Biological materials can release amino acids during the course of bacterial mutation testing. Low levels of released amino acids from soluble materials can cause moderate increases in the number of revertant colonies on the plate, whereas higher levels lead to overgrowth of the background lawn, making counting of revertant colonies impossible. For poorly soluble material, the released amino acids can be present at high levels in localized spots on the plate, leading to the growth of 'pseudorevertant' colonies. The 'treat and wash' modified preincubation method employed here is an adaptation of the treat and plate method (used for evaluation of antibiotics) and involves washing the bacteria free of test compound after a 90 min exposure prior to plating out on minimal plates. The MC overlay method is a modified version of the standard plate incorporation assay, in which a top overlay containing 4% high viscosity methylcellulose is used in place of agar to stabilize the test compound in solution, preventing precipitation and subsequent localized amino acid release. Both modified methods produce the expected results for negative and positive controls. Peptides [synthetic curtailed analogs of human parathyroid hormone, PTH(1-34) and Ostabolin-C] that produced false positive results or could not be evaluated owing to overgrowth of the background lawn using standard methods, showed no artifacts and no evidence of genotoxicity using the modified methods. It is concluded that the treat and wash and MC overlay methods are valid versions of the bacterial mutation test for avoiding complications associated with released amino acids.

There is widespread dietary exposure to capsaicin in the form of chili peppers, while capsaicin's analgesic qualities have led to increased use of a topical herbal remedy in various impure forms. Most recently, injection of pure capsaicin has been proposed as a means of relieving a variety of debilitating diseases, in which case tissues would receive relatively high and direct exposure. The purpose of the present study, where a series of standard assays were performed in accordance with the Organisation for Economic Cooperation and Development guidance, was to clarify earlier conflicting reports concerning potential genotoxicity of capsaicin prior to administering it to patients in an injectable form. The results confirm the absence of genotoxic activity of high-purity capsaicin in the bacterial mutation and chromosome aberration tests. In addition, no evidence of cytotoxicity or genotoxicity was seen in the rat bone marrow micronucleus test, where systemic exposure to pure capsaicin was achieved using the subcutaneous route and a rising dose toleration protocol. It is concluded that pure capsaicin is not active in the standard battery of genotoxicity assays recommended by the International Conference on Harmonisation for evaluation of new medicines; earlier reported in vitro genotoxic activity is probably associated with mutagenic impurities in commercial grades of the material.

The yeast two-hybrid system was used to identify peptide inhibitors of exotoxin A of Pseudomonas aeruginosa with the goal of using these to design peptide-based drugs against the toxin. A random peptide library consisting of 10(7) peptides ranging in length from 16 to 63 residues was screened for peptides that interact with the C-domain of exotoxin A. From the 10(7) transformants screened, three unique peptides of 63, 61 and 25 amino acids in length were found to specifically interact with the enzyme domain. The genes encoding these peptides were cloned and expressed as fusion proteins with the maltose-binding protein. In vitro inhibition measurements indicated that two of the peptides were modest inhibitors of toxin enzyme activity. These peptides now provide the basis for the development of more potent inhibitors, which will serve as lead inhibitors for evolution of potent peptide-based therapeutics.