A fluorescence-based analysis of aristolochic acid-derived DNA adducts

Aristolochic acids (AAs), major components of plant extracts from Aristolochia species, form (after metabolic activation) pro-mutagenic DNA adducts in renal tissue. The DNA adducts can be used as biomarkers for studies of AA toxicity. Identification of these adducts is a complicated and time-consuming procedure. We present here a fast, nonisotopic, fluorescence-based assay for the detection of AA-DNA adducts in multiple samples. This approach allows analysis of AA adducts in synthetic DNA with known nucleotide composition and analysis of DNA adducts formed from chemically diverse AAs in vitro. The method can be applied to compare AA-DNA adduct formation in cells and tissues.     

Ultra-performance liquid chromatography/tandem mass spectrometry for accurate quantification of global DNA methylation in human sperms

Aberrant DNA methylation in human sperms has been proposed to be a possible mechanism associated with male infertility. We developed an ultra-performance liquid chromatography/tandem mass spectrometry (UPLC-MS/MS) method for rapid, sensitive, and specific detection of global DNA methylation level in human sperms. Multiple-reaction monitoring (MRM) mode was used in MS/MS detection for accurate quantification of DNA methylation. The intra-day and inter-day precision values of this method were within 1.50-5.70%. By using 2-deoxyguanosine as an internal standard, UPLC-MS/MS method was applied for the detection of global DNA methylation levels in three cultured cell lines. DNA methyltransferases inhibitor 5-aza-2'-deoxycytidine can significantly reduce global DNA methylation levels in treated cell lines, showing the reliability of our method. We further examined global DNA methylation levels in human sperms, and found that global methylation values varied from 3.79% to 4.65%. The average global DNA methylation level of sperm samples washed only by PBS (4.03%) was relatively lower than that of sperm samples in which abnormal and dead sperm cells were removed by density gradient centrifugation (4.25%), indicating the possible aberrant DNA methylation level in abnormal sperm cells. Clinical application of UPLC-MS/MS method in global DNA methylation detection of human sperms will be useful in human sperm quality evaluation and the study of epigenetic mechanisms responsible for male infertility.     

Carcinogenic and nephrotoxic alkaloids aristolochic acids upon activation by NADPH : cytochrome P450 reductase form adducts found in DNA of patients with Chinese herbs nephropathy

Aristolochic acid (AA), a naturally occurring nephrotoxin and carcinogen, has been found to be implicated in an unique type of renal fibrosis, designated Chinese herbs nephropathy (CHN), and associated with the development of urothelial cancer in CHN patients. Understanding, which enzymes are involved in AA activation and/or detoxication is important in the assessment of individual susceptibility of humans to this natural carcinogen. Using the nuclease P1 version of the 32P-postlabeling assay we examined the ability of microsomal NADPH: CYP reductase to activate AA to metabolites forming DNA adducts. Renal and hepatic microsomes, containing NADPH:CYP reductase, generated AA-DNA adduct patterns reproducing those found in renal tissues in patients suffering from a renal fibrosis CHN and urothelial cancer. 7-(Deoxyadenosin-N6-yl)aristolactam I, 7-(deoxyguanosin-N2-yl)aristolactam I and 7-(deoxyadenosin-N6-yl)aristolactam II were identified as AA-DNA adducts formed by AAI. Two AA-DNA adducts, 7-(deoxyguanosin-N2-yl) aristolactam II and 7- (deoxyadenosin-N6-yl) aristolactam II, were generated from AAII. According to the structures of the DNA adducts identified, nitroreduction is the crucial pathway in the metabolic activation of AA. The identity of NADPH: CYP reductase as activating enzyme in microsomes has been proved with different cofactors and an enzyme inhibitor. Alpha-lipoic acid, a selective inhibitor of NADPH: CYP reductase, significantly decreased the amount of the adducts formed by microsomes. Likewise, only a cofactor of the enzyme, NADPH, supported the DNA adduct formation of AAI and AAII, while NADH was ineffective. These results demonstrate an involvement of NADPH: CYP reductase in the activation pathway of AAI and AAII in the microsomal system. Moreover, using the purified enzyme, the participation of this enzyme in the formation of AA-DNA adducts was confirmed. The results presented here are the first report demonstrating a reductive activation of natural nitroaromatic compounds, AA, by NADPH: CYP reductase.     

