1 Institute of Environmental and Occupational Toxicology, Casella Postale 108, 6780 Airolo, Switzerland, 2 Department of Environmental and Occupational Medicine, The Medical School, University of Newcastle Upon Tyne, Newcastle Upon Tyne, UK, 3 Walther-Straub-Institut fuer Pharmakologie und Toxikologie, Ludwig-Maximilians-Universitaet Muenchen, 80336 Muenchen, Germany and 4 Chinese Academy of Preventive Medicine, Nan Wei Road 29, Beijing, P. R. China 100050
5 To whom correspondence should be addressed Email: gabriele.sabbioni{at}bluewin.ch
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Abstract |
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Abbreviations: 2AA4AT, 2-acetylamino-4-aminotoluene; 4AA2AT, 4-acetylamino-2-aminotoluene; 2AA6AT, 2-acetylamino-6-aminotoluene; 2A4NT, 2-amino-4-nitrotoluene; 4A2NT, 4-amino-2-nitrotoluene; 2A6NT, 2-amino-6-nitrotoluene; d, deuterated; DNT, dinitrotoluenes; 26DNT, 2,6-dinitrotoluene; 24DNT, 2,4-dinitrotoluene; EI, electron impact ionization; Hb, hemoglobin; 2MA, 2-methylaniline; 3MA, 3-methylaniline; 4MA, 4-methylaniline; NCI, negative chemical ionization; NT, mononitrotoluenes; 2NT, 2-nitrotoluene; 4NT, 4-nitrotoluene; OR, odds ratio; PFPA, pentafluoropropionic anhydride; t-DNT, technical dinitrotoluene; 24TDA, 2,4-toluenediamine; 26TDA, 2,6-toluenediamine; TNT, trinitrotoluene
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Introduction |
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DNT and NT are metabolized by the reduction of the nitro group(s) and/or oxidation of the methyl group (reviewed in ref. 10). One or both of the nitro groups may be reduced to the corresponding aminonitrotoluenes, toluenediamines or aminotoluenes, while the methyl group is oxidized to a benzyl alcohol or benzoic acid. Aminonitrotoluenes, toluenediamines and aminotoluenes can be further N-oxidized to yield N-hydroxyarylamines. The N-hydroxyarylamines and benzylalcohols may undergo further conjugation with sulfate, glucuronide or acetyl. The secondary products of N-oxidation or methyl-oxidation are responsible for the genotoxic and cytotoxic effects of these compounds. The initiation of chemical carcinogenesis generally involves the covalent binding of xenobiotics, or their reactive metabolites, with nucleophilic DNA centres (11,12). Hemoglobin (Hb) in blood erythrocytes is a molecular target for reactive electrophilic species, and thus has been used as a surrogate dosimeter to measure the proportion of exposure, which attacks nucleophilic targets such as DNA (13,14, reviewed in ref. 15). Therefore, Hb adducts are an excellent tool to biomonitor exposed workers. Hb adducts result from N-hydroxyarylamines, which are oxidized to nitrosoarenes in the erythrocytes and then form sulfinamide adducts with cysteine residues in Hb (Figure 1). 2NT forms Hb and DNA adducts in rats (16). DNA adducts of DNT and TDA were investigated by Froines' group (1719). 24DNT, 26DNT and 2,4-toluenediamine (24TDA) bind to liver DNA but 26TDA does not. Hb adducts in rats have been investigated by Froines' group (1921) and Neumann's group (22) whereby Hb adducts were found in rats dosed orally with 24TDA, 26TDA, 26DNT and 24DNT. The results obtained by the two groups were very different. Therefore, for the present work the experiments with rats were repeated. The Hb adduct profile in rats will be compared with the adduct profile present in Hb from a collective of Chinese workers involved in the production of trinitrotoluene (TNT). Blood samples, work place description, air measurements and medical examinations were collected from Chinese workers and non-matching controls. Hb adducts were determined for all subjects. The Hb adduct levels were compared with the other measured parameters in order to find dose-related effects of nitrotoluene exposures.
