Heterocyclic ureas: inhibitors of acyl-CoA:cholesterol O-acyltransferase as hypocholesterolemic agents.

A series of diaryl-substituted heterocyclic ureas was prepared, and their ability to inhibit acyl-CoA: cholesterol O-acyltransferase (ACAT) in vitro and to lower plasma total cholesterol in cholesterol-fed animal models in vivo was examined. N-(2,6-Diisopropylphenyl)-N'-tetrazole or isoxazole-substituted heterocyclic ureas proved optimal. A carbon chain of 11-14 carbons substituted 1,3 with respect to the amine provided the optimal side chain. Substitution of the alkyl chain generally lowered activity. Tetrazole urea 2i dosed at 3 mg/kg lowered plasma total cholesterol (TC) 67% in an acute, cholesterol-fed (C-fed) rat model of hypercholesterolemia and 47% in C-fed dogs. Tetrazole 2i, dosed at 10 mg/kg, also lowered TC 52% and raised HDL cholesterol 113% in rats with pre-established hypercholesterolemia.

Toxicologic effects of a novel acyl-CoA:cholesterol acyltransferase inhibitor in cynomolgus monkeys.

PD 132301-2, an acyl-CoA: cholesterol acyltransferase (ACAT) inhibitor, was administered orally to cynomolgus monkeys for 2 wk at doses of 25, 50, 100, and 200 mg/kg to assess potential subacute toxicity. Sporadic episodes of soft feces and diarrhea increased in incidence from 100 to 200 mg/kg. Histopathologic alterations in adrenocortical cells of treated monkeys consisted of a dose-related decrease in cytoplasmic fine vacuolation and an increase in cytoplasmic eosinophilia most conspicuous in the zona fasciculata and reticularis. At 50, 100, and 200 mg/kg, a narrow discontinuous zone of cytotoxic cortical cell degeneration occurred in the outer zona fasciculata. Decreased fine vacuolation of cortical cells correlated ultrastructurally with reduced size and number of intracellular lipid vacuoles and biochemically with a dose-related decrease in adrenal total cholesterol (from 56 to 13% of control) and cholesteryl ester (from 51 to 3% of control) concentrations. Other ultrastructural changes noted in zona fasciculata cortical cells at all doses were an apparent increase in both smooth endoplasmic reticulum and variably sized autophagic vacuoles. Ovarian corpora lutea in some females at all doses had increased coarse vacuolation of luteal cells, foci of cellular degeneration, increased numbers of cholesterol clefts, and slight infiltrates of mononuclear cells. Sebaceous glands were atrophic in all treated monkeys due largely to a reduction in size and number of differentiated foam cells. Sebaceous gland reserve cells were hypertrophic and hyperplastic. Toxicity data from this study indicated that PD 132301-2 at 25-200 mg/kg targeted cholesterol-rich cells of the adrenals, ovaries, and skin adnexa.

Renal morphology and function in dogs after treatment with the angiotensin-converting enzyme inhibitor quinapril.

Angiotensin-converting enzyme (ACE) inhibitors have proven to be effective therapeutic agents for treatment of hypertension and congestive heart failure (CHF). Because of the role the renin-angiotensin system (RAS) plays in maintaining renal homeostasis, the effect these compounds have on renal function has been of interest. We assessed the effect of toxicologically significant doses of the new ACE inhibitor, quinapril, on renal function and morphology in dogs. Groups of 3 male beagle dogs were administered quinapril orally at daily doses of 0, 25, 125, or 250 mg/kg for 13 weeks. After treatment, animals were anesthetized and assessed for clinical pathologic and renal functional disturbances under normal conditions and after volume expansion and diuresis. Renal histopathology was conducted on perfusion-fixed kidney. No adverse effects on sensitive measures of renal function were detected; changes observed were consistent with the pharmacologic consequences of ACE inhibition. Decreased serum Na+ and Cl- (< 10%) and hematocrit at 125 and 250 mg/kg, twofold increases in serum creatinine and blood urea nitrogen (BUN) at 250 mg/kg, and decreased arterial blood pressure (BP) (20%) were observed at all doses. Under baseline conditions, urine flow increased 81-123% in quinapril-treated animals as compared with controls and urine specific gravities decreased 16% relative to controls at 125 and 250 mg/kg. Microscopically, juxtaglomerular hypertrophy was observed at all doses. At 250 mg/kg, minimal, widely scattered cortical tubular alterations were observed; glomerular lesions were not. No significant adverse effects of quinapril on renal morphology or function were observed at doses approximately 250 times the therapeutic dose.

