Download IJEB 48(4) 373-377

Indian Journal of Experimental Biology
Vol. 48, April 2010, pp. 373-377
Effect of antioxidant vitamins A, C, E and their analogues on azo-dye binding
protein in liver of rats treated with p-dimethylaminoazobenzene
A Antony Joseph Velanganni*
Department of Biochemistry, J.J. College of Arts and Science, Pudukkottai 622 422, India
and
C Balasundaram
Department of Animal Science, Bharathidasan University, Tiruchirappalli 620 024, India
Received 21 July 2009; revised 19 November 2009
p-Dimethylaminoazobenzene (DAB) is an azo-dye and known to cause liver tumour in rats. Azo-dye binding protein is a
specific cytosolic protein involved in the translocation of azo-dye carcinogen metabolites from liver cytoplasm into the
nucleus. Administration of vitamin A (40,000 and 50,000 IU), L-ascorbic acid (500 and 1,000 mg) and vitamin E succinate
(200–500 mg) reduced the amount of azo-dye binding protein in liver of rats treated with DAB. Supplementation of high
doses of vitamin A acetate, vitamin A palmitate, sodium ascorbate, ascorbyl palmitate and vitamin E acetate had no effect
on the quantity of azo-dye binding protein in liver. When the vitamin mixture was given, the level of azo-dye binding
protein decreased in the liver at all the studied doses, which may be due to their synergistic effect.
Keywords: Antioxidant vitamins A, C and E, Azo-dye binding protein, p-Dimethylaminoazobenzene
The pioneering work of Miller and Miller1 established
that aminoazo-dye, a known hepato carcinogen, binds
to liver protein in rats; further in vivo studies on
various animal models proved that the chosen
carcinogens bound to cellular protein in tissues
rendered them tissues susceptible to carcinogenic
action. Azo-dye binding protein is a specific cytosolic
protein involved in the translocation of azo-dye
carcinogen metabolites from liver cytoplasm into the
nucleus. The binding of azo-dye metabolites with
nuclear macromolecule necessitate further prior
processing that occur in the nucleus. The azo-dye
binding protein is found in all the highlydifferentiated hepatomas. It is not found in poorly
differentiated hepatomas, anaplastic carcinomas and
adenocarcinomas2. Thus the presence of azo-dye
binding protein in tumour indicates the degree of cell
differentiation. The hepatic binding protein is
involved in the translocation of azo-dye carcinogen
metabolites to the nucleus from liver cytoplasm.
Interaction of azo-dye metabolites with nuclear
macromolecules facilitates carcinogenesis that occurs
in the nucleus3. Covalent binding of azo-dye
metabolites to DNA occurs when the azo-dye
metabolites are incubated with liver nuclei and not
with isolated liver DNA. This is due to the specific
soluble protein that controls the translocation form
cytosol to the nucleus4.
The amount of protein-bound azo-dye is three
times more in mice after 2-3 weeks of DAB feeding;
in hamster, azo-dye binding to proteins increased
gradually up to 6 weeks, and then remained constant5.
Sub-fractionation of liver homogenates on
administration of azo-dyes shows that the dye is
bound principally with the microsomal fraction
(insoluble) and the soluble supernatant6-8. The soluble
fraction subjected to zone electrophoresis has shown
three protein fractions, namely a major basic protein
fraction and two minor mono-acidic protein
fractions9. The present study is concerned with the
soluble proteins that bind specifically to the azo-dye
so as to evaluate the role of antioxidant vitamins A, C,
E and their analogues in preventing the binding of
azo-dyes in DAB-treated rats.
——————
*Correspondent author
Telephone: +91-04322-260103
Fax: +91-04322-260103
E-mail: [email protected]
Materials and Methods
Animals—Male albino rats of Wistar strain (170 ±
20 g), reared and maintained in the animal house of
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INDIAN J. EXP BIOL., APRIL 2010
the Department of Biochemistry (under the
supervision
of
Institutional
Animal
Ethics
Committee), J. J. College of Arts and Science,
Pudukkottai, were used.
DAB and vitamins—The hepato carcinogen
p-dimethylaminoazobenzene (p-DAB; CAS No.
