Methylene Blue Protects Against Acute Ethanol-Induced Oxidative Stress and Organ Damage

Main Article Content

Omar M.E. Abdel-Salam
Eman R. Youness
Fatma A Morsy
Amany Ameen Sleem


Ethanol (EtOH) intake is an important global health problem which affects many organs such as the brain, liver and stomach. The aim of the study was to examine the effect of the redox dye methylene blue (MethyB) on oxidative stress and histologic damage to the liver, gastric mucosa, and brain induced high dose ethanol (EtOH). Male rats were treated with EtOH (2 ml/rat, 96%) via intragastric route (for two consecutive days). MethyB (20 or 40 mg/kg, intraperitoneally) was given immediately after EtOH administration. The control group received saline. Rats were euthanized three hours after the last treatment. Brain and liver levels of malondialdehyde (MDA), reduced glutathione (GSH), and paraoxonase-1 (PON-1) as well as brain 5-lipoxygenase (5-LOX) and butyrylcholinesterase (BChE) were determined. Histopathological assessment of brain, liver and gastric damage was done. Results indicated that compared to saline treated animals, EtOH caused significant increase in MDA, along with decreased GSH and PON-1 activity in brain and liver. Additionally, it significantly increased 5-LOX and decreased brain BChE activity. The EtOH group showed the presence of dead and red neurons, and damage of glial cells. The liver exhibited vacuolar degeneration, apoptotic hepatocytes and foci of necrosis. The gastric mucosa showed areas of tissue damage, mucosal atrophy, and loss of normal architecture of glandular cells. The EtOH induced biochemical and histopathological alterations were alleviated after treatment with MethyB at a dose-dependent manner. These results demonstrate that MethyB is able to protect against from acute effects of EtOH on brain, liver and gastric tissue via an antioxidant action. MethyB might be of value in reducing tissue injury in acute EtOH intoxication.

Article Details

How to Cite
O. M. Abdel-Salam, E. R. Youness, F. A. Morsy, and A. A. Sleem, “Methylene Blue Protects Against Acute Ethanol-Induced Oxidative Stress and Organ Damage”, Int.J.Halal.Res, vol. 3, no. 2, pp. 88-102, Sep. 2021.


Abdel-Salam, O.M.E., Omara, E.A., Youness, E.R., Khadrawy, Y.A., Mohammed, N.A., Sleem, A.A., 2014. Rotenone-induced nigrostriatal toxicity is reduced by methylene blue. J. Neurorestoratol. 2, 65–80.doi:

Abdel-Salam, O.M.E., Youness, E.R., Mohammed, N.A., Abu Elhamed, W.A., 2015. Nuclear factor-kappa B and other oxidative stress biomarkers in serum of autistic children. Open J. Mol. Integ. Physiol. 5, 18–27. doi: 10.4236/ojmip.2015.51002.

Abdel-Salam, O.M.E., Youness, E.R., Morsy, F.A., Yassen, N.N., Mohammed, N.A., Sleem AA., 2016a. Methylene blue protects against toluene induced brain damage: involvement of nitric oxide, NF-κB, and caspase-3. React Oxygen Species 2(5), 371–387.doi: 10.20455/ROS.2016.855.

Abdel-Salam, O.M.E., Youness, E.R., Esmail, R.S.E., Mohammed, N.A., Khadrawy, Y.A., Sleem, A.A., 2016b. Methylene blue as a novel neuroprotectant in acute malathion intoxication. React Oxygen Species 1(2), 165–177.

Abdel-Salam, O.M.E., Sleem, A.A., Youness, E.R., Mohammed, N.A., Shaffie, N., Yassen, N.N., 2018. Neuro- and hepatoprotective effects of methylene blue in rats treated with lipopolysaccharide endotoxin. React Oxygen Species 6(17), 325–337. doi: 10.20455/ros.2018.849.

Abdel-Salam, O.M.E., Sleem, A.A., Medhat, D., Salama, R.A.A., Morsy, F.A., Farrag, A.R.H., et al., 2019. Methylene blue protects against acidified sodium taurocholate-induced gastric mucosal damage. React Oxygen Species 7(20), 93–105.doi: 10.20455/ros.2019.815.

