Yukiko Tamura

Bisphenol‐A induces cellular transformation, aneuploidy and DNA adduct formation in cultured Syrian hamster embryo cells

Bisphenol‐A (BP‐A) is a major component of epoxy, polycarbonate and other resins. For an assessment of in vitro carcinogenicity and related activity of BP‐A, the abilities of this compound to induce cellular transformation and genetic effects were examined simultaneously using the Syrian hamster embryo (SHE) cell model. Cellular growth was reduced by continuous treatment with BP‐A at doses ≥100 μM. However, colony‐forming efficiencies were not decreased significantly following treatment with up to 200 μM BP‐A for 48 hr. Morphological transformation of SHE cells was induced by treatment of cells with BP‐A at 50 to 200 μM for 48 hr. BP‐A exhibited transforming activity at doses ≥50 μM but was less active than the benzo[α]pyrene used as a positive control. Over the dose range that resulted in cellular transformation, treatment of SHE cells with BP‐A failed to induce gene mutations at the Na+/K+ ATPase locus or the hprt locus. No statistically significant numbers of chromosomal aberrations were detected in SHE cells treated with BP‐A. However, treatment of cells with BP‐A induced numerical chromosomal changes in the near diploid range at doses that induced cellular transformation. 32P‐Postlabeling analysis revealed that exposure of cells to BP‐A also elicited DNA adduct formation in a dose‐dependent fashion. Our results indicate that BP‐A has cell‐transforming and genotoxic activities in cultured mammalian cells and potential carcinogenic activity. Int. J. Cancer 75:290–294, 1998. Published 1998 Wiley‐Liss, Inc. This article is a US Government work and, as such, is in the public domain in the United States of America.

Mammalian cell transformation and aneuploidy induced by five bisphenols

Bisphenol‐A (BP‐A), a monomer of plastics used in numerous consumer products and a xenoestrogen, induces cellular transformation and aneuploidy in Syrian hamster embryo (SHE) cells. In this study, the abilities of 4 other bisphenols to induce cellular transformation and genetic effects in SHE cells were examined and compared to BP‐A. Cellular growth was inhibited by all bisphenols in a concentration‐related manner. The growth inhibitory effect of the bisphenols ranked: BP‐5 > BP‐4 > BP‐3 > BP‐2 or BP‐A. Morphological transformation of SHE cells was induced by BP‐A, BP‐3, BP‐4 and BP‐5, and the induced‐transformation frequencies were highest with BP‐4. None of the bisphenols induced gene mutations at the Na+/K+ ATPase locus or the hprt locus, or chromosomal aberrations in SHE cells. By contrast, aneuploidy induction in the near‐diploid range was exhibited by BP‐A, BP‐3, BP‐4 or BP‐5, corresponding to the transforming activity of each compound. The results indicate that BP‐A, BP‐3, BP‐4 and BP‐5 exhibit transforming activity in SHE cells, while BP‐2 does not, and that aneuploidy induction may be a causal mechanism of the transforming activity. Int. J. Cancer 86:151–154, 2000.

Involvement of genotoxic effects in the initiation of estrogen‐induced cellular transformation: Studies using Syrian hamster embryo cells treated with 17β‐estradiol and eight of its metabolites

To examine a direct involvement of genotoxic effects of estrogens in the initiation of hormonal carcinogenesis, the abilities of 17β‐estradiol (E2) and 8 of its metabolites to induce cellular transformation and genetic effects were studied using the Syrian hamster embryo (SHE) cell model. Treatment with E2, estrone (E1), 2‐hydroxyestrone (2‐OHE1), 4‐hydroxyestrone (4‐OHE1), 2‐methoxyestrone (2‐MeOE1), 16α‐hydroxyestrone (16α‐OHE1), 2‐hydroxyestradiol (2‐OHE2), 4‐hydroxyestradiol (4‐OHE2) or estriol (E3) for 1 to 3 days inhibited SHE cell growth in a concentration‐dependent manner. Concentration‐dependent increases in the frequency of morphological transformation in SHE cells were exhibited by treatment for 48 hr with each of all estrogens examined, except for E3. The transforming activities of the estrogens, determined by the induced transformation frequencies, were ranked as follows: 4‐OHE1 > 2‐OHE1 > 4‐OHE2 > 2‐OHE2 ≥ E2 or E1 > 2‐MeOE1 or 16α‐OHE1 > E3. Somatic mutations in SHE cells at the Na+/K+ATPase and /or hprt loci were induced only when the cells were treated with 4‐OHE1, 2‐MeOE1 or 4‐OHE2 for 48 hr. Some estrogen metabolites induced chromosome aberrations in SHE cells following treatment for 24 hr. The rank order of the clastogenic activities of the estrogens that induced chromosome aberrations was 4‐OHE1 > 2‐OHE1 or 4‐OHE2 > 2‐OHE2 > E1. Significant increases in the percentage of aneuploid cells in the near diploid range were exhibited in SHE cells treated for 48 hr or 72 hr with each of the estrogens, except for 4‐OHE1 and E3. Our results indicate that the transforming activities of all estrogens tested correspond to at least one of the genotoxic effects by each estrogen, i.e., chromosome aberrations, aneuploidy or gene mutations, suggesting the possible involvement of genotoxicity in the initiation of estrogen‐induced carcinogenesis. Int. J. Cancer 86:8–14, 2000.

