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Department of Nutrition, College of Human Ecology and Agricultural Experiment Station, University of Tennessee, Knoxville, TN 37996-1900
2To whom correspondence should be addressed. E-mail: dsachan{at}utk.edu.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-aminobutyric
acid (GABA) were equally effective. Carnitine appears to interfere with
bioactivation of AFB1 and binding of
AFB1-epoxide to DNA. On the contrary, carnitine enhanced
the binding of AFB1 and its epoxide to microsomal proteins,
plasma proteins and bovine serum albumin. These results indicate that
carnitine diverts AFB1-epoxide away from DNA by promoting
binding to proteins. We conclude that modulation of AFB1
binding to proteins and DNA by carnitine alters the carcinogenic and
hepatotoxic potential of AFB1 and poses concerns about the
human AFB1-exposure data based on the
AFB1-albumin adduct concentrations as a biomarker.
KEY WORDS: • aflatoxin B1 • choline • L-carnitine • -aminobutyric acid • rats
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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and carcinogenic
(2)
of all naturally occurring aflatoxins. The effects of
AFB1 are mediated through its metabolites,
AFB1-8.9-epoxide, by covalent binding to cellular
proteins and nucleic acids (3)
.
AFB1-DNA adduct concentrations have been
correlated to incidences of liver cancer in animals (4)
and humans (5)
. Certain amounts of
AFB1-epoxide may be rendered harmless by
conjugation to various endogenous and exogenous compounds such as
glutathione (6)
.
After absorption from the small intestine, AFB1
readily binds to plasma albumin, which serves as the major transporter
of AFB1 in blood (7)
. It has been
postulated that some bioactivation of AFB1 occurs
in the intestinal mucosa (8
,9)
and in blood
(10)
; therefore, AFB1 metabolites
are also bound to albumin. The postabsorption binding of
AFB1 to albumin has been proposed to lessen the
toxicity of AFB1 (11)
.
L-Carnitine is a carrier of acyl groups, particularly the
long-chain fatty acids, across the cellular compartments
(12)
. It was reported recently that carnitine in
combination with coenzyme Q10 offered significant
protection against oxygen radicals induced by mycotoxins including
AFB1 in bacteria (13)
. When a
carnitine-supplemented diet (4 g/kg) was fed to rats for 6 wk
followed by a single oral dose of AFB1 (1 mg/kg),
there was significant reduction in the concentrations of
AFB1 adducts of hepatic DNA and RNA, 6 and
24 h after AFB1 dosing (14)
.
This study left a number of unanswered questions about the effects of
carnitine including the following: is it dose-dependent? Can it be
reproduced by other carnitine-like compounds? Is biotransformation
of AFB1 a prerequisite? Is there alteration in
intracellular partitioning of aflatoxin? We have attempted to answer
these questions using isolated hepatocytes and cell-free systems.
We believe that the results reported here bring us one step closer to
understanding the mechanism by which carnitine reduces
AFB1-DNA adducts.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The Animal Care and Use Committee of the University of Tennessee, Knoxville, approved the research protocol. Male Sprague-Dawley rats (Harlan Sprague Dawley, Indianapolis, IN) weighing 300–350 g were housed individually in suspended stainless steel cages in a cubicle of the animal facility and were given free access to Teklad 22/5 Rodent Diet (W) 8640 (Harlan, Indianapolis, IN) and water. These rats were used for obtaining hepatocytes, liver microsomes and blood plasma for the studies outlined below.
Liver perfusion and isolation of hepatocytes.
The buffers used for preperfusion, collagenase perfusion and incubation
were modified Hank’s balanced salt solutions (HBSS) as described by
Lotlikar et al. (15)
. All buffers were saturated with 95%
O2:5% CO2 gas before use. The perfusion
apparatus and procedures used followed the modified methods of Seglen
(16)
. The hepatocytes liberated by collagenase treatment
were washed, resuspended and tested for viability (90–95%) by trypan
blue exclusion.
Binding of [3H]AFB1 to DNA of hepatocytes.
The incubation of hepatocytes was carried out according to the method
by Lotlikar et al. (15)
with minor modifications. The
incubation mixtures contained 20 x 106 hepatocytes in
4 mL of modified HBSS (pH 7.4) with fatty acid–free bovine serum
albumin (BSA) (5 g/L), various concentrations of
L-carnitine (0–1.5 mmol/L), and
[3H]AFB1 (0.5 µmol/L, Sp.
