Chem. Res. Toxicol. 1989, 2, 162-168
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Decomposition and Quality Control Considerations in Biological Work with Fecapentaene Preparations Anthony J. Streeter,? Paul J. Donovan,? Takako Anjo,? Lena Ohannesian,* Pamela R. Sheffels,? Priscilla P. Wu,? Larry K. Keefer,*tt A. W. Andrews,t Wallace W. Bradford, 111,s Elmer J. Rei&,$ and Jerry M. Rice? Laboratory of Comparative Carcinogenesis, National Cancer Institute, Frederick Cancer Research Facility, Frederick, Maryland 21 701, Program Resources, Inc., National Cancer Institute, Frederick Cancer Research Facility, Frederick, Maryland 21 701, and S R I International, Menlo Park, California 94205 Received January 27, 1989
Solutions of synthetic fecapentaene 12 (FP-12) intended for carcinogenicity studies were found t o decompose extremely rapidly during customary dosage procedures. Apparent half-lives as short as 15 min were observed. While rates and even the qualitative course of decomposition were surprisingly variable in replicate experiments, high concentration and exposure to air were confirmed t o be especially important destabilizing influences. The results suggested a primary role for a radical decomposition mechanism in the presence of atmospheric oxygen. Consistent with this hypothesis, FP-12 solutions were significantly stabilized by the radical chain-breaking antioxidant vitamin E. On the other hand, dithiothreitol greatly destabilized FP-12, presumably because of its nucleophilicity. The diacetyl diester of FP-12 was more soluble than the parent diol, but its decomposition rates in the presence and absence of vitamin E were similar to those of unesterified FP-12. Ultraviolet irradiation of a n all-trans-FP-12 solution decreased its concentration by 70% in 0.5 min. T h e mutagenicities of the decomposition/isomerization products of FP-12, as studied in Salmonella t y p h i m u r i u m tester strain TA 100, ranged from negligible to comparable with all-trans-FP-12 itself. It is concluded that unchecked decomposition of fecapentaene preparations can profoundly affect biological tests therewith. While this can be largely controlled through the use of rigorous precautions, including protection from air, light, nucleophiles, and acids as well as selection of the lowest concentration compatible with the application a t hand, the data argue strongly for inclusion of appropriate quality control measures in all future dosing operations to prove that the biological activity reported is that of the fecapentaene itself rather than that of a decomposed dosing solution.
Introduction The fecapentaenes are a family of powerful mutagens produced by bacterial species found in human feces. These compounds are currently under intense scrutiny as possible causative agents in the development of colon carcinoma, a leading cause of cancer death in the United States (1-46). We have been engaged in characterizing their chronic genotoxic effects in mammals by repetitively administering large doses of synthetic fecapentaene 1 2 ( F P - 1 2 ) l to rodents for extended periods of time (16). In planning these experiments we noted previous reports of fecapentaene instability (1-15) and, therefore, needed to know the following: (a) to what extent the composition of an FP-12 sample actually changes during a typical bioassay application; (b) the extent to which any changes observed make a difference in the biological properties of the sample (rather than simply transforming it into a mixture of isomers or other derivatives of equivalent activity); and (c) what steps should be taken to ensure the chemical and toxicological integrity of the test substance throughout the dosing procedure. We have now measured the rates of F P - 1 2 decomposition under a variety of relevant conditions and have col-
lected mutagenicity data on the products in an effort to answer these questions. We believe the results, reported below, hold important implications for anyone seeking to design or interpret biological experiments with fecapentaenes.
Materials and Methods Caution! The materials used in these studies are mutagenic. Due precautions should be used during storage, manipulation, and disposal. Chemicals. FP-12 was synthesized by the method of Nicolaou et al. (20). DAFP-12 was prepared by acetylating a portion of the synthetic FP-12 according to the method described by Gupta et al. (4).Vitamin E (a-tocopherol, mixed isomers from vegetable oil) and trioctanoin were obtained from Sigma (St. Louis, MO), DMSO was supplied by Burdick and Jackson (Muskegon, MI), and ethanol (200 proof) was from US1 Chemicals Co. (Newark, NJ). Propylene glycol and dithiothreitol were from Aldrich (Milwaukee, WI). HPLC. Analytical HPLC was performed by using Waters equipment (Milford, MA) including a Model M-6000A pump operating at a flow rate of 1 mL/min, a Model U6K injector, and a Model 440 absorbance detector set at 340 nm. Where full spectra were required for each peak, a Hewlett-Packard Model 1040A flow-through diode array spectrophotometer was employed. The
t National Cancer Institute.
