Vol. 172, No. 2, 1990 October

BIOCHEMICAL

30, 1990

AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 767-774

EVIDENCE FOR INCREASED TRANSLATIONAL P450IIEl

BY SOLVENTS: ANALYSIS OF P450IIEl

Sang Geon Kiml,

'The

Institute

of Chemical

September

mRNA POLYRIBOSOMAL DISTRIBUTION

Stacey E. Shehinl, .I. Christopher and Raymond F. Novak1

Toxicology

Wayne State

Received

EFFICIENCY IN THE INDUCTION OF

and 2 Center

University,

for

Detroit,

States2

Molecular

Biology

MI 48201

4, 1990

The potential for enhanced translational processing of P450IIEl mRNA during the early phase of P450IIEl induction by pyridine or acetone was assessed by hybridization analysis of polyribosomal P450IIEl mRNA distribution in rat hepatic tissue. Optical absorbance profiles of polyribosomal fractions exhibited an apparent shift at 5 h following pyridine administration relative to control. Slot and Northern blot analyses for P450IIEl mRNA in the cytoplasmic extracts isolated from 5 h pyridine-treated rats demonstrated a shift in distribution of P450IIEl message toward heavier polyribosomal fractions and Northern blot analysis suggested the presence of different populations of P450IIEl mRNA. Slot blot analyses also demonstrated a shift in the polyribosomal distribution of P450IIEl mRNA at 12 h following pyridine treatment; in contrast, hybridization analysis for P450IAl revealed no shift in polyribosomal distribution of P450IAl m&IA. Acute acetone administration to animals also resulted in a similar shift in polyribosomal distribution of P4501IEl mRNA as compared to control. These data suggest that P4501IEl mRNA shifts toward larger polyribosomes following acute exposure of animals to pyridine or acetone and provide evidence that induction of P450IIEl at early times following acute pyridine or acetone administration involves enhanced translational efficiency through increased loading of ribosomes on P450IIEl 0 199u Acadrmlc Press, Ins. mRNA.

Pyridine (1,2) pyridine manner

is an efficacious

and elevation treatment (3).

has been

A similar

of IIEl

by acetone

acetone

induction

inducer

of P450IIEl

(4). of IIEl

time

content

of P450IIEl

shown to occur dependence

The rapid

in a time-

has been noted

time-response

was exploited

in both

and metabolic

maximally

rats

activity

and rabbits following

and dose-dependent recently

associated to examine

for with

induction

pyridine

and

potential

Abbreviations: P450IIE1, cytochrome P450IIEl (gene cyp 2El) is the nomenclature recommended for members of this gene family which include P-450j in rat and P-45OLM3, in rabbit (Nebert & A., 1989, DNA 8, 1-13); MOPS, 3-(N-morpholino)propanesulfonic acid; EDTA, ethylenediamine tetraacetic acid.

Vol.

172,

No.

2, 1990

molecular

mechanism(s)

revealed

that

increased

pyridine

rate

into

in the absence induction increased

responsible

for

administration of pyridine examined research

synthesis,

with revealed

translational of chemical

inducers

whether

a shift

following

ribosomal

of IIEl

in IIEl

mRNA toward

efficiency

Materials

complete

in order

animals,

inhibition

to examine

P450IIEl

expression

larger

in the induction

the

The effects

mRNA in polysomes

loading

protein.

mRNAs is

following

or acetone.

of P450IIEl

increased

incorporation

cycloheximide

such as pyridine

induction

an

or acetone-treated Moreover,

of IIEl

on the distribution

studies

through

by increased

of preexisting

phase

COMMUNICATIONS

of these

occurs

was initiated

efficiency

and rapid

translational

in pyridine-

was monitored

the early

the rapid

of IIEl

activation. study

RESEARCH

The results

as evidenced

protein

The present

to determine

of increased

induction

P450IIEl

or acetone

associated

and acetone

by pyridine (3).

