Mutation Research, 281 (1992) 193-202
193
© 1992 Elsevier Science Publishers B.V. All rights reserved 0165-7992/92/$05.00
MUTLET 00631
Mutagenicity of nitric oxide and its inhibition by antioxidants P.L. Arroyo, V. Hatch-Pigott, H.F. Mower and R.V. Cooney Unit~ersity of Hawaii, Cancer Research Center of Hawaii, 1236 Lauhala Street, Honolulu, HI 96813 (U.S.A.)
(Received 5 September 1991) (Accepted 15 October 1991)
Keywords: Nitric oxide; Nitrogen dioxide; /3-Carotene; Ames assay; Free radicals; DNA deamination
Summary Nitric oxide (NO) is produced both by macrophages in vivo as a physiological response to infection and by a variety of cell types as an intercellular messenger. In addition, N O and nitrogen dioxide (NO 2) are significant components of many combustion processes. The ubiquitous exposure of humans to nitrogen oxides (NOx), both endogenously and exogenously, may play a significant role in the carcinogenic process due to nitrosation of amines by NOx. We report here that exposure to low concentrations of NO, alone or in combination with N O 2, results in significantly enhanced mutation in Salmonella typhimurium TA1535 using a modified Ames Salmonella reversion assay. The observed mutagenicity requires that the bacteria be actively dividing at the time of exposure to NO or N O 2, suggesting that the nitrogen oxides, or their reaction products, function as direct-acting mutagens and that the induced lesion is easily repairable by non-dividing cells. Exposure to N O resulted in a time- and dose-dependent increase in the number of revertants approximately proportional to the square of the N O concentration from 0 to 20 ppm. N O was a more effective mutagen relative to NO2, however, the observed requirement for 0 2 suggests limited oxidation of N O (presumably to NO 2) is necessary. Numerous lipid- and aqueous-phase inhibitors of nitrosation, as well as a number of other general antioxidants and free-radical trapping agents, were examined for their effectiveness in blocking the mutagenic effects of NO. The mutagenic activity of N O was most effectively inhibited by/3-carotene and tocopherols. BHT, dimethyl sulfoxide and mannitol also blocked the mutagenic effects of N O x but appeared less effective than /3-carotene or vitamin E, while ascorbate was ineffective as an inhibitor of mutation resulting from N O exposure.
H u m a n s are exposed to nitrogen oxides (NO x) daily from sources such as automobile exhaust, cigarette smoke, domestic gas cooking and heat-
Correspondence: Dr. R.V. Cooney, University of Hawaii, Cancer Research Center of Hawaii, 1236 Lauhala Street, Honolulu, HI 96813 (U.S.A.).
ing (Lewis, 1980). Some of the biological effects associated with environmental exposure to nitric oxide (NO) and nitrogen dioxide (NO 2) are well established, such as the morphological and physiological changes in the pulmonary epithelium resulting in lung tissue damage (Nakajima et al., 1980). Toxicological evidence suggests that exposure to nitrogen oxides may increase susceptibil-
194
ity to viral respiratory infection and alter the alveolar epithelial permeability and transport properties resulting in functional deficits (Mustafa et al., 1980; Rose et al., 1989). Although the general toxic effects of nitrogen oxides have been thoroughly studied, only a few reports are available concerning the genotoxicity of these compounds (Sasaki et al., 1980; Kosaka et al., 1985; Biggart and Rinehart, 1987; Victorin and Stalberg, 1988; Gorsdorf et al., 1990). Because of its greater reactivity and acute toxicity, attention has been focused o n N O 2 despite the fact that in vivo (Marietta, 1989) and in many complex gas mixtures, NO is the main oxide of nitrogen formed (Sasaki et al., 1980). The mutagenicities of complex gas mixtures, such as welding fumes, have been compared with that of NO 2 gas, which is known to exist in high concentrations, yet NO 2 accounts for only a fraction of the mutagenicity observed (Biggart and Rinehart, 1987; Sasaki et al., 1980). The mutagenic role of NO, which is a major product of combustion, was not considered in these reports. Previous investigations have shown NO to have little or no mutagenic potential (Kosaka et al., 1985; Gorsdorf et al., 1990). Recent evidence that NO and NO 2 are required in combination for aqueous amine nitrosation (Cooney et al., 