Accepted Manuscript

Energy drink and alcohol combination leads to kidney and liver

alterations in rats

Marina Tuerlinckx Costa-Valle, Bruna Ducatti Tonieto, Louise

Altknecht, Camila D. Cunha, Nuryan Fão, Larissa V. Cestonaro,
Gabriela Göethel, Solange C. Garcia, Mirna Bainy Leal, Eliane
Dallegrave, Marcelo Dutra Arbo

PII: S0041-008X(18)30290-4
DOI: doi:10.1016/j.taap.2018.06.024
Reference: YTAAP 14315
To appear in: Toxicology and Applied Pharmacology
Received date: 7 February 2018
Revised date: 22 June 2018
Accepted date: 26 June 2018

Please cite this article as: Marina Tuerlinckx Costa-Valle, Bruna Ducatti Tonieto, Louise
Altknecht, Camila D. Cunha, Nuryan Fão, Larissa V. Cestonaro, Gabriela Göethel,
Solange C. Garcia, Mirna Bainy Leal, Eliane Dallegrave, Marcelo Dutra Arbo , Energy
drink and alcohol combination leads to kidney and liver alterations in rats. Ytaap (2018),
doi:10.1016/j.taap.2018.06.024

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 ACCEPTED MANUSCRIPT

Energy drink and alcohol combination leads to kidney and liver alterations in rats

Marina Tuerlinckx Costa-Vallea,b; Bruna Ducatti Tonietob; Louise Altknechtc; Camila D. Cunhac;

Nuryan Fãoc,d; Larissa V. Cestonaroc,d; Gabriela Göethelc,d; Solange C. Garciac,d; Mirna Bainy

Leala,b; Eliane Dallegravee; Marcelo Dutra Arboc,d*

a
Programa de Pós-Graduação em Ciências Biológicas: Neurociências, Instituto de Ciências

Básicas da Saúde, Universidade Federal do Rio Grande do Sul (UFRGS), Rua Sarmento Leite,

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500/209, 90046-900, Porto Alegre, Rio Grande do Sul, Brazil.

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b
Laboratório de Farmacologia e Toxicologia de Produtos Naturais, Departamento de

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Farmacologia, Instituto de Ciências Básicas da Saúde, Universidade Federal do Rio Grande do

Sul (UFRGS), Rua Sarmento Leite, 500/309, 90050-170, Porto Alegre, Rio Grande do Sul, Brazil.
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c
Laboratório de Toxicologia (LATOX), Departamento de Análises, Faculdade de Farmácia,
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Universidade Federal do Rio Grande do Sul (UFRGS), Av. Ipiranga 2752/605B, 90610-000, Porto

Alegre, Rio Grande do Sul, Brazil.

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d
Programa de Pós-Graduação em Ciências Farmacêuticas (PPGCF), Faculdade de Farmácia,
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Universidade Federal do Rio Grande do Sul (UFRGS), Av. Ipiranga 2752/1º andar, 90610-000,
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Porto Alegre, Rio Grande do Sul, Brazil.

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e
Departamento de Farmacociências, Universidade Federal de Ciências da Saúde de Porto
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Alegre (UFCSPA), Rua Sarmento Leite, 245, 90050-170, Porto Alegre, Rio Grande do Sul, Brazil.

*Corresponding author: Prof. Dr. Marcelo Dutra Arbo (marcelo.arbo@ufrgs.br)

Laboratório de Toxicologia (LATOX), Departamento de Análises, Faculdade de Farmácia,

Universidade Federal do Rio Grande do Sul (UFRGS), Av. Ipiranga 2752/605B, 90610-000, Porto

Alegre, Rio Grande do Sul, Brazil. Phone +55 51 3308 5297

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Abstract

The aim of this study was to evaluate the acute toxicity of the association of energy drink and

alcohol in male Wistar rats. Animals were treated by oral gavage with 10 ml/kg distilled water

(control); 10ml/kg energy drink (ED10); 3.2mg/kg caffeine + 40mg/kg taurine; 2g/kg alcohol

20%; 2g/kg alcohol 20% + ED10; and 2g/kg alcohol 20% + 3.2mg/kg caffeine + 40mg/kg taurine.

Behavioral alterations were observed for 6h after treatment. Animals presented significant

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differences in the frequency of rearing, ambulation, grooming, wakefulness and tachypnea

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along time. Caffeine+taurine increased the levels of TBARS and total thiols in kidneys. ED10

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increased lipoperoxidation in liver. The association of ED10+alcohol induced nephrotoxicity

observed by the increase of urinary N-acetyl-β-D-glucosaminidase (NAG) activity.

