- Email: [email protected]
Cell biology, trace elements and nuclear microscopy
Nuclear Instruments and Methods in Physics Research B 104 (1995)285-291
Beam Interactions with Materials & Atoms
Cell biology, trace elements and nuclear microscopy Ulf Lindh* Centrefor Metal Biology
in Uppsala, Uppsala University Box 53.5,
S-751 21 Upps& Sweden
Abstract
Essential trace elements often function as co-factors in enzymes, the most conspicuous being zinc for which more than 200 dependent enzymes have been found. Selenium is a trace element for which only a few enzymes have been discovered.
Quit recent findings of the importance of zinc-finger proteins in the transcription process in cells have attracted great interest. A protective effect against damage to base-pairs during this process is intriguing. Another exciting discovery is that the tumour-suppressor protein p53 also contains zinc. The development of the nuclear microscope has made possible intracellular studies of trace-element distributions. This paper discusses the progress of recent years concerning nuclear microscopy in cell biology. Progress in our laboratory concerning intracellular distributions of trace elements and the comparison with measurements on fractionated organelles will be presented.
1. Trace elements in cell biology
Except for iron and iodine our knowledge about the essential trace elements is only about 50 years old. About 98% of the human body mass is made up of nine nonmetallic elements. Another 1.89% are formed by the four main electrolytes sodium, magnesium, potassium and calcium. Eleven of the trace elements (Cr, Co, Cu, I, Fe, Mn, MO, Ni, Se, Sn and Zn) occupy just a tiny 0.012% or 8.6 g of the body weight. However, this small fraction exerts a tremendous influence on all body functions that could never have been imagined in earlier times. Whether an element is essential for life depends on its participation in one or several biochemical reactions. The term “essential” is borrowed from the early amino acid and protein chemistry. According to the recommendations of a WHO expert committee, this expression is not optimal when it is applied to trace elements [l]. According to a new, improved definition [2] an element is already useful to the organism and to the maintenance ofhealth when a measurable deficit in the diet reduces the growth and vitality of humans, animals or plants to a reproducible degree. If we start from this definition it becomes plausible that even well-known toxic elements, such as arsenic and lead are needed in minute quantities for the normal functioning of cell metabolism, although they are recognised as toxic in higher concentrations.
*Tel.: + 46-18-18 38 36. Fax: + 46-18-18 38 33. E-mail: ulf. [email protected].
Usually, the ions of trace elements act as coordination centres for building up or stabilising the structure of enzymes and proteins. Exceptions are iron, which is the central atom of haeme in cytochromes and haemoglobin, cobalt in the centre of vitamin Br2, and iodine, which is a constituent of the hormone thyroxine. Chromium seems to be essential for the biosynthesis of the glucose tolerance factor in man [3] which itself seems to be chromium free [4]. The absorption rates differ remarkably between the elements. Table 1 shows these rates for eight trace elements. Nutrients are taken up from water and soil by plants. The least quantity of most trace elements that accumulate is a consequence of their genetically determined cell structure and function that can vary somewhat between the species. However, trace elements that are accidentally present in the soil for geological reasons or by human deposition can accumulate in grass and food crops. They represent unforeseeable risks for the consumer. The minimum biological needs for trace elements of plants, animals and man are only proportionally but not fundamentally different. Even boron makes no exception, although boron is generally considered essential only for plants. Recent findings show that the hydroxylases which are responsible for the transformation of the vitamins Dz and D3 into their corresponding 1,25-dihydroxyderivates, and cholesterol into estradiol, strictly depend on borate ions. Therefore, boron may be a potent protective factor against post-menopausal osteoporosis in women [S].
0168-583X/95/$9.500 1995 Elsevier Science B.V. All rights reserved SSDI 0168-583X(95)00389-4
VII. LIFE SCIENCES
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(1. Lindhi Nucl. Instr. and Meth. in Phys. Res. B 104 (1995) 285-291
Table 1 Absorption rates for some trace elements Trace element in Food
Absorption rate (%)
Molybdenum Selenium Zinc Copper Iron Manganese Chromium (III)
7G30 or less Sk80 or less or more 2&40 or more 10-30 or more 7-15 or more 3-5 or less 0.5-l.Ob
“Depending on the dose, speciation, transformation, health status and interactions ‘Chromate absorption may be somewhat higher, especially in individuals in whom Cr (VI) is not reduced by gastric juice. Very little is still known about the details of uptake into cells and intracellular transport and storage of trace elements. In addition, there are innumerable potential metal-binding ligands in the intestinal lumen. Many nutritional studies have shown marked effects on uptake rates by synthetic chelating agents or naturally occurring metal-binding molecules e.g. citrate and lactoferrin. Binding to intestinal secretions e.g. pancreatic juice and mucus, may facilitate uptake whereas binding to the brush border glycocalyx and intestinal cell membrane may reduce availability [6]. Uptake of copper, iron and zinc appears to be regulated by feedback mechanisms since there is an increased uptake by deficient animals. Parallel effects on uptake of other metals are also frequently observed e.g. Cd and Pb in calcium deficiency and Zn in iron deficiency. However, these findings do not imply common carriers. In suckling animals there is pinocytic uptake of proteins and metal-protein adjuncts in the distal jejunum and ileum [7]. Many studies have suggested that paracellular uptake of metals across the intestine may also be important [S]. Furthermore, there is increasing evidence to suggest that some metals may cross cellular membranes by an uncontrolled, non-selective diffusion process as their neutral lipid-soluble complexes. Examples of such complexes are CdCli and methyl-HgCl. Paracellular uptake across the intestinal epithelium also appears to occur for many oxy-anions, including AsO:-, CrO:-, SeO:-, and VOi- [9].
2. A highlight of trace-element biology Zinc is an essential part of more than 200 enzymes, examples of which are the DNA- and RNA-polymerases. Recently it was discovered that Zn-finger proteins are involved in the cellular transcription process and that they probably also protect base pairs during this process against attack from reactive chemical species. Yet another fascinating discovery is the protein ~53 that is
a tumour suppressor protein. It is believed that inactivation of this protein through mutations is associated with many human cancers [lo]. The three-dimensional structure of this suppressor protein was very recently unveiled by the crystal structure of the p53 core domain [ll] and the solution structure of its oligomerisation domain [ 121. This important molecule is yet another zinc protein.
