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Absence of genotoxic activity of refined smoke flavor (RSF) in two bacterial short-term tests
Mutation Research, 204 (1988) 235-238 Elsevier
235
MTR 01258
Absence of genotoxic activity of refined smoke flavor (RSF) in two bacterial short-term tests Jacek Jenek 1,2 Philippe Quillardet 1, Danuta Chomiak 3, Andrzej Borys 3 and Maurice Hofnung 1 1 Unit~ de Programmation Mol~culaire et Toxicologie GOn~tique, CNRS LA271, I N S E R M U. 163, Institut Pasteur, 28, rue du Dr Roux, 75015 Paris (France), 2 Department of Medical Microbiology, Institute of Microbiology and Infectious Diseases, Academy of Medicine, ul. Wieniawskiego 3, 61-712 Poznah (Poland) and 3 Institute of Meat and Fat Industry, ul, Rakowiecka 36, 02-532 Warszawa (Poland) (Received 21 January 1987) (Revision received 17 September 1987) (Accepted 5 October 1987)
Keywords: Refined smoke flavor; Genotoxic activity; Short-term tests.
Summary The genotoxic activities of refined smoke flavor (RSF) produced in Poland and used in food processing were investigated in 2 bacterial short-term tests. Its mutagenic activity was examined in the Salmonella/ histidine plate assay and its SOS-inducing capacity in the SOS Chromotest both without and with 'activation' by a rat liver homogenate. No genotoxic activity was detected using these 2 bacterial tests.
Among the large number of short-term tests used to detect environmental genotoxins and consequently possible carcinogens, the bacterial tests constitute simple, rapid and economic systems (Hollstein and McCann, 1979). The Salmonella/ histidine test (Ames et al., 1975; Maron and Ames, 1983), the most popular of the bacterial assays, measures back mutations to prototrophy in several specially constructed his mutants of Salmonella. The SOS Chromotest (Quillardet et al., 1982; Quillardet and Hofnung, 1985) is a colorimetric assay of the induction by genotoxic agents of the gene sfiA in Escherichia coll. This gene is involved in one of the SOS functions (Walker, 1984). Its
Correspondence: Dr. J. Jenek, Department of Medical Microbiology, Institute of Microbiology and Infectious Diseases, Academy of Medicine, ul. Wieniawskiego 3, 61-712 Poznafi (Poland).
level of expression is monitored by means of a sfiA::lacZ genetic fusion. The SOS Chromotest has been validated on a series of known genotoxic agents (Quillardet et al., 1985; Ohta et al., 1984). In both tests a homogenate of rat liver can be added to the bacterial suspension as an approximation of mammalian metabolism. Both the Salmonella/histidine test and the SOS Chromotest were used to evaluate genotoxic activities of refined smoke flavor (RSF) produced in Poland and used in food processing in different commercial forms.
Materials and methods Refined smoke flavor (RSF) was produced from natural hard-wood smoke by condensation, distillation and rectification under reduced pressure according to the method described by Borys et al.
