PRV-1 mRNA expression discriminates two types of essential thrombocythemia.

Essential thrombocythemia (ET) is a heterogeneous disorder. For example, the growth of erythropoietin-independent erythroid colonies, termed "endogenous erythroid colonies (EECs)", has previously been observed in only 50% of ET patients. We have recently described the overexpression of a hematopoietic receptor, PRV-1 (polycythemia rubra vera-1), in patients with polycythemia vera (PV). Here, we compare PRV-1 expression and EEC formation in a cohort of 30 patients with ET; 50% of the ET patients in our cohort displayed EEC growth. Likewise, 50% of the ET patients overexpressed PRV-1. Remarkably, only the 15 ET patients displaying EEC growth showed elevated PRV-1 expression, while the 15 EEC-negative ET patients expressed normal PRV-1 levels. It has previously been reported that EEC-positive ET patients develop PV during long-term follow-up. Here, we show that 40% of the PRV-1-positive patients develop symptoms of PV during the course of their disease. In contrast, none of the 15 PRV-1-negative patients displayed such symptoms (p=0.017). Moreover, PRV-1-positive patients had a significantly higher number of thromboembolic or microcirculatory events (p=0.003). We propose that PRV-1-positive ET comprise a pathophysiologically distinct subgroup of patients, one that is at risk for the development of complications and for the emergence of PV.

Inhalation toxicity of propineb. Part II: Results of mechanistic studies in rats.

Previous repeated inhalation exposure studies revealed two independent organotropic effects of inhaled propineb dust: One was restricted to the lung, the other to muscle weakness of hindlimbs. These effects were believed to be causally related to the principle decomposition products of this type of dithiocarbamate in the biological milieu and related to zinc and carbon disulfide. Two mechanistic 1-wk inhalation studies were performed, each focusing on one of these findings. The 7 x 6-h/day repeated-exposure inhalation study analyzed whether the nature of the response occurring at the alveolar level is "adaptive" or "early adverse" and whether soluble zinc is the causative agent. Groups of 18 female rats were exposed nose-only to mean concentrations of 0, 1.1, 5.5, and 25.8 mg propineb/m(3) and 6.9 mg ZnO/m(3). On postexposure days 1, 3, and 15 the time course of responses was analyzed by bronchoalveolar lavage (BAL), including quantification of Zn and metallothionein (MT) in BAL cells. Clinical evidence of muscular weakness was investigated separately in 20 female Wistar rats exposed to 70 mg propineb/m(3) on 5 consecutive days (6 h/day), followed by a 2-wk postexposure period. Clinical signs, body weights, and feed and water consumption were recorded as frequently as technically feasible. Fifty percent of rats received an oral cysteine supplementation to verify/refute the hypothesis that the incapacitation observed in previous studies is the cause of emaciation and associated impairment of CS(2) detoxification. The findings of the first study are consistent with this hypothesis, namely, that soluble Zn triggers a series of pulmonary events that is consistent with the homeostasis of this essential metal. It is concluded, accordingly, that the adjusted maximal workplace level for ZnO is also valid for propineb to preclude Zn-mediated responses to occur in the lung. With respect to muscular effects, this mechanistic study demonstrates further that the increased detoxification capacity afforded by oral supplementation of cysteine mitigates markedly the toxic potency of propineb. Procedural variables specific to the inhalation bioassay appear to be decisive for the elicitation of muscular effects. The major variable is considered to be the large drop in body weights associated with each exposure session and the concomitantly decreased uptake of essential nutritional factors (e.g., cysteine) involved in the detoxification of this compound. Accordingly, the muscular deficits elicited by high concentrations of propineb are viewed to be secondary effects in an animal species likely to be more susceptible to this type of change than humans (Pauluhn & Rosenbruch, 2003).

In vitro genotoxicity assay of sidestream smoke using a human bronchial epithelial cell line.

