The control of glutathione and cytochrome p-45o concentrations of primary cultures of rat hepatocytes

~io~h=micoi

~hu~ma~logy,

Vol.

30,

No.

19,

pp.

2739-2742,

~-2952/81/192?39~~2.~/~ Pergamon Press Ltd.

1981.

Printed in Great Britain.

PREIJMINARY

COMMUNICATION

THE CONTROL OF ~LUTAT~IONE AND CYTOCHROME P-450 CONCENTRATIONS OF

PRr~R~

CULTURES OF RAT HEPATOCYTES

Christine M. Allen, Lesley J. Hockin and Alan J. Paine* M.R.C. Toxicology Unit, Medical Research Council Laboratories, Woodmansterne Road, Carshalton, Surrey SMS 4EF England. ( Received 10 July 1981 ; accepted 17 July 1981 ) The culture of rat hepatocytes is a popular model for the study of drug metabolism and toxicity (1,2).

An obstacle to the use of liver cell culture for these studies has

been the knowledge that the concentration of cytochrome P-450, a major system involved in activating drugs to reactive intermediates, falls by 60-70% during the first 24 hours of culture (3).

Recently we reported that this loss could be prevented by culturing

hepatocytes in a medium that does not contain cystine

(4).

However sulphur containing

amino acids are precursors of glutathione.tvhich plays an important role in the detoxification of reactive metabolites (5,6).

We have therefore examined the glutathione

content of hepatocytes cultured with and without cystine. MATERIALS

AND

METHODS

Isolation and culture of hepatocytes Hepatocytes were isolated and 20 x 10' cells were cultured in 150 mm diameter petri dishes in 20 ml of the respective medium as previously described (7).

The medium used

was based on Earle's balanced salt solution (Gibco Biocult Ltd., Paisley, Scotland) containing 1 ml "BME vitamin solution x 100 strength" (Gibco Biocult Ltd) per 100 ml medium and amino acids (BDH, Poole, Dorset, U.K.) at the same concentration as found in 1640 (8).

RPMT

-6 All media contained 5% foetal calf serum (Gibco Biocult), 10 M insulin,

lo-* M hydrocortisone-21-sodium

succinatc? (both from Sigma Chemical Co.,

Poole,

Dorset,

and 5 mg gentamicin (Flow Labs.Irvine, Scotland) per 300 ml medium.

U.K.)

"P-450 Maintenance Medium" constitutes the basic medium without cystine and supplemented with 100 PM 5-amino laevulinic acid (Sigma Chemical Co.) (4). Cytochrome P-450 and protein were determined as previously described (9). Glutathione (GSHL was determined as described by Hissin & Hilf (IO). nation

For this determi-

a 5% (w/v) homogenate of liver and isolated hepatocytes was made in 0.1 M sodium

phosphate buffer containing 5 mM EDTA pfl 8 by using an Ultra-Turrax TP18,&1 blender for 10 sets*

*

GSH was determined in cultured hepatocytes by scraping hepatocytes from

To whom correspwndence should be addressed. 2739

2740

2

Preliminary communication

x

150

mm

petri

diameter

dishes

into

5 mii EDTA pH 8 and homogenising tion

of 50 ug GSH/ml

tion

procedure

detected

caused

1 shows

containing

0.2

as found causes

of cytochrome

by removing

for

in medium

of that

Table

found

1

Effect and

Treatment

Intact

their

liver

(Table

from

have

+ 0.2

for

that

The

add

c-ould no1 tw

hepntoc-ytos.

hcpatocylcs

these

to Ihc

same

medi urn containing

This

loss,

medium

ill a mcxiium c:olt~'cntralio~,

0.2

mM c'ysl il~(t

of cytochromc~

P-1.50, <‘a,:

(4) but

a glutathione

in a '~O-T;II((~

for 23 hours

content

cullurc l)-

cells

rcsrllli

hcpatucytcs

~‘oncenIration

~‘nlturc~l

whi(.h ii, '%(I",

isolation

and

culture

conditions

on

their

glutathionc

concentrations.

Cytochrc,mc f’- &r,Oc’onc’n* (5% intnc‘t i ivcr 1

Glutathione conrn. (pg/mq protc in)

24 hr

9.5

+ 0.2

100

6.6

+ o.s*

100

8.3

+ 0.8

38 It J*

in:

mM cystine

* medium

3.7

- cystine

'10 + 0

1 0.5

* denotes The

significantly

results

from

six

intact

separate

liver

These

was

intact

how

and

examined

maintenance tion

individual

values

for

livers

(i.e.

raise

can

Reed

n = 6).

chemicals K Beatty

the effect medium"

two

of

(i.e.

of cultured

be maintained have

shown

in hepatocyte increasing medium

h'epatocytes.

