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In relation to carcinogenesis, aging and other pathologic conditions, urinary 8-hydroxydeoxyguanosine (8OHdG) is widely used as a marker for evaluating the effect of oxidative stress on DNA. Because no reports have described how 8OHdG is generated from DNA in vivo or by biological materials, and how it is excreted into urine, the authors investigated the generation of 8OHdG from DNA, using rat liver homogenate. Oxidatively damaged DNA samples containing different levels of 8OHdG were prepared using ultraviolet irradiation with three different concentrations of riboflavin. Following incubation of damaged DNA samples with rat liver homogenates, the generation of 8OHdG from the DNA was determined using high-performance liquid chromatography with electrochemical detection after ultrafiltration of the incubation mixtures. The generation of 8OHdG was also tested with an anti-8OHdG antibody. The quantity of 8OHdG generated from the DNA by rat liver homogenates was dependent on the 8OHdG levels in the DNA: almost all 8OHdG in the DNA was released as 8OHdG by rat liver homogenates. Generation of 8OHdG correlated with the degradation of DNA. Interestingly, the generated 8OHdG was stable in the presence of rat liver homogenates, whereas deoxyguanosine (dG) rapidly disappeared in the same conditions. Less than 1/10 000 of dG was converted to 8OHdG when dG was incubated with rat liver homogenate. Incubation of 8-hydroxyguanine with rat liver homogenates did not generate 8OHdG. These findings suggest that most of the 8OHdG in DNA is released as 8OHdG during DNA degradation and that, because of its stability, 8OHdG is excreted into urine, thus providing a convenient measure of oxidative damage to DNA. (Cancer Sci 2005; 96: 13–19)
Despite the presence of antioxidant defenses and DNA repair systems, oxidative damage to DNA is an inevitable consequence of metabolic activities, of ionizing radiation, and of environmental mutagens.(1−3) Such DNA damage is thought to play an important role in carcinogenesis, in aging and in a number of other pathological conditions.(4−6) Among the many types of oxidative base damage, 8-hydroxydeoxyguanosine (8OHdG) is the most extensively studied, both because of its mutagenicity,(7,8) and because its presence can be determined with high sensitivity.(9,10) In reactive oxygen species-related carcinogenesis, the level of 8OHdG in target tissues appears to play a critical role,(11,12) and this has led to 8OHdG being widely used as a marker of oxidative DNA damage.(13,14) However, because of the scantness of 8OHdG in DNA, and because of secondary formation during the analysis of 8OHdG in cellular DNA, urinary 8OHdG has been used to evaluate the level of 8OHdG in DNA, and a number of analytical methods have been developed with which to reliably measure 8OHdG in urine.(15−18) Furthermore, findings show that levels of urinary 8OHdG correlate well with many pathological conditions, particularly with carcinogenesis.(19−21)
Even so, although urinary excretion of 8OHdG has been proposed as a candidate biomarker of oxidative stress to DNA,(22) the ultimate source of urinary 8OHdG has not been clarified. In humans, urinary excretion of 8-hydroxyguanine (8OHG) and 8OHdG is reported to not depend on diet,(23) and may reflect the involvement of different repair mechanisms, namely base excision repair (BER) and nucleotide excision repair (NER).(24) BER is largely responsible for the removal of non-bulky base adducts, and involves specialized enzymes that recognize a specific repertoire of lesions. In this process, a number of glycosylases have been identified.(25,26) These enzymes, however, excise damaged bases, resulting in the excretion of damaged bases, rather than damaged nucleosides, into urine. Another set of human 8OHdG repair enzymes, endonucleases,(27) along with the NER process, which probably acts simply as a back-up system,(28) are likely to generate 8OHdG from DNA and thus contribute to the presence of 8OHdG in urine. No experimental evidence, however, has been provided to support this conjecture. Findings for several processes other than DNA repair indicate that other channels contribute to the background levels of 8OHdG that are excreted in urine. For example, even though proof of a defined role is still not forthcoming,(18) 8OHdG may derive from sanitation of the nucleotide pool by the action of human MutT homolog (MTH),(29,30) or from dead cells.(1) Potential sources of urinary 8OHdG have been collated in a comprehensive review.(31) Thus far, however, there have been neither reports that have described the generation of 8OHdG from DNA through incubation with tissue or cell extracts, nor have any researchers shown any correlation between the amount of 8OHdG generated and the 8OHdG levels in DNA.
In the present report, to more clearly elucidate the source of urinary 8OHdG, the authors investigated whether 8OHdG is generated from DNA by rat liver homogenate, and whether the amounts of generated 8OHdG correspond with the levels of oxidative damage in DNA.
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Urinary 8OHdG is widely used as a biological marker with which to evaluate oxidative stress in the body.(22,34,35) Its usefulness, however, has so far been limited because we do not know enough about how 8OHdG comes to be present in urine.(23,31) No evidence has been presented that 8OHdG is released from DNA by tissues, cells, or by their extracts. Here, the authors clearly show that 8OHdG is generated from DNA by rat liver homogenates (Fig. 1). Because other compounds that are present in rat liver homogenates might have, in HPLC analysis, produced peaks at the same position as 8OHdG, the authors tested with an anti-8OHdG antibody. The antibody absorbed the peak almost completely, indicating that 8OHdG was generated from DNA (Fig. 3). Our data also show that the quantity of 8OHdG generated from the DNA corresponds with the level of oxidative damage in the DNA (Fig. 2). These findings indicate that, in vivo, 8OHdG is generated from DNA, and that the amounts of generated 8OHdG are useful for evaluating oxidative damage in DNA. However, attention should be paid when determining the 8OHdG quantity (see Fig. 2).
