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Hepatoprotective and antioxidant effects of fish oil on isoniazid-rifampin induced hepatotoxicity in rats
Accepted Manuscript Title: Hepatoprotective and Antioxidant Effects of Fish Oil on Isoniazid-Rifampin Induced Hepatotoxicity in Rats Authors: Abdul Samad Basheer, Aisha Siddiqui, Yam Nath Paudel, Md. Quamrul Hassan, Mohd. Imran, Abul Kalam Najmi, Mohd. Akhtar PII: DOI: Reference:
S2213-4344(16)30068-8 http://dx.doi.org/doi:10.1016/j.phanu.2017.01.002 PHANU 97
To appear in: Received date: Revised date: Accepted date:
1-11-2016 23-1-2017 23-1-2017
Please cite this article as: Abdul Samad Basheer, Aisha Siddiqui, Yam Nath Paudel, Md.Quamrul Hassan, Mohd.Imran, Abul Kalam Najmi, Mohd.Akhtar, Hepatoprotective and Antioxidant Effects of Fish Oil on Isoniazid-Rifampin Induced Hepatotoxicity in Rats, PharmaNutrition http://dx.doi.org/10.1016/j.phanu.2017.01.002 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Hepatoprotective and Antioxidant Effects of Fish Oil on Isoniazid-Rifampin Induced
Hepatotoxicity in Rats.
Abdul Samad Basheer, Aisha Siddiqui, Yam Nath Paudel, Md. Quamrul Hassan,
Mohd Imran, Abul Kalam Najmi, Mohd Akhtar*
Department of Pharmacology, Faculty of Pharmacy, Jamia Hamdard (Hamdard University),
New Delhi-110 062, INDIA.
*Corresponding Author Dr.Mohd Akhtar Assistant Professor Department of Pharmacology Faculty of Pharmacy Jamia Hamdard (Hamdard University) New Delhi- 110 062, INDIA Mobile No +91- 9811777883 Email-Id- [email protected]; [email protected]
Graphical abstract Fish oil
Isoniazid- Rifampin
Reduced
Oxidative stress and Production of Hydrazine metabolite
Toxic metabolites generation Reduced (Antioxidant)
Reduced Hepatotoxicity Fish Oil
Hepatoprotective
HIGHLIGHTS
Isoniazid-Rifampin Induced Hepatotoxicity in Rats was observed
Demonstration of Hepatoprotective and Antioxidant Effects of Fish Oil on IsoniazidRifampin Induced Hepatotoxicity in Rats
Fish Oil may have therapeutic effects as well as food supplement in tuberculosis patients
Abstract In the present study hepatoprotective and antioxidant potential of fish oil (cod liver oil) against isoniazid and rifampin combination (INH-RMP)-induced toxicity was evaluated in rats. Administration of (50 mg INH+100 mg RMP/kg/day, intraperitonially [i.p]) for 14 daysproducedliver injury that was evident from elevated levels of serum marker enzymes, alanine aminotransferase (ALT), aspartate aminotransferase (AST) and alkaline phosphatase (ALP), and histopathological changes. The drug treatment caused significant changes in the cellular redox status as reflected by about 75% decrease in the level of serum total antioxidant capacity (TAC), a marked decrease in reduced glutathione (GSH) level, an increased in lipid peroxidation (LPO) in the liver. Fish oil treatment (4 ml/kg/day, i.p.) 5-6 hrprior to (INH-RMP) dose, markedlyprevented the rise in serum enzymes levelselicited by the drugs. The histopathological alterations were also improved. Remarkably, there was a complete reversal of the changes in the levels of GSH and LPO, and partial recovery of TAC in the animals receiving fish oil along with (INH-RMP). These observations support the mechanistic role of strong antioxidant property of fish oil in the protection of hepatic injury. This study suggests the importance of fish oil as a hepatoprotective and antioxidant dietary
supplement, particularly in the patients receiving anti-tubercular therapy which are at a risk of hepatotoxicity.
Key Words Fish Oil, Antioxidant, Liver protection, Isoniazid-Rifampin, Hepatotoxicity
Running Title: Hepatoprotective and Antioxidant Effects of Fish Oil
1. Introduction Tuberculosis (TB), a disease caused by a bacillus Mycobacterium tuberculosis, is one of the oldest and deadliest diseases known to mankind. TB remains a serious public health problem among certain world populations in the developing countries even today [1]. With the advent of anti-tubercular chemotherapy, there has been a marked improvement in the natural history of the disease.Among the anti-tubercular drugs available, isoniazid (INH) and rifampin (RMP) are the first-line essential drugs and a mainstay of the anti-tubercular combination therapy along with second-line drugs, pyrazinamide, ethambutol and streptomycin.The most frequent adverse effects of anti-tubercular treatment are hepatitis, skin reactions and gastrointestinal upset [2,3].
Anti-tubercular drugs-induced hepatotoxicity causes substantial morbidity and mortality. The major deterrent to the prolonged use of both drugs is their potential to cause hepatotoxicity, which is further enhanced when the two drugs are used in combination [4, 5].Studies undertaken in the past have proposed a possible role of enhanced formation of toxic INH metabolites (acetylisoniazid, acetylhydrazine, hydrazine and their reactive products) by RMP and the resultant oxidative injury in the hepatotoxicity caused by these drugs combination [5,6]. Attempts have been made in the past to investigate the hepatoprotective potential of some herbal preparations, synthetic compounds or natural antioxidants against the toxicity of these drugs [7,24].Silymarin, an extract from the seeds of S. marianum, has been shown to protect ratliver against toxic effects of anti-tubercular drugs [7,8]. Fish oil (FO) can be obtained from eating fish or taking supplements available in the market. Effectiveness of fish oil has been shown in different pathological conditions such as heart disease, stroke, neurological disorders, osteoporosis, obesity, eye diseases, inflammation and even cancer [9]. However, some clinicalstudies do not support a beneficial role for omega-3 fatty acids supplementation in preventing cardiovascular disease, stroke or cancer[10].The benefits of fishoil (cod liver oil) have been attributed to high contents of omega-3 fatty acids (34%), eicosapentaenoic acid (EPA, 15%)and docosahexaenoic acid (DHA, 19%)and fat soluble vitamins A, D and E [11,12]. Long chain omega-3 fatty acids are essential as they are not synthesized by mammals and, therefore, must be supplemented in the diet for good health.Other rich sources of omega-3 fatty acids include edible seeds oils and walnut [13]. Prolonged feeding to the rats with fish oil results in omega-3 fatty acids incorporation into hepatic lipids, inhibition of de novolipogenesis and change in the hepatic fatty acid profile [14].
