Possible protective activity of n‑acetyl cysteine against cisplatin‑induced hepatotoxicity in rats

Özlem Coşkun1 · Özlem Öztopuz1 · Başak Büyük2

Abstract

CP is one of the most widely used antineoplastic agents. However, its clinical application is very limited due to its severe toxic effects. The present study aimed to reveal the effects of NAC, which exhibits broad biological activities in reducing CP-induced liver damage, in consideration of biochemical, genetic, and histopathological findings. Twenty-eight wistar rats were randomly divided into four groups of seven animals. A dose of saline was administered (i.p.) to the control group for 5 days. One dose of NAC (200 mg/kg) was administered to the NAC group for 5 days (i.p.). To the NAC + CP group, a dose of CP (7.5 mg/kg) was administered on days 2 and 5 of the experiment, a dose of NAC (200 mg/ kg) (i.p.) was administered for 5 day of the experiment. CP (7.5 mg/kg) was administered to the CP group on days 2 and 5 of the experiment. At the end of the experiment, the biochemical, histological, and mRNA expression analyses of the liver tissues isolated from all the rats were performed. A statistically significant decrease was observed in the AST and ALT enzyme activities in Group NAC + CP compared to Control and CP groups. In addition, it was determined that the NAC administration reduced CPinduced inflammation by increasing the level of NF-κB and decreased CP-caused oxidative stress by decreasing the GPx level. Moreover, the histopathological analyses showed that NAC improved liver morphology. It was revealed by Western blotting analysis that NAC promoted Bcl-2 signaling and decreased p53 signaling. The findings herein showed that NAC could help alleviate hepatotoxicity, a serious therapeutic complication, by reducing CP-induced oxidative stress and playing an effective part in the regulation of apoptotic markers.

Keywords Cisplatin · N-acetyl cysteine · Hepatotoxicity · Apoptosis · Oxidative stress

Introduction

Cisplatin (CP) is among the chemotherapeutic agents active against various tumors occurring in lungs, ovary, bladder, testicles, chest and head-and-neck. Despite its anti-tumor activity, CP’s adverse side effects such as nephrotoxicity, hepatotoxicity, medullary toxicity, gastrointestinal toxicity, neurotoxicity, and ototoxicity restrict its clinical use and therapeutic potential [1, 2]. Recent studies have revealed that hepatotoxicity is a major dose-limiting side effect of cisplatin-based chemotherapy and results in its restricted administration [3]. Even though the mechanisms of hepatotoxicity have not been thoroughly understood yet, a great many experimental models have investigated the correlation between toxicity and oxidative stress, tissue damage, and eventually cytonecrosis [4]. The main mechanism in hepatotoxicity is the damage caused by ROS build-up and oxidative stress. There is a great amount of research evidencing that CP is not tumor-specific and leads to cytotoxicity through promoting the normal cells’ division through ROS production by causing oxidative stress. Natural superoxide dismutase (SOD) and glutathione peroxidase (GPX) members of the antioxidant defense system play a notable role in oxidative stress. Decreased amounts of antioxidant enzymes leading to overly produced ROS result in apoptosis induced by DNA damage [5–7]. Liver cell death by apoptosis is a significant pathogenic indicator of acute and chronic liver damage. Especially, the Bcl-2 protein family has a crucial role in the integrity of mitochondrial membrane and hepatocyte survival [8]. NAC is a sulfur-containing antioxidant of the thiol group and medically used in the hepatotoxicity treatment, immune system modulation, cardiovascular diseases, and prevention of cancer. Its toxicity level is very low. NAC has been shown to be effective in the formation of glutathione (GSH) and by this way to exhibit direct antioxidant activity against cells. It has been reported that NAC suppresses cytokine expression and secretion and exhibits direct anti-inflammatory property by inhibiting the expression of NF-κB. Previous research also reports that it has anti-inflammatory and anti-apoptotic properties [9–11]. Through biochemical, gene expression, and histological analyses of what NAC’s effects would be on inflammation, oxidative stress, and apoptotic process, which are the possible mechanisms of CP-induced hepatotoxicity in rats, the present study intends to reveal whether NAC has a protective activity against CP-induced acute liver damage.

