Unilateral optical nerve hypoplasia in a Beagle dog.

Unilateral (left eye) optic nerve hypoplasia was detected in a six-month-old male Beagle dog. Vision testing indicated that the left eye had poor vision and testing the pupillary light reflex showed the left eye to have an absence of the afferent pathway of the reflex but it had a normal efferent pathway. Ophthalmoscopy revealed a small-sized optic disc, winding retinal artery and dilated retinal vasculature in the left globe. Electroretinography showed no abnormal findings even in the left globe. Histopathologically, the left optic nerve was markedly hypoplastic and was composed of sparse neural elements and a moderate amount of connective and glial tissues. In the retina of the left globe, the nerve fibre layer and the ganglion cell layer were reduced in thickness, although a small number of ganglion cells were still present. There were no abnormal findings detected in the right globe and the right optic nerve. The brain appeared normal macroscopically.

Effects of bleomycin on human tongue carcinoma cells as revealed by electron microscopy.

Five patients with tongue carcinoma were treated for 20 days with bleomycin, 15 mg/day, given by one-shot infusion through the superficial temporal artery. Specimens were taken 7, 14, and 20 days after the initial administration for light and electron microscope observations. The first alterations observed were the decrease of electron-dense chromatin clumps in nuclei and, simultaneously, the segregation of nucleolar components, i.e., amorphous protein component segregated from fibrillar and granular components. Subsequently, numerous nuclear bodies appeared in the nucleoplasm, which were observed for the first time in human tongue carcinoma cell nuclei in cases of bleomycin treatment. On the other hand, the cytoplasmic alterations were recognized by the decrease in number of mitochondria and by the appearance of numerous free ribosomes followed by the formation of tonofilaments. The cells enlarged because tonofilaments flocked together to make cancer cell nests, which were gradually together to make cancer cell nests, which were gradually transformed into a keratinic structure showing the so-called cancer pearl pattern. Finally, cancer cells were degenerated nearly to necrosis; there was no evidence of recurrence of cancer. Although the precise mechanism of the effect of bleomycin on nuclear activities of human tongue cancer cells remains unsettled because of the lack of cytochemical analysis, it is believed that bleomycin inhibits DNA synthesis and might also activate ribosomal RNA synthesis.

Rapid regression of squamous cell hyperplasia and slow regression of basal cell hyperplasia in the forestomach of F344 rats treated with N-methyl-N'-nitro-N-nitrosoguanidine and/or butylated hydroxyanisole.

Cell kinetics of reversible and persistent forestomach lesions induced by the genotoxic agent N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) and/or the nongenotoxic antioxidant butylated hydroxyanisole (BHA) was investigated. A total of 184 male F344 rats were divided into four groups: Group 1 given an initial single gastric intubation of MNNG received 2% BHA diet from the third wk to the 26th wk and then basal diet; Group 2 receiving 2% BHA without MNNG initiation; Group 3 given MNNG alone; and Group 4 serving as a nontreated control. Rats were sequentially sacrificed at 6, 16, 26, 30, and 46 wk. Bromodeoxyuridine was administered either as a single i.p. injection (100 mg/kg of body weight) 1 h before killing or continuously via an osmotic minipump (120 micrograms/h) for 1, 3, or 7 days prior to sacrifice, in each case labeled cells being detected by immunohistochemistry. Squamous cell hyperplasia (SCH) and basal cell hyperplasia (BCH), each characterized by different phenotypic keratin expression, were induced in Groups 1 to 3. After withdrawal of BHA, rapid regression of SCH and extremely slow regression of BCH were observed. Papillomas and squamous cell carcinomas developed irreversibly in Group 1 and 3, BHA significantly (P less than 0.01) enhancing the incidence of SCC in Group 1. Flash and continuous bromodeoxyuridine labeling revealed SCH to consist of cells of high mitotic activity and short life span, whereas BCH consisted of cells with low mitotic activity and long life span. In addition, highly labeled areas were observed in SCH after cessation of BHA feeding in Group 1 without regression, and similar lesions were also evident in Group 3. The results suggest that rapid regression of SCH and slow regression of BCH reflect different cell kinetic patterns and that highly labeled areas after release from stimulating agents might be preneoplastic changes related to cancer development.

