Hepatocytes have a great capability to adapt to functional demand by changing enzyme activity. In this report, ultrastructural characteristics were described in developing, regenerating and drug-exposed hepatocytes in which various enzymes are rapidly synthesized. A brief review was made on the mechanism as to how the enzymes are transported from the site of synthesis and assembled to the organelles.

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Hepatic injuries induced by two free-radical mediated xenobiotics such as CCl₄ and AAPH were examined with light and electron microscopes. Administration of CCl₄ to male ICR mice resulted in disappearance of glycogen in the hepatocytes of centrilobular region within 1 hr. Seven hours after the CCl₄ injection, marked fatty degeneration and necrosis were restricted to hepatocytes around the central vein. Ultrastructurally, dissociation of ribosomes from rough surfaced endoplasmic reticulum, disappearance of glycogen particles, and accumulation of fat droplets were seen exclusively in hepatocytes located in centrilobular region. Lining cells of hepatic sinusoid were affected less severe than the hepatocytes. In contrast to CCl₄-poisoning, mice given AAPH in high dose, showed hepatic injuries in centrilobular, midzonal and periportal areas in a similar grade. Sinusoidal lining cells were most severely affected. Occasionally, the degenerated lining cells desquamated from the sinusoid. Fat droplets and degenerated mitochondria were frequently seen in almost all hepatocytes. AAPH attacks not only the liver but also other organs such as the kidney, heart, and thymus. Differences between the pathological features of both free radicals are resposible to sites of free radical generation.

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The distribution of alkaline phosphatase (ALP) activity in neoplastic lesions of the rat stomach was examined by histochemistry and electron microscopic cytochemistry, and its isoenzyme activity was biochemically determined in an experimental model of gastric carcinogenesis using N-methyl-N-nitrosourea (MNU). In addition, [³H]1, 25-dihydroxyvitamin D₃ (1, 25(OH)₂D₃) binding activity into the gastric lesions was assayed. Histochemical positive reaction for ALP was conspicuous exclusively in the fibrous stroma of both intramucosal neoplastic lesions and invasive adenocarcinomas in the glandular stomach. Ultracytochemical examinations revealed that the reaction product of ALP activity was present on the plasma membrane of fibroblasts adjacent to neoplastic cells and collagenous matrices beneath the latter cells. Biochemically, the major component of total ALP activity, which showed 25- to 33-fold increase in advanced carcinomas as compared with the normal glandular stomach, was found to be of tissue-unspecific (bone/liver/kidney) type, while the intestinal isoenzyme formed a minor proportion. Extraordinarily increased binding activity of 1, 25(OH)₂D₃ to carcinomas appeared consistent with marked production of ALP in the stroma of neoplastic lesions in the present study. The preferential localization of enhanced ALP activity in the fibrous stroma of neoplasms may be involved in some epithelial-stromal interactions or signal transduction mechanism during gastric carcinogenesis.

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The optimum condition for gastric carcinomas to be induced by N-methyl-N-nitrosourea (MNU) selectively in the glandular portions of the rat stomach was investigated. MNU in drinking water at various dose levels with shielding from light was administered ad libitum to a group of inbred male F344 rats for 15 weeks and thereafter switched to tap water to maintain them for further 25 weeks. The rats received MNU at the concentration of more than 200 ppm resulted in high mortality because of chronic toxicity by 25 weeks after withdrawal of MNU. Administration of MNU at 100 ppm followed by at least 25-week maintenance without the compound was concluded to be the optimal condition for a rat new model of gastric carcinogenesis, because the experiment under such a condition yielded a high incidence (95.2%) of carcinomas in the glandular stomach with no death at the end of the experiment. Almost all neoplastic lesions occurred in the pyloric regions; well differentiated adenocarcinoma with submucosal invasion accounted for approximately 50% of all the neoplastic lesions. Cell kinetics analysis using BrdU-immunohistochemical technique during MNU administration revealed 2-fold increase of DNA-synthesizing cells at 3 weeks after the start of administration as compared to the controls of normal stomach. These BrdU-positive cells, however, gradually decreased in number with time, approximating to the control value by 15 weeks. The results may indicate some suppressive effect of lengthy treatment of MNU on the growth of MNU-initiated atypical epithelium of the stomach.

