T. Tillmann

Sex- and strain-specific induction of renal tumors by ochratoxin A in rats correlates with DNA adduction

Ochratoxin A (OTA), a nephrotoxic and carcinogenic mycotoxin, has been implicated as an etiologic agent in the Balkan endemic nephropathy (BEN), a chronic disease affecting populations in the Balkans. Compared with unaffected individuals, patients suffering from BEN and/or urinary tract tumors were more frequently found to have a capacity for rapid debrisoquine (DB) metabolism, a metabolic reaction related mostly to cytochrome P450 (CYP) 2D in humans. Earlier studies, using female DA and Lewis rats phenotyped as poor or extensive DB metabolizers respectively, revealed a parallelism between DB‐4 hydroxylation and OTA‐4 hydroxylation. To investigate whether genetic polymorphism modifies tumor induction, we have compared both OTA‐induced renal carcinogenicity and DNA adducts in DA and Lewis rats of both sexes. OTA induced renal adenocarcinoma, DA male rats being most responsive, while DA females were resistant. Lewis rats showed an intermediate renal tumor response. OTA also induced malignant transitional cell carcinomas of the bladder in DA male rats only. DNA adducts in the kidney, as judged by the nature of spots and prevalence in OTA‐treated animals, were significantly correlated with renal carcinogenicity of OTA, being highest in DA males and lowest in DA females. A parallelism between karyomegalies and tumors of the kidney was observed. In conclusion, our results classify OTA as a genotoxic carcinogen in rats, with sex‐specific response controlled in part by drug‐metabolizing enzymes that convert OTA into reactive intermediates. Int. J. Cancer 77:70–75, 1998.© 1998 Wiley‐Liss, Inc.

Subcutaneous soft tissue tumours at the site of implanted microchips in mice

An experiment using 4279 CBA/J mice of two generations was carried out to investigate the influence of parental preconceptual exposure to X-ray radiation or to chemical carcinogens. Microchips were implanted subcutaneously in the dorsolateral back for unique identification of each animal. The animals were kept for lifespan under standard laboratory conditions. In 36 mice a circumscribed neoplasm occurred in the area of the implanted microchip. Females were significantly more frequently affected than male mice. An influence of age or different treatment on the s.c. tumour incidence in two mice generations could not be observed. Macroscopically, firm, pale white nodules up to 25 mm in diameter with the microchip in its center were found. Microscopically, soft tissue tumours such as fibrosarcoma and malignant fibrous histiocytoma were detected.

Ki-ras gene mutations and absence of p53 gene mutations in spontaneous and urethane-induced early lung lesions in CBA/J mice.

Ki‐ras and p53 genes are involved in human lung carcinogenesis; however, the role of these genes in experimental lung tumors is not well known. In our study, the CBA/J mouse strain was used to investigate the presence of Ki‐ras and p53 alterations in lung carcinogenesis of spontaneous tumors and tumors induced with high and low doses of urethane (ethyl carbamate). To study the presence of these alterations in the early stages of lung carcinogenesis and in very small lung tumors, restriction fragment length polymorphism and single‐strand conformation polymorphism analyses were performed on polymerase chain reaction–amplified DNA from microdissected tumoral and normal lung samples. Ki‐ras gene mutations in codons 12 and 61 were detected in all types of lung lesions, even in small and preneoplastic lesions, and their incidence increased with progression from lung hyperplasias (18%) to adenomas (75%) and to carcinomas (80%). Urethane exposure, in both high and low doses, increased the incidence of Ki‐ras mutations in lung tumors, especially in adenomas. The presence of Ki‐ras gene mutations in very small urethane‐induced lung tumors and the absence of hyperplasias among the treated‐group lesions may indicate that urethane accelerates tumoral progression. No p53 mutations were detected in exons 5–8 in any of the epithelium‐derived lung tumors. Only one p53 mutation in exon 5 was found in a spontaneous lymphoma. Therefore, p53 mutations do not seem to cooperate with Ki‐ras gene mutations or represent an alternative molecular pathway in murine carcinogenesis. Mol. Carcinog. 21:251–260, 1998.

Spectrum and age-related incidence of spontaneous tumours in a colony of Han:AURA hamsters

One-hundred-and-forty-four male and 184 female untreated Syrian golden hamsters (strain Han:AURA) were kept for life under standard laboratory conditions. They were examined with regard to spontaneously occurring tumours in relation to their survival periods. The mean survival rate of the males was 106 +/- 26 weeks and that of the females 97 +/- 20 weeks. Tumours were found in 71% of males and 67% of females. Adenomas and carcinomas of the adrenal glands were the most frequently observed tumours in both sexes (male: 66%; female: 38%) and in the early stages of life. Malignant lymphoma (8%), adenomas and carcinomas of pancreatic islet-cells (8%) and papillomatous benign and malignant squamous cell tumours of the forestomach (7%) showed relatively high incidences in males, whilst in females, leiomyoma (10%) and endometrial adenocarcinoma (7%) of the uterus and adenomas and carcinomas in the pars distalis of the pituitary gland (9%) occurred frequently.

