Distribution of glutathione peroxidase 1 in liver tissues of healthy and diabetic rats treated with capsaisin T Deprem1, SE Yıldız2, EK Sari1, SA Bingol2, SK Tasci1, S Aslan1, M Sozmen3, G Nur4 1Department

of Histology-Embryology, Faculty of Veterinary Medicine, University of Kafkas, 2School of Health Sciences, University of Kafkas, 36100 Kars, 3Department of Pathology, University of Ondokuz Mayis, 55139, Samsun, and 4Directorate of Food Control Laboratory, 31030, Hatay, Turkey

Accepted February 26, 2014

Abstract We investigated the immunohistochemical localization of glutathione peroxidase 1 (GPx 1) and the structural changes that occur in the livers of healthy and diabetic rats that were treated with capsaisin (CAP). Fifty female rats were divided into five groups: group 1, sham; group 2, untreated control; group 3, CAP-treated; group 4, streptozotocin (STZ) diabetic; group 5, STZ diabetic ⫹ CAP-treated. STZ was administered to groups 4 and 5; after verifying diabetes, CAP was administered daily for 2 weeks to groups 3 and 5. Diffuse, microvesicular and some macrovesicular fatty degeneration were observed in the cytoplasms of hepatocytes in the livers of the diabetic group. In the CAP-treated diabetic group, fat degeneration in the livers decreased slightly by day 7. Irregularity of the external contours of nuclei of the hepatocytes, swelling of the nuclei, and slight anisocytosis and anisokaryosis were observed in the hepatocytes of the diabetic group. In the CAP-treated diabetic groups, the severity of anisocytosis and anisokaryosis decreased slightly by day 7. In all groups, GPx 1 showed similar immunolocalization, but in the diabetic and diabetic ⫹ CAP groups, GPx 1 immunoreactivity was less than in the other groups. GPx 1 immunoreactivity in the CAP-treated diabetic group was weaker than in the diabetic group. In all groups, GPx 1 immunoreactivity was diffusely cytoplasmic in some of the hepatocytes, and diffusely cytoplasmic and diffusely nuclear in other hepatocytes. Also, GPx 1 immunoreactivity in the liver was more intense in the hepatocytes around Kiernan’s space. We found that CAP caused a decrease in GPx 1. Key words: capsaisin, diabetes mellitus, glutathione peroxidase 1, GPx 1, immunohistochemistry, liver Diabetes mellitus is a metabolic disorder that affects protein, fat and carbohydrate metabolism due to failure of insulin secretion or impaired tissue response to insulin (Wolff 1993). In diabetes, the balance between free radicals and the antioxidant defense system (Cross et al. 1987) become unbalanced in favor of free radicals (Maritim et al. 2003).

Correspondence: Turgay Deprem, Department of Histology and Embryology, Faculty of Veterinary Medicine, University of Kafkas, Kars, Turkey. Phone: ⫹ 90 0474 242 68 00-5299, Fax: ⫹ 90 0474 242 68 53, E-mail: [email protected] © 2014 The Biological Stain Commission Biotechnic & Histochemistry 2015, 90(1): 1–7.

DOI: 10.3109/10520295.2014.919024

The most typical indicator of oxidative stress is lipid oxidation (Gutteridge 1995). Superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), and melatonin (MEL) are part of the natural antioxidant defense systems against free radicals (Cross et al. 1987). SOD prevents the oxidative damage of lipid oxidation; the enzyme catalase aids this process and decreases lipid peroxides. Excess quantities of lipid peroxides that are formed in the tissues enter the serum and are metabolized in the liver by GPx (Hasegawa et al. 1992). GPx catalyzes conversion of hydrogen peroxide or organic hydroperoxides to water or alcohol by reduced glutathione (Akkus 1