0165-1218/88/$03.50 © 1988 Elsevier Science Publishers B.V. (Biomedical Division)
236 (1982). It was p r e p a r e d in Rzeped~ (Poland). R S F m a i n l y consists of organic acids, neutral, c a r b o n y l a n d phenolic c o m p o u n d s . T h e S a l m o n e l l a / h i s t i d i n e p l a t e assay was perf o r m e d as d e s c r i b e d b y M a r o n a n d A m e s (1983) with strains TA98, TA100, TA102, TA1535 a n d TA1537. I n each e x p e r i m e n t the tester strains were checked using k n o w n m u t a g e n s (see T a b l e 1). The e x p e r i m e n t s were c o n s i d e r e d valid w h e n b o t h the s p o n t a n e o u s m u t a t i o n rate a n d the reversion rate i n d u c e d b y the c o n t r o l m u t a g e n s were similar to those of M a r o n a n d A m e s (1983). Triplicate plates were used. T h e SOS C h r o m o t e s t was p e r f o r m e d as d e s c r i b e d b y Q u i l l a r d e t a n d H o f n u n g (1985). I n each e x p e r i m e n t the tester strain was c h e c k e d using a k n o w n S O S - i n d u c i n g c o m p o u n d as a control ( 4 N Q O , 100 ng p e r p r o b e in e x p e r i m e n t s w i t h o u t m e t a b o l i c activation, a n d A F B 1, 100 ng p e r p r o b e in e x p e r i m e n t s with m e t a b o l i c activation). A p r e p a r a t i o n of rat fiver 9000 x g s u p e r n a t a n t ($9) was used as a m e t a b o l i z i n g m i x t u r e in b o t h
tests. T h e rats were p r e t r e a t e d with A r o c l o r 1254. P r e p a r a t i o n s of the $9 fractions a n d c o m p o s i t i o n of the c o f a c t o r s o l u t i o n (mix) were p r e p a r e d as p r e v i o u s l y d e s c r i b e d ( M a r o n a n d A m e s , 1983; Q u i l l a r d e t a n d H o f n u n g , 1985). I n each experiment, the activity of the $9 mix was c h e c k e d b y e x a m i n i n g the m u t a g e n i c i t y or S O S - i n d u c i n g capacities of a f l a t o x i n B], a g e n o t o x i n k n o w n to require m e t a b o l i s m to b e active. R S F was d i s s o l v e d in d i m e t h y l - s u l f o x i d e ( D M S O ) a n d v a r i o u s c o n c e n t r a t i o n s were tested in b o t h tests until a toxic effect was observed. W h e n n o significant increase (i.e., less t h a n d o u b l i n g o f the b a c k g r o u n d level) in the n u m b e r of m u t a n t s or in the i n d u c t i o n factor was detectab l e in the p r e s e n c e of the c o m p o u n d , a ' l e s s than' value was c a l c u l a t e d in the 2 tests ( M c C a n n et al., 1975; Q u i l l a r d e t a n d H o f n u n g , 1985). I n the S a l m o n e l l a / h i s t i d i n e assay, this value corres p o n d e d to the s p o n t a n e o u s n u m b e r of revertants d i v i d e d b y the highest n o n - t o x i c d o s e tested. In the SOS C h r o m o t e s t , this value c o r r e s p o n d e d to 0.5 d i v i d e d b y the highest dose which d i d n o t
TABLE 1 ABSENCE OF MUTAGENIC ACTIVITY OF RSF IN THE SALMONELLA/HISTIDINE ASSAY Amount of RSF (ttg)
TA1535 _
+
TA100 _
+
TA98 _
0 0.01 0.1 1 10 100
22 20 21 27 22 20
25 31 22 29 26 26
147 156 156 163 160 163
172 165 184 167 180 175
36 40 40 36 44 40
Mutagenic potency (rev.//z g/plate)
0.2
0.25
Positive reference mutagen
6 x 103
_
1.5 2 450
1.7
0.4 453
TA1537 _
+ 47 56 53 54 58 42 0.5 1793
10 10 9 11 12 11 0.1 721
+
TA102 _
+
12 11 15 12 18 14
260 250 146 237 240 245
297 332 380 337 312 356
0.1
2.6 1708
3 -
For each strain (TA1535, TA100, TA98, TA1537 and TA102) and each quantity of compound per plate, the number of revertants per plate is given. It is the average number of 4 independent experiments for TA98 and TA100 and 2 independent experiments for TA1535, TA1537 and TA102, both with (+) and without ( - ) metabolic activation. A compound is considered mutagenic in the test when the number of revertants obtained is more than 2-fold the number of spontaneous revertants and a dose-response curve is obtained. Positive reference mutagens: EMS (ethyl methanesulfonate, Sigma) 10 /~1 per plate for TA1535; MMS (methyl methanesulfonate, Kodak) 1/d per plate for TA100; 4NQO (4-nitroquinoline-N-oxide, Fluka) 0.5/~g per plate for TA98 without metabofic activation; AFB1 (aflatoxin B1, Serva) 0.1 /~g per plate for TA98 with metabolic activation; ICR191 (2-methoxy-6-chloro-9-[3-]2chloroethyl[aminopropylamino]acridine. 2 HC1, Terochem Labs, Canada) 1/zg per plate for TA1537; and mitomicin C (Sigma) 0.5 /xg per plate for TA102.
