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Micro-PIXE analysis of macro- and trace elements in blood cells and tumors of patients with breast cancer
Nuclear Instruments and Methods North-Holland, Amsterdam
MICRO-PIXE ANALYSIS TUMORS OF PATIENTS Erland
JOHANSSON”,
in Physics
Research
B22 (1987)
OF MACRO- AND TRACE WITH BREAST CANCER Ulf LINDH”,
Henry
179-183
179
ELEMENTS
JOHANSSON2’
IN BLOOD
and Christer
CELLS
AND
SUNDSTROM3’
L’Gustaf Werner Institute. Box 531, S-75121 Uppsala, Sweden “Departments of Surgery and “Pathology3 University Hospital, S-75185 Uppsala. Sweden
neutrophil granulocytes and cancerous tissue from breast cancer patients, stage I-IV, Elemental profiles of erythrocytes, displayed significant alterations in some essential and nonessential elements. Aluminium and titanium concentrations were increased in cancerous tissue. In the erythrocytes the iron, zinc and magnesium concentrations were about half the normal levels. The hemoglobin value was within the lower normal range. Titanium was observed in the erythrocytes and the neutrophils from the group examined but not in the control group. The neutrophils of the cancerous group displayed significantly increased concentrations of iron, copper and manganese but lowered concentrations of magnesium and zinc. Micro-PIXE might be a useful complement in the detecting of the imbalance of essential and nonessential elements in cancer.
1. Introduction The role of metals in the initiation, progression and activation of cancer has of late gathered much interest. Carroll and Tullis [l] examined cells from lymphoma patients. The cells examined showed an increased concentration of titanium compared with controls. Santoliquido et al. [2] observed increased concentrations of magnesium and zinc and in one patient increased levels of aluminium. Titanium was sought for but was not found. O’Brien et al. [3] demonstrated the presence of titanium and iron in macrophage tumor cells. Altered concentrations of essential and nonessential elements in neoplastic tissue have been demonstrated by several authors. De Jorge [4] observed that the copper concentration increased without a parallel increase of the phosphorous concentration in breast cancer tissue and suggested that accumulation of copper was one of the first tissue disorders in carcinoma of the breast. Mulay et al. [5] showed that the concentrations of copper, manganese, zinc and magnesium were increased in tumor tissue from breast cancer patients. They also demonstrated increased levels of aluminium and titanium in the tissues examined. Altered elemental profiles in erythrocytes and neutrophil granulocytes have been observed in chronic lymphatic leukemia, chronic myeloid leukemia and acute myeloid leukemia by Johansson and Lindh [6], using micro-PIXE. The elemental profiles in erythrocytes displayed increased concentrations of titanium, aluminium and lowered concentrations of zinc. These observations encouraged further investigation as to whether the altered elemental profiles of essential
0168-583X187/$03.50 0 Elsevier Science Publishers (North-Holland Physics Publishing Division
B.V
elements and of aluminium elements), might be involved e.g. breast cancer.
and titanium (or other in other forms of cancer
2. Materials and methods The test group consisted of 13 women operated on for breast carcinoma (stage I-IV) at the Department of Surgery, University Hospital, Uppsala. The mean age was 60 years (range 31-85 years). Blood samples were drawn immediately before surgery (mastectomy). The patients were not subjected to medication before the surgical treatment. The disease was diagnosed at the Pathological Department of the University Hospital of Uppsala. Blood samples of 18 women, agematched, apparently healthy women who had not had any medication served as controls.
3. Cell isolation Venous blood (lOm1) was drawn in Venoject tubes (Terumo) with 0.10 ml of 0.38 M K,EDTA as an anticoagulant, at the same hour in the morning to lessen the effects of diurnal rythms. The separation of blood cells was started within 0.5 h after venepuncture. The various cell types were isolated by centrifugation at different speeds. The erythrocytes were fractioned at 15Og, the platelets at 1OOOg and the neutrophils at 400g. The isolated cells were suspended in 0.32M sucrose. Less than 0.5 ~1 of the suspension were then put on thin Formvar films, 20-40 nm thick, The cells were freeze-dried before analysis. Fifteen II. BIOLOGICAL/MEDICAL
APPLICATIONS
E. Johansson
I80
rf al.
