Ameliorating activity of ginger (Zingiber officinale) extract against lead induced renal toxicity in male rats (2024)

Materials

The ginger was obtained from a local market, Anantapur, India. The ginger was cut into small pieces and air-dried for 5 to 6days. The dried ginger was ground to fine powder using electronic grinder. Ginger powder (50g) was extracted in 250ml of ethanol for 18h in a soxhlet apparatus following the method of Misra et al. (2005). The extract was dried at reduced pressure and stored at 0–4°C until further experiments. The chemicals and solvents used in this study were of analytical grade.

Animals and treatment

Male Wistar rats of body weight weighing 160 ± 10g were procured from Sri Ragavendra Enterprises, Bangalore, India. The rats were maintained at 24 ± 2°C and 12h light/dark cycles. Rats were fed on standard rat diet and water was supplied ad libitum. Before experimentation rats were acclimatized for 2weeks. Animals were handled according to the rules and regulations of Institutional Animal Ethics committee (IAEC), Sri Krishnadevaraya University, Anantapur, India. Animals were divided into five groups of six animals each. Group I treated with vehicle (distilled water) was kept as control. Group II treated with lead nitrate (300mg/kg BW) daily for 1week, group III was treated with lead nitrate (300mg/kg BW) plus ginger extract (150mg/kg BW) daily for 1week. Group IV treated with lead nitrate (300mg/kg BW) daily for 3weeks, Group V was treated with lead nitrate (300mg/kg BW) plus ginger extract (150mg/kg BW) daily for 3weeks. Lead nitrate was dissolved in distilled water and given through gastro intestinally with the help of a gavage. The ethanolic extract of ginger was dissolved in 5% tween-80 and administered through gastro intestinally with the help of a gavage after 6h of lead nitrate incubation.

Animal sacrifice and organ collection

After one week of experimental period, three rats from group I, all six rats from group II and group III were sacrificed. From group I remaining three rats and from group IV and V all six rats were sacrificed after 3weeks of exposure. Animals were sacrificed by cervical dislocation and immediately kidneys were removed and washed with ice-cold saline to remove blood and suspended in 0.1M potassium chloride (KCl) in polypropylene container, sealed with parafilm, labeled and frozen at −80°C until assays were carried out.

Preparation of tissue extraction for glutathione estimation

Ten percent tissue hom*ogenate was prepared in 0.15M KCl using a hom*ogenizer at 0°C. The whole hom*ogenate was used for estimation of glutathione.

Sample preparation for antioxidant enzyme assays

Ten percent kidney hom*ogenate in 0.15M KCl was prepared using a hom*ogenizer at 0°C and centrifuged in cold (0–4°C) at 12000rpm for 45min. The supernatant thus obtained was distributed into eppendorf tubes, labeled and stored at −20°C and all the antioxidant enzymes were assayed at the earliest.

Glutathione (GSH)

Total reduced glutathione content was measured by following the method of Ellman (1959). This method is based on the development of a yellow color, when 5, 5′-dithio-2-nitrobenzoic acid (DTNB) reacts with the compounds containing sulfhydryl groups with a maximum absorbance at 412nm. Ten percent kidney hom*ogenate (0.5ml) was deproteinized with 3.5ml of 5% Trichloroacetic acid (TCA) and centrifuged at 4000rpm for 5min. To 0.5ml of supernatant, 3.0ml 0.2M phosphate buffer (pH 8.0) and 0.5ml of Ellman’s reagent were added and the yellow color developed was measured at 412nm. A series of standards (4–20μg) were treated in a similar manner along with a blank and values were expressed as μg of GSH/mg protein.

Glutathione peroxidase (GPX)

A known amount of the enzyme preparation was allowed to react with H₂O₂ in the presence of GSH for specific time period according to the method of Rostruck et al. (1973) and remaining GSH was measured by following the method of Ellman (1959) as described for GSH estimation. To 0.5ml 0.4M phosphate buffer (pH 7.0), 0.2ml of 10% kidney hom*ogenate, 0.2ml of GSH and 0.1ml of H₂O₂ were added and incubated at room temperature (25 ± 2°C) for 10min along with a control tube containing all reagents except enzyme source. The reaction was arrested by adding 0.5ml of TCA, centrifuged at 4000rpm for 5min and GSH content in 0.5ml of supernatant was estimated. The activity was expressed as μg of GSH consumed/min/mg protein.

