Ochratoxin cell culture

Faucet-Marquis, V., Pont, F., Stormer, F.C., Rizk, T., Castegnaro, M. Rohl-Leszkowicz, A. (2006) Evidence of a new dechlorinated ochratoxin A derivative formed in opossum kidney cell cultures after pretreatment by modulators of glutathione pathways correlation with DNA adduct formation. Mol. Nutr. Food Res. 50, 530-542. 

Zurich, M.G., Lengacher, S., Braissant, 0., Monnet-Tschudi, F., Pellerin, L. Honegger, P. (2005) Unusual astrocyte reactivity caused by the food mycotoxin ochratoxin A in aggregating rat brain cell cultures. Neuroscience 134, 771-782. 

There have been several studies that underscore the importance of unbound concentration in cell-based studies of receptor function. In a model study of the effect of plasma protein binding on the renal transport of organic anions using the expression of various organic anion transporters (OATPs) in Xenopus oocytes, the transport of ochratoxin A, methotrexate, and estrone sulfate was found to be strongly inhibited by the addition of human serum albumin to the culture medium [16]. Similarly, the addition of oq-acid glycoprotein was found to reverse the blockade of sodium-ion current by cocaine in a preparation of cardiac myocytes [17]. 

Whether these requirements are better met by primary cultures or renal cell lines is still subject of debate and will depend on the type of investigation. Techniques for the isolation and culture of primary cells of the renal tubular epithelium, glomerular mesangial cells, podocytes and endothelial cells have been developed for various species including human. Although cells in primary culture tend to dedifferentiate, the characteristics of those cells are usually closer to the in vivo situation than are animal cell tines, at least for a limited culture period. Primary cultures have been used successfully to study the in vitro effects of numerous nephrotoxins including, cyclosporine A (CsA), gentamicin, mercuric chloride and Ochratoxin A [38-42]. 

Schwerdt G, Flolzinger FI, Sauvant C, Konigs M, Flumpf FlU, and Gekle M. Long-term effects of ochratoxin A on fibrosis and cell death in human proximal tubule or fibroblast cells In primary culture.Toxicology 232 57-67, 2007. 

Groves CE, Nowak G, and Morales M. Ochratoxin A secretion in primary cultures of rabbit renal proximal tubule cells. J Am Soc Nephrol 10 13-20,1999. 

Schwerdt, G., Freudinger, R., Mildenberger, S., Silbernagl, S. and Gekle, M. (1999) The nephrotoxin ochratoxin A induces apoptosis in cultured human proximal tubule cells, Cell Biol. Toxicol., 15 (6), 405—415. 

As summarized previously by this Committee, there was no evidence of DNA repair as a result of possible DNA damage in bacteria, whereas DNA single-strand breaks were consistently induced in cultured mammalian cells and were also observed in vivo in spleen, liver and kidney cells of mice after intraperitoneal injection of ochratoxin A. DNA repair, manifested as unscheduled DNA synthesis, was observed in most studies with primary cultures of rat and mouse hepatocytes, porcine epithelial cells from bladder and human urothelial cells (Annex 1, reference 153). 

In cultured rat aggregated brain cells, 10-20 nmol ochratoxin A/l was shown to increase the expression of genes involved in the brain inflammatory system (messenger ribonucleic acid [mRNA] of peroxisome proliferator-activated receptor, haem oxygenase-1 and inducible nitric oxide synthase) and to reduce the expression of glial fibrillary acidic protein, a constituent of the intermediate filaments in astrocytes (Zurich et al., 2005). 

In cultured rat embryonic midbrain cells, 0.5 and 1 pg ochratoxin A/ml caused a reduction in the number of viable cells, an induction of transcription factors activator protein-1 and nuclear factor-kappa B (NF-kB) activation as well as an inhibition of neurite outgrowth at the higher concentration (Hong et al., 2002). 

Induction of oollagen secretion (a marker of fibrosis) has been shown in the OK proximal tubular cell line and in cultured primary human renal proximal tubular oells exposed to ochratoxin A. Collagen seoretion was both time and dose dependent, as was the induction of cell toxicity (Sauvant et al., 2005a). 

In a cultured human renal proximal tubular cell line, HK-2, a concentration-related increase in the level of ROS compared with controls was observed after 6 h exposure to ochratoxin A at 50-600 pmol/l. Pretreatment with A/-acetyl-L-cysteine, an antioxidant ROS scavenger, decreased the level of ROS and increased cell survival. A/-Acetyl-L-cysteine also reduced DNA damage at 50-200 pmol/l, as measured by the comet assay, but not at higher ochratoxin A concentrations of >400 pmol/l, at which the generation of ROS outpaced the reduction by A/-acetyl-L-cysteine (Arbillaga et al., 2007a). 

Dorrenhaus, A. Follmann, W. (1997) Effects of ochratoxin A on DNA repair in cultures of rat hepatocytes and porcine urinary bladder epithelial cells. Arch. Toxicol. 71, 709-713. 

Flieger, A., Dorrenhaus, A., Golka, K., Schulze, H. Fdllman, W. (1998) Genotoxic effect of the mycotoxin ochratoxin A in cultured human urothelial cells. Occup. Hyg. 4, 297-307. 

Hong, J.T., Lee, M.K., Park, K.S., Jung, K.M., Lee, R.D., Jung, H.K., Park, K.L, Yang, K.J. Chung, Y.S. (2002) Inhibitory effect of peroxisome proliferator-activated receptor gamma agonist on ochratoxin A-induced cytotoxicity and activation of transcription factors in cultured rat embryonic midbrain cells. J. Toxicol. Environ. Health A 65, 407—418. 

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