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Papers by Peter Foiles

Research paper thumbnail of Expression pattern of proteins that bind to the ultraviolet-responsive element (TGACAACA) in human keratinocytes

Molecular Carcinogenesis, 1993

We previously identified an ultraviolet (UV)‐responsive element (URE; TGACAACA) that plays a role... more We previously identified an ultraviolet (UV)‐responsive element (URE; TGACAACA) that plays a role in both transcription and replication of polyoma sequences. Mouse polyclonal antibodies were raised against affinitypurified URE‐bound proteins to characterize their expression patterns. These antibodies specifically recognized two of four URE‐bound proteins, of 40 and 68 kDa. The 68‐kDa protein was constitutively expressed in human keratinocytes, while the expression of the 40‐kDa protein was induced by UV irradiation. Of the two, the 68‐kDa protein bound to the URE with greater affinity than the 40‐kDa protein, as determined by southwestern analysis. The expression of the 40‐kDa protein increased as early as 1 h after UV irradiation of both rat fibroblasts and human keratinocytes and correlated with increased binding to the URE in an electrophoretic mobility shift assay. Other types of damage, as well as heat shock and serum stimulation, also induced the expression of this protein, suggesting that it may play a role in cellular response to stress or damage. The 40‐kDa protein was expressed at the highest levels in the S phase of the cell cycle and was induced by aphidicolin, suggesting that it has a role in DNA replication. All together, these results suggest that exposure of human keratinocytes to damage‐ and stress‐inducing agents modulates the expression of proteins that may play a role in regulating cellular response to DNA damage.

Research paper thumbnail of Analysis of mutagenic activity and ability to induce replication of polyoma DNA sequences by different model metabolites of the carcinogenic tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone

Mutation research, May 1, 1992

Research paper thumbnail of Biomarkers for human uptake and metabolic activation of tobacco-specific nitrosamines

PubMed, Apr 1, 1994

Tobacco-specific nitrosamines are a group of carcinogens formed from nicotine and related tobacco... more Tobacco-specific nitrosamines are a group of carcinogens formed from nicotine and related tobacco alkaloids. Two of these compounds, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and N'-nitrosonornicotine, are believed to be involved as causative agents for cancers of the lung, oral cavity, esophagus, and pancreas associated with the use of tobacco products. The goal of the studies described here is to develop biomarkers which will allow us to understand the uptake, metabolic activation, and detoxification of these carcinogens in humans. Two metabolites of NNK, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol and its glucuronide, have been identified and quantified in human urine. These metabolites allow assessment of NNK uptake in smokers, tobacco chewers, and people exposed to environmental tobacco smoke. NNK and N'-nitrosonornicotine form hemoglobin and DNA adducts upon metabolic activation by alpha-hydroxylation. These adducts release 4-hydroxy-1-(3-pyridyl)-1-butanone (HPB) upon hydrolysis. The released 4-hydroxy-1-(3-pyridyl)-1-butanone can be quantified by gas chromatography-mass spectrometry. A subset of smokers and most tobacco chewers have hemoglobin adduct levels which are higher than detected in nonsmokers. 4-Hydroxy-1-(3-pyridyl)-1-butanone-releasing DNA adducts are higher in lung tissue from smokers than from nonsmokers. These data indicate that some smokers and tobacco chewers are capable of metabolically activating NNK or N'-nitrosonornicotine to intermediates which bind to cellular macromolecules and are, therefore, at potentially higher risk for cancer development. The application of these biomarkers to studies on cancer induction by tobacco products is discussed.

Research paper thumbnail of Mass spectrometric analysis of tobacco-specific nitrosamine hemoglobin adducts in snuff dippers, smokers, and nonsmokers

PubMed, Sep 1, 1990

Hemoglobin adducts of the carcinogenic tobacco-specific nitrosamines 4-(methylnitrosamino)-1(3-py... more Hemoglobin adducts of the carcinogenic tobacco-specific nitrosamines 4-(methylnitrosamino)-1(3-pyridyl)-1-butanone and N'-nitrosonornicotine were quantified in blood samples collected from snuff dippers, smokers, and nonsmokers. Mild base treatment of hemoglobin adducted by 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone or N'-nitrosonornicotine releases 4-hydroxy-1-(3-pyridyl)-1-butanone (HPB). HPB was enriched by solvent partitioning and derivatized to its pentafluorobenzoate. After purification by high performance liquid chromatography, HPB-pentafluorobenzoate was analyzed by capillary column gas chromatography with detection by negative ion chemical ionization mass spectrometry and selected ion monitoring. [4,4-D2]HPB was used as internal standard. The detection limit for HPB-pentafluorobenzoate was approximately 100 amol/injection or 5 fmol/g hemoglobin. Mean adduct levels (fmol HPB/g hemoglobin) were 517 +/- 538 (SD) in snuff dippers, 79.6 +/- 189 in smokers, and 29.3 +/- 25.9 in nonsmokers. Adduct levels in snuff dippers and in a subgroup of smokers were higher than would have been predicted solely based on estimates of exposure to tobacco-specific nitrosamines. The results of this study provide the first measurements of tobacco-specific nitrosamine hemoglobin adducts in humans and suggest new approaches to understanding the metabolic activation of 4-(methyl-nitrosamino)-1-(3-pyridyl)-1-butanone and N'-nitrosonornicotine in humans.

Research paper thumbnail of Development of monoclonal antibodies specific for 1,<i>N</i><sup>2</sup>-ethenodeoxyguanosine and <i>N</i><sup>2</sup>,3-ethenodeoxyguanosine and their use for quantitation of adducts in G12 cells exposed to chloroacetaldehyde

Carcinogenesis, 1993

ABSTRACT Monoclonal antibodies specific for N2,3-ethenodeoxyguanosine (N2,3-epsilondGuo) and 1,N2... more ABSTRACT Monoclonal antibodies specific for N2,3-ethenodeoxyguanosine (N2,3-epsilondGuo) and 1,N2-ethenodeoxyguanosine (1,N2-epsilondGuo) were developed. In a competitive ELISA, 50% inhibition of binding of the N2,3-epsilondGuo specific antibody (ETH1) was achieved with 18 fmol of N2,3-epsilondGuo. Fifty per cent inhibition of the 1,N2-epsilondGuo-specific antibody (ETH2) required 11 pmol 1,N2-epsilondGuo. Immunoassays for N 23-epsilondGuo and 1,N2-epsilondGuo in single-stranded DNA were developed using these antibodies. The immunoassays could detect as little as 48 fmol of N2 3-epsilondGuo or 340 fmol 1,N2-epsilondGuo in 25 mug of single stranded DNA. These assays and previously developed immunoassays for 1,N6-ethenodeoxyadenosine (1,N6-epsilondAdo) and 3,N4-ethenodeoxycytidine (3,N4-epsilondCyd) were used to measure etheno adduct levels in DNA of cells exposed to chloroacetaldehyde. The cells used were V79 cells with an inactivated hprt gene and a single copy of the bacterial gpt gene (G12 cells). The most abundant etheno adduct was 1,N6-epsilondAdo, followed by 3,N4-epsilondCyd and N2,3-epsilondGuo. 1,N2-epsilondGuo was not detected in chloroacetaldehyde-treated G12 cells. Chloroacetaldehyde was also shown to be mutagenic in these same cells.

