Acute phase response in toxicity studies. II. Findings in beagle dogs injected with endotoxin or subjected to surgical operation.

Occurrence of characteristic transient changes in WBC counts and fibrinogen values in beagle dogs subjected to single-dose toxicity studies was pointed out in the previous survey (Hoshiya et al., 2001). These changes were thought to belong to the category of "Acute Phase Response (APR)". The purpose of the present study is to compare the APR found in the single-dose toxicity studies surveyed in our previous report with those experimentally produced by intravenous injection of 1 microgram/kg endotoxin (Experiment 1), and surgical treatment (Experiment 2) (intravenous indwelling catheterization). The animals used in Experiment 2 were intravenously injected with 1 microgram/kg endotoxin 2 weeks after the operation (Experiment 3), and the results were compared with those of Experiments 1 and 2. Each experimental group consisted of 5 dogs, and clinical, hematological and blood chemical examinations were performed. Essentially the same changes were observed in response to the intravenous injection with endotoxin and the surgical operation for intravenous indwelling catheterization in beagle dogs. The most remarkable changes common to both treatments were transient increases in the fibrinogen values and WBC counts during the 2 days from Day 1 to Day 2 of the treatment. These changes were preceded by decreases in WBC counts and fibrinogen in Experiments 1 and 3. Increased erythrocyte sedimentation rates were recorded in parallel with the increase in fibrinogen. The results obtained in the present study were similar to those found in dogs treated with various xenobiotic substances in our laboratory. These changes due to different causes were thought to belong to the category of "APR" with the same biological significance as a non-specific defense mechanism.

Acute phase response in toxicity studies. I. Survey of beagle dogs subjected to single-dose toxicity studies.

In the field of routine single-dose toxicity studies, we occasionally meet with transient leukocytosis associated with an increase in fibrinogen in beagle dogs within a few days after treatment with the test article. Only a little is known, however, about the toxicological significance of these changes. However, these changes were thought to belong to the category of "Acute Phase Response, APR," which has been known for a long time in connection with injury, trauma or infection. Aiming at proper understanding of these experiences, we surveyed 25 single-dose toxicity studies (7 intravenous bolus, 5 intravenous infusion, 12 oral and 1 subcutaneous treatment, hereafter referred to simply as i.v. bolus, i.v. infusion, oral and s.c.) in beagle dogs, provided with data from hematological examinations. We set the following criteria as a positive response in the present survey: increases of 50% or more in either or both WBC or fibrinogen compared to the predosing value, transiently from Day 1 to Day 3 of the study. Among 25 studies surveyed, about 1/2 of the studies exhibited increases of 50% or more in either or both fibrinogen or WBC counts compared to the predosing values showing dose-dependency transiently on Day 1 or Day 2. These changes were remarkable after intravenous application. Oral application produced similar effects, although the incidence and severity were low compared to the i.v. routes. Regarding blood chemical and hematological changes other than changes in fibrinogen and WBC counts, there were no essential differences between the groups of studies with and without the changes in fibrinogen and WBC counts. These changes were thought to be characteristic and to have occurred as incidents unrelated to other changes. The reported changes seen in single-dose toxicity studies may belong to the category of APR as the non-specific mechanism of living bodies as stated by Burns et al. (1996).

Maternal placenta-like endometrial hyperplasia in a beagle dog (Canine deciduoma).

Three thumb-sized segmental swellings were found in both uterine horns of a 15 month-old, non-pregnant and non-pseudopregnant female beagle dog. Histopathological examination of the uterus revealed a marked proliferation of the endometrium that was characterized by two distinct parts, an inner tightly-folded banded layer and an outer dilated spongy layer, quite similar to the maternal placenta except for the fetus and chorion in the lumen. Because the plasma level of progesterone was unusually high at autopsy, this hormonal disorder might be related to the pathogenesis of the endometrial hyperplasia in the present case.

Postnatal development of the auditory brainstem response (ABR) in beagles.

