9 1991 by the Humana Press, Inc. All rights of any nature, whatsoever, reserved. 0163-4984/91/2803q)213 $02.00
Effect of Nickel on Oxygen Free Radical Metabolism Inhibition of Superoxide Dismutase and Enhancement of Hydroxydopamine Autoxidation R.
G. M. B E R L Y N E * The Nephrology Section, Brooklyn VA /Vledical Center, Brooklyn, NY 11209
SHAINKIN-KESTENBAUM, C. C A R U S O , AND
Received February 21, 1990; Accepted March 16, 1990
ABSTRACT The effect of nickel on superoxide dismutase activity (SOD), as well as on rate of hydroxydopamine oxidation, was studied in vitro since lipid peroxidation has been implicated in cell damage by nickel, whose toxicity and carcinogenicity are well established. Nickel strongly inhibits SOD activity. The degree of inhibition is directly proportion to the nickel concentration (tested range 0.066 to 0.33 ixg/mL in the reaction mixture); to the substrate concentration (tested range 0.4 x 10-4M to 1.1 x 10-4M 6-hydroxydopamine); and to reaction mixture. Autoxidation of 6-hydroxydopamine was increased by nickel concentrations higher than 15 txg/mL. The combination of excessive oxygen free radical production and inhibition of their elimination by inhibition of SOD activity may contribute to the nickel toxicity that has been reported in industrial accidents, as well as to the high incidence of cancer occurring in nickel workers. It may also contribute to many complications in uremic patients, in w h o m increased serum nickel levels were reported to be in a similar range to those inhibiting SOD. Index Entries: Nickel; superoxide dismutase; oxygen free radi-
cals. *Author to whom all correspondence and reprint requests should be addressed.
Biological Trace Element Research
2]3
VoL 28, 1991
Shainkin-Kestenbaum, Caruso, and Berlyne
214
INTRODUCTION In recent years, the carcinogenicity of nickel compound has become of increasing concern. The high incident of pulmonary cancer occurring in nickel workers was first noticed in 1937 by Baunder (1) and since then, extensive studies were performed concerning the relations of nickel to carcinogenesis, mainly pulmonary carcinogenesis (2-4). In addition, nickel accidents were reported in plastic production, refinery, chemicals, and so on (5). Lipid peroxidation was already suggested as a molecular mechanism for cell damage induced by nickel (6). Since oxygen-free radicals (OFR) can promote a series of chemical reactions, such as, membrane lipid peroxidation, disrupting sulphydryl bonds in protein and enzymes, degradation of DNA molecules, and so on (7). Superoxide dismutase (SOD) is one of the key enzymes protecting aerobic organisms against oxidative damage. It is widely distributed in all body organs and cells, and acts as a catalyst in the dismutation of superoxide radicals (8,9). Studies on the mechanism of action of SOD, suggest that certain arrangements of electrostatic charges are necessary for optimal activity (10). It is interesting to determine if nickel compounds, that may interfere with the specific electrostatic charge of SOD, could affect SOD activity, and thus, cause severe toxicity leading to cell damage.
~TERIALS
AND M E T H O D S
Superoxide dismutase activity was assessed by an indirect negative assay, that determines the degree to which there is inhibition of oxygen radical induced oxidation of 6-hydroxydopamine. A modification of the method of Heikkla et al. (11), as described by us (12), was used, with hepes buffer pH 7.4 0.05M. The reaction mixture contained 3 l~g/mL of bovine erythrocyte SOD (Sigma, St. Louis, MO) and 0.4 x 10-4M - 1 x 10-4M 6-hydroxydopamine hydrochloride as substrate (Sigma, St. Louis, MO). All solutions were prepared in trace element quality water. The reaction was monitored continuously for 10 min at 490nm, using a Beckman DU-7 spectrophotometer (Beckman Instruments, Fullerton, CA). Enzymatic activity was expressed as A SOD at 490nm, where & SOD represents the difference in optical density between the substrate absorbance in the presence of and in the absence of SOD. The nickel effect on SOD activity and on 6-hydroxydopamine oxidation was determined by preincubation of nickel with concentrated enzyme (5 or 10 ~g nickel with 90U enzyme/mL) for 10 m at room temperature. Aliquots of 0.1 mL of enzyme solutions, that had been treated similarly in the presence and absence of nickel were added each to 3 mL reaction mixture and enzymatic activities were determined simultaneously as described above when the nickel effect on 6-hydroxydopamine oxidation was tested. Oxidation was followed immediately after nickel addition at 490nm, simultaneously Biological Trace Element Research
Vol. 28, 1991
Effect of Nickel on Oxygen Free Radical Metabolism
215
with a control of 6-hydroxydopamine. Nickel was prepared from nickel nitrate certified reference stock solution of 1000 p~g/mL (Fisher Scientific, Fair Lawn, NJ). The stock solution was diluted first (1 : 10) with the hepes buffer pH 7.4 0.02M, and then further with the same buffer pH 7.4 0.05M (as in reaction mixture). After testing the pH, appropriate amounts were taken for studying its effect on SOD activity. Nickel effects were analyzed statistically by Student's t-test for nonpaired data.
