Medhat D, El-Khayat Z, El-Banna M, Abdel-Latif Y, Morsy S, El-Daly S. M, Hussein J. S. Protective Effect of Polyunsaturated Fatty Acids Against Experimental Lung Injury Induced by Acute Ethanol Inhalation. Biomed Pharmacol J 2019;12(2).

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Manuscript received on :15-Apr-2019
Manuscript accepted on :16-May-2019
Published online on: 11-06-2019

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Dalia Medhat, Zakaria El-Khayat, Mona El-Banna, Yasmin Abdel-Latif, Safaa Morsy, Sherien M. El-Daly and Jihan Seid Hussein*

Medical Biochemistry Department, Medical Division, National Research Centre, Dokki, Giza, Egypt.

Corresponding Author E-mail: jihan_husein@yahoo.com

DOI : https://dx.doi.org/10.13005/bpj/1672

Abstract

Ethanol-induced diseases include oxidative mechanisms by which prolonged endoplasmic reticulum (ER) stress results in genesis and accumulation of cytotoxic total fatty acid ethyl esters (FAEEs, non-oxidative metabolites of ethanol). FAEEs participate in the pathogenesis of alcoholic lung disease. Polyunsaturated fatty acids (PUFA) offer a possible protective effect against damage induced by ethanol inhalation. The present study aimed to investigate the protective effect of flaxseed and fish oils administration against toxicity induced by ethanol inhalation. Forty healthy female albino rats were divided into four groups (control, ethanol, flaxseed and fish oils). Lung superoxide dismutase (SOD) and malondialdehyde (MDA) were measured. Plasma advanced oxidation end product (AOPP) and phosphatidylinositol 3- kinase (PI₃K) were determined. Erythrocyte membrane fatty acids were extracted and fractionated by HPLC. Ethanol inhalation results in significant increase in lung MDA, plasma AOPP and erythrocyte membrane arachidonic acid (AA), linolenic acid (LA), and oleic acid (OA) along with a significant decrease in erythrocyte membrane alpha-linolenic acid (ALA), lung SOD, and plasma PI₃K while pretreatment with flaxseed and fish oils daily (1.2 ml/kg) significantly attenuated these parameters. Supplementation of marine PUFAs reduced the oxidative stress induced by ethanol inhalation in experimental animals.

Keywords

Advanced Oxidation Protein Product; Arachidonic Acid; Cell Membrane; HPLC; Phosphatidylinositol 3-Kinase

Introduction

Chronic administration of alcohol is one of the most prevalent and expensive type of drug abuse with significant multi-systemic pathophysiological consequences.¹ Hasin et al., reported that about 7 per cent of the U.S. adult population attains the diagnostic standard for alcohol abuse and/or alcoholism.²

Alcohol is a major component of many products such as solvents, antifreeze, varnish, shellac, canned fuel, and industrial and commercial cleaners. Alcohol toxicity occurs from various routes of administration including dermal application, inhalation, or methanol ingestion. Obviously, exposure to alcohol via any course leads to significant metabolic acidosis, ocular, central nervous system and respiratory alterations.³

Vapour inhalation is considered as a modality of alcohol administration that stimulates alcohol reliance in rodents.⁴ Romero et al., (2014) found an increase in oxidative stress and inflammatory markers in rats’ lung were fed chronically fed Lieber-De Carli ethanol liquid diets.⁵

Moderate exposure to alcohol results in metabolic and immunologic impacts. Chronic alcohol consumption, formation and aggregation of ethanol metabolites in the lungs results in lung and airway disorders, including asthma and pneumonia and consider a potential hazard agent for lung infection.⁶

Lung parenchyma is believed to be a subject of infectious and environmental factors; this sensitiveness is increased in the state of reduced or abnormalities of innate or adaptive immunity. Ethanol and its metabolites affect redox homeostasis, antioxidants, and immune defence and lead to immunosuppression thus accelerating the lung infection.⁶

n–3 polyunsaturated fatty acids (PUFAs) are a group of structurally related FAs. ALA is an 18-carbon n–3 PUFA and is a precursor of the other n–3 PUFAs can be produced through a series of metabolic steps. LA and ALA are considered to be an essential nutrient for humans and most animals. DHA is the final end product of ALA elongation and desaturation.⁷

Hussein et al., demonstrated that integration of omega-3 fatty acids modulates inflammatory and immune reactions, it is considered as a potential therapeutic agent for inflammatory and autoimmune diseases. In addition, n-3 PUFA inhibits the generation of transforming growth factor-α (TGF-α) due to n-3 PUFA–derived lipid mediators^.8

According to these mechanisms, we aimed to determine the possible protective effect of PUFAs against lung toxicity induced by ethanol inhalation in the experimental animal.

