EXPLOSIVES AS TOXIC ENVIRONMENTAL POLLUTANTS: THE L EVEL OF CONTAMINATION, TOXICITY, AND ITS MECHANISMS

Narimantas Č÷nas*, Aušra Nemeikait÷-Č÷nien÷**, Audron ÷ Marozien÷*,

Jonas Šarlauskas*,Valentina Vilutien÷**, Juozas Baublys**

* Institute of Biochemistry, Mokslininkų 12, LT-08662 Vilnius, Lithuania

** The General Jonas Žemaitis Military Academy of Lithuania, Šilo 5a, LT- 10322 Vilnius, Lithuania, E-mail: ncenas@bchi.lt, hidrazon@bchi.lt,

ivk@lka.lt

Abstract. The survey of literature analyzes the levels of the environment contamination by explosives, and their toxic effects to humans. It is possible to conclude that among modern explosives, pentaerythritol tetranitrate (PETN) is the least toxic for humans. The impact of hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) on humans is unclear. Since 2,4,6-trinitrotoluene (TNT) is the most widespread explosive, its toxicity (methemoglobinemia, cataract, liver disorders) has been reported most intensively. The mechanisms of toxicity of TNT and other nitroaromatic explosives involve flavoenzyme-catalyzed single- and/or two-electron reduction (redox cycling of free radicals and/or formation of alkylating hydroxylamines), and the direct oxidation of oxyhemoglobin. In general, the toxicity of nitroaromatic explosives increases with an increase with their electron-accepting potency. Thus, some explosives of novel generation, e.g., 5-nitro-1,2,4-triazol-3-one (NTO), 5-nitro-1,2,4-triazol-3-amine may be less toxic to humans and other mammalian species than TNT. The experimental data of the present work show that xanthine oxidoreductase and cytochromes P-450 may be involved in the activation of nitroaromatic explosives in mammalian cells. Besides, some amino- metabolites of TNT may be more efficient than expected methemoglobinemia-inducing agents in erythrocytes. Keywords: explosives, toxicity, mechanism, negative influence, impact, redox, methemoglobin, oxyhemoglobin.

1. Introduction.

Currently, the research project “Analysis, evaluation and modeling of the impact on the environment of explosion products coming from explosive substances and ammunition used in training areas during military training” is being carried out at the General Jonas Žemaitis Military Academy of Lithuania, partly in cooperation with the Institute of Biochemistry. The explosives and ammunition can make a significant impact on the environment, when they are exploded at training areas during the military training. One of the objectives of this work is to analyze the impact of explosion products on the environment. This will enable us to foresee the scope of the impact, measures for mitigating the impact and possibilities of the long-term usage of the training areas.

In this paper, we present the literature survey on the environment contamination with explosives and their residues, their toxicity, and diseases caused to humans. A second part of the paper is devoted to the clarification of

some mechanistic aspects of toxicity of 2,4,6-trinitrotoluene (TNT), its degradation products, and other nitroaromatic explosives (Fig. 1) towards the mammalian cells in vitro.

2. The importance of the problem (literature survey). 2.1. The contamination of the environment with explosives and their residues.

Because of the military activities, the development of

military industry, tests of armaments in training areas and the civil use of explosives, nitroaromatic and nitroaliphatic explosives, their residues and degradation products have contaminated large areas of soil and natural water sources. At present, there are several hundred locations in Germany alone that are dangerously contaminated with explosives [1]. We think that this problem is relevant to Lithuania as well (Pabrade, Rukla and other training areas).

The most widely-spread explosive 2,4,6-trinitrotoluene (TNT) (Fig. 1) has been used since 1902. At the end of the 20th century, about 1,000 tons of it were produced per year. The toxicity of TNT was noticed as early as 1919 [2].

The harmless concentration of TNT in the soil and natural water sources is < 30 mg/kg and < 0.14 mg/l, respectively. However, the contamination of the territory of training areas and explosive plants by TNT reaches 12-20 g/kg and 0.1 g/l [3, 4]. It is suggested that the harmless TNT concentration in the drinking water that the population of the neighboring areas are supposed to use throughout their lives is 2 mkg/l [5]. TNT biodegrades relatively slowly under natural conditions, and some biodegradation products (hydroxylamines, amines) are also toxic. It appeared that 96 % of the explosive residue in the soil in “historically-formed” training areas, are relatively coarse particles (> 3 mm in diameter) [6]. This complicates the degradation of explosives. Depending on the conditions, the ratio of TNT and its degradation products monoamino-dinitrotoluenes in the soil of training areas ranges from 1:0.07 to 1:0.4 (expressed in g/kg of the soil) [7]. In ammunition plants and mining industry, employees come into contact with TNT and its vapor. The recommended concentrations in the air are 0.5-1.5 in the USA and 1 mg/m³ in China, however the actual air contamination is greater [8].

It is suggested that a single standard tetryl production line in the USA emits 16 kg of tetryl per 24 hours [9], however, there are no data about the possible amount of tetryl residues in the soil and water.