A semiquantitative assay of 8OHG using the 8OHdG detection system showed that the quantities of 8OHG produced from DNA were approximately 1/25 of the quantities of 8OHdG after incubation of DNA with rat liver homogenates, and that the proportion did not vary with the 8OHdG levels in DNA (data not shown). It might be thought that just 70% of 8OHdG was released from DNA after a 24-h incubation period (Table 1). However, Figure 5C shows that 75% of 8OHdG could be recovered after the same incubation time in the presence of rat liver homogenates. Thus, in this system, the authors considered that most of the 8OHdG in damaged DNA was released as 8OHdG.
The authors were surprised that most of the 8OHdG was released from DNA by the rat liver homogenates. At first, it was considered that 8OHdG was generated during the DNA repair process, because dG, a DNA degradation product,(1) was barely detectable in the ultrafiltrates. The result of electrophoresis (Fig. 4), however, indicated that the generation of 8OHdG co-occurred during DNA degradation. In the presence of rat liver homogenates, it is possible that dG rapidly disappeared, which was confirmed as shown in Figure 5A. When DNA was incubated with rat liver homogenates for 1 h, the electrophoretic mobility of oxidatively damaged DNA was decreased, probably due to the interaction between the DNA and the proteins in the homogenate.
It is also possible that 8OHdG is generated from dG or 8OHG. In particular, generation from dG has been reported in the co-presence of oxidants.(9,36,37) Commercially available dG preparations usually contain 1–5 molecules of 8OHdG per 100 000 of dG (data not shown). When dG was incubated with rat liver homogenates, however, <1/10 000 of the dG was converted to 8OHdG during 6 h of incubation (Fig. 5A). Meanwhile, 8OHdG was not generated when 8OHG was incubated with rat liver homogenates (Fig. 5B). These findings indicate that, during DNA degradation, 8OHdG was generated directly from DNA. The authors suggest that 8OHdG is generated when oxidative stress causes tissue or cell destruction: in such conditions, both oxidative DNA damage and tissue or cell homogenates could be produced. Because oxidative stress induces apoptosis,(38,39) and DNA is extensively degraded during apoptosis,(40) apoptotic cells might also be sources of 8OHdG. The authors are now investigating whether living cells could also generate 8OHdG from DNA using a cell culture system.
Liver contains many types of nuclease(41−44) that degrade DNA to nucleotides. In turn, these can be dephosphorylated to nucleosides by the phosphatases that are also present in the liver.(45,46) Some nucleases in the liver are reported to be sensitive to NaCl,(47,48) and when NaCl was added to the incubation mixtures, NaCl at concentrations of more than 150 mmol/L inhibited the generation of 8OHdG (data not shown). The finding further supports our conclusion that 8OHdG generation is coupled with DNA degradation. Thus it seems plausible that, in the present experiment, the nucleases and phosphatases present in the liver were responsible for the generation of 8OHdG from DNA. Additionally, in support of this conclusion, the technique for determining 8OHdG in DNA uses nuclease P1, an exonuclease, and alkaline phosphatase.(11,33,49) Further study, however, is required to identify which enzyme or enzymes are responsible for the generation of 8OHdG from DNA. Furthermore, investigation as to which organ most efficiently generates 8OHdG may eventually make it possible to use urinary 8OHdG to evaluate organ-specific oxidative stress. In contrast to rat liver homogenates, Fpg protein, a bacterial homolog of oxoguanine glycosylase that acts as a DNA BER enzyme,(50,51) generated 8OHG from DNA, but not 8OHdG (data not shown).
It is interesting that, while dG rapidly disappeared under the same conditions, in the presence of rat liver homogenates more than 75% of 8OHG and 8OHdG remained unchanged up to 24 h of incubation (Fig. 5). These findings suggest that 8OHdG and 8OHG are stable in the body and in the circulation, and so may be excreted into urine unchanged, whereas most of dG undergoes breakdown and may not be detectable in urine as intact dG. This hypothesis is supported by the finding that the quantities of 8OHdG and 8OHG in urine are greatly disproportionate to the quantity of dG in urine.(18,22,52) It is also interesting that 8OHdG and 8OHG seem not to be metabolized or reused, suggesting the presence of mechanisms that do not allow the naturally occurring damaged base to be incorporated into nucleic acids. Our discovery of the stability of 8OHdG in the presence of rat liver homogenates suggests a useful substrate that could be used to study nucleases. Because 8OHdG is a stable product of nuclease reaction and can be determined with high sensitivity, DNA with 8OHdG seems to be a better substrate than DNA without 8OHdG.
In conclusion, 8OHdG is released from DNA by rat liver homogenates in quantities that correspond with the levels of oxidative damage in the DNA. Because 8OHdG is stable in the presence of rat liver homogenates, it is likely that 8OHdG is stable enough in circulation to be excreted into urine. Thus, urinary 8OHdG, if determined at appropriate times or with 24-h urine testing, is a useful marker of oxidative DNA damage that is induced by oxidative stress, particularly oxidative stress that leads to the organ or cell destruction, or apoptosis. Although the present results do not show the in vivo generation of 8OHdG from DNA directly, they show that 8OHdG is generated from DNA by a biological material, rat liver homogenate.