Aflatoxin metabolism by liver microsomal preparations of two different species

BIOCHEMICAL

Vol. 33, No. 5, 1968

Aflatoxin

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Metabolism by Liver Microsomal Preparations of Two Different Species

R. S. Portman, K. M. Plowman, and T. C. Campbell Virginia Polytechnic Institute Blacksburg, Virginia, 24061

Received October 2, 1968 Aflatoxin,

the well known carcinogen produced by the mold Aspergillus

flavus,

has been shown to relatively

toxicity

(Platonow, 1964) and liver

in mice.

ineffective

in producing characteristic

cancer symptoms (Newberne et al.,

1965)

Studies were undertaken to evaluate the reasons for this

resistance shownby mice as opposed to rats,

since subtle differences

between these species may be suggestive of the toxicologic

and/or

carcitiogenic mechanism. Previous work done in this lab and reported elsewhere (Portman s al., faster liver

aflatoxin slices.

1968) showedthat rat liver

slices promoted a

B1 disappearance from the incubating mediumthan did mouse However, it was not determined whether this rate of

disappearance was a reflection further

of uptake by the cells

or metabolic rate,

per se.

Therefore,

experiments were designed to evaluate the

relative

rates of metabolism in these two species using liver

microsomal

preparations. The metabolism of aflatoxin derivative

similar

to many other polycyclic

1964; Holzapfel et al., preparations, reaction

B1 proceeds through a hydroxylated

1966).

In these experiments using microsomal

we measured the kinetic

shown in Figure 1.

hydrocarbons (de Iongh et al.,

properties

These results

show a much faster rate of

metabolism with thi mousemicrosomal preparation, to the greater resistance of mice. aflatoxin

B1

as the more toxic

of the hydroxylation

which may contribute

These data would therefore

component.

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Vol. 33, No. 5, 1968

BIOCHEMICAL

Figure 1.

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Hydroxylation

14 C-methyl-labeled

of Aflatoxin

= 57.6 C/ mole) 14 was purchased from Tracerlab and used to synthesize C-uniformlylabeled aflatoxin

acetate (specific

Bl.

activity

B1 according to the method of Adye and Mateles (1964).

Nicotinamide adenine dinucleotide

phosphate (disodium salt)

and yeast

glucose-6-phosphate dehydrogenase were obtained from Calbiochem and glucose-6-phosphate was purchased from Sigma Chemicals. aflatoxin

Bl was prepared and purified

Unlabeled

according to the method of Hanna

and Campbell (1968). Two-month old Sprague-Dawley derived rats and ICR derived mice were used in these studies. lab chow from weaning until excised, weighed, chilled, phosphate buffer centrifuged

Livers of both species were

pooled and homogenized in 2 volumes of Homogenateswere

at 20,000 x g for 20 minutes to remove the mitochondrial The microsomes were subsequently collected

of the 105,000 x g centrifugation

The microsomal pellet buffer

sacrifice.

(pH 7.4) with 0.25 M sucrose.

and nuclear fractions. a pellet

All animals were maintained on commercial

of the above supernatant.

was resuspended and washed 3 times in phosphate

(pH 7.4) and finally

microsomes equivalent

as

resuspended to give a concentration

of

to 1 gm of liver/ml.

Each incubation flask contained 1.2 ml phosphate buffer with 0.25 M sucrose (pli 7.4),

and 0.3 ml of the microsomal preparation.

Concentrations of 5.9 M glucose 6-P, 0.53 mMNADPand 3 units of

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BIOCHEMICAL

Vol. 33, No. 5, 1968

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

glucose-6-P dehydrogenase together with the appropriate 14C-labeled aflatoxin

Bl were also included.

preparation was diluted with cold aflatoxin activity

The labeled aflatoxin to give a specific

of 6.24 pC/pmole and from this stock solution,

hot to cold dilutions of approximately

appropriate

were made to give adequate counting statistics

15,000 cpm per flask.

Each aflatoxin

was added to the flask in 0.01 ml of ethanol. carried

amounts of

All

out at 37' in a Dubnoff metabolic shaker.

concentration

incubations were Reactions were

stopped with 5 ml of chloroform which was subsequently used to extract

the unmetabolized Bl and Ml product.

