Science of the Total Environment 414 (2012) 701–707

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Formaldehyde and tobacco smoke as alkylating agents: The formation ofN-methylenvaline in pathologists and in plastic laminate workers

Roberto Bono a,⁎, Valeria Romanazzi a, Valentina Pirro b, Raffaella Degan a, Cristina Pignata a, Elisa Suppo a,Marco Pazzi b, Marco Vincenti b

a Department of Public Health and Microbiology, University of Torino, Torino, Italyb Department of Analytical Chemistry, University of Torino, Torino, Italy

⁎ Corresponding author at: Department of Public Healtof Torino, Italy, Via Santena 5 bis, 10126 Torino, Italy. Tel.011 236 5818.

E-mail address: roberto.bono@unito.it (Bono R.).

0048-9697/$ – see front matter © 2011 Elsevier B.V. Alldoi:10.1016/j.scitotenv.2011.10.047

a b s t r a c t

a r t i c l e i n f o

Article history:

Received 5 April 2011Received in revised form 20 October 2011Accepted 21 October 2011Available online 21 November 2011

Keywords:FormaldehydeN-MethylenvalineTobacco smokeAlkylating agentsOccupational exposition

Objective: Aim of this study was to investigate the relationships between the concentration of formaldehydein air and the alkylation of hemoglobin to form a terminal N-methylenvaline residue in three occupationallyexposed groups: a) technicians of pathologywards, b) workers of the plastic laminates industry, and c) a controlgroup. All subjects recruited in this study were also tested on their smoking habits.Methods: Formaldehyde adsorbed on passive air samplers was quantified by HPLC with UV detection (360 nm),cotinine was quantified by GC–MS. Terminal hemoglobin N-methylenvalinewas determined by treating globineunder reducing conditions with pentafluorophenyl isothiocyanate to yield a derivative, subsequently detectedby GC–MS. One-way analysis of variance was performed to compare among the three groups the biomarkersconsidered in this study.Results: For air-FA and N-methylenvaline a difference between the three groups was detected (pb0.0001)and a significant higher concentration in the two professionally exposed groups was proved. Mean values

for FA (μg/m3): group a) 188.6, group b) 210.1, and group c) 41.4; mean values for N-methylenvaline(nmol/g of globin): group a) 377.9, group b) 342.8, and group c) 144.8. Conversely, the comparison betweenthe two professionally exposed groups, a) vs b), does not show any significant difference highlighting similarexposition to FA and, consequently, similar biological response. Tobacco smoke proves to have a minor impacton the formation of N-methylenvaline molecular adduct.Conclusions: A positive correlation was demonstrated between professional exposition to air-formaldehyde andhemoglobin alkylation to form N-methylenvaline molecular adduct in two occupationally exposed groups ofsubjects considered in the present study. In comparison with occupational exposition, tobacco smoke provedto have a minor impact on the formation of N-methylenvaline molecular adduct.

© 2011 Elsevier B.V. All rights reserved.

1. Introduction

Formaldehyde (FA) is a common environmental and occupationalcontaminant, emitted in air through various waste streams during theproduction of resins. FA is also contained in various disinfectants, preser-vatives and several other chemical formulations; it is present in tobaccosmoke, paint, garments, diesel and gasoline exhaust, and in numerousmedical and industrial products (Bono et al., 2010a; Saito et al., 2005). Ex-position to air-FA can induce local irritations, acute and chronic toxic ef-fects, genotoxic and carcinogenic activity (Schmid and Speit, 2007;Speit et al., 2007). Toxic and carcinogenic activity of FA was confirmedby (i) an increased incidence of nasopharyngeal cancer in industrial

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workers, embalmers, and pathologist (Duhayon et al., 2008;Hauptmann et al., 2004), (ii) the relationship demonstrated between FAand leukemia in a recent meta analysis (McGwin et al., 2010; Zhanget al., 2009), and (iii) a significant positive association betweenFA exposition and childhood asthma (McGwin et al., 2010). As anoutcome of these studies, FA was re-evaluated for carcinogenic ef-fects and reclassified as “Carcinogenic to humans” (Group 1) (IARC,2006). FA is currently considered as a xenobiotic of major environmen-tal and public health concern and its presence in occupational environ-ments has been regulated and periodically revised by the AmericanConference Governmental Industrial Hygienists (ACGIH) until the actu-al threshold limit value, ceiling limit (TLV-C) of 0.3 ppm (0.370 mg/m3)(Heck and Casanova, 1990; Nielsen and Wolkoff, 2010).

