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Quantitative relationship between carcinogenecity and mutagenicity of polyaromatic hydrocarbons in Salmonnella typhimurium mutants
Mutation Research, 31 (1975) 97-IO2 © Elsevier Scientific Publishing Company, Amsterdam--Printed in The Netherlands
97
Q U A N T I T A T I V E R E L A T I O N S H I P B E T W E E N C A R C I N O G E N I C I T Y AND M U T A G E N I C I T Y OF POLYAROMATIC H Y D R O C A R B O N S IN S A L M O N E L L A T Y P H I M U R I U M MUTANTS
KIYOSHI TERANISHI,
K O K I C H I I-IAMADA AND H I R O M U W A T A N A B E
Public Health Institute of Hyogo Prefecture, 2-1 Aratacho, Hyogoku, Kobe (Japan) (Received O c t o b e r I 4 t h , 1974)
SUMMARY
Mutagenic activities of various polyaromatic hydrocarbons (PAHs) in air pollutants, which are different in carcinogenic activities from each other, were examined with a set of tour strains of Salmonella typhimurium (TAI535 series;deep rough strains without excision repair). All the compounds tested were converted to frameshitt mutagens when they were metabolized by rat liver homogenate. There was a clear quantitative correlation between carcinogenicity and mutagenicity of PAHs tested in strain TAI538 using the rat liver enzyme induced with both dibenz(a,h)anthracene and phenobarbital. On the other hand, such a correlation was not obvious in strain TAI537.
INTRODUCTION
The various PAHs in air pollutants are known to be a cause of pulmonary cancer. The detection of carcinogenic potentials ot pure chemicals or extracts of airborne materials has been undertaken on various systems such as whole animals12,14, cultured cells 6,10, or microorganisms 8,13. However, these systems are not satisfactory in practice with respect to rapidity, simplicity and sensitivity. Recently, a simpler and more sensitive test system for determining the mutagenic activity of well-known carcinogens has been developed by AMES et al. using histidine-requiring mutants of Salmonella typhimurium 3. Those authors found that almost all the carcinogenic compounds tested are converted to mutagens when they are metabolized by human or rat liver homogenate 4. They therefore proposed that those carcinogens and m a n y others that are mutagens cause cancer by somatic cell mutations. Using their test system, we investigated the mutagenic potentials in relation to the carcinogenic activities judged by an animal test system 5,11. In this paper we report Abbreviations: B(a) A, benz (a) anthracene; B(a) P, benz(a)pyrene; B(e)P, benzo(e)pyrene; DB (a,h) A, dibenz(a,h)anthracene; DB(a,i)P, dibenzo(a,i)pyrene; DB(a,e)P, dibenzo(a,e)pyrene; DMSO, dimethyl sulfoxide; 3-MC, 3-nlethylcholanthrene; PAH, polyaromatic hydrocarbon; PB, phenobarbital.
98
K. TERANISHI et al.
that the mutagenic potentials of PAHs determined by the revertant colonies of frameshift m u t a n t are proportional to the carcinogenic activities expressed as IBALL index which is based on epithelial or subcutaneous tumor incidence in mice u. The results suggest the existence of a quantitative correlation between carcinogenicity and mutagenicity in PAHs. These results support the proposal of AMES and his coworkers and further suggest that their test system would be valid for quantitative determination of the carcinogenic potential in air pollutants. MATERIALS AND METHODS
Chemicals 3-Methylcholanthrene (3-MC) and dibenzo(a,i)pyrene (DB(a,i)P) were purchased from Sigma Chemical Co., dibenzo(a,i)pyrene (DB(a,e)P) and dibenz(a,h)anthracene(DB(a,h)A) were from Schnchardt (Munich) G m b H and Co., benz(a)anthracene (B(a)A) and benzo(e)pyrene (B(e)P) were from Ishizu Pharmaceutical Co. Ltd, benzo(a)pyrene (B(a)P) was from Wako Pure Chemical Industries, Ltd, NADP was from Boehringer Mannheim GmbH, and glucose-6-phosphate was from Sigma Chemical Co. All polyaromatie hydrocarbons were checked for purity b y silica gel thin-layer chromatography in hexane, o-dichlorobenzene and pyridine(Io :I :0.5 v/v/v), and found to have less than 1% impurities. All the PAHs were dissolved in dimethylsulfoxide (DMSO) (I mg/ml). A given concentration of each P A H was prepared by dilution with DMSO before use.
Bacterial strains Strains used were histidine-requiring mutants of Salmonella typhimurium LT-2 (TAI535, TAI536, TAI537, TAI538 ) given by AMES3. In addition to the histidine mutation each tester strain has two additional mutations: one causes loss of the excision repair system (uvr-) and the other causes loss of the lipopolysaccharide barrier that coats the surface of bacteria (rfa). Strain TAI535 can be used to detect mutagens causing base-pair substitution and the other three to detect various kinds of frameshift mutagen.
