Distinct enzymatic responses in mice exposed to a range of low doses of ozone.

Short-term exposure of mice to low O3 doses, as defined by the product of concentration and exposure time (ct), was observed to induce alterations in two enzyme systems: first, that leading to changes in hepatic reduced ascorbic acid (RAA) content, and second to changes in plasma creatine phosphokinase (CPK) activity. RAA alterations were noticed immediately, 30 min and 120 min after termination of the exposure period, whereas CPK showed alterations immediately and 15 min after termination of the exposure. Later determinations, i.e., 24 hr after O3 exposure for RAA and 30 min after 03 exposure for CPK, revealed no significant differences when compared to control animals. Although differences in sensitivity existed, the dose response curves for both systems were more or less similar, showing a short decrease for the initial very low O3 doses, followed by a profound rise and a gradual decrease to control levels for subsequent ct doses. Exceptions were the 30 min curve for RAA and the immediate curve for CPK in so far as that both showed an additional depression. Neither plasma histamine nor plasma lactic acid dehydrogenase (LDH3) were observed to be altered by the range of O3 doses employed. These findings were explained on the basis of adaptation of the organism to a potentially noxious O3 stimulus by enhanced metabolic processes: a weak stimulus leading to only a small adjustment, and stronger stimuli to elevated enzyme activity as well. With increasing doses of O3 this elevation in enzyme activity was found to be gradually diminished, possibly due to a steadily growing demand, leaving the overshoot becoming continually smaller until a balanced state is achieved.

Radiomimetic activity of ozone (03), first described by Brinkman and Lamberst (1) and supported by the suggestion of Goldstein et al. (2)(3)(4) that 03 action in living organisms may be mediated by the formation of free radicals, might have led investigators to consider every O3 action as being harmful regardless of the 03 concentration and the duration of exposure (5,6). In the opinion of these authors there is no threshold value (no-effect level), and the dose-effect curve starts at zero. However, in the same way as with ionizing irradiation, in order to visualize effects, dosages of 03 have to be explored which in general clearly exceed the permissible *Laboratoryfor Radiopathology, State University of Groningen, Bloemsingel  Exposure times of days or weeks have often been used under experimental conditions. There is no doubt that, in the higher concentration time ranges, 03 causes toxic symptoms. The linearity of the dose-effect relationship of hazardous 03 action, down to zero, must be disputed, as it has been for ionizing irradiation (7). Even more so, since nonanthropogenic 03 is a natural ambient constituent for which an adaptive mechanism to lower values might be supposed to be present. Currently, such a mechanism has actually been suggested in the results of a number of studies, including those done in man (8)(9)(10)(11).
The results of the present study, where mice are exposed to 03 doses in a range regularly occurring in ambient air, are in favor of the existence of such an adaptive process.

Materials and Methods
Groups of 15-20 male mice of an inbred grey strain (Gron), as well as of the hybrid of this strain with inbred C57bl mice (F1) were exposed to 03, with five mice to a cage. Similar groups of mice were concurrently sham-exposed. Cages were placed in a stainless steel cabinet. The animals had full access to water, but they were deprived of food for 16 hr prior to and in the penod of 03 exposure. 03 was generated by an adjustable silent-discharge ozonizer (Air-cos Switzerland) situated at the bottom of the cabinet.
The oxidant was monitored by a Dasibi type 1003AH ozonometer (Dasibi, USA) operating on the principle of UV absorption by 03. Blood for investigation was drawn from the orbital sinus under light ether anaesthesia.
Reduced ascorbic acid (RAA) in the liver was determined as previously described (12). Creatine phosphokinase (CPK) in blood plasma was assayed with the CPK test-combinations of Boehringer-Mannheim (W. Germany). Plasma histamine was determined according to the single-isotope method described by Beaven et al. (13), with some minor modifications. In our study, the sensitivity of this method was approximately 1 ng/ml. Total lactic acid dehydrogenase (LDH) in blood plasma was assessed with the LDH test-combinations of Boehringer-Mannheim (W. Germany). LDH isoenzymes were separated by disc gel electrophoresis (14), and the gels quantitatively scanned by means of a Gilford model 2400 S automatic spectrophotometer with linear transport equipment operating at 500 nm (Gilford, Ohio, USA). Results were expressed in percentages of those found in the controls and plotted against the product of concentration (c) and exposure time (t). As has been demonstrated earlier, when c and t are adapted so that the product ct remains constant, identical results are obtained (15). Student's t-test and a test based on normal approximation were used for statistical evaluation of the results. The level of significance was set at 5%. The standard error of the percentage difference between experimental and control values at each ct value was calculated as 100 times the square root of the expression: where SE1 = standard error of the mean of control values, SE2 = standard error of the mean of experimental values, X1 = mean of control data, and X2 = mean of experimental data.

