Project 2:  Mutation and Recombination in Mice Exposed to Toxic Metals

Research Goal: To increase the understanding of the genotoxic effects of chromium and arsenic in mammals, when these metals are introduced either alone, or combined with benzo[a]pyrene.

Overview: The research design employs two novel mouse strains designed to detect frameshift and recombination events. One set of such mice uses a human PLAP (Placental Alkaline Phosphatase) gene. The PLAP gene was rendered inactive by insertion of 7 G:C base pairs. The frameshifted PLAP transgene reverts; active PLAP enzyme is produced and deposited on the surface of the cell, where it is detected by a histochemical stain. This approach provides information about where mutant cells arise in different tissues. Mice that use PLAP expression to detect deletional recombination between direct repeats (PLAP-del mice) are under construction. Researchers will also study mitotic recombination by using another mouse strain they have developed. These mice are heterozygous for the aprt gene. Cells that have undergone mitotic recombination at any point between aprt and the centromere on chromosome 8 can be selected because such cells lack APRT function and survive in 2,6 diaminopurine. The following hypotheses are being investigated: 1) Chromium and arsenic cause frameshifts and recombination in diverse tissues of mice. 2) Combining one or another of these metals with benzo[a]pyrene has a synergistic effect on mutation and recombination.

Progress to Date: We found that exposure cultured cells carrying the frameshifted PLAP transgene to either arsenic or chromium lead to an increase in mutation. Mice that are heterozygous for the aprt gene have been exposed to different doses of arsenic and benzo[a]pyrene. These experiments are in progress.

Application of Knowledge: The genetic changes underlying the adverse health effects of exposure to complex mixtures of carcinogenic metals and polycyclic aromatic hydrocarbons (PAHs) are not understood. If recombination is involved, these experiments will detect this phenomenon.

Future Directions: Traditional tests would suggest that these metals are not very mutagenic, but these tests would not detect increases in mitotic recombination. Should we find that this process is stimulated by these metals, then it will be possible to determine why this happens. The most likely suspect would be breaks in chromosomes.

Techniques Incorporated:
Transgenic mice
Cell culture
DNA analysis

Keywords:
Arsenic
Chromium
Mutation
Transgenic Mice
Mitotic recombination