Illustration: Joseph Tart
A century ago, coal miners carried caged canaries into
underground mines to alert them to the presence of carbon monoxide
gas. Canaries are more sensitive to many gases than humans, and if
the birds died, the miners knew to evacuate the mine. Today,
organizations concerned with water quality, including the EPA and
the U.S. Army, are searching for an environmental sentinel to alert
them to the hazards of potentially carcinogenic chemicals in the
nation's waters. NIEHS chemist Jim Burkhart, working with the U.S.
Army and scientists at various research institutes, is developing a
recoverable bacteriophage inserted into a transgenic fish that may
be just what they are looking for. This combination promises to
provide a tool that can be used in both laboratory and field studies
to make a rapid determination of genetic health hazards to human and
animal populations.
Mutant mummichogs. Transgenic Fundulus
heteroclitus may indicate risks to human health.
Photo: Steve
McCaw, Image Associates, Inc.
There are any number of pathogens or chemicals which, if present
in a body of water in high enough concentrations, will kill fish and
cause humans to get sick. Scientists have established safe levels of
tolerance for most of these agents and governments have incorporated
these levels into the appropriate regulations. But there are many
other chemicals that have far more subtle effects on living
organisms, such as inducing genetic mutations that may lead to
cancer and other diseases over a lifetime of exposure. Science does
not have a clear understanding of dose-effect relationships for most
of these chemicals and therefore few standards have been set.
There are few methods available that can be used to assess
genetic hazards for aquatic species, or that focus on the study of
particular gene mutations as they occur in DNA in vivo. And
even where scientists have been able to demonstrate induced genetic
effects for aquatic species, they have not been able to compare that
to the situation for humans. There are enough differences in
exposure, metabolism, and gene expressivity to make correlations
between fish and humans a hazardous enterprise.
Added to the problem of cross-species comparisons are the sheer
numbers of observations required for studies of mutagenesis in
aquatic species and mammals. The cost, time, and physical
requirements of such studies can make them impractical, if not
impossible, in many circumstances. "Take the example of a base pair
substitution that can alter a gene's function," Burkhart says. "That
event happens at a very low in vivo frequency . . . and
requires a large number of observations to detect mutations (often
in the tens of thousands) which means that a large number of animals
must be used. And it requires unambiguous detection of mutants among
the total number of genes analyzed. The technology does not exist at
this time to realistically meet these experimental conditions with
natural chromosomal gene sequences, especially in conditions where
there has been little genetic analysis at the DNA level."
Along with the need to use fewer animals is the need to use
phylogenetically lower species than mammals. Space requirements,
cost, and ethical standards argue against the use of large numbers
of rodents or higher animals for mutagenic studies. As such, there
is a need for a model that can be used to quickly and easily detect
environmentally induced mutation in aquatic species, and to
accurately correlate that data with potential hazards to mammalian
species including man.
The Transgenic Approach
Scientists have recognized that the complexities involved in
studies of mutagenesis across species could be reduced by using the
same gene marker for mutation in a variety of species. A target for
mutation analysis that is independent of any requirement for
expression, growth, or selection in tissue could be introduced into
different host species. The resulting transgenic animals could be
exposed and the transgene later recovered to observe mutations.
Comparison of mutation rates could then be made between somatic
tissues and correlated with other endpoints. Identical transgenes
introduced into rodents, fish, or cultured human cells would
simplify the cross-species comparison.
NIEHS researchers have paved the way in the development of
transgenic mouse models for detection of mutations and understanding
of how alterations of certain critical genes may be involved in
disease. Beginning in 1991, Burkhart, in collaboration with H. V.
Malling of the NIEHS, Rebecca Van Beneden, then of Duke University,
and colleagues set out to develop an in vivo mutation
detection system using transgenic fish with a chromosomally
integrated and recoverable bacteriophage, named
X174.
"X174 is a bacteriophage that has markers for mutation that
are already well-characterized," Burkhart says. "It is not normally
expressed in eukaryotes, so we don't have the problem of different
phenotypic expressions across individuals or species. It's very
small, which permits the insertion of many copies in the host genome
with less chance of chromosomal disruption than larger constructs."
Mutant mummichogs. Transgenic Fundulus
heteroclitus may indicate risks to human health.
Photo: Steve
McCaw, Image Associates, Inc.
Burkhart has chosen two species of fish for transgenic
development, the medaka (Oryzias latipes), a freshwater
species, and the mummichog (Fundulus heteroclitus), an
estuarine species. Both are ideal for development as
transgenic fish because of their small size, manipulable eggs, and
short embryogenesis. Both have been used extensively for studies of
chemical carcinogenesis and toxicology.