Comparative nephrotoxicity of a novel platinum compound, cisplatin, and carboplatin in male Wistar rats.

The nephrotoxicity of three platinum-containing antitumor agents was compared at doses that approximate the LD10 (cisplatin) or the LD50 (CI-973, carboplatin) doses. Male Wistar rats were administered single iv doses of 45 mg/kg CI-973, 6.5 mg/kg cisplatin, or 65 mg/kg carboplatin and observed for 4 days. Cisplatin treatment increased blood urea nitrogen (4x), creatinine (3x), glucose, and fractional electrolyte excretions, and decreased creatinine clearance by Day 4. These parameters were not significantly altered in CI-973- and carboplatin-treated animals. Cisplatin increased urinary excretion of LDH (six-fold), GGT (twofold), and NAG (twofold); CI-973 and carboplatin increased GGT excretion (approximately twofold). Cisplatin induced the following functional changes as a consequence of direct nephrotoxicity: decreases in GFR (84%), ERPF (97%), ERBF (96%), and ERTS (95%), and increases in FF (fivefold). Functional changes, attributed to prerenal effects of CI-973, included a decrease in ERPF (35%) and an increase in FF (48%). No changes were seen following carboplatin treatment. All cisplatin-treated rats had proximal tubular necrosis in the outer stripe of the outer medulla, extending multifocally into inner cortical medullary rays. No renal lesions were detected by light or electron microscopy in the control or CI-973- or carboplatin-treated rats. Cisplatin produced marked nephrotoxicity as determined by biochemical, functional, and histopathologic endpoints. CI-973 and carboplatin were significantly less nephrotoxic than cisplatin.

Subacute and subchronic toxicology studies of CI-986, a novel anti-inflammatory compound.

CI-986 (5-[3,5-bis(1,1-dimethylethyl)-4-hydroxyphenyl]-1,3,4-thiadiazole-2(3H)- thione-2-hydroxy-N,N,N-trimethylethanaminium salt) is a novel anti-inflammatory compound classified as a dual inhibitor of cyclooxygenase and 5-lipoxygenase. Studies were undertaken to characterize the preclinical toxicology of the compound. CI-986 was administered to rats for 2 weeks (0, 50, 250, 750, and 1500 mg/kg) or 13 weeks (0, 20, 250, 500, and 1000 mg/kg), dogs for 2 weeks (0, 50, 150, and 500 mg/kg) or 13 weeks (0, 20, 100, and 200 mg/kg), and to monkeys for 2 weeks (0, 50, 250, and 1000 mg/kg). No drug-related deaths resulted. Mild clinical signs of toxicity were noted in rats given doses of 250 mg/kg and above. Drug-related emesis and diarrhea were absent at the low dose in the dog and monkey but increased in incidence and severity at higher doses. Severe clinical signs in monkeys (emesis and diarrhea) necessitated the lowering of the top dose to 500 mg/kg/day (administered b.i.d.) during the second week of the monkey study. Slight decreases (< 23%) in serum protein and/or albumin were noted in all studies at the higher doses. A dose-related increase in alkaline phosphatase was noted in both dog studies, with no other drug-related effect on clinical pathology parameters. A gastric ulcer occurred in one rat administered 500 mg/kg CI-986 for 13 weeks. Gastrointestinal ulcers were not noted at any other dose in rats or at any dose in dogs or monkeys. A dose-related eosinophilia of glandular stomach submucosa was noted in rats after 2 and 13 weeks of drug administration but not in dogs or monkeys. In the 2-week rat study, mean combined sex plasma drug concentrations monitored 2 hr after dose on Day 14 were 0.59, 1.10, 2.64, and 3.43 micrograms/ml for the 50, 250, 750, and 1,500 mg/kg dose groups, respectively. In the 2-week dog studies, maximum plasma drug concentrations on Day 10 or Day 11 were achieved within 2 hr of dose with mean combined sex Cmax values of 0.73, 2.05, and 2.62 micrograms/ml for the 50, 250, and 750 mg/kg groups, respectively. Hepatic microsomal induction characterized by increased microsomal protein, increased microsomal cytochrome P450 content, and increased p-nitroanisole O-demethylation activity was noted in dogs and monkeys but not rats. CI-986 was well tolerated in rats and dogs at the doses employed and in monkeys at doses up to 500 mg/kg (b.i.d.).(ABSTRACT TRUNCATED AT 250 WORDS)

