Int. J. Biometeor., 1978, vol. 22, number 1, pp. 43-52.

Effects of Atmospheric Small Negative Ions on the Oxygen Consumption of Mouse Liver Cells

by Bhartendu* and I. A. Menon**

ABSTRACT. - - The effects of small negative air ions on the oxygen uptake of isolated mouse liver cells were studied by exposing the liver cells to varying ion concentrations. For concentrations of the order of 1-2 x 105 ions/cm 3, the oxygen uptake was always higher than in the normal atmospheric conditions of 3-8 x 102/ions/cm 3. For interme- diate concentrations varying effects of activation and inhibition were observed. A statis- tical analysis showed that the oxygen uptake increased by approximately 14% when liver cells were exposed to ion concentrations of values 1-9 times the normal, by approximately 9% when exposed to 10-99 times the normal, and by approximately 38% when exposed to 100-999 times the normal. The significance and possible implications of the results are discussed.

Atmospheric small ions are considered biologically active (Krueger et al., 1962; Wehner, 1969) and up-to-date reviews on the subject have been published by Krueger (1972) and Krueger and Reed (1976). Reports on the growth stimulation as well as growth inhibi- tion of cell cultures and bacteria by small air ions have been published in the literature (Worden and Thompson, 1956; Krueger, Smith and Go, 1957; Krueger et al., 1975; Worden, 1961). Elkiey et al. (1977) have shown that positive air ions reduce the lesions of the net blotch disease of barley. Several workers have reported air ion-induced physiological changes in animals (Bachman, Krueger, McDonald and Lorentz, 1966; Minkh and Anisimov, 1972; Krueger, 1976), while others (Anderson, 1972) have not observed significant changes. Small air ions have also been considered to produce changes in some metabolic processes like decrease in serotonin levels in mice and rabbit tissues (Krueger & Smith, 1960a, b; Krueger and Reed, 1976), stimulation of cyto- chrome-c biosynthesis in several seedlings (Krueger, Kotaka and Andriese, 1963), and the oxygen consumption of barley seedlings (Kotaka et al. 1965) and of rat liver slices in vitro (Lotmar, 1972). Kotaka, Krueger and Andriese (1968) have also reported that air ions enhanced the ATP (adenosine triphosphate) - associated swelling and shrinking of isolated chloroplasts.

Lotmar (1972) observed that negative ions increased the oxygen consumption of rat liver slices by 11 to 14% whereas positive ions did not have any effect. Lotmar (1972) did not obtain accurate measurements of ion densities in some experiments and did not report normal (ambient) atmospheric ion concentrations. The only measured value of 2.2 x 102 ions/cm 3 is within the range of normal atmospheric concentrations in urban air.

* Atmospheric Environment Service, 4905 Dufferin Street, Downsview, Ontario, Canada.

** Clinical Science Division, Medical Sciences Building, University of Toronto, Toron- to, Ontario, Canada. Received 30 March 1977

44

Hence it is impossible to assess the importance of Lotmar's results. Lotmar also does not mention other meteorological parameters and it is not clear how they varied. Krueger and Reed (1976) have stressed the importance of these factors.

A wide variety of metabolic processes is dependent on the availability of utilizable energy (high energy compounds). The biosynthesis of these compounds is dependent upon the oxidative processes (electron transfer processes) or oxygen consumption. In view of the central role of oxygen consumption in cellular metabolism, the effect of ions on the oxygen uptake by liver cells was studied. This paper reports the results of a study in which the isolated rat liver cells were exposed to various concentrations of small negative ions and the oxygen consumption was measured.

MATERIALS AND METHODS

Generation and Counting of Ions A 65 cm long and 8 cm wide linear ion generator manufactured by Nuclear Radiation Developments, Inc., using a strip of radioactive foil of about 54 mCi of 210po secured in an insulated electrode and protected by an aluminium housing with a protective grid, was used as the ion source. The ion densities could be varied by adjusting the separator potential from 400 to 2000 V and by varying the distance of the ion generator from the liver tissue kept near the ion counter.

