Biomedical subjects

Peter B Farmer

Publications and source records attributed to Peter B Farmer.

At least 19 recordsLinked to original sources

PubMed2006

Development of a novel site-specific mutagenesis assay using MALDI-ToF MS (SSMA-MS).

We have developed and validated a novel site-specific mutagenesis assay, termed SSMA-MS, which incorporates MALDI-ToF mass spectrometry (MALDI-MS) analysis as a means of determining the mutations induced by a single DNA adduct. The assay involves ligating an adducted deoxyoligonucleotide into supF containing pSP189 plasmid. The plasmid is transfected into human Ad293 kidney cells allowing replication and therefore repair or a mutagenic event to occur. Escherichia coli indicator bacteria are transformed with recovered plasmid and plasmids containing the insert are identified colormetrically, as they behave as frameshift mutations. The plasmid is then amplified and digested using a restriction cocktail of Mbo11 and Mnl1 to yield 12 bp deoxyoligonucleotides, which are characterized by MALDI-MS. MALDI-MS takes advantage of the difference in molecular weight between bases to identify any induced mutations. This analysis method therefore provides qualitative and quantitative information regarding the type and frequency of mutations induced. This assay was developed and validated using an O(6)-methyl-2'-deoxyguanosine adduct, which induced the expected GC-->AT substitutions, when replicated in human or bacterial cells. This approach can be applied to the study of any DNA adduct in any biologically relevant gene sequence (e.g. p53) in human cells and would be particularly amenable to high-throughput analysis.

Cell Line↗

PubMed2006

Evaluation of enzyme-linked immunosorbent assay and liquid chromatography-tandem mass spectrometry methodology for the analysis of 8-oxo-7,8-dihydro-2'-deoxyguanosine in saliva and urine.

While ELISA is a frequently used means of assessing 8-oxo-7,8-dihydro-2-deoxyguanosine (8-oxodG) in biological fluids, differences in baseline urinary 8-oxodG levels, compared to chromatographic techniques, have raised questions regarding the specificity of immunoassays. Recently, ELISA of salivary 8-oxodG has been used to report on periodontal disease. We compared salivary 8-oxodG levels, determined by two commercial ELISA kits, to liquid chromatography-tandem mass spectrometry (LC-MS/MS) with prior purification using solid-phase extraction. While values were obtained with both ELISA kits, salivary 8-oxodG values were below or around the limit of detection of our LC-MS/MS assay. As the limit of detection for the LC-MS/MS procedure is much lower than ELISA, we concluded that the assessment of salivary 8-oxodG by ELISA is not accurate. In contrast to previous studies, ELISA levels of urinary 8-oxodG (1.67 +/- 0.53 pmol/mumol creatinine) were within the range reported previously only for chromatographic assays, although still significantly different than LC-MS/MS (0.41 +/- 0.39 pmol/mumol creatinine; p = 0.002). Furthermore, no correlation with LC-MS/MS was seen. These results question the ability of ELISA approaches, at present, to specifically determine absolute levels of 8-oxodG in saliva and urine. Ongoing investigation in our laboratories aims to identify the basis of the discrepancy between ELISA and LC-MS/MS.

8-Hydroxy-2'-Deoxyguanosine↗

PubMed2006

Pilot study of oral silibinin, a putative chemopreventive agent, in colorectal cancer patients: silibinin levels in plasma, colorectum, and liver and their pharmacodynamic consequences.

