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Studies of irradiated lysosomes with particular reference to the action of calcium
Q 1967 by .4cndemic Press Inc.
Experimental
Cell Reseurch 45, 487-506 (1967)
487
PRELIMINARY
NOTES
STUDIES OF IRRADIATED PARTICULAR
REFERENCE
LYSOSOMES WITH
TO THE ACTION OF CALCIUM
J. W. HARRIS A.I.N.S.E.
Postdoctoral
Commission
Fellow,
Research
Radiation
Establishment, Received
Biology
Group, Australian
Lucas Heights,
Atomic
Energy
N.S. W., Australia
July 22, 1966l
ISOLATED lysosomes are not usually affected by doses of ionizing radiation which are lethal for most mammals and for many mammalian cells (i.e., below 1000 rads). While this fact argues against a primary role for these organelles in radiation death, the possibility exists that latent damage (not involving enzyme release) imparted to intracellular lysosomes by irradiation might predispose the structures to rupture at a later time, thus contributing to the total radiation effect. Lysosomes held in the cold for various periods after irradiation (5-10 Krads) showed “latent” damage, the same relative effect being observed upon incubation after storage as immediately post-irradiation (Fig. 1). Since intracellular lysosomes are stable at body temperature, activation of latent damage in situ, if it occurred, might be accomplished by some agent other than heat; while irradiated lysosomes disclosed no proclivity towards differential lysis by Triton X-100 [6], agents acting upon the membrane in other ways might disclose weaknesses. Isolated leucocyte lysosomes were used to investigate this possibility. Lysosomes were prepared from rabbit polymorphonuclear leucocytes (PMN) obtained from peritoneal exudates as described earlier [3]. The final suspensions were adjusted to an optical density of 1.5 (520 m,u) and irradiated with 1000 rads of gamma-radiation from spent reactor fuel-elements; the suspensions were kept cold during this procedure. Within 5 min after irradiation 0.2 ml of various compounds (dissolved in sucrose solution) were added to 2.8 ml of lysosome suspension and the tubes held in the cold for a further 60 min. The suspensions were then cleared by centrifugation (50,000 g for 20 min) and the amount of protein in the supernatant determined. Protein leakage from control granules parallels, but slightly exceeds, leakage of ,%glucuronidase and aryl sulfatase (Fig. 2). In most cases, the compounds to be tested (chosen because of their known reactivity with other biological membranes) were added at concentrations which did not, by themselves, cause protein leakage from control lysosomes. Radiation alone did not cause significant protein leakage (2.6f0.9 S.E. per cent above controls in 11 experiments). Negative results were obtained with most agents tested. Post-irradiation treatment with Triton X-100 (0.001-0.05 per cent) or with buffers (pH 5.0-7.0) affected control and irradiated lysosomes similarly. The following compounds, at the final 1 Revised version received October 18, 1966. 31* - 671812
Experimental
Cell Research 45
J. W. Harris concentrations indicated, had little or no differential effect under the conditions studied: HCl (0.005 N), NaOH (0.005 N), L-thyroxine (10m2, 10-4, 1O-B mM), mercuric chloride (lo-l, 1O-3 mM), and reduced and oxidised glutathione (10m2m&f). Protamine sulfate at 0.05 mg/ml (0.025 mg/ml after correction for neutralization by the amount of heparin present) had little effect (9.4k2.4 S.E. per cent above controls in 6 experiments). At higher concentrations of protamine larger effects 100
1
80
sMINUTES
HOURS
Fig. 1.
Fig. 2.
Fig. I.-Decrease in optical density of irradiated lysosomes during incubation at 28°C. Incubation was begun immediately (O-O), 1 hr (O-O), or 2 hr ( l -- 0) post-irradiation. The ordinate is expressed in arbitrary units, as the absolute value is dependant upon the dose and preparatory procedures used [4]. Fig. 2.-Release of protein ( l - l ), aryl sulfatase leucocyte lysosomes during incubation at 37°C.
