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Bulgarian Journal of Veterinary Medicine, 2018, 21, No 4, 461–469
ISSN 1311-1477; DOI: 10.15547/bjvm.2010
Original article
EFFECTS OF BUTYLATED HYDROXYTOLUENE ON BLOOD
LIVER ENZYMES AND LIVER GLUTATHIONE AND
GLUTATHIONE-DEPENDENT ENZYMES IN RATS
S. MEAN1, Y. DEĞER2 & S. YILDIRIM3
1District and Research Hospital of Van, Brain Research Service, Van, Turkey;
2Department of Biochemistry, Faculty of Veterinary Medicine, Yüzüncü Yıl University,
Van, Turkey; 3Department of Pathology, Faculty of Veterinary Medicine,
Atatürk University, Erzurum, Turkey
Summary
Mean, S., Y. Değer & S. Yildirim, 2018. Effects of butylated hydroxytoluene on blood liver
enzymes and liver glutathione and glutathione-dependent enzymes in rats. Bulg. J. Vet.
Med., 21, No 4, 461–469.
This study was aimed to detect the effect of butylated hydroxytoluene (BHT) on the liver glutathione
level and glutathione-dependent enzyme activities in female Wistar albino rats. BHT was adminis-
tered by oral gavage at a dose of 250 mg/kg (Group I) and 500 mg/kg (Group II) for 28 days, 1000
mg/kg (Group III) and 1500 mg/kg (Group IV) for 4 days. The serum ALT, AST and LDH activities
were measured on an autoanalyzer, and liver gluthathione (GSH), gluthathione peroxidase (GPx),
gluthathione S-transferase (GST), and gluthathione reductase (GR) activities were analysed with
commercial ELISA kits. The ALT activity was significantly higher in Groups III and IV (P<0.05 and
P<0.001, respectively) compared to the control group. Blood AST and LDH activities were signifi-
cantly increased in Group IV (P<0.05). The GSH, GPx, GST and GR in the liver tissue were deter-
mined to be statistically low in Groups II, III and IV (P<0.001) in comparison with control group. In
microscopic examination, BHT caused histopathological changes in the rat liver tissue in Groups II,
III and IV depending on the dose and duration of exposure. It can be concluded that BHT plays a role
in producing liver damage in rats with depressed hepatic antioxidant defense. The hepatotoxic re-
sponse seemed to be dose- and time-dependent.
Key words: antioxidant enzymes, butylated hydroxytoluene, glutathione, histopathology,
liver
INTRODUCTION
Butylated hydroxytoluene (2,6-di-tert-
butyl-p-kresol or 2,6-di-tert-butyl-4-me-
thyl phenol; BHT) is a synthetically pre-
pared phenolic antioxidant, which is main-
ly used in food containing solid and liquid
fat, packing material and cosmetic prod-
ucts. Fat and fat-containing foods are sus-
ceptible to fermentation and other oxida-
tion reactions. BHT acts as radical scav-
enger against lipid oxidation resulting of
Effects of butylated hydroxytoluene on blood liver enzymes and liver glutathione and ….
BJVM, 21, No 4
462
free radical chain reaction and thereby
protects foods from deterioration (Lanigan
& Yamarik, 2002). BHT is generally
recognised as a safe food additive (E
321). The Joint Expert Committee on
Food Additives of the FAO/WHO appro-
ved an acceptable daily intake for man of
0.5 mg BHT/kg body weight. It is rapidly
absorbed from the gastrointestinal tract
and distributed to liver and body fat (Pan-
icker et al., 2014).
BHT is primarily metabolised in the
liver microsomes. Rat liver BHT is me-
tabolised to a variety of sulfate and glu-
curonic acid conjugates eventually ex-
creted in the urine and faeces and to some
extent through bile (Panicker et al., 2014).
Cytochrome P450 mediated metabolism in
liver causes the formation of a reactive
and electrophilic metabolite BHT quinone
methide (BHT-QM; 2,6-di-tert- butyl-
4methylenecyclohexa- 2,5-dienone), an
intermediate metabolite of BHT which
can covalently bind to various cellular
nucleophiles, especially those containing
sulfhydryl groups such as cysteine and
glutathione which consequently leads to
acute hepatotoxicity (Devi et al., 2003).
