Takeki Tsutsui

Benzene-, catechol-, hydroquinone- and phenol-induced cell transformation, gene mutations, chromosome aberrations, aneuploidy, sister chromatid exchanges and unscheduled DNA synthesis in Syrian hamster embryo cells.

Benzene is a human carcinogen present naturally in petroleum and gasoline. For the simultaneous assessment of benzene-induced carcinogenicity and mutagenicity, benzene and its principal metabolites, phenol, catechol and hydroquinone were examined for their ability to induce cell transformation and genotoxic effects using the same mammalian cells in culture. Each of the four compounds induced morphological transformation of Syrian hamster embryo (SHE) cells. Catechol was the most potent, inducing transformation at concentrations of 1-30 microM, followed by hydroquinone (3-30 microM), phenol (10-100 microM) and benzene (only at 100 microM). Gene mutations at two loci in SHE cells were induced by all four compounds, with catechol being the most potent; both ouabain-resistant and 6-thioguanine-resistant mutant frequencies were increased. Chromosomal aberrations in SHE cells were especially induced by catechol, lesser by hydroquinone, and to a marginal extent by phenol at only the 100 microM concentration, whereas sister chromatid exchanges in SHE cells occurred with hydroquinone (1-30 microM), catechol (10-30 microM) and phenol (1000-3000 microM). Aneuploidy in the near diploid range of SHE cells was significantly induced by benzene and catechol. All three metabolites induced unscheduled DNA synthesis in SHE cells, whereas benzene did not. This is the first report that the cell transforming activity and mutagenicity of benzene and its metabolites were assessed with the same mammalian cells in culture. The results provide evidence that benzene and several of its metabolites are cell transforming and genotoxic to cultured mammalian cells.

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.

Cytotoxicity, chromosome aberrations and unscheduled DNA synthesis in cultured human diploid fibroblasts induced by sodium fluoride

The effects of exposure of cultured human diploid fibroblasts (JHU-1 cells) to sodium fluoride have been studied with respect to cytotoxicity and induction of chromosome aberrations and unscheduled DNA synthesis (UDS) Cytotoxicity of NaF on JHU-1 cells, as determined by a decrease in colony-forming ability, linearly increased with increasing dose of NaF (50-150 micrograms/ml) or exposure time (1-24 h). Treatment of the cells with 50 micrograms/ml NaF for 24 h resulted in a lethality (approximately 70%) similar to that obtained with 100 micrograms/ml for 12 h. A linear increase in cytotoxicity was observed as a fraction of the product of NaF treatment time and dose. JHU-1 cells treated with 20-50 micrograms/ml NaF for 12 or 24 h were analyzed for chromosome aberrations. A significant increase in the frequency of chromosome aberrations at the chromatid level was observed in treated cells in a dose-dependent manner. For detection of UDS, confluent JHU-1 cells were cultured with medium containing low serum and then exposed to NaF in the presence of 10 mM hydroxyurea. Treatment with 100-400 micrograms NaF/ml for 4-24 h reproducibly elicited UDS in a dose-related fashion as determined by direct scintillation counting of [3H]thymidine incorporated into DNA during repair synthesis. These results suggest that NaF causes DNA damage in human diploid fibroblasts in culture.

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.

Transformation by inorganic arsenic compounds of normal Syrian hamster embryo cells into a neoplastic state in which they become anchorage-independent and cause tumors in newborn hamsters†

Arsenic is a known human carcinogen, but little evidence exists for its carcinogenicity in animals. In order to investigate the ability of inorganic arsenics to transform normal cells into a neoplastic state, mass cultures of normal, diploid Syrian hamster embryo (SHE) cells exposed to various concentrations of sodium arsenite or sodium arsenate for 48 hr were continually passaged and tested for neoplastic transformation, as determined by anchorage‐independent growth in semisolid agar and tumorigenicity in newborn hamsters. Twenty‐one of 22 (96%) untreated, control cultures senesced by 20 passages. While 1 culture escaped senescence, it did not acquire the ability to either grow in semisolid agar or form tumors in animals. Ten of 14 (71%) cultures exposed to sodium arsenite or sodium arsenate escaped senescence. Nine of the 10 (90%) arsenic‐treated immortal cultures acquired the anchorage‐independent phenotype. Five of 5 anchorage‐independent cultures examined were tumorigenic. Two of 3 morphologically transformed colonies induced by sodium arsenate also acquired the ability to grow in semisolid agar when isolated. Amplification of the c‐myc or c‐Ha‐ras oncogene was detected in 3 of 5 and 4 of 5 tumorigenic cell lines, respectively. Both c‐myc and c‐Ha‐ras were amplified even in a preneoplastic, anchorage‐dependent cell line, but neither was amplified in 6 of 9 anchorage‐independent cell lines. Overexpression of c‐myc and c‐Ha‐ras mRNA was observed in most of the neoplastically transformed cell lines but not in the preneoplastic cell line. Experiments using the methylation‐sensitive restriction endonuclease isoschizomers HpaII and MspI revealed hypomethylation of c‐myc and c‐Ha‐ras in the 5′‐CCGG sequence of arsenic‐exposed cell lines but not in the parental SHE cells or a spontaneously transformed cell line. Thus, inorganic arsenics induce neoplastic transformation of normal, diploid mammalian cells. Overexpression of oncogenes by DNA hypomethylation may participate in the arsenic‐induced neoplastic transformation of mammalian cells. Published 2002 Wiley‐Liss, Inc.

