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G N Wogan

Publications and source records attributed to G N Wogan.

At least 19 recordsLinked to original sources
PubMed1998

Mutagenesis associated with nitric oxide production in macrophages.

To better understand the mechanisms through which persistent infections/inflammation increase cancer risks, we assessed the potential genotoxic properties of NO produced by macrophages. We recently showed that mouse macrophage RAW264.7 cells were capable of resuming exponential growth after stimulation for NO production by interferon-gamma (IFN-gamma) and/or lipopolysaccharide. Here, we report that increases in mutant fraction (MF) in the endogenous, X-linked, hprt gene of the cells are associated with NO exposure. Cells stimulated with 100 units/ml IFN-gamma continuously for 14 and 23 days produced a total of 9.8 and 14 micromol of NO per 10(7) cells, respectively. MFs in the hprt gene of NO-producing cells were 16.6 and 31.3 x 10(-5), respectively, compared with 2.2 and 2.5 x 10(-5) in untreated cells. Addition of an NO synthase inhibitor, N-monomethyl-L-arginine, to the culture medium decreased NO production and MF by 90% and 85%, respectively. Reverse transcription-PCR and DNA sequencing revealed that NO-associated hprt mutations did not differ significantly from those arising spontaneously, with the exception that certain small deletions/insertions and multiple exon deletions were observed only in the former. MF also increased significantly in cells stimulated for only 4 days with lipopolysaccharide plus IFN-gamma for higher rates of NO production. The types and proportion of hprt mutations induced under these conditions were strikingly similar to those associated with long-term NO exposure. These results indicate that NO exposure results in gene mutations in RAW264.7 cells through mechanisms yet to be identified and may also contribute to spontaneous mutagenesis.

Animals↗
PubMed1998

Mutations induced in the supF gene of pSP189 by hydroxyl radical and singlet oxygen: relevance to peroxynitrite mutagenesis.

We previously showed that the oxidant peroxynitrite (ONOO-) was strongly mutagenic in the supF shuttle vector pSP189 replicated in bacteria or human cells. Qualitative characteristics of the mutational spectra induced by ONOO- differed significantly from those reportedly caused by hydroxyl radical (OH.) in other experimental systems but showed similarities to spectra reportedly produced by singlet oxygen (1O2). The molecular mechanisms of ONOO--mediated DNA damage are unknown. The objective of the present set of experiments was to characterize mutational effects induced in the supF gene of pSP189 by OH* and 1O2 to permit direct comparison with mutational spectra induced by ONOO- in this system. Base substitutions were the major form of mutation induced in plasmids replicated in human (AD293) cells by ONOO- (84%) and 1O2 (71%), whereas OH* induced fewer of them (49%). In plasmids replicated in bacteria (Escherichia coli MBL50), frequencies of base substitutions induced by the three treatments were similar. G:C-to-T:A transversions were the most common form of base substitution induced by ONOO- (75% and 67%, respectively, in AD293- and MBL50-replicated plasmids) and 1O2 (68% and 71%); they were induced at lower frequencies by OH. (51% and 47%). G:C-to-C:G transversions or G:C-to-A:T transitions were induced at almost equal frequencies by both ONOO- and 1O2, whereas OH* induced these mutations at different frequencies in the AD293 system. Collectively, our results confirm that in several important respects mutational spectra induced by ONOO- have greater similarity to spectra induced by 1O2 than to those induced by OH* and suggest that genotoxic derivatives of ONOO- are likely to include species that have DNA-damaging properties resembling those of 1O2 in selectivity for guanine but not identical in sequence specificity.

Amino Acid Sequence↗
PubMed1997

Growth and viability of macrophages continuously stimulated to produce nitric oxide.

