0099-2240/78/0035-0006$02.00/0 APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Jan. 1978, p. 6-10 Copyright © 1978 American Society for Microbiology
Vol. 35, No. 1 Printed in U.S.A.
Breaks Induced in the Deoxyribonucleic Acid of Aerosolized Escherichia coli by Ozonized Cyclohexene G. DE MIK* AND IDA DE GROOT Medical Biological Laboratory TNO, Rijswijk 2100, The Netherlands Received for publication 2 May 1977
The inactivation of aerosolized Escherichia coli by ozone, cyclohexene, and ozonized cyclohexene was studied. The parameters for damage were loss of reproduction and introduction of breaks in the deoxyribonucleic acid (DNA). Aerosolization of E. coli in clean air at 80% relative humidity or in air containing either ozone or cyclohexene hardly affected survival; however, some breaks per DNA molecule were induced, as shown by sucrose gradient sedimentation of the DNA. Aerosolization of E. coli in air containing ozonized cyclohexene at 80% relative humidity decreased the survival by a factor of 103 or more after 1 h of exposure and induced many breaks in the DNA.
The general occurrence of a germicidal agent in the air has been demonstrated in Britain (9) and in The Netherlands (6). The properties of this so-called open air factor (OAF) are similar to those of the biologically active products of ozone-olefin reactions. The activity of OAF seems to be more intense in urban air than in rural air, and the occurrence depends on the ozone concentration. Ozone is a natural constituent of clean air at high altitudes and is generated in the stratosphere by the reaction of oxygen atoms, formed by photolysis, with molecular oxygen. Ozone is brought down by turbulence and mixes with air in the lower atmosphere, giving maximum concentrations of up to 100 ,ug/m3 (50 ppb [ppb is used throughout to mean 1 part in 109]) at ground level. However, in different parts of the world, such as Los Angeles, The Netherlands, and England, (1, 3, 11, 16), concentrations in excess of 50 ppb are regularly recorded. This ozone is formed in polluted atmospheres by the photolysis of NO2 in the presence of hydrocarbons. The main sources of reactive hydrocarbons are traffic and industrial complexes, such as oil refineries and chemical plants. One of the reactions involved in the formation of photochemical smog is the ozone-olefin reaction. The products of this reaction include highly reactive compounds that have a strong toxic effect on plants (2, 5) and bacteria (4). In previous papers (7, 8) the inactivation mechanism of bacteriophage 4,X174 by ozonized cyclohexene was described. It was shown that the reactive product(s) reacts with the deoxyribonucleic acid (DNA) of the phage, inducing many breaks. Since Escherichia coli is also very sensitive to ozonized cyclohex-
ene and OAF, the question arose as to whether these products also induce lesions in the DNA of E. coli. The experiments presented in this paper may contribute to an understanding of the biological activity of ozonized olefins on bacteria. MATERIALS AND METHODS Bacterial strain. The strain of E. coli used was MRE 162, obtained from the Microbiological Research Establishment, Porton, England. Labeling procedure. Bacterial cells were labeled during an overnight incubation in tryptone medium supplemented with 10 iLCi of [methyl-3H]thymidine (specific activity, 17 Ci/mmol; The Radiochemical Centre, Amersham, England) per ml. The cells were washed by filtration on HA 045 membrane filters (Millipore Corp., Bedford, Mass.), suspended in tryptone medium, and incubated for 1 h at 37°C. Subsequently the cells were filtered again, resuspended in spray medium, and kept in ice until spraying. The final concentration of the cells was about 8 x 109 per ml. Aero8ol equipment and exposure to different atmospheres. The aerosol chamber was, in essence, a coliapsible bag made of 0.1-mm polyethylene film with a volume of approximately 1,500 liters. The bag was blown up with air, which was filtered with activated charcoal, dried with silica gel, and humidified to the desired relative humidity (RH). When the bag was filled with air, E. coli was aerosolized with a three-jet Collison nebulizer for 3 min, giving a concentration in the bag of approximately 3 x 10' cells per m3.
