DOI 10.1007/s10517-015-2850-8 Bulletin of Experimental Biology and Medicine, Vol. 158, No. 6, April, 2015 GENERAL PATHOLOGY AND PATHOPHYSIOLOGY
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Changes in Peripheral Blood Monocytes and Liver Macrophages in Male Rats after Benzo(a)pyrene Injection S. I. Kolesnikov, S. V. Michurina*, and S. A. Arkhipov* Translated from Byulleten’ Eksperimental’noi Biologii i Meditsiny, Vol. 158, No. 12, pp. 696-699, December, 2014 Original article submitted February 16, 2014 Intraperitoneal injections of benzo(a)pyrene to male rats in a total dose of 60 mg/kg modified the production of ROS and the phagocytic potential of blood monocytes by modulating their potential bactericidal activity. The lysosomal system (particularly the secondary lysosomes) of liver macrophages was activated, which promoted fusion of the hydrolytic potentials of macrophages and monocytes. These results indicated that the toxin modulated the cellular immune homeostasis and the level of general nonspecific resistance. Key Words: liver; Kupffer’s cells; monocytes; benzo(a)pyrene; phagocytic activity
Wide use and high toxicity of benzo(a)pyrene (BP) make it one of the most hazardous environmental pollutants for human health. It is present in many foodstuffs, in city air, cigarette smoke [11,12]. Irrespective of the route of its entry, BP penetrates into the blood, circulates in animal and human body, and modifies tissues and organs in general. Experiments have demonstrated the influence of BP on the cellular, humoral, and congenital immunity, each of these implies direct participation of the phagocytic cells [13]. Studies of the molecular characteristics of BP immunotoxicity have shown that the targets of this polycyclic aromatic carbohydrate are macrophages. The toxicant activates 96 of the 100 target genes in macrophages [14]. The liver as the main organ of homeostasis and detoxification suffers most of all in case of intoxication. Kupffer’s cells, components of the mononuclear phagocyte system, are directly involved in realization of homeostasis and detoxification. These cells constitute 70% of the entire macrophage population in the organism and up to 40% of all liver cells [5,7]. The liver is an immunoactive organ: its blood-lymph barrier includes numerous resident macrophages, T cells (Pit cells). Passing through the liver, T cells survive several stages of their differentiation [3], up to Research Center of Family Health Problems and Human Reproduction, Irkutsk; *Research Institute of Clinical Immunology and Experimental Lymphology, Novosibirsk, Russia. Address for correspondence: [email protected]. S. I. Kolesnikov
apoptosis. Presumably, the liver serves as a sort of a “trap” for T cells, these processes realized with direct involvement of the liver macrophages [8]. We study the morphology and functions of the blood monocytes and the ultrastructure of liver macrophages under conditions of BP exposure.
MATERIALS AND METHODS The study was carried out on adult male Wistar rats (180-220 g) kept in vivarium on standard ration. Adult animals (group 2, n=20) were daily (at 10.00) injected (intraperitoneally) with BP (20 mg/kg in 0.2-0.3 ml olive oil) for 3 days (summary dose (60 mg/kg). Controls (group 1, n=20) received olive oil according to the same protocol. The animals were sacrificed 24 h after the last injection of the toxicant. The study was carried out in accordance with the humanity philosophy presented in the EEC directives (86/609/EEC) and the Helsinki Declaration. The leukocyte suspension for culturing and in vitro studies was prepared by spontaneous sedimentation of erythrocytes in gelatin-containing saline [6]. The percentage of monocytic cells with phagocytic activity towards corpuscular staphylococcal reagent (phagocytic index) and the number of liophylization-inactivated staphylococcal bacterial bodies, phagocytosed by the blood mononuclear phagocytes (phagocytic number), were evaluated after 1 h of cell incubation in medium
0007-4888/15/15860735 © 2015 Springer Science+Business Media New York
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Bulletin of Experimental Biology and Medicine, Vol. 158, No. 6, April, 2015 GENERAL PATHOLOGY AND PATHOPHYSIOLOGY
containing phagocytosis objects [4]. ROS generation by blood monocytes was evaluated by the NBT test 1 h after addition of NBT to the cultures [2,4]. Liver specimens for ultrastructural studies were fixed with 2.5% glutaraldehyde in 0.1 M phosphate buffer (pH 7.4) and postfixed in buffered osmium fixative, dehydrated, and embedded in epon-812. The sections were sliced on an LKB-8800 ultratome. Ultrathin sections were contrasted with saturated water solutions of uranyl acetate and lead citrate and examined in JEM-7A and JEM-100S/SEC electron microscopes. Five blocks of the organ were prepared from each animal. Quantitative parameters of the structural organization of cells were evaluated in accordance with the main stereological rules [1]. Morphometric analysis of liver macrophage ultrastructure was carried out using open test systems consisting of squares with 0.252 μ distance between the points at the final magnification of ×59,500 by projecting the negative image to the test system. The volume, surface, and numerical densities of lysosomal structures were evaluated. A total of 15-30 profiles of Kupffer’s cell sections were examined at each stage of the study, because of high number of these cells in the liver. Equal numbers of cells from each animal were analyzed. The data were statistically processed by ANOVA (Microsoft Excel, Statistica 5.0 software) using the arithmetic means, standard errors in the arithmetic means, and Student and Mann–Whitney tests. The differences between the control and experimental groups were considered significant at p