Although FA is rapidly absorbed from the gastrointestinal andrespiratory system, the exposition to it is not easily assessable bydirect measurement in biological fluids, because the bio-availableportion of FA is rapidly metabolized and its metabolites are either

702 Bono R. et al. / Science of the Total Environment 414 (2012) 701–707

incorporated into macromolecules via the one-carbon pool pathway oreliminated with the expired air (CO2) and urine (Goulding, 1989).

FA is an extremely reactive chemical toward amines and amidesforming methylene bridges and producing covalently cross-linked com-plexes with protein and DNA. DNA-protein cross-links (DPCs) can bemeasured in blood or cells of exposed subjects (Chaw et al., 1980; Heckand Casanova, 1990; Lu et al., 2010). Since adducts between FA and pro-teins are not subjected to repairmechanisms, their presence correspondsto the life of the protein and their abundance is proportional to exposi-tion. The scientific interest is directed not only to major protein adducts,such as hemoglobin and albumin (half-lifetime in men, 120 and 23 days,respectively), but also to histones and collagen, as potential indicators oflong-term FA exposure (10–20 years) (Miraglia et al., 2004).

FA is able to bind to human serum albumin (HSA), forming the FA-HSA, amolecular adduct showing antigenic properties. The antibody re-sponse against this adduct could also provide an indirect measure of FAexposition (Thrasher et al., 1988; Thrasher et al., 1990), even if the for-mation of IgG and IgE antibodies against FA is minimal (Doi et al., 2003;Kim et al., 1999). Consequently, the evaluation of IgG antibodies forma-tion represents a useful indication of occupational exposition to air-FAonly for non-smokers (Carraro et al., 1997). DPCs were found in the tis-sues directly exposed to FA, more rarely in tissues not exposed to air(Goulding, 1989). With respect to air-FA exposition monitoring, DPCslevels were measured in the blood of subjects working in pathologywards and professionally exposed to FA, proving to be higher than incontrols (Shaham et al., 1996; Shaham et al., 2003; Zhitkovich, 1992).

Kautiainen (Kautiainen et al., 1989) demonstrated the formationof a covalent molecular adduct between FA and primary aminogroups of the globin, forming the corresponding Schiff's base. TheN-terminal valine of globin on 2α and 2β chains may also undergo al-kylation, forming N-methylenvaline. The formation of this early bio-logical marker is dependent on the exposition to various alkylatingagents, among which FA represent a significant example, describedin a previous study (Bono et al., 2006).

R–CHO þ H2N–Val–Hb ⟷ R–CH ¼ N–Val–Hbþ H2O

aldehydesþ N–terminalðvalineÞ ⟷ N–terminal ðvalineÞ adductsPathologists and plastic laminate workers are two important classes

of professionally exposedworkers. Pathology laboratories use FA as a pre-servant and disinfectant in pathology laboratories. In particular, thehigher levels of exposition to FA in pathology wards are recorded in re-duction rooms, where FA is directly used to fix the biological tissues(Bono et al., 2010). Plastic laminate workers are exposed to FA releasedby pressed-wood products used in home construction, in furnishingscontaining urea-formaldehyde resins (e.g. particleboard, hardwood ply-wood, medium density fiberboard and paneling) and phenol-formaldehyde resin (e.g. softwood plywood, oriented strand board)(Kelly et al., 1999b). Therefore, the widespread use of FA in many work-ing contexts may represent a potential health risk whenever this pollut-ant is breathed or assumed by direct skin contact. These twooccupational environments represent typical cases where theworkers can potentially show biological response to FA exposition.The first aim of the present study was to investigate the relationshipbetween air-FA exposition and N-methylenvaline formation in bloodhemoglobin, taking into account tobacco smoke habits as a possibleconfounding factor. Secondly, we intended to compare the extentof N-methylenvaline formation in two populations of workers ex-posed to air-FA, namely pathologists and plastic laminate workers.

