In silico approaches to predicting cancer potency for risk assessment of genotoxic impurities in drug substances.

The current risk assessment approach for addressing the safety of very small concentrations of genotoxic impurities (GTIs) in drug substances is the threshold of toxicological concern (TTC). The TTC is based on several conservative assumptions because of the uncertainty associated with deriving an excess cancer risk when no carcinogenicity data are available for the impurity. It is a default approach derived from a distribution of carcinogens and does not take into account the properties of a specific chemical.

Comparison of gene expression changes induced in mouse and human cells treated with direct-acting mutagens.

Exposure to DNA-damaging agents can elicit a variety of stress-related responses that may alter the gene expression of numerous biological pathways. We used Affymetrix microarrays to detect gene expression changes in mouse lymphoma (L5178Y) and human lymphoblastoid (TK6) cells in response to methyl methanesulfonate (MMS; a prototypical alkylating agent) and bleomycin (a prototypical oxidative mutagen). Cells were treated for 4 hr, and RNA was isolated either at the end of the treatment or after a 20-hr recovery period.

Variation in photodynamic efficacy during the cellular uptake of two phthalocyanine photosensitizers.

A decrease in the efficacy of photodynamic therapy (PDT) with phthalocyanine photosensitizers was observed for lymphoblastic murine and human cell lines as the time between the addition of the photosensitizer, aluminum phthalocyanine (AIPc), to the culture medium and exposure to light was increased from 4 h to 18 h. The total intracellular concentration of photosensitizer did not decrease significantly during this 18 h interval. For the murine cell lines, the maximum cytotoxic and mutagenic effects were observed when the time between addition of the photosensitizer and irradiation was between 1 and 4 h.

Mutagenicity of photodynamic therapy as compared to UVC and ionizing radiation in human and murine lymphoblast cell lines.

The mutagenicity of photodynamic therapy (PDT) using red light and either Photofrin (porfimer sodium) (PF) or aluminum phthalocyanine (AlPc) as the photosensitizer was determined at the thymidine kinase (TK) locus in the human lymphoblastic cell lines, TK6 and WTK1, and was compared to the mutagenicity of UVC and X-radiation in these cells as well as the mutagenicity of PDT in murine L5178Y lymphoblastic cell lines. Photodynamic therapy was found not to be mutagenic in TK6 cells, which possess an active p53 gene and which are relatively deficient in recombination and repair of DNA double-strand breaks. In contrast, PDT with either sensitizer was significantly mutagenic in WTK1 cells, which harbor an inactivating mutation in the p53 gene and are relatively efficient in recombination and double-strand break repair as compared to TK6 cells.

Large mutagenic lesions are induced by photodynamic therapy in murine L5178Y lymphoblasts.

Mutagenic lesions at the thymidine kinase locus (tk) in mouse lymphoma L5178Y (LY) cells treated with red light and either Photofrin (PF) or chloroaluminum phthalocyanine (AlPc) as the photosensitizer were compared in the relatively photodynamic therapy (PDT)-sensitive strain LY-R16 and the relatively resistant strains LY-S1 and LY-SR1. Southern blot analysis revealed that 92% (36/39) of the PDT-induced thymidine kinase (TK-/-) mutants of strains LY-R16 and LY-SR1 lost the entire active tk allele. (Strain LY-S1 lacks a known tk polymorphism and has not been analyzed for loss of the active tk allele.

Activity of 1,1,1- and 1,1,3-trichloroacetones in a chromosomal aberration assay in CHO cells and the micronucleus and spermhead abnormality assays in mice.

1,1,1- and 1,1,3-trichloroacetones (TCA) result from the disinfection of municipal water supplies with chlorine, and are direct-acting mutagens in the Ames/Salmonella assay. The objective of this study was to further investigate the genotoxicity of these compounds in mammalian cells using an in vitro chromosomal aberration assay in Chinese hamster ovary (CHO) cells and the micronucleus and spermhead abnormality assays in mice. Both compounds induced significant increases in structural chromosomal aberrations in CHO cells in the presence and in the absence of rat S9 metabolic activation (MA).