Neoplasia in zebrafish (Danio rerio) treated with 7,12-dimethylbenz[a]anthracene by two exposure routes at different developmental stages.

Using zebrafish, Danio rerio, initial pioneering work in the 1960s revealed carcinogen responsiveness of fish, yet very few subsequent tumorigenesis investigations have utilized this species. We exposed embryos (60 hours postfertilization) and fry (3 week posthatch) to 7,12-dimethylbenz[a]anthracene (DMBA) by immersion in aqueous solutions for 24 hours, at concentrations of 0-1 or 0-5 ppm (mg/L), respectively. Juvenile zebrafish 2 months posthatch were fed a diet containing 0-1,000 ppm DMBA for 4 months. Fish were sampled for histologic evaluation at 7-12 months after the onset of carcinogen treatment. Fry were most responsive to DMBA and showed the widest diversity of target tissues and histologic types of neoplasia, having several types of epithelial, mesenchymal, and neural neoplasia. The principal target tissues for carcinogenic response were liver following embryo or fry exposure, with gill and blood vessel the second and third most responsive tissues in fry. Intestine was the primary target and gill a secondary target in fish that received dietary DMBA as juveniles. These studies indicate that young zebrafish are most responsive to DMBA, showing a greater diversity of neoplasm types than rainbow trout. Thus, zebrafish are a valuable model system in which to study mechanistic aspects of the carcinogenesis process.

Neoplasia in zebrafish (Danio rerio) treated with N-methyl-N'-nitro-N-nitrosoguanidine by three exposure routes at different developmental stages.

We exposed embryos (83 hours postfertilizaton) and fry (3 weeks posthatch) to N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) by immersion in aqueous solutions of 0-10 ppm for 1 hour (embryo) or 0-2 ppm for 24 hours (fry). Zebrafish embryos were microinjected with MNNG at levels of 0 or 96 ng/egg. Diets containing 0-2,000 ppm MNNG were fed to juvenile zebrafish for 3 months beginning at 2 months posthatch. Fish were sampled for histopathologic study at 6-12 months after initiation of carcinogen exposure. Embryos and fry were both quite responsive to MNNG; however, juvenile zebrafish were remarkably refractory to MNNG-induced neoplasia. Principal target organs in zebrafish treated as embryos with MNNG were liver and testis, with hepatocellular adenoma the most prevalent hepatic neoplasm. A variety of mesenchymal neoplasms occurred in zebrafish following embryo exposure to MNNG, including chondroma, hemangioma, hemangiosarcoma, leiomyosarcoma, and rhabdomyosarcoma. Testis and blood vessels were primary target organs for MNNG following fry exposure, with seminoma, hemangioma, hemangiosarcoma, and various other epithelial and mesenchymal neoplasms occurring. The zebrafish is a responsive, cost-effective lower vertebrate model system in which to study mechanisms of carcinogenesis.

N-methyl-N'-nitro-N-nitrosoguanidine-induced neoplasms in medaka (Oryzias latipes).

To test the sensitivity of the small fish species Oryzias latipes to the direct-acting carcinogen N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), medaka were exposed at 15 days of age to 30 mg/L for 1 hr and followed for up to 16 mo. One hundred neoplasms were diagnosed in 84 of 213 exposed fish, with approximately equal percentages in males and females. Many neoplasms (62%) were of mesenchymal origin and were categorized as blood vascular neoplasms (hemangioma and hemangiosarcoma), invasive sarcomas, and scale-associated neoplasms. Invasive sarcomas included rhabdomyosarcoma, fascial sarcoma, hemangiopericytoma, and undifferentiated sarcoma. A scale-associated neoplasm, termed lepidocytoma, was an unusual neoplasm of scale anlage. Thyroid follicular neoplasms, with a 100% incidence in males, and pancreatic acinar carcinoma were the most common epithelial tumors. Neoplasms of the gills, swim bladder, and olfactory epithelium were also seen as well as teratoma with mixed epithelial and mesenchymal components. The study showed a broad range of neoplasms induced by MNNG in medaka, with a tissue distribution that might support direct action on exposed tissues.

Enhancing effect of vitamin E on murine intestinal tumorigenesis by 1,2-dimethylhydrazine dihydrochloride.

The effect of the antioxidant vitamin E on the tumor-inducing ability of 1,2-dimethylhydrazine dihydrochloride (1,2-DMH) was investigated in randomly bred Swiss mice. Three groups of mice that were 6 weeks of age at the beginning of the experiment received the following treatments: a) vitamin E acetate [DL-alpha-tocopheryl acetate (TA)] at a 4% dose level in a powdered diet for life; b) 1,2-DMH, 10 weekly sc injections at 20 micrograms/g body weight; c) combination of a and b treatments. The administration of TA enhanced the tumorigenicity of 1,2-DMH, as evidenced by statistically significant incidences of tumors in the duodenum, cecum, colon, rectum, and anus. The present finding apparently is in contrast with the reported inhibitory effect of TA on colon carcinogenesis by 1,2-DMH.

