Mutagenesis Advance Access originally published online on August 4, 2005
Mutagenesis 2005 20(5):375-379; doi:10.1093/mutage/gei050
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3-Chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone [MX] shows initiating and promoting activities in a two-stage BALB/c 3T3 cell transformation assay
1Genetic Toxicology Group, Biosafety Research Center, Foods, Drugs and Pesticides, 582-2, Shioshinden, Fukude-cho, Iwata-gun Shizuoka 437-1213, Japan, 2Laboratory of Food Hygiene, School of Food and Nutritional Sciences, COE Program in 21st Century University of Shizuoka, 52-1, Yada, Shizuoka 422-8526, Japan and 3Division of Genetics and Mutagenesis, National Institute of Health Sciences, 1-18-1, Kamiyoga, Setagaya-ku, Tokyo 158-8501, Japan
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Abstract |
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A transformation assay using BALB/c 3T3 cells was conducted on 3-chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone (MX) to assess initiation and promotion activities of MX carcinogenesis. Statistically significant positive responses were obtained compared with the corresponding solvent controls in both the initiation assay post-treated with 12-O-tetradecanoylphorbol 13-acetate (TPA) and the promotion assay pretreated with 3-methylcholanthrene (MCA). Both TPA and MX inhibited metabolic cooperation in an assay using co-culture of V79 6-thioguanine (6-TG) sensitive and insensitive cells. However, cells isolated from transformed foci in the initiation assay did not induce any nodules after inoculation to BALB/c mice, the strain of mouse from which the transformation assay cells were derived. Although the study was carried out for 2–3 weeks, this might have been too short to develop nodules under the conditions of this experiment. This in vitro cell transformation study with MX adds supportive information to studies showing MX carcinogenicity and tumour promoter activity, and adds mechanistic understanding of the action of MX.
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Introduction |
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3-Chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone (MX), an organic chlorinated contaminant commonly found in tap water as a disinfection by-product (1
), induces malignant tumours in the rat thyroid and mammary glands (2) and shows promoting activity in the rat stomach (3). Concerning mutagenicity, MX shows positive results in several in vitro assays, including the bacterial reverse mutation assay (Ames test) and the cultured cell mutation assay regardless of the presence of an exogenous metabolic activation system. Therefore, MX is classified as a direct acting mutagen (4–9). However, conflicting results have been reported for MX activity in in vivo genotoxicity assays (4,5,10–12). We have also performed several in vivo assays, namely, detection of 8-hydroxy-deoxyguanosine formation, gene mutation assay using transgenic mice (MutaTMMouse), and the comet assay, and almost all of the assays gave negative responses (M. Nakajima, S. Masumori, M. Kikuchi, S. Inagaki, J. Tanaka, Y. Furuya, M. Hayashi and N. Kinae, manuscript in preparation). Thus, the genotoxicity findings have not provided a conclusive mechanism for MX carcinogenicity.
Among the several types of cell transformation assays, we selected the focus assay using the BALB/c 3T3 established cell line for the present study because of its good reproducibility (13) and because the assay has been widely used in the elucidation of carcinogenicity mechanisms. We used A31-1-1 clone, isolated by Kakunaga and Crow (14) and thought to be sensitive to many chemicals, and employed a protocol with modified culture medium and shortened exposure period (15). The modified method can assess the potential initiation and promotion activities of test chemicals.
Many tumour promoters inhibit gap junctional intercellular communication (GJIC). For example, phorbol esters such as the strong promoter 12-O-tetradecanoylphorbol 13-acetate (TPA) inhibit GJIC in metabolic cooperation assays (16–18). As such, the metabolic cooperation assay has been widely used as a tool for detection of promoters and for providing information about the mechanism of carcinogenicity. Therefore, we applied the assay using V79 cells to assess the promoting potential of MX.
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Materials and methods |
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Test compounds and positive control substance
MX (CAS No. 77439-76-0, purity: 99.2%) was synthesized at the Laboratory of Food Hygiene, University of Shizuoka according to a modified method (19
). It was dissolved in physiological saline, Japanese Pharmacopoeia (Otsuka Pharmaceutical Factory, Inc.). 3-Methylcholanthrene (CAS No. 56-49-5; MCA; Wako Pure Chemical Industries Ltd, Osaka, Japan), the initiator, was dissolved in dimethylsulfoxide (DMSO; purity: 99.7% or higher: Merck KGaA, Darmstadt, Germany) and used at a concentration of 0.2 µg/ml. TPA (CAS No. 16561-29-8; Wako Pure Chemical Industries Ltd), the promoter, was dissolved in DMSO and used at 0.1 µg/ml.