Material and method

Experimental assay

The female Wistar Albino rats of 200–250 g were obtained from the Experimental Research Application and Research Center of Çanakkale Onsekiz Mart University and kept under proper feeding conditions in special cages. The rats were allowed to feed and drink at standard temperature (22 ± 2 °C) and under lighting-controlled conditions (light: 08:00–20:00, dark: 20:00–08:00). 28 rats were raised under the same conditions all of which here exhibitited the same physical characteristics. They randomly placed in four groups and the duration of the administration time was restricted for 5 days (Table 1).
Intraperitoneal administration of a dose of CP (7.5 mg/kg) on day 2 and 5 only (n = 7) [13]. CP and NAC were obtained from Sigma Chemical Co. (Missouri, USA). At the end of day 5, all the rats were anesthetized with 40 mg/kg ketamine (i.m.) + 10 mg/kg xylazine (i.p.). Their abdomens were shaved and sterilized with 10% polyvinylpyrrolidone–iodine complex. The harvested liver tissues were stored at −80 °C until the analyses. The serum was isolated by centrifugation from the collected blood samples at 2500 rpm at 4 °C for 10 min. The serum samples were stored at − 80 °C until the analyses.

Determination of serum ALT and AST levels

The serum from the blood samples was isolated at 2500 rpm at 4 °C for 10 min. The ALT and AST activities were determined with commercial test kits (Roche Diagnostics GMBH Sandhofer STR. 116 D-68305). The measurements were performed on an auto-analyzer (Roche/Hitachi Cobas 6000 C501).

Biochemical analysis

The GPx and NF-κB levels in the rats’ serum samples were measured with the ELISA kit (SunRed).

Gene expression

The total RNA was isolated from 10 to 30 mg liver tissue using a QIAamp RNA spin column (AmbionPure LinkRNA Mini Kit) according to the manufacturer’s recommended protocol. The quality and amount of the RNA were examined by determining the 260/280 absorbance ratio using a NanoDrop ND-1000 Spectrophotometer. The reverse transcription was performed using a kit (High Capacity cDNA Reverse Transcription Kit). All the samples were amplified using Taqman probe PCR master mix (Applied Biosystems). The synthesized cDNA samples were used for the quantitative Real-Time PCR (ABI Stepone) study. The gene expression levels were analyzed by using Taqman probe. β-actin was used for the normalization of the genes. The primary ID number of Bcl2, p53, Bax, and β-actin were Rn99999125_g1, Rn00755717_m1, Rn01480161_g1, Rn00667869_m1 (ThermoFisher), respectively.

Histopathologic analysis (Hematoxylin and eosin staining)

The liver tissues sampled during the surgical operation were rapidly placed in 10% neutral buffered formalin. After they were fixed in formalin for 24–48 h, routine tissue monitoring procedures were followed and the tissues were embedded in paraffin blocks. Sections of 4 µm from the paraffin blocks were placed on glass slides and then stained by routine hematoxylin and eosin staining. The stained preparations were assessed according to the assessment criteria, i.e. inflammatory cell infiltration, sinusoidal congestion, and hydropic degeneration, as described in the related literature [14]. The assessment was performed by using a cameraattached microscope (Olympus CX43). The assessment results were graded [15].
The liver tissue samples were homogenized by using PRO‐ PREP™ protein extraction solution (IntronBiotechnology). The post-centrifuge protein concentration of the supernatant was measured with Bradford assay kit (Sigma). The lysates (~ 50 µg) were eluted by using 4–12% Bis-Tris SDS-PAGE gel observing the Laemmli protocol. Then, the proteins were transferred to nitrocellulose membrane by using iBlot® Dry Blotting System (Thermo Fischer). The nitrocellulose membrane was placed on iBind™ Automated Western System (ThermoFisher) and incubated with anti-mouse p53, Bcl-2 primary antibodies (Thermo Fisher), and anti-rabbit IgG-HRP (Horseradish Peroxidase)-conjugated secondary antibodies (Thermo Fisher) for 150 min. After this process, the nitrocellulose membrane was scanned with C-DiGit Chemiluminescence Blot Scanner (LI-COR, NE, ABD) by using Western blotting luminol reagents. The band densities were normalized to the β-actin density to as control group.

Statistical analysis

SPSS (Social Sciences Statistics Packages, SPSS for Windows, Version 18.0, Chicago, IC, USA) was used for data analysis. The biochemical, genetic, and histopathological results of the mean and standard error values were used to present the descriptive data. Tukey’s test was used for posthoc analyses and then One-Way ANOVA test to analyze the differences between the groups. A p value of less than 0.05 was considered statistically significant.

