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Human & F, xplrimenta! Toxicology {1997) 16, 449-~.59 ~) 1997 Stockton Press NI rights reserved 0~4<--5952/97 $12.00 Biomonitoring exposure to environmental tobacco smoke (ETS): A critical reappraisal The most frequently used biomarkers for ex~posure to environmental tobacco smoke lETS) are cotinine and thiocyanate in body fluids, carboxyhaemoglobin in red blood cells (COHb) and carbon monoxide in the expired air. Although not ideal, cotinine in blood, saliva or urine is an established biomarkar for ETS exposure within the past 1-3 days. Comparison with cotinine concentrations in cigarette smokers reveals that passive smokers take up less than 1/100 of the nicotine dose of smokers. Biomon.itoring data availabte for the ETS-retated exposure to genotoxic substances comprise uptake of benzene, poiycycltc aromatic hydrocarbons (PAH), aromatic amines, tobacco-specific nitrosamiues {TSNA), eleclrophilic compounds giving rise to ur- inary thioethers, mutagens causing urinary mutageuic activity and the formation of various DNA adducts. With the exception of TSNA, these biomarkers are related to chemicals occurring ubiquitously in the environment and in the food. As a consequence, the background levels in unexposed nonsmokers are high compared to the observed increases (if any) associated with ETS exposure. Some markers of biological effects, which, by defini- tion, are non-specific with regard to the underlying exposure, have also been investigated in relation to ETS exposure. These markers comprise cytogenefic effects, aryi hydrocarbon hydroxylase {AI-H~ induc. tion, urinary hydroxTproline excretion and various factors indicative of cardiovascular risks. The avail- able data suggest that passive smoking is associated with a small induction of placental A_I-Ht and also with effects on cardlovascv.[ar risk markers. The latter findings in particular may be confounded by other risk factors, which have been observed to be more frequent in passive smokers than in unexposed nonsmokers. Keywords: btomonitodns; en~d_ronmental tobacco smoke; coti- nine; thiocy~nate; carbon monoxide; benzene In~oducfion Quant/tatfve risk estimates for ETS-exposed non- smokers published by various authorities rely al- most exclusively on data from epidemiological studies,t It is the purpose of this review to summarize available data on the internal dose of tobacco smoke constituents and their possible effects in nonsmokers, exposed to ETS under real- life conditions. We feel that biological monitoring with nonsmokers exposed to ETS in their normal environment can provide objective data in this controversial field of research. Surrogate biomarkers for ETS exposure Surrogate biomarkers for ETS exposure are markers related to substahces in tobacco smoke, which are assumed not to be implicated in toxicologically Covzespondence: G Scherer Received 3 March 1997; revised 2 April 1997; accepted 4 April 1997 relevant processes, e.g. cancer, cardiovascular d/s- eases or respiratory dysfunctions. The concen~ra- t.ions of precursors of surrogate biomarkers in ETS should be directly proportional to those of toxico- logically relevant constituents in ETS. Suitable biom~rkers should be specific for the exposure, their biological half-life should be long enough and the analytical method should be specific, precise, and not too labourious and expensive.2 Strictly, these criteria are not met by any of the most commonly used surrogate biomarkers such as carboxyhaemoglobin or carbon monoxide in the exhalate, as well as cotinine and thiocyanate in body fluids (Table 1). Carboxyhaemo~obin (COHb) and exhaled CO (COax) Carbon monoxide (CO) is generated during all incomplete combustions of organic materials (for example cooking, heating or vehicle exhaust). In addition, CO is endogenously formed in mammals during catabolism of haem moieties.' Therefore, the biomarkers COHb and COax are by no means specific for tobacco smoke exposure. Normal or This article is for individual use only and may not be further reproduced or stored electronically without written permission from the copyright holder. Unauthorized reproduction may result in financial and other penalties. (c) STOCKTON PRESS ENGLAND

