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PIXE microanalysis in human cells: physiology and pharmacology
Nuclear Instruments and Methods in Physics Research B75 (1993) 511-517 North-Holland
PIXE microanalysis Ph. Moretto,
Y. Llabador,
Centre d’Etudes Nu&aires
Beam Interactions with Materials 8 Atoms
in human cells: physiology and pharmacology R. Ortega,
de Bordeaux-Gradignan,
M. Simonoff
and L. Razafindrabe
33175 Gradignan cedex, France
The micro-PIXE technique has been regularly carried out for more than two years to provide elemental distributions in human cells. Using this technique in the framework of cellular biology, two research axes have been developed: the cellular pharmacology f chemotherapeutic agents and the physiology of ionic cellular exchanges. These studies are based upon in vitro experimental models of human cells, either under the form of isolated cultured cells or as part of well-structured tissues. The aim of this paper is to present the experimental procedures and methodological aspects of cellular and subcellular quantitative mapping. Cell processing, identification of intracellular structures, quantitatives results and beam damage will be discussed and illustrated by examplesissuing from the above-mentioned studies.
1. Introduction
During the three last decades, the development of modern molecular cell biology has coincided with technological improvements in cell exploration techniques. The availability of molecular markers, either radioactive or fluorescent, and the use of the specific antibodies for immunolabeling, are certainly important steps forward in this advance. The probing of tagged molecules within cells has now become a routine task for immunofluorescence, flow cytometry, microspectrofluorometry [l] or autoradiography methods. But there is a field of research where we have still to make progress: the microanalysis of subcellular elemental chemical distributions. The detection of intracellular essential trace-elements or toxic heavy metals requires a compromise between spatial resolution and sensitivity that has been achieved by very few techniques. Among them, mass spectrometry and X-ray fluorescence, carried out with ion or electron microbeams are the basic principle of electron microprobes, SIMS [2] and nuclear microprobes. The use of an ion beam is certainly a great advantage of the micro-PIXE technique. This allows of course focusing for microbeam utilization, but also the simultaneous application of associated techniques such as RBS, STIM [3] and secondary electrons detection. Conjointly applied, these methods can provide valuable help in concentrations normalization, organelle localization and fast topography determination of analysed regions. In most studies of experimental biology, the human cell is used as an experimental model. The purpose is to check in vitro, consequently in reproductible conditions, a cell function or the effect of molecular species on cell metabolism. This can be achieved culturing an 0168-583X/93/$06.00
0 1993
Elsevier
Science
Publishers
eukariotic cell line or on highly differentiated cells maintained in good survival conditions following recent sampling. We have used these two approaches during the last two years in order to develop hvo fields of investigation using the micro-PIXE analysis: The cellular pharmacology of cytotoxic drugs used in chemotherapy and the physiology of ionic cellular exchanges. The aim of this report is to review the specific experimental difficulties encountered in the microanalysis of isolated cells. The applied solutions will be discussed on the basis of examples extracted from ours experiments.
2. Cellular
pharmacology
Cancer chemotherapy is founded upon the cytotoxic action of drugs on malignant cells. But this effect is not entirely specific and secondary toxicity for the whole organism is a great limiting factor of treatment efficiency. This is the reason why a careful determination of doses and a good understanding of its mechanism of action are necessary. The last stage before the clinical experimentation is constituted by an in vitro exposure of cultured tumor cells to the drug. Information about the DNA-drug interaction and a precize quantification of the drug uptake can then be achieved with macroanalytical techniques [4]. But the survey of the molecule diffusion within the cell remains a major difficulty. This point becomes of extreme importance when a resistance of tumor cells to the drug arises during therapy. This emergence can therefore limit the clinical usefulness of the antineoplasic agent. Several mechanisms which can affect the drug distribution within the cell have been reported to contribute to this resistance: Decrease of drug accumula-
B.V. All rights reserved
VIII. MICROBEAM PIXE
512
Ph. Moretto et al. / Human cells: physiology and pharmacology
tion either due to a more efficient drug efflux or a lower membrane permeability and detoxifying action founded upon drug chelation. In all cases, the drug localization, especially in the nucleus region, and the eventual effects on essential trace elements distribution have to be elucidated using elemental mapping. From this point of view, a simple comparison between a sensible and a resistant cell line can provide valuable information. For that purpose, we have applied the micro-PIXE analysis to investigate the properties of cisplatin, an important antineoplasic agent, used in ovarian and testicular cancer treatment. A human ovarian adenocarcinoma cell line, IGROVl-p, has been cultured for in vitro drug exposure [5]. This sensitive line was compared with a selected drug resistant subpopulation, IGROVl-1~. Experimental details and first results have been already published elsewhere [6].
