INTRODUCTION Contamination due to heavy metals and metalloids including the toxic non-essential elements like arsenic (As), mercury (Hg), cadmium (Cd) and lead (Pb) is wide spread1. These heavy metals persistently accumulate and are not metabolized in other intermediate compounds due to which these metals easily enter the food chain and hence pose a great threat to public health. Amongst these metals, arsenic is possibly the most abundant pollutant having a complex metabolism and is classified as a potential human carcinogen2. Arsenic poisoning is one of the most important causes of heavy metal poisoning in canines and bovines3. World Health Organization (WHO)4 estimated that about 130 million people worldwide are exposed to arsenic concentration(s) above 50 μg L−1. In India, groundwater arsenic content is high in West Bengal, Gangetic plains and Punjab5. Human population is mostly exposed to arsenic in both acute and chronic forms through ingestion of arsenic contaminated water, foods, drugs, wines, cigarette smoke and fossil fuels6. In recent times, more people are attracted to various indigenous medicinal herbs as conventional medicine having one or other side effect which is causing detrimental effect to the human and animal population. |
Centella asiatica (Indian pennywort/Brahmi), a herbaceous perennial plant of Apiaceae family, is one such which has been used to treat a variety of ailments for thousands of years including leprosy, eczema, psoriasis, respiratory infections, ulcers, colds, hepatitis, epilepsy, fatigue, fevers, asthma and syphilis7. However, till date except for a single study based on effect of effect of C. asiatica on arsenic induced oxidative stress and metal distribution in rats8, the effect of this herbal drug has not been used to ameliorate pathomorphological changes due to arsenic toxicity. Thus, the present study was undertaken to assessthe ameliorative effect of Centella asiatica on arsenic induced toxicity in Wistar rats through pathomorphological studies. |
Top MATERIALS AND METHODS Animals and treatments The study protocol was approved by the Institutional Animal Ethics Committee (IAEC) of Guru Angad Dev Veterinary and Animal Sciences University (GADVASU), Ludhiana. Ninety (90) healthy adult male and female Wistar albino rats were procured from Small Animal Colony Department of Livestock Production and Management, College of Veterinary Science, GADVASU, Ludhiana. The rats were maintained individually in laboratory cages and were provided ad libitum feed and drinking water. The rats were kept for 7 days to acclimatize in laboratory condition prior to start of sampling protocols. On day 8, the rats were divided randomly into 6 groups (15 rats in each group) in cages in a 12 hr light and 12 hr dark cycles (Table 1). Shade dried leaves of Centella asiatica were crushed in mixer and converted to powder form. Powdered Centella asiatica was mixed with feed at a dose rate of 200 ppm. The rats were fed sodium arsenite (LOBA CHEMIE Pvt. Ltd. Mumbai) and Centella asiatica for 60 days as per the given table. The animal experiment was duly approved by Institutional Animal Ethics Committee (IAEC) vide order no. VMC/2010/IAEC/1976–92 GADVASU, Ludhiana. The rats were sacrificed on day 60 of experiment and necropsy was performed and relevant tissues were collected in 10% buffered formalin saline for histopathological studies. Pathomorphological studies The experimental animals sacrificed at the end of experiment were subjected to detailed necropsy examination and gross changes, if any, were recorded. Absolute weights of different organs, such as testes, brain, liver and kidneys and weight of each organ in relation to whole body weights (relative weight) were recorded at the end of experiment. Small representative pieces (approximately 0.5 cm thickness) of various organs/tissues (testes, liver, kidneys, spleen, brain, heart, pancreas, skin, thymus, ovary and intestines) were collected from each sacrificed animal and fixed in 10% neutral buffered formalin. The tissues were further routinely processed and embedded in paraffin. 5-μm thick sections were stained with hematoxylin and eosin. Scoring of the lesions was performed by categorizing the significant and consistent lesions in different organs into mild (m), moderate (M) and severe (S) and were allotted scores of 1,2 and 3, respectively. These were multiplied by the number of animals in a particular group to derive the final score of different lesions of that group. Top RESULTS Centella asiatica ameliorates arsenic induced gross lesions Gross lesions were observed on various organs due to the arsenic toxicity. Liver was pale in arsenic fed groups (Group II and Group V). However, the intensity of paleness was less in group VI as compared to group II and V. Normal coloration was evident in group III, IV and control group (Group I). Mild meningeal congestion was observed on brains of rats of group V. No such lesion was evident in any of the other groups. The absolute weight of testes in group V and VI were observed to be decreased and differed significantly from the average weight of the testes of control group. However, the absolute weights of liver and kidney did not vary in all