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This review covers chemical carcinogenesis studies in nonhuman primates performed by the National Cancer Institute, USA, to provide hitherto unavailable information on their susceptibility to compounds producing carcinogenic effects in rodents. From autopsy records of 401 breeders and untreated controls, incidences of spontaneous malignant tumors were found to be relatively low in cynomolgus (1.9%) and rhesus monkeys (3.8%), but higher in African green monkeys (8%). Various chemical compounds, and in particular 6 antineoplastic agents, 13 food-related compounds including additives and contaminants, 1 pesticide, 5 N-nitroso compounds, 3 heterocyclic amines, and 7 "classical" rodent carcinogens, were tested during the 34 years period, generally at doses 10 approximately 40 times the estimated human exposure. Results were inconclusive in many cases but unequivocal carcinogenicity was demonstrated for IQ, procarbazine, methylnitrosourea and diethylnitrosamine. Furthermore, negative findings for saccharine and cyclamate were in line with results in other species. Thus susceptibility to carcinogens is at least partly shared by nonhuman primates and rodents.

平成13年4月, A農場 (酪農, 飼養頭数: 220頭; 長野県) で, 流産した牛1頭にネオスポラ抗体が確認されたため, 浸潤状況と損失を分析し, 対策を検討した。その結果, 搾乳牛130頭を調査し, 陽性率は24.6%で全国平均 (推定) 5.7%より高かった。A農場での伝播の主因は水平感染と考えられたが, 陽性牛の一部に血縁関係が認められ, 垂直感染の関与も考えられた。流死産率は陽性群と陰性群に有意差はなく, 流死産を繰り返す牛もなかった。観察された全ての流死産のうちネオスポラを原因とするものは1.8%と推定され, A農場全体の年間損失額は57, 317円と推定された。以上のことから, 早急な陽性牛淘汰は経済的に困難であると判断されたため, 陽性牛を後継に残さないことを目的とし, (1) 陽性牛でのF1生産, (2) 流死産発生時の抗体検査, (3) 後産および流産胎子の後始末の徹底, (4) 犬科動物と牛との接触防止, を指導した。一般にネオスポラ感染牛は流産を起こしやすいとされるが, 本症例におけるA農場での損失は少なかった。経済的に効果的な対策の実施には, 個々の症例ごとに十分な浸潤状況と損失を分析し, 慎重な対策を検討する必要があると考えられた。

Twenty-one monkeys (cynomolgus, rhesus, African green) were fed cyclamate (100 mg/kg and 500 mg/kg) in the diet five times per week from a few days after birth and continuing for up to 24 years. Malignant tumors were diagnosed in three 24-year-old cyclamate monkeys; these were metastatic colon carcinoma (rhesus; 500 mg/kg), metastatic hepatocellular carcinoma (cynomolgus; 500 mg/kg), and a small, well differentiated adenocarcinoma of the prostate (cynomolgus; 100 mg/kg). Benign tumors were found at necropsy in three females; these were adenoma of the thyroid gland (rhesus; 100 mg/kg) and two cases of leiomyoma of the uterus (rhesus; 100 mg/kg and 500 mg/kg). No tumors were detected in an age-matched control group of 16 monkeys. Examination of the testes revealed complete testicular atrophy in one of the old cyclamate monkeys, and focal germ cell aplasia (Sertoli-only tubules) in two other cyclamate monkeys. Focal spermatogenic interruption (maturation arrest) at various germ cell levels mixed with normal spermatogenesis was observed in both the cyclamate-treated and the control monkeys, all of which were over 20 years old. Measurements of terminal cyclohexylamine concentrations showed that three of the males dosed with cyclamate at 500 mg/kg were high converters, with plasma concentrations comparable to the levels that produce testicular atrophy in rats. However, only one of the three high converters showed histologic evidence of irregular spermatogenesis. The overall conclusion is that the testicular abnormalities and the sporadic cases of different malignancies found after more than 20 years of dosing do not provide clear evidence of a toxic or carcinogenic effect of sodium cyclamate in monkeys.

