Toxicology, 64 (1990) 1--17 Elsevier Scientific Publishers Ireland Ltd.
Nickel induced lipid peroxidation in the rat: correlation with nickel effect on antioxidant defense systems Manoj Misra, Ricardo E. Rodriguez and Kazimierz S. Kasprzak Laboratory of Comparative Carcinogenesis, Division of Cancer Etiology, National Cancer Institute, Frederick Cancer Research Facility, Frederick, MD 21701 (U.S.A.) (Received March 14th, 1990 ; accepted June 1st, 1990)
Summary Lipid peroxidation (LPO) and alterations in cellular systems protecting against oxidative damage were determined in the liver, kidney and skeletal muscle of male F344/NCr rats, 1 h to 3 days after a single intraperitoneal (i.p.) injection of 107 /~mol nickel(II)acetate per kg body weight. At 3 h, when tissue nickel concentrations were highest, the following significant (at least, P < 0.05) effects were observed: in kidney, increased LPO (by 43°70), increased renal iron (by 24%), decreased catalase (CAT) and glutathione peroxidase (GSH-Px) activities (both by 15%), decreased glutathione (GSH) concentration (by 20 %), decreased glutathione reductase (GSSG-R) activity (by 10%), and increased glutathione-S-transferase (GST) activity (by 44 070); the activity of superoxide dismutase (SOD) and yglutamyl transferase (GGT), as well as copper concentration, were not affected. In the liver, nickel effects included increased LPO (by 30%), decreased CAT and GSH-Px activities (both by 15070), decreased GSH level (by 33%), decreased GSSG-R activity (by 10 070) and decreased GST activity (by 35070); SOD, GGT, copper, and iron remained unchanged. In muscle, nickel treatment decreased copper content (by 43070) and the SOD activity (by 30070) with no effects on other parameters. In blood, nickel had no effect on CAT and GSH-Px, but increased the activities of alanine-(ALT) and aspartate-(AST) transaminases to 330070 and 240% of the background level, respectively. In conclusion, nickel treatment caused profound cell damage as indicated by increased LPO in liver and kidney and leakage of intracellular enzymes, ALT and AST to the blood. The time pattern of the resulting renal and hepatic LPO indicated a possible contribution to its magnitude from an increased concentration of nickel and concurrent inhibition of CAT, GSH-Px and GSSG-R, but not from increased iron or copper levels. The oxidative damage expressed as LPO was highest in the kidney and lowest in the muscle, which concurs with the corresponding ranking of nickel uptake by these tissues.
Key words: Nickel; Lipid peroxidation; Cellular systems; Defense systems
Introduction
The toxicity and carcinogenesis of nickel compounds in man and experimental Address all correspondence to: Manoj Misra, Ph.D., National Cancer Institute, Frederick Cancer Research Facility, Building 538, Room 205E, Frederick, MD 21701, U.S.A. Printed and Published in Ireland
animals have been well documented [1,2]. The exact mechanism of their action, however, remains unknown. One possible mode by which nickel causes cell death and/or damage to the genetic material may involve oxidative reactions such as nickel-induced lipid peroxidation (LPO) [2--4] or nickel-catalyzed oxidative modification of some proteins [5]. Inhibition by nickel of cellular defense systems including glutathione (GSH), glutathione-S-transferase (GST), glutathione reductase (GSSG-R) [6,7], catalase (CAT) and glutathione peroxidase (GSH-Px) [8], may further increase the oxidative damage. LPO constitutes a free radical oxidation process in which polyunsaturated fatty acids of the cell membrane decompose to yield, among others, highly reactive lipid hydroperoxides, H202, hydroxyl radical (OH.), and malondialdehyde [9,10]. Malondialdehyde has been shown to cause cross-linking and polymerization of membrane components [I1] and may contribute to the mutagenic, genotoxic and carcinogenic effects [12]. Oxygen radicals (superoxide, 02-; OH" etc.,) have been proposed to play a crucial role in cell neoplastic transformation. Reactive oxygen species cause extensive strand breakage of DNA [13], protein-DNA crosslinks [14] and oxidative modification of DNA bases [15]. Oxidative damage from these radicals can also be enhanced by decreased activity of the cell's defensive systems. H202 can cause DNA degradation [16]. Its action can be enhanced by reduction to the extremely reactive OH" radical with various reductive agents like Fe2÷, Cu ~÷ etc. and result in cellular damage [17]. In biological systems, there exist enzymes which are capable of controlling the cytotoxic effects of active oxygen species, including CAT, GSH-Px and superoxide dismutase (SOD) [18] which can protect the cells from accumulating H202, hydroperoxides, and superoxide. These three enzymes appear to work in conjunction with each other with the purpose of minimizing the cell's exposure to reactive intermediates of dioxygen reduction. To elucidate the biochemical mechanism of oxidative damage due to acute nickel intoxication in rodents, we examined the effects of nickel on cellular iron and copper, cellular enzymes (ALT, AST), oxidation defense systems (GSH, GST, GSSG-R, GSH-Px, CAT, and SOD) and LPO in order to correlate the severity of LPO with changing metal concentrations and activities of the defense systems. Materials and methods Chemicals Bovine liver catalase, F.W. = 250 000 was purchased from Worthington Biochemicals, Freehold, NJ. Trizma buffer, phosphate buffer, 30°7o hydrogen peroxide (H202) , glutathione reductase (GSSG-R), xanthine oxidase, sodium azide (NAN3), reduced glutathione (GSH), oxidized glutathione (GSSG), sulfosalicylic acid (SSA), 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB), 1-chloro-2,4-dinitroben zene (CDNB), fl-nicotinamide adenine dinucleotide, reduced form (NADH), /3nicotinamide adenine dinucleotide phosphate, reduced form (NADPH), bovine serum albumin (BSA), thiobarbituric acid (TBA), nitrotetrazolium blue (NBT), diethylenetriamine pentaacetic acid (DETAPAC), glycylglycine and Triton X-100 were procured from Sigma Chemical Co., St. Louis, MO. Nickel and sodium acetates were obtained from J.T. Baker Chemical Co., Phillipsburg, NJ. L-}'-glu-
tamyl-p-nitroanilide (GGPNA) and 1,1',3,3'-tetramethoxypropane (TMP) were obtained from Aldrich Chemical Co. Inc., Milwaukee, WI.
Animals Male Fischer (F344 N/Cr) rats weighing 125--150 g (NCI-FCRF, Animal Production Area, Frederick, MD) were exposed to 12 h light/dark cycles in an environment maintained at 22 _+ 2°C and allowed free access to food (NIH-31 open formula 6% Modified, Zeigler Brothers Gardners, PA) and water. The animals were housed in polycarbonate cages on a sawdust bedding (Sanichips, P.G. Murphy Forest products, Co., Mountsville, N J). Treatment regimen All injections of nickel acetate or sodium acetate were made between 0900 h and 1000 h to minimize circadian variation in xenobiotic metabolism [19]. Animals were divided into five control and five experimental groups of five to six rats each. Control animals received i.p. sodium acetate (214/amol/kg) and experimental animals received i.p. nickel acetate (107/amol/kg). One control group and one experimental group was then sacrificed by overexposure to CO 2 at 1, 3, 24, 48 and 72 h after injection. Tissue preparation Plasma and erythrocytes: Blood withdrawn from the portal vein of euthanized rats was put into heparinized tubes and centrifuged for 10 min at 1000 g. Plasma was removed by aspiration and the sedimented erythrocytes were washed three times by resuspension in isotonic (0.9%) saline followed by recentrifugation and removal of the supernatant layer. Ceils were lysed by diluting 0.5 g of material to 10 ml with ice-cold distilled water and diluted again 1"11 for enzymatic assays. Liver, kidney and muscle: Individual rat livers, kidneys and muscles (M. gastrocnemius) were removed, weighed and homogenized in ice-cold isotonic 0.25 M sucrose (1"10 dilution). The homogenates were centrifuged for 20 min at 9000 g to obtain a post-mitochondrial (PMS) fraction. A portion of the PMS was then centrifuged at 100 000 g for 60 min to obtain a cytosolic fraction for enzyme analysis. Biochemical assays Cell membrane damage was assessed by measuring ALT and AST enzyme activities in plasma [20]. LPO (in vivo) [21], SOD (total) [22], GSH [23] and GGT [24] were measured in whole tissue homogenate. CAT in the erythrocytes and PMS fraction, and GSH-Px (total) in erythrocytes and cytosolic fraction of tissues were determined by the method of Aebi [25] and Lee et al. [26], respectively. GST [27] and GSSG-R [28] activities were measured in cytosolic fraction of tissues. Metal determination Concentrations of iron and copper were determined by flame atomic absorp tion spectrophotometry. Nickel was similarly determined with the use of the graphite furnace atomic absorption technique.