Ozone was generated by passing a part of the conditioned air over an ultraviolet lamp when filling the bag. The concentration of ozone in the bag, measured with an ozone meter (IG-TNO, model G 373, Delft, The Netherlands) directly before spraying, was 40 ppb. Cyclohexene (BDH Chemicals, England) was evaporated simultaneously with the E. coli by adding 10 6
DNA DAMAGE BY OZONIZED CYCLOHEXENE
VOI,. 35, 1978
,il to the spray suspension. The concentration of cyclohexene in the bag was estimated to be about 1,000 ppb. The aerosol was sampled with the lower stage of May's multistage liquid impinger (12) containing 10 ml of peptone water. The sampling time was 1 or 5 min, at a rate of 55 liters per min. Survival measurements. Surviving E. coli cells were counted by making dilutions in peptone water and plating 0.1-ml samples on tryptone agar in quadruplicate. The plates were incubated for 18 h at 37°C before counting. DNA extraction and centrifugation. Various methods of DNA extraction are available that were developed with the aim of minimizing the breakage of DNA by shearing and by nuclease action. The following procedure was employed in our experiments (13). E. coli was suspended in 1 ml of icecold 0.2 N NaOH containing 20 mM ethylenediaminetetraacetate and 0.5% sodium dodecyl sarcosinate (Sarkosyl NL-97, Geigy, Switzerland). The suspension was incubated at 370C for 20 min and after cooling was layered on top of a sucrose gradient. The induction of single-stranded breaks was studied by determining the sedimentation properties of the [3H]DNA in linear alkaline sucrose gradients (5 to 23% sucrose, adjusted to pH 12.2 with concentrated NaOH) containing 1 M NaCl and 0.02 M sodium citrate. Centrifugation was carried out in a Spinco L3 ultracentrifuge for 3 h at 27,000 rpm and 50C, using an SW27 rotor. After centrifugation, fractions were collected and the radioactivity present in each fraction was determined. Radioactivity assay. The radioactive fractions were counted by using 10 ml of tritosol per fraction (10). To obtain clear solutions, the fractions were neutralized with HCl before adding the scintillation cocktail. Tritosol had the following composition: 3.0 g of 2,5-diphenyloxazole, 257 ml of Triton X-100, 37 ml of ethylene glycol, 106 ml of ethanol, and finally xylene to make 1,000 ml. All counting was performed with a Nuclear-Chicago Mark II scintilation counter. Media. Tryptone medium contained 20 g of tryptone (Oxoid Ltd., London, England), 3 g of NaCl, 5 ml of 1 M K2HPO4, 1 ml of 1 M ferric citrate, 4 ml of 1 M MgSO4, and 1 ml of 1 M CaCl2 per liter. The pH was adjusted to 7.2 with H2SO4. Peptone water contained 1% peptone (Oxoid Ltd.) in tap water, pH 6.5. Tryptone agar contained 1% tryptone (Difco Laboratories, Detroit, Mich.), 0.5% NaCl, and 1.5% agar (Difco Laboratories). After sterilization, glucose was added to a concentration of 1%. The spraying medium consisted of equal parts of spent E. coli culture fluid (tryptone) and distilled water.
RESULTS
Surival in clean and contaminated atmospheres. The survival of E. coli MRE 162 aerosolized in clean air or in air containing ozone, cyclohexene, or both ozone and cyclohexene at 80% RH is shown in Fig. 1. The airborne cells were sampled at the indicated times for 1 min.
The data show that E. coli MRE 162 is stable
7
100 50
"
10
5
05
O
20
40 60 AEROSOL AGE (min.)
FIG. 1. Survival of E. coli MRE 162 aerosolized at 80% RH in various atmospheres and sampled for 1 min in 1% peptone water. Symbols: 0, clean air; A, air containing about 1,0O[Xppb of cyclohexene; A, air containing 40 ppb of ozone; 0, air containing both ozone and cyclohexene. Vertical bars represent standard error of the mean.
at 80% RH. Cyclohexene had no effect on the survival, whereas ozone slightly decreased the survival. The effect of ozonized cyclohexene was manifest, the survival decreasing by a factor of 103 in 1 h. Sedimentation of DNA from cells in the spray suspension. In the collison spray, the prmary aerosol is blown against the glass wall, and the remaining suspension recirculates. A small decrease of viable count in the suspension is often observed during aerosolization. To decide whether in the spray damage of the bacterial DNA also occurs, the suspension was analyzed before and after spraying. Typical sedimentation profiles of the isolated denatured DNA are shown in Fig. 2. The bulk of the DNA sedimented in the same way before and after aerosolization. However, after aerosolization, sometimes a peak of broken DNA, representing about 12% of the total radioactivity, was seen at the top of the gradient. This phenomenon was also sometimes observed when the bacteria were exposed to an atmosphere containing ozone, cyclohexene, or both. Probably some cells are damaged or killed
8
Apm,. ENVIIION. MICROBIOL.
DE MIK AND DE GROOT
c
0.2
0.4
FIG. 2. Alkaline sucrosegradient sedimentation of DNA from E. coli MRE 162 before and after aerosolization in the spray suspension. The radioactivity in disintegrations per min (DPM) is given for each fraction as the percentage of the total activity present in the gradient. ( ) DNA from cells in the spray suspension before aerosolization; (-----) DNA from cells from the suspension after aerosolization.
0.6
0.8
bottom
top
FIG. 3. Alkaline sucrose gradient sedimentation of DNA from E. coli MRE 162 sprayed in clean air at 80% RH. ( ) DNA from untreated cells; (-----) DNA from cells collected after 10 to 15 min of expo) DNA from cells colsure-survival, 47%; ( lected after 40 to 45 min of exposure-survival, 25%. DPM, Disintegrations per minute. .