Blomonitorin¢ of ETS exposure G Scherer and E Richter 450 Table 1 Surrogate biomarkers for ETS exposure~ Prec~J~'$or Biomarker in ETS O~her sources Significant association the extent of real- COHb, COex CO Incomplete No~°~'11° combustions, endogenous for- mation Thiocyanate in H~---'N Diet No body fluids Coti~nine in Nicotine Diet[?} Yeslz'~t2a~3 body fluids ~Abbreviatinns: COHb: carboxyhaernogloblin; COex: carbon monoxide in exhaled a~:. bLiterature cited is not cornprehensive: for a review, see Benowitz.I~ only marginally increased levels of COHb or COex were observed in passive smokers as compared to nonsmokers. This is compatible ~vith the finding that the ETS-related increase of CO indoors amounts to only 0.5-1 p.p.m.~ Thiocyanate in body fluids Thiocyanate is the detoxification product of cya- nide with a half-life of 10-14 days.' The precursor of thiocyanate in tobacco smoke is hydrogen cyanide. Hydrogen cyanide levels in mainstream smoke (MS) of cigarettes vary between 300 and 550 pg/cigarette and the ratio of sidestream smoke (SS) to MS is 0.19-0.37.~ In ETS, hydrogen cyanide occurs almost exclusively in the gaseous phase. Other sources for the intake of cyanide are almonds, pulses and maize. Cyanide is also formed in the colon by bacteria. Preformed thiocyanates are found in cabbage, turnips, mustard and cow milk.' There- fore, thiocyanate in body fluids is not specific for exposure to tobacco smoke. Although thiocyanate can be easily measured, and its long biological half- life prevents large fluctuations in body fluids, it is not specific enough to be suitable as a biomazker for ETS exposure under real-life conditions. Cotinine in body fluids Cotinine is the main metabolite of nicotine, the principal alkaloid of the tobacco plant. The nicotine yield of cigarette MS is about 1 rag, and the SS/MS ratio amounts to 2.6-8.3." Nicotine occurs almost completely in the particulate phase of mainstream smoke and in the gaseous phase of ETSJ Average nicotine concentrations in indoor environments where smoking occurs are usually in the range of l-lO~g/m',~." In addition, some .solanaceae- derived food items such as tomatoes, potatoes and egg plants as well as tea contain small amounts of nicotine.'° The biological half-life of cotinine is considerably longer than that of nicotine (16-20 h vs I h}.~ Therefore, cotinine is a much more reliable biomarker for active smoking, as well as ETS exposure, than is nicotine. In almost all studies, a statistically significant difference in cotinine levels between ETS-exposed and nonexposed nonsmokers was observed (Table 1). In addition, a significant relationship between the cotinine concentration in body fluids and the extent (frequency, duration and intensity) of ETS exposure was found. ETS expo- sure at home was often found to be more important than ETS exposure at the workplace or other places."~" The ratio between the cotinine concentra- tions in ETS-exposed nonsmokers and smokers is usually < 1/100. However, there are general limita- tions for comparisons of active smoking with ETS exposure.~" A major difference is the fact that cotinine in smokers is an indicator of the nicotine uptake with the particulate matter of mainstream smoke, whereas cotinine in nonsmokers indicates the exposure to the ETS gaseous phase." The longer biological half-life of cotinine in nonsmokers as compared to smokers would lead to an overestima- tion of the exposure in passive smokers relative to smokers.~ However, other researchers found simi- lar pharmacokinetics of nicotine and cotinine in smokers and nonsmokersJ~ On the other hand, the significantly shorter half-life of airborne nicotine compared to other ETS components could lead to an underestimation of ETS exposure when based on cotinine concentrations in body fluids." Airborne nicotine shows a high degree of adsorption to surfaces in indoor environments, which is respon- sible for the high decay rates of nicotine. Adsorbed nicotine can be released into the air so that nicotine, but not other ETS constituents, are measurable in environments in the absence of sine'king.~" This, together with possible dietary nicotine intake and transdermal nicotine absorption from nicotine- polluted surfaces might lead to an overestimation of cotinine-based ETS exposure. Biomarkers for potentially genotoxic com- pounds related to ETS exposure In contrast to biomonitoring for surrogate markers, which can give only indirect evidence of the uptake of substances of toxicological relevance, biomoni- taring for potent/ally genotoxic compounds directly reflects the internal dose of substances possibly implicated in the process of carcinogenesis. How- ever, with the exception of tobacco-specific nitro- samines, the genotoxic substances found in tobacco smoke occur almost ubiquitously in the environ- ment and are taken up from various sources. Hence, for evaluating the contribution of ETS exposure to the total body burden, it is knportant to assess the background levels of these biomarkers. ETS ex- posure-related biomonitoring data for specific geno- toxic compounds such as benzene, polycyclic This a~cle is for individual use only and may not be further reproduced or stored electronically without written permission from the copyright holder. 