For almost 20 years, the field of phytoremediation has explored ways to use plants to extract toxic materials from soil. Recent advances may offer the promise of economical alternatives to the traditional, labor-intensive phytoremediation technique of removing and incinerating contaminated earth. Most researchers in the field are focused on identifying and refining naturally occurring plants that concentrate pollutants, typically heavy metals, in their cells. But recent work with fission yeast may lead to another potential route--albeit a long one--through which genetic engineering would result in new strains of high-yield, metal-accumulating plants. This work also may be useful for identifying plants that can better tolerate and store metals, says David Ow, a molecular geneticist at the Plant Gene Expression Center of the U.S. Department of Agriculture's Agricultural Research Service in Berkeley, California, and the University of California at Berkeley.
Ow's group has identified the mechanism and the gene responsible for the capability of a fission yeast,
Schizosaccharomyces pombe
, to move the heavy metal cadmium across its cell membranes. For some time, scientists have known that
S. pombe
behaves much like certain plants and fungi that have developed coping strategies for surviving in metal-rich environments fatal to most organisms.
S. pombe
responds to cadmium by producing small peptides called phytochelatins that are rich in the amino acid cysteine. These phytochelatins bond with the cadmium ions, which allows them to transport the ions across cell membranes and into the yeast's vacuole, where the metal accumulates. Once in the vacuole, the ion-peptide complex stabilizes as a crystallite. This "cellular trashbag" can swell with metals until cadmium accounts for as much as 90% of the cell's volume, Ow says.
To isolate the gene responsible for this behavior, Ow compared normal
S. pombe
to cadmium-sensitive
S. pombe
mutants. The mutant yeast failed to chelate, it turned out, because they lack a single gene--dubbed
HMT1
, for "heavy metal tolerance"--which codes for the critical peptide. Now that Ow knows how
S. pombe
triggers the production of the phytochelatins, he is investigating exploiting this cellular "pump" through two angles. One way may be to bioengineer the yeast gene into plants that already tolerate heavy metals well enough to survive in polluted soil, but that aren't so-called "hyperaccumulators" of metals. Another way may be to identify an equivalent gene already present in metal-tolerant plants.
"The neat thing about it in yeast is that if you overproduce this [peptide], you get an increased rate of transport," Ow explains. "[The yeast] also ends up accumulating more metals in the vacuole, while at the same time becoming more resistant to cadmium. So the hunt is now on to look for a similar protein in plants. Assuming that the whole system is analogous, there should be a similar protein. If we can clone it out and overproduce it so that the plant makes lots more of these proteins, we may be able to pump more metals into the vacuole. Therefore, the plant can pick up more metals as a whole."
Genetically engineering such plants is important, Ow says, because although unaltered hyperaccumulators concentrate heavy metals at high levels (up to 1% of dry weight for cadmium and 5% for zinc and nickel), the plants themselves are too small to extract significant quantities of pollutants.
Other researchers, however, aren't so sure. "I can do more phytoremediation with natural metal-hyperaccumulator plants than they have any hope of doing with these plants that don't have the genetic capability," says research agronomist Rufus Chaney of the U.S. Department of Agriculture's environmental chemistry lab in Beltsville, Maryland. Engineering plants to collect more metals is not useful if those metals collect in the roots, which cannot be harvested practically, Chaney says. Furthermore, he continues, it's not necessarily a simple matter to switch the metal collection site from the plant's roots to its shoots. Finally, he says, "Obtaining expression of this gene at high levels in the membrane of xylem parenchyma cells to pump metals from the cytoplasm into the xylem would [require] further novel bioengineering . . . to make this gene relevant to phytoremediation rather than [merely] trapping cadmium in the roots."
