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Protective effect of treatment with thiamine or benfotiamine on liver oxidative damage in rat model of acute ethanol intoxication
Life Sciences 162 (2016) 21–24
Contents lists available at ScienceDirect
Life Sciences journal homepage: www.elsevier.com/locate/lifescie
Protective effect of treatment with thiamine or benfotiamine on liver oxidative damage in rat model of acute ethanol intoxication Guilherme Vannucchi Portari a,⁎, Paula Payão Ovidio b, Rafael Deminice c, Alceu Afonso Jordão Jr. a b c
b
Department of Nutrition, Health Sciences Institute, Federal University of Triangulo Mineiro, Brazil Department of Internal Medicine, Faculty of Medicine of Ribeirão Preto, University of São Paulo, Brazil Department of Physical Education, Faculty of Physical Education and Sport, State University of Londrina, Brazil
a r t i c l e
i n f o
Article history: Received 13 May 2016 Received in revised form 15 August 2016 Accepted 17 August 2016 Available online 18 August 2016 Keywords: Thiamine Benfotiamine Oxidative stress Acute alcohol intoxication
a b s t r a c t Aims: The aim of this study was to evaluate possible beneficial effects of treatment with thiamine or benfotiamine in an animal model of acute ethanol intoxication. Main methods: Thirty male Wistar rats were separated at random into three groups of 10 animals each: Ethanol (E), Ethanol treated with thiamine (T) and Ethanol treated with benfotiamine (BE). Rats were gavaged with single dose of ethanol (5 g/kg, 40% v:v). After 30 min of ethanol gavage the animals were treated with thiamine or benfotiamine. Six hours after first gavage, the animals were euthanized and blood and liver samples were collected for ethanol and oxidative stress biomarkers quantification. Key findings: Serum ethanol levels were higher in animals treated with thiamine or benfotiamine while hepatic alcohol levels were higher in animals of the group treated with benfotiamine comparing to controls or thiamine treated groups. The lipid peroxidation biomarkers were diminished for the groups treated with thiamine or benfotiamine comparing to E animals. Concerning protein oxidative damage parameters, they were enhanced for animals treated with benfotiamine in relation to other groups. Significance: In conclusion, the treatment with thiamine or benfotiamine even 30 min after the massive dose of ethanol has proven to be beneficial against liver damage. Improved results were obtained with benfotiamine in relation to oxidative damage from aqueous compartments. © 2016 Elsevier Inc. All rights reserved.
1. Introduction Ethanol metabolism produces free radicals and reactive oxygen species with consequent consumption of antioxidants leading to an imbalance between oxidants/antioxidants, known as oxidative stress. Several research groups have focused on the study of liver diseases caused by alcohol, since this is the organ responsible for about 95% of its catabolism [1]. The hepatic metabolism of ethanol is carried via three enzymatic pathways: pathway of alcohol dehydrogenase, the microsomal ethanol oxidation system (CYP2E1), and catalase [2,3]. While these metabolic pathways are well elucidated in the literature, little attention is given to the quantification of ethanol in liver although this can be an important finding to know the metabolizing status of alcohol in this organ. During the transformation of ethanol to acetaldehyde via CYP2E1 there is an overproduction of free radicals and reactive oxygen species,
⁎ Corresponding author at: Departamento de Nutrição, Universidade Federal do Triângulo Mineiro, Rua Getúlio Guaritá, 159 - sala 333, CEP: 38025-440 Uberaba, MG, Brazil. E-mail address: [email protected] (G.V. Portari).
http://dx.doi.org/10.1016/j.lfs.2016.08.017 0024-3205/© 2016 Elsevier Inc. All rights reserved.
mainly in the forms of superoxide anion (O‐2), hydrogen peroxide (H2O2) and hydroxyethyl radical (CH3CH(•)\\OH) [4–6]. These molecules are highly reactive and, as acetaldehyde, attack macromolecules, causing impairment or loss of function and, ultimately, cell death [7]. The use of antioxidants may help balance the hepatic antioxidant system reducing the deleterious effects caused by oxidative stress [8]. Thiamine, the vitamin B1, is a water soluble vitamin that plays essential role on energetic metabolism from carbohydrates. Benfotiamine is a weak soluble in water pro-vitamin B1 substance that displays better absorption and bioavailability compared with common pharmaceutical form available, thiamine hydrochloride, when administered orally, even after massive ethanol administration [12]. Thiamine therapeutic replacement is postulated in patients with Wernicke encephalopathy, especially alcoholics, due to deficiency of this vitamin in this particular group, because of poor dietary habits and a reduced absorption due to changes in the gastrointestinal system [9,10]. Hypothetically, thiamine supply could increase the metabolism of ethanol by restoring the microsomal ethanol oxidizing system (MEOS). Studies from our group have previously demonstrated some beneficial effects on intravenous administration of thiamine in an animal model of acute ethanol intoxication and more recently the high
22
G.V. Portari et al. / Life Sciences 162 (2016) 21–24
bioavailability of benfotiamine in rats acutely alcoholized [11,12]. In humans, benfotiamine has been tested as oral treatment of alcoholic polyneuropathy with great improvement of the symptoms [13]. The National Institute on Alcohol Abuse and Alcoholism (NIAAA) defines binge drinking as a pattern of drinking that brings blood alcohol concentration (BAC) levels to 0.8 g/L, typically occurring after 4 drinks for women and 5 drinks for men in about 2 h [14,15]. In experimental models this can be achieved by forced administration of a massive dose of ethanol by gavage since animals do not voluntarily consume alcohol at concentrations that produce intoxication [15]. It has been showed that, as the chronic models, the acute ethanol intoxication produces an increase in hepatic oxidative stress. In the present study we evaluate the effects of the administration of benfotiamine or thiamine on the improvement of hepatic oxidative damage and ethanol influx in a model of acute ethanol intoxication
were subjected to alkaline hydrolysis following proposed protocol by Cighetti et al. [18] with the following modifications: in the test tube 100 mg of liver (or 200 μL serum) were homogenized with 1 mL of 1.15% KCl. Then, it was added 3 mL of Milli-Q water and 0.5 mL of 2 M NaOH. After stirring the tubes they were heated at 60 °C for 30 min and then neutralized with 2 M HCl to follow the reaction with thiobarbituric acid. The lipid hydroperoxides were determinated using the ferrous oxidation-xylenol orange (FOX) assay as described by Sodergren et al. [19]. 2.5. Protein damage tests The extent of protein damage in serum and liver has been accessed by carbonyl content and advanced oxidation protein products (AOPP) assay following methodologies of Odetti et al. [20] and Witko-Sarsat et al. [21], respectively.