Act. 68.5 x 106 Bq/µmol) dissolved in
dimethyl sulfoxide (DMSO) (final concentration of 20 mL DMSO/L). The
incubations were carried out in triplicate in Erlenmeyer flasks.
Carnitine was preincubated for 15 min before the addition of
[3H]AFB1 and incubated further for 60 min at
37°C. The reaction was stopped by a quick chill of the flask in ice
water. Cellular DNA was extracted according to procedures of
Gross-Bellard et al. (17)
, and the concentration was
determined by the colorimetric method using calf thymus DNA as standard
(18)
. Radioactivity (disintegrations per minute) in 0.5 mL
extract was determined in a liquid scintillation counter (Beckman
Instrument, Irving, CA).
Determination of AFB1 partitioning into hepatocytes.
This determination followed the modified method of Jennings et al.
(19)
. Briefly, after incubation with carnitine and
[3H]AFB1, the hepatocytes were separated from
the incubation medium by centrifugation at 500 x g, washed with incubation buffer without BSA,
homogenized and centrifuged at 600 x g for 10 min
to pellet the nuclei. The free AFB1 was extracted with
chloroform/ethylacetate (1:1) from the postnuclear supernatant, the
nuclear pellet, the incubation medium and washed cells suspension. An
aliquot of each fraction was counted for radioactivity in a liquid
scintillation counter.
Preparation of liver microsomes.
Rats were anesthetized with Metofane (Pitman-Moore, Mundelein, IL); the
portal vein was cannulated and the liver was perfused in situ with 100
mL ice-cold physiologic saline. The perfused liver was removed and
homogenized in 0.154 mol/L KCl buffer containing 0.01 mol/L
KH2PO4 (pH 7.4). The homogenate (250 g/L) was
centrifuged at 10,000 x g for 20 min at 4°C. The
resulting supernatant was centrifuged at 100,000 x g for 60 min at 4°C. The microsomal pellet was
suspended in the glycerol/0.05 mol/L phosphate (1:1) buffer, pH 7.4,
and the concentration of protein in microsomes was determined
(20)
.
[3H]AFB1 binding to calf thymus DNA mediated by microsomal enzymes.
The DNA-binding method of Allameh et al. (21)
as
modified by Hasler et al. (22)
was used in these
experiments. The calf thymus DNA (Sigma Chemical, St. Louis, MO) was
incubated with the [3H]AFB1 as follows:
1) without microsome and carnitine; 2)
without microsome but with carnitine; 3) with microsome
but no carnitine; and 4) with both microsome and
carnitine. The 1-mL incubation mixtures contained 0.1 mol/L phosphate
buffer, microsome equivalent to 1.0 mg protein, 2 mmol/L NADPH, 2
nmol/L [3H]AFB1 dissolved in DMSO, 0.1 mg
calf thymus DNA, 1.2 mmol/L L-carnitine,
L-acetylcarnitine, choline, -aminobutyric acid (GABA) or
glycine (pH 7.0), and double-deionized water (DDW). The samples
were incubated in triplicate at 37°C for 30 min and replicated five
times using microsomes from five rats.
At the end of the incubation time, 1 volume of 5 mol/L NaCl was added to produce a mixture containing 1 mol/L NaCl followed by 2 mL of chloroform/isoamyl alcohol (24:1, v/v) and 0.9 mg of calf thymus DNA as a carrier. The tubes were shaken and centrifuged at 10,000 x g for 10 min. The rest of the procedure for DNA extraction and determination of concentration or radioactivity was as described earlier.
[3H]AFB1 binding to microsomes.
The incubation medium and procedure were similar to those used in the AFB1 binding to BSA described above. There were two groups in this experiment, control and carnitine. The medium contained 0.1 mol/L phosphate buffer, microsomes equivalent to 1.0 mg protein, 1.2 mmol/L carnitine (pH 7.0) for the carnitine group, 2 mol/L NADPH, 2 nmol [3H]AFB1 dissolved in DMSO and DDW to a total volume of 1.0 mL. After the incubation, the mixtures were quickly chilled in ice water and microsomes isolated by centrifugation at 100,000 x g for 1 h at 4°C. The supernatant was removed and microsomes were suspended in 1 mL KOH (1 mol/L) and extracted with 2 mL of chloroform/ethylacetate (1:1, v/v) to remove free AFB1. The aqueous top layer was transferred to new tubes and AFB1 extraction repeated. The protein concentrations and radioactivity were determined as described earlier.
[3H]AFB1 binding to BSA.