According to Ilya Raskin, a Rutgers University molecular biologist, current phytoremediation techniques don't depend heavily on the process Ow has identified. Instead, workers treat contaminated soil to dissolve metals and produce a soil solution that metal-resistant plants can draw in through their roots, concentrating metals that are then harvested. Says Raskin, "It's a collection system and it doesn't rely on intricate cellular processes of metal transport." Still, he says, "Only history will tell whether [the cadmium research] will . . . have any relevance to phytoremediation.
Everyone knows that saccharin causes cancer, right? Wrong, according to the National Toxicology Program (NTP), which is expected to delist the chemical from the ninth Report on Carcinogens, where it has been classified as "Reasonably Anticipated to Be a Human Carcinogen" since 1981. In the same review, to be held 30-31 October 1997 at the NIEHS, the NTP Board of Scientific Counselors' Report on Carcinogens Subcommittee will also examine the toxicity data on 13 other substances, and will expand the traditional scope of substances eligible for consideration for listing in the Report on Carcinogens to include chemical mixtures (such as in smokeless tobacco products) and exposure circumstances (such as UV radiation).
The NTP is required by law to prepare a report that contains a list of all substances that are either known to be human carcinogens or may reasonably be anticipated to be human carcinogens and to which a significant number of persons residing in the United States are exposed. The law also states that these reports should provide available information on the nature of exposure, the estimated number of persons exposed, and the extent to which the implementation of federal regulations decreases the risk to public health from exposures to these chemicals. The eighth volume of this report is nearing completion and is scheduled to be published later this year.
The preparation of the ninth report differs from previous reports in several significant ways. Traditionally, the Report on Carcinogens, unlike the Monographs on the Evaluation of Carcinogenic Risks to Humans prepared by the International Agency for Research on Cancer (IARC), have not examined and discussed evidence for the carcinogenicity of manufacturing processes and occupational exposures. This has been due in part to the difficulty of placing into categories human exposures that range from single chemicals to complex mixtures and occupational exposure circumstances. It also stems from the ambiguous language in the mandatory statute that requires the listing of carcinogenic "substances" in the Report on Carcinogens.
However, a recent series of events has led the NTP to conclude that it would benefit public health to expand the scope of what is eligible for consideration for listing. First, the question of whether the Report on Carcinogens should specifically address manufacturing processes and occupational exposures was considered during the recent public evaluation of the draft report of the eighth Report on Carcinogens. The Board of Scientific Counselors and the various ad hoc and federal committees that participated in this evaluation agreed that the Report on Carcinogens should include reference to the fact that IARC has examined those manufacturing processes or occupations that have been determined to pose a cancer threat to exposed workers, and that the NTP will also examine them in the future. The committees also felt strongly that any inclusion in the report of complex exposure circumstances should be accompanied by statements indicating that such circumstances may differ in various countries or may change over time.
Note: This corrected table (12/2/97) replaces the incorrect version in the hard copy of the October issue of EHP on page 1041. See the 1997 December issue of EHP for an explanation. Sulfuric acid mists, tamoxifen, tabacco smoke, and UV radiation were nominated for listing as "Known to be a human carcinogen" instead of "Reasonably anticipated to be a human carcinogen".
In addition, the NTP recently requested an opinion from the Department of Health and Human Services general counsel on whether manufacturing processes and occupational exposures were legally eligible for consideration for formal listing in the Report on Carcinogens. The opinion of the general counsel was that the NTP has broad latitude to consider exposures to other than single chemicals in the determination of carcinogenic threats to humans.
Finally, increased technological capabilities in exposure assessment and epidemiologic evaluations means that the best scientific criteria can be brought to bear on questions of carcinogenicity. Nominations for listing in the ninth Report on Carcinogens reflect the NTP's new attitude, and summary data are provided for agents, substances, mixtures, and exposure circumstances. For example, in addition to such chemicals as cadmium (which is used in batteries and alloys), 1,3-butadiene (which is used in the manufacture of rubber), and the drug phenolphthalein (which is used in laxatives), nominations also include inorganic sulfuric acid mists (used in metal smelting and manufacturing), UV radiation from both solar and artificial sources, and both smokeless tobacco and tobacco smoking.