Cell-transforming activity and mutagenicity of 5 phytoestrogens in cultured mammalian cells.

For the simultaneous assessment of in vitro carcinogenicity and mutagenicity of phytoestrogens, the abilities of 5 phytoestrogens, daidzein, genistein, biochanin A, prunetin, and coumestrol, to induce cell transformation and genetic effects were examined using the Syrian hamster embryo (SHE) cell model. Cellular growth was inhibited by all phytoestrogens in a concentration‐related manner. The growth inhibitory effect of the compounds was ranked: genistein, prunetin > coumestrol > biochanin A > daidzein, which did not correspond to their apoptosis‐inducing abilities. Morphological transformation in SHE cells was elicited by all phytoestrogens, except, prunetin. The transforming activities were ranked as follows: genistein > coumestrol > daidzein > biochanin A. Somatic mutations in SHE cells at the Na+/K+ ATPase and hprt loci were induced only by genistein, coumestrol, or daidzein. Chromosome aberrations were induced by genistein or coumestrol, and aneuploidy in the near diploid range was occurred by genistein or biochanin A. Genistein, biochanin A or daidzein induced DNA adduct formation in SHE cells with the abilities: genistein > biochanin A > daidzein. Prunetin was negative for any of these genetic endpoints. Our results provide evidence that genistein, coumestrol, daidzein and biochanin A induce cell transformation in SHE cells and that the transforming activities of these phytoestrogens correspond to at least 2 of the mutagenic effects by each phytoestrogen, i.e., gene mutations, chromosome aberrations, aneuploidy or DNA adduct formation, suggesting the possible involvement of mutagenicity in the initiation of phytoestrogen‐induced carcinogenesis.

Cell‐transforming activity and genotoxicity of phenolphthalein in cultured Syrian hamster embryo cells

Phenolphthalein is a cathartic agent widely used in non‐prescription laxatives. For the simultaneous assessment of in vitro carcinogenicity and mutagenicity of phenolphthalein, the ability of this chemical to induce cell transformation and genetic effects was examined using the Syrian hamster embryo (SHE) cell model. Cell growth was reduced by treatment with phenolphthalein at 10–40 μM in a dose‐related manner. Treatment with phenolphthalein for 48 hr induced a dose‐dependent increase in morphological transformation of SHE cells. Over the dose range that resulted in cell transformation (10–40 μM), treatment of SHE cells with phenolphthalein induced gene mutations at the hprt locus but not at the Na+/K+ ATPase locus. A statistically significant level of chromosomal aberrations was elicited in SHE cells treated with phenolphthalein at the highest dose (40 μM). Meanwhile, neither numerical chromosomal changes nor DNA adduct formation, analyzed by the nuclease P1 enhancement version of 32P‐post‐labeling, were induced by treatment with phenolphthalein at any concentrations examined. We thus report cell‐transforming activity and mutagenicity of phenolphthalein assessed with the same mammalian cells in culture. Our results provide evidence that phenolphthalein has cell‐transforming and genotoxic activity in cultured mammalian cells. The mutagenic and clastogenic activities of phenolphthalein could be a causal mechanism for carcinogenicity in rodents. Int. J. Cancer 73:697–701, 1997. Published 1997 Wiley‐Liss, Inc.

