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Citation: Banc, R.; Popa, D.-S.;
Cozma-Petru¸t, A.; Filip, L.; Kiss, B.;
F˘arca¸s, A.; Nagy, A.; Miere, D.;
Loghin, F. Protective Effects of Wine
Polyphenols on Oxidative Stress and
Hepatotoxicity Induced by
Acrylamide in Rats. Antioxidants
2022,11, 1347. https://doi.org/
10.3390/antiox11071347
Academic Editor: Paolo Bergamo
Received: 10 June 2022
Accepted: 6 July 2022
Published: 10 July 2022
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4.0/).
antioxidants
Article
Protective Effects of Wine Polyphenols on Oxidative Stress and
Hepatotoxicity Induced by Acrylamide in Rats
Roxana Banc 1, † , Daniela-Saveta Popa 2, † , Anamaria Cozma-Petru¸t 1, * , Lorena Filip 1, * , Béla Kiss 2,
Anca Fărca¸s 3, Andras Nagy 4, Doina Miere 1and Felicia Loghin 2
1Department of Bromatology, Hygiene, Nutrition, “Iuliu Ha¸tieganu” University of Medicine and Pharmacy,
6 Pasteur Street, 400349 Cluj-Napoca, Romania; roxana.banc@umfcluj.ro (R.B.); dmiere@umfcluj.ro (D.M.)
2Department of Toxicology, “Iuliu Ha¸tieganu” University of Medicine and Pharmacy, 6 Pasteur Street,
400349 Cluj-Napoca, Romania; dpopa@umfcluj.ro (D.-S.P.); kbela@umfcluj.ro (B.K.); floghin@umfcluj.ro (F.L.)
3Department of Mathematics-Informatics, “Iuliu Ha¸tieganu” University of Medicine and Pharmacy,
6 Pasteur Street, 400349 Cluj-Napoca, Romania; anca.farcas@umfcluj.ro
4Department of Veterinary Toxicology, University of Agricultural Sciences and Veterinary Medicine,
3-5 Mănă¸stur Street, 400372 Cluj-Napoca, Romania; andras.nagy@usamvcluj.ro
*Correspondence: anamaria.cozma@umfcluj.ro (A.C.-P.); lfilip@umfcluj.ro (L.F.);
Tel.: +40-(74)-5693208 (A.C.-P.); +40-(74)-0210135 (L.F.)
† These authors contributed equally to this work.
Abstract:
In recent years, it has been increasingly suggested that the consumption of natural polyphe-
nols, in moderate amounts, is beneficial for health. The aim of this study was to investigate the efficacy
of a red wine (the administered dose of 7 mL/kg/day being equivalent to ~16.5 mg/kg/day total
polyphenols) compared to a white wine (the administered dose of 7 mL/kg/day being equivalent to
~1.7 mg/kg/day total polyphenols), on the prevention of acrylamide-induced subacute hepatic injury
and oxidative stress in Wistar rats. Hepatic damage due to acrylamide intoxication (the administered
dose being 250
µ
g/kg body weight, for 28 days, by intragastric gavage) was assessed by employing
biochemical parameters (aspartate aminotransferase (AST) and alanine aminotransferase (ALT)) and
by histopathological studies. Markers of oxidative damage were measured in terms of plasma malon-
dialdehyde (MDA), hepatic Thiobarbituric Acid Reactive Substances (TBARS) and glutathione (GSH)
levels, and liver antioxidant enzyme (superoxide dismutase (SOD), catalase (CAT) and glutathione
peroxidase (GPx)) activities. Regarding hepatic enzyme activities, treatment with red wine signifi-
cantly decreased the AST values (p< 0.05), while for the ALT values only a normalization tendency
was observed. Treatment with red wine and white wine, respectively, significantly prevented the
increase in MDA and TBARS levels (p< 0.05), as well as the depletion of GSH (
p< 0.05
). Red wine
treatment normalized the activities of the antioxidant enzymes CAT and SOD in rats intoxicated with
acrylamide, while supplementing the diet with white wine did not produce significant differences in
the antioxidant enzyme activities. Histopathological findings revealed a moderate protective effect
of red wine after four weeks of daily consumption. Our findings provide evidence that red wine,
having a higher phenolic content than white wine, has a significant protective effect on oxidative
stress and liver injury induced by acrylamide in rats, through its antioxidative activity.
Keywords:
polyphenols; red wine; white wine; acrylamide; hepatotoxicity; rats; oxidative stress;
antioxidant activity
1. Introduction
Among the alcoholic beverages widely consumed worldwide, wine remains a very
popular beverage, having been consumed for hundreds of years [
1
–
3
]. In adults, moderate
consumption of red wine is one of the typical elements of the Mediterranean diet [
3
–
6
]. The
association of a moderate consumption of wine with numerous health benefits is based on
its content being rich in antioxidant phenolic compounds, which have both a functional
Antioxidants 2022,11, 1347. https://doi.org/10.3390/antiox11071347 https://www.mdpi.com/journal/antioxidants
Antioxidants 2022,11, 1347 2 of 23
role, acting against free radicals, and a physiological role, by increasing the antioxidant
capacity of the human body [1,2,4–7].
While moderate wine intake is associated with significant health benefits, excessive al-
cohol consumption is considered a major risk factor for many diseases (including alcoholic
liver disease, cirrhosis of the liver and cancer) and deaths among adults [
1
,
6
,
8
]. Alcohol,
consumed in excess, exerts toxic effects on the hepatocellular level and free radicals gen-
erated in excess from hepatocyte destruction cause alteration of proteins and lipids, with
the consequence of intensification of oxidative stress and lipid peroxidation, and, overall,
a negative effect on the cellular antioxidant defense system [
9
–
11
]. Therefore, oxidative
stress plays a key role in the pathophysiological processes that underlie liver damage
related to excessive alcohol consumption [
9
,
10
]. However, the results of several studies
associate regular consumption of wine in moderate amounts with beneficial cardiovascular
effects and a low incidence of deaths from atherosclerosis and coronary heart disease,
effects related to increased levels of high-density lipoprotein (HDL), but which have not
always been confirmed in the case of white wine [
2
,
6
,
12
–
15
]. Despite numerous studies
referring to the beneficial effects of red wine, Radeka et al., have shown that white wines,
not just red wines, have produced a decrease in systolic and diastolic blood pressure, total
cholesterol and LDL levels, and an increase in HDL levels and serotonin and dopamine
levels, following regular consumption for six weeks [
16
]. Moreover, moderate wine con-
sumption (200–300 mL/day), both for red and white wine, is generally associated with a
reduction in all-cause mortality, with studies highlighting some of the beneficial effects,
such as regulating blood pressure, cholesterol and lipids, anti-inflammatory and anti-tumor
effects, preventing diabetes, obesity, atherosclerosis, cardiovascular and neurodegenerative
diseases [
2
,
6
,
14
–
16
]. Although chronic exposure to ethanol induces up-regulation of hepatic
antioxidant enzymes in mice, the beneficial effects of red wine are thought to exceed those
attributed especially to alcohol [
17
,
18
]. Therefore, this increased protection is attributed to
the presence of polyphenols, powerful antioxidants, more abundant in red wines [
4
,
18
,
19
].
Acrylamide (ACR) is a chemical contaminant that naturally forms during heat pro-
cessing, at temperatures above 120
◦
C, by frying, roasting, toasting, grilling or baking of
foods rich in carbohydrates and low in proteins [
20
–
25
]. It is abundant in fried and roasted
potato, vegetable crisps, cocoa and roasted coffee, bread and pastry, cookies, as well as
in tobacco smoke, and, in lower concentrations, in breakfast cereals, biscuits, crackers,
roasted nuts, homemade food, human breast milk, infant milk powder and a range of baby
foods [
21
,
23
,
25
–
27
]. ACR can be absorbed through the digestive tract, respiratory system or
through the skin and after exposure can be rapidly distributed to many tissues and organs,
such as the thymus, thyroid, liver, heart, brain, spleen, kidneys, as well as the human
placenta and breast milk [
21
,
25
,
27
,
28
]. It is further transformed by an enzymatic reaction
involving cytochrome P450 2E1 into a more toxic and reactive epoxide, glycidamide, which
is widely distributed into the tissues [
21
,
24
,
26
,
29
]. Although both ACR and glycidamide
can react with proteins (e.g., haemoglobin) to form covalent adducts, only glycidamide
forms covalent adducts with DNA amino groups, which cause genetic mutations and
damage chromosomes [
26
,
27
,
29
,
30
]. Both Hb adducts and covalent DNA adducts of gly-
cidamide are used as important biomarkers of AA exposure [
29
]. Studies have shown
that ACR is neurotoxic and genotoxic and has been classified as “probably carcinogenic to
humans”, carcinogenicity Group 2A, by the International Agency for Research on Cancer,
based on its carcinogenicity in rodents [
21
,
23
,
24
,
31
]. In addition, in 2015, the European
Food Safety Authority (EFSA) confirmed that the presence of acrylamide in foods is a
public health concern, requiring continued efforts to reduce its exposure [
20
,
29
]. In this
context, Regulatory Agencies have provided recommendations for reducing the formation
of ACR in food, without setting mandatory limits for maximum acceptable levels of AA in
food [
25
]. Among the recommendations made by the Spanish Agency for Consumer Affairs,
Food Safety, and Nutrition (AECOSAN), in 2015, are: cooking food in the microwave or
baking instead of frying, reducing the time and temperature of frying food (<175
◦
C) and
avoiding reuse of frying oil [
25
]. At European Union level, “Commission Regulation (EU)
Antioxidants 2022,11, 1347 3 of 23
2017/2158” was issued in 2017, setting mitigation measures and benchmark levels to reduce
the presence of AA in food [32].
Oxidative stress is one of the main mechanisms explaining the neurotoxicity and
hepatotoxicity following ACR exposure [
28
,
30
]. Increased ROS production, as a result
of ACR exposure, may adversely affect cell survival, due to cell membrane damage by
oxidative degradation of lipids, proteins, and irreversible DNA modification [
21
,
25
,
33
].
