Development and application of the physiologically-based toxicokinetic (PBTK) model for ochratoxin A (OTA) in rats and humans.

Ochratoxin A (OTA) is a common fungal toxin frequently detected in food and human plasma samples. Currently, the physiologically based toxicokinetic (PBTK) model plays an active role in dose translation and can improve and enhance the risk assessment of toxins. In this study, the PBTK model of OTA in rats and humans was established based on knowledge of OTA-specific absorption, distribution, metabolism, and excretion (ADME) in order to better explain the disposition of OTA in humans and the discrepancies with other species. The models were calibrated and optimized using the available kinetic and toxicokinetic (TK) data, and independent test datasets were used for model evaluation. Subsequently, sensitivity analyses and population simulations were performed to characterize the extent to which variations in physiological and specific chemical parameters affected the model output. Finally, the constructed models were used for dose extrapolation of OTA, including the rat-to-human dose adjustment factor (DAF) and the human exposure conversion factor (ECF). The results showed that the unbound fraction (Fup) of OTA in plasma of rat and human was 0.02-0.04% and 0.13-4.21%, respectively. In vitro experiments, the maximum enzyme velocity (Vmax) and Michaelis-Menten constant (Km) of OTA in rat and human liver microsomes were 3.86 and 78.17 µg/g min-1, 0.46 and 4.108 µg/mL, respectively. The predicted results of the model were in good agreement with the observed data, and the models in rats and humans were verified. The PBTK model derived a DAF of 0.1081 between rats and humans, whereas the ECF was 2.03. The established PBTK model can be used to estimate short- or long-term OTA exposure levels in rats and humans, with the capacity for dose translation of OTA to provide the underlying data for risk assessment of OTA.

Subcellular spatio-temporal intravital kinetics of aflatoxin B1 and ochratoxin A in liver and kidney.

Local accumulation of xenobiotics in human and animal tissues may cause adverse effects. Large differences in their concentrations may exist between individual cell types, often due to the expression of specific uptake and export carriers. Here we established a two-photon microscopy-based technique for spatio-temporal detection of the distribution of mycotoxins in intact kidneys and livers of anesthetized mice with subcellular resolution. The mycotoxins ochratoxin A (OTA, 10 mg/kg b.w.) and aflatoxin B1 (AFB1, 1.5 mg/kg b.w.), which both show blue auto-fluorescence, were analyzed after intravenous bolus injections. Within seconds after administration, OTA was filtered by glomeruli, and enriched in distal tubular epithelial cells (dTEC). A striking feature of AFB1 toxicokinetics was its very rapid uptake from sinusoidal blood into hepatocytes (t1/2 ~ 4 min) and excretion into bile canaliculi. Interestingly, AFB1 was enriched in the nuclei of hepatocytes with zonal differences in clearance. In the cytoplasm of pericentral hepatocytes, the half-life (t1/2~ 63 min) was much longer compared to periportal hepatocytes of the same lobules (t1/2 ~ 9 min). In addition, nuclear AFB1 from periportal hepatocytes cleared faster compared to the pericentral region. These local differences in AFB1 clearance may be due to the pericentral expression of cytochrome P450 enzymes that activate AFB1 to protein- and DNA-binding metabolites. In conclusion, the present study shows that large spatio-temporal concentration differences exist within the same tissues and its analysis may provide valuable additional information to conventional toxicokinetic studies.

Synergistic Interaction of Ochratoxin A and Acrylamide Toxins in Human Kidney and Liver Cells.

Ochratoxin A (OTA) is a mycotoxin produced by Aspergillus and Penicillium, and it is found in many foods. Acrylamide (AA) can be produced in foods treated at high temperatures. In this study, we investigated the combined toxicity of OTA and AA against human renal and hepatic cells in vitro. The concentration at which the synergistic effect of OTA and AA occurs was determined using the combination index obtained from the cell viability results for OTA and AA individually or in combination. The synergistic toxicity of both substances was evaluated by cell viability and the production of reactive oxygen species. In addition, apoptosis-related markers were significantly upregulated by OTA and AA individually or in combination. To determine the combined toxic effects of OTA and AA on the cells, the levels of enzymes involved in the phase I reaction and apoptosis-related markers were determined using quantitative (q)PCR and Western blot. The expression levels of CYP enzymes CYP1A1 and CYP1A2 involved in the phase I reaction significantly increased when the cells were treated with OTA and AA in combination. The expression of apoptosis-related markers, Bcl2-associated X protein (Bax) and caspase 3, also increased when the cells were treated with OTA and AA in combination. Therefore, the synergistic toxicity of OTA and AA suggests that such effects may contribute to nephrotoxicity and hepatotoxicity.