2. Material and methods 2.6. Status of antioxidants 2.1. Animals and experimental protocol Thirty male Wistar rats, weighing 270–333 g, were obtained from the Central Animal Facilities of the Ribeirão Preto Campus, University of São Paulo, and allowed to acclimate for 1 week in the animal facilities of the Department of Internal Medicine, Faculty of Medicine of Ribeirão Preto, University of São Paulo, under controlled conditions of a 12-h light: dark cycle and temperature of 24 ± 2 °C in individual cages with free access to food and water. The experimental protocol was approved by the Animal Research Ethics Commission (protocol no. 152). The animals were separated at random into three groups of 10 animals each: Ethanol (E + S), Ethanol treated with thiamine (E + T) and Ethanol treated with benfotiamine (E + BE). In the dark period of the previous day (10:00 p.m.) the chow was withdrawal to ensure that the stomach was not filled preventing reflux and aspiration of fluid into lungs and, mimic the eating habits frequently encountered in alcoholics. Next day, at 6:00 a.m. the rats were gavaged with single dose of 40% (w:v) ethanol in aqueous solution (5 g/kg of body weight) and, after 30 min each group received a second gavage to delivery 100 mg/kg of thiamine (E + T), in an aqueous solution, or benfotiamine (E + BE), in an aqueous dispersion, except for E + S group that received a saline sham gavage. Animals were left in cages with food and water ad libitum but due to sedation caused by ethanol none of them sought for food. Six hours after the first gavage, the animals were euthanized and blood and liver samples were collected, weighed, frozen immediately in liquid nitrogen and stored at −40 °C until analysis. 2.2. Serum and hepatic ethanol quantification Ethanol was quantified in serum and liver homogenates by previous validated gas chromatography method [16]. The hepatic ethanol concentration was multiplied by the respective liver mass to achieve total content of ethanol in this organ. Then, the percentual of ethanol relative to the initial doses was calculated. 2.3. Aspartate aminotransferase (AST) and alanine aminotransferase (ALT) To check for liver damage were measured enzymes AST and ALT by colorimetric reaction and spectrophotometric reading (UV–Vis Mod Q98U Quimis®, Diadema, SP, Brazil), using commercial kit (Labtest Diagnostica, Lagoa Santa, MG, Brazil). 2.4. Lipid peroxidation Lipid peroxidation in serum and liver homogenates was measured by thiobarbituric acid reactive substances [17]. To quantify the malondialdehyde bound to the macromolecules serum or homogenate
The status of antioxidants was accessed by determination of vitamin E (exogenous) and free (mainly glutathione) and total thiols (endogenous). Vitamin E was quantified in liver homogenates by HPLC following methodology described by Arnaud et al. [22] and the obtained values were corrected for lipid content. Thiols (free and total) in liver homogenates were determined by colorimetric assay after 5,5′-dithiobis-(2-nitrobenzoic acid) reaction [23]. 2.7. Fat content in liver homogenates For the quantification of total fat in liver homogenates the method proposed by Bligh and Dyer [24] was used. 2.8. Statistical analysis Differences between groups were determined by one-way analysis of variance (ANOVA) using a multiple comparison procedure (Tukey). A P value of b 0.05 was considered significant. Data are given as mean ± standard deviation of the mean. 3. Results In Table 1 we note that the groups receiving treatment with thiamine and benfotiamine had higher serum ethanol (P b 0.05) than group E + S. The same behavior was observed for liver ethanol concentrations, but with E + BE group reaching values significantly higher (P b 0.05) than the E + T group. When we analyze the percentage of hepatic ethanol relative to the initial dose, a significant difference (P b 0.05) among the three groups that received ethanol was found, with values of 1.3 ± 1.3%, 2.5 ± 0.9% and 3.6 ± 1.2% for groups E + S, E + T and E + BE, respectively. The ALT and AST levels (Table 2) were lower (P b 0.05) in groups which received treatment with thiamine and benfotiamine. Table 1 Characterization of the experimental model as the ethanol concentrations in serum and liver. Parameter
Serum ethanol (g/L) Hepatic ethanol (mg/g of tissue) Hepatic percentage (%) of ethanol in relation to initial dose
Groups E+S
E+T
E + BE
1.08 ± 0.85 1.32 ± 1.12 1.3 ± 1.3
1.99 ± 0.75⁎ 3.34 ± 1.38⁎ 2.5 ± 0.9⁎
1.90 ± 0.39⁎ 5.13 ± 1.83⁎,⁎⁎ 3.6 ± 1.2⁎,⁎⁎
Groups: E + S (Ethanol), E + T (Ethanol treated with thiamine), E + BE (Ethanol treated with benfotiamine). ⁎ P b 0.05 relative to E + S group. ⁎⁎ P b 0.05 relative to E + T group.
G.V. Portari et al. / Life Sciences 162 (2016) 21–24 Table 2 Serum ALT and AST levels. Groups
E+S
E+T
E + BE
ALT (U/L) AST (U/L)
67.1 ± 5.5 196.3 ± 15.8
54.7 ± 7.3⁎ 142.0 ± 23.1⁎
55.2 ± 5.5⁎ 131.2 ± 8.9⁎
Groups: E + S (Ethanol), E + T (Ethanol treated with thiamine), E + BE (Ethanol treated with benfotiamine). ⁎ P b 0.05 relative to E + S group.
