DNA adducts of ortho-toluidine in human bladder.

BACKGROUND: 4-Aminobiphenyl (4-ABP) and o-toluidine are known human bladder carcinogens, but only 4-ABP-releasing DNA adducts are known. METHODS: Determination of 4-ABP and o-toluidine-releasing DNA adducts in epithelial and submucosal bladder tissues of sudden death victims (SDV: n=46), and bladder tumours (n=12) by gas chromatography/mass spectrometry. RESULTS: Above background, 4 and 11 of 12 tumour samples contained adducts of 4-ABP (0.057 ± 0.125 fmol/µg DNA) and o-toluidine (8.72 ± 4.49 fmol/µg DNA), respectively. Lower adduct levels were present in both epithelial and submucosal bladder tissues of SDV (4-ABP: 0.011 ± 0.022 and 0.019 ± 0.047 fmol/µg DNA; o-toluidine: 0.24 ± 0.63 and 0.27 ± 0.70 fmol/µg DNA). CONCLUSION: Detection of o-toluidine-releasing DNA adducts support the carcinogenicity of o-toluidine in the human bladder.

Biotransformation of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone in lung tissue from mouse, rat, hamster, and man.

Exposure to the tobacco-specific N-nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is considered to be an important etiological risk factor for lung cancer in tobacco users. The metabolism of NNK via carbonyl reduction to 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL), alpha-hydroxylation to form both DNA methylating and pyridyloxobutylating intermediates, and detoxification by pyridyl N-oxidation and glucuronide formation are well-characterized in laboratory animals but less so in man. The in vitro kinetics of 0.03-250 microM [5-(3)H]NNK metabolism were determined under identical experimental conditions using female A/J mouse, male Fischer 344 rat, female Syrian golden hamster, and human lung tissue explants in tissue culture. The concentration-dependent percentage contribution of the three major pathways of NNK metabolism (carbonyl reduction, alpha-hydroxylation, and N-oxidation) showed large interspecies variation. Quantitatively, in mouse, carbonyl reduction to NNAL increased steadily with an increasing substrate concentration (10-74% total NNK metabolism), while concurrent decreases occurred in end products of alpha-hydroxylation (60 to 18%) and N-oxidation (42 to 5%). In rat lung, there were no apparent concentration-dependent trends (NNAL, 42 +/- 4%; alpha-hydroxylation, 35 +/- 2%; and N-oxidation, 24 +/- 3%). In hamster lung, a clear concentration-dependent increase in the contribution of NNAL to total NNK metabolism (from 47 to 87%) was paralleled by a steady decline in end products of alpha-hydroxylation (31 to 11%) and N-oxidation (22 to 2%). Human lung metabolism showed no concentration-dependent tendencies (NNAL, 89 +/- 1%; alpha-hydroxylation, 8.8 +/- 1.1%; and N-oxidation, 2.1 +/- 0.3%). The major alpha-hydroxylation product in human lung was 4-hydroxy-1-(3-pyridyl)-1-butanone (keto alcohol), thus supporting the potential pyridyloxobutylation of lung DNA. Metabolism to 4-(3-pyridyl)-4-oxobutanoic acid (keto acid), which could result in lung DNA methylation, was only sporadically seen in human lung but present to a far greater extent in rodent lung. No evidence for glucuronidation was found in any species. Generally, the rate of formation of all NNK metabolites showed two different enzyme kinetics, resulting in large differences between apparent K(m) and V(max) values in the low (up to 2.8 microM) and high substrate concentration ranges. The metabolism of NNK by alpha-hydroxylation is considerably lower in human lung as compared to that observed in rodent species, suggesting that extrapolation of in vitro rodent data to man may result in invalid conclusions about the capacity of the human lung to activate NNK under realistic conditions of NNK exposure expected to occur in man.

Analysis of myosmine, cotinine and nicotine in human toenail, plasma and saliva.

