Environ. Sci. Technol. 2001, 35, 3576-3583
The Epigenetic Toxicity of Pyrene and Related Ozonation Byproducts Containing an Aldehyde Functional Group H O L L Y A . H E R N E R , †,§ JAMES E. TROSKO,‡ AND S U S A N J . M A S T E N * ,† Department of Civil and Environmental Engineering and Department of Pediatrics and Human Development, Michigan State University, East Lansing, Michigan 48824-1326
Gap junction intercellular communication (GJIC) was used to assess the epigenetic toxicity of pyrene, pure byproducts of pyrene ozonation, and other compounds similar in chemical structure. Byproduct mixtures collected from HPLC were also evaluated using GJIC. Of the 11 pure compounds studied, five inhibited GJIC completely. Two inhibiting compounds contained four rings and were the only compounds studied with greater than three rings. The remaining three compounds contained either two or three rings, and all three contained an aldehyde group. Toxicological evaluation and GC/MS of impure byproduct mixtures showed that two common compounds were found in inhibiting fractions. These common compounds contained both a bay region and at least one aldehyde group.
Introduction Polycyclic aromatic hydrocarbons (PAHs) are derived from the incomplete combustion of organic matter and are found in soil, air, and water (1-3). Sixteen PAHs have been identified as priority pollutants and eight as carcinogens and possible carcinogens (2, 3). These compounds are recalcitrant to conventional treatment and persist in the environment (3). However, some PAHs have been successfully degraded using chemical and biological technologies (4-7). Ozone has been used in the degradation of PAHs and other compounds resistant to conventional treatment methods (3). Although ozone is capable of the degradation of PAHs to carbon dioxide, water, and straight chain aliphatics, studies have shown that certain byproducts of PAH ozonation can be as or more harmful than the parent compounds themselves (4, 5, 8, 9). A dosage that will degrade the parent compound and the harmful byproducts but still offers process efficiency and low cost must be used. Pyrene, a four-ringed PAH, was almost entirely degraded (>90%) by ozone at a dosage of 1.6 mol of ozone/mol of pyrene (4). However, the mixture was still toxic as determined using inhibition of gap junction intercellular communication (GJIC) to monitor epigenetic toxicity (9). Epigenetic toxicity is defined as the alteration of the expression of genes, either at the transcriptional level (turning genes “on” or “off”), the * Corresponding author phone: 517-353-8539; fax: 517-355-0250; e-mail:
[email protected]. † Department of Civil and Environmental Engineering. ‡ Department of Pediatrics and Human Development. § Present address: RMT Engineering, 1143 Highland Drive, Suite B, Ann Arbor, MI 48108-2237. 3576
9
ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 35, NO. 17, 2001
translational level (modulating the stability of the gene’s message), or the posttranslational level (modifying the gene product or protein by phosphorylation, glycosylation, nitrosylation, etc.). At a dosage of 4.5 mol of ozone/mol of pyrene, all byproducts inhibitory to GJIC were eliminated (4). It is apparent that successful remediation must be defined by the removal of all potentially toxic compounds, not just the parent compounds responsible for the original risk. Upham et al. (9) showed that certain four- and five-ringed PAHs were more inhibitory to GJIC than certain three-ringed PAHs. In addition, compounds with a bay region were more inhibitory than those compounds without a bay region (9). The term bay region is used to describe the cove area created when multiple benzene rings create a sterically hindered C-shaped region. Phenanthrene, a three-ringed PAH with a bay region, was more inhibitory to GJIC than anthracene, a three-ringed PAH without a bay region (11). In another study, the addition of a methyl group to anthracene, at a position creating a bay-like region, caused inhibition of GJIC, whereas adding the same functional group at another position on the compound (without the bay-like region resulting) caused no inhibition (10). A common theory links the bay region, which many PAHs contain, to carcinogenesis (10-14). Functional groups such as the hydroxy, the diol-epoxide, and the methyl group that are adjacent to or part of the bay region have also been shown to enhance the activity of PAHs (12, 15-18). The ozonation of pyrene creates two types of byproducts: phenanthrene and biphenyl-type compounds, with aldehyde, carboxylic acid, and hydroxy functional groups. In the first portion of the study, we used ozone to degrade pyrene and to produce both phenanthrene and byphenyl-type byproducts. The epigenetic toxicity of pyrene, the byproducts of pyrene ozonation, and six commerically available byproducts similar to pyrene ozonation byproducts was evaluated using in vitro bioassays of intercellular communication in rat liver epithelial cell culture. The results of the toxicity evaluation were compared to each compound’s chemical structure. The first portion of the study focused on byproducts that constituted a majority of the mass. In the second portion of the study, impure byproduct fractions were evaluated to determine whether a byproduct, constituting a small percentage of the total mass, was contributing to the increased toxicity documented in previous studies. Two solutions containing pyrene were ozonated at differing dosages and fractionated. A toxicity evaluation and compound identification using GC/MS was performed for each fraction. The chemical structures for each fraction were compared to the respective toxicity. The objective was to determine whether any trends existed between chemical structure and epigenetic toxicity as measured by inhibition of GJIC.
