Further Information

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Metabolites of a Tobacco-Specific Lung Carcinogen in Nonsmoking Casino Patrons - Let us debunk this right here and now | Kristin E. Anderson,1,2 Jen Kliris,1 Lois Murphy,Steven G. Carmella, Shaomei Han, Carrie Link,Robin L. Bliss, Susan Puumala, Sharon E. Murphy, Stephen S. Hecht
Article Published: 2004

Type: Articles and Dissertations
Published By: Cancer Epidemiol Biomarkers Prev. 2003 Dec;12(12):1544-6

Further Information

On January 5, 2004 the ink was not yet dry on the latest misleading piece on passive smoke, and the mass media  already rushed to spread the disinformation all over the world. This was the latest “study” by the University of Minnesota on passive smoking. Just as an example this is how ABC network reported it:

“Researchers at the University of Minnesota, Minneapolis, found elevated levels of a cancer-causing agent, NNAL, in the urine of nonsmokers after they spent just four hours in a commercial casino. Researchers also found elevated levels of cotinine, a byproduct of nicotine, in the samples. Both NNAL and cotinine are specific to tobacco and were not found in the nonsmokers' urine before their casino visit.” See the stored copy of the piece here.

This is not how things were, and the following analysis demonstrates how the mass media and the University of Minnesota misrepresented the “dangers” of passive smoking once again.

Analysis - Nitrosamines

Firstly, no one can state that nicotine-derived nitrosamines (NNK, NNAL-Gloc, NNAL-N-oxide) cause cancer in humans, and specifically lung cancer in smokers. Nicotine-derived nitrosamines have caused cancer in rats and mice at much higher doses than could be possibly experienced by humans. Still, according to the WHO’s  International Agency for Research on Cancer and the US National Toxicology Program, there is no data proving that said nitrosamines can cause cancer in humans. However, an evident proof that those nitrosamines do not have a demonstrable effect in humans is that their concentration in those who chew “snuff” is greater than in those who smoke, while there is no increase of lung cancer in snuff chewers. Indeed those chewers may have an elevated chance of cancer of the mouth and pharynx (when compared to non smokers) – but less of a chance than those who smoke. Steven Hecht, whose name appears in the Anderson et al. article, is at the head of a group that, for years, has cashed huge sums from the nitrosamines issue, presenting it as a scarecrow on the basis of the pure conjecture that tobacco-derived nitrosamines may cause cancer in humans.

Indeed, the work of Anderson et al. is certainly no novelty in spite of the media propaganda, because the same academic group had previously published three papers or more on non smokers exposed to passive smoke. (2, 4, 6)

That tobacco derived nitrosamines can indeed be found in non-smokers exposed to passive smoke is obvious, and it cannot certainly be passed as an extraordinary event. But the key question is: at what dosage? And here is where the perception fraud kicks in. May the lay reader forgive the two short following paragraphs of technicalities, but they will suit those who are trained in such matters.

Anderson at al. have found the nicotine-derived nitrosamine NNAL at a rate of 0.018 pM/mg of creatinine (picomoles per mg of creatinine) in the 24-hour urine after exposure to passive smoke, which represents the total NNAL dose from passive smoke. The average urine volume of 24 hrs is about 1,000 ml, and the normal creatinine concentration is about 1 mg/100 ml of urine. It follows that the total average dose of NNAL from the subjects exposed to passive smoke gravitates around 0.18 pM for an average 70-Kg individual.

Another study from the Hecht group (Upadhyaya P, Kenney PM, Hochalter JB, Wang M, Hecht SS Tumorigenicity and metabolism of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol enantiomers and metabolites in the A/J mouse. (Carcinogenesis. 1999 Aug;20(8):1577-82) tells that lung cancer in A/J mice is obtained with a single intraperitoneal injection of 20 µM (microMoles) of NNAL. Since a mouse weighs an average of 20 grams, the equivalent dosage for a 70-Kg man would be 3,500 µM of NNAL.

The cascade of values is  1µM = 1,000 nM (nanoMoles) = 1,000,000 pM, thus:

3,500 µM = 3,500,000,000 pM to cause a hypothetical lung cancer.

