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ELSEVIER The Science of the Total Environment 199 (1997) 247-254 the Science of the Total Env/ronment ! ] Urinary 1-hydroxypyrene as a marker of exposure to pyrene: an epidemiological survey on a general population group C. Roggia, C. Minoiab'*, G.F. Sciarrac, P. Apostolid, L. Maccarinia, S. Magnaghiu, A. Cennic, A. Fontee, G.F. Nidasioe, G. Micolib ~ Di~artimento di Medicina Prevenfiva, Occupazionale e di Comunit~, Sezione di Igiene, Universith di Pavia, Pavia, Italy Laboratorio di Igiene Ambientale e Tossicologia Industrial~ Fondazione 'S. Maugeri', Clinica del Lavor~ e della Riabilitazione, IRCCS 27100 Pavia, via Almia 29, Italy Clstituto di Medicina del Lavoro, Unioersit?~ di Siena, Siena, Italy dCattedra di Medicina del Lavoro, Universita di Brescia, Brescia, Italy ePMIP, Unit~ Operativa Chimica, USSL 44, Pavia, Italy Received 2 December 1996; accepted 18 February 1997 Abstract Urinary levels of 1-hydroxypyrene in a general adult population group are studied. Experimental data are not normally distributed; statistical analysis required a base 10 logarithmic transformation of data. The concentrations of urinary 1-hydroxypyrene measured were expressed as ~g g-1 urinary creatinine and are comparable with those reported by other authors, both for smoker and non-smoker subgroups. Multiple regression analysis shows that, for smokers, the number of cigarettes smoked per day and the body mass index (BMI) significantly influence the levels of urinary 1-hydroxypyrene expressed as /.~g g-1 urinary creatinine, whereas no personal or behavioural variable (age, sex, alcohol consumption, dietary intake of pyrene, BMI) modified the 1-hydroxypyrene levels for non-smokers. © 1997 Elsevier Science B.V. Keywords: Epidemiological survey; Urinary 1-hydroxypyrene; Pyrene; General population 1. Introduction - Polynuclear aromatic hydrocarbons are re- garded as ubiquitary pollutants formed either '] during incomplete combustion processes or from * Corresponding author. organic material pyrolysis or, less commonly, syn- thesized by some vegetable species [1-6]. Expo- sure for humans can therefore be both occupatio- nal (coke plants, petrol refineries, tar plants, as- phalting, etc.) and due to non-occupational sources (tobacco smoke, breathing polluted air, consumption of contaminated food and beverages or of smoked, roasted or toasted food). 0048-9697/97/$17.00 © 1997 Elsevier Science B.V. All rights reserved. Pll S0048-9697(97) 05458-2 This article is for individual use only and may not be further reproduced or stored electronically without written permission from the copyright holder. U.am~or~':~ea reprod'ucffon may resut'~ fn fi'nanciaf an~ other pena/itie~. (c) ELSEVIER ~CIENCE

248 C. Roggi et al. / The Science of the It has been shown that these substances can be absorbed at the gastrointestinal level or via lungs and skin [7-12]. Pyrene is metabolized to 1-hy- droxypyrene in all animal species including man. 1-Hydroxypyrene is mainly excreted via feces or urine. Recent studies show that pyrene is ex- creted not only as 1-hydroxypyrene but also as the 1,2-dihydroxy derivative [13]. Several PAHs are regarded as possible carcino- genic agents, therefore are assuming greater im- portance in preventive medicine for both exposed workers and the general population. PAH metabolic pathways involve formation of epoxides and diolepoxides, which are considered to be responsible for the carcinogenicity of some hydrocarbons [14]. Given the numerous sources of PAH exposure and the numerous PAH metabolites, it is difficult to identify a single metabolite as a reliable marker. Pyrene is always present at relevant concentrations in PAH mix- tures, despite the emission source, and 1-hydroxy- pyrene is always present in human urine, for all subjects [15]. Several authors have suggested the use of 1-hydroxypyrene as a biomarker of expo- sure to pyrene having revealed statistically sig- nificant correlations between total environmental PAH concentrations, environmental pyrene con- centration and urinary excretion of 1-hydroxy- pyrene [16-19]. Urinary 1-hydroxypyrene is com- monly used as a biological marker of exposure to PAHs [20,21]. A correlation between tobacco smoke and 1- hydroxypyrene excretion has been reported by different authors: the urinary levels