Analyses of DNA adducts formed by ochratoxin A and aristolochic acid in patients with Chinese herbs nephropathy.

Chinese herbs nephropathy (CHN), a unique type of nephropathy has been associated with the intake of weight-reducing pills containing the Chinese herb Aristolochia fangchi. Moreover, an association between the use of A. fangchi and urothelial cancer in CHN patients has been reported indicating that aristolochic acid (AA) the major alkaloid of A. fangchi might be the causal agent. Similarities of CHN to the Balkan endemic nephropathy (BEN) have led to the hypothesis of a common etiological agent for both diseases. Evidence has accumulated that BEN is an environmentally-induced disease strongly associated with the fungal mycotoxin ochratoxin A (OTA). Both, AA and OTA are nephrotoxic and carcinogenic and induce the formation of DNA adducts. As OTA has been suspected as fungal contaminant in the herbal batches used for the preparation of the weight-reducing pills we analysed tissues from CHN patients by the 32P-postlabeling procedure for the presence of DNA adducts related to both OTA and AA exposure. Whereas, AA-specific DNA adducts were detected in all five urinary tract tissues from five patients (total RAL: 32-251 adducts per 10(9) nucleotides), OTA-related DNA adducts were detectable in two kidneys and one ureter only (total RAL: 1.5-3.7 adducts per 10(9) nucleotides). Thus, OTA-related DNA adduct levels were about 50 times lower than AA-DNA adduct levels. In female and male rats that were treated with the slimming regimen in the same way like the CHN patients except that the amount of Chinese herbs was 10 times higher, AA-DNA adducts were found in kidney tissues (total RAL ranging from 51 to 83 adducts per 10(9) nucleotides) but adducts derived from OTA were not observed. These results demonstrate that OTA-related DNA adducts do not play a key role in CHN or CHN-associated urothelial cancer.     

Speciation of oxaliplatin adducts with DNA nucleotides

This paper describes a set of fast and selective high performance liquid chromatography (HPLC) methods coupled to electro-spray ionisation linear ion trap mass spectrometry (ESI-MS), sector-field inductively coupled plasma mass spectrometry (SF-ICP-MS) and UV detection for in vitro studies of the bifunctional adducts of oxaliplatin with mono-nucleotides, di-nucleotides and cellular DNA. The stationary phases and the optimised conditions used for each separation are discussed. Interaction of oxaliplatin with A and G mono-nucleotides resulted in the formation of five bifunctional platinum diaminocyclohexane (DACHPt) adducts. These were two isomers of the A-DACHPt-A and A-DACHPt-G adducts, and one G-DACHPt-G adduct, as confirmed by MS/MS spectra obtained by collision induced dissociation. These adducts were also characterised by UV absorption data and SF-ICP-MS elemental (195)Pt and (31)P signals. Further, interaction of oxaliplatin with AG and GG di-nucleotides resulted in the formation of three adducts: DACHPt-GG and two isomers of the DACHPt-AG adduct, as confirmed by ESI-MS and the complementary data obtained by UV and SF-ICP-MS. Finally, a very sensitive LC-ICP-MS method for the quantification of oxaliplatin GG intra-strand adducts (DACHPt-GG) was developed and used for monitoring the in vitro formation and repair of these adducts in human colorectal cancer cells. The method detection limit was 0.14 ppb Pt which was equivalent to 0.22 Pt adduct per 10(6) nucleotides based on a 10 μg DNA sample. This detection limit makes this method suitable for in vivo assessment of DACHPt-GG adducts in patients undergoing oxaliplatin chemotherapy.     