Materials and Methods Chemicals. Pyrene, neutral red dye, and lucifer yellow dye were purchased from Sigma Chemical Co. (St. Louis, MO). Diphenic acid, 2-biphenyl carboxylic acid, 4-biphenyl carboxylic acid, 4-biphenyl carboxaldehyde, and 37% formaldehyde were all purchased from Aldrich Chemical Co. (Milwaukee, WI). 4-Carboxy-5-phenanthrene carboxaldehyde, 1,2,3,4-tetrahydro-9-phenanthrene carboxaldehyde, and 9-oxo-1-fluorene carboxaldehyde were purchased from Sigma-Aldrich’s Library of Rare Chemicals (Milwaukee, WI). Compound Q, 4-carboxypenathrene, and 4-carboxy-5phenanthrene carboxaldehyde, byproducts of pyrene ozonation (Figure 1), were generated in our laboratory. Acetonitrile and sodium chloride were purchased from EM Science 10.1021/es0106117 CCC: $20.00
2001 American Chemical Society Published on Web 07/31/2001
distance the dye traveled. The migration of the dye in the PAH treated and untreated cells was reported as a fraction of the distance the dye traveled in the control. The value of the control was 1.00, and any value less than 0.3 was considered completely inhibitory to GJIC. A value of 0.3 is commonly used to represent completion inhibition and is equal to the width of one row of cells with no dye having migrated beyond its boundary. A value between 0.9 and 0.3 was considered partially inhibitory to GJIC since a value greater than 0.9 is often difficult to distinguish statistically from the controls.
FIGURE 1. Pyrene and laboratory-generated byproducts. Pyrene was purchased commercially and used during ozonation experiments for generation of pyrene ozonation byproducts. Compound Q, 4CP, and 4C5P were obtained by chromatographic separation of the mixture of ozonation byproducts (ozonation conducted at pH ) 2 with ozone dosage of 1.6 mM ozone/mM pyrene, 0.5 M phosphate buffer solution). (Gibbstown, NJ). Sodium phosphate and ammonium persulfate were purchased from Columbus Chemical Industries (Columbus, WI) and Life Technologies (Gaithersburg, MD), respectively. Phosphoric acid and sodium tetraborate were purchased from J. T. Baker (Phillipsburg, NJ). Cell Culture Techniques. The GJIC assay was used because it has been shown to be not only a reliable, simple, and inexpensive in vitro assay, but also to be a very good predictor of epigenetic or nonmutagenic toxicants (19). WBF344 rat liver epithelial cells were obtained from Dr. J. W. Grisham and Dr. M. S. Tsao of the University of North Carolina (Chapel Hill, NC) (13). The WB-344 cell line was used because it is a diploid, nontumorgenic cell line, derived from the strain of rat on which many of the environmental toxicants and carcinogenic, or potentially carcinogenic, chemicals are tested. Therefore, there would be a large amount of toxicity and cancer data of in vivo tested chemicals with which our results could be compared. While the use of human diploid, nontumorgenic liver cells would have been ideal, primary hepatocytes quickly lose their biochemical phenotypes in vitro, and no normal human epithelial liver cells, equivalent to these rat liver cells, exist. Cells were cultured in 2 mL of D-medium (formula no. 78-5470EG, GIBCO Laboratories, Grand Island, NY) and were supplemented with 5% fetal bovine serum (GIBCO Laboratories, Grand Island, NY). Cells were prepared for experimentation in 35 mm2 plastic Petri dishes (Corning, Corning, NY). Cells were incubated at 37 °C in a humidified atmosphere containing 5% CO2 and 95% air. Bioassays were conducted on confluent cultures obtained after 2 days’ growth. Cell cultures were photographed using a Nikkon Diaphot-TMD epifluorescence phase-contrast microscope illuminated with an Orsam HBO 200W lamp and equipped with a 35-mm FA camera (Nikkon, Japan). In Vitro Bioassays of GJIC. The scrape loading/dye transfer (SL/DT) technique was adapted from El-Fouly et al. (20). Cell cultures were incubated with target compounds for various time periods with constant dosage or constant time periods with varied dosage. GJIC assays were conducted at noncytotoxic levels as determined by the neutral red dye uptake assay (21). Following chemical treatment, cells were rinsed five times with phosphate buffer solution (PBS). Approximately, 2 mL of lucifer yellow dye were added to cell cultures scraped using a steel surgical blade. Following a 3-min incubation, the dye was removed from cell cultures that were again rinsed with PBS five times. Cell cultures were fixed using 0.5 mL of 4% formalin. An inverted fluorescence microscope equipped with a camera was used to record the