RESULTS Effect of Nickel on SOD Acti~'ty--(see Fig. l ) Preincubation of 5 ~g of nickel with 90 U SOD reduced enzymatic activity and increased the rate of autoxidation (E,F in comparison to D), but those levels of nickel did not affect substrate oxidation in the absence of enzyme(C), thus, indicating inhibition of SOD by nickel.
Effect of Varying Nickel Concentration on SOD Enzyme Activity During the first 10 min of reaction, the inhibition by nickel increased with time elapsing, and was proportional to nickel concentrations when 5 and 10 ~g/mL were incubated with 90 U of enzyme, with 0.166 and 0.333 p~g/Nickel/mL in reaction mixture (Fig. 2). Both nickel concentrations significantly inhibited enzymatic activity from the third minute, with t values increased with time as shown in Table 1.
Effect of Various Nickel Levels at Various Substrate Concentrations The effect of varying Ni levels at different substrate concentrations during 5 and 10 min is expressed in Table 2(a,b). Inhibition by nickel is related to the following parameters: 1. Nickel Concentration--t values increase proportionately to the Ni concentration; 2. Subtrate Concentration--t values increase proportionately to the substrate concentration; and 3. Reaction Time--t values increase proportionately to the reaction time starting from the third minute. The effect was not significant at 1 and 2 min. Effect of Nickel on S u b s t r a t e Autoxidation---(see Fig. 3) Twenty ~g of nickel enhanced hydroxydopamine oxidation and shortened the time for achieving maximal autoxidation from 5 to 1 min. But whereas a nickel concentration of 0.066 I~g/mL of the reaction mixture Biological Trace Element Research
Vol. 28, 1991
Shainkin-Kestenbaum, Caruso, and Berlyne
216
.........
ii .........i.........i........................... i~...~.....0.~..... ~i........ =
~ 0. OGG~o
2.0
4.g
B.B
Tlme(mln)
8.g
10
0.~}3
Fig. 1. Nickel effect on SOD activity. A-Blank; B-Substrate -0.55 x 10-4 M 6-hydroxydopamine; C-Substrate 4- 5 l~g
nickel; D-Substrate + SOD; and E, F-Duplicates of substrate + SOD in the presence of nickel.
0.08'
E tO ,r
0.06'
0.04 '
O 0.02
0.00
,
2
9
Tim~
iml'n)~
1'0
12
Fig. 2. Nickel effect on SOD activity 90 U of SOD preincubated for 10 min with 5 or 10 I~g of nickel (0.166, 0.333 I~g nickel/rnL of reaction mixture). Control = open squares; Nickel 5 t~g = closed circles; and Nickel 10 p,g = closed squares. was n e e d e d for d e t e c t i n g inhibition of e n z y m a t i c activity, m o r e t h a n 15 ~g of Ni/mL of r e a c t i o n m i x t u r e w e r e n e e d e d for e n h a n c e m e n t of 6-hyd r o x y d o p a m i n e a u t o x i d a t i o n in the s a m e s y s t e m .
DISCUSSION Nickel is p r e s e n t in small a m o u n t s in the earth's crust, a n d is a n essential trace e l e m e n t for m a m m a l i a (5,19). H o w e v e r , i n c r e a s e d nickel levels carry a h i g h risk of toxicity a n d mortality. OFR are i m p l i c a t e d in a variety of d i s o r d e r s in the initiation a n d t h e p r o m o t i o n of carcinogenic Biological Trace Element Research
VoL 28, 1991
Effect of Nickel on Oxygen Free Radical Metabolism
217
Table 1 Nickel Effect on SOD Activity Time (minutes)
Nickel 5 ug
Nickel 10 ug
o
0.1
t
p
D.8
n.s
2
1 .75
Effect of Nickel on Oxygen Free Radical Metabolism Inhibition of Superoxide Dismutase and Enhancement of Hydroxydopamine Autoxidation R.
