Protein alterations associated with N-methyl-N-nitrosourea exposure of preimplantation mouse embryos transferred to surrogate mothers.

Mouse preimplantation embryo functions have been shown to be disrupted by in vitro exposure to N-methyl-N-nitrosourea (MNU) with subsequent transfer to the uteri of pseudopregnant surrogate mothers. Increased gross malformations and decreased fetal body lengths in the midgestational period have been previously documented. Protein extracts were isolated from day 12 mouse fetuses developed from MNU- or solvent-exposed blastocysts and analyzed by two-dimensional electrophoresis. The electrophoretic patterns reveal six protein alterations in day 12 fetal tissue induced by MNU treatment at the blastocyst stage. Five of these alterations involve shifts in isoelectric point (pI) and the other alteration involves a quantitative increase in a protein. The possibility that two of the proteins which exhibit a shift in pI following MNU exposure represent the cell adhesion molecules, N-CAM and L-CAM (based on similar Mr values), was investigated by Western blot analysis. No pI alteration in L-CAM or N-CAM expression is seen after MNU exposure. These results demonstrate that in vitro MNU exposure of preimplantation embryos results in protein alterations in midgestational fetuses. Thus, the effects of MNU exposure on preimplantation embryos may be manifest long after exposure, and subtle, non-lethal mutations may play a role in poor fetal outcome after early chemical exposure.

Effects of estradiol on estrogen receptor, progesterone receptor, and tyrosinase in hamster melanoma transplanted into athymic mice.

Nuclear estrogen binding was characterized in HM-1, a malignant hamster melanoma cell line transplanted into male and female athymic mice following acute, subchronic, and chronic injection of estradiol. Nuclear binding was saturable, of high affinity (10(10) M-1) and readily soluble in low salt buffer. Saturation analyses revealed that [3H]estradiol in excess of 5.0 nM apparently bound to a second class of lower affinity (10(9) M-1), higher capacity cytosol sites. Enzyme-linked immunoassay with a specific monoclonal antibody (H222 Sp gamma) directed against the human estrogen receptor protein was in excellent agreement (r = 0.93) with values obtained using hydroxyapatite to separate bound from free ligand. Nuclear estrogen receptor content in HM-1 cells was increased maximally 1 h after acute s.c. injection of a low dose (0.1 microgram) of estradiol. The increase in nuclear receptor content was accompanied by an apparent rapid reduction in cytosol binding. Subchronic (3 days) and chronic exposure (35 days) to estradiol also produced a significant, dose-related increase in tumor nuclear estrogen receptor content. Cytosol binding for progestin was low (less than or equal to 2 fmol) to absent in HM-1 xenografts not exposed to estradiol. Subchronic and chronic exposure to estradiol induced a dose-related, specific, high affinity (10(9) M-1) cytosol binding protein for progestin(s) in HM-1 xenografts carried in male and female athymic mice. In contrast, progestin binding to nuclear receptor was not increased in estrogen-primed animals, nor did acute injection of progesterone (100 micrograms s.c.) increase the amount of saturable, high affinity (10(9) M-1) nuclear progestin receptor in control or estradiol-primed athymic mice. In contrast to the induction of progestin binding, tyrosinase activity was not altered by a similar exposure to estradiol when assayed at a saturating concentration of tyrosine. These observations suggest that the estrogen receptor in HM-1 cells may be functional but that pigmentary changes observed in mammals following chronic exposure to estradiol may not be mediated by a direct effect on the rate limiting enzyme of melanin synthesis.

Inhibition of hamster melanoma growth by estrogen.

A malignant hamster melanoma cell line HM-1 derived from the heterogenous malignant hamster melanoma MM1 contains a specific, high affinity binding protein for estrogens. Partial purification of the binding protein with ammonium sulfate (40% saturation) increased mean binding content (3.1 +/- 1.2 (SD) fmol/mg protein) 15-fold without any change in affinity (10(10) M-1). The binding protein sedimented at 8-9S on 10-30% low salt sucrose gradients and 9-10S in the presence of 20 mM molybdate ion. Addition of 0.4 M KCl shifted the 8S peak to 4S. Binding was specific, saturable, and indicative of a single class of high affinity sites over a concentration range of 0.01-10.0 nM [3H]estradiol. Estradiol produced a dose related inhibition of HM-1 growth in vitro without altering the growth of an additional line (HM-2) which did not bind estrogen. The antiestrogen tamoxifen (10(-7) M) also significantly inhibited HM-1 melanoma growth in vitro, which was reversed by the addition of estradiol (10(-9) M). HM-1 xenografts grew faster in female BALB/c-nu/nu mice than male mice while there was no sex difference in HM-2 growth. Pharmacological doses of estradiol and the antiestrogen nafoxidine significantly inhibited HM-1 growth without altering tumor incidence or latency. Our observations suggest that HM-1 cell lines bind estrogens specifically and with high affinity and that hamster melanoma cells positive for this binding protein respond to estrogen.