Environ. Sci. Technol. 1984, 18,362-364
Identification of Benz[g ]isoquinoline-5,l O-dione as an Insect Teratogen in Commercial Acridine Cheng Yu Ma,+Chuen-hue1 Ho,+ Barbara T. Walton,' Glenn L. Kao,+ and Mlchael R. Guerin"+ Analytical Chemistry Division and Environmental Sciences Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee 37830
rn Benz[g]isoquinoline-5,10-dione is shown to be a primary contributor to the insect teratogenicity associated with commercial reagent-grade acridine. Confirmation is based upon spectroscopic, chromatographic, and bioassay results. The contribution of this chemical to coal oil insect teratogenicity is questioned. Introduction Cricket (Acheta domesticus) eggs incubated in sand contaminated with commercial reagenbgrade acridine have been previously reported (1)to yield nymphs with extra compound eyes, extra antennae, branched antennae, and extra heads. Similar effects have been observed for sand contaminated with coal-derived oil samples (2) and for eggs topically dosed (3)with test chemicals. The cricket system promises to be an effective bioassay for assessing an impact of terrestrial contamination on the insect population. The relationship of this bioassay to other measures of ecological impact or to human health impact is not yet known. It is necessary to identify the agents responsible for the observed teratogenicity in order to interpret the observations and to provide the active ingredient for testing in other bioassay systems. Work reported here identifies benz[g]isoquinoline-5,10-dione as a primary contributor to the cricket teratogenicity of impure commercial reagent-grade acridine. The relationship of this finding to the teratogenicity observed for coal oil is discussed. Materials and Methods Approximately 10-g quantities of reagent-grade acridine (Aldrich Chemical Co., lot no. 082987TL) were loaded on a column (3.8 cm 0.d. X 90 cm long) slurry packed with 750 g of basic alumina (100/200 mesh, AG-10, Bio-Rad Laboratories, Richmond, CA, used as received) in benzene. The column was eluted with 6 L of benzene to remove acridine and then with 3 L of 30% (V/V) 2-propanol/ methylene chloride to obtain the impurities. A total of approximately 100 g of acridine was processed in this manner. New columns were prepared for each batch separated. The combined isolate was subjected to normal- and reversed-phase high-performance preparative scale chromatography. The separations were performed on a Beckman Model 330 isocratic system equipped with a preparative pump head. In the normal-phase separation, a preparative scale (21.2 mm i.d. X 25 cm length) Zorbax CN column (Du Pont, Wilmington, DE) was first eluted at 8.4 mL/min with 50% (v/v) methylene chloride/hexane for 40 min and then with 100% methylene chloride for approximately 30 min. In the reversed-phase separation, a preparative scale (21.2 mm i.d. X 25 cm length) Zorbax ODS column (Du Pont, Wilmington, DE) was eluted at 7 mL/min with 80% (v/v) methanol/water. Approximately 100 mg of sample was subjected to chromatography for each separation. The effluent of the column was monitored with a UV detector at 254 nm. t Analytical t
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Gas chromatography was carried out on a Perkin-Elmer Model 3920 chromatograph equipped with a hydrogen flame ionization detector and a glass column (3.2 mm 0.d. X 6.1 m in length) packed with 3% Dexsil400 on 100/200 mesh Chromosorb 750. Column temperature was held at 100 "C for 16 min and then programmed to 320 "C a t 4 "C/min. Inlet and detector temperatures were maintained at 285 and 340 "C, respectively. Gas chromatographic/ mass spectrometric analyses were performed on a Hewlett-Packard 5985B GCMS. A fused silica capillary column (25 m; J & W Scientific, Inc.) coated with SE-52 was used for gas chromatography. The column temperature was maintained at 80 "C for 5 min and programmed to 280 "C at 3 "C/min. High-resolution mass spectra were generated on a Kratos MS50/DS50 mass spectrometer/data system at a resolution of approximately 