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Histopathological analysis showed the presence of congestion and hydropic and hyaline

degenerations in the livers of ED10+alcohol treated rats, and hemorrhage in the liver of
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alcohol+caffeine+taurine group. In kidneys, hyaline degeneration was observed in ED10;

ED10+alcohol; caffeine+taurine; and alcohol+caffeine+taurine. Hemorrhage was present in the

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kidneys of all groups. The combination of energy drinks and alcohol is not safe for the
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consumers. Therefore, precautionary measures should be disseminated among risk

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populations, especially the teenagers.

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Keywords: energy drinks, alcohol, acute toxicity, oxidative stress, nephrotoxicity.

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1. Introduction

Energy drinks became recognized worldwide when they reached the American market

in 1997 under the Red Bull® brand (Pennay and Lubman, 2012). Generally, their ingredients are

caffeine, taurine, guarana, sugar, sodium and vitamin B6 (Ishak et al. 2012). Moreover, some

brands add glucuronolactone, ginseng, Gingko biloba, among others (Higgins et al., 2010).

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Among the ingredients, caffeine (1,3,7-trimethylxanthine) is the most relevant

component, acting as a stimulant of the cardiovascular and central nervous systems (Boeck et

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al., 2009; Glade et al., 2010; Wolk et al., 2012). Red Bull® contains approximately 32 mg of

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caffeine per 100 ml, and it is commercialized in 250 ml (80 mg of caffeine) or 355 ml (113.6 mg

of caffeine) cans. In contrast, Cola-type soft drinks contain approximately 40 mg caffeine per
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can (Gunja and Brown, 2012). Comparatively a cup of coffee presents between 50 and 300 mg
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of caffeine, depending on coffee roast and mode of preparation (Ludwig et al., 2014).

Another substance found in energy drinks is taurine (2-aminoethanesulfonic acid),

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which is the most abundant amino acid in the central nervous system, obtained through diet
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or synthesized from cysteine in the liver (Hussy et al., 2000; Vitvitsky et al., 2011; Lambert et
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al., 2015). Even though it does not belong to the mammalian protein structure, it has
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important physiological functions as an antioxidant, osmoregulator, membrane stabilizer and

neurotransmitter. Taurine acts as an inhibitory neurotransmitter through GABAergic and

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glycine receptors, exhibiting anxiolytic effect in mice and rats (Chen et al., 2004; Kong et al.,

2006; Murakami and Furuse, 2010). Besides, taurine weakens the stimulatory effects caused

by caffeine, and it increases the fatigue state (Childs et al, 2014).

The consumption of energy drinks is high among adolescents and young adults, who

often associate them with alcoholic beverages, such as vodka and whisky (Pennay and

Lubman, 2012). It has been estimated that around 46% of the recreational use of energy drinks

has been in association with alcoholic beverages (Gunja and Brown, 2012). The association of
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alcohol and energy drinks leads to an increase in alcohol consumption, increasing the

probability of users to suffer accidents (McKetin et al., 2015). It is believed that the stimulant

effect of caffeine could possibly lead to an increase in alcohol consumption, consequently

increasing its toxic effects (Ferreira et al., 2004; Howland et al., 2010; Hann et al., 2012).

Increased risk of accidents and violence, as well as association with other drugs and low school

performance are also observed (Berger et al., 2013, Tucker et al., 2016). In addition, alcohol

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users are more likely to consume cocaine, ecstasy, marijuana, and non-prescription drugs

(Brache and Stockwell, 2011; Snipes et al., 2013), and some psychiatric disorders such as

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anxiety, psychosis and hyperactivity have been associated with energy drinks consumption

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(Wolk et al., 2012). Furthermore, a decrease in the perception of headache, xerostomia and
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impairment of motor coordination have been evidenced when energy drinks are consumed in

association with alcohol compared to alcohol consumption alone (Verster et al., 2012). The
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concomitant effects of alcohol and taurine administration are inconclusive about the

possibility of taurine enhancing the euphoric effects caused by alcohol (Ginsburg and Lamb,
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2008).
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Alcohol consumption increases free radical production and the development of

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oxidative stress, which is directly related to the products of ethanol oxidation (Reinke et al.,
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1997). The levels of lipid peroxidation in plasma, liver, kidney, and heart increase, as well as

blood pressure, after alcohol consumption. However, when consumption is associated with
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taurine, the lipid peroxidation levels decrease. Furthermore, the normalization of the

antioxidant defense system was implicated in this reduction (Pushpakiran, et al. 2004).

