Polynuclear aromatic hvdrocarbons J
New understanding of their mutagenic and carcinogenic mechanisms is accompanying an improved ability-mostly through chromatographyto detect them in extremely small concentrations
For several years, it has been generally accepted that polynuclear aromatic hydrocarbons (PAHs) have mutagenic and carcinogenic effects. Now, however, more light is being shed on the biochemical mechanism of these effects. In addition, we have better methods of analyzing for PAH concentrations, even below parts per billion (ppb). As Anthony Dennis of Battelle’s Columbus Laboratories expressed it, “There have been great advances in PAH detection/analysis techniques to answer current needs, as well as in the application of those techniques to delineating effects in biological systems.” These improved analytical techniques are being applied in order to learn the origin, transport. fate, and metabolism of PAHs. These and related topics were discussed in great depth at the Fifth International Symposium on Polynuclear Aromatic Hydrocarbons. Held in October as part of an ongoing series, this symposium was sponsored by Battelle, EPA, and the Electric Power Research Institute, and was cochaired by Dennis, and Marcus Cooke, also of Battelle. Sources
PAHs in the environment originate from vehicle exhaust (especially diesel), refuse, carbon black, creosote, soot, residual oil, and other sources. In the future, PAHs may come from
Coore and Uennis how new techniques m e applied 20
Environmental Science (L Technology
plants that produce synthetic fuels from coal or oil shale. As Willie May of the National Bureau of Standards (NBS) stated, the literature reports that a commercial synfuel plant, if not properly controlled, could emit as much as IO4 kg/d of PAHs. Instruments that can detect PAHs to sub-ppb levels have aided in the understanding of the mutagenic and carcinogenic biochemistry of PAHs. They have shown, for example, that PAHs themselves are not always the culprits. Rather, researchers blame chemical reactions and their metabolic products. Some earlier work In 1978, a t an ACS press conference, James Pitts of the University of California-Riverside explained how the mutagenic or carcinogenic potential of PAHs in air was increased when these materials were reacted with some common air pollutants. Under controlled conditions, for instance, benzo[a]pyrene (BaP) was reacted with ozone and N02. Direct-acting mutagens, such as hydroxybenzo[a]pyrene and l,3-, and 6-nitrobenzo[a]pyrenes were formed. Moreover, filters that were coated with about 2 mg of BaP, exposed to ambient air for several days, and then tested, contained direct mutagens in the form of BaP dihydrodiols, diphenols, phenols, and quinones. In discussing some of Pitts’s findings, Thomas Hughes et al. of the Research Triangle Institute spoke of an even more serious problem: a reaction of perylene and N02, both nonmutagens, which formed 3-nitroperylene, a potent mutagen.
Dihydrodiols Could parallel reactions in living tissue transform PAHs into mutagens or carcinogens? Perhaps. For example, scientists are scrutinizing dihydrodiol metabolites of PAHs for oncogenic
potential. M. Coombs of the Imperial Cancer Research Fund (England) described studies of chemicals of the cyclopenta[a]phenanthrene (CPAP) series. These studies found, in principle, that the unsubstituted hydrocarbon was essentially inactive as a carcinogen. A related compound, 12ketocholanic acid, had weak carcinogenic potential. With a keto group on the cyclopenta portion of some CPAPs, however, in vitro and in vivo metabolism experiments revealed that these compounds were changed into various diols and triols. Coombs noted that the 3,4-diols seemed to be especially active oncogenically. H e added that they could further evolve into epoxy compounds that can bind to a portion of a DNA helix. Coombs also described strong oncogenic potential in certain 1 I-alkoxy-1 7-ketone derivatives of CPAP. For instance, if the alkoxy is methoxy, the compound is very active-90% of test animals developed neoplasia over 30 days. However, as the alkyl chain becomes longer, activity decreases sharply, particularly when the alkyl chain is unsubstituted and unbranched. Metabolites of BaP, generated in an isolated rabbit lung system, were evaluated for mutagenicity by Rita Schoeny of the University of Cincinnati. These metabolites, extracted from pulmonary blood and tissue with a mixture of toluene and other solvents, were identified and quantitated by high-pressure liquid chromatography (HPLC). Among substances found were unmetabolized BaP, along with 4,s-, 7,8-, and 9,10-diols, various phenols and quinones, and a 4,S-epoxide or its derivative. When the lung is exposed to BaP and particulate matter, pulmonary macrophages appear to accumulate the BaP, but do not metabolize it to any significant degree. When particles are not present, these
