OBJECTIVE To determine the effect of subchronic oral exposure to zearalenone (ZEA) at a daily dose of 50 μg of ZEA/kg of body weight (an environmentally relevant concentration) on the reproductive system of rabbit bucks.
ANIMALS 8 healthy sexually mature New Zealand White rabbits.
PROCEDURES During the experimental period (March to June), each rabbit underwent a 7-week control protocol and then a 7-week treatment protocol. Water (0.5 mL) or ZEA solution (50 μg/kg [0.5 mL]) was administered orally once daily during the control and treatment period, respectively; ejaculates were collected weekly. Studied end points included semen quality variables (spermatozoa kinetics, morphology, viability, and DNA fragmentation), serum testosterone concentration, and results of histologic examination of the testes and epididymides following euthanasia at the end of the experimental period.
RESULTS Treatment with ZEA solution resulted in significant increases in spermatozoa beat-cross frequency, in the percentages of spermatozoa with head and midpiece abnormalities, and in the percentages of DNA-fragmented spermatozoa, compared with effects of the control treatment. Serum testosterone concentration, other spermatozoa velocity variables, and percentages of progressive and total motility, rapidly or slowly moving spermatozoa, and live spermatozoa did not differ significantly between the 2 periods. Histologic examination revealed no patterns of abnormal findings in the testes and epididymides.
CONCLUSIONS AND CLINICAL RELEVANCE Oral treatment with ZEA solution at an enviromentally relevant concentration caused minor interference with rabbit bucks' sperm quality. Although mostly considered mild, the sperm quality changes warrant further investigation in terms of fertilizing capacity impairment.
OBJECTIVE To investigate the cytotoxic effects of azathioprine, 6-mercaptopurine, and 6-thioguanine on canine hepatocytes.
SAMPLE Commercially available cryopreserved canine primary hepatocytes.
PROCEDURES The study consisted of 2 trials. In trial 1, hepatocytes were incubated with azathioprine, 6-mercaptopurine, or 6-thioguanine at 1 of 6 concentrations (0.468, 0.937, 1.875, 3.750, 7.500, or 15.000 μmol/L) for 24, 48, or 72 hours. At each time, cell viability and lactate dehydrogenase (LDH) activity were determined for each thiopurine-concentration combination, and alanine aminotransferase (ALT) activity was determined for cells incubated with each thiopurine at a concentration of 15 μmol/L. In trial 2, hepatocytes were incubated with azathioprine, 6-mercaptopurine, or 6-thioguanine at 1 of 3 concentrations (18.75, 37.50, or 75.00 μmol/L) for 24 hours, after which the free glutathione concentration was determined for each thiopurine-concentration combination and compared with that for hepatocytes incubated without a thiopurine (control).
RESULTS Incubation of hepatocytes with each of the 3 thiopurines adversely affected cell viability in a time- and concentration-dependent manner; however, this decrease in cell viability was not accompanied by a concurrent increase in LDH or ALT activity. Likewise, free glutathione concentration for hepatocytes incubated for 24 hours with supratherapeutic thiopurine concentrations (> 18.75 μmol/L) did not differ significantly from that of control cells.
CONCLUSIONS AND CLINICAL RELEVANCE Results indicated that thiopurines adversely affected the viability of canine hepatocytes in a time- and concentration-dependent manner but had a nonsignificant effect on the LDH and ALT activities and free glutathione depletion of those hepatocytes.
Objective—To identify biomarkers of P-glycoprotein (P-gp) substrate neurotoxicity in transgenic mice expressing the mutant canine ABCB1 gene (ABCB1-1Δ).
Animals—8 ABCB1 knock-in and knock-out transgenic mice expressing the ABCB1-1Δ gene and 8 control mice expressing the wild-type canine ABCB1 gene (ABCB1-WT).
Procedures—Groups including 2 ABCB1-1Δ mutant mice and 2 ABCB1-WT mice were administered the P-gp substrates ivermectin (10 mg/kg, SC), doramectin (10 mg/kg, SC), moxidectin (10 mg/kg, PO), or digoxin (1.53 mg/kg, SC). A toxicogenomic approach based on DNA microarrays was used to examine whole-genome expression changes in mice administered P-gp substrates.