The amount of hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) and octahidro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine (HMX) (Fig. 1) in the soil of training areas may reach 20-50 mg/kg [7], and in the run-off waters of ammunition plants it exceeds 1.5 mg/l [10], reaching 12 mg/l in separate cases [11]. It is suggested that the harmless concentration of RDX or HMX in the drinking water that the population of the neighboring areas is supposed to use throughout their lives, is 0.1 mg/l [12].

Explosives and their residues in the soil and water are usually detected by means of high-pressure liquid chromatography, sometimes in conjunction with the mass-spectroscopy analysis (HPLC/ms) [9].

During recent years, attempts have been made to detect explosive vapor in the air by using different semiconductor sensors (electronic noses) [13] and its residues in water and other biological liquids by using immunosensors [14, 15].

In 2002 the data about the use of enzymatic biosensor in TNT analysis was published [16]. Enterobacter cloacae PB2 nitroreductase is immobilized on the surface of the electrode in the conductive polymer matrix. The enzyme is electrocatalytically reduced by the immobilized polymer component – 4.4’dialkylbipyridine derivative, and reoxidized back by TNT added to the medium. This results in an increase in the reduction current. The linear part of the response is 60 µM (13.6

mg/ml) of TNT. This would correspond to the dilution of TNT-contaminated waters by several times.

Fig. 1. Formulae of modern explosives

2.2. Toxicity and diseases caused by the explosives in humans

In discussing the toxicity of explosives and their negative influence on humans, one may distinguish two cases: first, the toxicity and explosives-induced diseases of persons working with explosives and second, the influence on the neighboring population.

Personnel working with explosives (producers of explosives, military personnel, miners) are most often diagnosed with methemoglobinemia, cataract, dermatitis, liver malfunctions (including liver cirrhosis), and tumor formation. Sometimes respiratory and digestive disorders are observed. All these phenomena are observed when the amount of TNT in the air is 1.5-0.5 mg/m³. In separate cases slight disorders (decrease in the amount of hemoglobin and erythrocytes is observed already at 0.2 mg/m³ of TNT [17]. Therefore, it has long been recommended to limit the permissible amount of TNT in the air for an 8-hour shift to 0.5 mg/m³.

TNT-induced methemoglobinemia (the formation of methemoglobin, i.e., the oxidized hemoglobin that is unable to bind oxygen) has been known since 1919 [2].

After the end of the contact with TNT, methemoglobin remains in the blood for 2-5 days.Research carried out in Israel indicated that the explosive plant employees of the Mediterranean origin which characteristically had a lower amount of antioxidant enzyme glucose-6-phosphate dehydrogenase in their erythrocytes, frequently experienced an acute hemolytic crisis even after a 2-4 day contact with TNT [18, 19]. In several cases, decrease in hemoglobin, increase in methemoglobin up to 1.5-8.6 % of the total amount of hemoglobin, decrease in hematocrit (erythrocyte concentration, v/v) to 17-24 % (normally 40-50 %), and an increase in the concentration of reticulocytes, bilirubin, urobilinogen in urine were observed.

Another mode of impact of explosives on hemoglobin, the significance of which has not been fully understood yet, but which can be used as a biomarker, is the formation of covalent TNT adduct with hemoglobin [20, 21]. After the precipitation and hydrolysis of hemoglobin in blood samples, the concentration of mono-aminodinitrotoluene is determined by HPLC/ms. The authors point out that these adducts have not been detected in the blood of the workers of a German explosive plant, whereas in a Chinese plant they found 3.7-522 ng compound/g hemoglobin.

Among the employees aged 39.5 ± 8.9 who came into contact with TNT during the span of 6.8 ± 4.7 years, peripheral, both-sided, medium-complexity cataract having no influence on the sharpness of the eyesight and its scope was observed in 6 out of 12 cases [22]. In China, among the 413 employees that had a contact with TNT for longer than 3 years, 143 (34.6 %) cases of cataract were found, and in a separate group that had a contact with TNT for longer than 20 years the frequency of cataract is 88.4 % [23]. A link between the development of cataract and the concentration of TNT-hemoglobin adduct in blood has been observed in the explosive plants [20].

In comparing 61 employees of the explosive plant that worked with organic nitrates with 56 employees of the control group, 63 % of the former group (18 times more than in the control group) were diagnosed with asymptomatic light-form cataract [24].

Skin irritation is more frequent among miners that come into contact with TNT than among the control group [25]. Sometimes melanoderma is observed. To avoid the latter antioxidant therapy is recommended [26]. Both TNT and tetryl cause dermatitis [27].

Several persons that had a long-term contact (about 35 years) with TNT were diagnosed with liver cirrhosis; however, it is not clear whether TNT had been the immediate cause of this disease [28]. Other authors did not detect a direct link between liver cirrhosis and the contact with TNT, claiming that the liability of persons contacting with TNT to liver cirrhosis increased due to alcohol abuse [29].

Mutagenic 4-aminodinitrotoluene is formed in the organisms of workers of explosive plants. After a working shift, 9.7 ± 7.9 mg/l of aminodinitrotoluene (0.1-44 mg/l, n = 219) is detected in the urine. In most cases,

this amount decreases during the weekend; however, in 8 out of 9 people traces of TNT metabolites are detected even on the 17th day after work [30, 31]. It is interesting to note that these amounts of aminodinitrotoluene (0.2-15 mg/l) can be determined by the simple colorimetric method known since the World War II [32]. It is believed that only 40 % of TNT that enters the human organism is excreted as aminodinitrotoluene through the urine.