cells and other tissues metabolize the ’BaP to a number of mutagenic forms. One scientist at the symposium speculated that the presence of such diol groups on the BaP molecule allows the formation of epoxides at nearby molecular positions. These epoxides, in turn, can be transformed into carbonium ions which can attack cell D N A electrophilically . The tie that binds Most study results suggest that PAH epoxides or arene oxides are indeed the electrophilic chemical species which initiate BaP-mediated carcinogenesis. Stephen Safe of the University of Guelph (Ontario) discussed the metabolism of BaP and pointed out the many competing pathways for potentially mutagenic PAH carbonium (C+) ions. The intra- and extracellular competition for C+ species plays an important role in the activity of PAHs in short-term in vitro test systems. Suppose a reactive epoxide group is formed on a PAH. Further biochemical action may transform the epoxide into a C+ ion which actually attacks a D N A or R N A site electrophilically. Binding to the site disturbs the genetic makeup of the victim cell and may allow its reproduction by the wild, uncontrolled mitosis that characterizes a malignancy. Safe suggested research to ascertain whether there may be any mechanism that can detoxify carcinogens and their C+ ions. Also, even if cells are altered, can such cells be detected and destroyed by antibodies before they evolve into tumors? Steric formations In addition to the chemical content of PAH metabolites, steric structure may also play a role in the carcinogenic process. Kenneth Miller of Rensselaer Polytechnic Institute explained that, for example, each isomer of the benzo[a]pyrenediol epoxides (BPDE) interacts differently with DNA. When introduced into the D N A helix, the conformation BPDE II(-) is in a favorable position to bring about phosphorylation of the helix. Moreover, D N A stereoselectivity permits only one isomer, BaP triol carbonium (BPTC I(+)) in the diaxial conformation, to approach closely enough for covalent binding to N 2 on guanine. However, the D N A must be “unwound” from the B-DNA portion of the helix to permit oncogen access to the binding site. Miller said that results of theoretical calculations of steric effects correlate with mouse tumorogenesis, with the orientation of the
chromophore to the helical axis, and with the observed unwinding of DNA. Substituent effects Dibenzo[a,h]pyrene (DBahP) and dibenzo [a,i] pyrene (DBaiP), or their metabolites, seem to be strong carcinogens; the DBaiP isomer, in feedings as small as 6 pg, causes sarcomas in mice, noted Stephen Hecht of the American Health Foundation (Valhalla, N.Y.). Hecht postulated that 3,4-diols with a nearby epoxide structure may be the “proximate carcinogen.” He also said that when these highly toxic PAHs were substituted with fluorine (the halogen, as opposed to the PAH fluorene), carcinogenic activity declined. With sufficient fluorine substitution, carcinogenicity apparently fell to near zero. “Natural choice” Given the biochemical effects of PAHs, detection to sub-ppb levels is a must, and chromatography is “the natural choice” to detect trace amounts, said Elena Katz of PerkinElmer Corp. (Norwalk, Conn.). She recommended liquid chromatography, HPLC, and reversed-phase liquid chromatography with fluorescence detection. One group using liquid chromatography is a research team headed by Richard Luthy of Carnegie-Mellon University (CMU). That team’s goal is to establish methods of detecting and determining PAHs-listed by EPA as priority pollutants-in synfuel plant effluents. Luthy and team member Richard Walters explained that PAHs may be found primarily as adsorbed or absorbed species on suspended matter in the plant’s effluent water. If correct, that evaluation may affect wastewater treatment facility design. Since PAHs have low aqueous solubilities, suspended material removal would have to be considered. To conduct analyses, Luthy and his team extract PAHs with methylene chloride. Prior to this extraction, samples are separated into liquid and suspended solid phases by filtration. Alumina column chromatography cleans the extract, and HPLC with fluorescence does the actual detection. The analytical standard materials are obtained from a commercial source. The C M U team looks for the presence of 11 PAHs in samples from a coke plant and a coal gasification plant. Of these, chrysene (CHR) is listed as “uncertain” or “weakly carcinogenic,” while BaP and DBahA are
