Detrusor myopathy in young beagle dogs

Hematoxylin and eosin-stained sections of urinary bladder were examined microscopically from 449 male and female beagle dogs after 2- to 4-week toxicology studies. Degenerative lesions (detrusor myopathy) of the urinary bladder muscular tunic were present in 59 of 449 (13%) dogs. Myopathic lesions consisted of focal to multifocal areas of smooth muscle fiber atrophy with cytoplasmic basophilia and vacuolation, individual cell necrosis, enlarged smooth muscle nuclei and nucleoli, and occasional mitotic figures. Adjacent areas of arteritis and periarteritis were present in 10 of 59 (17%) beagles with detrusor myopathy suggesting a possible ischemic pathogenesis of the smooth muscle lesions. There was no significant difference in prevalence of myopathy in beagles administered vehicle or various test compounds. Prior urinary catheterization procedures appeared to augment the prevalence of myopathy; some lesions were adjacent to minor, iatrogenically traumatized urinary bladder mucosa. Muscle lesions were not observed in urinary bladders from mongrel dogs, monkeys, cats, rats, or microswine. When compared to crossbred dogs and other laboratory species, the beagle dog thus appears to be sensitive to development of detrusor myopathy.     

Uptake of dehydroascorbic acid in high-GSH and normal-GSH dog erythrocytes

Uptake of dehydroascorbic acid (DHA) was studied in two types of dog erythrocytes with high GSH and normal GSH levels. Compared with ascorbic acid uptake, DHA produced a much greater ascorbic acid accumulation in dog erythrocytes. Both dog erythrocytes showed a concentration dependence of DHA uptake, and cellular ascorbic acid concentrations were significantly higher in high-GSH cells than in normal-GSH cells. Glucose and cytochalasin B inhibited DHA uptake. This suggests that DHA enters dog erythrocytes predominantly by the facilitated glucose transporter, particularly by the Glut 1 glucose transporter. The rate of glucose uptake was quite similar in the two types of cells. Compared with normal-GSH cells, high-GSH cells were more resistant to oxidative stress induced by high concentration of DHA. As a rapid entry of DHA inflicts on cells a heavy demand for GSH for its reduction to ascorbic acid, high-GSH cells containing a larger reserve of GSH have an advantage over normal-GSH cells in both ascorbic acid accumulation and resisting oxidative stress produced by DHA.     

Rheological properties of young and aged human erythrocytes

Summary Rheological properties were studied of young and old human erythrocytes from healthy adults. Viscosity measurements of packed erythrocyte suspensions as well as filtration of cells through polycarbonate sieves show that young cells are more flexible than aged ones. Since deformability of erythrocytes is the product of cell shape, flexibility of the membrane and fluidity of the intracellular hemoglobin, we studied the manner in which these factors are relevant to the diminished flexibility of aged erythrocytes. The biconcave cell shape is maintained during the process of aging. The viscosity of packed ghost suspensions from aged erythrocytes is increased versus that of young ones. The diminished flexibility of old ghosts correlates well with their smaller cell volume. The fluidity of the hemoglobin in the interior of the cells is decreased as indicated by an increased hemoglobin content of the isolated ghosts. We conclude that aged erythrocytes loose their deformability as a result of both a decreased fluidity of the intracellular hemoglobin and a diminished flexibility of the membrane.This work was supported by the Deutsche Forschungsgemeinschaft     

Distinct mechanisms of transport of ascorbic acid and dehydroascorbic acid in intestinal epithelial cells (IEC-6)