Much less information is available on the effectiveness of fish oil in the liver diseases and in particular hepatic injury induced by drugs and other chemicals. Previous studies have shown hepatoprotective potential of fish oil againsttoxicity inducedby sodium nitrite in rats and carbon tetrachloride in rabbit [12, 15]. Another study showed a decrease in paracetamolinduced hepatic lipid peroxidation in the rats receiving fish oil for 7 days [16].The current study was therefore, designed to evaluate the hepatoprotective and antioxidant potential of fish oil against liver injury induced by INH and RMP combination in rats in order to understand the mechanism of hepatoprotective activity of fish oil. 2. Materials and Methods 2.1.
Drugs and Chemicals
A standard cod liver oil formulation (Seacod®, marketed by Sanofi-France in India) was used. Silymarinan extract from the seeds ofS. Marianum (marketed by Zenith Nutrition, USA in India) was commercially available. Isoniazid and rifampin (Active Pharmaceutical Ingredient) were purchased from Sisco Research Laboratories Pvt. Ltd., India. The assay kitsof ALT, AST and ALP were purchased from Span Diagnostics,Surat, India. All other chemicals used were of the analytical grade. 2.2.
Animals and Treatments
All the experimental procedures involving the use of laboratory animals were approvedby the Institutional Animal Ethics Committee of Hamdard University, New Delhi. Wistar albino rats of either sex weighing 180-250 g were procured from Central Animal House Facility of the University. The animals were housed in polypropylene cages for one week to acclimatize to the standard conditions (12 h light: dark cycle; temperature, 25±2 ºC) and provided ad libitum diet. After acclimatization, the rats were divided into six groups, each comprising of six animals (n=6), and treated as followed.Hepatic injury in rats was produced by intraperitoneal (i.p)
administration of a mixed dose of 50 mg INH + 100 mg RMP (dissolved in saline) for 14 consecutive days [7]. Animals were treated with fish oil (4 ml/kg, i.p.) 5-6 hr prior to (INHRMP) administration for 14 days [16]. Silymarin was administered orally to rats at a dose of 200 mg/kg, 5-6 hr prior to (INH-RMP) administration for 14 days [7]. Control groups of animals received normal saline, fish oil or silymarin alone and proceed under similar experimental conditions. Group 1: Control-normal saline, intraperitoneally (i.p.) for 14 consecutive days. Group 2: Fish Oil (FO) per se-4 ml/kg, i.p. for 14 days. Group 3: Silymarin (Sily) per se- 200 mg/kg, orally for 14 days. Group 4: (INH-RMP)-50 mg INH-100 mg RMP in saline/kg, i.p. daily for 14 days. Group 5: (INH-RMP)+FO - 4 ml/kg, i.p. 5-6 hr prior to 50 mg INH-100 mg RMP/kg, i.p. for 14 days. Group 6 : (INH-RMP)+Sily- 200 mg/kg, orally 5-6 hr prior to 50 mg INH-100 mg RMP/kg, i.p. for 14 days.
2.3.
Blood Collection and Tissue Preparation
Blood was collected by sinocular puncture 18-20 hr after the last drug treatment and centrifuged at 3,000g for 15 min to obtain serum for estimation of serum marker enzymes for liver function and total antioxidant capacity (TAC). After blood collection, rats were sacrificed by decapitation and the liver of each animal was removed, washed with ice-cold normal saline and processed separately for further investigations. A small piece of liver was fixed in 10% formaldehyde for histopathological examination. Remaining tissue was used for analysis of GSH and LPO as described below. 2.4.
Estimation of Serum EnzymesMarkers of Hepatotoxicity
The activities of serum marker enzymes, alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were estimated spectrophotometrically by the method of Reitman and Frankel [17] using commercially available kits. The activity ofalkaline phosphatase (ALP) was determined by following the formation of 4nitrophenol
from
4-nitrophenyl
phosphate
(substrate)
in
alkaline
medium
spectrophotometricallyat wavelength of 405 nm [18]. 2.5.
Estimation of Markers of Redox Status
The total antioxidant capacity (TAC) was estimated in the serum by the method of Koracevic et al [19] as described earlier [20].The method was based on the principle that Fe-EDTA complex reaction with hydrogen peroxide (Fenton reaction) generates hydroxyl radical, which degrades benzoate resulting in the release of thiobarbituric acid reactive substances (TBARS). The inhibitory potential of serum against TBARS production is then determined as antioxidant activity in mmole/l usinguric acid as a standard antioxidant.The method for TAC estimation is given below. The reaction mixture containing 0.5 ml of phosphate buffer (100 mmole/l, pH 7.4), 0.5 ml of sodium benzoate (10 mmole/l), 0.2 ml of Fe-EDTA (2 mmole/l EDTA + 2 mmole/l of Fe[NH4]2SO4, 0.2 ml 0f H2O2 (10mmole/l) and 0.01 ml of serum was incubated for 60 minutes at 37ºC. The reaction was stopped by addition of 1 ml of 20% acetic acid. One ml of thiobarbituric acid (TBA) solution (0.8% in 50 mmole/l NaOH) was added, and the solution was heated for 10 minutes at 100ºC. The absorbance of the pink color thus formed was estimated spectrophotometrically at 532 nm. 0.01 ml of uric acid (1mmole/l in 5 mmole/l NaOH) was used as the standard antioxidant for determining the antioxidant activity (AOA) of unknown samples. Proper blanks were run under similar experimental conditions. The AOA of each sample was calculated as follows:
(CU) (K-A) AOA (mmole/l) =
(K- UA)
Where K= absorbance of control; A= absorbance of sample; UA= absorbance of uric acid solution; CU= concentration of uric acid (mmole/l). Protein was determined by the method of Lowry et al. using bovine serum albumin as standard [21]. The content of GSH was measured as non-protein sulfhydryl group using Ellman’s reagent, 5, 5´-dithio (2-nitrobenzoic acid) in the liver was determined as described earlier by Sedlak and Lindsay [22]. Sulfhydryl content was measured in the supernatant obtained after deprotenization of tissue homogenate with trichloroacetic acid and detected by reacting with the Ellman’s reagent. For lipid peroxidation (LPO) estimation 10% liver homogenate was prepared in ice-cold 1.15% KCl with the help of Potter-Elvehjem homogenizer. The homogenates were centrifuged separately at 9,000 g for 20 min at 4 0C and the supernatant fractions thus obtained were used for measuring lipid peroxidation in terms of endogenous thiobarbituricacid reactive substances (TBARS) formed using malondialdehyde (MDA) as a standardby the method reported earlie r[23].
