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Effects of clonidine and idazoxan on tetrathiomolybdate-induced copper and lysosomal enzyme excretion into sheep bile
Research in Veterinary Science 92 (2012) 456–461
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Effects of clonidine and idazoxan on tetrathiomolybdate-induced copper and lysosomal enzyme excretion into sheep bile S.R. Gooneratne ⇑ Faculty of Agriculture and Life Sciences, P.O. Box 84, Lincoln University, Lincoln 7647, New Zealand
a r t i c l e
i n f o
Article history: Received 18 November 2010 Accepted 15 April 2011
Keywords: a2-Adrenergic agonists Bile Copper Tetrathiomolybdate
a b s t r a c t This study investigated the effects of intravenous (IV) administration of tetrathiomolybdate (TTM), and a2-adrenergic agonist clonidine (CLO) and a2-antagonist idazoxan (IDA), alone or in combination with TTM, on sheep fed low (LCu) and high (HCu) copper diets. Effects on bile flow, biliary Cu concentration and excretion, plasma Cu concentration, and lysosomal enzyme b-glucuronidase (b-GLU) activity in bile and plasma were determined. Tetrathiomolybdate alone or with CLO or IDA significantly enhanced biliary Cu excretion most likely by removing Cu from hepatocyte lysosomes as evidenced by a significant increase in b-GLU enzyme activity in bile. A significant increase in plasma b-GLU concentration occurred only in sheep treated with CLO in combination with TTM. Because of the lytic nature of the lysosomal enzymes, caution is advocated in use of drugs, especially a2-adrenergic agonists, to further enhance TTM-induced biliary Cu excretion in the treatment of chronic Cu poisoning in sheep. Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction Although sheep hepatocytes respond to copper (Cu) loading by sequestering excess Cu into an increased number and size of lysosomes, paralleled by an increase in lysosomal enzyme activity in Cu-loaded livers (Gooneratne et al., 1980), that there is also direct removal of Cu from hepatocyte lysosomes into bile has remained speculation until we reported this in sheep (Gooneratne et al., 1989b), noting also that increase in dietary Cu does not necessarily greatly increase biliary Cu excretion in sheep (Gooneratne and Christensen, 1997). However, excretion of Cu into bile can be markedly increased by administration of tetrathiomolybdate (TTM) (Gooneratne et al., 1985, 1989b,c; Ke and Symonds, 1986, 1989; Symonds and Ke, 1989). On recommendation based on our initial studies (Gooneratne et al., 1981a,b), TTM is now used in the treatment and prevention of chronic Cu poisoning (CCP) in sheep (Humphries et al., 1986). Tetrathiomolybdate is also now used by some physicians to treat Wilson’s disease, a genetically acquired Cu storage disease in humans (Walshe, 1987). Studies have shown that biliary Cu excretion is increased during anaesthesia- and surgery-induced stress (Caple and Heath, 1978), but the mechanism for this is still not clear. Cortisol concentration, which increases in blood during surgery, has been suggested as a likely candidate (Caple and Heath, 1978). Symonds and Ke (1989) reported that liver Cu excretion could be enhanced by xylazine (Rompun; an a2-adrenergic agonist with ⇑ Tel.: +64 3 325 3803; fax: +64 3 325 3851. E-mail address: [email protected] 0034-5288/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.rvsc.2011.04.013
some a1 activity) and this effect was abolished by the a2-antagonist idazoxan (IDA), showing that increased biliary Cu excretion was due to the a2-agonistic action of xylazine. The present study was undertaken to further evaluate this effect by injecting clonidine (CLO; a potent a2-agonist with some a1 activity) and IDA (a2-antagonist) with/without TTM to sheep fed low or high Cu diets. Since xylazine enhanced the effect of TTM-induced biliary Cu excretion (Symonds and Ke, 1989), it was suspected that perhaps a2-agonists may act by modulation of the lysosomal membrane resulting in release of additional liver Cu into bile. To test this hypothesis, lysosomal b-glucuronidase (b-GLU) enzyme activity in both bile and plasma was also monitored. The aim of this study was to examine the influence of a2-adrenergic agonist/ antagonist drugs in the therapeutic use of TTM in CCP in sheep, and the roles of TTM and the a2-adrenergic agonist/antagonist on the integrity of the lysosomal membrane. 2. Materials and methods 2.1. Animals Four 6-month-old female lambs weighing 26.9–29.8 kg were fed a maintenance diet [1.2 kg/day dry matter (DM)] formulated to meet nutrient requirements (National Research Council, 1985) supplemented with Cu to provide low (LCu; 6.3 mg/kg DM) or high Cu (HCu; 41.6 mg/kg DM) in the diet offered at 9 h daily throughout the study. After 2 weeks, the sheep were surgically modified by ligation of the bile duct and cannulation of the gall bladder and duodenum, to allow collection of bile into bile bags harnessed to
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Table 1 Copper concentration (mg/L; mean ± SEM) in bile samples collected for 6 h following a single IV administration of different combinations of tetrathiomolybdate (TTM) and a2adrenergic agonist/antagonists (CLO/IDA) to sheep fed low (LCu) and high copper (HCu) diets. DietB
LCu (n = 2) (6.3 mg/kg/day DM) HCu (n = 2) (41.6 mg/kg/day DM)
TreatmentA 1 BASAL (control)
2 TTM
3 CLO
4 IDA
5 TTM + CLO
6 TTM + IDA
0.08a ± 0.01 0.12a ± 0.02
0.70b ± 0.14 2.19b⁄⁄ ± 0.13
0.12a ± 0.04 0.12a ± 0.02
0.09a ± 0.02 0.31a ± 0.14
0.87b1 ± 0.09 3.54b1⁄⁄ ± 0.07
0.48b ± 0.03 2.27b⁄⁄ ± 0.35
For each treatment, within each column, asterisks indicate significant differences in bile Cu concentration between HCu and LCu diet groups: ⁄P < 0.05, ⁄⁄P < 0.01. A Samples were collected at 0.25, 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 5, 6 h from sheep following administration of TTM (2.5 mg/kg bodyweight (BW)), CLO = clonidine (2 lg/kg BW) or IDA = idazoxan (1.2 mg/kg BW) either alone or in combination. The six treatments were administered consecutively at weekly intervals. B For each diet, within each row, different superscript letters indicate significant differences in bile Cu concentration between treatments compared with the BASAL value (control): aNS, bP < 0.05, b1P < 0.01.
their side as described by Caple and Heath (1972) and for bile to flow into the duodenum directly (via an external valve) when not sampling. 2.2. Chemicals Thiomolybdate, a specific Cu chelating agent (Gooneratne and Christensen, 1997) was prepared as described by Gooneratne et al. (1981a). Tetrathiomolybdate was identified as the predominant component (>90% purity) from absorption spectra (Aymonino et al., 1969). Clonidine was purchased from Boehringer Ingelheim (NZ) Ltd. (Manukau City, NZ) and Idazoxan from Research Biochemicals Inc. (Natick, MA).