Myosmine is a minor tobacco alkaloid with widespread occurrence in the human diet. Myosmine is genotoxic in human cells and is readily nitrosated and peroxidated yielding reactive intermediates with carcinogenic potential. For biomonitoring of short-term and long-term exposure, analytical methods were established for determination of myosmine together with nicotine and cotinine in plasma, saliva and toenail by gas chromatography-mass spectrometry (GC/MS). Validation of the method with samples of 14 smokers and 10 non-smokers showed smoking-dependent differences of myosmine in toenails (66 +/- 56 vs 21 +/- 15 ng g(-1), p <0.01) as well as saliva (2.54 +/- 2.68 vs 0.73 +/- 0.65 ng ml(-1), p <0.01). However, these differences were much smaller than those with nicotine (1971 +/- 818 vs 132 +/- 82 ng g(-1), p <0.0001) and cotinine (1237 +/- 818 vs <35 ng g(-1)) in toenail and those of cotinine (97.43 +/- 84.54 vs 1.85 +/- 4.50 ng ml(-1), p <0.0001) in saliva. These results were confirmed in plasma samples from 84 patients undergoing gastro-oesophageal endoscopy. Differences between 25 smokers and 59 non-smokers are again much lower for myosmine (0.30 +/- 0.35 vs 0.16 +/- 0.18 ng ml(-1), p <0.05) than for cotinine (54.67 +/- 29.63 vs 0.61 +/- 1.82 ng ml(-1), p <0.0001). In conclusion, sources other than tobacco contribute considerably to the human body burden of myosmine.

Ultrasensitive method for the determination of 4-hydroxy-1-(3-pyridyl)-1-butanone-releasing DNA adducts by gas chromatography-high resolution mass spectrometry in mucosal biopsies of the lower esophagus.

4-Hydroxy-1-(3-pyridyl)-1-butanone (HPB)-releasing DNA adducts are formed by metabolic activation of the tobacco-specific nitrosamines 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and N'-nitrosonornicotine (NNN). NNK and NNN are considered carcinogenic to humans by the International Agency for Research on Cancer. Existing analytical methods for determination of HPB-releasing DNA adducts require 0.3-2.0 g of human target tissues such as lung and esophagus. For adduct determination in milligram amounts of biopsy samples, an ultrasensitive and specific method is presented using capillary gas chromatography coupled to a high-resolution mass spectrometer operated in the negative chemical ionization mode (GC-NCI-HRMS). The method has a limit of detection of 4.6 fmol HPB, a limit of quantification of 14.9 fmol HBP and a recovery of 45 +/- 15%. Intra- and inter-day imprecision for N = 6 samples were calculated with coefficients of variation of <3.1%. Method applicability was evaluated with biopsies of esophageal mucosa (N = 14) yielding 5.6 +/- 1.9 mg tissue and a mean adduct level of 6.13 +/- 9.35 pmol HPB/mg DNA.

Nicotine induces DNA damage in human salivary glands.

The tobacco alkaloid nicotine is responsible for addiction to tobacco and supposed to contribute to tobacco carcinogensis, too. Recently, genotoxic effects of nicotine have been reported in human cells from blood and upper aerodigestive tract. Because of nicotine accumulation in saliva, the study of possible in vitro genotoxic effects of nicotine have been extended to human salivary gland cells. Specimens of parotid glands of 10 tumor patients were obtained from tumor-free tissue. Single cells were prepared by enzymatic digestion immediately after surgery and exposed for 1h to 0.125-4.0mM of nicotine. Possible genotoxic effects were determined by the Comet assay using the % DNA in tail (DT) as a reliable indicator of DNA damage. Nicotine induced a significant dose-dependent increase of DNA migration in parotid gland single-cells. The mean DT was 1.12-fold (0.125mM) to 2.24-fold (4.0mM) higher compared to control. The lowest concentration eliciting significant DNA damage within 1h, 0.25mM nicotine, is only 10-fold higher than maximal concentrations of nicotine reported in saliva after unrestricted smoking. Although conclusive evidence for a carcinogenic potential of nicotine is still lacking, the safety of long-term nicotine replacement therapy should be carefully monitored.