3. Development of nuclear microscopy in cell biology The technical and methodological arsenal in the hands of biologists for the study of cells and biomolecules is steadily expanding. Electron microscopy has been and still is one very important technique. Its versatility in detecting elemental concentrations and distributions has been strengthened by the introduction of electron energy loss spectroscopy. In addition, we have witnessed the slow but gradual introduction of the nuclear microscope in the arsenal of modern cell biology. Without having the ambition of presenting a complete review, some illustrative examples are given here. In a series of studies, Moretto et al. [13-l 51 have used the nuclear microscope to study cellular pharmacology. In these studies they have achieved intracellular elemental mapping. To visualise the cell nucleus the group used the higher phosphorus content of nuclear material. However, these authors point out that the phosphorus contrast between nucleus and cytoplasm is low and show that Scanning Transmission Ion Microscopy provides far better contrast. In the latter case, it was even possible to map the nuclear membrane [13]. They have extended their study with the human amniotic membrane as an experimental model [14]. In this work they emphasised the beam damages and the consequences for the intracellular distribution of metal ions, the so-called electrolytes, of incubation of the membrane. In the most recent study, Moretto et al. [ 151 focused on the cellular pharmacology of the cancer drug cisplatin. A problem with this kind of therapy is that cancer cells acquire drug resistance. It was not possible to find any elemental differences between resistant and non-resistant cells. However, when cells were exposed to cisplatin, significant differences in trace elements were obvious. Other approaches to cellular pharmacology have been undertaken by the Melbourne group. They used human peripheral lymphocytes from healthy blood donors and a continuous line of human T-lymphocytes. This experimental model was used to test anti-AIDS drugs containing cobalt and tungsten. By intracellular elemental mapping it was shown that the drug accumulated in the cells [16]. By STIM it was shown that the irradiation to extract elemental information induced beam damages that should not influence the results. Another approach was to use a line of African green monkey kidney cells and V79 hamster lung cells to test two classes of drugs
U. Lindh /Nucl. Instr. and h4eth. in Phys. Res. 3 104 (1995) 2855291
against the HIV and flavi viruses. The drugs were inorganic polyanions and coordination complexes of chromium in oxidation states (III), (V) or (VI) and their uptake was tested by nuclear microscopy [17]. To study zinc in biological membranes Zhang et al. [18] used both the scanning proton microprobe and synchrotron-radiation induced X-ray fluorescence. Hepatocytes, nerve cells and cardiac cells were exposed to synchrotron light for total zinc concentrations, and the nuclear microprobe was used to get the positional information. The group used membranes of red cells to unveil the zinc concentration in the lipid and protein fractions, respectively. Following the current hypothesis of free-radical generation in ischaemic heart damage, Bas et al. [19] studied ion movements in ischaemic cardiac tissue both by proton- and electron-induced X-ray microanalysis. These authors put special emphasis on preserving both elemental integrity and ultrastructure. The Singapore group has undertaken several interesting studies in cell biology. The involvement of iron, and other trace elements in atherosclerosis was studied in a rabbit model. The highlight of this investigation was that a fivefold increase of iron in atherosclerotically lesioned tissue could be detected by nuclear microscopy [20]. This finding is in support of iron toxicity by radical generation [21]. Another aspect of radical-induced damages was investigated by this group. Parkinsonism was induced in the right hemisphere of rats by injecting a neurotoxin. In this way each animal was its own control. Sections containing the substantia nigra, where the dopaminergic cells are situated, were analysed both on the lesioned side and the control side. This investigation showed an increase of iron in the parkinsonian side [22]. Calcium and activation of cells with adenosine triphosphate were studied in an experimental cellular model (human Chang liver cells). A twenty fold increase in copper was established in activated cells. Blood cells from the ascidian Phallusia mammillata have the amazing capability to accumulate vanadium from the sea water by a factor of 100000 to 1000000. Such cells were submitted to nuclear microscopy by this group confirming earlier results [23]. Palsgard et al. [24] used both electron microscopy, X-ray microanalysis, and nuclear microscopy to study the polarisation phenomena induced in RINmSF- and isolated mouse B-cells by K+-stimulation. This group also made novel use of multivariate statistics to differentiate between intracellular free and bound ions.
4. Other techniques or combinations of techniques providing elemental information with cellular resolution
To be of the best value to cell biology, any elemental technique should be able to provide chemical informa-
287
tion. PIXE and the analytical techniques usually employed in nuclear microscopy seldom offer such possibilities. In certain cases, the use of multivariate statistics may improve the elemental information from PIXE [24]. Often, however, other methods have to be considered. Although the magnesium ion (Mg’+) plays an important role in cell activation, information has until now been limited by the lack of suitable methods for measuring cytosolic free Mgzf. Matsuno et al. [25] measured the concentration of free magnesium ions in resting and activated human platelets using a new fluorescent magnesium-ion indicator mag-fura-2. The concentration of free magnesium ions in resting platelets was 13.13 f 3.40 mg/l and in activated platelets 22.37 + 9.00 mg/l. A technique called diagnostic X-ray spectrometry (DXS), based on X-ray fluorescence, was used to quantify the multiple elemental composition of washed, intact human platelets [26]. They determined the potassium, calcium and zinc concentrations to 3.08 + 1.00, 1.18 f 0.29 mg/g and 35 _I 9 ug/g, respectively. In addition they used X-ray microanalysis in an electron microscope to determine the subcellular concentrations and found zinc to be distributed in the alpha-granules (40%) and the cytoplasm (60%). The importance of cytosolic-free calcium level in lymphocyte activation inspired Tan et al. [27] to investigate changes in [Ca’+] i in T cells caused by mercury compounds, which have been shown to have immunomodulatory and immunotoxic properties. Using fura- as fluorescent Ca” indicator they found that both methyl-mercury and mercuric chloride increased the cytosolic-free calcium concentration in lymphocytes from rat spleen in a concentration-dependent manner [27]. Aluminium toxicity in the rat liver and brain was studied by a combination of PIXE and EELS. PIXE was used to determine elemental concentrations in homogenised cells and isolated nuclei, and by EELS the subcellular distributions were assessed. Aluminium injected in health rats enters the brain through the blood-brain barrier and aluminium was incorporated in cell nuclei both in the brain and in the liver. In the liver parenchymal cells, aluminium was not detected in the cytoplasm but exclusively in the heterochromatin of nuclei [28].
