Pergamon Corn/~. Bior/~mt. P/r.wio/. Vol. I IOC. No. I, pp. 35-40. 1995

Copyright ‘(‘ 1995 Elsevier Science Ltd Printed in Great Brxtain. All rights reserved

0742-X413/95 $9.50 + 0.00

A study of gender, strain and age differences in mouse liver glutathione-S-transferase

Eliann Egaas, * J. Greg Falls? and Walter C. DautermanJf

*Norwegian Plant Protection Institute, Fellesbygget, N-1432 As, Norway; and TNorth Carolina State University, Department of Toxicology, Box 7633, Raleigh, NC 27695, U.S.A.

The hepatic cytosolic glutathione S-transferase (GST) activity in four strains of the mouse and one strain of the rat was studied with the substrates 1-chloro-2,4_dinitrobenzene (CDNB), 1,2-dichloro- 4-nitrobenzene (DCNB), ethachrynic acid (ETHA), cumene hydroperoxide (CU) and atrazine as the in vitro substrates. In the mouse, significant gender, strain and age-related differences in the GST activity towards CDNB and atrazine were found between adolescent and sexually mature males and females of the CD-l, C57BL/6, DBA/2 and Swiss-Webster strains, and the differences were larger with atrazine as the substrate. With DCNB and CU a similar tendency was observed, however not significant for all strains. The GST activity towards ETHA was also gender and strain specific, but revealed no age-related differences. The herbicide atrazine seems to be a useful substrate in the study of strain and age-related differences in the mouse GST class Pi.

Key words: Mouse hepatic glutathione S-transferase; Gender, Strain; Age; Atrazine; GST class Pi.

Comp. Biochem. Physiol. IlOC, 3540, 1995.

Introduction

The GSTs are a multigene family of detoxifi- cation enzymes that protect the body against chemical carcinogenesis (reviewed by Hinson and Kadlubar, 1988). Most of the exploratory work on GSTs was performed on the rat, thus, they are the best understood and provide the standards by which other species are compared. A species-independent classification of the dimeric GST into four classes; Alpha, Mu, Pi and Theta, has been suggested (Mannervik et al., 1985; Meyer et al., 1992). The class Pi has received considerable interest as a possible early marker for cancer, as it markedly increased in preneoplastic nodules in the rat liver (for a review see Sato et al., 1990). In normal hepatic tissues, the male mouse and Salmonoid fish are rich sources of GST class Pi (McLellan and Hayes, 1987; Dominey et al., 1991).

Correspondence 10: E. Egaas. Norwegian Plant Protection Institute, Fellesbygget, N-1432 As, Norway. Tel. 64949308; Fax 44949226.

Received 9 May 1994; accepted 28 September 1994.

The mouse isozymes are homodimers charac- terized by different substrate specificities: GST MI (class Alpha) has a comparatively high activity with cumene hydroperoxide (CU), GST MI1 (class Pi) with ethacrynic acid (ETHA) and GST MI11 (class Mu) with 1,2-dichloro-4-nitro- benzene (DCNB) (Mannervik et al., 1985). In a comparative study of the GST activity towards CDNB, DCNB, ETHA and atrazine in 10 species (including fish, insects, mammals and one plant), the GST activities towards ETHA and atrazine were similarly distributed between the species, indicating that atrazine may be a specific substrate for the GST class Pi (Egaas et al., 1993). Furthermore, the mouse hepatic GST activity towards ETHA and atrazine was 5510-fold higher than in the other species covered by the investigation.

In the present study, the GST-mediated metabolism of atrazine in the mouse has been further investigated. Conflicting reports regard- ing strain-specific variations in the mouse GST have been published (Wheldrake et al., 1981;

35

36 E. Egaas et al.

Borroz et al., 1991; McLellan and Hayes, 1987). Thus, four strains of mouse, separated accord- ing to gender, were included in the present study. Since in our previous study (Egaas et al., 1993) a low activity of atrazine metabolism was observed in the rat, one strain of rat was also included. Furthermore, interesting differences between the genders develop at maturity in the mouse (Hatayama et al., 1986) and the rat (Ketterer et a/., 1988; Igarashi et ul., 1987). Thus, all strains were represented two times after delivery; at day 44 (mouse) or day 40 (rat). when they generally had reached puberty and were considered late adolescent, and at day 88, after onset of sexual maturity.

Materials and Methods Chemicals

ETHA was recrystallized from toluene. DCNB and CDNB were recrystallized from ethanol. PAA-gels and SDSPAGE buffer strips for PhastSystems were purchased from Pharmacia, Uppsala, Sweden. “C-ring-labelled atrazine was a gift from the Ciba-Geigy Corp. (Greensboro, NC). All other chemicals were purchased from Sigma Chemical Co (St. Louis, MO) or from Merck, Darmstadt or Aldrich- Chemie GmbH & Co (Steinheim, Germany).