Because of reports on tumorigenic activity in different animal species exposed to DDT a decision was made in 1969 to evaluate the long-term effects of DDT on 24 cynomolgus and rhesus monkeys. DDT (20 mg/kg) was given in the diet for 130 months, followed by an observation period that ended in 1994. The two cases of malignant tumor detected in the DDT group included a metastatic hepatocellular carcinoma in a 233-month-old male and a well-differentiated adenocarcinoma of the prostate in a 212-month-old monkey. Benign tumors detected in the DDT group included three cases of leiomyoma, two of which were uterine and one, esophageal. No tumor was detected in the control group of 17 monkeys. Fatty changes in the liver were observed in 52.9% of the DDT group and 29.4% of the control group. More specific signs of hepatotoxicity were documented microscopically in seven DDT monkeys. Severe tremors and histological evidence of CNS and spinal cord abnormalities were observed in six DDT monkeys. The present findings show clear evidence of hepatic and CNS toxicity following long-term DDT administration to cynomolgus and rhesus monkeys. However, the two cases involving malignant tumors of different types are inconclusive with respect to a carcinogenic effect of DDT in nonhuman primates.

Background: It was observed in the early 1970s that saccharin produced bladder cancer in rats. However, it has been unclear whether sodium saccharin when consumed by humans poses a substantial carcinogenic hazard. Numerous epidemiologic studies have not shown any evidence of increased urothelial proliferation associated with ingestion of sodium saccharin. Purpose: Our purpose was to determine the effects of long-term feeding of sodium saccharin to three species of nonhuman primates. Methods: Twenty monkeys of three species (six African green, seven rhesus, six cynomolgus, and one hybrid [of rhesus male and cynomolgus female parentage]) were treated with sodium saccharin (25 mg in the diet/kg body weight daily for 5 days a week) beginning within 24 hours after birth and continuing for up to 24 years. Sixteen monkeys (seven rhesus and nine cynomolgus) served as controls. During their last 2 years of life, urine was collected from selected treated and control animals and evaluated for various urinary chemistries and for the presence of calculi, microcrystalluria, and precipitate. Urinary bladders were examined by light microscopy and by scanning electron microscopy. Results: Sodium saccharin treatment had no effect on the urine or urothelium in any of these monkeys. There was no evidence of increased urothelial cell proliferation, and there was no evidence of formation of solid material in the urine. Conclusion: Although the dose of sodium saccharin administered to these monkeys was only five to 10 times the allowable daily intake for humans, the results provide additional evidence that sodium saccharin is without a carcinogenic effect on the primate urinary tract. [J Natl Cancer Inst 1998;90:19‐25]

The binding of benzo(a)pyrene (BP) to DNA in the presence of liver microsomal preparations from 3-methylcholanthrene-treated rats was increased 28.5% (P < 0.001) by addition of UDP-glucuronic acid to the incubation mixture at a 100 microM concentration of BP. However this increase was not observed at lower concentrations of BP. Analysis of DNA-hydrolysates by column chromatography on Sephadex LH-20 indicated that this increase was due to greater binding of benzo(a)pyrene-7,8-dihydrodiol-9,10-epoxide. The amount of bound adducts derived from 9-hydroxybenzo(a)pyrene (9-OH BP) was not significantly affected by UDP-glucuronic acid. Addition of UDP-glucuronic acid to incubation mixtures containing microsomes from untreated rat liver caused only a slight increase in total binding. These findings suggest that UDP-glucuronyl transferase may be involved in the activation of BP.

2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), a carcinogen found in cooked meat, was determined to be a mammary carcinogen in female Sprague-Dawley rats on a high fat diet. Forty-three-day-old female Sprague-Dawley rats received 10 doses of PhIP (75 mg/kg, p.o., days 1-5 and 8-12). Two days after the last dose of PhIP, animals were placed on a high polyunsaturated fat diet (23.5% corn oil) or a standard low fat diet (5% corn oil). After 25 weeks on the defined diet, mammary tumor incidence (average tumor mass +/- SE) was 53% (5.7 +/- 1.3 g) and 16% (2.4 +/- 0.9 g) in rats on a high fat and standard low fat diet, respectively. The histological differences in mammary gland tumors found in animals on the standard low fat diet and the high fat diet were striking. Mammary gland tumors found in PhIP-treated rats on the low fat diet were all histologically benign. The histopathological changes in these tumors included hypertrophic changes resembling the normal mammary gland, fibrocystic changes, and sclerosing adenosis. However, 80% of the mammary gland tumors found in PhIP-treated rats on a high fat diet were histologically malignant. These tumors had several malignant phenotypes including intraductal carcinoma (papillary, cribriform, and comedotype), tubular adenocarcinoma, and infiltrating duct carcinoma. The data indicate that a high fat diet in combination with a heterocyclic amine carcinogen derived from cooked meat may enhance the incidence and severity of mammary gland cancer.