Research paper thumbnail of Formation of cyclic deoxyguanosine adducts in Chinese hamster ovary cells by acrolein and crotonaldehyde

Carcinogenesis, Nov 1, 1990

Acrolein and crotonaldehyde are alpha,beta-unsaturated carbonyl compounds that form 1,N2-propanod... more Acrolein and crotonaldehyde are alpha,beta-unsaturated carbonyl compounds that form 1,N2-propanodeoxyguanosine adducts when reacted with DNA in vitro. These compounds are mutagenic in Salmonella, and crotonaldehyde is tumorigenic in rats. This study used immunoassay and 32P-postlabeling methods to determine if acrolein and crotonaldehyde form these adducts in cultured mammalian cells. Adduct levels were highest in Chinese hamster ovary cells exposed to acrolein (1 mM) with 162 mumol adduct/mol deoxyguanosine. Crotonaldehyde (10 mM) formed adduct at a level of 75 mumol/mol deoxyguanosine. 32P-Postlabeling analysis confirmed the presence of adducts in crotonaldehyde-treated cells. Persistence studies showed that adduct levels were unchanged if the cells were cultured for 6 h before DNA isolation. Mutagenicity studies were performed to determine the biological consequences of these adducts. Mutations were not observed due to the toxicity of the compounds.

Research paper thumbnail of Study of DNA methylation by tobacco-specific N-nitrosamines

Environmental Health Perspectives, Oct 1, 1985

An enzyme-linked immunosorbent assay (BA-ELISA) involving use of biotin-labeled anti-rabbit IgG a... more An enzyme-linked immunosorbent assay (BA-ELISA) involving use of biotin-labeled anti-rabbit IgG and avidin-labeled horseradish peroxidase was developed for the measurement of 06-methyl-2'-deoxyguanosine (06-MedGuo). Up to 5 jg of methylated DNA was enzymatically hydrolyzed, and the extent of inhibition of binding of immobilized 06-MedGuo-bovine serum albumin to rabbit anti-O6-MedGuo was measured. Fifty percent inhibition of antigen-antibody binding was achieved with 2.5 pmole of of 06-MedGuo. Separation of 0-MedGuo from unmodified nucleosides by high-performance liquid chromatography (HPLC-BA-ELISA) allowed detection of 700 fmole 06-MedGuo in 1 mg of DNA. Among the tobacco-related carcinogens, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is one of the most potent. In F344 rats it induces nasal cavity, lung and liver tumors. Four hours after a single IV injection of NNK to F344 rats (87 mg/kg body weight), 0'-MedGuo was present in target organs (,umole 0'-MedGuo/mole dGuo) (nasal mucosa, 219; lung, 13.2; and liver, 34.5) but was not detectable in nontarget organs. F344 rats receiving daily IP injections of NNK (40 mg/kg body weight) for 14 days were sacrificed 24 hr after the last injection. The levels of (06-Medguo/dGuo) were 7.9 and 11.4 ,umole/mole in the nasal mucosa and lung, respectively. In the liver no 06-MedGuo was detected, but 1050 ,umole of 7-MeGua/mole Gua was measured by HPLC-fluorimetry. No DNA methylation was observed in the nasal mucosa or liver of F344 rats treated with the nicotine-derived carcinogen N'-nitrosonornicotine. Reduction of the carbonyl of NNK is a major metabolic pathway, giving rise to 4-(methylnitrosamino)-1-(3-pyridyl)-1-butan-1-ol (NNAI). Nasal mucosae were cultured in vitro with NNK or NNAI. After 1 hr, methylation at the 06-dGuo and 7-dGuo sites were observed with NNK but not with NNAI. Methylation by NNAI after 24 hr was associated with the conversion of NNAI to NNK. These results suggest that NNAI is not associated with the activation of NNK to DNA methylating species.

Research paper thumbnail of Evaluation of <sup>32</sup>P-postlabeling analysis of DNA from exfoliated oral mucosa cells as a means of monitoring exposure of the oral cavity to genotoxic agents

Carcinogenesis, 1989

Development of oral cavity cancer in man has been linked to alcohol consumption and use of tobacc... more Development of oral cavity cancer in man has been linked to alcohol consumption and use of tobacco products. In order to understand the underlying carcinogenic mechanisms in the oral cavity a method is needed to monitor exposure of this site to various environmental insults. In this pilot study we evaluate the use of the 32P-postlabeling assay to detect adducts in DNA from exfoliated oral mucosa cells. Exfoliated cells were collected from the cheek and tongue of 27 men aged 35-69 years. DNA was extracted from the cells and analyzed by the enhanced 32P-postlabeling technique using butanol extraction. A variety of adduct spots were detected but none was consistently associated with exposure to alcohol or tobacco products. Some of the adducts detected had migration patterns in TLC very similar to the major deoxyguanosine adducts formed by the diol epoxides of benzo[a]pyrene and 5-methylchrysene, suggesting that they may have been formed from polynuclear aromatic hydrocarbons. Adduct spots with migration patterns similar to polynuclear hydrocarbon adducts accounted for only about one third of the total adduct spots observed. Relative adduct labeling (RAL) values were determined for samples from 12 of the 27 individuals. RAL values ranged from 1.6 X 10(-6) to 7.7 X 10(-11) adducts per nucleotide. The RAL values for adducts from the cheek or tongue were not significantly different. Adduct levels in smokers (median RAL of 4.8 X 10(-8) were significantly higher (P less than 0.001) than adduct levels in non-smokers (median RAL of 2.9 X 10(-9). Adduct levels in drinkers (median RAL of 9.1 X 10(-10) were significantly lower (P less than 0.001) than adduct levels in non-drinkers (median RAL of 3.7 X 10(-8). Four of the subjects in this study have subsequently developed squamous cell carcinoma of the oral cavity. 32P-Postlabeling analysis of DNA from the oral cavity of these subjects did not demonstrate unique patterns or RAL values. Lack of information on the structure of the majority of adducts observed in this study was a serious limitation. Further improvements in adduct identification will be needed before 32P-postlabeling can be a useful tool for monitoring exposure of the oral cavity to carcinogens.

Research paper thumbnail of Inhibition of PKC and Pka by Chemopreventive Organoselenium Compounds

International Journal of Oncology, Sep 1, 1995

Numerous efficacy studies in rodents revealed that l,4-phenylenebis(methylene)-selenocyanate (p-X... more Numerous efficacy studies in rodents revealed that l,4-phenylenebis(methylene)-selenocyanate (p-XSC) is a more effective chemopreventive organoselenium compound and less toxic than benzyl selenocyanate (BSC) or the inorganic compound Na 2 Se0 3. To explore mechanisms which mediate chemopreventive activities of p-XSC we have tested its effect on protein kinase A and C using in vitro and cell culture systems. While p-XSC did completely inhibit PKC and PKA activity, BSC was less active and Na 2 Se0 3 had no effect. Comparative EC 50 revealed values of 0.1, 1 and >10 fiM for p-XSC, BSC and Na 2 Se0 3 , respectively. p-XSC was also capable of inhibiting protein phosphorylation in cultures of primary human fibroblasts and altered morphology of rat fibroblast (R6) cells. When combined, sub-optimal doses of p-XSC and staurosporine yielded an additive effect on cell morphology. The ability of p-XSC and BSC to inhibit protein kinase A and C activities may in part account for the mechanism(s) by which these agents mediate their chemo preventive effects.

