Superfund Research Program
Analysis of Causes of Mortality in the Aberjona and Mystic Valley Populations: Comparison to Massachusetts and United States Experience
Project Leader: William G. Thilly
Grant Number: P42ES004675
Funding Period: 1995 - 2000
The intended use of mitochondrial DNA mutational spectra as an indicator of exposure and related genetic change required reexamination as a result of these observations. Investigators had already perfected the proposed technology to observe mitochondrial point mutational spectra in human cells and tissues. In several public meetings project investigators described the technology and its use in studies of Woburn and neighboring towns.
Samples of tissue from different organs in different persons yielded clear reproducible mutational spectra containing 17 separate hotspots. They were, however, essentially the same. The researchers next showed the same spectrum arose in cultured human cells permitting the conclusion that mitochondrial mutations have endogenous and not exogenous causes. These data appeared in a PNAS in December 1997. The mitochondrial mutational spectra in twins discordant for smoking were checked and showed cigarette smoking had no effect on mitochondrial mutational kind or number. In addition to showing that cigarette smoke chemicals did not directly induce mitochondrial mutations, these data support the hypothesis that smoking did not increase the tissue turnover rate since this effect would have been expected to increase the number but not the kind of mutants observed.
The sampling and analyses of children's hair samples has disproved the hypothesis that children drinking water from Woburn's wells G & H were exposed to significantly increased quantities of heavy metals including arsenic, chromium and mercury.
Assumptions were made based on fate and transport data that wells G & H water created a condition of exposure to one specific chemical, chromate(+6), in concentrations which would be expected to raise mutation rates in peripheral T cells above those observed in unexposed populations and would, therefore, give rise to a recognizable mutational spectrum in mitochondrial and/or nuclear genes. It is now clear that such an exposure did not take place. Further research determined that human mitochondrial mutational spectra are essentially identical in all tissues and donors, are not induced even in smokers' bronchial epithelium, are essentially identical to those seen in unexposed human cells in culture, and are recreated by copying the DNA sequence studied with cloned human gamma DNA polymerase which is responsible for replication of mitochondrial DNA. Investigators have now found that mitochondrial mutations can be induced with the mutagen MNNG, but only under conditions far exceeding those to which human tissues can be exposed.
Investigators developed means to observe point mutations in single copy genes at fractions at and somewhat below 10-6. This is a significant technological advance that places NIEHS at the forefront of world research in human somatic genetic change. This advance goes beyond the proposed application to multi-copy nuclear genes. The advance depended on discovering and eliminating the source of nonspecific DNA "carryover" in the use of biotin labeled probes for isolating specific DNA sequences from the necessarily large DNA samples.
Additionally, related studies in solid organs in animals and humans have revealed that nearly all mutant copies are present in clusters of more than 100 copies each. Investigators interpreted this to mean that only mutations in stem cells are "remembered" in human aging and that the clusters represent the mutant stem cell and its descendent transition and terminal cells in a tissue turnover unit. This advance is important to understanding basic tissue physiology and the quantitative pathways for multiple mutation requiring diseases such as cancers.
On the negative side, the studies of a number of multi-copy sequences including LINE element subsets, rRNA and histone genes have revealed a sufficient degree of inherited intraindividual genetic variation among copies to block application of the investigators' approach. Additional possibilities are being explored. Furthermore, the ability to measure single copy mutants at 10-6 while extremely useful in the study of anatomically distinct sectors of human organs, is of limited value in studying peripheral blood samples in which a typical 10 ml sample contains about 4 x 107 copies of an autosomal allele or only 40 copies of a strong hotspot present at 10-6. Since cells from many "turnover units" are mixed in a blood sample, the strategy of finding clusters of mutants in anatomically distinct samples cannot be applied.
Researchers discussed the progress and limitations of these studies at public meetings in the Aberjona Basin and concluded that study of mitochondrial or nuclear mutational spectra in this population would not yield data of sufficient public health value to justify the public concern that would be engendered if we continued our donor recruitment efforts.
Instead, project investigators proposed a study of the mortality records in the Aberjona and Mystic Watersheds using a new approach to their analyses developed in this MIT NIEHS Center that reflected an "engineering-style" quantitative analysis missing from the present public discussion of health statistics for communities.