Agents and Actions, vol. 33, 3/4 (1991)
0065-4299/91/040247-07 $1.50 +0.20/0 9 1991 Birkh/iuser Verlag, Basel
Effects of acute and chronic prednisolone treatment on serum zinc levels in rats with adjuvant arthritis J. Fontaine 1, J. N6ve 2, A. Peretz 3, P. Capel 4 and J.P. Famaey 3 x Laboratory of Pharmacology, Institute of Pharmacy, Universit6 Libre de Bruxelles, Campus Plaine, 205-7, B-1050 Bruxelles, Belgium, 2 Laboratory of Medicinal Chemistry, Institute of Pharmacy, Universit6 Libre de Bruxelles, 3 Department of Rheumatology and Physical Medicine, University Hospital Saint-Pierre, and 4 Laboratory of Hematology, University Hospital Saint-Pierre
Abstract
Several studies in animals and humans have independently demonstrated that zinc metabolism is significantly affected either by inflammation or by glucocorticoid administration. The relative importance of these two factors was assessed in this study by the investigation of the effects on serum zinc concentrations of acute and chronic prednisolone treatments in adjuvant arthritis rats and in healthy controls animals. Acute steroid administration (3 mg/kg, i.p.) caused a rapid drop in serum zinc followed by a quick recovery, regardless to the fact that these concentrations were normal (healthy animals) or already reduced by the inflammatory process. However, the modification occurred faster in inflamed animals. Chronic steroid administration (0.58 to 0.78 mg/kg/day during 1 to 4 weeks) had a more complex effect. A previous experiment in healthy rats demonstrated that such a treatment only induced a slight decrease in serum zinc. In adjuvant arthritis animals, the early steroid treatment of the induced process promoted a further decrease in serum zinc level while a delayed treatment did not result in additional changes.
Introduction
Zinc is an essential trace element intimately involved in various processes relevant to inflammation such as prostaglandin biosynthesis [1], lymphocyte's stimulation and immune responses [2], scavenging of oxygen free radicals [3], and lysosomal enzyme release [4]. It takes also part in the metabolism of connective tissues [5] and bones [6]. Modifications in zinc homoeostasis, mainly hypozincemia, were described in localized inflammation [7] and in systemic infections diseases [8, 9]. They are apparently associated with an increased hepatic zinc uptake [7, 8], which was documented in animals both during the acute carrageenan induced pleurisy of the rat [10] and in adjuvant 1 Author for correspondence.
arthritis [11]. Patients suffering from rheumatoid arthritis [12, 13] or psoriatic arthritis [13], as well as rats with established adjuvant arthritis [14-16] also exhibit low plasma zinc levels which were attributed to the inflammatory process and implicated in some pathological features. The first trial of oral zinc supplementation in rheumatoid arthritis patients resulted in beneficial clinical effects [17] that were however not confirmed in subsequent studies [18, 19]. Several investigators demonstrated that zinc metabolism is also affected by corticosteroid administration [20-22]. These drugs seem to induce synthesis of hepatic metallothionein, a powerful ligand for the element, causing accumulation of zinc in the liver with a resulting alteration in plasma levels. As a matter of fact, oral long term treatment with glucocorticoids as well as acute ad-
248 ministration of intravenous high doses to rheumatoid arthritis patients caused significant decreases in plasma zinc levels [23, 24]. This change may be beneficial for the defence of the organism by potentiating the functional capacity of phagocyte ceils [25, 26], but long-term treatment is liable to induce a zinc deficiency state [27]. Previous studies by our group both in humans [23] and in animals [15, 28] were devoted to some of these factors liable to affect zinc homoeostasis. In rats, we carefully described the evolution of several indicators of zinc status (including plasma zinc) after the induction of adjuvant arthritis [15]. In another investigation, we characterized a slight significant lowering in plasma zinc after 5 weeks (but not after 3 weeks) chronic oral prednisolone administration to healthy rats [28]. In the present study, the effects on serum zinc concentrations of both acute and chronic prednisolone treatments were investigated in adjuvant arthritis rats and compared to those in healthy control animals.