Postnatal development of the auditory brainstem response (ABR) in beagles was studied by repeated ABR recordings from birth to 16 weeks of age. In ABR recording, waves I, II and III appeared at the same time 13 to 17 days of age, while wave IV during 13 to 20 days of age and wave V during 20 to 35 days of age, respectively. The latencies of waves II, III and IV became close to the control level at 6 weeks of age, whereas wave I had a similar latency to the control at 3 weeks of age. The latency of wave V became close to the control level at 8 weeks of age. I-III interpeak latency stabilized at 4 weeks of age, however, I-V and III-V interpeak latencies stabilized during 8 to 10 weeks of age. There was a marked individual difference in amplitude of ABR components in beagles. Wave I amplitude reached the plateau level at 3 weeks of age, and wave II, III and IV at 6 weeks of age. No further development in ABR was observed on and after 10 weeks of age. It may be concluded, from electrophysiological point of view, that the peripheral auditory pathway matures at 3 weeks of age and the auditory rely system in brainstem at 10 weeks of age in beagles.

Retinal dopamine and its metabolite contents in zitter rats and spontaneously epileptic rats.

In order to investigate the relationship between visual dysfunction and retinal DA metabolism in zitter rats and spontaneously epileptic rats (SER), we measured the amounts of retinal dopamine (DA) and its metabolites, 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillate (HVA). DA, DOPAC and HVA contents were markedly decreased in zitter rats as compared with the controls, Kyo:Wistar or Sprague-Dawley rats. In contrast, in SER, retinal DA and its metabolite contents were not significantly different from those of Kyo:Wistar rats. SER showed higher levels of DA and its metabolites in comparison with Sprague-Dawley rats. Thus, it is suggested that the debilitation of retinal DA synthesis is at least partly related to the visual dysfunction observed in zitter rats, but not that of SER.

Pathology of chemically induced chronic active hepatitis in mice.

Female Swiss mice were treated for 24 weeks, with 3-hydroxy-4-pyrone (Py) added to their powdered diet at 0.5% (wt/wt), and the effects of this agent on the liver were examined. Serum transaminases (especially GPT) rose continuously, while the GOT/GPT ratio remained at approximately 1.0 throughout the study period. The characteristic changes found from 8 weeks onward were piecemeal necrosis and bridging necrosis of the hepatocytes with dense lymphocytic infiltration. Proliferation of collagen fibers in the portal tracts and formation of narrow fibrous septa dividing the lobules into pseudolobules were also noted from 12 weeks onward. A large number of the infiltrating lymphocytes were identified as T cells by immunohistochemical and electron microscopic studies. These lymphocytes often surrounded or were closely attached to degenerating hepatocytes. Focal apoptosis and necrosis accompanied by a granulomatous reaction of the centrilobular hepatocytes were noted as early changes in the liver. Our findings indicate that the hepatic changes produced in mice by long-term Py administration have characteristics in common with those of human chronic active hepatitis. Immunological cytotoxic mechanisms, especially T cell-mediated ones, appear to play an essential role in the development of hepatic lesions in this murine model of chronic active hepatitis.

Postnatal behavioral development in methylazoxymethanol-induced microcephalic rats--a behavioral teratology study.

Behavioral testings in methylazoxymethanol (MAM)-induced microcephalic rats were conducted. Pregnant Sprague-Dawley rats were treated intraperitoneally with 0, 20 or 40 mg/kg of MAM once a day on day 14 of gestation and were allowed to delivery. Male pups from each litter were examined for open field test at 6 weeks of age and shuttle-box avoidance test at 7 weeks or more of age. In the open field activity of pups, the counts of ambulation and locomoting distance in 40 mg/kg group have increased significantly as compared with those in control group. In the shuttle-box avoidance test, the avoidance response rate was dose-dependently high in the session of the 1st day. As to the interaction between the avoidance response rate and sequence of sessions, however, the avoidance response rate in 40 mg/kg group was significantly low. Rate of the rats with errors and number of response during the intertrial interval was significantly high in 40 mg/kg group. Thus, we could demonstrate functional disturbance in the memory retaining ability in utero MAM-exposed rats.

