Acute cytogenetic effect of benzene on rat bone marrow cells in vivo and the effect of inducers or inhibitors of drug-metabolizing enzymes

Mutation Research, 298 (1992) 81-90 © 1992 Elsevier Science Publishers B.V. All rights reserved 0165-1218/92/$05.00

81

MUTGEN 01829

Acute cytogenetic effect of benzene on rat bone marrow cells in vivo and the effect of inducers or inhibitors of drug-metabolizing enzymes Kimiko Fujie

a

Yoshiaki Ito b and Sakan Maeda c

a Department of Natural Science, Osaka Women's University, Daisen-cho, Sakai, Osaka 590, Japan, b Public Health Research Institute of Kobe City, Minatofima-Nakamachi, Chuo-ku, Kobe 650, Japan and c Department of Pathology. Kobe University School of Medicine, Kusunoki-cho, Chuo-ku, Kobe 650, Japan (Received 5 April 1992) (Revision received 8 June 1992) (Accepted 8 June 1992)

Keywords: Benzene; Chromosome aberration; Drug metabolization Summary

Acute cytogenetic effects of benzene in LE rat bone marrow cells in vivo were studied. Chromosome aberrations (CA) induced by benzene consisted mainly of gaps and breaks. Cells with exchanges were rarely observed. The incidence of benzene-induced CA was at its maximum level 12 h after the p.o. or i.p. administration of benzene, dependent on the dose of benzene administered, and higher in male rats than in female rats. However, the sex difference was not observed in the repeated inhalation experiment. Chromosome damage was higher with the p.o. than the i.p. administration. LE rats were more sensitive than Wistar and SD rats to the clastogenic action of benzene. Phenobarbital and Sudan lII are well known as inducers of drug-metabolizing enzymes. The peak percentage of benzene-induced CA in the rats pretreated with phenobarbital was observed 6 h after the benzene injection, and it occurred at a higher level than in the rats given only benzene. On the other hand, Sudan III pretreatment suppressed benzene-induced CA at all periods after the benzene injection. SKF-525A (a cytochrome P-450 inhibitor) and cyclohexene oxide (an epoxide hydrasc inhibitor) pretreatment also suppressed benzene-induced CA.

Correspondence: Dr. K. Fujie, Department of Natural Science, Osaka Women's University, Daisen-cho, Sakai, Osaka 590, Japan. Abbreviations: CA, chromosome aberration(s); LE rats, Long-Evans rats; p.o., oral gavage; i.p., intraperitoneal; PB, phenobarbital; CHO, cyclohexene oxide; DMBA, 7,12-dimethylbenzlalanthracene; AFB1, aflatoxin B j; BNU, 1-butyl1-nitrosourea; MMS, methyl methanesulfonate; GST, glutathione S-transferase; PCB, polychlorinated biphenyl(s); DMSO, dimethyl sulfoxide; 3-MC, 3-methylcholanthrene; SD rats, Sprague-Dawley rats.

Benzene has been extensively used in industry as a solvent or a starting material for the synthesis of other chemicals since 1888. Furthermore, benzene contained in gasoline is one of the most widely distributed environmental pollutants. The major toxic effect of benzene is hemopoietic toxicity (Hamilton, 1922; Goldstein, 1977). Since chronic exposure of humans to low levels of benzene in industrial settings is associated with aplastic anemia and leukemia, benzene has been

82

classified as a carcinogen for humans (Vigliani, 1964; Hernberg, 1966). Benzene is metabolized into compounds such as phenol, hydroquinone, pyrocatechol, benzoquinone, 1-phenylmercapturic acid, and trans, trans-muconic acid (Parr, 1954; Snyder, 1975; Dean, 1978) by mixed function oxidase enzymes which are found predominantly in the liver, but also in the bone marrow which is the putative target organ of benzene toxicity (Kinoshita, 1981; Bhat, 1988). It is clear that benzene toxicity or carcinogenicity is caused by benzene metabolites (Parr, 1954; Snyder, 1975; Dean, 1978). However, the chemical nature of benzene metabolites ~esponsible for its toxicity or carcinogenicity remains to be fully elucidated. Moreover, the information available on benzene-induced chromosome aberrations in rat bone marrow cells is limited, as compared with that in mice (Dean, 1985). In this paper, we have studied (1) the acute cytogenetic effects of benzene in rat bone marrow cells in vivo, (2) the sex differences in cytogenetic

effects, (3) the effects of the route and timing of benzene administration, (4) the strain differences and (5) the effects on benzene-induced CA of inducers or inhibitors of drug-metabolizing enzymes, such as PB, Sudan III, SKF-525A and CHO. Materials and methods