5. Nuclear microscopy in cell biology-capabilities and problems To be of good value to cell biologists, any elemental technique has to allow subcellular resolution. As it has been pointed out by several authors through the years, resolution is one merit of nuclear microscopy. Onemicron beam resolution was achieved roughly simultaneously in Oxford and Melbourne around 1980 [29]. In this sense, the resolution should be interpreted as
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Instr. and Meth. in Phys. Res. B 104 (1995) 285-291
resolution with analytically meaningful particle intensity. Only a few groups have attained sub-micron resolution. Among these are the Oxford group keeping the lead by 0.4 urn [30]. In Uppsala, 0.7~urn resolution was achieved with the same probe-forming lenses at Oxford [31]. With the similar equipment, the group in Singapore has showed 0.6~pm resolution [32]. Neither of these achievements, however, is on routine basis. This slowing down of the development of nuclear microscopes has, of course, hampered the introduction into cell biology. However, l-urn resolution should be adequate for some cell-biological work. Here is presented some attempts by the Uppsala group to exploit the nuclear microscope in cell biology. Lymphocytes [33] and neutrophil (polymorphonuclear) granulocytes [34] were isolated from venous samples of healthy volunteers according to procedures presented earlier. These cell types represent different functions in the circulating blood. They are different also in morphology as granulocytes have granules in the cytoplasm. Other differences are size and presence of organelles in the cytoplasm. Our attempt was to measure trace elements in a few of the cell organelles that are possible to identify in nuclear microscopy and to compare these results with those from analysis of cellular fractions. The choice was to investigate nuclei and mitochondria. Backing this choice was that nuclei are often easily identified in unstained samples and that mitochondria challenge the resolution of nuclear microscopy. Though the methods employed in nuclear microscopy are multielemental, we have chosen to discuss only the values for zinc. This is due to problems associated with other techniques used to assess elemental concentrations in fractionated cells and the fractionation procedure itself. Nuclear microscopy demands unstained objects and this makes identification very difficult. In freeze-dried unstained tissue sections it may be possible to identify individual cells but very seldom any further subcellular details. Working with isolated individual cells that vary in thickness, the problem becomes more severe. In cells trapped onto a backing (formvar, pioloform, etc.) it is often easy to identify the nucleus. Using light directed in, say 45” to the specimen surface, it may be possible to identify other organelles such as mitochondria. To reduce the uncertainty in identification, we have used densitometry on the samples. From the investigation by nuclear microscopy are shown elemental maps of one lymphocyte (Fig. la) and one granulocyte (Fig. 2a). To ease the interpretation, drawings (Figs. lb and 2b) pointing to the important cellular features are incorporated. To prepare highly purified nuclei in high yield, the procedure described by Ford and Graham was used [35]. It involves the use of calcium and magnesium compounds to preserve the integrity of nuclei. The mitochondria were
Mitochondtion
Nucleus
-.~~ .-._,....., ..h
(b)
..,....\
~
‘......
-.___....-
.../’
,-’
Fig. 1. (a) &-map of one lymphocyte from a healthy volunteer. (b) Drawing showing the essential features (nucleus and mitochondrion) of the lymphocyte.
prepared according to the procedure used by Ford and Graham. A Coulter counter was used to count the number of cells in the suspensions. These were adjusted to contain 10” cells per injection volume, which was introduced into the graphite furnace of an atomic absorption spectrophotometer for zinc determination. The results of these determinations are presented in Table 2. In situ measurements by nuclear microscopy of five cells of each type are presented in Table 3. However, the results achieved in both these ways are not directly comparable because AAS measurements present concentrations by wet weight and the nuclear microscopy by dry weight. TO get an idea about the dry weight fraction of white cells, preparations of thrombocyte and plasma free mononuclear and polymorophonuclear white cells were made according to the procedures mentioned above. The resulting volumes were weighed and then oven dried at 120°C overnight. The remaining mass was assumed to represent the dry weight fraction, which was 25-28% for both cell types.
289
U. Lindh iNuc1. Instr. and Meth. in Phys. Rex B 104 (1995) 285-291
Table 2 Concentrations of Zn @g/g d.w.) in fractionated cell organelles from lymphocytes.and neutrophil granulocytes. Values are presented as median values f interquartile ranges for n = 5 measurements Cell type
Nuclear fraction
Mitochondrial fraction
Lymphocytes Neutrophil granulocytes
10.7 f 1.6 13.1 * 2.1
7.2 f 1.4 9.8 + 1.9
Table 3 Concentrations of Zn @g/g d.w.) in cell organelles measured in situ in five cells of each type. Values are presented as median values f interquartile ranges for n = 5 cells ,_,,_....
,,,...’/”
,’
2’
_._ ...____,I .,.. ““‘..,
\ \
Y..
-.._
Segmented nucleus
(b) Fig. 2. (a) Zn-map of one neutrophil granulocyte from a healthy volunteer. (b) Drawing showing the essential features (nucleus and two mitochondria) of the granulocyte.
For the nuclear fraction, the results are in good agreement. This is, however, not so for the mitochondrial fraction. There are several possible explanations to the disagreement. As the nucleus is a far greater part of the cell than a mitochondrion, it is natural that identification is easier. Furthermore, any part of the cytoplasm covering the nucleus should mean fewer errors than for mitochondria. The identification of mitochondria is uncertain, though densitometry was used to aid the localisation. It is not possible to know how much other cell material is present under the beam. In the worst case, other organelles or parts of organelles could be within the irradiated volume. If the cells are intact, the influence from the plasma membrane cannot be avoided.