Cell cultures were exposed to PAH stock solutions in the range of 0 to 200 µM. The equivalent volumes of acetonitrile added to cell cultures were used as vehicle controls. Vehicle controls using acetonitrile were run in conjunction with all cell culture experiments. The volume of acetonitrile in each control was equivalent to the volume of acetonitrile required to deliver the compound in the test cultures. All experiments were conducted in triplicate (three plates per dosage or time exposure), and values were reported as an average ( 1 SD. A statistical analysis was performed to determine significance between controls and test cultures. A t-test was performed to determine whether target averages differed significantly from that of the controls at p e 0.01. Ozonation. Ozone was generated by corona discharge using a Polymetrics model T-408 ozone generator (San Jose, CA). Oxygen was first dried using a molecular sieve. A 250mL gas washing bottle was used for the ozonation reactor. Ozone was bubbled into 200 mL of an acetonitrile and water (90:10 v/v) solution containing 5 mM dissolved pyrene. In an aqueous environment, water is a participating solvent in the degradation of pyrene using ozone (22). A 10% water solution ensures the reaction is not water limited but is dilute enough to avoid solubility problems due to the nonpolar characteristics of pyrene. The pH of the solution in the reactor was acidified to approximately 2 using a phosphoric acid solution (0.5 M) or increased to approximately 9.5 using a borate buffer solution (16 mM) as prepared by Adams (23). The flow of ozone was regulated at 100 mL/min with a side track flow controller (Sierra Instruments Inc., Monterey, CA). The ozonated solutions were continuously mixed with a magnetic stirrer and stir bar. Influent and effluent gaseous ozone were monitored spectrophotometrically at 258 nm using a UV-Vis spectrophotometer (model 1201 Shimadzu, Scientific Instruments, Japan). Excess effluent gaseous ozone was trapped in an aqueous 2% (w/v) potassium iodide solution. HPLC Analysis. Ozonated materials were evaporated in vacuo (Buchii RE111 Rotovapor, Brinkman, Westbury, NY) at 35 °C and weighed prior to separation by semipreparative high-pressure liquid chromatography. Ozonation byproduct mixtures were fractionated using a semipreparative HPLC (Perkin-Elmer, series 200, Cupertino, CA) using a Phenomenex, Capcell Pak, C18 (5 µM) column (i.d. 10 × 250 mm). A gradient solvent system using acteonitrile and water was employed with a flow rate of 1.5 mL/min. A 70/30% (v/v) acetonitrile/water solution at the time of injection was ramped linearly to 75/25% (v/v) acetonitrile/water over 23 min. At 23 min, the solvent system was increased to 90/10% (v/v) acetonitrile/water for 15 min. At 38 min after injection, the solvent system was decreased to 70/30% (v/v) acetonitrile/ water for 10 min to yield a total run time of 48 min. The eluent was monitored at both 240 and 254 nm (Waters, model 2487, Milford, MA). Fractionated materials were dried in vacuo at 35 °C and resuspended to make stock solutions ranging in concentration from 0 to 20 mM as pyrene. Aliquots ranging from 0 to 30 µL were added to the cell cultures used for GJIC assays. VOL. 35, NO. 17, 2001 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
9
3577
FIGURE 2. Commercially obtained compounds. These seven compounds, with structures similar to those generated during ozonation of pyrene, were purchased and evaluated for their ability to inhibit gap junction intercellular communication.
FIGURE 3. Dose response curves for pyrene and byproducts of pyrene ozonation. Concentrations of chemicals ranging from 80 to 200 µM were applied to cell cultures. Cell cultures were exposed to chemicals for 30 min. A GJIC value of 1 is indicative of total communication, while a GJIC value of 0.3 is indicative of complete inhibition of communication. Each data point represents an average ( 1 SD of the distance the dye traveled in three plates. PY and CMP Q were found to differ significantly from the control at p e 0.01 as determined by a two-tailed t-test.