The Anderson et al. study gives a dose of  0.18 pM per individual human, which means around a twenty billion times dosage difference between the mouse and the human! One could object that mice are exposed once, while people are exposed repeatedly, but even this objection does not stand a most elementary consideration. There are, in fact, about 25,000 days in the average 70-year life, and let’s say that a person is exposed to passive smoke for 20,000 days of his life. Well, even in that case, in order to “get” a dose equivalent to that of the mouse, the exposed person should live for about one million years. No matter how we turn the terms of this confounded argument in an attempt to justify it, the claims of the paper’s authors are absolutely unverifiable. They wacky comparisons only underline the absurdity of this (and this kind of) study, and of the conclusions reached by the authors.

Analysis - Cotinine

The second issue is cotinine, which is a by-product of nicotine that turns out to be good for memory improvement and for the protection of brain cells.  The presence of cotinine in urine, therefore, is nothing negative and simply indicates exposure to passive smoke (our nostrils can sense that without diverting big dollars into the pockets of professional scaremongers), but the exposure in question is to a substance (passive smoke) for which there is no scientific evidence whatsoever of harm to human health. In fact, there is not even the demonstrated existence of a risk for human health, as the maximum risk elevation ever concocted with artificial boosting of statistical data is 20%, while epidemiology itself demands a 200-300% statistical risk elevation just to begin thinking that a risk may exist. Even if the accepted epidemiological threshold were reached, risk does not mean something that has happened or will happen: each time we go down the stairs there is the risk that we will stumble, roll down and break our necks. That simply means that the accident is possible, although quite improbable – and at any rate the existence of the risk does not suggest the need to ban stairs.


It is preposterous that those “scientists” who promote "studies" such as this are not exposed for what they really are. Instead, they pass for “scholars” dedicated to saving humanity, and they get big dollars and media credence. The devastating part is that this incredible distortion is not an isolated case, but today it is almost the standard used for the most disparate issues, from pesticides, to plastic toys, to passive smoke, to food.

In past times of greater ethical and moral rectitude, “studies” like this would have been punished with ridicule, and those reporting it as serious stuff would lose their credibility at once. In fact, it would not finish there: investigations would be started and the guilty would be punished.



1 - Cancer Epidemiol Biomarkers Prev. 2003 Dec;12(12):1544-6

Metabolites of a tobacco-specific lung carcinogen in nonsmoking casino patrons (see header)

2 -
Cancer Epidemiol Biomarkers Prev. 2003 Nov;12(11 Pt 1):1257-61
Analysis of total 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) in human urine (click on the link to download study)

Carmella SG, Han S, Fristad A, Yang Y, Hecht SS.

University of Minnesota Cancer Center, Minneapolis, Minnesota 55455, USA.

A new method was developed for the analysis of metabolites of the tobacco-specific lung carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) in human urine. The metabolites are 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) and its glucuronides (NNAL-O-Gluc and NNAL-N-Gluc). The sum of these metabolites, total NNAL, was measured with this method. Urine was treated with beta-glucuronidase, which converts NNAL-O-Gluc and NNAL-N-Gluc to NNAL. After solvent partitioning and further purification on a liquid-liquid extraction cartridge and by high-performance liquid chromatography, total NNAL was quantified by gas chromatography with nitrosamine selective detection. The new method is accurate and precise, and the results are in good agreement with those obtained using the traditional method, which quantifies NNAL and its glucuronides separately. Levels of total NNAL +/- SD (pmol/mg creatinine) were 2.60 +/- 1.30 (n = 41) in smokers, 3.25 +/- 1.77 (n = 55) in snuff-dippers, and 0.042 +/- 0.020 (n = 18) in nonsmokers exposed to environmental tobacco smoke. The new method is faster and more sensitive than the traditional method and should greatly facilitate studies on human uptake of NNK.

3 - Carcinogenesis. 2002 Jun;23(6):907-22

Human urinary carcinogen metabolites: biomarkers for investigating tobacco and cancer (click on the link to download study)

Hecht SS.

University of Minnesota Cancer Center, Minneapolis, MN 55455, USA.