237 TABLE 2 ABSENCE OF SOS-INDUCING ACTIVITY OF RSF IN THE SOS CHROMOTEST WITHOUT METABOLIC ACTIVATION Amount of RSF (/t g)
fl-gal units
P-ase units
fl-gal units
1.60 1.63 1.77 1.70 1.69 1.73 2.13
19.59 18.07 19.75 20.27 19.47 22.23 16.04
0.082 0.090 0.089 0.084 0.087 0.077 0.133
1 1.1 1.1 1.0 1.1 0.9 1.6
18.04
1.31
12.48
0 0.001 0.01 0.1 1 10 100 4NQO (100 ng)
23.6
P-ase units
Induction factor
SOS-inducing potency (per/tg)
0.05
fl-Galactosidase (fl-gal) units represent the expression of the SOS gene sfiA. Phosphatase alkaline (P-ase) units are a control for protein synthesis inhibition. They were calculated according to Quillardet and Hofnung (1985). The ratio of fl-gal units reflects the induction of the sfiA gene even when some inhibition of protein synthesis occurs. It is divided by its value in the absence of compound to obtain the induction factor. A compound is considered an SOS inducer when the induction factor reaches a value more than 1.5-2 and a dose-response curve is obtained.
i n h i b i t p r o t e i n symthesis. W e consider that a n increase of 0.5 or more i n the SOS i n d u c i n g factor (Quillardet a n d H o f n u n g , 1985) is significant.
Results and discussion The results o b t a i n e d with refined smoke flavor (RSF) tested in the S a l m o n e l l a / h i s t i d i n e plate assay are shown in T a b l e 1. N o significant increase in the n u m b e r of His + revertant colonies could be observed, either in the absence or i n the presence of m e t a b o l i z i n g mixture, with the 5 tester strains TA98, TA100, TA102, TA1535 a n d TA1537, whatever the c o n c e n t r a t i o n of RSF. These
results suggest that R S F is n o t m u t a g e n i c for bacteria. A t a n y rate, f r o m the d a t a it is possible to calculate that the m u t a g e n i c p o t e n c y of R S F is less than: 0.25 m u t a n t s p e r / ~ g per plate i n strain TA1535; 1.7 i n TA100; 0.12 in TA1537; 0.47 in TA98 a n d 3 in T A 1 0 2 (see T a b l e 1). The results o b t a i n e d with R S F tested i n the SOS C h r o m o t e s t are shown in Tables 2 a n d 3. N o significant increase in the i n d u c t i o n factor could be observed, either in the absence or in the presence of m e t a b o l i z i n g mixture, a n d whatever the c o n c e n t r a t i o n of RSF. These results indicate that, u n d e r our conditions, R S F c a n n o t i n d u c e the SOS f u n c t i o n sfiA in E. coli. Since SOS f u n c t i o n s are
TABLE 3 ABSENCE OF SOS-INDUCING ACTIVITY OF RSF IN THE SOS CHROMOTEST WITH METABOLIC ACTIVATION Amount of RSF (~ g) 0 0.001 0.01 0.1 1 10 100 AFB1 (100 ng) See footnote to Table 2.