Materials and methods
Animals and diets Groups of male Wistar rats (from Proefdieren centrum, Leuven, Belgium), 4 4 - 50 days old at the beginning of the experiments, were used. They stayed 2 weeks in the laboratory before any treatment. They were kept alone in makrolon cages on a 12 h light-dark cycle at constant temperature (2022 ~ and humidity (40-60%) and had free access to food and desionised water. Solid food was a complete maintenance diet from the Usine d'alimentation rationelle, Villemoisson, France (ref: A04), containing 85 mg zinc/kg, and representing for the animals a daily zinc intake of between 1.7 and 2.1 mg, which is considered as adequate [29, 30]. Contribution of water to zinc nutriture is negligible. Some of the animals (see further) were fed with the same diet enriched with prednisolone (Pfizer), 8 mg/kg. Such as in previous experiments [15, 28], food intake and weight of animals were monitored daily.
Induction of adjuvant arthritis and determination of clinical indices Arthritis was induced by subcutaneous injection into the tail of a suspension containing 0.7 mg of
Agents and Actions, vol. 33, 3/4 (1991) heat killed Mycobacterium Butyricum (Freund's adjuvant, Difco) in 0.1 ml light mineral oil. Importance of joint involvement (number of affected joints and swelling) was estimated on an arbitrary scale (scores 1 to 5) just before sacrifice. Simultaneously, volumes of both hind paws (ml/100 g animal) were measured by plethysmography (Ugo Basile apparatus) in ether-anaesthetized rats.
Acute prednisolone treatment Two experiments were conducted on the same pattern. The first one included a group of 90 healthy rats (weighing 200_+ 5 g) which were injected with prednisolone 30 mg/kg i.p. (0.1 ml/100 g rat of a laboratory-made preparation containing 30 mg prednisolone, 1 mg tween 80, and 5 mg polyethylene glycol 4000 in I ml isotonic saline), as well as 90 control healthy rats (206_ 5 g) which were injected with the vehicle alone. The second one was performed with 60 rats with adjuvant arthritis, 2 weeks after injection of the Freund's adjuvant (229 ___8 g), which received the same prednisolone preparation, and of 60 other adjuvant arthritis animals (238 +_11 g), which served as control arthritic animals for the injection of the vehicle. In these two experiments, subgroups of 10 rats were sacrificed at various times after injection: after 2, 4, 6, 7, 9, 12, 16, 24 and 72 h for healthy rats, and after 4, 6, 7, 9, 12, and 19 h for arthritic animals. Beside these animals, two groups of 20 healthy or arthritic animals (same characteristics than before), which did not receive any treatment, were simultaneously sacrificed to determine initial (time 0) values. Great care was taken in the procedures to synchronize injections and rat sacrifices in order to strictly adhere to the fixed time intervals. Moreover, prednisolone treated animals and vehicle-injected controls were alternatively sacrificed in each series.
Chronic prednisolone treatment In this experiment involving 150 animals, groups of rats with adjuvant arthritis induced since either 1 (n=45) or 2 weeks (n=30) were chronically treated by oral prednisolone for a maximum of 4 weeks. The treatment was initiated by replacing their ordinary diet by the prednisolone-enriched one. According to the mean daily food consumption, which varied during the experiment, they
Agents and Actions, vol. 33, 3/4 (1991) consumed between 0.58 and 0.78 m g prednisolone/ kg/day. A n o t h e r group o f arthritic animals (n = 60) did not receive any treatment and served as time controls for the disease process. Subgroups o f 15 animals were sacrificed 1, 2, 3 or 5 weeks after disease induction. Beside these animals, 15 healthy rats were sacrificed at the beginning o f the experiment (time 0). Other d a t a such as the weight o f animals are mentioned later. Sacrifice o f animals and analytical determinations
Diet o f animals was withdrawn 16 h before sacrifice. Unless indicated (in the experiments with acute prednisolone treatment), ether-anaesthetized rats were killed between 9 a n d 10.30a.m. by a o r t a cutting. Their blood was collected in dry p o l y p r o p y l e n e tubes previously acid washed in order to avoid trace element contamination. 0.9 ml b l o o d was immediately collected in 0.1 ml citrate for fibrinogen determination [31]. After b l o o d clotting, serum was separated by centrifugation and kept frozen until analysis. Zinc was determined by flame atomic a b s o r p t i o n spectroscopy (Perkin Elmer 2100 apparatus) after dilution in 0.1 N HC1 [32]. A c c u r a c y o f the p r o c e d u r e was established by analysis o f a reference material m a n u f a c t u r e d by J. Versieck, Belgium [33]. Results are expressed as m e a n concentrations in each subgroup-+ s t a n d a r d deviation (SD), in gg zinc/dL serum, a n d c o m p a r e d by the Student's t-test.
