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Effects of Concurrent Administration of Monensin and Selenium on Erythrocyte Glutathione

Peroxidase Activity and Liver Selenium Concentration

in Broiler Chickens M. Z. KHAN, 1 J. SZAREK, *'1'2 E. MARCHALUK, 3

A. MACIG, 2 AND P. M. BARTLEWSKI 1

1Department of Forensic Veterinary Medicine and Veterinary Medicine Administration, University of Agriculture

and Technology, ul. Oczapowskiego 13, Olsztyn, Poland; 2Department of Veterinary Pathology, University of Agriculture,

Faisalabad, Pakistan; and 3Department of Biochemistry, Medical Academy, Bydgoszcz, Poland

Received March 30, 1994; Accepted May 31, 1994

ABSTRACT

Different toxic doses of selenium and monensin preparations were administered to broiler chickens. The two substances were given by oral route, alone or concurrently, for variable periods. Erythrocyte glutathione peroxidase (GSH-Px) activity was found to be elevated after the administration of the drugs. This increase was considerably higher when selenium and monensin were administered concurrently, indicating the occurrence of strong interaction between them. Admin- istration of selenium led to a rapid increase in the liver selenium con- centration. This increase, in turn, was enhanced by concurrent application of monensin. Monensin given alone did not have any sig- nificant effect on the changes of liver selenium concentration. Further results suggest that administration of monensin increases erythrocyte GSH-Px activity, even in the absence of supplemental selenium or during increased liver selenium concentration.

*Author to whom all correspondence and reprint requests should be addressed.

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130 Khan et al.

Index Entries: Selenium; selenium status; selenium toxicosis; monensin; monensin toxicosis; chicken broilers; erythrocyte glu- tathione peroxidase (GSH-Px) activity.

INTRODUCTION

Monensin, a metabolic product of Streptomyces cinnamonensis, a sodium-selective ionophore (polyether antibiotic) is commonly used as an anticoccidial drug in poultry. It is highly effective against all species of coccidia in the field. Additional growth-promoting properties of this antibiotic have been exploited Monensin is used exclusively in cattle beef as a dietary growth promoter. The mode of action of this antibiotic is chiefly to interfere with the transport of negatively charged ions through biological membranes, which favors an influx of cations. The amount of the drug absorbed by the host organism has limited therapeutic value, but it may result in changes of deposition rates for ions in different vis- ceral organs. Monensin has a low therapeutic index and may be fatal in certain species when administered in excessive doses (1). A n interaction between monensin and other organic and inorganic compounds has been reported. When monensin is used along with tiamulin and both sub- stances are applied at therapeutic doses, they appear to induce an acute toxic response (2,3). Selenium plus vitamin E at therapeutic levels par- tially ameliorated the toxic effect of monensin in swine (3), whereas mon- ensin, also at a therapeutic level, aggrevated the selenium toxicity in sheep (4). Recent studies carried out in our departments showed that hematological and biochemical parameters in broiler chickens exhibited more pronounced changes following concurrent oral administration of monensin and selenium (5).

A peculiar property of monensin was observed when erythrocyte GSH-Px activity was measured in sheep after administration of the drug. An increase in erythrocyte GSH-Px activity was detected as a result of monensin supplementation at therapeutic doses, and its further increase was observed in the presence of selenium, as compared with controls treated with selenium exclusive of the antibiotic (6). An increase in ery- throcyte GSH-Px activity in liver tissue of broiler chicks following an intake of toxic doses of monensin was also reported (7). All of these reports suggest the existence of monensin-dependent lipid peroxidase metabolism, as well as Se-dependent antioxidative defense mechanisms. Selenium is known to enhance erythrocyte GSH-Px activity. The latter is strongly correlated with amounts of selenium absorbed, regardless of possible harmfulness of the dose, e.g., it tends to increase either at high therapeutic, subtoxic, or toxic levels of the element (8-11). However, the effects of concurrent treatment with selenium and monensin on erythro- cyte GSH-Px activity remained ill-understood.