1991) and the demonstration of deamination of D N A bases by NO (Nguyen et al., 1991; Wink et al., 1991) suggest that NO a n d / o r its oxidation products could either act as, or yield direct-acting mutagens in vivo. Endogenous production of NO by several cell types including endothelial cells (Moncada et al., 1988), neutrophils (Wright et al., 1989), and macrophages (Stuehr et al., 1989) has been demonstrated. The small molecular size, lipophilic nature, chemical instability and rapid reaction with oxygen and oxygen-derived radicals account for its pharmacokinetic properties as a trans-cellular messenger (Ignarro, 1991). The cytotoxic action of macrophage-derived NO on internalized organisms appears to require oxidation of NO to N O 2 (Iyengar et al., 1987; Leaf et al., 1991). Such a mixture of NO x could generate N20 3 which is capable of nitrosating amines at neutral pH (Challis et al., 1978; Kosaka et al., 1989) or react in a two step free radical reaction to produce an N-nitrosamine (Cooney et al., 1991). The forma-
tion of a primary N-nitrosamine on a DNA base would result in rapid deamination (Ridd, 1961) with the possibility of a point mutation occurring at this site in the absence of repair. To investigate the mutagenic activity of nitrogen oxides, a modified Ames test was used. The data presented in this paper show that exposure to NO, alone or in combination with NO 2, results in significantly enhanced mutation in Salmonella typhimurium TA1535. The use of antioxidants as effective inhibitors of the mutagenic activity of several carcinogens in the Salmonella assay has been examined previously (Rosin and Stich, 1979; Santamaria et al., 1984). In the present study the role of /3-carotene, tocopherols (a, y and ~), BHT, DMSO, ascorbate and mannitol in preventing genetic damage by NO alone, or in combination with NO 2 was assessed by measuring the frequency of reversion to histidine prototrophy in Salmonella typhimurium TA1535. Materials and methods
(1) Chemicals Mannitol, DMSO, BHT, fl-carotene, morpholine, a-, y- and 3-tocopherols and sodium ascorbate were purchased from Sigma Chemical Co., St. Louis, MO. Tetrahydrofuran (THF) containing 0.025% BHT as an antioxidant was purchased from Aldrich Chemical Co., St. Louis, MO. Optimal grade acetone and methylene chloride were from Fisher Scientific, Kent, Washington. NNitrosomorpholine and N-nitrosopyrrolidine standards were purchased from Chem Service Inc., Westchester, PA. Tanks of compressed gases containing NO and N O 2 in nitrogen were EPA grade from Scott Specialty gases, San Bernardino, CA. Certified purity for NO 2 was 55 ppm with contamination by NO of 3.5 ppm. The NO tank was certified to contain 91.00 ppm NO, with contamination from other oxides of nitrogen of 0.08 ppm (balance N2). Certified N 2 (99.999%) from Big Three Co., Honolulu, Hawaii was used to dilute the NO x gases. No nitric oxide was detectable in the N 2 source (limit of detection = 0.1 ppm). Gas flow rates were measured by individually calibrated flow meters. The gases were dry prior to exposure in the glove bag, however, there was some humid-
195 ity in the exposure chamber due to the agar plates. NO concentrations were measured using a Thermal Energy Analyzer (TEA) to verify the calculated gas concentration (Cooney et al., 1991).
(2) Exposure system NO, NO 2 or a combination of the two gases, was diluted with N 2 gas to the desired concentration by adjusting the flow rates of the individual gases with a rotameter (Scott Specialty Gases, San Bernardino, CA) to a total flow rate of 100 m l / m i n . The gas mixture was then allowed to fill the exposure chamber, consisting of an evacuated polyethylene glove bag (Inst. Res. Ind. Inc., Cheltenham, PA). Prior to changing the concentration of the gas mixture, the glove bag was fully emptied and then exposed for 30 min to the new nitrogen oxide concentration. The exposure chamber was cleaned and disinfected with ethanol once a week and the glove bag replaced after one month of use. In a typical experiment, culture dishes containing the bacteria were placed in the glove bag, lids were removed and the contents exposed to the desired concentration of NO, NO 2 or a combination of the two gases for 30 min. The bacterial plates were then recovered and transferred to an incubator and maintained as described below.
