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. 2024 Sep 10;16(9):389.
doi: 10.3390/toxins16090389.

Combinatory Effects of Acrylamide and Deoxynivalenol on In Vitro Cell Viability and Cytochrome P450 Enzymes of Human HepaRG Cells

Julia Beisl  1 , Kristina Jochum  1   2 , Yi Chen  3 , Elisabeth Varga  1   4 , Doris Marko  1
Affiliations

Combinatory Effects of Acrylamide and Deoxynivalenol on In Vitro Cell Viability and Cytochrome P450 Enzymes of Human HepaRG Cells

Julia Beisl et al. Toxins (Basel). .

Abstract

Acrylamide (AA) can be formed during the thermal processing of carbohydrate-rich foods. Deoxynivalenol (DON), a mycotoxin produced by Fusarium spp., contaminates many cereal-based products. In addition to potential co-exposure through a mixed diet, co-occurrence of AA and DON in thermally processed cereal-based products is also likely, posing the question of combinatory toxicological effects. In the present study, the effects of AA (0.001-3 mM) and DON (0.1-30 µM) on the cytotoxicity, gene transcription, and expression of major cytochrome P450 (CYP) enzymes were investigated in differentiated human hepatic HepaRG cells. In the chosen ratios of AA-DON (10:1; 100:1), cytotoxicity was clearly driven by DON and no overadditive effects were observed. Using quantitative real-time PCR, about twofold enhanced transcript levels of CYP1A1 were observed at low DON concentrations (0.3 and 1 µM), reflected by an increase in CYP1A activity in the EROD assay. In contrast, CYP2E1 and CYP3A4 gene transcription decreased in a concentration-dependent manner after incubation with DON (0.01-0.3 µM). Nevertheless, confocal microscopy showed comparably constant protein levels. The present study provided no indication of an induction of CYP2E1 as a critical step in AA bioactivation by co-occurrence with DON. Taken together, the combination of AA and DON showed no clear physiologically relevant interaction in HepaRG cells.

Keywords: acrylamide; deoxynivalenol; food processing; hepatocytes; metabolism; mixtures; process contaminants.

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Conflict of interest statement

The authors declare no conflicts of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.

Figures

Figure 1
Figure 1
Cell viability of differentiated HepaRG cells after incubation with acrylamide (AA, dark grey), deoxynivalenol (DON, light grey), their respective combination (light grey, dashed), and the calculated combined effects (white, dashed) measured in technical triplicates by the neutral red assay after 24 h at a ratio of AA to DON of 10:1 (A) and 100:1 (C), as well as after 48 h at a ratio of AA to DON of 10:1 (B) and 100:1 (D). The results of the 4–12 biological replicates are presented as means + SD, normalized to the solvent control displayed by dashed lines. The calculated combined effects were determined by the Bliss Independence model [40,41,42]. The no-effect levels were determined by one sample Student’s t tests. Statistical differences with the no-effect levels were calculated by ANOVA (p < 0.05, Bonferroni post hoc test) and marked with the respective letters (a = AA, b = DON, c = measured combined effects, d = calculated combined effects).
Figure 2
Figure 2
Relative quantities of the mRNA transcript levels of CYP1A1 (A), CYP2E1 (B), and CYP3A4 (C) observed in the HepaRG cells after 24 h of incubation with acrylamide (AA, dark grey), deoxynivalenol (DON, light grey), and their respective combination (light grey, dashed) using quantitative real-time PCR. Data evaluation using the 2−∆∆Ct method, according to Livak and Schmittgen [43]. All target genes were normalized to the housekeeping genes ALAS1 and HPRT1. The results are shown as means + SD of n = 5–8 independent experiments measured in technical duplicates. Statistical differences with the solvent control were calculated by the two-sample Student’s t-test (* p < 0.05). Dashed lines indicate the values obtained for the solvent controls.
Figure 3
Figure 3
Ethoxyresorufin-O-deethylase activity in HepaRG cells after 24 h of incubation with acrylamide (AA, dark grey), deoxynivalenol (DON, light grey), and their respective combination (light grey, dashed) assessed with EROD assay and bicinchonic acid protein assay. Results are presented as means + SD of 5–8 biological replicates measured in technical triplicates, normalized to solvent controls (SC). Statistical differences to the solvent control were determined with the one-sample Student’s t-test (* p < 0.05).
Figure 4
Figure 4
Immunofluorescence staining and subsequent quantitative analysis of CYP2E1 enzymes after 24 h (A,C) and 48 h (B,D) incubation of HepaRG cells with acrylamide (AA, dark grey), deoxynivalenol (DON, light grey), and their respective combination (light grey, dashed). Microscopy panels (A,B) show single-channel pictures of each enzyme in first column, merge pictures with nuclei (stained with DAPI) in second column and merge pictures with nuclei and actin filaments (stained using phalloidin) in last column. Quantitative analysis is depicted in bar charts (C,D) with dashed lines displaying values of solvent controls. Results are presented as mean + SD of five biological replicates with 16–20 analyzed optical fields per condition for 24 h and four biological replicates with 13–16 analyzed optical fields per condition for 48 h. Statistical differences from the solvent control were calculated with the one-sample Student’s t-test (* p < 0.05). Statistical differences between single substances and their respective combination were calculated with the two-sample Student’s t-test (* p < 0.05).
Figure 5
Figure 5
Immunofluorescence staining and subsequent quantitative analysis of CYP3A4 enzymes after 24 h (A,C) and 48 h (B,D) incubation of HepaRG cells with acrylamide (AA, dark grey), deoxynivalenol (DON, light grey), and their respective combination (light grey, dashed). Microscopy panels (A,B) show single-channel pictures of each enzyme in first column, merge pictures with nuclei (stained with DAPI) in second column, and merge pictures with nuclei and actin filaments (stained using phalloidin) in last column. Quantitative analysis is depicted in bar charts (C,D) with dashed lines displaying the values of solvent control. Results are presented as mean + SD of five biological replicates with 17–20 analyzed optical fields per condition for 24 h and four biological replicates with 15–16 analyzed optical fields per condition for 48 h. Statistical differences to solvent control were calculated with the one-sample Student’s t-test (* p < 0.05). Statistical differences between single substance and combination were calculated with the two-sample Student’s t-test (* p < 0.05).
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