8-oxo-7,8-dihydro-2'-deoxyguanosine as a biomarker of oxidative damage in oesophageal cancer patients: lack of association with antioxidant vitamins and polymorphism of hOGG1 and GST

Oesophageal cancer remains an important public health concern worldwide with an estimated burden of 500, 000 new cases in 2005 [1]. The two major histological types of oesophageal cancers, squamous cell carcinoma (SCC) and adenocarcinoma (ADC) differ substantially in their underlying patterns of incidence and key etiologic factors. Alcoholism and smoking are the major established risk factors for SCC, whereas Barrett's oesophagus or gastro-oesophageal reflux disease (GORD) are consistently associated with an increased risk of ADC.

Oxidative stress and reactive oxygen species (ROS) are thought to play a role in oesophageal carcinogenesis. ROS may result from external factors such as smoking, and alcohol metabolism, or may be produced endogenously via inflammatory conditions such as oesophagitis or GORD or may also be due to precancerous lesions (Barrett's oesophagus), as has been shown experimentally in rats [2, 3]. Diet influences incidence of oesophageal cancers. An adequate diet of fruits and vegetables is associated with a decreased incidence [4], presumably due to a better supply of antioxidants.

Among the various markers of oxidative stress, 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) is particularly popular. It is generated by the oxidation of DNA under physiopathological conditions or environmental stress, but is also a by-product of normal cellular metabolism. It is a premutagenic oxidized-DNA lesion as it is able to mispair with adenine, thus generating G:C to T:A transversion mutations, unless the lesion is repaired prior to DNA replication [5]. Moreover, affordable analytical methods are available for its quantification. In population studies, 8-oxodG is often determined in DNA extracted from white blood cells, peripheral blood mononuclear cells (PBMC) or lymphocytes, although PBMCs being mostly lymphocytes, the distinction is rarely made. These cells are considered to be representative of the whole organism in terms of the level of exposure of to oxidative stress. However, it has been suggested that the apparent high levels of 8-oxodG could be due to artefactual oxidation of DNA during the treatment of the samples. The European Standards Committee on Oxidative DNA Damage (ESCODD) has now been set up within the European laboratory network to improve and harmonise 8-oxodG measurement methods [6‐9].

In a previous study [10], we have described the optimisation of an analytical procedure to measure 8-oxodG in PBMCs by using HPLC coupled with electrochemical detection (HPLC-ED). In that study [10], the protocol was applied to the analysis of 8-oxodG in PBMCs of subjects (n = 60) from a case-control study that included both, SCC and ADC cases. Control samples (n = 43) exhibited 4.9 ± 1.9 molecules of 8-oxodG per 10⁶ unaltered guanosines, levels which correspond to the median values reported by the latest ESCODD trial for HPLC measurement in lymphocytes from healthy young men [11]. In comparison, oesophageal cancer patients (n = 17) showed higher oxidative DNA damage as indicated by the 8-oxodG levels of 7.2 ± 2.6 per 10⁶, 2'-dG (Student's t-test, P < 0.001). This difference remained significant even after technical (storage, sampling period, 2'-dG levels) and individual (age, sex, smoking, alcohol) confounding factors were taken into account (P < 0.0001, generalized linear regression model). Moreover, data on smoking habits and alcohol consumption of the volunteers were available, and could be correlated with the observed levels of oxidatively-damaged DNA.

The aim of the present study was to characterize the relationship between the levels of oxidative stress, antioxidant vitamins and genetic constitution in oesophageal cancers. An elevated level of oxidative DNA lesions could be related to exogenous or endogenous parameters. Therefore, factors that may influence the extent of oxidative DNA damage such as the nutritional status and genetic polymorphisms were included in this study.

Antioxidant vitamins, such as vitamin A and vitamin E are effective free radical scavengers and can also be useful markers of antioxidant status. Presumably, a higher production of ROS due to severe oxidative stress, characteristic of oesophageal cancers, could lead to a higher metabolic consumption of the antioxidant vitamins, and this would be reflected in their lower serum levels. This "antioxidant hypothesis" was examined in the subjects included in our study by determining the serum concentrations of vitamins A and E.

Oxidatively damaged bases in DNA are preferentially repaired by base excision enzymes. The hOGG1 gene encodes the human 8-oxo-guanine DNA glycosylase that cleaves the 8-oxo-guanine base from damaged DNA. The single-nucleotide polymorphism at codon 326 (Ser 326, rs 1052133) is the most well-studied polymorphism of hOGG1. Homozygous carriers of the variant form of the hOGG1 Ser 326Cys gene appear to have a reduced capacity to repair oxidised DNA lesions [12‐14], although this is controversial [15]. A genetic susceptibility toward SCC of the oesophagus linked to the Ser 326Cys polymorphism in the hOGG1 gene has been described [16]. We have measured this polymorphism in our population and correlated it with the corresponding 8-oxodG level.