fl-gal units
P-ase units
fl-gal units P-ase units
Induction factor
2.7 2.44 2.41 2.28 2.11 2.36 2.67
14.8 16.43 20.65 21.36 20.00 19.44 16.45
0.182 0.148 0.117 0.107 0.106 0.121 0.162
1 0.8 0.6 0.6 0.6 0.7 0.9
34.25
10.04
3.41
21.32
SOS-inducing potency (per/t g)
0.05
238 i n d u c e d for the m o s t p a r t b y D N A - d a m a g i n g agents, we m a y c o n c l u d e that R S F does n o t c o n tain powerful DNA-damaging compounds. The SOS i n d u c i n g p o t e n c y is less t h a n 0.05 p e r / ~ g (see T a b l e s 2 a n d 3). T h u s we have p r o d u c e d s t r o n g e v i d e n c e t h a t R S F is n o t g e n o t o x i c i n bacteria. Since there is a correlation between genotoxic activity in bacteria a n d c a r c i n o g e n i c activity for a large n u m b e r of c h e m i c a l s ( H o l l s t e i n a n d M c C a n n , 1979), the a b sence of g e n o t o x i c activity of R S F i n b a c t e r i a m i g h t be a n i n d i c a t i o n of a n a b s e n c e of c a r c i n o genic activity. H o w e v e r , m o r e d a t a h a v e to b e p r o v i d e d for the a b s e n c e of c a r c i n o g e n i c i t y of RSF.
References Ames, B.N., J. McCann and E. Yamasaki (1975) Methods for detecting carcinogens and mutagens with the Salmonella/ mammalian microsome mutagenicity test, Mutation Res., 31, 347-364. Borys, A., D. Chomiak, T. Wierzbicki, Z. Kr61ik and B. Pasiorowski (1982) Spos6b i urzodzenie do otrzymywania
preparatu dymu w~dzarniczego o modulowanych w/asnogciach sensorycznych. Patent pending P-238017. Hollstein, M., and J. McCann (1979) Short-term tests for carcinogens and mutagens, Mutation Res., 65, 133-226. Maron, D.M., and B.N. Ames (1983) Revised methods for the Salmonella mutagenicity test, Mutation Res., 113, 173-215. McCann, J., E. Choi, E. Yamasaki and B.N. Ames (1975) Detection of carcinogens as mutagens in the Salmonella/ microsome test: assay of 300 chemicals, Proc. Natl. Acad. Sci. (U.S.A.), 72, 5135-5139. Ohta, T., N. Nakamura, M. Moriya, Y. Shirasu and T. Kada (1984) The SOS function-inducing activity of chemical mutagens in Escherichia coli, Mutation Res., 131, 101-109. Quillardet, P., O. Huisman, R. D'Ari and M. Hofnung (1982) The SOS Chromotest, a direct assay of induction of an SOS function in Escherichia coli K12 to measure genotoxicity, Proc. Natl. Acad. Sci. (U.S.A.), 79, 5971-5975. Quillardet, P., and M. Hofnung (1985) The SOS Chromotest, a colorimetric bacterial assay for genotoxins: procedures, Mutation Res., 147, 65-78. Quillardet, P., C. De Bellecombe and M. Hofnung (1985) The SOS Chromotest, a colorimetric bacterial assay for genotoxins: validation study with 83 compounds, Mutation Res., 147, 79-95. Walker, G.C. (1984) Mutagenesis and inducible responses to deoxyribonucleic acid damage in Escherichia coli, Microbiological Rev., 48, 60-93.
235
MTR 01258
Absence of genotoxic activity of refined smoke flavor (RSF) in two bacterial short-term tests Jacek Jenek 1,2 Philippe Quillardet 1, Danuta Chomiak 3, Andrzej Borys 3 and Maurice Hofnung 1 1 Unit~ de Programmation Mol~culaire et Toxicologie GOn~tique, CNRS LA271, I N S E R M U. 163, Institut Pasteur, 28, rue du Dr Roux, 75015 Paris (France), 2 Department of Medical Microbiology, Institute of Microbiology and Infectious Diseases, Academy of Medicine, ul. Wieniawskiego 3, 61-712 Poznah (Poland) and 3 Institute of Meat and Fat Industry, ul, Rakowiecka 36, 02-532 Warszawa (Poland) (Received 21 January 1987) (Revision received 17 September 1987) (Accepted 5 October 1987)
Keywords: Refined smoke flavor; Genotoxic activity; Short-term tests.
