Investigation of new cytogenetic biomarkers specific to high-LET radiation using in vivo and in vitro exposed human lymphocytes.

PURPOSE: To find detectable cytogenetic biomarkers that can offer information about the radiation quality of in vivo exposure retrospectively. MATERIALS AND METHODS: Chromosome-type aberrations of peripheral lymphocytes of uterine cancer patients that received internal gamma- and external X-ray therapy or carbon beam therapy and of victims severely exposed to neutrons and gamma-rays in a criticality accident that occurred in Tokai-mura, Japan were analysed. Data obtained from in vitro irradiation experiments using 60Co gamma-rays and 10 MeV neutrons were compared with the in vivo exposure data. RESULTS: The ratio of acentric rings to dicentric chromosomes (termed RaD ratio) and that of excess fragments to dicentrics (termed EfD ratio) showed significant (p < 0.05) differences between the two groups of cancer patients, and these ratios for accidental victims were in between the values of the two groups of cancer patients. The in vitro studies using doses equivalent to 1 - 3 Gy of gamma-rays have confirmed that the EfD ratios were increased with the high LET (linear energy transfer) and RaD ratios decreased. CONCLUSION: The present data show that the RaD and EfD ratios can be used as cytogenetic biomarkers of exposure to high-LET radiation at least within a few years of exposure.

Existence of N-nitroso-N-propylurea target cells in the thymus of F344 rats in thymic lymphomagenesis.

There are two hypotheses for location of first transformation of cells of T-cell lineage into preneoplastic cells from studies of leukemogenesis in mice; one is the bone marrow and another is the thymus. N-Nitroso-N-propylurea [(NPU) CAS: 816-57-9] induces high incidence of thymic lymphoma in F344 rats. In the present experiments, the location of NPU-target cells was examined in F344 rats. In the first experiment, bone marrow cells from NPU-treated male rats were inoculated into sublethally irradiated female rats. However, neither thymic nor other types of leukemias were induced in these rats. In the following experiment, thymectomized male rats received grafts sc with normal thymuses of age-matched female F344 rats. Continuous administration of NPU to the rats successfully induced 9 thymic lymphomas in the grafted thymuses. In 8 thymic lymphomas analyzed, 6 consisted of donor cells and the other 2 consisted of recipient cells. The present results from these 2 experiments strongly suggested that NPU-induced rat thymic lymphomas originate from intrathymic cells but not from bone marrow cells. In other words, target cells of leukemogenic activity of the chemical carcinogen NPU probably exist in the thymus of F344 rats.

Morphologic characteristics of thymic lymphomas induced by N-nitroso-N-propylurea in F344 rats.

Eighty-two thymic tumors induced by N-nitroso-N-propylurea in inbred F344/DuCrj rats were examined by light and electron microscopy. These tumors were diagnosed as malignant lymphomas and classified according to light microscopic features into three types: 1) lymphoblastic (57%), 2) large cell (32%), and 3) pleomorphic (11%). Electron microscopy revealed no epithelial cells in all 82 malignant lymphomas, except for 2, in which one sheet of epithelial cells was found under the capsule. This finding confirmed that all of these thymic tumors were malignant lymphomas, not thymomas. The tumors of the lymphoblastic and large cell types consisted of lymphoid cells with a few macrophages. Lymphoid cells of the lymphoblastic type were medium sized and contained a moderate amount of polyribosomes and a few clustered dense bodies; cells of the large cell type were much larger than those of the lymphoblastic type and contained many more polyribosomes and larger nucleoli. The tumors of the pleomorphic type consisted of lymphoid cells with severely infolded nuclei and interdigitating reticulum cells that were thought to be nonneoplastic in nature. No viral particle was found in these cells among the three types of thymic lymphomas.

Induction of Leukemias and digestive tract tumors in Donryu rats by 1-propyl-1-nitrosourea.