Cell type
Nuclei
Mitochondria
Lymphocytes Neutrophil granulocytes
14.2 _+2.4 15.8 _+2.8
15.3 f 4.2 14.2 f 4.5
Other causes of the disagreement observed in this experiment could be that the centrifugation and reagents used have influenced the zinc concentration. The fractionation procedure has been designed to maintain organelle integrity from the biological point of view. This does not necessarily mean that the trace-element integrity is well preserved. Identification of cell organelles should be eased by using STIM, but the problem with interference from other organelles in the irradiated volume still remains. The obvious solution to this problem would be sectioning of embedded cells. Evaluations of this approach warrants further experimental studies. Another example of comparisons is the one undertaken in a systematic study of acute intermittent porphyria (AIP). The acute hepatic porphyrias comprise a group of hereditary disqrders of haeme metabolism. In some porphyria gene carriers attacks of generalised neuropathy occur, not seldom with disablement as a result, and in occasional cases with fatal outcome. Since no cure for the genetic condition is yet available, the therapeutic strategy is concentrated on prophylactic measures aimed at avoiding porphyrinogenic environmental factors of notorious significance. In a study presented in detail elsewhere [36] a broad survey of clinical chemical measurements together with assessment of trace-element status was made for patients with AIP. One of the most important discriminating factors between health and disease was the manganese concentration in granulocytes. The latter was determined in single isolated white blood cell by nuclear microscopy. These results were compared
VII. LIFE SCIENCES
290
U. Lindh j Nucl. Instr. and Meth. ilz Phys. Res. 3 IO4 (1995) 285-29:
Table 4 Manganese concentration in neutrophil granulocytes from AIP patients and healthy controls (n = 5) as assessed by nuclear microscopy on single cells or XRF on cell fractions. Concentrations are given as median values + interquartile ranges and are based on dry weight Method
AIP patients
Healthy controls
Nuclear microscopy XRF on cell suspension XRF on cell pellet
3.5 f 0.5 4.1 If: 0.6 3.3 + 0.4
1.2 & 0.3 0.9 2 0.2 0.8 i: 0.3
with measurement of the manganese concentration by X-ray fluorescence. Two approaches were used. Either samples of 50 pl with a known cell concentration (2-3 x lo6 cells) were put onto formvar foils and air-dried or packed pellets of centrifuged granulocytes were weighed and dissolved in nitric acid, diluted and added with an internal standard and eventually put on formvar foils. The results of manganese determination using single cells or cell fractions are shown in Table 4. Estimations of dry weight fractions were done as described above. In the case of manganese total concentration in polymorphonuclear blood cells there is a good agreement between the various analytical procedures used. The conclusions drawn from nuclear microscopy, therefore lies on solid ground.
6. Conclusions For nuclear microscopy to be of real value in cell biology, techniques for preparation and identification of morphology, i.e. organelles, have to be improved. Our experience suggests that individual intact cells e.g. from a culture, is mainly useful for assessing the total concentration. Such information may be beneficial in those cases where skew distributions of trace elements are encountered. If good-quality data on trace-element concentrations are to be extracted from subcellular parts, sectioning of the cells is probably necessary. There is certainly a need for further investigations and exploitations of techniques from X-ray microanalysis carried out in electron microscopes.
Acknowledgements The financial support for the Slim-Up nuclear microscope in Uppsala from the Knut and Alice Wallenberg
foundation is greatfully acknowledged. Thanks go also to Dr. T. Sunde for skilful operation of the Slim-Up.
References Cl1 WHO, Trace elements in human nutrition, Tech. Rep. Ser. No. 532 (Geneva) (1973). PI W. Mertz and W.E. Cornatzer, Newer Trace Elements in Nutrition (Marcel Dekker, New York, 1971). c31J.A. Vinson and K.-H. Hsiao, Nutr. Rep. Int. 32 (1985) 1. M F. Kieffer, in: Metals and their compounds in the environment, ed. E. Merian (VCH Verlagsgesellschaft mbH, Weinheim, 1991) p. 481. PI F.H. Nielsen, FASEB J. 1 (1987) 394. C61M.I. Menard and R.J. Cousins, J. Nutr. 113 (1983) 1434. c71S.A. Keller and R.A. Doherty, Am. J. Physiol. 339 (1980) G114. L-81M.E. Hilburn, J.A. Blair, M.J. Coogan, J.L. Porter and J.R.F. Walters, in: Proc. Internat. Conf. on Heavy Metals in the Environment (CEP Consultants, Edinburgh, 1981). c91M.V. Bell, K.F. Kelly and J.R. Sargent, J. Exp. Biol. 102 (1983) 295. DOI S. Friend, Science 265 (1994) 334. Cl11Y. Cho, S. Gorina, P.D. Jeffrey and N.P. Pavletich, Science 265 (1994) 346. Cl21G.M. Clore, J.G. Omichinski, K. Sakaguchi, N. Zambrano, H. Sakamoto, E. Appella and A.M. Gronenborn, Science 265 (1994) 386. Cl31Ph. Moretto, Y. Llabador, R. Ortega, M. Simonoff and L. Razafindrabe, Nucl. Instr. and Meth. B 75 (1993) 511. Cl41Ph. Moretto, Y. Llabador, M. Simonoff, L. Razafindrabe, M. Bara and A. Guiet-Bara, Nucl. Instr. and Meth. B 77 (1993) 275. WI Ph. Moretto, R. Ortega, Y. Llabador, M. Simonoff and J. Bknard, these proceedings (ICNMTAPB, Nucl. Instr. and Meth. B 104 (1995) 292. Cl61M. Cholewa, G.J.F. Legge, H. Weigold, G. Holan and C. Birch, Nucl. Instr. and Meth. B 77 (1993) 282. Legge,H. Weigold, S.M. Cl71M. Cholewa, I.F. Turnbul1,G.J.F. Marcuccio, G. Holan, E. Tomlinson, P.J. Wright, C.P. Dillon, P.A. Lay and A.M. Bonin, these proceedings (ICNMTA’94), Nucl. Instr. and Meth. B 104 (1995) 317. iI181Jing-Xia Zhang, Ben-Jie Wu, Ping Huang, Gui-Fen Yu, Hui-Ying Yao, Ju-Xiang Pan, Zhi-Yu Chao, Jie-Qing Zhu, Xian-Kang Wu, Ying-Mei Gu and Rong-Rong Lu, presented at this conference. (ICNMTA’94). Cl91B.A.W. Verhoef, P.M. Frederik, P.H.H. Bomans, G.J. van der Vusse and M.J.A. de Voigt, these Proceedings (ICNMTA’94), Nucl. Instr. and Meth. B 104 (1995) 311. PO1 P.S.P. Thong, M SC. Thesis, National University of Singapore (1994); c211F. Watt, M. Selley and P.S.P. Thong, these proceedings (ICNMTA’94), Nucl. Instr. and Meth. B 104 (1995) 356. c221F. Watt, T. Lee and P.S.P. Thong, these proceedings (ICNMTA’94), Nucl. Instr. and Meth. B 104 (1995) 361. ~231 P.S.P. Thong, N.C. Law, I. Orlic, D.J.W. Lane and F. Watt, these Proceedings (ICNMTAP4), Nucl. Instr. and Meth. B 104 (1995) 365.
