Wolfram Föllmann

Sister chromatid exchange frequency in cultured isolated porcine urinary bladder epithelial cells (PUBEC) treated with ochratoxin A and alpha.

The mycotoxin ochratoxin A (OTA) and its metabolite ochratoxin alpha (OT-alpha) were investigated, to examine their potency to induce sister chromatid exchages (SCE) in cultured poricine urinary baldder epithedial cells (PUBEC) (primary cluture). Serum-free cultured PUBEC were incubated for 5 h with either OTA or OT-alpha, respectively, and subsequently cutured in the presence of 5-bromo-2-deoxyuridine (BrdU). After two cell cycles, mitosis was inhibited by the colchicine derivative Colcemid, cells were fixed and chromosomes were prepared for SCE analysis. For OTA, a dose-dependent increase in SCE frequency was measured in concentrations between 100 pM and 100 nM OTA. At 100nM OTA, SCE frequency increased by about 41%, compared to the base SCE level (7.27 SCEs per chromosome set, solvent control). Higher concentrations of OTA were cytotoxic. The metabolite OT-alpha also increased SCE frequency, but at higher concentrations. At a concentration of 10μM OT-alpha, an increase of about 55% was detected. OT-alpha showed no cytotoxic effect. There results indicate that OTA is genotoxic in this in vitro system, which represents the urinary bladder epithelium, a target organ of OTA in vivo. It could also be shown that OT-alpha, which is said to be non-toxic, is genotoxic in this assay at higher concentrations.

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.

Primary cell cultures of bovine colon epithelium: isolation and cell culture of colonocytes.

Epithelial cells from bovine colon were isolated by mechanical preparation combined with an enzymatic digestion from colon specimens derived from freshly slaughtered animals. After digestion with collagenase I, the isolated tissue was centrifuged on a 2% D-sorbitol gradient to separate epithelial crypts which were seeded in collagen I-coated culture flasks. By using colon crypts and omitting the seeding of single cells a contamination by fibroblasts was prevented. The cells proliferated under the chosen culture conditions and formed monolayer cultures which were maintained for several weeks, including subcultivation steps. A population doubling time of about 21 hr was estimated in the log phase of the corresponding growth curve. During the culture period the cells were characterized morphologically and enzymatically. By using antibodies against cytokeratine 7 and 13 the isolated cells were identified as cells of epithelial origin. Antibodies against vimentin served as negative control. Morphological features such as microvilli, desmosomes and tight junctions, which demonstrated the ability of the cultured cells to restore an epithelial like monolayer, were shown by ultrastructural investigations. The preservation of the secretory function of the cultured cells was demonstrated by mucine cytochemistry with alcian blue staining. A stable expression of enzyme activities over a period of 6 days in culture occurred for gamma-glutamyltranspeptidase, acid phosphatase and NADH-dehydrogenase activity under the chosen culture conditions. Activity of alkaline phosphatase decreased to about 50% of basal value after 6 days in culture. Preliminary estimations of the metabolic competence of these cells revealed cytochrome P450 1A1-associated EROD activity in freshly isolated cells which was stable over 5 days in cultured cells. Then activity decreased completely. This culture system with primary epithelial cells from the colon will be used further as a model for the colon epithelium in toxicological studies in vitro.

Effects of ochratoxin A on DNA repair in cultures of rat hepatocytes and porcine urinary bladder epithelial cells

Abstract In cultured rat hepatocytes the mycotoxin ochratoxin A (OTA) induced unscheduled DNA synthesis (UDS) only in a narrow concentration range. Using a culture medium supplemented with 1% fetal calf serum, at 750 nM OTA a weak induction and at 1 μM OTA a marked induction of DNA repair was observed (15 ± 11 and 38 ± 24% cells in repair, respectively). Concentrations >1 μM OTA were cytotoxic, and <750 nM no induction occurred. In cultures of cells from the urinary bladder (porcine urinary bladder epithelial cells; PUBEC), a target organ of the mycotoxin, OTA induced UDS in a concentration-dependent manner. To inhibit the proliferation of the cultured epithelial cells, which would counteract the detection of DNA repair, epidermal growth factor was omitted and an arginine-deficient medium (ADM) was used. Under these serum-free culture conditions the amount of cells undergoing DNA repair in PUBEC control cultures was ∼7±4%, a value also comparable to those of control cultures of rat hepatocytes. At concentrations between 250 nM and 1 μM OTA a concentration-dependent increase of cells in repair was observed. Above 1 μM OTA was cytotoxic. At this concentration a maximum of ∼61±9% of the cells undergo DNA repair. This amount is comparable to control cultures incubated with 5 or 10 mM ethylmethane-sulphonate (EMS) (49±9 and 69±10% cells in repair, respectively), used as a positive control. These results show that in cultured rat hepatocytes induction of UDS is relatively weak whereas in urothelial cells this effect was significant. Whether this effect is due to OTA metabolites formed locally in the urothelium cannot be excluded since PUBEC have been shown to be able to metabolize xenobiotics independently from the liver.

