Biomedicine & Pharmacotherapy 103 (2018) 75–86

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Biomedicine & Pharmacotherapy

journal homepage: www.elsevier.com/locate/biopha

Betaine treatment protects liver through regulating mitochondrial function T

and counteracting oxidative stress in acute and chronic animal models of
hepatic injury
⁎
Reza Heidaria, , Hossein Niknahada,b, Ala Sadeghib, Hamidreza Mohammadib,
Vahid Ghanbarinejadb, Mohammad Mehdi Ommatia, Arghavan Hosseinib, Negar Azarpirac,
Forouzan Khodaeib, Omid Farshada,b, Elaheh Rashidib, Asma Siavashpourb, Asma Najibib,
⁎
Asrin Ahmadib, Akram Jamshidzadeha,b,
a
Pharmaceutical Sciences Research Center, Shiraz University of Medical Sciences, Shiraz, Iran
b
Department of Pharmacology and Toxicology, School of Pharmacy, Shiraz University of Medical Sciences, Shiraz, Iran
c
Transplant Research Center, Shiraz University of Medical Sciences, Shiraz, Iran

A R T I C LE I N FO A B S T R A C T

Keywords: Betaine is a derivative of the amino acid glycine widely investigated for its hepatoprotective properties against
Amino acid alcoholism. The protective properties of betaine in different other experimental models also have been docu-
ATP mented. On the other hand, the exact cellular mechanism of cytoprotection provided by betaine is obscure. The
Bile acid current study was designed to evaluate the hepatoprotective effects of betaine and its potential mechanisms of
Bioenergetics
hepatoprotection in two animal models of acute and chronic liver injury. Bile duct ligation (BDL) was used as a
Cholestasis
Liver injury
model of chronic liver injury and thioacetamide (TAA)-induced hepatotoxicity was applied as the acute liver
Oxidative stress injury model. Severe increase in serum markers of liver tissue damage along with significant liver tissue his-
topathological changes were evident in both acute and chronic models of hepatic injury. It was also found that
tissue markers of oxidative stress were significantly increased in BDL and TAA-treated animals. Moreover, liver
mitochondrial indices of functionality were deteriorated in both investigated models. Betaine supplementation
(10 and 50 mg/kg, i.p) ameliorated hepatic injury as judged by decreased liver tissue histopathological altera-
tions, a significant decrease in tissue markers of oxidative stress, and mitigation of serum biomarkers of hepa-
totoxicity. On the other hand, betaine (10 and 50 mg/kg, i.p) protected hepatocytes mitochondria in both
chronic and acute models of hepatotoxicity. These data indicate that the antioxidative and mitochondria reg-
ulating properties of betaine could play a primary role in its mechanisms of hepatoprotection.

1. Introduction mitochondrial dysfunction has also been reported in several human

Betaine (Trimethylglycine) enters the human body through different
dietary sources [1,2]. Some physiological roles including osmor-
egulatory properties have been attributed to betaine [1,2]. This che-
mical also participates in many critical biochemical pathways as a
“methyl-donor” [1,2]. The hepatoprotective properties of betaine have
repeatedly been mentioned in different experimental models [3–6].
Betaine is also a well-known agent which protect the liver against al-
coholism [5,7,8].
Previous investigations have provided compelling evidence that
oxidative stress and its associated events play a pivotal role in hepa-
totoxicity with different etiologies [9–13]. On the other hand,
pathological conditions and xenobiotics-induced cytotoxicity [14].
Mitochondrial injury and inability to maintain sufficient cellular ATP
level could lead to cell death [15]. Oxidative stress and mitochondrial
dysfunction are involved in the pathogenesis of liver injury in different
experimental models [10,16–19]. It has been revealed that TAA model
of the acute liver injury is associated with severe oxidative stress and
mitochondrial dysfunction [20,21]. On the other hand, accumulation of
the cytotoxic bile acid during bile duct obstruction is associated with
mitochondrial injury and oxidative stress in the liver [22–25].
It has been found that betaine supplementation might blunt oxida-
tive stress and its consequences in different experimental models
[3,4,6,8,26]. Betaine also has been reported to prevent mitochondria-

⁎
Corresponding authors at: Pharmaceutical Sciences Research Center, Shiraz University of Medical Sciences, Shiraz, Iran.
E-mail addresses: rheidari@sums.ac.ir (R. Heidari), ajamshid@sums.ac.ir (A. Jamshidzadeh).

https://doi.org/10.1016/j.biopha.2018.04.010
Received 24 February 2018; Received in revised form 29 March 2018; Accepted 2 April 2018
0753-3322/ © 2018 Elsevier Masson SAS. All rights reserved.
R. Heidari et al.
dependent cell death and apoptosis [7,27,28]. Hence, this chemical
might provide promising protective properties in animal models of
hepatic injury.
Although the beneficial properties of betaine in different experi-
mental models have been well-studied, the precise mechanism(s) of
cytoprotection behind remained obscure [3,4,8]. The current study was
designed to evaluate the effect of betaine supplementation on liver
mitochondrial function and oxidative stress in two experimental models
of acute (Thioacetamide; TAA treatment) and chronic (Bile duct liga-
tion; BDL) liver injury. The data could help to clear the mechanism of
hepatoprotection provided by betaine as well as developing new ther-
apeutic strategies against liver injury with different etiologies.
2. Materials and methods
2.1. Chemicals
4,2-Hydroxyethyl,1-piperazineethanesulfonic acid (HEPES), 3-(N-
morpholino)propanesulfonic acid (MOPS), Dimethyl sulfoxide (DMSO),
D-mannitol, Bovine serum albumin (BSA), Thiobarbituric acid (TBA), 3-
[4,5dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT), di-
thiobis-2nitrobenzoic acid (DTNB), Glutathione (GSH), 2′,7′-
Dichlorofluorescein diacetate (DCFH-DA), Betaine (Tri-methyl glycine),
Malondialdehyde (MDA), Sucrose, n-Propanol, n-Butanol, Sodium ci-
trate, Potassium chloride, di-Sodium hydrogen phosphate (Na2HPO4),
Sodium succinate, Glacial acetic acid, Magnesium chloride,
Dithiothreitol, Rhodamine123 (Rh 123), Coomassie brilliant blue,
Ethyleneglycol-bis (2-aminoethylether)-N,N,N′,N′-tetraacetic acid
(EGTA), and Ethylenediaminetetraacetic acid (EDTA) were purchased
from Sigma Chemical Co. (St. Louis, MO, USA). Sodium bicarbonate,
Trichloroacetic acid (TCA), Sodium acetate, and Hydroxymethyl amino
methane-hydrochloride (Tris-HCl) were purchased from Merck
(Darmstadt, Germany). All salts for preparing buffer solutions were of
analytical grade and prepared from Merck (Darmstadt, Germany).
2.2. Animals
Male Sprague Dawley rats (200–250 g) were obtained from Animal
Breeding Center of Shiraz University of Medical Sciences, Shiraz Iran.
Rats were housed in cages on wood-chip bedding at a temperature of
23 ± 2 °C and relative humidity ≈40%. Animals had free access to
food (Behparvar®, Tehran, Iran) and tap water. Animals were handled
according to the guidelines approved by a local ethics committee at
Shiraz University of Medical Sciences, Shiraz, Iran (95-01-36-12046).
2.3. Animal surgery and bile duct ligation (BDL) as the model of chronic
liver injury
Animals were anesthetized (A mixture of Ketamine 80 mg/kg and
Xylazine 10 mg/kg, i.p), a midline incision was made, and the common
bile duct was localized, doubly ligated, and cut between these ligatures
[29]. The sham operation comprised laparotomy and bile duct identi-
fication and manipulation without ligation. The treatments in chronic
liver injury model were as follow: 1) Sham-operated; 2) BDL; 3)
BDL + Betaine (10 mg/kg/day, i.p); 4) BDL + Betaine (50 mg/kg/day,
i.p).
2.4. Animal model of acute liver injury
Thioacetamide-induced hepatotoxicity is extensively used as an
animal model of acute liver injury [30]. In the current investigation,
thioacetamide-induced liver injury was achieved by i.p injection of
thioacetamide (200 mg/kg) to rats [31]. Betaine (10 and 50 mg/kg, i.p)
was administered for three consecutive days before thioacetamide
challenge. The treatments were as follow: 1) Control (Vehicle-treated);
2) Thioacetamide; 3) Betaine (10 mg/kg, i.p) + Thioacetamide; 4)
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Biomedicine & Pharmacotherapy 103 (2018) 75–86
Betaine (50 mg/kg, i.p) + Thioacetamide.
Animals were anesthetized (Thiopental, 80 mg/kg, i.p) and their
blood and liver samples were collected. Supportive therapy by admin-
istering 5% dextrose-containing 0.45% sodium chloride and 0.2% po-
tassium chloride (2.5 ml/kg body weight, S.C), was given to avoid hy-
poglycemia and renal failure [32]. Control animals (Vehicle-treated)
received normal saline as the thioacetamide solvent.
2.5. Serum biochemistry
Animals were anesthetized (Thiopental, 50 mg/kg, i.p) and their
blood, liver, and kidney samples were collected. Blood was collected
from the abdominal aorta, transferred to standard tubes
(Improvacuter®; gel and clot activator-coated tubes; Guangzhou, China)
and centrifuged (3000 g, 10 min, 4 °C) to prepare serum. Mindray BS-
200® autoanalyzer (Mindray chemistry analyzers for low-volume la-
boratories, Guangzhou, China) was used to analyze serum biochem-
istry. Standard kits (Pars Azmun®, Tehran, Iran) were employed to as-
sess serum gamma-glutamyl transpeptidase (γ-GT), alkaline
phosphatase (ALP), Creatinine (Cr), glucose, phosphate, calcium, uric
acid and blood urea nitrogen (BUN) [33].
2.6. Reactive oxygen species formation
Reactive oxygen species (ROS) in the liver was estimated as pre-
viously described [20,34,35]. Briefly, liver tissue (500 mg) was homo-
genized in 5 ml of ice-cooled Tris-HCl buffer (40 mM, pH = 7.4, 4 °C).
Samples of the resulted tissue homogenate (100 μL) were mixed with
Tris-HCl buffer (pH = 7.4, 1 mL) and 2′, 7′dichlorofluorescein diacetate
(DCF-DA; Final concentration 10 μM). The mixture was incubated in the
dark (37 °C; 15 min). Finally, the fluorescence intensity of the samples
was assessed using a FLUOstar Omega® multi-functional microplate
reader with λ excitation = 485 nm and λ emission = 525 nm [34,36].
2.7. Lipid peroxidation
The thiobarbituric acid reactive substances (TBARS) were measured
as an index of lipid peroxidation in the liver tissue [20]. Briefly, the
reaction mixture was consisted of 500 μL of liver tissue homogenate
(10% w/v in KCl, 1.15% w/v), 1 mL of thiobarbituric acid (0.375%, w/
v), and 3 mL of phosphoric acid (1% w/v, pH = 2) [37]. Samples were
mixed well and heated (100 °C). After the incubation period (45 min),
the mixture was cooled, and then 2 mL of n-butanol was added. Samples
were vigorously mixed and centrifuged (10,000 g for 10 min) [38].
Finally, the absorbance of developed color in n-butanol phase was
measured at λ = 532 nm using an Ultrospec 2000®UV spectro-
photometer (Pharmacia Biotech, Uppsala, Sweden) [20].
2.8. Hepatic glutathione content
Liver samples (500 mg) were homogenized in 8 ml of ice-cooled
(4 °C) EDTA solution (0.04 M). Then, 5 mL of the prepared homogenate
were added to 4 mL of distilled water (4 °C) and 1 mL of trichloroacetic
acid (50% w/v; 4 °C). The mixture was vortexed and centrifuged
(10,000 g, 4 °C, 15 min) [37]. Then, 2 mL of the supernatant was mixed
with 4 mL of Tris-HCl buffer (40 mM, pH = 8.9), and 100 μl of DTNB
(10 mM in methanol) [38,39]. The absorbance of the developed color
was measured at λ = 412 nm using an Ultrospec 2000®UV spectro-
photometer (Pharmacia Biotech, Uppsala, Sweden) [20].
2.9. Ferric reducing antioxidant power (FRAP) of liver tissue
The FRAP assay measures the formation of a blue colored Fe2+-
tripyridyl-triazine compound from the colorless oxidized Fe3+ form by
the action of electron-donating antioxidants [40]. In the current study,
the working FRAP reagent was prepared by mixing 10 volumes of
R. Heidari et al. Biomedicine & Pharmacotherapy 103 (2018) 75–86