2. Materials and methods

2.1. Epidemiological sample

44 pathologists and 51 workers of an industry of plastic laminateswere recruited as subjects potentially exposed to air-FA. 78 subjects

were enrolled as controls from some scientific labs and officeswhere FA is not used (3 hospitals, 1 industry, 1 university and severaloffices in Piedmont region, Italy). All samplings were executed in2009. For each subject, an air-FA sample was passively collected foran entire working shift (i.e. from 6 a.m. to 2 p.m.; about 8 h) in themiddle of the working week (Wednesday). Information on personalmedical history, smoking habits, and drug intake were also collectedthrough a questionnaire administered at the end of the workingshift, when a sample of venous blood and a spot of urine were collectedas well. Immediately after – at about 4 p.m. – air sample, urine, andblood where taken to the lab refrigerated at +4 °C and immediatelyprocessed as follows: a) air samples were eluted in 3 ml of toluene,shaken and stored into a vial at−20 °C until analysis, b) urine were ali-quoted and then stored at −80 °C until analysis, and c) blood sampleswere treated to produce dried globin storable at −80 °C. In all threecases analysis was performed within few days.

The description of smoking habits for all subjects was establisheda-priori. Both the never-smokers and the former smokers who hadceased smoking from at least 1 month were classified as “non-smokers”, while “smokers” were the ones who smoked at least onecigarette per day. All subjects were informed about the objective ofthe study and voluntarily gave a written consent.

2.2. Personal air-FA

FA air sample was collected from each subject for the whole work-ing shift (8 h) using a passive, personal air sampler working with ra-dial symmetry (Radiello), clipped near the breathing zone of thesubject. Samplers were equipped with a specific sorbent tube con-taining 35–50 florisil mesh coated with 2,4-dinitrophenylhydrazine(DNPH). DNPH reacts with FA yielding 2,4-dinitrophenylhydrazonewhich was subsequently quantified using a HPLC with UV detectionat 360 nm: NIOSH Method no. 2016 (Eller, 1984). The instrumentwas set as follows: Perkin-Elmer series 200 binary pump, Perkin-Elmer LC 295 UV–vis detector, Gilson model 401 dilutor and model231 automatic sampler, 10 m LiChro CART 250-4 HPLC column andcartridge. The elution conditions were set as follows: mobile phase,45% acetonitrile and 55% water; 1 ml/min flow; injection volume:20 μl. The estimate limit of detection (LOD) was 0.05 μg/ml. Thecalibration curve was prepared using a calibration standard (2,4dinitrophenilhydrazone), with a certificated concentration of 3.83 μg/ml,which was diluted to yield working solution with concentrationsranging between 0.05 and 3 μg/ml.

2.3. Questionnaire

A questionnaire was administered to each subject to acquireinformation about individual and clinical features (age, sex, placeof residence, hobbies, and therapies), smoking habits, profession(qualifications, seniority, and job-specific work), and the presenceand use of environmental and personal devices to prevent air ex-position and health risks.

2.4. Blood sampling and N-methylenvaline analysis

For each subject, 10 ml of venous blood was collected in a heparin-ized tube at the end of the working shift, taken to the lab and imme-diately processed for the quantification of N-methylenvaline, asdescribed in a previous study (Bono et al., 2006). Briefly, the collectedblood was centrifuged and washed with NaCl. Then, ultrapure waterwas added to lise the erythrocytes. The emolysate was centrifugedand dialyzed in water. Acidic 2-propanol, ethyl-acetate, and pentanewere added to precipitate the globin, which was subsequently driedand stored at −20 °C. By applying the Edman degradation method,modified by Tornqvist (Tornqvist et al., 1986), the globin was

Table 1General description of some individual characteristics of all the 173 volunteers.

Exposed to formaldehyde Controls

Pathologists Plastic laminate workers

Subjects (n) 19 51 103Age (mean of years)a 38 40,9 39.1Gender (m/f) 8/11 51/0 30/73Smoking habits (yes/no) 6/13 23/28 24/79

a ANOVA test p=not significant.

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derivatized and the reduction was performed by the Kautiainenmethod (Kautiainen et al., 1989).

For each sample the procedure was applied twice. In the firstapplication, 50 mg of dry globin was diluted in formamide andbuffered with NaOH to obtain the N-methylvaline (N-methylvalinebackground). In the second application of the procedure, the globindiluted with formamide was followed by addition of NaBH4 inbuffered water (Tornqvist and Kautiainen, 1993).

