http://informahealthcare.com/rnf ISSN: 0886-022X (print), 1525-6049 (electronic) Ren Fail, 2015; 37(3): 497–504 ! 2015 Informa Healthcare USA, Inc. DOI: 10.3109/0886022X.2015.1006085
LABORATORY STUDY
Renoprotection of Kolaviron against benzo (A) pyrene-induced renal toxicity in rats Isaac A. Adedara, Yetunde M. Daramola, Joshua O. Dagunduro, Motunrayo A. Aiyegbusi, and Ebenezer O. Farombi
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Drug Metabolism and Toxicology Research Laboratories, Department of Biochemistry, College of Medicine, University of Ibadan, Ibadan, Nigeria
Abstract
Keywords
Benzo(a)pyrene (B[a]P), a polycyclic aromatic hydrocarbon generally formed from incomplete combustion of organic matter, reportedly causes renal injury and elicits a nephropathic response. The present study investigated the modulatory effect of Kolaviron, an isolated bioflavonoid from the seed of Garcinia kola, on renal toxicity induced by B[a]P in Wistar rats. Benzo[a]pyrene was administered at a dose of 10 mg/kg alone or in combination with Kolaviron at 100 and 200 mg/kg for 15 d. Administration of B[a]P alone resulted in significant increase in plasma levels of urea and creatinine in the treated rats. Moreover, B[a]P exposure significantly decreased the activities of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx) and glutathione-S-transferase (GST) as well as glutathione (GSH) level in the kidneys of treated rats. Conversely, myeloperoxidase (MPO) activity, hydrogen peroxide (H2O2) and malondialdehyde (MDA) levels were markedly elevated in kidneys of B[a]P-treated rats compared with control. Further, B[a]P exposure significantly decreased the circulatory concentrations of triiodothyronine (T3) and T3/T4 ratio without affecting thyroxine (T4) in the treated rats. Light microscopy revealed tubular lumen with numerous protein casts in kidneys of rats exposed to B[a]P alone. Kolaviron co-treatment significantly improved the renal antioxidant status, thyroid gland function and restored the renal histology, thus demonstrating the protective effect of Kolaviron in B[a]P-treated rats. Dietary inclusion of Kolaviron could exert protective effects against renal toxicity resulting from B[a]P exposure.
Benzo[a]pyrene, Kolaviron, kidney, thyroid hormones, rats
Introduction Benzo(a)pyrene (B[a]P, Figure 1A) is a global environmental contaminant belonging to a member of polycyclic aromatic hydrocarbon (PAH) family. Environmental contamination by PAH occur as complex mixtures. The principal environmental sources of B[a]P includes diesel exhaust, industrial incineration products, incomplete combustion of carbonaceous materials by humans for energy and barbeque.1–3 Human exposure to B[a]P is principally through ingestion of contaminated food and water as well as inhalation of particulates.4 Exposure to B[a]P is known to cause renal injury and elicits a nephropathic response.5 Previous toxicological studies demonstrated the expression of aryl hydrocarbon receptor (AHR) in the renal tissues, thus reinforcing the possible metabolic activation of B[a]P to reactive intermediates similar to the liver cells.6,7 Moreover, B[a]P-induced nephropathy was demonstrated to involve disruption of glomerular cell–cell and cell–matrix interactions in rats.8 A single oral administration of B[a]P (125 mg/kg) Address correspondence to Dr. Isaac A. Adedara, Drug Metabolism and Toxicology Research Laboratories, Department of Biochemistry, College of Medicine, University of Ibadan, Ibadan 20005, Nigeria. Tel: +2347032962608; Fax: 234-2-8103043. E-mail: dedac2001@ yahoo.co.uk
History Received 7 September 2014 Revised 23 November 2014 Accepted 11 December 2014 Published online 23 January 2015
caused renal toxicity and loss of DNA integrity in mice.9 Intrauterine exposure of mice to B[a]P resulted in sustained deficits of renal structure and function that compromise organ function long after birth.7 The metabolic activation of B[a]P which releases reactive oxygen species (ROS) that produce oxidative stress is a well-established mechanism of toxicity in various experimental animal models.9–12 The link between chemical injury and nephropathy is best demonstrated by the strong association between aromatic hydrocarbon exposures and glomerular diseases.13,14 In Nigeria, exposure to B[a]P for the general population could occur through consumption of contaminated food or groundwater from frequent spillage of petroleum (with aromatic hydrocarbons accounting for approximately 45% of total hydrocarbons),15 at refineries, underground storage tanks at gas stations and poorly managed waste sites. End-stage renal disease accounts for 8% of all medical admissions and 42% of renal admissions in Nigeria.16,17 Kolaviron (Figure 1B) is an isolated bioflavonoid from the seeds of Garcinia kola Heckel (Family Guttiferae) widely used in African Traditional Medicine. It is a potent phytochemical with established antioxidant,18 antiinflammatory,19,20 and anti-genotoxic,21,22 properties. Besides its protection against hepatic oxidative damage induced by 2-acetylaminofluorene, carbon-tetrachloride, aflatoxin
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Figure 1. Chemical structures of tested compounds. (A) Benzo[a]pyrene (B[a]P) and (B) Kolaviron (KV).