* Program Resources, Inc.
Abbreviations: FP-12, fecapentaene 12; DAFP-12, diacetylfecapentaene 12; DMSO, dimethyl sulfoxide.
* SRI International.
0893-228x/89/2702-0162$01.50/0
0 1989 American Chemical Society
Chem. Res. Toxicol., Vol. 2, No. 3, 1989 163
Use of Fecapentaenes i n Biological Studies system was completed by a Spherisorb S5 ODS2 column (25 cm x 4.6 mm, 5 pm, Anspec Co., Ann Arbor, MI), which was eluted with methanol/water (75/25) for FP-12 and methanol/water (85/15) for DAFP-12. For the photolysis experiment, preparative HPLC was carried out by using a Partisil M9 10/25 ODS-2 column (7.9 mm X 25 cm, Whatman) eluted with methanol/water (75/25) at a flow rate of 1.0 mL/min. An injection of 0.8 mg of FP-12 in DMSO (50 pL) was made, and 2.0-mL fractions were collected for 30 min. Photoisomerization was performed, on fractions containing the later eluting isomers of FP-12, by irradiating the samples in a clear glass vial with a hand-held Model UVGL-25 Multiband UV254/366 Mineralight lamp (UVP, Inc., San Gabriel, CA). Aliquots of 10 pL were injected into the analytical HPLC system before and after irradiation. Mutagenicity Investigations. T o study the mutagenicities of the various chromatographic fractions, 90 pg of FP-12 in DMSO (6 pL) was injected into the analytical HPLC system and 1.0-mL fractions were collected for 22 min. From each fraction 200 pL was added to 1.8mL of methanol for measurement of a complete UV spectrum using a Hewlett-Packard Model 8451A diode array spectrophotometer. An additional volume of 50 pL of each fraction (or a 50-pL aliquot of a 1:4,1:16,1:64, or 1:256 dilution in DMSO) was added to 2.0 mL of top agar and 200 WLof Salmonella typhimurium (TA 100)suspension to carry out the mutagenicity assay of Ames et al. (47) as modified by Andrews et al. (48). Sodium azide was used as the positive control. Backgrounds were subtracted for spontaneous revertants, and the sum of all the fractions was calculated for comparison with the original solution. Metabolic activation (S9) was not necessary as the fecapentaenes are direct-acting mutagens. Kinetic Studies. Rates of FP-12 decomposition were measured by injecting aliquots of the solution being studied into the analytical HPLC system described above. The amount remaining undecomposed was inferred from the height of the HPLC peak a t longest retention time (see Figure 1).The influence of oxygen was determined by comparing decomposition rates in nitrogencontaining septum-stoppered versus unstoppered vials. Solubility Determinations. The solvents studied were initially deoxygenated, either by distillation under argon atmosphere or by bubbling argon gas through the solvent for 20 min. After deoxygenation was complete, sufficient material was added so that a portion remained undissolved after stirring under argon for 10 min. An ultraviolet spectrum of the “saturated” solution was obtained, and an extinction coefficient of 106000 L/(mol.cm) was used to calculate the concentration in solution. Storage and Use Procedures. Bulk samples were stable when stored at -70 “C or lower in the absence of light and in an inert atmosphere. In our experience, bulk storage is best accomplished by storing a deaerated solution of FP-12 in ethanol or DMSO in a vial, flushed with argon and sealed with a Teflon-lined cap, which is in turn placed in a secondary container also flushed with argon, capped, and stored over liquid nitrogen. If a secondary container is not used, stability is variable. As far as possible, all operations with FP-12 including dilution, dosing, and storage were conducted a t the lowest practical temperature in amber vessels (or a t least in subdued or yellow light) with the rigorous exclusion of air. If a stock solution had to be subdivided into smaller portions required over a period of time, the subdivision was performed all a t once the first time the bulk sample was thawed, with the resulting smaller vials being returned to the freezer in flushed amber septum vials until they were individually needed; repeated freeze-thaw cycling of a sample can markedly increase decomposition. Glove bags (e.g., the two-hand Atmosbag, Aldrich Chemical Co. Inc., Milwaukee, WI) filled with nitrogen or argon have proven to be convenient vehicles for subdivision and other manipulation of FP-12 solutions. Solvents were always deoxygenated before mixing with FP-12, e.g., by bubbling argon through them. Syringes, volumetric flasks, and other vessels were flushed with inert gas before contact with FP-12.
