Secondary Logo

Journal Logo

Original articles

Biochemical and ultrastructural studies on the protective effect of ginger extract against cisplatin-induced hepatotoxicity in adult male albino rats

Ali, Doaa A.

Author Information
The Egyptian Journal of Histology: June 2011 - Volume 34 - Issue 2 - p 231-238
doi: 10.1097/01.EHX.0000396639.02881.79
  • Free

Abstract

Introduction

Cisplatin [cis-(NH3)2PtCl2] is one of the most effective antineoplastic drugs, and it is used extensively for the treatment of a variety of solid tumors [1]. However, its clinical utility is limited due to some adverse side effects. Recent studies around the world suggested that hepatotoxicity is a major dose-limiting side effect in cisplatin-based chemotherapy [2–6].

Recently, attention has been drawn to natural products and their active principles as sources for new drug. It is well established that herbs and spices are used safely and effectively against various human ailments [7]. Ginger (Zingiber officinale Rosc.), belonging to Family Zingiberaceae, has been cultivated for thousands of years as a spice and for medicinal purposes [8]. The underground stem or rhizome of this plant has been used as a medicine in Asian, Indian, and Arabic herbal traditions since ancient times [9]. Phytochemical studies showed that ginger is rich in a large number of polyphenolic constituents, including gingerols, paradol, shogaols, and zingerone [10,11]. These compounds display diverse biological activities such as antioxidant [10], anti-inflammatory [12,13], and anticarcinogenic properties [14].

The aim of this study was to investigate the hepatoprotective effect of ginger on liver biochemical and ultrastructural changes induced by cisplatin chemotherapy.

Materials and methods

Chemicals

The rhizomes of Z. officinale were brought from Metro market in El-Gomhoria Street, Mansoura, Egypt. They were shade dried at room temperature and were crushed to powder. One hundred twenty-five grams of the powder was macerated in 200 ml of distilled water for 12 h at room temperature and filtered to obtain the final aqueous extract (120 mg/ml) as previously described [15]. Cisplatin was purchased from Merck (Rodleben, Germany); it was provided as 50 mg/50 ml saline.

Experimental animals

Healthy adult male albino rats (Rattus rattus; 120±5 g) were housed and maintained on 12-h light/dark cycle under a constant temperature of 25±1°C with free access to food and drinking water. Animals were acclimatized to laboratory conditions for 1 week before the experiments. They were randomly divided into four groups (n=6 per group) as follows: (i) group 1 (control group): the animals were injected with 0.9 normal saline intraperitoneally for 3 consecutive days (vehicle); (ii) group 2 (ginger-treated group): the animals were orally administered with 1 ml of ginger extract (120 mg/kg) every other day for 4 weeks; (iii) group 3 (cisplatin-treated group): the animals were injected intraperitoneally with cisplatin dissolved in 0.9 normal saline (3.3 mg/kg bwt /day; LD10) for 3 consecutive days as previously described [16]. They reported that this dose scheduling was less toxic than a single injection in terms of loss of body weight and drug-related deaths; and (iv) group 4 (cisplatin+ginger-treated group): in addition to Cisplatin treatment (similar to group 3), these animals were orally administered with 1 ml of ginger extract (120 mg/ml) every other day for 4 weeks. At the end of the treatment, animals were killed and blood samples and liver tissues were collected.

Biochemical determinations

Serum samples were separated for the measurement of indices of hepatotoxicity. Serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels were estimated as previously reported [17] using a BIOMRIEUX kit (bioMe´rieux, Marcy l'Etoile, France). In addition, albumin was determined using the human colorimetric kit method [18].

Electron microscopy investigation

The liver tissue was fixed for 2 h in 2.5% glutaraldehyde buffered in 0.1 mol/l cacodylate buffer (pH 7.2) at 4°C and then post fixed in 1% cold osmium tetraoxide in 0.1 mol/l cacodylate at pH 7.2 for 3 h. Ultrathin sections were obtained from specimens embedded in a Lowicryl K4M resin after dehydration through graded ethanol series, substitution, and polymerization at −20°C. Ultrathin sections were obtained using an Ultracut S microtome (Leica, Vienna, Austria). Sections were mounted on 400-mesh cellodion-carbon-coated nickel grids and examined with a Joel Electron Microscope (Jeol Ltd., Tokyo, Japan ) operating at 60 kV.