Polymorphism also exists in genes encoding enzymes involved in the metabolism of xenobiotics that act as an indirect source of free radicals. Genetic polymorphisms in genes involved in detoxification such as glutathione-S-transferases (GST), GSTM1, GSTT1 and GSTP1 could potentially affect the susceptibility of an individual to the adverse effects of environmental risk factors involved in oesophageal cancer. The above three genes, are expressed in the oesophageal mucosa. We have earlier reported a significant increase in the risk of oesophageal cancers correlated with the null genotypes of GSTM1 and GSTT1 but not with the GSTP1 Ile/Val polymorphism [17]. The polymorphisms in the GST genes were analysed according to their histological status, among controls and cases of oesophageal cancers. These polymorphisms were revisited in the present study to investigate their correlation with the levels of 8-oxodG.

Oxidative damage to DNA is considered to be an important risk factor for carcinogenesis. 8-oxodG is a key biomarker in this process because it is one of the most frequently encountered product of oxidatively-damaged DNA and also one that can be easily detected in samples of tissues or urine [26‐30]. We have previously reported a significantly higher level of 8-oxodG in circulating blood cells from oesophageal cancer patients compared to control subjects [10]. Similar observations have been made for colorectal carcinoma [31], lung cancer [22, 24, 32] and leukaemia [33, 34]. In our study, none of the individual variables such as smoking, alcohol, sex or age, was shown to influence 8-oxodG concentrations. The aim of the present study was to identify other factors that could modulate 8-oxodG levels. We have attempted to characterize the relationship between oxidative stress, evaluated in terms of levels of 8-oxodG in PBMCs, and the levels of antioxidant vitamins and the genetic constitution, in a population consisting of healthy volunteers and oesophageal cancer patients.

Vitamin C, vitamin E, carotenoids, and other antioxidants present in fruits and vegetables could contribute to cancer prevention by protecting DNA from oxidative damage, according to the "antioxidant hypothesis". By inference, the endogenous levels of these antioxidant vitamins in the serum of oesophageal cancer patients are expected to be low. Likewise, under conditions of severe oxidative stress also, their serum levels may be low as these would be consumed in redox reactions involving ROS.

Many recent epidemiological studies have confirmed that a high intake of fruits and vegetables is associated with a decreased risk of upper aero-digestive tract cancers [4, 35‐37]. One of the possible mechanisms of this protective effect is the antioxidant activity of vitamins A, C and E. These vitamins are effective antioxidants in vitro, and might be expected to protect against cancer. Calişkan-Can et al. [24] found lower levels of β-carotene and vitamins A, C and E in lung cancer patients compared to healthy controls. Foksinski et al. [23] observed that the mean levels of all the measured antioxidant vitamins were significantly lower in smokers in comparison with non-smokers. Gackowski et al. [22] reported that vitamin E levels were significantly reduced in the plasma of lung cancer patients (smokers and ex-smokers) compared to healthy smokers and that the levels of vitamins A and E in plasma of colorectal carcinoma patients were lower than in the control group [31]. In contrast, other studies found no significant differences between healthy smokers and non-smokers for either serum vitamin A or vitamin E [22, 38, 39]. Also, several large-scale antioxidant supplementation trials have failed to show any clear evidence for a decrease in cancer risk [30, 40]. In our study, we found that the endogenous serum levels of vitamins A and E were similar in oesophageal cancer patients and in controls. Notably, despite the fact that our study subjects come from the same geographical area, there was substantial intersample variability, especially for the cancer cases. These differences could reflect the balance between absorption and tissue secretion, and may also be genetically determined. A recording of dietary habits (fruit and vegetable consumption) could have added a complementary and an interesting feature to our study. Determination of the two major water-soluble antioxidants, ascorbate and glutathione would also have brought complementary information. However, as particular conditions are required for sample collection, processing and storage to prevent their oxidation and degradation, these could not be analysed in this retrospective study.

Correlation between the levels of vitamins and 8-oxodG has been reported. In their analyses of 30 cross-sectional studies, Moller & Loft [41] identified 12 studies showing an inverse correlation between oxidatively damaged DNA and antioxidant levels, 16 reporting no correlation and two, a positive correlation. A lack of a correlation between 8-oxodG and antioxidant vitamins has also been reported by others [22, 35, 42]. In a recent paper, Sram et al. [43] found a negative correlation between 8-oxodG and β-carotene and vitamin E but a weak positive association with vitamin A. Similar positive correlations were reported for vitamin A in chemical workers exposed to vinyl chloride monomer [44], carotenoids and vitamin E [45]. We did not find any correlation between the levels of 8-oxodG and vitamins in our study group (cases and controls combined).

