Toxic pit could hold the unlikely key to blockbuster drug discoveries

by Jason Zasky. Published Tue 06 Dec 2017 21:19, Last updated: 2017-12-06
NASA pic of the Berkeley Pit

“To go to Berkeley Pit Lake, you have to complete a 40-hour Hazmat programme — and that’s just to stand next to the water,” advises Andrea Stierle, a research professor at the University of Montana - Missoula, who began studying samples from the Pit sixteen years ago.

And when employees of the Montana Bureau of Mines and Geology venture out onto the lake, they do so in a boat that’s made of fibreglass (as opposed to aluminium), “because they don’t want it to dissolve before they get back to shore,” she continues.

It’s probably best that the privately-owned Berkeley Pit - a mile by a mile-and-a-half across, and encircled by a barbed-wire fence - is off-limits to all but a select few.

After all, it’s an abandoned open pit copper mine filled with an estimated forty billion gallons of acidic, metal-contaminated water - part of the largest Environmental Protection Agency (EPA) Superfund site in the United States, and an ongoing liability for its “responsible parties,” the Atlantic Richfield Company (which merged into British Petroleum) and Montana Resources.

Though it might seem an irredeemable place, it turns out that the Pit - located in the mining town of Butte, Montana, and operational between 1955 and 1982 - is proving to be a rich source of unusual extremophilic microorganisms, which have produced novel and compelling bioactive metabolites.

In other words, the water is filled with a hardy assortment of fungi, algae, protozoans, and bacteria, many of which have shown great promise as producers of potential anti-cancer agents and anti-inflammatories.

Yet as late as 1995, local microbiologists assumed that the environment was too toxic for much of anything to survive, much less thrive. That is, until that same year, when Andrea and her husband Donald (also now a research professor in the department of Biomedical and Pharmaceutical Sciences at UM-Missoula) were provided water samples by a Bureau of Mines and Geology hydrogeologist and found some “fascinating compounds,” including one that has the potential to prevent migraine headaches.

Despite a lack of funding, the Stierles (at the time full-time residents of Butte) decided to take a chance and continue “bioprospecting.” If nothing else, the Pit was conveniently located, and there was zero competition from fellow scientists.

“No one was going to arm wrestle us to go look in the Berkeley Pit for microbes that produce anti-cancer compounds,” notes Andrea, who describes herself and her husband as marine natural products chemists with a bent toward drug discovery. (“We are taking what the natural world offers, and giving it the western science flair,” she elaborates.)

Yet it wasn’t long before they made their first remarkable find, one which occurred in the wake of a tragic incident that took place within the confines of the Pit.

November 5, 1995, is a well-remembered day in Butte, not so much for the blizzard-like weather conditions, but for the hundreds of snow geese that arrived with the storm. Though the geese likely viewed the Pit lake as an oasis - a place to stop and eat, drink, and rest during their migration south - it turned out to be their graveyard.

After the storm subsided, observers found 342 bird carcasses, most floating in the Pit, their gullets and gastrointestinal tracts eroded. The incident provided tangible evidence of how dangerous it could be to drink Pit water, which is not only acidic (pH 2.5, about the same as Cola), but contaminated with high concentrations of metal sulphates, including iron, copper, aluminium, and zinc.

Notably, however, when the Stierles were provided a fresh set of water samples by the Bureau the following spring, they found completely different microbes than they had discovered in their initial examination the year before. Specifically, they found an uncommon yeast - an especially shocking discovery considering that yeast shouldn’t grow in that pH.

“We checked the genus of the species and it had been identified before,” begins Andrea, “but the only place it had ever been seen was in the rectal swabs of geese. We think that the birds inoculated the Pit, and that the yeast was able to over-winter,” she continues.

As it turns out, the birds that perished during that fateful storm may not have died in vain. “The yeast - when you grow it in Pit water - actually absorbs about 87 percent of the metals in the water,” explains Andrea. “We were able to patent it, and we have a partner who is looking at this organism, to see if it can be used for secondary ore recovery or toxic waste clean-up. So we’re very grateful to the snow geese,” she concludes.

Meanwhile, in the wake of the mass die-off, the two responsible parties under federal Superfund law agreed to implement a waterfowl mitigation program, which is aimed at locating birds in the area and encouraging them to fly away.

