International Journal of Advancements in Research & Technology, Volume 1, Issue 5, October-2012
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Review: DNA oxidation, its consequences and efficacy of GC-MS
and SPME-GC-MS for In Vitro quantification of DNA oxidative
products
Himansha Singh1, Abhishek Udawat2, Tony Franklin3 and Sai Partha Sarathi4
1
School of Translational Medicine, University of Manchester, Oxford Road, Manchester M139PT, United Kingdom, 2School of Life Sciences, University of Salford,
The Crescent M54WT, United Kingdom, 3 School of Life Sciences, University of Glasgow, Glasgow G128QQ, United Kingdom, 4School of Life Sciences, University
of Salford, The Crescent M54WT, United Kingdom
E-mail: himansha.singh@gmail.com
ABSTRACT
DNA oxidation could be one of the main factors contributing to DNA damage, eventually leading to carcinogenesis, mutations
or non-carcinogenic diseases such as Parkinson’s and Alzheimer’s. Only recently has the focus turned towards identifying oxidative products of DNA and their consequences. Metabolism activities in vitro produce reactive radicals, which can break DNA
strands to cause lesions. These lesions could also act as biomarkers for diagnostic purposes. This review provides an insight of
the DNA oxidation mechanism, its harmful consequences and the advantages/disadvantages of available techniques to quantify
such DNA oxidative products, focussing mainly on the use GC-MS along with derivatization reaction. In addition, the review
also discusses the use of Solid Phase Micro Extraction (SPME) before conducting GC-MS as a potential assay to overcome the
discrepancies involved in using GC-MS alone for the identification of DNA oxidative products.
Keywords : DNA Oxidation, GC-MS, SPME, Derivatization, DNA repair enzymes,
1.0 INTRODUCTION
O
ver the last few decades, DNA damage has intrigued
researchers for diagnosis and understanding of carcinogenesis or non-carcinogenic diseases. Damage to the
gene codons subsequently caused mutations or modifications/perturbations to cellular processes. Many studies have
been directed towards involvement of DNA damage as the
leading contributor to cancer development [1], [2], [3], [4], [5],
[6] and a few other non-carcinogenic diseases such as Parkinson's. Today, analytical chemistry has shown dramatic improvements in identifying and analyzing oxidative DNA adducts. Free radicals have been identified in vivo to play an important role in DMA damage [7], [8]. Oxidation of oxygen results in free radical species which attack DNA strands or bases
[9]. However, these radicals do not break DNA strands under
physiological conditions, instead the toxicity arises due to
their conversion into highly reactive hydroxyl radical (•OH)
[10], [11], [12], [13], [14], [15]. Alternatively, oxidation of proteins and lipids can generate an intermediate, which aids in
DNA oxidation [16].
Free radicals are produced in the form of reactive oxygen species (ROS), reactive chlorine species (RCS) and reactive nitrogen species (RNS). These species may arise from endogenous
factors (metabolic) or by exogenous factors (carcinogenic
compounds and ionizing radiations) [17]. Once oxidized,
DNA loses its genetic integrity and vital functions such as protein translation, which directly or indirectly affects the normal
biological functions of the body. Within the body these damages may be fixed by various repair mechanisms such as base
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excision repair.[18]. However, when the adverse effects outweigh the repair system, it becomes carcinogenic.
Due to these reasons, the oxidation of DNA and its products
could be used for studying mutagenesis or carcinogenicity.
Detection of the oxidized DNA products could, therefore, be
used as a prognostic biomarker to identify DNA damage and
its corresponding oxidative stress [19], [20], [21], [22]. It is also
important to incorporate sensitive analytical techniques for
accurate and rapid detection [23], [24], [25], [26].
