Excretion of loline alkaloids in urine and faeces of sheep dosed with meadow fescue (Festuca pratensis) seed containing high concentrations of loline alkaloids.

AIM: To determine the effect of oral dosing of sheep with loline alkaloids on their excretion in urine and faeces, and to monitor for any toxic effects. METHODS: In Experiment 1, six 9-month-old ewe lambs were given a single oral dose of loline alkaloids (52 mg/kg bodyweight (BW); acute exposure) as a suspension of ground meadow fescue (Festuca pratensis) seed in water. In Experiment 2, on six consecutive days, six ewe lambs were given three doses of loline (68 mg/kg BW/day; chronic exposure). Blood was collected at variable intervals up to 72 h in Experiment 1, and up to 8 days in Experiment 2, for haematology and measurement of alkaline phosphatase, aspartate aminotransaminase, creatine kinase and γ-glutamyl transferase in plasma. Urine and faecal samples were collected at similar times for measurement of creatinine in urine and loline alkaloid analysis. A post mortem with histopathology was carried out on two animals at the end of each experiment. RESULTS: The loline alkaloids, N-acetyl norloline, N-formyl loline, N-acetyl loline, N-methyl loline and loline base were detected in urine within 15 minutes after the single dosing. N-formyl loline and loline base were the predominant metabolites in urine in both experiments. The total quantity of lolines excreted in both urine and faeces was 10% and 4% of the amount dosed in Experiments 1 and 2, respectively. In both experiments, the clinical chemistry parameters in blood and urine were within normal ranges. Post-mortem and histopathological examination did not show any abnormalities. CONCLUSIONS: This is the first report of loline alkaloid profiles in both urine and faeces of sheep. The appearance of loline alkaloids and the loline base in urine within 15 minutes suggests rapid uptake, metabolism and excretion. Loline alkaloids were non-toxic to sheep at the concentrations they are exposed to under New Zealand grazing conditions. The low recovery of loline alkaloids in urine and faeces in the absence of toxicity signs suggests lolines are extensively metabolised; probably to forms other than N-formyl loline, N-methyl loline, N-acetyl loline, N-acetyl norloline, and loline base in the digestive tract of sheep prior to absorption, and/or in the liver or other tissues following absorption.

Effects of clonidine and idazoxan on tetrathiomolybdate-induced copper and lysosomal enzyme excretion into sheep bile.

This study investigated the effects of intravenous (IV) administration of tetrathiomolybdate (TTM), and α(2)-adrenergic agonist clonidine (CLO) and α(2)-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 ß-glucuronidase (ß-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 ß-GLU enzyme activity in bile. A significant increase in plasma ß-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 α(2)-adrenergic agonists, to further enhance TTM-induced biliary Cu excretion in the treatment of chronic Cu poisoning in sheep.

Changes in concentrations of lysergol in urine and prolactin in plasma, rectal temperature and respiration rate in sheep selected for resistance or susceptibility to ryegrass staggers and fed ergovaline.

AIM: To determine the effects of feeding ryegrass seed containing ergovaline to sheep selected for resistance or susceptibility to ryegrass staggers on concentration of lysergol (a metabolite of ergovaline) in urine, prolactin in plasma, rectal temperature and respiration rate. METHODS: Two experiments were carried out using 12 Romney crossbred ewe lambs aged 9 months, comprising animals resistant (n=4), susceptible (n=4) or outcross (n=4) to ryegrass staggers. In Experiment 1, sheep were given either a single (Part A) or six (Part B) feed (s) of endophyte-infected seed containing ergovaline at 30 mg/kg dry matter (DM), at 42 µg ergovaline/kg bodyweight (BW), to simulate acute and chronic exposure to ergovaline, respectively. The concentration and excretion of lysergol in urine and concentration of prolactin in plasma were measured over 3 and 12 days, for Parts A and B respectively. In Experiment 2, after a recovery period of 7 days, the same sheep were fed with ergovaline at 67 µg/kg of BW for 7 days. Soon after the seventh feed the sheep were moved to a hothouse at 36.5°C and 60% humidity, and 3 h later their rectal temperatures and respiration rates were measured. RESULTS: The concentration of lysergol and excretion in urine increased to a peak between 6 and 9 h after exposure to ergovaline whereas the concentration of prolactin in plasma was markedly reduced. Differences in concentration and rate of excretion of lysergol in urine between animals resistant, outcross and susceptible to ryegrass staggers were not significant (p>0.1). The animals resistant to ryegrass staggers had a lower rectal temperature (p<0.05) and a faster respiration rate than the outcross and susceptible groups when exposed to high ambient temperature and high humidity. CONCLUSIONS: This study showed that excretion of lysergol in urine increased with each exposure of sheep to ergovaline. Animals genetically resistant to ryegrass staggers exhibited a lower rectal temperature and a faster respiration rate than those susceptible, demonstrating the possible cross resistance of sheep to ergovaline in a population originally selected for resistance to ryegrass staggers. Hence potential exists to select animals resistant to ryegrass staggers that are also resistant to ergovaline.