Protein determination Protein determinations were made by using the Bradford method [29]; bovine serum albumin was used as standard.
Statistical analysis All data were analyzed using the unpaired Student's t-test to compare means. The results are expressed as mean _+ S.E. The level set for statistical significance was P < 0.05.
Results
Effect o f nickel on A L T and A S T Control rats treated with sodium acetate showed low circulatory values of ALT and AST activities in plasma that did not change between 3 and 72 h after treatment (Fig. 1). Nickel treatment produced a significant 2--3-fold increase in both ALT and AST levels at 3, 24, 48 and 72 h after injection.
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Time (hr) Fig. 2. Level of hepatic, renal and muscular LPO in male F344 rats at various times after intraperitoheal injection of nickel acetate (107/~mol/kg body weight). Values (expressed as nmol TMP formed/g tissue) represent the mean _+ S.E. of five to six rats. Star indicates values that are significantly different (P < 0.05) from controls (Student's t-test).
Effect o f nickel on L P O and iron and copper Figure 2 depicts the time d e p e n d e n t effects o f nickel o n hepatic, renal a n d m u s c u l a r levels o f L P O . T a b l e I shows the i r o n a n d copper c o n t e n t in liver, kidney a n d muscle at different time intervals after t r e a t m e n t . I n liver, the a m o u n t o f L P O was f o u n d to be increased to 129070, 133070 a n d 114070 of c o n t r o l value at 3,
TABLE I E F F E C T OF N I C K E L ON H E P A T I C , R E N A L A N D M U S C U L A R C O N T E N T OF IRON A N D C O P P E R AT VARIOUS T I M E INTERVALS Tissue Iron:
Treatment
Metal content (/ag l r o n / g fresh wt of tissue) after following time intervals
3 h
24 h
48 h
72 h
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Control Treated
51.52 ± 0.55 51.74 ± 1.85
51.52 ± 3.37 69.06 ± 0.95*
51.52 ± 3.37 52.92 ± 1.78
51.52 ± 1.87 58.24 ± 2.06*
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Control Treated
31.25 ± 1.60 38.88 ± 1.47"
31.25 ± 1.27 38.19 ± 1.74"
31.25 ± 1.27 34.16 ± 0.83
31.25 ± 2.93 34.34 ± 1.41
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Control Treated
19.25 ± 1.20 18.28 ± 0.96
19.25 ± 0.60 20.20 ± 0.10
19.25 __. 0.06 20.20 ± 0.39
19.25 ± 0.99 18.81 ± 0.78
Control Treated
Metal content (/ag C o p p e r / g fresh weight of tissue) 3.18 ± 0.026 3.18 ± 0.07 3.18 ± 0.07 3.83 ± 0.05* 3.97 ± 0.12" 3.51 _ 0.08*
Copper: Liver
3.18 ± 0.06 3.91 ± 0.08*
Kidney
Control Treated
6.14 ± 0.17 6.37 ± 0.27
6.14 ± 0.36 5.48 ± 0.28
6.14 ± 0.36 6.31 ± 0.43
6.14 ± 0.22 5.31 ± 0.44
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9.92 ± 0.35
9.92 ± 0.35
9.92 ± 0.47
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6.16 ± 0.52*
8.95 ± 0.47
Control rats received sodium acetate (214/~mol/kg, i.p.) and treated animals received nickel acetate (107/amol N i / k g , i.p.). Each value represents the mean _ S.E. of five to six rats.
TABL E 11 DIST RIBUT ION OF N I C K E L IN BLOOD, LIVER, K ID N EY A N D MU S C LE AT VARIOUS TIME INTERVALS Tissue"
Metal content (/ag Nickel/g fresh weight of tissue) after following time intervals.
3 h Blood Liver Kidney Muscle
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24 h ± 0.05 ± 0.16 ___ 2.90 ± 0.13
0.33 0,50 13.65 0,56
48 h ± ± ± ±
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0.23 0.28 1.66 0.51
72 h ± ± ± ±
0.02 0.04 0.22 0.12
0.18 0.13 1.66 0.43
± _ ± ±
0.02 0.03 0.26 0.40
"Animals received a single nickel acetate injection (107 /amol N i / k g , i.p.). Each value represents the mean _ S.E. of five to six rats. Control animals received 214 /amol sodium a c e t a t e / kg, i.p. Nickel concentrations in tissues of these rats were below detection limit of analysis (0.045/ag/ml).
24 and 48 h of nickel treatment, respectively. Hepatic iron was elevated to 134070 and 113°70 of control at 24 and 72 h and hepatic copper increased to 125070, 110070 and 123070 of control at 24, 48 and 72 h, respectively. In kidney, LPO levels were increased to 136070, 111070 and 111070 of control at 3, 24 and 48 h respectively after nickel administration. Renal iron content increased to 12407o and 122070 of control at 3 and 24 h while no change was observed in renal copper content. However, in muscle, no significant changes were observed in LPO and iron levels whereas copper was depleted significantly to 57°7o, 53070 and 6207o at 3, 24 and 48 h post-injection. Hepatic, renal and muscular nickel retention Nickel retention in liver, kidney and muscle as well as nickel levels in blood at various time intervals are shown in Table II. The concentrations of nickel was greatest at 3 h post-injection in all the tissue and gradually decreased with time.
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Effect of nickel on CA T, GSH-Px and SOD CAT, GSH-Px and SOD activities were measured at 3, 24, 48, and 72 h in liver, kidney, muscle and blood (Figs. 3, 4 and 5, respectively). In the liver, the only significant effect o f nickel on CAT activity was observed at 24 h when the activity decreased to 86070 o f the control. In contrast, the only effect on the hepatic GSH-Px was observed when its activity increased to 129°70 of the control at 48 h post-injection. The hepatic SOD activity was not affected by nickel injection. In the kidney, CAT activity significantly decreased to 87070 o f control at 3 h followed by an increase to 120070 o f control at 24 h. Then the renal CAT decreased to 84070 of control at 48 h and 69070 at 72 h. There was a decrease in renal GSH-Px activity to 87070 only at 3 h post-injection. No statistically significant change in renal SOD was observed at any o f the time intervals. In the skeletal muscle, CAT activity significantly increased to 136°70 vs. control at 48 h. However, at 72 h post-injection, CAT activity dropped to 73070 o f control. GSHPx in muscle was significantly increased at 24 h (108070) and 48 h (154070) vs. con-
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Time (hr) Fig. 4. Activity o f hepatic, renal, muscular and erythrocyte GSH-Px in male F344 rats at various times after intraperitoneal injection o f nickel acetate (107 /~mol/kg body wt). Values (expressed as /~mol N A D P H c o n s u m e d / m i n / m g protein) represent the mean _+ S.E. of five to six rats. Star indicates values that are significantly different (P < 0.05) from controls (Student's t-test).
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Time (hr) Fig. 5. Activity o f hepatic, renal and muscular SOD in male F344 rats at various times after intraperitoneal injection of nickel acetate (107/amol/kg body wt). Values (expressed as % inhibition/mg protein) represent the mean ± S.E. o f five to six rats. Star indicates values that are significantly different (P < 0.05) from controls (Student's t-test).
trol values. A significant decrease to 72°70 and 81070 of control at 3 and 24 h was observed in muscle SOD activity (Fig. 5). Erythrocyte CAT activity was significantly increased (126070) only at 72 h after injection o f nickel. No significant changes were observed in erythrocyte GSH-Px activity at any of the time points.