Considering all these effects, the association between alcohol and energy drinks was

considered not safe by the FDA (Food and Drug Administration) in 2010 (Marczinski et al.,

2015). In 2014, the World Health Organization (WHO) stated that as energy drink sales are

rarely regulated by age, unlike alcohol and tobacco, they are potentially a significant public
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health problem in the future since they pose a proven negative effect on children (WHO,

2014).

Due to the popularity of the consumption of energy drinks mainly associated with

alcohol, and considering that the toxicological impact of this excessive consumption is

unknown, the aim of this work was to evaluate the acute effects induced by energy drink and

its main components caffeine and taurine, associated with alcohol in Wistar rats.

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2. Material and methods

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2.1. Animals NU
Adult male Wistar rats (60 days), obtained from the Center for Reproduction and

Laboratory Animal Experimentation at UFRGS (CREAL-UFRGS) were used. The experiments

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were approved by the University Ethics Committee (Number 26689) and were carried out in

accordance with current guidelines for the care of laboratory animals. Before starting the
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experiments, the rats were adapted for 15 days in the animal facility of the Department of
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Pharmacology, Institute of Basic Health Sciences (ICBS) - UFRGS. They were kept in
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polypropylene boxes (41 x 34 x 16 cm, 4 rats per box) with free access to water and food in a
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light/dark cycle of 12h (7am - 7pm), controlled temperature (22±2°C), and monitored

humidity. The number of animals per group followed the literature standard for acute toxicity
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testing, fixed in 5 animals (OECD 420), with a total of 30 animals being used (Caballero et al.

2011, Pandolfo et al, 2013).

2.2. Drugs and treatments

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Caffeine and taurine were purchased from Sigma-Aldrich (St. Louis, MO, USA). The

energy drink (Red Bull®, Fuschl, Austria) was purchased from local markets. Drugs were

solubilized in distilled water. In all the experiments distilled water (10 ml/kg) was used as

control and administered by oral gavage as well as the other treatments. Alcohol (ethanol,

Nuclear, Diadema, SP, Brazil) was diluted daily with distilled water to prepare a 20% w/v

solution and administrated by gavage in a volume of 2 g/kg of body weight.

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Doses of energy drink (ED), caffeine (3.2 mg/kg) and taurine (40 mg/kg) were set

through the calculation of relative doses usually ingested by consumers. The amounts of

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caffeine and taurine contained in a 250 ml energy drink can consumed by a 70 kg adult are

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1000 mg taurine/can and 80 mg caffeine/can. The volumes administered were equivalent to 3
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cans (10 ml/kg, ED 10) (Ferreira et al, 2013). The dose of ED at 10 ml/kg contained taurine at

40 mg/kg and caffeine at 3.2 mg/kg (Ferreira et al, 2004). Treatment was administered orally
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to the following groups of five animals each (table 1): 10 ml/kg distilled water (control),

10ml/kg energy drink (ED 10), 3.2 mg/kg caffeine + 40 mg/kg taurine, 2 g/kg ethanol 20%, 2
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g/kg alcohol 20% + 10 ml/kg energy drink (ED 10); and 2 g/kg alcohol 20% + 3.2 mg/kg caffeine
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+ 40 mg/kg taurine.
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2.3. Acute Toxicity Test

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The test was based on the fixed dose acute toxicity method by the Organization for

Economic Cooperation & Development (OECD 420). Immediately after treatment, each animal

was observed for 1 minute in the periods of 0, 15, 30, 60, 120, 180, 240, 300, 360 min after

dosing for the presence of respiratory, digestive and neurological alterations. After 24 hours of

treatments administration, the animals were anesthetized with a combination of thiopental

(100 mg/kg) and lidocaine (10 mL/kg) by the intraperitoneal route; abdomen was incised and

the blood was collected and animals were euthanized by exsanguination. Urine was collected
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directly from the urinary bladder. The vital organs were removed and observed for

macroscopical alterations.

2.4. Blood collection

Blood samples were collected from the vena cava and transferred to tubes (BD

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Vacutainer®) containing EDTA or heparin as anticoagulants and to tubes without anticoagulant.

