Ring-shaped cataract and urinary metabolites among 2,4,6-trinitrotoluene exposed population of Pakistan.

BACKGROUND: Safety protocols are usually neglected among most of the trinitrotoluene (TNT)-exposed population, therefore, rendering the community prone to various occupational hazards. The current study highlights ring-shaped cataract and urinary metabolites of TNT among TNT-exposed population (n = 26) against a control group (n = 20). METHOD: An observational case-control study was carried out in two groups: subjects exposed to TNT in Dir and Bajour Agency, Pakistan, and a control group from the base hospital. We determined the presence of ring-shaped cataract and urine metabolites of TNT using slit-lamp biomicroscope and gas chromatography-mass spectrometric analysis, respectively. RESULTS: Results substantiate a high level of urine metabolites for exposed subjects compared to the control group (p < 0.001). Age had no significant effect (p > 0.05) on the presence of ring-shaped cataract and the level of urinary metabolites of TNT, while duration of exposure showed significant effect (p < 0.001). Females showed high incidence of ring-shaped cataract and urinary metabolites of TNT than men ( p < 0.001). The mean age of the exposed subjects was 51 ± 14.38 (Mean ± SD) years. The mean year of exposure was 49 ± 5 (Mean ± SD) years. CONCLUSION: This study showed TNT as a risk factor for the presence of ring-shaped cataract among TNT-exposed group in Pakistan. It is important to screen exposed community for the presence of ring-shaped cataract, and pre-clinical identification of TNT adducts to prevent systemic complications.

A novel method development and validation for determination of 2,4,6-Trinitrotoluene and its metabolites on LC-MS/MS.

LC-MS/MS has recently emerged as the best practice for simultaneous analysis of 2, 4, 6 Trinitrotoluene (TNT) and its metabolites. We have developed and validated an LC-MS/MS method for simultaneous quantification of 2, 4, 6 Trinitrotoluene (TNT) and its metabolites 4-ADNT, 2-ADNT, 2,4-DNT, and 2,6-DNT in urine samples. These four metabolites were acid hydrolyzed using 1 mL of urine followed by extraction using n-Hexane and ethyl acetate as an extracting solvent. Separation was achieved by centrifugation, and the supernatant was dried under nitrogen, reconstituted with water and acetonitrile, and then filtered. Chromatographic separation was achieved on Agilent Poroshel 120 EC-C18 column (2.1 mm × 75 mm × 2.7 µm) utilizing two mobile phases 0.1% formic acid in water and 0.1% formic acid in acetonitrile in gradient flow. The validated AMR of TNT and its metabolites was 7.8-1000 ng/mL. The method showed an excellent correlation (>0.99) for TNT and its metabolites. Accuracy and within/between day precision of TNT and its metabolites were within ±15%. The integrity of diluted samples was maintained for each dilution factor. The method was found stable after storage and freeze-thaw cycle. The presented method can be used for TNT screening in occupationally exposed ordnance factory workers.

Development of a microextraction by packed sorbent with gas chromatography-mass spectrometry method for quantification of nitroexplosives in aqueous and fluidic biological samples.

A new method for quantification of 12 nitroaromatic compounds including 2,4,6-trinitrotoluene, its metabolites and 2,4,6-trinitrophenyl-N-methylnitramine with microextraction by packed sorbent followed by gas chromatography and mass spectrometric detection in environmental and biological samples is developed. The microextraction device employs 4 mg of C18 silica sorbent inserted into a microvolume syringe for sample preparation. Several parameters capable of influencing the microextraction procedure, namely, number of extraction cycles, washing solvent, volume of washing solvent, elution solvent, volume of eluting solvent and pH of matrix, were optimized. The developed method produced satisfactory results with excellent values of coefficient of determination (R2  > 0.9804) within the established calibration range. The extraction yields were satisfactory for all analytes (> 89.32%) for aqueous samples and (> 87.45%) for fluidic biological samples. The limits of detection values lie in the range 14-828 pg/mL.

Exposure to trinitrotoluene and health effects among workers in an artillery and ammunition plant.