60-11-7) was purchased from Sigma Chemical Co.,
St. Louis, USA. Vitamin A, C and E and their
analogues vitamin A acetate, vitamin A palmitate,
sodium ascorbate, ascorbyl palmitate, vitamin E
acetate and vitamin E succinate with research grade
purity were used for the present work.
Individual vitamin treatment —Rats (275) were
divided into 3 groups. First group (n = 25) served as
normal control. The second group (n = 25) received
0.06% of DAB2 for 4 months followed by basal diet
alone for an additional 2 months and served as DAB
control. The third group consisting of the remaining
225 rats was further subdivided into subgroups A, C
and E (n = 75) based on the vitamins used. The
subgroups A, C and E were further divided into
subsets of A1, A2 and A3, C1, C2 and C3 and E1, E2
and E3 respectively consisting of 25 rats each.
Finally, all the subsets were further divided into
5 sub-subsets (Sub-subsets I to V) of 5 rats each.
All the rats were acclimatized to laboratory
conditions for one week and fed ad libitum with a
basal diet [containing 660 g glucose, 180 g milk
casein, 40 g salt mixture10, 100 g corn oil, 20 g cod
liver oil, 1.5 g choline chloride, 50 mg vitamin K3,
20 mg riboflavin, 20 mg thiamine, 20 mg pyridoxine,
60 mg calcium pantothenate, 50 mg nicotinamide,
1.8 mg folic acid, 0.6 mg biotin, 100 mg inositol,
50 mg p-aminobenzoate, 40 μg cyanocobalamin per
kg diet11. Animals of second and third groups were
fed with 0.06% DAB supplemented diet2. Control rats
were continued to be fed on the basal diet for
6 months. The third group was administered chosen
doses of vitamins (per kg body wt) based on a
previous study12, once in a week by gavage route
{A1: DAB + vit. A (10,000-50,000 IU), A2: DAB +
vit. A acetate (10,000-50,000 IU), A3: DAB + vit. A
palmitate (10,000-50,000 IU), C1: DAB + L-ascorbic
acid (75-1000 mg), C2: DAB + sodium ascorbate
(75-1000 mg), C3: DAB + ascorbyl palmitate
(75-1000 mg) and E1: DAB + vit. E (50-500 mg), E2:
DAB + vit. E acetate (50-500 mg) and E3: DAB + vit.
E succinate (50-500 mg)} for a period of 4 months.
After this period, the vitamin enriched basal diet was
offered for an additional period of 2 months.
Multiple vitamin treatment—Multiple vitamin
treatment study was performed with mixture13-15 of
vitamin A, L-ascorbic acid and vitamin E succinate.
For this study, 35 rats were divided into 7 groups of
5 rats each for normal, DAB, DAB + vitamin A
(10,000 IU) + vitamin C (75 mg) + vitamin E
(50 mg), DAB + vitamin A (20,000 IU) + vitamin C
(150 mg) + vitamin E (100 mg), DAB + vitamin A
(30,000 IU) + vitamin C (250 mg) + vitamin E
(200 mg), DAB + vitamin A (40,000 IU) + vitamin C
(500 mg) + vitamin E (400 mg), DAB + vitamin A
(50,000 IU) + vitamin C (1000 mg) + vitamin E
(500 mg). The mixture of vitamins was given to all
the rats by gavage route once in a week for 6 months.
Isolation of basic azo-dye binding protein—
Animals administered with 0.06% of DAB were
sacrificed with ether after 6 months and used for the
isolation of basic azo-dye binding protein.
Immediately, excised livers of rats were perfused with
20 ml cold 0.25 M sucrose. All the sub cellular
particles were removed by centrifuging at 30,000 rpm
(65,390 g) for 120 min. The resulting soluble cell
supernatant was used to isolate azo-dye binding
protein as per Ketterer et al16.
Amino acid analysis—Azo-dye binding protein
was hydrolysed in constant boiling HCl at 105oC for
16 h and hydrolysate was analysed by
chromatography on Whatman no.1 filter paper with
the solvent butanol-acetic acid-water (12:3:5).
Ninhydrin reagent (100 mg/ml of acetone) was used
to spot the amino acids and the amino acids present in
the sample were then identified by comparing the Rf
values with that of the authentic amino acids,
co-chromatographed.