A second extraction

with 5 ml of chloroform was repeated and combined with the first extract.

. These extracts

were then spotted on thin layer chromatography

plates coated with 750 p Adsorbosil-1

(Applied Science Labs) and

developed in the benzene rich phase of benzene:ethanol:water The appropriate

(46:35:19).

B1 and Ml adsorbosil spots were removed from the plates,

added to 15 ml of counting solution containing counted in a scintillation For the rat,

1 ml of methanol and

counter.

velocities

and for the mouse, velocities

were found to be linear

for 20 minutes;

were found to be linear for 10 minutes.

The data were analyzed using the computer program of Cleland, V = VmA/K + A 11963). Results and Discussion The apparent Michaelis

constants and maximal velocities

these two species are shown in Table 1. these kinetic

different

It should be emphasized that

parameters represent 'apparent'

are not involved

and only aflatoxin

for

values since pure enzymes

was varied.

For the mouseboth the V and K are significantly max m from the corresponding values for the rat. Both parameters

for the mousepreparation

are such that a much greater rate of metabolism is

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Vol. 33, No. 5, 1968

BIOCHEMICAL

Table

AND BIOPHYSI~L

RESEARCH COMMUNICATIONS

1. Comparative Rinetic Data of Aflatoxin Metabolism by Two-month Old Rat and Mouse Liver Microsomes.

Rat

Bl

Mouse

358 k 55 21.2

44.7

16.9 f. 0.8

% a Substrate b

+ 2.2

concentration

+ 8.4

2.9 f. 0.3

in mM.

Appearance of ML (hydroxylated derivative) in mpmoles/300 mg liver/hr (these data are not expressed per unit weight of protein since equivalent concentrations of crude microsomal protein would not necessarily imply equivalent levels of active enzyme).

achieved,

whatever

the concentration

Previously of disappearance

in these

we had shown

of aflatoxin

was relatively

faster

with

experiments,

disappearance

rates

are

imply

faster

Taken

together,

these

dual

advantage

of not

more

slowly

but

the aflatoxin

Whether

1968)

Bl from media

incubated

with

preparations.

Prom the

it

that

is

apparent to rates

across

once

liver

data

those

liver

show that

the rate slices

reported

aflatoxin

of metabolism

the rat

two observations

that

cell

B1

and therefore

membrane.

the mouse enjoys

transporting

aflatoxin

into

the

the aflatoxin

is within

the cell,

liver

the cell

of metabolizing

more rapidly.

species the rate

data

suggest

that

toxicity,

of B1 uptake

by the

are, totally

between

species

these

also

of greatest

B1 metabolism

to compare

et al.,

not equivalent

only

used.

(Portman

of transport

als,

These molecular

rat

however,

rates

of aflatoxin

such activities

as opposed cells

responsible is not yet with

the parent

for

known, acute

714

aflatoxin

to’ the metabolites.

and the

rate

the

difference

although

it

--in vivo

B1 is

would

toxicities.

of subsequent in resistance be interesting

the

Vol. 33, No. 5, 1968

8lOCHEMlCAL

AND 8lOPHYSlCAL

RESEARCH COMMUNICATIONS

References Adye, J. and Mateles, R. I.,

Biochim. Biophys. Acta 86, 418 (1964).

Cleland, W. W., Nature 198, 463 (1963). de Iongh, H., Vles, R. O., and van Pelt,

J. G., Nature, 202, 466 (1964).

Hanna, K. L. and Campbell, T. C., J. Assoc. Offic. Holzapfel,

Anal. Chem. Nov. (1968).

C. W., Steyn, P. S., and Purchase, I. F. I-l., Tetrahedron Letters

25, 2799 (1966). Newberne, P. M. 3

G. N. Wogan (ea.) mcotoxins

in Foodstuffs,

M.I.T.

Press, Cambridge, Mass. pp. 187-208 (1965). Platonow, N., Vet. Rec. 76, 589 (1964). Portman, R. S., Campbell, T. C., and Plowman, K. M., Fed. Proc. 27, 839 (1968).

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