Age-related incidence of spontaneous non-neoplastic lesionsin a colony of Han:AURA hamsters

In toxicologic testing or experimental studies using animals, an adequate knowledge of spontaneously occurring lesions is required. 144 male and 184 female untreated Syrian golden hamsters (strain Han:AURA) were kept for life under standard laboratory conditions and an investigation of non-neoplastic lesions in relationship to the lifespan was performed. The average lifespan of the males was 106 weeks and that of the female hamsters 97 weeks. While cartilage degeneration of the sternum and fatty degeneration of the femoral bone marrow occurred already in the first half of life with high incidence, the majority of lesions were observed in the second half. The most frequent non-neoplastic changes in various organs were fatty change, calcification, cystic change, hyperplasia and amyloidosis. Such spontaneous lesions were discussed in connection with the same alterations which can also be induced by chemical or hormonal agents.

Ito Cell Tumor: Immunohistochemical Investigations of a Rare Lesion in the Liver of Mice

In 2 lifespan transgeneration experiments using a total of 4,682 CBA/J mice, we observed uncommon lipomatous lesions in the livers of 8 mice independent of the treatment. Macroscopically, the lesions were described as pale white areas (2) or nodules (6) during necropsy. The lesions ranged from 1 to 15 mm in diameter. Microscopically, the lesions consisted of nodular aggregations of round to spindle-shaped cells that partly caused distinct compression of the adjacent hepatic parenchyma. The tumor cells were smaller than hepatocytes and had dark oval nuclei. Many of the more spherical cells contained clear vacuoles of various sizes, which were shown to be lipid droplets by oil red O staining. In addition to Gomoris silver and Massons trichrome staining, several immunohistochemical stains were used to characterize the origin of the proliferating cells. Tumor cells were labeled by vimentin, actin, desmin, and proliferating cell nuclear antigen. The 2 cell phenotypes showed similar staining characteristics. Increased amounts of laminin and tenascin, 2 extracellular matrix proteins of the liver, were detected within these neoplasms. Summarizing, we suggest that these tumors are of Ito cell origin.

Quality control of three methods for lung tumorigenesis studies

Many variables influence experimental results obtained from laboratory animal studies. One of the variables is tissue sampling for the detection of lesions. The contribution of different levels of sampling to the variability in reported tumour rates was evaluated in a tumorigenesis study using 1872 CBA/J mice. The number of lung neoplasms was estimated by three methods and the results compared. These methods were: 1. counting the macroscopically visible nodules, 2. microscopical examination of macroscopically-detected nodules and one histological section of each lung lobe, cut at the level of the bronchi (common method) and 3. microscopical examination as in method number 2 and additional microscopic examination of step-sections (200 microm interval) of the remaining lung tissue beginning at the level of the bronchi. Analysis using only macroscopic examination (method 1) showed that 40% (747/1872) of the animals had single or multiple nodules (i.e. tumour suspicious areas) in the lungs. When combined with microscopic examination (method 2), primary lung tumours were diagnosed in only 586 animals (31%). Evaluation by gross examination alone therefore gave an apparent overestimation of lung tumours compared to microscopic evaluation of grossly visible nodules. This was found to be due to a significant number of mice having nodules formed by processes other than primary lung tumours (i.e. non-specific inflammation, alveolar histiocytosis, focal hyperplasia of the alveolar epithelium, lymphoma infiltration or tumour metastases). On the other hand, in the more thorough sectioning of the lungs (method 3), primary lung tumours were detected in 712/1872 animals (38%). Additionally, these three different methods influenced the results with regard to the tumour multiplicity in each tumour-bearing animal.

Sex- and Strain-Specific Induction of Renal Tumors by Ochratoxin A in Rats Correlates With DNA Adduction

Ochratoxin A (OTA), a nephrotoxic and carcinogenic mycotoxin, has been implicated as an etiologic agent in the Balkan endemic nephropathy (BEN), a chronic disease affecting populations in the Balkans. Compared with unaffected individuals, patients suffering from BEN and/or urinary tract tumors were more frequently found to have a capacity for rapid debrisoquine (DB) metabolism, a metabolic reaction related mostly to cytochrome P450 (CYP) 2D in humans. Earlier studies, using female DA and Lewis rats phenotyped as poor or extensive DB metabolizers respectively, revealed a parallelism between DB-4 hydroxylation and OTA-4 hydroxylation. To investigate whether genetic polymorphism modifies tumor induction, we have compared both OTA-induced renal carcinogenicity and DNA adducts in DA and Lewis rats of both sexes. OTA induced renal adenocarcinoma, DA male rats being most responsive, while DA females were resistant. Lewis rats showed an intermediate renal tumor response. OTA also induced malignant transitional cell carcinomas of the bladder in DA male rats only. DNA adducts in the kidney, as judged by the nature of spots and prevalence in OTA-treated animals, were significantly correlated with renal carcinogenicity of OTA, being highest in DA males and lowest in DA females. A parallelism between karyomegalies and tumors of the kidney was observed. In conclusion, our results classify OTA as a genotoxic carcinogen in rats, with sex-specific response controlled in part by drug-metabolizing enzymes that convert OTA into reactive intermediates.