Research paper thumbnail of Measurement of O6-methyldeoxyguanosine in DNA methylated by the tobacco-specific carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone using a biotin-avidin enzyme-linked immunosorbent assay

Carcinogenesis, 1985

Rabbit antibodies raised against O6-methyl-2&#39;-deoxyguanosine (O6-MedGuo) were used to dev... more Rabbit antibodies raised against O6-methyl-2&#39;-deoxyguanosine (O6-MedGuo) were used to develop a competitive Biotin-Avidin enzyme-linked immunosorbent assay (BA-ELISA) for the quantitation of this adduct in DNA from animals exposed to tobacco-specific N-nitrosamines. The assay was able to detect as little as 30 mumol O6-MedGuo per mol deoxyguanosine. The specificity of the assay was at least 10 000-fold greater for O6-MedGuo than for unmodified nucleosides or 7-methyl-2&#39;-deoxyguanosine. The sensitivity of the assay was increased to 1 mumol O6-MedGuo per mol deoxyguanosine by the prior separation of O6-MedGuo from unmodified nucleosides by h.p.l.c. This assay was used to study the methylating ability of the tobacco-specific nitrosamine 4-(methyl-nitrosamino)-1-(3-pyridyl)-1-butanone (NNK) in F344 rats. Four hours after exposure to a single i.v. dose of NNK O6-MedGuo was detected in the target tissues nasal mucosa, lung and liver (219, 13.2 and 34.5 mumol/mol deoxyguanosine) but not in the non-target tissues esophagus, spleen, heart or kidney. Chronic exposure by 14 daily i.p. injections of NNK also resulted in detectable amounts of O6-MedGuo in the lung and nasal mucosa (11.4 and 7.9 mumol/mol deoxyguanosine). These results demonstrate that NNK is capable of methylating DNA in vivo. The methodology developed in these studies will allow us to examine in detail the role of DNA methylation in NNK-mediated carcinogenesis.

Research paper thumbnail of Approaches to the development of assays for interaction of tobacco-specific nitrosamines with haemoglobin and DNA

PubMed, 1988

The tobacco-specific, nicotine-derived nitrosamines 4-(N-nitrosomethylamino)-1-(3-pyridyl)-1-buta... more The tobacco-specific, nicotine-derived nitrosamines 4-(N-nitrosomethylamino)-1-(3-pyridyl)-1-butanone (NNK) and N'-nitrosonornicotine (NNN) are among the most important carcinogens in tobacco and tobacco smoke. Treatment of Fischer 344 rats with these carcinogens resulted in alkylation of haemoglobin and DNA by the 4-(3-pyridyl)-4-oxobutyl group formed during their metabolism. This alkyl group can be detached from globin or DNA under mild hydrolytic conditions as 4-hydroxy-1-(3-pyridyl)-1-butanone, which appears to be a potentially useful dosimeter for human exposure to, and activation of, tobacco-specific nitrosamines.

Research paper thumbnail of Modulation of Carcinogen-Induced Polyoma Dna-Replication by Organoselenium and Organosulfur Chemopreventive Agents

International Journal of Oncology, Mar 1, 1993

Both chemical and physical carcinogens are potent inducers of asynchronous replication of various... more Both chemical and physical carcinogens are potent inducers of asynchronous replication of various DNA tumor viruses. Employing H3 cells that carry an integrated copy of polyoma virus we have evaluated potential effects of known chemopreventive agents on carcinogen-induced polyoma DNA replication. The ability of well established organoselenium and organosulfur chemopreventive agents, in laboratory animals, to modulate polyoma DNA replication induced by AMMN or NNKOAc which are direct acting carcinogens derived from the tobacco carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) was examined. We demonstrate that both benzyl selenocyanate (BSC) and benzyl isothiocyanate (BITC) are capable of reducing the level of polyoma DNA replication induced by NNK-model compounds. BITC also reduced the transcriptional activity of polyoma sequences as measured through chloramphenicol-acetyl-transferase (CAT) assays using the polyoma regulatory region cloned upstream of the CAT gene. These results suggest that the mechanisms by which BSC and BITC exert their protective effects involve changes in the expression of cellular proteins which regulate transcription and replication of polyoma DNA sequences.

Research paper thumbnail of Application of an immunoassay for cyclic acrolein deoxyguanosine adducts to assess their formation in DNA of <i>Salmonella typhimurium</i> under conditions of mutation induction by acrolein

Carcinogenesis, 1989

Acrolein has been shown to form cyclic deoxyguanosine adducts when it reacts with DNA in vitro. I... more Acrolein has been shown to form cyclic deoxyguanosine adducts when it reacts with DNA in vitro. In this study, we have used a recently developed immunoassay for these adducts to study their formation in DNA from Salmonella typhimurium exposed to acrolein. Acrolein--deoxyguanosine adducts were formed in a dose-dependent fashion in Salmonella tester strains TA100 and TA104, reaching levels as high as 5 mumol adduct per mol deoxyguanosine. Using the liquid pre-incubation assay, acrolein-induced mutations were also found in strains TA100 and TA104. The correlation between acrolein--deoxyguanosine adduct concentration and acrolein-induced mutations in TA100, which contains GC base pairs at the site of reversion, suggests that the acrolein--deoxyguanosine adduct is a promutagenic lesion. That mutations are also seen in TA104 which contains AT base pairs at the site of reversion suggest that adducts of bases other than deoxyguanosine may also be important in the mutagenic activity of acrolein.

Research paper thumbnail of The relationship between <i>N</i>-nitrosodimethylamine metabolism and DNA methylation in isolated rat hepatocytes

Carcinogenesis, 1996

The metabolism of JV-nitrosodimethylamine (NDMA) and its methylation of DNA were simultaneously d... more The metabolism of JV-nitrosodimethylamine (NDMA) and its methylation of DNA were simultaneously determined in hepatocytes isolated from untreated and saline-and pyrazole-treated male Sprague-Dawley rats. Metabolism of NDMA was directly measured by monitoring its disappearance via gas chromatography coupled with a sensitive and specific detector for iV-nitrosamines. DNA methylation was determined in the same cells employed in the metabolism studies using a monoclonal antibody-based competitive ELISA procedure specific for O*-methyldeoxyguanosine (6-Me-dG). The apparent K m and V^^, for NDMA metabolism are 61 pM and 56 pmol/min/10* cells respectively for hepatocytes isolated from untreated rats. It was found that the addition of pyrazole to the in vitro hepatocyte incubations caused a dose-dependent inhibition of both metabolism and DNA methylation. However, when DNA methylation is expressed as a function of NDMA metabolized, there is no significant difference between hepatocyte incubations without or with pyrazole, with an average value of 79 nmol 6-Me-dG/mol dG/nmol NDMA metabolized. Based on the pyrazole inhibition studies, cytochrome P450IIE1 is responsible for at least 60% of the DNA methylation in rat hepatocytes. In pyrazole-pretreated rats there was an inconsistent increase in NDMA metabolism, but when metabolism was elevated so was DNA methylation. In contrast, microsomes isolated from pyrazole-pretreated rats consistently showed elevated metabolism of NDMA. Based on the simultaneous determination of adduct levels and metabolism, there is-1 6-Me-dG adduct formed/133 000 NDMA molecules metabolized in the uninduced hepatocytes.

Research paper thumbnail of Inhibition of repair of O6-methyldeoxyguanosine and enhanced mutagenesis in rat-liver epithelial cells

Mutation Research: Fundamental And Molecular Mechanisms Of Mutagenesis, Jun 1, 1990

The pro-mutagenicity of chemically-induced methylation of DNA at the O6 position of deoxyguanosin... more The pro-mutagenicity of chemically-induced methylation of DNA at the O6 position of deoxyguanosine was studied in cultured adult rat liver epithelial cells. To modify the level of O6-methyldeoxyguanosine (O6-medGuo) resulting from exposure to an alkylating agent, partial depletion of the O6-alkylguanine-DNA alkyltransferase (AGT) repair system was produced by pretreatment of ARL 18 cells with a non-toxic dose of exogenous O6-methylguanine (O6-meG). Exposure of cells to 0.6 mM O6-meG for 4 h depleted AGT activity by about 40%. Intact and pretreated cells were exposed to a range of doses of N-methyl-N&#39;-nitro-N-nitrosoguanidine (MNNG), and mutagenesis at the hypoxanthine-guanine phosphoribosyl transferase (HGPRT) locus was quantified by measurement of 6-thioguanine-resistant mutants. The mutagenicity of MNNG was dose dependent and was greater in O6-meG pretreated cultures than in intact cultures. Immunoslot blot measurement of O6-medGuo employing a mouse monoclonal antibody demonstrated that MNNG produced O6-medGuo and that the intact liver cells were efficient in eliminating this lesion from their DNA. Since depletion of AGT would be expected to affect the rate of elimination of only O6-medGuo, it is concluded that this lesion is highly pro-mutagenic.

