ISSN 00063509, Biophysics, 2015, Vol. 60, No. 4, pp. 553–558. © Pleiades Publishing, Inc., 2015. Original Russian Text © V.I. Bruskov, L.S. Yaguzhinsky, Z.K. Masalimov, A.V. Chernikov, V.I. Emelyanenko, S.V. Gudkov, 2015, published in Biofizika, 2015, Vol. 60, No. 4, pp. 673–680.
MOLECULAR BIOPHYSICS
The Continuous Generation of Hydrogen Peroxide in Water Containing Very Low Concentrations of Unsymmetrical Dimethylhydrazine V. I. Bruskova, L. S. Yaguzhinskyb, Z. K. Masalimova, A. V. Chernikova, V. I. Emelyanenkoa, and S. V. Gudkova, c, d a
Institute of Theoretical and Experimental Biophysics, Russian Academy of Sciences, ul. Institutskaya 3, Pushchino, Moscow oblast, 142290 Russia bMoscow State University, Moscow, 119991 Russia c Prokhorov General Physics Institute, Russian Academy of Sciences, ul. Vavilova 38, Moscow, 119991 Russia dLobachevsky State University of Nizhny Novgorod, pr. Gagarina 23, Nizhny Novgorod, 603950 Russia email:
[email protected] Received May 20, 2015
Abstract—Continuous generation of hydrogen peroxide catalyzed by low concentrations of 1,1dimethylhy drazine (heptyl), a rocket fuel component, in airsaturated water was shown by the method of enhanced chemiluminescence in a luminolp–iodophenol–peroxidase system. The concentration dependence and the influence of heat and light on the formation of hydrogen peroxide in water under the influence of dimethyl hydrazine at concentrations that are considerably lower than the maximum allowable concentrations were studied and the physicochemical mechanism of this process was considered. It is supposed that dimethylhy drazine at ultralow concentrations is associated with air nanobubbles and represents a longlived complex, which catalyzes the hydrogen peroxide formation under the influence of heat and light. We propose a new concept of the toxicity of dimethylhydrazine at very low concentrations due to impaired homeostasis of the formation of reactive oxygen species in aqueous solutions that enter humans and animals. Keywords: unsymmetrical dimethylhydrazine (heptyl), water, heat, light, molecular oxygen, reactive oxygen species, hydrogen peroxide DOI: 10.1134/S0006350915040065
INTRODUCTION Unsymmetrical 1,1dimethylhydrazine (UDMH) or heptyl is widely used as a fuel component in Russian 1
and worldwide rocket technology. When launching rockets, the incomplete combustion of UDMH results in its subsequent release into the atmosphere as aero sol. It gradually precipitates and is distributed in the direction of movement of air masses over long dis tances [1]. UDMH is a highly toxic substance of the first danger class. The World Health Organization included UDMH in the list of highly hazardous chem icals [2,3]. The problem of environmental pollution by components of rocket fuel and the purpose of the safety of people near launch sites and the landing places of separating parts of rockets are very impor tant. The amount of UDMH that is released into the environment in rocket launch areas because of acci dental spills is estimated at 300 t/year and the total area with local pollution reaches 1 million hectares 1 Abbreviations:
UDMH, unsymmetrical 1,1dimethylhydrazine; MAC, maximum allowable concentration.