10
due to the aerosolization procedure, after which the DNA of these damaged cells is broken down mechanically or enzymatically. Sedimentation of DNA from E. coli exposed to clean air. The sedimentation profile of DNA from E. coli MRE 162 exposed to clean air at 80% RH is shown in Fig. 3. The cells were sprayed in clean air and collected after an exposure time of 10 to 15 min and after 45 to 50 min. The percent survival measured in the samples was 47 and 25, respectively. As shown in Fig. 3, the DNA from the exposed cells sedimented slower than the DNA from the untreated control cells, indicating that exposure to clean air results in the production of some strand breakage. Sedimentation of DNA from E. coli exposed to cyclohexene. Figure 4 shows the sedimentation profile of DNA from E. coli MRE 162 exposed to air containing about 1,000 ppb of cyclohexene at 80% RH. The cells were collected after an exposure time of 80 min. The percent survival was 43. This sedimentation pattern did not differ from that after exposure of the cells to clean air. A comparison of these profiles suggests that the sedimentation velocity is related to the percent survival. Sedimentation of DNA from E. coli exposed to ozone. The sedimentation of DNA from E. coli MRE 162 exposed to air containing 40 ppb of ozone is shown in Fig. 5. The aerosol was sampled from 30 to 35 min and from 40 to 45 min (the samples were obtained from differ-
8
top
bottom
FIG. 4. Alkaline sucrosegradient sedimentation of DNA from E. coli MRE 162 sprayed in air containing about 1,(K00 ppb of cyclohexene at 80% RH. ( ) DNA from untreated cells; (-----) DNA from cells sampled after 75 to 80 min of exposure. The survival in the sample was 43%. DPM, Disinlegrations per minute.
ent experiments). The survival percentages were 74 and 37, respectively. As shown in Fig. 1, ozone seems to affect the survival slightly. The results
of sedimentation analysis allowed no conclusion as to whether ozone induced additional breaks since aerosolization in clean air or in air containing cyclohexene gave comparable results. Sedimentation of DNA from E. coli exposed to ozonized cyclohexene. The sedimentation of DNA from E. coli MRE 162 exposed to air containing both ozone and cyclo-
VOL. 35, 1978
DNA DAMAGE BY OZONIZED CYCLOHEXENE
9
10
0
8
61
0
top
02
04
0.6
08
1
bottom
FIG. 5. Alkaline sucrose gradient sedimentation of DNA from E. coli MRE 162 sprayed in air contain) DNA from ing 40 ppb of ozone at 80o RH.( untreated cells; (-) DNA from cells collected at 30 to 35 min. The survival measured in this sample was 74%. (... ) DNA from cells samnpled at 40 to 45 min during another experiment. The survival was 37% in this case. DPM, Disintegrations per minute.
FIG. 6. Alkaline sucrosegradient sedimentation of DNA from E. coli MRE 162 sprayed in air containing both ozone (40 ppb) and cyclohexene (1,000 ppb) at 80% RH. ( ) DNA from untreated cells; (-----) DNA from cells with a survival of 4% and sampled from 30 to 35 min; (. ) DNA from cells with a survival of 0.02%, sampled at 75 to 80 min. DPM, Disintegrations per minute.
hexene at 80% RH is shown in Fig. 6. The concentrations used were the same as before. The aerosol was sampled from 30 to 35 min tration of 1,000 ppb does not affect the survival (survival, 4%) and from 75 to 80 min (survival, of E. coli MRE 162 and that an effect of ozone 0.02%). These results show an important dea concentration of 40 ppb is hardly noticeable. crease in single-stranded DNA molecular at On the other hand, ozonized cyclohexene inacthe slower weight; the higher the inactivation, tivates E. coli MRE 162 by a factor of about the sedimentation of the DNA. 300 in 1 h (Fig. 1). Since the ozone reacts rapidly with the cyclohexene that is in large excess (2), DISCUSSION the inactivation must be ascribed to the reaction The present study was undertaken to investi- products of ozone and cyclohexene and not to gate the inactivation mechanism of E. coli MRE the effect of ozone alone. The inactivation data presented are in good 162 by ozonized cyclohexene, which is supposed to be a model of the OAF. The only way to agreement with those published by Dark and observe the germicidal effect of ozonized olefins Nash (4), who used the microthread technique is to expose the organism in the aerosol state. to expose E. coli MRE 162 to various ozonized However, this implies that unknown factors are olefins. Alkaline sucrose gradient sedimentation has introduced since aerosolization, aerosol storage, and collection, to mention only a few factors, been used successfully to detect the production may inactivate the organism. In this study the and repair of single-stranded breaks in DNA experimental circumstances were chosen in such after irradiation or chemical treatment of DNA a way as to minimize the damage to the control or cells. However, it is known that alkali not cells, whereas the effect of ozonized cyclohexene only exposes preexisting single-stranded breaks was large enough to study the inactivation mech- by denaturing the DNA, but also produces anism. As was shown in a previous paper (6), breaks in the DNA molecule when alkali-labile E. coli MRE 162 is stable at high RH values lesions have been introduced. Our gradient studies show that although the when sprayed from 50% spent medium. For this study, the E. coli was sprayed at 80% RH, an survival in clean air is rather high, the DNA is RH at which the survival in clean air is high damaged since it sediments distinctly slower and the effect of ozonized cyclohexene reaches than the control DNA. DNA damage has been suggested as an important cause of death of a maximum as shown by Dark and Nash (4). The data show that cyclohexene in a concen- gram-negative bacteria in aerosols (14, 15). The
10
Ap)I.. ENVIIION. MICROBIOI.