Preparation of test cells and culture medium
BALB/c 3T3 A31-1-1 cells were supplied by Showa Denko K.K. Cells were cultured in a CO2 incubator (FORMA and SANYO Electric Medica Systems Co. Ltd) under 5% CO2 atmosphere at 37°C. Eagles-MEM liquid medium (Asahi Techno Glass Corporation, Funabashi, Chiba) supplemented with 10% fetal bovine serum (FBS; Moregate BioTech, Bulimba, Australia) and 60 mg/ml Kanamycin sulfate (Invitrogen Corp., NY) (MEM) was used throughout the experiment unless otherwise indicated. Low serum concentration medium (DMEM:F12) used was DMEM:HAM's F-12 liquid medium (Asahi Techno Glass Corporation) supplemented with 5.2 ml Daigo's ITES (insulin, transferrin, ethanolamine, sodium selenite; Wako Pure Chemical Industries Ltd) per 500 ml and 2% FBS. The 500 ml extracting medium for absorption measurement was composed of 4.48 g sodium citrate dihydrate, 97.5 ml of 0.1 mol/l HCl, and 250 ml ethanol made to 500 ml with distilled water. For the metabolic cooperation assay using V79 cells, the medium used was Eagles-MEM liquid medium supplemented with 3% fetal bovine serum, 0.1% Eagle's non-essential amino acids (Invitrogen Corp.), 0.1% pyruvic acid and 0.1% glutamic acid.
Dose range-finding cytotoxicity test
For cytotoxicity testing with the initiation assay protocol, 1 x 103 cells were seeded into 24-well plates and treated 24 h later with MX at 1.42, 1.90, 2.53, 3.38, 4.50, 6.00 and 8.00 µg/ml. The culture medium was removed 72 h later, and the cells were fixed with 10% neutral buffered formalin and stained with 0.1% crystal violet for 30 min. For cytotoxicity testing with the promotion assay protocol, cells were seeded as above and MEM was replaced with DME/F12 medium 48 h after seeding. The cells were then treated with 1.68, 2.10, 2.62, 3.28, 4.10, 5.12 and 6.40 µg/ml of MX, and fixed and stained 96 h after cell seeding in the same manner as for the initiation assay cytotoxicity test. Extractant (1.5 ml) was placed in each well for 10 min and then absorption was measured with a spectrophotometer set at 580 nm. Cell survival at each dose was calculated relative to the negative vehicle control group. The MX concentration that inhibited cell growth by 50% (IC50 value) was calculated using the Probit method and approximately twice the IC50 was selected as the highest concentration for the cell transformation assays.
Cell transformation assays
In the initiation assay, 1.2 x 104 cells were seeded into 60 mm diameter culture dishes, 12 dishes per concentration, and control groups. After 24 h incubation, DMSO as negative control, MX as the initiator (1.64, 2.05, 2.56, 3.20 and 4.00 µg/ml) or MCA as a positive control (0.2 µg/ml) was added. Seventy-two hours after treatment, MEM was replaced with fresh DME/F12. On the 7th day after the beginning of treatment, TPA (0.1 µg/ml) or DMSO was added to cultures as the first promoter treatment. For the second and third promoter treatments, TPA or DMSO was added on the 11th and 14th day, respectively.
In the promotion assay, MCA (0.2 µg/ml, as the initiator) or DMSO was added 24 h after seeding 1.2 x 104 cells per dish. On the 4th day, MEM was replaced with fresh DME/F12. On the 7th day, saline, MX (0.156, 0.313, 0.625, 1.25 and 2.50 µg/ml) or TPA (positive control; 0.1 µg/ml) was added to the culture. MX and TPA were also added on the 11th and 14th day.
The cells were fixed with methanol and stained with 2.5% Giemsa solution on the 25th day for both assays. The foci that met the following criteria were counted as transformant: (i) 2 mm or more in diameter, (ii) criss-cross growth pattern, (iii) layering of cells and (iv) deep basophilic staining.