3. The physiology of ionic cellular exchanges The plasma membrane of the cell is a selectively permeable barrier between the cytoplasm and the extracellular fluid. This membrane is permanently crossed by ion currents under the effect of electrochemical gradients [7]. During this passive diffusion, when the membrane is at rest, sodium gets into the cell whereas potassium leaves it, crossing the wall. In order to maintain the ionic concentrations of the intracellular milieu at a constant level, mechanisms of active transport (Na/K ATPase) are acting to reject sodium from the cell compartment. These exchanges are a vital function for the cell. Their regulation is extremely complicated on account of the numerous acting factors. In order to investigate these cellular pathways, electrophysiological studies are generally carried out in vitro on isolated cells by mean of microelectrodes introduced in the cytoplasm. Electric parameters, such as membrane potential or conductance, can then be measured with the aim to deduce information about ionic transfers [8,9]. We have used the monocellular epithelial layer of the human amniotic membrane, a classical model developed by electrophysiologists, to investigate the stabilizing action of magnesium on membranes [lo]. PIXE is indeed one of the few microanalysis multielemental techniques which allow one to reveal simultaneously the distribution of most minerals involved in cellular transmembrane currents. The accurate measurement of minerals concentrations in the intracellular compartment after incubation in physiological fluids permits one to check the subsequent migration of ionic species. The effect on the membrane permeability of extracellular fluid components such as magnesium salt or other nutrients can then be studied.
4. Cell preservation The aim of in vitro experimentation is to study the cell in a metabolic state as close as possible to the in vivo situation. We particularly have to reduce the consequences of the initial trauma of sampling procedures. From this point of view, cell culture seems to offer the best guarantees. The explanted cells are only a precursor of those which are actually used for assays. The fact that they meanwhile replicate is certainly the best viability test. Continuous cell lines, such as the neoplasic cell line we studied, proliferate as a monolayer in standardized conditions. Minor deviations would significantly alter their growing kinetic. To avoid extracting the cell from their growing medium, we cultured them directly on the polymer film we intended to use for irradiation, a very thin (15 ug/cm’) sterilised FormvarR film. In order to insure the cell growth, the substrates were precoated with collagen, an attachment factor. Physical and chemical methods, generally used to make the surface of cultur polymer flasks wettable and suitable for cell attachment, could not be applied here on account of the substrate fragility. The epithelial cells of the amniotic membrane are, contrary to cancer cells, fully differentiated with a main natural function of mother to foetus exchange. They would consequently normally not proliferate. They were simply maintained in survival conditions by the interaction with their initial amnion tissue and especially by the experimental conditions of incubation: temperature, oxygenation and isotonic&y of bathing fluid. In these electrophysiological studies, cell preservation has a particular meaning. The goal is to preserve mainly the exchange function, even though the intracellular ionic concentrations are deeply modified. These epithelial cells belongs to a class of leaky membrane. In those cell species, the passive diffusion is the main component of ionic transmembrane transport. The intracellular homeostasis consequently depends on the ionic concentrations in the extracellular milieu. As a general rule, the physiological shock occurs during the first minutes of incubation. The membrane is submitted to violent ionic unbalance and has to be left in the medium for more than half an hour to retrieve a normal at rest potential. We noticed during this essay a potassium release from the cells whereas sodium and chlorine was found to penetrate into the cell compartment [ll]. Following the essays, the second critical stage is to bring the cells under vaccum for analysis. The cells have then first to be fixed. Cryofixation, according to all contributors, is assumed to give the best results [12]. But this technique can be used for two differents purposes: to preserve the vital functions or to preserve
Ph. Moretto et al. / Human cells: physiology and pharmacology
the morphology and the elemental distributions. The two procedures are not necessarily compatible. In the first case, the freezing must be operated with a slow cooling rate and with the addition of a cryoprotective agent, such as dimethyl sulphoxide (DMSO), in order to minimise cell membranes injury due to ice crystallisation. These crystallisations first occur in the extracellular milieu. Following the subsequent modifications of osmotic gradients, the volume of the intracellular compartment decreases. When a critical volume is reached, the membrane phospholipid bilayer can disrupt. The role of cryoprotectants is to limit the water release and to stabilize the membranes. But the morphology of cells and especially the elemental intracellular components are greatly affected. In the second case, the freezing must be very quick in order to reach vitrification. To enhance the cooling rate, cryoliquids such as isopentane or freon are preferable to liquid nitrogen. They allow one to overcome boiling phenomena. We have used the two approaches, the first one for usual cell banking and the second for analysis purposes. The cultured cells were quench frozen in isopentane directly on the analysis substrate and freezed dried at -30°C during overnight. The structural preservation was checked using scanning electron microscopy. Fig. 1 displays a comparison of cells processed using isopentane and nitrogen freezing. In the analysis of the amnion epithelial cells, the fixation of diffusible electrolytes, Na+, K+ and Ca’+, was the main difficulty. The ionic membrane conductances can therefore be activated very quickly when cells are removed from their natural milieu. The amnion strips were quench frozen in isopentane, cut at low temperature (-30°C) in a cryostat and freeze dried.
