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 . 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 . Ginger (Zingiber officinale Rosc.), belonging to Family Zingiberaceae, has been cultivated for thousands of years as a spice and for medicinal purposes . The underground stem or rhizome of this plant has been used as a medicine in Asian, Indian, and Arabic herbal traditions since ancient times . 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 , anti-inflammatory [12,13], and anticarcinogenic properties .
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
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 . Cisplatin was purchased from Merck (Rodleben, Germany); it was provided as 50 mg/50 ml saline.
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 . 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.
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  using a BIOMRIEUX kit (bioMe´rieux, Marcy l'Etoile, France). In addition, albumin was determined using the human colorimetric kit method .
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.
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).
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).
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).
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).
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).
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).
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 . 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 . 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 . 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 .
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 .
Ginger is one of the widely used spices and has been used in traditional oriental medicines . 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 .
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 . It is now well recognized that removal of damaged precancerous cells through apoptosis provides an important strategy for the treatment of cancer [46,47].
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.
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