Agrawal, N.M., Godiwala, T., Arimura, A., Dajani, E.Z., 1986. Cytoprotection by a synthetic prostaglandin against ethanol-induced gastric mucosal damage. A double-blind endoscopic study in human subjects. Gastrointest. Endosc. 32(2), 67-70. doi: 10.1016/s0016-5107(86)71757-4.doi:

Aksu, B., Umit, H., Kanter, M., Guzel, A., Aktas, C., Civelek, S., et al., 2010. Effects of methylene blue in reducing cholestatic oxidative stress and hepatic damage after bile-duct ligation in rats. Acta Histochemica 112(3), 259—269. doi: 10.1016/j.acthis.2008.12.002.

Atamna, H., Nguyen, A., Schultz, C., Boyle, K., Newberry, J., Kato, H., et al., 2008. Methylene blue delays cellular senescence and enhances key mitochondrial biochemical pathways. FASEB. J. 22(3), 703-712. doi: 10.1096/fj.07-9610com.

Blanco, A.M., Guerri, C., 2006. Alcohol and neuroinflammation: Involvement of astroglial cells and TLR4/IL-1RI receptors. Inmunología 25(3),188-200.

Bolkent, S., Arda-Pirincci, P., Bolkent, S., Yanardag, R., Tunali, S., Yildirim, S., 2006. Influence of zinc sulfate intake on acute ethanol-induced liver injury in rats. World J. Gastroenterol. 12(27), 4345-4351. doi: 10.3748/wjg.v12.i27.4345.

Bradberry, S.M., 2003. Occupational methaemoglobinaemia: mechanisms of production, features, diagnosis and management including the use of methylene blue. Toxicol. Rev. 22(1), 13‒27. doi: 10.2165/00139709-200322010-00003.

Budygin, E.A., Phillips, P.M., Robinson, D.L., Kennedy, A.P., Gainetdinov, R.R., Wightman, R.M., 2001. Effect of acute ethanol on striatal dopamine neurotransmission in ambulatory rats. J. Pharmacol. Exp. Ther. 297, 27–34.

Ceni, E., Mello, T., Galli, A., 2014. Pathogenesis of alcoholic liver disease: Role of oxidative metabolism. World J. Gastroenterol. 20(47), 17756-17772. doi: 10.3748/wjg.v20.i47.17756.

Chen, J.L., Dai, L., Zhang, P., Chen, W., Cai, G.S., Qi, X.W., et al., 2015. Methylene blue attenuates acute liver injury induced by paraquat in rats. International Immunopharmacology 28 (1), 808–812.

Clifton, J., 2nd, Leikin, J.B., 2003. Methylene blue. Am. J. Ther. 10(4), 289–291. doi: 10.1097/00045391-200307000-00009.

Comporti, M., Signorini, C., Leoncini, S., Gardi, C., Ciccoli, L., Giardini, A., et al, 2010. Ethanol-induced oxidative stress: basic knowledge. Genes Nutr. 5(2), 101–109. doi: 10.1007/s12263-009-0159-9.

Darvesh, S., Hopkins, D.A., Geula, C., 2003. Neurobiology of butyrylcholinesterase. Nat. Rev. Neurosci. 4(2), 131-138. doi: 10.1038/nrn1035.

Eckerson, H.W., Wyte, C.M., La Du, B.N., 1983. The human serum paraoxonase/arylesterase polymorphism. Am. J. Hum. Genet. 35(6), 1126–1138.

Ellithey, M., El Awdan, S.A.W., Abdel Jaleel, G., Shaffie. N., Abdel-Salam O.M.E., 2019. Origanum majorana water extract protects against ethanol and indomethacin-induced gastric mucosal damage via decreasing oxidative stress and cytokine release. J. Basic. Pharmacol. Toxicol. 3(1), 19-28.

Ellman, G.L., 1959. Tissue sulfhydryl groups. Arch. Biochem. Biophys. 82(1), 70–77.

Ellman, G.L., Courtney, K.D., Andres, V Jr., Feather-Stone, R.M., 1961. A new and rapid colorimetric determination of acetylcholinesterase activity. Biochem. Pharmacol. 7, 88-95.

Elshennawy, A.T.M., Sayed, S.R., Saber, E.A., Rifaai, R.A., 2015. Histopathological and histochemical assessment of the protective effects of zinc on ethanol-induced acute hepatotoxicity in adult albino rats. J. Cytol. Histol. 6, 3. doi: 10.4172/2157-7099.1000321.