Lipid peroxidation, estimated through the level of substances reacting with thiobarbituric
acid and of hydroperoxides, as well as products of proteins’ oxidation, such as carbony-
lated proteins, are markers of oxidative degradation produced by ROS [
33
]. In addition,
oxidative damage is aggravated by decreased antioxidant enzyme activities, such as those
of superoxide dismutase (SOD), catalase (CAT), glutathione S-transferase (GST) and glu-
tathione peroxidase (GPx), which act as free radical traps in conditions associated with
oxidative stress [
21
,
25
,
33
,
34
]. Since both ACR and glycidamide can be conjugated to
glutathione (GSH) as a detoxifying pathway of ACR, forming GSH adducts that are subse-
quently converted to mercapturic acids, which are used as biomarkers of ACR exposure,
the consequence is GSH depletion [
25
,
28
,
29
]. Therefore, proteomics studies suggest that
ACR-induced toxicity may be mediated by cellular oxidative stress following extensive
GSH consumption [
25
,
28
]. Free radicals are produced continuously
in vivo
and protective
antioxidant enzymes (SOD, CAT, GST, GPx), as well as reduced GSH, have roles in coping
with these toxic substances [33,35].
Since it is vital to know the role of naturally occurring antioxidants in preventing the
negative health effects of ACR exposure, several recent studies have evaluated these issues.
Thus, Sayed et al., showed that pomegranate peel has anti-inflammatory, anti-apoptotic,
and free radical scavenging activity and strong antioxidant activity that protects against
ACR toxicity [
21
]. In another study by Hamdy et al., hesperidin and tiger nut demonstrated
an antioxidant defense against ACR toxicity in breast, liver and kidney tissues [
34
]. Qu et al.
also showed that digested jackfruit flake provides increased protection against oxidative
damage caused by ACR, significantly reducing cytotoxicity and excessive ROS production
in cells, thus mitigating mitochondrial disorders [36].
In order to reveal a new strategy for the prevention of oxidative stress-caused diseases,
it is important to take into account that, although the health effects of phenolic compounds
are supported by their powerful antioxidant activity, their influence on health will depend
on the amount consumed and their bioavailability. Moreover, despite the promising po-
tential of any compound/element/extract demonstrated
in vitro
, the efficacy/beneficial
effects and the level of safety can only be established through
in vivo
toxicological stud-
ies [
37
,
38
]. As there are currently insufficient studies in chronic models to demonstrate
the relationship between the bioavailability of phenolic compounds and their antioxidant
effects, this experiment aimed to assess the relationship between
in vitro
and
in vivo
wine
functionality, namely whether the
in vivo
antioxidant response of wines correlates with
the
in vitro
antioxidant activity of wines. Thus,
in vitro
wine functionality was tested
in a previous study, which characterized white and red wines obtained from Romanian
grape cultivars. Based on the results obtained previously, the white wine and the red wine,
respectively, with the richest phenolic content and the highest
in vitro
antioxidant activity,
were selected for the present study [39].
Since the generation of ACR in food is unavoidable, a modern approach is to include
functional foods in the diet, thus taking advantage of the protective effects of their bio-
logically active components. Therefore, the present study was designed to evaluate the
protective effects of wine phenolic compounds against oxidative stress and liver toxicity
experimentally induced by ACR in Wistar rats. For this purpose, wine was administered
to rats, in the quantity and with the periodicity that corresponded to the most frequent
consumption behavior in Mediterranean countries: two to three glasses of wine with 12.5%
ethanol, during a day. Thus, in this study, the wine was administered both to rats fed
with a standard diet and to rats treated with ACR, in addition to the standard diet, as an
experimental model for inducing oxidative stress.
Antioxidants 2022,11, 1347 4 of 23
2. Materials and Methods
2.1. Wine Samples and Reagents
The two wine samples used in this study were local varieties produced from old
Romanian vine varieties and were purchased from a local supermarket. As we reported
before, FeteascăNeagrăred wine (FN
Toh2010
) came from the Viticultural Region of the
Muntenia and Oltenia Hills, Dealu Mare Vineyard, Tohani Wine Center, while Fetească
Regalăwhite wine (FR
Jid2011
) came from the Transylvanian Plateau Wine Region, Târnave
Vineyard, Jidvei Wine Center [
39
]. The bottles were opened, immediately separated into
tubes and stored at −80 ◦C.
The total phenolic content (TPC) and
in vitro
antioxidant activity of the wines used in
this study are presented in Table 1.
Table 1.
Total phenolic content (TPC), DPPH radical scavenging activity (%) and total antioxidant
activity (TAA) of the two wine samples used in this study (mean value (n= 3)).
White Wine Sample TPC
(mg GAE/L)
DPPH Radical Scavenging
Activity (%)
TAA
(mM TE/L)
FRJid2011 245 * 51 * 0.93 *
Red Wine Sample TPC
(mg GAE/L)
DPPH Radical Scavenging
Activity (%)
TAA
(mM TE/L)
FNToh2010 2359 * 95 * 9.84 *
GAE: Gallic acid equivalents; TE: Trolox equivalents. * Data from Banc et al. [39].
Acrylamide (C
3
H
5
NO, purity > 99%) was purchased from Merck (Merck, Darm-
stadt, Germany) and 1,1,3,3-tetraethoxypropane (TEP) was purchased from Sigma-Aldrich
(Sigma-Aldrich, Steinheim, Germany). HPLC grade reduced and oxidized glutathione
were obtained from Fluka (Fluka, Buchs SG, Switzerland). Acetonitrile, methanol, formic
acid, acetic acid, acetone and HPLC grade hexane were purchased from Merck (Merck,
Darmstadt, Germany). All other chemicals were of analytical grade and were obtained
from Merck (sodium hydroxide, hydrochloric acid, sulfuric acid, perchloric acid, 2,4-
dinitrophenylhydrazine (DNPH), sodium tetraborate decahydrate, o-phthalaldehyde, ac-
etaldehyde, TRIS hydrochloride (Tris hydroxymethyl aminomethane hydrochloride) and
1,4-dithiothreitol (DTT)).
Deionized water was obtained using a Milli-Q water purification system (Millipore,
Milford, MA, USA). All other reagents used to determine the biochemical parameters were
of analytical grade and were purchased from Merck and Sigma.
2.2. Experimental Animals
A community of 60 healthy white rats, Charles River Wistar (Crl:WI) strain, males,
with an initial weight of 150
±
14 g, was used. The animals were provided by the Center for
Experimental Medicine and Practical Skills of the “Iuliu Ha
t
,
ieganu” University of Medicine
and Pharmacy Cluj-Napoca (Romania). The rats were housed in large polypropylene
cages (5 rats per cage) and kept in a controlled environment: the temperature of the
animal storage room was 21
±
1
◦
C, with a relative humidity of at least 60% and the
daily cycle of animals was 12 h of light and 12 h of darkness, with an air change ratio
of 10–20/h. Throughout the experiment, the animals had free access to the standard dry
pellet diet (Cantacuzino Institute, Bucharest, Romania) and drinking water ad libitum. All
procedures and treatments applied to animals in this study were performed in accordance
with European Union Directive (2010/63/EU) for animal experiments and were approved
by the Ethics Commission of the “Iuliu Ha
t
,
ieganu” University of Medicine and Pharmacy
Cluj-Napoca (Romania) (protocol number 219/11.06.2014).
Antioxidants 2022,11, 1347 5 of 23
2.3. Experimental Design
The study lasted 33 days, with an animal acclimatization period of 5 days and an
effective experimental period of 28 days. The animals were randomly divided into six
groups, each group consisting of ten animals. All experiments on rats were conducted in
the morning (in accordance with ethical principles for conducting painful experiments on
animals and with the current guidelines for the care of laboratory animals). The exper-
imental groups, the feeding regime, and the manipulations performed are presented in
Table 2.
Table 2. Experimental design.
Group Number of Animals Intragastric Gavage Diet
Control (C) 10 12.5% (v/v) hydroalcoholic solution standard
Positive control (PC) 10 12.5% (v/v) hydroalcoholic solution + acrylamide
250 µg/kg of weight, 1% (m/v) aqueous solution standard
White wine (WW) 10 FRJid2011 white wine standard
White wine + acrylamide
(WW + ACR) 10
FR
Jid2011
white wine + acrylamide 250
µ
g/kg of weight,
1% (m/v) aqueous solution standard
Red wine (RW) 10 FNToh2010 red wine standard
Red wine + acrylamide
(RW + ACR) 10 FNToh2010 red wine+ acrylamide 250 µg/kg of weight,
1% (m/v) aqueous solution standard
The volumes of wine or 12.5% hydroalcoholic solution administered were calculated
based on a wine consumption of 500 mL/70 kg body weight/day, and an equivalent
volume of 7 mL/kg/day (~16.5 mg/kg/day total polyphenols in the case of red wine)
was administered. Doses were adjusted daily according to the weight of the animals.
Intragastric gavage was performed once a day at the same hour using flexible feeding
needles (17 Gauge, 85 mm length and 2.4 mm tip diameter) (Fine Science Tools). No
samples were taken during the experiment.
The animals were observed daily for 4 weeks. It was observed whether the animals
showed signs of toxicity, such as the following: pathological changes in the skin and
mucous membranes, changes in appearance and fur condition, damage to other systems
(respiratory system, central nervous system) or changes in behavior and general condition.
2.4. Collection of Biological Material
After 4 weeks, the rats were deprived of food for 12 h, and blood samples were
collected, in tubes treated with ethylenediaminetetraacetic acid (EDTA), from the retro-
orbital venous sinus, after instillation of the eye drops with local anesthetic, oxybuprocaine
0.4% (Benoxi 4 mg/mL). Plasma was separated by centrifugation for 15 min at 1600
×
g
and 4
◦
C and was used immediately to determine reduced and total GSH. The rest of the
plasma samples were frozen in liquid nitrogen and stored at
−
80
◦
C for further analysis
(determination of total plasma level of malondialdehyde (MDA) and determination of
transaminases).
The animals were sacrificed by dislocating the cervical spine under isoflurane general
anesthesia and the liver was taken and weighed. Liver tissue samples were taken immedi-
ately, some being fixed in 10% formalin solution, buffered at neutral pH, for incorporation
into paraffin, while others were washed three times in 0.9% ice-cold saline solution to
remove blood, wiped individually on filter paper and quickly frozen in liquid nitrogen and
stored at −80 ◦C, being subsequently used for the preparation of liver homogenates.