of 1-hydroxy- pyrene are higher for smokers than for non- smokers although the reported difference is not always statistically significant [22]. Experimental data show that tobacco smoke has an effect on the P450 microsomial system and hence on hepatic metabolism of PAHs [23]. Since the effect of alcohol is similar to that of smoke, questions may arise on the possible combined effect of cigarette smoke and alcohol consump- tion [19,24,25]. Besides dietary intake other variables such as body fat can influence the tissue distribution of pyrene which is excreted as 1-hydroxypyrene. Variables such as age and sex also influence body Total Environment 199 (1997) 247-254 metabolism. Ageing leads to a reduced oxidation of xenobiotic substances, due to decreased blood flow to liver. Small differences exist between cy- tochrome P450 in males and females that could influence oxidation processes. In this work the urinary levels of 1-hydroxypyrene in relation to age, sex, BMI, diet, smoking and alcohol con- sumption are investigated to evaluate the use of 1-hydroxypyrene as a reliable marker of exposure for the general population [26]. 2. Materials and method 21. Sampling The work focused on a population group (age between 20 and 79) living in Rovescala (Pavia, Italy), and subjects were randomly chosen from the county register. A total of 517 persons were selected and 419 of them participated in the survey (81%; 183 males and 236 females). Table 1 and Table 2 summarize the features of the sur- veyed population. 2.2. Analytical method 1. Urinary 1-hydroxypyrene was quantitated ac- cording to the method proposed by Jongenee- len et al. [27]. 10 mL urine samples under- went enzymatic hydrolysis and 1-hydroxy- pyrene was separated on LC-DIOL solid phase extraction columns (SUPELCO, Su- pelco Park, Bellefonte, PA, USA). Elution was carried out with methanol. The organic phase was concentrated under nitrogen and injected on a C18 HPLC column. Recovery ranged between 88 and 98%; the detection limit was 0.05 /.~g 1-1; at a concentration of 0.8 tzg 1-1 the variation coefficient was 5.9%. Seronom TM Trace Elements Urine was used as reference material, 1-hydroxypyrene con- centration was 1 ~g 1-1. 2. Diet and alcohol consumption were assessed according to the 'dietaly story' technique [28,29]. Each subject involved in the survey was interviewed by our technical staff on the weekly consumption of food and alcoholic This article is for individual use only and may not be further reproduced or stored electronically without written permission from the copyright holder.

! I C. Roggi et al. / The Science of the Total Environment 199 (1997) 247-254 Table 1 Data of subjects taking part in the survey 249 Parameters Mean + S,D. Min Max Median 5* perc. 9° perc. Age (years) 56.4 + 15.7 22 81 59 26 79 Weight (kg) 69.8 + 13.3 40 118 69 49 93 Height (era) 162.8 + 9.4 140 187 163 148 178 BMI (kg/m~) 26.3 + 4.4 17.4 44.4 25.8 20.2 34.6 Males (N- 183) Age (years) 54.3 + 15.3 23 81 58 25 78 76.9 12.5 47 118 75 58.5 102 Weight (kg) Height (cm) 170.6 5:6.6 153 187 171 159.5 182.5 BMI (kg/m2) 26.4 + 3.9 17.7 44,8 25.8 21.3 34.1 Females (N - 236) Age (years) 58.2 + 15.8 22 81 62 26 79.8 Weight (kg) 64.3 5:11.6 40 107 63 45 83.9 Height (cm) 156.7 + 6.4 140 172 156 146 167.9 BMI (kg/m2) 26~2 5:4.6 17.5 42.1 25.8 19.5 34.6 weekly consumption of food and alcoholic beverages. Interviews were preferred to ques- tionnaires to avoid omissions. Quantitation of the dietary intake was obtained using a speci- fic photo-book (an atlas of different portions and different glasses) of the typical food and beverages of the surveyed area, to be selected according to the weekly consumption of each individual. Ethanol intake was estimated adding up the alcoholic content of beer, wine and spirits portions [30]. Persons who did not consume alcoholic beverages were classified as teetotal. The validation of the method is reported in the literature [31]. All interviewed subjects answered all questions.On the basis of the interviews a list of the typical foods of the surveyed population was obtained, the weight~ of the different portions were calcu- lated and the cooking techniques were identi- fied. The diet was described according to the 'market basket' method, foods with a pur- chase frequency lower than 1% were not taken into account. More