Table 1
Serum level of liver injury biomarkers in bile duct ligated (BDL) and thioacetamide (TAA)-treated rats.
Treatment Serum ALT (U/l) Serum AST (U/l) Serum LDH (U/l) Serum ALP (U/l) Serum Bilirubin (mg/dl) Serum γGT (IU/l) Serum Albumin (g/dl)

Control 33 ± 3 92 ± 4 433 ± 25 1055 ± 115 0.08 ± 0.006 39 ± 7 2.98 ± 0.21

BDL 342 ± 28* 220 ± 12* 623 ± 71* 3814 ± 115* 11.8 ± 0.60* 265 ± 16* 1.57 ± 0.41*
BDL + Betaine 10 mg/kg 267 ± 14 a 154 ± 10 a 687 ± 26 2548 ± 173 9.2 ± 1.40 272 ± 25 2.42 ± 0.12a
BDL + Betaine 50 mg/kg 190 ± 11 a 119 ± 11 a 447 ± 16 a 3335 ± 308 9.5 ± 1.14 190 ± 22 2.3 ± 0.05
TAA 1103 ± 144* 1532 ± 133* 1043 ± 115* 2553 ± 514* 2.3 ± 0.2* 44 ± 7 1.9 ± 0.07*
TAA + Betaine 10 mg/kg 723 ± 58 b 320 ± 40 b 660 ± 69 b 2409 ± 184 1.0 ± 0.07 b 30 ± 4 2.4 ± 0.1b
TAA + Betaine 50 mg/kg 560 ± 55 b 377 ± 62 b 402 ± 70 b 2643 ± 240 0.67 ± 0.15 b 59 ± 8 2.6 ± 0.08 b

Data are given as Mean ± SEM (n = 6).

* Indicates significantly different as compared with control group (P < 0.001).
a
Indicates significantly different as compared with BDL group (P < 0.01).
b
Indicates significantly different as compared with TAA group (P < 0.01).

Fig. 1. Markers of oxidative stress in the liver tissue of cirrhotic rats. BDL: Bile duct ligation.
Data are given as Mean ± SEM (n = 6).
***Indicates significantly different as compared with sham-operated group (P < 0.001).
a
Indicates significantly different as compared with BDL group (P < 0.01).
ns: not significant.

acetate buffer (300 mmol/L, pH = 3.6), with 1 vol of TPTZ (10 mmol/L
in 40 mmol/L hydrochloric acid) and 1 vol of ferric chloride (20 mmol/
L). All solutions were freshly-prepared and stored in the dark (4 °C).
Liver tissue (500 mg) was homogenized in an ice-cooled Tris-HCl buffer
(250 mM Tris-HCl, 200 mM sucrose and 5 mM DTT, pH = 7.4, 4 °C)
[39]. Then, 50 μL of tissue homogenate and 150 μL of deionized water
was added to 1.5 mL of the FRAP reagent [21]. The reaction mixture
was incubated at 37 °C for 5 min. Finally, the absorbance of developed
color was measured at λ = 595 nm using an Ultrospec 2000® UV
spectrophotometer (Pharmacia Biotech, Uppsala, Sweden) [35,41].
2.10. Liver hydroxyproline level
Liver hydroxyproline content was assessed as an index of tissue fi-
brosis. Briefly, 500 μl of the tissue homogenate (20% w/v in phosphate
buffered saline; PBS; pH = 7.4) was digested in 1 ml of hydrochloric
acid (6 N) at 120 °C for at least 12 h. An aliquot of the digested
homogenate (25 μl) was added to 25 μl of citrate-acetate buffer