BIOPHYSICAL

induction.

of transcriptional

administration whether

AND

of P450IIEl

of protein

of [14C]leucine of IIEl

BIOCHEMICAL

on IIEl

The results polyribosomes

was

mRNA is of this in support

of P4501IEl.

and Methods

Materials: Reagents in the molecular studies were purchased from Sigma Chemical Co. (St. Louis, MO). T[~~P]ATP (>5000 Ci/mmol) was obtained from Amersham (Arlington Heights, IL). Supported nitrocellulose transfer membranes were obtained from Schleicher & Schuell (Keene, NH). Analysis of polvsomes: Polysomal fractions were isolated using a sucrose-density gradient according to Kleene et al. (5) and modified as Male Sprague-Dawley rats (150-200 g) were injected with either follows. pyridine (100 mg/kg, i.p.) or acetone (2.4 g/kg, i.p.) and the livers excised from untreated rats and pyridineor acetone-treated rats at 5 and 12 h following treatment and rinsed in ice-cold saline. One gram of tissue was minced in 5 ml of HKM (HKM: 20 mM Hepes buffer, pH 7.6, 100 mM KC1 and 1.5 mM MgC12) buffer containing 6 mM P-mercaptoethanol and 10 mM ribonucleoside vanadyl complexes at 0-4°C. The tissues were homogenized at 4°C with 10 strokes of a motor driven Teflon-glass homogenizer. The homogenate was centrifuged at 15,000 rpm for 20 min at 4°C in a SS-34 Sorvall rotor to The membranes in the microsomal sediment the nuclei and mitochondria. fraction were dissolved by adding sodium deoxycholate and Nonidet P-40, each to a final concentration of 0.5%. An aliquot of ribosomal fraction was loaded onto a 10 ml lo-50% linear sucrose (w/w) density gradient prepared in HKM buffer with a 0.5 ml 60% sucrose cushion and centrifuged for 3.5 h at 40,000 The fractions were isolated by aspiration from rpm in a Beckman SW-40 rotor. the bottom of the tubes with a peristaltic pump and the absorbance at 260 nm was monitored. Slot blot hybridization analvsis of polvsomal RNA: Aliquots of the polysomal fractions were diluted with 20X SSC and 37% formaldehyde, heated at 65°C for 15 min and applied to nitrocellulose sheets using a mini-fold II slot blot apparatus according to the manufacturer's protocol (Scheicher & Schuell). Prehybridization and hybridization of the nitrocellulose membranes with the radiolabelled oligonucleotide probe for IIEl (5'-d(CAAAGCCAACTGTGACAGG)-3') or IA1 (5'-d(TCTGGTGAGCATCCAGGACA)-3') mRNA was performed as described previously AutoThe nitrocellulose membranes were subjected to autoradiography. (3). densitometer. radiographs of the X-ray films were scanned with a LKB Ultroscan 768

Vol.

BIOCHEMICAL

172, No. 2, 1990

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

Northern blot analvsis of oolvsomal RNA: Northern blot analysis was performed as described by Sambrook a A. (6). Aliquots of the polysomal fractions were mixed with 5X formaldehyde gel-running buffer consisting of 0.1 The samples M MOPS (pH 7.0), 40 mM sodium acetate and 5 mM EDTA (pH 8.0). were incubated for 15 min at 65°C and mixed with the gel loading buffer (50% glycerol, 1 mM EDTA and 0.25% bromophenol blue). The polysomal RNA was fractionated on a 1% formaldehyde denaturing agarose gel and the fractionated Prehybridization and RNA was transferred to a nitrocellulose membrane. hybridization of the nitrocellulose membranes with the radiolabelled oligonucleotide probe for IIEl mRNA were performed as described previously The nitrocellulose membranes were exposed at -80°C to Kodak X-Cmat AR (3). film for autoradiography. Results Optical

absorbance

P450IIEl

induction

increase

rapidly

the hepatic

of IIEl

distribution tissue,

were

gradient

fractions

towards

observed

at 5 h following

shown).

The shift

Slot

performed

pyridine-treated analyses.

Both

(20-358

amounts

of IIEl

distribution mRNA.

and Northern associated

slot

number

in the more dense

sucrose

This

ribosomes

The slot

blot

gradient)

from (Fig.

with

isolated

fractions

intensities

is associated

results

was found

with fractions being

1A) clearly

using

packed

and Northern enhanced

polyribosomal rats

from untreated

of ribosomes

as

animals.

represent fractions

the relative

and the

loaded

considered

toward the bottom more closely along

shows a shift 769

slot

and

from pyridine-treated

per mRNA is (i.e.