Media Heart-infusion broth (Nissui) was used to grow the tester strains. Minimal medium used for the mutagenesis test was prepared according to AMES1.
Induction of liver enzyme system Random-bred (14o-17o g) or Spraque-Dawley-JCL (17o-19o g) male rats were used. A week before the rats were killed, their drinking water was made o.1% with phenobarbital (PB). Another group of rats were given the PB-containing drinking water as above, and then injected intraperitoneally once at 48 h or twice at 34 h and 48 h before being killed, with 3-MC or with DB(a,h)A which were dissolved in corn oil.
Preparation of liver homogenate fraction "S-9" This preparation was carried out according to AMES et aI. ~. All steps were performed at 0-4 ° with cold and sterile solutions and glassware. The liver was washed in an equal volume of o.15 M KC1, minced with sterile scissors in three volumes of
99
MUTAGENICITY OF POLYAROMATIC HYDROCARBONS
o.15 M KC1, and homogenized with a Potter-Elvehjem apparatus having a Teflon pestle. The homogenate was centrifuged for io min at 9ooo×g. The supernatant ("S-9") was decanted and saved. The fresh S- 9 fraction was distributed in 4 ml portions in sterile glass tubes and stored at --80 ° in a Revco freezer.
Mutagenesis test with the S-9 fraction To a test-tube containing 2 ml of molten top agar at 45-5 °o were added o.I ml of bacterial culture, up to o.I ml DMSO solution of the compound to be tested, and 0.5 ml S- 9 mixture. The tube was rotated quickly and the contents poured onto the agar plate. The S- 9 mixture contained, per ml : 0.3 ml S-9, 8/,moles MgC12, 33/~moles KC1, 5/,moles glucose-6-phosphate, 4 #moles NADP and IOO ,umoles sodium phosphate (pH 7-4).
Determination of surviving cell number in the mutagenesis test An appropriate dilution of fully grown culture of a tester strain was mixed with a given chemical to be tested and S- 9 fraction exactly as in the mutagenesis test and poured onto a minimal agar plate containing an excess (3 #moles/plate) of histidine. TABLE
1
MUTAGENICITY OF VARIOUS POLYAROMATIC HYDROCARBON METABOLITES BY PB-INDUCED ENZYME The enzyme used here was the preparative liver homogenate of random bred male rats which were g i v e n d r i n k i n g w a t e r c o n t a i n i n g o . I % p h e n o b a r b i t a l f o r 7 d a y s . E a c h v a l u e w a s t h e a v e r a g e of duplicate plates.
Compound added (5o t,g/plate)
Revertant colonies per plate on tester strains TA1535 Td1536 TA1537 Td~538
3-Methylcolanthrene Dibenzo(a,i)pyrene Benzo(a) pyrene Dibenzo(a,e)pyrene D i b e n z (a, h) a n t h r a c e n e Benz(a) anthracene Benzo(e)pyrene Dimethyl sulfoxide (control)
18 io iI IO 6 8 9 5
I o o o o i o I
123 9 36 I3 Ii 31 15 I3
203 i o2 77 55 4° 29 47 38
RESULTS AND DISCUSSION
The mutagenic activities of seven PAHs with different grades of carcinogenic activity judged by the IBALL test in the mouseS, II were investigated with S. typhimurium LT-2 mutants to examine the quantitative correlation with the mutagenic potential. In a preliminary experiment (Table I), no mutagenic activity was detected with TAI535 or TAI536 used as tester strains in all the PAHs studied, even if metabolized by PB-induced liver enzyme. In TAI537 or TAI538, however, 3-MC, DB (a,i) P and B(a)P,which are known to exhibit highest carcinogenicities, gave rise to significant numbers of his+ revertants. Neither DB(a,h)A, B(e)P, nor B(a)A metabolites having relatively low carcinogenicities did so in any of these strains. The administration of a small dose of P A H to a rat induces a marked increase in activity of hydroxylating enzymes for PAHs in the liverT; and an additive effect on the induction of hydroxylase activity is known to occur among PB and various PAHs in fetal rat hepatocytes 9. Hence two kinds of PAH, DB(a,h)A and 3-MC, were used as inducers of the liver enzyme in addition to PB. First, the dependence of revertant