(3) Mutagenicity assay Salmonella strain TA1535 was used in these experiments. Cultures were maintained and grown as described by Ames and co-workers (Ames et al., [975). As in the standard plate-incorporation assay, 0.1 ml of an overnight (16 h) bacterial culture (approximately 2 × 108 bacteria) was mixed with 2.0 ml soft agar containing traces of histidine and biotin. The mixture was poured onto minimal glucose agar plates (30 ml agar per 100 mm petri dish). The Ames Salmonella assay as modified by Barber et al. (1983) was used to maximize the detection of short-lived, directacting mutagens. The plated bacteria were incubated at 37°C for 8 h prior to exposure to NO,. The plates were then placed in a polyethylene glove bag, lids were removed and the bacteria were exposed to the desired concentration of NO, NO 2 or a combination of the two gases in N 2. Control plates were exposed to a N 2 atmo-
sphere. At the end of the exposure period, usually 30 min, dishes were returned to the incubator and maintained at 37°C for 48 h, prior to colony counting using an automated colony counter, Biotran II (New Brunswick Scientific Co. Inc., Edison, N J). Positive controls utilizing sodium azide (2.5 ~ g / d i s h ) were run simultaneously yielding an average of 541 _+ 85 revertant colonies (n = 48) per dish for all the experiments described herein. Negative controls were included in every experiment for measurement of spontaneous revertants (mean = 12 _+ 4 revertants/dish, n = 48) and the number of revertant colonies was corrected by subtracting this background spontaneous mutation. The ability of antioxidants to inhibit mutation was assessed by adding 0.1 ml of various concentrations of the antioxidant to 2 ml of the top agar along with 0.1 ml of the bacterial culture. /3Carotene dissolved in T H F (final T H F concentration = 0.5%) was added to bacterial cultures prior to the 16-h incubation. 100 ILl of the bacterial suspensions were then added to 2.0 ml of the molten agar at 45°C as described above. All control cultures received the same amount of solvent as the treated samples. /3-Carotene was solubilized with T H F (Bertram et al., 1991), tocopherols and BHT with acetone (0.1% final solvent concentration), and DMSO and mannitol with water. T H F and acetone had no effect on the number of spontaneous revertants or any effect on mutagenicity by NO x at the concentrations used.
(4) Nitrosamine analysis The in vitro reactivity of amines with N O / N O 2 was assessed using a defined exposure system described by Cooney et al. (1987) in which a solution of morpholine (10 mM) at pH 7.4 was bubbled with 10 ppm NO and 5 ppm NO 2 in N 2 at a flow rate of 60 m l / m i n for 10 rain. NNitrosomorpholine was then extracted and analyzed by GC-TEA. The organic phase reaction was carried out in the manner described by Cooney et al. (1991) using 50 /~M morpholine in methylene chloride as the reaction solvent. The reaction was stopped by removing the tube from the gas stream and adding 100 p.1 of methanol containing 200 ng of N-nitrosopyrrolidine as an
196
internal standard. Aliquots were then injected for analysis on the GC-TEA.
LU F--
Although preliminary experiments indicated that N O was mutagenic in the standard A m e s assay, we sought to maximize the number of observed revertants by following the modifications described by Barber et al. (1983). Exponential growth normally occurs after approximately 4 - 8 h of incubation in agar at 37°C. It is during this period of log-phase growth that the bacteria are most susceptible to direct-acting, short-lived mutagens. The effect of incubation time on reversion frequency for strain TA1535 exposed to N O x for 30 min is shown in Fig. 1. The maximum mutation rate for 10 ppm N O alone or 8 ppm N O in combination with 5 ppm NO 2 is observed after 8 h growth of the test bacteria on the plate at 37°C. Subsequent experiments, therefore, followed this experimental protocol using an 8-h incubation prior to exposure to NO~. The mutagenic effects of N O and N O 2 were both time-dependent (Fig. 2), and dose-depen-
250
EL
200
Co I-Z i,i Cd :=~
LU LJ O~
•
100 5O 0
A,~, I 0
I
I
P
I
20
40
60
80
I
b
-
100 120 140
E X P O S U R E TIME ( r n i n )
Fig. 2. Dependence on incubation time for reversion of strain TA1535 by NO. Plated bacteria were incubated for 8 h at 37°C in culture dishes as described in Fig. 1 and Methods, then exposed to 10 ppm nitric oxide for the indicated periods of time. After subtraction of spontaneous revertants, each value represents the mean number of revertant colonies consisting of triplicate culture dishes + the standard deviation.
dent (Fig. 3). Fig. 3 shows dose-dependent mutagenicity after exposure of Salmonella typhimurium TA1535 to various concentrations of N O and N O 2 for 30 min. The numbers of revertants in the presence of NO 2 increased as a function of the NO 2 concentration up to 10 ppm, while in
200
700
EL co Fz
250
5
Results
LLI
193
© 1992 Elsevier Science Publishers B.V. All rights reserved 0165-7992/92/$05.00
MUTLET 00631
Mutagenicity of nitric oxide and its inhibition by antioxidants P.L. Arroyo, V. Hatch-Pigott, H.F. Mower and R.V. Cooney Unit~ersity of Hawaii, Cancer Research Center of Hawaii, 1236 Lauhala Street, Honolulu, HI 96813 (U.S.A.)