Table 3 gives all measured biomarkers of oxidative stress. With respect to free TBARS in liver, represented by aldehydes from lipid peroxidation not linked to macromolecules, there was a significant increase (P b 0.05) in group E + S (18.0 ± 3.7 nmol/g protein) compared to other groups with significant drop for the E + T group (9.7 ± 3.2 nmol/g protein) and E + BE (10.0 ± 2.5 nmol/g protein). Analyzing the total hepatic TBARS, representing the sum of aldehydes from lipid peroxidation (linked to macromolecules plus free aldehydes), there was a significant reduction (P b 0.05) in the treated groups (E + T 5.4 ± 2.9 mmol/g protein and E + BE - 3.8 ± 1.3 mmol/g protein) compared to group E + S (10.9 ± 3.1 mmol/g protein). Serum analysis of free and total TBARS show similar behavior with a significant decrease (P b 0.05) in their concentrations for the E + BE and E + T groups compared to group E + S. With respect to serum hydroperoxides there was a significant increase (P b 0.05) in group E + S (44.8 ± 8.2 μmol/L) in comparison to other groups with significant drop for the group E + T (27.9 ± 7.3 μmol/L) and E + BE (30, 5 ± 4.5 μmol/L). The results of oxidative protein damage analyzed as AOPP and carbonyl content were lower for group E + BE. The hepatic and serum values of AOPP were significantly lower (P b 0.05) for E + BE group compared to group E + S and also group E + T. Similar values were observed in the values of the hepatic content of carbonyls but with differences between the three groups. No differences were observed in the analysis of serum content of carbonyls. With respect to vitamin E, an exogenous antioxidant mainly of lipid environment, there were differences between the three groups (P b 0.05) with values of 0.57 ± 0.13 μg/mg of fat (group E + BE), 0.92 ± 0.17 (group E + T) and 1.30 ± 0.37 μg/mg of fat (group E + S). The liver free thiol content was significantly diminished (P b 0.05) in E + BE and E + T groups compared to group E + S. No differences were observed in the analysis of hepatic total thiols. 4. Discussion This study aimed to evaluate the benefits of treatment with thiamine or benfotiamine against damage caused by acute ethanol administration
23
in rats, an experimental model that produces effects similar to those caused by the massive intake (“binge drinking”) of ethanol in humans. Although there are no reports in the literature, liver ethanol values can be auxiliary in the elucidation of the deleterious effects of hepatic metabolism of the substance. Thus, treatment with thiamine and benfotiamine raised the serum and liver concentrations of ethanol compared with the group that received only ethanol. The higher presence of ethanol intact, i.e., not metabolized, has two possible explanations. The first one would be an increase in metabolism via microsomal ethanol oxidizing system (MEOS), which would increase the flow of different aqueous compartments of the body to blood and, consequently, a higher influx to the liver. This explanation is consistent with the work of Tabake and Itokawa [25,26]. These authors found that acute administration of ethanol reduces the concentration of thiamine [25]. In mice deficient in thiamine they found a decreased activity of MEOS which was restored with the administration of thiamine or thiamin diphosphate [26]. The other possibility, contrary to the findings of Tabake and Itokawa, is that the ethanol remains circulating in blood with low metabolization over the tested period of 6 h after treatment with both forms of vitamin B1. This possible explanation comes against our previous findings that although found a drop in serum alcohol levels in rats treated with ethanol and thiamine we showed an increased urinary excretion of unchanged ethanol compared to rats that received only ethanol, indicating no metabolism of alcohol. It is well documented that acute intoxication models for ethanol lead to liver damage due to oxidative stress caused by the formation of free radicals and reactive oxygen species and consequent consumption of antioxidants [5,7,11,27,28]. This finding can be verified by the values of ALT and AST increased in the group that received only ethanol. It is also verified that the groups receiving thiamine or benfotiamine had normal values of these markers of liver damage. Added to this, there was an increase in serum and liver TBARS and hydroperoxides representing an index of lipid peroxidation. However, these rates were always lower in the groups that received treatment with any of the forms of thiamine indicating an antioxidant role even with a decrease in vitamin E content. Some studies have shown an antioxidant role of thiamine and benfotiamine. Thus Lukienko et al. [29] have postulated that the thiamine interacts directly with free radicals and hydroperoxides being oxidized to thiochrome and thiamine disulfide. Although the antioxidant role observed in the parameters of lipid peroxidation are similar for treatments with thiamine and benfotiamine, when we observed the protein oxidation parameters, benfotiamine treatment showed better results than thiamine. Importantly, the proteins are mainly in aqueous environment as well as
Table 3 Serum and hepatic biomarkers of oxidative stress. Groups
E+S
E+T
E + BE
Serum Free serum TBARS (nmol/L) Total serum TBARS (μmol/L) Serum hydroperoxides (μmol/L) Serum AOPP (μmol/L) Serum carbonyl (nmol/L)
86.4 ± 22.2 1.30 ± 0.67 44.8 ± 8.2 23.4 ± 10.2 76.8 ± 6.1
66.0 ± 11.6⁎ 0.80 ± 0.08⁎ 27,9 ± 7,3⁎ 16.1 ± 7.8 76.2 ± 4.8
59.6 ± 11.3⁎ 0.83 ± 0.12⁎ 30.5 ± 4.5⁎ 10.8 ± 3.6⁎ 72.2 ± 7.7
Liver Free hepatic TBARS (nmol/g of protein) Total hepatic TBARS (mmol/g of protein) Hepatic AOPP (μmol/g of protein) Hepatic carbonyl (μmol/g of protein) Hepatic α-tocopherol (μg/mg of fat) Free hepatic thiol (μmol/g of protein) Total hepatic thiol (μmol/g of protein)
18.0 ± 3.7 10.9 ± 3.1 21.2 ± 5.1 24.5 ± 5.8 1.30 ± 0.37 4.1 ± 1.4 15.4 ± 3.6
9.7 ± 3.2⁎ 5.4 ± 2.9⁎ 16.9 ± 4.2 22.9 ± 5.0⁎
10.0 ± 2.5⁎ 3.8 ± 1.3⁎ 14.1 ± 4.9⁎ 17.7 ± 3.1⁎,⁎⁎ 0.57 ± 0.13⁎,⁎⁎ 2.8 ± 0.8⁎
Groups: E + S (Ethanol), E + T (Ethanol treated with thiamine), E + BE (Ethanol treated with benfotiamine). TBARS: thiobarbituric acid reactive substances. AOPP: advanced oxidation protein products. ⁎ P b 0.05 relative to E + S group. ⁎⁎ P b 0.05 relative to E + T group.
