Bogdan Prokopczyk

Identification of benzo[a]pyrene metabolites in cervical mucus and DNA adducts in cervical tissues in humans by gas chromatography-mass spectrometry

Epidemiological studies indicate that cigarette smoking increases the risk of cervical cancer. To address questions regarding possible mechanisms of tobacco-related cervical carcinogenesis, in a pilot study, using supercritical fluid extraction and a gas chromatographic-mass spectrometric (GC-MS) technique, we detected and characterized benzo[a]pyrene and its metabolites, namely B[a]P-dihydrodiols, phenols and tetraols in cervical mucus samples from eight smokers and non-smokers. Twenty-eight epithelial and stromal cervical tissue samples from seventeen patients undergoing surgery for non-malignant disease were quantitatively analyzed for BPDE-DNA adducts by a GC-MS technique. BPDE-DNA adducts were found in 25 samples. The mean level of BPDE-DNA adducts in epithelial cervical tissues of smokers was nearly two-fold greater than that in self-reported non-smokers; P = 0.02. The mean number of BPDE-adducts (+/- SD) in epithelial cervical tissues of smokers was 3.5 +/- 1.06 adducts/10(8) nucleotides while that in non-smokers was 1.8 +/- 0.96 adducts/10(8) nucleotides. The mean number of BPDE-DNA adducts in stromal cervical tissues of the same subjects was 1.8 +/- 0.96 adducts/10(8) nucleotides in smokers and that in the stromal tissues of non-smokers was 1.4 +/- 1.1 adducts/10(8). These results suggest that polynuclear aromatic hydrocarbons (PAHs) from tobacco smoke and other environmental sources can be transported to the cervix where they are metabolized in the cervical epithelium to ultimate carcinogenic agents, although transport of ultimate carcinogenic metabolites from other organs to the cervix cannot be ruled out. Exposure of cervical epithelia to PAHs and their carcinogenic metabolites suggests a potential role of such carcinogens in the pathogenesis of cervical cancer in humans.

Formation and analysis of tobacco-specific N-nitrosamines

Chemical-analytical studies during the past 4 years led to several new observations on the formation of tobacco-specific N-nitrosamines (TSNA) and their occurrence in smokeless tobacco, mainstream smoke (MS), and sidestream smoke (SS) of American and foreign cigarettes. When snuff was extracted by means of supercritical fluid extraction with carbon dioxide containing 10% methanol, analysis of this material confirmed that the extraction with organic solvents had been partially incomplete. Epidemiological studies in the northern Sudan showed a high risk for oral cancer for users of toombak, a home-made oral snuff. Toombak contains 100-fold higher levels of TSNA than commercial snuff in the U.S. and Sweden. The TSNA content in the saliva of toombak dippers is at least ten times higher than that reported in the saliva of dippers of commercial snuff. Biomarker studies have shown corresponding high levels of hemoglobin adducts with metabolites of NNN and NNK as well as for urinary metabolites of NNK. These data supported the epidemiological findings. The analyses of MS of U.S. and foreign cigarettes smoked under FTC conditions revealed comparable data for the smoke of nonfilter cigarettes and filter cigarettes except in the case of low- and ultralow-yield cigarettes, which showed reduced TSNA yields. The MS of cigarettes made from Burley or dark tobacco is exceptionally high in TSNA, primarily because of the high nitrate content of those tobacco types. Taking puffs of larger volume and drawing puffs more frequently, practices observed among most smokers of cigarettes with low nicotine yield, results in high TSNA values in the MS. The formation of the lung carcinogen NNK is favored during the smoldering of cigarettes, between puffs, when SS is generated. Consequently, in most samples from indoor air polluted with environmental tobacco smoke (ETS), the highest concentration of an individual TSNA is that of NNK. When nonsmokers had remained for up to 2 h in a test laboratory with high ETS pollution, they excreted measurable amounts of NNK metabolites in the urine, indicative of the uptake of TSNA.