ity using box plots. Where data were skewed, a log transformation was used. When ANOVA indicated significant effects, pairs of means were compared using Fisher’s LSD test (a = 0.05 or 0.01). Within each diet, the results from each treatment were compared with the respective initial BASAL value (of either LCu or HCu). To examine the effect of diet, the results from the LCu and HCu diets were compared within each treatment. The relationship between biliary Cu concentration and b-GLU activity was analysed using simple linear regression (GenStat version 2). Because data were not normally distributed, they were log transformed. The correlation coefficients (r) were compared using a Tukey-type multiple comparison test (a = 0.05). 3. Results
2.3. Treatments After surgery, bile flow was allowed to stabilize for 2 weeks before sheep were subjected to six treatments at weekly intervals. Because of the very short biological half-lives of the drugs used in this study, 7 days was regarded as a suitable recovery period to prevent any carryover effect between treatments. In Treatment 1 (BASAL), 12 samples each of blood and bile were collected at 0, 0.25, 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 5 and 6 h to obtain baseline values of biliary Cu concentration, plasma Cu concentration, and b-GLU activity in bile and plasma. After each sampling, an equal volume of pooled bile from untreated sheep (collected previously) was replaced into the duodenum to avoid interruption of enterohepatic circulation. Sheep were then administered IV one dose each of TTM [Treatment 2; 2.5 mg/kg bodyweight (BW)], CLO (Treatment 3; 2 lg/kg BW), IDA (Treatment 4; 1.2 mg/kg BW), CLO plus TTM (Treatment 5), and IDA plus TTM (Treatment 6)] in six consecutive weekly treatments, each followed by a 6-h sampling of blood and bile as described for Treatment 1. 2.4. Analytical techniques Copper in plasma and bile was determined on an atomic absorption spectrophotometer (Perkin Elmer, model 5000) after appropriate dilution with deionized water. b-GLU activity in plasma and bile was measured by the method of Fishman (1963) and expressed as phenolphthalein produced (lg/mL) during a 30-min incubation. 2.5. Statistics Results are expressed as mean ± SEM. The data were analysed using GenStat version 12. The dependent variable was the mean of 12 samples from each sheep for bile flow, plasma Cu concentration, plasma b-GLU activity, biliary Cu concentration and excretion, and biliary b-GLU activity and excretion. Results were analysed using two-way analysis of variance (ANOVA) with diet and treatments as explanatory variables. All data were analysed for normal-
Clinically, all sheep appeared normal throughout the treatments, consuming all food offered and gaining weight at 0.9– 1.2 kg a week except in the week after surgery. The Cu level in the diet did not appear to affect feed intake. 3.1. Bile flow The baseline bile flow (Treatment 1) varied from 428 ± 28 mL/ 6 h in sheep on the LCu diet to 288 ± 40 mL/6 h in sheep on the HCu diet. Bile flow was similar in Treatments 2–4 but declined slightly in Treatments 5 and 6 during the first 3 h after TTM + CLO/IDA administration, returning to normal thereafter. This affected biliary Cu and b-GLU excretion, which otherwise would have followed biliary Cu concentration and b-GLU activity trends. Bile flow between each treatment and between diets was not significantly different. 3.2. Plasma copper Dietary Cu concentration did not affect baseline plasma Cu concentration in sheep on the BASAL diet in Treatment 1, the values being 1.05 ± 0.1 mg/L (mean ± SEM) for sheep on the LCu diet and 1.12 ± 0.08 mg/L for sheep on the HCu diet. Neither CLO nor IDA affected plasma Cu concentration to any great extent, but TTM either alone or in combination with CLO/IDA slightly increased concentration within 30 min, reached a peak at 2–2.5 h, but declined to baseline values by the end of each treatment. In sheep on the HCu diet, the plasma Cu concentration increased to maxima of 1.20, 1.23 and 1.27 mg/L within 2.5 h of administration of TTM + IDA (Treatment 4), TTM alone (Treatment 2) and TTM + CLO (Treatment 3), respectively. 3.3. Biliary copper Tetrathiomolybdate either alone or with CLO/IDA produced a significant increase in biliary Cu concentration (Table 1) and hence an increase in biliary Cu excretion, 8- to 10-fold in sheep on the
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LCu diet (P < 0.05) and 18- to 30-fold in sheep on the HCu diet (P < 0.01) compared with the control (BASAL diet) (Fig. 1), and was highest in sheep on the HCu diet given TTM + CLO. In sheep on the HCu diet, IDA (Treatment 4) but not CLO (Treatment 3) increased biliary Cu concentrations and hence biliary Cu excretion (Fig. 1). Following TTM administration alone or with CLO/IDA, biliary Cu concentration peaked within 2.5 h (Fig. 2), after which it fluctuated and/or declined slowly but was still slightly above baseline levels by the end of the 6-h sampling period. There was a significant interaction between treatment and diet for biliary Cu concentration (F5,274 = 14.72; P < 0.001) and biliary Cu excretion (F5,274 = 3.06; P < 0.011).
significantly elevated only in sheep given TTM with CLO (Table 2). There was a significant interaction between treatment and diet for b-GLU activity in bile (F5,274 = 6.94; P < 0.001). The regression coefficients showed good correlation between biliary Cu concentration and b-GLU activity in bile in sheep for all treatments (Table 3). The regression equations for this relationship for each treatment are also shown in Table 3. The comparison of the r values in a Tukey-type multiple comparison test shows that the correlation between the two variables is strongest and significant in the TTM + CLO and TTM + IDA treatments.
3.4. b-Glucuronidase activity
Biliary b-GLU excretion (mg/6 h; Fig. 1) was also significantly greater in sheep administered TTM alone and with CLO/IDA, and this was highly significant (P < 0.01) in sheep on the HCu diet. Biliary b-GLU excretion was also significantly higher in HCu diet sheep given IDA compared with the respective BASAL value and also the corresponding LCu group (Treatment 4). There was a significant interaction between treatment and diet for b-GLU excretion (F5,274 = 4.26; P < 0.001).
b-Glucuronidase activity in plasma and bile is shown in Table 2 and for one sheep on the HCu diet in Fig. 2. In general, all treatments significantly (P < 0.05) increased b-GLU activity in bile (Table 2) in sheep on the LCu diet compared with the control, but the values were less than 70 lg phenolphthalein/mL/30 min, except in the TTM + CLO group (Treatment 5), which was in excess of 100 lg phenolphthalein/30 min, and this was highly significant (P < 0.01). In sheep on the HCu diet also, b-GLU activity increased significantly (P < 0.05) for each treatment compared with the control, but was most marked in sheep given TTM alone (6-fold) or in combination with either CLO (50-fold) or IDA (17-fold). In spite of the increased biliary b-GLU excretion, b-GLU activity in plasma was
Fig. 1. Total excretion of copper (mg) and b-glucuronidase (phenolphthalein, mg) (mean ± SEM) in bile over a 6-h sampling period in sheep on low (h) and high ( ) copper diets following treatment with tetrathiomolybdate (TTM) and a2-adrenergic agonist clonidine (CLO), and antagonist idazoxan (IDA), either alone or in combination. Different letters indicate significant differences in treatments from the control (BASAL) for each diet: b, P < 0.05; b1, P < 0.01. Asterisks indicate significant differences per treatment between high- and low-copper-diet groups: ⁄ P < 0.05; ⁄⁄P < 0.01.