R � CH ¼ N� Val� HbN−terminal valineð Þ adducts

→Reduction NaBH4ð Þ

R � CH2NH� Val� Hbreduced N�terminal valineð Þ adducts

This procedure achieved the reduction of the targetN-methylenvaline to stable N-methylvaline which adds up to back-ground N-methylvaline (N-methylvaline background+N-methylen-valine reduced to N-methylvaline). The target N-methylenvaline,produced by the alkylating activity of FA, is obtained by subtractingthe endogenous N-methylvaline from the total amount of N-methylvaline arising from the reduction procedure. In each sampleand for both procedures, N-methyl-L-isoleucinewas dissolved in form-amide and used as the internal standard. Subsequently, each samplewas derivatized with pentafluorophenyl isothiocyanate (PFPITC) andthe products were extracted with diethyl ether. The extract was dried,re-dissolved in toluene and washed with ultra pure water andNa2CO3. Lastly, the N-methylvaline-pentafluorophenyl thiohydan-toin derivative (N-methylvaline-PFPTH) was isolated togetherwith the internal standard by evaporation. The sample purificationwas executed by C-18 cartridges, before GC/MS analysis.

A calibration curve was prepared by dissolving 50 mg of globin informamide and adding appropriate concentrations of both calibratorand internal standard (Bono et al., 1999; Bono et al., 2005).

2.5. GC–MS analysis

The dried samples were dissolved in 100 μl of toluene and im-mediately analyzed. Analyses were performed using a Perkin Elmer(Norwalk, CT, USA) Turbomass single quadrupole GC–MS, operating inthe electron-capture negative chemical ionization mode. The injectionvolume was 1 μl, in the pulsed splitless mode. The capillary columnused was a Phenomenex, Zebron ZB-5, 30 m×0,25 mm×0,25 μm filmthickness. Initial column temperature was 80 °C, and increased by22 °C/min to 190 °C, then 5 °C/min to 235 °C and lastly by 20 °C/minto 300 °C; isothermal at 300 °C for 7 min. The transfer-line temperaturewas set at 200 °C. Carrier gas was ultrapure He (1.0 ml/min).

The mass analyzer was operated in the Selected Ion Monitoring(SIM) mode, by selecting m/z=290, 310 for N-methylvaline andm/z=314, 334 for N-methyl-L-isoleucine. The resulting chromato-graphic profiles were integrated to calculate the quantitative results.Coefficients of variation (CV,%) calculated to test repeatability were4,8%, for the internal standard derivative and 4,3% for N-methylvaline.

2.6. Urine collection and cotinine analysis

A spot of fresh urine was collected in the early morning andapproximately at the same time from each volunteer, and storedat −80 °C. Urinary cotinine was measured in order to consider thepossible role played by tobacco smoke in FA global intake for thesubjects under study. 10 ml of urine was transferred into a glasstube and 4 g of NaCl, 500 μl of NaOH (5 M) and 10 μl of cotinine-d3([2H3]-Cotinine) (internal standard) were added. Subsequently, cotin-ine was extracted from urine by repeating the following proceduretwice: addiction of 2 ml of trichloromethane (CHCl3), liquid–liquid ex-traction in a shaking wheel for 15 min, centrifugation for 10 min at1000 g. The two resulting organic phases were collected together andevaporated to dryness in a rotary evaporator. The cotinine calibrationcurvewasbuilt by fortifying a blank urinepool of non-smoking subjects,

to obtain a concentration range from 10 to 100 ng/ml. The fortifiedurine was extracted as for the samples.

The dry residue was dissolved in 200 μl of CHCl3 and transferredinto a conical vial. GC–MS analysis was performed using an AgilentTechnologies 6890 gas chromatograph, interfaced to a 5973 MSDInert Agilent mass spectrometer. A Gerstel CIS4 PTV injection systemutilized an initial temperature of 50 °C followed by heating at 10 °C/s;with a final temperature of 300 °C, held for 10 min. The injection vol-ume was 1 μl in the split mode. The capillary column used was aHP-5MS 30 m×0.25 mm×0.25 μm film thickness. The initial columntemperature was 50 °C, increased at 15 °C/min up to 300 °C. The carriergas was ultrapure Helium (1.0 ml/min). The transfer-line temperaturewas set at 280 °C. The mass spectrometer operated in electron impactand SIM mode. The monitored m/z values for cotinine were: 98.0;118.0; 176.0, while the ones for the internal standard were: 101.0;121.0; 179.0. Coefficients of variation (CV,%) calculated to test repeat-ability were below 5% for both cotinine and the internal standard.

