Year : 2022, Volume : 46, Issue : 1
First page : ( 49) Last page : ( 58)
Print ISSN : 0250-4758. Online ISSN : 0973-970X. Published online : 2022  28.
Article DOI : 10.5958/0973-970X.2022.00007.4

Effect of Lead toxicity on liver and kidney in Wistar rats and its amelioration with Linum usitatissimum (flaxseed) and Emblica officinalis (amla)

Paul Amitava¹, Sujatha K.^1,*, Srilatha C.H.¹, Kumar N. Vinod²

¹Department of Veterinary Pathology, College of Veterinary Science, Sri Venkateshwara Veterinary University, Tirupati-517502, Andhra Pradesh, India.

²Department of Veterinary Microbiology, College of Veterinary Science, Sri Venkateshwara Veterinary University, Tirupati-517502, Andhra Pradesh, India.

^*Address for Correspondence, Dr K. Sujatha, Centre for Continuing Veterinary Education and Communication, Sri Venkateshwara Veterinary University, Tirupati-517502, Andhra Pradesh, India, E-mail: karamalasujatha@gmail.com

Online Published on 28 July, 2022.

Received:  07  ,  2021; Accepted:  22  November,  2021.

Abstract

The present study was conducted to elucidate the possible protective effect of Linum usitatissimum (Flaxseed) and Emblica officinalis (Amla) on lead induced hepatic and renal toxicity in wistar rats. A total of 108 adult wistar albino rats were randomly assigned to 6 groups with 18 rats in each group. Group I served as vehicle control and they received distilled water, whereas rats in group II-@ 60 mg/kg b.wt. lead acetate, group III-Emblica officinalis @ 100 mg/rat/day, group IV-Linum usitatissimum @ 300 mg/kg b.wt, group V-lead acetate @ 60 mg/kg b.wt + Emblica officinalis @ 100 mg/rat/day, and group VI-lead acetate @ 60 mg/kg b.wt + Linum usitatissimum @ 300 mg/kg b. wt. were administered through oral gavage for 45 days. The results indicated that rat treated with lead acetate showed significant elevation in thiobarbituric acid reactive substances (TBARS) levels compared with the healthy control ones while there were significant reduction in antioxidant enzymes like catalase (CAT), superoxide dismutase (SOD) and glutathione peroxidase (GPx) levels in liver and kidney. Histopathology of liver revealed degenerative changes like micro vesicular fatty changes, focal loss of hepatocytes and bile ductular hyperplasia and in kidney desquamated renal tubular epithelium, disruption of renal tubules and inter tubular haemorrhages were evident in lead treated group. The levels of all above parameters were significantly improved in the ameliorated group (Group V and VI). These experimental results strongly indicate the protective effect of Amla and Flaxseed against lead induced different pathological manifestations on liver and kidney. In addition, the ameliorating effect of Emblica was found to be relatively better than that of Flaxseed in most of the parameters.

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Keywords

Amla, CAT, Flaxseed, GPx, Lead, SOD, TBARS.

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Introduction

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Materials And Methods

Procurement of experimental animals, aqueous extract of Emblica offi cinalis and lead acetate

Wistar albino rats with body weight around 150 to 200 gms were procured from Sri Venkateshwara Agencies, Bangalore and after one week of acclimatization the rats were grouped randomly and housed in standard polypropylene rat cages (three rats/cage) during the experimental period. They were maintained at 25° C ± 10°C and a 12:12 hour interval light and dark cycle during the 45 days of experimental period by maintaining standard laboratory hygienic conditions with ad libitum supply of laboratory animal feed and water. The permission of the institutional animal ethical committee (IAEC) was obtained earlier to commencement of the experiment. The Lead acetate was procured from the Qualigens Fine Chemicals Company, product code No.27645 with 97% purity. Aqueous extract of Amla (Emblica offi cinalis) with product code C/SVU/EMOF-01 was procured from Chemiloids Company, Vijayawada, India.