Though the 2002 Consent Decree between the EPA and BP-Arco/Montana Resources (MR) states that “birds exposed to Berkeley Pit water for less than 4-6 hours should not be at substantial risk,” the plan calls for MR personnel to make hourly observations for birds during spring and fall migrations (reduced to 5-6 per day during non-migratory seasons) and to use rifles and shotguns to scare birds into the air.

In addition, staffers also have three Phoenix Wailers (high-tech devices that emit predator and electronic sounds) at their disposal, not to mention a collection of heavy metal music. And if all other hazing activities fail, personnel can and will take their boat onto the lake to displace birds that don’t elicit the predictable response to noise, or those - like grebes and loons - that tend to dive underwater when alarmed.

Even so, mitigation efforts aren’t always successful, as was the case in October 2007, when 37 birds - 17 snow geese, 19 ducks, and a swan - were found dead at the Pit after a weekend of dense fog.

“It usually happens when it’s cold - when the temperature drops real fast,” says Donald about the sinister fog that periodically forms on Berkeley Pit Lake, then rolls into downtown Butte, just a few blocks away. Though unnerving, it appears that so-called Pit Fog isn’t dangerous.

“Water is the predominant liquid in the Pit, and at different temperatures and atmospheric pressures, that water is going to boil off and create condensation,” he explains. In other words, it’s just pure water evaporating from the Pit, much like any other fog.

In fact, a chemist at Montana Tech once explored the idea of increasing the evaporative rate of the water in the Pit as part of a clean-up strategy. “He thought it would be cool to get a bunch of big mirrors and train the sun on the Pit and evaporate the water,” begins Andrea. “The volume of the water would become less and less and there would be more and more of a pure sludge, at which point you could do secondary ore recovery.

“There was a cost-benefit analysis and [even if the evaporative rate could be increased sufficiently] the cost of making the mirrors was prohibitive. So it wasn’t going to work, but it was a great idea,” she concludes.

So, for the moment at least, the primary focus of all responsible parties is on containment, no small concern considering that the volume of water in the 1,780-foot-deep Pit has been increasing for the better part of the last thirty years. (On Earth Day 1982 Arco announced it was suspending operations in Butte and shutting down the pumps in the [Kelley] mine, which had been preventing the vertical shafts, horizontal work ways, and the Pit itself from filling with acidic water.)

Fortunately, at current levels the water - now rising at a mere six to eight inches per month - is essentially contained in its own steep-walled repository. But if the water level were to rise above the 5,410-foot mark - well below the lowest point on the rim at 5,509 feet above sea level, and not terribly far above the May 2017 water level of 5,296 feet - the Pit would no longer function like a big sink drawing water towards a drain.

At that point water would begin migrating out into the surrounding aquifers and surface waters, a frightening prospect for the residents of Butte (not to mention BP-Arco and MR, which would be subject to staggering fines).

With this in mind, in 2002-03 BP-Arco and MR constructed an $18 million water treatment plant about 600 feet east of the Pit, which is designed to treat up to seven million gallons of water a day.

Visible from the Berkeley Pit viewing stand, which overlooks the water and is open to the public (admission fee: $2 per person), the Horseshoe Bend Water Treatment Plant currently treats some of the water that goes into the Pit, effectively slowing the rate of fill.

Ultimately, though, as the water nears the critical 5,410-foot mark circa 2023, the plant will begin treating Pit water itself, ostensibly meeting all EPA discharge standards for contaminants of concern, then sending the treated water into nearby Silver Bow Creek and the Clark Fork River.

Meanwhile, MR is mining copper from the waters in the Pit, recovering approximately 200,000 pounds per month, which has helped the company offset water treatment costs. Naturally, ore recovery activities have also changed the water chemistry of the Pit, which now features much less copper and much more iron than in the past, a potentially advantageous change in terms of future clean-up efforts, as copper is considerably more toxic than iron.

Between the copper mining, the introduction of new microbes, and the occasional slope failure (in October 1998, part of the Pit wall collapsed, causing a tidal wave that washed out the boat and dock and initiated “lake mixing”), the ecosystem in the Pit is constantly evolving.

In the course of their ongoing investigation, the Stierles are always finding new, and often-times unique, organisms in the water.

They are also discovering different microbes in samples collected at different depths, so that some of the extremophiles they’ve identified might also be classified as piezophiles (capable of living at extreme pressure), acidophiles (able to survive at pH 3 or lower), or metallotolerants (capable of tolerating high levels of dissolved heavy metals).