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Effects of Monensin on Se Status 131

With this study, we aimed to corraborate an effect of various toxic levels of monensin and selenium given concurrently to broiler chickens on eventual changes of erythrocyte GSH-Px activity and, additionally, liver selenium concentrations. This article bears the answer to whether and at what stages of monensin and/or selenium toxicosis an increase in erythrocyte GSH-Px activity owing to administration of monensin is influenced by the selenium status of the birds.

MATERIALS AND METHODS

Broiler chickens (Astra B breed) aged 2 wk were divided into four preliminary experimental groups (A, B, C, and E), and kept on feed with addition of selenium (15 mg/kg feed) as sodium selenite, selenium (15 mg/kg feed) plus vitamin E (200 mg/kg feed) as 0~ tocopherol, and mon- ensin (240 mg/kg feed) as monensin sodium (Elanco Laboratories, Italy) (Fig. 1). The major group E was kept on basal feed free of any toxic sub- stances as a control group. The purpose of the first experiment was to induce a state of acute monensin or selenium toxicosis. At first, we allowed 12 d of adaptation period during which the birds were fed as described above. The birds were then divided for the second time into two final groups numbering 13 birds each, excluding controls. Half of these groups were administered a second toxic chemical different from that already present in the diet. The second chemical was also given by oral route, but this time as a single oral dose to individual birds. A dosage of selenium and monensin was 8 mg and 400 mg/kg body mass, respectively. In this way, each bird was treated with two toxic substances, one supplied in feed and the second as a single oral dose. Remaining groups derived from the primary groups were not given the second toxic chemical. Finally, the birds of group E were divided into three sub- groups. Two of them were intoxicated with either selenium or monensin, and only one group was sustained as an untoxicated control (E0). A con- cise scheme of experiments is presented in Fig. 1. After 24 h postintoxi- cation (pi), meaning after administration of the second toxic chemical, three birds from each group were randomly selected and sacrificed for subsequent biochemical analysis. All remaining birds were kept under observation for next 2 wk.

In the second experiment, in order to induce the state of subacute toxicosis, an adaptation period was extended up to 4 wk. Formation of final groups in this experiment was, in comparison to the first experi- ment, essentially the same. The second toxic substance was being admin- istered to individual birds orally on alternate days for the next 4 wk at the following dosage: selenium--1 mg and monensin---40 mg/kg body mass. Three birds from each experimental group were sacrificed at weekly intervals for pathological examinations. Erythrocyte GSH-Px or

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132

Preliminary dietary groups (dietary additions / kg feed)

Khan et al.

Adaptation period Second Final (12 days) toxic chemical group

A ~ Monensin ~ AM (Selenium 15 m ~ ~ Nil ~ AO

B ~ Monensin ~ BM (Selenium 15 mg ~ Nil ~ BO

+ vit.E 200 rag)

C ~ Selenium ~ CS (Monensln 240 m ~ ~ Nil ~ CO

Selenium I ~ - ES Monensin ~ EM E

( Nil ) ~ Nil ~ EO

Fig. 1. Schematic representation of grouping and allocation of sec- ond toxic chemicals to birds in first and second experiments.

red blood cell (RBC) glutathione peroxidase (GSH-Px, EC 1.11.1.9) activ- ity was measured by the method of Paglia and Valentine (12) as modi- fied by Hopkins and Thudhope (13) using t-butyl as a substrate. One unit of enzyme activity was defined as equal to the number of NADPH ~tmol- oxidized/min, and final results were expressed in u /g of hemoglobin (Hb). Selenium concentration in examined livers was determined by atomic absorption spectrometry (Pye Unicam Spectrometer) using the hydride generation method (14). The data were subject to analysis of variance and mean values for different groups were compared by Dun- can's multiple-range test (15).