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T i m e (hr) Fig. 6. Level of hepatic, renal and muscular GSH in male F344 rats at various times after intraperitoneal injection of nickel acetate (107 /amol/kg body wt). Values (expressed as/amol GSH/g wet wt of tissue) represent the mean _+ S.E. of five to six rats. Star indicates values that are significantly different (P < 0.05) from controls (Student's t-test).
Effect o f nickel on GSH, GST, GSSG-R and G G T T h e e f f e c t s o f i n t r a p e r i t o n e a l l y i n j e c t e d n i c k e l o n G S H in rats a r e s h o w n in F i g . 6. N i c k e l s i g n i f i c a n t l y d e p l e t e d t h e h e p a t i c G S H level t o 77°70, 68°70 a n d 76°70 o f c o n t r o l v a l u e s at 1, 3, a n d 24 h a f t e r t r e a t m e n t , r e s p e c t i v e l y . T h e n , G S H levels
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Time (hr) Fig. 7. Activity of hepatic, renal and muscular GSSG-R in male F344 rats at various times after intraperitoneal injection of nickel acetate (107/a-nol/kg body wt). Values (expressed as/~mol NADPH oxidized/min/mg protein) represent the mean :l: S.E. of five to six rats. Star indicates values that are significantly different (P < 0.05) from controls (Student's t-test).
increased significantly to 132070 o f c o n t r o l at 72 h o f t r e a t m e n t . A c t i v i t y o f hepatic G S S G - R was elevated to 113°70 o f c o n t r o l at 1 h a n d d e p l e t e d to 90070 o f c o n t r o l at 3 h o f t r e a t m e n t (Fig.7). H e p a t i c G S T was significantly r e d u c e d to 6 6 % a n d 84°70 o f c o n t r o l activity at 3 a n d 24 h o f t r e a t m e n t , respectively (Fig. 8). A c t i v i t y o f h e p a t i c G G T was n o t c h a n g e d at a n y o f the time intervals (Fig. 9). A s t h a t in liver, renal G S H was d e p l e t e d to 82070 o f c o n t r o l value at b o t h 1
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Time (hr) Fig. 8. Activity of hepatic, renal and muscular GST in male F344 rats at various times after intraperitoneal injection of nickel acetate (107 /amol/kg body wt). Values (expressed as nmol conjugate f o r m e d / m i n / m g protein) represent the mean ± S.E. o f five to six rats. Star indicates values that are significantly different (P < 0.05) from controls (Student's t-test).
and 3 h after treatment. At 24 and 48 h after nickel G S H concentrations increased to 148070 and 138070 of control value, respectively (Fig. 6). Renal GSSGR activity was reduced to 92070, 89°7o and 8407o of control at 3, 24 and 48 h (Fig. 7). Renal GST was significantly elevated, to 145°70, 11207o and 12007o over control
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Time (hr) Fig. 9. Activity of hepatic, renal and muscular GGT in male F344 rats at various times after intraperitoneal injection of nickel acetate (107 /amol/kg body wt). Values (expressed as /amol p-nitroanaline released/min/mg protein) represent the mean _+ S.E. o f five to six rats. Star indicates values that are significantly different (P < 0.05) from controls (Student's t-test).
value at 3, 24 and 72 h o f nickel treatment, respectively (Fig. 8). Renal GGT activity did not change at any time intervals after nickel treatment (Fig. 9). Results in Figs. 6 - - 9 show the effects o f nickel treatment on GSH, GSSG-R, GST and GGT in muscle. Except for initial enhancement in GSH content at 1 h o f treatment (118% vs. control), no changes were observed at any time point.
13
Discussion
Alterations in serum levels of ALT, which is liver specific and AST have been considered a tool to study varying cell viability and changes in cell membrane permeability [30]. Increased levels of these enzymes in blood plasma after nickel treatment in the present investigation thus reflect damaging effect of nickel on the cell membrane, leading to increased permeability and increased leakage of intracellular enzymes. Thus, our results are consistent with earlier reports [6]. It has been established that there is a direct relationship between L P O and tissue iron and copper content, involving reactive oxygen species [31,32]. Such species are generated in situ by interaction of various hydroperoxides with transition metal catalysts, particularly iron in both ferrous and ferric forms [32]. Recently, Inoue and Kawanishi [33] reported the possible production of superoxide, hydroxyl radical and singlet oxygen from H202reacting with the nickel(II) complex of glycylglycyl-L-histidine. Thus, in addition to iron and copper, which catalyze hydroxyl radical formation, nickel, depending on its chemical form, may also assist in generation of activated oxygen species. These active oxygen species may in turn, damage lipids, cell membranes and DNA. In the present experiment, nickel treatment that by itself supplied the tissues with high concentrations of a potential metal oxidation catalyst, also tended to increase iron and copper contents in liver and iron in kidney. The exact cause of this increase is unknown; perhaps, nickel disturbed homeostatis of these two essential metals resulting in their inter-organ relocation. Elevated amounts of the two latter metals might be expected to further enhance LPO in liver and kidney. Comparison of the time course of L P O in liver and kidney with corresponding concentrations of either metal reveals a possible simple correlation only with nickel. However, the magnitude of LPO was not proportional to nickel concentration in the respective organ. Both organs showed the same level, 110--130%, of L P O increase while the concentration of nickel in kidney was ten fold higher than in liver. This result might depend on different lipid contents in these organs. LPO in kidney and liver decreased concurrently with the decrease of nickel levels, but not with levels of iron or copper. Thus, our results do not confirm the direct contribution of iron a n d / o r copper to L P O proclaimed by others [32]. Perhaps, the nickel-induced shifts in tissue iron and copper observed in this study were too small to produce additional oxidative effects. The high level of LPO in liver and kidney during the first 24 h post-injection may result in increased concentration of H202in tissues affected by nickel. In liver, this increase concurred with transiently decreased activity of HEO2-scavenging enzymes, CAT and GSH-Px, that are known to be inhibited by nickel [8]. These two effects combined could augument the potential of oxidative cell damage. At latter time intervals, with decreasing nickel, the activity of hepatic GSHPx and CAT was restored and L P O decreased. This should lower the potential of oxidative damage to the ceils [18]. However, in kidney, the concurrence between changes in LPO and CAT was lost after 24 h when CAT activity dropped again with no LPO increase. The reason for this highly significant CAT drop is not clear. Its relevance to renal toxicity of nickel deserves further investigations. The
14
