Histological evaluation of rat mammary tumours after treatment with retinoic acid analogues — phytol, TTNPB and vitamin D3 analogue seocalcitol

The effects of retinoic acid analogue — phytol (precursor of retinoic X receptors ligand), TTNPB (retinoic acid receptor agonist) and seocalcitol (EB1089, analogue of vitamin D₃) in mammary tumours of Sprague-Dawley rats induced with 1-methyl-1-nitrosourea (MNU) were investigated. Treatment with phytol, TTNPB and seocalcitol may have some protective and therapeutical effects on malignant processes. Treatment with these components in combination of TTNPB and phytol or seocalcitol and phytol inhibited progression of MNU-induced tumours of the rat mammary gland, and also induced decrease of tumour burden and volume in comparison with treated control group. Treatment of rats with the above compounds had no effect on malignity and invasiveness of carcinomas.     

Reduction of 1-methyl-1-nitrosourea-induced mammary gland carcinoma by in vivo application of immunostimulatory CpG motifs in Sprague-Dawley rats

In the present work the role of 13-cis retinoic acid and CpG oligodeoxynucleotides (CpG-ODN) in a 1-methyl-1-nitrosourea (MNU)-induced mammary gland carcinoma animal model was investigated. Treatment with both components, applied either alone or in combination, induced a significant decrease of the tumour burden and the volume of tumours only in rats that received CpG-ODN (p = 0.046, compared to the MNU control group). The data indicate that the Th-1 biased immunostimulatory capacities of CpG motifs may play a significant role in induction of protective immune responses against mammary gland tumours in Sprague-Dawley rats.     

Zinc accumulation in N-methyl-N-nitrosourea-induced rat mammary tumors is accompanied by an altered expression of ZnT-1 and metallothionein

Zinc is essential for cell proliferation. Several human studies have shown that in breast cancer tissues, zinc concentration expressed on a per tissue weight basis is higher than that in normal breast tissues. However, the mechanisms involved are unknown. N-methyl-N-nitrosourea (MNU)-induced rat mammary tumorigenesis is one of the most widely used rodent mammary tumorigenesis models for studying human breast cancer due to their similarities in hormone dependency, pathogenesis, histological classification, and immunocytochemical markers. This study was to establish if there was an accumulation of zinc in MNU-induced rat mammary tumors and, if there was, to explore the possible mechanisms involved. Sprague-Dawley rats were sham-treated or MNU-treated (50 mg/kg; n = 12) for 100 days. In MNU-induced mammary tumors (mammary tumors), zinc concentration expressed on a per dry weight basis was 12 times of that in normal mammary glands. Moreover, the mRNA level of ZnT-1 (a transporter involved in zinc efflux) in mammary tumors was reduced by 55% as compared with that in normal mammary glands. The mRNA level of Nramp2 (a divalent cation importer) and ZnT-4 (another transporter involved in zinc efflux) was unaffected by MNU-induced mammary tumorigenesis. The mRNA and protein levels of metallothionein (a putative zinc storage protein) in mammary tumors were 1.3 and 3.5 times of that in normal mammary glands, respectively. Collectively, our observations showed that zinc is accumulated in MNU-induced rat mammary tumors and this accumulation is accompanied by an altered expression of ZnT-1 and metallothionein, suggesting that zinc homeostasis might be altered in MNU-induced rat mammary tumorigenesis. Because zinc is essential to cell proliferation and cell proliferation is increased in mammary tumors, zinc accumulation is likely a part of an integrated effort to ensure sufficient zinc supply to sustain tumor growth.     

Quantification of epithelial cell differentiation in mammary glands and carcinomas from DMBA- and MNU-exposed rats

