Species-specific pharmacology of antiestrogens: role of metabolism

The nonsteroidal antiestrogen tamoxifen exhibits a paradoxical species-specific pharmacology. The drug is a full estrogen in the mouse, a partial estrogen/antiestrogen in humans and the rat, and an antiestrogen in the chick oviduct. Inasmuch as tamoxifen has antiestrogenic effects in vitro, differential metabolism of tamoxifen to estrogens might occur in the species in which it has an estrogenic pharmacology. Tamoxifen or its metabolite 4-hydroxytamoxifen could lose the alkylaminoethane side chain to form the estrogenic compound metabolite E or bisphenol. Sensitive metabolic studies with [3H]tamoxifen in chicks, rats, and mice identified 4-hydroxytamoxifen as the major metabolite, but no potentially estrogenic metabolites were observed. Athymic mice with transplanted human breast tumors can be used to study the ability of tamoxifen to stimulate target tissue or tumor growth. Estradiol caused the growth of transplanted MCF-7 breast cancer cells into solid tumors and a uterotrophic response. However, tamoxifen does not support tumor growth when administered alone, although it stimulates uterine growth. Since a similar profile of metabolites is sequestered in human and mouse tissues, these studies strongly support the concept that the drug can selectively stimulate or inhibit events in the target tissues of different species without metabolic intervention.     

Tamoxifen and vitamin E treatments delay symptoms in the mouse model of Niemann-Pick C

Niemann-Pick C disease (NPC) is an irreversible neurodegenerative disorder without current treatment. It is the result of deficient intracellular cholesterol movement. We investigated the effects of tamoxifen and vitamin E (D-alpha tocopherol) treatment on patterns of weight loss and motor function in the mouse model of Niemann-Pick C disease (Npc1-/- mice). Tamoxifen has multiple metabolic effects, including reducing oxidative damage, while vitamin E primarily has this property. Npc1-/- mice were identified and treatment was initiated at an approximate age of 21 days. Tamoxifen suspended in peanut oil was administered via intraperitoneal injection (weekly, at a dose calculated to deliver 0.023 microg/g/day). Vitamin E (25 IU) was administered orally via gavage once a week. Weight loss and Rota-Rod performance were analyzed by using Kaplan-Meyer survival curves. Tamoxifen treatment by itself significantly delayed weight loss (an endpoint of neurodegeneration) in male and female mice compared to untreated controls. Motor function was evaluated by performance on a Rota-Rod. Tamoxifen maintained Rota-Rod performance for about an extra week. Vitamin E treatment significantly delayed weight loss in females only. Rota-Rod performance was maintained slightly longer in mice treated with vitamin E. Simultaneous use of both treatments did not delay weight loss longer than tamoxifen-only treatment but had a greater effect than either treatment alone on Rota-Rod performance and demonstrated a significant positive effect on the early "learning curve" portion of the Rota-Rod evaluations. We found significant but relatively small improvements in rate of disease progression by treating Npc1-/- mice with tamoxifen and/or vitamin E. Some sex differences in response and an early improvement in Rota-Rod performance suggest areas for further study.     

Possibility of anti-estrogen action from estriol specific binding sites in rabbit uterus

1. This study was designed to investigate the clomiphene or tamoxifen binding to receptor sites for estradiol-17 beta (E2R) and estriol (E3R) in the rabbit uterus. 2. Those so-called anti-estrogenic compounds tended to inhibit E2-E2R and E3-E3R bindings equally. 3. The inhibitor constant of clomiphene for E2R was approximately 3.8 x 10(-8) M at 4 degrees C and that for E3R approximately 1.8 x 10(-8) M at 4 degrees C in a given case, determined by charcoal assay. 4. It is suggested that the anti-estrogenic compounds demonstrate their effects after binding either to E2R or to E3R. 5. There were some tissue differences of the contents between E2R and E3R. For example, the uterus and the cortex contained E2R, and the pituitary E3R more than the other.     