1.1 Mechanism of oxidative DNA damage
The oxidative damage of DNA is a complex process influenced by charge transport and by reactions that are controlled
by combination of enthalpic, entropic, steric, and compositional factors. These reactions occur over broad distributions
of energy, time, and spatial scale. Past studied showed that the
stacked aromatic base pairs of duplex DNA could provide a
pathway for efficient movement of charge. These studies eventually led to the emergence of three general views on the
mechanism of long-distance charge transport in DNA: (i) Super-exchange, which includes a one-step coherent transport of
charge by long-distance tunneling from “donor” to “acceptor”
through the intervening “bridging” nucleotide bases; (ii) An
incoherent multistep random walk from donor to acceptor
consisting of short-distance tunneling intervals linked by base
sequences that serve as charge “resting” sites; (iii) Classical
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hopping, where the charge resides on a single base or a small
number of adjacent bases and thermal fluctuations activate its
motion from one base to another [26], [27], [28]. Under oxidizing conditions, DNA loses one electron from a single aromatic
nucleo-base. It causes on electron hole damage to duplex
DNA, which is irreversible [29]. Such oxidation is normally
caused by hydroxyl radical, hydrogen peroxide or singlet oxygen. Five major types of OH radical mediated damages are
seen in oxidized bases, abasic sites, DNA–DNA intra-strand
adducts, DNA strand breaks and DNA–protein cross-links
[30].
Oxidative stress is studied widely as one of the leading reasons behind oxidative damage. It is the inevitable consequence
of the inefficiency of cellular enzymes to reduce reactive free
radicals from cells, especially reactive oxygen species (ROS).
ROS can be formed due to ‘leak’ of electrons from electron
chain cycle or due to redox cycle of endogenous chemicals to
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other oxygen molecules in cells resulting in the formation of
super oxides (O2•–) [31]. Ultraviolet radiations or ionizations
could also result in formation of ROS. Irrespective of their
origin, ROS may interact with cellular biomolecules, such as
DNA, leading to modifications and potentially serious consequences for the cells [32]. Of the ROS, the most reactive species
is studied to be the hydroxyl group (•OH), which can easily
attack the double bond of DNA bases and abstract hydrogen
from the methyl group of thymine and C-H bonds of 2’deoxyribose. [32] Addition to double bonds of DNA bases
occur at or near diffusion controlled rates with rate constants
from 3 - 10 x 109 M–1 s –1; the rate constant of H abstraction
amounts to 2 x 109M–1 s–1 [33]. Addition to C-5 and C-6 bonds
generates C5-OH and C6-OH, where C5-OH is reducing and
C6-OH is oxidizing [34]. Hydroxyl radicals attack the methyl
group of thymine and forms allyl radical. Figure 1 illustrates
the commonly formed products by DNA oxidation.
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Figure 1. Products of DNA Oxidation [33], [34], [35], [36]
1.1.1 Pyrimidine oxidation
Pyrimidine forms several radicals upon oxidation depending
on redox properties/environment and reaction partners [33],
[34], [35], [36]. Pyrimidine oxidation products primarily depend on the availability of oxygen 37], [38]. Under the absence
or insufficiency of oxygen, -OH group addition to C5-OH radicals would generate cytosine and thymine glycols (Fig. 2). The
effect of oxygen on allyl radicals may result in the 5hydroxymethyluracil formation [33], [35], [36]. In the presence
of oxygen, C5-OH reacts to give C5-OH-6-hydroxy-peroxyl
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radicals at a diffusion-controlled rate. Once formed, these radicals can eliminate O2•-, and addition of -OH (in form of water) results in formation of thymine and cytosine glycols [33],
[35]. In the case of allyl radicals, oxygen attacks to form 5hydroxymethyluracil and 5-formyluracil. Thymine peroxyl
radicals undergo reduction, followed by addition of hydrogen
to give hydroxyhydroperoxides. This further decomposes to
yield thymine glycol, 5-hydroxymethyluracil, 5-formyluracil,
and 5-hydroxy-5-methylhydantoin [38].
Cytosine products are prone to deamination and dehydration.
Their deamination results in the formation of products such as
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uracil glycol, 5-hydroxycytosine and 5-hydroxyuracil (Fig. 2)
[36], [37], [39], [40], and [41]. These products are also seen in
the gamma-irradiated cytosine suggesting that these are simultaneously present in the damaged DNA. C5-OH-6-peroxyl
and C6-OH-5-peroxyl radicals of cytosine are further reduced
to form 4-amino-5-hydroxy-2, 6(1H, 5H)-pyrimidinedione and
4-amino-6-hydroxy-2, 5(1H, 6H)-pyrimidinedione. These
could then deaminate to give dialuric acid and isodialuric acid
respectively. Oxygen oxidizes dialuric acid to alloxan which
has been confirmed by studies carried out on Escherichia coli
Nth protein [40], [42]. Acidic treatment of alloxan leads to its
decarboxylation, which yields 5-hydroxyhydantoin. Intramolecular cyclization of cytosine
C5-OH-6-hydroperoxide gives rise to trans-1-carbamoyl-2-oxo4, 5-dihydroxyimidazolidine as a major product [35], [41].
