Biomonitoring of Mycotoxins in Urine: Pilot Study in Mill Workers.

Contamination of grains with mycotoxins results in a dietary background exposure of the general population. In occupational settings such as during processing of raw materials as in milling, an additional mycotoxin exposure by inhalation is possible. Biomonitoring is an integrative approach to assess human exposure from various sources and by all routes. To investigate possible workplace exposure to mycotoxins, a pilot study was conducted that compared levels of urinary biomarkers in mill workers to those in a control group with dietary mycotoxin intake alone. Workers (n = 17) from three grain mills in North Rhine Westphalia, Germany, provided spot urines during shift; volunteers (n = 13, IfADo staff) with matched age structure served as control group. The mycotoxins selected for biomarker analysis were citrinin (CIT) deoxynivalenol (DON), ochratoxin A (OTA), and zearalenone (ZEN). Immunoaffinity columns (CIT, DON, ZEN) or liquid-liquid extraction (OTA) was employed for urine sample cleanup prior to targeted analysis by liquid chromatography with tandem mass spectrometry (LC-MS/MS) or by high-performance liquid chromatography (HPLC). In addition, mycotoxin metabolites that may be formed in the organism were analyzed, including deepoxy-deoxynivalenol (DOM-1), ochratoxin alpha (OTα), dihydrocitrinone (DH-CIT), and α- and ß-zearalenol (α- and ß-ZEL), as well as phase II metabolites that were hydrolyzed with ß-glucuronidase/arylsulfatase prior to sample cleanup. All analyte concentrations were adjusted for creatinine (crea) content in the spot urine samples. Citrinin, DON, OTA, and ZEN were detected in nearly all urine samples from mill workers and controls. Interestingly, DH-CIT was found at higher mean levels than the parent compound (~0.14 and 0.045 µg/g crea, respectively), suggesting an effective metabolism of CIT in humans. Other metabolites DOM-1, OTα, and α- and ß-ZEL were detected less frequently in urine. Deoxynivalenol was detected at the highest concentrations (mean ~6 µg/g crea), followed by OTA (mean ~0.08 µg/g crea); ZEN (mean ~0.03 µg/g crea) and its metabolites appeared in urine at lower levels. Mycotoxin biomarker levels in urine from mill workers and controls were not significantly different. From these results it is concluded that biomarker levels measured in urine samples from the two cohorts reflect mainly dietary mycotoxin exposure. An additional occupational (inhalational) exposure of mill workers, if any, is apparently low at the investigated workplaces.

Protective effect of boric acid on lead- and cadmium-induced genotoxicity in V79 cells.

The toxic heavy metals cadmium (Cd) and lead (Pb) are important environmental pollutants which can cause serious damage to human health. As the metal ions (Cd(2+) and Pb(2+)) accumulate in the organism, there is special concern regarding chronic toxicity and damage to the genetic material. Metal-induced genotoxicity has been attributed to indirect mechanisms, such as induction of oxidative stress and interference with DNA repair. Boron is a naturally occurring element and considered to be an essential micronutrient, although the cellular activities of boron compounds remain largely unexplored. The present study has been conducted to evaluate potential protective effects of boric acid (BA) against genotoxicity induced by cadmium chloride (CdCl2) and lead chloride (PbCl2) in V79 cell cultures. Cytotoxicity assays (neutral red uptake and cell titer blue assay) served to determine suitable concentrations for subsequent genotoxicity assays. Chromosomal damage and DNA strand breaks were assessed by micronucleus tests and comet assays. Both PbCl2 and CdCl2 (at 3, 5 and 10 µM) were shown to induce concentration-dependent increases in micronucleus frequencies and DNA strand breaks in V79 cells. BA itself was not cytotoxic (up to 300 µM) and showed no genotoxic effects. Pretreatment of cells with low levels of BA (2.5 and 10 µM) was found to strongly reduce the genotoxic effects of the tested metals. Based on the findings of this in vitro study, it can be suggested that boron provides an efficient protection against the induction of DNA strand breaks and micronuclei by lead and cadmium. Further studies on the underlying mechanisms for the protective effect of boron are needed.

