Modifications of Ames Test for Assessing the Mutagenicity of Traditional Chinese Medicines

Traditional Chinese Medicines (TCM) are histidine-containing biological samples, they are commonly orally taken in the form of water extracts. False-positive results of TCMs’ water extracts may get when using standard Ames test, therefore the interference resulted from histidine within the samples must be eliminated or reduced, and the standard Ames test needs to be modified. This mini-review summarizes the main modifications of Ames test, by providing the modification strategies and pointing out the advantages and disadvantages of their application on TCMs’ mutagenic test, and wish it giving beneficial references to the related researches in the future. False-Positive Result of Standard Ames Test when assessing the Mutagenicity of a Traditional Chinese Medicine Ames test is a widely employed biological assay that uses bacteria to assess the mutagenic potential of chemical compounds [1]. The test serves as a quick and convenient in vitro method to estimate the carcinogenic potential of a compound because standard carcinogen assays on mice and rats are very time-consuming and expensive. However, false-positives are known, [2] especially when evaluating the mutagenicity of samples containing histidine and/or its precursors [3-19]. Ames test uses several strains of the bacterium Salmonella typhimurium that carry mutations in genes involved in histidine synthesis. These strains are auxotrophic mutants (his-), they require histidine for their growth. The method tests the capability of the tested substance in creating his-→his+ mutation that result in his+ revertants, these revertant cells can grow on a histidine-free medium or histidinelimited medium. So, the amount of histidine or histidine-related compounds in the test media is a main factor influencing the results of the standard Ames test [12,13]. If the tested samples containing histidine, external his+ revertants would potentially occur due to the growth-promoting capacities of histidine in the tested samples, and therefore giving a positive false result [3-9]. Traditional Chinese medicines (TCM) are histidine-containing biological samples, their water extracts are commonly taken orally. Many prepared Chinese medicines are mainly composed of the water extracts of TCMs. Such water extracts of TCMs are complex mixtures containing a series of known and unknown chemical compounds. When evaluating the mutagenicity of such TCMs’ water extracts using standard Ames test, false-positive results may get, too [8,12-18]. So that, the interference of histidine and its precursors of the samples must be eliminated or reduced, and the Ames test be modified. Modifications of Ames Test for Assessing the Mutagenicity of TCMs In order to avoid false-positive results owing to the histidine and its precursors in tested samples, a variety of modification strategies have been developed. Excepting some researchers using other bacterial organisms and other mutations to assess the mutagenicity, such as the forward mutation to 8-azaguanine resistance in Salmonella typhimurium TM6779, [19] reverse mutation in Escherchia coli tryptophan-deficient strain WP2, [4] or neomycin-resistant mutation in Vibrio harveyi, [20,21] the following sections focus on the modifications about Ames test. The first modification is the “washing” method [19,22-26]. The common step of these methods is washing the bacteria free of test compound after a exposure prior to plating out on minimal plates, in order to avoid false positive responses and overgrowth of the background bacterial lawn resulting from the free histidine contained in the samples, while producing clear positive responses with appropriate positive controls. For example, the "treat and wash" method by Thompson et al. in 2005, [22] washing the bacteria free of test compound after a 90 min exposure prior to plating out on minimal plates. The shortcoming of these methods is the pre-exposure time with the test samples is not long enough to let the mutagenic components in the samples fully display their mutagenic capacity. The second modification is using extraction methods to remove histidine as well as its precursors from tested samples, [4,9,27,28] and the extraction methods usually use sorbants and solvents, such as alcohols, esters and aliphatic hydrocarbons [29,30]. This is not suitable for TCMs, because some active ingredients in TCMs may lose during the extraction process, and therefore the final test results may be misinterpret, because the mutagenicity of the lost active compositions of TCMs will be unknown [12,18]. The third modification is a "modified plate" method [12-16,18]. The histidine concentration of the tested samples needs be determined previously. Then, a series of modified negative control groups are set. In each control group, same amount of histidine corresponding to the Journal of Clinical Toxicology Jin, J Clin Toxicol 2016, 6:2 http://dx.doi.org/10.4172/2161-0495.1000285 Review Article open access J Clin Toxicol ISSN:2161-0495 JCT, an open access Volume 6 • Issue 2 • 1000285 test sample concentrations is added into the test media. So, the Mutagenicity may be estimated by comparing the ratio of the number of his+ revertants from experimental group to those from the modified negative control group. Becuase the histidine in the experimental group and modified negative control group are same, and thereby the interference resulted from the histidine of the tested samples can be eliminated or reduced. The forth modification is a "suspension assay", reported by Verhagen et al. in 1994, [21] and promoted by Jin et al. [12,18]. These methods are based on a hypothesis that the growth-promoting capacities of histidine in the tested samples might be overcome, by using very rich growth conditions and thereby overwhelming any growth-enhancing constituents present in a tested samples. The fifth modification is a "modified suspension assay using a new judge criterion"-relative reversion frequency (RRF) [17]. The reversion frequency is the ratio of the number of his+ revertants to the total cells showed as colony forming units (CFUs). The relative reversion frequency is the ratio of the number of his+ revertants to the total cells showed as OD600nm of the suspension. Here, rich growth conditions are used, and the growth-promoting capacities of histidine in tested samples can be overcome, because both the numerator (his+ revertants) and denominator (CFU or OD600nm) of RRF increase with similar times. Comparing the Modifications of Ames Test In my opinion, for assessing the mutagenecities of TCMs, the first type of modification, i.e. “treat and wash” method, may increase the probability to get false negative results. Because the short exposure period maybe impede the mutagenic components in the samples to fully display their mutagenic capacity. The second type of modification, i.e. using extraction methods to remove histidine from tested samples, may increase the probability to get false negative results, too. The third type of modification, i.e. a "modified plate" method, is not directly removing histidine from tested samples, but reducing the interference of histidine on the final Ames test results by addition a set of negative control experiments, so this method is somewhat laborious. In both of the forth and the fifth type of modifications, rich growth conditions is used, and the growth-promoting capacities of histidine in tested samples can be overcome. These methods have good repeatability, should be recommended to use in the related researches in the future.