Discussion On Methods For The Assessment Of Potential Carcinogenicity Of Environmental Factors

Cancer has been one of the greatest threats to human health;despite the mechanism of its occurrence and formation has not yet been fully explained,a consensus has been already obtained that most cancers are caused due to some environmental factors. Some environmental factors can change the expression levels of some cell growth factors and their receptors,which leads to the increase in mutation rates of DNA and occurrence of cancer cells.Therefore,the investigation on the ability of environmental factors to induce gene mutation is an effective way to estimate their potential to cause cancer.From the mathematical point of view,this involves the probability and statistical inference,that is,the possibility of an incident is estimated based on a seriety of experiment results and data.It is crucial for decreasing the chance of misjudgment to adopt the appropriate detection and analysis methods,and the results from inappropriate methods will inevitably lead to wrong misjudgments.The Ames test is based on the principle that mutagenic compounds can increase the reversion frequencies of histidine-deficient strains,thereby speculating their potential carcinogenicity.The potential carcinogenicity of pure compounds can be well estimated with the Ames test;however,as to complex mixtures,it is inappropriate to adopt the Ames test to monitor their potential carcinogenicity.Similarly,it is inappropriate to adopt the Ames test to monitor the potential carcinogenicity of traditional Chinese medicine because traditional Chinese medicine is a mixture,too.Not long ago,the National Development and Reform Commission issued the high-tech industries development during 11th five-year plan,16 major projects including the 'modem traditional Chinese medicine' project will be implemented.So, whether the mutagenicity of traditional Chinese medicine can be accurately determined will have direct affects on not only the national policy on the traditional Chinese medicine industry,but also the utilization of traditional Chinese medicine. Therefore,it is necessary to develop a novel mutagenicity detection method for the complex mixtures including traditional Chinese medicine.The bacterial reversion assays such as the Ames test were affected by multiple factors;at the same time,the environmental factors can change not only a specific gene but also many other genes,which is related to specificity or random.Therefore, the mutation detection method at DNA level may be a way to solve the above issues.The study in this paper started with these problems mentioned above,and the following results were obtained in the past three years.1.Analysis of the factors leading to uncertainty of Ames test resultsThe Ames test is used world-wide as an initial screen to determine the mutagenic potential of new chemicals and drugs;however it involves many uncertain factors. The estimation of the spontaneous and induced mutation rate of a microbial group is complex from the point of view of the genetic theory and experimental technique, which involves a number of factors.The estimation of induced mutation rate is related to the mixing of two normal distributions,which needs lots of samples;unfortunately, the number of samples in the Ames test is small,as a result the parameter of the mixed distribution can not be estimated with conditional moment closure model.1.1 Only small-scale mutations such as base substitution and one or two nucleotidesinsertion or deletion can be detected by the tester strains used in the Ames test,as a result many other types of mutation can not be monitored.1.2 Only the mutations occurring at specific locuses such as hiG46 locus can be monitored,many more mutations occurring at other locuses can not be detected with the Ames test. 1.3 The Ames test is based on the minimal medium,which made this method unsuitable for the detection of mutagenicity of nutrient-rich materials. Additionally,this barren environment might also lead to the adaptive mutation, which much more enhanced the uncertainty of test results.1.4 The revertant colony numbers are the function of incubation time,so the time to count the revertant colony numbers(48 h) is relatively arbitrary,lacking of scientific connotation.1.5 Various types of revertants grew at different rates in the selective medium; thereby the revertants with a higher growth rate were probably counted compared with those with lower growth rates.That is to say,not all the revertants could be counted,which was a vital defect to the Ames test.2.A novel potential eareinogenieity detection method for traditional Chinese medicineThe Ames test is based on the test system that contains trace amounts of histidine (15.52μg/plate),and the amounts ofhistidine in the test groups and the control groups are same.Many traditional Chinese medicines were estimated to be mutagenic with the Ames test,but these results on the mutagenicity of these traditional Chinese medicines might be questionable due to considerable amounts of histidine in them. Several traditional Chinese medicines were estimated with the Ames test,and all of them were found to be mutagenic.However,these samples were nonmutagenic after deducting the revertant numbers resulting from the histidine in the samples.Additional excessive external histidine did not affect the growth of TA100 when TA100 was cultured in LB broth;furthermore,dose-response relationships occurred between the mutagen concentrations and the relative reversion frequencies of the tester strains during the declining phase.According to these results,a novel strategy based on liquid LB broth to estimate the mutagenicity of samples containing much free and/or protein-bound histidine was developed.This strategy could be expected to avoid the false-positive results when adopting the Ames test or other methods to estimate the mutagenicity of these samples containing considerable amounts of histidine.The mutagenicity of these traditional Chinese medicines was assayed with the new method.The RRFs of tester strains in all test groups were not significantly beyond those in the negative control groups(t test;p＞0.05);furthermore,no dose-response relationships appeared between the RRFs of tester strains and the concentrations of traditional Chinese medicine.Thus,the results in the new method indicated that all these traditional Chinese medicines were nonmutagenic.These traditional Chinese medicines were also proved to be nonmutagenic with mammalian bone marrow chromosome aberration test.These results were in accordance with the results in the new method,which suggested that the new method was effective to monitor the mutagenicity of traditional Chinese medicine.3.A novel mutation detection assay based on MutS and real-time fluorescent quantitative PCR assayMutS is known to be an important component of mismatch repair(MMR) systems and play a vital role in maintaining the fidelity of genetic information in living cells. In vivo,it specifically recognizes and binds all the base pair mismatches and some small-scale insertion/deletion mutations.However,under in vitro conditions,MutS does not bind specifically to heteroduplex DNA.Whether MutS binds to homoduplex DNA or not is closely correlated with the molar ratio of MutS to DNA.A novel,sensitive mutation detection strategy was developed by performing real-time fluorescent quantitative PCR assay and utilising the binding property of MutS.The mutants as low as 2 percent among the normal cells can be detected with this method and this method can be applied to detect the unknown mutations within the whole genome.4.Establishing a detection method for low-proportion mutantsA variety of methods such as DNA sequencing and SSCP for detecting DNA fragment polymorphism and mutation were established,but these methods can not be adopted to detect low-proportion mutants for lacking of high sensitivity.Some other methods such as DNA chip can be used to detect the low-proportion mutants,but these methods are not widely adopted because of high costs.In this study, based on the difference in the denaturation temperatures between heteroduplex DNA and homoduplex DNA,a novel PCR procedure was proposed to improve the relative and absolute contents of mutant.Afterwards,samples were analyzed by single-strand conformation polymorphism with capillary electrophoresis and laser-induced fluorescence detector(CE-LIF-SSCP).Some low-proportion mutants were believed to be monitored by this method.