Three groups of female Donryu rats were continuously given 600, 300, or 150 ppm solution of 1-propyl-1-nitrosourea in their drinking water. Leukemias developed in 62 of 109 (57%) rats surviving for more than 17 weeks and tumors developed in the digestive tracts of 31 (28%) animals. Of the leukemias, the differentiated myelocytic type was the most frequent, followed by myeloblastic leukemia and erythroleukemia. Tumors in the digestive tract, predominantly in the glandular stomach and duodenum, were both epithelial and nonepithelial. The other induced tumors were mainly in the mammary glands, ear ducts, and thymus, though the incidence was less than 15%.

Relation between development of leukemia and duration of N-nitroso-N-ethylurea treatment in DONRYU rats.

N-Nitroso-N-ethylurea (NEU; CAS: 759-73-9) is a strong leukemogen that induces erythroblastic leukemia in inbred DONRYU rats. In the present experiments, relationships between development of leukemia, duration of NEU treatment, and sequential changes in the hematopoietic organs during carcinogen administration were examined. In experiment 1, groups of rats were given a 400-ppm NEU solution for 0, 2, 4, 6, 8, or 10 weeks, and the resultant incidence of leukemias was 0, 26, 40, 75, 95, and 100%, respectively. Of the various types of leukemia, the erythroblastic type was observed in 0, 0, 20, 40, 95, and 90% of rats, respectively. The average latent period showed an inverse correlation with the duration of NEU treatment. In experiment 2 the animals were divided into carcinogen-treated and control groups, and rats were sacrificed periodically for histopathologic examination. In the experimental group, the bone marrow became hypoplastic soon after commencement of NEU treatment and at the 6th week became severely aplastic, thereafter recovering slightly. At the 10th week, 2 rats out of 5 examined were leukemic. Relationships between incidence of leukemia, duration of NEU treatment, and sequential changes of the bone marrow during carcinogen administration are discussed.

Induction of duodenal tumors in F344 rats by continuous oral administration of N-ethyl-N-nitrosourea.

Forty male and 40 female inbred F344 rats were given a solution of 400 mg N-ethyl-N-nitrosourea/liter in their drinking water. Digestive tract tumors were induced in 32 males (an incidence rate of 80%) and in 28 females (an incidence rate of 70%). Among these digestive tract neoplasms, duodenal tumors occurred most frequently. Most were of the epithelial type, such as adenoma or adenocarcinoma. Tumors in hematopoletic organs were also found in 15 males (38% incidence) and in 17 females (43% incidence).

Development of thymic lymphomas by oral administration of N-nitroso-N-propylurea and establishment of transplantable lines of thymic lymphoma in F344 rats.

Forty-eight female F344/DuCrj rats were given a 400-ppm solution of N-nitroso-N-propylurea (CAS: 816-57-9) continuously in their drinking water. Thymic lymphomas were induced most frequently (85%) followed by duodenal tumors (48%). Sixteen tumors were examined by the immunofluorescence inhibition test for murine leukemia virus-related antigens; 15 were negative and the other was weakly positive. Twenty-six tumors were intraperitoneally and subcutaneously transplanted syngeneically; 25 (96%) were successfully transplanted intraperitoneally and 24 (92%) subcutaneously. The serial intraperitoneal transplantation was continued, and 22 lines of transplantable lymphoma in an ascites form were established. In almost all tumor lines, tumor cells took in a high percentage of recipient rats and caused the death of the recipients within 12-23 days. The thymus, liver, spleen, greater omentum, and lymph nodes were frequently invaded by transplanted tumor cells. The tumor lines were considered to be of T-cell lineage.

Long-term studies on carcinogenicity and promoting effect of phenylbutazone in DONRYU rats.