K. TERANISHI at al.
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Carcinogenicity Fig. I. Effect of B(a)P concentration on mutagenesis of TAI538. o.i nil of fully grown culture of strain TAI538 was mixed with the indicated amount of B(a)P together with (or without) the hydroxylation system which consisted of rat liver homogenate (see below), glucose-6-phosphate and NADP. The whole mixture was spread on a minimal agar plate containing a limited amount of histidine (o.i #mole/plate); his + colonies were scored after 48 h incubation at 37 °. A, No liver homogenate; O, liver homogenate from rats (SD-1CL strain) which were given drinking water containing o.i % phenobarbital for 7 days; ©, liver homogenate from rats (SD-JCL) given tile PB-containing drinking water as above, and then injected intraperitoneally with DB(a,h)A at 24 h and also 48 h before being killed (55 mg/kg for each time). Each value is the average of duplicate plates. Fig. 2. The correlation between carcinogenicity and mutagenicity in TAI537. The procedure was as described in the legend to Fig. i except t h a t random bred rats were used in place of SD-JCL strain. Each chemical was used at a concentration of 5o #g/plate; a, 3-methylcholanthrene; b, d i b e n z o ( a , i ) p y r e n e ; c, benzo(a)pyrene; d, dibenzo(a,e)pyrene ; e, d i b e n z ( a , h ) a n t h r a c e n e ; f. benz(a)anthracene; g, benzo(e)pyrene; h, dimethylsulfoxide (control). O, liver homogenate from rats given drinking water containing o.i % phenobarbital for 7 days; {), liver homogenate from rats given the PB-containing drinking water as above, and then injected intraperitoneally with 3-MC at 48 h before being killed (14o nlg/kg) ; ©, liver homogenate from rats given the PB-containing drinking water as above, and then injected intraperitoneally with D B ( a , h ) A at 48 h (14o nlg/kg). Each value is the average of duplicate plates.
frequencies on drug concentration was studied using PB- plus DB(a,h)A-induced ratliver enzyme together with PB-induced enzyme. As shown in Fig. I, a plateau was reached at a concentration of B (a) P o t about 25 fig/plate. Similar results were obtained also in DB(a,i)P, 3-Me, DB(a,e)P or DB(a,h)A by using PB- or PB- plus DB(a,h)Ainduced liver enzyme (results not shown). Based on these results, we adopted a concentration of 5o fig of each chemical per plate for their quantitative comparison in mutagenicity in the following experiments. The mutagenic activities of all the PAHs tested were increased in strains TAI537 or TAI538 by using the 3-MC- or DB(a,h)A-induced rat-liver enzyme over the value obtained with the liver enzyme induced by PB alone (Figs. 2 and 3). The increase was more marked tor PAHs known to have low carcinogenic potentials such as DB(a,h)A, B(a)A or B(e)P, especially when TAI537 was used (Fig. 2). Again no significant mutagenicity was seen when TAI535 or TAI536 was used as tester strain even with 3-MC- or DB(a,h)A-induced liver enzyme. As shown in Fig. 3, a correlation be-
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Fig. 3. The correlation between carcinogenicity and mutagenicity in TAI538. The procedures were as described in the legend to Fig.i except t h a t random bred rats were used in place of SD-JCL strain. Each chemical was used at a concentration of 5 ° #g/plate; a, 3-methylcholanthrene; b, dibenzo(a,i)pyrene; c, benzo(a)pyrene; d, dibenzo(a,e)pyrene; e, dibenz(a,h)anthracene; f, benz(a)anthracene; g, benzo(e)pyrene; h, dimethylsulfoxide (control). The enzyme used and the symbols were the same as described in the legend to Fig. 2. Each value was the average of duplicate plates. t w e e n t h e m u t a g e n i c p o t e n t i a l a n d c a r c i n o g e n i c i t y e x p r e s s e d as IBALL i n d e x w a s c l e a r l y o b s e r v e d in s t r a i n T A I 5 3 8 b y u s i n g t h e D B ( a , h ) A - i n d u c e d e n z y m e s y s t e m . O n t h e o t h e r h a n d , s u c h a c o r r e l a t i o n w a s n o t o b v i o u s i n s t r a i n T A 1537 (Fig. 2). R e v e r s i o n t o his + w i t h D B ( a , i ) P w a s less f r e q u e n t t h a n t h a t w i t h B ( a ) P o r D B ( a , h ) A , b o t h of w h i c h a r e less p o t e n t i n c a r c i n o g e n i c i t y t h a n D B ( a , i ) P . T h e d i f f e r e n c e b e t w e e n T A I 5 3 7 a n d T A I 5 3 8 i n t h e m u t a b i l i t y r e s p o n s e t o P A H h a s also b e e n o b s e r v e d b y TABLE II S U R V I V A L T E S T O F TAI538 AT T H E S A M E C O N C E N T R A T I O N HYDROCARBON M E T A B O L I T E S B Y T H E P B - plus
(5 °/zg/PLATE)
OF VARIOUS POLYAROMATIC-
DB(a,h)A-INDUCED E N Z Y M E
The enzyme used here was the preparative liver homogenate from random-bred male rats given drinking water containing o. 1% phenobarbital for 7 days, and then injected intraperitoneally with DB (a,h)A at 24 h before being killed (14o mg/kg). Each value was the average of duplicate plates.