/
Elementul
cells of each
type were selected for nuclear microThe preparation was described in detail by Lindh et al., and Lindh and Johansson [7.8]. probe
analysis
of blood
and tumor.5
6. Assay of glutathione
peroxidase
analysis.
4. Tissue sampling and preparation Breast cancer tissue was obtained from 6 of the patients from which blood cells were also prepared. The samples were frozen. freeze-dried and embedded in paraffin. Sections, 25 pm thick cut with a glass knife were placed over an aperture of 2 mm of a special plexiglass holder. Blood vessels in the tissue were avoided. Five different areas. 200 x 200 pm?. were examined in every sample, the periphery being avoided in case of contamination. Routine histopathologic examination verified the diagnosis of breast cancer in each case.
5. Elemental
analysis of individual
cells and tissues
The microprobe at Studsvik permits the use of 3 X 3 km’ beams of protons of l-5 MeV at a current of 100 pA. The elemental characterisation relies on particle induced X-rays (micro-PIXE). The simultaneous detection of elements from sodium to uranium is possible if present in concentrations higher than 0.5 pgig in cells or tissue. The detection limit is a smoothly varying function of the atomic number but is on the average less than 1 pgig. Determination of the actual mass under the probe was performed using the continuous background radiation being proportional to the mass [9], A technical account of the analytical instrument has been published elsewhere by Lindh and Sunde [lo]. The induced spectra of the characteristic X-rays were collected by a mutichannel analyser for 300-1800 s per point and stored on tape for later treatment. The data reduction followed the scheme presented by Lindh [ 111. This is an interactive program comprising data smoothing, background subtraction and peak identification. The X-ray spectra generated by the proton bombardment are summed and used to yield elemental profiles of lo-15 elements in individual cells and tissues. The concentration data derived from this process were treated by nonparametric statistical techniques. Hence, median values were used as a measure of central tendency and interquartile ranges as the deviation measure. This makes it possible to include measurements resulting in concentrations below the detection limit. The elemental data were then treated by the Cram&r-von Mises test to reveal statistically significant differences. This test is a modification of the Kolmogorov-Smirnov test [ 121.
Glutathione peroxidase (GSH-Px) activity was determined on whole blood after Paglia and Valentine [ 131 with cumene peroxide as a substrate. The cancerous group and the control group (n = 61) was assayed in the same way. The within and between assay precisions for the range of activity was 2.4 and 7.5%.
7. Results and discussion 7. I. from
Compnrision breast
of
cancer
elementul
patients
profiles
with
of
those
of
erythrocytes controls
The elemental profiles of macro- and trace elements in individual blood cells of patients with breast cancer are not fully known. We have examined the elemental profiles of erythrocytes, thrombocytes and neutrophil granulocytes in blood samples from breast cancer patients, stages I-IV. The thrombocytes presented no significant alteration compared with controls and the results are not reported here. Alterations of the elemental profiles were observed in the erythrocytes (fig. 1). Aluminium, vanadium, lanthanum, tellurium, rubidium and bromine were sought but not found, (~0.5 Fgig). The concentrations of magnesium, iron and zinc were about half the normal values. The mean hemoglobin value was 123 g/l which is within the lower normal range. Lowered magnesium and zinc concentration might impair cell metabolism by reducing ATP and the GSHIGSH-Px protection chain [ 141. The copper concentration was significantly higher (P < 0.001) in the erythrocytes of the cancerous group than in erythrocytes of the control group. The procedure of the cell preparation and analysis was the same in both groups. Higher copper values in the erythrocytes are known to induce hemolysis [ 15,161. The
Mg Ca
,
Ti
Ml-l
CU Zn
-0
Mahqn COhOl LMalign Con’ro’iL 1
_‘_
2
~
173I,
5
Concentration
~10 In ug/g
20
~- -A 50 x)0
(* l/10 n-g/g)
Fig. 1. Elemental profiles of erythrocytes from breast cancer patients, stages I-IV, compared with controls. The concentrations are given as median values and the arrows indicate interquartile ranges, pg/g dry tissue.
