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Matysik S.I., KuzminovB.P., Ostapiv D.D. Cytotoxic action of hepatoprotector antral on bull sperm (на англ. яз.; Матысик С.И., Кузьминов Б.П., Остапив Д.Д. Цитотоксическое действие гепатопротектора антраля на сперму быков)
DOI: http://dx.doi.org/10.33029/0016-9900-2020-99-2-206-209 Оригинальная статья
© AUTHOR COLLECTIVE, 2020
Matysik S.I.1, Kuzminov B.P.1, Ostapiv D.D.2
Cytotoxic action of hepatoprotector Antral on bull sperm
'Danylo Halytsky Lviv National Medical University Department of hygiene and prophylactic toxicology, 79010, Lviv, Ukraine;
institute of animal biology of NAAS, Lviv, 79034, Ukraine
Introduction. According to the European legislation and requirements, industrial production of medication shall be provided with well-defined regulations of the quality of such preparation.
Material and methods. Cytotoxic action of hepatoprotector Antral on bull sperm was evaluated. Samples of sperm were divided into control and two experimental samples: control one was diluted with phosphate-buffered saline (PSB), which included NaCl - 0.8 g, KCl - 0.02 g, Na2HPO4 - 0.11 g, KH2PO4 - 0.02 g, MgCl2 - 0.01 г, Н2О till 100ml; in the first experimental sample PSB was added with antral in the dose 1/500 LD50 (2.77 mg); in the second - PSB with Antral in the dose 1/100 LD50 (13.87 mg) was added. The survival of spermatozoa was defined until termination of rectilinear forward movement in sperm, which was preserved in temperature of 2-5 °C; respiratory activity was defined (by polarography (ng-atom 0/0.1 ml of semen (S) • min) in thermostated sell (temperature of 38.5 °C), with the volume of 1.0 ml with the automatic registration of process flow by potentiometer, the proportion of decrease of which was evaluated in accordance to impacted dose of antral in substrate and restorative activity was defined potentiometrically (mV/0.1ml C • min) using a system of pen microelectrodes that were inserted in thermostatedpolarographic cell).
Results. In the experimental samples of semen in comparison with the control sample the respiratory and restorative activities were reduced; dose 1/500 LD50 to 58.8% and dose 1/100 LD50 to 68.5%; restorative activity - to 66.7% and 54.5% correspondingly.
Conclusion. Antral reduces the survival of sperm being irrespective of dose. Keywords: Antral; cytotoxic action; bull sperm.
For citation: Matysik S.I., Kuzminov B.P., Ostapiv D.D. Cytotoxic action of hepatoprotector Antral on bull sperm. Gigiena i Sanitaria (Hygiene and Sanitation, Russian journal). 2020; 99(2): 206-209. (In Russ.). DOI: http://dx.doi.org/10.33029/0016-9900-2020-99-2-206-209
For correspondence: Svetlana I. Matysik, Assistant at the Department of hygiene and prophylactic toxicology of the Danylo Halytsky Lviv National Medical University, Lviv, 79010, Ukraine. E-mail: lana77matysik@gmail.com
Information about authors: Matysik S.I., https://orcid.org/0000-0002-2251-4948
Conflict of interest. The authors declare no conflict of interest. Acknowledgment. The study had no sponsorship.
Contribution: Research concept and design - Ostapiv D.D.; Statistical polishing of the material - Matysik S.I.; Writing of the text - Matysik S.I., Kuzminov B.P.; Manuscript text editing - Kuzminov B.P.
Received: August 21, 2018 Accepted: December 12, 2019 Published: March 23, 2020
Матысик С.И.1, Кузьминов Б.П.1, Остапив Д.Д.2
Цитотоксическое действие гепатопротектора антраля на сперму быков
'Львовский национальный медицинский университет им. Данила Галицкого, 79010, Львов, Украина;
2Институт биологии животных НААН, 79034, Львов, Украина
Введение. Согласно с Европейским законодательством и требованиями, промышленный выпуск препарата должен быть обеспечен чётко очерченным регламентом качества проведения такого вида производства, что обусловливает проведение обязательной токсикологической оценки.
Материал и методы. Проведена оценка цитотоксического влияния гепатопротектора антраля на спермиях быков. Пробы спермы делили на контрольную и две опытные: контрольную пробу разжижали фосфатно-солевым буфером (ФСБ), в состав которого входят NaCl - 0,8 г, KCl - 0,02 г, Na2HPO4 - 0,11 г, KH2PO4 - 0,02 г MgCl2 - 0,01 г, Н2О - до 100 мл; в первую опытную пробу добавляли ФСБ с антралем в дозе 1/500 LD50 (2,77мг), во вторую - ФСБ с антралем в дозе 1/100 LD50 (13,87 мг). В пробах определяли выживаемость спермиев (ч), которые хранились при температуре 2-5 °С, по времени прекращения их прямолинейно-поступательного движения; дыхательную активность (полярографически (нг-атом 0/0,1 мл спермы (С) • мин) в термостатированной ячейке (температура 38,5 °С) объёмом 1 мл с автоматической регистрацией течения процесса потенциометром; пропорциональность, снижение которой оценивали в соотношении к введённой дозе антраля в субстрате), восстановительную активность (потенциометрически (mV/0,1 мл с • мин) с использованием системы открытых микроэлектродов, которые вставляли в термостатированную полярографическую ячейку.