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BiomonRodn£ of LeTS exposure G $cherer and E Richter aromatic hydrocarbons (PAH], aromatic amines and tobacco-specific nitrosamines (TSNA) as well as group-selective analyses for various DNA adducts, urinary thioethers and mutagenic acitivity are available (Table 2). Benzene occurs ubiquitously in the environment, with traffic exhaust being the most important source.'' Average benzene concentrations in rural and urban environments of 1-10 and 10-20 m3, respectively, have been reported in Ger- many.2°':~ Cigarettes were found to emit 30- 50 #g/cigarette in MS and 345-653/~g/cigarette of benzene in SS.=2 In a household survey including 230 homes in Germany, the median benzene concentrations were 6.9 pglm~ in house- holds wit~ nonsmokers and 9.3 pg/m~ in homes with at least ore smoker.=~ Corresponding levels reported for the USA were 7 and 10.5 pg/m respectively.=~ Various biomarkers can be used for the biological monitoring of benzene exposure.== Determination of benzene in blood=6 and exha- late=7 is suitable for biomonitoring acute exposure. Due to the longer half-lives of 6-12 h, the urinary benzene metabolites trans, trans-muconic acid and phenylmercapturic acid should prefer- ably be used in field studies.~'.~6 However, trans, trans-muconic acid is also a metabolite of the food preservative sorbic acid, and thus may not be a specific biomarker for low-level environ- mental benzene exposure3° Investigations under real-life conditions showed no, or only a marginal contribution, of ETS exposure to the background of benzene in nonsmokers (Table 2}. Analysis of variance in one study showed that at most 15 To of the variation is explained by ETS exposure." This is in good agreement with an air monitoring survey in 49 homes, which apportioned. 11% of indoor air benzene to ETS2~ Taken together, the biomonitortng data show that ETS exposure is only a minor source of the total benzene burden. Polycyclic aromatic hydrocarbons (PAH) PAH are formed during incomplete combustion of organic materials and are ubiquitously distributed in the environment. The main source of intake of PAH is the diet. In particular, fried, grilled, smoked and cured foods as well as leafy vegetables contain PAH mostly in the upper p.p.b, range with benzo[a]pyrene occurring in the lower p.p.b. range?~ It is estimated that >90 % of the total PAH body burden originates from the diet3=.~ In the MS and SS of a cigarettes about 10 and 100 ng of benzo[a]pyrene, respectively, axe emitted3~ Indoor air concentrations of benzo[a]pyrene were found to T~ble ~ Biomonltorlng for exposure to genotoxic compounds" Precursor Significant increase after in ETS Other sources Biomarker z~al.life ETS exposure Benzene Tra~c exhaust. combustion, fuels Sorbic acid. PAH Combustion, diet, ~nbient a~r 4-ABP Gas or kezosene burners, diesel exhaust NNN Polycyclic Diet, ambient air, aromatics endogenous NDMA, Diet, ambient air, NNK, endognous (NDEA) formation ? Oxidative stress (exogenous aRd endogenous) Ca~bonyLs Diet (acroleJn) Aromatic Diet aminesC?) Benzene in blood or Notre/Yess'~s exhalate t,t-MA in urine DNA adducts in WBC {Yes~7}b Albumin adducts NTo~/Yesss l-Hydroxypyrena inNos~er~' ~ urine 4-ABP-haemoglobin No4e'~'o/'Yes44'~':~z adducts ~'qNAL and NNAL- ~es~)= glucumnide in urine HBP-haemoglobin No~ adducts Bulky DNA adduct~ ia Noe4'e~'e~'eel(Yesee)d WBC and placenta 3-Methyl-13-Ethyl- (No'Z]" adenine in urine S-OHdG in placenta Nose Thioethers in urine No~e Mutagenlc activity in No~-7~/Yasz~ "Abbreviations~ t,t-.'V..A: transJzans-muconlc acid; PAH: polycycl~c aromatic hydroc~bons; WBC: white blood cells; 4-A~P: 4- .mi~obiphenyh NNK: 4-(methylnltrosamino)-1-(3-pyridyl}-l-butanone; NNAL: 4-(methyl-nitzossrn~o)-1-C3-pyrldyl)-l-butanol; NNN: .,V- nitrosouornicotine; HPB: 4-by ~dc~oXy-1-{3-pyridyl}-1-butanone; N'DMA: N-nitrosodimethy]~m¢~e; NDEA: N-nitrosodiethylamine; S-OHdG- 8.hydtoxy-2'-deoxyguanosine. =Significant correlation between score for ETS exposure and. DNA adducts. (Only 4 of ~I samples had detectable adduct levels,) CARet high experimental exposure to sidestzeam smoke foz 3 h. aNo systematic assessment of ETS exposure. "After h/gh experimental exposure to ETS for 8 h. 451 This article is for individual use only and may not be further reproduced or stored electronically without written permission from the copyright holder. Unauthorized reproduction may result in financial and other p~nalties. (c) STOCKTON PRESS ENGLAND

