* Division of Applied Pharmacology Research, Office of Testing and Research, Office of Pharmaceutical Sciences, and
Division of Biometrics II, Office of Biostatistics, Office of Pharmacoepidemiology and Statistical Science, Center for Drug Evaluation and Research, Food and Drug Administration, Laurel, Maryland 20708
Received December 20, 2002; accepted April 9, 2003
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ABSTRACT |
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Key Words: Tg.AC; gadd153; TPA; DMSO; solvent.
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INTRODUCTION |
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Among the goals of the ILSI-sponsored collaboration was the evaluation of the response of the alternative assays to species-specific carcinogens and to noncarcinogens (Robinson and MacDonald, 2001). Sulfisoxazole was the only noncarcinogen tested in the Tg.AC mouse by the ILSI consortium. Our study was designed to complement the ILSI effort through the additional testing of noncarcinogens chosen by screening for potential "false positive" activity in the Tg.AC assay. To develop an in vitro assay system that could detect gene induction patterns correlative with in vivo activity in the Tg.AC assay, 24 Tg.AC tested compounds were analyzed in four rapid throughput in vitro reporter gene assays (Thompson et al., 2000
). These assays were three CAT-Tox (L) assays that measured induction of the gadd153 promoter, c-fos promoter, and p53 response elements in HepG2 cells and a fourth assay that measured induction of the
-globin promoter in K562 cells. Of the four assays, the gadd153-CAT assay showed the strongest overall concordance (81%) with activity in the Tg.AC assay. The
-globin promoter assay correctly classified only 64% of Tg.AC positive and 58% of Tg.AC negative compounds.
To prioritize selection of drugs for testing Tg.AC assay specificity, 99 pharmaceuticals that had tested negative for carcinogenic activity in male and female rats and mice, and were available from commercial or internal sources were screened for their ability to induce the gadd153 promoter in vitro (see accompanying article). Approximately 10% of the screened drugs induced the gadd153 promoter by four-fold or more. Several criteria were used to select among this subset of nine drugs for the three best candidates for subsequent in vivo testing in a Tg.AC assay: whether drug solubility in acetone or ethanol was sufficient to elicit systemic toxicity; the level of gadd153, -globin, and c-fos promoter inductions by the drug; the potency of the drug in the in vitro assays; and the cost of the drug required for six months of dosing. Based on these criteria, amiloride, dipyridamole, and pyrimethamine were selected for testing the specificity of the Tg.AC assay towards pharmacologically active, noncarcinogenic drugs.
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MATERIALS AND METHODS |
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Chemicals and solvents.
12-O-tetradecanoylphorbol-13-acetate (TPA; 99% purity) was from Alexis Corp., San Diego, CA. Pyrimethamine, amiloride hydrochloride hydrate, and dipyridamole were all purchased from Sigma, St. Louis, MO. A.C.S. reagent grade ethanol (99.5%, Aldrich), molecular biology grade dimethyl sulfoxide (DMSO; >99.9%, Sigma), HPLC grade acetone (Thomas Scientific), and Millipore-filtered house distilled H2O were used as solvents.
Dose range finding study.
Groups of 10 FVB/N mice of each sex were dosed with each of the test solutions or vehicle alone 5 days per week for 4 weeks. Doses were extrapolated from maximum tolerated doses (MTDs) in 2-year bioassays. The MTD was converted from mg/kg/day to mg/mouse/day, assuming a 25 g mouse, multiplied by 7 to derive the total weekly exposure, and then divided by 5 to derive the daily skin paint high dose. The daily skin paint target dose was divided by 200 µl (the constant skin paint delivery volume) to calculate the concentration of the high-dose application solution needed.
Stability studies indicated that the dosing solutions could be prepared weekly and stored at 4°C (data not shown). Dipyridamole was prepared as a 25 mg/ml solution in ethanol and applied once or twice daily. For the low dose group, the dipyridamole stock solution was diluted 1:1 in ethanol. To prepare the high dose solution, amiloride was dissolved in DMSO to 150 mg/ml and diluted 1:33 with ethanol. The middle and low dose solutions were prepared by diluting the high dose solution 1:2.5 or 1:5 with ethanol. The high-dose pyrimethamine solution was prepared by first adding 70% ethanol and then acidifying with 1N HCl until dissolved. This solution was applied once or twice a day for the middle and high dose groups, respectively. The high-dose pyrimethamine solution was diluted 1:3 in ethanol to prepare the low dose formulation.
At necropsy, blood was collected by cardiac puncture after CO2 anesthesia for hematology and clinical chemistry measurements. Kidney, liver, spleen, thymus, and skin were collected for histopathology.
Twenty-six-week carcinogenesis study.
