Lung cancer due to passive smoking--a review.

OBJECTIVES: Even from the scientific literature it is difficult to conclude whether the increased risk of lung cancer due to exposure to environmental tobacco smoke (ETS), as reported in many epidemiological studies, is based on sound data from reliable studies, or rather on passionate assertions derived from unsound investigations. To shed some light on this matter the differences between cigarette mainstream smoke (MS)--inhaled by the smoker- and ETS--inhaled by everyone exposed-, the concentration of ETS under real life conditions, the internal dose of toxic compounds due to ETS exposure, and the risk of lung cancer as found in epidemiological studies are discussed. RESULTS: MS and ETS differ considerably in their physical, chemical and toxicological characteristics because of the different conditions under which they are generated, the dilution in air, and the degree of ageing. Based on toxicological data, a very low internal dose of potentially genotoxic compounds can be measured in people after ETS exposure. The epidemiological data suggest a slightly increased risk of lung cancer in non-smokers chronically exposed to ETS. However, it is equally well known, that none of these studies is free from bias and confounding effects. CONCLUSION: The average intake of toxic and genotoxic compounds due to ETS exposure is that low that it is difficult, if not impossible, to explain the increased risk of lung cancer as found in epidemiological studies. The uncertainty is further increased because the validity of epidemiological studies on passive smoking is limited severely by numerous bias and confounding factors which cannot be controlled for reliability. The question of whether or not ETS exposure is high enough to induce and/or promote the carcinogenic effects observed in epidemiological studies thus remains open, and the assumption of an increased risk of lung cancer due to ETS exposure is, at present, more a matter of opinion than of firm scientific evidence.

Misclassification of smoking in a follow-up population study in southern Germany.

Smoking prevalence in southern Germany was studied in 1984-1985 using a representative cohort of 4022 subjects aged 25 to 64 years, with 3753 reinterviewed in 1987-1988. Data were available for analysis from interviews on self-reported smoking behavior and from serum cotinine measurements in both investigations. More men than women reported current smoking, and particularly heavy smoking. Serum cotinine levels increased steadily with the daily number and nicotine yield of cigarettes smoked. Mean cotinine levels in ex-smokers were higher than those in never smokers, suggesting that a higher percentage of current smokers are misclassified as ex-smokers than never smokers. Using cotinine rather than self-reported smoking data increased the proportion of true smokers in the subgroup of self-reported smokers by about 3% in males and by about 1% in females. Data from the reinterviews revealed that reported smoking status confirmed by cotinine measurement in 1987-1988 conflicted in a number of cases with the data obtained in 1984-1985 using the same procedure. For example, 0.1% of those who stated they were current regular smokers, 4.3% of those who stated they were current occasional smokers, and 17.6% of those who stated they were ex-smokers in 1984-1985 claimed in 1987-1988 to have never smoked. This misclassification of ex-smokers was higher in women. Altogether the true proportion of ex-smokers among self-reported never smokers was about 9.7% (17.8% in men and 6.7% in women). The widely variable uptake of tobacco smoke by smokers, as well as the misclassification of true smokers and ex-smokers as never smokers, needs to be considered in epidemiological studies evaluating the health risks from both active and passive smoking.

DNA adducts in human placenta in relation to tobacco smoke exposure and plasma antioxidant status.