Rat mammary carcinogenesis models have been used extensively to study breast cancer initiation, progression, prevention, and intervention. Nevertheless, quantitative molecular data on epithelial cell differentiation in mammary glands of untreated and carcinogen-exposed rats is limited. Here, we describe the characterization of rat mammary epithelial cells (RMECs) by multicolor flow cytometry using antibodies against cell surface proteins CD24, CD29, CD31, CD45, CD49f, CD61, Peanut Lectin, and Thy-1, intracellular proteins CK14, CK19, and FAK, along with phalloidin and Hoechst staining. We identified the luminal and basal/myoepithelial populations and actively dividing RMECs. In inbred rats susceptible to mammary carcinoma development, we quantified the changes in differentiation of the RMEC populations at 1, 2, and 4 weeks after exposure to mammary carcinogens DMBA and MNU. DMBA exposure did not alter the percentage of basal or luminal cells, but upregulated CD49f (Integrin α6) expression and increased cell cycle activity. MNU exposure resulted in a temporary disruption of the luminal/basal ratio and no CD49f upregulation. When comparing DMBA- or MNU-induced mammary carcinomas, the RMEC differentiation profiles are indistinguishable. The carcinomas compared with mammary glands from untreated rats, showed upregulation of CD29 (Integrin β1) and CD49f expression, increased FAK (focal adhesion kinase) activation especially in the CD29hi population, and decreased CD61 (Integrin β3) expression. This study provides quantitative insight into the protein expression phenotypes underlying RMEC differentiation. The results highlight distinct RMEC differentiation etiologies of DMBA and MNU exposure, while the resulting carcinomas have similar RMEC differentiation profiles. The methodology and data will enhance rat mammary carcinogenesis models in the study of the role of epithelial cell differentiation in breast cancer.     

Transforming c-Ki-ras mutation is a preneoplastic event in mouse mammary carcinogenesis induced in vitro by N-methyl-N-nitrosourea.

Mouse mammary epithelial cells can be transformed in primary cultures to preneoplastic and neoplastic states when treated with N-methyl-N-nitrosourea (MNU). Mammary carcinomas arising from MNU-induced hyperplastic alveolar nodules (a type of mouse mammary preneoplastic lesion) contained transforming c-Ki-ras genes when examined by the NIH 3T3 focus assay. Hybridization of allele-specific oligonucleotides to c-Ki-ras sequences amplified by the polymerase chain reaction demonstrated the presence of a specific G-35----A-35 point mutation in codon 12 in each of the NIH 3T3 foci as well as the mammary carcinomas. This mutation resulted in the substitution of the normal glycine with an aspartic acid. Furthermore, this mutation in the c-Ki-ras proto-oncogenes was also detected in 9 of 10 hyperplastic alveolar nodules. These results demonstrate that the specific c-Ki-ras mutation is a preneoplastic event in MNU-induced mouse mammary carcinogenesis.     

Proteomic evaluation of sheep serum proteins

Background

Mammary tumours frequently develop in female domestic cats being highly malignant in a large percentage of cases. Chemokines regulate many physiological and pathological processes including organogenesis, chemotaxis of inflammatory cells, as well as tumour progression and metastasization. In particular, the chemokine/receptor pair SDF-1/CXCR4 has been involved in the regulation of metastatic potential of neoplastic cells, including breast cancer. The aim of this study was the immunohistochemical defininition of the expression profile of CXCR4 in primary and metastatic feline mammary carcinomas and the evaluation of the role of SDF-1 in feline mammary tumour cell proliferation.

Results

A total of 45 mammary surgical samples, including 33 primary tumours (31 carcinomas and 2 adenomas), 6 metastases, and 4 normal mammary tissues were anlyzed. Tumor samples were collected from a total number of 26 animals, as in some cases concurrent occurrence of neoplasm in more than one mammary gland was observed. Tissues were processed for standard histological examination, and all lesions were classified according to the World Health Organization criteria. CXCR4 expression in neoplastic cells was evaluated by immunohistochemistry. The level of CXCR4 immunoreactivity was semi-quantitatively estimated as CXCR4 score evaluating both the number of positive cells and the intensity of staining. Six primary, fibroblast-free primary cultures were obtained from fresh feline mammary carcinomas and characterized by immunofluorescence for CXCR4 and malignant mammary cell marker expression. SDF-1-dependent in vitro proliferative effects were also assayed. CXCR4 expression was observed in 29 out of 31 malignant tissues with a higher CXCR4 score observed in 4 out of 6 metastatic lesions than in the respective primary tumours. In 2 benign lesions analyzed, only the single basaloid adenoma showed a mild positive immunostaining against CXCR4. Normal tissue did not show CXCR4 immunoreactivity. CXCR4 score was statistically significantly associated with the histological features of the samples, showing an increase accordingly with the degree of neoplastic transformation (from normal tissue to metastatic lesions). Finally, in the primary cultures obtained from 6 primary feline mammary carcinomas CXCR4 expression was detected in all cells and its activation by SDF-1 in vitro treatment caused a significant increase in the proliferation rate in 5 out of 6 tumours.