2.5. Histopathological Evaluation of Liver Tissue
For the histological exam, the hepatic samples were fixed in 10% buffered neutral
formalin and embedded in paraffin. The sections were made with a high-precision micro-
tome Leica RM 2125 RT, at 5-
µ
m thick, and stained by the hematoxylin–eosin (HE) and
Sirius red (SR) methods. The slides were examined under a BX51 Olympus microscope,
Antioxidants 2022,11, 1347 6 of 23
Olympus DP 25 digital camera and processed using the Olympus Cell B image acquisition
and processing program.
2.6. Evaluation of Liver Function
Transaminases, aspartate aminotransferase (AST) and alanine aminotransferase (ALT),
which are useful markers for assessing the level of liver cell damage, were determined in
plasma samples by spectrophotometric tests using commercial kits provided by Randox
Laboratories (Ardmore, Nothern Ireland, Great Britain).
2.7. Preparation of Hepatic Tissue Homogenates
Each pre-weighed frozen liver tissue was homogenized on an ice bath for 10 min
in a Polytron PT 1200 E homogenizer with 10 volumes of 50 mM Tris buffer containing
10 mM EDTA (pH 7.5). The homogenate was centrifuged for 10 min at 1000
×
gand
4
◦
C to remove all cellular debris and the resulting supernatant was used to determine
oxidative stress parameters (Thiobarbituric Acid Reactive Substances (TBARS), GSH) and
antioxidant enzymes (SOD, CAT and GPx). The expression of the parameters determined
in the supernatant was done per mg of protein.
2.8. Protein Quantification in Liver Tissue Homogenates
The protein content in liver homogenates was quantified in the supernatant by the
method described by Bradford [
40
]. This method is based on the color reaction of proteins
with an acidic solution of Coomassie Brilliant Blue G-250, called Bradford reagent. Samples
of tissue homogenate were diluted so that their protein content was 5–100
µ
g protein/100
µ
L
homogenate. A volume of up to 0.1 mL (adjusted to 0.1 mL with phosphate buffered saline)
was taken from this diluted tissue homogenate, to which 5 mL of Bradford reagent were
added. After 5 min, the absorbance was read at 595 nm against the blank (prepared from
0.1 mL of buffer and 5 mL of Bradford reagent) and the protein content (expressed in
mg/mL) was determined from the calibration curve.
2.9. Evaluation of Oxidative Stress by Means of Biomarkers Determined in Plasma
2.9.1. Determination of Lipid Peroxidation by Quantification of Malondialdehyde in
Rat Plasma
The degree of lipid peroxidation was assessed by quantitative analysis of MDA using
a Waters Acquity Ultra Performance Liquid Chromatography (UPLC) system coupled with
a Waters Acquity PDA (Waters, Milford, USA). For this purpose, 75
µ
L rat plasma sample
were used. The sample preparation consisted in a hydrolysis step at 60
◦
C in a waterbath,
in the presence of 25
µ
L 6M NaOH, followed by protein precipitation with 63
µ
L 35%
perchloric acid and derivatization with 100
µ
L 5mM 2,4-dinitrophenylhydrazine (in 2N
HCl). The final derivatization product was extracted in 1.2 mL n-hexane, followed by the
evaporation of the organic layer under a stream of nitrogen. The residue was dissolved in
100
µ
L mobile phase (a mixture of 1% formic acid/acetonitrile, 62/38, v/v) and subjected
to UPLC-PDA analysis.
Chromatographic separation was achieved on a BEH C18 column (50 mm
×
2.1 mm
i.d., 1.7
µ
m) from Waters (Waters, Milford, USA) preceded by a 0.2
µ
m online filter. A
7.5 min gradient elution was performed with a mixture of 1% formic acid/acetonitrile as
the mobile phase. The flow rate was 0.3 mL/min and the absorbance of the eluent was
monitored at 301 nm [41,42].
2.9.2. Determination of Reduced and Total Glutathione Plasma Levels
Reduced and total GSH were determined in plasma samples by UPLC with flu-
orescence detection. A Waters Acquity UPLC system coupled with a Waters Acquity
Fluorescence detector was used (Waters, Milford, CT, USA).
Antioxidants 2022,11, 1347 7 of 23
For the quantification of reduced GSH, 100
µ
L of rat plasma samples were needed.
The sample preparation involved a simple protein precipitation step with sulfosalicylic
acid, followed by derivatization for 1 min with ortho-phtalaldehyde.
In case of the total GSH, an additional step was needed. After deproteinization, the
oxidized GSH was reduced by incubation with 100 mM DTT, for 5 min at room temperature.
Chromatographic separation was carried out using a BEH C18 column (
100 mm ×2.1 mm
i.d., 1.7
µ
m) from Waters (Waters, Milford, CT, USA) preceded by a 0.2
µ
m online filter.
For reduced GSH, isocratic elution was performed at a flow rate of 0.3 mL/min, with a
mixture of methanol and 0.25% acetic acid (pH 6.9, with 6 M NaOH) as the mobile phase.
In the case of the total GSH, the same mixture was used as the mobile phase, but a gradient
elution was needed. The selected excitation and emission wavelengths were 350 nm and
420 nm, respectively [42,43].
2.10. Evaluation of Oxidative Stress in Liver Tissue
2.10.1. Determination of Lipid Peroxidation by Quantifying TBARS in Liver Tissue
Liver tissue lipid peroxidation was measured by the fluorimetric method for the deter-
mination of TBARS, described by Conti et al. [
44
]. This method depends on the formation of
MDA, as the final product of lipid peroxidation, which reacts with thiobarbituric acid and
forms a fluorescent adduct, which can be measured spectrofluorimetrically at 534 nm. The
TBARS method is nonspecific for MDA, fatty peroxide-derived decomposition products
other than MDA being thiobarbituric acid positive. For the quantification, 50
µ
L of tissue
homogenate were mixed with 1 mL of 10 mM solution of 2-thiobarbituric acid in 75 mM
solution of K
2
HPO
4
(pH 3.0), followed by stirring for 5 s, then the mixture was heated
in a water bath at 95
◦
C for an hour. After sudden cooling on ice, the reaction product
was extracted into 5 mL of n-butanol. Its concentration was determined in the organic
phase, after separation by centrifugation for 10 min at 1500
×
gand 4
◦
C. Emission intensity
measurement was performed at 534 nm with a Perkin Elmer LS 45 spectrofluorimeter (Nor-
walk, CT, USA), using a synchronous fluorescence technique at a wavelength difference
between excitation and emission (
∆λ
) of 14 nm. The concentrations were expressed in
nmol/mg protein.
2.10.2. Determination of Reduced Glutathione Levels in Liver Tissue
The fluorimetric method described by Hu [
45
] was used to measure reduced GSH
levels in liver tissue. This method of quantifying GSH by fluorescence is based on the
reaction of GSH with o-phthalaldehyde, resulting in the formation of a fluorescent product
that can be measured spectrofluorimetrically. Briefly, a volume of 0.5 mL tissue homogenate
was added to 0.5 mL of 10% (m/v) trichloroacetic acid solution. After being kept on ice
for 10 min, the mixture was centrifuged for 10 min at 3000
×
gand 4
◦
C and 0.2 mL of
supernatant were mixed with 1.7 mL of phosphate buffer and 0.1 mL of o-phthalaldehyde.
After 15 min, the emission intensity was measured at 420 nm, at an excitation of 350 nm,
against a blank containing deionized water, instead of the tissue homogenate, using a
Perkin Elmer LS 45 spectrofluorimeter (Norwalk, CT, USA). Concentrations were expressed
in nmol/mg protein.
2.10.3. Determination of Antioxidant Enzymes in Liver Tissue
The determination of superoxide dismutase activity was performed by the cytochrome
C reduction test, described by Flohéand Otting [
46
]. Superoxide dismutase catalyzes the
dismutation of the superoxide radical (O
2−•
) in hydrogen peroxide (H
2
O
2
) and oxygen
(O
2
), thus being a defense agent against the toxic effects of superoxide. The superoxide
radical (O
2−•
) is generated by the xanthine-xanthine oxidase system in the presence of
oxygen. O
2−•
reacts with ferricytochrome C, which can be continuously monitored by
recording the absorbance at 550 nm. In the presence of SOD, the reduction of cytochrome
C is inhibited, due to the decrease in the concentration of superoxide ions. The SOD
concentration in the sample could, thus, be calculated from the degree of inhibition of
Antioxidants 2022,11, 1347 8 of 23
cytochrome C reduction, using a calibration curve obtained using SOD standards of known
concentrations. Absorbance measurements were recorded with a Jasco V 530 UV-Vis
spectrophotometer (Tokyo, Japan).
A unit (U) of SOD activity was defined as the amount of enzyme capable of inhibiting
by 50% the rate of reduction of cytochrome C under the conditions specified. The results
were expressed in U SOD/g protein.
The determination of catalase activity was performed by the method described by
Pippenger et al. [
47
]. Catalase is considered to be an enzyme with an antioxidant role
because it regulates the level of H
2
O
2
which, in its absence, could increase and lead to
the appearance of an excess of highly reactive
•
OH radicals. The method consisted of
measuring the change of the absorbance of a solution of 10 mM H
2
O
2
in 0.05 M phosphate
buffer (pH 7.4) at 240 nm. A unit of enzymatic activity was defined as the amount of
catalase that induced a reduction in absorbance of 0.43 times over a period of 3 min, at
25
◦
C. The activity was expressed in U/mg protein, being calculated according to the
following formula:
CAT = A240/0.43 ×0.02 (mg/mL),
where A
240
is the absorbance at 240 nm. Absorbance changes were monitored using the
Jasco V 530 UV-Vis spectrophotometer (Tokyo, Japan).