4-Hydroxy-1-(3-pyridyl)-1-butanone-releasing DNA adducts in lung, lower esophagus and cardia of sudden death victims.

4-Hydroxy-l-(3-pyridyl)-l-butanone (HPB)-releasing adducts are formed by metabolic activation of N'-nitrosonornicotine and 4-(methylnitrosamino)-l-(3-pyridyl)-l-butanone and have been proposed as specific biomarkers for exposure to tobacco smoke. However, in several studies hemoglobin adducts releasing HPB were on average less than threefold higher in smokers compared to nonsmokers. Using an improved analytical method we have recently found a sevenfold difference in DNA adduct levels in the lung from smoking and nonsmoking lung cancer patients. In the present study we extended the determination of HPB-releasing DNA adducts by gas chromatography-negative ion chemical ionization-mass spectrometry (GC-NICI-MS) to samples of peripheral lung, lower esophagus and cardia from tumor-free sudden death victims (primarily road traffic accidents, suicide and sudden cardiac arrest). The donors were classified as either current smokers or nonsmokers based on cotinine in either blood or urine (cut-off values for active smoking: >15 ng cotinine/ml blood or >100 ng cotinine/ml urine). Contrary to our expectation, DNA adduct levels (fmol HPB/mg DNA) in lung tissue from tumor-free smokers (N=32, 92+/-148) were not significantly different from values in nonsmokers (N=56, 61+/-66). The values in tumor-free smokers were on average more than fourfold lower compared to smoking lung cancer patients in our previous study. Adduct levels in the mucosa of esophagus (N=82; 133+/-160) and cardia (N=30; 108+/-102) of sudden death victims did not show any difference according to the current smoking status. HPB-releasing DNA adduct levels in cardia and esophagus were significantly correlated (N=29; Spearman r=0.609; p<0.001). In contrast, adduct levels in lung did not correlate with either esophagus (77 cases) or cardia (28 cases). Further studies are necessary to elucidate the discrepancies in lung DNA adduct levels in smokers with or without lung cancer and to identify obvious additional sources other than tobacco for these adducts.

Mass spectrometric analysis of 4-hydroxy-1-(3-pyridyl)-1-butanone-releasing DNA adducts in human lung.

An improved analytical method was developed for the analysis of 4-hydroxy-1-(3-pyridyl)-1-butanone (HPB)-releasing DNA adducts in lung samples of patients undergoing surgery for lung cancer. HPB-releasing adducts can be formed by metabolic activation of the tobacco-specific nitrosamines 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone and N'-nitrosonornicotine, and have been reported to play an important role in tobacco carcinogenesis. [2,2,3,3-D(4)]HPB (D(4)-HPB) was used as an internal standard, and HPB released by acid hydrolysis of DNA was determined by gas chromatography/mass spectrometry in the negative ion chemical ionisation mode. The method is sensitive with a limit of detection of 5.9 fmol HPB and a limit of quantification of 15.2 fmol HBP/mg DNA. The recovery of HPB was 82+/-17% and the background response was 10.1+/-1.8 fmol HPB/sample. The concentration of HPB-releasing lung DNA adducts was significantly higher (p<0.0001) in 21 self-reported smokers compared to in 11 self-reported nonsmokers (404+/-258 fmol versus 59+/-56 fmol HPB/mg DNA, respectively). HPB-releasing hemoglobin adduct concentrations were only marginally higher in a subset of 12 smokers compared to in 7 nonsmokers (63+/-53 fmol versus 42+/-34 fmol HPB/g hemoglobin; p=0.36). No correlation was found between HPB-releasing adducts in DNA and hemoglobin (p=0.074).