the arsenic groups as compared to control group at the end of experiment (Table 2). Further, the relative weight of liver in group V and VI, were observed to be significantly increased as compared to control group. The relative weight of testes in group V were decreased significantly as compared to the relative weight of testes in control group. Centella asiatica ameliorates arsenic induced histopathological changes In the present experiment, although the consistent lesions were observed in group II, III, V and VI but the histopathological lesions in organs were observed in a dose dependent manner. The histopathological scores for different organs in different groups are presented in table 4. Group V showed the highest histopathological score followed by groups VI, II and III. In group V, histopathological sections of kidney revealed severe tubular degeneration (Fig. 1) and prominence of basement membrane due to sloughing of epithelial cells. There was presence of proteinaceous cast and swollen tubular epithelial cells almost occluding the lumen. Marked shrinkage of glomeruli and increase in Bowman‘s space were evident (Fig. 2). However, group II (Fig. 3) and VI showed moderate tubular degeneration and sloughing of the tubular epithelial cells. In group III, very mild tubular degeneration along with mild congestion was observed. Group IV and I did not revealed any adverse histopathological lesions. In group V, liver showed severe degenerative changes and almost complete loss of normal parenchyma (Fig. 4). Cytoplasm of hepatocytes were granular which varied from fine to coarse. Plenty of hepatocytes revealed karyomegaly. Hepatocytes were swollen without prominent cell margins which at places were clustered and practically disappearance of nucleus was seen. Vacuolar degeneration was also a consistent finding in group V (Fig. 5). The sinusoids were engorged in majority of cases. In few cases, double nuclei were also seen. Moderate hepatocytic degeneration along with single cell necrosis was evident in group II andVI. However, in group III these changes were of very mild intensity indicating protective action of Centella asiatica (Fig. 6). Group IV and control group I showed normal architecture of liver. Testes of group V revealed intensive decrease in spermatogenic activity and number of spermatogonial cells with increased intertubular spaces. The desquamated epithelial cells were found in the lumen of tubules. Epididymal ductules showed decrease number of spermatozoa and detached lining epithelium cells from the basement membrane (Fig. 7). In group II, moderate spermatogonial degeneration and decrease number of spermatogonia along with loss of whirling movement were evident (Fig. 8). In group III, the seminiferous tubules were uniform in shape and size, with little intertubular spaces. In group VI, the intertubular spaces were increased with marked decrease in spermatogenic activity and spermatogonial cells. However, the histopathological sections of testes of group IV and I (Fig. 9) revealed normal architecture. Brain revealed neuronal degeneration, oedema, gliosis and degeneration of Purkinje cells in rats of group V, II and VI in a dose dependent manner. The nature of changes were very severe in group V (Fig. 10) and moderate in group II and VI. In few cases, purkinje cells were almost absent in the molecular layer of cerebellum. In group III changes were present but were of very mild intensity (Fig. 11). Heart in group V showed massive necrosis of myocardium, resembling Zenker‘s necrosis. Congestion was present in heart of group V. Focal areas of necrosis of myocardial muscles resembling Zenker‘s necrosis were seen in sections of group II (Fig. 12). In VI, moderate type of degeneration was present where normal architecture of the heart muscles were lost. Very mild myocardial degeneration was present in group III. Group I and IV normal histological structure. In few cases of group V, stomach showed deep crate like ulcers where the stratified squamous epithelium of stomach was eroded along with infiltration of inflammatory cells. Spleen showed severe congestion of the red pulp along with lymphoid depletion (Fig. 13). Moderate congestion of red pulp along with lymphoid depletion was observed in group II and VI. Very mild lymphoid depletion was present in group III as compared to normal histology in Group IV and group I (Fig. 14). Further, moderate to severe degeneration of pancreatic acini were evident in group V followed by lower intensity in group II and VI. In group III, no lesion of degeneration was found indicating ameliorating effect of Centella asiatica on induced arsenic toxicity. In arsenic treated animals, sloughing of villi, depletion of lymphoid follicles in payer patches, goblet cell hyperplasia were evident. However, the severity of lesions were dose dependent. No significant lesions were evident in group IV and I. Only in few cases of group V, keratitis was observed with infiltration of inflammatory cells in the connective tissue stroma of eye. No histopathological alterations were observed in ovaries and uterus of females. Top DISCUSSION Arsenic induced toxicity is mainly caused by increased oxidative stress in tissue and this arsenic mediated oxidative stresses are indicated for changing the organ degeneration during exposure9. Moreover, arsenic-induced pathological changes may be caused by oxidative DNA damage other than nitrative DNA damage10. Centella asiatica is used as medicine in Ayurvedic tradition of India. Various biochemical compounds are present in C. asiatica which includes flavonoids, alkaloids, saponin, glycosides, phenol, triterpenoids etc. Flavonoids and triterpenoids are antioxidant11. |
