Biol Trace Elem Res DOI 10.1007/s12011-014-9899-5
Effects of Boron on Structure and Antioxidative Activities of Spleen in Rats Qianqian Hu & Shenghe Li & Enmei Qiao & Zhongtao Tang & Erhui Jin & Guangming Jin & Youfang Gu
Received: 27 September 2013 / Accepted: 14 January 2014 # Springer Science+Business Media New York 2014
Abstract In order to determine the relationship between boron and development of the spleen, especially in the promoting biological effects, we examined the effects of different levels of boron on weight, organ index, microstructure, and antioxidative activities of the spleen in rats. Sprague-Dawley (SD) rats were selected and treated with different concentrations of boron, and then, the organs were resected and weighed. One half of the tissue was fixed and embedded in paraffin to observe tissue structure changes. The other half of the tissue was homogenated for determining the antioxidant activities. The results showed that 40 mg/L of boron could increase weight, organ indexes, and antioxidant capacity of spleens and improve the spleen tissue structure, while the boron concentration above 80 mg/L could decrease weight, organ indexes, and antioxidant capacity of spleens and damage the spleen tissue structure. The higher the concentration, the more serious the damage was. Especially at the concentration of 640 mg/L, it could significantly inhibit the development of the spleen and even exhibit toxic effect. Hence, low boron concentration played a protective role in the development of the spleen, while high boron concentration could damage the organs and even produce toxic effect. Keywords Boron . Rats . Spleen . Antioxidative activity . Immune function . Microstructure
Introduction Boron is a bioactive food component with beneficial effects on the metabolism of many biological compounds (glucose, amino acids, triglycerides, macrominerals, and estrogen) and Q. Hu : S. Li (*) : E. Qiao : Z. Tang : E. Jin : G. Jin : Y. Gu Anhui Science and Technology University, Fengyang, China e-mail: [email protected]
the function of the tissues (the brain, bone, and immune system) [1–6]. Armstrong [7, 8] confirmed that boron enhanced fragment c (Fc) receptor expression and interleukin-6 production in the cultured mammalian macrophages. Although borates are generally viewed as relatively nontoxic chemicals, it is recognized that excessive exposure to boron can be associated with significant adverse health effects [9–11]. Recent studies [12, 13] showed that the relatively large quantity of dietary boron caused reproductive and developmental defects in rodents, especially the defects of male reproductive function [14]. The mechanism of boron toxicity is unknown, but it has attracted researchers to study the toxic effects of boron on the organ tissue even on the biomolecular level. Geyikoğlu [5] reported that high dose of boron decreased the concentration of glucose, glucogen, lactic acid, and ATP in pectoral muscle fiber and damaged mitochondrion structure in muscle fibers. Gu et al. [15] demonstrated that the consumption of drinking water containing 400 mg/L boron increased the thymus interstitial component, degenerated parenchyma, decreased thymocytes, and enlarged or increased thymic corpuscle. As harmful compounds with strong oxidization in the body, free radicals can damage the tissues and cells, thereby causing chronic diseases and aging effects. The free radicals were indirectly studied by detecting various antioxidants, such as superoxide dismutase (SOD), hydrogen peroxidase (CAT), and glutathione peroxidase (GSH-Px) [16]. The effects of boron on animal and human have become the hot spot for boron which is involved in the functions of the immune system, the activity of antioxidant defense system, and the inhibition of lipid peroxidation (LPO). Previous studies demonstrated that boron supplementation to a low-boron diet decreased paw swelling in adjuvant-induced arthritic rats [17, 18] and increased total antibody concentration in rats [19]; supplementation of boron compounds in diet exhibited potent hepatoprotective effects on carbon tetrachloride (CCl(4))-induced liver damage in mice; likely, both result to
Hu et al.