The methodology employed by Burkhart involves linearizing the
circular bacteriophage DNA and ligating it to form a ladder of
X174 catenates. Low numbers of phage sequences per host
genome contribute to low recovery of the phage. Therefore, in the
production of transgenic animals, the copy number is increased by
catenating the DNA. A solution of either a single gene copy or
catenated
X174 DNA is physically injected or electroporated into fish
eggs, which are then allowed to hatch. The fish are then analyzed
for integration of the transgene and are used to establish a
transgenic line. At the chosen stage in the fishes' development, the
vector DNA sequence is recovered from the hosts' genomic DNA. The
phage DNA is transfected into a specialized strain of E. coli
for packaging, then plated with various selective indicator bacteria
to determine the total number of progeny phage recovered and the
number of mutants. In experiments run over the past five
years, Burkhart and his colleagues have successfully produced
transgenic medaka, mummichog, and mice, as well as cultured human,
hamster, mouse, and fish cells.
Graphic: James
Burkhart
In the development of transgenic animals, scientists have
observed that methylation, a natural mechanism of DNA modification,
can be a major obstacle to comparisons of DNA sequences across
different species. Methylation varies as a function of tissue, sex,
age, and parental origin. Any molecular approach that responds to
natural methylation, independent of actual mutation, would be
seriously limited in its utility for comparing data between
different species. Studies with transgenic mice using
X174 showed extensive methylation after two generations,
which resulted in reduced recovery of the phage. The problem was
solved for mice by producing a new E. coli strain for
recovery of the methylated phage DNA. Fortunately, this method has
also proven effective for fish, allowing for a correlation to be
made between species without the unknown contribution of variable
methylation.
Spontaneous mutations occur among phages recovered from the DNA
of untreated transgenic animals, as well. Burkhart has measured the
range of spontaneous mutation frequencies in the transgene to be
between 1.5 x 10-7 and 3.7 x 10-7 for fish,
mice, and cultured cells.
Initial experiments have been run exposing transgenic mice and
fish to mutagens including N-ethyl-N-nitrosourea (ENU)
and polycyclic aromatic hydrocarbons (PAHs), pollutants commonly
found in U.S. waters. Burkhart has measured the induced mutation
frequencies for the transgene and is assembling data to allow
comparison across species.
"Taken together, our current results indicate that
X174 may be useful as an identical gene target in aquatic
species, in laboratory animals, and in cell cultures with a
potential for comparative mutagenesis studies in both basic and
applied research," Burkhart says. "The advantages of this approach
are that it measures mutagenic effect at the DNA level, it is not
limited by gene expression to a cell type, and it has target
specificity combined with a numerical power not practically
available in other systems."
Fish out of Water
The transgenic approach to detection of mutations in aquatic
species could have direct applications in a number of areas. Gary
Boorman, deputy director of the Office of Special Programs in the
NIEHS's Laboratory of Experimental Pathology, sees great use for
Burkhart's system in understanding the potential toxicity of
chemicals used to disinfect drinking water.
"Disinfection of drinking water is one of the greatest public
health advances of this century," Boorman says. "But in the last few
years, studies have shown that when used at 100,000-fold
concentrations to normal exposure, byproducts of these disinfectants
cause cancer in mouse liver tumors. If disinfectants are going to
protect human health, they are going to have some toxicity. The
question is what are the best levels or chemicals to protect human
health. [Burkhart's] model will be helpful in giving comparative
toxicities and at more sensitive levels than is presently available.
With the proper knowledge, utility engineers could switch the
balance of disinfectants to make more or less of different classes
of byproducts."
Fisheye view. Jim Burkhart gets a
close-up of the fish of his labors.
Steve McCaw, Image
Associates, Inc. |
Scientists at the Army's Research Institute of Environmental
Medicine are currently cosponsoring research with the NIEHS on the
use of the transgenic fish approach for assessing human health
hazards in aquatic environments. They are hoping that such a system
can be used to evaluate hazardous waste sites, effluents, and
drinking water supplies that might be used by the military. They are
particularly excited at the prospect of water supplies being tested
in mobile units with this system. "We want quicker, cheaper, and
more relevant ways to test complex environmental mixtures," says
Hank Gardner, director of the U.S. Army's Biomedical Research and
Development Laboratory. "We've been using standard mutagenicity
assays, which are in vitro cell-based assays. The opportunity
with [this] model is that it provides in vivo data and that
can be related to mammalian species as well."
Burkhart is quick to point out that studies with the
X174 bacteriophage have provided parallel mutation data
among fish and mammals for only a limited number of chemicals and
target genes. Further research will be needed to verify the system's
efficacy for a larger number of genes and with a variety of
environmental agents. Gardner, for one, is hopeful about the
prospects. "No model can predict with absolute certainty the risk to
human health," he says. "But [this] approach offers the best
opportunity that I know."
John Manuel
|
Anderson SL, Harrison FL. Predicting the ecological
significance of exposure to genotoxic substances in aquatic
orgnaisms. In: In situ evaluation of biological hazards
of environmental pollutants (Sandu SS, Lower WR, de Serres FJ,
Suk WA, Tice RR, eds). New York:Plenum Press, 1990;81-93.
Burkhart JG, Gardner HS. Nonmammalian and environmental
sentinels in human health: "back to the future?" human
ecological risk assessment. In press.
Malins DC, Ostrander GK. Aquatic toxicology: molecular,
biochemical and cellular perspectives. Boca Raton, FL:CRC
Press, 1994. |
[Table of
Contents]
Last Update: July 29, 1997