0.92 ± 0.17⁎ 2.8 ± 1.0⁎ 13.9 ± 3.4
11.5 ± 2.0
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thiamine. As demonstrated in our previous publication [12], benfotiamine administration increased by 100% plasma levels of thiamine and 25-fold the erythrocyte content. Added to this is the fact discussed above that possibly the groups treated with both forms of thiamin may metabolized less ethanol, which decreased formation of free radicals as consequence. Our study has some limitations. We could not exploit the hepatic metabolism of ethanol, for example, the quantification of acetaldehyde, as we could not quantify the urinary ethanol since the animals were prostrate and without urination. Thus, we can only hypothesize, supported in the literature, our finding of hepatic ethanol quantification. 5. Conclusion The treatment with thiamine or benfotiamine even 30 min after the massive dose of ethanol has proven to be beneficial against liver damage. Better results were obtained with benfotiamine related to oxidative damage from aqueous compartments. References [1] P.D. Masalkar, S.A. Abhang, Oxidative stress and antioxidant status in patients with alcoholic liver disease, Clin. Chim. Acta 355 (1–2) (2005) 61–65. [2] C.S. Lieber, The discovery of the microsomal ethanol oxidizing system and its physiologic and pathologic role, Drug Metab. Rev. 36 (3–4) (2004) 511–529. [3] C.S. Lieber, Metabolism of alcohol, Clin Liver Dis 9 (1) (2005) 1–35. [4] J.C. Fernandez-Checa, N. Kaplowitz, A. Colell, C. Garcia-Ruiz, Oxidative stress and alcoholic liver disease, Alcohol Health Res World 21 (4) (1997) 321–324. [5] P. Navasumrit, T.H. Ward, N.J. Dodd, P.J. O'Connor, Ethanol-induced free radicals and hepatic DNA strand breaks are prevented in vivo by antioxidants: effects of acute and chronic ethanol exposure, Carcinogenesis 21 (1) (2000) 93–99. [6] D. Wu, A.I. Cederbaum, Alcohol, oxidative stress, and free radical damage, Alcohol Res Health 27 (4) (2003) 277–284. [7] V.B. Patel, S. Worrall, P.W. Emery, V.R. Preedy, Protein adduct species in muscle and liver of rats following acute ethanol administration, Alcohol Alcohol. 40 (6) (2005) 485–493. [8] C.S. Lieber, Relationships between nutrition, alcohol use, and liver disease, Alcohol Res Health 27 (3) (2003) 220–231. [9] C. Lemos, I. Azevedo, F. Martel, Effect of red wine on the intestinal absorption of thiamine and folate in the rat: comparison with the effect of ethanol alone, Alcohol Clin. Exp. Res. 29 (4) (2005) 664–671. [10] C.M. Tallaksen, A. Sande, T. Bohmer, H. Bell, J. Karlsen, Kinetics of thiamin and thiamin phosphate esters in human blood, plasma and urine after 50 mg intravenously or orally, Eur. J. Clin. Pharmacol. 44 (1) (1993) 73–78.
[11] G.V. Portari, J.S. Marchini, H. Vannucchi, A.A. Jordao, Antioxidant effect of thiamine on acutely alcoholized rats and lack of efficacy using thiamine or glucose to reduce blood alcohol content, Basic Clin Pharmacol Toxicol 103 (5) (2008) 482–486. [12] G.V. Portari, H. Vannucchi, A.A. Jordao Jr., Liver, plasma and erythrocyte levels of thiamine and its phosphate esters in rats with acute ethanol intoxication: a comparison of thiamine and benfotiamine administration, Eur. J. Pharm. Sci. 48 (4–5) (2013) 799–802. [13] H. Woelk, S. Lehrl, R. Bitsch, W. Kopcke, Benfotiamine in treatment of alcoholic polyneuropathy: an 8-week randomized controlled study (BAP I Study), Alcohol Alcohol. 33 (6) (1998) 631–638. [14] (NIAAA) TNIoAAaA, Drinking Levels Defined, 2016. [15] J.C. Crabbe, R.A. Harris, G.F. Koob, Preclinical studies of alcohol binge drinking, Ann. N. Y. Acad. Sci. 1216 (2011) 24–40. [16] G.V. Portari, J.S. Marchini, A.A. Jordao, Validation of a manual headspace gas chromatography method for determining volatile compounds in biological fluids, Labmedicine 39 (1) (2008) 42–45. [17] J.A. Buege, S.D. Aust, Microsomal lipid peroxidation, Methods Enzymol. 52 (1978) 302–310. [18] G. Cighetti, S. Debiasi, R. Paroni, P. Allevi, Free and total malondialdehyde assessment in biological matrices by gas chromatography–mass spectrometry: what is needed for an accurate detection, Anal. Biochem. 266 (2) (1999) 222–229. [19] E. Sodergren, J. Nourooz-Zadeh, L. Berglund, B. Vessby, Re-evaluation of the ferrous oxidation in xylenol orange assay for the measurement of plasma lipid hydroperoxides, J. Biochem. Biophys. Methods 37 (3) (1998) 137–146. [20] P. Odetti, S. Garibaldi, G. Gurreri, I. Aragno, D. Dapino, M.A. Pronzato, U.M. Marinari, Protein oxidation in hemodialysis and kidney transplantation, Metabolism 45 (11) (1996) 1319–1322. [21] V. Witko-Sarsat, M. Friedlander, C. Capeillere-Blandin, T. Nguyen-Khoa, A.T. Nguyen, J. Zingraff, P. Jungers, B. Descamps-Latscha, Advanced oxidation protein products as a novel marker of oxidative stress in uremia, Kidney Int. 49 (5) (1996) 1304–1313. [22] J. Arnaud, I. Fortis, S. Blachier, D. Kia, A. Favier, Simultaneous determination of retinol, alpha-tocopherol and beta-carotene in serum by isocratic high-performance liquid chromatography, J. Chromatogr. 