Results and Discussion Pure Compound Evaluation. A series of compounds were assessed for inhibition of GJIC. Pyrene, three byproducts of pyrene ozonation, and seven commercially obtained compounds similar to byproducts of pyrene ozonation were studied. The compounds ranged from two to four rings and contained a variety of functional groups. Figures 1 and 2 contain structures and abbreviations for all compounds. Of the compounds studied, dose response experiments showed that five compounds were strong inhibitors of GJIC, and the remaining six were partial inhibitors. The parent compound pyrene, the largest PAH in size investigated, was a strong inhibitor, more so than pyrene ozonation byproducts 4CP and 4C5P (Figure 3). 4CP and 4C5P were both partial inhibitors of GJIC. 4CP and 4C5P were not evaluated at higher doses due to their aqueous insolubility. Similar dose response 3578
9
ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 35, NO. 17, 2001
curves were obtained for commercial and laboratory generated 4C5P, although commercial 4C5P was more inhibitory than the laboratory isolated version. The commercially obtained 4C5P was evaluated by HPLC and found to contain impurities, one of which was pyrene. Residual pyrene, a commercial compound impurity, is most likely responsible for the increased activity noted in the 4C5P purchased commercially. Phenanthrene, which has the same base structure without any functional groups, does completely inhibit GJIC (9). Both 4CP and 4C5P are phenanthrene-type compounds, each with a bay region and an acid group. 4C5P also contains an aldehyde group. However, 4C5P does exist in two configurations, one of which is a pseudo-four-ringed structure where the acid and aldehyde functional groups interact to create a fourth nonaromatic ring (Figure 1). This alternate configuration may play a role in the lack of activity observed for the 4C5P. Interestingly, 4C5P is the most abundant byproduct of pyrene ozonation (24). Of the laboratory-generated byproducts, only compound Q (CMP Q), a three-ringed aromatic compound with a fourth nonaromatic ring, almost completely inhibited GJIC but at a higher dosage than pyrene (Figure 3). Although this compound was inhibitory to GJIC, the results could not be compared with pyrene from a parent-byproduct compound standpoint since methylations encountered during purification translated the structure of compound Q from its original oxidized form. However, compound Q was one of the largest PAHs studied (four rings) and had a unique structure. Toxicity data for compound Q were evaluated and used as if it were just another PAH, unrelated to pyrene. Previous studies have shown that some byproducts of PAH ozonation can be as or more toxic than the parent compound (4, 8, 9). Although none of the byproducts during this study exhibited that characteristic, it is still likely to be the case. Only a limited number of byproducts were isolated due to the difficulty of separation of compounds with such similar structures and due to the small masses generated for many of the byproducts. It is very possible that one of the byproducts which constitutes a small amount of the byproduct mass is responsible for the more inhibitory results observed in studies by Upham et al. (4). During byproduct isolation studies, byproducts constituting a large percentage of the total mass were the focus. In the second part of this study, impure byproduct fractions characterized by GC/MS, were investigated to address this possibility. Interest in the identification of relationships between structure and chemical activity prompted the investigation of six additional commercial compounds (Figure 2). The breakdown of pyrene occurs with the formation of phenanthrene-type compounds initially followed by their subsequent breakdown and formation of biphenyl type compounds. Therefore, two phenanthrene-type PAHs and four biphenyltype PAHs that were commercially available were evaluated in the same manner. These six compounds resembled the byproducts in ring number and functional groups. Both threeringed compounds, TPC and OFC, inhibited GJIC completely at a low dose, with responses comparable to pyrene (Figure 4). Both compounds possess a bay-like region and an aldehyde group but also have subtle differences (Figure 2). Although, several explanations for the observed activity could be proposed, the results strongly suggest that the aldehyde group is at least partially responsible. In this study, OFC, fluorene with an aldehyde group, completely inhibited GJIC, whereas fluorene, studied previously, did not (9). And although phenathrene is inhibitory to GJIC, TPC, phenanthrene with an aldehyde group, was more inhibitory to GJIC (9). The last four commercial compounds evaluated were all two-ringed, biphenyl-type compounds with three containing
