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Induction of umuC gene expression by nitrogen dioxide in Salmonella typhimurium
Mutation Research, 142 (1985) 99-102 Elsevier
99
MRLett. 0659
Induction of umuC gene expression by nitrogen dioxide in Salmonella
typhimurium Hiroaki
Kosaka*,
Yoshimitsu
Oda and Mitsuro Uozumi
Division o f Environmental Health Research, Osaka Prefectural Institute o f Public Health, 1-3-69 Nakamichi, Higashinari-ku, Osaka 53 7 (Japan) (Accepted 22 November 1984)
Summary Gaseous nitrogen dioxide ( N O 2 ) w a s found to induce umuC gene expression in Salmonella typhimurium carrying the umuC-lacZ fusion plasmid. The induction level of the umu operon responsible for inducible mutagenesis was measured by the level of/3-galactosidase in the cell, encoded by the fusion gene. N O 2 gas was bubbled into bacterial suspensions at 10, 30 and 90 ~1/1 for 30 min at a flow rate of 100 ml/min. Expression of the umuC gene varied with the concentration, flow rate and bubbling time of the NO2 gas. Although NO2 gas induces SOS functions, mutagenesis due to it was not detectable in Salmonella typhimurium TA100 and TA102. Nitric oxide gas (NO) did not induce any umuC gene expression.
Nitrogen oxides (NOx) are major air pollutants in large cities, and as nitrogen dioxide (NO2) is considered to be the most toxic, considerable attention has been devoted to assessing its biological effect. Tsuda et al. (1981) reported that nitrogen dioxide gas (NOD could induce chromosome aberrations in cultured Chinese hamster V79-H3 cells. Isomura et al. (1984) reported that NO2 induced mutagenicity in Salmonella typhimurium TA100 and TA1535 and that mutations and chromosome aberrations were induced in lung cells following in vivo exposure of rats to N O z . However, no evidence of malignant tumors developing in animals exposed to NO2 has been reported (Freeman et al., 1968), i.e. whether or not N O 2 is carcinogenic remains to be determined. To check the mutagenic activity of NO2, we in*To whom correspondence should be addressed.
vestigated the inducibility of SOS functions in bacteria by NO2 gas. The SOS functions include mutagenesis, increased DNA-repair capacity, prophage induction, and inhibition of cell division (Witkin, 1976; Little and Mount, 1982). In the regulatory system of SOS functions, the lexAcoded protein represses a set of unlinked SOS genes, including the umuC gene. The recA-coded protein (which is activated as a protease by DNAdamaging treatments) specifically inactivates repressors such as the LexA protein, and can also inactivate some prophage repressors by proteolytic cleavage (Little and Mount, 1982). Therefore, if N O 2 gas induce SOS functions, it is probably mutagenic. Of the SOS functions, we tried to detect the levels of the UmuC protein, using a plasmid carrying umuC-lacZ fusion gene. This gene was described by Shinagawa et al. (1983) and the principle of the UmuC protein detection
0165-7992/85/$ 03.30 © 1985 Elsevier Science Publishers B.V. (Biomedical Division)
100 method by Yamamoto et al. (1984). The umuC gene product is directly involved in the induction of mutagenesis (Kato and Shinoura, 1977). We used the strain in which the umuC-lacZ plasmid pSK1002 had been introduced into S. typhimurium TA1535; its membrane permeability is higher than that of E. coli. When a bacterium carrying the umuC-lacZ plasmid (pSK1002) was treated with UV or chemical mutagens, the umu operon was reported to be inducible by the mutagens (Shinagawa et al., 1983; Yamamoto et al., 1984). Our study using S. typhimurium carrying umuC-lacZ fusion showed that gaseous NO2 induces SOS functions. Since the umuC-coded gene product is considered to be responsible for the induction of mutagenesis (Kato and Shinoura, 1977), we speculated that NO2 gas is mutagenic and examined this hypothesis. Materials and methods
