Comparative Induction of 28S Ribosomal RNA Cleavage by Ricin and the Trichothecenes Deoxynivalenol and T-2 Toxin in the Macrophage

Abstract

Ribosome-inactivating proteins (RIPs) and sesquiterpenoid trichothecene mycotoxins are known to bind to eukaryotic ribosomes, inhibit translation and activate mitogen-activated protein kinases. Here we compared the capacities of the RIP ricin to promote 28S ribosomal RNA (rRNA) cleavage with that of the trichothecenes, deoxynivalenol (DON), and T-2 toxin (T-2). In a cell-free model, exposure to ricin at 300 ng/ml for 30 min depurinated yeast 28S rRNA, however, neither DON (< or = 4 microg/ml) nor T-2 (< or = 2 microg/ml) exhibited this N-glycosidase activity. Incubation of RAW 264.7 macrophages with ricin (20-320 ng/ml), DON (250-5000 ng/ml), or T-2 (2-80 ng/ml) for 6 h, however, generated 28S rRNA-specific products consistent with cleavage sites near the 3' terminal end of murine 28S rRNA. Oligonucleotide extension analysis of treated RAW 264.7 cells revealed that ricin evoked 28S rRNA damage at one site in the alpha-sarcin/ricin (S/R)-loop (A4256) and two other sites (A3560 and A4045) in the peptidyl transferase center. Although DON or T-2 did not damage the S/R loop, these trichothecenes did promote cleavage at A3560 and A4045. In addition, incubation of the cells with ricin (> or = 20 ng/ml), DON (> or = 250 ng/ml), or T-2 (> or = 10 ng/ml) induced RNase activity as well as RNase L mRNA and protein expression. These data suggest that only ricin directly damaged 28S rRNA under cell-free conditions but that ricin, DON, and T-2 promoted intracellular 28S rRNA cleavage, potentially by facilitating the action of endogenous RNases and/or by upregulating RNase expression.

Effect of deoxynivalenol on the levels of toll-like receptors 2 and 9 and their mRNA expression in enterocytes in the porcine large intestine: a preliminary study

“…After binding with ribosomes, DON inhibits ribosomal peptidyl transferase, which prevents the initiation and elongation of the translation process (Shifrin and Anderson 1999). The inhibition of protein synthesis induced by DON can also result from the degradation of 28S rRNA (Li and Pestka 2008). Zhou et al (2003) demonstrated that DON can activate double-stranded RNA-associated protein kinase (PKR) which is responsible for the phosphorylation of the eukaryotic initiation factor 2α (eIF2α) and inhibition of translation.…”

A genome-wide screen in Saccharomyces cerevisiae reveals a critical role for the mitochondria in the toxicity of a trichothecene mycotoxin

“…In the macrophage, DON induced mobilization of MAPKs to the ribosome (13) and promoted 28S rRNA cleavage at the peptidyltransferase center (14), suggesting that ribosome interactions of trichothecenes play an important role in their cytotoxicity.…”

GC-TOF/MS-based metabolomics approach to study the cellular immunotoxicity of deoxynivalenol on murine macrophage ANA-1 cells

“…This toxin has a broad spectrum of effects on the gastrointestinal, reproductive and neuroendocrine systems in mammals and especially the immune system [2,3]. DON is known to inhibit protein synthesis by binding to the peptidyltransferase region of the 28S ribosomal RNA resulting in ribotoxic stress [4]. It has a major effect on actively dividing cells including bone marrow, spleen and thymus cells [5].…”

“…With respect to the strongest correlation to phenotype, metabolites (unlike genes and proteins, whose function is determined by epigenetic regulation or posttranslational modification) are the end products of cellular regulatory processes and their levels can be regarded as the ultimate response of a biological system to genetic or environmental changes [9].Metabolomics represents the global assessment of metabolites in a biological sample and defines the information closest to the M A N U S C R I P T A C C E P T E D ACCEPTED MANUSCRIPT 4 phenotype of the biological system under study [10].The metabolome is defined as the full complement of small-molecule metabolites found in a specific cell, organ, or organism [11]. It is an excellent indicator of phenotypic heterogeneity and has been recognized as a key factor in rare-cell survival when populations are subjected to major chemical or environmental challenges [12].…”