Drug metabolizing enzyme activities in porcine urinary bladder epithelial cell cultures (PUBEC)

Abstract Drug metabolizing enzyme activities have been determined in cultured porcine urinary bladder epithelial cells (PUBEC) in order to evaluate this system as an in vitro model for studies of urinary bladder carcinogens. Activities of several phase I and II enzymes were measured in cells cultured for various periods and compared with the activities determined in freshly isolated PUBEC. Prostaglandin H synthase mediated production of prostaglandin E2 was found both in freshly isolated and in cultured PUBEC, whereas cytochrome P450 1A1-associated EROD activity was only detectable in freshly isolated bladder cells. The latter activity was not inducible by benz(a)anthracene or 3-methylcholanthrene in PUBEC cultures. N-acetyltransferase (NAT) activity measured with p-aminobenzoic acid, a diagnostic substrate for human NAT-1, was stable and even higher during the culture period compared to freshly isolated cells. In contrast, isoniazid (a substrate for NAT-2) was not acetylated either in fresh or cultured PUBEC. Glutathione S-transferases activity determined with 1-chloro-2,4-dinitrobenzene decreased gradually to 50% after 1 week and to 20% after 4 weeks in culture compared to fresh cells. A similar decline was also observed for UDP-glucuronyltransferase activities measured with 1-naphthol. In accordance with the reported lack of sulfotransferases in pigs, no sulfation of 1-naphthol or 2-naphthylamine was detected in PUBEC. Our results show that cultured porcine urinary bladder epithelial cells maintain several enzyme activities required for the biotransformation of xenobiotics. In future investigations on the mechanism of action of bladder carcinogens PUBEC cultures may thus provide a useful in vitro model for this target tissue.

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 IC20 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.

Expression of cytochrome P450 enzymes CYP1A1, CYP1B1, CYP2E1 and CYP4B1 in cultured transitional cells from specimens of the human urinary tract and from urinary sediments

Expression of cytochromes P450 CYP1A1, CYP1B1, CYP2E1 and CYP4B1 was analysed on the transcript level in human urothelial cells obtained by various methods. As a source of urothelial cells, exfoliated cells in urine samples were used. Their expression profiles were determined either immediately after centrifugal enrichment (n=4) or after their cultivation and propagation (n=8). Another source of urothelial cells were ureter specimens from surgical subjects (n=4). Generally, expression was most prominent for CYP1B1 and CYP4B1 among the CYP transcripts analysed. CYP1B1 mRNA was detected in all samples investigated except for one ureter specimen. CYP4B1 mRNA was present in cell cultures from three out of eight healthy subjects, in three out of four directly investigated urinary sediments and in the cells of all five ureter specimens of four donors investigated after resection and subsequent cell culture. In most cases, CYP2E1 transcript levels were lower than those of CYP1B1 and CYP4B1. CYP2E1 mRNA was detected in cell cultures of six out of eight healthy subjects, in one out of four urinary sediments and in three out of five ureter specimens. CYP1A1 mRNA was clearly observed only in cells from resected ureters. In cell cultures the relative mRNA expression levels varied with subjects interindividually, intraindividually and also during the time of cell culture. The study demonstrates constitutive mRNA expressions of xenobiotic metabolising CYP enzymes in human urothelial cells obtained by different methods. In particular, transcripts of CYP1B1 and CYP4B1 are present, coding for enzymes which are active in the metabolism of polycyclic aromatic hydrocarbons and arylamines, respectively.

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.

Biomonitoring of Mycotoxins in Urine: Pilot Study in Mill Workers

ABSTRACT 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 β-zearalenol (α- and β-ZEL), as well as phase II metabolites that were hydrolyzed with β-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 β-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.

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.