The DNA adduct 8-hydroxy-2'-deoxyguanosine (8-OHdG) has been widely used as a biomarker for oxidative stress. Bulky DNA adducts, which are detectable by the 32P-postlabelling method, provide evidence for exposure to and metabolic activation of large, mainly apolar compounds, e.g. polycyclic aromatic hydrocarbons. We determined both types of adducts in placental tissues of 30 term pregnancies and related the adduct levels to the exposure to tobacco smoke and the plasma antioxidant status. Urine and plasma continine concentrations were used to select 10 nonsmokers, 9 nonsmokers exposed to environmental tobacco smoke (ETS) and 11 smoking women. Placental levels of 8-OHdG were 0.84 +/- 0.11, 0.90 +/- 0.21 and 0.83 +/- 0.20/10(5) deoxyguanosine bases (dG) for nonsmokers, nonsmokers exposed to ETS and smokers, respectively. The differences between the groups were not significant. Smoking women had significantly lower plasma vitamin C and beta-carotene concentrations than nonsmoking women or nonsmoking women exposed to environmental tobacco smoke. The 8-OHdG adduct level in placental DNA was inversely correlated with the plasma vitamin E concentration (r = -0.47, P < 0.05). There was no association between placental 8-OHdG adducts and vitamin A, C and beta-carotene in plasma. In total, 15 different adducts could be identified in the 30 placenta samples by the 32P-postlabelling method. There was a strong inter-individual variation in both the number of adducts and adduct intensities. No smoking-related or vitamin-related effects on adduct patterns or intensities were found. Our findings suggests that, within the limits of the methods used, tobacco smoke exposure during pregnancy does not lead to a measurable increase in placental DNA adduct levels and that vitamin E appears to have a protective effect on placental 8-OHdG formation.

trans,trans-muconic acid as a biomarker of non-occupational environmental exposure to benzene.

Excretion of trans,trans-muconic acid (2,4-hexadienedioic acid; t,t-MA), a potential biomarker of low-level exposure to benzene, was determined in 32 smokers and 82 nonsmokers. In smokers the median background excretion of t,t-MA was 0.13 (0.06-0.39) mg/g creatinine and was significantly higher (P < 0.05) than the value of 0.065 (0.02-0.59) mg g creatinine in nonsmokers. For nonsmokers, the correlation between t,t-MA excretion and environmental exposure to benzene in ambient air, which was determined during the 8-day study period by personal diffusion samplers, was not significant (r = 0.164, P = 0.18). Nonsmokers living in the city tended to have higher t,t-MA excretion rates than nonsmokers living in the suburbs. However, the difference was only significant for nonsmokers from nonsmoking homes. For the same location (suburb or city), smoking at home leads to a marginal increase in t,t-MA excretion of the nonsmoking members of the household. In a further study with eight nonsmokers we found that dietary supplementation with 500 mg sorbic acid significantly increased (P < 0.001) the mean urinary t,t-MA excretion from 0.08 (0.04-0.12) to 0.88 (0.57-1.48) mg/24 h. Under study conditions 0.12% of the sorbic acid dose was excreted in urine as t,t-MA, thereby indicating that a typical dietary intake of 6-30 mg/day sorbic acid accounts for 10-50% of the background t,t-MA excretion in nonsmokers, and for 5-25% in smokers. As a consequence, sorbic acid in the diet is a significant confounding factor in assessing low-level benzene exposure if t,t-MA excretion in urine is used as a biomarker.

Urinary mutagenicity and thioethers in nonsmokers: role of environmental tobacco smoke (ETS) and diet.

The urinary excretion of mutagens and thioethers was investigated in a controlled diet study and in two field studies. A diet containing charcoal-broiled meat and other items rich in mutagenic compounds increased the urinary mutagenicity as assessed in Salmonella typhimurium strain TA98 with metabolic activation approximately 46-fold compared to a diet low in mutagens. The excretion of thioethers after ingestion of the diet rich in mutagens also increased significantly when compared to the diet low in mutagens. The increase was associated with the content of preformed thioethers in the diet. In the first field study with 21 nonsmokers, urinary mutagenicity as assessed in Salmonella typhimurium strain TA98 and excretion of thioethers showed no relation to either the self-reported exposure to environmental tobacco smoke (ETS) or to serum cotinine concentrations used as an objective marker for ETS exposure. In the second field study, urinary mutagenicity was determined with a tobacco-smoke sensitive Salmonella typhimurium strain YG1024 with metabolic activation. No correlation was found between the mutagenic activity in urine and ETS exposure duration, nicotine on the personal sampler, cotinine in saliva and cotinine in urine. Our results suggest that real-life ETS exposure does not measurably increase either urinary mutagen or urinary thioether excretion. Furthermore, diet seems to be the most important source for both urinary mutagen and thioether excretion in nonsmokers.