452 BiomonJtoring of ETS exposure G Scherer and E Richter be 5 ng/m3 in homes with smokers and 3 ng/m3 in homes with nonsmokers.~' The ultimate carcino- genic metabolites of benzo[a]pyrene and other PAH can bind to DNA or proteins. The long life-time of erythrocytes (120 days) leads to the accumulation of haemoglobin adducts and makes them suitable biomarkers reflecting exposure during the last 4 months. Because of the shorter half-life of albumin [20-24 days), its adducts reflect more recent exposure. Protein adducts can be taken as an index of the biologically effective dose of a carcinogen. Urinary excretion or monohydroxy phenanthrenes and 1-hydroxypyrene are also used for biomonitor- ing PAH exposure by passive smokers?6 In most investigations, no increase in PAH biomarkers was found after ETS exposure. The two studies''.3. showing significant ETS-related increases (Table 2] remain doubtful in view of the fact that (a) other working groups found only a small, if any, difference in protein PAH adduct levels between smokers and nonsmokers~g-'', [b) even extremely high exposure to ETS did not increase the urinary excretion of PAH metabolites~* and (c) the estimated intake of PAH suggested an overwhelming role of diet2~ Summarizing the available evidence for PAH exposure by passive smoking, it can be stated that the high background exposure to PAH from other sources, particularly from diet, excludes biomoni- toting of these substances from being of any value in ETS risk assessment. Aromatic amines Haemoglobin adducts of 4-aminobiphenyl (4-ABP) and 3-aminobiphenyl (3-A_BP] have been used to determine exposure to these substances by tobacco smoke. MS yields of 4-ABP and 3-ABP were found to be 2,4-4.6 and 2.7-5.0 ng/cigarette, respec- tively.'2.'~ SS was found to emit 143 and 132 ng/ cigarette of 4-ABP and 3-ABP, respectively.6 In an experimental room containing an ETS-related carbon monoxide concentration of 8 p.p.m., a level of about 5 ng/m~ of 4-ABP was measured (Grimmer, personal communication). As yet, no other sources for 4- and 3-ABP have been identified, apart from the fact that ~-ABP was used in the dye industry decades ago." However, 3- and 4-nitrobiphenyl, which are emitted by kerosene heaters and gas burners?s as well as by diesel engines, form the same haemoglobin adduct .as do 3- and 4- ABP and must, therefore, be considered confounding factors. Controversial results on an effect of ETS exposure on the 4- ABP haemoglobin adduct level were reported (Table 2). In the most extensive study,'* no ETS- related increase was observed. Nonsmokers living in cities were found to have 4-ABP-haemoglobin adduct levels 10-30 pg/g higher than nonsmokers living in ru.ral areas.''.'a This suggests that sources other than ETS are also important. Tobacco-specij~c ~itrosamines (TSNA} TSNA are mainly formed from the tobacco alkaloids nicotine, nornicotine, anabasine and anatabine during fermentation of tobacco leaves. 4-(Methylni- trosamino)-l-(3-pyridyl)-l-butanone (NNK) and N- nitrosonornicotine (NNN) are the biologically most important TSNA.~ MS yields of commercial cigar- ettes ware reported to be 17-306 ng/cigarette for NNK and 34-675 ng/cigarette for NNN.~° Co~e- sponding SS yields were 180-671 rig/cigarette and 141 - 348 ng/cigarette, respectively.~ Reported con- centtations of TSNA in rooms where smoking occurs, range from 0.2 to 29.3 ng/m* for NNK and 0.7-23 ng/m' for NNN.*'-** As biomarkers for NNK exposure, urinary NNAL, the corresponding alcohol of NNK, and its glucuronide as well as pyridylox- obutylated haemoglobin and DNA have been used." DNA and haemoglobin adducts, which also indicate exposure to NNN, release 4-hydroxy-l-(3-pyridyl)- 1-butanone (HPB) upon hydrolysis. There is no systematic investigation on the influence of every- day ETS exposure on urinary excretion of NNAL and NNAL glucuronide. In an experimental study, Hecht et al," exposed five nonsmokers for 3 h in a small chamber of 16 m~ air volume to the extremely high SS concentrations of up to 230 Hg/m' nicotine and up to 263 ng/m' NNK. Under these conditions, the urinary excretion of free and conjugated NNAL increased from 31_+.41 pmol/d before exposure to 127_+74 pmol/d after exposure. The authors state that NNK uptake in smokers is about 120 times higher than in nonsmokers exposed to sidestream smoke.'