Commercial fatty acid–free BSA (Sigma Chemical) was incubated with the
[3H]AFB1 as follows: 1)
without microsome or carnitine; 2) without microsome but
with carnitine; 3) with microsome without carnitine; or
4) with both microsome and carnitine. In general, the
incubation medium and conditions were similar to those described above
for the AFB1-DNA adduct formation. After the incubation,
the mixtures were quickly chilled in ice water and then centrifuged at
100,000 x g for 1 h at 4°C to pellet the
microsome. The supernatant was extracted with chloroform/ethylacetate
(1:1, v/v), and centrifuged at 2000 x g at 4°C
for 10 min to remove free AFB1. The aqueous (top layer) was
reextracted and protein concentrations were determined
(20)
. Radioactivity was measured by adding 0.2 mL of the
protein fraction to 5 mL Aquasol-2 (Dupont-NEN Research Products,
Boston, MA), with 0.04 mL of glacial acetic acid.
Noncovalent binding of AFB1 to BSA and plasma proteins measured by membrane ultrafiltration method.
The incubation was done in glass culture tubes. The mixture contained
BSA (0.8 µg) or rat plasma (20 µL
equivalent to 0.8 µg albumin), with or without 1.2
mmol/L carnitine (pH 7.0) and 2 nmol [3H]AFB1
dissolved in DMSO; the total volume with DDW was 300
µL. The separation of bound vs. unbound
[3H]AFB1 to proteins was according to the
modified method of Lipford et al. (23)
. The mixture was
allowed to stand at room temperature (
25°C) for 10 min and then
transferred into the upper chamber of a prewashed Ultrafree-MC
filter unit (Millipore, Bedford, MA). The nominal molecular weight
limit of the Ultrafree-MC regenerated cellulose membrane used was
30,000. The filter unit was then centrifuged at 2000 x g for 20 min. The filtrate was removed, and the
concentrate in the upper chamber was washed once with 400
µL HBSS and centrifuged again at 2000 x g for 30 min. The upper chamber that retained the
protein was transferred into scintillation vials and counted for
radioactivity.
Statistics.
The values are reported as means ± SEM of a minimum of 5 rats. Statistical analysis of the data was performed using the SAS statistical program (SAS version 6.11, SAS Institute, Cary, NC). The differences between two groups were analyzed by Student’s t test. For comparison of several groups, the general linear model procedures or two-way crossed ANOVA was used when appropriate. When significant, Duncan’s Multiple Range test or Contrast statement was used to compare difference between means. The regression procedure of SAS was used for regression analysis, and the regression curve was plotted using Microsoft Excel 97 software (Redmond, WA). The level of significance was set at P < 0.05.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). Regression analysis showed that as the concentrations of carnitine
increased from 0 to 1500 µmol/L, the concentrations of
AFB1-DNA adducts decreased linearly in the
isolated rat hepatocytes (r = -0.681; P
= 0.0002). The partitioning of AFB1 into the
incubation medium and hepatocytes or into the nuclear and extranuclear
(postnuclear supernatant) fractions of hepatocytes was not
significantly affected by carnitine (Fig. 2
).
|
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. There was very little AFB1 radioactivity in the
calf thymus DNA in the absence of microsomes in the incubation mixture,
and carnitine had no effect on the formation of adducts in the absence
of microsomes. On the other hand, in the presence of microsomes, there
was significantly higher AFB1-DNA adduct
formation which was significantly lower in the presence of carnitine.
The specificity of the carnitine effect on
AFB1-DNA adduct formation in the cell
free–system with liver microsomes was tested using a few
carnitine-like substances (Figure 4
). The concentrations of AFB1-DNA adducts (pmol/mg
DNA) in the presence of carnitine (170 ± 12), acetylcarnitine
(154 ± 8) and GABA (164 ± 9) were significantly lower
compared with the control (216 ± 13), choline (203 ± 11)
and glycine (212 ± 14) treatments, but were not different from
one another.
