CC BY
7PACS 61.48.+C
Dmitriy S. Shevchenko1, Olga V. Rakhimova2, Nikita E. Podolsky3, Nikolay A. Charykov4, Konstantin N. Semenov5, Victor A. Keskinov6, Zhasulan K. Shaimardanov7, Batagoz K. Shaimardanova8, Natalie A. Kulenova9, Zhanar S. Onalbaeva10
TOXICITY OF WATER SOLUBLE OCTO-ADDUCT OF FULLERENE C60 AND
ARGININE 60 C60(C6H12NaN4O2)8H8
St. Petersburg Electrotechnical University «LETI», ul. Professora Popova 5, 197376, St. Petersburg, Russia St. Petersburg State Institute of Technology (Technical University), Moskovsky pr., 26 Saint-Petersburg, 190013, Russia
St Petersburg State University,7/9 Universitetskaya emb., Saint-Petersburg, 199034, Russia D. Serikbayev East Kazakhstan state technical university, A.K. Protozanov Street, 69, Ust-Kamenogorsk city, 070004, Republic of Kazakhstan. e-mail: keskinov@mail.ru Pavlov First St Petrsburg State Medical University, L.Tolstoy st. 6-8, St. Petersburg, 195176, Russia
Toxicity of water soluble octo-adduct of light fullerene Ceo with L-arginine is reported. The aim of this work was to study the biological activity of aqueous solutions of the adduct against Paramecium Caudatum (infusorians). Biotest method for determining the toxicity of water extracts was used. The parameters of the behavioral test based on
УДК 541.11/.118
Д.С. Шевченко1, О.В. Рахимова2, Н.Е.Подольский3, Н.А. Чарыков4, К.Н. Семенов5, В.А. Кескинов6, Ж.К. Шаймарданов7, Б.К.Шаймарданова8, Н.А. Куленова9, Ж.С. Оналбаева 10
ТОКСИЧНОСТЬ ВОДОРАСТВОРИМОГО ОКТО-АДДУКТА ФУЛЛЕРЕНА C60 И
АРГИНИНА 60
C60(C6H12NaN4O2)8H8
Санкт-Петербургский государственный электротехнический университет «ЛЭТИ», ул. Профессора Попова, 5, Санкт-Петербург, 197376, Россия Санкт-Петербургский государственный технологический институт (технический университет), Московский пр. 26, Санкт-Петербург, 190013, Россия Санкт-Петербургский государственный университет, Университетская наб., 7/9, Санкт-Петербург, 199034, Россия
Восточно-Казахстанский государственный технический университет им. Д Серикбаева, ул. Протозанова, 69, г. Усть-Каменогорск, 070004, Республика Казахстан e-mail: keskinov@mail.ru
Первый Санкт Петербургский медицинский университет им. И.П.Павлова. ул. Л. Толстого, 6-8, Санкт-Петербург, 195176, Россия
Сообщается о токсичности водорастворимого окто-аддукта легкого фуллерена С60 с L-аргинином. Целью данной работы было изучение биологической активности водных растворов аддукта по отношению к Paramecium Caudatum (инфузориям). Использован метод биотестирования для определения токсичности
1. Dmitriy S. Shevchenko, student of group 3508 Saint Petersburg Electrotechnical University "LETI", e-mail: skarp.ru@yandex.ru Шевченко Дмитрий Сергеевич, студент гр. 3508, СПбГЭТУ «ЛЭТИ»,
2. Olga V. Rakhimova Ph.D (Chem.), Associate Professor St. Petersburg State Electrotechnical University "LETI", e-mail:olga-18061963@yandex.ru
Рахимова Ольга Викторовна, канд. хим. наук, доцент, СПбГЭТУ «ЛЭТИ»
3. Nikita E. Podolsky, post-graduate Chemical Institute of S-Petersburg State University
Подольский Никита Евгеньевич аспирант Химический институт Санкт-Петербургского государственного университета
4. Чарыков Николай Александрович д-р хим. наук, профессор, СПбГТИ(ТУ) e-mail: ncharykov@yandex.ru Nikolay A. Charykov Dr Sci. (Chem.), Professor, St. Petersburg State Institute of Technology (Technical University)
5. Семенов Константин Николаевич, д-р хим. наук, профессор, СПбГУ, СПбГТИ(ТУ), ПСПбГМУ им. И.П. Павлова e-mail: semenov1986@yandex.ru
Konstantin N. Semenov,. Dr Sci. (Chem.), Professor, St. Petersburg State University: St. Petersburg State Institute of Technology (Technical University), Pavlov First St Petersburg State Medical University
6. Кескинов Виктор Анатольевич, канд. хим. наук, доцент, СПбГТИ(ТУ), e-mail: keskinov@mail.ru
Victor A. Keskinov, Ph.D (Chem.), Associate Professor, St. Petersburg State Institute of Technology (Technical University)
7. Шаймарданов Жасулан Кудайбергенович, д-р биол. наук, профессор, ректор Восточно-Казахстанского государственного технического университета им. Д. Серикбаева
Zhasulan K. Shaimardanov, Dr Sci., (Biol.), Professor, Rector, D. Serikbayev East Kazakhstan state technical university