Silibinin, a flavonolignan from milk thistle, has intestinal cancer chemopreventive efficacy in rodents. It is a strong antioxidant and modulates the insulin-like growth factor (IGF) system by increasing circulating levels of IGF-binding protein 3 (IGFBP-3) and decreasing levels of IGF-I. Here, the hypothesis was tested that administration of oral silibinin generates agent levels in human blood and colorectal and hepatic tissues consistent with pharmacologic activity. Patients with confirmed colorectal adenocarcinoma received silibinin formulated with phosphatidylcholine (silipide) at dosages of 360, 720, or 1,440 mg silibinin daily for 7 days. Blood and biopsy samples of normal and malignant colorectum or liver were obtained before dosing, and blood and colorectal or hepatic tissues were collected at resection surgery after the final silipide dose. Levels of silibinin were quantified by high-pressure liquid chromatography-UV, and plasma metabolites were identified by liquid chromatography-mass spectrometry. Blood levels of IGFBP-3, IGF-I, and the oxidative DNA damage pyrimidopurinone adduct of deoxyguanosine (M1dG) were determined. Repeated administration of silipide was safe and achieved levels of silibinin of 0.3 to 4 micromol/L in the plasma, 0.3 to 2.5 nmol/g tissue in the liver, and 20 to 141 nmol/g tissue in colorectal tissue. Silibinin monoglucuronide, silibinin diglucuronide, silibinin monosulfate, and silibinin glucuronide sulfate were identified in the plasma. Intervention with silipide did not affect circulating levels of IGFBP-3, IGF-I, or M1dG. The high silibinin levels achieved in the human colorectal mucosa after consumption of safe silibinin doses support its further exploration as a potential human colorectal cancer chemopreventive agent.

Administration, Oral↗

PubMed2006

Searching for biomarkers of heart failure in the mass spectra of blood plasma.

We have developed a technique for analysing blood plasma using MALDI-MS with subsequent data analysis to identify significant and specific differences between heart failure (HF) patients and healthy individuals. A training dataset comprising 100 HF patients and 100 healthy individuals was used to search for biomarkers (m/z range 1000-10,000). EWP cartridges when used in tandem with microcon centrifugal filters were found to give the best results. A data management chain including event binning, background subtraction and feature extraction was developed to reduce the data, and statistical analysis was used to map feature intensities on to a common scale. Various mathematical approaches including a simple cumulative score, support vector machines (SVM) and genetic algorithms (GAs) were then used to combine the results from individual features and provide a robust classification algorithm. The SVM gave the most promising results (accuracy 95%, receiver operating characteristic (ROC) score of 0.997 using 18 selected features). Finally, a test dataset comprising a further 32 HF patients and 20 controls was used to verify that the 18 putative biomarkers and classification algorithms gave reliable predictions (accuracy 88.5%, ROC score 0.998).

Biomarkers↗

PubMed2006

A novel 14C-postlabeling assay using accelerator mass spectrometry for the detection of O6-methyldeoxy-guanosine adducts.

Accelerator mass spectrometry (AMS) is currently one of the most sensitive methods available for the trace detection of DNA adducts and is particularly valuable for measuring adducts in humans or animal models. However, the standard approach requires administration of a radiolabeled compound. As an alternative, we have developed a preliminary 14C-postlabeling assay for detection of the highly mutagenic O6-methyldeoxyguanosine (O6-MedG), by AMS. Procedures were developed for derivatising O6-MedG using unlabeled acetic anhydride. Using conventional liquid chromatography/mass spectrometry (LC/MS) analysis, the limit of detection (LOD) for the major product, triacetylated O6-MedG, was 10 fmol. On reaction of O6-MedG with 14C-acetic anhydride, using a specially designed enclosed system, the predominant product was 14C-di-acetyl O6-MedG. This change in reaction profile was due to a modification of the reaction procedure, introduced as a necessary safety precaution. The LOD for 14C-di-acetyl O6-MedG by AMS was determined as 79 amol, approximately 18,000-fold lower than that achievable by liquid scintillation counting (LSC). Although the assay has so far only been carried out with labeled standards, the degree of sensitivity obtained illustrates the potential of this assay for measuring O6-MedG levels in humans.

Acetic Anhydrides↗

PubMed2006

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.

Alkylating Agents↗

PubMed2006

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.

Animals↗

PubMed2006

Biomarkers for isocyanate exposure: Synthesis of isocyanate DNA adducts.

