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.

Human cervical tissue metabolizes the tobacco-specific nitrosamine, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone, via alpha-hydroxylation and carbonyl reduction pathways.

We determined the ability of human epithelial cervical cells, human cervical microsomes and cytosol to metabolize 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK). All preparations metabolized NNK by alpha-hydroxylation, demonstrated by the presence of 4-oxo-4-(3-pyridyl)butyric acid (keto acid), and by carbonyl reduction, illustrated by the formation of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL). Cervical cells metabolized NNK by the oxidative pathway to an extent comparable to that by the reductive pathway. In both human cervical cytosol and microsomes, the concentration of alpha-hydroxylation products ranged from undetectable to 10 times lower than those of NNAL. An apparent K(m) and V(max) of 7075 microM and 650 pmol/mg/min, respectively, were determined for the keto acid in one microsomal preparation. NNAL was formed in all preparations at the highest levels, ranging from 16.9 to 35.5 pmol/10(6) cells in incubations with ectocervical cells and 6.2 pmol/10(6) cells in incubations with endocervical cells. NNAL levels were 1.88-4.95 and 1.44-2.08 pmol/mg/min in human cervical microsomes and cytosolic fractions, respectively. An apparent K(m) of 739 microM and a V(max) of 1395 pmol/mg/min for NNAL formation were established in the same microsomal preparation used for the keto acid kinetics study. The stereochemistry of the NNAL formed in incubations of NNK with human cervical cells and subcellular fractions was determined by derivatization with (S)-(-)-methylbenzyl isocyanate. Human cervical cells and microsomes both formed the (R)-enantiomer of NNAL almost exclusively; incubations with human cervical cytosol resulted predominantly in the formation of the (S)-enantiomer. Substrates for 11 beta-hydroxysteroid dehydrogenase, cortisone, glycyrrhizic acid and metyrapone all inhibited the formation of NNAL in incubations with human cervical microsomes; the inhibition ranged from 16% to 80%. These studies illustrate that human cervical tissue can metabolize NNK by both oxidative and reductive pathways and that 11 beta-HSD may, in part, be responsible for the carbonyl reduction of NNK.

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.

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.

Elevated 8-hydroxy-2'-deoxyguanosine levels in lung DNA of A/J mice and F344 rats treated with 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone and inhibition by dietary 1,4-phenylenebis(methylene)selenocyanate.

1,4-Phenylenebis(methylene)selenocyanate (p-XSC) is an effective chemopreventive agent against 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK)-induced lung adenoma in female A/J mice. While p-XSC can effectively inhibit NNK-induced DNA methylation in female A/J mice and in male F344 rats, its effect on NNK-induced oxidative DNA damage had not been determined. Thus, the effect of p-XSC on the levels of 8-hydroxy-2'-deoxyguanosine (8-OH-dG) in lung DNA from A/J mice and F344 rats treated with NNK was examined. Mice were given NNK by gavage (0.5 mg/mouse in 0.2 ml corn oil, three times per week for 3 weeks) or by a single i.p. injection (2 mg/mouse in 0.1 ml saline) while maintained on a control diet (AIN-76A) or control diet containing p-XSC at 10 or 15 p.p.m. (as Se) starting 1 week before NNK administration and continuing until termination. Mice were killed 2 h after the last NNK gavage in the multiple administration protocol or 2 h after the single i.p. injection. Treatment with NNK by gavage significantly elevated the levels of 8-OH-dG in lung DNA of A/J mice from 0.7 +/- 0.1 to 1.6 +/- 0.2 adducts/10(5) 2'-deoxyguanosine (dG) (P < 0.001), while dietary p-XSC (at 10 p.p.m. Se) prevented significant elevation of the levels of this lesion caused by NNK, keeping them at 0.9 +/- 0.1 adducts/10(5) dG (P < 0.003). Injection of NNK in saline also significantly increased the levels of 8-OH-dG in lung DNA of A/J mice from 1.2 +/- 0.6 to 3.6 +/- 0.8/10(5) dG adducts (P < 0.01), while dietary p-XSC (at 15 p.p.m. Se) kept these levels at 1.9 +/- 0.5 adducts/10(5) dG (P < 0.03). Rats were given a single i.p. injection of NNK (100 mg/kg body wt) in saline while being maintained on control diet (AIN-76A) or control diet containing p-XSC (15 p.p.m. as Se) starting 1 week before NNK administration and continuing until termination. The rats were killed 2 h after injection. Treatment with NNK using this protocol significantly elevated the levels of 8-OH-dG in lung DNA of F344 rats from 2.6 +/- 0.5 to 3.5 +/- 0.5 adducts/10(5) dG (P < 0.03), while dietary p-XSC (at 15 p.p.m. Se) kept the levels of this lesion at 2.2 +/- 0.6 adducts/10(5) dG (P < 0.01). Our findings suggest that the chemopreventive efficacy of p-XSC against NNK-induced lung tumorigenesis in A/J mice and F344 rats may be due in part to inhibition of oxidative DNA damage.

