NOTE BREVI
Non-destructive computerized assessment of yeast metabolism
evidences memory and recovery ability following coherent
UV irradiation
M. COSTATO(1), L. FERRARO(2), M. MILANI(2)(*) and S. MORSELLI(1)
(1) Dipartimento di Fisica, Università di Modena - Via Campi 213-a, 4100 Modena, Italy (2) Dipartimento di Scienza dei Materiali, Università di Milano
Via Emanueli 15, 20126 Milano, Italy
(ricevuto il 19 Maggio 1997; approvato il 21 Luglio 1997)
Summary. — The dynamics of long-run glycolytic metabolism is assessed in
nitrogen-laser-(337.1 nm)–irradiated yeast cells for different doses at various time lags (from one to fourteen days) after the insult. A non-monotonic memory effect is evident from the enhancement in metabolic-enzyme activity on the first and the last day of observation and inhibition on the tenth day. Recovery from inhibited to normal state is shown to occur upon a subsequent “treatment” of insulted cells with a HeNe laser (632.8 nm) radiation with no noticeable difference depending on its state of coherence.
PACS 87.80 – Biophysical instrumentation and techniques. PACS 87.90 – Other topics in biophysics and medical physics.
UV-induced damage on cells is an up-to-date subject in order to assess the various facets associated with the so-called “ozone layer depletion”. Most studies are concerned with the cell genetic aspects where, e.g., i) ring openings, base fragmentation, single-and double-strsingle-and breaks are analyzed, ii) DNA repair time is assessed single-and quantitative evaluation of the number of damaged cells vs. number of incident photons are used to validate threshold models [1]. Furthermore not only chromosome structural rearrangements are among the important genetic damage in cells exposed to radiation, but it also seems that these damages are directly related to the critical event associated with cell killing, mutation and malignant transformation [2, 3]. However, despite the extensive investigations, the mechanism by which energy-absorbing process is related to DNA damage induction still remains an open problem [3].
(*) Also at INFM (Istituto Nazionale di Fisica della Materia).
On the other side, DNA is functionally more stable than two other classes of cellular macromolecules, RNA and proteins. It is understood that this stability can be attributed to the double-helical structure, which carries information in duplicate, and to the fact that in order to transfer information, one only needs the primary structure of DNA [4].
Moreover UV photons are not the only source of DNA modification, in fact DNA is a highly reactive species, and therefore it is the target of numerous physical and chemical agents, such as heat and the activated oxygen species generated during oxidative metabolism. Among the enzymes involved in the various DNA repairs there are photo-activated one (photolyases). These enzymes catalyze a unique reaction in which the energy of light is utilized to repair pyrimidine dimers in DNA. The involved photon wavelength ranges from 300 to 500 nm [4], therefore, low-energy UV photons may be good candidates for DNA-repair too. It must be stressed that the identification of the above enzymes requires crude cell extracts.
From the above it is understood that the investigation of the damage produced by ionising radiation by current methods (and used by most investigators) mostly requires the cell dissection. Cell life seems to be maintained only for the studies of population survival. This means that by standard methods the cell life is irreversibly stopped (at a given time after the insult) and no further information can be gathered on its time evolution, including the important genetic effects which dynamically accompaign the cell life. Moreover the interesting information on cellular dynamics are not only those associated with the integral over the time domain, but—when possible—also the temporal follow-up of metabolic parameters.
Here we present a totally non-invasive method which permits to follow up continuosly the cell life way after the moment of insult (UV-A radiation) and to keep track of its dynamics from the point of view of its energetic needs, giving rise to a wealth of novel information. In fact we use the time evolution of the cells glycolytic metabolism as a direct testimony of the energy needs of the cell, and as an evidence of the performance of the relevant enzymes controlling this metabolism.
UV irradiation. We have taken yeast cells (Saccharomyces cerevisiae); in
particular batch cultures are followed up where the microorganism are growing under controlled conditions requiring also aseptic techniques to keep the culture pure. We have used a number of samples in the typical cuvettes for use in spectrophotometry (4 mL) each containing the same number of cells (12 3 107). The concentration is the typical one in biology, and it is sufficiently low to validate the negligibility of multiple light scattering effects, so that the delivered and absorbed doses can be calculated as if the cells were deposited on a single layer orthogonal and fully illuminated by the impinging homogeneous photon flux. Radiation was obtained from a nitrogen laser (337.1 nm wavelength, thus in the UV-A range) delivering 1 mJ in 0.5 ns pulses with a repetition rate of 1 Hz (average power 1 mW, peak power 2 3 106W, average power density 0.25 mW/cm2
, peak power density 0.5 3 106W/cm2) with a spot light identical to the cuvette cross-section. Exposure times ranged from five to twenty minutes. The laser was built in our laboratory according to the scheme of Blumlein [5], it works at atmospheric pressure, with a 1 m cavity length and 3.2 3 1025m3
cavity volume. Irradiation procedure on all samples was concentrated on a single day every time. We have thereafter mantained the cells (in parallel to controls which did undergo exactly the same above procedure except irradiation) in their original suspension
medium (i.e. simply distilled water), in the dark, at room temperature, all in the same cabinet of our laboratory, for a long period of time up to 14 days for each experiment set.