(not

to be reproducible

revealed

the heavier

gradient

the number

to control

from untreated

in the autoradiograph

of ribosomes

was

and IA1 mRNA: The relative

analyses

isolated

sucrose

density)

of animals. fractions

blot

in -45

in the polysomal

as compared

of IIEl

slot

mRNA in the linear

The average

sets

intensities same polysomal

lA,

shift sucrose

at 5 and 12 h was examined

sucrose

to the

In figure

different

from

and collected

(greater

profile

hvbridization

from hepatic

at 5 or 12 h

A notable

administration

mRNA in polysomal

animals

autoradiographic compared

with blot

polysomes density

the

from each of the sucrose

at 260 nm.

heavier

isolated

gradients

aliquot

of blot

simultaneously

rats

sucrose

of an equal

pyridine

of P450IIEl

fractions

on lo-50%

i.p.)

(3).

and Northern

To compare

isolated

of

of P450IIEl

and distribution

and slot

fractions

in the optical

and Northern

distribution

profile

absorbance

course

dose of pyridine

supernatants

was monitored

profile

in experiments

a single

(100 mg/kg,

on the time

and activities

mRNA in polysomal

sedimented

The absorbance

absorbance

levels

absorbance

optical

and pyridine-treated

fractions.

Studies

mRNA at 5 and 12 h posttreatment.

of P450IIEl

posttreatment

blot

polysomal using

the post-mitochondrial

untreated

the

3 to 16 h following

mRNA was examined

analyses

of polvribosomes:

have shown that from

Consequently, IIEl

profile

onto

the IIEl

to be greater of sucrose gradient) the mRNA molecule.

in the distribution

of IIEI

I@UVA

Vol.

172,

No.

2, 1990

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

Figure 1. (A) Slot blot hybridization analysis showing the polysomal distribution of IIEl WA from untreated rats (left panel) and 5 h pyridine-treated rats (right panel). B and T represent the bottom (heavier fraction, polysomes) and top (lighter fraction, monosomes) of the sucrose gradient, respectively. (B) Scanning densitometry of the slot blot hybridization showing an apparent shift of P450IIEl mRNA toward the larger polysomes in fractions from pyridine-treated rats as compared to control.

from lighter

to heavier

pyridine-treated control

in the livers

suggests

of signal

intensity

Northern polysomes.

analysis

1A) also

This

is

consistent

to the P450IIEl

the existence

of different

of IIEl

at 12 h posttreatment

the larger lower

oligonucleotide

polysomes

density

fractions. versus

signal

in the lower

intensity

in larger

To examine was unique

for

3).

control

animals density

blot

hybridization.

in the Northern in polysomes

A noticable pattern

increase with

analysis Moreover,

blot

suggests

for

IIEl

with

was also

in IIEl

a loss

at 12 h illustrates fractions

blot

mENAs.

in conjunction

The densitometric

pyridine-treated intensity

probe

mRNA distribution

monitored

in the slot to larger

from the slot blot

loss

to control.

employed

obtained

of P450IIEl

(Fig.

is also

illustrated

a concomitant

mRNA distribution

of the slot

populations

analysis

fractions

mRNAs

Scanning

lB,

with

of

IIEl

pyridine.

as compared

the results

the specificity

hybridization

blot

with

onto

shown in Fig.

in IIEl

of

the livers

loaded

with

fractions

a shift

from

polysomes

of the polyribosomal

2) and demonstrates

The slot

lA,

the larger

density

revealed

are

treatment

in Fig.

from livers

isolated

more ribosomes

mRNA into

in the lower

blot

(Fig.

examined

that

at 5 h following

of IIEl

isolated

to polysomes

of the autoradiographs

the shift

analysis

in polysomes

as compared

of animals

densitometry

(Fig.

This

animals.

further

fractions

animals

mRNA in

of signal

in the

mRNA distribution the loss

a concomitant

of IIEl gain

in mFUA

of signal

polyribosomes.

whether P4501IE1,

the

increase

a replicate

in IIEl

signal

intensity

membrane was probed 770

in larger with

polysomes

a radiolabelled

Vol.

172,

No.

2, 1990

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

Figure 2. Northern blot hybridization analysis showing the polysomal distribution of the P450IIEl mRNA in the hepatic tissue at 5 h following pyridine treatment. The polysomal mRNA from the gradient fractions was electrophoresed on a formaldehyde denaturing 1% agarose gel and transferred to Hybridization was performed with a supported nitrocellulose membrane. radiolabelled 19-mer P450IIEl oligonucleotide probe. The lanes in the blot proceed left to right from the higher density (larger polysomes) to the lower density fractions. The lanes contain, in an alternating array, polysomal mFSA from untreated (arrows) and pyridine-treated animals at 5 h posttreatment. The probe appeared to hybridize to two populations of mRNA and the enhanced autoradiographic intensities in the high-density fractions from pyridine-treated animals as compared to control is associated with increased P450IIEl mRNA in the larger polysomes.

