Assessment and characterisation of yeast-based products intended to mitigate ochratoxin exposure using in vitro and in vivo models.

The aim of this paper was to evaluate the capacity of several yeast-based products, derived from baker's and brewer's yeasts, to sequester the mycotoxin ochratoxin A (OTA) and to decrease its rate of absorption and DNA adduct formation in vivo. The experimental protocol included in vitro binding studies using isotherm models, in vivo chicken experiments, in which the serum and tissue concentrations of OTA were analysed in the absence and presence of the test compounds, and the profile of OTA-derived metabolites and their associated DNA adducts were determined. Additionally in vitro cell culture studies (HK2 cells) were applied to assess further the effects for yeast cell product enriched with glutathione (GSH) or selenium. Results of the in vitro binding assay in a buffer system indicated the ability of the yeast-based products, as sequester of OTA, albeit at a different level. In the in vitro experiments in chickens, decreased serum and tissue concentrations of treated animals confirmed that yeast-based products are able to prevent the absorption of OTA. A comparison of the binding affinity in a standard in vitro binding assay with the results obtained in an in vivo chicken experiment, however, showed a poor correlation and resulted in a different ranking of the products. More importantly, we could show that yeast-based products actively modulate the biotransformation of OTA in vivo as well as in vitro in a cell culture model. This effect seems to be attributable to residual enzymatic activities in the yeast-based products. An enrichment of yeast cell wall products with GSH or selenium further modulated the profile of the generated OTA metabolites and the associated pattern of OTA-induced DNA adducts by increasing the conversion of OTA into less toxic metabolites such as OTA, OTB and 4-OH-OTA. A reduced absorption and DNA adduct formation was particularly observed with GSH-enriched yeast, whereas selenium-enriched yeasts could counteract the OTA-induced decrease in cell viability, but at the same time increased the OTA-DNA adducts formation. These findings indicate the need for an in-depth characterisation of yeast-based products used as mycotoxin-mitigating feed additives, in in vivo models with target animal species taking into account not only their ability to sequester toxins in the gastrointestinal tract but also their potential effects on the biotransformation of mycotoxins.

In vitro tests aiding ecological risk assessment of ciprofloxacin, tamoxifen and cyclophosphamide in range of concentrations released in hospital wastewater and surface water.

Ciprofloxacin (CIP), tamoxifen (TAM) and cyclophosphamide (CP) which are often used in anticancer treatment are released in hospital effluent and into the environment. Although the concentrations are low (from ng/L to µg/L), no data exist concerning their ecotoxicological impact. In this study two biomarkers of early effect were performed on hepatic cells (HepG2): cell viability and genotoxicity (DNA breaks) using cell proliferative assay and comet assay, respectively. These data were compared with two standardized ecotoxicological tests: algaltoxkit F™ and microtox®. Cells were exposed to an increasing amount of an individual drug or in a mixture for 24, 48 or 72h. The time-exposure of bacteria and algae ranged between 5 and 30min and 72h, respectively. A non-monotonic dose-response on cell viability was observed when HepG2 cells were exposed to TAM alone or in the presence of CIP. The same scheme was observed with microtox® when the bacteria were exposed to the mixtures. On the other side, an individual drug does not induce any DNA breaks on hepatic cells, whereas a mixture leads to a dose dependent increase of DNA breaks. Similarly a positive response was observed with algaltoxkit F™ only with mixtures. Synergistic effects observed when drugs are in a mixture highlight the importance of investigating the ecotoxicological effects of contaminants at low concentrations and in mixtures.

Occurrence of ochratoxin A and citrinin in Czech cereals and comparison of two HPLC methods for ochratoxin A detection.

The aims of the study were to obtain information about the occurrence of ochratoxin A (OTA) and citrinin (CIT) in cereals harvested in the Czech Republic and to compare two analytical procedures for detecting OTA. A total of 34 cereal samples, including two matrix reference materials (R-Biopharm, Germany), were analysed. The results were compared with the limit for raw cereal grains used as a foodstuff according to Commission Regulation No. 1881/2006, which allows a maximum OTA level of 5 µg kg(-1). Compared were two methods based on the high-performance liquid chromatography principle, one using the immunoaffinity columns OchraTest (VICAM) and the second based on solvent partition (PART), both followed by fluorescence detection. The highest OTA contents were found in two barley samples. According to the method employed, the results for the first sample (malting barley) were VICAM = 31.43 µg kg(-1) and PART = 44.74 µg kg(-1). For the second sample (feeding barley) they were VICAM = 48.63 µg kg(-1) and PART = 34.40 µg kg(-1). Two samples of bread wheat had an OTA content approaching the legal limit (VICAM = 4.71 µg kg(-1) and PART = 6.03 µg kg(-1); VICAM = 4.12 µg kg(-1) and PART = 3.95 µg kg(-1)). CIT was analysed using the PART method only, and its highest content (93.64 µg kg(-1)) was found for the malting barley sample with high OTA content (44.74 µg kg(-1) as analysed using PART).

