Sage Journals: Discover world-class research

Abstract

Adverse complications associated with antineoplastic drug-based cancer therapy are the major clinical drawbacks. Oxidative stress and inflammation play a major role in the damage due to cancer therapy. In the current study, we investigated the modulatory effect of vitamin C (Vit. C) on liver toxicity induced by 5-fluorouracil (5-FU) in rats. Animals were divided into four groups. Animals in group I received vehicle. Oral gavage of Vit. C (500 mg kg⁻¹ body weight (b.wt.)) was given to the animals in group III and group IV. 5-FU (150 mg kg⁻¹ b.wt.) was injected intraperitoneally to the animals in group II and group III. Findings of the present study revealed that oral administration of Vit. C significantly ameliorated the level of lipid peroxidation and the activity of myeloperoxidase. Vit. C administration markedly reduced the activation of nuclear factor κB and expression of cyclooxygenase 2, whereas nuclear translocation of nuclear factor erythroid 2–related factor 2 was increased. Hepatic histopathological analyses further supported the protective effect of Vit. C. Findings of the current study demonstrate that the toxic free radicals and inflammatory mediators generated due to chemotherapy play a critical role in 5-FU–induced hepatic damage. Attenuating action of Vit. C may be due to the modulation of redox-sensitive transcription factors and associated target molecules.

Keywords

5-Fluorouracil liver toxicity redox-sensitive transcription factors

Introduction

Cancer is the leading cause of death worldwide. The available therapeutic measures are not adequate as the incidence and mortality is not reducing at large. Chemotherapy is one of the most commonly used measures to treat various forms of cancer. However, this is associated with severe toxic effects. 5-Fluorouracil (5-FU), a pyrimidine analog, is one of the commonly used chemotherapeutic drugs for the treatment of neoplasms.¹ One of the important limitation of 5-FU use is its toxicity on normal proliferating cells. The adverse effect of 5-FU on different major and minor vital organs of the body is well documented.^2,3
–5 Metabolic activation of 5-FU results in the formation of three major reactive metabolites such as 5-fluoro-uridine-5′-monophosphate, 5-fluoro-uridine-5′-triphosphate, and 5-fluoro-2′-deoxyuridine-5′-triphosphate. These metabolites of 5-FU exert a cytotoxic action on cancerous as well as normal cells by disruption of nucleic acid synthesis and the action of thymidylate synthase.¹

The toxic action of 5-FU is dose dependent and also varies from patient to patient, which sometimes may lead to cessation of the therapy. Common unbearable, serious, and painful side effects of 5-FU–based chemotherapy are mucositis, hepatorenal toxicity, diarrhea, myelosuppression, cardiotoxicity, dermatitis, and toxicity of genital organs.⁶

It has been well documented that overproduction of reactive oxygen species and inflammatory mediators play a vital role in 5-FU–induced toxic manifestations.³ Use of the agents or compounds with antioxidant and anti-inflammatory potential have been suggested to reduce the chemotherapy-associated adverse effects. A number of combinational approaches have been used along with 5-FU to minimize the associated adverse effects without compromising its antineoplastic action.⁷

Ascorbic acid (vitamin C (Vit. C)) is a major water-soluble vitamin with strong antioxidant and anti-inflammatory potential. It is not only important for cellular physiology but also provide protection against various toxic insults. Both preclinical and clinical observations demonstrated that Vit. C has shown strong abrogative potential in the mitigation of various disease conditions due to its native strong antioxidant and anti-inflammatory properties.^8

–12 Since it is well documented that Vit. C has modulatory potential on array of signaling pathways, the present study was designed to investigate the abrogative potential of Vit. C against antineoplastic drug 5-FU–induced hepatic damage.

Materials and methods

Chemicals and other reagents

Potassium dihydrogen phosphate, dipotassium hydrogen phosphate, o-dianisidine dihydrochloride, hexadecyl trimethyl ammonium bromide, hydrogen peroxide (H₂O₂), Vit. C, Tris, trichloroacetic acid (TCA), thiobarbituric acid (TBA), and Tween 20 were purchased from Sigma Aldrich (St Louis, Missouri, USA). 5-FU was supplied by ICN Biomedicals Inc. (Aurora, Ohio, USA).

