Comparative Histopathological and Biochemical Study Of The Effect Of Alcoholic Beverages On The Liver Of Adult Wistar Rats
Idorenyin U. Umoh*, Samuel J. Umanah, Patience Udoh
Department of Human Anatomy, Faculty of Basic Medical Sciences, University of Uyo, Nigeria.
*Corresponding Author
Idorenyin U. Umoh,
Department of Human Anatomy, Faculty of Basic Medical Sciences, University of Uyo, Nigeria.
Tel: 080336743886
Email: Idorenyinumoh@uniuyo.edu.ng
Received: April 10, 2024; Accepted: May 08, 2024; Published: May 23, 2024
Citation:Idorenyin U. Umoh, Samuel J. Umanah, Patience Udoh. Comparative Histopathological and Biochemical Study Of The Effect Of Alcoholic Beverages On The Liver Of Adult
Wistar Rats. Int J Aeronautics Aerospace Res. 2024;11(1):301-307.
Copyright: Idorenyin U. Umoh© 2024. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution and reproduction in any medium, provided the original author and source are credited.
Abstract
This study was designed to evaluate the histopathological and biochemical effect of different alcoholic beverages; brandy,
beer, soured wine and dry gin, on the liver of adult Wistar rats. Sixty-five (65) rats weighing between 180-230g were used
for this experiment. They were randomly divided into 13 groups of five (5) animals each. Group 1 was the normal control
group. Group 2-13 were used as the experiment group. Group 2,3 and 4 were treated with 1.23mg/kg, 2.45mg/kg and
3.68mg/kg bodyweight of brandy respectively. Group 5, 6 and 7 were treated with 17.32mg/kg, 34.64mg/kg and 51.96mg/
kg body weight of beer respectively. Group 8,9 and 10 were treated with 12.25mg/kg, 24.96mg/kg and 36.74mg/kg bodyweight
of soured wine respectively. Group 11, 12 and 13 were treated with 1.73mg/kg, 3.46mg/kg and 5.20mg/kg bodyweight
of dry gin respectively. Administration was done daily for 28 days and orally using orogastric tube. On the 29th day,
the animals were sacrificed using chloroform inhalation anaesthesia. The blood samples were aspirated via cardiac puncture
and liver tissues were harvested, fixed in 10% buffered formalin, processed, and stained with haematoxylin and eosin. Body
weight showed significant (p<0.05) increase in brandy administered groups compared to control, and no significant difference
in liver weight. Histology showed haemorrhagic central and portal vein; atrophied, dilated and vacuolated sinusoids,
dilated bile ducts and general distortion incyto-architecture of administered groups compared to control. AST, total and
conjugated bilirubin showed significant (p<0.01, 0.001) decrease in high dose of brandy, beer and soured wine compared
to control respectively. ALT showed significant (p<0.01) decrease in high dose of beer compared to group C and Total
protein and Albumin showed no significant difference. In conclusion, brandy and dry gin caused more detrimental effect to
the liver than the other alcoholic beverages.
2.Dynamic Lorentz Forces
3.Signal Modulation and Field Strengths
4.Thermal Velocities and Plasma Density
5.Measurement Examples
6. Increased Plasma Densities
7. Partly and Fully Ionized Ions
8. Fusion Temperatures
9. Conclusion
10. References
Keywords
Alcoholic Beverages; Liver; Histology; Liver Function.
Introduction
Alcohol (ethanol or ethyl alcohol) is the ingredient found in beer,
wine and spirits which causes drunkenness [22]. At low doses,
alcohol can act as a stimulant, inducing feelings of euphoria and
talkativeness, but drinking too much alcohol at one session can
lead to drowsiness, respiratory depression, coma or even death
[12]. As well as its acute and potentially lethal sedative effect at
high doses, alcohol has effects on every organ in the body, and
these effects depend on the blood alcohol concentration (BAC)
over time [30]. Most of the metabolism, or breaking down of
alcohol from a toxic substance to water and carbondioxide is performed
by the liver, with the rest excreted through the lungs (allowing
alcohol breath tests), through the kidneys (into urine) and
in sweat [30]. Chronic heavy alcohol use can damage the liver,
causing alcoholic liver disease. This occurs across a spectrum
from fatty liver, to acute alcoholic hepatitis, to cirrhosis [3].
Beer is a fermented alcoholic carbonated alcoholic beverages
produced from malted barley using yeast as biological catalyst
and hops (HumulusLupulus) as spice which gives bitterness to
the beer [4]. The specific mechanisms through which beer and its
minor components may affect the liver are not fully understood
and poorly elucidated. Experimental studies in humans showed
that there are only limited data from human studies investigating
the effect of beer drinking on liver enzymes [24]. Wine is made
by fermenting the juices of grapes or other fruits such as apples
(cider), cherries, berries, or plums [1]. Brandy is strong alcoholic
spirit made from fruit juice or distilled wine. Brandy is derived
from wine, yet it is aged in oak barrels, which increases the alcohol content and also gives it a unique colour [18]. Brandy can affect
normal functioning of the liver [5].