77
R. Heidari et al.
Fig. 2. Effect of betaine treatment on the liver tissue markers of oxidative stress in an animal model of acute liver injury. TAA: Thioacetamide.
Data are given as Mean ± SEM (n = 6).
***Indicates significantly different as compared with control group (P < 0.001).
a
Indicates significantly different as compared with TAA group (P < 0.01).
ns: not significant.
(pH = 6). Afterward, 500 μl of chloramines-t-solution (56 mM) was
added, and the mixture was left at room temperature for 20 min. Then,
500 μl of freshly-prepared Ehrlich's reagent (15 g of p-dimethyl amino
benzaldehyde in n-propanol/perchloric acid; 2: 1 v: v) was added, and
the mixture was incubated at 65 °C for 15 min [42]. After cooling, the
intensity of developed color was measured at λ = 550 nm (Ultrospec
2000® UV spectrophotometer; Pharmacia Biotech, Uppsala, Sweden)
[20].
2.11. Liver mitochondria isolation
Rat liver mitochondria were isolated based on a previously de-
scribed method [43]. Briefly, animals were anesthetized (Thiopental
80 mg/kg, i.p) and their liver was excised and washed with ice-cold
saline (sodium chloride 0.9%) [43,44]. The liver was homogenized in
isolation buffer (220 mM sucrose, 70 mM mannitol, 0.5 mM ethylene
glycol-bis(2-aminoethyl ether)-N,N,N´,N´-tetraacetic acid (EGTA),
2 mM N-(2-hydroxyethyl) piperazine-N,N´-(2-ethane sulfonic acid)
(HEPES), 0.1% essentially fatty acid-free bovine serum albumin;
pH = 7.4) at a 10:1 buffer to tissue (v/w) ratio [43]. Afterward, tissue
homogenate was centrifuged at 1000 × g for 10 min at 4 °C to remove
intact cells and nuclei. The supernatants were further centrifuged at
15,000 × g (4 °C for 10 min) to precipitate the heavy membrane frac-
tions (mitochondria). This step was repeated at least three times using
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Biomedicine & Pharmacotherapy 103 (2018) 75–86
fresh buffer medium. As mentioned, all procedures were performed at
0–4 °C or on ice to minimize mitochondrial damage during the mi-
tochondria isolation process [43].
2.12. Mitochondrial dehydrogenases activity (MTT assay)
The 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide
(MTT) assay was applied as a colorimetric method for determination of
mitochondrial succinate dehydrogenase activity in isolated mice liver
mitochondria [39,45]. Mitochondrial suspension in a buffer containing
320 mM sucrose, 1 mM EDTA, and 10 mM Tris-HCl; pH = 7.4, was in-
cubated with MTT (0.4% w: v; 37 °C for 30 min in the dark). The pro-
duct of purple formazan crystals was dissolved in 1 mL dimethyl sulf-
oxide (DMSO). Then, 100 μl of dissolved formazan was added to 96 well
plate, and the optical density (OD) at λ = 570 nm was measured with
an EPOCH plate reader (BioTek Instruments, Highland Park, USA) [46].
2.13. Reactive oxygen species (ROS) in isolated liver mitochondria
The fluorescent probe DCFH-DA was used to assess mitochondrial
ROS [43,47]. Briefly, isolated liver mitochondria were placed in re-
spiration buffer (125 mM sucrose, 65 mM KCl, 10 mM HEPES, 5 mM
Sodium succinate, 20 μM Ca2+, pH = 7.2) [22,43]. Following this step,
DCFH-DA was added (Final concentration, 10 μM) to mitochondria and
R. Heidari et al.
Fig. 3. Mitochondrial indices of functionality in bile duct ligated (BDL) rats.
Data are given as Mean ± SEM (n = 6).
***Indicates significantly different as compared with sham-operated group (P < 0.001).
a
Indicates significantly different as compared with BDL group (P < 0.01).
ns: not significant.
then incubated for 30 min (37 °C in the dark). Then, the fluorescence
intensity of DCF was measured using a FLUOstar Omega® multi-
functional microplate reader (λ excitation = 485 nm and λ emission
= 525 nm) [43].
2.14. Mitochondrial depolarization
Mitochondrial uptake of the cationic fluorescent dye, rhodamine
123, has been used for the estimation of mitochondrial depolarization
[43,48]. For this purpose, the mitochondrial fractions (0.5 mg protein/
mL) were incubated with 10 μM of rhodamine 123 in a buffer con-
taining 125 mM sucrose, 65 mM KCl, 10 mM HEPES, dissolved in
double distilled water, pH = 7.2 (30 min, 37 °C, in the dark). Samples
were centrifuged (15,000 g, 10 min, 4 °C) and the fluorescence intensity
of supernatant was monitored using a FLUOstar Omega® multi-
functional microplate reader at the excitation and emission wavelength
of λ = 485 nm and λ = 525 nm, respectively [43,49].
2.15. Mitochondrial swelling assay
Mitochondrial swelling was measured based on the light scattering
method as previously described [43,50]. Briefly, the mitochondria
(0.5 mg protein/ml) were suspended in swelling buffer (125 mM su-
crose, 65 mM KCl, 10 mM Hepes-KOH, 20 μM Ca2+, pH = 7.2). Light
absorbance at λ = 540 nm was monitored (Constant temperature of
30 °C) [43,51]. It is accepted that a decreased light absorbance is con-
sistent with an increase in mitochondrial volume. Hence, as mi-
tochondria are more swelled, the differences between light absorbance
of two-time points are higher. The differences between the absorbance
of samples were assessed (ΔOD540 nm) and compared in different
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Biomedicine & Pharmacotherapy 103 (2018) 75–86
experimental groups and reported as maximal mitochondrial swelling
amplitude [43].
2.16. Mitochondrial ATP content
A luciferase-luciferin-based kit (Enliten® from Promega Corp.,
Madison, WI, USA) was used to assess mitochondrial ATP content
[21,52]. Samples and buffer solutions were prepared based on the kit
instructions. Briefly, 500 μL of mitochondrial samples (1 mg protein/
mL) were treated with 200 μL of ice-cooled TCA solution (0.3% w: v in
double distilled water) and centrifuged (15,000 g, 15 min). Afterward,
100 μL of the supernatant was treated with 100 μL of ATP kit content,
and the luminescence intensity of samples was measured λ = 560 nm
using a FLUOstar Omega® multi-functional microplate reader. For
standardization of data, samples protein concentrations were de-
termined using the Bradford method [53].
2.17. Statistical analysis
Data are given as the Mean ± SEM. Data comparison was per-
formed by the one-way analysis of variance (ANOVA) with Tukey’s
multiple comparison test as the post hoc. Differences were considered
statistically significant when P < 0.05.
3. Results
Severe tissue damage was evident in both acute and chronic models
of hepatotoxicity as judged by the significant increase in serum bio-
markers of liver injury (Table 1). It was found that betaine treatment
(10 and 50 mg/kg, i.p) mitigated serum markers of hepatotoxicity
R. Heidari et al.
Fig. 4. Mitochondrial dysfunction in the liver of thioacetamide (TAA)-treated rats.
Data are given as Mean ± SEM (n = 6).
***Indicates significantly different as compared with control group (P < 0.001).
a
Indicates significantly different as compared with TAA group (P < 0.01).
(Table 1). On the other hand, betaine supplementation (10 and 50 mg/
kg, i.p) did not significantly change some markers of liver and bile duct
injury (e.g., bilirubin, γ-GT, and ALP) (Table 1).
The markers of oxidative stress were significantly increased in cir-
rhotic BDL rats (Fig. 1). Severe ROS formation, along with an increase
in liver tissue lipid peroxidation was detected in BDL group (Fig. 1).
Moreover, liver glutathione reservoirs were depleted, and tissue anti-
oxidant capacity was significantly decreased in BDL rats (Fig. 1). It was
found that betaine supplementation (10 and 50 mg/kg, i.p) alleviated
liver tissue markers of oxidative stress in BDL animals (Fig. 1). It is
noteworthy to mention that the effect of betaine on oxidative stress
biomarkers was not dose-dependent in cirrhotic BDL animals in the
current study (Fig. 1).
Oxidative stress markers were significantly changed in TAA-treated
rats as the animal model of acute liver injury (Fig. 2). It was found that
betaine treatment (10 and 50 mg/kg, i.p) abated markers of oxidative
stress in TAA-treated animals (Fig. 2). The higher dose of betaine
(50 mg/kg, i.p) seems to have more significant effect against oxidative
stress in comparison with the lower dose in TAA model of acute liver
injury (Fig. 2).
It was found that bile duct obstruction leads to significant dete-
rioration in the liver mitochondrial function (Fig. 3). Mitochondrial
ATP content and dehydrogenases activity were significantly decreased
where mitochondrial ROS formation and swelling were increased in
BDL group (Fig. 3). Moreover, the significant collapse of mitochondrial
membrane potential was detected in the liver mitochondria isolated
from BDL animals (Fig. 3). It was found that betaine (10 and 50 mg/kg,
i.p) preserved mitochondrial indices of functionality in cirrhotic rats
(Fig. 3). The effect of betaine on liver mitochondrial function was not
dose-dependent in cirrhotic animals (Fig. 3).
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Biomedicine & Pharmacotherapy 103 (2018) 75–86
A significant decrease in mitochondrial function was also detected
in the liver of TAA-treated rats (Fig. 4). On the other hand, it was found
that betaine treatment (10 and 50 mg/kg, i.p) prevented TAA-induced
mitochondrial injury (Fig. 4). The mitochondrial protecting properties
of betaine seem to be dose-dependent at the mitochondrial level in the
animal model of acute liver injury (Fig. 4).
Liver tissue histopathological changes were evident as severe ne-
crosis, inflammation, ballooning degeneration, and fatty changes in
BDL animals (Fig. 5 and Table 2). On the other hand, significant tissue
fibrosis and collagen deposition were detected in BDL rats (Fig. 6 and
Table 2). It was found that betaine administration in both doses of 10
and 50 mg/kg, alleviated liver tissue histopathological alterations in
cirrhotic animals (Figs. 5 and 6, Table 2).
Significant liver tissue necrosis, inflammation, and fatty changes
were also detected in TAA-treated (Fig. 7 and Table 2). It was found
that betaine (10 and 0 mg/kg, i.p) significantly decreased histopatho-
logical alterations in TAA-induced hepatotoxicity (Fig. 7 and Table 2).
4. Discussion
The current investigation was designed to evaluate the potential
mechanisms of hepatoprotection provided by betaine in two experi-
mental models of acute and chronic liver injury. It was found that be-
taine supplementation (10 and 50 mg/kg) significantly decreased
markers of oxidative stress and efficiently preserved mitochondrial
function and energy metabolism in both acute and chronic hepato-
toxicity models. Hence, the mitochondrial protection and antioxidative
properties could serve as fundamental mechanisms for the hepatopro-
tective effects of betaine.
Previous investigations have provided compelling evidence that
R. Heidari et al. Biomedicine & Pharmacotherapy 103 (2018) 75–86

