Different Toxicity Mechanisms for Citrinin and Ochratoxin A Revealed by Transcriptomic Analysis in Yeast

Abstract

Citrinin (CIT) and ochratoxin A (OTA) are important mycotoxins, which frequently co-contaminate foodstuff. In order to assess the toxicologic threat posed by the two mycotoxins separately or in combination, their biological effects were studied here using genomic transcription profiling and specific live cell gene expression reporters in yeast cells. Both CIT and OTA cause highly transient transcriptional activation of different stress genes, which is greatly enhanced by the disruption of the multidrug exporter Pdr5. Therefore, we performed genome-wide transcription profiling experiments with the pdr5 mutant in response to acute CIT, OTA, or combined CIT/OTA exposure. We found that CIT and OTA activate divergent and largely nonoverlapping gene sets in yeast. CIT mainly caused the rapid induction of antioxidant and drug extrusion-related gene functions, while OTA mainly deregulated developmental genes related with yeast sporulation and sexual reproduction, having only a minor effect on the antioxidant response. The simultaneous exposure to CIT and OTA gave rise to a genomic response, which combined the specific features of the separated mycotoxin treatments. The application of stress-specific mutants and reporter gene fusions further confirmed that both mycotoxins have divergent biological effects in cells. Our results indicate that CIT exposure causes a strong oxidative stress, which triggers a massive transcriptional antioxidant and drug extrusion response, while OTA mainly deregulates developmental genes and only marginally induces the antioxidant defense.

Keywords: Citrinin; Ochratoxin A; Saccharomyces cerevisiae; Transcriptome; dose response; mycotoxins; oxidative stress.

Figure 1

Ochratoxin A (OTA) and citrinin (CIT) activate stress gene expression independently and with different dose response profiles. (A) OTA and CIT induction of the stress-activated genes GRE2 (methylglyoxal reductase) and SOD2 (superoxide dismutase). Live cell reporter fusions with destabilized luciferase were used in yeast wild type cells and the induction of both genes was measured in real time upon the indicated mycotoxin doses. (B) The deletion of the Pdr5 multidrug exporter increases the transcriptional response to both OTA and CIT. The expression profiles for the GRE2 and SOD2 genes are compared for wild type and the pdr5 deletion mutant upon the indicated mycotoxin doses. (C) OTA and CIT do not activate stress gene expression in a synergistic manner. The dose response profiles of (A) and (B) are represented here as the maximal activity (A_max) for each mycotoxin dose. Additionally (purple columns at the right of each plot), a constant concentration of CIT (50 ppm = 200 μM) was combined with growing concentrations of OTA (50 ppm = 124 μM; 200 ppm = 497 μM; 400 ppm = 994 μM) as indicated. All gene expression experiments were performed on three independent culture aliquots; the Standard Deviation was <15%; error bars are not included in the graphs in order to make the figure clearly visible.

Figure 2

Ochratoxin A and citrinin activate largely nonoverlapping gene sets in the yeast genome. Venn diagram comparing the >5-fold induced transcripts of the yeast genome upon OTA and CIT exposure. The exclusively upregulated genes by one mycotoxin (CIT or OTA) and the commonly upregulated genes are depicted in the table. The main functional groups associated with each gene cluster are given.

Figure 3

Citrinin, but not ochratoxin A, toxicity is exacerbated in mutants with a defective antioxidant response or multidrug export. The indicated yeast strains were treated or not with 400 μM CIT (upper panel) or 400 μM OTA (lower panel) for the indicated time. Serial dilutions 1:1, 1:10, and 1:100 of the yeast cultures were then assayed for survival on yeast extract peptone dextrose (YPD) agar plates without mycotoxins.

Figure 4

CIT, as opposed to OTA, induces a sensitive oxidative and general stress response in yeast cells. (A) OTA and CIT induction of the PDR5–, GRE2– and AP1–luciferase reporters. Live cell reporter fusions with destabilized luciferase were used in yeast wild type cells and the induction of both genes was measured in real time upon the indicated mycotoxin doses. The data are derived from three independent culture aliquots and had an error of <15%. (B) Dose-response profiles of the different luciferase reporters. The maximal steady-state activity (A_max) was calculated for each reporter strain and toxin dose and plotted against the mycotoxin concentration. A_max for the highest toxin exposure was arbitrarily set to 100.