than 150 foods were collected and were cooked in a fully equipped laboratory according to local habits. Ready to eat dishes were grouped according to affinity criteria (different cooking techniques and subsequent formation of PAHs). As a whole, 32 groups were formed and PAIl analyses were carded out. The analytical procedure for PAH determina- tion in food is carded out inthree steps: Table 2 • PAH extraction with acetone using a Composition of the four population subgroups Soxhlet apparatus for freeze dried food Class Smokersa Drinkers" No. of No. Alcohol (12 h) or liquid-liquid extraction for water subjects cigarettes/ (g ethanol/ and beverages (10 g samples). day day) • Filtration on anhydrous Na~SO4 and PAH " clean up with dimethylsulphoxide and/or 1 0 0 134 0 0 2 0 1 191 0 31.4 silica gel column chromatography. 3 1 0 29 12.3 0 • Instrumental analysis on a HPLC-fluo- 4 1 1 65 15.5 45.1 rescence apparatus. • 0 - no; 1 = yes. This article is for indiyidual use only and may not be/hrther reproduced or stored electronically without written permission from the copyright holder.

250 C. Roggi et al. / The Science of the Total Environment 199 (1997) 247-254 The detection limit was 0.08 ng g-i and pyrene recoveries ranged between 75 and 95% [32,33]. Table 3 Distribution of subjects according to sex and urinary creati- nine classes (percentual frequencies) 2.3. Cigarette smoking and other variables Urinary creatinine (g/l) 0.001-0.49 0.5-3.0 > 3.0 Males 0.053 0.931 0.016 Females 0.206 0.794 -- • Cigarette smoke: smoking habits and number of cigarettes smoked per day were investi- gated. Nobody in the surveyed population smoked cigars or pipe. • All persons involved in the survey were inter- viewed to assess other variables: sex, age, oc- cupation, residential area. 2.4. Body mass index (BMI) The BMI was calculated as weight/height2 (kg/m2). ]Each person was weighed undressed and the weight was rounded up to the next 500 g. Height was measured barefoot and rounded up to the next cm. 2.5. Statistical analysis Statistical analyses w.ere performed using SPSS/PC+ software [34]. Urinary 1-hydroxy- pyrene levels were not normally distributed, data were transformed in base 10 logarithm. Shapiro- Wilks test gave a result of 0.991. Analysis of variance and the t-test group was performed on normalized data. A multiple regression analysis (stepwise method) was carried out on both smokers and non-smokers subgroups to point out the be- havioural variables that can influence the urinary 1-hydroxypyrene levels. 3. Results ification class and 1.6% males to the third strati- fication class. A variance analysis applied to the above men- tioned urinary creatinine ciasses did not show statistically significant variables in the urinary 1- hydroxypyrene log values, no subject was there- fore excluded from our statistical analysis. When expressing the urinary 1-hydroxypyrene log values in/zg g- 1 urinary creatinine, no statis- tically significant differences are indicated between males and females, the subsequent elaboration was therefore performed on the sam- pie as a whole. Age does not seem to be a statistically significant variable, whereas differ- ences appeared between smokers and non- smokers (P < 0.001) and between drinkers and teetotals (P < 0.05). Table 4 shows the concentrations of urinary 1-hydroxypyrene expressed in/zg g-1 urinary ere- atinine for the surveyed population divided into four classes: 1. Subjects who do not smoke and do not drink. 2. Subjects who drink and do not smoke. 3. Subjects who smoke and do not drink. 4. Subjects who drink and smoke. Table 4 Urinary 1-hydroxypyrene concentrations (/~g/g urinary crea- tinine) in the four classes (alcohol consumption and cigarette smoke) Table 3 reports the distribution percentage fre- Class N SmokeI AlcoholI Mean Median 5* perc. 95* pete. quencies of the sample stratified according to sex 1 134 0 0 0.18 0.15 0.04 0_34 and urinary creatinine classes (0.001 + 0.49 g/l; 2 191 0 1 0.27 0.15 0.05 0.91 0.5 + 3.0 g/l; > 3.0 g/l). 3 29 1 0 0.34 0.28 0.09 1.09 In the surveyed population of Rovescala 5.3% 4 65 1 1 0.44 0.36 0.06 1.09 males and 20.6% females belong to the first strat- "0 m no; 1 -yes. This article is for individual use only and may not be further reproduced or stored electronically without written permission from the copyright holder.