Results—Compared with control ABCB1-WT mice, ABCB1-1Δ mutant mice developed neurotoxic signs including ataxia, lethargy, and tremors similar to those reported for dogs with the ABCB1-1Δ mutation. Microarray analysis revealed that gene expression was altered in ABCB1-1Δ mutant mice, compared with findings for ABCB1-WT mice, following administration of the same P-gp substrates. Gene pathway analysis revealed that genes with a ≥ 2-fold gene expression change were associated with behavior and nervous system development and function. Moreover, 34 genes were altered in the ABCB1-1Δ mutant mice in all 4 drug treatment groups. These genes were also associated with behavior, which was identified as the top-ranked gene network.
Conclusions and Clinical Relevance—These study data have facilitated understanding of the molecular mechanisms of neurotoxicosis in ABCB1-1Δ mutant mice following exposure to various P-gp substrates. Some genes appear to be potential biomarkers of P-gp substrate neurotoxicity that might be used to predict the safety of those drugs in dogs with the ABCB1-1Δ mutation.
Objective—To develop in genetically engineered mice an alternative screening method for evaluation of P-glycoprotein substrate toxicosis in ivermectin-sensitive Collies.
Animals—14 wild-type C57BL/6J mice (controls) and 21 genetically engineered mice in which the abcb1a and abcb1b genes were disrupted and the mutated canine ABCB1 gene was inserted.
Procedures—Mice were allocated to receive 10 mg of ivermectin/kg via SC injection (n = 30) or a vehicle-only formulation of propylene glycol and glycerol formal (5). Each was observed for clinical signs of toxic effects from 0 to 7 hours following drug administration.
Results—After ivermectin administration, considerable differences were observed in drug sensitivity between the 2 types of mice. The genetically engineered mice with the mutated canine ABCB1 gene had signs of severe sensitivity to ivermectin, characterized by progressive lethargy, ataxia, and tremors, whereas the wild-type control mice developed no remarkable effects related to the ivermectin.
Conclusions and Clinical Relevance—The ivermectin sensitivity modeled in the transgenic mice closely resembled the lethargy, stupor, disorientation, and loss of coordination observed in ivermectin-sensitive Collies with the ABCB1–1Δ mutation. As such, the model has the potential to facilitate toxicity assessments of certain drugs for dogs that are P-glycoprotein substrates, and it may serve to reduce the use of dogs in avermectin derivative safety studies that are part of the new animal drug approval process.
Objective—To determine the toxicokinetics of N-(methylsuccinimido)anthranoyllycoctonine–type low larkspur alkaloids in beef cattle.
Animals—5 Black Angus steers and 35 Swiss Webster mice.
Procedures—Low larkspur (Delphinium andersonii) was collected, dried, ground, and administered to 5 steers via oral gavage to provide a dose of 12 mg of N-(methylsuccinimido)-anthranoyllycoctonine alkaloids/kg. Steers were housed in metabolism crates for 96 hours following larkspur administration; heart rate was monitored continuously, and blood samples were collected periodically for analysis of serum concentrations of 16-deacetylgeyerline, methyllycaconitine, geyerline, and nudicauline and assessment of kinetic parameters. The LD50 of a total alkaloid extract from D andersonii was determined in Swiss Webster mice.
Results—The alkaloids were quickly absorbed, with a maximum serum concentration achieved within 18 hours after administration. Geyerline and nudicauline coeluted as 1 peak and were considered together for toxicokinetic analysis. Mean ± SD elimination half-life was 18.4 ± 4.4 hours, 15.6 ± 1.5 hours, and 16.5 ± 5.1 hours for 16-deacetylgeyerline, methyllycaconitine, and geyerline and nudicauline, respectively. There were significant differences in maximum serum concentration, amount absorbed, and distribution half-life among the 4 alkaloids. The mouse LD50 was 9.8 mg/kg.