B1,22–24 Kolaviron has been reported to protect against potassium bromate-induced nephrotoxicity,25 inhibit renal apoptosis by ethylene glycol monoethyl ether,26 and ameliorate hyperglycemia-mediated renal oxidative damage in diabetic rats.27 However, there is no report on the effect of Kolaviron on renal toxicity due to B[a]P exposure. Considering the widespread availability of B[a]P in the environment and the beneficial health effects of Kolaviron, we have investigated the influence of Kolaviron on B[a]P-induced renal toxicity using Wistar rats as the animal model.
Materials and methods
Animal model Adult male Wistar rats (8 weeks old; 158 ± 3 g) obtained from the Department of Biochemistry, University of Ibadan, Ibadan were allowed to acclimate for 1 week prior to the commencement of the experiment. The animals were fed rat pellets, given drinking water ad libitum, and subjected to natural photoperiod of a 12 h alternating light–dark cycle. Animal care and experimental protocols were executed according to the approved guidelines set by the University of Ibadan Ethical Committee, which is in agreement with the Guide for the Care and Use of Laboratory Animals’ prepared by the National Academy of Science (NAS) and published by the National Institute of Health.
Chemicals Benzo[a]pyrene (B[a]P), trichloroacetic acid, thiobarbituric acid, 1-chloro-2,4-dinitrobenzene, hydrogen peroxide, glutathione, epinephrine and 50 ,50 -dithio-bis-2-nitrobenzoic acid were procured from Sigma Chemical Co. (St. Louis, MO). All other reagents were of analytical grade and were purchased from the British Drug Houses (Poole, Dorset, UK). Isolation of Kolaviron Isolation of Kolaviron from the seeds of Garcinia kola was carried out according to published procedure.28,29 Briefly, the fresh Garcinia kola seeds were sliced, air dried and powdered. Extraction of the powdered seeds was done using light petroleum ether (bp 40–60 C) in a soxhlet for 24 h. The defatted dried product was repacked and extracted with acetone. The extract was concentrated, diluted to twice its volume with distilled water and subsequently extracted with ethyl acetate (6 300 mL). The concentrated ethyl acetate yielded a golden yellow solid termed Kolaviron which was identified by direct comparison of the 1H nuclear magnetic resonance (NMR), 13C NMR and electron ionization (EI)mass spectral result with previously published data.28 The purity and identity of Kolaviron was determined by subjection to thin-layer chromatography using Silica gel GF 254-coated plates with a solvent consisting of a mixture of methanol and chloroform in a ratio 1:4 (v/v). Purity of isolated Kolaviron was 96%.
Experimental design The animals were randomly assigned to four groups of 10 rats each and were treated for 15 d as follows: Group I rats were orally administered 2 mL/kg of corn oil alone and served as control. Group II rats were orally administered corn oil solution containing benzo[a]pyrene (B[a]P) alone at 10 mg/kg bw according to Liang et al.30 Groups III and IV rats were orally co-administered with BaP and Kolaviron at 100 (KV1) and 200 (KV2) mg/kg, respectively, according to Adedara and Farombi.31 Twenty-four hours after the last treatment, the rats were sacrificed by cervical dislocation, and blood was collected from retro-orbital venous plexus using heparin containing tubes. The kidneys were quickly excised, weighed and subsequently processed for biochemical assays and histology. Thyroid and renal functional parameters Plasma samples obtained by centrifugation of the blood at 3000 g for 10 min were subsequently used to estimate the plasma levels of urea and creatinine with commercially available kits (Randox Laboratory Limited, Antrim, UK). The total plasma triiodothyronine and thyroxine concentrations were determined using the commercial enzyme immunoassay kits (DiaSorin, Sauggia, Italy) according to the manufacturer’s instructions. Sensitivity of the assays was 60 pg/dL for total thyroxine and 231 pg/dL for total triiodothyronine. Intra-assays coefficients of variation for total thyroxine were
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DOI: 10.3109/0886022X.2015.1006085
3.0–3.4% whereas for total triiodothyronine, 3.5–4.8%. All the samples were assayed on the same day to avoid the inter-assay variation. The total plasma triiodothyronine and thyroxine concentrations were expressed as ng/dL and mg/dL, respectively.