RESULTS

In the first experiment, the birds kept on diets containing selenium (group A) or selenium plus vitamin E (group B) had significantly higher erythrocyte GSH-Px activity as well as liver selenium concentration than those in the control group (Table 1). At 24 h pi during acute monensin toxicosis, no significant changes of erythrocyte GSH-Px activity were recorded in the experimental group of the birds kept on diets containing selenium (group AM) and selenium plus vitamin E (group BM)-Table 2. Administration of monensin, however, was accompanied by an increase

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Effects of Monensin on Se Status 133

Table 1 Erythrocyte GSH-Px Activity and Liver Selenium Levels 24 H After Acute

Monensin Toxicosis in Birds Fed Different Diets (Means _+ SD)

Erythrocyte Group, Monensin* GSH-Px, Selenium in liver tissue, dietary additions mg U / g Hb ~tg/g wm**

E0 (-) - - 68.13 _+ 6.94a 0.58 -+ 0.01 a EM (-) 400 86.37 + 9.47a, b 1.61 + 0.22 c A0 (Se) - - 125.35 _+ 41.33b 3.04 + 0.16d AM (Se) 400 108.27 _+ 11.75b 3.94 _+ 0.08 e B0 (Se, E)*** - - 114.44 _+ 19.76b 7.10 + 0.08f BM (Se, E) 400 115.59 + 19.76b 1.31 _+ 0.07b

*Given orally per kg body mass. **Liver wet mass. ***Selenium plus vitamin E. Values appearing in the same column with different superscripts are statistically sig-

nificant (p < 0.05).

Table 2 Erytrocyte GSH-Px Activity and Liver Selenium Levels 24 H After Acute

Selenium Toxicosis in Birds Fed Different Diets (Means + SD)

Erytrocyte Group, Selenium,* GSH-Px, dietary additions mg U / g Hb

Selenium in liver tissue, ~tg/g wm**

E0 (-) - - 68.13 + 06.94 0.58 + 0.01 a ES (-) 8 72.29 + 07.86 2.23 + 0.11b CO (Mon)*** - - 54.64 _+ 15.61 0.57 _+ 0.10 a CS (Mon) 8 40.67 _+ 12.93 4.08 + 0.17 c

*Given orally per kg body mass one time only. **Liver wet mass. ***Monensin. Values in same column with different superscripts are significantly different (p <

0.05).

in liver se len ium concent ra t ion in the birds kept on basal diet (group EM). At the same time, a certain increase in liver se len ium concent ra t ion was observed in the birds previous ly t reated wi th se len ium (group AM) as compared wi th their untoxica ted counterpar t s (Group AO)-see also Table 1. A single toxic dose of se len ium admin i s t e red to the birds kept ei ther on feed conta ining monens in (group CS) or addi t ive-free basal feed (group ES) d id not have an inf luence on the RBC GSH-Px activity at 24 h pi. Liver se len ium concentra t ion was, however , significantly h igher in g roup CS as compared wi th group ES, wh ich in tu rn was sig- nif icantly h igher than in controls (Table 2). Concern ing the second exper- iment , e ry throcy te GSH-Px activity as well as liver se len ium levels were

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134 Khan et al.

significantly higher in the birds kept for 5 wk on feed with addition of selenium (group A0) or selenium plus vitamin E (group B0). Subsequent administration of monensin, on alternate days, to the birds fed with grower mash containing selenium plus vitamin E (group BM) resulted in a significant increase in RBC GSH-Px activity and liver selenium levels at 4 wk pi. Administration of monensin had a similar effect on the birds kept on basal feed (group EM) resulting in a significant increase in RBC GSH-Px activity as compared with the control group at 4 wk pi. This increase, however, was considerably lower than that recorded earlier in groups AM and BM. Liver selenium levels determined in the birds in group EM were not significantly different from controls (Table 3).

Feeding of monensin in the diet (chickens of group CO) for 6 and 8 wk resulted in increased arythrocyte GSH-Px activity. Liver selenium level of those birds, however, remained insignificant from control. Administration of selenium on alternate days to the chickens fed the diet containing monensin (group CS) and also those kept on basal feed (group ES) resulted in a significant increase of erythrocyte GSH-Px activ- ity on the second and fourth weeks pi. The liver selenium level in those birds was also constantly higher compared with group CO and control (E0). However, chickens of group CS exhibited a significantly higher liver selenium concentration compared with that of group ES at the first and third weeks pi (Table 4).