Deregulated production of nitric oxide (NO) has been implicated in the development of certain human diseases, including cancer. We sought to assess the damaging potential of NO produced under long-term conditions through the development of a suitable model cell culture system. In this study, we report that when murine macrophage-like RAW264.7 cells were exposed continuously to bacterial lipopolysaccharide (LPS) or mouse recombinant interferon-gamma (IFN-gamma) over periods of 21-23 days, they continued to grow, but with doubling times 2 to 4 times, respectively, longer than the doubling time of unstimulated cells. Stimulated cells produced NO at rates of 30 to 70 nmol per million cells per day throughout the stimulation period. Within 24 hr after removal of stimulant, cells resumed exponential growth. Simultaneous exposure to LPS and IFN-gamma resulted in decreased cell number, which persisted for 2 days after removal of the stimulants. Exponential growth was attained only after an additional 4 days. Addition of N-methyl-L-arginine (NMA), an NO synthase inhibitor, to the medium inhibited NO production by 90% of all stimulated cells, partially reduced doubling time of cells stimulated with LPS or IFN-gamma, and partially increased viability and growth rates in those exposed to both LPS and IFN-gamma. However, when incubated with LPS and IFN-gamma at low densities both in the presence and in the absence of NMA, cells grew at a rate slower than that of unstimulated cells, with no cell death, and they resumed exponential growth 24 hr after removal of stimulants. Results from cell density experiments suggest that macrophages are protected from intracellularly generated NO; much of the NO damaging activity occurred outside of the producer cells. Collectively, results presented in this study suggest that the type of cellular toxicity observed in macrophages is markedly influenced by rate of exposure to NO: at low rates of exposure, cells exhibit slower growth; at higher rates, cells begin to die; at even higher rates, cells undergo growth arrest or die. The ability of RAW264.7 cells to produce NO over many cell generations makes the cell line a useful system for the study of other aspects of cellular damage, including genotoxicity, resulting from exposure to NO under long-term conditions.

Animals↗
PubMed1997

An intercalation inhibitor altering the target selectivity of DNA damaging agents: synthesis of site-specific aflatoxin B1 adducts in a p53 mutational hotspot.

Aflatoxin B1 (AFB1) is a potent human carcinogen implicated in the etiology of hepatocellular carcinoma. Upon metabolic activation to the reactive epoxide, AFB1 forms DNA adducts primarily at the N7 position of guanines. To elucidate more fully the molecular mechanism of AFB1-induced mutagenesis, an intercalation inhibitor was designed to probe the effects of intercalation by AFB1 epoxide on its reaction with DNA. DNA duplexes were prepared consisting of a target strand containing multiple potentially reactive guanines and a nontarget strand containing a cis-syn thymidine-benzofuran photoproduct. Because the covalently linked benzofuran moiety physically occupies an intercalation site, we reasoned that such a site would be rendered inaccessible to AFB1 epoxide. By strategic positioning of this intercalation inhibitor in the intercalation site 5' to a specific guanine, the adduct yield at that site was greatly diminished, indicating that intercalation by AFB1 epoxide contributes favorably to adduct formation. Using this approach it has been possible to simplify the production of site-specifically modified oligonucleotides containing AFB1 adducts in the sequence context of a p53 mutational hotspot. Moreover, we report herein isolation of site-specifically AFB1-modified oligonucleotides in sequences containing multiple guanines. Use of intercalation inhibitors will facilitate both investigation of the ability of other carcinogens to intercalate into DNA and the synthesis of specific carcinogen-DNA adducts.

Aflatoxin B1↗
PubMed1997

Nitrotyrosine formation, apoptosis, and oxidative damage: relationships to nitric oxide production in SJL mice bearing the RcsX tumor.

In SJL mice, growth of RcsX lymphoma cells results in activation of macrophages in the spleen and lymph nodes to produce high levels of NO radical (NO.). We used this experimental model system to study the toxicology of NO. in vivo. To characterize spatial relationships between sites of NO. production and tissue damage, immunohistochemical techniques were developed for simultaneous detection of inducible NO. synthase (iNOS), 3-nitrotyrosine, and apoptosis in spleen and lymph nodes of tumor-bearing animals. Elevated expression of iNOS, presumed to reflect increased NO. production, was associated with a significant increase in frequency of apoptotic nuclei. Both apoptotic nuclei and 3-nitrotyrosine staining were found in cells juxtaposed to iNOS-expressing (ie., NO.-producing) macrophages and also within the macrophages themselves. To assess the extent of DNA damage associated with the response, 8-oxoguanine levels were quantified in DNA extracted from spleens of tumor-bearing mice. No increase in levels of this marker of oxidative DNA damage was found in tissues in which apoptosis and 3-nitrotyrosine levels were highly elevated within specific subsets of cells. Collectively, our results indicate that under the pathophysiological conditions existing in the RcsX tumor-bearing SJL mouse, cellular damage caused by NO. and/or other reactive species produced by activated macrophages is highly localized within cells in close proximity to the activated macrophages.

Animals↗
PubMed1997

UV-induced mutagenesis of human p53 in a vector replicated in Saccharomyces cerevisiae.