Summary The genotoxic activities of refined smoke flavor (RSF) produced in Poland and used in food processing were investigated in 2 bacterial short-term tests. Its mutagenic activity was examined in the Salmonella/ histidine plate assay and its SOS-inducing capacity in the SOS Chromotest both without and with 'activation' by a rat liver homogenate. No genotoxic activity was detected using these 2 bacterial tests.
Among the large number of short-term tests used to detect environmental genotoxins and consequently possible carcinogens, the bacterial tests constitute simple, rapid and economic systems (Hollstein and McCann, 1979). The Salmonella/ histidine test (Ames et al., 1975; Maron and Ames, 1983), the most popular of the bacterial assays, measures back mutations to prototrophy in several specially constructed his mutants of Salmonella. The SOS Chromotest (Quillardet et al., 1982; Quillardet and Hofnung, 1985) is a colorimetric assay of the induction by genotoxic agents of the gene sfiA in Escherichia coll. This gene is involved in one of the SOS functions (Walker, 1984). Its
Correspondence: Dr. J. Jenek, Department of Medical Microbiology, Institute of Microbiology and Infectious Diseases, Academy of Medicine, ul. Wieniawskiego 3, 61-712 Poznafi (Poland).
level of expression is monitored by means of a sfiA::lacZ genetic fusion. The SOS Chromotest has been validated on a series of known genotoxic agents (Quillardet et al., 1985; Ohta et al., 1984). In both tests a homogenate of rat liver can be added to the bacterial suspension as an approximation of mammalian metabolism. Both the Salmonella/histidine test and the SOS Chromotest were used to evaluate genotoxic activities of refined smoke flavor (RSF) produced in Poland and used in food processing in different commercial forms.
Materials and methods Refined smoke flavor (RSF) was produced from natural hard-wood smoke by condensation, distillation and rectification under reduced pressure according to the method described by Borys et al.
0165-1218/88/$03.50 © 1988 Elsevier Science Publishers B.V. (Biomedical Division)
236 (1982). It was p r e p a r e d in Rzeped~ (Poland). R S F m a i n l y consists of organic acids, neutral, c a r b o n y l a n d phenolic c o m p o u n d s . T h e S a l m o n e l l a / h i s t i d i n e p l a t e assay was perf o r m e d as d e s c r i b e d b y M a r o n a n d A m e s (1983) with strains TA98, TA100, TA102, TA1535 a n d TA1537. I n each e x p e r i m e n t the tester strains were checked using k n o w n m u t a g e n s (see T a b l e 1). The e x p e r i m e n t s were c o n s i d e r e d valid w h e n b o t h the s p o n t a n e o u s m u t a t i o n rate a n d the reversion rate i n d u c e d b y the c o n t r o l m u t a g e n s were similar to those of M a r o n a n d A m e s (1983). Triplicate plates were used. T h e SOS C h r o m o t e s t was p e r f o r m e d as d e s c r i b e d b y Q u i l l a r d e t a n d H o f n u n g (1985). I n each e x p e r i m e n t the tester strain was c h e c k e d using a k n o w n S O S - i n d u c i n g c o m p o u n d as a control ( 4 N Q O , 100 ng p e r p r o b e in e x p e r i m e n t s w i t h o u t m e t a b o l i c activation, a n d A F B 1, 100 ng p e r p r o b e in e x p e r i m e n t s with m e t a b o l i c activation). A p r e p a r a t i o n of rat fiver 9000 x g s u p e r n a t a n t ($9) was used as a m e t a b o l i z i n g m i x t u r e in b o t h