Chemoprevention of lung tumorigenesis induced by a mixture of benzo(a)pyrene and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone by the organoselenium compound 1,4-phenylenebis(methylene)selenocyanate

We evaluated the chemopreventive efficacy of the organoselenium compound 1,4-phenylenebis(methylene)selenocyanate (p-XSC) against the development of tumors of the lung and forestomach induced by a mixture of benzo(a)pyrene (B(a)P) and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), two of the major lung carcinogens present in tobacco smoke. A/J mice (20 mice/group) were given intragastric doses of a mixture of B(a)P (3 micromol/mouse) and NNK (3 micromol/mouse) in cottonseed oil (0.1 ml) once a week for eight consecutive weeks. Mice were fed either AIN-76A control diet or control diet containing p-XSC (10 ppm selenium), either during or after carcinogen administration. Dietary p-XSC significantly reduced lung tumor multiplicity, regardless of whether it was given during or after carcinogen administration. p-XSC was also an effective inhibitor of tumor development in the forestomach. To provide some biochemical insights into the protective role of p-XSC, its effect on selected phase I and II enzyme activities involved in the metabolism of NNK and B(a)P was also examined in vivo in this animal model. Dietary p-XSC significantly inhibited the activities of the phase I enzymes, methoxyresorufin O-dealkylase (MROD) and N-nitrosodimethylamine N-demethylase (NDMAD), in mouse liver, but it had no effect on ethoxyresorufin O-dealkylase (EROD), pentoxyresorufin O-dealkylase (PROD), and erythromycin N-demethylase (ERYTD). Total glutathione S-transferase (GST) enzyme activity, as well as GST-pi and GST-mu enzyme activities, were significantly induced by dietary p-XSC in both the lung and liver. Glutathione peroxidase (GPX) activity was also induced by p-XSC in mouse lung, but not in the liver. Dietary p-XSC had no effect on selenium-dependent glutathione peroxidase (GPX(Se)), GST-alpha, and UDP-glucuronosyl transferase (UDPGT) enzyme activities in either the lung or the liver. These studies suggest that the chemopreventive efficacy of p-XSC, when fed during carcinogen administration, may be, in part, due to the inhibition of certain phase I enzymes involved in the metabolic activation of these carcinogens, and the induction of specific phase II enzymes involved in their detoxification. The mechanisms that account for the effect of p-XSC when fed after carcinogen administration remain to be determined.

The Impact of Fish Oil on the Chemopreventive Efficacy of Tamoxifen against Development of N-Methyl-N-Nitrosourea- Induced Rat Mammary Carcinogenesis

The antiestrogen tamoxifen reduces breast cancer incidence in high-risk women but is unable to inhibit the development of hormone-independent tumors. Omega-3 polyunsaturated fatty acids (n-3 PUFA), known ligands of the peroxisome proliferator activated receptor-γ (PPARγ), generally exert tumor-suppressive effects. Based on the known crosstalk between the estrogen and the PPARγ receptors, we tested the hypothesis that the combination of tamoxifen with n-3 PUFA results in a superior antitumor action over the individual interventions. In this study, we report for the first time that the combination of a fish oil diet rich in n-3 PUFA and tamoxifen seemed to inhibit N-methyl-N-nitrosourea–induced mammary carcinogenesis, tumor multiplicity, and volume to a greater extent than the individual interventions. The potential superiority of the combination was particularly evident at a suboptimal dose of tamoxifen, which, by itself, was unable to significantly decrease tumor development. Because activation of PPARγ is known to inhibit oxidative stress, we examined the effects of our interventions on circulating and tumor levels of glutathione, a major intracellular antioxidant. Our results indicate that reduction in the level of oxidative stress may be a potential mechanism by which the n-3 PUFA–rich diet potentiated the tumor-suppressive effect of tamoxifen. Our interventions were well tolerated without evidence of toxicity. Combined administration of tamoxifen and n-3 PUFA is a promising new approach to breast cancer prevention. Because of its safety, this combination can quickly be translated to the clinic if its superiority can be supported by future studies. Cancer Prev Res; 3(3); 322–30

Administration of omega-3 fatty acids and Raloxifene to women at high risk of breast cancer: interim feasibility and biomarkers analysis from a clinical trial.

Background/Objectives:The antiestrogen, Raloxifene (Ral) is an effective breast cancer chemopreventive agent. Omega-3 fatty acids (n-3FA) may inhibit mammary carcinogenesis. On the basis of their mechanisms of action, we test the hypothesis that a combination of n-3FA and Ral may be superior in reducing select biomarkers of breast cancer risk in women.Subjects/Methods:Postmenopausal women at increased risk for breast cancer (breast density ⩾25%) were randomized to: (1) no intervention; (2) Ral 60 mg; (3) Ral 30 mg; (4) n-3FA (Lovaza) 4 g and (5) Lovaza 4 g+Ral 30 mg for 2 years. Reduction in breast density is the primary end point of the study. We report preliminary data on feasibility, compliance and changes in secondary end points related to IGF-I signaling, estrogen metabolism, oxidative stress and inflammation in the first group of 46 women who completed 1 year of the study.Results:All interventions were well tolerated with excellent compliance (96±1% overall) by pill count and also supported by the expected rise in both serum n-3FA and n-3FA/Omega-6 fatty acids (n-6FA) ratio in women randomized to groups 4 and 5 (P<0.05). Lovaza decreased serum triglycerides and increased high-density lipoprotein (HDL) cholesterol compared with control (P<0.05 for both). Ral reduced serum IGF-1 in a dose-dependent manner (P<0.05) while Lovaza did not. Lovaza had no effect on IGF-1 or IGFBP-3. None of the other biomarkers were affected by our treatment.Conclusion:The combination of Lovaza and Ral is a feasible strategy that may be recommended in future breast cancer chemoprevention trials.