Fenn, A.M., Skendelas, J.P., Moussa, D.N., Muccigrosso, M.M., Popovich, P.G., Lifshitz, J., et al., 2015. Methylene blue attenuates traumatic brain injury-associated neuroinflammation and acute depressive-like behavior in mice. J. Neurotrauma. 32(2), 127-138. doi: 10.1089/neu.2014.3514.

Gönenç S., Uysal N., Açikgöz O., Kayatekin B. M., Sönmez A., Kiray, M., et al., 2005. Effects of melatonin on oxidative stress and spatial memory impairment induced by acute ethanol treatment in rats. Physiol. Res. 54(3), 341-348.

Gordon, M.J., O'Brien, P., Skillman, J.J., Silen, W., 1975. The effect of carbenoxolone on changes in canine and human gastric mucosa caused by taurocholate and ethanol. Surgery 77(5), 707-714.

Grunwald, F., Schrock, H., Biersack, H.J., Kuschinsky, W., 1993. Changes in local cerebral glucose utilization in the awake rat during acute and chronic administration of ethanol. J. Nucl. Med. 34 (5), 793-798.

Gutteridge, J.M., 1995. Lipid peroxidation and antioxidants as biomarkers of tissue damage. Clin. Chem. 41 (12 Pt 2), 1819–1828.

Haorah, J., Knipe, B., Leibhart, J., Ghorpade, A., Persidsky, Y., 2005. Alcohol-induced oxidative stress in brain endothelial cells causes blood-brain barrier dysfunction. J. Leukoc. Biol. 78, 1223–1232. doi:

Haorah, J., Ramirez, S.H., Floreani, N., Gorantla, S., Morsey, B., Persidsky, Y., 2008. Mechanism of alcohol-induced oxidative stress and neuronal injury. Free Radic. Biol. Med. 45(11), 1542–1550. doi:10.1016/j.freeradbiomed.2008.08.030.

Iaquinto, G., Giardullo, N., Taccone, W., Leandro, G., Pasquale, L., De Luca, L., et al., 2003. Role of endogenous endothelin-1 in ethanol-induced gastric mucosal damage in humans. Dig. Dis. Sci. 48 (4), 663–669. doi: 10.1023/a:1022864120761.

Ji, C., 2012. Mechanisms of alcohol-induced endoplasmic reticulum stress and organ injuries. Biochem. Res. Int. art no 216450. doi:

Kelner, M.J., Bagnell, R., Hale, B., Alexander, N.M., 1988. Potential of Methylene Blue to Block Oxygen Radical Generation in Reperfusion Injury. In: Simic, M.G., Taylor, K.A., Ward, J.F., von Sonntag, C. (eds) Oxygen Radicals in Biology and Medicine. Basic Life Sciences, vol 49. Springer, Boston, MA.

Keskin, M., Dolar, E., Dirican, M., Kiyici, M., Yilmaz, Y., Gurel, S., et al., 2009. Baseline and salt-stimulated paraoxonase and arylesterase activities in patients with chronic liver disease: relation to disease severity. Intern. Med. J. 39(4), 243-248. doi: 10.1111/j.1445-5994.2009.01793.x.

Knoll, M.R., Kolbel, C.B., Teyssen, S., Singer, M.V., 1998. Action of pure ethanol and some alcoholic beverages on the gastric mucosa in healthy humans: a descriptive endoscopic study. Endoscopy 30(3), 293-301. doi: 10.1055/s-2007-1001257.

Kvietys, P.R., Twohig, B., Danzell, J., Specian, R.D., 1990. Ethanol-induced injury to the rat gastric mucosa. Role of neutrophils and xanthine oxidase-derived radicals. Gastroenterology 96(4), 909-920. doi: 10.1016/0016-5085(90)90015-s.

La Du, B.N., 1992. Human serum paraoxonase/arylesterase. In: Kalow, W. (ed) Pharmacogenetics of drug metabolism. Pergamon, New York, pp 51–91.

Lieber, C.S., 2004. New concepts of the pathogenesis of alcoholic liver disease lead to novel treatments. Curr. Gastroenterol. Rep. 6(1), 60–65. doi: 10.1007/s11894-004-0027-0.

Lin, A.L., Poteet, E., Du, F., Gourav, R.C., Liu, R., Wen, Y., et al., 2012. Methylene blue as a cerebral metabolic and hemodynamic enhancer. PLoS One 7(10), e46585. doi: 10.1371/journal.pone.0046585.