Tumourigenicity of transformed cells
Six-week-old male BALB/c CR mice were purchased from Japan Slc, Inc. (Shizuoka, Japan), and were quarantined and acclimated to the testing facility for 1 week. They were given pelleted diet (MF: Oriental Yeast Co., Ltd) and tap water ad libitum through the acclimation and assay periods.
At the end of the transformation assay with MX (4.0 µg/ml as initiator and 2.5 µg/ml as promoter; experimental data not shown), cultures were washed once with Dulbecco's PBS. Cells were isolated from transformed foci by trypsinization and mass cultured. An aliquot of 0.2 ml of cell suspension (1 x 106 cells for Experiment 1 and 1.5 x 106 cells for Experiment 2) was injected subcutaneously into the cervical region of the BALB/c CR mice. In both experiments, the cells isolated from the transformed foci in the negative control groups were inoculated into three animals and cells isolated from MX-induced transformed foci were inoculated into four animals. All animals were examined 2 weeks (Experiment 1) or 3 weeks (Experiment 2) after inoculation.
Metabolic cooperation assay
6-Thioguanine (6-TG) sensitive V79 cells (6-TGs; 4 x 105 cells) and 6-TG resistant cells (6-TGr; 200 cells) were co-cultured to evaluate the inhibition of metabolic cooperation (5 dishes per concentration). For calculating cytotoxicity 200 V79 [6-TGs or 6-TGr] cells alone were plated (3 dishes per concentration). Cells were treated with either MX or TPA 4 h after seeding. The 6-TG (10 µg/ml) was added 15 min after MX or TPA treatment and cells cultured for an additional 3 days before the medium was replaced with fresh medium containing only 6-TG; the cells were cultured another 4 days. The cells were fixed in ethanol and stained with 0.1% crystal violet for 10 min. Colonies with 50 or more cells were counted. These colonies developed from cells that were either not in GJIC contact with 6-TGs cells or were in contact but then ‘rescued’ by GJIC inhibition from test chemical action. The assay is based on toxicity of 6-TG to 6-TGsV79 cells (HGPRT+), non-toxicity of 6-TG to 6-TGr mutant V79 cells (HGPRT–), with toxicity to these latter cells if in GJIC contact with HGPRT+ cells, which transfer the HGPRT-catalysed toxic 6-TG metabolite via gap junctions to the HGPRT– cells. Inhibition of GJIC rescues the contacting mutant cells to allow their clonal expansion (20).
Statistical analysis
The percentage of dishes with foci and the mean number of foci per dish were analysed using Fisher's exact test and the Wilcoxon's rank sum test, respectively.
In the metabolic cooperation assay, the number of 6-TGr colonies was analysed for difference from the negative control group using Dunnett's test.
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Results |
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Initiation assay
In the cytotoxicity assay for dose-range finding, a concentration-dependent decrease in cell survival was observed with MX treatment (Figure 1). An IC50 value using Probit's method was calculated to be 1.92 µg/ml. In the negative control group (saline initiation–TPA promotion) the mean number of transformed foci per dish was 0.50 and the percentage of dishes with foci was 41.7% (5 of 12 dishes, Table I). When MX was used as an initiator at 1.64, 2.05, 2.56, 3.20 and 4.00 µg/ml, and with DMSO post-treatment, no significant increase in transformation was observed. In the five groups treated with 1.64–4.00 µg/ml MX (as initiator) and TPA (0.1 µg/ml, as promoter) the numbers of foci per dish were 0.58, 1.25, 1.92, 1.82 and 3.64, respectively. A statistically significant increase (P < 0.05) was observed at MX
2.56 µg/ml compared with the negative control group. The number of dishes with foci also increased in a concentration-dependent manner and foci were observed in all dishes at the highest concentration (4.00 µg/ml) group. A large number of foci were induced in the positive control and MCA initiation-TPA promotion group; the mean number of foci per dish was 7.58, and all dishes contained foci.
