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Characterization of ochratoxin A-induced apoptosis in primary rat hepatocytes

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Abstract

The main target organ of the mycotoxin ochratoxin A (OTA) in mammals is the kidney but OTA has also been shown to be hepatotoxic in rats and to induce tumors in mouse liver. Even at very low concentrations, OTA causes perturbations of cellular signaling pathways as well as enhanced apoptosis. OTA has been extensively studied in kidney cell systems. Since this substance also affects liver health, we focused our work on apoptosis-related events induced by OTA in primary rat hepatocytes. We performed pathway-specific polymerase chain reaction arrays to assess the expression of genes involved in apoptosis. Treatment with 1 µM OTA for 24 h caused marked changes in apoptosis-related gene expression. Genes as apaf1, bad, caspase 7, polb (DNA polymerase beta, performs base excision repair), and p53, which are marker genes for DNA damage, were upregulated. FAS and faslg were also markedly induced by treatment with OTA. Treatment of hepatocytes with OTA led to a concentration-dependent inhibition of protein biosynthesis. Apoptosis-inducing factor was released from mitochondria following OTA treatment; the mycotoxin induced the activity of caspases 8, 9, and 3/7 and caused chromatin condensation and fragmentation. Caspase inhibition led to a significant but not complete reduction of OTA-induced apoptosis. Our data suggest that not only OTA leads to p53-dependent apoptosis in rat hepatocytes but it also hints to other mechanisms, independent of caspase activation or protein biosynthesis, being involved.

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Abbreviations

AIF:

Apoptosis-inducing factor

CAD:

Caspase-activated DNase

CHX:

Cycloheximide

DAPI:

4′, 6-Diamidino-2-phenylindole

DMSO:

Dimethyl sulfoxide

DTT:

Dithiothreitol

DFF:

DNA fragmentation factor

ICAD:

Inhibitor of caspase-activated DNase

OTA:

Ochratoxin A

PIPES:

Piperazine-N,N′-bis(2-ethanesulfonic acid)

PBS:

Phosphate-buffered saline

PVDF:

Polyvinylidene fluoride

qPCR:

Quantitative polymerase chain reaction

ROS:

Reactive oxygen species

c T :

Threshold cycle

TBS:

Tris-buffered saline

VDAC:

Voltage-dependent anion channel

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  1. Institute of Food Chemistry and Toxicology, University of Kaiserslautern, Erwin Schrödinger Straße 52, 67659, Kaiserslautern, Germany

    Martin Chopra, Pascal Link, Christine Michels & Dieter Schrenk

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  1. Martin Chopra
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  2. Christine Michels
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  3. Dieter Schrenk
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Correspondence to Dieter Schrenk.

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Chopra, M., Link, P., Michels, C. et al. Characterization of ochratoxin A-induced apoptosis in primary rat hepatocytes. Cell Biol Toxicol 26, 239–254 (2010). https://doi.org/10.1007/s10565-009-9131-0

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