Hemoglobin adducts of the human bladder carcinogen o-toluidine after treatment with the local anesthetic prilocaine.

Prilocaine, a widely used local anesthetic, is metabolized to o-toluidine which is classified as human carcinogen. We aimed to assess the impact of prilocaine-treatment on hemoglobin adducts from o-toluidine. Blood samples were obtained before and 24h after receiving prilocaine local anesthesia (Xylonest, 100mg) from 20 head and neck surgery patients and 6 healthy volunteers. Hemoglobin adducts of o-toluidine and 4-aminobiphenyl were determined by gas chromatography/mass spectrometry. Hemoglobin adducts of o-toluidine were significantly increased 24h after 100mg prilocaine-treatment by 21.6+/-12.8ng/g hemoglobin (mean+/-S.D., N=26; P<0.0001). This corresponds to a 6-360-fold increase of o-toluidine adduct levels in 25 patients from 0.54+/-0.95ng/g before treatment to 22.0+/-13.2ng/g 24h after surgery (mean+/-S.D.). Because of an extremely high background level the increase was only 1.6-fold in one patient (40.9ng/g before and 64.4ng/g 24h after prilocaine injection). Current smoking had no influence on background values and on the increase of o-toluidine adducts. No treatment-related differences were seen in mean hemoglobin adduct levels of 4-aminobiphenyl which were significantly higher in smokers, 0.149+/-0.096ng/g (mean+/-S.D., N=8) as compared to nonsmokers 0.036+/-0.035ng/g (mean+/-S.D., N=16; P<0.01). In conclusion, prilocaine anesthesia leads to a massive increase of hemoglobin adducts of the carcinogenic arylamine o-toluidine. This implies a carcinogenic risk which should be taken into account in preventive hazard minimization.

Genotoxic effects of myosmine in a human esophageal adenocarcinoma cell line.

The incidence of esophageal adenocarcinoma is rapidly rising in Western populations. Gastroesophageal reflux disease (GERD) is thought to be one of the most important risk factors. However, the mechanisms by which GERD enhances tumor formation at the gastroesophageal junction are not well understood. Myosmine is a tobacco alkaloid which has also a wide spread occurrence in human diet. It is readily activated by nitrosation and peroxidation giving rise to the same hydroxypyridylbutanone-releasing DNA adducts as the esophageal carcinogen N'-nitrosonornicotine. Therefore, the genotoxicity of myosmine was tested in a human esophageal adenocarcinoma cell line (OE33). DNA damage was assessed by single-cell gel electrophoresis (Comet assay). DNA strand breaks, alkali labile sites and incomplete excision repair were expressed using the Olive tail moment (OTM). The Fapy glycosylase (Fpg) enzyme was incorporated into the assay to reveal additional oxidative DNA damage. DNA migration was determined after incubation of the cells for 1-24h. Under neutral conditions high myosmine concentrations of 25-50mM were necessary to elicit a weak genotoxic effect. At pH 6 genotoxicity was clearly enhanced giving a significant increase of OTM values at 5mM myosmine. Lower pH values could not be tested because of massive cytotoxicity even in the absence of myosmine. Co-incubation of 25 mM myosmine with 1mM H(2)O(2) for 1h significantly enhanced the genotoxicity of H(2)O(2) but not the oxidative lesions additionally detected with the Fpg enzyme. In the presence of the peroxynitrite donor 3-morpholinosydnonimine (SIN-1) a dose-dependent significant genotoxic effect was obtained with 1-10mM myosmine after 4h incubation. NS-398, a selective inhibitor of cyclooxygenase 2, did not affect the SIN-1 stimulated genotoxicity of myosmine. Finally, the 23 h repair of N-methyl-N'-nitro-N-nitrosoguanidine-induced DNA lesions was significantly inhibited in the presence of 10mM myosmine. In conclusion, myosmine exerts significant genotoxic effects in esophageal cells under conditions which may prevail in GERD such as increased oxidative and nitrosative stress resulting from chronic inflammation.