Lin, Z.H., Wang, S.Y., Chen, L., Zhuang, J.Y., Ke, Q.F., Xiao, D.R., et al., 2017. Methylene blue mitigates acute neuroinflammation after spinal cord injury through inhibiting NLRP3 inflammasome activation in microglia. Front. Cell. Neurosci. 11, 391. doi: 10.3389/fncel.2017.00391. eCollection 2017.

Liu, H., Zheng, W., Yan, G., Liu, B., Kong, L., Ding, Y., et al., 2014. Acute ethanol-induced changes in edema and metabolite concentrations in rat brain. BioMed. Research. International. Article ID 351903. doi:

Louvet, A., Mathurin, P. 2015. Alcoholic liver disease: mechanisms of injury and targeted treatment. Nat. Rev. Gastroenterol. Hepatol.12(4):231-42. doi: 10.1038/nrgastro.2015.35.

Manghelli, J., Brown, L., Tadros, H.B., Munfakh, N.A., 2015. A reminder of methylene blue's effectiveness in treating vasoplegic syndrome after on pump cardiac surgery. Tex. Heart. Inst. J. 42(5), 491-494.

Medina, D.X., Caccamo, A., Oddo, S., 2011. Methylene blue reduces abeta levels and rescues early cognitive deficit by increasing proteasome activity. Brain Pathol. 21(2), 140–149. doi: 10.1111/j.1750-3639.2010.00430.x.

Menini, T., Gugliucci, A., 2014. Paraoxonase 1 in neurological disorders. Redox Rep. 19(2), 49-58. doi: 10.1179/1351000213Y.0000000071.

Morsy, F.A., Abdel-Salam, O.M.E., Farrag A.R.H., Shabana, M.E., Sleem, A.A., 2019. Protection by methylene blue alone or with vitamin C on gastric mucosal damage and brain histopathological changes caused by indomethacin in rats. J. Basic. Pharmacol. Toxicol. 3(2), 6-16.

Motawi, T.K., Hamed, M.A., Hashem, R.M., Shabana, M.H., Ahmed, Y.R., 2012. Protective and therapeutic effects of argyreia speciosa against ethanol-induced gastric ulcer in rats. Z. Naturforsch. C. J. Biosci. 67(1-2), 47-57.doi: 10.1515/znc-2012-1-207.

Nair, V., Turner, G.A., 1984. The thiobarbituric acid test for lipid peroxidation: structure of the adduct with malondialdehyde. Lipids 19, 804-805.

Ng, D.S., Chu, T., Esposito, B., Hui P., Connelly, P.W., Grosset, P.L., 2008. Paraoxonase-1 deficiency in mice predisposes to vascular inflammation, oxidative stress, and thrombogenicity in the absence of hyperlipidemia. Cardiovasc. Pathol. 17(4), 226-32. doi: 10.1016/j.carpath.2007.10.001.

Obernier, J.A., Bouldin, T.W., Crews, F.T., 2002. Binge ethanol exposure in adult rats causes necrotic cell death. Alcohol. Clin. Exp. Res. 26(4), 547–557. doi:10.1111/j.1530-0277.2002.tb02573.x

Osna, N.A., Donohue, T.M., Jr., Kharbanda, K.K., 2017. Alcoholic liver disease: Pathogenesis and current management. Alcohol Res. 38(2), 147–161.

Radmark, O., Werz, O., Steinhilber, D., Samuelsson, B., 2007. 5-Lipoxygenase: regulation of expression and enzyme activity. Trends Biochem. Sci. 32(7),332-341.doi: 10.1016/j.tibs.2007.06.002.

Ramezani, A., Goudarzi, I., Lashkarboluki, T., Ghorbanian, M.T., Abrari, K., Salmani, M.E., 2012. Role of oxidative stress in ethanol-induced neurotoxicity in the developing cerebellum. Iran. J. Basic. Med. Sci. 15(4), 965-974.

Rojas, J.C., Bruchey, A.K., Gonzalez-Lima, F., 2012. Neurometabolic mechanisms for memory enhancement and neuroprotection of methylene blue. Prog. Neurobiol. 96(1), 32-45. doi: 10.1016/j.pneurobio.2011.10.007.

Schirmer, R.H., Adler, H., Pickhardt, M., Mandelkow, E., 2011. "Lest we forget you--methylene blue.". Neurobiol. Aging. 32(12), 2325.e7-16. doi: 10.1016/j.neurobiolaging.2010.12.012.