Mutagenic heterocyclic amines in cooked foods were carcinogenic to mice, rats and/or monkeys, when they were given orally continuously. The most common target organ was the liver, but in CDF1 mice lung tumors, forestomach tumors, lymphomas/leukemias, and blood vessel tumors in the brown adipose tissues were also induced. In F344 rats, in addition to liver tumors, tumors in the Zymbal gland, skin, clitoral gland, small and large intestines, oral cavity, and mammary gland were also induced. Monkeys given IQ developed metastatic hepatocellular carcinomas.

A signet ring cell carcinoma in the gastric antrum of a Cynomolgus monkey induced by N‐ethyl‐N′‐nitro‐N‐nitrosoguanidine was sequentially studied by endoscopy, biopsy, and autopsy. The carcinoma was first detected on the angulus of the stomach at the 38th month as a slightly elevated lesion. Sixty‐one months later this tumor was found to be still in the “early”(intramucosal) stage. Another, independent, initial gastric cancer was also discovered. This is the first example of an induced gastric carcinoma remaining in the “early’stage during a six‐year follow‐up period after the initial histologic diagnosis.

The carcinogenic potential of 2‐amino‐3‐methylimidazo[4,5f]quinoline (IQ) was evaluated in cynomolgus monkeys. Monkeys received IQ, beginning at the age of one year, at doses of 10 or 20 mg/kg by gavage. Thus far, IQ has induced hepatocellular carcinoma in three monkeys with a latent period of 27 to 37 months. Metastases to the lung occurred in two of the three monkeys. Microscopically, the hepatocellular carcinoma in all three cases demonstrated a trabecular pattern. These data demonstrate that IQ is a potent carcinogen in nonhuman primates and support the idea that it is a potential carcinogen for humans.

In recent years, there are several reports that amblyopia may be associated with changes in retinal function at the level of production of the P-ERG(pattern-reversal ERG), which may be pre-ganglionic. Among them, Arden et al3)have recorded P-ERG in amblyopic children and concluded as follows: PI amplitude of amblyopic eyes to that of fellow eyes was less than 0.8 in amblyopia but was higher than 0.8 in those children whose vision was improved by the subsequent orthoptic treatment.

†Background and Aims In response to inclination stimuli, gymnosperm trees undergo corrective growth during which compression wood develops on the lower side of the inclined stem. High compressive growth stress is generated in the compression wood region and is an important factor in righting the stem. The aims of the study were to elucidate how the generation of compressive growth stress in the compression wood region is involved in the righting response and thus to determine a righting mechanism for tree saplings. †Methods Cryptomeria japonica saplings were grown at inclinations of 08 (vertical) to 508. At each inclination angle, the growth stress on the lower side of the inclined stem was investigated, together with the degree of compression-wood development such as the width of the current growth layer and lignin content, and the upward bending moment. †Key Results Growth stress, the degree of compression wood development, and the upward moment grew as the stem inclination angle increased from 0 to 308, but did not rise further at inclinations .308. †Conclusions The results suggest the following righting mechanism for gymnosperm saplings. As the stem inclination is elevated from 0 to 308, the degree of compression wood development increases to force the sapling back to its original orientation; at inclinations .308, the maximum degree of compression wood is formed and additional time is needed for the stem to reorient itself.

