METABOLIC RESPONSES OF CULTURED CELLS TO OXYGENATED PERFLUOROCARBON

Abstract

Protoplast-derived cells of albino Petunia hybrida cv. Comanche were used as a model, nonphotosynthetic, eukaryotic plant system to study changes in (1) the rate of oxygen consumption as measured by a Clark-type oxygen microelectrode, (2) mitochondrial membrane potential (MMP) as assessed by Rhodamine 123 fluorescence, and (3) intracellular activities of superoxide dismutases (SOD, EC 1.15.1.1) and catalases (CAT, EC 1.11.1.6), following culture for up to 14 d in aqueous nutrient medium overlaying oxygen-gassed perfluorodecalin (Flutec® PP5; F2 Chemicals, Preston, UK). The mean (±s.e.m., n=7) rate of oxygen consumption of Petunia cells after 24 h of culture in the presence of oxygenated PFC was 14.3±1.6 μmol O₂ ml⁻¹ min⁻¹, compared to 9.7±0.8 μmol O₂ ml⁻¹ min⁻¹ for untreated (control) cells (P<0.05). Similarly, culture of cells with oxygenated PFC for 24 h resulted in a significant (P<0.05) increase of over 50% in the mean MMP, compared to control. Culture of protoplasts with oxygenated PFC also produced significant (P<0.05) increases in both mean SOD and CAT activities after 3–7 d of culture, the former comparable to that reported previously for protoplasts of Salpiglossis sinuata cultured with oxygenated PFC.

INTRODUCTION

Inert, respiratory gas-dissolving perfluorochemical (PFC) liquids can regulate the supply of oxygen and carbon dioxide in both prokaryotic and eukaryotic (including human) cultured cells, but the underlying biochemical changes in such cells, particularly those relating to oxygen-sensitive pathways, are poorly understood. The use of PFCs as oxygen carriers enhances mitosis during the culture of isolated protoplasts of several plant species [[1]], with subsequent increases in biomass production from protoplast-derived cells. Although oxygen availability is vital in aerobic systems, its reduction can generate highly reactive oxygen species, such as superoxide (O₂⁻), which can have deleterious effects on cellular metabolism. However, there have been few studies on changes in cellular metabolism or on the activities of enzymes responsible for removal of reactive oxygen species during culture of cells in the presence of PFCs. Aerobic organisms have evolved enzyme systems for scavenging oxygen radicals, thus protecting against oxidative damage. One such group of enzymes, the superoxide dismutases (SOD), react with superoxide anions to produce hydrogen peroxide that, in turn, is converted by catalases (CAT) to H₂O and oxygen. This study has evaluated the beneficial effects of PFC-facilitated oxygen enhancement on changes in (1) the rate of oxygen consumption, as assessed by a Clark-type oxygen microelectrode, (2) mitochondrial membrane potential (MMP) assessed using Rhodamine 123 fluorescence, and (3) cellular SOD (EC 1.15.1.1) and CAT (EC 1.11.1.6) activities.

MATERIALS AND METHODS

Source Material and Assessment of Mitotic Division in Protoplast-Derived Cells

Protoplasts were isolated enzymatically from cell suspensions of albino Petunia hybrida cv. Comanche [[2]]. Protoplasts were suspended at a density of 2×10⁵ ml⁻¹ in 3 ml of aqueous KM8P medium based on the formulation of Kao and Michayluk [[3]], as modified by Gilmour et al. [[4]], and cultured in 30 ml bottles either alone (control), overlaying 6 ml of ungassed perfluorodecalin (Flutec® PP5; F2 Chemicals, Preston, UK), or overlaying 6 ml of oxygen-gassed (10 mbar; 15 min) perfluorodecalin. Cell growth was monitored for up to 14 d of culture (22±2°C, dark). Division frequency was measured as the number of viable protoplast-derived cells, determined using fluorescein diacetate [[5]], which had undergone mitotic division.

Measurement of Oxygen Consumption

The rate of oxygen consumption of protoplast-derived cells was determined using a Clark-type micro-oxygen electrode (737 Microelectrode; Diamond General Development Corporation, Ann Arbor, MI) attached to an oxygen meter (Model 781; Strathkelvin Instruments, Glasgow, UK). Assessments were undertaken following a 24 h culture period. Cultures containing viable protoplasts were analyzed polargraphically and changes in oxygen concentrations assessed over a 5 min period. Cellular oxygen consumption was assessed in protoplast-derived cells cultured in medium alone (controls) and in the presence of ungassed or oxygenated PFC.