Conclusions

These results indicate that malignant feline mammary tumours commonly express CXCR4, with a higher level in malignant tumours, and, in most of the cases analysed, metastatic cells display stronger immunoreactivity for CXCR4 than the corresponding primary tumours. Moreover, CXCR4 activation in primary cultures of feline mammary carcinomas causes increase in the proliferative rate. Thus, SDF-1/CXCR4 system seems to play a tumorigenic in feline mammary gland malignancy and in vitro cultures from these tumour samples may represent an experimental model to investigate the biological and pharmacological role of this chemokinergic axis.     

Localization of endogenous lectins in normal human breast, benign breast lesions and mammary carcinomas

Specific antisera against three mammalian beta-galactoside-specific lectins of apparent molecular weights 14.5 kDa, 18 kDa and 29 kDa have been used to localize these lectins in normal breast, and in benign and malignant mammary lesions. In normal breast tissue discrete localization of two lectins (Mrs 14.5 kDa and 18 kDa) was demonstrated in fibroblasts, smooth muscle cells, myoepithelial cells and capillary endothelium. Extracellular localization of one lectin (Mr 14.5 kDa) in collagen was apparent. The third lectin (Mr 29 kDa) labelled preferentially luminal cells and their secretory product. Two benign tumours (an analyzed fibroadenoma and a papilloma) revealed strong staining with two lectins (Mrs 18 kDa and 29 kDa). Of the 24 mammary carcinomas examined, the lectin (Mr 14.5 kDa) was expressed by only occasional tumour cells, the lectin (Mr 18 kDa) occurred in many tumour cells and the lectin (Mr 29 kDa) labelled tumour cells in nearly all cases. The expression of these beta-galactoside-specific endogenous lectins therefore appears to be regulated differently in normal breast compared with mammary tumours.     

The 3-dimensional organization of the nucleoli of benign and malignant tumor cells from different human organs]

The method of ultrathin serial sections was used to perform a comparative ultrastructural and 3-dimensional analysis of nucleoli for the following variants of human tumours: benign (fibroadenoma) and malignant (infiltrating ductal carcinoma) tumours of one organ (mammary gland); malignant tumours of epidermal genesis in different organs (squamous cell carcinomas of skin, larynx, lung, gullet, uterus); two forms of malignant tumours (squamous cell and small cell carcinomas) of one organ (lung). The spatial models of nucleoli in these tumour cells are given. The specific signs in architecture of tumour nucleoli was found. Nucleoli of fibroadenomas have well pronounced 1-4 fibrillar centres forming a united system with a lacunar component and intranucleolar chromatin. Unlike benign tumour cells, nucleoli of infiltrating ductal carcinomas are characterized by large, prominent nucleoli containing giant, multiform fibrillar centres with a complicated surface, a well developed granular component and an unusually organized lacunar system. In squamous cell carcinomas of various localization, active, hypertrophied nucleoli with pseudonucleolonemal organization were found. The small cell carcinoma of lung differs from the squamous cell cancer of the same organ by dense, fibrillar nucleoli with a small amount of granular component located on the periphery of the nucleolar body. Nucleolar type reflecting the functional state of malignization process may serve as an additional diagnostic criterion for tumour identification.     

Mammary gland carcinoma-related increase of type I iodothyronine 5'-deiodinase activity in Sprague-Dawley rats.

Type I, iodothyronine 5'-deiodinase (5'-DI) catalyses deiodination of the prohormone thyroxine (T4) to the metabolically active 3,5,3'-triiodo-L-thyronine (T3). The present study was undertaken to investigate the activity of 5'-DI in rat mammary gland tumours representing various combinations of histologically defined papillary, cribriform or comedo patterns of ductal carcinomas. Female Sprague-Dawley rats were given two doses 50 mg x kg(-1) 1-methyl-1-nitrosourea (MNU) in abdominal parts on the 52nd day and 113th day of age. We have found that in comparison with non-lactating mammary gland, the activity of 5'-DI in all mammary gland tumours studied was significantly (p < 0.0001) increased and that the 5'-DI activity, expressed as pmol of 125I- released per min and per mg of protein, in malignant mammary gland tumours was found to be at least two order higher than that of intact mammary non-lactating gland. From our data, we suggest that thyroid hormone in mammary gland tumours might play a significant role to support high energetic expenditure of neoplastic tissues.     