tests. T h e rats were p r e t r e a t e d with A r o c l o r 1254. P r e p a r a t i o n s of the $9 fractions a n d c o m p o s i t i o n of the c o f a c t o r s o l u t i o n (mix) were p r e p a r e d as p r e v i o u s l y d e s c r i b e d ( M a r o n a n d A m e s , 1983; Q u i l l a r d e t a n d H o f n u n g , 1985). I n each experiment, the activity of the $9 mix was c h e c k e d b y e x a m i n i n g the m u t a g e n i c i t y or S O S - i n d u c i n g capacities of a f l a t o x i n B], a g e n o t o x i n k n o w n to require m e t a b o l i s m to b e active. R S F was d i s s o l v e d in d i m e t h y l - s u l f o x i d e ( D M S O ) a n d v a r i o u s c o n c e n t r a t i o n s were tested in b o t h tests until a toxic effect was observed. W h e n n o significant increase (i.e., less t h a n d o u b l i n g o f the b a c k g r o u n d level) in the n u m b e r of m u t a n t s or in the i n d u c t i o n factor was detectab l e in the p r e s e n c e of the c o m p o u n d , a ' l e s s than' value was c a l c u l a t e d in the 2 tests ( M c C a n n et al., 1975; Q u i l l a r d e t a n d H o f n u n g , 1985). I n the S a l m o n e l l a / h i s t i d i n e assay, this value corres p o n d e d to the s p o n t a n e o u s n u m b e r of revertants d i v i d e d b y the highest n o n - t o x i c d o s e tested. In the SOS C h r o m o t e s t , this value c o r r e s p o n d e d to 0.5 d i v i d e d b y the highest dose which d i d n o t
TABLE 1 ABSENCE OF MUTAGENIC ACTIVITY OF RSF IN THE SALMONELLA/HISTIDINE ASSAY Amount of RSF (ttg)
TA1535 _
+
TA100 _
+
TA98 _
0 0.01 0.1 1 10 100
22 20 21 27 22 20
25 31 22 29 26 26
147 156 156 163 160 163
172 165 184 167 180 175
36 40 40 36 44 40
Mutagenic potency (rev.//z g/plate)
0.2
0.25
Positive reference mutagen
6 x 103
_
1.5 2 450
1.7
0.4 453
TA1537 _
+ 47 56 53 54 58 42 0.5 1793
10 10 9 11 12 11 0.1 721
+
TA102 _
+
12 11 15 12 18 14
260 250 146 237 240 245
297 332 380 337 312 356
0.1
2.6 1708
3 -
For each strain (TA1535, TA100, TA98, TA1537 and TA102) and each quantity of compound per plate, the number of revertants per plate is given. It is the average number of 4 independent experiments for TA98 and TA100 and 2 independent experiments for TA1535, TA1537 and TA102, both with (+) and without ( - ) metabolic activation. A compound is considered mutagenic in the test when the number of revertants obtained is more than 2-fold the number of spontaneous revertants and a dose-response curve is obtained. Positive reference mutagens: EMS (ethyl methanesulfonate, Sigma) 10 /~1 per plate for TA1535; MMS (methyl methanesulfonate, Kodak) 1/d per plate for TA100; 4NQO (4-nitroquinoline-N-oxide, Fluka) 0.5/~g per plate for TA98 without metabofic activation; AFB1 (aflatoxin B1, Serva) 0.1 /~g per plate for TA98 with metabolic activation; ICR191 (2-methoxy-6-chloro-9-[3-]2chloroethyl[aminopropylamino]acridine. 2 HC1, Terochem Labs, Canada) 1/zg per plate for TA1537; and mitomicin C (Sigma) 0.5 /xg per plate for TA102.