The tobacco alkaloid nicotine demonstrates genotoxicity in human tonsillar tissue and lymphocytes.

Recent studies suggest a direct contribution of nicotine, the addictive component of tobacco and tobacco smoke, to human carcinogenesis. To assess the genotoxicity of nicotine, the DNA-damaging effect on human lymphocytes and target cells from lymphatic tissue of the palatine tonsils from 10 healthy patients was tested with the alkaline single-cell microgel electrophoresis (Comet) assay. The degree of DNA migration, a measure of possible DNA single strand breaks, alkali labile sites, and incomplete excision repair sites, was expressed as the Olive tail moment, the percentage of DNA in the tail, and the tail length. One hour exposure to nicotine at 0.125, 0.25, 0.5, 1, 2, and 4 mM induced a statistically significant dose-dependent increase of DNA migration up to 3.8-fold and 3.2-fold in tonsillar cells and lymphocytes, respectively. The lowest concentration eliciting significant DNA damage was 0.5 mM nicotine. The genotoxic effect was confirmed in a second series of experiments using nicotine of high purity from two different suppliers. There were no significant differences between the two series, excluding artifacts from the source of nicotine. Finally, DNA damage by nicotine was compared in cells incubated in medium strictly adjusted to neutral pH, with non-adjusted medium becoming alkaline with increasing nicotine concentrations. Again no differences in DNA migration were observed. The data indicate that nicotine expresses significant direct genotoxic effects in human target cells in vitro. However, no differences in DNA damage were observed in cells from smokers and nonsmokers incubated without nicotine. The lack of higher DNA damage in smokers compared to nonsmokers could be a question of nicotine dose, rapid DNA repair, or interactions with other smoke constituents. These results require further investigations on the contribution of nicotine to tobacco carcinogenesis.

Genotoxicity of nicotine in mini-organ cultures of human upper aerodigestive tract epithelia.

The direct role of nicotine in tobacco carcinogenesis is still controversial. Recently, DNA damage by nicotine has been demonstrated in isolated human tonsillar tissue cells. Presently, these effects were investigated using mini-organ cultures (MOC) of human nasal epithelia. Intact MOC were repeatedly exposed to 2 and 4 mM nicotine for 1 h on culture days 7, 9, and 11. N-Methyl-N'-nitro-N-nitrosoguanidine (MNNG) served as a positive control. DNA damage was examined by Comet assay either directly after exposure or following a 24-h recovery period. Cell viability was not reduced by any treatment. On day 7, 1 h exposure to 2 and 4 mM nicotine caused a significant dose-dependent 3.3- and 5.6-fold increase in DNA damage compared to solvent controls. Although there was no evidence of significant repair within 24 h recovery, DNA damage was not further increased by nicotine on days 9 and 11. After double and triple exposure to 4 mM nicotine a significant reduction in DNA damage following 24 h recovery was observed. In contrast, treatment with MNNG resulted in a highly significant and cumulative increase in DNA migration up to 110-fold compared to controls. During recovery periods, MNNG-induced DNA damage was significantly repaired, leading to a 1.5- to 1.8-fold reduction in DNA migration within 24 h. These results confirm genotoxic effects of nicotine on human nasal epithelia. Further studies are needed to explain the lack of cumulative DNA-damaging effects of nicotine and the absence of significant DNA repair. These studies should include a battery of assays with multiple end points.

Acute and subacute effects of tobacco alkaloids, tobacco-specific nitrosamines and phenethyl isothiocyanate on N'-nitrosonornicotine metabolism in rats.