Research paper thumbnail of The <i>in Vitro </i>Methylation of DNA by a Minor Groove Binding Methyl Sulfonate Ester

Chemical Research in Toxicology, 1996

ABSTRACT The preparation of sequence and groove specific DNA methylating agents based on N-methyl... more ABSTRACT The preparation of sequence and groove specific DNA methylating agents based on N-methylpyrrolecarboxamide subunits appended with an O-methyl sulfonate ester functionality (MeOSO2(CH2)2-Lex) has previously been described [Zhang, Y., Chen, F.-X., Mehta, P., and Gold, B. (1993) Biochemistry 32,7954-7965]. In contrast to simple methyl sulfonate esters, e.g., methyl methanesulfonate (MMS), which predominantly methylate at 7-guanine, MeOSO2-(CH2)2-Lex affords N3-methyladenine (3-MeAde) as its major adduct. Using competitive ELISA determinations, the methylation at major and minor groove sites in calf thymus DNA by MeOSO2(CH2)2-Lex has been precisely quantitated. The yields of N7-methylguanine (7-MeGua), 3-MeAde, and O6-methyldeoxyguanosine (6-Me-dGuo) are 0.424, 3.195, and 0.0027 mmol of adduct/mol of DNA, respectively, using 10 microM MeOSO2(CH2)2-Lex and 100 microM DNA. This compares to 0.773, 0.072, and 0.0033 mmol of adduct/mol of DNA for 7-MeGua, 3-MeAde, and 6-Me-dGuo, respectively, using MMS. The increase in the yield of 3-MeAde due to the minor groove equilibrium binding properties of MeOSO2(CH2)2-Lex is approximately 40-fold relative to MMS.

Research paper thumbnail of Tobacco-specific nitrosamine adducts: studies in laboratory animals and humans

Environmental Health Perspectives, Mar 1, 1993

This paper describes quantitation of human hemoglobin and DNA adducts of the carcinogenic tobacco... more This paper describes quantitation of human hemoglobin and DNA adducts of the carcinogenic tobacco-specific nitrosamines 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and N'-nitrosonornicotine (NNN). NNK and NNN are believed to be involved in cancers ofthe lung, esophagus, onl cavity, and pancreas in people who use tobacco products. The adduct dosimetry method employs GC-MS for quantitation of 4-hydroxy-1-(3-pyridyl)-1-butanone (HPB) released by mild base hydrolysis of hemoglobin or acid hydrolysis of DNA as a biochemical marker of the pyridyloxobutylation metabolic activation pathway. Approximately 22% of smokers (n = 101) had elevated levels of HPB released from hemoglobin (range, 200-1600 fmole/g Hb). Adduct levels in snuff dippers ranged from 2001800 fmole/g Hb. HPB levels in nonsmokers were generally below the detection limit. Acid hydrolysis of lung and tracheal DNA obtained at autopsy and analysis for released HPB revealed levels ranging up to 50 fmole/mg DNA in smokers; the adduct was not detected in nonsmokers. These findings are consistent with data generated in studies of adduct formation by NNK in rats. The biological significance of the HPB-releasing DNA pyridylksobutylation pathway was compared to that of the DNA methylation pathway in the A/J mouse. These studies demonstrated that the persistence of O'-methylguanine in lung DNA is critical for tumorigenesis by NNK and that pyridylaobutylation enhances both peristence of O-methylguanine and tumorigenesis by acetoxymethylmethylnitrosamine. In the rat, the relative roles of methylation and pyridyloxobutylation in lung tumorigenesis by NNK are not as clearly defined. Although the biological significance of DNA methylation in NNK tumorigenesis is well characterized, dosimetry studies of tobacco-specific nitrosamines in humans should be carried out using biochemical markers of the pyridyloxobutylation pathway because of their specificity to tobacco products.

Research paper thumbnail of Mass spectrometric analysis of tobacco-specific nitrosamine-DNA adducts in smokers and nonsmokers

Chemical Research in Toxicology, May 1, 1991

A gas chromatography, negative ion chemical ionization mass spectrometry (GC-NICI-MS) based assay... more A gas chromatography, negative ion chemical ionization mass spectrometry (GC-NICI-MS) based assay for tobacco-specific nitrosamine adducts of DNA is described. The assay is based on the observation that acid hydrolysis of DNA from animals treated with tobacco-specific nitrosamines releases 4-hydroxy-l-(3-pyridyl)-l-butanone (HPB). HPB and the internal standard [4,4-D2]HPB are derivatized with pentafluorobenzoyl chloride and the resulting HPB-pentafluorobenzoate is purified by high-performance liquid chromatography prior to GC-NICI-MS analysis. DNA from human peripheral lung and tracheobronchial tissue, collected at autopsy, was analyzed for acid-released HPB. The mean H P B level (fmol/mg of DNA) for peripheral lung DNA was 11 f 16 (SD, n = 9) for smokers and 0.9 f 2.3 (n = 8) for nonsmokers. Mean adduct levels in tracheobronchus were 16 f 18 (n = 4) for smokers and 0.9 f 1.7 (n = 4) for nonsmokers. These are the first measurements of tobacco-specific nitrosamine-DNA adducts in humans. Further studies comparing the levels of DNA and globin adducts w i l l provide a better understanding of the metabolic activation of tobacco-specific nitrosamines in humans and may provide a more accurate indication of an individual's risk of developing tobacco-related cancer.

Research paper thumbnail of Partial reconstitution of TNP-Ficoll responses and IgG3 expression in Xid mice undergoing graft-vs-host reaction

The Journal of Immunology

In uence of lymphokines on the anti-TNP-Ficoll response of normal and X-linked B lymphocyte-defec... more In uence of lymphokines on the anti-TNP-Ficoll response of normal and X-linked B lymphocyte-defective mice.

Research paper thumbnail of Inhibition of repair of O6-methyldeoxyguanosine and enhanced mutagenesis in rat-liver epithelial cells

Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis, 1990

The pro-mutagenicity of chemically-induced methylation of DNA at the O6 position of deoxyguanosin... more The pro-mutagenicity of chemically-induced methylation of DNA at the O6 position of deoxyguanosine was studied in cultured adult rat liver epithelial cells. To modify the level of O6-methyldeoxyguanosine (O6-medGuo) resulting from exposure to an alkylating agent, partial depletion of the O6-alkylguanine-DNA alkyltransferase (AGT) repair system was produced by pretreatment of ARL 18 cells with a non-toxic dose of exogenous O6-methylguanine (O6-meG). Exposure of cells to 0.6 mM O6-meG for 4 h depleted AGT activity by about 40%. Intact and pretreated cells were exposed to a range of doses of N-methyl-N&#39;-nitro-N-nitrosoguanidine (MNNG), and mutagenesis at the hypoxanthine-guanine phosphoribosyl transferase (HGPRT) locus was quantified by measurement of 6-thioguanine-resistant mutants. The mutagenicity of MNNG was dose dependent and was greater in O6-meG pretreated cultures than in intact cultures. Immunoslot blot measurement of O6-medGuo employing a mouse monoclonal antibody demonstrated that MNNG produced O6-medGuo and that the intact liver cells were efficient in eliminating this lesion from their DNA. Since depletion of AGT would be expected to affect the rate of elimination of only O6-medGuo, it is concluded that this lesion is highly pro-mutagenic.

