The authors have no acknowledgements.

The experimental procedures and ethical treatment of animals was approved by the University of Pittsburgh IACUC (protocol #1104674).
1. Preparation of G6PD Staining Mixture
NOTE: Ensure that the final concentration of each reagents is as follows; 5 mM glucose-6-phosphate (G6P); 2 mM NADP, 5 mM MgCl2, 0.35 mM 1-methoxy-5-methylphenazinium methyl sulfate (MMPMS) and 0.8 mM NBT.1,8,9 For each reagent the initial concentration was derived for a 40 ml final volume. Solut...

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Often the genotoxic effect of a compound is determined by its ability to modify DNA. This is normally done by isolating the tissue and measuring the global level of DNA adducts. For AOM, this would involve quantifying O6-methylguanine adducts in the colon. Using this approach information on damage within specific cell types, such as in the stem cell niche, is lost. In addition, a DNA adduct is not the same as a mutation since only a small subset of adducts are eventually converted into fixed mutations.12<...

Colon cancer is thought to involve exposure to environmental agents and dietary components that can damage DNA and produce activating somatic mutations in oncogenes (e.g., &#223;-catenin) or inactivating mutations in tumor suppressor genes (e.g., APC). It is assumed that these critical mutations occur in colonic stem cells. Because of the unique crypt architecture of the colonic epithelium, it is possible to measure stem cell mutations in the colon after exposing animals to chemicals associated with colon carcinogenesis. Several X-linked enzymes can serve as indicators for mutations that occur in stem cells, as the mutations will be present in all cells within a crypt.
Previously, a procedure was published demonstrating that chemicals that induce colon tumors in mice also generated somatic stem cell mutations in the colon via analysis of mutations in the X-linked glucose-6-phosphate dehydrogenase (G6PD) gene.1-3 The method quantifies the incidence of random somatic mutations in colonic stem cells without any selection pressure. The procedure involves the production of unfixed frozen colon sections from treated and control mice and the identification of crypts devoid of cells that produce functional G6PD activity. These mutated crypts, which appear white, indicate that the mutation occurred in a stem cell that gave rise to progeny also harboring a mutated G6PD gene. In the enzymatic assay, G6PD deficient mutant crypts cannot oxidize glucose-6-phosphate, which is required for the reduction of nitro blue tetrazolium (NBT). When the G6PD enzyme is functional, the NBT in the staining mixture is reduced to insoluble formazan and precipitates, accumulating at the location of the enzyme and &#8220;staining&#8221; cells blue. Cells that are G6PD mutants remain whitish in contrast to blue stained wild type crypts. This method measures mutations that have a &#8220;null&#8221; phenotype. Because enzymes, such as G6PD, can diffuse out of the cells on an unfixed tissue section if the sections are placed in an aqueous solution, it is necessary to stabilize the enzyme in the tissue sections to be analyzed.4 The stabilization of the enzyme in the tissue must not interfere with diffusion of small reagent molecules necessary for the G6PD enzymatic reaction.
We have made a number of significant changes to the original procedure. The medium for the critical enzymatic staining has been changed from polyvinyl alcohol to Optimal Cutting Temperature Compound (OCT), which facilitates pH control and solubilization of the components used in the assay. The staining is performed using 0.4 &#8211; 0.5 ml wells made of steel washers so that each tissue section received the same volume of the G6PD reaction mixture. A procedure was developed to estimate the number of crypts in an imaged section that provides a large sampling of the colonic tissue without having to manually count &#62;105 crypts for each colon. The analysis of adjacent 7 &#181;m tissue sections affords visualization of a mutant crypt on more than one slide, which reduces potential staining artifacts. These changes make the procedure more efficient and reproducible.
Using this revised protocol, we have quantified the mutation frequency in colonic stem cells after exposure of C57Bl/6 mice to azoxymethane, a well-known colon carcinogen in rodents.5-7

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>reagents</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>nitroblue tetrazolium</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>NADP</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>glucose-6-phosphate</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>1-methoxy-5-methylphenazinium methyl sulfate</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>dimethyl formamide</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>phosphate buffer (pH 7.4</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Optimal Cutting Temperature Compound (OCT)&#160;</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>light microscope equipped with 5 megapixel camera</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>cryostat</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>warm room&#160;</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

quantification of  colonic stem cell mutations

The ability to measure stem cell mutations is a powerful tool to quantify in a critical cell population if, and to what extent, a chemical can induce mutations that potentially lead to cancer. The use of an enzymatic assay to quantify stem cell mutations in the X-linked glucose-6-phosphate dehydrogenase gene has been previously reported.1 This method requires the preparation of frozen sections and incubation of the sectioned tissue with a reaction mixture that yields a blue color if the cells produce functional glucose-6-phosphate dehydrogenase (G6PD) enzyme. If not, the cells appear whitish. We have modified the reaction mixture using Optimal Cutting Temperature Compound (OCT) medium in place of polyvinyl alcohol. This facilitates pH measurement, increases solubilization of the G6PD staining components and restricts diffusion of the G6PD enzyme. To demonstrate that a mutation occurred in a stem cell, the entire crypt must lack G6PD enzymatic activity. Only if a stem cell harbors a phenotypic G6PD mutation will all of the progeny in the crypt lack G6PD enzymatic activity. To identify crypts with a stem cell mutation, four consecutive adjacent frozen sections (a level) were cut at 7 &#181;m thicknesses. This approach of making adjacent cuts provides conformation that a crypt was fully mutated since the same mutated crypt will be observed in adjacent sections. Slides with tissue samples that were more than 50 &#181;m apart were prepared to assess a total of &#62;104 crypts per mouse. The mutation frequency is the number of observed mutated (white) crypts &#247; by the number of wild type (blue) crypts in a treatment group.

The ability to measure colonic stem cell mutations in animals provides a unique way to correlate mutations to cancer induction. Normally, it is assumed that the critical step in carcinogenesis involves activating mutations in oncogenes and/or inactivating mutations in tumor suppressor genes. We injected C57Bl/6 mice with 200 &#181;l of PBS or 10 mg/kg AOM in 200 &#181;l of PBS. AOM is a known colon carcinogen.5-7 At 90 days the colons were analyzed for stem cell mutations. This time is required to allow stem c...

Watch this Scientific Journal Video about Quantification of  Colonic Stem Cell Mutations at JoVE.com

Quantification of  Colonic Stem Cell Mutations

We report significant improvements for the reproducible measurement of somatic colonic stem cell mutations after exposure of mice to potential DNA damaging agents.

The ability to measure stem cell mutations is a powerful tool to quantify in a critical cell population if, and to what extent, a chemical can induce ...

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