Early indicators for carcinogenesis in sex-hormone-sensitive organs

Hormones induce tumours in various target tissues in different species of laboratory animals in long-term toxicity studies. Examples of such tumours are: mammary gland tumours in beagle dogs after long-term treatment with progestogens or progestogen/oestrogen combinations; pituitary and mammary gland tumours in rats and mice after long-term treatment with oestrogens or progestogens with an oestrogenic partial effect; interstitial cell tumours in rats after chronic overstimulation by endogenous luteinising hormone; endometrial carcinomas in rats after chronic treatment with dopamine agonists. As a rule every hormone when given in excessive doses over prolonged periods can induce a tumour in the relevant target organs. Drugs or chemicals which stimulate or inhibit the endogenous hormone production of certain endocrine organs can have the same effect. Tumour induction can be a direct or indirect effect involving specific regulatory mechanisms. In general, the induction is preceded by excessive hyperplasia of the target tissue concerned or with regard to the pituitary where excess production of the stimulating hormone occurs. Tumour induction in chronic toxicity studies can usually be predicted by determining hormone levels in short-term studies. Hormones and drugs or chemicals which induce tumours when given in doses high enough to induce hyperplasia are unlikely to do so by a genotoxic mechanism.     

Isolation of rat mammary epithelial cells for polyamine analysis.

Mammary epithelial cells were isolated from either abdominal-inguinal glands or mammary tumours of rats, after enzymic digestion of the tissues, and were analysed for polyamine content. Optimum conditions were developed for the isolation of cells in sufficient yield for the analysis of polyamines from 1 g of mammary gland or 0.5 g of tumour tissue. Complete recoveries of the polyamines in the tissues were achieved in the isolated epithelial cells.     

Dietary prevention of hormone refractory prostate cancer in Lobund-Wistar rats: a review of studies in a relevant animal model

Lobund-Wistar (LW) rats, which have high testosterone levels, are predisposed to develop hormone-refractory prostate cancer (HRPC) spontaneously and by methylnitrosourea (MNU) induction, and the development of HRPC progresses through 2 stages. This paper reviews several studies in which LW rats were placed on soy-containing diets and were evaluated for development of either spontaneous or MNU-induced prostate cancer. The premalignant, testosterone-dependent stage is inhibited by testosterone deprivation. In the absence of testosterone deprivation, tumorigenesis progresses spontaneously to the testosterone-independent refractory stage. In LW rats: moderate caloric restriction prevented development of spontaneous prostate cancer; dietary 4-hydroxyphenylretinamide prevented MNU-induced prostate cancer; and dietary supplementation with soy protein isolate with high isoflavones prevented spontaneous and induced tumors and led to moderate reduction of serum testosterone. In rats 12 mo of age and younger, changing from the control diet to the soy+isoflavone diet significantly prevented progression of spontaneous tumors to the refractory stage of disease. Tumors that developed spontaneously and after MNU induction showed similar developmental stages and morphology, but MNU-induced tumors had shorter latency periods before development. The accumulated data indicate that soy-based diets are effective in the prevention of prostate cancer.     

High susceptibility of transgenic rats carrying the human c-Ha-ras proto-oncogene to chemically-induced mammary carcinogenesis

A rat line carrying three copies of the human c-Ha-ras proto-oncogenes, including its own promoter region, was established and designated as Hras128. Expression of the transgene was detected in all organs by Northern blot analysis. To examine its influence on susceptibility to mammary carcinogenesis, female rats were treated with N-methyl-N-nitrosourea (MNU) or 7,12-dimethylbenz[a]anthracene (DMBA) at 50 days of age. With MNU, all the transgenic rats rapidly developed multiple mammary carcinomas within as short as 8 weeks (14.1 tumors/rat), in contrast to 0.46 tumors/rat in non-transgenic rats. PCR-RFLP analysis and direct sequencing for the transgene indicated that the large majority of carcinomas (38/44, 86.4%) contained cells with mutations at codon 12 in exon 1. However, comparison of the signal densities of the mutated band to dilution scale bands revealed that the cells with the mutated transgene were not in the majority. By PCR-SSCP analysis for codons 12 and 61 of the rat endogenous c-Ha-ras gene, no mutations were detected. Similarly, with DMBA, almost all (13/14, 92.9%) the transgenic rats developed multiple mammary carcinomas (9.39 tumors/rat) within 16 weeks, and 4 out of 12 (33.3%) non-transgenic rats had only small tumors (0.83 tumors/rat). A lower incidence of mutation of the transgene was found in codon 12 (5/25, 25%) than in MNU-induced tumors, but mutations were detected in codon 61 (7/20, 35%). No mutations were detected in the rat endogenous gene. No mutation was found in the rat endogenous c-Ha-ras gene in non-transgenic rats. As observed in both the MNU- and DMBA-induced tumor cases, the population of cells with the mutated transgene were in the minority. The results thus indicate that rats carrying the transduced human c-Ha-ras proto-oncogene are highly susceptible to MNU- and DMBA-induced mammary carcinogenesis and that this is not primarily due to mutations of the transgene or endogenous c-Ha-ras gene. Furthermore, irrespective of the mechanism of enhanced susceptibility, the Hras128 transgenic rats can be utilized for the screening of mammary carcinogens.     

