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Mechanisms of inactivation of bacteriophage phiX174 and its DNA in aerosols by ozone and ozonized cyclohexene.

The mechanisms of inactivation of aerosolized bacteriophage phiX174 in atmospheres containing ozone, cyclohexene, or ozonized cyclohexene were studied by using 32P-labelled phage. The inactivation of the aerosolized phage in clear air or in air containing cyclohexene is due to damage of the protein coat since the deoxyribonucleic acid (DNA) extracted from the inactivated phage retains its biological activity. Inactivation of the phage in air containing ozonized cyclohexene is due both to protein and DNA damage. Sucrose gradient analysis shows that aerosolized inactivated phiX174 releases unbroken DNA. In contrast, the DNA from phage phiX174 inactivated by ozonized cyclohexene is broken. The inactivation of aerosolized phage phiX174-DNA was studied in the same atmospheres using 32P-labelled DNA. phiX174-DNA aerosolized in clear air or air containing cyclohexene at 75% r.h. is inactivated by a factor of 2 in 30 min. The inactivated DNA is broken. Ozone as well as ozonized cyclohexene inactivates KNA very fast causing breaks in the molecule. This is in contrast with the intact bacteriophage in which ozone does not produce breaks in the DNA.

Aerosols

[Ozone-oxygen therapy for gynecologic carcinomas. The effect of parenteral-ozone oxygen mixture administration on free fatty acids and triglycerides in patients with gynecologic carcinomas].

As some authors suspect that ozone influences the metabolic process of fat, we tried to analyse the influence of an ozone-oxygen gas which was applied parenterally. 40 patients with gynecological cancer received 10 ml ozone-oxygen gas with a content of 450 gamma ozone and venous blood was removed before and 10 minutes after application. The serum was lyophilized and the level of fatty acids and triglycerids was determined by the method of Randerath (1965). a statistically significant decrease of the concentrations was observed after application of ozone. Different theories as to the cause of this action are discussed.

Clinical Trials as Topic

Reactions of ozone with fatty acid monolayers: a model system for disruption of lipid molecular assemblies by ozone.

Ozone concentrations of 0.03 ppm are shown to initiate physical disruption of both oleic and linoleic acid spread monolayers in a few seconds. Approximately equal yields of water soluble products in nitrogen- and oxygen-containing environments as well as in the two acid systems suggest that the dominant reaction pathway involves direct attack on fatty acid double bonds and not oxidative chain reactions. A possible explanation for the observed loss of monolayer material is provided by the Criegee mechanism for ozonide rearrangement following ozone attachment to double bonds. In this scheme polar intermediates are generated which could pass into the aqueous subphase before recombination can occur. alpha-Tocopherol exhibits a protective effect only when present at a high fraction in the fatty acid monolayer (6 mole%). These systems provide an efficient elementary model for studying the physical effects of ozone on lipid membrane structure.

Fatty Acids

[Animal experiment studies on the use of ozone in irradiated and non-irradiated tumors. I. Intravenous ozone therapy of Crocker's sarcoma 180 and Ehrlich carcinoma in the white mouse].

The fractionated tangential local X-ray therapy, in cases of sarcoma 180 in white mice with a total dose of 1500 R, effects a shrinking of the tumours as well as a certain number of cures. Intravenous administration of ozone in a total dose of 0.15 times 10(-7)g, 1.5 times 10(-7)g, and 15 times 10(-7)g - by itself or together with irradiation - has as consequence neither an inhibitory effect on the tumour growth, nor an increase in radiation effect that are surpassing the diffusion width of the model and are reproducible in an exact manner. It has been possible to prove an inhibition of the tumour growth in the solid Ehrlich's carcinoma of white mice after a fractionated 2000 R irradiation, but also in these cases, no effect of the ozone application was stated.

Animals

[Comparative studies on the action of chlorine and ozone on polioviruses in the reprocessing of drinking water in Essen (author's transl)].