finding of titanium is interesting in view of the increased titanium concentration in the erythrocytes in patients with leukemia [h]. Changes in the GSHIGSHPx chain might impair the oxygen metabolism and necessitate a nonenzymatic protection system, e.g. an inorganic scavenger like titanium which is known to create stable forms of hydrogen peroxides [6]. Ionic aluminium and titanium are also good scavengers of O”- which might stabilize the effects of this oxygen metabolite. The increased concentrations of the nonessential elements might thus be secondary effects due to disturbed oxygen metab~~lism. Titanium and ajuminiLim are not considered to be elements essential to man. GSH-Px is a selenium dependent enzyme which, together with catalase and superoxide, dismutases demetabolites (hydrogen peroxides, toxify oxygen superoxides). GSH-Px activity of whole blood of the cancerous group (mean activity) was (307 _f 30) pkatal/l. A control group assayed in the same way resulted in (315 26.6) pkatal/l (B. Jones, personal communication). The increase in the activity was unexpected f17]. When using cumeneperoxide the selenium dependent and nonselenium dependent enzyme will contribute both to the total activity. Provided the selenium dependent GSH-Px activity is decreased in cancer of the mammae, the observed increased activity might be a compensation reaction of S-transferases [ 181. Further investigations are essential to separate the contribution of GSH-Px and S-transfcrases. 7.2. Comparison of elemental profiles of neutrophils from bred cancer patients with those qf controls En the neutrophi~s of the test group si~ni~cant altions were observed for copper (Y<0.05). iron (P< O.OOl), manganese (P < 0.001). zinc (P < O.OUl) and magnesium in comparison with the control group (fig. 2). Aluminium, vanadium, lanthanum, tellurium.
w Ca Mn Fe Cu Zn
Concentratron
in ug,‘g
Fig. 2. Elemental profiles of neutrophil granulocytes of breast cancer patients. stages I-IV, compitred with controls. The concentrations of the elements are given as median values, pgig dry weight.
bromine and rubidium were sought for but not found (41.5 @g/g). The increased concentrations of iron, manganese and copper and lowered concentrations of magnesium and zinc might indicate activated cells [19.20]. Gawlik et al. [21] showed recently that an increased supply of aluminium in the diet decreased the concentrations of selenium and zinc in the plasma of rats (blood cells were not examined). This observation is of great interest because a low selenium status might be an early indicator of an infectious episode in man (22,231. Hence a low selenium c~)ncentration in man may not necessarily be presumptive evidence of cancer. 7.3. Elernentnl profiles of mulignant tissue - irnhnlante of essential und nonessential elements Tumor tissue from six patients with breast cancer were analysed by the nuclear microprobe for essential and nonessential elements. Increased concentrations (in comparison with literature values) of copper, aluminium, titanium were observed (241 (fig. 3). It is interesting to note that the concentrations of elements in noncancer tissue from one patient operated on for a were altered: copper: 2.2 /Lgig, second time aiuminium: 1 fig/g. lanthanum: 1 pg!g; but titanium was not detectable (4.5 /*g/g, dry weight). These data for the nonessential elements aluminium and titanium in noncancer tissue are similar to the literature values. Aluminium is known to compete with magnesium [25,26] and thus decrease the Mg-ATP production e.g. by interacting with hexokinase. Aluminium is also known to bind to chromatin in the nucleus 1271. Provided afuminium binds to the cell nucleus, severai interactions are possible. The genetic response might be impaired by gene conjugati~~ns which the repair systems cannot handle. If aluminium is introduced into the DNA strands, crosslinking might occur and the cell would not be able to perform the genetic program. The concentration of rubidium in cancerous tissue was increased compared with the literature value. This increase is in accordance with the findings of Rizk and Sky-peck [ZS]. The biochemical role of rubidium in tissue is not known. Vandenhaute et al. [20] reported recently a low rubidium concentration in liver. spleen and thymus of mice that were on a selenium-deficient diet. It is well known that the Na/K flux is carefully regulated in the cells. There may be a similar regulation of r~lbidium/cesium which has been altered in cancer tissue. The results indicated similarities between nonessential element profiles of blood cells in leukemic patients and breast cancer tissue. In leukemia, aluminium and titanium were found in the erythrocytes and the neutrophils. In breast cancer patients, titanium was obserII. BIOLOGICAL/MEDICAL