I I C. Roggi et al. / The Sc(ence of the Total Enoironment 199 (1997) 247-254 251 Table 5 Urinary 1-hydroxypyrene concentrations (/~g/g urinary creatinine) for all subjects (distribution according to cigarette smoke) N Mean S.D. G.M G.M.S.D. Median Mode 95° pore. 5°perc. Entire population 419 0.273 0.396 0.184 2.254 0.173 0.192 0.744 0.054 Smokers 92 0.408 0.312 0.313 2.138 0.333 0.250 1.059 0.076 Non-smokers 327 0.233 0.411 0.143 2.438 0.149 0.192 0.654 0.052 A variance analysis applied to the above men- tioned four classes (Ran-Tukey test) shows a sta- tistically significant difference between classes 1, 2 (non-smokers) and classes 3, 4 (smokers): smoke is therefore identified as the major influencing factor as far as urinary 1-hydroxypyrene is concerned. Table 5 reports the concentrations of urinary 1-hydroxypyrene expressed in/zg g-x urinary ere- atinine for the general population stratified ac- cording to cigarette smoking. A very good correlation is found between the number of cigarettes smoked per day and urinary 1-hydroxypyrene (R -- 0.38; P < 0.001). Alcohol consumption has a minor effect on the urinary 1-hydroxypyrene concentration, acting therefore as an uncertainty factor. 3.1. Pyrene exposure We estimated the pyrene intake due to cigarette smoking and diet on the basis of the information collected from each participant. 3.1.1. Smoke Pyrene intake was calculated on the basis of the number of cigarettes smoked per day and their tar content. The tar content of the cigarettes smoked by the reference population ranged between 0.7 and 15 mg with an average value of 8.62 rag. Pyrene intake ranged between 0.12 and 3.62 /~g/day and the average value was 0.87 /~g/day, calculated according to the following formula [21]: Pyrene intake = N × (0.043 × tar content + 0.027) × DP where pyrene intake is the pyrene intake due to smoke as particulate (nmol/day); N is the aver- age number of cigarettes smoked daily; tar con- tent is the tar content of one cigarette (mg); DP is the percent of the total inhaled quantity of pyrene that is absorbed in the lungs (75%). 3.1.2. Dietary intake Foods belonging to the 32 groups were ana- lyzed. The dietary intake of pyrene was estimated from the pyrene concentrations in foods and the data on food consumption. Fig. 1 shows the average concentrations of pyrene in the 32 food groups. Table 6 reports the estimate of the daily pyrene intake due to food and smoke. 3.1.3. Multiple regression analysis A stepwise method used for multiple regression analysis evaluated the relation between the con- centration of 1-hydroxypyrene expressed as g-1 creatinine and the individual parameters of the surveyed subjects: age, sex, alcohol consump- tion, BMI, dietary pyrene intake and number of cigarettes smoked per day. Among smokers, variables such as number of cigarettes smoked per day (multiple R = 0.30) and BMI (multiple R--0.41) turned out to be significant, whereas no variable had any statisti- cally significant influence among non-smokers. 4. Discussion The distribution of urinary 1-hydroxypyrene levels of the reference population are not Gauss- ian. Normalization can be obtained when express- ing experimental data as a base 10 logarithm. The levels of urinary 1-hydroxypyrene ex- This article is for individual use only and may not be further reproduced or stored electronically without written permission from the copyright holder.