Tumour necrosis factor alpha (TNF-alpha) transgenic mice, which overexpress TNF-alpha in the lungs, develop interstitial pneumonitis resembling idiopathic pulmonary fibrosis (IPF) in humans. Transgenic mice were used to study molecular pathogenesis of interstitial pneumonitis with regard to sequential histological changes and cytokine network induced by TNF-alpha. The authors divided the histological process of interstitial pneumonitis into three stages: early stage with lymphocytic infiltration in alveolar septa, middle stage with recruitment of macrophages, and late stage with hyperplasia of epithelial cells and mild fibrosis. As for cytokine network, prolonged overexpression of TNF-alpha along with increasing interleukin 6 (IL-6) were associated with the progression of interstitial pneumonitis. Increasing IL-1 was found only in the early stage, the beginning of lymphocyte proliferation. The mRNA level of an anti-inflammatory cytokine, IL-10, was constantly enhanced in the lungs of transgenic mice. However, transforming growth factor beta 1 (TGF-beta 1) protein decreased, which is closely associated with prolonged TNF-alpha synthesis, resulting in development of chronic inflammation and less severe fibrosis in the lungs of this animal model, analogous to inflammatory stage of human IPF. TNF-alpha transgenic mice enabled the analysis of the sequential process of interstitial pneumonitis as a model of IPF pathogenesis in humans, the results of which will give rise to new therapeutic measures for human IPF.

The most reliable means of identifying the hormone- secreting cells in the pituitary gland is immunohistochemical staining. This method has been used by various investigators to classify human and animal pituitary tumors. For many years, the classification of pituitary tumors into three types - acidophilic, basophilic, and chromophobic - was based on their staining by dyes, such as hematoxylin and eosin (H and E), periodic acid Schiff (PAS), orange G, aldehyde fuchsin, and erythrosin. It was believed that the chromophobic tumors did not produce hormone. However, immunohistochemical methods have clearly shown that many chromophobic adenomas do produce hormones; for example, almost all prolactin-secreting tumors are chromophobic (Osamura and Watanabe 1976; Osamura et al. 1978, 1980).

For the purpose of investigating the time of development and myelination of the optic nerve, we applied immunohistochemical stains to the optic nerves of 26 human embryos and fetuses aged 6–39 weeks. The minimum diameter of the optic nerve's cross section increased with the development of embryos; 0.15 mm at the 9th week, 0.4 mm at the 11th week, 1.5 mm at the 24th week, and 2.1 mm at the 32nd week. With regard to myelin basic protein (MBP), the localization could not be observed at 28 gestational weeks. The MBP was proved to be positive in oligodendrocytes of the optic nerve at the age of the 32nd gestational week. The localization of glial fibrillary acidic protein (GFAP) was proved partially in astrocytes around the optic nerve in the case of 24 gestational weeks. The remarkable localization of GFAP-positive astrocytes inside the nerve fibers was proved at the 27th gestational week. The histological appearance of GFAP-positive cells advanced to that of MBP-positive ones after 8 weeks interval. The degree of MBP staining was less marked than that of GFAP in examined fetal cases. These results suggest that astrocytes and oligodendrocytes correlate to the developmental growth and myelination of the optic nerves during the fetal development.

Three thyroid carcinogens, 2,4-diaminoanisole sulfate (DAAS), N,N'-diethylthiourea (DETU) and 4,4'-thiodianiline (TDA), were simultaneously administered in the diet to male F344 rats at one-third the concentration of the reported TD50 levels (610, 200 and 46 p.p.m. respectively) for 52 weeks. The resultant incidence of thyroid adenocarcinomas in the combined treatment group (18/18: 100%) was significantly higher than those in each individual treatment group (DAAS, 0/21; DETU, 1/21; TDA, 2/20). Hepatocellular carcinomas (9/18, 50%) and lung adenomas (6/18, 33%) were also induced by the combined treatment, the incidences being higher than in the TDA group (3/20, 15% and 4/20, 20% respectively) to which these tumors were limited in the three groups treated with single chemicals. Thus, the results indicated that the three carcinogens exert apparent synergism in thyroid carcinogenesis when administered simultaneously.

2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), which is produced during cooking and is mutagenic to bacteria and cultured mammalian cells, was found to induce high incidences of colon and mammary carcinomas in F344 rats when administered at a concentration of 400 p.p.m. in the diet for 52 weeks. Since PhIP is the most abundant of the mutagenic heterocyclic amines in cooked meat and fish, the compound might be related to malignancies of the colon and breast in humans.

The ultrastructure of gap junctions throughout the human foetal lens was observed. By freeze-fracture analysis, we observed numerous gap junctions in both lens cortex and lens nucleus. Comparison between lens cortex and lens nucleus showed that the gap junctions of lens nucleus are characterized by extreme mosaics of closely apposed P- and E-faces in junctional areas, though no significant difference in the area of gap junctions was observed between lens cortex and lens nucleus. In addition, some morphological variations, such as the smooth domains without particles or pits in junctional areas and the reticulated figures of gap junctions, were observed only in the lens nucleus. We also observed by thin-section electron microscopy that cell membranes of human foetal lens nucleus, as observed in the lens cortex, are mainly composed of continuous lipid bilayer and junctional structures. We concluded that characteristic morphology of lens gap junctions, as observed in the cortex of human foetal lens, is mostly preserved in the human foetal lens nucleus, although some depth-dependent alterations were also observed.