Preparation of aqueous methanolic extract of Linum usitatissimum (Flaxseed)

Seeds of Linum usitatissimum (Flax seeds) were procured from a local herbal shop. After drying in shade, seeds were ground into powder form, which then used for the preparation of aqueous methanolic extract. Flaxseed lignans was prepared as per the method of Zhang et al.¹³. Flaxseeds (800g) were ground using a grinder to attain a fine powder form. The powder form of flaxseeds was defatted by blending with hexane (1:6 w/v, 12 h) in room temperature. Defatting of flaxseed is a pre-extraction procedure and it was done to remove undesirable components so that pure form of flaxseed lignans can be extracted. The defatted flaxseeds powder was air dried for around 12 hours. 200 grams of this defatted powder was again blended with 1.2 Litres complex solution of ethanol and water (7:3 v/v) for 24 hours at ambient temperature (25 °C). The extract was filtered into flask, and then filtered product was concentrated at 50 °C using a Rotary evaporator (Buch, Model 462, Germany) @ 90 rpm. Light yellow color syrup of flaxseed lignans extract was obtained.

Ethical matters

The ethics governing the use and conduct of experiments on laboratory animals were strictly observed and the experimental protocol was approved by Institutional Animal Ethical Committee (IAEC), Sri Venkateswara Veterinary University, Tirupati, Andhra Pradesh, India (Reference number 281/go/ReBi/S/2000/CPCSEA/CVSC/TPTY/018/VPP/2016-17). Institutional Animal Ethical Committee (IAEC), Sri Venkateswara Veterinary University, Tirupati, Andhra Pradesh, is affiliated under The Committee for the Purpose of Control and Supervision of Experiments on Animal (CPCSEA, India) with the Registration number-281/go/ReBi/S/2000/CPCSEA.

Study design

A total of 108 no. adult wistar albino rats were assigned to 6 groups randomly with 18 rats in each group. Group I served as vehicle control and they received distilled water, whereas rats in group II-@ 60 mg/kg b.wt. lead acetate, group III-Emblica offi cinalis @ 100 mg/rat/day, group IV-Linum usitatissimum @ 300 mg/kg b.wt, group V-lead acetate @ 60 mg/kg b.wt + Emblica offi cinalis @ 100 mg/rat/day, and group VI-lead acetate @ 60 mg/kg b.wt + Linum usitatissimum @ 300 mg/kg b. wt. were administered through oral gavage for 45 days. The dose rate of lead (Pb), amla and flaxseed for the present study were selected according to method describe by previous researchers^3,4,5,12. Six rats from each group were sacrificed at fourteen days intervals to study the pathological changes and to estimate the changes in antioxidant enzymes and lipid peroxidation level in a duration dependant manner in liver and kidney.

Histopathology

A detailed post-mortem examination was carried out on the sacrificed rats of all the trial groups. The gross changes were documented and tissue pieces were collected and well-preserved in 10% NBF for histopathological studies, fixed tissues were processed by standard paraffin embedding technique. 5-6 microns thick tissue sections were cut and section were stained with Haematoxylin and Eosin stain (H&E)¹⁴.

Lipid peroxidation (TBARS) assay and Antioxidant profile

For oxidative stress liver and kidney were collected and stored at-20° C until use. Small tissue piece of liver and kidney were minced in separate container and homogenized in 0.05 M ice cold phosphate buffer (pH 7.4) and make 10% homogenate by using a virtis homogenizer. 0.2 ml of the 10% homogenate was utilized for lipid peroxidation assay¹⁵. The residual part of homogenate then mixed with 10% trichloroacetic acid in 1:1 ratio, then centrifuged @ 5000 g at 4°C for 10 min and supernatant was collected and was used for valuation of reduced glutathione¹⁶. The residual portion of the homogenate was centrifuged for 60 min. at 15,000 g at 4°C and the supernatant attained was used for valuation of superoxide dismutase, catalase and glutathione peroxidase in liver and kidney of all rats in all groups^17,18,19.