Effect of dietary zinc intake on N-methyl-N-nitrosourea-induced mammary tumorigenesis in rats

Numerous studies have shown that zinc nutrition influences the growth of several types of tumor. However, the influence of zinc nutrition on mammary tumorigenesis is not known. To study the effects of dietary zinc intake on N-methyl-N-nitrosourea (MNU)-induced mammary tumorigenesis, female Sprague-Dawley rats were fed an egg-white-based diet providing 3 (Z3), 12 (Z12), or 31 (Z31) mg zinc/kg diet ad libitum. In addition, two pair-fed controls, PFZ12 and PFZ31, were also included. Fourteen weeks after MNU injection, cumulative tumor incidence and total number of tumors were lower in Z3 rats than in Z12 and Z31 rats. Cumulative tumor incidence and total number of tumors were lower in Z3 rats than in PFZ12 rats, but were the same as in PFZ31 rats. Cumulative tumor incidence and total number of tumors were also lower in pair-fed controls than in their corresponding ad libitum controls, but were the same between the ad libitum controls. Overall, the results showed that the effect of marginal zinc deficiency on MNU-induced mammary tumorigenesis in rats was primarily the result of a reduced feed intake associated with marginal zinc deficiency rather than zinc per se.     

Mammary cancer promotion by ovarian hormones involves IGFR/AKT/mTOR signaling

In a previous study, we observed that N-methyl-N-nitrosourea (MNU)-induced mammary lesions are promoted to overt mammary cancers by exogenous administration of estradiol (E) and progesterone (P). The purpose of the present study was to identify the early molecular events occurring during the hormonal promotion of mammary carcinogenesis and persistent activation of molecular pathways responsible for tumor growth. Seven-week-old female Copenhagen (COP) rats, which are resistant to MNU-induced mammary carcinogenesis, were intraperitoneally administered a single dose of MNU (50 mg/kg body weight). Six weeks after carcinogen administration, the rats were treated with E+P, killed at 15th week and 43rd week to obtain mammary lesions and tumor tissues and the molecular analysis were performed. Quantitative RT-PCR experiments showed increased mRNA expression of Igfr, Grb2, Sos1, and Shc1 in mammary lesions and tumors. Immunoblot data also showed increased protein levels of IGFR, GRB2 and SHC1 in mammary lesions and tumors, which is in correlation with their respective RT-PCR data. Activation of AKT and ERK1/2 were up regulated in E+P treated mammary lesions and tumors. Molecular analysis of mTOR pathway proteins revealed increased phosphorylation of p70S6K and 4EBP1 in the hormone treated tumors indicating the activation of mTOR signaling. E+P treatment reduced the protein expression of BAX and increased BCL2 expression along with down regulation of active caspase 3 and 8. Together, these data demonstrate that ovarian hormones promote the lesions to mammary tumors by enhancing IGFR and Akt/mTOR signaling along with inhibition of apoptotic stimuli.     

Effects of astaxanthin supplementation on chemically induced tumorigenesis in Wistar rats

ABSTRACT: BACKGROUND: Astaxanthin (ASTA) is a fat-soluble xanthophyll with powerful antioxidant functions. It is extracted from e.g. salmon, an important food source for certain human populations known to have a reduced risk of tumor development. It is possible that ASTA plays a role in cancer chemoprevention in such populations. The purpose of this study was to investigate the effects of dietary ASTA on chemically induced mammary tumorigenesis using N-methyl-N-nitroso-urea (MNU) in immature Wistar rats. METHODS: Thirty-six 37 days old juvenile female Wistar rats were at random allocated to 4 groups of which Groups 1 and 2 received a single dose of 55 mg MNU/kg body weight. The effects of ASTA was evaluated by giving rats of Groups 2 and 4 a dose of 50 mg ASTA/kg/day for the entire duration of the study. Group 3 rats received feed added alimentary oil.Necropsy and histopathological examinations were carried out on each rat 14 months after the administration of MNU. Haematological values and antioxidative status were determined. Oxidative stress was evaluated by monitoring superoxide dismutase (SOD) and glutathione peroxidase (GPx) activities in hepatic tissue. Lipid peroxidation and carbonylation of proteins was determined in protein extracts from the liver. RESULTS: Tumor development occurred only in rats of Groups 1 and 2, i.e. MNU exposed animals. Frequency of tumor development in general and average number of tumors per animal were insignificant between these two groups. Mammary gland tumors developed in equal frequencies in Group 1 and 2 rats, respectively. Although only rather few tumors were found in the mammary glands, a substantial number of other tumors were found in Group 1 and 2 rats, but at equal rates.Biochemical analyses showed significant higher levels of GPx, malondialdehyde and dinitrophenylhydrazine in Group 1 rats that for rats in all other groups thus indicating protective effects of ASTA on MNU induced hepatic oxidative stress. CONCLUSIONS: Supplementation with ASTA did not reduce tumorigenesis induced by MNU in Wistar rats. However, supplementation with ASTA seemed to have anti-inflammatory effects.     