Compound added (5 ° I~g/plate)
Survival (io -~ dil.) Plating Colony volume (ml) counts
Mutation (undiluted) Plating Revertant volume (ml) colonies
3-Methylcholanthrene Dibenzo (a, i) pyrene Dibenzo (a, h) anthracene Dimethyl sulfoxide (control)
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102
K. TERANISHI et al.
AMES et al.2. Th e result in T a b l e I I indicates t h a t no difference in cell killing effect was observed a m o n g three P A H s tested in our m u t a g e n i c i t y test. W e assume therefore t h a t the n u m b e r of his + r e v e r t a n t colonies is p r o p o r t i o n a l to the m u t a t i o n f r e q u e n c y in the present test. I n accord with AMES et al. 4, the present results indicate t h a t all t h e carcinogenic P A H s are the frameshift mutagens. W e suppose t h a t the carcinogenic p o t e n t i a l of P A H or o t h er chemicals in air can be q u a n t i t a t i v e l y d e t e c t e d b y strain T A I 5 3 8 r a t h e r t h a n strain T A I 5 3 7 using the P B - plus DB(a,h)A-induced e n z y m e system. In a p r e l i m i n a r y e x p e r i m e n t , we found t h a t b e n z e n e - e x t r a c t e d materials (50 /~g/plate) s t a r t i n g f r o m ab o u t 25 m a air in the v i c i n i t y of Kobe gave significant r e v e r t a n t colonies in T A I 5 3 8 over the control b y the P B - plus DB(a,h)A-indueed e n z y m e system. The q u a n t i t a t i v e d e t e c ti o n of the m u t a g e n i c p o t e n t i a l of air pollutants and also identification of the c a u s a t iv e agents are now in progress in our l ab o r at o r y . ACKNOWLEDGEMENTS The au t h o rs are grateful to Dr. BRUCE AMES of t h e U n i v e r s i t y of California for the gift of tester strain T A I 5 3 5 series and for criticism of the present work, and to Dr. TOSHIO ~'UKASAWA of the I n s t i t u t e for P r o te in Research of Osaka U n i v e r s i t y for re ad i n g the m an u s c r i p t . REFERENCES I AMES, B. N., in A. HOLLAENDER(Ed.), Chemical Mutagens: Principles and Methods for Their Detection, Vol. I, Plenum, New York, 1971, p. 267. 2 AMES,B. N., P. SIMSAND P. L. GROVER, Epoxides of carcinogenic polycyclic hydrocarbons are frameshift mutagens, Science, 176 (1972) 47. 3 AMES,B. N., F. D. LEE AND W. E. DURSTON,An improved bacterial test system for detection and classification of mntagens and carcinogens, Proc. Natl. Acad. Sci. (U.S.), 7° (1973) 782. 4 AMES, B. ~5~., W. E. DURSTON, E. YAMASAKI AND F. D. LEE, Carcinogens are mutagens: a simple test system combining liver homogenates for activation and bacteria for detection, Pro& Natl. Aead. Sci. (U.S.), 7° (1973) 2281. 5 ARCOS, J. C., AND M. F. ARGUS, Molecular geometry and carcinogenic activity of aromatic compounds, Adv. Cancer Res., i i (1968) 305 ` 6 BERWALD, Y., AND L. SACHS, In vitro transformation of normal cells to tumor cells by carcinogenic hydrocarbons, J. Natl. Cancer Inst., 35 (1965) 641. 7 DAO, T. L., AND H. YOGO, Effects of polynuclear hydrocarbons on benzopyrene hydroxylase activity in rats, Proc. Soc. Exptl. Biol, Med., 116 (1964) lO48. 8 EPSTEIN, S. S., I. ]~. SAPOROSHETZE, M. SMALL, W. PAARK AND •. MANTEL, A simple bioassay
9
IO
Ii 12 13
14
for antioxidants based on protection of Tetrahymena pyriformis from the photodynamic toxicity of benzo(a)pyrene, Nature, 208 (1965) 655. GIELEN, J, E., AND D. W. •EBERT, Aryl hydrocarbon hydroxylase induction in mammalian liver cell culture: stimulation of enzyme activity in nonhepatic cells and in hepatic cells by phenobarbital, polycyclic hydrocarbons and 2,2-bis(p-chlorophenyl)-i,I,I-trichloroethane, J. Biol. Chem., 246 (1971) 5189 . HUBERMAN, E., L. ASPIRAS, C. HEIDERBERGER, P. L. GROVER AND P. SIMS, Mutagenicity to mammalian cells of epoxides and other derivatives of polycyclic hydrocarbons, Proc. Natl. Acad. Sci. (U.S.), 68 (1971) 3195 . IBALL, J., The relative potency of carcinogenic compounds, Am. J. Cancer, 35 (I939) 186. LEITER, J., M. B. SHIMKIN AND M. J. SHEAR, Production of subcutaneous sarcomas in mice with tars extracted from atomospheric dusts, J. Natl. Cancer Inst., 3 (1942) 155. SCHERR, G. H., M. FISHMAN AND R. H. WEAVER, The mutagenicity of some carcinogenic compounds for Escherichia coli, Genetics, 39 (1954) 141. SUGIYAMA, T., Chromosomal aberration and carcinogenesis by various benz(a)anthracene derivatives, Gann, 64 (1973) 637.
