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CYTOCHROME

P-450 IN A CULTURED

HUMAN LYMPHOCYTE

Hira L. Gurtoo, Harvey Departments

.I. Freedman,

of Experimental

Roswell

especially

carcinogens,

which are metabolized somal mixed including

monocytes

five years:

smokers.

who compared

lymphocyte

limited

availability

studies

on the mechanism

population.

Ig-synthesizing

normal

B-lymphocyte

of carcinogens, enzymes,

activate

containing

10% heat inactivated

and 50 pg/ml streptomycin. C02:35%

a period

lag period

of exponential

harvested maximal

a

AHH activity

[IO] as modified

It is well established induction

Purposes

with a of the

and detailed

we investigated

and under proper

75 stable

of the mixed

culture

cell

con-

(RPMI-1788),

and inducibility

an

compara-

of the cell line makes

of cells that are required

for detailed

function

stud-

oxygenases

and

by which

these

studies on the mechanisms

carcinogens

and most other xenobiotics. of 0.5 x lo6 cells/ml

fetal calf serum were incubated

in WMI-1640

(FCS), 100 units/ml

penicillin

at 37' in an atmosphere

in cell growth of 12 hrs was observed; until 50-60 hrs in five different

the initiation

which was determined

by Gurtoo c

et al. [8],

of 5%

humidity.

growth

at 46-48 hrs after

by Paigen

of AHH, and also because

This property

at a density

The cultures

air and 100% relative

Initially

drugs,

the past

Rasco -et al. [7]

of lung cancer patients

oxygenases,

purification

cells were suspended

during

and lung cancer risk

One of these cell lines

and for detailed

and/or detoxify

The RPMI-1788

[3,4].

attention

AHH activity

human tissues,

So far we have found three lymphocyte

[8,9].

source for large quantities

related

lymphocytes

cell line, has AHH activity

lymphocytes

hydrocarbons of the micro-

in various

for enzyme purification

function

AHH activity.

ies on the metabolism

medium

required

culture.

metabolically enzymes

of the progeny

of mitogenic-prestimulation

able to fresh mitogen-activated it a convenient

aromatic

have not been confirmed

of the significance

tissues

appreciable

environmental

(AHH) activity

has received

lymphocyte

these reports

of human mixed

in the absence

possess

between

Because

of human

of numerous

[S], Trell -et al. [6], and more recently

cell lines in permanent

lines which, ditions,

&..

1978)

has been reported

lymphocytes

AHH inducibility

control

16 August

[2] and mitogen-activated

correlation

However,

matched

NY 14263, U.S.A.

hydroxylase

AHH activity

[1], macrophages

g

and Immunology,

(BP) and other polycyclic

in mitogen-activated

in cigarette

lymphocyte

Buffalo,

Biology

is focused on the metabolism

oxygenases.

a positive

and Jun Minowada

Molecular

Institute,

benzo(a)pyrene

Kellermann

have reported

Paigen

by the aryl hydrocarbon

function

AHH activity

Beverly

.I. Marine110

10 July 1978; accepted

much attention

Currently,

Anthony

Therapeutics,

Park Memorial

(Received

CELL LINE

a.

of the cultures,

by the fluorometric

this was followed

experiments.

Cells were

since this corresponds method

by

to

of Nebert and Gelboin

[9].

that AHH is inducible

the cells in culture

by many of its substrates

were exposed 2659

[ll].

to 0.3 uM dibenz(a,h)anthracene

For (DBA)

Preliminary Communications

0.016

0008

-RotPB .“.“.._.-. RQ, m -----

RPM1 - I778

i

01

I

1

425

,

475

450 WAVELENGTH

Fig.

Difference

1. Cells

spectra

{4x109) were

of cytochrome

suspended

centrifuged

in a Beckman

microsomal

ter.

on an Aminco

Using an extinction

concentration microsomes under

was calculated

from phenobarbital

identical

conditions

previously

the contents coefficient

to be 0.2 pmole

KPMI-1788 obtained

cells leads us to suggest from studies

to humans;

instead,

calibrated

to both cuvettes;

The

buffer

The resulting

pellet

1 mM sodium cyanide. was

with homium oxide carbon monaxide

fi'l.-

was

of the sample cuvctte and the spectrum was -1 -1 cm for the cytochrome P-450, the cytochrome

F'-451/106 cells.

of the spectral that mechanistic,

on human cytochrome

For rat liver

rats, spectrum was recorded The concentration

phenobarbital

wirh rodent liver cytochromes

information

phosphate

of equal light absorbance

chat cyanide was omitted.

interpretation

x g for 60 minutes.

containing

or Zknethylcholanthrene-treated

except

was

of 91 mM

liver microsomal protein used was as follows: cholanthrene (MC) 0.8 mg/ml. The significant

at 105,000

x g for 60 minutes.

were added

was centrifuged

The 15,000 x g supernatant

The baseline

DW,f2 spectrophotometer

for 30 set anl..ythrough

recorded.

mM EDTA and homogenized,

The homogenate

(8 mg protein/ml)

to two cuvettes.

A few mg of sodium hydrosulfite

bubbled

cells.

in 35 ml of 0.1 M potassium

again at 105,OUO

in 7 ml. of the above buffer

Three ml were transferred recorded

respectively.

L5-50 Model ultracentrifuge

pel.let was suspended

fpW 7.4) and was centrifuged was suspended

P-4!% in RPMI-1788

in 60 ml of 0.25 M sucrose-l

twice at 15,000 x g for 15 and 5 minutes,

I

5ooo

(nm)

fitted with a Teflon pestle.

using a glass homogenizer,

resulting

CELLS

(PB) 0.6 mg/ml;

properties physical

of rat 3-methyl-

of the cytochrome and biochemical

information

F-450 might not be directly P-450 should be obtained

P-451 in

applicable

to explain

2662

hw

~r~~irninary ~ornrnul~i~~ti~ns

humans metabolize

al variations to explain

the eEfects

lismofdrugs

of xenobiotics

of various

for detailed

of mixed

Acknowledgements Project

Grants

H.J. Freedman

oxygenases

CA-14413

was supported

Pharmacology

Department,

Finally,

Yale University,

examination

factors on the metabo-

as well as may prove useful related

F32-CA-05928.

we would

at

ior the

enzymes.

Grant CA-13038

and

Cancer

Institute.

Reprint

requests

Ill-. should

like to thank Dr. John K. Schenkman,

for his valuable

of the RPMI-1788

involved

source of human cells in

from the National

by the NIH fellowship

to Dr. H.1,. Gurroo.

spectral

a convenient

in part by the Program

and CA-18542

arc

cell line is very useful,

and metabolically

- This work was supported CA-17538,

bases of interindividumechanisms

and physiological

studies on metabolism,

function

be addressed

difference

environmental

as it can provide

least for some of the studies,

purification

and what specific

To this end, the RPMI-1788

and carcinogens.

large quantities

what are the genetic

drugs and carcinogens,

in the metabolism

suggestions

concerning

the

cell microsomes.

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2.

E.T. Cantrell, 1884

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Tnhncrrt.7 ‘LILY **A c.!?.. Sh&d, Prnr hnw_Jr Aci20’” rnnrclr !?‘%C A1VL. I...‘-.. . II”Y”I. yy.L._-- &.__,. D.A. ““.‘.llC