Antigenic heterogeneity of sialodacryoadenitis virus isolates.

Seventeen field isolates of sialodacryoadenitis (SDA) virus had been isolated from the lung of rats with clinical SDA during epizootics of SDA from 1976 to 1978 in the research laboratory. Based on their neutralization patterns against antisera to strains 681, 930-10, Lu-3, Lu-4 and Lu-7, these isolates were divided into 3 antigenic groups. The first group consisted of 8 isolates which were neutralized by 4 out of 5 antisera at high serum dilution. The second group consisted of 6 isolates which were neutralized by only 2 antisera at high serum dilution. The third group exhibited intermediate neutralization pattern and 3 isolates belonged to it. Considering the time course of virus isolation, it was concluded that three antigenically different SDA viruses had been spread irregularly and ocassionally two had been spread simultaneously in an animal house of rats during the several epizootics of SDA.

Auditory brainstem response (ABR) in spontaneously epileptic rats (SER) and their parent mutants.

Postnatal development of auditory brainstem response (ABR) in spontaneously epileptic rats (SER; zi/zi, tm/tm), a double mutant, their parent mutants, tremor rats (tm/tm) and zitter rats (zi/zi), and Kyo:Wistar rats (control), was studied by repeated ABR recordings from 2 to 13 weeks of age. In tremor rats and Kyo:Wistar rats at 2 weeks of age, ABR pattern was almost the same and 2 or 3 waves were identified. However, no further development in ABR was observed in tremor rats and prolongation of latency and lowering of amplitude were marked. Only wave I with prolonged latency and lowered amplitude was recognized in zitter rats, SER and SER-N (zitter phenotype without epilepsy; zi/zi, tm/+ or zi/zi, +/+). ABR pattern was almost the same from 2 to 13 weeks of age in them. Electrocochleography revealed delay of N1 latency of compound action potential in SER. Vacuolation was observed in the cochlear nuclei and the brainstem of tremor rats and SER from 3 weeks of age in histopathological examination. The organ of Corti and cochlear nerve of tremor rats and SER exhibited no remarkable histopathological findings. The deafness in SER and its parent mutants was considered to occur as a result of impairment of inner ear, cochlear nerve and brainstem as well.

Pathological evaluation of anti-rheumatic drugs on type II collagen-induced arthritis in DBA/1J mouse.

The effects of anti-rheumatic drugs (lobenzarit (CCA); 10 and 50mg/kg, cyclophosphamide (CP); 5 mg/kg and dexamethasone (DM); 0.25mg/kg) were evaluated immunologically and histopathologically on DBA/1J mice that develop polyarthritis after immunization by the intradermal injection of type II collagen. Serum anti-type II collagen IgG levels in the groups treated with CP and DM were significantly suppressed to 1/2 and 1/10 as compared to those of the positive control group, respectively. Those of both groups treated with CCA were not different from those of the positive control group. Histopathological examination revealed that treatment with CP and DM markedly reduced or suppressed inflammatory changes and resulted in low incidence of arthritis. From the standpoint mentioned above, treatment with anti-rheumatic drugs suppressed the development of arthritis in this model, and we could confirm that this model was useful for evaluation of the effect of anti-rheumatic drugs.

The spontaneously epileptic rat (SER), a zitter*tremor double mutant rat: histopathological findings in the central nervous system.

