Search

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.

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.

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.

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.

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.

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.

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.

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 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.

The amount of DNA in cell nuclei was determined by microspectrophotometry on Feulgen-stained preparations using basal cells from the back skin of mice after painting with 3-methylcholanthrene (3-MC), croton oil and turpentine, and from skin after a burn. In the hyperplastic epithelium of the skin painted with 3-MC, the amount of DNA per cell nucleus was the same as that of normal skin but the mean amount of DNA in papilloma and atypical hyperplasia was slightly higher. However, no significant difference was found from that of the normal. In contrast to the above lesions, the amount of DNA in squamous cell carcinomas was elevated and the range of modal distribution was wider. In the hyperplastic epithelium induced by painting with croton oil or turpentine, the amount of DNA was also near the normal range. In the regenerating hyperplastic epithelium after a burn, the number of cells with an increased amount of DNA was higher for 2–5 days after the burn but no essential difference was observed from the normal.

Three kinds of cells were observed on intraocular lenses (IOLs) by specular microscope: fibroblast-like cells, small round cells and foreign body giant cells. With PMMA IOL implantation with good postoperative course, only a small number of these cells appeared immediately after surgery and they tended to disappear after 3 months. However, in cases with prolonged postoperative inflammation, a large number of cell proliferations was observed and these remained on the IOL surface for a longer time. With silicone posterior chamber IOLs, fewer cell reactions were observed. Judging from the clinical signs and visually, and also by observation of the cells by specular microscope, PMMA IOLs and silicone IOLs showed the same level of safety and good results when observed for as long as 2 1/2 years.

Experiments on the effect of transplacental initiation with 7,12-dimethylbenz(a)anthracene (DMBA) and postnatal promotion with teleocidin were carried out in mice. The percentage of tumor-bearing mice among females treated with DMBA transplacentally on day 17 of gestation and postnatally by topical application of teleocidin to the skin of the back was 73.3% in week 30, whereas that among females treated with DMBA on day 10 of gestation and postnatally by topical application of teleocidin was 20.0%. This indicates that teleocidin shows potent tumor promoting activity on mouse skin in a transplacental initiation and postnatal promotion protocol. Furthermore, in the males treated with DMBA transplacentally on day 17 of gestation and given diet containing 0.01% teleocidin postnatally five tumors of the forestomach were found in 5 of 19 effective mice (26.3%) in week 52. One of these five tumors was a squamous cell carcinoma, and the others were papillomas. This indicates that teleocidin also has tumor promoting activity in the forestomach of mice.

The mutagenic compound, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), originally isolated from cooked beef, was tested for carcinogenicity in CDF1 mice of both sexes. When PhIP was given in the diet at a concentration of 0.04%, high incidences and early appearances of lymphomas were observed in both sexes during the 579-day experiment.

A mutagenic compound, 2-amino-3,4-dimethylimidazo[4,5-f]quinoline, which was first isolated from broiled sardines and was shown to be carcinogenic to mice, was also found to be carcinogenic to F344 rats. It induced tumors in various organs, such as the Zymbal gland, oral cavity, colon, skin and mammary gland of male and female rats when given at 0.03% in the diet. However, it was noteworthy that tumors were not observed in the liver. Most of the tumors in the Zymbal gland, oral cavity and skin were squamous cell carcinomas, and most of the mammary gland tumors were adenocarcinomas. The colon tumors were identified as adenomas or adenocarcinomas. In control rats no tumors developed in these organs during the 40-week experiment.

The growth responses of hamster dermal fibroblasts (HDF cells) to platelet-derived growth factor (PDGF) during chemical carcinogenesis in vitro were investigated. Normal HDF cell grew in medium with whole blood serum (WBS), but not in medium with plasma-derived serum (PDS). However, they grew in PDS medium when PDGF was added. In contrast to control cultures which finally stopped proliferating, 7 out of 8 cell strains treated with 4-nitroquinoline-1-oxide (4NQO) or N-methyl-N'-nitro-nitrosoguanidine changed to cell lines. Later, 5 of these 7 cell lines became able to grow in PDS medium in association with ability to grow in soft agar. When clonal cell lines were isolated at early stages of carcinogenesis while parental cell lines were still PDGF-dependent, most of them gradually became PDGF-independent as well. Dialysed cell lysates of transformed HDF cells showed strong growth stimulating activity on normal HDF cells in PDS medium. Thus, this conversion of PDGF-dependent cells to PDGF-independent cells was correlated with the appearance of a growth factor(s) produced specifically by transformed HDF cells.