97
Q U A N T I T A T I V E R E L A T I O N S H I P B E T W E E N C A R C I N O G E N I C I T Y AND M U T A G E N I C I T Y OF POLYAROMATIC H Y D R O C A R B O N S IN S A L M O N E L L A T Y P H I M U R I U M MUTANTS
KIYOSHI TERANISHI,
K O K I C H I I-IAMADA AND H I R O M U W A T A N A B E
Public Health Institute of Hyogo Prefecture, 2-1 Aratacho, Hyogoku, Kobe (Japan) (Received O c t o b e r I 4 t h , 1974)
SUMMARY
Mutagenic activities of various polyaromatic hydrocarbons (PAHs) in air pollutants, which are different in carcinogenic activities from each other, were examined with a set of tour strains of Salmonella typhimurium (TAI535 series;deep rough strains without excision repair). All the compounds tested were converted to frameshitt mutagens when they were metabolized by rat liver homogenate. There was a clear quantitative correlation between carcinogenicity and mutagenicity of PAHs tested in strain TAI538 using the rat liver enzyme induced with both dibenz(a,h)anthracene and phenobarbital. On the other hand, such a correlation was not obvious in strain TAI537.
INTRODUCTION
The various PAHs in air pollutants are known to be a cause of pulmonary cancer. The detection of carcinogenic potentials ot pure chemicals or extracts of airborne materials has been undertaken on various systems such as whole animals12,14, cultured cells 6,10, or microorganisms 8,13. However, these systems are not satisfactory in practice with respect to rapidity, simplicity and sensitivity. Recently, a simpler and more sensitive test system for determining the mutagenic activity of well-known carcinogens has been developed by AMES et al. using histidine-requiring mutants of Salmonella typhimurium 3. Those authors found that almost all the carcinogenic compounds tested are converted to mutagens when they are metabolized by human or rat liver homogenate 4. They therefore proposed that those carcinogens and m a n y others that are mutagens cause cancer by somatic cell mutations. Using their test system, we investigated the mutagenic potentials in relation to the carcinogenic activities judged by an animal test system 5,11. In this paper we report Abbreviations: B(a) A, benz (a) anthracene; B(a) P, benz(a)pyrene; B(e)P, benzo(e)pyrene; DB (a,h) A, dibenz(a,h)anthracene; DB(a,i)P, dibenzo(a,i)pyrene; DB(a,e)P, dibenzo(a,e)pyrene; DMSO, dimethyl sulfoxide; 3-MC, 3-nlethylcholanthrene; PAH, polyaromatic hydrocarbon; PB, phenobarbital.
98
K. TERANISHI et al.
that the mutagenic potentials of PAHs determined by the revertant colonies of frameshift m u t a n t are proportional to the carcinogenic activities expressed as IBALL index which is based on epithelial or subcutaneous tumor incidence in mice u. The results suggest the existence of a quantitative correlation between carcinogenicity and mutagenicity in PAHs. These results support the proposal of AMES and his coworkers and further suggest that their test system would be valid for quantitative determination of the carcinogenic potential in air pollutants. MATERIALS AND METHODS
Chemicals 3-Methylcholanthrene (3-MC) and dibenzo(a,i)pyrene (DB(a,i)P) were purchased from Sigma Chemical Co., dibenzo(a,i)pyrene (DB(a,e)P) and dibenz(a,h)anthracene(DB(a,h)A) were from Schnchardt (Munich) G m b H and Co., benz(a)anthracene (B(a)A) and benzo(e)pyrene (B(e)P) were from Ishizu Pharmaceutical Co. Ltd, benzo(a)pyrene (B(a)P) was from Wako Pure Chemical Industries, Ltd, NADP was from Boehringer Mannheim GmbH, and glucose-6-phosphate was from Sigma Chemical Co. All polyaromatie hydrocarbons were checked for purity b y silica gel thin-layer chromatography in hexane, o-dichlorobenzene and pyridine(Io :I :0.5 v/v/v), and found to have less than 1% impurities. All the PAHs were dissolved in dimethylsulfoxide (DMSO) (I mg/ml). A given concentration of each P A H was prepared by dilution with DMSO before use.
Bacterial strains Strains used were histidine-requiring mutants of Salmonella typhimurium LT-2 (TAI535, TAI536, TAI537, TAI538 ) given by AMES3. In addition to the histidine mutation each tester strain has two additional mutations: one causes loss of the excision repair system (uvr-) and the other causes loss of the lipopolysaccharide barrier that coats the surface of bacteria (rfa). Strain TAI535 can be used to detect mutagens causing base-pair substitution and the other three to detect various kinds of frameshift mutagen.
Media Heart-infusion broth (Nissui) was used to grow the tester strains. Minimal medium used for the mutagenesis test was prepared according to AMES1.