5. Identification
of cellular and subcellular
structures
One of the major problems in PIXE microanalysis of mammalian tissue sections is the identification of analysed structures. This difficulty can be overcome, at the tissue scale, performing light microscopy of stained adjoining sections. Sometimes, it is even possible to elucidate the tissue architecture on the freeze dried section itself. But the practical search of a particular cell among others cannot be achieved on the stained slide because the thickness of this section is of about the order of magnitude of the cellular dimensions. Before going further, we can nevertheless distinguish between the cellular and the subcellular level, i.e. the identification of organelles. For morphology examination purpose, elemental distributions can play the role of natural intracellular markers. In order to discern cell boundaries from con-
513
Fig. 1. Test of cryofiiation. Scanning electron microscopy cultured cells following liquid nitrogen freezing (WD27) and isopentane freezing (WD31). In nitrogen, cells shrank and membranes were injured.
(X 1000) of IGROVl
nective tissue, several well-known intracellular minerals, such as potassium and phosphorus, can provide valuable help. That means, of course, that the tissue was correctly fixed to avoid electrolytes migration. This is what we carried out during the study of the human amniotic membrane. Using elemental mapping, the monocellular epithelial layer was determined by a high phosphorus concentration. The phospholipid bilayer of plasma membranes contributed undoubtedly to this high phosphorus level because of the numerous convolutions which increase the wall surface for cellular exchanges. Moreover, the compact lamina was clearly delineated by sulphur and chlorine distributions [lo]. The explanation of this phenomenon is more difficult. After incubation in a physiological Hanks’ solution, this effect was reinforced by a massive fixation of sodium and chlorine, the main buffer components, on cell nucleus and fibers of the compact lamina (fig. 2). VIII. MICROBEAM PIXE
514
Ph. Moretto et al. / Human cells: physiology and pharmacology
Large vacuoles appeared in the epithelial cells allowing one thus to discern more easily the nucleus and cell walls.
60p.m
55pm FIBROBLASI’
CONNE-CTIVE
COM-PACT
TISSUE
LAMINA
EPITHELILJM
Fig. 3. PIXE microanalysis of phosphorus in a polynuclear IGROVl tumor cell. The three intracellular structures which appear on the figure are the nucleus. Scan dimension 60 X 60 pm’.
The carbon distribution, following RBS analysis, can likewise give interesting information about the organic density of the specimen. The dense fibrous composition of the amnion compact lamina allowed us to localize this layer and consequently the basal pole of epithelial cells. To localize a single cell in a well-structured monolayer grown in culture does not set any problem for light microscopy with suitable magnification. But the identification of the subcellular ultrastructure is more critical because of the opacity of freeze dried cells. The cell size, compared with the spatial resolution of nuclear microprobes, is certainly a limiting factor. But the major difficulty remains still the lack of physical exploitable information. This morphology examination must be carried out prior to the analysis, either with a specific technique such as densitometry or with specific markers detected during the PIXE analysis. In the study of the ovarian adenocarcinoma cell line, the point was to localize the cell nucleus in order to assess the drug spatial repartition between nucleus
Fig. 2. (top and middle) PIXE microanalysis of the chlorine distribution in the human amniotic membrane after incubation in Hanks’ solution. Single epithelial cells can be easily localized on the right part of the figure. (bottom) Three-dimensional plot of chlorine within the same area. The sharp peaks on the left part of the map define the nuclear regions of fibroblasts. Dimension of the scan: 55 x 55 pm2.
515
Ph. Moretto et al. / Human cells: physiology and pharmacology
Fig. 4. PIXE microanalysis of an IGROVl-17 cell after multidrug exposure to cisplatin and iododeoxyrubicin. A strong spatial correlation between iron and iodine appears in the assumed nucleus region. Platinum is homogeneously distributed in the whole cell compartment. Dimension of the scan: 80 x 80 pm’.
Fig. 5. PIXE microanalysis of an IGROVl-p cell after multidrug exposure to cisplatin and iododeoxyrubicin. Despite the small dimensions of the cell (15 pm), structures in the elemental distributions of iodine and iron can be noticed. Scan dimension: 25 x 25 um2. VIII. MICROBEAM
PIXE
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Ph. Moretto et al. / Human cells: physiology and pharmacology
S.T.I.M.