Equipment

Centrifugations were carried out in a Sorvall centrifuge, Model RC-SB and a Centricon 2070 centrifuge with a 70.28 rotor (Kontron, Switzerland). A thermostatted Uvicon 860 (Kontron, Switzerland) spectrophotometer was used for enzyme activity and protein concen- tration measurements.

Animds

Male and female rats, SpragueeDawley, and mice, CD-l (CDl), C57BL/6 (C57), DBA/2 (DBA) and Swiss-Webster (SW) at an age of about 30 days old were obtained from Charles- River Laboratories (Raleigh, NC). The animals were divided into groups according to strain, gender and age and allowed food (Prolab ani- mal diet) and water ad libitum. Housing was under controlled temperature and lighting con- ditions. At the appropriate age (mice; 44 and 88 days old, rats 40 5 2 days and 88 + 1 day old), the animals were euthanized by CO> asphyxi- ation, livers (pooled from seven mice or four rats) were excised, rinsed with homogenization buffer (50 mM potassium phosphate, pH 7.5 with 0.1 mM EDTA and 1.15% KCI) (Levi and Hodgson, 1983) and homogenized with buffer (4 volumes of liver wet weight) in a Polythron” homogenizer. The samples were centrifuged at 10,OOOg for 15 min. The resulting supernatant

was filtered through glass wool and re- centrifuged for 1 hr at 100,OOOg. The post- microsomal supernatant was stored at -80°C.

Enzyme assays

GST activities were measured in the 100,000 g supernatant in 0.1 M Na-phosphate buffer con- taining 1 mM EDTA, with CDNB, DCNB and ETHA (Habig et al., 1974) CU (Lawrence and Burke, 1976) or atrazine (Guddewar and Dauterman, 1979) as the substrate. When using CDNB and DCNB, the optimum pH was 7.5, whereas ETHA, atrazine and CU were measured at pH 6.5, 7.4 and 7.0, respectively. The incubation temperature was 37’ C except for CU, which was measured at room temperature. All assays were duplicated twice and then run three times, always corrected for non-enzymatic conjugation. Protein concentration was deter- mined by the Bio-Rad method (Bradford, 1976). or by the method of Schaffner and Weissman (1973) with bovine serum albumin as the standard.

Statistical method

Statistical methods were carried out with the Mann-Whitney test for the difference between two population means (Ben-Horim and Levy, 1984) using a significance level of P < 0.05.

Results and Discussion

The levels of GST activity in the hepatic cytosols from male and female mice of the strains CDl, DBA, C57 and SW at day 44 and 88 measured with 5 different substrates are presented in Table 1. The GST activities towards CDNB, ETHA and atrazine were sig- nificantly higher in the males than in the females of the corresponding strain and age, except for the CDNB activity in the 44-day-old SW strain. With DCNB, a slightly similar tendency was observed; however, this was not significant for the 44-day-old CD1 and C57. Furthermore, the DCNB activity in the female of the SW strain was less than half the corresponding levels of other strains in both age groups. Again except for the SW strain, the glutathione peroxidase activity (measured with CU as a substrate) was generally lower in the males than the females.

The percentage difference between the GST activity of males and females varied greatly with the substrate used. Thus, with DCNB and CU, the GST activities of one gender (respectively females and males) were in the range 68890% of the opposite gender. With CDNB and ETHA the GST activities in the females were usually below 50% of the activity in the corresponding males, and with atrazine usually below 20% (Table 1).

Stra

in

Tab

le

1. C

luta

thio

ne

S-tr

ansf

eras

e (G

ST)

activ

ity

in

the

hepa

tic

cyto

sol

of

mou

se

and

rat

CD

NB

(p

mol

/mg

Pjm

in)

DC

NB

(n

mol

/mg

Pjm

in)

ET

HA

(nm

ol/m

g Pj

min

) A

traz

in

(pm

ol/m

g P/

hr)

CU

(n

mol

/mg

Pjm

in)

Gen

der

LA

SM

L

A

SM

LA

SM

L

A

SM

LA

SM

M

CD

1 F

9.1

1.0.

0 bc

def

9,

>1

2.9

_t 0

.5

M

6‘2

2 0.

6 D

BA

a,

d.e,

f F

2.6

i_ 0

.8

M

c57

F M

SW

F M

SD

F

7.0

F 0.

2

a,d,

e,f

3.5

5 0.