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Biochemical assays The kidney samples for biochemical parameters were homogenized in 50 mM Tris–HCl buffer (pH 7.4) containing 1.15% potassium chloride. The homogenate was centrifuged at 10,000 g for 15 min at 4 C and the supernatant was collected to determine superoxide dismutase (SOD) activity according the method of Misra and Fridovich.32 Catalase (CAT) activity was assayed using hydrogen peroxide as substrate according to the method of Clairborne.33 Glutathione peroxidase (GPx) activity was determined by the method of Rotruck et al.34 Glutathione-S-transferase (GST) activity was determined according to the method of Habig et al.35 Myeloperoxidase (MPO) activity was determined according to the method of Granell.36 Reduced glutathione (GSH) level was determined by the method described by Jollow et al.37 Protein concentration was determined by the method of Lowry et al.38 Hydrogen peroxide generation was measured by the method of Wolff.39 Lipid peroxidation was quantified as malondialdehyde (MDA) according to the method described by Farombi et al.24 and expressed as micromoles of MDA per mg protein.
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taken with a Sony DSC-W 30 Cyber-shot (Sony, Tokyo, Japan) by pathologists who were blinded to control and treatment groups. Statistical analyses Statistical analyses were carried out using one-way analysis of variance (ANOVA) to compare the experimental groups followed by Bonferroni’s post-hoc test to identify significantly different groups (SPSS for Windows, version 17, SPSS Inc., Chicago, IL). Values of p50.05 were considered significant.
Results Biomarkers of renal and thyroid toxicity The renal functionality was confirmed by estimating the levels of urea and creatinine whereas the functionality of the thyroid gland was confirmed by measuring the plasma levels of T3 and T4, and calculating the T3/T4 ratio. As depicted in Figures 2 and 3, a significant (p50.05) elevation in urea and creatinine was accompanied by significant decrease in T3 and T3/T4 ratio in plasma of B[a]P-treated rats when compared with the control rats. The level of T4 was not affected in all the treatment groups. However, co-administration of B[a]Ptreated rats with Kolaviron significantly restored the levels of these biomarkers to near normalcy. Renal antioxidant status
Histological examination Biopsies of the kidney were fixed in 10% neutral-buffered formalin and processed for histology according to standardized procedure.40 Briefly, the fixed tissues were dehydrated using ascending concentrations of alcohol, cleared by xylene and embedded in paraffin wax. The tissues were subsequently cut into 4–5 mm sections by a microtome, fixed on the slides and stained with hematoxylin and eosin. The slides were examined under a light microscope (Olympus CH; Olympus, Tokyo, Japan) and photomicrographs were
Figures 4–6 represent the effects of B[a]P exposure and Kolaviron treatment on antioxidant defense systems and lipid peroxidation in the kidney of experimental animals. Administration of B[a]P caused a significant (p50.05) decrease in the SOD, CAT, GPx and GST activities and the level of GSH in the kidney of treated rats. Conversely, co-administration of Kolaviron ameliorated the decrease in the activities of these antioxidant enzymes and GSH level, and restored their normalcy in B[a]P-exposed rats. Moreover, there was a significant elevation in the renal MPO activity
Figure 2. Plasma concentrations of urea (A) and creatinine (B) in experimental rats. KV1, 100 mg/kg Kolaviron; KV2, 200 mg/kg Kolaviron. Each bar represents mean ± SD of 10 rats per group after 15 d treatment period. a: p50.05 against control. b: p50.05 against B[a]P.
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Figure 3. Effects of Kolaviron on circulatory levels of triiodothyronine (T3), thyroxine (T4) and T3/T4 ratio in B[a]P-treated rats. KV1, 100 mg/kg Kolaviron; KV2, 200 mg/kg Kolaviron. Each bar represents mean ± SD of 10 rats per group after 15 d treatment period. a: p50.05 against control. b: p50.05 against B[a]P.
Figure 4. Effect of Kolaviron on the activities of superoxide dismutase (SOD) and catalase (CAT) in kidneys of B[a]P-treated rats. KV1, 100 mg/kg Kolaviron; KV2, 200 mg/kg Kolaviron. Each bar represents mean ± SD of 10 rats per group after 15 d treatment period. a: p50.05 against control. b: p50.05 against B[a]P.
and H2O2 and MDA levels in B[a]P-exposed rats. Co-administration of Kolaviron markedly decreased the MPO activity and levels of H2O2 and MDA to near control in the kidneys of the treated rats.