Toxicity of the mycotoxin citrinin and its metabolite dihydrocitrinone and of mixtures of citrinin and ochratoxin A in vitro.

Citrinin (CIT) and ochratoxin A (OTA) are mycotoxins produced by several species of the genera Aspergillus, Penicillium and Monascus. Both can be present as contaminants in various food commodities and in animal feed. The occurrence and toxicity of OTA and human exposure have been intensively studied, but for CIT such data are scarce by comparison. Recently, dihydrocitrinone (DH-CIT) was detected as main metabolite of CIT in human urine, and co-occurrence of CIT and OTA was shown in human blood plasma (Blaszkewicz et al. in Arch Toxicol 87:1087-1094, 2013). In light of these new findings, we have now investigated the toxicity of the metabolite DH-CIT in comparison with CIT and analysed the effects of mixtures of CIT and OTA in vitro. The cytotoxic potency of DH-CIT (IC50 of 320/200 µM) was distinctly lower compared with CIT (IC50 of 70/62 µM) after treatment of V79 cells for 24 and 48 h. Whereas CIT induced a concentration-dependent increase in micronucleus frequencies at concentrations ≥30 µM, DH-CIT showed no genotoxic effect up to 300 µM. Thus, conversion of CIT to DH-CIT in humans can be regarded as a detoxification step. Mixtures of CIT and OTA exerted additive effects in cytotoxicity assays. The effect of CIT and OTA mixtures on induction of micronuclei varied dependent on the used concentrations between additive for low µM concentrations and more-than-additive for high µM concentrations. Effects on cell cycle were mostly triggered by OTA when both mycotoxins were used in combination. The implications of our and related in vitro studies are discussed with respect to in vivo concentrations of CIT and OTA, which are found in animals and in humans.

Effects of cigarette smoke condensate on primary urothelial cells in vitro.

Cigarette smoking is a risk factor for bladder cancer. Since urothelial cells express phase I and II enzymes these cells are able to metabolize precarcinogens into DNA reactive intermediates. Cigarette smoke is a complex mixture containing at least 80 known carcinogens. In this context especially aromatic amines and polycyclic aromatic hydrocarbons are discussed as being responsible for bladder-carcinogenicity. Cell cultures of primary porcine urinary bladder epithelial cells (PUBEC) have been useful models for studies on bladder-specific effects. These cells are metabolically competent and found to be a valuable tool for examining effects of cigarette smoke constituents. In the present study PUBEC were utilized to investigate the effects of the complex mixture cigarette smoke condensate total particulate matter (CSC TPM) with emphasis on induction of cytochrome P-450 1A1 (CYP1A1) and genotoxic effects. CYP1A1 induction was investigated by Western blot and flow cytometry. The most pronounced effects were found after 24 h of incubation with 1-10 µg/ml CSC TPM. Maximal induction was observed at 5 µg/ml by flow cytometry and at 10 µg/ml by Western blot analysis. Genotoxic effects were investigated by means of alkaline single-cell gel electrophoresis ("comet assay") with and without the use of the DNA repair enzyme formamidopyrimidine-DNA glycosylase (Fpg) and the micronucleus (MN) test. A numerical concentration-dependent increase in Fpg-sensitive sites indicating oxidative DNA damage and a quantitative rise in MN formation were noted. The CSC utilized in this study contained low amounts of benzo[a]pyrene, 4-aminobiphenyl, and 2-naphthylamine. With regard to the observed CYP1A1 induction, these substances cannot explain the CYP1A1 inducing effect of CSC TPM. It is possible that other compounds within CSC TPM contribute to CYP1A1 induction in our cellular model.

N-Acetylation of p-aminobenzoic acid and p-phenylenediamine in primary porcine urinary bladder epithelial cells and in the human urothelial cell line 5637.