Animals Non-inbred LE rats at 4 weeks of age were used. Each experimental group consisted of three rats. They were kept at 22°C in an air-conditioned room and given MF (Oriental Ferment Co., Tokyo) and water ad libitum. SD and Wistar rats were purchased from Oriental Ferment Co., Tokyo. Route of administration In this study we examined three routes of benzene administration: i.p., p.o. administration, and inhalation exposure. In the third route, the animals were exposed to atmospheres of 10, 20,

TABLE 1 DOSE- A N D T I M E - D E P E N D E N T B E N Z E N E - I N D U C E D C H R O M O S O M E A B E R R A T I O N IN RAT BONE M A R R O W CELLS A F T E R AN I N T R A P E R I T O N E A L I N J E C T I O N a Sex

Dose (tzl/kg)

Treatment (h)

Number of cells with Gap Break

Exchanges

Number of aberrations/cell (means 5: SD)

Percentage of aberrant cells b (means + SD)

Male

0 300 100 300 500 300 300

0 (control) 6 12 12 12 18 24

4 12 9 11 12 6 4

3 41 26 52 74 29 8

0 0 0 0 0 0 0

0.01 + 0.00 0.14 _+0.02 0.10 + 0.03 0.20 + 0.01 0.28 + 0.03 0.12+0.04 0.03 +_0.00

1.0 _+0.0 13.7 + 1.7 8.7 +_2.6 17.3 5:2.5 24.7 -+_1.7 9.7_+ 1.7 2.7 + 0.5

Female

0 300 100 300 500 300 300

0 (control) 6 12 12 12 18 24

1 14 7 7 11 7 2

2 24 21 31 47 19 7

0 0 0 0 0 0 0

0.01 + 0.01 0.08 ± 0.01 0.08+0.01 0.11 _+0.02 0.19 5:0.01 0.07 +_0.03 0.02 + 0.01

0.7 +_0.5 8.0 +_0.8 7.0_+0.8 10.3 + 1.2 15.7 _+1.2 6.3 + 1.2 2.3 -+_0.5

Chromosome specimens were prepared 6-24 h after various doses of benzene were given intraperitoneally. Each group consisted of 3 rats. 100 cells were examined per rat. b Values are means + SD for 3 rats. Not included are the cells with gaps. * Significantly different from female at P < 0.05. * * Significantly different from female at P < 0.01. a

83

or 60 ppm benzene for 2 h/day, 5 days/week for 2 weeks. Food and water were not given to the rats during the 2-h exposures. Rats were exposed in a 6-1 glass chamber (Sugiyamagen MC type, Tokyo) with 0.2 I/rain air flow. The benzene concentration in the air was first analyzed with a model GC-9AM :Simazu Gas Chromatograph equipped with a flame ionization detector, then it was monitored in the chamber every 30 rain with a gas analyzer (Cosmotector XP-336).

Chemicals Benzene (CAS No. 71-43-2) was obtained from Wako Pure Chemicals Co., Osaka, Japan. Sudan III, 1-(p-phenylazophenylazo)-2-naphthol (Chroma, Stuttgart, Germany) was recrystallized from appropriate solvents and dissolved in sesame oil by gentle heating. SKF-525A, diethylaminoethyl 2,2-diphenylvalerate HCI (Smith Kline & French Laboratories, PA, USA, distributed by Smith Kline & Fujisawa, Tokyo, Japan) was dissolved in physiological saline. CHO (Wako) was

70-

dissolved in sesame oil. PB, obtained from Wako, was dissolved in physiological saline.