APPLICATIONS
E. Johansson
182
et al. I Elemental analysis of blood and tumors TISSUE
1
2
5
10
20
50
100 L.R.’
Al
0.5
Mg
46
Ca
150
TI
0.37
v
ND
Mn
0.06
Fe
14
CU
1.5
zn *
4
Br
22
Rb
9.2
La Te
113
1
2
5
10
20
50
100
Fig. 3. Tumor tissue from patients with breast cancer (n = 6, stages I-IV). The concentrations are presented as median values of 5 examined areas 200 x 200 pm’. All data in pgig, dry weight. L.R. means literature references [24,2X]. All values are mean values except Br which was presented as a median value. The Br and Tc literature data were converted to dry weight basis.
ved in the erythrocytes only, but both aluminium and identified in cancerous tissue. titanium were Aluminium might be a marker of malignancy because in the cells examined in acute leukemia, aluminium was observed in a greater frequency than in the chronic leukemias. The results indicate directions for further investigations with micro-PIXE. Comparison of elemental profiles of blood cells and tissue might be of diagnostic as well as therapeutic value to monitor different stages in cancer.
Acknowledgements Financial support from the Swedish Board for Technical Development and the Swedish Natural Science Research Council is gratefully acknowledged.
References
111K.G. PI P.M.
Carroll and J.L. Tullis, Nature 217 (1968) 1172. Santoliquido, H.W. Southwick and J.H. Olwin, Surgery Gynec. Obstet. 142 (1976) 65. G.J.F. Legge, T.R. Bradley and G.S. 131 P.M. O’Brien, Hodgson, Australas. Phys. Engin. Sci. in Med. 5 (1982) 30. I41 F.B. de Jorge, G.J. Sampalo Jr, J.L. Diurdes and A.B. Cintra, Clin. Chim. Acta 12 (1965) 403. ISI I.L. Mulay, R.R. Ray, B.E. Knox and N.H. Suhr, J. Natl. Cancer Inst. 47 (1971) 1.
[6] E. Johansson and U. Lindh, Nucl. Instr. and Meth. B3 (1984) 637. [7] U. Lindh, E. Johansson and L. Gille, Nucl. Instr. and Meth. B3 (1984) 631. [S] U. Lindh and E. Johansson, Biol. Trace Elem. Res.. in press. [9] U. Lindh, Nucl. Instr. and Meth. 193 (1982) 343. [lo] U. Lindh and T. Sunde, Nucl. Instr. and Meth. BlOill (1985) 703. Ill] U. Lindh, Anal. Chim. Acta 150 (1983) 233. [12] W.J. Conover, Practical Nonparametric Statistics, 2nd ed. (Wiley, New York, 1980). [13] D.E. Paglia and W.N. Valentine, J. Lab. Clin. Med. 70 (1967) 158. [14] E. Beutler, Red Cell Metabolism, 2nd ed. (Grune and Stratton, New York. 1975). [15] H.K. Chuttani, P.S. Gupta, S. Gulatti and D.N. Gupta, Am. J. Med. 39 (1965) X49. [16] V.F. Fairbanks, Ann. Int. Med. 120 (1967) 428. 1171 M.F. Robinson. P.J. Godfrey, C.D. Thomson, H. Rea and A.M. Rij, Am. J. Clin. Nutr. 32 (1979) 1477. [18] R.F. Burk, K. Nischki, R.A. Lawrence and B. Chance, J. Biol. Chem. 253 (1978) 43. [19] U. Lindh and E. Johansson, Neurotoxicol. 4 (1983) 177. [20] E. Johansson. U. Lindh, T. Alanen, T. Westermarck, H. Heiskala. P. Santavuori and I. Elovara, Med. Biol. 62 (1984) 139. [21] R. Gawlik. P. Bratter, W. Gatschke, W. Meyer-Sabellek, Trace Element Analytical Chemistry in Medicine and Biology, Abstract 4th GSF Conf. (20-23 April, 1986) Munich, FRG. 1221 R.E. Serfass and H.E. Ganther. Nature 255 (1975) 640. 1231 R. Boyne and J.R. Arthur, J. Comp. Path. 91 (1981) 271.