Results
A c u t e prednisolone treatment in healthy rats
Table 1 reports the mean serum zinc levels in the various examined subgroups. D a t a for healthy controls (injected with the vehicle alone) show large fluctuations according to m o m e n t o f b l o o d collection. As a m a t t e r o f fact, in this experiment, b l o o d was not collected in the different groups at the same period o f the day. This p h e n o m e n o n is linked to the physiological circadian variation in serum zinc previously observed b o t h in animals [34] a n d in h u m a n s [35]. However, to allow comparison with d a t a from prednisolone treated animals, rats were always sacrificed at the same m o ment o f the d a y than their respective time controls (see Materials a n d methods). In these conditions, some significant differences between values for the
249 Table 1 Serum zinc in a group of healthy rats and in a group of rats with adjuvant arthritis (induced since 2 weeks) treated with i.p. prednisolone, 30 mg/kg, or with its vehicle(controls) and sacrificedat various times after treatment. Results are expressedin gg Zn/dL as mean + SD, n = 10, except for t = 0 (no treatment) wheren = 20. Time (h)
Healthy rats
0 2 4 6 7 9 12 16
129_+17 132_+13 134+_12 126+_20 122_+18 106_ 6 87_+19"* 110-+19 77+ 8*** 117_+ 7 79+_19"** 103_+10 98_+10 130+_13 140_+ 8*
19
24 72
Controls (vehicle)
-
118_+10 138+_11
Arthritis rats Prednisolone treatment
-
115_+12 149+_ 9*
Controls (vehicle)
Prednisolone treatment
93_+14 93_+12 77-1-20" 87__12 62-+13"** 804-14 60_+15" 95+__ 7 76+_12"* 75_+19 80_+14 92+_11
-
88_+I5 -
* Significant differences between prednisolone treated animals and controls: *p
0065-4299/91/040247-07 $1.50 +0.20/0 9 1991 Birkh/iuser Verlag, Basel
Effects of acute and chronic prednisolone treatment on serum zinc levels in rats with adjuvant arthritis J. Fontaine 1, J. N6ve 2, A. Peretz 3, P. Capel 4 and J.P. Famaey 3 x Laboratory of Pharmacology, Institute of Pharmacy, Universit6 Libre de Bruxelles, Campus Plaine, 205-7, B-1050 Bruxelles, Belgium, 2 Laboratory of Medicinal Chemistry, Institute of Pharmacy, Universit6 Libre de Bruxelles, 3 Department of Rheumatology and Physical Medicine, University Hospital Saint-Pierre, and 4 Laboratory of Hematology, University Hospital Saint-Pierre
Abstract
Several studies in animals and humans have independently demonstrated that zinc metabolism is significantly affected either by inflammation or by glucocorticoid administration. The relative importance of these two factors was assessed in this study by the investigation of the effects on serum zinc concentrations of acute and chronic prednisolone treatments in adjuvant arthritis rats and in healthy controls animals. Acute steroid administration (3 mg/kg, i.p.) caused a rapid drop in serum zinc followed by a quick recovery, regardless to the fact that these concentrations were normal (healthy animals) or already reduced by the inflammatory process. However, the modification occurred faster in inflamed animals. Chronic steroid administration (0.58 to 0.78 mg/kg/day during 1 to 4 weeks) had a more complex effect. A previous experiment in healthy rats demonstrated that such a treatment only induced a slight decrease in serum zinc. In adjuvant arthritis animals, the early steroid treatment of the induced process promoted a further decrease in serum zinc level while a delayed treatment did not result in additional changes.
