Identification of mutagenic heterocyclic amines (IQ, Trp-P-1 and AalphaC) in the water of the Danube river.

Three mutagenic heterocyclic amines, 2-amino-3-methylimidazo-[4, 5-f]quinoline (IQ), 3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indole (Trp-P-1) and 2-amino-9H-pyrido[2,3-b]indole (AalphaC), were isolated and identified in water from the Danube River in Vienna. Heterocyclic amines were extracted from river water by the blue rayon hanging method, and analyzed by gas chromatography with a nitrogen-phosphorous detector (GC-NPD) and GC-mass spectrometry (GC-MS) after conversion into their N-dimethylaminomethylene derivatives. Identity of IQ, Trp-P-1 and AalphaC in the river water was confirmed by GC-MS. The contents of IQ, Trp-P-1 and AalphaC were estimated by GC-NPD at 1.78+/-0.17, 0.14+/-0.02 and 0.44+/-0.02 ng/g blue rayon equivalent (n=3), respectively. The total amounts of these amines accounted for 26% of the mutagenicity of blue rayon extracts evaluated by the Ames test using TA98 with metabolic activation.

Use of metabolically competent human hepatoma cells for the detection of mutagens and antimutagens.

The human hepatoma line (Hep G2) has retained the activities of various phase I and phase II enzymes which play a crucial role in the activation/detoxification of genotoxic procarcinogens and reflect the metabolism of such compounds in vivo better than experimental models with metabolically incompetent cells and exogenous activation mixtures. In the last years, methodologies have been developed which enable the detection of genotoxic effects in Hep G2 cells. Appropriate endpoints are the induction of 6-TGr mutants, of micronuclei and of comets (single cell gel electrophoresis assay). It has been demonstrated that various classes of environmental carcinogens such as nitrosamines, aflatoxins, aromatic and heterocyclic amines and polycyclic aromatic hydrocarbons can be detected in genotoxicity assays with Hep G2 cells. Furthermore, it has been shown that these assays can distinguish between structurally related carcinogens and non-carcinogens, and positive results have been obtained with rodent carcinogens (such as safrole and hexamethylphosphoramide) which give false negative results in conventional in vitro assays with rat liver homogenates. Hep G2 cells have also been used in antimutagenicity studies and can identify mechanisms not detected in conventional in vitro systems such as induction of detoxifying enzymes, inactivation of endogenously formed DNA-reactive metabolites and intracellular inhibition of activating enzymes.

Development of test systems for the detection of compounds that prevent the genotoxic effects of heterocyclic aromatic amines: preliminary results with constituents of cruciferous vegetables and other dietary constituents.

Over the past decades, strong efforts have been made to identify dietary constituents that protect against the genotoxic effects of heterocyclic aromatic amines (HAAs). However, most of the methods that have been used, in particular in vitro assays that require the addition of exogenous enzyme homogenates, have only a limited predictive value because important protective mechanisms are not adequately represented and may give misleading results. Therefore, we attempted to develop improved test systems, namely assays, with human hepatoma cells and single-cell gel electrophoresis (SCGE) tests with rats. Genotoxicity tests with human derived Hep G2 cells reflect the genotoxic effects of HAAs better than other in vitro systems. They also enable the detection of protective effects since the human derived hepatoma cells possess phase I and phase II enzymes that are involved in the activation/ detoxification of the amines. The most appropriate endpoint for experiments with Hep G2 cells appears to be micronucleus induction, but protocols for other endpoints are available as well. The second promising model is the SCGE ("comet") assay with rats that was used successfully to measure protective effects of constituents of cruciferous vegetables against 2-amino-3-methylimidazo[4,5-flquinoline (IQ) in the liver and in the colon mucosa. The present study describes the experimental design of the new approaches, as well as results obtained with various dietary constituents.

Search for compounds that inhibit the genotoxic and carcinogenic effects of heterocyclic aromatic amines.

Over the last 30 years approximately 160 reports have been published on dietary compounds that protect from the mutagenic and carcinogenic effects of heterocyclic aromatic amines (HAAs). In the first section of this review, the current state of knowledge is briefly summarized. Based on the evaluation of the available data, various protective mechanisms are described, and the use of different methodologies for the detection of protective effects is critically discussed. In most antimutagenicity studies (>70%) bacterial indicators (predominantly Salmonella strain TA98) were used, and about 600 individual compounds and complex mixtures have been identified that attenuate the effects of HAAs. The most frequently used in vivo method to detect protective effects are adduct measurements; anticarcinogenic dietary factors were identified by aberrant crypt foci assays and liver foci tests with rats. The mechanisms of protection include inactivation of HAAs and their metabolites by direct binding, inhibition of enzymes involved in the metabolic activation of the amines, induction of detoxifying enzymes, and interaction with DNA repair processes. The detection spectrum of conventional in vitro mutagenicity assays with metabolically incompetent indicator cells is limited. These procedures reflect only simple mechanisms such as direct binding of the HAAs to pyrroles and fibers. It has been shown that these compounds are also effective in rodents. More complex mechanisms, namely, interactions with metabolic activation reactions are not adequately represented in in vitro assays with exogenous enzyme homogenates, and false-negative as well as false-positive results may be obtained. More appropriate approaches for the detection of protective effects are recently developed test systems with metabolically competent cells such as the human Hep G2 line or primary hepatocytes. SCGE tests and DNA adduct measurements with laboratory rodents enable the detection of antigenotoxic effects in different organs, including those that are targets for tumor induction by the amines. Medium term assays based on aberrant crypt foci in colon and liver foci tests have been used to prove that certain compounds that prevented DNA damage by HAAs also reduced their carcinogenic effects. These experiments are costly and time consuming and, due to the weak induction capacity of the amines, only pronounced anticarcinogenic effects can be detected. Over the years, a large bulk of data on HAA protective compounds has accumulated, but only for a few (e.g., fibers, pyrroles, constituents of teas, and lactic acid bacteria) is there sufficient evidence to support the assumption that they are protective in humans as well.