Currently, the underlying mechanism of oxaliplatin (OXA) induced live injury is unclear. In addition, there is no standard clinical treatment for OXA-induced acute liver injury (ALI). In this study, we established an animal model of OXA-induced ALI, and studied the role of oxidative stress in OXA-induced ALI and the impacts of reduced glutathione (GSH) treatment on OXA-induced ALI. To establish an OXA-induced ALI model, KM mice received intraperitoneal injection of OXA (8 mg/kg) for 4 days. Serum alanine aminotransferase (ALT), aspartate aminotransferase levels (AST), hepatic pathology and oxidative stress indicators in liver tissues were analyzed. To study the impact of GSH treatment on OXA-induced ALI, mice were treated with GSH (400 mg/kg, i.p). In this ALI mouse model, ALT and AST levels were significantly increased (P<0.01). Liver pathological examination revealed varying degrees of liver cell turbidity and degeneration, even balloon-like changes and focal necrosis, and sinusoidal hemorrhage in some cells. Compared with control group, the malondialdehyde (MDA) and GSH levels were significantly increased in OXA-treated group (P<0.01), while the superoxide dismutase SOD and GSH-peroxidase levels were decreased after OXA withdrawal (P<0.01). When GSH was used to treat OXA-induced ALI mice, the pathological injury of liver tissues was alleviated, and serum ALT and AST were significantly decreased. In addition, GSH treatment could reduce the OXA-induced increase of MDA level (P<0.05) in liver tissues, but had no impact on SOD level (P>0.05). We have successfully established an OXA-induced ALI model. Using this model, we discover that oxidative stress plays an important role in OXA-induced ALI. GSH-based hepatoprotective therapy can partially inhibit oxidative stress and alleviate OXA-induced ALI.
Oxaliplatin (OXA) is a third-generation platinum compound and OXA-based chemotherapy is a widely used treatment for solid organ malignancies. The combination of OXA with other chemotherapy agents, including 5-fluorouracil/folic acid (FOLFOX) and capecitabine, is a first-line therapy for colorectal cancer (
Currently, the underlying mechanism of OXA-induced liver toxicity is unclear. One hypothesis is that OXA-induced liver damage may be associated with oxidative stress (
However, prior clinical and animal studies have focused on studying chronic liver injuries caused by long-term use (4–8 weeks) of OXA-based chemotherapy. Currently, few studies are performed using animal models of OXA-induced acute liver injury (ALI). In addition, there are limited reports available regarding the pathological changes in patients with ALI receiving OXA-based chemotherapy. Due to ethical issues and unwillingness of patients to receive a liver needle biopsy, it is difficult to perform clinical studies on OXA-induced ALI.
At present, there is no standard clinical treatment for OXA-induced ALI. Clinicians can only use experience to select one or a combination of various hepatoprotective drugs, one of which is reduced glutathione (GSH). GSH is a bioactive peptide and important non-enzymatic antioxidant widely present in living organisms (
All animal studies were performed according to the guidelines of the Chinese Council on Animal Care and were approved by the Affiliated Tumor Hospital of Guangxi Medical University Committees on Animal Experimentation (Nanning, China).
OXA for injection (no. 13092615; Jiangsu Hengrui Medicine Co., Ltd.); alanine aminotransferase (ALT) kit (no. 2014007; Changchu Huli Biotech Co., Ltd.); aspartate aminotransferase (AST) kit, GSH kit, SOD kit, glutathione peroxidase (GSH-px) kit, malondialdehyde (MDA) kit and total protein quantification kit (BCA method) (all 6 kits are no. 20140402; Jiangcheng Bioengineering Institute (Nanjing, China).
Twenty male KM mice (aged 8–10 weeks and weighing 26–28 g) were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. (Beijing, China). All mice were housed under standardized conditions with one cage for every 5 mice,
Liver tissues were fixed in 4% paraformaldehyde, and then embedded in paraffin. After sectioning, the liver specimens were stained with H&E. As observed via optical microscopy, the pathological changes associated with liver injury included liver cell turbidity and degeneration, balloon-like changes and necrosis. According to the coverage of abnormal liver cells, liver injuries were graded as follows: Level 0, normal, no liver cell degeneration; level 1, mild, the ratio of hepatic lobule lesion <1/3; level 2, moderate, the ratio of hepatic lobule lesion was between 1/3 and 2/3; level 3, serious, the ratio of hepatic lobule lesion >2/3 (+++).