Introduction
Zinc is an essential trace element intimately involved in various processes relevant to inflammation such as prostaglandin biosynthesis [1], lymphocyte's stimulation and immune responses [2], scavenging of oxygen free radicals [3], and lysosomal enzyme release [4]. It takes also part in the metabolism of connective tissues [5] and bones [6]. Modifications in zinc homoeostasis, mainly hypozincemia, were described in localized inflammation [7] and in systemic infections diseases [8, 9]. They are apparently associated with an increased hepatic zinc uptake [7, 8], which was documented in animals both during the acute carrageenan induced pleurisy of the rat [10] and in adjuvant 1 Author for correspondence.
arthritis [11]. Patients suffering from rheumatoid arthritis [12, 13] or psoriatic arthritis [13], as well as rats with established adjuvant arthritis [14-16] also exhibit low plasma zinc levels which were attributed to the inflammatory process and implicated in some pathological features. The first trial of oral zinc supplementation in rheumatoid arthritis patients resulted in beneficial clinical effects [17] that were however not confirmed in subsequent studies [18, 19]. Several investigators demonstrated that zinc metabolism is also affected by corticosteroid administration [20-22]. These drugs seem to induce synthesis of hepatic metallothionein, a powerful ligand for the element, causing accumulation of zinc in the liver with a resulting alteration in plasma levels. As a matter of fact, oral long term treatment with glucocorticoids as well as acute ad-
248 ministration of intravenous high doses to rheumatoid arthritis patients caused significant decreases in plasma zinc levels [23, 24]. This change may be beneficial for the defence of the organism by potentiating the functional capacity of phagocyte ceils [25, 26], but long-term treatment is liable to induce a zinc deficiency state [27]. Previous studies by our group both in humans [23] and in animals [15, 28] were devoted to some of these factors liable to affect zinc homoeostasis. In rats, we carefully described the evolution of several indicators of zinc status (including plasma zinc) after the induction of adjuvant arthritis [15]. In another investigation, we characterized a slight significant lowering in plasma zinc after 5 weeks (but not after 3 weeks) chronic oral prednisolone administration to healthy rats [28]. In the present study, the effects on serum zinc concentrations of both acute and chronic prednisolone treatments were investigated in adjuvant arthritis rats and compared to those in healthy control animals.
Materials and methods
Animals and diets Groups of male Wistar rats (from Proefdieren centrum, Leuven, Belgium), 4 4 - 50 days old at the beginning of the experiments, were used. They stayed 2 weeks in the laboratory before any treatment. They were kept alone in makrolon cages on a 12 h light-dark cycle at constant temperature (2022 ~ and humidity (40-60%) and had free access to food and desionised water. Solid food was a complete maintenance diet from the Usine d'alimentation rationelle, Villemoisson, France (ref: A04), containing 85 mg zinc/kg, and representing for the animals a daily zinc intake of between 1.7 and 2.1 mg, which is considered as adequate [29, 30]. Contribution of water to zinc nutriture is negligible. Some of the animals (see further) were fed with the same diet enriched with prednisolone (Pfizer), 8 mg/kg. Such as in previous experiments [15, 28], food intake and weight of animals were monitored daily.
Induction of adjuvant arthritis and determination of clinical indices Arthritis was induced by subcutaneous injection into the tail of a suspension containing 0.7 mg of
Agents and Actions, vol. 33, 3/4 (1991) heat killed Mycobacterium Butyricum (Freund's adjuvant, Difco) in 0.1 ml light mineral oil. Importance of joint involvement (number of affected joints and swelling) was estimated on an arbitrary scale (scores 1 to 5) just before sacrifice. Simultaneously, volumes of both hind paws (ml/100 g animal) were measured by plethysmography (Ugo Basile apparatus) in ether-anaesthetized rats.
Acute prednisolone treatment Two experiments were conducted on the same pattern. The first one included a group of 90 healthy rats (weighing 200_+ 5 g) which were injected with prednisolone 30 mg/kg i.p. (0.1 ml/100 g rat of a laboratory-made preparation containing 30 mg prednisolone, 1 mg tween 80, and 5 mg polyethylene glycol 4000 in I ml isotonic saline), as well as 90 control healthy rats (206_ 5 g) which were injected with the vehicle alone. The second one was performed with 60 rats with adjuvant arthritis, 2 weeks after injection of the Freund's adjuvant (229 ___8 g), which received the same prednisolone preparation, and of 60 other adjuvant arthritis animals (238 +_11 g), which served as control arthritic animals for the injection of the vehicle. In these two experiments, subgroups of 10 rats were sacrificed at various times after injection: after 2, 4, 6, 7, 9, 12, 16, 24 and 72 h for healthy rats, and after 4, 6, 7, 9, 12, and 19 h for arthritic animals. Beside these animals, two groups of 20 healthy or arthritic animals (same characteristics than before), which did not receive any treatment, were simultaneously sacrificed to determine initial (time 0) values. Great care was taken in the procedures to synchronize injections and rat sacrifices in order to strictly adhere to the fixed time intervals. Moreover, prednisolone treated animals and vehicle-injected controls were alternatively sacrificed in each series.