8. Batagoz K. Shaimаrdanova Dr Sci., (Biol.), professor, D. Serikbayev East Kazakhstan state technical university Шаймарданова Ботагоз Касымовна д-р биол. наук, профессор, Восточно-казахстанский государственный технический университет
9. Куленова Наталья Анатольевна, канд. хим. наук, зав.кафедрой ХМиО, Восточно-Казахстанский государственный технический университет им. Д. Серикбаева, e-mail: NKulenova@ektu.kz
Natalie A. Kulenova, Ph.D (Chem.), Head of the Department of ChMiO, D. Serikbayev East Kazakhstan state technical university
10. Оналбаева Жанар Сагидолдиновна, канд. хим. наук, доцент, Восточно-Казахстанский государственный технический университет им. Д. Серикбаева
Zhanar S. Onalbaeva, Ph.D (Chem.), Associate Professor, D. Serikbayev East Kazakhstan state technical university Дата поступления - 16 января 2019 года
the chemotaxis reaction of infusorians was recorded with the help of the device "Biotester-2".
Key words: fullerene C60, arginine, octo-adduct, bio-testing, Paramecium Caudatum, infusoria, chemotaxis reaction, device "Biotester-2".
Introduction
This article continue the cycle of publications, concerning the synthesis, identification and properties investigation of the adducts of light fullerenes and amino-acids [1-23], herewith some publications [1-3] were devoted to the investigation of octo-adduct of light fullerene C60 with L-arginine, namely.
Toxicity of fullerenes and their derivatives were investigated and discussed widely. In one of the last works [24] a detailed and complete overview is given. Bibliography on the theme of fullerene toxicity [25-52] covers quite extensive number of works in 20 last years. The main conclusions are the following:
-Numerous further studies have also not shown any adverse or toxic effects of fullerene on organism. According to the Toxicological classification of substances exhibiting toxicity at doses above 1 g/kg, belong to the class of non-toxic substances. A long experiment was conducted in rats the diet which added fullerene in the form of solution in olive oil ( - Olive oil) [28]. The experiment lasted 5.5 years, as a control diet with the addition of just olive oil and water. Fullerene almost doubled the life expectancy of rats. Different diets did not affect the dynamics of animal weight, which also indicates the absence of toxic effects in .
- It is shown in [25] that fullerene in the form of water colloidal dispersion also does not show toxic properties, but only shows the properties of antioxidant. This conclusion is based on more than ten years of biological tests of fullerene dispersion in various experiments in vitro and in vitro. It did not reveal any toxic effects (at concentrations from 10-9 to 10-4 mol/l and at total doses up to 25 mg/kg).
- The toxicology of water-soluble derivatives of fullerene has been the subject of many discussions, but the vast majority of works have shown their low toxicity. So, water-soluble fullerene derivatives do not exhibit acute toxicity in vivo, even at sufficiently high doses. For example, the value for fullerenol in intraperitoneal mice is 1.2 g/kg [50].
is semi-lethal dose or the average dose of the substance, causing the death of half of the members of the test group. Parenteral administration of the amino acid derivative to mice at a dose of 80 mg/kg had no effect on the behavior and viability of mice for 6 months [28].
Materials
Authors used the following reagents and preparations:
- octo-adduct of light fullerene C60 with L-arginine C60(C6H13N4O2)8H8, which was synthesized by the method, described in [1, 3]. Adduct was purified by triple recrystal-lization (precipitation by methanol and solution in water) with subsequent washing on Soxhlet extractor by methanol. Adduct was identified by the methods of high performance liquid phase chromatography, Infrared and electronic spectroscopy, element C-H-N-O analysis, thermal
водных экстрактов. Параметры тестирования, основанные на реакции хемотаксиса инфузорий, фиксировались с помощью прибора "Biotester-2".
Ключевые слова: фуллерен С60, аргинин, окто-аддукт, биотестирование, Paramecium Caudatum, инфузории, хемотаксис , "Biotester-2".
complex analysis [1-3]. We appreciate the purity of octo-adduct Сю(СбН13Л4С2)8Л8 is equal to 97.5+0.7 mass.%.