[Toxicological characteristics of Ochratoxin A and its impact on male reproduction].

Ochratoxin A (OTA) is found not only nephrotoxic, teratogenic, neurotoxic, and immunotoxic, but also reprotoxic for human and animals. In the recent decade, more attention has been paid to the impact of OTA on human reproduction and the studies of its underlying mechanisms. Many studies show that OTA affects the function of the reproductive system by acting as an endocrine disrupter and, as a testicular toxin, decreases sperm quality and even induces testis cancer. This review summarizes the toxicological characteristics and toxicokinetic process of OTA as well as recent progress in the studies of various toxic effects of OTA and their underlying mechanisms, hoping to call the attention from more people to the toxicity of OTA to male reproductive health.

Short communication: Toxicokinetics of ochratoxin A in dairy ewes and carryover to milk following a single or long-term ingestion of contaminated feed.

Ruminal microbes have the capacity to inactivate ochratoxins, rendering ruminants less sensitive to this fungal contaminant found in cereal feeds. However, ochratoxin A has been reported in milk surveys. The objective of this study was to assess the toxicokinetics, excretion, and transmission into milk of ochratoxin A using doses similar to those of naturally occurring field contaminations. Six Lacaune dairy ewes in late lactation were separated into 2 groups that received a single dose of contaminated wheat containing 5 or 30 µg of ochratoxin A/kg of body weight. After administration, toxicokinetics and excretion were monitored for 48 h. Subsequently, ewes were administered the corresponding toxin dose daily for 24 d followed by a second toxicokinetics and excretion monitoring period for this long-term exposure. The doses used did not affect production or health of ewes. After a single dose, ochratoxin A and its main metabolite, ochratoxin α, were found in blood 1h postexposure. The maximum blood concentrations of ochratoxin A and α, respectively, were dose dependent and were observed, on average, 6 and 8h after exposure. Long-term exposure increased the maximum concentration of ochratoxin A detected in blood, whereas ochratoxin α was not affected. In contrast, the time to reach the maximum concentration was reduced to 3h for both molecules. Ochratoxins, essentially ochratoxin α, were mainly excreted in feces. Ochratoxin A and α were detected in milk at concentrations that were dose dependent but with a low carryover rate (<0.02%). Chronic administration did not increase the concentration of toxin in milk. Even though ochratoxin A can escape ruminal degradation and traces were found in milk of experimentally exposed ewes, the low carryover of ochratoxin A in milk minimizes the risk to consumers.

Ochratoxin A exposure biomarkers in the Czech Republic and comparison with foreign countries.

Among ochratoxins, ochratoxin A (OTA) occupies a dominant place and represents significant risk for human and animal health which also implies economic losses around the world. OTA is nephrotoxic, hepatotoxic, teratogenic and immunotoxic mycotoxin. OTA exposure may lead to formation of DNA adducts resulting to genotoxicity and carcinogenicity (human carcinogen of 2B group). Now it seems that OTA could be "a complete carcinogen" which obliges to monitor its presence in biological materials, especially using the suitable biomarkers. In this article, OTA findings in urine, blood, serum, plasma and human kidneys (target dose) in the Czech Republic and comparison with foreign countries are presented.

Functional characterization of mouse organic anion transporting peptide 1a4 in the uptake and efflux of drugs across the blood-brain barrier.