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[24] E. Palsgbrd, U. Lindh, L. Juntti-Berggren, P.-O. Berggren, G.M. Roomans and G.W. Grime, Scanning Microsc., suppl. 8 (1994) 325. [ZS] K. Matsuno, M. Koyama, H. Takeda, K. Abe, K. Sakaruda, T. Miyazaki and K. Kobayashi, Thrombosis Res. 69 (1993) 131. [26] R. Gorodetsky, X.D. Mou, A. Blankenfeld and G. Marx, Am. J. Hematol. 42 (1993) 278. [27] X.X. Tan, C. Tang, A.F. Castoldi, L. Manzo and L.G. Costa, J. Toxicol. Environ. Health 38 (1993) 159. [ZS] S. Yumoto, H. Ohashi, H. Nagai, S. Kakimi, A. Ishikawa, K. Kobayashi, Y. Ogawa and K. Ishii, Nucl. Instr. and Meth. B 75 (1993) 188. [29] G.W. Grime and F. Watt, Nucl. Instr. and Meth. B 30 (1988) 227.
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G.J.F. Legge, J.S. Laird, L.M. Mason, A. Saint, M. Cholewa and D.N. Jamieson, Nucl. Instr. and Meth. B 77 (1993) 153. [3 l] T. Sunde, personal communication. [32] F. Watt, personal communication. [33] E. Johansson, U. Lindh, T. Westermarck, H. Heiskala and P. Santavouri, J. Trace Elem. Electrolytes Health Dis. 4 (1990) 139. [34] U. Lindh and E. Johansson, Biol. Trace Elem. Res. 12 (1987) 351. [35] T.C. Ford and J.M. Graham, in: An Introduction to Centrifugation (BIOS Scientific Publishers, Oxford, 1991) p. 80. [36] S. Thunell, C. Andersson, B. Carlmark, Y. Floderus, S.-O. Griinquist, P. Harper, A. Henrichson and U. Lindh, Europ. J. Clin. Chem. Clin. Biochem. 33 (1995) 179.
VII. LIFE SCIENCES
Beam Interactions with Materials & Atoms
Cell biology, trace elements and nuclear microscopy Ulf Lindh* Centrefor Metal Biology
in Uppsala, Uppsala University Box 53.5,
S-751 21 Upps& Sweden
Abstract
Essential trace elements often function as co-factors in enzymes, the most conspicuous being zinc for which more than 200 dependent enzymes have been found. Selenium is a trace element for which only a few enzymes have been discovered.
Quit recent findings of the importance of zinc-finger proteins in the transcription process in cells have attracted great interest. A protective effect against damage to base-pairs during this process is intriguing. Another exciting discovery is that the tumour-suppressor protein p53 also contains zinc. The development of the nuclear microscope has made possible intracellular studies of trace-element distributions. This paper discusses the progress of recent years concerning nuclear microscopy in cell biology. Progress in our laboratory concerning intracellular distributions of trace elements and the comparison with measurements on fractionated organelles will be presented.
1. Trace elements in cell biology
Except for iron and iodine our knowledge about the essential trace elements is only about 50 years old. About 98% of the human body mass is made up of nine nonmetallic elements. Another 1.89% are formed by the four main electrolytes sodium, magnesium, potassium and calcium. Eleven of the trace elements (Cr, Co, Cu, I, Fe, Mn, MO, Ni, Se, Sn and Zn) occupy just a tiny 0.012% or 8.6 g of the body weight. However, this small fraction exerts a tremendous influence on all body functions that could never have been imagined in earlier times. Whether an element is essential for life depends on its participation in one or several biochemical reactions. The term “essential” is borrowed from the early amino acid and protein chemistry. According to the recommendations of a WHO expert committee, this expression is not optimal when it is applied to trace elements [l]. According to a new, improved definition [2] an element is already useful to the organism and to the maintenance ofhealth when a measurable deficit in the diet reduces the growth and vitality of humans, animals or plants to a reproducible degree. If we start from this definition it becomes plausible that even well-known toxic elements, such as arsenic and lead are needed in minute quantities for the normal functioning of cell metabolism, although they are recognised as toxic in higher concentrations.
*Tel.: + 46-18-18 38 36. Fax: + 46-18-18 38 33. E-mail: ulf. [email protected].
Usually, the ions of trace elements act as coordination centres for building up or stabilising the structure of enzymes and proteins. Exceptions are iron, which is the central atom of haeme in cytochromes and haemoglobin, cobalt in the centre of vitamin Br2, and iodine, which is a constituent of the hormone thyroxine. Chromium seems to be essential for the biosynthesis of the glucose tolerance factor in man [3] which itself seems to be chromium free [4]. The absorption rates differ remarkably between the elements. Table 1 shows these rates for eight trace elements. Nutrients are taken up from water and soil by plants. The least quantity of most trace elements that accumulate is a consequence of their genetically determined cell structure and function that can vary somewhat between the species. However, trace elements that are accidentally present in the soil for geological reasons or by human deposition can accumulate in grass and food crops. They represent unforeseeable risks for the consumer. The minimum biological needs for trace elements of plants, animals and man are only proportionally but not fundamentally different. Even boron makes no exception, although boron is generally considered essential only for plants. Recent findings show that the hydroxylases which are responsible for the transformation of the vitamins Dz and D3 into their corresponding 1,25-dihydroxyderivates, and cholesterol into estradiol, strictly depend on borate ions. Therefore, boron may be a potent protective factor against post-menopausal osteoporosis in women [S].