However, this compound is formed as a minor product in
DNA [41], [43], [44], [45].
Figure 2 illustrates the products of thymine after hydroxyl
radical-mediated oxidation. Using NMR 18, the main modified
nucleosides have been isolated and characterized as a result of
oxidation in an aerated aqueous solution.
Figure 2 Hydroxyl radical-mediated oxidation products of the
thymine moiety of DNA. 6-hydroperoxy-5-hydroxy-5, 6dihydrothymidine
[8],
5-hydroperoxy-6-hydroxy-5,
6dihydrothymidine [9], 5-hydroxyperoxymethyl-2’-deoxyuridine
[10], N-2-deoxy-β-D-erythro-pento-furanosyl, formylamine [11],
5,6-dihydroxy-5, 6-dihydro-thymidine [12], 1-2-deoxy-β-Derythro-pentofuranosyl.-5-hydroxy-5methyl hydantoin [13], 1-2deoxy-β-D-erythro-pentofuranosyl.-5-hydroxy 5-methyl barbituric acid [14], 5- hydroxymethyl-2’-deoxyuridine (15) 5-formyl-2’deoxyuridine [16] [Adapted from 4]
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1.1.2 Purine oxidation
Purines are attached by hydroxyl radicals at C4, C8 and C5
positions and generate C4-OH-, C5-OH-, and C8-OH-adduct
radicals. Adenine forms two radicals; C4-OH and C5-OH,
which undergo dehydration to produce a purine (-H)• radical.
This may later be reduced and protonated to reconstitute purines [46], [47], [48], [49], [50], [51], [52]. C4-OH adduct radicals
are oxidizing in nature; whereas C5-OH and C8-OH adduct
radicals are reducing in nature. Dehydration reactions of C4OH adduct radicals of guanine and adenine follows a rate
constant of 1.5 x 105S-1 and 6 x 103 S-1 respectively. Elimination
of –OH from C4-OH adduct radical of guanine results in the
formation of guanine radical cation, which may deprotonate to
give guanine (-H)•. The radical cation of C8-OH may react
with 2’-deoxyribose in DNA by hydrogen abstraction thereby
5
breaking DNA strands [53]. Hydration of guanine•+ results in
the formation of 8-OH-Gua (Fig. 3) [54]. C8-OH radicals of
purines are oxidized by oxygen and unlike C4-OH radicals,
they are controlled by diffusion [55]. Oxidation of C8-OH
competes with the unimolecular opening of the imidazole
ring. The single electron reduction of the ring-opened radical
leads
to
formation
of
2,6-diamino-4-hydroxy-5formamidopyrimidine (Fapy-Gua) from guanine and 4,6diamino-5-formamidopyrimidine (Fapy-Ade) from adenine
[37], [61] (Fig. 1 and Fig. 3). The single electron reduction of
C8-OH-adduct radicals without ring opening gives rise to give
7-hydro-8-hydroxypurines (Fig. 3) [56]. Research has shown
that due to the low ionization potential of guanine amongst
nucleobases, it becomes the most susceptible target of oxidation [57], [58], [59], [60].
Figure 3. The formation of guanine products from the C8-OH adduct radical, which is formed by the attack of •OH to the C8position of guanine [61]
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1.2 Repair mechanisms
The effect of anti-oxidants in the body have been studied and
documented over recent times. The study of oxidative damage
repair of DNA, although fairly new, has been gaining importance and has seen a significant increase in efforts to unravel the mechanism.
