The mutagenic and clastogenic activity of tobacco smoke

Mu,'ation Research, 208 (1988) 237-241

237

Elst.vier

MTR 01308

The mutagenic and clastogenic activity of tobacco smoke * Roumen

M. Balansky, Penka M. Blagoeva and Zvetanka

I. M i r c h e v a

Laborato O' of Chemical Carcinogenesis and Testing, Institute of Ontology, Sofia-1156 (Bulgaria) (Received 15 September 1987) (Revision received 15 February 1988) (Accepted 2 March 1988)

Keywords': Tobacco smoke; Mutagenicity; Clastogenicity; Mouse

Summary Employing the Salmonella/microsome mutagenicity assay it was established that the mutagenic effect of tobacco smoke (TS) (240 cm 3 in a 16-1 glass chamber, at 1 min or 5 min exposure time) in S. typhimurium TA98 depended on the type of $9 mix used. Addition of $9 mix obtained from the liver of 3-methylcholanthrene- or Aroclor-1254-pretreated rats but not from the liver of phenobarbital-pretreated or untreated rats was required to demonstrate the mutagenic activity of TS. One might suggest that pol2rcyclic aromatic hydrocarbons were involved in TS-induced mutagenesis in S. typhimurium TA98. In addition, treatment of BDF 1 mice with TS (600 cm 3 TS in a 14-1 glass chamber, 2 - 6 exposures of 30 min eacl~ with a l-rain interval between them during which a total change of the air was made) caused an up to 3.5-fold increase of the number of micronucleated polychromatic crythrocytes (PCE) in mouse bone marrow detected 24 h after the TS exposure. Furthermore, a stable 2-5-fold elevation of the number of micronucleated normochromatic erythrocytes (NCE) was detected in the peripheral blood of mice treated daily (2 × 30 min) with TS, starting 48 h after the first TS exposure. The application of the micronucleus test in mouse peripheral blood, a more convenient and useful approach for detecting the chronic clastogenic activity of TS, allowed us to establish the cumulative genotoxic effect of TS in mice.

The mutagenic and clastogenic activities of tobacco smoke (TS) were demonstrated in previous investigation employing the Salmonella/microsome mutagenicity assay and the micronucleus test in mouse bone marrow (Balansky et al., 1987). The direct exposure of S. typhimurium TA98 to TS

Correspondence: Dr. R.M. Balansky, Institute of Ontology, Sofia-l156 (Bulgaria). * This study was supported by a grant (N 317) from the Committee of Sciences to the Council of Ministers of the People's Republic of Bulgaria.

caused a 3-9-fold increase of the number of his ÷ revertant colonies. An addition of microsomal metabolic activation system ($9 mix) to the top agar was required to detect this effect (Balansky et al., 1987). It was of some interest to clarify to what extent pretreatment with different enzyme inducers would influence the metabolic activation of mutagenic species in TS. This information might also shed some light on the type of putative mutagens involved in the TS-induced mutagenesis in S. typhimuriurn TA98. In recent studies, applying the standard micronucleus test, a 2-fold elevation of the number of micronuclcated polychromatic erythrocytes (MN

0165-7992/88/$03.50 ~' 1988 Elsevier Science Publishers B.V. (Biomedic',d Division)

238 PCE) in mice bone marrow was detected 24 h after the TS treatment but not 48 h after, when the level of MN PCE dropped to the control values (Balansky et ai., 1987). In addition, no cumulative effect was observed in mice exposed to TS repeatedly during 28 consecutive days (Balansky et al., 1987). A suggestion was made that the lack of cumulation as well as the disappearance of MN PCE from the bone marrow might be linked to their entering the peripheral blood circulation. Consequently, one might expect an elevation of the level of MN peripheral erythrocytes because, unlike other species, the life-span of MN erythrocytes in mouse peripheral blood is similar to those typical for normal erythrocytes (MacGregor et al., 1980; Schlegel and MacGregor, 1982, 1983; Barale et al., 1985). The data presented in this paper describe the dependence of TS-induced mutagenesis in S. typhimurium TA98 on the type of activation of the liver microsomal metabolizing enzymes used for $9 preparation as well as the clastogenic effect of TS in mouse peripheral erythrocytes. Materials and methods

Treatment of animals Female BDF a (C57B1 × DBA2) mice 7-10 weeks of age and weighing 15-18 g were used. The animals had free access to standard rodent chow and water. The treatment of mice with TS was carried out daily (1-42 days) in a 14-1 glass chamber containing 600 cm 3 TS as described earlier (Balansky et al., 1987). Each TS treatment lasted 60 min (2 exposures of 30 min each with a 1-min interval between them during which a whole air change was made). In several series of experiments up to 180 min (6 × 30 rain) exposure of mice to TS was used.