FIGURE 4. Dose response curves for pyrene and commercially obtained compounds similar structurally to pyrene ozonation byproducts. Concentrations of chemical ranging from 150 to 200 µM were applied to cell culture. Cell cultures were exposed to chemicals for 30 min. A GJIC value of 1 is indicative of total communication, while a GJIC value of 0.3 is indicative of complete inhibition of communication. Each data point represents an average ( 1 SD of the distance the dye traveled in three plates. OFC, TPC, and 4BCH were found to differ significantly from the control at p e 0.01 as determined by a two-tailed t-test. one or more acid functional groups and one compound containing an aldehyde functional group. Of the four biphenyl-type compounds, only 4BCH inhibited GJIC but at a much higher concentration than pyrene (Figure 3). The maximum concentration evaluated was dictated by solubility for all compounds. Generally, biphenyl-type compounds are not strong inhibitors of GJIC, which may explain the high dosage of 4BCH required for inhibition of GJIC. Although the concentrations examined were much higher than that used for the three- and four-ringed compounds, the concentrations studied are not unreasonable since biphenyltype compounds are more soluble in aqueous solutions and could be found in greater concentrations in the environment, should they be produced. For each of the five compounds that inhibited GJIC completely, the inhibiting dose was applied to cell cultures for varying time periods and GJIC observed (Figure 5). Pyrene inhibited GJIC completely after approximately 20 min and communication between adjacent cells remained blocked after 70 min. Although compound Q inhibited GJIC after approximately 20 min, partial restoration of communication was observed after 60 min (Figure 5). Both TPC and OFC blocked cell-cell communication within about 20 min and also displayed partial recovery of communication after 2 h (Figure 5). TPC regained almost 100% communication within 4 h of exposure. Although 4BCH required a high concentration for inhibition of GJIC, the response was observed after only 1 min but recovered partial communication after only 45 min (Figure 5). Time response data for all other compounds are reported in Herner (25). All five compounds that inhibited GJIC, inhibited quickly within 30 min, but interestingly, all five compounds also displayed partial recovery of GJIC without the removal of the test compound. Several possible explanations for the transient nature in which the compounds blocked GJIC exist. It is possible that the chemical indirectly inhibits GJIC by way of a protein kinase (PK) activation (26). In this case, a PK such as protein
FIGURE 5. Time response curves for compounds inhibitory to GJIC. The time of exposure varied from 2 to 350 min. The chemical dosage for each compound was equal to the dosage at which GJIC was completely inhibited during dose response experiments. A GJIC value of 1 is indicative of total communication while a GJIC value of 0.3 is indicative of complete inhibition of communication. Each data point represents an average ( 1 SD of the distance the dye traveled in three plates. All averages at 350 min (except PY) were found to differ significantly from the averages at the inhibited state at p e 0.01 as determined by a two-tailed t-test, indicating partial recovery. kinase C (PKC) phosphorylates the gap junction protein (connexin 43) and blocks cell-cell communication. At the same time, however, PKC is also phosphorylating phosphatases which dephosphorylate the gap junction protein and restore communication. There is a lag time before the phosphatases are activated which allows the inhibition of GJIC to be observed. If this process is indeed the mechanism by which the chemical inhibits GJIC, the addition of a phosphatase inhibitor would prohibit the recovery of GJIC. It is also possible that the inhibiting compounds work in a direct manner in their attack (26). Membrane fluidity and other conditions of the cell’s homeostasis may be altered which causes the initial inhibition of GJIC. The recovery of GJIC may be due to the cell’s ability to adapt to the new conditions. Finally, metabolism of the chemical may explain the return of cell-cell communication. Certain cells produce enzymes that can metabolize PAH compounds. However, it is unlikely that the undifferentiated rat liver epithelial cells used in this study produce such enzymes. The indirect inhibition by PK production may also be the proper explanation of the observed recovery. For example, 12-o-tetradecanoylphorbol-13-acetate (TPA), a known inhibitor of GJIC, works by this mechanism (27). In addition to the dephosphorylation of the gap junction, phosphatase dephosphorylates PKC, which initially causes inhibition of GJIC (27). Studies showed that after communication was restored, the addition of more TPA did not cause inhibition of GJIC. In this case, the PKC has been completely inactivated and the TPA cannot act again by the indirect mechanism to inhibit GJIC until the PKC has been replenished, which takes approximately 24 h (27). Cell cultures exposed to compounds inhibitory to GJIC completely recovered cell-cell communication when the respective compound was removed from cell culture (Figure 6). In most cases, GJIC was restored completely after approximately 2 h. This phenomena is consistent with the VOL. 35, NO. 17, 2001 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
9
3579