Gin is a colourless spirit obtained by distilling an aqueous mixture
of alcohol together with aromatic plant materials, generally juniper
berries (juniperuscommunis L.), to which water and alcohol
and at times fruit juices, extracts, and/or essential oils of fruits
may be added [14]. Gin has a lethal effect on the liver cells and
duration of this alcohol consumption is a major determinant of
the degree of alcoholic liver disease [15]. This study was carried
out in order to estimate or see the damaging effect of different
alcohol beverages on the histology and function of the liver.
Materials and Method
Materials
The materials that was used in this experiment include the following:
Experimental rats (60 male wistar rats), well ventilated
wooden cages, feed, feeder, wood shavings, tissue, 2ml and 5ml
syringes, cannula, beaker, sample bottles, cotton wool, dissecting
board, dissecting blade, light microscope, wooden block, rotary
microtome, weighing balance, forceps, hand gloves, masking tape,
markers, embedding mold, electric water bath, detergent, 10%
buffered formalin, chloroform, normal saline, alcohol (Absolute,
95%, 70%), xylene, DPX mountant, haematoxylin and eosin. Alcoholic
beverages included: brandy, soured wine, dry gin and beer.
Ethical Approval
The experimental procedure was approved by the AkwaIbom
State Ministry of Health Ethical Committee, Uyo, Nigeria. The
protocol was in accordance to National Guidelines for care and
use of laboratory animals (National Research Council, 2011).
Animal Care and Protocol
Sixty-five (65) wistar rats weighing 180g-230g were used for the
study. They were obtained from animal house, Faculty of Pharmacology
and were acclimatized for two weeks. They were housed
in wooden cages under standard housing conditions (Ventilated
room with 12/12 hour light/dark cycle at 24 ± 2°C). The rats will
be fed with standard rat chow and water given ad-libitum.
Drug Preparation and Administration
There were four (4) different alcoholic beverages used for this
research. The beverages were obtained at a wine store in Uyo City
of AkwaIbom State, Nigeria. The alcoholic beverages include:
Brandy (Red Label), Sour Wine (Lambrusco), Dry Gin (Seaman)
and Beer (Heineken). The alcoholic beverages were administered
orally through an orogastric tube.
Determination of the Median Lethal Dose (LD50)
In determining the LD50 of the different alcoholic beverages, the
Lorke’s method was used. Sixty (60) mice weighing between 15g-
25g were collected and grouped into four (4) groups according to
the number of alcoholic beverages used. Each group consisted
of 3 mice which were well labelled. All animals were observed
for restlessness, increased heartbeat, excitation of tissues and
death within 24 hours. The LD50 was calculated as the geometric
means of the maximum dosage producing 0% mortality (A) and
the minimum dosage producing 100% mortality or the dosage in
which half of the animals show signs of toxicity and die.
LD50=√AB(Lorke, 1983).
Experimental Design
Table 1.
Termination of Experiment
On 24 hours after stoppage of administration, the animals were
sacrificed by inhalation of chloroform intraperitoneally on day 29.
The liver tissues were harvested for histological studies and blood
aspirated for biochemical analysis.
Morphometric Analysis
The weight of the kidney was assessed with the aid of a weighing
balance.
Histopathology studies
The liver was excised and immediately transferred into 10% neutral
buffered formalin and processed for light microscopic study,
using an automatic tissue processor machine (Shandon 2000,
Leica, Frankfurt, Germany). Tissues were dehydrated in various
grades of alcohol then cleared in two changes of xylene, infiltrated
in two changes of wax bath and finally embedded in paraffin
wax. Five microns thick paraffin sections were obtained, which
were finally stained using the Hematoxylin and Eosin staining
procedure and the sections mounted with DPX and examined
microscopically by means of ×10 objective lenses [2].
Biochemical Analysis
Venous blood samples were collected at days 0, 10 and 30 and
used for biochemical analysis. The parameters measured included
aspartate aminotransferase (AST), alanine aminotransferase
(ALT), alkaline phosphatase (ALP), total protein, albumin and
bilirubin (total and conjugated). AST, ALT, ALP, total protein
and albumin were analyzed using commercial diagnostic kits from
Randox laboratory, United Kingdom. The kits employed the procedure
of [21] for the analysis of AST, ALT and ALP, while total
protein was estimated using the procedure of [28]. Albumin and
bilirubin (total and conjugated)was estimated using the method
of Grant (1987).