In the present study, C. asiatica treatment showed attenuation of arsenic induced histomorphological changes. Kidneys and livers are susceptible to xenobiotic toxicity mainly due to kidney being the major excretory organ and liver being the main detoxification organ. In the present study, liver and kidneys showed severe arsenic toxicity. Kidney which is an organ rich in phospholipids and thus oxidative damage in kidney due to arsenic induced lipid peroxidation may deteriorate the function of the kidneys9. In this study, kidney of arsenic treated group revealed severe tubular degeneration with marked shrinkage of glomeruli and these changes was attenuated by treatment with C. asiatica. C. asiatica has been used to reduce urinary protein excretion in the treatment of kidney diseases for thousands of years in China. C. asiatica may reduce swelling, stimulate circulation, and promote diuresis12. It has been stated that asiaticoside, a compound present in C. asiatica exerts immunomodulatory, antiinflammatory functions, promotion of wound healing and nephroprotective13. |
The increase in relative weight of liver might be due to the decrease in percentage gain in whole body weight and degenerative changes as observed histopathologically in the liver of group V. Significant increase in the relative weight of liver due to arsenic toxicity has been reported earlier14. Further, liver of arsenic treated groups showed degenerative changes, vacuolar degeneration as well as swollen without prominent cell margins hepatocytes. Those finding were similar to that of other workers9,15. Arsenicals are known to produce oxidative stress as a mechanism of hepatotoxicity. Arsenic caused hepatic degeneration may be due to imbalance between pro-oxidation and antioxidant homeostasis in liver system, primarily by arsenic induced increased generation of free radicals9. Centella asiatica treated mice showed hepatoprotective effect. This may be due to the active antioxidant ingredients of C. asiatica (flavonoids and triterpenoids) which may contributed to the hepatoprotective effect through increasing the levels of antioxidant enzymes and reducing the levels of inflammatory mediators in rats16. |
Inorganic arsenics have suppressive effect on spermatogenesis and androgenesis in male reproductive system. Arsenic causes testicular toxicity by germ cell degeneration and inhibits androgen production in adult male rats probably by affecting pituitary gonadotrophins17. In the present study, the relative weight of testes in arsenic group were decreased significantly and testes revealed intensive decrease in spermatogenic activity and number of spermatogonial cells. Similar changes were observed by other workers17-18. Decrease in testicular weight may be due tospermatotoxicity, sperm cells degeneration, inhibit the spermatogenesis and sperm development17,19. However these changes were attenuated in C. asiatica treated group. C. asiatica showed a beneficial role by significant increase in the weights of reproductive organs, counteracting oxidative stress and restoring the suppressed reproduction in male rats20. |
Arsenic freely crosses blood-brain barrier and thus brain is a soft target for arsenic toxicity21. In the present study, brain revealed neuronal degeneration, oedema, gliosis and degeneration of Purkinje cells in arsenic treated rats a dose dependent manner. Degeneration of Purkinje cells due to arsenic toxicity, swelling and vacuolation of nerve cells and degenerative changes (nuclear pyknosis) in the brain of arsenic intoxicated rats were reported22,23. In arsenic-induced neurotoxicity, oxidative stress, induction of thiamine deficiency, and inhibitions of pyruvate decarboxylase, acetyl cholinesterase, reduction in biogenic monoamines seem to play a pivotal role24. In Ayurveda C. asiatica is stated to have cognitive properties as a brain tonic, in the treatment of mental disorders, and as a memoryenhancing agent. Neuroprotective effect of C. asiatica may be due to inhibition of acetylcholinesterase activity25. Asiaticoside from C. asiatica showed neuroprotective effects in animal model26. |
Many studies showed that there is an association of arsenic and cardiovascular disease. Arsenic causes inflammation in vascular tissues and activates oxidative signalling27. Arsenic plays a role in the development of arrhythmias and coronary heart disease through vascular endothelial dysfunction and free radical injury and also induces interstitial myocardial fibrosis28. In the present study arsenic caused necrosis of the myocardium which was attenuated in C. asiatica administered group. Cardioprotective effects of C. asiatica may be due to an augmentation of the endogenous antioxidants and inhibition of lipid peroxidation of membrane29. |