Effects of 1,4-phenylenebis(methylene)selenocyanate and selenomethionine on 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone-induced tumorigenesis in A/J mouse lung.

We reported earlier that continuous feeding of 1,4-phenylenebis(methylene)selenocyanate (p-XSC) inhibited lung tumor induction by the tobacco-specific nitrosamine, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) in the A/J mouse (El-Bayoumy et al., Carcinogenesis, 14, 1111-1113, 1993). The present investigation was designed to determine whether p-XSC inhibits pulmonary neoplasia induced by NNK in female A/J mice during the initiation phase of carcinogenesis or during the post-initiation phase. The naturally occurring selenomethionine was also included in this study. Doses higher than 4 p.p.m. of selenomethionine can induce toxic effects, therefore, dietary supplementation of this compound was selected at a dose level of 3.75 p.p.m. However, we were able to give p-XSC at selenium levels of 7.5 and 15 p.p.m., as mice can tolerate such doses in this form without any adverse effects. NNK was given by a single i.p. injection at dose of 10 micromol in 0.1 ml of saline. Selenomethionine did not show chemopreventive activity when administered in either phase of tumorigenesis. In contrast, p-XSC significantly reduced lung tumor multiplicity regardless of whether it was given during the initiation phase of tumorigenesis (P = 0.0009 at both levels of selenium) or post-initiation (P = 0.0009 at 15 p.p.m. and P = 0.036 for 7.5 p.p.m.). This is the first report describing that the synthetic organoselenium compound, p-XSC, can effectively block and suppress chemically (NNK)-induced lung tumor development in mice.

Identification of tobacco-specific carcinogen in the cervical mucus of smokers and nonsmokers.