The objectives of this paper are to describe 1) the development of new research systems for biochemical comparison of cellular traits between normal and tumorous pigment cells of fish origin, 2) the similarity and dissimilarity between these two categories of pigment cells with regard to growth, differentiation and pigment translocating activities and 3) the potentials of these tumorous pigment cells to manifest multiple differentiation. The development of research systems has been achieved by the establishment of 1) methods to obtain homogenous populations of viable, cultivatable xanthophores (erythrophores) and melanophores from goldfish skins, 2) permanent cell lines from goldfish erythrophoromas (tumors derived from erythrophores) and from Nibe croaker irido-melanophoromas (tumors composed of mixed populations of iridophore- and melanophore-like cells) and 3) procedures to induce differentiation in normal and tumorous stem cells (including the formation of pigments and ability to undergo pigment translocation in response to cAMP, to the neurotransmitter epinephrine and to the hormones ACTH and melatonin). Two kinds of tumorous pigment cell lines examined herein have the ability to form, in addition to various pigments, structures similar to dermal skeletons and lentoid bodies. These findings strongly suggest the possibility that these fish pigment cell tumors are neural crest stem cell tumors in nature.

Hamster sternal chondrocytes were transfored morphologically in vitro 29-51 days after exposure to 4-nitroquinoline 1-oxide for 3 h. The transformed chondrocytes were randomly oriented, piled up and grew continuously. These cells produced nodules when transferred to the cheek pouch of a hamster. Histologically, these nodules revealed chondromatous patterns. Untreated cells in the control cultures failed to produce nodules.

Sequential histologic changes of the stomach during carcinogenesis induced by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG; CAS: 70-25-7) were studied in susceptible ACI and resistant BUF strain rats. Rats were given MNNG at a concentration of 83 micrograms/ml in their drinking water for 32 weeks and then tap water and were sacrificed sequentially between weeks 1 and 57. In ACI rats, erosions, regenerative changes, focal and slightly atypical changes, and diffuse and severe atypical changes were observed sequentially in the pyloric region during the period of MNNG administration, where adenocarcinomas were observed after the cessation of MNNG treatment. In BUF rats, the main histologic changes induced by MNNG were erosions and hyperplasia of the glandular portion of pyloric glands at the margin of erosions. After the cessation of MNNG treatment, the hyperplasia of the pyloric glands subsided and was followed by atrophy of these glands. The results suggested that the responses of the gastric mucosa to MNNG in ACI and BUF rats were qualitatively different.

Epithelial-mesenchymal interactions during development of the forestomach and glandular stomach in fetal mice were investigated by recombination experiments in vitro. Stomach epithelium could not survive when cultivated alone, but its development was supported by the presence of homologous or heterologous mesenchyme. The developmental fate of the epithelium was not affected by recombination with heterologous mesenchyme, but the expression of epithelial differentiation was influenced by the type of mesenchyme. The rate of keratinization of the forestomach epithelium was significantly greater on recombination with homologous mesenchyme than on recombination with heterologous mesenchyme. Moreover, the rate of formation of glandular structures in the glandular stomach epithelium was significantly greater on recombination with 16.5-day stomach mesenchyme than on recombination with 14.5- or 18.5-day stomach mesenchyme.

The effects of serum on the metabolism of benzo[a]pyrene (BP) by liver microsomes from 3-methylcholanthrene-treated rats were studied. In the presence of serum, the aryl hydrocarbon hydroxylase activities were enhanced to 160-570% of the control value. Enhancement of BP-metabolism by serum was also revealed by high performance liquid chromatography, but the relative amounts of a series of BP-metabolites were not changed appreciably by addition of serum. Binding of BP-metabolites to DNA was influenced by serum: adducts derived from two stereo-isomeric BP-7,8-di-hydrodiol-9,10-oxides were increased, while those from 9-hydroxybenzo[a]pyrene-4,5-oxide were reduced.

