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Final Progress Reports: University of Cincinnati: Mutation and Recombination in Mice Exposed to Toxic Metals

Superfund Research Program

Mutation and Recombination in Mice Exposed to Toxic Metals

Project Leader: James R. Stringer
Grant Number: P42ES004908
Funding Period: 2001 - 2006

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Final Progress Reports

Year:   2005 

The goal of the Stringer Laboratory is to improve the understanding of mutation in somatic cells of mammals through the use of new methods.

 

One such method is the PLAP mouse model, which employs a transgene, PLAP, that, when mutated, confers a cellular phenotype that can be detected in cells in mouse tissues in situ because cells with PLAP enzyme activity stain dark purple.  This model allows direct detection of mutant cells in all mouse tissues.  One application of the PLAP model has been to study the effects of arsenic in mice when the metal is introduced either alone, or combined with a polycyclic aromatic hydrocarbon (PAH), benzo[a]pyrene (B[a]P).  Arsenic and chromium are often found in complex mixtures with PAHs such as B[a]P at Superfund sites. This year, the Stringer lab completed studies on the effects of arsenic in the PLAP model.  In collaboration with core members Stambrook and Talaska, the Stringer lab utilized the PLAP mouse model to detect mutant cells in skin of mice exposed to arsenic in drinking water.  Arsenic can generate reactive oxygen species, suggesting that oxidation of DNA may play a role in carcinogenesis. Oxidization of guanosines in polyG tracts is known to cause frameshift mutations, and such events can be detected in situ using the G11 placental alkaline phosphatase (PLAP) transgenic mouse model, which reports frameshift mutations in a run of 11 G:C basepairs by generating cells containing heat-resistant alkaline phosphatase activity. PAH can also induce frameshift mutations. In the study described here, FVB/N mice carrying the G11 PLAP transgene were crossed to C57Bl/6 mice. Half of the hybrid mice were given drinking water with sodium arsenite (10mg/L) for 10 weeks. Half of the arsenic treated mice were also exposed to BaP by skin painting (500nmol/week) for 8 weeks. Another group of mice was exposed to BaP but not arsenic. The effect on frameshift mutation was assessed by staining sections of skin tissue to detect cells with PLAP activity. Arsenic alone had no significant effect. On average, mice given BaP alone had approximately three times more PLAP-positive (PLAP(+)cells. By contrast, mice exposed to both arsenic and BaP exhibited 10-fold more PLAP(+) cells in the skin, and these cells were often arranged in large clusters, suggesting derivation from stem cells. Whereas combined treatment produced more PLAP(+) cells, stable BaP adduct levels and arsenic burdens were not higher in mice exposed to both agents compared to mice exposed to either one agent or the other.

 

Whereas point mutations are clearly an important aspect of genetic instability and cancer, loss of heterozygosity (LOH) via recombination and nondisjunction is at least as important.  The rate at which LOH events occur in normal cells of the body is not clear. LOH in normal cells of the body would be detectable if this event were to confer an obvious phenotype.  Therefore, the Stringer lab produced mice that carry two different fluorescent protein genes as alleles at the ubiquitously expressed ROSA26 locus on chromosome 6.  These mice should make it possible to mitotic recombination in mouse tissues.  As a first step in assessing the utility of this approach, two ES cell lines were derived and analyzed by FACS, which showed that the vast majority of cells expressed both fluorescent proteins, yellow and cyan.  However, monochrome cells were readily detectable and isolated.  Some of these monochromatic cells were used to establish clonal lines. Examination by fluorescent microscopy confirmed that most clones expressed only one of the fluorescent proteins.  Genetic analysis of the locus by PCR and RFLP showed that 83 of 83 spontaneously occurring stably monochromatic clones had lost one marker gene.  These data suggest that the R26CY biallelic model has utility for studying LOH in embryonic stem cells as well as in other cells in the mouse.  Preliminary studies on mouse tissues have also been encouraging, with both fluorescent proteins seen in all tissues examined.   

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