Research paper thumbnail of Expression pattern of proteins that bind to the ultraviolet-responsive element (TGACAACA) in human keratinocytes

Molecular Carcinogenesis, 1993

We previously identified an ultraviolet (UV)‐responsive element (URE; TGACAACA) that plays a role... more We previously identified an ultraviolet (UV)‐responsive element (URE; TGACAACA) that plays a role in both transcription and replication of polyoma sequences. Mouse polyclonal antibodies were raised against affinitypurified URE‐bound proteins to characterize their expression patterns. These antibodies specifically recognized two of four URE‐bound proteins, of 40 and 68 kDa. The 68‐kDa protein was constitutively expressed in human keratinocytes, while the expression of the 40‐kDa protein was induced by UV irradiation. Of the two, the 68‐kDa protein bound to the URE with greater affinity than the 40‐kDa protein, as determined by southwestern analysis. The expression of the 40‐kDa protein increased as early as 1 h after UV irradiation of both rat fibroblasts and human keratinocytes and correlated with increased binding to the URE in an electrophoretic mobility shift assay. Other types of damage, as well as heat shock and serum stimulation, also induced the expression of this protein, suggesting that it may play a role in cellular response to stress or damage. The 40‐kDa protein was expressed at the highest levels in the S phase of the cell cycle and was induced by aphidicolin, suggesting that it has a role in DNA replication. All together, these results suggest that exposure of human keratinocytes to damage‐ and stress‐inducing agents modulates the expression of proteins that may play a role in regulating cellular response to DNA damage.

Research paper thumbnail of Analysis of mutagenic activity and ability to induce replication of polyoma DNA sequences by different model metabolites of the carcinogenic tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone

Mutation research, May 1, 1992

Research paper thumbnail of Biomarkers for human uptake and metabolic activation of tobacco-specific nitrosamines

PubMed, Apr 1, 1994

Tobacco-specific nitrosamines are a group of carcinogens formed from nicotine and related tobacco... more Tobacco-specific nitrosamines are a group of carcinogens formed from nicotine and related tobacco alkaloids. Two of these compounds, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and N'-nitrosonornicotine, are believed to be involved as causative agents for cancers of the lung, oral cavity, esophagus, and pancreas associated with the use of tobacco products. The goal of the studies described here is to develop biomarkers which will allow us to understand the uptake, metabolic activation, and detoxification of these carcinogens in humans. Two metabolites of NNK, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol and its glucuronide, have been identified and quantified in human urine. These metabolites allow assessment of NNK uptake in smokers, tobacco chewers, and people exposed to environmental tobacco smoke. NNK and N'-nitrosonornicotine form hemoglobin and DNA adducts upon metabolic activation by alpha-hydroxylation. These adducts release 4-hydroxy-1-(3-pyridyl)-1-butanone (HPB) upon hydrolysis. The released 4-hydroxy-1-(3-pyridyl)-1-butanone can be quantified by gas chromatography-mass spectrometry. A subset of smokers and most tobacco chewers have hemoglobin adduct levels which are higher than detected in nonsmokers. 4-Hydroxy-1-(3-pyridyl)-1-butanone-releasing DNA adducts are higher in lung tissue from smokers than from nonsmokers. These data indicate that some smokers and tobacco chewers are capable of metabolically activating NNK or N'-nitrosonornicotine to intermediates which bind to cellular macromolecules and are, therefore, at potentially higher risk for cancer development. The application of these biomarkers to studies on cancer induction by tobacco products is discussed.

Research paper thumbnail of Mass spectrometric analysis of tobacco-specific nitrosamine hemoglobin adducts in snuff dippers, smokers, and nonsmokers

PubMed, Sep 1, 1990

Hemoglobin adducts of the carcinogenic tobacco-specific nitrosamines 4-(methylnitrosamino)-1(3-py... more Hemoglobin adducts of the carcinogenic tobacco-specific nitrosamines 4-(methylnitrosamino)-1(3-pyridyl)-1-butanone and N'-nitrosonornicotine were quantified in blood samples collected from snuff dippers, smokers, and nonsmokers. Mild base treatment of hemoglobin adducted by 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone or N'-nitrosonornicotine releases 4-hydroxy-1-(3-pyridyl)-1-butanone (HPB). HPB was enriched by solvent partitioning and derivatized to its pentafluorobenzoate. After purification by high performance liquid chromatography, HPB-pentafluorobenzoate was analyzed by capillary column gas chromatography with detection by negative ion chemical ionization mass spectrometry and selected ion monitoring. [4,4-D2]HPB was used as internal standard. The detection limit for HPB-pentafluorobenzoate was approximately 100 amol/injection or 5 fmol/g hemoglobin. Mean adduct levels (fmol HPB/g hemoglobin) were 517 +/- 538 (SD) in snuff dippers, 79.6 +/- 189 in smokers, and 29.3 +/- 25.9 in nonsmokers. Adduct levels in snuff dippers and in a subgroup of smokers were higher than would have been predicted solely based on estimates of exposure to tobacco-specific nitrosamines. The results of this study provide the first measurements of tobacco-specific nitrosamine hemoglobin adducts in humans and suggest new approaches to understanding the metabolic activation of 4-(methyl-nitrosamino)-1-(3-pyridyl)-1-butanone and N'-nitrosonornicotine in humans.

Research paper thumbnail of Development of monoclonal antibodies specific for 1,<i>N</i><sup>2</sup>-ethenodeoxyguanosine and <i>N</i><sup>2</sup>,3-ethenodeoxyguanosine and their use for quantitation of adducts in G12 cells exposed to chloroacetaldehyde

Carcinogenesis, 1993

ABSTRACT Monoclonal antibodies specific for N2,3-ethenodeoxyguanosine (N2,3-epsilondGuo) and 1,N2... more ABSTRACT Monoclonal antibodies specific for N2,3-ethenodeoxyguanosine (N2,3-epsilondGuo) and 1,N2-ethenodeoxyguanosine (1,N2-epsilondGuo) were developed. In a competitive ELISA, 50% inhibition of binding of the N2,3-epsilondGuo specific antibody (ETH1) was achieved with 18 fmol of N2,3-epsilondGuo. Fifty per cent inhibition of the 1,N2-epsilondGuo-specific antibody (ETH2) required 11 pmol 1,N2-epsilondGuo. Immunoassays for N 23-epsilondGuo and 1,N2-epsilondGuo in single-stranded DNA were developed using these antibodies. The immunoassays could detect as little as 48 fmol of N2 3-epsilondGuo or 340 fmol 1,N2-epsilondGuo in 25 mug of single stranded DNA. These assays and previously developed immunoassays for 1,N6-ethenodeoxyadenosine (1,N6-epsilondAdo) and 3,N4-ethenodeoxycytidine (3,N4-epsilondCyd) were used to measure etheno adduct levels in DNA of cells exposed to chloroacetaldehyde. The cells used were V79 cells with an inactivated hprt gene and a single copy of the bacterial gpt gene (G12 cells). The most abundant etheno adduct was 1,N6-epsilondAdo, followed by 3,N4-epsilondCyd and N2,3-epsilondGuo. 1,N2-epsilondGuo was not detected in chloroacetaldehyde-treated G12 cells. Chloroacetaldehyde was also shown to be mutagenic in these same cells.