Blood samples from the mice were centrifuged at 300 × g for 8 min at 37°C, and the supernatants were measured using an alanine aminotransferase (ALT) kit (HuiLi Biotech Co., Ltd., Changchun, China) and an aspartate aminotransferase (AST) kit (Jiancheng Bioengineering Institute, Nanjing, China), according to the manufacturer's protocols. The results are represented as units/l.
Proteins were extracted from whole liver tissues in RIPA buffer and quantified using a Bradford assay (Nanjing Jiangcheng Bioengineering Institute). The GSH, GSH-Px, SOD and MDA content of liver tissues were detected using the kits obtained from the Nanjing Jiangcheng Bioengineering Institute, according to the protocols provided by the manufacturer.
All statistical analyses were performed using SPSS version 10 (SPSS, Inc., Chicago, IL, USA). All experiments were performed using 3–5 mice per experimental group and repeated at least three times to assess reproducibility. Differences were analyzed using Student's t-test or one-way analysis of variance, followed by Tukey's post hoc test. Cumulative survival time was calculated using the Kaplan-Meier method and was analyzed by the log-rank test. Data are presented as the mean ± standard deviation. P<0.05 was considered to indicate a statistically significant difference.
To establish a mouse model of OXA-induced ALI, KM mice were treated with OXA (i.p.) for 4 days. Following 2 days of OXA treatment, mice exhibited a reduced appetite and mild diarrhea, which were aggravated with an increase in OXA treatment. A number of mice experienced severe diarrhea, and ultimately died. No abnormal pathological changes were observed in the control mice (
To evaluate OXA-induced liver toxicity in the mouse model, changes in the serum AST and ALT levels were detected. Compared with the control mice, OXA-treated mice showed significantly elevated serum ALT and AST levels (P<0.05) after 2 days and 1 day of OXA treatment, respectively. With the increase in the number of OXA treatments, these elevations were enhanced, and the high serum AST and ALT levels persisted for 4 days following OXA withdrawal (
Evidence from various patient studies suggests that liver injuries induced by OXA-based chemotherapy, including FOLFOX-induced SOS, are associated with increased oxidative stress in the liver (
To examine whether GSH therapy has a protective effect on OXA-induced ALI, OXA-treated mice received GSH treatment 30 min prior to each OXA injection for 4 days. Optical microscopy and H&E staining indicated clear liver cell injury in OXA-treated mice, including liver cell swelling and degeneration (mainly moderate and severe), balloon-like changes and focal necrosis (
The antioxidative effect of GSH on liver injury was investigated. As presented in
Chemotherapy-associated liver injury can include steatosis, liver cell necrosis, severe steatohepatitis and SOS. Distinct types of liver injuries may be associated with specific chemotherapy drugs (
A mouse model of OXA-induced ALI was successfully established in the current study. In this model, elevated ALT and AST levels characterized OXA-induced ALI during the early stage of OXA treatment. Hepatic histopathology of the OXA-induced ALI demonstrated varying degrees of liver cell turbidity and degeneration, even balloon like changes and focal necrosis, and sinusoidal hemorrhage in certain individuals. These hepatic pathological changes in OXA-induced ALI were different from the pathology of chronic liver injuries induced by multi-cycle OXA-based chemotherapy reported in clinical observation and animal studies, in which the primary characteristics of liver injury are liver sinusoidal injury and SOS (
Recently, it has been suggested that oxidative stress is an important contributing factor to hepatotoxicity induced by long-term OXA chemotherapy (
Under physiological conditions, the liver can resist oxidative stress through GSH synthesis in hepatocytes. In the present study, mice treated with OXA and GSH exhibited high GSH-Px levels and low MDA levels, which indicated a reduction of oxidative stress and is accompanied by decreased tissue injury, ALT and AST levels. GSH can directly scavenge radicals and peroxides via mixed disulfide formation or oxidization to generate oxidized glutathione (
In the present study, MAD levels in the GSH treatment group remained higher than in the control group, and no significant impact on SOD level downregulation was observed following GSH treatment. Therefore, although GSH treatment exerted a significant protective effect against OXA-induced liver injury in the present study, hepatic oxidative stress continues to occur. In addition, the ALT and AST levels in OXA and GSH-treated mice did not recover to within the normal range, indicating that GSH alone is insufficient for suppressing oxidative stress during OXA-induced ALI. Perhaps combining GSH with other drugs, such as antioxidants, may further alleviate OXA-induced liver injury. Indeed, various endogenous of dietary antioxidants are capable of ameliorating steatohepatitis and OXA-induced neurotoxicity via reducing oxidative stress. Besides oxidative stress, prior studies determined that other mechanisms are also involved in OXA-induced liver injury. These mechanisms include the activation of inflammation-associated pathways (
As observed in the present study, GSH treatment alone cannot reduce OXA-induced mortality. Histopathological examination detected no liver failure, and the cause of mortality was determined to be severe diarrhea. Compared with the OXA-treated mice, OXA and GSH-treated mice exhibited no significant difference in body weight loss, appetite reduction and diarrhea (data not presented), indicating that GSH treatment has no significant ameliorative effect on OXA-induced liver injury. Therefore, during treatment of the liver injury caused by OXA chemotherapy, other OXA-induced toxicities, including neurotoxicity, gastrointestinal toxicity and hematological toxicity, must also be considered.