Determination of molecular weight by SDSPAGE—Sodium dodecyl sulphate-poly acrylamide
gel electrophoresis (SDS-PAGE) was performed with
10% poly acrylamide gel following the procedure of
Laemmli17.
Solution of basic azo-dye binding protein in PBS
(10mg/2 ml) was prepared and applied in SDS-PAGE.
The molecular weight of azo-dye binding protein was
determined using SDS-PAGE in which protein
marker of known molecular weight and the azo-dye
binding protein fraction were run simultaneously.
Sample containing 100 µg protein was mixed with
an equal volume of sample buffer and loaded in the
wells of stacking gel. Electrophoresis was performed
at 50 V initially and then at 90 V until bromophenol
blue moved nearer to the bottom of the gel. The
VELANGANNI & BALASUNDARAM: ANTIOXIDANT VITAMINS ETC. & AZO-DYEBINDING PROTEIN
proteins present in the gel were stained for 4 h, using
0.2% Coomassie brilliant blue R-250 (Sigma) in 50%
methanol and 7% acetic acid. The gel was later destained using destaining solution (7% acetic acid and
30% methanol) until the background was clear.
Estimation of bound azo-dye—The liver of rats
administered 0.06% DAB were treated in a blender
with 4 times its weight of distilled water until the
tissue became a frothy pulp. The liver pulp was boiled
for 5 min in acetate buffer and adjusted to pH 5 with
acetic acid (5 ml for buffer /g of liver). After cooling,
the coagulated protein was centrifuged and washed
with buffer and then with 95% ethanol until no further
colour could be extracted. Any remaining unbound
azo-dye and lipid material was removed from the
protein by extraction in a soxhlet apparatus with 95%
alcohol for 48 h at 60oC. The protein mass was broken
up and the ethanol was allowed to evaporate from the
powder, which was then dried over P2O5 in a vacuum
desicator2,18.
Chemical assay—The liver protein powder was
hydrolysed to release bound azo-dye and the released
azo-dye was extracted with pentan-1-ol. The extracts
were poured into 5 volumes of light petroleum and
10 ml of a freshly prepared mixture of concentratedHCl and acetone (2:1, v/v) was added. The mixture
was shaken and allowed to stand for 30 min. The
lower layer containing the dye was transferred to a
measuring cylinder and its extinction was measured at
520 nm in a spectrophotometer. The colour intensity
became maximal after 30 min and remained constant
for several hours. The extraction was corrected to a
final volume of 10 ml and the bound azo-dye was
expressed in arbitrary units of E520/100 mg of
protein18. Statistical analysis of the data was
performed with Student’s ‘t’ test.
Results
The amino acid analysis of hydrolysates of the
basic azo-dye binding protein shows the presence of
following 17 amino acids: alanine (Ala), arginine
(Arg), aspartic acid (Asp), cysteine (Cys), glutamic
acid (Glu), glycine (Gly), histidine (His), isoleucine
(Ile), leucine (Leu), lysine (Lys), methionine (Met),
phenylalanine (Phe), proline (Pro), serine (Ser),
tyrosine (Tyr), threonine (Thr) and valine (Val).
Amino acids such as Asn, Gln and Trp were not found
in azo-dye binding protein (since tryptophan would
have been destroyed during acid hydrolysis16, it is not
been included in the list). The amino acid analysis of
hydrolysates of basic azo-dye binding peptide after
375
being digested with protease contains Ala, Asp, Glu,
Gly, Ser, Thr, other amino acids were destroyed
during digestion with protease. Isolated azo-dye
binding protein had a molecular weight of 45 KDa as
evident from the SDS-PAGE study (Fig. 1).
The amount of bound azo-dye in DAB-treated rats
was found to be 0.41 arbitrary units/mg protein18.
However, administration of vitamin A (40,000 and
50,000 IU), L-ascorbic acid (500 and 1,000 mg) and
vitamin E succinate (200–500 mg) reduced the
amount of azo-dye binding protein in liver (Fig. 2 to
4). Individual supplementation of high doses of
vitamin A acetate, vitamin A palmitate, sodium
ascorbate, ascorbyl palmitate and vitamin E acetate
have no effect on quantity of azo-dye binding protein
in liver (Fig. 5). When the vitamin mixture was given,
the level of azo-dye binding protein decreased in the
liver at all the studied doses (Fig. 6).