the inhibition LPO and the increase of the antioxidant defense system activity and vitamin status [20, 21]. The spleen is the largest lymphoid organ in which the innate and adaptive immune systems are combined in a unique organization way. For the spleen can remove elder erythrocytes from the circulation and allow the efficient removal of blood-borne microorganisms and cellular debris, the spleen is the most important organ for antibacterial and antifungal immune reactivity [22]. The purpose of this study is to investigate the effects of six levels of boron on the antioxidative activities of the spleen tissue in Sprague-Dawley (SD) rats. In addition, the effects of boron on the spleen tissue structure were studied.
physiological saline and dried with a filter paper. Samples were homogenated in ice bath. After 10 % homogenate was prepared, it was centrifuged at 3,000 rpm for 15 min. Then, supernatant fluid was stored at –80 °C. The other half of the tissue was subjected to bouin solution fixation, gradient alcohol dehydration, and methylsalicylate pellucidum, and then, paraffin blocks were prepared. The sections of about 6 μm thickness were cut off. The sections were stained with hematoxylin and eosin (HE) to study the general structure of organs and were then observed and photographed with Olympus CH30 microphotography system. Index Determination
Materials and Methods Animals, Reagents, and Diets All the 96 Sprague-Dawley rats (48 females and 48 males, 28±2 days old, 55.0±5.0 g) were obtained from Qinglong Mountain Experiment Breeding Farm (Nanjing City) and were numbered on the body surface with trinitrophenol. Boric acid is analytical grade, with purity of ≥99.5 % and content of ≥17.4 % (Chinese Medicine Group Chemical Reagent Co., Ltd.). SOD, malondialdehyde (MDA), CAT, GSH-Px, and Coomassie blue reagent were obtained from Nanjing Jiangcheng Biotech Institute. The basal rodent diet (from Qinglong Mountain Experiment Breeding Farm) contains 1.43 % Ca, 0.87 % P, 0.05 % Mg, 1.96 mg/kg boron, ≥18.15 % crude protein, ≥4.03 % crude fat, ≥5.12 % crude fiber, ≥7.94 % crude ash, and ≤10 % water. Groups and Breeding Ninety-six clean grade SD rats were acclimated for 7 days and then randomly divided into control group and experiment groups I, II, III, IV, and V, and the six groups of rats were fed with drinking water with different levels of boric acid (boron concentrations 0, 40, 80, 160, 320, and 640 mg/L) for 60 days. Every group had an equal number of female and male rats and had four repeats with 16 animals. All rats were placed in individually ventilated cages (IVCs) maintained at 24–28 °C and 50–65 % relative humidity with the normal 12:12-h light/dark cycle and provided with enough distilled drinking water and a commercial pelleted diet. Samples and Sampling Procedures After overnight food deprivation, all rats were injected with 10 % chloral hydrate and then immediately dissected to obtain the spleen. One half of the tissue was washed with cool
The spleen was weighed, and then, the spleen organ index was calculated by dividing the body weight by the spleen mass (the body weight data has been presented by Feng et al. [23]). Protein level of the spleen was determined with Coomassie blue reagent kit; SOD activity was determined according to the hydroxylamine method; MDA content was assayed according to thiobarbituric acid (TBA) method; GSH-Px activity was analyzed with thiodinitrobenzoic acid. Every procedure was carried out according to antioxygen kit manuals. The average area of white pulp splenic follicle, the average thickness of periarterial lymphatic sheath, and marginal zone were measured under Olympus CH30 microphotography system. Statistical Analysis Single factor variance analysis was performed by SPSS 11.5. The difference was considered to be significant at the 0.05 level, and testing results were expressed as mean value ± standard deviation (x SD ). Spleen weight, organ index, and antioxidation index were shown in a curve trend via Excel 2003.