The Gerdien type aspirator ion counter initially designed by Saxer and Sigrist (1966) was modified by changing the input resistors to measure ion concentrations up to the maximum valffe of 106 ions/cm 3.

MEASUREMENTS OF SFERICS AND OTHER METEOROLOGICAL VARIABLES

A VLF analyser (Heydt, 1967; Frisius and Heydt, 1968), was continuously operated at the Atmospheric Environment Service Station for Amospheric Experiments, situated at a distance of about 30 km from the laboratory in which the measurements of oxygen uptake were made. This analyser continuously gave hourly averages of the 5 kHz sferics activity and was found to provide a good measure of North American thunderstorm activity.

The atmospheric pressure was measured by a MSC type B barograph, and the tempe- rature and humidity were measured by a Lambrecht Thermohygrograph. These meteoro- logical elements were measured in the laboratory.

PREPARATION OF MOUSE LIVER CELLS

C57BL/6J mice, 4 to 6 months old, weighing 20 to 26 g, and kept under controlled conditions with free access to food and water, were anaesthetized with ether and exsanguinated. No significantdifference in control experiments was found between the oxygen uptake of livers from mice killed by decapitation and by the use of ether. The livers were quickly removed and chilled in a beaker over ice. The livers from two mice were combined and the tissue was weighed, minced with scissors and dropped into 4 layers of gauze suspended in 0.9% NaCI. The livers were dispersed gently using a teflon homogenizer. The liver cells were sedimented by centrifugation at 2000 g for 2 min. and resuspended in medium 199. The measurement of oxygen uptake was commenced immediately and the stock suspension of the cells was always kept immersed in ice.

TREATMENT OF MOUSE LIVER CELLS WITH AIR IONS. - - 0.2 ml of the suspension of liver cells (0.5 g/ml) was mixed with 1.8 ml of the medium 199 which was manufactured by the Grand Island Biological Company. The diluted suspension was

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taken in a 20 ml beaker and kept stirred for 15 min at room temperature with a magnetic stirrer in an atmosphere of the specific ion concentration. The stirring was carried out near the inlet of the ion counter.

The objective of this study was to know whether or not atmospheric ions affect the oxygen consumption of liver cells and hence only the ion density measurements commonly used to describe the ionized state of the air in atmospheric electricity were made. A more precise measurement for studying the mechanism would be to measure the number of ions reaching the tissue Surface. However, this measurement would not reveal the air ion concentration; a parameter more important for practical purposes. Future experiments should attempt to incorporate the measurements of ions reaching the tissue by using an ion probe.

MEASUREMENT OF OXYGEN UPTAKE. - - After stirring, the cell suspension was transferred into the reaction cell having an oscillating platinum cathode of an oxygraph mfinufactured by Gilson Medical Electronics and the oxygen uptake was measured. The initial rare of the oxygen uptake was calculated from the early portion of the curve and is expressed as taM oxygen consumed per minute.

CALCULATION OF THE DATA. - - Control experiments (at the normal ambient ion concentration) were performed in the beginning and at the end of the experiment. These values usually did not vary more than 10%. The mean of all these results was taken as the vaIue for the control. In each experiment 3 to 6 readings using separate portions of liver cell suspension were obtained for each ion concentration. The mean, standard deviation (SD) and standard error (SE) were calculated for each ion concentration. The result for each concentration was compared with the corresponding control. The statistical significance of each result was calculated using the t-test. Seven experiments were carried out and the statistical significance of all combined experiments was deter- mined. The details of this analysis would be available from the authors.