1995). Therefore, GPx prevents lipid oxidation and protects cell membranes from oxidative damage (Czuczejko et al. 2003). GPx is widely dispersed in mammalian tissues and the liver is a rich source (Stadtman 1977). Capsaicin (CAP) is the active substance of chili pepper (Lembeck 1987). Effects of CAP include increased carbohydrate metabolism and increased liver enzyme activity, and breakdown of lipid in fatty tissues by stimulating lipid metabolism and increasing serum glucose and insulin levels (Monsereenusorn 1983). Srinivasan et al. (2004) reported that some spices enhance catabolism of cholesterol to bile acids, which involves hydroxylation of cholesterol by the hepatic mixed function oxygenase system. Suresh and Srinivasan (2006) showed that capsaicin increased the amount of aryl hydroxylase enzyme in rats. The effects of CAP have been investigated in diabetes as a dietary supplement (Nevius et al. 2012), by topical application (Webster et al. 2011) and by subcutaneous (van de Wall et al. 2005) and intraperitoneal (Guillot et al. 1996, Zhang and Ritter 2012) injection. Guillot et al. (1996) reported that intraperitoneal administration of CAP in neonatal STZ-diabetic rats improved glucose tolerance without changing insulin secretion and Webster et al. (2011) reported that topical application of CAP may be beneficial for diabetic nephropathy. We investigated the immunohistochemical localization of GPx 1 in the livers of healthy and diabetic rats to which CAP had been administered. We also investigated the effects of CAP and structural changes in the liver.

Material and methods Experimental animals Our experimental protocol was approved by the Kafkas University Animal Experiments Local Ethical Committee. Animals were obtained from Erzurum Veterinary, Control and Investigation Institute Experiment Animals Center. We used 50 8–12 week-old female Sprague-Dawley rats. The rats were kept in standard cages at 22 ⫾ 2° C, 50 ⫾ 5% humidity and a 12 h dark:12 h light cycle. Animals were fed standard rat feed and provided water ad libitum. The rats were divided into five groups of 10: group 1, sham; group 2, untreated control; group 3, CAP-treated; group 4, STZ diabetic; group 5, STZ diabetic ⫹ CAP-treated. STZ (Sigma, St. Louis, MO) dissolved 0.1 M in fresh citrate buffer (pH 4.5) was injected intraperitoneally (i.p.) into animals of groups 4 and 5 as a single dose of 45 mg/kg 2

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(Das et al. 1996, Zafar et al. 2009, 2010). Citrate buffer 0.1 M was administered to the sham group and the control group was untreated. The day STZ was injected was defined as day 0 of the experiment. The rats were weighed after fasting for 8 h on days 0, 3, 7 and 14. Blood glucose values were determined from blood taken from the tail veins using a hand glucometer (Accu-Chek-Go, Roche, Switzerland). Fasting blood glucose levels of all groups were measured at 72 h; animals with levels ⬎ 200 mg/dl were considered diabetic (Kanitkar and Bhonde 2004). On the day that diabetes was verified, 1 mg/kg CAP dissolved in a solution of 10% ethanol, 1% Tween 20 and 80% distilled water (Sigma) was administered subcutaneously to groups 2 and 5 every day for 2 weeks (Moran et al. 2003). Tissue samples Animals were euthanized by cervical dislocation under ether anesthesia and liver samples were excised on days 7 and 14. After the weights of the livers were normalized by the body weights (liver weight/body weight x 100) (Sechi et al. 1997), they were used for statistical analysis. Histology examination The liver samples were fixed with Bouin’s solution. Tissue samples were embedded in paraffin after routine histologic tissue processing and 6 μm sections were cut. To examine the histological structure of the livers, the sections were stained with Crossman’s triple stain, hematoxylin & eosin (H & E) and periodic acid-Schiff (PAS) stains. (Luna 1968). Evaluation of liver damage, including the severity and prevalence of degeneration, were graded subjectively as 0, normal; 1, minimal; 2, slight; 3, medium; 4, significant; 5, severe. Immunohistochemistry examination The avidin-biotin-peroxidase (ABC) technique (Hsu et al. 1981) was used to examine the immunohistochemical distribution of GPx 1 in the liver tissues. After deparaffinization and rehydration, the sections were incubated in 3% H202 for 10 min to block endogenous peroxide activity. All tissues were processed in 0.1 M citrate buffer, pH 6.0, in an 800 watt microwave oven for 10 min to expose antigenic sites. After the sections were washed with phosphate buffered saline (PBS), they were incubated in blocking buffer (Ultra V Block, Fremont, CA) (10%) for 10 min to prevent nonspecific binding. The sections then were incubated with anti-GPx 1 (ab22604; Abcam,

Table 1. Comparison of the blood glucose levels among groups Days 0 3 7 14 a,b,c : A,B,C:

Sham

Control

CAP

Diabetes

Diabetes ⴙ CAP

107.90 ⫾ 4.92a 109.40 ⫾ 4.43c 120.90 ⫾ 1.04c 116.00 ⫾ 2.04b

105.50 ⫾ 4.37ab 105.50 ⫾ 4.37c 116.90 ⫾ 2.47c 117.60 ⫾ 6.56b

96.80 ⫾ 2.69abcB 96.00 ⫾ 3.11cB 120.90 ⫾ 1.15cA 118.60 ⫾ 9.71bA

87.00 ⫾ 3.56cB 428.50 ⫾ 24.00aA 423.10 ⫾ 13.18bA 441.40 ⫾ 53.25aA

94.20 ⫾ 4.14bcC 357.50 ⫾ 20.50bB 510.60 ⫾ 16.57aA 491.20 ⫾ 21.07aA

values with different letters in the same line were significantly different from each other. differences in the values with different letters in the same column were statistically significant.

Cambridge, MA) (1:2000) for 1 h at room temperature. After washing with PBS, biotinized secondary antibody (510.991.2800; Ultravision detection system anti-rabbit, biotinylated goat anti-rabbit, Lab Vision, Fremont, CA) that was against the type for which anti-GPx 1 was produced was applied to the sections for 30 min. After washing with PBS, streptavidin horseradish peroxidase (Lab Vision) was added to the sections and they were kept at room temperature for 30 min. Sections then were washed with PBS. The DAB-H2O2 (Lab Vision) technique (Shu et al. 1988) was used to visualize the reaction. Hematoxylin was used to stain nuclei (Bancroft and Cook 1984). The preparations were examined using a BX-051 Olympus, Tokyo, Japan) research microscope and the images were photographed. Statistical analysis SPSS version 12.0 was used for statistical analysis (SPSS). Significant differences were determined using Student’s t-test and ANOVA test. Values for p ⱕ 0.05 were considered significant.

Results Blood glucose values The blood glucose levels were higher in the diabetic and diabetic ⫹ CAP groups than in the other groups (p ⬍ 0.05). Also, the diabetic and diabetic ⫹ CAP groups showed significantly increased blood glucose levels each day (p ⬍ 0.05) (Table 1). We found that changes in blood glucose levels each day were not statistically significant for the control and sham groups. Blood glucose levels in the control, sham and CAP groups were within the normal range.

Body weights A significant increase in body weights was observed only in the diabetic group (p ⬍ 0.05) (Table 2); the body weights of the sham, control and CAP groups were not significantly different. No increase in body weight was observed in the diabetic ⫹ CAP group.

Table 2. Comparison of body weights among groups Days 0 3 7 14

Sham

Control

CAP

Diabetes

Diabetes ⴙ CAP

184.16 ⫾ 2.84a 186.31 ⫾ 2.67a 188.86 ⫾ 2.81a 195.25 ⫾ 4.34a

168.28 ⫾ 12.31ab 169.37 ⫾ 12.76ab 180.26 ⫾ 13.31a 182.47 ⫾ 20.40ab

162.13 ⫾ 11.35abc 163.20 ⫾ 11.74abc 178.67 ⫾ 12.30a 180.10 ⫾ 16.49ab

138.92 ⫾ 6.16cB 136.63 ⫾ 6.42cB 161.62 ⫾ 8.19abA 165.18 ⫾ 9.29abA

146.17 ⫾ 6.12bc 147.69 ⫾ 7.63bc 146.11 ⫾ 7.19b 147.23 ⫾ 2.66b

a,b,c: A,B:

values with different letters in the same line were significantly different from each other. differences in the values with different letters in the same column were statistically significant.

Table 3. Comparison of normalized liver weights by body weights (%) among groups Days 7 14

Sham

Control

CAP

Diabetes

Diabetes ⴙ CAP

4.55 ⫾ 0.51abc 4.62 ⫾ 0.83b

4.25 ⫾ 3.75bc 4.34 ⫾ 3.36b

4.15 ⫾ 2.04c 4.62 ⫾ 2.81b

4.82 ⫾ 1.73abB 5.45 ⫾ 1.27aA

5.03 ⫾ 0.68aB 5.34 ⫾ 0.73aA

a,b,c: A,B:

values with different letters in the same line were significantly different from each other. differences in the values with different letters in the same column were statistically significant.