Ascorbic acid (AsA) is an essential nutrient for humans as they lack its biosynthesizing key enzyme. Its absorption mechanism in small intestinal epithelial cells still remains to be resolved. In this study, the transport mechanisms functioning on the uptake of AsA and its oxidized form, dehydroascorbic acid (DHA), were investigated using rat small intestinal epithelial cell line IEC-6. Both AsA and DHA were accumulated in the cells in time- and concentration-dependent manners, but their absorption kinetics were apparently different. The saturability of AsA uptake was shown at a considerably lower concentration in IEC-6 cells as well as other mammalian cells, indicating that this absorption was mediated by a specific transporting carrier. The absorption efficiency of AsA was about 1/5-1/10 that of DHA at the same concentration range and, moreover, the uptake of DHA was almost comparable to that of 2-deoxy-D-glucose, an alternative of glucose. The uptake of AsA was diminished by the removal of sodium ion, but not by the addition of glucose, whereas that of DHA was sodium ion-independent and effectively inhibited by glucose. In addition, phlorizin and cytochalasin B, which are blockers of glucose transporters, interfered the uptake of DHA more efficiently than that of AsA. These results indicate that there are at least two distinct transport systems of vitamin C in rat small intestinal epithelial cells; AsA is transported by a specific transporter and DHA is mainly transported by glucose transporter(s).     

Drug-induced protoporphyria in beagle dogs

As part of regulatory safety testing program, a 13-week oral toxicity study with a new antipsychotic drug candidate was performed in beagle dogs. During this study, dark red/brown feces were recorded in treated dogs and increases in liver parameters (alanine aminotransferase, alkaline phosphatase, bilirubin) were measured biochemically. At the end of the study, livers of high-dose (50 mg/kg) animals were (mottled) dark brown, sometimes with pale foci. Histopathological examination of these livers showed dark globular pigment deposits in the hepatocellular cytoplasm and within the bile canaliculi. Varying numbers of inflammatory cell infiltrates were additionally present in association with the deposits. These pigment deposits showed birefringency with characteristic "Maltese Cross"-like structures under polarized light. Electronmicroscopy revealed the typical, so-called "sunburst" pattern with radiating double-lined crystalline structures. These morphologic characteristics strongly indicated at the presence of porphyrins, which was definitely confirmed biochemically. Published reports of drug-induced hepatic porphyria in dogs are rare. The possible underlying mechanism in the dog and man is discussed.     

Aging and aortic impedance in beagle dogs

Previous studies of the effect of aging on aortic impedance have proved inconclusive. Studies in man could be affected by clinically inapparent atherosclerotic effects which cannot be separated from aging effects. Therefore, to more directly examine the role of aging, hemodynamic studies were carried out in awake, resting young and senescent non-atherosclerotic beagles before and after the administration of various vasoactive agents. On separate days graded infusions of a rapid acting beta-adrenergic agonist and antagonist (isoproterenol and propranolol), and alpha-adrenergic agonist and antagonist (phenylephrine and phenoxybenzamine), combined alpha- and beta-adrenergic antagonists (propranolol plus phenoxybenzamine), and a non-specific vasodilator (nitroprusside) were administered. Aortic impedance was calculated from high-fidelity pressure and flow signals using standard Fourier analysis methods. Under baseline conditions no age-associated differences in any of the hemodynamic parameters were determined to be likely. The only significant age-associated difference in response was to graded doses of isoproterenol wherein there was a larger increase in flow and a greater decrease in resistance in the young compared to the senescent dogs. There were no age-associated differences in response to any of the other drugs. Thus, these data are further evidence in an intact circulation for an age-associated decrease in another beta-adrenergically mediated response.     

Spontaneous and age-related testicular findings in beagle dogs

This study was conducted to characterize spontaneous testicular and epididymal microscopic findings in eighty control beagle dogs from toxicity studies. Hypospermatogenesis, characterized by randomly scattered missing spermatids and/or spermatocytes within seminiferous tubules, was observed in 75% of dogs six to seven months of age and declined to fewer than 10% in dogs over eleven months of age. Atrophy/hypoplasia of seminiferous tubules, characterized by subcapsular triangular clusters of tubules containing no germ cells, was observed in 25 to 40% of dogs under twelve months old, decreasing with age to 14 to 17% in dogs twelve to thirty-six months old. Retained spermatids, multinucleate giant cells, intracytoplasmic vacuoles (presumably in Sertoli cells), and swollen spermatocytes were common findings of minimal severity. Six- and seven-month-old dogs had lower testicular weights, less filling of the epididymal tails with sperm, and a two-fold higher incidence of abnormal epididymal content compared to dogs more than eight months of age. Most male beagles were histologically sexually mature by eight to nine months of age. This study confirms published reports that dogs at least ten months of age at necropsy usually are adequate for routine microscopic evaluation of the testes. If evaluation of spermatogenesis is critical, the incidental findings can be minimized by using males over twelve months of age.     