Statistical analysis

Data were presented as mean±standard error of the mean of at least triplicates or replicates from three experiments and the data were analyzed statistically using Student's t-test using SPSS software (SPSS, version 15.0, Chicago, IL, USA).

Results

Biochemical investigations

In this study, cisplatin injection resulted in a significant increase in serum levels of AST and ALT and a significant decrease in serum albumin levels compared with the control group. However, ginger was able to prevent liver function alteration as demonstrated by significant decreases in serum levels of AST and ALT in the combination group compared with the cisplatin group (Table 1).

Table 1
Table 1:
Effect of cisplatin, ginger, and their combination on serum AST, ALT, and albumin in male albino rats

Electron microscopy examination

In this study, the hepatic cells from the control group show normal large rounded nucleus with electron-lucent euchromatin and scattered areas of heterochromatin. The cytoplasm contains a profuse amount of rough endoplasmic reticulum (RER) wrapping the rounded mitochondria. The hepatic sinusoids are extremely thin walled with only one discontinuous layer of endothelial lining cells. Microvilli of the hepatic cells project into the lumen of the bile canaliculi and into the space of Disse (Figs 1–3).

Figure 1
Figure 1:
Electron micrograph of hepatocyte from control rat showing round nucleus (N), numerous mitochondria (M), and rough endoplasmic reticulum (RER).Figure 1. ×7.500.
Figure 2
Figure 2:
Electron micrograph of adjacent hepatic cell borders from control rat showing the bile canaliculus (BC) and microvilli (MV). Note the presence of mitochondria (M) and rough endoplasmic reticulum (RER) in the cytoplasm of the hepatocytes.Figure 2. ×15.000.
Figure 3
Figure 3:
Electron micrograph of blood sinusoid from control rat illustrating the space of Disse (DI), numerous microvilli (MV), endothelial cells (EN), and red blood cells. Note the presence of mitochondria (M) and rough endoplasmic reticulum (RER) in the adjacent hepatocytes.Figure 3. ×10.000.

The hepatic cells, bile canaliculi, and blood sinusoids of the ginger-treated animals are quite normal as those described of the control group (Figs 4–6).

Figure 4
Figure 4:
Electron micrograph of hepatocyte from ginger-treated rats showing part of the nucleus (N), mitochondria (M), and rough endoplasmic reticulum (RER).Figure 4. ×7.500.
Figure 5
Figure 5:
Electron micrograph of bile canaliculus (BC) from ginger-treated rat.Figure 5. ×15.000.
Figure 6
Figure 6:
Electron micrograph of blood sinusoid from ginger-treated rats showing a wide space of Disse (DI), endothelial cells (EN), and microvilli (MV).Figure 6. ×13.000.

However, in cisplatin-treated rats, the liver tissue showed signs of the hepatotoxicity. The nuclei were round-to-ovoid with marginated heterochromatin. The cytoplasm was found to contain extensive profiles of dilated and vesiculated RER, especially around the nuclear envelope and between the mitochondria as shown in (Figs 7–9). In addition, the mitochondria showed different pathological forms. Some mitochondria fused with each other forming megamitochondria (Figs 8–10), whereas others showed high-amplitude starting with swelling of the mitochondria and ended with myelin figure formation (Fig. 11). In addition, the bile canaliculi were more or less similar to those of the control counterpart (Fig. 12), whereas blood sinusoids were wide and discontinuous with absence of endothelial cells and detachment of Kupffer cells (Fig. 10).