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Materials and methods |
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Methods
Questionnaire and medical examination. The study was performed in accordance with the principles embodied in the declaration of Helsinki (www.wma.net/e/policy/b3.htm). Informed consent was obtained from each worker. Blood collection, medical examination and questionnaire were all performed in the same week. Each participant was interviewed, using the questionnaire, about their general status, exposure history, smoking and alcohol consuming habits, previous medical record and present symptoms. The Medical Department of the Chinese Academy of Preventive Medicine performed the following examinations: (i) physical examinations: blood pressure, cardiovascular system, nervous system and heart rate. (ii) Routine blood and urine tests, liver function test: glutamic pyruvic transaminase, alkaline phosphatase, total protein, albumin, total bilirubin. (iii) Electrocardiogram. (iv) Ultrasonic type B examination for liver and spleen. (v) Serological assays of hepatitis B antigens and antibodies were conducted, because hepatitis B is common in China, and liver damage can also be caused by some of the nitroarenes.
Collection of blood samples. Blood samples were collected from employees manufacturing dinitrotoluenes and TNT in a factory situated in Liaoning (Liaoning Province, China). In total, 160 blood samples were collected from the DNT factory workers. Of these, 99 were from exposed workers and 61 were from non-matched, non-exposed controls working in the same factory. Blood samples were collected in EDTA tubes. The erythrocytes were separated from the plasma by centrifugation. The erythrocytes were washed three times with an equal amount of 0.9% saline buffer. The erythrocytes were stored below 20°C and shipped to Europe on dry ice. The erythrocytes were lysed by adding 4 vol of water. Cell debris was removed by centrifugation. Hb was precipitated with ethanol from the lysed erythrocytes. The precipitate was washed with ethanolwater (8:2), ethanol (2 times), ethanoldiethyl ether (3:1) and diethyl ether. The washed Hb was dried in a dessicator.
Hb hydrolysis. Hb (100 mg human Hb or 20 mg rat Hb) was dissolved in 0.1 M NaOH (2 ml) by vortex mixing in glass tubes (100 x 22 mm). The hexane solution (10 µl) with the internal standards (1 ng/10 µl of d4-2MA, d4-3MA, d4-4MA, d6-24TDA, d6-26TDA, d6-4A2NT, d6-2A6NT and 2 ng/10 µl d6-4AA2AT) was added to the basic Hb solution (pH >12). After 1 h in a shaking bath at room temperature the Hb was extracted with CH2Cl2 (3 ml). The mixture was vortex mixed for 1 min then separated by centrifugation at 2000 g for 10 min. The samples were frozen in liquid nitrogen, then thawed at room temperature to facilitate phase separation. The organic layer was dried through a pipette containing anhydrous Na2SO4 (1 g) and washed with CH2Cl2 (1.5 ml). The dried organic phase was collected in a graduated tapered tube (98 x 15 mm). Aliquots of PFPA (5 µl) were added to each sample and left to fully derivatize for 10 min at room temperature. The samples were evaporated carefully under a stream of nitrogen to 150 µl then transferred to 200 µl micro-inserts for autosampler vials (32 x 12 mm). The samples were carefully evaporated to dryness under a stream of nitrogen and the residue reconstituted in EtOAc (15 µl).
Quantification of the Hb adducts. The analysis was performed on a Hewlett-Packard gas chromatographer (HP 5890II) equipped with an autosampler (HP 7673) and interfaced to a mass spectrometer (HP 5989A). The PFPA derivatives of the aromatic amines were analyzed by splitless injection (2 µl) onto a fused-silica capillary column (Phenomenex ZB5, 15 m x 0.25 mm i.d., 0.5 µm film thickness) with a 1 m x 0.25 mm i.d. methyl-silyl retention gap (SupelCo). In all cases the initial oven temperature, the injector temperature and the transfer line temperature were set at 50, 280 and 280°C, respectively. The oven temperature was kept at 50°C for 1 min then increased at a rate of 50°C/min to 200°C, held for 1.2 min, then heated at 50°C/min to 280°C, and held for 1 min. Helium was used as a carrier gas with a flow rate of 1.5 ml/min. For negative chemical ioniziation (NCI), with methane as the reagent gas, the source pressure was typically 160 Pa, the electron energy was 150 eV, the emission current was 300 mA, the quadrupole temperature was 100°C and the source temperature was 200°C. For EI, the electron energy was 70 eV, the emission current was 300 µA, and the source temperature was 200°C. Additional sensitivity and specificity were obtained by monitoring individual ions for each of the analytes. The elution times and the major mass fragments detected are listed in Table I. The dwell times for analyte masses: m/z = 233, 237, 374, 394, 398, 399, 284, 278 and 298, in group 1 (3.005.50 min), were 40 ms; and for analyte masses: m/z = 295, 296, 289, and 290, in group 2 (5.507.00 min), were 80 ms for GC-MS-NCI-SIM. The level of hydrolyzable Hbarylamine adducts present in each Hb (100 mg) was calculated from a calibration line of known standards, spiked into bovine Hb and taken through the assay procedure. The ion abundance of each amine peak, [M-HF], detected in the single ion chromatogram was related to that of the deuterated internal standard. The ratio was then compared with the abundance ratio determined for the calibration line. For quantification of 2MA, 3MA and 4MA, concentrations of 0, 100 and 300 pg/100 mg Hb were used in the calibration line. For 26TDA and 24TDA, concentrations of 0 and 100 pg/100 mg were used. For 2A6NT and 2A4NT, concentrations of 0, 100, 300, 500 and 800 pg/100 mg Hb and 0, 100, 500, 2500 and 5000 pg/100 mg Hb were used, respectively. All regression lines fitted with R2 >0.98.