Discussion
Amino acid analysis of azo-dye binding peptide
revealed the presence of Ala, Asp, Glu, Gly, Ser and
Thr. The earlier studies on azo-dye bound peptide
revealed that it contained Pro, Leu/Ile, Val and Gly as
major components and Glu, Phe, Ser, Ala and Asp as
minor components19. Terayama and Takeuchi20
isolated a fraction on hydrolysis of azo-dye binding
protein which was found to have Phe, Ser, Gly, Pro,
Val, Glu and Asp. The discrepancies between the
earlier results and the present study may be due to the
fact that more than one azo-dye binding protein exists
and the possibility of the azo-dye to bind with
different types of amino acid sequences in each
protein gives several different azo-dye bound
peptides16.
Fig. 1 — SDS-Page profile of purified basic azo-dye-binding
protein [Lane 1: marker proteins, Lane 2: protein bound azo-dye]
376
INDIAN J. EXP BIOL., APRIL 2010
Figs 2-6 — Protein bound azo-dye in liver [2—a: DAB, b: DAB + vit. A (10,000 IU), c: DAB + vit. A (20,000 IU), d: DAB + vit. A
(30,000 IU), e: DAB + vit. A (40,000 IU), f: DAB + vit. A (50,000 IU). 3—a: DAB, b: DAB + vit. C (75 mg), c: DAB + vit. C (150 mg),
d: DAB + vit. C (250 mg), e: DAB + vit. C (500 mg), f: DAB + vit. C (1,000 mg). 4—a: DAB, b: DAB + vit. E (50 mg), c: DAB + vit. E
(100 mg), d: DAB + vit. E (200 mg), e: DAB + vit. E (400 mg), f: DAB + vit. E (500 mg). 5— a: DAB, b: DAB + vit. A (50,000), c:
DAB + vit. A acetate (50,000), d: DAB + vit. A palmitate (50,000), e: L-ascorbic acid (1,000), f: DAB + sodium ascorbate (1,000), g:
DAB + ascorbyl palmitate (1,000), h: DAB + vit. E (500), i: DAB + vit. E acetate (500), j: DAB + vit. E succinate (500). 6— a: DAB, b:
vit. A (10,000 IU) + vit. C (75 mg) + vit. E (50 mg), c: vit. A (20,000 IU) + vit. C (150 mg) + vit. E (100 mg), d: vit. A (30,000 IU) + vit.
C (250 mg) + vit. E (200 mg), e: vit. A (40,000 IU) + vit. C (500 mg) + vit. E (400 mg), f: vit. A (50,000 IU) + vit.C (1,000 mg) + vit. E
(500 mg).*P<0.05 vs DAB]
The diet rich in riboflavin increased the induction
period of liver tumours by azo-dyes21. Similary, rats
fed with both riboflavin and cupric oxyacetate
hexahydrate delayed the formation of liver tumour
induced by azo-dyes18. The results of the present
study also indicate that supplementation of vitamin
A, C and E offer protection against DAB by delaying
the binding of the azo-dye. The binding of protein
with azo-dye was lowered in vitamin treated rats.
DAB can be inactivated in the liver by an enzyme
system, azo-dye reductase, that reductively cleaves
the molecule to non-carcinogenic amine moieties and
that the coenzyme required is FAD5,22. The protection
given by vitamins is through the increased enzymatic
destruction of the azo-dye. Dyes were bound with the
proteins from the high-sulfur-protein area or hightyrosine-protein area, depending on the level of dye
dissolution23. The passage of azo-dye binding protein
through an immobilized protease column reduced the
azo-dye
metabolite
translocation
by
65%,
concomitant with the degradation of proteins24.
Translocation from the cytoplasm into nucleus was
not observed with protein-free metabolites and
addition of protein promotes the metabolite
translocation in rat liver24. In the present study,
supplementation of vitamin mixture inhibits the
translocation of azo-dye binding proteins in liver of
rats treated with DAB. In conclusion, antioxidant
vitamin mixture prevents the binding of azo-dye in
the liver by reducing the level of azo-dye binding
protein.
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