Results Synthesis. M a t e r i a l synthesized for the biological s t u d i e s proved to be a m i x t u r e of m a n y discrete compounds. A typical H P L C trace of a freshly prepared FP-12
5
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Wavelength (nm) Figure 1. UV spectra of HPLC peaks observed for freshly synthesized FP-12 solution (60 mM in DMSO). The solution was diluted with DMSO such that 3 pg in 10 pL was injected into the analytical HPLC system described under Materials and Methods with diode array detection. Methanol/water (70/30) was the mobile phase. solution is shown at the t o p of Figure 1. The peaks with the longest retention times (D-H) had similar spectra indicative of the conjugated pentaenol ether chromophore. The faster moving p e a k s (A-C) displayed different UV spectra, lacking fine structure and i n o n e case showing a new m a x i m u m at s h o r t e r wavelengths; these s p e c t r a are also shown i n Figure 1 a n d indicate the presence of decomposition products. Decomposition of FP-12. The newly synthesized material described above suffered rapid chemical changes in t h e course of n o r m a l handling procedures considered “good laboratory practice” for other test substances. Some typical results a r e shown in Figures 2 and 3, i n which the height of the principal H P L C p e a k (Figure 1, p e a k H) is plotted semilogarithmically as a function of time. It was very difficult t o reproduce these rate plots, consecutive experiments u n d e r w h a t were t h o u g h t t o b e identical conditions o f t e n giving substantially different kinetics. Nevertheless, exclusion of air could be shown t o improve dramatically t h e stability of the F P - 1 2 solutions, as t h e data of Figure 2 illustrate. Additionally, despite the imprecision i n the d a t a , a clear concentration d e p e n d e n c e
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Figure 2. Effect of oxygen on the decomposition at room temperature of a 6.0 mM solution of FP-12 in ethanol/DMSO (9/1): in a sealed amber vial under a nitrogen atmosphere ( 0 ) ;or under
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could be established; as suggested in Figure 3, the more dilute solutions proved to be more stable. Decomposition also occurred when FP-12 solutions were photolyzed; the height of the all-trans-FP-12 peak in a methanollwater (75/25) solution that had been freshly isolated by preparative HPLC decreased by 70% during irradiation for 30 s in a clear glass vial with an ultraviolet lamp. Finally, decomposition could be extremely rapid. In the more concentrated DMSO solution studied in the experiment depicted in Figure 2, an apparent half-life of 15 min was observed on standing in air. The HPLC trace of an FP-12 solution after standing in air for a prolonged period was shown in a previous publication (16). Compared with the chromatogram of Figure 1for freshly prepared material, the following changes were noted. Two of the peaks (A and B) increased in height by a factor of severalfold. One of the constituents of the
mixture (peak C) appeared not to change in concentration. Most importantly, all constituents having the fecapentaene ultraviolet spectrum (peaks D-H of Figure 1)disappeared from the solution. Mutagenicity of Decomposition Products. The decomposition of FP-12 in solutions of differing concentrations as noted in Figure 3 was accompanied by similar rates of disappearance of the mutagenicity of the solutions toward S. typhimurium strain TA 100. Figure 4 illustrates that the mutagenicity decreased very rapidly in a 65 mM sample exposed to air but remained essentially unchanged during 3 h when diluted 3000-fold. In a separate experiment, preparative HPLC was used to isolate the various constituents of a solution of FP-12 in DMSO (59 mM) which had been stored over liquid nitrogen for a period of several months. As can be seen from Figure 5, the solution contained a mixture of several components, a number of which proved to be mutagenic. The fractions that contained mutagenic activity were also found to possess UV characteristics indicative of the conjugated pentaenol ether chromophore, suggesting that the compounds are cis-trans isomers of the all-tram-FP-12 initially present in the solution; several of these isomers have recently been tentatively identified by Kingston et al. (40). Standardization of the mutagenic activity to the UV absorbance at 334 nm revealed that the major components all had similar mutagenic potencies. This observation is in agreement with recent reports that FP-12 isomers with at least one cis double bond are similar in mutagenic activity to all-trans-FP-12 (35,40). No mutagenic activity was associated with the early eluting compounds, which did not possess the FP-12 spectrum and represent the ultimate decomposition products. In all, 65% of the mutagenicity of the FP-12 solution initially