10 000. Infrared spectra were generated on a Digilab FTS-2O/C Fourier transform infrared spectrometer equipped with a diffuse reflectance attachment (JASCO Model DRA-100) and operated at a resolution of 4 cm-l. Benz[g]isoquinoline-5,10-dione was synthesized by using the method of Phillips ( 4 ) . Briefly, reaction of 3,4pyridinedicarboxylic acid with thionyl chloride yielded its acid anhydride, which was then converted to 3-benzoylisonicotinic acid by Friedal-Craft reaction in the presence of aluminum chloride and benzene. The final product was obtained by dehydration of 3-benzoylisonicotinicacid with concentrated sulfuric acid. The dione was purified by preparative reversed-phase HPLC as described above. Its identity was confirmed by high-resolution mass spectrometry, infrared spectrometry, and nuclear magnetic resonance spectrometry. Teratogenicity and embryotoxicity were tested by using the method of topical application as previously described ( 3 , 5 ) . In summary, gravid female A. domesticus crickets oviposit in moist sand, and eggs are harvested by flotation in 0.5 M sucrose solution. Individual eggs are treated 16-24 h postoviposition with the test agent in 0.05 pL of dimethyl sulfoxide using a Hamilton syringe operated by a power-driven micrometer. Twenty similarly treated eggs are transferred to moistened filter paper in a Petri dish, and the covered dish is incubated at 30 "C. Typically, each agent is tested at three dose levels, and each dose level involved triplicate plating (60 eggs, 20 eggs per plate). Eggs are scored for multiple eyespots beginning approximately day 7 , and nymphal emergence is recorded beginning on day 10. Historical controls show (5) no multiple eyespots for over 50 untreated and/or dimethyl sulfoxide treated eggs. Untreated and dimethyl sulfoxide treated controls exhibited (5) 8 2 4 7 % survival as compared to 21-76% survival depending on the dose applied for eggs treated with the compound reported here. Results and Discussion Total impurities in the commercially purchased acridine represented less than 2% of the original material. To isolate enough of the teratogenic component for spectroscopic identification, approximately 100 g of commercial acridine was fractionated on basic alumina columns to generate 1.9 g of the impurity fraction. Teratogenicity
0013-936X/84/0918-0362$01.50/0
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Figure 2. (a) High-resolution mass spectra of the isolated and synthesized dione. (b) Fourier transform Infrared spectra of the isolated and synthesized dione.
evaluation of this fraction by topical application to cricket eggs confirmed earlier observations ( I ) of teratogenicity following egg development in a contaminated sand substrate. Morphological abnormalities (5)of embryos were observed (6) a t a dose of 114 ngfegg of the impurity fraction. No abnormalities were observed in the vehicle control groups. Gas chromatographic analysis (Figure 1A) and preparative liquid chromatographic separation (Figure 1A') of the impurity isolate revealed the presence of multiple impurities. Analysis of the isolate by gas chromatography/mass spectrometry (GCIMS) suggested that the major components of the chromatograms were simple structural isomers of acridine with molecular weights of 179. Four of the eight structural isomers were obtained commercially and assayed for teratogenicity, but none of these proved to be teratogenic (3). The remaining four isomers have now been synthesized and tested for teratogenic activity; none are teratogenic in the cricket embryos (3). Minor components detected in the impurity fraction exhibited mlz values of 180, 193, and 209 by GC/MS analysis. One acridine-related compound which could show molecular ion at mlz 209 is the dicarbonyl derivative. Such compounds might be present in the impurity fraction especially after exposure to air and sunlight, or to other oxidizing conditions (7). As a consequence of this observation, benz[g]isoquinoline-5,lO-dione(I) was synthesized as a candidate for study.
