Activation of tamoxifen and its metabolite α-hydroxytamoxifen to DNA-binding products: comparisons between human, rat and mouse hepatocytes

The metabolic activation of tamoxifen and its metabolite α-hydroxytamoxifen in primary cultures of rat, mouse and human hepatocytes has been compared. The extent of formation of DNA adducts in these cells was measured by 32P-postlabelling, using either nuclease P1 digestion or sorbent extraction of DNA digests to enhance the sensitivity of the assay. DNA adducts were readily detected in rat hepatocytes treated with 1 or 10 μM tamoxifen (mean levels 18.2 and 89.8 adducts/108 nucleotides respectively). Similar levels of adducts were formed by mouse hepatocytes (15.0 ± 1.8 adducts/108 nucleotides, 10 μM tamoxifen). However DNA adducts were not detected in tamoxifen-treated human hepatocytes with a detection limit for the assay of 4 adducts/1010 nucleotides. Treatment of rat cells with α-hydroxytamoxifen resulted in 15- to 63-fold higher levels of adducts than with comparable concentrations of tamoxifen. A similar level of adducts was also seen in mouse hepatocytes treated with α-hydroxytamoxifen at the 1 μM concentration (173.9 ± 4.1 adducts/108 nucleotides). Treatment of human cells with α-hydroxytamoxifen resulted in DNA adduct formation at levels (1.94 ± 0.89 and 18.9 ± 17.9 adducts/108 nucleotides at 1 and 10 μM respectively) ∼300-fold lower than those in rat hepatocytes. The presence of α-hydroxytamoxifen in the culture medium from experiments where cells were incubated with tamoxifen was monitored by mass spectrometry. Concentrations were found to be ∼50-fold lower in the medium from human hepatocytes than from rat and mouse hepatocytes.