Research paper thumbnail of Formation of cyclic deoxyguanosine adducts in Chinese hamster ovary cells by acrolein and crotonaldehyde

Carcinogenesis, Nov 1, 1990

Acrolein and crotonaldehyde are alpha,beta-unsaturated carbonyl compounds that form 1,N2-propanod... more Acrolein and crotonaldehyde are alpha,beta-unsaturated carbonyl compounds that form 1,N2-propanodeoxyguanosine adducts when reacted with DNA in vitro. These compounds are mutagenic in Salmonella, and crotonaldehyde is tumorigenic in rats. This study used immunoassay and 32P-postlabeling methods to determine if acrolein and crotonaldehyde form these adducts in cultured mammalian cells. Adduct levels were highest in Chinese hamster ovary cells exposed to acrolein (1 mM) with 162 mumol adduct/mol deoxyguanosine. Crotonaldehyde (10 mM) formed adduct at a level of 75 mumol/mol deoxyguanosine. 32P-Postlabeling analysis confirmed the presence of adducts in crotonaldehyde-treated cells. Persistence studies showed that adduct levels were unchanged if the cells were cultured for 6 h before DNA isolation. Mutagenicity studies were performed to determine the biological consequences of these adducts. Mutations were not observed due to the toxicity of the compounds.

Research paper thumbnail of Study of DNA methylation by tobacco-specific N-nitrosamines

Environmental Health Perspectives, Oct 1, 1985

An enzyme-linked immunosorbent assay (BA-ELISA) involving use of biotin-labeled anti-rabbit IgG a... more An enzyme-linked immunosorbent assay (BA-ELISA) involving use of biotin-labeled anti-rabbit IgG and avidin-labeled horseradish peroxidase was developed for the measurement of 06-methyl-2'-deoxyguanosine (06-MedGuo). Up to 5 jg of methylated DNA was enzymatically hydrolyzed, and the extent of inhibition of binding of immobilized 06-MedGuo-bovine serum albumin to rabbit anti-O6-MedGuo was measured. Fifty percent inhibition of antigen-antibody binding was achieved with 2.5 pmole of of 06-MedGuo. Separation of 0-MedGuo from unmodified nucleosides by high-performance liquid chromatography (HPLC-BA-ELISA) allowed detection of 700 fmole 06-MedGuo in 1 mg of DNA. Among the tobacco-related carcinogens, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is one of the most potent. In F344 rats it induces nasal cavity, lung and liver tumors. Four hours after a single IV injection of NNK to F344 rats (87 mg/kg body weight), 0'-MedGuo was present in target organs (,umole 0'-MedGuo/mole dGuo) (nasal mucosa, 219; lung, 13.2; and liver, 34.5) but was not detectable in nontarget organs. F344 rats receiving daily IP injections of NNK (40 mg/kg body weight) for 14 days were sacrificed 24 hr after the last injection. The levels of (06-Medguo/dGuo) were 7.9 and 11.4 ,umole/mole in the nasal mucosa and lung, respectively. In the liver no 06-MedGuo was detected, but 1050 ,umole of 7-MeGua/mole Gua was measured by HPLC-fluorimetry. No DNA methylation was observed in the nasal mucosa or liver of F344 rats treated with the nicotine-derived carcinogen N'-nitrosonornicotine. Reduction of the carbonyl of NNK is a major metabolic pathway, giving rise to 4-(methylnitrosamino)-1-(3-pyridyl)-1-butan-1-ol (NNAI). Nasal mucosae were cultured in vitro with NNK or NNAI. After 1 hr, methylation at the 06-dGuo and 7-dGuo sites were observed with NNK but not with NNAI. Methylation by NNAI after 24 hr was associated with the conversion of NNAI to NNK. These results suggest that NNAI is not associated with the activation of NNK to DNA methylating species.

Research paper thumbnail of Evaluation of <sup>32</sup>P-postlabeling analysis of DNA from exfoliated oral mucosa cells as a means of monitoring exposure of the oral cavity to genotoxic agents

Carcinogenesis, 1989

Development of oral cavity cancer in man has been linked to alcohol consumption and use of tobacc... more Development of oral cavity cancer in man has been linked to alcohol consumption and use of tobacco products. In order to understand the underlying carcinogenic mechanisms in the oral cavity a method is needed to monitor exposure of this site to various environmental insults. In this pilot study we evaluate the use of the 32P-postlabeling assay to detect adducts in DNA from exfoliated oral mucosa cells. Exfoliated cells were collected from the cheek and tongue of 27 men aged 35-69 years. DNA was extracted from the cells and analyzed by the enhanced 32P-postlabeling technique using butanol extraction. A variety of adduct spots were detected but none was consistently associated with exposure to alcohol or tobacco products. Some of the adducts detected had migration patterns in TLC very similar to the major deoxyguanosine adducts formed by the diol epoxides of benzo[a]pyrene and 5-methylchrysene, suggesting that they may have been formed from polynuclear aromatic hydrocarbons. Adduct spots with migration patterns similar to polynuclear hydrocarbon adducts accounted for only about one third of the total adduct spots observed. Relative adduct labeling (RAL) values were determined for samples from 12 of the 27 individuals. RAL values ranged from 1.6 X 10(-6) to 7.7 X 10(-11) adducts per nucleotide. The RAL values for adducts from the cheek or tongue were not significantly different. Adduct levels in smokers (median RAL of 4.8 X 10(-8) were significantly higher (P less than 0.001) than adduct levels in non-smokers (median RAL of 2.9 X 10(-9). Adduct levels in drinkers (median RAL of 9.1 X 10(-10) were significantly lower (P less than 0.001) than adduct levels in non-drinkers (median RAL of 3.7 X 10(-8). Four of the subjects in this study have subsequently developed squamous cell carcinoma of the oral cavity. 32P-Postlabeling analysis of DNA from the oral cavity of these subjects did not demonstrate unique patterns or RAL values. Lack of information on the structure of the majority of adducts observed in this study was a serious limitation. Further improvements in adduct identification will be needed before 32P-postlabeling can be a useful tool for monitoring exposure of the oral cavity to carcinogens.

Research paper thumbnail of Inhibition of PKC and Pka by Chemopreventive Organoselenium Compounds

International Journal of Oncology, Sep 1, 1995

Numerous efficacy studies in rodents revealed that l,4-phenylenebis(methylene)-selenocyanate (p-X... more Numerous efficacy studies in rodents revealed that l,4-phenylenebis(methylene)-selenocyanate (p-XSC) is a more effective chemopreventive organoselenium compound and less toxic than benzyl selenocyanate (BSC) or the inorganic compound Na 2 Se0 3. To explore mechanisms which mediate chemopreventive activities of p-XSC we have tested its effect on protein kinase A and C using in vitro and cell culture systems. While p-XSC did completely inhibit PKC and PKA activity, BSC was less active and Na 2 Se0 3 had no effect. Comparative EC 50 revealed values of 0.1, 1 and >10 fiM for p-XSC, BSC and Na 2 Se0 3 , respectively. p-XSC was also capable of inhibiting protein phosphorylation in cultures of primary human fibroblasts and altered morphology of rat fibroblast (R6) cells. When combined, sub-optimal doses of p-XSC and staurosporine yielded an additive effect on cell morphology. The ability of p-XSC and BSC to inhibit protein kinase A and C activities may in part account for the mechanism(s) by which these agents mediate their chemo preventive effects.