Mutation of the p53 tumor suppressor gene is the most common genetic alteration identified to date in human cancers. Similarities of p53 mutations found in human cancers with those induced in experimental systems have been interpreted as evidence supporting a causative role for environmental carcinogens in certain tumor types. We have developed and validated a method for generation of mutation spectra and measurement of mutation frequency directly on human p53 cDNA in a vector following treatment with mutagens and replication in yeast. Mutants that had lost the DNA binding/transcription activation function of p53 were detected by yeast colony color, isolated, and sequenced. UV light was used to characterize and validate the system, and a dose-dependent increase in mutation frequency was seen following exposure of the plasmid to increasing doses of UV, resulting in an 18-fold increase over the spontaneous frequency (3.2 x 10(-4)) at the highest level tested (300 J/m2). Sequence analysis of p53 in the mutants revealed that the types of mutations induced were similar to those obtained in previous studies of UV mutagenesis in other model systems, and the types and positions of mutations were also similar to those found in human skin tumors. This experimental system will be useful in further evaluation of the importance of environmental agents as risk factors for cancer.

Base Sequence↗
PubMed1997

Review of the toxicology of tamoxifen.

Tamoxifen was carcinogenic to the liver of male and female rats, inducing hepatocellular carcinomas when administered daily by gavage or fed continuously in the diet. It also acted as a promoting agent in a two-stage model of carcinogenesis in rat liver. In contrast, tamoxifen acted as a protective agent in abrogating estrogen-induced hepatotoxicity and hepatocarcinogenesis in hamsters. Tamoxifen did not induce malignancies in mice when administered according to dosing protocols that are effective in inducing hepatocellular carcinomas in rat liver. In the rat, tamoxifen is metabolized to alpha-hydroxytamoxifen, which is further activated to a product that binds principally to the exocyclic amino group of deoxyguanosine in DNA. The same adduct pattern is formed in mouse hepatocytes treated with tamoxifen or its alpha-hydroxylated derivative, and in human hepatocytes exposed to the latter metabolite. However, available data indicate that human cells have a substantially lower capacity than rodent cells for activation of tamoxifen. Tamoxifen also induces aneuploidy in rat hepatocytes in vivo. This evidence has led some investigators to characterize tamoxifen as a carcinogen that acts through both genotoxic and nongenotoxic mechanisms, with the implication that women treated with the drug may be consequently subjected to elevated risk of cancer, particularly of the endometrium. Critical examination of the evidence, however, indicates that extrapolation of these experimental data to humans is subject to very substantial uncertainty. Available data clearly indicate major differences between women and rats with respect to the activation of tamoxifen and formation of DNA adducts, and bring into question the validity of direct extrapolation of data generated in the single susceptible species, the rat, to women in assessing potential risks attendant to tamoxifen administration.

Animals↗
PubMed1996

Mutagenesis associated with nitric oxide production in transgenic SJL mice.

We recently reported development of an experimental model for the study of nitric oxide (NO.) toxicology in vivo. SJL mice were injected with superantigen-bearing RcsX (pre-B-cell lymphoma) cells, which migrated to the spleen and lymph nodes, where their rapid growth induced activation of macrophages to produce large amounts of NO. over a period of several weeks. In the experiments described here, we used this model to investigate mutagenesis in splenocytes exposed to NO. during RcsX cell growth. Transgenic mice were produced by crossbreeding animals of the pUR288 transgenic C57BL/6 and SJL strains. RcsX cells were injected into F1 mice and NO. production was confirmed by quantification of urinary nitrate, the ultimate metabolite of NO. Mutant frequency in the lacZ gene of the pUR288 plasmid was determined in DNA isolated from spleen (target) and kidney (nontarget) tissues. A significant elevation in mutant frequency was found in the spleen, but not in the kidney, of tumor-bearing mice. Furthermore, increases in mutant frequency in the spleen as well as NO. production were abrogated by administration of N-methylarginine, a NO. inhibitor, to mice following injection of RcsX cells. These results indicate that NO. had mutagenic activity in RcsX tumor-bearing mice and thus support a possible role for its involvement in the carcinogenic process.

Animals↗
PubMed1996

Nitric oxide production in SJL mice bearing the RcsX lymphoma: a model for in vivo toxicological evaluation of NO.