Glutathione-s-transferase (GST)

Glutathione-s-transferase activity was measured by monitoring the increase in the absorbance at 340nm using 1-chloro-2,4-dinitrobenzene (CDNB) as a substrate according to the method of Habig et al. (1974). To 1.7ml phosphate buffer (pH 6.5), 0.2ml of GSH and 0.04ml of kidney hom*ogenate (40μg protein) were added and the reaction was initiated by the addition of 0.06ml CDNB. The change in absorbance was recorded at 1min intervals at 340nm for 5min and the activity was calculated using extinction coefficient of CDNB-GSH conjugate as 9.6mM ^-1 cm ^-1 and expressed as m moles of CDNB-GSH conjugate formed/min/mg protein.

Catalase (CAT)

Catalase catalyses the breakdown of H₂O₂ to H₂O and O₂ and the rate of decomposition of H₂O₂ was measured spectrophotometrically at 240nm following the method of Beers and Sizer (1952). To 1.9ml of phosphate buffer (pH 7.0), 1.0ml of H₂O_2.was added and then the reaction was initiated by the addition of 0.1ml kidney hom*ogenate (45μg protein). The decrease in absorbance was monitored at 1min intervals for 5min at 240nm and activity was calculated using a molar absorbance coefficient of H₂O₂ as 43.6M^-1cm^-1. The activity was expressed as m moles of H₂O₂ decomposed/min/mg protein.

Histology

The histology sections of the kidneys of male rats were taken by adopting the procedure as described by Humason (1972). The tissues were isolated and gently rinsed with saline solution to remove mucus and other debris adhering to them. They were fixed in Bouin’s fluid for 24h and the fixative was removed by washing through running tap water for overnight. Then the tissues were processed for dehydration. The tissues were passed through successive series containing 30%, 50%, 70%, 80%, 90%, 95% and absolute alcohols. Then the tissues were cleaned in methyl benzoate and embedded in paraffin wax. Sections of 5μ thickness were made using rotary microtome. The sections were stained with Harris hematoxylin (Harris 1900) and counter stained with eosin, dissolved in 95% alcohol. After dehydration and cleaning, the sections were mounted in Canada balsam. Photomicrographs of the section preparations were taken using Olympus photomicrography equipment (PM-6 model, Japan).

Statistical analysis

Statistical analysis was carried out using SPSS 11.0 for Windows (SPSS Inc., Chicago, IL). Each experiment repeated in triplicates and all data were represented as mean ± SD. Duncan’s multiple range test was used to determine the statistical significance between groups. The significance is presented at level of P < 0.05.

Glutathione

Glutathione is an essential biofactor synthesized in all living cells. It forms an important substrate for GPX, GST and several other enzymes, which are involved in free radical scavenging. It can act as a nonenzymatic antioxidant by direct interaction of the SH groups with ROS or it can be involved in the enzymatic detoxificatin reactions for ROS as a cofactor (Ding et al. 2000). In the present study GSH levels were decreased in lead treated rat group when compared to control group. The GSH levels of control group was 1.05μg of GSH/mg protein, and in 1week lead exposed group was 0.95μg of GSH/mg protein, and in 3weeks lead exposed group was 0.88μg of GSH/mg protein. Several studies reported that lead toxicity decreases GSH levels (Bechara 2004; Patra et al. 2001; Sivaprasad et al. 2003). In 3weeks lead plus ginger treated group the GSH level was increased (0.94μg of GSH/mg protein). As the GSH is substrate for GPX, GST and several other enzymes, it is believed that GSH level is also increased when these enzymes activity is increased. Kikuzaki and Nakatani (1993) and Lee and Ahn (1985) have also reported that GSH depended antioxidant activity is increased by ginger extracts.