The effects of Brij 58 on microsome-mediated benzo[a]pyrene (BP)-metabolism were investigated. At a concentration of 0.01%, which did not decompose the microsomal mixed-function oxidase system, Brij 58 increased UDP-glucuronyl transferase activity on 3-hydroxy-BP to 560% of the control level. Quantitative changes of BP-metabolites by glucuronidation were found by high performance liquid chromatography. On incubation in the presence of UDP-glucuronic acid, the amounts of phenols decreased but the amounts of dihydrodiols did not change. Brij 58 enhanced the decrease in phenol metabolites. Unexpectedly, the binding of BP-metabolites to DNA was found to be higher in the presence of UDP-glucuronic acid, and Brij 58 also enhanced the binding in the presence of UDP-glucuronic acid. The ratio of bound adducts with BP-7,8-dihydrodiol-9,10-oxide to those with 9-hydroxy-BP-4,5-oxide, which were separated by Sephadex LH-20 column chromatography, was increased by UDP-glucuronic acid and further enhanced by Brij 58. These results are discussed in relation to the role of glucuronidation in metabolism of BP.

3-Amino-1-methyl-5H-pyrido[4,3-b]indole (Trp-P-2), a mutagenic principle in tryptophan pyrolysates, binds to DNA after metabolic activation by rat liver enzymes. The enzymes for activation of Trp-P-2 were found in both microsomes and the cytosol. The reaction required NADPH and ATP, metabolic and was inhibited by 7,8-benzoflavone. Considerable binding was observed with only microsomes as enzyme source, but further addition of cytosol enhanced the binding, enhancement depending on the amount of cytosol added. Inducers for microsomal mixed-function oxidases induced activating enzyme(s) for Trp-P-2, 3-methylcholanthrene being most effective, followed by polychlorinated biphenyls and then phenobarbital.

The in vitro carcinogenic activities of 3-(N-salicyloyl)amino-1,2,4-triazole (SAT) and its two components, 3-amino-1,2,4-triazole and phenyl salicylate were examined in a transformation assay with cryopreserved hamster embryo cells. SAT induced morphological transformation at certain doses between 10 μg ml and 100 μg ml in each of 5 repeated experiments, but gave a negative result in the Salmonella mutation assay. 3-Amino-1,2,4-triazole also induced transformation in 3 repeated experiments. Phenyl salicylate did not induce transformation at any of the doses tested in 3 consecutive experiments.

Long-term in vivo carcinogenicity tests of potassium bromate (KBrO3), sodium hypochlorite (NaClO), and sodium chlorite (NaClO2) have been conducted in Japan from 1977 to 1985. In these investigations, groups of approximately 50 male and 50 female F344 rats or B6C3F1 mice were given solutions of the compounds as their drinking water ad libitum at two dose levels determined on the basis of preliminary 13-week tests. Control animals were given distilled water. The carcinogenic potential of KBrO3 was tested by administering doses of 500 or 250 ppm to rats for 110 weeks. Significantly elevated incidences of renal cell tumors in males and females and mesotheliomas of the peritoneum in males as compared to controls were observed. When female mice were given KBrO3 at doses of 1000 or 500 ppm for 78 weeks, no significant differences in tumor incidences between experimental and control groups were apparent. NaClO was administered to male and female rats, respectively, at doses of 1000 or 500 ppm and 2000 or 1000 ppm for 104 weeks. In mice, NaClO was given at doses of 1000 or 500 ppm to either sex for 103 weeks. The incidences of tumors in NaClO-treated and control animals of both sexes were not significantly different in both rat and mouse studies. NaClO2 was given to rats of both sexes at a dose of 600 or 300 ppm for 85 weeks. No statistically significant differences were observed in the incidences of tumor formation between NaClO2-treated and control groups of both sexes. NaClO2 was administered to mice at a concentration of 500 or 250 ppm for 85 weeks. In males, the combined incidences of hyperplastic nodules and hepatocellular carcinomas of the liver in a low-dose group, and adenomas and adenocarcinomas of the lung in a high-dose group, were marginally increased compared to controls (p less than 0.05). However, these incidences in treated males were within the range of values of historical control data in our program. We concluded that KBrO3 was carcinogenic in rats of both sexes. NaClO was not carcinogenic in either rats and or mice under the conditions of the present studies. Although NaClO2 was shown to be noncarcinogenic in rats, the results for mice were evaluated as inconclusive. Also the results of two-stage mouse skin carcinogenesis using KBrO3, NaClO, and NaClO2 are presented. The necessity for further testing of oxidant chemicals to determine potential carcinogenic and/or promoting effects is suggested in view of the recently proposed role of active oxygen species in carcinogenesis. Images FIGURE 3. FIGURE 4.