Histopathological observations Figure 7 represents photomicrographs of kidneys from the experimental groups. The kidney of control rats appeared
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Figure 5. Effect of Kolaviron on glutathione (GSH) level and activities glutathione peroxidase (GPx) and glutathione S-transferase (GST) in kidneys of B[a]P-treated rats. KV1, 100 mg/kg Kolaviron; KV2, 200 mg/kg Kolaviron. Each bar represents mean ± SD of 10 rats per group after 15 d treatment period. a: p50.05 against control. b: p50.05 against B[a]P.
structurally and functionally normal. The glomeruli show a well-preserved morphology. The kidneys of rats exposed to B[a]P alone showed some tubular lumen with numerous protein casts (arrow). Conversely, the kidneys of rats cotreated with Kolaviron at 100 mg/kg (B[a]P + KV1) appear normal but with few protein casts in the lumen. The kidneys of rats co-treated with Kolaviron at 200 mg/kg (B[a]P + KV2) appeared structurally and functionally normal.
Discussion The present investigation revealed significant increase in plasma creatinine and urea levels in B[a]P-treated rats, thus indicating renal dysfunction in the animals. Elevated plasma urea signifies decreased reabsorption at the renal epithelium whereas high plasma creatinine revealed impairment in the renal functions, mostly for glomerular filtration rate in the B[a]P-treated rats.41 Interestingly, co-treatment with Kolaviron remarkably reversed the B[a]P-mediated increase in the plasma levels of renal functional indices. The restoration of these biomarkers indicates a protective effect of Kolaviron against renal toxicity resulting from B[a]P exposure in the experimental rats. The inter-relationship between the kidney and thyroid functions indicates that the kidney regulates the metabolism
and elimination of thyroid hormones whereas the kidney is an important target organ for thyroid hormones actions.42,43 Renal function has been reported to be regulated by the thyroid status in both animal models and human studies.44 In the present study, B[a]P exposure significantly decreased the plasma levels of T3 without affecting T4 and consequently decreased the T3/T4 level in the experimental animals. The decrease in T3, the biologically active form of thyroid hormones, indicates inhibition of its synthesis and/or acceleration of T3 metabolism. Moreover, lower circulating level of T3/T4 indicates a defective thyroid pathway to activate the negative feedback to the hypothalamus and pituitary. Our data confirmed the previous reports that PAHs exposure altered thyroid hormone levels in both human and animals.45,46 The observed hypothyroidism in B[a]P-treated rats could result in decreased tubular function, renal blood flow, glomerular filtration rate, electrolytes homeostasis and altered kidney structure.47 However, the restoration of T3 and T3/T4 levels to near normalcy following co-treatment with Kolaviron indicates an improvement in the function of thyroid system in the treated rats. SOD is known to accelerate the conversion of endogenous cytotoxic superoxide radicals to H2O2 whereas CAT acts to eliminate H2O2 thereby protecting the cells. Moreover, GSH and GSH-dependent enzymes namely GPx and GST
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Figure 6. Effect of Kolaviron on MPO activity and levels of H2O2 and LPO in kidneys of B[a]P-treated rats. KV1, 100 mg/kg Kolaviron; KV2, 200 mg/kg Kolaviron. Each bar represents mean ± SD of 10 rats per group after 15 d treatment period. a: p50.05 against control. b: p50.05 against B[a]P. Figure 7. Photomicrographs of kidneys from the experimental groups. The kidneys of control rats showing normal histology. The kidneys of B[a]P-treated rats showing some tubular lumen with numerous protein casts (arrow). The kidneys of rats co-treated with Kolaviron at 100 mg/kg (B[a]P + KV1) appear normal however with few protein casts in the lumen. The kidneys of rats cotreated with Kolaviron at 200 mg/kg (B[a]P + KV2) appeared structurally and functionally normal and comparable to control. Original magnification: 240.
Control
BaP alone
BaP + KV1
BaP + KV2
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DOI: 10.3109/0886022X.2015.1006085
participate in the glutathione redox cycle by converting H2O2 and lipid peroxides to non-toxic products.35,41,48 MPO is an index of inflammation. MDA level, a biomarker of lipid peroxidation, is a well-established indicator of oxidative stress in cells and tissues. In the present study, B[a]P-treated rats showed marked diminution in renal GSH level, activities of antioxidant enzymes with significant elevation in MPO activity and H2O2 and MDA levels. These observations clearly indicate a compromised antioxidant defense system, inflammation and a state of oxidative stress in the renal tissues of B[a]P-exposed rats. The present observations are in agreement with the previous reports that B[a]P exposure induced oxidative stress and inflammation in rodents.49–50 The increased intra-tubular protein cast indicates nephrotic glomerular dysfunction in the B[a]P-treated rats. Interestingly, Kolaviron co-treatment significantly improved the antioxidant status and ameliorated renal histology, thus demonstrating the protective effect of Kolaviron in B[a]P-treated rats. In conclusion, Kolaviron amelioration of B[a]P-induced renal toxicity is attributed to the enhancement antioxidant defense mechanism and improvement in the thyroid gland function. Thus, dietary inclusion of Kolaviron could exert protective effects against renal toxicity resulting from B[a]P exposure.
