzed enzymatically with T4 polynucleotide kinase to the 5′-monophosphates of HNE-dG or treated with sodium borohydride to yield the ring-opened derivatives and followed by comigration with the corresponding UV standards in a reversed phase HPLC. Details of the assay were published previously (27). Statistical significance was determined using Student’s t test. 8-OxodG Analysis. The 8-oxodG was analyzed on an HPLC system equipped with an electrochemical detector (Bioanalytical System Inc.) (28, 29). To a 30-50 µg DNA sample in 50 µL of H2O was added both 15 µL of 0.5 M sodium acetate (pH 5.1) and 7.5 units of nuclease P1. The mixture was incubated in the dark at 37 °C for 30 min and further digested with 4.5 units of alkaline phosphatase for 1 h at 37 °C in the dark after addition of 120 µL of 0.4 M Tris-HCl (pH 7.5). After filtration, the hydrolysate was analyzed with an HPLC system equipped with a 5 µm 4.6 mm × 250 mm C18 reverse phase column eluted at a flow rate of 1 mL/min with 5% methanol containing 12.5 mM citric acid, 25 mM sodium acetate, 30 mM NaOH, and 10 mM acetic acid (pH 5.1). The effluent was routed through a UV detector for quantification of dG and then through the EC detector with +600 mV applied on the analytical electrode and 1.0 nA set as the analysis range. The standard curves for 8-oxodG (0-10 pmol, R2 > 0.95) and dG (0-50 nmol, R2 > 0.99) were constructed and used for quantification.
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Table 1. Effects of BSO on GSH, HNE-dG, and 8-oxodG Levels in Liver of F344 Rats treatment
GSH (µmol/ g tissue)
HNE-dG (nmol/molG)
8-oxodG (µmol/molG)
control BSO treated
6.3 ( 0.3a 1.5 ( 0.1b
6.2 ( 2.2 28.5 ( 16.1c
5.7 ( 1.6 11.1 ( 7.6d
a Data are obtained from four rats. b p < 0.01. c p < 0.05. 0.21.
d
p)
Figure 3. Typical HPLC chromatograms obtained in this study for the detection of HNE-dG adducts in rat liver DNA using an HPLC-based 32P-postlabeling method (27).
Figure 2. Body weights of the BSO-treated and the control groups during the 2 week treatment period.
Results and Discussion After 10 mM BSO was administered in drinking water for 2 weeks, the hepatic GSH levels of F344 rats were reduced by more than 77%, from 6.3 ( 0.3 to 1.5 ( 0.1 µmol/g tissue (Table 1). In a separate study, we found that the depletion of GSH was evident within 3 days of BSO administration; the levels of GSH on day 3 were similar to those found on day 14 (data not shown). Our previous study showed that the administration of 30 mM BSO in drinking water for 2 weeks resulted in an 84% depletion of hepatic GSH, only slightly higher than that obtained with 10 mM BSO. The body weight gains in that study, however, were reduced by as much as 15%, as compared to those in the control group, therefore raising questions about possible toxicity (22). In this study, the BSO concentration was reduced by 3-fold. While there was no overt sign of toxicity during the treatment, we still observed losses in body weight gain, although less than 10% (Figure 2). It is conceivable that a further decrease in BSO concentration in drinking water would deplete hepatic GSH to a comparable level, while causing a minimal loss of body weight. Concomitant with the depletion of hepatic GSH, we found a significant increase in the levels of HNE-dG adducts in liver DNA of rats treated with BSO as compared to the rats in the control group. The total levels of HNE-dG adducts, four adduct isomers combined, were increased by more than 4-fold, from 6.2 ( 2.2 nmol/mol G in the control rats to 28.5 ( 16.1 nmol/mol G (P < 0.05) (Table 1). The increase in HNE adducts was not stereospecific, because all four adduct isomers appeared to increase as a result of BSO treatment. Figure 3 shows typical HPLC chromatograms for the detection of HNEdG adducts in liver DNA of rats obtained in this study. The sharp rise of HNE adducts in tissue after the BSO treatment indicates that they are of an endogenous origin. Because HNE is an oxidation product of ω-6 PUFAs, these results support the notion that lipid peroxidation is a likely source of their formation and,
more importantly, that GSH plays a pivotal role in protecting tissue DNA from cyclic adduction by HNE. In the same liver DNA samples, we also found that the formation of 8-oxodG was increased by 2-fold with the BSO treatment, from 5.7 ( 1.6 in the control group to 11.1 ( 7.6 µmol/mol G (Table 1). However, this increase was not statistically significant due to small sample size and relatively large variability. These results, nevertheless, are in agreement with the general notion that GSH depletion can cause an increase of oxidative stress (3). In this context, the simultaneous increases in cyclic DNA adducts and the oxidized bases by BSO treatment offer some clues that these DNA lesions, one from enals and the other from hydroxyl radicals, are, at least partially, formed by a common pathway. A similar parallel effect was previously reported in the formation of cyclic etheno adducts and 8-oxodG in liver DNA of rodents under oxidative stress (30). The in vivo cyclic DNA adducts derived from enals, therefore, appear to be specific markers of oxidative DNA damage originated from fatty acids. Several mechanisms can be envisioned for the increased formation of HNE-dG adducts by GSH depletion. As an antioxidant, GSH suppresses the oxidation of lipids and, consequently, reduces the HNE generated. Alternatively, GSH can conjugate with enals and the conjugation reaction represents a major route to remove enals (31), although enals can also be metabolized to the corresponding carboxylic acid and alcohol derivatives (3, 32, 34). Recent studies showed that HNE adducts are repaired by the NER pathway, and the NER activity is compromised by HNE (35, 36). This may also contribute to the increase of HNE adduct levels in tissue DNA by GSH depletion. It is not clear whether one or the combination of these mechanisms is involved in the elevated HNE-dG adduct levels observed after depleting GSH. However, the increased levels of 8-oxodG in DNA in the liver of BSO-treated rats seem to support the importance of the antioxidant activity of GSH. Although the two types of in vivo cyclic PdG adducts, those derived from the short-chain enals, such as Acr and Cro, that predominantly come from ω-3 PUFAs, and those from the long-chain HNE derived only from ω-6 PUFAs, are ubiquitously detected in tissues at a relatively high occurrence, their significance in tumorigenesis remains unclear. In this study, we have demonstrated that like Acr and Cro adducts, the in vivo formation of HNE adducts is also sharply increased by GSH depletion,
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suggesting a universal role of GSH in protecting tissue DNA against cyclic DNA modifications by enals. The upregulation of cyclic PdG adduct levels in tissue DNA by BSO may provide a means to study their roles in tumorigenesis.
Acknowledgment. We thank Chang-In Choi and Joel Reinhardt in the Research Animal Facility for their help in conducting the animal study. This work was supported by NIH Grant CA43159.
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