Tamoxifen for induction of Cre-recombination may confound fibrosis studies in female mice

A variety of conditional knock-out mice relying on Tamoxifen-driven ERT2/Cre -mediated recombination are available and have been used to study involvement of specific genes in kidney disease. However, recent data suggest that Tamoxifen itself might attenuate fibrosis when administered during experimental models of kidney disease. It has remained unclear whether this still applies also if kidney damage is initiated after a wash-out period has been implemented. Here we report that the commonly applied regimen of administration of 4 alternate day doses of 1mg Tamoxifen per mouse until 14 days prior to start of the actual experiment, in this case the induction of obstructive nephropathy by Unilateral Ureteral Obstruction (UUO), still attenuated fibrosis in female obstructed mouse kidneys, whereas this effect was not seen in male obstructed kidneys. Attenuation of fibrosis was accompanied by a reduction in nuclear ERα positivity despite absence of detectable levels of the active tamoxifen metabolite endoxifen throughout the UUO experiment. In conclusion, these results indicate that the Tamoxifen dosing regimen commonly applied in conditional gene targeting experiments might have prolonged confounding effects in female mice through attenuation of renal fibrosis independent of modulation of the expression of the targeted gene(s).     

Effects of 9-ene-tetrahydrocannabinol on uterine estrogenicity in the mouse.

Several early (Phase I) and late (Phase II) estrogenic effects of 9-ene-tetrahydrocannabinol (THC) were examined in the adult mouse uterus. An injection of THC (2.5 or 10 mg/kg body wt) in ovariectomized mice neither stimulated uterine water imbibition or accumulation of [125I]bovine serum albumin (Phase I responses) at 6 h, nor antagonized these Phase I responses elicited by estradiol-17 beta (E2). With respect to Phase II responses, although single injections of THC (2.5, 5.0 and 10 mg/kg body wt) alone were ineffective in influencing uterine weight at 24 h or incorporation of [3H]thymidine at 18 h, this drug interfered with these responses elicited by E2 in a dose-dependent manner. In contrast, an injection of THC in progesterone (P4)-primed ovariectomized mice modestly enhanced (61%) uterine incorporation of [3H]thymidine. However, E2-stimulated uterine thymidine incorporation in P4-primed ovariectomized mice was antagonized by THC treatment. Effects of THC on blastocyst implantation were examined. Single or multiple injections of various doses of THC neither induced implantation in P4-primed delayed implanting mice, nor interfered with E2-induced implantation. Furthermore, daily injections of THC (10 mg/kg body wt) during the peri-implantation period had no apparent adverse effects on implantation, or on experimentally induced decidualization (deciduomata). The data suggest that THC is neither pro- nor antiestrogenic with respect to Phase I responses. However as regards Phase II responses, THC is modestly pro-estrogenic in the P4-treated uterus, but is anti-estrogenic in the presence of E2. These estrogen agonistic/antagonistic effects of THC on uterine Phase II responses do not adversely affect the process of implantation and decidualization.     

Inducible gene manipulations in brain serotonergic neurons of transgenic rats

The serotonergic (5-HT) system has been implicated in various physiological processes and neuropsychiatric disorders, but in many aspects its role in normal and pathologic brain function is still unclear. One reason for this might be the lack of appropriate animal models which can address the complexity of physiological and pathophysiological 5-HT functioning. In this respect, rats offer many advantages over mice as they have been the animal of choice for sophisticated neurophysiological and behavioral studies. However, only recently technologies for the targeted and tissue specific modification of rat genes - a prerequisite for a detailed study of the 5-HT system - have been successfully developed. Here, we describe a rat transgenic system for inducible gene manipulations in 5-HT neurons. We generated a Cre driver line consisting of a tamoxifen-inducible CreERT2 recombinase under the control of mouse Tph2 regulatory sequences. Tissue-specific serotonergic Cre recombinase expression was detected in four transgenic TPH2-CreERT2 rat founder lines. For functional analysis of Cre-mediated recombination, we used a rat Cre reporter line (CAG-loxP.EGFP), in which EGFP is expressed after Cre-mediated removal of a loxP-flanked lacZ STOP cassette. We show an in-depth characterisation of this rat Cre reporter line and demonstrate its applicability for monitoring Cre-mediated recombination in all major neuronal subpopulations of the rat brain. Upon tamoxifen induction, double transgenic TPH2-CreERT2/CAG-loxP.EGFP rats show selective and efficient EGFP expression in 5-HT neurons. Without tamoxifen administration, EGFP is only expressed in few 5-HT neurons which confirms minimal background recombination. This 5-HT neuron specific CreERT2 line allows Cre-mediated, inducible gene deletion or gene overexpression in transgenic rats which provides new opportunities to decipher the complex functions of the mammalian serotonergic system.     