In addition to the changes in the substance of the Report on Carcinogens, the process by which the report is prepared and reviewed is also being broadened to promote more public input.
Touted as a way to protect public health by decreasing carbon monoxide (CO) emissions from automobiles, oxygenated fuels containing methyl tertiary butyl ether (MTBE) have been blamed anecdotally for causing headaches, dizziness, eye irritation, burning of the nose and throat, disorientation, and nausea in motorists. In addition, studies have shown that MTBE can cause cancer in rats and mice, though it appears to be a less potent carcinogen than many of the other chemicals found in gasoline and exhaust. Most recently, wells that supply drinking water to Santa Monica, California, were shut down due to high levels of MTBE contamination.
Problems at the pump?
Findings of a recent federal report on oxygenated fuels do little to dispel debate over the health effects of MTBE.
Concerns about the health risks of MTBE, combined with doubts about its ability to significantly lower CO emissions, have caused many to question the usefulness of the chemical as a fuel additive. However, proponents point to decreases in nationwide CO levels as evidence that the 1990 amendments to the Clean Air Act, which sparked the widespread use of MTBE by requiring the use of oxygenated fuels in areas with high CO levels, have led to greatly improved air quality.
Disagreement over the safety and practicality of using MTBE in fuels spurred the EPA to request that the White House National Science and Technology Council review studies done on oxygenated fuels and compile the
Interagency Assessment of Oxygenated Fuels
, which was published in June 1997. The report considers the effects that using MTBE-treated fuel may have on air quality, water quality, fuel economy, engine performance, and human health.
However, the report does not make any policy recommendations about the future of MTBE as a fuel additive. Ronald Melnick, a toxicologist with the NIEHS who contributed to the interagency report, says, "As far as the total picture of exposure and health effects of oxygenated fuels versus nonoxygenated fuels, there is just not enough evidence right now to draw any definitive conclusion on comparative cancer risk."
As for the effectiveness of MTBE, the authors of the report did find evidence that the chemical, under certain conditions, can decrease CO levels, but they also point out that its performance has not met expectations. "The effectiveness of MTBE was based on models that predicted a 25% decline in CO emissions, which we just have not seen," says Melnick. "CO emissions are decreasing, but it's incorrect to say that this is only a result of using oxygenated fuels. A large part of the decrease is due to improved emission control technology." The report notes that MTBE also cuts other harmful emissions such as benzene and possibly 1,3-butadiene, which is 100 times more carcinogenic than the additive, but that it increases emissions of aldehydes such as the metabolite formaldehyde, which the EPA and the IARC have labeled a genotoxin and probable human carcinogen.
How such changes in emissions and in the composition of fuel vapors will affect motorists is still uncertain, the report concludes. "Complaints have been raised and have not been dismissed about acute health effects like dizziness, headaches, [and] nausea," says Melnick. "There seems to be some consistency in these reports." But the authors of the interagency report could not find enough evidence to support the contention that MTBE, as used in the winter oxygenated fuels program, is significantly increasing these effects at levels over background levels.
According to the report, the interagency assessment "found that chronic noncancer health effects (neurological, developmental, or reproductive) would not likely occur at environmental or occupational exposures to MTBE." However, inhalation of MTBE has been shown to cause cancer in multiple organ sites in rats and mice. "The EPA is working on a health advisory on MTBE that will be issued in the near future," says Robert Hitzig, the technical lead for the EPA's Office of Underground Storage Tanks. "But it's uncertain now whether [MTBE] will be classified as a possible human carcinogen, a probable human carcinogen, or what its classification will be." However, the interagency report points out that the cancer risk to humans posed by MTBE is similar or slightly less than that posed by untreated gasoline vapors.