(O--
l ), and B-glucuronidase
(0
-
0)
from
have been observed, but these are variable and further experiments will be needed to confirm the extent of this action. Exposure of irradiated suspensions to hypertonic sucrose produced a slight differential effect [4]. With calcium chloride there was some evidence of an effect, but the results were somewhat puzzling. At 0.1 mM, calcium released a small but significant amount of protein from irradiated (but not control) lysosomes; in 7 experiments this release amounted to 32.Okl.5 per cent above controls. However, when the concentration of added calcium was increased tenfold (to 1 mM) no leakage was observed. Concentration-dependant effects of calcium were also observed when control lysosomes were incubated at 28°C for 1 hr. In 4 experiments the presence of 0.1 mM calcium during incubation caused a small (10 per cent) but consistent additional decline in optical density and increase in protein release; I mM calcium had little or no effect on these parameters. The effects of calcium were not observed in the presence of NaCl or KC1 (10 m&f); these ions transiently increase the stability of lysosomes during incubation, irradiation, and mechanical stress [4]. It is possible that the increased ionic strength at 1 mM calcium chloride was sufficient to prevent the labilizing action of the cation. Experimental
Cell Research 45
Irradiated
lysosomes
489
Calcium protects cells against radiation damage by preventing leakage of materials through the membrane [5]. However, the cation may act quite differently on intracellular membranes [9]; calcium is a well-known mitochondrial swelling agent and it increases water-loss from phospholipid suspensions consequent to osmolarity changes in the medium [7]. Under certain conditions calcium increases the permeability of lysosomal membranes [8]; since injured cells accumulate calcium [a], this might form a basis for lysosome activation in cellular radiation death. However, intact PMN cells suspended in tris-buffered Hank’s solution and irradiated with 1000 rads did not take up 45Ca from the medium during 2 hr incubation at 37°C; other doses and time periods were not investigated. Varying the calcium concentration of the medium over a wide range had no consistent effect on the degree of lysosomal enzyme release in these cells. The dose used in these experiments (1000 rads) caused no increase in nigrosin staining over the 2 hr observation period, however, and higher doses or more radiosensitive cells might profitably be used in further investigations. Direct ultraviolet microbeam irradiation of eosinophil granules has been shown to produce rapid nuclear degenerative changes [ 11. In other experiments, no differential enzyme release could be observed when various fractions of control or irradiated cells were incubated with irradiated lysosomes. These results provide no support for the suggestion that latent lysosomal membrane damage might contribute to cell death at radiation doses below 1000 rads, at least within the limitations of the methods used. Participation of such mechanisms is not, however, completely excluded by the experiments reported here. While the results indicate that irradiated leucocyte lysosomes may, under certain conditions, be more “fragile” than controls, the effects are small and no correlation with in uivo events is apparent, at least within the hours immediately following irradiation. The interest of Dr G. Watson and the technical assistance of Miss Beverley Koch are gratefully acknowledged. REFERENCES 1. AMENTA, P. S., Anaf. Rec. 142, 81 (1962). 2. CAMERON, R. and SPECTOR, W. G., in The Chemistry of the
Springfield, 1961. 3. HARRIS, J. W., Radiation
Injured Cell, p. 16. Chas.Thomas,
Res. 28, 766 (1966). 4. __ Intern. J. Radiation Biof. In press. 5. MYERS, D. K. and DE WOLFE, D. E., Intern. .7. Radiation Biof. 5, 195 (1962). 6. RAHMAN, Y. E., Radiation Res. 20, 741 (1963). 7. RENDI, R., J. Cell Biol. 27, 83A (1965). 8. SAWANT, P. L., DESAI, I. D. and TAPPEL, A. L., Arch. Biochem. Biophys. 105, 247 9. STREFFER, C. and WILLIAMSON, D. H., Biochem. J. 95, 552 (1965).