The determination of glutathione peroxidase activity was performed by an indirect
method, described by Flohéand Günzler [
48
]. GPx is a selenoprotein that catalyzes the
reaction between a hydroperoxide (e.g., H
2
O
2
) and GSH, as an electron donor, leading
to the formation of oxidized glutathione (GSSG) and water. The method used was based
on monitoring the decrease in NADPH concentration, in the presence of which GSSG
formed in the reaction was converted to GSH by glutathione reductase (GSSG-R). GSSG
formed during the GPx reaction was reduced instantly and continuously by an excess
activity of glutathione reductase, ensuring a constant level of GSH. Concomitant oxidation
of NADPH was monitored photometrically. The reduction of absorption to 340 (or 365) nm
was monitored for 6 min, using the Jasco V 530 UV-Vis spectrophotometer (Tokyo, Japan).
The non-enzymatic reaction rate was assessed by replacing the test sample with buffer.
The activity of the enzyme was defined as the amount of GPx which induced a net
decrease in GSH of 10% of the initial concentration, in one minute, at 37
◦
C and at pH 7.
The calculation took into account the stoichiometry of the reaction and the molar extinction
coefficient of NADPH. Thus,
A = 0.868 (∆[NADPH]/[GSH]0t)(Vi/Vs),
where [NADPH] is the molar concentration of NADPH, [GSH]
0
-the initial concentration of
GSH, t-duration of reaction, Vi-the volume of incubation mixture, and Vs-the volume of the
sample to be analyzed. Activity was reported at 1 mg protein for liver tissue homogenates.
2.11. Statistical Analysis
Statistical analysis of data was performed using the SPSS statistical program (version
13.0). All data were expressed as mean
±
SEM (standard error of the mean). To evaluate
the data variations, the analysis of the unifactorial variance (One-Way ANOVA) was used,
followed by the Bonferoni test as a post-test for multiple comparisons. The level of statistical
significance was set for a value p≤0.05.
3. Results
3.1. General Toxicity: Evolution of Animal Body Weight, Absolute and Relative Weight of the Liver
All animals survived throughout the experimental period. There were no changes in
the external appearance of the animals, but in terms of their behavior, a state of agitation
and hypermotility of the animals in the groups treated with ACR (PC, WW + ACR, RW
+ ACR) was observed from the 17th experimental day, compared to those who were not
given ACR (C, WW, RW).
Antioxidants 2022,11, 1347 9 of 23
The results regarding the evolution of body weight, at the end of the 28 day experimen-
tal period, for the 6 groups of animals included in the study, are presented in Figure 1. The
initial body weight and the final body weight of the rats from the 6 experimental groups
are presented in Supplementary Table S1.
Antioxidants 2021, 10, x FOR PEER REVIEW 9 of 24
2.11. Statistical Analysis
Statistical analysis of data was performed using the SPSS statistical program (version
13.0). All data were expressed as mean ± SEM (standard error of the mean). To evaluate
the data variations, the analysis of the unifactorial variance (One-Way ANOVA) was used,
followed by the Bonferoni test as a post-test for multiple comparisons. The level of statis-
tical significance was set for a value p ≤ 0.05.
3. Results
3.1. General Toxicity: Evolution of Animal Body Weight, Absolute and Relative Weight of the
Liver
All animals survived throughout the experimental period. There were no changes in
the external appearance of the animals, but in terms of their behavior, a state of agitation
and hypermotility of the animals in the groups treated with ACR (PC, WW + ACR, RW +
ACR) was observed from the 17th experimental day, compared to those who were not
given ACR (C, WW, RW).
The results regarding the evolution of body weight, at the end of the 28 day experi-
mental period, for the 6 groups of animals included in the study, are presented in Figure
1. The initial body weight and the final body weight of the rats from the 6 experimental
groups are presented in Supplementary Table S1.
Figure 1. The evolution of the average body weight of the animals from the 6 groups, during the
entire experimental period. C−hydroalcoholic solution group; PC−hydroalcoholic solution + acryla-
mide group; WW−white wine group; WW + ACR−white wine + acrylamide group; RW−red wine
group; RW + ACR−red wine + acrylamide group.
At the end of the 4 experimental weeks, the final body weight differed significantly
only for rats in the RW + ACR group, not for those in the WW + ACR group, when com-
pared to the PC group. No significant differences were observed in terms of weight gain
in the animals in the WW + ACR group and the RW + ACR group, respectively, when
compared to the PC group, nor in the animals in the WW group and the RW group, re-
spectively, when compared to the control group.
Table 3 shows the absolute and relative liver weight of rats in each experimental
group.
Figure 1.
The evolution of the average body weight of the animals from the 6 groups, during the entire
experimental period. C
−
hydroalcoholic solution group; PC
−
hydroalcoholic solution + acrylamide
group; WW
−
white wine group; WW + ACR
−
white wine + acrylamide group; RW
−
red wine group;
RW + ACR−red wine + acrylamide group.
At the end of the 4 experimental weeks, the final body weight differed significantly
only for rats in the RW + ACR group, not for those in the WW + ACR group, when
compared to the PC group. No significant differences were observed in terms of weight
gain in the animals in the WW + ACR group and the RW + ACR group, respectively,
when compared to the PC group, nor in the animals in the WW group and the RW group,
respectively, when compared to the control group.
Table 3shows the absolute and relative liver weight of rats in each experimental group.
Table 3.
Effects of white/red wine on absolute liver weight and relative liver weight (% of body
weight) of rats in the 6 experimental groups at the end of the experiment.
Experimental Groups
C PC WW WW + ACR RW RW + ACR
Absolute liver weight (g)
4.78 ±0.15 a6.04 ±0.17 b4.66 ±0.18 a6.02 ±0.15 b4.79 ±0.17 a5.99 ±0.03 b
Relative liver weight (%) 2.58 ±0.08 a3.15 ±0.09 b2.39 ±0.09 a2.94 ±0.08 b2.46 ±0.09 a2.82 ±0.02 a,b
Values are expressed as mean
±
SEM (n= 10).
a,b
Mean values not sharing the same superscript letter within a
row are different at p< 0.05. C
−
hydroalcoholic solution group; PC
−
hydroalcoholic solution + acrylamide group;
WW
−
white wine group; WW + ACR
−
white wine + acrylamide group; RW
−
red wine group; RW + ACR
−
red
wine + acrylamide group.
At the end of the experimental period, the relative liver weight of rats in the PC group
was significantly higher (p< 0.05) than that of the rats in the C group. Also, the relative
liver weight was significantly increased (p< 0.05) in the WW + ACR group compared to
the C group. In contrast, red wine supplementation in the RW + ACR group prevented
a significant increase in the relative liver weight of rats compared to the C group. No
significant differences in relative liver weight were obtained between the WW + ACR and
RW + ACR groups, respectively, and the PC group.
3.2. Histopathological Examination of Rat Liver
Histological studies of the liver of rats from the 6 experimental groups are illustrated
in Figure 2.
Antioxidants 2022,11, 1347 10 of 23
Antioxidants 2021, 10, x FOR PEER REVIEW 10 of 24
Table 3. Effects of white/red wine on absolute liver weight and relative liver weight (% of body weight) of rats in the 6
experimental groups at the end of the experiment.
Experimental Groups
C PC WW WW + ACR RW RW + ACR
Absolute liver
weight (g) 4.78 ± 0.15
a
6.04 ± 0.17
b
4.66 ± 0.18
a
6.02 ± 0.15
b
4.79 ± 0.17
a
5.99 ± 0.03
b
Relative liver
weight (%) 2.58 ± 0.08
a
3.15 ± 0.09
b
2.39 ± 0.09
a
2.94 ± 0.08
b
2.46 ± 0.09
a
2.82 ± 0.02
a,b
Values are expressed as mean ± SEM (n = 10).
a,b
Mean values not sharing the same superscript letter within a row are
different at p < 0.05. C−hydroalcoholic solution group; PC−hydroalcoholic solution + acrylamide group; WW−white wine
group; WW + ACR−white wine + acrylamide group; RW−red wine group; RW + ACR−red wine + acrylamide group.
At the end of the experimental period, the relative liver weight of rats in the PC group
was significantly higher (p < 0.05) than that of the rats in the C group. Also, the relative
liver weight was significantly increased (p < 0.05) in the WW + ACR group compared to
the C group. In contrast, red wine supplementation in the RW + ACR group prevented a
significant increase in the relative liver weight of rats compared to the C group. No sig-
nificant differences in relative liver weight were obtained between the WW + ACR and
RW + ACR groups, respectively, and the PC group.
3.2. Histopathological Examination of Rat Liver
Histological studies of the liver of rats from the 6 experimental groups are illustrated
in Figure 2.
Figure 2. Hepatic histology in rats from different experimental groups (HE stain). Control group
treated with 12.5% hydroalcoholic solution (A), showing perivascular lymphohistiocytic inflamma-
tory infiltrate; Positive control group treated with 12.5% hydroalcoholic solution and acrylamide
(B), presenting a focus of hepatic fibrosis and lymphohistiocytic inflammatory infiltrate; Group
treated with Fetească Regală white wine (C), showing focal lymphohistiocytic infiltrate; Group
treated with Fetească Regală white wine and acrylamide (D), showing large focus of hepatic fibrosis
and lymphohistiocytic inflammatory infiltrate; Group treated with Fetească Neagră red wine (E),
showing minimal lymphohistiocytic inflammatory cell infiltrate in the portal space; Group treated
with Fetească Neagră red wine and acrylamide (F), presenting focus of fibrosis, minimal lympho-
histiocytic inflammatory infiltrate.
Figure 2.
Hepatic histology in rats from different experimental groups (HE stain). Control group
treated with 12.5% hydroalcoholic solution (
A
), showing perivascular lymphohistiocytic inflammatory
infiltrate; Positive control group treated with 12.5% hydroalcoholic solution and acrylamide (
B
),
presenting a focus of hepatic fibrosis and lymphohistiocytic inflammatory infiltrate; Group treated
with FeteascăRegalăwhite wine (
C
), showing focal lymphohistiocytic infiltrate; Group treated
with FeteascăRegalăwhite wine and acrylamide (
D
), showing large focus of hepatic fibrosis and
lymphohistiocytic inflammatory infiltrate; Group treated with FeteascăNeagrăred wine (
E
), showing
minimal lymphohistiocytic inflammatory cell infiltrate in the portal space; Group treated with
FeteascăNeagrăred wine and acrylamide (
F
), presenting focus of fibrosis, minimal lymphohistiocytic
inflammatory infiltrate.
