Determination of cisplatin 1,2-intrastrand guanine-guanine DNA adducts in human leukocytes by high-performance liquid chromatography coupled to inductively coupled plasma mass spectrometry.

Platinum-containing drugs are widely used to treat cancer in a variety of clinical settings. Their mode of action involves the formation of DNA adducts, which facilitate apoptosis in cancer cells. Cisplatin binds to the N7 position of the purine DNA bases forming intrastrand cross-links between either two adjacent guanines [cis-Pt(NH(3))(2)d(pGpG), 1,2-GG] or an adjacent adenine and guanine [cis-Pt(NH(3))(2)d(pApG), 1,2-AG)]. The cytotoxic efficacy for each of the different types of DNA adducts and the relationship between adduct levels in tumor cells and blood are not well understood. By using these Pt-containing adduct species as biomarkers, information on a patient's response to chemotherapy would be directly related to the mode of action of the drug. This type of analysis requires the most sensitive and specific methods available, to facilitate detection limits sufficient to measure the DNA adduct in the limited sample quantities available from patients. This was achieved in the current study by coupling a highly specific enzyme-based adduct isolation method with a sensitive detection system based on HPLC coupled to inductively coupled plasma mass spectrometry to measure the 1,2-GG cisplatin adducts formed in DNA. The method was developed and validated using calf thymus DNA and two different adenocarcinoma cell lines. The values for the limit of detection (LOD) and the limit of quantitation determined for the 1,2-GG cisplatin adduct were 0.21 and 0.67 fmol per microg DNA, respectively. This corresponds to an absolute LOD of 0.8 pg as Pt for the 1,2-GG adduct. Cisplatin-sensitive (H23) and -resistant (A549) tumor cells were exposed to the drug, and the 1,2-GG adduct levels were measured over a 24 h time period. The results showed a statistically significant (P < 0.05) higher concentration in the sensitive cells as compared to the resistant cells after repair for 7 h. Although the adduct concentration present fell at subsequent time points (12 and 24 h), the levels in each cell line were broadly similar. The protocol was then applied to the analysis of patient samples taken before and then 1 h after treatment. The 1,2-GG cisplatin adduct was present in the range from 113 to 1245 fg Pt per microg DNA in all of the patient samples taken after treatment. Although the adduct was not present at levels greater than the LOD in the initial pretreatment samples, trace amounts were discernible in some patient samples on their third treatment cycle.

Phosphotriester adducts (PTEs): DNA's overlooked lesion.

In addition to reacting with DNA base moieties, many chemical genotoxins also react with the oxygen atoms of the internucleotidic phosphodiester linkages to form phosphotriester adducts (PTEs). In view of their stability under physiological conditions, it has been suggested that PTEs may be useful biomarkers for measuring cumulative genotoxin exposure. The methodology for their determination is varied and still not completely developed but includes determination of hydrolysis products and (32)P-postlabelling approaches. More recently, transalkylation and direct mass spectrometry techniques have been devised, which give extra chemical information on the structures of the PTEs. The proportion of DNA damage formed as PTEs is much greater with SN1 compared to SN2 alkylating agents, and it has been shown in DNA that the formation of PTEs is partially sequence dependent. PTEs have been considered to be refractory to repair in mammalian cells but repair mechanisms have been found in prokaryotic cells, e.g. PTEs in Escherichia coli are repaired by O(6)-methylguanine-DNA methyltransferase (O(6)-MGT or Ada protein). However, studies on in vivo persistence of PTEs in mammalian systems have not ruled out the possibility of a contribution from an active repair process for PTEs. The biological significance of PTEs is largely unstudied and unknown, although effects of PTEs on DNA polymerases, and some exo- and endonucleases have been observed. Also site-specific PTEs impair the repair processing of adjacent sites of DNA damage, which may be a biological mechanism of importance for these lesions. In this review, we will consider the analytical methods available for the determination of PTEs, their stability in vitro and in vivo, the mechanisms for their repair, their possible biological significance and their potential role as biomarkers in human molecular epidemiology studies.

Development of a liquid chromatography-electrospray ionization tandem mass spectrometry method for detecting oxaliplatin-DNA intrastrand cross-links in biological samples.

Cellular resistance, both intrinsic and acquired, poses a problem in the effectiveness of platinum-based chemotherapy. The cytotoxic activity of Pt-based chemotherapeutic agents is derived from their ability to react with cellular DNA. Oxaliplatin binds to the N7 position of the purine DNA bases, forming mainly intrastrand cross-links between either two adjacent guanines (GG), an adjacent adenine and guanine (AG), or two guanines separated by an unmodified nucleotide (GNG). We report the development of a liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) method for measuring GG and AG intrastrand cross-links formed by oxaliplatin. The limits of detection for GG-oxPt and AG-oxPt were 23 and 19 adducts per 10 (8) nucleotides, respectively. We compare the formation and persistence of intrastrand cross-links between wild-type and glutathione transferase P null mice (GSTP null) treated with oxaliplatin. No significant difference was observed in the level of intrastrand cross-links formed by oxaliplatin between the mouse strains in liver, kidney, and lung DNA. Adduct levels were greatest in liver and lowest in lung tissue.

Detection and quantitation of benzo[a]pyrene-derived DNA adducts in mouse liver by liquid chromatography-tandem mass spectrometry: comparison with 32P-postlabeling.