RESULTS

Figures 1 to 3 show the relationship between the concentration of small negative ions and the oxygen uptake of mouse liver cells. Because of large variations, the ion concen- trations aye plotted on logarithmic scale. The lowest ion concentration in each experi- ment represents the control (normal ambient atmospheric ion concentration) in the figures. No single relationship is found between oxygen consumption and ion concen- tration. Experiments 1 and 7 shown in Fig. 1 indicate that oxygen consumption decrea- sed with the increase of ion concentrations from 3-8 x 102-ions/cm 3 (control values) to 2-5 x 103 ions/cm 3. As the ion concentration was further increased, the oxygen uptake increased at different rates. On the other hand, experiments 2, 3 and 6 illustrated in Fig. 2 show that with the increase in ion concentrations from 4-8 x 102 ions/cm 3 (control values) to 2-5 x 103 ions/cm 3, oxygen consumption increased. With the further increase of ion concentrations to 5 x 103 - 3 x 104 ions/cm 3, the oxygen uptake decreased at different rates. Afterwards the oxygen uptake increased with further increase of ion concentrations at different rates. The experiments 4 and 5 presented in Fig. 3 show that the oxygen consumption increased with the increase of ion concentration from 3-5 x 102 ions/cm 3 (control values) to 1-2 x 105 ions/cm 3. Because of the unavailability of the intermediate observations, it is not possible to study the relationship for the middle ranges.

The ion concentration for control (normal atmospheric conc.) varied from experi- ment to experiment. Also, it was not possible to maintain a constant ion concentration from the generator for different readings for each ion concentration range. Further- more, the relationship of the oxygen uptake with the ion concentration was different in various experiments as shown in the Fig. 1 to 3. Therefore, ratios of ion concentrations with respect to those for the controls were calculated and the observations were grouped

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irito four categories. The first one was called control (Ic) and the remaining three I 1/Ic, I2/Ic, I3~Ic had ratios between 1-9 (same order of magnitude), 10-99 (! order of magni- tude higher, and 100-999 (2 orders of magnitude higher) respectively. These are referred to as Control, Group I, II, and III, respectively. The oxygen consumptions for these four groups for each experiment along with the necessary meteorological data are tabulated in Table 1.

In Group I, out of our experiments, two experiments showed activation (increase in oxygen uptake) and one showed inhibition (decrease in oxygen uptake), significant at 10% probability level. In Group II, out of 6 experiments, two experiments showed acti- vation at 10% level of significance and one experiment showed inhibition at 1% level. In Group III, none showed inhibition. Out of 7 experiments in this group, 4 experiments showed activation significant at 10% level, of which two were significant even at 1% level.

The statistical significance of all the combined experiments for these three groups I, II and III is shown in the bottom of Table 1. The combined results of oxygen uptake in Group I showed an activation of 14% with a standard error of 6%. This result is signi- ficant at 10% level. The Group II showed an activation of 9% with a standard error of 4%, a result significant at 10% level. Group III showed an activation of 38% with a stan- dard error of 16%. This result is significant at 1% level.

The variation in the oxygen uptake due to ion concentration was not dependent on the meteorological elements in our experimental conditions, as seen from the values of temperature, relative humidity and pressure. The barometric pressure for experiment 5 was 100.9 kpa and no significant result was obtained for the ion concentration in Group III, while the experiment 2, for which the second highest pressure of 100.5 kpa was observed, shows significant results for Groups I and III. For experiment 3, when atmospheric pressure was 99.4 kpa, no significant results were observed, while for experiment 7, when the pressure was 98.4 kpa, all results were significant. Also, the results of experiments 4 and 5, when the relative humidity was same, were different. The temperature variation for these experiments was only I°C and hence it can not be considered important.

The variations in the effect of ions on the oxygen uptake were not due to the changes of sferics activity of 5 kHz as evident from the Table 1 and Fig. 4. The 5 kHz activity changed by five orders of magnitude as shown in Fig. 4 without any significant relation-

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ship with the effects of ions on oxygen uptake. Moreover, there was no simple relation- ship apparent between the sferics activity and the oxygen uptake for the controls for various experiments; this is in disagreement with the findings of Lotmar, Ranschto Froemsdorff and Weise (1969), where respiration rates of mouse liver tissue showed significant (42%) decrease in simulated cyclonic weather conditions.

Several similar experiments were performed using an ion generator which also produced ozone and hence the results of these measurements are not included in this

50

paper. An experiment was also conducted in which many readings of the oxygen consumption were taken throughout the day under normal atmospheric conditions; no significant variation was observed among these readings. As all mice in our experiments were killed in the morning between 9:30 and 10:30 h effects of daily rhythm on the oxygen consumption were considered unimportant.