The carcinogenicity and promoting effect of phenylbutazone were investigated in inbred DONRYU rats. In the carcinogenicity study, both sexes were administered the chemical at dietary levels of 0 (control), 0.125, or 0.25% for 2 years. Toxic lesions were associated with phenylbutazone treatment in the kidney and digestive tract, appearing to have an adverse effect on life expectancy. Various tumors were detected in all groups including the controls. With the exception of pheochromocytoma in the female high-dose group, no statistically significant increase in yield of any tumors, including leukemia, was apparent in the treated groups of either sex when the data were analyzed by Fisher's exact probability and/or chi-square tests. Application of an age-adjusted statistical analysis revealed a slight positive effect regarding the occurrence of pheochromocytomas, neoplastic liver nodules, and leukemias in females. However, these tumors are commonly observed to develop spontaneously in this rat strain, and no such effect was apparent in the male groups. In addition, no differences in incidences of relevant preneoplastic lesions were evident between control and treated groups. Thus phenylbutazone showed no carcinogenic activity in DONRYU rats when given continuously in the diet for 2 years. For the investigation of promoting effect, phenylbutazone was given as a dietary supplement for 2 years subsequent to initiation with N-ethyl-N-nitrosourea or N-propyl-N-nitrosourea. No enhancement of nitrosourea-induced leukemogenesis was apparent, although a slight promoting effect was demonstrated for renal and thyroid tumorigenesis.

Reduced fidelity of DNA synthesis in cell extracts from chemically induced primary thymic lymphomas of mice.

To examine whether the fidelity of DNA synthesis is reduced in tumor cells, M13 mp2-based fidelity assays were carried out using 15 samples of whole-cell extracts from primary mouse thymic lymphomas induced by alkylating agents. We found that DNA synthesis activities of thymic lymphomas, detected as incorporation of [3H]TTP into acid-insoluble materials, were 2- to 10-fold higher compared to those of normal thymus. Furthermore, mutant frequencies in the forward mutation assay of DNA synthesis were increased 2- to 7-fold in cell extracts from thymic lymphomas compared to those from normal thymus. As the DNA polymerase beta (pol beta) activity was extremely high in the thymic lymphomas, we screened mutations in the pol beta gene to examine the possibility of involvement of mutated pol beta in reduction of the fidelity of DNA synthesis. Of 20 lymphomas, one case of point mutation (T to A) was found by reverse transcription-PCR single-strand conformation polymorphism analysis. These results suggest that the mutagenic DNA synthesis is involved in murine thymic lymphoma genesis, although mutation of the pol beta gene is not a major causal event.

Experimental induction of ovarian Sertoli cell tumors in rats by N-nitrosoureas.

Spontaneous ovarian tumors are very rare in ACI, Wistar, F344 and Donryu rats; the few neoplasms found are of the granulosa/theca cell type. Ovarian tumors were also rare in these strains of rats when given high doses of N-alkyl-N-nitrosoureas continuously in the drinking water for their life-span; however, relatively high incidences of Sertoli cell tumors or Sertoli cell tumors mixed with granulosa cell tumors were induced in Donryu rats after administration of either a 400 ppm N-ethyl-N-nitrosourea solution in the drinking water for 4 weeks or as a single dose of 200 mg N-propyl-N-nitrosourea per kg body weight by stomach tube. Typical Sertoli cell tumors consisted of solid areas showing tubular formation. The tubules were lined by tall, columnar cells, with abundant, faintly eosinophilic, often vacuolated cytoplasm, and basally oriented, round nuclei, resembling seminiferous tubules in the testes. In some cases, Sertoli cell tumor elements were found mixed with areas of granulosa cells. The induction of ovarian Sertoli cell tumors in Donryu rats by low doses of nitrosoureas may provide a useful model for these tumors in man.

Cytogenetic studies on rat thymic lymphomas induced by N-propyl-N-nitrosourea.