7 d

3.6

+ 1.

2 a,

b,c,

f 2.

4 k

0.3

c,f

1.9 F

0.4

e,f

2.6

k 0.

7

11.1

*

1.3

d,e

3.5

rt; 0

.1

b 9.

5 2

2.4

d,e

3.2

+ 0.

2

9.8;

1.

1

d,e

3.5

* 0.

3

6.9

f 0.

5

a,b,

c,e

3.0

+ 0.

4

2.6

+ 0.

2

2.5

+ 0.

I

54+

10

46 &

3

f 54

f

2

d,e

43 t

I

CJ-

59

+

7

49 *

5

d,f

61 +

2

b,e

17 &

8

C

44+3

e 50

+

5 f

69 +

2

54:

I b,

d 61

‘L

9

49ei

2

67 &

2

61e+

7 d

61 _

t7

l,“,, a,c

51 +

6

,,‘,I

17 i

_ 2

c,d,

e 29

+ II

9411

7

d,e

27+

14

94 _

c 1

d,e

32&

21

41 i

_ 17

a,

b,c,

e 17

18

l9+2

e,f

29 +

3

f

71+

6

d,e

19&

3

90 5

20

d,

e 21

&9

91 &

18

d,e

24 +

9

57 r

t: I4

a,b,

c,e

1855

IlkI

16:

I

634

+ 33

b,

e,f

169+

I

b 13

27 &

319

a,

d,e

229

+ 2

a,d,

f I.

516

+ 18

4 a,

c,e

260

2 40

d

444+

II

0 b,

c,e,

f 14

1 i:

22

b,c

60 ;

t 20

80 +

0

1434

i

122

d,e

180+

74

1553

f

15

c&e

l88&

7 d l3

98&

44

d,e

212*

21

d 82

6 +

I4

a,b,

c,e

134+

9 b,

c

8Ok2

0

70+

IO

549

+ 32

530

f 37

556

_+ I3

541;

9 e,f

598f

17

b.

f

245

+ 22

e,

f 37

2 +

32

f

366

+ 8

b,c,

d,e

520

+ IO

b.

c,d

460

+ 18

a,

c,d,

e 58

3 _+

I

a,c,

d 61

5 +

_ 27

a,b

654

k 22

a,b

654

+ IO

a,b,

e 61

8 +

21

a,b

376)

10

376:

42

GST

ac

tivity

to

war

ds

I-ch

loro

-2,4

-din

itrob

enze

ne

(CD

NB

).

1,2-

dich

loro

-4-n

itrob

enze

ne

(DC

NB

),

etha

chry

nic

acid

(E

TH

A),

at

razi

ne

and

cum

ene

hydr

oper

oxid

e (C

U)

in h

epat

ic

cyto

sols

fr

om l

ate

adol

esce

nt

(LA

) an

d se

xual

ly m

atur

e (S

M)

mal

es a

nd

fem

ales

of

the

mou

se,

CD

-I

(CD

]),

DB

Aj2

(D

BA

), C

57B

L/6

(C

57)

and

Swis

s-W

ebst

er

(SW

) an

d ra

t, Sp

ragu

e-D

awle

y (S

D).

All

anal

yses

wer

e pe

rfor

med

in

dup

hcat

e,

and

repl

icat

ed

337

times

. St

atis

tical

ly s

igni

fica

nce

at P < 0.05.

a,b,

c,d:

di

ffer

ent

from

re

spec

tivel

y th

e C

DI,

D

BA

, C

57

and

SW s

trai

n;

e: d

iffe

rent

fr

om

the

fem

ales

of

the

sam

e st

rain

: f:

dif

fere

nt

from

the

sex

ually

mat

ure

of t

he s

ame

stra

in

and

sex.

38 E. Egaas et ul.

A difference in GST activity between day 44 and 88 was observed with some of the substrates (Table 1). Thus, with CDNB, the activity of males of all the strains was significantly higher at day 88 compared to the day 44 mouse. Furthermore, with DCNB as the substrate, only the females (except SW) increased in activity from day 44 to 88. In contrast, no change in activity was observed between day 44 and 88 with ETHA as substrate. For atrazine, the outbred CD1 and SW males doubled in activity from day 44 to day 88, whereas the males of the inbred strains DBA and C57 did not change. For CU, the C57 strain only increased sightly in activity between day 44 and day 88.