Subacute toxicity of a novel inhibitor of acyl-CoA: cholesterol acyltransferase in beagle dogs.

PD 132301-2 is a substituted urea hypolipidemic and antiatherosclerotic agent that is a potent inhibitor of acyl-CoA:cholesterol acyltransferase (ACAT). To determine its subacute toxicity, PD 132301-2 was administered orally to beagle dogs at 0, 6, 12, 25, 50, 200, 400, or 800 mg/kg/day for 2 weeks. Clinico-pathologic evaluations were completed on all dogs. Liver and adrenal total and esterified cholesterol concentrations, adrenocorticotrophic hormone (ACTH) responsiveness, and adrenal ultrastructure were determined at 0, 6, 12, and 25 mg/kg. At 12 mg/kg or greater, salivation, epiphora, conjunctivitis, emesis, anorexia or decreased food consumption, and soft to mucoid feces and/or diarrhea were noted. Suppression of ACTH response occurred by Day 6 at all doses. Adrenocortical degeneration and/or necrosis in zona fasciculata and reticularis was seen at all doses; adrenal free and esterified cholesterol were normal at 6 mg/kg and decreased at 12 and 25 mg/kg. Increases in serum alanine aminotransferase (2- to 15-fold), aspartate aminotransferase (2- to 12-fold), and alkaline phosphatase (2- to 7-fold) were noted at 50 mg/kg or greater. Periportal hepatocellular hypertrophy and hypereosinophilia occurred at 50 mg/kg or greater; hepatic cholesterol values were not significantly affected by treatment. Dose-dependent ultrastructural alterations in adrenocortical cells included decreased numbers of mitochondria and smooth endoplasmic reticulum profiles, qualitative and quantitative changes in lipid globules, and increased numbers of autolysosomes. PD 132301-2 or one of its metabolites has potent adrenocorticolytic properties and limited hepatotoxic properties by mechanism(s) that are likely independent of systemic ACAT inhibition.

Morphogenesis of a zone-specific adrenocortical cytotoxicity in guinea pigs administered PD 132301-2, an inhibitor of acyl-CoA:cholesterol acyltransferase.

PD 132301-2, a novel inhibitor of acyl-CoA:cholesterol acyltransferase, is adrenotoxic to several laboratory animal species. Morphogenesis of a zona fasciculata-specific cytotoxicity was evaluated in male Hartley guinea pigs administered 100 mg/kg of PD 132301-2 for up to 7 days. Reversibility of adrenal effects was assessed after a 14-day drug withdrawal period (day 21). Serum cortisol concentrations were determined under basal conditions and after administration of adrenocorticotrophic hormone (ACTH) on days 1, 2, 4, 7, and 21. Isolated foci of cortical cell degeneration and necrosis were apparent in outer zona fasciculata by 2 hr and throughout the zona fasciculata at 6 hr. Early degenerative ultrastructural changes included aggregation of smooth endoplasmic reticulum (SER), variable condensation of mitochondrial matrices and swelling of cristae, partitioning of organelles, autophagosome formation, and disruption of lipid globules. Lesions progressed to locally extensive or diffuse zonal necrosis on days 1 and 2 and near complete ablation of zona fasciculata by day 4. Fasciculata cells remaining on day 4 had reduced numbers and increased size of lipid globules, increased lysosomes, and, occasionally, aggregates of SER and mitochondria. On day 7, SER proliferation and lipid depletion were apparent in remaining cells. ACTH responses were attenuated 24 hr after the first dose, and reduction in basal cortisol levels were seen by 24 hr after the second dose with both effects maximal on day 7. After a 14-day withdrawal period, ACTH responses and adrenal morphology returned to normal. It was concluded that PD 132301-2 induced rapid, reversible, zone-specific, morphologic, and functional adrenocortical effects. Furthermore, mitochondria and SER represented early subcellular targets of toxicity.