Synthesized dione I was found to be teratogenic to Acheta domesticus (L.) eggs when administered 24-h postoviposition a t doses of 20-0.1 ngfegg (5, 6). Morphological abnormalities of embryos consisted of one or two extra compound eyes and were similar to those observed ( I , 2) after treating eggs with the impurity fraction isolated from commercial acridine. We proceeded to determine whether dione I is present in the acridine impurity fraction. Results of GC/MS analysis along with a comparison of GC retention times suggested that dione I was present in the impurity fraction but at a very low level (Figure 1A). Isolation of trace dione started with repeated fractionation of the impurity fraction on a preparative scale Zorbax CN column (Figure lA', B'). Gradual enrichment of dione in the subfractions collected between two major peaks was evident in their GC profiles (Figure lB, C). Further separation (Figure IC') using a preparative scale Zorbax ODS column eluted with 80120 methanol/water yielded an isolate consisting of approximately 75% dione (Figure 1D). A total of 1.8 mg of the suspected dione (Figure 1E) was finally purified by using the same HPLC system (Figure 1D'). This quantity corresponds to an initial concentration of 20 ppm in the original commercial acridine, assuming quantitative recovery during the isolation steps. Identification is based on excellent agreement of chromatographic retention time and high-resolution mass spectra (HRMS) of the isolated dione and synthesized benz[g]isoquinoline-5,lO-dione.The exact masses of the molecular ion as well as fragment ions (Figure 2a) determined by HRMS agreed with those of the theoretical values within a range of 0.2 millimass unit. Final confirmation on positional isomeric structure was established on the basis of an excellent match of FTIR spectra of the isolated and synthesized dione (Figure 2b). A distinctive spectral feature in the fingerprint region and the unique
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Flgure 3. Fourier transform infrared spectra of related diones.
aromatic carbonyl absorption band (1678 cm-l) of benz[g]isoquinoline-5,10-dione corroborate the assignment. As is illustrated in Figure 3, FTIR spectra of related diones are readily distinguished from the suspect isomer. Comparable embryotoxic activity and teratogenic activity were observed when both the isolate and authentic benz[g]isoquinoline-5,10-dionewere tested simultaneously. The LD,, of dione I was calculated (5) from experimental data to be 13.2 ng/egg (95% confidence limits of 12.6 and 14.2 ng/egg), and the LD,, of the isolate was calculated to be 12.9 (95% confidence limits of 12.0 and 13.8 ng/egg). Five percent of the embryos developed extra eyes after treatment with 4 ng of dione I/egg, whereas 6.7% abnormals occurred at the same dose of the acridine isolate. No abnormalities were detected in the control groups. Finally, thin-layer chromatographic analysis of the synthetic dione (I) on silica gel G eluted with a mixture of chloroform, methanol, and acetic acid ( 9 0 5 5 v/v) ( I ) yielded a nonfluorescence spot at Rf 0.74. This agrees with the earlier report ( I ) that only one (Rf0.74) of 10 spots isolated from the impurity fraction exhibited teratogenic activity. While dione I is not fluorescent, it coelutes with benzo[flquinoline under these conditions, and benzo[flquinoline does fluoresce. We conclude that benzo[g]isoquinoline-5,10-dioneis a primary contributor to the cricket teratogenicity exhibited by commercial reagent grade acridine. While it is not currently possible to rule out contributions to teratogenicity by other acridine diones, the similarity between the activity of dione I and that of the impurity mixture as a whole suggests that dione I is the primary causative agent. Interest in identifying the acridine impurity responsible for cricket teratogenicity stems in part from similar biological observations on coal-derived petroleum substitutes. Coal oils have been reported (2) to exhibit embryotoxicity in the cricket system, and the alkaline fraction of a coal oil has been reported ( I , 2) to exhibit teratogenicity similar
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to that exhibited by commercial acridine. Acridine and its unsubstituted benzquinoline isomers would be expected to concentrate in the alkaline fraction of coal oils if they are present. The relationship between these observations is futher heightened by the fact ( I , 8) that commercial reagent-grade acridine is sometimes produced by the vacuum distillation of coal tar. The implication of these concerns is that the transport and handling of coal-derived petroleum substitutes are accompanied by a unique biological hazard. Two qualitative studies have been carried out here to assess whether dione I is likely to be responsible for the reported cricket teratogenicity of coal oils. First, the distribution of dione I between the chemical class fractions produced for an earlier (2) teratogenicity evaluation has been determined. Second, dione I has been used to develop a separations method that isolates the dione from coal oils for quantitative analysis. It has been found that dione I distributes preferentially (2:l) into the neutral fraction rather than the alkaline fraction reported (2) to exhibit teratogenicity. Quantitative analyses of the fraction expected to concentrate the dione show the dione to be undetectable (