|
|
. There was some binding of AFB1 to BSA (13.4
pmol/mg BSA in group 1, and 16.2 pmol/mg BSA in group 2). These are
noncovalent bindings because no microsomes were added to the incubation
mixtures to produce the AFB1-epoxide that binds
covalently to macromolecules. Carnitine had no significant effect
(contrast P-value = 0.171) on the noncovalent binding
of AFB1 to BSA (group 1 vs. group 2). In the
presence of microsomes, however, there were significantly more
AFB1-BSA adducts formed and carnitine enhanced
formation (Fig. 5
, groups 3 and 4). The BSA adducts formed were 4.8
fold higher in group 3 than in group 1, and 4.5 fold higher in
group 4 than in group 2. The concentrations of
AFB1-BSA adducts in the presence of microsomes
were higher primarily because of the covalent and some noncovalent
binding. The average amount of covalent AFB1-BSA
bindings in group 3 was 64.1 pmol/mg BSA; in group 4, it was 72.7
pmol/mg BSA (calculated by subtracting the adduct concentrations of
group 1 from group 3, and group 2 from 4). Therefore, covalent bindings
constituted the higher portion of the AFB1-BSA
adduct shown in group 3 and 4. The presence of carnitine in the
incubations containing microsomes significantly increased (P
= 0.0005) the AFB1-BSA adduct formation
(group 4 vs. group 3), clearly indicating that carnitine promoted
covalent binding of AFB1-epoxide to BSA.
|
. There was some binding of AFB1 to the microsomal
component, and carnitine significantly increased (P = 0.007) this binding. This AFB1 binding to
microsomal protein was about two times the binding of
AFB1 to BSA (Fig. 5
; groups 1 and 2). In
microsomal systems, it is not easy to separate covalent and noncovalent
binding, but the latter is most likely minimal.
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and
B
, respectively. The carnitine group had significantly greater binding of
AFB1 to plasma proteins than the control group
(P = 0.022). The AFB1-BSA adduct
formation was also greater in the carnitine group than in the control
group (P = 0.018). Compared with rat plasma, more
protein adducts were formed when the protein was BSA. This is most
likely related to the purity of BSA protein because plasma has
components other than albumin.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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was demonstrated in
the isolated hepatocytes from a group of rats fed a nonpurified diet.
As shown in Figure 1
, in vitro addition of carnitine to the freshly
isolated hepatocytes decreased AFB1-DNA adduct
formation in a dose-dependent manner with a strong negative
correlation. The attenuation of AFB1-DNA adducts
could not have been due to the effect of carnitine on
AFB1 uptake by hepatocytes or the differential
partitioning of AFB1 among the nuclear and
cytosolic fractions (Fig. 2)
. The formation of
AFB1-RNA adducts in the isolated hepatocytes was
not significantly affected by carnitine under these conditions (data
not shown); therefore, AFB1-RNA adducts were no
longer pursued.
In the intact rats (6)
, it was difficult to distinguish
whether carnitine was affecting binding of AFB1
or AFB1-epoxide to DNA. The speculation was that
carnitine may have suppressed bioactivation of
AFB1 in a manner similar to that suggested for
crocetin (18)
. The in vitro system provided an opportunity
to evaluate this hypothesis.
The hepatic microsomal enzymes metabolize AFB1 to
AFB1-epoxide which covalently binds to the
electrophilic centers of DNA (6)
. In the cell-free
system, binding of AFB1 to calf-thymus DNA
was not significantly affected by carnitine (Fig. 3)
. However, when
microsomes were added to the cell-free in vitro incubation system
containing purified calf thymus DNA, AFB1-DNA
adducts were formed, and these were significantly decreased in the
presence of carnitine (Fig. 3)
. This indicates that carnitine
interferes with AFB1-bioactivation in a manner
analogous to the inhibition of the formation of oxygen-free
radicals (24
,25)
. The highly reactive nature of epoxides
results in rapid formation of AFB1-DNA adducts,
which have a half-life of only 12 h. It is also possible that
carnitine prevented binding of the epoxide to DNA, resulting in reduced
AFB1-DNA adducts. Carnitine has the quarternary
nitrogen similar to the electrophilic center of guanine of DNA and is
available for electrophilic attack by
AFB1-epoxide. The present data preclude
separation of the effect of carnitine on bioactivation of
AFB1 to AFB1-epoxide and
electrophilic binding of epoxide to the guanine of DNA.
The inhibition of AFB1-DNA adduct formation by
carnitine in the cell-free system was mimicked by acetylcarnitine
and GABA but not by choline and glycine (Fig. 4)
. The important
distinction between inhibitors (carnitine, acetylcarnitine, GABA) and
noninhibitors (choline, glycine) is the carbon chain length because
amino and trimethylamino groups are common to both groups of compounds.