BACKGROUND: In 1996, an estimated 15,700 new cases of cancer of the uterine cervix and 4,900 deaths from this disease were expected to occur in the United States. In a recent international study, human papillomavirus DNA was found in more than 90% of cervical tumor specimens examined, irrespective of the nationality of the patients from whom the samples were obtained. Although infection with human papillomavirus is the major known risk factor for the development of cervical cancer, it alone is not sufficient. Other etiologic factors that have been associated with this disease include deficiencies in micronutrients, lower socioeconomic status, oral contraceptive use, and cigarette smoking. Several compounds from cigarette smoke (nicotine and its major metabolite, cotinine) have been identified in cervical mucus, and the occurrence of smoking-related DNA damage in the cervical epithelium has been documented. PURPOSE: This investigation was conducted to determine for the first time whether carcinogenic tobacco-specific N-nitrosamines are present in the cervical mucus of cigarette smokers and of nonsmokers (most likely as a result of environmental exposure). METHODS: Cervical mucus specimens from 15 smokers and 10 nonsmokers were subjected to supercritical fluid extraction with the use of carbon dioxide that contained 10% methanol, and the resultant extracts were analyzed for tobacco-specific nitrosamines by use of a very sensitive method that involved gas chromatography and mass spectroscopy analyses. RESULTS: In a total of 16 samples obtained from 15 women who were current smokers (two samples from the same woman), we detected the tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) at concentrations that ranged from 11.9 to 115.0 ng/g of mucus. Only one of a total of 10 cervical mucus specimens obtained from 10 women who claimed to be nonsmokers did not contain detectable NNK, and NNK concentrations ranged from 4.1 to 30.8 ng/g of mucus in the specimens from the remaining nine women. The concentrations of NNK in specimens from cigarette smokers were significantly higher than those from nonsmokers (mean +/- standard deviation: 46.9 +/- 32.5 ng/g of mucus versus 13.0 +/- 9.3 ng/g of mucus; two-tailed Student's t test, P = .004). CONCLUSION: The cervical mucus of cigarette smokers contains measurable amounts of the potent carcinogen NNK. This compound represents the first tobacco-specific carcinogen identified in this physiologic fluid of women who smoke cigarettes. The presence of NNK in the cervical mucus of nonsmokers is likely due to environmental exposure or to the fact that some of the subjects in this study may not have revealed that they occasionally smoked cigarettes. IMPLICATIONS: The presence of NNK in human cervical mucus further strengthens the association between cervical cancer and tobacco smoking.

Effects of dietary 1,4-phenylenebis(methylene)selenocyanate on 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone-induced DNA adduct formation in lung and liver of A/J mice and F344 rats.

1,4-Phenylenebis(methylene)selenocyanate (p-XSC) was tested for its ability to inhibit DNA adduct formation induced by the tobacco-specific N-nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) in the liver and lung of A/J mice and F344 rats. Dietary p-XSC, providing a dose of 5 p.p.m. selenium, significantly inhibited the formation of 7-methylguanine (7-mGua) induced by a single i.p. injection of 10 mumol of NNK(12.8% inhibition at 4 h and 19.9% at 96 h) and O6-methylguanine (O6-mGua) (16.5% at 4 h and 34.8% at 96 h) in the liver of A/J mice. Dietary supplements of p-XSC providing 15 p.p.m. of selenium reduced the levels of 7-mGua by 17.3% (4 h) and 33.6% (96 h). The formation of O6-mGua was inhibited by 69.5% (4th) and 73.8 (96h). In A/J mouse lung DNA the most significant reduction was observed in levels of O6-mGua. Dietary p-XSC at 5 p.p.m. as selenium inhibited the formation of this adduct by 73.1% (4 h). Ninety-six hours after NNK injection, and at both time points with p-XSC providing 15 p.p.m. selenium, O6-mGua was not detected. Although levels of 7- mGua in mouse lung DNA were also reduced, this was significant only 4 h after carcinogen administration. In general, selenite at a5 p.p.m. as selenium had no significant effect on the levels of these lesions; however, it inhibited O6-mGua in the liver only 4 h after NNK administration. These effects may explain why there is chemopreventive activity for p-XSC, but not for selenite, in NNK-induced lung carcinogenesis in A/J mice. Moreover, these findings raised our interest in determining the potential chemopreventive activity of p-XSC against NNK-induced lung adenocarcinomas in male F344 rats by first determining its effects on NNK-induced DNA methylation in the lungs of rats. Diet supplemented with 10 p.p.m. selenium as p-XSC did indeed inhibit the formation of adducts in pulmonary DNA of F344 rats treated with four consecutive injections of 81 mg/kg of NNK. Statistically significant inhibition of O6-mGua formation was observed 4 h after carcinogen treatment in both pulmonary (49.1% inhibition) and hepatic (39.8%) DNA. Statistically significant inhibition of 7-mGua formation was also measured in lung DNA isolated 24 h after the last NNK injection (45.0%) and in liver DNA 4 h after carcinogen treatment (31.8%). These results suggest that p-XSC would also inhibit induction of lung adenocarcinoma in male F344 rats by NNK.

Effects of dietary fat content on the metabolism of NNK and on DNA methylation induced by NNK.

