The Metallothionein System in Tetrahymena thermophila Is Iron-Inducible

Abstract

Metallothioneins are multifunctional proteins implicated in various cellular processes. They have been used as biomarkers of heavy metal exposure and contamination due to their intrinsic ability to bind heavy metals and their transcriptional response to both physiological and noxious metal ions such as cadmium (Cd) and mercury (Hg). In this study, we aimed to clarify the role of iron and reactive oxygen species (ROSs) in the induction of the metallothionein system (Mtt) in the ciliate protozoan Tetrahymena thermophila. We investigated the relative mRNA abundances of the metallothionein genes Mtt1, Mtt2/4, and Mtt5, revealing for the first time their responsiveness to iron exposure. Furthermore, by using inhibitors of superoxide dismutase (SOD) and catalase (CAT), alone or in combination with iron, we highlighted the roles of superoxide ion and endogenous hydrogen peroxide, as well as the complex interplay between the metal and ROSs. These results enhance our understanding of the metallothionein system in ciliates and suggest that ROSs may be a primary evolutionary driver for the selection of these proteins in nature.

Keywords: aquatic system; evolution; heavy metals; iron homeostasis; oxidative stress; pollution; protist.

Figure 1

Metallothionein mRNA relative abundances (box plot) after exposure to 100 μM H₂O₂ and 100 μM H₂O₂ plus 200 μM FeCl₃ at 24 h (left column) and 72 h (right column). (a) Mtt1 at 24 h, (b) Mtt2/4 at 24 h, (c) Mtt5 at 24 h, (d) Mtt1 at 72 h, (e) Mtt2/4 at 72 h and (f) Mtt5 at 72 h. The asterisks above each bar indicate the statistical significance of the difference in gene expression compared to control non-exposed cells. The inlet horizontal lines link the conditions that were further compared in contrasts using ANOVA. [**** p < 0.0001; (ANOVA, Tukey’s multiple comparison test)].

Figure 2

Metallothionein mRNA relative abundances (box plots) after 24 h (left column) and 72 h (right column) exposure to 20 μM CdCl₂, 200 μM FeCl₃, 200 μM sodium azide (SAN), sodium azide plus iron (SAN + Fe), 100 μM sodium diethyldithiocarbamate (DTN) and sodium diethyldithiocarbamate plus FeCl₃; (a) Mtt1 24 h; (b) Mtt2/4 24 h; (c) Mtt5 24 h; (d) Mtt1 72 h; (e) Mtt2/4 72 h and (f) Mtt5 72 h. The asterisks above each bar indicate the statistical significance of the difference in gene expression compared to control non-exposed cells. The inlet horizontal lines link the conditions that were further compared in contrasts using ANOVA. Significance levels: [**** p < 0.0001; *** p < 0.001; ** p < 0.01; * p < 0.05; ° p < 0.1 ((a–c) Welch’s ANOVA, Dunnett’s T3 multiple comparison test; (d–f) ANOVA, Tukey’s multiple comparison test)]. Cadmium was used as a positive control, and was not included in contrasts.

Figure 3

Metallothionein mRNA relative abundances (box plots) after 2 h (left column), 24 h (center column) and 72 h (right column) exposure to iron. (a) Mtt1 2 h; (b) Mtt2/4 2 h; (c) Mtt5 2 h; (d) Mtt1 24 h; (e) Mtt2/4 24 h; (f) Mtt5 24 h; (g) Mtt1 72 h; (h) Mtt2/4 72 h and (i) Mtt5 72 h. Significance levels: the asterisks above each bar indicate the statistical significance of the difference in gene expression compared to control non-exposed cells [** p < 0.001; * p < 0.05; ° p < 0.1 (Welch’s ANOVA, Dunnett’s T3 multiple comparison test vs. non-exposed reference control)].