Some investigations on the mechanism of
BHT-induced plasma transaminase activi-
ty were used to indicate toxicity (Naka-
gawa, 1987).
The endogenous glutathione (GSH),
synthesised mainly in the liver, plays an
important role in the cell defense system.
GSH, being a thiol compound, acts in
cells as an antioxidant. GSH, as a co-
factor of glutathione peroxidase (GPx),
participates in the reduction of peroxides
with concomitant formation of oxidised
glutathione disulfide (GSSG). At normal
physiological conditions GSSG is reduced
to GSH by glutathione reductase (GR) at
the expense of reduced nicotinamide ade-
nine dinucleotide phosphate (NADPH),
thereby forming a redox cycle (Parke &
Piotrowski, 1996). The glutathione
peroxidase/glutathione reductase redox
cycle is responsible for the maintenance of
proper GSH concentration (Dringer,
2000). GSH is also involved in deto-
xication of many xenobiotics through the
formation of S-conjugates with toxic me-
tabolites in the second phase of biotrans-
formation. GSH forms S-conjugates also
with products of lipid peroxidation. The
reaction of S-conjugation can be sig-
nificantly accelerated by glutathione S-
transferase (GST) (Jurczuk et al., 2006).
Butylated hydroxytoluene has been
subjected to extensive toxicological stu-
dies in various animals. The LD50 value of
BHT in rats when administered orally is
>2930 mg/kg. At high doses it is known to
cause haemorrhagic death because of the
inhibition of hepatic prothrombin syn-
thesis. BHT has been shown to induce
reversible mixed function oxidases and
liver enlargement in rats and peliosis,
hepatocellular vacuolation, degeneration
and necrosis in the liver of mice and
found to increase the mitotic activity of
liver cells in rats (Devi et al., 2003).
BHT is used to treat herpes in humans
and is found to be effective against a wide
variety of lipid coated viruses like
Newcastle disease virus. BHT can act
both as promoter and antipromotor of
carcinogenesis (Panicker et al., 2014).
Despite the antioxidant and antiviral ef-
fects, many questions about the use of
BHT remain unanswered. Therefore, the
present study was carried out to evaluate
the toxic effects of BHT in rat liver.
MATERIALS AND METHODS
Animals
Fourty seven female Wistar Albino rats
(200–250 g on average) were used in the
S. Mean, Y. Değer & S. Yildirim
BJVM, 21, No 4 463
study. They were housed in the cages on a
12 h light-dark cycle at 22 ± 2 °C. They
were maintained on food pellets and
drinking water ad libitum. The study was
approved by the Animal Use Ethics
Commision (31.03.2014) of Yüzüncü Yıl
Universty.
Experimental design
The rats were randomly divided into five
groups as control (n=7) and BHT groups
(n=10). The rats in the control group were
given only corn oil.
BHT (Sigma-B1378, E321) was dis-
solved in corn oil and administered by
oral gavage to the different treatment
groups as followed: Group I (250 mg/kg
for 28 days), Group II (500 mg/kg for 28
days), Group III (1000 mg/kg for 4 days)
and Group IV (1500 mg/kg for 4 days)
(Powell et al.,1986).
Sample collection and analysis
At the end of the trial period, intraperi-
toneal 75 mg/kg ketamine HCI was in-
jected, and the blood was collected by
heart puncture. Then, the collected blood
was centrifuged at +4 °C, 3000 rpm for 5
min to obtain serum. ALT, AST and LDH
activities in the extracted serum were
measured on an autoanalyser (Roche/Hi-
tachi) using commercial kits.
The rats were immediately sacrificed
by bloodless method under ketamine
anaesthesia, and the liver was excised.
Samples of liver tissue were homogenised
in cold 0.1 mM phosphate buffer (pH
7.4), and centrifuged at 10,000 rpm at
4 °C for 15 min. The GSH level and GPx,
GR, GST activities in the collected super-
natants were measured by ELISA (Zenyth
200 rt) using commercial kits (Cayman
Chemical Company, Ann Arbor, MI,
2014).
