Metabolic influences for mutation induction curves after exposure to Sudan-1 and para red.

Sudan-1 and para red are industrial dyes that have been illegally added to some foodstuffs, leading to withdrawal of the adulterated products throughout the UK since 2003. This resulted in international concern that arose because Sudan-1 is classified by International Agency for Research on Cancer as a Category 3 carcinogen. However, little is known about the dose response of this chemical at low, more biologically relevant, doses. The study therefore aimed to characterize the dose response for gene mutation and chromosomal damage induced by two azo dyes, namely Sudan-1 and para red. Gene mutations were analysed using the hypoxanthine phosphoribosyltransferase forward mutation assay and chromosomal damage was measured using the cytokinesis-blocked micronucleus assay. Two cell lines were used in these investigations. These were the AHH-1 cell line, which inducibly expresses CYP1A1, and the MCL-5 cell line derived from a subpopulation of AHH-1 cells that expresses a particularly high level of CYP1A1 activity. The MCL-5 cell line has also been transfected with two plasmids that stably express CYP1A2, CYP2A6 and CYP3A4 and all four of these CYP enzymes are known to metabolically activate Sudan-1. AHH-1 cells were used to investigate the dose response of the azo dyes, and MCL-5 cells were used to see if the dose response changed with increased metabolism. Sudan-1 induced a non-linear dose-response curve for gene mutation and chromosomal damage in AHH-1 cells. The genotoxic activity of Sudan-1 was greatly increased in MCL-5 cells. This indicated that the oxidation metabolites from Sudan-1 were both more mutagenic and more clastogenic than the parent compound. Para red also demonstrated a non-linear dose response for both gene mutation and chromosome damage in AHH-1 cells, and an increase in micronuclei induction was observed after increased oxidative metabolism in MCL-5 cells. Sudan-1 and para red are genotoxic chemicals with non-linear dose responses in AHH-1 but not in MCL-5 cells, and oxidative metabolism increases the genotoxic effect of both compounds.

Non-linear dose-response of DNA-reactive genotoxins: recommendations for data analysis.

Until recently, there has only been a limited amount of data available on the kinetics of mutation induction in the low dose region of exposure. In our publication Doak et al. [S.H. Doak, G.J. Jenkins, G.E. Johnson, E. Quick, E.M. Parry, J.M. Parry, Mechanistic influences for mutation induction curves after exposure to DNA-reactive carcinogens, Cancer Res. 67 (2007) 3904-3911] we showed that the two alkylating agents methyl-methanesulfonate (MMS) and ethyl-methanesulfonate (EMS) possess non-linear dose-response curves with no observed effect levels (NOEL) for mutation or chromosomal damage in vitro. These experiments were carried out in the AHH-1 human lymphoblastoid cell line, using the hypoxanthine phosphoribosyl transferase (HPRT) assay and the cytokinesis-block micronucleus (CBMN) assay, respectively. We have now carried out more advanced statistical analyses to define threshold values, which is critical as it has a dramatic impact on hazard and risk assessment. To do this, we re-analysed the data to see if the linear model or a more complex model (hockey stick or quadratic) gave a significant better fit of the data. For both EMS and MMS cytokinesis-block micronucleus data sets, the hockey stick model gave the most significant fit. The same was true for EMS, MMS and surprisingly ethylnitrosourea (ENU) in the HPRT assay in human AHH-1 cells. However, methylnitrosourea (MNU) was linear in both assays. These further analyses have shown that EMS and MMS have clear thresholds for both gene mutation and chromosome damage, as does ENU for gene mutation in AHH-1 cells. MNU was linear for gene and chromosome mutation and so was ENU for chromosome mutations at the concentrations tested. These findings correlate closely with those in vivo findings of Gocke et al. [E. Gocke, L. Müller, In vivo studies in the mouse to define a threshold for the genotoxicity of EMS and ENU, Mutat. Res. (this issue)] and together these data show a true threshold for EMS both in vitro and in vivo. In this report, we will discuss the approaches that were taken to investigate potential threshold dose-response curves for DNA-reactive genotoxic compounds, with recommendations for further studies.

No-observed effect levels are associated with up-regulation of MGMT following MMS exposure.