Fig. 5. Liver tissue histopathological changes

(H&E stain) in cirrhotic animals. BDL: Bile duct
ligated.
Severe tissue histopathological changes in-
cluding necrosis, inflammation, ballooning
degeneration, and fatty changes were detected
in the liver of BDL animals (Table 2). Betaine
supplementation (10 and 50 mg/kg, i.p) alle-
viated BDL-associated liver injury in cirrhotic
rats (Table 2).

Table 2
Effect of betaine supplementation on the liver histopathological alterations in acute and chronic models of liver injury.
Pathological assessment Confluent Necrosis Focal Necrosis Portal Inflammation InterfaceHepatitis Ballooning Total Ishak stage of liver
Treatment degeneration grade fibrosis

Control (vehicle-treated rats) 0 0 0 0 0 0 –

Bile duct ligated (BDL) rats
BDL + Betaine 10 mg/kg
BDL + Betaine 50 mg/kg
TAA
TAA + Betaine 10 mg/kg
TAA + Betaine 50 mg/kg
4 3
0 0
0 0
3 0
2 0
1 0
3 2 1 13 4
2 1 1 4 2
1 1 1 3 1
1 0 2 9 –
0 0 1 7 –
0 0 1 4 –

BDL: Bile duct ligation; TAA: Thioacetamide.

oxidative stress and its consequences play a critical role in different
liver diseases [9–13,54]. The crucial role of oxidative/nitrosative stress
in the pathogenesis of acute and chronic models of hepatic injury has
also been widely investigated [9,55,56]. The role of oxidative stress in
the mechanism of liver injury and fibrogenesis also has largely been
revealed [9,12,54]. Hence, antioxidant supplementation might be of
value against these complications.
It has been found that the accumulation of cytotoxic bile acids in the
rat model of BDL-induced cholestasis/cirrhosis is a well-documented
pathogenic factor associated with vast tissue injury [23,25,57–62].
Hydrophobic bile acids are detergents which severely disrupt bio-
membrane lipids, and defect the nature and function of cellular proteins
[25,58–62]. Changes in the liver tissue antioxidant status has also been
documented in acute and chronic animal models of hepatotoxicity [63].
In line with previous studies, the data obtained in the current in-
vestigation mentioned the occurrence of oxidative stress and its asso-
ciated complications in liver tissue of acute and chronic models of liver
injury (Fig. 1 and 2). All these data could mention the crucial role of
antioxidant therapy. On the other hand, increasing evidence also sug-
gest that mitochondrial dysfunction can also contribute to the
development of tissue fibrosis [64]. Cellular mitochondria dysfunction
and its associated events play a critical role in the cell death process
[65–68]. Mitochondrial dysfunction also involved in the mechanism of
a wide range of xenobiotics-induced liver injury [11,16,18–20,69,70].
Hence, targeting this vital organelle could serve as a crucial pharma-
cological point of intervention.
Betaine is abundantly found in daily food intake [1,2]. This che-
mical is also endogenously synthesized in cellular mitochondria by the
oxidative metabolism of choline [1,2]. Betaine is then partially trans-
ported from mitochondria to the cytoplasm [1,2]. On the other hand,
betaine transporters are predominantly expressed in the liver which is
responsible for betaine accumulation in hepatocytes [2,71]. The hepa-
toprotective properties of betaine have been shown against different
complications including fatty liver, alcoholism–associated liver injury,
tissue fibrosis, and cellular necrosis [5–7,26,72–74]. However, the
precise cellular mechanisms behind betaine cytoprotection remain
mostly unknown.
The antioxidant property of betaine is the dominant mechanism
proposed for the cytoprotection provided by this chemical
[5,72,75–79]. The role of betaine against oxidative stress and its

81
R. Heidari et al. Biomedicine & Pharmacotherapy 103 (2018) 75–86

Fig. 6. Effect of betaine supplementation on the liver tissue fibrosis and hydroxyproline content in cirrhotic rats (Masson Trichrome stain). BDL: Bile duct ligated.
Liver tissue score of fibrosis (Blue color) is given in Table 2. Data for liver hydroxyproline content and collagen deposition are given as Mean ± SEM (n = 6).
***Indicates significantly different as compared with control (P < 0.001).a Indicates significantly different as compared with BDL group (P < 0.01). ns: not
significant.

consequences has repeatedly been mentioned in different experimental
models [5,72,75–79]. The effects of betaine on enzymatic and non-
enzymatic cellular antioxidants have also been reported in previous
investigations [5,72,75–79]. In the current study, we found that betaine
treatment efficiently abated oxidative stress and its associated compli-
cations in both acute and chronic liver injury.
Previous investigations also have shown that in addition to the
antioxidative properties, betaine serves as an ideal osmolyte in plant or
animal cells [1,28,80,81]. The osmoprotective function of betaine has
also been reported in different biological systems [2,80]. Although it
has not been evaluated in the current study, the osmoregulatory
properties of betaine might protect hepatocytes in the extreme condi-
tions of bile duct obstruction or acute liver injury.
Some investigations revealed that betaine increases hepatocytes S-
Adenosyl methionine (SAM) level [82,83]. SAM plays an essential role
in the reconstitution of damaged biological targets (e.g., membrane
phospholipids, DNA) [84,85]. SAM also is essential for biomembrane
lipids biosynthesis [85,86]. Hence, improving the biomembrane
synthesis might play a crucial role in the cytoprotective properties of
betaine. These data mention that a significant part of hepatoprotection
provided by betaine might be associated with SAM production in the
liver and sustaining the function of the vital cell organelles such as
mitochondria. SAM is also known as a radical scavenger and a chemical
which participates in boosting cellular antioxidant defense mechanisms
[5,84,85,87–89]. Therefore, a part of the antioxidant properties of be-
taine in the liver tissue might be attributed to its SAM metabolite.
Betaine also efficiently alleviated liver fibrosis in different experi-
mental models [72,79,90]. In accordance with these studies, we found
that betaine treatment of BDL animals significantly alleviated liver
tissue collagen deposition and hydroxyproline content (Fig. 6). The