Statistical analysis

The results were analysed statistically by performing one way ANOVA²⁰.

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Results

Lipid peroxidation – Thiobarbituric acid reactive substances (TBARS)

There was significant (P< 0.05) increase in mean liver and kidney TBARS levels in lead acetate treated rats (Group II) when compared to the control rats (Group I). The mean liver TBARS values from group I to VI were 517.63, 1682.5, 511.5, 514.2, 532.46 and 539.53 respectively (nM of MDA/g of tissue) and the mean kidney TBARS values from Group I to VI were 477.06, 1002.76, 476.23, 479.43, 495.7 and 506.53 (nM of MDA/g of tissue) and are shown in Table 1 and Table 2. Statistically significant improvement was noticed in mean liver and Kidney TBARS levels in emblica (Group V) and flaxseed (Group VI) ameliorated rats when compared to the lead acetate treated rats (Group II). There was no significant alteration in lipid peroxidation (TBARS) level was observed in liver and kidney of Emblica (Group III) and Flaxseed (Group IV) treated rats.

Antioxidant profile Catalase (CAT)

Statistically significant (P< 0.05) decrease in mean liver and kidney catalase activity was noticed in lead acetate treated rats (Group II) when compared to the control rats (Group I). The mean liver catalase activity from group I to VI were 0.32, 0.21, 0.33, 0.32, 0.30, 0.28 (nM of H₂O₂ decomposed/min/mg of protein) and the mean kidney catalase activity from Group I to VI were 0.28, 0.12, 0.29, 0.28, 0.25 and 0.23 (nM of H₂O₂ decomposed/min/mg of protein) respectively and are shown in Table 3 and Table 4. A significant increase in mean liver catalase activity was noticed in ameliorated group rats (Group V and Group VI) when compared to the lead acetate treated rats (Group II). No significant changes were observed in CAT level in liver and kidney of Emblica (Group III) and Flaxseed (Group IV) treated rats.

Superoxide dismutase (SOD)

Significant (P< 0.05) reduction was observed in mean liver and Kidney SOD activity in lead acetate treated rats (Group II) when compared to the control rats (Group I). The mean liver SOD activity in group I to VI were 16.23, 8.86, 16.3, 16.13, 15.56,15.4 (U/min/mg of protein) and the mean kidney SOD activity from group I to VI were 15.13, 7.7, 15.16, 15.03, 14.6 and 14.26 (U/min/mg of protein) respectively and presented in Table 5 and Table 6. Statistically significant improvement was noticed in mean liver SOD activity in Emblica ameliorated rats (Group V) and flaxseed ameliorated rats (Group VI) when compared to the lead acetate treated rats (Group II). No significant alteration was observed in SOD in liver and kidney of Emblica (Group III) and flaxseed (Group IV) treated rats.

Glutathione peroxidase (GP_x)

The mean liver and kidney GP_x activity from Group I to VI were 30.06, 17.95, 29.90, 29.81, 26.27 and 25.27 (μ of glutathione utilized/min/mg protein) and 26.39, 14.47, 26.32, 26.16, 22.63 and 22.24 (μ of glutathione utilized/min/mg protein) respectively and presented in Table 7 and Table 8. There was significant (P< 0.05) decrease in mean kidney GP_x activity in lead acetate treated rats (Group II) when compared to the control rats (Group I). Statistically significant increase in mean kidney GP_X activity was noticed in emblica ameliorated rats (Group V) and flaxseed ameliorated rats (Group VI) when compared to the lead acetate treated rats (Group II). There was no significant alteration in GPx level in kidney and liver of emblica (Group III) and flaxseed (Group IV) treated rats.

