The multipotential carcinogenic action of N-ethyl-N-nitrosourea administered neonatally to mice.

Newborn A, C57BL, DBAf and IF mice were injected s.c. with a range of doses of N-ethyl-N-nitrosourea (ENU). A high proportion of treated mice developed tumours, particularly hepatomata, pulmonary adenomata and carcinomata, and malignant lymphomata of thymus and spleen. Liver tumours occurred most frequently in C57BL and DBAf mice, lung tumours in A mice, and lymphomata in A and DBAf mice. A small proportion of C57BL, DBAf and IF mice developed tumours of the nervous system. The results are discussed with reference to the ready induction of nervous system tumours in similarly treated rats, and their relevance for human cancer.     

Genetic transmission of mammary tumour virus in the DBAf mouse strain.

MTV antigens were demonstrable by radioimmunoassay in milk samples from individual DBAf mice, and in samples from (male BALB/c X female DBAf) F1 mice. Although some samples collected during the first lactation periods of these mice were virus-negative, all samples of later lactation periods were virus-positive. From 75 mice of the [ male BALB/c X female (male BALB/c X female DBAf)]Bc I population, milk samples were collected during one or more lactation periods and tested for the presence of viral antigens; the samples of 42 mice were virus-positive. In the ([BALB/c X (BALB/c X DBAf)] X BALB/c)Bc II population two groups were distinguished. In the first group, the progeny of virus-positive Bc I mothers, 37 out of 62 mice had detectable levels of viral antigen in the milk. None of the 43 samples from mice of the second group, derived from MTV-negative Bc I females, were virus-positive. These data suggest that the presence of viral antigens in the milk of DBAf mice is controlled by a single dominant gene; evidence for linkage of this gene and the albino locus was obtained (recombination percentage: 20).     

7,12-Dimethylbenz[a]anthracene-induced perinatal carcinogenesis and its modulation by butylated hydroxyanisole in mice

We discuss the transplacental and transmammary carcinogenicity of 7,12-dimethylbenz[a]anthracene (DMBA) in Swiss albino mice and its modulation by butylated hydroxyanisole (BHA). Transmission of the carcinogen by either route elicits the development of tumour in F1 individuals, and in either situation the incidence of tumours is dependent upon the dose of DMBA administered to gestating or lactating mothers or foster mothers. However, for a given dose of carcinogen, its transplacental carcinogenicity is much greater than its transmammary carcinogenicity. Transmammary carcinogenicity is evident in F1 progeny whether they are nursed by DMBA-exposed mothers, syngenic foster mothers (Swiss albino strain) or allogenic foster mothers (C57BL/6 strain), but the incidence of tumours is appreciably lower when allogenic females are the foster mothers. DMBA administered to females during gestation appears to remain as a residue, then to find its way through the transmammary route into normal F1 individuals being foster-nursed, and to produce tumours. We have also shown the influence of age, but not of parity, of foster mothers on DMBA-induced transmammary carcinogenesis in F1 individuals. In these experiments, BHA has a chemopreventive action against DMBA-induced transplacental and transmammary carcinogenesis in mice.     

Carcinogenicity studies with deltamethrin in mice and rats

The pesticide Deltamethrin, a synthetic pyrethroid, was studied for carcinogenicity in long-term experiments in mice and rats. Mice were given Deltamethrin by gavage in arachis oil at 0, 1, 4 or 8 mg/kg body wt for 2 years. A group of untreated controls was also available. Rats received 0, 3 or 6 mg/kg body wt. Deltamethrin in arachis oil for 2 years. In mice, an increased incidence of lymphomas was observed in the groups receiving 1 and 4 mg/kg body wt., but not in the group treated with 8 mg/kg body wt. Deltamethrin. In rats, an increased incidence of thyroid tumours was noted, but, no clear dose-response relationship was shown. Deltamethrin does not appear to be carcinogenic in mice or rats, but further studies are needed on the group of compounds to which this substance belongs.     