DE MIK AND DE GROOT
lesions in the DNA observed here support this suggestion. Under the conditions used, cyclohexene had no effect on E. coli MRE 162. Ozone in a concentration of 40 ppb did not induce more breaks in DNA than those observed in the control cells in clean air. A complicating factor in the present experiments was that under the experimental conditions breaks were introduced in DNA of cells exposed to clean air. To study additional effects of ozone on E. coli MRE 162, higher concentrations of ozone would be necessary. Ozonized cyclohexene induced many breaks in E. coli DNA (Fig. 6); the higher the inactivation, the more breaks detected. It was demonstrated in an earlier paper (6) that ozonized cyclohexene causes breaks in aerosolized free DNA. The same phenomenon was observed when bacteriophage 4X174 was exposed to ozonized cyclohexene. The phage protein gave only scant protection. The DNA of E. coli is protected much better against exogenous reactants than the DNA of OX174. It cannot be excluded, therefore, that the breaks in E. coli DNA are the results of a secondary effect. It is assumed that the reactive product in the reaction between cyclohexene and ozone is the peroxide dipolar ion originating from a splitting of the double bond of cyclohexene (4): H
C=/O
C
1
+
03
--+
C+
O-O-
This product or one of its secondary reaction products reacts with the DNA, giving breaks or alkali-labile lesions that are detected as breaks in the sucrose gradients. The fact that these products affect E. coli before being inactivated by the walls of the plastic bag shows their high reactivity.
ACKNOWLEDGMENTS We thank K. C. Winkler and H. C. Bartlema for their stimulating discussions.
LITERATURE CITED 1. Anlauf, K. G., M. A. Lusis, H. A. Wiebe, and R. D. S. Stevens. 1975. High ozone concentrations measured in the vicinity of Toronto, Canada. Atmos. Environ. 9:1137-1139. 2. Arnolds, W. N. 1959. The longevity of the phytotoxicant produced from gaseous ozone-olefin reactions. Int. J. Air Pollut. 2:167-174. 3. Atkins, D. H. F., R. A. Cox, and A. E. J. Eggleton. 1972. Photochemical ozone and sulphuric acid aerosol formation in the atmosphere over Southern England. Nature (London) 235:372-376. 4. Dark, F. A., and T. Nash. 1970. Comparative toxicity of various ozonized olefins to bacteria suspended in air. J. Hyg. 68:245-252. 5. Darley, E. F., E. R. Stephens, J. T. Middleton, and P. L. Hanst. 1959. Oxidant plant damage from ozoneolefin reactions. Int. J. Air Pollut. 1:155-162. 6. de Mik, G., and Y. de Groot. 1977. The germicidal effect of the open air in different parts of The Netherlands. J. Hyg. 78:175-187. 7. de Mik, G., and Y. de Groot. 1977. Mechanism of inactivation of the bacteriophage 4X174 and its DNA in aerosols by ozone and ozonized cyclohexene. J. Hyg. 78:199-211. 8. de Mik, G., Y. de Groot, and J. L. F. Gerbrandy. 1977. Survival of aerosolized bacteriophage 4X174 in air containing ozone-olefin mixtures. J. Hyg. 78:189-198. 9. Druett, H. A., and K. R. May. 1968. Unstable germicidal pollutant in rural air. Nature (London) 220:395-396. 10. Fricke, U. 1975. Tritosol: a new scintillation cocktail based on TRITON X-100. Anal. Biochem. 63:555-558. 11. Guicherit, R., R. Jeltes, and F. Lindqvist. 1972. Determination of the ozone concentration in outdoor air near Delft, The Netherlands. Environ. Pollut. 3:91-110. 12. May, K. R. 1966. Multistage liquid impinger. Bacteriol. Rev. 30:559-570. 13. Okazaki, R. 1971. Demonstration of replicated short DNA chains. Methods Enzymol. 21D:298-299. 14. Webb, S. J. 1967. Mutation of bacterial cells by controlled desiccation. Nature (London) 213:1137-1139. 15. Webb, S. J., and J. L. Walker. 1968. The effects of mutation and nucleic acid base analogues on the sensitivity of E. coli to partial dehydration. Can. J. Microbiol. 14:557-563. 16. Wisse, J. A., and C. A. Velds. 1970. Preliminary discussion on some oxidant measurements at Vlaardingen, The Netherlands. Atmos. Environ. 4:79-85.