Research paper thumbnail of Measurement of O6-methyldeoxyguanosine in DNA methylated by the tobacco-specific carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone using a biotin-avidin enzyme-linked immunosorbent assay

Carcinogenesis, 1985

Rabbit antibodies raised against O6-methyl-2&#39;-deoxyguanosine (O6-MedGuo) were used to dev... more Rabbit antibodies raised against O6-methyl-2&#39;-deoxyguanosine (O6-MedGuo) were used to develop a competitive Biotin-Avidin enzyme-linked immunosorbent assay (BA-ELISA) for the quantitation of this adduct in DNA from animals exposed to tobacco-specific N-nitrosamines. The assay was able to detect as little as 30 mumol O6-MedGuo per mol deoxyguanosine. The specificity of the assay was at least 10 000-fold greater for O6-MedGuo than for unmodified nucleosides or 7-methyl-2&#39;-deoxyguanosine. The sensitivity of the assay was increased to 1 mumol O6-MedGuo per mol deoxyguanosine by the prior separation of O6-MedGuo from unmodified nucleosides by h.p.l.c. This assay was used to study the methylating ability of the tobacco-specific nitrosamine 4-(methyl-nitrosamino)-1-(3-pyridyl)-1-butanone (NNK) in F344 rats. Four hours after exposure to a single i.v. dose of NNK O6-MedGuo was detected in the target tissues nasal mucosa, lung and liver (219, 13.2 and 34.5 mumol/mol deoxyguanosine) but not in the non-target tissues esophagus, spleen, heart or kidney. Chronic exposure by 14 daily i.p. injections of NNK also resulted in detectable amounts of O6-MedGuo in the lung and nasal mucosa (11.4 and 7.9 mumol/mol deoxyguanosine). These results demonstrate that NNK is capable of methylating DNA in vivo. The methodology developed in these studies will allow us to examine in detail the role of DNA methylation in NNK-mediated carcinogenesis.

Research paper thumbnail of Approaches to the development of assays for interaction of tobacco-specific nitrosamines with haemoglobin and DNA

PubMed, 1988

The tobacco-specific, nicotine-derived nitrosamines 4-(N-nitrosomethylamino)-1-(3-pyridyl)-1-buta... more The tobacco-specific, nicotine-derived nitrosamines 4-(N-nitrosomethylamino)-1-(3-pyridyl)-1-butanone (NNK) and N'-nitrosonornicotine (NNN) are among the most important carcinogens in tobacco and tobacco smoke. Treatment of Fischer 344 rats with these carcinogens resulted in alkylation of haemoglobin and DNA by the 4-(3-pyridyl)-4-oxobutyl group formed during their metabolism. This alkyl group can be detached from globin or DNA under mild hydrolytic conditions as 4-hydroxy-1-(3-pyridyl)-1-butanone, which appears to be a potentially useful dosimeter for human exposure to, and activation of, tobacco-specific nitrosamines.

Research paper thumbnail of Modulation of Carcinogen-Induced Polyoma Dna-Replication by Organoselenium and Organosulfur Chemopreventive Agents

International Journal of Oncology, Mar 1, 1993

Both chemical and physical carcinogens are potent inducers of asynchronous replication of various... more Both chemical and physical carcinogens are potent inducers of asynchronous replication of various DNA tumor viruses. Employing H3 cells that carry an integrated copy of polyoma virus we have evaluated potential effects of known chemopreventive agents on carcinogen-induced polyoma DNA replication. The ability of well established organoselenium and organosulfur chemopreventive agents, in laboratory animals, to modulate polyoma DNA replication induced by AMMN or NNKOAc which are direct acting carcinogens derived from the tobacco carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) was examined. We demonstrate that both benzyl selenocyanate (BSC) and benzyl isothiocyanate (BITC) are capable of reducing the level of polyoma DNA replication induced by NNK-model compounds. BITC also reduced the transcriptional activity of polyoma sequences as measured through chloramphenicol-acetyl-transferase (CAT) assays using the polyoma regulatory region cloned upstream of the CAT gene. These results suggest that the mechanisms by which BSC and BITC exert their protective effects involve changes in the expression of cellular proteins which regulate transcription and replication of polyoma DNA sequences.

Research paper thumbnail of Application of an immunoassay for cyclic acrolein deoxyguanosine adducts to assess their formation in DNA of <i>Salmonella typhimurium</i> under conditions of mutation induction by acrolein

Carcinogenesis, 1989

Acrolein has been shown to form cyclic deoxyguanosine adducts when it reacts with DNA in vitro. I... more Acrolein has been shown to form cyclic deoxyguanosine adducts when it reacts with DNA in vitro. In this study, we have used a recently developed immunoassay for these adducts to study their formation in DNA from Salmonella typhimurium exposed to acrolein. Acrolein--deoxyguanosine adducts were formed in a dose-dependent fashion in Salmonella tester strains TA100 and TA104, reaching levels as high as 5 mumol adduct per mol deoxyguanosine. Using the liquid pre-incubation assay, acrolein-induced mutations were also found in strains TA100 and TA104. The correlation between acrolein--deoxyguanosine adduct concentration and acrolein-induced mutations in TA100, which contains GC base pairs at the site of reversion, suggests that the acrolein--deoxyguanosine adduct is a promutagenic lesion. That mutations are also seen in TA104 which contains AT base pairs at the site of reversion suggest that adducts of bases other than deoxyguanosine may also be important in the mutagenic activity of acrolein.

Research paper thumbnail of The relationship between <i>N</i>-nitrosodimethylamine metabolism and DNA methylation in isolated rat hepatocytes

Carcinogenesis, 1996

The metabolism of JV-nitrosodimethylamine (NDMA) and its methylation of DNA were simultaneously d... more The metabolism of JV-nitrosodimethylamine (NDMA) and its methylation of DNA were simultaneously determined in hepatocytes isolated from untreated and saline-and pyrazole-treated male Sprague-Dawley rats. Metabolism of NDMA was directly measured by monitoring its disappearance via gas chromatography coupled with a sensitive and specific detector for iV-nitrosamines. DNA methylation was determined in the same cells employed in the metabolism studies using a monoclonal antibody-based competitive ELISA procedure specific for O*-methyldeoxyguanosine (6-Me-dG). The apparent K m and V^^, for NDMA metabolism are 61 pM and 56 pmol/min/10* cells respectively for hepatocytes isolated from untreated rats. It was found that the addition of pyrazole to the in vitro hepatocyte incubations caused a dose-dependent inhibition of both metabolism and DNA methylation. However, when DNA methylation is expressed as a function of NDMA metabolized, there is no significant difference between hepatocyte incubations without or with pyrazole, with an average value of 79 nmol 6-Me-dG/mol dG/nmol NDMA metabolized. Based on the pyrazole inhibition studies, cytochrome P450IIE1 is responsible for at least 60% of the DNA methylation in rat hepatocytes. In pyrazole-pretreated rats there was an inconsistent increase in NDMA metabolism, but when metabolism was elevated so was DNA methylation. In contrast, microsomes isolated from pyrazole-pretreated rats consistently showed elevated metabolism of NDMA. Based on the simultaneous determination of adduct levels and metabolism, there is-1 6-Me-dG adduct formed/133 000 NDMA molecules metabolized in the uninduced hepatocytes.

Research paper thumbnail of Inhibition of repair of O6-methyldeoxyguanosine and enhanced mutagenesis in rat-liver epithelial cells

Mutation Research: Fundamental And Molecular Mechanisms Of Mutagenesis, Jun 1, 1990

The pro-mutagenicity of chemically-induced methylation of DNA at the O6 position of deoxyguanosin... more The pro-mutagenicity of chemically-induced methylation of DNA at the O6 position of deoxyguanosine was studied in cultured adult rat liver epithelial cells. To modify the level of O6-methyldeoxyguanosine (O6-medGuo) resulting from exposure to an alkylating agent, partial depletion of the O6-alkylguanine-DNA alkyltransferase (AGT) repair system was produced by pretreatment of ARL 18 cells with a non-toxic dose of exogenous O6-methylguanine (O6-meG). Exposure of cells to 0.6 mM O6-meG for 4 h depleted AGT activity by about 40%. Intact and pretreated cells were exposed to a range of doses of N-methyl-N&#39;-nitro-N-nitrosoguanidine (MNNG), and mutagenesis at the hypoxanthine-guanine phosphoribosyl transferase (HGPRT) locus was quantified by measurement of 6-thioguanine-resistant mutants. The mutagenicity of MNNG was dose dependent and was greater in O6-meG pretreated cultures than in intact cultures. Immunoslot blot measurement of O6-medGuo employing a mouse monoclonal antibody demonstrated that MNNG produced O6-medGuo and that the intact liver cells were efficient in eliminating this lesion from their DNA. Since depletion of AGT would be expected to affect the rate of elimination of only O6-medGuo, it is concluded that this lesion is highly pro-mutagenic.