Chromosome analysis At various periods after treatment with each chemical, the animals were killed to study the frequency of aberrant metaphase cells in femoral bone marrow. The rats were injected with colchicine (0.3 mg dissolved in 0.3 ml of physiological saline) 1 h before various harvest times. Chromosome specimens were prepared from the femoral bone marrow by the conventional method (Sugiyama, 1971), stained in 2% Giemsa solution (pH 6.8) for 15 min, and microscopically analyzed under code. CA consisted of chromatid breaks, gaps and exchanges: gaps were defined as complete interruptions of the continuity of one or both chromatids not exceeding the width of a chromatid, and breaks as a discontinuity greater than the width of a chromatid, irrespective of whether or not the distal fragment was dislocated. Cells with gaps were not included in the

70.

(a)

(b)

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m

60.

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50.

8 ~ 40

40-

g 30

30-

g- 20-

o_ 20-

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10-

10

0 0

6 12 18 Benzene t~atment (ha

24

0

150 500 Dose of benzene (~l/kg)

7B&

Fig. 1. (a) Variation in the incidence of aberrant cells in rat bone marrow cells over time after 500 p.l b e n z e n e / k g b.w. was orally given. Peak percentages for male and female are 44.3 ± 9.9 and 28.7 ± 6.6 respectively. (b) Relationship between benzene dose and the percentage of aberrant cells in rat bone marrow cells 12 h after oral administration. Each point represents the m e a n ± SD for 3 rats. o, male; A, female.

84 30- (b)

_~ 3O (Q) g

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&to c

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; 1;o s;0 12 ]g 24 Dose of benzene (#l/kg) Benzene treatment (hr) Fig. 2. (a) Variation in the incidence of aberrant cells in rat bone marrow cells over time after 300/xl benzene/kg b.w. was injected i.p. (b) Relationship between benzene dose and the percentage of aberrant cells in rat bone marrow cells 12 h after i.p. injection. Each point represents the mean + SD for 3 rats. e, male; A, female. 0

category of d a m a g e d cells. 100 m e t a p h a s e cells per rat were examined, and the p e r c e n t a g e of aberrant cells was calculated. T h e statistical significance was d e t e r m i n e d by the X Z-test. Results

Benzene-induced CA B e n z e n e - i n d u c e d C A consisted mainly of gaps and breaks (Table 1). N o exchanges were observed in either the i.p. or the r e p e a t e d inhalation experiments. Exchanges were rarely observed following b e n z e n e administration. T h e incidence of aberrant cells increased progressively after the p.o. or i.p. b e n z e n e administration and r e a c h e d a m a x i m u m level after 12 h (Figs. la, 2a). T h e r e a f t e r it decreased with time and r e a c h e d the control level after 24 h. A dose-response relationship was also clearly observed with all administration routes (Figs. lb, 2b, 3). T h e incidence of aberrant cells was higher with the p.o. than with the i.p. route. Male rats were m o r e sensitive than females to the c h r o m o s o m e - b r e a k ing effect of benzene. This sex difference was observed in both the p.o. and the i.p. b e n z e n e experiments but not in the r e p e a t e d inhalation one.

The effect of PB pretreatment on benzene-induced CA PB p r e t r e a t m e n t greatly increased the clastogenic activity of b e n z e n e 6 h after the b e n z e n e

injection but decreased it after the lapse of 12 or 18 h (Fig. 4a). These results show that the peak percentage of b e n z e n e - i n d u c e d C A in the rats pretreated with PB o c c u r r e d earlier and at a higher level than in the rats given only benzene. T h e effect of PB p r e t r e a t m e n t on the clastogenic activity of benzene was d e p e n d e n t on the n u m b e r of times of t r e a t m e n t (Fig. 4b), that is, the incidence of aberrant cells in the rats pretreated with PB increased in p r o p o r t i o n to the dose of PB when administered 6 h after the b e n z e n e injection, but decreased w h e n administered 12 h after the injection.

30dl) O

20..Q

glo-

N

~

0

i

o

2'o

i

6o

Benzene exposure (ppm) Fig. 3. Relationship between benzene exposure dose and the percentage of aberrant cells in rat bone marrow cells 12 h after inhalation. Each point represents the mean+_SD for 3 rats. e, male; A, female.

