Metabolism of tibolone and its metabolites in uterine and vaginal tissue of rat and human origin.

In postmenopausal women, tibolone shows clear tissue differences in its stimulatory effects on the vagina and uterus. In rats, however, it has stimulatory effects on both tissues, with a different, more estrogenic, effect on the uterus than in humans. This may be due to differences in local metabolism. Therefore, in the present study, the metabolism of tibolone was analyzed in incubations of uterine and vaginal tissue from postmenopausal women and ovariectomized rats using radiolabeled tibolone in order to understand the tissue- and species-specific metabolism. In the rat, tibolone (50 nM) was mainly 3alpha-reduced to the estrogenic 3alpha-OH-tibolone in the uterus and vagina. The 3beta-OH tibolone can be isomerized to 3alpha-OH-tibolone with tibolone as intermediate. In contrast, in the same tissues from postmenopausal women, the progestagenic Delta4-isomer and estrogenic 3beta-OH-tibolone were the major metabolites of tibolone. The formation of the Delta4-isomer was higher in uterine tissue. The 3beta-hydroxysteroid dehydrogenase (HSD) inhibitor epostane had no effect on tibolone metabolism in human uterine and vaginal tissue microsomes and HEK293 cells expressing the human 3beta-HSD types 1 and 2 isoforms did not metabolize tibolone. Moreover, the 3beta-reduction of tibolone to 3beta-OH-tibolone was NADPH dependent, while the isomerization of tibolone to the Delta4-isomer did not require a cofactor. It was therefore concluded that human 3beta-HSD isoforms are not involved in the metabolism of tibolone, and that the 3beta-reduction and the Delta5-10 to Delta4 isomerization may be catalyzed by different enzymes. In conclusion, we showed that, in hormone therapy target tissues of the rat as compared with the human, different metabolic pathways for tibolone exist and therefore result in metabolites with different pharmacological properties. The rat is therefore a poor model to predict the effects of tibolone on the uterus in postmenopausal women.

Metabolism of norethisterone and norethisterone derivatives in rat uterus, vagina, and aorta.

The 19-nor-progestogen norethisterone is used as a progestogen component in contraceptives and in continuous- and sequential combined hormone replacement therapy (HRT) in postmenopausal women. Metabolism of norethisterone in HRT target tissues may play a role in its biological response. The aim of this study was to investigate which steroid-metabolizing enzymes are present in rat uterus, vagina, and aorta, three HRT target tissues. Next, the ability of the tissues to metabolize norethisterone was assessed. Furthermore, to investigate the effect of substituents at the 7- and 11-position, the metabolism of Org OM38 (7alpha-methyl-norethisterone), Org 4060 (11beta-ethyl-norethisterone), and Org 34694 (7alpha-methyl,11-ethylidene-norethisterone) was studied. Using radiolabeled progesterone, the presence of 20alpha-hydroxysteroid dehydrogenase, 5alpha-reductase, and 3alpha-hydroxysteroid dehydrogenase activity could be demonstrated in uterus, vagina, and to a lesser extent in aorta. The combined action of the latter two enzyme activities resulted in 3alpha-OH,5alpha-H-norethisterone as the major metabolite of radiolabeled norethisterone in uterus (26.9%), vagina (37.1%), and aorta (1.4%). The norethisterone derivatives, however, were metabolized to a much lesser extent (1.0-7.6%). No formation of 5alpha-reduced forms of Org 4060, Org OM38, or Org 34694 was found, while formation of minor amounts of 3alpha-OH-Org 4060 and 3alpha-OH-Org OM38 could be demonstrated in both uterus, vagina, and aorta. These findings confirm the role of 5alpha-reductase as a rate-limiting step in the metabolism of norethisterone derivatives and show important inhibitory effects of substituents at the 7alpha- and 11-position of the steroid skeleton on 5alpha-reduction.

Metabolism of estradiol, ethynylestradiol, and moxestrol in rat uterus, vagina, and aorta: influence of sex steroid treatment.