Each study group contained equal numbers of male and female mice. The negative control groups had 15 animals per sex, the positive control groups had 10 animals per sex, and the test compound groups had 15 animals per sex. All doses were applied in a volume of 200 µl. The following doses and dosing regimens were used for the 26-week study. The solvent control vehicle consisted of 87% ethanol, 2.4% DMSO, 10.6% H2O, and was applied three times/week. One positive control group received 2 µg TPA in acetone three times/week. The second positive control group received 2 µg TPA in solvent control vehicle three times/week. High (3.6 mg/ml) and low (1.8 mg/ml) doses of amiloride were applied five times/week. Amiloride was solubilized in 100% DMSO at 150 mg/ml and diluted with ethanol to make a 3.6 mg/ml solution in 2.4% DMSO, 97.6% ethanol. The low-dose amiloride solution was prepared by diluting the high dose solution 1:1 with ethanol. Initially, pyrimethamine (5 or 10 mg/ml in 87% ethanol, 13% acidified H2O) was applied once daily to the low and high dose groups, respectively. Dipyridamole was prepared as a 25 mg/ml solution in 100% ethanol and applied once or twice daily to the low and high dose groups, respectively. All dosing solutions were prepared and stored in the dark in amber vials to minimize light exposure.
Animals were observed twice daily for mortality and moribundity; body weights and detailed clinical findings including test site papilloma counts were recorded weekly. Papillomas were counted as present if observed for three consecutive weeks. The upper limit of papillomas counted per animal was 24. Necropsy was performed at the end of the study on all animals. Organ weights were taken at necropsy for brain, heart, kidney, liver, and ovaries or testis with epididymis. A complete selection of tissues from negative control, all high dose groups, and the low-dose pyrimethamine group were paraffin-embedded, sectioned, and examined microscopically.
Statistical analyses.
Pairwise comparisons using two-sample nonparametric statistical procedures (Wilcoxon rank-sum and Mann-Whitney tests) were used for testing significance of differences in weekly skin papilloma counts between pairs of treatment groups. Statistical significance of clinical chemistry parameters, hematology parameters, and mean body and organ weights between vehicle control and treatment groups were determined by analysis of variance using Dunnetts method.
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RESULTS |
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Toxicity after four weeks of drug treatment was assessed using histopathology and standard clinical chemistry and hematology indicators. No treatment-related effects on mortality, body weight, major organ weight, or gross pathology were observed in any of the treatment groups. Female mice in the high-dose amiloride group had elevated mean blood urinary nitrogen (BUN) levels (36.9 ± 12.1 vs. 20.8 ± 5.3 for the control group, p < 0.05) and elevated serum potassium levels (p < 0.05). Hyperkalemia and transient elevations in BUN are reported side effects of amiloride therapy (McEvoy, 1998). Moderate to severe renal lesions were observed in 5 of 10 males in the high-dose amiloride group. In the dipyridamole treatment groups, the only indication of an adverse effect was a statistically significant elevation in mean serum glucose levels in female mice receiving the middle dose (5 mg/day) of dipyridamole for four weeks. Alterations in several hematologic parameters were observed in male mice in the high-dose pyrimethamine group. The mice in this group had decreased albumin to globulin ratios, decreased white blood cell counts, and elevated mean corpuscular volume and hemoglobin content (MCV and MCH) values (p < 0.05). Although toxicity was observed in the high dose group, the middle and high doses of pyrimethamine may still have been incompletely delivered. It was discovered late into the dose range finding study that the vehicle originally chosen to deliver 15 mg/ml of pyrimethamine (68% ethanol/32% acidified H2O; pH 5.0) did not uniformly wet the surface of the skin upon application (see Fig. 1). With subsequent testing, we observed that 14% was the maximal concentration of water that could be added to ethanol and applied to shaved mouse skin in a uniformly wettable manner. A vehicle containing 70% ethanol/30% water did not spread evenly over the application site.
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Twenty-Six-Week Skin Paint Study
The 26-week skin paint study was conducted in groups of male and female hemizygous Tg.AC mice with amiloride, dipyridamole, and pyrimethamine at two dose groups per drug. Amiloride was administered five times per week at doses of 0.9 or 1.8 mg in 97.6% ethanol/2.4% DMSO. Dipyridamole was administered five days a week at a dose of 5 mg once or twice a day in 100% ethanol. Initially, pyrimethamine was given five times per week at doses of 1 or 2 mg in 87% ethanol/9.75% H2O/3.25% 1N HCl, pH 5. During the second week of the study, a sudden onset of mortality was observed among male mice in the low and high-dose pyrimethamine groups and in female mice in the high-dose pyrimethamine (see Fig. 2). Male mice were more sensitive to pyrimethamine toxicity than female mice. Necropsy failed to reveal the cause of death. Doses of pyrimethamine were lowered until mortality stabilized (at week 6). From weeks 6 to 26, male mice received pyrimethamine at doses of 0.15 or 0.3 mg five days per week and female mice received 1 or 1.5 mg five times per week (see Table 2
).