This study investigated the role of a multispecific organic anion transporter, Oatp1a4/Slco1a4, in drug transport across the blood-brain barrier. In vitro transport studies using human embryonic kidney 293 cells expressing mouse Oatp1a4 identified the following compounds as Oatp1a4 substrates: pitavastatin (K(m) = 8.3 microM), rosuvastatin (K(m) = 12 microM), pravastatin, taurocholate (K(m) = 40 microM), digoxin, ochratoxin A, and [d-penicillamine(2,5)]-enkephalin. Double immunohistochemical staining of Oatp1a4 with P-glycoprotein (P-gp) or glial fibrillary acidic protein demonstrated that Oatp1a4 signals colocalized with P-gp signals partly but not with glial fibrillary acidic protein, suggesting that Oatp1a4 is expressed in both the luminal and the abluminal membranes of mouse brain capillary endothelial cells. The brain-to-blood transport of pitavastatin, rosuvastatin, pravastatin, and taurocholate after microinjection into the cerebral cortex was significantly decreased in Oatp1a4(-/-) mice compared with that in wild-type mice. The blood-to-brain transport of pitavastatin, rosuvastatin, taurocholate, and ochratoxin A, determined by in situ brain perfusion, was significantly lower in Oatp1a4(-/-) mice than in wild-type mice, whereas transport of pravastatin and [D-penicillamine(2,5)]-enkephalin was unchanged. The blood-to-brain transport of digoxin was significantly lower in Oatp1a4(-/-) mice than in wild-type mice only when P-gp was inhibited by N-(4-[2-(1,2,3,4-tetrahydro-6,7-dimethoxy-2-isoquinolinyl)ethyl]-phenyl)-9,10-dihydro-5-methoxy-9-oxo-4-acridine carboxamide (GF120918). Taken together, these results show that Oatp1a4 can mediate the brain-to-blood and blood-to-brain transport of its substrate drugs across the blood-brain barrier. The brain-to-plasma ratio of taurocholate, pitavastatin, and rosuvastatin was close to the capillary volume in wild-type mice, and it was not affected by Oatp1a4 dysfunction. Whether Oatp1a4 can deliver drugs from the blood to the brain remains controversial.

Evaluation and validation of two chromatographic methods (HPLC-fluorescence and LC-MS/MS) for the determination and confirmation of ochratoxin A in pig tissues.

Two different analytical methods for the determination and confirmation of ochratoxin A (OTA) in blood serum, kidney, and liver of pigs have been compared. Sample cleanup was based on liquid-liquid phase extraction. The detection of OTA was accomplished with high-performance liquid chromatography (HPLC) combined either with fluorescence detection (FLD) or electrospray ionization (ESI+) tandem mass spectrometry (MS/MS). The comparative method of evaluation was based on the investigation of 90 samples of blood serum, kidney, and liver per animal originating from different regions of Serbia. The analytical results are discussed in view of the respective method validation data and the corresponding experimental protocols. In general, analytical data obtained with liquid chromatography (LC)-MS/MS detection offered comparable good results in the sub-ppb concentration level indicating that the electrospray tandem mass spectrometric (LC-MS/MS) method was more selective and sensitive for the analysis and confirmation of OTA in pig tissues than the HPLC method after the methylation of OTA.

Review. Ochratoxin A: presence in human plasma and intake estimation.

Ochratoxin A (OTA) is a fungal toxic secondary metabolite that can be found in several foodstuffs and thereby ingested by humans. One way to assess exposure of humans to OTA is the determination of the levels of this mycotoxin in blood plasma from a certain population. Such studies have been done in many countries, both in healthy people and nephropathy patients. Relationships with individual characteristics were investigated in several cases. Thus, most studies found no correlation with age, either with gender. However, the few studies that found correlation between OTA plasma levels and gender showed that men presented the highest values. When sampling was done over more than one season, the highest OTA plasma levels were found mostly in summer. Differences within regions of a country were related to dietary habits of each area. OTA levels of group populations showed variations from year to year, whereas intraindividual repetitions showed no specific trend. Daily intake of the toxin can be estimated from OTA plasma concentrations by the Klaassen equation. OTA toxicokinetics are considered in this review. Calculated daily intake of OTA by different studies did not overpass the proposed tolerable daily intakes of OTA.

Occurrence and bioacessibility of mycotoxins in fish feed.

In this study Aflatoxin B1 (AFB1), ochratoxin A (OTA) and zearalenone (ZEN) occurrence in fish feed, regarding its chemical composition, were investigated. Besides, AFB1 bioaccessibility to fish was evaluated by in vitro digestion. Mycotoxins were extracted by QuEChERS and quantified by HPLC-FLD. Results showed that 93.3% of the samples were contaminated at maximum levels of 16.5, 31.6, and 322 µg/kg in the cases of AFB1, OTA, and ZEN, respectively. A positive correlation between OTA, ZEN contamination, and lipid content was observed. Risk estimation of feed consumption by fish at the highest levels of AFB1, OTA, and ZEN shows that the younger the fish, the higher the risk of exposure to mycotoxins. The AFB1 bioaccessibility assay showed that 85% of this mycotoxin may be absorbed by fish. Therefore, establishing maximum levels in the fishing sector is fundamental to contribute to feed quality and nutritional safety of fish species.

Modulation of key regulators of mitosis linked to chromosomal instability is an early event in ochratoxin A carcinogenicity.

