formaldehyde S-hydroxymethyl-glutatione S-formylglutatione Formic acid CO 2 + H 2 O (exhaled) Na + HCOO - (urine)

Transcription

1 Roberto Bono Biological monitoring of exposure to formaldehyde. Different mechanisms of action; formation of protein adduct and evidence of oxidative stress formaldehyde Although formaldehyde is rapidly absorbed, the exposition to it is not easily assessable by direct measurement in biological fluids. This is because the bio-available portion of formaldehyde is rapidly metabolized and its metabolites are either incorporated into macromolecules via the onecarbon pool pathway or eliminated with the expired air (CO 2 ) and urine. S-hydroxymethyl-glutatione CO 2 + H 2 O (exhaled) S-formylglutatione Formic acid Na + HCOO - (urine)

2 This well-known figure of prof. Bolt reminds us that formaldehyde also may reacts non - enzymatically with sulfhydryl groups, urea, or amino groups: formaldehyde is an extremely reactive chemical forming methylene bridges and producing covalently Crosslinked complexes with Protein and DNA (DPCs) which can be measured in blood or cells of exposed subjects. Apart from the excreted component, Since adducts between formaldehyde and proteins are not subjected to repair mechanisms, their presence corresponds to the life of the protein and their abundance is proportional to exposition.

3 Kautiainen published in 1989 the demonstration that the aldehydes bind reversibly to primary amino groups such as, the N-terminal valine of hemoglobin, through the formation of the corresponding Schiff base. The N-terminal valine of globin may also undergo alkylation, forming N- methylenvaline. The formation of this early biological marker is dependent on the exposition to various alkylating agents, among which formaldehyde represent a significant example. R-CHO + H 2 N-Val-Hb R-CH = N-Val-Hb + H 2 O Aldehyde + N-terminal valine N-terminal valine adduct Kautiainen, A., Törnqvist, M., Svensson, K., Osterman-Golkar, S., November Adducts of malonaldehyde and a few other aldehydes to hemoglobin. Carcinogenesis 10 (11),

4 To allow accurate determination, the unstable Schiff base should be transformed to secondary amine by reduction with sodium borohydride (NaBH 4 ). R-CH = N-Val-Hb N-terminal (valine) adduct reduction (NaBH 4 ) R-CH 2 NH-Val-Hb reduced N-terminal (valine) adduct In practice, formaldehyde reacts with hemoglobin to form the N-methylenvaline (unstable), which is subsequently reduced to N-methylvaline, stable and measurable. reduction (NaBH 4 ) FA + Hb N-methylenvaline N-methylvaline CH 2 = N-Val-Hb CH 3 NH-Val-Hb For each sample the procedure to measure the methylenvaline is applied twice: In the first application, 50 mg of dry globin are used to obtain the background N-methylvaline In the second application, the globin is reduced to obtain the total N-methylvaline

5 reduction (NaBH 4 ) formaldehyde + Hb N-methylenvaline N-methylvaline unstable (non well measurable) stable (measurable) N-methylvaline, obtained by the reduction of N-methylenvaline coming from the alkylating activity of formaldehyde, is obtained by subtracting the endogenous (background) N-methylvaline measured the first time from the total N-methylvaline resulting from the reduction measured the second time. N-methylvaline corresponding to N-reduced methylenvaline = total N-methylvaline (background N-methylvaline + N-methylenvaline reduced to N-methylvaline) - background (endogenous) N-methylvaline a numerical example 25 = 35 10

6 Pathologists and plastic laminate workers are two important classes of professionally workers exposed to formaldehyde In pathology labs formaldehyde is used as a preservative and disinfectant. In particular, the higher levels of exposition in pathology wards are recorded in reduction rooms, where formaldehyde is directly used to fix the biological tissues. Plastic laminate workers are exposed to formaldehyde released by pressed-wood products used in home construction, in furnishings containing urea-formaldehyde and phenol-formaldehyde resins (e.g. particleboard, hardwood plywood, medium density fiberboard and paneling - softwood plywood, oriented strand board)

7 These two occupational environments represent typical cases where the workers can potentially show biological response to formaldehyde exposition and to a consequent potential health risk. The first aim of the study was to investigate the relationship between exposition to formaldehyde and N- methylenvaline formation in blood hemoglobin, taking into account tobacco smoke habits as a possible confounding factor. Secondly, we intended to compare the extent of N- methylenvaline formation in the two populations of workers exposed to formaldehyde.