Estrogen replacement therapy for postmenopausal women consists of an estrogenic and a progestagenic compound. The treatment has a positive estrogenic effect on bone, the cardiovascular system, and vagina but is dependent of the estrogen-progestagen balance in uterus to prevent unwanted proliferation. We were interested in the influence of estrogens and progestagens on estrogen metabolism in target tissues of estrogen replacement therapy. Therefore, we studied the metabolism of estradiol, 17alpha-ethynylestradiol, and moxestrol (11beta-methoxy-17alpha-ethynylestradiol) in rat uterus, vagina, and aorta. In uterus and vagina, estradiol was converted to estrone, estradiol-3-glucuronide, and estrone-3-glucuronide. These metabolites demonstrate the presence of 17beta-hydroxysteroid dehydrogenase (17beta-HSD) and UDP-glucuronosyl transferase (UDP-GT) in uterus and vagina. We found that the conversion of estradiol by 17beta-HSD in uterus was increased in animals treated with estradiol or with a combination of estradiol and progesterone. The conversion of estradiol in uterus by UDP-GT was estradiol-induced and in contrast, progesterone-suppressed. In the vagina, steroid hormone treatment had no effect on estradiol conversion by 17beta-HSD or UDP-GT. Ethynylestradiol was glucuronidated only, and this was not affected by steroid treatment. Moxestrol was not converted in any of the three organs that were studied, indicating that the 11beta-methoxy substituent renders it a poor substrate for glucuronidation. Overall, the estrogen metabolism, and its regulation by sex steroids, in rat uterus is different compared with human uterus. Therefore, the rat may not be the best-suited model to investigate uterine effects of estradiol-progestagen combined treatment.

Profound variation in dihydropyrimidine dehydrogenase activity in human blood cells: major implications for the detection of partly deficient patients.

Dihydropyrimidine dehydrogenase (DPD) is responsible for the breakdown of the widely used antineoplastic agent 5-fluorouracil (5FU), thereby limiting the efficacy of the therapy. To identify patients suffering from a complete or partial DPD deficiency, the activity of DPD is usually determined in peripheral blood mononuclear cells (PBM cells). In this study, we demonstrated that the highest activity of DPD was found in monocytes followed by that of lymphocytes, granulocytes and platelets, whereas no significant activity of DPD could be detected in erythrocytes. The activity of DPD in PBM cells proved to be intermediate compared with the DPD activity observed in monocytes and lymphocytes. The mean percentage of monocytes in the PBM cells obtained from cancer patients proved to be significantly higher than that observed in PBM cells obtained from healthy volunteers. Moreover, a profound positive correlation was observed between the DPD activity of PBM cells and the percentage of monocytes, thus introducing a large inter- and intrapatient variability in the activity of DPD and hindering the detection of patients with a partial DPD deficiency.

Development of human cytochrome P450-expressing cell lines: application in mutagenicity testing of ochratoxin A.

With the aim to assess the involvement of distinct forms of cytochrome P450 enzymes in the activation of procarcinogens, we have developed by means of retroviral infection a series of NIH/3T3 cell lines stably expressing human CYP1A1, CYP1A2, CYP2C10, CYP2D6, and CYP2E1 cDNA. The levels of cytochrome P450 enzyme activities were determined using specific substrates. An increase in specific catalytic activity could be observed in all cell lines compared to background activity in vector-infected cells. Furthermore, we developed a test system in which we are able to combine P450-expressing cells with a shuttle vector containing the lacZ' gene, which serves as a reporter gene for mutations. Using this system, we investigated the cytotoxicity and mutagenicity of the mycotoxin ochratoxin A. Natural occurrence of ochratoxin A in food commodities has been linked to an increased incidence of urinary tract tumors in certain geographic regions. Although biotransformation seems to play a crucial role in ochratoxin A toxicity, the possible contribution of metabolites to genotoxicity and carcinogenicity remained unelucidated. We have demonstrated that the mutation frequency of ochratoxin A was increased dependent upon concentration in NIH/3T3 cell lines, stably expressing human CYP1A1, CYP1A2, CYP2C10, and CYP3A4. In contrast, neither in vector-infected NIH/3T3 cells nor in CYP2D6- and CYP2E1-expressing cells was an increase of mutation frequency observed.

Toxicity and metabolism of ochratoxin A.

The frequent occurrence of ochratoxin A (OA) in food and feed commodities and the high incidence of human exposure, as confirmed by different surveillance studies initiated several investigations devoted to elucidating the molecular mechanisms underlying OA toxicity. Previous studies indicated that the primary effects of OA are the inhibition of tRNA synthetase, the inhibition of mitochondrial respiration, and a disturbance of intracellular calcium homeostasis inherent to lipid peroxidation processes. We here report the effect of OA on a number of toxicological endpoints including cytotoxicity in different cell lines and effects on macromolecular synthesis and cell proliferation in primary cultures of hepatocytes at concentrations corresponding to overall exposure levels. These studies provide evidence that prominent toxicological effects might be linked to biotransformation processes. Analysis of hepatic biotransformation resulted in the detection of a number of distinct stable OA-metabolites. As metabolic activation has also been identified as an essential step in OA mutagenicity, the biological relevance on this mechanistic data is discussed.