Temporal dissection of K-ras(G12D) mutant in vitro and in vivo using a regulatable K-ras(G12D) mouse allele

Animal models which allow the temporal regulation of gene activities are valuable for dissecting gene function in tumorigenesis. Here we have constructed a conditional inducible estrogen receptor-K-ras(G12D) (ER-K-ras(G12D)) knock-in mice allele that allows us to temporally switch on or off the activity of K-ras oncogenic mutant through tamoxifen administration. In vitro studies using mice embryonic fibroblast (MEF) showed that a dose of tamoxifen at 0.05 μM works optimally for activation of ER-K-ras(G12D) independent of the gender status. Furthermore, tamoxifen-inducible activation of K-ras(G12D) promotes cell proliferation, anchor-independent growth, transformation as well as invasion, potentially via activation of downstream MAPK pathway and cell cycle progression. Continuous activation of K-ras(G12D) in vivo by tamoxifen treatment is sufficient to drive the neoplastic transformation of normal lung epithelial cells in mice. Tamoxifen withdrawal after the tumor formation results in apoptosis and tumor regression in mouse lungs. Taken together, these data have convincingly demonstrated that K-ras mutant is essential for neoplastic transformation and this animal model may provide an ideal platform for further detailed characterization of the role of K-ras oncogenic mutant during different stages of lung tumorigenesis.     

Temporal Cre-mediated recombination exclusively in endothelial cells using Tie2 regulatory elements

Summary: The versatility of the bacteriophage Cre/LoxP system is dependent on the availability of a spectrum of tissue-specific Cre transgenic mice to address a host of biological questions. In this paper, we report on the generation of an inducible Tie2Cre transgenic mouse line that facilitates gene targeting exclusively in endothelial cells. The temporal manner of recombination is feasible through the use of a Cre-estrogen receptor fusion protein ER(T2) and was, in practical terms, achieved by feeding the animals the estrogen antagonist tamoxifen orally for 5 weeks. High efficiency of recombination was found in the vast majority of endothelial cell populations examined, as monitored by an EGFP reporter mouse line. Critically, no EGFP expression was observed in any uninduced mice. This inducible Cre line will be a very beneficial asset to investigating the role of endothelial specific genes in the adult mouse and to induce transgenes in the endothelium in an extremely efficient manner. genesis 33:191-197, 2002.     

Coumestrol antagonizes neuroendocrine actions of estrogen via the estrogen receptor alpha

The phytoestrogen coumestrol has estrogenic actions on peripheral reproductive tissues. Yet in the brain this compound has both estrogenic and anti-estrogenic effects. We used estrogen receptor alpha knockout mice (ERalphaKO) to determine whether coumestrol has estrogenic actions in mice and also if these effects are mediated by the classic ERalpha. Female wild-type (WT) and ERalphaKO mice were ovariectomized and treated with estradiol (E2), dietary coumestrol, both, or neither compound. Ten days later the animals were sacrificed, blood was collected, and brain tissues were perfused. Fixed brains were sectioned and immunocytochemistry was employed to quantify progesterone receptors (PR) in the medial preoptic (POA) and ventromedial nucleus of the hypothalamus (VMN). Plasma was assayed for luteinizing hormone (LH). Estrogen treatment induced PR immunoreactivity in both regions in brains of WT females. In ERalphaKO mice, lower levels of PR were induced. The stimulatory effects of E2 on PR were attenuated in the POA by cotreatment with coumestrol, and the same trend was noted in the VMN. WT ovariectomized females treated with E2 had low levels of LH, while LH was high in untreated females and even higher in ovariectomized females treated with coumestrol. ERalphaKO females in all treatment groups had high levels of LH. Taken together, the results show that coumestrol has anti-estrogenic actions in the brain and pituitary and that ERalpha mediates these effects.     