Measurement of human urinary carcinogen metabolites is a practical approach for obtaining important information about tobacco and cancer. This review presents currently available methods and evaluates their utility. Carcinogens and their metabolites and related compounds that have been quantified in the urine of smokers or non-smokers exposed to environmental tobacco smoke (ETS) include trans,trans-muconic acid (tt-MA) and S-phenylmercapturic acid (metabolites of benzene), 1- and 2-naphthol, hydroxyphenanthrenes and phenanthrene dihydrodiols, 1-hydroxypyrene (1-HOP), metabolites of benzo[a]pyrene, aromatic amines and heterocyclic aromatic amines, N-nitrosoproline, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol and its glucuronides (NNAL and NNAL-Gluc), 8-oxodeoxyguanosine, thioethers, mercapturic acids, and alkyladenines. Nitrosamines and their metabolites have also been quantified in the urine of smokeless tobacco users. The utility of these assays to provide information about carcinogen dose, delineation of exposed vs. non-exposed individuals, and carcinogen metabolism in humans is discussed. NNAL and NNAL-Gluc are exceptionally useful biomarkers because they are derived from a carcinogen- 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK)- that is specific to tobacco products. The NNAL assay has high sensitivity and specificity, which are particularly important for studies on ETS exposure. Other useful assays that have been widely applied involve quantitation of 1-HOP and tt-MA. Urinary carcinogen metabolite biomarkers will be critical components of future studies on tobacco and human cancer, particularly with respect to new tobacco products and strategies for harm reduction, the role of metabolic polymorphisms in cancer, and further evaluation of human carcinogen exposure from ETS.

4 - J Natl Cancer Inst. 2001 Oct 17;93(20):1575-7.
Metabolites of a tobacco-specific lung carcinogen in nonsmoking women exposed to environmental tobacco smoke (click on the link to download study)

Anderson KE, Carmella SG, Ye M, Bliss RL, Le C, Murphy L, Hecht SS.

Division of Epidemiology, University of Minnesota, Minneapolis 55454, USA. anderson_k@epi.umn.edu

BACKGROUND: Environmental tobacco smoke (ETS) is associated with lung cancer in nonsmokers. Most epidemiologic studies find a higher risk for lung cancer in nonsmoking women married to smokers than in those married to nonsmokers. We measured metabolites of a tobacco-specific lung carcinogen in urine from healthy, nonsmoking women exposed to ETS. METHODS: We recruited women and their partners through advertisements. Couples completed questionnaires on smoking history and demographics, and both partners provided 100 mL of urine; 23 women had male partners who smoked in the home (i.e., exposed women), and 22 women had male partners who did not smoke (i.e., unexposed women). Urine samples were analyzed for nicotine, for cotinine, for 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) and its glucuronide (NNAL-Gluc), as well as for creatinine. NNAL and NNAL-Gluc are metabolites of the tobacco-specific lung carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK). Unpaired Student's t tests were conducted on log-transformed values. All statistical tests are two-sided. RESULTS: Urinary levels of nicotine, cotinine, NNAL, and NNAL-Gluc were statistically significantly higher in exposed women than in unexposed women. Geometric means for these compounds in exposed versus unexposed women, respectively, were as follows: nicotine, 0.050 nmol/mg of creatinine (95% confidence interval [CI] = 0.033 to 0.076) versus 0.008 nmol/mg of creatinine (95% CI = 0.004 to 0.014); cotinine, 0.037 nmol/mg of creatinine (95% CI = 0.022 to 0.061) versus 0.007 nmol/mg of creatinine (95% CI = 0.004 to 0.011); NNAL, 0.013 pmol/mg of creatinine (95% CI = 0.007 to 0.024) versus 0.004 pmol/mg of creatinine (95% CI = 0.002 to 0.007); and NNAL-Gluc, 0.027 pmol/mg of creatinine (95% CI = 0.016 to 0.045) versus 0.004 pmol/mg of creatinine (95% CI = 0.003 to 0.006). CONCLUSIONS: Nonsmoking women exposed to ETS take up and metabolize the tobacco-specific lung carcinogen NNK, which could increase their risk of lung cancer. Within couples, the NNAL plus NNAL-Gluc level in exposed women compared with that of their smoking partners averaged 5.6%. Notably, epidemiologic studies have estimated the excess risk for lung cancer in nonsmoking women exposed to ETS as 1%-2% of that in smokers.

5 - Cancer Res. 1999 Aug 1;59(15):3602-5.
Stereochemistry of metabolites of a tobacco-specific lung carcinogen in smokers' urine (click on the link to download study)

Carmella SG, Ye M, Upadhyaya P, Hecht SS.

University of Minnesota Cancer Center, Minneapolis 55455, USA.