Declaration of interest The authors report no conflicts of interest. This research was done without specific grant from any funding agency in the public, commercial, or not-for-profit sectors.
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LABORATORY STUDY
Renoprotection of Kolaviron against benzo (A) pyrene-induced renal toxicity in rats Isaac A. Adedara, Yetunde M. Daramola, Joshua O. Dagunduro, Motunrayo A. Aiyegbusi, and Ebenezer O. Farombi
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Drug Metabolism and Toxicology Research Laboratories, Department of Biochemistry, College of Medicine, University of Ibadan, Ibadan, Nigeria
Abstract
Keywords
Benzo(a)pyrene (B[a]P), a polycyclic aromatic hydrocarbon generally formed from incomplete combustion of organic matter, reportedly causes renal injury and elicits a nephropathic response. The present study investigated the modulatory effect of Kolaviron, an isolated bioflavonoid from the seed of Garcinia kola, on renal toxicity induced by B[a]P in Wistar rats. Benzo[a]pyrene was administered at a dose of 10 mg/kg alone or in combination with Kolaviron at 100 and 200 mg/kg for 15 d. Administration of B[a]P alone resulted in significant increase in plasma levels of urea and creatinine in the treated rats. Moreover, B[a]P exposure significantly decreased the activities of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx) and glutathione-S-transferase (GST) as well as glutathione (GSH) level in the kidneys of treated rats. Conversely, myeloperoxidase (MPO) activity, hydrogen peroxide (H2O2) and malondialdehyde (MDA) levels were markedly elevated in kidneys of B[a]P-treated rats compared with control. Further, B[a]P exposure significantly decreased the circulatory concentrations of triiodothyronine (T3) and T3/T4 ratio without affecting thyroxine (T4) in the treated rats. Light microscopy revealed tubular lumen with numerous protein casts in kidneys of rats exposed to B[a]P alone. Kolaviron co-treatment significantly improved the renal antioxidant status, thyroid gland function and restored the renal histology, thus demonstrating the protective effect of Kolaviron in B[a]P-treated rats. Dietary inclusion of Kolaviron could exert protective effects against renal toxicity resulting from B[a]P exposure.
Benzo[a]pyrene, Kolaviron, kidney, thyroid hormones, rats
Introduction Benzo(a)pyrene (B[a]P, Figure 1A) is a global environmental contaminant belonging to a member of polycyclic aromatic hydrocarbon (PAH) family. Environmental contamination by PAH occur as complex mixtures. The principal environmental sources of B[a]P includes diesel exhaust, industrial incineration products, incomplete combustion of carbonaceous materials by humans for energy and barbeque.1–3 Human exposure to B[a]P is principally through ingestion of contaminated food and water as well as inhalation of particulates.4 Exposure to B[a]P is known to cause renal injury and elicits a nephropathic response.5 Previous toxicological studies demonstrated the expression of aryl hydrocarbon receptor (AHR) in the renal tissues, thus reinforcing the possible metabolic activation of B[a]P to reactive intermediates similar to the liver cells.6,7 Moreover, B[a]P-induced nephropathy was demonstrated to involve disruption of glomerular cell–cell and cell–matrix interactions in rats.8 A single oral administration of B[a]P (125 mg/kg) Address correspondence to Dr. Isaac A. Adedara, Drug Metabolism and Toxicology Research Laboratories, Department of Biochemistry, College of Medicine, University of Ibadan, Ibadan 20005, Nigeria. Tel: +2347032962608; Fax: 234-2-8103043. E-mail: dedac2001@ yahoo.co.uk
History Received 7 September 2014 Revised 23 November 2014 Accepted 11 December 2014 Published online 23 January 2015
caused renal toxicity and loss of DNA integrity in mice.9 Intrauterine exposure of mice to B[a]P resulted in sustained deficits of renal structure and function that compromise organ function long after birth.7 The metabolic activation of B[a]P which releases reactive oxygen species (ROS) that produce oxidative stress is a well-established mechanism of toxicity in various experimental animal models.9–12 The link between chemical injury and nephropathy is best demonstrated by the strong association between aromatic hydrocarbon exposures and glomerular diseases.13,14 In Nigeria, exposure to B[a]P for the general population could occur through consumption of contaminated food or groundwater from frequent spillage of petroleum (with aromatic hydrocarbons accounting for approximately 45% of total hydrocarbons),15 at refineries, underground storage tanks at gas stations and poorly managed waste sites. End-stage renal disease accounts for 8% of all medical admissions and 42% of renal admissions in Nigeria.16,17 Kolaviron (Figure 1B) is an isolated bioflavonoid from the seeds of Garcinia kola Heckel (Family Guttiferae) widely used in African Traditional Medicine. It is a potent phytochemical with established antioxidant,18 antiinflammatory,19,20 and anti-genotoxic,21,22 properties. Besides its protection against hepatic oxidative damage induced by 2-acetylaminofluorene, carbon-tetrachloride, aflatoxin
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Figure 1. Chemical structures of tested compounds. (A) Benzo[a]pyrene (B[a]P) and (B) Kolaviron (KV).