Among the employees contacting with nitroaromatic explosives, cases of myelodysplastic syndrome (indicator of the liability to myelocytic leukemia) are more frequent [33]. In 1984-1997, among the 500 copper mine workers of the former GDR (7-37 years of contact with explosives) 6 cases of urinary tract tumors and 14 cases of kidney tumors were registered. It is by 4.5 and 14.3 times higher than the respective national average of the former GDR [34]. The occurrence of urinary tract tumors correlated with the intensity of the contact with nitro-aromatic explosives, e.g. dinitrotoluene; however, the link has not been detected in the case of kidney tumors. It is also interesting to note, that some Chinese workers contacting with TNT complained of the reproductive disorders and/or impotence. They were diagnosed with the decrease in sperm volume and quantity of mature spermatozoa, also with different spermatozoan damage [35, 8].

Concerning the negative effects of explosives on the neighboring population, one may note that in the land of Hesse (Germany), the morbidity with acute and chronic leukemia in 1983-1989 was more frequent in one Marburg community where the underground TNT plant was operating during WWII, than in neighboring communities. It was linked to the increased contamination of soil and underground water with TNT and products of its production [36]. The risk of chronic leukemia among men but not among women in this locality exceeded the national average by 10 times. Later research did not reject this opinion by confirming that a small group of persons living adjacent to the plant around 1940, actually contracted leukemia much more often [37]. However, the authors were unable to explain why leukemia morbidity grew considerably around 1980.

3. The mechanistic studies of toxicity of explosives The mechanisms of toxicity of explosives were assessed through the studies of laboratory animals, cell cultures in vitro, and the relevant enzymatic reactions. First, nitroaliphatic esters pentaerythritol tetranitrate (PETN) and trinitroglycerol (TNG) (Fig. 1) were found to be nontoxic to rats and mice [38]. Second, the administration of RDX (Fig. 1) (30-300 mg/kg daily, 13 weeks) to rats caused hypotriglycidiremia, convulsions, and death [39]. These symptoms were different from those caused by TNT (methemoglobinemia, liver and spleen damage). However, the latter symptoms were similar to those induced in humans by chronic TNT intoxication. The mechanisms underlying toxicity of RDX and HMX remain undisclosed so far. However, the

toxicity of nitroaromatic explosives has been studied more extensively ([40-42], and references cited therein). Briefly, their toxicity may be manifested through several mechanisms:

a) the redox cycling of free radicals of nitroaromatics. Free radicals are formed in the single-electron reduction of nitroaromatic compounds by flavoenzymes dehydrogenases-electrontransferases, e.g. liver NADPH: cytochrome P-450 reductase (EC 1.6.2.4):

Enzyme (reduced) + 2 TNT →

→ Enzyme (oxidized) + 2 TNT−• (1)

Subsequently, the radicals are reoxidized by oxygen: TNT−• + O2 → TNT + O2

−• (superoxide). (2) Further, superoxide participates in Haber-Weiss reaction: 2 O2

−• + 2 H+ + Fe2+ → O2 + OH− + Fe3+ + OH•. (3) The radical of hydroxyl (OH•) is very active oxidant

that damages cell proteins, nucleic acids and phospholipids.

b) the two-electron reduction of TNT to nitroso- and, subsequently, to hydroxylamino-dinitrotoluenes under the action of two electron-transferring flavoenzyme DT-diaphorase (EC 1.6.99.2) in mammalian cells, or under the action of ‘oxygen-insensitive’ nitroreductases in gastrointestinal tract bacteria ([41,43], and references cited therein). The toxicity of aromatic hydroxylamines is attributed to their reactions with DNA.

Our studies on the cytotoxicity of nitroaromatic explosives towards bovine leukemia virus-transformed lamb kidney fibroblasts (line FLK) [40-42] revealed that their toxicity in general increases with an increase in their electron-accepting potency, i..e., tetryl, pentryl > tetranitrocarbazole > TNT, tetranitrobenzimidazolone > 4,6-dinitrobenzofuroxan (DNBF) > hydroxylamino- and amino- metabolites of TNT > 5-nitro-1,2,4-triazol-3-one (NTO), 5-nitro-1,2,4-triazol-3-amine (ANTA). These data in general correlate with the studies on the toxicity of several explosives in rats and in mice when given perorally: teryl (LD50 ≥ 300 mg/kg [44]), TNT (LD50 = 1300-600 mg/kg [45]), and NTO (LD50 > 5 g/kg [46]). The toxicity is partly prevented by the antioxidants N,N‘ -diphenyl-p-phenylene diamine and desferrioxamine, and potentiated by the alkylating agent 1,3-bis-(2-chloroethyl)-1-nitrosourea. These findings taken together with a cytotoxicity increase with an increase in the electron-accepting potency of explosives, show that the cytotoxicity is caused mainly by the redox cycling mechanism (reactions 1-3). However, the cytotoxicity of amino- and hydroxylamino- metabolites of TNT was higher than one may expect from their electron-accepting properties. This points to the posibility of the additional mechanisms of their toxicity. Besides, the protective effects of the inhibitor of DT-diaphorase, dicumarol, show that the formation of toxic hydroxylamine products

under the action of this enzyme is also partly responsible for the cytotoxicity of nitroaromatic explosives.

