The Effect of Dissolved Oxygen Partial Pressure on the Growth and Carbohydrate Metabolism of Mouse Ls Cells

J. Cell Sci. 4, 25-37 (1969) 25 Printed in Great Britain

THE EFFECT OF DISSOLVED OXYGEN PARTIAL PRESSURE ON THE GROWTH AND CARBOHYDRATE METABOLISM OF MOUSE LS CELLS

D. G. KILBURN,' M. D. LILLY, DAPHNE A. SELF AND F. C. WEBB Biochemical Engineering Section, Department of Chemical Engineering, University College London, England

SUMMARY Batch cultures of mouse LS cells were grown in suspension at controlled dissolved oxygen partial pressures (pOt). At low pOt (i-6 mmHg) the growth and respiration rates and the final cell population were all limited. At high pOt (320 mmHg), cell division was inhibited after an initial doubling of the cell number. At intermediate values of pOt, the growth rate was constant but the final cell population varied. Within the^>O2 range of 40-100 mmHg, the final cell population was constant and maximal at 1-2 x 10s viable cells/ml. Except at 320 mmHg pOt, about 90 % of the glucose consumed served as an energy source and could be accounted for as lactate and CO,. In the culture at 320 mmHg, only 60 % of the glucose consumed could be accounted for in this way. During growth the production of lactate and pyruvate was highest at low pOt. A sharp increase in lactate production was observed as logarithmic growth ceased in each culture, except at high pO% (160 mmHg). These observations indicate that pOt markedly influences cell growth and carbohydrate metabolism in these cells.

INTRODUCTION The role of oxygen in the regulation of the metabolism of animal cells is not yet fully understood. It has been found that anaerobic conditions generally halt or severely depress cell growth (Clark, 1964; Dales, i960; Danes, Broadfoot & Paul, 1963). At the other extreme, high oxygen partial pressure (pO2) also inhibits replication but does not necessarily interfere with the synthesis of macromolecules (Brosemer & Rutter, 1961; Rueckert & Mueller, i960). Between these limits an optimumpO2 range for cell growth must exist. Although several workers have demonstrated a beneficial effect of moderately low^O2 (Zwartouw & Westwood, 1958; Cooper, Burt & Wilson, 1958; Cooper, Wilson & Burt, 1959; Pace, Thompson & Van Camp, 1962; Jones & Bonting, 1956), there is little quantitative information on the influence of pO2 on cellular growth and metabolism except at the extreme pO2 values. Specific studies on the effect of dissolved oxygen partial pressure on animal cell growth or metabolism have been made both in static and suspension cultures. The results relating to static monolayer cultures are difficult to interpret, because the static cell can establish its own micro-environment, which may vary considerably • Present address: Vaccine Department, Rijks Instituut voor de Volksgezondheid, Bilthoven, The Netherlands. 26 D. G. KiUnirn and others from conditions in the bulk of the medium. Culture of cells in suspension permits the pO2 environment to be specified much more exactly, although one cannot assume that the gaseous and liquid phases of these cultures are in equilibrium and hence the pO2 of the medium must be measured (Kilburn & Webb, 1966). The presence in the literature of a number of conflicting reports on the effects of dissolved oxygen may have resulted from the assumption that the measured oxygen partial pressure in the gas phase can be equated with the liquid pO2. Our previous work on the measurement of pO2 in suspension cultures of animal cells led to the development of a culture apparatus in which cultures could be grown at controlled pO2 values. The cultivation of cells at a number of discrete pO2 values between the inhibiting levels at high and low pO2 has permitted the influence of dissolved oxygen on cellular metabolism to be studied in much more detail than has previously been possible. It has been found that maximal populations of mouse LS fibroblasts are produced when the pO2 in the culture is controlled at values from about 40 to 100 mmHg (Kilburn & Webb, 1968). The present paper presents further information on the effect of pO2 on growth rate, maximum cell concentration and carbohydrate metabolism.

METHODS Organism The mouse LS cell used in this work was kindly supplied by Dr J. Paul. This cell grows spontaneously in suspension, showing little tendency to attach to glass and has been maintained in our laboratory for 3 years.

Cell culture technique All experiments were with 3 1. suspension cultures in Eagle's minimal essential medium (Eagle, 1955) supplemented with 2% (v/v) horse serum, 2-5 g/1. lactalbumin hydrolysate and 1 g/1. carboxymethyl cellulose. The glucose concentration in the medium was increased to 2 g/1. The temperature of the cultures was controlled at 35 +0-2° C and the pH was controlled at 7-4 + 0-1 by the addition of 0-5 N NaOH. Details of the culture vessel and the methods used to control pH and dissolved oxygen partial pressure have been described elsewhere (Kilburn & Webb, 1968). Viable cell counts were determined directly by counting a 1:1 dilution of cell suspension with stain (0-05 % trypan blue in o-8 % saline) on a haemocytometer (Fuchs-Rosenthal ruling, Cristalite, Gallenkamp, London). Cells which did not take up trypan blue were termed 'viable'.