Pathology

Gross Pathology

In the present study, during the initial experimental period (2^nd week), no specific changes were noticed in liver and kidney of lead acetate treated group. Noticeable gross lesions like paleness of liver (Fig. 1) and congested kidneys (Fig. 2) were observed from of 4^th week onwards of experiment and same was evident throughout the experimental period in group-II. Whereas during 4^th week of experiment, slight paleness of liver and congested kidneys was noticed in lead acetate + emblica (Group V) and lead acetate + flaxseed (Group VI) ameliorated rats. By the end of experiment, livers and kidneys of ameliorated group (Group V and VI) were regained almost normal appearance similar to control group. Grossly no significant lesion was observed in liver and kidney of group-III and group-IV rats during the entire experimental period.

Histopathology

Liver

Histopathology of livers of group II revealed, degenerative changes in hepatocytes, dilated and congested sinusoids, mild infiltration of MNCs in portal area (Fig. 3) by the end of 2^nd week of experiment. In addition to the above, focal loss of hepatocytes with infiltration of MNCs, severe dilatation of blood vessels with thrombus formation (Fig. 4), mild to moderate micro vesicular fatty change of hepatocytes (Fig. 5), moderate to severe sinusoidal dilatation and congestion, and edema, mild to moderate fibroblast proliferation along with MNCs infiltration in portal area, and binucleated hepatocytes were evident by the end of 4^th week of experiment. By the end of 6^th week of experiment, severe dilated and congested blood vessels, infiltration of MNCs and bile ductular hyperplasia in portal area (Fig. 6), hyperplastic changes of bile duct epithelium with infiltration of MNCs, severe dilation of sinusoidal space with congestion (Fig. 7) and moderate fibroblast proliferation in portal area, multi focal loss of hepatocyte with infiltration of MNCs (Fig. 8), more number of binucleated hepatocytes (Fig. 9) and degenerated hepatocyte with karyomegaly (Fig. 10) were more evident.

In group V (Lead + Amla) similar lesions like group II with mild intensity were observed up to 4^th weeks. Later, the changes were gradually reduced in its intensity like mild dilation of sinusoidal space with congestion (Fig. 11), mild infiltration of MNCs and mild congestion of blood vessels in portal area (Fig. 12) and liver regained its near to normal appearance by the end of 6^th week. In group VI the lesions as described in group II with less intensity were noticed up to 4^th weeks. Afterwards, the changes were reduced like mild congestion of blood vessels, mild dilation of sinusoidal space, portal area mild MNCs infiltration (Fig. 13) and liver appeared as almost normal (Fig. 14) by the end of the experiment. There was no pathological alteration was observed in liver of Emblica (Group III) and flaxseed (Group IV) treated rats.

Kidney

Light microscopic study of kidney of the group II animals revealed mild to moderate degenerated renal tubular epithelium and congested glomerulus (Fig. 15), mild perivascular MNCs infiltration and pockets of haemorrhages in inter tubular spaces (Fig. 16) by the end of 2^nd week of trial period. By the end of 4^th week moderate degenerated renal tubules, variation in size of glomerulus, congested, degenerated and atrophied glomerulus, perivascular infiltration of MNCs in interstitial space (Fig. 17), severe intertubular hemorrhages and edema were evident. In addition to the above changes, cystic glomerulus (Fig. 18), periglomerular fibroblast proliferation, severe degenerated and desquamation of tubular epithelium, severe infiltration of MNCs in interstitial spaces (Fig. 19) along with severe pockets of haemorrhages. Complete disruption of renal tubules and intertubular edema with degenerated glomerulus (Fig. 20), thickened blood vessels, perivascular MNCs infiltration and intertubular fibroblast proliferation, atrophied glomerulus with periglomerular fibroblast proliferation (Fig. 21) was more conspicuous by the end of 6^th week.