N-Acetyltransferases (NAT) are important enzymes in the metabolism of certain carcinogenic arylamines, as N-acetylation decreases or prevents their bioactivation via N-hydroxylation. To study such processes in the bladder, cell culture models may be used, but metabolic competence needs to be characterized. This study focused on the N-acetylation capacity of two urothelial cell systems, using p-aminobenzoic acid (PABA) and the hair dye precursor p-phenylenediamine (PPD), two well-known substrates of the enzyme NAT1. The constitutive NAT1 activity was investigated using primary cultures of porcine urinary bladder epithelial cells (PUBEC) and in the human urothelial cell line 5637 to assess their suitability for further in vitro studies on PABA and PPD-induced toxicity. N-Acetylation of PABA and PPD was determined by high-performance liquid chromatography (HPLC) analysis in cytosols of the two cell systems upon incubation with various substrate levels for up to 60 min. The primary PUBEC revealed higher N-acetylation rates (2.5-fold for PABA, 5-fold for PPD) compared to the 5637 cell line, based on both PABA conversion to its acetylated metabolite and formation of mono- and diacetylated PPD. The urothelial cell systems may thus be useful as a tool for further studies on the N-acetylation of aromatic amines via NAT1.

Cytotoxic potency of mycotoxins in cultures of V79 lung fibroblast cells.

In addition to dietary mycotoxin intake, exposure by inhalation is possible and may result in local effects in the lung. As a first approach to assess the potential local impact of inhaled mycotoxins, the cytotoxicity of 14 different mycotoxins was determined in V79 cell cultures, which served as an in vitro surrogate for lung cells. Cell viability was measured by the neutral red (NR) uptake assay after 48 h of exposure to graded concentrations of structurally diverse compounds: beauvericin, citrinin, enniatin B, moniliformin, ergocornine, ergotamine, fumonisin B1, ochratoxin A, patulin, the trichothecenes deoxynivalenol, HT-2, and T-2 toxin, and zearalenone, and α-zearalenol. The 14 mycotoxins show a wide range of cytotoxic potency, encompassing 7 orders of magnitude, with IC(20) values (concentration reducing cell viability by 20%) of 4.3 mM for moniliformin, the least potent mycotoxin, and 2.1 nM for T-2 toxin, the most potent agent. Thus, when inhaled in sufficient quantities, local adverse effects in lung cells cannot be excluded, in particular for highly cytotoxic mycotoxins.

Primary bovine colonic cells: a model to study strain-specific responses to Escherichia coli.

The parasitic or commensal lifestyle of bacteria in different hosts depends on specific molecular interactions with the respective host species. In vitro models to study intestinal bacteria-host interactions in cattle are not available. Bovine primary colonocyte (PC) cultures were generated from colon crypt explants. Up to day 4 of culture, the vast majority of cells were of epithelial phenotype (i.e., expressed cytokeratin but not vimentin). PCs harboured mRNA specific for Toll-like receptors (TLR) 1, TLR3, TLR4 and TLR6 but not for TLR2, TLR5, TLR7, TLR8, TLR9 and TLR10. Six hours after inoculation of PC cultures with Escherichia coli (E. coli) prototype strains representing different pathovars (enterohaemorrhagic E. coli [EHEC], enteropathogenic E. coli [EPEC], enterotoxic E. coli [ETEC]), bacteria were found attached to the cells. EPEC adhesion was accompanied by intracellular actin accumulation. An attenuated laboratory strain (E. coli K12 C600) and a bovine commensal E. coli strain (P391) both did not adhere. Bacterial or LPS challenge of PC cultures resulted in specific increases in mRNA transcripts for IL-8, GRO-alpha, MCP-1, RANTES, and IL-10. The level of mRNA transcripts for TGF-beta stayed constant, while IL-12 mRNA was not detectable. Short-term cultures of PCs, maintaining epithelial cell properties, interacted with commensal and pathogenic bacteria in a strain-specific manner and have proven to be a useful in vitro model to study the interaction of bacteria with the bovine intestinal mucosa.

The Fusarium toxin enniatin B exerts no genotoxic activity, but pronounced cytotoxicity in vitro.

