Elsevier

Toxicology in Vitro

Volume 17, Issue 4, August 2003, Pages 441-448
Toxicology in Vitro

Effects of the mycotoxin ochratoxin A and some of its metabolites on human kidney cell lines

https://doi.org/10.1016/S0887-2333(03)00053-5Get rights and content

Abstract

The modulations of complement-regulating surface proteins on a human embryonic and a renal carcinoma cell line are described regarding the effects of ochratoxin A and some of its metabolites on the surface markers CD46, CD55 and CD59. Membrane integrity, cell proliferation and metabolic activity were reduced to different extents, depending on the kind of mycotoxin and the dosage, which was ranging from 10 to 1000 ng/ml. The number of cells carrying surface markers was suppressed significantly at 1000 ng/ml, in some cases even at 100 ng/ml, whereas the intensity of receptor expression on the positive cells was found to be stimulated. The fraction RE2 (OTC) isolated from an OTA-containing crude toxin surpassed the effects of all other ochratoxin metabolites. Apart from well-known cytotoxic and genotoxic effects modulation of cell surface marker expression by low concentrations of OTA and OTC deserves more attention with regard to its immuno-pathogenic importance. Furthermore, occurrence and impact of the mycotoxin OTC should be studied more into detail.

Introduction

Tests on laboratory animals and livestock showed the capacity of the mycotoxin ochratoxin A (OTA) to trigger immuno-modulations even at levels far below the threshold of toxicity (Müller et al., 1995, Müller et al., 1999). Remarkable variations of the influence of ochratoxin on individual animals, animal groups and species as well as on different organ systems leave many questions open concerning the mechanisms of action. As a consequence, an increasing number of in vitro experiments has been carried out in addition to animal tests, where the immunological effects of mycotoxins on cells were investigated (Marin et al., 1996, Wong et al., 1998, Moon et al., 1999, Heller et al., 2000, Berek et al., 2001).

On the basis of animal tests in different species, OTA is to be considered nephrotoxic (Baudrimont, 1997, Dortant et al., 2001, Gekle et al., 1998, Krogh et al., 1988, Shlosberg et al., 1997, Stoev et al., 2000, Stoev et al., 2001). Epidemiological research (Creppy, 1999, Frank, 1991), the detection of OTA in food (Vrabcheva et al., 2000) and in human blood (Wafa et al., 1998), as well as findings in slaughter pigs (Curtui et al., 2001, Stoev et al., 1998a, Stoev et al., 1998b) indicate a relation between the Balkan nephropathy as well as urothelian tumours in humans and a higher intake of OTA in this region, although there has been no exact proof so far.

According to the assessment of SCOOP (2001), the intake of OTA on the basis of the mean food consumption amounts to 0.2–3.2 ng/kg b.w. per day for adults in the EC, depending on the country. The tolerable intake with regard to nephrotoxic effects has been set by the JECFA (2001) to 100 ng/kg b.w. per week. However, a daily intake of only 1.2–5.7 ng/kg b.w. is discussed to be the minimum for carcinogenic effects (Kuiper-Goodman, 1996).

In Germany, an OTA concentration of 0.45 ng/ml (90% percentil) has been found in human serum (Wolff et al., 2000). On the basis of this value, a mean OTA intake of 0.9 ng/kg b.w./d was calculated. This indicates, that a remarkable amount of OTA passes the human kidney with the blood flow.

The regulation of complement activation by a group of proteins is crucial for immunological processes. The most important proteins are membrane cofactor protein (MCP/CD46), decay-accelerating factor (DAF/CD55) and the homologous restriction factor 20 (CD59), all of which bind and act differently in the complement system (Gorter et al., 1996, Hourcade et al., 2000). CD55 is modulated on tumour cells by the cytokines IL-1α, TNF-α and IFN-γ (Varsano et al., 1998). According to Gorter et al. (1996) several tumour cell lines express these proteins in order to prevent a complement-mediated lysis. It is possible that changes in the expression of these proteins influence the degeneration of cells as well as their elimination by immunological effector mechanisms. Therefore, it was the aim of this paper to examine the influence of OTA compared to an OTA-containing crude toxin and some of its metabolites at low concentrations on the expression of complement-regulating surface proteins in a human embryonic and a renal carcinoma cell line. Additionally, effects on cell viability were studied, as assessed by cell proliferation, the MTT assay and propidium iodide dye exclusion.

Section snippets

Chemicals and immunoreagents

OTA was purchased from Sigma (Taufkirchen, Germany). We are grateful to Dr. Ackerman and Dr. Horak (CSIR, Pretoria, South Africa) for providing us with ochratoxin pure and crude products. 3-(4,5-dimethyl thiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) and propidium iodide were supplied by Sigma and l-glutamine by Serva (Heidelberg, Germany). We received the cell cultivation media MEM and Dulbecco's MEM from Gibco (Life Technologies, Eggenstein, Germany), foetal calf serum, penicillin,

Influence on cell proliferation

The changes of cell proliferation of both kidney cell lines caused by ochratoxins are shown in Table 1. OTA and the crude toxin influenced the net proliferation of both cell lines only at 1000 ng/ml, for the A 498 cell line significantly, whereas OTC and the fraction RE2 reduced it significantly already at 10–100 ng/ml. The carcinoma cell line proved more sensitive. After exposure to 100–1000 ng/ml of the toxins in some cases smaller cell numbers were found at the end of the experiments than at

Discussion

Because of the nephrotoxic effects of ochratoxin in animal tests, numerous experiments on kidney cell cultures have been carried out in order to broaden the knowledge about the mechanisms of action of the toxins in these cell systems. Kidney cells are sensitive indicators for nephrotoxic mycotoxins (Baudrimont, 1997, Bondy, and Armstrong, 1998). Gekle et al. (1998) gave an overview of the literature data about the different effects on kidney cells caused by high and low doses of OTA. Low-dose

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