Materials and Method

Chemicals

Ethanol and standards of fatty acids, chloroform, methanol and ethyl ether are all HPLC grade and were purchased from Sigma-Aldrich (St. Louis, MO, USA). 100% natural cold pressed flaxseed oil was purchased from Imtenan Company (El Obour City 1^st Industrial Area, Egypt). Fish oil was purchased from SEDICO Pharmaceutical Company (6 October City, Egypt).

Ethanol Consumption

Rats were kept in cages and exposed to 5000 ml/m³ ethanol for the duration of 15 minutes daily for 14 days, the dose was modified from Mullin and Krivanek.⁹

Flaxseed oil and Fish oil Administration

Flaxseed and fish oils were administrated (1.2 ml/kg body weight) daily for two months by gavage according to Hussein et al.,⁸

Animals

Forty Swiss female albino rats (180 ± 20 g) purchased from Animal House, National Research Centre (NRC), Giza, Egypt. Animals were kept in clean cages of polypropylene and maintained in controlled room temperature (37º C) with light and dark cycle (12h:12h) , given a standard diet and water ad libitum along the experimental period. Animal procedures were performed in accordance with the Ethics Committee of the National Research Centre and followed the recommendations of the National Institutes of Health Guide for Care and Use of Laboratory Animals (Publication No. 85-23, revised 1985).

Methods

Experimental Design

Rats were assigned into four groups (10 rats each) as follows:

Group (I): Control group: Healthy rats received saline.

Group (II): Ethanol group: Healthy Rats exposed to ethanol for 14 days.

Group (III): Flaxseed group: Healthy Rats received flaxseed oil (1.2 ml/kg body weight) daily for two months by oral gavage then exposed to 5000 ml/m³ ethanol for the duration of 15 minutes daily for 14 days, the dose was modified from Mullin and Krivanek (1982).⁹

Group (IV): Fish oil group: Healthy Rats received fish oil (1.2 ml/kg body weight) daily for two months by oral gavage then exposed to 5000 ml/m³ ethanol for the duration of 15 minutes daily for 14 days, the dose was modified from Mullin and Krivanek⁹

After finalizing the experiment; rats were kept fasting overnight (12 hours). Under anaesthetic blood withdrawn in heparinized tubes; centrifuged (at 3000 rpm) using cooling centrifuge (Laborzentrifugen, 2K15, and Sigma, Germany); plasma was separated and stored at -20 °C for biochemical analysis. Packed red blood cells were used for the estimation of erythrocyte membrane fatty acids fractions. Lung was removed from each rat quickly; washed with ice-cold saline, homogenized in phosphate buffer (pH 7.4), and centrifuged (at 4000 rpm) for 15 minutes at 4°C and the supernatant was separated for biochemical analysis.⁸

Determination of Lung Superoxide Dismutase (SOD) and Malondialdehyde (MDA)

Lung SOD and MDA levels were determined colourimetrically according to the methods described by Nishikimi et al.,¹⁰ Uchiyama and Yamaguchi¹¹ respectively.

Determination of Plasma Advanced Oxidation Protein Product (AOPP) and Phosphatidylinositol 3-Kinase (PI₃K)

Plasma AOPP and PI₃K were estimated by Enzyme-linked immunosorbent assay kits according to Deschamps -Latscha et al. ¹² and the manufacturing kit.

Determination of Erythrocyte Membrane Fatty Acids by High-Performance Liquid Chromatography (HPLC)

Sample Treatment

Cell membrane was homogenized in 2% acetic acid- ethyl ether mixture (2:1 volume ratio). The solution was then filtered and centrifuged at 500 xg, the organic phase was evaporated to dryness. The extract was dissolved in 200 μl acetonitrile.^13-14

HPLC Conditions

HPLC system (Agilent technologies 1100 series) supplied by a quaternary pump (Quat Pump, G131A model) and C18 column (260 X 4.6, particle size 5 μm) was used. Mixture of acetonitrile/water (70/30) v/v used as mobile phase and delivered by isocratic elution with flow rate 1 ml/min and 200 nm wavelength. Standards serial dilutions were injected and their peak areas were determined. A linear standard curve was constructed by plotting peak areas versus the corresponding concentrations. Samples concentration was obtained from the standard curve.