45 pm
and cytoplasm. Hopefully, in the IGROVl-1~ resistant cell subpopulation under investigation, morphologically different cells appeared, no doubt following mutational events. These cells were differentiated from others because of a larger size (> 50 urn) and a polynuclear phenotype. This subpopulation allowed us to investigate nuclear markers without any problem of beam spatial resolution. In some particular cases, mapping of intracellular phosphorus and potassium permitted us to depict the nucleus region (fig. 3). But the nucleo/cytoplasmic concentration ratio was too low to achieve a good contrast. This contrast can be exceptionally improved when the cells were partially dissolved following plasma membrane injury. Molecules known for their ability to fix on DNA were then tested for nucleus labelling. Following an unsuccessful attempt with bromodeoxyuridine, we investigated 4’-iododeoxyurubicin, an intercalating agent used in cancer chemotherapy. We noticed an effective fixation of this molecule in the nuclear region but still with problems of reproducibility (fig. 5). Fig. 4 displays the PIXE elemental distributions in an IGROVl-ly cell simultaneously exposed to cisplatin and iododeoxyrubicin for 2 h. The respective drug concentrations in the incubation medium were 200 and 100 kg/ml. We can notice the quite different behavior of cisplatin and iododeoxyrubicin when they diffuse into the cell. Platinum is homogeneously distributed in the whole cell whereas iodine is located in a narrow region we suppose to be the nucleus. These observations agree with the fact that cisplatin binds to numerous cellular ligands such as cytosolic proteins or nucleic acids [4]. But the most promising method seems to be the scanning transmission ion microscopy [3]. The application of this technique to biological samples is founded upon the variation of the slowing down of beam particles according to the organic matrix density and to its chemical composition. This technique is especially more interesting because it can be carried out with a very low beam intensity of about 1000 particles/s. This allows one to achieve a better spatial resolution (of less than 1 urn) using a smaller collimator aperture, and more particularly to reduce sample damage. The STIM
Fig. 6. (top) Identification of subcellular structures in an IGROVl cell without any drug exposure. STIM is compared to PIXE elemental mapping of potassium. A single nucleolus surrounded by the nuclear membrane can be clearly idendified on the STIM three-dimensional plot. On the potassium map, we can remark the smooth structure in the nucleolus region. Dimension of the scan: 45 x45 urn’. (bottom) Scale map of the STIM distribution displayed above. The thickness of the nuclear membrane appeares more clearly.
517
Ph. Moretto et al. / Human cells: physiology and pharmacology
can thus be used before PIXE microanalysis without any sample injury. Recent trials on IGROVl cells allowed us to reveal not only the nucleus boundaries but also suborganelles such as nucleolus. The fact that most of the cell’s ribosomal RNA is synthesized in the nucleolus can probably explain this high density. The double nuclear membrane bounding the nucleus is clearly displayed in fig. 6.
available on nuclear microprobes can plete set of valuable information. The of the biological activity of new metal chemotherapy could provide numerous cations.
provide a comcharacterization compounds for potential appli-
References [II M. Gigli, T.W.D. Rasoanaivo,
6. Conclusion We did not deal with the problem of sample damage which was extensively reviewed by numerous contributors [3,12], but it will certainly be the main limitation for future applications of microbeam analysis. This problem becomes particularly acute when very narrow areas are scanned during single cell analysis. In that case, even high speed scanning does not avoid the disastrous effects of temperature rise. We did not likewise deal with the obtainment of quantitative results. The capability to calculate, for instance, the absolute platinum amount within a single cell is certainly a serious argument in favour of PIXE microanalysis. This allows one to easily cross-check the drug uptake values obtained using other techniques and expressed in unit of kg per million of cells. At last, another interesting feature is the sample mass measurement by means of RBS analysis with the aim to normalize the concentrations in terms of dry mass of scanned tissue. For that purpose, we are developing a simulation program of RBS spectra in inhomogeneous organic samples in order to achieve accurate measurements of carbon, nitrogen and oxygen, the main components of a biological matrix. The potentialities of PIXE microanalysis are extremely well suited to isolated cell analysis. The combined utilization of PIXE and the various techniques
J.M. Millot, P. Jeannesson, V. Rizzo, J.C. Jardillier, F. Arcamone and M. Manfait, Cancer Res. 49 (1989) 560. Dl P. Fragu, J. KIijanienko, D. Gandia, S. Halpem and J.P. Armand, Cancer Res. 52 (1992) 974. 131 M. Cholewa, G. Bench, B.J. Kirby and G.J.F. Legge, Nucl. Instr. and Meth. B54 (1991) 101. [41 J. Reedijk Pure and Appl. Chem. 59 (1987) 181. Dl J. BCnard, J. Da Silva, M.C. De Blois, P. Boyer, P. Duvillard, E. Chiric and G. Riou, Cancer Res. 45 (1985) 4970. 161 Ph. Moretto, R. Ortega, M. Simonoff, Y. Llabador, G. Simonoff, J. Robert and J. Benard, in: Metal Ions in Biology and Medicine, vol. 2, eds. J. Anastassopoulou, Ph. Collery, J.C. Etienne and Th. Theophanides (Libbey, London, 1992) p. 125. 171 E. Coraboeuf, Am. J. Physiol. 234 (1978) 101. [Sl M. Bara, A. Guiet-Bara and J. Durlach, Magnesium Bull. 6 (1984) 36. Med. Sci. Res. 15 (1987) [91 M. Bara and A. Guiet-Bara, 975. M. Simonoff, M. Bara, A. [lOI Ph. Moretto, Y. Llabador, Guiet-Bara, Y. Rayssiguier and J. Durlach, Magnesium Res. 4 (1991) 221. Ull Ph. Moretto, Y. Llabador, M. Simonoff and L. Razafindrabe, Quantitative mapping of intracellular cations in the human amniotic membrane, Proc. 3rd Int. Conf. on Nuclear Microprobe Technology and Applications, Uppsala, 1992, Nucl. Instr. and Meth. B, to be published. iI21 U. Lindh, Nucl. Instr. and Meth. B54 (1991) 160. [131 Y. Llabador, D. Bertault, J.C. Gouillaud and Ph. Moretto, Nucl. Instr. and Meth. B49 (1990) 435.