* We found in four of nine nonsmokers detectable NNAL and NNAL glucuronide levels (detection limit about 5 pmol/1) with an average excretion rate of 41 + 52 pmol/d,s~ These results are comparable to the findings with nonsmokers before experimental SS exposure in the study of Hecht et a/,s' and suggest that the ETS-related NNK exposure under real-life conditions amounts to about 1% of the NNK dose in smokers. HPB-releasing haemoglobin adducts could be tobacco-specific biomarkers of chronic exposure to NNK and NNN. Mean adduct levels for smokers and nonsmokers were 80 and 29 fmol/g haemoglobin, respectively, as reported by the working group of Hecht" and 69 and 34 fmol/g haemoglobin as found by the working group of Richter.'" There was a large overlap be~veen smokers and nonsmokers in both studies. The small differences in the HPB-adducts found between smokers and nonsmokers are remarkable. According to the NNK and NNN levels in MS and ETS as well as the urinary NNAL excretion rates discussed above, smokers should have HPB adduct levels at least two orders of magnitude higher than ETS-eXposed nonsmokers. The lack of such a large difference might be explained by induction of TSNA detoxification and/or inhibition of TSNA activation in smokers. 0 This article is for individual use only and may not be further reproduced or stored electronically without written permission from the copyright holder. Unauthorized reproduction may result in financial and other penalties. (c) STOCKTON PRESS ENGLAND

Evidence for the latter hypothesis is provided by a recent fnding suggesting that nicotine in smoking doses can inhibit NNK activation in rats.'~ In pregnant women, however, no relationship between ETS exposure (either based on self-reported ex- posure or classified by urinary cotinlne levels] and HPB-haemoglobin adduct levels was observed.~ In theory, NNK may also be formed endogenously by nitrosation of nicotine. No evidence for nitrosation of nicotine or cotinine to the possible nitrosation product 4-[N-methylnitrosarnino)-4-[3 -pyridyl]bu- tyric acid (iso-NNAC) was observed.~ In addition, the nitrosoproline test, which reveals an increased endogenous nitrosation capacity of smokers,~ did not show elevated nitrosation in nonsmokers experimentally exposed to high ETS doses.~'~2 The two biomarkers for TSNA exposure, namely urinary NNAL and the HPB-haemoglobin adducts, need further validation before conclusions on ETS- related TSNA doses and possibly implicated r~sks can be drawn. Various DNA adducts Cigarette smoke condensate (CSC), when darmally applied to mice, was found to form various, as yet unidentified DNA adducts detectable by the postlabelling method.~s In human biomonitoring studies, available tissues and cells are limited to leucocytes, oral mucosa, exfollated epithelial blad- der cells, bronchial lavage cells and placenta. We are aware of only a few investiga~/ons where experimental~.e~ or real-life exposure to ETS~-'~ has been considered causative for the formation of DNA adducts detectable by the ~'P-postlabelling method. In one study, it was suggested that passive smoking might lead to the formation of a tobacco smoke-related adduct with placental DNA.~ How- ever, the evidence was weak, since ETS exposure was only reported for those nonsmokers who had detectable levels of this adduct. In another study with systematic assessment of ETS exposure in pregnant women, no increase in placental DNA adducts in passive or active smokers was found.~ No additional DNA adducts or increases in DNA adduct levels after ETS exposure ware observed in peripheral monocytes,~ lymphocytes~ or white blood cells.~' In addition to NNK, N-nitrosodimethylamine (NDMA) is another methylating agent in tobacco smoke. MS and SS yields were reported to be 0.1- 20 and 143-1040 ng/cigarette, respectiveIy.~3 ETS concentrations of NDMA in rooms with moderate smoking were 20-50 ng/m~.~° N-Nitrosodiethyla- mine (NDEA), N-nitrosomethylethylamine (NMEA) and ethyihalogenides are potential ethylating agents in tobacco smoke. However, only trace amounts of these nitrosamines are detectable in tobacco smoke/° whereas about 1/~g of ethylchtor- ida was found in MS.'~ In an experimental study, no Siomonitoring of ETS exposure G Scherer and E Richter increase in the urinary excretion of 3-methylade- nine or 3-ethyladenine was observed in nonsmokers exposed to high ETS concentrations.'' The promutagenic DNA adduct 8-hydroxy-2'- deoxyguanosine (8-OHdG) formed by reactive oxy- gen species is an established biomarker for oxidative DNA damage,r~ In vitro experiments have shown that the MS of cigarettes can induce 8-OHdG adducts after metabolic activation, the gaseous phase being responsible for this effect." In a study aimed at systematically assessing ETS exposure in pregnant women, no ETS exposure-related increases in placental 8-OHdG adduct levels were found.'