Differential activities of rat and human lung glutathione S-transferase isoenzymes towards benzo(a)pyrene epoxides

Vol. 133, No. 3, 1985 December 31, 1985

BIOCHEMICAL

DIFFERENTIAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 863-867

ACTIVITIES OF RAT AND HUMAN LUNG CLUTATHIONE S-TRANSFERASE ISOENZYMES TOWARDS BENZO(a)PYRENE EPOXIDES Yogesn C. Awasthi

and Shivendra

V. Singh

Department of Human Biological Chemistry and Genetics, The University of Texas Medical Branch, Galveston, TX 77550 Mukul Das and Hasan Mukhtar Department of Dermatology, Case Western Reserve University and Veterans Administration Medical Center, Cleveland, OH 44106 Received

November

4, 1985

SUMMARY: The isoenzymes of human and rat lung glutathione S-transferase (GST) differ among themselves in their activities towards the epoxides of benzo(a)pyrene (BP). The Ya’ and Yc-type subunits of rat lung GST exhibit maximum activities towards BP-4,5-oxide and BP-7,8-oxide suggesting that these two subunits are preferentially involved in the detoxification of highly reactive epoxides and diolepoxides of polycyclic aromatic hydrocarbons (PAH). The studies with human lung CST isoenzymes indicate that BP-4,5-oxide, and BP-7,8-oxide are preferred substrates for the cationic (p1 8.3) form of the enzyme. Identification of compounds which can selectively induce these isoenzymes of GST could prove useful as inhibitors of PAH induced neoplasia. 0 1985 Academic Press, Inc.

Polycyclic ubiquitous

aromatic

hydrocarbons

environmental

organs of rodents

pollutants

act

as precarcinogens

epoxides

(2).

These epoxides

to diol-epoxides

as the ultimate

carcinogenic

are detoxified

through

compounds

such as butylated

forms

protection

and an identification

PAH-epoxides

to produce

first

undergo

are then converted

conjugation (GST) (3).

to dihydrodiols

(2).

glutathione

Enhancement

(GSH),

PAH-induced which

neoplasia

which

to

are further

have been implicated

Epoxides

and diol epoxides

a reaction

catalyzed

in target

by

tissues by

hydroxytoluene

have been

(4).

in multiple

GST exist

posses maximum

of compounds

and

activation

which

of GST activity

and butylated

are

biologically

metabolic

of PAH

with

(BP)

in lung and other

inert

diol epoxides

of isoenzymes

may lead to a search

cancer

Bay region

hydroxyanisole against

as benzo(a)pyrene

BP, are relatively

must

metabolites

S-transferase

shown to provide

that

(2).

glutathione

such

and are known

The PAH, including

(I).

essentially

metabolized

(PAH)

activities

preferentially

towards

induce

these

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BIOCHEMICAL

Vol. 133, No. 3, 1985 isoenzymes.

Such compounds

would

be expected

to provide

of protection

against

PAH-induced

neoplasia.

the mechanism present least

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

in rat lung and these forms arise from the different five

structurally

isoenzymes

and functionally

of rat lung

specificities

as well

as human

of these isoenzymes

subunit

of rat

lung

activity

towards

distinct

towards

and cationic

form

understanding

Six forms

dimeric

subunits

lung

better

Using

we have investigated

BP epoxides.

We provide

(p1 8.3) of human

lung

of GST are

combinations

(6,s).

on

of at

purified

GST

the substrate evidence

GST have

that Ya’ maximum

BP-epoxides.

MATERIALS

AND METHODS

Sources of most of the chemicals used in this study were the same as reported by us previously (5-8). [3I-il-Labelled and unlabelled BP-4,5-oxide and BP-7,8-oxide were obtained from the Cancer Research Program of the National Cancer Institute, Division of Cancer Cause and Prevention, Bethesda, MD. Rat and human lung CST were purified according to previously published procedures (5,9). The purity of these isoenzymes, designated by us previously as GST I (PI 8.8), GST II (PI 7.2), GST III (pl 6.8), GST IV (PI 6.0), GST V (PI 5.3), and GST VI (p1 4.8) of rat lung was established by gels electrophoresis in the system of Davis (IO), Gabriel (1 I) and in reduced-denaturin using the system of Laemmli (12). Results of urea/SDS/2-mercaptoethanol Bpolyacrylamide-slab-gel electrophoresis indicated that the subunit compositions (Table 1) of these isoenzymes correspond to those reported previously (5,8). The GST activity using BP-4,5-oxide and BP-7,8-oxide as substrate was determined according to the method of Mukhtar and Bend (13). A typical reaction mixture in a total volume of 0.9 ml contained 120 ,umol Hepes buffer (pH 7.85), 4.5 pmol GSH, 100 ~1 of purified GST. The reaction was initiated by the addition of 9 n mol of BP-4,5-oxide or BP-7,8-oxide in 1 p1 methanol. After 1 min. of incubation at 37OC the reaction was stopped by the The extraction was repeated twice and the aqueous addition of 2.6 ml of ethylacetate. solution containing the GSH conjugate was counted. The protein content was determined according to the method of Bradford (14). RESULTS The specific six isoenzymes nomenclature

activities of rat lung

studies

ourselves subunits Data towards subunit

with

of rat tissues

nomenclature

those

BP epoxides

GST are presented

for GST subunits

have used the previous these

towards

AND DISCUSSION

in Table

observations

nomenclature

1 indicate

BP epoxides. which

appear

that

the GST isoenzyme

As reported to be specifically

prFviously expressed 864

Even

in order (8).

compositions though,

a revised

recently

to correlate Also,

of the

(15) we

the results

we have

of

refrained

(15) because as yet the Ya’ and and Yc’

of lung GST (6,7) have not been assigned

in Table

1.

have been suggested

system (16-18)

of previous

from the use of revised

and the subunit

names in the new nomenclature. VI (PI 4.8) has maximum

(6,s) this

isoenzyme

in lung.