activity of CAT and GSH-Px in muscle were least affected by nickel with no alterations in LPO. Interestingly, under the present experimental conditions, no apparent involvement of SOD in the observed toxic effects of nickel was noticed. Activity of this enzyme that converts superoxide radical into H202 remained practically unchanged in all tissues tested. Thus, undisturbed generation of H202 at times of decreased CAT and GSH-Px activity may add to the toxic effect of nickel. Our results differ from those of NoveUi and Rodriguez [34], who observed an increase in Cu-Zn SOD activity in the pancreas of rats given nickel chloride. Perhaps, nickel effect on SOD is tissue specific. In the present experiment, we have found that there is a possible direct relationship between nickel-induced LPO and effects of this metal on the GSH/GSHPx activity. However, the results do not provide enough information about the degree of interdependence of these effects. The peroxidative activity of metal ions has generally been related to their ability to deplete the cellular GSH content which is known to maintain the cellular redox potential [7]. Here we report that nickel injection caused immediate deficiency of hepatic GSH (and GSH-Px) thus lowering the cell's ability to protect itself against LPO at earlier time points. The levelling of LPO at later time points, when most of the nickel dose is excreted, might be assisted by a concurrent increase of GSH. In the kidney, the concurrence of the LPO and GSH changes was similar to that in the liver, with, however, some time-related and quantitative differences which most likely resulted from different retention of nickel and different basal concentration of GSH in these organs. The turnover of GSH mainly depends upon the levels of various GSH synthesis and metabolism enzymes, including GSSG-R, GGT, and GST. The overall effect of nickel on GSH can be explained by the concept that nickel binds to GSH and thus initially depletes preexisting GSH level. In liver, at the same time, nickel treatment lowers the activity of GST, which might be due to the lesser availability of GSH or to the direct effect of nickel on the enzyme. However, in the kidney, the opposite is true. Thus, nickel effect on GST is more complex and indirect. The observed pattern in GSSG-R response to nickel exposure was biphasic. The initial enhancement of GSSG-R activity at 1 h might be due to the biological response to nickel insult. The rapid depletion of GSSG-R, that followed (3 h), might result from decreased production of NADPH in the presence of nickel [35]. This depletion, however, may not be enough to affect regeneration of GSH from GSSG. The increased hepatic GSH level at later times might thus reflect both regeneration and enhanced de novo synthesis of GSH [36]. Likewise, the secondary rise in renal GSH level despite significant depletion of GSSG-R may indicate increased intracellular GSH synthesis. In conclusion, LPO may result in the production of various hydroperoxides (ROOH) and organic radicals (R., RO2-). GSH is an efficient substrate for enzymatic reduction of peroxides via GSH-Px [37]. Therefore, depletion of intracellular GSH and GSH-Px following nickel treatment would make tissues more vulnerable to peroxides as observed in the present study. The increased level of active oxygen species in the tissues ultimately may cause damage to the genetic material [13] and thus contribute to carcinogenic effects of nickel. 15
References 1 2 3 4 5 6 7
8 9 10 11 12
13 14 15 16 17
.18 19
20
21 22 23
16
T.P. Coogan, D.M. Latta, E.T. Snow and M. Costa, Toxicity and carcinogenicity of nickel compounds. CRC Crit. Rev. Toxicol., 19 (1989) 341. F.W. Sunderman, Jr., Mechanisms of nickel carcinogenesis. Scand. J. Work Environ. Health, 15 (1989) 1. F.W. Sunderman, Jr. Lipid peroxidation as a mechanism of acute nickel toxicity. Toxicol. Environ. Chem., 15 (1987) 59. F.W. Sunderman, Jr., A. Marzouk, S.M. Hopfer, O. Zaharia, and M.C. Reid, Increased lipid peroxidation in tissue of nickel chloride-treated rats. Ann. Clin. Lab. Sci., 15 (1985) 229. K.S. Kasprzak and R.B. Bare, in vitro polymerization of histones by carcinogenic nickel compounds. Carcinogenesis, 10 (1989) 621. M. Misra, M. Athar, S.K. Hasan and R.C. Srivastava, Alleviation of nickel-induced biochemical alterations by chelating agents. Fundam. Appl. Toxicol., l 1 (1988) 285. M. Athar, S.K. Hasan and R.C. Srivastava, Role of glutathione metabolizing enzymes in nickel mediated induction of hepatic glutathione. Res. Commun. Chem. Pathol. Pharmacol., 57 (1987) 421. R.E. Rodriguez and K.S. Kasprzak, Nickel(II) inhibition of catalase, glutathione peroxidase/ glutathione reductase system. The Toxicologist, 9 (1989) 134. E.N. Frankel and W.E. Neff, Formation of malonaldehyde from lipid peroxidation products. Biochim. Biophys. Acta, 754 (1983) 264. B. Halliwell and J.M.C. Gutteridge, Oxygen toxicity, oxygen radicals, transition metals and disease. Biochem. J., 219 (1984) 1. P. Hochstein and S.K. Jain, Association of lipid peroxidation and polymerization of membranes proteins with erythrocyte aging. Fed. Proc., 40 (1981) 183. R.P. Bird and H.H. Draper, Effect of malonaldehyde and acetaldehyde on cultured mammalian cells: growth, morphology and synthesis of macro molecules. J. Toxicol. Environ. Health, 6 (1980) 811. R.B. Ciccareli and K.E. Wetterhahn, Molecular basis for the activity of nickel. IARC Sci. Publ., 53 (1984) 201. S.R. Patierno and M. Costa, DNA-protein-crosslinks induced by nickel compounds in intact cultured mammalian cells. Chem.-Biol. Interact., 55 (1985) 75. K.S. Kasprzak and L. Hernandez, Enhancement of hydroxylation and deglycosylation of 2deoxyguanosine by carcinogenic nickel compounds. Cancer Res., 49 (1989) 5964. M.J. Olson, DNA strand breaks induced by hydrogen peroxide in isolated rat hepatocytes. J. Toxicol. Environ. Health, 23 (1988) 407. M. Athar, S.K. Hasan and R.C. Srivastava, Evidence for the involvement of hydroxyl radicals in nickel mediated enhancement of lipid peroxidation: implications for nickel carcinogenesis. Biochem. Biophys. Res. Commun., 5 (1987) 1276. B.M. Babior, Oxygen-dependent microbial killing by phagocytes: Part I. N. Engl. J. Med., 1978 (1987) 659. R.C. Schnell, H.P. Bozigian, M.H. Davies, B.A. Merrick, and K. Johnson, Circadian rhythms in acetaminophen toxicity: Role of non-protein sulphydryl. Toxicol. Appl. Pharmacol., 71 (1983) 353. J.H. Wilkinson, D.N. Baron, D.W. Moss and P.G. Walker, Standardization of clinical enzymes assays: a reference method for aspartate and alanine transaminases. J. Clin. Pathol., 25 (1972) 940. M. Shlafer and B.M. Shepard, A method to reduce interference by sucrose in the detection of thiobarbituric acid-reactive substances. Anal. Biochem., 137 (1984) 269. L.W. Oberly and D.R. Spitz, Assay of superoxide dismutase activity in tumor tissue. Methods Enzymol., 105 (1984) 457. D.J. Jollow, J.R. Mitchell, Z. Zampaglione and J.R. Gillette, Bromobenzene induced liver necrosis. Protective role of glutathione and evidence for 3,4-bromobenzene oxide as the hepatotoxic metabolite. Pharmacology, 11 (1974) 151.
24 25 26 27 28 29 30 31 32 33
34 35 36
37
A. Meister, S.S. Tate and O.W. Griffith, y-Glutamyl Transpeptidase. Methods Enzymol., 77 (1981) 237. H. Aebi, Catalase in vitro. Methods Enzymol., 105 (1984) 121. Y.H. Lee, D.K. Layman and R.R. Bell, Glutathione peroxidase activity in iron-deficient rats. J. Nutr., 111 (1981) 194. W.H. Habig, M.J. Pabst and W.B. Jakoby, Glutathione-S-transferase. The first enzymatic step in mercapturic acid formation. J. Biol. Chem., 249 (1974) 7130. E. Racker, Glutathione reductase from baker yeast and beef liver. J. Biol. Chem., 217 (1955) 855. M.M. Bradford, A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem., 72 (1976) 248. B. Vonen and J. Morland, Isolated rat hepatocytes in suspension: potential hepatotoxic effects of six different drugs. Arch. Toxicol., 56 (1984) 33. J.M.C. Gutteridge, Tissue damage by oxy-radicals: The possible involvement of iron and copper complexes. Med. Biol., 62 (1984) 101. C.E. Thomas, L.A. Morehouse and S.D. Aust, Ferritin and superoxide-dependent lipid peroxidation. J. Biol. Chem., 260 (1985) 3275. S. Inoue and S. Kawanishi, ESR evidence for superoxide, hydroxyl radicals and singlet oxygen produced from hydrogen peroxide and nickel(lI) complex of glycylglycyl-L-histidine. Biochem. Biophys. Res. Commun., 159 (1989) 445. E.L.B. Novelli and N.L. Rodriguez, Effect of nickel chloride on streptozotocin-induced diabetes in rats. Can. J. Physiol. Pharmacol., 66 (1988) 663. L. Cartana and A.A. Romeu, Characterization of the inhibition effect induced by nickel on glucose-6-phosphate dehydrogenase and glutathione reductase. Enzyme, 41 (1989) 1. A. Chung and M.D. Maines, Effect of selenium on glutathione metabolism. Induction of y-glutamyl cysteine synthetase and glutathione reductase in the rat liver. Biochem. Pharmacol., 30 (1981) 3217. A.G. Splittgerber and A.L. Tappel, Inhibition of glutathione peroxidase by cadmium and other metal ions. Arch. Biochem. Biophys., 197 (1979) 534.