Analyses of culture medium Culture samples taken aseptically from the fermentor were centrifuged at 350^ for 10 min. The supernatant was deproteinized with perchloric acid and analysed for glucose, lactate and pyruvate. Glucose was analysed by the method of Marks (1959), using D glucose oxidase (EC 1.1.3.4) and peroxidase (EC 1.11.1.7) with o-toluidine as the chromogenic Effect of oxygen on growth of mouse LS cells 27 oxygen acceptor. Lactate was determined spectrophotometrically by measuring NAD+ reduction during the oxidation of lactate to pyruvate in the presence of lactate dehydrogenase (EC1.1.1.27) (Olson, 1962). Pyruvate was determined by measuring the oxidation of NADH in the reverse reaction (Segal, Blair & Wyn- gaarden, 1956).

Materials 'Fermcozyme' glucose oxidase preparation and peroxidase (60 units/mg) were supplied by Hughes and Hughes Ltd., London, W. 1. Nicotinamide adenine dinucleotide (NAD+) and dihydronicotinamide adenine dinucleotide (NADH) were obtained from Seravac Laboratories, Maidenhead, Berks., and lactate dehydrogenase from Koch-Light Laboratories Ltd., Colnbrook, Bucks.

Determination of respiration rate The rate of cellular respiration in the culture vessel under the controlled conditions of pH and^>02 was determined from the rate of CO2 evolution measured using tubing probes as described previously (Kilburn & Webb, 1968). An independent estimate of respiration rate in the presence of excess oxygen was obtained by following the depression of pO2 in a cell suspension taken from the fermentor and sealed in the cuvette of a Rank Oxygen electrode (Rank Bros., Bottisham, Cambridge). Samples taken from cultures at low pO2 were aerated before the cuvette was closed so that a significant pO2 drop with time could be measured. In several experiments, 2,4- dinitrophenol (DNP) was added to the Rank electrode to determine the increase in respiration rate effected by the uncoupling agent. In most cases the maximum uncoupling occurred with 5 x io~6 M DNP.

RESULTS

The effect of pO2 on cell growth As reported previously (Kilburn & Webb, 1966), the maximum concentration of LS cells reached in batch cultures grown at controlled dissolved oxygen partial pressures (j>02) was optimal in thepO2 range from about 40 to 100 mmHg. Figure 1 shows the batch growth curves for four cultures at different controlled values of dissolvedpO2, and illustrates three separate effects: (1) at low^O2 (i-6 mmHg) both the growth rate and the maximum population level are limited; (2) at high pO2 (320 mmHg) cell division is inhibited; (3) at intermediate values of pO2 the growth rates are constant (initially the cultures at 48 and 140 mmHg follow the same growth curve) but the maximum cell population varies.

Cultures grown within the range of pO2 from 12 to 160 mmHg exhibited the same exponential growth rate equivalent to a mean generation time of 27-5 + 2-5 h.

The effect of pO2 on respiration rate

The average respiration rates, calculated from the rate of CO2 production during the rising portion of the growth curve, for cells growing at controlled pO2 levels are 28 D. G. Kilburn and others shown in Table i. These results agree well with the rates of oxygen consumption measured with the Rank oxygen electrode, except for the culture at i-6 mmHg.

Within the range of pO2 from 32 to 160 mmHg the respiration rate is constant. At 320 mmHg the rate is higher, but this is probably associated with the increased

Days Fig. 1. Batch growth curves for LS cells cultured at controlled dissolved oxygen partial pressures (pO2): A, i-6 mmHg; O, 48 mmHg; •, 140 mmHg; A, 320 mmHg.

Table 1. Average respiration rates for cultures of mouse LS cells at controlled pO2

Respiration rate 8 Controlled pOt (mmHg) (mmole O,/h io cells)

0-130 12 0-166 32 0175 40 0180 96 018 160 018 320 0-228

dry weight of the cells. At 12 mmHg respiration may be slightly depressed by the

low/>O2, while at i-6 mmHg the cells are definitely oxygen-limited. This is confirmed 6 by the increase in respiration rate from 0-13 mmoles/h io cells at^>02 = i-6 mmHg Effect of oxygen on growth of mouse LS cells 29 to 0-21 mmoles/h io6 cells when respiration was measured in the Rank electrode with oxygen in excess.