Evidence for reductive activation of carcinogenic aristolochic acids by prostaglandin H synthase -- (32)P-postlabeling analysis of DNA adduct formation

Aristolochic acid (AA), a naturally occurring nephrotoxin and carcinogen, is implicated in an unique type of renal fibrosis, designated Chinese herbs nephropathy (CHN), which can develop to urothelial cancer. Understanding which enzymes are involved in AA activation and/or detoxication is important in the assessment of an individual susceptibility to this natural carcinogen. We examined the ability of prostaglandin H synthase (PHS) to activate AA to metabolites forming DNA adducts with the nuclease P1 and 1-butanol extraction enrichment procedure of the (32)P-postlabeling assay. PHS is a prominent enzyme in the kidney and urothelial tissues. Ram seminal vesicle (RSV) microsomes, which contain high levels of PHS, generated AA-DNA adduct patterns reproducing those found in renal tissues in CHN patients. 7-(Deoxyadenosin-N(6)-yl)aristolactam I, 7-(deoxyguanosin-N(2)-yl)aristolactam I and 7-(deoxyadenosin-N(6)-yl)aristolactam II were identified as AA-DNA adducts formed by AAI. Two adducts, 7-(deoxyguanosin-N(2)-yl)aristolactam II and 7-(deoxyadenosin-N(6)-yl)aristolactam II, were generated from AAII. According to the structures of the DNA adducts identified, nitroreduction is the crucial pathway in the metabolic activation of AA. The identity of PHS as the activating enzyme in RSV microsomes was proven with different cofactors and inhibitors. Only indomethacin, a selective inhibitor of PHS, significantly decreased the amount of adducts formed by RSV microsomes. The inhibitor of NADPH:CYP reductase (alpha-lipoic acid) and some selective inhibitors of cytochromes P450 (CYP) were not effective. Likewise, only cofactors of PHS, arachidonic acid and hydrogen peroxide, supported the DNA adduct formation of AAI and AAII, while NADPH and NADH were ineffective. These results demonstrate a key role of PHS in the activation pathway of AAI and AAII in the RSV microsomal system and were corroborated with the purified enzyme, namely ovine PHS-1. The results presented here are the first report demonstrating a reductive activation of nitroaromatic compounds by PHS-1.     

Dissection of Targeting Molecular Mechanisms of Aristolochic Acid-induced Nephrotoxicity via a Combined Deconvolution Strategy of Chemoproteomics and Metabolomics

Aristolochic acid (AA), mainly derived from herbal Aristolochia and Asarum plants, was listed as a human carcinogen class I in 2002. Aristolochic acid nephropathy (AAN) is a rapidly progressive tubulointerstitial nephritis and urothelial cancer caused by AA. However, the targeting molecular mechanisms of AAs-induced nephrotoxicity are largely unclear. This study aims to dissect targeting molecular mechanisms of AA-induced nephrotoxicity. Activity-based protein profiling (ABPP) in combination with cellular thermal shift assay (CETSA) was performed to identify the AAs binding target proteins. Our data indicated that several key enzymes in the metabolic process and mitochondrial respiration including IDH2 and MDH2 (Krebs cycle), PKM and LDH (aerobic respiration), FASN (fatty acid beta-oxidation), HK2 (glucose metabolism), and ATP synthase were identified as directly binding targets of AAs. Metabolomics and oxygen consumption rate (OCR) experiments further confirmed that AAs targeting proteins disrupted metabolic biosynthesis processes and impaired mitochondrial functions. Ultimately, AAs induced renal cells apoptosis by disturbing various biological processes. Cumulatively, AAs may directly bind to key proteins involved in the metabolic process and mitochondrial homeostasis, and finally induce aristolochic acid nephropathy. Our findings provide novel insight into underlying mechanisms of AAs-induced kidney toxicity, which may help to develop therapeutic strategies for AAN.     

Quantification of aristolochic acid-derived DNA adducts in rat kidney and liver by using liquid chromatography-electrospray ionization mass spectrometry

Aristolochic acid (AA), derived from the herbal genus Aristolochia and Asarum, has recently been shown to be associated with the development of nephropathy. Upon enzyme activation, AA is metabolized to the aristolactam-nitrenium ion intermediate, which reacts with the exocyclic amino group of the DNA bases via an electrophilic attack at its C7 position, leading to the formation of the corresponding DNA adducts. The AA-DNA adducts are believed to be associated with the nephrotoxic and carcinogenic effects of AA. In this study, liquid chromatography coupled with electrospray ionization mass spectrometry (LC-MS) was used to identify and quantify the AA-DNA adducts isolated from the kidney and liver tissues of the AA-dosed rats. The deoxycytidine adduct of AA (dC-AA) and the deoxyadenosine-AA adduct (dA-AA) were detected and quantified in the tissues of rats with one single oral dose (5mg or 30mg AA/kg body weight). The deoxyguanosine adduct (dG-AA), however, was detected only in the kidney of rats that were dosed at 30mg AA/kg body weight for three consecutive days. The amount of AA-DNA adducts found in the rats correlated well with the dosage.     