DISCUSSION

An increase in selenium levels deposited in body tissues followed by a detectable increase in erythrocyte GSH-Px activity, as observed in the present study, has been reported in birds, sheep, and cattle. Many exper- imentors proved the existence of such a correlation in natural and exper- imental conditions. This correlation leads to indirect estimation of selenium status of farm animals based on evaluation of RBC GSH-Px activity in examined tissues (8). Accessory effects of vitamin E responsi- ble for enhanced production of erythrocyte GSH-Px during selenium and/or monensin toxicosis have not been reported earlier. Vitamin E acts as a free radical scavenger and inhibits formation of lipid hydroperoxide, whereas RBC GSH-Px catalyzes reduction of hydrogen peroxide and all hydroperoxides containing reduced glutathione as a cofactor (9). How- ever, the mechanism by which vitamin E controlled the activity of the enzyme was not ascertained in our study. It is likely that vitamin E decreases peroxidation of lipids by sequestering free radicals and thereby favors the availability of selenium for RBC GSH-Px synthesis. A failure to record statistically significant changes of RBC GSH-Px activity at 24 h pi during selenium toxicosis might have been owing to a long life-span of erythrocytes. This result supports an observation that erythrocyte GSH-Px activity should be considered a "detector" of a permanent or at

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Effects of Monensin on Se Status 1 3 5

Table 3 Erythrocyte GSH-Px Acitvi ty and Liver Selenium Levels Dur ing

Subacute Monens in Toxicosis in Birds of Groups Fed Different Diets (Mean _+ SD)

Erythrocyte Selenium in Group , Monesin* GSH-Px, liver tissue, d ie tary addi t ions m g U / g Hb ~tg/g wm**

1 w k pi EO (-) EM (-) A0 (Se) AM (Se) B0 (Se.E)*** BM (Se.E)

2 w k pi EO (-) EM (-) AO (Se) AM (Se) BO (Se.E) BM (Se.E)

3 w k pi EO (-) EM (-) A0 (Se) AM (Se) B0 (Se.E) BM (Se.E)

4 w k pi EO (-) EM (-) A0 (Se) AM (Se) B0 (Se.E) BM (Se.E)

- - 102.39 + 13.28 a 0.48 + 0.01a, b 40 146.15 + 13.29a, b 0.38 + 0.06a, b - - 303.66 -+ 07.71b,c 4.06 _+ 0.43d 40 313.52 _+ 40.86c 4.71 _+ 0.41/ - - 506.28 +_ 4.97 + 0.06f 40 628.14 _+ 80.31d 2.86 +_ 0.17c

- - 86.42 _+ 7.99 a 0.71 _+ 0.15 a 40 84.27 _+ 47.16 b 0.35 +_ 0.10 a - - 315.09 _+ 69.35 b 3.88 +_ 0.46e 40 297.3 _+ 62.43b 2.80 _+ 0.17b,c - - 350.91 _+ 68.63b 2.93 _+ 0.11c,d 40 321.31 _+ 31.42b 3.37 _+ 0.46d

- - nd 0.17 + 0.01a 40 nd 0.52 _+ 0.09a - - nd 3.67 _+ 0.51r 40 nd 2.54 +_ 0.52b - - nd 3.64 +_ 0.61c 40 nd 3.12 + 0.2@;

- - 81.73 _+ 5.63 a 0.51 + 0.05 a 40 204.72 _+ 3.48 b 0.62 _+ 0.11 a - - 619.12 + 188.15 c 5.29 _+ 0.67c 40 768.60 _+ 75.56 c 5.47 _+ 0.82 c - - 1127.95 _+ 222.05 d 2.89 _+ 0.14b 40 1370.69 + 19.71e 4.49 _+ 0.63c

*Given orally per kg body mass one time only. **Liver wet mass. ***Selenium plus vit. E. nd: not determined. Values in same column with different superscripts are significantly dif-

ferent (p < 0.05).

l eas t l o n g - t e r m s e l e n i u m s t a t u s of t he b o d y , r a t h e r t h a n a n " i n d i c a t o r " of the r e c e n t s e l e n i u m i n t a k e (9). In s h e e p , c o n c u r r e n t a d m i n i s t r a t i o n of safe d o s e s of m o n e n s i n a n d d o s i n g of t he toxic a m o u n t of s e l e n i u m led to a

s t o r a g e of the e l e m e n t in d i f f e r e n t v i s c e r a l o r g a n s . O t h e r w i s e , w h e n m o n -

e n s i n a n d s e l e n i u m w e r e u s e d s e p a r a t e l y (4,) t h e r e w a s n o s i gn i f i c an t

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136 Khan et al.