Isocyanates are important intermediates in industrial manufacturing. DNA adducts and protein adducts are important tools to biomonitor people exposed to xenobiotics. In the present work, the formation of DNA adducts deriving from 4-chlorophenyl isocyanate (4CPI) and 4-methylphenyl isocyanate (4MPI) were explored. The adducts of 4CPI and/or 4MPI with 2'-deoxyadenosine, 2'-deoxyguanosine, and 2'-deoxycytidine were synthesized and characterized by NMR and MS. For low level detection, an LC-MS/MS method was developed. The formation of DNA adducts was confirmed in in vitro reactions with DNA.

Biomarkers↗

PubMed2005

Influence of environmental exposure to PAHs on the susceptibility of lymphocytes to DNA-damage induction and on their repair capacity.

The influence of occupational exposure to environmental carcinogenic polycyclic aromatic hydrocarbons (c-PAHs) on DNA damage detected in lymphocytes of exposed people (city policemen) was studied. The cellular susceptibility to the induction of the DNA damage and the repair capacity of exposed donors are presented in comparison with matched controls. Monitoring was performed and blood samples (164 donors) were collected in Prague, Czech Republic, during the winter and summer seasons. The single-cell gel electrophoresis (SCGE) assay with an internal standard was applied to evaluate the DNA damage. A challenging dose of 2Gy of X-rays was used to study cellular capacities. In the results of studies of the DNA damage induced in vivo or as an immediate response to the challenging treatment no significant difference was found between exposed and unexposed subgroups. The percentage of non-repaired X-ray-induced DNA damage (residual damage, RD) overall in both seasons was significantly higher in lymphocytes of policemen exposed to c-PAHs than in matched controls (RD(T-DNA), %DNA in the comet tail: winter 36.4+/-22.1 versus 22.7+/-10.8, p < 0.001; summer 47.7+/-22.9 versus 34.7+/-15.2, p < 0.001). The results suggest that occupational exposure to environmental c-PAHs significantly reduces the cellular capacity to repair the DNA damage induced by a challenging treatment. A significant decrease of repair efficiency in donors occupationally exposed to environmental c-PAHs was also observed when subgroups were stratified according to smoking history. In conclusion, our results suggest that environmental exposure to c-PAHs affects the cellular repair processes and can lead to harmful effects hazardous to human health.

Adult↗

PubMed2005

Liquid chromatography-electrospray ionization-mass spectrometry: the future of DNA adduct detection.

Over the past 40 years considerable emphasis has been placed on the development of accurate and sensitive methods for the detection and quantitation of DNA adducts. The formation of DNA adducts resulting from the covalent interaction of genotoxic carcinogens with DNA, derived from exogenous and endogenous sources, either directly or following metabolic activation, can if not repaired lead to mutations in critical genes such as those involved in the regulation of cellular growth and subsequent development of cancer. The major analytical challenge has been to detect levels of DNA adducts at the level of 0.1-1 adducts per 10(8) unmodified DNA bases using only low microgram amounts of DNA, and with high specificity and accuracy, in humans exposed to genotoxic carcinogens derived from occupational, environmental, dietary and life-style sources. In this review we will highlight the merits as well as discuss the progress made by liquid chromatography coupled to electrospray ionization mass spectrometry as a method for DNA adduct detection.

Carcinogens↗

PubMed2005

A synthetic approach to the generation of quercetin sulfates and the detection of quercetin 3'-O-sulfate as a urinary metabolite in the rat.

To study the biological effects of quercetin, authentic products of quercetin metabolism are required as standards. The synthesis of quercetin sulfate standards is thus described. Quercetin was reacted with a 10-fold molar excess of sulfur trioxide-N-triethylamine, and the products were analyzed by HPLC and mass spectrometry. Four monosulfates and three disulfates were identified, and structural inferences were drawn by 1H NMR spectrometry of HPLC peak isolates. Analysis of the urine of rats that had received quercetin (1.9 g/kg po) yielded a single peak, which by comparison with the products of the reaction between quercetin and sulfur trioxide-N-triethylamine was identified as quercetin 3'-O-sulfate.

Animals↗

PubMed2005

The human apoptosis-inducing protein AMID is an oxidoreductase with a modified flavin cofactor and DNA binding activity.