2.7. Statistical analysis

The data of airborne chemicals and urinary biomarkers showed anon-normal distribution as proved by the Kolmogorov–Smirnov testfor all three populations (pathologists, workers in the plastic lami-nates industry, and controls) suggesting a log-transformation of datain order to stabilize the variance and normalize the distributions.Student's t-test and one-way analysis of variance (ANOVA) were thenapplied to compare two or more groups of independent samples,while Pearson's correlations were used to test the possible associationsbetween the variables. For ANOVA test, the equal variance of Tukeywasassumed for post-hoc multiple comparison. Lastly, a p value of ≤0.05(two-tailed) was considered significant for all tests. All the statisticalanalyses were performed using SPSS Package, version 17.0 (www.spss.com).

3. Results

Among the 44 pathologists recruited for the present study, theones working in reduction rooms (n. 19), where FA is directly used,showed a statistically significant higher level of air-FA (pb0.0001),unlike the ones working in other services (n. 25). Conversely, air-FAlevels measured for the latter group of pathologists show similarvalues than those recorded for the subjects recruited as controls.Consequently, they were included into the control group. The resultingfinal composition of groups comprised 19 pathologists (exposed to FA),51 plastic laminate workers (exposed to FA), and 103 controls. Table 1shows some general epidemiological aspects of the three resultinggroups of volunteers.

It is worth noting that the mean age of these three groups werecomparable, and any statistical differences were recorded excluding“age of the subjects” a possible confounding factor.

Tobacco smoke is a known source of air pollutants, including FAand many other alkylating agents (Bono et al., 1997; Bono et al.,2005). To quantify the exposition level to this important confoundingfactor, the urinary cotinine (i.e. the main nicotine metabolite) wasmeasured. Cotinine is a sensitive and specific biomarker of passive

Table 2Air-FA, N-methylenvaline and urinary cotinine concentrations in the three groups ofstudy. In brackets the log-transformed data are reported.

Exposed to formaldehyde Controlsa

Pathologists Plastic laminate workers

Number 19 51 103

FA μg/m3 (log-transformed data)Min 14.9 (1.172) 49.1 (1.691) 5.3 (0.725)Max 558.4 (2.747) 444.5 (2.648) 134.6 (2.129)Median 189.6 (2.278) 195.1 (2.290) 29.8 (1.474)Mean 188.6 (2.110) 210.1 (2.265) 41.4 (1.507)SD 144.2 (0.442) 104.5 (0.235) 29.4 (0.321)p ANOVA b0.0001p (19 vs 51) Tukey N.S.p (19 vs 103) Tukey b0.0001p (51 vs 103) Tukey b0.0001p (70 vs 103) t-test b0.0001

N-methylenvaline nmol/g of globin (log-transformed data)Min 22.13 (1.345) 30.80 (1.489) 0.7 (−0.156)Max 1606 (3.206) 1242 (3.094) 894.3 (2.951)Median 369.3 (2.567) 278.4 (2.445) 65.8 (1.818)Mean 377.9 (2.365) 342.8 (2.420) 144.8 (1.739)SD 362.7 (0.501) 259.2 (0.338) 204.8 (0.689)p ANOVA b 0.0001p (19 vs 51) Tukey N.S.p (19 vs 103) Tukey b0.0001p (51 vs 103) Tukey b0.0001p (70 vs 103) t-test b0.0001

Cotinine ng/ml (log-transformed data)Min 3.1 (0.491) 1.0 (0) 0.8 (−0.097)Max 1963 (3.293) 3306 (3.519) 1644 (3.216)Median 8.0 (0.903) 45.0 (1.653) 10.0 (1)Mean 409.1 (1.521) 704.3 (1.794) 129.6 (1.164)SD 706.0 (1.076) 954.2 (1.278) 307.4 (0.867)p (ANOVA) 0.002p (19 vs 51) Tukey N.S.p (19 vs 103) Tukey N.S.p (51 vs 103) Tukey 0.001p (70 vs 103) t-test 0.001

a Reference category.