Cyclophosphamide removal from water by nanofiltration and reverse osmosis membrane.

The rejection of cyclophosphamide (CP) by nanofiltration (NF) and reverse osmosis (RO) membranes from ultrapure (Milli-Q) water and membrane bioreactor (MBR) effluent was investigated. Lyophilization-extraction and detection methods were first developed for CP analysis in different water matrices. Experimental results showed that the RO membrane provided excellent rejection (>90%) under all operating conditions. Conversely, efficiency of CP rejection by NF membrane was poor: in the range of 20-40% from Milli-Q water and around 60% from MBR effluent. Trans-membrane pressure, initial CP concentration and ionic strength of the feed solution had almost no effect on CP retention by NF. On the other hand, the water matrix proved to have a great influence: CP rejection rate by NF was clearly enhanced when MBR effluent was used as the background solution. Membrane fouling and interactions between the CP and water matrix appeared to contribute to the higher rejection of CP.

Further arguments in favour of direct covalent binding of Ochratoxin A (OTA) after metabolic biotransformation.

Ochratoxin A (OTA) is nephrotoxic to all animal species, carcinogenic for rats and mice and probably implicated in human Balkan endemic nephropathy and the associated urothelial tract tumour. Controversial results concerning genotoxicity and biotransformation of OTA have been generated. By (32)P post-labelling technique, a dose- and time-dependent DNA adduct formation is observed in vivo and in vitro. Use of several inducers or inhibitors of biotransforming enzymes (including cytochrome P 450, cyclooxygenase, lipoxygenase, glutathione-S-transferase), demonstrated that OTA is biotransformed into genotoxic derivatives damaging for DNA. Authentic C8dG-OTA standards have been synthesized by photo-oxidation. Both of them (C-C8 & O-C8) co-migrate on TLC with two adducts formed by in vitro incubation of OTA in the presence of kidney microsomes, and in vivo in kidney of pig or rodent fed OTA as well as in kidney and bladder tumour of humans exposed to OTA. Several OTA metabolites have been isolated from tissues or cells treated by OTA. The open ring lactone (OP-OTA) and quinone OTA (OTQ) are genotoxic.

Safety of Oxygreen, an ozone treatment on wheat grains. Part 1. A four-week toxicity study in rats by dietary administration of treated wheat.

The Oxygreen process is a new treatment approved by The French Food Safety Authority (AFSSA) as a processing aid for flour quality improvement, based on treatment by ozone, in a closed sequential batch reactor. This treatment takes place in the classical milling sequence, after the grain-cleaning step and before milling. The Oxygreen process could also be used for its properties in wheat grain decontamination (insects, fungi, bacteria, mycotoxins, storage insecticides residues). The aim of this study was to determine if Oxygreen treatment could induce in the grain the formation of processing-related substances, able to provoke adverse effects, after ingestion of the wheat and/or derived products, and to establish the safety of the Oxygreen process for animals and consumers. A four-week toxicity study, according to OECD guideline No. 407, was performed on Dark agouti rats fed exclusively with wheat grains, treated or untreated with Oxygreen. Clinical, haematological, blood biochemical, urinary and histopathological parameters were investigated during the study. The few modifications observed in animals given treated wheat were an increase of rectal temperature in females, a slight decrease of calcium concentration in males and slight decrease of certain blood cell number without clinical significances. This work shows that wheat treated by Oxygreen does not induce adverse effects in Dark agouti rats after oral administration. Therefore wheat and derived products from wheat, after Oxygreen treatment on grain, could be considered as safe for the consumer.

Measurement of DNA adducts and strand breaks in dab (Limanda limanda) collected in the field: effects of biotic (age, sex) and abiotic (sampling site and period) factors on the extent of DNA damage.