AMID (apoptosis-inducing factor-homologous mitochondrion-associated inducer of death; also known as PRG3 (p53-responsive gene 3)) is a human caspase-independent pro-apoptotic protein with some similarity to apoptosis-inducing factor. AMID was purified from a recombinant bacterial host, enabling biochemical analysis of the protein. AMID is a flavoprotein; possesses NAD(P)H oxidase activity; and catalyzes NAD(P)H-dependent reduction of cytochrome c and other electron acceptors, including molecular oxygen. NADPH binds approximately 10-fold tighter than NADH. AMID binds 6-hydroxy-FAD (a cofactor that accumulates only adventitiously and at low abundance in other flavoprotein enzymes) to form a stoichiometric cofactor.protein complex. AMID has a distinctive electronic spectrum due to the modified flavin. NAD(P)+ binding perturbed the spectrum, enabling determination of K(d) values for these coenzymes. 6-Hydroxy-FAD could be removed from AMID and the apoprotein reconstituted with FAD. FAD was converted to 6-hydroxy-FAD in reconstituted AMID during aerobic turnover with NADPH. AMID is a DNA-binding protein that lacks apparent DNA sequence specificity. Formation of the protein.DNA complex (i) effected a major protein conformational change and (ii) was prevented in the presence of nicotinamide coenzyme. Apo-AMID retains DNA binding activity. Our studies establish a link between coenzyme and DNA binding that likely impacts on the physiological role of AMID in cellular apoptosis.

Apoptosis↗

PubMed2005

Mutation spectra induced by alpha-acetoxytamoxifen-DNA adducts in human DNA repair proficient and deficient (xeroderma pigmentosum complementation group A) cells.

Tamoxifen, a breast cancer drug, has recently been approved for the chemoprevention of this disease. However, tamoxifen causes hepatic carcinomas in rats through a genotoxic mechanism and increases the risk of endometrial tumors in women. DNA adducts have been detected at low levels in human endometrium, and there is much interest in determining whether DNA damage plays a role in tamoxifen-induced endometrial carcinogenesis. This study investigates the mutagenicity of tamoxifen DNA adducts formed by alpha-acetoxytamoxifen, a reactive ester producing the major DNA adduct formed in livers of tamoxifen-treated rats. pSP189 plasmid DNA containing the supF gene was treated with alpha-acetoxytamoxifen and adduct levels (0.5-8.0 adducts per plasmid) determined by (32)P-postlabeling. Adducted plasmids were transfected into nucleotide excision repair proficient (GM00637) or deficient (GM04429, XPA) human fibroblasts. After replication, plasmids were recovered and screened in indicator bacteria. Relative mutation frequencies increased with the adduct level, with 1.3-3.6-fold higher numbers of mutations in the XP cells compared to the GM00637 cells, indicating that NER plays a significant role in the removal of these particular tamoxifen DNA adducts. The majority of sequence alterations (91-96%) occurred at GC base pairs, as did mutation hotspots, although the type and position of mutations was cell-specific. In both cell lines, as the adduct level increased, the proportion of GC --> AT transitions decreased and GC --> TA transversions, mutations known to arise from the major tamoxifen adducts, increased. Given the high mutagenicity of dG-N(2)-tamoxifen adducts, if not excised, they may potentially contribute to the initiation of endometrial cancer in women.

Base Sequence↗

PubMed2005

Genotoxicity of the benzene metabolites para-benzoquinone and hydroquinone.

Our interest in benzene-DNA adduct formation and their consequence has led us to develop a number of sensitive methods for their analysis. A HPLC method for the analysis of 32P-postlabelled benzene-DNA adducts was developed and used to detect adducts formed from the reaction of DNA or individual deoxynucleotides with the metabolites para-benzoquinone (p-BQ) and hydroquinone (HQ). Reaction of DNA with BQ yielded four adducts, the major product being a deoxycytidine adduct. HQ formed a single detectable deoxyguanosine DNA adduct, which was a minor product of the reaction of DNA with p-BQ. The supF forward mutation assay was used to assess the mutagenicity of p-BQ and HQ after transfection of treated plasmid in the human kidney cell line, Ad293. Single base substitution mutations at GC base pairs (bp) predominated for each treatment. However, when the mutation spectra achieved for each treatment were compared they were shown to be significantly different (p=0.004). These results may suggest either a possible role for the minor benzene-deoxyguanosine adducts in benzene genotoxicity or that HQ is causing DNA modification via a different mechanism, such as oxidative damage.