2.6.
MorphologicExamination of Liver
The liver tissue fixed in 10% formaldehyde solution was processed by the procedure described earlier [24]. Thin sections obtained from paraffin wax block of the tissue were stained with haematotoxylin and eosin (H&E) and mounted on glass slides for study of changes in liver histology. 2.7.
Statistical Analysis
The results were expressed as mean ± SEM and analysed using the one-way analysis of the variance test (ANOVA) followed by Dunnett’s t-test. For statistical analysis we used GraphPad InStat-3 and Microsoft Office Excel 2010. A probability level of P<0.05 was chosen as the criterion of statistical significance. 3.
Results
3.1.
Effect ofFish Oil on Serum Enzyme Markers
Treatment of rats (Group 4) with a combined dose of (INH-RMP) for 14 consecutive days caused a significant increase (p<0.01) in the activities of serum marker enzymes, ALT (68%), AST (126%) and ALP (79%), compared to control values, indicating liver damage (Table 1). Administration of fish oil to Group 5 rats (4ml/kg/day, i.p.) for 14 days, 5-6hr prior to (INHRMP) dose markedlyprevented the rise in serum enzymes levels. Likewise, under these experimental conditions there was a significant reduction (p<0.01) in the (INH-RMP)induced increase in the serum enzymes levels in the rats (Group 6) receiving 200 mg silymarin/kg/day, orally for 14 days, a standard hepatoprotective agent. The serum enzymes levels observed in the rats treated with only fish oil per se (Group 2) or silymarin per se (Group 3) were not significantly different from the normal valuesrecorded in the control group receiving saline alone (Group 1) (Table 1).
3.2.
Effect of Fish Oil on Oxidative Stress Markers
The effects of (INH-RMP) combination and fish oil on oxidative stress were assessed in the rats by measuring the TAC in serum (Table 1) and the levels of GSH and lipid peroxidation in liver (Table 2). There was about 75% decrease in the serumTAC activity in the rats (Group 4) treated with (INH-RMP), when compared to that of control group (Group 1). The concentration of GSH in the liver of these rats was reduced by 55%. The depletion of
antioxidant defences resulted in liver injury as was reflected by about 189% increase in lipid peroxidation, measured as TBARS, in the liver of drugs treated rats (Table 2). Co-administration of fish oil with (INH-RMP) to rats (Group 5 markedly contained the lowering of serum TAC and hepatic GSH levels invoked by hepatotoxic dosage of the drugs. There was significantly reduced (p<0.01) lipid peroxidation in the liver of these animals compared to those receiving the drugs only (Group 4). These effects were in line with those elicited by the standard hepatoprotective agent, silymarin. Administration of silymarin together with (INH-RMP) significantly (p<0.05) reversed the changes in the levels of TAC, GSH, and lipid peroxidation induced by the drugs. The levels of redox parameters observed in the rats receiving fish oil per se (Group 2) and silymarin per se (Group 3) were not significantly different from normal values recorded in Control group (Group 1)(Table 2).
3.3.
Effect of Fish Oil on Liver Morphology
The morphology of the rat liver samples representing the six groups were shown in Figure 1 A to F. Photomicrographs of the liver of Control (normal), Fish Oil per se and Silymarin per se groups showed normal arrangement of hepatocytes around central vein and portal triads (Fig. 1A, B & C). The liver of rats (Group 4) treated with (INH-RMP) showed enlarged hepatocytes, vacuolation, inflammatory cells infiltration and a few hepatocytes containing centrally located nucleus with reticular boundary (Fig. 1D). Co-administration of fish oil with (INHRMP) protected the hepatocytes with relatively less histopathological alterations (Fig. 1E). Administration of silymarin with (INH-RMP) also resulted in the recovery of hepatocytes in Group 6 rats (Fig. 1F) as compared to the animals treated with drugs only (Group 4).