The effect of low pO2

In the batch culture at i-6 mmHg pO2 the growth rate and maximum cell count were both less than in cultures at higher pO2. It seems unlikely that the low final cell count in this culture can be attributed entirely to an increased requirement for some limiting nutrient; particularly since the cell viability, measured by trypan blue exclusion, was always lower than in cultures at higher pO2. Possibly some cells were unable to adapt to growth at low pO2. The presence of dead cells could then create toxic conditions, e.g. by releasing proteolytic enzymes, leading to low viability. The exponential growth rate at i-6 mmHg was about half that observed for cells grown in the intermediate pO2 range. Presumably this decreased growth rate reflects the lower rate of energy production under oxygen-limited conditions.

The effect of high pO2

The results from the culture at 320 mmHg/>O2 can be summarized as follows: (a) Growth rate was sharply inhibited after about 18 h. There was a slight increase in the total cell count over the next 48 h, but the viable count remained constant (during this period the percentage viability dropped about 10 %). (b) The dry weight/cell was about 30 % higher than normal. (c) The respiration rate/cell was about 25 % higher than normal. (d) Very little lactate was formed.

Table 2. The effect of DNP on the respiration rate of cells grown at high pO2 Respiration ratef

Measured Multiplication* No DNP DNPJ Uncoupling

Sample pOt (mmHg) in 2 days , * , factor

1 145 3-3 0-122 0-277 2-3 2 290 2-i 0-191 0348 i-8 3 270 24 0186 0-372 20 , ,. . 2-day viable count • Multiplication = . . . .—r-r~, . initial viable count 8 6 f Respiration rate = mmoles Os/h io cells. % DNP concentration = 5 x io~ M. § Uncoupling factor = respiration rate with DNP respiration rate without DNP

The reason why cell division is blocked while other cell processes proceed unchanged (initially at least) is still obscure. The continued operation of glycolytic enzymes, containing functional sulphydryl groups, makes it unlikely that the oxidation of such groups was extensive. At high pO2 cell division could be blocked for lack of energy, if the generation of ATP was uncoupled from NADH oxidation. To test this proposition, a series of 30 D. G. Kilburn and others cultures at high pO2 was set up in 500-ml bottles rotated at 40 rev/min on a set of rollers. Previous measurements showed that this type of culture gave good equi- libration between the gaseous and liquid phases. After 2 days the respiration rate of the cells in these bottles was measured with, and without, added 2,4-dinitrophenol (DNP). Table 2 shows the results of these experiments. In a similar experiment, using static bottle cultures, the uncoupling factor was 2-0 for cells grown at 150 mmHg pO2 and 2-2 for cells grown at 290 mmHg pO2. Thus the uncoupling factors appear to be independent of pO2, which suggests that the degree of coupling at different pO2 is the same. If cells at high/>02 were appreciably uncoupled, one would not expect a large increase in respiration rate upon adding DNP.

Variation of carbohydrate metabolism with pO2

Fate of glucose. If it is assumed that the C02 and lactate produced during a culture are derived mainly from glucose and not from other components of the medium, it is possible to estimate the amount of glucose oxidized for energy production and the amount incorporated into the cells. Except for the culture at 320 mmHg pO2, about 90 % of the glucose consumed during the growth phase of a culture (i.e. to the peak of the growth curve) could be accounted for as lactate and C02. This is consistent with the findings of Papacon- stantinou & Colowick (1961). During the growth phase the assumption that only glucose is oxidized appears justified, because it was found that during this period the alkali consumed for pH control was equivalent to the increases in C02 and lactate. If significant amino acid oxidation had occurred this balance would have been upset. After the peak in the growth curve the amount of C02 and lactate produced some- times exceeded the amount of glucose consumed, indicating that other constituents of the medium were being oxidized. In the culture at 320 mmHg pO2 almost 40 % of the glucose consumed was incorporated into the cells. In this culture cell division was inhibited after the initial population had doubled, although the cell dry weight continued to increase to 1-3 times its normal value. This is in accord with the observations of Rueckert & Mueller (i960) and Brosemer & Rutter (1961), who found that the synthesis of macromolecules continued after cell division was halted. In the present work the soluble protein content per cell was the same as in cultures at lower pO2, so it is possible that the cell composition changed. This might relate to the finding of King, Edward & Bensch (1959), that under conditions in which protein synthesis and cell division were inhibited, L-cells accumulated considerable quantities of lipid. Alternatively, the cells may have begun to store glycogen. Lactate production. Figure 2 shows the lactate produced by cultures at different pO2 values. The rate of production is clearly a function of pO2; being very high at low pO2, but dropping as pO2 is increased. A sharp increase in rate occurs in the final stages of the cultures (except those at high pO2). This probably results from the breakdown of the citric acid cycle, and hence its inability to accept pyruvate. Effect of oxygen on growth of mouse LS cells 31 Failure of the citric acid cycle could be caused by an increased demand for its intermediates, perhaps for amino acid synthesis when amino acids in the medium approach exhaustion. Support for this hypothesis comes from an experiment in which glutamine was added to a culture after growth had stopped, shown in Fig. 3. Glutamine halted

