Superfund Research Program

Mutational Fingerprinting to Find Causes of Mutations in Humans

Release Date: 01/21/1998

Many of the areas that are now designated as Superfund sites were at one time a source of chemical contaminants that led to significant human exposures. One such site is located within the Aberjona River watershed. In the early 1960's, chromium in various chemical forms, arsenicals, and other chemicals were discharged in large quantities into the Aberjona River from lagoons ruptured during land reclamation activities. In the period between 1969 and 1985 a town through which the Aberjona flows experienced what was considered by some epidemiologists to be a childhood leukemia cluster.

This situation is not an uncommon one in American towns and cities. Field studies demonstrate that the environment - in this case ground, river and pond water - has been contaminated with toxic chemicals for an extended period. An apparently higher rate of some disease is observed in the local population. But no means exists to determine if the environmental contamination increased the disease rates.

Because it was known that human cancers involve mutations in normal cells and that different chemicals produce specific patterns of mutations in laboratory experiments, researchers at the Massachusetts Institute of Technology (MIT) developed a molecular technology known as "mutational spectrometry" in order to see the patterns of mutation in human tissues. The idea was that if a chemical, such as chromate, caused a major portion of the mutations in a person, then the mutational pattern induced by chromium should be found in that person's tissues. Now they are using this technology to better understand the relationships, if any, between chemical exposure and mutations that lead to human disease.

Mutational spectrometry in human tissues required advances in technology with sensitivity well beyond that practiced even in modern molecular biology or human genomic laboratories. The technique couples constant denaturing capillary gel electrophoresis, which separates mutant from wild type sequences of DNA, with a high fidelity polymerase chain reaction (PCR) procedure. PCR amplifies the amount of DNA so that it can be more easily detected. With their advances they can measure mutations that occur once in more than a million human cells.

The MIT scientists first optimized mutational spectrometry to detect and measure patterns of mitochondrial DNA point mutations in blood cell or tissue samples. Their original plan was to examine mitochondrial mutational spectra in persons living in and outside of the Aberjona Valley to discover any differences in mutational spectra that could be attributed to the contaminants found at exceptionally high levels in stream and pond sediments.

However, during the analysis of the very first few tissue samples from several organs and donors, the MIT investigators discovered that mitochondrial mutations had the SAME pattern throughout the different samples and were the same as spontaneous mutations found in human cells grown under laboratory conditions. Using a 100 base pair sequence of the human mitochondrial genome, a set of 17 hot-spot mutations have been found, 15 of which are transition mutations. This set of hot-spots was found in all human tissues examined and in the mitochondrial DNA of human cells grown in culture under presumably pristine environmental conditions.

However, mutations that lead to cancer and most inherited diseases occur in nuclear genes. Thus, the investigation is currently focused on applications of mutational spectrometry to nuclear genes. The scientists have extended their technology in order to observe the mutational spectra in the cellular nucleus especially in those genes which must mutate first to cause cancer. They believe they are now in a position to determine if human nuclear mutations are spontaneous or exogenously induced. If the latter, the project will then reinitiate a study of the effects of age, gender, home location, smoking and dietary habits on the mutations accumulated in human tissues with emphasis on comparison of Aberjona Basin residents with other Massachusetts populations.

This research is significant for providing a valuable tool to discover the amount and kinds of mutations in human populations and to identify environmental causes, if any, of genetic changes. Because molecular spectrometry offers the means to contrast the mutational spectra of chemically exposed and unexposed DNA, it can readily determine whether the kinds and positions of genetic changes in exogenously induced mutations differ from the "spontaneous" patterns observed in human cells. This capability to discriminate between spontaneous and induced genetic change in humans may assist in a more accurate determination of the risks associated with human exposures to mutagenic contaminants from Superfund and other hazardous waste sites.

For More Information Contact:

William G. Thilly
Massachusetts Institute of Technology
50 Ames Street, Bldg. E18-666
Cambridge, Massachusetts 02139
Phone: 617-253-6220

To learn more about this research, please refer to the following sources:

  • Khrapko K, Coller HA, Andre PC, Li X, Hanekamp JS, Thilly WG. 1997. Mitochondrial mutational spectra in human cells and tissues. Proc Natl Acad Sci U S A 94(25):13798-13803. PMID:9391107

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