Inhibition of mammary gland carcinogenesis by green tea catechins and other naturally occurring antioxidants in female Sprague-Dawley rats pretreated with 7,12-dimethylbenz[alpha]anthracene.

Effects of the naturally occurring antioxidants on mammary gland carcinogenesis were examined in female Sprague-Dawley rats pretreated with 7,12-dimethylbenz[alpha]anthracene (DMBA). Groups of 15-16 7-week-old rats received a 50 mg/kg body weight intra-gastric dose of DMBA, and starting one week thereafter placed on diet containing 0.4% catechol, 1.0% gamma-oryzanol, 2.0% phytic acid, 1.0% green tea catechins (GTC), 1.0% tannic acid or basal diet alone for 35 weeks. Although the final incidences and multiplicities of mammary tumors were not significantly different between DMBA-treated groups, the numbers of survivors in the antioxidant-treated groups at the end of the experiment at week 36 were significantly higher than in the basal diet group. In particular, the survival rate of the GTC group at 93.8% strongly contrasted with that of only 33.3% for rats on the basal diet. At the end of week 18, when all the animals were still alive, the average size of palpable mammary tumors was significantly smaller in the catechol, phytic acid and catechins groups. These results indicate that antioxidants, and GTC in particular, inhibit rat mammary gland carcinogenesis after DMBA initiation.

Medium-term bioassay for the hepatocarcinogenicity of hexachlorobenzene.

Hexachlorobenzene (HCB) is an important environmental contaminant derived mainly from industrial and agricultural sources. It is carcinogenic in mice, rats and hamsters. It has now been studied in a medium-term bioassay for carcinogenicity based on the induction of preneoplastic lesions in the liver. We report here that the bioassay can rapidly detect carcinogenic doses of HCB and that there is a clear dose-response relationship. At the lowest dose of HCB administered, the incidence of preneoplastic lesions in the liver was no different from that in controls.

Modulating effects of ellagic acid, vanillin and quercetin in a rat medium term multi-organ carcinogenesis model.

Effects of dietary supplementation with the antioxidants ellagic acid, quercetin and vanillin were examined using a medium term multi-organ carcinogenesis model in rats. Groups of 10-15 male F344 rats were given i.p. injections of diethylnitrosamine (DEN, 100 mg/kg body wt.) and N-methylnitrosourea (MNU, 20 mg/kg body wt), s.c. injections of 1,2-dimethylhydrazine (DMH, 40 mg/kg body wt.), together with 0.05% N-butyl-N-(4- hydroxybutyl)nitrosamine (BBN) and 0.1% 2,2'-dihydroxy-di-n-propylnitrosamine (DHPN), both in the drinking water, for a total multiple initiation period of 4 weeks (DMBDD) treatment). Ellagic acid, quercetin or vanillin, each at a dose of 1% each in the diet were administered from 1 day before and throughout the carcinogen exposure period, or after completion of the initiation regimen. All surviving animals were sacrificed at the end of week 36, and major organs were examined histopathologically. In the small intestine, significant reductions in the incidence and number of tumors (adenomas and carcinomas) were observed in the groups administered ellagic acid during (8%, 0.08 +/- 0.29) or after (8%, 0.08 +/- 0.29) DMBDD treatment, and those receiving quercetin after DMBDD treatment (0%) compared to the control value (57%, 1.07 +/- 1.21). Although the incidences were not statistically significant, slightly decreased numbers of small intestinal tumors were found in the groups receiving vanillin during (0.33 +/- 0.72), or after (0.40 +/- 0.83) DMBDD treatment. The incidence of large intestinal carcinomas in the group treated with vanillin during DMBDD treatment was significantly higher (73%) than the control value (21%). These results indicated that while ellagic acid and quercetin exerted potent chemopreventive action in both the initiation and promotion stages in the present experimental system, their beneficial effects were restricted to the small intestine. Since small intestinal carcinomas are very infrequent in humans, the advantages of these phenolic compounds for human application as chemopreventors should not be overestimated.

Green tea catechins enhance tumor development in the colon without effects in the lung or thyroid after pretreatment with 1,2-Dimethylhydrazine or 2,2'-dihydroxy-di-n-propylnitrosamine in male F344 rats.