E. Johansson
et al. I Elemental analysis of blood and tumors
[24] G.V. Iyengar, W.E. Kollmer and H.J.M. Bowen, The Elemental Composition of Human Tissues and Body Fluids (Verlag Chemie. Weinheim, New York, 1978). [25] W. Harrison, E. Codd and R.M. Gray, Lancet II (1972) 277. [26] F.C. Womack and S.P. Colowick, Proc. Nat]. Acad. Sci. 76 (1979) 5080. [27] U. de Boni, M. Seger and D.R. Crapper McLachlan, Neurotoxicol. 1 (1980) 65.
183
[28] S.L. Rizk and H.H. Sky-Peck, Cancer Res. 44 (1984) 5390. [29] J. Vandenhaute, W. Maenhaut, H.A. Van Rinsvelt, R.W. Hurd and J.M. Andres, Trace Element Analytical Chemistry in Medicine and Biology, Abstract 4th GSF Conf. (20-23 April, 1986) Munich, FRG.
II. BIOLOGICAL/MEDICAL
APPLICATIONS
MICRO-PIXE ANALYSIS TUMORS OF PATIENTS Erland
JOHANSSON”,
in Physics
Research
B22 (1987)
OF MACRO- AND TRACE WITH BREAST CANCER Ulf LINDH”,
Henry
179-183
179
ELEMENTS
JOHANSSON2’
IN BLOOD
and Christer
CELLS
AND
SUNDSTROM3’
L’Gustaf Werner Institute. Box 531, S-75121 Uppsala, Sweden “Departments of Surgery and “Pathology3 University Hospital, S-75185 Uppsala. Sweden
neutrophil granulocytes and cancerous tissue from breast cancer patients, stage I-IV, Elemental profiles of erythrocytes, displayed significant alterations in some essential and nonessential elements. Aluminium and titanium concentrations were increased in cancerous tissue. In the erythrocytes the iron, zinc and magnesium concentrations were about half the normal levels. The hemoglobin value was within the lower normal range. Titanium was observed in the erythrocytes and the neutrophils from the group examined but not in the control group. The neutrophils of the cancerous group displayed significantly increased concentrations of iron, copper and manganese but lowered concentrations of magnesium and zinc. Micro-PIXE might be a useful complement in the detecting of the imbalance of essential and nonessential elements in cancer.