In group V Similar lesions as described in Group II with reduced intensity were noticed up to 4^th weeks. Later the changes were less severe like mild hemorrhages in the cortex (Fig. 22), slight variation in size of glomerulus with mild congestion and mild degenerative changes in renal tubules (Fig. 23) Kidney regained its normal appearance by the end of 6^th week. In group VI similar lesions like Group II with mild intensity were observed up to 4^th weeks and later stages the changes were gradually reduced in its severity like mild degenerated and desquamated renal tubular epithelium (Fig. 24) and intertubular haemorrhages and kidney regained almost near to normal appearance by the end of 6^th week. There was no significant pathological alteration was observed in Kidney of emblica (Group III) and flaxseed (Group IV) treated rats.

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Discussion

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Conclusion

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Figures

Fig. 1.: Photograph showing pale coloured liver in group-II and almost normal appearance of liver in other groups (6th week) ; TopBack

Fig. 2.: Photograph showing congested kidneys in group-II, and rest of the groups’kidney appeared normal (6th week) ; TopBack

Fig. 3.: Section showing periportal infiltration of mononuclear cells in portal areas in liver (Group II, H&E x400) ; TopBack

Fig. 4.: Section showing congestion and thrombus formation in blood vessels in portal area in liver (Group II, H&E x400) ; TopBack

Fig. 5.: Section showing micro vesicular type of fatty change in hepatocytes (Group II, H&E x100) ; TopBack

Fig. 6.: Section showing bile ductular hyperplasia (Group II, H&E x400) TopBack

Fig. 7.: Section showing sinusoidal dilatation and congestion (Liver. Group II, H&E x400) ; TopBack

Fig. 8.: Section showing focal loss of hepatocytes with infiltration of mononuclear cells (Group II, H&E x400) ; TopBack

Fig. 9.: Section showing binucleated Hepatocyte (Group II, H&E x1000) ; TopBack

Fig. 10.: Section showing karyomegaly of hepatocyte (Group II, H&E x1000) ; TopBack

Fig. 11.: Section showing mild sinusoidal dilation with congestion (Liver. Group V, H&E x400) ; TopBack

Fig. 12.: Section showing mild infiltration of cells in portal area (Liver, Group V, H&E x40) TopBack

Fig. 13.: Note mild congestion of blood vessels (Liver, Group VI, H&E x40) ; TopBack

Fig. 14.: Section showing almost near to normal appearance of liver (Group VI, H&E x40) ; TopBack

Fig. 15.: Section showing degenerated renal tubules (kidney, Group II, H & E x 400) ; TopBack

Fig. 16.: Section showing perivascular MNCs infiltration and pockets of haemorrhages in interstitial spaces (Kidney, Group II, H & E x100) ; TopBack

Fig. 17.: Section showing Perivascular infiltration of MNCs (Kidney, Group II, H & E x 400) ; TopBack

Fig. 18.: Photograph showing cystic glomerulus (Kidney, Group II, H & E x 400) TopBack

Fig. 19.: Photograph showing severe infiltration of MNCs in intertubular space (Kidney, Group II, H&E x400) ; TopBack

Fig. 20.: Section showing complete disruption of renal tubules, intertubular edema, degenerated glomerulus and pockets of haemorrhages (kidney, Group II, H&E x400) ; TopBack

Fig. 21.: Section showing atrophied glomerulus with periglomerular fibroblast proliferation (Group II, H&E x400) ; TopBack

Fig. 22.: Section showing mild haemorrhages in the cortex in kidney (Group V, H&E x40) ; TopBack

Fig. 23.: Section showing normal appearance of kidney except mild congestion of glomerulus and intertubular hemorrhages (Group V, H&E x400) ; TopBack

Fig. 24.: Section showing mild degenerative changes in renal tubules (Kidney, Group VI, H&E x400) TopBack

Tables

Acknowledgements

The authors are thankful to Sri Venkateshwara Veterinary University, Tirupati, Andhra Pradesh, India for providing various facilities during the research period.

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