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Promotion assay
In the cytotoxicity assay, a concentration-dependent decrease in cell survival with MX treatment was observed (Figure 2). The IC50 value was 3.37 µg/ml. The experimental doses for promotion assay included at least 2 doses expecting to have
90% cell survival rate. In the negative control group (MCA initiation–saline promotion), the mean number of foci per dish was 0.25 and the percentage of dishes with foci was 25% (3 out of 12 dishes, Table II). After initiation treatment with DMSO, cells were treated with MX at 0.156, 0.313, 0.625, 1.25 and 2.50 µg/ml as the promoter. The numbers of foci were 0.00, 0.08, 0.00, 1.10 and 0.90 per dish, respectively, with only the highest dose eliciting a significant increase in dishes with foci (Table II). In the groups treated with MCA (0.2 µg/ml) and MX at the above five concentrations, the numbers of foci were 0.25, 0.92, 1.33, 3.40 and 6.18 per dish, respectively. A statistically significant increase (P < 0.05) was observed at concentrations 0.625 µg/ml compared with the negative control group. The number of dishes with foci also increased concentration-dependently, and foci were observed in all dishes of the 1.25 and 2.50 µg/ml groups. The positive control group (MCA initiation–TPA promotion) confirmed the effectiveness of MCA/TPA in this cell transformation assay.
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Tumourigenicity assay
Gross examination of the mice necropsied in tumourigenicity experiments 1 and 2 did not reveal any visible nodules or tissue masses in any organs of any animals.
Inhibition of metabolic cooperation assay
The mean number of 6-TGr colonies increased dose-dependently in the MX-treated groups (Figure 3), indicating an inhibition of GJIC by MX. This occurred at non-cytotoxic concentrations. The mean number of 6-TGr colonies at 1.12 µg/ml of MX was 188.4 (=94.2%) compared with 47.6 (=23.8%) in the negative control. In the positive control (TPA-treated) group the mean number of 6-TGr colonies was 159.4 (=79.7%).
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Discussion |
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From a public health viewpoint it is important to understand the toxicology of MX. This is particularly evident since long-term animal studies have shown carcinogenic and tumour promoting activity of MX (2
,3). In the present study, we conducted transformation assays on MX using BALB/c 3T3 cells to give any supportive data for the carcinogenic potential of this chemical as revealed in the long-term and promotion bioassays.
MX induced statistically significant positive responses in both initiation and promotion cell transformation assays. These findings agree with positive outcomes using a C3H 10T1/2 cell transformation assay system (21) and also give supportive information to the carcinogenicity of MX (2). However, tumours were not observed after in vivo inoculation of a large number of transformed cells harvested from the initiation assay in which MX was used as the initiator and TPA as the promoter. This may be owing to the fact that the BALB/c 3T3 cells of the transformation assay were derived from a BALB/c mouse strain as used for the tumourigenicity assay, or may be related to the rather short in vivo expression period. We did not perform a similar experiment using immune-deficient nude mice. However, within 2–4 months Boone and Jacobs reported the induction of tumours in BALB/c mice by inoculation of transformed cells (22). Although we carried out the study for 2–3 weeks, the period might have been too short to develop nodules. Cells isolated from transformed foci in the initiation assay did not induce any nodules after inoculation to BALB/c mice, the strain of mouse from which the transformation assay cells were derived. Taken together, we could not adjudge the malignancy of transformed cells induced by MX when used as an initiator.
The possibility of an in vivo promoting effect of MX was revealed by the positive result in the promotion assay using BALB/c 3T3 cells. Moreover, this result was supported by the demonstration that MX inhibited GJIC, which is a characteristic of many tumour promoters evaluated using the metabolic cooperation assay (8). The major role of GJIC is considered to be the maintenance of homeostasis in multicellular organisms, and it is believed that second messenger transfer through GJIC is important for cell growth control (23,24). Tumour-promoting chemicals such as TPA and analogues, DDT and aldrin inhibit GJIC (25–27), and this in vitro test for tumour promoters is recommended as a useful tool for detecting non-genotoxic carcinogens (28). This activity of MX in the current GJIC assay is consistent with a recent report on GJIC inhibition in BALB/c 3T3 cells (29).
MX appears to have weak genotoxicity in mammalian systems in vivo, and it is probable that the tumour promoting activity of MX is important for explaining its carcinogenic activity. Although many regulatory bodies assess chemical safety based on the dogma that genotoxic carcinogens do not have any threshold, we propose that risk assessment of MX takes into account the chemical's likely threshold as a tumour promoter.