17
Nickel induced lipid peroxidation in the rat: correlation with nickel effect on antioxidant defense systems Manoj Misra, Ricardo E. Rodriguez and Kazimierz S. Kasprzak Laboratory of Comparative Carcinogenesis, Division of Cancer Etiology, National Cancer Institute, Frederick Cancer Research Facility, Frederick, MD 21701 (U.S.A.) (Received March 14th, 1990 ; accepted June 1st, 1990)
Summary Lipid peroxidation (LPO) and alterations in cellular systems protecting against oxidative damage were determined in the liver, kidney and skeletal muscle of male F344/NCr rats, 1 h to 3 days after a single intraperitoneal (i.p.) injection of 107 /~mol nickel(II)acetate per kg body weight. At 3 h, when tissue nickel concentrations were highest, the following significant (at least, P < 0.05) effects were observed: in kidney, increased LPO (by 43°70), increased renal iron (by 24%), decreased catalase (CAT) and glutathione peroxidase (GSH-Px) activities (both by 15%), decreased glutathione (GSH) concentration (by 20 %), decreased glutathione reductase (GSSG-R) activity (by 10%), and increased glutathione-S-transferase (GST) activity (by 44 070); the activity of superoxide dismutase (SOD) and yglutamyl transferase (GGT), as well as copper concentration, were not affected. In the liver, nickel effects included increased LPO (by 30%), decreased CAT and GSH-Px activities (both by 15070), decreased GSH level (by 33%), decreased GSSG-R activity (by 10 070) and decreased GST activity (by 35070); SOD, GGT, copper, and iron remained unchanged. In muscle, nickel treatment decreased copper content (by 43070) and the SOD activity (by 30070) with no effects on other parameters. In blood, nickel had no effect on CAT and GSH-Px, but increased the activities of alanine-(ALT) and aspartate-(AST) transaminases to 330070 and 240% of the background level, respectively. In conclusion, nickel treatment caused profound cell damage as indicated by increased LPO in liver and kidney and leakage of intracellular enzymes, ALT and AST to the blood. The time pattern of the resulting renal and hepatic LPO indicated a possible contribution to its magnitude from an increased concentration of nickel and concurrent inhibition of CAT, GSH-Px and GSSG-R, but not from increased iron or copper levels. The oxidative damage expressed as LPO was highest in the kidney and lowest in the muscle, which concurs with the corresponding ranking of nickel uptake by these tissues.
Key words: Nickel; Lipid peroxidation; Cellular systems; Defense systems
Introduction
The toxicity and carcinogenesis of nickel compounds in man and experimental Address all correspondence to: Manoj Misra, Ph.D., National Cancer Institute, Frederick Cancer Research Facility, Building 538, Room 205E, Frederick, MD 21701, U.S.A. Printed and Published in Ireland
animals have been well documented [1,2]. The exact mechanism of their action, however, remains unknown. One possible mode by which nickel causes cell death and/or damage to the genetic material may involve oxidative reactions such as nickel-induced lipid peroxidation (LPO) [2--4] or nickel-catalyzed oxidative modification of some proteins [5]. Inhibition by nickel of cellular defense systems including glutathione (GSH), glutathione-S-transferase (GST), glutathione reductase (GSSG-R) [6,7], catalase (CAT) and glutathione peroxidase (GSH-Px) [8], may further increase the oxidative damage. LPO constitutes a free radical oxidation process in which polyunsaturated fatty acids of the cell membrane decompose to yield, among others, highly reactive lipid hydroperoxides, H202, hydroxyl radical (OH.), and malondialdehyde [9,10]. Malondialdehyde has been shown to cause cross-linking and polymerization of membrane components [I1] and may contribute to the mutagenic, genotoxic and carcinogenic effects [12]. Oxygen radicals (superoxide, 02-; OH" etc.,) have been proposed to play a crucial role in cell neoplastic transformation. Reactive oxygen species cause extensive strand breakage of DNA [13], protein-DNA crosslinks [14] and oxidative modification of DNA bases [15]. Oxidative damage from these radicals can also be enhanced by decreased activity of the cell's defensive systems. H202 can cause DNA degradation [16]. Its action can be enhanced by reduction to the extremely reactive OH" radical with various reductive agents like Fe2÷, Cu ~÷ etc. and result in cellular damage [17]. In biological systems, there exist enzymes which are capable of controlling the cytotoxic effects of active oxygen species, including CAT, GSH-Px and superoxide dismutase (SOD) [18] which can protect the cells from accumulating H202, hydroperoxides, and superoxide. These three enzymes appear to work in conjunction with each other with the purpose of minimizing the cell's exposure to reactive intermediates of dioxygen reduction. To elucidate the biochemical mechanism of oxidative damage due to acute nickel intoxication in rodents, we examined the effects of nickel on cellular iron and copper, cellular enzymes (ALT, AST), oxidation defense systems (GSH, GST, GSSG-R, GSH-Px, CAT, and SOD) and LPO in order to correlate the severity of LPO with changing metal concentrations and activities of the defense systems. Materials and methods Chemicals Bovine liver catalase, F.W. = 250 000 was purchased from Worthington Biochemicals, Freehold, NJ. Trizma buffer, phosphate buffer, 30°7o hydrogen peroxide (H202) , glutathione reductase (GSSG-R), xanthine oxidase, sodium azide (NAN3), reduced glutathione (GSH), oxidized glutathione (GSSG), sulfosalicylic acid (SSA), 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB), 1-chloro-2,4-dinitroben zene (CDNB), fl-nicotinamide adenine dinucleotide, reduced form (NADH), /3nicotinamide adenine dinucleotide phosphate, reduced form (NADPH), bovine serum albumin (BSA), thiobarbituric acid (TBA), nitrotetrazolium blue (NBT), diethylenetriamine pentaacetic acid (DETAPAC), glycylglycine and Triton X-100 were procured from Sigma Chemical Co., St. Louis, MO. Nickel and sodium acetates were obtained from J.T. Baker Chemical Co., Phillipsburg, NJ. L-}'-glu-
tamyl-p-nitroanilide (GGPNA) and 1,1',3,3'-tetramethoxypropane (TMP) were obtained from Aldrich Chemical Co. Inc., Milwaukee, WI.
Animals Male Fischer (F344 N/Cr) rats weighing 125--150 g (NCI-FCRF, Animal Production Area, Frederick, MD) were exposed to 12 h light/dark cycles in an environment maintained at 22 _+ 2°C and allowed free access to food (NIH-31 open formula 6% Modified, Zeigler Brothers Gardners, PA) and water. The animals were housed in polycarbonate cages on a sawdust bedding (Sanichips, P.G. Murphy Forest products, Co., Mountsville, N J). Treatment regimen All injections of nickel acetate or sodium acetate were made between 0900 h and 1000 h to minimize circadian variation in xenobiotic metabolism [19]. Animals were divided into five control and five experimental groups of five to six rats each. Control animals received i.p. sodium acetate (214/amol/kg) and experimental animals received i.p. nickel acetate (107/amol/kg). One control group and one experimental group was then sacrificed by overexposure to CO 2 at 1, 3, 24, 48 and 72 h after injection. Tissue preparation Plasma and erythrocytes: Blood withdrawn from the portal vein of euthanized rats was put into heparinized tubes and centrifuged for 10 min at 1000 g. Plasma was removed by aspiration and the sedimented erythrocytes were washed three times by resuspension in isotonic (0.9%) saline followed by recentrifugation and removal of the supernatant layer. Ceils were lysed by diluting 0.5 g of material to 10 ml with ice-cold distilled water and diluted again 1"11 for enzymatic assays. Liver, kidney and muscle: Individual rat livers, kidneys and muscles (M. gastrocnemius) were removed, weighed and homogenized in ice-cold isotonic 0.25 M sucrose (1"10 dilution). The homogenates were centrifuged for 20 min at 9000 g to obtain a post-mitochondrial (PMS) fraction. A portion of the PMS was then centrifuged at 100 000 g for 60 min to obtain a cytosolic fraction for enzyme analysis. Biochemical assays Cell membrane damage was assessed by measuring ALT and AST enzyme activities in plasma [20]. LPO (in vivo) [21], SOD (total) [22], GSH [23] and GGT [24] were measured in whole tissue homogenate. CAT in the erythrocytes and PMS fraction, and GSH-Px (total) in erythrocytes and cytosolic fraction of tissues were determined by the method of Aebi [25] and Lee et al. [26], respectively. GST [27] and GSSG-R [28] activities were measured in cytosolic fraction of tissues. Metal determination Concentrations of iron and copper were determined by flame atomic absorp tion spectrophotometry. Nickel was similarly determined with the use of the graphite furnace atomic absorption technique.