In this work, we further clarified the mechanism of cytotoxicity of TNT and its amino-metabolites towards FLK cells, showing that their action is partly prevented by the inhibitors of cytochromes P-450, α-naphthoflavone and izoniazide, and the inhibitor of xanthine oxidoreductase (XOR, EC 1.1.3.22), allopurinol (Table 1). Table 1. The protective effects of α-naphthoflavone (5.0 µM),

izoniazide (1.0 mM), and allopurinol (100 µM) towards the toxicity of 2,4,6-trinitrotoluene (TNT, 25 µM), 2-amino-4,6-dinitrotoluene (2-NH2-DNT, 450 µM), and 4-amino-2,6-dinitrotoluene (4-NH2-DNT, 320 µM) in FLK cells (n = 3, p < 0.02). The cytotoxicity experiments were performed as described in [40,41].

No. Compound Additions Cell viability

(%) 1 TNT None 45.2±3.5 2. TNT α-Naphthoflavone 60.8±4.8 3. TNT Izoniazide 64.5±5.2 4. TNT Allopurinol 63.5±4.7 5. 2-NH2-DNT None 40.5±3.0 6. 2-NH2-DNT α-Naphthoflavone 56.6±4.5 7. 2-NH2-DNT Izoniazide 60.1±5.0 8. 2-NH2-DNT Allopurinol 57.6±4.0 9. 4-NH2-DNT None 53.6±2.0 10. 4-NH2-DNT α-Naphthoflavone 66.4±3.5 11. 4-NH2-DNT Izoniazide 62.0±3.0

The data of Table 1 confirm the suggestion that

cytochromes P-450 may perform N-hydroxylation of NH2-DNTs, converting them into toxic hydroxylamines [41,47]. The protective effects of α-naphthoflavone and izoniazide against the cytotoxicity of TNT indicate that a fraction of TNT may be intracellularly reduced into NH2-DNTs. This may take place in partly anaerobic cell compartments with high concentration of reducing flavoenzymes ([42], and references cited therein). The data on the protective effects of allopurinol (Table 1) show that XOR may be involved in the reductive activation of TNT and other nitroaromatic compounds. Because most flavoenzymes do not possess specific inhibitors, this finding extends our understanding on the involvement of individual flavoenzymes in the bioreductive activation of nitroaromatic explosives.

Methemoglobinemia, i.e., methemoglobin (Hb−Fe3+) formation from oxyhemoglobin (Hb−Fe2+−O2) in human blood erythrocytes is another important mechanism of toxicity of nitroaromatic explosives ([48], and references cited therein): Hb−Fe2+−O2 + TNT → Hb-Fe3+ + O2

−• + TNT−• (4)

Using a number of compounds, we have concluded that the rate constant of Hb-Fe2+-O2 oxidation by nitroaromatic explosives and the extent of Hb-Fe3+ formation in intact erythrocytes in general increases with an increase in their electron accepting potency [48]. In

this work, we extended these studies, using in particular the amino- metabolites of TNT (Table 2).

Table 2. The rate constants of oxidation (k) of oxyhemoglobin by nitroaromatic explosives and their metabolites in lysed human erythrocytes (pH 7.0, 37 oC), the amount of methemoglobin (Hb-Fe3+) formed during 24 h incubation of erythrocytes with 300 µM of corresponding compounds during 24 h at 37 oC and pH 7.0 (n = 3), and the single-electron reduction potentials (E1

7(calc.)) of nitroaromatic explosives [49]. The experiments were performed as described in [48]. The concentration of Hb-Fe3+ in control erythrocytes was 0.4±0.1 %.

No. Compound k

(M-1s-1) [Hb-Fe3+]

(%) E1

7(calc.) (V)

1. Tetryl 8.9±0.5 42.5±1.2 -0.156 2. TNT 3.3±0.2 48.1±6.3 -0.254 3. TNT (100 µM) 35.6±1.2 4. DNBF (100 µM) 12±1.2 2.4±0.5 -0.258 5. 2-NH2-DNT 1.0±0.2 8.3±0.2 -0.423 6. 4-NH2-DNT 0.3±0.1 0.9±0.2 -0.453 7. 2,4-(NH2)2-NT 0.4±0.3 10.5±2.1 -0.467 8. ANTA 0.12±0.02 2.1±0.3 -0.466 9. NTO ≤0.1 1.2±0.3 -0.509

Our current results suggest that although an increase

in the electron-accepting potency of nitroaromatic compounds (E1

7(calc.), Table 2) in general increases Hb-Fe2+-O2 oxidation rate, it does correlate with the extent of Hb-Fe3+ formation in erythrocytes during long lasting incubation. This discrepancy is most pronounced comparing the efficiency of Hb-Fe3+ formation by 2-NH2-DNT, diamino- metabolite of TNT, 2,4-(NH2)2-NT, and ANTA (Table 2). Possibly, the other reactions of amino-metabolites of TNT in erythrocytes, e.g., their N-hydroxylation by various redox forms of hemoglobin, may substantially increase the extent of methemoglobin formation under the chronic intoxication conditions [50]. 5. Conclusions

The presented survey of literature reflects the most important data about the contamination of the environment with explosives, their negative influence on personnel and the neighboring population. It is possible to state that among modern explosives PETN is the least toxic for humans. At present it is being tested as a possible remedy for the ischemic disease (a substitute for trinitroglycerol) [51].