Results Effects of Boron on Spleen Weight and Organ Index of Rats Compared with the average spleen weight and organ index of control group, average spleen weight (1.83±0.15 g) and organ index (0.59×10–2 ±0.04) of experiment group I were respectively increased by 6.6 % (p=0.412) and 15.3 % (p=0.139), and the difference was not significant (p>0.05). Experiment groups II–V were decreased significantly or extremely significantly and showed significant dose-effect relationship. The average spleen weight of groups II–V was respectively decreased by 21.3 % (p=0.038), 25.1 % (p=0.026), 41.0 % (p= 0.011), and 68.3 % (p
Effects of Boron on Structure and Antioxidative Activities of Spleen in Rats Qianqian Hu & Shenghe Li & Enmei Qiao & Zhongtao Tang & Erhui Jin & Guangming Jin & Youfang Gu
Received: 27 September 2013 / Accepted: 14 January 2014 # Springer Science+Business Media New York 2014
Abstract In order to determine the relationship between boron and development of the spleen, especially in the promoting biological effects, we examined the effects of different levels of boron on weight, organ index, microstructure, and antioxidative activities of the spleen in rats. Sprague-Dawley (SD) rats were selected and treated with different concentrations of boron, and then, the organs were resected and weighed. One half of the tissue was fixed and embedded in paraffin to observe tissue structure changes. The other half of the tissue was homogenated for determining the antioxidant activities. The results showed that 40 mg/L of boron could increase weight, organ indexes, and antioxidant capacity of spleens and improve the spleen tissue structure, while the boron concentration above 80 mg/L could decrease weight, organ indexes, and antioxidant capacity of spleens and damage the spleen tissue structure. The higher the concentration, the more serious the damage was. Especially at the concentration of 640 mg/L, it could significantly inhibit the development of the spleen and even exhibit toxic effect. Hence, low boron concentration played a protective role in the development of the spleen, while high boron concentration could damage the organs and even produce toxic effect. Keywords Boron . Rats . Spleen . Antioxidative activity . Immune function . Microstructure
Introduction Boron is a bioactive food component with beneficial effects on the metabolism of many biological compounds (glucose, amino acids, triglycerides, macrominerals, and estrogen) and Q. Hu : S. Li (*) : E. Qiao : Z. Tang : E. Jin : G. Jin : Y. Gu Anhui Science and Technology University, Fengyang, China e-mail: [email protected]
the function of the tissues (the brain, bone, and immune system) [1–6]. Armstrong [7, 8] confirmed that boron enhanced fragment c (Fc) receptor expression and interleukin-6 production in the cultured mammalian macrophages. Although borates are generally viewed as relatively nontoxic chemicals, it is recognized that excessive exposure to boron can be associated with significant adverse health effects [9–11]. Recent studies [12, 13] showed that the relatively large quantity of dietary boron caused reproductive and developmental defects in rodents, especially the defects of male reproductive function [14]. The mechanism of boron toxicity is unknown, but it has attracted researchers to study the toxic effects of boron on the organ tissue even on the biomolecular level. Geyikoğlu [5] reported that high dose of boron decreased the concentration of glucose, glucogen, lactic acid, and ATP in pectoral muscle fiber and damaged mitochondrion structure in muscle fibers. Gu et al. [15] demonstrated that the consumption of drinking water containing 400 mg/L boron increased the thymus interstitial component, degenerated parenchyma, decreased thymocytes, and enlarged or increased thymic corpuscle. As harmful compounds with strong oxidization in the body, free radicals can damage the tissues and cells, thereby causing chronic diseases and aging effects. The free radicals were indirectly studied by detecting various antioxidants, such as superoxide dismutase (SOD), hydrogen peroxidase (CAT), and glutathione peroxidase (GSH-Px) [16]. The effects of boron on animal and human have become the hot spot for boron which is involved in the functions of the immune system, the activity of antioxidant defense system, and the inhibition of lipid peroxidation (LPO). Previous studies demonstrated that boron supplementation to a low-boron diet decreased paw swelling in adjuvant-induced arthritic rats [17, 18] and increased total antibody concentration in rats [19]; supplementation of boron compounds in diet exhibited potent hepatoprotective effects on carbon tetrachloride (CCl(4))-induced liver damage in mice; likely, both result to
Hu et al.
the inhibition LPO and the increase of the antioxidant defense system activity and vitamin status [20, 21]. The spleen is the largest lymphoid organ in which the innate and adaptive immune systems are combined in a unique organization way. For the spleen can remove elder erythrocytes from the circulation and allow the efficient removal of blood-borne microorganisms and cellular debris, the spleen is the most important organ for antibacterial and antifungal immune reactivity [22]. The purpose of this study is to investigate the effects of six levels of boron on the antioxidative activities of the spleen tissue in Sprague-Dawley (SD) rats. In addition, the effects of boron on the spleen tissue structure were studied.