Histopathogical analysis
For histopathological evaluation, the liver
tissue samples were fixed in 10% formalin
solution for 48 h, and then washed in tap
water for 8 h. During the routine tissue
control stage, they were treated with alco-
hol (70°, 80°, 90°, 96°, 100°) and xylene,
embedded in paraffin. Slides of 4 m
thickness were cut from each block,
stained with haematoxylin-eosin (H&E),
investigated under light microscope and
the relevant areas were photographed
(Luna, 1968; Taylor & Cote, 1994).
Statistical analysis
The Kolmogorov-Smirnov test was used
to analyse the normal distribution of con-
tinuous data. For the variables showing
normal distribution, one way analysis of
variation (one-way ANOVA) was used for
the comparison of the groups. The level of
significance was set up to P<0.05,
P<0.001. All data were analysed by using
SPSS 20.0 software.
RESULTS
Biochemical results
Blood ALT activity was significantly
increased in in Groups III and IV (P<0.05
and P<0.001, respectively) with regard to
the control group, while no statistically
significant change was observed between
the other two groups and the control
group. The AST and LDH activities were
found to be significantly increased in
Group IV (P<0.05) compared to all other
groups (Table 1). The liver tissue GSH
level and GPx, GST, GR activities were
significantly decreased in Groups II, III
and IV when compared to the control
group (P<0.001), and no significant dif-
ference was detected between Group I
and the control group (Table 2).
Effects of butylated hydroxytoluene on blood liver enzymes and liver glutathione and ….
BJVM, 21, No 4
464
Histopathological results
The liver tissue was normal in the control
group (Fig. 1A) and Group I (Fig. 1B);
hepatic congestion and mild degeneration
were detected in central hepatocytes in
Group II (Fig. 1C); congestion, degene-
ration, many necrotic fields and haemor-
rhagic foci in central hepatocytes were
observed in Group III (Fig. 1D). Around
the necrotic foci and in the portal region
in Group IV (Fig. 1E,F), many necrotic,
haemorrhagic fields in the central region
and lympho-plasmocytic cell infiltration
were detected. Although the histopatho-
logical findings in the Groups III and IV
were similar, necrotic haemorrhagic chan-
ges in Group IV were more severe.
Table 1. Effect of butylated hydroxytoluene on the activities of ALT, AST and LDH in rat serum.
Values are presented as mean ±SD (n=7 for controls, n=10 for all treatment groups)
Groups ALT (U/L) AST (U/L) LDH (U/L)
Control 36.62 ± 2.10a 126.18 ± 13.79a 1041.57 ± 150.48a
Group I
250 mg/kg; 28 days 39.12 ± 2.14a 160.55 ± 12.24ab 1156.57 ± 185.79ab
Group II
500 mg/kg; 28 days 61.47 ± 6.70ab 176.95 ± 19.49ab 1212.28 ± 107.87ab
Group III
1000 mg/kg; 4 days 82.62 ± 3.77bc* 218.20 ± 11.80ab 1221.00 ± 220.09ab
Group IV
1500 mg/kg; 4 days 98.51 ± 13.84c** 306.24 ± 90.74b* 2280.85 ± 543.78b*
The difference among the group averages with different letters in the same column is statistically
significant, * P<0.05, ** P<0.001.
Table 2. Effect of butylated hydroxytoluene on the level of rat liver gluthathione (GSH),
gluthathione peroxidase (GPx), gluthathione S-transferase (GST), and gluthathione reductase (GR).
Values are presented as mean ±SD (n=7 for controls, n=10 for all treatment groups)
Groups GSH
(nmol/min/mg)
GPx
(nmol/min/mg)
GST
(nmol/min/mg)
GR
(nmol/min/mg)
Control 4.43 ± 0.04a 210.00 ± 3.40a 289.85 ± 2.63a 198.00 ± 1.91a
Group I
250 mg/kg; 28 days 4.27 ± 0.03a 202.71 ± 1.86ab 273.71 ± 6.78ab 190.42 ± 2.51a
Group II
500 mg/kg; 28 days 1.72 ± 0.06b** 193.85 ± 2.14b** 260.85 ± 6.13b** 178.14±1.95b**
Group III
1000 mg/kg; 4 days 1.01 ± 0.05c** 159.28 ± 1.12c** 197.57 ± 4.05c** 173.14±1.69b**
Group IV
1500 mg/kg; 4 days 0.45 ± 0.01d** 81.57 ± 2.47d** 143.00 ± 6.06d** 130.71±1.92c**
The difference among the group averages with different letters in the same column is statistically
significant, * P<0.05, ** P<0.001.