Interpretation of these correlative data must be made with extreme caution because the precise effects of antioxidants on mutagenesis and carcinogenesis remain unclear. An antioxidant, including a vitamin antioxidant, is essentially a redox (reduction-oxidation) agent that provides protection against free radicals, but may promote free radical generation under certain circumstances or may exert pro-oxidant effects. Conversely, recent meta-analysis on supplementation trials indicates increased risk of mortality [40], suggesting a pro-oxidant activity at high doses or in cancer-risk subjects (smokers and workers exposed to asbestos). Further, besides vitamins, several food-derived phytochemicals or bioreactive substances in natural products likely contribute to the protection of DNA from damage and thus influence the generation of 8-oxodG in vivo. The number of such antioxidants exceeds that of antioxidant vitamins. The availability of these unidentified antioxidants in individual diet could thus affect the correlation between levels of 8-oxodG and antioxidant vitamins. Some dietary components also could up-regulate DNA repair without having any recognised antioxidant function. Interestingly, a positive association was observed in our study between the levels of 8-oxodG and those of the two vitamins, but only in the cases and not in the controls. However, this observation should be interpreted with caution, in the light of the foregoing discussion. Moreover, to arrive at a more convincing conclusion, our data would have to be expanded and adjusted for possible confounders such as age which can become the predominant, independent determinant of oxidative damage as has been discussed recently [43].

In view of the conflicting reports in the literature and the results of the present study, the "antioxidant hypothesis" seems open to criticism. Is there indeed a relationship between antioxidant vitamins and oxidatively-damaged DNA? Secondly, are the concentrations of antioxidants and 8-oxodG in the blood representative measures of the situation in the target tissue of the carcinogenesis and a true reflection of overall cellular DNA damage? Thirdly, do we have reliable tools to examine this correlation? The choice and reliability of biomarkers such as 8-oxodG has also been debated [28, 30, 46]. The reliability of 8-oxodG is influenced by its method of detection since its artefactual production is a serious concern. Notably, the values of 8-oxodG reported in this study are low and reach the background level of 8-oxodG recommended by ESCODD for HPLC-ED measurement, indicating that these were not an artefact.

It is known that individuals have different responses to oxidative damage and that the risk for oxidative stress-related cancer varies according to both, the environmental exposure and the genetic background. The human 8-oxoguanine DNA glycosylase1 (hOGG1) is one of the major enzymes involved in DNA base excision repair (BER). A positive relationship between hOGG1 mRNA expression and 8-oxodG suggests that the expression level of hOGG1 may be interpreted as a biomarker of exposure to oxidative DNA damage [47, 48]. On the other hand, some studies indicated that there was no interaction between these parameters [12, 49, 50], which could be explained by the fact that hOGG1 is weakly expressed in certain tissues such as the aerodigestive tract tissue [51].

The activity of hOGG1 can be impaired by a polymorphic mutation at codon 326, the hOGG1 Ser 326Cys polymorphism. However, the phenotypic impact of hOGG1 Ser 326Cys polymorphism is unclear. Recent data have shown that the hOGG1 Ser 326Cys polymorphism is associated with a reduced DNA repair capacity for oxidatively-damaged DNA [52], whereas according to others, the converse is true [15, 53, 54]. This led to the conclusion that both, wild type and the hOGG1Cys326 variant-encoded proteins should be functional and probably do not exhibit significant differences in repair activities and hence the polymorphism at codon 326 would probably be neutral [53, 55].

Many epidemiological studies have investigated the association of the Ser 326Cys polymorphism in the hOGG1 gene indicating an increased risk for head and neck cancers but the reports are conflicting [51, 56, 57]. Studies on the prevalence of this polymorphism in susceptibility to oesophageal cancer also show conflicting results. Xing et al. [16] reported a positive association between the Cys 326 variant and oesophageal cancer risk in Asians population whereas Tse et al. [58] reported no association in Caucasians. In the present study, the small number of samples did not allow us to make a comparison of the genotype distribution between cases and controls in order to determine whether the hOGG1^326Cys allele contributed to the risk of oesophageal cancer. However, the distribution of hOGG1 Ser 326Cys genotype in our controls (0.44) is in agreement with the frequencies previously described in Caucasian population. This frequency is classically lower than that in Asians [21, 51, 59], suggesting that this allele may be differently distributed among ethnic groups and may not confer a particular susceptibility to oesophageal cancer in Caucasian population. The allelic distribution of this polymorphism in our combined population followed Hardy-Weinberg equilibrium.

Besides DNA repair activity, enzymes involved in the detoxification of xenobiotics such as glutathione S-transferases may influence the extent of oxidative damage in humans. We genotyped our study population for the GSTM1, GSTT1 and GSTP1 genes. Our results indicate no association between GSTM1 and GSTT1 null polymorphisms and 8-oxodG levels in DNA from PBMCs. On the other hand, we found a statistically significant association between GSTP1 Val/Val homozygote carriers and a high level of 8-oxodG (Figure 2). However, as no obvious relationship was found between the frequency of the Val allele (Val/Val and Ile/Val combined) and the level of 8-oxodG, we consider this result questionable. Indeed, correlation of GST polymorphisms with 8-oxodG levels in WBCs or lymphocytes varies with the context of exposure: polycyclic aromatic hydrocarbons [60, 61], benzene [62], fine particulate matters [63] and hyperbaric oxygen [64].

In conclusion, although the power of our study is limited, it seems likely that vitamin levels in serum and polymorphisms in the hOGG1 or GST genes are not important modulators of 8-oxodG levels. Further studies in a larger cohort of patients using other biomarkers of oxidative damage and antioxidant status are required to better understand the possible involvement of oxidative stress in oesophageal cancer.