Metallothionein colon crypt immuno-positivity as a rapid in vivo essay for drug efficacy studies

Metallothionein immune positivity indices are considered as representative of crypt stem cell mutations. The frequency and size of MT- immunopositive foci, as well as the total number of MT immuno positive crypts were assessed here in a short term in vivo assay. Drug efficacy was tested on early mutated crypt in colon of Balb-c mice 30 days after induction with a single dose of the mutagen dimethylhydrazine. The different drugs used (MS 275, vioxx, 5-fluorouracil, aminophylline, 5-azadeoxycytine) affected metallothionein - immunopositive crypt frequency according to their predicted efficacy on this specific model of mouse colon carcinogenesis. This preliminary validation study of metallothionein - immunopositive crypt frequency strengthens the evidence that metallothionein immuno-positivity indices could be used as short term markers to assess the capability of different pro-drugs to counteract crypt invasion and clonal expansion of mutated stem cell progeny. This rapid in vivo test (30 days) based on metallothionein immunopositivity indices can be assayed in paraffin-fixed tissue sections and has been validated against the Glucose 6 phosphate Dehydrogenase assay. To quantify metallothionein immunopositivity indices, we devised a novel fast analysis protocol based on Zeiss Axiovision software for image processing. Metallothionein (MT) crypt-restricted immunopositivity is a colonic crypt stem cell mutation marker that may be induced early and in abundance after mutagen treatment. Persistent MT overexpression within single crypts has been tested as a biomarker of colonic crypt stem cell mutation in the morphologically normal appearing mucosa. Jasani et al. (2) found that the frequency and time course of crypt conversion to MT immunopositivity in mice treated with Dimethylhydrazine was similar to that of the Glucose 6 phosphate Dehydrogenase assay (2). Cook et al. (1) demonstrated a strong correlation between the MT and G6PD assays (r>0.9) in Balb/c mice treated with N-ethyl-N-nitrosourea (1). Stable, crypt restricted immunopositivity for MT thus correlates with an established mutation marker (G6PD) in the mouse colon. This is considered the result of a mutation affecting expression of the MT gene in colonic stem cells (3). Morphologically normal- appearing mucosa from human colorectal carcinoma resection specimens and from the colons of ageing laboratory mice contain scattered single crypts whose cells exhibited uniformly increased MT immunostaining. This suggests that MT overexpression arises directly from random crypt stem cell somatic mutation, followed by colonization of the clonal unit by the mutated progeny (3-5). The oncological relevance of MT crypt restricted immunopositivity indices (MTCRII) was also recently proved (6).