The present study was performed in order to evaluate comparatively the inactivation of polio virus type I strains (wild type and attenuated) by means of chlorine and ozone. Polio virus type I was chosen with regard to its epidemiological behaviour and high stability in drinking-water and sewage lines. In view of the lack of propagation techniques, hepatitis viruses A and B, unfortunately, could not be used for these experiments. The experiments were done under laboratory conditions only, and not in the water recovery plant because of hygienic reasons. Defined quantities of disinfectants were examined for their virus-inactivating effect in water without redox-potential (double-distilled water), water with low defined redox-potential (double-distilled water + KOH), previously chlorinated water with a residual chlorine content of 0.03 mg chlorine per liter (tap water) and water with a high redox-potential (well water from the drinking-water plant). Time-course studies were performed, both with chlorine and ozone, in order to evaluate the characteristics of the inactivation procedure. The experimental conditions chosen varied from experiment to experiment to obtain relevant conclusions for the practice. On the basis of our results, and taking into account the quantitative differences in effect, chlorine and ozone principially can be considered equivalent in their action of virus-disinfection. Both, the initial rate and the kinetics of virus disinfection are really identical. Both disinfectants are dependant on the condition of the water (redox-potential, pH etc.) to a great extent in their efficacy. Therefore, a decision of whether or not ozone should substitute for chlorine for the drinking-water supply in Essen cannot be drawn on the basis of virological experiments. This decision, then, depends more or less on other questions - such as relative costs and practicability of the ozonization on a large technical scale. The safety risk and technical reliability of the ozonization process is of particular significance. In the present condition of the Essen reservoir water, a good virus disinfection can be expected already with 1.0 to 1.5 mg ozone/liter (dissolved!); such a concentration guarantees very little residual ozone and, thus, makes then this procedure technically feasible. Continuous checking of the redox-potential and the amount of the ozone added is necessary. With regard to a continuous supply of ozone, the dependence on current supply must be guaranteed. Ozonization of water, probably by the cleavage of humic acid, promotes bacterial recontamination of the drinking-water in the city taps(Stalder und Klosterkötter, 45). Therefore only a combined pre-ozonization with subsequent chlorination would guarantee, both, safety and improvement of the cosmetical conditions of the drinking-water. Such a combination would be feasible with highly reduced amounts of ozone and chlorine.

Chlorine

Asthma and respiratory irritants (ozone).

Asthmatics appear to be more susceptible to the effects of air pollutants than nonasthmatics. The present studies were undertaken to examine the effects of exposing asthmatics to ozone concentrations that occur in the environment. Seventeen well-documented male and female asthmatics have been exposed for 2 hr in an environmental chamber to 0.25 ppm of ozone on one occasion (ozone) and to air on another occasion (air). Effects were assessed by measurements of pulmonary function obtained prior to (0 hr), every half-hour during and at the end of all exposures (2 hr). Paired t-test analysis of lung volumes, forced expiratory volume in 1 sec (FEV1.0), and maximum expiratory flow rates at 50% of vital capacity (V50%VC) showed no significant changes (p greater than 0.05) when the following comparisons were made: 0 hr air vs 0 hr ozone, 0 hr air vs. 2 hr air, 0 hr ozone vs. 2 hr ozone, 2 hr air vs. 2 hr ozone. There was variability in severity of asthma and pulmonary function status; most subjects were taking some form of medication at the time of study. Some asthmatics showed no change or improvement with both air and ozone and others developed greater reductions in pulmonary function with ozone than with air. Approximately one-third of the asthmatics demonstrated greater changes in V50%VC with exposure to 0.25 ppm of ozone relative to air exposure. These studies indicate that acute exposures to ozone at realistic concentrations in the environment can produce adverse responses in some asthmatics.

Adult

Health effects of ozone exposure in asthmatics.