Effects of dietary Cu, Mo and S on urinary Cu and Zn excretion in Simmental and Angus cattle.

This study determined the effects of dietary copper (Cu), molybdenum (Mo) and sulphur (S) on urinary Cu and zinc (Zn) excretion in cattle. Four Simmental and four Angus heifers were fed low (L) or high (H) levels (mg/kg DM) of Cu (5,40), Mo (1,10) and S (0.2,0.5%). Initially two of each breed was fed either LCu or HCu (2 mo). Then all eight animals were fed sequentially LCuHS (1.5 mo), HCuHS, HCuHMo and HCuHMoHS (2 mo each). Simmental had a higher urine flow, increased concentration and total excretion of urinary Cu and Zn compared to Angus, but only total Zn excretion was significantly higher. Urinary Cu excretion was greatest with the HCuHMoHS diet. Urinary Zn excretion significantly increased with HS but not HS in combination with HMo and/or HCu. This study, together with previously reported biliary excretion, allows a direct comparison of urinary and biliary Cu and Zn excretion responses to dietary Cu, Mo and S.

Acute and long-term effects of exposure to sodium monofluoroacetate (1080) in sheep.

Acute and long-term effects of a single, relatively high oral dose (0.25 and 0.30 mg/kg) of sodium monofluoroacetate (1080) on the survival and productivity of sheep were evaluated to establish a better understanding of 1080 poisoning and identify more specific changes diagnostic of toxicosis. In survivors, clinical signs of acute 1080 toxicosis such as salivation and lethargy were generally very mild. Fasted animals were more prone to 1080 toxicity. In animals that died, more severe signs, including tachypnoea, dyspnoea, and tremors occurred for 15-20 min prior to death. 1080 concentrations were highest in the blood > heart > skeletal muscle > liver. 1080 could not be detected in any of these organs of the animals that survived. Serum citrate concentrations were elevated for 4 days after dosing. No clinical or biochemical abnormalities were found in any animal after 4 days. Histopathological lesions were most marked in the heart and lung with inflammation, necrosis, and scattered foci of fibrous tissue in the myocardium, pulmonary oedema and inflammation of the lung. No adverse long-term effects on general health or reproductive performance were observed in any sheep that survived the first 4 days following exposure to 1080. The most reliable diagnostic indicators of 1080 exposure in sheep were measurement of its residues in blood, skeletal muscle and ruminal contents, increased serum citrate concentration, elevated heart rate, and characteristic electrocardiograph changes (up to 4 days after exposure). Death from 1080 is most likely to occur within 96 h, and animals that survived this period appeared normal.

Anal fin morphology and gonadal histopathology in mosquitofish (Gambusia holbrooki) exposed to treated municipal sewage effluent.

Feral mosquitofish living in undiluted treated municipal sewage in Queensland, Australia, were examined for morphologic abnormalities indicative of reproductive dysfunction. Male and female mosquitofish were captured at two sites receiving undiluted treated sewage and compared with those captured at a reference site. Several morphologic end points were examined, including length of the fourth and sixth anal fin rays and gonadal histology. Both male and female mosquitofish at one site exhibited minor elongation of the fourth anal fin ray consistent with exposure to androgenic stimulation, although the spatial extent of the effect was limited and not significant at other sites further downstream. No incidences of intersex were found. These findings suggest that the level of treatment of domestic sewage at the two sewage treatment plants assessed is adequate to prevent reproductive abnormalities in exposed mosquitofish populations.

Herbicide spray drift odor: measurement and toxicological significance.