In the Eastern English Channel, the potential application of the comet assay and post-labelling technique in dab was evaluated for genotoxicity monitoring of the marine environment. The effects of biotic (age, sex) and abiotic (sampling site and period) factors on the extent of DNA lesions were also studied. Female and male dab of two class of size (juvenile and adult) were collected by trawling in different sites in Seine Bay and Somme Bay during September 2001. Single-strand breaks and adducts were, respectively, measured in erythrocytes and the liver. Results obtained for the adult female were compared with those collected during a first cruise in March 2001 [Akcha et al., Mutat Res. 534 (1-2) (2003) 21]. Significant effects of sex and age were demonstrated on the level of strand breaks. Moreover, a significant interaction between age and sex was shown that might indicate the complex influence of other factors on the extent of DNA damage (i.e. reproduction status). In the adult dab, the level of breaks is higher in the male than in the female, whereas the opposite trend was observed for the juvenile. Whatever the sex, the number of DNA breaks is higher in the adult than in the juvenile. For the female dab, significant differences were observed with the comet assay between the Seine Bay and the Somme Bay in March but not in September. This may be due to seasonal variations in the formation of DNA lesions related to variations in lipid content and levels of biotransformation activities and/or to spawning cycles. The presence of genotoxic substances in the study areas was also confirmed by the detection of DNA adducts in each sample analysed. Whereas no effect was shown on the total level of adducts for the tested biotic and abiotic factors, qualitative differences in adduct profiles were observed for each of these factors. For the female dab, comparison of adduct profiles obtained in March and September with one generated by hepatic microsomal activation in dab of a PAH mixture indicated a PAH contamination of the study areas in autumn. These results show the importance of studying the effects of biotic and abiotic factors on the genotoxic endpoints considered to correctly assess the contribution of chemical contamination to the measured biological responses.

Evaluation of toxic and genotoxic potential of stabilized industrial waste and contaminated soils.

Artificial aqueous samples (eluates, percolates, immersion waters) were obtained from contaminated soils and stabilized industrial wastes. The toxicity and genotoxicity of these aqueous fractions have been evaluated in vivo in the aquatic larvae of the amphibian Xenopus laevis. Four biotests have been applied: a test of subchronic toxicity and three biomakers: (1) measurement of the activity of ethoxyresorufine-o-dealkylase in the liver, (2) detection of DNA adducts in the liver and the blood, and (3) measurement of the rate of micronuclei in the erythrocytes. Biological datas were completed through a chemical analysis. The main conclusions of this study are: The importance of integrating different toxicity criterias into a biological battery (phenotypic and genotypic criterias). Some aqueous extracts did not seem to be very toxic, whereas their genotoxic effects were rather significant [e.g. the stabilized Municipal Solid Waste (MSW) ashes]. The importance of coupling together chemical and biological approaches to refine the impact. Actually, some eluates (lixiviation or percolation) coming from polluted soils appeared to be very poorly loaded with pollutants, whereas the toxic and genotoxic impact of these complex matrices were rather noticeable. In addition, when applying the leaching standardized procedure, the hazardous potential of the two analysed soils may be underestimated if the results on percolates and on eluates have been compared. This study highligths the importance of coupling the tools of characterization and preparation of samples to be analysed according to the objectives to be reached.

Balkan endemic nephropathy and associated urinary tract tumours: a review on aetiological causes and the potential role of mycotoxins.

A series of publications in the 1950s described a kidney disease in Bulgaria, the former Yugoslavia and Romania that became known as Balkan endemic nephropathy (BEN). The disease was qualified by World Health Organisation (WHO) experts as 'progressive and very gradually developing renal failure with insidious onset.... The last stage shows marked fibrosis...'. BEN is characterized by tubular degeneration, interstitial fibrosis and hyalinization of glomeruli accompanied by enzymuria and impaired renal function without nephrotic syndrome. Later, an association between BEN and tumours of the kidney pelvis and ureter was recognized, so that the problem of BEN became not only nephrological, but also oncological. There may also be an association with increased urinary bladder cancer incidence, although many confounding factors may interfere in the analysis of data for this organ. In view of the very intimate association between BEN and the urinary tract tumours (UTT), the term 'endemic uropathy' has been proposed. Several hypothesis concerning the aetiology of these diseases has been investigated, which include: predisposing genes factors, environmental factors (heavy metals, minerals, bacteria, leptospira, viruses, fungal toxins and, most recently, pliocene lignites). This paper reviews the different hypotheses about the aetiology of endemic uropathy and pays particular attention to the role of fungal toxins.

Bitumen fumes: review of work on the potential risk to workers and the present knowledge on its origin.