In summary, an animal model of OXA-induced ALI was successfully established. The results suggest that oxidative stress serves an important role in the pathogenesis of OXA-induced ALI, and that GSH treatment can attenuate OXA-induced ALI by suppressing oxidative stress in the liver.
The present study was partially supported by the Guangxi Natural Science Foundation (grant no. 2016GXNSFBA380218), the Guangxi Key Laboratory of Molecular Medicine in Liver Injury and Repair (grant no. 16-140-46-18), the Guangxi Basic Ability Promotion Project of Middle-aged and Young Teachers in Colleges and Universities (grant no. 2017KY0121), the Youth Science Foundation of Guangxi Medical University (grant no. GXMUYSF201336), the Self-Raised Funds of Guangxi Health Department (grant no. Z2016438 and grant no. Z2013423), The Medication and Health Care Research Program of Guangxi (grant no. S201418-03) and the Key Planning Development Research Program of Guangxi (grant no. guikeAB16380215).
OXA-induce ALI in mice. KM mice were treated with OXA (8 mg/kg, i.p.) for 4 days. Following OXA withdrawal, the mice were observed for 4 days. The mice treated with 5% glucose (i.p.) for 4 days were used as the control group. (A-F) Representative images of the histological evaluation of H&E stained liver tissues (×100). (A) Control liver tissue. (B) Liver cell turbidity and degeneration. (C) Certain liver cells exhibited balloon-like degeneration. (D) Dot-like liver cell necrosis. (E) The liver tissue at 2 days following OXA withdrawal. (F) The liver tissue of deceased mice. (G) The survival rate of the OXA-treated group. (H and I) Time course study of ALT and AST serum levels in the OXA-treated group. Results are presented as the means ± standard deviation from five mice in each group. *P<0.05. OXA, oxaliplatin; ALI, acute liver injury; ALT, alanine aminotransferase; AST, aspartate aminotransferase levels; H&E, hematoxylin and eosin.
Oxidative stress during OXA-induced hepatotoxicity in the mouse model. Time course study of the levels of the oxidative indicator MDA (A) and the antioxidative indicators SOD (B), GSH (C) and GSH-Px (D) in the liver tissues of OXA-treated mice. Results are presented as the means ± standard deviation from five mice in each group. *P<0.05. OXA, oxaliplatin; GSH-Px, glutathione peroxidase; MDA, malondialdehyde; ALT, alanine aminotransferase; AST, aspartate aminotransferase levels; SOD, superoxide dismutase.
Treatment with GSH attenuated OXA-induced ALI in mice. The OXA group were treated with OXA for 4 days, the GSH group were treated with OXA for 4 days and with GSH every day from the first day of OXA administration until the end of the experiment, and the control group were administered with 5% glucose (i.p.) for 4 days. The samples (blood and liver tissue) from each group were collected 3 days after the final dose of OXA. (A) The liver histopathology was examined in each group (H&E staining, original magnification, ×100). (B) The serum ALT and AST levels of each group 3 days after the final dose of OXA. (C) The body weights of each group 3 days after the final dose of OXA. For (B) and (C), the results are presented as the means ± standard deviation from five mice in each group. *P<0.05 and **P<0.01, compared with the control group. #P<0.05, compared with the OXA group. (D) The survival rates of the three groups were observed. OXA, oxaliplatin; GSH, glutathione; ALI, acute liver injury; ALT, alanine aminotransferase; AST, aspartate aminotransferase levels; H&E, hematoxylin and eosin.
GSH treatment suppressed OXA-induced oxidative stress. (A) MDA, (B) GSH, (C) GSH-Px and (D) SOD in the liver tissues of the OXA, GSH and control groups. The results presented are the mean ± standard deviation of three independent experiments performed in triplicate. *P<0.05 and **P<0.01, compared with the control group. #P<0.05, compared with the OXA group. OXA, oxaliplatin; GSH-Px, glutathione peroxidase; SOD, superoxide dismutase; MDA, malondialdehyde.