- Lozina-Lozinsky mineral environment (composition is represented in Table 1):
Table 1. Composion of Lozin Lozinsky mineral environment for laboratory-pure cultures of Paramecium caudatum infusorium __production [56].
Salt concentration (g/dm3) (reagent brand "pure for the analysis") pH, a.u. Temperature, oC
NaCl KCl CaCl2 MgCl2 NaHCÜ3
0.10 0.01 0.01 0.01 0.01 6.8 - 7.2 22-26
- Solution for sharp mobility reduction of Paramecium caudatum - 10 mass. % NaCl in distilled water.
- Si-Mo HPA heteropolyanions as thickener.
- Baking yeast for Paramecium caudatum cultivation.
- Test odject Paramecium caudatum (infusorians). In Toxicological studies as test-objects using Paramecia (Paramecium caudatum Ehrenberg) is a common free-living ciliated infusoria, prefer alpha-mesosaprobic conditions. The temperature optimum lies in the range 24 to 28 °C, prefers a pH close to neutral (6.5 to 7.5). The main recorded indicators are survival and reproduction rate. The choice of Paramecium as a test object is determined by the following criteria. Through a combination of in Para-mecium characteristics of the cell and of the organism it is possible to study both cellular and organismic forms of reaction to the toxic effects. The possibility of cultivation in a wide range of temperatures allows them to be used for experimental work at any time of the year. The short life cycle, the speed of reproduction allow us to trace the reaction to intoxication in a relatively short time in a long series of generations. Using the principle of cloning, it is possible to obtain a large amount of genetically homogeneous material. The typical linear dimensions of Parame-cium caudatum is 0.1-0.3 mm (see Fig.1).
Fig.1. Appearance of Paramecium caudatum (permission 1 *102, optical stereoscopic microscope MBS-12).
Experimental method
Principe of the method. The method of determining toxicity is based on the ability of test objects, in this case Paramecium caudatum, to react to the presence in aqueous extracts of substances that are dangerous to their life, and directed to move along the gradient of concentrations of these substances (towards concentration
reduction), avoiding their harmful effects - as it is named "chemotaxis reaction". Such a gradient is created by layering in a vertical cuvette on a suspension of the infusoria, located in the thickener, the tested liquid. In this case, a stable interface boundary is formed in the measuring cuvette, which remains during the whole time of biotesting. This boundary does not prevent the free movement of the infusoria in their preferred direction and thus prevents the mixing of liquids from the lower and upper zones. After the creation of two zones in the cell within 30 min there is a redistribution of infusoria by zones. An important feature of the behavioral response of the infusoria is the mass movement of organisms into the upper layers of the liquid. If the analyzed sample does not contain toxic substances, the concentration of cells of infusoria in the upper zone will be observed in the cuvette. The presence of toxic substances in the analyzed sample leads to a different character of redistribution of infusoria in the cuvette, namely: the higher the toxicity of the sample, the smaller the proportion of infusoria moves to the upper zone (analyzed sample).
The criterion of toxic effect is a significant difference in the number of cells of infusoria observed in the upper zone of the cell in the idle solution (control sample) based on the Lozina-Lozinsky mineral medium, compared with this indicator observed in the analyzed sample. The quantitative assessment of the test reaction parameter characterizing the toxic effect is made by calculating the ratio of the number of cells of infusoria observed in the control and analyzed samples, and is expressed in the form of a dimensionless value — the toxicity index (T), the calculation formula of which is presented below:
T (cont ^sam)/^cont (1),
where: Icon, Isam - the instrument Biotest-2 reading, that is directly proportional to the infusoria concentrations in control sample and analyzed samples, correspondingly.
According to the value of the index T, the analyzed samples are classified according to their toxicity into 3 groups:
- the acceptable degree of toxicity (T < 0.40);
- moderate toxicity (0.40 < T< 0.70);
- high toxicity (T > 0.70).
In the case of very toxic samples, where the value Ttakes a value of 1 (i.e., when in the upper area of the cuvette with the analyzed sample motile ciliates are not detected), the toxicity index may not clearly describe the true level of toxicity of the sample. The sample should be diluted to such an extent that the toxicity index value does not reach one [53].
Concentration determination with the help of Biotester-2. Measurement and indication of the average values of Icon, Isam was determined from five counts, each measurement lasts 34 seconds. All serial measurements were repeated triply. All our probes sufficiently transparent (not turbid) for our measurements. Physical principle of Icont, Isam values calculation is based on the determination of optical densities in infusoria-containing dispersions (nephelometry). Infusoria concentrations in Biotester-2 investigations are expressed conditional-arbitrary units (a.u.).