Spontaneous testicular lesions in purpose-bred beagle dogs

Spontaneous testicular lesions were assessed in 50 control purpose-bred male beagle dogs. They were selected from 13 toxicology studies conducted over the period 1988-1999. Age of the dogs at study termination varied from 8-20 months with an average age of 13 months. Regardless of age, the most significant finding was bilateral segmental hypospermatogenesis in 15/50 (30%) of the dogs. Cross sections of tubules with hypospermatogenesis were distributed randomly throughout the testes and were characterized by reduced proportions of germ cells, tubular shrinkage, and Sertoli cell prominence. These changes were occasionally associated with giant cells, with cellular debris, and in 6/15 (40%) with atrophic tubules devoid of germ cells, indicating a degenerative process. Focal subcapsular tubular atrophy or hypoplasia (tubules lined by Sertoli cells only) was also found in 9/35 (26%) of dogs without hypospermatogenesis. Inhibited spermiation with retention of mature sperm in tubules was seen in 6/50 dogs, 3 of which also showed hypospermatogenesis. Other findings of high incidence but low prevalence included tubules with multinucleated giant cells, swollen spermatocytes, or apoptotic germ cells. These latter changes are probably a constituent of normal spermatogenesis. In conclusion, about 30% of control beagle dogs show segmental hypospermatogenesis, which may be associated with degenerative changes, and an additional 18% of the dogs exhibit focal tubular atrophy/hypoplasia in otherwise normal testes. These changes have to be distinguished from compound-related toxic effects.     

p-Aminobenzoic acid transport by normal and Plasmodium falciparum-infected erythrocytes.

De novo folate biosynthesis is required for the growth of malarial parasites and is inhibited by several important antimalarial agents. We show here that exogenous p-aminobenzoic acid (pABA) can be utilized by malaria parasites to synthesize folates. The transport of pABA into parasite infected red cells was therefore characterized. Normal red cells transport pABA in a saturable and energy-dependent manner, with a dissociation constant of 83 nM. pABA transport in parasite-infected red cells may use the same mechanism, as demonstrated by similarities in time course, concentration-response, and dissociation constant (111 nM). The transport capacity of red cells is temperature-, energy- and pH-dependent. It is inhibited by the proton ionophore, carbonylcyanide m-chlorophenylhydrazone (CCCP), but not by the sodium ionophores nigericin and monensin. p-Aminosalicylic acid (PAS) inhibits pABA transport competitively, with a inhibition constant of 378 nM. Phloritin, flufanamic acid, and 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DITS), which are inhibitors of the anion transporter (band 3), and oxalic acid, a substrate of this transporter, partially inhibit pABA transport into both normal and infected red cells. Interestingly, in both normal and infected red cells, the inhibitory effects of PAS and the anion transport inhibitors are additive, suggesting the involvement of 2 independent mechanisms.     