572 (1–2) (1991) 103–116. [23] J. Sedlak, R.H. Lindsay, Estimation of total, protein-bound, and nonprotein sulfhydryl groups in tissue with Ellman's reagent, Anal. Biochem. 25 (1) (1968) 192–205. [24] E.G. Bligh, W.J. Dyer, A rapid method of total lipid extraction and purification, Can J Biochem Physiol 37 (8) (1959) 911–917. [25] M. Takabe, Y. Itokawa, Thiamin depletion after ethanol and acetaldehyde administration to rabbits, J. Nutr. Sci. Vitaminol. (Tokyo) 29 (5) (1983) 509–514. [26] M. Takabe, Y. Itokawa, Participation of thiamin in hepatic microsomal ethanol oxidizing system, Int. J. Vitam. Nutr. Res. 52 (3) (1982) 260–265. [27] A.A. Jordao Jr., P.G. Chiarello, M.R. Arantes, M.S. Meirelles, H. Vannucchi, Effect of an acute dose of ethanol on lipid peroxidation in rats: action of vitamin E, Food Chem. Toxicol. 42 (3) (2004) 459–464. [28] Y.M. Li, S.H. Chen, C.H. Yu, Y. Zhang, G.Y. Xu, Effect of acute alcoholism on hepatic enzymes and oxidation/antioxidation in rats, Hepatobiliary Pancreat Dis Int 3 (2) (2004) 241–244. [29] P.I. Lukienko, N.G. Mel'nichenko, I.V. Zverinskii, S.V. Zabrodskaya, Antioxidant properties of thiamine, Bull. Exp. Biol. Med. 130 (9) (2000) 874–876.
Contents lists available at ScienceDirect
Life Sciences journal homepage: www.elsevier.com/locate/lifescie
Protective effect of treatment with thiamine or benfotiamine on liver oxidative damage in rat model of acute ethanol intoxication Guilherme Vannucchi Portari a,⁎, Paula Payão Ovidio b, Rafael Deminice c, Alceu Afonso Jordão Jr. a b c
b
Department of Nutrition, Health Sciences Institute, Federal University of Triangulo Mineiro, Brazil Department of Internal Medicine, Faculty of Medicine of Ribeirão Preto, University of São Paulo, Brazil Department of Physical Education, Faculty of Physical Education and Sport, State University of Londrina, Brazil
a r t i c l e
i n f o
Article history: Received 13 May 2016 Received in revised form 15 August 2016 Accepted 17 August 2016 Available online 18 August 2016 Keywords: Thiamine Benfotiamine Oxidative stress Acute alcohol intoxication
a b s t r a c t Aims: The aim of this study was to evaluate possible beneficial effects of treatment with thiamine or benfotiamine in an animal model of acute ethanol intoxication. Main methods: Thirty male Wistar rats were separated at random into three groups of 10 animals each: Ethanol (E), Ethanol treated with thiamine (T) and Ethanol treated with benfotiamine (BE). Rats were gavaged with single dose of ethanol (5 g/kg, 40% v:v). After 30 min of ethanol gavage the animals were treated with thiamine or benfotiamine. Six hours after first gavage, the animals were euthanized and blood and liver samples were collected for ethanol and oxidative stress biomarkers quantification. Key findings: Serum ethanol levels were higher in animals treated with thiamine or benfotiamine while hepatic alcohol levels were higher in animals of the group treated with benfotiamine comparing to controls or thiamine treated groups. The lipid peroxidation biomarkers were diminished for the groups treated with thiamine or benfotiamine comparing to E animals. Concerning protein oxidative damage parameters, they were enhanced for animals treated with benfotiamine in relation to other groups. Significance: In conclusion, the treatment with thiamine or benfotiamine even 30 min after the massive dose of ethanol has proven to be beneficial against liver damage. Improved results were obtained with benfotiamine in relation to oxidative damage from aqueous compartments. © 2016 Elsevier Inc. All rights reserved.
1. Introduction Ethanol metabolism produces free radicals and reactive oxygen species with consequent consumption of antioxidants leading to an imbalance between oxidants/antioxidants, known as oxidative stress. Several research groups have focused on the study of liver diseases caused by alcohol, since this is the organ responsible for about 95% of its catabolism [1]. The hepatic metabolism of ethanol is carried via three enzymatic pathways: pathway of alcohol dehydrogenase, the microsomal ethanol oxidation system (CYP2E1), and catalase [2,3]. While these metabolic pathways are well elucidated in the literature, little attention is given to the quantification of ethanol in liver although this can be an important finding to know the metabolizing status of alcohol in this organ. During the transformation of ethanol to acetaldehyde via CYP2E1 there is an overproduction of free radicals and reactive oxygen species,
⁎ Corresponding author at: Departamento de Nutrição, Universidade Federal do Triângulo Mineiro, Rua Getúlio Guaritá, 159 - sala 333, CEP: 38025-440 Uberaba, MG, Brazil. E-mail address: [email protected] (G.V. Portari).
http://dx.doi.org/10.1016/j.lfs.2016.08.017 0024-3205/© 2016 Elsevier Inc. All rights reserved.