ATP depletion is associated with cytotoxicity of a novel lipid regulator in guinea pig adrenocortical cells.

A novel lipid regulator (PD132301-2) produces degeneration and necrosis of adrenal fasciculata in guinea pigs. Primary adrenocortical cell cultures from male Hartley guinea pigs were utilized to investigate potential mechanisms of this toxicity. Concentration-dependent loss of viability, measured by neutral red (NR) accumulation or MTT reduction, was observed within 6 hr at concentrations of 0.01 to 10 microM PD132301-2. At 10 microM, NR and MTT indices were 50% of those of control after 6 hr exposure. Maximal decreases in NR and MTT indices to 20% of control values occurred by 24 hr at > or = 1 microM PD132301-2. Adenine nucleotide analysis after PD132301-2 challenge indicated that ATP depletion preceded loss of viability. At 10 microM PD132301-2, ATP levels were 80% of those of control after 30 min and 25% of those of control after 6 hr. Supplementation of glucose-free buffer with 20 mM fructose protected adrenocortical cells from PD132301-2-induced toxicity. Fructose protection was blocked by inhibiting glycolysis with 1 mM sodium fluoride. Pretreatment of cultures with 100 microM metyrapone, an inhibitor of cytochrome P-450, did not block cytotoxicity induced by 10 microM PD132301-2, but did block cytotoxicity of 100 microM o,p'-DDD. In adrenocortical mitochondrial preparations, inhibition of respiration by PD132301-2 was site II-specific. Both state 3 and state 4 respiration were inhibited 50-75% at 1-30 microM PD132301-2. Thus, ATP depletion resulting from direct inhibition of mitochondrial respiration is a critical early event in adrenocortical cytotoxicity of PD132301-2.

Renal structure and function in rats after suprapharmacologic doses of quinapril, an angiotensin-converting enzyme inhibitor.

Angiotensin-converting enzyme (ACE) inhibitors have adverse effects on renal function in some hypertensive patients, and some of them produce renal tubular lesions in animals at high doses. To assess the effect of quinapril on renal function and structure, a 4-week time-course study was conducted in male Wistar rats with daily oral gavage doses of 0, 10, 100, or 400 mg/kg. Glomerular filtration rate (GFR) estimated as creatinine clearance and fractional electrolyte excretion values were derived from urinalysis and blood chemistry data obtained at days 1, 7, 14, and 28. Renal sections were collected on these days for histopathologic evaluation, and cortical slices were obtained to assess organic ion transport in vitro. Expected pharmacologic effects of an ACE inhibitor were observed at all doses and included increased urine output, increased water consumption, decreased serum aldosterone (65 or 25% of control at 10 or 400 mg/kg, respectively, on day 28), increased plasma renin activity (PRA, up to two- to threefold higher than controls at day 28), and hypertrophy of the juxtaglomerular apparatus. Despite these expected class effects, quinapril administration to male rats for 28 days produced no functional alterations or renal tubular lesions suggestive of renal toxicity at doses up to 400-fold higher than the effective antihypertensive dose in rats.

Subacute toxicity of a halogenated pyrrole hydroxymethylglutaryl-coenzyme A reductase inhibitor in Wistar rats.