Fig. 2. Variation of extracellular lactate concentration in cultures grown at controlled pO2 values: O, i-6 mmHg; •, 12 mmHg; A, 96 mmHg; •, 160 mmHg. The corresponding growth curves are shown in the lower part of the figure. lactate production and restored respiration, presumably by entering the citric acid cycle via glutamic acid and a-ketoglutarate, or alternatively by reducing the demand for TCA intermediates. There was no increase in the lactate dehydrogenase activity at the inflexion point, except in the culture at i-6 mmHg/>02. In the other cultures, lactate dehydrogenase must have been in large excess. Pyruvate production. Figure 4 shows the extracellular pyruvate concentration for three batch cultures at controlled dissolved pO2. With the exception of the experiment at i-6 mmHg pO2, the pyruvate in the medium was approximately constant. The build-up of pyruvate in the culture medium at i-6 mmHg suggests again that the 32 D. G. Kilburn and others

Fig. 3. Batch culture at 96 mmHg pO2) showing the effect of glutamine addition (shown by arrow) on lactate production: O, viable cells per mix io~6; •» lactate in medium, mmoles/1.

Days Fig. 4. Variation of extracellular pyruvate concentration in cultures grown at controlled pOt values: O, 1-6 mmHg; A, 96 mmHg; •, 320 mmHg. The corresponding growth curves are shown in the lower part of the figure. Effect of oxygen on growth of mouse LS cells 33 lactate dehydrogenase was operating at near maximum rate (the Km of lactate dehydrogenase for pyruvate is about o-i rruvi). Lactate to pyruvate (L/P) ratio. Assuming that lactate and pyruvate equilibrate with their environment by diffusion, then their concentrations in the external environment should relate to the intracellular values, and the ratio L/P should be proportional to the NADH/NAD+ ratio in the cytoplasm of the cell.

Fig. 5. Variation of extracellular lactate/pyruvate molar ratio in cultures grown at controlled pO2: O, i-6 mmHg; •, 12 mmHg; A, 96 mmHg. The corresponding growth curves are shown in the lower part of the figure.

Figure 5 shows the variations of L/P ratio for three batch experiments at different pO2. The high L/P ratio for the culture at i-6 mmHg pO2 indicates that the cells were relatively more reduced than in the cultures at higher pO2. The sharp increase in L/P ratio at the end of the logarithmic phase of growth reflects the rise in lactate production discussed previously. If this ratio is a true representation of the redox 3 Cell. Sci. 4 34 D. G. KiUmrn and others potential in the cytoplasm, one must conclude that highly reducing conditions occur at the end of the growth cycle. Figure 6 shows the variation of the L/P ratio with the pO2 at which the culture is grown. An average L/P ratio has been taken from the early part of the cultures (i.e. the flat part of the curves in Fig. 5). It can be seen that the L/P ratio is high only at very low pO2 and drops to a more or less constant value from 32-320 mmHg pO2. The values in the range from 50-100 mmHg pO2 may be slightly lower, i.e. more oxidized. It is of interest that the same cytoplasmic redox potential can be maintained at 320 mmHg pO2 as at 32 mmHg pO2. In contrast to the other cultures, there was no appreciable rise in the L/P ratio at the end of the culture grown at 320 mmHg^>02.

30*

100 200 300 (mmHg) Fig. 6. Variation of the average extracellular lactate/pyruvate molar ratio during exponential growth of LS mouse cells at controlled pO2 values.