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The modifying activities of butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT) and tertiary butylhydroquinone (TBHQ) and of paired combinations of these compounds on urinary bladder carcinogenesis in male F344 rats pretreated with N-butyl-N-(4-hydroxybutyl) nitrosamine (BBN) were investigated. Six weeks old animals were given 0.05% BBN in their drinking water for 4 weeks and then BHA, BHT or TBHQ, alone (0.8% each) or in pairs (0.4% each) in their diet for 32 weeks. Control rats received no further treatment after BBN administration. Surviving animals were killed at the end of week 36 of the experiment. The incidence of preneoplastic papillary or nodular hyperplasia (PN hyperplasia) was significantly higher in the group treated with BHA plus TBHQ after treatment with 0.05% BBN than in the group given the initiating carcinogen alone. The densities of PN hyperplasia (number per 10cm of basement membrane) were also significantly increased in all treated groups. However, no synergistic enhancing effects were observed. The incidences and densities of papillomas or carcinomas were not significantly different between any treated group or as compared to the controls. Thus the present experiments indicated that while BHA, BHT, and TBHQ all exerted enhancing effects in BBN-induced urinary bladder carcinogenesis in rats. no synergism regarding this promotion occurs, an important consideration for human risk assessment.

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Chronic toxicity of diphenyl and its ability to promote carcinogenesis by N-ethyl-N-hydroxyethylnitrosamine (EHEN) in the kidney, were studied in Wistar rats. In the study of chronic toxicity, diphenyl at doses of 0, 0.25, and 0.5% was given in the diet to Wistrar rats of both sexes for 75 weeks. Diphenyl dose-dependently induced urolithiasis, and stones, accompanied by hematuria, were observed in the kidney, ureter, and urinary bladder as early as 16 weeks after the beginning of the experiment. At the dose of 0.5% of diphenyl, the incidence of stones in the kidney and urinary bladder was 46.2 and 15.4%, respectively, in females and 31.9 and 27.7%, respectively, in males. Kidney stones were black and composed of protein, but urinary bladder stones were yellowish-white and composed of ammonium magnesium phosphate. In the promotion experiment, EHEN, at a dose of 0.1%, was given in the diet for 2 weeks as the initiator of carcinogenesis, and then diphenyl, at doses of 0, 0.125, and 0.5% was supplied in the diet for 34 weeks, to male Wistar rats. Neither doses of diphenyl affected the incidences of dysplastic foci and renal cell tumors induced by EHEN in the kidney. However, 0.5% of diphenyl, in spite of inducing urolithiasis, significantly decreased the mean numbers of both dysplastic foci and renal cell tumors per cm² of the kidney. These results indicated that diphenyl induced urolithiasis but exhibited rather inhibitory effect of the induction of kidney carcinogenesis by EHEN in rats.

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Potential modifying effects of dietary high fat on 3, 2'-dimethyl-4-aminobiphenyl (DMAB)-induced rat prostate and colon carcinogenesis were investigated in male F344 rats using a two stage, initiation-promotion protocol. Starting at 3 weeks of age, animals were given s.c. injections of DMAB (150mg/kg body weight/week for 3 consecutive weeks). Starting fifteen weeks after the last injection of DMAB, rats were fed semipurified diet containing 20% corn oil (high fat) or 4.5% corn oil (normal fat) for 42 weeks and then all animals were sacrificed for histopathological investigation. No significant effects of the high fat diet on the resultant incidences of atypical hyperplasia and carcinoma of the prostate or of tumor development in the colon were observed.

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Chronological changes in hemodynamics and histopathological lesions of the brain were investigated in rats given a single dose of hypertonic glucose solution (osmotic ratio: 6.5) intravenously or intraperitoneally. After administration, dehydration developed rapidly and markedly but recovery was noted on the following day. An excess dosage of the hypertonic solution caused cerebral lesions with meningeal hemorrhage showing nervous signs. Shrinkage of neurons, and loosening and vacuolation of the ground substance with swollen astrocytes (spongy appearance) were seen mainly in CA4 subfield of the hippocampus. The spongy appearance occurred in cases which died 130 min or later post-administration (PA), while the ischaemic change consisting of necrosis and loss of neurons with glial proliferation was seen mainly in CA1 and CA4 of cases sacrificed 14 days PA. The cerebral lesions showed selective vulnerability and were considered to result from ischaemia caused by hemodynamic changes.

