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Translational step inhibited in vivo by aflatoxin B1 in rat-liver polysomes
Abstract:
Aflatoxin B1 strongly inhibits protein synthesis in rat liver cells. We previously demonstrated that this inhibition could be divided into two steps: up to 5 h aflatoxin blocks protein synthesis directly and specifically at the polysome level; beyond 7 h protein synthesis inhibition appears chiefly as a consequence of transcription impairment due to drug action. This paper confirms the foregoing results and represents an attempt to localize the translational step inhibited in vivo by aflatoxin B1. We used the simulation study developed by Li, Kisilevsky, Wasan and Hammond, 1972 (Biochim. Biophys. Acta, 272, 451-462) to determine precisely the site inhibited in vivo after drug intoxication. This analysis is based on two parameters: the kinetics of polysome labeling to follow the nascent peptide synthesis, and the kinetics of supernatant labeling to follow the completed protein synthesis. Up to 5 h after dosing, aflatoxin specifically inhibits the elongation and/or termination steps during protein synthesis; after longer periods of time inhibition occurs essentially at the initiation step. When the intracellular concentration of aflatoxin is too high, particularly 2 h after dosing, each step of protein synthesis is blocked. Polypeptide synthesis by the postmitochondrial supernatants isolated from aflatoxin-treated animals is impaired in the same proportion as protein synthesis in vivo. The damage caused by aflatoxin is mostly observed on microsomes. However, purified polysomes isolated from aflatoxin-treated rats synthesize proteins in vitro to the same extent as those from controls. These results suggest that aflatoxin metabolite(s) are bound to polysomes with noncovalent bonds. These active metabolites are probably lost during polysome isolation procedures. Finally, relationships between protein metabolism and aflatoxin carcinogenesis are discussed.
Insights
Aflatoxin B1 impairs protein synthesis in rat liver cells by inhibiting elongation/termination early on, and initiation later. High concentrations block all steps, with damage primarily on microsomes.
Area of Science:
- Biochemistry
- Molecular Biology
- Toxicology
Background:
- Aflatoxin B1 is a potent mycotoxin known to inhibit protein synthesis in mammalian cells.
- Previous studies indicated a biphasic inhibition: direct polysomal blockade early, and transcriptional impairment later.
- The precise translational step affected by aflatoxin B1 in vivo remained to be localized.
Purpose of the Study:
- To precisely localize the translational step inhibited in vivo by aflatoxin B1.
- To investigate the time-dependent effects of aflatoxin B1 on protein synthesis at the polysomal level.
- To elucidate the mechanism of aflatoxin B1-induced protein synthesis inhibition.
Main Methods:
- Utilized a simulation study based on polysome and supernatant labeling kinetics.
- Analyzed nascent peptide synthesis (polysome labeling) and completed protein synthesis (supernatant labeling).
- Compared in vitro protein synthesis by isolated postmitochondrial supernatants and purified polysomes from treated and control rats.
Main Results:
- Aflatoxin B1 initially inhibits elongation and/or termination steps of protein synthesis (up to 5 hours).
- After longer exposure (beyond 7 hours), inhibition shifts to the initiation step.
- High aflatoxin B1 concentrations block all protein synthesis steps; damage is mainly on microsomes, but purified polysomes remain active in vitro, suggesting non-covalent binding of metabolites.
Conclusions:
- Aflatoxin B1 exhibits time-dependent inhibition of protein synthesis, targeting different translational steps.
- The active aflatoxin B1 metabolite(s) likely bind non-covalently to polysomes and are lost during isolation.
- Understanding these mechanisms is crucial for exploring aflatoxin B1's role in carcinogenesis.