P450IAl

oligonucleotide.

observed

in the polysomal

significant

polysomal

the densitometric however,

unpublished).

[14C]leucine administration

of acetone research incorporation, (3).

on rat

mRNA

in signal

oolvsomal

hepatic

to be elevated

of acetone was also

synthesis,

the slot

results

(Kim,

of P450IIEl monitored

at 5 h following

administration

examined

4A),

mRNA does occur

in these distribution

IIEl

(Fig.

in either

in P4501Al

and is reflected

heoatic that

intensity

animals

was noted

An increase

appeared

The effect

of P450IIEl

intensity

4B).

treatment

showed

increase

from pyridine-treated

of signal (Fig.

Novak,

Previous

distribution

shift

pattern pyridine

The effect

a generalized

fractions

following

Reddy and m:

Although

acetone

on hepatic

at 5 h following

via polysomal

acetone

was no blot

o

Vol.

172, No. 2, 1990

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

T

B

T

Figure 3. (A) Slot blot hybridization analysis showing the polysomal distributionof IIEl mRNA from untreated rats (left panel) and 12 h pyridine-treated rats (right panel). B and T represent the bottom and (B) Scanning densitometry of the a sucrose gradient, respectively. slot blot hybridization showing a significant shift of P450IIEl mIWA toward the larger polysomes in the gradient fractions isolated from pyridine-treated rats as compared to control.

treatment.

Slot

blot

hybridization

and B, respectively, polysomes

isolated

clearly

analyses

and densitometric

show a significant

from acetone-treated

shift

animals

of

scanning,

of P450IIEl

as compared

top

mF3A in

to controls.

B

T

T

B

Figure 4. (A) Slot blot hybridization analysis showing the polysomal distribution of P450IAl mF9A in the replicate membrane from control (left panel) and 12 h pyridine-treated animals (right panel) employed in Figure 3. B and T represent fractions taken from the bottom and top of the sucrose (B) Scanning densitometry of the slot blot respectively. gradient, hybridization reveals no significant shift of P450IAl message in the polysomes from pyridine-treated rats relative to control.

772

figure An

5A

Vol.

172,

No.

BIOCHEMICAL

2, 1990

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

Figure 5. (A) Slot blot hybridization analysis showing the polysomal distribution of IIEl m!UA from untreated rats (left panel) and 5 h acetone-treated rats (right panel). B and T represent the bottom and top of a sucrose gradient, respectively. (B) Scanning densitometry of the intensity of slot blot hybridization shows a shift of P450IIEl message toward the larger polysomes in the gradient fraction isolated from acetone-treated rats as compared to control.

of IIEl

increase

concomitant appears

loss

to occur

mRNA in heavier

polyribosomal

of hybridizable

IIEl

following

acetone

fractions

mRNA signal

along

with

in the lower

a

density

fractions

treatment.

Discussion P450IIEl including

has been pyridine,

shown to be increased

acetone,

in the TIE1 mRNA level. evidence

of protein

performed

at later

regimen).

times

at early

of transcriptional

ethanol

(8).

radiolabelled

apparent

enhanced

unpublished).

rate

Similar

in rabbit

similar

semi-purification of IIEl

synthesis

studies

in rabbits

tissue. ethanol

administration

(3).

the potential

These

data for

(7),

data

Moreover,

also

have been

(Kim

revealed a decrease

were

and

reported

increased

increased

utilization

from

confirmed

an

Novak,

in poly(A+)

suggested

for

fractions

an increased

(9) and was observed

to be

in the

analysis

treatment

773

was found

enriched

pyridine

studies

5 to 10 day exposure in rats

by autoradiographic by

these

administration

of P450IIEl

followed

present

study,

or acetone

(3);

in vivo

hepatic

following

pyridine

activation

Subsequent

proteins

been reported

following

(i.e.

synthesis

elevation

has provided

of IIEl

treatment

of P450IIEl

inducers

a concomitant

work by others

chronic

the rate

to many organic

without

in the induction

following

times

in response

and benzene previous

stabilization

absence

of IIEl

Although

More recently,

increased

ethanol

band

following

translational of P450IIEl

intensity

mRNA has pyridine

efficiency. mRNA (i.e.