Processing of bone marrow The animals were killed by cervical dislocation 24 h after the TS exposure or after 42 days of treatment. The bone marrow smears were prepared and stained with M a y - G ~ n w a l d - G i e m s a according to Schmid (1975). From each mouse (10 mice per group) 1000 PCE were screened for micronuclei.

Processing of blood At different sampling times (see Table 4) peripheral blood smears from the tail vein were prepared and stained with M a y - G r i ~ n w a l d Giemsa stain (Barale et al., 1985). From each mouse (10 mice per group) 10000 peripheral normochromatic erythrocytes (NCE) were scored.

Salmonella/microsome mutagenicity assay Salmonella tvphimurium strains TA98

and T A I 0 0 kindly provided by Prof. B.N. Ames (Berkeley, CA) were grown overnight in nutrient broth (Difco). The mutagenicity test was performed according to the standard protocol recommended by Prof. B.N. Ames (Maron and Ames, 1983) as described earlier (Balansky et ai., 1987).

$9 fraction As a source of $9 fraction, livers of male BD6 rats (160-180 g) pretreated with one of the following inducers of microsomal oxidation were used, namely: (a) Aroclor-1254, i.p., 500 m g / k g b.w.. 5 days before the animals were killed; (b) 3-methyicholanthrene (MC), 40 m g / k g , i.p., 48 h and 24 h before the animals were killed and (c) phenobarbital (PB), i.p., 70 m g / k g , 3 consecutive days before sacrificing the rats. The livers of untreated rats were also used for the preparation of $9 in some series of experiments. The cofactor concentrations in the $9 mix were as recommended by Maron and Ames (1983).

Treatment of bacteria S. typhimurium TA98 was treated with TS for 1 min or 5 rain respectively in a 16-1 glass chamber containing 240 cm 3 of TS, as described earlier (Balansky et al., 1987). In several series of experiments 3,4-benzo[a]pyrene (BP) 10 ~ g / p l a t e (Fluka) was added to the top agar as a mutagen together with S. typhimurium strains TA98 or TA100. Being an ubiquitous environmental mutagen/carcinogen also detected in TS, we used BP, which requires metabolic activation to exert its genotoxic effect when controlling the metabolic capacities of the microsomal metabolic activation systems used in the present experiments. After plating bacteria cells the plates were incubated for 48 h at 3 7 ° C and then reveriant (his +) colonies were counted.

239

Statistical analysis of data obtained was performed according to Student's t test. Results and di~ussion

The data presented in Table 1 confirm earlier findings (Balansky et al., 1987) showing that the direct treatment of S. tvphimuriurn TA98 with TS in the presence of $9 mix obtained from Aroclor1254-pretreated rats caused an increase of the number of his + revertant colonies. Furthermore, it was established that the $9 fraction obtained from the liver of untreated rats was unable to metabolically activate promutagens in TS that could be detected with the Salmonella/microsome mutagenicity assay. When an $9 fraction obtained from the liver of PB-pretreated rats was used in the composition of $9 mix, a slightly increased number of his + revertant colonies was established, but the data are inconsistent and could not be considered a result of the mutagenic activity of TS. It seen'ks likely that PB was ineffective in inducing the appropriate profile of metabolic enzymes in the rat liver to ensure the activation of promutagens in TS. In contrast, when an $9 fraction from the liver of Aroclor-1254-pretreated or of MC-pretreated rats were used in $9 mix, the mutagenic effect of TS was strongly expressed (Table 1). The effect observed was more pronounced in some series of experiments with $9 from MC-pretreated animals. Taking into account the specificity of MC as an inducer of cytochrome