3.5. Biliary b-glucuronidase excretion
4. Discussion This study reports for the first time an increase in b-GLU activity in bile and its relationship to enhanced biliary Cu excretion in sheep given TTM. The study confirms our previous finding that in control sheep, biliary Cu excretion does not increase significantly with increased dietary Cu concentration (Gooneratne et al., 1989b,c). Although most previous studies (Gooneratne et al., 1985, 1989a,b,c) hypothesised TTM-induced liver lysosomal Cu release into bile, this study is the first to show that TTM influences Cu stored in liver lysosomes by expelling excess Cu and the lysosomal enzyme b-GLU into bile. Although high TTM doses can induce molybdenum (Mo) toxicity and cause deaths in sheep (Gooneratne, 1979), this study confirmed that although TTM administration can increase bile b-GLU activity, a significant amount of the b-GLU enzyme from the liver does not spill into the bloodstream. Hence TTM is relatively non-toxic to sheep at low doses (2.5 mg/kg BW), i.e. plasma b-GLU in TTM-administered sheep (Treatment 2) was only slightly elevated, with most values similar to the control (Treatment 1). In contrast, CLO, which is a potent a2-agonist, did not increase biliary Cu concentration to any great extent when given alone, but significantly increased b-GLU activity in both bile and plasma when given in combination with TTM in all sheep and was most marked in sheep on the HCu diet. It is interesting to note that IDA, an a2-antagonist when given alone, significantly increased biliary Cu excretion and this was accompanied by an increase in biliary b-GLU activity and excretion in sheep on the HCu diet. All these changes are reflected in a strong relationship between biliary Cu concentration and b-GLU activity in bile (Table 3), with TTM + CLO and TTM + IDA significantly different to the BASAL, TTM and CLO values. It is possible that the increase in biliary Cu excretion and b-GLU activity in HCu sheep in Experiments 5 and 6 during weeks 9 and 10 may have occurred due to liver damage caused by continuous exposure to a HCu diet for over 9 weeks. However, it is unlikely because the amounts of biliary Cu and b-GLU excreted in week 10 were less than in week 9 and Kumaratilake et al. (1981) have shown that the increase in the lysosomal enzyme acid phosphatase in serum, even during continuous dosing of sheep, is minimal during the pre-haemolytic phase and is markedly increased only during the haemolytic crises of CCP. Gooneratne (1979) and Gooneratne et al. (1979) also have shown in liver fractionation studies that acid phosphatase and b-GLU are only slightly
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TTM+IDA
IDA
8 6 4 2 0
1600 1200 800 400 0
4 3 2 1 0
μ
400 300 200 100 0
TTM+CLO
CLO 400 300 200 100 0
4 3 2 1 0
8 6 4 2 0
1600 1200 800 400 0
β
Basal
TTM
400 300 200 100 0
4 3 2 1 0 0
2
4
8
1600 1200 800 400 0
6
6 4 2 0 0
2
4
6
Fig. 2. Profile comparison between biliary copper (Cu) concentration (mg/L) (d) and b-glucuronidase activity (lg phenolphthalein/mL/30 min) in bile (1;4) and plasma (10;h) over a 6-h sampling period (0, 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 5 and 6 h) in one sheep (sheep #95) on a high Cu diet. Treatments are: (1) control (BASAL), (2) tetrathiomolybdate (TTM), (3) clonidine (CLO), (4) idazoxan (IDA), (5) TTM and CLO, and (6) TTM and IDA. See text for details.
Table 2 b-Glucuronidase (b-GLU) activity (lg phenolphthalein produced/mL/30 min) in bile and plasma (mean ± SEM) in samples collected for 6 h following a single IV administration of different combinations of tetrathiomolybdate (TTM) and a2-adrenergic agonist/antagonists (CLO/IDA) from sheep fed low (LCu) and high copper (HCu) diets. b-GLU activity
Bile (n = 2 / diet) Plasma (n = 2 / diet)
Diet
LCu HCu LCu HCu
TreatmentA 1 BASAL (control)
2 TTM
3 CLO
4 IDA
5 TTM + CLO
6 TTM + IDA
9.0 ± 0.3a 17.8 ± 2.8a 5.5 ± 0.5a 11.5 ± 1.8a
68.9 ± 31.4b1 120.8 ± 51.3b 7.1 ± 0.5a 11.1 ± 0.9a
38.4b ± 21.5 39.0b ± 12.8 11.7 ± 0.2a 10.9 ± 2.1a
19.0 ± 0.9b 75.8 ±.1b 5.4 ± 0.4a 5.7 ± 0.7a
128.0 ± 21.4b1 796.8 ± 139.2b1⁄⁄ 17.1 ± 1.7b 26.7 ± 5.6b⁄
24.9 ± 5.9b 300.8 ± 17.9b1⁄⁄ 9.5 ± 2.1a 6.6 ± 1.5a
For each diet, within each row, different superscript letters indicate significant differences between treatments compared with the BASAL value (control): aNS, bP < 0.05; b1 P < 0.01. For each treatment, within each column, superscript asterisks indicate significant differences between diet groups for each parameter (b-GLU activity in bile or plasma): ⁄ P < 0.05; ⁄⁄P < 0.01. A Samples were collected at 0.25, 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 5, 6 h from sheep following administration of TTM (2.5 mg/kg bodyweight (BW)), CLO = clonidine (2 lg/kg BW) or IDA = idazoxan (1.2 mg/kg BW) either alone or in combination. The six treatments were administered consecutively at weekly intervals.
Table 3 Relationship between b-glucuronidase activity in bile (x; lg phenolphthalein produced/mL/30 min) and biliary Cu concentration (y; mg/L) in samples collected from sheep (n = 4) for 6 h following a single IV administration of different combinations of tetrathiomolybdate (TTM) and a2-adrenergic agonist/antagonists (CLO/IDA)A.
A B
Treatment No.
TreatmentA
Linear regression equation
Correlation coefficient (r)B
P
1 2 3 4 5 6
BASAL TTM CLO IDA TTM + CLO TTM + IDA
Log y = 0.015 + 0.047 Log y = 0.178 + 0.291 Log y = 0.029 + 0.054 Log y = 0.122 + 0.129 Log y = 0.426 + 0.373 Log y = 0.324 + 0.353
0.68a 0.61a 0.70a,b 0.90b,c 0.92c,d 0.94c,d
<0.001 <0.001 <0.001 <0.001 <0.001 <0.001
(log x) (log x) (log x) (log x) (log x) (log x)
The treatments are: control (BASAL) and tetrathiomolybdate (TTM), clonidine (CLO), and idazoxan (IDA), either alone or in combination. Details as for Tables 1 and 2. Correlation coefficients with different superscript (a, b and c) are significantly different (Tukey-type multiple comparison test; P < 0.05).
increased during the pre-haemolytic phase. The sheep used in the present study did not show any symptoms of a haemolytic crisis. In addition, TTM, a specific Cu chelating agent, administered in Exper-
iments 2 (week 6), 5 (week 9) and 6 (week 10), would have removed any excess liver Cu that may have accumulated from continuous exposure to the HCu diet.