mainly in the forms of superoxide anion (O‐2), hydrogen peroxide (H2O2) and hydroxyethyl radical (CH3CH(•)\\OH) [4–6]. These molecules are highly reactive and, as acetaldehyde, attack macromolecules, causing impairment or loss of function and, ultimately, cell death [7]. The use of antioxidants may help balance the hepatic antioxidant system reducing the deleterious effects caused by oxidative stress [8]. Thiamine, the vitamin B1, is a water soluble vitamin that plays essential role on energetic metabolism from carbohydrates. Benfotiamine is a weak soluble in water pro-vitamin B1 substance that displays better absorption and bioavailability compared with common pharmaceutical form available, thiamine hydrochloride, when administered orally, even after massive ethanol administration [12]. Thiamine therapeutic replacement is postulated in patients with Wernicke encephalopathy, especially alcoholics, due to deficiency of this vitamin in this particular group, because of poor dietary habits and a reduced absorption due to changes in the gastrointestinal system [9,10]. Hypothetically, thiamine supply could increase the metabolism of ethanol by restoring the microsomal ethanol oxidizing system (MEOS). Studies from our group have previously demonstrated some beneficial effects on intravenous administration of thiamine in an animal model of acute ethanol intoxication and more recently the high
22
G.V. Portari et al. / Life Sciences 162 (2016) 21–24
bioavailability of benfotiamine in rats acutely alcoholized [11,12]. In humans, benfotiamine has been tested as oral treatment of alcoholic polyneuropathy with great improvement of the symptoms [13]. The National Institute on Alcohol Abuse and Alcoholism (NIAAA) defines binge drinking as a pattern of drinking that brings blood alcohol concentration (BAC) levels to 0.8 g/L, typically occurring after 4 drinks for women and 5 drinks for men in about 2 h [14,15]. In experimental models this can be achieved by forced administration of a massive dose of ethanol by gavage since animals do not voluntarily consume alcohol at concentrations that produce intoxication [15]. It has been showed that, as the chronic models, the acute ethanol intoxication produces an increase in hepatic oxidative stress. In the present study we evaluate the effects of the administration of benfotiamine or thiamine on the improvement of hepatic oxidative damage and ethanol influx in a model of acute ethanol intoxication
were subjected to alkaline hydrolysis following proposed protocol by Cighetti et al. [18] with the following modifications: in the test tube 100 mg of liver (or 200 μL serum) were homogenized with 1 mL of 1.15% KCl. Then, it was added 3 mL of Milli-Q water and 0.5 mL of 2 M NaOH. After stirring the tubes they were heated at 60 °C for 30 min and then neutralized with 2 M HCl to follow the reaction with thiobarbituric acid. The lipid hydroperoxides were determinated using the ferrous oxidation-xylenol orange (FOX) assay as described by Sodergren et al. [19]. 2.5. Protein damage tests The extent of protein damage in serum and liver has been accessed by carbonyl content and advanced oxidation protein products (AOPP) assay following methodologies of Odetti et al. [20] and Witko-Sarsat et al. [21], respectively.
2. Material and methods 2.6. Status of antioxidants 2.1. Animals and experimental protocol Thirty male Wistar rats, weighing 270–333 g, were obtained from the Central Animal Facilities of the Ribeirão Preto Campus, University of São Paulo, and allowed to acclimate for 1 week in the animal facilities of the Department of Internal Medicine, Faculty of Medicine of Ribeirão Preto, University of São Paulo, under controlled conditions of a 12-h light: dark cycle and temperature of 24 ± 2 °C in individual cages with free access to food and water. The experimental protocol was approved by the Animal Research Ethics Commission (protocol no. 152). The animals were separated at random into three groups of 10 animals each: Ethanol (E + S), Ethanol treated with thiamine (E + T) and Ethanol treated with benfotiamine (E + BE). In the dark period of the previous day (10:00 p.m.) the chow was withdrawal to ensure that the stomach was not filled preventing reflux and aspiration of fluid into lungs and, mimic the eating habits frequently encountered in alcoholics. Next day, at 6:00 a.m. the rats were gavaged with single dose of 40% (w:v) ethanol in aqueous solution (5 g/kg of body weight) and, after 30 min each group received a second gavage to delivery 100 mg/kg of thiamine (E + T), in an aqueous solution, or benfotiamine (E + BE), in an aqueous dispersion, except for E + S group that received a saline sham gavage. Animals were left in cages with food and water ad libitum but due to sedation caused by ethanol none of them sought for food. Six hours after the first gavage, the animals were euthanized and blood and liver samples were collected, weighed, frozen immediately in liquid nitrogen and stored at −40 °C until analysis. 2.2. Serum and hepatic ethanol quantification Ethanol was quantified in serum and liver homogenates by previous validated gas chromatography method [16]. The hepatic ethanol concentration was multiplied by the respective liver mass to achieve total content of ethanol in this organ. Then, the percentual of ethanol relative to the initial doses was calculated. 2.3. Aspartate aminotransferase (AST) and alanine aminotransferase (ALT) To check for liver damage were measured enzymes AST and ALT by colorimetric reaction and spectrophotometric reading (UV–Vis Mod Q98U Quimis®, Diadema, SP, Brazil), using commercial kit (Labtest Diagnostica, Lagoa Santa, MG, Brazil). 2.4. Lipid peroxidation Lipid peroxidation in serum and liver homogenates was measured by thiobarbituric acid reactive substances [17]. To quantify the malondialdehyde bound to the macromolecules serum or homogenate
The status of antioxidants was accessed by determination of vitamin E (exogenous) and free (mainly glutathione) and total thiols (endogenous). Vitamin E was quantified in liver homogenates by HPLC following methodology described by Arnaud et al. [22] and the obtained values were corrected for lipid content. Thiols (free and total) in liver homogenates were determined by colorimetric assay after 5,5′-dithiobis-(2-nitrobenzoic acid) reaction [23]. 2.7. Fat content in liver homogenates For the quantification of total fat in liver homogenates the method proposed by Bligh and Dyer [24] was used. 2.8. Statistical analysis Differences between groups were determined by one-way analysis of variance (ANOVA) using a multiple comparison procedure (Tukey). A P value of b 0.05 was considered significant. Data are given as mean ± standard deviation of the mean. 3. Results In Table 1 we note that the groups receiving treatment with thiamine and benfotiamine had higher serum ethanol (P b 0.05) than group E + S. The same behavior was observed for liver ethanol concentrations, but with E + BE group reaching values significantly higher (P b 0.05) than the E + T group. When we analyze the percentage of hepatic ethanol relative to the initial dose, a significant difference (P b 0.05) among the three groups that received ethanol was found, with values of 1.3 ± 1.3%, 2.5 ± 0.9% and 3.6 ± 1.2% for groups E + S, E + T and E + BE, respectively. The ALT and AST levels (Table 2) were lower (P b 0.05) in groups which received treatment with thiamine and benfotiamine. Table 1 Characterization of the experimental model as the ethanol concentrations in serum and liver. Parameter
Serum ethanol (g/L) Hepatic ethanol (mg/g of tissue) Hepatic percentage (%) of ethanol in relation to initial dose
Groups E+S
E+T
E + BE
1.08 ± 0.85 1.32 ± 1.12 1.3 ± 1.3
1.99 ± 0.75⁎ 3.34 ± 1.38⁎ 2.5 ± 0.9⁎
1.90 ± 0.39⁎ 5.13 ± 1.83⁎,⁎⁎ 3.6 ± 1.2⁎,⁎⁎
Groups: E + S (Ethanol), E + T (Ethanol treated with thiamine), E + BE (Ethanol treated with benfotiamine). ⁎ P b 0.05 relative to E + S group. ⁎⁎ P b 0.05 relative to E + T group.