[4]. According to the published data [5], the UDMH concentration in some craters formed by falling rocket stages reaches values that significantly exceed the maximum allowable concentrations (MAC) even in a year. Medical, social, and environmental issues related to the environmental pollution by toxic components of rocket fuel are considered in the review [2]. The most important problem is the increased incidence of people who live in vast territories that are adjacent to the areas of the landing of spent rocket stages with remaining fuel. A significant excess of various types of diseases has been identified in children of different age groups in comparison with the control area [6]. Disor ders of bilirubin metabolism, anemia of pregnancy, the birth of “yellow children,” the development of immu nodeficiency, etc. are observed in adults. Experimental studies with animals have shown that these pathologies are caused by the toxic action of UDMH [7, 8]. The MAC value for UDMH in water is taken as 0.3 μM [9]. The current approximate allowable con centration of UDMH in water is 0.06 μg/L, i.e., 300 times lower than the above value [10]. UDMH has
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high solubility in water (1 g/mL) [11]. However, UDMH is quickly oxidized in water under the action of air oxygen and hydrogen peroxide with the forma tion of various products [12, 13]. Without the catalytic decomposition, the half life of UDMH at concentra tions of several MAC values is more than 24 h and approximately 10 h under the action of air oxygen and hydrogen peroxide, respectively [14]. The efficiency of UDMH oxidation increases with rising temperature and increasing pH. The products of the UDMH deg radation contain supermutagenic and carcinogenic nitrosodimethylamine and a number of other toxic compounds [12]. The maximum allowable concentra tion of nitrosodimethylamine in water is 1.4 ⋅ 10–9 g/L, which is much lower than that of UDMH [15]. Nitrosodimethylamine is a stable compound that can accumulate in the environment [16]. The toxicity of UDMH at the molecular level is based on its ability to inhibit glutamate decarboxylase and a number of other enzymes [17]. It is also assumed that the toxicity of UDMH is due to the formation of reactive oxygen species [18], which can lead to the development of oxidative stress in biological systems and to remote pathological changes [1, 4, 19]. The goal of this work was to study the continuous generation of hydrogen peroxide catalyzed by UDMH at low concentrations in airsaturated water. We con sidered a possible physicochemical mechanism of this process and proposed a new concept of the toxic ity of heptyl at very low concentrations caused by the formation of hydrogen peroxide in air micronano bubbles in water, which enter humans and animals. MATERIALS AND METHODS Materials. We used hydrogen peroxide, horserad ish peroxidase, tris(hydroxymethyl)aminomethane (Sigma, United States), 4iodophenol (Aldrich, United States), luminol (AppliChem, Germany), 1,1 dimethylhydrazine (Econ, Russia), and bidistilled water saturated with atmospheric air for 24 h. The spe cific electric conductivity of the water was 120 μS/m and the pH was 5.6. The concentration of molecular oxygen in the water was 270 μM. We used the previ ously assumed MAC value of UDMH (18 μg/L, 0.3 μM) [9]. The effects of visible light and heat. A Comtech electric lamp bulb (Comtech, Ukraine) with a power of 100 W and a luminous flux of ~1500 lumens was used as an artificial light source. Irradiation of water (10 mL) was carried out in 20mL vials (Beckman, United States) in the dark at room temperature (20– 22°C). The luminous flux spectral characteristics were determined by an automated spectrometric MDR41 complex (Spectrum, Russia) in the range of 200– 1000 nm. The luminous flux electrical characteristics were evaluated using a CMP3 pyranometer (Kipp
and Zonen, Netherlands) with a constant spectral sen sitivity in the range of 310–2800 nm. The samples were heated in a BT2021 thermostat (Termex, Russia) at different temperatures with an accuracy of ±0.1°C during different periods of time. Evaluation of the hydrogen peroxide concentration. The hydrogen peroxide concentration in water was evaluated by the enhanced chemiluminescence method in a luminol4iodophenolhorseradish per oxidase system [20, 21]. A liquid scintillation Beta1 counter (MedaApparatura, Ukraine) for the measure ment of βradiation in singlephoton counting mode (without the scheme of coincidence) [22] was used as a chemiluminometer. A counting solution (0.5 mL) containing 10 mM trisHCl (pH 8.5), 50 μM 4iodophenol, 50 μM luminol, and horseradish per oxidase [23] was added to the samples immediately prior to the measurement of the H2O2 content. The concentration of hydrogen peroxide that was formed in solutions that contain low UDMH concentrations under the influence of heat and light was evaluated using the calibration plots of the dependence of chemiluminescence in solutions on the hydrogen per oxide concentration [24]. The initial H2O2 concentra tion used for calibration was measured spectrophoto metrically at 240 nm taking the absorption molar coef ficient value of 43.6 M–1 cm–1 [25] into account. The sensitivity of the method allows one to evaluate H2O2 at a concentration of 0.1 nM [26]. RESULTS The influence of UDMH on H2O2 generation in dis tilled water under heating. The influence of UDMH at different concentrations on the formation of hydrogen peroxide in bidistilled water at 40°C is presented in Fig. 1. The maximum formation of hydrogen peroxide is observed in a certain range of ultralow concentra tions of heptyl. The heptyl concentration of ~0.3 μM (1 MAC) under the given heating conditions leads to an approximately fourfold increase in the efficiency of the H2O2 formation in water compared to the samples without UDMH. The most intensive generation of H2O2 occurs at UDMH concentrations that are two– three orders of magnitude less than the MAC. In this case, an approximately tenfold increase in the H2O2 formation was observed compared with the control. The decrease in the UDMH concentration by four orders of magnitude relative to the MAC led to a two fold increase in the H2O2 formation. Similar results were obtained in the case where UDMH was dissolved in a phosphate buffer (pH 7.4; 1 mM) and in solutions that contain sodium chloride, bicarbonate, or nitrate (1 mM). It is known that two physical factors, viz., heat and light, constantly influence water in all ecosystems. Moreover, heat is a constantly acting factor of the internal medium in homoiothermic animals. The BIOPHYSICS
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Fig. 1. The dependence of H2O2 formation in water on UDMH concentrations under the action of heat (3 h, 40°C). The mean values and their standard errors of mean for four independent measurements are presented. The hatched area indicates the limits of the formation of hydrogen peroxide in water under the action of heat in the absence of UDMH. The xaxis is logarithmic.