Histological examination of the liver samples was performed on HE and SR stained
slides. Liver sections from the control group (Group C) treated with 12.5% hydroalcoholic
solution showed minimal lymphohistiocytic inflammatory infiltrate around the portal
spaces, as well as minimal perivascular collagen deposition in the same areas. Animals
from the positive control group (Group PC) treated with 12.5% hydroalcoholic solution
and ACR presented several foci of hepatic fibrosis and lymphohistiocytic inflammatory
infiltrate, as well as minimal perisinusoidal collagen deposition. The hepatic sections in rats
from the group treated with FeteascăRegalăwhite wine (Group WW), presented rare foci
of lymphohistiocytic infiltrate and minimal collagen deposition around the portal spaces.
Histological examination of the hepatic sections from the group treated with Fetească
Regalăwhite wine and ACR (Group WW + ACR) showed minimal lymphohistiocytic
infiltration in the portal areas, and several foci of lymphohistiocytic infiltrate and small
foci of hepatic fibrosis throughout the parenchyma. The sections from the animals of the
group treated with FeteascăNeagrăred wine (Group RW) showed normal histology, the
only modification observed was a minimal mononuclear inflammatory cell infiltrate in
the portal areas. The hepatic sections from the group treated with FeteascăNeagrăred
wine and ACR (Group RW + ACR) showed mild to severe lymphohistiocytic inflammatory
infiltrate around portal spaces. In the parenchyma scattered foci of fibrosis with minimal
lymphohistiocytic inflammatory infiltrate was observed.
Antioxidants 2022,11, 1347 11 of 23
3.3. Biochemical Parameters of Liver Damage
The effects of intragastric administration of white wine and red wine, respectively, on
the plasma levels of AST and ALT enzymes, are presented for all experimental groups in
Table 4.
Table 4.
Effects of white/red wine on aspartate aminotransferase and alanine aminotransferase levels
in the plasma of rats from the 6 experimental groups.
Experimental Groups
C PC WW WW + ACR RW RW + ACR
AST (U/mL plasma) 101.29 ±4.96
162.06
±
23.15
a
116.31
±
15.06
130 ±12.29 105.17 ±6.45 b108.3 ±10.06 b
ALT (U/mL plasma) 60.34 ±9.71 88.93 ±10.27 a52.93 ±3.83 b70.77 ±14.79 44.94 ±5.56 b62.68 ±11.61
Values are expressed as mean
±
SEM (n= 10).
a
There are significant differences when compared to the control
group (C) at the significance level p< 0.05.
b
There are significant differences when compared to the positive control
group (PC) at the significance level p< 0.05. AST
−
aspartate aminotransferase; ALT
−
alanine aminotransferase.
C
−
hydroalcoholic solution group; PC
−
hydroalcoholic solution + acrylamide group; WW
−
white wine group;
WW + ACR
−
white wine + acrylamide group; RW
−
red wine group; RW + ACR
−
red wine + acrylamide group.
The plasma levels of AST in the PC group, treated with ACR, were significantly
increased (p< 0.05) compared to those of group C, which received only 12.5% (v/v) hy-
droalcoholic solution. Also, ACR treatment led to an increase in the plasma level of ALT
in group PC compared to group C (p< 0.05). Supplementation with red wine in the RW
+ ACR group significantly prevented the increase of the AST level compared to the PC
group, while observing a trend of normalization of the ALT value, statistically insignificant
compared to the PC group. For the groups treated only with white wine and red wine,
respectively, no significant differences in plasma levels of AST and ALT were obtained
compared to the control group.
3.4. Effects of Wine Polyphenols on Acrylamide-Induced Oxidative Stress Assessed by Plasma
Concentration of Biomarkers
3.4.1. Determination of Lipid Peroxidation by Quantification of Malondialdehyde in
Rat Plasma
The influence of intragastric administration of white wine and red wine, respectively,
on the total level of MDA in the plasma of the animals from the 6 experimental groups is
presented in Figure 3A. A representative chromatogram for the determination of the total
MDA level in a rat plasma sample is shown in Supplementary Figure S1.
ACR treatment led to an increase in plasma MDA in the PC group compared to the
control group (p< 0.05). The MDA value was lower in both the WW + ACR group and the
RW + ACR group, compared to the PC group, but the MDA reduction was only statistically
significant in the case of the first group, WW + ACR (p< 0.05). The groups that received one
of the 2 wine samples ‘in addition to the standard diet, without inducing their oxidative
stress with ACR, showed a low plasma level of MDA compared to the control group, with
significant differences between the WW and C groups (p< 0.05), and without significant
differences between RW and C.
Antioxidants 2022,11, 1347 12 of 23
Antioxidants 2021, 10, x FOR PEER REVIEW 12 of 24
3.4. Effects of Wine Polyphenols on Acrylamide-Induced Oxidative Stress Assessed by Plasma
Concentration of Biomarkers
3.4.1. Determination of Lipid Peroxidation by Quantification of Malondialdehyde in Rat
Plasma
The influence of intragastric administration of white wine and red wine, respectively,
on the total level of MDA in the plasma of the animals from the 6 experimental groups is
presented in Figure 3A. A representative chromatogram for the determination of the total
MDA level in a rat plasma sample is shown in Supplementary Figure S1.
Figure 3. Effects of white/red wine on: total MDA plasma level (nmol/mL) (A), reduced GSH plasma
level (nmol/mL) (B), and the reduced GSH/total GSH ratio (C) in rats from ACR-treated groups
compared to untreated ones (n = 10). Bars marked with the same letter do not differ significantly (p
< 0.05). C−hydroalcoholic solution group; PC−hydroalcoholic solution + acrylamide group;
WW−white wine group; WW + ACR−white wine + acrylamide group; RW−red wine group; RW +
ACR−red wine + acrylamide group.
ACR treatment led to an increase in plasma MDA in the PC group compared to the
control group (p < 0.05). The MDA value was lower in both the WW + ACR group and the
RW + ACR group, compared to the PC group, but the MDA reduction was only statisti-
cally significant in the case of the first group, WW + ACR (p < 0.05). The groups that re-
ceived one of the 2 wine samples `in addition to the standard diet, without inducing their
oxidative stress with ACR, showed a low plasma level of MDA compared to the control
group, with significant differences between the WW and C groups (p < 0.05), and without
significant differences between RW and C.
Figure 3.
Effects of white/red wine on: total MDA plasma level (nmol/mL) (
A
), reduced GSH plasma
level (nmol/mL) (
B
), and the reduced GSH/total GSH ratio (
C
) in rats from ACR-treated groups
compared to untreated ones (n= 10). Bars marked with the same letter do not differ significantly
(p< 0.05). C
−
hydroalcoholic solution group; PC
−
hydroalcoholic solution + acrylamide group;
WW
−
white wine group; WW + ACR
−
white wine + acrylamide group; RW
−
red wine group; RW +
ACR−red wine + acrylamide group.
3.4.2. Determination of Reduced and Total Glutathione Plasma Levels
The effects of intragastric administration of white wine and red wine, respectively, on
the plasma levels of reduced GSH and on the ratio of reduced GSH/total GSH are presented
for all experimental groups in Figure 3B and Figure 3C, respectively. A representative
chromatogram of total GSH and reduced GSH, respectively, from a rat plasma sample are
shown in Supplementary Figure S2 and S3, respectively.
The reduced GSH plasma level in the PC group, treated with ACR, was not statistically
different compared to that of the control group, which received only 12.5% (v/v) hydroalco-
holic solution. Wine supplementation significantly prevented the reduction of the reduced
GSH level in the WW + ACR and RW + ACR groups (p< 0.05) compared to the PC group.
Treatment of animals in the PC group with ACR did not produce a significant decrease in
the reduced GSH/total GSH ratio compared to the control group. Wine supplementation in
ACR-treated groups only significantly prevented the reduction of the reduced GSH/total
GSH ratio in the RW + ACR group (p< 0.05), compared to the PC group. For the groups
treated only with white wine and red wine, respectively, no significant differences, in terms
of the reduced GSH plasma levels or in the reduced GSH/total GSH ratio, were obtained,
compared to the control group.
Antioxidants 2022,11, 1347 13 of 23
3.5. Effects of Wine Polyphenols on Acrylamide-Induced Oxidative Stress in Liver Tissue
3.5.1. Effects of Wine Polyphenols on Lipid Peroxidation
The influence of intragastric administration of white wine and red wine, respectively,
on the hepatic level of TBARS, for the experimental groups included in the study, is
presented in Figure 4A.
Antioxidants 2021, 10, x FOR PEER REVIEW 13 of 24
3.4.2. Determination of Reduced and Total Glutathione Plasma Levels
The effects of intragastric administration of white wine and red wine, respectively,
on the plasma levels of reduced GSH and on the ratio of reduced GSH/total GSH are pre-
sented for all experimental groups in Figure 3B and Figure 3C, respectively. A representa-
tive chromatogram of total GSH and reduced GSH, respectively, from a rat plasma sample
are shown in Supplementary Figure S2 and S3, respectively.
The reduced GSH plasma level in the PC group, treated with ACR, was not statisti-
cally different compared to that of the control group, which received only 12.5% (v/v) hy-
droalcoholic solution. Wine supplementation significantly prevented the reduction of the
reduced GSH level in the WW + ACR and RW + ACR groups (p < 0.05) compared to the
PC group. Treatment of animals in the PC group with ACR did not produce a significant
decrease in the reduced GSH/total GSH ratio compared to the control group. Wine sup-
plementation in ACR-treated groups only significantly prevented the reduction of the re-
duced GSH/total GSH ratio in the RW + ACR group (p < 0.05), compared to the PC group.
For the groups treated only with white wine and red wine, respectively, no significant
differences, in terms of the reduced GSH plasma levels or in the reduced GSH/total GSH
ratio, were obtained, compared to the control group.
3.5. Effects of Wine Polyphenols on Acrylamide-Induced Oxidative Stress in Liver Tissue
3.5.1. Effects of Wine Polyphenols on Lipid Peroxidation
The influence of intragastric administration of white wine and red wine, respectively,
on the hepatic level of TBARS, for the experimental groups included in the study, is pre-
sented in Figure 4A.