Ochratoxin A (OTA) is a potent renal carcinogen, but little is known regarding the mechanism of OTA carcinogenicity. Early histopathological alterations induced by OTA in rat kidney include single cell death, stimulation of cell proliferation and prominent karyomegaly indicative of blocked nuclear division during mitosis. Based on these observations, it has been suggested that disruption of mitosis by OTA may be the principal cause of cell death and subsequent trigger for cell proliferation to compensate for cell loss. To gain further insight into the molecular mechanism of OTA toxicity, we used targeted quantitative real-time polymerase chain reaction arrays to investigate the expression of genes involved in cell cycle control and mitosis in kidneys of male F344 rats treated with 0, 21, 70 and 210 microg/kg body wt OTA for up to 90 days. Treatment with OTA resulted in overexpression of key regulators of mitosis, including the mitotic protein kinases Polo-like kinase 1, Aurora B and cyclin-dependent kinase 1 (Cdk1Cdc2), several cyclins and cyclin-dependent kinase inhibitors, topoisomerase II and survivin. Immunohistochemical analysis confirmed upregulation of Cdk1, p21(WAF1/CIP1), topoisomerase II and survivin in S3 proximal tubule cells, from which OTA-induced tumors in rats arise, and demonstrated increased phosphorylation of histone H3, a target of Aurora B. Importantly, many of the genes found to be deregulated in response to OTA have been linked to chromosomal instability and malignant transformation, supporting the hypothesis that aberrant mitosis, resulting in blocked or asymmetric cell division, accompanied by an increased risk of aneuploidy acquisition, may play a critical role in OTA carcinogenicity.

Low doses of ochratoxin A upregulate the protein expression of organic anion transporters Oat1, Oat2, Oat3 and Oat5 in rat kidney cortex.

Mycotoxin ochratoxin A (OTA) is nephrotoxic in various animal species. In rodents, OTA intoxication impairs various proximal tubule (PT) functions, including secretion of p-aminohippurate (PAH), possibly via affecting the renal organic anion (OA) transporters (Oat). However, an effect of OTA on the activity/expression of specific Oats in the mammalian kidney has not been reported. In this work, male rats were gavaged various doses of OTA every 2nd day for 10 days, and in their kidneys we studied: tubule integrity by microscopy, abundance of basolateral (rOat1, rOat3) and brush-border (rOat2, rOat5) rOat proteins by immunochemical methods, and expression of rOats mRNA by RT-PCR. The OTA treatment caused: a) dose-dependent damage of the cells in S3 segments of medullary rays, b) dual effect upon rOats in PT: low doses (50-250 microg OTA/kg b.m.) upregulated the abundance of all rOats, while a high dose (500 microg OTA/kg b.m.) downregulated the abundance of rOat1, and c) unchanged mRNA expression for all rOats at low OTA doses, and its downregulation at high OTA dose. Changes in the expression of renal Oats were associated with enhanced OTA accumulation in tissue and excretion in urine, whereas the indicators of oxidative stress either remained unchanged (malondialdehyde, glutathione, 8-hydroxydeoxyguanosine) or became deranged (microtubules). While OTA accumulation and downregulation of rOats in the kidney are consistent with the previously reported impaired renal PAH secretion in rodents intoxicated with high OTA doses, the post-transcriptional upregulation of Oats at low OTA doses may contribute to OTA accumulation and development of nephrotoxicity.

Effects of probiotic bacteria on the bioaccessibility of aflatoxin B(1) and ochratoxin A using an in vitro digestion model under fed conditions.

In the present study, we aimed at determining the release of aflatoxin B(1) (AFB(1)) and ochratoxin A (OTA) from different food products in the gastro-intestinal tract in the absence and presence of probiotics, a possible adsorbent. The average bioaccessibility of AFB(1) and OTA without probiotics was about 90%, and 30%, respectively, depending on several factors, such as food product, contamination level, compound and type of contamination (spiked versus naturally contaminated). The six probiotic bacteria showed varying binding capacity to AFB(1) and OTA depending on the bacterial strain, toxin studied, type of food and contamination level. A reduction to a maximum of 37% and 73% as observed for the bioaccessibility of AFB(1) and OTA in the presence of probiotic bacteria, respectively. This is the first report on the effect of probiotic bacteria on reducing the fraction of mycotoxins available for absorption in the gastrointestinal tract from different food products.

Determination of Ochratoxin-A in the brain microdialysates and plasma of awake, freely moving rats using ultra high performance liquid chromatography fluorescence detection method.