1. Introduction The role of metals in the initiation, progression and activation of cancer has of late gathered much interest. Carroll and Tullis [l] examined cells from lymphoma patients. The cells examined showed an increased concentration of titanium compared with controls. Santoliquido et al. [2] observed increased concentrations of magnesium and zinc and in one patient increased levels of aluminium. Titanium was sought for but was not found. O’Brien et al. [3] demonstrated the presence of titanium and iron in macrophage tumor cells. Altered concentrations of essential and nonessential elements in neoplastic tissue have been demonstrated by several authors. De Jorge [4] observed that the copper concentration increased without a parallel increase of the phosphorous concentration in breast cancer tissue and suggested that accumulation of copper was one of the first tissue disorders in carcinoma of the breast. Mulay et al. [5] showed that the concentrations of copper, manganese, zinc and magnesium were increased in tumor tissue from breast cancer patients. They also demonstrated increased levels of aluminium and titanium in the tissues examined. Altered elemental profiles in erythrocytes and neutrophil granulocytes have been observed in chronic lymphatic leukemia, chronic myeloid leukemia and acute myeloid leukemia by Johansson and Lindh [6], using micro-PIXE. The elemental profiles in erythrocytes displayed increased concentrations of titanium, aluminium and lowered concentrations of zinc. These observations encouraged further investigation as to whether the altered elemental profiles of essential
0168-583X187/$03.50 0 Elsevier Science Publishers (North-Holland Physics Publishing Division
B.V
elements and of aluminium elements), might be involved e.g. breast cancer.
and titanium (or other in other forms of cancer
2. Materials and methods The test group consisted of 13 women operated on for breast carcinoma (stage I-IV) at the Department of Surgery, University Hospital, Uppsala. The mean age was 60 years (range 31-85 years). Blood samples were drawn immediately before surgery (mastectomy). The patients were not subjected to medication before the surgical treatment. The disease was diagnosed at the Pathological Department of the University Hospital of Uppsala. Blood samples of 18 women, agematched, apparently healthy women who had not had any medication served as controls.
3. Cell isolation Venous blood (lOm1) was drawn in Venoject tubes (Terumo) with 0.10 ml of 0.38 M K,EDTA as an anticoagulant, at the same hour in the morning to lessen the effects of diurnal rythms. The separation of blood cells was started within 0.5 h after venepuncture. The various cell types were isolated by centrifugation at different speeds. The erythrocytes were fractioned at 15Og, the platelets at 1OOOg and the neutrophils at 400g. The isolated cells were suspended in 0.32M sucrose. Less than 0.5 ~1 of the suspension were then put on thin Formvar films, 20-40 nm thick, The cells were freeze-dried before analysis. Fifteen II. BIOLOGICAL/MEDICAL
APPLICATIONS
E. Johansson
I80
rf al.
/
Elementul
cells of each
type were selected for nuclear microThe preparation was described in detail by Lindh et al., and Lindh and Johansson [7.8]. probe
analysis
of blood
and tumor.5
6. Assay of glutathione
peroxidase
analysis.
4. Tissue sampling and preparation Breast cancer tissue was obtained from 6 of the patients from which blood cells were also prepared. The samples were frozen. freeze-dried and embedded in paraffin. Sections, 25 pm thick cut with a glass knife were placed over an aperture of 2 mm of a special plexiglass holder. Blood vessels in the tissue were avoided. Five different areas. 200 x 200 pm?. were examined in every sample, the periphery being avoided in case of contamination. Routine histopathologic examination verified the diagnosis of breast cancer in each case.
5. Elemental
analysis of individual
cells and tissues
The microprobe at Studsvik permits the use of 3 X 3 km’ beams of protons of l-5 MeV at a current of 100 pA. The elemental characterisation relies on particle induced X-rays (micro-PIXE). The simultaneous detection of elements from sodium to uranium is possible if present in concentrations higher than 0.5 pgig in cells or tissue. The detection limit is a smoothly varying function of the atomic number but is on the average less than 1 pgig. Determination of the actual mass under the probe was performed using the continuous background radiation being proportional to the mass [9], A technical account of the analytical instrument has been published elsewhere by Lindh and Sunde [lo]. The induced spectra of the characteristic X-rays were collected by a mutichannel analyser for 300-1800 s per point and stored on tape for later treatment. The data reduction followed the scheme presented by Lindh [ 111. This is an interactive program comprising data smoothing, background subtraction and peak identification. The X-ray spectra generated by the proton bombardment are summed and used to yield elemental profiles of lo-15 elements in individual cells and tissues. The concentration data derived from this process were treated by nonparametric statistical techniques. Hence, median values were used as a measure of central tendency and interquartile ranges as the deviation measure. This makes it possible to include measurements resulting in concentrations below the detection limit. The elemental data were then treated by the Cram&r-von Mises test to reveal statistically significant differences. This test is a modification of the Kolmogorov-Smirnov test [ 121.