The toxicological significance of exposure of members of the public to spray drift odors of four herbicide formulations (three 2, 4-dichlorophenoxyacetic [2,4-D] acid derivatives and one MCPA [4-methyl-2-chlorophenoxyacetic acid] derivative) has been studied using a combination of novel odor measurement and classic residue analysis techniques. The mean odor concentrations, generated during the spraying of the commercial herbicide formulations under simulated aerial application conditions, were about twofold higher for 2,4-D ethylhexyl ester (22,500 OU(c)/m(3)) and MCPA (30,100 OU(c)/m(3)) than for 2,4-D butyl ester (12,400 OU(c)/m(3)) and 2,4-D amine (11,800 OU(c)/m(3)). Detailed investigations determined that the odors are due to trace manufacturing impurities and additives in the commercial formulations, whereas the herbicide active ingredients are odorless. Measured airborne herbicide active ingredient concentrations under the simulated aerial application conditions were all below their respective occupational safety and health TLV-TWA values, indicating that exposures of toxicological significance as a result of spray drift are unlikely.

Effect of chelating agents on the excretion of copper, zinc and iron in the bile and urine of sheep.

The effect of tetrathiomolybdate (TM), disodium calcium ethylenediamine tetraacetate (EDTA), D-penicillamine (PEN), 2-3 dimercapto-1-propanol (BAL) and dimethyl dithiocarbamate (DDC) administration on biliary and urinary excretion of copper (Cu), zinc (Zn) and iron (Fe) was investigated in sheep on a low Cu diet (Group A) and a high Cu diet (Group B). Only biliary Cu excretion increased significantly (P<0.01) with TM treatment. Urinary Cu excretion increased (P<0.01) following PEN treatment. TM, EDTA, PEN, BAL and DDC adminstration increased Cu excretion via bile and urine by 254, 11, 266, 46 and 16%, respectively in Group A sheep, and by 354, 13, 196, 20 and (-) 31% in Group B sheep. Urinary Zn excretion increased (P<0.01) following EDTA and PEN treatments. Only urinary Fe excretion increased (P<0.01) with EDTA treatment. The results show that TM and PEN are the most efficient decoppering agents, but PEN unlike TM also removes Zn. The major routes of excretion of Cu chelates by TM and PEN are different. TM increases Cu excretion significantly (P<0.05 in Group A and P<0.01 in Group B) in bile with only a slight increase in urinary Cu whereas PEN increases Cu excretion significantly (P<0.01) in urine. Therefore from a therapeutic view, a combination therapy of TM and PEN would be useful to maximize Cu removal from the body.

Persistence of sodium monofluoroacetate in rabbits and risk to non-target species.

1. Sodium monofluoroacetate (1080), a vertebrate pesticide used in New Zealand, was administered orally to rabbits at two dose levels (sub-lethal and lethal) to determine how long 1080 would persist in plasma, liver, kidney, and muscle so that the risk of consumption of meat from lethally or sub-lethally poisoned rabbits by non-target species could be assessed. 2. The plasma elimination half-life in rabbits receiving a sub-lethal dose was 1.1 h. Retention of 1080 in tissue was greater in rabbits dosed with a lethal dose than in those that received a sub-lethal dose. Irrespective of the dose level, concentration of 1080 in muscle, kidney, and liver was substantially lower than in the plasma. 3. Poisoning of dogs is possible because of their extreme susceptibility to 1080. Poisoning of birds is less likely. The risk of secondary poisoning is reduced as the concentration of 1080 declines in putrefying carcasses.

Role of thiamine status in sulphur induced polioencephalomalacia in sheep.

The effects of excess dietary sulphur were studied in sheep supplemented and unsupplemented with thiamine. The diets contained either 0.19 per cent sulphur (LS) or 0.63 per cent sulphur (HS) in combinations with 14 mg kg-1 thiamine (LB1) or 243 mg kg-1 thiamine (HB1). A total of 56 two-month-old lambs were used. Groups consisting of nine, nine, 22 and 16 lambs were fed LS-LB1, LS-HB1, HS-LB1 and HS-HB1 diets, respectively for 14 weeks. Out of 22 lambs fed the HS-LB1 diet, seven lambs developed neurological signs between the third and eighth week of the trial. Two of these lambs died, three that were in extremis were euthanased, and two recovered completely. All clinically affected animals had extensive malacic lesions in the cerebral cortex, midbrain and brainstem. None of the lambs from the LS groups or HS-HB1 group developed clinical signs. Several clinically normal lambs from the HS-LB1 group had necrotic lesions in their brains at gross and microscopic examination. Supplementation with dietary thiamine prevented development of clinical signs, but did not totally prevent development of microscopic brain lesions. Brain thiamine concentration, transketolase activity and thiamine pyrophosphate (TPP) effect were not different (P greater than 0.05) among groups. There was a strong effect (P less than 0.0001) of dietary thiamine supplementation on blood thiamine concentration and TPP effect. Blood thiamine concentration was higher whereas TPP effect was lower in the thiamine supplemented sheep. Blood and tissue thiamine concentrations in sheep exposed to high dietary sulphur did not indicate either systemic or local thiamine deficiency per se. Increased TPP effect in sheep fed the HS-LB1 diet indicated mild to moderate metabolic thiamine deficiency. Thiamine inadequacy may be an effect of an increased requirement for thiamine in animals exposed to excess dietary sulphur.