Modifying effects of green tea catechins (GTCs) on the post-initiation stage of colon, lung and thyroid carcinogenesis were examined in F344 male rats. Groups of 20 animals were given subcutaneous injections of 40 mg/kg body wt of 1,2-dimethylhydrazine twice a week for 2 weeks or oral administration of 0.1% 2,2'-dihydroxy-di-n-propylnitrosamine (DHPN) in the drinking water for 2 weeks for initiation. They then received diet containing 1 or 0.1% green tea catechin or basal diet alone for 33 weeks. Histopathological examination after final sacrifice showed that although total incidence and multiplicity of colon tumors were not significantly different from controls, values for colon adenomas were decreased while those for carcinomas and the average size of tumors were significantly increased in the 0.1% GTC group. A similar tendency was observed for the 1% GTC group. Incidences and/or multiplicity of lung hyperplasia and tumors, and thyroid lesions did not significantly vary among the DHPN-treated groups. These results indicate that GTCs do not inhibit, but rather may enhance colon carcinogenesis, while not influencing lung and thyroid carcinogenesis under the present experimental conditions.

Enhancement by non-mutagenic pesticides of GST-P positive hepatic foci development initiated with diethylnitrosamine in the rat.

The potential hepatocarcinogenicity of seven pesticides was examined using a rapid bioassay based on the induction of glutathione S-transferase placental form positive foci in the rat liver. Rats were initially injected with diethylnitrosamine and two weeks later were fed on diet supplemented with one of the pesticides for 6 weeks and then killed; all rats were subjected to a partial hepatectomy at week 3. Positive results were seen with chlorobenzilate (2000 ppm), vinclozolin (2000 ppm), malathion (4000 ppm), tecnazene (2000 ppm) and isoproturon (2000 ppm). S,S,S-tributylphosphorotrithioate (DEF, 200 ppm) and dicloran (2000 ppm) were negative in both number and area analyses. Although chlorobenzilate is carcinogenic in mice, malathion and vinclozolin have been reported as non-carcinogens in both rats and mice. Since the present system is based on the two-stage carcinogenesis hypothesis, it is possible that the chemicals showing positive results in this system possess at least tumor-promoting activity in the rat liver. This is very significant, as most carcinogens show tumor-promoting activity in their target organs.

Effects of catechol, sodium chloride and ethanol either alone or in combination on gastric carcinogenesis in rats pretreated with N-methyl-N'-nitro-N-nitrosoguanidine.

Combined effects of catechol, sodium chloride (NaCl) and ethanol on the post-initiation stage of gastric carcinogenesis were examined in rats pretreated with N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). F344 male rats were given a single intragastric dose of 150 mg/kg b.w. MNNG at 6 weeks of age. Starting 1 week thereafter, groups of 15 rats were administered 0.8% catechol, 5% NaCl and 10% ethanol either individually or in combination, or basal diet alone for 51 weeks. Further groups of animals were similarly treated with these chemicals without the MNNG pretreatment. All rats were killed at the end of week 52 for histopathological examination. In the forestomach, treatment with catechol alone after MNNG initiation caused a 100% incidence of papillomas (versus 67% in the controls) as well as carcinomas (versus 0% in the controls). On the other hand, the treatment with ethanol alone significantly lowered the incidence of papillomas (13 versus 67% in the controls). The combined treatment with catechol, NaCl and ethanol significantly lowered the incidence of squamous cell carcinomas (57%) as compared to the catechol alone group value (100%). In the glandular stomach, catechol enhanced the development of adenocarcinomas (73 versus 0% in the controls), but this was decreased to 29% by the combined treatment with ethanol and NaCl. NaCl without MNNG pretreatment slightly enhanced epithelial cell proliferation in the forestomach. These results indicate that combined treatment with NaCl and ethanol exerts protective effects against catechol-induced forestomach and glandular stomach carcinogenesis, this apparently being largely due to the ethanol.

Inosamycin, a complex of new aminoglycoside antibiotics. I. Production, isolation and properties.