Table 4 Erythrocyte GSH-Px Activity and Liver Selenium Levels

During Subacute Selenium Toxicosis in Birds Fed Different Diets (Mean _+ SD)

Erythrocyte Selenium in Group, Selenium* GSH-Px liver tissue, dietary additions mg U / g Hb ~g /g wm**

1 wk pi E0 (-) - - 102.39 _+ 13.28 0.48 _+ 0.01a ES (-) 1 187.22 + 89.42 1.41 + 0.18b CO (Mon)*** - - 137.54 _+ 21.61 0.51 + 0.06a CS (Mon) 1 96.65 + 23.32 1.95 + 0.18c

2 wk pi E0 (-) - - 86.42 + 7.99a 0.61 _+ 0.03 a ES (-) 1 164.32 _+ 37.05b 1.47 + 0.30b CO (Mon) - - 93.14 + 36.10 a 0.74 _+ 0.12 a CS (Mon) 1 213.15 _+ 27.91b 1.23 + 0.37b

3 wk pi E0 (-) - - nd 0.47 + 0.01a ES (-) 1 nd 1.33 _+ 0.06b CO (Mon) - - nd 0.53 _+ 0.04a CS (Mon) 1 nd 1.58 _+ 0.15c

4 wk pi E0 (-) - - 81.72 _+ 5.63 a 0.51 _+ ES (-) 1 211.52 + 12.86 b 1.10 _+ 0.33 b CO (Mon) - - 197.29 _+ 58.75 b 0.64 _+ CS (Mon) 1 1116.15 + 94.30 c 1.20 + 0.27b

*Given orally per kg body mass on alternate days. **Liver wet mass. ***Monensin. nd: not determined. Values in same column with different superscripts are significantly different

(p < 0.05).

increase in se len ium up take and its accumula t ion in viscera. Similar b u t inconsis tent results were recorded in the present study. Fol lowing treat- m e n t wi th monens in and selenium, w h e n bo th subs tances were app l i ed at toxic doses , liver se len ium concentrat ions exhibi ted a w i d e range of var ia t ions over a no rma l level.

There is good ev idence that monens in affects e ry throcyte GSH-Px act ivi ty in more than one metabol ic pa thway. Exclusive adminis t ra t ion of monens in caused nonsignif icant var ia t ions of liver se len ium levels in intoxicated chicks at different intervals dur ing the course of our experi- ments , b u t resul ted in increased RBC GSH-Px activity. These data are in ag reemen t wi th the those repor ted b y Salyi et al. (7). Se- independen t metabol ic p a t h w a y s for RBC GSH-Px synthesis have also been repor ted

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Effects o f M o n e n s i n on S e S t a t u s 137

(16). According to other authors, RBC GSH-Px belongs to the enzymic group having activity of gluthatione transferase and gives no reaction with hydrogen peroxide, but becomes active in the presence of organic hydroperoxide substrates (17,18). Monensin may possibly stimulate the production of these enzymes.

CONCLUSIONS

Erythrocyte GSH-Px activity increased following the oral adminis- tration of toxic levels of both selenium and monensin. Administration of selenium resulted in elevated liver selenium concentration, whereas monensin had no significant effect on liver selenium level.

Concurrent administration of selenium and monensin applied at toxic doses resulted in a significant increase in erythrocyte GSH-Px activ- ity, as well as liver selenium concentration. Neither selenium nor mon- ensin alone resulted in a similar increase.

All these results suggest that monensin may burgeon the absorption of selenium from the intestine, but the mechanism by which monensin regulates the erythrocyte GSH-Px activity remains unknown.

ACKNOWLEDGMENT

The experiment was performed in the framework of research project 4.100.201.

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