Point mutation of K-ras gene in cisplatin-induced lung tumours in A/J mice

The risks of secondary lung cancer in patients with early stage non-small and small cell lung cancers are estimated to be 1-2% and 2-10% per patient per year, respectively. Surprisingly, the incidence of second primary cancer in locally advanced non-small cell lung cancer at 10 years, following cisplatin-based chemotherapy with concurrent radiotherapy, increases to 61%. Those patients, on the road to being cured, cannot overlook the possibility of developing a second primary cancer. We developed a second primary lung cancer model using cisplatin as a carcinogen in A/J mice to screen for chemopreventive agents for a second malignancy. In the primary lung tumour model, 4-(methyl-nitrosamino)-1-(3-pyridyl)-1-butanone (NNK), benzo(a)pyrene (BaP), urethane induces specific K-ras mutations in codon 12, codon 12, and codon 61, respectively, in the A/J mice. In this study, we investigated the mechanisms of carcinogenicity by cisplatin in the A/J mice. In the cisplatin-induced tumours, we found no K-ras codon 12 mutation, which is the major mutation induced by NNK or BaP. K-ras gene mutations in codon 13 and codon 61 were found in one tumour (4%) and five tumours (17.8%), respectively. These findings suggest that cisplatin is partially related to K-ras codon 61 mutations, and that the mechanism of carcinogenicity by cisplatin is different from that by NNK or BaP.     

HEXACHLOROBUTADIENE: HAZARD CHARACTERIZATION AND EXPOSURE–RESPONSE ANALYSIS

Hexachlorobutadiene (HCBD) has been assessed as a Priority Substance under the Canadian Environmental Protection Act. Based on results of studies conducted in experimental animals, the proximal tubules of the kidney are the principal target sites for HCBD-induced non-neoplastic lesions. Kidney tumours have also been observed in rats following long-term exposure to HCBD, but only at doses associated with significant nephrotoxicity. A tolerable intake of 0.34 μg/kg body weight per day has been derived, based upon the benchmark dose for renal tubular regeneration in female mice. This value is also considered protective for potential carcinogenicity.     

Thyroid tumours in rats and hepatomas in mice after griseofulvin treatment.

Griseofulvin, an antibiotic used to treat dermatophystosis, was tested for carcinogenicity in mice, rats and hamsters. Three groups of mice and rats were given the drug in powdered diet in alternating 5-week periods for life, at dose levels of 3.0%, 1.5% and 0.3% (mice) and 2.0%, 1.0% and 0.2% (rats). A group of mice and 3 groups of hamsters received continuous daily treatment for life with griseofulvin at 3.0%, 1.5%, 0.3% and 0.1% dose levels respectively. A significant incidence of hepatic tumours was observed at the 2 higher treatment levels in mice. Also, statistically significant rates (P less than or equal to 0.001 and/or P less than or equal to 0.020) of thyroid tumours, indicating a dose-response, were recorded in male rats at the 2.0%, 1.0%, and 0.2% dose levels, and in females at the 2.0% and 1.0% dose levels. Hamsters did not develop neoplasms in response to treatment at any level.     

Effect of glycerol on local and systemic carcinogenicity of topically applied tobacco condensate

When glycerol was added to tobacco smoke condensate in acetone solvent, the topical carcinogenicity and the ability to produce epithelial hyperplasia in mice was reduced. Two doses of condensate were applied, combined with 2 concentrations of added glycerol. Age-standardized results show that glycerol reduced the incidence of tumours and malignant tumours and of hyperplasia in animals not developing skin tumours. The relative incidences of malignant tumours, benign tumours, hyperplasia and unaffected skin suggest that there is a sequential relationship (i.e. normal skin to hyperplasia to benign neoplasia to malignant neoplasia) which is impeded by glycerol. There was no systemic effect attributable to the condensate.     

