Exposure to pentachlorophenol (PCP) strongly intensifies the formation of mouse hepatic DNA adducts elicited by oral administration of tamoxifen (TAM), as previously shown by 32P-postlabeling. To explain this effect, PCP was proposed to interfere with the detoxication by sulfate conjugation of an as yet unidentified hydroxylated proximate TAM metabolite. A comparison of the present and earlier results shows that the hepatic TAM adduct pattern in female ICR mice depended on the route of administration of TAM (120 mumol/kg), with oral administration primarily eliciting formation of more polar adducts (termed group I adducts), while after i.p. administration less polar adducts (group II) predominated over group I adducts by a factor of 17.5. All these adducts were also formed in female Sprague-Dawley rats after i.p. dosing with TAM, but total adduct levels were 3.5- to 5-fold higher than in mice. After four daily i.p. treatments, TAM adducts accumulated in mouse liver DNA in a non-linear fashion. Adduct levels were 30-50 times lower in mouse kidney and lung than in liver. The phenolic metabolite 4-hydroxy TAM (120 mumol/kg) exclusively led to formation of polar (group I) hepatic adducts, and this process was stimulated 8-fold by co-administration of PCP (75 mumol/kg). Co-administration of PCP with the parent compound led to an 11-fold enhancement of group I adduct formation; simultaneously, levels of group II adducts were suppressed 6-fold. Another inhibitor of sulfate conjugation, 2,6-dichloro-4-nitrophenol, unlike PCP, had no effect on group I adducts, but it reduced group II adduct formation 2.2-fold. The PCP metabolite 2,3,5,6-tetrachlorohydroquinone (75 mumol/kg) did not significantly affect any major TAM adduct, suggesting that PCP itself was the active compound. Similar to group II TAM adducts, the formation of hepatic safrole-DNA adducts was inhibited in female ICR mice by both sulfotransferase inhibitors, consistent with the proposal that metabolic alpha-hydroxylation of the ethyl group of TAM followed by sulfate conjugation represented a mechanism of TAM activation. On the other hand, the strong intensification of group I adducts by PCP and the lack of this effect by 2,6-dichloro-4-nitrophenol suggested that inhibition of sulfate conjugation may not have been the primary mechanism underlying the intensification of group I adducts formed from TAM or 4-hydroxy TAM. The results presented herein demonstrate conclusively that TAM was activated to DNA-reactive compounds along two distinct pathways which contrasted in their responses to metabolic inhibitors.
Intranasal administration of hormone replacement therapy presents an original plasma kinetic profile with transient estrogen levels giving rise to the concept of pulsed therapy. To further understand the molecular effects of this new therapy, we have compared the effects of pulsed and continuous estradiol treatments on two critical aspects of estradiol action: gene expression and cell proliferation. Cells were stimulated with estradiol as 1-h pulsed or 24-h continuous treatments at concentrations such that the 24-h exposure (concentration×time) was identical in both conditions. In MCF7 cells, the transcriptional activity of estrogen receptors (ER) on a transiently transfected responsive estrogen response element-luciferase reporter construct was shown to be drastically (∼10-fold) and similarly stimulated after both treatments. Moreover, the increased mRNA expression of three representative estradiol-sensitive genes (pS2, cathepsin D, progesterone receptor), evaluated by Northern blot, was identical after 1-h pulse with 7 nM estradiol or continuous treatment with 0·29 nM estradiol with the same kinetic profile over 48 h. Proliferation was quantified by a histomorphometric method on primary cultures of human normal breast cells from reduction mammoplasties and using a fluorescence DNA assay in six human breast cancer cell lines which were ER positive or negative. After a 7-day treatment period, estradiol had no effect on the proliferation of the three ER negative cell lines (BT20, MDA MB231, SK BR3) but significantly stimulated the proliferation of the normal cells and of the three tumoral hormone-sensitive cell lines (MCF7, T47D, ZR 75-1); both hormone treatments producing the same increases in cell growth. In conclusion, we have shown that the genomic or proliferative effects of estradiol were identical with pulsed or continuous treatments, thus indicating that estrogenic effects are not strictly related to concentrations but rather to total hormone exposure.
Although negative in assays for mutagenicity, the clinically important antiestrogen tamoxifen induces hepatic DNA adduct formation in mice, rats and hamsters, as indicated by 32P-postlabeling, and is a potent hepatocarcinogen in rats. Both phenolic and alcoholic metabolites of tamoxifen have been reported. As these metabolites are potential candidates for sulfate conjugation, we examined whether the sulfotransferase inhibitor pentachlorophenol, a ubiquitous environmental contaminant, modulates hepatic tamoxifen adduct formation in vivo. Female ICR mice were given tamoxifen (45 mg/kg) daily per os for up to 4 days, with and without i.p. pretreatment with pentachlorophenol (20 mg/kg) 1 h before dosing with tamoxifen. At days 1, 2 and 4, liver DNA was analyzed 5 h after tamoxifen administration by a modified monophosphate version of the 32P-postlabeling assay. At day 4, pentachlorophenol pretreatment led to a large increase (13- to 17-fold) of the levels of four tamoxifen adduct fractions, while two adducts appeared unaffected, resulting in an approximately 7-fold enhancement of overall adduct formation. Significant pentachlorophenol related increases were also observed at day 1 and day 2. The mechanism of this effect has not yet been determined, but may involve the inhibition of sulfation of a tamoxifen metabolite(s) involved in the detoxication of the drug to nonelectrophilic derivatives. It was also apparent that there are two pathways of metabolic activation of tamoxifen, one being sensitive and the other resistant to pentachlorophenol.
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