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Statistical analyses
Statistical analyses were performed with the program SPSS 10.0, Sigma Stat 2.0, and Sigma Plot 3.0. The results of the questionnaire and of the medical examination were not known to the scientists performing the Hb adduct analyses. All results were disclosed at the end of the analyses. First univariate logistic regression analysis was used to compare the disease with the log-transformed Hb adduct data. The inclusion of a constant was allowed. Confounding factors like age, smoker status and gender were introduced stepwise using a multivariate logistic regression analysis. The likelihood ratio statistic based on the maximum partial likelihood estimate was used for variable entry. The criterion for entry was set at P < 0.05.
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Results and discussion |
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Determination of Hb adducts in Chinese workers exposed to mono- and dinitrotoluenes
Exposure to mixed nitrotoluenes in workers was determined by measurement of the level of arylamine-cleavage products released from Hb following mild base hydrolysis. The full scan mass spectra m/z 50500 of PFPA derivatized standards, 2MA, 3MA, 4MA, 26TDA, 24TDA, 2A4NT, 4A2NT, 2A6NT, 2AA4AT, 4AA2AT and 2AA6AT were acquired in both NCI and EI modes. For analysis of low levels of the above amines in human Hb samples, increased sensitivity was achieved by selection of the most abundant characteristic fragments, determined in the full scan spectra of authentic standards. These selected ions were acquired in single ion monitoring and have been presented in Table I.
The PFPA derivatized arylamines were identified by their characteristic mass fragments in the NCI mode, and by their retention times with respect to authentic deuterated internal standard (Table I). Single ion chromatograms, acquired in the NCI mode, have been presented in Figure 2. The presented chromatograms were from CH2Cl2 extracts of the Hb hydrolyzates from an exposed worker and a control worker. The methylanilines 2MA, 3MA and 4MA were observed at 3.27, 3.37 and 3.42 min, respectively. The toluenediamines, 26TDA and 24TDA, were observed at 4.41 and 4.50 min, respectively. The aminonitrotoluenes, 2A6NT and 4A2NT, were observed at 4.33 and 4.66 min and the acetylated adduct 4AA2AT at 5.97 min.
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There were a number of arylamine adducts identified in the organic extracts of base hydrolyzed Hb from workers exposed to mixed nitrotoluenes. These included (i) methylanilines, 2MA, 3MA and 4MA, which were indicative of exposure to 2NT, 3NT and 4NT, (ii) the toluenediamines, 26TDA and 24TDA and (iii) the aminonitrotoluenes, 2A6NT and 4A2NT, which were indicative of exposure to 24DNT and 26DNT. Only one acetylated arylamine, 4AA2AT, was observed in the extracts of Hb hydrolyzates from exposed workers. The aminonitrotoluene, reduced at the ortho position (2A4NT), and the acetylated amines, 2AA4AT and 2AA6AT, were not identified in the single ion chromatograms of exposed workers.
Quantification of arylamines covalently bound to Hb was based upon the analytical procedure described by Sabbioni and Beyerbach (25,26). The level of hydrolyzable Hbarylamine adducts present in each Hb (100 mg) was calculated from a calibration line of known standards spiked into bovine Hb and taken through the assay procedure. The ion abundance of each amine peak, [M-HF], detected in the single ion chromatogram was related to that of the deuterated internal standard. The ratio was then compared with the abundance ratio determined for the calibration line.