Wistar rats received an hydroxymethylglutaryl-coenzyme A (HMG-CoA) reductase inhibitor, a halogenated pyrrole designated PD 123244-15, orally by gavage for 14 days at 10, 50, 150, 300, and 600 mg/kg. Doses of 150-600 mg/kg caused death and marked systemic toxicity involving stomach, esophagus, liver, gonads, lymphoid tissues, and skeletal muscle. Histopathologic findings included hyperkeratosis in esophagus and forestomach, increased hepatic mitotic activity, ovarian follicular necrosis, testicular atrophy and arrested spermatogenesis, and skeletal muscle necrosis and regeneration. Elevated serum aspartate aminotransferase correlated with muscle necrosis and hepatocellular damage. Marked systemic effects associated with high plasma concentrations were consistent with toxicity defined for other HMG-CoA reductase inhibitors, with the exception of pathologic alterations in the esophagus and ovaries. Direct mucosal irritation may have contributed to forestomach and esophageal lesions induced by this halogenated pyrrole.

Mesenteric lymph node hemangiomas of Wistar rats.

Vascular tumors in rodent mesenteric lymph nodes are uncommon. Fifty-seven of these neoplasms were identified in control and treated Wistar rats from 6 tumor bioassays. Tumor incidence ranged from 0.75% to 5.50% and was higher in males than females (2:1). Lesions, noted as incidental necropsy findings or in routine histologic sections, were typically solitary and restricted to nodal and perinodal tissue. Additional solitary vascular tumors were identified in skin of 3 rats and pararenal lymph node of 1 rat. Distinct metastases were not evident. When apparent grossly, affected nodes were red to purple, hemorrhagic, and/or enlarged. Histologically, all tumors were composed of variably sized, endothelial-cell-lined, blood-filled spaces separated by variable amounts of poorly cellular stroma. Nodal effacement was common in larger tumors. Approximately half of the tumors had features of typical cavernous hemangiomas. The remaining tumors had slightly more aggressive features consisting of single or multiple foci of lymph node capsule invasion, presence of tumor cells in muscular blood vessels, or cellular atypia with variable mitotic activity. Death due to tumor rupture and consequent hemoperitoneum occurred in 1 rat only.

Alpha 2u-globulin nephropathy without nephrocarcinogenesis in male Wistar rats administered 1-(aminomethyl)cyclohexaneacetic acid.

Alpha 2u-Globulin (alpha 2u) nephropathy is a male rat-specific condition caused by a diverse group of xenobiotics. Features of this nephropathy include hyaline droplet accumulation in proximal tubules, tubular epithelial necrosis and regeneration, exacerbation of spontaneous renal disease, and induction of renal epithelial tumors. Nephrocarcinogenicity of compounds that cause this nephropathy may be a consequence of increased proximal tubular proliferation resulting from cell injury. These studies document alpha 2u nephropathy without primary renal epithelial tumors in male Wistar rats administered 1-(aminomethyl)cyclohexaneacetic acid (gabapentin), a therapeutic agent with antiepileptic/anticonvulsant properties. In a series of preclinical studies gabapentin was administered to rats at the following doses and durations: 50 and 2000 mg/kg for 2 weeks; 250, 1000, 2000, and 3000 mg/kg for 13 weeks; 250, 1000, and 2000 mg/kg for 52 and 104 weeks. Renal effects were evaluated by biochemical, immunocytochemical, histopathologic, and ultrastructural techniques. Reversible increases in size and distribution of hyaline droplets within proximal tubular epithelium occurred through 1 year of treatment at a severity that was dose-dependent. In males given 2000 mg/kg, alpha 2u accumulation, degeneration, and necrosis of the P2 segment and intraluminal cellular casts were seen after 2 days of treatment. In the 2-week study, the size and number of phagolysosomes containing alpha 2u and the renal tissue alpha 2u increased with increasing dose and time. By Day 7, polymorphic crystalline inclusions were abundant in phagolysosomes of 2000 mg/kg males. In subchronic and chronic studies, spontaneous glomerulonephrosis was exacerbated in males given 2000 mg/kg, and, interestingly, no drug-related effect on renal tumor incidence was observed. To the best of our knowledge, this is the first documentation of the absence of nephrocarcinogenic effect in male rats treated for up to 104 weeks with a compound that causes acute and chronic lesions of alpha 2u nephropathy.