H2S concentrations in the arterial blood during H2S administration in relation to its toxicity and effects on breathing

Our aim was to establish in spontaneously breathing urethane-anesthetized rats, the relationship between the concentrations of H2S transported in the blood and the corresponding clinical manifestations, i.e., breathing stimulation and inhibition, during and following infusion of NaHS at increasing rates. The gaseous concentration of H2S (CgH2S, one-third of the total soluble form) was computed from the continuous determination of H2S partial pressure in the alveolar gas, while H2S, both dissolved and combined to hemoglobin, was measured at specific time points by sulfide complexation with monobromobimane (CMBBH2S). We found that using a potent reducing agent in vitro, H2S added to the whole blood had little interaction with the plasma proteins, as sulfide appeared to be primarily combined and then oxidized by hemoglobin. In vivo, H2S was undetectable in the blood in its soluble form in baseline conditions, while CMBBH2S averaged 0.7 ± 0.5 μM. During NaHS infusion, H2S was primarily present in nonsoluble form in the arterial blood: CMBBH2S was about 50 times higher than CgH2S at the lowest levels of exposure and 5 or 6 times at the levels wherein fatal apnea occurred. CgH2S averaged only 1.1 ± 0.7 μM when breathing increased, corresponding to a CMBBH2S of 11.1 ± 5.4 μM. Apnea occurred at CgH2S above 5.1 μM and CMBBH2S above 25.4 μM. At the cessation of exposure, CMBBH2S remained elevated, at about 3 times above baseline for at least 15 min. These data provide a frame of reference for studying the putative effects of endogenous H2S and for testing antidotes against its deadly effects.

Residential environmental exposures and other characteristics associated with detectable PAH-DNA adducts in peripheral mononuclear cells in a population-based sample of adult females.

The detection of polycyclic aromatic hydrocarbon (PAH)-DNA adducts in human lymphocytes may be useful as a surrogate end point for individual cancer risk prediction. In this study, we examined the relationship between environmental sources of residential PAH, as well as other potential factors that may confound their association with cancer risk, and the detection of PAH-DNA adducts in a large population-based sample of adult women. Adult female residents of Long Island, New York, aged at least 20 years were identified from the general population between August 1996 and July 1997. Among 1556 women who completed a structured questionnaire, 941 donated sufficient blood (25+ ml) to allow use of a competitive ELISA for measurement of PAH-DNA adducts in peripheral blood mononuclear cells. Ambient PAH exposure at the current residence was estimated using geographic modeling (n=796). Environmental home samples of dust (n=356) and soil (n=360) were collected on a random subset of long-term residents (15+ years). Multivariable regression was conducted to obtain the best-fitting predictive models. Three separate models were constructed based on data from : (A) the questionnaire, including a dietary history; (B) environmental home samples; and (C) geographic modeling. Women who donated blood in summer and fall had increased odds of detectable PAH-DNA adducts (OR=2.65, 95% confidence interval (CI)=1.69, 4.17; OR=1.59, 95% CI=1.08, 2.32, respectively), as did current and past smokers (OR=1.50, 95% CI=1.00, 2.24; OR=1.46, 95% CI=1.05, 2.02, respectively). There were inconsistent associations between detectable PAH-DNA adducts and other known sources of residential PAH, such as grilled and smoked foods, or a summary measure of total dietary benzo-[a]-pyrene (BaP) intake during the year prior to the interview. Detectable PAH-DNA adducts were inversely associated with increased BaP levels in dust in the home, but positively associated with BaP levels in soil outside of the home, although CIs were wide. Ambient BaP estimates from the geographic model were not associated with detectable PAH-DNA adducts. These data suggest that PAH-DNA adducts detected in a population-based sample of adult women with ambient exposure levels reflect some key residential PAH exposure sources assessed in this study, such as cigarette smoking.