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Notes |
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* To whom correspondence should be addressed. Tel: +81 538 58 3572; Fax: +81 538 58 1368; Email: nakajima{at}anpyo.or.jp
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1. Meier,J.R., Knohl,R.B., Coleman,W.E., Ringhand,H.P., Munch,J.W., Kaylor,W.H., Streicher,R.P. and Kopfler,F.C. (1987) Studies on the potent bacterial mutagen, 3-chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone: aqueous stability, XAD recovery and analytical determination in drinking water and in chlorinated humic acid solutions. Mutat. Res., 204, 363–373.
2. Komulainen,H., Kosuma,V.M., Vaittinen,S.L., Vartiainen,T., Kaliste-Korhonen,E., Lotjonen,S., Tuominen,R.K. and Tuomisto,J. (1997) Carcinogenicity of 3-chloro-4-(dichloromethyl)-5-hydroxy-2[5H]-furanone in the rat. J. Natl Cancer Inst., 89, 848–856.
3. Nishikawa,A., Kinae,N., Furukawa,F., Mitsui,M., Enami,T., Hasegawa,T. and Takahashi,M. (1994) Enhancing effects of 3-chloro-4-(dichloromethyl)-5-hydroxy-2[5H]-furanone (MX) on cell proliferation and lipid peroxidation in the rat gastric mucosa. Cancer Lett., 85, 151–157.[CrossRef][Web of Science][Medline]
4. Meier,J.R., Blazak,W.F. and Knohl,R.B. (1987) Mutagenic and clastogenic properties of 3-chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone: a potent bacterial mutagen in drinking water. Environ. Mol. Mutagen., 10, 411–424.[Web of Science][Medline]
5. Tikkanen,L. and Kronberg,L. (1990) Genotoxic effects of various chlorinated butenoic acids identified in chlorinated drinking water. Mutat. Res., 240, 109–116.[CrossRef][Web of Science][Medline]
6. Brunborg,G., Holme,J.A., Soderlund,E.J., Hongslo,J.K., Vartiainen,T., Lotjonen,S. and Becher,G.H. (1991) Genotoxic effects of the drinking water mutagen 3-chloro-4-(dichloromethyl)-5-hydroxy-2[5H]-furanone (MX) in mammalian cells in vitro and in rats in vivo. Mutat. Res., 260, 55–64.[CrossRef][Web of Science][Medline]
7. Jansson,K. and Hyttinen,J.M. (1994) Induction of gene mutation in mammalian cells by 3-chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone (MX), a chlorine disinfection by-product in drinking water. Mutat. Res., 322, 129–132.[CrossRef][Web of Science][Medline]
8. Matsumura,H., Watanabe,M., Matsumoto,K. and Ohta,T. (1994) 3-Chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone (MX) induces gene mutations and inhibits metabolic cooperation in cultured Chinese hamster cells. J. Toxicol. Environ. Health, 43, 65–72.[Web of Science][Medline]
9. Miyamae,Y., Iwasaki,K., Kinae,N., Tsuda,S., Murakami,M., Tanaka,M. and Sasaki,Y.F. (1997) Detection of DNA lesions induced by chemical mutagens using the single-cell gel electrophoresis (comet) assay, 2. Relationship between DNA migration and alkaline condition. Mutat. Res., 393, 107–113.[Web of Science][Medline]
10. Nunn,J.W., Davies,J.E. and Chipman,J.K. (1997) Production of unscheduled DNA synthesis in rodent hepatocytes in vitro, but not in vivo, by 3-chloro-4-(dichloromethyl)-5-hydroxy-2[5H]-furanone (MX). Mutat. Res., 373, 67–73.[Web of Science][Medline]
11. Sasaki,Y.F., Nishidate,E., Izumiyama,F., Watanabe-Akanuma,M., Kinae,N., Matsusaka,N. and Tsuda,S. (1997) Detection of in vivo genotoxicity of 3-chloro-4-(dichloromethyl)-5-hydroxy-2[5H]-furanone (MX) by the alkaline single cell gel electrophoresis (Comet) assay in multiple mouse organs. Mutat. Res., 393, 47–53.[Web of Science][Medline]
12. Holme,J.A., Haddeland,U., Haug,K. and Brunborg,G. (1999) DNA damage induced by the drinking water mutagen 3-chloro-4-(dichloromethyl)-5-hydroxy-2[5H]-furanone (MX) in mammalian cells in vitro and in mice. Mutat. Res., 441, 145–153.[Web of Science][Medline]
13. Kakunaga,T. and Yamasaki,H. (eds) (1985) Transformation assay of established cell lines: mechanisms and application. IARC Scientific Publication No. 67, IARC, Lyon.