Antioxidants 2021, 10, x FOR PEER REVIEW 14 of 24
Figure 4. Effects of white/red wine on hepatic TBARS level (nmol/mg protein) (A) and reduced GSH
hepatic level (nmol/mg protein) (B) in rats from ACR-treated groups compared to untreated ones
(n = 10). Effects of white / red wine on superoxide dismutase activity (U/mg protein) (C), catalase
activity (U/mg protein) (D), and glutathione peroxidase activity (U/mg protein) (E) in liver tissue of
rats from ACR-treated groups compared to untreated ones (n = 10). Bars marked with the same letter
do not differ significantly (p < 0.05). C−hydroalcoholic solution group; PC−hydroalcoholic solution
+ acrylamide group; WW−white wine group; WW + ACR−white wine + acrylamide group; RW−red
wine group; RW + ACR−red wine + acrylamide group.
As can be seen from Figure 4A, hepatic TBARS levels increased significantly in the
PC group treated with ACR compared to the control group (p < 0.05), while supplemen-
tation with wine in the groups WW + ACR and RW + ACR significantly prevented (p <
0.05) the increase in TBARS level compared to the PC group. For the groups treated only
with white wine, or with red wine, respectively, the decrease of the TBARS value was only
significant in the case of the RW group (p < 0.05) compared to the control group.
3.5.2. Effects of Wine Polyphenols on Reduced Glutathione Hepatic Levels
The effects of intragastric administration of white wine and red wine, respectively,
on the hepatic level of reduced GSH are shown in Figure 4B.
The hepatic level of reduced GSH in the PC group, treated with ACR, was not statis-
tically different from the control group, which received only 12.5% (v/v) hydroalcoholic
solution. In the case of the RW + ACR group, a significantly increased hepatic level of GSH
was obtained (p < 0.05) compared to the PC group, while between the WW + ACR and PC
groups the differences were not statistically significant. In the groups treated only with
white wine and red wine, respectively, the hepatic level of reduced GSH was significantly
increased for the groups WW and RW (p < 0.05) compared to the control group.
3.5.3. Effects of Wine Polyphenols on Liver Antioxidant Enzymes Activities
The results of the in vivo study regarding the influence of wine samples on the activ-
ity of the antioxidant enzymes SOD, CAT and GPx in the liver are presented in Figure 4C–
E.
A significant decrease (p < 0.05) in SOD activity was observed in rats from the PC
group, treated with ACR, compared to those in the control group, which received only
12.5% (v/v) hydroalcoholic solution. Wine intragastric administration in groups of rats
with ACR-induced oxidative stress led to a significant increase in SOD activity only for
the RW + ACR group (p < 0.05) compared to the PC group. In the WW and RW groups,
treated only with wine, there were no significant differences in SOD activity compared to
the control group.
A significant reduction (p < 0.05) in the level of CAT activity was evident for the PC
group compared to the control group. For the RW + ACR group, a significantly increased
Figure 4.
Effects of white/red wine on hepatic TBARS level (nmol/mg protein) (
A
) and reduced GSH
hepatic level (nmol/mg protein) (
B
) in rats from ACR-treated groups compared to untreated ones
(n= 10). Effects of white/red wine on superoxide dismutase activity (U/mg protein) (
C
), catalase
activity (U/mg protein) (
D
), and glutathione peroxidase activity (U/mg protein) (
E
) in liver tissue
of rats from ACR-treated groups compared to untreated ones (n= 10). Bars marked with the same
letter do not differ significantly (p< 0.05). C
−
hydroalcoholic solution group; PC
−
hydroalcoholic
solution + acrylamide group; WW
−
white wine group; WW + ACR
−
white wine + acrylamide group;
RW−red wine group; RW + ACR−red wine + acrylamide group.
Antioxidants 2022,11, 1347 14 of 23
As can be seen from Figure 4A, hepatic TBARS levels increased significantly in the PC
group treated with ACR compared to the control group (p< 0.05), while supplementation
with wine in the groups WW + ACR and RW + ACR significantly prevented (p< 0.05) the
increase in TBARS level compared to the PC group. For the groups treated only with white
wine, or with red wine, respectively, the decrease of the TBARS value was only significant
in the case of the RW group (p< 0.05) compared to the control group.
3.5.2. Effects of Wine Polyphenols on Reduced Glutathione Hepatic Levels
The effects of intragastric administration of white wine and red wine, respectively, on
the hepatic level of reduced GSH are shown in Figure 4B.
The hepatic level of reduced GSH in the PC group, treated with ACR, was not statis-
tically different from the control group, which received only 12.5% (v/v) hydroalcoholic
solution. In the case of the RW + ACR group, a significantly increased hepatic level of GSH
was obtained (p< 0.05) compared to the PC group, while between the WW + ACR and PC
groups the differences were not statistically significant. In the groups treated only with
white wine and red wine, respectively, the hepatic level of reduced GSH was significantly
increased for the groups WW and RW (p< 0.05) compared to the control group.
3.5.3. Effects of Wine Polyphenols on Liver Antioxidant Enzymes Activities
The results of the
in vivo
study regarding the influence of wine samples on the activity
of the antioxidant enzymes SOD, CAT and GPx in the liver are presented in Figure 4C–E.
A significant decrease (p< 0.05) in SOD activity was observed in rats from the PC
group, treated with ACR, compared to those in the control group, which received only
12.5% (v/v) hydroalcoholic solution. Wine intragastric administration in groups of rats
with ACR-induced oxidative stress led to a significant increase in SOD activity only for
the RW + ACR group (p< 0.05) compared to the PC group. In the WW and RW groups,
treated only with wine, there were no significant differences in SOD activity compared to
the control group.
A significant reduction (p< 0.05) in the level of CAT activity was evident for the PC
group compared to the control group. For the RW + ACR group, a significantly increased
value (p< 0.05) of CAT activity was obtained, compared to the PC group. No significant
differences were observed between the WW and RW groups, respectively, and the control
group in terms of CAT activity levels.
GPx activity was significantly reduced (p< 0.05) in the animals from the PC group
versus the animals from the control group. No significant differences were observed
between the WW + ACR and RW + ACR groups, respectively, and the PC group in terms
of GPx activity levels. Wine intragastric administration for the RW group has led to a
significant increase in GPx activity (p< 0.05) compared to the control group and to a
decrease in enzyme activity for the WW group.
4. Discussion
The use of ACR as an inducer of oxidative stress in this study was based on the fact
that, although ACR poses a significant risk to human health, its widespread presence has
been found in a range of processed foods, leading to an estimate of dietary exposure of
0.4
µ
g ACR/kg body weight/day [
49
,
50
]. Since Yousef and El-Demerdash [
51
], in their
study, reported disorders of oxidative status and enzymatic activities produced by ACR
in rats, the effects being pronounced at high doses, we chose to use the minimum dose of
ACR (250
µ
g/kg body weight), at which these authors observed clinical signs of general
toxicity and a risk of organ damage.
Numerous studies have suggested that regular and moderate wine consumption
can reduce oxidative stress and inflammation, leading to a reduction in the incidence of
coronary heart disease [
2
,
6
,
52
], these effects being attributed to phenolic compounds present
in wines. In order to evaluate the hypothesis that the effects of wines on oxidative stress are
dependent on their polyphenolic content and on their
in vitro
antioxidant activity, the diet
Antioxidants 2022,11, 1347 15 of 23
of rats was supplemented with two different wine samples, white and red, respectively,
and the oxidative stress conditions were induced by ACR-treatment. As shown above
(Table 1), the two wine samples chosen for this study were the most representative, having
the richest phenolic content and the highest
in vitro
antioxidant activity, among red and
white wines, respectively, characterized in our previous study [
39
]. In comparison, the two
wine samples showed significant differences in terms of total and individual content of
phenolic compounds and in terms of in vitro antioxidant activity.
Although the intake of phenolic compounds associated with red wine (~16.5 mg total
polyphenols/kg body weight/day) in the diet of animals from the RW + ACR group was
much higher than that associated with white wine (~1.7 mg total polyphenols/kg body
weight/day) to animals in the WW + ACR group, after 4 weeks neither of the two wines
contributed to increased weight gain, compared to rats from the PC group.
Hepatotoxicity caused by ACR led to an increase in liver size in groups treated with
ACR. This enlargement of the liver may have been due to hepatocyte proliferation as a
result of chemically-induced hyperplasia, due to ACR and alcohol, or to an increase in the
volume of individual hepatocytes. These results are explained by the fact that both ACR
and alcohol are enzyme inducers, primarily of CYP2E1, but also of GST, which can lead to
increased liver weight [
53
–
56
]. Regarding the absolute and relative weight of the liver, the
intragastric administration of white wine and red wine, respectively, did not significantly
protect against increase in liver weight, caused by ACR, in the WW + ACR and RW + ACR
groups, respectively, compared to the PC group.
The results obtained suggest that the concomitant administration of wine samples to
ACR-treated rats did not result in a change in relative and absolute liver weights in the
direction of the values obtained for the control group.
The histopathological examination aimed at identifying morphological changes in the
liver (considered the possible target organ) following the administration of alcohol in a
concentration of 12.5% (in the form of hydroalcoholic solution, with either white wine or
red wine), or 12.5% alcohol associated with acrylamide, respectively.
The histopathological study of the liver for the control group revealed histo-architectonic
features similar to that of the groups that received white wine and red wine, respectively.
The morphological changes in these 3 groups were discrete, following subacute alcohol
consumption. The images obtained following the histopathological evaluation in the
animals from the groups treated with ACR showed slight morphological changes, the
differences observed between the 3 groups being minor. Thus, the most affected were the
PC and WW + ACR groups, which showed micro-foci of hepatocyte lysis, lymphohistiocytic
infiltrate in the portal spaces and minimal collagen deposition. In the ACR-treated group,
whose diet was supplemented with red wine, the severe lymphohistiocytic inflammatory
infiltrate in the portal spaces suggests that the administration of red wine failed to visibly
protect the liver from the effects of subacute intoxication with low doses of ACR. In contrast,
collagen deposition, was lower in the RW + ACR group compared to the PC group. This
proliferation of collagen fibers in ACR-treated groups could have been a result of hepatic
oxidative stress which, according to Li et al. [52], stimulates collagen synthesis.