NO2, 2000 /A/l, or NO, 15 000 /zl/l, (Seitetsu Kagaku Works, Ltd.) was diluted with air using a standard gas dilution system Model 302 (Seitetsu Kagaku Works, Ltd.). The flow rate of the diluted gas monitored by a flowmeter was kept constant by a flow apparatus consisting of a pressureregulating valve (model 6600, Kojima Flow Instruments Corp.) and a teflon capillary. NO and NO2 concentrations were monitored with an NOx analyzer (Denki Kagaku Keiki Ltd.). Nitrite was measured as described previously (Kosaka et al., 1979). S. typhimurium TA1535 carrying plasmid pSK1002 was kindly supplied by Dr. H. Shinagawa, Osaka University. The plasmid pSK1002 which has a umuC-lacZ fusion gene has been described previously (Shinagawa et al., 1983). S. typhimurium TA1535 carries the umuC-lacZ plasmid, pSK1002 and has a deletion for the lactose operon so that 3-galactosidase activity is strictly dependent on umuC expression. To increase the response to diverse DNA-damaging agents, the strain was made deficient in excision repair (uvrA), and to allow better diffusion of mutagens into the cell it was made lipopolysac-
charide deficient (rfa). S. typhimurium strains TA100 and TAI02 were used (Ames, 1971) to investigate the mutagenicity of NO2. When NO2 was bubbled through TGA (trypton glucose ampicillin) medium, the color changed to yellow, as tyrosine, tryptophan and phenylalanine each contain a benzene ring which causes the xanthoprotein reaction. When these components were excluded, the medium color did not change. We used this medium, devoid of these amines (hereafter called the minimum medium). We grew each test strain overnight at 37°C. The test strain suspension was then diluted 100-fold with the minimum medium and the diluted suspension was allowed to grow exponentially for 2 h. Next, the suspension was divided into 3-ml fractions and NO or NO2 gas was bubbled into each through a stainless steel tube (i.d. 2 mm) for 30 min at room temperature (around 25°C). These samples were incubated for 120 rain at 37°C and then assayed for 3-galactosidase by Miller's method (1972). NOz gas was bubbled into the bacterial suspension (TA100 or TA102 was suspended in 0.1 M phosphate buffer) for 30 min at a constant flow of 100 ml/min. Cells had been mixed with soft agar and spread onto the agar plates containing SEM (semi-enriched medium) to allow counting of the His + revertants. In an alternative procedure, SEM plates with soft agar containing TA100 or TA102 were placed in a desiccator and exposed to 1 1/min of NO2 gas. Results
Gaseous NO2 induced 3-galactosidase synthesis at 10-90 ~1/1 as shown in Fig. 1, which shows the activity ratio normalized to its value in the absence of the test compound to allow comparison with NO and NO2. The normalized value was called the SOS induction factor. 90 ~1/1 NOz increased /3galactosidase activity 2-3-fold. With the same method, 2-fold increase in 3-galactosidase activity was detected with 0.08 t~g/ml 4-nitroquinoline 1-oxide, 0.6 #g/ml N-methyl-N' -nitro-Nnitrosoguanidine and 27 #g/ml methyl
101
300
2o0
5 100 i
20
40 60 80 NOx ( .ulll )
100
50
Fig. 1. Effect o f NO2 (O) or NO (©) on the induction o f umuC gene expression. The gas (100 ml/min) was bubbled into each bacterial suspension for 30 min.
methanesulfonate. In contrast to NOz, NO did not induce the production of any B-galactosidase. The amount of ~-galactosidase increased with the exposure time (10-90 min) to 30 ~1/1 NOz at a flow rate of 100 m l / m i n . It also increased when the flow rate of the 30 ~1/1 NO2 gas rose f r o m 10 to 160 m l / m i n , although the level remained constant from 160 to 300 m l / m i n (Fig. 2). This suggests that the capacity of bacterial membrane to pass the d i s s o l v e d N 0 2 is limited. Assuming that the induction of SOS functions b y N O 2 is due to the nitrite formed during the bubbling of NOz gas, we examined the induction of SOS functions by nitrite. As illustrated in Fig. 3, high concentrations of 50 and 100 mM nitrite were required to induce SOS functions, although we did note that when 90 ~1/1 NOz was passed through the
Fig. 3. Induction of u m u C gene expression by nitrite.