Though the health effects of MTBE ingestion are less understood than the effects of inhalation, the appearance of the additive in drinking water across the nation has caused concern over this route of exposure. "MTBE absorbs weakly in soil and not very biodegradable," explains Melnick, "so when there are leakages from underground gasoline storage tanks, it travels further in the ground water and persists for long periods of time." Also, MTBE that enters the atmosphere through exhaust and evaporation can fall to earth and flow into surface water reservoirs with precipitation. Recent studies found MTBE in 7% of urban storm water samples and in 5% of well water samples from across the United States. "The health hazards of MTBE in water are debatable," says Hitzig, "but it's not debatable that there are aesthetic problems with it. MTBE has a very low taste and smell threshold, so that if it's in people's water, they probably know it."
It is also unknown if the presence of MTBE in water is taking a toll on aquatic life. Studies so far have focused only on water used for human consumption, but the persistence of the chemical in the environment makes it of particular concern. Also, notes Hitzig, "There's no way of telling how much MTBE has leaked into groundwater. There have been over 300,000 confirmed releases of petroleum from underground storage tanks since 1988, but we don't know what percentage of them had MTBE in them."
MTBE, which is derived from methanol, is now the most widely used oxygenate in the United States. From 1984 to 1995, production has increased by about 26% annually, with 8 billion kg produced in 1995. Essentially all of the MTBE produced or imported in the United States is used in the oxygenation of gasoline. Gasoline can contain up to 15% MTBE by volume.
In light of the concerns raised over the use of MTBE, many wonder why the oxygenated fuel program was implemented without further research being conducted first. "Congress required the use of oxygenates in the Clean Air Act," says Hitzig. "The EPA is simply following the statute that was passed. There was a lot known about the risks of MTBE at the time, but maybe the people working on the legislation didn't know [that]."
"A program should be evaluated for [MTBE's] effectiveness as well as its health effects before it is put into use," says Melnick. "There are obviously serious questions about MTBE's effectiveness and its role as an environmental contaminant. So the question is, are we gaining sufficient benefits to make this program worthwhile." Much more research will be needed before this question may be answered with any certainty.
One more step toward understanding the uncontrolled proliferation of immortal cancer cells was taken in August as two separate groups identified the putative human telomerase gene for the first time. These results were published almost simultaneously in the scientific journals
Science
and
Cell
.
The gene produces the enzyme necessary to maintain telomeres, the structures at the end of each chromosome. When cells divide they lose, through the normal process of proliferation, a few of the special base pairs that make up the telomeres. For most cells, this is presumably part of the natural aging process of the cell. When these base pairs run out, the cell can no longer divide properly.
However, for several specialized normal cells in the body it is imperative to be able to continue to divide past the limits normally enforced by the length of the telomeres. Two examples include gamete cells, which carry genetic material from one generation to the next, and the cells in a developing embryo. In the delicate mechanics of the cell, the enzyme telomerase is turned on and off as it is needed to maintain the length of the telomeres.
In contrast to specialized cells, normal cells do not express telomerase. In almost all tumor cells, however, telomerase is expressed. When telomerase is turned on inappropriately, the cell can then become immortal, meaning it can continue to divide indefinitely. The discovery of the gene-encoding part of the telomerase enzyme will allow scientists to probe deeper into the mystery of how the correct and incorrect expression of telomerase is linked to the development of cancer.
The first group to describe the gene was headed by Toru Nakamura and included Nobel Prize winner Thomas Cech of Colorado University at Boulder and the Howard Hughes Medical Institute, as well as six additional authors, five of whom work for Geron Corporation of Menlo Park, California. Their findings were published in the 15 August 1997 issue of
Science
. "We hope the cloning of this gene will lead to the discovery and use of new drugs in the fight against cancer," said Greg Morin of Geron, one of the coauthors.
In the 22 August 1997 issue of
Cell
, a second group, led by Matthew Meyerson of the Massachusetts Institute of Technology (MIT), announced their discovery of the same gene. The group also included Robert Weinberg of the Whitehead Institute of Biomedical Research at MIT and eleven others.