Vol. 35, No. 1 Printed in U.S.A.
Breaks Induced in the Deoxyribonucleic Acid of Aerosolized Escherichia coli by Ozonized Cyclohexene G. DE MIK* AND IDA DE GROOT Medical Biological Laboratory TNO, Rijswijk 2100, The Netherlands Received for publication 2 May 1977
The inactivation of aerosolized Escherichia coli by ozone, cyclohexene, and ozonized cyclohexene was studied. The parameters for damage were loss of reproduction and introduction of breaks in the deoxyribonucleic acid (DNA). Aerosolization of E. coli in clean air at 80% relative humidity or in air containing either ozone or cyclohexene hardly affected survival; however, some breaks per DNA molecule were induced, as shown by sucrose gradient sedimentation of the DNA. Aerosolization of E. coli in air containing ozonized cyclohexene at 80% relative humidity decreased the survival by a factor of 103 or more after 1 h of exposure and induced many breaks in the DNA.
The general occurrence of a germicidal agent in the air has been demonstrated in Britain (9) and in The Netherlands (6). The properties of this so-called open air factor (OAF) are similar to those of the biologically active products of ozone-olefin reactions. The activity of OAF seems to be more intense in urban air than in rural air, and the occurrence depends on the ozone concentration. Ozone is a natural constituent of clean air at high altitudes and is generated in the stratosphere by the reaction of oxygen atoms, formed by photolysis, with molecular oxygen. Ozone is brought down by turbulence and mixes with air in the lower atmosphere, giving maximum concentrations of up to 100 ,ug/m3 (50 ppb [ppb is used throughout to mean 1 part in 109]) at ground level. However, in different parts of the world, such as Los Angeles, The Netherlands, and England, (1, 3, 11, 16), concentrations in excess of 50 ppb are regularly recorded. This ozone is formed in polluted atmospheres by the photolysis of NO2 in the presence of hydrocarbons. The main sources of reactive hydrocarbons are traffic and industrial complexes, such as oil refineries and chemical plants. One of the reactions involved in the formation of photochemical smog is the ozone-olefin reaction. The products of this reaction include highly reactive compounds that have a strong toxic effect on plants (2, 5) and bacteria (4). In previous papers (7, 8) the inactivation mechanism of bacteriophage 4,X174 by ozonized cyclohexene was described. It was shown that the reactive product(s) reacts with the deoxyribonucleic acid (DNA) of the phage, inducing many breaks. Since Escherichia coli is also very sensitive to ozonized cyclohex-
ene and OAF, the question arose as to whether these products also induce lesions in the DNA of E. coli. The experiments presented in this paper may contribute to an understanding of the biological activity of ozonized olefins on bacteria. MATERIALS AND METHODS Bacterial strain. The strain of E. coli used was MRE 162, obtained from the Microbiological Research Establishment, Porton, England. Labeling procedure. Bacterial cells were labeled during an overnight incubation in tryptone medium supplemented with 10 iLCi of [methyl-3H]thymidine (specific activity, 17 Ci/mmol; The Radiochemical Centre, Amersham, England) per ml. The cells were washed by filtration on HA 045 membrane filters (Millipore Corp., Bedford, Mass.), suspended in tryptone medium, and incubated for 1 h at 37°C. Subsequently the cells were filtered again, resuspended in spray medium, and kept in ice until spraying. The final concentration of the cells was about 8 x 109 per ml. Aero8ol equipment and exposure to different atmospheres. The aerosol chamber was, in essence, a coliapsible bag made of 0.1-mm polyethylene film with a volume of approximately 1,500 liters. The bag was blown up with air, which was filtered with activated charcoal, dried with silica gel, and humidified to the desired relative humidity (RH). When the bag was filled with air, E. coli was aerosolized with a three-jet Collison nebulizer for 3 min, giving a concentration in the bag of approximately 3 x 10' cells per m3.