Silman, I., Sussman, J.L., 2005. Acetylcholinesterase: ‘classical’ and ‘nonclassical’ functions and pharmacology. Curr. Opin. Pharmacol. 5(3), 293-302. doi: 10.1016/j.coph.2005.01.01.

Sontag, E.M., Lotz, G.P., Agrawal, N., Tran, A., Aron, R., Yang, G., et al., 2012. Methylene blue modulates huntingtin aggregation intermediates and is protective in Huntington's disease models. J. Neurosci. 32(32), 11109–19. doi: 10.1523/JNEUROSCI.0895-12.2012.

Souli, A,, Sebai, H., Chehimi, L., Rtibi, K., Tounsi, H., Boubaker, S., et al., 2013. Hepatoprotective effect of carob against acute ethanol-induced oxidative stress in rat. Toxicol. Ind. Health. 31(9), 802-10.doi: 10.1177/0748233713475506.

Strate, L.L., Singh, P., Boylan, M.R., Piawah, S., Cao, Y., Chan, A.T., 2016. A prospective study of alcohol consumption and smoking and the risk of major gastrointestinal bleeding in men. PLOS ONE. doi:10.1371/journal.pone.0165278.

Sullivan, E.V., Pfefferbaum, A., 2005. Neurocircuitry in alcoholism: A substrate of disruption and repair. Psychopharmacology (Berl) 180(4),583-594.doi: 10.1007/s00213-005-2267-6.

Szabo, S., Trier, J.S., Brown, A., Schnoor, J., 1985. Early vascular injury and increased vascular permeability in gastric mucosal injury caused by ethanol in the rat. Gastroenterology 88(1 Pt 2), 228-236.doi: 10.1016/s0016-5085(85)80176-1.

Talley Watts, L., Long, J.A., Chemello, J., Van Koughnet, S., Fernandez, A., Huang, S., et l., 2014. Methylene blue is neuroprotective against mild traumatic brain injury. J. Neurotrauma. 31(11), 1063–1071.

Tarnawski, A.J., Hollander, D., 1985. Ethanol-induced gastric mucosal injury. Sequential analysis of morphologic and functional changes. Gastroenterol. Clin. Biol. 9(12 Pt 2),88-92.

Tretter, L., Horvath, G., Hölgyesi, A., Essek, F., Adam-Vizi, V., 2014. Enhanced hydrogen peroxide generation accompanies the beneficial bioenergetic effects of methylene blue in isolated brain mitochondria. Free. Radic. Biol. Med. 77, 317-730. doi: 10.1016/j.freeradbiomed.2014.09.024.

Turner, A.R., Duong, C.D., Good, D.J., 2003. Methylene blue for the treatment and prophylaxis of ifosfamide-induced encephalopathy. Clinical Oncology 15, 435–439. doi:10.1016/S0936-6555(03)00114-6.

Vonghia, L., Leggio, L., Ferrulli, A., Bertini, M., Gasbarrini, G., Addolorato, G., Alcoholism Treatment Study Group., 2008. Acute alcohol intoxication. Eur. J. Intern. Med. 19(8), 561-7. doi: 10.1016/j.ejim.2007.06.033.

Wang, J., Du, H., Jiang, L., Ma, X., de Graaf, R.A., Behar, K.L., et al., Oxidation of ethanol in the rat brain and effects associated with chronic ethanol exposure. Proc. Natl. Acad. Sci. USA 110 (35), 14444–14449. doi:10.1073/pnas.1306011110.

Whittle, B.J.R., Oren-Wolman, N., Guth, P.H., 1985. Gastric vasoconstrictor actions of leukotriene C4, PGF2,, and thromboxane mimetic U-46619 on rat submucosal microcirculation in vivo. Am. J. Physiol. 248(5 Pt 1), G580-6. doi: 10.1152/ajpgi.1985.248.5.G580.

Yonei, Y., Guth, P.H., 1991. Ethanol-induced gastric injury. Rrole of submucosal venoconstriction and leukotrienes. Dig. Dis. Sci. 36(5), 601-8. doi: 10.1007/BF01297026.

Zahr, N.M., Mayer, D., Rohlfing, T., Hasak, M.P., Hsu, O., Vinco, S., et al., 2010. Brain injury and recovery following binge ethanol: Evidence from in vivo magnetic resonance spectroscopy. Biol. Psychiatry. 67(9), 846-54.doi: 10.1016/j.biopsych.2009.10.028.