85

Suppression of benzene-induced CA by Sudan III Rats pretreated with Sudan 1II 24 h before the benzene injection displayed a considerably suppressed incidence of benzene-induced CA in their bone marrow ceils. The suppression was observed at all periods after the b e n z e n e injection (Fig. 5a). The suppressive effect of Sudan III on the clastogenic activity of benzene was proportional to the Sudan 1II p r e t r e a t m e n t dose in the range of 2 - 2 0 m g / k g body weight (Fig. 5b). Sudan III pretreatment 2 or 12 h before the benzene injection did not significantly suppress benzene-induced CA, and the maximum suppression was observed in the rats pretreated with Sudan III 24 h before the benzene injection (Fig. 5c).

with SKF-525A 6 h or 12 h before the benzene injection and in female rats pretreated with SKF525A 12 h before the injection (Fig. 6c). On the other hand, C H O pretreatment 7 h before the benzene injection increased the incidence of aberrant cells, although C H O pretreatment 2 h before the injection suppressed it (Fig. 7c).

Comparison of the sensitiuity of different rat strains to benzene-induced CA Table 2 shows that LE rats are more sensitive than Wistar or SD rats to the clastogenic action of other chemical carcinogens, especially to that of the direct-acting carcinogens such as BNU or MMS, which do not need metabolic activation (data not shown).

Suppression of benzene-induced CA by SKF-525A or CHO

Discussion

SKF-525A or C H O pretreatment of male rats 6 h or 2 h before the injection suppressed the incidence of aberrant cells at all periods after the benzene injection (Figs. 6a, 7a). The suppressive effect of SKF-525A or C H O on the clastogenic activity was dependent on the SKF-525A or C H O dose, respectively (Figs. 6b, 7b). The maximum suppression was observed in male rats pretreated

The present study demonstrates that the administration of benzene to LE rats induced signicant increases of CA in their bone marrow cells. Benzene-induced CA consisted mainly of gaps and breaks. The incidence of aberrant cells increased after p.o. or i.p. benzene administration and reached a maximum level after 12 h. A dose-related response was also observed after

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Benzene treatment (hr) Dose of PB (mg/kg) Fig. 4. (a) Time-dependent variations in the 300/xl benzene/kg b.w.-induced incidence of aberrant cells in bone marrow cells of rats pretreated with PB. PB was administered at 75 mg/kg b.w.i.p., 3 times with 24-b intervals before the benzene i.p. injection, o, male rats given only benzene; o, female rats given only benzene; A, male rats given both PB and benzene; zx, female rats given both PB and benzene. (b) Dose-dependent variation in the incidence of aberrant cells induced by benzene in bone marrow cells of rats pretreated with PB. Chromosome specimens were prepared 12 h after 300 /~1 benzene/kg b.w. was injected i.p. Each point represents the mean+SD for 3 rats. o, A, male; o, zx female; solid line, after 12 h benzene treatment; dashed line, after 6 h benzene treatment.

86 30

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Sudan ]]I treatment (hr) Fig. 5. (a) Variations in time course of the benzene (300 /xl/kg)-induced incidence of aberrant cells in bone marrow cells of rats pretreated with Sudan III, 20 mg/kg b.w. administered p.o. 24 h before the benzene i.p. injection. Each point represents the mean ± SD for 3 rats. e, male; o, female; solid line, rats given only benzene; dashed line, rats given both Sudan III and benzene. (b) Suppression of benzene (300 /xl/kg)-induced chromosome aberration by various doses of Sudan III administered p.o. 24 h before benzene injection. (c) Suppression of benzene (300 /zl/kg)-induced chromosome aberration by Sudan III administered at various times before the benzene injection. Sudan III was administered orally at 20 mg/kg b.w.. Chromosome specimens were prepared 12 h after the benzene injection. Each point represents the mean ± SD for 3 rats. e, male; A, female.

a.