The available data support the concept that high-fat diets increase cytochrome P-450 activities in the liver, leading to increased rates of carcinogen metabolism and, in some instances, DNA adduct formation. Therefore we investigated whether a high-fat diet can also influence DNA methylation by the tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) in the lungs of rats. Male F344 rats were fed a regular AIN-76A low-fat (5% corn oil) or AIN-76A high-fat (23.5% corn oil) diet. After three weeks on this dietary regimen, the animals were injected subcutaneously once daily for four days with NNK at 0.39 mmol/kg body wt. Groups of rats were sacrificed 4 and 24 hours after the last NNK administration; livers and lungs were excised for DNA isolation. We found that the high-fat diet significantly enhanced the formation of O6-methylguanine (O6-mGua) in the rat lung four hours (p < 0.01) after the last carcinogen administration. This may, in part, account for our previous finding in regard to the enhancing effect of the high-fat diet on NNK-induced lung carcinogenesis. There was no effect on O6-mGua or 7-mGua in the rat liver at either time point. To further elucidate the enhancing effect of the high-fat diet on DNA methylation by NNK in the lung, we determined its effect on the in vitro and in vivo metabolism of NNK. The in vitro data indicated that dietary fat has no measurable effect on liver and lung microsomal mixed-function oxidase in catalyzing the metabolic activation of NNK. The results of the metabolism study of NNK in vivo appear to be consistent with the in vitro finding, in that fat had no effect on the excretion pattern of NNK or on the distribution pattern of its urinary metabolites. It is apparent that the enhancing effect of the high-fat diet on O6-mGua in the lung of rats that was measured four hours after NNK injection requires future investigations.

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.

Assessment of chlorinated pesticides and polychlorinated biphenyls in adipose breast tissue using a supercritical fluid extraction method.

A precise and highly reproducible analytical method was developed for the assessment of organochlorinated pesticide and polychlorinated biphenyl residues in adipose tissue (> or = 50 mg). The method can be utilized for epidemiological studies on the significance of these environmental pollutants in the etiology of breast cancer. Supercritical fluid extraction (SFE) with CO2 and modified CO2 (addition of 5% dichloromethane) is employed to remove incurred pesticide residues from adipose tissues that have been surgically removed from breast cancer patients and controls. An alumina sorbent, placed in the extracting vessel together with a specimen, removes the bulk of co-extracted lipids; a subsequent purification of the SFE extracts by column chromatography on alumina removes the remaining traces of lipids that would interfere with the gas chromatographic analysis with electron capture detection. The method was tested by analyzing a Certified Reference Material 430 pork fat with known amounts of pesticide residues that are commonly found in fat or in foods with a high fat content. The recoveries of analytes ranged from 73.4% for endrin to 115% for alpha-, beta- and gamma-hexachlorocyclohexane, hexachlorobenzene and dieldrin, with standard deviations of 4-12% for individual analytes. The analysis of adipose tissue for organochlorinated compounds on the basis of this new method suggested that the pesticide levels were higher in breast cancer patients than in controls. However, the small number of samples analyzed in this study (n = 5, both groups) precludes definitive conclusions. The most abundant compounds in both cases and controls were p, p-DDE (379 +/- 286 and 160 +/- 149 p.p.b.) and PCB (223 +/- 145 and 124 +/- 65.7 p.p.b.), followed by the termiticide chlordane residues oxychlordane and transnonachlor.

Toombak: a major risk factor for cancer of the oral cavity in Sudan.