Microbiological methods of thiamine measurement in biological material.

Semiautomated, microbiological methods for the determination of thiamine in a variety of biological material are presented. Thiamine content was measured in bovine, ovine and rat blood, plasma, bile, urine, duodenal fluid, liver, kidney and brain. Thiamine was extracted from samples by mild acid digestion (trans aconitate pH 4.0) at 115 degrees C for 20 min. Microbiologic assays utilizing Lactobacillus fermenti and Ochromonas danica were performed on 96-well microplates and growth, as monitored by optical density, was directly read on a microplate reader at 650 nm. Particular attention was given to the validation of the two methods in terms of accuracy, reproducibility and recovery. The microbiological methods described here are specific, sensitive, and ensure good reproducibility. The methods can be used to analyse a large number of samples rapidly and at low cost.

The effects of thiamine and EDTA on biliary and urinary lead excretion in sheep.

Relative efficacy of thiamine (B1) and/or calcium disodium ethylinediaminetetraacetate (EDTA) administration on lead (Pb) excretion via bile and urine was studied in Pb-loaded sheep. The sheep were administered B1 s.c., 75 mg/kg body wt., EDTA i.v., 110 mg/kg body wt., and a combination of B1-EDTA at rates as above. Each treatment was followed by 69 h sampling of blood, bile, and urine. Urinary Pb excretion increased following B1-EDTA greater than EDTA greater than B1 administration. Thiamine and B1-EDTA treatments increased biliary Pb excretion. Overall, B1, EDTA and B1-EDTA administration increased Pb excretion via bile and urine by 72%, 595%, and 842% respectively over basal level (mean +/- SE: 20.1 +/- 2.9 micrograms Pb/h). It has been concluded that thiamine enhances elimination of Pb from the body and this feature may be beneficial in chelation therapy.

Ovine polioencephalomalacia associated with dietary sulphur intake.

Fifty-six female crossbred two-month-old lambs were housed in individual cages, and fed a basic ration of barley (59%), soybean meal (5%), and alfalfa (32%) prepared to meet NRC nutrient requirements. Four percent of the diet contained a standard salt mix to which the factors inorganic sulphur (S) and thiamine (B1) were added. Four treatment groups were used: low sulphur and normal thiamine (0.19% S, 13.7 mg/kg B1) low sulphur and high thiamine (0.19% S, 243 mg/kg B1), high sulphur and normal thiamine (0.63% S, 13.7 mg/kg B1), high sulphur and high thiamine (0.63% S, 243 mg/kg B1). All animals had free access to water and were offered 1 kg/animal/day of diet for 14 weeks, when necropsy was undertaken. Seven lambs fed unsupplemented (normal B1) diets containing added sulphur developed clinical symptoms of polioencephalomalacia (PEM) between the 3rd and 7th week of the trial. Morbidity (P less than 0.013) and mortality (P = 0.08) differences were attributed to S administration. None of the B1 supplemented lambs developed clinical signs of PEM. Body weight and relative organ weights did not differ among treatment groups. Serial sections of all brains were examined grossly and microscopically. Nonparametric statistical analysis revealed sulphur related effects in the cerebrum, midbrain and hindbrain (P less than 0.0001), no thiamine-related effects or interaction between the factors were seen, except in the amygdaloid body. It was concluded that inorganic sulphur was associated with polioencephalomalacia, and that dietary thiamine may decrease the severity of lesions in some affected areas of the central nervous system.

Effects of diets of high sulphur content and varied concentrations of copper, molybdenum and thiamine on in vitro phagocytic and candidacidal activity of neutrophils in sheep.