0168-583X/95/$9.500 1995 Elsevier Science B.V. All rights reserved SSDI 0168-583X(95)00389-4
VII. LIFE SCIENCES
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(1. Lindhi Nucl. Instr. and Meth. in Phys. Res. B 104 (1995) 285-291
Table 1 Absorption rates for some trace elements Trace element in Food
Absorption rate (%)
Molybdenum Selenium Zinc Copper Iron Manganese Chromium (III)
7G30 or less Sk80 or less or more 2&40 or more 10-30 or more 7-15 or more 3-5 or less 0.5-l.Ob
“Depending on the dose, speciation, transformation, health status and interactions ‘Chromate absorption may be somewhat higher, especially in individuals in whom Cr (VI) is not reduced by gastric juice. Very little is still known about the details of uptake into cells and intracellular transport and storage of trace elements. In addition, there are innumerable potential metal-binding ligands in the intestinal lumen. Many nutritional studies have shown marked effects on uptake rates by synthetic chelating agents or naturally occurring metal-binding molecules e.g. citrate and lactoferrin. Binding to intestinal secretions e.g. pancreatic juice and mucus, may facilitate uptake whereas binding to the brush border glycocalyx and intestinal cell membrane may reduce availability [6]. Uptake of copper, iron and zinc appears to be regulated by feedback mechanisms since there is an increased uptake by deficient animals. Parallel effects on uptake of other metals are also frequently observed e.g. Cd and Pb in calcium deficiency and Zn in iron deficiency. However, these findings do not imply common carriers. In suckling animals there is pinocytic uptake of proteins and metal-protein adjuncts in the distal jejunum and ileum [7]. Many studies have suggested that paracellular uptake of metals across the intestine may also be important [S]. Furthermore, there is increasing evidence to suggest that some metals may cross cellular membranes by an uncontrolled, non-selective diffusion process as their neutral lipid-soluble complexes. Examples of such complexes are CdCli and methyl-HgCl. Paracellular uptake across the intestinal epithelium also appears to occur for many oxy-anions, including AsO:-, CrO:-, SeO:-, and VOi- [9].
2. A highlight of trace-element biology Zinc is an essential part of more than 200 enzymes, examples of which are the DNA- and RNA-polymerases. Recently it was discovered that Zn-finger proteins are involved in the cellular transcription process and that they probably also protect base pairs during this process against attack from reactive chemical species. Yet another fascinating discovery is the protein ~53 that is
a tumour suppressor protein. It is believed that inactivation of this protein through mutations is associated with many human cancers [lo]. The three-dimensional structure of this suppressor protein was very recently unveiled by the crystal structure of the p53 core domain [ll] and the solution structure of its oligomerisation domain [ 121. This important molecule is yet another zinc protein.
3. Development of nuclear microscopy in cell biology The technical and methodological arsenal in the hands of biologists for the study of cells and biomolecules is steadily expanding. Electron microscopy has been and still is one very important technique. Its versatility in detecting elemental concentrations and distributions has been strengthened by the introduction of electron energy loss spectroscopy. In addition, we have witnessed the slow but gradual introduction of the nuclear microscope in the arsenal of modern cell biology. Without having the ambition of presenting a complete review, some illustrative examples are given here. In a series of studies, Moretto et al. [13-l 51 have used the nuclear microscope to study cellular pharmacology. In these studies they have achieved intracellular elemental mapping. To visualise the cell nucleus the group used the higher phosphorus content of nuclear material. However, these authors point out that the phosphorus contrast between nucleus and cytoplasm is low and show that Scanning Transmission Ion Microscopy provides far better contrast. In the latter case, it was even possible to map the nuclear membrane [13]. They have extended their study with the human amniotic membrane as an experimental model [14]. In this work they emphasised the beam damages and the consequences for the intracellular distribution of metal ions, the so-called electrolytes, of incubation of the membrane. In the most recent study, Moretto et al. [ 151 focused on the cellular pharmacology of the cancer drug cisplatin. A problem with this kind of therapy is that cancer cells acquire drug resistance. It was not possible to find any elemental differences between resistant and non-resistant cells. However, when cells were exposed to cisplatin, significant differences in trace elements were obvious. Other approaches to cellular pharmacology have been undertaken by the Melbourne group. They used human peripheral lymphocytes from healthy blood donors and a continuous line of human T-lymphocytes. This experimental model was used to test anti-AIDS drugs containing cobalt and tungsten. By intracellular elemental mapping it was shown that the drug accumulated in the cells [16]. By STIM it was shown that the irradiation to extract elemental information induced beam damages that should not influence the results. Another approach was to use a line of African green monkey kidney cells and V79 hamster lung cells to test two classes of drugs
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against the HIV and flavi viruses. The drugs were inorganic polyanions and coordination complexes of chromium in oxidation states (III), (V) or (VI) and their uptake was tested by nuclear microscopy [17]. To study zinc in biological membranes Zhang et al. [18] used both the scanning proton microprobe and synchrotron-radiation induced X-ray fluorescence. Hepatocytes, nerve cells and cardiac cells were exposed to synchrotron light for total zinc concentrations, and the nuclear microprobe was used to get the positional information. The group used membranes of red cells to unveil the zinc concentration in the lipid and protein fractions, respectively. Following the current hypothesis of free-radical generation in ischaemic heart damage, Bas et al. [19] studied ion movements in ischaemic cardiac tissue both by proton- and electron-induced X-ray microanalysis. These authors put special emphasis on preserving both elemental integrity and ultrastructure. The Singapore group has undertaken several interesting studies in cell biology. The involvement of iron, and other trace elements in atherosclerosis was studied in a rabbit model. The highlight of this investigation was that a fivefold increase of iron in atherosclerotically lesioned tissue could be detected by nuclear microscopy [20]. This finding is in support of iron toxicity by radical generation [21]. Another aspect of radical-induced damages was investigated by this group. Parkinsonism was induced in the right hemisphere of rats by injecting a neurotoxin. In this way each animal was its own control. Sections containing the substantia nigra, where the dopaminergic cells are situated, were analysed both on the lesioned side and the control side. This investigation showed an increase of iron in the parkinsonian side [22]. Calcium and activation of cells with adenosine triphosphate were studied in an experimental cellular model (human Chang liver cells). A twenty fold increase in copper was established in activated cells. Blood cells from the ascidian Phallusia mammillata have the amazing capability to accumulate vanadium from the sea water by a factor of 100000 to 1000000. Such cells were submitted to nuclear microscopy by this group confirming earlier results [23]. Palsgard et al. [24] used both electron microscopy, X-ray microanalysis, and nuclear microscopy to study the polarisation phenomena induced in RINmSF- and isolated mouse B-cells by K+-stimulation. This group also made novel use of multivariate statistics to differentiate between intracellular free and bound ions.