Determination of low level exposure to volatile aromatic hydrocarbons and genotoxic effects in workers at a styrene plant.

OBJECTIVES: Low exposures to volatile aromatic hydrocarbons and cytogenetic effects in peripheral white blood cells were determined in 25 healthy workers employed in different areas of a styrene production plant in the former German Democratic Republic. The results were compared with 25 healthy unexposed controls (matched for age and sex) employed in the same company. METHODS: The concentrations of aromatic hydrocarbons determined from active air sampling in all areas of the factory (styrene: 73-3540 micrograms/m3 (< 0.01-0.83 ppm); ethylbenzene 365-2340 micrograms/m3 (0.08-0.53 ppm); benzene 73-3540 micrograms/m3 ( < 0.02-1.11 ppm); toluene 54-2960 micrograms/m3 (0.01-0.78 ppm); xylenes 12-94 micrograms/m3 ( < 0.01-0.02 ppm)) were considerably lower than in the pump house ( > 4000 micrograms/m3 styrene, ethylbenzene, benzene, and toluene; > 500 micrograms/m3 xylenes), which was only intermittently occupied for short periods. Passive personal monitoring, biomonitoring of exhaled air and metabolites (mandelic, phenylglyoxylic, trans, trans-muconic, hippuric, o-, m- and p-methylhippuric acids, and phenol) in urine samples collected before and after an eight hour working shift was used to assess individual exposure. Questionnaires and examination of company records showed that the historical exposure was far higher than that measured. Genotoxic monitoring was performed by nuclease P1-enhanced 32P-postlabelling of DNA adducts in peripheral blood monocytes, and DNA single strand breaks, sister chromatid exchange, and micronuclei in lymphocytes. The content of kinetochores in the micronuclei was determined by immunofluorescence with specific antibodies from the serum of CREST patients. RESULTS: No genotoxic effects related to exposure were detected by DNA adducts or DNA single strand breaks and sister chromatid exchange. The only effect related to exposure was an increase in kinetochore positive micronuclei in peripheral lymphocytes; the frequency of total micronuclei in peripheral lymphocytes did not change. Smoking was confirmed by measurement of plasma cotinine, and no confounding effect was found on any of the cytogenetic variables. CONCLUSIONS: Low occupational exposure to styrene, benzene, and ethylbenzene did not induce alterations of genotoxicological variables except kinetochore positive micronuclei. This is the first reported use of the CREST technique for an in vivo study in occupational toxicology, which thus could serve as a valuable and sensitive technique for toxicogenic monitoring.

Determination of urinary trans,trans-muconic acid by gas chromatography-mass spectrometry.

A sensitive and specific method for the determination of trans,trans-muconic acid (t,t-MA) in urine is described. After clean-up on an anion-exchange cartridge, t,t-MA was derivatized with BF3-methanol to the dimethyl ester and analyzed by gas chromatography-mass spectrometry (GC-MS), with 2-bromohexanoic acid as an internal standard. The limit of detection was 0.01 mg/l, the coefficient of variation for duplicate analysis in a series of urine samples (n = 50) was 2.6% and the recovery rate ranged from 93.3 to 106.3%. The between-day and within-day precision for the analysis were 7.4 and 14.6%, respectively. The method was applied to the determination of t,t-MA in urine samples from smokers and non-smokers. The mean concentration of t,t-MA in urine of 10 smokers was 0.09 +/- 0.04 mg/g creatinine and was significantly (p = 0.012) higher than that found in urine of 10 non-smokers (0.05 +/- 0.02 mg/g creatinine). In contrast to the results obtained with the commonly used high-performance liquid chromatographic ultraviolet detection (HPLC-UV) methods, no interference between t,t-MA and other urinary compounds was found. This GC-MS method is both specific and sensitive for biomonitoring of low environmental benzene exposure.