SJL mice spontaneously develop pre-B-cell lymphoma that we hypothesized might stimulate macrophages to produce nitric oxide (NO.). Transplantation of an aggressive lymphoma (RcsX) was used to induce tumor formation. Urinary nitrate excretion was measured as an index of NO. production and was found to increase 50-fold by 13 days after tumor injection. NO. production was prevented by the addition of a nitric oxide synthase (NOS) inhibitor. The expression of inducible NOS (iNOS) in various tissues was estimated by Western blot analysis and localized by immunohistochemistry. The synthase was detected in the spleen, lymph nodes, and liver of treated but not control mice. To assess whether the iNOS-staining cells were macrophages, spleen sections from ResX-bearing animals were costained with anti-iNOS antibody and the anti-macrophage antibody moma-2. Expression of iNOS was found to be limited to a subset of the macrophage population. The concentration of gamma-interferon, a cytokine known to induce NO. production by macrophages, in the serum of tumor-bearing mice, was measured and found to be elevated 25-fold above untreated mice. The ability of ResX-activated macrophages to inhibit splenocyte growth in primary culture was estimated and macrophage-derived NO. was found to inhibit cell division 10-fold. Our findings demonstrate that ResX cells stimulate NO. production by macrophages in the spleen and lymph nodes of SJL mice, and we believe this experimental model will prove useful for study of the toxicological effects of NO. under physiological conditions.

Animals↗
PubMed1996

Peroxynitrite-induced mutation spectra of pSP189 following replication in bacteria and in human cells.

Peroxynitrite is a powerful oxidant formed through reaction of nitric oxide with superoxide. Because activated macrophages can produce both nitric oxide and superoxide, it has been proposed that peroxynitrite may contribute to cytotoxicity and increased cancer risks associated with the inflammatory response during chronic infections. We therefore investigated mutagenicity of peroxynitrite in the supF gene of the pSP189 shuttle vector as a mutation target. The plasmid was exposed to 2.5 mM peroxynitrite in vitro, then replicated in Eschericia coli MBL50 and in human AD293 cells. Mutation frequency increased 21-fold in pSP189 replicated in E. coli and 9-fold in plasmid replicated in human cells. Mutations were clustered within the 5' region of the supF gene in plasmids replicated in bacteria. The hot spots were located at positions 108, 113, 116, 124, 126 and 141; more than 25% of all mutations occurred at position 124. Following replication in human cells, mutations were more widely distributed over the gene, with hot spots at positions 113, 124, 133, 156 and 164; 15% occurred at position 124. In both systems, the majority of mutations occurred at G:C base pairs, predominantly involving G:C-->T:A transversions (65% when replication was in bacteria and 63% when in human cells). G:C-->C:G transversions were observed at lower frequency (28% in MBL50 and 11% in AD293 cells), and 11% of mutations found in vectors replicated in AD293 cells were G:C-->A:T transitions. A greater number of large deletions, insertions, tandem and multiple mutations occurred in plasmid replicated in AD293 cells. Differences in mutation spectra following replication in the two systems may be attributable to differences in recognition and repair of the lesions and/or properties of the replication apparatus.

Base Sequence↗
PubMed1996

Percutaneous absorption and tissue distribution of [3H]diacetoxyscirpenol (anguidine) in rats and mice.

Acute toxicity, absorption, excretion, and tissue distribution of topically administered diacetoxyscirpenol (DAS, anguidine) were studied in Fischer rats and CD-1 mice. Mortality (75%) was observed in rats treated with a single dose of 2.625 mg of DAS to 1.44 cm2 of skin, whereas the proportionate (0.75 mg to 0.42 cm2 of skin) and even higher doses were not lethal to mice. Histopathological lesions induced in the rat were similar to those observed by administration through other routes, mainly involving lymphohematopoietic tissues and the gastrointestinal tract. Although lesions in internal organs were less severe, the skin of the mouse was more severely damaged at the application site than that of the rat. During the 90-min period after topical application of a single dose of [3H]DAS, the rat absorbed and retained more [3H]DAS and excreted less radioactivity through urine and feces than the mouse. By 24 hr after treatment, the rat had absorbed, excreted, and retained about twice as much [3H]DAS as had the mouse (p < 0.05 or < 0.005). At 7 days posttreatment, the rat had absorbed more than four times the amount of [3H]DAS than had the mouse (13.1 vs 57.5%; p < 0.005). However, tissues of the mouse retained a higher proportion of administered radioactivity (4.1%) than those of the rat (1.0%; p < 0.05). Total excretion of radiolabel by the rat was approximately sixfold higher than that of the mouse (56 to 9%; p < 0.005). The ratio of excretion in urine to that in feces in the rat was about 2 to 1 (37 to 18%) and in the mouse was about 3.5 to 1 (7 to 2%). Significant differences in the time course of tissue distribution of [3H]DAS in the rat and mouse were found when data were expressed as the percentage of absorbed dose present in tissues or as specific radioactivity (dpm) per gram tissue. These results demonstrated a significant interspecies difference in acute percutaneous toxicity of DAS and different patterns of absorption, excretion, and tissue distribution of topically administered [3H]DAS in rats and mice.