B1,22–24 Kolaviron has been reported to protect against potassium bromate-induced nephrotoxicity,25 inhibit renal apoptosis by ethylene glycol monoethyl ether,26 and ameliorate hyperglycemia-mediated renal oxidative damage in diabetic rats.27 However, there is no report on the effect of Kolaviron on renal toxicity due to B[a]P exposure. Considering the widespread availability of B[a]P in the environment and the beneficial health effects of Kolaviron, we have investigated the influence of Kolaviron on B[a]P-induced renal toxicity using Wistar rats as the animal model.
Materials and methods
Animal model Adult male Wistar rats (8 weeks old; 158 ± 3 g) obtained from the Department of Biochemistry, University of Ibadan, Ibadan were allowed to acclimate for 1 week prior to the commencement of the experiment. The animals were fed rat pellets, given drinking water ad libitum, and subjected to natural photoperiod of a 12 h alternating light–dark cycle. Animal care and experimental protocols were executed according to the approved guidelines set by the University of Ibadan Ethical Committee, which is in agreement with the Guide for the Care and Use of Laboratory Animals’ prepared by the National Academy of Science (NAS) and published by the National Institute of Health.
Chemicals Benzo[a]pyrene (B[a]P), trichloroacetic acid, thiobarbituric acid, 1-chloro-2,4-dinitrobenzene, hydrogen peroxide, glutathione, epinephrine and 50 ,50 -dithio-bis-2-nitrobenzoic acid were procured from Sigma Chemical Co. (St. Louis, MO). All other reagents were of analytical grade and were purchased from the British Drug Houses (Poole, Dorset, UK). Isolation of Kolaviron Isolation of Kolaviron from the seeds of Garcinia kola was carried out according to published procedure.28,29 Briefly, the fresh Garcinia kola seeds were sliced, air dried and powdered. Extraction of the powdered seeds was done using light petroleum ether (bp 40–60 C) in a soxhlet for 24 h. The defatted dried product was repacked and extracted with acetone. The extract was concentrated, diluted to twice its volume with distilled water and subsequently extracted with ethyl acetate (6 300 mL). The concentrated ethyl acetate yielded a golden yellow solid termed Kolaviron which was identified by direct comparison of the 1H nuclear magnetic resonance (NMR), 13C NMR and electron ionization (EI)mass spectral result with previously published data.28 The purity and identity of Kolaviron was determined by subjection to thin-layer chromatography using Silica gel GF 254-coated plates with a solvent consisting of a mixture of methanol and chloroform in a ratio 1:4 (v/v). Purity of isolated Kolaviron was 96%.
Experimental design The animals were randomly assigned to four groups of 10 rats each and were treated for 15 d as follows: Group I rats were orally administered 2 mL/kg of corn oil alone and served as control. Group II rats were orally administered corn oil solution containing benzo[a]pyrene (B[a]P) alone at 10 mg/kg bw according to Liang et al.30 Groups III and IV rats were orally co-administered with BaP and Kolaviron at 100 (KV1) and 200 (KV2) mg/kg, respectively, according to Adedara and Farombi.31 Twenty-four hours after the last treatment, the rats were sacrificed by cervical dislocation, and blood was collected from retro-orbital venous plexus using heparin containing tubes. The kidneys were quickly excised, weighed and subsequently processed for biochemical assays and histology. Thyroid and renal functional parameters Plasma samples obtained by centrifugation of the blood at 3000 g for 10 min were subsequently used to estimate the plasma levels of urea and creatinine with commercially available kits (Randox Laboratory Limited, Antrim, UK). The total plasma triiodothyronine and thyroxine concentrations were determined using the commercial enzyme immunoassay kits (DiaSorin, Sauggia, Italy) according to the manufacturer’s instructions. Sensitivity of the assays was 60 pg/dL for total thyroxine and 231 pg/dL for total triiodothyronine. Intra-assays coefficients of variation for total thyroxine were
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DOI: 10.3109/0886022X.2015.1006085
3.0–3.4% whereas for total triiodothyronine, 3.5–4.8%. All the samples were assayed on the same day to avoid the inter-assay variation. The total plasma triiodothyronine and thyroxine concentrations were expressed as ng/dL and mg/dL, respectively.