In the present study gastric ulcerative lesions were observed in the stomach of arsenic treated mice which were not observed in C. asiatica administered rats. Aqueous extract of C. asiatica was found to be effective in inhibiting gastric lesions induced by ethanol administration30. The authors concluded that the C. asiatica extract presumably strengthened the gastric mucosal barrier and reduced the damaging effects of free radicals30. |
Lymphoid depletion in the spleen in the present experiment may be due to the immuno suppressive effect of arsenic. Lymphoid depletion in the spleen indicates cytotoxic effect of arsenic on the antibody producing cells leading to immune suppression as a possible outcome of chronic arsenic intoxication18. Arsenic induced islet cell injury with characteristic shrinkage of islets cells, reduction of cell numbers and increase in number and diameter of capillaries has already been reported31. |
In conclusion, kidney, liver, testes and brain were the prime target organ of arsenic and the toxic effect of arsenic when given @2.5 ppm was ameliorated by C. asiatica as compared to high doses of arsenic (5 ppm) group. Moreover, at the given dose, C. asiatica did not cause any toxicity and is safe if to be used in amelioration. |
Top Figures | Fig. 1.: Group V: Sections of kidney revealed severe tubular degeneration, There was presence of proteinaceous cast and swollen tubular epithelial cells almost occluding the lumen. H&E ×200
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| | Fig. 2.: Group V: Kidney showing marked shrinkage of glomeruli and increase in Bowman‘s space. H&E ×400
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| | Fig. 3.: Group III: Kidney showing moderate tubular degeneration and sloughing of the tubular epithelial cells, very mild tubular degeneration along with mild congestion. H&E ×100
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| | Fig. 4.: Group V: Liver evincing severe degenerative changes and almost complete loss of normal parenchyma. H&E ×400
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| | Fig. 5.: Group V: Liver showing degeneration and vacuolar degeneration. H&E ×100
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| | Fig. 6.: Group III: Liver showing mild degenerative changes. H&E ×400
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| | Fig. 7.: Group V: Testes depicting intensive decrease in spermatogenic activity, number of spermatogonial cells and increased intertubular spaces. H&E ×400
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| | Fig. 8.: Group II; Testes: moderate spermatogonial degeneration and decrease number of spermatogonia along with loss of whirling movement was evident. H&E ×100.
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| | Fig. 9.: Group I: Testes showing well developed and compactly packed seminiferous tubules and normal spermatogonial activity along with whirling movement. H&E ×100
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| | Fig. 10.: Group V; Cerebellum: purkinje cells were almost absent in the molecular layer of cerebellum. H&E ×100.
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| | Fig. 11.: Group III: Cerebellum: Section showing mild loss of purkinje cells. H&E ×100
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| | Fig. 12.: Group II: Heart: Focal areas of necrosis of myocardial muscles resembling Zenker‘s necrosis.H&E ×400
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| | Fig. 13.: Group V; Spleen: Marked lymphoid depletion. H&E ×100
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| | Fig. 14.: Group I: Spleen: Well developed splenic corpuscles with densely packed lymphocytes. H&E ×100.
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Tables | Table 1.: Experimental animal groups and treatments given.
| | Group No. | No. of rats | Treatment | | Group I | 15 | Control group with normal feed and water | | Group II | 15 | Group given Sodium arsenite @ 2.5 ppm in drinking water | | Group III | 15 | Group given Sodium arsenite @ 2.5 ppm in drinking water plus Centella asiatica @ 200 ppm | | Group IV | 15 | Group given only Centella asiatica @ 200 ppm | | Group V | 15 | Group given Sodium arsenite @ 5 ppm in drinking water | | Group VI | 15 | Group given Sodium arsenite @ 5 ppm plus Centella asiatica @ 200 ppm |
| | | Table 2.: Effect of sodium arsenite on absolute weights of different organs.
| | Group | Liver | Kidney | Testes | | Group I | 9.53±0.41a | 1.88±0.033a | 4.91±0.13cd | | Group II | 9.10±0.69a | 1.78±0.065a | 4.31±0.15bc | | Group III | 8.87±0.46a | 1.974±0.12a | 4.84±0.12cd | | Group IV | 10.05±0.57a | 2.01±0.12a | 5.12±0.27d | | Group V | 10.21±0.45a | 1.75±0.038a | 3.29±0.28a | | Group VI | 10.33±0.22a | 1.78±0.035a | 3.99±0.037ab |
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| * The values (Mean ± S.E.) in a column having different superscript differ significantly from each other at 5% level of significance. * The values (Mean ± S.E.) in a column having same superscript do not differ significantly from each other at 5% level of significance. | | | Table 3.: Effect of sodium arsenite on relative weights of different organs.