Induction of liver enzyme system Random-bred (14o-17o g) or Spraque-Dawley-JCL (17o-19o g) male rats were used. A week before the rats were killed, their drinking water was made o.1% with phenobarbital (PB). Another group of rats were given the PB-containing drinking water as above, and then injected intraperitoneally once at 48 h or twice at 34 h and 48 h before being killed, with 3-MC or with DB(a,h)A which were dissolved in corn oil.
Preparation of liver homogenate fraction "S-9" This preparation was carried out according to AMES et aI. ~. All steps were performed at 0-4 ° with cold and sterile solutions and glassware. The liver was washed in an equal volume of o.15 M KC1, minced with sterile scissors in three volumes of
99
MUTAGENICITY OF POLYAROMATIC HYDROCARBONS
o.15 M KC1, and homogenized with a Potter-Elvehjem apparatus having a Teflon pestle. The homogenate was centrifuged for io min at 9ooo×g. The supernatant ("S-9") was decanted and saved. The fresh S- 9 fraction was distributed in 4 ml portions in sterile glass tubes and stored at --80 ° in a Revco freezer.
Mutagenesis test with the S-9 fraction To a test-tube containing 2 ml of molten top agar at 45-5 °o were added o.I ml of bacterial culture, up to o.I ml DMSO solution of the compound to be tested, and 0.5 ml S- 9 mixture. The tube was rotated quickly and the contents poured onto the agar plate. The S- 9 mixture contained, per ml : 0.3 ml S-9, 8/,moles MgC12, 33/~moles KC1, 5/,moles glucose-6-phosphate, 4 #moles NADP and IOO ,umoles sodium phosphate (pH 7-4).
Determination of surviving cell number in the mutagenesis test An appropriate dilution of fully grown culture of a tester strain was mixed with a given chemical to be tested and S- 9 fraction exactly as in the mutagenesis test and poured onto a minimal agar plate containing an excess (3 #moles/plate) of histidine. TABLE
1
MUTAGENICITY OF VARIOUS POLYAROMATIC HYDROCARBON METABOLITES BY PB-INDUCED ENZYME The enzyme used here was the preparative liver homogenate of random bred male rats which were g i v e n d r i n k i n g w a t e r c o n t a i n i n g o . I % p h e n o b a r b i t a l f o r 7 d a y s . E a c h v a l u e w a s t h e a v e r a g e of duplicate plates.
Compound added (5o t,g/plate)
Revertant colonies per plate on tester strains TA1535 Td1536 TA1537 Td~538
3-Methylcolanthrene Dibenzo(a,i)pyrene Benzo(a) pyrene Dibenzo(a,e)pyrene D i b e n z (a, h) a n t h r a c e n e Benz(a) anthracene Benzo(e)pyrene Dimethyl sulfoxide (control)
18 io iI IO 6 8 9 5
I o o o o i o I
123 9 36 I3 Ii 31 15 I3
203 i o2 77 55 4° 29 47 38
RESULTS AND DISCUSSION
The mutagenic activities of seven PAHs with different grades of carcinogenic activity judged by the IBALL test in the mouseS, II were investigated with S. typhimurium LT-2 mutants to examine the quantitative correlation with the mutagenic potential. In a preliminary experiment (Table I), no mutagenic activity was detected with TAI535 or TAI536 used as tester strains in all the PAHs studied, even if metabolized by PB-induced liver enzyme. In TAI537 or TAI538, however, 3-MC, DB (a,i) P and B(a)P,which are known to exhibit highest carcinogenicities, gave rise to significant numbers of his+ revertants. Neither DB(a,h)A, B(e)P, nor B(a)A metabolites having relatively low carcinogenicities did so in any of these strains. The administration of a small dose of P A H to a rat induces a marked increase in activity of hydroxylating enzymes for PAHs in the liverT; and an additive effect on the induction of hydroxylase activity is known to occur among PB and various PAHs in fetal rat hepatocytes 9. Hence two kinds of PAH, DB(a,h)A and 3-MC, were used as inducers of the liver enzyme in addition to PB. First, the dependence of revertant
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Carcinogenicity Fig. I. Effect of B(a)P concentration on mutagenesis of TAI538. o.i nil of fully grown culture of strain TAI538 was mixed with the indicated amount of B(a)P together with (or without) the hydroxylation system which consisted of rat liver homogenate (see below), glucose-6-phosphate and NADP. The whole mixture was spread on a minimal agar plate containing a limited amount of histidine (o.i #mole/plate); his + colonies were scored after 48 h incubation at 37 °. A, No liver homogenate; O, liver homogenate from rats (SD-1CL strain) which were given drinking water containing o.i % phenobarbital for 7 days; ©, liver homogenate from rats (SD-JCL) given tile PB-containing drinking water as above, and then injected intraperitoneally with DB(a,h)A at 24 h and also 48 h before being killed (55 mg/kg for each time). Each value is the average of duplicate plates. Fig. 2. The correlation between carcinogenicity and mutagenicity in TAI537. The procedure was as described in the legend to Fig. i except t h a t random bred rats were used in place of SD-JCL strain. Each chemical was used at a concentration of 5o #g/plate; a, 3-methylcholanthrene; b, d i b e n z o ( a , i ) p y r e n e ; c, benzo(a)pyrene; d, dibenzo(a,e)pyrene ; e, d i b e n z ( a , h ) a n t h r a c e n e ; f. benz(a)anthracene; g, benzo(e)pyrene; h, dimethylsulfoxide (control). O, liver homogenate from rats given drinking water containing o.i % phenobarbital for 7 days; {), liver homogenate from rats given the PB-containing drinking water as above, and then injected intraperitoneally with 3-MC at 48 h before being killed (14o nlg/kg) ; ©, liver homogenate from rats given the PB-containing drinking water as above, and then injected intraperitoneally with D B ( a , h ) A at 48 h (14o nlg/kg). Each value is the average of duplicate plates.