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Blood collected in EDTA tubes was centrifuged at 1500g for 10 min and the plasma was

removed. Serum, plasma and total blood with heparin were stored in microcentrifuge tubes at

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−80 °C until analysis. NU
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2.5. Serum biochemistry

Serum obtained after centrifugation at 1500×g for 10 min was used for biochemical
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analysis, including urea, creatinine, alanine aminotransferase (ALT), aspartate

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aminotransferase (AST), γ-glutamyltranspeptidase (γ-GT), alkaline phosphatase (ALP), and

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lactate dehydrogenase (LDH) using commercial laboratory kits (BioClin®, Belo Horizonte,
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Brazil). All the analysis were performed in the automatized equipment BS-120 (Mindray Co.,

Shenzhen, China).
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2.6. Oxidative stress evaluation in tissue

Tissues (liver, kidney, heart, and spleen) were quickly removed and washed with saline

solution to remove blood, then weighed, placed on ice, and homogenized in cold 0.1 M Tris-

HCl buffer, pH 7.4. The homogenates were centrifuged at 2400×g for 15 min to yield the low-

speed supernatant fraction that was used for analysis. Lipid peroxidation was evaluated in the
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homogenates through the thiobarbituric acid reactive substances (TBARS) analysed at 532 nm,

according to Esterbauer & Cheeseman (1990).

For the levels of total non-protein thiols, the tissue was homogenized in cold 1.0 M PBS

buffer (pH 7.4) and centrifuged at 3000×g for 10 min at 4°C. The supernatants were analysed

based on the protocol develop by Ellman (1959) with modifications.

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2.7. Oxidative status evaluation in blood

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Lipid peroxidation was determined by the measurement of malondialdehyde (MDA) by

high performance liquid chromatography with a visible detector (HPLC-Vis, Knauer, Berlin,
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Germany), as described by Grotto et al. (2007) on plasma samples. This method quantifies

MDA levels after alkaline hydrolysis and extraction with n-butanol, MDA levels were expressed
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as μM.
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The levels of reduced GSH were measured as non-protein thiols based on the protocol
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developed by Ellman (1959) with modifications. Aliquots (0.3 ml) of erythrocytes were added
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to a phosphate buffer 0.3 mol/l (0.85 ml), pH 7.4 and the reaction was read in

spectrophotometer at 412 nm after addiction of 0.05 ml 10 mM 5-5’-dithio-bis(2-nitrobenzoic)

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acid (DTNB). The results were expressed as mM.

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2.8. NAG activity

Kidney function was evaluated through the N-acetyl-beta-D glucosaminidase (NAG)

activity in urine, according to Horak et al. (1981). Levels of creatinine were quantified by using

commercial laboratory kits (Doles Reagents, Goiânia, GO, Brazil). All the spectrophotometrical

analyses were realized in an UV–VIS Hitachi spectrophotometer model U-1800 (Tokyo, Japan).
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2.9. Comet assay

Comet assay was performed to assess potential DNA damage in treated rats. Blood

samples (sodium heparin tubes) were transported to the laboratory under refrigeration and

processed immediately. Comet assay was performed under alkaline conditions (pH > 13)

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according to Göethel et al. (2014). GelRed-stained slides were examined at ×500·magnification

in a fluorescence microscope (Olympus BX60F-3, Olympus Optical, Japan). Images of 100

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randomly selected cells (50 cells from each of the two replicated slides) were analysed from

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each sample. The comet parameter to measure DNA damage in the cells was the percentage of

DNA in the tail (% Tail DNA).

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2.10. Histopathological analysis

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Liver and kidney were placed in a 10% formaldehyde buffer for 7 days for fixation. The
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organs were cut and included in paraffin blocks, which were then sectioned by a microtome in
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4-micron thick cuts for the liver and 3-micron for the kidney. The slices were stretched at 51°C

and then prepared on slices. Hematoxylin/eosin staining was used. The reading was
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performed in an optical microscope at 400x.

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2.11. Statistical analysis

The signs of toxicity were expressed as percentage of animals that presented the

behavior related to toxicity. The signs of toxicity and the histopathological analyses were

analyzed through the chi-square test. All other results were analyzed through one-way ANOVA,

followed by Bonferroni post-hoc test for multiple comparisons. Normal distribution of data
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was evaluated with the Kolmogorov-Smirnov test. All data were analyzed using GraphPad

Prism (GraphPad Software, Inc.) version 5.0. Data are presented as mean ± standard error of

the mean. The level of significance was set at p<0.05.

3. Results

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3.1. Acute toxicity test

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According to the results presented in table 2, an expected increase in exploratory

activity in the first 30 min was observed in control group, which was decreased along the time.

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However, a significant increase in the locomotor activity was observed in the ED10 treated
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group after 1 h post-dosing. Even 2 h after treatment, the caffeine + taurine and alcohol +

ED10 groups presented a significant increase in locomotion (p<0.01, Chi-square test). All
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treated groups presented a significant increase in rearing (p<0.01, Chi-square test) compared

to control after 1 and 2 h of treatment. Tachypnea was present in the alcohol, caffeine +
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taurine, and alcohol + caffeine + taurine treated animals. After 2 h of treatment, the animals
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from ED10 and caffeine + taurine groups were awake, a behavior that was not so intense in the
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other groups. The animals from alcohol groups were not as awake as others after 1 h of
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treatment. After 2 h of observation, no significant changes in rodent’s behavior were

observed.
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3.2. Serum biochemistry

No alterations in urea, creatinine, ALT, AST, γ-GT, ALP, and LDH were observed in

serum 24 h after the treatments (data not shown).