Benzene↗

PubMed2005

The use of S-phenylmercapturic acid as a biomarker in molecular epidemiology studies of benzene.

S-Phenylmercapturic acid (S-PMA), is a urinary metabolite of benzene, thought to be derived from the condensation product of benzene oxide with glutathione. S-PMA may be determined by GC, HPLC (UV or fluorescence detection), GC-MS, LC-MS/MS or immunoassays. The limit of sensitivities of most of these techniques is 1 microg/l urine or below. It has been suggested that S-PMA may have value as a biomarker for low level human exposure to benzene, in view of the facts that urinary excretion of S-PMA has been found to be related to airborne benzene in occupationally exposed workers, and that only low background levels of S-PMA have been found in control subjects. We have evaluated the use of S-PMA as a biomarker, using a commercially available analytical service, in a multicentre European study of populations exposed to varying levels of benzene, in Italy (Milan, Genoa) and in Bulgaria (Sofia). These were filling station attendants, urban policemen, bus drivers, petrochemical workers and referents (a total of 623 subjects). S-PMA was measured at the end of the work shift by an immunoassay procedure. Urinary benzene (in Milan only) and the benzene metabolite trans,trans-muconic acid (t,t-MA) were measured before and after the work shift. Air-borne benzene was measured as a monitor of exposure. Urinary benzene was the most discriminatory biomarker and showed a relationship with airborne benzene at all levels of exposure studied (including groups exposed to <0.1 ppm benzene), whereas t,t-MA and S-PMA, as determined by immunoassay, were suitable only in the highest exposed workers (petrochemical industry, geometric mean 1765 microg/m3 (0.55 ppm) benzene). All three biomarkers were positively correlated with smoking as measured by urinary cotinine).

Acetylcysteine↗

PubMed2005

Inhibition of the respiratory burst by resveratrol in human monocytes: correlation with inhibition of PI3K signaling.

trans-Resveratrol (t-RVT) has been shown to have a wide range of anti-inflammatory properties, some of which have been suggested to contribute to the molecular explanation of the French Paradox, a possible reason for the low incidence of heart disease in France. The ability of t-RVT to inhibit the production of reactive oxygen species (ROS) from monocytes (differentiated U937) was investigated using isoluminol, luminol, lucigenin, and 2',7'-dichlorofluorescein (DCF). t-RVT (0.1-50 microM) was found to significantly inhibit cellular ROS production stimulated by f-Met-Leu-Phe (fMLP), 12-phorbol 13-myristate, and arachidonic acid after a 1-h preincubation. The efficacy of t-RVT could be increased if it was added directly into the assay. NADPH-dependent superoxide production was measured in cell homogenates and t-RVT (10-50 microM) was found to have no effect on this activity. The majority of these redox probes require a peroxidase to be oxidized; therefore, the inhibitory effect of t-RVT on ROS measured by these probes is complicated by its ability to be oxidized by peroxidase enzymes and thus compete with the probe. t-RVT, known to be oxidized by the horseradish peroxidase (HRP)/H(2)O(2) system, was found to inhibit the HRP-dependent oxidation of the fluorescent probe DCF and the chemiluminescent probe isoluminol. However, using a redox probe that did not require oxidation by a peroxidase (lucigenin), significant inhibition was still observed. Moreover, the inhibitory effects of t-RVT on fMLP-induced ROS production correlated with significant inhibitory effects on fMLP-induced phosphatidylinositol 3-kinase (PI3K) activity at 50 microM and Akt phosphorylation (10-50 microM). Other known inhibitors of both PI3K and Akt were also found to inhibit this response. Therefore, inhibition of signaling through the PI3K to NADPH oxidase by t-RVT might represent an important anti-inflammatory mechanism.