DISCUSSION

The ability to cultivate mammalian cells at specific pO2 levels affords an excellent opportunity to study cellular metabolism and its regulation. From the present results it can be seen that carbohydrate metabolism is markedly influenced by^>02, particularly at the lower levels. In the intermediate range of pO2 values (32-96 mmHg) the exponential growth rate is almost constant and this is reflected in the respiration rate and pattern of carbohydrate metabolism. The interpretation of the observations on the variation of maximum cell populations is complicated by the lack of exact knowledge of the growth-limiting factors in the medium. On the basis of the studies of Griffiths & Pirt (1967) it seems likely that at least for the cultures in the intermediate range of pO2 values the primary factor limiting growth was glutamine, although other amino acids also may have become limiting. The variation in maximum Effect of oxygen on growth of mouse LS cells 35 cell population with pO2 (except at i-6 and 320 mmHg) probably reflects a change in the pattern of amino acid utilization in response to pO2. This might be associated with the production of specific proteins or mucopolysaccharides. An effect of this kind might be expected if, as has been suggested, the specialized functions of some cells are oxygen-dependent (Brosemer & Rutter, 1961; Paul, 1965). We have some evidence that extracellular protein production was initiated in continuous culture when the pO2 was less than 80 mmHg. This phenomenon was not detected in batch cultures, suggesting that the function is also dependent on the growth rate. In the culture grown at i-6 mmHgpO2 the respiration rate measured by the Rank electrode was higher than that of cells grown in the intermediate pO2 range, suggesting that the cytochrome content of cells grown at i-6 mmHg was higher than normal. Increases in the cytochrome contents of bacteria and yeast in response to low pO2 have been reported by Herbert (1965) and Moss (1956). The pO2 value at which respiration rate is half maximal has been compared to the K,n of the Michaelis-Menten equation for enzyme kinetics (Longmuir, 1957; Froese, 1962). Reported values of 'Km' for oxygen for animal cells range from o-1 to 2-0 mmHg pO2', approximately the same as the range of the Km for oxygen with cytochrome oxidase (Hayaishi, 1962). Johnson (1967) has shown that the half-maximal respiration rate of bacteria is a function of the growth conditions and is often not related to an enzyme Km. These arguments apply equally well to the present animal-cell cultures at low pO2, hence one cannot define the saturation level of the enzyme system coupled to oxygen. It can only be concluded that at i-6 mmHg pO2 (and possibly 12 mmHg pO2) the cellular respiration rate was oxygen limited. Although the culture at 1-6 mmHg pO2 grew at a reduced rate, replication was still exponential, whereas without pO2 control, cultures grew linearly after reaching a very low pO2. This demonstrates an important distinction between oxygen-limited cultures. In the first case the controlled pO2 is constant but at such a low level that growth rate is limited. Nevertheless, each succeeding generation of cells encounters the same pO2 conditions and hence growth is exponential. The amount of oxygen consumed per cell remains constant, while the amount of oxygen consumed by the culture increases as the cell number increases. In the second case the cells depress the pO2 to a very low growth-limiting level, after which the amount of oxygen transferred to the medium remains constant. As the cell number increases, the amount of oxygen available per cell decreases, and the growth rate drops progressively, with the result that the cell number increases linearly. The results of the experiments at high pO2 are in general agreement with previous reports in the literature but do little to clarify the mechanism by which oxygen blocks cell division. Rueckert & Mueller (i960) found that the replication of Hela cells grown with 95 % O2 in the gas phase was inhibited after 24 h; DNA, RNA and protein synthesis continued for a further 12 h, after which the viability dropped sharply. In contrast to the present results, they found that glucose was converted entirely to lactate. Perhaps in their experiments the citric acid cycle was inoperative due to

3-2 36 D. G. Kilburn and others inactivation of lipoic acid by oxygen, as suggested by Thomas, Neptune & Sudduth (1963). This clearly did not occur at the lower pO2 used in the present work, because the respiration rate (and presumably the citric acid cycle activity) was higher than normal. The increase was in proportion to the increase in cell size so that, on a weight basis, respiration was unchanged. The rapid decrease in viability after 36 h reported by Rueckert & Mueller was not observed, but this again can probably be attributed to the different pO2 used. Brosemer & Rutter (1961) found no change in the pattern of glucose utilization when AH cells were incubated with 45 % oxygen in the gas phase. Cell division was blocked but protein synthesis continued. It can be seen that there is a need for more research on the effect of moderately high pO2. The mechanisms might be much more subtle than those acting at high pO2, e.g. complete oxidation of sulphydryl groups appears to be ruled out at moderate pO2. It would be of interest to determine the virus-producing capacity of cells grown at high pO2, and also to study the effect of moderately high pO2 on virus replication. This might show whether functions such as nucleic acid synthesis are abnormal. Brosemer & Rutter showed that 32P incorporation into nucleic acid was unchanged but they did not show that the nucleic acid was functional. If pO2 operates only on the cell division mechanism one might expect virus replication to be unhampered by hyperbaric pO2. The authors wish to thank the Wellcome Foundation for their support of this project.

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