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3.3. Oxidative status

After 24 h of treatments, no alterations in plasma MDA levels were found (data not

shown). Interestingly, when TBARS were evaluated in kidney, a 54% and a 17% significant

increase were observed in ED10, and caffeine + taurine treated groups, respectively (p<0.05,

ANOVA/Bonferroni; Figure 1).

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In erythrocytes, non-protein thiol levels did not present any alteration (data not

shown). However, similarly to what was observed in lipoperoxidation, a 90% significant

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increase in total thiols was observed in the kidney of the caffeine + taurine treated group

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(p<0.05, ANOVA/Bonferroni; Figure 2).
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3.4. Nephrotoxicity
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NAG activity is an early marker of tubular proximal cells damage. After 24 h of

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treatment, a 76% significant increase in NAG activity was observed in the urine of alcohol +
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ED10 group (p<0.05, ANOVA/Bonferroni), evidencing kidney damage (Figure 3).

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3.5. DNA damage

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Induction of DNA damage was assessed by the comet assay. No single or double breaks

of DNA were seen 24 h after treatments (data not shown).

3.6. Histopathological analysis

After 24 h of the treatment, congestion and hyaline degeneration were observed in

livers of alcohol-treated animals. Hydropic degeneration, hyaline degeneration, and

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congestion were observed in the livers of the animals treated with alcohol + ED10. The

animals treated with caffeine + taurine and alcohol + caffeine + taurine presented hyaline

degeneration, hydropic degeneration, congestion and hemorrhage (p<0.05; Chi-square, Figure

4).

In kidney, hyaline degeneration was observed in ED10; alcohol + ED10, caffeine +

taurine; and alcohol + caffeine + taurine. Hemorrhage was present in the kidneys of all groups,

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except the control (p<0.05; Chi-square). The images of kidney histopathological analysis are

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presented in figure 5.

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4. Discussion

Due to the high consumption of energy drinks, mainly associated with alcoholic
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beverages, this work aimed to evaluate the effects of this association in a 24 h period. Besides

behavioral alterations, the treatment with the association of alcohol and energy drink led to
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kidney damage and oxidative imbalance in this tissue.

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Based on caffeine and taurine mechanism in the central nervous system, it is possible

to assume that these substances modulate the effects of alcohol, mainly through the
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stimulating effects of caffeine and the influence of taurine in GABA-mediated

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neurotransmission (Ferreira et al., 2004). In Swiss mice, the administration of energy drink

(10,71 mg/kg) reduced the alcohol (2,5 g/kg) depressant effect (Ferreira et al., 2004). In

another study, 1 h after the administration of energy drink (8 ml/kg) and alcohol (4 g/kg) to

mice, an increase in locomotor activity in open-field test was observed (Krahe et al., 2017).

This is also related to the be awake behavior, which was observed in the animals treated with

energy drink and the association of caffeine + taurine, suggesting that the stimulant activity of

such components keep the individuals awake even after long periods of time. Interestingly,
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this could be also related to our previous study, where an increased attention in the ox-maze

test was observed in rats treated with energy drink and the combination caffeine + taurine, in

the same doses of this study (Costa-Valle et al., 2017).

In humans, the energy drink (500 ml and 750 ml) attenuated the impairment of

psychomotor function caused by alcohol (0.05 and 0.08%) (Peacock et al., 2013). Furthermore,

a study has reported the positive effect of caffeine (20 mmol/kg) + taurine (15 mg/kg) after 1

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week on physical resistance performance (Imagawa et al., 2009). In the consumers, the desired

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effects are the reduction of sleepiness and fatigue and the increase in alertness (Ishak et al.,

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2012). However, the main adverse effect due to the amount of caffeine present in energy

drinks is the insomnia (Grandner et al., 2014), which is also related to our results. Besides,
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tachypnea was present along all the experiment in the animals treated with the alcohol +

caffeine + taurine association. In accordance with Kozik et al. (2016), the stimulant properties
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of the energy drinks and their components (caffeine) are related to an increase in heart rate

and blood pressure, causing arrhythmias.

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Oxidative stress is attributed to the etiology of several diseases such as diabetes,

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cancer, and cardiovascular disorders. However, reactive oxygen species (ROS) may have

beneficial functions in terms of cellular signaling and combating microorganism in cells.