Animals

Male Sprague Dawley rats (weighing 150–200 g and aged 6–8 weeks), were obtained from the Animal House Facility of Research Center, Prince Sultan Military Medical City (PSMMC), Riyadh, Saudi Arabia. All animals were housed in the animal care facility under room temperature at 22–25°C with 12-h light/12-h dark cycle in standard cages and were given free access to standard laboratory diet and water ad libitum. The animals received humane care in accordance with the Guide for the Care and Use of Laboratory Animals, published by the Ethics and Scientific Research Committee of PSMMC, Riyadh, Saudi Arabia.

Treatment regimen

Rats were randomly divided into four groups. Each group comprises six animals. The animals of all the four groups received normal standard diet and water throughout the experimental period. Animals in group I received only vehicle (0.9% saline intraperitoneally) on day 8 and serve as the control group. Animals of the group II were given single intraperitoneal injection of 5-FU (150 mg kg⁻¹ body weight (b.wt.)) on day 8 and served as positive control. Animals in group III were given oral gavage of Vit. C (500 mg kg⁻¹ b.wt.) for 10 days (day 1 to day 10) and single intraperitoneal injection of 5-FU (150 mg kg⁻¹ b.wt.) on day 8. Animals of the group IV received only oral gavage of Vit. C (500 mg kg⁻¹ b.wt.) for 10 days. Animals of the all four experimental groups were killed under mild anesthesia on day 11.

Tissue processing

At the end of the experimental period, the animals were killed by cervical dislocation under mild anesthesia. Liver tissues were excised and washed with ice-cold sodium chloride (0.9%). A piece of liver tissue was preserved in 10% neutral-buffered formalin (NBF) for histological observation.

Lipid peroxidation

Lipid peroxidation was determined according to the method described by Utley et al.¹³ by estimating malondialdehyde (MDA) with slight modification. Briefly, 0.25 ml of tissue homogenate (in 10% phosphate buffer, pH 7.4) was incubated at 37°C in water bath. After an hour of incubation, 0.25 ml of TCA (5%) and 0.5 ml of TBA (0.67%) were added to the sample and centrifuged at 3000g (Eppendorf 5810 R) for 10 min. The clear supernatant was transferred to another tube and placed in a boiling water bath for 10 min. Finally, the tubes were cooled, and the color intensity was measured at 535 nm. The MDA concentration in the samples was calculated using the molar extinction coefficient of 1.56 × 10⁻⁵ M⁻¹ cm⁻¹ for MDA-TBA colored complex and expressed as micromoles of MDA formed per gram of tissue.

MPO activity

Activity of myeloperoxidase (MPO) was assayed by the method of Bradley et al.¹⁴ Tissue homogenate was prepared in 50 mM potassium phosphate buffer (pH 6.0)-containing 0.5% hexadecyl trimethylammonium bromide. Samples were subjected to three cycles of sonication and freezing-thawing procedures, followed by spinning at 14,000 rpm (Eppendorf 5810 R) for 25 min at 4°C. In 0.1 ml of supernatant, MPO activity was measured using 2.9 ml phosphate buffer (50 mM, pH 6.0) containing 0.167 mg ml⁻¹ o-dianisidine dihydrochloride and 1% H₂O₂ as substrates at 460 nm for 3 min. MPO activity is defined as the quantity of enzyme degrading one micromole of H₂O₂ per minute at 25°C and are expressed as units per minute per gram.

Immunohistochemical detection of Nrf2, NF-kB, and Cox-2 expression

Five-micrometer-thick liver sections were cut onto poly l-lysine–coated glass slides. Sections were deparaffinized 3 times (5 min each) in xylene, followed by dehydration in graded ethanol and finally rehydrated in running tap water. For antigen retrieval, the sections were boiled in 10 mM citrate buffer (pH 6.0) for 15–20 min. Next, the sections were incubated with protein block (Abcam, UK) for 10 min at room temperature followed by Tris-buffered saline with Tween 20 wash. Normal goat serum (ab138478) was used as the blocker for 2 h prior to primary antibodies treatment for overnight at 4°C. Further processing was done using mouse and rabbit-specific horseradish peroxidase (ABC) Detection IHC kit (ab93697) from Abcam. Primary antibodies were rabbit anti- nuclear factor κB (NF-κB) p105/p50 (phospho S927’; dilution1:150, Abcam, ab131493), rabbit anti- nuclear factor erythroid 2–related factor 2 (Nrf2; Abcam, ab31164) and rabbit anti-cyclooxygenase 2 (COX-2; Abcam, ab21704).The peroxides complex was visualized with 3,3′-diaminobenzidine. Lastly, the slides were counterstained with hematoxylin and cleaned in water, xylene, and ethanol. After mounting with DPX, microscopic (BX53 microscope, Olympus, Japan) analysis was performed.