4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), a tobacco-specific lung carcinogen, is believed to be important as a causative agent for lung cancer in smokers. NNK is extensively metabolized to its carbonyl reduction product 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL), which, in turn, can be glucuronidated, producing [4-methylnitrosamino)-1-(3-pyridyl)but-1-yl]-beta-O-D-glucosiduronic+ ++ acid (NNAL-Gluc). Metabolism of NNK to NNAL produces a chiral center. A recent study demonstrated that (R)-NNAL is more tumorigenic in mice than (S)-NNAL and that these enantiomers have substantially different metabolic pathways. Therefore, it is important to determine the stereochemistry of NNAL and NNAL-Gluc in smokers. In this study, we used chiral stationary phase-gas chromatography-nitrosamine-selective detection with confirmation by liquid chromatography-tandem mass spectrometry to determine the stereochemistry of NNAL and NNAL-Gluc in smokers' urine. The two methods agreed well. The results of analyses of urine samples from 30 smokers demonstrated that the enantiomeric distribution of NNAL in urine was 54% (R) and 46% (S) +/- 7.0 (SD), whereas the diastereomeric distribution of NNAL-Gluc was 68% (R) and 32% (S) +/- 8.1. These results conclusively demonstrate that both (R)- and (S)-NNAL are formed metabolically from NNK in smokers. These data are essential for furthering our understanding of the role of NNK as a cause of lung cancer in smokers.

6 - Cancer Epidemiol Biomarkers Prev. 1998 Mar;7(3):257-60.

A metabolite of the tobacco-specific lung carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone in the urine of hospital workers exposed to environmental tobacco smoke (click on the link to download study)

Parsons WD, Carmella SG, Akerkar S, Bonilla LE, Hecht SS.

Ste. Anne's Hospital, Ste-Anne-de-Bellevue, Quebec, Canada.

We analyzed the urine of nonsmoking hospital workers exposed to environmental tobacco smoke for [4-(methylnitrosamino)-1-(3-pyridyl)but-1-yl]-beta-O-D-glucosiduronic acid (NNAL-Gluc), a metabolite of the tobacco-specific lung carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone. Samples were collected three times on a single day from nine workers. Quantitative analysis was carried out by combined gas chromatography-nitrosamine-selective detection. The identity of NNAL-Gluc was confirmed by combined gas chromatography-tandem mass spectrometry. The results demonstrated the presence of NNAL-Gluc in the urine of the exposed subjects. The mean level of NNAL-Gluc +/- SD was 0.059 +/- 0.028 pmol/ml urine (23 pg/ml urine); range, 0.005-0.11 pmol/ml urine. Levels of NNAL-Gluc per milliliter of urine correlated with those of cotinine (r = 0.51; P = 0.029). These results demonstrate for the first time that NNAL-Gluc, a metabolite of the lung carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone, is present in the urine of nonsmokers exposed to environmental tobacco smoke under field conditions.

7- Cancer Epidemiol Biomarkers Prev. 1997 Feb;6(2):113-20.

Analysis of human urine for pyridine-N-oxide metabolites of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone, a tobacco-specific lung carcinogen (click on the link to download study)

Carmella SG, Borukhova A, Akerkar SA, Hecht SS.

American Health Foundation, Valhalla, New York 10595, USA.

4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is a potent pulmonary carcinogen in rodents and is believed to be a causative factor for lung cancer in smokers. NNK also may be involved in oral cancer etiology in users of smokeless tobacco products. Pyridine-N-oxidation of NNK and its major metabolite, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL), produces NNK-N-oxide and NNAL-N-oxide, respectively, which are detoxification products of NNK metabolism and are excreted in the urine of rodents and primates. Our goal is to develop a panel of urinary biomarkers to assess the metabolic activation and detoxification of NNK in humans. In this study, we developed methodology to analyze human urine for NNK-N-oxide and NNAL-N-oxide. The key step in the method was conversion of the N-oxides to NNK and NNAL by treatment with Proteus mirabilis. The resulting samples were then analyzed essentially by methods that we have described previously. 4-(Methylnitrosamino)-4-(3-pyridyl-N-oxide)-1-butanol (iso-NNAL-N-oxide) was used as internal standard. Levels of NNAL-N-oxide in smokers' urine ranged from 0.06 to 1.4 pmol/mg creatinine, mean +/- SD 0.53 +/- 0.36 pmol/mg creatinine. Its presence was confirmed by high performance liquid chromatography-electrospray ionization-tandem mass spectrometry. NNK-N-oxide was not detected in smokers' urine. Levels of NNAL-N-oxide in the urine of smokeless tobacco users ranged from 0.02 to 1.2 pmol/mg creatinine, mean +/- SD 0.41 +/- 0.35 pmol/mg creatinine. The amounts of NNAL-N-oxide in urine were less than 20% of those of [4-(methylnitrosamino)-1-(3-pyridyl)but-1-yl]-beta-O-D-glucosiduronic acid (NNAL-Gluc) and were approximately 50% as great as those of free NNAL. These results demonstrate that pyridine-N-oxidation is a relatively minor detoxification pathway of NNK and NNAL in humans. The method was applied to analysis of urine from 11 smokers who consumed a diet containing watercress. In an earlier study (S.S. Hecht et al., Cancer Epidemiol., Biomarkers & Prev., 4: 877-884, 1995), we showed that consumption of watercress, a source of phenethyl isothiocyanate (PEITC), caused an increase in urinary excretion of NNAL plus NNAL-Gluc. This was attributed to inhibition of alpha-hydroxylation of NNK by PEITC, as seen in rodents in which PEITC also inhibits the pulmonary carcinogenicity of NNK. However, PEITC also could have inhibited pyridine-N-oxidation of NNK and NNAL. The urine of these smokers was analyzed for NNAL-N-oxide. The results demonstrated that watercress consumption had no effect on levels of NNAL-N-oxide in urine, supporting the conclusion that PEITC does inhibit the metabolic activation of NNK in humans.