The impact of RDX and HMX on humans is unclear, as well as the mechanisms of their toxicity in other mammalian species. Particular attention should be paid to the results of long-term contact with RDX and HMX, including the possible penetration of the vapor and particles of these combinations into the organism through the skin and the respiratory tract.

Since TNT is the most widespread explosive, its toxicity has been investigated most thoroughly. Particular attention should be paid to TNT-induced

cataract and damage to reproductive system. However, the comparative data of German and Chinese explosive plants indicate that the regulation of TNT amount in the air might partly mitigate its toxic effects.

On the other hand, research carried out in TNT contaminated location in Germany in reference to the susceptibility to leukemia indicates that particular attention should be paid to the acceleration of degradation of TNT and related nitroaromatic explposives.

The mechanisms of toxicity of TNT and other nitroaromatic explosives involve flavoenzyme-catalyzed single- and two-electron reduction, and the direct oxidation of oxyhemoglobin. In general, their action increases with an increase with their electron-accepting potency. Thus, some explosives of novel generation (NTO, ANTA) may be less toxic to humans and other mammalian species than TNT. The data of the present work show that xanthine oxidoreductase and cytochromes P-450 may be involved in the activation of explosives in mammalian cells. Besides, some amino- metabolites of TNT may be more efficient than expected methemoglobinemia-inducing agents in erythrocytes.

Acknowledgements

This work was supported in part by the EC Leonardo da Vinci Programme EUExcert and the Agency for International Science and Technology Development Programmes in Lithuania (COST Action CM-0603).

References 1. Preus J., Haas R. Die Standorte der Pulver−,

Sprengstoff−, Kampf− und Nebelerzeugung im ehemaligen deutschen Reich (1987) Geogr. Rundschau 39, p. 378−584.

2. Voegtlin C., Hooper C.W., Johnson J.M. Trinitrotoluene poisoning (1919) US Public Health Rep. 34, p. 1307−1313.

3. Fernando T., Bumpus J.A., Aust S.D. Biodegradation of TNT (2,4,6−trinitrotoluene) by Phanerochaete chrysosporum (1990) Appl. Environ. Microbiol. 56, p. 1666−1671.

4. Breitung J., Bruns−Nagel D., Steinbach K., Kaminski L., Gemsa D., von Low E. Bioremediation of 2,4,6−trinitrotoluene−contaminated soils by two different aerated compost systems (1996) Appl. Microbiol. Biotechnol. 44, p. 795−800.

5. Ross R.H., Hartley W.R. Comparison of water quality criteria and health advisories for 2,4,6−trinitrotoluene (1990) Regul. Toxicol. Pharmacol. 11, p. 114−117.

6. Radtke C.W., Gianotto D., Roberto F.F. Effects of particulate explosives on estimating contamination at a historic explosives testing area (2002) Chemosphere 46, p. 3−9.

7. Tuomi E., Coover M.P., Stroo H.F. Bioremediation using composting or anaerobic treatment for ordnance−contaminated soils (1997) Ann. NY Acad. Sci. 829, p. 160−178.

8. Liu H.X., Qin W.H., Wang G.R., Yang Z.Z., Chang Y.X., Jiang Q.G. Some altered concentrations of

elements in semen of workers exposed to trinitrotoluene (1995) Occup. Environ. Med. 52, p. 842−845.

9. Harvey S.D., Fellows R.J., Cataldo D.A., Bean R.M. Analysis of the explosive 2,4,6− trinitrophenylmethyl−nitramine (tetryl) in bush bean plants (1993) J. Chromatogr. 630, p. 167−177.

10. Best E.P., Sprecher S.L. Larson S.L., Frederickson H.L., Bader D.F. Environmental behavior of explosives in groundwater from the Milan Army Ammunition Plant in aquatic and wetland plant treatments (1999) Chemosphere 38, p. 3383−3396.

11. Hawari J., Halasz A., Sheremata T., Beaudet S., Groom C., Paquet L., Rhofir C., Ampleman G., Thiboutot S. Characterization of metabolites during biodegradation of hexahydro−1,3,5−trinitro− 1,3,5− triazine (RDX) with municipal anaerobic sludge (2000) Appl. Environ. Microbiol. 66, p. 2652−2657.

12. Binks P.R., Nicklin S., Bruce N.C. Degradation of hexahydro − 1,3,5−trinitro−1,3,5−triazine (RDX) by Stenotrophomonas maltophila PB1 (1995) Appl. Environ. Microbiol. 61, p. 1318−1322.

13. Pardo M., Benussi G.P., Niederjaufner G., Falia G. Detection of TNT vapours with the Pico−1 nose (2000) in: 7th International Symposium on Olfaction and Electronic Noses (Gardner J.W., Persaud K.C., Eds.), IOP Publishing, Brighton, p. 67−74.