frequencies on drug concentration was studied using PB- plus DB(a,h)A-induced ratliver enzyme together with PB-induced enzyme. As shown in Fig. I, a plateau was reached at a concentration of B (a) P o t about 25 fig/plate. Similar results were obtained also in DB(a,i)P, 3-Me, DB(a,e)P or DB(a,h)A by using PB- or PB- plus DB(a,h)Ainduced liver enzyme (results not shown). Based on these results, we adopted a concentration of 5o fig of each chemical per plate for their quantitative comparison in mutagenicity in the following experiments. The mutagenic activities of all the PAHs tested were increased in strains TAI537 or TAI538 by using the 3-MC- or DB(a,h)A-induced rat-liver enzyme over the value obtained with the liver enzyme induced by PB alone (Figs. 2 and 3). The increase was more marked tor PAHs known to have low carcinogenic potentials such as DB(a,h)A, B(a)A or B(e)P, especially when TAI537 was used (Fig. 2). Again no significant mutagenicity was seen when TAI535 or TAI536 was used as tester strain even with 3-MC- or DB(a,h)A-induced liver enzyme. As shown in Fig. 3, a correlation be-
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Fig. 3. The correlation between carcinogenicity and mutagenicity in TAI538. The procedures were as described in the legend to Fig.i except t h a t random bred rats were used in place of SD-JCL strain. Each chemical was used at a concentration of 5 ° #g/plate; a, 3-methylcholanthrene; b, dibenzo(a,i)pyrene; c, benzo(a)pyrene; d, dibenzo(a,e)pyrene; e, dibenz(a,h)anthracene; f, benz(a)anthracene; g, benzo(e)pyrene; h, dimethylsulfoxide (control). The enzyme used and the symbols were the same as described in the legend to Fig. 2. Each value was the average of duplicate plates. t w e e n t h e m u t a g e n i c p o t e n t i a l a n d c a r c i n o g e n i c i t y e x p r e s s e d as IBALL i n d e x w a s c l e a r l y o b s e r v e d in s t r a i n T A I 5 3 8 b y u s i n g t h e D B ( a , h ) A - i n d u c e d e n z y m e s y s t e m . O n t h e o t h e r h a n d , s u c h a c o r r e l a t i o n w a s n o t o b v i o u s i n s t r a i n T A 1537 (Fig. 2). R e v e r s i o n t o his + w i t h D B ( a , i ) P w a s less f r e q u e n t t h a n t h a t w i t h B ( a ) P o r D B ( a , h ) A , b o t h of w h i c h a r e less p o t e n t i n c a r c i n o g e n i c i t y t h a n D B ( a , i ) P . T h e d i f f e r e n c e b e t w e e n T A I 5 3 7 a n d T A I 5 3 8 i n t h e m u t a b i l i t y r e s p o n s e t o P A H h a s also b e e n o b s e r v e d b y TABLE II S U R V I V A L T E S T O F TAI538 AT T H E S A M E C O N C E N T R A T I O N HYDROCARBON M E T A B O L I T E S B Y T H E P B - plus
(5 °/zg/PLATE)
OF VARIOUS POLYAROMATIC-
DB(a,h)A-INDUCED E N Z Y M E
The enzyme used here was the preparative liver homogenate from random-bred male rats given drinking water containing o. 1% phenobarbital for 7 days, and then injected intraperitoneally with DB (a,h)A at 24 h before being killed (14o mg/kg). Each value was the average of duplicate plates.
Compound added (5 ° I~g/plate)
Survival (io -~ dil.) Plating Colony volume (ml) counts
Mutation (undiluted) Plating Revertant volume (ml) colonies
3-Methylcholanthrene Dibenzo (a, i) pyrene Dibenzo (a, h) anthracene Dimethyl sulfoxide (control)
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K. TERANISHI et al.