Conclusions and Clinical Relevance—Results suggested that clinical poisoning was likely to be most severe approximately 18 hours after exposure. Cattle should be closely monitored for at least 36 hours after initial exposure. Additionally, a withdrawal time of approximately 7 days would be required to clear > 99% of the toxic alkaloids from the serum of cattle that have ingested low larkspur.
Objective—To compare the toxic effects of a Delphinium occidentale chemotype containing N-(methylsuccinimido) anthranoyllycoctonine (MSAL)—type alkaloids and a D occidentale chemotype lacking MSAL-type alkaloids in mice and cattle.
Animals—225 male Swiss Webster mice and 11 Black Angus steers.
Procedures—4 collections of larkspur containing MSAL-type alkaloids and 4 collections of larkspur lacking MSAL-type alkaloids were used. From each collection, total alkaloid extracts (0.05 to 0.20 mL) were administered via tail-vein injection in 27 to 29 mice. Dried, finely ground plant material from 1 collection with and 1 collection without MSAL-type alkaloids (doses equivalent to 37.6 mg of total alkaloids/kg) were each administered to 8 cattle via oral gavage in a crossover experiment; 3 cattle received a single dose equivalent to 150.4 mg of total alkaloids/kg (no MSAL-type alkaloids). In mice, clinical effects were monitored; in cattle, heart rate was monitored before (baseline) and 24 hours after treatment. At the 24-hour time point, cattle were exercised as a measure of muscle weakness.
Results—In mice, mean LD50 associated with alkaloid extracts prepared from plants that did or did not contain MSAL-type alkaloids was 2.3 and 54.2 mg/kg, respectively. In cattle at 24 hours after treatment, plant material containing MSAL-type alkaloids significantly increased heart rate from baseline and was associated with exercise-induced collapse; plant material lacking MSAL-type alkaloids had no similar effects.
Conclusions and Clinical Relevance—Taxonomic classification of D occidentale alone was not a good indicator of the toxic risk to grazing cattle.
Objective—To determine the contribution of 7,8-methylenedioxylycoctonine (MDL)–type alkaloids to the toxic effects of tall larkspur (Delphinium spp) consumption in cattle.
Animals—Sixteen 2-year-old Angus steers.
Procedures—Plant material from 3 populations of tall larkspur that contained different concentration ratios of MDL-type-to-N-(methylsuccinimido) anthranoyllycoctonine (MSAL)–type alkaloids was collected, dried, and finely ground. For each plant population, a dose of ground plant material that would elicit similar clinical signs of toxicosis in cattle after oral administration was determined on the basis of the plants' MSAL-type alkaloid concentration. Cattle were treated via oral gavage with single doses of ground plant material from each of the 3 populations of tall larkspur; each animal underwent 1 to 3 single-dose treatments (≥ 21-day interval between treatments). Heart rate was recorded immediately before (baseline) and 24 hours after each larkspur treatment.
Results—Tall larkspur populations with a lower MDL-type-to-MSAL-type alkaloid concentration ratio required a greater amount of MSAL-type alkaloids to cause the expected clinical signs of toxicosis (including increased heart rate) in cattle.
Conclusions and Clinical Relevance—Results indicated that the typically less toxic MDL-type alkaloids contributed in a significant manner to the toxic effects of tall larkspur in steers. Consequently, both the concentration of MSAL-type alkaloids and the total concentration of MSAL- and MDL-type alkaloids should be determined when assessing the relative toxicity of tall larkspur populations. These results provide valuable information to determine the risk of toxicosis in cattle grazing on tall larkspur–infested rangelands.
Objective—To determine whether signs of avermectin (AVM)-milbemycin (MB) toxicosis would be evident in AVM-MB–sensitive Collies after treatment with an experimental formulation of spinosad alone or spinosad combined with MB 5-oxime (MBO) at doses up to 5 and 10 times the MBO maximum label dose.
Animals—20 adult Collies homozygous or heterozygous for the MDR1 gene mutation that had signs of toxicosis after oral administration of ivermectin.