The superior effect of acetylcarnitine on
AFB1-DNA adduct formation is analogous to the
effect of this molecule on the inhibition of ethanol metabolism in
hepatocytes (26)
. Acetylcarnitine competitively inhibited
binding of NAD+ to alcohol dehydrogenase in the
cell-free system (27)
. A variety of acylcarnitines,
including acetylcarnitine, can be produced in microsomal systems
(28)
. The fact that the magnitude of decrease in
AFB1-DNA adducts in the presence of GABA was
equal to that of carnitine is intriguing and requires additional
studies. The common features of carnitine and GABA are the 4-carbon
length, carboxyl group on carbon-1 and amino nitrogen on carbon-4. The
lack of an effect of choline on AFB1-DNA adducts
is analogous to that seen in ethanol metabolism (29)
. The
reports on choline-AFB1 interactions are
conflicting. For example, choline deficiency had no effect on the liver
AFB1-DNA adduct concentration in rats given a
single dose of AFB1; however, multiple doses of
AFB1 markedly elevated
AFB1-DNA adduct formation (30)
. On
the other hand, rats fed a diet marginally deficient in lipotropes
(methionine, choline and folacin) and given a single dose of
AFB1 showed suppression of hepatic
AFB1-DNA adduct formation (31)
.
Recently a choline and methionine–deficient diet was shown to have no
significant effects on serum biochemistry or liver pathology due to a
dose of AFB1 in rats (32)
.
AFB1 and its metabolites, particularly
AFB1-epoxide, are known to bind to plasma
proteins (33)
, and this binding may be altered by the pH
and fatty acid concentrations (34)
. Carnitine
significantly increased the covalent binding of
microsomal-activated AFB1 to BSA, a purified
serum protein (Fig. 5)
. Plasma contains 7–8% proteins, and albumin
constitutes 50–60% of plasma proteins (35)
. Albumin
has been shown to be the main plasma protein that binds
AFB1 and thus serves as the major transporter of
AFB1 in the blood (7)
. Carnitine
also increased the binding of inactivated (nonmetabolized)
AFB1 to BSA and plasma proteins of rats (Fig. 7)
.
It has been shown that >95% of the AFB1 found
in rat plasma proteins was noncovalently bound, and 80% of it was
associated with the albumin fraction (36)
. The binding of
AFB1 was nearly 5 times higher in the presence of
microsomes than in absence of microsomes, suggesting that
AFB1 metabolites have a higher affinity for BSA;
however, the effect of carnitine was significant.
The AFB1 binds to serum albumin and hepatic DNA
in a dose-dependent manner (10)
and carnitine
attenuates the latter (Fig. 1)
. AFB1 has been
shown to have the highest affinity for liver microsomes followed by the
cytosol, mitochondria and nuclei (36)
. The binding of
AFB1 to microsomes is increased by carnitine
(Fig. 6)
. It has been suggested that binding of
AFB1 to plasma albumin offers protection to the
liver, which has a high capability to draw free
AFB1 from the blood (11)
. Because
carnitine increases the AFB1 retention by plasma
proteins, it is possible that less free AFB1 will
be available for uptake and metabolism by liver cells in the intact
animal. We know that under in vitro conditions, carnitine does not
significantly alter the entry of AFB1 into the
hepatocytes or its partitioning among the subcellular fractions (Fig. 2)
. It may be argued that carnitine increases covalent binding of
AFB1 to cellular proteins in a manner similar to
that seen for plasma proteins (Figs. 5
, 7)
, resulting in the reduced
binding of AFB1 to nuclear DNA.
AFB1-albumin adduct concentration in plasma has
been widely used as an acceptable biomarker for evaluating exposure of
humans to AFB1 (37
,38)
. Our findings
indicate that caution should be taken in the interpretation of the
AFB1-albumin data. Higher concentrations of
AFB1-albumin adduct in the blood may not always
mean a higher intake of AFB1 but may be a
reflection of the modulation by dietary nutrients such as carnitine and
perhaps others not yet identified. Dirr (34)
reported that
the increase in plasma concentrations of long-chain fatty acids
considerably increases the AFB1-albumin
concentration in plasma, which may be modulated by acylcarnitines.
We conclude that carnitine lowers binding of AFB1 to DNA in isolated hepatocytes as well as to calf thymus DNA in a cell-free system. The carnitine effect is dose dependent but not highly specific because acetylcarnitine and GABA were equally effective. Carnitine enhances binding of AFB1 and its metabolites to various proteins (BSA, rat serum, microsomal) and thereby reduces the formation of AFB1-DNA adducts.
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FOOTNOTES |
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3 Abbreviations used: AFB1, aflatoxin B1; BSA, bovine serum albumin; DDW, double deionized water; DMSO, dimethyl sulfoxide; GABA, -aminobutyric acid; HBSS, Hank’s balanced salt solutions.
Manuscript received December 8, 2000. Initial review completed December 29, 2000. Revision accepted April 20, 2001.
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