82
R. Heidari et al.
antifibrotic properties of betaine could be mediated through a range of
mechanisms. As mentioned, oxidative/nitrosative stress and its asso-
ciated complications play a crucial role in the initiation and propaga-
tion of tissue fibrosis [9,12,54]. Hence, the antioxidative properties and
modulation of cellular redox environment (e.g., by boosting cellular
antioxidant defense mechanisms) could play a significant role in the
antifibrotic properties of betaine [5].
The role of inflammatory cells and mediators in the progression of
liver injury and tissue fibrosis has been well-documented in previous
models of hepatotoxicity [57,91,92]. Interestingly, it has been revealed
that betaine could abate inflammatory responses [76,93,94]. Although
not evaluated in the current study, the anti-inflammatory properties of
betaine might play a role in its hepatoprotective properties especially in
chronic liver injury and cirrhosis.
The effect of betaine on tissue fibrosis also has been revealed in
previous studies [72,95]. Stellate cells play a crucial role in liver fi-
brogenesis [96,97]. Therefore, the inhibitory effects of betaine on
stellate cells might also play a role in its mechanisms of hepatopro-
tection and anti-fibrogenesis. The anti-inflammatory effects of betaine
might also provide another possible explanation for its hepatoprotec-
tive properties in acute or chronic liver injury. Hence, the effect of
betaine on inflammatory cytokines and its relevance to the hepato-
protective properties of this chemical deserve further investigations.
Interestingly, it has been mentioned that betaine supplementation
could prevent liver mitochondrial morphological alterations in rats
[98]. Recently, the direct effect of betaine on mitochondrial energy
metabolism also has been reported in an in vitro system [99]. The
protective effects of betaine on mitochondrial function and mitochon-
dria-mediated cell death process in other experimental models also
have been revealed [28,100–103]. All these data indicate that mi-
tochondrial protecting properties of betaine could play a role in its
mechanism of cytoprotection. On the other hand, the impairment of
83
Biomedicine & Pharmacotherapy 103 (2018) 75–86
Fig. 7. Effect of betaine supplementation on
liver tissue histopathology in the animal model
of acute liver injury (H&E stain). TAA:
Thioacetamide.
Liver tissue histopathological changes in
thioacetamide-treated group revealed as in-
flammation, necrosis, and ballooning degen-
eration. Betaine supplementation (10 and
50 mg/kg respectively) mitigated thioaceta-
mide hepatotoxicity. The grade of histopatho-
logical changes in the acute model of hepatic
injury is given in Table 2.
mitochondrial function and decreased cellular ATP content could also
impair the cellular repairing processes and tissue regeneration. In the
current study, we found that mitochondrial ATP content was higher in
betaine-treated groups in animal models of acute and chronic liver in-
jury (Fig. 3 and 4). Therefore, sufficient ATP might be available for liver
tissue regeneration in betaine-treated animals.
Hydrophobic bile acids are well-known mitochondrial toxins which
their accumulation might be a causative factor for oxidative stress and
damage to hepatocytes during cholestasis/cirrhosis [9,23–25,62,104].
These chemicals severely affect mitochondrial function and impair
energy metabolism [9,23–25,62,104]. Bile acids induce mitochondrial
permeability transition, dissipate mitochondrial membrane potential,
and finally cause the release of cell death mediators from mitochondria
[9,23–25,62,104]. The inhibitory effects of bile acids on mitochondrial
respiratory complexes also has been revealed [9,23–25,62,104]. On the
other hand, the relevance of mitochondrial dysfunction to the hepatic
injury in animal models of cholestasis also has been mentioned in
previous studies [9,23–25,62,104]. Hence, preserving normal mi-
tochondrial function could serve as a potential therapeutic point of
intervention during cholestasis. Our results showed the same pattern
and revealed significant impairment of mitochondrial function in an-
imal models of chronic or acute liver injury (Fig. 3 and 4). It was found
that betaine supplementation preserved mitochondrial indices of func-
tionality both in acute or chronic animal models of liver injury (Fig. 3
and 4). These data mention the mitochondrial protection as a funda-
mental mechanism for the hepatoprotection provided by betaine. On
the other hand, oral administration of betaine also prevented ROS
formation and lipid peroxidation. Moreover, betaine preserved liver
tissue glutathione content and antioxidant capacity (Fig. 1 and 2).
As mentioned, the effect of betaine on oxidative stress and its con-
sequences has been mentioned in several investigations [5,8,72,76]. On
the other hand, oxidative stress and mitochondrial dysfunction are two
R. Heidari et al.
mechanistically related events [105]. Hence, mitochondrial protecting
properties of betaine might also play a role in its ability to alleviate
oxidative stress in the liver.
In the current study, the insignificant changes in some markers of
the bile duct and liver injury (e.g., ALP, γ-GT, and bilirubin) upon be-
taine treatment (Table 1) might be due to the permanent nature of bile
duct obstruction in BDL model.
The data presented in the current study suggest that betaine might
provide hepatoprotection through regulating mitochondrial function,
membrane stabilizing action, and antioxidative effects in animal models
of acute or chronic liver injury. Further investigations should be per-
formed to clarify the effects of betaine on tricarboxylic acid cycle (TCA;
Krebs cycle) enzymes and mitochondrial respiration complexes. On the
other hand, other vital organs and tissues (e.g., kidneys, skeletal muscle,
and heart) could be affected by conditions such as cholestasis/cirrhosis
[106,107]. Hence, investigating the potential protective properties of
the molecules such as betaine against these complications could be the
subject of further research.
Declaration of interest
The authors declare no conflicts of interest.
Acknowledgment
The authors thank Pharmaceutical Sciences Research Center of
Shiraz University of Medical Sciences for providing technical and fi-
nancial support of the current investigation (Grant number: 12046/
10930).
References
[1] S.A.S. Craig, Betaine in human nutrition, Am. J. Clin. Nut. 80 (3) (2004) 539–549.
[2] V. Preedy, Betaine: Chemistry, Analysis, Function and Effects, Royal Society
Chemistry, 2015.
[3] M. Ahn, J.S. Park, S. Chae, S. Kim, C. Moon, J.W. Hyun, T. Shin, Hepatoprotective
effects of Lycium chinense Miller fruit and its constituent betaine in CCl4-induced
hepatic damage in rats, Acta Histochem. 116 (6) (2014) 1104–1112.
[4] J. Balkan, F.H. Parldar, S. Dogru-Abbasoglu, G. Aykaç-Toker, M. Uysal, The effect
of taurine or betaine pretreatment on hepatotoxicity and prooxidant status in-
duced by lipopolysaccharide treatment in the liver of rats, Eur. J. Gastroenterol.
Hepatol. 17 (9) (2005) 917.
[5] Y.S. Jung, S.J. Kim, D.Y. Kwon, C.W. Ahn, Y.S. Kim, D.W. Choi, Y.C. Kim,