OBJECTIVE: To determine urinary trinitrotoluene (TNT), 2-amino-4, 6-dinitrotoluene (2ADNT) and 4-amino-2, 6-dinitrotoluene (4ADNT) and health effects upon workers in an ammunition plant. MATERIAL AND METHOD: The urine samples from forty munition workers and forty office workers were monitored for TNT and its metabolites by gas chromatography-mass spectrometry. The workers were interviewed with a questionnaire. RESULTS: The median levels of TNT and its metabolites were 112.84 and ranged from non-detectable (ND) to 1,833.81 mg/L. Median levels for 2ADNT were 11.66, ranging from ND to 360.89 mg/L. Median levels for 4ADNT were 19.95 and ranged from ND to 314.28 mg/L. There were significant correlations between TNT and 2ADNT in urine (r = 0. 700, p-value < 0.001), as well as for TNT and 4ADNT (r = 0.783, p-value < 0.001). Exposed workers reported eye, nose and throat irritations, weakness and headaches with considerably higher frequency than non-exposed workers. CONCLUSION: TNT levels in urine were strongly associated with 4ADNT and 2ADNT levels. Workers exposed to TNT complained of nose, throat and eye irritation, along with overall weakness and headaches.

Biomonitoring of workers cleaning up ammunition waste sites.

2,4,6-Trinitrotoluene (TNT) is an important occupational and environmental pollutant. In TNT-exposed humans, notable toxic manifestations have included aplastic anaemia, toxic hepatitis, cataracts, hepatomegaly and liver cancer. Therefore, it is important to develop protection measures and to monitor workers involved in the clean-up of ammunition sites. Haemoglobin (Hb) adducts of TNT, 4-amino-2,6-dinitrotoluene (4ADNT) and 2-amino-4,6-dinitrotoluene (2ADNT), and the urine metabolites of TNT, 4ADNT and 2ADNT were found in 22-50% of the exposed workers, but not in the control group. The exposed workers were wearing protective equipment. The levels of erythrocytes, haemoglobin, creatinine, serum glutamic pyruvic transaminase and lymphocyte levels were significantly lower in the exposed workers than in the non-exposed workers. The levels of blood urea and reticulocytes were significantly higher in the exposed workers than in the non-exposed workers. Headache (26%), mucous membrane irritation (16%), sick leave (18%), lassitude (8%), anxiety (6%), shortness of breath (3%), nausea (5%) and allergic reactions (8%) were reported by the exposed workers. In a further analysis the U-4ADNT levels and the Hb-adduct levels were compared to the blood parameter and the health effects. The blood parameters were not significantly different between the U-4ADNT positive and U-4ADNT-negative group. Headache, mucous membrane irritation, sick leave, lassitude, anxiety, shortness of breath and allergic reactions were statistically not different between the two groups. Also in the workers with Hb-4ADNT adducts no significant negative changes were seen in regards to the changes of the blood parameters or the health effects. According to the results of the present study, it appears that the blood parameter changes and the health effects are more influenced by other factors than by the internal exposure to TNT.

Comparison of biomarkers in workers exposed to 2,4,6-trinitrotoluene.

2,4,6-Trinitrotoluene (TNT) is an important occupational and environmental pollutant. In TNT-exposed humans, notable toxic manifestations have included aplastic anaemia, toxic hepatitis, cataracts, hepatomegaly, and liver cancer. Therefore, methods were developed to biomonitor workers exposed to TNT. The workers were employed in a typical ammunition factory in China. The external dose (air levels and skin exposure), the internal dose (urinary metabolites), the biologically effective dose (haemoglobin adducts, urinary mutagenicity), biological effects (chromosomal aberrations and health effects), and individual susceptibility (genotypes of xenobiotic-metabolizing enzymes) were determined. Haemoglobin-adducts of TNT, 4-amino-2,6-dinitrotoluene (4ADNT) and 2-amino-4,6-dinitrotoluene (2ADNT), and the urinary metabolites of TNT, 4ADNT and 2ADNT, were found in all workers and in some controls. The levels of the haemoglobin-adducts or the urinary metabolites correlated weakly with the skin or air levels of TNT. The urinary mutagenicity determined in a subset of workers correlated strongly with the levels of 4ADNT and 2ADNT in urine. The haemoglobin-adducts correlated moderately with the urinary metabolites and with the urinary mutagenicity. The genotypes of glutathione S-transferases (GSTM1, GSTT1, GSTP1) and N-acetyltransferases (NAT1, NAT2) were determined. In general, the genotypes did not significantly influence the haemoglobin-adduct levels and the urine metabolite levels. However, TNT-exposed workers who carried the NAT1 rapid acetylator genotype showed an increase in urinary mutagenicity and chromosomal aberrations as compared with slow acetylators. The haemoglobin adduct 4ADNT was significantly associated with a risk of hepatomegaly, splenomegaly and cataract; urine metabolites and genotypes were not associated with health effects. These results indicate that a set of well-selected biomarkers may be more informative regarding exposure and effect than routinely performed chemical measurements of pollutants in the air or on the skin.