N-Propyl-N-nitrosourea (PNU) was proved to be a strong leukemogen, which induces myelogenous leukemia or thymic lymphoma in rats. BUF/Mna rats and F344 rats were the strain most susceptible to thymic lymphomagenic activity of PNU. In addition, F1 rats between BUF/Mna and WKY rats were also susceptible to PNU-lymphomagenic activity. In the present experiment, karyotypes of 31 thymic lymphomas induced by PNU in BUF/Mna rats and in F1 rats between BUF/Mna and WKY rats were analysed for chromosomal abnormalities. Although no specific chromosomal abnormalities were observed throughout all lymphomas, del(11q) and dup(2q) were observed frequently in BUF/Mna rat lymphomas. Breakpoints and/or fusion-points were frequently observed in chromosome 11, followed by chromosomes 2, 5 and 6. Trisomy of chromosome 7, on which c-myc oncogene is mapped, was observed in seven cases, and monosomy of chromosomes 12, 18, 19, 20 and X was seen in seven or eight cases each, though these changes were generally observed in minor cell population in each case.

Induction of lung tumors and peritoneal mesotheliomas in F344 rats given intragastric N-propyl-N-nitrosourea and histochemical, immunohistochemical, and ultrastructural characteristics of induced mesotheliomas.

N-Propyl-N-nitrosourea is a strong leukemogen that induces myelogenic leukemia in Donryu rats and thymic lymphoma in F344 rats when administered in drinking water. In the present study, a single or multiple doses of PNU (total 500 mg/kg body weight) was given to young male and female F344 rats via a stomach tube. The results demonstrated that the percentage of tumor-bearing rats was 100% in all PNU-treated male groups, while that of the control group was 46%. Predominant tumors induced by PNU in male rats were lung adenoma/adenocarcinoma followed by peritoneal mesothelioma, and forestomach papilloma. In females, the tumor incidence of PNU-treated groups varied between 58% and 92% while that of the control group was 42%. Although pituitary tumor was the most frequent tumor in PNU-treated female rats, it was thought to be spontaneous since its incidence in each experimental group was not statistically different from that of the control group. Lung tumors and forestomach papillomas were also induced by PNU in female rats. No thymic lymphoma, however, was found in any of the PNU-treated groups of either sex. Lung tumors developed in almost all PNU-treated male rats and in about one-third of PNU-treated female rats. Mesothelioma was induced only in male rats, and its incidence depended on the treatment schedule. Induced mesotheliomas were extensively examined histologically, histochemically, immunohistochemically, and electron microscopically.

DNA strand breaks and death of thymocytes induced by N-methyl-N-nitrosourea.

N-Methyl-N-nitrosourea (MNU) is a potent carcinogen in various sites of experimental animals and induces thymic lymphoma in rats, which has long been hard to induce by any carcinogen. To analyze the action of MNU on thymocytes, DNA strand breaking in thymocytes from the MNU-treated rat and that in MNU-treated cultured thymocytes were assayed. Fluorometric analysis of DNA unwinding (FADU assay), first reported by Birnboim and Jevcak to detect X-ray-induced DNA damage, was modified and applied to detect DNA damage in thymocytes treated with MNU in vitro or in vivo. In the present modified method, cell lysate was admixed with 0.15 M sodium hydroxide, and DNA unwinding was processed at pH 12.0 for up to 2 h at 0 degree C in iced water. Double-stranded DNA remaining after alkaline reaction was detected by binding ethidium bromide and measuring its fluorescence. The severity of DNA damage, both in vivo and in vitro, depended on the MNU concentration. In addition, the sequential survival rate and cell-size distribution of thymocytes treated with MNU in vitro were investigated. A close relationship between the severity of DNA damage and cell death was demonstrated in MNU-treated thymocytes, and DNA damage by a non-cell-killing dose of MNU was detected with this FADU assay. MNU-induced cell death is not programmed as in apoptosis, which is caused in thymocytes physiologically, immunologically and by X-ray irradiation or corticoids.

Comparison of dose-dependent enhancing effects of gamma-ray irradiation on urethan-induced lung tumorigenesis in athymic nude (nu/nu) mice ac (nu/+) littermates.