The conversion of 2-amino-6-methyldipyrido[1,2-a:3' ,2' -d]-imidazole (Glu-P-1), a highly mutagenic principle in a pyrolysate of glutamic acid, to protein-bound metabolites in vitro was examined with microsomes from various tissues of female F344 rats. Addition of NADPH to the incubation mixture containing microsomes and [14C]Glu-P-1 increased the binding of its metabolites to microsomal proteins linearly with time for up to 30 min, while on addition of arachidonic acid the binding increased linearly only for the first 2-4 min of incubation and then levelled off. However, due to the initial rapid binding, addition of arachidonic acid resulted in 6-fold greater binding of metabolites to small intestinal microsomes than addition of NADPH on incubation for 4 min, and with microsomes from liver and colon, arachidonic acid was found to be a better cofactor than NADPH for activation of Glu-P-1. Indomethacin significantly inhibited the increase in binding by arachidonic acid. Additions of linoleic and linolenic acids also increased the binding, but addition of oleic acid had no influence. With hepatic microsomes from 3-methylcholanthrene-treated rats, binding within 4 min after addition of arachidonic acid was greater than that after addition of NADPH and the reverse on further incubation. These findings suggest that prostaglandin synthetase may serve as an alternative enzyme to cytochrome P-450 monooxygenases for conversion of Glu-P-1 to active intermediates in all the rat tissues investigated.

Betel quid, a masticatory widely used in Sri Lanka, consists of Jaffna tobacco, the nuts and leaves of betel plants and calcium hydroxide. An ethyl acetate extract of Jaffna tobacco induced morphological transformation of hamster embryo cells. The extract also induced sister chromatid exchanges in virally transformed and phytohaemagglutinin (PHA)-stimulated human lymphocytes. The induction of sister chromatid exchanges was potentiated by addition of rat liver homogenate. The extract did not induce ouabain-resistant mutation of V79 cells. Extracts of betel nuts and leaves gave negative results in assay of morphological transformation, sister chromatid exchange and mutagenesis.

2-Amino-6-methyldipyrido[1,2-a:3',2'-d]imidazole and 2- aminodipyrido [1,2-a:3',2'-d]imidazole, potent mutagens from glutamic acid pyrolysate, were given orally to CDF1 mice of both sexes at concentrations of 0.05% in pellet diet. 2-Amino-3-methyl-9H-pyrido[2,3-b]indole and 2-amino-9H-pyrido[2,3-b]indole, potent mutagens from soybean globulin pyrolysate, were given to CDF1 mice of both sexes at concentrations of 0.08%. Hepatocellular carcinomas and hemangioendothelial sarcomas in the brown adipose tissue were induced in high incidence by all these compounds.

Two new gamma-carboline derivatives, 3-amino-1,4-dimethyl-5H pyrido[4,3-b]indole (Trp-P-1) and 3-amino-1-methyl-5H pyrido[4,3-b]indole (Trp-P-2), isolated from the pyrolysis products of tryptophan, were found to be potent mutagens. When weanling rats were injected with Trp-P-1 and then given diet containing phenobarbital, they developed enzyme-altered islands in the liver. Since these islands are considered to be formed by immediate progeny of "initiated cells" and cell precursors in hepatocarcinogenesis, Trp-P-1 may well be carcinogenic in rat liver.

A potent mutagenic compound, 2-amino-3-methylimidazo-[4,5-f]quinoline (IQ), isolated from broiled sardines, cooked beef and beef extract was tested for carcinogenicity in CDF1 mice of both sexes. Mice were given diet containing 0.03% IQ or control diet for up to 675 days. Tumors were observed mainly in the liver, forestomach and lung. In the mice given IQ, the incidences of these tumors were as follows: liver tumors - 41% in males and 75% in females; tumors of the forestomach - 41% in males and 31% in females; lung tumors - 69% in males and 42% in females. In the control mice, incidences of these tumors were as follows: liver tumors - 9% in males and 8% in females; tumors of the forestomach - 3% in males and 0% in females; lung tumors - 21% in males and 18% in females. The incidences of tumors in the liver, forestomach and lung were significantly higher in mice given IQ than in control mice.