Job-related exposure to mono- and dinitrotoluenes
Hb adducts were found in the exposed workers and in parts in the controls working in the same factory. The box plots, presented in Figure 3 show the median Hb adduct levels of 2MA, 3MA, 4MA, 26TDA, 24TDA, 2A6NT and 4A2NT, with the 10th, 25th, 75th and 90th percentiles presented as vertical boxes and error bars. 2MA, 3MA and 4MA were found in all exposed and factory controls. 4A2NT, 2A6NT, 24TDA and 26TDA were found in 99, 96, 100 and 85% of the exposed workers, respectively. In the factory controls 4A2NT, 2A6NT, 24TDA and 26TDA were present in 62, 31, 64 and 62% of the workers. 4AA2AT was found in 20% of the exposed workers. This is probably due to the limit of determination, which is 40 times higher compared with the other compounds, and to the chromatographic properties of 4AA2AT, which yields broad peaks.
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The workers were grouped according to their job description as follows: group leader (n = 14), NT-tank (n = 15), DNT-tank (n = 7), TNT-tank (n = 19), analysis laboratory (n = 18), transportation of TNT to packaging (n = 2), packaging (n = 7), control room (n = 12), disposal of waste acid (n = 2) and disposal of waste H2O (n = 3), and control workers (n = 61). For workers grouped into waste acid, waste H2O or transportation of TNT, statistical analysis was not performed because the sample number was too low (n < 5). In general the adduct levels decrease in the following order among the worker groups: analysis > TNT-tank > NT-tank > DNT-tank > group leaders > control room workers > packing > factory controls. The relative levels of each DNT Hb adduct are closely paralleled between the differently grouped workers, such that high levels of 4A2NT in analysis workers are associated with high levels of 24TDA, 2A6NT or 26TDA in the same analysis workers. The most significant differences among the groups were found for adducts of 2MA, 2A6NT, 4A2NT and 24TDA using the MannWhitney test. From 28 possible comparisons between worker groups six, seven, nine and nine comparisons were not significantly different for 24TDA, 4A2NT, 2A6NT and 2MA, respectively. In Figure 4, the adduct levels of 4A2NT are presented as an example. All the differences between the groups are significant (P < 0.05) for 4A2NT adducts, except for the following seven comparisons: factory controls versus packers; analysis versus TNT-tank workers; TNT-tank versus DNT-tank workers; TNT-tank versus NT-tank workers; DNT-tank versus NT-tank workers; DNT-tank workers versus group leaders.
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The adduct levels of the major adducts found in over 96% of the exposed workers were compared using correlation analysis to determine to what extent one amine could predict the internal exposure dose of another amine. The major adducts found in the workers were 2MA, 24TDA, 2A6NT and 4A2NT. Linear regression analysis was performed on the raw Hb adduct data (pmol/g Hb) and on the log-transformed data (log base 10 of Hb data pmol/g Hb). In addition, the data were compared by the Spearman rank test. The statistical significance associated with each correlation has been presented along with the correlation coefficients in Table II. The correlation coefficients obtained from the log-transformed data are almost identical to the correlation coefficients obtained from the Spearman rank test. In contrast the correlation coefficients obtained from the linear regression of the raw data differ substantially in some cases, indicating that outliers skew the analyses. In the following text the correlation coefficients of the log-transformed data are discussed. There is a high correlation between the aminonitrotoluenes, 2A6NT and 4A2NT (r = 0.94, Figure 5). The association between these amines and the total level of combined Hb adducts of 24DNT and 26DNT is very strong (r = 0.96 for 2A6NT and 0.99 for 4A2NT) and confirms that these amines strongly predict the total level of hydrolyzable Hb adducts of 24DNT and 26DNT. These amines give a good indication of the absorbed dose following exposure to 24DNT and 26DNT in exposed workers. There is a high correlation between the level of 24TDA and 2A6NT (r = 0.82) or 4A2NT (r = 0.87). For 4A2NT, 2A6NT and 24TDA, correlation coefficients of the log-transformed data are very close to the Spearman rank test, and comparable with the raw data, which confirms that the raw data are not adversely skewed, and no one data point takes too much weight in the regression line.