Determination of DNA single-strand breaks in lymphocytes of smokers and nonsmokers exposed to environmental tobacco smoke using the nick translation assay.

The detection of DNA single-strand breaks (SSB) in human mononucleated white blood cells (MWBC) using a modified version of the nick translation assay is presented. This assay allows rapid and sensitive examination of SSB using only 5 ml heparinized blood for an eightfold determination. The assay was standardized by incubation of MBWC in vitro with N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), a known genotoxic agent. In vitro incubation of MWBC with MNNG induced a dose-dependent increase in DNA-SSB at doses between 5 and 500 microM MNNG. The detection limit for the assay was 5 microM MNNG. To assess the suitability of this assay to detect SSB in vivo a controlled study was performed in which volunteer smokers (n = 5), nonsmokers (n = 5) exposed to environmental tobacco smoke (ETS), and nonsmokers controls (n = 5) were compared. The study lasted 4 experimental days, 2 control and 2 exposure days. On control days (days 1 and 3) smokers and nonsmokers sat in an unventilated 45 m3 room for 8 h. On the exposure days (days 2 and 4) each of the five smokers smoked 24 cigarettes in 8 h, while the five nonsmokers were exposed to the ETS generated by the smoking volunteers. High exposure to tobacco smoke was confirmed by dosimetry of carboxyhemoglobin (CO-Hb), plasma nicotine and cotinine levels. Blood was drawn before and after each exposure on all 4 experimental days for determination of DNA-SSB in lymphocytes immediately after isolation of blood cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Acute effects of smoking and high experimental exposure to environmental tobacco smoke (ETS) on the immune system.

Controversial results have been published on the immune response to cigarette smoking while the effects of exposure to environmental tobacco smoke (ETS) have not yet been reported. In a controlled study, acute effects of smoking and of a high environmental exposure to ETS on immunological parameters have been investigated. The study consisted of four experimental days, two control and two exposure days. On control days, 1 and 3, smokers (n = 5) and nonsmokers (n = 5) sat in an unventilated 45 m3 room for 8 h. On the exposure days, 2 and 4, each of the smokers smoked 24 cigarettes in 8 h, while the nonsmokers were exposed to the ETS generated by the smoking volunteers. Blood was drawn before and after each exposure session on all four experimental days for dosimetry of tobacco smoke exposure and determination of the immune response. Flow cytometry using monoclonal antibodies was used to determine CD3+ cells (whole T cells), CD19+ cells (B lymphocytes), CD16+ and CD56+ cells (natural killer cells), CD4+ cells (T-helper cells), CD8+ cells (T-suppressor cells), the CD4+/CD8+ (helper/suppressor ratio), and Fc receptors on granulocytes. Serum was analyzed for soluble CD14 receptors (sCD14), interleukin 1, interleukin 6 and prostaglandin E2 (PGE2). Functional stimulation assays were performed to determine the basal and induced level of reactive oxygen intermediate (ROI) production by polymorphic neutrophils. Exposure to tobacco smoke in both groups was confirmed by dosimetry of carboxyhemoglobin, plasma nicotine, and cotinine levels. In comparison to nonsmokers, smokers had elevated granulocyte cell counts, increased CD16+ and CD56+ cell levels and decreased CD3+ and CD19+ levels. Acute smoking, but not exposure to ETS, resulted in a slight decrease in the number of CD19+ cells and an increase in the number of granulocytes; the latter was restricted to one subject. Acute smoking and exposure to high experimental concentrations of ETS resulted in a slight increase in CD16+ and CD56+ cells. None of the changes determined in immunological parameters after either acute smoking or exposure to ETS reached statistical significance. Serum sCD14, cytokine and PGE2, functional stimulation of in vitro ROI production, and changes in Fc receptors were not affected by acute smoking or exposure to ETS. Although no clear guidelines exist to assess immunotoxicity in man, our data do not favor immunosuppression and the possibility of increased risk of infection in nonsmokers exposed to ETS under real-life conditions.