0

-4'8

-24

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SKF-525A treatment (hr) Fig. 6. (a) Variations in time course of the benzene (300 /xl/kg)-induced incidence of aberrant cells in bone marrow cells of rats pretreated with SKF-525A, 10 mg/kg b.w. administered i.p. 6 h before the benzene injection. Each point represents the m e a n ± S D for 3 male rats. Solid line, rats given only benzene; dashed line, rats given both SKF-525A and benzene. (b) Suppression of benzene (300/zl/kg)-induced chromosome aberrations by various doses of SKF-525A administered i.p. 6 h before the benzene injection. (c) Suppression of benzene (300/xl/kg)-induced chromosome aberrations by SKF-525A administered at various times before the benzene injection. SKF-525A was administered i.p. at 10 mg/kg b.w.. Chromosome specimens were prepared 12 h after the benzene injection, e, male; A, female.

87 TABLE 2 C O M P A R I S O N O F CA I N D U C E D BY FIVE C H E M I C A L S IN T H R E E R A T S T R A I N S Sex

Male Male Female Male Female

Chemical

Benzene DMEA AFB~ BNU MMS

Treatment

Dose

i.p. i.p. i.p. p.o. i.p.

300/z l / k g 100 m g / k g 10 m g / k g 200 m g / k g 75 m g / k g

Time

Percentage of aberrant cells (mean _+SD)

(h)

LE

Wistar

SD

12 24 18 18 18

17.3_+ 2.5 26.8-+ 7.4 54.3_+ 4.2 40.8_+ 5.0 65.4_+10.6

11.0_+2.9 24.2_+6.5 30.0_+5.9 23.5_+5.9 35.0_+7.7

10.3+3.0 16.3_+3.0 31.3_+5.8 32.2_+5.8 39.4_+2.2

Each experimental group consisted of three rats of 4 weeks old. 100 cells were examined per rat,

p.o. or i.p. treatment. Chromosome damage was more severe with p.o. than with i.p. benzene administration. These results are consistent with the data obtained with CD-1 mice by Meyne and Legator (1980). We observed a sex difference in the clastogenic response to benzene for both p.o. and i.p. benzene administration. These results are consis-

tent with the report by Siou et al. (1981) demonstrating that male mice were about twice as sensitive as females to the clastogenic effect of benzene. This sex difference may be due to the hormonal influence on the metabolism of benzene, as pointed out by Siou et al. However, the sex difference was not observed in our repeated inhalation experiment. Styles and Richardson

TABLE 3 E F F E C T S O F I N D U C E R A N D I N H I B I T O R ON B E N Z E N E - I N D U C E D C A 12 h A F T E R I N T R A P E R I T O N E A L T R E A T MENT " Experimental group

Dose (mg/kg)

Sex

PB + b e n z e n e

75 × 3

M

F

Treatment (h) 6 12 18 6 12 18

N u m b e r of cells examined

N u m b e r of cells with Gap Break Exchanges

Number of aberrations/cell (means -+ SD)

Percentage of aberrant cells (means _+SD)

300 300 300 300 300 300

8 13 6 17 7 8

80 24 18 57 14 16

1 0 0 0 0 0

0.36_+0.06 0.08_+0.01 0.07_+0.01 0.27_+0.08 0,07_+ 0.06 0.07 _+0.01

27.0_+3.7 8.0_+0.8 6.0_+0.8 19.2_+2.9 4.7_+3.1 5.3 -+ {).5

3 - M C + benzene

30

M F

-24 -24

300 300

1 5

13 11

0 0

0.05_+0.01 0.03-+0.04

4.3_+0.5 3.7_+ 1.8

Sudan I l l + b e n z e n e

20

M F

-24 - 24

300 300

7 2

12 7

0 0

0.04-+0.01 0.03 + 0.02

4.0_+0.8 2.3 -+ 1.7

S K F + benzene

10

M

- 6 -12 - 6 - 12

300 300 300 300

6 4 6 3

16 16 20 12

0 0 0 0

0.06 -+ 0.02 0.06-+0.02 0.08 _+0.02 0.05-+0.02

5.3 -+ 0.5 5.3_+1.2 6.7 + 1.2 4.0_+0.8

-2 -7 -2 - 7

300 300 300 300

12 8 11 8

24 65 15 46

0 0 0 0

0.12_+0.06 0.29_+0.01 0.06_+0.02 0.21 +_0.01

8.0+2.2 21.7_+1,9 5.0_+0,8 15.3 + 1,2

F C H O + benzene

3.69 mmole

M F

a Chromosome specimens were prepared 12 h after various doses of benzene given intraperitoneally. Each group consisted of 3 rats.