listed as “strongly carcinogenic.” Associated with the suspended particles in untreated wastewater samples, C H R was detected at 800 pg/L, while BaP and DBahA were found at 280 pg/L and 150 pg/L, respectively. Concentrations for these and other PAHs in treated wastewater streams were on the order of pg/L (ppb) or less. In related studies, Luthy’s group found that phenol recovery by solvent extraction from heavily contaminated coal gasification process condensates removes PAHs to less than detection limits. The CMU group is commencing studies on treatment and fate of organics in oil shale and tar sand effluent samples. Another H P L C user is Dilip Choudhury of the New York State Department of Health, who reported on the application of real-time ultraviolet (UV) spectral determination of HPLC eluates to unambiguous characterization of PAHs. The vidiconbased instrument enables the UV spectrum of the HPLC eluate to be observed every 32 ms; several scans can be integrated to obtain a welldefined spectrum. The detector has excellent sensitivity (low-ng for most PAHs), and is particularly attractive for confirming the identity of an HPLC-separated PAH and, to a certain extent, for its quantitation. Choudhury demonstrated that isomeric PAHs can be differentiated and confirmed even when they coelute, an important consideration because mutagenic and carcinogenic properties of a PAH are isomer-specific. Spot checks Other techniques may also prove useful for PAH detection and analysis. For example, Takeo Sakuma described a trace atmospheric gas analyzer (TAGATM) now being marketed by SCIEX INC. (Thornhill, Ontario, Canada). It tests samples from ambient air, stack gas, fly ash, soot, exhaust, sediment, and other PAH sources, and utilizes ion/molecule reactions initiated by corona discharge. PAH present in hot gas streams can be analyzed in 1-3 minutes by transporting the hot gas through heated glass tubing before PAHs have a chance to condense onto particulate matter. The hot gas is analyzed by a TAGATM-which utilizes the principle of atmospheric pressure chemical ionization-followed by mass analysis and ion detection using a quadrupole mass analyzer. With this technique, over 200 PAHs and other combustion products were tentatively identified in a coal-fired cement kiln on a real-time basis. Volume 15, Number 1, January 1981
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Another approach-one which acts as a spot check or test-uses sensitized fluorescence. As an added feature, it can save a lot of money by eliminating the need for more sophisticated analytical techniques when no PAHs are present. This method involves making three spots on a circle of filter paper, such as Whatman 42. One spot is a blank, containing only sensitizer. Another is the analyte, and the third is the analyte mixed with the sensitizer. The analyte-sensitizer mixture should dry on the paper for about 30 s. The spotted paper is then placed in
a UV cabinet, where PAH material appears as a bright spot, explained Raymond Merrill, Jr., of EPA (Research Triangle Park, N.C.). The spot test can detect the presence of many PAHs, such as BaP, fluorene, fluoranthene, and acenaphthene. For most PAHs with molecular weights greater than 200, this type of UV spot test is sensitive to amounts as low as IO- IO0 pg, Merrill said. Spot tests were run on roof pitch and on coke-oven off-gas samples. The roof pitch test predicted 6-60% PAHs; subsequent analyses showed that 80%
SeOarSIi""
was the correct figure. The coke-oven spot test detected and predicted 100% PAH-related material. Corroborative analyses also indicated the presence of PAHs, PAH-related heterocyclics, and other such materials. Merrill also listed compounds that show negative spot tests. For instance, p-terphenyl, biphenyl, isoquinoline, carbazol, and pp'-DDE show no sensitized fluorescence. However, one must be on guard, since TNT, benzothiazol, or a PCB, such as ArocloP 1254, in high concentration, can cause interference with the sensitizer. Other compounds, such as phthalates, absorb UV, but do not interfere with the fluorescence-sensitized spot test. Metabolite analysis These analytical techniques lend themselves very well to analysis of samples of air, synfuel effluent, pitches and creosotes, and other such materials. But what about PAH metabolites from living organisms? That was a problem that Usha Varanasi of the National Marine Fisheries Service (Seattle, Wash.) faced when she ran experiments on commercial fish, such as English sole and starry flounder. She fed them PAHs at the rate of 2 mg/kg of fish body weight. The PAH was dissolved in corn oil, after being labeled with '4C.
alumina column
In order to separate BaP metabolites more clearly-in this case, those in liver lipids-Varanasi developed two-dimensional thin-layer chromatography (TLC). This involved slicing liver tissue into such thin sections that they appeared to be flat. BaP metabolites were detected a t the 4 5 , 7,8-,and 9,lO-sites. Also found were quinones and other unidentified compounds. Varanasi reminded the symposium that sole and flounder are members of the same family and that both are bottom feeders. They may therefore encounter BaP and other PAHs in oil-contaminated bottom sediments while hunting for food. She also noted that the PAH experiments she conducted were run under controlled laboratory conditions, rather than on fish taken from suspected contaminated habitats. Mitigation? The information being developed through P A k research will, hopefully, lead to improved ways of minimizing their discharge to the environment. Perhaps the future will even bring new techniques to mitigate the adverse health effectsof the toxic ones. -Julian Josephson
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Environmental Science & Technology