medium at 100 m l / m i n , 0.25 mM nitrite was produced for 30 min. N 0 2 induced the transcription of umuC (Fig. 1). Because the umuC gene is considered to be responsible for the induction of mutagenesis, we examined the mutagenicity of NO2 by the Ames test. Neither S. typhimurium TA100 nor TA102 showed mutagenicity at 45, 90, 180 txl/1 NO2 which was bubbled through the medium as shown in Table 1. We tried another method of exposing the bacteria to NOz gas, as N O 2 m a y have interacted with the component in the SEM agar to form a complex contributing to mutagenicity. Plates were seeded with bacteria and placed in a desiccator through which NOz was passed. However, NOz TABLE 1 R E V E R T A N T S PER P L A T E BY NOz E X P O S U R E NOz (gl/l) 0
TAI00 90 84
10 = v3
o
"6 100
300
Fig. 2. Dependence of u m u C gene expression on the flow rate of 30 #1/1 NO2 gas for 30 rain.
TAI02
238 205
205
45
89 107
332 208
222 214
90
100 97
358 272
240
180
129 84
0 0
100 200 Flow rate ( m l / m i n )
aTA100
259 253
200
00
100
No, NO2 ( mM )
179 242
NO2 gas was bubbled into the solution at 100 rnl/min for 30 min. aNO2 gas was passed through the desiccator containing the T A I 0 0 plates.
102
(10, 45, 90 t~l/1, 1 1/min) did not increase the revertants even by this method as shown in Table 1.
Discussion This study demonstrates that production of the u m u C gene can be induced by gaseous NO2 at 30 and 90 tA/I NOz given at flow rate of 100 m l / m i n for 30 min. The induction of u m u C gene expressions depended upon the concentration, exposure time and flow rate of gaseous NOz. To our knowledge, this is the first report of significant induction of SOS functions by gaseous NOz but no induction of u m u C gene expression by NO. This result agrees with the fact that there has been no report demonstrating the mutagenicity of NO. We found that nitrite induced u m u C gene expression (Fig. 3). NO2 gas dissolves in water to yield nitrite which is suspected of producing the above-mentioned effects of gaseous N O 2 . This possibility was checked by measuring the nitrite concentrations in the medium after bubbling NO2 gas. Even at 90 tA/1 NOz, the nitrite concentration was 0.25 raM, which did not increase the induction of u m u C gene expression. We thus concluded that the effect of gaseous NO2 in the induction of u m u C gene expression could not be ascribed only to nitrite. Why and how gaseous NOz induces the expression of u m u C gene remain unanswered questions. Some of the damaging effects of NOz may be related to its capability to initiate free-radical reaction including initiation of lipid peroxidation (Thomas et al., 1967; Menzel, 1976). The products of lipid peroxidation in bacterial membranes may induce D N A lesions, leading to the induction of u m u C gene expression. Since U m u C protein is thought to be responsible for the induction of mutagenesis (Kato and Shinoura, 1977), the mutagenicity of NO2 gas should have been detected in the present assay systems. However, we were not able to detect any mutagenicity with the Ames test using TA100 and TA102. The reason for this is not clear. Some critical experimental conditions may be necessary for the induction of mutagenicity of NOz gas.
Acknowledgements This work was supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science and Culture of Japan. We are grateful to Dr. K. Y a m a m o t o for helpful suggestions. We thank Miss T. Kimura for her excellent technical assistance.