BACKGROUND: Snuff dipping as practiced in North America and Western Europe is causally associated with cancer of the oral cavity and pharynx. In the Sudan, natives use local Nicotiana rustica, a tobacco species with high levels of nicotine and nornicotine, to prepare their own snuff which they call toombak. The finely ground tobacco leaves are mixed with sodium bicarbonate, water is added, and a paste is made. The resulting "saffa" is placed in the oral vestibule where it remains up to several hours. In general, a saffa is replaced 10-30 times per day. METHODS: A cross-sectional survey was initiated for establishing the extent of toombak dipping in the Northern Sudan. Using earlier published methods, toombak and the saliva of toombak dippers were analyzed for the major carcinogens, the nicotine- and nornicotine-derived tobacco-specific N-nitrosamines. RESULTS: Survey data on the use of toombak in the Nile Province in the Northern Sudan encompass 2,000 households with 5,500 adults. About 40% of the males dip toombak, including 9% who are also cigarette smokers. The toombak habit is especially prevalent (> 45%) among males ages 40 years or older. Among women, toombak use is popular only in the older age groups, where up to 10% engage in the habit, whereas cigarette smoking is uncommon (< 1.5%). So far, only one descriptive study has dealt with toombak dipping and oral cancer. Among 62 patients with oral cancer 50 were toombak users; the majority of these had tumors at the site of contact with the tobacco, or in adjacent areas. Chemical analytical studies revealed that toombak contains at least 100-fold higher concentrations of the tobacco-specific N-nitrosamines (TSNA) than US and Swedish commercial snuff brands. The TSNA are by far the most powerful and most abundant carcinogens in snuff. Their concentrations in the saliva of toombak dippers are about the same as those in a solution that had been assayed in rats by twice daily swabbing of the oral cavity and had induced tumors of the cheek and palate. CONCLUSION: The need for in-depth epidemiological studies and further laboratory research to prove that toombak chewing causes cancer is discussed. The data at hand point to the urgent need for educational programs and preventive measures against the use of toombak.

Formation of the acrolein-derived 1,N2-propanodeoxyguanosine adducts in DNA upon reaction with 3-(N-carbethoxy-N-nitrosamino)propionaldehyde.

3-(Methylnitrosamino)propionaldehyde (MNPA) is a carcinogenic nitrosamine formed by nitrosation of arecoline, a major alkaloid in areca nut which is a constituent of betel quid. While DNA adducts of its analogue, 3-(methylnitrosamino)propionitrile, have been characterized, little is known about the structures of DNA adducts by MNPA. In this paper, we report that the acrolein-derived 1,N2-propanoguanine adducts are formed upon incubating deoxyguanosine or DNA with 3-(N-carbethoxy-N-nitrosamino)propionaldehyde, a stable carbamate precursor of the metabolically activated MNPA. The identities of these adducts were confirmed by HPLC co-migration, by their NMR and UV spectra, and by chemical properties as compared with those of the synthetic standards. Analogous results were obtained from the reaction of the carbamate with calf thymus DNA. Upon acid or enzyme hydrolysis of the carbamate-modified DNA, acrolein-guanine adducts were detected, and the levels were quantitated. Again, the identities of the adducts were verified by co-chromatography with the standards, by UV spectroscopy, or by the ring-opening with NaOH/NaBH4. Consistent with its ability to modify DNA, the carbamate was found to be mutagenic in Salmonella tester strains. These results show that acrolein is a likely metabolite from the activation of MNPA and that the formation of 1,N2-propanoguanine adducts may contribute to the mutagenicity of the carbamate of MNPA.

Tobacco-specific N-nitrosamines and Areca-derived N-nitrosamines: chemistry, biochemistry, carcinogenicity, and relevance to humans.