Enniatin B, a fungal metabolite produced by various Fusarium strains, is a frequent contaminant in cereals used for human foods and animal feeds, but, so far very limited data are available on its toxicity. The aim of this study was to investigate the effects of enniatin B in a battery of short-term tests to evaluate its genotoxic potential. In Salmonella typhimurium assays (Ames assay) with the strains TA 98, TA 100, TA 102, and TA 104, both in the presence and absence of an external metabolizing enzyme system (rat liver S9), no mutagenicity was detected up to toxic levels (100 microM) of enniatin B. Likewise, mutagenicity tests in mammalian cells, i. e., the hypoxanthin-guanin-phosphoribosyl-transferase (HPRT) assay with V79 cells performed with and without S9 mix, did not reveal a significant increase in mutant frequency for enniatin B up to 30 microM, a cytotoxic concentration. Additional tests on other types of genotoxicity, i. e., clastogenicity and chromosomal damage, were conducted in V79 cells, applying the alkaline single cell gel electrophoresis (Comet assay with and without FPG, formamidopyrimidine DNA glycosylase, enzyme) and the micronucleus assay. None of these assays revealed a significant genotoxic potential of enniatin B. However, enniatin B exerts pronounced cytotoxic effects in V79 cells as determined by neutral red uptake assay for 48 h exposure: The IC(20) and IC(50) values of 1.5 and 4 microM, are higher than those of the more potent Fusarium toxin deoxynivalenol (IC(20) 0.6 microM, IC(50) of 0.8 microM), but in a similar range as values reported for cytotoxicity of enniatin B in various tumor cell lines. In summary, despite an apparent lack of genotoxic activity, enniatin B can exert biological activity at low micromolar concentrations in mammalian cells.

Distinct subtypes of urinary bladder epithelial cells with inducible and non-inducible cytochrome P450 1A1.

Cultured primary porcine urinary bladder epithelial cells (PUBEC) represent an adequate and easy to handle in vitro system for studies of urothelial toxicity. PUBEC maintain in vivo-like metabolic activities and physiological functions. They express inducible cytochrome P4501A isoenzymes, which are of particular relevance, since they contribute to activation of bladder carcinogens. A possible drawback of PUBEC is their isolation from common domestic pigs that do not represent an inbred strain. In order to further establish PUBEC as a standard in vitro toxicity test system we analysed possible interindividual differences in CYP1A1 inducibility. Interestingly, we observed by flow cytometry that PUBEC obtained from individual pigs consist of two distinct subpopulations with inducible and non-inducible cells. A strong, concentration-dependent CYP1A1 induction was observed in the responsive subpopulation when incubated with benzo[a]pyrene (B[a]P) in a concentration range between 1 and 10 muM. In contrast, no CYP1A1 induction was obtained in the non-responsive subpopulation up to the highest tested concentrations of 100 muM. The fraction of responsive cells showed large interindividual differences ranging from 10 to 65% of the total cell number. For practical purposes it might be reasonable to analyse pools of PUBEC from five pigs which substantially reduce batch to batch variability. In conclusion, we have identified two functionally distinct subpopulations of urinary bladder epithelial cells. It will be interesting to study whether the CYP1A inducible subtype is more susceptible to bladder carcinogens.

The emerging Fusarium toxin enniatin B: in-vitro studies on its genotoxic potential and cytotoxicity in V79 cells in relation to other mycotoxins.