G. M. B E R L Y N E * The Nephrology Section, Brooklyn VA /Vledical Center, Brooklyn, NY 11209
SHAINKIN-KESTENBAUM, C. C A R U S O , AND
Received February 21, 1990; Accepted March 16, 1990
ABSTRACT The effect of nickel on superoxide dismutase activity (SOD), as well as on rate of hydroxydopamine oxidation, was studied in vitro since lipid peroxidation has been implicated in cell damage by nickel, whose toxicity and carcinogenicity are well established. Nickel strongly inhibits SOD activity. The degree of inhibition is directly proportion to the nickel concentration (tested range 0.066 to 0.33 ixg/mL in the reaction mixture); to the substrate concentration (tested range 0.4 x 10-4M to 1.1 x 10-4M 6-hydroxydopamine); and to reaction mixture. Autoxidation of 6-hydroxydopamine was increased by nickel concentrations higher than 15 txg/mL. The combination of excessive oxygen free radical production and inhibition of their elimination by inhibition of SOD activity may contribute to the nickel toxicity that has been reported in industrial accidents, as well as to the high incidence of cancer occurring in nickel workers. It may also contribute to many complications in uremic patients, in w h o m increased serum nickel levels were reported to be in a similar range to those inhibiting SOD. Index Entries: Nickel; superoxide dismutase; oxygen free radi-
cals. *Author to whom all correspondence and reprint requests should be addressed.
Biological Trace Element Research
2]3
VoL 28, 1991
Shainkin-Kestenbaum, Caruso, and Berlyne
214
INTRODUCTION In recent years, the carcinogenicity of nickel compound has become of increasing concern. The high incident of pulmonary cancer occurring in nickel workers was first noticed in 1937 by Baunder (1) and since then, extensive studies were performed concerning the relations of nickel to carcinogenesis, mainly pulmonary carcinogenesis (2-4). In addition, nickel accidents were reported in plastic production, refinery, chemicals, and so on (5). Lipid peroxidation was already suggested as a molecular mechanism for cell damage induced by nickel (6). Since oxygen-free radicals (OFR) can promote a series of chemical reactions, such as, membrane lipid peroxidation, disrupting sulphydryl bonds in protein and enzymes, degradation of DNA molecules, and so on (7). Superoxide dismutase (SOD) is one of the key enzymes protecting aerobic organisms against oxidative damage. It is widely distributed in all body organs and cells, and acts as a catalyst in the dismutation of superoxide radicals (8,9). Studies on the mechanism of action of SOD, suggest that certain arrangements of electrostatic charges are necessary for optimal activity (10). It is interesting to determine if nickel compounds, that may interfere with the specific electrostatic charge of SOD, could affect SOD activity, and thus, cause severe toxicity leading to cell damage.
~TERIALS
AND M E T H O D S
Superoxide dismutase activity was assessed by an indirect negative assay, that determines the degree to which there is inhibition of oxygen radical induced oxidation of 6-hydroxydopamine. A modification of the method of Heikkla et al. (11), as described by us (12), was used, with hepes buffer pH 7.4 0.05M. The reaction mixture contained 3 l~g/mL of bovine erythrocyte SOD (Sigma, St. Louis, MO) and 0.4 x 10-4M - 1 x 10-4M 6-hydroxydopamine hydrochloride as substrate (Sigma, St. Louis, MO). All solutions were prepared in trace element quality water. The reaction was monitored continuously for 10 min at 490nm, using a Beckman DU-7 spectrophotometer (Beckman Instruments, Fullerton, CA). Enzymatic activity was expressed as A SOD at 490nm, where & SOD represents the difference in optical density between the substrate absorbance in the presence of and in the absence of SOD. The nickel effect on SOD activity and on 6-hydroxydopamine oxidation was determined by preincubation of nickel with concentrated enzyme (5 or 10 ~g nickel with 90U enzyme/mL) for 10 m at room temperature. Aliquots of 0.1 mL of enzyme solutions, that had been treated similarly in the presence and absence of nickel were added each to 3 mL reaction mixture and enzymatic activities were determined simultaneously as described above when the nickel effect on 6-hydroxydopamine oxidation was tested. Oxidation was followed immediately after nickel addition at 490nm, simultaneously Biological Trace Element Research
Vol. 28, 1991
Effect of Nickel on Oxygen Free Radical Metabolism
215
with a control of 6-hydroxydopamine. Nickel was prepared from nickel nitrate certified reference stock solution of 1000 p~g/mL (Fisher Scientific, Fair Lawn, NJ). The stock solution was diluted first (1 : 10) with the hepes buffer pH 7.4 0.02M, and then further with the same buffer pH 7.4 0.05M (as in reaction mixture). After testing the pH, appropriate amounts were taken for studying its effect on SOD activity. Nickel effects were analyzed statistically by Student's t-test for nonpaired data.