N'-Nitrosonornicotine (NNN) was the first tobacco-specific nitrosamine (TSNA) identified as carcinogen in tobacco smoke, but no data exist on in vivo interactions between NNN and other tobacco alkaloids, TSNA or phenethyl isothiocyanate (PEITC) which have been demonstrated in various studies on 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK). Acute effects on NNN metabolism were tested in male Fischer F344 rats injected s.c. with 30nmol/kg body weight (bw) [5-(3)H]NNN either alone or simultaneously with 15mumol/kg bw nicotine, nornicotine, anatabine, or anabasine, 150mumol/kg bw cotinine, 3mumol/kg bw myosmine, or 300nmol/kg bw of either N'-nitrosoanatabine or N'-nitrosoanabasine. Another group of rats was fed a diet supplemented with PEITC at 1mumol/g diet starting 24h before NNN treatment. Within 24h more than 80% and about 10% of the radioactivity was excreted with urine and feces, respectively. Urinary metabolites were separated by reversed-phase radio-HPLC and identified by co-chromatography with UV standards. In two sets of experiments with control rats treated with NNN only, 4-hydroxy-4-(3-pyridyl)butanoic acid (hydroxy acid, 44.4/44.8%), 4-oxo-4-(3-pyridyl)butanoic acid (keto acid, 32.4/31.5%), NNN-N-oxide (5.0/3.8%), 4-(3-pyridyl)butane-1,4-diol (diol, 1.1/1.0%) and norcotinine (2.3/1.0%) were consistently detected besides unmetabolised NNN (4.7/3.3%). Co-treatment with nicotine, cotinine, nornicotine and PEITC shifted the contribution of the two major metabolites significantly in favor of hydroxy acid (108-113% of control) as compared to keto acid (86-90% of control). The same treatments also increased norcotinine (135-170% of control). These changes are consistent with a decreased metabolic activation of NNN. In subacute studies rats received NNN in drinking water for 4 weeks at a daily dose of 30 nmol/kg bw with or without nornicotine at 15 micromol/kg bw or myosmine at 3 micromol/kg bw. On the last day of the experiment all rats received [5-(3)H]NNN at 30 nmol/kg bw with a contaminated apple bite followed by collection of urine and feces for 18h. Most of the radioactivity, 87-96% of the dose, was recovered in urine and only minor amounts have been excreted in feces or persisted in blood. In urine of the NNN-control group keto acid (32.2%) and unmetabolised NNN (3.9%) were present in identical amounts as in the acute experiment whereas hydroxy acid (41.4% of total radioactivity in urine, 93% of acute NNN control) was reduced in expense of the minor NNN metabolites. Co-administration of nornicotine resulted in a small but significant rise of keto acid (107% of control) and a significant decrease in NNN-N-oxide (76% of control). After co-treatment with myosmine the increase of keto acid (104% of control) was even less but still significant whereas NNN-N-oxide and diol were significantly reduced to 72% and 79% of control, respectively. Our experiments with rats indicate significant mutual effects of some of the major tobacco alkaloids and most relevant TSNA. Further studies on the impact on smokers and the inhibitory effects of isothiocyanates are needed for a final risk assessment.

Biomonitoring of human exposure to arylamines.

Extensive industrial use of arylamines started in the middle of the 19th century in the dye industry. Because of the high incidence of bladder cancer, arylamines belong to the first and most intensively studied occupational and environmental carcinogens. In workers, biomonitoring of exposure to arylamines including ortho-toluidine started in the first half of the 20th century. This review highlights the many gaps in our knowledge on the human carcinogen ortho-toluidine.

Effect of nicotine, cotinine and phenethyl isothiocyanate on 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) metabolism in the Syrian golden hamster.