The DNA oxidation process leads to the formation of a DNA
base lesion. The repair mechanism involves two processes; (i)
Base Excision Repair (BER), which involves removal of single
lesions by glycosylase action and (ii) Nucleotide Excision Repair (NER), which includes removal of complex lesions containing an oligonucleotide [62]. Figure 4 illustrates, in brief,
the pathways and proteins involved in DNA repair.
8-OH-Gua is considered to be a common purine derived oxi-
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dative. The formation of its lesion in DNA leads to pairing in
two ways i.e. 8-OH-Gua:C pair and 8-OH-Gua:A pair [62],
[63], [64], [65]. These pairs are substrates for two proteinsOGG1 and OGG2 respectively. OGG1 uses the BER mechanism of lesion removal by glycolytic action and uses an internal lysine residue [63], [64]. OGG2 also works in a similar
manner but acts on 8-OH-Gua:A pair, formed due to miscorporation of 8-OH-Gua in nascent DNA [65].
Two other enzymes namely MutY homologue (MYH) and
MutT homologue 1 (MTH1) may also be involved in repair.
The former removes adenine from 8-OH-Gua:A pair and helps
OGG1 [66], [67] and the latter acts at earlier stages to stop the
incorporation of 8-OH-Gua to DNA and degrading it to 8-OHdG for excretion [68]. NEIL1 (Nei-like glycosylases 1), among
newly discovered DNA glycosylases, removes the 8-Oh-Gua
mispairs with G and A [69].
Figure 4. Overview of the main pathways and proteins involved in DNA repair [5]
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Another lesion that is a potential target for studying the repair
process is 8-OH-Ade, however, its mechanism is yet to be understood completely. OGG1 is thought to be involved in this
process but the clear pathway is not yet elusive [70]. Another
protein named Cockayne Syndrome B (CSB) is important for
the repair of 8-OH-Ade. However, it does not support the glycolytic removal of this lesion because this activity is not present in CSB [71].
NER enzyme also plays an important role in repair mechanism. Cyclo-dA, a substrate for NER, is classically associated
with the repair of helix distortion and bulky adducts [72], [73],
[74]. These lesions are formed in 5'S and 5'R diasteriomeric
forms. Among these, 5'R cyclo-dA is more efficiently cured by
NER. 8-OH-Gua lesion is also seen to be removed by NER and
proves that this removal pathway has a much broader range
than previously thought. However, if both NER and BER are
exposed to free substrate competition, BER will repair more
number of 8-OH-Gua unlike NER, which acts on negligible
substrates [75], [76]. NER may be more useful when other
pathways are compromised [77].
Similarly, the NTH1 enzyme is known for its effect on pyrimidine-derived lesion. Tg is considered among the main substrates of NTH1 [78], [79]. Other substrates of NTH1 derived
by cytosine oxidation are 5-hydroxycytosine (preferentially
when it’s paired opposite to guanine) and 5, 6dihydroxycytosine [79]. 5-hydroxycytosine undergoes deamination and forms 5-hydroxyuracil which is a substrate of
NEIL2 enzyme [80]. Other enzymes such as NEIL1, UMG,
SMUGI, MYH and glycosylase are vital in the repair mechanism. However, the role of SMUGI in repair activity of 5OHMeUra is still awaiting concrete evidence for confirmation
[5]. Further research is yet required to suggest evidence-based
literature on repair mechanisms to extrapolate the fate of DNA
lesions.
1.3 Harmful effects of DNA Damage
One thoroughly explored and well-known consequence of the
persistence of DNA lesions is mutation and carcinogenicity.
Although, the presence of DNA lesions is mainly observed in
carcinogenic tumors and are widely studied in cancer cells, it
is a very controversial subject matter [56]. It is also a possibility that DNA damage is just an outcome of the pathophysiological process or the high metabolism and cell turnover rate in tumor formation. The oxidative DNA damage can
lead to carcinogenesis only if the damage occurs in the stem
cells or undifferentiated cells and if this occurs in the coding
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region of the DNA. This is the reason why we can't really
summarize that DNA damage can always elicit cancer [56].