14. Kakunaga,T. and Crow,J.D. (1980) Cell variants showing differential susceptibility to ultraviolet light-induced transformation. Science, 209, 505–507.
15. Tsuchiya,T. and Umeda,M. (1995) Improvement in the efficiency of the in vitro transformation assay method using BALB/3T3 A31-1-1 cells. Carcinogenesis, 16, 1887–1894.
16. Murray,A.W. and Fitzgerald,D.J. (1979) Tumour promoters inhibit metabolic cooperation in cocultures of epidermal and 3T3 cells. Biochem. Biophys. Res. Commun., 91, 395–401.[CrossRef][Web of Science][Medline]
17. Yotti,L.P., Chang,C.C. and Trosko,J.E. (1979) Elimination of metabolic cooperation in Chinese hamster cells by a tumour promoter. Science, 206, 1089–1091.
18. Stedman,D.B. and Welsch,F. (1985) Effects of 6-thioguanine on communication competence and factors affecting reversibility of phorbol ester inhibition in V79 cells measured by the metabolic cooperation assay and by dye coupling. Carcinogenesis, 6, 1599–1605.
19. Padmapriya,A.A., Just,G. and Lewis,N.G. (1985) Synthesis of 3-chloro-4-(dichloromethyl)-5-hydroxy-2[5H]-furanone, a potent mutagen. Can. J. Chem., 63, 828–832.[CrossRef]
20. Yamasaki,H., Krutovskikh,V., Mesnil,M., Columbano,A., Tsuda,H. and Ito,N. (1993) Gap junctional intercellular communication and cell proliferation during rat liver carcinogenesis. Environ. Health Perspect., 101, 191–197.
21. Laaksonen,M., Maki-Paakkanen,J., Kronberg,L. and Komulainen,H. (2003) Effects of chlorohydroxyfuranones on 3-methylcholanthrene-induced neoplastic transformation in the two-stage transformation assay in C3H 10T1/2 cells. Arch. Toxicol., 77, 594–600.[CrossRef][Web of Science][Medline]
22. Boone,C.W. and Jacobs,J.B. (1976) Sarcomas routinely produced from putatively nontumorigenic Balb/3T3 and C3H/10T1/2 cells by subcutaneous inoculation attached to plastic platelets. J. Supramol. Struct., 5, 131–137.[CrossRef][Web of Science][Medline]
23. Loewenstein,W.R. (1979) Junctional intercellular communication and the control of growth. Biochim. Biophys. Acta, 560, 1–65.[Medline]
24. Yamasaki,H. (1990) Gap junctional intercellular communication and carcinogenesis. Carcinogenesis, 11, 1051–1058.
25. Fitzgerald,D.J. and Murray,A.W. (1983) A new intercellular communication assay: its use in studies on the mechanism of tumour promotion. Cell Biol. Int. Rep., 6, 235–242.[CrossRef]
26. Bohrman,J.S., Burg,J.R., Elmore,E., Gulati,D.K., Barfnecht,T.R., Niemeier,R.W., Dames,B.L., Toraason,M. and Langenbach,R. (1988) Interlaboratory studies with the Chinese hamster V79 cell metabolic cooperation assay to detect tumor-promoting agents. Environ. Mol. Mutagen., 12, 33–51.[Web of Science][Medline]
27. Noda,K. (1984) Effects of various chemicals including tumor promoters on metabolic cooperation. Yokohama Med. J., 35, 407–420.
28. Sakai,A., Iwase,Y., Nakamura,Y., Sasaki,K., Tanaka,N. and Umeda,M. (2002) Use of a cell transformation assay with established cell lines, and a metabolic cooperation assay with V79 cells for the detection of tumor promoters: a review. Altern. Lab. Anim., 30, 33–59.[Medline]
29. Hakulinen,P., Maki-Paakkanen,J., Naarala,J., Kronberg,L. and Komulainen,H. (2004) Potent inhibition of gap junctional intercellular communication by 3-chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone (MX) in BALB/c 3T3 cells. Toxicol. Lett., 151, 439–449.[CrossRef][Web of Science][Medline]
Received on January 5, 2005; revised on June 8, 2005; accepted on July 15, 2005.
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