Bitumens fumes contain polycyclic aromatic compounds (PAC). There is a possibility of long-term health effects following chronic exposure by inhalation or skin contamination in asphalt road pavers and highway maintenance workers. Epidemiological and experimental studies on this topic are reviewed and the possible causes of cancer discussed with a primary focus on heterocyclic polyaromatic compounds. In 2001, the results of the IARC epidemiological study confirmed an excess of lung cancer despite a lower cancer mortality. In vitro genotoxicity and mechanistic studies demonstrated a mutagenic effect of bitumen fume condensates (BFC) and some data suggested that the polycyclic aromatic hydrocarbons (PAH) analysed were not the major genotoxic compounds in bitumen fume condensates. Other compounds such as nitrogen-, sulfur- and/or oxygen-containing PAH or their alkyl substituted analogues, mutagenic in the Ames mutation assay, may be involved in the genotoxic effect of BFC. After skin painting with BFC, DNA adducts were found in skin, lung and lymphocytes of all the treated animals. Differences in the adduct patterns were also observed, but a more polar adduct was common to the three tissues and not observed in those from rats treated with coal-tar fume condensates (CTFC). Rat inhalation experiments with bitumen fumes confirmed the presence of a DNA-adduct in the lungs with the same Rf as the previous polar adduct. This adduct therefore merits further investigation as a potential biomarker in lymphocyte DNA to follow exposed workers. All the analytical data and the mechanistic data are complementary and suggest the potential role of thiophenes in the genotoxicity of bitumen fumes. Some thiophenes have lower mutagenic activity than their isosteric PAH, whereas others are very potent carcinogens. Generally, the sulfur analogues of PAH (SPAH) in bitumen fumes have a higher concentration than the PAH of similar molecular weight, whereas the SPAH in coal-tar fumes have a much lower concentration than the corresponding PAH. This may explain why the more polar adducts have been detected only in animals exposed to bitumen fume. In a skin carcinogenicity study of condensed asphalt roofing fumes, it has been demonstrated that the most active fractions were those containing a variety of aromatic SPAH. In conclusion to this review, there is an interest in determining the chemical identity of the major DNA adducts induced by BFC. This would allow experimental studies on the carcinogenic potency of these compounds and their validation as potential biomarkers. These compounds could thus merit further analytical investigation in preference to the PAH included in the list of the US Environmental Protection Agency that are currently being analysed by the industry in field studies.

The mycotoxin ochratoxin A alters intestinal barrier and absorption functions but has no effect on chloride secretion.

Ochratoxin A (OTA) is a mycotoxin that contaminates cereals and animal feed and causes nephropathy to a variety of animal species. OTA is also known as a potent immunotoxic, teratogenic, and carcinogenic mycotoxin. In addition, OTA ingestion induces intestinal injuries, including inflammation and diarrhea. With the aim to study the cellular mechanisms associated with the intestinal toxicity of OTA, two human epithelial intestinal cell lines (HT-29-D4 and Caco-2-14 cells), widely used as in vitro models for the intestinal epithelium, were incubated with OTA. The main effects of the mycotoxin were an inhibition of cellular growth and a dramatic decrease of transepithelial resistance in both cell lines. Since transepithelial resistance reflects the organization of tight junctions over the cell monolayer, these data may suggest that OTA could potentiate its own absorption through paracellular pathways. OTA induced a 60% decrease of sodium-dependent glucose absorption but increased the absorption of fructose and L-serine in HT-29-D4 cells. Moreover, the mycotoxin did not inhibit the cAMP-dependent chloride secretion through the cystic fibrosis transmembrane conductance regulator channel. The inhibitory effect of OTA on active glucose transport was partially antagonized by L-phenylalanine, but not by alpha-tocopherol, suggesting that the toxicity of OTA could result from an inhibition of protein synthesis, rather than an induction of lipid peroxidation. In particular, OTA affected the protein content of plasma membrane microdomains, which are known to regulate tight junction assembly and intestinal transport activity. Taken together, these data showed that OTA alters both barrier and absorption functions of the intestinal epithelium.

Analyses of DNA adducts formed by ochratoxin A and aristolochic acid in patients with Chinese herbs nephropathy.