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In conclusion, 4A2NT is the best marker to monitor workers exposed to DNT: 4A2NT is the major adduct and present in 99% of the workers, and 4A2NT correlates with the other major adducts resulting from DNT exposure. For exposure to NT, 2MA is the most appropriate marker, as it is the major adduct and is present in 100% of the workers.
Identification and quantification of Hb adducts in dosed rats
Experiments to identify Hb adducts in rats (n = 3) dosed with 0.5 mmol/kg 24TDA, 26TDA, 24DNT and 26DNT were performed in order to corroborate the Hb adducts identified in the DNT workers. The average level of each arylamine detected in the PFPA-derivatized extracts of base hydrolyzed Hb (three rats per compound analyzed in duplicate), has been presented in Table III. From these results the Hb binding index (HBI) was determined for each cleavage product and also for the total amount of compound bound to Hb. Hydrolyzable Hb adducts were found for each of the compounds investigated. The same spectrum of adducts was observed in the rat Hb extracts as determined in the Hb extracts from workers exposed to 24DNT and 26DNT.
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Hydrolysis of Hb from rats dosed with 26TDA yields two amines, 26TDA and 2AA6AT. The levels of 2AA6AT are 5-fold higher than 26TDA. Hydrolysis of Hb from rats dosed with 26DNT yields three amines, 2A6NT, 26TDA and 2AA6AT. The level of 26TDA is equivalent in rats dosed with either 26TDA or 26DNT. This was in contrast to the results obtained with rats dosed with either 24TDA or 24DNT, where the level of 24TDA is 31-fold higher in the rats administered 24DNT. The level of the acetylated amine, 2AA6AT, found in rats dosed with 26DNT is 5.7-fold lower than with 26TDA.
Hb adduct levels are higher in rats dosed with the dinitrotoluenes compared with rats dosed with toluenediamines. Hb adduct levels in rats dosed with 24DNT are higher than adduct levels in rats dosed with 26DNT. In contrast Hb adduct levels in rats dosed with 24TDA are lower than in rats dosed with 26TDA.
Generally, less oxidizable arylamines bind, to a larger extent, to Hb (28,29). The adduct levels found for 24TDA, 24DNT, 26TDA and 26DNT follow this rule. For 24TDA more 4AA2AT is present than 24TDA. 4AA2AT is less oxidizable than 24TDA as demonstrated by the calculation of the stability of the intermediate oxidation product, the nitrenium ion (data not shown). The same argument explains the higher levels of 2AA6AT than 26TDA. For 24DNT, 4A2NT is less oxidizable than 4AA2AT and 24TDA. This corresponds with the highest adduct level found for 4A2NT. 2A4NT adducts are not present. Nitro groups, which are co-planar with the aromatic ring, like the nitro group in the 4 position of 24DNT, are more easily reduced than nitro groups in the ortho position to a methyl group forced out of the plane. The reducibilty of nitro groups can be estimated with the LUMO calculated with semi-empirical programs (AM1, PM3). This explains also the difference between the total adduct levels in rats given 24DNT and 26DNT. 24DNT is better reducible than 26DNT. The oxidizability also explains the higher levels of 26TDA compared with 24TDA. 26TDA is less oxidizable than 24TDA.
As shown in Table III there are major differences between the results of the three different studies: the present study, Wilson et al. (1921) and Zwirner-Baier et al. (22). The results of Wilson et al. (1820) were obtained in a different rat strain (male Fischer rats) and using a different work-up. Wilson did not use an internal surrogate standard, but an internal volumetric standard 2,6-dimethylaniline, which was added after the hydrolysis and extraction of Hb. Therefore, any differences in extraction or derivatization of 24TDA and 26TDA, would not be accounted for using an internal volumetric standard. In addition Wilson et al. used hydrolysis conditions (concentrated NaOH, 37°C), which would cleave all monoacetylated amines to the parent diamines. In addition, it appears that Wilson et al. did not look for 4A2NT, 2A4NT and 2A6NT. Therefore, only the analyses of the diamines 26TDA and 24TDA were reported. Zwirner-Baier et al. (22) used the same animals and dose regimen to those reported in the present study. The major differences are the extraction procedure, the internal standards and the analysis procedure [HPLC with electrochemical detection (ECD)]. Unfortunately the internal standard used was not specified in the publication (22). In our work we used the deuterated analogs for each arylamine. Therefore, differences in relative recovery of the Hb adducts relative to the internal standard where accounted for in our study. In addition HPLC-ECD methods have in general a higher determination limit than GC-MS-NCI analyses. Therefore, some adducts in animals given 24TDA, 24DNT and 26DNT were missed in the earlier study (22). In summary, only our method allows the analysis of all possible Hb adducts.