Krebs durch Chemikalien

Aristolochia as human carcinogen Aristolochic acid (AA), the active principle of the old drug Aristolochia ssp. has recently been classified as human carcinogen. The elucidation of the molecular mechanism of carcinogenesis of AA in animals led to the establishment of two specific biomarkers which were used to prove a causative role of AA in human cancers. These are AA‐DNA adducts, biomarkers of exposure to AA and AT→TA transversion mutations induced by these DNA adducts, biomarkers of effect. By detecting both biomarkers in individuals who ingested Aristolochia herbs during a weight‐reduction regimen in Belgium and in farmers in the Balkans where Aristolochia ssp are growing as weeds in cereal fields AA was identified as the cause for urothelial cancer and a disease of the kidney (AAN).     

Second-tier test for quantification of underivatized amino acids in dry blood spot for metabolic diseases in newborn screening

The quantitative analysis of amino acids (AAs) in single dry blood spot (DBS) samples is an important issue for metabolic diseases as a second-tier test in newborn screening. An analytical method for quantifying underivatized AAs in DBS was developed by using liquid chromatography coupled with tandem mass spectrometry (LC–MS/MS). The sample preparation in this method is simple and ion-pairing agent is not used in the mobile phase that could avoid ion suppression, which happens in mass spectrometry and avoids damage to the column. Through chromatographic separation, some isomeric compounds could be identified and quantified, which cannot be solved through only appropriate multiple reactions monitoring transitions by MS/MS. The concentrations of the different AAs were determined using non-deuterated internal standard. All calibration curves showed excellent linearity within test ranges. For most of the amino acids the accuracy of extraction recovery was between 85.3 and 115 %, and the precision of relative standard deviation was <7.0 %. The 35 AAs could be identified in DBS specimens by the developed LC–MS/MS method in 17–19 min, and eventually 24 AAs in DBS were quantified. The results of the present study prove that this method as a second-tier test in newborn screening for metabolic diseases could be performed by the quantification of free AAs in DBS using the LC–MS/MS method. The assay has advantages of high sensitive, specific, and inexpensive merits because non-deuterated internal standard and acetic acid instead of ion-pairing agent in mobile phase are used in this protocol.     

Detection and quantitation of N-(deoxyguanosin-8-yl)-2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine adducts in DNA using online column-switching liquid chromatography tandem mass spectrometry

The heterocyclic aromatic amine, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) is formed by the grilled cooking of certain foods such as meats, poultry and fish. PhIP has been shown to induce tumours in the colon, prostate and mammary glands of rats and is regarded as a potential human dietary carcinogen. PhIP is metabolically activated via cytochrome P450 mediated oxidation to an N-hydroxylamino-PhIP intermediate that is subsequently converted to an ester by N-acetyltransferases or sulfotransferases and undergoes heterolytic cleavage to produce a PhIP-nitrenium ion, which reacts with DNA to form the N-(deoxyguanosin-8-yl)-2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP-C8-dG) adduct. Thus far, the detection and quantification of PhIP-DNA adducts has relied to a large extent on ³²P-postlabelling methodologies. In order to expand the array of available techniques for the detection and improved quantification of PhIP-C8-dG adducts in DNA we have developed an online column-switching liquid chromatography (LC)–electrospray ionization (ESI)-tandem mass spectrometry (MS/MS) selected reaction monitoring (SRM) method incorporating an isotopically [¹³C₁₀]-labelled PhIP-C8-dG internal standard for the analysis of DNA enzymatically hydrolysed to 2′-deoxynucleosides. A dose-dependent increase was observed for PhIP-C8-dG adducts when salmon testis DNA was reacted with N-acetoxy-PhIP. Analysis of DNA samples isolated from colon tissue of mice treated by oral gavage daily for 5 days with 50 mg/kg body weight of PhIP resulted in the detection of an average level of 14.8 ± 3.7 PhIP-C8-dG adducts per 10⁶ 2′-deoxynucleosides. The method required 50 μg of hydrolysed animal DNA on column and the limit of detection for PhIP-C8-dG was 2.5 fmol (1.5 PhIP-C8-dG adducts per 10⁸ 2′-deoxynucleosides). In summary, the LC–ESI-MS/MS SRM method provides for the rapid automation of the sample clean up and a reduction in matrix components that would otherwise interfere with the mass spectrometric analysis, with sufficient sensitivity and precision to analyse DNA adducts in animals exposed to PhIP.     