In this study, a simple, efficient and rapid Ultra High Performance Liquid Chromatography method with fluorescence detection (UHPLC-FLD) has been developed and validated for the determination of Ochratoxin-A (OTA) in rat brain microdialysates and plasma samples. Six adult male wistar rats were used in the study and a single dose (5 mg/kg b.w.) of OTA was given by intraperitoneal (i.p.) injection. Rat blood and microdialysate samples were collected simultaneously after i.p. injection in awake freely moving rats, over a twelve-hour period. An UHPLC analysis was performed on a Zorbax Eclipse Plus C8 (150 mm × 3.0 mm ID × 1.8 µm particles) column with a mobile phase of acetonitrile:water:phosphoric acid (50:50:0.1, v/v) using a flow rate of 0.6 mL/min. The fluorescence detector was set at 330 nm excitation and 460 nm emission wavelengths. Diflunisal (DIF) was used as an internal standard (IS). OTA and IS were separated within 5 min under these conditions. The method was validated in terms of linearity, precision, accuracy, limit of detection, limit of quantification, and stability. Calibration curves obtained with spiked biological matrices show good linearity with high correlation coefficients. The intra- and inter-day assay variability was <5% for the OTA. The limit of detection and the limit of quantification values were found to be 0.490 ng/mL and 1.48 ng/mL for plasma; 0.0900 ng/mL and 0.270 ng/mL for microdialysate samples, respectively. This method was successfully applied for the monitoring of OTA levels in the rat brain and plasma samples.

Human Study on the Kinetics of 2'R-Ochratoxin A in the Blood of Coffee Drinkers.

SCOPE: The aim of this study is to obtain a deeper knowledge of the kinetics of 2'R-ochratoxin A (2'R-OTA), the thermal degradation product of the mycotoxin ochratoxin A (OTA). To investigate the correlation between the amount of this compound in roasted coffee and human blood samples, a human study is performed. METHODS AND RESULTS: An 18-week human study is carried out. During the first eight weeks, all known 2'R-OTA-containing food sources are excluded from the diet and the reduction of 2'R-OTA in venous blood is analyzed. Afterwards, participants are allowed to consume coffee with known OTA and 2'R-OTA concentrations. On a biweekly scale, 2'R-OTA and OTA blood levels are determined. After eight weeks of fasting on 2'R-OTA-containing foods, the 2'R-OTA blood concentration decreased by about 10%. Based on this, a long biological half-life of over seven months is estimated. In the 24 h urine samples collected before and after the coffee fasting period, only traces of 2'R-OTA are detected. CONCLUSION: Results show that 2'R-OTA has a more than seven-fold higher biological half-life in human blood compared to OTA (approx. 35 days). The reason for the long persistence of 2'R-OTA in human blood is still unclear and further research is needed.

Distribution of ochratoxin A in plasma and tissues of rats fed a naturally contaminated diet amended with micronized wheat fibres: effectiveness of mycotoxin sequestering activity.

The effectiveness of micronized wheat fibres (MWF) alone or in association with yeast cell walls (YCW) as active adsorbents to decrease, in vivo, the levels of ochratoxin A (OTA) was checked in a total of 48 rats, equitably distributed into four groups: (1) control; (2) OTA naturally contaminated diet (2.2 microg/g); (3) OTA naturally contaminated diet (2.2 microg/g) amended with MWF (2%); (4) OTA naturally contaminated diet (2 microg/g) amended with MWF (1.8%) in association with YCW (0.2%). A 4 week experimental period corresponding to a daily intake in the range of 132.2-146.1 microg OTA/kg bw decreased the rat body weight gains, as compared to the controls. The adsorbents did not significantly alleviate the growth depression caused by the contaminated diet. However, a significant protective effect of MWF was observed in terms of OTA concentration in plasma (40.5% decrease), kidney (28.1% decrease) and liver (38.8% decrease). Mixing this sorbent with the YCW did not significantly improve its protective activity against OTA. The faecal OTA concentrations were higher for the MWF and MWF+YCW treated animals, as compared to the positive control (group II). Taken together, these results suggest that MWF are a promising tool to counteract the toxic effects of OTA naturally contaminated diets.

Interpretation of the pharmacokinetics of ochratoxin A in blood plasma of rats, during and after acute or chronic ingestion.