Experimental
(1964).
Cell Research 45
Experimental
Cell Reseurch 45, 487-506 (1967)
487
PRELIMINARY
NOTES
STUDIES OF IRRADIATED PARTICULAR
REFERENCE
LYSOSOMES WITH
TO THE ACTION OF CALCIUM
J. W. HARRIS A.I.N.S.E.
Postdoctoral
Commission
Fellow,
Research
Radiation
Establishment, Received
Biology
Group, Australian
Lucas Heights,
Atomic
Energy
N.S. W., Australia
July 22, 1966l
ISOLATED lysosomes are not usually affected by doses of ionizing radiation which are lethal for most mammals and for many mammalian cells (i.e., below 1000 rads). While this fact argues against a primary role for these organelles in radiation death, the possibility exists that latent damage (not involving enzyme release) imparted to intracellular lysosomes by irradiation might predispose the structures to rupture at a later time, thus contributing to the total radiation effect. Lysosomes held in the cold for various periods after irradiation (5-10 Krads) showed “latent” damage, the same relative effect being observed upon incubation after storage as immediately post-irradiation (Fig. 1). Since intracellular lysosomes are stable at body temperature, activation of latent damage in situ, if it occurred, might be accomplished by some agent other than heat; while irradiated lysosomes disclosed no proclivity towards differential lysis by Triton X-100 [6], agents acting upon the membrane in other ways might disclose weaknesses. Isolated leucocyte lysosomes were used to investigate this possibility. Lysosomes were prepared from rabbit polymorphonuclear leucocytes (PMN) obtained from peritoneal exudates as described earlier [3]. The final suspensions were adjusted to an optical density of 1.5 (520 m,u) and irradiated with 1000 rads of gamma-radiation from spent reactor fuel-elements; the suspensions were kept cold during this procedure. Within 5 min after irradiation 0.2 ml of various compounds (dissolved in sucrose solution) were added to 2.8 ml of lysosome suspension and the tubes held in the cold for a further 60 min. The suspensions were then cleared by centrifugation (50,000 g for 20 min) and the amount of protein in the supernatant determined. Protein leakage from control granules parallels, but slightly exceeds, leakage of ,%glucuronidase and aryl sulfatase (Fig. 2). In most cases, the compounds to be tested (chosen because of their known reactivity with other biological membranes) were added at concentrations which did not, by themselves, cause protein leakage from control lysosomes. Radiation alone did not cause significant protein leakage (2.6f0.9 S.E. per cent above controls in 11 experiments). Negative results were obtained with most agents tested. Post-irradiation treatment with Triton X-100 (0.001-0.05 per cent) or with buffers (pH 5.0-7.0) affected control and irradiated lysosomes similarly. The following compounds, at the final 1 Revised version received October 18, 1966. 31* - 671812
Experimental
Cell Research 45
J. W. Harris concentrations indicated, had little or no differential effect under the conditions studied: HCl (0.005 N), NaOH (0.005 N), L-thyroxine (10m2, 10-4, 1O-B mM), mercuric chloride (lo-l, 1O-3 mM), and reduced and oxidised glutathione (10m2m&f). Protamine sulfate at 0.05 mg/ml (0.025 mg/ml after correction for neutralization by the amount of heparin present) had little effect (9.4k2.4 S.E. per cent above controls in 6 experiments). At higher concentrations of protamine larger effects 100
1
80
sMINUTES
HOURS
Fig. 1.
Fig. 2.
Fig. I.-Decrease in optical density of irradiated lysosomes during incubation at 28°C. Incubation was begun immediately (O-O), 1 hr (O-O), or 2 hr ( l -- 0) post-irradiation. The ordinate is expressed in arbitrary units, as the absolute value is dependant upon the dose and preparatory procedures used [4]. Fig. 2.-Release of protein ( l - l ), aryl sulfatase leucocyte lysosomes during incubation at 37°C.