DISCUSSION

It is evident from the results of this study that the relationship between the small nega- tive air ions and the oxygen uptake of mouse liver cells is complex. The oxygen con- sumption is found to increase or decrease with increase in ion concentrations as high as one order of magnitude above the normal atmospheric concentration (control). When the ion concentration is increased to a value two orders of magnitude above the normal atmospheric concentration, oxygen uptake always increased, although the statistical significance of this activation differs from experiment to experiment. These results show the variable nature of the effects of ion on biological systems and this explains, at least partially, the controversy in the literature (Anderson, 1972; Krueger and Reed, 1976). This also questions the growing popular belief regarding the role of ions in possible advantages in health by moving to certain environments where ion concentrations are within the same order of magnitude.

It is important to compare the present results with the only published work (Lotmar, 1972) on the effect of ions on the oxygen uptake. The significant differences between the two experiments are: (1) Rat liver slices were employed in Lotmar's experiments while suspension of dispersed mouse liver mostly as single cells was used in the present study (2). In Lotmar's work oxygen uptake was measured using the manometric method and the period of oxygen measurement was 75 min, in the present study the oxygen uptake was measured using an oxygen electrode and the period of oxygen measurement was 10 minutes. (3). The mode of aeration was different in the two experiments. In Lotmar's experiment the medium was bubbled with air whereas in the current study the medium was stirred with a magnetic stirrer. (4). In our experiments, the minimum value of 3 x 102 ions/cn9 of the concentration in normal atmosphere is even higher than the value of 2.2 x 102 ions/cm 3 of the ion concentration generated in Lotmar's experiments. The ion concentrations were continuously monitored in our study and the highest con- centration produced was 2 x 105 ions/cm 3.

In view of the variability of the results described above, it is important that accurate measurement of ion concentrations should be made continuously during the entire experiment. Also, varying ion concentrations should be used to ensure a complete understanding. Lotmar's work is deficient in these respects.

The mechanism by which the ions affect the oxygen consumption of isolated cells is not known. It is, also, difficult to extrapolate these results to in vivo respiration. In order to understand the effects of ions on the in vivo metabolism, it is necessary to know the effects of ions in vitro on various isolated systems such as tissue slices, cell suspension, isolated organelles, particulate and soluble enzymes from various organs of several species of animals. Only then the implications of small air ions on the human health and well being can be understood.

This study is concerned only with the negative small ions. Future studies should take into consideration the positive ions also. It would be also useful to control the meteoro- logical variables in the future work so that the ion effects could be studied under varying meteorological conditions.

ACKNOWLEDGEMENTS We would like to thank Dr. N. Barthakur, Macdonald Campus of McGill University, Ste. Anne de Bellevue, Quebec, for making the linear ion generator available for this study. The advice of Dr. R P. Bhargava, Department of Measurement and Evaluation,

51

Ontario Institute of Studies in Education, in the statistical analysis is gratefully acknow- ledged.

REFERENCES

ANDERSON, I. (1972): Effects of natural and artificially generated air ions on mammals. Biometeorology 5, S. W. Tromp, W. H. Weihe and J. J. Bouma (ed.). Suppl. Int. J. Biometeor. 16, Part II, 229-238.

BACHMAN, C. H., McDONALD, R. D. and LORENZ, C. J. (1966): Some effects of air ions on the activity of rats. Int. J. Biometeor., 10: 36-46.

ELKIEY, T. M., PELLETIER, R. L., BHARTENDU, and BARTHAKUR, N. (1977): Effects of small ions on net blotch disease of barley. Int. J. Biometeor., 21: 1-6.

FRISIUS, J. and HEYDT, G. (1968): Spectral parameters of the VLF radio noise observed as functions of the azimuth. Radio Sci., 3: 1004-1009.