Figure 7
Figure 7:
Electron micrograph of hepatocyte from cisplatin-treated rat showing large rounded nucleus (N), extensive profiles of dilated and vesiculated rough endoplasmic reticulum (RER), and mitochondria (M).Figure 7. ×7.500.
Figure 8
Figure 8:
Electron micrograph of hepatic cells from cisplatin-treated rat showing oval nucleus (N), dilated rough endoplasmic reticulum (RER), and aggregated mitochondria (M) around the fat droplets (FD).Figure 8. ×5.000.
Figure 11
Figure 11:
Electron micrograph of hepatocyte from cisplatin-treated rat showing large myelin figure (MF), high amplitude mitochondria (M), secondary lysosome (LY), and part of the nucleus (N).Figure 11. ×10.000.
Figure 9
Figure 9:
Electron micrograph of hepatocyte from cisplatin-treated rat showing megamitochondria (MM) wrapped by the dilated and vesiculated rough endoplasmic reticulum (RER) with detached ribosomes.Figure 9. ×20.000.
Figure 12
Figure 12:
Electron micrograph of adjacent hepatic cell borders from cisplatin-treated rat showing bile canaliculus (BC), microvilli (MV), and zonula occludents (ZO).Figure 12. ×13.000.
Figure 10
Figure 10:
Electron micrograph of very wide blood sinusoid from cisplatin-treated rat showing absence of endothelial cells (arrow), detachment of Kupffer cell (KC), and very narrow space of Disse (DI). Note the fusion of mitochondria forming megamitochondria (MM) in the adjacent hepatocytes.Figure 10. ×7.500.

In this study, cisplatin-treated rats in combination with ginger extract exhibit remarkable improvements. The liver tissue ultrastructure showed oval nuclei with normal distribution of heterchromatin (Fig. 13). Large number of mitochondria, RER, and disappearance of the fat droplets were observed in cytoplasm (Figs 13 and 14). In addition, the bile canaliculi showed the normal construction of those of control group (Fig. 14). Moreover, the hepatic sinusoid seemed to be normal with respect to that of the control group (Fig. 15). However, on other foci, many hepatocytes underwent apoptosis in which the cytosol was digested by the lysosomal enzymes, which increased in these cells, and the cell organoids were not clear. The RER and smooth ER were disintegrated and the mitochondria were mostly rounded with electron-lucent matrix (Fig. 16).

Figure 13
Figure 13:
Electron micrograph of hepatocyte from cisplatin and ginger-treated rat showing oval nucleus (N), large number of mitochondria (M), and rough endoplasmic reticulum (RER).Figure 13. ×7.500.
Figure 14
Figure 14
Figure 16
Figure 16:
Electron micrograph of hepatocyte from cisplatin and ginger-treated rat showing the dissolution of the cytoplasm.Figure 16. ×10.000.
Figure 15
Figure 15:
Electron micrograph of blood sinusoid from cisplatin and ginger-treated rat showing space of Disse (DI), endothelial cells (EN), and Kupffer cells (KC).Figure 15. ×7.500.

Discussion

In this study, the biochemical and ultrastructural investigations revealed that ginger did not cause any side effects or organ toxicities. This is in good agreement with findings of previous investigators [19–23].

However, in cisplatin-treated rats, significant elevation in serum levels of AST and ALT were obtained compared with the control group. In addition, a significant decrease in serum albumin level was observed with respect to the control group. Thus, these data indicate that cisplatin impairs the liver function and confirm those previously reported [24–28]. The alterations in the activity of these enzymes could be a secondary event following cisplatin-induced liver damage, with the consequent leakage from hepatocytes.

Furthermore in this study, the ultrastructure of the hepatocyte cytoplasm of the cisplatin-treated group showed dilated and vesiculated RER. Such findings are parallel to previous results reporting that damage to the RER is the earliest microscopic evidence of cytotoxicity [29]. In addition, the dilatation and vesiculation of the RER due to ingress of water and solutes into the cell is part of cloudy swelling, a ubiquitous change observed in cells subjected to various noxious influences [30]. RER damage is one of the essential factors responsible for the lowered activity of liver cells in protein synthesis as this cell organelle plays a major role in this function. Hence, the significant decrease in serum albumin level could be attributed to damage of the RER.

In addition, the mitochondria showed different pathological forms. Such results are confirmed through previous studies [31,32], which found that the mitochondrion is the primary target for cisplatin-induced oxidative stress, resulting in loss of mitochondrial protein-SH, inhibition of calcium uptake, and reduction in the mitochondrial membrane potential. Moreover, cisplatin has been described as one of the most active cytotoxic agents used in the treatment of cancer and induces mitochondrial dysfunctions, particularly the inhibition of the electron transfer system, thereby resulting in enhanced reactive oxygen species production and subsequent tissue damage [33–36].