Nicotine and the minor tobacco alkaloids give rise to tobacco-specific N-nitrosamines (TSNA) during tobacco processing and during smoking. Chemical-analytical studies led to the identification of seven TSNA in smokeless tobacco (< or = 25 micrograms/g) and in mainstream smoke of cigarettes (1.3 micrograms TSNA/cigarette). Indoor air polluted by tobacco smoke may contain up to 24 pg/L of TSNA. In mice, rats, and hamsters, three TSNA, N'-nitrosonornicotine (NNN), 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL), are powerful carcinogens; two TSNA are moderately active as carcinogens; and two TSNA appear not to be carcinogenic. The TSNA are procarcinogens, agents that require metabolic activation. The active forms of the carcinogenic TSNA react with cellular components, including DNA, and with hemoglobin (Hb). The Hb adducts in chewers and smokers serve as biomarkers for the uptake and metabolic activation of carcinogenic TSNA and the urinary excretion of NNAL as free alcohol and as glucuronide for the uptake of TSNA. The review presents evidence that strongly supports the concept that TSNA contribute to the increased risk for cancer of the upper digestive tract in tobacco chewers and for the increased risk of lung cancer, especially pulmonary adenocarcinoma, in smokers. The high incidence of cancer of the upper digestive tract especially among men on the Indian subcontinent has been causally associated with chewing of betel quid mixed with tobacco. In addition to the TSNA, the betel quid chewers are exposed to four N-nitrosamines that are formed during chewing from the Areca alkaloids, two of these N-nitrosamines are carcinogens. The article also reviews approaches toward the reduction of the carcinogenic potency of smokeless tobacco, betel quid-tobacco mixtures, and cigarette smoke. Although the safest way to reduce the risk for tobacco-related cancers is to refrain from chewing and smoking, modifications of smokeless tobacco and of cigarettes are indicated to lead to less toxic products. Another more recent approach for reducing the carcinogenic effect of tobacco products is the application of chemopreventive agents, primarily of micronutrients. Future aspects in tobacco carcinogenesis, especially as it relates to TSNA, are expected in the field of molecular biochemistry and in biomarker studies, with the goal of identifying those tobacco and betel quid chewers and tobacco smokers who are at especially high risk for cancer.

Supercritical fluid extraction in the determination of tobacco-specific N-nitrosamines in smokeless tobacco.

A new approach to the analysis of the carcinogenic, tobacco-specific N-nitrosamines (TSNA) in moist snuff tobacco is based on the extraction of tobacco with methanol-modified supercritical carbon dioxide. Extracted TSNA are trapped across a glass cartridge filled with Tenax GR, from which they are subsequently released by thermal desorption and analyzed by capillary gas chromatography with a thermal energy analyzer. The analytical recoveries for the major TSNA range from 83 to 98%; the detection limits are below 2 ng/g. The methodology is fast, reproducible, highly selective, and sensitive. The supercritical fluid extraction (SFE) releases up to 7 times more of the highly carcinogenic 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) from tobacco than has been determined after conventional solvent extraction. Studies have confirmed that this is not an artifact. In contrast, the cyclic N-nitrosamines, N'-nitrosonornicotine, N'-nitrosoanabasine, and N'-nitrosoanatabine, showed no significant quantitative differences whether determined by the SFE method or the conventional solvent extraction method.

Bioavailability of tobacco-specific N-nitrosamines to the snuff dipper.

Use of supercritical carbon dioxide extraction in the analysis of smokeless tobacco for tobacco-specific N-nitrosamines revealed the presence of higher levels of 4-(methylnitros-amino)-1-(3-pyridyl)-1-butanone (NNK) than had been determined with conventional methods. Whether human saliva may be similarly capable of releasing apparently bound NNK during chewing or snuff dipping was tested by incubating smokeless tobacco with enzymatically active saliva and with heat-treated saliva respectively. Enzymatically active human saliva was found to liberate up to twice the amount of the highly carcinogenic NNK than did heat-treated saliva. This is of major consequence for the cancer risk assessment of snuff dippers and tobacco chewers.

A study of betel quid carcinogenesis--VIII. Carcinogenicity of 3-(methylnitrosamino)propionaldehyde in F344 rats.

