Antioxidant and immune potential marker enzymes assessment in the various tissues of rats exposed to indoleacetic acid and kinetin: A drinking water study

Abstract

In the present study, the influence of two different PGRs, indoleacetic acid (IAA) and kinetin (Kn) on immune potential enzymes, adenosine deaminase (ADA) and myeloperoxidase (MPO), and antioxidant defense enzymes such as glutathione peroxidase (GPx), glutathione reductase (GR), and superoxide dismutase (SOD) in various tissues of rats were investigated during the treatment as a drinking water model. 100 ppm of IAA and Kn as drinking water were administered orally to rats (Sprague–Dawley albino) ad libitum for 21 days continuously. The PGRs treatments caused different effects on the immune potential and antioxidant defense enzymes of experimented rats compared to controls. Results show that IAA caused a significant decrease in GR activity in the lungs and liver and an increase in the spleen. Also, IAA caused a significant decline in GPx activity in the lungs and an increase in the heart. SOD was significantly reduced in the heart, while increased in the lungs. Furthermore, IAA caused a significant decrease in ADA activity in the heart and blood whereas an increase in the kidney and spleen. MPO activity was also significantly increased in the heart by IAA treatment. The activity of enzymes were also seriously affected by Kn; GR activity decreased in the lungs, brain, and blood while GPx activity decreased in the spleen, brain, and heart. ADA activity was also significantly reduced in the blood whereas MPO activity rose in the spleen. In addition, SOD activity lowered in all tissues except for lungs where a significant increment was determined. As a conclusion, the results indicate that PGRs might affect on antioxidant and immune potential enzymes. These data, along with the determined changes suggest that PGRs produced substantial systemic organ toxicity in the erythrocyte, liver, brain, heart, lungs, spleen, and kidney during the period of a 21-day subacute exposure.

Introduction

Many chemicals are currently used in agriculture, and PGRs¹ are among those widely employed. The amounts of these substances placed into the environment may soon exceed those of insecticides [1]. Although PGRs are used for pest control and giving rise product on a wide variety of crops, little is known about the biochemical or physiological effects in mammalian organisms.

There is increased oxidative stress in obtained from a polluted area containing high concentrations of polyaromatic hydrocarbons, polychlorinated biphenyls, plant growth regulators (PGRs) and pesticides; however, little is known of how different PGRs classis contribute to the oxidative stress induced by these xenobiotics.

Antioxidant defenses, present in all aerobic organisms, include antioxidant enzymes and free-radical scavengers whose function is to remove reactive oxygen species (ROS), thus protecting the functions of organisms from oxidative stress [2]. The sensitivity of cell to oxidants is attenuated by antioxidant defense system such as GSH, GST, CAT, SOD, GPx, GR, and glucose-6-phosphate dehydrogenase (G6PD). The antioxidant defense system maintains a relatively low rate of the reactive and harmful OH. Oxidative stress occurs as a result of the effect of xenobiotics causing the disturbances in the antioxidant enzymes system [3]. Among these, GPx, through reduction of both hydrogen peroxide and organic hydroperoxides, provide an efficient protection against oxidative damage and free radicals in the presence of reduced glutathione (GSH). Previously oxidized GSH is regenerated by GR. SOD catalyses dismutation of superoxide anion radicals, whereas CAT eliminates hydrogen peroxide. Antioxidant enzymes play a crucial role in maintaining cell homeostasis. Another group of enzymes, GST act as catalyst of a wide variety of conjugation reactions of glutathione with xenobiotic compounds containing electrophilic center. Additionally, there are glutathione-independent enzymes that act as part of the cellular defense system against toxicity originated by active oxygen forms [4]. Oxidative stress may produce DNA damage, enzymatic inactivation, and peroxidation of cell constituents, especially lipid peroxidation when antioxidant defenses are impaired or overcome [5]. Oxidative stress is defined as a loss of the normal balance between reactive oxygen species (ROS) production and the antioxidant system [3]. Indeed, in addition to an excess generation of ROS, xenobiotics have a decreased or increased antioxidant capacity, which causes oxidative damage to cells. Superoxide anion $(\mathrm{O}_2{}^{-})$ is a major ROS and can be converted to hydrogen peroxide, which can be used by activated myeloperoxidase (MPO) to produce the strong oxidant hypochlorite [6].

In the literature, it is reported that fecundity, longevity, and egg viability have been changed in different insects by PGRs treatment [7], [8], [9], [10], [11]. Hsiao et al. [12] results’ show that kinetin has effective free radical-scavenging activity in vitro and antithrombotic activity in vivo. Furukawa et al. [13] findings indicate that IAA might induce the neuronal apoptosis in the S phase and lead to microencephaly. Also, de Melo et al. [14] determined that incubation for 24 h in the presence of IAA (1 mM) showed increase in the activities of SOD, CAT, and GPx in rat neutrophils and lymphocytes. The addition of exogenous antioxidant enzymes (SOD and CAT) prevented the loss of cell membrane integrity induced by IAA. John et al. [15] observed that IAA possesses teratogenic effects in mice and rats. El-Mofty and Sakr [16] found that gibberellic acid (GA₃) induced liver neoplasm in Egyptian toads, and they suggested that the tumors could be diagnosed as hepatocellular carcinomas. IAA and Kn also affects on the hematological and biochemical parameters of rats [17]. Ozmen et al. [18] observed that abscisic acid and gibberellic acid affect on sexual differentiation and some physiological parameters of laboratory mice. Also, it is reported that PGRs causes increase in the number of splenic plaque forming cells and circulating white blood cells, hematocrit values, and thymus weight in young deer mice [19]. On the other hand, some PGRs have been shown to affect the antioxidant defense systems and MDA content [20]. The effects of IAA and Kn were also investigated on human serum enzymes in vitro. IAA was found to inhibit aspartate aminotransferase (AST) and activate amylase, creatine phosphokinase (CPK), and lactate dehydrogenase (LDH). Kn inhibited muscle creatine kinase (CK-MB), while it activated AST and alanine aminotransferase (ALT) [21]. In the other hand, we have found that the level of IAA AST, LDH, and CPK were increased significantly by IAA, and the level of AST, LDH, and CPK were increased significantly by Kn in another study [22]. In addition, PGRs may induce oxidative stress, leading to generation of free radicals and cause lipid peroxidation as one of the molecular mechanisms involved in PGRs-induced toxicity [23]. According to The US Environmental Protection Agency (EPA), toxic xenobiotic chemicals are irritating to the eyes, skin and mucous membrane and since it is easily absorbed dermally, orally or by inhalation, can injure liver, kidney, muscle, and brain tissues.

In spite of reason above, there is still great contradiction between results. Therefore, to achieve a more rational design of PGRs, it is necessary to clarify the mechanism of toxicity for PGRs and develop an understanding of structure–toxicity relationships. For this aim, the treatment of PGRs was done orally because the effect of chemicals represents a well characterized in vivo toxicity model system. The tissues were chosen due to its important role during detoxification in degradation and bioactivation of PGRs.