On several occasions, other non-carcinogenic diseases are also
found to be associated with oxidative DNA lesions. For instance, apart from being involved with carcinogenicity, enhanced levels of 8-OH-Gua lesion has also shown to be associated with Parkinson's disease, Colorectal cancer, Cardiovascular disease and Rheumatoid arthritis, to name a few. Although
it is not the most abundant product of DNA oxidation, it is
widely and extensively studied due to its high importance as
disease biomarkers owing to its easy detection [66], [81], [82],
[83]. Site-specific approaches have also been used to demonstrate mutagenesis by 8-oxo-adenine, thymine glycol and 5hydroxyuracil [68]. Other studies have confirmed the high
mutagenic properties of 5-hydroxyuracil and uracil glycol [84].
Clearly, the involvement of DNA oxidative products in serious illnesses can’t be ignored and it is important to develop
assays for the identification of such derivatives, keeping in
mind the efficacy, cost and convenience of its usage.
1.4 Measurement of oxidative DNA damage
Up until the mid-1980s, researchers were focused on DNA
damage by exogenous factors such as polycyclic hydrocarbons, aromatic amines, nitrosamines etc. The analytical techniques used in such research must have good sensitivity and
specificity to measure the oxidative adducts. The accurate assessment of oxidative DNA damage in samples should be a
valuable marker, not only of considering the risk in occurrence
of carcinogenic or non-carcinogenic events, but also the overall
level of oxidative stress within the body. The measurement of
oxidized and modified bases plays very important role in
studying DNA damage and main marker being used is 8oxoguanine. The development of a simple and reliable method
for analysis of multiple DNA base-damage products is important. DNA damage in normal cell size would usually range
to be one single lesion per 105–106 [90]. Various analytical
techniques are used to measure the oxidative damage to DNA.
These include immunochemical techniques, post-labeling assays, comet assay, alkaline elution with the use of DNA repair
enzymes, high-performance liquid chromatography (HPLC)
with electrochemical detection (ECD) and GC/MS,
LC/MS/MS, and LC/MS [85], [91] [92], [93]. The main concern in such studies remains to be the identification of the extent of DNA oxidation that actually occurs within the DNA.
There is a lot of variance in the amount of oxidation taking
place in these studies [85]. Concerns regarding the formation
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tion of the sample remain, in order to make it optimum for
GC-MS studies [86], [87]. Research has shown that the limitation of measuring lesions is not the sensitivity of the assay but
the possibility that artifactual DNA oxidation gives overestimated results [99], [100], [101], [102].
8-oxo-G can be formed from dG by the silylation process used
for making volatile derivatives in GC-MS. This raises the question as to whether a pre-purification step should be employed.
It is even more concerning whilst dealing with a low amount
of DNA. The formation of repair enzymes does support the
presence of lesions (8-oxo-G) but it does quantify them [88].
Therefore, the process of evaluation has to be made more sensitive. Recent studies have been carried out towards quantifying another such residue, namely thymine glycol, by the use of
a monoclonal antibody specific to it. A secondary fluorescently
labeled antibody makes this process extremely sensitive with a
possibility to detect as low as 3x10-21 mol of thymine glycol
[89]. This approach may be used for other lesions as well but
would require a specific monoclonal antibody. Up until then,
thymine glycol is considered to be the main biomarker used
for the study of oxidative damage to DNA [88].
The measurement of oxidative DNA lesions can be carried out
either by direct or indirect methods. The direct method involves the isolation of cellular DNA, followed by the enzyme
or chemical hydrolysis, which is then, studied using chromatographic analysis. On the other hand, the indirect method
involves breakage of strands where comet assay, alkaline elution or DNA unwinding assay carries out the measurement.
PCR and antibody detection technique may also be used for
measurement.
Alkaline Elution (AE) involves cell lysis, followed by DNA
elution using alkaline solution [103]. The rapidity of DNA elution depends on the amount of DNA collected and is analysed
using a fluorescent dye. It is also dependent on the length of
the DNA fragment and on the number of DNA strand-breaks
[104].
Comet Essay involves addition of cells onto an agarose gel
plate, which is hydrolysed by alkaline solution. The movement is then observed during electrophoresis. Movement towards the anode proves the presence of DNA, which are then
observed under a fluorescence microscope forming loops like
the tail of a comet [105], [106], [107]. The percentage of DNA in
the tail of the comet is proportional to the oligonucleotide
break frequency.Alkaline unwinding is another fairly new
technique that uses alkaline pH to unwind DNA [108].