Experimentally induced malignant hypertension in beagle dogs

The pathology of malignant hypertension in dogs induced either purposely or inadvertently by the Goldblatt procedure has not been previously reported. Malignant hypertension was experimentally produced in beagle dogs by a modified Goldblatt procedure; in a single surgical procedure, one kidney was removed and the blood flow to the remaining kidney was reduced by 50%. A sudden onset of severe clinical signs developed within one to three weeks after surgery. The dogs were markedly depressed or in shock, were vomiting, and had either bloody feces or bloody diarrhea. Hematologic changes compatible with a diagnosis of microangiopathic hemolytic anemia consisted of hemolysis, thrombocytopenia, and the presence of burr cells and schistocytes. Some dogs had neutrophilia and slight to moderate increases in blood urea nitrogen and creatinine. At necropsy, there were gross hemorrhages in the heart, brain, urinary bladder, and gastrointestinal tract. Histologic findings consisted of multifocal parenchymal hemorrhage, fibrinoid necrosis of arterioles, medial smooth muscle hyperplasia, adventitial fibroplasia and mononuclear cell infiltrates, and microthrombi. The vascular clamp most likely protected the kidney from the systemic hypertension since the remaining kidney was largely not remarkable by light or electron microscopy. The dog appears to be a good model to study the pathology of malignant hypertension and microangiopathic hemolytic anemia.     

Chronic effects of flutamide in male beagle dogs

Flutamide, a potent nonsteroidal antiandrogen, was administered orally to male beagle dogs for 2, 3, or 4 years at doses of 10, 20, or 40 mg/kg/day. At each study interval, the results of clinical pathology examinations, organ weight determinations, necropsy, and histopathologic examinations generally were similar and included atrophy of the prostate gland, testicular interstitial cell hyperplasia, and seminiferous tubular atrophy and degeneration. After 3 years of drug exposure, there were 3 dogs with testicular interstitial cell adenomas and a few dogs with 1 or more enlarged mammary gland nipples. Based upon the pharmacologic activity of flutamide, these findings were expected and considered the consequence of long-term blocking of testosterone receptors and an exaggerated compensatory response to increased secretion of luteinizing hormone. The findings of this study were consistent with other examples of dysregulated hormone stimulation of target tissues noted during the nonclinical safety assessment of flutamide. In consideration of the clinical indication of flutamide for advanced prostatic carcinoma and based upon reports of minimal flutamide-related adverse clinical responses, the findings of this study pose no concern for human risk assessment.     

Ionic transport in individual renal epithelial cells from adult and young rats

Renal epithelial cells were isolated from the outermost superficial cortex of adult and young rats. The cells, likely of proximal origin, were plated on silicon pieces, and cultured during 1-3 days. Intracellular content and concentrations of K, Na, Cl, and P, and the kinetics of change in intracellular content, after inhibition of Na-K ATPase by incubation with ouabain or in K-free medium, were measured in individual cells in small populations using electron probe analysis. In control medium, concentrations in mM were approximately: K, 130; Na, 15; Cl, 28; P, 140. After 6 h inhibition of Na-K ATPase, cells exchanged all K for Na, and the intracellular Na concentration increased to 139 mM in K-free medium. The Cl concentration increased at most to 46 mM. The sum of intracellular K + Na + Cl did not increase more than 25% after 24 h incubation in K-free medium. There were no differences in intracellular K, Na, and Cl for adult and young rat cells in similar conditions. The half-times of K efflux and Na influx after inhibition of Na-K ATPase measured in adult rat were approximately 16-20 min. In the absence of serum, in K-free medium, the half-times of K efflux and Na influx in young rat cells were approximately 30 min, significantly higher than the half-time in the presence of serum, and with ouabain, being approximately 13 min. Histograms of distributions of K and Na content showed that the cells behaved as a single functional population. Ouabain Ki was estimated to be 10(-4) M. After 24 h preincubation in K-free medium, when returned in 5 mM K-containing medium, adult rat cells recovered rapidly normal intracellular K and Na concentrations. Using this approach, expression of the kinetics ionic transport properties of renal epithelial cells during development, and the hormonal influences on terminal differentiation may be studied.     

Renal hemodynamics and proteinuria in running and swimming beagle dogs

Summary In beagle dogs swimming, in contrast to treadmill running, was found to cause an increase in urine flow and urinary protein excretion. Renal blood flow measured by electromagnetic flow probes decreased by 13.0±4.9% when the treadmill gradient was 15% and arterial pressure was elevated by 11.6±4.9%. Immersion resulted in an immediate decrease in renal blood flow of 8.8±5.1% and a 24.6±6.9% increase in arterial pressure. Acid-base status indicated a respiratory alkalosis in all running experiments, no net change in five swimming experiments in which hyperventilation occured, but a metabolic acidosis in eight swimming experiments without hyperventilation. During running there was a threefold increase in oxygen consumption. We conclude that swimming possibly induces more sympathetic nervous activity than treadmill running in dogs, while an alkalosis is consistently present during running, but acid-base response is variable during swimming.     