impact of visible light along with heating leads to H2O2 formation [27]. Therefore, we studied the generation of hydrogen peroxide under the light in the presence of UDMH. The influence of UDMH on H2O2 generation in dis tilled water under light. The formation of hydrogen per oxide under the action of an artificial light source. A household incandescent light bulb (100 W) was used as an artificial light source. The table presents the values of irradiance at different distances from the light source. This lamp provided the irradiance of a surface of up to 800 W/m2, which is comparable with the mag nitude of the light energy that is generated by sunlight in the summer in Central Russia. The kinetics of the formation of hydrogen peroxide in the presence and in the absence of UDMH under visible light are presented in Fig. 2. In the absence of UDMH, H2O2 accumulated in water during the first 2 h of the lighting and reached the value of ~15 nM at its initial concentration of ~4 nM. The H2O2 concen tration increased insignificantly during the next 2 h. In the presence of UDMH, similar lighting conditions led to a higher rate of the increase in the H2O2 concen tration, which reached values of more than 30 nM by the end of the second hour. The H2O2 concentration increased insignificantly during the next 2 h. The influence of UDMH at different concentra tions on the generation of H2O2 under the action of light is shown in Fig. 3. The presence of heptyl at con centration of approximately 1 MAC (0.3 μM) caused an approximately twofold increase in the H2O2 con centration compared to the control. A significantly more intensive generation of hydrogen peroxide (by factors of 4–5) in comparison with the experiment in BIOPHYSICS
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Fig. 2. The influence of UDMH (0.3 µM) on the kinetics of the H2O2 formation in bidistilled water under the action of visible light (irradiance, 83.3 W/m2). The mean values of three independent experiments and their standard errors of mean are presented.
the absence of heptyl was observed at the UDMH con centrations that were less than 1 MAC by two–four orders of magnitude. A twofold increase in the H2O2 formation was observed even if the UDMH concen tration decreased by four orders of magnitude relative to the MAC. Similar results were obtained in the case where UDMH was dissolved in a phosphate buffer (pH 7.4; 1 mM). The time dependence of the change in the hydrogen peroxide content in water after a single introduction of UDMH. We studied the influence of a single addition of UDMH to water to the final concentration of 0.3 μM on the continuous generation of H2O2 (tem perature, 20°C; day/night light conditions, 12 h/12 h) (Fig. 4). The H2O2 concentration was measured dur ing 15 days at the same time. The concentration of H2O2 in water that contained no UDMH increased The values of irradiance (E) depending on distance (R) between light bulb coil and object1 R, m
E, W/m2
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774.6 ± 0.9 385.3 ± 0.5 227.0 ± 0.4 148.1 ± 0.5 105.8 ± 0.6 83.3 ± 0.4 67.3 ± 0.3 52.2 ± 0.2 42.0 ± 0.2
1 The mean values and their standard errors of mean for four inde pendent measurements are presented.
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Fig. 3. The dependence of H2O2 formation in water on UDMH concentrations under the action of visible light for 2 h (irradiance, 83.3 W/m2). The mean values of H2O2 concentrations and their standard errors of mean for three independent measurements are presented. The hatched area indicates the limits of the formation of hydrogen per oxide in water in the absence of UDMH. The xaxis is log arithmic.