Histological analysis

For histopathological studies, a portion of liver tissue was fixed in freshly prepared 10% NBF immediately after killing. Then, tissue was embedded in paraffin wax, and blocks were prepared. Sections of 5-µm thickness were cut onto poly-l-lysine–coated microscopic slides and stained with hematoxylin and eosin (H&E). Histological changes were monitored under light microscope (BX53 microscope, Olympus, Japan) at least in six different regions.

Statistical analysis

The data from individual treatment groups are presented as the means ± standard error of the mean (SEM). Differences between groups were analyzed using one-way analysis of variance (ANOVA), followed by Tukey–Kramer multiple comparisons test. Data were considered statistically significant when the values of p < 0.05.

Results

Effect of Vit. C on 5-FU–induced hepatic oxidative stress (MDA level)

Role of oxidative stress is well documented in hepatic damage. We observed a significant increase in MDA level of liver tissue after 5-FU injection to the animals of group II as compared to vehicle-treated group I animals (p < 0.001). Treatment of Vit. C together with 5-FU in group III animals showed significant depletion of MDA level as compared to 5-FU–treated group II animals (p < 0.001). No significant difference in MDA level was observed between vehicle-treated group I animals and Vit. C-treated group IV animals (Figure 1).

Figure 1.

Effect of Vit. C and 5-FU on hepatic MDA level. Bars represent the mean ± SEM per treatment group (n = 6). ***p < 0.001 shows significant difference in 5-FU–treated group II animals as compared to vehicle-treated animals (group I); ^###p < 0.001 shows that Vit. C administration significantly inhibited the level of MDA in group III animals as compared to only 5-FU–injected group II animals. Vit. C: vitamin C; 5-FU: 5-fluorouracil; MDA: malondialdehyde; SEM: standard error of the mean.

Effect of Vit. C on MPO activity in the liver after 5-FU injection

MPO is one of the key enzymes secreted by immunoregulatory cells and plays a vital role in toxic manifestations. Significantly increased MPO activity was observed in 5-FU–injected group II animals as compared to vehicle alone-injected group I animals (p < 0.05). Supplementation of Vit. C with 5-FU in group III animals diminished the MPO activity significantly as compared to group II animals (p < 0.05).There was no remarkable difference in MPO activity was detected in Vit. C-administered group IV animals as compared to vehicle-injected group I animals (Figure 2).

Figure 2.

Effect of Vit. C and 5-FU on hepatic MPO activity. Bars represent the mean ± SEM per treatment group (n = 6). *p < 0.05 shows significant difference in 5-FU–treated group II animals as compared to vehicle-treated animals (group I). ^#p < 0.05 shows that Vit. C administration significantly inhibited MPO activity in group III animals as compared to only 5-FU–injected group II animals. Vit. C: vitamin C; 5-FU: 5-fluorouracil; MPO: myeloperoxidase; SEM: standard error of the mean.

Effect of Vit. C on nuclear translocation of redox-sensitive transcription factors (NF-κB and Nrf2) and expression of Cox-2

Redox-sensitive transcription factors and their signaling targets play vital role in adverse complications associated with chemotherapy. Immunohistochemical analysis for the expression of NF-κB, Nrf2, and Cox-2 in liver tissue are presented in Figures 3, 4, and 5, respectively. Increased hepatic expression of Nrf2, NF-κB, and Cox-2 was seen in 5-FU–injected group II animals as compared to vehicle-injected group I animals. Supplementation of Vit. C in group III animals markedly reduced the expression of NF-κB and Cox-2, while activation of Nrf2 was increased as compared to 5-FU-injected group II animals. There was no remarkable difference found in the expression of NF-κB and Cox-2 in group IV animals as compared to vehicle-injected group I animals, while the expression of Nrf2 was increased in group IV animals as compared to vehicle-injected group I animals. For immunohistochemical analyses, brown color was indicative of the specific immunostaining of NF-κB, Nrf2, and Cox-2 and light blue color of hematoxylin staining. Original magnification: 40×

Figure 3.