Procedures—On the basis of AVM-MB sensitivity score, each dog was assigned in a randomized block design to 1 of 5 treatment groups (control group, 300 mg of spinosad/kg [5 times maximum label dose], 180 mg of spinosad/kg with 3 mg of MBO/kg [3 times maximum MBO label dose], 300 mg of spinosad/kg with 5 mg of MBO/kg, and 300 mg of spinosad/kg with 10 mg of MBO/kg). Treatments were administered orally as a sequence of single doses during 5 consecutive days. After a 28-day washout period, treatment sequences were repeated. Posttreatment observation and scoring by blinded observers were conducted to specifically include neurologic abnormalities typical of AVM-MB toxicosis, such as signs of depression, ataxia, mydriasis, and hypersalivation.
Results—No signs of AVM-MB toxicosis were attributed to treatment in any dog during the study.
Conclusions and Clinical Relevance—Results indicated that oral administration of spinosad at 300 mg/kg alone or in combination with MBO at doses up to 10 mg/kg did not cause signs of AVM-MB toxicosis in AVM-MB–sensitive dogs with the MDR1 gene mutation.
Objective—To determine whether larkspur-derived N-(methylsuccinimido) anthranoyllycoctonine (MSAL)-type alkaloids alter heart rate and electrically evoked electromyographic (eEMG) response of the external anal sphincter (EAS) in cattle and whether these effects can be reversed by acetylcholinesterase inhibitors.
Animals—12 beef heifers and 4 cows.
Procedures—3 or 4 heifers were used in 1 or 2 of 7 dose-response experiments; heart rate and EAS eEMG response were assessed before and 24 hours after oral treatment with larkspur (doses equivalent to 0.5 to 15 mg of MSAL-type alkaloids/kg). In 3 subsequent experiments, 3 heifers (1 of which was replaced with another heifer in the control experiment) each received 10 mg of MSAL-type alkaloids/kg and were injected IV with physostigmine (0.04 mg/kg), neostigmine (0.04 mg/kg), or saline (0.9% NaCl) solution 24 hours later, prior to assessment. Additionally, EAS eEMG response was measured in 4 cows before and after epidural administration of 2% lidocaine hydrochloride.
Results—Larkspur-treated heifers developed dose-related increases in heart rate and decreases in EAS eEMG response. Twenty-four hours after administration of MSAL-type alkaloids, neostigmine decreased heart rate but did not affect eEMG response, whereas physostigmine did not affect heart rate but caused a 2-fold increase in eEMG response. In cows, epidural anesthesia did not alter eEMG response, suggesting that transdermal stimulation of the EAS pudendal innervation did not occur.
Conclusions and Clinical Relevance—In cattle, cardiac effects and muscle weakness or loss of EAS eEMG response induced by larkspur-derived MSAL-type alkaloids were reversed by neostigmine or physostigmine, respectively. Treatment with anticholinesterase inhibitors may alter the clinical effects of larkspur poisoning in cattle.
Objective—To evaluate the safety and efficacy of an ocular insert designed to provide controlled release of apomorphine for the induction of emesis in dogs.
Animals—5,001 dogs treated with ocular apomorphine inserts and 32 dogs treated with IV administration of apomorphine.
Procedures—Data collected on a case report form included breed, body weight, time to emesis after placement of the insert, and any information available regarding the nature of the toxicosis and clinical signs. A list of potential adverse effects was provided, and attending clinicians graded their occurrence by use of a subjective scale. Similar report forms were used for dogs that received apomorphine IV. Treatment was considered successful if emesis occurred within 15 minutes of administration. Safety was assessed by evaluation of the frequency and severity of adverse effects.
Results—For the ocular insert and IV injection groups, the success rates were 83.5% and 90.6% respectively, and were not significantly different. Adverse effects were more frequent in the IV group, whereas ocular irritation was most frequent in the insert group.
Conclusions and Clinical Relevance—Overall, the ocular inserts provided an alternative to parenteral administration of apomorphine with comparable efficacy and a lower prevalence of adverse effects.