G.V. Portari et al. / Life Sciences 162 (2016) 21–24 Table 2 Serum ALT and AST levels. Groups
E+S
E+T
E + BE
ALT (U/L) AST (U/L)
67.1 ± 5.5 196.3 ± 15.8
54.7 ± 7.3⁎ 142.0 ± 23.1⁎
55.2 ± 5.5⁎ 131.2 ± 8.9⁎
Groups: E + S (Ethanol), E + T (Ethanol treated with thiamine), E + BE (Ethanol treated with benfotiamine). ⁎ P b 0.05 relative to E + S group.
Table 3 gives all measured biomarkers of oxidative stress. With respect to free TBARS in liver, represented by aldehydes from lipid peroxidation not linked to macromolecules, there was a significant increase (P b 0.05) in group E + S (18.0 ± 3.7 nmol/g protein) compared to other groups with significant drop for the E + T group (9.7 ± 3.2 nmol/g protein) and E + BE (10.0 ± 2.5 nmol/g protein). Analyzing the total hepatic TBARS, representing the sum of aldehydes from lipid peroxidation (linked to macromolecules plus free aldehydes), there was a significant reduction (P b 0.05) in the treated groups (E + T 5.4 ± 2.9 mmol/g protein and E + BE - 3.8 ± 1.3 mmol/g protein) compared to group E + S (10.9 ± 3.1 mmol/g protein). Serum analysis of free and total TBARS show similar behavior with a significant decrease (P b 0.05) in their concentrations for the E + BE and E + T groups compared to group E + S. With respect to serum hydroperoxides there was a significant increase (P b 0.05) in group E + S (44.8 ± 8.2 μmol/L) in comparison to other groups with significant drop for the group E + T (27.9 ± 7.3 μmol/L) and E + BE (30, 5 ± 4.5 μmol/L). The results of oxidative protein damage analyzed as AOPP and carbonyl content were lower for group E + BE. The hepatic and serum values of AOPP were significantly lower (P b 0.05) for E + BE group compared to group E + S and also group E + T. Similar values were observed in the values of the hepatic content of carbonyls but with differences between the three groups. No differences were observed in the analysis of serum content of carbonyls. With respect to vitamin E, an exogenous antioxidant mainly of lipid environment, there were differences between the three groups (P b 0.05) with values of 0.57 ± 0.13 μg/mg of fat (group E + BE), 0.92 ± 0.17 (group E + T) and 1.30 ± 0.37 μg/mg of fat (group E + S). The liver free thiol content was significantly diminished (P b 0.05) in E + BE and E + T groups compared to group E + S. No differences were observed in the analysis of hepatic total thiols. 4. Discussion This study aimed to evaluate the benefits of treatment with thiamine or benfotiamine against damage caused by acute ethanol administration
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in rats, an experimental model that produces effects similar to those caused by the massive intake (“binge drinking”) of ethanol in humans. Although there are no reports in the literature, liver ethanol values can be auxiliary in the elucidation of the deleterious effects of hepatic metabolism of the substance. Thus, treatment with thiamine and benfotiamine raised the serum and liver concentrations of ethanol compared with the group that received only ethanol. The higher presence of ethanol intact, i.e., not metabolized, has two possible explanations. The first one would be an increase in metabolism via microsomal ethanol oxidizing system (MEOS), which would increase the flow of different aqueous compartments of the body to blood and, consequently, a higher influx to the liver. This explanation is consistent with the work of Tabake and Itokawa [25,26]. These authors found that acute administration of ethanol reduces the concentration of thiamine [25]. In mice deficient in thiamine they found a decreased activity of MEOS which was restored with the administration of thiamine or thiamin diphosphate [26]. The other possibility, contrary to the findings of Tabake and Itokawa, is that the ethanol remains circulating in blood with low metabolization over the tested period of 6 h after treatment with both forms of vitamin B1. This possible explanation comes against our previous findings that although found a drop in serum alcohol levels in rats treated with ethanol and thiamine we showed an increased urinary excretion of unchanged ethanol compared to rats that received only ethanol, indicating no metabolism of alcohol. It is well documented that acute intoxication models for ethanol lead to liver damage due to oxidative stress caused by the formation of free radicals and reactive oxygen species and consequent consumption of antioxidants [5,7,11,27,28]. This finding can be verified by the values of ALT and AST increased in the group that received only ethanol. It is also verified that the groups receiving thiamine or benfotiamine had normal values of these markers of liver damage. Added to this, there was an increase