~ Ele~trophilic compounds Electrophilic chemicals are considered potential toxicants, mutagens and/or carcinogens because they may covalently bind to cellular macromole- cutes such as proteins, RNA and DNA.'~ Conjugation of electrophites to glutathione [GSHI, either sponta- neously or enzymatically by means ofglutathione S- transferases (GST), in most cases indicates a detox- iftcation process. The GSH adducts are further metabolized to form S-substituted N-acetyl-L-cy- stains conjugates (thioethers, mercapturic acids), which are renally excreted. Urinary thioethers have been used as group-selective biomarkers for the exposure to electrophilas,re Passive smoking under real-life conditions did not lead to a measurable increase in urinary thioether excretion (Table 2)." In an experimental study under dietary control and exposure to high ETS concentrations over a period of 8 h, a significant increase in excretion as compared to sham exposure was observed.'~ This effect was found to be caused by exposure to the gaseous phase of ETS. As a specific thioether, 3-hydroxypropyl mercapturic acid was identified, which is probably related to acrolein exposure. Mutagens The mutagenic properties of CSC in short-term tests after metaboIic activation are well established.~ Measurements o£ the urinary mutagenicity in nonsmokers experimentally exposed to high doses of ETS yielded controversial results2~ The influence of real-life ETS exposure on urinary mutagenicity has been determined in five populations.~-z' No significant influence of ETS exposure was found in all but one investigaton.~ More extensive studies are needed in order to quantify the contribution of ETS exposure to urinary mutagenicity, which is primarily dominated by dietary factors.~° Markers for bioIogical effects related to ETS exposure By definition, biological effect markers are not specific for the underlying exposure but reflect 453 This article is for individual use only and may not be further reproduced or stored electronically without written permission from the copyright holder. Unauthorized reproduction may result in financial and other penalties. 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454 B~omonitor|ng of ETS exposure G $cherer an~t E Richter physiological or toxicological responses to a great number of factors. Therefore, it is extremely important to control for confounding factors when applying these markers in population studies. Data about various biological effect markers in relation to ETS exposure are summarized in Table 3. C.~togenetic effects Tobacco smoke and its condensate have been shown to induce various cytogenetic damages and effects such as chromosomal aberrations (CA), sister chromatid exchanges (SCE) and micronuclei (MN) in vitro and in rive."2.'I The influence of ETS exposure on cytogenetic para- meters has been investigated in three limited studies''.s~,s~ and one more extensive approach,at No effect of passive smoking was found, probably because these tests were not sensitive enough to detect effects arising from low exposures, such as passive smoking. Aryl hydrocarbon hydroxylase (AI-iT-l) induction It is well documented that cigarette smoking can induce, via the Ah receptor, enzymes involved in the activation (e.g. cytochrome P4501A1) and detoxification (glutathione S-transferase, UDP-gtu- curonyltransferase) of xenobiotics. Induction is particularly high in the placenta,as The role of exposure to ETS in AHH induction has been investigated in human placenta at the enzymess'a' and messenger RNA level.'a The results suggest that passive smoking may exert an inducing effect in the placenta. Further investigations are needed to clarify, this possible effect of ETS exposure. Whether induction of the AHH enzyme system may increase the risk after exposure to PAH or other chemicals which are activated by cyto- chrome P450 enzymes or whether induction may be protective, as suggested by Remmer,'" is an open question. Urinary hydroxjrproline (HOP) excretion Increased urinary excretion of HOP is an estab- lished biomarker for certain osteopathic destruc- tions, some endocrinological disorders and severe burns,g° Since HOP is a degradation product of lung collagen and elastin induced by exposure to nitrogen dioxide (NO~), this marker has also been used to study the effect of low NO~ exposures arising from environmental aqtomobile exhausts as well as acitive and passive smoking.