Metabolic activation of carcinogenic aristolochic acid, a risk factor for Balkan endemic nephropathy

Aristolochic acid (AA), a naturally occurring nephrotoxin and carcinogen, is associated with tumor development in patients suffering from Chinese herbs nephropathy (now termed aristolochic acid nephropathy, AAN) and may also be a cause for the development of a similar type of nephropathy, the Balkan endemic nephropathy (BEN). Major DNA adducts [7-(deoxyadenosin-N6-yl)-aristolactam and 7-(deoxyguanosin-N2-yl)aristolactam] formed from AA after reductive metabolic activation were found in renal tissues of patients with both diseases. Understanding which human enzymes are involved in AA activation and/or detoxication is important in the assessment of an individual's susceptibility to this plant carcinogen. This paper reviews major hepatic and renal enzymes responsible for AA-DNA adduct formation in humans. Phase I biotransformation enzymes play a crucial role in the metabolic activation of AA to species forming DNA adducts, while a role of phase II enzymes in this process is questionable. Most of the activation of AA in human hepatic microsomes is mediated by cytochrome P450 (CYP) 1A2 and, to a lower extent, by CYP1A1; NADPH:CYP reductase plays a minor role. In human renal microsomes NADPH:CYP reductase is more effective in AA activation. Prostaglandin H synthase (cyclooxygenase, COX) is another enzyme activating AA in human renal microsomes. Among the cytosolic reductases, NAD(P)H:quinone oxidoreductase (NQO1) is the most efficient in the activation of AA in human liver and kidney. Studies with purified enzymes confirmed the importance of CYPs, NADPH:CYP reductase, COX and NQO1 in the AA activation. The orientation of AA in the active sites of human CYP1A1, -1A2 and NQO1 was predicted from molecular modeling and explains the strong reductive potential of these enzymes for AA detected experimentally. We hypothesized that inter-individual variations in expressions and activities of enzymes activating AA may be one of the causes responsible for the different susceptibilities to this carcinogen reflected in the development of AA-induced nephropathies and associated urothelial cancer.     

Measurement of DNA adducts in cells exposed to cisplatin

The two main cisplatin-induced DNA lesions, G--G and A--G, have been measured in cells exposed to the drug. (G--G and A--G denote the intrastrand bifunctional adducts formed between adjacent purine bases.) It has proven feasible, using liquid chromatography-tandem mass spectrometry (LC-MS/MS), observe the G--G and A--G lesions in mouse fibroblast cells exposed for 1 h to a 120 microM concentration of cisplatin. After extraction of the DNA from the cells, the lesions were enzymatically isolated from the DNA in the form of modified dinucleoside monophosphates with the phosphodiester bond intact. MS/MS detection of the modified dinucleoside monophosphates in the negative ion mode manifests two transitions; from the negative ion to the loss of one NH(3) group and from the ion less one NH(3) group to the loss of both NH(3) groups. The multiple reaction monitoring capability of LC-MS/MS was used to measure the three most abundant isotopes of the two main lesions for both transitions of each lesion (i.e., 12 MS/MS values in toto). Ion currents could be detected for all 12 pairs of MS/MS values in the DNA from exposed cells. Although this protocol results in some overlap of MS/MS values between the two lesions, a slight difference in elution times clearly distinguishes between them.     