252 C. Roggi et al. / The Science of the Total Environment 199 (1997) 247-254 25 20 15 IO 0 Fig. 1. Pyrene average concentration in 32 food groups. pressed as ~g g-~ creatinine are in good accor- dance with data reported by other authors both for smokers and non-smokers [17,25,35] and are increased by cigarette smoke. Within these stud- ies it is not easy to compare data of urinary 1-hydroxypyrene excretion due to differences in number and tar content of smoked cigarettes. This study confirms the influence of smoke on the urinary levels of 1-hydroxypyrene given that its excretion is dose-dependent on the number of cigarettes smoked daily. A minor effect seems to be related to alcohol consumption, in presence of subjects with smok- ing habits the effect of alcohol is masked, in accordance with what has already been reported Table 6 Estimate of'the daily pyrene intake from food and cigarette smoke(mg/day) Food Smoke Smokers Mean 3.47 0.87 Median 3.28 0.91 5° and 95* pert. 2.12-5.79 0.17-1.57 Non-smokers Mean 3.57 Median 3.34 5* and 95* l~rc. 1.86-5.73 for a population of occupationally exposed work- ers by Van Rooij et al. [36]. Differences between excretion levels in drinkers and teetotal subjects are therefore related to the uncertainty of the effect of smoke. Our statistical analysis shows a significant effect of BMI on urinary excretion of 1-hydroxypyrene among smokers. The same result has been recently achieved by Van Rooij et ah [36]. Both age and diet do not influence the data distribution, contrary with other reported data. We measured the pyrone dietary intake which ranged between 1.8 and 5.8 /xg/day, in accor- dance with reported data ranging between 0.3 and 5.1 /~g/day [36-38]. When using urinary 1-hydroxypyrene as a mea- sure of cancer risk due to PAH exposure it is necessary to know the relative concentrations of the different polynuclear aromatic hydrocarbons. Jongeneelen has related the urinary levels of 1- hydroxypyrene and the epidemiological data on lung cancer risk for workers of a coke plant to the PAH concentration in the working environment: a biological exposure limit (BEL) of 2.3 mmol mo1-1 creatinine leads to a lung cancer risk "fac- tor of 1.3 [39]. As far as oral exposure is concerned, the widest number of data regards benzo(a)pyrene for which several ~ut~ho.rs _report a variation range from 9 0 O~ ~0 0 This article is for individual use only and may not be fi~rther reproduced or stored electronically without written pennission from the copyright holder. Uaet~tt&~ci~red t'¢~,cod~tetioa aray ¢-¢~,u1¢ itr ~aaa¢ial and other ~¢naliti¢~, (¢~ ELSEVIER SCIE?qCE BV

! ! C. Roggi et al. / The Science of the ng/day to 2160 ng/day [37]. The high variability of data is to be related to the different interview- ing techniques and meal preparation. Our study evaluates the usual dietary intake of pyrene in- stead of the punctual exposure; this could be the reason for the lack of correlation with urinary excretion of 1-hydroxypyrene. A significant corre- lation was found when voluntary subjects were given a highly rich meal in PAHs [40]. Recent reports describe variations of urinary 1-hydroxy- pyrene from 6-7 ng/h to 60-89 ng/h in relation to minimum and maximum BaP dietary intake. Such an increase does not have a statistical lin- earity, probably due to an unreliable evaluation of the PAH excretion pathways [41]. Considerably large differences are reported for the dietary intake in different seasons [42]. 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