Effect of Vit. C supplementation on 5-FU–induced hepatic NF-κB activation. Representative photomicrographs (magnification ×40; scale bar 200 µm) depicting immunohistochemical activation of NF-κB: (a) vehicle-treated animals showing minimal or no nuclear staining, (b) 5-FU–treated animals showing remarkable nuclear staining, (c) Vit. C + 5-FU–treated animals showing moderate nuclear staining, and (d) only Vit. C-treated animals showing minimal or no nuclear staining. Brown color indicates immunopositive staining of NF-κB and blue color indicates hematoxylin staining. Vit. C: vitamin C; 5-FU: 5-fluorouracil; NF-κB: nuclear factor κB.

Figure 4.

Effect of Vit. C treatment on 5-FU–induced Nrf2 activation in the liver. Representative photomicrographs (magnification ×40; scale bar 200 µm) depicting immunohistochemical expression of Nrf2: (a) vehicle-treated animals showing minimal nuclear and more cytoplasmic staining, (b) 5-FU–treated animals showing median nuclear staining, (c) Vit. C + 5-FU–treated animals showing high nuclear staining, and (d) Vit. C only showing more nuclear staining as compared to vehicle-treated animals. Brown color indicates immunopositive staining of Nrf2 and blue color indicates hematoxylin staining. Vit. C: vitamin C; 5-FU: 5-fluorouracil; Nrf2: nuclear factor erythroid 2–related factor 2.

Figure 5.

Effect of Vit. C supplementation on 5-FU–induced expression of COX-2 in liver. Representative photomicrographs (magnification ×40; scale bar 200 µm) depicting immunohistochemical activation of COX-2: (a) vehicle-treated animals showing minimal or no staining, (b) 5-FU–treated animals showing intense staining, (c) Vit. C + 5-FU–treated animals showing low staining, and (d) Vit. C only-treated animals showing minimal or no staining. Brown color indicates immunopositive staining of COX-2 and blue color depicts hematoxylin staining. Vit. C: vitamin C; 5-FU: 5-fluorouracil; COX-2: cyclooxygenase 2.

Effect of Vit. C on the histological alterations in liver after 5-FU injection

Protective action of Vit.C was further evaluated by the microscopic examination of liver tissue. Histological analysis of liver tissue of vehicle-injected animals showed normal histological pattern, whereas 5-FU injected animals showed deformities in liver tissue such as infiltration of neutrophils, cellular vocalization, loss of cellular integrity, and necrosis. Treatment with Vit. C markedly suppressed these histological anomalies in group III animals as compared to 5-FU–injected animals in group II. No remarkable histological difference was observed between the animals treated with Vit. C and the animals of vehicle-injected group (Figure 6). Original magnification: 10× and 20×

Figure 6.

Effect of Vit.C on 5-FU–induced hepatic histological alterations. Representative photomicrographs (magnification ×10 and ×20; scale bar 200 µm): (a) vehicle only-treated animals showing normal histology, (c) 5-FU–treated animals showing histological deformities, (e) Vit. C + 5-FU–treated animals showing suppressed histological alterations, and (g) Vit. C only-treated animals showing normal histology. Insets (b, d, f and h) at the right panel show a magnified view (×20 magnifications) of the pictures (a, c, e and g) showed at the left panel (×10 magnifications). Vit. C: vitamin C; 5-FU: 5-fluorouracil.

We also observed that the rats injected with 5-FU showed marked cellular regeneration anomalies as compared to vehicle-injected animals. Normal cellular proliferation was also distorted after 5-FU injection, whereas Vit. C supplementation has markedly ameliorated these cellular distortions (Figure 7). Original magnification: 40×

Figure 7.