S. Mean, Y. Değer & S. Yildirim
BJVM, 21, No 4 465
Fig. 1. Histopathological structure of the liver tissue of rats (H&E staining). A. Control group (bar:
20 µm). B. Group I. The liver tissue structure is normal (bar: 20 µm). C. Group II. Hepatic conges-
tion in the liver tissue and degeneration in the hepatocytes (bar: 20 µm). D. Group III. Congestion in
the liver tissue, degeneration in the hepatocytes (black arrows), a number of necrotic areas and hae-
morrhagic foci (white arrows), (bar: 20 µm). E. Group IV. Severe necrotic, haemorrhagic areas in the
liver tissue and lymphocytic cell infiltration around these necrotic foci and in the portal area (bar:
50 µm). F. Group IV. Severe necrotic, haemorrhagic areas in the liver tissue (bar: 20 µm).
Effects of butylated hydroxytoluene on blood liver enzymes and liver glutathione and ….
BJVM, 21, No 4
466
DISCUSSION
Toxicological studies with BHT supple-
mentation to animal feed found that ad-
verse effects were dose-dependent. BHT
amounts in excess of 526 mg/kg/day re-
sulted in pleural and peritoneal haemor-
rhage in laboratory rats (Takahashi & Hi-
raga, 1978) A human consumes approxi-
mately 0.1 mg/kg/day of BHT. Research
shows that 500 times (50 mg/kg/day) this
amount yields no deleterious effects (Bra-
nen, 1975).
AST, ALT and LDH are intra-cellular
enzymes that play a role in amino acid and
carbohydrate metabolism. These enzymes
are present in high concentrations in the
liver, muscle and brain. During liver dam-
age, transport functions of hepatocytes are
impaired and serum enzyme activity in-
creases due to leakage from the plasma
membrane. Elevated activities of AST,
ALT and LDH in the serum indicate liver
necrosis or disease (Jang et al., 1999; Lin
et al., 2007).
We observed that serum AST, ALT
and LDH activities increased in all groups
following BHT administration. The in-
crease in AST, ALT and LDH activities
were statistically significant in Groups III
and IV. The increase in liver enzyme
activities occurred in a dose dependent
manner. The present findings correlate
with the reports of many studies using
different doses of BHT (Nakagawa et al.,
1984; Nakagawa, 1987; Ip & Ko, 1996;
Devi et al., 2003; Farag et al., 2006; El-
Anany & Ali, 2013; Panicker et al.,
2014). On the other hand, some studies
reported that none of tested BHT doses
led to difference in serum transaminase
activities (Jaeschke & Wendel, 1985;
1986; Jang et al., 1999). Yamamoto et al.
(1995), also did not observe any differ-
ence in AST and ALT activities in the
blood when rats were fed a diet containing
0.2 % BHT.
The liver has many defense and adap-
tation mechanisms against oxidants and
xenobiotics. It has known that GST ca-
talyses the conjugation of the biological
nucleophile GSH with many electrophilic
compounds. That is to say, GSH and GST
play an important role in the detoxi-
fication of xenobiotic compound contain-
ing the carcinogen (Nakagawa et al.,
1981). Maintenance of mitochondrial
GSH redox is of great importance in order
to prevent chemical hepatic injury as the
decrease in mitochondrial GSH-related
protein thiols is critical for cell viability
(Ip & Ko, 1996). The activated metabolite
of BHT formed by the microsomal
monooxygenase system is bound to the
thiol compounds (Mizutani et al., 1987;
Witschi et al., 1989; Devi et al., 2003).
The induction threshold of the glutathione
conjugation system in liver is greater than
or equal to 100 mg/kg for BHT (Jaeschke
& Wendel, 1986).
It has been reported that oral admini-
stration of 500 mg/kg of BHT (Nakagawa
et al., 1981) and dietary intake of 5%
BHT (Cha & Heine, 1982) elevated the
total level of GSH and the activity of
GST, but has not effect on GPx activity
(Nakagawa et al., 1981). It was observed
that liver GSH level was decreased and
did not alter GST activity after the appli-
cation of BHT (Nakagawa et al., 1984).