The Fusarium metabolite enniatin B is now recognized as a frequent contaminant of grains used for human foods and animal feeds. Yet, so far very limited data are available on its toxicity and that of other emerging Fusarium mycotoxins (Jestoi M, 2008, Crit Rev Food Sci Nutr 48:21-49). Thus, the mutagenic/genotoxic potential of enniatin B was investigated in a battery of short-term tests, and its cytotoxicity compared with that of several other mycotoxins. No mutagenicity was detected in the Ames assay with four Salmonella typhimurium strains, and in the HPRT (hypoxanthine guanine phosphoribosyl transferase) assay with V79 cells, in either the presence or absence of an external metabolizing enzyme system (rat liver S9). For other types of genotoxicity, i.e., clastogenicity and chromosomal damage, studied in V79 cells by means of alkaline single-cell gel electrophoresis (Comet) assay and micronucleus assay, no significant genotoxic potential of enniatin B was revealed. However, the Fusarium metabolite exerts pronounced time- and concentration-dependent cytotoxic effects in V79 cells as determined by Alamar Blue reduction and by neutral red uptake assays. For instance, IC20 and IC50 values determined for enniatin B by neutral red assay for 48-h exposure are 1.5 µM and 4 µM. These values are higher than those of the more potent Fusarium toxin deoxynivalenol (IC20 0.7 µM, IC50 of 0.8 µM), but clearly lower than the IC values of several other mycotoxins tested in parallel. Their ranking of cytotoxicity in V79 cells was as follows: deoxynivalenol > enniatin B > patulin > ochratoxin A > zearalenone > citrinin. Moreover, enniatin B was found to induce nuclear fragmentation, a sign of apoptosis, already at low submicromolar concentrations. In summary, despite an apparent lack of mutagenic and genotoxic activity, enniatin B can cause pronounced cytotoxicity in mammalian cells, detectable at low micromolar concentrations.

Synergism of aromatic amines and benzo[a]pyrene in induction of Ah receptor-dependent genes.

Aromatic amines have been shown to cause bladder cancer. However, epithelial cells of the urinary bladder, cells of origin of bladder cancer, may be exposed to numerous substances besides aromatic amines. In the present study, we analysed possible interactions between the aromatic amines 4-aminobiphenyl (4-ABP) as well as 2-naphthylamine (2-NA) and the polycyclic aromatic hydrocarbon benzo[a]pyrene (B[a]P). For this purpose we incubated primary porcine urinary bladder epithelial cells (PUBEC) with concentrations of 1 to 50 microM 4-ABP with and without co-exposure to B[a]P. As expected B[a]P increased mRNA expression of cytochrome P450 1A1 (CYP1A1), whereas 4-ABP had no effect. However, when co-exposed 4-ABP enhanced the induction of CYP1A1 by B[a]P. This result was confirmed by Western blot analysis of CYP1A1 protein expression. A similar effect as for CYP1A1 was also observed for cyclooxygenase-2 (COX-2) and UDP-glucuronosyltransferase 1 (UGT1). Next, we studied co-exposures of 2-NA and B[a]P. Similar as for 4-ABP also 2-NA enhanced B[a]P-mediated induction of CYP1A1. Our results demonstrate that some aromatic amines may enhance the influence of B[a]P on Ah receptor-dependent genes.

Analysis of CYP1A1 induction in single cells of urothelial cell populations by flow cytometry.

As carcinogenesis is a process starting at the single-cell level it is desirable to study carcinogen-mediated effects in individual cells. A primary step in chemically induced carcinogenesis is the formation of reactive DNA-binding metabolites by cytochromes P450 (CYP). We applied indirect immunofluorescence to stain CYP1A1 in urothelial cells for quantification by flow cytometry. Our studies were carried out with metabolically competent primary porcine urinary bladder epithelial cells (PUBECs) and the human urothelial cell line 5637 for which we have previously demonstrated CYP1A1 mRNA induction by the polycyclic aromatic hydrocarbon (PAH) benzo[a]pyrene (B[a]P) applying real-time RT-PCR. Flow cytometric analysis revealed that for PUBEC and 5637 cells the fraction of CYP1A1-induced cells increased with B[a]P concentration. Furthermore, in 5637 cells this effect was time-dependent, being more pronounced after 48 h than after 24 h. However, CYP1A1 induction could not be detected in all analyzed PUBEC and 5637 cells after treatment with up to 50 muM B[a]P. The reason for this remains unknown at the moment. Overall, B[a]P-treated cells could be divided into fractions of clearly CYP1A1-induced and clearly uninduced cells. Another fraction of "unclear" CYP1A1-induced cells and one of unclassifiable cells remained, as quantification of CYP1A1 induction by flow cytometry was hampered by B[a]P-related fluorescence. This is ascribed to phenolic B[a]P metabolites formed by CYP1A1 and which are known to fluoresce at wavelengths above 500 nm, whereas B[a]P does not. Overall, the method permits the detection of CYP1A1 protein level in large numbers of individual cells, thereby providing an adequate basis for statistical analyses. Flow cytometric detection of CYP1A1 induction in individual cells allows further insight into the metabolic competence of single cells and therefore could be a valuable tool for toxicological studies.

