Felix A. de la Iglesia

Juvenile canine drug-induced arthropathy: Clinicopathological studies on articular lesions caused by oxolinic and pipemidic acids

Abstract Oxolinic acid and pipemidic acid were administered orally to young beagle dogs for 14 days. Dogs receiving oxolinic acid were hyperactive throughout the study and showed transient hindlimb stiffness on Day 12. In contrast, dogs treated with pipemidic acid exhibited marked suppression of spontaneous activity and by Day 4 were unable to stand and ambulated painfully. Both compounds induced gross alterations of the articular cartilage in major synovial joints, including solitary or multiple vesicles and erosions. Histopathologically, these changes were characterized by cartilage matrix rarefaction, cavitations, cartilage fibrillation, and chondrocyte clustering predominantly in the intermediate zone. Hemorrhagic synovitis was a coincident finding in pipemidic-acid-treated dogs. Using lesion incidence and a scoring system which provided an overall estimation of both gross and microscopic findings, the arthropathic index was higher in dogs receiving pipemidic acid, thus demonstrating the greater arthropathogenic potential of this chemical. Lowered serum alkaline phosphatase values paralleled the arthropathies in each group.

Teratogenesis of Calcium Valproate in Rats

Studies were conducted to determine the teratogenic potential of the calcium salt of valproic acid in rats when given orally at doses of 600, 150, and 50 mg/kg on days 6--15 of gestation. The sodium salt of valproic acid was used as a reference agent at a dose level of 600 mg/kg. The administration of 600 mg/kg/day of either calcium or sodium valproate resulted in transient, severe sedation in the dams. Four dams receiving 600 mg/kg of either salt died during the experiment, with deaths occurring between day 7 and 11 of gestation. Food consumption and body weight gain were significantly reduced during the dosing period with both salts at dose levels of 600 mg/kg. Embryotoxicity at the high doses (600 mg/kg) with either salt was manifested by increases in fetal resorption, reduced body weights, and significantly increased incidence of supernumerary ribs and bifid vertebral centra among the surviving fetuses. A teratogenic effect was evident at 600 mg/kg with either salt of valproic acid. Seven of 16 fetuses from dams given the calcium salt were abnormal. Findings included one with omphalocele and six others with skeletal malformations. Eleven of 24 fetuses from dams given the sodium salt were abnormal: three littermates had bilateral ectrodactyly of the rear feet and malformed vertebral centra and eight others had skeletal malformations. No teratogenic effect was evident among the fetuses from dams given 150 mg/kg calcium salt. Embryotoxicity was demonstrated by a significant increase in the incidence of supernumerary ribs. No adverse effect was observed among the fetuses from dams given 50 mg/kg of the calcium salt.

Immunotoxicologic studies with CI-959, a novel benzothiophene cell activation inhibitor

CI-959 is an orally effective inhibitor of cellular activation in both in vitro and animal models. To assess the effects of CI-959 on immune function, male Fischer 344 rats were evaluated for splenic T- and B-lymphocyte populations, antibody-forming cell response to sheep red blood cells (sRBC), concanavalin A and pokeweed mitogen-induced lymphocyte proliferation, Natural Killer cell activity, and reticuloendothelial system clearance of sRBC. Host resistance was measured in female B6C3F1 mice using Listeria monocytogenes, Streptococcus pneumonia, and B16F10 melanoma models. CI-959 was administered to both species of rodents at 25, 50, and 75 mg/kg/day for 14 days. A vehicle control and two positive controls (cyclophosphamide and dexamethasone) were run concurrently. CI-959 generally did not suppress immunological responses in rats at doses lower than those which also altered body weight gain and reduced spleen and thymus weights. Natural Killer cell activity was significantly reduced at 50 and 75 mg/kg CI-959. At 75 mg/kg rats also exhibited a reduction in ability to make anti-sRBC antibody. The number of T- and B-lymphocytes, proliferative response to mitogens, and macrophage activity of the reticuloendothelial system were not affected by CI-959. CI-959 also did not alter resistance of mice to Listeria monocytogenes, Streptococcus pneumoniae, or B16F10 melanoma cells. Based on these ex vivo and in vivo assays, the rodent immune system does not appear to be a sensitive or toxicologically important target for CI-959.