GPx 1 in capsaisin treated diabetic rats 3

Liver weights We determined that the liver weights in the diabetic and diabetic ⫹ CAP groups were greater than in the other groups and that the liver weights were increased in the diabetic and diabetic ⫹ CAP groups (p ⬍ 0.05) (Table 3). Histology assessments Normal histology was observed in the livers of the rats in the control, sham and CAP groups (Fig. 1). Diffuse, mostly microvesicular and a small amount of macrovesicular fatty degeneration was observed in the cytoplasms of the hepatocytes of the diabetic group, (Fig. 2a, b). Although the amount of fat degeneration in this group on days 7 and 14 appeared similar, the lesions observed on day 7 were more apparent than on day 14. We observed that fat degeneration in the livers decreased slightly by day 7 in the CAP-treated diabetic group (Fig. 3). We observed irregular external contours of the hepatocyte nuclei, nuclear swelling, and limited anisocytosis and anisokaryosis in the diabetic groups. We observed that the severity of anisocytosis and anisokaryosis decreased somewhat by day 7 in the CAP-treated diabetic group. We observed bile duct hyperplasia in the diabetic groups that was similar on days 7 and 14. In the CAP-treated diabetic groups, bile duct hyperplasia was slightly decreased. Immunochemistry assessments GPx 1 immunoreactivity was observed in all groups. On day 7, GPx 1 immunoreactivity in the control, sham and CAP-treated groups was similar (Fig. 4a).

Fig. 1. Histology of rat liver of control group on day 14. H & E. Bar ⫽ 200 μm.

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Fig. 2. a) Severe, widespread liver lipidosis in the rat livers of the diabetic group on day 7. PAS. Bar ⫽ 200 μm. b) Liver lesion in rat liver of diabetic group on day 7. Diffuse lipid deposition in the hepatocytes in the liver parenchyma (Arrow). Arrowhead, vena centralis. H & E. Bar ⫽ 100 μm.

Fig. 3. Normal histology of CAP-treated diabetic rat liver on day 14. H & E. Bar ⫽ 200 μm.

Fig. 4. a) GPx 1 immunoreactivity in rat liver of CAP group on day 7. Arrows, cytoplasmic GPx 1 immunoreactivity; arrowhead, cytoplasmic and nuclear GPx 1 immunoreactivity. Bar ⫽ 50 μm. b) GPx 1 immunoreactivity in rat liver of diabetic group on day 7. Bar ⫽ 50 μm. c) GPx 1 immunoreactivity in rat liver of diabetic ⫹ CAP group on day 7. Bar ⫽ 50 μm.

On the same day, however, GPx 1 immunoreactivity in the diabetic and diabetic ⫹ CAP groups was less than in the other groups (Fig. 4b, c). Moreover, GPx 1 immunoreactivity in the CAP-treated diabetic group was slightly weaker than in the diabetic group (Fig. 4c). On day 14, GPx 1 immunoreactivity in the liver was similar to that on day 7. GPx 1 immunoreactivity was diffusely cytoplasmic in some of the hepatocytes, and both diffusely cytoplasmic and nuclear in others in all groups (Fig. 4a). We also observed that GPx 1 immunoreactivity in

Fig. 5. GPx 1 immunoreactivity in hepatocytes around Kiernan’s space in rat liver of sham group on day 7. Arrows, Kiernan’s spaces; arrowhead, vena centralis. Bar ⫽ 200 μm.

the liver was more intense in the hepatocytes near Kiernan’s space (Fig. 5).