Effect of Vit.C on 5-FU–induced hepatic cellular regeneration. Representative photomicrographs (magnification ×40; scale bar 200 µm): (a) vehicle only-treated animals showing normal cellular pattern, (b) 5-FU–treated animals showing marked cellular deformities with necrosis and loss of cellular integrity, (c) Vit. C + 5-FU–treated animals showing suppressed hepatocytes deformities, and (d) Vit. C only-treated animals showing normal hepatic pattern. Vit. C: vitamin C; 5-FU: 5-fluorouracil.

Discussion

Chemotherapy, apart from increasing the survival of cancer patients, is also associated with severe adverse effects. Various preclinical and clinical observations have demonstrated the adverse effects of chemotherapy on different vital organs as well as on behavior of cancer patients.^15

–18 Liver damage is a critical issue associated with chemotherapy-based neoplasm treatment.^19,20 In the present study, we investigated the modulatory action of Vit. C against 5-FU–induced liver damage in rats. Vit. C was selected due to its strong antioxidant and anti-inflammatory potential with minimal or no side effects.

5-FU is one of the most commonly used antineoplastic drugs for cancer treatment but is also associated with various forms of organ toxicity.^2,6,21 Liver damage is one of the complications associated with 5-FU –based neoplasm therapy.^19,22

–25 Role of oxidative stress has been well documented in the toxic action of 5-FU because of the interference of the reactive oxygen species (ROS) in the various signaling pathways.^2,26 One of the findings of the current study showed that 5-FU injection causes massive production of ROS, which was evident from the increased level of MDA formation, while Vit.C administration diminished the formation of lipid peroxides possibly by boosting the endogenous antioxidant machinery. This action of Vit. C has been previously demonstrated by different research groups that support the strong attenuation action of Vit.C.^27
–29

Keeping in view the well documented role of oxidative stress in the chemotherapy-associated complications, we further assessed the expression of redox-sensitive transcription factors after the administration of 5-FU and Vit.C. Nrf2 is an important redox-sensitive transcription factor that plays a vital role in cellular protection by regulating the cellular antioxidant defense machinery at genetic level.^30,31 Oxidative stress can activate Nrf2 via conformational modification of its suppressor KEAP1, resulting in the translocation of Nrf2 from cytosol to inside the nucleus.^30,32,33 Our findings demonstrate that 5-FU causes the activation of Nrf2 via oxidative stress as earlier reported by Numazawa et al.³⁴ Supplementation of Vit. C showed protection, which might be due to oxidative modification of KEAP1 and thus Nrf2 induced increased expression of antioxidant responsive element. Similar action of Vit. C in the upregulation of cytoprotective machinery via Nrf2-dependent pathway was observed by different investigators which authenticates our finding.^35
–37

NF-κB is another important redox-sensitive transcription factor involved in the development of liver anomalies.^38,39 NF-κB, which is abundantly present inside the cell, plays a major role in different processes including immunity, cell division and survival, inflammation, immune response, and apoptosis.⁴⁰ In normal conditions, it is present in the cytoplasm along with its suppressor. A number of stimuli, such as oxidative stress, cytokines, and lipopolysaccharides cause its translocation inside the nucleus where it regulates the transcriptional activation of battery genes.^38,39 The findings of the current study suggest that Vit. C has strong protective potential against 5-FU–induced hepatic damage as it reduced the nuclear translocation of NF-κB. Our findings are in line with earlier reports that demonstrated the inhibitory action of Vit. C on the activation of NF-κB.^41

–45The underlying mechanism behind this effect of Vit. C might be due to its modulatory action on signaling pathways which lead to the suppression of the NF-κB nuclear translocation via inhibition of nuclear factor κB kinase.^42,46,47 We further assessed the role of Vit. C on the modulation of Cox-2 expression. Cox-2 is one of the important stress response proteins that plays vital role in the toxicity and disease development during chemotherapy.^48,49 A number of earlier studies and the findings of the current study suggest that an elevated expression of Cox-2 may be one of the causative factors for liver damage.^50,51 In this study, we have observed that the mitigatory potential of Vit. C against antineoplastic drug-induced liver injury might be due to its free radical scavenging property and modulation of NF-κB and other signaling pathways, which lead to the inhibition of Cox-2.^45,52 Histological observations of the present study corroborated with the biochemical and molecular findings. Supplementation with Vit. C suppressed the histological changes observed in the liver tissue after 5-FU injection, which further supports the antioxidant and anti-inflammatory action of Vit. C. Although the overall findings of the current study demonstrate remarkable scientific basis for the protective action of Vit. C against liver damage induced by 5-FU, deciphering the deep molecular action of Vit. C on different molecular pathways by exploiting modern techniques may further elucidate the mitigatory action of Vit. C.

