Exposure to ethinyl estradiol prenatally and/or after sexual maturity induces endometriotic and precancerous lesions in uteri and ovaries of mice.

Unrecognizable exposure to estrogenic substance may cause estrogen-dependent diseases, endometriosis and cancer. Pregnant mice (ICR/Jcl, CLEA) were exposed to 0.01 mg ethinyl estradiol (EE2 )/kg per day or vehicle (olive oil) through oral intubation from day 11 to 17 of gestation. They delivered their offspring and raised them. When the experimental female F1 mice were at 8 weeks of age, they were not exposed to EE2 or to the same dose of EE2 or to vehicle twice a week until 20 weeks of age. The control female F1 mice were exposed to the same dose of EE2 or vehicle alone, similarly. All mice were killed at 28 weeks of age. The resected uteri and ovaries were processed for microscopic examinations and for determination of the aromatase mRNA levels and aromatase protein through quantitative RT-PCR and Western blotting, respectively. Adenomyosis and adenocarcinomatous changes were significantly discernible in the EE2 -exposed uteri, and incidence of ectopic glands and serous cysts were significantly increased in the prenatally EE2 -exposed ovaries as compared with respective controls. Significant upregulation of the aromatase mRNA was seen in the prenatally EE2 -exposed uteri and in the EE2 -exposed ovaries. The aromatase protein was identified in all ovaries examined, and in EE2 -exposed uteri but not in controls and confirmed its localization in eutopic and ectopic glands, abnormally proliferated lesions and the lining of the cysts. Taken together, continuous EE2 exposure may cause endometriotic and precancerous lesions due to excessive estrogen synthesis in both target organs.

Death-resistant and nonresistant malignant human cell lines under anoxia in vitro.

BACKGROUND: Erythropoietin supports the survival of erythroblasts. We previously demonstrated that 24 malignant human cell lines expressed erythropoietin and its receptor and that erythropoietin secretion was enhanced under anoxia. In this study, we examined the viability of 22 of these cell lines excluding two leukemia cell lines under anoxia. METHODS: Twenty-two cancer cell lines of various origins were cultured under anoxia or normoxia for 4 days, and their viability was examined at 1-day intervals. The levels of lactate and ATP were measured. The expressions of hypoxia-inducible transcription factor 1alpha (HIF-1alpha) and Bcl-2 family proteins were examined by western blotting analysis. The cellular and mitochondrial features were examined by microscopy. RESULTS: Eleven of the 22 cancer cell lines examined showed 80% to 100% cell viability after 4 days under anoxia; 2 cell lines showed similar viability for 3 days, 3 cell lines showed similar viability for 2 days, and 6 cell lines showed similar viability for 1 day or less. These 11 death-resistant cell lines, which secrete various amounts of erythropoietin under anoxia, produced significantly more lactate during 2 days under anoxia than under normoxia, with ATP levels about 60% of those before anoxia. ATP returned to the normal level when normoxia was restored after 4 days of anoxia. However, the nonresistant cell lines responded to anoxia by yielding significantly more lactate without a reduction of the ATP level. The expression patterns of Bcl-2 family proteins revealed that apoptosis-inhibiting signals predominated over proapoptotic signals in the death-resistant cells under anoxia. CONCLUSION: The majority of the cancer cell lines examined survived under anoxia in vitro, through the Pasteur effect, in a dormant state without direct support of erythropoietin.

Localization of Erythropoietin and Erythropoietin-Receptor in Postimplantation Mouse Embryos: (erythropoietin/erythropoietin receptor/universal morphogen/neurogenesis/mouse embryo).

We used RT-PCR (reverse transcription-polymerase chain reaction) and immunocytochemical methods to demonstrate the presence of erythropoietin (Ep) and its receptor (EpR) in postimplantation mouse embryos from the egg-cylinder to the unturned stage. Expression of mRNA for EpR was detected in total RNA from embryos and decidua in all these stages, but Ep mRNA was confined to embryos in the primitive streak stage and beyond and was not detected in the decidua. Staining of Ep and EpR was seen in all tissues, embryo proper and extra-embryonic. Moreover, regions of marked staining of Ep and EpR were detected in the extra-embryonic endoderm, embryonic ectoderm, neural folds and yolk sac, chronologically. No conspicuous differences were present in the staining patterns between Ep and EpR until primitive streak stage; however, after this stage, Ep was predominantly present in the nucleus and EpR on the surface of almost all cells; in the visceral yolk sac endoderm EpR was also detected in adsorption vacuoles and lipid droplets. These studies suggest that Ep first of exogenous and then endogenous origin and EpR of endogenous origin are involved not only in embryogenesis but also in neurogenesis and hematopoiesis in early postimplantation mouse embryos.