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The data obtained for hepatic RAA for three different post-exposure time intervals, i.e., for 0, 30, and 120 min, are plotted in Figure 1. Each of the three curves starts with a short negative period, followed by a sharp rise to values significantly above zero. Thereafter the curves level off to normal at the higher ct values tested. A few measurements performed 24 hr after 03 exposure reveal RAA values falling within the normal range. Similar curves were obtained for plasma CPK immediately and 15 min after 03 exposure (Fig. 2). this enzyme appears to be more sensitive, in that lower ct values are capable of evoking alterations in the enzyme activity. Furthermore, the effect of 03 on CPK disappears more rapidly; 30 min after termination of the 03 supply, significant alterations were no longer observed. Values obtained 15 min after 03 exposure also failed to show significant changes, with the exception of the CPK value obtained at ct = 0.8.
The histamine content of blood plasma remained unaltered after treatment of mice with a series of 03 concentrations for various time periods (Table   1). In addition, both total LDH as well as its isoenzyme remaining within normal limits ( Table  2).

Discussion
Since RAA is a product of a chain of enzymatic activities, and since 03-induced changes in RAA are of an enzymatic nature (12), we  recorded alterations in two enzymatic systems in this study. Although differences in sensitivity between the two systems occur, the alterations are principally similar. They are restricted to the lower 03 doses tested. This, together with the rapid reversibility of the effects may indicate that we are dealing with a physiological phenomenon rather than with cellular injury. In this sense, the initial negative part of the curves obtained might just represent a certain metabolic adjustment due to weak 03 action. Subsequent higher doses may stimulate enzyme activity (an all-or-none effect) (12) in concert with increasing consumption until a new equilibrium may be achieved where experimental and control values are similar. Such a course with increasing 03 doses might be characterized as adaptive and noninjurious. This is supported by the finding that plasma histamine and LDH3 are not significantly changed. A decrease in histamine has been demonstrated in the lungs of mice and rats exposed to oxidants (16)(17)(18), whereas increased levels of plasma LDH3 are suggested to be indicative of lung tissue damage (19).
The use of ct values seems justified under the conditions employed in this study, where low 03 concentrations and short exposure times are combined. The method has been validated for toxicological research (20) and has also been used in air-pollutant investigations (21). We have also verified this method for some enzymes using a single ct product with different c and t values and the product was found to be constant (15,22). The sensitivity of different enzyme systems in reacting to an adverse stimulus appears to be different. At least, lower ct doses are capable of inducing alterations in plasma CPK compared with those leading to modified RAA values of the liver.
Other enzymes have been observed to show altered activity after 03 exposure, for example, acetylcholinesterase (Ach-ase) activity is decreased in the erythrocytes of mice after exposure to 8 ppm 03 for 4 hr (3).
G-6-PDH as well as LDH are enhanced, whereas Ach-ase is lowered in human erythrocytes after exposure to 0.5 ppm 03 for 2.75 hr. Serum GSHR is concomitantly decreased (8).
Ishiwatari (26) has also found an increase in pulmonary GSHP in rabbits and mice exposed to at least 5 ppm O3 for 4 hr. Glucose-6-phosphatase is concurrently lowered in murine lungs, whereas  ATP-ase shows lower values in the lungs of rabbits exposed to 10 ppm 03 for 2 hr per day on five successive days. Lee et al. (27) have treated rats with 0.2, 0.5, and 0.8 ppm 03 for 1 to 30 days and obtained elevated levels of lung succinate oxidase, cytochrome-c reductase and also G-6-PDH.
In man the values for Ach-ase, G-6-PDH and phosphokinase in red blood cells are unaltered after exposure to 0.2 ppm during a normal working day, but LDH and a-hydroxybutyrate dehydrogenase levels decrease (25).
In most cases cited above, the 03 doses used were significantly higher than those for which we observed altered enzyme activity, but the reversibility of the reaction has not been considered. Although elevated blood levels of some enzymes are indicative of tissue injury, and most authors tend to explain their findings to be those of an injurious nature, one may question whether the possibility of adaptation has been sufficiently taken into account, as was done by Buckley et al. (8) and Hackney et al. (9).
The second depression in two ofour curves, at ct = 0.8 and at ct = 2.0 for CPK, determined immediately, and for RAA 30 min after 03 exposure respectively, cannot be precisely interpreted. They certainly point to metabolic imbalance. In both cases later values (at 15 min and 2 hr after exposure respectively) show a positive overshoot.
Increased RAA levels have been claimed (12) to contribute to the organism's defense. CPK is involved in the generation of ATP from creatinephosphate, and in this way supports the easily available energy source of the organism. This might also be considered to be part of the defensive potency of the organism. In fact, mobilization of defense may imply a scala of diverse reactions which will stabilize at a certain level if the noxious stimulus is not too strong. At that point the organism reaches the adaptive state.
We explored O3 doses. equal to or slightly higher than those occurring in environmental air. To conclude that these O3 doses can be safely borne is premature, since we tested only one animal species. Moreover, 03 iS frequently present in combination with other pollutants and our knowledge of combined effects is fragmentary (12,(29)(30)(31). The occurrence of mutational events under influence of air pollutants is still under discussion (32,33).