704 Bono R. et al. / Science of the Total Environment 414 (2012) 701–707

and active tobacco smoke intake. In fact, correlation between num-bers of cigarettes smoked per day and cotinine levels among all 173subjects, calculated by means of the Pearson test, showed strongand highly significant correlation: r=0.814, pb0.0001 (Fig. 1).

To compare the biomarkers considered in this study among thethree groups, one-way ANOVA was performed, whose results areshown in Table 2. For air-FA and N-methylenvaline, a general differencebetween the three investigated groups was observed (pb0.0001). Asignificantly higher concentration of these markers for the twogroups of professionally exposed workers (N=19 and 51) in com-parison to controls (N=103) was demonstrated by the Tukeypost-hoc test. Conversely, the comparison between the two groupsof professionally exposed workers (N=19 vs. 51) does not showsignificant differences, in air-FA and N-methylenvaline concentra-tions. A peculiar aspect of the data reported in Table 2 is that similarlevels of exposition to FA produce similar biological response despitethe remarkable differences between the two professions.

Finally, by means of the t-test, the merged data for the two profes-sions (N=51+19=70) showed a significantly higher levels of FAand N-methylenvaline than for controls. Statistical analysis of urinarycotinine on the whole population underlines a general difference be-tween the three levels of exposition investigated (pb0.002) (Table 2).In detail, by comparing the groups of 19 and 51 FA-exposed workers,the post-hoc Tukey test does not show significant differences in thetobacco smoke habit, so as between the groups of 19 pathologistand 103 controls. Conversely, the group of 51 plastic laminate workersexhibits a significantly higher tobacco smoke habit than the controlgroup.

This observation can be possibly attributed to sociological reasons,indicating a larger use of tobacco in blue collars (i.e., the plastic lam-inate workers) than white collars (i.e., the pathologists and controls).Accordingly, the t-test confirms a higher level of tobacco consumptionin exposed subjects (N=70) than in controls (N=103) (p=0.001).

In order to separate the contribution of tobacco smoke in the for-mation of N-methylenvaline from the one due to the FA professionalexposition, the statistical analysis was repeated by excluding the ac-tive smokers, so as to consider only the 120 non-smokers (Table 3).The last group exhibits a negligible level of cotinine (10.6 ng/ml) sig-nificantly lower to that of active smokers (1093 ng/ml), pb0.0001.Also in the case of non-smokers a general difference among thethree levels of exposition investigated for air-FA (pb0.0001) and forN-methylenvaline (pb0.0001) is clearly evident. In detail, post-hocTukey test demonstrates a higher exposition to air-FA for the plasticlaminated workers than for pathologists (p=0.025) and a higher ex-position in the two professionally exposed groups than in controls: 13

Fig. 1. Correlation between urinary cotinine and number of cigarettes/d

versus 79 and 28 versus 79 (pb0.0001). Post-hoc Tukey test shows forN-methylenvaline no difference between the two types of profession-al exposition to FA, but significant differences were evidenced be-tween each group of FA-exposed and control group: 13 versus 79(p=0.023) and 28 versus 79 (pb0.0001). Finally, t-test confirms ageneralized higher level of N-methylenvaline in exposed (n. 70)

ie referred to the whole population (n. 173). pb0.0001, r=0.814.

Table 3Air-FA and N-methylenvaline in the three category of air-FA exposition of the 120 non-smokers. In brackets the log-transformation.

Exposed to formaldehyde Controlsa

Pathologists Plastic laminate workers

n=120 13 28 79

FA μg/m3 (log-transformed data)2Min 14.9 (1.173) 49.10 (1.691) 5.300 (0.725)Max 335.5 (2.536) 444.5 (2.648) 134.6 (2.129)Median 117.9 (2.072) 190.5 (2.279) 29.80 (1.474)Mean 136.1 (1.966) 208.5 (2.248) 40.44 (1.502)SD 106.7 (0.439) 115.9 (0.263) 28.33 (0.312)p ANOVA b0.0001p (13 vs 28) Tukey 0.025p (13 vs 79) Tukey b0.0001p (28 vs 79) Tukey b0.0001p (41 vs 79) t-test b0.0001

