Re: Yang, J. and Duerksen-Hughes, P. (1998) A new approach to identifying genotoxic carcinogens: p53 induction as an indicator of genotoxic damage. Carcinogenesis, 19, 1117–1125.

Ulla Stenius and Johan Högberg

Institute of Environmental Medicine, Karolinska Institutet, Box 210, S-171 77 Stockholm, Sweden

Dear Sir,

In a recent issue of the Journal, Yang and Duerksen-Hughes present a new in vitro approach for identifying genotoxic carcinogens. In this study they used p53 induction in vitro as an indicator of genotoxic damage. The authors suggest that this endpoint could be used as an effective tool for identifying environmental carcinogens.

In our opinion, p53 induction has clear weaknesses as an endpoint for in vitro genotoxicity testing. It is induced by many types of DNA damage and the most serious problem, which has not been addressed by the authors, are the data on p53 stabilizing mechanisms not related to DNA damage. Thus, p53 can be induced by factors interfering with the degradation of p53, and it was recently shown that inhibition of the ubiquinone pathway by proteasome inhibitors induces p53 and p53-dependent apoptosis (1). In addition, there are mechanisms involving alterations in p53 binding proteins. For example, inhibition of MDM2 gene expression by antisense oligonucleotide treatment (2) or altered MDM2 phosphorylation (3) may influence p53 induction and apoptosis. Also, hypoxia, a known p53 stabilizing factor, induces p53 as a result of hypoxia-inducible factor 1{alpha} activation (4). Furthermore, ribonucleotidyl depletion induces p53 in the absence of detectable DNA damage (5).

Considering this plethora of p53 inducing mechanisms and the wide range of effects that has been ascribed to toxins, the suggested endpoint seems far too unspecific. To illustrate our point, we can use a simple inorganic compound, selenite. The redox cycling metabolites of selenite may have at least two effects of interest: they may produce superoxide which can damage DNA and they may induce hypoxia (6). In addition, selenite may inhibit protein synthesis, which can attenuate any form of induction (7).

It has long been known that additional in vitro tests for genotoxicity do not necessarily add predictive power when testing chemicals for carcinogenicity (8). In order to improve in vitro testing, it seems most reasonable to develop specific tests, ideally one test for each type of DNA damage.

It may be added that data on in vivo genotoxicity is often lacking in risk assessment. A critical question is whether a compound, shown to be genotoxic in in vitro tests, induces genotoxicity in the target organ or in the target cell type. It is possible that histological data on in vivo p53 expression might prove useful in this situation, especially negative data suggesting lack of induction. Wild-type p53, stabilized by DNA damage, can readily be visualized in many organs, including liver, by sensitive immunohistochemical techniques (9).

References

  1. Lopes,U.G., Erhardt,P., Yao,R. and Coopers,G.M. (1997) p53-dependent induction of apoptosis by proteasome inhibitors. J. Biol. Chem., 272, 12893–12896.[Abstract/Free Full Text]
  2. Chen,L., Agrawal,S., Zhou,W., Zhang,R. and Chen,J. (1998) Synergistic activation of p53 by inhibition of MDM2 expression and DNA damage. Proc. Natl Acad. Sci. USA, 95, 195–200.[Abstract/Free Full Text]
  3. Mayo,L.D., Turchi,J.J. and Berberich,S.J. (1997) Mdm-2 phosphorylation by DNA-dependent protein kinase prevents interaction with p53. Cancer Res., 57, 5013–5016.[Abstract]
  4. An,W.G., Kanekal,M., Simon,M.C., Maltepe,E., Blagosklonny,M.V. and Neckers,L.M. (1998) Stabilization of wild-type p53 by hypoxia-inducible factor 1{alpha}. Nature, 392, 405–408.[ISI][Medline]
  5. Linke,S.P., Clarkin,K.C., DiLeonardo,A., Tsou,A. and Wahl,G.M. (1996) A reversible, p53-dependent G0/G1 cell cycle arrest induced by ribonucleotide depletion in the absence of detectable DNA damage. Genes Dev., 10, 934–947.[Abstract]
  6. Garberg,P., Ståhl,A., Warholm,M. and Högberg,J. (1991) Studies on the role of DNA fragmentation in selenium toxicity. Biochem. Pharmacol., 37, 3401–3406.
  7. Vernie,L., Bont,W. and Emmelot,P. (1974) Inhibition of in vitro amino acid incorporation by sodium selenite. Biochemistry, 13, 337–341.[ISI][Medline]
  8. Tennant,R.W., Margolin,B.H., Shelby,M.D. et al. (1987) Prediction of chemical carcinogenicity in rodents from in vitro genetic toxicity assays. Science, 236, 933–941.[ISI][Medline]
  9. Lennartsson,P., Högberg,J. and Stenius,U. (1998) Wild-type p53 expression in liver tissue and in enzyme-altered foci: an in vivo investigation on diethylnitrosamine-treated rats. Carcinogenesis, 19, 1231–1237.[Abstract]

 

Response

P. Duerksen-Hughes

Dear Sir,

Drs Stenius and Högberg raise concerns regarding the specificity of p53 induction as an accurate indicator of genotoxic damage. Specifically, they point out that p53 levels can rise following many types of DNA damage and that some compounds may be able to induce p53 without damaging cellular DNA.

We agree that p53 levels do rise following many types of DNA damage. However, for the purpose we envision, we see this as an advantage, not a disadvantage. We propose the use of this method as a simple, cost-effective screening technique designed to identify potential mammalian genotoxins. In this context, it is desirable to detect DNA damaging agents with a wide range of actions.

The possibility that some substances may induce p53 without damaging cellular DNA brings up the issue of accuracy. Every assay has some frequency of false positives and false negatives; substances such as proteosome inhibitors could potentially fall in the category of a `false positive' for this assay. The relevant question in the context of its intended use is how many of the environmental substances for which this assay would be used would actually function as proteosome inhibitors, inhibit mdm2 gene expression, etc., without damaging cellular DNA. We suspect this frequency will be quite low. The only way to get a realistic estimate of the frequency of such false positives is to test a larger number of environmental substances using this assay and to compare the results with those obtained by other methods. We are currently in the process of testing the 30 Predictive Toxicology Evaluation 2 chemicals (http://dir.niehs. nih.gov/dirlecm/pte2.htm#sources) using this method and hope that the results will give us some further idea of its utility. Of course, the use of animal models to confirm significant findings from this assay would significantly strengthen the evidence that a particular substance is or is not a mammalian genotoxin.

We assume that Drs Stenius and Högberg refer to the ubiquitin pathway, not the `ubiquinone pathway' in their letter.





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