A less obvious hepatic protective effect of red wine may be the consequence of the
experimental study period of four weeks being of too short a duration, such that the
administration of wine was subacute, especially given that the hepatic protective effects of
polyphenols have been reported following longer-term consumption of red wine. Thus,
Uzma et al. [
57
] achieved a significant attenuation of carbon tetrachloride-induced liver
damage in rats by supplementing the diet with red wine for 8 weeks.
The hepatocellular cytoplasmic enzymes AST and ALT serve as indicators of liver
function and integrity, being released into the systemic circulation in the case of liver
damage, such as hepatocellular necrosis, with cell membrane degradation [
21
,
53
,
58
]. The
activities of these enzymes are usually increased in acute hepatotoxicity or mild hepatocel-
lular damage but tend to decrease with prolonged intoxication due to liver damage [
58
]. In
our study, the evolution of ALT and AST values showed significant increases (p< 0.05) of
Antioxidants 2022,11, 1347 16 of 23
the activities of these enzymes in the PC group compared to the control group, suggesting
hepatocyte membrane permeability and ALT and AST migration in the intercellular space.
Permeabilization of the hepatocyte membrane may have occurred as a consequence of
the damage caused by the binding of acrylamide or metabolites, such as glycidamide,
to membrane proteins [
21
]. The values obtained for AST showed a significant decrease
(
p< 0.05
) in the case of the ACR-treated group that received additional red wine, compared
to the PC group. The results obtained for AST indicated that red wine polyphenols had
a protective effect against the hepatotoxicity of acrylamide in the RW + ACR group. This
protection of red wine was not fully confirmed in the case of the values obtained for ALT in
the
RW + ACR
group, compared to the PC group, In this case only a trend of normalization
of ALT values in the direction of the values obtained for the control group was observed.
The dynamics of transaminases values were consistent with data published by other au-
thors only in case of the AST. The lower hepatoprotective effect may have been due either
to supplementing the diet with wine for too short a time, the effects of polyphenols being
visible only after chronic wine administration, or to an insufficient polyphenolic intake to
ensure liver protection, even in the case of red wine. Thus, Alturfan et al. [
49
] reported
significantly low levels of transaminases following dietary supplementation of rats with
resveratrol, although most likely the hepatic protective effect was due to the administration
of resveratrol in high doses (30 mg/kg body weight/day).
Oxidative stress is a phenomenon caused by an imbalance between the production
and accumulation of ROS in cells and tissues and the antioxidant defense capacity, resulting
in the oxidative damage of important cellular macromolecules, such as lipids, DNA and
proteins [
33
,
59
–
61
]. Besides DNA damage, oxidative stress mediated by ROS may result
in lipid peroxidation [
33
,
59
]. For instance, an excess of hydroxyl radical and peroxynitrite
can cause lipid peroxidation, thus damaging cell membranes and lipoproteins [
59
]. Among
several available markers of lipid oxidation, MDA, a volatile
β
-scission product formed
from the peroxidation of polyunsaturated fatty acids is very popular. Since MDA is toxic
and mutagenic, it is one of the most studied products of peroxidative damage [33,59].
The data obtained in this study showed that ACR produced an increase in lipid
peroxidation, expressed by an increase in plasma MDA levels. The decrease in plasma MDA
concentrations observed in the study in the WW + ACR and RW + ACR groups suggests
that the whole lipid peroxidation process was diminished by regular supplementation of the
wine diet. Our data are consistent with those obtained by Montilla et al. [
62
], who reported
a significant reduction in lipid peroxidation, assessed by the MDA level determined in
the plasma of rats with induced oxidative stress, due to dietary supplementation with red
wine. In contrast to these results, Macedo et al. [
63
] did not obtain significant differences in
plasma MDA levels after administration of 3 samples of red wine, having low, medium
and high in vitro antioxidant activity.
GSH is the main cellular antioxidant and one of the most important parameters for
assessing oxidative damage [
25
,
33
]. The antioxidant effects of wines were demonstrated by
the results obtained for the two parameters, reduced GSH and reduced GSH/total GSH
ratio, in animal plasma. Thus, a significant increase (p< 0.05) in the reduced GSH plasma
level was evident in both the WW + ACR group and the RW + ACR group compared to the
PC group, while the reduced GSH/total GSH ratio was significantly increased (p< 0.05)
only in the RW + ACR group. This showed that wine ingestion resulted in a protective
effect, due to the phenolic antioxidants present in the two types of wine, but the protective
effect was significantly more pronounced for the red wine, which has a higher phenolic
content than the white wine. Similar results were reported by Montilla et al. [
62
], whose
study on rats with induced oxidative stress obtained a significant increase in plasma GSH
following the administration of red wine.
In this study, plasma levels of reduced GSH decreased while MDA levels increased
in ACR-treated rats. The increase in MDA levels, the indicator marker of the degree
of lipid peroxidation, was in agreement with the findings of Filipovic et al. [
25
], which
Antioxidants 2022,11, 1347 17 of 23
suggested that intensified lipid peroxidation is a consequence of GSH depletion as a result
of oxidative stress.
The generation of ROS in rat liver was found to be significantly increased (p< 0.05) in
the PC group treated with ACR compared to C, WW and RW groups, which did not receive
ACR. These results confirmed the harmfulness of ACR in producing an oxidative stress
state, with changes of all the following evaluated oxidative stress markers being observed:
TBARS, GSH and the activities of antioxidant enzymes (SOD, GPx and CAT).
ACR is able to interact with vital cell nucleophiles that possess -SH or -NH
2
groups,
due to its
α
,
β
-unsaturated carbonyl structure [
21
,
64
]. Thus, ACR is oxidized to glycidamide,
a reactive epoxide, which is conjugated to GSH [
25
]. Glycidamide also forms adducts with
amino groups in DNA [
26
,
27
]. Increased lipid peroxidation levels occur as a result of
GSH depletion to certain critical levels [
25
]. Therefore, by the reaction of ACR with GSH,
S-conjugates of GSH are formed, which represent the first step in the biotransformation of
electrophilic substances into mercapturic acids [
27
,
29
,
49
]. In the present study, reduction
of hepatic GSH levels, and increase of lipid peroxidation, respectively, in the ACR-treated
group compared to the control group, could be explained by the reaction between ACR and
GSH. At the same time, with the administration of wines, it was observed that there was
reduction of TBARS levels and increase of GSH values, due to the protective antioxidant
effects of the constituent polyphenols. Thus, supplementation with both white and red
wine of the diet of rats treated with ACR significantly reduced (p< 0.05) the hepatic level
of TBARS in the 2 groups, WW + ACR and RW + ACR, compared to the PC group, treated
only with ACR. These findings demonstrate that the oxidative lesions induced by ACR in
the liver were ameliorated by treatment with both white and red wine, but especially red
wine. Macedo et al. [
63
] also reported a reduction in hepatic TBARS level by supplementing
the diet of rats with red wine with high antioxidant activity. Similar data were reported by
Alturfan et al. [
49
], who induced oxidative stress with acrylamide in rats, and the protective
effect was conferred by dietary supplementation with resveratrol.
Regarding hepatic GSH levels, a significant difference (p< 0.05) was observed between
the different experimental groups, namely, the RW group compared to the control group,
and the RW + ACR group compared to the PC group, suggesting that wine ingestion
affected the homeostasis of the animals’ bodies. Increasing GSH levels in experimental
groups given red wine might suggest an antioxidant response, even in the case of inducing
oxidative stress with acrylamide. Similar results were reported by Alturfan et al. [
49
], while
Gris et al. [
65
] did not obtain significant differences between the hepatic GSH levels of their
different experimental groups, that were given eight different red wine samples, without
inducing oxidative stress. In contrast to the results obtained for TBARS, where white
wine supplementation conferred a protective effect against ACR, in the case of the hepatic
GSH, the effect of white wine supplementation was weak, without significant increase
in GSH level to provide protection. The favorable effects following the administration
of wine, especially red wine, seem to be due to the presence of flavonoids and other
phenolic compounds in wine, and are similar to those obtained in previous studies by
Uzma et al. [
57
], where red wine relieved the hepatic oxidative stress induced by carbon
tetrachloride in rats. The phenolic compounds present in wine may be responsible for the
protective effects observed against ACR.
Antioxidant enzymes protect major molecules, such as lipids, proteins, and DNA
from oxidative damage by inactivating oxidants. These antioxidant enzymes can act in a
coordinated way to protect living tissues from oxidative damage [
66
]. Low levels of the
enzymatic activities of CAT, SOD and GPx were recorded in the liver tissues of rats in
the PC group treated with ACR compared to the control group, suggesting acute lesions
caused by ACR. The harmful effects of ACR in the liver decreased with the administration
of wines, with a change in the level of CAT and SOD to the normal level, depending on the
type of wine administered.
Statistical analysis showed that the type of wine administered influenced the activity
of SOD and CAT but did not show any significant effect on GPx. Thus, supplementing the
Antioxidants 2022,11, 1347 18 of 23
diet with red wine in the RW + ACR group resulted in a significant increase (p< 0.05) in
the enzymatic activity of SOD and CAT, compared to the PC group, while supplementing
the diet with white wine did not produce significant differences in the activities of these
enzymes. The positive effects of the red wine were more pronounced than those determined
by the white wine, due to red wine having higher polyphenolic content compared to
white wine. Rocha et al. [
67
] also obtained a significant increase in SOD activity when
supplementing the diet of rats with resveratrol, while Uzma et al. [
57
] reported a significant
increase in CAT activity in rats with oxidative stress induced by carbon tetrachloride due
to dietary supplementation with red wine.
Regarding GPx activity, supplementing the diet with white or red wine did not
produce significant differences between the groups treated with ACR, suggesting that
in this case neither of the 2 types of wine was able to exert a protective effect against
ACR. Similar results were reported by Macedo et al. [
63
], who did not obtain significant
differences in GPx activity when supplementing the diet of rats with red wine with high
antioxidant activity.
In general, when cells are exposed to eustress, they increase the activity and expression
of antioxidant enzymes as a compensation mechanism to better protect them from free
radical-induced damage by the activation of the nuclear factor erythroid 2-related factor
2/electrophile-responsive elements (Nrf2/EpRE) signaling pathway, which is a very impor-
tant cytoprotective mechanism, ROS acting in low concentrations as cell mediators [
68
,
69
].