Result

Biochemical data

Serum ALT and AST are biomarkers of liver damage. The detection of significantly higher serum AST and ALT levels in Group CP in comparison to the Control group indicates CP-induced liver damage (p < 0.001). Administration of NAC was found to decrease significantly the AST and ALT levels in group NAC + CP compared with CP group (p < 0.001). Only NAC administration did not cause a significant change in ALT, AST levels compared to the control group (p = 0.206; p = 0.212 respectively) (Fig. 1).
The NF-κB and GPx levels in the serum were investigated in the Control, NAC, NAC + CP, and CP groups and the within-group values of these parameters were compared with each other. It is revealed that the NF-κB level was significantly increased in Group CP compared to the Control, NAC, and NAC + CP groups (p < 0.001). The NF-κB level in Control, NAC, and NAC + CP groups was determined approximetally similar (Fig. 2a). The GPx enzyme activity was observed to decrease significantly in Group CP in comparison with the NAC, and NAC + CP groups (p < 0.007; p < 0.001 respectively (Fig. 2b).

Gene expression levels

Gene expression levels of Bcl-2, p53 and Bax were analyzed from liver tissues. In both gene expression and Western blot analyses β-actin was used as housekeeping gene/protein to normalize the expression data. The significant differences were observed in the NAC + CP and CP groups compared to the C and NAC groups in all genes studied (p < 0.05). Comparison of the C group with the CP group showed more than two-time increase in Bcl-2, p53 and Bax expression levels (p < 0.001, p < 0.001, p < 0.001) respectively (Fig. 3a–c). When the CP group was compared to the NAC + CP group, p53 and Bax expression levels increased in CP group. NAC + CP group compared to the group CP, the expression levels of p53 and Bax increased in the CP group, while the expression level of Bcl-2 decreased. In our immunoblotting results, an increase in Bcl-2 protein levels was observed in the NAC + CP group compared to the CP group (Fig. 3a). Also, a significant increase in p53 protein levels was observed in the CP group compared to the NAC + CP group (Fig. 3b).

Histopathological results

The histological sections from the groups were assessed in terms of inflammatory cell infiltration, sinusoidal congestion, and hydropic degeneration. For this hence the normal liver microscopy was monitored in Group C in terms of radial arrangement of the hepatocytes, sinusoidal structures, and portal areas. The sections of Group NAC were seen to be similar to those of Group C. A significant increase was detected in Group CP in comparison to Group C in terms of the three parameters (p < 0.05 for inflammatory cell infiltration; p < 0.05 for sinusoidal congestion; p < 0.05 for hydropic degeneration). The comparison of Group NAC + CP with Group CP indicated a significant amelioration in terms of the three histopathologic parameters (p < 0.05 for inflammatory cell infiltration, p = 0.002 for sinusoidal congestion, and p < 0.05 for hydropic degeneration). The obtained results showed that NAC ameliorated liver morphology in the CP-induced hepatotoxicity (Fig. 4).