Genotoxic effects of air contaminants, such as gaseous or particulate compounds, have been difficult to investigate due to inefficient methods for exposing cell cultures directly to these substances. New cultivation and exposure techniques enable treatment of epithelial cells with sample atmospheres with subsequent in vitro assays, as demonstrated by a new system called CULTEX (CULTEX: patent No. DE 19801763; PCT/EP99/00295), which uses a transwell membrane technique for direct exposure of complex mixtures, for example sidestream cigarette smoke, at the air/liquid interface. The sensitivity and susceptibility of human bronchial epithelial cells to this complex mixture have already been shown for cytotoxic endpoints. In this study, genotoxic effects of sidestream cigarette smoke at different concentrations were assessed using the alkaline comet assay. HFBE 21 cells were exposed for 1 h to clean air, nitrogen dioxide or sidestream smoke. Exposure of the cells to sidestream cigarette smoke induced DNA strand breaks in a dose-dependent manner. The combination of gas phase exposure and the comet assay provides a realistic and efficient model for sensitive detection of DNA strand breaks induced by airborne and inhalable compounds.

Inhalation toxicity of 4-ethoxyaniline (p-phenetidine): critical analysis of results of subacute inhalation exposure studies in rats.

This article addresses results from a single 4-h and repeated 1- and 4-wk inhalation exposure studies in Wistar rats with vapor and/or aerosol atmospheres of 4-ethoxyaniline (p-phenetidine). Groups of 10 rats/sex were exposed nose-only to mean analytical concentrations of 11.1, 86.2, and 882.6 mg p-phenetidine/m(3) using an exposure regimen of 6 h/day, 5 days/wk for 4 wk. Concentrations were selected based on results from a pilot study in which rats were exposed under identical conditions on 5 consecutive days for 6 h/day to mean analytical concentrations of 38.2, 133.0, and 1247.6 mg/m(3). In repeated exposure studies, the focus of endpoints was on hematotoxicity. The LC50 was not determined, but no rats died following a single 4-h exposure to 5085 mg/m(3) as a mixture of vapor and aerosol. No mortality was observed either in the 1- or 4-wk studies. Rats exposed to 882.6 mg/m(3) and above evoked characteristic signs of toxicity that included cyanosis, with no apparent progression of findings during the exposure period. Animals exposed to 86.2 mg/m(3) and above exhibited a concentration-dependent, significant increase in blood methemoglobin and reticulocyte counts as well as a significant decrease in hemoglobin, hematocrit, and red blood cell counts. Spleen weights were significantly increased in groups exposed to 133.0 mg/m(3) and above. Microscopic changes demonstrated an increased hematopoiesis (bone marrow smears) and splenic hemosiderosis at 86.2 and 882.6 mg/m(3) and a hepatic hemosiderosis only at 882.6 mg/m(3). These data suggest that the toxicity of p-phenetidine is similar to that of its structural analog aniline. Based on the erythrocytotoxicity occurring at 86.2 mg/m(3) and above, including the apparent reactive changes in bone marrow (increased erythropoiesis) and spleen (increased erythroclasia), the no-observed-adverse-effect level (NOAEL) of the 4-wk study was 11.1 mg/m(3) air and that of the 1-wk study was 38.2 mg/m(3) air. This difference in NOAELs is considered to be related to the selection of exposure concentrations rather than cumulative toxicity.

Assessment of the morphology and significance of the lymph nodal and hepatic lesions produced in rats by the feeding of certain mineral oils and waxes. Proceedings of a pathology workshop held at the Fraunhofer Institute of Toxicology and Aerosol Research Hannover, Germany, May 7-9, 2001.