Pancreatic acinar cell neoplasia in male Wistar rats following 2 years of gabapentin exposure

Gabapentin, an anticonvulsant agent designated chemically as 1-(aminomethyl)-cyclohexaneacetic acid, was evaluated in a 2-year tumor bioassay in male Wistar rats. Three groups of 50 rats were fed gabapentin at 250, 1000 and 2000 mg/kg in the diet for 104 weeks. A fourth group was fed diet without drug. All rats were subjected to full histopathological evaluation. Body weight gain suppression occurred at 1000 and 2000 mg/kg. Survival was comparable across all groups. There was a treatment-related increase in the number of pancreatic acinar cell carcinomas; 0, 4, 3 and 8 of these carcinomas were observed in the control, 250, 1000 and 2000 mg/kg groups, respectively. There were no other increases in other tumor types, and there were no tumor increases in female rats. The frequency of pancreatic acinar cell hyperplasia was similar in treated and control groups. Biologically, the pancreatic carcinomas were not invasive, did not metastasize, were of late onset and did not compromise survival. Thus, gabapentin was a carcinogen in male Wistar rats. However, the tumorigenic response was of low-grade because it constituted a late tumor response which required very high doses. We reported recently that mice treated with gabapentin had no increase in pancreatic tumors. Therefore, neoplastic development was confined to the pancreas in a single sex and species of rodent. Consequently, gabapentin at therapeutic doses poses a low carcinogenic risk to humans.

Flow cytometric characterization of lymphocyte subpopulations in the cynomolgus monkey (Macaca fascicularis)

Characterization of immune cell subpopulations in the cynomolgus monkey was performed using a direct immunofluorescence technique adaptable for routine and repeated monitoring. This whole blood procedure is faster and requires less volume than conventional density gradient isolation methods. Low intra- and inter-animal variations were seen in hematology parameters and in CD4, CD8, and CD20 lymphocyte subtypes. CD4 values were 28% of lymphocytes in males and 30% in females. Fifty-six percent were CD8+ in males and 54% in females. CD4:CD8 ratios were approximately 0.5 in both sexes. This proportion is the reverse of that observed in humans, but appears normal for the cynomolgus. Consistent with values reported for humans, approximately 12% of cynomolgus peripheral blood lymphocytes were CD20+. Greater than 95% of the lymphocytes present in blood were identified as CD4, CD8, or CD20 positive.

Systemic Proliferative Changes and Clinical Signs in Cynomolgus Monkeys Administered a Recombinant Derivative of Human Epidermal Growth Factor

Epidermal growth factor (EGF) effects have been explored extensively in vivo in rodents, but little is known about trophic responses in nonhuman primates. A previous publication reports the hyperplastic epithelial/parenchymal changes noted in the digestive tract (tongue, esophagus, stomach, intestine, liver, gallbladder, pancreas, and salivary glands) of adult cynomolgus monkeys treated with recombinant human EGF1-48 (rhEGF1-48). This report documents clinical findings and structural effects in the remaining epithelium-containing tissues of these animals. Two monkeys/sex/dose received rhEGF1-48 by intravenous bolus at 0 (vehicle), 10, 100, 500 (females only), or 1,000 μg/kg/day (males only) daily for up to 2 weeks. Treatment- and dose-related clinical findings included emesis, fecal alterations (soft feces and diarrhea), lacrimation, nasal discharge, hypoactivity, transient hypotension, and salivation after dosing. Male monkeys administered 1000 μg/kg became moribund after 5 days of treatment and were necropsied. All other monkeys completed the 2-week treatment period. Necropsy findings in nongastrointestinal tissues were: enlarged, pale kidneys at 100 μ g/kg and greater; small thymuses seen sporadically at all doses; and enlarged adrenals and small thyroids in males at 1,000 μg/kg. Respective organ-to-brain weight ratios at 500 and 1,000 μg/kg for kidneys were 1.5- and 2.6-fold greater and for heart were 1.7- and 1.3-fold greater than controls. Microscopically, pronounced dose-related epithelial hypertrophy and hyperplasia were evident in kidney, urinary bladder, skin (epidermis and adnexa), mammary gland, prostate, seminal vesicles, epididymis, uterus, cervix, vagina, thyroid, thymus, tonsillar crypts, cornea, trachea, and pulmonary airways. Epitheliotrophic effects were conspicuous in many tissues at 100 to 1,000 μg/kg. Changes to renal collecting ducts were present at 10μg/kg, suggesting that kidneys were a relatively sensitive target. Proliferative alterations were not apparent in testes, intraocular structures, brain ependyma and choroid plexus at any dose. Aside from the noted exceptions, rhEGF1-48 was a pantrophic epithelial mitogen in cynomolgus monkeys when used intravenously at suprapharmacologic doses.

Monitoring simultaneous subcellular events in vitro by means of coherent multiprobe fluorescence

Monitoring simultaneous subcellular events in vitro by means of coherent multiprobe fluorescence

Cynomolgus monkeys (Macaca fascicularis) in preclinical immune function safety testing: development of a delayed-type hypersensitivity procedure