Acute cardiovascular toxicity induced by an adenosine agonist-antihypertensive in beagles.

An adenosine agonist, designated chemically as (R)-N-(2,3-dihydro-1H-inden- 1-yl) adenosine, or CI-947, was administered to 3 male and 3 female beagles in oral doses of 5 mg/kg body weight. Multiple episodes of arrhythmia were recorded electrocardiographically with Holter monitors in 2 males and 2 females monitored up to 48 hr. One male and 1 female were necropsied at 24 hr and the remaining dogs were necropsied at 48 hr post-dosing. At 48 hr, multifocal perivascular epicardial and myocardial hemorrhage was noted grossly in 1 female. Microscopic coronary arterial alterations were present in all treated dogs irrespective of the occurrence of arrhythmias. At 24 hr, proteinic material and red cells were present in the media accompanied by minimal adventitial accumulation of neutrophils. At 48 hr, coronary arterial lesions progressed to media vacuolation, transmural necrosis, and perivascular accumulation of neutrophils. Ultrastructural alterations included: endothelial retraction, subendothelial accumulation of fibrin and platelets, necrosis of smooth muscle cells, and mural infiltration of granulocytes and monocytes. Coronary vascular injury may be due to altered hemodynamics associated with the coronary vasodilator properties of adenosine agonist compounds.

Proliferative bone lesions in rats given anticancer compounds.

Proliferative endosteal lesions were observed in metaphysis and diaphysis of femur and sternebra of Wistar (CRL:[WI]BR) rats administered 3 chemically-distinct anticancer compounds with dissimilar mechanisms of action: trimetrexate glucuronate, an antifolate; pentostatin, an adenosine deaminase inhibitor; and CI-980, a mitotic inhibitor. Islands of woven bone, often circumscribed by conspicuous myelostromal proliferation, were seen on Days 8-28 in rats given trimetrexate glucuronate daily by gavage, and on Day 4 but not Day 29 in rats given a single intravenous dose of pentostatin. Intravenous administration of CI-980 for 1 or 5 days resulted in marrow necrosis, marked centripetal new bone formation, and myelostromal proliferation on Days 4 and 8, respectively. These lesions were not present at the termination of these latter studies (Days 29 and 35, respectively). In conclusion, anticancer compounds induced local bone marrow injury and the release of local inflammatory mediators which may have provided the stimulus for bone formation and myelostromal proliferation.

Ultrastructural juxtaglomerular cell changes in normotensive rats treated with quinapril, an inhibitor of angiotensin-converting enzyme.

Sequential histologic and ultrastructural changes in juxtaglomerular apparatus (JGA) were defined in male rats treated with quinapril, an inhibitor of angiotensin-converting enzyme (ACE). Doses of 0, 10, 100, and 400 mg/kg were administered daily by gavage for up to 4 weeks. Granular juxtaglomerular (JG) cells were normal or hypogranular on Day 1 at all doses and were hypergranular by Day 7 in rats given 100 and 400 mg/kg relative to age-matched controls. Histologically, JGA hypertrophy was apparent by Day 7 at all doses and was most pronounced by Day 14 in intermediate and deep cortical zones of rats given 100 and 400 mg/kg. Ultrastructurally, hypertrophic JG cells had abundant rough endoplasmic reticulum and free ribosomes, and prominent Golgi complexes associated with numerous cytoplasmic coated vesicles. Dose-dependent increases in numbers of protogranules, altered granules, and cytoplasmic vacuoles occurred in association with decreased size and increased pleomorphism of mature secretory granules. Granule alterations included vesicular to lamellar membranous matrical inclusions, irregular patterns of osmiophilia, matrical vacuolation, and flocculent to coarsely granular matrix of greater density than mature granules. We concluded that JG cell hypertrophy and hyperplasia occurred rapidly in response to subchronic ACE inhibition. Further, ultrastructural changes in JG cells were indicative of stimulated renin synthesis by a regulated pathway, renin secretion by exocytosis and cytoplasmic solubilization of granules, and autophagy of granules as a mechanism whereby JG cells regulate levels of stored renin under conditions of excessive stimulation.