The spontaneously epileptic rat (SER), a mutant homozygous for both zitter and tremor genes, exhibits absence-like seizures and tonic convulsions without external stimulation from 7 to 8 weeks of age. Histopathological studies of the central nervous system revealed the following abnormalities. The 35-day-old SERs which exhibit body tremor, and which have never shown seizures, had marked vacuolation and hypomyelination in the brainstem and cerebellum. The vacuoles were produced by splitting of the myelin sheaths and swelling of the dendrites and were related to primary swelling of the astrocytes. The 2- to 3-month-old SERs with staggering gait and seizures showed focal axonal swelling ('torpedo') and advanced vacuolation in the granular cell layer of the cerebellum in addition to the abnormalities observed at 35 days of age. Degenerative neurons and spheroidal bodies were observed in the substantia nigra and ventral tegmental nucleus. These brain areas are known to be related to tonic convulsions in the several experimental models. The SER is believed to be a useful tool for the investigation of the relationship between the structure and function of the central nervous system in epilepsy. It is probable that the more severe changes in the cerebellum are responsible for the staggering gait.

Behavioural characteristics of wild jumping or running episodes in the double mutant spontaneously epileptic rat.

Spontaneously epileptic rats (SER) are homozygous for two mutations, zitter (zi) and tremor (tm), and spontaneously exhibit both absence-like seizure and tonic convulsion. In addition, wild jumping or running episodes sometimes appear without any previous stimuli. We recorded the behaviour on a video camera and/or a vibration response apparatus to examine the behavioural characteristics. All wild jumping or running episodes appeared within 27 s after termination of tonic convulsion with a frequency of 2.1%, and the episode could be detected even in the dark by using the vibration response apparatus. A rapid increase of high motor activity around the episodes was recorded as a single peak with high amplitude by the vibration response apparatus. The wild jumping or running episode appeared in all 4 SER at 8-14 weeks of age with a variable frequency, 0-9 times in the light period and 0-6.7 times in the dark period 8 h each week. Thus, the wild jumping or running episode was found to occur not only during the light period but also during the dark period without any significant difference in frequency.

Sensitivity of spontaneously epileptic rats to external stimuli that induce seizures.

The sensitivity of spontaneously epileptic rats (SER), double homozygotes of zitter and tremor mutations, to external stimuli that induce seizures was studied in comparison with tremor (tm/tm) and zitter (zi/zi) rats, and with normal Kyo: Wistar and F 344/N rats. Touching their body, a blinking light (1200 lux, 1 sec interval) or a big sound (8 and 12 kHz, 95 dB) induced tonic extension only in the SER. The response frequency was 22 to 44% at 9 weeks of age and 75 to 100% at 13 weeks of age. Electric stimulus at 30 mA induced tonic-clonic convulsions in all Kyo: Wistar and F 344/N rats. At 20 mA the incidence of seizures decreased with age, from 100% at 5 weeks of age to 33% at 13 weeks of age in Kyo: Wistar rats and from 100 to 71% in F344/N rats. In SER, 10-mA stimuli induced tonic extension at 9 and 13 weeks of age, and 20 and 30 mA induced tonic convulsions, generalized or partial convulsions, and wild jumping or running episodes at 5, 9 and 13 weeks of age. At 30 mA, the incidence of convulsive seizures decreased with age in both tremor (tm/tm) and zitter (zi/zi) rats. Apparently external stimuli acted as simple triggers in the induction of tonic extension, since characteristic tonic extension is induced in the SER by each of the stimuli used in the present study, and induced convulsions closely resembled spontaneously occurring convulsions. The threshold of external stimuli in the induction of tonic extension became lower with aging in the SER, indicating that they are appropriate models for evaluating anticonvulsant drugs, as reported previously.

Outbreak of neonatal canine herpesvirus infection in a specific pathogen-free beagle colony.

Nine out of 19 newborn pups from 4 litters died or euthanatized on day 10 to 17 after birth in a specific-pathogen-free beagle colony which had been established by cesarean section and bottle-feeding. Necropsy showed congestion and edema of the lungs, congestion of the liver and splenomegaly, as well as many subcapsular petechiae and wedge-shaped areas of ecchymosis on the cut surface of the kidneys. A cytopathic agent isolated from the lungs, kidneys and liver was identified as a canine herpesvirus. The bitches in the animal house were seroconverted into positive for the isolate after the outbreak of the disease. Histopathological lesions in six cases were characterized by multiple necrosis and hemorrhage in various organs. In addition, fibrinoid necrosis was seen in the arterial walls in wedge-shaped necrotic areas of the kidneys. The outbreak appeared to be temporary, since the same disease has not occurred in the colony up to the present time.