4. Discussion In the present study hepatotoxicity was produced in rats by intraperitoneal administration of anti-tubercular drugs combination of INH and RMP for 14 days. The extent of liver injury was evident from about two fold increase in serum marker enzymes and histopathological changes in liver, which was in line with the observations reported in previous studies where this model has been used to elucidate the mechanism of hepatotoxicity produced by antitubercular drugs and its protection by a variety of synthetic and herbal preparations [7,25]. Multiple mechanisms have been proposed to explain the toxicity of these drugs. Recent studies have suggested that the toxic metabolites of INH, mainly CYP 2E1-mediated reactive oxidative products of hydrazine and acetylhydrazine, play a crucial role in liver injury [26]. The combined use of INH and RMP has been shown to enhance the risk of hepatotoxicity in both clinical and experimental studies. Rifampin alone may also invoke hepatocellular dysfunction, but earlier its mechanism of hepatotoxicity was unknown [27]. Later on it was reported that rifampin particularly, activates cytochrome P450 (CYP3A4) via hepatocyte xeno sensing pregnane X receptor (PXR) which leads to increased metabolism of isoniazid and thus formation of toxic metabolites [28]. Rifampin, a potent inducer of xenobiotic metabolising enzymes, has been reported to increase the metabolism of isoniazid by a hydrolase and CYP 2E1 to reactive toxic metabolites, resulting in enhanced INH toxicity [7, 26, 28].The reactive species thus generated can produce oxidative stress and trigger membrane lipid peroxidation, deplete cellular antioxidant and bind/or react covalently with cellular components resulting in hepatocyte damage [28]. Moreover, a strong correlation between oxidative stress and hepatotoxicity has been demonstrated in experimental animals treated with anti-tubercular drugs [29, 30]. Lower plasma GSH level and higher malondialdehyde concentration were found in tuberclosis (TB) patients with anti-tubercular drug induced hepatotoxicity [26]. In the current
study INH+RMP-induced hepatotoxicity was associated with a marked decrease in the levels of serum TAC (75%) and hepatic GSH (55%), and an increase in lipid peroxidation in liver (189%). Depletion of cellular antioxidant defences and/or excessive production of reactive oxygen species can lead to increased membrane lipid peroxidation and cell damage. Co-administration of fish oil (cod liver oil) with (INH-RMP) combination for 14 days in the present study resulted in marked protection against the drugs induced hepatotoxicity. Notably such treatment restored the drug induced changes in the redox parameters resulting in decreased oxidative stress. This was further supported by positive histopathological alterations by fish oil treatment.The hepatoprotective and antioxidant effects of silymarin (milk thistle), a standard hepatoprotective agent, was also investigated in this study and was found to be comparable to those reported earlier [7,8]. The hepatoprotective effect of fish oil has been demonstrated earlier against liver injury induced by carbon tetrachloride in rabbits [15]. Another study has reported a significant decrease in paracetamol induced hepatic lipid peroxidation in the rats pre-treated with fish oil at a dose of 4 ml/kg/day, i.p. for 7 days [16]. Administration of cod liver oil treatment (5 ml/kg, orally) to rats for 12 weeks alleviated sodium nitrite induced hepatic injury through multiple mechanisms including mitigation of oxidative stress, blocking proinflammatory monocyte chemoattractant protein (MCP)-1 and reduction of DNA fragmentation [12]. Moreover, the hepatoprotective effects of omega-3 fatty acids (a mixture of eicosapentaenoic acid [EPA] and docosahexaenoic acid [DHA] in the ratio 3:2) against thioacetamide-induced injury in rats has been shown to be mediated through suppression of oxidative stress and inhibition of Nuclear Factor Kappa B (NF-KB), a key transcriptional factor regulator implicated in the pathogenesis of hepatic inflammation and fibrosis [31]. In another study chronic DHA supplementation was found to be effective in attenuating BDL-induced cholestatic liver injury and associated oxidative stress, inflammation and fibrosis [32]. In
addition cod liver oil is also a good source of vitamin A and E supplementation, which is also likely to contribute to the beneficial effects of fish oil in liver diseases. Antioxidant and hepatoprotective effects of vitamins A and E have been demonstrated against hepatic injury induced by gasoline [33] and cadmium [34] in rats. Conclusion: The observations of the present study suggest that the underlying mechanism of hepatoprotective action of fish oil and its ingredients involves suppression of oxidative stress, reinforcement of cellular antioxidant defences, cell membrane stabilization and that fish oil may be used as a hepatoprotective and antioxidant dietary supplement, particularly in the patients receiving anti-tubercular therapy which are at a risk of hepatotoxicity.
Disclosure Statement The authors declare that they do not have any conflicts of interest. Acknowledgement Thanks are due to All India Council for Technical Education for awarding scholarship to one of the author during the course of this study.
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Figure Legends Fig. 1.Photomicrographs of the liver (H & E; 100×), The histopathological changes observed in the rat liver representing the six groups were shown in Figure 1 A to F. Photomicrographs of the liver of Control (normal), Fish Oil per se and Silymarin per se groups showed normal arrangement of hepatocytes around central vein and portal triads (Fig. 1A, B & C). The liver of rats treated with (INH-RMP) showed enlarged hepatocytes, vacuolation, inflammatory cells infiltration and a few hepatocytes containing centrally located nucleus with reticular boundary (Fig. 1D). Co-administration of fish oil with (INH-RMP) protected the hepatocytes with relatively less histopathological alterations (Fig. 1E). Administration of silymarin with (INH-RMP) also resulted in the recovery of hepatocytes in Group 6 rats (Fig. 1F) as compared to the animals treated with drugs only (Group 4).
Table 1.Effect of Fish Oil on Serum Enzyme Markers in Isoniazid-Rifampin Induced Hepatotoxicity in Rats.
Groups
ALT
AST
ALP
TAC
( IU/L)
(IU/L)
(IU/L)
(mmoles/l)
Control
35.2±1.5
18.7±0.5
271±9
1.21±0.07
Fish Oil per se
40.6±2.6
20.5±0.7
290±10
1.27±0.03
Silymarinper se
36.0±1.3
20.0±2.1
293±8
1.30±0.02
(INH-RMP)
59.2±3.1##
42.2±1.6##
486±10##
0.30±0.02##
(INH-RMP)+ FO
43.3±0.9**
21.7±1.3**
306±9**
0.85±0.09**
(INH-RMP)+ Sily
45.6±0.6**
30.7±0.5**
311±12**
0.55±0.05*
Values were mean ± SEM from 6 rats (n=6) in each group analysed by using the one-way analysis of the variance test (ANOVA) followed by Dunnett’s t-test. ##
P<0.01: Values were significantly different from that of control.
*
P<0.05, **P<0.01: Values are significantly different from that of (INH-RMP) group.
P>0.05: Values of FO per se and silymarinper se groups are not significantly different from that of control. INH- isoniazid, RMP- rifampin, FO- fish oil, Sily- silymarin
Table 2.Effect of Fish Oil on Oxidative Stress Markers in Isoniazid-Rifampin Induced Hepatotoxicity in Rats.
Groups
GSH (µmoles/mg protein)
LPO (nmoles MDA/mg protein)
Control
0.182±0.015
0.214±0.005
Fish Oil per se
0.211±0.006
0.276±0.023
Silymarinper se
0.167±0.004
0.266±0.018
(INH-RMP)
0.082±0.011##
0.618±0.068##
(INH-RMP)+ FO
0.176±0.008**
0.282±0.015**
(INH-RMP)+ Sily
0.127±0.015*
0.362±0.004**
Values were mean ± SEM from 6 rats (n=6) in each group analysed by using the one-way analysis of the variance test (ANOVA) followed by Dunnett’s t-test. ##
P<0.01: Values are significantly different from that of control.