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Early morphological changes in the kidney of Wistar rats given 5% diethylene glycol (DEG) in drinking water for 2, 4 or 6 days were studied. The kidney was fixed by perfusion with Karnovsky's solution and examined by a light microscopy and electron microscopy. The histological change appeared mainly in proximal convoluted tubules in cortex. At day 2, eosinophilic granules in the cytoplasm of proximal convoluted tubular epithelium increased in number and also lysosomal-like dense bodies increased in number. At day 4, focal necrosis with vacuoles usually appeared in the inner cortex, mitochondrias in proximal convoluted tubular epithelium were small or irregular sizes and high electron dense. At day 6, most of the cytoplasm of proximal convoluted tubular epithelium were occupied by various sizes of cystic vacuoles, and a few organelle located near cellar membrane on one side.

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The incidence and histopathological features of spontaneous thyroid, parathyroid, and pancreatic islet tumors encountered in long-term oncogenicity studies using F344 rats or B6C3F₁ mice were investigated. Spontaneous C-cell tumors of the thyroid were found at high incidence in both sexes of F344 rats, whereas pancreatic islet-cell tumors were only observed at high incidence in males. All tumors were usually found in old animals, and were rare in young and middle aged rats. In general, the incidences of endocrine tumors in B6C3F₁ mice were very much lower, although follicular cell tumors of the thyroid were sometimes observed. In the endocrine system, the follicular cell of the thyroid is the most susceptible to chemical carcinogens. Carcinogen-induced tumors can be distinguished from spontaneous lesions due to their higher incidence, earlier induction, multiplicity, and increased evidence of invasion and metastasis.

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Initially, morphological and endocrinological features of the thyroid gland are reviewed in view of the recent progress in thyroidology. The second part of this article deals with morphology and hormone measurement in the rat following drug-induced alteration in thyroid function. Single and short-term injection of hypothalamic thyrotropin-releasing hormone (TRH) resulted in an increase in serum TSH concentration but not in thyroid hormone concentration. Electron microscopic observation revealed that augmented TSH stimulation after administration of TRH, induced follicular exocytosis, followed by endocytosis. The increase in colloid droplet counts after TRH-treatment differed between follicles as well as between follicular cells in the same thyroid. Treatment with the antithyroid drug, propylthiouracil (PTU) caused a progressive decrease in serum thyroid hormone levels and an elevation of serum TSH. The earliest morphological changes in follicular cells after short-term administration of PTU were the appearance of subapical vesicles and colloid droplet formation in response to the elevated level of serum TSH. Conversely, prolongation of goitrogen-treatment led to the disappearance of exocytotic and endocytotic vesicles. Substained decrease of serum TSH after thyroxine (T₄) treatment led to colloid and thyroglobulin (Tg) accumulation in the follicular lumen and to the disappearance of exocytotic and endocytotic vesicles. In the T₄-treated rats hypothalamic stimulation (TRH injection) resulted in a rise in the serum level of TSH, accompanied by apparent colloid droplet formation in the follicular cells.

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The incidence, nomenclature, and diagnostic criterion of spontaneous tumors of the pituitary and adrenal glands in rats and mice were reviewed. From these reviews, diagnostic problems on these proliferative lesions encountered sometimes in routine long-term toxicity and carcinogenicity studies were pointed out. In these organs, the main diagnostic problems were the difficulty to differentiate between focal hyperplasia and small adenoma and to separate carcinoma from adenoma. Most of these problems might be due to the fact that the biological features of these proliferative lesions were not completely clarified in these animals. Criteria in the differential diagnosis proposed in the literature, therefore, seemed to be somewhat arbitrary. More practical methodology for adequate histopathological evaluation was discussed on the carcinogenicity of endocrine organs.

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A melanocytic tumor was found in the skin or subcutaneous tissue in a BDF₁ mouse maintained up to 109 weeks. The tumor was solid and well-demarcated from the surrounding tissue, and showed an extensive metastasis to lymph node, liver, and lung. The tumor was composed of both oval or polygonal cells with abundant melanin pigments, and spindle cells mixed with the oval or polygonal cells. These blackish pigments in the tumor cells disappeared by bleaching and more blackened by Fontana-Masson stain. The tumor cell showed poor cellular pleomorphism and rare mitosis. Numerous mature, stage IV-melanosomes were observed in the cytoplasms of the tumor cells by electron-microscopical examination. It was suggested that the origin of the present tumor was a dermal melanocyte by its histological features, though no definite conclusion was obtainable because of ulceration in the central area of the skin covering the nodule.

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