P450-dependent monooxygenases, i.e., the mixedfunction oxidases and in particular the cytochrome P450-dependent aryl hydrocarbon hydroxylase, it seems likely that the putative promutagen(s) detected in TS in the present experiments employing the Salmonella/microsome mutagenicity assay (S. typhimurium TA98) i s / a r e metabolically activated by cytochrome P448. This hemoprotein has a high catalytic activity for the biotransformation of BP and other polycyclic aromatic hydrocarbons to their proximate and ultimate m u t a g e n i c / c a r c i n o g e n i c metabolites (Conney, 1982). According to Poland (1982), ' MC induces one or more species of cytochrome P450 distinct from those induced by PB, and associated with this is an increase in enzyme activity towards a more limited spectrum of substrates.' Hence, aromatic polycyclic hydrocarbons might be involved in the TS-induced mutagenesis in S. typhirnurium TA98. This suggestion might be indirectly supported by the data summarized in Table 2 showing a similar pattern of metabolic activation towards BP. MC and Aroclor-1254 but not PB were effective inducers of metabolic activation of BP. The results presented in Table 3 are an additional confirmation of the previously established data that the treatment of mice in vivo with TS causes a dose-dependent elevation of the number of MN PCE in mouse bone marrow (Balansky et al., 1987). In addition we studied the appearance of MN N C E in the peripheral blood after matura-

TABLE 1 DEPENDENCE OF TS-INDUCED FRACTION USED

MUTAGENIC

EFFECT

IN S. typhimurium TA98 " O N

THE TYPE OF

Treatraent

$9

TS, 1 rain

TS, 5 min

I S , 249 cm 3 in 16 1 TS, 2 4 0 c m 3 in 16 1 TS, 240 cm 3 in 16 1

U n t r e a t e d liver PB-treated liver Aroclor-1254-treated liver

40 + 3.2 b 56_+ 1.6 183 ± 5.1

35 + 2.1 41 _+ 3.8 293 _+41.6

TS, TS, TS, TS,

U n t r e a t e d liver PB-treated liver Aroclor-1254-treated liver M C - t r e a t e d liver

6 6 + 5.1 97_+ 6.7 212 -+ 59.2 257 -+ 40.3

70.- 5.4 93 -+ 8.6 230 _+ 28.8 198 +_41.4

240 cm 3 240 cm 3 241) cm ~ 241) cm ~

in in in in

16 16 16 16

I 1 1 1

his " revertant colonies per plate

" S p o n t a n e o u s m u t a t i o n rate in untreated control (S. typhlrnurium TA98): 2 7 - 3 6 his ~ revertant colonies per plate. h Mean_+ S.E. D a t a from 6 samples.

L I V E R $9

240 TABLE 2 DEPENDENCE OF 3,4-BENZO[a]PYRENE-INDUCED MUTAGENIC EFFECTS IN S. typhtmurium TA98 AND TA100 ON THE TYPE OF LIVER $9 FRACTION USED S. t)'phimuriurn strain

Treatment

$9

his ' revertant colonies per plate

TA98 TA98 TA98 TA98 TA100 TA100 TA100 TA100 TAIO0 TA100 TA100

Untreated control Untreated control BP BP Untreated control Untreated control BP BP BP BP BP

No Phosphate buffer Phosphate buffer Aroclor-1254-treated liver No Aroclor-1254-treated liver Phosphate buffer Untreated liver PB-treated liver Aroclor-1254-treated liver MC-treated liver

42+ 2.8 ~ 34± 3.1 46+ 3.2 270 ± 10.7 160 + 18.4 187 + 32.5 209 ± 22.8 216 + 1.5 190 + 13.0 990 ± 32.2 1 350 + 50.0

Mean + S.E. Data from 3 samples.

TABLE 3

tion of the erythrocytes and

INDUCTION OF MICRONUCLEI IN MOUSE BONE MARROW BY TOBACCO SMOKE (TS) AND ITS DEPENDENCE ON EXPOSURE TIME

peripheral circulation. The results obtained (Table 4) s h o w e d t h a t 24 h a f t e r T S e x p o s u r e t h e r e w a s

Treatment

Number of MN PCE per 1000 PCE 24 h a

Untreated control "IS, 60 min treatment TS, 120 rain treatment TS, 180 min treatment

1.5 ±0.48 h 3.3 + 0.59 3.5 ± 0.45 5.2 ± 0.61

their entering the

n o d i f f e r e n c e b e t w e e n t h e levels o f M N

N C E in

the peripheral blood of control and T S - e x p o s e d m i c e . H o w e v e r , a l r e a d y at 48 h a f t e r T S t r e a t m e n t an about 2-fold elevation of peripheral MN NCE level w a s e s t a b l i s h e d . T h e d a i l y e x p o s u r e o f m i c e t o T S c a u s e d a f u r t h e r i n c r e a s e in t h e n u m b e r o f

p < 0.05 p < 0.05 p < 0.05

M N N C E a p p r o x i m a t e l y u p t o d a y 14 w h e n a n a b o u t 4 - f o l d i n c r e a s e in t h e n u m b e r o f M N N C E