A Panel of medical and veterinary pathologists reviewed published and unpublished reports dealing with studies of various white mineral oils and waxes in F344 and Sprague-Dawley rats. They also had available and studied histologic slides from both subchronic and chronic studies of certain mineral hydrocarbons (90-day oral study of low melting point wax (LMPW) in female Fischer 344 and Sprague-Dawley rats; 90-day studies of P15H* and P70H white oil and high melting point wax (HMPW) in male and female F344 rats and 24 month study of P70H white oil in male and female F344 rats. The Panel also reviewed mineral oil-induced alterations in tissues of human patients (liver, hepatic lymph node and spleen). The Panel agreed that certain of the mineral hydrocarbons produced lesions in the mesenteric lymph nodes and liver of the F344 rat and these lesions were best described as microgranulomas/granulomas. The lesions were fundamentally similar in both organs, although varying in severity with dose and type of mineral hydrocarbons. The Panel agreed that hepatic lesions with inflammatory cell infiltration, necrosis, and fibrosis were produced only by feeding of LMPW and the lesions were confined to F344 rats and not found in Sprague-Dawley rats. The most severe granulomatous lesions in the mesenteric lymph nodes were found in high dose LMPW-fed F344 rats. The microgranulomas were similar in subchronic and chronic studies. Also, little difference existed between controls and treated F344 rats in the incidence and severity of the lesions after 2 years of feeding P70H white oil. The Panel agreed that some slight reversibility existed for these lesions, but also agreed that complete resolution was unlikely as regression of the lesions in the rat would likely be slow. The Panel agreed that a minimal severity infiltrate of mononuclear inflammatory cells occurred in the base of the mitral valve in a slightly increased incidence in F344 rats fed LMPW. The Panel concluded that these mitral valve alterations had little if any toxicologic significance as the focal infiltrate was minimal in severity, occurred in controls, occurred in association with murine cardiomyopathy, and were unlike the responses in the liver and mesenteric lymph nodes. The Panel agreed that the lesions observed in the liver and mesenteric lymph nodes of F344 rats exposed to MHCs, especially the LMPW, were different morphologically from changes observed in lymph node, liver, and spleen of humans that were mineral oil-users. These changes in humans are usually found incidentally in tissues taken at biopsy or autopsy. The MHC-induced lesions can be considered incidental and inconsequential in humans.

Age-related incidence of spontaneous non-neoplastic lesions in 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.

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.

Expression of cyclin D1 and p53 and its correlation with proliferative activity in the spectrum of esophageal carcinomas induced after duodenal content reflux and 2,6-dimethylnitrosomorpholine administration in rats.

Alterations in expression of the p53 and cyclin D1 genes have been implicated in the development of esophageal carcinomas in both humans and animal models. We hypothesize that altered expression of cyclin D1 and p53 may be involved in the sequential development of esophageal carcinomas with glandular differentiation induced by the carcinogen, 2,6-dimethylnitrosomorpholine (DMNM) in rats with duodenal content reflux esophagitis. In the present study Sprague-Dawley rats were given DMNM 15 days after performing an esophago-jejunostomy in order to induce chronic duodenal content reflux esophagitis. Expression and localization of p53, cyclin D1 and Ki-67 were examined by immunohistochemical analyses. Twenty of 24 animals developed different types of esophageal carcinomas, including pure squamous carcinoma, adenosquamous carcinoma and pure adenocarcinoma. Undifferentiated basaloid areas were frequently observed in these tumors. Cyclin D1 overexpression was observed in hyperplastic lesions and increased through dysplasia and in undifferentiated areas of infiltrating carcinoma. Cyclin D1 expression coincided with increased Ki-67 expression and decreased along with cell differentiation. The p53 immunohistochemical pattern was parallel to that of cyclin D1, although the percentage of positive cells was usually smaller in all lesions and increased p53 expression started at the dysplastic stage. These findings suggest that overexpression of cyclin D1 may be an early event in DMNM-induced rat esophageal tumorigenesis, causing increased proliferation of esophageal stem cells. Abnormal p53 expression may then be required to promote the development of neoplastic transformation from dysplastic epithelium through invasive phenotype, being more evident in cancer cells with squamous differentiation.

Rabbit cytochrome P450 4B1: A novel prodrug activating gene for pharmacogene therapy of hepatocellular carcinoma.

