Oxidative stress and lipid peroxidation-derived DNA-lesions in inflammation driven carcinogenesis

Abstract

During chronic inflammatory processes an excess of free radicals and DNA-reactive aldehydes from lipid peroxidation (LPO) are produced, which deregulate cellular homeostasis and can drive normal cells to malignancy. Etheno (ɛ)-modified DNA bases are generated by reactions of DNA with a major LPO product, trans-4-hydroxy-2-nonenal. We investigated steady state levels of ɛ-DNA adducts in organs, blood or urine from patients with cancer prone diseases, especially when related to persistent inflammatory processes. We have developed sensitive and specific methods for adduct detection in vivo. Hepatic etheno-adduct levels were significantly elevated in patients with Wilson's disease and primary hemochromatosis. Excess storage of copper/iron causing oxidative stress and LPO-derived DNA-damage, are implicated in disease pathogenesis as confirmed by studies in LEC-rats, a model for Wilson's disease. When patients with alcohol related hepatitis, fatty liver, fibrosis and cirrhosis were compared with asymptomatic livers, excess hepatic DNA-damage was seen in all patients, except those with hepatitis. Etheno-deoxyadenosine excreted in urine was measured in HBV-infected patients diagnosed with chronic hepatitis, cirrhosis and hepatocellular carcinoma: as compared to controls, patients had 20–90-fold increased urinary levels. In conclusion: ɛ-DNA adducts may serve as potential markers for assessing progression of inflammatory cancer-prone diseases. Also the efficacy of human chemopreventive interventions could be verified by using our non-invasive urine assay. Mechanisms and host-factors that influence the steady-state levels of ɛ-DNA adducts in cancer prone tissues are under investigation.

Introduction

An article published in The Lancet in 2001 [1], celebrating the German surgeon Rudolph Virchow's 100th anniversary, stated: “Over the past 10 years, our understanding of the inflammatory microenvironment of malignant tissues has supported Virchow's hypothesis, and the links between cancer and inflammation are starting to have implications for prevention and treatment”. Hence, the article title ‘Inflammation and cancer: back to Virchow?’, a connection he had already suggested in 1863. Indeed, current attention is directed to persistent oxidative and nitrosative stress and excess lipid peroxidation (LPO), which are induced by inflammatory processes, impaired metal transport or dietary imbalance, causing accumulation of massive DNA damage together with deregulation of cell homeostasis (Fig. 1). As these events appear to play an important role in human chronic disease pathogenesis [2], [3], [4] DNA damage caused by ROS, RNS and LPO endproducts provides promising markers for risk prediction and targets for preventive measures. We and others have contributed to this paradigm by quantifying modified DNA base adducts in human tissues and body fluids that are generated by reactions of DNA with two major LPO-endproducts, trans-4-hydroxy-2-nonenal (HNE) to yield etheno (ɛ)-adducts, and malondialdehyde (MDA) to form the MDA-derived DNA adduct such as M₁dG. The DNA-reactive LPO-products HNE and MDA are increasingly implicated in the carcinogenesis process [5]. These intermediates can react with DNA bases to form exocyclic DNA adducts of which several have been characterized as propano- and etheno (ɛ)-DNA-base adducts [6], [7]. Of the latter, 1,N⁶-ethenodeoxyadenosine (ɛdA), 3,N⁴-ethenodeoxycytidine (ɛdC) and N²,3-ethenodeoxyguanosine have been detected in vivo (Fig. 2). These promutagenic, chemically stable markers appear to be useful for assessing oxidative stress-derived DNA damage. They are also formed from the carcinogens vinyl chloride and urethane [8] via their reactive oxirane intermediates, where they are considered as the initiating carcinogenic DNA-lesions. An additional pathway via a lipid hydroperoxide (13-HPODE, see Fig. 2) has recently been suggested from in vitro experiments [9]. Nitric oxide via peroxynitrite-induced stress can also produce LPO-derived DNA modifications such as etheno (ɛ)-DNA adducts, demonstrated in a mouse model whereby NO overproduction in vivo led to a concomitant increase in ɛ-adduct levels [10]. These initial results suggested that oxidative and LPO-derived DNA damage could play a major role in the development of human cancers especially those that have an inflammatory component in their etiopathogenesis.

In order to study the role of oxidative stress and LPO in human carcinogenesis, we have developed ultrasensitive methods that allow detection of ɛ-DNA adducts in vivo at levels as low as a few adducts per cell: ɛdA and ɛdC can be quantified by an ultrasensitive immunoaffinity-³²P-postlabelling method [11]. ɛdA excreted in a few millilitres of urine can be measured by an immuno-enriched HPLC-fluorescence method [12]. ɛdA in cells/tissue sections can be visualized by immunohistochemistry [13], [14].

We have also recently developed a sensitive and specific method to quantitate M₁dG in small human samples requiring <10 μg DNA with a detection limit of ∼6 adducts/10⁹ [15]. We have shown the suitability of this method for human biomonitoring in tissues and WBC, allowing simultaneous measurement of ɛ-DNA adducts and M₁dG in the same sample. These comparative analyses are important as MDA is formed not only by LPO but also by additional biochemical pathways, namely from prostaglandin-endoperoxide breakdown and DNA oxidation.

Using our ultrasensitive detection procedures we could unambiguously and quantitatively reveal the existence of highly variable background levels of ɛ-adducts in liver and other tissues from unexposed rodents and humans [16]. This likely reflects the physiological level of LPO, causing DNA damage with which the cell can cope. We then investigated which pathological conditions or cancer risk factors could significantly increase these background adduct levels (for details see earlier reviews [7], [17]). We could show that steady state adduct levels were markedly elevated in humans by: (i) a high ω-6 PUFA diet, (ii) metal (Cu/Fe) storage diseases and (iii) by chronic infections and inflammatory processes, like inflammatory bowel diseases and chronic pancreatitis. In this report, we summarize our recent results on DNA damage arising from inflammatory processes in human liver caused by viral infection and alcohol abuse; we then compare the extent of endogenously produced damage with levels seen in the liver of asymptomatic controls and patients with Wilson's disease and primary hematochromatosis.