Matysik S.I., Kuzminov B.P., Ostapiv D.D. Cytotoxic action of hepatoprotector Antral on bull sperm
DOI: http://dx.doi.org/10.33029/0016-9900-2020-99-2-206-209
Original article
Результаты. В опытных пробах по сравнению с контрольной уменьшилась как дыхательная, так и восстановительная активность спермы: доза 1/500 LDsg на 58,8% и доза 1/100 LDsg - на 68,5%; восстановительная активность - на 66,7 и 54,5% соответственно.
Заключение. Антраль снижает выживаемость сперматозоидов независимо от дозы. Ключевые слова: антраль; цитотоксическое действие; спермии быков.
Для цитирования: Матысик С.И., Кузьминов Б.П., Остапив Д.Д. Цитотоксическое действие гепатопротектора антраля на сперму быков. Гигиена и санитария. 2020; 99 (2): 206-209. DOI: http://dx.doi.org/10.33029/0016-9900-2020-99-2-206-209
Для корреспонденции: Матысик Светлана Игоревна, ассист. каф. гигиены и профилактической токсикологии Львовского национального медицинского университета им. Данила Галицкого, 79010, Львов, Украина. E-mail: lana77matysik@gmail.com
Конфликт интересов. Авторы заявляют об отсутствии конфликта интересов. Финансирование. Исследование не имело спонсорской поддержки.
Участие авторов: концепция и дизайн исследования - Остапив Д.Д.; статистическая обработка материала - Матысик С.И.; написание текста - Матысик С.И., Кузьминов Б.П.; редактирование - Кузьминов Б.П.
Поступила: 21.08.18 Принята к печати: 12.12.19 Опубликована: 23.03.2020
Relevance
The influence of the environmental pollution factor, is burdened with the difficult economic situation, wide-spread harmful habits among population, popularization and unsubstantiated use of medications, biologically active substances, stimulants, useless food products, stipulates increase of the number of liver disease, which, as the main organ of detoxification, is affected first. According to the data of the WHO, the annual growth rate of liver disease reaches 2billion in the world, that requires the use of effective drugs with a selective action on a hepatobiliary system of a person [4]. One of such drugs in modern therapy is domestic hepatoprotector antral, which also has an-tiulcer and antioxidant effects. Use of this drug in the field of hepatol-ogy and gastroenterology is stipulated by its impact on the respiratory and restorative activity of cells, reducing oxygen consumption [13].
According to the European legislation and requirements [12], industrial production of medication shall be provided with well-defined regulations of the quality of such production, what stipulates conduction of compulsory toxicological assessment of the influence of antral (in this case) on the organisms of people working, with compulsory substantiation of the safe reference level of impact.
The present modern list of compulsory methods of the assessment of potential hazards of toxic substances on the health of population and environment stipulates for engagement of medication, highly-informative, non-invasive and express methods, which include study of the impact of medication on alternative test-systems - cell cultures Daphnia magnastraus, bull sperm, etc. [6,7]. Such methods enable to study specificity and the direction of the toxic influence of the active substance on the culture of cells from different organs and systems of the organism in isolation or in model environments, and to assess efficiency of the action of the medications in regards to their phar-macokinetics and pharmacodynamics in experiments short enough [2,5].Having high sensitivity to the influence of toxic substances and being easily standardized, bull sperm suspension is widely used in the practice of toxicological studies [1, 3, 5].
The purpose of the work was the assessment of the cytotoxic action of antral on bull sperm.
Material and methods
Antral is a synthetic hepatoprotector, which is an active pharmaceutical ingredient, containing tris-{N-(2,3-dimethylphenyl)an-thranilate} aluminium - 0,1 or 0,2g. The medication is moderately soluble in chloroform, and practically insoluble in water, 96% ethyl alcohol, acetone and hexanes.The median lethal dose (LD50) for white rates - 1387,5 mg/kg [27].