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Enhanced production of ROS or depletion of antioxidant defense system can cause an

imbalance between oxidants and antioxidants (Ilaiyaraja et al., 2011). Overexpression of ROS
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causes damages to lipids, proteins, and DNA structure (Bloomer et al., 2013). Besides, ethanol

consumption is highly recognized as increasing the production of ROS and enhancing oxidative

damage in many tissues (Jing et al., 2012; Reedy et al., 2013). Oxidative imbalance has already

been described in the liver and brain of male Sprague-Dawley rats treated for 14 days with

energy drinks (3.5 and 7g/kg) associated or not to alcohol (1 g/kg). Interestingly, energy drink

has promoted oxidative damage as well as alcohol, but this damage was greater in the animals
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treated with the association of energy drink and alcohol (Reis et al., 2017). In another study,

the association of energy drink (7.5 ml/kg) and alcohol (2.5 g/kg) induced oxidative and

inflammatory response in the temporal cortex and hippocampus of male Wistar rats after 60

days of treatment (Diaz et al., 2016). In a previous study of our group, the oral subchronic

neurotoxicity of energy drinks and their constituents was evaluated in rats. The results showed

that the groups treated with caffeine (3.2 mg/kg) and taurine (40 mg/kg), at doses equivalent

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to the consumption of 3 cans of energy drink for 28 days, had a better performance in memory

tests. However, they presented an increase of the oxidative imbalance in pre-frontal cortex,

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hippocampus and striatum. Surprisingly, it was evident in all trials that the association of

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caffeine and taurine at concentrations similar to the highest dose of energy drink was different
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from the effects of the administration of the energy drink itself (Costa-Valle et al., 2017).

The effects of caffeine and taurine on parameters related to oxidative stress have
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generated conflicting results. The administration of caffeine (4.7 mg/kg) to male Wistar rats

had an antioxidant effect after 4 weeks of treatment, lowering the level of NO in serum and
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tissues (brain, kidney and liver), increasing TAS (total antioxidant status) in serum and tissues
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(brain, kidney and liver) and decreasing TBARS in serum, brain, kidney, and liver (Stepniak and
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Czarnowski, 2010). The chronic consumption of coffee in the diet (3% and 6%) as well as
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caffeine (0.04% and 0.08%) for 21-90 days in male Wistar rats was able to reduce the lipid

peroxidation measured by TBARS levels, and increase glutathione (GSH), glutathione reductase
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(GR), and superoxide dismutase (SOD) in the brain (Abreu et al., 2011). On the contrary, a

study conducted with female BALB/c mice showed an increase of the lipoperoxidation in

kidneys after administration of caffeine in different doses (1, 4, 16, and 32 mg/kg) at 4 hours

after treatment and one day of treatment, respectively (Pohanka, 2014). In animal studies,

taurine (2%, w/v) had a protective effect on the liver of male Wistar rats treated with alcohol

(6 g/kg/day) by decreasing TBARS and normalizing thiol levels (Devi and Anuradha, 2010). In

humans, taurine (6 g/kg/day) administered to chronic alcoholic patients (for 3 months)

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decreased the serum enzymatic activity of AST and ALT, and TBARS levels (Hsieh et al., 2014).

In this study, the kidneys were particularly affected by oxidative stress, leading to an increase

in TBARs and total thiol levels. This could be related to the vulnerability to damage caused by

ROS, due to the abundance of long-chain polyunsaturated fatty acids in the composition of

renal lipids (Ozbek, 2012).

Alcohol is a non-genotoxic carcinogen, however long-term consumption of alcoholic

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beverages was confirmed to increase the risks for cancers of the upper digestive tract, liver,

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female breast and colorectum in humans. After ingestion, alcohol is metabolized by alcohol

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dehydrogenase to acetaldehyde, which is a known genotoxic compound, causing genomic

instability (Kotova et al., 2013). In previous experiments, alcohol did not exhibit genotoxicity
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(Nakagawa et al., 2015), which is in agreement with our study. Similarly, no DNA damage by

the comet assay was observed after 30 days treatment with caffeine in young mice. On the
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contrary, a reduction in DNA damage was observed in peripheral blood of aged mice treated

with caffeine (Damiani et al., 2017). Additionally, in an animal experiment, taurine also exerted
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genoprotective effects against arsenic-induced DNA damage (Zheng et al., 2017). In both cases,
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the results agree with our findings, which show that caffeine and taurine, isolated or combined
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to alcohol, did not increase DNA damage in peripheral blood of rats.