Assessment of MMP

Changes in MMP were assessed in protoplasts (density 1×10⁶ ml⁻¹) following 24 h culture in medium alone (controls) and in the presence of nonoxygenated and oxygenated PFC. Protoplast-derived cells were equilibrated for 60 min with a 10 μg ml⁻¹ solution of Rhodamine 123 (Sigma, Poole, UK), washed in CPW13M solution [[6]] and analyzed by flow cytometry (EPICS 541; Coulter Electronics Ltd., Luton, UK). Protoplasts were exposed to a 100 mW of 488 nm light and fluorescence emissions (550–600 nm) detected with a photo-multiplier tube, converted to digital format and analyzed by computer. Histograms of fluorescence intensity per protoplast were constructed and the mean Rhodamine 123 fluorescence per protoplast calculated.

SOD Assay

SOD was extracted from protoplast-derived cells, following freeze-thaw lysis, after 1, 3, 7, and 14 d of culture and assayed spectrophotometrically (560 nm). Briefly, 6×10⁵ protoplast-derived cells were harvested by centrifugation and the pelleted cells resuspended in 1.0 ml of sterile distilled water. Cells were frozen for 1 h at −20°C, thawed, and cellular debris removed by centrifugation. One hundred μl aliquots of supernatant were added to a reaction mixture (3 ml) consisting of 50 mM K₂HPO₄ 3H₂O–KH₂PO₄ buffer (pH 7.8), 0.1 μM EDTA, 3.9×10 μ⁻³ M xanthine, 5.03×10⁻³ M nitro-blue tetrazolium and sufficient xanthine oxidase [typically 0.25 units (U) per assay] to produce a reaction rate of 0.025 absorbance U min⁻¹. The baseline assay was followed in a cuvette of 1-cm path length at 25°C. One U of SOD enzyme activity was defined as that required to inhibit the reduction of nitro-blue tetrazolium by 50% (0.0125 absorbance U min⁻¹) through the xanthine-xanthine oxidase system [[7]]. SOD concentrations were determined using a standard curve generated using commercially available SOD (Sigma, Poole, UK) under identical conditions.

Catalase Assay

Catalase was extracted from protoplast-derived cells following cell lysis, as described for the SOD assay. Catalase activity was measured spectrophotometrically at 240 nm [[8]]. One U of enzyme activity was defined as that needed to decompose 1 μM of H₂O₂ min⁻¹ at 25°C. The reaction was followed in a 1-cm path-length cuvette containing 3 ml of a reaction system consisting of 100 μl sample, 3.5 μM H₂O₂ in 50 mM phosphate buffer (pH 7.8). A standard curve was constructed using commercially available catalase (Sigma).

Statistical Methods

Means and standard errors (s.e.m.) were used throughout; statistical significance between mean values was assessed using conventional ANOVA and Student's t-test, as appropriate [[9]]. A probability of P<0.05 was considered significant.

RESULTS

The mean (n=7) rate of oxygen consumption of Petunia cells after 24 h of culture in the presence of oxygenated PFC was 14.3±1.6 μmol O₂ ml⁻¹ min⁻¹, compared to 9.7±0.8 μmol O₂ ml⁻¹ min⁻¹ for untreated (control) cells (P<0.05; Table 1). There was no significant difference in the rate of oxygen consumption when protoplast-derived cells were cultured with ungassed PFC or in medium alone Table 1.

Table 1. Oxygen Consumption Rate in Protoplast-Derived Cells of P. hybrida Cultured with Oxygenated PFC

Treatment	Oxygen Consumption Rate (O2 mol ml^−1 min^−1)
Medium alone (control)	4.4±0.8
PFC	5.1±0.6
PFC+O2	4.7±0.6
Media+protoplasts	9.7±0.81^*
PFC+protoplasts	9.6±0.71^*
PFC+O2+protoplasts	14.3±1.61^*

Values are mean±s.e.m., n=7 throughout.

^*P<0.05 compared to control (medium alone) mean value.

Culture of protoplast-derived cells with oxygenated PFC for 24 h resulted in a significant (P<0.05) increase of over 50% in the mean (n=7) MMP, expressed as percentage fluorescence, compared to that for cells in unsupplemented medium (38±4% fluorescence) or cells cultured in the presence of ungassed PFC (40±1% fluorescence).