Induction of angiogenic tumors in the duodenum of female Donryu rats by continuous oral administration of N-isobutyl-N-nitrosourea.

Four groups of female Donryu rats were continuously given 400, 200, 100 or 0 ppm N-isobutyl-N-nitrosourea (iso-BNU) in their drinking water, and were examined for the development of tumors. The incidence of digestive tract tumors was 25/28 (89%), 14/24 (58%), 6/25 (24%) and 0/17 (0%), in the 400, 200, 100 and 0 ppm groups, respectively. The predominant type of digestive tract tumor was angiogenic, and a few were of the epithelial type. A dose-effect relationship was clearly demonstrated not only in the incidence of digestive tract tumors but also in the average survival period of rats with these tumors.

The carcinogenic effect of intracolonic administration of 1,2-dimethylhydrazine dihydrochloride in mice.

1,2-Dimethylhydrazine dihydrochloride (1,2-DMH) was given to Swiss mice in 1 or 10 weekly intracolonic instillations of 60 and 40 micrograms/g body weight, respectively. In mice that received a single treatment, the tumor incidences in the lungs, blood vessels and subcutis were 34% (p less than 0.006), 8 and 25 in females and 2, 20 and 0% in males, respectively. In mice treated repeatedly with the carcinogen, the corresponding tumor incidences were 34% (p less than 0.006), 38% (p less than 0.000001) and 12% (p less than 0.03) in females and 34, 10 and 0% in males, respectively. In control mice the tumor incidences in lungs, blood vessels and subcutis were 15, 8 and 2% in females and 22, 5 and 2% in males, respectively. The induction of large intestinal cancer, which was the main objective of this investigation, however, failed to materialize.

A carcinogenicity dose response study by continuous administration of 1,2-dimethylhydrazine dihydrochloride in mice.

A dose response study in carcinogenesis by 1,2-dimethylhydrazine dihydrochloride (1,2-DMH) in Swiss mice was performed. The compound was administered continuously in drinking water for life from 6 weeks of age at dose levels of 0.002, 0.001, 0.0005, 0.00025, 0.000125, 0.0000625, 0.00003125 and 0.000015625%. A positive correlation was established between the dose levels of 1,2-DMH and the yield of blood vessel tumors. In addition, the latency periods for these tumors diminished when the dose levels of the carcinogen decreased. Although some of the treatments induced lung tumors, no association was observed between the dose levels of the carcinogen and the lung tumor incidence. The study thus provides an example in which the continuous lifespan administration of a carcinogen resulted in a partial dose response effect. This method of administration closely resembles some of the human exposure situation.

Carcinogenicity of 1,2-dimethylhydrazine dihydrochloride in BALB/c mice. Influence of the route of administration and dosage.

The carcinogenicity of 1,2-dimethylhydrazine dihydrochloride (DMH) by oral, intragastric and subcutaneous administration was examined in 339 BALB/c mice. Subcutaneous injection of DMH induced intestinal tumors in the lower colon of all mice. After oral administration it induced a high incidence of vascular tumors in the liver and soft tissues, but colon tumors were found in only 2 mice when given at a high dosage. On intragastric administration, it induced a fairly high incidence both of colon and vascular tumors. The sites and incidences of vascular tumors and squamous cell carcinomas of the perianal glands were also described.

In vivo studies in Syrian golden hamsters: a transplacental bioassay of ten nitrosamines.

The carcinogenic effects of low doses of 10 nitrosamines were determined in pregnant Syrian golden hamsters and their offspring. Compounds studied included dimethylnitrosamine, di-n-propylnitrosamine, di-n-butylnitrosamine, nitrosopiperidine, nitrosohexamethyleneimine, 2-dydroxypropyl-propyl-nitrosamine, 2-oxopropyl-propyl-nitrosamine, methylpropylnitrosamine, di(2-hydroxypropyl) nitrosamine, and 4-hydroxybutyl-butyl-nitrosamine. Tumor incidences of all organ systems were almost always higher and latencies shorter in the mothers than in the offspring. Exceptions occurred in the respiratory system in which several compounds induced a low incidence of tumors in the offspring but none in the mothers. Fetal susceptibility appeared greatest toward the end of gestation. For purposes of bioassay, transplacental exposure was less efficient than conventional adult treatment.