In conclusion, the findings of the present study provide substantial biochemical, molecular, and histological evidence about the protective action of Vit. C against antineoplastic drug 5-FU–induced hepatic damage. Additional research is required to further understand the protective action of Vit. C so that substantial progress can be made in the management of complications associated with chemotherapy.

Footnotes

Acknowledgment

The authors thank the Prince Sultan Military Medical City hospital and research center for providing all necessary facilities to conduct this study.

Declaration of Conflicting Interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) received no financial support for the research, authorship, and/or publication of this article.

References

Longley

Harkin

Johnston

. 5-fluorouracil: mechanisms of action and clinical strategies. Nat Rev Canc 2003; 3: 330–338.

Rashid

Ali

Nafees

, et al. Mitigation of 5-fluorouracil induced renal toxicity by chrysin via targeting oxidative stress and apoptosis in Wistar rats. Food Chem Toxicol 2014; 66: 185–193.

Conklin

. Chemotherapy-associated oxidative stress: impact on chemotherapeutic effectiveness. Integr Cancer Ther 2004; 3: 294–300.

Polk

Vistisen

Vaage-Nilsen

, et al. A systematic review of the pathophysiology of 5-fluorouracil-induced cardiotoxicity. BMC Pharmacol Toxicol 2014; 15: 47.

Garg

Lincz

Adler

, et al. Predicting 5-fluorouracil toxicity in colorectal cancer patients from peripheral blood cell telomere length: a multivariate analysis. Br J Canc 2012; 107: 1525–1533.

Al-Asmari

Al-Zahrani

Khan

, et al. Taurine ameliorates 5-flourouracil-induced intestinal mucositis, hepatorenal and reproductive organ damage in Wistar rats: a biochemical and histological study. Hum Exp Toxicol 2016; 35: 10–20.

Agbarya

Ruimi

Epelbaum

, et al. Natural products as potential cancer therapy enhancers: a preclinical update. SAGE Open Med 2014; 2: 2050312114546924.

Chambial

Dwivedi

Shukla

, et al. Vitamin C in disease prevention and cure: an overview. Indian J Clin Biochem 2013; 28: 314–328.

Hoda

Sinha

. Vitamin C-mediated minimisation of Rogor-induced genotoxicity. Mut Res 1993; 299: 29–36.

10.

Rana

Bera

Das

, et al. Supplementation of ascorbic acid prevents oxidative damages in arsenic-loaded hepatic tissue of rat: an ex vivo study. Hum Exp Toxicol 2010; 29: 965–972.

11.

Nishi

Campos

Bergamaschi

, et al. Vitamin C prevents DNA damage induced by renovascular hypertension in multiple organs of Wistar rats. Hum Exp Toxicol 2010; 29: 593–599.

12.

Sokmen

Basaraner

Yanardag

. Combined effects of treatment with vitamin C, vitamin E and selenium on the skin of diabetic rats. Hum Exp Toxicol 2013; 32: 379–384.

13.

Utley

Bernheim

Hochstein

. Effect of sulfhydryl reagents on peroxidation in microsomes. Arch Biochem Biophys 1967; 118: 29–32.

14.

Bradley

Priebat

Christensen

, et al. Measurement of cutaneous inflammation: estimation of neutrophil content with an enzyme marker. J Invest Dermatol 1982; 78: 206–209.

15.

Plenderleith

. Treating the treatment: toxicity of cancer chemotherapy. Can Fam Phys 1990; 36: 1827.

16.

Livshits

Rao

Smith

. An approach to chemotherapy-associated toxicity. Emerg Med Clin North Am 2014; 32: 167–203.

17.

Hedigan

. Cancer: herbal medicine reduces chemotherapy toxicity. Nat Rev Drug Discov 2010; 9: 765.

18.