Glutathione peroxidase (GSH-Px) activity was determined on whole blood after Paglia and Valentine [ 131 with cumene peroxide as a substrate. The cancerous group and the control group (n = 61) was assayed in the same way. The within and between assay precisions for the range of activity was 2.4 and 7.5%.
7. Results and discussion 7. I. from
Compnrision breast
of
cancer
elementul
patients
profiles
with
of
those
of
erythrocytes controls
The elemental profiles of macro- and trace elements in individual blood cells of patients with breast cancer are not fully known. We have examined the elemental profiles of erythrocytes, thrombocytes and neutrophil granulocytes in blood samples from breast cancer patients, stages I-IV. The thrombocytes presented no significant alteration compared with controls and the results are not reported here. Alterations of the elemental profiles were observed in the erythrocytes (fig. 1). Aluminium, vanadium, lanthanum, tellurium, rubidium and bromine were sought but not found, (~0.5 Fgig). The concentrations of magnesium, iron and zinc were about half the normal values. The mean hemoglobin value was 123 g/l which is within the lower normal range. Lowered magnesium and zinc concentration might impair cell metabolism by reducing ATP and the GSHIGSH-Px protection chain [ 141. The copper concentration was significantly higher (P < 0.001) in the erythrocytes of the cancerous group than in erythrocytes of the control group. The procedure of the cell preparation and analysis was the same in both groups. Higher copper values in the erythrocytes are known to induce hemolysis [ 15,161. The
Mg Ca
,
Ti
Ml-l
CU Zn
-0
Mahqn COhOl LMalign Con’ro’iL 1
_‘_
2
~
173I,
5
Concentration
~10 In ug/g
20
~- -A 50 x)0
(* l/10 n-g/g)
Fig. 1. Elemental profiles of erythrocytes from breast cancer patients, stages I-IV, compared with controls. The concentrations are given as median values and the arrows indicate interquartile ranges, pg/g dry tissue.
finding of titanium is interesting in view of the increased titanium concentration in the erythrocytes in patients with leukemia [h]. Changes in the GSHIGSHPx chain might impair the oxygen metabolism and necessitate a nonenzymatic protection system, e.g. an inorganic scavenger like titanium which is known to create stable forms of hydrogen peroxides [6]. Ionic aluminium and titanium are also good scavengers of O”- which might stabilize the effects of this oxygen metabolite. The increased concentrations of the nonessential elements might thus be secondary effects due to disturbed oxygen metab~~lism. Titanium and ajuminiLim are not considered to be elements essential to man. GSH-Px is a selenium dependent enzyme which, together with catalase and superoxide, dismutases demetabolites (hydrogen peroxides, toxify oxygen superoxides). GSH-Px activity of whole blood of the cancerous group (mean activity) was (307 _f 30) pkatal/l. A control group assayed in the same way resulted in (315 26.6) pkatal/l (B. Jones, personal communication). The increase in the activity was unexpected f17]. When using cumeneperoxide the selenium dependent and nonselenium dependent enzyme will contribute both to the total activity. Provided the selenium dependent GSH-Px activity is decreased in cancer of the mammae, the observed increased activity might be a compensation reaction of S-transferases [ 181. Further investigations are essential to separate the contribution of GSH-Px and S-transfcrases. 7.2. Comparison of elemental profiles of neutrophils from bred cancer patients with those qf controls En the neutrophi~s of the test group si~ni~cant altions were observed for copper (Y<0.05). iron (P< O.OOl), manganese (P < 0.001). zinc (P < O.OUl) and magnesium in comparison with the control group (fig. 2). Aluminium, vanadium, lanthanum, tellurium.