VIII. MICROBEAM PIXE
PIXE microanalysis Ph. Moretto,
Y. Llabador,
Centre d’Etudes Nu&aires
Beam Interactions with Materials 8 Atoms
in human cells: physiology and pharmacology R. Ortega,
de Bordeaux-Gradignan,
M. Simonoff
and L. Razafindrabe
33175 Gradignan cedex, France
The micro-PIXE technique has been regularly carried out for more than two years to provide elemental distributions in human cells. Using this technique in the framework of cellular biology, two research axes have been developed: the cellular pharmacology f chemotherapeutic agents and the physiology of ionic cellular exchanges. These studies are based upon in vitro experimental models of human cells, either under the form of isolated cultured cells or as part of well-structured tissues. The aim of this paper is to present the experimental procedures and methodological aspects of cellular and subcellular quantitative mapping. Cell processing, identification of intracellular structures, quantitatives results and beam damage will be discussed and illustrated by examplesissuing from the above-mentioned studies.
1. Introduction
During the three last decades, the development of modern molecular cell biology has coincided with technological improvements in cell exploration techniques. The availability of molecular markers, either radioactive or fluorescent, and the use of the specific antibodies for immunolabeling, are certainly important steps forward in this advance. The probing of tagged molecules within cells has now become a routine task for immunofluorescence, flow cytometry, microspectrofluorometry [l] or autoradiography methods. But there is a field of research where we have still to make progress: the microanalysis of subcellular elemental chemical distributions. The detection of intracellular essential trace-elements or toxic heavy metals requires a compromise between spatial resolution and sensitivity that has been achieved by very few techniques. Among them, mass spectrometry and X-ray fluorescence, carried out with ion or electron microbeams are the basic principle of electron microprobes, SIMS [2] and nuclear microprobes. The use of an ion beam is certainly a great advantage of the micro-PIXE technique. This allows of course focusing for microbeam utilization, but also the simultaneous application of associated techniques such as RBS, STIM [3] and secondary electrons detection. Conjointly applied, these methods can provide valuable help in concentrations normalization, organelle localization and fast topography determination of analysed regions. In most studies of experimental biology, the human cell is used as an experimental model. The purpose is to check in vitro, consequently in reproductible conditions, a cell function or the effect of molecular species on cell metabolism. This can be achieved culturing an 0168-583X/93/$06.00
0 1993
Elsevier
Science
Publishers
eukariotic cell line or on highly differentiated cells maintained in good survival conditions following recent sampling. We have used these two approaches during the last two years in order to develop hvo fields of investigation using the micro-PIXE analysis: The cellular pharmacology of cytotoxic drugs used in chemotherapy and the physiology of ionic cellular exchanges. The aim of this report is to review the specific experimental difficulties encountered in the microanalysis of isolated cells. The applied solutions will be discussed on the basis of examples extracted from ours experiments.