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All the aforementioned assays lead to strand breaks. When
exposed to DNA N-Glycosylase, a repair enzyme, the lesions
are recognized and quantified. This quantification is carried
out by subtracting the number of strand breaks following glycosylase incubation from the number in absence of enzymes.
Indirect assays are advantageous as they are more sensitive,
have lower background values and need lower number of
cells for performing the assay. However, they do have their
disadvantages as well; (i) if two lesions are present in close
proximity, it is difficult to identify them as separate entities,
(ii) they require highly specific DNA glycosylases to avoid
negative results and (iii) only a small number of lesions can be
identified, which means that the assays are only effective if the
magnitude ranges from 0.1 to 10 lesions per million nucleosides [109].
On the other hand, the direct approach involves isolation of
cellular DNA, which is then hydrolysed and separated to
DNA constituents to aid the detection of lesions. Direct approaches have been known to generate potential artifactual
DNA oxidation and explain the origin of discrepancies between reported levels of 8-oxodGua in different literatures
even though detected with same method [109]. To avoid this, a
chemical precursor of labelled singlet oxygen is used to generate 18O-labeled 8-oxoGuo in cellular DNA, which is considered an internal standard [110], [111]. The then labelled/unlabelled 8-0xoGuos are measured by MS. Any decomposition of oxidised nucleosides can be monitored by
looking at the levels of labelled/unlabelled entities. HPLC-EC,
HPLC-MS/MS, GC MS and 32P post labelling are used in direct approach. Oxidative DNA Damage is measured in whole
DNA as well as extracted DNA. Methods involved in measuring that of extracted DNA include HPLC, GC or capillary electrophoresis (CE). Floyd proposed HPLC technique for the
measurement of 8-oxoguanine [94].
1.41 Assay development using DNA repair enzymes
Some assays involve the use of DNA repair enzymes and require no need of DNA extraction. Here, DNA repair endonucleases play an important role in in situ analysis of the cells
and reveal the strand breaks at modified bases [97].
Methods used for this purpose include (i) the comet assay single cell electrophoretic method, sedimentation techniques including (ii) the alkaline elution assay and (iii) the alkaline unwinding. DNA repair enzymes mainly include the Fpg protein, endonuclease III and exonuclease III to monitor the formation of modified purine bases, oxidized pyrimidine bases
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and abasic sites respectively. The main advantage of comet
and elution assays is that they minimize the auto oxidation
processes, which are responsible for artifactual oxidation
caused by exonuclease digestion of DNA. The compact nature
of DNA and shorter incubation periods are the main reasons
behind this reduced auto oxidation. The use of immunefluorescent detection directed to the target is also advantageous in the comet assay technique [49], [52], [97]. The noninvasive assays aid the evaluation of oxidative stress on human DNA. The accurate amount of oxidized bases and nucleosides (used as biomarkers) that are released in urine are
quantified using HPLC or GC/MS assays, which include direct measurement of 8-oxo-7, 8-dihydroguanine or 5hydroxymethyl uracil.
1.4.2 Assay Development by HPLC-EC
HPLC-EC runs on the principle that oxidised purines have a
redox potential below that of the normal nucleoside. The EC
detector oxidises 8-oxo-Guo specifically and such oxidation
liberates electrons, which are specifically detected. The natural
nucleotides are not detected here as they are not oxidised at
the defined potential and thus makes the technique sensitive.
However, since the separation is to be done in isocratic conditions, it is almost impossible to detect more than one nucleotide in a single injection [95]. Application of this assay has extended to a few other electroactive oxidized lesions including
8-oxo-7,
8-dihydroadenine,
5-hydroxycytosine,
5hydroxyuracil and the corresponding 2’ deoxyribonucleosides
[95].
1.4.3 GC-MS in combination with derivatization for the
identification of oxidative DNA products
GC-MS was initially developed to detect lesions formed by the
oxidation of DNA. This technique, being more sensitive and
versatile, makes it possible to study a large amount of DNA
base products simultaneously. The earlier use of GC in combination with MS saw several comebacks in terms of combining
the two. This was solved by jet separators, which used the
principle that the sample with the carrier gas coming out of
the GC can be concentrated to a small exit nozzle, which
pushes the sample to the vacuum region of MS but with a gap
between the two, which causes the carrier gas to disperse [113]
However, theses separators faced several maintenance issues..