Comparative aspects of contraceptive steroids--effects observed in beagle dogs

The effects of oral contraceptives have been studied in the beagle bitch for periods up to 7 yr. High doses of these potent estrogen: progestogen (E:P) combinations have been shown to promote tumors in the mammary glands, smooth muscle of the tubular genitalia, and occasionally in the transitional epithelium of the neck/trigone area of the urinary bladder. The contraceptive formulations used in humans are balanced with an E:P ratio of about 1:5 to 1:80 to produce a desired decidual response in the uterus. The corresponding ratio for producing the decidual reaction in the dog is 1:1,000 to 1:3,000 with the result that the dog is grossly overdosed with estrogens when given the human formulation at the usual multiples of up to 25 times the human dose. Smooth muscle tumors of the tubular reproductive tract are common sequelae to estrogen overstimulation in the dog and are known to occur in other species, including the humans. The dog also has major differences in hormonal control and sensitivity when compared to humans. Progestogens stimulate synthesis and release of growth hormone (GH) in dogs which in turn is the major stimulant (with progestogens) of mammary growth and tumors. Evidence is accumulating which indicates that most if not all progestogens can produce mammary tumors in the dog if given by the correct route and at high enough dosage. In contrast, GH in humans is not increased nor does it have any significant mammotrophic role. Mammary tumors in dogs related to oral contraceptives are now widely considered to be irrelevant as a model or predictor for human tumors. Transitional cell tumors in the urinary bladder seem to be a species specific phenomenon seen on occasion in the dog, but not in the rat, monkey, or human. The usual location in the neck/trigone area may be related to the embryologic origin of this portion of the bladder, which derives from tissues more closely related to the genital organs than does the rest of the bladder.     

Renaut bodies in the sciatic nerve of beagle dogs

182 control Beagle dogs from 23 historical studies (14 chronic, 9 subchronic) were reviewed histologically for the presence of Renaut bodies in the sciatic nerve. Renaut bodies were found in 36.1 percent of the subchronic-study dogs and in 46.4 percent of the chronic-study dogs. The Renaut bodies most often resided in the distal sections of the sciatic nerve, specifically in the tibial branch as it traversed the knee joint in situ. There was no sex predilection. Renaut bodies were located predominately in the endoneurium, in the center of the nerve sections. There was no associated axonal degeneration, reactive gliosis, or encapsulation. The Renaut bodies were characterized as large (20 to 500 microns diameter in cross section), well-demarcated elliptical structures with an onion-skin arrangement of loosely textured, filamentous strands intermixed with sparse numbers of dark spindle-shaped nuclei. Occasionally the core displayed a more dense, intensely eosinophilic arrangement of fibers. Histochemical results included: positive acidic alcian blue, Gomori's trichrome, and Verhoeff Van Gieson's; and negative Periodic-acid Schiff, Congo Red, and Luxol fast blue/cresyl violet. Immunohistochemical results included: positive vimentin and collagen (subtypes I, II, and VI); and negative NSE, S-100, GFAP, amyloid A component, desmin, alpha-sarcomeric actin, pancytokeratin, EMA, and von Willebrand factor. Transmission electron microscopy revealed loosely arrayed, circumferentially oriented collagen fibers intermixed with varying amounts of amorphous substance and finely fibrillar material. Most of the cells comprising the Renaut body were identified as fibroblasts. No nerve fibers entered or left the Renaut body, and nearby nerves appeared to be normal structurally. Based on this characterization of Renaut bodies and in conjunction with the past literature, Renaut bodies appear to have little or no pathological significance, but rather are suggestive of a physiological adaptation in response to mechanical stress imposed on nerves.