*
P<0.05, **P<0.01: Values are significantly different from that of (INH-RMP) group.
P>0.05: Values of FO per se and silymarinper se groups are not significantly different from that of control. INH- isoniazid, RMP- rifampin, FO- fish oil, Sily- silymarin
S2213-4344(16)30068-8 http://dx.doi.org/doi:10.1016/j.phanu.2017.01.002 PHANU 97
To appear in: Received date: Revised date: Accepted date:
1-11-2016 23-1-2017 23-1-2017
Please cite this article as: Abdul Samad Basheer, Aisha Siddiqui, Yam Nath Paudel, Md.Quamrul Hassan, Mohd.Imran, Abul Kalam Najmi, Mohd.Akhtar, Hepatoprotective and Antioxidant Effects of Fish Oil on Isoniazid-Rifampin Induced Hepatotoxicity in Rats, PharmaNutrition http://dx.doi.org/10.1016/j.phanu.2017.01.002 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Hepatoprotective and Antioxidant Effects of Fish Oil on Isoniazid-Rifampin Induced
Hepatotoxicity in Rats.
Abdul Samad Basheer, Aisha Siddiqui, Yam Nath Paudel, Md. Quamrul Hassan,
Mohd Imran, Abul Kalam Najmi, Mohd Akhtar*
Department of Pharmacology, Faculty of Pharmacy, Jamia Hamdard (Hamdard University),
New Delhi-110 062, INDIA.
*Corresponding Author Dr.Mohd Akhtar Assistant Professor Department of Pharmacology Faculty of Pharmacy Jamia Hamdard (Hamdard University) New Delhi- 110 062, INDIA Mobile No +91- 9811777883 Email-Id- [email protected]; [email protected]
Graphical abstract Fish oil
Isoniazid- Rifampin
Reduced
Oxidative stress and Production of Hydrazine metabolite
Toxic metabolites generation Reduced (Antioxidant)
Reduced Hepatotoxicity Fish Oil
Hepatoprotective
HIGHLIGHTS
Isoniazid-Rifampin Induced Hepatotoxicity in Rats was observed
Demonstration of Hepatoprotective and Antioxidant Effects of Fish Oil on IsoniazidRifampin Induced Hepatotoxicity in Rats
Fish Oil may have therapeutic effects as well as food supplement in tuberculosis patients
Abstract In the present study hepatoprotective and antioxidant potential of fish oil (cod liver oil) against isoniazid and rifampin combination (INH-RMP)-induced toxicity was evaluated in rats. Administration of (50 mg INH+100 mg RMP/kg/day, intraperitonially [i.p]) for 14 daysproducedliver injury that was evident from elevated levels of serum marker enzymes, alanine aminotransferase (ALT), aspartate aminotransferase (AST) and alkaline phosphatase (ALP), and histopathological changes. The drug treatment caused significant changes in the cellular redox status as reflected by about 75% decrease in the level of serum total antioxidant capacity (TAC), a marked decrease in reduced glutathione (GSH) level, an increased in lipid peroxidation (LPO) in the liver. Fish oil treatment (4 ml/kg/day, i.p.) 5-6 hrprior to (INH-RMP) dose, markedlyprevented the rise in serum enzymes levelselicited by the drugs. The histopathological alterations were also improved. Remarkably, there was a complete reversal of the changes in the levels of GSH and LPO, and partial recovery of TAC in the animals receiving fish oil along with (INH-RMP). These observations support the mechanistic role of strong antioxidant property of fish oil in the protection of hepatic injury. This study suggests the importance of fish oil as a hepatoprotective and antioxidant dietary
supplement, particularly in the patients receiving anti-tubercular therapy which are at a risk of hepatotoxicity.
Key Words Fish Oil, Antioxidant, Liver protection, Isoniazid-Rifampin, Hepatotoxicity
Running Title: Hepatoprotective and Antioxidant Effects of Fish Oil
1. Introduction Tuberculosis (TB), a disease caused by a bacillus Mycobacterium tuberculosis, is one of the oldest and deadliest diseases known to mankind. TB remains a serious public health problem among certain world populations in the developing countries even today [1]. With the advent of anti-tubercular chemotherapy, there has been a marked improvement in the natural history of the disease.Among the anti-tubercular drugs available, isoniazid (INH) and rifampin (RMP) are the first-line essential drugs and a mainstay of the anti-tubercular combination therapy along with second-line drugs, pyrazinamide, ethambutol and streptomycin.The most frequent adverse effects of anti-tubercular treatment are hepatitis, skin reactions and gastrointestinal upset [2,3].
Anti-tubercular drugs-induced hepatotoxicity causes substantial morbidity and mortality. The major deterrent to the prolonged use of both drugs is their potential to cause hepatotoxicity, which is further enhanced when the two drugs are used in combination [4, 5].Studies undertaken in the past have proposed a possible role of enhanced formation of toxic INH metabolites (acetylisoniazid, acetylhydrazine, hydrazine and their reactive products) by RMP and the resultant oxidative injury in the hepatotoxicity caused by these drugs combination [5,6]. Attempts have been made in the past to investigate the hepatoprotective potential of some herbal preparations, synthetic compounds or natural antioxidants against the toxicity of these drugs [7,24].Silymarin, an extract from the seeds of S. marianum, has been shown to protect ratliver against toxic effects of anti-tubercular drugs [7,8]. Fish oil (FO) can be obtained from eating fish or taking supplements available in the market. Effectiveness of fish oil has been shown in different pathological conditions such as heart disease, stroke, neurological disorders, osteoporosis, obesity, eye diseases, inflammation and even cancer [9]. However, some clinicalstudies do not support a beneficial role for omega-3 fatty acids supplementation in preventing cardiovascular disease, stroke or cancer[10].The benefits of fishoil (cod liver oil) have been attributed to high contents of omega-3 fatty acids (34%), eicosapentaenoic acid (EPA, 15%)and docosahexaenoic acid (DHA, 19%)and fat soluble vitamins A, D and E [11,12]. Long chain omega-3 fatty acids are essential as they are not synthesized by mammals and, therefore, must be supplemented in the diet for good health.Other rich sources of omega-3 fatty acids include edible seeds oils and walnut [13]. Prolonged feeding to the rats with fish oil results in omega-3 fatty acids incorporation into hepatic lipids, inhibition of de novolipogenesis and change in the hepatic fatty acid profile [14].