RESULTS Effect of Nickel on SOD Acti~'ty--(see Fig. l ) Preincubation of 5 ~g of nickel with 90 U SOD reduced enzymatic activity and increased the rate of autoxidation (E,F in comparison to D), but those levels of nickel did not affect substrate oxidation in the absence of enzyme(C), thus, indicating inhibition of SOD by nickel.
Effect of Varying Nickel Concentration on SOD Enzyme Activity During the first 10 min of reaction, the inhibition by nickel increased with time elapsing, and was proportional to nickel concentrations when 5 and 10 ~g/mL were incubated with 90 U of enzyme, with 0.166 and 0.333 p~g/Nickel/mL in reaction mixture (Fig. 2). Both nickel concentrations significantly inhibited enzymatic activity from the third minute, with t values increased with time as shown in Table 1.
Effect of Various Nickel Levels at Various Substrate Concentrations The effect of varying Ni levels at different substrate concentrations during 5 and 10 min is expressed in Table 2(a,b). Inhibition by nickel is related to the following parameters: 1. Nickel Concentration--t values increase proportionately to the Ni concentration; 2. Subtrate Concentration--t values increase proportionately to the substrate concentration; and 3. Reaction Time--t values increase proportionately to the reaction time starting from the third minute. The effect was not significant at 1 and 2 min. Effect of Nickel on S u b s t r a t e Autoxidation---(see Fig. 3) Twenty ~g of nickel enhanced hydroxydopamine oxidation and shortened the time for achieving maximal autoxidation from 5 to 1 min. But whereas a nickel concentration of 0.066 I~g/mL of the reaction mixture Biological Trace Element Research
Vol. 28, 1991
Shainkin-Kestenbaum, Caruso, and Berlyne
216
.........
ii .........i.........i........................... i~...~.....0.~..... ~i........ =
~ 0. OGG~o
2.0
4.g
B.B
Tlme(mln)
8.g
10
0.~}3
Fig. 1. Nickel effect on SOD activity. A-Blank; B-Substrate -0.55 x 10-4 M 6-hydroxydopamine; C-Substrate 4- 5 l~g
nickel; D-Substrate + SOD; and E, F-Duplicates of substrate + SOD in the presence of nickel.
0.08'
E tO ,r
0.06'
0.04 '
O 0.02
0.00
,
2
9
Tim~
iml'n)~
1'0
12
Fig. 2. Nickel effect on SOD activity 90 U of SOD preincubated for 10 min with 5 or 10 I~g of nickel (0.166, 0.333 I~g nickel/rnL of reaction mixture). Control = open squares; Nickel 5 t~g = closed circles; and Nickel 10 p,g = closed squares. was n e e d e d for d e t e c t i n g inhibition of e n z y m a t i c activity, m o r e t h a n 15 ~g of Ni/mL of r e a c t i o n m i x t u r e w e r e n e e d e d for e n h a n c e m e n t of 6-hyd r o x y d o p a m i n e a u t o x i d a t i o n in the s a m e s y s t e m .
DISCUSSION Nickel is p r e s e n t in small a m o u n t s in the earth's crust, a n d is a n essential trace e l e m e n t for m a m m a l i a (5,19). H o w e v e r , i n c r e a s e d nickel levels carry a h i g h risk of toxicity a n d mortality. OFR are i m p l i c a t e d in a variety of d i s o r d e r s in the initiation a n d t h e p r o m o t i o n of carcinogenic Biological Trace Element Research
VoL 28, 1991
Effect of Nickel on Oxygen Free Radical Metabolism
217
Table 1 Nickel Effect on SOD Activity Time (minutes)
Nickel 5 ug
Nickel 10 ug
o
0.1
t
p
D.8
n.s
2
1 .75