°' The results reported in the literature on the effects of active and passive smoking on urinary HOP excretion are controversial: while Kasuga~' found significantly increased levels of this marker in smokers and passive smokers, these findings were not confirmed by others.~.g~ In a recent study, no relationship between personal NO~ and ETS exposure and urinary excretion of HOP or desmosine, a catabolic product of elastin, was found."" Effect markers for cardiovascular diseases The pathomechanism by which active or passive smokin8 might increase the risk of cardiovascular disease is not completely understood. Possible effects include reduced oxygen supply, endothelial injury, reduction in high-density lipoprotein (HDL) cholesterol, increase in low-density lipoprotein (LDL) cholesterol, increased LDL oxidation with subsequent foam cell formation, increased platelet activation and coagulation.°~-"~ Physiological changes in parameters related to these effects can be used as biological effect markers. In a couple of studies, the influence of ETS exposure under experimental and real-life conditions on possible effect markers has been investigated (Table 3). In a small ETS field study, no effect of ETS exposure on either urinary 2,3-dinorthromboxane B~ or 2,6-dinor-6-ketoprostaglandin F,, excretion was found.~a Sinzinger and coworkers reported a significant reduction in platelet sensitivity to Table 3 Monitoring of biological effects related to ETS exposure" Significant effect of ETS Biolo~cai effect or ETS component possibly exposure under real-life biomarker implicated conditions SCE, CA ia lymphocytas ETS particulate matter (?] No:'~'~''s'~ O AHH induction in PAH, others Yes~e'a~ 0"~ placenta 0"~ Hydroxyproline in urine N0z (?] No°Z'~S/Yess~ Total cholesterol • NolO~.~te/Yes~ 03 . 03 H~L in plasma ? NoZO~.US/yes~o~,l~ ~ LDL ia plasma ? No~°"'~°'~° d~ Platelet aggregation ? Yes~°z 00 Fibrinogen in plasma ? Yes~°~ Tx-M and PGI-M in urine 7 No~a Carotid wall thickness ? Yes~0~.w~ ~Abbreviations: SCE: sister chromatid exchanges; CA: chromosomal aberratlon~; A.I-IH: aryl hydrocarbon hydroxylase; PAH: poiycycllc aromatic hydrocarbons: I-~L: high-density lipoprotein; LDL: low-density Iipoprotein; Tx-M: 2,3-dlnorthromboxane Bz; PGI-M: 2,6-dinor- 6-ketoprostaglandin This article is for individual use only and may not be further reproduced or stored elecb'onically without written permission from the copyright holder. Unauthorized reproduction may result in financial and other penalties. (c) STOCKTON PRESS ENGLAND

antiaggregatory prostacyclins after acute passive smoking,gin-'°1 However, these authors exposed nonsmokers to ETS from 30 cigarettes over 15 rain in a 18 me chamber, stating that this exposure would 'resemble that in discos, restaurants and the like'.~g Unfortunately, no concentrations of ETS components in the chamber were reported. Accord- ing to our experience, this ETS exposure far outreaches the levels in real-life conditions. In addition, no sham exposed control group was investigated in order to exclude unspecific, possibly stress-related effects. Davis et el,:°2 reported a reduced platelet aggregate ratio after passive smok- ing, which would reflect an increased formation of platelet aggregates. In addition, these authors found an increase in the counts of anuclear endothelial cell carcasses, suggesting endothelial damage after ETS exposure. These findings need confirmation with larger groups of subjects exposed under well defined experimental conditions. In two large population studies, ETS exposure ~vas found to significantly increase the carotid wall thickness'a3,~°~ and the plasma fibrinogen concentra- tion2°~ Although the observed changes are small and the biological significance in terms of cardio- vascular risk is unclear, the effects are relatively large in relation to the effect observed in active smokers. Both studies claim to have controlled for the major cardiovascular risk factors such as age, body mass index, ethanol intake, hypertension, diabetes, and total fat intake. However, since it is well established that the passive smoking status can be associated with an unfavourable constellation of a great number of cardiovascular risk factorsJ~ it seems doubtful whether statistical control of con- founding factors can be complete. Conclusions During the past 10-20 years, a large amount of data on biomonitoring of ETS exposure has accumu- lated. Cotinine in body fluids of nonsmokers has proven to be an