Gene therapy using vector-mediated transfer of prodrug activating genes is a promising treatment approach for malignant tumors. As demonstrated recently, the novel prodrug activating gene coding for rabbit cytochrome P450 4B1 (CYP4B1) is able to induce tumor cell death at low micromolar concentrations in glioblastoma cells after treatment with the prodrug 4-ipomeanol (4-IM) in vitro and in vivo. The rabbit CYP4B1 converts this prodrug and other furane analogs and aromatic amines, such as 2-aminoanthracene, to highly toxic alkylating metabolites, whereas the human isoenzyme exhibits only minimal enzymatic activity. In the present study, the cDNA encoding rabbit CYP4B1 was used for pharmacogene therapy of hepatocellular carcinoma (HCC). Cell clones derived from the human HCC cell lines Hep3B, HuH-7, and HepG2 and stably expressing the chimeric protein CYP4B1-EGFP (the CYP4B1 coding sequence fused to the enhanced green fluorescent protein (EGFP) gene) were selected. HCC clones expressing EGFP served as controls. 4-IM rapidly induced tumor cell death in CYP4B1-EGFP-expressing clones at low concentrations (a 50% lethal dose of between 0.5 and 2 microg/mL). No signs of toxicity were found in control cells expressing EGFP even at high prodrug concentrations (20 microg/mL). Cell death occurred by apoptosis and was independent of functional p53. A pronounced direct bystander effect was observed in Hep3B cells, whereas bystander HepG2 and HuH-7 cells were highly resistant to toxic 4-IM metabolites. These results demonstrate that the CYP4B1/4-1M system efficiently and rapidly induces cell death in HCC cells, and that a cell line-specific mechanism may exist that limits the extent of the bystander effect of this novel prodrug activating system.

Incidence and spectrum of spontaneous neoplasms in male and female CBA/J mice.

Although CBA mice are occasionally used in biomedical research, little is known about their life-data and diseases ("background pathology"). Therefore, it was the aim of this study to determine the life expectancy, spectrum and incidence of spontaneous neoplasms of the inbred CBA/J mouse strain. A total of 631 untreated mice (293 females; 338 males) were kept from birth to death under standard laboratory conditions. A complete histological examination was performed on all organs/tissues. In female CBA mice, the average lifespan was 94 weeks, while the mean age in males was 85 weeks. Neoplastic lesions were observed in 70% (238/338) of the males and 51% (150/293) of the female CBA/J mice. Tumours of the liver, lung and haematopoietic tissue were common in males, while tumours of the lung, ovary and haematopoietic tissue were the most frequent neoplasms in female CBA/J mice.

CULTEX--an alternative technique for cultivation and exposure of cells of the respiratory tract to airborne pollutants at the air/liquid interface.

The assessment of cytotoxicity of air contaminants such as gaseous or particulate compounds and complex mixtures has traditionally involved animal experiments, due to the difficulties in exposing cell cultures directly to these substances. New cultivation and exposure techniques enhance the efficiency of in vitro studies, as demonstrated by a new experimental system called CULTEX which allows direct exposure of cells at the air/liquid interface. In this case, human bronchial epithelial cells are cultivated on porous transwell membranes in a device allowing intermittent medium supply. The medium is pumped into a special modular culture unit through the transwell membrane supporting the cells. At certain time intervals, the medium is completely removed and the cells can be maintained and exposed at the air/liquid interface until the next medium supply without loss of viability. In comparison to conventional submersed culture conditions, the cells have been grown on transwell membranes using the new pulse submersion technique. There are no deleterious effects on cell viability due to the direct exposure to airborne pollutants. Thus, the introduction of these new cultivation and exposure techniques offers new testing strategies for the toxicological evaluation of inhalable soluble and inert substances as well as complex mixtures.

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 Gomori's silver and Masson's 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.

Possible carcinogenic effects of X-rays in a transgenerational study with CBA mice.

A lifetime experiment using 4279 CBA/J mice was carried out to investigate whether the pre-conceptual exposure of sperm cells to X-ray radiation or urethane would result in an increased cancer risk in the untreated progeny, and/or increased susceptibility to cancer following exposure to a promoting agent. The study consisted of four main groups, namely a control group (saline), a urethane group (1 mg/g body wt) and two X-ray radiation groups (1 Gy, 2 Gy). At 1, 3 and 9 weeks after treatment, the males of these four parental groups were mated with untreated virgin females. The offspring of each parental group was divided into two subgroups: one received s.c. urethane (0.1 mg/g body wt once) as a promoter, the other saline, at the age of 6 weeks. All animals were evaluated for the occurrence of tumours. K-ras oncogene and p53 tumour suppressor gene mutations were investigated in frozen lung tumour samples. The female offspring of male parents exposed to X-rays 1 week before their mating showed a trend towards a higher tumour incidence of the haematopoietic system than the F1 controls. In addition, a higher percentage of bronchioloalveolar adenocarcinomas in male offspring born to irradiated paternals mated 1 week after X-ray treatment points to a plausible increased sensitivity of post-meiotic germ cell stages towards transgenerational carcinogenic effects. On the other hand, no increased tumour incidence and malignancy were observed in the offspring born to irradiated paternals mated 3 and 9 weeks after X-ray treatment. Paternal urethane treatment 1, 3 and 9 weeks prior to conception did not result in significantly altered incidence or malignancy of tumours of the lung, liver and haematopoietic tissue in the offspring. K-ras mutations increased during tumour progression from bronchioloalveolar hyperplasia to adenoma. Codon 61 K-ras mutations were more frequent in lung tumours of urethane-promoted progeny from irradiated parents than from control parents. P53 mutations were absent from these lung alterations.