To investigate whether ambient air quality standards for ozone adequately protect high-risk populations, we assessed pulmonary and biochemical responses of 22 asthmatic volunteers to 2-hour controlled exposures to ozone at concentrations approximating 0.2 ppm, with secondary stresses of heat and intermittent exercise. All subjects had physician-diagnosed asthma; clinically, they covered a range from minimal wheezing to persistent marked abnormality in forced expiratory performance. Control experiments included repeated sham exposures (to purified air with no ozone added) as well as brief exposures to the odor of ozone followed by purified air. No meaningful changes in forced expiratory measures, lung volumes, or single-breath N2 indices were found after ozone exposure relative to control. Symptoms, scored semiquantitatively, increased slightly but not significantly with exposure to ozone. Small but significant (P is less than 0.05) group mean blood biochemical changes occurred with exposure to ozone; these included increased glucose-6-phosphate dehydrogenase and lactate dehydrogenase activities, increased erythrocyte fragility, and decreased concentration of reduced glutathione. Hemoglobin concentration and acetylcholinesterase activity decreased with ozone and decreased to a lesser extent in control studies. Concentrations of ozone readily attainable in smog episodes thus appear to be capable of affecting blood biochemistry in at least some asthmatic persons, in the absence of obvious adverse pulmonary responses. Whether the biochemical effects represent harm to health or a normal response to stress remains to be determined.

Acetylcholinesterase

Ozonation of mutagenic and carcinogenic alkylating agents, pesticides, aflatoxin B1, and benzidine in water.

The effect of ozonation on the mutagenicity of selected chemicals in water was determined. The use of the Salmonella-microsome assay for mutagensis allowed kinetic studies to be performed on the ozonation of all chemicals tested. The results indicate that the mutagenicity of certain pesticides, including captan and Dexon, was inactivated by short periods of ozonation. The mutagenicity of certain alkylating agents including bis(2-chloroethyl)amine and sodium azide was rapidly inactivated by ozonation while other alkylating agents such as beta-propiolactone, propanesultone, and N-methyl-N'-nitro-N-nitrosoguanidine were unaffected by treatment with ozone. The mutagenicity of aflatoxin B1 was rapidly inactivated by treatment with ozone. Three chemicals were shown to be converted to direct mutagens by ozone treatment. Under certain conditions, dimethylhydrazine could be converted to a mutagen that was stable for 3 weeks. A similar chemical, 2-hydroxyethylhydrazine, was converted to an unstable mutagen that was inactive after 24 hr at room temperature. When benzidine was treated with ozone, there was a transient increase in mutagenicity which was lost after longer treatment with ozone.

Aflatoxins

Effect of ozone on bronchomotor response to inhaled histamine aerosol in dogs.

To study the effect of ozone on bronchial reactivity to inhaled histamine diphosphate aerosol, we performed 14 experiments on 5 dogs anesthetized with pentobarbital sodium (25-30 mg/kg, iv) and ventilated with a Harvard respirator. Prior to ozone exposure, inhalation of histamine aerosol (2% solution; 5 breaths) increased total pulmonary resistance (RL) 5.1 +/- 0.5 cmH2O/1 per s (mean +/- SE). One day after ozone exposure (0.7-1.2 ppm; 2 h), the base-line RL was not significantly changed (P greater than 0.05), but the increase of RL caused by histamine (10.7 +/- 1.1 cmH2O/1 per s) was greater than in the control state (P less than 0.01). When the dogs were pretreated with atropine sulfate aerosol (1.5% solution; 10 breaths), the increase of RL after histamine was decreased to 3.8 +/- 0.3 cmH2O/1 per s before ozone, and this was not significantly different after ozone (4.5 +/- 1.1 cmH2O/1 per s; P greater than 0.5). Cooling blockade of conduction in the vagus nerves diminished the increase of RL after histamine to 3.9 +/- 0.5 cmH2O/1 per s before ozone, and this was not significantly different after ozone (4.4 +/- 0.6 cmH2O/1 per s; P less than 0.5). Since both atropine and vagal cooling abolished the ozone-induced bronchial hyperirritability, we conclude that it is mediated via vagal cholinergic pathways.

Aerosols

Characterization of an episode with elevated ozone concentrations.