Jerry Shay, a professor of cell biology and neurosciences at University of Texas Southwestern Medical Center in Dallas and a recognized leader in telomerase research, said of the simultaneous cloning of this gene, "This is important because we can now begin to understand how telomerase works. If we can't find a cure for cancer, we need to start detecting it earlier so we can stop it--or control it--before it spreads. Telomerase can be used as an early definitive marker of cancer."
Also newsworthy is the way in which the telomerase gene was cloned. "It should have taken a couple of years to clone," said Shay, "but because the Human Genome Project clones ESTs [expressed sequence tags] the groups were able to pick it out through its homology to the yeast telomerase gene." An EST is a 400- or 500-base-pair fragment that is identified through random sequencing of cDNA libraries. The groups were able to look at these ESTs and identify those that showed homology, or identical sequence, with the already identified yeast telomerase gene. Therefore, the work of the Human Genome Project accelerated by years the cloning and identification of an unknown gene.
"This is all early, basic, science-type stuff. We are a long way from being able to extend life and cure cancer," said Shay. "But these are profound things to even be contemplating."
A question of cancer
. New evidence shows that environmental tobacco smoke may have serious health effects for women including a strong link to cervical cancer.
|
New evidence provides support for the argument that smoking may cause cervical cancer, not just in smokers but also in nonsmoking women who are exposed to environmental cigarette smoke. The study, reported in the 18 June 1997 issue of the
Journal of the National Cancer Institute
, is the first to find a tobacco-specific carcinogen in samples of cervical mucus taken from both smoking and nonsmoking women. The carcinogen is 4-(methylnitrosamino) -1-(3-pyridyl)-1- butanone (NNK), an
N
-nitrosamine that is formed during the processing and burning of tobacco products. NNK is one of the most potent carcinogens found in tobacco smoke. It has been detected in the sidestream smoke of cigarettes (the smoke that wafts from a smoldering cigarette), which means that not only smokers but also nonsmokers who breathe in secondary cigarette smoke are exposed to NNK.
The study was conducted by Bogdan Prokopczyk, head of the section of bio-organic chemistry in the division of cancer etiology and prevention at the American Health Foundation in Valhalla, New York, and Steven E. Waggoner, an assistant professor of gynecologic oncology at the University of Chicago Medical Center, and colleagues. The study cohort consisted of 25 women, 15 of whom smoked and 10 of whom did not smoke. The women were aged 18-45, were free of any active genital tract disease, and were not currently using oral contraceptives. The scientists collected cervical mucus samples from the women and used highly sensitive gas chromatography-mass spectrometry analyses to identify and quantify the NNK in the samples. Of 26 samples taken (one woman gave two samples), only one--taken from one of the nonsmoking women--did not contain some measurable amount of NNK. Among the other 9 nonsmokers, NNK concentrations ranged from 4.1 to 30.8 nanograms per gram (ng/g) of mucus, approximately one-third lower than the amounts found in the samples taken from the smokers, which ranged from 11.9 to 115.0 ng/g.
Scientists have known for some time that there is a link between cigarette smoke and cervical cancer, but the precise nature of the link is still uncertain. Prior research has determined that noncarcinogenic compounds from cigarette smoke, such as nicotine and its metabolite cotinine, can be detected in the cervical mucus. There is also evidence that cigarette smoke is capable of causing damage to DNA in cervical epithelial tissue. But the new findings are the first time that a carcinogen specific to tobacco has been found in the cervical mucus of women who smoke. The link between smoking and cervical cancer has been greatly strengthened by these latest findings, which, according to the report's authors, lend "biologic plausibility in support of the association between cigarette smoking and [cervical cancer]."