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Evaluating the effects of the energy drinks and alcohol, Khayyat et al. (2012)

demonstrated that the administration of Red Bull® (1.5 ml/100 g) to male Wistar rats for 4
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weeks elicited hepatotoxicity, assessed by elevation of serum ALT and AST activities, and

histological alteration in liver, such as loss of architecture and hepatocyte necrosis. In another

study, the treatment of male Wistar rats with energy drink (3.75 ml/kg and 7.5 ml/kg) and

alcohol (1.0 g/kg and 2.0 g/kg) for 30 days elevated serum urea, creatinine, total bilirubin, and

potassium levels, as well as hepatic enzymes activities (Ugwuja, 2014). However, all the results

from these studies could also be related to the alcohol effects. In a study by Raj and co-
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workers (2009), alcohol (0.5 g/100g body weight) was capable of increasing AST, ALT, and ALP

enzymatic activities and the TBARS levels in liver and kidneys of male rats 12 h after treatment.

Herein, no changes were observed in ALT, AST, γ-GT, ALP, and LDH activities, therefore the

effects observed are related to the treatment with energy drink and their components,

associated or not to alcohol, and not to the alcohol itself.

In our study, no biochemical alterations or oxidative damage was noted in liver.

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However, morphological alterations were observed in histopathological analysis, such as

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hydropic degeneration. This is a result of an electrolytic imbalance and accumulation of water

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in the cytoplasm (Abdelhalim and Jarra, 2011), and it was observed in the livers of caffeine +

taurine, alcohol + caffeine + taurine, and alcohol + ED10 treated groups. Hyaline degeneration
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detects the accumulation of extracellular protein material (Abbas et al., 2010) and it was

detected in all groups, except for control and ED10. In a study conducted by Khayyat et al.
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(2012), rats receiving energy drink (1.5 ml/kg) for 14 and 28 days presented infiltration,

vacuolization and necrosis, which were not observed in our study. However, differently from
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this study, our effects were evaluated 24 hours after treatment. Hepatic toxicity of alcohol is
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well reported and it is related to alcohol metabolism in hepatocytes, leading to lipid

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peroxidation and consequent damage due to the production of reactive oxygen species (ROS)
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(Louvet and Mathurin, 2015). In this study, alcohol presented histological damage (hyaline

degeneration and congestion) as expected, but the damage was more pronounced when
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alcohol was associated to the energy drink.

Histological analysis of rat kidney tissues after treatment with energy drinks (10 and 20

ml/kg) for 21 days did not show any damages (Akande and Banjoko, 2011). Interestingly, in a

case-report, a young adolescent presented acute tubular necrosis of kidneys with subsequent

increase of creatinine, urinary sodium concentration and lower urine osmolality requiring

hemodialysis after the consumption of 3l energy drink in association with 1l vodka (Schoffl et
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al., 2011). In another report, a 40-year-old man in abstinence from alcohol for 3 months, who

used to ingest 6 of the 20-oz Red Bull drinks per day in the past months, presented increased

serum creatinine, blood urea nitrogen, and glucose, leading to an acute renal failure (Greene

et al., 2014). In our work, in addition to the hyaline degeneration and hemorrhage observed in

kidneys, an oxidative imbalance was noted after 24 h treatment with energy drink and their

components. Surprisingly, the most prominent kidney alteration was the increased NAG

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excretion, which is one of the lysosomal enzymes abundantly present in the proximal tubular

cells. Therefore, increased activity of this enzyme in urine may suggest injury to tubular cells,

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serving as a specific urinary marker for tubular cell function. In the course of active kidney

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disease, urinary NAG levels remain persistently elevated. The increase in urinary NAG activity
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indicates damage to tubular cells, although it may also reflect increased lysosomal activity

without cellular damage (Liangos et al., 2007). Increased urinary NAG excretion has been
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reported in acute renal disease of varying etiology, especially induced by toxic agents (Price,

1992).
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5. Conclusions
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In summary, our results indicated behavioral alterations following energy drink and

alcohol treatments. Additionally, oxidative imbalance was observed in kidneys after 24 h of

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treatment with energy drink and their components caffeine and taurine. Moreover, a potential

transitory nephrotoxicity was observed when energy drink was combined with alcohol. Mild

histopathological alterations were also observed in liver. These results demonstrate that the

association of alcohol and energy drinks requires a better safety evaluation in humans,

especially in teenagers, who are the main consumers of these beverages. Therefore, a risk

evaluation and the potential nephrotoxic effects of alcohol and energy drink combination

should be better characterized.

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Conflict of interest

The authors declare that there are no conflict of interest.