2. Cellular
pharmacology
Cancer chemotherapy is founded upon the cytotoxic action of drugs on malignant cells. But this effect is not entirely specific and secondary toxicity for the whole organism is a great limiting factor of treatment efficiency. This is the reason why a careful determination of doses and a good understanding of its mechanism of action are necessary. The last stage before the clinical experimentation is constituted by an in vitro exposure of cultured tumor cells to the drug. Information about the DNA-drug interaction and a precize quantification of the drug uptake can then be achieved with macroanalytical techniques [4]. But the survey of the molecule diffusion within the cell remains a major difficulty. This point becomes of extreme importance when a resistance of tumor cells to the drug arises during therapy. This emergence can therefore limit the clinical usefulness of the antineoplasic agent. Several mechanisms which can affect the drug distribution within the cell have been reported to contribute to this resistance: Decrease of drug accumula-
B.V. All rights reserved
VIII. MICROBEAM PIXE
512
Ph. Moretto et al. / Human cells: physiology and pharmacology
tion either due to a more efficient drug efflux or a lower membrane permeability and detoxifying action founded upon drug chelation. In all cases, the drug localization, especially in the nucleus region, and the eventual effects on essential trace elements distribution have to be elucidated using elemental mapping. From this point of view, a simple comparison between a sensible and a resistant cell line can provide valuable information. For that purpose, we have applied the micro-PIXE analysis to investigate the properties of cisplatin, an important antineoplasic agent, used in ovarian and testicular cancer treatment. A human ovarian adenocarcinoma cell line, IGROVl-p, has been cultured for in vitro drug exposure [5]. This sensitive line was compared with a selected drug resistant subpopulation, IGROVl-1~. Experimental details and first results have been already published elsewhere [6].
3. The physiology of ionic cellular exchanges The plasma membrane of the cell is a selectively permeable barrier between the cytoplasm and the extracellular fluid. This membrane is permanently crossed by ion currents under the effect of electrochemical gradients [7]. During this passive diffusion, when the membrane is at rest, sodium gets into the cell whereas potassium leaves it, crossing the wall. In order to maintain the ionic concentrations of the intracellular milieu at a constant level, mechanisms of active transport (Na/K ATPase) are acting to reject sodium from the cell compartment. These exchanges are a vital function for the cell. Their regulation is extremely complicated on account of the numerous acting factors. In order to investigate these cellular pathways, electrophysiological studies are generally carried out in vitro on isolated cells by mean of microelectrodes introduced in the cytoplasm. Electric parameters, such as membrane potential or conductance, can then be measured with the aim to deduce information about ionic transfers [8,9]. We have used the monocellular epithelial layer of the human amniotic membrane, a classical model developed by electrophysiologists, to investigate the stabilizing action of magnesium on membranes [lo]. PIXE is indeed one of the few microanalysis multielemental techniques which allow one to reveal simultaneously the distribution of most minerals involved in cellular transmembrane currents. The accurate measurement of minerals concentrations in the intracellular compartment after incubation in physiological fluids permits one to check the subsequent migration of ionic species. The effect on the membrane permeability of extracellular fluid components such as magnesium salt or other nutrients can then be studied.
4. Cell preservation The aim of in vitro experimentation is to study the cell in a metabolic state as close as possible to the in vivo situation. We particularly have to reduce the consequences of the initial trauma of sampling procedures. From this point of view, cell culture seems to offer the best guarantees. The explanted cells are only a precursor of those which are actually used for assays. The fact that they meanwhile replicate is certainly the best viability test. Continuous cell lines, such as the neoplasic cell line we studied, proliferate as a monolayer in standardized conditions. Minor deviations would significantly alter their growing kinetic. To avoid extracting the cell from their growing medium, we cultured them directly on the polymer film we intended to use for irradiation, a very thin (15 ug/cm’) sterilised FormvarR film. In order to insure the cell growth, the substrates were precoated with collagen, an attachment factor. Physical and chemical methods, generally used to make the surface of cultur polymer flasks wettable and suitable for cell attachment, could not be applied here on account of the substrate fragility. The epithelial cells of the amniotic membrane are, contrary to cancer cells, fully differentiated with a main natural function of mother to foetus exchange. They would consequently normally not proliferate. They were simply maintained in survival conditions by the interaction with their initial amnion tissue and especially by the experimental conditions of incubation: temperature, oxygenation and isotonic&y of bathing fluid. In these electrophysiological studies, cell preservation has a particular meaning. The goal is to preserve mainly the exchange function, even though the intracellular ionic concentrations are deeply modified. These epithelial cells belongs to a class of leaky membrane. In those cell species, the passive diffusion is the main component of ionic transmembrane transport. The intracellular homeostasis consequently depends on the ionic concentrations in the extracellular milieu. As a general rule, the physiological shock occurs during the first minutes of incubation. The membrane is submitted to violent ionic unbalance and has to be left in the medium for more than half an hour to retrieve a normal at rest potential. We noticed during this essay a potassium release from the cells whereas sodium and chlorine was found to penetrate into the cell compartment [ll]. Following the essays, the second critical stage is to bring the cells under vaccum for analysis. The cells have then first to be fixed. Cryofixation, according to all contributors, is assumed to give the best results [12]. But this technique can be used for two differents purposes: to preserve the vital functions or to preserve
Ph. Moretto et al. / Human cells: physiology and pharmacology
the morphology and the elemental distributions. The two procedures are not necessarily compatible. In the first case, the freezing must be operated with a slow cooling rate and with the addition of a cryoprotective agent, such as dimethyl sulphoxide (DMSO), in order to minimise cell membranes injury due to ice crystallisation. These crystallisations first occur in the extracellular milieu. Following the subsequent modifications of osmotic gradients, the volume of the intracellular compartment decreases. When a critical volume is reached, the membrane phospholipid bilayer can disrupt. The role of cryoprotectants is to limit the water release and to stabilize the membranes. But the morphology of cells and especially the elemental intracellular components are greatly affected. In the second case, the freezing must be very quick in order to reach vitrification. To enhance the cooling rate, cryoliquids such as isopentane or freon are preferable to liquid nitrogen. They allow one to overcome boiling phenomena. We have used the two approaches, the first one for usual cell banking and the second for analysis purposes. The cultured cells were quench frozen in isopentane directly on the analysis substrate and freezed dried at -30°C during overnight. The structural preservation was checked using scanning electron microscopy. Fig. 1 displays a comparison of cells processed using isopentane and nitrogen freezing. In the analysis of the amnion epithelial cells, the fixation of diffusible electrolytes, Na+, K+ and Ca’+, was the main difficulty. The ionic membrane conductances can therefore be activated very quickly when cells are removed from their natural milieu. The amnion strips were quench frozen in isopentane, cut at low temperature (-30°C) in a cryostat and freeze dried.
