No abstract

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.

No abstract

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.

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.

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 M 4-ABP with and without coexposure to B[a]P. As expected B[a]P increased mRNA expression of cytochrome P450 1A1 (CYP1A1), whereas 4-ABP had no eVect. However, when co-exposed 4-ABP enhanced the induction of CYP1A1 by B[a]P. This result was conWrmed by Western blot analysis of CYP1A1 protein expression. A similar eVect 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 inXuence of B[a]P on Ah receptor-dependent genes.

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.

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.