Metabolic investigation. After checking that the cell population parameters,
controls and irradiated ones, did not change in this time interval with respect to their initial value (the day of insult) and verifying under the microscope that no samples did exhibit a state of evident stress (at least at the morphological level) we have studied the dynamics of their glycolytic (a-D-glucose was used as a nutrient) metabolism on different days (1, 3, 5, 8, 10, 14) after the irradiation in sealed flasks at constant volume. In particular the culture is incubated at a given temperature; the pH of the medium is known and often controlled using buffers. The ad hoc computerized apparatus used [6] continuously follows the time evolution of the metabolic process, collecting data (measure of pressure increase in flasks) every second in a procedure lasting several hours, without any external perturbation to the cell population involved. Such a novel technique permits to evidence several interesting features of cell metabolism, besides those here described, e.g., the onset of a population oscillatory behaviour and chiral properties depending on the nutrient used [6]. Furthermore, preliminary investigation shows that this technique can as well be succesfully applied to lymphocyte cultures. Finally it is worth mentioning that it allows to gather on-line information on the cell dynamics (including rapidly changing properties) in contrast with the currently used techniques. In fact it is known that the glycolytic metabolism produces CO2 which mainly passes from the liquid to the gaseous phase: in the present case CO2molecules are added in the gaseous phase of the sealed flasks where the cells are suspended in the liquid culture medium, producing a measurable pressure increase [6]. We have double-checked it by sampling the gaseous phase at various times of the metabolic process, through a mass spectrometer. Furthermore we have also found that the nitrogen-to-oxygen ratio did remain constant, a fact we here interpret as follows: the metabolic process under study did actually undergo mainly an anaerobic process (fermentation with no oxygen consumption). This permits to greatly simplify the picture of the relevant energy-dependent enzymatic mechanisms at work in the cells excluding aerobic-mechanism contribution to metabolism [7]. Thereafter it will be implied that when we consider an increase in pressure (with respect to controls) as a result of the CO2 production, it is consequence of an enhancement of the metabolic activity with respect to controls, which is directly related to the (increasing) energy needs of the cell population. This consideration is also supported by cell counting and sugar consumption monitoring.
The full set of techniques and results will be published elsewhere: let us here summarize the most relevant facts.
Memory effect: the cells, although perfectly surviving (as checked both by
microscopic morphological appearance, and by vitality), do exibit a long-time memory of the irradiation by showing a metabolic activity different from controls, this effect lasting up to the last day (the fourteenth).
Non-monotonic effect: the metabolic activity vs. controls, has different fundamental
moments. Maximum increase in metabolic activity (up to about 20%) occurs the first and last day (the 14th). In the range from the 3rd to the 8th day, irradiated samples tend to behave like the controls, converging about the 5th day. On the 10th day they exhibit a minimum of the metabolic activity with a marked inhibition with respect to
Fig. 1. – Per cent variation in irradiated cells (three), with respect to controls, of the measured pressure due to CO2 production in the glycolytic metabolism of nitrogen laser UV-irradiated
(337.1 nm) yeast cells (Saccharomyces cerevisiae) as a function of the day after the insult in which the metabolic run was made. Points refer to the total CO2produced after 104s from the start of the
metabolic run. (!absorbed photons per cell, y = 267 3 107absorbed photons per cell.)
controls down to about 2 25%, which we shall call “state of highest crisis” (see fig. 1). In all days except the 10th the effect seems to be dose independent (in the range from 53.4 3 107 to 267 3 107 UV-A photons absorbed per cell). On the other hand, the occurrence of highest crisis on the 10th day has been double-checked within a three months interval, therefore we have decided to go into detail concerning this range. We have made special runs on this very day, increasing the range of UV-A doses up to 801 3 107 photons absorbed per cell, and we found that on the 10th day the CO2 production, which is proportional to the metabolic activity, (typically at 6 000 seconds from the beginning of the fermentation run) has a structured (inhibited) response with two minima at 160.2 3 107and at 534 3 107photon absorbed per cell as shown in fig. 2 full curve.