Tumor induction with the N'-acetyl derivative of 4-hydroxymethyl-phenylhydrazine, a metabolite of agaritine of Agaricus bisporus.

N'-Acetyl-4-(hydroxymethyl)phenylhydrazine was administered as a 0.0625% solution in drinking water continuously for the life span of Swiss mice, from 6 weeks of age. Compared to that in untreated controls, in treated animals the lung tumor incidence rose from 15 to 34% in females and 22 to 48% in males, whereas the incidence of blood vessel tumors increased from 8 to 32% in females and from 5 to 30% in males. Histopathologically, the tumors were classified as adenomas and adenocarcinomas of the lungs and angiomas and angiosarcomas of the blood vessels. The commonly eaten mushroom Agaricus bisporus contains beta-N-[gamma-L(+)-glutamyl]-4-hydroxymethylphenylhydrazine, which under certain conditions yields 4-hydroxymethylphenylhydrazine and L-glutamic acid. Since 4-hydroxymethylphenylhydrazine is relatively unstable, its acetyl derivative was synthesized for this study. The possible environmental significance of the findings is discussed.

Tumorigenic effect of 4-methylphenylhydrazine hydrochloride in Swiss mice.

4-Methylphenylhydrazine hydrochloride was administered as 10 weekly subcutaneous injections of 140 microgram/g body weight and as 7 weekly intragastric instillations of 250 microgram/g body weight in physiological saline to randomly bred Swiss mice. Treatments given subcutaneously resulted in induction of lung tumors in incidences of 36% in females and 44% in males, while intragastric treatment caused a 40% incidence in females. In addition, it gave rise to blood vessel tumors by intragastric route in incidences of 32% in females and 18% in males. In the two physiological saline-treated control groups, the lung tumor incidence (combined) was 20% in females and 21% in males, while the blood vessel tumor incidence (combined) was 7% in females and 6% in males. Histopathologically, the lesions were classified as adenomas and adenocarcinomas of the lungs, and angiomas and angiosarcomas of blood vessels. 4-Methylphenylhydrazine was postulated to be a metabolite of 4-hydroxymethylphenylhydrazine, an ingredient of the commonly eaten mushroom Agaricus bisporus. The implications are discussed with respect to the tumorigenesis data.

Tumorigenic effects of chronic administration of benzylhydrazine dihydrochloride and phenylhydrazine hydrochloride in Swiss mice.

Separate solutions of 0.015% benzylhydrazine dihydrochloride and 0.01% phenylhydrazine hydrochloride were given continuously in the drinking water of 6- and 5-week-old randomly bred Swiss mice for the remainder of their life. The consumption of benzylhydrazine dihydrochloride significantly increased the lung tumor incidence from 21 to 42% in the females, while in phenylhydrazine hydrochloride-treated mice, the incidence of blood vessel tumors rose significanly from 5 to 22% in females and from 6 to 20% in males, as compared with the controls. Histopathologically, the tumors were classified as adenomas and adenocarcinomas of lungs and angiomas and angiosarcomas of blood vessels. The study thus proves for the first time the tumorigenicity of benzylhydrazine dihydrochloride. It also confirms the tumor inducing ability of phenylhydrazine hydrochloride, which is used in medicine for treatment of polycythemia vera.

Blood vessel tumorigenesis by 1,2-dimethylhydrazine dihydrochloride (symmetrical). Gross, light and electron microscopic descriptions. I.

Administration of 0.001% 1,2-dimethylhydrazine dihydrochloride, symmetrical, in the drinking water of 7-week-old randomly bred Swiss mice for the remainder of their lifetime induced blood vessel tumors and enhanced the incidence of lung neoplasms. Ninety-eight percent of the females and 92% of the males developed vascular lesions, whereas among the controls the incidence was 3% in the females and 1% in the males. In addition, the incidence of lung tumors rose from 12 to 44% in the females and from 10 to 24% in the males, as compared with the controls. The occurrence of the vascular tumors in order of decreasing frequency was as follows: muscle, pararenal, fat, liver, parametrial, paraepididymal tissues, etc. Gross, light and electron microscopic examinations of vascular lesions revealed the characteristic appearance of angiosarcomas. The type and extent of macroscopic and histologic involvements of the various tissues by the tumors are presented. The ultrastructural descriptions of hemorrhagic areas, vascular spaces, neoplastic endothelial cells, their cytoplasms and organelles are illustrated in detail.In conclusion, whereas hydrazine enhanced the development of lung tumors, when the dimethyl group was attached to it at symmetrical positions, it evoked vascular tumors. Thus, the present study provides evidence for the possible relationship between chemical structure and tumor induction at specific organ sites.