References Ames, B.N. (1971) Chemical Mutagens, Principles and Methods for Their Detection, Vol. 1, Plenum, New York, pp. 267-282. Freeman, G., R.J. Stephens, S.C. Crane and N.J. Furiosi (1968) Lesion of the lung in rats continuously exposed to two parts per million of nitrogen dioxide, Arch. Environ. Health, 17, 181-192. Isomura, K., M. Chikahira, K. Teranishi and K. Hamada (1984) Induction of mutations and chromosome aberrations in lung cells following in vivo exposure of rats to nitrogen oxides, Mutation Res., 136, 119-125. Kato, T. and Y. Shinoura (1977) Isolation and characterization of mutants of Escherichia coli deficient in induction of mutations by ultraviolet light, Mol. Gen. Genet., 156, 121-131. Kosaka, H., K. Imaizumi, K. lmai and I. Tyuma (1979) Stoichiometry of the reaction of oxyhemoglobin with nitrite, Biochim. Biophys. Acta, 581, 184-188. Little, J.W., and D.W. Mount (1982) The SOS regulation system of Escherichia coli, Cell, 29, 11-22. Menzel, D.B. (1976) The role of free radicals in the toxicity of air pollutants (nitrogen oxides and ozone), in: A.W. Pryor (Ed.), Free Radicals in Biology, Vol. 2, pp. 181-200. Miller, J.H. (1972) Experiments in Molecular Genetics, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, pp. 352-355. Shinagawa, H., T. Kato, T. Ise, K. Makino and A. Nakata (1983) Cloning and characterization of the umu operon responsible for inducible mutagenesis in Escherichia coli, Gene, 23, 167-174. Thomas, H.V., P.K. Mueller and P.L. Lyman (1967) Lipoperoxidation of lung lipids in rats exposed to nitrogen dioxide, Science, 159, 532-534. Tsuda, H., A. Kushi, D. Yoshida and F. Goto (1981) Chromosomal aberrations and sister-chromatid exchanges induced by gaseous nitrogen dioxide in cultured Chinese hamster cells, Mutation Res., 89, 303-309. Witkin, E.M. (1976) Ultraviolet mutagenesis and inducible DNA repair in Escherichia coli, Bacteriol. Rev., 40, 869-907. Yamamoto, K., T. Hiramoto, H. Shinagawa and Y. Fujiwara (1984) Genetic activity of bleomycin in Escherichia coli, Chem.-Biol. Interact., 48, 145-152.
99
MRLett. 0659
Induction of umuC gene expression by nitrogen dioxide in Salmonella
typhimurium Hiroaki
Kosaka*,
Yoshimitsu
Oda and Mitsuro Uozumi
Division o f Environmental Health Research, Osaka Prefectural Institute o f Public Health, 1-3-69 Nakamichi, Higashinari-ku, Osaka 53 7 (Japan) (Accepted 22 November 1984)
Summary Gaseous nitrogen dioxide ( N O 2 ) w a s found to induce umuC gene expression in Salmonella typhimurium carrying the umuC-lacZ fusion plasmid. The induction level of the umu operon responsible for inducible mutagenesis was measured by the level of/3-galactosidase in the cell, encoded by the fusion gene. N O 2 gas was bubbled into bacterial suspensions at 10, 30 and 90 ~1/1 for 30 min at a flow rate of 100 ml/min. Expression of the umuC gene varied with the concentration, flow rate and bubbling time of the NO2 gas. Although NO2 gas induces SOS functions, mutagenesis due to it was not detectable in Salmonella typhimurium TA100 and TA102. Nitric oxide gas (NO) did not induce any umuC gene expression.
Nitrogen oxides (NOx) are major air pollutants in large cities, and as nitrogen dioxide (NO2) is considered to be the most toxic, considerable attention has been devoted to assessing its biological effect. Tsuda et al. (1981) reported that nitrogen dioxide gas (NOD could induce chromosome aberrations in cultured Chinese hamster V79-H3 cells. Isomura et al. (1984) reported that NO2 induced mutagenicity in Salmonella typhimurium TA100 and TA1535 and that mutations and chromosome aberrations were induced in lung cells following in vivo exposure of rats to N O z . However, no evidence of malignant tumors developing in animals exposed to NO2 has been reported (Freeman et al., 1968), i.e. whether or not N O 2 is carcinogenic remains to be determined. To check the mutagenic activity of NO2, we in*To whom correspondence should be addressed.