Comparison of rat hemoglobin with human Hb binding
The Hb-cleavage products detected in hydrolyzed Hb from workers exposed to DNT were qualitatively comparable with the Hb adducts reported in rats administered 24DNT or 26DNT. The predominant Hb-cleavage product in workers exposed to 24DNT was 4A2NT. Low levels of 24TDA, and in some cases similar amounts of the acetylated product, 4AA2AT, were also detected. As in the case of rats administered 24DNT (Table III), mainly reduction of the nitro group para to the methyl group was observed.
In humans the ratio between 4A2NT and 24TDA was very different compared with rats. In humans the ratio for 4A2NT and 24TDA was 24:1 and in rats it was 4:1. Quantitatively, 4A2NT was not as prevalent in rats as in humans. This difference indicates that the concomitant reduction of both nitro groups in man was less prevalent than in rat.
The spectrum of Hb adducts formed in rats and the workers exposed to 26DNT were similar although the acetylated adduct, 2AA6AT was not found in the workers. The quantitative differences between levels of each 26DNT Hb-cleavage product were similar to that described for the 24DNT isomer. In humans the ratio for 2A6NT and 26TDA was 14:1; however, in rats it was 2.1:1. Therefore, also in the case of 26DNT, the concomitant reduction of both nitro groups was more prevalent in rats than in the workers.
To date, only one other group has published data on the fate of DNT in the blood tissues of humans. Neumann et al. (30) quantified DNT-derived Hb adducts in residents living in an area containing soil contaminated with explosives waste and in a control population. 2A4NT, 4A2NT and 2A6NT were analyzed and found at similar levels in the exposed residents and in the control population. The reported adduct levels were higher than in the present study of Chinese workers exposed to DNT. It is peculiar that the general environmental exposure in Germany appears to be higher than the occupational exposure in a Chinese factory. Unfortunately, no experimental details were given for the German study (30,31) and as such the results cannot be reproduced and cannot be discussed further.
To date, Hb adduct determinations had not been described for humans exposed to mono-nitrotoluenes (2NT, 3NT or 4NT) although Hb adducts in rats have been described. The Hb-cleavage products identified in rats administered 0.5 mmol/kg 2NT, 3NT or 4NT were 2MA, 3MA and 4MA (32). The same Hb-cleavage products were identified in hydrolyzed Hb from the Chinese workers.
Relationship between internal dose of NT and DNT and health effects in Chinese workers
In previous studies where the health status of workers exposed to DNT was assessed, the most common complaints recorded were due mainly to the ability of DNT to induce MetHb, the secondary effects of which were non-specific health effects such as headache, dizziness, nausea and drowsiness (32,33).
A full medical examination was performed on each of the Chinese workers by doctors from the Institute of Occupational Medicine, at the Chinese Academy of Preventative Medicine. Each worker was examined for non-specific adverse health effects linked to exposure to DNT, the results of which have been presented in Table IV. Of particular interest was the investigation of doseresponse relationships in the Chinese workers exposed to NT and DNT. We were interested in defining whether biomarkers of recent exposure or chronic exposure correlated with the risk of suffering from one or more of the health conditions, symptomatic of toxic exposure to nitroarenes. Logistic regression analysis was used to predict the presence or absence of a particular condition in the Chinese workers. The logistic regression was performed using the log-transformed data of Hb-cleavage products. The obtained odds ratios (OR) are listed in Table V. The OR indicates the odds of suffering for the various health effects of subjects with one log-unit more of each adduct relative to the odds of subjects with one log-unit less. The results presented in Table V show that there is a significant doseresponse relationship between the log-transformed values of the aminonitrotoluene Hb-cleavage products and the risk of suffering from inertia, somnolence, dizziness or nausea. The odds of suffering from inertia were 3.2 times higher when the level of 4A2NT Hb adducts increased by one log-unit (P < 0.001). Similar ORs were observed with somnolence (3.1), nausea (2.4) and dizziness (5.5). The OR of suffering from inertia, somnolence, dizziness or nausea increased by a factor of 3.2, 3.3, 7.8 and 2.7, respectively, based upon the 2A6NTHb adduct levels. These results were tested for confounding factors like age, smoker status and gender using stepwise forward logistic regression analysis. In the case of nausea, age is a borderline confounder. The age-adjusted OR increases only by 4, 3.5 and 8.7% for the adducts of 4A2NT, 2A6NT and 24TDA, respectively. Therefore, the crude ORs have been listed in Table V. These results inferred that quantification of DNTHb adducts provided an effective biomarker of toxicity and could be used to estimate the risk associated with a particular exposure to DNT.