237 TABLE 2 ABSENCE OF SOS-INDUCING ACTIVITY OF RSF IN THE SOS CHROMOTEST WITHOUT METABOLIC ACTIVATION Amount of RSF (/t g)
fl-gal units
P-ase units
fl-gal units
1.60 1.63 1.77 1.70 1.69 1.73 2.13
19.59 18.07 19.75 20.27 19.47 22.23 16.04
0.082 0.090 0.089 0.084 0.087 0.077 0.133
1 1.1 1.1 1.0 1.1 0.9 1.6
18.04
1.31
12.48
0 0.001 0.01 0.1 1 10 100 4NQO (100 ng)
23.6
P-ase units
Induction factor
SOS-inducing potency (per/tg)
0.05
fl-Galactosidase (fl-gal) units represent the expression of the SOS gene sfiA. Phosphatase alkaline (P-ase) units are a control for protein synthesis inhibition. They were calculated according to Quillardet and Hofnung (1985). The ratio of fl-gal units reflects the induction of the sfiA gene even when some inhibition of protein synthesis occurs. It is divided by its value in the absence of compound to obtain the induction factor. A compound is considered an SOS inducer when the induction factor reaches a value more than 1.5-2 and a dose-response curve is obtained.
i n h i b i t p r o t e i n symthesis. W e consider that a n increase of 0.5 or more i n the SOS i n d u c i n g factor (Quillardet a n d H o f n u n g , 1985) is significant.
Results and discussion The results o b t a i n e d with refined smoke flavor (RSF) tested in the S a l m o n e l l a / h i s t i d i n e plate assay are shown in T a b l e 1. N o significant increase in the n u m b e r of His + revertant colonies could be observed, either in the absence or i n the presence of m e t a b o l i z i n g mixture, with the 5 tester strains TA98, TA100, TA102, TA1535 a n d TA1537, whatever the c o n c e n t r a t i o n of RSF. These
results suggest that R S F is n o t m u t a g e n i c for bacteria. A t a n y rate, f r o m the d a t a it is possible to calculate that the m u t a g e n i c p o t e n c y of R S F is less than: 0.25 m u t a n t s p e r / ~ g per plate i n strain TA1535; 1.7 i n TA100; 0.12 in TA1537; 0.47 in TA98 a n d 3 in T A 1 0 2 (see T a b l e 1). The results o b t a i n e d with R S F tested i n the SOS C h r o m o t e s t are shown in Tables 2 a n d 3. N o significant increase in the i n d u c t i o n factor could be observed, either in the absence or in the presence of m e t a b o l i z i n g mixture, a n d whatever the c o n c e n t r a t i o n of RSF. These results indicate that, u n d e r our conditions, R S F c a n n o t i n d u c e the SOS f u n c t i o n sfiA in E. coli. Since SOS f u n c t i o n s are
TABLE 3 ABSENCE OF SOS-INDUCING ACTIVITY OF RSF IN THE SOS CHROMOTEST WITH METABOLIC ACTIVATION Amount of RSF (~ g) 0 0.001 0.01 0.1 1 10 100 AFB1 (100 ng) See footnote to Table 2.
fl-gal units
P-ase units
fl-gal units P-ase units
Induction factor
2.7 2.44 2.41 2.28 2.11 2.36 2.67
14.8 16.43 20.65 21.36 20.00 19.44 16.45
0.182 0.148 0.117 0.107 0.106 0.121 0.162
1 0.8 0.6 0.6 0.6 0.7 0.9
34.25
10.04
3.41
21.32
SOS-inducing potency (per/t g)
0.05
238 i n d u c e d for the m o s t p a r t b y D N A - d a m a g i n g agents, we m a y c o n c l u d e that R S F does n o t c o n tain powerful DNA-damaging compounds. The SOS i n d u c i n g p o t e n c y is less t h a n 0.05 p e r / ~ g (see T a b l e s 2 a n d 3). T h u s we have p r o d u c e d s t r o n g e v i d e n c e t h a t R S F is n o t g e n o t o x i c i n bacteria. Since there is a correlation between genotoxic activity in bacteria a n d c a r c i n o g e n i c activity for a large n u m b e r of c h e m i c a l s ( H o l l s t e i n a n d M c C a n n , 1979), the a b sence of g e n o t o x i c activity of R S F i n b a c t e r i a m i g h t be a n i n d i c a t i o n of a n a b s e n c e of c a r c i n o genic activity. H o w e v e r , m o r e d a t a h a v e to b e p r o v i d e d for the a b s e n c e of c a r c i n o g e n i c i t y of RSF.
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