Awasthi et al. (1983) reported that 0.4
w/w BHT feeding increased liver GST
activity and did not alter GPx activity
In a study investigating the effect of
adding BHT in two different rodent spe-
cies, the liver GR activity increased
significantly only in rats and GST activity
– in both mice and rats (Jaeschke &
Wendel, 1985). Also, other studies repor-
ted that the addition of 4000 and 1000
S. Mean, Y. Değer & S. Yildirim
BJVM, 21, No 4 467
ppm BHT significantly enhanced GST
activity of turkeys’ liver (Klein et al.,
2002; Coulombe et al., 2005). It was re-
ported that 3 mmol/kg BHT application
decreased liver GSH level and increased
GR activity (Ip & Ko, 1996). The liver
GPx and GST activities were increased
after dietary supplementation of 0.05%
and 0.5% BHT (Jang et al., 1999). The
liver GPx activity decreased following
application of BHT at 1000 mg/kg dosage
(Lin et al., 2007).
In the our study, twenty eight days
after the administration of 250 mg/kg of
BHT, no noteworthy change was observed
in GSH level and the GPx, GST and GR
activities in rat liver. The lack of agree-
ment among various studies including our
results with respect to antioxidant parame-
ters may be likely due to differences in
dosage, age, periods of experiment, even
animal strain and gender (Egaas et al.,
1995). However, twenty eight or four days
after the administration of 500, 1000 and
1500 mg/kg of BHT the GSH level and
GPx, GST, GR activities significantly
decreased. The decreased liver activity of
GPx due to BHT administration might
result from the involvement of this
enzyme in the dismutation of peroxides
generated during BHT biotransformation.
Because GSH is utilised in the GPx-
mediated reactions, an enhanced intensity
of these reactions might also be a cause of
decrease in GSH concentration in the liver
of the BHT treated rats. In a reaction
catalysed by GPx, an oxidised form of
GSH: GSSG is formed. Under the influen-
ce of GR and reduced nicotinamide ade-
nine dinucleotide phosphate (NADPH),
GSSG is reduced to GSH (Moniuszko-
Jakoniuk et al., 2007). The decrease in
liver GR activity in rats treated with BHT
might result from this enzyme utilisation
for GSSG reduction or from NADPH
deficiency. GST catalyses reactions of
toxic substances conjugation with GSH
(Jurczuk et al., 2006). The decreased GST
activity in the liver of rats exposed to
BHT alone suggests that the reduction of
GSH concentration in this organ might
result from GSH utilisation in antioxi-
dative reactions. These liver biochemical
alterations were accompanied by amelio-
ration of histopathological degenerative
changes seen in liver of BHT administred
rats.
Sevaral studies indicated that BHT
caused liver damage (Nakagawa et al.,
1981; Lanigan & Yamarik, 2002). The
hydropic degeneration (Klein et al.,
2003), vacuolar degeneration and hepato-
cellular degeneration including fuzzy
swelling (El-Anany & Ali, 2013) and ne-
crosis (Powell et al., 1986) were reported.
Contrary to the above mentioned studies,
Jang et al., (1999) and Lin et al., (2007)
showed that BHT caused no histo-
pathological changes in liver. In this
study, the livers of rats in the Group I had
normal appearance. The histopathological
degenerative changes in liver tissue were
observed as congestion, degeneration,
necrosis, haemorrhages in the other BHT-
treated groups. The severety of these
changes increased as a function of applied
dose of BHT.
As a result, it can be said that BHT
plays a role in producing liver damage in
rats with depressed hepatic antioxidants.
The hepatotoxic response seemed to be
dose- and time- dependent.
ACKNOWLEDGEMENTS
This study was supported by the Yüzüncü Yıl
University Scientific Research Projects Fund
and registered with project No: 2014-SBE-
YL137.
Effects of butylated hydroxytoluene on blood liver enzymes and liver glutathione and ….
BJVM, 21, No 4
468
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Paper received 03.01.2017; accepted for
publication 07.04.2017
Correspondence:
Sinan Mean
District and Research Hospital of Van,
Brain Research Service,
Van, Turkey
e-mail: sinanmean@hotmail.com