N-methylenvaline nmol/g globin (log-transformed data)Min 22.1 (1.345) 32.4 (1.511) 0.7 (−0.156)Max 626.2 (2.797) 1214.0 (3.084) 894.3 (2.951)Median 130.1 (2.114) 342.2 (2.254) 51.1 (1.708)Mean 244.7 (2.182) 380.6 (2.476) 133.6 (1.694)SD 211.7 (0.489) 258.9 (0.332) 197.5 (0.694)P b0.0001p (13 vs 28) Tukey N.S.p (13 vs 79) Tukey 0.023p (28 vs 79) Tukey b0.0001p (41 vs 79) t-test b0.0001

a Reference category.

Fig. 2. Box-plots of the four groups of N-methylenvaline data according to the fourquartiles of air-FA exposition in μg/m3: I quartile=159.2–558.4, II=66 one-wayANOVA test: p=0.0001.

705Bono R. et al. / Science of the Total Environment 414 (2012) 701–707

than in controls (N=103) (p=0.001). To evaluate the possible rela-tionships between the three biomarkers, Pearson's correlation testswere carried out after log-transformation of data. By analyzing thewhole population, the air-FA appears to be significantly correlatedwith N-methylenvaline (r=0.678; pb0.0001) and weakly correlatedto cotinine (r=0.291; p=b0.0001). A similar correlation was foundbetween air-FA and N-methylenvaline (r=0.658, pb0.0001) whenthe statistical analysis was restricted to non-smokers. No correlationwas recorded between air-FA and cotinine, as expected.

In order to assess the sensitivity of the N-methylenvaline markerin responding to an increased exposition to air-FA, independentlyfrom the profession of the subjects, a one-way ANOVA test was carriedout by dividing the 173 subjects into four quartiles according to themeasured air-FA values (Fig. 2). The corresponding N-methylenvalineconcentrations are represented by the average value and box-plotfor each quartile. The test provides high statistical significance(pb0.0001) demonstrating direct responsibility of air-FA exposi-tion in the N-methylenvaline formation. In particular, the post-hocTukey test underlines that the response of this biomarker as a func-tion of FA exposition is linear, particularly at low FA-levels. Actually,significant differences in N-methylenvaline concentration were foundbetween fourth and third quartile (pb0.0001), third and second(pb0.0001), fourth and second (pb0.0001), but not between secondand first quartiles (p=N.S.).

4. Discussion and conclusions

Both pathologists and workers in plastic laminates production,make use of formaldehyde despite the remarkable differences intheir professions. In the present work, it is demonstrated that the bi-ological effects of FA-exposure are clearly evident and comparable,no matter what are the conditions under which this professional ex-posure occurred. Taking into consideration the toxicological and car-cinogenic properties of FA, its widespread use in many working (andlife) contexts represents a potential risk factor for the human healthwhen it is breathed. As previously mentioned, the actual ceiling limitvalue (TLV-C) suggested by ACGIH is 0.3 ppm (0.370 mg/m3).

Our air-FA measurements showed that, during the sampling day,at least one pathologist and two workers of the plastic laminate in-dustry were exposed to air-FA at higher concentration than therecommended limit, as already described by several other authors(Costa et al., 2008; Orsiere et al., 2006; Pala et al., 2008). It can assumethat these three exceeding values may be possibly due to isolated ep-isodes of improper behavior or temporary hoods malfunction. Also forthe pathologists, the exposition level depends on their specific tasksand, possibly, by not perfectly ventilated indoor environments. Inparticular, the pathologists are subjected to a higher FA-expositionwhen they carry out the tissues reduction in direct contact with FA.While less hazardous substances are expected to replace FA, whenev-er possible, air-FA exposition must be reduced in both occupationalenvironments through better aspiration of exhausts and environmen-tal ventilation. Particularly, in a pathological wards the preventionappears easier, by both reducing the quantity of FA adopted for reduc-tion and treating under vacuum the anatomical tissues removed bythe patients in the operating room (Di Novi et al., 2010). These pre-ventive actions appear necessary, because the biological effect ofhemoglobin alkylation is clearly evident even at much lower air-FA concentrations than the ACGIH suggested limit, as the presentstudy demonstrated.