Of course, what is really important to the Stierles is the activity demonstrated by the microbes.

“Most of our research in the past few years has been focused on selective anti-cancer agents,” says Donald. “Once we finish with a compound we send it off to the National Cancer Institute (NCI) and they test it against their cell lines. What we have found is that some of the compounds have selective anti-cancer activity associated with them,” he continues. For instance, a novel spiroketal they discovered, Berkelic acid, has been shown to be very potent against OVCAR-3, an ovarian cancer cell line in the NCI cell line screen.

“Some of the microbes we’ve found have been isolated from other places and some of them haven’t,” elaborates Donald, the aforementioned goose yeast being a prime example of the former.

If the organism has been isolated previously, that allows the Stierles to look at how the environment in the Pit is influencing its biochemistry. “If we find an organism that has a genus and a species that is known, we buy that organism from American Type Culture Collection (ATCC) and grow it side by side, under the same conditions, and examine the chemistry and find that our Pit-derived organism is producing very different secondary metabolites,” begins Andrea. “So even if you are looking at the same organism, it still seems to be unique.

“With the Berkeley Pit we have found more unique secondary metabolites per workday that we have in any other environment we’ve looked at,” she concludes.

Of course, just because the Stierles find a promising anti-cancer agent, that doesn’t ensure it will become clinically relevant.

“There are so many leads that come out of small labs like ours, and 99.99 percent of them are not going to go anywhere,” explains Andrea. To be sure, there are countless possible barriers: It might not be a compound that is relevant to what a particular pharmaceutical company is looking for. “Or they might find that there is activity, but it’s cytotoxic as hell, so before it could be therapeutically relevant it would kill the patient,” says Andrea, naming just two potential stumbling blocks.

On the other hand, the Stierles have already found upwards of 70 compounds that might be medically useful. “If a fungus or bacteria is going to make these compounds they probably have a use. It’s our job to find out what is therapeutic about them,” begins Andrea. “We may never know. But if somebody can find the right biological assay system and pipeline it to find the clinical relevance, they all have some beneficial potential,” she concludes.

Most notably, one of the enzymes the Stierles work with turns out to be a key component of inflammation. Coincidentally, and perhaps fortuitously, Dr. Andrij Holian’s research lab at the University of Montana is working on a conglomeration of proteins called the inflammasome.

“One of the post-docs in the lab, Dr. Teri Girtsman, can induce inflammasome production in certain cells. The inflammasome binds and activates an enzyme called caspase-1, which then releases inflammatory chemicals. We can now study compounds that inhibit this enzyme,” says Andrea. “If we can find compounds that knock back inflammation, we may be able to prevent the onset and proliferation of certain cancers, as well as rheumatoid arthritis, Huntington’s disease, and ALS [Amyotrophic Lateral Sclerosis]. Many diseases seem to have their inception in chronic inflammation,” she notes.

So even if the Stierles are never directly involved in the development of a drug, it’s very possible that something they discover will have a ripple effect and inspire a compound. If nothing else, “we can use these molecules as tools to study particular systems,” emphasizes Donald. “Now we can study the cellular processes that happen under normal cellular conditions and under disease cellular conditions. As we know more about that process, we have a better idea how to change it - how we can take a disease state and return it to a normal state,” he says.

Meanwhile, the Stierles have high hopes for the potential of their goose yeast to contribute to the clean-up of the Pit water, which in a jar doesn’t look as ominous as its reputation might suggest. In fact, it looks a little like Bud Light, except for its orange-yellow tinge (which comes from the oxidized iron that’s present), and the funky black bits that settle at the bottom.

“There is nothing intrinsically - I’ll use the word - poisonous about the water,” says Andrea. “Once I spilled some on my hand and the skin got red and inflamed and it burned but it wasn’t like I ended up in the hospital,” she continues.

But cleaning up the water in the Pit presents a daunting challenge, in part because of the acidity, and in part because the particles in the water will rapidly clog any filtration system. That’s where the slime from the yeast comes in.

“It takes those [bits] and drops them out of solution and completely encapsulates them; the term I use is flocculation,” says Donald. Then you can decant the water and you have gotten rid of eighty percent of the iron hydroxide fines in about thirty seconds. If you let the organism continue to grow, over the course of a week it will start absorbing about eighty percent of the other metals,” hinting at the method’s promise as a clean-up agent.

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