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Symonds and Ke (1989) reported that the ability of xylazine to increase biliary Cu excretion and also augment the effect of TTM on biliary Cu excretion is due to the a2-adrenergic agonist effect of the drug. However, the present study has shown that CLO, also an a2adrenergic agonist, when given alone (Treatment 3) did not significantly increase biliary Cu excretion. Possible explanations for this discrepancy are proposed. First, the increase in biliary Cu excretion associated with xylazine administration may not be related to a2agonist activity as proposed by Symonds and Ke (1989). Second, the ratio of a2 to a1-agonist activity in CLO is probably different to that of xylazine. Third, the dose of CLO used in this study (2 lg/kg BW) may have been inadequate to produce a significant a2-agonist activity. Fourth, a higher a1-agonist activity of CLO (than in xylazine) may have had a negative impact on biliary Cu excretion. But it should be noted that even at this low dosage, CLO and also IDA when given with TTM significantly increased bGLU enzyme activity in bile with high r values indicative of increased permeability of the lysosomal membrane in the liver and/or of lysosomal damage, but it is only CLO when given with TTM that resulted in b-GLU enzyme leakage into the plasma. It has previously been shown that rupture of liver lysosomes during Cu loading can cause liver necrosis (Gooneratne, 1979; Gooneratne et al., 1980). Moderate leakage of the lysosomal enzyme into the liver can induce apoptosis (Bursch, 2001), while pronounced leakage causes necrosis (Wang et al., 2006). Leakage of lysosomal enzyme from the liver can spill over into the bloodstream and may be one of the causes of red cell lysis in CCP in sheep (Kumaratilake et al., 1981). It is therefore hypothesised that plasma b-GLU may also be increased in CCP in sheep with liver hepatocyte lysosomal damage, and therefore, both b-GLU and acid phosphatase (Kumaratilake et al., 1981), in combination with increased plasma and red blood cell Cu (Gooneratne et al., 1981a,b), may be responsible for haemolytic crisis, a feature of CCP in sheep. A limitation of this study is the small sample size in each treatment. However, the trends in the profiles of parameters monitored, namely, biliary Cu concentration and excretion, plasma Cu concentration, and b-GLU enzyme activity in bile and plasma, were consistent in all the animals in the LCu or HCu diet group for each treatment. This study involved surgery, intensive sampling, and returning bile to the duodenum to avoid interruption to the enterohepatic circulation. This required significant resources including a large number of personnel. Therefore, the number of animals had to be limited. Although TTM has been used to treat CCP in sheep successfully (Gooneratne et al., 1981b; Humphries et al., 1986), the mechanisms by which Cu homeostasis is altered in ruminants apart from the increased biliary and urinary Cu excretion (Gooneratne et al., 1989a,b,c) continues to be debated (Suttle 2002, 2003a,b; Telfer et al., 2003, 2004). There is concern about the potential toxic effects of excess Mo in TTM because TTM in high doses can retard the growth of the epiphyseal plate in the long bones (Read et al., 1986), cause atrophy of the pituitary gland (Haywood et al., 2004) and reduce steroidogenesis (Kendall et al., 2003). Symonds and Ke (1989) suggested the use of an a2-adrenergic agonist in combination with low TTM doses to augment liver Cu removal via bile, but the present study has shown that although the a2agonist CLO alone has little effect on biliary Cu excretion, potential complications may arise if it is used in combination with TTM because of the significant increase in plasma b-GLU activity in Culoaded sheep, which by itself may compromise red blood cell integrity. IDA when given with TTM also increased bile but not plasma b-GLU activity. Therefore caution is advocated in the use of other drugs, especially a2-agonists including CLO, in combination with TTM until there is more evidence that the increase in plasma b-GLU has minimal effect on red blood cell integrity, to support such combination therapy. It would be interesting to
investigate the properties of xylazine other than the a2-agonist activity that may have contributed to increased biliary Cu excretion as reported by Symonds and Ke (1989). 5. Conclusions Clonidine alone, given IV, did not significantly increase biliary Cu excretion. TTM consistently increased biliary Cu concentration with or without CLO or IDA and this was most significant in sheep on the HCu diet given TTM + CLO, although biliary Cu excretion was higher in TTM > TTM + CLO > TTM + IDA because of the lower bile flow in sheep given CLO and IDA with TTM. This was also associated with an increase in biliary excretion of the lysosomal enzyme b-GLU, especially in sheep on the HCu diet, and was consistently significant only in the HCu diet group given TTM + CLO > TTM + IDA > TTM. However, significant spillage of biliary b-GLU from the liver into plasma occurred only in the TTM + CLO group. In view of this, the role of other drugs, especially a2-agonists, to augment TTM-induced biliary Cu excretion requires further clarification. Conflict of interest The author of this paper has no financial or personal relationship with other people or organizations that could inappropriately influence or bias the content of this paper. Acknowledgements This study was funded by a grant from the Lincoln University Research Committee. The author thanks the staff at Johnstone Memorial Laboratory for care of animals, Alex Familton for bile duct cannulation surgery, Peter Isherwood for Cu analysis and measurement of b-GLU activity, and Martin Wellby for preparation of graphics, Christine Bezar and Neville Grace for editorial comments, and James Ross for statistical analysis. References Aymonino, P.J., Ranade, A.C., Miller, A., 1969. Evidence for the existence of MoO3S 2 and WoO3S 2 ions in aqueous solution. Zeitschrift für Anorganische und Allgemeine Chemie 371, 295–298. Bursch, W., 2001. The autophagosomal–lysosomal compartment in programmed cell death. Cell Death and Differentiation 8, 69–81. Caple, I.E., Heath, T.J., 1972. Regulation of output of electrolytes in bile and pancreatic juice in sheep. Australian Journal of Biological Sciences 25, 155–165. Caple, I.E., Heath, T.J., 1978. Regulation of excretion of copper in bile of sheep: effect of anaesthesia and surgery. Comparative Biochemistry and Physiology 61A, 503–507. National Research Council, 1985. Nutrient Requirements of Sheep, sixth ed. National Academy Press, Washington DC, USA. Fishman, W.H., 1963. D-Glucuronidase. In: Bergmeyer, H.U. (Ed.), Methods of Enzymatic Analysis. Winheim/Bergstr Verlag Chemie, New York, pp. 869–874. Gooneratne, S.R., 1979. Morphometric, electron microscopic, biochemical and pathological investigation of liver of normal and copper poisoned sheep. PhD thesis, Murdoch University, Western Australia. Gooneratne, S.R., Christensen, D.A., 1997. Effect of chelating agents on the excretion of copper, zinc and iron on the bile and urine of sheep. The Veterinary Journal 153, 171–178. Gooneratne, S.R., Howell, J.McC., Gawthorne, J.M., 1979. Intracellular distribution of copper in the liver of normal and copper loaded sheep. Research in Veterinary Science 27, 30–37. Gooneratne, S.R., Howell, J.McC., Cook, R.D., 1980. An ultrastructural and morphometric study of the liver of normal and copper-poisoned sheep. American Journal of Pathology 99, 429–450. Gooneratne, S.R., Howell, J.McC., Gawthorne, J.M., 1981a. Intravenous administration of thiomolybdate for the prevention and treatment of chronic copper poisoning in sheep. British Journal of Nutrition 46, 457–467. Gooneratne, S.R., Howell, J.McC., Gawthorne, J.M., 1981b. An investigation of the effects of intravenous administration of thiomolybdate on copper metabolism in chronic Cu-poisoned sheep. British Journal of Nutrition 46, 469–480. Gooneratne, S.R., Christensen, D.A., Chaplin, R., Trent, A.M., 1985. Copper: biliary excretion in copper-supplemented and thiomolybdate-treated sheep. In: Mills, D.F., Bremner, I., Chesters, C.K. (Eds.), Proceedings of the 5th International
S.R. Gooneratne / Research in Veterinary Science 92 (2012) 456–461 Symposium on Trace Elements in Man and Animals. Commonwealth Agriculture Bureaux, Slough, pp. 342–346. Gooneratne, S.R., Chaplin, R., Trent, A.M., Christensen, D.A., 1989a. Effect of thiomolybdate administration on the excretion of copper, zinc, iron and molybdenum in sheep bile. British Veterinary Journal 145, 62–72. Gooneratne, S.R., Laarveld, B., Chaplin, R.K., Christensen, D.A., 1989b. Profiles of 67Cu in blood, bile, urine and faeces from 67Cu primed lambs: Effect of 99Mo-labelled tetrathiomolybdate on the metabolism of recently stored tissue 67Cu. British Journal of Nutrition 61, 355–371. Gooneratne, S.R., Laarveld, B., Chaplin, R.K., Christensen, D.A., 1989c. Profiles of 67Cu in blood, bile, urine and faeces from 67Cu primed lambs: Effect of 99Mo-labelled tetrathiomolybdate on the metabolism of 67Cu after long-term storage. British Journal of Nutrition 61, 373–385. Haywood, D., Dincer, Z., Jasani, B., Loughran, M.J., 2004. Molybdenum-associated pituitary endocrinopathy in sheep treated with ammonium tetrathiomolybdate. Journal of Comparative Pathology 130, 21–31. Humphries, W.R., Mills, C.F., Greig, A., Roberts, L., Inglis, D., Halliday, G.L., 1986. Use of ammonium tetrathiomolybdate in the treatment of copper poisoning in sheep. Veterinary Record 119, 596–599. Ke, Y., Symonds, H.W., 1986. The effect of molybdate compounds on the biliary excretion of copper by sheep. In: Proceedings of the Nutrition Society vol. 46. p. 69A. Ke, Y., Symonds, H.W., 1989. Enhancement of tetrathiomolybdate-induced biliary copper excretion in sheep by general anaesthesia and the effect on copper excretion in urine and bile. Research in Veterinary Science 46, 344–348. Kendall, N.R., Marsters, P., Scaramuzzi, R.J., Campbell, B.K., 2003. Expression of lysyl and effect of copper chloride and ammonium tetrathiomolybdate on bovine ovarian follicle granulose cells cultured in serum-free media. Reproduction 125, 657–665.