vestigated the inducibility of SOS functions in bacteria by NO2 gas. The SOS functions include mutagenesis, increased DNA-repair capacity, prophage induction, and inhibition of cell division (Witkin, 1976; Little and Mount, 1982). In the regulatory system of SOS functions, the lexAcoded protein represses a set of unlinked SOS genes, including the umuC gene. The recA-coded protein (which is activated as a protease by DNAdamaging treatments) specifically inactivates repressors such as the LexA protein, and can also inactivate some prophage repressors by proteolytic cleavage (Little and Mount, 1982). Therefore, if N O 2 gas induce SOS functions, it is probably mutagenic. Of the SOS functions, we tried to detect the levels of the UmuC protein, using a plasmid carrying umuC-lacZ fusion gene. This gene was described by Shinagawa et al. (1983) and the principle of the UmuC protein detection
0165-7992/85/$ 03.30 © 1985 Elsevier Science Publishers B.V. (Biomedical Division)
100 method by Yamamoto et al. (1984). The umuC gene product is directly involved in the induction of mutagenesis (Kato and Shinoura, 1977). We used the strain in which the umuC-lacZ plasmid pSK1002 had been introduced into S. typhimurium TA1535; its membrane permeability is higher than that of E. coli. When a bacterium carrying the umuC-lacZ plasmid (pSK1002) was treated with UV or chemical mutagens, the umu operon was reported to be inducible by the mutagens (Shinagawa et al., 1983; Yamamoto et al., 1984). Our study using S. typhimurium carrying umuC-lacZ fusion showed that gaseous NO2 induces SOS functions. Since the umuC-coded gene product is considered to be responsible for the induction of mutagenesis (Kato and Shinoura, 1977), we speculated that NO2 gas is mutagenic and examined this hypothesis. Materials and methods
NO2, 2000 /A/l, or NO, 15 000 /zl/l, (Seitetsu Kagaku Works, Ltd.) was diluted with air using a standard gas dilution system Model 302 (Seitetsu Kagaku Works, Ltd.). The flow rate of the diluted gas monitored by a flowmeter was kept constant by a flow apparatus consisting of a pressureregulating valve (model 6600, Kojima Flow Instruments Corp.) and a teflon capillary. NO and NO2 concentrations were monitored with an NOx analyzer (Denki Kagaku Keiki Ltd.). Nitrite was measured as described previously (Kosaka et al., 1979). S. typhimurium TA1535 carrying plasmid pSK1002 was kindly supplied by Dr. H. Shinagawa, Osaka University. The plasmid pSK1002 which has a umuC-lacZ fusion gene has been described previously (Shinagawa et al., 1983). S. typhimurium TA1535 carries the umuC-lacZ plasmid, pSK1002 and has a deletion for the lactose operon so that 3-galactosidase activity is strictly dependent on umuC expression. To increase the response to diverse DNA-damaging agents, the strain was made deficient in excision repair (uvrA), and to allow better diffusion of mutagens into the cell it was made lipopolysac-
charide deficient (rfa). S. typhimurium strains TA100 and TAI02 were used (Ames, 1971) to investigate the mutagenicity of NO2. When NO2 was bubbled through TGA (trypton glucose ampicillin) medium, the color changed to yellow, as tyrosine, tryptophan and phenylalanine each contain a benzene ring which causes the xanthoprotein reaction. When these components were excluded, the medium color did not change. We used this medium, devoid of these amines (hereafter called the minimum medium). We grew each test strain overnight at 37°C. The test strain suspension was then diluted 100-fold with the minimum medium and the diluted suspension was allowed to grow exponentially for 2 h. Next, the suspension was