4. Other techniques or combinations of techniques providing elemental information with cellular resolution
To be of the best value to cell biology, any elemental technique should be able to provide chemical informa-
287
tion. PIXE and the analytical techniques usually employed in nuclear microscopy seldom offer such possibilities. In certain cases, the use of multivariate statistics may improve the elemental information from PIXE [24]. Often, however, other methods have to be considered. Although the magnesium ion (Mg’+) plays an important role in cell activation, information has until now been limited by the lack of suitable methods for measuring cytosolic free Mgzf. Matsuno et al. [25] measured the concentration of free magnesium ions in resting and activated human platelets using a new fluorescent magnesium-ion indicator mag-fura-2. The concentration of free magnesium ions in resting platelets was 13.13 f 3.40 mg/l and in activated platelets 22.37 + 9.00 mg/l. A technique called diagnostic X-ray spectrometry (DXS), based on X-ray fluorescence, was used to quantify the multiple elemental composition of washed, intact human platelets [26]. They determined the potassium, calcium and zinc concentrations to 3.08 + 1.00, 1.18 f 0.29 mg/g and 35 _I 9 ug/g, respectively. In addition they used X-ray microanalysis in an electron microscope to determine the subcellular concentrations and found zinc to be distributed in the alpha-granules (40%) and the cytoplasm (60%). The importance of cytosolic-free calcium level in lymphocyte activation inspired Tan et al. [27] to investigate changes in [Ca’+] i in T cells caused by mercury compounds, which have been shown to have immunomodulatory and immunotoxic properties. Using fura- as fluorescent Ca” indicator they found that both methyl-mercury and mercuric chloride increased the cytosolic-free calcium concentration in lymphocytes from rat spleen in a concentration-dependent manner [27]. Aluminium toxicity in the rat liver and brain was studied by a combination of PIXE and EELS. PIXE was used to determine elemental concentrations in homogenised cells and isolated nuclei, and by EELS the subcellular distributions were assessed. Aluminium injected in health rats enters the brain through the blood-brain barrier and aluminium was incorporated in cell nuclei both in the brain and in the liver. In the liver parenchymal cells, aluminium was not detected in the cytoplasm but exclusively in the heterochromatin of nuclei [28].
5. Nuclear microscopy in cell biology-capabilities and problems To be of good value to cell biologists, any elemental technique has to allow subcellular resolution. As it has been pointed out by several authors through the years, resolution is one merit of nuclear microscopy. Onemicron beam resolution was achieved roughly simultaneously in Oxford and Melbourne around 1980 [29]. In this sense, the resolution should be interpreted as
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resolution with analytically meaningful particle intensity. Only a few groups have attained sub-micron resolution. Among these are the Oxford group keeping the lead by 0.4 urn [30]. In Uppsala, 0.7~urn resolution was achieved with the same probe-forming lenses at Oxford [31]. With the similar equipment, the group in Singapore has showed 0.6~pm resolution [32]. Neither of these achievements, however, is on routine basis. This slowing down of the development of nuclear microscopes has, of course, hampered the introduction into cell biology. However, l-urn resolution should be adequate for some cell-biological work. Here is presented some attempts by the Uppsala group to exploit the nuclear microscope in cell biology. Lymphocytes [33] and neutrophil (polymorphonuclear) granulocytes [34] were isolated from venous samples of healthy volunteers according to procedures presented earlier. These cell types represent different functions in the circulating blood. They are different also in morphology as granulocytes have granules in the cytoplasm. Other differences are size and presence of organelles in the cytoplasm. Our attempt was to measure trace elements in a few of the cell organelles that are possible to identify in nuclear microscopy and to compare these results with those from analysis of cellular fractions. The choice was to investigate nuclei and mitochondria. Backing this choice was that nuclei are often easily identified in unstained samples and that mitochondria challenge the resolution of nuclear microscopy. Though the methods employed in nuclear microscopy are multielemental, we have chosen to discuss only the values for zinc. This is due to problems associated with other techniques used to assess elemental concentrations in fractionated cells and the fractionation procedure itself. Nuclear microscopy demands unstained objects and this makes identification very difficult. In freeze-dried unstained tissue sections it may be possible to identify individual cells but very seldom any further subcellular details. Working with isolated individual cells that vary in thickness, the problem becomes more severe. In cells trapped onto a backing (formvar, pioloform, etc.) it is often easy to identify the nucleus. Using light directed in, say 45” to the specimen surface, it may be possible to identify other organelles such as mitochondria. To reduce the uncertainty in identification, we have used densitometry on the samples. From the investigation by nuclear microscopy are shown elemental maps of one lymphocyte (Fig. la) and one granulocyte (Fig. 2a). To ease the interpretation, drawings (Figs. lb and 2b) pointing to the important cellular features are incorporated. To prepare highly purified nuclei in high yield, the procedure described by Ford and Graham was used [35]. It involves the use of calcium and magnesium compounds to preserve the integrity of nuclei. The mitochondria were
Mitochondtion
Nucleus
-.~~ .-._,....., ..h
(b)
..,....\
~
‘......