Ketamine prevents ischemic neuronal injury.

The dissociative anesthetic ketamine hydrochloride antagonizes the excitotoxic action of excitatory amino acids in the central nervous system. Proposals that the excitatory amino acid neurotransmitters may become excitotoxic and contribute to the pathophysiology of ischemic brain injury prompted us to examine ketamine in a model of global cerebral ischemia in gerbils. Pretreatment with anesthetic doses of ketamine ameliorated in a dose-dependent manner both behavioral and histopathological assessments of ischemic neuronal injury. These neuroprotective effects are proposed to result from a specific antiexcitotoxic rather than general anticonvulsant drug action. There may be clinical situations in which the neuroprotective actions of ketamine would be of therapeutic importance.

Turbinate osteoporosis in pigs following intranasal inoculation of purified Pasteurella toxin: histomorphometric and ultrastructural studies.

Turbinate osteoporosis, induced by intranasal inoculation of purified toxin isolated from serotype D Pasteurella multocida, was investigated in 3- to 5-week-old, caesarean-derived, colostrum-deprived, isolation-reared pigs. Marked bilateral reduction in relative volume of trabecular bone occurred in osseous cores of turbinates of toxin-treated pigs relative to control pigs on post-inoculation day (p.i.d.) 3, 6, 9, 12, and 15. The fractional resorptive surface along turbinate bone was greater in toxin-treated pigs when compared to controls on p.i.d. 3 and 6. A significant decrease in resorptive surface occurred over time in toxin-treated pigs, whereas the fractional resorptive surface was constant over time in control pigs. Osteoclasts in medullary spaces separating bony trabeculae of turbinates were abundant in toxin-treated pigs and scant in controls on p.i.d. 3, 6, and 9. Degeneration and necrosis of bone forming cells, principally osteoblasts, were progressively more extensive with time and were associated with decreased mineralization and reduced thickness of osteoid and woven bone matrix. Osteoclasts along resorptive surfaces of turbinate bone in toxin-treated pigs had more abundant, more highly vacuolated cytoplasm, a more prominent microvillous border, and a greater number of nuclei per cell than osteoclasts from control pigs on p.i.d. 3 and 6. We conclude that this Pasteurella toxin stimulates osteoclastic osteolysis and inhibits osteogenesis in turbinates by causing degeneration and death of osteoblasts.

Turbinate atrophy in gnotobiotic pigs intranasally inoculated with protein toxin isolated from type D Pasteurella multocida.

Three doses (75 micrograms, 25 micrograms, and 25 micrograms) of purified toxin isolated from a toxigenic strain of type D Pasteurella multocida were given (by atomizer) into the right nasal cavities of each of 10 gnotobiotic pigs on the 21st, 24th, and 27th days of age, respectively. Inoculated pigs (usually 2) and 1 noninoculated control pig each were necropsied on 3, 6, 9, 12, and 15 days after inoculations were given. Severe bilateral atrophy of turbinates occurred in all toxin challenge-exposed pigs. Atrophy was more severe in the inoculated nasal cavity than that in the noninoculated side in 2 of the 10 pigs. Microscopic changes in turbinates of toxin challenge-exposed pigs were more severe in pigs killed at later dates. Dominant changes included degeneration and necrosis of osteoblasts, markedly accelerated osteoclastic osteolysis, replacement of the osseous core by a highly cellular mesenchymal stroma, and multifocal atrophy of submucosal glands. Seemingly, a protein toxin isolated from toxigenic type D strains of P multocida produced rapid atrophy of turbinates and may be a contributing factor in development of clinical progressive atrophic rhinitis in swine.