FEATURE
A Renaissance for Genotoxicity Testing? Test results are driving a rebirth of interest in the use of the technique to evaluate pollution. DEBORAH lthough genotoxicity tests—bioassays that measure a substance's potential to induce mutations or other types of genetic damage—have been used for more than two decades in evaluating environmental samples, they have yet to be formally incorporated into regulations controlling air emissions, wastewater effluents, or contaminated soils. Now, however, new findings from applications of genotoxicity assays are spurring heightened interest in more fully understanding its practical utility. Environmental toxicologist Paul White, now a visiting sicentitst at EPA, and aquatic ecologist Joe Rasmussen of McGill University in Montreal reported this year (i) that on the basis of a regional analysis of genotoxicity inputs to the Saint Lawrence River, the genotoxic loading of municipal wastewater treatment plant effluent far outdistanced genotoxic inputs from industrial effluents. And in a current review of the genotoxicity of industrial wastes and effluents (2), genetic toxicologists Larry Claxton, Virginia Houk, and Thomas Hughes, of EPA's National Health and Environmental Effects Research Laboratory in Research Triangle Park, N.C. noted that bioremediation in some instances, can increase the genotoxicity of contaminated soils. The research incorporates a biological approach into environmental assessment and is motivated by a shortcoming of chemical monitoring: Even the most indepth characterizations cover only a fraction of the thousands of compounds that may be present in complex environmental mixtures. Moreover, toxicological information is often missing for the set of substances found, and interactions between different chemicals are not accounted for. Yet while genotoxicity testing, including the widely used Ames mutagenicity test, has long played an important regulatory role in evaluating the toxicity, particularly the carcinogenicity, of single substances chemical products and pharmaceuticals (see sidebar on page 501A) direct regulatory applications in pollution control have been limited.
A
4 9 8 A • NOV. 1, 1998 / ENVIRONMENTAL SCIENCE & TECHNOLOGY / NEWS
SCHOEN A major obstacle for using the method within a regulatory framework has been the difficulty of evaluating the real risks for humans from exposure to complex environmental mixtures shown to be genotoxic in bacterial or mammalian cell test systems. Nonetheless, researchers remain optimistic about the resolution of this issue. For Houk, a positive genotoxic response from a compound or mixture sends a red flag that indicates the substance can cause genetic damage. "But we must use our collective knowledge about exposure to that chemical and about our body's ability to repair genetic damage before we can draw conclusions about true risk " she said. In this respect recent applications of molecular techniques that allow sequencing of induced mutations provide a new avenue for monitoring exposure of people to complex environmental mixtures (3) The full potential of genotoxicity testing, however, may well lie in its application to problems other than h u m a n cancer. With increasing interest in the conservation of biodiversity and advances in molecular techniques for detecting DNA damage, ecotoxicologists interested in the impacts of chronic exposure to pollutants are focusing on genotoxic effects in organisms and the associated detrimental outcomes for exposed populations. To the extent that such adverse effects in natural populations can be causally related to the presence of genotoxic substances, testing for genotoxicity may well play a much greater role in environmental policy in the future.