MN PCE, micronucleated polychromatic erythrocytes. b Mean±S.E. Data from 10 animals, 1000 PCE scored per animal.

was detected. However, the continuous treatment o f m i c e u p t o d a y 42 d i d n o t r e s u l t in a n y f u r t h e r e l e v a t i o n . T h e s e d a t a m i g h t i m p l y t h a t t h e life-

TABLE 4 FREQUENCIES OF MICRONUCLEATED NORMOCHROMATIC ERYTHROCYTES (MN NCE) IN PERIPHERAL BLOOD OF MICE TREATED WITH TOBACCO SMOKE (TS) Treatment

Untreated control TS, 2 × 30 rain, daily

Peripheral blood MN NCE per 1 000 NCE 24 h

48 h

Day 7

Day 14

Day 42

0.68+0.048 a 0.56-t-0.038

0.51+0.015 1.16+0.110 c (+127.5%) *

0.75+0.054 1.51+0.080 ~ (+101.3%)

0.38-t-0.045 2.17+0.110 J (+471.1%)

0.55-t-0.050 2.37±0.180 'j (+330.9%)

Mean +_S.E. Data from 10 mice, 10000 NCE scored per animal. b Mean + S.E. Data from 10 mice, 1000 PCE scored per animal. " p < 0.01. d p < 0.001. ¢ Elevation of the number of MN NCE compared to the corresponding control data.

Bone marrow MN PCE per 1000 PCE Day 42 3.2±0.66

5.2±1.17

~'

241 span of T S - i n d u c e d M N N C E in this particular mouse strain is a b o u t 1 4 - 2 0 days. In c o m p a r i s o n with the clear-cut 3 - 4 - f o l d elevation of the n u m ber of peripheral M N erythrocytes, the increase of the n u m b e r of M N PCE in b o n e marrow of the same animals treated with TS for 42 consecutive days was less p r o n o u n c e d (Table 4). It should be m e n t i o n e d that the daily exposure of mice to TS for at least 7 days or more d u r i n g the present experiments resulted in a b o u t 10% reduction of their body weight, indicating systemic toxicity. Thc data presented emphasized the utility of the S a l m o n e l l a / m i c r o s o m e m u t a g e n i c i t y assay as well as the micronucleus test in s t u d y i n g the genotoxic activity of TS. This refers in particular to the application of the micronucleus test in mouse peripheral blood. This approach i n t r o d u c e d by M a c G r e g o r a n d coworkers (1980) for the detection of the clastogenic activity of weak m u t a gens given chronically to mice proved useful in the present experiments with TS.

References Balansky, R.M., P.M. Blagoeva and Z.I. Mircheva (1987) Investigation of the mutagenic activity of tobacco smoke. Matation Res., 188, 13-19.

Barale, R., F. Giorgelli, L. Migliore, R. Ciranni, D. Casini, D. Zucconi and N. Loprieno (1985) Benzene induces micronuclei in circulating erythrocytes of chronically treated mice, Mutation Res., 144, 193-196. Conney, A.H. (1982) Induction of microsomal enzymes by foreign chemicals and carcinogenesis by polycyclicaromatic hydrocarbons: G.H.A. Clowes memorial lecture, Cancer Res., 42, 4875-4917. MacGregor, J.T., C.M. Wehr and D.H. Gould (1980) Clastogen-induced micronuclei in peripheral blood erythrocytes: the basis of an improved micronucleus test, Environ. Mutagen.. 2, 509-514. Maron, D.M., and B.N. Ames (1983) Revised method for the S',dmoneUamutagenicity test, Mutation Res., 113, 173-215. Poland. A. (1982) Induction of the drug-metabolizing enzymes, in: H. Bartsch and B. Armstrong (Eds.), Host Factors in Human Carcinogenesis, IARC Sci. Publ., 39, Lyon, pp. 351 - 364. Schlegel, R., and J.T. MacGregor (1982) The persistence of micronuclei in peripheral blood erythrocytes, Mutation Res., 104, 367-369. Schlegel, R., and J.T. MacGregor (1983) A rapid screen for cumulative chromosomal damage in mice: accumulation of circulating micronucleated erythrocytes, Mutation Res., 113, 481-487. Schmid, W. (1975) The micronucleus test, Mutation Res., 31, 9-15.