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Comparative Study
, 105 (1), 67-78

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

Affiliations
Comparative Study

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

Maoxiang Li et al. Toxicol Sci.

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.

Figures

FIG. 1.
FIG. 1.
Ricin evokes depurination of yeast 28S rRNA in vitro. Yeast ribosomes were treated with indicated concentrations of (A) ricin, (B) DON, or (C) T-2 for 30 min at 37°C. RNA was extracted, treated with aniline acetate for 30 min at 30°C, and subjected to electrophoresis on acrylamide. Arrow indicates aniline-dependent 370 nt 28S rRNA fragment induced by ricin (300 ng/ml). Results are representative of six separate experiments.
FIG. 2.
FIG. 2.
DON induces rRNA degradation in RAW 264.7 cells. Cells were treated with DON (2 μg/ml) for indicated time intervals. Total RNA was isolated and subjected to agarose gel electrophoresis under denaturing conditions. Arrows indicate 28S rRNA cleavage fragments. Results are representative of four separate experiments.
FIG. 3.
FIG. 3.
Induction of 18S rRNA cleavage in RAW 264.7 cells by DON, T-2 and ricin. Cells were treated with DON (A), T-2 (C), or ricin (E) at indicated concentrations for 6 h or with DON (2 μg/ml) (B), T-2 (80 ng/ml) (D), or ricin (160 ng/ml) (F) for indicated time intervals. Northern blots were performed on total RNA with biotin-dUTP labeled probes and streptavidin-labeled IRDye 800CW and analyzed for infrared fluorescence. Arrows indicate 18S rRNA cleavage products. Results are representative of two separate experiments.
FIG. 4.
FIG. 4.
Induction of 28S rRNA cleavage in RAW 264.7 cells by DON, T-2 and ricin. Cells were treated with DON (A), T-2 (C), or ricin (E) at indicated concentrations for 6 h or with DON (2 μg/ml) (B), T-2 (80 ng/ml) (D), or ricin (160 ng/ml) (F) for indicated time intervals and analyzed as described in Figure 3 legend. Arrows indicate 28S rRNA cleavage products. Results are representative of four separate experiments.
FIG. 5.
FIG. 5.
Oligonucleotide extension analysis of toxin-induced cleavage of 28S rRNA S/R loop in RAW 264.7 cells. Cells were incubated with DON, T-2, and ricin RAW cells were treated with DON (1000 ng/ml), T-2 (80 ng/ml), and ricin (160 ng/ml) for 4 h. Reverse transcription reaction was performed on total RNA using FAM-labeled primer 1–specific for mouse 28S rRNA from 4300 to 4281 (depicted in Fig. 6). Resultant cDNA extension fragments were sized and quantified. Results are representative of two separate experiments.
FIG. 6.
FIG. 6.
Peptidyl transferase target sites of ricin, DON and T-2. Secondary structure of the peptidyl transferase region of mouse rRNA based on Michot et al. (1984) using base pairings described by Larsson et al. (2002). Structure was drawn with PseudoViewer web application (http://pseudoviewer.inha.ac.kr/) developed by Byun and Han (2006). Filled circles indicates primer sites and dotted arrow show direction of primer extension. Solid arrows indicate targets for ricin, DON and T-2 identified by oligonucleotide extension.
FIG. 7.
FIG. 7.
Oligonucleotide extension analysis of toxin-induced 28S rRNA peptidyl transferase cleavage in RAW 264.7 cells. Cells were treated with DON (1000 ng/ml), T-2 (80 ng/ml), and ricin (160 ng/ml) for 0, 0.5, 1, 2, and 4 h. Reverse transcription reaction was performed on total RNA using FAM-labeled (A) primer 2 specific for mouse 28S rRNA specific, from 4107 to 4091) or (B) primer 3 specific for mouse 28S rRNA from 3622 to 3605 (depicted in Fig. 6). Resultant cDNA extension fragments were sized and quantified. Results are representative of two separate experiments.”
FIG. 8.
FIG. 8.
DON, T-2, and ricin induce RNase activity in RAW 264.7 cells. Cells were treated with DON (A), T-2 (C), or ricin (E) at indicated concentrations for 1 h or with DON (1000 ng/ml) (B), T-2 (160 ng/ml) (D), or ricin (320 ng/ml) (F) for indicated time intervals Total cellular proteins were extracted and RNase activity determined. Data are means ± SEM (n = 6) pooled from three separate experiments. Bars without same letter differ (p < 0.05).
FIG. 9.
FIG. 9.
DON, T-2, and ricin concentration-dependently modulate RNase mRNA expression in RAW 264.7 cells. Cells were treated with DON, (A, D) T-2, (B, E) and ricin (C, F) for 2 h at the indicated concentrations. Total RNA was analyzed for RNase 6 and RNase L expression by real-time PCR. Data are means ± SEM (n = 8) pooled from two separate experiments. Bars without same letter differ (p < 0.05).
FIG. 10.
FIG. 10.
DON, T-2, and ricin time-dependently alter RNase mRNA expression in RAW 264.7 cells. Cells were treated with DON (1000 ng/ml), (A, D) T-2 (160 ng/ml), (B, E) and ricin (320 ng/ml) (C, F) for indicated time interval. Total RNA was analyzed for RNase 6 and RNase L expression analyzed by real-time PCR. Data are means ± SEM (n = 8) pooled from two separate experiments. Bars without same letter differ (p < 0.05).
FIG. 11.
FIG. 11.
DON, T-2 and ricin altered RNase L protein expression in RAW 264.7 cells. Cells were treated with indicated concentration of DON, ricin, or T-2 for 1 h or with (A) 500 ng/ml of DON, 160 ng/ml of ricin, or 80 ng/ml of T-2 for indicated time interval (B). RNase L protein was probed by Western blotting. Data were representative of four separate experiments.

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