Discussion

In order to reduce the toxicity occurring as a side effect of chemotherapeutic drugs, the potential use of protective agents come into prominence. The focus of the present study is to investigate the possible protective activity of NAC against CP-induced acute hepatic injury in rats. This study revealed that NAC played an effective role in protecting liver tissues against CP-induced liver damage by reducing free radical production and apoptotic and inflammatory response. Free radicals emerging in cells due to unmetabolized, exogenously introduced drugs and toxic agents lead to liver damage [16]. CP is among the most powerful chemotherapeutic agents commonly used in cancer treatment. It is exploited in the treatment of solid cancers, e.g. in testis, ovary, bladder, lung, cervical, head-and-neck, stomach cancer and other cancers [17, 18]. Despite many studies on CP-induced nephrotoxicity, there is little research on hepatotoxicity [19]. ALT and AST are the best biomarkers of hepatotoxicity. Kim et al. report that CP overdose (45 mg/kg) causes liver dysfunction characterized by increased AST and ALT activities [20].
Similarly, the present study detected significantly higher levels of serum AST and ALT in Group CP in comparison to the control, NAC, and NAC + CP groups, which reveals that CP may cause to liver damage. NAC administration in the NAC + CP group caused a decrease in ALT, AST levels Fig. 3 Between-group variations in the gene expression levels of Bcl-2 (a), p53 (b) and Bax (c). Protein expression levels of Bcl-2 and p53 detected by western blot. The data were presented as 2 − (ΔΔCT) relative expression after the mRNA levels were normalized with β-actin. All the results are presented as mean ± standard error for seven rats in each group. Bcl-2 gene expression; a compared to C, NAC, and CP (p < 0.001, p < 0.001, p < 0.001 respectively); b compared to C and NAC (p < 0.001, p < 0.001 respectively). p53 gene expression; a: compared to C, NAC, and CP (p < 0.001, p < 0.001, p < 0.001 respectively); b: compared to C and NAC (p < 0.001, p < 0.001 respectively). Bax gene expression; a compared to C, NAC, and CP (p < 0.001, p < 0.001, p < 0.001 respectively); b: compared to C and NAC (p < 0.001, p < 0.001 respectively) compared to the CP group. This suggests that it may be due to the protective effect of NAC.
Antioxidants offer protection against oxidative stress. Endogenous antioxidants, such as GSH, GPx, SOD, and CAT, function as free radical scavengers [21]. Many research studies are available on the amelioration of CP toxicity by using antioxidants against CP-caused toxicity. The related literature incorporates a multitude of works reporting that CP, which exerts hepatotoxic effects, produces highly reactive oxygen species that potentially lead to oxidative damage in liver by intervening in the antioxidant defense system [22]. GPx is the most effective antioxidant against oxidative
The grade results of hydropic degeneration, inflammatory cell infiltration, and sinusoidal congestion were scored as follows: 0 = no damage, 1 = mild damage, 2 = moderate damage, 3 = severe damage. All the results are presented as mean ± standard error for seven rats in each group a Compared to NAC+CP, CP p < 0.05 b Compared to NAC+ CP p = 0.002 c Compared to NAC+CP, CP p < 0.05
stress by protecting the cell membrane against peroxidation [23]. A study reports that the administration of Tangeretin, which exhibits antioxidant property, protects hepatic tissues against oxidative stress by decreasing GSH and GPx and playing a critical role in defense against oxidative stress and cellular damage in rats [24].
Nasr et al. report that an intraperitoneal administration of CP of 7.5 mg/kg causes hepatotoxic effects owing to the changes in lipid peroxidation biomarker Malondialdehyde (MDA), catalase (CAT), superoxide dismutase (SOD), GPx, Glutathione S-transferase (GST), and histological parameters [25]. A similar study notes that CP administration triggers lipid peroxidation and leads to acute hepatic damage by reducing GSH and GPx levels [26]. This study too revealed significantly decreased GPx activity in the CP-administered group as a result of oxidative stress caused by CP-induced acute hepatotoxicity. NAC, an antioxidant agent, exerts its effect on hepatotoxicity by increasing the synthesis of glutathione an important factor, directly acting as a free oxygen radical scavenger or producing stable nitrosyl derivatives [27, 28].
Substantiating previous research, the present study found that NAC administration protected liver tissue against CPinduced damage by significantly increasing GPx level with its antioxidant property.NF-κB is reported to play a protective role in oxidative stress by suppressing ROS build-up [29]. Moreover, Hayakawa et al. remark that NAC inhibits NF-κB activation independently of its antioxidant function. It has been also reported that NAC can downregulate cytokines by inhibiting NF-κB and thus possibly reverse liver damage [30, 31]. NAC + CP group was not significantly different from the control and NAC groups in this study. Oxidative stress and p53-mediated apoptosis are noted to play a role in CP-induced liver damage [7]. Prevention of oxidative stress and protein p53’s deactivation by NAC are reported to mitigate CP-induced toxicity [32]. In the present research, a significant decrease was observed in the expression of p53 as a result of NAC in comparison with the control, NAC + CP and CP groups. A previous study has shown that the expression of p53, caspase-3, and Bax is higher and the Bcl-2 level is lower in the CP-administered group in comparison to the control group [33]. The obtained data herein evidenced that CP induced apoptotic cell death in hepatic tissues through modulation of the Bax and Bcl-2 expression and that NAC inhibited pro-apoptotic Bax and increased anti-apoptotic Bcl-2, which in turn reduced apoptosis in liver tissue (Table 2).
As a result of histopathologic analyses, the study by Koç et al. reports changes in circumcellular central veins, hepatocellular vacuolization, and sinusoidal dilatations in CP-induced liver damage. They also report that CP administration causes various microscopically observable damages in liver. They present a hepatic cellular morphology characterized by various Kupffer cell activations, degeneration of hepatocytes, and slight dilatation of sinusoids [34]. Another study, by İşeri et al. reports severe liver damage having been caused by a single dose of CP (2,5 mg kg/l) [35].
The present study histologically showed that CP led to serious damage in liver and NAC served as an active agent in reversing this damage. This recovery was observed in restored cellular pathology as in hydropic degeneration and restored morphological structure as in sinusoidal congestion and inflammatory cell infiltration.