acceptable, although nonideal biomarker for assessing ETS exposure dating back I-3 days. This marker has been and will be almost routinely used in field studies in addition to or for validation of self-reported exposure to ETS. Non- smokers, chronically exposed to ETS under real-life conditions, show coO.nine concentrations in blood, saliva and urine at least two orders of magnitude lower than those of current cigarette smokers. Although nicotine uptake by active and passive smoking differs in many aspects, this comparison gives a good estimate for the dose difference between smoking and ETS exposure. COHb and CO in the exhalate as well as thiocyanate in body fluids, which all are also used as biomarkers for ETS exposure, are less suitable due to their low Biomonltorlng of ETS exposure G Scherer ~nd E Richter specificity for tobacco smoke exposure. However, COHb and exhaled CO have proven to be use~l biomarkers for acute tobacco smoke exposure under well defined experimental conditions. Biomonitoring for exposure to ETS-related gone- toxic compounds might provide more relevant information for risk assessment than do the surrogate biomarkers cotinine, COHb and thiocya- nate. Data are available for exposure to benzene, PAH, aromatic amines and tobacco-specific nitro- samines. In addition, various DNA adducts in nucleated blood cells and placenta as well as urinary thioether excretion and mutagenic activity have been investigated in relation to ETS exposure. The results obtained are not conclusive, but suggest that every-day ETS exposure, if at all, only margin- ally increases the levels of these markers above bacl~round levels. Three major difficulties have to be considered when interpreting these data: (1) it is yet unknown which carcinogens in tobacco smoke are responsible for tumor induction in humans. As a consequence, it is not possible to focus the biological monitoring on the appropriate chemicals or classes of chemicals. (2) Population biomonitor- ing is limited to readily available body fluids [blood, urine, saliva), cells (blood cells, oral mucosa cells, exfoliated bladder cells) and tissues (skin, nails, hair, placenta). With respect to the most widely discussed cancer risk attributed to ETS exposure, namely lung cancer, these materials represent no target cells or tissues, but allow only the determina- tion of surrogate biomarkers. (3) With the exception of TSNA, the biomarkers for exposure to genotoxic compounds applied today are not related to substances unique for tobacco smoke, but occur ubiquitously in the environment and in food. Therefore, the existing background exposure to these substances has to be considered when evaluating an ETS-related increase in risk. As discussed above, the most promising biomarkers for TSNA exposure, i.e. urinary I~rNAL and the TSNA-related haemoglobin adducts, need further validation before being applicable for the biomoni- toting of ETS exposure. ETS exposure was net found to exert cytogenetic effects such as SCE or CA in peripheral lympho- cytes. However, these biomarkers may not be sensitive enough to indicate effects after low exposure, such as passive smoking. Controversial results were reported on the effect of ETS exposure on urinary hydroxyproline (HOPI excretion. The findings of Kasuga,~ who found a significant dose-related effect of ETS exposure on urinary HOP excretion, could not be confirmed by other investigators.~2-°* Further studies are needed to clear this discrepancy. A small AHH-inducing effect of ETS exposure in human placenta was found. The biological signifi- cance of this finding is unclear, because induction 455 This article is for individual use only and may not be further reproduced or stored electronically without wdtten permission from the copyright ~older. Unauthorized reproduction may result in finan~al and other penalties. (c) STOCKTON PRESS ENGLAND

Blomon|toring of EI'S exposure G Scherer a,~d E Richter comprises most probably enzymes involved in both activating and detoxi~ing pathways of carcinogens. It would be of interest to investigate ETS exposure- related enzyme induction in other relevant organ systems (e.g. the lung). Significant, unfavourable effects of ETS exposure were reported for some biomarkers, which are regarded to be indicative for the development of cardiovascular diseases. These include total serum cholesterol, HDL, plasma fibrinogen, platelet aggre- gation and carotid wall thickness. The extent of these effects was surprisingly high as compared to that observed with smokers. 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