Much less information is available on the effectiveness of fish oil in the liver diseases and in particular hepatic injury induced by drugs and other chemicals. Previous studies have shown hepatoprotective potential of fish oil againsttoxicity inducedby sodium nitrite in rats and carbon tetrachloride in rabbit [12, 15]. Another study showed a decrease in paracetamolinduced hepatic lipid peroxidation in the rats receiving fish oil for 7 days [16].The current study was therefore, designed to evaluate the hepatoprotective and antioxidant potential of fish oil against liver injury induced by INH and RMP combination in rats in order to understand the mechanism of hepatoprotective activity of fish oil. 2. Materials and Methods 2.1.
Drugs and Chemicals
A standard cod liver oil formulation (Seacod®, marketed by Sanofi-France in India) was used. Silymarinan extract from the seeds ofS. Marianum (marketed by Zenith Nutrition, USA in India) was commercially available. Isoniazid and rifampin (Active Pharmaceutical Ingredient) were purchased from Sisco Research Laboratories Pvt. Ltd., India. The assay kitsof ALT, AST and ALP were purchased from Span Diagnostics,Surat, India. All other chemicals used were of the analytical grade. 2.2.
Animals and Treatments
All the experimental procedures involving the use of laboratory animals were approvedby the Institutional Animal Ethics Committee of Hamdard University, New Delhi. Wistar albino rats of either sex weighing 180-250 g were procured from Central Animal House Facility of the University. The animals were housed in polypropylene cages for one week to acclimatize to the standard conditions (12 h light: dark cycle; temperature, 25±2 ºC) and provided ad libitum diet. After acclimatization, the rats were divided into six groups, each comprising of six animals (n=6), and treated as followed.Hepatic injury in rats was produced by intraperitoneal (i.p)
administration of a mixed dose of 50 mg INH + 100 mg RMP (dissolved in saline) for 14 consecutive days [7]. Animals were treated with fish oil (4 ml/kg, i.p.) 5-6 hr prior to (INHRMP) administration for 14 days [16]. Silymarin was administered orally to rats at a dose of 200 mg/kg, 5-6 hr prior to (INH-RMP) administration for 14 days [7]. Control groups of animals received normal saline, fish oil or silymarin alone and proceed under similar experimental conditions. Group 1: Control-normal saline, intraperitoneally (i.p.) for 14 consecutive days. Group 2: Fish Oil (FO) per se-4 ml/kg, i.p. for 14 days. Group 3: Silymarin (Sily) per se- 200 mg/kg, orally for 14 days. Group 4: (INH-RMP)-50 mg INH-100 mg RMP in saline/kg, i.p. daily for 14 days. Group 5: (INH-RMP)+FO - 4 ml/kg, i.p. 5-6 hr prior to 50 mg INH-100 mg RMP/kg, i.p. for 14 days. Group 6 : (INH-RMP)+Sily- 200 mg/kg, orally 5-6 hr prior to 50 mg INH-100 mg RMP/kg, i.p. for 14 days.
2.3.
Blood Collection and Tissue Preparation
Blood was collected by sinocular puncture 18-20 hr after the last drug treatment and centrifuged at 3,000g for 15 min to obtain serum for estimation of serum marker enzymes for liver function and total antioxidant capacity (TAC). After blood collection, rats were sacrificed by decapitation and the liver of each animal was removed, washed with ice-cold normal saline and processed separately for further investigations. A small piece of liver was fixed in 10% formaldehyde for histopathological examination. Remaining tissue was used for analysis of GSH and LPO as described below. 2.4.
Estimation of Serum EnzymesMarkers of Hepatotoxicity
The activities of serum marker enzymes, alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were estimated spectrophotometrically by the method of Reitman and Frankel [17] using commercially available kits. The activity ofalkaline phosphatase (ALP) was determined by following the formation of 4nitrophenol
from
4-nitrophenyl
phosphate
(substrate)
in
alkaline
medium
spectrophotometricallyat wavelength of 405 nm [18]. 2.5.
Estimation of Markers of Redox Status
The total antioxidant capacity (TAC) was estimated in the serum by the method of Koracevic et al [19] as described earlier [20].The method was based on the principle that Fe-EDTA complex reaction with hydrogen peroxide (Fenton reaction) generates hydroxyl radical, which degrades benzoate resulting in the release of thiobarbituric acid reactive substances (TBARS). The inhibitory potential of serum against TBARS production is then determined as antioxidant activity in mmole/l usinguric acid as a standard antioxidant.The method for TAC estimation is given below. The reaction mixture containing 0.5 ml of phosphate buffer (100 mmole/l, pH 7.4), 0.5 ml of sodium benzoate (10 mmole/l), 0.2 ml of Fe-EDTA (2 mmole/l EDTA + 2 mmole/l of Fe[NH4]2SO4, 0.2 ml 0f H2O2 (10mmole/l) and 0.01 ml of serum was incubated for 60 minutes at 37ºC. The reaction was stopped by addition of 1 ml of 20% acetic acid. One ml of thiobarbituric acid (TBA) solution (0.8% in 50 mmole/l NaOH) was added, and the solution was heated for 10 minutes at 100ºC. The absorbance of the pink color thus formed was estimated spectrophotometrically at 532 nm. 0.01 ml of uric acid (1mmole/l in 5 mmole/l NaOH) was used as the standard antioxidant for determining the antioxidant activity (AOA) of unknown samples. Proper blanks were run under similar experimental conditions. The AOA of each sample was calculated as follows:
(CU) (K-A) AOA (mmole/l) =
(K- UA)
Where K= absorbance of control; A= absorbance of sample; UA= absorbance of uric acid solution; CU= concentration of uric acid (mmole/l). Protein was determined by the method of Lowry et al. using bovine serum albumin as standard [21]. The content of GSH was measured as non-protein sulfhydryl group using Ellman’s reagent, 5, 5´-dithio (2-nitrobenzoic acid) in the liver was determined as described earlier by Sedlak and Lindsay [22]. Sulfhydryl content was measured in the supernatant obtained after deprotenization of tissue homogenate with trichloroacetic acid and detected by reacting with the Ellman’s reagent. For lipid peroxidation (LPO) estimation 10% liver homogenate was prepared in ice-cold 1.15% KCl with the help of Potter-Elvehjem homogenizer. The homogenates were centrifuged separately at 9,000 g for 20 min at 4 0C and the supernatant fractions thus obtained were used for measuring lipid peroxidation in terms of endogenous thiobarbituricacid reactive substances (TBARS) formed using malondialdehyde (MDA) as a standardby the method reported earlie r[23].