Evaluation of 4-(N-methylnitrosamino)-4-(3-pyridyl)butyric acid as a potential monitor of endogenous nitrosation of nicotine and its metabolites.

The potential endogenous nitrosation of nicotine and cotinine to yield 4-(N-methylnitrosamino)-4-(3-pyridyl)butyric acid (Iso-NNAC) has been studied in smokers and non-smokers. Following i.v. administration of 100 micrograms Iso-NNAC to rats, excretion in urine (67.4 +/- 25.4%) and feces (6.1 +/- 1.6%) occurred within 24 h. The urinary excretion of nitrate, nicotine, cotinine and Iso-NNAC were determined in 24 h urine samples from 19 smokers and 10 non-smokers. Iso-NNAC excretion was found on four occasions (44, 65, 74 and 163 ng/day) in smokers; non-smokers did not excrete Iso-NNAC. Oral administration of nicotine (n = 8; 12-40 mg) and cotinine (n = 3; 40-60 mg) to abstinent smokers did not result in Iso-NNAC excretion, even after oral nitrate (150 mg) supplementation. However, Iso-NNAC was found in cigarette tobacco (10-330 ng/g) and mainstream cigarette smoke (1.1-5.5 ng/cig.). Our studies suggest that the occasional presence of Iso-NNAC in smokers' urine results from exogenous exposure to the preformed compound in mainstream cigarette smoke and not from endogenous nitrosation of nicotine and its metabolites.

Detection of DNA single-strand breaks in lymphocytes of smokers.

In a controlled study, ten male volunteers (five smokers and five nonsmokers) were subjected to different smoking conditions and compared to five nonsmokers, not exposed to cigarette smoke. During the 4 days of the study, nonsmoking periods were strictly controlled. On the first day the ten subjects were sham exposed. On the second day the five smokers smoked 24 cigarettes in 8 h, while the five nonsmokers were exposed to the environmental tobacco smoke. After another day of sham exposure the smoke exposure was repeated under the same conditions. Blood was drawn before and after exposure and DNA single-strand breaks (SSBs) were analyzed in lymphocytes immediately (1 h) after isolation of cells and after 4 h incubation at 37 degrees C, using a modified assay based on the nick translation reaction. Base levels of unscheduled DNA synthesis (UDS) and UDS levels were determined after 1 h incubation with methyl methanesulfonate. Duplicate analysis using the same method was performed in a second laboratory after transportation of blood samples at 0 degree C on a train from Munich to Hamburg. Tobacco smoke exposure of the subjects increased COHb and plasma cotinine levels. SSBs could be detected in all probands with some interindividual day-to-day and morning-to-evening variations. In four of five active smokers, SSB increases were found after smoking. In nonsmokers exposed to tobacco smoke no exposure-related variation in SSB levels could be detected.(ABSTRACT TRUNCATED AT 250 WORDS)

Uptake of tobacco smoke constituents on exposure to environmental tobacco smoke (ETS).

For the purpose of risk evaluation, passive smoking is frequently regarded as low-dose cigarette smoking. However, since the physical, chemical and biological properties of mainstream smoke (MS), which is inhaled by the smoker and environmental tobacco smoke (ETS), which is breathed by the passive smoker are quite different, risk extrapolation from active smoking to passive smoking is of doubtful value. In a series of experimental exposure studies we compared the uptake of tobacco smoke constituents by active and passive smoking. The results show that biomarkers which were found to be elevated after experimental ETS exposure, such as nicotine and cotinine in plasma and urine as well as thioethers in urine, indicate gas-phase exposure in passive smokers, but particle-phase exposure in active smokers. Biomarkers which should indicate the uptake of particle-bound, genotoxic substances with ETS, such as urinary mutagenicity, metabolites of polycyclic aromatic hydrocarbons (PAH) and DNA adducts, were not found to be elevated even after extremely high ETS exposure. From these results we conclude that a risk evaluation for passive smoking on the basis of dosimetric data is currently not possible.