HEYDT, G. (1967): Peilanlagen zur Messung yon spektralen Amplitudenverteilungen, Amplitudenverh~iltnissen und Gruppenlaufzeitdifferenzen yon Atmospherics- Technischer Bericht Nr. 90 (Heinrich-Hertz lnstitut for Schwingungs- forschung Berlin-Charlottenburg. West Germany) 73 pp.

KOTAKA, S., KRUEGER, A.P. and ANDRIESE, P. C. (1968): The effect of air ions on light-induced swelling and dark induced shrinking of isolated chloroplasts. Int. J. Biometeor., 12: 85-92.

KOTAKA, S., KRUEGER, A. P., NISHIZAWA, N., OHUCHI, T., TOKENOBU, M., KOGURE, Y. and ANDRIESE, P. C. (1965): Air ion effects on the oxygen consumption of barley seedlings. Nature (Lond.), 208:1112-1113.

KRUEGER, A. P. (1972): Are air ions biologically significant? A review of a contro- versial subject. Int. J. Biometeor., 16: 313-322.

KRUEGER, A. P (1976): Biological effects of ionization of the air on animals. In: Progress in Biometeorology. S. W Tromp (ed.), Division B. Progress in Animal Biometeorology. Chapter 5, Section 1, 155-162. Swets and Zeitlinger, B. V Amsterdam.

KRUEGER, A. P., BECKETT, J. C., ANDRIESE, P. C. and KOTAKA, S. (1962): Studies on the effectsof gaseous ions on plant growth, II. The construction and operation of an air purification unit for use in studies on the biological effects of gaseous ions. J. gen. Physiol., 45: 897-904.

KRUEGER, A. P., KOTAKA, S., and ANDRIESSE, P. (1962): Gaseous-ion-induced stimulatioia of cytochrome c biosynthesis, Nature (Lond.), 200: 707-708.

KRUEGER, A. P. and REED, E. J. (1976): Biological impact of small air ions. Science 193: 1209-1213.

KRUEGER, A. P., REED, E. J., BROOK, K. B. and DAY, M. B, (1975): Air ion action on bacteria. Int. J. Biometeor., 19: 65-71.

KRUEGER, A. P., SMITH, R. F. and GO, I. G. (1957): The action of air-ions on bacteria, I. Protective and lethal effects on suspension of staphylococci in droplets, J. gen. Physiol., 41: 359-381.

KRUEGER, A. P. and SMITH, R. F. (1956a): The biological mechanisms of air ion action. I. 5-Hydroxytryptamine as the endogenous mediator of positive air ion effects on the mammalian trachea. J. gen. Physiol., 43: 533-540.

KRUEGER, A. P. and SMITH, R. F. (1960b): The biological mechanisms of air ion action. II. Negative air ion effects on the concentration and metabolism of 5-hydroxytryptamine in the mammalian respiratory tract. J. gen. Physiol., 44: 269-276.

LOTMAR, R. (1972): Oxygen consumption of rat liver tissue in substrate soluti~n treated with ionized air. Int. J. Biometeor., 16: 323-327.

52

LOTMAR, R., RANSCHT-FROEMSDORFF, W. R. and WEISE, H. (1969): D~impfung der Gewebeatmumg (QO2) von M~iuseleber durch kdnstliche Impulsstrahlung. Int. J. Biometeor., 13: 231-238.

MINKH, A. A. and ANISIMOV, B.V. (1972): Regularities in the physiological effects produced by ionized air. Vestnik Akademii Medilsinskikh Nauk SSSR-C1STI, 27:3-13 (in Russian).

SAXER, L. and SIGRIST, W. (1966): Die luftelektrische Station in Aarau, Schweiz. Mittl. Aargau. Naturforsch. Ges. 187-224.

WEHNER, A. P. (1969): Special review - electro-aerosols, air ions and physical medi- cine. Amer. J. phys. Med., 48:119-149.

WORDEN, J. L. (1961): proliferation of mammalian cells in ion controlled environ- ments. J. nat. Cancer Inst., 26: 801-811.

WORDEN, J. L. and THOMPSON, J. R. (1956): Air ion concentration and the growth of cells in vitro. Anat. Rec., 124:500.