Fig. 4. The influence of a single introduction of UDMH into water to the final concentration of 0.3 µM on contin uous generation of H2O2 under close to natural conditions (20°C; day/night light conditions, 12 h/12 h; irradiance, 83.3 W/m2). The mean values of H2O2 concentrations and their standard errors of mean for three independent mea surements are presented.
from 4–5 nM to 16–17 nM for the first 24 h and was almost constant for the next 15 days. In the case of the presence of UDMH in water, the H2O2 concentration increased almost linearly during the first 9 days, reached 100–110 nM, and decreased to 50 nM after 11 days.
light is the transition of oxygen from the triplet to the singlet state, which leads to local electromagnetic dis turbances and, thereby, to spatial heterogeneity and bubston collapse. The hydroxyl and hydroperoxide radicals that form as a result of the bubston collapse are converted to hydrogen peroxide.
DISCUSSION It was previously shown in our laboratory that visi ble light and heat cause the formation of reactive oxy gen species, particularly, longlived hydrogen peroxide in airsaturated water [27–30]. We studied the phys icochemical mechanism of the H2O2 formation in water under the constant action of these physical fac tors and formulated the concept of the water–air sys tem as an open active nonequilibrium environmental medium [31]. Air molecules are hydrophobic and exist in a polar system, such as water, in the form of so called bubstons (from ‘bubble stabilized by ions’), i.e., microbubbles (1–2 μm), which, in turn, are clusters of nanosized bubbles (70–90 nm) [32, 33]. It is assumed that this system is sensitive to weak physicochemical impacts and its activity is due to the accumulation of additional free energy in the form of surface tension in bubstons [32, 33]. This energy can be released under the action of heat and light as a result of cavitation col lapse of bubstons, which is accompanied by local heat ing to extremely high temperatures in the air nanobub ble volume. This process is also accompanied by the formation of reactive oxygen and nitrogen species similar to that under the influence of ultrasound [34, 35]. The starting stage of the formation of reactive oxygen species in water under the action of heat and
It was established in this work that UDMH at con centrations of two–three orders of magnitude less than the MAC is extremely efficient as a generator of hydrogen peroxide in water and aqueous solutions under the action of moderate heating and lighting (Figs. 1⎯3). Under these conditions, heptyl at the above concentrations generates intensive H2O2 forma tion, i.e., heptyl plays the role of a catalyst of this pro cess. In this case, the autocatalytic activity of heptyl and its possible decomposition products is maintained for a long time (up to 10 days) (Fig. 4). The accumu lation of H2O2 in water for 10 days may be explained by the continuous high autocatalytic activity of UDMH in the formation of hydrogen peroxide in air nanobub bles because UDMH itself, as a strong reducing agent, promotes the rapid decomposition of H2O2. Let us consider the possible physicochemical mechanism of the continuous autocatalytic process of hydrogen peroxide formation in the presence of low concentrations of UDMH. UDMH has amphiphilic surfaceactive properties due to the polar amino group and two hydrophobic methyl groups. One can assume, therefore, that it can be found at low concentrations on the surface of the nanobubbles in water. This small portion of waterdissolved heptyl is probably stored for a long time in these nanobubbles, which are microre actors of the autocatalytic generation of H2O2. BIOPHYSICS
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Our results could be explained by the following mechanism. The reaction of singleelectron reduction of oxygen with the formation of the UDMH and hydroperoxide radicals occurs at certain concentra tions of UDMH [36]: ( CH 3 ) 2 –N–NH 2 + O 2 •
•
= ( CH 3 ) 2 –N–NH + HO 2 . Disproportionation of the hydroperoxide radical leads to the formation of hydrogen peroxide: •
2HO 2 = H 2 O 2 + O 2 . It should be noted that radicals are formed in water due to cavitation collapse of nanobubbles, which is accompanied by high temperature and pressure [37, 38]: •
•
H 2 O → H + OH . This process occurs in water under the influence of heat and light, which leads to the reaction: •
( CH 3 ) 2 –N–NH + H
•