735
Changes in Peripheral Blood Monocytes and Liver Macrophages in Male Rats after Benzo(a)pyrene Injection S. I. Kolesnikov, S. V. Michurina*, and S. A. Arkhipov* Translated from Byulleten’ Eksperimental’noi Biologii i Meditsiny, Vol. 158, No. 12, pp. 696-699, December, 2014 Original article submitted February 16, 2014 Intraperitoneal injections of benzo(a)pyrene to male rats in a total dose of 60 mg/kg modified the production of ROS and the phagocytic potential of blood monocytes by modulating their potential bactericidal activity. The lysosomal system (particularly the secondary lysosomes) of liver macrophages was activated, which promoted fusion of the hydrolytic potentials of macrophages and monocytes. These results indicated that the toxin modulated the cellular immune homeostasis and the level of general nonspecific resistance. Key Words: liver; Kupffer’s cells; monocytes; benzo(a)pyrene; phagocytic activity
Wide use and high toxicity of benzo(a)pyrene (BP) make it one of the most hazardous environmental pollutants for human health. It is present in many foodstuffs, in city air, cigarette smoke [11,12]. Irrespective of the route of its entry, BP penetrates into the blood, circulates in animal and human body, and modifies tissues and organs in general. Experiments have demonstrated the influence of BP on the cellular, humoral, and congenital immunity, each of these implies direct participation of the phagocytic cells [13]. Studies of the molecular characteristics of BP immunotoxicity have shown that the targets of this polycyclic aromatic carbohydrate are macrophages. The toxicant activates 96 of the 100 target genes in macrophages [14]. The liver as the main organ of homeostasis and detoxification suffers most of all in case of intoxication. Kupffer’s cells, components of the mononuclear phagocyte system, are directly involved in realization of homeostasis and detoxification. These cells constitute 70% of the entire macrophage population in the organism and up to 40% of all liver cells [5,7]. The liver is an immunoactive organ: its blood-lymph barrier includes numerous resident macrophages, T cells (Pit cells). Passing through the liver, T cells survive several stages of their differentiation [3], up to Research Center of Family Health Problems and Human Reproduction, Irkutsk; *Research Institute of Clinical Immunology and Experimental Lymphology, Novosibirsk, Russia. Address for correspondence: [email protected]. S. I. Kolesnikov
apoptosis. Presumably, the liver serves as a sort of a “trap” for T cells, these processes realized with direct involvement of the liver macrophages [8]. We study the morphology and functions of the blood monocytes and the ultrastructure of liver macrophages under conditions of BP exposure.
MATERIALS AND METHODS The study was carried out on adult male Wistar rats (180-220 g) kept in vivarium on standard ration. Adult animals (group 2, n=20) were daily (at 10.00) injected (intraperitoneally) with BP (20 mg/kg in 0.2-0.3 ml olive oil) for 3 days (summary dose (60 mg/kg). Controls (group 1, n=20) received olive oil according to the same protocol. The animals were sacrificed 24 h after the last injection of the toxicant. The study was carried out in accordance with the humanity philosophy presented in the EEC directives (86/609/EEC) and the Helsinki Declaration. The leukocyte suspension for culturing and in vitro studies was prepared by spontaneous sedimentation of erythrocytes in gelatin-containing saline [6]. The percentage of monocytic cells with phagocytic activity towards corpuscular staphylococcal reagent (phagocytic index) and the number of liophylization-inactivated staphylococcal bacterial bodies, phagocytosed by the blood mononuclear phagocytes (phagocytic number), were evaluated after 1 h of cell incubation in medium
0007-4888/15/15860735 © 2015 Springer Science+Business Media New York
736
Bulletin of Experimental Biology and Medicine, Vol. 158, No. 6, April, 2015 GENERAL PATHOLOGY AND PATHOPHYSIOLOGY
containing phagocytosis objects [4]. ROS generation by blood monocytes was evaluated by the NBT test 1 h after addition of NBT to the cultures [2,4]. Liver specimens for ultrastructural studies were fixed with 2.5% glutaraldehyde in 0.1 M phosphate buffer (pH 7.4) and postfixed in buffered osmium fixative, dehydrated, and embedded in epon-812. The sections were sliced on an LKB-8800 ultratome. Ultrathin sections were contrasted with saturated water solutions of uranyl acetate and lead citrate and examined in JEM-7A and JEM-100S/SEC electron microscopes. Five blocks of the organ were prepared from each animal. Quantitative parameters of the structural organization of cells were evaluated in accordance with the main stereological rules [1]. Morphometric analysis of liver macrophage ultrastructure was carried out using open test systems consisting of squares with 0.252 μ distance between the points at the final magnification of ×59,500 by projecting the negative image to the test system. The volume, surface, and numerical densities of lysosomal structures were evaluated. A total of 15-30 profiles of Kupffer’s cell sections were examined at each stage of the study, because of high number of these cells in the liver. Equal numbers of cells from each animal were analyzed. The data were statistically processed by ANOVA (Microsoft Excel, Statistica 5.0 software) using the arithmetic means, standard errors in the arithmetic means, and Student and Mann–Whitney tests. The differences between the control and experimental groups were considered significant at p