8 Subjects recruitment: 95 subjects professionally exposed to formaldehyde 78 Controls not professionally exposed to formaldehyde For each subject, an formaldehyde sample was passively collected with a personal sampler for an entire working shift pathologists from 3 hospital wards in Piedmont region, Italy 51 workers of an industry of plastic laminates in Piedmont region Workers in hospital Workers in University Workers in offices Information on personal medical history, smoking habits, and drug intake were also collected through a questionnaire administered at the end of the working shift, when a sample of venous blood and a spot of urine were collected as well. All subjects were informed about the objective of the study and voluntarily gave a written consent.

9 Materials and methods MEDIA OR SAMPLING SYSTEMS questionnaire ANALYTE individual and clinical features, smoke, profession, and presence and use of devices. ANALYTICAL TECHNIQUE The questionnaire was administered by one interviewer Radiello FA air-samplers breathed formaldehyde HPLC with UV-Vis venous blood urine N-methylenvaline Cotinine Turbomass single quadrupole GC MS, operating in the electroncapture negative chemical ionization mode GC MS operated in electron impact and SIM mode At the end of working shift air sample, urine, and blood where taken (refrigerated at +4 C) to the lab and immediately processed: air samples were eluted in toluene, shaken and stored at 20 C until analysis, urine were stored at 80 C until the cotinine analysis, blood samples were treated to produce dried globin and stored at 80 C.

10 Among the 44 pathologists, the 19 working in reduction rooms, where formaldehyde is directly used, showed a statistically significant higher exposure (p<0.0001), unlike the ones working in other services (n. 25). Conversely, these 25 last subjects show similar values than controls. Consequently, they were included into the control group = 103 The resulting final composition of groups comprised 19 pathologists, 51 plastic laminate workers, and 103 controls. the mean age of these three groups were comparable, and any statistical differences were recorded Any significant differences were appreciated among the endogenous N-methylvaline of all the 173 subjects.

11 Correlation between numbers of cigarettes smoked per day and cotinine levels among the all173 subjects (Pearson test) showed strong and highly significant correlation: r = 0.814, p < , as expected.

12 µg/m 3 (log-transf.) Exposed to formaldehyde Pathologists 19 Plastic laminate workers 51 Controls a 103 A general difference between the three investigated groups was observed (p < ) for formaldehyde and N- methylenvaline. median (2.747) (2.290) 29.8 (1.474) mean (2,110) (2.265) 41.4 (1.507) ANOVA p< Tukey:(19 vs 51) N.S. - (19 vs 103) < (51 vs 103) < (70 vs 103) t-test < N mol/g globin (log-transf.) Exposed to formaldehyde Pathologists 19 Plastic laminate workers 51 Controls a 103 median (2.567) (2.445) 65.8 (1.818) Mean (2.365) (2.420) (1.739) ANOVA p< Tukey: (19 vs 51) N.S. - (19 vs 103) < (51 vs 103) < (70 vs 103) - t-test < a reference category A significantly higher concentration of these markers for the exposed workers (N=19 and 51) versus controls (N=103) was demonstrated for both the two markers. Conversely, the comparison between the two groups of professionally exposed workers (N = 19 vs. 51) does not show significant differences for both twe two markers. similar levels of exposition to formaldehyde produce similar biological response despite the remarkable differences between the two professions.

13 Exposed to formaldehyde urinary cotinine on the whole population underlines a general difference between the three levels of exposition investigated (p < 0.002) ng/ml (log-transf.) Pathologists 19 Plastic laminate workers 51 Controls a 103 median 8.0 (0.903) 45.0 (1.653) 10.0 (1) Mean (1.521) (1.794) (1.164) S.D (1.076) (1.278) (0.867) ANOVA p = Tukey:(19 vs 51) N.S. - (19 vs 103) N.S. - (51 vs 103) = (70 vs 103) t-test p = a reference category Tukey test does not show significant differences in the tobacco smoke habit with the exclusion of the 51 plastic laminate workers which exhibit a significantly higher tobacco smoke habit than the control group. This observation can be possibly attributed to sociological reasons, indicating a larger use of tobacco in blue collars (the 51 plastic laminate workers) than white collars (the pathologists and controls). On the whole, a higher level of tobacco consumption is confirmed in exposed subjects than in controls (p = 0.001)