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Kumaratilake, J.S., Howell, J.McC., Gooneratne, S.R., 1981. Blood copper, sorbitol dehydrogenase and acid phosphatase in copper poisoning. In: Howell, J.M.C.C., Glawthorne, J.M., White, C.L. (Eds.), Proceedings of the 4th International Symposium on Trace Element Metabolism in Man and Animals. Australian Academy of Sciences, Canberra, pp. 457–460. Read, R., Sutherland, J., Gosh, P., 1986. The matrix components of the epiphyseal growth plate and articular cartilage for dogs treated with ammonium tetrathiomolybdate, a copper antagonist. Australian Journal of Experimental Biology and Medical Sciences 64, 545–562. Suttle, N.F., 2002. Copper deficiency – How has the disease and its diagnosis changed in the last 15 years? Cattle Practice 10, 275–278. Suttle, N.F., 2003a. Copper deficiency – How has the disease and its diagnosis changed in the last 15 years? Cattle Practice 11, 193–194. Suttle, N.F., 2003b. Copper deficiency in grazing livestock. The Moredun Foundation Newsheet 3 (19), 1–12. Symonds, H.W., Ke, Y., 1989. Enhancement of tetrathiomolybdate-induced copper excretion in bile of sheep by the alpha 2-agonistic action of xylazine. Research in Veterinary Science 46, 349–353. Telfer, S.B., Kendall, N.R., Illingworth, D.V., Mackenzie, A.M., 2003. Copper deficiency or molybdenum toxicity? Diagnosis and treatment requires a new perspective. Cattle Practice 11, 190–192. Telfer, S.B., Kendall, N.R., Illingworth, D.V., Mackenzie, A.M., 2004. Molybdenum toxicity in cattle: an underestimated problem. Cattle Practice 12, 259–263. Walshe, J.M., 1987. Tetrathiomolybdate (MOS4) as an ‘anti copper’ agent in man. In: Scheinberg, I.H., Walshe, J.M. (Eds.), Orphan Disease/Orphan Drugs. Manchester University Press, UK, pp. 76–85. Wang, J.W., Sun, L., Hu, J.S., Li, Y.B., Zhang, G.J., 2006. Effects of phospholipase A2 on lysosomal ion permeability and osmotic sensitivity. Chemistry and Physics of Lipids 144, 117–126.
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Research in Veterinary Science journal homepage: www.elsevier.com/locate/rvsc
Effects of clonidine and idazoxan on tetrathiomolybdate-induced copper and lysosomal enzyme excretion into sheep bile S.R. Gooneratne ⇑ Faculty of Agriculture and Life Sciences, P.O. Box 84, Lincoln University, Lincoln 7647, New Zealand
a r t i c l e
i n f o
Article history: Received 18 November 2010 Accepted 15 April 2011
Keywords: a2-Adrenergic agonists Bile Copper Tetrathiomolybdate
a b s t r a c t This study investigated the effects of intravenous (IV) administration of tetrathiomolybdate (TTM), and a2-adrenergic agonist clonidine (CLO) and a2-antagonist idazoxan (IDA), alone or in combination with TTM, on sheep fed low (LCu) and high (HCu) copper diets. Effects on bile flow, biliary Cu concentration and excretion, plasma Cu concentration, and lysosomal enzyme b-glucuronidase (b-GLU) activity in bile and plasma were determined. Tetrathiomolybdate alone or with CLO or IDA significantly enhanced biliary Cu excretion most likely by removing Cu from hepatocyte lysosomes as evidenced by a significant increase in b-GLU enzyme activity in bile. A significant increase in plasma b-GLU concentration occurred only in sheep treated with CLO in combination with TTM. Because of the lytic nature of the lysosomal enzymes, caution is advocated in use of drugs, especially a2-adrenergic agonists, to further enhance TTM-induced biliary Cu excretion in the treatment of chronic Cu poisoning in sheep. Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction Although sheep hepatocytes respond to copper (Cu) loading by sequestering excess Cu into an increased number and size of lysosomes, paralleled by an increase in lysosomal enzyme activity in Cu-loaded livers (Gooneratne et al., 1980), that there is also direct removal of Cu from hepatocyte lysosomes into bile has remained speculation until we reported this in sheep (Gooneratne et al., 1989b), noting also that increase in dietary Cu does not necessarily greatly increase biliary Cu excretion in sheep (Gooneratne and Christensen, 1997). However, excretion of Cu into bile can be markedly increased by administration of tetrathiomolybdate (TTM) (Gooneratne et al., 1985, 1989b,c; Ke and Symonds, 1986, 1989; Symonds and Ke, 1989). On recommendation based on our initial studies (Gooneratne et al., 1981a,b), TTM is now used in the treatment and prevention of chronic Cu poisoning (CCP) in sheep (Humphries et al., 1986). Tetrathiomolybdate is also now used by some physicians to treat Wilson’s disease, a genetically acquired Cu storage disease in humans (Walshe, 1987). Studies have shown that biliary Cu excretion is increased during anaesthesia- and surgery-induced stress (Caple and Heath, 1978), but the mechanism for this is still not clear. Cortisol concentration, which increases in blood during surgery, has been suggested as a likely candidate (Caple and Heath, 1978). Symonds and Ke (1989) reported that liver Cu excretion could be enhanced by xylazine (Rompun; an a2-adrenergic agonist with ⇑ Tel.: +64 3 325 3803; fax: +64 3 325 3851. E-mail address: [email protected] 0034-5288/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.rvsc.2011.04.013
some a1 activity) and this effect was abolished by the a2-antagonist idazoxan (IDA), showing that increased biliary Cu excretion was due to the a2-agonistic action of xylazine. The present study was undertaken to further evaluate this effect by injecting clonidine (CLO; a potent a2-agonist with some a1 activity) and IDA (a2-antagonist) with/without TTM to sheep fed low or high Cu diets. Since xylazine enhanced the effect of TTM-induced biliary Cu excretion (Symonds and Ke, 1989), it was suspected that perhaps a2-agonists may act by modulation of the lysosomal membrane resulting in release of additional liver Cu into bile. To test this hypothesis, lysosomal b-glucuronidase (b-GLU) enzyme activity in both bile and plasma was also monitored. The aim of this study was to examine the influence of a2-adrenergic agonist/ antagonist drugs in the therapeutic use of TTM in CCP in sheep, and the roles of TTM and the a2-adrenergic agonist/antagonist on the integrity of the lysosomal membrane. 2. Materials and methods 2.1. Animals Four 6-month-old female lambs weighing 26.9–29.8 kg were fed a maintenance diet [1.2 kg/day dry matter (DM)] formulated to meet nutrient requirements (National Research Council, 1985) supplemented with Cu to provide low (LCu; 6.3 mg/kg DM) or high Cu (HCu; 41.6 mg/kg DM) in the diet offered at 9 h daily throughout the study. After 2 weeks, the sheep were surgically modified by ligation of the bile duct and cannulation of the gall bladder and duodenum, to allow collection of bile into bile bags harnessed to
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Table 1 Copper concentration (mg/L; mean ± SEM) in bile samples collected for 6 h following a single IV administration of different combinations of tetrathiomolybdate (TTM) and a2adrenergic agonist/antagonists (CLO/IDA) to sheep fed low (LCu) and high copper (HCu) diets. DietB
LCu (n = 2) (6.3 mg/kg/day DM) HCu (n = 2) (41.6 mg/kg/day DM)
TreatmentA 1 BASAL (control)
2 TTM
3 CLO
4 IDA
5 TTM + CLO
6 TTM + IDA
0.08a ± 0.01 0.12a ± 0.02
0.70b ± 0.14 2.19b⁄⁄ ± 0.13
0.12a ± 0.04 0.12a ± 0.02
0.09a ± 0.02 0.31a ± 0.14
0.87b1 ± 0.09 3.54b1⁄⁄ ± 0.07
0.48b ± 0.03 2.27b⁄⁄ ± 0.35
For each treatment, within each column, asterisks indicate significant differences in bile Cu concentration between HCu and LCu diet groups: ⁄P < 0.05, ⁄⁄P < 0.01. A Samples were collected at 0.25, 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 5, 6 h from sheep following administration of TTM (2.5 mg/kg bodyweight (BW)), CLO = clonidine (2 lg/kg BW) or IDA = idazoxan (1.2 mg/kg BW) either alone or in combination. The six treatments were administered consecutively at weekly intervals. B For each diet, within each row, different superscript letters indicate significant differences in bile Cu concentration between treatments compared with the BASAL value (control): aNS, bP < 0.05, b1P < 0.01.