Nowadays, the capillary column is mostly inserted directly
into the ion source of the MS [114]. GC-MS instruments generate approximately 600,000 numbers per minute. High-speed
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computers are used to convert this data into mass peaks in real
time and further into mass intensity pairs [115]. For qualitatively analyzing this information, the approach uses a three
dimensional mass spectra of the intensities and masses plotted
against the retention time that they are obtained in [116].
The pre-condition for using a sample in GC is that it has to be
volatile, organic and should be in form of a solution before
injection [112]. The sample used should also have a vapour
pressure of more than 10-10 torr. As for compounds with lower
pressures, derivatization is required using chemicals such as
BSTFA, which adds a trimethylsilyl (TMS) group to the compound. Trimethylsilylation is the most common derivatization
reaction used [95], [96].
Derivatization is a technique used to enhance chromatographic sensitivity. It also helps improve the performance of GC for
compounds carrying a reactive hydrogen or oxygen atom. In
other words, it is a technique which aids in improving the
conditions for GC by increasing the volatility of the otherwise
non-volatile compounds. Derivatization also helps when the
compound of interest goes undetected or does not produce a
desirable peak [118]. Compounds with reactive hydrogen/oxygen can infer with the normal analysis by GC by reacting with the surface of the injection port or the analytical
column. It could result in tailing of peaks or a low response.
Such compounds are also highly soluble in sample phase
which causes poor partitioning into headspace. Derivatization
can improve their volatility, as well as reduce the potential for
surface adsorption once they enter the GC system. Therefore,
derivatization is a useful tool to detect compounds in complex
matrix and is widely used in forensic, medicinal and environmental chemistry [119].
The low volatility of compounds may be due to their sizes.
Larger compounds are held by dispersion forces and smaller
ones by strong intermolecular forces. The volatility of these
compounds may be improved with derivatization [120]. Sample vial is used as the reaction vessel for derivatization. Although, it has been pointed out that derivatization is essential
to improve GC analysis performance, it could also introduce
some problems into the analytical scheme. For instance, byproducts of derivatization could be volatile, and therefore interfere with the test compounds by eluting with similar retention times as theirs, causing partial or complete co-elution.
Temperature and pressure should also be maintained optimum to the samples. Special attention is given to pressure
inside the sample vials and specially designed caps allow excess pressure to be vented during these reactions.
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Techniques used in derivatization include esterification, acetylation, silylation and alkylation. Past studies show that silylation is the most prevalent technique amongst others mentioned [121], [122], [123]. Various studies have been documented over the years to validate the use of trimethylsilyl derivatives for GC-MS [118]. Silylation mainly occurs via SN2nucleophilic attack of trimethylsilyl (TMS) group on the test
compound, which eventually leads to the replacement of hydrogen [124]. It results into production of more volatile, thermally stable compound. The leaving group should have low
basicity and the ability to stabilize the negative charge produced in the transition state of SN2 nucleophilic reaction [119].
In addition, there should be very little or negligible π back
bonding between the leaving and silicon group. Another factor to involve is the use of reagents which shouldn’t be harmful for column. BSTFA {N, O-bis (trimethylsilyl) trifluoroacetamide} is a commonly used reagent in trimethylsilylation of
alcohols, alkaloids, amines, biogenic amines, carboxylic acids,
phenols and steroids. This reagent silylates a wide range of
non-sterically hindered functional groups [97]. The mass spectrometer may be set in the selective ion monitoring mode in
order to detect only ions corresponding to the major peaks in
the mass spectrum of the compound of interest. This also provides a specific and sensitive detection. BSTFA has previously shown success as a derivatization agent with biological matrices on hypnotics like benzodiazepines [134], antiinflammatory drugs like naproxen [126], hydroxylated polycyclic aromatic hydrocarbons [127], pharmaceuticals [128],
contaminants like hydroxylated polycyclic biphenyls [129] and
flavonoids [130].