Ozone was generated by passing a part of the conditioned air over an ultraviolet lamp when filling the bag. The concentration of ozone in the bag, measured with an ozone meter (IG-TNO, model G 373, Delft, The Netherlands) directly before spraying, was 40 ppb. Cyclohexene (BDH Chemicals, England) was evaporated simultaneously with the E. coli by adding 10 6
DNA DAMAGE BY OZONIZED CYCLOHEXENE
VOI,. 35, 1978
,il to the spray suspension. The concentration of cyclohexene in the bag was estimated to be about 1,000 ppb. The aerosol was sampled with the lower stage of May's multistage liquid impinger (12) containing 10 ml of peptone water. The sampling time was 1 or 5 min, at a rate of 55 liters per min. Survival measurements. Surviving E. coli cells were counted by making dilutions in peptone water and plating 0.1-ml samples on tryptone agar in quadruplicate. The plates were incubated for 18 h at 37°C before counting. DNA extraction and centrifugation. Various methods of DNA extraction are available that were developed with the aim of minimizing the breakage of DNA by shearing and by nuclease action. The following procedure was employed in our experiments (13). E. coli was suspended in 1 ml of icecold 0.2 N NaOH containing 20 mM ethylenediaminetetraacetate and 0.5% sodium dodecyl sarcosinate (Sarkosyl NL-97, Geigy, Switzerland). The suspension was incubated at 370C for 20 min and after cooling was layered on top of a sucrose gradient. The induction of single-stranded breaks was studied by determining the sedimentation properties of the [3H]DNA in linear alkaline sucrose gradients (5 to 23% sucrose, adjusted to pH 12.2 with concentrated NaOH) containing 1 M NaCl and 0.02 M sodium citrate. Centrifugation was carried out in a Spinco L3 ultracentrifuge for 3 h at 27,000 rpm and 50C, using an SW27 rotor. After centrifugation, fractions were collected and the radioactivity present in each fraction was determined. Radioactivity assay. The radioactive fractions were counted by using 10 ml of tritosol per fraction (10). To obtain clear solutions, the fractions were neutralized with HCl before adding the scintillation cocktail. Tritosol had the following composition: 3.0 g of 2,5-diphenyloxazole, 257 ml of Triton X-100, 37 ml of ethylene glycol, 106 ml of ethanol, and finally xylene to make 1,000 ml. All counting was performed with a Nuclear-Chicago Mark II scintilation counter. Media. Tryptone medium contained 20 g of tryptone (Oxoid Ltd., London, England), 3 g of NaCl, 5 ml of 1 M K2HPO4, 1 ml of 1 M ferric citrate, 4 ml of 1 M MgSO4, and 1 ml of 1 M CaCl2 per liter. The pH was adjusted to 7.2 with H2SO4. Peptone water contained 1% peptone (Oxoid Ltd.) in tap water, pH 6.5. Tryptone agar contained 1% tryptone (Difco Laboratories, Detroit, Mich.), 0.5% NaCl, and 1.5% agar (Difco Laboratories). After sterilization, glucose was added to a concentration of 1%. The spraying medium consisted of equal parts of spent E. coli culture fluid (tryptone) and distilled water.
RESULTS
Surival in clean and contaminated atmospheres. The survival of E. coli MRE 162 aerosolized in clean air or in air containing ozone, cyclohexene, or both ozone and cyclohexene at 80% RH is shown in Fig. 1. The airborne cells were sampled at the indicated times for 1 min.
The data show that E. coli MRE 162 is stable
7
100 50
"
10
5
05
O
20
40 60 AEROSOL AGE (min.)
FIG. 1. Survival of E. coli MRE 162 aerosolized at 80% RH in various atmospheres and sampled for 1 min in 1% peptone water. Symbols: 0, clean air; A, air containing about 1,0O[Xppb of cyclohexene; A, air containing 40 ppb of ozone; 0, air containing both ozone and cyclohexene. Vertical bars represent standard error of the mean.
at 80% RH. Cyclohexene had no effect on the survival, whereas ozone slightly decreased the survival. The effect of ozonized cyclohexene was manifest, the survival decreasing by a factor of 103 in 1 h. Sedimentation of DNA from cells in the spray suspension. In the collison spray, the prmary aerosol is blown against the glass wall, and the remaining suspension recirculates. A small decrease of viable count in the suspension is often observed during aerosolization. To decide whether in the spray damage of the bacterial DNA also occurs, the suspension was analyzed before and after spraying. Typical sedimentation profiles of the isolated denatured DNA are shown in Fig. 2. The bulk of the DNA sedimented in the same way before and after aerosolization. However, after aerosolization, sometimes a peak of broken DNA, representing about 12% of the total radioactivity, was seen at the top of the gradient. This phenomenon was also sometimes observed when the bacteria were exposed to an atmosphere containing ozone, cyclohexene, or both. Probably some cells are damaged or killed
8
Apm,. ENVIIION. MICROBIOL.
DE MIK AND DE GROOT
c
0.2
0.4
FIG. 2. Alkaline sucrosegradient sedimentation of DNA from E. coli MRE 162 before and after aerosolization in the spray suspension. The radioactivity in disintegrations per min (DPM) is given for each fraction as the percentage of the total activity present in the gradient. ( ) DNA from cells in the spray suspension before aerosolization; (-----) DNA from cells from the suspension after aerosolization.
0.6
0.8
bottom
top
FIG. 3. Alkaline sucrose gradient sedimentation of DNA from E. coli MRE 162 sprayed in clean air at 80% RH. ( ) DNA from untreated cells; (-----) DNA from cells collected after 10 to 15 min of expo) DNA from cells colsure-survival, 47%; ( lected after 40 to 45 min of exposure-survival, 25%. DPM, Disintegrations per minute. .