divided into 3-ml fractions and NO or NO2 gas was bubbled into each through a stainless steel tube (i.d. 2 mm) for 30 min at room temperature (around 25°C). These samples were incubated for 120 rain at 37°C and then assayed for 3-galactosidase by Miller's method (1972). NOz gas was bubbled into the bacterial suspension (TA100 or TA102 was suspended in 0.1 M phosphate buffer) for 30 min at a constant flow of 100 ml/min. Cells had been mixed with soft agar and spread onto the agar plates containing SEM (semi-enriched medium) to allow counting of the His + revertants. In an alternative procedure, SEM plates with soft agar containing TA100 or TA102 were placed in a desiccator and exposed to 1 1/min of NO2 gas. Results
Gaseous NO2 induced 3-galactosidase synthesis at 10-90 ~1/1 as shown in Fig. 1, which shows the activity ratio normalized to its value in the absence of the test compound to allow comparison with NO and NO2. The normalized value was called the SOS induction factor. 90 ~1/1 NOz increased /3galactosidase activity 2-3-fold. With the same method, 2-fold increase in 3-galactosidase activity was detected with 0.08 t~g/ml 4-nitroquinoline 1-oxide, 0.6 #g/ml N-methyl-N' -nitro-Nnitrosoguanidine and 27 #g/ml methyl
101
300
2o0
5 100 i
20
40 60 80 NOx ( .ulll )
100
50
Fig. 1. Effect o f NO2 (O) or NO (©) on the induction o f umuC gene expression. The gas (100 ml/min) was bubbled into each bacterial suspension for 30 min.
methanesulfonate. In contrast to NOz, NO did not induce the production of any B-galactosidase. The amount of ~-galactosidase increased with the exposure time (10-90 min) to 30 ~1/1 NOz at a flow rate of 100 m l / m i n . It also increased when the flow rate of the 30 ~1/1 NO2 gas rose f r o m 10 to 160 m l / m i n , although the level remained constant from 160 to 300 m l / m i n (Fig. 2). This suggests that the capacity of bacterial membrane to pass the d i s s o l v e d N 0 2 is limited. Assuming that the induction of SOS functions b y N O 2 is due to the nitrite formed during the bubbling of NOz gas, we examined the induction of SOS functions by nitrite. As illustrated in Fig. 3, high concentrations of 50 and 100 mM nitrite were required to induce SOS functions, although we did note that when 90 ~1/1 NOz was passed through the
Fig. 3. Induction of u m u C gene expression by nitrite.
medium at 100 m l / m i n , 0.25 mM nitrite was produced for 30 min. N 0 2 induced the transcription of umuC (Fig. 1). Because the umuC gene is considered to be responsible for the induction of mutagenesis, we examined the mutagenicity of NO2 by the Ames test. Neither S. typhimurium TA100 nor TA102 showed mutagenicity at 45, 90, 180 txl/1 NO2 which was bubbled through the medium as shown in Table 1. We tried another method of exposing the bacteria to NOz gas, as N O 2 m a y have interacted with the component in the SEM agar to form a complex contributing to mutagenicity. Plates were seeded with bacteria and placed in a desiccator through which NOz was passed. However, NOz TABLE 1 R E V E R T A N T S PER P L A T E BY NOz E X P O S U R E NOz (gl/l) 0
TAI00 90 84
10 = v3
o
"6 100
300
Fig. 2. Dependence of u m u C gene expression on the flow rate of 30 #1/1 NO2 gas for 30 rain.
TAI02
238 205
205
45
89 107
332 208
222 214
90
100 97
358 272
240
180
129 84
0 0
100 200 Flow rate ( m l / m i n )
aTA100
259 253
200
00
100
No, NO2 ( mM )
179 242
NO2 gas was bubbled into the solution at 100 rnl/min for 30 min. aNO2 gas was passed through the desiccator containing the T A I 0 0 plates.
102
(10, 45, 90 t~l/1, 1 1/min) did not increase the revertants even by this method as shown in Table 1.
