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Toxicity mechanisms of the food contaminant citrinin: application of a quantitative yeast model.

Pascual-Ahuir A, Vanacloig-Pedros E, Proft M - Nutrients (2014)

Bottom Line: Additionally, genes in various multidrug resistance transport systems are functionally involved in the resistance to citrinin.Our study identifies the antioxidant defense as a major physiological response in the case of citrinin.In general, our results show that the use of live cell gene expression reporters in yeast are a powerful tool to identify toxicity targets and detoxification mechanisms of a broad range of food contaminants relevant for human nutrition.

View Article: PubMed Central - PubMed

Affiliation: Department of Biotechnology, Instituto de Biología Molecular y Celular de Plantas, Universidad Politécnica de Valencia, Ingeniero Fausto Elio s/n, 46022 Valencia, Spain. apascual@ibmcp.upv.es.

ABSTRACT
Mycotoxins are important food contaminants and a serious threat for human nutrition. However, in many cases the mechanisms of toxicity for this diverse group of metabolites are poorly understood. Here we apply live cell gene expression reporters in yeast as a quantitative model to unravel the cellular defense mechanisms in response to the mycotoxin citrinin. We find that citrinin triggers a fast and dose dependent activation of stress responsive promoters such as GRE2 or SOD2. More specifically, oxidative stress responsive pathways via the transcription factors Yap1 and Skn7 are critically implied in the response to citrinin. Additionally, genes in various multidrug resistance transport systems are functionally involved in the resistance to citrinin. Our study identifies the antioxidant defense as a major physiological response in the case of citrinin. In general, our results show that the use of live cell gene expression reporters in yeast are a powerful tool to identify toxicity targets and detoxification mechanisms of a broad range of food contaminants relevant for human nutrition.

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Related in: MedlinePlus

The yeast Pdr5 multidrug transporter is important for the citrinin dose response and sensitivity. (a) Fusions of the stress inducible GRE2 (left panel) or SOD2 (right panel) promoters with destabilized luciferase were used as a real time reporter for gene expression. The reporter activity was measured in yeast wild type and pdr5 mutants upon addition of the indicated concentrations of citrinin. Data shown are mean values from three independent biological samples. SD < 15%; (b) pdr5 mutants show increased sensitivity to citrinin. The indicated citrinin doses were applied to yeast wild type, pdr1, pdr5 and snq2 mutants for three hours in YPD culture medium. Surviving cells were then assayed on a fresh YPD plate; (c) Yeast wild type and pdr5 mutant cells were incubated for one hour with the indicated amounts of citrinin in KP buffer. The amount of living cells was then quantified by staining with FDA. Data are mean values from three independent biological replicas. Error bars are SD. The asterisks refer to p < 0.05 different from wt in the same condition according to the Student’s t-test. The value for mock treated cells was arbitrarily set to 100 for both yeast strains.
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nutrients-06-02077-f004: The yeast Pdr5 multidrug transporter is important for the citrinin dose response and sensitivity. (a) Fusions of the stress inducible GRE2 (left panel) or SOD2 (right panel) promoters with destabilized luciferase were used as a real time reporter for gene expression. The reporter activity was measured in yeast wild type and pdr5 mutants upon addition of the indicated concentrations of citrinin. Data shown are mean values from three independent biological samples. SD < 15%; (b) pdr5 mutants show increased sensitivity to citrinin. The indicated citrinin doses were applied to yeast wild type, pdr1, pdr5 and snq2 mutants for three hours in YPD culture medium. Surviving cells were then assayed on a fresh YPD plate; (c) Yeast wild type and pdr5 mutant cells were incubated for one hour with the indicated amounts of citrinin in KP buffer. The amount of living cells was then quantified by staining with FDA. Data are mean values from three independent biological replicas. Error bars are SD. The asterisks refer to p < 0.05 different from wt in the same condition according to the Student’s t-test. The value for mock treated cells was arbitrarily set to 100 for both yeast strains.

Mentions: We next wanted to gain insights into the molecular mechanisms of citrinin extrusion and its effect on its toxicitiy. The yeast genome encodes a large family of multidrug transporters which are pleiotropically involved in the export of many xenobiotic chemicals from the cytosol. Two genes of this transporter family, PDR5 and SNQ2, have been identified in yeast as up-regulated upon citrinin exposure [12]. Additionally we included a yeast strain, Δpdr1, in the citrinin study, which lacks one of the principal transcriptional activators of the multidrug resistance gene family [19]. As shown in Figure 4, the pdr5 mutant strain showed the highest degree of sensitivity to citrinin in a growth assay in rich medium (Figure 4B). The enhanced toxicity of citrinin in a pdr5 mutant was further confirmed by an independent assay using FDA as a live cell stain upon acute citrinin stress in potassium phosphate buffer (Figure 4C). These results indicated that Pdr5 was a major citrinin export activity in yeast and we postulated that pdr5 mutant cells were more sensitive to the mycotoxin because of greater accumulation of citrinin in the cell interior. We therefore measured the adaptive response to citrinin in pdr5 mutants and compared it to wild type. As shown in Figure 4A, the dose dependent response of two citrinin inducible luciferase reporters, GRE2 and SOD2, was largely enhanced in the pdr5 mutant strain. Therefore we can confirm that the lack of the Pdr5 multidrug transporter leads to hypersensitivity to citrinin and a more sensitive adaptive response to the toxin, presumably caused by overaccumulation of citrinin inside the cell.