A delayed-type hypersensitivity (DTH) test commonly used for humans was adapted for use with cynomolgus monkeys (Macaca fascicularis). Pilot experiments showed naive animals had poor response rates and inconsistent reactivity to the antigens. In an exploratory phase, it was determined that monkeys could be experimentally sensitized by immunization with commercially available antigens. Animals were then sensitized with various concentrations of diphtheria and tetanus toxoids, Candida, and Trichophyton in the dose-response phase. Antigens were injected intradermally (i.d.) 3 times over a 7-day period and monkeys were tested 14 days after the last injection. Responses were measured 24, 48, and 72 h post-challenge, with skin biopsies taken from two animals per group at the 24 h interval. Optimal concentrations were 1.2 Lf diphtheria, 6 Lf tetanus, 1000 PNU Candida, and 1000 PNU Trichophyton. These concentrations produced the best balance between DTH responses, homogeneity of dermal mononuclear cell infiltrate and lowest frequency of undesirable skin reactions. Positive responses were seen at 24 and 48 h post-challenge and were waning by 72 h. DTH responses were inhibited by topical corticosteroids. The final phase of these studies assessed whether sensitization of naive animals could be achieved using subcutaneous (s.c.) administration of the optimal antigen concentrations. Comparable responses to i.d. sensitization were obtained and skin sores did not develop at injection sites. These studies show that the DTH test adapted to monkeys was reproducible, minimally invasive, did not require sacrifice of the test animal, allowed repeated measurements and paralleled the reactions observed in humans.

Elucidation of mitochondrial effects by tetrahydroaminoacridine (tacrine) in rat, dog, monkey and human hepatic parenchymal cells

Abstract Tetrahydroaminoacridine (tacrine) causes morphological and functional changes in the endoplasmic reticulum, ribosomes, and mitochondria in the liver of humans and animals. In order to investigate species differences as well as to understand the morphological changes, we examined the effects of tacrine on respiration and electron transport in mitochondria isolated from rat, dog, monkey, and human liver. Tacrine produced significantly decreased respiratory control ratios (RCR) in all species at concentrations ranging from 5 to 25 μg/ml. Human mitochondria were more sensitive to tacrine effects with RCR decreased 24% at 5 μg/ml while other species were unaffected at this concentration. The tacrine effects were characterized by increased hepatic mitochondrial State 4 respiration in rats and decreased State 3 respiration in humans. Mitochondria from aged rats were more sensitive to the effects of tacrine than mitochondria from young animals, with significantly decreased RCR at 10 μg/ml in aged rats while mitochondria from young rats were unaffected at this concentration. Concomitant with the respiratory changes, mitochondrial DNA synthesis was impaired. Since tacrine undergoes extensive biotransformation, we also explored the possibility that metabolites could exert detrimental effects. The ranking order of potency for decreasing RCR caused by monohydroxylated metabolites was: tacrine >4-OH and 7-OH >2-OH, 1-OH, and velnacrine with the latter group of metabolites having no effect on mitochondrial respiration at concentrations up to 50 μg/ml. In vivo administration of 20 mg/kg tacrine to rats for up to 20 days caused a paradoxical increase in RCR and P/O on Day 1 and decreased RCR on Days 9 and 20, the later findings being consistent with in vitro data. From these data we propose that tacrine does not necessarily have to be metabolized to exert effects on mitochondria at different sites in the electron transport chain that differ among species. These effects are exacerbated in mitochondria from older animals and humans appear to be more sensitive than the laboratory animals studied.

Bacterial and mammalian cell mutagenesis, sister-chromatid exchange, and mouse lung adenoma bioassay with the antineoplastic acridine derivative amsacrine

Amsacrine is a DNA intercalating agent with antineoplastic properties in lymphoproliferative disorders. This report describes a group of short-term tests with multiple endpoints to characterize the mutagenic and carcinogenic properties of this drug. In vitro studies included bacterial and mammalian cell mutagenesis, and sister-chromatid exchange and chromosome aberrations in mammalian cells. In vivo, mice were given amsacrine for 7 wk at 2, 5, and 10 mg/kg and were observed for an additional 17 wk. The standard bacterial assay revealed cytotoxicity at 2000 and 5000 micrograms/plate in the preincubation assay. No significant increase in revertants occurred in Salmonella strains, except for TA1537 in the activation phase. Amsacrine at 4.0 micrograms/ml was cytotoxic to V-79 cells in the cell mutation assay, and at lower dose levels was a direct-acting mutagen for the HGPRT locus. Sister-chromatid exchange rate of Chinese hamster ovary cells was increased more than twofold at 2 micrograms/ml without metabolic activation. Cell anomalies included changes in metaphase cell kinetics and chromosome damage. Mice in the lung adenoma bioassay failed to show increased numbers of tumors, while indicating lack of tolerance and survival beyond 5 mg/kg. The results indicate clear genotoxicity to mammalian cell systems with a spectrum of changes from point mutation and SCE induction to cell-cycle alterations, irrespective of exogenous metabolic activation. These results corroborate previous findings in animal and human cell systems in vitro. The reduction of genotoxicity in bacterial assays after exogenous metabolic activation may suggest some detoxification, and the magnitude of effects observed in mammalian cells indicates that exogenous metabolic activation is not required to manifest amsacrines activity. The lack of tumor-inducing potential in mice may be attributed to strong cytotoxic effects in this species, or to an insensitivity of the target organ, or to assay systems that may mask the carcinogenic potential.