In assays of Areca-specific N-nitrosamines, 3-(methylnitrosamino)propionaldehyde (MNPA) exhibits higher cytotoxicity than nitrosoguvacine (NGC), nitrosoguvacoline (NG) and 3-(methylnitrosamino)propionitrile (MNPN). NGC is not mutagenic. However, NG is a weak carcinogen in F344 rats while MNPN is a potent carcinogen; MNPA had thus far not been tested. In this study MNPA was injected s.c. at a dose of 6.57 mg three times weekly for 15 weeks (total dose 2.6 mmol/rat). During the 100 weeks of the bioassay, the treated F344 rats, and especially the females, showed significantly less weight gain than the control animals, indicating high toxicity for MNPA at the tested dose. Upon termination of the bioassay, the MNPA-treated animals were found to have tumors of the lung, liver, nasal cavity, forestomach and kidneys. The control animals showed no tumors in these organs. The incidence of lung tumors in the MNPA group was statistically significant (P less than 0.025). The results of this study show that MNPA is a carcinogen in F344 rats.

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.

Cyanoethylation of DNA in vivo by 3-(methylnitrosamino)propionitrile, an Areca-derived carcinogen.

2-Cyanoethyldiazohydroxide is a likely product of metabolic alpha-hydroxylation of 3-(methylnitrosamino)propionitrile (MNPN). The reaction of 2-(N-carbethoxy-N-nitrosamino)propionitrile, a stable precursor of 2-cyanoethyldiazohydroxide, with deoxyguanosine, catalyzed by porcine liver esterase, was investigated. Two major deoxyguanosine adducts were produced. They were isolated by high-performance liquid chromatography and characterized by their UV spectra, mass spectra, and proton magnetic resonance spectra. On the basis of these spectral data, the structures of the two adducts were assigned as 7-(2-cyanoethyl)guanine and O6-(2-cyanoethyl)deoxyguanosine. The potential of MNPN to cyanoethylate DNA in F344 rats was evaluated by measuring 7-(2-cyanoethyl)guanine and O6-(2-cyanoethyl)guanine in the liver, nasal mucosa, and esophagus. The highest levels were detected in the nasal cavity, which is one of the major target organs for the carcinogenic effects of MNPN.

Induction of lung and exocrine pancreas tumors in F344 rats by tobacco-specific and Areca-derived N-nitrosamines.

The tobacco-specific N-nitrosamines 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL), as well as the Areca-derived N-nitrosoguvacoline (NG) were assayed for carcinogenicity in male F344 rats by lifetime administration in the drinking water. Groups of 30 to 80 rats were treated with 0.5 ppm, 1.0 ppm, or 5.0 ppm of NNK; 5.0 ppm of NNAL, 20 ppm of NG, a mixture of 20 ppm of NG and 1 ppm of NNK, and water only in the control group. The approximate total doses of the nitrosamines (mmol/kg of body weight) in these groups were: NNK, 0.073, 0.17, and 0.68; NNAL, 0.69; NG, 4.1; NG and NNK, 4.1 and 0.17. As in previous assays in which NNK was tested by s.c. injection, the lung was its principle target organ. Lung tumor incidences in the 0.5-, 1.0-, and 5.0-ppm groups were nine of 80, 20 of 80, and 27 of 30 compared to six of 80 in the control rats. This trend was significant, P less than 0.005. Significant incidences of nasal cavity and liver tumors were observed only in the rats treated with 5.0 ppm of NNK. In contrast to the results of the s.c. bioassays of NNK, tumors of the exocrine pancreas were observed in five of 80 and nine of 80 rats treated with 0.5 and 1.0 ppm. This trend was significant, P less than 0.025. This is the first example of pancreatic tumor induction by a constituent of tobacco smoke. It is also the first finding of duct-like carcinomas in the rat pancreas, including one tumor containing epidermoid, keratin-generating tissue. NNAL, the major metabolite of NNK, induced lung tumors in 26 of 30 rats and pancreatic tumors in eight of 30 rats. It appears to be the proximate pancreatic carcinogen of NNK. NG induced pancreatic tumors in four of 30 rats, P less than 0.05. This finding requires confirmation. The mixture of NG and NNK induced lung tumors in eleven of 30 rats. There were no apparent synergistic interactions of NG and NNK. The observation of benign and malignant tumors of the lung and pancreas of rats treated with the tobacco-specific nitrosamines NNK and NNAL is discussed in respect to the causal association between cigarette smoking and cancer of the lung and pancreas.