88 30reO

{a)

20-

glo. e-

n

0

i

0

30.

rD .Z3

i

i

1

6 12 18 Benzene treatment (hr)

i

24

(b)

20.

"6 lo-

a_

0

i

(]

i

1.23 2.46 Dose of CHO (mmole/kg)

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30- ~c)

~, 20. "6

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a_

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CHO treatment (hr) Fig. 7. (a) Variations in time course of the benzene (300 ~l/kg)-induced incidence of aberrant cells in bone marrow cells of rats pretreated with CHO, 3.69 mmole/kg b.w. administered p.o. 2 h before the benzene injection. Each point represents the mean + SD for 3 male rats. (b) Suppression of benzene (300/~l/kg)-induced chromosome aberrations by various doses of CHO administered p.o. 2 h before the benzene injection. (c) The effects of pretreatment with CHO at various times before the benzene injection on the incidence of benzene (300 /~l/kg)-induced aberrant cells. CHO was administered p.o. at 3.69 mmole/kg b.w. Chromosome specimens were prepared 12 h after the benzene injection, o, male; A, female.

(1984) have r e p o r t e d that the r e p e a t e d e x p o s u r e of rats to b e n z e n e over a p e r i o d of 5 days d i d not i n c r e a s e the f r e q u e n c y of a b e r r a t i o n s over that s e e n a f t e r a single e x p o s u r e and t h a t the freq u e n c y o f C A o b s e r v e d after 2 h i n h a l a t i o n exposure was significantly h i g h e r t h a n t h a t s e e n after 6 h e x p o s u r e to t h e s a m e c o n c e n t r a t i o n . M o r e over, N o r p o t h et al. (1974) have r e p o r t e d t h a t t h e e x p o s u r e of rats to b e n z e n e v a p o r at 450 p p m for 10 days i n c r e a s e d c y t o c h r o m e P-450 levels 65%. T h e r e f o r e , the a b s e n c e of sex d i f f e r e n c e o b s e r v e d in o u r i n h a l a t i o n e x p e r i m e n t m a y be d u e to the e n z y m e i n d u c t i o n by the r e p e a t e d b e n z e n e exposure t h a t r e d u c e s the g e n e r a t i o n of c l a s t o g e n i c metabolites from benzene. T a b l e 2 shows that L E rats w e r e m o r e sensitive t h a n W i s t a r or S D rats to the c l a s t o g e n i c action of b e n z e n e . H u g g i n s et al. (1965) also d e s c r i b e d that L E rats have a special v u l n e r a b i l i t y to c a r c i n o g e n i c a r o m a t i c h y d r o c a r b o n s . T h e d a t a on t h e c y t o g e n e t i c effects of b e n z e n e in rat b o n e m a r r o w a r e few as c o m p a r e d with t h o s e in mice. This s e e m s to b e why rats, except L E rats, are less sensitive t h a n mice to the c l a s t o g e n i c action of b e n z e n e . T h e rats p r e t r e a t e d with PB s h o w e d t h e p e a k p e r c e n t a g e of b e n z e n e - i n d u c e d a b e r r a n t cells 6 h after the b e n z e n e injection, this p e a k b e i n g e a r l i e r a n d at a h i g h e r level t h a n in the rats given only b e n z e n e . This m a y m e a n t h a t PB p r e treatment increases the rate of benzene metabolism. W e have previously r e p o r t e d (1984) t h a t G S T activity or the c y t o c h r o m e P-450 c o n t e n t of L E rat liver was e n h a n c e d by p r e t r e a t m e n t with PB, PCB o r S u d a n III. G o n a s u n et al. (1973) d e t e r m i n e d t h a t PB p r e t r e a t m e n t i n c r e a s e d the cytochrorne P-450 c o n t e n t of m o u s e liver microsomes, b u t d i d not i n c r e a s e t h e m e t a b o l i c rate of b e n z e n e . In contrast, Post a n d S n y d e r (1983) d e m o n s t r a t e d that PB i n c r e a s e d n o t only t h e c y t o c h r o m e P-450 c o n t e n t of rat liver microsomes, b u t also t h e m e t a b o l i c r a t e of b e n z e n e . O u r results with rats a r e c o n s i s t e n t with those results. This m a y be e x p l a i n e d by t h e d i f f e r e n t r e s p o n s e of rats a n d mice to PB. On the other hand, Sudan III suppressed benz e n e - i n d u c e d C A at all p e r i o d s a f t e r t h e b e n z e n e injection. T h e level of s u p p r e s s i v e effect corres p o n d e d with t h e d o s e of S u d a n III, t h e maxi-