their side as described by Caple and Heath (1972) and for bile to flow into the duodenum directly (via an external valve) when not sampling. 2.2. Chemicals Thiomolybdate, a specific Cu chelating agent (Gooneratne and Christensen, 1997) was prepared as described by Gooneratne et al. (1981a). Tetrathiomolybdate was identified as the predominant component (>90% purity) from absorption spectra (Aymonino et al., 1969). Clonidine was purchased from Boehringer Ingelheim (NZ) Ltd. (Manukau City, NZ) and Idazoxan from Research Biochemicals Inc. (Natick, MA).
ity using box plots. Where data were skewed, a log transformation was used. When ANOVA indicated significant effects, pairs of means were compared using Fisher’s LSD test (a = 0.05 or 0.01). Within each diet, the results from each treatment were compared with the respective initial BASAL value (of either LCu or HCu). To examine the effect of diet, the results from the LCu and HCu diets were compared within each treatment. The relationship between biliary Cu concentration and b-GLU activity was analysed using simple linear regression (GenStat version 2). Because data were not normally distributed, they were log transformed. The correlation coefficients (r) were compared using a Tukey-type multiple comparison test (a = 0.05). 3. Results
2.3. Treatments After surgery, bile flow was allowed to stabilize for 2 weeks before sheep were subjected to six treatments at weekly intervals. Because of the very short biological half-lives of the drugs used in this study, 7 days was regarded as a suitable recovery period to prevent any carryover effect between treatments. In Treatment 1 (BASAL), 12 samples each of blood and bile were collected at 0, 0.25, 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 5 and 6 h to obtain baseline values of biliary Cu concentration, plasma Cu concentration, and b-GLU activity in bile and plasma. After each sampling, an equal volume of pooled bile from untreated sheep (collected previously) was replaced into the duodenum to avoid interruption of enterohepatic circulation. Sheep were then administered IV one dose each of TTM [Treatment 2; 2.5 mg/kg bodyweight (BW)], CLO (Treatment 3; 2 lg/kg BW), IDA (Treatment 4; 1.2 mg/kg BW), CLO plus TTM (Treatment 5), and IDA plus TTM (Treatment 6)] in six consecutive weekly treatments, each followed by a 6-h sampling of blood and bile as described for Treatment 1. 2.4. Analytical techniques Copper in plasma and bile was determined on an atomic absorption spectrophotometer (Perkin Elmer, model 5000) after appropriate dilution with deionized water. b-GLU activity in plasma and bile was measured by the method of Fishman (1963) and expressed as phenolphthalein produced (lg/mL) during a 30-min incubation. 2.5. Statistics Results are expressed as mean ± SEM. The data were analysed using GenStat version 12. The dependent variable was the mean of 12 samples from each sheep for bile flow, plasma Cu concentration, plasma b-GLU activity, biliary Cu concentration and excretion, and biliary b-GLU activity and excretion. Results were analysed using two-way analysis of variance (ANOVA) with diet and treatments as explanatory variables. All data were analysed for normal-
Clinically, all sheep appeared normal throughout the treatments, consuming all food offered and gaining weight at 0.9– 1.2 kg a week except in the week after surgery. The Cu level in the diet did not appear to affect feed intake. 3.1. Bile flow The baseline bile flow (Treatment 1) varied from 428 ± 28 mL/ 6 h in sheep on the LCu diet to 288 ± 40 mL/6 h in sheep on the HCu diet. Bile flow was similar in Treatments 2–4 but declined slightly in Treatments 5 and 6 during the first 3 h after TTM + CLO/IDA administration, returning to normal thereafter. This affected biliary Cu and b-GLU excretion, which otherwise would have followed biliary Cu concentration and b-GLU activity trends. Bile flow between each treatment and between diets was not significantly different. 3.2. Plasma copper Dietary Cu concentration did not affect baseline plasma Cu concentration in sheep on the BASAL diet in Treatment 1, the values being 1.05 ± 0.1 mg/L (mean ± SEM) for sheep on the LCu diet and 1.12 ± 0.08 mg/L for sheep on the HCu diet. Neither CLO nor IDA affected plasma Cu concentration to any great extent, but TTM either alone or in combination with CLO/IDA slightly increased concentration within 30 min, reached a peak at 2–2.5 h, but declined to baseline values by the end of each treatment. In sheep on the HCu diet, the plasma Cu concentration increased to maxima of 1.20, 1.23 and 1.27 mg/L within 2.5 h of administration of TTM + IDA (Treatment 4), TTM alone (Treatment 2) and TTM + CLO (Treatment 3), respectively. 3.3. Biliary copper Tetrathiomolybdate either alone or with CLO/IDA produced a significant increase in biliary Cu concentration (Table 1) and hence an increase in biliary Cu excretion, 8- to 10-fold in sheep on the
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LCu diet (P < 0.05) and 18- to 30-fold in sheep on the HCu diet (P < 0.01) compared with the control (BASAL diet) (Fig. 1), and was highest in sheep on the HCu diet given TTM + CLO. In sheep on the HCu diet, IDA (Treatment 4) but not CLO (Treatment 3) increased biliary Cu concentrations and hence biliary Cu excretion (Fig. 1). Following TTM administration alone or with CLO/IDA, biliary Cu concentration peaked within 2.5 h (Fig. 2), after which it fluctuated and/or declined slowly but was still slightly above baseline levels by the end of the 6-h sampling period. There was a significant interaction between treatment and diet for biliary Cu concentration (F5,274 = 14.72; P < 0.001) and biliary Cu excretion (F5,274 = 3.06; P < 0.011).
significantly elevated only in sheep given TTM with CLO (Table 2). There was a significant interaction between treatment and diet for b-GLU activity in bile (F5,274 = 6.94; P < 0.001). The regression coefficients showed good correlation between biliary Cu concentration and b-GLU activity in bile in sheep for all treatments (Table 3). The regression equations for this relationship for each treatment are also shown in Table 3. The comparison of the r values in a Tukey-type multiple comparison test shows that the correlation between the two variables is strongest and significant in the TTM + CLO and TTM + IDA treatments.
3.4. b-Glucuronidase activity
Biliary b-GLU excretion (mg/6 h; Fig. 1) was also significantly greater in sheep administered TTM alone and with CLO/IDA, and this was highly significant (P < 0.01) in sheep on the HCu diet. Biliary b-GLU excretion was also significantly higher in HCu diet sheep given IDA compared with the respective BASAL value and also the corresponding LCu group (Treatment 4). There was a significant interaction between treatment and diet for b-GLU excretion (F5,274 = 4.26; P < 0.001).
b-Glucuronidase activity in plasma and bile is shown in Table 2 and for one sheep on the HCu diet in Fig. 2. In general, all treatments significantly (P < 0.05) increased b-GLU activity in bile (Table 2) in sheep on the LCu diet compared with the control, but the values were less than 70 lg phenolphthalein/mL/30 min, except in the TTM + CLO group (Treatment 5), which was in excess of 100 lg phenolphthalein/30 min, and this was highly significant (P < 0.01). In sheep on the HCu diet also, b-GLU activity increased significantly (P < 0.05) for each treatment compared with the control, but was most marked in sheep given TTM alone (6-fold) or in combination with either CLO (50-fold) or IDA (17-fold). In spite of the increased biliary b-GLU excretion, b-GLU activity in plasma was
Fig. 1. Total excretion of copper (mg) and b-glucuronidase (phenolphthalein, mg) (mean ± SEM) in bile over a 6-h sampling period in sheep on low (h) and high ( ) copper diets following treatment with tetrathiomolybdate (TTM) and a2-adrenergic agonist clonidine (CLO), and antagonist idazoxan (IDA), either alone or in combination. Different letters indicate significant differences in treatments from the control (BASAL) for each diet: b, P < 0.05; b1, P < 0.01. Asterisks indicate significant differences per treatment between high- and low-copper-diet groups: ⁄ P < 0.05; ⁄⁄P < 0.01.