5. Identification
of cellular and subcellular
structures
One of the major problems in PIXE microanalysis of mammalian tissue sections is the identification of analysed structures. This difficulty can be overcome, at the tissue scale, performing light microscopy of stained adjoining sections. Sometimes, it is even possible to elucidate the tissue architecture on the freeze dried section itself. But the practical search of a particular cell among others cannot be achieved on the stained slide because the thickness of this section is of about the order of magnitude of the cellular dimensions. Before going further, we can nevertheless distinguish between the cellular and the subcellular level, i.e. the identification of organelles. For morphology examination purpose, elemental distributions can play the role of natural intracellular markers. In order to discern cell boundaries from con-
513
Fig. 1. Test of cryofiiation. Scanning electron microscopy cultured cells following liquid nitrogen freezing (WD27) and isopentane freezing (WD31). In nitrogen, cells shrank and membranes were injured.
(X 1000) of IGROVl
nective tissue, several well-known intracellular minerals, such as potassium and phosphorus, can provide valuable help. That means, of course, that the tissue was correctly fixed to avoid electrolytes migration. This is what we carried out during the study of the human amniotic membrane. Using elemental mapping, the monocellular epithelial layer was determined by a high phosphorus concentration. The phospholipid bilayer of plasma membranes contributed undoubtedly to this high phosphorus level because of the numerous convolutions which increase the wall surface for cellular exchanges. Moreover, the compact lamina was clearly delineated by sulphur and chlorine distributions [lo]. The explanation of this phenomenon is more difficult. After incubation in a physiological Hanks’ solution, this effect was reinforced by a massive fixation of sodium and chlorine, the main buffer components, on cell nucleus and fibers of the compact lamina (fig. 2). VIII. MICROBEAM PIXE
514
Ph. Moretto et al. / Human cells: physiology and pharmacology
Large vacuoles appeared in the epithelial cells allowing one thus to discern more easily the nucleus and cell walls.
60p.m
55pm FIBROBLASI’
CONNE-CTIVE
COM-PACT
TISSUE
LAMINA
EPITHELILJM
Fig. 3. PIXE microanalysis of phosphorus in a polynuclear IGROVl tumor cell. The three intracellular structures which appear on the figure are the nucleus. Scan dimension 60 X 60 pm’.
The carbon distribution, following RBS analysis, can likewise give interesting information about the organic density of the specimen. The dense fibrous composition of the amnion compact lamina allowed us to localize this layer and consequently the basal pole of epithelial cells. To localize a single cell in a well-structured monolayer grown in culture does not set any problem for light microscopy with suitable magnification. But the identification of the subcellular ultrastructure is more critical because of the opacity of freeze dried cells. The cell size, compared with the spatial resolution of nuclear microprobes, is certainly a limiting factor. But the major difficulty remains still the lack of physical exploitable information. This morphology examination must be carried out prior to the analysis, either with a specific technique such as densitometry or with specific markers detected during the PIXE analysis. In the study of the ovarian adenocarcinoma cell line, the point was to localize the cell nucleus in order to assess the drug spatial repartition between nucleus
Fig. 2. (top and middle) PIXE microanalysis of the chlorine distribution in the human amniotic membrane after incubation in Hanks’ solution. Single epithelial cells can be easily localized on the right part of the figure. (bottom) Three-dimensional plot of chlorine within the same area. The sharp peaks on the left part of the map define the nuclear regions of fibroblasts. Dimension of the scan: 55 x 55 pm2.
