Elsevier

Free Radical Biology and Medicine

Volume 148, 20 February 2020, Pages 128-139
Free Radical Biology and Medicine

Ccrn4l as a pre-dose marker for prediction of cisplatin-induced hepatotoxicity susceptibility

https://doi.org/10.1016/j.freeradbiomed.2020.01.003Get rights and content

Highlights

  • We used a pre-biopsy method to discover the genetic factors for DIH susceptibility.

  • Cisplatin caused hepatotoxicity with increased hepatic CYP2E1 and H2O2 levels.

  • Ccrn4l was found to be lower before drug exposure in cisplatin-sensitive animals.

  • Ccrn4l expressions were positively correlated with catalase expressions in individuals.

  • Ccrn4l-siRNA silencing aggravated cisplatin-induced cytotoxicity.

Abstract

Clinical cisplatin use is often limited by its drug-induced liver injury (DILI). Particularly, individual differences in susceptibility to DILI can cause life-threatening medical conditions. This study aimed to uncover the inherent genetic factors determining individual variations in hepatotoxicity susceptibility. Rats were subjected to liver biopsy and a 3-week postoperative recovery period before cisplatin administration. At 2 days post-treatment with cisplatin, the rats exhibited histopathological and serum biochemical alterations in the liver, and changes in hydrogen peroxide and cytochrome P450-2E1 levels. Based on these results of liver-related biochemical markers, 32 rats were grouped into the susceptible (top five) and resistant (bottom five) groups. Using RNA-sequencing, we compared gene expressions in the liver pre-biopsied from these two groups before cisplatin treatment and found 161 differently expressed genes between the Susceptible and Resistant groups. Among them, the clock-controlled Ccrn4l responsible for ‘rhythmic process’ was identified as a common gene downregulated inherently prior to drug exposure in both cisplatin- and acetaminophen-sensitive animals. Additionally, low Ccrn4l levels before cisplatin treatment in the Susceptible group were maintained even after treatment, with decreased antioxidants, increased nitration, and apoptosis. The relationship of Ccrn4l with catalase and mitochondrial RNAs in the liver was confirmed by correlation of their hepatic levels among individuals and similar patterns of circadian variation in their mRNA expression. Remarkably, Ccrn4l knockdown promoted cisplatin-induced mitochondrial dysfunction in WB-F344 cells with antioxidant catalase and apoptosis-related Bax changes. Inherent individual hepatic Ccrn4l level might be a novel factor affecting cisplatin-induced hepatotoxicity susceptibility, possibly through regulation of mitochondrial and antioxidant functions.

Introduction

Drug-induced liver injury (DILI) is a problem of increasing significance as a frequent cause of liver damage. Underreporting and underestimation of adverse drug reactions have led to DILI in humans [1]. In general, DILI incidence varies according to the drug, ranging from 5–20/1000 with isoniazid and chlorpromazine to 1–10/1 million with minocycline and statins [2]. The process of drug research and development from target identification to drug approval takes over 12 years and often much longer [3], and the costs per new drug introduction continue to rise, with the recent average costs estimated at approximately $2.6 billion [4]. The occurrence of DILI with hepatic failure in patients receiving commercialized drugs after their release to the market can cause post-marketing drug withdrawals and substantial financial losses [2]. More importantly, DILI can be a serious, life-threatening medical condition in humans; DILI-associated acute liver failure can result in significant morbidity, high mortality, and high healthcare costs [5]. All drug candidates are rigorously tested in in vitro and in vivo nonclinical and clinical studies to determine their DILI risk; however, DILI still occurs after the administration of marketed drugs. Based on recent examples, including troglitazone and bromfenac [2], conventional nonclinical and clinical toxicology studies cannot provide complete identification of drugs that may potentially cause hepatotoxicity, including idiosyncratic hepatotoxicity, despite providing reliable information from standardized liver tests. In our opinion, this relatively low accuracy of DILI prediction might be due to individual differences in susceptibility factors.

Cisplatin is a platinum-based potent anticancer drug used to treat various solid tumors [6]. However, its clinical application is often limited by its significant side effects, including hepatotoxicity, nephrotoxicity, ototoxicity, gastrotoxicity, myelosuppression, neurotoxicity, and allergic reactions [[7], [8], [9], [10]]. In fact, there have been rare case reports of cisplatin-induced liver injury in humans. In one instance associated with hepatotoxicity, steatosis and necrosis were found with marked liver enzyme elevations at 4 weeks after starting a regimen of cisplatin. In another case, hepatocellular liver injury was described. Nonetheless, the severity was generally mild and the outcome benign when it does occur. More importantly, there is likely to be cross sensitivity to hepatotoxicities of various platinum coordination complexes [11]. Despite clear evidence of hepatotoxicity associated with this drug, the factors influencing the differences in susceptibility to cisplatin-induced hepatotoxicity have not been fully identified. Because DILI is affected by complex interactions between genetic and environmental risk factors, accurate prediction is not easy; unexpected fatal hepatotoxicity may occur depending on each individual, even after rigorous toxicity testing [2,12,13].

Here, we examined whether inter-individual differences in susceptibility to cisplatin-induced hepatotoxicity are due to intrinsic inter-individual variability in hepatic gene expression. For this purpose, we used a pre-biopsy method, in which liver tissues were biopsied before cisplatin administration. Using these tissues biopsied prior to cisplatin treatment, we identified differences in the pre-dosing expression of inherent genes among individuals who showed different degrees of cisplatin-induced hepatotoxicity. The findings of this study may provide an important clue to identify a common key genetic marker for application to the treatment of major DILIs.

Section snippets

Animal experiment

Male Sprague-Dawley rats purchased from Orient Bio (Sungnam, Korea) were maintained under controlled normal atmospheric temperature (22 ± 2 °C) and relative humidity (55 ± 5%) during a 12-h light-dark cycle. The rats were fed a natural ingredient diet Harlan 2918C (Raon Bio, Yongin, Korea) and tap water ad libitum. Animals were used for research after 3 days acclimatization. All experimental procedures were approved by the Institutional Animal Care and Use Committee at The Catholic University

Cisplatin caused hepatotoxicity with increased hepatic H2O2 and CYP2E1

Based on the results of the first experiment, the administration of cisplatin at a dose of 25 mg/kg to rats led to a significant decrease in body weight and a significant increase in relative liver weight (Fig. 1B). Along with significant increases in serum markers of liver function (ALT, AST, and γGT) (Fig. 1C), cisplatin also induced liver histological changes that ranged from hepatocellular hypertrophy and regeneration, which have been considered as the liver's attempt of adaptation [15], to

Discussion

Numerous attempts have been made to discover biomarkers for predicting DILI outcome. The gene expression profiles associated with pathological or physiological changes during and after hepatotoxicity have been reported [24]. However, the hepatotoxicity of prescription drugs continues to occur frequently with varying severity despite rigorous nonclinical and clinical toxicity testing [2,12,13]. The reason why it is difficult to predict the hepatotoxicity of commercial drugs through classical

Declaration of competing interest

All authors declare that they have no competing financial interests with respect to this manuscript.

Acknowledgements

The author would like to thank Dr. Xingen Lei (Cornell University, Ithaca, NY, USA) for critical review of the manuscript. This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (2017R1D1A1B03031604) and the Bio & Medical Technology Development Program of the NRF funded by the Korean government, MSIP (NRF-2016M3A9D5A01952416). This work was supported by the Catholic University of Korea, Research

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