A study of betel quid carcinogenesis. IX. Comparative carcinogenicity of 3-(methylnitrosamino)propionitrile and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone upon local application to mouse skin and rat oral mucosa.

The Areca-derived 3-(methylnitrosamino)propionitrile (MNPN) was tested for its tumor initiating activity on mouse skin and for its tumorigenic potential in the oral mucosa of rats. On mouse skin, like the otherwise strongly carcinogenic, tobacco-specific 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), MNPN showed only weak local tumor initiator activity. However, the application of MNPN to mouse skin led also to multiple distant tumors in the lungs of the animals. Twice daily swabbing of the oral cavity of rats with aqueous solutions of MNPN or NNK for up to 61 weeks led only to one oral tumor in each group of 30 animals. Yet, these N-nitrosamines proved again to be strong organ-specific carcinogens. Thus, MNPN induced nasal tumors in 80% of the rats and lung adenomas in 13%, liver tumors in 10% and papillomas of the esophagus in 7%. NNK induced lung adenoma and/or adenocarcinoma in 90%, nasal tumors in 43% and liver adenomas/adenocarcinomas in 30% of the rats. These results confirm previous observations that, independent of the site and mode of application, MNPN and NNK remain strong organ-specific carcinogens in laboratory animals.

In-Vivo Interactions Between Cobalt or Ferric Compounds and the Pools of Sulphide in the Blood During and After H2s Poisoning

Hydrogen sulphide (H2S), a chemical hazard in oil and gas production, has recently become a dreadful method of suicide, posing specific risks and challenges for the first responders. Currently, there is no proven effective treatment against H2S poisoning and its severe neurological, respiratory or cardiac after-effects. We have recently described that H2S is present in various compartments, or pools, in the body during sulphide exposure, which have different levels of toxicity. The general goals of our study were to (1) determine the concentrations and kinetics of the various pools of hydrogen sulphide in the blood, i.e., gaseous (CgH2S) versus total sulphide, i.e., reacting with monobromobimane (CMBBH2S), during and following H2S exposure in a small and large mammal and (2) establish the interaction between the pools of H2S and a methemoglobin (MetHb) solution or a high dose of hydroxocobalamin (HyCo). We found that CgH2S during and following H2S infusion was similar in sedated sheep and rats at any given rate of infusion/kg and provoked symptoms, i.e., hyperpnea and apnea, at the same CgH2S. After H2S administration was stopped, CgH2S disappeared within 1 min. CMBBH2S also dropped to 2-3μM, but remained above baseline levels for at least 30 min. Infusion of a MetHb solution during H2S infusion produced an immediate reduction in the free/soluble pool of H2S only, whereas CMBBH2S increased by severalfold. HyCo (70 mg/kg) also decreased the concentrations of free/soluble H2S to almost zero; CgH2S returned to pre-HyCo levels within a maximum of 20 min, if H2S infusion is maintained. These results are discussed in the context of a relevant scenario, wherein antidotes can only be administered after H2S exposure.

Validation and Calibration of a Model Used to Reconstruct Historical Exposure to Polycyclic Aromatic Hydrocarbons for Use in Epidemiologic Studies

Objectives We previously developed a historical reconstruction model to estimate exposure to airborne polycyclic aromatic hydrocarbons (PAHs) from traffic back to 1960 for use in case–control studies of breast cancer risk. Here we report the results of four exercises to validate and calibrate the model. Methods Model predictions of benzo[a]pyrene (BaP) concentration in soil and carpet dust were tested against measurements collected at subjects’ homes at interview. In addition, predictions of air intake of BaP were compared with blood PAH–DNA adducts. These same soil, carpet, and blood measurements were used for model optimization. In a separate test of the meteorological dispersion part of the model, predictions of hourly concentrations of carbon monoxide from traffic were compared with data collected at a U.S. Environmental Protection Agency monitoring station. Results The data for soil, PAH–DNA adducts, and carbon monoxide concentrations were all consistent with model predictions. The carpet dust data were inconsistent, suggesting possible spatial confounding with PAH-containing contamination tracked in from outdoors or unmodeled cooking sources. BaP was found proportional to other PAHs in our soil and dust data, making it reasonable to use BaP historical data as a surrogate for other PAHs. Road intersections contributed 40–80% of both total emissions and average exposures, suggesting that the repertoire of simple markers of exposure, such as traffic counts and/or distance to nearest road, needs to be expanded to include distance to nearest intersection.