HeNe irradiation. There is a wide literature in various facets of biophysics, biochemistry and medicine that indicate repair processes being put at work on injured biosystems by a subsequent irradiation by monochromatic light in the red portion of the visible spectrum (usually an HeNe laser, 632.8 nm, at low power, low power
Fig. 2. – Pressure variation due to CO2production in the glycolytic metabolism of nitrogen laser
UV-irradiated (337.1 nm) yeast cells (Saccharomyces cerevisiae) as a function of the absorbed energy per cell, as measured on the tenth day after the insult. The lower and upper curves refer to the total CO2produced, respectively, after 4000 s and 6000 s from the beginning of the metabolic
run. Full lines refer to cells which did undergo UV irradiation only. The broken line refers to cells which were originally UV irradiated, and on the 10th day were also exposed to HeNe radiation (632.8 nm, 5600 3 107absorbed photons per cell) just before the metabolic run.
densities and low energies delivered). However, it is not easy to correlate different results, and it is not often easy to reproduce published data, for which a complete consensus is not yet at hand [8]. One of the major problems in these light-driven recovery investigations is the definition of the “pathological” state which of course implies difficulties in defining the recovery potential of a “therapy”. In this experiment we have a pretty controllable and reproducible induction of stress (or pathology) together with a wide range of monitoring parameters. Consequently we have checked whether that was also the case on our set-up of UV-A–irradiated cells on the day of highest crisis, by irradiating on that very day our samples with a HeNe laser (0.7 mW/cm2, irradiation time 20 minutes, 5 600 3 107 absorbed photons per cell), and obtained the results shown by the broken line of fig. 2. Here it is seen that, well beyond the measure incertitude, the two minima (at 160.2 3 107
and at 534 3 107 photon absorbed per cell) in CO2 production (thus in the metabolic activity) disappear as an
Fig. 3. – Per cent variation with respect to controls of the measured pressure due to CO2
production in the glycolytic metabolism yeast cells (Saccharomyces cerevisiae) on the tenth day after the insult as a function of time from the beginning of the metabolic run. != cells UV-irradiated with a dose of 267 3 107 absorbed photons per cell ten days before the metabolic
assess. The full line is a linear best fit. b 4 the same cells as above, which on the 10th day after the UV insult were also exposed to HeNe radiation (632.8 nm) made partially incoherent (1620 3 107
absorbed photons per cell) before the start of the metabolic run.{= the same cells as above [!] which on the 10th day after the UV insult were also exposed to HeNe radiation (632.8 nm) in its normal coherent state (1620 3 107absorbed photons per cell) before the start of the metabolic run.
The full line is a linear best fit.
effect of the HeNe irradiation. The present experimental set-up also allows us to investigate the possible role of radiation coherence. We have made our laser source partially incoherent, as described in [9] where as an effect of this procedure the power delivered in both the coherent and the incoherent state was reduced down to 0.8 mW (thus 0.2 mW/cm2). We have repeated the experiment for longer periods of the metabolic activity (up to 7 hours) in order to have an even better spectrum of the response of the system. Results are summarized in fig. 3 where on the 10th day it is seen that the activity for UV-A–irradiated samples (lower curve, 267 3 107 absorbed photons per cell) is highly depressed up to more than 30% with respect to controls, and that in a metabolic long run it gets nearer to the normal state, but still remains inhibited up to about 15% [10]. On the contrary, the subsequent “treatment” with HeNe
laser irradiation (upper curve), made on the 10th day after the UV-A insult, makes the metabolic activity resume the normal level of controls (of course in the same interval of metabolic run), with no relevant difference between coherent or non-coherent radiation.
Conclusions. The reported experiment shows the effectiveness of UV-A radiation in
the modification of the metabolism of yeast cell cultures and provides a good laboratory set-up for cross correlated quantitative analysis both on the system itself and on its response to various external parameters. The above is germane to more sophisticated studies of mammalian cells where comparison can be made between the effectiveness of UV-A radiation and UV-B and UV-C. Finally it proves to be a good tool for the investigation of the energy balance in cells from immune systems as a quantitative index of pathological states.
* * *
G. BARONI, A. CONTI, S. COZZI, S. HUGES and F. SALSI, are thanked for providing
support and useful discussions.
R E F E R E N C E S
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[10] All reported data in photons absorbed per cell can be derived from fluence. In the UV-A case the cells were exposed to about 0.15 kJ/m2
3 min, while in the HeNe case to about 0.042 kJ/m2
3 min and 0.014 kJ/m23 min respectively. Figures on photons absorbed per cell were preferred because our aim is to acheive a quantitative analysis of the response of the metabolic process to an external stimulus at a single-cell level.