Acknowledgements

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The research was supported by the following Brazilian funding agencies: National

Council for Scientific and Technological Development - CNPq; CAPES Foundation, and

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Universidade Federal do Rio Grande do Sul (UFRGS). The authors are grateful to MSc. Jessica

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Nardi for the revision of the manuscript.

Conflict of interest Statement

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The authors declare that there are no conflict of interest.
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Tables

Behavior (express in percentage of animals)

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Table 1. Experimental design

Treatment groups
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Alcohol + Caffeine + Alcohol + Caffeine
Control ED10 Alcohol ED10 Taurine + Taurine
Energy
drink 10 Caffeine 3.2 Caffeine 3.2 mg/kg
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Distilled water Energy drink 20% Ethanol ml/kg mg/kg + Taurine 40 mg/kg
10 ml/kg 10 ml/kg 2 g/kg + 20% + Taurine 40 + 20% Ethanol
Ethanol 2 mg/kg 2g/kg
g/kg
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Table 2: Acute effects of the treatment with energy drink, alcohol, and their components
isolated or in association. Data are expressed as percentage of animals that presented the
effect after 30, 60 and 120 min (n=5). *p<0.01 vs control, Chi-square test.
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Increase in Rearing Grooming Tachypnea Awake

locomotor
activity (%) (%) (%) (%) (%)
Groups Time Time (minutes) Time Time (minutes) Time (minutes)
(minutes) (minutes)
3 60 12 30 60 12 30 6 12 30 60 12 3 60 120
0 0 0 0 0 0 0
Control 4 0 0 20 0 0 4 - 20 0 0 - 10 60
0 80 0 0
ED 10 60 0 10 60 - 0 0 20 - 10 100
0 * 0 80* * 40 0 0 *

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* * *
Caffein 6 40 80 10 100 60 0 2 - 20 20 - 10 100
e+ 0 * * 0 * * 0 40 0 *

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Taurine *
Alcohol 2 20 40 40 4 - 20 0 - 60
0 * 20 80* * 60 0 60 60

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* * * *
Alcohol 4 60 60 80 60 0 2 - 20 20 20 - 80 80
+ ED 10 0 * * 60* * 0
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Alcohol 2 0 0 0 40 20 2 - - 80
+ 0 40 * 0 80 60 60 60
Caffein * * * * *
e+
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Legends of figures

Figure 1. TBARS levels in kidney, liver, spleen, and heart measured 24 h after treatment with

ED10, alcohol, alcohol + ED10, caffeine + taurine, or alcohol + caffeine + taurine. Each column

represents the mean ± SEM (n = 4–5). Statistical comparisons were made using one-way

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ANOVA/Bonferroni post-hoc test (*p<0.05; **p<0.01 vs control).

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Figure 2. Non protein thiol levels in kidney, liver, spleen, and heart measured 24 h after

treatment with ED10, alcohol, alcohol + ED10, caffeine + taurine, or alcohol + caffeine +
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taurine. Each column represents the mean ± SEM (n = 4–5). Statistical comparisons were made

using one-way ANOVA/Bonferroni post-hoc test (*p<0.05 vs control).

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Figure 3. Urinary NAG activity measured 24 h after treatment with ED10, alcohol, alcohol +
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ED10, caffeine + taurine, or alcohol + caffeine + taurine. Each column represents the mean ±
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SEM (n = 4). Statistical comparisons were made using one-way ANOVA/Bonferroni post-hoc
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test (*p<0.05 vs control).

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Figure 4. Histopathological analysis of livers from Wistar rats 24h after treatment. A-Control, B-

ED 10, C- Caffeine + taurine, D- Alcohol, E- Alcohol + ED10, F- Alcohol + caffeine + taurine.

Dotted arrows represent hyaline degeneration, white arrows represent hemorrhage, fine black

arrows represent hydropic degeneration, and thick black arrows represent congestion. All

images are in 400x optical magnification.

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Figure 5. Histopathological analysis of kidneys from Wistar rats 24h after treatment. A-Control,

B- ED 10, C- Caffeine + taurine, D- Alcohol, E- Alcohol + ED10, F- Alcohol + caffeine + taurine.

Dotted arrows represent hyaline degeneration and the white arrows represent hemorrhage.

All images are in 400x optical magnification.

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Energy drink and alcohol combination lead to kidney and liver alterations in rats

Highlights:

 The consumption of energy drinks associated with alcohol could be harmful.

 Behavioral effects after the association of alcohol and energy drink are distinct.

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 Energy drinks and their constituents induce oxidative damage in the kidney of rats.

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 The combined consumption of energy drink and alcohol induced nephrotoxicity to

rats.

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Figure 1
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Figure 4
Figure 5