Vichaya

Chiu

Krukowski

, et al. Mechanisms of chemotherapy-induced behavioral toxicities. Front Neurosci 2015; 9: 131.

19.

King

Perry

. Hepatotoxicity of chemotherapy. Oncologist 2001; 6: 162–176.

20.

Maor

Malnick

. Liver injury induced by anticancer chemotherapy and radiation therapy. Int J Hepatol 2013; 2013: 815105.

21.

Al-Asmari

Khan

Al-Qasim

, et al. Ascorbic acid attenuates antineoplastic drug 5-fluorouracil induced gastrointestinal toxicity in rats by modulating the expression of inflammatory mediators. Toxicol Rep 2015; 2: 908–916.

22.

Moertel

Fleming

Macdonald

, et al. Hepatic toxicity associated with fluorouracil plus levamisole adjuvant therapy. J Clin Oncol 1993; 11: 2386–2390.

23.

Mossa

Osman

El-Sayed

, et al. Distribution and toxicity of 5-fluorouracil after intraperitoneal and anal submucosal administration. J Pharm Belg 1991; 47: 129–134.

24.

El-Sayyad

Ismail

Shalaby

, et al. Histopathological effects of cisplatin, doxorubicin and 5-flurouracil (5-FU) on the liver of male albino rats. Int J Biol Sci 2009; 5: 466.

25.

Zorzi

Laurent

Pawlik

, et al. Chemotherapy-associated hepatotoxicity and surgery for colorectal liver metastases. Br J Surg 2007; 94: 274–286.

26.

Yoshino

Yoshida

Nakajima

, et al. Alteration of the redox state with reactive oxygen species for 5-fluorouracil-induced oral mucositis in hamsters. PLoS One 2013; 8: e82834.

27.

Chen

Wei

, et al. Potential protection of vitamin C against liver-lesioned mice. Int Immunopharmacol 2014; 22: 492–497.

28.

Mozhdeganloo

Jafari

Koohi

, et al. Methylmercury-induced oxidative stress in rainbow trout (Oncorhynchus mykiss) liver: ameliorating effect of vitamin C. Biol Trace Elem Res 2015; 165: 103–109.

29.

Raina

Baba

Verma

, et al. Hepatotoxicity induced by subchronic exposure of fluoride and chlorpyrifos in Wistar rats: mitigating effect of ascorbic acid. Biol Trace Elem Res 2015; 166: 157–162.

30.

Klaassen

Reisman

. Nrf2 the rescue: effects of the antioxidative/electrophilic response on the liver. Toxicol Appl Pharmacol 2010; 244: 57–65.

31.

Copple

Goldring

Kitteringham

, et al. The Nrf2–Keap1 defence pathway: role in protection against drug-induced toxicity. Toxicology 2008; 246: 24–33.

32.

Kensler

Wakabayashi

Biswal

. Cell survival responses to environmental stresses via the Keap1-Nrf2-ARE pathway. Annu Rev Pharmacol Toxicol 2007; 47: 89–116.

33.

Itoh

Tong

Yamamoto

. Molecular mechanism activating Nrf2–Keap1 pathway in regulation of adaptive response to electrophiles. Free Rad Biol Med 2004; 36: 1208–1213.

34.

Numazawa

Sugihara

Miyake

, et al. Possible involvement of oxidative stress in 5-fluorouracil-mediated myelosuppression in mice. Basic Clin Pharmacol Toxicol 2011; 108: 40–45.

35.

Singh

Bhat

. Superoxide dismutase 3 is induced by antioxidants, inhibits oxidative DNA damage and is associated with inhibition of estrogen-induced breast cancer. Carcinogenesis 2012; 33: 2601–2610.

36.

Olalekan Lawal

Folusho Lawal

Ologundudu

, et al. Antioxidant effects of heated garlic juice on cadmium-induced liver damage in rats as compared to ascorbic acid. J Toxicol Sci 2011; 36: 549–557.

37.

Kim

, et al. Ascorbic acid reduces HMGB1 secretion in lipopolysaccharide-activated RAW 264.7 cells and improves survival rate in septic mice by activation of Nrf2/HO-1 signals. Biochem Pharmacol 2015; 95: 279–289.

38.