[Histopathological study of arthritic lesions induced by immunization with type II collagen in DBA/1J mouse].

Eight male DBA/1J mice immunized twice by intradermal injection of type II collagen were autopsied 12 weeks after the first immunization and analyzed for anti-type II collagen antibody level, and the limb joints were examined radiologically and histopathologically. Clinical onset of swelling and erythema in the limb joints occurred about 5 weeks after the first immunization and deformity of the limbs was observed in a few animals about 5 weeks later. Although there were marked individual differences, serum anti-type II collagen antibody levels were elevated in all animals. Histopathologically, the changes were similar to those seen in human rheumatoid arthritis and were characterized by proliferation of synovial lining cells, formation of granulation tissue with destruction of cartilage and subchondral bone, and ankylosis. Systematic examination of various joints of the fore- and hind-limbs revealed definitely that the sequence of arthritic lesions was not uniform. The knee joint was involved most frequently, but smaller joints such as the phalangeal joints were involved less frequently but exhibited severe changes. The significance of histopathological examinations in the evaluation of effects of anti-rheumatic drugs was discussed with reference to this model.

Delayed postnatal behavioral development in spontaneously epileptic rats and tremor rats, and poor operant performance in spontaneously epileptic rats.

Postnatal behavioral development and learning ability of operant performance were examined in spontaneously epileptic rats (SER: zi/zi, tm/tm), and the original tremorous mutant strains of rats, tremor rats (tm/tm) and zitter rats (zi/zi) and their controls. Before the eyes opened, the increase in body weight and the age of achieving the righting reflex on a surface were no significantly different between the SER and their littermates without epileptic seizures (SER-N: zi/zi, tm/+ or zi/zi, +/+), and between tremor rats and the original strain Kyo: Wistar rats. After the eyes opened, the increase in body weight, age of achieving the righting reflex in air and traction performance, and the development of rotarod performance, were delayed in SER and tremor rats in comparison with other groups of rats. The zitter rats were apparently inferior in their development of rotarod performance in comparison with the same zitter homozygous SER-N. Operant performance was more inferior in SER than in SER-N and in tremor rats than in Kyo: Wistar rats. The differences were much more marked between SER and SER-N than between tremor and Kyo: Wistar rats. Thus, homologous tm genes and the coexistence of homologous tm and zi genes have an inhibitory effect on postnatal behavioral development and learning ability.

Spontaneous expulsion of micronuclei by enucleation in the micronucleus assay.

The effect of enucleation on the frequency of micronuclei induced by mitomycin C (MMC) and vincristine (VCR) was examined in mouse L-929 cells enucleated with cytochalasin B (Cyt-B). Approximately 30% of the L-929 cells became enucleated cells during the 8-h incubation in medium containing 8 micrograms/ml of Cyt-B. Using this enucleation technique, we estimated the reduction rate of 2 mutagen-induced micronuclei by enucleation. Treatment with MMC caused a dose-dependent induction of micronuclei in L-929 cells, with the reduction rate being 38.6% at the lowest dosage (0.0125 microgram/ml), which induced mostly mono-micronuclei in L-929 cells, and 6.8% at the highest dosage (0.1 microgram/ml), which induced many multi-micronuclei. Furthermore, VCR also induced micronuclei in a dose-dependent way in L-929 cells, and the same tendency for micronucleus reduction as with MMC was observed. The reduction rate of micronucleated cells by enucleation was estimated to be about 31-39% when the micronucleated cells contain mono-micronuclei. Therefore, the rate of reduction is affected by the number of micronuclei per cell, and the reduction depends on the increase in the number of micronuclei per cell.