Role of Aneuploidy in Early and Late Stages of Neoplastic Progression of Syrian Hamster Embryo Cells in Culture

There is increasing evidence in support of the somatic mutation theory of carcinogenesis (2,6,8,12–15,21,36,49,50,56,57,60,70). The recent demonstrations of the involvement of point mutations in the activation of the ras oncogenes (49,60), the associations of chromosomal rearrangements with proto-oncogenes (50,70), and the amplification of oncogenes (36,56,57) in certain tumors add to other lines of evidence, such as the demonstration of DNA as a critical target in neoplastic transformation (6,8) and the correlation between mutagenesis and carcinogenesis (2,8,12,13) with most chemicals (see Ref. 8, 41, and 51 for discussion of the arguments against the somatic mutation theory).

Induction of unscheduled DNA synthesis in cultured human oral keratinocytes by sodium fluoride

The effect of treatment of cultured human oral keratinocytes with sodium fluoride (NaF) has been investigated with respect to induction of unscheduled DNA synthesis (UDS). Oral keratinocytes were isolated from excised buccal mucosa of normal individuals by trypsinization at 4 degrees C overnight, followed by separation of the epithelium of mucosa from lamina propria mucosae with forceps. Isolated cells were cultured in vitro and all experiments were performed with secondary cultures. For detection of UDS, the keratinocytes were cultivated with medium containing 1% fetal calf serum (FCS) for 2 days and then treated with 100-300 micrograms/ml NaF for 4 h in medium containing 1% FCS and 10 mM hydroxyurea (1% FCS-HU medium). Following treatment with NaF, UDS was measured by direct scintillation counting of [3H]thymidine incorporated into DNA of the cells in 1% FCS-HU medium. Significant levels of UDS were induced in a dose-related fashion by NaF treatment. The results suggest that NaF causes DNA damage in cultured human oral keratinocytes.

Estrogen-induced cell transformation and DNA adduct formation in cultured Syrian hamster embryo cells

To study the possible involvement of DNA damage in cell transformation induced by estrogens, we examined whether DNA adduct formation is elicited in cultured Syrian hamster embryo (SHE) cells treated with estrogens and their derivatives by means of the 32P‐postlabeling assay. Morphological transformation of the cells was induced by treatment with diethylstilbestrol (DES) at 1–10 μg/mL for 24 h but not by treatment with its derivatives trans, trans‐dienestrol (α‐DIES) or cis, cis‐dienestrol (β‐DIES) at 1–10 μg/mL for 24 h. Similarly, DNA adduct formation was elicited by exposure of SHE cells to DES at 1–10 μg/mL for 24 h but not by either α‐DIES or β‐DIES. Treatment of SHE cells with DES at 1–10 μg/mL for 2 h in the presence of exogenous metabolic activation with rat liver post‐mitochondrial supernatant enhanced morphological transformation in a dose‐dependent manner. Our previous studies have demonstrated that exposure of SHE cells to DES under the same conditions with exogenous metabolic activation induces somatic mutations at the Na+/K+ ATPase locus. Therefore, we examined whether with exogenous metabolic activation DES induced DNA adduct formation in SHE cells. DNA adducts were not detected when SHE cells were treated with DES at 1–10 μg/mL for 2 h in the presence of exogenous metabolic activation. Treatment with 17β‐estradiol (E2), 2‐hydroxyestradiol (2‐OH E2), or 4‐hydroxyestradiol (4‐OH E2) at 1 μg/mL for 24 h induced DNA adduct formation in the cells, in parallel with the induction of cell transformation. The rank order of DNA adduct formation was 4‐OH E2 > 2‐OH E2 > E2. The results indicate that estrogens induce DNA adduct formation in cultured SHE cells, but the induction may not be the only mechanism relevant to the initiation of cell transformation.

IMMORTALIZATION OF NORMAL HUMAN EMBRYONIC FIBROBLASTS BY INTRODUCTION OF EITHER THE HUMAN PAPILLOMAVIRUS TYPE 16 E6 OR E7 GENE ALONE

The ability of the human papillomavirus type 16 (HPV‐16) E6 or E7 gene to induce immortalization of normal human embryonic fibroblast WHE‐7 cells was examined. WHE‐7 cells at 9 population doublings (PD) were infected with retrovirus vectors encoding either HPV‐16 E6 or E7 alone or both E6 and E7 (E6/E7). One of 4 isolated clones carrying E6 alone became immortal and is currently at >445 PD. Four of 4 isolated clones carrying E7 alone escaped from crisis and are currently at >330 PD. Three of 5 isolated clones carrying E6/E7 were also immortalized and are currently at >268 PD. The immortal clone carrying E6 only and 2 of the 3 immortal clones carrying E6/E7 expressed a high level of E6 protein, and all the immortal clones carrying E7 alone and the other immortal clone carrying E6/E7 expressed a high level of E7 protein when compared to their mortal or precrisis clones. The immortal clones expressing a high level of E6 or E7 protein were positive for telomerase activity or an alternative mechanism of telomere maintenance, respectively, known as ALT (alternative lengthening of telomeres). All the mortal or precrisis clones were negative for both phenotypes. All the immortal clones exhibited abrogation of G1 arrest after DNA damage by X‐ray irradiation. The expression of INK4a protein (p16INK4a) was undetectable in the E6‐infected mortal and immortal clones, whereas Rb protein (pRb) was hyperphosphorylated only in the immortal clone. The p16INK4a protein was overexpressed in all the E7‐infected immortal clones and their clones in the pre‐crisis period as well as all the E6/E7‐infected mortal and immortal clones, but the pRb expression was downregulated in all of these clones. These results demonstrate for the first time to our knowledge that HPV‐16 E6 or E7 alone can induce immortalization of normal human embryonic fibroblasts. Inactivation of p16INK4a/pRb pathways in combination with activation of a telomere maintenance mechanism is suggested to be necessary for immortalization of normal human embryonic fibroblasts by these viral oncogenes. The susceptibility of human cells to immortalization may be related to the state of differentiation of the cells.