An investigation was conducted to determine whether a mixture of low doses of forty carcinogens that target different organs, including the liver, intestine, thyroid, urinary bladder, and skin, is effective for tumor induction in F344/DuCrj rats. The dose of each carcinogen in the diet was 1/50 of the TD50 value, treatment being continued for 102 weeks. Significant numbers of neoplastic nodules of the liver and follicular cell tumors of the thyroid developed in the animals exposed to the carcinogen mixture, although the question of whether the observed carcinogenic effects were synergistic or additive could not be answered. The results serve to evaluate carcinogenic risk in the search for causes of human cancer.

Modulation of microsome mediated benzo[a]pyrene (BP) metabolism by oleic and linoleic acids was studied. Oleic and linoleic acids did not influence the apparent activity of aryl hydrocarbon hydroxylase or the relative ratios of BP-7,8-dihydrodiol to phenol metabolites on high performance liquid chromatography. The total binding of BP-metabolites to DNA was not influenced much by the presence of oleic acid, but was inhibited significantly by linoleic acid (p less than 0.05). The quantity of BP-metabolite bound adducts was determined by Sephadex LH-20 column chromatography with a methanol gradient. The amount of adducts bound with the anti-isomer of BP-7,8-dihydrodiol-9,10-oxide (diol-epoxide) was more than that with 9-hydroxybenzo[a]pyrene-4,5-oxide in the absence of fatty acids, and vice versa in the presence of fatty acids. The presence of 1, 2 or 5 microliters/ml oleic acid decreased the amounts of adducts with the anti- and syn-isomers of the diol-epoxide but not those with 9-hydroxybenzo[a]pyrene-4,5-oxides, whereas the presence of 0.08 - 2 microliters/ml of linoleic acid decreased the amounts of all these adducts but to different extents, resulting in predominance of 9-hydroxybenzo[a]pyrene-4,5-oxide derived adducts over anti-isomer adducts of the diol-epoxide. These observations suggest that endogenous materials that do not have any enzyme activity may have important influences on the metabolism of chemical carcinogens.

The inhibitory effects of hemin and related compounds on the mutagenicity of benzo[a]pyrene (BP) were investigated in Chinese hamster V79 cells co-cultivated with X-irradiated hamster embryo cells. Mutant V79 cells were selected by their resistance to ouabain. The mutation frequency induced by BP was substantially inhibited dose dependently by hemin. The mutagenicity of BP (1 microgram/ml) on V79 cells was reduced to 6.5% by hemin, 52% by biliverdin, 73% by protoporphyrin and 85% by chlorophyllin at the highest concentration of the compounds tested (15 microM).

The mutagenecities of dimethylnitrosamine (DMN) and diethylnitrosamine (DEN) on Chinese hamster V79 cells were examined by cocultivation of the cells with primary cultures of rat hepatocytes or hamster hepatocytes. Mutations in V79 cells were selected by resistance to ouabain. The mutation frequencies induced by all doses of DMN (1, 5, 10 and 50 μg/ml) and DEN (10, 50, 100 and 500 g/ml) were substantially higher on cocultivation with hamster hepatocytes than with rat hepatocytes. Thus it is proposed that hamster hepatocytes are useful for detecting mutagenesis in V79 cells induced by nitrosamines.

Massive abdominal enlargement was discovered in 8 adult carp (Cyprinus carpio) 4-6 years old, living in breeding ponds of carp fisheries in northern Japan. The abdominal enlargements rapidly progressed and the affected fish died within a few months. At necropsy, single or multiple tumors up to 20 cm in diameter were found in the abdominal cavities. Ovarian tissue identified within the tumor capsule in 4 fish supported the impression, based on gross anatomic features, that the neoplasm arose in the ovaries. Histologically, the tumors were composed mainly of various types of mesenchymal cells. In one sample, striated muscle was identified by electron microscopy; in another, squamous cell nests were intermixed with mesenchymal elements. Although some tumors had areas of cellular pleomorphism suggesting a malignant character, no evidence of invasion or metastasis was found. These neoplasms were classified tentatively as dysontogenetic tumors of the ovary, possibly teratoid in nature.