AMES et al.2. Th e result in T a b l e I I indicates t h a t no difference in cell killing effect was observed a m o n g three P A H s tested in our m u t a g e n i c i t y test. W e assume therefore t h a t the n u m b e r of his + r e v e r t a n t colonies is p r o p o r t i o n a l to the m u t a t i o n f r e q u e n c y in the present test. I n accord with AMES et al. 4, the present results indicate t h a t all t h e carcinogenic P A H s are the frameshift mutagens. W e suppose t h a t the carcinogenic p o t e n t i a l of P A H or o t h er chemicals in air can be q u a n t i t a t i v e l y d e t e c t e d b y strain T A I 5 3 8 r a t h e r t h a n strain T A I 5 3 7 using the P B - plus DB(a,h)A-induced e n z y m e system. In a p r e l i m i n a r y e x p e r i m e n t , we found t h a t b e n z e n e - e x t r a c t e d materials (50 /~g/plate) s t a r t i n g f r o m ab o u t 25 m a air in the v i c i n i t y of Kobe gave significant r e v e r t a n t colonies in T A I 5 3 8 over the control b y the P B - plus DB(a,h)A-indueed e n z y m e system. The q u a n t i t a t i v e d e t e c ti o n of the m u t a g e n i c p o t e n t i a l of air pollutants and also identification of the c a u s a t iv e agents are now in progress in our l ab o r at o r y . ACKNOWLEDGEMENTS The au t h o rs are grateful to Dr. BRUCE AMES of t h e U n i v e r s i t y of California for the gift of tester strain T A I 5 3 5 series and for criticism of the present work, and to Dr. TOSHIO ~'UKASAWA of the I n s t i t u t e for P r o te in Research of Osaka U n i v e r s i t y for re ad i n g the m an u s c r i p t . REFERENCES I AMES, B. N., in A. HOLLAENDER(Ed.), Chemical Mutagens: Principles and Methods for Their Detection, Vol. I, Plenum, New York, 1971, p. 267. 2 AMES,B. N., P. SIMSAND P. L. GROVER, Epoxides of carcinogenic polycyclic hydrocarbons are frameshift mutagens, Science, 176 (1972) 47. 3 AMES,B. N., F. D. LEE AND W. E. DURSTON,An improved bacterial test system for detection and classification of mntagens and carcinogens, Proc. Natl. Acad. Sci. (U.S.), 7° (1973) 782. 4 AMES, B. ~5~., W. E. DURSTON, E. YAMASAKI AND F. D. LEE, Carcinogens are mutagens: a simple test system combining liver homogenates for activation and bacteria for detection, Pro& Natl. Aead. Sci. (U.S.), 7° (1973) 2281. 5 ARCOS, J. C., AND M. F. ARGUS, Molecular geometry and carcinogenic activity of aromatic compounds, Adv. Cancer Res., i i (1968) 305 ` 6 BERWALD, Y., AND L. SACHS, In vitro transformation of normal cells to tumor cells by carcinogenic hydrocarbons, J. Natl. Cancer Inst., 35 (1965) 641. 7 DAO, T. L., AND H. YOGO, Effects of polynuclear hydrocarbons on benzopyrene hydroxylase activity in rats, Proc. Soc. Exptl. Biol, Med., 116 (1964) lO48. 8 EPSTEIN, S. S., I. ]~. SAPOROSHETZE, M. SMALL, W. PAARK AND •. MANTEL, A simple bioassay
9
IO
Ii 12 13
14
for antioxidants based on protection of Tetrahymena pyriformis from the photodynamic toxicity of benzo(a)pyrene, Nature, 208 (1965) 655. GIELEN, J, E., AND D. W. •EBERT, Aryl hydrocarbon hydroxylase induction in mammalian liver cell culture: stimulation of enzyme activity in nonhepatic cells and in hepatic cells by phenobarbital, polycyclic hydrocarbons and 2,2-bis(p-chlorophenyl)-i,I,I-trichloroethane, J. Biol. Chem., 246 (1971) 5189 . HUBERMAN, E., L. ASPIRAS, C. HEIDERBERGER, P. L. GROVER AND P. SIMS, Mutagenicity to mammalian cells of epoxides and other derivatives of polycyclic hydrocarbons, Proc. Natl. Acad. Sci. (U.S.), 68 (1971) 3195 . IBALL, J., The relative potency of carcinogenic compounds, Am. J. Cancer, 35 (I939) 186. LEITER, J., M. B. SHIMKIN AND M. J. SHEAR, Production of subcutaneous sarcomas in mice with tars extracted from atomospheric dusts, J. Natl. Cancer Inst., 3 (1942) 155. SCHERR, G. H., M. FISHMAN AND R. H. WEAVER, The mutagenicity of some carcinogenic compounds for Escherichia coli, Genetics, 39 (1954) 141. SUGIYAMA, T., Chromosomal aberration and carcinogenesis by various benz(a)anthracene derivatives, Gann, 64 (1973) 637.