In the

Vol.

172,

No.

2, 1990

increased

translational

was examined polysomal data

by monitoring

show that

pyridine research

primary

mechanism

pyridine,

Evidence of mRNAs is (12-14).

exposure report

induction

mechanism

for

the first

in

treatment.

in the liver

Our cells

by inducing

agents

such as

(2,10,11). shows that

the utilization

of pre-existing conditions

ferritin

to pyridine

contains

of IIEl

synthesis

(18).

agen

These results along with those of translation of IIEl mRNA may be a

protein

(15),

to inducing

mRNA content

xenobiotic

polyribosomes

of mRNA for

storage

of cytochrome

and IIEl

storage

proteins

of animals

to larger

COMMUNICATIONS

in response

h) following

in increased

include

from either

and attractive induction

involved

reductase

proto-oncogene polysomes

or ethanol

profiles

(5-12

more efficient

the rapid

RESEARCH

hepatocytes

administration.

that

from many studies

These

ribonucleotide

times

or acetone suggest

acetone

in rat

mRNA is shifted

for

BIOPHYSICAL

polyribosomal

at early

IIEl

following

AND

efficiency)

fractions

previous

this

BIOCHEMICAL

heat

under

Mobilization or less

or acetone an immediate evidence

(16),

of existing

efficiently cellular for

tubulin IIEl

translated

would a role

of induction

and transferrin

shock proteins

appear

receptors (17)

(14),

and

mRNA to larger pools

following

to represent

response

stores

to these

of translational

an efficient agents

and

efficiency

P450.

by NIH Grant AcknowledPment : Supported Institute of Environmental Health Sciences.

ES03656

from

the National

References l.Kaul, K.L. and Novak, R.F. (1987) J. Pharmacol. Exp. Ther. 243, 384-390. 2.Kim, S.G., Williams, D.E., Schuetz, E.G., Guzelian, P.S., and Novak, (1988) J. Pharmacol. Exp. Ther. 246, 1175-1182. R.F. 3.Kim, S.G. and Novak, R.F. (1990) Biochem. Biophys. Res. Commun. 166, 1072-1079. and Ingelman-Sundberg, M. (1989) Xenobiotica 19, 4. Ronis, M.J.J. 1161-1165. 5.Kleene, K.C., Distel, R.J., and Hecht, N.B. (1984) Dev. Biol. 105, 71-79 E.F. and Maniatis, T. (1989) In: Molecular 6.Sambrook, J., Fritsch, Cloning, A Laboratory Manual, 2nd ed. pp. 7.43-7.48. 7.Song, B.J., Veech, R.L., Park, S.S., Gelboin, H.V., and Gonzalez, F.J. (1989) 3. Biol. Chem. 264, 3568-3572. C.S. and Lasker, J.M. (1990) FASEB J. 8.Ito, Y., Tsutsumi, M., Lieber, 4, A1622. g.Porter, T.D., Khani, S.C., and Coon, M.J. (1989) Mol. Pharmacol. 36, 61-65. M.E., Veech, R.L. (1984) J. Biol. Chem. 259, 231-236. lO.Casazza, J.P., Felver, ll.Ryan, D.E., Ramanathan, L., Iida, S., Thomas, P.E., Haniu, M. Shively, Lieber, C.S., and Levin, W. (1985) J. Biol. Chem. 260, J.E., 6385-6393. lZ.Theil, E.C. (1990) J. Biol. Chem. 265, 4771-4774. 13.Zahringer, J., Baliga, B.S. and Munro, H.N. (1976) Proc. Nat. Acad. USA 73, 857-861. Sci., 14.Thei1, E.C. (1987) In: Translational Regulation of Gene Expression Plenum Publishing Corp., New York. (Ilan, J., ed) pp. 141-163, 15.Standart, N.M., Bray, S.J., George, E.L., Hunt, T., and Ruderman, J. (1985) J. Cell Biol 100, 1968-1976. 16.Lindquist, S. (1986) Annu. Rev. Biochem. 55, 1151-1191. 17.Yen, T.J., Machlin, P.S., and Cleveland, D.W. (1988) Nature 334, 580-585. 18.Shaw, G. and Kamen, R. (1986) Cell 46, 659-667. 774

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