Experiments designed to reveal aspects of delivery of circulating ochratoxin A (OTA) to kidneys showed maximum plasma concentration within 3h of acute gavage, but this persisted for 4 days before decline. During long-term daily administration in feed, plasma values stabilised, proportional to dose and, for Fischer males, immediately followed an 8-10 day half-life upon ceasing OTA intake. In mature adult males, plasma OTA accumulated during the month after commencing daily intake of 100 microg. By comparison, in Dark Agouti males, lower steady state plasma values occurred and there was much shorter plasma OTA half-life (2-3 days). In F1 hybrid rats (Sprague Dawley x Fischer) males ingesting 100 microg OTA daily the steady state plasma value stabilised as in Fischer males, but females on the same diet accumulated OTA in plasma to nearly twice the value for males after only 5 months' exposure. Although OTA causes renal tumours in Fischer and Dark Agouti male rats in spite of marked difference in OTA elimination rates presently shown, understanding comparison of re-uptake along nephrons in rat and man in vivo is vital in applying rat toxicological data to assessment of risk of dietary OTA to humans.

Efficacy of a new ochratoxin-binding agent (OcraTox) to counteract the deleterious effects of ochratoxin A in laying hens.

An experiment was conducted to evaluate the efficacy of a new ochratoxin-binding agent (Ocra-Tox, 5 g/kg of feed) in offsetting the toxic effects of ochratoxin A (OTA, 2 mg/kg of feed) in laying hen diets. Performance, serum biochemistry, OTA residue in the liver and eggs, and egg quality parameters were evaluated. Twenty-eight Hisex Brown laying hens, 47 wk of age, were allocated to 1 of 4 experimental treatments for 3 wk: control, OTA (containing 2 mg of OTA/kg of feed), OcraTox (containing 5 g of OcraTox/kg of feed), and OTA + OcraTox (containing 2 mg of OTA and 5 g of OcraTox/kg of feed). Laying hens fed OcraTox showed results similar to the control hens (P > 0.05). The OTA diet significantly (P < 0.05) reduced daily feed consumption, egg mass production, and serum triglyceride concentrations, and increased the relative liver weight, the serum activity of alkaline phosphatase, and the serum concentration of uric acid as compared with the control diet. Addition of OcraTox to the contaminated diet alleviated (P < 0.05) the negative effects resulting from OTA, reaching values not significantly different from the control diet for most of the parameters except the relative weight of the liver. Birds fed the OTA treatment showed a greater content of OTA in the liver (15.1 microg/kg) than those fed the control diet (<0.05 microg/kg). Supplementing the contaminated diet with OcraTox (OTA + OcraTox) reduced the values to 12.0 microg/kg. Residues of OTA were not detected above our detection limit (0.05 microg/kg) in any of the analyzed eggs. In conclusion, our results indicated that addition of OcraTox can counteract the deleterious effects caused by OTA in laying hens.

Ochratoxin A: 13-week oral toxicity and cell proliferation in male F344/n rats.

Ochratoxin A (OTA) is nephrotoxic and a potent renal carcinogen. Male rats are most susceptible to OTA toxicity, and chronic administration of OTA (70 and 210 microg/kg bw) for 2 years has been shown to induce high incidences of adenomas and carcinomas arising from the straight segment of the proximal tubule epithelium. In contrast, treatment with a lower dose of 21 microg/kg bw did not result in increased tumor rates, suggesting a nonlinear dose response for renal tumor formation by OTA. Since the mechanism of OTA carcinogenicity is still largely unknown, this study was conducted to investigate early functional and pathological effects of OTA and to determine if sustained stimulation of renal cell proliferation plays a role. Male F344/N rats were treated with OTA for up to 13 weeks under conditions of the National Toxicology Program (NTP) bioassay. Cell proliferation in the renal cortex and outer stripe of the outer medulla (OSOM) was determined using bromodeoxyuridine incorporation and immunohistochemistry. Histopathological examination showed renal alterations in mid- and high-dose-treated animals involving single-cell death and prominent nuclear enlargement within the straight proximal tubules. Treatment with OTA at doses of 70 and 210 microg/kg bw led to a marked dose- and time-dependent increase in renal cell proliferation, extending from the medullary rays into the OSOM. No effects were evident in kidneys of low-dose-treated animals or in the liver, which is not a target for OTA carcinogenicity. A no observed effect level in this study was established at 21 microg/kg bw, correlating with the dose in the NTP 2-year bioassay that did not produce renal tumors. The apparent correlation between enhanced cell turnover and tumor formation induced by OTA indicates that stimulation of cell proliferation may play an important role in OTA carcinogenicity and provides further evidence for an epigenetic, thresholded mechanism.