Glutathione peroxidase (GPX)

Glutathione peroxidase (GPX) is an antioxidant enzyme that catalyzes the hydrogen peroxide-reduced glutathione reaction. GPX is an important component in the enzymatic defense system against the increase of free radicals (Crack et al. 2001). In this study, the activity of glutathione peroxidase was reduced in lead treated groups (1.25 and 1.08μg of GSH consumed/min/mg protein, respectively for 1week and 3weeks lead treated groups) when compared to control group (1.34μg of GSH consumed/min/mg protein). Farmand et al. (2005) reported that the decrease in GPX activity was due to lead toxicity. Lead plus ginger extract treated groups showed a significant increase of kidney GPX activity. The results obtained in present study further support that ginger can promote glutathione peroxidase as one of the antioxidant activity (Kikuzaki and Nakatani 1993; Lee and Ahn 1985).

Glutathione-s-transferase (GST)

Glutathione-s-transferase (GST) is an important enzymatic system of the cellular mechanism of detoxification that protects cells against reactive oxygen metabolites due to the conjugation of glutathione with electrophillic compounds. In this investigation the activity of renal GST was found to be 19.04, 17.13 and 15.02μ moles of GSH-CDNB formed/min/mg protein, respectively for control, 1week and 3weeks lead treated groups. Significant improvement in the activity of GST was noticed in ginger supplemented groups. These findings further support the studies that reported the enhancement of GST activity by ginger extracts (Misra et al. 2005; Kikuzaki and Nakatani 1993).

Catalase (CAT)

Catalase (CAT) is a metalloprotein that protects the tissues from highly reactive hydroxyl radicals by reducing hydrogen peroxide. In this study the activity of catalase was found to be significantly (P < 0.05) increased in kidneys of lead plus ginger extract treated groups when compared to lead treated groups. These findings coincide with earlier studies that showed a significant increase in CAT activity due to ginger treatment (Misra et al. 2005; Kikuzaki and Nakatani 1993). The CAT activity was significantly (P < 0.05) decreased in lead treated groups when compared to control group. The decrease in CAT activity might be due to the inhibition of haembiosynthesis of catalase by lead (Patil et al. 2006). Mohammad et al. (2008) have also reported that decrease in CAT activity was due to lead toxicity.

Histopathology

The results of histopathological examination of rat kidney tissue sections are presented in the Fig.2. Kidney sections of 1week lead exposed rat group showed architectural changes of glomerulus and tubular epithelial cells (Fig.2b). The changes observed were dispersed in intratubular region. Whereas in kidney of lead exposed plus ginger extract treated rat group mild changes were observed when compared to the kidney of 1week lead exposed rat group (Fig.2c). Necrotic regions were observed and it seems to be fatty changes in between glomeruli and intratubular region. The kidney sections of 3weeks lead exposed rat group showed severe changes than that of 1week lead exposed group (Fig.2d). Infiltration of inflammatory cells was seen in intratubular region. Tubular epithelial cells appear to be degenerative with mild necrotic changes. Whereas in 3weeks lead plus ginger extract treated rat group kidney indicated lesser changes than 3weeks lead exposed rat group (Fig.2e).

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Fig. 2

Comparison of photomicrographs of kidney tissue sections of a Control group, bLead treated group for 1week, c Lead plus ginger extract treated group for 1week, d Lead treated group for 3weeks, e Lead plus ginger extract treated group for 3weeks

The histopathological results obtained in the present investigation indicated that lead induced many histopathological changes in the kidney of rats treated with lead for 1week and 3weeks. Lead potentially induces oxidative stress and causes pathological changes of tissues like kidney (Ercal et al. 2000; Farmand et al. 2005; Khalil-Manesh et al. 1992). Physiological studies of lead transport in the kidney have shown this metal is taken up in proximal tubule cells (Vander et al. 1977; Vander et al. 1979). Exposure to high lead levels can produce renal tubular damage with glycosuria and aminociduria (Loghman-Adham 1997; Ehrlich et al. 1998). The results obtained in the present study showed that treating rats with lead plus ginger extract improved the histopathological changes induced in the kidney by lead. This suggested the effectiveness of ginger extract in prevention of lead induced renal toxicity.

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