A strain of Streptomyces hygroscopicus No. J296-21 (ATCC 39150) was found to produce a complex of new antibiotics, called inosamycins, which consisted of components A, B, C, D and E. They are novel aminocyclitol antibiotics structurally related to neomycin, paromomycin and ribostamycin. The antibiotic complex and each component of inosamycin exhibit a broad antibacterial spectrum but they are inactive against most of the aminoglycoside-resistant organisms. The antibacterial activity of inosamycin A, the major component of the complex, is comparable to that of neomycin but its acute toxicity is significantly lower (ca. 1/3) than that of neomycin.

[A 13-week subcutaneous toxicity study of prednisolone farnesylate (PNF) in rats].

The toxicity of Prednisolone farnesylate (PNF), a synthetic glucocorticoid, was investigated in the Sprague-Dawley rat. PNF was injected subcutaneously at doses of 0.03, 0.3, 3 and 30 mg/kg/day for 13 weeks. In addition, 18.7 mg/kg/day prednisolone (PN), which is approximate to 30 mg/kg/day PNF in prednisolone molarity, was also administered to the rat for comparison. The results are summarized as follows: 1. All animals from the PN 18.7 mg/kg/day group, and four(4) out of ten(10) males and three(3) out of ten(10) females from the PNF 30 mg/kg/day group died having shown weakened condition such as unkempt fur and emaciation. Histopathologically, systemic suppurative inflammation, as shown by pyeronephritis and abscess formation in many organs and tissues, was observed and it was considered that the administration of steroid induced weakened condition and systemic suppuration which resulted in death. In addition, atrophy was noted in the adrenal glands, lymphatic organs and skin, and histopathological lesions were also observed in the lungs, liver, pancreatic islets, bone, bone marrow and mammary glands. 2. Surviving animals in the PNF 30 mg/kg/day group showed almost the same changes as those observed in the dead animals that died. Hematological examination revealed an anemic change and a decrease in lymphocytes with an increase in segmented neutrophils and eosinophils. In the urinalysis and blood chemistry, the changes suggesting damages to the liver and kidneys were mainly observed. 3. In the PNF 3 and 0.3 mg/kg/day groups, several changes such as atrophy of the adrenal glands, lymphatic organs and skin were noted in a dose dependent manner. 4. In the PNF 0.03 mg/kg/day group, ther were no toxic signs. 5. Based on these results, it was concluded that the overt toxic dose of PNF was 0.3 mg/kg/day and the non-toxic dose was 0.03 mg/kg/day in the present study.

[A 13-week dermal toxicity study of prednisolone farnesylate (PNF) gel in rats with a recovery period of 5 weeks].

The toxicity of Prednisolone farnesylate (PNF) gel, a synthetic glucocorticoid, was investigated in the Sprague-Dawley rat. PNF gel was administered dermally to the rats at doses of 0.25, 1, 4 and 16 mg/kg/day for 13 weeks, then the drug was withdrawn for 5 weeks to evaluate the reversibility. In addition, 10 mg/kg/day prednisolone gel (PN gel), which is approximate to 16 mg/kg/day PNF gel in prednisolone molarity, was also administered to the rats for comparison. The results are summarized as follows: 1. In the PNF gel 16 mg/kg/day group, temporary erythema at the application site, retarded body weight gains, a decrease in the white blood cell count and lymphocyte ratio with an increase in the segmented neutrophil ratio, an elevation of serum AIP activity were observed. The pathological examinations revealed atrophy of the adrenal glands, lymphatic organs and skin. In addition, histopathological lesions were also found in the liver, pancreatic islets, bone, bone marrow and mammary glands. 2. In the PNF gel 4 mg/kg/day group, retarded body weight gains were observed, and histopathological lesions were noted in the adrenal glands, lymphatic organs, skin at the application site, liver and bone marrows. 3. In the groups that received less than 1 mg/kg/day of PNF gel, there were no toxic signs induced by the drug. 4. In the PN gel 10 mg/kg/day group, drug-related changes were almost similar to those of the PNF gel group, but the severity of the lesions was stronger than in the PNF gel group. 5. After the 5-week recovery period, the above changes almost completely disappeared and so it was demonstrated that the changes were reversible. 6. Based on these results, it was concluded that the overt toxic dose of PNF gel was 4 mg/kg/day and the non-toxic dose was 1 mg/kg/day in the present study.

