Protein synthesis toxin detection

Complete elucidation of the mechanisms by which ricin kills target cells remains an area of active study, but it is clear that the A -glycosidase activity of RTA is the essential triggering event. Inhibition of protein synthesis precedes other detectable alterations in target cell biochemistry. Ricin blocks amino acid incorporation in cmde microsome preparations before changes occur in energy metabohsm or oxidative phosphorylation the toxin has essentially no effect on mitochondrial respiration in isolated mitochondria or tumor cells (Waller et al., 1966 Dirheimer et al., 1968 Lin et al., 1971). Likewise, the first observable cytotoxic effect of ricin in cell culture is typically the inhibition of protein synthesis, followed by a reduction in DNA synthesis (Lin et al., 1970 Lin et al., 1971 Onozaki et al., 1972 Refsnes et al., 1974 Nicolson et al., 1975 Olsnes et al., 1976 Refsnes et al., 1977). 

The toxic efiects of the 12,13-epoxytrichothecenes are associated with the more highly oxidized members such as T-2 toxin (9.8) and vomitoxin (deoxynivalenol) (9.6) together with their macrocychc esters. The symptoms of alimentary toxic aleukia in test animals (cats) were detected on administration of T-2 toxin at a level of 0.08 mg kg per day. Structure activity studies on T-2 toxin revealed a requirement for the presence of the 12,13-epoxide and the 9-ene. The compounds exert their activity by the inhibition of protein synthesis. The trichothecenes interfere with the peptidyl transferase in the ribosomes. 

Another example of the use of mass spectrometry to delect toxins is to identify ricin, a highly toxic protein that inhibits cell protein synthesis. Ricin is produced from the seeds of Ricinus communis plants (known conunonly as castor beans) [73]. Structurally, ricin is made of A- and B-chains linked by a disulfide bridge. The toxicity of ricin is due primarily to the A-chain, which acts as an RNA A-glycosidase, which leads to ribosome incapacitation and ultimately to cell death. Ricin was identified from crude castor bean extracts using LC-MS/MS. The extract was denatured, reduced, and alkylated prior to trypsin digestion. Ricin identification was based on the detection of marker peptides in the digest. These markers include T5, T7, Til, T12, and T13 from the A-chain and T3, T5, T14, T19, and T20 from the B-chain. MS/MS can provide the amount and sequence of each marker for irrefutable evidence. For quick screening of ricin in crude extracts, MALDI-MS can be used to provide the molecular mass profile of the marker peptides. 

Fan et al report on a method for detecting toxins that inhibit protein synthesis in Chapter 15. To detect a toxin, a group of proteins are simultaneously synthesized in a microfluidic array device. The production yields of these proteins are inhibited differentially by the toxin. The toxin can thus be identified based on the unique response pattern (or signature) of the array. The limit of detection for ricin, a bioterrorism agent, is determined at 10 pM. In... 

A novel concept for toxin detection is presented based on toxin s inhibition of biological protein synthesis. We demonstrated the feasibility of the concept by confirming differential inhibitory effects of two toxin simulants on the expression yields of three proteins. The unique response pattern is obtained from the 3 X 4 IVT sensor array for each toxin simulant. 

In recent years there has been increasing interest in the use of primary and established animal and human cell lines in which mycotoxin toxicity is determined on morphological criteria and end-points like protein or DNA synthesis. The development of immortalised human cell lines is now being actively pursued to serve as standardised bioassays for the detection of food toxins including mycotoxins (Lewis et al., 1998). 

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