Analyses of (1-chloroethenyl)oxirane headspace and hemoglobin N-valine adducts in erythrocytes indicate selective detoxification of (1-chloroethenyl)oxirane enantiomers

Chloroprene (2-chloro-1,3-butadiene, CAS 126-99-8, CP) is a colorless volatile liquid used in manufacture of polychloroprene, a synthetic rubber polymer. National Toxicology Program inhalation studies of CP in rats and mice gave clear evidence of carcinogenic activity. CP is metabolized by CYP2E1 to electrophilic epoxides, including R- and S-(1-chloroethenyl)oxirane (CEO), which form adducts with nucleic acids and other nucleophiles including glutathione and hemoglobin. As detection of these epoxide metabolites in vivo is technically challenging, measurements of CEO-Hb adducts may provide biomarkers of exposure to bioactivated metabolites of CP. The present studies involved exposure of C57BL/6 mouse erythrocytes (RBC) in vitro to pure enantiomers of CEO. Headspace analysis of CEO using Cyclodex-B capillary GC/MS with selected ion monitoring enabled separation, specific detection, and quantification of CEO enantiomers as reactions proceeded in vitro with RBC. These analyses indicated that R-CEO was much more persistent when incubated in vitro with RBC, while S-CEO disappeared rapidly. After periods of exposure of RBC to various concentrations of R- or S-CEO, erythrocytes were lysed and globin isolated. Covalent adducts, formed by reaction of CEO with N-terminal valine in Hb, were analyzed following Edman cleavage and trimethylsilylation. SIM-GC/MS analyses using a 5%-phenyl-dimethylsiloxane capillary column enabled quantification of CEO-Hb adducts. These analyses produced two chromatographic peaks of CEO-valine adduct derivatives, which were tentatively identified from mass spectra, reaction, and abundance data to be 1-(3-chloro-2-trimethylsilyloxybut-3-en-1-yl)-5-isopropyl-3-phenyl-2-thiohydantoin and 1-[2-chloro-1-(trimethylsilyloxymethyl)prop-2-en-1-yl]-5-isopropyl-3-phenyl-2-thiohydantoin. Analyses quantified significantly greater levels of adducts formed from R-CEO than from S-CEO. Studies involving pretreatment of RBC with glutathione-depleting diethyl maleate diminished the selective detoxification of S-CEO, and suggest enantiomeric selectivity of mouse glutathione-S-transferase as a mechanism of differential detoxification of CEO enantiomers. These results indicate more rapid detoxification of S-CEO by mouse RBC in vitro, while R-CEO may persist to react with cellular nucleophiles.     

Analysis of cholesteryl esters and diacylglycerols using lithiated adducts and electrospray ionization-tandem mass spectrometry

Cholesteryl ester (CE) and diacylglycerol (DAG) molecular species are important lipid storage and signaling molecules. Mass spectrometric analyses of these lipids are complicated by the presence of isobaric molecular ions shared by these lipid classes and by relatively poor electrospray ionization, which is a consequence of an inherently weak dipole moment in these lipid classes. The current study demonstrates that lithiated adducts of CE and DAG molecular ions have enhanced ionization and lipid class-specific fragmentation in tandem mass spectrometry (MS/MS) scan modes, thereby allowing the implementation of strategies capable of lipid class-specific detection. Using neutral loss (NL) mode for the loss of cholestane from cholesterol esters (NL 368.5) and specific selected reaction monitoring for DAG molecular species, the response of specific molecular species to that of internal standards was determined. CE and DAG molecular species were quantified in human coronary artery endothelial cells (HCAECs) incubated with both palmitic acid and oleic acid. Furthermore, NL 368.5 spectra revealed the oxidation of the aliphatic fatty acid residues of CE molecular species. Taken together, these studies demonstrate a new analytical approach to assessing CE and DAG molecular species that exploits the utility of lithiated adducts in conjunction with MS/MS approaches.     