Statistical Analysis

All data were expressed as mean ± SE. Distribution of the data was verified to be normal using Tests of Normality (SPSS package, version 16). Statistical significance was tested by one-way analysis of variance (ANOVA). For all tests, a probability (P < 0.05) was considered significant.

Results and Discussion

Severe ethanol exposure is a leading factor in the progression of liver, cardiovascular, and pulmonary diseases.¹⁵  A lot of alcohol administration methods have been used in experimental animals to investigate the potential mechanisms of alcohol to induce alcoholic liver, lung, and cardiovascular diseases in addition to offering diverse merits to researchers, including the invalidation of rat’ natural aversion to alcohol, and the reliable achievement of consistently high blood alcohol levels with relative ease.¹⁶

In this study, rats exposed to ethanol showed a significant decline in lung SOD with significant elevation in lung MDA (table 1).

Table 1: Lung SOD and MDA in different studied groups.

P^a value significant difference compared to control group.

P^b value significant difference compared to ethanol group.

Chronic ethanol consumption boosts the levels of activated transforming growth factor- β₁ (TGF-β₁) that activates NADPH oxidase (Nox). Nox₄ upregulates Nox₁ and Nox₂ causing the generation of reactive oxygen species (ROS) and reduction in antioxidants thus impair the function of alveolar macrophages.¹⁷

In agreement with our results, D’Onofrio et al., reported that alcohol consumption depletes levels of antioxidant in the lungs which results in chronic oxidative stress, and then impairs alveolar macrophage functions. Thus, decreasing both cellular-based microbial lung clearance and alveolar macrophage cell viability and resulting in increase in oxidative stress.¹⁸

The current study showed the elevation of AOPP level in alcohol group compared to the control group. AOPP is a novel described protein marker formed under oxidative damage status and it is not only a marker for oxidative stress but also represent a modern class of pathogenic mediator via redox-dependent pathway in addition it is implicated in oxidation-associated diseases. AOPP is strongly associated with overexpression of Nox which is known as the major cytosolic source of ROS thus results in cell damage and involved in diseases progression.¹⁹

As were observed in this study. Indeed, PI₃K was significantly decreased in ethanol group compared to the control group (table 2). Cederbaum, (2012) suggested that PI3K/Akt signaling has been described to activate NADPH oxidase (Nox), therefore producing ROS. Alcohol-associated oxidative stress in the lungs is associated with alcohol-driven changes in Nox enzymes functions and depletion of antioxidants including GSH and SOD that protect the cells against oxidative stress.²⁰

Table 2: Plasma AOPP and PI3K in different studied groups.

P^a value significant difference compared to control group.

P^b value significant difference compared to ethanol group.

Liver alcohol dehydrogenase (ADH) and microsomal mono-oxygenases (cytochrome P450 2E1 isozyme) are part of ethanol oxidative detoxification. Over consumption of ethanol results in impairment of the detoxification mechanisms and related to production of acetaldehyde that binds to cellular proteins and DNA results in oxidation of proteins that enhancing oxidative stress.²¹

Kaphalia and Calhoun (2013) reported that ethanol oxidation, ER stress, and generation of FAEEs directly lung inflammation through bronchial circulation.⁵ Cederbaum, (2012) reported another potential metabolic pathway in which chronic ethanol exposure motivate hepatic cytochrome P450 2E1, forming different profile of metabolites, which may have overlapping or distinct toxicities such as FAEEs.²⁰ On the other hand, suppression of hepatic ADH expedite the FAEEs formation through non-oxidative metabolism of ethanol catalyzed by FAEE synthase, which occurs in target organs including lungs of mammalians  exposed to ethanol.²¹

Our data revealed that rats exposed to ethanol showed a significant decrease in erythrocyte membrane ALA and LA along with an increase in AA and OA compared to the control group table (3).

Table 3: Erythrocyte membrane fatty acids in different studied groups.

P^a value significant difference compared to control group.

P^b value significant difference compared to the ethanol group.