Research paper thumbnail of The <i>in Vitro </i>Methylation of DNA by a Minor Groove Binding Methyl Sulfonate Ester

Chemical Research in Toxicology, 1996

ABSTRACT The preparation of sequence and groove specific DNA methylating agents based on N-methyl... more ABSTRACT The preparation of sequence and groove specific DNA methylating agents based on N-methylpyrrolecarboxamide subunits appended with an O-methyl sulfonate ester functionality (MeOSO2(CH2)2-Lex) has previously been described [Zhang, Y., Chen, F.-X., Mehta, P., and Gold, B. (1993) Biochemistry 32,7954-7965]. In contrast to simple methyl sulfonate esters, e.g., methyl methanesulfonate (MMS), which predominantly methylate at 7-guanine, MeOSO2-(CH2)2-Lex affords N3-methyladenine (3-MeAde) as its major adduct. Using competitive ELISA determinations, the methylation at major and minor groove sites in calf thymus DNA by MeOSO2(CH2)2-Lex has been precisely quantitated. The yields of N7-methylguanine (7-MeGua), 3-MeAde, and O6-methyldeoxyguanosine (6-Me-dGuo) are 0.424, 3.195, and 0.0027 mmol of adduct/mol of DNA, respectively, using 10 microM MeOSO2(CH2)2-Lex and 100 microM DNA. This compares to 0.773, 0.072, and 0.0033 mmol of adduct/mol of DNA for 7-MeGua, 3-MeAde, and 6-Me-dGuo, respectively, using MMS. The increase in the yield of 3-MeAde due to the minor groove equilibrium binding properties of MeOSO2(CH2)2-Lex is approximately 40-fold relative to MMS.

Research paper thumbnail of Tobacco-specific nitrosamine adducts: studies in laboratory animals and humans

Environmental Health Perspectives, Mar 1, 1993

This paper describes quantitation of human hemoglobin and DNA adducts of the carcinogenic tobacco... more This paper describes quantitation of human hemoglobin and DNA adducts of the carcinogenic tobacco-specific nitrosamines 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and N'-nitrosonornicotine (NNN). NNK and NNN are believed to be involved in cancers ofthe lung, esophagus, onl cavity, and pancreas in people who use tobacco products. The adduct dosimetry method employs GC-MS for quantitation of 4-hydroxy-1-(3-pyridyl)-1-butanone (HPB) released by mild base hydrolysis of hemoglobin or acid hydrolysis of DNA as a biochemical marker of the pyridyloxobutylation metabolic activation pathway. Approximately 22% of smokers (n = 101) had elevated levels of HPB released from hemoglobin (range, 200-1600 fmole/g Hb). Adduct levels in snuff dippers ranged from 2001800 fmole/g Hb. HPB levels in nonsmokers were generally below the detection limit. Acid hydrolysis of lung and tracheal DNA obtained at autopsy and analysis for released HPB revealed levels ranging up to 50 fmole/mg DNA in smokers; the adduct was not detected in nonsmokers. These findings are consistent with data generated in studies of adduct formation by NNK in rats. The biological significance of the HPB-releasing DNA pyridylksobutylation pathway was compared to that of the DNA methylation pathway in the A/J mouse. These studies demonstrated that the persistence of O'-methylguanine in lung DNA is critical for tumorigenesis by NNK and that pyridylaobutylation enhances both peristence of O-methylguanine and tumorigenesis by acetoxymethylmethylnitrosamine. In the rat, the relative roles of methylation and pyridyloxobutylation in lung tumorigenesis by NNK are not as clearly defined. Although the biological significance of DNA methylation in NNK tumorigenesis is well characterized, dosimetry studies of tobacco-specific nitrosamines in humans should be carried out using biochemical markers of the pyridyloxobutylation pathway because of their specificity to tobacco products.

Research paper thumbnail of Mass spectrometric analysis of tobacco-specific nitrosamine-DNA adducts in smokers and nonsmokers

Chemical Research in Toxicology, May 1, 1991

A gas chromatography, negative ion chemical ionization mass spectrometry (GC-NICI-MS) based assay... more A gas chromatography, negative ion chemical ionization mass spectrometry (GC-NICI-MS) based assay for tobacco-specific nitrosamine adducts of DNA is described. The assay is based on the observation that acid hydrolysis of DNA from animals treated with tobacco-specific nitrosamines releases 4-hydroxy-l-(3-pyridyl)-l-butanone (HPB). HPB and the internal standard [4,4-D2]HPB are derivatized with pentafluorobenzoyl chloride and the resulting HPB-pentafluorobenzoate is purified by high-performance liquid chromatography prior to GC-NICI-MS analysis. DNA from human peripheral lung and tracheobronchial tissue, collected at autopsy, was analyzed for acid-released HPB. The mean H P B level (fmol/mg of DNA) for peripheral lung DNA was 11 f 16 (SD, n = 9) for smokers and 0.9 f 2.3 (n = 8) for nonsmokers. Mean adduct levels in tracheobronchus were 16 f 18 (n = 4) for smokers and 0.9 f 1.7 (n = 4) for nonsmokers. These are the first measurements of tobacco-specific nitrosamine-DNA adducts in humans. Further studies comparing the levels of DNA and globin adducts w i l l provide a better understanding of the metabolic activation of tobacco-specific nitrosamines in humans and may provide a more accurate indication of an individual's risk of developing tobacco-related cancer.

Research paper thumbnail of Partial reconstitution of TNP-Ficoll responses and IgG3 expression in Xid mice undergoing graft-vs-host reaction

The Journal of Immunology

In uence of lymphokines on the anti-TNP-Ficoll response of normal and X-linked B lymphocyte-defec... more In uence of lymphokines on the anti-TNP-Ficoll response of normal and X-linked B lymphocyte-defective mice.

Research paper thumbnail of Inhibition of repair of O6-methyldeoxyguanosine and enhanced mutagenesis in rat-liver epithelial cells

Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis, 1990

The pro-mutagenicity of chemically-induced methylation of DNA at the O6 position of deoxyguanosin... more The pro-mutagenicity of chemically-induced methylation of DNA at the O6 position of deoxyguanosine was studied in cultured adult rat liver epithelial cells. To modify the level of O6-methyldeoxyguanosine (O6-medGuo) resulting from exposure to an alkylating agent, partial depletion of the O6-alkylguanine-DNA alkyltransferase (AGT) repair system was produced by pretreatment of ARL 18 cells with a non-toxic dose of exogenous O6-methylguanine (O6-meG). Exposure of cells to 0.6 mM O6-meG for 4 h depleted AGT activity by about 40%. Intact and pretreated cells were exposed to a range of doses of N-methyl-N&#39;-nitro-N-nitrosoguanidine (MNNG), and mutagenesis at the hypoxanthine-guanine phosphoribosyl transferase (HGPRT) locus was quantified by measurement of 6-thioguanine-resistant mutants. The mutagenicity of MNNG was dose dependent and was greater in O6-meG pretreated cultures than in intact cultures. Immunoslot blot measurement of O6-medGuo employing a mouse monoclonal antibody demonstrated that MNNG produced O6-medGuo and that the intact liver cells were efficient in eliminating this lesion from their DNA. Since depletion of AGT would be expected to affect the rate of elimination of only O6-medGuo, it is concluded that this lesion is highly pro-mutagenic.