Discussion STZ-induced diabetes and insulin deficiency cause increased blood glucose levels (Saeed et al. 2008). Complications of diabetes include diabetic nephropathy and cardiovascular disorders. The relationship between diabetes and liver functions, however, is not fully understood (Arkkila et al. 2001). We investigated the immunohistochemical localization of GPx 1in the livers of CAP-treated healthy and diabetic rats as well as structural changes that occurred in the liver. Consistent with the literature, we found that STZ-induced diabetes was characterized by elevated blood glucose levels (Bolkent et al. 2008, Omoruyi et al. 2001, Satav and Katyare 2004, Zafar et al. 2010). Loss of body weight has been reported in STZinduced diabetic animals (Bolkent et al. 2008, Omoruyi et al. 2001, Ravi et al. 2004, Satav and Katyare 2004, Zafar et al. 2010), but we found that the body weights of diabetic rats increased. It is possible that the disparity is due to gender differences and different experimental periods. The literature contains some reports that liver weights increased in diabetic animals (Cho et al. 2002, Deprem and Gulmez 2009, Zafar et al. 2010), although others reported that liver weights GPx 1 in capsaisin treated diabetic rats 5

decreased (Omoruyi et al. 2001, Satav and Katyare 2004). We found that liver weights increased in diabetic animals. It has been reported that the effects of diabetes on the liver include cytoplasmic vacuolization (Bolkent et al. 2008) and dilation of the central veins (Das et al. 1996, Liu et al. 2013, Zafar et al. 2009) and sinusoids (Bahceci et al. 2007, Bolkent et al. 2008, Das et al. 1996, Zafar et al. 2009). Zafar et al. (2009) and Bahceci et al. (2007) reported also that lipid droplets appeared in the cytoplasms of hepatocytes of diabetic rats. Our finding of microvesicular and limited macrovesicular lipidosis in the cytoplasms of hepatocytes of diabetic animals is consistent with the reports by Zafar et al. (2009) and Bahceci et al. (2007). In addition, we observed that fat degeneration decreased somewhat in CAP-treated diabetic rat livers by day 7. Moreover, Zafar et al. (2009) reported bile duct hyperplasia in diabetic rats, which is consistent with our observations. The hyperplasia was somewhat less in the CAP-treated diabetic group. Some investigators have reported that hepatocyte nuclear area was increased in the diabetic liver (Doi et al. 1997, Laguens et al. 1980) and others reported that the hepatocytes exhibited hypertrophy (Deprem and Gulmez 2009, Laguens et al. 1980). Using the STZ-induced diabetes model, Doi et al. (1997) reported that hepatocyte nuclei in diabetics were larger and had irregular boundaries compared to controls; our findings were consitent with theirs. We surmise that changes in liver enzyme levels could be affected by structural changes in hepatocytes. We demonstrated that GPx 1 immunoreactivity in the liver was localized diffusely in the cytoplasm of hepatocytes and staining was more intense in periphery of the hepatic lobe than in the zone around the central vein; this observation is consistent with published findings (Asayama et al. 1996, Deprem and Gulmez 2009, Yoshimura et al. 1980). Deprem and Gulmez (2009) and Yoshimura et al. (1980) reported no immunoreactivity in the Kupffer and endothelial cells around the sinusoids. GPx 1 immunoreactivity in the diabetic group was less than in other groups (Deprem and Gulmez 2009). We found less GPx 1 immunoreactivity in the CAPtreated diabetic group than in diabetic group. Capsaisin use in diabetic rats previously has been reported to regulate glucose tolerance without changing the secretion of insulin (Guillot et al. 1996). Moreover, topical administration of capsaicin has been shown to have positive effects on diabetic nephropathy (Webster et al. 2011). We have shown using immunohistochemistry that capsaicin treatment causes a reduction in the GPx1expression in 6

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the livers of diabetic rats. The route of capsaicin administration, dosage, duration and species differences appear to affect GPx 1 expression. Some investigations have shown that capsaicin may have differential effects on the hepatic mixed function oxygenase system (Suresh and Srinivasan 2006). Our study suggests that capsaicin may affect various antioxidant systems. Therefore, various antioxidant enzymes and other molecules should be studied to clarify this matter.

Acknowledgments This Project was supported by the Commission for the Scientific Research Projects of Kafkas Univertsity (Project No: 2012-VF-20). This study was presented at the 21st National Electron Microscope Congress, P:133, 28-31 May, 2013, Mersin, Turkey. Declaration of interest: The authors report no conflicts of interest. The authors alone are responsible for its contents.

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GPx 1 in capsaisin treated diabetic rats 7

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Distribution of glutathione peroxidase 1 in liver tissues of healthy and diabetic rats treated with capsaisin.

We investigated the immunohistochemical localization of glutathione peroxidase 1 (GPx 1) and the structural changes that occur in the livers of health...
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