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Biochemical assays The kidney samples for biochemical parameters were homogenized in 50 mM Tris–HCl buffer (pH 7.4) containing 1.15% potassium chloride. The homogenate was centrifuged at 10,000 g for 15 min at 4 C and the supernatant was collected to determine superoxide dismutase (SOD) activity according the method of Misra and Fridovich.32 Catalase (CAT) activity was assayed using hydrogen peroxide as substrate according to the method of Clairborne.33 Glutathione peroxidase (GPx) activity was determined by the method of Rotruck et al.34 Glutathione-S-transferase (GST) activity was determined according to the method of Habig et al.35 Myeloperoxidase (MPO) activity was determined according to the method of Granell.36 Reduced glutathione (GSH) level was determined by the method described by Jollow et al.37 Protein concentration was determined by the method of Lowry et al.38 Hydrogen peroxide generation was measured by the method of Wolff.39 Lipid peroxidation was quantified as malondialdehyde (MDA) according to the method described by Farombi et al.24 and expressed as micromoles of MDA per mg protein.
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taken with a Sony DSC-W 30 Cyber-shot (Sony, Tokyo, Japan) by pathologists who were blinded to control and treatment groups. Statistical analyses Statistical analyses were carried out using one-way analysis of variance (ANOVA) to compare the experimental groups followed by Bonferroni’s post-hoc test to identify significantly different groups (SPSS for Windows, version 17, SPSS Inc., Chicago, IL). Values of p50.05 were considered significant.
Results Biomarkers of renal and thyroid toxicity The renal functionality was confirmed by estimating the levels of urea and creatinine whereas the functionality of the thyroid gland was confirmed by measuring the plasma levels of T3 and T4, and calculating the T3/T4 ratio. As depicted in Figures 2 and 3, a significant (p50.05) elevation in urea and creatinine was accompanied by significant decrease in T3 and T3/T4 ratio in plasma of B[a]P-treated rats when compared with the control rats. The level of T4 was not affected in all the treatment groups. However, co-administration of B[a]Ptreated rats with Kolaviron significantly restored the levels of these biomarkers to near normalcy. Renal antioxidant status
Histological examination Biopsies of the kidney were fixed in 10% neutral-buffered formalin and processed for histology according to standardized procedure.40 Briefly, the fixed tissues were dehydrated using ascending concentrations of alcohol, cleared by xylene and embedded in paraffin wax. The tissues were subsequently cut into 4–5 mm sections by a microtome, fixed on the slides and stained with hematoxylin and eosin. The slides were examined under a light microscope (Olympus CH; Olympus, Tokyo, Japan) and photomicrographs were
Figures 4–6 represent the effects of B[a]P exposure and Kolaviron treatment on antioxidant defense systems and lipid peroxidation in the kidney of experimental animals. Administration of B[a]P caused a significant (p50.05) decrease in the SOD, CAT, GPx and GST activities and the level of GSH in the kidney of treated rats. Conversely, co-administration of Kolaviron ameliorated the decrease in the activities of these antioxidant enzymes and GSH level, and restored their normalcy in B[a]P-exposed rats. Moreover, there was a significant elevation in the renal MPO activity
Figure 2. Plasma concentrations of urea (A) and creatinine (B) in experimental rats. KV1, 100 mg/kg Kolaviron; KV2, 200 mg/kg Kolaviron. Each bar represents mean ± SD of 10 rats per group after 15 d treatment period. a: p50.05 against control. b: p50.05 against B[a]P.
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Figure 3. Effects of Kolaviron on circulatory levels of triiodothyronine (T3), thyroxine (T4) and T3/T4 ratio in B[a]P-treated rats. KV1, 100 mg/kg Kolaviron; KV2, 200 mg/kg Kolaviron. Each bar represents mean ± SD of 10 rats per group after 15 d treatment period. a: p50.05 against control. b: p50.05 against B[a]P.
Figure 4. Effect of Kolaviron on the activities of superoxide dismutase (SOD) and catalase (CAT) in kidneys of B[a]P-treated rats. KV1, 100 mg/kg Kolaviron; KV2, 200 mg/kg Kolaviron. Each bar represents mean ± SD of 10 rats per group after 15 d treatment period. a: p50.05 against control. b: p50.05 against B[a]P.
and H2O2 and MDA levels in B[a]P-exposed rats. Co-administration of Kolaviron markedly decreased the MPO activity and levels of H2O2 and MDA to near control in the kidneys of the treated rats.
Histopathological observations Figure 7 represents photomicrographs of kidneys from the experimental groups. The kidney of control rats appeared
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Figure 5. Effect of Kolaviron on glutathione (GSH) level and activities glutathione peroxidase (GPx) and glutathione S-transferase (GST) in kidneys of B[a]P-treated rats. KV1, 100 mg/kg Kolaviron; KV2, 200 mg/kg Kolaviron. Each bar represents mean ± SD of 10 rats per group after 15 d treatment period. a: p50.05 against control. b: p50.05 against B[a]P.
structurally and functionally normal. The glomeruli show a well-preserved morphology. The kidneys of rats exposed to B[a]P alone showed some tubular lumen with numerous protein casts (arrow). Conversely, the kidneys of rats cotreated with Kolaviron at 100 mg/kg (B[a]P + KV1) appear normal but with few protein casts in the lumen. The kidneys of rats co-treated with Kolaviron at 200 mg/kg (B[a]P + KV2) appeared structurally and functionally normal.