The role of immunological surveillance in carcinogenesis is still controversial. In our previous experiments, urethan-induced lung tumorigenesis in athymic (nu/nu) mice and euthymic (nu/+) littermates was examined, and it was concluded that immunosurveillance mediated by T cells could not be demonstrated. However, the reported enhancement of development of various tumors following ionizing radiation might be achieved through modulating the host immunological conditions. In the present experiment, nu/nu and littermate nu/+ mice were treated with 1-4 Gy gamma-rays alone at 6 weeks of age or treated with urethan at 0.5 mg/g body weight when aged 14 days followed by 1-4 GY gamma-rays 4 weeks later. Lung tumors were assessed at 6.5 months of age. Ionizing radiation itself caused a very low incidence of these lesions. On the other hand, multiplicities and incidences of lung tumors after urethan treatment at 0.5 mg/g body weight were similar between the two phenotypically different groups of mice (1.66 and 1.84 tumors/mouse, 73% and 80% incidences, for nu/nu and nu/+ cases respectively). This urethan-induced lung tumorigenesis was significantly enhanced by gamma-rays in both nu/nu and nu/+ mice, and the magnitude of tumor enhancement was somewhat higher in nu/+ mice than in nu/nu mice, especially with a 2-Gy dose. In conclusion, it may be said that lung tumorigenicity of gamma-ray irradiation itself and the enhancing effect of radiation on urethan-induced tumorigenesis are scarcely influenced by immunosurveillance mechanisms mediated by T cells.

Effect of Nocardia rubra cell wall skeleton and/or cyclophosphamide on leukemogenesis induced by N-ethyl-N-nitrosourea in Donryu rats.

The effect of Nocardia rubra cell wall skeleton (N-CWS) and/or cyclophosphamide (CP) on chemical carcinogenesis was examined in female Donryu rats exposed to N-ethyl-N-nitrosourea (ENU) in the drinking water for 6 weeks. Five administrations of N-CWS following ENU treatment caused a slight prolongation of the average survival of rats but did not reduce the incidence of leukemia. CP given on two occasions after ENU treatment caused a moderate prolongation of average survival period and a moderate reduction of the incidence of leukemia, but significant differences from ENU-treated control group values were not observed after statistical analysis. Combined treatment with N-CWS and CP after ENU treatment caused prolongation of the average survival period of rats and a statistically significant reduction in the incidence of leukemia. The present experiment indicates that combined treatment with N-CWS and CP effectively reduces induction of leukemia by ENU in rats, although other types of tumors were not affected.

Induction of digestive-tract tumors in F344 rats by continuous oral administration of N-butyl-N-nitrosourea.

Male and female F344/DuCrj rats were administered N-butyl-N-nitrosourea at a concentration of 400 ppm in their drinking water. By the 50th week of the experiment, the cumulative incidence of upper-digestive-tract tumors was as high as 35/39 (90%) and 34/39 (87%) in male and female rats, respectively. Among these, esophageal and forestomach tumors occurred most frequently. Except one female rat with fibroma, upper-digestive-tract neoplasms were of the epithelial type -- papilloma, squamous-cell carcinoma or adenocarcinoma. In female rats, vaginal tumors were induced in 16 (41%) animals. Ear-duct tumors and hematopoietic neoplasms were also induced in both sexes of rats, with incidence of less than 21%.

Organ-specific carcinogenicity of N-methyl-N-nitrosourea in F344 and ACI/N rats.