The effect of nicotine, cotinine and phenethyl isothiocyanate (PEITC) on metabolism of the tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) was studied in the Syrian golden hamster. Urinary metabolite profiles were determined in 24 h urine after a single subcutaneous (s.c.) administration of [5-(3)H]NNK (80 nmol/kg, s.c.). Co-administration of either a 500-fold higher dose of nicotine (40 micromol/kg, s.c.) or a 5000-fold higher dose of cotinine (400 micromol/kg, s.c.) significantly (P<0.001) reduced metabolic activation of NNK by alpha-hydroxylation to 85 and 71% of control, respectively. Co-administration of a 300-fold higher dose of PEITC (1 micromol/g diet) slightly reduced alpha-hydroxylation of NNK (94% of control). Metabolism of NNK by reduction to 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) was increased by nicotine (155%), and significantly increased by cotinine (670%, P<0.001) and PEITC (219%, P<0.01). Detoxification of NNAL by glucuronidation was also increased by all three test agents. Detoxification of NNK and NNAL by N-oxidation was marginally increased by nicotine, reduced by PEITC, and significantly reduced by cotinine. The urinary metabolite profiles suggest that nicotine, which occurs in concentrations up to 30000-fold higher than NNK in mainstream cigarette smoke, and cotinine, its proximal metabolite, may have a significant protective effect against in vivo metabolic activation of NNK.

Genotoxic effects of myosmine in human lymphocytes and upper aerodigestive tract epithelial cells.

Myosmine, 3-(1-pyrroline-2-yl)pyridine, is an alkaloid found in tobacco plants. Recently, it was also detected in various edibles and staple foods. Whereas other tobacco alkaloids such as nicotine and nornicotine and their nitrosation products, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and N'-nitrosonornicotine (NNN), have been widely discussed, the mutagenic impact of myosmine has not been investigated in detail. In the present study, possible genotoxic effects of myosmine were studied in human lymphocytes and nasal mucosal cells using the alkaline single cell microgel electrophoresis (Comet) assay. DNA single strand breaks, alkali labile sites and incomplete excision repair sites were expressed using the Olive tail moment (OTM). One hour incubation with myosmine at 0, 5, 10, 25 and 50 mM induced a low but significantly dose-dependent increase of DNA migration from 1.29 +/- 0.13 to 18.25 +/- 1.59 (OTM, mean +/- S.E., N=11) in lymphocytes. In nasal mucosal cells a similar although somewhat less extensive DNA damage from 1.17 +/- 0.12 to 21.67 +/- 2.97 (OTM, mean +/- S.E., N=10-11) was obtained after 1 h incubation with myosmine at 0, 10, 25, 50 and 100 mM. After prolonged incubation of human lymphocytes with 10mM myosmine for 1, 3, 6, and 24 h, a significant time-dependent increase of DNA migration from 3.45 +/- 0.43 to 57.77 +/- 8.24 (OTM, mean +/- S.E., N=4) was observed. Our data indicate that myosmine expresses significant genotoxic effects in human target cells of carcinogenesis. This result warrants further investigations on the impact of this dietary component on human health.

Biomonitoring of exposure to aromatic amines: haemoglobin adducts in humans.

Haemoglobin (Hb) adducts from aromatic amines (AAs) are well established biomarkers of exposure. Tobacco smoking and occupational exposure are major sources of AA Hb adducts. The origin of background levels in non-smokers and non-occupationally exposed humans are largely unknown. Here we examine the determination of AA Hb adducts, focussing on the analytical strategies for Hb isolation, removal of unbound AAs from Hb solutions, hydrolysis of the Hb bound AAs, extraction, preconcentration, clean-up and derivatisation of the free amines for determination by gas chromatography-mass spectrometry. Finally, a detailed summary of available results on the determination of AA Hb adducts is given.

New sources of dietary myosmine uptake from cereals, fruits, vegetables, and milk.