Discussion The present investigation revealed significant increase in plasma creatinine and urea levels in B[a]P-treated rats, thus indicating renal dysfunction in the animals. Elevated plasma urea signifies decreased reabsorption at the renal epithelium whereas high plasma creatinine revealed impairment in the renal functions, mostly for glomerular filtration rate in the B[a]P-treated rats.41 Interestingly, co-treatment with Kolaviron remarkably reversed the B[a]P-mediated increase in the plasma levels of renal functional indices. The restoration of these biomarkers indicates a protective effect of Kolaviron against renal toxicity resulting from B[a]P exposure in the experimental rats. The inter-relationship between the kidney and thyroid functions indicates that the kidney regulates the metabolism
and elimination of thyroid hormones whereas the kidney is an important target organ for thyroid hormones actions.42,43 Renal function has been reported to be regulated by the thyroid status in both animal models and human studies.44 In the present study, B[a]P exposure significantly decreased the plasma levels of T3 without affecting T4 and consequently decreased the T3/T4 level in the experimental animals. The decrease in T3, the biologically active form of thyroid hormones, indicates inhibition of its synthesis and/or acceleration of T3 metabolism. Moreover, lower circulating level of T3/T4 indicates a defective thyroid pathway to activate the negative feedback to the hypothalamus and pituitary. Our data confirmed the previous reports that PAHs exposure altered thyroid hormone levels in both human and animals.45,46 The observed hypothyroidism in B[a]P-treated rats could result in decreased tubular function, renal blood flow, glomerular filtration rate, electrolytes homeostasis and altered kidney structure.47 However, the restoration of T3 and T3/T4 levels to near normalcy following co-treatment with Kolaviron indicates an improvement in the function of thyroid system in the treated rats. SOD is known to accelerate the conversion of endogenous cytotoxic superoxide radicals to H2O2 whereas CAT acts to eliminate H2O2 thereby protecting the cells. Moreover, GSH and GSH-dependent enzymes namely GPx and GST
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Figure 6. Effect of Kolaviron on MPO activity and levels of H2O2 and LPO in kidneys of B[a]P-treated rats. KV1, 100 mg/kg Kolaviron; KV2, 200 mg/kg Kolaviron. Each bar represents mean ± SD of 10 rats per group after 15 d treatment period. a: p50.05 against control. b: p50.05 against B[a]P. Figure 7. Photomicrographs of kidneys from the experimental groups. The kidneys of control rats showing normal histology. The kidneys of B[a]P-treated rats showing some tubular lumen with numerous protein casts (arrow). The kidneys of rats co-treated with Kolaviron at 100 mg/kg (B[a]P + KV1) appear normal however with few protein casts in the lumen. The kidneys of rats cotreated with Kolaviron at 200 mg/kg (B[a]P + KV2) appeared structurally and functionally normal and comparable to control. Original magnification: 240.
Control
BaP alone
BaP + KV1
BaP + KV2
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participate in the glutathione redox cycle by converting H2O2 and lipid peroxides to non-toxic products.35,41,48 MPO is an index of inflammation. MDA level, a biomarker of lipid peroxidation, is a well-established indicator of oxidative stress in cells and tissues. In the present study, B[a]P-treated rats showed marked diminution in renal GSH level, activities of antioxidant enzymes with significant elevation in MPO activity and H2O2 and MDA levels. These observations clearly indicate a compromised antioxidant defense system, inflammation and a state of oxidative stress in the renal tissues of B[a]P-exposed rats. The present observations are in agreement with the previous reports that B[a]P exposure induced oxidative stress and inflammation in rodents.49–50 The increased intra-tubular protein cast indicates nephrotic glomerular dysfunction in the B[a]P-treated rats. Interestingly, Kolaviron co-treatment significantly improved the antioxidant status and ameliorated renal histology, thus demonstrating the protective effect of Kolaviron in B[a]P-treated rats. In conclusion, Kolaviron amelioration of B[a]P-induced renal toxicity is attributed to the enhancement antioxidant defense mechanism and improvement in the thyroid gland function. Thus, dietary inclusion of Kolaviron could exert protective effects against renal toxicity resulting from B[a]P exposure.
Declaration of interest The authors report no conflicts of interest. This research was done without specific grant from any funding agency in the public, commercial, or not-for-profit sectors.
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