DNA oxidative products are identified using acid hydrolysis
of the DNA followed by derivatization by trimethylsilyl
(TMS). MS has the advantage of detecting and quantifying
several DNA lesions simultaneously. The GC-MS assay was
first applied to the detection of 8oxoGua in isolated DNA that
was exposed to y-radiation in an aqueous solution. Subsequently, the method has been applied numerous times to the
measurement of various types of oxidative base damage in
isolated DNA. At times, DNA may be exposed to elevated
temperatures and prooxidant chemicals and therefore have
the potential to cause further artifactual oxidation of DNA
(especially of guanine residues), raising the apparent level of
base oxidation products and invalidating the measurement.
This can be reduced by lowering temperature of derivatization
process and addition of ethanediol [98]. The values obtained
by GC-MS in previous studies were found significantly higher
than those obtained by the HPLC-EC approach [95]. These
Copyright © 2012 SciResPub.
10
higher values were later concluded to be a result of artifactual
DNA oxidation produced due to the silylation step performed
at higher temperatures [109].
The result of getting errors in GC-MS may be due to several
reasons; sample contamination, sample decomposition, problems with the GC column, the GC/MS interface or even a
faulty data system. It’s therefore recommended to use standards before performing the experiments [117]. The use of Solid
Phase Micro Extraction (SPME) before conducting GC-MS
could also have a potential to overcome the discrepancies involved in using GC-MS alone for identification. SPME, which
comprises of a silica-based fibre coated with a polymer, is a
rapid sampling technique compatible with GC analysis [131],
[132]. It can use volatile/non-volatile compounds, Gas/liquid
samples and also eliminates the need of solvents and complex
apparatus for concentrating samples [133],[134],[135].SPME
can also use a wide concentration of analytes by extracting
them from the gas/liquid sample and adsorbing them on a
solid stationary phase. The principle behind this technique
relies on the equilibrium of the analytes in three phases; namely polymeric liquid coating, the headspace and the aqueous
solution. The SPME extraction is completed when the analyte
reaches equilibrium between sample matrix and fibre coating
[136], [137], and [138]. The fibre carrying the concentrated
analyte is then removed from the sample vial and inserted into
the injector port of the GC where the analyte is desorbed from
the fibre. The sample is then run in GC followed by the mass
spectrometer for identification [131].
2.0 CONCLUSION
Over the past couple of decades, DNA damage by oxidation
has intrigued several researchers. The metabolic activities in
cells produce reactive oxygen species, which in turn gives rise
to highly reactive free radicals. Hydroxyl radical are extremely
harmful and can attack nucleotides, proteins or lipids. DNA,
being the most sensitive and important macromolecule, produces several consequences. If oxidized by reactive radicals,
DNA bases can suffer severe lesions, which in turn may be
carcinogenic. The frequency of lesions is important in the development of mutations, carcinogenic states, and some degenerative diseases [139], [140], [141], [142]. Several reports of carcinogenic incidences and involvement of DNA damage in
Parkinson's have been presented. Such oxidative products can
be useful as bio-markers as their enhanced levels could help in
diagnosis of specific diseases. They also provide information
about oxidative stress within the body [143]. A simple, reliable
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International Journal of Advancements in Research & Technology, Volume 1, Issue 5, October-2012
ISSN 2278-7763
method for analyzing different DNA bases is important in
order to identify such products. Nevertheless, measurement of
a single oxidation product can give misleading results [144].
Although several techniques have been used, the traditional
GC-MS method has proven its credibility and successfully
shown to produce the oxidative products of DNA [145], [146],
[147], [148]. The addition of SPME technique prior to GC-MS
could also be efficient to improve the sensitivity of the assay.
Further experiments should be designed in order to completely understand the SPME-GC-MS, also keeping the parameters
suitable for the analysis. For instance, addition of salt, sample
agitation and fibre selection is very important for SPME. The
use of TMCS over BSTFA should also be explored to enhance
the derivatization capacity, and to increase the sensitivity of
the detection technique. Furthermore, coating the GC-MS fibre
with 5 % Phenyl methylsilicone gum may be helpful to permit
measurement of high sensitivity.
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