Protein determination Protein determinations were made by using the Bradford method [29]; bovine serum albumin was used as standard.
Statistical analysis All data were analyzed using the unpaired Student's t-test to compare means. The results are expressed as mean _+ S.E. The level set for statistical significance was P < 0.05.
Results
Effect o f nickel on A L T and A S T Control rats treated with sodium acetate showed low circulatory values of ALT and AST activities in plasma that did not change between 3 and 72 h after treatment (Fig. 1). Nickel treatment produced a significant 2--3-fold increase in both ALT and AST levels at 3, 24, 48 and 72 h after injection.
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Time (hr) Fig. 2. Level of hepatic, renal and muscular LPO in male F344 rats at various times after intraperitoheal injection of nickel acetate (107/~mol/kg body weight). Values (expressed as nmol TMP formed/g tissue) represent the mean _+ S.E. of five to six rats. Star indicates values that are significantly different (P < 0.05) from controls (Student's t-test).
Effect o f nickel on L P O and iron and copper Figure 2 depicts the time d e p e n d e n t effects o f nickel o n hepatic, renal a n d m u s c u l a r levels o f L P O . T a b l e I shows the i r o n a n d copper c o n t e n t in liver, kidney a n d muscle at different time intervals after t r e a t m e n t . I n liver, the a m o u n t o f L P O was f o u n d to be increased to 129070, 133070 a n d 114070 of c o n t r o l value at 3,
TABLE I E F F E C T OF N I C K E L ON H E P A T I C , R E N A L A N D M U S C U L A R C O N T E N T OF IRON A N D C O P P E R AT VARIOUS T I M E INTERVALS Tissue Iron:
Treatment
Metal content (/ag l r o n / g fresh wt of tissue) after following time intervals
3 h
24 h
48 h
72 h
Liver
Control Treated
51.52 ± 0.55 51.74 ± 1.85
51.52 ± 3.37 69.06 ± 0.95*
51.52 ± 3.37 52.92 ± 1.78
51.52 ± 1.87 58.24 ± 2.06*
Kidney
Control Treated
31.25 ± 1.60 38.88 ± 1.47"
31.25 ± 1.27 38.19 ± 1.74"
31.25 ± 1.27 34.16 ± 0.83
31.25 ± 2.93 34.34 ± 1.41
Muscle
Control Treated
19.25 ± 1.20 18.28 ± 0.96
19.25 ± 0.60 20.20 ± 0.10
19.25 __. 0.06 20.20 ± 0.39
19.25 ± 0.99 18.81 ± 0.78
Control Treated
Metal content (/ag C o p p e r / g fresh weight of tissue) 3.18 ± 0.026 3.18 ± 0.07 3.18 ± 0.07 3.83 ± 0.05* 3.97 ± 0.12" 3.51 _ 0.08*
Copper: Liver
3.18 ± 0.06 3.91 ± 0.08*
Kidney
Control Treated
6.14 ± 0.17 6.37 ± 0.27
6.14 ± 0.36 5.48 ± 0.28
6.14 ± 0.36 6.31 ± 0.43
6.14 ± 0.22 5.31 ± 0.44
Muscle
Control
9.92 ± 0.31
9.92 ± 0.35
9.92 ± 0.35
9.92 ± 0.47
Treated
5.69 ± 1.03"
5.31 ± 0.30*
6.16 ± 0.52*
8.95 ± 0.47
Control rats received sodium acetate (214/~mol/kg, i.p.) and treated animals received nickel acetate (107/amol N i / k g , i.p.). Each value represents the mean _ S.E. of five to six rats.
TABL E 11 DIST RIBUT ION OF N I C K E L IN BLOOD, LIVER, K ID N EY A N D MU S C LE AT VARIOUS TIME INTERVALS Tissue"
Metal content (/ag Nickel/g fresh weight of tissue) after following time intervals.
3 h Blood Liver Kidney Muscle
0.86 4.13 49.94 1.81
24 h ± 0.05 ± 0.16 ___ 2.90 ± 0.13
0.33 0,50 13.65 0,56
48 h ± ± ± ±
0.07 0.10 1.65 0.10
0.23 0.28 1.66 0.51
72 h ± ± ± ±
0.02 0.04 0.22 0.12
0.18 0.13 1.66 0.43
± _ ± ±
0.02 0.03 0.26 0.40
"Animals received a single nickel acetate injection (107 /amol N i / k g , i.p.). Each value represents the mean _ S.E. of five to six rats. Control animals received 214 /amol sodium a c e t a t e / kg, i.p. Nickel concentrations in tissues of these rats were below detection limit of analysis (0.045/ag/ml).
24 and 48 h of nickel treatment, respectively. Hepatic iron was elevated to 134070 and 113°70 of control at 24 and 72 h and hepatic copper increased to 125070, 110070 and 123070 of control at 24, 48 and 72 h, respectively. In kidney, LPO levels were increased to 136070, 111070 and 111070 of control at 3, 24 and 48 h respectively after nickel administration. Renal iron content increased to 12407o and 122070 of control at 3 and 24 h while no change was observed in renal copper content. However, in muscle, no significant changes were observed in LPO and iron levels whereas copper was depleted significantly to 57°7o, 53070 and 6207o at 3, 24 and 48 h post-injection. Hepatic, renal and muscular nickel retention Nickel retention in liver, kidney and muscle as well as nickel levels in blood at various time intervals are shown in Table II. The concentrations of nickel was greatest at 3 h post-injection in all the tissue and gradually decreased with time.
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Effect of nickel on CA T, GSH-Px and SOD CAT, GSH-Px and SOD activities were measured at 3, 24, 48, and 72 h in liver, kidney, muscle and blood (Figs. 3, 4 and 5, respectively). In the liver, the only significant effect o f nickel on CAT activity was observed at 24 h when the activity decreased to 86070 o f the control. In contrast, the only effect on the hepatic GSH-Px was observed when its activity increased to 129°70 of the control at 48 h post-injection. The hepatic SOD activity was not affected by nickel injection. In the kidney, CAT activity significantly decreased to 87070 o f control at 3 h followed by an increase to 120070 o f control at 24 h. Then the renal CAT decreased to 84070 of control at 48 h and 69070 at 72 h. There was a decrease in renal GSH-Px activity to 87070 only at 3 h post-injection. No statistically significant change in renal SOD was observed at any o f the time intervals. In the skeletal muscle, CAT activity significantly increased to 136°70 vs. control at 48 h. However, at 72 h post-injection, CAT activity dropped to 73070 o f control. GSHPx in muscle was significantly increased at 24 h (108070) and 48 h (154070) vs. con-
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Time (hr) Fig. 4. Activity o f hepatic, renal, muscular and erythrocyte GSH-Px in male F344 rats at various times after intraperitoneal injection o f nickel acetate (107 /~mol/kg body wt). Values (expressed as /~mol N A D P H c o n s u m e d / m i n / m g protein) represent the mean _+ S.E. of five to six rats. Star indicates values that are significantly different (P < 0.05) from controls (Student's t-test).
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Time (hr) Fig. 5. Activity o f hepatic, renal and muscular SOD in male F344 rats at various times after intraperitoneal injection of nickel acetate (107/amol/kg body wt). Values (expressed as % inhibition/mg protein) represent the mean ± S.E. o f five to six rats. Star indicates values that are significantly different (P < 0.05) from controls (Student's t-test).
trol values. A significant decrease to 72°70 and 81070 of control at 3 and 24 h was observed in muscle SOD activity (Fig. 5). Erythrocyte CAT activity was significantly increased (126070) only at 72 h after injection o f nickel. No significant changes were observed in erythrocyte GSH-Px activity at any of the time points.