Myosmine has been regarded as a specific tobacco alkaloid until investigations pointed out that nuts and nut products constitute a significant source of myosmine. In the present study it is shown that the occurrence of myosmine is widespread throughout a large number of plant families. Using a method for extraction practicable for all examined foods, quantitative analysis through internal standard addition showed nanograms per gram amounts. Positively tested edibles were staple foods such as maize, rice, wheat flour, millet, potato, and milk and also cocoa, popcorn, tomato, carrot, pineapple, kiwi, and apples. No myosmine was detectable in other vegetables and fruits such as lettuce, spinach, cucumber, onion, banana, tangerines, and grapes. Myosmine is easily nitrosated giving rise to a DNA adduct identical to the esophageal tobacco carcinogen N-nitrosonornicotine. Therefore, the role of dietary myosmine in esophageal adenocarcinoma should be further investigated.

Determination of caffeine, myosmine, and nicotine in chocolate by headspace solid-phase microextraction coupled with gas chromatography-tandem mass spectrometry.

The occurrence of the bioactive components caffeine (xanthine alkaloid), myosmine and nicotine (pyridine alkaloids) in different edibles and plants is well known, but the content of myosmine and nicotine is still ambiguous in milk/dark chocolate. Therefore, a sensitive method for determination of these components was established, a simple separation of the dissolved analytes from the matrix, followed by headspace solid-phase microextraction coupled with gas chromatography-tandem mass spectrometry (HS-SPME-GC-MS/MS). This is the first approach for simultaneous determination of caffeine, myosmine, and nicotine with a convenient SPME technique. Calibration curves were linear for the xanthine alkaloid (250 to 3000 mg/kg) and the pyridine alkaloids (0.000125 to 0.003000 mg/kg). Residuals of the calibration curves were lower than 15%, hence the limits of detection were set as the lowest points of the calibration curves. The limits of detection calculated from linearity data were for caffeine 216 mg/kg, for myosmine 0.000110 mg/kg, and for nicotine 0.000120 mg/kg. Thirty samples of 5 chocolate brands with varying cocoa contents (30% to 99%) were analyzed in triplicate. Caffeine and nicotine were detected in all samples of chocolate, whereas myosmine was not present in any sample. The caffeine content ranged from 420 to 2780 mg/kg (relative standard deviation 0.1 to 11.5%) and nicotine from 0.000230 to 0.001590 mg/kg (RSD 2.0 to 22.1%).

Nicotine causes genotoxic damage but is not metabolized during long-term exposure of human nasal miniorgan cultures.

Human nasal miniorgan cultures (MOC) are a useful tool in ecogenotoxicology. Repetitive exposure to nicotine showed reversible DNA damage, and stable CYP2A6 expression was demonstrated in nasal MOC in previous investigations. The aim of the present study was to evaluate the genotoxic effect of nicotine in nasal MOC after chronic nicotine exposure, and to monitor possible metabolism capacities. MOC were dissected from human nasal mucosa and cultured under standard cell culture conditions. MOC were exposed to nicotine for 3 weeks at concentrations of 1 µM and 1 mM. The concentrations were chosen based on nicotine plasma levels in heavy smokers, and possible concentrations used in topical application of nicotine nasal spray. DNA damage was assessed by the comet assay at days 7, 14 and 21. Concentrations of nicotine and cotinine were analyzed in cell culture medium by gas chromatography/mass spectrometry to determine a possible metabolism of nicotine by MOC. Distinct DNA damage in MOC could be demonstrated after 1 week of exposure to 1 µM and 1 mM nicotine. This effect decreased after 2 and 3 weeks with no statistically relevant DNA migration. No nicotine metabolism could be detected by changes in nicotine and cotinine concentrations in the supernatants. This is the first time genotoxic effects have been evaluated in nasal MOC after chronic nicotine exposure for up to 3 weeks. Genotoxic effects were present after 1 week of culture with a decrease over time. Down-regulation of nicotinic acetylcholine receptors, which are expressed in nasal mucosa, may be a possible explanation. The lack of nicotine metabolism in this model could be explained by the functional loss of CYP2A6 during chronic nicotine exposure. Further investigations are necessary to provide a more detailed description of the underlying mechanisms involved in DNA damage by nicotine.