Protoplast-derived cells cultured in the presence of oxygenated PFC showed a significant increase (P<0.05) in mean (n=5) SOD activity during the initial 3 d of culture (5.1±0.3 U mg⁻¹ protein min⁻¹), compared to ungassed PFC (1.9±0.2 U mg⁻¹ protein min⁻¹) or medium alone (0.9±0.2 U mg⁻¹ protein min⁻¹). Mean SOD activity declined progressively over the subsequent 11-d culture period and, after 14 d, was not significantly different from the mean initial value (data not shown).

A similar increase in mean (n=5) catalase activity was observed when protoplast-derived cells were cultured with oxygenated PFC. Such changes in catalase activity were significant (P<0.05) after both 3 d (0.44±0.01 U mg⁻¹ protein) and 7 d (0.52±0.01 U mg⁻¹ protein) of culture, compared to the corresponding values for ungassed PFC and unsupplemented medium (0.35±0.01 and 0.38±0.01 U mg⁻¹ protein, respectively).

DISCUSSION

The present results demonstrate that culture of protoplast-derived cells of P. hybrida with oxygenated perfluorodecalin stimulated the rate of cellular oxygen consumption, mitochondrial function, and the activity of both SOD and CAT. The observed increase in oxygen consumption was consistent with an enhanced cellular respiration rate in the presence of increased oxygen supplied by the PFC. The increase in MMP in response to culture with oxygenated PFC reflects increased energy metabolism in protoplast-derived cells. The method employed here was based on mitochondrial uptake of the lipophilic, cationic fluorochrome, Rhodamine 123, which is modulated by the transmembrane potential. Previous studies have utilized a similar approach for evaluating mitochondrial respiratory activity in yeast [[10]] and isolated mammalian nerve cells [[11]]. However, the effects on mitochondrial function of exposing cells to oxygenated PFC have been relatively poorly studied. Branch et al. [[12]] reported that the MMP and the rates of both ATP synthesis and ADP-stimulated respiration in mitochondria from mammalian liver cells were decreased following exposure to a commercial PFC emulsion consisting of perfluorotributylamine emulsified with the commonly used poloxamer surfactant, Pluronic® F-68. While this earlier study did not distinguish which of the components of the emulsion were responsible for these changes, it is likely that the surfactant was an active factor, since preliminary experiments using protoplast-derived cells of P. hybrida have similarly demonstrated a decrease in MMP following exposure to Pluronic® F-68 (Wardrop et al., unpublished observations). Further studies are needed to determine how mitochondrial function may be altered by exposure to increased oxygen flux in the presence of surfactants, such as Pluronic® F-68, that can have beneficial effects on plant cell growth in vitro, probably through alterations in membrane functions [[13]].

The present results, showing that protoplast-derived cells cultured in the presence of oxygenated PFC exhibited increased SOD and CAT activities that were maximal during the earlier stages of culture, provide further evidence for a biochemical response to the oxygen delivered by the PFC. In this respect, previous preliminary studies [[14]] have demonstrated that SOD activity was correlated closely with available oxygen and that biosynthesis of this enzyme in protoplasts of Salpiglossis sinuata can be induced by high levels of molecular oxygen. The present experiments significantly extend this earlier work by providing data on changes in both the SOD and catalase oxygen-detoxifying enzyme systems in response to culture of protoplast-derived cells with oxygenated PFC.

It has been noted previously [[14]] that the induced SOD activity arising from exposure of cultured plant cells to oxygen supplied by PFC appears to be sufficient to protect protoplasts and protoplast-derived cells from oxidative damage arising from any free radicals generated through prolonged exposure to increased levels of oxygen supplied by the PFC. Thus, the present approach enables a beneficial supply of oxygen to be maintained, resulting in improved in vitro respiration and subsequent cell division and growth. Indeed, related studies have shown that intact, fertile plants can be regenerated from protoplast-derived cells cultured for up to 21 days with oxygenated PFC [[15]], indicating that this cultural strategy has no long-term, deleterious effects on development.

ACKNOWLEDGMENTS

This study was supported by F2 Chemicals Ltd., UK (Contract FC/S/004). The authors thank Mr. N. W. Blackhall for assistance with flow cytometry.