Moreover, blood sinusoids were wide and discontinuous with the absence of endothelial cells and detachment of Kupffer cells. This confirm the finding that liver tissue displays cytoplasmic changes, especially around cells of central vein and hepatocellular vacualization and sinusoidal dilatations [4]. Furthermore, a single dose of cisplatin (2.5 mg/kg) induced severe hepatic damage characterized by severe activation of Kupffer cells, degenerated hepatocytes, and moderate enlargement of sinusoids [28].

The mechanism underlying the hepatotoxicity induced by cisplatin may be attributed to the combination of multiple ways, such as the generation of reactive species derived from oxygen and nitrogen, which could interfere with the antioxidant defense system, resulting in oxidative damage in different tissues as previously described [4,6,27,37] and the reaction with thiols in protein and glutathione resulting in a reduction in the level of antioxidant enzyme, which could cause cell dysfunction. In contrast, it has been proposed that the antitumor activity of cisplatin is due to its ability to form adducts with DNA by its positively charged and highly reactive hydrated electrophilic product, which causes cross-linking of DNA strands [38].

Ginger is one of the widely used spices and has been used in traditional oriental medicines [39]. In this study, cisplatin-treated rats, in combination with ginger extract, exhibit significant decreases in serum levels of AST and ALT and increase the serum level of albumin, indicating that ginger effectively improved liver function impairments induced by cisplatin. In addition, the protection with ginger inhibited most of the hepatopathological alterations induced by cisplatin chemotherapy. The potential hepatoprotective role of ginger may be associated with antioxidant constituents such as 6-gingerol, 6-shogoal, 6-paradol, zingerone, and some related phenolic ketone derivatives working individually or in synergy [7,40,41]. Moreover, zingerone, a compound isolated from ginger, has been shown to inhibit nitro blue tetrazolium reduction in a xanthine–xanthine oxidase system, providing the evidence that it scavenges superoxide anions [41].

In this study, the findings regarding the ultrastructural apoptotic changes observed in the liver of rats treated with cisplatin in combination with ginger are in good agreement with the previous results [42–44], which revealed that 6-paradol and 6-gingerol induce apoptosis. In addition, confirmation of these observations comes from the study reporting that treatment with cisplatin in combination with ginger resulted in a significant increase in the expression of the Bax proapoptotic protein in the liver tissue, which reflects the induction of apoptosis [45]. It is now well recognized that removal of damaged precancerous cells through apoptosis provides an important strategy for the treatment of cancer [46,47].

Conclusion

In conclusion, the results obtained provide in-vivo evidence, at biochemical and ultrastructural levels, of the chemoprotective effects of ginger against the hepatotoxicity induced by cisplatin chemotherapy, suggesting its potential use in chemoprevention of cancer in combination with chemotherapy.

Table
Table:
No title available.