Miniaturization of primary porcine urinary bladder epithelial cell cultures and application for gene expression studies after exposure to benzo[a]pyrene.

Benzo[a]pyrene (BaP) is an environmental pollutant used as a key marker substance for polycyclic aromatic hydrocarbons (PAHs). PAHs are believed to play a prominent role in the development of bladder cancer. A test system based on primary porcine urinary bladder epithelial cells (PUBEC) has been utilized as an in vitro model for urinary bladder epithelium. Recently in PUBEC cultures derived from pools of several bladders potent induction of CYP1A1 was detected after BaP treatment. Results from a modified approach using miniaturized PUBEC cultures for the analysis of individual bladder specimens with regard to cell growth and to BaP-mediated induction of CYP1A1 mRNA expression are presented herein. Two types of responses, low and high CYP1A1 induction among individual bladder specimens from eight donor animals, were detected. All of these tissue samples expressed the wild-type genotype of CYP1A1.

Improvements in culturing exfoliated urothelial cells in vitro from human urine.

Human bladder cancer is a common malignant tumor that may be produced by factors such as lifestyle, environment and occupation. The aim of this study was to evaluate parameters related to the viability of exfoliated urothelial cells. Exfoliated urothelial cells were obtained from 83 urine samples of 22 healthy participants (20-53 yr). From 67 of these samples, cells were transferred to collagen-coated 24-well plates. Parameters including sample volume, pH, osmolality and participant age and gender were examined on cell viability. In successive cultures, the numbers of cell colonies and cells per cell colony were determined. The number of viable cells in the urinary sediments of males varied from 0 to 6.5 x 10(3) cells per sample (mean 1 x 10(3)). Higher cell numbers in urine samples from females (6 x 10(3)) were due to considerable amounts of exfoliated vaginal cells. Cell numbers in males were positively related to volume, osmolality, and pH of the samples, as well as to the retention time of urine in the bladder. Cell proliferation was achieved in 25 out of 67 samples and was positively related to sample osmolality and pH. Participant age and content of urinary oxalates exerted negative effects on cell proliferation in vitro. The mean number of cell colonies per sample was 1.7. The mean cell number per colony was 11.7 x 10(3). It appears that high variability in individual excretion of urothelial cells able to proliferate is a limiting factor for routine use of these cells for in vitro toxicology.

Primary cultures of human urothelial cells for genotoxicity testing.

A cell culture system with human-derived urothelial cells was established based upon previous experience with cultures of porcine urinary bladder epithelial cells. Human tissue specimens used were derived from urinary bladders (n = 17) or ureters (n = 50) of patients undergoing urological operations. The epithelial origin and differentiation status was evaluated by an immunohistochemical staining for cytokeratins 7, 8, 18, 19, and 20 for isolated and cultured cells. Specimens from human ureters were better suited for primary cell cultures of the urothelium than specimens from human urinary bladders. Successful attachment and proliferation were reached by 98% of the ureter specimens (urinary bladder: 71%) and confluency was reached by 78% of the ureter cultures (urinary bladder: 18%). In the first 14 d of culture the cytokeratin patterns of cultured cells were comparable to those of native mucosa cells. During prolonged cell culture the cytokeratin patterns of the human urothelial cells (HUC) changed into a beginning dedifferentiation: Cytokeratin (CK) 18 was only detectable in cell cultures cultured for more than 29 d, whereas CK 19 was not detectable at d 29. Cell cultures of primary human urothelial cells may be used for in vitro testing of cytotoxic or genotoxic effects.