Novel approach for evaluation of estrogenic and anti-estrogenic activities of genistein and daidzein using B16 melanoma cells and dendricity assay

The effects of soy isoflavones, genistein and daidzein, which exhibit estrogenic, anti-estrogenic and/or tyrosine kinase inhibitory activity, on the dendritic morphology of B16 mouse melanoma cells were quantitatively evaluated and compared with those of 17 beta-estradiol (Est) and tyrphostin, a tyrosine kinase inhibitor. Dendricity was significantly stimulated in the order of Est > genistein > daidzein = tyrphostin, but not by glycosides of genistein and daidzein. In competition experiments, Est counteracted the stimulatory activity of genistein and daidzein, but enhanced the activity of tyrphostin additively, suggesting that genistein and daidzein agonized Est. In addition, when the concentration ratios of genistein/Est and daidzein/Est were higher than 5000 and 50,000, respectively, genistein and daidzein agonized Est. In contrast, when the ratio of daidzein/Est was lower than 500, daidzein antagonized Est. Furthermore, genistein and daidzein competed with each other in stimulatory activity. These observations suggest that: 1) dendricity is stimulated by agonists (genistein and daidzein) of Est and tyrosine kinase inhibitors (genistein and tyrphostin), 2) the concentration ratio of isoflavone aglycone/Est is very important as one regulatory factor for estrogenic and/or anti-estrogenic activity, and 3) daidzein antagonizes not only Est but also genistein. It is concluded that a quantitative and simple dendricity assay using B16 mouse melanoma cells is available to evaluate estrogenic and anti-estrogenic activity in vitro.     

The activation function-1 domain of estrogen receptor alpha in uterine stromal cells is required for mouse but not human uterine epithelial response to estrogen

The activation function-1 (AF-1) domain of the estrogen receptor alpha (ERalpha) in stromal cells has been shown to be required for epithelial responses to estrogen in the mouse uterus. To investigate the role of the stroma in estrogenic responses of human uterine epithelium (hUtE), human/mouse chimeric uteri composed of human epithelium and mouse stroma were prepared as tissue recombinants (TR) that were grown in vivo under the renal capsule of female nude mouse hosts. In association with mouse uterine stroma (mUtS), hUtE formed normal glands surrounded by mouse endometrial stroma and the human epithelium influenced the differentiation of stroma into myometrium, such that a histologically normal appearing uterine tissue was formed. The hUtE showed a similar proliferative response and increase in progesterone receptors (PR) in response to 17beta-estradiol (E2) in association with either human or mUtS, as TRs. However, under identical endocrine and micro-environmental conditions, hUtE required 5-7 days exposure to E2 rather than 1 day, as shown for mouse uterine epithelium, to obtain a maximal proliferative response. Moreover, this extended length of E2 exposure inhibited mouse epithelial proliferation in the presence of mouse stroma. In addition, unlike the mouse epithelium, which does not proliferate or show regulation of PR expression in response to E2 in association with uterine stroma derived from mice that are null for the AF-1 domain of ERalpha, hUtE proliferates and PR are up-regulated in response to E2 in association genetically identical ERalpha knock-out mouse stromal cells. These results clearly demonstrate fundamental differences between mouse and human uterine epithelia with respect to the mechanisms that regulate estrogen-induced proliferation and expression of PR. Moreover, we show that genetically engineered mouse models could potentially aid in dissecting molecular pathways of stromal epithelial interactions in the human uterus.     