2.6.
MorphologicExamination of Liver
The liver tissue fixed in 10% formaldehyde solution was processed by the procedure described earlier [24]. Thin sections obtained from paraffin wax block of the tissue were stained with haematotoxylin and eosin (H&E) and mounted on glass slides for study of changes in liver histology. 2.7.
Statistical Analysis
The results were expressed as mean ± SEM and analysed using the one-way analysis of the variance test (ANOVA) followed by Dunnett’s t-test. For statistical analysis we used GraphPad InStat-3 and Microsoft Office Excel 2010. A probability level of P<0.05 was chosen as the criterion of statistical significance. 3.
Results
3.1.
Effect ofFish Oil on Serum Enzyme Markers
Treatment of rats (Group 4) with a combined dose of (INH-RMP) for 14 consecutive days caused a significant increase (p<0.01) in the activities of serum marker enzymes, ALT (68%), AST (126%) and ALP (79%), compared to control values, indicating liver damage (Table 1). Administration of fish oil to Group 5 rats (4ml/kg/day, i.p.) for 14 days, 5-6hr prior to (INHRMP) dose markedlyprevented the rise in serum enzymes levels. Likewise, under these experimental conditions there was a significant reduction (p<0.01) in the (INH-RMP)induced increase in the serum enzymes levels in the rats (Group 6) receiving 200 mg silymarin/kg/day, orally for 14 days, a standard hepatoprotective agent. The serum enzymes levels observed in the rats treated with only fish oil per se (Group 2) or silymarin per se (Group 3) were not significantly different from the normal valuesrecorded in the control group receiving saline alone (Group 1) (Table 1).
3.2.
Effect of Fish Oil on Oxidative Stress Markers
The effects of (INH-RMP) combination and fish oil on oxidative stress were assessed in the rats by measuring the TAC in serum (Table 1) and the levels of GSH and lipid peroxidation in liver (Table 2). There was about 75% decrease in the serumTAC activity in the rats (Group 4) treated with (INH-RMP), when compared to that of control group (Group 1). The concentration of GSH in the liver of these rats was reduced by 55%. The depletion of
antioxidant defences resulted in liver injury as was reflected by about 189% increase in lipid peroxidation, measured as TBARS, in the liver of drugs treated rats (Table 2). Co-administration of fish oil with (INH-RMP) to rats (Group 5 markedly contained the lowering of serum TAC and hepatic GSH levels invoked by hepatotoxic dosage of the drugs. There was significantly reduced (p<0.01) lipid peroxidation in the liver of these animals compared to those receiving the drugs only (Group 4). These effects were in line with those elicited by the standard hepatoprotective agent, silymarin. Administration of silymarin together with (INH-RMP) significantly (p<0.05) reversed the changes in the levels of TAC, GSH, and lipid peroxidation induced by the drugs. The levels of redox parameters observed in the rats receiving fish oil per se (Group 2) and silymarin per se (Group 3) were not significantly different from normal values recorded in Control group (Group 1)(Table 2).
3.3.
Effect of Fish Oil on Liver Morphology
The morphology of the rat liver samples representing the six groups were shown in Figure 1 A to F. Photomicrographs of the liver of Control (normal), Fish Oil per se and Silymarin per se groups showed normal arrangement of hepatocytes around central vein and portal triads (Fig. 1A, B & C). The liver of rats (Group 4) treated with (INH-RMP) showed enlarged hepatocytes, vacuolation, inflammatory cells infiltration and a few hepatocytes containing centrally located nucleus with reticular boundary (Fig. 1D). Co-administration of fish oil with (INHRMP) protected the hepatocytes with relatively less histopathological alterations (Fig. 1E). Administration of silymarin with (INH-RMP) also resulted in the recovery of hepatocytes in Group 6 rats (Fig. 1F) as compared to the animals treated with drugs only (Group 4).