References

1. Rosenberg B. Fundamental studies with cisplatin. Cancer. 1985;55:2303–2316
2. Kim SH, Hong KO, Chung WY, Hwang JK, Park KK. Abrogation of cisplatin-induced hepatotoxicity in mice by xanthorrhizol is related to its effect on the regulation of gene transcription. Toxicol Appl Pharmacol. 2004;196:346–355
3. Liao YJ, Tang H, Jin YP. Study of toxic effects on hearing, kidney and liver of mice induced by anticancer agent of cisplatin and their mechanisms. Chin Pharm Bull. 2004;20:82–85
4. Koc A, Duru M, Ciralik H, Akcan R, Sogut S. Protective agent, erdosteine, against cisplatin-induced hepatic oxidant injury in rats. Mol Cell Biochem. 2005;278:79–84
5. Lu Y, Cederbaum AI. Cisplatin-induced hepatotoxicity is enhanced by elevated expression of cytochrome P450 2E1. Toxicol Sci. 2006;89:515–523
6. Pratibha R, Sameer R, Rataboli PV, Bhiwgade DA, Dhume CY. Enzymatic studies of cisplatin induced oxidative stress in hepatic tissue of rats. Eur J Pharmacol. 2006;532:290–293
7. Ajith TA, Hema U, Aswathy MS. Zingiber officinale Roscoe prevents acetaminophen-induced acute hepatotoxicity by enhancing hepatic antioxidant status. Food Chem Toxicol. 2007;45:2267–2272
8. Park EJ, Pezzuto JM. Botanicals in cancer chemoprevention. Cancer Metastasis Rev. 2002;21:231–255
9. Altman RD, Marcussen KC. Effects of a ginger extract on knee pain in patients with osteoarthritis. Arthritis Rheum. 2001;44:2531–2538
10. Masuda Y, Kikuzaki H, Hisamoto M, Nakatani N. Antioxidant properties of gingerol related compounds from ginger. Biofactors. 2004;21:293–296
11. Jolad SD, Lantz RC, Chen GJ, Bates RB, Timmermann BN. Commercially processed dry ginger (Zingiber officinale): composition and effects on LPS-stimulated PGE2 production. Phytochemistry. 2005;66:1614–1635
12. Young HY, Luo YL, Cheng HY, Hsieh WC, Liao JC, Peng WH. Analgesic and anti-inflammatory activities of [6]-gingerol. J.Ethnopharmacol. 2005;96:207–210
13. Lantz RC, Chen GJ, Sarihan M, Sólyom AM, Jolad SD, Timmermann BN. The effect of extracts from ginger rhizome on inflammatory mediator production. Phytomedicine. 2007;14:123–128
14. Shukla Y, Singh M. Cancer preventive properties of ginger: a brief review. Food Chem Toxicol. 2007;45:683–690
15. Kamtchouing P, Mbongue Fandio GY, Dimo T, Jatsa HB. Evaluation of androgenic activity of Zingiber officinale and Pentadiplandra brazzeana in male rats. Asian J Androl. 2002;4:299–301
16. Pace A, Savarese A, Picardo M, Maresca V, Pacetti U, Del Monte G, et al. Neuroprotective effect of vitamin E supplementation in patients treated with cisplatin chemotherapy. J Clin Oncol. 2003;21:927–931
17. Reitman S, Frankel S. A colorimetric method for the determination of serum glutamic oxalacetic and glutamic pyruvic transaminases. Am J Clin Pathol. 1957;28:56–63
18. Doumas BT, Watson WA, Biggs HG. Albumin standards and the measurement of serum albumin with bromcresol green. Clin Chim Acta. 1971;31:87–96
19. Sakamura F, Hayashi S. Constitution of the essential oil from rhizomes of Zingiber officinale Roscoe. Nihon Nogei Kogakkaishi. 1978;52:207–211
20. Smith RM, Robinson JM. The essential oil of ginger from Fiji. Phytochemistry. 1981;20:203–206
21. Nishimura O. Identification of the characteristic odorants in fresh rhizomes of ginger (Zingiber officinale Roscoe) using aroma extract dilution analysis and modified multidimensional gas chromatography-mass spectroscopy. J Agric Food Chem. 1995;43:2941–2945
22. Langner E, Greifenberg S, Gruenwald J. Ginger: history and use. Adv Ther. 1998;15:25–44
23. Bartley JP, Jacobs AL. Effects of drying on flavour compounds in Australian-grown ginger (Zingiber officinale). J Sci Food Agric. 2000;80:209–215
24. Dubskaia T, Vetoshkina TV, Gol'dberg VE. The mechanisms of the hepatotoxicity of complex platinum compounds. Eksp.Klin.Farmakol. 1994;57:38–41
25. Saad SY, Najjar TA, Daba MH, Al Rikabi AC. Inhibition of nitric oxide synthase aggravates cisplatin-induced nephrotoxicity: effect of 2-amino-4-methylpyridine. Chemotherapy. 2002;48:309–315