Statistical Analysis
The results were analysed using (SPSS) version 22. The data were
expressed using descriptive statistics and Analysis of Variance
(ANOVA). Multiple comparisons for the groups were done using
Post Hoc Turkey to test for the level of significance between
means. A p < 0.05 was considered to be statistically significant.
Values are expressed in Means ±Standard Deviation (M±SD). Superscript
c: indicates significant difference (p < 0.05).
Figure 4.1-4.13: Photomicrographs of the Liver showing PV= Haemorrhagic and dilated Portal Vein, CA= Haemorrhagic
Central Vein, HA= Hepatic Artery, Bd= Dilated Bile duct, S= Sinusoids, H= Hepatocytes. Stained with H&E at x100 magnification.
Legend: NC- Normal Control, BR-L= Brandy Low, BR-M= Brandy Middle, BR-H= Brandy High, BE-L= Beer Low, BEM=
Beer Middle, BE-H= Beer High, SW-L= Soured Wine Low, SW-M= Soured Wine Middle, SW-H= Soured Wine High,
DG-L= Dry Gin Low, DG-M= Dry Gin Middle, DG-H= Dry Gin High.
Results
Body Weight
The body weight of the rats was taken before the administration
commenced, every seven (70 days and at the last day after administration.
Student paired T-test was used as a statistical tool for
the analysis of the body weight before and after the administration.
There was a general marked difference in bodyweight of all
the groups but groups administered 1.23mg/kg, 2.46mg/kg and
3.69mg/kg bodyweight of brandy showed significant increase in
the final body weight compared to the initial weight at p<0.05
respectively. Only group 13 administered with 5.20mg/kg bodyweight
of dry gin showed a slight insignificants decrease in final
bodyweight when compared to initial bodyweight (Table 4.1).
Organ Weight
Results of the liver weight showed no marked difference in all
treated groups compared to control (Table 4.2).
Biochemical Analysis
Result of AST showed significant decrease in group administered
3.69mg/kg bodyweight of brandy compared to control at p <
0.001. Groups administered 51.96mg/kg bodyweight of beer and
36.74mg/kg bodyweight of soured wine were also significantly
lower compared to control at p < 0.01 respectively. ALT showed
significant decrease in groups administered with 51.96mg/kg
bodyweight of beer and 5.20mg/kg bodyweight of dry gin compared
to group administered with 3.69mg/kg bodyweight of
brandy at p < 0.01 respectively (Table 4.3). ALP showed a general
insignificant decrease in all treated groups compared to control
except group administered with 1.23mg/kg bodyweight of
brandy. There was a significant decrease in groups administered
3.69mg/kg bodyweight of brandy, 34.64 and 51.96mg/kg bodyweight
of beer, all treated groups of soured wine and 5.20mg/kg
bodyweight of dry gin compared to group administered 1.23mg/
kg bodyweight of brandy at p < 0.05, p < 0.01 and p < 0.001
respectively (Table 4.3).
Total protein and Albumin showed no marked difference in the
treated groups compared to control. Total bilirubin showed significant
decrease in groups administered 3.69mg/kg bodyweight
of brandy, 51.96mg/kg bodyweight of beer and 36.74mg/kg
bodyweight of soured wine compared to control at p < 0.01 and
p < 0.001 respectively. Conjugated bilirubin also showed significant decrease in groups administered 3.69mg/kg bodyweight of
brandy, 51.96mg/kg bodyweight of beer and 36.74mg/kg bodyweight
of soured wine compared to control at p < 0.01 and p <
0.001 respectively (Table 4.4).
Table 1.
Table 4.1: Showing body weight difference.
Table 4.2: Showing results of the liver weight.
Table 4.3: Showing results of Liver function tests (AST, ALT and ALP).
Table 4.4: Showing results of Liver function tests (T.P, Albumin, T.B, C.B).
Discussion
The liver is a critical organ in the human body and a hub for
numerous physiological processes [29] which include macronutrient
metabolism, blood volume regulation, immune system
support, endocrine control for growth signalling pathways, lipid
and cholesterol homeostasis, and the breakdown of xenobiotic
compounds, including many current drugs [29]. Thus, the liver
is a prime target for damage because it processes everything that
enters the mouth and is swallowed [27]. Liver disease is the major
cause of death every year; Approximately 29 million people suffer
from chronic liver condition [7]. The most common causes of
liver disease worldwide are alcohol, non-alcoholic steatohepatitis
associated with obesity and metabolic syndrome, chronic hepatitis
A [7-9].
Our findings showed that there was a general marked difference
in body weight of all the administered groups but however rats
administered with 1.23 mg/kg, 2.46 mg/kg and 2.69 mg/kg body
weight of brandy showed significant increase in body weightrespectively.