Cytotoxic action of tris-{N-(2,3-dimethylphenyl)anthranilate} aluminium in the form of the suspension was studied on fresh ejaculates of 4 bulls with the following physiological characteristics: sperm volume 4-5 ml, sperm cells concentration 0,7-1,2*109, cells/ ml, number of live spermatozoa amounted to 70-85 %. Samples of sperm were divided into control and two experimental samples: con-
trol one was diluted with phosphate-buffered saline (PSB), which included NaCl - 0,8 r, KCl - 0,02 r, Na2HPO4 - 0,11 r, KH2PO4 - 0,02 g, MgCl2 - 0,01 r, H20 till 100ml, in the first experimenta2 sample PSB was added with antral in the dose 1/500 LD50 (2,77 mg); in the second - PSB with antral in the dose 1/100 LD50 (13,87 mg) was added. The survival of spermatozoa was defined until termination of rectilinear forward movement in sperm, which was preserved in temperature 2-5 °C ; respiratory activity was defined (by polarography (ng-atom 0/0,1 ml of semen (S) • min) in thermostated sell (temperature 38,5 °C), with the volume 1,0 ml with the automatic registration of process flow by potentiometer, the proportion of decrease of which was evaluated in accordance to impacted dose of antral in substrate and restorative activity was defined potentiometrically (mV/0,1ml C • min) using a system of pen microelectrodes that were inserted in thermostated polarographic cell [15,16,17]). Statistical processing of study results was held by M.O.Plokhinskyi [10].
Results
In the experimental samples of semen in comparison with the control sample the respiratory and restorative activities were reduced; dose 1/500 LD50 was accompanied by oxygen reduction to 58,8% (p < 0,01), and dose 1/100 LD50 to 68,5% (p < 0,01); restorative activity reduced to 66,7% (p < 0,001) and 54,5% (p < 0,01) accordingly. No reliable dose-dependent changes of the studied biochemical parameters were determined at exposure to different doses of the medication.
The influence of antral on the intensity of oxidation processes is confirmed by strong correlational dependence between the dose of the pharmacological drug and the value of respiratory and restorative activity (n2 = 0,76 and 0,82 accordingly).
Reduction of the parameters of the oxidation processes in sperm under the influence of antral in dose 1/500 LD50did not affect cell survival, which amounted to 102,0 ± 9,95 and was at the control level (table). Instead low respiratory and restorative activity of spermatozoa in the sample with hepatoprotector in dose 1/100 LD5o, probably, lead to reduction of their survival time, which was lower for 30,0 hours ((27,2 %; p < 0,01) compared to the control and for 24 hours (25,6 %; p = 0,1) compared to the action of the medication in dose 1/500 LD50.
Correlation dependence between the influence of increasing doses of antral and survival of bull spermatozoa is weak (n2 = 0,438), what may be explained by the sufficiently wide range between the therapeutic and median lethal dose of the medication, determined by us in experiments on laboratory animals [9].
Discussion
Respiratory activity of reproductive cells is related exclusively to the functioning of mitochondria, thus, oxygen consumption by spermatozoa is a feature of their potential possibilities to PSB synthesis and ensuring of the filament movement. By means of PSB
Matysik S.I., Kuzminov B.P., Ostapiv D.D. Cytotoxic action of hepatoprotector antral on bull sperm (на англ. яз.; Матысик С.И., Кузьминов Б.П., Остапив Д.Д. Цитотоксическое действие гепатопротектора антраля на сперму быков)
DOI: http://dx.doi.org/10.33029/0016-9900-2020-99-2-206-209 Оригинальная статья
The intensity of oxidation processes in spermatozoa and their survival under the influence of different doses of antral (n = 4, M ± m)
Conditions of sperm incubation Activity Survival of spermatozoa, h
respiratory, ng-atom O/O,1ml С • min restorative, mV/0,1ml С • mon
Control (PSB) 7,04 ± 0,74 0,ЗЗ ± 0,01 108,0 ± 6,00
PSB + 1/500 LD50 2,90 ± 0,68* 0,11 ± 0,01* 102,0 ± 9,95
PSB + 1/100 LD50 2,22 ± 0,65* 0,15 ± 0,04* 78,0 ± 5,20*
Correlation ratio, n2 0.76 0.82 0.44
Note. * - probable compared to control (0,001 > p < 0,01).
energy, created in mitochondria as a result of the coupling of respiration and phosphorylation, cells perform specific functions, including, conduction of reaction-answer to the external influence. Although the cytotoxic influence of antral in dose 1/500 LD50 does not lead to changes of energy process in spermatozoa but probably does not disrupt the balance between the functional activity of the components of electron transport chain and anti-oxidant system of cells [18, 19, 22]. Excess of pharmacological drug (1/100 LD50) reduces the functioning of the respiratory chain of mitochondria, violates mitochondrial oxy-reduction [20, 21], accompanied by the creation of active forms of oxygen, the result of which is a movement of spermatozoa and period of their survival on the level of the reproductive system.
Conclusions
1. Cytotoxic action of hepatoprotector antral in dosesl/500 LD50 and 1/100 LD50 onoxidation processes in semen is characterized by the reduction of respiratory and restorative activity of spermatozoa.
2. Survival of the reproductive cells is preserved under the action of 1/500 LD50 of the drug and significantly reduces under the action of 1/100 LD50 of antral.
3. To substantiate the allowable level of antral content in the air of the working area, it is necessary to conduct the study of its gonadotoxic effects on laboratory animals.
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DOI: http://dx.doi.org/10.33029/0016-9900-2020-99-2-206-209
Original article
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