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Fig. 4. PIXE microanalysis of an IGROVl-17 cell after multidrug exposure to cisplatin and iododeoxyrubicin. A strong spatial correlation between iron and iodine appears in the assumed nucleus region. Platinum is homogeneously distributed in the whole cell compartment. Dimension of the scan: 80 x 80 pm’.
Fig. 5. PIXE microanalysis of an IGROVl-p cell after multidrug exposure to cisplatin and iododeoxyrubicin. Despite the small dimensions of the cell (15 pm), structures in the elemental distributions of iodine and iron can be noticed. Scan dimension: 25 x 25 um2. VIII. MICROBEAM
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and cytoplasm. Hopefully, in the IGROVl-1~ resistant cell subpopulation under investigation, morphologically different cells appeared, no doubt following mutational events. These cells were differentiated from others because of a larger size (> 50 urn) and a polynuclear phenotype. This subpopulation allowed us to investigate nuclear markers without any problem of beam spatial resolution. In some particular cases, mapping of intracellular phosphorus and potassium permitted us to depict the nucleus region (fig. 3). But the nucleo/cytoplasmic concentration ratio was too low to achieve a good contrast. This contrast can be exceptionally improved when the cells were partially dissolved following plasma membrane injury. Molecules known for their ability to fix on DNA were then tested for nucleus labelling. Following an unsuccessful attempt with bromodeoxyuridine, we investigated 4’-iododeoxyurubicin, an intercalating agent used in cancer chemotherapy. We noticed an effective fixation of this molecule in the nuclear region but still with problems of reproducibility (fig. 5). Fig. 4 displays the PIXE elemental distributions in an IGROVl-ly cell simultaneously exposed to cisplatin and iododeoxyrubicin for 2 h. The respective drug concentrations in the incubation medium were 200 and 100 kg/ml. We can notice the quite different behavior of cisplatin and iododeoxyrubicin when they diffuse into the cell. Platinum is homogeneously distributed in the whole cell whereas iodine is located in a narrow region we suppose to be the nucleus. These observations agree with the fact that cisplatin binds to numerous cellular ligands such as cytosolic proteins or nucleic acids [4]. But the most promising method seems to be the scanning transmission ion microscopy [3]. The application of this technique to biological samples is founded upon the variation of the slowing down of beam particles according to the organic matrix density and to its chemical composition. This technique is especially more interesting because it can be carried out with a very low beam intensity of about 1000 particles/s. This allows one to achieve a better spatial resolution (of less than 1 urn) using a smaller collimator aperture, and more particularly to reduce sample damage. The STIM
Fig. 6. (top) Identification of subcellular structures in an IGROVl cell without any drug exposure. STIM is compared to PIXE elemental mapping of potassium. A single nucleolus surrounded by the nuclear membrane can be clearly idendified on the STIM three-dimensional plot. On the potassium map, we can remark the smooth structure in the nucleolus region. Dimension of the scan: 45 x45 urn’. (bottom) Scale map of the STIM distribution displayed above. The thickness of the nuclear membrane appeares more clearly.
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can thus be used before PIXE microanalysis without any sample injury. Recent trials on IGROVl cells allowed us to reveal not only the nucleus boundaries but also suborganelles such as nucleolus. The fact that most of the cell’s ribosomal RNA is synthesized in the nucleolus can probably explain this high density. The double nuclear membrane bounding the nucleus is clearly displayed in fig. 6.
available on nuclear microprobes can plete set of valuable information. The of the biological activity of new metal chemotherapy could provide numerous cations.
provide a comcharacterization compounds for potential appli-
References [II M. Gigli, T.W.D. Rasoanaivo,
6. Conclusion We did not deal with the problem of sample damage which was extensively reviewed by numerous contributors [3,12], but it will certainly be the main limitation for future applications of microbeam analysis. This problem becomes particularly acute when very narrow areas are scanned during single cell analysis. In that case, even high speed scanning does not avoid the disastrous effects of temperature rise. We did not likewise deal with the obtainment of quantitative results. The capability to calculate, for instance, the absolute platinum amount within a single cell is certainly a serious argument in favour of PIXE microanalysis. This allows one to easily cross-check the drug uptake values obtained using other techniques and expressed in unit of kg per million of cells. At last, another interesting feature is the sample mass measurement by means of RBS analysis with the aim to normalize the concentrations in terms of dry mass of scanned tissue. For that purpose, we are developing a simulation program of RBS spectra in inhomogeneous organic samples in order to achieve accurate measurements of carbon, nitrogen and oxygen, the main components of a biological matrix. The potentialities of PIXE microanalysis are extremely well suited to isolated cell analysis. The combined utilization of PIXE and the various techniques
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