Alleviation of alcoholic liver injury by betaine involves an enhancement of anti-
oxidant defense via regulation of sulfur amino acid metabolism, Food Chem.
Toxicol. 62 (2013) 292–298.
[6] Z. Wang, T. Yao, M. Pini, Z. Zhou, G. Fantuzzi, Z. Song, Betaine improved adipose
tissue function in mice fed a high-fat diet: a mechanism for hepatoprotective effect
of betaine in nonalcoholic fatty liver disease, Am. J. Physiol. 298 (5) (2010)
G634–G642.
[7] C. Ji, N. Kaplowitz, Betaine decreases hyperhomocysteinemia, endoplasmic re-
ticulum stress, and liver injury in alcohol-fed mice, Gastroenterology 124 (5)
(2003) 1488–1499.
[8] J. Oliva, F. Bardag-Gorce, B. Tillman, S.W. French, Protective effect of quercetin,
EGCG, catechin and betaine against oxidative stress induced by ethanol in vitro,
Exp. Mol. Pathol. 90 (3) (2011) 295–299.
[9] B.L. Copple, H. Jaeschke, C.D. Klaassen, Oxidative stress and the pathogenesis of
cholestasis, Semin. Liver Dis. 30 (2) (2010) 195–204.
[10] H. Jaeschke, G.J. Gores, A.I. Cederbaum, J.A. Hinson, D. Pessayre, J.J. Lemasters,
Mechanisms of hepatotoxicity, Toxicol. Sci. 65 (2) (2002) 166–176.
[11] H. Jaeschke, M.R. McGill, A. Ramachandran, Oxidant stress, mitochondria, and
cell death mechanisms in drug-induced liver injury: lessons learned from acet-
aminophen hepatotoxicity, Drug Metab. Rev. 44 (1) (2012) 88–106.
[12] E. Mormone, J. George, N. Nieto, Molecular pathogenesis of hepatic fibrosis and
current therapeutic approaches, Chem.-Biol. Interact. 193 (3) (2011) 225–231.
[13] C. Stephens, R.J. Andrade, M.I. Lucena, Mechanisms of drug-induced liver injury,
Curr. Opin. Allergy Clin. Immunol. 14 (4) (2014) 286–292.
[14] M.R. Duchen, Mitochondria in health and disease: perspectives on a new mi-
tochondrial biology, Mol. Asp. Med. 25 (4) (2004) 365–451.
[15] S.W.G. Tait, D.R. Green, Mitochondria and cell death: outer membrane permea-
bilization and beyond, Nat. Rev. Mol. Cell Biol. 11 (9) (2010) 621–632.
[16] G. Labbe, D. Pessayre, B. Fromenty, Drug-induced liver injury through mi-
tochondrial dysfunction: mechanisms and detection during preclinical safety stu-
dies, Fundam. Clin. Pharmacol. 22 (4) (2008) 335–353.
[17] R. Heidari, H. Niknahad, A. Jamshidzadeh, M.A. Eghbal, N. Abdoli, An overview
on the proposed mechanisms of antithyroid drugs-induced liver injury, Adv.
84
Biomedicine & Pharmacotherapy 103 (2018) 75–86
Pharm. Bull. 5 (1) (2015) 1–11.
[18] D. Pessayre, B. Fromenty, A. Berson, M.-A. Robin, P. Lettéron, R. Moreau,
A. Mansouri, Central role of mitochondria in drug-induced liver injury, Drug
Metab. Rev. 44 (1) (2012) 34–87.
[19] S. Russmann, G.A. Kullak-Ublick, I. Grattagliano, Current concepts of mechanisms
in drug-induced hepatotoxicity, Curr. Med. Chem. 16 (23) (2009) 3041–3053.
[20] R. Heidari, H. Babaei, M.A. Eghbal, Amodiaquine-induced toxicity in isolated rat
hepatocytes and the cytoprotective effects of taurine and/or N-acetyl cysteine,
Res. Pharm. Sci. 9 (2) (2014) 97–105.
[21] A. Jamshidzadeh, R. Heidari, M. Abasvali, M. Zarei, M.M. Ommati, N. Abdoli,
F. Khodaei, Y. Yeganeh, F. Jafari, A. Zarei, Z. Latifpour, E. Mardani, N. Azarpira,
B. Asadi, A. Najibi, Taurine treatment preserves brain and liver mitochondrial
function in a rat model of fulminant hepatic failure and hyperammonemia,
Biomed. Pharmacother. 86 (2017) 514–520.
[22] R. Heidari, V. Ghanbarinejad, H. Mohammadi, A. Ahmadi, M.M. Ommati,
N. Abdoli, F. Aghaei, A. Esfandiari, N. Azarpira, H. Niknahad, Mitochondria pro-
tection as a mechanism underlying the hepatoprotective effects of glycine in
cholestatic mice, Biomed. Pharmacother 97 (Supplement C) (2018) 1086–1095.
[23] A.P. Rolo, P.J. Oliveira, A.J.M. Moreno, C.M. Palmeira, Bile acids affect liver
mitochondrial bioenergetics: possible relevance for cholestasis therapy, Toxicol.
Sci. 57 (1) (2000) 177–185.
[24] A. Arduini, G. Serviddio, A.M. Tormos, M. Monsalve, J. Sastre, Mitochondrial
dysfunction in cholestatic liver diseases, Front. Biosci. 4 (2012) 2233–2252.
[25] C.M. Palmeira, A.P. Rolo, Mitochondrially-mediated toxicity of bile acids,
Toxicology 203 (1–3) (2004) 1–15.
[26] E. Kathirvel, K. Morgan, G. Nandgiri, B.C. Sandoval, M.A. Caudill, T. Bottiglieri,
S.W. French, T.R. Morgan, Betaine improves nonalcoholic fatty liver and asso-
ciated hepatic insulin resistance: a potential mechanism for hepatoprotection by
betaine, Am. J. Physiol. 299 (5) (2010) G1068–G1077.
[27] D. Graf, A.K. Kurz, R. Reinehr, R. Fischer, G. Kircheis, D. Häussinger, Prevention of
bile acid-induced apoptosis by betaine in rat liver, Hepatology 36 (4 Pt. 1)) (2002)
829–839.
[28] Q. Garrett, N. Khandekar, S. Shih, J.L. Flanagan, P. Simmons, J. Vehige,
M.D.P. Willcox, Betaine stabilizes cell volume and protects against apoptosis in
human corneal epithelial cells under hyperosmotic stress, Exp. Eye Res. 108
(Supplement C) (2013) 33–41.
[29] L. Moezi, R. Heidari, Z. Amirghofran, A.A. Nekooeian, A. Monabati, A.R. Dehpour,
Enhanced anti-ulcer effect of pioglitazone on gastric ulcers in cirrhotic rats: the
role of nitric oxide and IL-1b, Pharmacol. Rep. 65 (134) (2013) 134–143.
[30] M.J. Tuñón, M. Alvarez, J.M. Culebras, J. González-Gallego, An overview of an-
imal models for investigating the pathogenesis and therapeutic strategies in acute
hepatic failure, W. J. Gastroenterol. 15 (25) (2009).
[31] S. Ali, K.A. Ansari, M.A. Jafry, H. Kabeer, G. Diwakar, Nardostachys jatamansi
protects against liver damage induced by thioacetamide in rats, J.
Ethnopharmacol. 71 (3) (2000) 359–363.
[32] R. Bruck, H. Aeed, H. Shirin, Z. Matas, L. Zaidel, Y. Avni, Z. Halpern, The hydroxyl
radical scavengers dimethylsulfoxide and dimethylthiourea protect rats against
thioacetamide-induced fulminant hepatic failure, J. Hepatol. 31 (1) (1999) 27–38.
[33] A. Jamshidzadeh, R. Heidari, S. Mohammadi-Samani, N. Azarpira, A. Najbi,
P. Jahani, N. Abdoli, A comparison between the nephrotoxic profile of gentamicin
and gentamicin nanoparticles in mice, J. Biochem. Mol. Toxicol. 29 (2) (2015)
57–62.
[34] R. Gupta, D.K. Dubey, G.M. Kannan, S.J.S. Flora, Concomitant administration of
Moringa oleifera seed powder in the remediation of arsenic-induced oxidative
stress in mouse, Cell Biol. Int. 31 (1) (2007) 44–56.
[35] R. Heidari, N. Esmailie, N. Azarpira, A. Najibi, H. Niknahad, Effect of thiol-re-
ducing agents and antioxidants on sulfasalazine-induced hepatic injury in nor-
motermic recirculating isolated perfused rat liver, Toxicol. Res. 32 (2) (2016)
133–140.
[36] D.J. Socci, K.B. Bjugstad, H.C. Jones, J.V. Pattisapu, G.W. Arendash, Evidence that
oxidative stress is associated with the pathophysiology of inherited hydrocephalus
in the H-Tx rat model, Exp. Neurol. 155 (1) (1999) 109–117.
[37] H. Niknahad, R. Heidari, R. Firuzi, F. Abazari, M. Ramezani, N. Azarpira,
M. Hosseinzadeh, A. Najibi, A. Saeedi, Concurrent inflammation augments anti-
malarial drugs-induced liver injury in rats, Ad. Pharm. Bull. 6 (4) (2016) 617–625.
[38] R. Heidari, A. Jamshidzadeh, H. Niknahad, F. Safari, H. Azizi, N. Abdoli,
M.M. Ommati, F. Khodaei, A. Saeedi, A. Najibi, The hepatoprotection provided by
taurine and Glycine against antineoplastic drugs induced liver injury in an ex vivo