Candidacidal and phagocytic tests were performed on 34 female sheep fed high sulphur diets containing varied concentrations of thiamine, copper, and molybdenum for 14 weeks. Tests were conducted at weekly intervals for five weeks during the latter half of the experiment. The ability of polymorphonuclear leucocytes from sheep on diets unsupplemented with thiamine to kill phagocytosed Candida albicans was lower than those on diets supplemented with thiamine. The ability of polymorphonuclear leucocytes to phagocytose C albicans was lower in animals on low copper diets than in animals on high copper diets. Factorial analysis showed that thiamine supplementation significantly increased (P less than 0.0001) candidacidal but not phagocytic activity. By contrast, copper supplementation significantly increased (P less than 0.0005) the phagocytic activity with no effect on candidacidal activity. The observed detrimental effect of high sulphur intake on in vitro polymorphonuclear leucocyte function can mean that ruminants in areas where large quantities of sulphur are taken in with water and feed have compromised immune function due to lower copper and thiamine status and hence are at risk of increased susceptibility to infections.

Effects of high dietary sulfur on brain functions using evoked potentials technique.

Brain stem auditory-evoked response (BAER) is a noninvasive technique used for detecting neurophysiological abnormalities of the brain stem along the auditory pathway. Brain stem auditory-evoked response recordings were obtained from subcutaneous skin electrodes from two control sheep and 22 other sheep fed high sulfur (S) diets with low or high concentration of thiamine (B1), copper (Cu), and molybdenum (Mo). At least four peaks (I,II,III,IV) of varied amplitude were observed in all animals. Neurophysiological abnormalities due to decreased conductivity and/or excitability of nerve fibers along the auditory pathway were found on the BAER recordings of sheep fed high S diet. Abnormalities of peaks and interpeak latencies within BAER recordings were related to histopathological observations of brain stem lesions. Lesions in the areas of the cochlear nuclei and lateral lemniscus were seen in conjunction with altered BAER components. However, abnormalities in BAER recordings and lesions in the brain stem also occurred in the absence of overt clinical signs. Analysis of interpeak latencies between peaks I and IV revealed significant differences among dietary groups. Sheep given diets low in Cu, Mo, and B1 were affected most. Factorial analysis indicated B1 and interactions among Cu, Mo, and B1 as significant factors influencing interpeak latencies.

Sulfur-induced polioencephalomalacia in sheep: some biochemical changes.

The effect of high dietary sulfur (S) supplementation on blood thiamine (B1) concentration, biochemical indices of liver, muscle and kidney damage and selected plasma electrolytes was studied in six sheep. Three of these sheep received an additional 230 mg thiamine/kg diet (Group 2). After approximately 2.5-3 weeks on this diet, all three sheep in the non-B1-supplemented group (Group 1) showed loss of appetite and developed mild neurological signs: depression, intermittent signs of excitation and head pressing. Increases in blood B1 concentration and plasma creatine kinase (CK) and aspartate aminotransferase (AST) were observed during this time in all affected animals. Clinical signs lasted only for two to five days. Sheep in group 2 were clinically normal throughout the experiment, but all of these animals also had elevated blood B1 concentrations and plasma CK activity at the 3 wk sampling. Plasma magnesium concentrations of group 1 sheep were elevated at the 2.5-3 wk and 6 wk samplings but they declined significantly (p less than 0.05) to low normal levels thereafter. Magnesium concentrations of group 2 sheep were low at the beginning but progressively increased during the course of the experiment. At necropsy, brain lesions suggestive of polioencephalomalacia (PEM) were observed in all sheep but were most marked in group 1. It is speculated that PEM may be caused by a direct toxic effect of S, S metabolites or B1 antimetabolites in the brain rather than by an in vivo B1 deficiency per se.

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.

1. The relative importance of excretory routes in the removal of recently stored 67Cu following tetrathiomolybdate (TTM) administration was studied. Lambs fed on either 5 mg Cu/kg dry matter (DM) or 35 mg Cu/kg DM, were primed intravenously (iv) with 67Cu and challenged 27 h later with 99Mo-labelled TTM given either iv or intraduodenally (id). The profiles of 67Cu and 99Mo and of Cu and Mo in blood, bile, urine and faeces were measured. 2. Level of dietary Cu and route of administration of 99Mo-TTM affected the amplitude of blood, bile and urine profiles of 67Cu and stable Cu, but not the pattern of the responses observed. 3. The present study describes for the first time increased excretion of endogenous 67Cu through gastrointestinal secretions other than the bile due to TTM administration. 4. Administration of TTM resulted in the immediate release of 67Cu from storage compartments in the body into the blood circulation. Changes in stable Cu levels in blood, bile, urine and faeces, and gut and systemic effects were evident. Biliary and urinary Cu excretion due to TTM was rapid and maximal within 24 h of injection. 5. Administration of 67Cu iv resulted in the immediate excretion of 67Cu in bile in a pulsatile, constant pattern. A similar pattern of 67Cu excretion into bile in synchrony with that of 99Mo was observed after 99Mo-labelled TTM administration. 6. The similar pattern of biliary 67Cu excretion observed after injection of 67Cu and after injection of 99Mo-labelled TTM 27 h later is discussed in relation to the times required to process the Cu through different hepatic pathways for excretion in bile.