Chem. Res. Toxicol., Vol. 2, No. 3, 1989 165
Use of Fecapentaenes in Biological Studies
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injected was found in the recovered HPLC fractions. Effect of Vitamin E. The increasing instability of FP-12 with increasing concentration and exposure to air suggested that decomposition of FP-12 in the presence of atmospheric oxygen occurred by a radical process. If correct, this concept suggests that a radical chain-breaking antioxidant capable of reacting with the intermediate odd-electron species might inhibit decomposition (49). T o test this hypothesis, a 2 mM solution of FP-12 in ethanol was divided into portions and a-tocopherol (vitamin E) was added to each in concentrations varying from 0 to 2 mM. A clear stabilizing effect of this antioxidant was seen, as shown in Figure 6. Effect of Dithiothreitol. A sample of FP-12 was purified by HPLC using 75% aqueous methanol. The eluate, estimated to be 99% pure all-trans-FP-12, showed no change in extinction coefficient or HPLC behavior when stored under argon for 24 h. To an identical fraction, sufficient dithiothreitol was added to make the solution 1% by weight in this compound, which also has been used as a protective agent for materials that are sensitive to oxidative decomposition (50). The resulting mixture was stored under argon for 24 h. HPLC indicated complete decomposition. This deleterious effect may have resulted from the nucleophilicity of the sulfhydryl groups of dithiothreitol, as FP-12 is known to be sensitive to destruction by thiols (10, 13). Solubilization by Esterification. The most concentrated solutions of synthetic FP-12 we have been able to prepare contained only 15 mg/mL in DMSO (our solvent of choice because of its excellent biocompatibility as well as solubilizing power for FP-12). Even acetone was capable of dissolving only 6 mg/mL at saturation. Since much higher concentrations were desired for the biological tests, derivatization to more lipophilic species was investigated. Acetylation by the procedure of Gupta et al. (4) gave a 60% yield of DAFP-12, an agent we were interested to study biologically because of its presumably greater solubility
Figure 6. Effect of vitamin E concentration on the decomposition rate of a solution of 2.3 mM FP-12 in ethanol/DMSO (23/2) exposed to air. The concentrationsof vitamin E were 0 mM (a), 0.02 mM (O),0.2 mM (A),and 1.9 mM (0). Table 1. Solubilities of FP-12 and DAFP-12 (in mg/mL) solute FP-12
DAFP-12
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>46
solvent trioctanoin 9 35
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and, not determined.
as well as ability to penetrate cell membranes and assumed susceptibility to esterase hydrolysis. Its solubility data are compared in Table I with those of FP-12. The diacetate proved to be severalfold more soluble than FP-12 in the solvents of interest. HPLC analysis of freshly prepared samples of DAFP-12 using the analytical system described under Materials and Methods revealed an essentially similar pattern to that of FP-12. The bulk of the material that absorbed at 340 nm was found in the final peak to emerge from the system. Again this peak was assumed to be the all-trans isomer of DAFP-12, and its height was used to monitor decomposition. Effect of Vitamin E on DAFP-12. A similar experiment was performed to that with FP-12 which showed that the decomposition rate of the diacetate approximated that of the diol, as did the stabilizing effect of vitamin E on that decomposition (Figure 7 ) . The small decreases in concentration occasionally observed upon freezing and thawing a 62.5 mM solution of DAFP-12 in DMSO were similar in magnitude and frequency to those seen with FP-12 under these conditions, and Vitamin E at a concentration of 4.9 mM had little effect on either process. As with FP-12, the disappearance of DAFP-12 from solution was accompanied by a parallel loss of mutagenicity, both in the presence and in the absence of vitamin E; the vitamin appeared to have little effect on the mutagenicity of FP-12 (Figure 8). As a preliminary to carcinogenicity studies with DAFP-12, we have determined that the maximum stabilization of a 30 mM DAFP-12 solution in DMSO could be achieved by using a 3.2 mM vitamin E concentration. No greater stability could be conferred by further increases in the amount of vitamin E, although DAFP-12 still decomposed (with an apparent half-life of 3 h) when exposed to air. DAFP-18 coformulated with Vitamin E is currently
166 Chem. Res. Tonicol., Vol. 2, No. 3, 1989
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Figure 7. Effect of vitamin E concentration on the decomposition rate of a solution of 2.9 mM DAFP-12 in ethanol/DMSO (25/1) exposed to air. The concentrations of vitamin E were 0 mM (a), 0.02 mM (O),0.2 mM (A),and 2.1 mM (0).