Carcinogenicity of a single administration of N-nitrosomethylurea: a comparison between newborn and 5-week-old mice and rats

N-Nitrosomethylurea (NMUrea) was given as a single intraperitoneal injection either to newborn or to 5-week-old (C57BL * C3Hf)F₁ mice and Wistar rats. Newborn mice were more susceptible than 5-week-old mice to the development of lymphosarcomas, lung adenomas and hepatomas, whereas newborn rats were more susceptible than their weaned counterparts to the development of renal anaplastic tumours. Other tumours occured with the same frequency in newborn and mature animals. Tumours of the forestomach in mice were more frequenty found in animals treated at 5 weeks than in those treated at birth. Since NMUrea persists for only a very short time and breaks down spontaneously it seems that the paucity of enzymes related to immaturity in newborns is not a major factor in determining the different susceptibility of newborn animals to NMUrea carcinogenicity.     

Sensitivity of the skin of different mouse strains to the promoting effect of 12-0-tetradecanoyl-phorbol-13-acetate

The sensitivity of the skin of Swiss DBA/2 and C57B1/6 mice was compared in two-stage carcinogenicity experiments. Groups of 30 mice were treated once with 7,12-dimethylbenz(a)anthracene (DMBA) (50 micrograms/mouse) which was applied to the shaved dorsal skin as initiator and, starting one week later, 12-0-tetradecanoyl-phorbol-13-acetate (TPA) was applied thrice-weekly at doses of either 0.04, 0.16 or 0.64 micrograms/mouse to the areas of initiated skin. Other groups of mice were similarly treated with TPA (0.64 micrograms) alone. The times at which papillomas and carcinomas first appeared were recorded. Overall the results show, in terms of the numbers of animals with tumours, the total numbers of tumours produced and the numbers of malignant tumours formed all in dose-response relationship, that the Swiss mice were the most sensitive and the B57B1/6 mice the least sensitive to the tumour-promoting effects of TPA with the DBA/2 mice occupying and intermediate position. This relationship also held in the control groups that were treated with TPA alone.     

Use of E mu-PIM-1 transgenic mice short-term in vivo carcinogenicity testing: lymphoma induction by benzo[a]pyrene, but not by TPA

E mu-pim-1 transgenic mice are predisposed to develop lymphomas. Due to their low spontaneous tumour incidence and their increased sensitivity towards the lymphomagen ethylnitrosourea these mice may present an interesting model for short-term carcinogenicity testing. Here, we report on the further exploration of this transgenic mouse model with two additional carcinogens known to have, among others, the lymphohaematopoietic system as target, i.e. benzo[a]pyrene (B[a]P) and 12-O-tetradecanoylphorbol-13-acetate (TPA). B[a]P, given three times a week (by gavage) for 13 weeks at 4.3, 13 or 39 mg/kg body weight, resulted in a dose-related increase in lymphomas up to a 90% incidence in E(mu)-pim-1 mice during the observation period of 40 weeks. B[a]P also induced tumours of the forestomach within this observation period, though at a lower incidence and apparently equally effective in wildtype and transgenic mice. TPA, on the other hand, was unable to induce lymphomas (or tumours in any other organ) in either transgenic or wildtype animals within the observation period of 44 weeks, when applied dermally at the maximum tolerated dose of 3 microg/mouse, twice a week for 35 weeks. Molecular analysis showed that B[a]P-induced lymphomas in transgenic mice were of T-cell origin, 80% of which had elevated levels of c-myc expression. None of the lymphomas had increased N-myc expression and mutation analysis of the ras-gene family revealed a K-ras mutation in only one out of eight tumours investigated. Also, none of the lymphomas showed aberrant expression of p53 as determined by immunohistochemistry. It is concluded that the E mu-pim-1 mouse model will not be very suitable for short-term carcinogenicity testing in general: only genotoxic chemicals that have the lymphohaematopoietic system as target for carcinogenesis in wild- type mice, appear to be efficiently identified.      