Chinese herbs nephropathy (CHN), a unique type of nephropathy has been associated with the intake of weight-reducing pills containing the Chinese herb Aristolochia fangchi. Moreover, an association between the use of A. fangchi and urothelial cancer in CHN patients has been reported indicating that aristolochic acid (AA) the major alkaloid of A. fangchi might be the causal agent. Similarities of CHN to the Balkan endemic nephropathy (BEN) have led to the hypothesis of a common etiological agent for both diseases. Evidence has accumulated that BEN is an environmentally-induced disease strongly associated with the fungal mycotoxin ochratoxin A (OTA). Both, AA and OTA are nephrotoxic and carcinogenic and induce the formation of DNA adducts. As OTA has been suspected as fungal contaminant in the herbal batches used for the preparation of the weight-reducing pills we analysed tissues from CHN patients by the 32P-postlabeling procedure for the presence of DNA adducts related to both OTA and AA exposure. Whereas, AA-specific DNA adducts were detected in all five urinary tract tissues from five patients (total RAL: 32-251 adducts per 10(9) nucleotides), OTA-related DNA adducts were detectable in two kidneys and one ureter only (total RAL: 1.5-3.7 adducts per 10(9) nucleotides). Thus, OTA-related DNA adduct levels were about 50 times lower than AA-DNA adduct levels. In female and male rats that were treated with the slimming regimen in the same way like the CHN patients except that the amount of Chinese herbs was 10 times higher, AA-DNA adducts were found in kidney tissues (total RAL ranging from 51 to 83 adducts per 10(9) nucleotides) but adducts derived from OTA were not observed. These results demonstrate that OTA-related DNA adducts do not play a key role in CHN or CHN-associated urothelial cancer.

Phenobarbital increases DNA adduct and metabolites formed by ochratoxin A: role of CYP 2C9 and microsomal glutathione-S-transferase.

Ochratoxin A (OTA), a mycotoxin that induces nephrotoxicity and urinary tract tumors, is genotoxic and can be metabolized not only by different cytochromes P450 (CYP) but also by peroxidases involved in the arachidonic cascade, although the exact nature of the metabolites involved in the genotoxic process is still unknown. In order to establish the relation between OTA genotoxicity and the formation of metabolites, we chose three experimental models: kidney microsomes from rabbit, human bronchial epithelial cells, and microsomes from yeast that specifically express the human cytochrome P450 2C9 or 2B6 genes. OTA-DNA adducts were analyzed by (32)P postlabeling and the OTA derivatives formed were isolated by HPLC after incubation of OTA in the presence of: (1) kidney microsomes from rabbit pretreated or not with phenobarbital (PB); (2) human pulmonary epithelial cells simultaneously pretreated (or not) with PB alone or in the presence of ethacrynic acid (EA); (3) microsomes expressing CYP 2B6 and 2C9. PB pretreatment significantly increased DNA adducts formed after OTA treatment, both in the presence of kidney microsomes and bronchial epithelial cells, and induced the formation of new adducts. Ethacrynic acid, which inhibits microsomal glutathione-S-transferase, reduced DNA adduct level. DNA adducts were detected when OTA were incubated with microsomes expressing human CYP 2C9 but not with those expressing CYP 2B6. Several metabolites detected by HPLC were increased after PB treatment. Some of them could be related to DNA-adduct formation. In conclusion, OTA biotransformation, enhanced by PB pretreatment, increased DNA-adduct formation through pathways involving microsomal glutathion-S-transferase and CYP 2C9.

Activation of mitogen-activated protein kinase by fumonisin B(1) stimulates cPLA(2) phosphorylation, the arachidonic acid cascade and cAMP production.

Activation of mitogen-activated protein kinase (MAPK) results in pleiotropic effects such as modulation of the transcription and activation of enzymes involved in signal transduction. One such enzyme is the cytoplasmic phospholipase A(2) (cPLA(2)), which releases arachidonic acid (AA). AA is the precursor of prostaglandins and leukotrienes, two inflammatory mediators, which regulate gene expression and protein kinase (PK) activity. Fumonisin B(1) (FB(1)) was shown to increase PKC translocation and stimulate MAPK. We have investigated the effect of FB(1) on the AA cascade in a human epithelial cell line and the signal transduction pathway regulating PLA(2) activation. We observed that FB(1) stimulated cPLA(2) activity and increased AA release by a mechanism independent of PKC activation and that the activation of cPLA(2) is a two-step process: the first is phosphorylation of cPLA(2) by MAPK; the second is a consequence of the increase in sphingosine inside and outside the cells after 2 h, which is known to induce a rise in intracellular free calcium. Overall, this suggests that the effect of FB(1) on cells is partially dependent on the action of FB(1) on the enzymes involved in the cell cycle, such as MAPK and PKA, and on bioactive fatty acids, such as the prostaglandins and leukotrienes, and also on disruption of sphingolipid metabolism. In addition, we have observed down-regulation of cPLA(2) activity and AA metabolism by a mechanism involving prostaglandin production, cAMP synthesis and PKA activation.