4. Discussion In the present study hepatotoxicity was produced in rats by intraperitoneal administration of anti-tubercular drugs combination of INH and RMP for 14 days. The extent of liver injury was evident from about two fold increase in serum marker enzymes and histopathological changes in liver, which was in line with the observations reported in previous studies where this model has been used to elucidate the mechanism of hepatotoxicity produced by antitubercular drugs and its protection by a variety of synthetic and herbal preparations [7,25]. Multiple mechanisms have been proposed to explain the toxicity of these drugs. Recent studies have suggested that the toxic metabolites of INH, mainly CYP 2E1-mediated reactive oxidative products of hydrazine and acetylhydrazine, play a crucial role in liver injury [26]. The combined use of INH and RMP has been shown to enhance the risk of hepatotoxicity in both clinical and experimental studies. Rifampin alone may also invoke hepatocellular dysfunction, but earlier its mechanism of hepatotoxicity was unknown [27]. Later on it was reported that rifampin particularly, activates cytochrome P450 (CYP3A4) via hepatocyte xeno sensing pregnane X receptor (PXR) which leads to increased metabolism of isoniazid and thus formation of toxic metabolites [28]. Rifampin, a potent inducer of xenobiotic metabolising enzymes, has been reported to increase the metabolism of isoniazid by a hydrolase and CYP 2E1 to reactive toxic metabolites, resulting in enhanced INH toxicity [7, 26, 28].The reactive species thus generated can produce oxidative stress and trigger membrane lipid peroxidation, deplete cellular antioxidant and bind/or react covalently with cellular components resulting in hepatocyte damage [28]. Moreover, a strong correlation between oxidative stress and hepatotoxicity has been demonstrated in experimental animals treated with anti-tubercular drugs [29, 30]. Lower plasma GSH level and higher malondialdehyde concentration were found in tuberclosis (TB) patients with anti-tubercular drug induced hepatotoxicity [26]. In the current
study INH+RMP-induced hepatotoxicity was associated with a marked decrease in the levels of serum TAC (75%) and hepatic GSH (55%), and an increase in lipid peroxidation in liver (189%). Depletion of cellular antioxidant defences and/or excessive production of reactive oxygen species can lead to increased membrane lipid peroxidation and cell damage. Co-administration of fish oil (cod liver oil) with (INH-RMP) combination for 14 days in the present study resulted in marked protection against the drugs induced hepatotoxicity. Notably such treatment restored the drug induced changes in the redox parameters resulting in decreased oxidative stress. This was further supported by positive histopathological alterations by fish oil treatment.The hepatoprotective and antioxidant effects of silymarin (milk thistle), a standard hepatoprotective agent, was also investigated in this study and was found to be comparable to those reported earlier [7,8]. The hepatoprotective effect of fish oil has been demonstrated earlier against liver injury induced by carbon tetrachloride in rabbits [15]. Another study has reported a significant decrease in paracetamol induced hepatic lipid peroxidation in the rats pre-treated with fish oil at a dose of 4 ml/kg/day, i.p. for 7 days [16]. Administration of cod liver oil treatment (5 ml/kg, orally) to rats for 12 weeks alleviated sodium nitrite induced hepatic injury through multiple mechanisms including mitigation of oxidative stress, blocking proinflammatory monocyte chemoattractant protein (MCP)-1 and reduction of DNA fragmentation [12]. Moreover, the hepatoprotective effects of omega-3 fatty acids (a mixture of eicosapentaenoic acid [EPA] and docosahexaenoic acid [DHA] in the ratio 3:2) against thioacetamide-induced injury in rats has been shown to be mediated through suppression of oxidative stress and inhibition of Nuclear Factor Kappa B (NF-KB), a key transcriptional factor regulator implicated in the pathogenesis of hepatic inflammation and fibrosis [31]. In another study chronic DHA supplementation was found to be effective in attenuating BDL-induced cholestatic liver injury and associated oxidative stress, inflammation and fibrosis [32]. In
addition cod liver oil is also a good source of vitamin A and E supplementation, which is also likely to contribute to the beneficial effects of fish oil in liver diseases. Antioxidant and hepatoprotective effects of vitamins A and E have been demonstrated against hepatic injury induced by gasoline [33] and cadmium [34] in rats. Conclusion: The observations of the present study suggest that the underlying mechanism of hepatoprotective action of fish oil and its ingredients involves suppression of oxidative stress, reinforcement of cellular antioxidant defences, cell membrane stabilization and that fish oil may be used as a hepatoprotective and antioxidant dietary supplement, particularly in the patients receiving anti-tubercular therapy which are at a risk of hepatotoxicity.
Disclosure Statement The authors declare that they do not have any conflicts of interest. Acknowledgement Thanks are due to All India Council for Technical Education for awarding scholarship to one of the author during the course of this study.
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Figure Legends Fig. 1.Photomicrographs of the liver (H & E; 100×), The histopathological changes observed in the rat liver representing the six groups were shown in Figure 1 A to F. Photomicrographs of the liver of Control (normal), Fish Oil per se and Silymarin per se groups showed normal arrangement of hepatocytes around central vein and portal triads (Fig. 1A, B & C). The liver of rats treated with (INH-RMP) showed enlarged hepatocytes, vacuolation, inflammatory cells infiltration and a few hepatocytes containing centrally located nucleus with reticular boundary (Fig. 1D). Co-administration of fish oil with (INH-RMP) protected the hepatocytes with relatively less histopathological alterations (Fig. 1E). Administration of silymarin with (INH-RMP) also resulted in the recovery of hepatocytes in Group 6 rats (Fig. 1F) as compared to the animals treated with drugs only (Group 4).
Table 1.Effect of Fish Oil on Serum Enzyme Markers in Isoniazid-Rifampin Induced Hepatotoxicity in Rats.
Groups
ALT
AST
ALP
TAC
( IU/L)
(IU/L)
(IU/L)
(mmoles/l)
Control
35.2±1.5
18.7±0.5
271±9
1.21±0.07
Fish Oil per se
40.6±2.6
20.5±0.7
290±10
1.27±0.03
Silymarinper se
36.0±1.3
20.0±2.1
293±8
1.30±0.02
(INH-RMP)
59.2±3.1##
42.2±1.6##
486±10##
0.30±0.02##
(INH-RMP)+ FO
43.3±0.9**
21.7±1.3**
306±9**
0.85±0.09**
(INH-RMP)+ Sily
45.6±0.6**
30.7±0.5**
311±12**
0.55±0.05*
Values were mean ± SEM from 6 rats (n=6) in each group analysed by using the one-way analysis of the variance test (ANOVA) followed by Dunnett’s t-test. ##
P<0.01: Values were significantly different from that of control.
*
P<0.05, **P<0.01: Values are significantly different from that of (INH-RMP) group.
P>0.05: Values of FO per se and silymarinper se groups are not significantly different from that of control. INH- isoniazid, RMP- rifampin, FO- fish oil, Sily- silymarin
Table 2.Effect of Fish Oil on Oxidative Stress Markers in Isoniazid-Rifampin Induced Hepatotoxicity in Rats.
Groups
GSH (µmoles/mg protein)
LPO (nmoles MDA/mg protein)
Control
0.182±0.015
0.214±0.005
Fish Oil per se
0.211±0.006
0.276±0.023
Silymarinper se
0.167±0.004
0.266±0.018
(INH-RMP)
0.082±0.011##
0.618±0.068##
(INH-RMP)+ FO
0.176±0.008**
0.282±0.015**
(INH-RMP)+ Sily
0.127±0.015*
0.362±0.004**
Values were mean ± SEM from 6 rats (n=6) in each group analysed by using the one-way analysis of the variance test (ANOVA) followed by Dunnett’s t-test. ##
P<0.01: Values are significantly different from that of control.
*
P<0.05, **P<0.01: Values are significantly different from that of (INH-RMP) group.
P>0.05: Values of FO per se and silymarinper se groups are not significantly different from that of control. INH- isoniazid, RMP- rifampin, FO- fish oil, Sily- silymarin