26. Kadikoylu G, Bolaman Z, Demir S, Balkaya M, Akalin N, Enli Y. The effects of desferrioxamine on cisplatin-induced lipid peroxidation and the activities of antioxidant enzymes in rat kidneys. Hum Exp Toxicol. 2004;23:29–34
27. Mansour HH, Hafez HF, Fahmy NM. Silymarin modulates cisplatin-induced oxidative stress and hepatotoxicity in rats. J Biochem Mol Biol. 2006;39:656–661
28. Işeri S, Ercan F, Gedik N, Yüksel M, Alican I. Simvastatin attenuates cisplatin-induced kidney and liver damage in rats. Toxicology. 2007;230:256–264
29. Rouiller C, Jezequel AMRouiller C. Electron microscopy of the liver. The liver. 2006 New York Academic Press:195–264
30. Ghadially FNKillam IW, Lindsay WS. Ultrastructural pathology of cell and matrix. A text and atlas of physiological and pathological alterations in the fine structure of cellular and extracellular components. 19974th ed London Butterworth-Heinemann:1184–1191
31. Li Ping X, Skrezek C, Wand H, Reibe F. Mitochondrial dysfunction at the early stage of cisplatin-induced acute renal failure in rats. J Zhejiang Univ Sci. 2000;1:91–96
32. Saad SY, Najjar TAO, Alashari M. Role of non-selective adenosine receptor blockade and phosphodiesterase inhibition in cisplatin-induced nephrogonadal toxicity in rats. Clin Exp Pharmacol Physiol. 2004;31:862–867
33. Baliga R, Ueda N, Walker PD, Shah SV. Oxidant mechanisms in toxic acute renal failure. Drug Metab Rev. 1999;31:971–997
34. Baek SM, Kwon CH, Kim JH, Woo JS, Jung JS, Kim YK. Differential roles of hydrogen peroxide and hydroxyl radical in cisplatin-induced cell death in renal proximal tubular epithelial cells. J Lab Clin Med. 2003;142:178–186
35. Fariss MW, Chan CB, Patel M, Van Houten B, Orrenius S. Role of mitochondria in toxic oxidative stress. Mol Interv. 2005;5:94–111
36. Chang KL, Seung HS, Kwang KP, Park JHY, Soon SL, Won YC. Isoliquiritigenin inhibits tumor growth and protects the kidney and liver against chemotherapy-induced toxicity in a mouse xenograft model of colon carcinoma. J Pharmacol Sci. 2008;106:444–451
37. Iraz M, Ozerol E, Gulec M, Tasdemir S, Idiz N, Fadillioglu E, et al. Protective effect of caffeic acid phenethyl ester (CAPE) administration on cisplatin-induced oxidative damage to liver in rat. Cell Biochem Funct. 2006;24:357–361
38. Hanigan MH, Devarajan P. Cisplatin nephrotoxicity: molecular mechanisms. Cancer Ther. 2003;1:47–61
39. Peng P. Pharmacological study of Zingiber officinale (willd). Rosc and its clinical use. Zhongguo Zhong Yao Za Zhi. 1992;17:370–373
40. Masuda T. Antioxidant activity of tropical ginger extracts and analysis of the contained curcuminoids. J Agric Food Chem. 1992;40:1337–1340
41. Afshari AT, Shirpoor A, Farshid A, Saadatian R, Rasmi Y, Saboory E, et al. The effect of ginger on diabetic nephropathy, plasma antioxidant capacity and lipid peroxidation in rats. Food Chem. 2006;101:148–153
42. Lee E, Surh YJ. Induction of apoptosis in HL-60 cells by pungent vanilloids, [6]-gingerol and [6]-paradol. Cancer Lett. 1998;134:163–168
43. Keum YS, Kim J, Lee KH, Park KK, Surh YJ, Lee JM, et al. Induction of apoptosis and caspase-3 activation by chemopreventive [6]-paradol and structurally related compounds in KB cells. Cancer Lett. 2002;177:41–47
44. Wang CC, Chen LG, Lee LT, Yang LL. Effects of 6-gingerol, an antioxidant from ginger, on inducing apoptosis in human leukemic HL-60 cells. In Vivo. 2003;17:641–645
45. Ali DA. Histopathological and immunohistochemical studies on the chemopreventive effects of ginger extract against the hepatotoxicity induced by cisplatin chemotherapy in male rats. Egypt J Zool. 2010;54:33–46
46. Fisher DE. Apoptosis in cancer therapy: crossing the threshold. Cell. 1994;78:539–542
47. Lotan R. Retinoids and apoptosis: implications for cancer chemoprevention and therapy. J.Natl Cancer Inst. 1995;87:1655–1657
Keywords:

albumin; cisplatin; electron microscopy; ginger; hepatotoxicity; liver enzymes

© 2011 The Egyptian Journal of Histology