10
due to the aerosolization procedure, after which the DNA of these damaged cells is broken down mechanically or enzymatically. Sedimentation of DNA from E. coli exposed to clean air. The sedimentation profile of DNA from E. coli MRE 162 exposed to clean air at 80% RH is shown in Fig. 3. The cells were sprayed in clean air and collected after an exposure time of 10 to 15 min and after 45 to 50 min. The percent survival measured in the samples was 47 and 25, respectively. As shown in Fig. 3, the DNA from the exposed cells sedimented slower than the DNA from the untreated control cells, indicating that exposure to clean air results in the production of some strand breakage. Sedimentation of DNA from E. coli exposed to cyclohexene. Figure 4 shows the sedimentation profile of DNA from E. coli MRE 162 exposed to air containing about 1,000 ppb of cyclohexene at 80% RH. The cells were collected after an exposure time of 80 min. The percent survival was 43. This sedimentation pattern did not differ from that after exposure of the cells to clean air. A comparison of these profiles suggests that the sedimentation velocity is related to the percent survival. Sedimentation of DNA from E. coli exposed to ozone. The sedimentation of DNA from E. coli MRE 162 exposed to air containing 40 ppb of ozone is shown in Fig. 5. The aerosol was sampled from 30 to 35 min and from 40 to 45 min (the samples were obtained from differ-
8
top
bottom
FIG. 4. Alkaline sucrosegradient sedimentation of DNA from E. coli MRE 162 sprayed in air containing about 1,(K00 ppb of cyclohexene at 80% RH. ( ) DNA from untreated cells; (-----) DNA from cells sampled after 75 to 80 min of exposure. The survival in the sample was 43%. DPM, Disinlegrations per minute.
ent experiments). The survival percentages were 74 and 37, respectively. As shown in Fig. 1, ozone seems to affect the survival slightly. The results
of sedimentation analysis allowed no conclusion as to whether ozone induced additional breaks since aerosolization in clean air or in air containing cyclohexene gave comparable results. Sedimentation of DNA from E. coli exposed to ozonized cyclohexene. The sedimentation of DNA from E. coli MRE 162 exposed to air containing both ozone and cyclo-
VOL. 35, 1978
DNA DAMAGE BY OZONIZED CYCLOHEXENE
9
10
0
8
61
0
top
02
04
0.6
08
1
bottom
FIG. 5. Alkaline sucrose gradient sedimentation of DNA from E. coli MRE 162 sprayed in air contain) DNA from ing 40 ppb of ozone at 80o RH.( untreated cells; (-) DNA from cells collected at 30 to 35 min. The survival measured in this sample was 74%. (... ) DNA from cells samnpled at 40 to 45 min during another experiment. The survival was 37% in this case. DPM, Disintegrations per minute.
FIG. 6. Alkaline sucrosegradient sedimentation of DNA from E. coli MRE 162 sprayed in air containing both ozone (40 ppb) and cyclohexene (1,000 ppb) at 80% RH. ( ) DNA from untreated cells; (-----) DNA from cells with a survival of 4% and sampled from 30 to 35 min; (. ) DNA from cells with a survival of 0.02%, sampled at 75 to 80 min. DPM, Disintegrations per minute.
hexene at 80% RH is shown in Fig. 6. The concentrations used were the same as before. The aerosol was sampled from 30 to 35 min tration of 1,000 ppb does not affect the survival (survival, 4%) and from 75 to 80 min (survival, of E. coli MRE 162 and that an effect of ozone 0.02%). These results show an important dea concentration of 40 ppb is hardly noticeable. crease in single-stranded DNA molecular at On the other hand, ozonized cyclohexene inacthe slower weight; the higher the inactivation, tivates E. coli MRE 162 by a factor of about the sedimentation of the DNA. 300 in 1 h (Fig. 1). Since the ozone reacts rapidly with the cyclohexene that is in large excess (2), DISCUSSION the inactivation must be ascribed to the reaction The present study was undertaken to investi- products of ozone and cyclohexene and not to gate the inactivation mechanism of E. coli MRE the effect of ozone alone. The inactivation data presented are in good 162 by ozonized cyclohexene, which is supposed to be a model of the OAF. The only way to agreement with those published by Dark and observe the germicidal effect of ozonized olefins Nash (4), who used the microthread technique is to expose the organism in the aerosol state. to expose E. coli MRE 162 to various ozonized However, this implies that unknown factors are olefins. Alkaline sucrose gradient sedimentation has introduced since aerosolization, aerosol storage, and collection, to mention only a few factors, been used successfully to detect the production may inactivate the organism. In this study the and repair of single-stranded breaks in DNA experimental circumstances were chosen in such after irradiation or chemical treatment of DNA a way as to minimize the damage to the control or cells. However, it is known that alkali not cells, whereas the effect of ozonized cyclohexene only exposes preexisting single-stranded breaks was large enough to study the inactivation mech- by denaturing the DNA, but also produces anism. As was shown in a previous paper (6), breaks in the DNA molecule when alkali-labile E. coli MRE 162 is stable at high RH values lesions have been introduced. Our gradient studies show that although the when sprayed from 50% spent medium. For this study, the E. coli was sprayed at 80% RH, an survival in clean air is rather high, the DNA is RH at which the survival in clean air is high damaged since it sediments distinctly slower and the effect of ozonized cyclohexene reaches than the control DNA. DNA damage has been suggested as an important cause of death of a maximum as shown by Dark and Nash (4). The data show that cyclohexene in a concen- gram-negative bacteria in aerosols (14, 15). The
10
Ap)I.. ENVIIION. MICROBIOI.
