Estrogenic/antiestrogenic activities of 14 polycyclic aromatic hydrocarbons (PAHs) and 63 monohydroxylated PAHs (OHPAHs) having 2 to 6 rings were evaluated by yeast two-hybrid assay expressing human estrogen receptor α. Relative effective potencies of estrogenic and antiestrogenic activities were calculated as the inverse values of the relative concentration of the test compound that gave the same activities of E 2 and 4-hydroxytamoxifen, respectively. PAHs did not show any estrogenic/antiestrogenic activity, but several OHPAHs having 3 to 5 rings showed activities. Especially, OHPAHs having 4 rings such as 3-, 4-and 10-hydroxybenz[a]anthracenes (3-, 4-and 10-OHBaAs) and 2-hydroxychrysene (2-OHCh) showed strongly estrogenic activity. Several other OHPAHs having 4 rings such as 2-and 3-hydroxybenzo[c]phenanthrenes (2-, 3-OHBcPhs), 2-OHBaA and 3-OHCh showed strongly antiestrogenic activity. The length-to-breadth (L/B) ratios of the rectangular van der Walls planes surrounding the ring molecules of estrogenic OHPAHs were in the narrow range from 1.599 to 1.734. The distances between the oxygen atom of the phenol group and farthest hydrogen atom (O-H distance) of the estrogenic OHPAHs ranged from 10.825 Å to 11.738 Å. The L/B ratios and O-H distances of antiestrogenic OHPAHs were in the wider ranges from 1.277 to 1.734 and from 8.47 Å to 11.681 Å, respectively. The partial charges (atomic unit) of the phenol group of both estrogenic and antiestrogenic OHPAHs were in the range from −0.250 atmic unit (au) to −0.253 au. The similarity of these values to those of E 2 and diethylstilbestrol suggested that the compositions of estrogenic OHPAHs were similar to them and that the compositional conditions of estrogenic OHPAHs were much smaller than those of antiestrogenic OHPAHs. These results raise the possibility of predicting the estrogenic/antiestrogenic activities of OHPAHs from their structural characteristics, although using only the above three parameters might not be enough for accurate estimations.

Estrogenic and antiestrogenic activities of 19 quinoid polycyclic aromatic hydrocarbons (PAHQs) and 9 ketone PAHs were evaluated by the yeast two-hybrid assay using yeast cells expressing estrogen receptor-α (ERα). Binding affinity of PAHQs to ERα was assayed by the polarized fluorescence method using Fluormone TM ES2. Ten PAHQs having 3-5 rings showed antiestrogenic activities. The most strongly antiestrogenic PAHQs were 1,4-chrysenequinone and 5,6-chrysenequinone. On the other hand, benzo [a]pyrene-3,6-quinone showed the strongest estrogenic activity. However, the other compounds tested did not show so strong estrogenic/antiestrogenic activities. Binding affinity to ER was required but not sufficient for estrogenic/antiestrogenic activities of PAHQs. The length-to-breadth ratios of the rectangular planes surrounding the ring molecules and the distances between the oxygen atom of the carbonyl group and farthest hydrogen atom of estrogenic/antiestrogenic PAHQs were in narrow ranges, suggesting a structure-activity relationship. As interactions between active PAHQ and ER, hydrogen bonding between carbonyl groups and amino acid residues and van der Waals forces were considered.

Bone marrow stroma contains progenitors of skeletal tissue components such as bone, cartilage, the hematopoietic supporting stroma and adipocytes.1,2) Among cell types, the adipocytes are perhaps the most abundant stromal cell phenotype in adult human bone marrow. Generally, adipocytes play an important role in systemic lipid metabolism and also contribute to adiposity, insulin resistance and atherogenesis.3) Gimble et al. have reviewed the function of bone marrow adipocytes and proposed four hypotheses: (1) a passive role, simply occupying excess space in the bone marrow cavity; (2) an active role in systemic lipid metabolism; (3) a localized energy reservoir in the bone marrow; (4) direct contribution to the promotion of hematopoiesis and an influence on osteogenesis.4) However, the function of adipocytes in bone marrow is still controversial.The stromal cell line MC3T3-G2/PA6 (PA6) was established from mouse clavaria as preadipocytes having the capacity to convert to mature adipose cells.5) Greenberger reported that corticosteroids accumulated lipids in primary cultured mammalian bone marrow preadipocytes, but insulin did not. 6,7) In addition, the adipogenesis in PA6 cells is also markedly accelerated by the addition of dexamethasone, but insulin is not required for the process. 5)Insulin is a major hormone controlling critical energy functions such as glucose and lipid metabolism. Its effects result from insulin binding to a membrane receptor (insulin receptor), which is expressed highly on insulin target tissues, such as the liver, muscles and adipocytes.8) The binding leads to its receptor autophosphorylation and then to tyrosine phosphorylation of substrate proteins, such as insulin receptor substrates (IRSs). These allow for the formation of macromolecular complexes close to the receptor. The two main transduction pathways are the phosphatidylinositol 3-kinase (PI3K) pathway and the mitogen-activated protein kinase (MAPK) pathway. [8][9][10] The MAPK pathway is considered to be involved in nuclear effects, proliferation and differentiation, while the PI3K pathway plays a major role in insulin functions, mainly via activation of the Akt cascade. 8,10) Activation of Akt stimulates glycogen synthesis, protein synthesis, cell survival, inhibition of lipolysis, and glucose uptake.11) However, nothing is known about insulin signaling in bone marrow adipocytes, which are unresponsive to insulin in the adipogenesis process.Therefore, we assessed insulin signaling and its functions, such as glucose uptake and the inhibition of lipolysis, in adipocytes differentiated from bone marrow preadipocyte PA6 cells. The stromal MC3T3-G2/PA6 ( MATERIALS AND METHODS Materials

Endocrine disrupting activities of three isomers of monohydroxylated 1-nitropyrene (1-NP) [3-, 6-, and 8-hydroxy-1-nitropyrenes (OHNPs)] were evaluated for the first time by yeast two-hybrid assay. OHNPs, which are not only metabolites of 1-NP but are also found in airborne particles, did not exhibit androgenic activity but exhibited estrogenic, antiestrogenic, and antiandrogenic activities. 6-OHNP showed the strongest estrogenic activity among the three OHNP isomers examined in this study. Concentrations of the OHNP isomers that gave 10% of activity of 1.0 × 10 −6 M 17β-estradiol (E 2 ) were as fol-

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