A detailed analysis has been made of a seven day period in 1973, during which elevated ozone levels were observed at Ottawa. In addition to the ozone data, results are also presented for oxides of nitrogen, carbon monoxide, methane, total hydrocarbons and particulate matter. The particulates were analyzed for seven constituents, including the cations ammonium, calcium, sodium, potassium and lead and the anions sulfate and nitrate. Also measured were several other properties of the particulates, including the size distribution. It was found that none of the other gaseous pollutants showed any increase in concentration when high ozone levels were present. In contrast, the particulate matter did increase and several constituents, notably ammonium and sulfate, showed a dramatic rise. A comparison with ozone data from Toronto and upper New York state showed that the high ozone concentrations were widespread. A meteorological analysis showed that elevated ozone levels were associated with a Tropical air mass with a moderate southwesterly flow of air. Trajectory calculations were carried out to show the origin of the air over Ottawa before, during and after the period of increased ozone.

Air Movements

Adaptations to ozone in reference to mucociliary clearance.

Adaptation to ozone in respiratory tract mucociliary clearance was investigated in this study. Eighty rats inhaled insoluble radioactively labeled particles in order to permit monitoring of clearance. The respiratory tract and the feces were counted for radioactivity at fixed intervals to determine clearance rates. A brief challenge to 1.2 ppm of ozone following particle deposition caused a substantial delay in rapid (mucociliary) clearance. This delay (or "ozone effect") however, was eliminated by brief pre-exposure to 0.8 ppm of ozone 3 days prior to deposition of particles. When a 13-day period intervened between the ozone pre-exposure and challenge, a substantial delay (or "ozone effect") was again seen. Thus, the pre-exposure to ozone appeared to afford essentially complete protection at 3 days, and no protection by 13 days.

Animals

Inactivation of viruses and bacteria by ozone, with and without sonication.

Selected organisms with public health significance were placed in a reaction chamber for treatment by ozonation, by ozonation and sonication, by sonication, or by sonication during oxygenation. Vesicular stomatitis virus, encephalomyocarditis virus, GDVII virus, Staphylococcus aureus, Pseudomonas fluorescens, Salmonella typhimurium, enteropathogenic Escherichia coli, Vibrio cholerae, and Shigella flexneri were inactivated by treatment with ozone. When microorganisms were suspended in phosphate-buffered saline, they were inactivated rapidly by treatment with ozone. However, microorganisms suspended in secondary effluent from a wastewater treatment plant required longer contact times with ozone for complete inactivation. Simultaneous treatments by ozonation and sonication reduced the contact time for complete inactivation of microorganisms in secondary effluent. Treatment by sonication alone or sonication and oxygenation did not inactivate microorganisms. Therefore, the simultaneous treatment of microorganisms in secondary effluent with ozone and sonication resulted in a synergistic effect.

Animals

Bronchial hyperirritability in healthy subjects after exposure to ozone.

We studied the effect of a 2-hour exposure to 0.6 ppm of ozone on bronchial reactivity in 8 healthy, nonsmoking subjects by measuring the increase in airway resistance (Raw) produced by inhalation of histamine diphosphate aerosol (1.6 per cent, 10 breaths). Before exposure to ozone, histamine increase the mean Raw from 1.2 to 1.8 cm H2O per liter per sec. Immediately after exposure to ozone, the mean baseline Raw was unchanged, but the mean response to histamine was significantly greater than the pre-ozone response (Raw = 3.3 cm H2O per liter per sec; P less than 0.05). For the group, this increase disappeared 1 day after exposure to ozone, although 2 subjects still had a significantly increased response to histamine for more than 1 week after exposure. In 4 subjects, pretreatment with atropine sulfate aerosol (0.1 to 0.2 mg per kg of body weight) blocked the increase in Raw produced by histamine after exposure to ozone. We concluded that brief exposure to 0.6 ppm of ozone produces bronchial hyperirritability via cholinergic postganglionic pathways, probably by damaging airway epithelium and thereby sensitizing bronchial irritant receptors.

Adult

Adaptation in human subjects to the effects of inhaled ozone after repeated exposure.

