Determination of multiple forms of cytochrome P-450 in microsomes from the digestive gland of Cryptochiton stelleri

Vol. 163, No. 1, 1989

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 476-480

August 30, 1989

Determination of Multiple Forms of Cytochrome P-450 in Microsomes From the Digestive Gland of Crvptochiton stelleri Daniel

Schlenk

and Donald

R. Buhler

Toxicology Program Oregon State University Corvallis, Ore 97331 Received

July

17,

1989

Polyclonal antibodies raised against purified trout cytochromes P-450 (P-450) IM2 (anti-IM2) and h!+i4b (anti-bM4b) were used in Western blot analyseswith digestive gland microsomes from control and /.3- naphthoflavone (BNF)-treated gumboot chitons Crvntochiton stelleri. An increase and decrease in staining intensity subsequent to treatment with anti-LM4b and anti-W2, respectively, was observed in digestive gland microsomes from BNF-treated chiton. Thus, there appears to be at least two forms of P-450 in microsomes from the digestive gland of Crvntochiton; one of which is induced by BNF and perhaps is involved in benzo(a)pyrene (BP) biotransformation, and another form which is inhibited by BNF. 0 1989 Academic Press,

Inc.

Phase well

I biotransformation

as

detoxify

bioactivation

to

xenobiotics

which

to lead

animal

same

either

essential the

of the

daes not

possess to more

with

detoxication

enzymatic

pathways

dynamic

number

of

unpublished

has

relatively

toxic In

data).

an attempt

copes with the toxic chemicals several xenobiotic biotransformation Cytochrome

(2). of

Crwtochiton

greater OOW291x/89 Copyright All rights

in

P-450 and

animals

to that

was observed pretreated

better

with

476

the

enzymes over

animals to

which

survive

contain

bromophenols how

a

(Yates, this

animal

the contribution of species was examined

from (BPH)

cytochrome

the

ecosystem.

on algae as

are

or has enzymes

understand

hydroxylase

toxicity

with

equipped

it encounters, systems in this

$1.50

0 1989 by Academic Press, Inc. of reproduction in any form reserved.

such

of

habitat

Consequently,

marine

feed

in microsomes

benzo(a)pyrene

the

an advantage

be better

as

ratio

a given

of detoxication

to

chemicals

the

equipped

species.

would

shown

in is

such as the

been

of

may have

toxic

environment

stelleri

to bioactivate

understanding

that

enzymes

chemicals

Crvntochiton

in

chemical

bioactivate

potential

two animals

animal

which

in a chemically

the

Determination

assuming

toxicant,

to detoxication

that

is

Therefore,

the

have

(1).

detoxication

of any xenobiotic. exposed

pathways

the

digestive

was

significantly

P-450

inducer,

gland j3-

BIOCHEMICAL

Vol. 163, No. 1, 1989

naphthoflavone

(BNF)

noncarcinogenic Since

the

multiple gland

of ,these

raised

against

two

trout

were

in

trout,

P-450

catalytic shown

forms

to

reactive

be

used

trout

liver

(8)

gene

family

(9).

MATERIALS

and

for

IM4b

for

the

are there

BNF.

oxidation

LM4b has been

possibly

representing

in most research

is

from

shown

the

possess

Isoform of

aflatoxin

livers two

LM2 has Bl

expressed

from

of forms

different to

the

to be the BNF-inducible

a protein

to

antibodies

These to

was

in microsomes

Polyclonal

shown

animal

any quantitative

immunoquantitation. been

a

hydrocarbon

present

isolated

specificities.

while

this

and if P-450

have

substrate

of

as

and BP.

enzyme with

of

used

to be inducible

of this

forms and

is

polyaromatic

purpose

treatment

as probes

IM2

responsible

epoxide

appears the

forms

purified

activities

inducing

of Crvntochiton after

chemical

as 3-methylcholanthrene

chiton,

whether

latter

P-450

activity

the

digestive

change rainbow

such

P-450

including

determine

of

for

contaminants cytochrome

species, from

This

(2).