The use of cultured primary bovine colon epithelial cells as a screening model to detect genotoxic effects of heterocyclic aromatic amines in the comet assay.

Isolated epithelial cells from the bovine colon were maintained in dividing monolayer cultures and used as a model system for colon tissue in in vitro toxicological studies. The cytotoxic effects of the heterocyclic aromatic amines 2-amino-3-methylimidazo [4,5-f]quinoline (IQ) and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhiP) were investigated in these cells and IC(50) values were determined by inhibition of neutral red uptake into the cultured cells. Although PhiP was not cytotoxic up to concentrations of 500 microM, IQ was cytotoxic above 300 microM. The induction of DNA strand breaks in cultured bovine epithelial colon cells was determined using the alkaline single-cell gel electrophoresis (Comet assay) technique, and subsequently, the DNA damage was used as a determinant of genotoxic effects of the heterocyclic aromatic amines in order to establish this system for detection of adverse effects of chemicals in a model system for the colon. In the absence of an external enzymatic metabolizing system (S9 mix) both amines did not induce DNA strand breaks. When S9 mix was used, PhiP induced DNA strand breaks above 10 microM whereas IQ did not show any significant effect at 300 microM. This cell culture system was found to be a useful screening system for testing of compounds that are considered to affect colonic tissue.

Evaluation of time dependence and interindividual differences in benzo[a]pyrene-mediated CYP1A1 induction and genotoxicity in porcine urinary bladder cell cultures.

Exposure to tobacco smoke is an established cause of cancer in humans and cigarette smoking is a risk factor for urinary bladder cancer development. Aromatic amines are believed responsible for the bladder-specific carcinogenic effect, but polycyclic aromatic hydrocarbons (PAHs) are also of potential relevance. Urothelial cells contain a number of xenobiotic-metabolizing enzymes, which enable them to convert pro-carcinogens into reactive intermediates. In a preceding study, it was demonstrated using cultured porcine urinary bladder epithelial cells (PUBEC) that CYP1A1 mRNA is induced in a potent manner by treatment with benzo[a]pyrene (BaP). In the present study, the time dependence of these effects was evaluated and whether PUBEC cultures derived from individual donors respond differently to BaP treatment was determined. CYP1A1 induction was analyzed by quantitative reverse-transcription polymerase chain reaction (RT-PCR), and genotoxic effects were studied using the Comet assay. Incubation of PUBEC with BaP increased CYP1A1 expression and induction of DNA strand breaks in a time-dependent manner. Interindividual differences were found between PUBEC cultures derived from several donor animals with respect to the response to BaP, such that the extent of CYP1A1 induction and magnitude of DNA damage was interrelated. Hence, individual differences in metabolic capacities and responsiveness to xenobiotics of urothelial cells from individual donors may be factors in susceptibility to genotoxic effects induced by PAHs.

Glutathione S-transferase polymorphisms and ochratoxin A toxicity in primary human urothelial cells.

The mycotoxin ochratoxin A (OTA) is a worldwide contaminant of human food. OTA is genotoxic, immunotoxic, teratogenic and carcinogenic in rodents and can cause nephropathy in pigs. High amounts of OTA can cause nephropathy in humans. Moreover, evidence has been accumulated that OTA is a genotoxic carcinogen. Nevertheless, the mechanism that leads to OTA toxicity has not been fully resolved and it is discussed if a bioactivation of OTA is necessary or not. In this study the genotoxicity of OTA was investigated in primary human urothelial cells by means of alkaline single cell gel electrophoresis (Comet assay). Primary cultured human urothelial cells derived from tissue specimens of urological patients were incubated with 100 microM OTA for 3 h. In contrast to recently published results in MDCK cell lines, the cell cultures showed great interindividual differences in the extent of DNA damage. To evaluate these great interindividual differences the influence of the genotype of the isoenzymes of glutathione S-transferase (GST), namely GSTT1, GSTM1 and GSTP1 on the genotoxic potential of OTA was examined. The genotypes of these polymorphic enzymes were determined by polymerase chain reaction (PCR) and the distributions of the genotypes were correlated with the extent of DNA damage. We found associations between the genotypes of the polymorphic GST isoenzymes and the extent of DNA damage between subgroups with and without OTA-related DNA damage. From these results we conclude that genetic predisposition has the potential to influence OTA genotoxicity.