FIGURE 6. Time of recovery curves for compounds inhibitory to GJIC. Cell cultures were exposed to an inhibiting dosage of chemical for 30 min and were observed up to 350 min after the chemical was removed. A GJIC value of 1 is indicative of total communication, while a GJIC value of 0.3 is indicative of complete inhibition of communication. Each data point represents an average ( 1 SD of the distance the dye traveled in three plates. All averages greater than 340 min were found to differ significantly from the averages at the inhibited state at p e 0.01 as determined by a two-tailed t-test, indicating recovery. theory that inhibition of GJIC by a tumor promoter is a reversible process (28). In addition, the recovery of cell-cell communication shows indirectly that the compounds tested were not cytotoxic at the inhibiting doses. Cytotoxicity experiments conducted for all compounds confirmed that none of the compounds evaluated at any concentrations studied were cytotoxic, even after 24 h of exposure (25). This result proved that the inhibition caused by certain compounds was not due to cell death. Pyrene Byproduct Fraction Evaluation. Although the isolated byproducts studied were not more inhibitory to GJIC than the parent compound, pyrene; there is a possibility that interaction between products might make the mixture more toxic than pyrene. During the second phase of this study, mixtures of byproducts generated by ozonation of pyrene at low and high pH were fractionated using liquid chromatography and tested for their ability to inhibit GJIC. Two solutions of pyrene (5 mM), were ozonated until the majority of the pyrene was oxidized (ozone dosage of approximately 1.6 mol of ozone/mole of pyrene) (4). Byproducts of pyrene ozonation were produced at both pH 2 and 9.5. Typically, ozonation experiments are conducted at a low pH to ensure that the majority of reactions are due to direct oxidation of pyrene by molecular ozone. However, there are some practical circumstances, such as in alkaline soils or water treatment, where the pH could be much higher and should be evaluated. Dose response experiments for byproduct mixtures generated at low and high pH showed that both mixtures completely inhibited GJIC. Byproducts from ozonation at low and high pH were fractionated by RP-HPLC into 10 and six fractions, respectively (Figure 7). At low pH, a watersoluble fraction was collected prior to fractionation using the RP-HPLC. Because of the impurity of the fractions, the bioassay concentrations were calculated based upon the molecular weight of the parent compound, pyrene. All fractions collected from RP-HPLC were evaluated for their 3580
9
ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 35, NO. 17, 2001
FIGURE 7. Chromatograms from separation of pyrene ozonation byproducts. (A) Chromatogram from high-pressure liquid chromatographic separation of pure products for first portion of this study. Details of the purification and solvent system are contained in Herner (24). Peaks 9 and 10 are 4-carboxy phenanthrene and 4-carboxy-5-phenanthrene carboxaldehyde, respectively. Compound Q was derived from peak 10 and methylated in the laboratory. (B) Chromatogram from high-pressure liquid chromatographic fractionation of byproduct mixture generated during ozonation of pyrene at low pH. (C) Chromatogram from high-pressure liquid chromatographic fractionation of byproduct mixture generated during ozonation of pyrene at high pH. ability to inhibit GJIC at 75 µM as pyrene, a concentration at which pyrene completely inhibits GJIC. Fractions collected from mixtures generated at both low and high pH were characterized using GC/MS, and results are shown in Tables 1 and 2. Figure 8 shows the chemical structures for the identified byproducts.
TABLE 1. GC/MS Characteristics for HPLC Fractions and Water-Soluble Fraction (low pH mixture) peak no.
compound I.D. (Figure 8)a
water-soluble fraction 1 2 3 4 5 6 7 8 9 10
J, K, L I, J, K, L A, C, E, I, J A, B, C, E, Gc, J, K, L, Rb C C, E C, D, E C C C, D, E, I, J, K, Lc A
a Mass spectra in ref 24. pound weakly present.
b
Mass spectrum located in ref 25. c Com-
TABLE 2. GC/MS Characteristics for HPLC Fractions (high pH mixture) peak no.
compound I.D. (Figure 8)a
1 2 3 4 5 6
Ac, C C, Ec B, C, D, E, Rb E c, S b C, Dc, Ec A
a Mass spectra in ref 24. pound weakly present.
b
Mass spectrum located in ref 25. c Com-
FIGURE 8. Previously identified byproducts of pyrene ozonation (23). Tentative structures were confirmed using GC/MS. Of the 11 fractions collected from the fractionation of the pH 2 solution, only fraction 10, which contained residual pyrene, was completely inhibitory to GJIC at 75 µM as pyrene (Figure 9). Fractions 1-4, 6, and 9 partially inhibited GJIC. Complete dose response experiments for fractions 6 and 9 showed these fractions were completely inhibitory to GJIC at an increased concentration of 100 µM as pyrene (Figure 10). The complete dose response curve for pyrene, fraction
FIGURE 9. Dose response results for byproduct fractions generated under low pH conditions during ozonation. Cell cultures were exposed to a concentration of 75 µM (as pyrene) of each byproduct fraction. Cell cultures were exposed to each byproduct fraction for 30 min. A GJIC value of 1 is indicative of total communication, while a GJIC value of 0.3 is indicative of complete inhibition of communication. Each data point represents an average ( 1 SD of the distance the dye traveled in three plates. Fractions 1-3, 6, 9, and 10 were found to differ significantly from the control at p e 0.01 as determined by a two-tailed t-test, indicating partial or full inhibition of GJIC. Note: FW is the water-soluble fraction and has a compound I.D. number of 1. Fractions are abbreviated consistent with the following example: Fraction 1 is F1 and has a compound I.D. number of 2.