substitute

environmental

AND BIOPHYSICAL RESEARCH COMMUNKATIONS

been more

form the

in

P450IB

AND METHODS

Chemicals fi-Naphthoflavone (BNF) was purchased from Aldrich Chemical and [G-3H]-Benzo(a)pyrene Company (Milwaukee, from ICN WI) (BP) Radiochemicals (Irvine, CA). [14C-7,10]-BP was obtained from Amersham (Arlington Heights, IL). All other biochemicals were purchased from Sigma Chemical Company (St. Louis, MO). For induction studies, 10 Animal treatment and microsome nrenaration animals (5 treated and 5 control), obtained by using SCUBA from subtidal rock jetties in Newport OR, were not fed for 2 weeks prior to treatment. Treated animals were dosed orally at 10 mg/kg with /3-naphthoflavone (BNF) in corn oil three times a week for three weeks. Control animals received oil. only corn Animals were sacrificed by freezing at -2OOC for approximately 20 min and dissected. Microsomes were prepared at 4OC by a modified procedure as described in Livingstone and Farrar (3). Tissues were placed in a 1:4 w/v ratio of 20 mM Tris-HCl buffer, pH 7.6, containing 0.5 M sucrose, 0.15 M KGl, 1 mM ethylenediaminetetraacetic acid (EDTA), 1 mM dithiothreitol and 0.1 mM CD=) were carried out phenylmethylsulfonylfluoride (PMSF). Homogenizations using a teflon Potter-Elvehjem homogenizer. The homogenate was centrifuged at 12,000 x g for 20 min in a Sorvall RC-PB refrigerated centrifuge. Following a second 12,000 x g spin of the supernatant, a final centrifugation at 100,000 x g for 90 min was performed. The microsomal pellet was resuspended in a storage buffer which contained 20 mM Tris-HCl pH 7.6, 20% w/v glycerol, 1 mM EDTA, 1 mM DTT, and 0.1 mM were stored at -8OOC until needed. PMSF. Unused microsomes Electrouhoresis and immunoblotting Electrophoresis of microsomal proteins was performed using 8.0 % separating and 3.0 % stacking polyacrylamide gels in the presence of sodium dodecyl sulfate (SDS) (4). Following electrophoresis, the separated proteins were transferred via electroblot to nitrocellulose sheets following a modification of the method of Towbin et al. (5) and stained by a modification of the method of Burnette (6) using polyclonal antibodies raised against purified trout cytochrome P-450s IM2 and I&b (7). The nitrocellulose was exposed to Kodak XAR-5 X-ray film for 7 days at - 80°C. Relative quantitation of autoradiograms was performed utilizing laser densitometry (Zeineh). 411

BIOCHEMICAL

Vol. 163, No. 1, 1989

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

RESULTS AND DISCUSSION Microsomes

from

purified

trout

observed

to

react

BNF-treated control

antibodies to

those

chitons

to

observed

possessed

antibodies chiton

greater

relative microsomes

with

trout

data

suggests

protein

m4b, at

observed shown

is,

be present

within

A similar

activity pretreatment

was

observed caused

in

in

and

gland

BNF-treated

in

from the

of for

the

I ,

Figure

1.

2.

this

from

P-450

the

chiton. content

content

and

(8).

BNF

livers form

23456 , ,,I

Western blot analysis of microsomes from the digestive glands of control and BNF treated chiton stained with anti-trout LM 4b' Lane l-3, control, 100 yg protein; Lane 4-6, BNF, 100 1J.g protein; Lane 7-10, purified trout LM4b, 2.0, 1.0, 0.5, 0.2 pm01 protein.

were

P-450

p-450

trout

the

BPH

activities

Western blot analysis of microsomes from the digestive glands of control and BNF-treated chiton stained with anti-trout LM2. Lane 1-3, control microsomes, 100 ng protein; Lane 4-6, BNF microsomes, 100 ng protein; Lane 7, control cytosol, 100 1.18 protein; Lane BNF cytosol, 100 ng protein; Lane 9-11, purified 0.5, 1.0 pm01 protein. trout LM2, 0.2,

478

then

augmented

LM2 and a

I

-54K Figure

from

and

while

between

IM4b

function

BP,

Crvntochiton

microsomes

rainbow

in

in

chitons,

constitutive

with protein

equivalent

associated

relationship

microsomes

a decrease

the

biotransformation

digestive

was

stained

P-450

bands darker

BNF-treated

when

responsible

anti-LM2

these

Assuming

exist

from

consistently

3).

is

in

a much

with

reaction

P-450

be

chitons. greater

(2).

in

with

Although is

and

microsomes

Conversely,

anti-IM4b

of

may

BPH was significantly unchanged

with

forms

daltons

BNF-treated

of (Figure

involved

multiple

60,000

in to

that

that

3). band

was

observed

along

staining

band control

was

of

samples.

reacted

also

(Figure

levels

both

gland

BNF-treated

which

60,000 anti-m2

each

controls

in

intensities

against

A single

Digestive

intensities,

to

antibodies

daltons

with 1).