Consequences of haemolysis without haptoglobin

Haptoglobin (Hp), a conserved plasma glycoprotein, forms very stable soluble complexes with free plasma haemoglobin. Haemoglobin binding by haptoglobin is thought to be important in the rapid hepatic clearance of haemoglobin from the plasma and in the inhibition of glomerular filtration of haemoglobin. It is thought to reduce haemoglobin-induced renal damage during haemolysis. To evaluate these functions, Hp knockout (Hp-/-) mice were created. The Hp-/- mouse was generated by a standard gene replacement technique in mouse embryonic stem cells. These mice were evaluated with and without haemolysis using several parameters: mortality, haemoglobin clearance, renal tissue damage and function. Hp-/- mice were viable but had a small, significant reduction in postnatal viability. The lack of Hp did not impair clearance of free plasma haemoglobin. Induction of severe haemolysis by phenylhydrazine caused extensive haemoglobin precipitation in the renal tubular cells. However, haemoglobin precipitation in the kidney was not increased in Hp-/- mice. Nevertheless, Hp-/- mice were more susceptible to phenylhydrazine with a mortality rate of 55% in Hp-/- mice versus 18% in Hp+/+ mice. In general, phenylhydrazine-treated Hp-/- mice suffered greater tissue damage, as evidenced by the induction of a hepatic acute phase response, resulting in increased plasma alpha1-acidic glycoprotein (AGP) levels and higher plasma malonaldehyde (MDA) and 4-hydroxy-2(E)-nonenal (HNE) levels. Gross pathological analysis indicated that the kidney was the most affected tissue in phenylhydrazine-treated Hp-/- and Hp+/+ mice, and Hp-/- mice were more severely affected. They had lower mitotic indices in their kidneys, higher basal levels of renal lipid peroxidation, as evidenced by levels of malonaldehyde and 4-hydroxy-2(E)-nonenal (MDA/HNE) and elevated levels of 8-hydroxyguanine (but not other products of oxidative DNA damage). There also was increased induction of haem oxygenase-1. The more severe renal damage in Hp-/- mice was also evident in the delayed erythropoietin gene expression and poorer renal clearance of [3H]-inulin. The reduction in glomerular filtration function in Hp+/+ and Hp-/- mice could be restored to baseline by vasodilators (prazosin or diazoxide), implicating renal vasoconstriction as a major mechanism of acute renal failure during induced haemolysis. These data suggest that Hp plays a pivotal role in reducing renal oxidative damage during haemolysis.     

Inhibition of human breast cancer cell proliferation with estradiol metabolites is as effective as with tamoxifen.

The anti-estrogenic substance tamoxifen is effective in the adjuvant therapy applied in human breast cancer. Since it partly exhibits estrogenic activity and has serious side-effects, however, pure anti-estrogenic compounds are being sought. In our experimental study, we compared the anti-proliferative effect of estradiol and 13 endogenous estradiol metabolites on human breast cancer cells with the effect of tamoxifen. We used MCF-7 and MDA-MB 231, the well-established estrogen receptor-positive and -negative cell lines. 4-hydroxytamoxifen, the active metabolite of tamoxifen, estradiol and 13 estradiol metabolites were tested in concentrations ranging from 3.1 to 100 microM. Incubation time was 4 days and cell proliferation was measured by means of the ATP chemosensitivity test. 4-hydroxytamoxifen showed an IC50 value of 27 microM and 18 microM in MCF-7 and MDA-MB 231 cells, respectively. Estradiol and its metabolites were anti-proliferative in both cell lines. A few A-ring metabolites were more effective in inhibiting cell proliferation than D-ring metabolites and the parent substance 17beta-estradiol. 4-OHE1, 2-MeOE1 and 2-MeOE2 were as effective in both cell lines as tamoxifen. For the first time it has been demonstrated that endogenous estradiol metabolites are equally anti-proliferative as tamoxifen in the context of human breast cancer cells. Since some of these metabolites exhibit no estrogenic activity, they are likely to be valuable in clinical studies of chemoprevention and adjuvant therapy of breast cancer.     