Immortalization of normal human gingival keratinocytes and cytological and cytogenetic characterization of the cells

Most in vitro studies of oral carcinogenesis in human cells are carried out with oral keratinocytes immortalized by human papillomavirus type 16 DNA. However, because various etiological factors for oral cancer are known, it is important to establish new human keratinocyte cell lines useful for studying the mechanism of oral carcinogenesis. Normal human gingival keratinocytes in secondary cultures grown in serum-free medium were either transfected with origin (−) SV40 DNA or sequentially transfected with origin (−) SV40 DNA and human c-fos. The transfected cells were continually passaged and analyzed for cytological and cytogenetic characterizations. Four immortal cell lines were grown for over 1100 days in culture and maintained a vigorous growth for over 250 population doublings. They expressed SV40 T antigen, cytokeratins 8 and 18, and E-cadherin, and overexpressed the c-Fos protein. The immortal cell lines had telomerase activity but lacked transformed phenotypes on soft agar or in nude mice. Each cell line had nonrandom chromosomal abnormalities and minisatellite alterations. One of the immortal cell lines, NDUSD-1, retained the capability to deposit calcium, which was also demonstrated in normal human gingival keratinocytes by alizarin red staining, indicating the possibility that NDUSD-1 cells may retain some natural characteristics of normal gingival keratinocytes. Because the oral ectoderm plays an important role in tooth development, these immortal cell lines may be useful in various experimental models for investigations of oral biology and oral carcinogenesis.

Modulation of Transforming and Clastogenic Activities of Catechol Estrogens by a Catechol-O-methyltransferase Inhibitor in Syrian Hamster Embryo Fibroblasts

Catechol estrogens (CEs) are considered critical intermediates in estrogen (E)-induced carcinogenesis. Previously, we demonstrated that estradiol (E2), estrone (E1), and four of their catechol estrogens, 2- and 4-OHE2 and 2- and 4-OHE1 induced morphological transformation in Syrian hamster embryo (SHE) cells, and their transforming activities varied as follows: 4-OHE1 > 2-OHE1 > 4-OHE2 > 2-OHE2 ≧ E1, E2, which are consistent with the genetic effects, i.e., chromosome aberrations and DNA adduct formation, of each E. To further elucidate the mechanism of hormonal carcinogenesis, we studied the effect of the catechol-O-methyltransferase (COMT) inhibitor Ro41-0960 on the transforming and clastogenic activities of the CEs using SHE cells. The frequencies of transformation and chromosome aberrations induced by 4-OHE1 were not affected by co-treatment with Ro-41-0960, but those induced by 2-OHE1 were markedly enhanced. The frequency of transformation induced by 4-OHE1 was markedly decreased by E2 in a concentration dependent manner, but this decrease was not inhibited by Ro41-0960. Cell treatment with E2, 2-OHE1, or 4-OHE1 alone induced apoptosis as detected by the TUNEL method. Additive effect on the induction of apoptosis was observed in cells treated with E2 + 2-OHE1 or 4-OHE1. The % apoptotic cells induced by E2 and 4-OHE1 decreased in the presence of Ro41-0960, while those induced by E2 and 2-OHE1 did not. These results suggest an important role of both the substrate specificity of COMT and the induction of apoptosis in CE-induced carcinogenesis.

Cellular Transforming Activity and Genotoxic Effects of 17β- Estradiol and its Eight Metabolites in Syrian Hamster Embryo Cells

The abilities of 17β-estradiol (E2) and its eight metabolites to induce cellular transformation and genetic effects were studied simultaneously using the Syrian hamster embryo (SHE) cell model. Treatment for 1–3 days with E2, estrone (E1), 2-hydroxyestrone (2- OHE1), 4-hydroxyestrone (4-OHEi), 2-methoxyestrone (2-MeOE1), 16α-hydroxyestrone (16α-OHE1), 2-hydroxyestradiol (2-OHE2), 4- hydroxyestradiol (4-OHE2), or estriol (E3) inhibited SHE cell growth in a dose-dependent manner. Morphological transformation in SHE cells were induced by exposure for 48 hr to each estrogen (E), except for E3. Somatic mutations in SHE cells at the Na+/K+ATPase and /or hprt loci were induced following treatment of cells with 4-OHE1, 2-MeOE1 or 4-OHE2 for 48 hr. Chromosome aberrations were elicited only in cells treated with 4-OHE1, 2- OHE1, 4-OHE2, 2-OHE2 or E1 for 24 hr. Aneuploidy induction in the near diploid range was observed in SHE cells treated with each E, except for 4-OHE1 and E3. The results indicate that the transforming activities of all Es tested correspond to at least one of genotoxic effects by each E, i.e., chromosome aberrations, aneuploidy or gene mutations, suggesting the involvement of genotoxicity in the initiation of E-induced carcinogenesis.