2-(2-Furyl)-3-(5-nitro-2-furyl)-acrylamide (AF-2) induced the malignant transformation of secondary cultures of Syrian golden hamster embryo cells prepared from cryopreserved primary cells. Transformed cells grew in semi-solid agar medium and formed sarcomas when inoculated subcutaneously into non-immunosuppressed suckling hamsters.

A rat hepatocellular carcinoma, IQ7, induced by 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) gave two transformants of NIH 3T3 cells on DNA mediated gene transfer. One of these transformants was examined further and secondary and tertiary transformants were obtained. The secondary transformant was tumorigenic in nude mice. The activated oncogene in this primary transformant was identified as rat c-raf by Southern blot analysis.

A mutagenic nitrosation product of tyramine, 4-(2-aminoethyl)-6-diazo-2,4-cyclohexadienone (3-diazotyramine, 3-DT) preferentially induced tumors of the oral cavity. Squamous-cell carcinomas of the mucosa of the oral cavity floor developed in 19 out of 28 male F344 rats administered 0.1% 3-DT in their drinking water. Tyramine and nitrite are found at fairly high concentrations in various foods. This demonstration of the carcinogenicity of 3-DT indicates that although the implications of 3-DT for human cancer are not clear, other nitrosable mutagen precursors need to be tested as possible risk factors in human cancer.

Induction of unscheduled DNA synthesis (UDS) (repair DNA synthesis) in stomach pyloric mucosa of the F344/- DuCrj rat was examined in in vitro organ cultures in the presence of tritiated thymidine ([3H]dThd) and hydroxyurea after administration of chemical carcinogens in vivo. The DNA fraction was extracted from the cultured tissue, and the incorporation of [3H]dThd into DNA was determined in a liquid scintillation counter. DNA concentration was determined spectrophotometrically with either diphenylamine or 3,5- diaminobenzoic acid. A good correlation between induction of UDS and site specificity of carcinogens was observed. The glandular stomach carcinogens N-methyl-N'-nitro-N-nitrosoguanidine (CAS: 70-25-7), N-ethyl-N'-nitro-N-nitrosoguanidine (CAS: 63885-23-4), N-propyl-N'-nitro-N-nitrosoguanidine, 4-nitroquinoline 1-oxide (CAS: 56-57-5), and N-nitroso-N-methylurethane (CAS: 615-53-2) induced UDS in the pyloric mucosa of the stomach. UDS could be detected 2-4 hours after administration of carcinogens in vivo by the present method. The forestomach carcinogens 1-methyl-1-nitrosourea (CAS: 684-93-5) and aristolochic acid (CAS: 1398-06-7) and the nongastric carcinogens 2-acetylaminofluorene (CAS: 53-96-3), 3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indole, 3-amino-1-methyl-5H-pyrido[4,3-b]indole, and dimethylnitrosamine (CAS: 62-75-9) did not induce UDS in the pyloric mucosa.

Sulfate conjugation of benzo[a]pyrene(BP) metabolites and derivatives was studied. The reaction sequence consisted of two steps; activation of sulfate ion to 3′-phosphoadenosine-5′-phosphosulfate and transfer of the activated sulfate to the BP-derivatives. Both reactions were carried out by enzymes located in the rat liver 105 000 g supernatant. The reactions required MgCl2. Phenol and quinone derivatives were generally good substrates for sulfate conjugation and different reactivities were observed with the dihydrodiol derivatives. Sulfate conjugates were more polar than their parent BP-derivatives and except for quinone conjugates were easily extracted with ethyl acetate. The role of sulfate conjugation in BP carcinogenesis is discussed.