The formation of water-soluble metabolites of tritium-labeled benzo[a]pyrene (BP) by cultured hamster embryo cells was studied. The ratio of the radioactivity in the aqueous phase to that in the organic phase increased with the incubation period. After incubation for 48 h with 3.75 nmol/ml of [3H]BP in the medium more than 90% of the 3H-radioactivity was found in the aqueous phase, whereas with 10-fold more BP about half the radioactivity remained in the organic phase. The main metabolites extracted from the medium at 37.5 nmol/ml BP with ethyl acetate by high pressure liquid chromatography (HPLC) were 9,10-diol and 7,8-diol; but after treatment of the medium with β-glucuronidase the main oxygenated metabolites were phenols, the amount of 9-OH BP being more than that of 3-OH BP. β-Glucuronidase also released 9,10-diol and 7,8-diol, but most of these diols were in the free form in the medium. The medium from cells treated with 3.75 nmol/ml BP has a quantitatively different profile, and most of the radioactivity obtained by extraction with organic solvent and digestion with β-glucuronidase was eluted in the regions of phenols. These results show that in hamster embryo cells BP is mainly metabolised to conjugates of phenols with glucuronic acid.

In vitro carcinogenicity of betel tobacco extracts was examined in a transformation assay with cryopreserved hamster embryo cells. The ethanol extract induced a transformed colony at 100 μg ml and the ethyl acetate extract induced 2 transformed colonies at 5 μg ml and 2 at 100 μg ml .

Activation of benzo[a]pyrene to bind to proteins by co-oxidation with prostaglandin synthesis was studied in rat liver and lung microsomes and cytosols. The kinetics of this activation showed a two-phase reaction; a rapid initial reaction for 2-4 min after addition of arachidonic acid and then a slow reaction or a plateau state. The reaction was linear only with low contents of the enzyme proteins, and was slower with more than 1 mg of enzyme per ml of incubation mixture. Other unsaturated fatty acids were also effective in activating benzo[a]pyrene with both microsomes and cytosols. With microsomal proteins linoleic acid was more effective than arachidonic acid, whereas with cytosolic proteins arachidonic acid was the best cofactor. Linolenic acid could also activate benzo[a]pyrene, though less efficiently, but oleic acid had no influence on the binding. Indomethacin did not inhibit the activation, but nordihydroguaiaretic acid and quercetin significantly reduced the binding. Addition of hematin significantly increased the binding. The NADPH-dependent bindings of benzo[a]pyrene to proteins with liver and lung microsomes were one-third and one-twelfth the values after incubation with arachidonic acid. Addition of glutathione or Ca2+ ion reduced the binding significantly. The present results suggest the importance of co-oxidation with lipoxygenase for activation of benzo[a]pyrene and the possible role of both the arachidonic acid cascade system and the NADPH-dependent cytochrome P-450 system in metabolic activation of chemical carcinogens.

Liver neoplasms were induced in medakas (Oryzias latipes) by the addition of diethylnitrosamine (DENA) to their aquarium water at levels of 15-135 ppm for 8 weeks. After 13 weeks, 21 to 32 fish had developed hepatomas. Medakas are useful for further studies because they are highly susceptible to the carcinogenic effect of DENA, and the time for tumor induction is relatively short. Histologic type differed in the lesions of different fish and also within individual tumors. Some were typical trabecular hepatomas, others were anaplastic hepatomas or cholangiomas, or mixtures of these. Electron microscopy revealed an extensive rough-surfaced endoplasmic reticulum in a lamellar pattern, many mitochondria, and several round lysosomes in tumor cells. A few fat droplets with occasional crystalline ghosts were sometimes in the cytoplasm. The Golgi apparatus was not conspicuous. Some cells had highly developed microvilli that showed differentiation toward structures resembling bile capillaries.

A system in which oral tissues of inbred F344 adult rats and Syrian golden hamster embryos were used in combination with autoradiography was developed for measurement of unscheduled DNA synthesis (UDS). For this, oral mucosa, submandibular gland, tooth germ and mandible in short-term organ cultures were treated with 4-nitroquinoline 1-oxide or N-methyl-N-nitrosourea plus (methyl-3H)thymidine. Significant numbers of silver grains, indicating UDS, were detected over the nuclei of cells of all these tissues except rat salivary gland after treatment with carcinogens. This autoradiographic method is suitable for detection of UDS in oral tissues in conditions mimicking those in vivo. Results obtained in this study indicated a potential use of this system for studies on the mechanism of carcinogenesis at a cellular level comparable to in vivo carcinogenesis studies on oral tissues.