Male and female F344 rats were continuously administered N-methyl-N-nitrosourea (MNU) in their drinking water at concentrations of 200 or 100 ppm, and both sexes of ACI/N rats were given MNU at a concentration of 200 ppm. By the 42nd week of the experiment, high incidences of brain/spinal cord tumors were observed in both strains of rats. Histologically, many of them were astrocytomas or anaplastic astrocytomas. In addition, malignant neurinomas were also detected in the spinal nerve roots and trigeminal nerves, although their incidences were rather low. There was no difference in the type and incidence of these neurogenic tumors between the two strains of rats. Tumors of the tongue and esophagus were mainly observed in the high-dose group of F344 rats and those of the glandular stomach were observed in the low-dose group of F344 rats. In ACI/N rats, tumors of the heart and renal pelvis were detected. The organ-specific carcinogenicity of MNU in these two strains of rats was compared with that of MNU in Donryu rats. It was demonstrated that organ specificity of MNU given orally was influenced not only by the strain of rats but also by the dose level.

Induction of tumors in the small intestine and mammary gland of female Donryu rats by continuous oral administration of N-carboxymethyl-N-nitrosourea.

The carcinogenicity of N-carboxymethyl-N-nitrosourea (CMNU), a naturally occurring N-nitroso compound, was tested in female Donryu rats. Four groups of female Donryu rats were given 400, 200, 100, or 0 ppm of CMNU solution continuously as drinking water. The incidence of tumors was highest and the mean survival time shortest in the 400 ppm group. A dose-effect relationship was observed in the tumor incidence and the mean survival time and the incidences of tumors in all experimental groups were significantly different from those in the control group. In the 400 ppm group, tumors were detected most frequently in the small intestine, followed by the mammary gland. In contrast, most tumors were observed in the mammary gland in the other two experimental groups, although dose-dependent induction of tumors of the small intestine was also detected in these two groups. The organ specificity of CMNU is compared with that of other N-alkyl-N-nitrosourea derivatives.

Mammary tumorigenic effect of a new nitrosourea, 1,3-dibutyl-l-nitrosourea (B-BNU), in female Donryu rats.

Four groups (groups 1-4) of female Donryu rats were given continuously 400, 200, 100, or 0 ppm solution of 1,3-dibutyl-l-nitrosourea (B-BNU) as their drinking water, and were studied for the development of tumors. The incidence of mammary tumors was 15/19 (79%), 20/24 (83%), 21/26 (81%), and 8/25 (32%) in groups 1, 2, 3, and 4, respectively. In addition, hematopoietic neoplasms, uterine tumors, and vaginal tumors developed in 13, 11, and six rats, respectively in 69 treated rats. Other tumors were infrequent.

Carcinogenicity of N-alkyl-N-(acetoxymethyl)nitrosamines after subcutaneous injections in F-344 rats.

As model compounds for metabolically activated N,N-dialkylnitrosamines, five N-alkyl-N-(acetoxymethyl)nitrosamines were synthesized and their carcinogenicity was tested in F-344 rats of both sexes. Compounds used in this study are N-methyl-(MAMN), N-ethyl-(EAMN), N-propyl-(PAMN), N-butyl-(BAMN), and N-isobutyl-N-(acetoxymethyl)nitrosamines (i-BAMN). All chemicals were dissolved in olive oil and rats received 10 weekly subcutaneous injections of these chemicals (10 X 5 mg MAMN or equimolar amounts of other chemicals) at the interscapular region. Subcutaneous tumors were detected in many rats of all groups treated with the chemicals, although no tumor was detected in the control group. Lung and/or thyroid tumors were also observed in many rats in the experimental groups. The incidence of subcutaneous tumors was highest in EAMN, followed in order by MAMN, PAMN, BAMN, and i-BAMN. On the contrary, the incidence of lung and thyroid tumors was highest in MAMN and decreased as the length of the alkyl chain of the chemicals increased. Histologically, almost all subcutaneous tumors were malignant fibrous histiocytomas. The results indicate that the chemicals possess systemic as well as local carcinogenicity in F-344 rats. The potent carcinogenic effects at the injection site of the alpha-acetoxy nitrosamines, coupled with their direct mutagenic activity reported previously, support the notion that these derivatives are useful as models for the ultimate form in the metabolic activation of N,N-dialkylnitrosamines.