Analysis of mammary tumors for cytochemical evidence of endogenous mammary peroxidase.

The purpose of this study was to determine whether or not endogenous mammary peroxidase can serve as a cytochemical marker to distinguish ovarian hormone-dependent from ovarian hormone independent mammary tumors. Spontaneous mammary tumors arising in virgin C3H and GR mice (hormone independent tumors) and hormone-dependent mammary tumors arising during pregnancy in GR mice were examined. None of these tumors contained mammary peroxidase. Mammary tumors induced in Sprague-Dawley rats with methylnitrousourea (MNU) and dimethylbenzanthracene (DMBA) were also examined. These tumors included hormone-dependent and hormone independent ones. Several of the DMBA-induced hormone-dependent tumors contained a few peroxidase-positive cells, but the hormone independent tumors were negative. All of the MNU-induced tumors examined were negative for mammary peroxidase. Twenty human breast tumors (malignant and non-malignant) removed from women at surgery, were also negative for mammary peroxidase. Our results indicate that endogenous mammary peroxidase cannot be used to distinguish hormone-dependent from hormone independent mammary tumors.     

Study of cytokines involved in the prevention of a murine experimental breast cancer by kefir

Previous studies have shown that compounds released during milk fermentation by Lactobacillus helveticus are implicated in the antitumour effect of this product. Here the effects of the consumption, during 2 or 7 days, of kefir or kefir cell-free fraction (KF) on the systemic and local immune responses in mammary glands and tumours using a murine hormone-dependent breast cancer model were studied. In the tumour control group, mice did not receive these products. At the end of the feeding period, mice were injected subcutaneously with tumour cells in the mammary gland. Four days post-injection, they received kefir or KF on a cyclical basis. Rate of tumour development, cytokines in serum; mammary gland tissue, and tumour isolated cells were monitored. Two-day cyclical administration of both products delayed tumour growth. Both kefir and KF increased IL-10 in serum and decreased IL-6(+) cells (cytokine involved in oestrogen synthesis) in mammary glands. Two-day cyclical administration of KF increased IL-10(+) cells in mammary glands and in tumours and decreased IL-6(+) cells in tumour. This study demonstrated the modulatory capacity of KF on the immune response in mammary glands and tumours and the importance of the administration period to obtain this effect.     

Etiology of MNU-induced melanomas in Xiphophorus hybrids

Genetic hybrids of the genus Xiphophorus have historically been useful models for study of the genetic aspects of tumor formation. In the most studied Xiphophorus tumor model, two-gene loci, XMRK and DIFF, are implicated as critical both to UV-induced and spontaneous melanoma formation in BC(1) hybrids of crosses between X. maculatus and X. helleri, with X. helleri as the recurrent backcross parent. In addition to UV, the direct-acting carcinogen N-methyl-N-nitrosourea (MNU) has been used to induce tumors in Xiphophorus BC(1) hybrids from several cross types. In the present study, we address the hypothesis that excess melanomas in MNU-treated BC(1) hybrids may have been generated by direct mutation of CDKN2AB, a candidate gene for DIFF. MNU treatment of F(1) and BC(1) hybrid fish significantly increased tumor incidence at 6 months; however, no association was found between MNU-induced tumor formation and zygosity of the candidate tumor tumor-suppressor CDKN2AB in BC(1) hybrids, consistent with previously reported results. Sequence analysis of the X. maculatus CDKN2AB locus of heterozygous individuals (both BC(1) and F(1) hybrids) did not reveal any mutations caused by MNU, suggesting that the mechanism of MNU-induced melanoma formation in this Xiphophorus model does not involve direct mutation of CDKN2AB but may result from mutation of other critical genes.