Conclusion

The data herein indicated that NAC had a positive effect on CP-induced oxidative stress and regeneration of apoptotic tissue damage. The limitation of our study is that Bax’s WB analysis cannot be made due to the lack of financial resources. For NAC to be suggested as a supportive agent in CP treatment, further research should be conducted to better understand the mechanisms causing hepatotoxicity in selective tissues.

References

1. Kaygusuzoglu E, Caglayan C, Kandemir FM, Yıldırım S, Kucukler S, Kılınc MA, Saglam YS (2018) Zingerone ameliorates cisplatininduced ovarian and uterine toxicity via suppression of sex hormone imbalances, oxidative stress, inflammation and apoptosis in female wistar rats. Biomed & Pharmacother 102:517–530
2. Perše M, Večerić-Haler Ž (2018) Cisplatin-induced rodent model of kidney injury: characteristics and challenges. BioMed Res Int 2018
3. Alrashed AA, El-Kordy EA (2019) Possible protective role of panax ginseng on cisplatin-induced hepatotoxicity in adult male albino rats (Biochemical and Histological Study). J Microsc Ultrastruct 7(2):84
4. Omar HA, Mohamed WR, Arab HH, Arafa ESA (2016) Tangeretin alleviates cisplatin-induced acute hepatic injury in rats: targeting MAPKs and apoptosis. PLoS One 11(3):e0151649
5. Kadikoylu G, Bolaman Z, Demir S, Balkaya M, Akalin N, Enli Y (2004) The effects of desferrioxamine on cisplatin induced lipid peroxidation and the activities of antioxidant enzymes in rat kidneys. Hum Exp Toxicol 23(1):29–34
6. Hakiminia B, Goudarzi A, Moghaddas A (2019) Has vitamin E any shreds of evidence in cisplatin-induced toxicity. J Biochem Mol Toxicol 33(8):e22349
7. Sun Y, Yang J, Wang LZ, Sun LR, Dong Q (2014) Crocin attenuates cisplatin-induced liver injury in the mice. Hum Exp Toxicol 33(8):855–862
8. Hu L, Chen L, Li L, Sun H, Yang G, Chang Y, Wang H (2011) Hepatitis B virus X protein enhances cisplatin-induced hepatotoxicity via a mechanism involving degradation of Mcl-1. J Virol 85(7):3214–3228
9. Güntürk İ, Yazici C, Köse SK, Dağlı F, Yücel B, Yay AH (2019) The effect of N-acetylcysteine on inflammation and oxidativestress in cisplatin-induced nephrotoxicity: a rat model. Turk J Med Sci 49(6):1789–99
10. Bishr A, Sallam N, Nour El-Din M, Awad AS, Kenawy SA (2019) Ambroxol attenuates cisplatin-induced hepatotoxicity and nephrotoxicity via inhibition of p-JNK/p-ERK. Can J Physiol Pharmacol 97(1):55–64
11. Kim KY, Rhim T, Choi I, Kim SS (2001) N-acetylcysteine induces cell cycle arrest in hepatic stellate cells through its reducing activity. J Biol Chem 276(44):40591–40598
12. Kheradpezhouh E, Panjehshahin MR, Miri R, Javidnia K, Noorafshan A, Monabati A, Dehpour AR (2010) Curcumin protects rats against acetaminophen induced hepatorenal damages and shows synergistic activity with N-acetylcysteine. J Pharmacol 628(1–3):274–281
13. Atasayar S, Gürer-Orhan H, Orhan H, Gürel B, Girgin G, Özgüneş H (2009) Preventive effect of amino guanidine compared to vitamin E and C on cisplatin-induced nephrotoxicity in rats. Exp Toxicol Pathol 61(1):23–32
14. Kaya H, Polat B, Albayrak A, Mercantepe T, Buyuk B (2018) Protective effect of an L-type calcium channel blocker, amlodipine, on paracetamol-induced hepatotoxicity in rats. Hum Exp Toxicol 37(11):1169–1179
15. Oztopuz O, Turkon H, Buyuk B, Coskun O, Sehitoglu MH, Ovali MA, Uzun M (2020) Melatonin ameliorates sodium valproateinduced hepatotoxicity in rats. Mol Biol Rep 47(1):317–325
16. Ayla Ş, Oktar H, Tanrıverdi G, Cengiz M, Özkılıç AÇ, Can G, Batur Ş (2009) Doksorubisin nedenli sıçan hepatotoksisitesine nikotinamidin (koruyucu) etkisi. Anadolu University Journal of Sciences & Technology 10(1)