Establishment of sperm associated antigen 6 gene knockout mouse model and its mechanism of deafness

To investigate the effects of knocking out the Sperm associated antigen6 (Spag6) gene on the auditory system of mice, the heterozygous type Spag6 knockout mouse model built in the previous period was used for mating and breeding, and homozygous type Spag6 gene knockout mouse (Spag−/−), heterozygous type Spag6 gene knockout mouse (Spag+/−) and wild type mouse (Spag+/+) were obtained. PCR technology was used to verify mouse models with different genotypes. After verification, the hearing threshold responses of Spag+/+ and Spag−/− genotype mice were detected. The localization of Spag6 gene in the basal membrane of the cochlea of the inner ear was detected by immunofluorescence staining. The changes of middle ear tissues were observed by H.E. staining sections. The relative expression of Prestin gene and Pgrn gene in different age mice was detected by fluorescence quantitative PCR. The relative expression of Prestin gene was detected by western blot. The results showed that Spag−/− mice had hearing impairment compared with Spag+/+ mice. And Spag6 protein is distributed in different genotypes of mouse hair cells; Spag−/− mice showed otitis media. The expression of Prestin mRNA and protein in Spag−/− mice was significantly higher than that in Spag+/+ mice (P < 0.01). The expression of Pgrn gene in Spag+/+ mice was significantly higher than that in Spag−/− mice (P < 0.05). It indicates that the loss of Spag6 gene would lead to the decline of hearing sense in mice. It is likely that the Spag6 gene could affect hearing by regulating the expression of Prestin gene. And the absence of the Spag6 gene causes otitis media in mice. The results of this study can lay a theoretical foundation for the follow-up studies of Spag6 gene in deafness diseases.     

Gnotobiotic IL-10-/-;NF-kappa B(EGFP) mice reveal the critical role of TLR/NF-kappa B signaling in commensal bacteria-induced colitis

Commensal bacteria and TLR signaling have been associated with the maintenance of intestinal homeostasis in dextran sodium sulfate-induced intestinal injury. The aim of this study was to determine the in vivo role of TLR/NF-kappaB activation in a model of commensal bacteria-induced T cell-mediated colitis. A NF-kappaB reporter gene mouse (NF-kappaBEGFP) (EGFP, enhanced GFP) was crossed to the colitogenic susceptible strain IL-10-/- and derived into germfree conditions using embryo-transfer technology. Germfree IL-10wt/wt;NF-kappaBEGFP and IL-10-/-;NF-kappaBEGFP mice (wt, wild type) were dual associated with the nonpathogenic commensal bacteria strains Enterococcus faecalis and Escherichia coli. EGFP was detected using macroimaging, confocal microscopy, and flow cytometry. IL-10-/-;MyD88-/- mice were used to assess E. faecalis/E. coli-induced TLR-dependent signaling and IL-23 gene expression. Dual-associated IL-10-/-;NF-kappaBEGFP mice developed severe inflammation by 7 wk. Macroscopic analysis showed elevated EGFP expression throughout the colon of bacteria-associated IL-10-/-;NF-kappaBEGFP mice. Confocal microscopy analysis revealed EGFP-positive enterocytes during the early phase of bacterial colonization (1 wk) in both IL-10wt/wt and IL-10-/- mice, while the signal shifted toward lamina propria T cells, dendritic cells, neutrophils, and macrophages in IL-10-/- mice during colitis (7 wk). The NF-kappaB inhibitor BAY 11-7085 attenuated E. faecalis/E. coli-induced EGFP expression and development of colitis. Additionally, E. faecalis/E. coli-induced NF-kappaB signaling and IL-23 gene expression were blocked in bone marrow-derived dendritic cells derived from IL-10-/-;MyD88-/- mice. We conclude that bacteria-induced experimental colitis involves the activation of TLR-induced NF-kappaB signaling derived mostly from mucosal immune cells. Blocking TLR-induced NF-kappaB activity may represent an attractive strategy to treat immune-mediated intestinal inflammation.     

Embryonic lethality and defective neural tube closure in mice lacking squalene synthase.

Squalene synthase (SS) catalyzes the reductive head-to-head condensation of two molecules of farnesyl diphosphate to form squalene, the first specific intermediate in the cholesterol biosynthetic pathway. We used gene targeting to knock out the mouse SS gene. The mice heterozygous for the mutation (SS+/-) were apparently normal. SS+/- mice showed 60% reduction in the hepatic mRNA levels of SS compared with SS+/+ mice. Consistently, the SS enzymatic activities were reduced by 50% in the liver and testis. Nevertheless, the hepatic cholesterol synthesis was not different between SS+/- and SS+/+ mice, and plasma lipoprotein profiles were not different irrespective of the presence of the low density lipoprotein receptor, indicating that SS is not a rate-limiting enzyme in the cholesterol biosynthetic pathway. The mice homozygous for the disrupted SS gene (SS-/-) were embryonic lethal around midgestation. E9.5-10.5 SS-/- embryos exhibited severe growth retardation and defective neural tube closure. The lethal phenotype was not rescued by supplementing the dams either with dietary squalene or cholesterol. We speculate that cholesterol is required for the development, particularly of the nervous system, and that the chorioallantoic circulatory system is not mature enough to supply the rapidly growing embryos with maternal cholesterol at this developmental stage.     