The polycyclic aromatic hydrocarbon, benzo[a]pyrene (B[a]P) is a proven animal carcinogen that is potentially carcinogenic to humans. B[a]P is an ubiquitous environmental pollutant and is also present in tobacco smoke, coal tar, automobile exhaust emissions, and charred food. A liquid chromatography-tandem mass spectrometry (LC-MS/MS) method using electrospray ionization and selected reaction monitoring (SRM) has been developed for the detection of 10-(deoxyguanosin-N(2)-yl)-7,8,9-trihydroxy-7,8,9,10-tetrahydrobenzo[a]pyrene (B[a]PDE-N(2)dG) adducts formed in DNA following the metabolic activation of B[a]P to benzo[a]pyrene-7,8-dihydrodiol-9,10-epoxide (B[a]PDE). The method involves enzymatic digestion of the DNA sample to 2'-deoxynucleosides following the addition of a stable isotope internal standard, [(15)N(5)]B[a]PDE-N(2)dG, and then solid phase extraction to remove unmodified 2'-deoxynucleosides prior to analysis by LC-MS/MS SRM. The limit of detection of the method was 10 fmol (approximately 3 B[a]PDE-N(2)dG adducts per 10(8) 2'-deoxynucleosides) using 100 microg of calf thymus DNA as the matrix. Calf thymus DNA reacted with B[a]PDE in vitro and mouse liver DNA samples at different time points following dosing intraperitoneally with 50, 100, and 200 mg/kg B[a]P was analyzed. Three stereoisomers of the B[a]PDE-N(2)dG adduct were detected following the reaction of calf thymus DNA with B[a]PDE in vitro. The levels of B[a]PDE-N(2)dG DNA adducts in the mice livers were found to increase in a dose-dependent manner with adducts reaching maximal levels at 1-3 days and then gradually decreasing over time but still detectable after 28 days. A very good correlation (r = 0.962, p < 0.001) was observed between the results obtained for the mouse liver DNA samples using LC-MS/MS SRM as compared to those obtained using a (32)P-postlabeling method. However, the levels of adducts observed following (32)P-postlabeling using butanol enrichment were approximately 3.7-fold lower. The LC-MS/MS method allowed the more precise quantitation of DNA adduct levels that were structurally characterized, in addition to a reduction in the time taken to perform the analysis when compared with the (32)P-postlabeling method.

Phosphate alkylation in different DNA substrates: the role of local DNA sequence and electrophile character in determining the nonrandom nature of phosphotriester adduct formation.

DNA phosphate oxygens are sites for alkylation leading to DNA phosphotriester adduct (PTE) formation. Previously, we have reported that the manifestation of PTEs was nonrandom in mouse liver DNA treated in vivo [Guichard et al. (2000) Cancer Res. 60, 1276-1282], and while further studies revealed possible PTE repair, this was determined not to play a role in the observed nonrandom manifestation in vivo [Le Pla et al. (2004) Chem. Res. Toxicol. 17, 1491-1500]. In the present study, to determine whether the nonrandom manifestation of PTEs in vivo was specifically due to their nonrandom formation, we have compared the in vitro formation of diethylsulfate (DES)-induced PTEs in h2E1/OR human B-lymphoblastoid cells, their isolated nuclei, and their isolated DNA, using the 5' nearest neighbor analysis postlabeling procedure developed by Le Pla et al.. Furthermore, to determine the role of electrophile character in PTE manifestation, prepared oligonucleotides ([dT](20)[dG](20):[dC](20)[dA](20)) were treated with three alkylating agents of differing electrophilic character (DES, methylnitrosourea, and ethylnitrosourea), and PTE manifestation was assessed by postlabeling. The formation of PTEs was determined to be nonrandom in the whole cells, nuclei, and DNA, with PTEs being formed to a greater extent 3' to pyrimidine moieties than 3' to purine moieties. The studies with the oligonucleotides confirm these observations and demonstrate that the nonrandom formation of PTEs is primarily determined by DNA sequence, and not by DNA packaging/chromatin factors, and that the extent of the nonrandom formation of PTEs is also governed by electrophile reactivity, with the more reactive electrophiles yielding a more random formation of PTEs. From our observations, we propose a model for the nonrandom formation of PTEs, which is governed by (i) the phosphate oxygens having to compete with adjacent nucleophilic sites for the alkylating electrophile and (ii) the electrophile's inherent reactivity.

Further development of 32P-postlabeling for the detection of alkylphosphotriesters: evidence for the long-term nonrandom persistence of ethyl-phosphotriester adducts in vivo.

DNA phosphate oxygens are sites for alkylation leading to phosphotriester adducts (PTEs). PTEs are reported to be both abundant and persistent and so may serve as long-term markers of genotoxicity. Previously, we reported a 32P-postlabeling assay for the specific detection of PTEs plus identification of nucleosides located 5' to PTEs. Using this, we demonstrated the nonrandom nature of ethyl-PTEs (Et-PTEs) in vivo, these results being suggestive of either the nonrandom formation of Et-PTEs in vivo or sequence specific Et-PTE repair. Presently, we report the further development and validation of the 32P-postlabeling assay, to permit the more straightforward determination of nucleosides 5' to PTEs and, using this, have investigated the long-term persistence of PTEs in vivo. Analysis of liver DNA of mice treated in vivo with N-nitrosodiethylamine reveals an initial decline in the level of Et-PTEs (t1/2<24 h) as well as their nonrandom persistence for the duration of the time course, with approximately 37 and approximately 15% of the initial Et-PTEs remaining 4 and 56 days after treatment, respectively. From this, we conclude that Et-PTEs are suitable as long-term markers of genotoxic exposure and that putative PTE repair is not responsible for their nonrandom manifestation. However, the possibility of active repair contributing to the initial decline of Et-PTEs is considered.