杏吧原创

Cleveland, Ohio

LAKE HARSHA in southern Ohio is not a place you would ever describe as
achingly beautiful, but it鈥檚 good enough to supply clean water to most of
Clermont County. Just 8 kilometres away, though, is a large hazardous waste dump
containing hundreds of kilograms of polychlorinated biphenyls鈥攊t was
closed by the Environmental Protection Agency after violating safety
regulations. These two sites are connected by a patchwork of intersecting
waterways. The potential for disaster is obvious.

鈥淚t would only take about a coffee cup full of PCBs to make Lake Harsha鈥檚
water unsafe,鈥� says biochemist Paul Russell, a former environmental consultant
to the county who has been part of a citizen鈥檚 group that鈥檚 been campaigning
against the hazardous waste dump for years. 鈥淧eople could be very sick before
the authorities wake up and discover they have a problem,鈥� he says.

What the lake needs is a monitoring system that will sound the alarm if even
a tiny amount of PCBs leaches into the water. And Russell thinks he鈥檚 found the
answer鈥攁 genetically engineered golden zebra fish that glows when exposed
to pollutants. It鈥檚 a veritable light bulb of eco-ingenuity that could be used
at thousands of sites across the US alone where toxic waste dumps threaten
drinking water supplies. So why won鈥檛 anybody buy the idea?

The notion of using animals as early-warning sentinels against environmental
hazards is scarcely new. 杏吧原创s in Japan in the 1950s discovered that fish
were tainted with mercury when an epidemic of neurologically poisoned 鈥渄ancing
cats鈥� swept coastal villages. Then there are the famous canaries in the coal
mines, which would keel over as soon as dangerous gases seeped into the air.
Their modern counterparts do a similar job for the US Army in Maryland:
electronic sensors monitor eight bluegill fish day and night to look for any
aberrant behaviour that would indicate toxins in the river upstream of Fort
Detrick鈥檚 water supply.

But now, transgenic techniques are propelling these living sentinels smack
bang into the 21st century. The flashiest of these brave new animals are the
zebra fish developed by Russell鈥檚 college lab partner from the 1950s,
environmental geneticist Daniel Nebert from the University of Cincinnati.

Three decades ago, Nebert had the idea of creating transgenic fish with
built-in toxin alarms designed to be quicker and more targeted than death and
disease, the traditional red flags of sentinel species. He had read a report
that fish living near a petroleum factory had higher levels of a particular
enzyme than the same type of fish living 3 miles away. Fish living 10 miles away
produced none of the enzyme. Nebert realised that it might be possible to splice
some sort of mechanism into the fish鈥檚 genetic machinery that would produce a
clear sign as soon as toxins prompted the genes to produce this enzyme. But at
that time genetic techniques were nowhere near advanced enough to do this.

It was the 1990s before geneticists had discovered enough for Nebert to test
his idea. In 1993 he teamed up with Michael Carvan III, an aquatic toxicologist
now at the University of Wisconsin-Milwaukee鈥檚 Great Lakes Water Institute to
create a benign 鈥渇rankenfish鈥� that would glow after swimming in water tainted
with certain toxins.

First, Carvan selected a gene from humans that is usually activated when PCBs
and dioxins are present. He spliced this together with the luciferase gene,
which produces one of the chemicals that make fireflies glow. He made similar
transgenic packages using luciferase and one of three other genes: a trout gene
expressed when heavy metals are present, a rat gene that responds to oxidants,
and a human gene sensitive to environmental oestrogens.

When he put these foreign DNA fragments into zebra fish cells grown in the
lab, they glowed in the presence of the various pollutants. After that it was
just a matter of injecting fertilised zebra fish eggs with plasmids containing
one of the transgenic packages. The result: 80 zebra fish that glowed faintly if
they had swum in water tainted with PCBs.

The beauty of this idea is that it takes advantage of the fish鈥檚 tendency to
accumulate and 鈥渂ioconcentrate鈥� certain toxic substances鈥攖he same feature
that makes fish caught in polluted waters so dangerous to eat. The fish acquire
the poisons much more quickly from their food and water than they can get rid of
them. Dioxin, for example, bioconcentrates in fish by a factor of 100,000
relative to the surrounding water.

As PCBs bioconcentrate in the transgenic zebra fish, they trip the genetic
switch for the production of luciferase. The resulting glow is too faint to be
seen by the naked eye, so the fish have to be scooped out of the tank and 鈥渞ead鈥�
by a luminometer. This portable device is about the size of a large toaster and
can be adapted to run off a car or boat battery. Each fish goes into a vial
where it is incubated with luciferin, which boosts the glow. The vial then fits
into a slot in the luminometer. 鈥淭he only difficult part of this process is
getting the fish from the net into the vial,鈥� says Russell.

During the summer of 2000, Russell kept 10 of these fish in an ordinary tank
at one of Clermont County鈥檚 water quality monitoring stations. Water from a
river downstream of the PCB dump flowed directly into the tank, which Russell
checked once a day to feed the fish and make sure they hadn鈥檛 been eaten by
snakes. He tested the fish twice over the summer to see if they showed any signs
of PCBs in the water鈥攖hey didn鈥檛鈥攂ut the real purpose of the
county-funded experiment was to see if the semi-tropical fish could survive
these fairly rugged field conditions. Eight hearty souls made it through.

The experiments have convinced Carvan and Nebert that sentinel zebra fish
have great potential as a first line of defence against pollution. If the fish
are commercialised, they won鈥檛 be as cheap as dime-store guppies but they will
be both less expensive and more efficient than much of the equipment used today.
What鈥檚 more, the fish are more sensitive to pollution than all but the most
sophisticated鈥攁nd enormously costly鈥� testing systems. 鈥淲hat shows up
in the fish is below detection by most lab instruments,鈥� says Nebert.

You could scarcely ask for more from a prospective environmental sentinel:
fish share most of their biochemistry and physiology with mammals, but produce
more offspring and mature faster than lab mice. They are less expensive to
maintain, and using them for this task provokes fewer outcries from
tender-hearted humans. In addition, embryonic fish are transparent enough for
researchers to use luminescence or fluorescence to check gene expression during
their development. And of course, their natural habitats are the lakes and
rivers where poisonous substances from run-off, air pollution and chemical
spills might accumulate and make their way to humans.

Another team is ready to use a different transgenic sentinel fish to detect
unknown mutagens in the sediments near New York鈥檚 Long Island Sound. But despite
the huge potential of this new technology, its enthusiasts are having a hard
time attracting funding. Neither Carvan nor Nebert have been able to get the
money they need to develop stable lines of glowing zebra fish. Doing this is
crucial if they are to turn the fish into reliable and well-calibrated chemical
detectors, generation after generation.

鈥淭his is a really cool idea with all sorts of applications, but there aren鈥檛
a lot of funders willing to give us what we need,鈥� Carvan sighs. 鈥淚 think it
sounds like science fiction to them.鈥� But then, today鈥檚 science fiction has a
habit of turning into tomorrow鈥檚 reality.