The alkylating agents methyl methanesulphonate (MMS) and ethyl methanesulphonate (EMS) have non-linear dose-response curves, with a no-observed effect level (NOEL) and a lowest observed effect level (LOEL) for both gross chromosomal damage and mutagenicity. However, the biological mechanism responsible for the NOEL has yet to be identified. A strong candidate is DNA repair as it may be able to efficiently remove alkyl adducts at low doses resulting in a NOEL, but at higher doses fails to fully remove all lesions due to saturation of enzymatic activity resulting in a LOEL and subsequent linear increases in mutagenicity. We therefore assessed the transcriptional status of N-methylpurine-DNA glycoslase (MPG) and O(6)-methylguanine DNA methyltransferase (MGMT), which represent the first line of defence following exposure to alkylating agents through the respective enzymatic removal of N7-alkylG and O(6)-alkylG. The relative MPG and MGMT gene expression profiles were assessed by real-time RT-PCR following exposure to 0-2 microg/ml MMS for 1-24h. MPG expression remained fairly steady, but in contrast significant up-regulation of MGMT was observed when cells were treated with 0.5 and 1.0 microg/ml MMS for 4h (2.5- and 6.5-fold increases respectively). These doses lie within the NOEL for MMS mutagenicity (LOEL is 1.25 microg/ml), thus this boost in MGMT expression at low doses may be responsible for efficiently repairing O(6)methylG lesions and creating the non-linear response for mutations. However, as the LOEL for MMS clastogenicity is 0.85 microg/ml, O(6)-alkylG is unlikely to be responsible for the clastogenicity observed at these concentrations. Consequently, at low doses N7-methylG is possibly the predominant cause of MMS clastogenicity, while O(6)-methylG is more likely to be responsible for MMS mutagenicity, with MGMT up-regulation playing a key role in removal of O(6)-alkylG lesions before they are fixed as permanent point mutations, resulting in non-linear dose-responses for direct acting genotoxins.

Do oestrogens induce chromosome specific aneuploidy in vitro, similar to the pattern of aneuploidy seen in breast cancer?

The study was concerned with investigating the specific effects of non-DNA reactive oestrogens at low "biologically relevant" doses and the causative role they may play in breast cancer through inducing aneuploidy. A review of previous studies identified a non-random pattern of aneuploidy seen in breast cancers. This information was used to select those chromosomes that undergo copy number changes in breast cancer and chromosomes that appear stable. A panel of centromeric specific probes were selected and centromeric specific fluorescence in situ hybridisation (FISH) was carried out on the human lymphoblastoid cell line, AHH-1, which had been pre-treated with the chemical aneugens 17-beta oestradiol, diethylstilbestrol (DES) and bisphenol-A (BP-A). The results suggest that oestrogens may play a causative role in breast cancer by inducing a specific pattern of aneuploidy similar to that seen in breast carcinomas. 17-beta oestradiol appears to induce changes most similar to those seen in breast tumours, BP-A induces the same pattern but at a lower frequency and DES appears to be less chromosome specific in its act.

Mechanistic influences for mutation induction curves after exposure to DNA-reactive carcinogens.

A mechanistic understanding of carcinogenic genotoxicity is necessary to determine consequences of chemical exposure on human populations and improve health risk assessments. Currently, linear dose-responses are assumed for DNA reactive compounds, ignoring cytoprotective processes that may limit permanent damage. To investigate the biological significance of low-dose exposures, human lymphoblastoid cells were treated with alkylating agents that have different mechanisms of action and DNA targets: methylmethane sulfonate (MMS), methylnitrosourea (MNU), ethylmethane sulfonate (EMS), and ethylnitrosourea (ENU). Chromosomal damage and point mutations were quantified with the micronucleus and hypoxanthine phosphoribosyltransferase forward mutation assays. MNU and ENU showed linear dose-responses, whereas MMS and EMS had nonlinear curves containing a range of nonmutagenic low doses. The lowest observed effect level for induction of chromosomal aberrations was 0.85 microg/mL MMS and 1.40 microg/mL EMS; point mutations required 1.25 microg/mL MMS and 1.40 microg/mL EMS before a mutagenic effect was detected. This nonlinearity could be due to homeostatic maintenance by DNA repair, which is efficient at low doses of compounds that primarily alkylate N(7)-G and rarely attack O atoms. A pragmatic threshold for carcinogenicity may therefore exist for such genotoxins.

The detection of genotoxic activity and the quantitative and qualitative assessment of the consequences of exposures.

A wide range of assays are now available which enable the effective detection of the mutagenic (the induction of gene and chromosomal changes) and more generally genotoxic (cellular interactions such as DNA lesion formation) activity of individual chemicals and mixtures. However, when genotoxic activity has been detected and human exposure occurs the critical questions relate to the qualitative and quantitative activity of the agent and the parameters such as routes of exposure, target organs and metabolism. Of major importance in hazard and risk estimation is the nature of the dose response relationship of each chemical and their potential interactions in mixtures. In this paper, we illustrate the methods available to produce quantitative and qualitative data in vitro using the micronucleus assay (as a measure of chromosomal structural and numerical mutations) and the HPRT assay (as a measure of induced gene and point mutations) and the current limitations (such as the large numbers of animals required) for obtaining such information in vivo. We recommend that in vivo studies should primarily focus upon confirmatory mechanistic analysis. For individual chemicals, profiles of the base changes induced can be obtained using the HPRT gene mutation assay and comparisons produced both in vitro and in vivo and thus allow identification of mechanistic differences between different modes of exposure.