The

interesting

(12)

Siudcnt's found

l-test,

from

inlact

in preparations

cytochromo

P-450

liver.

d~~rivccl

con<-cntsal ion of

protein.

the potential

of glutathione,

of cultllred

by

155 2 20 pmoles/mg

detoxify

liver?

precursor we

rat

(p < 0.05)

i: S.D.

observations

Firstly, activate

different

are means

i-

111c homogenisa-

in rcf.11,

cultured

i tlg

i II

1).

origin

cultured

of

the culture

hepatocytes

Hepatocytes

or

as

c-onta

blcndcr.

demonstrated

glutathione

P-450

(Table

P-450

an Ultra-Turrax

to isolale

However

buffer

DISCUSSION

culture

cystine

of hepatocyte

sample

AND

liver

Isolated

medium

without

cytochrome

and

RESULTS used

phosphate

determined

isolated

I).

cystine

in intact

GSSG,

The

(Table

with

liver,

restores

liver

be prevented 24 hours

of GSH.

the procedure

mM cystine

loss

IO sees

sodium

to homogenisation

(p < 0.05).

in intact

a 60%

prior

of intact

that

of glutathione

for

no loss

in homogenates

Table loss

buffer

1 Ff

5 ml 0.

hepatocytcs, at the that

the methionine cystine)

2 shows

that

same

as a model level

methionine

suspensions,

without Table

questions:-

than

as frxlnd in the

kc-ording1.y

is c-yslinr. of

the

the glutathionr

increasing

to

is a mot-c efficicn?.

concentration (4) on

system,

"P-150 r.oncentra-

lhe methioninc-

2741

Preliminary Communication

concentration cytes

Erom

cultured

for

of removing

cystine

hepatocytes

can

The causal

second

from

cytochrome

P-450

this

at the

by

same

between Such

medium

by

on the

since

glutathione

shown

concentration

of glutathione

of glutathione P-450.

it is possible

that

of cultured

is there

that

a of

maintains

maintains

the results both

the effect

the maintenance

medium

and medium

to maintain

Thus

of the medium.

1 is: and

because

However

liver.

of hepato-

concentration

in Table

is suggested

of cytochrome

concentration

in intact

the methionine

the concentration

in a loss

the case

as found

the results

a relationship

results

the glutathione

level

increasing

raised

in a loss

is not

m&l maintains

the culture

question

P-350?

results

to 0.5

24 hours

relationship

thione

mM

be overcome

cytochrome

that

0.1

gluta-

in Table

cytochrome

2 show

P--150 aiid

ylutathione. These the

observations

involvement

Table

2

may

useful

of glutathione

Effect

and

of methionine

in hepatocytes Meihionine

be

on

since

they provide

cytochrome

P-450

the concentration

cultured

for

concn.(mM)

24 hours

(wg/mg 0 .1

4.0

metabolism

of glutathione

concn.

and

maintenance

+ O.hX

2 0.2

0 ..5

11.0

1.c> 2.0

to dissociate and

toxicity.

cytochrome

P-l50

medium".

Cytochrome P-450 concn. (% isolated cells)

protein)

0.0

0.25

in drug

in "P-450

Glutathione

the opportunity

85 + 13

* 98It

6

+ 1.2

945

7

12.0

+ 0.8

101 2

i2

11.2

+ 0.9

87 ?: 70

* denotes The

significantly

results

three

are

separate

isolated

cells

means

different 2 S.D.

for

(i.e.

(p < 0.05)

by Student's

t-test,

individual

values

in preparations

rat

livers

n = 3).

was

160 rt k9 pmoles/mg

The

found

cytochrome

from

intact

liver.

derived

P--150 concentration

of

protein. REFERENCES

1. Grisharn, 2. Sirica,

J.W. A.E.

(1979) K Pitot,

3. Paine,

A-J.

& Legg,

4. Paine,

A.J.

& Hockin,

5. McLean, 6. Thor, 7. Paine, 8. Gihco 9.. Paine,

Int.Exp.Pathol.~,

A.E.M.

R.F.

& Day,

H. & Orrenius, A-J., Bio-Cult A-J.,

H.C.

Hockin, Tissue Williams,

(1980)

(1978)

123-210.

Pharmacol.Revs.

2,

X15-228.

Biochem.Kiopilys.Res.Commun.

8l,

t172-679.

L-J.

(1980)

Biochem.Pharmacol.

29,

3215-3218.

P.A.

(1975)

Riochem.Pharmacot.

E,

37-J2_

S.

(1980)

L-J.

Arch.Toxi(-ol.s,

& Legg,

Culture L.J.

R-F.

(1979)

Catalogue, K Legg,

R.F.

Cibco (1979)

111-43. Kiachem.3. Europe Life

l_&%, 401-103.

Ltd., Sci.

Paisley, 24,

Scotland.

2185-2192.

from

Preliminary Communization

2742

10. Hissin, 71. Bernt,

P.J.

& Hilf,

R.

E. K Bergmeyer,

(Bergmeyer

H.U.

H.U.

ed) pp

12. Reed,

D.J.

& Bentty,

(Sies

Hand

Wendel

(197h)

(1974)

1644-1647

P.W.

A eds)

Anal.Biochem.

(1978) pp.13-21

",

in Methods

Academic

Springer

of Enzymatic

Press

in Functions

213-226. Analysis

volume

4

London.

of Glutathione

Verlag

Berlin,

in Liver

Heidelberg

and Kidney and New

York.