BNF treated

from

at

stained

with

1 and 2).

60,000

(Figure greater

variable

reacted

A band

samples

possessed

have

at

2).

daltons

relative

appeared

gland (Figures

anti-IM4b

(Figure

at 54,000

chitons

than

with

BNF-treated

band

control

digestive

LM2 and w4b

samples and

darker

chiton

P-450s

8,

78 I

I

9 10 II

BIOCHEMICAL

Vol. 163, No. 1, 1989

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

: Errol P < 0.01 t

LM-2

LY-4b Enzyme

Figure

3.

Relative staining intensities of microsomes from the digestive glands of control and BNF-treated chitons stained with anti-trout LM2 and LM4b. Each value represents the mean of three sample replications from the same preparation +- S.D.

significant

increase

in

differences

in

content,

BNF-treated

and control

The

is

suggest

biosynthesis

when

antibodies

to

be structurally I&b

only

indiscriminate than against with probes

in

anti-l.M4b. a specific other

P45Os

as a more

In

accurate

studies,

P-450 measure

role

isozyme

to

the of

led

in

to

no

BPH between

and

primitive

Gonzalez in

to include

xenobiotic

steroid

metabolites.

Since

crossreacted that

the

from

invertebrates two bands, that

thus

(9)

a function

with

the

enzyme

must

to fish.

more

antibodies shown

P450 isozymes

anti-

may be more

recognizing

been

with

whereas

anti-I&i2

polyclonal have

a

to

sites raised

crossreact

use of mRNA quantitation

individual 479

P-450

appears

identification

led

Nebert

implication

It

mammalian has

which

chiton

plant

reacted

protein.

purified which

its

evolution

anti-m2

epitope

of

of

a strong

in

serving

encounter

forms

through

one its

already

to

is

that

nmol/mg)

hypothesis

trout

conserved

LH4b

differences

broadened

began there

recognized

the P-450,

purified

interesting

(0.11

evolution,

protein,

is

P-450

animals

form

significant

(8).

with

in

the

BP metabolizing but

cytochrome

early

metabolism

It

of

consistent that

invertebrate

the fish

content

large

herbivore, who

P-450

with (9).

cDNA At any

Vol. 163, No. 1, 1969

rate, with

the

two proteins

anti-LM2 The

must to

environments whether

induction

data

should

organic of

P-450

a better

in

some structural P-450

More

natural allow

chiton

as

(10).

of

observed of

serve

effect

AND BIOPHYSICAL RESEARCH COMMUNKATIONS

share

measurement

suggested

BIOCHEMICAL

a

digestive

research

in

marine

indicator

of

needs

chemicals can

occur

understanding

samples

stained

similarities.

induction

possible

gland

marine

has

pollution

to be performed on

in

organisms focusing

molluscs

nonpolluted of how marine

in to

environments. organisms

been marine on the

determine These cope with

xenobiotics. ACKNOWLEDGMENTS We would like to thank Dr. Lavern J. Weber of the OSU Hatfield Marine Science Center for the use of the facilities and Kenneth Yates for his collection and maintenance of the chitons. This research was supported by grants from the National Institutes of Environmental Health Sciences No. ES00210, ES03850 and ES07060. This paper was issued as Technical Paper No. 8935 from Oregon Agricultural Experiment Station. REFERENCES (1) Buhler, D.R. and Williams, D.E. (1988) Aquat. Toxicol. 11, 19-28. (2) Schlenk, D. and Buhler, D. R. (1988) Aquat. Toxicol. 13, 167-182. (3) Livingstone, D. R. and Farrar, S. V. (1984) Sci. Total Environ. 39, 209-235. (4) Laemmli, U. K. (1970) Nature 227, 680-685. (5) Towbin, H., Staehelin, T., and Gordon, J. (1979) Proc. Natl. Acad. Sci. USA, 76, 4350-4354. (6) Burnette, W. N. (1981) Anal. Biochem. 112, 195-203. (7) Miranda, C. L., Wang, J. L., Henderson, M. C., and Buhler, D. R. (1989) Arch. Biochem. Biophys. 268, 227-238. (8) Williams, D. E., and Buhler, D. R. (1984) Biochem. Pharm. 33, 37433753. (9) Nebert, D.W., and Gonzalez, F.J. (1987) Ann. Rev. Biochem. 56, 945-93. (10) Payne, J. F., Fancey, L. L., Rahimtula, A. D., and Porter, E. L. (1987) Comp. Biochem. Physiol. 86C, 233-245.

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