3.5. Biliary b-glucuronidase excretion
4. Discussion This study reports for the first time an increase in b-GLU activity in bile and its relationship to enhanced biliary Cu excretion in sheep given TTM. The study confirms our previous finding that in control sheep, biliary Cu excretion does not increase significantly with increased dietary Cu concentration (Gooneratne et al., 1989b,c). Although most previous studies (Gooneratne et al., 1985, 1989a,b,c) hypothesised TTM-induced liver lysosomal Cu release into bile, this study is the first to show that TTM influences Cu stored in liver lysosomes by expelling excess Cu and the lysosomal enzyme b-GLU into bile. Although high TTM doses can induce molybdenum (Mo) toxicity and cause deaths in sheep (Gooneratne, 1979), this study confirmed that although TTM administration can increase bile b-GLU activity, a significant amount of the b-GLU enzyme from the liver does not spill into the bloodstream. Hence TTM is relatively non-toxic to sheep at low doses (2.5 mg/kg BW), i.e. plasma b-GLU in TTM-administered sheep (Treatment 2) was only slightly elevated, with most values similar to the control (Treatment 1). In contrast, CLO, which is a potent a2-agonist, did not increase biliary Cu concentration to any great extent when given alone, but significantly increased b-GLU activity in both bile and plasma when given in combination with TTM in all sheep and was most marked in sheep on the HCu diet. It is interesting to note that IDA, an a2-antagonist when given alone, significantly increased biliary Cu excretion and this was accompanied by an increase in biliary b-GLU activity and excretion in sheep on the HCu diet. All these changes are reflected in a strong relationship between biliary Cu concentration and b-GLU activity in bile (Table 3), with TTM + CLO and TTM + IDA significantly different to the BASAL, TTM and CLO values. It is possible that the increase in biliary Cu excretion and b-GLU activity in HCu sheep in Experiments 5 and 6 during weeks 9 and 10 may have occurred due to liver damage caused by continuous exposure to a HCu diet for over 9 weeks. However, it is unlikely because the amounts of biliary Cu and b-GLU excreted in week 10 were less than in week 9 and Kumaratilake et al. (1981) have shown that the increase in the lysosomal enzyme acid phosphatase in serum, even during continuous dosing of sheep, is minimal during the pre-haemolytic phase and is markedly increased only during the haemolytic crises of CCP. Gooneratne (1979) and Gooneratne et al. (1979) also have shown in liver fractionation studies that acid phosphatase and b-GLU are only slightly
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TTM+IDA
IDA
8 6 4 2 0
1600 1200 800 400 0
4 3 2 1 0
μ
400 300 200 100 0
TTM+CLO
CLO 400 300 200 100 0
4 3 2 1 0
8 6 4 2 0
1600 1200 800 400 0
β
Basal
TTM
400 300 200 100 0
4 3 2 1 0 0
2
4
8
1600 1200 800 400 0
6
6 4 2 0 0
2
4
6
Fig. 2. Profile comparison between biliary copper (Cu) concentration (mg/L) (d) and b-glucuronidase activity (lg phenolphthalein/mL/30 min) in bile (1;4) and plasma (10;h) over a 6-h sampling period (0, 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 5 and 6 h) in one sheep (sheep #95) on a high Cu diet. Treatments are: (1) control (BASAL), (2) tetrathiomolybdate (TTM), (3) clonidine (CLO), (4) idazoxan (IDA), (5) TTM and CLO, and (6) TTM and IDA. See text for details.
Table 2 b-Glucuronidase (b-GLU) activity (lg phenolphthalein produced/mL/30 min) in bile and plasma (mean ± SEM) in samples collected for 6 h following a single IV administration of different combinations of tetrathiomolybdate (TTM) and a2-adrenergic agonist/antagonists (CLO/IDA) from sheep fed low (LCu) and high copper (HCu) diets. b-GLU activity
Bile (n = 2 / diet) Plasma (n = 2 / diet)
Diet
LCu HCu LCu HCu
TreatmentA 1 BASAL (control)
2 TTM
3 CLO
4 IDA
5 TTM + CLO
6 TTM + IDA
9.0 ± 0.3a 17.8 ± 2.8a 5.5 ± 0.5a 11.5 ± 1.8a
68.9 ± 31.4b1 120.8 ± 51.3b 7.1 ± 0.5a 11.1 ± 0.9a
38.4b ± 21.5 39.0b ± 12.8 11.7 ± 0.2a 10.9 ± 2.1a
19.0 ± 0.9b 75.8 ±.1b 5.4 ± 0.4a 5.7 ± 0.7a
128.0 ± 21.4b1 796.8 ± 139.2b1⁄⁄ 17.1 ± 1.7b 26.7 ± 5.6b⁄
24.9 ± 5.9b 300.8 ± 17.9b1⁄⁄ 9.5 ± 2.1a 6.6 ± 1.5a
For each diet, within each row, different superscript letters indicate significant differences between treatments compared with the BASAL value (control): aNS, bP < 0.05; b1 P < 0.01. For each treatment, within each column, superscript asterisks indicate significant differences between diet groups for each parameter (b-GLU activity in bile or plasma): ⁄ P < 0.05; ⁄⁄P < 0.01. A Samples were collected at 0.25, 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 5, 6 h from sheep following administration of TTM (2.5 mg/kg bodyweight (BW)), CLO = clonidine (2 lg/kg BW) or IDA = idazoxan (1.2 mg/kg BW) either alone or in combination. The six treatments were administered consecutively at weekly intervals.
Table 3 Relationship between b-glucuronidase activity in bile (x; lg phenolphthalein produced/mL/30 min) and biliary Cu concentration (y; mg/L) in samples collected from sheep (n = 4) for 6 h following a single IV administration of different combinations of tetrathiomolybdate (TTM) and a2-adrenergic agonist/antagonists (CLO/IDA)A.
A B
Treatment No.
TreatmentA
Linear regression equation
Correlation coefficient (r)B
P
1 2 3 4 5 6
BASAL TTM CLO IDA TTM + CLO TTM + IDA
Log y = 0.015 + 0.047 Log y = 0.178 + 0.291 Log y = 0.029 + 0.054 Log y = 0.122 + 0.129 Log y = 0.426 + 0.373 Log y = 0.324 + 0.353
0.68a 0.61a 0.70a,b 0.90b,c 0.92c,d 0.94c,d
<0.001 <0.001 <0.001 <0.001 <0.001 <0.001
(log x) (log x) (log x) (log x) (log x) (log x)
The treatments are: control (BASAL) and tetrathiomolybdate (TTM), clonidine (CLO), and idazoxan (IDA), either alone or in combination. Details as for Tables 1 and 2. Correlation coefficients with different superscript (a, b and c) are significantly different (Tukey-type multiple comparison test; P < 0.05).
increased during the pre-haemolytic phase. The sheep used in the present study did not show any symptoms of a haemolytic crisis. In addition, TTM, a specific Cu chelating agent, administered in Exper-
iments 2 (week 6), 5 (week 9) and 6 (week 10), would have removed any excess liver Cu that may have accumulated from continuous exposure to the HCu diet.