w Ca Mn Fe Cu Zn
Concentratron
in ug,‘g
Fig. 2. Elemental profiles of neutrophil granulocytes of breast cancer patients. stages I-IV, compitred with controls. The concentrations of the elements are given as median values, pgig dry weight.
bromine and rubidium were sought for but not found (41.5 @g/g). The increased concentrations of iron, manganese and copper and lowered concentrations of magnesium and zinc might indicate activated cells [19.20]. Gawlik et al. [21] showed recently that an increased supply of aluminium in the diet decreased the concentrations of selenium and zinc in the plasma of rats (blood cells were not examined). This observation is of great interest because a low selenium status might be an early indicator of an infectious episode in man (22,231. Hence a low selenium c~)ncentration in man may not necessarily be presumptive evidence of cancer. 7.3. Elernentnl profiles of mulignant tissue - irnhnlante of essential und nonessential elements Tumor tissue from six patients with breast cancer were analysed by the nuclear microprobe for essential and nonessential elements. Increased concentrations (in comparison with literature values) of copper, aluminium, titanium were observed (241 (fig. 3). It is interesting to note that the concentrations of elements in noncancer tissue from one patient operated on for a were altered: copper: 2.2 /Lgig, second time aiuminium: 1 fig/g. lanthanum: 1 pg!g; but titanium was not detectable (4.5 /*g/g, dry weight). These data for the nonessential elements aluminium and titanium in noncancer tissue are similar to the literature values. Aluminium is known to compete with magnesium [25,26] and thus decrease the Mg-ATP production e.g. by interacting with hexokinase. Aluminium is also known to bind to chromatin in the nucleus 1271. Provided afuminium binds to the cell nucleus, severai interactions are possible. The genetic response might be impaired by gene conjugati~~ns which the repair systems cannot handle. If aluminium is introduced into the DNA strands, crosslinking might occur and the cell would not be able to perform the genetic program. The concentration of rubidium in cancerous tissue was increased compared with the literature value. This increase is in accordance with the findings of Rizk and Sky-peck [ZS]. The biochemical role of rubidium in tissue is not known. Vandenhaute et al. [20] reported recently a low rubidium concentration in liver. spleen and thymus of mice that were on a selenium-deficient diet. It is well known that the Na/K flux is carefully regulated in the cells. There may be a similar regulation of r~lbidium/cesium which has been altered in cancer tissue. The results indicated similarities between nonessential element profiles of blood cells in leukemic patients and breast cancer tissue. In leukemia, aluminium and titanium were found in the erythrocytes and the neutrophils. In breast cancer patients, titanium was obserII. BIOLOGICAL/MEDICAL
APPLICATIONS
E. Johansson
182
et al. I Elemental analysis of blood and tumors TISSUE
1
2
5
10
20
50
100 L.R.’
Al
0.5
Mg
46
Ca
150
TI
0.37
v
ND
Mn
0.06
Fe
14
CU
1.5
zn *
4
Br
22
Rb
9.2
La Te
113
1
2
5
10
20
50
100
Fig. 3. Tumor tissue from patients with breast cancer (n = 6, stages I-IV). The concentrations are presented as median values of 5 examined areas 200 x 200 pm’. All data in pgig, dry weight. L.R. means literature references [24,2X]. All values are mean values except Br which was presented as a median value. The Br and Tc literature data were converted to dry weight basis.
ved in the erythrocytes only, but both aluminium and identified in cancerous tissue. titanium were Aluminium might be a marker of malignancy because in the cells examined in acute leukemia, aluminium was observed in a greater frequency than in the chronic leukemias. The results indicate directions for further investigations with micro-PIXE. Comparison of elemental profiles of blood cells and tissue might be of diagnostic as well as therapeutic value to monitor different stages in cancer.
Acknowledgements Financial support from the Swedish Board for Technical Development and the Swedish Natural Science Research Council is gratefully acknowledged.
References
111K.G. PI P.M.
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