8 -
Carcinogenesis. 1999 Aug;20(8):1577-82.  Related Articles, Links 
Tumorigenicity and metabolism of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol enantiomers and metabolites in the A/J mouse (click on the link to download study)

Upadhyaya P, Kenney PM, Hochalter JB, Wang M, Hecht SS.

University of Minnesota Cancer Center, Box 806 Mayo, 420 Delaware Street SE, Minneapolis, MN 55455, USA.

4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL), a major metabolite of the tobacco-specific pulmonary carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), has a chiral center but the tumorigenicity of the NNAL enantiomers has not been previously examined. In this study, we assessed the relative tumorigenic activities in the A/J mouse of NNK, racemic NNAL, (R)-NNAL, (S)-NNAL and several NNAL metabolites, including [4-(methylnitrosamino)-1-(3-pyridyl)but-(S)-1-yl] beta-O-D-gluco-siduronic acid [(S)-NNAL-Gluc], 4-(methylnitrosamino)-1-(3-pyridyl N-oxide)-1-butanol, 5-(3-pyridyl)-2-hydroxytetrahydrofuran, 4-(3-pyridyl)butane-1,4-diol and 2-(3-pyridyl) tetrahydrofuran. We also quantified urinary metabolites of racemic NNAL and its enantiomers and investigated their metabolism with A/J mouse liver and lung microsomes. Groups of female A/J mice were given a single i.p. injection of 20 micromol of each compound and killed 16 weeks later. Based on lung tumor multiplicity, (R)-NNAL (25.6 +/- 7.5 lung tumors/mouse) was as tumorigenic as NNK (25.3 +/- 9.8) and significantly more tumorigenic than racemic NNAL (12.1 +/- 5.6) or (S)-NNAL (8.2 +/- 3.3) (P < 0. 0001). None of the NNAL metabolites was tumorigenic. The major urinary metabolites of racemic NNAL and the NNAL enantiomers were 4-hydroxy-4-(3-pyridyl)butanoic acid (hydroxy acid), NNAL-N-oxide and NNAL-Gluc, in addition to unchanged NNAL. Treatment with (R)-NNAL or (S)-NNAL gave predominantly (R)-hydroxy acid or (S)-hydroxy acid, respectively, as urinary metabolites. While treatment of mice with racemic or (S)-NNAL resulted in urinary excretion of (S)-NNAL-Gluc, treatment with (R)-NNAL gave both (R)-NNAL-Gluc and (S)-NNAL-Gluc in urine, apparently through the metabolic intermediacy of NNK. (S)-NNAL appeared to be a better substrate for glucuronidation than (R)-NNAL in the A/J mouse. Mouse liver and lung microsomes converted NNAL to products of alpha-hydroxylation, to NNAL-N-oxide, to adenosine dinucleotide phosphate adducts and to NNK. In lung microsomes, metabolic activation by alpha-hydroxylation of (R)-NNAL was significantly greater than that of (S)-NNAL. The results of this study provide a metabolic basis for the higher tumorigenicity of (R)-NNAL than (S)-NNAL in A/J mouse lung, namely preferential metabolic activation of (R)-NNAL in lung and preferential glucuronidation of (S)-NNAL.

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