Maximum

activity

is a dimer affinity

of Ya’

of GST VI

Vol. 133, No. 3, 1985

Table

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

Human and Rat Lung CSH S-transferase activities towards Benzo(a)pyreneJQoxide (BP-4,5-oxide) and Benzo(a)pyrene-7,8-oxide (BP-7,8-oxide)

I:

Subunit Compositions (5-s)”

Specific Activitya BP 4 5 o~;ts/mg protein) BP-7,8-oxide - , -

Rat Lung CST: GST I (PI 8.8)

YCYC’

95.5 + 9.1

23.2 + 1.8

GST II (pl 7.2)

YaYa’

48.4 + 3.3

10.5 + 0.7

CST III (pl 6.8)c

YaYb

68.3 + 1.9

16.7 + 0.9

GST IV (PI 6.OF

YaYb

45.5 + 2.4

10.8 + 0.9

GST V (PI 5.3F

YaYb

94.4 + 2.0

22.5 + 1.4

GST VI (PI 4.8)

Ya’Ya’

164.4 2 7.0

38.7 + 2.3

Cationic (PI 8.3)

29.7 + 0.1

7.1 + 0.1

Anionic (pi 4.9)

9.4 + 0.2

2.1 + 0.1

Human Lung GST:

aQne unit of enzyme utilizes S.E.M.

I n mole substrate/min.

of three determinations;

composition

at 37%.

Values represent

b, These numbers denote the references

mean +

to our subunit

studies; c, The nature of Yb type subunits in these isoenzymes is not completely

understood.

The Ya type subunits of these isoenzymes are immunologically

distinct

from

the Ya’ subunits (5,7).

(Ya’ Ya’) for the binding

of BP and/or

activity

of this

indicate

that the Ya’ subunits

in the defense highest

isoenzyme

towards

CST I (p1 8.8).

activity

of lung GST could

towards

As demonstrated distinct

Yc-type

these studies,

therefore,

suggest

contribute

the toxic

have an important

effects

impair

activity.

This

reported

here

physiological

role

of BP is associated is a dimer

and also binds BP in vivo (8).

in the detoxification

would 865

imply

The next

with

rat lung

(Yc Yc’) of two The results

Yc’ subunits

of BP epoxides.

of BP in vivo

catalytic

of PAH carcinogens.

that the Yar and Yc and/or

that the binding

catalytic

BP-7,8-oxide

(7,8) this isoenzyme

subunits

(8) have demonstrated their

and

both the oxides earlier

immunologically

GST preferentially

in vivo (81, and maximum

BP-4,5-oxide

of this tissue against

catalytic

metabolites

of

of rat lung

Our earlier

studies

to rat lung GST I and VI does not that

both

these

isoenzymes

can

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Vol. 133, No. 3. 1985 simultaneously

detoxify

non-catalytic

BP and its epoxides by their removal from circulation

binding as well as by conjugating

through

them to GSH. The isoenzymes,

CST II

(p1 7.21, CST III (PI 6.8), and GST IV (pl 6.0) have relatively

lower specific

towards

of GST V (pl 5.3) may be

both the epoxides of BP. Some what higher activity

due to slight contaminations strongly

suggest

mechanisms

that

activities

of CST VI whose p1 is closer to that of GST V. This would

GST family

of isoenzymes

of lung in the detoxification

are important

in the defense

of highly toxic PAH and their metabolites,

and that the Ya’ and Yc type subunits appear to contribute

more efficiently

in this

process. In human lung two major isoenzymes Table 1 also show that cationic towaras

BP-4,5-oxide

indicate towards

enzyme has about three times higher specific activity

and BP-7,8-oxide

as compared to the anionic enyzme.

that even though the cationic substrate,

of GST have been described (9). Data in

enzyme has seemingly

1-chloro-2,4-dinitrobenzene

This may

very little

(P), it can more effectively

activity

participate

in the conjugation of BP epoxides with GSH in human lung. This could have important implications selectively epoxides

for

PAH-induced

human

lung

carcinogenesis.

Compounds

induce these subunits of GST in lung which preferentially could

prove

useful

as chemopreventive

agents

against

which

detoxify

PAH

PAH-induced

carcinogenesis. ACKNOWLEDGEMENTS This investigation

was supported in part by U.S. PHS grants CA 27967 and CA

38028 awarded by National Cancer Institute, Institute,

and grant GM 32304 awarded

grant EY 04396 awarded by National Eye by National

Institute

of General

Medical

Sciences. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8.

National Academy of Sciences, Committee on Biological Effects of Atmospheric Pollutants. Particulate Polycyclic Organic Matter (19821, Washington, D.C. Conney, A.H. (1982) Cancer Res., 42, 4875-4917. Gelboin, H.V. (1980) Physiol. Rev., a 1107-l 166. Wattenberg, L.W. (1983) Cancer Res., G 2448s-2453s. Partridge, C.A., Singh, S.V., Hong, T.D., Theodore, C., Dao, D.D., and Awashti, Y.C. (1985) Int. J. Biochem., 11, 331-340. Singh, S.V., Partridge, C.A. and Awasthi, Y.C. (1984) Biochem. J., a, 609-615. Singh, S.V., and Awasthi, Y.C. (1984) Biochem. J., 224, 335-338. Singh, S.V., Srivastava, S.K. and Awasthi, Y.C. (1985) FEBS Letters, 179, ill114.

Vol.

133,

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No. 3, 1985

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