14. Rabbany S.Y., Marganski W.A., Kusterbeck A.W., Ligler F.S. A membrane−based displacement flow immunoassay (1998) Biosens. Bioelectron. 13, p. 939−944.

15. Sapsford K.E., Charles P.T., Patterson C.H., Ligler F.S. Demonstration of four immunoassay formats using the array biosensor (2002) Anal. Chem. 74, p. 1061−1068.

16. Naal Z., Park J.H., Bernahrd S., Shapleigh J.P., Bati C.A., Abruna H.D. Amperometric TNT biosensor based on the oriented immobilization of a nitroreductase maltose binding protein fusion (2002) Anal. Chem. 74, p. 140−148.

17. Hathaway J.A. Trinitrotoluene: a review of reported dose−related effects providing documentation for a workplace standard (1977) J. Occup. Med. 19, p. 341−345.

18. Djerassi L.S., Vitany I. Haemolytic episode in G6PD deficient workers exposed to TNT (1975) Br. J. Ind. Med. 32, p. 54−58.

19. Djerassi L. Hemolytic crisis in G6PD−deficient individuals in the occupational setting (1998) Int. Arch. Occup. Environ. Health 71, p. 26−28.

20. Liu Y.Y., Yao M., Fang J.L., Wang Y.V. Monitoring human risk and exposure to trinitrotoluene (TNT) using haemoglobin adducts as biomarkers (1995) Toxicol. Lett. 77, p. 281−287.

21. Sabbioni G., Wei J., Liu Y.Y. Determination of hemoglobin adducts in workers exposed to 2,4,6−trinitrotoluene (1996) J. Chromatogr. B Biomed. Appl. 682, p. 243−248.

22. Harkonen H., Karki M., Lahti A., Savolainen H. Early equatorial cataracts in workers exposed to trinitrotoluene (1983) Am. J. Ophthalmol. 95, p. 807−810.

23. Zhou A.S. A clinical study of trinitrotoluene cataract (1990) Pol. J. Occup. Med. 3, p. 171−176.

24. Lewis-Younger C.R., Mamalis N., Egger M.J., Wallace D.O., Lu C. Lens opacificiations detected by

slitlamp biomicroscopy are associated with exposure to organic nitrate explosives (2000) Arch. Ophthalmol. 118, p. 1653−1659.

25. Vysochin V.I., Vysochina S.N. Skin lesions in miners in contact with trinitrotoluene (1991) Vrach. Delo 7, p. 107−109.

26. Cherkasskaia R.G., Razumov V.V., Semenikhin V.A., Briukhova A.R. Toxic melanoderma in chronic intoxication with trinitrotoluene (1993) Med. Tr. Prom. Ekol. 1, p. 39−41.

27. Goh C.L. Allergic contact dermatitis from tetryl and trinitrotoluene (1984) Contact Dermatitis 10, 108 p.

28. Garfinkel D., Sidi Y., Steier M., Rothem A., Marilus R., Atsmon A., Pinkhas J. Liver cirrhosis and hepatocellular carcinoma after prolonged exposure to TNT: causal relationship or mere coincidence (1988) Med. Interne 26, p. 287−290.

29. Li J., Jiang Q.C., Zhong W.D. Persistent ethanol drinking increase liver injury induced by trinitrotoluene exposure: an in−plant case−control study (1991) Hum. Exp. Toxicol. 10, p. 405−409.

30. Ahlborg G., Einisto P., Sorsa M. Mutagenic activity and metabolites in the urine of workers exposed to trinitrotoluene (TNT) (1988) Br. J. Ind. Med. 45, p. 353−358.

31. Woolen B.H., Hall M.G., Craig R., Steel G.T. Trinitrotoluene: assesment of occupational absorption during manufacture of explosives (1986) Br. J. Ind. Med. 43, p. 465−473.

32. Snyder R.K., von Oettingen W.F. A new test for the detection and the appraisal of exposure to trinitrotoluene (1942) J. Am. Med. Assoc. 123, p. 202−203.

33. Nisse C., Lorthois C., Dorp V., Eloy E., Haguenoer J.M., Fenaux P. Exposure to occupational and environmental factors in myelodysplastic syndromes. Preliminary results of a case−control study (1995) Leukemia 9, p. 693−699.

34. Bruning T., Chronz C., Thier R., Havelka J., Ko Y., Bolt H.M. Occurrence of urinary tract tumours in miners highly exposed to dinitrotoluene (1999) J. Occupat. Environ. Med. 41, p. 144−149.

35. Yi L., Quan-Guan J., Shou-Qi Y., Wei L., Gao-Jun T., Jing-Wei C. Effects of exposure to trinitrotoluene on male reproduction (1993) Biomed. Environ. Sci. 6, p. 154−160.

36. Kolb G., Becker N., Scheller, S., Zugmaier G., Pralle H., Wahrendorf J., Havemann K. Increased risk of acute myelogenous leukemia (AML) and chronic myelogenous leukemia (CML) in a county of Hesse, Germany (1993) Soz. Praeventivmed. 38, p. 190−195.