89

mum suppression being obtained in rats given Sudan III 24 h before the benzene injection. Sudan II1 is generally considered to be one of the enzyme inducers of the 3-MC type. Gad-E1-Karim et al. (1984) demonstrated that 3-MC pretreatmerit of CD-1 mice greatly promoted the clastogenic effect of benzene. We therefore examined the effect of 3-MC on the clastogenic activity of benzene. The administration of 3-MC 24 h before the benzene injection also suppressed benzeneinduced CA considerably (Table 3). The inconsistency between our results and Gad-El-Karim's data seems to be due to the different response of rat liver microsomes and mouse liver microsomes to enzyme inducers like 3-MC. This apparent difference between rats and mice is very interesting and worthy of further study. PB and Sudan IlI are well known as inducers of drug-metabolizing enzymes. The pretreatment with Sudan IlI suppressed the incidence of benzene-induced aberrant cells at all periods after the benzene injection, while the pretrcatment with PB just caused the peak incidence of benzene-induced aberrant cells to occur earlier and at a higher level. This difference between PB and Sudan III may be explained by the induction of different drug-metabolizing enzymes, especially a different set of cytochrome P-450 isoenzymes. Benzene is first metabolized into benzene oxide by mixed function oxidases and benzene oxide is further transformed through several pathways into various compounds, such as phenol, hydroquinone, pyrocatechol and benzoquinone (Snyder, 1981). There is considerable evidence (Snyder, 1975; Dean, 1978; Tunek, 1978; Brodfuehrer, 1990) that hydroquinone and benzoquinone are major benzene metabolites responsible for benzene toxicity, such as genotoxicity, hematoxicity and leukemogenesis. However, the chemical nature of benzene metabolites responsible for its toxicity remains to be fully elucidated. Next, we examined the effect of inhibitor drug-metabolizing enzymes on benzene-induced CA. Both SKF-525A and CHO suppressed benzene-induced CA, and these suppressive effects were dependent on their respective doses. However, the CHO administration of 3.7 mmole/kg body weight which is about 20-fold the SKF-525A