Only rats administered with 5.20 mg/kg body weight
of dry gin showed a slight insignificant decrease in final body
weight when compared to initial body weight. Alcohol is not only
an addictive substance; it is a high- caloric beverage that interferes
with metabolic function [8] andwhen consumed to intoxicating
levels, it can affect an individual’s weight status [10]. Results also showed no marked difference in liver weight ofthe rats inall the
administered groups compared to control.
The most common health-related consequences include alcoholic
liver disease (ALD), which is a condition associated with various
morphological changes in the liver, ranging from steatosis to advanced
steatosis accompanied by inflammation, fibrosis, and /or
cirrhosis [20, 13]. Histological findings showed normal histological
features in the liver of normal control and rats administered
with 1.23mg/kg body weight of Brandy; central vein with haemorrhage,
blood deposits and atrophied sinusoids in animals administered
with 2.46mg/kg body weight of Brandy;haemorrhagic
central vein, blood deposits, sinusoidsvacoulated, and general distortions
incytoarchitecture in 3.69 mg/kg body weight of Brandy
administered rats.
Rats administered with 17.32 mg/kg body weight of Beer showed
portal vein with haemorrhage; 34.64 mg/kg body weight of Beer
showed central vein with haemorrhage, blood deposits and vacuolatedsinusoids;
51.96 mg/kg body weight of Beer showed central
Vein with blood deposits, dilated sinusoids, and general distortions.
Animals administered with 12.25 mg/kg body weight of
Soured Wine showed normal central and portal vein, and dilated
sinusoids; 24.49 mg/kg body weight of Soured Wine showed
normal portal Vein, hepatic artery, bile duct, vacoulated portal
triad area and normal hepatocytes; 36.74 mg/kg body weight of
Soured Wine showed haemorrhagic portal vein, dilated bile duct
and blood droplets.Animals administered with 1.73 mg/kg body
weight of Dry Gin showed haemorrhagic portal vein, but normal
hepatic artery, bile duct and hepatocytes; 3.46 mg/kg body weight
of Dry Gin showed haemorrhagic portal vein, and dilated Sinusoids;
5.20 mg/kg body weight of Dry Gin showed haemorrhagic
and dilated portal vein, and dilated Sinusoids.
In the present study, the result of aspartate aminotransferase
(AST) showed significant decrease in groups administered
3.69mg/kg bodyweight of brandy, 51.96mg/kg bodyweight of
beer and 36.74mg/kg bodyweight of soured wine compared to
control respectively. Alanine aminotransferase (ALT) showed significant
decrease in groups administered with 51.96mg/kg bodyweight
of beer and 5.20mg/kg bodyweight of dry gin compared
to group administered with 3.69mg/kg bodyweight of brandy
respectively. Experimental studies in humans showed that there
are only limited data from human studies investigating the effect
of beer drinking on liver enzymes [24]. Two independent crossover
trials indicated that up to 4 beers/day do not affect liver
enzymes significantly. No evidence of an increase in gamma-glutamyltransferase
(GGT), aspartate animotranasferase (AST) and
alanine aminotransferase (ALT) after 3 weeks of daily intake of
4 beers was found in 11 men [24]. However, in a small cross-over
trial of 10 men consuming 4 beers/day and of a post-menopausal
women consuming 3 beers/day for a period of three weeks,
found a slight increase of GGT and ALT but only in women [23].
Alkaline Phosphatase (ALP) showed a general insignificant decrease
in all treated groups compared to control except group administered
1.23mg/kg bodyweight of brandy. There was a significant
decrease in groups administered 3.69mg/kg bodyweight of
brandy, 34.64mg/kg bodyweight of beer and 51.96mg/kg bodyweight
of beer, all treated groups of soured wine and 5.20mg/
kg of dry gin compared to administered 1.23mg/kg bodyweight
of brandy 1 respectively. Total protein and Albumin showed no
marked difference in the treated groups compared to control.
Total bilirubin and conjugated bilirubin showed significant decrease
in groups administered 3.69mg/kg bodyweight of brandy,
51.96mg/kg bodyweight of beer and 36.74mg/kg bodyweight of
soured wine compared to control respectively. One of the polyphenols
of wine is resveratrol [6], and study have showed that
oral administration of 20mg of resveratrol daily for six weeks can
significantly prevent the loss of liver weight and inhibit serum alanine
aminotransferase, aspartate aminotransferase, alkaline phosphatase,
and bilirubin levels [16].
Conclusion
From this research, it has been observed that alcohol has the ability
to cause histomorphological and biochemical alterations. The
biochemical parameters were altered.
Recommendation
Alcoholic beverages is proven to be of high toxicity and should
not be consumed for prolonged periods of time. It is pertinent
for more research to be conducted to ascertain its toxic effect using
special staining techniques and immunohistological methods.
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