37. Kilian P.H., Skrzypek S., Becker N., Havemann K. Exposure to armament wastes and leukemia: a case−control study within a cluster of AML and CML in Germany (2001) Leukemia Res. 25, p. 839−845.

38. Bucher, J.R., Huff, J., Haseman, J.K., Eustis, S.L., Lilja, H.S., Murthy, A.S. No evidence of toxicity or carcinogenicity of pentaerythritol tetranitrate given in the diet to F334 rats and B6C3F1 mice up to two years (1990) J. Appl. Toxicol. 10, p. 353-357.

39. Levine, B.S., Furedi, E.M., Gordon, D.E., Barkley, J.J., Lish, P.M. Toxic interactions of the munition compounds TNT and RDX in F344 rats (1990) Fundam. Appl. Toxicol. 15, 373-380.

40. Č÷nas, N., Nemeikait÷-Č÷niene, A., Sergedien÷, E., Nivinskas, H., Anusevičius, Ž., Šarlauskas, J.

Quantitative structure−activity relationships in enzymatic single-electron reduction of nitroaromatic explosives: implications for their cytotoxicity (2001) Biochim. Biophys. Acta 1528, p. 31−33.

41. Šarlauskas, J., Nemeikait÷-Č÷nien÷, A., Anusevičius, Ž., Misevičien÷, L., Martinez-Julvez, M., Medina, M., Gomez-Moreno, C., Č÷nas, N. Flavoenzyme-catalyzed redox cycling of hydroxylamino- and amino metabolites of 2,4,6-trinitrotoluene: implications for their cytotoxicity (2004) Arch. Biochem. Biophys. 425, p. 184-192.

42. Nemeikait÷-Č÷nien÷, A., Miliukien÷, V., Šarlauskas, J., Maldutis, E., Č÷nas, N. Chemical aspects of cytotoxicity of nitroaromatic explosives: a review (2006) Chemija 17 (2-3), p. 34-41.

43. Nivinskas, H., Koder, R.L., Anusevičius, Ž., Šarlauskas, J., Miller, A.-F., Č÷nas, N. Quantitative structure-activity relationships in two-electron reduction of nitroaromatic compounds by Enterobacter cloacae NAD(P)H:nitroreductase (2001) Arch. Biochem. Biophys. 385, p. 170-178.

44. Reddy, T.V., Olson, G.R., Wiechman, B., Reddy, G., Torsella, J., Daniel, F.B., Leach, G.J. Toxicity of tetryl (N-methyl-N,2,4,6-tetranitroaniline) in Fischer F344 rats (1999) Int. J. Toxicol. 18, p, 97-107.

45. Dilley, J.V., Tyson, C.A., Spangoord, R.J., Sasmore, D.P., Newell, G.W., Decre, J.C. Short-term otal

toxicity of 2,4,6-trinitrotoluene in mice, rats, and dogs (1982) J. Toxicol. Environ. Health. 9, p. 565-585.

46. London, J.O., Smith, D.M. A toxicological study of NTO (1985) Los Alamos National Laboratory Report LA-10533-MS.

47. Kim, D., Guengerich, F.P. Cytochrome P450 activation of arylamines and heterocyclic amines (2005) Annu. Rev. Pharmacol. Toxicol. 45, p. 27-49.

48. Marozien÷, A., Kliukien÷, R., Šarlauskas, J., Č÷nas, N. Methemoglobin formation in human erythrocytes by nitroaromatic explosives (2001) Z. Naturforsch. 56 C, p. 1157−1163.

49. Šarlauskas, J., Nivinskas, H., Anusevičius, Ž., Misevičien÷, L., Marozien÷, A., Č÷nas, N. Estimation of single-electron reduction potentials (E1

7) of nitroaromatic compounds according to the kinetics of their single-electron reduction by flavoenzymes (2006) Chemija 17 (1), p. 31-37.

50. Sabbioni, G., Jones, C.T. Biomonitoring of arylamines and nitroarenes (2002) Biomarkers 7, p. 347-421.

51. Hinz B., Kuntze U., Schroder H. Pentaerythrityl tetranitrate and its phase I metabolites are potent activators of cellular cyclic GMP accumulation (1998) Biochem. Biophys. Res. Commun. 253, p. 658−661.