Absorption↗
PubMed1996

Use of carcinogen-DNA and carcinogen-protein adduct biomarkers for cohort selection and as modifiable end points in chemoprevention trials.

Clinical cancer prevention studies that use disease as an end point are of necessity large, lengthy and extremely costly. Development of the field of cancer chemoprevention is being accelerated by the application of intermediate markers to preclinical and clinical studies. One class of potentially useful biomarkers comes from studies of cancer risks derived from exposure to environmental carcinogens of both endogenous and exogenous origins. Sensitive and specific analytical methods have been developed for detecting and quantifying levels of covalent adducts of several important classes of carcinogens with cellular DNA and blood proteins at ambient levels of exposure. Such biomarkers can be applied to the preselection of exposed individuals for study cohorts, thereby reducing study-size requirements. Levels of these carcinogen-DNA and carcinogen-protein adducts can be modulated by some classes of chemopreventive agents. In this regard, these markers can also be used to rapidly assess the efficacy of preventive interventions such as exposure abatement and chemoprevention. However, the successful application of these biomarkers to prevention trials will be dependent upon prior determination of the associative or causal role of the marker to the carcinogenic process, establishment of the relationship between dose and response, and appreciation of the kinetics of adduct formation and removal.

Anticarcinogenic Agents↗
PubMed1995

Nitric oxide production in relation to spontaneous B-cell lymphoma and myositis in SJL mice.

SJL mice spontaneously develop B cell lymphomas (historically described as reticulum cell sarcomas) by 12 months of age and inflammatory muscle disease (myositis) by 6 months of age. Tumors originate in mesenteric lymph nodes and in Peyer's patches and resemble human germinal center lymphomas. The growth of reticulum cell sarcomas is completely dependent on cytokine production by normal T cells. The spontaneous myositis, which resembles human idiopathic myositis, is characterized by various abnormalities in skeletal muscle, including infiltration with inflammatory cells consisting primarily of macrophages. The participation of different cytokines in the pathogenesis of the lymphoma and the massive invasion of macrophages into muscle tissues led us to investigate the possible involvement of nitric oxide (NO.), which is known to be synthesized by activated macrophages under inflammatory conditions. Elevated NO. production, measured by urinary nitrate excretion, by SJL mice in comparison with BALB/c control mice was observed as early as 7 weeks of age. Both aging and degree of spontaneous myositis correlated with increased nitric oxide production. Oral administration of N-monomethyl-L-arginine, an inhibitor of nitric oxide synthase (NOS), reduced urinary nitrate excretion and also the severity of myositis. Immunohistochemical analysis revealed the presence of inducible NOS (iNOS) in cells in the spleen, lymph nodes, and skeletal muscle. The iNOS is primarily responsible for the enhanced nitric oxide production. Morphology of cells that stained positive for iNOS was similar to that of macrophages infiltrating into the affected tissues. Chronic production of elevated amounts of nitric oxide by the SJL mice, therefore, provides a useful in vivo model for future studies of cellular damage resulting from endogenously produced NO.in combination with oxygen radicals.

Amino Acid Oxidoreductases↗
PubMed1995

Tissue distribution of DNA adducts in pre-weanling BLU:Ha mice treated with a tumorigenic dose of fluoranthene.

Fluoranthene (FA), a ubiquitous atmospheric pollutant, is tumorigenic to the lung when injected into BLU:Ha mice 1, 8, and 15 days after birth. DNA adducts were measured by a modified HPLC-32P-postlabeling method in target (lung) and non-target (liver, kidney, spleen plus thymus) organs of 16-day-old mice 24 h after the last treatment with a tumorigenic dose (3.5 mg/mouse) of FA. The anti-FADE adduct was the major DNA adduct and was present at levels ranging from 3.5 to 37 adducts per 10(8) nucleotides (106-1138 fmols/mg DNA) in all DNA samples from the organs of FA-treated mice. The lung contained anti-FADE adduct levels approximately 2, 2.5 and 5 times higher than those in kidney, liver, and spleen plus thymus, respectively. There was no significant difference between anti-FADE adduct levels in liver and kidney; however, adduct levels were significantly higher than those in spleen plus thymus. Significant variation was found among litters in anti-FADE adduct levels from lung, liver, and kidney, but not from spleen plus thymus. An unidentified peak was present in the adduct profile of the kidneys of treated but not control animals. Three additional unidentified radioactive peaks were present in adduct profiles of DNA from tissues of both treated and control animals, but their levels were significantly higher in the tissues of treated animals than in controls. Possible correlations between tumorigenic response and DNA adduct formation are discussed.