A system was developed in which organ culture of human bronchial epithelium was used in combination with autoradiography for quantitative measurement of unscheduled DNA synthesis (UDS) in bronchial epithelial cells. Human bronchi obtained at surgery were cut into small sections and treated with various carcinogens plus (methyl-/sup 3/H)thymidine in short-term organ culture. Significant numbers of silver grains, indicating UDS, were detected on the nuclei of epithelial cells of human bronchi treated with carcinogens, and the numbers were proportional to the concentrations of carcinogens. In this system seven representative carcinogens induced UDS. Four active metabolites of benzo(a)pyrene, and benz(a)anthracene also were found to induce very active UDS in human bronchial epithelium. These findings suggest that human bronchial epithelial cells can repair different types of DNA modification induced by chemical carcinogens.

Unscheduled DNA synthesis (UDS) was investigated by autoradiography in human oral mucosa treated with 10 representative chemical carcinogens. Small sections of gingiva in short-term organ culture were exposed for 2 hours to chemical carcinogens plus [methyl-3H]thymidine. Significant numbers of silver grains, indicating UDS, were detected over the nuclei of both epithelial cells and fibroblasts. All ultimate or proximate carcinogens tested induced UDS. Of five procarcinogens tested, only benzo[a]pyrene (BP) did not induce UDS, but the more activated metabolite of BP, (+/-)-trans-7 beta-8 alpha-dihydroxy-9 alpha, 10 alpha-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE I), was very effective in inducing UDS. Ultimate or proximate carcinogens induced higher levels of UDS than did procarcinogens. All the carcinogens that induced UDS showed clear dose-dependent effects. UDS levels were twofold to fivefold higher in the epithelial cells than in the fibroblasts, regardless of the type of carcinogen tested. Comparisons of the levels of UDS induced by 4-(hydroxyamino)quinoline 1-oxide, methyl methanesulfonate, or BPDE I did not reveal any significant differences. These findings indicate that human gingival epithelial UDS assay should be useful for short-term detection of environmental carcinogens that induce cancer in human oral mucosa.

A strong skin irritant, lyngbyatoxin A, isolated from the marine blue-green alga Lyngbya majuscula is structurally related to teleocidin. Since lyngbyatoxin A statisfied our short-term screening tests for possible tumor promoters, viz. irritation of mouse ear, induction of ornithine decarboxylase (ODC) in mouse skin, and adhesion of human promyelocytic leukemia cells (HL-60), a two-stage carcinogenesis experiment was carried out. Tumor incidences in the groups treated with 7,12-dimethylbenz(a)anthracene (DMBA) plus lyngbyatoxin A and with DMBA plus 12-O-tetradecanoylphorbol-13-acetate (TPA) were 86.7% and 93.3% in week 30, respectively. The average number of tumors per mouse was 3.7 in the former group and 10.5 in the latter group. This paper reports for the first time the potent tumor-promoting activity of lyngbyatoxin A and also the histological examination of tumors.

Attempts to establish permanent cell lines from spontaneous erythrophoromas (tumors derived from red pigment cells or erythrophores) of goldfish were made with the use of biopsy specimens from 12 tumors in 10 fish. Three cell lines were established that grew in vitro in synthetic medium (L-15 or Dulbecco's modified Eagle's minimum essential medium) supplemented with 20% fetal bovine serum for more than 8 months. One of these lines (GEM-81) with high proliferative activity was cultured for over 200 generations without an obvious change in growth rate. From this cell line, clonal cultures were obtained that formed clones with relatively intense yellow pigmentation. Descendants of these cell lines and clones contained low but detectable amounts of pteridine pigments (such as 7-hydroxybiopterin, biopterin, xanthopterin, and isoxanthopterin) and numerous cytoplasmic organelles analogous to pterinosomes. Both these characteristics are phenotypic markers of normal erythrophores and their neoplastic counterparts. After numerous subcultivations, these long-term cultures differed from those of the initial explants in having much lower contents of total pteridines and relatively lowered contents of 7-hydroxybiopterin. As manifestations of their neoplastic origins, all cell lines examined showed disoriented growth with dense focal mounding in monolayer cultures. The population doubling time of the uncloned GEM-81 cell line was 31 hours at 35 degrees C over a feeder cell layer and 3 days at 25 degrees C without feeder cells. Injection of cultured cells (1.5 x 10(7) cells/fish) into normal goldfish that had not been immunosuppressed did not give rise to tumors within 5 months.