dose (70 mg = 0.2 mmole/kg body weight) did not suppress as potently as SKF-525A. Furthermore, CHO administration 7 h before the benzene injection increased the incidence of aberrant cells. SKF-525A administration greatly reduced the generation of pyrocatechol, hydroquinone or benzoquinone, because SKF-525A, a cytochrome P-450 inhibitor (Gad-EI-Karim, 1984), blocks the metabolic pathway from benzene to benzene oxide (the initial metabolite). On the other hand, since CHO, an epoxide hydrase inhibitor (Guenther, 1986), blocks the metabolic pathway from benzene oxide to dihydrodiol, its administration greatly decreases the generation of dihydrodiol and pyrocatechol and may increase the generation of hydroquinone and benzoquinone. Therefore, these results suggest that hydroquinone and benzoquinone are major benzene metabolites responsible for its toxicity. References Bhat, R.V., V.V. Suhrahmanyam, A. Sadler and D. Ross (1988) Bioactivation of catechol in rat and human bone marrow cells. Toxicol. Appl. Pharmacol. 94, 297-304. Brodfuehrer, J.I., D.E. Chapman, T.J. Wilke and G. Powis (1990) Comparative studies of the in vitro metabolism and covalent binding of 14C-benzene by liver slices and microsomal fraction of mouse, rat and human. Drug Metabolism Dispos. 18, 20-27. Dean, B.J. (1978) Genetic toxicology of benzene, toluene, xylenes, and phenols. Mutation Res. 47, 75-97. Dean, B.J. (1985) Recent findings on the genetic toxicology of benzene, toluene, xylenes and phenols. Mutation Res. 154, 153-181. Gad-EI-Karim, M.M., B.L. Harper and M.S. Legator (1984) Modifications in the myeloclastogenic effect of benzene in mice with toluene, phenobarbital, 3-methylcholanthrene, Aroclor 1254 and SKF-525A. Mutation Res. 135, 225-243. Goldstein, B.D. (1977) Hematoxicity in humans. J. Toxicol. Environ. Health. Suppl. 2, 69-105. Gonasun, L.M., C. Witmer, J.J. Kocsis and R. Snyder (1973) Benzene metabolism in mouse liver microsomes. Toxicol. Appl. Pharmacol. 26, 398-406. Guenther, T.M. (1986) Selective inhibition and selective induction of multiple microsomal epoxide hydrolases. Biochem. Pharmacol. 35, 839-845. Hamilton, A. (1922) The growing menace of benzene (benzol) poisoning in American industry. J. Am. Med. Assoc. 78, 627-630. Hernberg, S., M. Savilanti, K. Ahlman and S. Aps (1966) Prognostic aspects of benzene poisoning. Br. J. Ind. Med. 23, 204-209. Huggins, C.B., E. Ford and E.V. Jensex (1965) Carcinogenic

90 aromatic hydrocarbons; Special vulnerability of rats. Science 147, 1153. Ito, Y., S. Maeda, K. Souno, N. Ueda and T. Sugiyama (1984) Induction of hepatic glutathion transferase and suppression of 7,12-dimethylbenz[a]anthracene-induced chromosome aberration in rat bone marrow cells by Sudan III and related azo dyes. J. Natl. Cancer Inst. 73, 177-183. Kinoshita, T., R. Santella, P. Pulkrabek and A.M. Jeffrey (1981) Benzene oxide genetic toxicity. Mutation Res. 91, 99-102. Meyne, T., and M.S. Legator (1980) Sex-related differences in cytogenetic effects of benzene in the bone marrow of Swiss mice. Environ. Mutagen. 2, 43-50. Norpoth, K., U. Witting and M. Springorum (1974) Induction of microsomal enzyme in the rat liver by inhalation of hydrocarbon solvents. Int. Arch. Arbeitsmed. 33, 315. Parre, D.V., and R.T. Williams (1954) Detoxication. XLIX. Metabolism of benzene containing t4C-benzene. Biochem. J. 55, 231-238. Post, G.B., and R. Snyder (1983) Fluoride stimulation of benzene metabolism. J. Toxicol. Environ. Health 11,799810.

Siou, G., L. Conan and M. El Haiten (1981) Evaluation of the clastogenic action of benzene by oral administration with 2 cytogenetic techniques in mouse and Chinese hamster. Mutation Res. 90, 273-278. Snyder, R., and J.J. Kocsis (1975) Current concepts of chronic benzene toxicity. CRC Crit. Rev. Toxicol. 3, 265-288. Snyder, R., S.L. Longacre, C.M. Witmer, J.J. Kocsis, L.S. Andrews and E.W. Lee (1981) Biochemical toxicology of benzene. Rev. Biochem. Toxicol. 3, 123-153. Styles, J.A., and C.R. Richardson (1984) Cytogenetic effects of benzene; dosimetric studies on rats exposed to benzene vapour. Mutation Res. 135,203-209. Sugiyama, T. (1971) Specific vulnerability of the largest telocentric chromosome of rat bone marrow cells to 7,12-dimethylbenz[a]anthracene, J. Natl. Cancer Inst. 47, 12671275. Tunek, A., K.F. Platt, P. Bentley and F. Oesch (1978) Microsomal metabolism of benzene to species irreversibly binding to microsomal protein and effects of modifications of this metabolism. Mol. Pharmacol. 14, 920. Vigliani, E.C., and G. Saita (1964) Benzene and leukemia. New Engl. J. Med. 271, 872-876.