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Symonds and Ke (1989) reported that the ability of xylazine to increase biliary Cu excretion and also augment the effect of TTM on biliary Cu excretion is due to the a2-adrenergic agonist effect of the drug. However, the present study has shown that CLO, also an a2adrenergic agonist, when given alone (Treatment 3) did not significantly increase biliary Cu excretion. Possible explanations for this discrepancy are proposed. First, the increase in biliary Cu excretion associated with xylazine administration may not be related to a2agonist activity as proposed by Symonds and Ke (1989). Second, the ratio of a2 to a1-agonist activity in CLO is probably different to that of xylazine. Third, the dose of CLO used in this study (2 lg/kg BW) may have been inadequate to produce a significant a2-agonist activity. Fourth, a higher a1-agonist activity of CLO (than in xylazine) may have had a negative impact on biliary Cu excretion. But it should be noted that even at this low dosage, CLO and also IDA when given with TTM significantly increased bGLU enzyme activity in bile with high r values indicative of increased permeability of the lysosomal membrane in the liver and/or of lysosomal damage, but it is only CLO when given with TTM that resulted in b-GLU enzyme leakage into the plasma. It has previously been shown that rupture of liver lysosomes during Cu loading can cause liver necrosis (Gooneratne, 1979; Gooneratne et al., 1980). Moderate leakage of the lysosomal enzyme into the liver can induce apoptosis (Bursch, 2001), while pronounced leakage causes necrosis (Wang et al., 2006). Leakage of lysosomal enzyme from the liver can spill over into the bloodstream and may be one of the causes of red cell lysis in CCP in sheep (Kumaratilake et al., 1981). It is therefore hypothesised that plasma b-GLU may also be increased in CCP in sheep with liver hepatocyte lysosomal damage, and therefore, both b-GLU and acid phosphatase (Kumaratilake et al., 1981), in combination with increased plasma and red blood cell Cu (Gooneratne et al., 1981a,b), may be responsible for haemolytic crisis, a feature of CCP in sheep. A limitation of this study is the small sample size in each treatment. However, the trends in the profiles of parameters monitored, namely, biliary Cu concentration and excretion, plasma Cu concentration, and b-GLU enzyme activity in bile and plasma, were consistent in all the animals in the LCu or HCu diet group for each treatment. This study involved surgery, intensive sampling, and returning bile to the duodenum to avoid interruption to the enterohepatic circulation. This required significant resources including a large number of personnel. Therefore, the number of animals had to be limited. Although TTM has been used to treat CCP in sheep successfully (Gooneratne et al., 1981b; Humphries et al., 1986), the mechanisms by which Cu homeostasis is altered in ruminants apart from the increased biliary and urinary Cu excretion (Gooneratne et al., 1989a,b,c) continues to be debated (Suttle 2002, 2003a,b; Telfer et al., 2003, 2004). There is concern about the potential toxic effects of excess Mo in TTM because TTM in high doses can retard the growth of the epiphyseal plate in the long bones (Read et al., 1986), cause atrophy of the pituitary gland (Haywood et al., 2004) and reduce steroidogenesis (Kendall et al., 2003). Symonds and Ke (1989) suggested the use of an a2-adrenergic agonist in combination with low TTM doses to augment liver Cu removal via bile, but the present study has shown that although the a2agonist CLO alone has little effect on biliary Cu excretion, potential complications may arise if it is used in combination with TTM because of the significant increase in plasma b-GLU activity in Culoaded sheep, which by itself may compromise red blood cell integrity. IDA when given with TTM also increased bile but not plasma b-GLU activity. Therefore caution is advocated in the use of other drugs, especially a2-agonists including CLO, in combination with TTM until there is more evidence that the increase in plasma b-GLU has minimal effect on red blood cell integrity, to support such combination therapy. It would be interesting to
investigate the properties of xylazine other than the a2-agonist activity that may have contributed to increased biliary Cu excretion as reported by Symonds and Ke (1989). 5. Conclusions Clonidine alone, given IV, did not significantly increase biliary Cu excretion. TTM consistently increased biliary Cu concentration with or without CLO or IDA and this was most significant in sheep on the HCu diet given TTM + CLO, although biliary Cu excretion was higher in TTM > TTM + CLO > TTM + IDA because of the lower bile flow in sheep given CLO and IDA with TTM. This was also associated with an increase in biliary excretion of the lysosomal enzyme b-GLU, especially in sheep on the HCu diet, and was consistently significant only in the HCu diet group given TTM + CLO > TTM + IDA > TTM. However, significant spillage of biliary b-GLU from the liver into plasma occurred only in the TTM + CLO group. In view of this, the role of other drugs, especially a2-agonists, to augment TTM-induced biliary Cu excretion requires further clarification. Conflict of interest The author of this paper has no financial or personal relationship with other people or organizations that could inappropriately influence or bias the content of this paper. Acknowledgements This study was funded by a grant from the Lincoln University Research Committee. The author thanks the staff at Johnstone Memorial Laboratory for care of animals, Alex Familton for bile duct cannulation surgery, Peter Isherwood for Cu analysis and measurement of b-GLU activity, and Martin Wellby for preparation of graphics, Christine Bezar and Neville Grace for editorial comments, and James Ross for statistical analysis. References Aymonino, P.J., Ranade, A.C., Miller, A., 1969. Evidence for the existence of MoO3S 2 and WoO3S 2 ions in aqueous solution. Zeitschrift für Anorganische und Allgemeine Chemie 371, 295–298. Bursch, W., 2001. The autophagosomal–lysosomal compartment in programmed cell death. Cell Death and Differentiation 8, 69–81. Caple, I.E., Heath, T.J., 1972. Regulation of output of electrolytes in bile and pancreatic juice in sheep. Australian Journal of Biological Sciences 25, 155–165. Caple, I.E., Heath, T.J., 1978. Regulation of excretion of copper in bile of sheep: effect of anaesthesia and surgery. Comparative Biochemistry and Physiology 61A, 503–507. National Research Council, 1985. Nutrient Requirements of Sheep, sixth ed. National Academy Press, Washington DC, USA. Fishman, W.H., 1963. D-Glucuronidase. In: Bergmeyer, H.U. (Ed.), Methods of Enzymatic Analysis. Winheim/Bergstr Verlag Chemie, New York, pp. 869–874. Gooneratne, S.R., 1979. Morphometric, electron microscopic, biochemical and pathological investigation of liver of normal and copper poisoned sheep. PhD thesis, Murdoch University, Western Australia. Gooneratne, S.R., Christensen, D.A., 1997. Effect of chelating agents on the excretion of copper, zinc and iron on the bile and urine of sheep. The Veterinary Journal 153, 171–178. Gooneratne, S.R., Howell, J.McC., Gawthorne, J.M., 1979. Intracellular distribution of copper in the liver of normal and copper loaded sheep. Research in Veterinary Science 27, 30–37. Gooneratne, S.R., Howell, J.McC., Cook, R.D., 1980. An ultrastructural and morphometric study of the liver of normal and copper-poisoned sheep. American Journal of Pathology 99, 429–450. Gooneratne, S.R., Howell, J.McC., Gawthorne, J.M., 1981a. Intravenous administration of thiomolybdate for the prevention and treatment of chronic copper poisoning in sheep. British Journal of Nutrition 46, 457–467. Gooneratne, S.R., Howell, J.McC., Gawthorne, J.M., 1981b. An investigation of the effects of intravenous administration of thiomolybdate on copper metabolism in chronic Cu-poisoned sheep. British Journal of Nutrition 46, 469–480. Gooneratne, S.R., Christensen, D.A., Chaplin, R., Trent, A.M., 1985. Copper: biliary excretion in copper-supplemented and thiomolybdate-treated sheep. In: Mills, D.F., Bremner, I., Chesters, C.K. (Eds.), Proceedings of the 5th International
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