(Received 5 September 1991) (Accepted 15 October 1991)
Keywords: Nitric oxide; Nitrogen dioxide; /3-Carotene; Ames assay; Free radicals; DNA deamination
Summary Nitric oxide (NO) is produced both by macrophages in vivo as a physiological response to infection and by a variety of cell types as an intercellular messenger. In addition, N O and nitrogen dioxide (NO 2) are significant components of many combustion processes. The ubiquitous exposure of humans to nitrogen oxides (NOx), both endogenously and exogenously, may play a significant role in the carcinogenic process due to nitrosation of amines by NOx. We report here that exposure to low concentrations of NO, alone or in combination with N O 2, results in significantly enhanced mutation in Salmonella typhimurium TA1535 using a modified Ames Salmonella reversion assay. The observed mutagenicity requires that the bacteria be actively dividing at the time of exposure to NO or N O 2, suggesting that the nitrogen oxides, or their reaction products, function as direct-acting mutagens and that the induced lesion is easily repairable by non-dividing cells. Exposure to N O resulted in a time- and dose-dependent increase in the number of revertants approximately proportional to the square of the N O concentration from 0 to 20 ppm. N O was a more effective mutagen relative to NO2, however, the observed requirement for 0 2 suggests limited oxidation of N O (presumably to NO 2) is necessary. Numerous lipid- and aqueous-phase inhibitors of nitrosation, as well as a number of other general antioxidants and free-radical trapping agents, were examined for their effectiveness in blocking the mutagenic effects of NO. The mutagenic activity of N O was most effectively inhibited by/3-carotene and tocopherols. BHT, dimethyl sulfoxide and mannitol also blocked the mutagenic effects of N O x but appeared less effective than /3-carotene or vitamin E, while ascorbate was ineffective as an inhibitor of mutation resulting from N O exposure.
H u m a n s are exposed to nitrogen oxides (NO x) daily from sources such as automobile exhaust, cigarette smoke, domestic gas cooking and heat-
Correspondence: Dr. R.V. Cooney, University of Hawaii, Cancer Research Center of Hawaii, 1236 Lauhala Street, Honolulu, HI 96813 (U.S.A.).
ing (Lewis, 1980). Some of the biological effects associated with environmental exposure to nitric oxide (NO) and nitrogen dioxide (NO 2) are well established, such as the morphological and physiological changes in the pulmonary epithelium resulting in lung tissue damage (Nakajima et al., 1980). Toxicological evidence suggests that exposure to nitrogen oxides may increase susceptibil-
194
ity to viral respiratory infection and alter the alveolar epithelial permeability and transport properties resulting in functional deficits (Mustafa et al., 1980; Rose et al., 1989). Although the general toxic effects of nitrogen oxides have been thoroughly studied, only a few reports are available concerning the genotoxicity of these compounds (Sasaki et al., 1980; Kosaka et al., 1985; Biggart and Rinehart, 1987; Victorin and Stalberg, 1988; Gorsdorf et al., 1990). Because of its greater reactivity and acute toxicity, attention has been focused o n N O 2 despite the fact that in vivo (Marietta, 1989) and in many complex gas mixtures, NO is the main oxide of nitrogen formed (Sasaki et al., 1980). The mutagenicities of complex gas mixtures, such as welding fumes, have been compared with that of NO 2 gas, which is known to exist in high concentrations, yet NO 2 accounts for only a fraction of the mutagenicity observed (Biggart and Rinehart, 1987; Sasaki et al., 1980). The mutagenic role of NO, which is a major product of combustion, was not considered in these reports. Previous investigations have shown NO to have little or no mutagenic potential (Kosaka et al., 1985; Gorsdorf et al., 1990). Recent evidence that NO and NO 2 are required in combination for aqueous amine nitrosation (Cooney et al., 1991) and the demonstration of deamination of D N A bases by NO (Nguyen et al., 1991; Wink et al., 1991) suggest that NO a n d / o r its oxidation products could either act as, or yield