Toxicity mechanisms of the food contaminant citrinin: application of a quantitative yeast model.

Pascual-Ahuir A, Vanacloig-Pedros E, Proft M - Nutrients (2014)

The yeast Pdr5 multidrug transporter is important for the citrinin dose response and sensitivity. (a) Fusions of the stress inducible GRE2 (left panel) or SOD2 (right panel) promoters with destabilized luciferase were used as a real time reporter for gene expression. The reporter activity was measured in yeast wild type and pdr5 mutants upon addition of the indicated concentrations of citrinin. Data shown are mean values from three independent biological samples. SD < 15%; (b) pdr5 mutants show increased sensitivity to citrinin. The indicated citrinin doses were applied to yeast wild type, pdr1, pdr5 and snq2 mutants for three hours in YPD culture medium. Surviving cells were then assayed on a fresh YPD plate; (c) Yeast wild type and pdr5 mutant cells were incubated for one hour with the indicated amounts of citrinin in KP buffer. The amount of living cells was then quantified by staining with FDA. Data are mean values from three independent biological replicas. Error bars are SD. The asterisks refer to p < 0.05 different from wt in the same condition according to the Student’s t-test. The value for mock treated cells was arbitrarily set to 100 for both yeast strains.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4042565&req=5

nutrients-06-02077-f004: The yeast Pdr5 multidrug transporter is important for the citrinin dose response and sensitivity. (a) Fusions of the stress inducible GRE2 (left panel) or SOD2 (right panel) promoters with destabilized luciferase were used as a real time reporter for gene expression. The reporter activity was measured in yeast wild type and pdr5 mutants upon addition of the indicated concentrations of citrinin. Data shown are mean values from three independent biological samples. SD < 15%; (b) pdr5 mutants show increased sensitivity to citrinin. The indicated citrinin doses were applied to yeast wild type, pdr1, pdr5 and snq2 mutants for three hours in YPD culture medium. Surviving cells were then assayed on a fresh YPD plate; (c) Yeast wild type and pdr5 mutant cells were incubated for one hour with the indicated amounts of citrinin in KP buffer. The amount of living cells was then quantified by staining with FDA. Data are mean values from three independent biological replicas. Error bars are SD. The asterisks refer to p < 0.05 different from wt in the same condition according to the Student’s t-test. The value for mock treated cells was arbitrarily set to 100 for both yeast strains.
Mentions: We next wanted to gain insights into the molecular mechanisms of citrinin extrusion and its effect on its toxicitiy. The yeast genome encodes a large family of multidrug transporters which are pleiotropically involved in the export of many xenobiotic chemicals from the cytosol. Two genes of this transporter family, PDR5 and SNQ2, have been identified in yeast as up-regulated upon citrinin exposure [12]. Additionally we included a yeast strain, Δpdr1, in the citrinin study, which lacks one of the principal transcriptional activators of the multidrug resistance gene family [19]. As shown in Figure 4, the pdr5 mutant strain showed the highest degree of sensitivity to citrinin in a growth assay in rich medium (Figure 4B). The enhanced toxicity of citrinin in a pdr5 mutant was further confirmed by an independent assay using FDA as a live cell stain upon acute citrinin stress in potassium phosphate buffer (Figure 4C). These results indicated that Pdr5 was a major citrinin export activity in yeast and we postulated that pdr5 mutant cells were more sensitive to the mycotoxin because of greater accumulation of citrinin in the cell interior. We therefore measured the adaptive response to citrinin in pdr5 mutants and compared it to wild type. As shown in Figure 4A, the dose dependent response of two citrinin inducible luciferase reporters, GRE2 and SOD2, was largely enhanced in the pdr5 mutant strain. Therefore we can confirm that the lack of the Pdr5 multidrug transporter leads to hypersensitivity to citrinin and a more sensitive adaptive response to the toxin, presumably caused by overaccumulation of citrinin inside the cell.

Bottom Line: Additionally, genes in various multidrug resistance transport systems are functionally involved in the resistance to citrinin.Our study identifies the antioxidant defense as a major physiological response in the case of citrinin.In general, our results show that the use of live cell gene expression reporters in yeast are a powerful tool to identify toxicity targets and detoxification mechanisms of a broad range of food contaminants relevant for human nutrition.

View Article: PubMed Central - PubMed

Affiliation: Department of Biotechnology, Instituto de Biología Molecular y Celular de Plantas, Universidad Politécnica de Valencia, Ingeniero Fausto Elio s/n, 46022 Valencia, Spain. apascual@ibmcp.upv.es.

ABSTRACT
Mycotoxins are important food contaminants and a serious threat for human nutrition. However, in many cases the mechanisms of toxicity for this diverse group of metabolites are poorly understood. Here we apply live cell gene expression reporters in yeast as a quantitative model to unravel the cellular defense mechanisms in response to the mycotoxin citrinin. We find that citrinin triggers a fast and dose dependent activation of stress responsive promoters such as GRE2 or SOD2. More specifically, oxidative stress responsive pathways via the transcription factors Yap1 and Skn7 are critically implied in the response to citrinin. Additionally, genes in various multidrug resistance transport systems are functionally involved in the resistance to citrinin. Our study identifies the antioxidant defense as a major physiological response in the case of citrinin. In general, our results show that the use of live cell gene expression reporters in yeast are a powerful tool to identify toxicity targets and detoxification mechanisms of a broad range of food contaminants relevant for human nutrition.

Show MeSH
Related in: MedlinePlus