5-Azacytidine carcinogenesis in BALB/c mice

5-Azacytidine, a drug used in the treatment of acute leukaemias and beta-thalassemia, was administered i.p. to BALB/c mice at a dose of 2.0 mg/kg body wt. once a week for 50 weeks to test its carcinogenicity. The treatment induced a significant increase in lung tumours (males P less than 0.001, females P less than 0.05), lymphomas (males P less than 0.01, females P less than 0.01), skin tumours (males P less than 0.05, females P less than 0.01) in both sexes and mammary carcinomas (P less than 0.01) and a variety of other tumours in female mice. These results, with other investigations reported in literature, suggest that 5-azacytidine is carcinogenic in mice.     

Thiophene analogues of the carcinogens benzidine and 4-aminobiphenyl: evaluation in vitro.

A biologically active molecule with one or more aromatic rings often retains its activity when one of these rings is replaced by an isosteric and/or isoelectronic aromatic ring. Consideration has been given to whether this effect can be expected to apply to aromatic organic carcinogens. The literature relevant to this topic has been reviewed and the thiophene analogues of the carcinogens benzidine and 4-aminobiphenyl have been synthesized and evaluated for potential carcinogenicity. The compounds prepared were 5-p-acetamidophenyl-2-thiophenamine hydrochloride (XIII), 5-phenyl-2-thiophenamine hydrochloride (XIV), N-(5-p-acetamido-phenylthiophen-2-yl)acetamide (XV) and N-(5-phenylthiophen-2-yl)-acetamide (XVI) (see Chart for structures). Each compound was evaluated in the Salmonella reverse-mutation assay of Ames and the cell-transformation assay of Styles. The activity profiles observed for these compounds in vitro were consistent with their known chemistry, and indicate potential carcinogenicity. However, their overall chemical and biological behaviour casts doubt upon whether they would be capable of eliciting tumours in vivo. Because it is important to establish the degree of reliance which can be placed upon in vitro predictions of potential carcinogenicity generated for structurally new compounds, one of the thiophene derivatives, N-(5-phenylthiophen-2-yl)acetamide ((XVI), is currently being evaluated for carcinogenicity in mice.     

Carcinogenicity study of the pesticide fenvalerate in mice

Fenvalerate is a widely used pesticide, which has been shown recently to be nonmutagenic. We studied its carcinogenicity in a long-term experiment in inbred C57Bl/6 mice given 0, 40 and 80 mg/kg body weight fenvalerate (99% pure) by gavage on 5 days/week for 104 weeks. Survival was decreased especially among females receiving the high dose. Exposure to fenvalerate resulted in a slight increase in the incidence of liver-cell tumours over that in controls only in male mice receiving the high dose. No significant difference in the incidence of other types of tumours was observed in treated groups when compared with controls. Fenvalerate-induced microgranulomas occurred concomitantly in the liver, spleen and lymph nodes of male and female mice, but their overall incidence did not increase with dose. In a separate experiment, groups of SJL/ola female mice were administered two different samples of fenvalerate (92% and 99% pure) once per week for 12 weeks. In animals that received 92% pure compound, the latent period for induction of lymphomas was shortened and their incidence increased, when compared with the group receiving 99% pure fenvalerate and with controls.     

