Involvement of p53 function in different magnitude of genotoxic and cytotoxic responses in in vitro micronucleus assays.
In in vitro micronucleus (MN) assays the sensitivity to MN induction or cytotoxicity can vary depending on the kind of cells employed. This study was conducted to examine the involvement of the p53 function in the different sensitivities between Chinese hamster lung (CHL) cells and human lymphoblastoid TK6 cells in MN assays. MN induction and cytotoxicity were compared using MN-inducing chemicals reported as DNA reactive clastogens, non-DNA reactive clastogens or aneugens. The study revealed that the maximum levels of MN induction in p53-compromised CHL cells were higher than those in p53-competent TK6 cells, but MN were significantly induced in TK6 cells at lower concentrations than in CHL cells. Most of the test chemicals produced a more severe cytotoxicity in TK6 cells, suggesting TK6 cells are more sensitive for cytotoxicity than CHL cells. An additional experiment with 9 MN inducers revealed that the magnitude of MN induction and cytotoxicity were comparable between p53-competent TK6 cells and its p53-null mutant NH32 cells at the same concentrations. Furthermore, the MN frequencies induced by methylmethane sulfonate, aphidicolin and hydroxyurea in NH32 cells were identical to those in TK6 cells at different recovery times. From these results, it is suggested that the p53 abrogation does not explain the difference in sensitivity to MN induction or cytotoxicity between CHL and TK6 cells. In this regard, p53 abrogated NH32 cells can be an option for the in vitro MN assay.
Hashimoto K
，Nakajima Y
，Uematsu R
，Matsumura S
，Chatani F
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Comparison of four different treatment conditions of extended exposure in the in vitro micronucleus assay using TK6 lymphoblastoid cells.
In the OECD Guideline 487, a total of four extended exposure treatment conditions are proposed for the in vitro micronucleus (MNvit) assay in the presence and absence of a cytokinesis block and with or without a recovery period. This guideline also states that rodent cell lines and human lymphocytes can be used as shown by many validated studies but that human cell lines such as TK6 and HepG2 are not yet validated. In this present study each extended exposure condition was characterized by investigation using TK6 cells and nine chemicals known to be able to induce micronucleus (MN) in rodent cell lines. The results revealed two concerns: six chemicals did not show significant MN induction in the 'cytokinesis block without recovery period'; two aneugens showed no dose-dependent cytotoxicity in the 'cytokinesis block with recovery period'. Further investigation revealed that 3-4 times higher spontaneous MN frequency than that in the other conditions is a possible reason for the low sensitivity, and this high spontaneous MN frequency was not observed in Chinese hamster lung cells under the identical treatment condition. With regard to the two conditions without cytokinesis block, two negative substances were evaluated and found to be negative, suggesting the validity of the TK6 test system for these conditions. Although our findings showed a few concerns for the treatment with cytokinesis block, the TK6 cells were considered to be a reliable cell line to be used for detecting potential inducers of MN in the in vitro micronucleus assay based on the overall results.
Hashimoto K
，Nakajima Y
，Matsumura S
，Chatani F
... -
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Reduction of misleading ("false") positive results in mammalian cell genotoxicity assays. I. Choice of cell type.
Current in vitro mammalian cell genotoxicity assays show a high rate of positive results, many of which are misleading when compared with in vivo genotoxicity or rodent carcinogenicity data. P53-deficiency in many of the rodent cell lines may be a key factor in this poor predictivity. As part of an European Cosmetics Industry Association initiative for improvement of in vitro mammalian cell assays, we have compared several rodent cell lines (V79, CHL, CHO) with p53-competent human peripheral blood lymphocytes (HuLy), TK6 human lymphoblastoid cells, and the human liver cell line, HepG2. We have compared in vitro micronucleus (MN) induction following treatment with 19 compounds that were accepted as producing misleading or "false" positive results in in vitro mammalian cell assays [6]. Of these, six chemicals (2-ethyl-1,3-hexandiol, benzyl alcohol, urea, sodium saccharin, sulfisoxazole and isobutyraldehyde) were not toxic and did not induce any MN at concentrations up to 10mM. d,l-Menthol and ethionamide induced cytotoxicity, but did not induce MN. o-Anthranilic acid was not toxic and did not induce MN in V79, CHL, CHO, HuLy and HepG2 cells up to 10mM. Toxicity was induced in TK6 cells, although there were no increases in MN frequency up to and above the 55% toxicity level. The other 10 chemicals (1,3-dihydroxybenzene, curcumin, propyl gallate, p-nitrophenol, ethyl acrylate, eugenol, tert-butylhydroquinone, 2,4-dichlorophenol, sodium xylene sulfonate and phthalic anhydride) produced cytotoxicity in at least one cell type, and were evaluated further for MN induction in most or all of the cell types listed above. All these chemicals induced MN at concentrations <10mM, with levels of cytotoxicity below 60% (measured as the replication index) in at least one cell type. The rodent cell lines (V79, CHO and CHL) were consistently more susceptible to cytotoxicity and MN induction than p53-competent cells, and are therefore more susceptible to giving misleading positive results. These data suggest that a reduction in the frequency of misleading positive results can be achieved by careful selection of the mammalian cell type for genotoxicity testing.
Fowler P
，Smith K
，Young J
，Jeffrey L
，Kirkland D
，Pfuhler S
，Carmichael P
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Relationships between p53 status, apoptosis and induction of micronuclei in different human and mouse cell lines in vitro: Implications for improving existing assays.
Accumulated evidence has shown that in vitro mammalian cell genotoxicity assays produce high frequencies of "misleading" positive results, i.e. predicted hazard is not confirmed in in vivo and/or carcinogenicity studies [1], raising the question of relevance to human risk assessment. A recent study of micronucleus (MN) induction [2] showed that commonly used p53-deficient rodent cell lines (CHL, CHO and V79) gave a higher frequency of "misleading" positive results with 9 non-DNA reactive, Ames-negative and in vivo negative chemicals [3] than human p53-competent cells (blood lymphocytes, TK6 and HepG2 cell lines). This raised the question of whether these differences were due to p53 status or species origin. This present study compared human versus mouse and p53-competent versus p53-mutated function. The same 9 chemicals were tested for induction of MN in mouse lymphoma L5178Y (mutated p53), human TK6 (functional p53) and WIL2-NS (TK6 related, with mutated p53) cells. Six chemicals provided clear positive increases in MN frequency in at least one cell type. L5178Y cells yielded clear positive responses with more chemicals than either TK6 or WIL2-NS, indicating origin rather than p53 functionality was most relevant. Apoptosis induction (measured via caspase-3/7) was also investigated with clear differences in the timing and extent of apoptosis induction between mouse and human cells noted. With curcumin in TK6 cells, induction of caspase-3/7 activity coincided with MN induction, whereas for L5178Y cells, MN induction occurred in the absence of increased caspase activity. By contrast, with MMS in TK6 cells, MN induction preceded increased caspase-3/7 activity. These data suggest that MN induction by "misleading positive" genotoxins in p53-competent human cell lines may result from apoptosis, whereas in p53-defective rodent cells such as L5178Y, MN induction may be independent of apoptosis.
Whitwell J
，Smith R
，Jenner K
，Lyon H
，Wood D
，Clements J
，Aschcroft-Hawley K
，Gollapudi B
，Kirkland D
，Lorge E
，Pfuhler S
，Tanir JY
，Thybaud V
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