Animals↗
PubMed1995

Formation and persistence of DNA adducts in organs of CD-1 mice treated with a tumorigenic dose of fluoranthene.

Fluoranthene (FA) is tumorigenic to the lung when injected i.p. into CD-1 mice 1, 8 and 15 days after birth (Wang, J.-S. and Busby, W.F. Jr, Carcinogenesis, 14, 1871-1874, 1993). Levels, tissue distribution and persistence of FA--DNA adducts detected by HPLC-32P-postlabeling were investigated during the course of lung tumorigenesis by FA. Anti-10b-N2-deoxyguanosin-1,2,3,-trihydroxy-1,2,3 10b-tetrahydrofluoranthene [sequence: see text] (anti-FADE adduct) was consistently the major adduct in DNA samples from lung, heart, liver and kidney of animals examined at different time points from 2 h to 165 days after the last treatment with the tumorigenic dose (3.5 mg/mouse) of FA. Several unidentified adducts were also detected. Lung, the target organ for FA tumorigenicity, contained higher levels of anti-FADE adduct than other tissues from 1-165 days after treatment. The anti-FADE adduct level decreased in a biphasic manner after reaching maximum values at 2 h in heart and spleen plus thymus and 3 days in lungs, liver and kidneys. About 10% of the maximum amount of anti-FADE adduct remained in lung, liver and heart 165 days after final FA treatment, at which time 44% of animals had developed lung adenomas. Significant inter-litter variations, but no sex differences in adduct levels were observed. These results indicated a positive correlation between anti-FADE adduct level and persistence in relation to target organ specificity for tumor formation.

Animals↗
PubMed1994

Molecular biomarkers for aflatoxins and their application to human cancer prevention.

The rapidly expanding understanding of the progressive processes of carcinogenesis provides opportunities for the identification of molecular biological markers reflecting events from exposure through clinical disease. These molecular biological markers can be classified into categories of markers of exposure reflecting the dose of toxic agents, markers of effect indicating a biological response to exposure, and markers of susceptibility providing information about the inherent sensitivity of individuals to the toxic agents. By definition some of these markers are chemical agent specific, such as a carcinogen-DNA or -protein adduct, while others are biological process specific, such as the altered expression of a gene. This article reviews the development and validation of molecular biomarkers of aflatoxins using experimental and human population studies. The development of molecular biomarkers for aflatoxins is based upon the extensive research database available about their metabolism, macromolecular adduct formation, and general mechanisms of action. The long-term goal of the research described in this paper is the application of aflatoxin biomarkers to the development of preventive interventions in human populations at high risk for liver cancer.

Aflatoxins↗
PubMed1994

Adduct detection by acylation with [35S]methionine: analysis of DNA adducts of 4-aminobiphenyl.

Reaction of synthetic N-(2'-deoxyguanosin-8-yl)-4-aminobiphenyl (dGuo-8-ABP) with t-butoxycarbonyl-L-[35S]methionine, N-hydroxysuccinimidyl ester (35S-labeled TBM-NHS), under optimized conditions produced mono-, bis-, and tris-TBM-acylated nucleosides that were separable by HPLC. Reaction of different amounts of N-(2'-deoxy-1',2'-[3H]guanosin-8-yl)-4-aminobiphenyl ([3H]dGuo-8-ABP) with 35S-labeled TBM-NHS established that total 35S content of acylated products was linearly related to adduct concentration (r = 0.992) over the range of 10 fmol to 30.6 pmol. Additionally, the N-(deoxyguanosin-8-yl)-4-[3H]aminobiphenyl (dGuo-8-[3H]ABP) adduct was isolated from calf thymus DNA adducted in vitro and from rat liver DNA adducted in vivo and similarly reacted with 35S-labeled TBM-NHS. Acylation products of dGuo-8-ABP from all three sources showed HPLC retention times identical to those of authentic TBM-dGuo-8-ABP, and 35S incorporation into acylated products was linearly related to amount of adduct reacted. These results indicate that the procedure, to which we have referred as adduct detection by acylation with methionine (ADAM), has potential applicability as an analytical procedure for detection and quantification of DNA adducts in human tissues in the molecular epidemiology of cancer.

Acylation↗