The differences of N-methylenvaline between exposed and controlsand between smokers and non-smokers have to be attributed to thedifferent N-methylenvaline formation not to different backgroundlevels of N-methylvaline, as it was verified that N-methylvalinebackground levels were the same (differences not significant) forall four groups mentioned above. Since FA represents an alkylatingpollutant present in both occupational and domestic environments,the formation of N-methylenvaline as an early biological effect result-ing from air-FA exposition was evaluated under critical conditions bycomparing two groups of voluntary subjects exposed to FA underdifferent professional activity and a control group. As a consequenceof their FA exposition, both for pathologists and workers of plasticlaminate a significant increase of hemoglobin N-methylenvalineconcentration was observed up to two times the control level(Table 2, the whole population). Conversely, the two occupationallyexposed groups recorded concentrations of the adduct of the sameorder of magnitude and not statistically different.

In order to evaluate the role of tobacco smoke (a possible con-founding factor) in the methylation of human hemoglobin and in theconsequent N-methylenvaline formation, the number of cigarettessmoked per day by each subject was correlated to his urinary cotinine.Fig. 1 demonstrated once more the high sensitivity and specificity ofthis internal dose marker. In Table 2, the analysis of cotinine in thethree groups evidenced a significant higher exposition to tobaccosmoke in the two categories of workers under study (Daher et al.,

706 Bono R. et al. / Science of the Total Environment 414 (2012) 701–707

2010). This aspect, even if apparently parallels the presence of formal-dehyde in tobacco smoke, does not seem to be due to absorption ofthe gas by this route but to other reasons. However among the pathol-ogists, the so low number of smokers (n. 6) does not allow to attributeany role to the profession in highest concentration of cotinine than con-trols (Tukey post-hoc test not significant). Instead, a significant higherconcentrations of cotinine in workers of plastic laminates (Tukeypost-hoc test: p=0.001) is probably dependent by their smokinghabits, larger as number of smokers and as number of cigarettessmoked per day (typical of the blue collars) than in controls (allwhite collars).

Even by excluding the smokers from the statistical evaluation (i.e.on a smaller population set), the data reported in Table 3 still show asignificantly higher concentration of N-methylenvaline for both pa-thologists (p=0.023) and plastic laminate workers with respect tothe control group.

In conclusion, a positive correlation was demonstrated betweenprofessional exposition to air-FA and hemoglobin alkylation to formN-methylenvaline molecular adduct in the two occupationally ex-posed groups of subjects considered. The one-way analysis of varianceunderlines a general positive significance of the model (pb0.0001)and evidences a minor role of tobacco smoke as a confounding factor.In general, the two occupationally exposed groups (pathologists andplastic laminate workers) present a higher level of exposition to FAthan the control group, but similar levels when compared with eachother. Some critical factors can be evidenced by considering: a) the ex-position variability, b) the different time described by the twomeasure-ments, namely 7 h for air-FA and 120 days for N-methylenvaline, thelife of the erythrocytes, and c) the N-methylenvaline variability(possible genetic polymorphisms).

Competing interest

All of authors declare to have no competing interests.

Fundings

This study was made possible by a grant from Regione Piemonte,Italy (grants 2008).

What this study adds

The novel knowledge provided by the present results includes (i)the evidence that exposition to formaldehyde induces early biologicaleffects in humans (i.e. formation of terminal N-methylenvaline inhemoglobin), as is demonstrated for the first time in two groups offormaldehyde-exposed workers; and (ii) the evidence that also to-bacco smoke can induce this biological response, independentlyfrom the occupational exposition to formaldehyde, but to a muchlower extent. Thus, the present study may provide a fundamentalcontribution to the design of a protective grid for workers andsmokers.

Acknowledgments

This study was made possible by a grant of from Piedmont Region,Italy (grants 2008). The authors kindly thank to doctors: Prof. Bussolati,Dr. P. De Giuli, Dr. G. Cera, heads of the pathology wards of Torino, Alba,and Mondovì, Dr. G. Monchiero, Dr. E. Laudani, Dr. D. Bogetti, Dr. P.Corino, Dr. M. Favilla, Mr. M. Cravero and all the workers (pathologists,plastic laminateworkers, and subjects non-exposed) for their voluntaryand generous collaboration.

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