The levels of GST activity towards CDNB, DCNB, ETHA and CU in the 88 day old DBA and C57 mouse shown in Table 1 were in accordance (20@60% lower) with the levels in hepatic cytosol from the 91 day old DBA and C57 published by McLellan and Hayes (1987) who presented evidence that some of these differences were related to a gender-specific expression of the GST MI1 in the mouse. This is supported by our observations of the changes with age in the mouse GST activities between day 44 (late adolescent) and day 88 (sexually mature). Thus, a selective androgen regulation of the expression of the mouse GST MT1 has been documented (Hatayama et al., 1986).

In the rat, the hepatic GST activity towards all substrates, except atrazine, was significantly different in males and females both at day 40 and day 88 (CDNB only at day 40) (Table 1). The GST activity towards atrazine was only just detectable. In contrast to the results in the mouse, only minor (however significant) gender related differences in activity were observed with CDNB (44 day only), DCNB, ETHA and CU as substrates. Minor, but significant age related GST differences in activity towards CDNB (male), DCNB (female) and ETHA and CU (both sexes) were observed.

A gender-specific regulation of the class Pi subunit in the rat has not been described, and is not indicated in our results. However, in both the rat and the mouse, the higher GST peroxidase activity in the females compared to the males and has been connected to a gender- specific regulation of the GST class Alpha (Igarashi et al., 1987; McLellan and Hayes, 1987) in the liver.

Since atrazine was the GST substrate that revealed the largest difference in GST activity between genders of mouse at both stages of sexual maturity, the data on atrazine from Table 1 were rearranged in Fig. 1 together with the corresponding values obtained for the rat. In the 44 day old male mouse (Fig. la) the atrazine values of the DBA and the C57 strains were

a

CD1 DBA c57 SW SD

b

1 L- A- 157 SW SD CD1 DBA (

m

L--l

Fig. I. GST activity towards atrazine (nmol/mg P/hr) in hepatic cytosols frdm (a) 44-day-old and (b) 88-day-old males and females of the mouse CD-1 (CDI). DBAi2 (DBA), C57BL/6 (C57) and Swiss+Webster (SW) and (a) 40-day-old and (b) 88-day-old males and females of the

rat (Sprague+Dawley, SD).

similar and 2-3 times higher than those of the CD1 and the SW strains. At day 88, the atrazine value was increased in all strains. At this point, the GST activity towards atrazine was highest in the DBA strain; the CDl, however, had more than doubled the activity since day 44 and was similar to the C57 strain, whereas the SW male still had only half the value of the other three. The same tendency between the strains, although at a much lower level of atrazine activity, was observed in the female mouse (for the significance of the data, see Table 1). How- ever, even the atrazine activity shown by the female from the SW strain was twice the corre- sponding activity in the rat.

The SW and the CD1 male, in which the GST activity towards atrazine doubled between days 44 and 88, are both outbred strains. Further- more, the CD1 strain originates from a group of Swiss mice that served as progenitors (Charles- River Laboratories, information sheet). This may explain the similarities in these strains at day 44. It may be a coincidence that the C57 and the DBA males, which had no similar dramatic increase in GST activity towards atrazine be- tween days 44 and 88, are both inbred strains. It is, however, surprising, as the two outbred

Gender, strain and age differences in the mouse 39

strains grow faster and are the heavier in this time interval (Charles-River Laboratories, in- formation sheet). However, the two inbred strains may have reached sexual maturity at a lower body weight than the outbred strains. Thus, genetic influences other than those affect- ing body weight contribute to the differential timing of pubertal events in DBA and C57 (Nelson et al., 1990).

In connection with its widespread use and persistence in the environment, atrazine has been exhaustively studied in terms of its toxico- logical properties, and has demonstrated low acute toxicity in a number of animal species (Seiler et al., 1992). In a long-term experiment, however, a mixture of atrazine and another triazine, simazine, was reported to induce a variety of tumors in Swiss albino mice (Donna et al., 1981). It has long been known that differences in the metabolism of atrazine by plants could be responsible for some of the observed differences in the sensitivity of certain plant species to this herbicide (Jensen, 1982). Thus, resistant inbred maize lines with a rela- tively high GST activity are protected against atrazine toxicity (for a review of the role of GST in triazine tolerance and resistance, see Dauterman, 1989). Since the results in the pre- sent paper indicate that the hepatic GST activity towards atrazine in the SW strain is significantly lower than in the other mouse strains covered by this investigation, it would be interesting to study whether the atrazine-mediated tumor induction observed by Donna et al. (198 1) in the mouse is strain dependent.

Acknowledgements-This project was funded by the Agri- cultural Research Council of Norway, the Norwegian State Pollution Control Agency and by PHS grant ES-00044 from the National Institutes of Environmental Health Sciences, U.S.A. We thank the Ciba-Geigy Co. for their generous gift of atrazine14C and atrazine.

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