Urinary metabolites of workers exposed to nitrotoluenes.

Nitrotoluenes are important intermediates in the chemical industry. 2,6-Dinitrotoluene (26DNT), 2,4-dinitrotoluene (24DNT) and 2-nitrotoluene (2NT) are carcinogenic in animals and possibly carcinogenic in humans. Thus, it is important to develop methods to biomonitor workers exposed to such chemicals. The authors have monitored the air and urine metabolite levels for a group of workers in China exposed to 24DNT, 26DNT, 2NT and 4-nitrotoluene (4NT). The metabolites 2,4-dinitrobenzylalcohol (24DNBAlc), 2-amino-4-nitrobenzoic acid (2A4NBA), 4-amino-2-nitrobenzoic acid (4A2NBA) and 2,4-dinitrobenzoic acid (24DNBA) resulting from exposure to 24DNT were found in 89, 88, 91 and 78% of the exposed workers, respectively. The metabolites 2,6-dinitrobenzylalcohol (26DNBAlc) and 2,6-dinitrobenzoic acid resulting from 26DNT exposure were found in 99 and 86% of the exposed workers, respectively. Quantitatively, 2A4NBA, 4A2NBA and 26DNBAlc were the major metabolites. The nitrobenzoic acids were the major metabolites resulting from exposure to 2NT and 4NT and were present in 96 and 73% of the exposed workers, respectively. Air concentrations of DNT and 2NT did not correlate with the levels of metabolites in the urine. In conclusion, the dinitrobenzyl alcohols and aminonitrobenzoic acids determined in the urine provided a good marker for recently absorbed dose and were intrinsically related to the bioactivation and detoxification pathways of DNT. Air measurements were not a good measure to predict internal exposure.

Determination of trinitrotoluene and metabolites in urine by means of gas-chromatography with mass detection.

Biological monitoring of chemicals and/or their metabolites has been a recent development in South Africa with the publication of the Hazardous Chemical Substance Regulations in 1995. Although trinitrotoluene (TNT) exposure has been included in recent legislative developments, only occupational exposure limits have been set. TNT does not have a biological exposure index in South Africa or internationally. This study was conducted to determine levels of TNT and its metabolite excretion in a sample of munition workers by using gas-chromatography with mass detection methodology. The metabolites 4-amino-2,6-dinitrotoluene (4ADNT) and 2-amino-4,6-dinitrotoluene (2ADNT) in urine of exposed subjects, as well as total TNT (the sum of 4ADNT and 2ADNT), were used to determine absorption and excretion due to TNT exposure. Results indicated an increased absorption of TNT during the working week as compared to other published international studies. Post-shift values exceeded pre-shift levels in all samples. More reliable and specific research is necessary to set a biological threshold limit for TNT and its metabolites since monitoring indicated no definite symptoms, signs or biological effects due to increased excretion.

Mutagenicity of HPLC-fractionated urinary metabolites from 2,4,6-trinitrotoluene-treated Fischer 344 rats.

The production and storage of explosives has resulted in the environmental accumulation of the mutagen 2,4,6-trinitrotoluene (TNT). In order to characterize the production of mutagenic urinary metabolites, 6-week old male Fischer 344 rats were administered 75 mg of TNT/kg or DMSO vehicle by gavage. The animals were placed into metabolism cages, and urine was collected for 24 hr. Following filtration, metabolites in the urine were deconjugated with sulfatase and beta-glucuronidase and concentrated by solid phase extraction. The eluate was fractionated by reverse-phase high-performance liquid chromatography (HPLC) using acetonitrile/water, and the fractions, were solvent exchanged in DMSO by nitrogen evaporation. Each HPLC fraction was bioassayed in strains TA98, TA98NR, TA100, and TA100NR without metabolic activation using a microsuspension modification of the Salmonella histidine reversion assay. Fractions 3, 5-18, 21, 22, and 24-26 contained mutagens detected by strain TA98. In the nitroreductase-deficient strain TA98NR, some mutagenic activity was lost; however, fractions 3, 6, 9-11, 15, and 25 clearly contained direct-acting mutagens. Fewer fractions were positive in strain TA100 (9-16, 19, 20, and 25) with less activity observed in the nitroreductase deficient strain TA100NR (fractions 3, 12, 14, 15, and 25). Although some mutagenic activity coeluted with known TNT metabolite standards, there were still many unidentified mutagenic peaks.