Chronic prednisolone treatment In this experiment involving 150 animals, groups of rats with adjuvant arthritis induced since either 1 (n=45) or 2 weeks (n=30) were chronically treated by oral prednisolone for a maximum of 4 weeks. The treatment was initiated by replacing their ordinary diet by the prednisolone-enriched one. According to the mean daily food consumption, which varied during the experiment, they
Agents and Actions, vol. 33, 3/4 (1991) consumed between 0.58 and 0.78 m g prednisolone/ kg/day. A n o t h e r group o f arthritic animals (n = 60) did not receive any treatment and served as time controls for the disease process. Subgroups o f 15 animals were sacrificed 1, 2, 3 or 5 weeks after disease induction. Beside these animals, 15 healthy rats were sacrificed at the beginning o f the experiment (time 0). Other d a t a such as the weight o f animals are mentioned later. Sacrifice o f animals and analytical determinations
Diet o f animals was withdrawn 16 h before sacrifice. Unless indicated (in the experiments with acute prednisolone treatment), ether-anaesthetized rats were killed between 9 a n d 10.30a.m. by a o r t a cutting. Their blood was collected in dry p o l y p r o p y l e n e tubes previously acid washed in order to avoid trace element contamination. 0.9 ml b l o o d was immediately collected in 0.1 ml citrate for fibrinogen determination [31]. After b l o o d clotting, serum was separated by centrifugation and kept frozen until analysis. Zinc was determined by flame atomic a b s o r p t i o n spectroscopy (Perkin Elmer 2100 apparatus) after dilution in 0.1 N HC1 [32]. A c c u r a c y o f the p r o c e d u r e was established by analysis o f a reference material m a n u f a c t u r e d by J. Versieck, Belgium [33]. Results are expressed as m e a n concentrations in each subgroup-+ s t a n d a r d deviation (SD), in gg zinc/dL serum, a n d c o m p a r e d by the Student's t-test.
Results
A c u t e prednisolone treatment in healthy rats
Table 1 reports the mean serum zinc levels in the various examined subgroups. D a t a for healthy controls (injected with the vehicle alone) show large fluctuations according to m o m e n t o f b l o o d collection. As a m a t t e r o f fact, in this experiment, b l o o d was not collected in the different groups at the same period o f the day. This p h e n o m e n o n is linked to the physiological circadian variation in serum zinc previously observed b o t h in animals [34] a n d in h u m a n s [35]. However, to allow comparison with d a t a from prednisolone treated animals, rats were always sacrificed at the same m o ment o f the d a y than their respective time controls (see Materials a n d methods). In these conditions, some significant differences between values for the
249 Table 1 Serum zinc in a group of healthy rats and in a group of rats with adjuvant arthritis (induced since 2 weeks) treated with i.p. prednisolone, 30 mg/kg, or with its vehicle(controls) and sacrificedat various times after treatment. Results are expressedin gg Zn/dL as mean + SD, n = 10, except for t = 0 (no treatment) wheren = 20. Time (h)
Healthy rats
0 2 4 6 7 9 12 16
129_+17 132_+13 134+_12 126+_20 122_+18 106_ 6 87_+19"* 110-+19 77+ 8*** 117_+ 7 79+_19"** 103_+10 98_+10 130+_13 140_+ 8*
19
24 72
Controls (vehicle)
-
118_+10 138+_11
Arthritis rats Prednisolone treatment
-
115_+12 149+_ 9*
Controls (vehicle)
Prednisolone treatment
93_+14 93_+12 77-1-20" 87__12 62-+13"** 804-14 60_+15" 95+__ 7 76+_12"* 75_+19 80_+14 92+_11
-
88_+I5 -
* Significant differences between prednisolone treated animals and controls: *p