→ ( CH 3 ) 2 –N–NH 2 with the formation of the initial UDMH molecule and hydrogen peroxide: 2OH• = H2O2. The ability of the decomposition products of UDMH to form initial UDMH was noted earlier [39]. Thus, in the whole reaction, which probably occurs in air micronanobubbles, heptyl is not subjected to the longterm decomposition but acts as a catalyst of the process: O2 + 2H2O = 2H2O2. Moreover, the reaction of UDMH with oxygen can lead to the formation of a peroxide complex, i.e., hydrazine hydroperoxide (CH3)2–N–NH(OOH). Hydrazine hydroperoxide can be converted into dimethyldiazen with the formation of hydrogen per oxide: ( CH 3 ) 2 –N–NH(OOH) → H 2 O 2 + CH 3 –N=N–CH 3 , or with the formation of Nnitrosodimethylamine and water: ( CH 3 ) 2 –N–NH(OOH) → ( CH 3 ) 2 –N–N=O + H 2 O. UDMH itself is a strong reducing agent [40], which causes the decomposition of H2O2: ( CH 3 ) 2 –N–NH 2 + H 2 O 2 → ( CH 3 ) 2 –N=O + H 2 O + H 2 . Thus, the simultaneous formation of different products can be due to radical mechanisms. The radi cal particles can occur during the decomposition of BIOPHYSICS
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both hydrogen peroxide and hydrazine hydroperoxide [13]. Tetramethyl tetrazene, (CH3)2–N–N=N– N(CH3)2, was also observed among the products of the reaction between UDMH and oxygen and H2O2 [14, 16]. The accumulation in the solution of this product, which is more hydrophobic than UDMH, may further lead to both the displacement of dimethyl hydrazine from air nanobubbles and the cessation of the H2O2 generation. Water is the largest part among the substances in any organism, including humans. Its constant flow into the body and renewal is extremely necessary. Dif ferent physical environmental factors lead to the for mation of reactive oxygen species in water including hydrogen peroxide, which plays an especially impor tant role as the most longlived form. Reactive oxygen species play a dual role in mammalian organisms. On one hand, they cause oxidative lesions in nucleic acids, proteins, lipids, and other biomolecules. The oxidative lesions in DNA are probably one of the causes of mutagenesis, carcinogenesis, aging, and cer tain diseases of the elderly. Different human patholo gies including oncological diseases are associated with continuous hyperproduction of reactive oxygen spe cies in the response to the impacts of different envi ronmental factors [41]. On the other hand, small amounts of the reactive oxygen species play an impor tant signalregulatory role in mammals [42]. There may be a false impression that heptyl at low concentrations catalyzes the formation of a small amount of H2O2, which cannot significantly influence biological processes. It should be noted, however, that the H2O2 concentration is evaluated in the experiment as the mean value in all of the volume of the solution at rather low concentrations of air microbubbles. The local hydrogen peroxide concentration in the vicinity of the surface of air microbubbles can be quite signifi cant. Zavilgelsky et al. [43] obtained direct evidence in support of the hypothesis that the biological effect of UDMH on bacteria is determined by the generation of H2O2 in water caused by UDMH. The UDMH toxicity at ultralow concentrations in water that enters humans and animals may be due to both the continuous generation of reactive oxygen species (predominantly in the form of longlived hydrogen peroxide) and the formation of the very mutagenic product of its decomposition, Nnitro sodimethylamine, and other toxic compounds. Thus, a new concept of the toxicity of heptyl at very low concentrations was proposed on the basis of our results. According to this concept, the toxicity of hep tyl is due to the impaired homeostasis of reactive oxy gen species in aqueous solutions that enter humans and animals. The results indicate that the currently adopted value of the maximum allowable concentra tion of UDMH should be decreased. However, this issue requires further research.
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ACKNOWLEDGMENTS
20. I. N. Shtarkman, S. V. Gudkov, A. V. Chernikov, et al., Biokhimiya 73, 576 (2008).
The authors thank Dr. S.F. Chalkin (Roskosmos) for the provision of UDMH and special instruments to work with this compound.
21. S. V. Gudkov, V. I. Bruskov, M. E. Astashev, et al., J. Phys. Chem. B 115, 7693 (2011).
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Translated by A. Levina BIOPHYSICS
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