The mutational signature of alpha-hydroxytamoxifen at Hprt locus in Chinese hamster cells.

Abstract

The anti-oestrogen tamoxifen is very effective in the treatment and prevention of breast cancer. Tamoxifen is not a pure antagonist, but possesses weak oestrogenic activity that may contribute to a slightly increased risk of endometrial cancer. Whilst this can be incorporated into risk-benefit analysis for the use of the drug, residual concerns exist over the exact mechanism of formation of these tumours. Tamoxifen is a potent hepatocarcinogen in the rat, probably via a genotoxic mechanism. Whilst tamoxifen does not appear to cause liver tumours in humans, DNA adducts have been found in endometrial tissue of women receiving the drug. Hence, there is still a need to establish the mechanism of formation of these tumours. We have therefore determined the molecular nature of mutations induced in vitro by alpha-hydroxytamoxifen, the putative proximate genotoxic metabolite, in a mammalian cell line (V79-rHSTa) with stable expression of rat hydroxysteroid sulfotransferase a, which catalyses the further metabolism of alpha-hydroxytamoxifen to its ultimate genotoxic product. DNA sequence alterations were examined at the Hprt gene in 50 mutant clones. Simple base substitutions, mainly GC-->TA transversions, predominated. However, single G:C base pair deletions and partial/complete exon skippings were also observed. All but one of the mutations involved guanine bases on the non-transcribed strand, probably indicating preferential repair of alpha-hydroxytamoxifen-induced guanine adducts from the transcribed strand. Nearest neighbour analysis of the mutations (on the non-transcribed strand) indicated that thymines (20/40) followed by guanines (13/40) were the most frequent 5' neighbours, with adenines or guanines the most frequent 3' neighbours. Many of the mutations occurred at TTGA/G sequences. Three mutational hot spots accounted for 11 GC-->TA transversions and another site for two single G:C base pair deletions. A search for these characteristic mutations in tumour-related genes of treated rats and humans should help in understanding the mechanism(s) of tamoxifen-induced carcinogenicity.