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.

1. The effectiveness of tetrathiomolybdate (TTM) in the removal of 67Cu from the long-term storage compartment in liver was studied. Lambs receiving 5 mg Cu/kg dry matter (DM) or 35 mg Cu/kg DM were primed intravenously (iv) with 67Cu and challenged 10 d later with 99Mo-labelled TTM given either iv or intraduodenally (id). The profiles of 67Cu and 99Mo and of Cu and of Mo with time were measured in blood, bile, urine and faeces. 2. The level of dietary Cu affected the amplitude of profiles of 67Cu and Cu in blood, bile and urine after administration of 99Mo-labelled TTM. TTM administration increased liver Cu removal and this was most marked in sheep given TTM iv. The liver Cu removal from the long-term storage Cu compartment was low and was not affected by the route of administration of TTM. Endogenous Cu excretion was higher in lambs given TTM id. 3. Excretion of 67Cu in bile through the transhepatocellular pathway after TTM administration appeared absent, while the transbiliary and hepatolysosomal pathways were operative. The potential reasons for this change are discussed. 4. TTM predominantly enhances the removal of Cu from the short-term storage compartment, but effects on the long-term storage compartment may still be of significance.

High sulfur related thiamine deficiency in cattle: A field study.

Following development of polioencephalomalacia in one of 105 cattle in a farm in southeastern Saskatchewan, a study was initiated to monitor thiamine (B(1)) and copper (Cu) status, and to evaluate interactive nutritional factors which may have been responsible for this occurrence. It was evident that a combination of high sulfur (S) and low Cu intake was responsible for the depletion of blood B(1) and plasma Cu. Supplementation with trace minerals alone resulted in a significant (p<0.05) improvement in both B(1) and Cu status of the herd. We recommend that herds exposed to high intakes of S be supplemented with Cu up to 50 mg/kg feed dry matter to alleviate potential deficiencies of B(1) and Cu.

Subcellular distribution of copper in the kidneys of normal, copper-poisoned, and thiomolybdate-treated sheep.

Twenty-seven sheep given either copper (Cu) and/or tetrathiomolybdate (TM) were used to study the subcellular distribution of Cu within the kidney and to monitor the location of lysosomes within the subcellular fractions using acid phosphatase (AP) as a marker enzyme. Copper dosing alone increased the Cu content in the liver and the kidneys. The administration of intravenous TM prevented the development of chronic copper poisoning (CCP) in sheep, reduced the rate of accumulation of Cu in the liver of Cu-dosed animals, but increased the Cu content of kidneys in both the control and Cu-dosed sheep. The total amount of Cu that accumulated in the kidneys of sheep given TM appears to depend on several factors: a) liver Cu concentration, b) Cu intake, and c) dosage of TM. Thus, the highest Cu concentration was found in the kidneys of sheep that continued to receive Cu orally at the same time as they were given TM. The intracellular distribution of Cu and AP in the kidneys showed that in the control sheep given neither Cu or TM, the highest proportion of Cu was in the cytosol fraction, and the highest specific activity of AP was in the light mitochondrial (lysosomal) fraction. Dosing with Cu markedly increased the Cu concentration and greatly elevated the total activity of AP in the heavier fractions, i.e., the nuclear (N) and heavy mitochondrial (MH). Thus, the increase in Cu observed in the N and MH fractions was not caused by an accumulation of Cu by nuclei and mitochondria, but was due to an accumulation of Cu by lysosomes that sedimented with the heavier fractions. The intracellular distribution of Cu in the kidneys of TM-treated sheep was similar to that seen in Cu-loaded sheep. Although Cu accumulated readily in the kidneys of animals receiving TM, kidney function tests showed neither glomerular nor tubular functional impairment.