In the United States in 1996, there were an estimated 15,700 new cases of cervical cancer and 4,900 deaths from the disease. According the
JNCI
study, cervical cancer is the top cause of death from cancer in women in developing countries, and is one of the most common cancers among U.S. women aged 15-54. The foremost risk factor for cervical cancer is infection with certain strains of the human papillomavirus (HPV). The DNA-level effects of such viruses are found in up to 93% of all examined cervical tumors, but these effects alone are not thought to be enough to induce carcinogenesis. Many other factors are associated with cervical cancer, including deficiencies in micronutrients such as beta carotene and folate, impaired immune status, early onset of sexual activity, and cigarette smoking.
All cancers, scientists have learned in the last 20 years, begin with a malfunction in the genetic machinery of a cell, causing it to duplicate itself without restraint. Such knowledge brings with it not only new hope for stopping these deadly diseases, but also a great challenge for scientists--to discern the location and function of the culprit genes among the over 100,000 in each human cell.
Now, a powerful new tool on the World Wide Web is making this task much more feasible. The Cancer Genome Anatomy Project (CGAP), which was launched in August 1997, provides scientists with data on the genes expressed in cancerous, precancerous, and normal cells from several different tissues and organs. The site, located at
http://www.ncbi.nlm.nih.gov/ncicgap/
, was developed by the National Cancer Institute and the National Library of Medicine. Information accumulated there will allow scientists to determine how gene expression changes as cells become cancerous, and use this knowledge to direct their research toward suspect genes or their protein products.
Such research could lead to new ways to stop malignant tumors, but even if fulfillment of that goal remains elusive, the genetic "fingerprints" in the CGAP gene index will serve other valuable functions. Using this information, doctors will be able to determine if cells are cancerous before a tumor forms by looking at what genes are being expressed in them. This will allow treatment regimens to be started earlier, quite possibly saving the lives of many patients. Doctors could also use this information to better match treatments to patients by using genetics to distinguish cancers that look nearly identical but respond differently to treatments.
Another goal of the CGAP is to promote the development of new technologies that will add to the breadth of information available on the site. Current technology, for example, makes it difficult to sequence the full lengths of the genes expressed in a cell, so much of the CGAP database is made up of expressed sequence tags--short sequences from randomly isolated portions of genes that can be used to identify genes but that do not necessarily contain all of their genetic information. To address this shortcoming, the CGAP has made $2.5 million in grants available to researchers to develop methods to efficiently map the full-length sequences of all the genes expressed in cells. Other CGAP grants support the development of technologies to scan the genomes of cancer cells and to evaluate molecular changes in tumor specimens.
From the CGAP home page, information on CGAP grants can be found by following the Information link on the left menu bar. Also on this menu bar is a link to a description of one of the newest technologies being used to gather data for the CGAP web site: clicking on the picture of the laser capture microdissection microscope brings users to an account of how this tool allows scientists to isolate only a few cells of interest from a tissue sample for genetic analysis, rather than using larger specimens that may contain several types of cells.
Except for the View Libraries link, all other links on the home page take users to other sites or to information on the methods used to create the CGAP database. By following the View Libraries link, users are led to the heart of the site, a list of the tissue libraries for which CGAP genetic data are available. Choosing a link in this list will lead to the genetic library page for that tissue. Each library page includes information about the tissue donor, a link to download all of the expressed sequence tags observed in that library, and brief descriptions of the genes found in the library that seem particularly interesting (either because they code for a large portion of the proteins found in the cell or because they are unique to that library or tissue).
Each gene of interest on the library page is a link to another page of additional data about that gene and the protein for which it codes. The gene pages contain links to maps that show the location of the gene on the human chromosomes, information on other libraries in which the gene is expressed, and links to the DNA sequence that composes that gene.
In many cases, the information on the CGAP site is incomplete--evidence of the enormous amount of work that has yet to be done. But using the Internet to coordinate the effort increases the likelihood that the CGAP will obtain its goal of achieving a comprehensive molecular characterization of the broad spectrum of normal, precancerous, and malignant cells.
Last Update: October 20, 1997