NMR-based metabonomic evaluation of livers from rats chronically treated with tamoxifen,mestranol, and phenobarbital

In this study, we look at the metabolic effects of long-term dosing with tamoxifen, mestranol or phenobarbital on the liver. Tamoxifen, mestranol and phenobarbital have all been reported to act as promoters of hepatic tumors. While tamoxifen and mestranol are known to have estrogenic activity, in the liver phenobarbital is a non-estrogenic compound. Aqueous and lipophilic liver extracts from control and chronically treated Fisher 344 rats were evaluated by nuclear magnetic resonance spectroscopy (NMR). In both the aqueous and lipophilic sample sets, the estrogenic action of mestranol appears to be responsible for the clustering of these samples with those animals treated with tamoxifen. Phenobarbital does not have estrogenic activity and, therefore, clusters away from the estrogenic and control groups. In the lipophilic samples, the fatty acid peak (CH₂)_n was higher in tamoxifen-treated rats than in control, phenobarbital- or mestranol-treated rats. In the aqueous samples, serine and choline levels were higher in phenobarbital-treated rats than controls, which may be an indication that the folate–homocysteine metabolic pathways were altered.     

Tamoxifen feeding method is suitable for efficient conditional knockout

The Cre-driver system is used to generate conditional knockout mice. Tamoxifen inducible Cre-driver mice can be used for spatiotemporal knockout by administration of the drug. A major tamoxifen administration is performed by intraperitoneal administration or oral administration. However, these forced administrations may be damaging to mice. Herein, we have demonstrated an improved method of administering tamoxifen with powdered food to mice. A mouse line expressing the tamoxifen-inducible Cre gene was used ubiquitously in this experiment to evaluate the efficiency of Cre recombination in the whole body. Our method also achieved efficient recombination without causing injury to mice. The X-gal staining intensity of the feeding method was equivalent to that of the intraperitoneal administration method. Furthermore, this method can be used for recombination before birth, or during the fetal period. We recommend researchers to employ this feeding method to administer tamoxifen to minimize the risk of injury to mice.     

Opposite regulation of cAMP concentration in the quail oviduct and the mouse uterus by tamoxifen. Correlation with estrogen-antagonist and estrogen-agonist activity

The ability of estradiol and tamoxifen to regulate cAMP levels and cAMP phosphodiesterase activities has been determined in the quail oviduct and in the mouse uterus. In the quail, tamoxifen (1 mg/kg daily for 3 days) had no effect on oviducal growth but significantly increased cAMP concentration (+49%). Injected concurrently with estradiol, tamoxifen completely inhibited oviduct growth as well as the increase of cAMP phosphodiesterase activity induced by the hormone alone and increased cAMP concentration (+229% over estradiol treated group). In the mouse, estradiol and tamoxifen displayed uterotrophic activity and increased cAMP phosphodiesterase activity. In both groups, cAMP concentration was greatly reduced (−76% in estradiol treated group; −86% in tamoxifen treated group). The opposite regulation of cAMP levels in the quail oviduct and the mouse uterus by tamoxifen reflected large differences in the contribution of calmodulin-dependent and -independent forms of phosphodiesterase to the hydrolysis of cAMP in the two models and the fact that tamoxifen stimulated the activity of the calmodulin-independent isoenzyme, while it competitively inhibited the activation of the calmodulin-dependent isoenzyme by calmodulin. Several lines of evidence strongly suggest that the regulation of cAMP levels is involved in growth-inhibiting or growth-promoting activity of tamoxifen.