(O--
l ), and B-glucuronidase
(0
-
0)
from
have been observed, but these are variable and further experiments will be needed to confirm the extent of this action. Exposure of irradiated suspensions to hypertonic sucrose produced a slight differential effect [4]. With calcium chloride there was some evidence of an effect, but the results were somewhat puzzling. At 0.1 mM, calcium released a small but significant amount of protein from irradiated (but not control) lysosomes; in 7 experiments this release amounted to 32.Okl.5 per cent above controls. However, when the concentration of added calcium was increased tenfold (to 1 mM) no leakage was observed. Concentration-dependant effects of calcium were also observed when control lysosomes were incubated at 28°C for 1 hr. In 4 experiments the presence of 0.1 mM calcium during incubation caused a small (10 per cent) but consistent additional decline in optical density and increase in protein release; I mM calcium had little or no effect on these parameters. The effects of calcium were not observed in the presence of NaCl or KC1 (10 m&f); these ions transiently increase the stability of lysosomes during incubation, irradiation, and mechanical stress [4]. It is possible that the increased ionic strength at 1 mM calcium chloride was sufficient to prevent the labilizing action of the cation. Experimental
Cell Research 45
Irradiated
lysosomes
489
Calcium protects cells against radiation damage by preventing leakage of materials through the membrane [5]. However, the cation may act quite differently on intracellular membranes [9]; calcium is a well-known mitochondrial swelling agent and it increases water-loss from phospholipid suspensions consequent to osmolarity changes in the medium [7]. Under certain conditions calcium increases the permeability of lysosomal membranes [8]; since injured cells accumulate calcium [a], this might form a basis for lysosome activation in cellular radiation death. However, intact PMN cells suspended in tris-buffered Hank’s solution and irradiated with 1000 rads did not take up 45Ca from the medium during 2 hr incubation at 37°C; other doses and time periods were not investigated. Varying the calcium concentration of the medium over a wide range had no consistent effect on the degree of lysosomal enzyme release in these cells. The dose used in these experiments (1000 rads) caused no increase in nigrosin staining over the 2 hr observation period, however, and higher doses or more radiosensitive cells might profitably be used in further investigations. Direct ultraviolet microbeam irradiation of eosinophil granules has been shown to produce rapid nuclear degenerative changes [ 11. In other experiments, no differential enzyme release could be observed when various fractions of control or irradiated cells were incubated with irradiated lysosomes. These results provide no support for the suggestion that latent lysosomal membrane damage might contribute to cell death at radiation doses below 1000 rads, at least within the limitations of the methods used. Participation of such mechanisms is not, however, completely excluded by the experiments reported here. While the results indicate that irradiated leucocyte lysosomes may, under certain conditions, be more “fragile” than controls, the effects are small and no correlation with in uivo events is apparent, at least within the hours immediately following irradiation. The interest of Dr G. Watson and the technical assistance of Miss Beverley Koch are gratefully acknowledged. REFERENCES 1. AMENTA, P. S., Anaf. Rec. 142, 81 (1962). 2. CAMERON, R. and SPECTOR, W. G., in The Chemistry of the
Springfield, 1961. 3. HARRIS, J. W., Radiation
Injured Cell, p. 16. Chas.Thomas,
Res. 28, 766 (1966). 4. __ Intern. J. Radiation Biof. In press. 5. MYERS, D. K. and DE WOLFE, D. E., Intern. .7. Radiation Biof. 5, 195 (1962). 6. RAHMAN, Y. E., Radiation Res. 20, 741 (1963). 7. RENDI, R., J. Cell Biol. 27, 83A (1965). 8. SAWANT, P. L., DESAI, I. D. and TAPPEL, A. L., Arch. Biochem. Biophys. 105, 247 9. STREFFER, C. and WILLIAMSON, D. H., Biochem. J. 95, 552 (1965).
Experimental
(1964).
Cell Research 45