FIGURE 10. Dose response curves for byproduct fractions F6 and F9 generated under low pH conditions during ozonation. Cell cultures were exposed to each byproduct fraction for 30 min at varying dosages (concentration as pyrene). A GJIC value of 1 is indicative of total communication, while a GJIC value of 0.3 is indicative of complete inhibition of communication. Each data point represents an average ( 1 SD of the distance the dye traveled in three plates. Fractions 6 and 9 were found to differ significantly from the control at p e 0.01 as determined by a two-tailed t-test. Note: Fraction 6 and fraction 9 are abbreviated as F6 and F9, respectively. 10, is shown in Figure 2. Fractions 6 and 9 both contained three common compounds, C, D, and E. Fraction 10 contained residual pyrene. Pure compound C was studied VOL. 35, NO. 17, 2001 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
9
3581
FIGURE 11. Dose response results for byproduct fractions generated under high pH conditions during ozonation. Cell cultures were exposed to a concentration of 75 µM (as pyrene) of each fraction. Cell cultures were exposed to each byproduct fraction for 30 min. A GJIC value of 1 is indicative of total communication, while a GJIC value of 0.3 is indicative of complete inhibition of communication. Each data point represents an average ( 1 SD of the distance the dye traveled in three plates. The MIX, PY, and fractions 3 and 6 were found to differ significantly from the control at p e 0.01 as determined by a two-tailed t-test, indicating partial or full inhibition of GJIC. Note: MIX is the byproduct mixture prior to fractionation and has a compound I.D. number of 1. Fractions are abbreviated consistent with the following example: fraction 1 is F1 and has a compound I.D. number of 3. in detail and was not found to exhibit inhibitory characteristics. Compound E was found in fractions other than 6 and 9, and these fractions were not active. Compound D, containing both a bay region and an aldehyde group, was present only in fractions 6 and 9 and appears to be a good candidate for the inhibition noted. In general, byproducts generated at high pH were consistent with the byproducts generated at low pH. Of the six fractions collected from the pH 9.5 solution, fractions 3 and 6 completely inhibited GJIC, and fractions 1, 2, 4, 5 partially inhibited GJIC at 75 µM as pyrene (Figure 11). The mixture prior to RP-HPLC separation (crude) and pyrene are also shown for reference. Complete dose response experiments for fraction 3 and fraction 6 (containing residual pyrene) showed that fraction 3 was slightly more epigenetically toxic than the parent compound, pyrene (Figure 12). Fraction 3 contained compounds B, C, D, E, and R. From test results of low pH fractions, compounds C and E may be individually ruled out as the likely cause of the inhibition. Compound R was unlikely as well since it was present only in trace amounts. Compounds B and D appeared to be the likely cause of the inhibition, although fraction 5, which was not inhibitory to GJIC, contained a trace amount of compound D. Compound D has both a bay region and an aldehyde group. Compound B has the same basic structure with the addition of two aldehyde groups instead of one. In addition, commercial compounds, OFC, TPC, and 4BCH all contained bay regions and aldehyde functional groups and were inhibitory to GJIC where as their counterparts without aldehyde groups were not. These results are consistent with hypotheses drawn from the study conducted by Eberius et al. (29). Although individually it appears that byproduct compounds containing both a bay region and an aldehyde group caused the observed inhibition of GJIC, it is possible that 3582
9
ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 35, NO. 17, 2001
FIGURE 12. Dose response curves for byproduct fractions F3 and F6 generated under high pH conditions during ozonation. Cell cultures were exposed to each byproduct fraction for 30 min at varying dosages (concentration as pyrene). A GJIC value of 1 is indicative of total communication, while a GJIC value of 0.3 is indicative of complete inhibition of communication. Each data point represents an average ( 1 SD of the distance the dye traveled in three plates. Fractions 3 and 6 were found to differ significantly from the control at p e 0.01 as determined by a two-tailed t-test. Note: Fraction 3 and fraction 6 are abbreviated as F3 and F6, respectively. synergy between two or more compounds was the cause. It is difficult to assess how a compound will behave when present as a mixture with other compounds. In this case, the behavior of many of these compounds individually was unknown as was the exact concentration of each compound with respect to the others in the mixture. To positively identify compounds such as compounds B and D as the cause of the inhibition of GJIC, additional studies of these individual compounds would be required. Because these compounds are not commercially available and difficult to isolate in the laboratory, it will be necessary to synthesize these compounds for individual study.
Acknowledgments We would like to acknowledge the following sources of funding: NIESH-Superfund Grant 2P42ES04911-05 and the National Science Foundation. We would also like to acknowledge Dr. Long Lee, Dr. Muraleedharan Nair, Dr. Russel Ramsewak, Dr. Jehng-Jyun Yao, Dr. Zhi-Heng Huang, Dr. Beverly Chamberlain, and Alisa Rummel for their technical assistance during this project.