direct-acting mutagens in vivo. Endogenous production of NO by several cell types including endothelial cells (Moncada et al., 1988), neutrophils (Wright et al., 1989), and macrophages (Stuehr et al., 1989) has been demonstrated. The small molecular size, lipophilic nature, chemical instability and rapid reaction with oxygen and oxygen-derived radicals account for its pharmacokinetic properties as a trans-cellular messenger (Ignarro, 1991). The cytotoxic action of macrophage-derived NO on internalized organisms appears to require oxidation of NO to N O 2 (Iyengar et al., 1987; Leaf et al., 1991). Such a mixture of NO x could generate N20 3 which is capable of nitrosating amines at neutral pH (Challis et al., 1978; Kosaka et al., 1989) or react in a two step free radical reaction to produce an N-nitrosamine (Cooney et al., 1991). The forma-
tion of a primary N-nitrosamine on a DNA base would result in rapid deamination (Ridd, 1961) with the possibility of a point mutation occurring at this site in the absence of repair. To investigate the mutagenic activity of nitrogen oxides, a modified Ames test was used. The data presented in this paper show that exposure to NO, alone or in combination with NO 2, results in significantly enhanced mutation in Salmonella typhimurium TA1535. The use of antioxidants as effective inhibitors of the mutagenic activity of several carcinogens in the Salmonella assay has been examined previously (Rosin and Stich, 1979; Santamaria et al., 1984). In the present study the role of /3-carotene, tocopherols (a, y and ~), BHT, DMSO, ascorbate and mannitol in preventing genetic damage by NO alone, or in combination with NO 2 was assessed by measuring the frequency of reversion to histidine prototrophy in Salmonella typhimurium TA1535. Materials and methods
(1) Chemicals Mannitol, DMSO, BHT, fl-carotene, morpholine, a-, y- and 3-tocopherols and sodium ascorbate were purchased from Sigma Chemical Co., St. Louis, MO. Tetrahydrofuran (THF) containing 0.025% BHT as an antioxidant was purchased from Aldrich Chemical Co., St. Louis, MO. Optimal grade acetone and methylene chloride were from Fisher Scientific, Kent, Washington. NNitrosomorpholine and N-nitrosopyrrolidine standards were purchased from Chem Service Inc., Westchester, PA. Tanks of compressed gases containing NO and N O 2 in nitrogen were EPA grade from Scott Specialty gases, San Bernardino, CA. Certified purity for NO 2 was 55 ppm with contamination by NO of 3.5 ppm. The NO tank was certified to contain 91.00 ppm NO, with contamination from other oxides of nitrogen of 0.08 ppm (balance N2). Certified N 2 (99.999%) from Big Three Co., Honolulu, Hawaii was used to dilute the NO x gases. No nitric oxide was detectable in the N 2 source (limit of detection = 0.1 ppm). Gas flow rates were measured by individually calibrated flow meters. The gases were dry prior to exposure in the glove bag, however, there was some humid-
195 ity in the exposure chamber due to the agar plates. NO concentrations were measured using a Thermal Energy Analyzer (TEA) to verify the calculated gas concentration (Cooney et al., 1991).
(2) Exposure system NO, NO 2 or a combination of the two gases, was diluted with N 2 gas to the desired concentration by adjusting the flow rates of the individual gases with a rotameter (Scott Specialty Gases, San Bernardino, CA) to a total flow rate of 100 m l / m i n . The gas mixture was then allowed to fill the exposure chamber, consisting of an evacuated polyethylene glove bag (Inst. Res. Ind. Inc., Cheltenham, PA). Prior to changing the concentration of the gas mixture, the glove bag was fully emptied and then exposed for 30 min to the new nitrogen oxide concentration. The exposure chamber was cleaned and disinfected with ethanol once a week and the glove bag replaced after one month of use. In a typical experiment, culture dishes containing the bacteria were placed in the glove bag, lids were removed and the contents exposed to the desired concentration of NO, NO 2 or a combination of the two gases for 30 min. The bacterial plates were then recovered and transferred to an incubator and maintained as described below.
