Although the application of the concept of a threshold to risk assessment is widespread, there remains little experimental evidence for the existence of thresholds for genotoxic compounds, other than aneugens. The clastogenicity of topoisomerase inhibitors is believed to result from the transient stabilization of the topoisomerase enzyme with DNA during the catalytic cycle. This leads to the formation of a stabilized cleavage complex, which, in turn, may result in the formation of a DNA strand break. This indirect mechanism of clastogenicity is the basis for the concept of threshold for this class of drug. Using micronucleus induction in L5178Y mouse lymphoma cells as a genotoxic end-point, a three pronged approach was used to examine whether the concept of a threshold for clastogenicity could be demonstrated for topoisomerase type II inhibitors in vitro. This involved (i) the study of mechanism (TARDIS assay), (ii) hypothesis testing versus estimation (i.e. scoring up to 10,000 cells/treatment at concentrations immediately above and below the NOEL for micronucleus induction) and (iii) statistical modelling of the concentration-response curves for micronucleus induction. Several topoisomerase type II inhibitors were investigated with varying clastogenic potencies (etoposide = doxorubicin < genistein < ciprofloxacin). Pragmatic thresholds for clastogenicity in L5178Y cells were defined at 0.00236 microg/ml for etoposide, 0.00151 microg/ml for doxorubicin, 1 microg/ml for genistein and 50 microg/ml for ciprofloxacin. In addition, it was demonstrated that etoposide-induced clastogenicity was concentration and time dependent. These results, along with mechanistic data showing that all of the compounds induced concentration-dependent increases in the formation of topoisomerase II stabilized cleavage complexes, provide a weight of evidence to support a threshold concept for clastogenicity with topoisomerase II poisons.
The GADD45a-GFP (GreenScreen HC) reporter assay detects genotoxic damage in the human lymphoblastoid TK6 cell line and gives positive results for all classes of genotoxin, including mutagens, aneugens and clastogens. In this study, a collection of 75 marketed pharmaceuticals were tested in the assay. Compounds in the collection represent a broad range of chemical structures, pharmacologies and therapeutic indications, including neoplasia and viral infection where positive genotoxicity results are often associated with the pharmacological activity. Based on the results of this study, two main conclusions can be drawn: (i) the GreenScreen HC is more predictive of in vivo genotoxicity (88%) and genotoxic carcinogenicity (93%) data than the any of the other regulatory in vitro genotoxicity assay and (ii) no compounds were uniquely positive in the GADD45a-GFP assay. This analysis therefore provides additional evidence to support the use of the GADD45a-GFP assay as an effective tool either in early genotoxic liability identification or non-clinical safety assessment of candidate pharmaceuticals during development.
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