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The absorbed dose per day was calculated from the Hb adduct levels using the equation for the steady-state level of Hb adducts. In the rat experiments with 24DNT, 26DNT and 2NT (37), 0.0398, 0.0075 and 0.010435% of the dose were bound to Hb. Assuming that Hb binding in rats and man was comparable, the daily exposure dose of 24DNT, 26DNT and 2NT in workers was estimated. The 95th percentile adduct level of 24DNT, 26DNT and 2NT corresponded to a daily dose of 20.53, 11.02 and 3.46 µg/kg/day, respectively. An excess lifetime cancer risk (ELCR) for exposure to these chemicals was estimated using the formula published by the US regulatory agencies agency (38,39; http://risk.lsd.ornl.gov): (ELCR) = (cancer slope factor) x (human dose). Workers are not exposed 7 days/week and 52 weeks/year to the work place contaminant. As we deduced the external dose from the internal dose, days without exposure were included. Therefore, the dose was not corrected for the days without exposure. As workers are not exposed for a lifetime of 70 years to the occupational pollutants, but for 40 years of work life, the dose was corrected with the factor 40/70. The cancer slope factors for 24DNT (40; www.epa.gov/iris), 26DNT (40) and 2NT (41) are 0.68, 0.68 and 0.23 (mg/kg/day)1, respectively. Therefore, the excess cancer risk for lifetime exposure to 24DNT, 26DNT and 2NT are 8 in 1000, 4.3 in 1000 and 4.6 in 10 000. Adding up the risks yields 1.3 in 100 for a lifetime exposure and the worst-case scenario. Taking the average adduct levels of the exposed workers the risk for 24DNT, 26DNT and 2NT are 2.5 in 1000, 1.3 in 1000 and 2.5 in 100 000, respectively. A risk of 1 in 106 is perceived as the virtual safe dose. In any case the risk resulting from 24DNT and 26DNT exposure is far above these levels. Therefore, the exposures in this group of workers should be drastically reduced. This is in accordance with the German Research Commission (42) who classified DNT mixtures as a Category 2 carcinogen. No MAK value (maximum concentrations at the workplace) was listed as a safe concentration range cannot be given. This is in contrast to the permissible exposure limit (PEL) set by the US Occupational Safety and Health Administration (43). For 24DNT and 26DNT the PEL was set at 1.5 and 1.5 mg/m3 as an 8-h time-weighted average, respectively. Assuming that a 70 kg worker inhales 9.6 m3 air/day, and assuming 100% absorption of the dose, the daily permissible exposure dose would be 206 µg/kg/day for 24DNT, and/or 26DNT. Such exposure levels would yield a risk of 8 in 100. Also for other compounds the PEL values appear to be too high when compared with the cancer risk deduced from the animal experiments (34).
The calculated carcinogenic risk was compared with organ-specific changes. The prevalence of tissue changes (hepatomegaly) in the liver of the Chinese workers exposed to nitrotoluenes was evaluated: only 2 out of 105 exposed workers showed a deviation from the norm (>1.0 cm). This ratio was not statistically different with that reported in factory control workers (1 out of 14). For tissue changes in the spleen (splenomegaly) only 1 out of 105 exposed workers compared with 1 out of the 14 factory controls showed a deviation from the norm with regard to spleen size. For these spleen and liver investigations the number of controls was only 14 and not 61 as for the other analyses. However, the analyses of 14 controls showed that the prevalence is not lower than in exposed workers. Based upon the high-calculated carcinogenic risk we would have expected to see a higher number of workers with tissue changes in the liver or spleen, although it was apparent from the epidemiological data that the urinary tract was the principle target tissue in humans. Possibly the studied population was not large enough and/or humans are not so sensitive to these compounds.
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Acknowledgments |
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References |
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