The mouse-skin carcinogenicity of a mutagenic fraction from beech wood dusts

A life-time mouse-skin carcinogenicity assay was conducted usingfemale NMRI mice to evaluate the possible direct carcinogenicactivity of a mutagenic fraction isolated from beech wood dusts.The samples of untreated beech wood dusts were extracted withmethanol at pH3 and were purified from the inhibitory compoundstoxic to bacteria, using silica-gel cohimn chromatography. Thefraction obtained after passing through the column was testedfor mutagenicity in the Ames assay employing Salmonella typhimuriumTA100 in the presence of Aroclor-treated rat-liver-S9. Usingacetone as the vehicle, this mutagenic fraction was tested forcarcinogenicity on an area of 1 - 1.5 cm shaved skin of miceon the lower back. The mice were treated with half of each dose,twice a week, for only 3 months. The total doses applied perweek were 2.5, 5, 7.5 or 10 g equivalent dust/mouse. No substancewas used as promoter. No statistically significant differencewas found when the life spans of treated and untreated animalswere compared. TTie observed carcinogenic effect was based ontumours and lesions found only on the ate of application ofthe test material. Of 210 mice (effective number, 129) servingas the negative controls, three developed skin lesions but notumours. Of 280 treated animats (effective number, 188) 34 developeddifferent types of tumours and 20 had a uniform type of precancerousskin lesion. Of 34 tumours observed 21 were originated fromthe skin, 12 from the mammary glands beneath the site of application,and one was a lymphoma. Comparing the negative controls withthe treated animals, the overall carcinogenic effect observedwas dose-dependent and statistically significant. Excludingthe mammary tumours and a lymphoma found beneath the site oftreatment, the overall induction of skin tumours was still significant.However, the dose-dependent increase in the number of skin tumoursalone was not statistically significant. These results suggestthat beech wood dust contains mutagenic and carcinogenic constituent(s).     

Carcinogenicity of ochratoxin A in experimental animals

The carcinogenicity of ochratoxin A, a naturally occurring mycotoxin of the fungal genera Aspergillus and Penicillium, was evaluated in three strains of mice and in one strain of rats. The kidney, and in particular the tubular epithelial cells, was the major target organ for ochratoxin A-induced lesions. In male ddY and DDD mice, atypical hyperplasia, cystadenomas and carcinomas of the renal tubular cells were induced, as were neoplastic nodules and hepatocyte tumours of the liver. In B6C3F1 mice, tubular-cell adenomas and carcinomas of the kidneys were induced in male mice, and the incidences of hepatocellular adenomas and carcinomas were increased in male and female mice. In male and female F344 rats, ochratoxin A induced nonneoplastic (degeneration, karyomegaly, proliferation, cytoplasmic alteration, hyperplasia) and neoplastic effects (adenomas, and carcinomas with metastases) in the kidneys; the incidence of fibroadenomas of the mammary glands was also increased in female rats. Other studies on ochratoxin A were considered inadequate for evaluating the presence or absence of a carcinogenic effect; however, these are mentioned and referenced below. The collective experimental findings, together with accumulating evidence in humans, forecast further toxic and carcinogenic effects in humans exposed to ochratoxin A, mainly via foodstuffs.     

The sensitivity of the skin of hairless mice to chemical carcinogenesis.

The sensitivity of hairless mice to cutaneous chemical carcinogenesis has been compared with that of normal mice of the same strain with hair. A single application of 125 mug methylcholanthrene in benzene was given to 48 hairless male mice (hr/hr Oslo strain) and to 96 male mice of the same strain with hair. Among hairless mice there were 94% papilloma-bearing animals with a total of 5.9 tumors per animal after 18 months of observation, compared to 22% papilloma-bearing animals with an average of 0.3 tumors per animal among the mice with hair. The hairless mice included 31% carcinoma-bearing and 23% sarcoma-bearing animals, whereas only 1% of the mice with hair were carcinoma bearing and 3% were sarcoma bearing. Hairless mice of the hr/hr Oslo strain are thus not refractory to chemical carcinogenesis, but under the experimental conditions used in this study they are significantly more sensitive than are mice from the same strain with hair. Giovanella et al. reported almost opposite results in 1970 and came to the general conclusion that hairless mice are refractory to chemical carcinogenesis due to lack of hair follicles. Since hairless mice always have some hair follicles and rudimentary pilosebaceous appendages, comparisons between chemical carcinogenesis in hairless mice and mice with hair can neither strengthen nor weaken any theory about the hair follicle origin of epidermoid carcinomas of mouse skin.     

Genetic analysis of mammary tumor induction and expression of mammary tumor virus antigen in hormone-treated ovariectomized GR mice.