| | Group | Liver | Kidney | Testis | | Group I | 4.49±0.19a | 0.89±0.015a | 2.31±0.06b | | Group II | 4.76±0.36ab | 0.93±0.034a | 2.25±0.08b | | Group III | 4.28±0.22a | 0.95±0.06a | 2.33±0.06b | | Group IV | 4.71±0.27ab | 0.94±0.055a | 2.40±0.12b | | Group V | 5.69±0.25b | 0.98±0.021a | 1.83±0.15a | | Group VI | 5.67±0.12b | 0.98±0.019a | 2.19±0.02ab |
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| * The values (Mean ± S.E.) in a column having different superscript differ significantly from each other at 5% level of significance. * The values (Mean ± S.E.) in a column having same superscript do not differ significantly from each other at 5% level of significance. | | | Table 4.: Histopathological score based on severity of lesions in different organs in different groups.
| | Organ | Histopathological lesions | Group 1 | Group 2 | Group 3 | Group 4 | Group 5 | Group 6 | | Kidney | PCT degeneration | -- | 4m,11M | 11m,4M | -- | 12S,3M | 9S,6M | | Increased Peri glomerular space | -- | 5m,10M | 11m,4M | -- | 10S,5M | 7S,6M,2m | | Protein Cast | -- | 8m,7M | 13m,2M | -- | 9S,6M | 12M,3m | | DCT degeneration | -- | 6m,9M | 12m,3M | -- | 11S,4M | 7S,8M | | Blood vessel congestion | -- | 7m,8M | 13m,2M | -- | 8S,7M | 10M,5m | | Total | -- | 120 | 90 | -- | 200 | 163 | | Liver | Hepatocyte degeneration | -- | 5m,10M | 10m,5M | | 12S,3M | 10S,3M,2m | | Fatty change | -- | 4m,11M | 12m,3M | | 13S,2M | 11S,4m | | Swollen sinusoids | -- | 7m,8M | 11m,4M | | 9S,6M | 8S,4M,3m | | Congested sinusoidal space | -- | 8m,7M | 13m,2M | | 8S,7M | 7S,5M,3m | | Total | -- | 96 | 74 | | 162 | 144 | | Brain | Oedema | -- | 6m,9M | 11m,4M | -- | 10S,5M | 7S,6M,2m | | Neuronal degeneration | -- | 2S,10M,3m | 10m,5M | -- | 12S,3M | 9S,6M | | Purkinje cell degeneration | -- | 12M,3m | 11m,4M | -- | 14S,1M | 11S,4M | | Total | -- | 80 | 58 | -- | 126 | 115 | | Spleen | Red pulp congestion | -- | 10M,5m | 12m,3M | -- | 12S,3M | 9S,6M | | Lymphoid depletion | -- | 12M,3m | 10m,5M | -- | 13S,2M | 10S,3M,2m | | Total | -- | 52 | 38 | -- | 85 | 67 | | Testis | Decrease in spermatogenic activity | -- | 11M,4m | 12m,3M | -- | 14S,1M | 10S,5M | | Number of spermatogonial cells | -- | 10M,5m | 11m,4M | -- | 13S,2M | 10S,4M,1m | | Increased intertubularspaces | -- | 12M,3m | 12m,3M | -- | 10S,5M | 8S,6M,1m | | Total | -- | 78 | 55 | -- | 127 | 116 | | Intestine | Goblet cell hyperplasia | -- | 8M,7m | 4M,11m | -- | 8S,7M | 5S,11M | | Depletion in payer patches | -- | 10M,5m | 3M,12m | -- | 9S,6M | 6S,9M | | Total | -- | 48 | 37 | -- | 77 | 73 | | Stomach | Ulcer | -- | -- | -- | -- | 2M | -- | | Total | -- | -- | -- | -- | 4 | -- | | Eye | Keratitis | -- | -- | -- | -- | 2m | -- | | Total | -- | -- | -- | -- | 2 | -- | | Heart | Zenker necrosis | -- | 6M,9m | -- | -- | 11S,4M | 4S,8M,3m | | Congestion | -- | 4M,11m | -- | -- | 7S,8M | 10M,5m | | Total | -- | 40 | -- | -- | 78 | 56 | | Pancreas | Degeneration of acini | -- | 7 M, 8m | 12m,3M | -- | 10S,5M | 5S,8M2m | | Total | -- | 22 | 18 | -- | 40 | 28 |
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| S - Severe (score = 3); M - Moderate (score = 2), m - mild (score = 1) | |
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