Lipid Peroxidation-induced Etheno-DNA Adducts in the Liver of Patients with the Genetic Metal Storage Disorders Wilson’s Disease and Primary Hemochromatosis1

To assess DNA damage caused by lipid peroxidation due to copper and iron storage disorders in the human liver, the formation of the etheno adducts 1,N -ethenodeoxyadenosine (#{128}dA) and 3,N -ethenodeoxycytine (#{128}dC) was measured in liver DNA from normal subjects and from patients with Wilson’s disease (WD) and primary hemochromatosis. The mean #{128}d.A and edC levels per 10 parent nucleotides in normal liver were 19.3 ± 4.9 and 27.5 ± 10.0, respectively. The mean #{128}dA and #{128}dC levels per iO parent nucleotides in WD were 61.03 ± 7.95 and 91.50 ± 36.02, and in primary hemochromatosis, they were 46.62 ± 32.83 and 64.32 ± 11.55, respectively, two to three times higher than those in the normal liver. The etheno adduct levels were highly correlated with the copper content of the liver in the normal and ‘ND samples. This study demonstrates for the first time the formation of promutagenic etheno adducts in humans in association with copper and iron storage-induced lipid peroxidation. Thus, the etheno adducts are implicated as initialing DNA damage in copper/Iron-induced carcinogenesis In hwnans and should also be explored as biomarkers in disease progression and prevention trials. Introduction WD3 is an autosomal recessive disorder of copper transport, resulting in copper accumulation in the liver and brain. The WD Received 10/7/97; revised 2/13/98; accepted 2/26/98. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. I Supported in part by Contract EV5V-CT94-0409 with the European Union. This study was presented in part at the 87’s’ Annual Meeting of the American Association for Cancer Research. Washington, D.C., April 14, 1996. 2 To whom requests for reprints should be addressed, at Division of Toxicology and Cancer Risk Factors, Deutsches Krebsforschungszentrum (DKFZ), Im Neuenheimer Feld 280, D-69l20 Heidelberg, Germany. 3 The abbreviations used are: WD, Wilson’s disease; LEC; Long Evans cinnamon; HCC, hepatocellular carcinoma; PH, primary hemochromatosis; adA, I .N”ethenodeoxyadenosine; #{128}dC, 3,W’-ethenodeoxycytine; dA, deoxyadenosine; dC, deoxycytidine. gene (ATP7B gene) has been mapped to human chromosome l3ql4.3, and its product is characterized as a copper-binding P-type ATPase protein, which acts as an AlP-dependent transporter of copper within hepatocytes (1-3). Copper accumulation in WD patients, if not treated by chelating therapy, leads to liver injury. In LEC rats, an animal model for WD, copper accumulation leads to hepatitis and HCC (4). Similar to LEC rats, HCC has been reported in a WD patient who was poorly compliant to pharmacotherapy (5). PH is a genetic disorder characterized by a progressive accumulation of iron, leading to liver cirrhosis (6). A candidate hemochromatosis gene has been located on chromosome 6, which is related to the MHC class I family (7). PH is 100 times more common than WD in Caucasians (8). A 90-240-fold increase in the relative risk for primary liver cancer has been reported in patients with PH (9-1 1). The DNA damage that leads to liver cancer in WD and PH is poorly understood, although evidence exists that the damage is caused by oxidative stress resulting from a high concentration of monovalent copper and bivalent iron ions acting as prooxidants (12, 13). The metal-induced oxidative stress can lead to the formation of hydroxyl radicals via the Fenton and HaberWeiss reactions. A similar process involving transition metal ion-mediated oxygen free radical-dependent DNA damage has been suggested to be responsible for bulky DNA lesions detected in the livers of humans with WD and PH (14). The increased DNA damage may be caused by hydroxyl radicals or via the formation of reactive aldehydes such as trans-4hydroxy-2-nonenal and malondialdehyde as a result of lipid peroxidation (15). Etheno-bridged nucleic acid bases have been shown to be formed as a consequence of lipid peroxidation in vitro, presumably via a reaction between peroxidized trans-4hydroxy-2-nonenal and DNA (16, 17). In LEC rats, ageand copper-dependent increased formation of the etheno adducts edA and #{128}dC has been reported (18). To determine whether similar DNA damage could occur in patients with WD and PH, we have measured the levels of edA and #{128}dC in the liver DNA of patients with these disorders and compared them with the levels formed in normal human livers. The copper and iron content of the livers was also determined to investigate the relationship between the etheno adduct levels and metal ion concentrations in the liver. Materials and Methods Tissue Accrual. WD and PH liver samples were obtained from patients undergoing orthotopic liver transplant at the Queen Elizabeth Hospital (Birmingham, United Kingdom). Control samples were from normal liver donor tissue excess of surgical requirements from the pediatric segmental liver transplant program. Tissue samples were frozen at -85#{176}Cuntil use. on October 29, 2017. © 1998 American Association for Cancer Research. cebp.aacrjournals.org Downloaded from A B 436 Etheno Adducts in WD/PH Fig. 1. Autoradiograms of 32Plabeled human liver DNA samples. A, standard compounds; B, normal liver; C, WD liver; D, PH liver. DI, 1 M acetic acid (pH 3.5); D2, saturated ammonium sulfate (pH 3.5). Spot identification: I, 5’-#{128}dAMP; 2, 5’-#{128}dCMP; 3, 5-dUMP (internal standard). The other spots on the autoradiograms are unremoved normal nucleotides from immunoaffinity purifications and impurities in [‘y-32PIATP and T4 polynucleotide kinase. Analysis of Copper and Iron. Copper and iron were determined by atomic absorption spectrometry, after the digestion of tissue with nitric and perchloric acids, using established procedures (19). Analysis of #{128}dA and #{128}dC. DNA was prepared from the liver tissue by hydroxylapatite purification with the modification of RNase treatments as described previously (20). A highly sensitive method using immunoaffinity purification with 32P-postlabeling was used to quantitate edA and #{128}dC in the liver DNA samples as their 5’-monophosphate (20). In brief, -25 g of DNA were hydrolyzed to deoxynucleoside 3’-monophosphates using micrococcal endonuclease and calf spleen phosphodiesterase. dA and dC were determined in enzymatic hydrolysate by high-performance liquid chromatography. edA and #{128}dC were enriched on immunoaffinity columns (2 1 ) that were prepared from monoclonal antibodies (22). The dried samples were labeled with [‘y-32P]ATP and T4 polynucleotide kinase in the presence of 3’-dUMP as an internal standard to nucleoside 5’-monophosphates and resolved on polyethyleneimine cellulose TLC plates using two-directional chromatography (20). The spots corresponding to edA, #{128}dC, and 3’-dUMP were quantitated by autoradiography and liquid scintillation counting. The number of adducts/parent nucleotides was obtained from the ratio of the quantity of the etheno adducts measured by TLC:the quantity of the parent nucleotides in the sample obtamed from high-performance liquid chromatography analysis