Single exposures to low concentrations of ozone (0.4 to 0.5 ppm) have resulted in decrements in forced vital capacity and specific airway conductance. To establish whether adaptation might occur with repeated exposure, 14 normal human subjects were exposed on 5 consecutive days to 0.4 ppm of ozone for 3 hours per day in an environmental chamber. Measurements of forced vital capacity and specific airway conductance obtained after exposure to ozone were compared to corresponding control values obtained during the previous week, when the same subjects breathed filtered air in the environmental chamber for 3 hours per day on 5 consecutive days at the same time of day. The forced vital capacity was significantly lower than the control value on the first 3 days of exposure to ozone, but there was no significant difference on the fourth and fifth days. Specific airway conductance was significantly lower than the control value on the first and second days of exposure to ozone; no significant difference was noted on the third, fourth, or final day. All subjects were symptomatic on the first and second days of exposure to ozone. Symptoms resolved thereafter, with only one subject remaining symptomatic on the final day of exposure to ozone.

Adaptation, Biological

Ozonation of mutagenic and carcinogenic polyaromatic amines and polyaromatic hydrocarbons in water.

The Salmonella-microsome assay for mutagenesis was used to determine the effect of ozone on the mutagenesis of selected carcinogens and mutagens in water. Short periods of ozonation were shown to completely inactivate the mutagenicity of several polyaromatic amine mutagens including acriflavine, proflavine, and beta-naphthylamine. Selected polyaromatic hydrocarbons were also sensitive to ozonation. Kinetic studies revealed that the mutagenicity of benzo(a)pyrene, 3-methylcholanthrene, and 7,12-dimethylbenz(a)anthracene was destroyed after short periods of ozonation. To correlate loss of mutagenicity with loss of carcinogenicity, two polyaromatic hydrocarbons were treated with ozone, extracted from water with hexane, and tested for carcinogenicity in mice. When 7,12-dimethyl-benz(a)anthracene and 3-methyl-cholanthrene were treated with ozone, there was a substantial reduction in carcinogenicity compared to control groups treated with oxygen alone. However, a small number of tumors developed in the group of animals receiving a hexane extract of ozonated 7,12-dimethylbenz(a)anthracene. This activity may be due to breakdown products of 7,12-dimethylbenz(a)anthracene that are not mutagenic.

2-Naphthylamine

[Ozone and gynecologic radiotherapy].

A short survey is given of the historical development and the physical basis of ozone therapy. Ozone is used in medicine as well as in other spheres. Papers reporting good results of ozone treatment in carcinoma seemed of particular interest. The efficacy of ozone as an adjuvant to the irradiation of carcinosarcomas of rats was confirmed by us. On account of this fact ozone was introduced by us as an adjuvant to the irradiation of women with gynaecological cancer and appeared to give good results. The mechanism of action of ozone is not yet fully clarified and several theories are discussed. Investigations are currently being undertaken in respect to the behaviour of several substances in the organism during ozone therapy.

Animals

The pulmonary and extrapulmonary effects of ozone.

The toxicity of ozone is solely due to its action as an oxidant. It is an extremely reactive gas which rapidly forms intermediate oxidizing derivatives after inhalation. High concentrations cause death from pulmonary oedema. Both pulmonary and extrapulmonary toxicity have been observed at lower concentrations of ozone, including those currently present in urban air. Pulmonary cellular and subcellular membranes appear to be particularly susceptible. A primary mechanism of this effect is the oxidative decomposition of polyunsaturated fatty acids, which has been demonstrated in rodent lungs after inhalation of ozone. Supporting evidence includes the potentiation of ozone toxicity by vitamin E deficiency and an increased use of this antioxidant vitamin during repetitive exposure to ozone. Other membrane effects include oxidation of thiol groups and, perhaps, of tryptophan. Microsomal alterations include a loss of lung cytochrome P450 which may also be related to lipid peroxidation. Extrapulmonary toxicity is not directly due to ozone but may represent in effect due to lipid peroxide decomposition products, particularly malonaldehyde. This three-carbon dialdehyde has been shown to alter cell membrane fluidity and to have mutagenic properties; the latter perhaps due to cross-linkage of DNA to histone.

Amino Acids