-.___....-
.../’
,-’
Fig. 1. (a) &-map of one lymphocyte from a healthy volunteer. (b) Drawing showing the essential features (nucleus and mitochondrion) of the lymphocyte.
prepared according to the procedure used by Ford and Graham. A Coulter counter was used to count the number of cells in the suspensions. These were adjusted to contain 10” cells per injection volume, which was introduced into the graphite furnace of an atomic absorption spectrophotometer for zinc determination. The results of these determinations are presented in Table 2. In situ measurements by nuclear microscopy of five cells of each type are presented in Table 3. However, the results achieved in both these ways are not directly comparable because AAS measurements present concentrations by wet weight and the nuclear microscopy by dry weight. TO get an idea about the dry weight fraction of white cells, preparations of thrombocyte and plasma free mononuclear and polymorophonuclear white cells were made according to the procedures mentioned above. The resulting volumes were weighed and then oven dried at 120°C overnight. The remaining mass was assumed to represent the dry weight fraction, which was 25-28% for both cell types.
289
U. Lindh iNuc1. Instr. and Meth. in Phys. Rex B 104 (1995) 285-291
Table 2 Concentrations of Zn @g/g d.w.) in fractionated cell organelles from lymphocytes.and neutrophil granulocytes. Values are presented as median values f interquartile ranges for n = 5 measurements Cell type
Nuclear fraction
Mitochondrial fraction
Lymphocytes Neutrophil granulocytes
10.7 f 1.6 13.1 * 2.1
7.2 f 1.4 9.8 + 1.9
Table 3 Concentrations of Zn @g/g d.w.) in cell organelles measured in situ in five cells of each type. Values are presented as median values f interquartile ranges for n = 5 cells ,_,,_....
,,,...’/”
,’
2’
_._ ...____,I .,.. ““‘..,
\ \
Y..
-.._
Segmented nucleus
(b) Fig. 2. (a) Zn-map of one neutrophil granulocyte from a healthy volunteer. (b) Drawing showing the essential features (nucleus and two mitochondria) of the granulocyte.
For the nuclear fraction, the results are in good agreement. This is, however, not so for the mitochondrial fraction. There are several possible explanations to the disagreement. As the nucleus is a far greater part of the cell than a mitochondrion, it is natural that identification is easier. Furthermore, any part of the cytoplasm covering the nucleus should mean fewer errors than for mitochondria. The identification of mitochondria is uncertain, though densitometry was used to aid the localisation. It is not possible to know how much other cell material is present under the beam. In the worst case, other organelles or parts of organelles could be within the irradiated volume. If the cells are intact, the influence from the plasma membrane cannot be avoided.
Cell type
Nuclei
Mitochondria
Lymphocytes Neutrophil granulocytes
14.2 _+2.4 15.8 _+2.8
15.3 f 4.2 14.2 f 4.5
Other causes of the disagreement observed in this experiment could be that the centrifugation and reagents used have influenced the zinc concentration. The fractionation procedure has been designed to maintain organelle integrity from the biological point of view. This does not necessarily mean that the trace-element integrity is well preserved. Identification of cell organelles should be eased by using STIM, but the problem with interference from other organelles in the irradiated volume still remains. The obvious solution to this problem would be sectioning of embedded cells. Evaluations of this approach warrants further experimental studies. Another example of comparisons is the one undertaken in a systematic study of acute intermittent porphyria (AIP). The acute hepatic porphyrias comprise a group of hereditary disqrders of haeme metabolism. In some porphyria gene carriers attacks of generalised neuropathy occur, not seldom with disablement as a result, and in occasional cases with fatal outcome. Since no cure for the genetic condition is yet available, the therapeutic strategy is concentrated on prophylactic measures aimed at avoiding porphyrinogenic environmental factors of notorious significance. In a study presented in detail elsewhere [36] a broad survey of clinical chemical measurements together with assessment of trace-element status was made for patients with AIP. One of the most important discriminating factors between health and disease was the manganese concentration in granulocytes. The latter was determined in single isolated white blood cell by nuclear microscopy. These results were compared
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U. Lindh j Nucl. Instr. and Meth. ilz Phys. Res. 3 IO4 (1995) 285-29:
Table 4 Manganese concentration in neutrophil granulocytes from AIP patients and healthy controls (n = 5) as assessed by nuclear microscopy on single cells or XRF on cell fractions. Concentrations are given as median values + interquartile ranges and are based on dry weight Method
AIP patients
Healthy controls
Nuclear microscopy XRF on cell suspension XRF on cell pellet
3.5 f 0.5 4.1 If: 0.6 3.3 + 0.4
1.2 & 0.3 0.9 2 0.2 0.8 i: 0.3
with measurement of the manganese concentration by X-ray fluorescence. Two approaches were used. Either samples of 50 pl with a known cell concentration (2-3 x lo6 cells) were put onto formvar foils and air-dried or packed pellets of centrifuged granulocytes were weighed and dissolved in nitric acid, diluted and added with an internal standard and eventually put on formvar foils. The results of manganese determination using single cells or cell fractions are shown in Table 4. Estimations of dry weight fractions were done as described above. In the case of manganese total concentration in polymorphonuclear blood cells there is a good agreement between the various analytical procedures used. The conclusions drawn from nuclear microscopy, therefore lies on solid ground.
6. Conclusions For nuclear microscopy to be of real value in cell biology, techniques for preparation and identification of morphology, i.e. organelles, have to be improved. Our experience suggests that individual intact cells e.g. from a culture, is mainly useful for assessing the total concentration. Such information may be beneficial in those cases where skew distributions of trace elements are encountered. If good-quality data on trace-element concentrations are to be extracted from subcellular parts, sectioning of the cells is probably necessary. There is certainly a need for further investigations and exploitations of techniques from X-ray microanalysis carried out in electron microscopes.
Acknowledgements The financial support for the Slim-Up nuclear microscope in Uppsala from the Knut and Alice Wallenberg
foundation is greatfully acknowledged. Thanks go also to Dr. T. Sunde for skilful operation of the Slim-Up.
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