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.

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.

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.

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.

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.