17. Güntürk I, Yazici C, Köse SK, Dağlı F, Yücel B, Yay AH (2019) The effect of N-acetylcysteine on inflammation and oxidative stress in cisplatin-induced nephrotoxicity: a rat model. Turk J Med Sci 49(6):1789–1799
18. Ghosh S (2019) Cisplatin: The first metal based anticancer drug. Bioorg Chem 88:102925
19. Abd Rashid N, Hussan F, Hamid A, Ridzuan NRA, Lin TS, Budin SB (2019) Preventive effects of polygonum minus essential oil on cisplatin-induced hepatotoxicity in sprague Dawley Rats. Sains Malaysiana 48(9):1975–1988
20. Kim SH, Hong KO, Chung WY, Hwang JK, Park KK (2004) Abrogation of cisplatin-induced hepatotoxicity in mice by xanthorrhizol is related to its effect on the regulation of gene transcription. Toxicol Appl Pharmacol 196(3):346–355
21. Karadeniz A, Simsek N, Karakus E, Yildirim S, Kara A, Can I, Kisa E, Emre H, Turkeli M (2011) Royal jelly modulates oxidative stress and apoptosis in liver and kidneys of rats treated with cisplatin. Oxid Med Cell Longev 2011
22. Mansour HH, Hafez HF, Fahmy NM (2006) Silymarin modulates cisplatin-induced oxidative stress and hepatotoxicity in rats. BMB Reports 39(6):656–661
23. Erenel G, Erbaş D, Arıcıoğlu A (1992) Serbest radikaller ve antioksidan sistemler. Gazi Med J 3(4)
24. Arab HH, Mohamed WR, Barakat BM, Arafa ESA (2016) Tangeretin attenuates cisplatin-induced renal injury in rats: impact on the inflammatory cascade and oxidative perturbations. Chem Biol Interact 258:205–213
25. Nasr AY (2014) Protective effect of aged garlic extract against the oxidative stress induced by cisplatin on blood cells parameters and hepatic antioxidant enzymes in rats. Toxicol Rep 1:682–691
26. Bentli R, Parlakpinar H, Polat A, Samdanci E, Sarihan ME, Sagir M (2013) Molsidomine prevents cisplatin-induced hepatotoxicity. Arch Med Res 44(7):521–528
27. Aydoğdu N, Kaymak K, Yalçın Ö (2005) Sıçanlarda Böbrek İskemi/Reperfüzyon Hasarında N-Asetilsisteinin Etkileri. Fırat Tıp Dergisi 10(4):151–155
28. Thong-Ngam D, Samuhasaneeto S, Kulaputana O, Klaikeaw N (2007) N acetylcysteine attenuates oxidative stress and liver pathology in rats with non-alcoholic steatohepatitis. World J Gastroenterol 13(38):5127
29. Lingappan K (2018) NF-κB in oxidative stress. Curr Opin Toxicol 7:81–86
30. Reyes-Gordillo K, Segovia J, Shibayama M, Vergara, P, Moreno MG, Muriel P (2007) Curcumin protects against acute liver damage in the rat by inhibiting NF-κB, proinflammatory cytokines production and oxidative stress. Biochim Biophys Acta (BBA) Gen Subj 1770(6): 989-996
31. Hayakawa M, Miyashita H, Sakamoto I, Kitagawa M, Tanaka H, Yasuda H, Kikugawa K (2003) Evidence that reactive oxygen species do not mediate NF-κB activation. EMBO J 22(13):3356–3366
32. Huang S, You J, Wang K, Li Y, Zhang Y, Wei H, Liu Y (2019) N-acetylcysteine attenuates cisplatin-induced acute kidney injury by inhibiting the C5a receptor. BioMed Res Int 2019
33. Kandemir FM, Yildirim S, Caglayan C, Kucukler S, Eser G (2019) Publisher’s Note Springer Nature remains neutral with regard to Protective effects of zingerone on cisplatin-induced nephrotoxicity jurisdictional claims in published maps and institutional affiliations. in female rats. Environ Sci Pollut Res 26(22):22562–22574
34. Koc A, Duru M, Ciralik H, Akcan R, Sogut S (2005) Protective agent, erdosteine, against cisplatin-induced hepatic oxidant injury in rats. Mol Cell Biochem 278(1–2):79–84
35. İşeri S, Ercan F, Gedik N, Yüksel M, Alican I (2007) Simvastatin attenuates cisplatin-induced kidney and liver damage in rats. Toxicology 230(2–3):256–264