Muriel

. NF-κB in liver diseases: a target for drug therapy. J Appl Toxicol 2009; 29: 91–100.

39.

Luedde

Schwabe

. NF-κB in the liver—linking injury, fibrosis and hepatocellular carcinoma. Nat Rev Gastroenterol Hepatol 2011; 8: 108–118.

40.

Hayden

Ghosh

. Signaling to NF-kappaB. Genes Dev 2004; 18: 2195–2224.

41.

Wang

W-G

Xiu

R-J

Z-W

, et al. Protective effects of Vitamin C against spinal cord injury-induced renal damage through suppression of NF-κB and proinflammatory cytokines. Neurol Sci 2015; 36: 521–526.

42.

Cárcamo

Pedraza

Bórquez-Ojeda

, et al. Vitamin C suppresses TNFα-induced NFκB activation by inhibiting IκBα phosphorylation. Biochemistry 2002; 41: 12995–13002.

43.

Son

E-W

S-J

Rhee

D-K

, et al. Vitamin C blocks TNF-α-induced NF-kB activation and ICAM-1 expression in human neuroblastoma cells. Arch Pharm Res 2004; 27: 1073–1079.

44.

Abhilash

Harikrishnan

Indira

. Ascorbic acid suppresses endotoxemia and NF-κB signaling cascade in alcoholic liver fibrosis in guinea pigs: a mechanistic approach. Toxicol Appl Pharmacol 2014; 274: 215–224.

45.

Liang

Chen

, et al. Vitamin C exerts beneficial hepatoprotection against Concanavalin A-induced immunological hepatic injury in mice through inhibition of NF-κB signal pathway. Food Function 2014; 5: 2175–2182.

46.

Bowie

O’Neill

. Vitamin C inhibits NF-κB activation by TNF via the activation of p38 mitogen-activated protein kinase. J Immunol 2000; 165: 7180–7188.

47.

Cárcamo

Pedraza

Bórquez-Ojeda

, et al. Vitamin C is a kinase inhibitor: dehydroascorbic acid inhibits IκBα kinase β. Mol Cell Biol 2004; 24: 6645–6652.

48.

Dubois

Abramson

Crofford

, et al. Cyclooxygenase in biology and disease. FASEB J 1998; 12: 1063–1073.

49.

Yeoh

Gibson

Yeoh

, et al. A novel animal model to investigate fractionated radiotherapy-induced alimentary mucositis: the role of apoptosis, p53, nuclear factor-κB, COX-1, and COX-2. Mol Canc Therap 2007; 6: 2319–2327.

50.

Horrillo

Planagumà

González-Périz

, et al. Comparative protection against liver inflammation and fibrosis by a selective cyclooxygenase-2 inhibitor and a nonredox-type 5-lipoxygenase inhibitor. J Pharmacol Exp Therap 2007; 323: 778–786.

51.

K-Q

. Cyclooxygenase 2 (COX2)-prostanoid pathway and liver diseases. Prostaglandins Leukotr Essent Fatty Acids 2003; 69: 329–337.

52.

Lee

Kang

Jung

, et al. Vitamin C suppresses proliferation of the human melanoma cell SK-MEL-2 through the inhibition of cyclooxygenase-2 (COX-2) expression and the modulation of insulin-like growth factor II (IGF-II) production. J Cell Physiol 2008; 216: 180–188.

Mitigation of 5-fluorouracil–induced liver damage in rats by vitamin C via targeting redox–sensitive transcription factors

Abstract

Keywords

Introduction

Materials and methods

Chemicals and other reagents

Animals

Treatment regimen

Tissue processing

Lipid peroxidation

MPO activity

Immunohistochemical detection of Nrf2, NF-kB, and Cox-2 expression

Histological analysis

Statistical analysis

Results

Effect of Vit. C on 5-FU–induced hepatic oxidative stress (MDA level)

Effect of Vit. C on MPO activity in the liver after 5-FU injection

Effect of Vit. C on nuclear translocation of redox-sensitive transcription factors (NF-κB and Nrf2) and expression of Cox-2

Effect of Vit. C on the histological alterations in liver after 5-FU injection

Discussion

Footnotes

Acknowledgment

Declaration of Conflicting Interests

Funding

References