Being a major component of the cell membrane, decreased amount of PUFAs affects cell surface and intracellular receptors and thus impairs the anti-inflammatory gene expression. PUFAs are a critical factor in lung inflammation in COPD patients.²²

Our results suggested that prophylactic treatment with flaxseed and fish oils significantly ameliorate oxidative stress and inflammatory markers compared to the ethanol group.

Omega-3 and omega-6 fatty acids have competitive interactions that are critical in modifying inflammation.²³ Studies showed an inverse association between dietary intake of PUFAs and systemic inflammation across different populations including people with chronic obstructive pulmonary disease (COPD).²⁴

Since the mechanisms of inflammation comprise activation of both nuclear factor кB (NF-кB) and peroxisome proliferator agonist receptors (PPAR) which known as a ligand-activated transcription factor. Interestingly, it was found that n-3 PUFAs binding effectively to PPAR. PUFAs-PPAR binds to peroxisome proliferator response elements on DNA to control gene expression, prevent the energizing of NF-кB, which can prohibit genes encoding for inflammatory factors including interleukin 6 (IL-6) and tumour necrosis factor α (TNF-α). This explains the anti-inflammatory and antioxidant mechanisms of n-3 PUFAs.²⁵

Titz et al., (2016) suggested a complex interplay between smoke exposures, lung disease, and systemic alterations in serum lipid profiles and reported that high dietary intake of anti-inflammatory ω-3 PUFAs was associated with a possible protective effect against smoking-related COPD.²⁶

Several studies reported that supplementation of PUFAs attenuate cell membrane fatty acids disturbance and inhibit inflammation progression as well as oxidative stress.^{14, 27}

Conclusion

We concluded that PUFAs may protect the lung from toxicity induced by ethanol inhalation through incorporation into the cell membrane thus increasing arachidonic acid and reducing protein glycation and oxidative stress.

Acknowledgements

Authors are grateful to the National Research Centre, Egypt for supporting this work.

Conflict of Interest

There is no conflict of interest.