model of normothermic recirculating isolated perfused rat liver, Trend. Pharm.
Sci. 2 (1) (2016) 59–76.
[39] R. Heidari, M. Rasti, B. Shirazi Yeganeh, H. Niknahad, A. Saeedi, A. Najibi,
Sulfasalazine-induced renal and hepatic injury in rats and the protective role of
taurine, BioImpacts 6 (1) (2016) 3–8.
[40] V. Katalinic, D. Modun, I. Music, M. Boban, Gender differences in antioxidant
capacity of rat tissues determined by 2,2’-azinobis (3-ethylbenzothiazoline 6-sul-
fonate; ABTS) and ferric reducing antioxidant power (FRAP) assays, Comp.
Biochem. Physiol. 140 (1) (2005) 47–52.
[41] M. Alía, C. Horcajo, L. Bravo, L. Goya, Effect of grape antioxidant dietary fiber on
the total antioxidant capacity and the activity of liver antioxidant enzymes in rats,
Nutr. Res. 23 (9) (2003) 1251–1267.
[42] R. Heidari, L. Moezi, B. Asadi, M.M. Ommati, N. Azarpira, Hepatoprotective effect
of boldine in a bile duct ligated rat model of cholestasis/cirrhosis,
PharmaNutrition 5 (3) (2017) 109–117.
[43] A.A. Caro, L.W. Adlong, S.J. Crocker, M.W. Gardner, E.F. Luikart, L.U. Gron, Effect
of garlic-derived organosulfur compounds on mitochondrial function and integrity
in isolated mouse liver mitochondria, Toxicol. Lett. 214 (2) (2012) 166–174.
R. Heidari et al.
[44] P. Zhao, T.F. Kalhorn, J.T. Slattery, Selective mitochondrial glutathione depletion
by ethanol enhances acetaminophen toxicity in rat liver, Hepatology 36 (2) (2002)
326–335.
[45] T. Mosmann, Rapid colorimetric assay for cellular growth and survival: applica-
tion to proliferation and cytotoxicity assays, J. Immunol. Methods 65 (1) (1983)
55–63.
[46] R. Heidari, F. Jafari, F. Khodaei, B. Shirazi Yeganeh, H. Niknahad, The mechanism
of valproic acid-induced Fanconi syndrome involves mitochondrial dysfunction
and oxidative stress in rat kidney, Nephrology (Carlton, Vic.) 23 (4) (2018)
351–361.
[47] R. Heidari, V. Taheri, H.R. Rahimi, B.S. Yeganeh, H. Niknahad, A. Najibi,
Sulfasalazine-induced renal injury in rats and the protective role of thiol-re-
ductants, Ren. Fail. 38 (1) (2016) 137–141.
[48] A. Jamshidzadeh, H. Niknahad, R. Heidari, M. Zarei, M.M. Ommati, F. Khodaei,
Carnosine protects brain mitochondria under hyperammonemic conditions: re-
levance to hepatic encephalopathy treatment, PharmaNutrition 5 (2) (2017)
58–63.
[49] H. Niknahad, A. Jamshidzadeh, R. Heidari, Z. Hosseini, K. Mobini, F. Khodaei,
M.M. Ommati, N. Abdoli, N. Keshavarz, M. Bazyari, A. Najibi, Paradoxical effect of
methimazole on liver mitochondria: in vitro and in vivo, Toxicol. Lett. 259 (2016)
108–115.
[50] H. Niknahad, A. Jamshidzadeh, R. Heidari, M. Zarei, M.M. Ommati, Ammonia-
induced mitochondrial dysfunction and energy metabolism disturbances in iso-
lated brain and liver mitochondria, and the effect of taurine administration: re-
levance to hepatic encephalopathy treatment, Clin. Exp. Hepatol. 3 (3) (2017)
141–151.
[51] M.M. Ommati, R. Heidari, A. Jamshidzadeh, M.J. Zamiri, Z. Sun, S. Sabouri,
J. Wang, F. Ahmadi, N. Javanmard, K. Seifi, S. Mousapour, B.S. Yeganeh, Dual
effects of sulfasalazine on rat sperm characteristics, spermatogenesis, and ster-
oidogenesis in two experimental models, Toxicol. Lett. 284 (2018) 46–55.
[52] M.M. Ommati, N. Tanideh, B. Rezakhaniha, J. Wang, S. Sabouri, M. Vahedi,
B. Dormanesh, O. Koohi Hosseinabadi, F. Rahmanifar, S. Moosapour, A. Akhlaghi,
R. Heidari, M.J. Zamiri, Is immunosuppression, induced by neonatal thymectomy,
compatible with poor reproductive performance in adult male rats? Andrology 6
(1) (2018) 199–213.
[53] M.M. Bradford, A rapid and sensitive method for the quantitation of microgram
quantities of protein utilizing the principle of protein-dye binding, Analyt.
Biochem. 72 (1) (1976) 248–254.
[54] M. Pinzani, K. Rombouts, Liver fibrosis: from the bench to clinical targets, Dig.
Liver Dis. 36 (4) (2004) 231–242.
[55] C. Ara, H. Kirimlioglu, A.B. Karabulut, S. Coban, S. Ay, M. Harputluoglu,
V. Kirimlioglu, S. Yilmaz, Protective effect of resveratrol against oxidative stress in
cholestasis, J. Surg. Res. 127 (2) (2005) 112–117.
[56] H. Shapiro, M. Ashkenazi, N. Weizman, M. Shahmurov, H. Aeed, R. Bruck,
Curcumin ameliorates acute thioacetamide-induced hepatotoxicity, J.
Gastroenterol. Hepatol. 21 (2) (2006) 358–366.
[57] C.G. Tag, S. Sauer-Lehnen, S. Weiskirchen, E. Borkham-Kamphorst, R.H. Tolba,
F. Tacke, R. Weiskirchen, Bile duct ligation in mice: induction of inflammatory
liver injury and fibrosis by obstructive cholestasis, J. Vis. Exp. (2015) 96.
[58] E. Krones, M. Wagner, K. Eller, A.R. Rosenkranz, M. Trauner, P. Fickert, Bile acid-
induced cholemic nephropathy, Dig. Dis. 33 (3) (2015) 367–375.
[59] T. Li, J.Y.L. Chiang, Bile acid-induced liver injury in cholestasis, Cellular Injury in
Liver Diseases, Springer, Cham, 2017, pp. 143–172.
[60] R.H. Palmer, Bile acids, liver injury, and liver disease, Arch. Intern. Med. 130 (4)
(1972) 606–617.
[61] R.J. Sokol, M. Devereaux, R. Dahl, E. Gumpricht, “Let there be bile”–under-
standing hepatic injury in cholestasis, J. Pediatr. Gastroenterol. Nutr. 43 (Suppl. 1)
(2006) S4–S9.
[62] A.P. Rolo, C.M. Palmeira, K.B. Wallace, Mitochondrially mediated synergistic cell
killing by bile acids, Biochim. Biophys. Acta 1637 (1) (2003) 127–132.
[63] A. Jamshidzadeh, R. Heidari, Z. Latifpour, M.M. Ommati, N. Abdoli, S. Mousavi,
N. Azarpira, A. Zarei, M. Zarei, B. Asadi, M. Abasvali, Y. Yeganeh, F. Jafari,
A. Saeedi, A. Najibi, E. Mardani, Carnosine ameliorates liver fibrosis and hyper-
ammonemia in cirrhotic rats, Clin. Res. Hepatol. Gastroenterol. 41 (4) (2017)
424–434.
[64] M. Jain, S. Rivera, E.A. Monclus, L. Synenki, A. Zirk, J. Eisenbart, C. Feghali-
Bostwick, G.M. Mutlu, G.R.S. Budinger, N.S. Chandel, Mitochondrial reactive
oxygen species regulate transforming growth factor-β signaling, J. Biol. Chem. 288
(2) (2013) 770–777.
[65] M.J. Parsons, D.R. Green, Mitochondria in cell death, Essays Biochem. 47 (2010)
99–114.
[66] A.P. Halestrap, E. Doran, J.P. Gillespie, A. O’Toole, Mitochondria and cell death,
Biochem. Soc. Trans. 28 (1) (2000) 11.
[67] P. Bernardi, L. Scorrano, R. Colonna, V. Petronilli, F. Di Lisa, Mitochondria and
cell death, Eur. J. Biochem. 264 (3) (1999) 687–701.
[68] M.R. Duchen, Mitochondria and calcium: from cell signalling to cell death, J.
Physiol. 529 (1) (2000) 57–68.
[69] N. Abdoli, R. Heidari, Y. Azarmi, M.A. Eghbal, Mechanisms of the statins cyto-
toxicity in freshly isolated rat hepatocytes, J. Biochem. Mol. Toxicol. 27 (6) (2013)
287–294.
[70] R. Heidari, H. Niknahad, A. Jamshidzadeh, N. Abdoli, Factors affecting drug-in-
duced liver injury: antithyroid drugs as instances, Clin. Mol. Hepatol. 20 (3)
(2014) 237–248.
[71] S.A. Kempson, Y. Zhou, N.C. Danbolt, The betaine/GABA transporter and betaine:
roles in brain, kidney, and liver, Front. Physiol. 5 (2014).
[72] İ. Bingül, A.F. Aydın, C. Başaran-Küçükgergin, I. Doğan-Ekici, J. Çoban, S. Doğru-
85
Biomedicine & Pharmacotherapy 103 (2018) 75–86
Abbasoğlu, M. Uysal, High-fat diet plus carbon tetrachloride-induced liver fibrosis
is alleviated by betaine treatment in rats, Int. Immunopharmacol. 39 (2016)
199–207.