9
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T i m e (hr) Fig. 6. Level of hepatic, renal and muscular GSH in male F344 rats at various times after intraperitoneal injection of nickel acetate (107 /amol/kg body wt). Values (expressed as/amol GSH/g wet wt of tissue) represent the mean _+ S.E. of five to six rats. Star indicates values that are significantly different (P < 0.05) from controls (Student's t-test).
Effect o f nickel on GSH, GST, GSSG-R and G G T T h e e f f e c t s o f i n t r a p e r i t o n e a l l y i n j e c t e d n i c k e l o n G S H in rats a r e s h o w n in F i g . 6. N i c k e l s i g n i f i c a n t l y d e p l e t e d t h e h e p a t i c G S H level t o 77°70, 68°70 a n d 76°70 o f c o n t r o l v a l u e s at 1, 3, a n d 24 h a f t e r t r e a t m e n t , r e s p e c t i v e l y . T h e n , G S H levels
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Time (hr) Fig. 7. Activity of hepatic, renal and muscular GSSG-R in male F344 rats at various times after intraperitoneal injection of nickel acetate (107/a-nol/kg body wt). Values (expressed as/~mol NADPH oxidized/min/mg protein) represent the mean :l: S.E. of five to six rats. Star indicates values that are significantly different (P < 0.05) from controls (Student's t-test).
increased significantly to 132070 o f c o n t r o l at 72 h o f t r e a t m e n t . A c t i v i t y o f hepatic G S S G - R was elevated to 113°70 o f c o n t r o l at 1 h a n d d e p l e t e d to 90070 o f c o n t r o l at 3 h o f t r e a t m e n t (Fig.7). H e p a t i c G S T was significantly r e d u c e d to 6 6 % a n d 84°70 o f c o n t r o l activity at 3 a n d 24 h o f t r e a t m e n t , respectively (Fig. 8). A c t i v i t y o f h e p a t i c G G T was n o t c h a n g e d at a n y o f the time intervals (Fig. 9). A s t h a t in liver, renal G S H was d e p l e t e d to 82070 o f c o n t r o l value at b o t h 1
11
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Time (hr) Fig. 8. Activity of hepatic, renal and muscular GST in male F344 rats at various times after intraperitoneal injection of nickel acetate (107 /amol/kg body wt). Values (expressed as nmol conjugate f o r m e d / m i n / m g protein) represent the mean ± S.E. o f five to six rats. Star indicates values that are significantly different (P < 0.05) from controls (Student's t-test).
and 3 h after treatment. At 24 and 48 h after nickel G S H concentrations increased to 148070 and 138070 of control value, respectively (Fig. 6). Renal GSSGR activity was reduced to 92070, 89°7o and 8407o of control at 3, 24 and 48 h (Fig. 7). Renal GST was significantly elevated, to 145°70, 11207o and 12007o over control
12
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Time (hr) Fig. 9. Activity of hepatic, renal and muscular GGT in male F344 rats at various times after intraperitoneal injection of nickel acetate (107 /amol/kg body wt). Values (expressed as /amol p-nitroanaline released/min/mg protein) represent the mean _+ S.E. o f five to six rats. Star indicates values that are significantly different (P < 0.05) from controls (Student's t-test).
value at 3, 24 and 72 h o f nickel treatment, respectively (Fig. 8). Renal GGT activity did not change at any time intervals after nickel treatment (Fig. 9). Results in Figs. 6 - - 9 show the effects o f nickel treatment on GSH, GSSG-R, GST and GGT in muscle. Except for initial enhancement in GSH content at 1 h o f treatment (118% vs. control), no changes were observed at any time point.
13
Discussion
Alterations in serum levels of ALT, which is liver specific and AST have been considered a tool to study varying cell viability and changes in cell membrane permeability [30]. Increased levels of these enzymes in blood plasma after nickel treatment in the present investigation thus reflect damaging effect of nickel on the cell membrane, leading to increased permeability and increased leakage of intracellular enzymes. Thus, our results are consistent with earlier reports [6]. It has been established that there is a direct relationship between L P O and tissue iron and copper content, involving reactive oxygen species [31,32]. Such species are generated in situ by interaction of various hydroperoxides with transition metal catalysts, particularly iron in both ferrous and ferric forms [32]. Recently, Inoue and Kawanishi [33] reported the possible production of superoxide, hydroxyl radical and singlet oxygen from H202reacting with the nickel(II) complex of glycylglycyl-L-histidine. Thus, in addition to iron and copper, which catalyze hydroxyl radical formation, nickel, depending on its chemical form, may also assist in generation of activated oxygen species. These active oxygen species may in turn, damage lipids, cell membranes and DNA. In the present experiment, nickel treatment that by itself supplied the tissues with high concentrations of a potential metal oxidation catalyst, also tended to increase iron and copper contents in liver and iron in kidney. The exact cause of this increase is unknown; perhaps, nickel disturbed homeostatis of these two essential metals resulting in their inter-organ relocation. Elevated amounts of the two latter metals might be expected to further enhance LPO in liver and kidney. Comparison of the time course of L P O in liver and kidney with corresponding concentrations of either metal reveals a possible simple correlation only with nickel. However, the magnitude of LPO was not proportional to nickel concentration in the respective organ. Both organs showed the same level, 110--130%, of L P O increase while the concentration of nickel in kidney was ten fold higher than in liver. This result might depend on different lipid contents in these organs. LPO in kidney and liver decreased concurrently with the decrease of nickel levels, but not with levels of iron or copper. Thus, our results do not confirm the direct contribution of iron a n d / o r copper to L P O proclaimed by others [32]. Perhaps, the nickel-induced shifts in tissue iron and copper observed in this study were too small to produce additional oxidative effects. The high level of LPO in liver and kidney during the first 24 h post-injection may result in increased concentration of H202in tissues affected by nickel. In liver, this increase concurred with transiently decreased activity of HEO2-scavenging enzymes, CAT and GSH-Px, that are known to be inhibited by nickel [8]. These two effects combined could augument the potential of oxidative cell damage. At latter time intervals, with decreasing nickel, the activity of hepatic GSHPx and CAT was restored and L P O decreased. This should lower the potential of oxidative damage to the ceils [18]. However, in kidney, the concurrence between changes in LPO and CAT was lost after 24 h when CAT activity dropped again with no LPO increase. The reason for this highly significant CAT drop is not clear. Its relevance to renal toxicity of nickel deserves further investigations. The
14