Mutagenic activity and metabolites in the urine of workers exposed to trinitrotoluene (TNT).

Urine samples taken after work and after a free weekend from 50 workers employed in various activities in a chemical plant manufacturing explosives were analysed. On the basis of hygienic surveys, the subjects were divided into three categories of exposure to trinitrotoluene (TNT). The urine analyses consisted of gas chromatographic identification of TNT and its two metabolites, 4-ADNT and 2-ADNT, and a determination of the mutagenic activity. Two frame shift detector strains of Salmonella typhimurium were used, TA 98 and TA 98 NR, the latter being deficient in endogenous nitroreductase activity. On the basis of previous results on TNT mutagenicity, no exogeneous metabolic system was used to test the urine concentrates. Both tester strains showed that the mean urinary mutagenic activity was higher in the after work samples than in post weekend samples from the same subjects, showing that bacterial nitroreductase activity was not significantly responsible for the mutagenicity, although the response was higher with strain TA 98 than with TA 98 NR. The interindividual variation in urine mutagenicity was high, however, and the difference between the two sampling times was statistically significant (p less than 0.05) only for the high exposed group (workers in trotyl foundry and sieve house). Correlation between urinary mutagenicity and concentration of TNT in urine was poor; correlation was significant only with the urinary concentration of 4-ADNT. The correlation between urinary TNT and both metabolites was good (p less than 0.001). These results suggest that analysis of 4-ADNT in urine would be a sufficient biological measure for controlling exposure to TNT.

Diazo-positive metabolites in urine from workers exposed to aromatic nitro-amino compounds.

The amount of diazo-positive compounds in urine from workers at a chemical plant producing pharmaceuticals and explosives was determined in samples collected after work and after a holiday. Forty-five persons working with aromatic nitro-amino compounds (ANA) showed a statistically significant (p less than 0.01) increase in the exposed samples (1.21 +/- 1.40 mmol/mol creatinine) compared to the unexposed samples (0.56 +/- 0.31 mmol/mol creatinine). No increase in the level of diazo-positive metabolites was found in the 25 workers not exposed to ANA compounds. In a follow-up study, 32 trinitrotoluene (TNT) workers were divided into three exposure categories and seemed to show a dose-dependent increase in the level of urinary diazo-positive metabolites. However, there was a considerable interindividual variation. The method seems suitable for the biological assessment of exposure to ANA compounds--at least on a group level. This may be valuable, especially in situations where significant dermal uptake is expected.

Applications of liquid chromatography-mass spectrometry in metabolic studies of explosives.

Liquid chromatography-mass spectrometry was used for the detection and identification of metabolites of 2,4,6-trinitrotoluene (TNT) in urine and blood. The metabolites were found in the urine of rats and in the blood of rabbits fed with TNT, in the urine of rats exposed to TNT by skin absorption and in the urine of TNT munition workers. The detected metabolites, formed by reduction processes, included 2-amino-4,6-dinitrotoluene, 4-amino-2,6-dinitrotoluene, 2,4-diamino-6-nitrotoluene and 2,6-diamino-4-nitrotoluene, in addition to untransformed TNT.

Metabolic studies of explosives. 5. Detection and analysis of 2,4,6-trinitrotoluene and its metabolites in urine of munition workers by micro liquid chromatography/mass spectrometry.

Metabolites of 2,4,6-trinitrotoluene (TNT) were found in the urine of a group of TNT munition workers. The urine extracts were analysed by micro liquid chromatography/mass spectrometry. The metabolites found included 2-amino-4,6-dinitrotoluene, 4-amino-2,6-dinitrotoluene, 2,4-diamino-6-nitrotoluene, 2,6-diamino-4-nitrotoluene and untransformed TNT. The detection limit of the metabolites in urine was 0.1 ng/ml for 20 ml urine samples.

Identification of urinary metabolites of 2,4,6-trinitrotoluene in rats by liquid chromatography-mass spectometry.

Metabolites of 2,4,6-trinitrotoluene (TNT) were found in the urine of rats fed with TNT. The urine extracts were analysed by liquid chromatography-mass spectrometry (LC-MS). Metabolites found included TNT itself as well as 2-amino-4,6-dinitrotoluene, 4-amino-2,6-dinitrotoluene and 2,4-diamino-6-nitrotoluene, indicating that reduction processes are responsible for the formation of these metabolites.