[73] R. Deminice, R.Pd. Silva, S.G. Lamarre, K.B. Kelly, R.L. Jacobs, M.E. Brosnan,
J.T. Brosnan, Betaine supplementation prevents fatty liver induced by a high-fat
diet: effects on one-carbon metabolism, Amino Acids 47 (4) (2015) 839–846.
[74] M.F. Abdelmalek, P. Angulo, R.A. Jorgensen, P.B. Sylvestre, K.D. Lindor, Betaine, a
promising new agent for patients with nonalcoholic steatohepatitis: results of a
pilot study, Am. J. Gastroenterol. 96 (9) (2001) 2711–2717.
[75] H. Hagar, W. Al Malki, Betaine supplementation protects against renal injury in-
duced by cadmium intoxication in rats: role of oxidative stress and caspase-3,
Environ. Toxicol. Pharmacol. 37 (2) (2014) 803–811.
[76] H. Hagar, A.E. Medany, R. Salam, G.E. Medany, O.A. Nayal, Betaine supple-
mentation mitigates cisplatin-induced nephrotoxicity by abrogation of oxidative/
nitrosative stress and suppression of inflammation and apoptosis in rats, Exp.
Toxicol. Pathol. 67 (2) (2015) 133–141.
[77] F. Erman, J. Balkan, U. Çevikbaş, N. Koçak-Toker, M. Uysal, Betaine or taurine
administration prevents fibrosis and lipid peroxidation induced by rat liver by
ethanol plus carbon tetrachloride intoxication, Amino Acids 27 (2) (2004)
199–205.
[78] B. Ganesan, S. Buddhan, R. Anandan, R. Sivakumar, R. AnbinEzhilan, Antioxidant
defense of betaine against isoprenaline-induced myocardial infarction in rats, Mol.
Biol. Rep. 37 (3) (2009) 1319–1327.
[79] S.K. Kim, J.M. Seo, Y.R. Chae, Y.S. Jung, J.H. Park, Y.C. Kim, Alleviation of di-
methylnitrosamine-induced liver injury and fibrosis by betaine supplementation in
rats, Chem.-Biol. Interact. 177 (3) (2009) 204–211.
[80] S. Cayley, B.A. Lewis, M.T. Record, Origins of the osmoprotective properties of
betaine and proline in Escherichia coli K-12, J. Bacteriol. 174 (5) (1992)
1586–1595.
[81] M. Wettstein, D. Haussinger, Cytoprotection by the osmolytes betaine and taurine
in ischemia- reoxygenation injury in the perfused rat liver, Hepatology 26 (6)
(1997) 1560–1566.
[82] A.J. Barak, H.C. Beckenhauer, M. Junnila, D.J. Tuma, Dietary betaine promotes
generation of hepatic S-adenosylmethionine and protects the liver from ethanol-
induced fatty infiltration, Alcoholism: Clin. Exp. Res. 17 (3) (1993) 552–555.
[83] S.K. Kim, Y.C. Kim, Effects of betaine supplementation on hepatic metabolism of
sulfur-containing amino acids in mice, J. Hepatol. 42 (6) (2005) 907–913.
[84] J.M. Mato, F.J. Corrales, S.C. Lu, M.A. Avila, S-Adenosylmethionine: a control
switch that regulates liver function, FASEB J. 16 (1) (2002) 15–26.
[85] Q.M. Anstee, C.P. Day, S-Adenosylmethionine (SAMe) therapy in liver disease: a
review of current evidence and clinical utility, J. Hepatol. 57 (5) (2012)
1097–1109.
[86] R.L. Jacobs, J.N. Veen, D.E. Vance, Finding the balance: the role of S-adeno-
sylmethionine and phosphatidylcholine metabolism in development of nonalco-
holic fatty liver disease, Hepatology 58 (4) (2013) 1207–1209.
[87] A.A. Caro, A.I. Cederbaum, Antioxidant properties of S-adenosyl-l-methionine in
Fe2+-initiated oxidations, Free Radic. Biol. Med. 36 (10) (2004) 1303–1316.
[88] M. Rizzardini, M. Carelli, M.R. Cabello Porras, L. Cantoni, Mechanisms of en-
dotoxin-induced haem oxygenase mRNA accumulation in mouse liver: synergism
by glutathione depletion and protection by N-acetylcysteine, Biochem. J. 304 (Pt
2) (1994) 477–483.
[89] Z. Ming, Y.-j. Fan, X. Yang, W.W. Lautt, Synergistic protection by S-adeno-
sylmethionine with vitamins C and E on liver injury induced by thioacetamide in
rats, Free Radic. Biol. Med. 40 (4) (2006) 617–624.
[90] M.-T. Tsai, C.-Y. Chen, Y.-H. Pan, S.-H. Wang, H.J. Mersmann, S.-T. Ding,
Alleviation of carbon-tetrachloride-induced liver injury and fibrosis by betaine
supplementation in chickens, Evid. Based Complement. Alternat. Med. (2015).
[91] R.A. Roth, J.P. Luyendyk, J.F. Maddox, P.E. Ganey, Inflammation and drug idio-
syncrasy—is there a connection? J. Pharmacol. Exp. Ther. 307 (1) (2003) 1–8.
[92] W. Zou, R.A. Roth, P.E. Ganey, A.V. Lyubimov, Animal models of idiosyncratic,
drug-induced liver injury: emphasis on the inflammatory stress hypothesis,
Encyclopedia of Drug Metabolism and Interactions, John Wiley & Sons, Inc., 2011.
[93] D.H. Kim, B. Sung, Y.J. Kang, J.Y. Jang, S.Y. Hwang, Y. Lee, M. Kim, E. Im, J.-
H. Yoon, C.M. Kim, H.Y. Chung, N.D. Kim, Anti-inflammatory effects of betaine on
AOM/DSS‑induced colon tumorigenesis in ICR male mice, Int. J. Oncol. 45 (3)
(2014) 1250–1256.
[94] E.K. Go, K.J. Jung, J.Y. Kim, B.P. Yu, H.Y. Chung, Betaine suppresses proin-
flammatory signaling during aging: the involvement of nuclear factor-κB via nu-
clear factor-inducing kinase/IκB kinase and mitogen-activated protein kinases, J.
Gerontol. 60 (10) (2005) 1252–1264.
[95] R.R. Watson, Foods and Dietary Supplements in the Prevention and Treatment of
Disease in Older Adults, Academic Press, 2015.
[96] R.K. Moreira, Hepatic stellate cells and liver fibrosis, Arch. Pathol. Lab. Med. 131
(11) (2007) 1728–1734.
[97] R. Safadi, S.L. Friedman, Hepatic fibrosis–role of hepatic stellate cell activation,
Med. Gen. Med. 4 (3) (2002) 27.
[98] M. Junnila, T. Rahko, A. Sukura, L.A. Lindberg, Reduction of carbon tetrachloride-
induced hepatotoxic effects by oral administration of betaine in male Han-Wistar
rats: a morphometric histological study, Vet. Pathol. 37 (3) (2000) 231–238.
[99] I. Lee, Betaine is a positive regulator of mitochondrial respiration, Biochem.
Biophys. Res. Commun. 456 (2) (2015) 621–625.
[100] B. Ganesan, R. Rajesh, R. Anandan, N. Dhandapani, Biochemical studies on the
protective effect of betaine on mitochondrial function in experimentally induced
myocardial infarction in rats, J. Health Sci. 53 (6) (2007) 671–681.
[101] A.R. Im, Y.-H. Kim, M.R. Uddin, S. Chae, H.W. Lee, Y.H. Kim, Y.S. Kim, M.-Y. Lee,
Betaine protects against rotenone-induced neurotoxicity in PC12 cells, Cell. Mol.
R. Heidari et al.
Neurobiol. 33 (5) (2013) 625–635.
[102] K.K. Kharbanda, S.L. Todero, A.L. King, N.A. Osna, B.L. McVicker, D.J. Tuma,
J.L. Wisecarver, S.M. Bailey, Betaine treatment attenuates chronic ethanol-in-
duced hepatic steatosis and alterations to the mitochondrial respiratory chain
proteome, Int. J. Hepatol. (2011) 2012.
[103] D. Nash, L.G. Paleg, J.J. Wiskich, Effect of proline, betaine and some other solutes
on the heat stability of mitochondrial enzymes, Funct. Plant Biol. 9 (1) (1982)
47–57.
[104] B. Yerushalmi, R. Dahl, M.W. Devereaux, E. Gumpricht, R.J. Sokol, Bile acid‐-
induced rat hepatocyte apoptosis is inhibited by antioxidants and blockers of the
86
Biomedicine & Pharmacotherapy 103 (2018) 75–86
mitochondrial permeability transition, Hepatology 33 (3) (2001) 616–626.
[105] P.S. Brookes, Y. Yoon, J.L. Robotham, M.W. Anders, S.-S. Sheu, Calcium, ATP, and
ROS: a mitochondrial love-hate triangle, Am. J. Physiol. 287 (4) (2004)
C817–C833.
[106] M. Kalafateli, C. Konstantakis, K. Thomopoulos, C. Triantos, Impact of muscle
wasting on survival in patients with liver cirrhosis, W. J. Gastroenterol. 21 (24)
(2015) 7357–7361.
[107] E. Krones, M. Wagner, K. Eller, A.R. Rosenkranz, M. Trauner, P. Fickert, Bile acid-
induced cholemic nephropathy, Dig. Dis. 33 (3) (2015) 367–375.