Complex Relationships between Occupation,Environment, DNA Adducts,Genetic Polymorphisms and Bladder Cancer in a Case-Control Study Using a Structural Equation Modeling

DNA adducts are considered an integrate measure of carcinogen exposure and the initial step of carcinogenesis. Their levels in more accessible peripheral blood lymphocytes (PBLs) mirror that in the bladder tissue. In this study we explore whether the formation of PBL DNA adducts may be associated with bladder cancer (BC) risk, and how this relationship is modulated by genetic polymorphisms, environmental and occupational risk factors for BC. These complex interrelationships, including direct and indirect effects of each variable, were appraised using the structural equation modeling (SEM) analysis. Within the framework of a hospital-based case/control study, study population included 199 BC cases and 213 non-cancer controls, all Caucasian males. Data were collected on lifetime smoking, coffee drinking, dietary habits and lifetime occupation, with particular reference to exposure to aromatic amines (AAs) and polycyclic aromatic hydrocarbons (PAHs). No indirect paths were found, disproving hypothesis on association between PBL DNA adducts and BC risk. DNA adducts were instead positively associated with occupational cumulative exposure to AAs (p = 0.028), whereas XRCC1 Arg 399 (p<0.006) was related with a decreased adduct levels, but with no impact on BC risk. Previous findings on increased BC risk by packyears (p<0.001), coffee (p<0.001), cumulative AAs exposure (p = 0.041) and MnSOD (p = 0.009) and a decreased risk by MPO (p<0.008) were also confirmed by SEM analysis. Our results for the first time make evident an association between occupational cumulative exposure to AAs with DNA adducts and BC risk, strengthening the central role of AAs in bladder carcinogenesis. However the lack of an association between PBL DNA adducts and BC risk advises that these snapshot measurements are not representative of relevant exposures. This would envisage new scenarios for biomarker discovery and new challenges such as repeated measurements at different critical life stages.     

Electrospray ionisation-cleavable tandem nucleic acid mass tag-peptide nucleic acid conjugates: synthesis and applications to quantitative genomic analysis using electrospray ionisation-MS/MS

The synthesis and characterization of isotopomer tandem nucleic acid mass tag-peptide nucleic acid (TNT-PNA) conjugates is described along with their use as electrospray ionisation-cleavable (ESI-Cleavable) hybridization probes for the detection and quantification of target DNA sequences by electrospray ionisation tandem mass spectrometry (ESI-MS/MS). ESI-cleavable peptide TNT isotopomers were introduced into PNA oligonucleotide sequences in a total synthesis approach. These conjugates were evaluated as hybridization probes for the detection and quantification of immobilized synthetic target DNAs using ESI-MS/MS. In these experiments, the PNA portion of the conjugate acts as a hybridization probe, whereas the peptide TNT is released in a collision-based process during the ionization of the probe conjugate in the electrospray ion source. The cleaved TNT acts as a uniquely resolvable marker to identify and quantify a unique target DNA sequence. The method should be applicable to a wide variety of assays requiring highly multiplexed, quantitative DNA/RNA analysis, including gene expression monitoring, genetic profiling and the detection of pathogens.     

An ultra performance liquid chromatography-tandem MS assay for tamoxifen metabolites profiling in plasma: first evidence of 4'-hydroxylated metabolites in breast cancer patients

There is increasing evidence that the clinical efficacy of tamoxifen, the first and most widely used targeted therapy for estrogen-sensitive breast cancer, depends on the formation of the active metabolites 4-hydroxy-tamoxifen and 4-hydroxy-N-desmethyl-tamoxifen (endoxifen). Large inter-individual variability in endoxifen plasma concentrations has been observed and related both to genetic and environmental (i.e. drug-induced) factors altering CYP450s metabolizing enzymes activity. In this context, we have developed an ultra performance liquid chromatography-tandem mass spectrometry method (UPLC-MS/MS) requiring 100 μL of plasma for the quantification of tamoxifen and three of its major metabolites in breast cancer patients. Plasma is purified by a combination of protein precipitation, evaporation at room temperature under nitrogen, and reconstitution in methanol/20 mM ammonium formate 1:1 (v/v), adjusted to pH 2.9 with formic acid. Reverse-phase chromatographic separation of tamoxifen, N-desmethyl-tamoxifen, 4-hydroxy-tamoxifen and 4-hydroxy-N-desmethyl-tamoxifen is performed within 13 min using elution with a gradient of 10 mM ammonium formate and acetonitrile, both containing 0.1% formic acid. Analytes quantification, using matrix-matched calibration samples spiked with their respective deuterated internal standards, is performed by electrospray ionization-triple quadrupole mass spectrometry using selected reaction monitoring detection in the positive mode. The method was validated according to FDA recommendations, including assessment of relative matrix effects variability, as well as tamoxifen and metabolites short-term stability in plasma and whole blood. The method is precise (inter-day CV%: 2.5-7.8%), accurate (-1.4 to +5.8%) and sensitive (lower limits of quantification comprised between 0.4 and 2.0 ng/mL). Application of this method to patients' samples has made possible the identification of two further metabolites, 4'-hydroxy-tamoxifen and 4'-hydroxy-N-desmethyl-tamoxifen, described for the first time in breast cancer patients. This UPLC-MS/MS assay is currently applied for monitoring plasma levels of tamoxifen and its metabolites in breast cancer patients within the frame of a clinical trial aiming to assess the impact of dose increase on tamoxifen and endoxifen exposure.     