DE MIK AND DE GROOT
lesions in the DNA observed here support this suggestion. Under the conditions used, cyclohexene had no effect on E. coli MRE 162. Ozone in a concentration of 40 ppb did not induce more breaks in DNA than those observed in the control cells in clean air. A complicating factor in the present experiments was that under the experimental conditions breaks were introduced in DNA of cells exposed to clean air. To study additional effects of ozone on E. coli MRE 162, higher concentrations of ozone would be necessary. Ozonized cyclohexene induced many breaks in E. coli DNA (Fig. 6); the higher the inactivation, the more breaks detected. It was demonstrated in an earlier paper (6) that ozonized cyclohexene causes breaks in aerosolized free DNA. The same phenomenon was observed when bacteriophage 4X174 was exposed to ozonized cyclohexene. The phage protein gave only scant protection. The DNA of E. coli is protected much better against exogenous reactants than the DNA of OX174. It cannot be excluded, therefore, that the breaks in E. coli DNA are the results of a secondary effect. It is assumed that the reactive product in the reaction between cyclohexene and ozone is the peroxide dipolar ion originating from a splitting of the double bond of cyclohexene (4): H
C=/O
C
1
+
03
--+
C+
O-O-
This product or one of its secondary reaction products reacts with the DNA, giving breaks or alkali-labile lesions that are detected as breaks in the sucrose gradients. The fact that these products affect E. coli before being inactivated by the walls of the plastic bag shows their high reactivity.
ACKNOWLEDGMENTS We thank K. C. Winkler and H. C. Bartlema for their stimulating discussions.
LITERATURE CITED 1. Anlauf, K. G., M. A. Lusis, H. A. Wiebe, and R. D. S. Stevens. 1975. High ozone concentrations measured in the vicinity of Toronto, Canada. Atmos. Environ. 9:1137-1139. 2. Arnolds, W. N. 1959. The longevity of the phytotoxicant produced from gaseous ozone-olefin reactions. Int. J. Air Pollut. 2:167-174. 3. Atkins, D. H. F., R. A. Cox, and A. E. J. Eggleton. 1972. Photochemical ozone and sulphuric acid aerosol formation in the atmosphere over Southern England. Nature (London) 235:372-376. 4. Dark, F. A., and T. Nash. 1970. Comparative toxicity of various ozonized olefins to bacteria suspended in air. J. Hyg. 68:245-252. 5. Darley, E. F., E. R. Stephens, J. T. Middleton, and P. L. Hanst. 1959. Oxidant plant damage from ozoneolefin reactions. Int. J. Air Pollut. 1:155-162. 6. de Mik, G., and Y. de Groot. 1977. The germicidal effect of the open air in different parts of The Netherlands. J. Hyg. 78:175-187. 7. de Mik, G., and Y. de Groot. 1977. Mechanism of inactivation of the bacteriophage 4X174 and its DNA in aerosols by ozone and ozonized cyclohexene. J. Hyg. 78:199-211. 8. de Mik, G., Y. de Groot, and J. L. F. Gerbrandy. 1977. Survival of aerosolized bacteriophage 4X174 in air containing ozone-olefin mixtures. J. Hyg. 78:189-198. 9. Druett, H. A., and K. R. May. 1968. Unstable germicidal pollutant in rural air. Nature (London) 220:395-396. 10. Fricke, U. 1975. Tritosol: a new scintillation cocktail based on TRITON X-100. Anal. Biochem. 63:555-558. 11. Guicherit, R., R. Jeltes, and F. Lindqvist. 1972. Determination of the ozone concentration in outdoor air near Delft, The Netherlands. Environ. Pollut. 3:91-110. 12. May, K. R. 1966. Multistage liquid impinger. Bacteriol. Rev. 30:559-570. 13. Okazaki, R. 1971. Demonstration of replicated short DNA chains. Methods Enzymol. 21D:298-299. 14. Webb, S. J. 1967. Mutation of bacterial cells by controlled desiccation. Nature (London) 213:1137-1139. 15. Webb, S. J., and J. L. Walker. 1968. The effects of mutation and nucleic acid base analogues on the sensitivity of E. coli to partial dehydration. Can. J. Microbiol. 14:557-563. 16. Wisse, J. A., and C. A. Velds. 1970. Preliminary discussion on some oxidant measurements at Vlaardingen, The Netherlands. Atmos. Environ. 4:79-85.
