286
Ecotoxicological Issues
In addition, one of the main limiting factors is the occurrence of autoxidation
reactions during the work-up of DNA which may induce a significant level of
oxidized base damage. This is particularly true for 8-oxodG (8), whose formation
is significantly enhanced under Fenton reaction conditions (see above). In
addition to the measurement of damage within isolated cells, two main approaches
are currently developed for singling out specific oxidative DNA damage.
The available assays for monitoring oxidative base damage within whole DNA or
after hydrolysis of the biopolymer have been critically reviewed in two recent
surveys [18, 19]. The main methods which in most cases require either enzymatic
digestion or chemical hydrolysis of DNA are presented in Table 2.
The most sensitive methods of detection currently available involve radioactive
postlabeling of nucleoside 3'-monophosphates and dinucleoside monophosphates
which are susbtrates for polynucleotide kinases.
One example is illustrated by the HPLC_32P-postlabeling assay that was recently
devised for the measurement of adenine N-l oxide in cellular DNA exposed to
hydrogen peroxide and UV A radiation [20]. The sensitivity of the method is of
one modification per 10 7 normal bases in a sample size of 1 Ilg of DNA.
Similar assays have been developed for the detection of 5-hydroxymethyluracil,
8-oxo-7 ,8-dihydro-guanine, 5,6-dihydroxy-5,6-dihydrothymine, phosphoglycolate residues and abasic sites [for a recent review, see [21]].
It should be added that chemical postderivatization involving radioactive and
fluorescence detections are also available [21]. The most widely used assay deals
with the measurement of 8-oxodG 8 by electrochemical detection after high
performance liquid separation (HPLC).
Another interesting assay involves the combined use of gas chromatography (GC)
with mass spectrometry. However, the method despite its high intrinsic sensitivity,
suffers from the fact that about 200 Ilg of DNA are required [18].
3
NON INVASIVE ASSAYS
Evaluation of the effects of oxidative stress on DNA in humans requires the
development of non-invasive assays. Attempts are currently being made to use the
release of oxidized bases and nucleosides in urine as an index of DNA damage.
For this purpose, HPLC-EC and GC-MS assays have been applied to the
measurement of several oxidized DNA compounds including mostly
8-oxo-7,8-dihydroguanine, 5-hydroxymethyluracil and their corresponding
nucleosides [22, 23].
A significant increase in the release of 8-oxodG (8) was observed in human urine
and leukocytes of cigarette smokers [for a review, see [15]].
These few examples illustrate the potentiality of using oxidized bases and related
nucleosides as indicators for epidemiological studies aimed at correlating dietary
and life habits with cancer risk.
Ecotoxicological Issues
In addition, one of the main limiting factors is the occurrence of autoxidation
reactions during the work-up of DNA which may induce a significant level of
oxidized base damage. This is particularly true for 8-oxodG (8), whose formation
is significantly enhanced under Fenton reaction conditions (see above). In
addition to the measurement of damage within isolated cells, two main approaches
are currently developed for singling out specific oxidative DNA damage.
The available assays for monitoring oxidative base damage within whole DNA or
after hydrolysis of the biopolymer have been critically reviewed in two recent
surveys [18, 19]. The main methods which in most cases require either enzymatic
digestion or chemical hydrolysis of DNA are presented in Table 2.
The most sensitive methods of detection currently available involve radioactive
postlabeling of nucleoside 3'-monophosphates and dinucleoside monophosphates
which are susbtrates for polynucleotide kinases.
One example is illustrated by the HPLC_32P-postlabeling assay that was recently
devised for the measurement of adenine N-l oxide in cellular DNA exposed to
hydrogen peroxide and UV A radiation [20]. The sensitivity of the method is of
one modification per 10 7 normal bases in a sample size of 1 Ilg of DNA.
Similar assays have been developed for the detection of 5-hydroxymethyluracil,
8-oxo-7 ,8-dihydro-guanine, 5,6-dihydroxy-5,6-dihydrothymine, phosphoglycolate residues and abasic sites [for a recent review, see [21]].
It should be added that chemical postderivatization involving radioactive and
fluorescence detections are also available [21]. The most widely used assay deals
with the measurement of 8-oxodG 8 by electrochemical detection after high
performance liquid separation (HPLC).
Another interesting assay involves the combined use of gas chromatography (GC)
with mass spectrometry. However, the method despite its high intrinsic sensitivity,
suffers from the fact that about 200 Ilg of DNA are required [18].
3
NON INVASIVE ASSAYS
Evaluation of the effects of oxidative stress on DNA in humans requires the
development of non-invasive assays. Attempts are currently being made to use the
release of oxidized bases and nucleosides in urine as an index of DNA damage.
For this purpose, HPLC-EC and GC-MS assays have been applied to the
measurement of several oxidized DNA compounds including mostly
8-oxo-7,8-dihydroguanine, 5-hydroxymethyluracil and their corresponding
nucleosides [22, 23].
A significant increase in the release of 8-oxodG (8) was observed in human urine
and leukocytes of cigarette smokers [for a review, see [15]].
These few examples illustrate the potentiality of using oxidized bases and related
nucleosides as indicators for epidemiological studies aimed at correlating dietary
and life habits with cancer risk.