in serum and liver TBARS and hydroperoxides representing an index of lipid peroxidation. However, these rates were always lower in the groups that received treatment with any of the forms of thiamine indicating an antioxidant role even with a decrease in vitamin E content. Some studies have shown an antioxidant role of thiamine and benfotiamine. Thus Lukienko et al. [29] have postulated that the thiamine interacts directly with free radicals and hydroperoxides being oxidized to thiochrome and thiamine disulfide. Although the antioxidant role observed in the parameters of lipid peroxidation are similar for treatments with thiamine and benfotiamine, when we observed the protein oxidation parameters, benfotiamine treatment showed better results than thiamine. Importantly, the proteins are mainly in aqueous environment as well as
Table 3 Serum and hepatic biomarkers of oxidative stress. Groups
E+S
E+T
E + BE
Serum Free serum TBARS (nmol/L) Total serum TBARS (μmol/L) Serum hydroperoxides (μmol/L) Serum AOPP (μmol/L) Serum carbonyl (nmol/L)
86.4 ± 22.2 1.30 ± 0.67 44.8 ± 8.2 23.4 ± 10.2 76.8 ± 6.1
66.0 ± 11.6⁎ 0.80 ± 0.08⁎ 27,9 ± 7,3⁎ 16.1 ± 7.8 76.2 ± 4.8
59.6 ± 11.3⁎ 0.83 ± 0.12⁎ 30.5 ± 4.5⁎ 10.8 ± 3.6⁎ 72.2 ± 7.7
Liver Free hepatic TBARS (nmol/g of protein) Total hepatic TBARS (mmol/g of protein) Hepatic AOPP (μmol/g of protein) Hepatic carbonyl (μmol/g of protein) Hepatic α-tocopherol (μg/mg of fat) Free hepatic thiol (μmol/g of protein) Total hepatic thiol (μmol/g of protein)
18.0 ± 3.7 10.9 ± 3.1 21.2 ± 5.1 24.5 ± 5.8 1.30 ± 0.37 4.1 ± 1.4 15.4 ± 3.6
9.7 ± 3.2⁎ 5.4 ± 2.9⁎ 16.9 ± 4.2 22.9 ± 5.0⁎
10.0 ± 2.5⁎ 3.8 ± 1.3⁎ 14.1 ± 4.9⁎ 17.7 ± 3.1⁎,⁎⁎ 0.57 ± 0.13⁎,⁎⁎ 2.8 ± 0.8⁎
Groups: E + S (Ethanol), E + T (Ethanol treated with thiamine), E + BE (Ethanol treated with benfotiamine). TBARS: thiobarbituric acid reactive substances. AOPP: advanced oxidation protein products. ⁎ P b 0.05 relative to E + S group. ⁎⁎ P b 0.05 relative to E + T group.
0.92 ± 0.17⁎ 2.8 ± 1.0⁎ 13.9 ± 3.4
11.5 ± 2.0
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thiamine. As demonstrated in our previous publication [12], benfotiamine administration increased by 100% plasma levels of thiamine and 25-fold the erythrocyte content. Added to this is the fact discussed above that possibly the groups treated with both forms of thiamin may metabolized less ethanol, which decreased formation of free radicals as consequence. Our study has some limitations. We could not exploit the hepatic metabolism of ethanol, for example, the quantification of acetaldehyde, as we could not quantify the urinary ethanol since the animals were prostrate and without urination. Thus, we can only hypothesize, supported in the literature, our finding of hepatic ethanol quantification. 5. Conclusion The treatment with thiamine or benfotiamine even 30 min after the massive dose of ethanol has proven to be beneficial against liver damage. Better results were obtained with benfotiamine related to oxidative damage from aqueous compartments. References [1] P.D. Masalkar, S.A. Abhang, Oxidative stress and antioxidant status in patients with alcoholic liver disease, Clin. Chim. Acta 355 (1–2) (2005) 61–65. [2] C.S. Lieber, The discovery of the microsomal ethanol oxidizing system and its physiologic and pathologic role, Drug Metab. Rev. 36 (3–4) (2004) 511–529. [3] C.S. Lieber, Metabolism of alcohol, Clin Liver Dis 9 (1) (2005) 1–35. [4] J.C. Fernandez-Checa, N. Kaplowitz, A. Colell, C. Garcia-Ruiz, Oxidative stress and alcoholic liver disease, Alcohol Health Res World 21 (4) (1997) 321–324. [5] P. Navasumrit, T.H. Ward, N.J. Dodd, P.J. O'Connor, Ethanol-induced free radicals and hepatic DNA strand breaks are prevented in vivo by antioxidants: effects of acute and chronic ethanol exposure, Carcinogenesis 21 (1) (2000) 93–99. [6] D. Wu, A.I. Cederbaum, Alcohol, oxidative stress, and free radical damage, Alcohol Res Health 27 (4) (2003) 277–284. [7] V.B. Patel, S. Worrall, P.W. Emery, V.R. Preedy, Protein adduct species in muscle and liver of rats following acute ethanol administration, Alcohol Alcohol. 40 (6) (2005) 485–493. [8] C.S. Lieber, Relationships between nutrition, alcohol use, and liver disease, Alcohol Res Health 27 (3) (2003) 220–231. [9] C. Lemos, I. Azevedo, F. Martel, Effect of red wine on the intestinal absorption of thiamine and folate in the rat: comparison with the effect of ethanol alone, Alcohol Clin. Exp. Res. 29 (4) (2005) 664–671. [10] C.M. Tallaksen, A. Sande, T. Bohmer, H. Bell, J. Karlsen, Kinetics of thiamin and thiamin phosphate esters in human blood, plasma and urine after 50 mg intravenously or orally, Eur. J. Clin. Pharmacol. 44 (1) (1993) 73–78.
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