being administered to rodents in this laboratory to determine whether the stabilizer might unmask any latent carcinogenic activity of the fecapentaene, which we assume might be rapidly formed on esterase hydrolysis of DAFP12 in vivo. If the lack of activity in the prior tests (16) resulted from rapid decomposition of the FP-12 after dosage but before reaction with target molecules in the cell critical to initiation could occur, the vitamin might be expected to produce an observable potentiating effect. As a reviewer has pointed out, on the other hand, a chainbreaking antioxidant could further inhibit expression of carcinogenicity if the mechanism of FP-12 action is mediated by free radicals. The results of the long-term studies now in progress will be reported in due course.
Discussion The present study was designed with three purposes in mind: to estimate the extent of decomposition of fecapentaene solutions during typical biological experiments; to assess the effects of such decomposition on the toxicological properties of the solution; and to determine how to use the results in the design, conduct, and interpretation of carcinogenicity and other biological experiments with fecapentaene preparations. We found FP-12 to be more unstable and difficult to handle than any other substance we have as yet attempted to test for carcinogenic activity. One dosing solution used in a previous study was later found to contain no detectable FP-12, and several others were at least half decomposed at the time of dosing (16). Figure 2 shows that half the FP-12 in a sample initially 1.5 mg/mL can decompose in only 15 min on exposure to air at room temperature. When we divided a solution of FP-12 into four separate
Figure 8. Disappearance of major HPLC peak (solid lines) and mutagenicity (dashed lines) over time from a 63 mM DAFP-12 solution in DMSO exposed to air in the presence (0 and A) and absence (A and 4) of 4.3 mM vitamin E.
aliquots and stored them under seemingly identical conditions (data not shown), the solute was found to decompose at different rates in the four vials and even yielded different arrays of decomposition products. The results reinforce recent reports of rapid fecapentaene decomposition under conditions modeling those of a biological test in which up to 80% of the FP-12 in solution decomposed in 20 min (38, 43). Decomposition of FP-12 changes its genotoxic potential. Figure 5 shows that apparent isomerization yields products whose mutagenic potencies differ little from that of the all-trans starting material but that decomposition (as opposed to isomerization) products are devoid of mutagenicity. The variable but often remarkably rapid deterioration of FP-12 demands that special precautions and quality control procedures be used in conjunction with any dosage regimen to ensure that what is being tested is actually the fecapentaene and not its decomposition products (1-4, 8-12,15,38,39,43). The precautions include the following: storage in amber vessels; avoidance of strong light, nucleophiles, and acidic conditions; working with the minimum concentration compatible with the desired biological application; and maximum protection from air. Continuous low-temperature storage is also recommended, as measurable changes in the chromatogram of FP-12 were sometimes noted after a single freeze-thaw cycle. The use of protective agents such as vitamin E (49) can also be recommended wherever compatible with the application at hand, since it does significantly retard decomposition upon exposure to air. Butylated hydroxytoluene has also been recommended for this purpose (1, 4, 38). As to quality control procedures, we have found it best to check the composition of the dosing solution by UV and/or HPLC both before and after application and would recommend that others do so too, reporting the chemical data as a critical adjunct of the biological results. Such
Use of Fecapentaenes in Biological S t u d i e s
measurements would both permit verification of the sample’s suitability for test before it is applied and allow determination of the extent of decomposition (if any) incurred during the dosing procedure. Our experience suggests that such measurements be made routine during toxicological studies with fecapentaenes. We thus strongly support the recent quality control recommendations made by Pfaendler et al. concerning biological work with these “extremely sensitive” compounds (39). The crucial importance of confirming the purity and concentration of fecapentaene preparations during each application to a biological system cannot be overstated.