Discussion This study demonstrates that production of the u m u C gene can be induced by gaseous NO2 at 30 and 90 tA/I NOz given at flow rate of 100 m l / m i n for 30 min. The induction of u m u C gene expressions depended upon the concentration, exposure time and flow rate of gaseous NOz. To our knowledge, this is the first report of significant induction of SOS functions by gaseous NOz but no induction of u m u C gene expression by NO. This result agrees with the fact that there has been no report demonstrating the mutagenicity of NO. We found that nitrite induced u m u C gene expression (Fig. 3). NO2 gas dissolves in water to yield nitrite which is suspected of producing the above-mentioned effects of gaseous N O 2 . This possibility was checked by measuring the nitrite concentrations in the medium after bubbling NO2 gas. Even at 90 tA/1 NOz, the nitrite concentration was 0.25 raM, which did not increase the induction of u m u C gene expression. We thus concluded that the effect of gaseous NO2 in the induction of u m u C gene expression could not be ascribed only to nitrite. Why and how gaseous NOz induces the expression of u m u C gene remain unanswered questions. Some of the damaging effects of NOz may be related to its capability to initiate free-radical reaction including initiation of lipid peroxidation (Thomas et al., 1967; Menzel, 1976). The products of lipid peroxidation in bacterial membranes may induce D N A lesions, leading to the induction of u m u C gene expression. Since U m u C protein is thought to be responsible for the induction of mutagenesis (Kato and Shinoura, 1977), the mutagenicity of NO2 gas should have been detected in the present assay systems. However, we were not able to detect any mutagenicity with the Ames test using TA100 and TA102. The reason for this is not clear. Some critical experimental conditions may be necessary for the induction of mutagenicity of NOz gas.
Acknowledgements This work was supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science and Culture of Japan. We are grateful to Dr. K. Y a m a m o t o for helpful suggestions. We thank Miss T. Kimura for her excellent technical assistance.
References Ames, B.N. (1971) Chemical Mutagens, Principles and Methods for Their Detection, Vol. 1, Plenum, New York, pp. 267-282. Freeman, G., R.J. Stephens, S.C. Crane and N.J. Furiosi (1968) Lesion of the lung in rats continuously exposed to two parts per million of nitrogen dioxide, Arch. Environ. Health, 17, 181-192. Isomura, K., M. Chikahira, K. Teranishi and K. Hamada (1984) Induction of mutations and chromosome aberrations in lung cells following in vivo exposure of rats to nitrogen oxides, Mutation Res., 136, 119-125. Kato, T. and Y. Shinoura (1977) Isolation and characterization of mutants of Escherichia coli deficient in induction of mutations by ultraviolet light, Mol. Gen. Genet., 156, 121-131. Kosaka, H., K. Imaizumi, K. lmai and I. Tyuma (1979) Stoichiometry of the reaction of oxyhemoglobin with nitrite, Biochim. Biophys. Acta, 581, 184-188. Little, J.W., and D.W. Mount (1982) The SOS regulation system of Escherichia coli, Cell, 29, 11-22. Menzel, D.B. (1976) The role of free radicals in the toxicity of air pollutants (nitrogen oxides and ozone), in: A.W. Pryor (Ed.), Free Radicals in Biology, Vol. 2, pp. 181-200. Miller, J.H. (1972) Experiments in Molecular Genetics, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, pp. 352-355. Shinagawa, H., T. Kato, T. Ise, K. Makino and A. Nakata (1983) Cloning and characterization of the umu operon responsible for inducible mutagenesis in Escherichia coli, Gene, 23, 167-174. Thomas, H.V., P.K. Mueller and P.L. Lyman (1967) Lipoperoxidation of lung lipids in rats exposed to nitrogen dioxide, Science, 159, 532-534. Tsuda, H., A. Kushi, D. Yoshida and F. Goto (1981) Chromosomal aberrations and sister-chromatid exchanges induced by gaseous nitrogen dioxide in cultured Chinese hamster cells, Mutation Res., 89, 303-309. Witkin, E.M. (1976) Ultraviolet mutagenesis and inducible DNA repair in Escherichia coli, Bacteriol. Rev., 40, 869-907. Yamamoto, K., T. Hiramoto, H. Shinagawa and Y. Fujiwara (1984) Genetic activity of bleomycin in Escherichia coli, Chem.-Biol. Interact., 48, 145-152.