(3) Mutagenicity assay Salmonella strain TA1535 was used in these experiments. Cultures were maintained and grown as described by Ames and co-workers (Ames et al., [975). As in the standard plate-incorporation assay, 0.1 ml of an overnight (16 h) bacterial culture (approximately 2 × 108 bacteria) was mixed with 2.0 ml soft agar containing traces of histidine and biotin. The mixture was poured onto minimal glucose agar plates (30 ml agar per 100 mm petri dish). The Ames Salmonella assay as modified by Barber et al. (1983) was used to maximize the detection of short-lived, directacting mutagens. The plated bacteria were incubated at 37°C for 8 h prior to exposure to NO,. The plates were then placed in a polyethylene glove bag, lids were removed and the bacteria were exposed to the desired concentration of NO, NO 2 or a combination of the two gases in N 2. Control plates were exposed to a N 2 atmo-
sphere. At the end of the exposure period, usually 30 min, dishes were returned to the incubator and maintained at 37°C for 48 h, prior to colony counting using an automated colony counter, Biotran II (New Brunswick Scientific Co. Inc., Edison, N J). Positive controls utilizing sodium azide (2.5 ~ g / d i s h ) were run simultaneously yielding an average of 541 _+ 85 revertant colonies (n = 48) per dish for all the experiments described herein. Negative controls were included in every experiment for measurement of spontaneous revertants (mean = 12 _+ 4 revertants/dish, n = 48) and the number of revertant colonies was corrected by subtracting this background spontaneous mutation. The ability of antioxidants to inhibit mutation was assessed by adding 0.1 ml of various concentrations of the antioxidant to 2 ml of the top agar along with 0.1 ml of the bacterial culture. /3Carotene dissolved in T H F (final T H F concentration = 0.5%) was added to bacterial cultures prior to the 16-h incubation. 100 ILl of the bacterial suspensions were then added to 2.0 ml of the molten agar at 45°C as described above. All control cultures received the same amount of solvent as the treated samples. /3-Carotene was solubilized with T H F (Bertram et al., 1991), tocopherols and BHT with acetone (0.1% final solvent concentration), and DMSO and mannitol with water. T H F and acetone had no effect on the number of spontaneous revertants or any effect on mutagenicity by NO x at the concentrations used.
(4) Nitrosamine analysis The in vitro reactivity of amines with N O / N O 2 was assessed using a defined exposure system described by Cooney et al. (1987) in which a solution of morpholine (10 mM) at pH 7.4 was bubbled with 10 ppm NO and 5 ppm NO 2 in N 2 at a flow rate of 60 m l / m i n for 10 rain. NNitrosomorpholine was then extracted and analyzed by GC-TEA. The organic phase reaction was carried out in the manner described by Cooney et al. (1991) using 50 /~M morpholine in methylene chloride as the reaction solvent. The reaction was stopped by removing the tube from the gas stream and adding 100 p.1 of methanol containing 200 ng of N-nitrosopyrrolidine as an
196
internal standard. Aliquots were then injected for analysis on the GC-TEA.
LU F--
Although preliminary experiments indicated that N O was mutagenic in the standard A m e s assay, we sought to maximize the number of observed revertants by following the modifications described by Barber et al. (1983). Exponential growth normally occurs after approximately 4 - 8 h of incubation in agar at 37°C. It is during this period of log-phase growth that the bacteria are most susceptible to direct-acting, short-lived mutagens. The effect of incubation time on reversion frequency for strain TA1535 exposed to N O x for 30 min is shown in Fig. 1. The maximum mutation rate for 10 ppm N O alone or 8 ppm N O in combination with 5 ppm NO 2 is observed after 8 h growth of the test bacteria on the plate at 37°C. Subsequent experiments, therefore, followed this experimental protocol using an 8-h incubation prior to exposure to NO~. The mutagenic effects of N O and N O 2 were both time-dependent (Fig. 2), and dose-depen-
250
EL
200
Co I-Z i,i Cd :=~
LU LJ O~
•
100 5O 0
A,~, I 0
I
I
P
I
20
40
60
80
I
b
-
100 120 140
E X P O S U R E TIME ( r n i n )
Fig. 2. Dependence on incubation time for reversion of strain TA1535 by NO. Plated bacteria were incubated for 8 h at 37°C in culture dishes as described in Fig. 1 and Methods, then exposed to 10 ppm nitric oxide for the indicated periods of time. After subtraction of spontaneous revertants, each value represents the mean number of revertant colonies consisting of triplicate culture dishes + the standard deviation.
dent (Fig. 3). Fig. 3 shows dose-dependent mutagenicity after exposure of Salmonella typhimurium TA1535 to various concentrations of N O and N O 2 for 30 min. The numbers of revertants in the presence of NO 2 increased as a function of the NO 2 concentration up to 10 ppm, while in
200
700
EL co Fz
250
5
Results
LLI