Literature Cited (1) Yoshikawa, T.; Ruhr, L. P.; Flory, W.; Giamalva, D.; Church, D. F.; Pryor, W. Toxicol. Appl. Pharm. 1985, 79, 218-226. (2) Sontag, J. M. In Carcinogens in Industry and the Environment; Marcel Dekker: New York, 1981; p 169. (3) Masten, S. J.; Davies, S. H. R. Environ. Sci. Technol. 1994, 28 (4), 181A-185A. (4) Upham, B. L.; Yao, J. J.; Trosko, J. E.; Masten, S. J. Environ. Sci. Technol. 1995, 29 (12), 2923-2928. (5) Mahro, B.; Schaefer, G.; Kastner, M. In Bioremediation of Chlorinated and PAH Compounds; Hinchee, R. E., Leeson, A., Semprini, L., Ong, S. K., Eds.; Lewis Publishers: Boca Raton, 1994; pp 203-217. (6) Corless, C. E.; Reynolds, G. L.; Graham, N. J. D.; Perry, R. Wat. Res. 1990, 24 (9), 1119-1123. (7) Yang, Y.; Chen, R. F.; Shiaris, M. P. J. Bacteriol. 1994, 176 (8), 2158-2164.
(8) Sasaki, J.; Arey, J.; Harger, W. Environ. Sci. Technol. 1995, 29, 1324-1335. (9) Upham, B. L.; Masten, S. J.; Lockwood, B. R.; Trosko, J. E. Fundam. Appl. Toxicol. 1994, 23, 470-475. (10) Upham, B. L.; Weis, L. M.; Rummel, A. M.; Masten, S. J.; Trosko, J. E. Fundam. Appl. Toxicol. 1996, 34, 260-264. (11) Yamasaki, H. Carcinogenesis 1990, 11 (7), 1051-1058. (12) Lehr, R. E.; Wood, A. W.; Levin, W.; Conney, A. H.; Thakker, D. R.; Yagi, H.; Jerina, D. M. Polynuclear Aromatic Hydrocarbons: Physical and Biological Chemistry, Sixth International Symposium; Battelle Press: Columbus, 1982; pp 21-37. (13) Wood, A. W.; Levin, W.; Chang, R. L.; Yagi, H.; Thakker, D. R.; Lehr, R. E.; Jerina, D. M.; J. M.; Conney, A. H. Polynuclear Aromatic Hydrocarbons - Third Symposium on Chemistry, Biology, Carcinogenesis, and Mutagenesis; Ann Arbor Science Publishers: Ann Arbor, 1979; pp 531-551. (14) Flesher, J. W.; Kadry, A. M.; Chien, M.; Stansburg, K. H.; Gairola, C.; Sydnor, K. L. Polynuclear Aromatic Hydrocarbons: Formation, Metabolism and Measurement; Seventh International Symposium; Battelle Press: Columbus, 1983; pp 505-515. (15) Friesel, H.; Schope, K. B.; Hecker, E. Polynuclear Aromatic Hydrocarbons: Formation, Metabolism and Measurement; Seventh International Symposium; Battelle Press: Columbus, 1983; pp 515-530. (16) Hoffmann, D.; Lavoie, E. J.; Hecht, S. S. Polynuclear Aromatic Hydrocarbons: Physical and Biological Chemistry; Sixth International Symposium; Battelle Press: Columbus, 1982; pp 1-19. (17) Melikian, A. A.; Lavoie, E. J.; Hecht, S. S.; Hoffmann, D. Polynuclear Aromatic Hydrocarbons: Formation, Metabolism, and Measurement; Seventh International Symposium; Battelle Press: Columbus, 1982; pp 861-875.
(18) Silverman, B. D.; Lowe, J. P. Polynuclear Aromatic Hydrocarbons: Physical and Biological Chemistry; Battelle Press: Columbus; pp 743-753. (19) Rosenkranz, H. S.; Pollack, N.; Cunningham, A. R. Carcinogenesis 2000, 21, 1007-1011. (20) El-Fouly, M. H.; Trosko, J. E.; Chang, C. C. Exp. Cell Res. 1987, 168, 422-430. (21) Borenfreund, E.; Puerner, J. A. Toxicology Lett. 1985, 24, 119124. (22) Bailey, P. S. Ozonation in Organic Chemistry, Volume II. Nonolefinic Compounds; Academic Press: New York, 1982. (23) Adams, V. D. In Water and Wastewater Manual; Lewis Publishers: Chelsea, 1991. (24) Yao, J. J. Ph.D. Dissertation, Michigan State University, 1997. (25) Herner, H. A. Ph.D. Dissertation, Michigan State University, 1999. (26) Trosko, J. E., personal communication; Michigan State University, February, 1999. (27) Oh, S. Y.; Madhukar, B. V.; Trosko, J. E. Carcinogenesis 1988, 9 (1), 135-139. (28) Trosko, J. E.; Madhukar, B. V.; Chang, C. C. Life Sci. 1993, 53, 1-19. (29) Eberius, M.; Berns, A.; Schuphan, I. Fresenius J. Anal. Chem. 1997, 359, 2274-279.
Received for review February 5, 2001. Revised manuscript received June 15, 2001. Accepted June 19, 2001. ES0106117
VOL. 35, NO. 17, 2001 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
9
3583