The reduction in the activities of antioxidant enzymes in groups of animals whose oxidative
stress has been induced with ACR, might be due to the rapid consumption and deple-
tion of these enzymes in combating free radicals generated during the development of
oxidative stress. Given that ethanol can participate in free radical reactions, producing
alkoxyl and hydroxyl radicals which increase cellular oxidative stress by producing O
2−
and H
2
O
2
[
70
,
71
], the association of alcohol with ACR could have generated a higher
amount of O
2−
in hepatocytes. In this case, the cells responded by modifying the activity of
antioxidant enzymes, depending on the contribution of each phenolic compound and also
on the synergism of the constituent compounds of the wines, to neutralize the excess ROS.
Due to the fact that the present study evaluated the effects of wine polyphenols, and
taking into account the fact that alcohol is an enzyme inducer, all groups received the same
concentration of alcohol, in the form of wine, that being 12.5% hydroalcoholic solution.
Therefore, the differences in activity obtained between groups, namely the increase in the
activity of antioxidant enzymes in animals from wine-treated groups, were attributed to
the polyphenolic components of wines, rather than their ethanol content.
The main mechanisms of acrylamide hepatotoxicity are oxidative damage and mi-
tochondrial dysfunction [
72
,
73
]. Regarding the first mechanism, oxidative stress, lipid
peroxides and impaired antioxidant defense in the liver are linked to the activation of
cytochrome P450 (CYP) 2E1, which functions as a central pathway in the formation of high
levels of ROS, being also the only enzyme involved in biotransformation of ACR in glyci-
damide [
53
]. Therefore, CYP2E1-catalyzed ACR metabolism causes an imbalance between
ROS production and elimination, in the sense of excessive ROS production, then results in
lipid peroxidation and increases oxidative stress, which is related to liver damage [
53
,
73
,
74
].
Since ACR is not only a substrate, but also an inducer of CYP2E1, with CYP2E1 overexpres-
sion being induced by ACR intoxication and associated with increased oxidant production,
the prevention and treatment of ACR-induced toxicity can be supported by antioxidants
that have the ability to inhibit or to downregulate CYP2E1 [
53
]. Regarding the second
mechanism of ACR hepatotoxicity, studies have confirmed the apoptotic property of ACR
in a dose- and time-dependent manner in the liver, with long-term exposure to ACR causing
mitochondrial collapse and leading to apoptosis [
53
,
73
,
74
]. Therefore, ACR treatment alters
the potential of the mitochondrial membrane of hepatocytes and may alter the expression
of nuclear factor-erythroid 2-related factor 2 (Nrf2) [
74
,
75
]. In addition, studies show that
mitochondrial dysfunction causes the production of large amounts of ROS [75].
Antioxidants 2022,11, 1347 19 of 23
Polyphenols are known to be natural antioxidants with important antioxidant proper-
ties, which can significantly inhibit oxidative stress and inhibit the onset of mitochondrial
dysfunction [
74
]. It is known that the mechanism of action of polyphenols is related to
their ability to eliminate ROS, chelate metals, and influence the activity of enzymes, cell
signal transduction pathways and gene expression [
4
,
69
]. When acting as direct antioxi-
dants, polyphenols scavenge and neutralize free radicals involved in the etiology of various
diseases, and also scavenge the 2,2-diphenyl-1-picrylhydrazyl radical used in the
in vitro
DPPH assay; thus preventing the oxidative damage caused by ROS and blocking the
cascade of reactions in lipid peroxidation [
69
]. On the other hand, by modulating the
nuclear factor erythroid 2-related factor 2/electrophile-responsive elements (Nrf2/EpRE)
signaling pathway, polyphenols increase the activity of some antioxidant and detoxifying
enzymatic systems and down-regulate the Nuclear Factor kappa B (NF-
к
B) system [
69
].
As polyphenols have a very complex antioxidant activity, it is important that the results
support its exercise through several mechanisms of action. Therefore, through the markers
used both
in vitro
(DPPH) and
in vivo
(SOD, CAT, GPx), wine polyphenols were able to
demonstrate their protective capacity exerted by several mechanisms, mentioned above.
Thus, flavonoids, the main polyphenols contained in wine, are known to prevent lipid
peroxidation and low-density lipoprotein oxidative changes due to their antioxidant prop-
erties [
4
,
6
]. They have antioxidant action through electron transfer and formation of elec-
trophilic metabolites, while ortho- or para-dihydroxyphenols can be oxidized to quinones
which act by increasing Nrf2 [
69
]. Considering the flavonoid-rich content (flavan-3-ols,
such as catechin, epicatechin gallate, gallocatechin, but also flavonols, such as derivatives
of myricetin, quercetin, isorhamnetin, laricitrin, syringetin and kaempferol) of the wines
tested in this study [
39
], the results obtained support a positive association between the
intake of wine flavonoids and the reduction of oxidative stress and its consequences.
Along with flavonoids, the anthocyanins (derivatives of cyanidin, petunidin, delfini-
din, peonidin and malvidin) contained in the red wine tested in the study [
39
] contribute to
the reduction of lipid peroxidation, resulting from oxidative stress, as reported by Kolota
et al. [
4
]. The red wine fraction containing anthocyanins has been shown to be the most
effective in its ability to trap ROS and inhibit LDL oxidation, compared to two other frac-
tions containing phenolic acids, flavonols, procyanidins and catechins. The properties of
anthocyanins are due to their peculiar chemical structure, being very reactive towards ROS
because of their electron deficiency [
76
]. In addition, anthocyanins have shown cellular
antioxidant mechanisms comparable to, or greater than, other micronutrients, such as
vitamin E [
76
]. Regarding the ability of flavonoids and anthocyanins to prevent lipid perox-
idation, a study in a human population supports a positive association between regular
consumption of red wine and a reduction in serum MDA levels, demonstrating that lipid
peroxidation was reduced following a regular consumption of 100 mL of red wine/day,
through a mechanism involving the intestinal microbiota [6].
The antioxidant activity demonstrated
in vivo
by the tested wines was also due to
their stilbenes content, particularly of resveratrol, the main representative, which have
capacity to inhibit the so-called “oxidative burst” (production of O
2−
and H
2
O
2
), to regulate
CAT, SOD, GPx, glutathione reductase, GST activities, as well as to induce endogenous
antioxidant defenses, such as the Nrf2 pathway [69,77].
In summary, the results of recent research given in the literature show that ACR
toxicity is due to oxidative stress induced by ROS generation and activation of the tran-
scription factor NF-
κ
B, involved in the generation of the inflammatory response by the
release of cytokines, including IL-6, TNF-
α
, and IL-1
β
[
21
,
78
]. Furthermore, a number of
studies have confirmed this hypothesis by the fact that various antioxidant compounds,
such as sulforaphane [
79
], blueberry anthocyanins [
80
], or N-acetylcysteine [
81
], have the
ability to reverse the toxicity of ACR, by activating the transcription factor Nrf2 and its
Nrf2/ARE signaling pathway, and, consequently, inhibit NF-
κ
B. It is known that several
wine polyphenols, such as resveratrol, catechins, quercetin, ellagic acid and ellagitannins,
gallic acid and gallotannins, etc., have significant antioxidant activity, exerted by several
Antioxidants 2022,11, 1347 20 of 23
mechanisms of action, including activation of Nrf2 and inhibition of NF-
κ
B [
69
]. Indeed,
previous phytochemical analysis of the wines we tested in this study showed that they
have a high content of such polyphenols, and the results obtained
in vivo
show that these
wine samples administered to rats decrease ACR toxicity by reducing oxidative stress.
5. Conclusions
In conclusion, the administration of ACR (250
µ
g/kg body weight over a period of
28 days) induces hepatotoxicity in rats, by altering MDA, TBARS and GSH levels, antioxi-
dant enzyme activities, liver enzyme activities and by producing hepatic histopathological
changes. Our results showed that wine polyphenols increased GSH content, normalized
the activities of the antioxidant enzymes CAT and SOD, inhibited lipid peroxidation in the
liver, improved the AST level and attenuated morphological changes, especially in the case
of red wine. Therefore, the antioxidant properties of wine polyphenols may be considered
to be primary mechanisms in protection against ACR-induced oxidative stress and toxicity.
Supplementary Materials:
The following supporting information can be downloaded at: https:
//www.mdpi.com/article/10.3390/antiox11071347/s1, Figure S1: Chromatogram of a rat plasma
sample from RW group for determination of MDA; Figure S2: Chromatogram of a rat plasma sample
from PC group for determination of total GSH; Figure S3: Chromatogram of a rat plasma sample
from RW + ACR group for determination of reduced GSH; Table S1: The average body weight of rats
from the 6 experimental groups at the beginning and at the end of the 28 experimental days.
Author Contributions:
Conceptualization, R.B., D.-S.P., A.C.-P., L.F. and F.L.; methodology, R.B.,
D.-S.P., A.C.-P., L.F., B.K., A.F., A.N., D.M. and F.L.; formal analysis, R.B., D.-S.P., A.C.-P., L.F., B.K.,
A.F., A.N., D.M. and F.L.; writing—original draft preparation, R.B., D.-S.P., A.C.-P., L.F., B.K., D.M.
and F.L.; writing—review and editing, R.B., D.-S.P., A.C.-P., L.F. and F.L.; supervision, F.L. All authors
have read and agreed to the published version of the manuscript.
Funding: This research received no external funding.
Institutional Review Board Statement:
The animal study protocol was approved by the Ethics
Committee of “Iuliu Ha
t
,
ieganu” University of Medicine and Pharmacy Cluj-Napoca, Romania
(Protocol number 219/2014, 11 June 2014).
Informed Consent Statement: Not applicable.
Data Availability Statement: The data presented in this study are available in this manuscript.
Acknowledgments:
This work was granted by project PDI-PFE-CDI 2021, entitled Increasing the
Performance of Scientific Research, Supporting Excellence in Medical Research and Innovation,
PROGRES, no. 40PFE/30.12.2021.
Conflicts of Interest: The authors declare no conflict of interest.
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