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.

Sarcocystis muris--a rare case in laboratory mice.

In a lifespan transgeneration study under standard laboratory conditions using a total of 4682 CBA/J mice, unusual intramuscular inclusions were found in the diaphragm, heart and skeletal muscle of one mouse using light microscopy. Located within the myocytes, they caused no visible tissue reaction. Cross-sections of these spherical and cystic lesions showed numerous banana-shaped structures, identified as permanent parasitic bradyzoites, which permitted these infections to be diagnosed microscopically as sarcocystosis.

Sex- and strain-specific expression of cytochrome P450s in ochratoxin A-induced genotoxicity and carcinogenicity in rats.

Ochratoxin A (OTA), a nephrotoxic and carcinogenic mycotoxin, is implicated in the etiology of Balkan endemic nephropathy (BEN), a chronic disease affecting populations in the Balkans. Patients suffering from Balkan endemic nephropathy, urinary-tract tumors, or both are more frequently extensive metabolizers of debrisoquine than persons unaffected by these conditions. As shown previously (Castegnaro et al., Int J Cancer, 77:70-75, 1998), OTA induction of renal tumors is markedly sex- and strain-specific in Dark Agouti (DA) and Lewis rats, with DA males being most responsive and DA females being resistant; only DA females were phenotyped as slow debrisoquine metabolizers. Formation of OTA-related DNA adducts in the kidney was closely correlated with renal carcinogenicity. To elucidate the critical biotransformation enzymes involved in OTA genotoxicity and carcinogenicity, the expression of cytochrome P450s (CYPs) 1A, 2A, 2B, 2C, 2D, and 3A in the kidney and liver microsomes of untreated and OTA-treated DA and Lewis rats (both sexes) was determined by western blot analysis. Chronic OTA treatment was found to modify CYP expression in kidney and liver. In the animals most susceptible to renal OTA carcinogenicity and DNA adduct formation, the OTA-toxifying isoforms (CYPs 2C11, 1A2, and 3A) were highly expressed in the liver, and little of the OTA-detoxifying isoforms (CYPs 1A1 and 2A) was detected. CYP2D was not expressed in DA rats and therefore is not involved in these phenomena. Our results confirm that the strain- and sex-specific genotoxic response of OTA is controlled, in part, by CYP-mediated metabolic reactions that convert OTA into DNA-reactive intermediates.

Correlation between molecular genetic analyses and immunohistochemical evaluation of the epidermal growth factor receptor and p185HER2.

Several methods have been developed for the measurement of gene amplification and expression. This study compared different molecular genetic analyses (Southern blot analysis (SBA) and polymerase chain reaction (PCR)) with immunohistochemical (IHC) evaluation of the corresponding protein content. PCR may be used as a semi-quantitative analysis of gene amplification and allows DNA extraction from paraffin-embedded blocks. SBA is more accurate than PCR to measure the exact degree of amplification, but only DNA extracted from frozen or fresh tissue can be used. We examined 23 breast tumors and 16 lung tumors, where the genes HER-1 coding for the epidermal growth factor receptor (EGFR) and HER-2 coding for p185HER-2 were analysed. Furthermore, PCR performed on DNA from frozen tissue was compared to PCR on DNA extracted from paraffin-embedded blocks. The results showed correlation between the different analyses, especially when the gene copy number was highly amplified. Some breast tumors showed moderately increased gene copy number of HER-1 by SBA, but no increased protein content by IHC evaluation. This probably reflects that minor degrees of genetic aberrations are not sufficient to cause major biological disturbances, because regulatory cellular pathways are still operating.

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.