SPROGMENYS KAIP TOKSIŠKI APLINKOS TARŠALAI: UŽTERŠT UMO LAIPSNIS, TOKSIŠKUMAS IR JO MECHANIZMAI N. Č÷nas, A. Nemeikait÷-Č÷nien÷, A. Marozien÷, J. Šarlauskas, V. Vilutien÷, J. Baublys S a n t r a u k a Literatūros apžvalgoje analizuojami aplinkos užterštumo sprogmenų likučiais laipsnis, ir jų toksiškumas žmon÷ms. Galima teigti, kad tarp šiuolaikinių sprogmenų mažiausiai toksiškas yra pentaeritritolio tetranitratas (PETN). Heksahidro-1,3,5-trinitro-1,3,5-triazino (RDX) poveikis žmon÷ms n÷ra aiškus. Kadangi 2,4,6-trinitrotoluenas (TNT) yra labiausiai paplitęs sprogmuo, jo sukeliami toksiniai efektai (methemoglobinemiia, katarakta, kepenų sutrikimai) yra detaliauisiai aprašyti. TNT ir kitų nitroaromatinių sprogmenų citotoksikumo mechanizmai yra flavininių fermentų katalizuojama vien- ir/ar dvielektronin÷ redukcija (laisvųjų radikalų cikliniai redoks procesai ir/ar alkilinančių hidroksilaminų susidarymas), ir tiesiogin÷ oksihemoglobino oksidacija. Kaip taisykl÷, nitroaromatinių junginių toksiškumas did÷ja, did÷jant jų elektronoakceptorin÷ms savyb÷ms. Tod÷l kai kurie naujos kartos sprogmenys, pvz. 5-nitro-1,2,4-triazol-3-onas (NTO), 5-nitro-1,2,4-triazol-3-aminas gali būti mažiau toksiški žmogui ir kitiems žinduoliams nei TNT. Šio darbo eksperimentiniai duomenys rodo, kad nitroaromatinių sprogmenų aktyvacijoje ląstel÷je dalyvauja ir ksantinoksidoreduktaz÷, bei citochromai P-450. Be to, paaišk÷jo, kad kai kurie TNT amino metabolitai netik÷tai gali labai efektyviai sukelti methemoglobino susidarymą eritrocituose. Raktiniai žodžiai: sprogstamosios medžiagos, toksiškumas, poveikio mechanizmas, methemoglobinas, oksihemoglobinas. ВЗРЫВЧАТЫЕ ВЕЩЕСТВА KAK T ОКСИЧНЫЕ ЗАГРЯЗНИТЕЛИ СРЕДЫ: УРОВНИ ЗАГРЯЗНЕНИЯ, ТОКСИЧНОСТЬ И МЕХАНИЗМ ВОЗДЕЙСТВИЯ Н. Ченас, А. Немейкайте-Ченене, А. Марозене, Й. Шарлаускас, В. Вилутене, Ю. Баублис Р е з ю м е В литературном обзоре анализируются данные о загрязнении среды взрывчатыми веществами (ВВ), их токсичном воздествии, и механизмах токсичности. Можно заключить, что из современных ВВ тетранитрат пентаэритрита (PETN) является наименее токсичным. Воздействие гексагидро-1,3,5-тринитро-1,3,5-триазина (RDX) на людей не изучено. Так как 2,4,6-тринитротолуол (TNT) является наиболее распространенным ВВ, с ним связанные токсичные эффекты (метгемоглобинемия, катаракта, цирроз печени) известны в лучщей степени. Токсичность TNT и других нитроароматических ВВ связана с их одно- или двухэлектронным восстановлением флавиновыми ферментами (циклирование свободных радикалов или образование алкилирующих гидроксиламинов), и прямое окисление оксигемоглобина. Как правило, токсичность нитроароматических соединений возрастает с увеличением их электроноакцепторных свойств. Поэтому некоторыйе ВВ нового поколения, напр. 5-нитро-1,2,4-триазол-3-он (NTO), 5-нитро-1,2,4-триазол-3-амин могут бытъ менее токсичны чем TNT. В экспериментальной части работы показано, что в

активации нитроароматических ВВ в клетке участвуют ксантиноксидоредуктаза и цитохромы P-450. Кроме того, выяснилось, что некоторые амино-метаболиты TNT особенно активно вызывают образование метгемоглобина в эритроцитах.

Narimantas Č÷nas Biochemijos institutas, Ksenobiotikų biochemijos skyrius, Mokslininkų 12, LT-08662 Vilnius Telefonas: (8 5) 272 90 42 El. paštas: ncenas@bchi.lt Aušra Nemeikait÷-Č÷nien÷ Generolo Jono Žemaičio Lietuvos karo akademija, Taikomųjų mokslų katedra, Šilo 5a, LT- 10322 Vilnius Telefonas: (8 5) 210 35 65 El. paštas ceniene@imi.lt Jonas Šarlauskas Biochemijos institutas, Ksenobiotikų biochemijos skyrius, Mokslininkų 12, LT-08662 Vilnius Telefonas: (8 5) 272 90 42 El. paštas: hidrazon@bch.lt EUExcert nacionalinis koordinatorius Lietuvai Audron÷ Marozien÷ Biochemijos institutas, Ksenobiotikų biochemijos skyrius, Mokslininkų 12, LT-08662 Vilnius Telefonas: (8 5) 272 90 42 El. paštas: hidrazon@bchi.lt Valentina Vilutien ÷ Generolo Jono Žemaičio Lietuvos karo akademija, Inžinerin÷s vadybos katedra, Šilo 5a, LT- 10322 Vilnius Telefonas: (8 5) 210 35 52 Faksas: (8 5) 212 73 18 El. paštas: ivk@lka.lt Atsakinga už korespondenciją su žurnalo redakcijos kolegija Juozas Baublys Generolo Jono Žemaičio Lietuvos karo akademija, Inžinerin÷s vadybos katedra, Šilo 5a, LT- 10322 Vilnius Telefonas: (8 5) 210 35 52 Faksas: (8 5) 212 73 18 El. paštas: juozas.baublys@lka.lt