14 The non-smokers In order to separate the contribution of tobacco smoke in the formation of N-methylenvaline, the statistical analysis was repeated considering only the 120 non-smokers. µg/m 3 (log-transf.) Exposed to formaldehyde Pathologists 13 Plastic laminate workers 28 Controls a 79 median (2.072) (2.248) 29.8 (1.474) mean (1.966) (0.263) 40.4 (1.502) ANOVA p < Tukey: (13 vs 28) (13 vs 79) < (28 vs 79) < (41 vs 79) t-test < a reference category A general difference is clearly evident (p < ) among the three levels of exposition to formaldehyde. Tukey test demonstrates a higher exposition to formaldehyde for the plastic laminated workers than for pathologists (p = 0.025) and a higher exposition in the two professionally exposed groups than in controls.

15 N mol/g globin (log-transf.) Exposed to formaldehyde Pathologists 13 Plastic laminate workers 28 Controls a 79 median (2.11) (2.25) 51.5 (1.71) Mean (2.18) (2.48) (1.7) The non-smokers A general difference among the three levels of exposition is clearly evident also for N- methylenvaline (p<0.0001). ANOVA p < Tukey: (13 vs 28) N.S. - (13 vs 79) = (28 vs 79) < (41 vs 79) - t-test < a reference category Tukey test shows no difference between the two types of exposition to formaldehyde (13 vs 28), but significant differences were evidenced between each group of professionally exposed to formaldehyde and control group (13 vs 79 and 28 vs 79)

16 In order to assess the sensitivity of the N- methylenvaline marker in responding to an increased exposition to formaldehyde, independently from the profession of the subjects, a ANOVA test was carried out by dividing all the 173 subjects into four quartiles according to the measured formaldehyde. One-way ANOVA p < IV quartile III quartile II quartile I quartile (µg/m 3 ) The corresponding N-methylenvaline concentrations are represented by the average value and box-plot for each quartile. The test provides high statistical significance (p < ) demonstrating direct responsibility of formaldehyde exposition in the N-methylenvaline formation. In particular, the response of this biomarker as a function of formaldehyde is linear at low levels

17 Discussion 1/3 1. Despite the remarkable differences in the 2 professions, we demonstrated that the biological effects of formaldehyde are clearly evident and comparable, no matter what are the conditions under which this professional exposure occurred. 2. Three subjects were exposed to higher concentrations than the limit suggested (0.370 mg/m 3 ) It can assume that these exceeding values may be possibly due to isolated episodes of improper behavior or temporary hoods malfunction. 3. While less hazardous substances are expected to replace formaldehyde, it must be reduced in both occupational environments through better aspiration of exhausts and environmental ventilation. Particularly, in a pathological wards the prevention appears easier, by both reducing the quantity of formaldehyde adopted for reduction and treating under vacuum the anatomical tissues removed by the patients in the operating room. 4. These preventive actions appear necessary, because the biological effect of hemoglobin alkylation is clearly evident even at much lower formaldehyde concentrations than the actual suggested limit

18 Discussion 2/3 1. The demonstrated differences of N-methylenvaline have to be attributed to the different N- methylenvaline formation, not to different background levels of N- methylvaline. 2. cotinine evidenced a significant higher exposition to tobacco smoke in the professionally exposed to formaldehyde. This aspect, even if apparently parallels the presence of formaldehyde in tobacco smoke, does not seem to be due to absorption of the gas by this route but to other reasons. 3. A significant higher concentrations of cotinine in workers of plastic laminates is probably dependent by their smoking habits, larger as number of smokers and as cigarettes/day, typical of the blue collars, than in controls. 4. Even by excluding the smokers from the evaluation, data reported still show a significantly higher concentration of N-methylenvaline for both pathologists and plastic laminate workers with respect to the controls.

19 Discussion 3/3 Some critical factors can be evidenced by considering: a) the exposition variability, b) the different time described by the two measurements, namely 7 h for formaldehyde and 120 days for N-methylenvaline, the life of the erythrocytes, and c) the N-methylenvaline variability (possible genetic polymorphisms).