Polycyclic aromatic hydrocarbon-DNA adducts in humans: relevance as biomarkers for exposure and cancer risk

The methodology applied for DNA adducts in humans has become more reliable in recent years, allowing to detect even background carcinogenic adduct levels in environmentally exposed persons. Particularly, combinations of the various methods now allow the elucidation of specific adduct structures with detection limits of 1 adduct in 10⁸ unmodified nucleotides or even lower. The quantification of polycyclic aromatic hydrocarbon-DNA (PAH-DNA) adducts in human tissues and cells has been achieved with a number of highly sensitive techniques: immunoassays and immunocytochemistry using polyclonal or monoclonal antisera specific for DNA adducts or modified DNA, the ³²P-postlabelling assay, and adduct identification using physicochemical instrumentation. The results summarized in this review show that PAH-DNA adducts have been detected in a variety of human tissues, including target organs of PAH- and tobacco-associated cancers. Although dosimetry has not always been precise, a large number of data now clearly show that lowering exposure to carcinogenic PAH results in decreasing PAH-DNA adduct levels. In most studies, however, bulk DNA of a certain tissue or cell type has been examined, and there were relatively few studies in which mutations as a consequence of DNA damage at specific genes have been investigated. Promising as these biomarker studies seem for epidemiology and health surveillance, future biomonitoring and molecular epidemiological studies should be directed to combine several endpoint measurements: i.e., adduct formation (preferably at specific sites), mutational spectra in cancer-relevant genes, and genetic markers of (cancer) susceptibility in a number of cancer-predisposing genes.     

Use of DNA adducts to identify human health risk from exposure to hazardous environmental pollutants: the increasing role of mass spectrometry in assessing biologically effective doses of genotoxic carcinogens

The carcinogens to which humans are exposed are normally in the form of complex mixtures, and much effort has gone into determining the nature of the most significant carcinogenic components in these mixtures and their mechanisms of action. Essential to achieving this aim in exposed populations is the use of biomarkers, which can characterize the chemical nature of the carcinogens involved and identify key biological effects that result from the exposure. DNA adducts are particularly appropriate as biomarkers in the case of genotoxic carcinogens as they indicate the biologically effective dose of the genotoxin in the target tissue under study. This review considers in particular the use of mass spectrometry (MS), which is having an increasing role in the determination of DNA adducts. Compared to other existing DNA damage detection methods, such as 32P-postlabeling, HPLC-fluorescence or electrochemical detection, immunoassay-based techniques and modified Comet assays, MS provides improved structural characterization of adducts. Greater selectivity in the analyses is achieved by the use of tandem MS with selected reaction monitoring or constant neutral loss of ions. Use of capillary/nano liquid chromatography and micro/nano electrospray ionization improves the analytical sensitivity and higher throughput may be obtained by the use of online-column switching. The application of microfluidics technology offers exciting new possibilities for interfacing sample preparation to the mass spectrometer. Despite these improvements in the use of MS for adduct detection, the main current requirement is to validate these methods both analytically and in molecular epidemiology studies. More knowledge of the stability of stored samples is required. Development of sensitive mass spectrometric DNA adductomic screening systems, and of long-term biomarkers (e.g., phosphotriester adducts that are not repaired efficiently) seems important areas for the future assessment of the effects of human exposure to environmental genotoxins, together with studies of dose-response relationships at low doses.