The effect of benzo(a)pyrene on porcine urinary bladder epithelial cells analyzed for the expression of selected genes and cellular toxicological endpoints.

Consumption of tobacco products is the most relevant risk factor for the development of bladder cancer beside occupational contributions. In order to investigate mechanisms of tobacco smoke components in bladder carcinogenesis we have introduced a primary epithelial cell culture system derived from porcine urinary bladder as a suitable representative for the corresponding human tissue under physiological conditions. Two independent readouts were selected as markers for genotoxic events. Changes in the expression level of several toxicologically relevant genes should serve as indicators for early response, while classical genotoxic endpoints monitored manifested damages. Here, we present the first results of our study with benzo(a)pyrene (BaP) as a member of polycyclic aromatic hydrocarbons (PAHs) found in tobacco smoke. Cells treated with BaP show a dramatic increase in the expression of CYP1A1 that appears to be both indicator of and contributor for BaP toxicity. Genes coding for other proteins relevant in xenobiotic metabolism, signal transduction or tumor suppression show moderate effects or no enhancement of their expression levels. Comet assay and micronucleus test did show a significant, dose-dependent increase in DNA damages or aberrations after cell division. While these effects are conforming to the response at the mRNA expression level, they are less pronounced and require rather higher dosages of the chemical.

Comparative metabolic activation of benzidine and N-acetylbenzidine by prostaglandin H synthase.

Benzidine and N-acetylbenzidine are activated to genotoxic metabolite(s) within the urothelial target tissue, with phase-I and phase-II enzymes being relevant. In principle, both benzidine and N-acetylbenzidine are activated by prostaglandin H synthase (PHS) to reactive intermediates. However, the relative impacts of benzidine and N-acetylbenzidine in this process remain unclear. Two experimental in vitro systems were used in the present comparative investigation: ram seminal vesicle microsomes rich in PHS and porcine urinary bladder epithelial cells (PUBEC) as a model system mimicking the general metabolic situation within the human urothelium. Benzidine, N-acetylbenzidine and N,N'-diacetylbenzidine were incubated with ram seminal vesicle microsomes and arachidonic acid and control incubations were performed with heat-inactivated microsomes. The metabolic disappearance of benzidine, N-acetylbenzidine or N,N'-diacetylbenzidine indicated a rapid turnover by PHS of benzidine and a slower turnover of N-acetylbenzidine. There was almost no PHS-associated metabolism of N,N'-diacetylbenzidine, suggesting that diacetylation of benzidine could represent a pathway of biological inactivation. Under similar conditions, incubations were performed with ram seminal vesicles and benzidine or N-acetylbenzidine upon addition of calf thymus DNA. After re-isolation of the DNA and 32P-postlabeling, with benzidine 2 distinct adducts were found of unknown nature, and with N-acetylbenzidine a single adduct appeared with co-migrated with the N'-(3'-monophosphodeoxyguanosin-8-yl)-N-acetylbenzidine. PUBEC cells were also incubated with benzidine or N-acetylbenzidine. No DNA adduct was found with benzidine, but a total of five adducts was produced from N-acetylbenzidine. The major adduct again co-migrated with N'-(3'-monophosphodeoxyguanosin-8-yl)-N-acetylbenzidine. When benzidine was incubated with PUBEC cells N-acetylbenzidine and, with some delay, N,N'-diacetylbenzidine were formed. Application of Lineweaver-Burk plots for the formation of N-acetylbenzidine from benzidine revealed a K(m) of 56.4 microM and a Vmax of 7.05 nmol/h per 10(6) PUBEC cells. The investigations generally support a key role of N-acetylbenzidine at the target site of the urothelium.