physiological saline and dried with a filter paper. Samples were homogenated in ice bath. After 10 % homogenate was prepared, it was centrifuged at 3,000 rpm for 15 min. Then, supernatant fluid was stored at –80 °C. The other half of the tissue was subjected to bouin solution fixation, gradient alcohol dehydration, and methylsalicylate pellucidum, and then, paraffin blocks were prepared. The sections of about 6 μm thickness were cut off. The sections were stained with hematoxylin and eosin (HE) to study the general structure of organs and were then observed and photographed with Olympus CH30 microphotography system. Index Determination
Materials and Methods Animals, Reagents, and Diets All the 96 Sprague-Dawley rats (48 females and 48 males, 28±2 days old, 55.0±5.0 g) were obtained from Qinglong Mountain Experiment Breeding Farm (Nanjing City) and were numbered on the body surface with trinitrophenol. Boric acid is analytical grade, with purity of ≥99.5 % and content of ≥17.4 % (Chinese Medicine Group Chemical Reagent Co., Ltd.). SOD, malondialdehyde (MDA), CAT, GSH-Px, and Coomassie blue reagent were obtained from Nanjing Jiangcheng Biotech Institute. The basal rodent diet (from Qinglong Mountain Experiment Breeding Farm) contains 1.43 % Ca, 0.87 % P, 0.05 % Mg, 1.96 mg/kg boron, ≥18.15 % crude protein, ≥4.03 % crude fat, ≥5.12 % crude fiber, ≥7.94 % crude ash, and ≤10 % water. Groups and Breeding Ninety-six clean grade SD rats were acclimated for 7 days and then randomly divided into control group and experiment groups I, II, III, IV, and V, and the six groups of rats were fed with drinking water with different levels of boric acid (boron concentrations 0, 40, 80, 160, 320, and 640 mg/L) for 60 days. Every group had an equal number of female and male rats and had four repeats with 16 animals. All rats were placed in individually ventilated cages (IVCs) maintained at 24–28 °C and 50–65 % relative humidity with the normal 12:12-h light/dark cycle and provided with enough distilled drinking water and a commercial pelleted diet. Samples and Sampling Procedures After overnight food deprivation, all rats were injected with 10 % chloral hydrate and then immediately dissected to obtain the spleen. One half of the tissue was washed with cool
The spleen was weighed, and then, the spleen organ index was calculated by dividing the body weight by the spleen mass (the body weight data has been presented by Feng et al. [23]). Protein level of the spleen was determined with Coomassie blue reagent kit; SOD activity was determined according to the hydroxylamine method; MDA content was assayed according to thiobarbituric acid (TBA) method; GSH-Px activity was analyzed with thiodinitrobenzoic acid. Every procedure was carried out according to antioxygen kit manuals. The average area of white pulp splenic follicle, the average thickness of periarterial lymphatic sheath, and marginal zone were measured under Olympus CH30 microphotography system. Statistical Analysis Single factor variance analysis was performed by SPSS 11.5. The difference was considered to be significant at the 0.05 level, and testing results were expressed as mean value ± standard deviation (x SD ). Spleen weight, organ index, and antioxidation index were shown in a curve trend via Excel 2003.
Results Effects of Boron on Spleen Weight and Organ Index of Rats Compared with the average spleen weight and organ index of control group, average spleen weight (1.83±0.15 g) and organ index (0.59×10–2 ±0.04) of experiment group I were respectively increased by 6.6 % (p=0.412) and 15.3 % (p=0.139), and the difference was not significant (p>0.05). Experiment groups II–V were decreased significantly or extremely significantly and showed significant dose-effect relationship. The average spleen weight of groups II–V was respectively decreased by 21.3 % (p=0.038), 25.1 % (p=0.026), 41.0 % (p= 0.011), and 68.3 % (p