Methodics of experiment consists of the following stages:
1. Preparation of infusoria culture with the working concentration of C = 1000 ± 500 cells/ml at the beginning of the stationary growth phase;
2. Preparation of the analyzed basic aqueous solution (with concentration 0.5 g octo-adduct/dm3);
4. Preparation with the help of consequent dilution the series of analyzed samples with the dilution: 1/1, 1/10, 1/1000, 1/1000, 1/10000;
5. Introducing 2 ml of suspension of infusoria in the vertical cuvette types 2T3.993.065 (13x13x45 mm) or 2TZ.993.066 (12.5x12.5x45 mm);
6. Creation of a time-stable gradient of chemical concentrations in the cell by alternately layering 1.6 ml of aqueous solutions of Si-Mo HPA (heteropolyanion) with different concentrations;
7. The exposure of the cell under standard laboratory conditions for 30 min for the occurrence of chemotactic response of infusoria;
8. Registration of the infusoria, released in the upper part of the cuvette, using a specialized pulse spectrophotometer series " Biotester-2" (5 experiments for each sample);
9. Determination of toxicity index-quantitative assessment of the test reaction parameter characterizing the toxic effect of the analyzed substances on the infusoria;
10. Statistical evaluation of the obtained data;
11. Classification according to the degree of toxicity;
12. Analysis of the results.
Results and discussion
Toxicity data for water soluble octo-adduct of light fullerene Ce0 with L-arginine Ce0(CeH13N4O2)8H8 are represented in Table 2 and in Fig.2
Table 2. Toxicity data for water soluble octo-adduct of light fullerene C with L-arginine
qoCQMsMQOsH
Dilution of initial solution (0.5 g/dm3)
Average device reading I (a.u.)
Average toxicity index T (a.u.)
Boardes of toxicity index*
T-T /T-T (a.u0
(a.u.)
Convergence
T - T„
T-T^ (a.u)
Toxicity level
Control 1/0
56.92
absence
1/1
32.33
0.43
0.03/0.04
0.19
satisfactory
moderate
1/10
47.56
0.16
0.01/0.01
0.07
satisfactory
acceptable
1/100
50.11
0.12
0.01/0.01
0.05
satisfactory
acceptable
1/1000
53.17
0.07
0.07/0.06
0.05
satisfactory
acceptable
1/10000
50.22
0.06
0.08/0.06
0.05
satisfactory
acceptable
*Tmax Tmin - maximal and minimal T values in series of measurements for the same sample, correspondingly.
**Operational control standard for convergence r = 0.43 T.
Fig.2. Toxicity index against -ig of the dilution of basic C60(C6H13N4O2)8H8 with the concentration 0.5 g/dm3.
One can see, that all water solutions of octo-adduct Q0(GHi3N4C2)8H8 are characterized by acceptable (other words low) toxicity and the only one most concentrated solution (with concentration (0.25 g/dm3) can be characterized by moderate toxicity (at the cower limit). But if one will determine the mass of infusoria in 1 dm3 of the solution he will get the value several orders of magnitude less than mass of octo-adduct in the same volume of the solution. So, these values of toxicity index (T = 0.43), also characterize practically non-toxicity.
Conclusions
One can see, that all water solutions of octo-adduct are characterized by acceptable (other words low) toxicity and the only one most concentrated solution (with concentration (0.25 g/dm3) can be characterized by moderate toxicity (at the cower limit). But if one will determine the mass of infusoria in 1 dm3 of the solution he will get the value several orders of magnitude less than mass of octo-adduct in the same volume of the solution. So, these values of toxicity index (T=0.43), also characterize practically non-toxicity.
Заключение
Можно видеть, что все водные растворы окто-аддукта С60(С6И13М402)8Н8характеризуются приемлемой (другими словами низкой) токсичностью и только один наиболее концентрированный раствор (с концентрацией (0.25 г/дм3) может характеризоваться умеренной токсичностью (на нижней границе умеренной токсичности). Однако, если определить массу инфузорий в 1 дм3 раствора, то получится величина на несколько порядков меньшая, чем масса окто-аддукта в том же объеме. Таким образом, эти значения индекса токсичности (T = 0.43), также характеризуют практическую нетоксичность раствора.
Acknowledgements
This work was supported by Russian Foundation for Basic Research (RFBR) (Projects № 18-08-00143 A, 19-015-00469A, and 19-016-00003A).
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