activity of CAT and GSH-Px in muscle were least affected by nickel with no alterations in LPO. Interestingly, under the present experimental conditions, no apparent involvement of SOD in the observed toxic effects of nickel was noticed. Activity of this enzyme that converts superoxide radical into H202 remained practically unchanged in all tissues tested. Thus, undisturbed generation of H202 at times of decreased CAT and GSH-Px activity may add to the toxic effect of nickel. Our results differ from those of NoveUi and Rodriguez [34], who observed an increase in Cu-Zn SOD activity in the pancreas of rats given nickel chloride. Perhaps, nickel effect on SOD is tissue specific. In the present experiment, we have found that there is a possible direct relationship between nickel-induced LPO and effects of this metal on the GSH/GSHPx activity. However, the results do not provide enough information about the degree of interdependence of these effects. The peroxidative activity of metal ions has generally been related to their ability to deplete the cellular GSH content which is known to maintain the cellular redox potential [7]. Here we report that nickel injection caused immediate deficiency of hepatic GSH (and GSH-Px) thus lowering the cell's ability to protect itself against LPO at earlier time points. The levelling of LPO at later time points, when most of the nickel dose is excreted, might be assisted by a concurrent increase of GSH. In the kidney, the concurrence of the LPO and GSH changes was similar to that in the liver, with, however, some time-related and quantitative differences which most likely resulted from different retention of nickel and different basal concentration of GSH in these organs. The turnover of GSH mainly depends upon the levels of various GSH synthesis and metabolism enzymes, including GSSG-R, GGT, and GST. The overall effect of nickel on GSH can be explained by the concept that nickel binds to GSH and thus initially depletes preexisting GSH level. In liver, at the same time, nickel treatment lowers the activity of GST, which might be due to the lesser availability of GSH or to the direct effect of nickel on the enzyme. However, in the kidney, the opposite is true. Thus, nickel effect on GST is more complex and indirect. The observed pattern in GSSG-R response to nickel exposure was biphasic. The initial enhancement of GSSG-R activity at 1 h might be due to the biological response to nickel insult. The rapid depletion of GSSG-R, that followed (3 h), might result from decreased production of NADPH in the presence of nickel [35]. This depletion, however, may not be enough to affect regeneration of GSH from GSSG. The increased hepatic GSH level at later times might thus reflect both regeneration and enhanced de novo synthesis of GSH [36]. Likewise, the secondary rise in renal GSH level despite significant depletion of GSSG-R may indicate increased intracellular GSH synthesis. In conclusion, LPO may result in the production of various hydroperoxides (ROOH) and organic radicals (R., RO2-). GSH is an efficient substrate for enzymatic reduction of peroxides via GSH-Px [37]. Therefore, depletion of intracellular GSH and GSH-Px following nickel treatment would make tissues more vulnerable to peroxides as observed in the present study. The increased level of active oxygen species in the tissues ultimately may cause damage to the genetic material [13] and thus contribute to carcinogenic effects of nickel. 15
References 1 2 3 4 5 6 7
8 9 10 11 12
13 14 15 16 17
.18 19
20
21 22 23
16
T.P. Coogan, D.M. Latta, E.T. Snow and M. Costa, Toxicity and carcinogenicity of nickel compounds. CRC Crit. Rev. Toxicol., 19 (1989) 341. F.W. Sunderman, Jr., Mechanisms of nickel carcinogenesis. Scand. J. Work Environ. Health, 15 (1989) 1. F.W. Sunderman, Jr. Lipid peroxidation as a mechanism of acute nickel toxicity. Toxicol. Environ. Chem., 15 (1987) 59. F.W. Sunderman, Jr., A. Marzouk, S.M. Hopfer, O. Zaharia, and M.C. Reid, Increased lipid peroxidation in tissue of nickel chloride-treated rats. Ann. Clin. Lab. Sci., 15 (1985) 229. K.S. Kasprzak and R.B. Bare, in vitro polymerization of histones by carcinogenic nickel compounds. Carcinogenesis, 10 (1989) 621. M. Misra, M. Athar, S.K. Hasan and R.C. Srivastava, Alleviation of nickel-induced biochemical alterations by chelating agents. Fundam. Appl. Toxicol., l 1 (1988) 285. M. Athar, S.K. Hasan and R.C. Srivastava, Role of glutathione metabolizing enzymes in nickel mediated induction of hepatic glutathione. Res. Commun. Chem. Pathol. Pharmacol., 57 (1987) 421. R.E. Rodriguez and K.S. Kasprzak, Nickel(II) inhibition of catalase, glutathione peroxidase/ glutathione reductase system. The Toxicologist, 9 (1989) 134. E.N. Frankel and W.E. Neff, Formation of malonaldehyde from lipid peroxidation products. Biochim. Biophys. Acta, 754 (1983) 264. B. Halliwell and J.M.C. Gutteridge, Oxygen toxicity, oxygen radicals, transition metals and disease. Biochem. J., 219 (1984) 1. P. Hochstein and S.K. Jain, Association of lipid peroxidation and polymerization of membranes proteins with erythrocyte aging. Fed. Proc., 40 (1981) 183. R.P. Bird and H.H. Draper, Effect of malonaldehyde and acetaldehyde on cultured mammalian cells: growth, morphology and synthesis of macro molecules. J. Toxicol. Environ. Health, 6 (1980) 811. R.B. Ciccareli and K.E. Wetterhahn, Molecular basis for the activity of nickel. IARC Sci. Publ., 53 (1984) 201. S.R. Patierno and M. Costa, DNA-protein-crosslinks induced by nickel compounds in intact cultured mammalian cells. Chem.-Biol. Interact., 55 (1985) 75. K.S. Kasprzak and L. Hernandez, Enhancement of hydroxylation and deglycosylation of 2deoxyguanosine by carcinogenic nickel compounds. Cancer Res., 49 (1989) 5964. M.J. Olson, DNA strand breaks induced by hydrogen peroxide in isolated rat hepatocytes. J. Toxicol. Environ. Health, 23 (1988) 407. M. Athar, S.K. Hasan and R.C. Srivastava, Evidence for the involvement of hydroxyl radicals in nickel mediated enhancement of lipid peroxidation: implications for nickel carcinogenesis. Biochem. Biophys. Res. Commun., 5 (1987) 1276. B.M. Babior, Oxygen-dependent microbial killing by phagocytes: Part I. N. Engl. J. Med., 1978 (1987) 659. R.C. Schnell, H.P. Bozigian, M.H. Davies, B.A. Merrick, and K. Johnson, Circadian rhythms in acetaminophen toxicity: Role of non-protein sulphydryl. Toxicol. Appl. Pharmacol., 71 (1983) 353. J.H. Wilkinson, D.N. Baron, D.W. Moss and P.G. Walker, Standardization of clinical enzymes assays: a reference method for aspartate and alanine transaminases. J. Clin. Pathol., 25 (1972) 940. M. Shlafer and B.M. Shepard, A method to reduce interference by sucrose in the detection of thiobarbituric acid-reactive substances. Anal. Biochem., 137 (1984) 269. L.W. Oberly and D.R. Spitz, Assay of superoxide dismutase activity in tumor tissue. Methods Enzymol., 105 (1984) 457. D.J. Jollow, J.R. Mitchell, Z. Zampaglione and J.R. Gillette, Bromobenzene induced liver necrosis. Protective role of glutathione and evidence for 3,4-bromobenzene oxide as the hepatotoxic metabolite. Pharmacology, 11 (1974) 151.
24 25 26 27 28 29 30 31 32 33
34 35 36
37
A. Meister, S.S. Tate and O.W. Griffith, y-Glutamyl Transpeptidase. Methods Enzymol., 77 (1981) 237. H. Aebi, Catalase in vitro. Methods Enzymol., 105 (1984) 121. Y.H. Lee, D.K. Layman and R.R. Bell, Glutathione peroxidase activity in iron-deficient rats. J. Nutr., 111 (1981) 194. W.H. Habig, M.J. Pabst and W.B. Jakoby, Glutathione-S-transferase. The first enzymatic step in mercapturic acid formation. J. Biol. Chem., 249 (1974) 7130. E. Racker, Glutathione reductase from baker yeast and beef liver. J. Biol. Chem., 217 (1955) 855. M.M. Bradford, A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem., 72 (1976) 248. B. Vonen and J. Morland, Isolated rat hepatocytes in suspension: potential hepatotoxic effects of six different drugs. Arch. Toxicol., 56 (1984) 33. J.M.C. Gutteridge, Tissue damage by oxy-radicals: The possible involvement of iron and copper complexes. Med. Biol., 62 (1984) 101. C.E. Thomas, L.A. Morehouse and S.D. Aust, Ferritin and superoxide-dependent lipid peroxidation. J. Biol. Chem., 260 (1985) 3275. S. Inoue and S. Kawanishi, ESR evidence for superoxide, hydroxyl radicals and singlet oxygen produced from hydrogen peroxide and nickel(lI) complex of glycylglycyl-L-histidine. Biochem. Biophys. Res. Commun., 159 (1989) 445. E.L.B. Novelli and N.L. Rodriguez, Effect of nickel chloride on streptozotocin-induced diabetes in rats. Can. J. Physiol. Pharmacol., 66 (1988) 663. L. Cartana and A.A. Romeu, Characterization of the inhibition effect induced by nickel on glucose-6-phosphate dehydrogenase and glutathione reductase. Enzyme, 41 (1989) 1. A. Chung and M.D. Maines, Effect of selenium on glutathione metabolism. Induction of y-glutamyl cysteine synthetase and glutathione reductase in the rat liver. Biochem. Pharmacol., 30 (1981) 3217. A.G. Splittgerber and A.L. Tappel, Inhibition of glutathione peroxidase by cadmium and other metal ions. Arch. Biochem. Biophys., 197 (1979) 534.
17