Acknowledgment. Research was funded in part through Department of Health and Human Services contracts N01-CO-74102 to Program Resources, Inc., and N01-CP-71108 to SRI International. We thank Corinthia Brown and Paul Johnson for their expert technical assistance in carrying out the mutagenesis assays. Registry No. FP-12, 91423-46-0; FP-12 diacetyl diester, 120789-74-4; DMSO, 67-68-5; 02,7782-44-7; trioctanoin, 538-23-8; propylene glycol, 57-55-6; a-tocopherol, 59-02-9.
References (1) Wilkins, T. D., Lederman, M., Van Tassell, R. L., Kingston, D. G. I., and Henion, J. (1980) Characterization of a mutagenic bacterial product in human feces. Am. J. Clin. Nutr. 33, 2513-2520. (2) Hirai, N., Kingston, D. G. I., Van Tassell, R. L., and Wilkins, T. D. (1982) Structure elucidation of a potent mutagen from human feces. J. Am. Chem. SOC.104, 6149-6150. (3) Bruce, W. R., Baptista, J., Che, T., Furrer, R., Gingerich, J. S., Gupta, I., Krepinsky, J. J., Grey, A. A., and Yates, P. (1982) General structure of “fecapentaenes”-the mutagenic substances in human faeces. A preliminary report. Naturwissenschaften 69, 557-558. (4) Gupta, I., Baptista, J., Bruce, W. R., Che, C. T., Furrer, R., Gingerich, J. S., Grey, A. A., Marai, L., Yates, P., and Krepinsky, J. J. (1983) Structures of fecapentaenes, the mutagens of bacterial origin isolated from human feces. Biochemistry 22, 241-245. (5) Wilkins, T. D., and Van Tassell, R. L. (1983) Production of intestinal mutagens. In Human Intestinal Microflora in Health and Disease (Hentges, D. J., Ed.) pp 265-288, Academic Press, New York. (6) de Wit, P. P., van Schaik, T. A. M., and van der Gen, A. (1984) A convenient synthesis of fecapentaene-12 by the Horner-Wittig reaction. Recl.: J . R. Neth. Chem. SOC. 103, 369-370. (7) Gupta, I., Suzuki, K., Bruce, W. R., Krepinsky, J. J., and Yates, P. (1984) A model study of fecapentaenes: Mutagens of bacterial origin with alkylating properties. Science (Washington,D.C.) 225, 521-523. (8) Baptista, J., Gupta, I., and Krepinsky, J. J. (1985) Modification of a commercial preparative HPLC system allowing for UV detection of small amounts of materials with high extinction coefficients. Chromatographia 20, 117-119. (9) Hirai, N., Kingston, D. G. I., Van Tassell, R. L., and Wilkins, T. D. (1985) Isolation and structure elucidation of fecapentaenes-12, potent mutagens from human feces. J. N a t . Prod. 48, 622-630. (10) de Wit, P. P., van der Steeg, M., and van der Gen, A. (1986) Remarkable electrophilic properties of the pentaenol ether system of fecapentaene-12. Tetrahedron Lett. 27,6263-6266. (11) Van Tassell, R. L., Schram, R. M., and Wilkins, T. D. (1986) Microbial biosynthesis of fecapentaenes. In Genetic Toxicology of the Diet (Knudsen, I . , Ed.) pp 199-211, Alan R. Liss, New York. (12) Van Tassell, R. L., and Wilkins, T. D. (1986) The precursors of fecapentaenes. A preliminary report. Ann. Zst. Super. Sanita 22, 933-942. (13) Wattenberg, L. W., Galbraith, A. R., and Hochalter, J. B. (1986) Inhibitory effects of 4-methoxybenzenethio1(4-MBT) and sodium thiosulfate (STS) on direct acting carcinogens. Proc. Am. Assn. Cancer Res. 27, 123. (14) Baptista, J., Krepinsky, J. J., and Pfaendler, H. R. (1987) Natural fecapentaene-14 and one fecapentaene-12 component are
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