Acridines↗

PubMed2005

Innovative agents in cancer prevention.

There are many facets to cancer prevention: a good diet, weight control and physical activity, a healthy environment, avoidance of carcinogens such as those in tobacco smoke, and screening of populations at risk to allow early detection. But there is also the possibility of using drugs or naturally occurring compounds to prevent initiation of, or to suppress, tumour growth. Only a few such agents have been used to date in the clinic with any success, and these include non-steroidal anti-inflammatory drugs for colon, finasteride for prostate and tamoxifen or raloxifene for breast tumours. An ideal chemopreventive agent would restore normal growth control to a preneoplastic or cancerous cell population by modifying aberrant signalling pathways or inducing apoptosis (or both) in cells beyond repair. Characteristics for such an agent include selectivity for damaged or transformed cells, good bioavailability and more than one mechanism of action to foil redundancy or crosstalk in signalling pathways. As more research effort is being targeted towards this area, the distinction between chemotherapeutic and chemopreventive agents is blurring. Chemotherapeutic drugs are now being designed to target over- or under-active signalling molecules within cancer cells, a philosophy which is just as relevant in chemoprevention. Development of dietary agents is particularly attractive because of our long-standing exposure to them, their relative lack of toxicity, and encouraging indications from epidemiology. The carcinogenic process relies on the cell's ability to proliferate abnormally, evade apoptosis, induce angiogenesis and metastasise to distant sites. In vitro studies with a number of different diet-derived compounds suggest that there are molecules capable of modulating each of these aspects of tumour growth. However, on the negative side many of them have rather poor bioavailability. The challenge is to uncover their multiple mechanisms of action in order to predict their efficacy, to learn how to use them effectively in combination, and in some cases to redesign them to improve potency or bioavailability. These ideas are illustrated by dietary agents such as indole-3-carbinol (I3C), epigallocatechin gallate (EGCG), curcumin and resveratrol, all of which appear to have a number of different molecular targets, impinging on several signalling pathways. Ultimately it may be possible not only to suppress tumours and to extend quality of life by administering appropriate diet-derived molecules, but also to refine the definition of a cancer chemopreventive diet.

Antineoplastic Agents↗

PubMed2005

Detection of DNA damage derived from a direct acting ethylating agent present in cigarette smoke by use of liquid chromatography-tandem mass spectrometry.

Cigarette smoke contains a complex mixture of chemicals, including some that are genotoxic. A number of epidemiological and clinical studies have reported the association of increased DNA adduct levels with the development of lung cancer in smokers. The majority of chemicals present in cigarette smoke require cytochrome P450-mediated metabolic activation to form the ultimate reactive species that covalently binds with DNA. We have investigated the presence of a direct-acting ethylating agent present in cigarette smoke by studying the formation of N-7 ethylguanine (N-7EtG) following exposure of DNA to cigarette smoke in vitro. A sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method with multiple reaction monitoring (MRM) was developed for the detection of N-7EtG in DNA. DNA samples were subjected to thermal hydrolysis to selectively release the N-7EtG, which was then quantified by LC-MS/MS MRM using a stable isotope internal standard [15N5]N-7EtG. The limit of detection of the method for N-7EtG was 2.0 fmol injected on column with 100 microg of calf thymus DNA as the matrix (0.6 N-7EtG adducts per 10(8) nucleotides). A linear dose-response was observed for the formation of N-7EtG in calf thymus DNA treated with diethyl sulfate at concentrations ranging from 1 to 1000 microM. Calf thymus DNA treated with smoke generated from 1, 5, and 10 commercially available cigarettes resulted in the formation of 1.3, 3.6, and 8.4 N-7EtG adducts per 10(8) nucleotides, respectively. There was a positive correlation between the formation of N-7EtG and the number of cigarettes (r = 0.9938). These results confirm the presence of an as yet unidentified direct acting ethylating agent in cigarette smoke, which is present at levels that can produce DNA damage that could ultimately have adverse implications for human health, particularly in the case of the development of lung cancer.

Alkylating Agents↗