Biological monitoring exposure of workers from plant producing carbon electrodes: quantification of benzo[a]pyrene DNA-adducts in leukocytes, by a 32P-postlabelling method and an immunoassay.

The levels of benzo[a]pyrene were monitored for blood DNA-benzo[a]pyrene adducts in 17 workers from a plant producing carbon electrodes, with high exposure to benzo[a]pyrene (575-902-1149 ng m(-3)). Two different techniques, a 32P-postlabelling method and a competitive immunoassay using polyclonal antibodies obtained from rabbits immunised with DNA modified by benzo[a]pyrene-trans-7,8-dihydrodiol-9,10-epoxide were used. For each worker, urinary 1-hydroxypyrene, a potential indicator of exposure to polycyclic aromatic hydrocarbons, was measured. The effect of tobacco by urinary cotinine measurement was also considered. The postlabelling and immunoassay detection limits for DNA-benzo[a]pyrene adducts were respectively 0.15 and 10 fmol 50 microg(-1) of DNA. The results obtained by the two methods demonstrated a good detection of DNA-benzo[a]pyrene adducts, but no direct relationship between the quantity of adducts and the concentration of benzo[a]pyrene in air-borne was noted in the studied plant. The levels of DNA-benzo[a]pyrene adducts obtained by immunoassay were significantly higher than those obtained by the 32P-postlabelling (P < 0.001). For several workers, variations due to professional or non professional factors must be taken into account in interpreting the results. In conclusion, the two methods used proved very efficient in determining DNA-benzo[a]pyrene adducts, and may be useful in monitoring human exposure to known and previously unidentified environmental genotoxic agents.

Roles of cyclooxygenase and lipoxygenases in ochratoxin A genotoxicity in human epithelial lung cells.

The roles of constitutive prostaglandin-H-synthetase (PGHS) and lipoxygenases in ochratoxin A (OTA) genotoxicity, as reflected by DNA adduct formation, have been investigated in vitro: (1) in culture of human epithelial cells and (2) by incubation in presence of pig seminal vesicle microsomes. Indomethacin (0.1 µM), which inhibits PGHS and significantly increases leukotriene C(4) production by enhancement of lipoxygenases, enhanced formation of OTA-DNA adducts tenfold. At highest dose of 10 µM, indomethacin inhibited all pathways (PGHS and lipoxygenases) and thus prevented OTA-DNA adduct formation. Nordihydroguaiaretic acid, which inhibits lipoxygenases, suppressed OTA-DNA adduct formation. The OTA metabolites formed were analysed by HPLC. OTα, 4[R]- and 4[S]-hydroxy-OTA and a unidentified derivative were formed in control cells. After pre-incubation with indomethacin (0.1 µM), further unidentified metabolites were obtained. They were similar to those obtained in presence of pig seminal vesicle microsomes. These data demonstrate that OTA is biotransformed into genotoxic metabolites via a lipoxygenase, whereas PGHS decreases OTA genotoxicity.

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.

Formation of DNA adducts in tissues of mouse progeny through transplacental contamination and/or lactation after administration of a single dose of ochratoxin A to the pregnant mother.

Ochratoxin A (OTA) is a mycotoxin which has been detected in foods of plant origin, in edible animal tissues, and in human sera, urine, and milk in many countries. OTA is nephrotoxic and carcinogenic in mice and rats and is suspected to play a key role in the etiology of Balkan endemic nephropathy and/or associated urinary tract tumors. In the present study, some early signs of genetic impairment, including the presence of DNA adducts in target tissues from the progeny of mice after administration of a single OTA dose during late pregnancy, have been investigated. By the 32P-postlabeling method, several characteristic DNA adducts with the same Rf values were detected in kidney and liver of both the OTA-treated mice and their progeny the fetus and the offspring. No adduct was found in tissues from control animals. Different adducts were most important in kidney and liver DNA and some were organ-specific. High levels of DNA adducts were detected in the kidneys of male progeny, whereas in the female progeny and the mothers they were detected almost exclusively in the liver. This result correlates well with the carcinogenicity in mice: the target organ for males is the kidney, while for females it is the liver. High levels of DNA adducts were also found in fetuses. These results provide evidence for a direct genotoxic action of OTA in the progeny through transplacental contamination, which constitutes a new serious health hazard of exposure to this toxin.

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.