Importance of exposure to gaseous and particulate phase components of tobacco smoke in active and passive smokers.

The uptake of tobacco smoke constituents from gaseous and particulate phases of mainstream smoke (MS), inhaled by smokers, and of environmental tobacco smoke (ETS), breathed in by non-smokers, was investigated in two experimental studies. Tobacco smoke uptake was quantified by measuring carboxyhemoglobin (COHb), nicotine and cotinine in plasma and urine and the data obtained were correlated with urinary excretion of thioethers and of mutagenic activity. An increase in all biochemical parameters was observed in smokers inhaling the complete MS of 24 cigarettes during 8 h, whereas only an increase in COHb and, to a minor degree, in urinary thioethers was found after smoking the gas phase of MS under similar conditions. Exposure of non-smokers to the gaseous phase of ETS or to whole ETS at similar high concentrations for 8 h led to identical increases in COHb, plasma nicotine and cotinine as well as urinary excretion of nicotine and thioethers which were much lower than in smokers. Urinary mutagenicity was not found to be elevated under either ETS exposure condition. As shown by our results, the biomarkers most frequently used for uptake of tobacco smoke (nicotine and cotinine) indicate on the one hand the exposure to particulate phase constituents in smoking but on the other hand the exposure to gaseous phase constituents in passive smoking. Particle exposure during passive smoking seems to be low and a biomarker which indicates ETS particle exposure is as yet not available. These findings emphasize that risk extrapolations from active smoking to passive smoking which are based on cigarette equivalents or the use of one biomarker (e.g. cotinine) might be misleading.

Significance of exposure to benzene and other toxic compounds through environmental tobacco smoke.

In order to assess the uptake of benzene from environmental tobacco smoke (ETS) and to estimate its contribution to the total body burden of benzene observed in non-smokers, two experimental studies have been conducted. Controlled exposure to high levels of ETS equivalent to 10 ppm CO for 9 h and 20 ppm for 8 h resulted in a nonsignificant increase in blood benzene levels and a significant increase in exhaled CO, COHb and cotinine in serum and urine. The slightly rising blood concentration of benzene following experimental ETS exposure was paralleled by an increased exhalation of benzene and aromatic hydrocarbons and in contrast to blood levels, this increase was significant. The blood levels of benzene obtained during exposure were comparable to those observed at the time of admission to the laboratory, when biomarkers of ETS uptake, e.g. cotinine in serum and urine, were at the limit of detection, thus demonstrating that these background levels were not from ETS exposure. No difference in the urinary excretion of phenol, the main metabolite of benzene, was found during the experimental periods. The background levels of urinary phenol in unexposed nonsmokers were rather high, demonstrating that phenol excreted in urine must be formed from several endogenous and exogenous precursors. In the light of our findings it is highly questionable whether exposure to benzene from ETS under real life conditions poses a cancerogenic risk to the general population, which is measurable today or in the future by toxicological or epidemiological methods.

Dietary influences on urinary excretion of hydroxyphenanthrenes, thioethers and mutagenicity in man.

Our study indicates that large differences in dietary polycyclic aromatic hydrocarbon (PAH) content in humans are not reflected by urinary and faecal excretion of hydroxyphenanthrenes, although significant increases in 3-hydroxybenzo[a]pyrene and 3-hydroxychrysene in faeces were observed after consumption of a diet rich in PAHs. The question therefore arises whether urinary hydroxyphenanthrenes are a reliable marker for exposure to PAHs. As expected, the elevated mutagenicity of the diet rich in PAHs led to increased mutagenic activity in urine. The increased urinary excretion of thioethers after this diet was probably due to its higher thioether content. Therefore, an elevated thioether excretion does not always indicate exposure to electrophilic compounds.