[A 13-week subacute oral toxicity study of 6-amidino-2-naphthyl 4-[(4,5-dihydro-1H-imidazol-2-yl) amino] benzoate dimethanesulfonate (FUT-187) in rats].

The toxicity of FUT-187, a synthetic protease inhibitor, was investigated in Sprague-Dawley rats. FUT-187 was given orally to the rats at doses of 2, 10, 50, 250 and 1250 mg/kg/day for 13 weeks, then the drug was withdrawn for 5 weeks for recovery. The results are summarized as follows: In the 1250 mg/kg/day group, 9 out of 20 males died with decreased body weight and exhaustion. Histopathological examination revealed renal papillary necrosis, ulcer in the urinary bladder, hemostatic lesions in the lungs and liver, ulcer or erosion in the stomach, duodenum and jejunum. The surviving animals in this group showed swelling of the limbs due to synovitis, transient salivation immediately after administration, suppression of growth with decreased food consumption. Urinalysis revealed a low pH, increased ketones and bilirubin excretion, dark yellowish change in color, the appearance of "leaflet-shaped" crystals and increased red blood cells and epithelial cells in the urinary sediment, increased water intake, decreased specific gravity and decreased sodium, potassium and chloride in the urine. Hematologically, there was an increase in the white blood cell count. A biochemical analysis of the blood revealed decreased amylase activity, glucose and total protein levels and increased GOT activity and inorganic phosphorus levels. Pathological changes were observed in the pancreas, kidney, digestive tract, urinary bladder and liver. The pancreas showed macroscopical enlargement and increased organ weight. Histopathologically, there were several alterations in the acinar cells, such as vacuolization due to increased fat droplets, nuclear irregularity, prominent nucleoli, irregular arrangement and vesiculation of rough endoplasmic reticulum (rER), dilatation of developed Golgi apparatus and increased free ribosomes. In the kidney, increased weight and pigmentation in the proximal tubular epithelium were noted. Electron microscopically, these pigments were recognized as secondary lysosomes containing filamentous material and electron dense granules within a lucent matrix. In the digestive tract, ulcer or erosion in the stomach and duodenum, and villous proliferation in the small intestine were observed. Furthermore, hyperplasia and vacuolization were noted in the mucosal epithelium of the urinary bladder. In addition, loss of perilobular fat droplets in the liver and increased adrenal weight without histological change were observed. After a 5-week recovery period, these changes disappeared almost completely. In the 250 mg/kg/day group, slight suppression of growth the appearance of "leaflet-shaped" crystals in the urinary , sediment, increased water intake and decreased sodium in the urine were observed. The pancreas showed enlargement, increased weight, acinar cell hypertrophy with increased zymogen granules, fine vacuolization, slight derangement and vesicular of rER, and dilatation of Golgi apparatus.(ABSTRACT TRUNCATED AT 400 WORDS)

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).

A rapid in vivo bioassay for the carcinogenicity of pesticides.

Eight pesticides were tested in a bioassay based on the induction of preneoplastic lesions in the liver. Rats were given diethylnitrosamine intraperitoneally at 200 mg/kg bw and two weeks later were treated with pesticides for six weeks and then killed; all rats had a partial hepatectomy at week 3. Hepatocarcinogenic potential was assessed by comparing the number and area of glutathione s-transferase (placental form) -positive foci in the liver with those of controls given diethylnitrosamine alone. Positive results were seen with Chinomethionat, Phosmet and Propiconazole; the results obtained with Captan and Prochloraz were borderline; Benomyl, Daminozide and Folpet gave negative results. Our findings provide enough experimental evidence to indicate that great care should be exercised in the use of these compounds.

[Compound therapy including PSK for malignant head and neck cancer].

The effect of protein-bound polysaccharide preparation, PSK was studied in 44 cases with head and neck cancer. 1) Eight cases with laryngeal cancer received radiation therapy and PSK as immunotherapy, and eight cases as control received radiation alone. During radiation, the mean values of peripheral lymphocyte counts showed statistically significant difference (P less than 0.05) between the cases with immunotherapy and those without immunotherapy. 2) Twenty-eight cases received adjuvant immunochemotherapy with Carboquone and PSK. 22 out of 28 evaluable cases in the group received adjuvant immunochemotherapy were judged as effective. These studies suggest that PSK is one of the valid immunopotentiators.