Palytoxon, which is a toxin with a molecular weight of 2681 daltons isolated from a marine coelenterate, is a potent skin irritant. However, it did not induce ornithine decarboxylase in mouse skin, or adhesion of human promyelocytic leukemia cells (HL-60). Moreover, it did not inhibit the specific binding of [3H]12-O-tetradecanoylphorbol-13-acetate (TPA) to a mouse skin particulate fraction or activate protein kinase C isolated from mouse brain in vitro. Since palytoxin showed strong irritation on mouse ear in one short-term screening test for a promoter, it was examined in a two-stage carcinogenesis experiment. The incidence of tumors in a group of mice treated with 7,12-dimethylbenz[a]anthracene plus palytoxin was 62.5% in week 25. These tumors were identified histologically as seven papillomas and one carcinoma. This paper reports the potent tumor-promoting activity of palytoxin, which is classified as a non-TPA-type tumor promoter.

A system in which the retinal tissues of noninbred Wistar rats were used in combination with autoradiography was developed for measurement of DNA repair synthesis in ganglion cells of the central nervous system. Retinal tissues in short-term organ culture were treated with various carcinogens plus tritiated thymidine ([methyl-3H]dThd) or were irradiated with UV light and then treated with [methyl-3H]dThd. Preliminary study with retinal tissues from rats at various ages revealed no age-associated changes in the levels of unscheduled DNA synthesis in ganglion cells.

A poly(ADP-ribosyl)ation reaction is suggested to be involved in DNA repair, mutagenesis, cell transformation, and cell differentiation [1–3].

2-Amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx), which is a mutagenic compound present in fried beef and beef extracts, was given orally to CDF1 mice at a concentration of 0.06% in the diet for 84 weeks. Liver tumors were induced in 43% of males and 91% of females fed MeIQx. The incidences of liver tumors in mice of both sexes were significantly higher in groups fed MeIQx than in control groups. The incidences of lung tumors in females fed MeIQx and of lymphomas and leukemias in both sexes fed MeIQx were also significantly higher than in the respective controls.

Summary Addition of sodium nitrite to cell cultures obtained from newborn Syrian hamsters resulted in transformation. The shortest time required for morphological transformation after this treatment was 21 days. Two of 5 transformed cultures produced progressively growing tumors when injected into animals. These tumors were diagnosed as fibrosarcomas. In two control cultures of the same experimental groups, transformed cells appeared 10 weeks or more later than in treated cultures.

Microspectrophotometric evaluation of the DNA content of cell nuclei and 3H-thymidine autoradiography were carried out on the cells from cerebellums, thymus, glandular stomach, liver, and kidney of embryonic, suckling, and adult Syrian hamsters and Buffalo rats. The results obtained from this study were as follows: 1) In both hamsters and rats, the cells with diploidDNA (2C) showed the main modal peak in various organs examined of embryonic to 6-month-old animals. 2) In liver tissues, tetraploid cells (4C) began to appear in the 3-day-old animals and increased gradually in number until 6 months old, 40% or more of the cells showing tetraploid value of DNA. 3) No cells with intermediate or extra DNA were found in tissues examined during the growth period of animals, and neither octaploid nor higher nuclei appeared even in liver tissues of the adults. 4) 3H-thymidine labeling index showed high percentages in organs of embryo to 1 day after birth. 5) 3H-thymidine labeling index rapidly declined 1 week after birth in all the organs except in the cells of glandular stomach. 6) There is a close correlation between labeling and mitotic indices and the distribution pattern of DNA value. From these data it would be supposed that the appearance of the cells with extra or intermediate DNA value is due to the existence of the cell in S-phase.