Early stages of mammary tumors (EMT) were induced with a combined treatment of progesterone (P) and estrone (E) in ovariectomized adult GRS/A (GR) mice, a strain of European origin and with a high incidence of mammary cancer. The mammary tumors were comparable to the pregnancy-dependent tumors of breeding females of this strain. The hormone treatment did not lead to EMT in a variety of other strains and only occasionally in the RIII an C3H strains. Treatment with P or E alone di not lead to EMT in GR mice, but treatment with the steroid compound 17 alpha-ethynyl-19-nortestosterone (ANT) did mimic the combined effe(t of P and E. Since EMT could be induced by ANT in all ovariectomized adult GR mice within 3 weeks, this tumor-induction method was suitable for analysis of the gene responsible for palpable, pregnancy-dependent mammary tumors of this strain. Another argument for the usefulness of this test for genetic analysis was the fact that, though mouse mammary tumor virus (MuMTV) of the GR strain was introduced into BALB/c and tmas mice by foster-nursing, ANT treatment did not lead to EMT. First and second backcross analyses showed that one gene was responsible for EMT induction. There was a strong (orrelation between the presence of EMT and MuMTV antigens in the mammary glands and milk of several first backcross populations between GR and other strains such as C57BL, BALB/c, DBAf, and C3Hf. This suggested that the expression of MuMTV antigens was also controlled by the EMT gene. Two types of resistance phenomena were observed. Neither type could prevent EMT after hormone treatment; however, they could delay EMT development. One resistance factor for EMT induction was noticeable and dominant in reciprocal hybrids of the GR and DBAf strains, whereas another resistance factor was detected in the backcross population only [i.e., in the C57BL X (C57BL X Gr) ba(kcross] and not in hybrids; therefore, this factor was recessive. Until now, linkage experiments with 18 markers to locate the gene for EMT induction in the map of the mouse were unsuccessful.     

Carcinogenicity of some pyrrolic pyrrolizidine alkaloid metabolites and analogues

Repeated topical doses of dehydromonocrotaline, a putative primary toxic metabolite of the pyrrolizidine alkaloid monocrotaline, followed by repeated treatments with croton oil, produced malignant tumours of the skin in LACA mice. A chemically similar alkylating agent, 2,3-bistrimethylace-toxymethyl-1-methylpyrrole, was carcinogenic without use of a promoter, confirming a preliminary report. Its high activity was due to the intact ester, not to its hydrolysis products. Dehydroretronecine (DHR), a less reactive secondary metabolite of monocrotaline, was also carcinogenic to LACA mouse skin, but it was less active than previously reported in experiments with another strain. Similar carcinogenicity was shown by the synthetic 2,3-bishydroxymethyl-1-methylpyrrole, but not by 2,3-bis-hydroxymethyl-5-methyl-1-phenylpyrrole, which has a high level of antimitotic activity.     

Strain A mouse pulmonary tumor test results for chemicals previously tested in the National Cancer Institute carcinogenicity tests

Sixty-five chemicals were coded and examined for their ability to induce lung tumors in strain A/St (laboratory A) or strain A/J (laboratory B) mice. Thirty-five chemicals were tested in laboratory A only, 6 in laboratory B only, and 24 in both laboratories. Two-year carcinogenicity test results as well as genotoxicity test data are available for most of these chemicals. There was poor interlaboratory agreement in strain A test results for the 24 chemicals tested in both laboratories. In addition, there was poor agreement between strain A test results from either laboratory and 2-year carcinogenicity test results or genotoxicity results. Possible explanations for these findings include selection of a large number of aromatic amines in the group of chemicals submitted for strain A testing, differences in strain A testing protocols and in statistical analysis of results from the two laboratories, low sensitivity of the strain A/St mice used in this particular study, and general problems inherent in comparing any relatively short-term animal tumor model with 2-year carcinogenicity tests. Since there is no absolute reference for carcinogenicity, no one test system is better than another. Carcinogenicity test data are relevant only to the test model employed.