Genotoxicity of effluents and wastes Among the environmental samples extensively characterized for genotoxicity are industrial effluents, for which there is a large body of past research on pulp and paper mills (4). More recently, White worked with Rasmussen and toxicologist Christian Blaise of Environment Canada's Saint-Lawrence Center in Montreal to explore the sources and fate of genotoxins discharged to the Saint Lawrence River. Their regional-scale study 0013-936X/98/0932-498AS15.00/0 © 1998 American Chemical Society
involved using the SOS Chromotest to measure the genotoxicity of 42 industrial discharges (5). This assay (see photo at right) detects DNA-damaging agents by using the error-prone repair pathway of Escherichia coli bacteria, known as the SOS response, rather than by directly detecting mutants, as in the Ames test. The SOS Chromotest advantageously provides same-day results for a large number of samples, while producing measurements of genotoxic potency that are highly correlated with Ames test results. Consistent with previous investigations using the Ames test (4), the most genotoxic effluents found were those from organic chemical production plants, whereas pulp and paper, metal refining and founding, and petroleum refining facilities also exhibited high potency (5). When White examined genotoxic loadings (potency x flow rate), however, he found that municipal wastewater treatment plant effluent accounted for over 85% of the local increase in genotoxicity in St. Lawrence River water in the Montreal region (i). "In terms of potency, municipal wastewaters are not very high compared to some industrial effluents," explained White, "but genotoxic potency of the whole effluents did not vary that heavily, only about fivefold, whereas the discharge rate of the municipal treatment facility may be several orders of magnitude greater than industrial discharges." Looking for sources of genotoxicity in municipal wastewaters, White estimated the contribution of the industrial effluents discharged to the Montreal Urban Community sewer system (see photo above) and found that they accounted for only about 8% of the municipal effluent loading value, leading him to speculate that substances contributing most to the genotoxicity in surface waters are domestic in origin. Though the compounds remain unidentified, analytical data indicate that they are direct-acting (not requiring liver enzyme activation), relatively water-soluble organic compounds and have a low affinity for suspended particulate matter. Other data (i), however, do not always support this characterization for the predominant genotoxins in surface waters and domestic wastewaters. White noted that different testing methodologies may account for differences in results and variability in wastewater t r e a t m e n t processes
Treatment processes can greatly affect the genotoxicity of wastes and effluents, although often in unpredictable ways. For example, biotreatment of whole pulp and paper mill effluent in an aerated lagoon can reduce mutagenic potency by several orders of magnitude (4). In contrast, and consistent with other reported observations (6), toxicologist Christoph Helma at the University of Vienna in Austria and colleagues observed the formation of direct-acting Salmonella mutagens in the activated sludge treatment of municipal wastewater in Krakow, Poland. Similarly (2), genotoxicity increased during the bioremediation of coal tarcontaminated soils, even as polycyclic aromatic compunds were reduced. But in other instances such as the bioremediation of the crude oil spilled by the Exxon Valdez the genotoxicity of contaminated soil sam~ ples has been effectively reduced through treatment (2) Given the inconsistency between the results of chemical monitoring for specific substances and geno-
The effluent plume of the Montreal Urban Community wastewater treatment plant is one of many contributors of genotoxic substances to the Saint Lawrence River system. Researchers attribute over 85% of the increase in genotoxicity of the river in the Montreal region to municipal wastewater treatment plant effluent. (Courtesy Saint-Lawrence Center, Environment Canada.) Inset: The SOS Chromotest genotoxicity assay measures the level of induction of the SOS response (errorprone DNA repair pathways) in Escherichia coli. Two columns of the microplate indicate a dark blue gradient, confirming genotoxic activity in two samples assessed at a range of concentrations. (Courtesy Saint-Lawrence Center, Environment Canada).
toxicity measurements, many researchers advocate incorporating genotoxicity assays into environmental monitoring of remediated sites. Kirby Donnelly and colleagues at Texas A&M University in College Station, Tex., found in a study of an abandoned solvent recovery site that, even after all visible sources of contamination (paint sludges) were removed and cleanup criteria were met, samplesfroma drainage area downgradient from the major waste storage area had mutagenic activity over 30 times greater than uncontaminated background soil (7). Donnelly has also combined genotoxicity testing with chemical analysis to assess the differences between solvent and aqueous extraction procedures in removing chemicals from soil (8) and to investigate the capability of microbial cultures and indigenous organisms to detoxify a soil contaminated with a complex mixture. "A combined chemical and biological testing protocol provides the most accurate information from which to assess risk. Bioassays are capable of detecting chemical interactions and chemicals, which may not be well characterized regarding their potential toxicity. However chemical analysis is important to eliminate false positives or negatives that may be detected in bioassays" he said Bioassay-directed chemical analysis Environmental scientists have long recognized the value of combining chemical and biological approaches in the evaluation of complex environmental mixtures, notably through bioassay-directed chemical analysis—a protocol used to identify specific chemicals or classes NOV. 1, 1998 / ENVIRONMENTAL SCIENCE & TECHNOLOGY / NEWS • 4 9 9 A
Genotoxic potencies of complex mixtures