References

1. Molina PE, Hoek JB, Nelson S, Guidot DM, Lang CH, Wands JR, Crawford JM. Mechanisms of alcohol-induced tissue injury. Alcohol Clin Exp Res, 2003; 27(3):563–575. doi: 10.1097/01.ALC.0000057946.57330.F7.
2. Hasin DS, Stinson FS, Ogburn E, Grant BF. Prevalence, correlates, disability, and comorbidity of DSM-IV alcohol abuse and dependence in the United States: results from the National Epidemiologic Survey on Alcohol and Related Conditions. Arch Gen Psychiatry, 2007; 64 (7):830–842. doi: 10.1001/archpsyc.64.7.830.
3. FreniaL. and Schauben J.L. Methanol inhalation toxicity. Annals of Emergency Medicine, 1993; 22(12):1919–1923.
4. McCool BA, Chappell AM. Chronic intermittent ethanol inhalation increases ethanol self-administration in both C57BL/6J and DBA/2J mice. Alcohol, 2015; 49(2):111–120. doi10.1016/j.alcohol.2015.01.003.
5. Romero F, Shah D, Duong M, Stafstrom W, Hoek JB, Kallen CB, Lang CH, Summer R. Chronic alcohol ingestion in rats alters lung metabolism, promotes lipid accumulation, and impairs alveolar macrophage functions. Am J Respir Cell Mol Biol, 2014;51(6):840–849.
6. Kaphalia L and  Calhoun Alcoholic Lung Injury: Metabolic, Biochemical and Immunological Aspects. Toxicol Lett, 2013; 24: 222(2).
7. Brenna JT. The efficiency of conversion of α-linolenic acid to long-chain n-3 fatty acids in man. Curr Opin Clin Nutr Metab Care, 2002; 5:127–32.
8. Hussein J, Abo Elmatty D, Medhat D, Mesbah N, Farrag AR and Fahmy H. Flaxseed oil attenuates experimental liver hepatitis. Der Pharmacia Lettre, 2016; 8 (8):142-150.
9. Mullin LS, Krivanek ND. Comparison of unconditioned reflex and conditioned avoidance tests in rats exposed by inhalation to carbon monoxide, 1,1,1-trichloroethane, toluene or ethanol. Neurotoxicology, 1982; 3:126–137.
10. Nishikimi M, Rao NA, Yagi K: The occurrence of superoxide anion in the reaction of reduced phenazine methosulphate and molecular oxygen. Biochemical and Biophysical Research Communications, 1972; 46 (2): 849–854.
11. Uchiyama S, Yamaguchi M. Alteration in serum and bone component findings induced in streptozotocin-diabetic rats is restored by zinc acexamate. International Journal of Molecular Medicine, 2003; 12: 949-954.
12. Deschamps-Latscha B, Witko-Sarsat  V, Nguyen-Khoa  T, Nguyen  AT, Gausson V, Mothu N. Advanced oxidation protein products as risk factors for atherosclerotic cardiovascular events in nondiabetic predialysis patients. Am J Kidney Dis, 2005; 45(1): 39-47.
13. El-khayat Z., Abo el-matty, D., Rasheed, W., Hussein, J., Shaker, O. and Raafat, J. Role of cell membrane fatty acids in insulin sensitivity in diabetic rats treated with flaxseed oil. International journal of pharmacy and pharmaceutical sciences, 2013; 5 (2): 146-151.
14. Medhat D, El-Bana MA, Ashour MN, Badawy E, Diab Y, Hussein J. New approaches in protecting against atherosclerosis in an experimental model of postmenopause. Journal of Applied Pharmaceutical Science, 2017; 7(11): 090-096.
15. Szabo G. Gut-liver axis in alcoholic liver disease. Gastroenterology, 2015; 148(1):30–36. doi: 10.1053/j.gastro.2014.10.042.
16. Shamseer L, Adams D, Brown N, Johnson JA, Vohra S. Antioxidant micronutrients for lung disease in cystic fibrosis. Cochrane Database Syst Rev, 2010; CD007020.
17. Yeligar SM, Harris FL, Hart CM, and  Brown Ethanol induces oxidative stress in alveolar macrophages via upregulation of NADPH oxidases. J Immunol, 2012; 15:188(8): 3648–3657.
18. D’Onofrio N, Servillo L, Giovane A, Casale R, Vitiello M, Marfella R, et al. Ergothioneine oxidation in the protection against high-glucose induced endothelial. Free Radical Biology and Medicine, 2016; 96: 211-222.
19. Browning EA, Chatterjee S, Fisher AB. Stop the flow: a paradigm for cell signaling mediated by reactive oxygen species in the pulmonary endothelium. Annu Rev Physiol, 2012; 74:403-24.
20. Cederbaum AI. Alcohol metabolism. Clinics in Liver Disease, 2012; 16 (4): 667–685.
21. Manautou J, Buss N, Carlson G. Oxidative and non-oxidative metabolism of ethanol by the rabbit lung. Toxicol letter, 1992;62:93–99.
22. Deckelbaum RJ, Worgall TS, Seo T. n-3 fatty acids and gene expression. Am J Clin Nutr, 2006 ; 83(6):1520S–1525S.
23. Romieu I, Torrent M, Garcia-Esteban R et al., Maternal fish intake during pregnancy and atopy and asthma in infancy. Clinical and Experimental Allergy, 2007; 37(4): 518–525.
24. Julia C, Touvier M, Meunier N, et al., Intakes of PUFAs were inversely associated with plasma C-reactive protein 12 years later in a middle-aged population with vitamin E as an effect modifier. J Nutr, 2013; 143:1760–1766.
25. Li K, Huang T, Zheng J, et al., Effect of marine-derived n-3 polyunsaturated fatty acids on C-reactive protein, interleukin 6 and tumor necrosis factor alpha: a meta-analysis. PLoS One, 2014; 9:e88103.
26. Titz B, Luettich K, Leroy P,  Boue S,  Vuillaume G, Vihervaara T, Ekroos K,  Martin F,  Peitsch M .C, and  Hoeng Alterations in Serum Polyunsaturated Fatty Acids and Eicosanoids in Patients with Mild to Moderate Chronic Obstructive Pulmonary Disease (COPD). Int J Mol Sci, 2016; 17(9): 1583.
27. El-khayat Z, AbasOA, Medhat D, Elghreeb M, Farrag AR and Mostafa N. Biochemical studies on the effect of flaxseed and corn oils on cell membrane phospholipids in Ehrlich ascites carcinoma and solid tumor in mice. Der Pharmacia Lettre, 2016; 8 (9):90-101.