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All male mice and 80% of female mice treated with TPA in acetone developed a maximal tumor burden (Fig. 3). The two females that developed no papillomas in the positive control group were further analyzed for evidence of alterations in transgene DNA. One animal showed the most commonly observed nonresponder genotype in which there is loss of palindromic transgene promoter sequence. This nonresponder genotype, which is diagnosed by Southern blot hybridization of BamHI-digested genomic DNA (Thompson et al., 1998
), typically occurs at a rate of 1.51.6% per generation in the breeding of Tg.AC hemizygous mice (Cannon et al., 2001
). The other female nonresponder had no observable loss of palindromic transgene sequence although she and her offspring were refractory to TPA (data not shown).
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DISCUSSION |
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Pyrimethamine, administered dermally at doses that produced systemic toxicity, failed to induce papillomas in Tg.AC mice. Although pyrimethamine has a clear genotoxic effect in rodent bone marrow micronucleus tests (Ono et al., 1997; Vijayalaxmi and Vishalakshi, 2000
), this drug tested negative in female mice and rats of both sexes in a carcinogenesis bioassay (National Toxicology Program, 1978
). Reduced survival in both control and test groups prevented assessment of the carcinogenic potential of this drug to male mice. Inhibition of dihydrofolate reductase by high doses of pyrimethamine can cause depletion of deoxyribonucleotide pools, which increases the error rate during DNA synthesis, promoting the formation of DNA lesions such as fragile sites, structural chromosomal aberrations, and sister chromatid exchanges (Egeil, 1998
). Although the Tg.AC assay has reduced sensitivity in detecting mutagenic carcinogens (Sistare et al., 2002
), less evidence is available on how this assay responds to clastogens other than benzene. The negative result seen in this study with maximally tolerated doses of pyrimethamine suggests that noncarcinogenic, bone marrow clastogens do not produce false positive results in the Tg.AC assay.
A vehicle containing 2.4% DMSO had a strongly inhibitory effect on papilloma induction by TPA in this study. The same concentration of DMSO was used in the vehicle used to deliver the high dose of amiloride, which may have confounded interpretation of the true tumorigenic potential of amiloride in the Tg.AC assay. Inhibition by DMSO has been seen in the two-stage skin carcinogenesis model in studies with SENCAR mice that examined the effect of solvent on TPA induction of papillomas after initiation with 7,12-dimethylbenz(a)anthracene (DMBA; Slaga and Fischer, 1983). In this system, the concentration of DMSO in the vehicle was directly proportional to the degree of inhibition observed. Based on these results, it was reasonable to assume that the use of small amounts of DMSO would have minimal inhibitory effects on papilloma formation in Tg.AC mice and we designed our study accordingly. However, a recent study of the effects of vehicle composition on the activity of TPA in Tg.AC mice by Stoll et al. has shown that DMSO concentration has an inverse relationship to inhibition of papilloma formation in this model. A vehicle containing 20% DMSO was more inhibitory to papilloma induction by TPA than was 100% DMSO (Stoll et al., 2001
). Although the Tg.AC mouse has the phenotype of preinitiated mouse skin, the relationship between dose of DMSO and degree of inhibition is reversed between Tg.AC mice and DMBA-initiated SENCAR mice. In both models, papilloma formation is follicular in origin and associated with activating mutations in the Ha-ras gene (Binder et al., 1998
; Hansen and Tennant, 1994
), but the results with DMSO suggest that there may be mechanistic differences between these two models. The process by which DMSO is inhibitory is not known but it is not thought to be through altering the delivery of TPA to the skin. Preapplication of DMSO immediately or up to 1 h before promotion with TPA in acetone is inhibitory to papilloma formation in a two-stage model of skin carcinogenesis using CD-1 mice (Jacoby and Weiss, 1986
). In addition, limited evidence suggests that the inhibitory effect of DMSO on papilloma formation is not specific to TPA, because the use of DMSO as a vehicle also has a dampening effect on methylcholanthrene-induced skin carcinogenesis in hairless mice (Iversen et al., 1981
). Therefore, this solvent should best be avoided in Tg.AC studies because of its ability to reduce a papilloma response, which could confound results with weak tumorigens. The use of a vehicle containing 20% DMSO/80% ethanol in the ILSI sponsored evaluation of the Tg.AC assay was not associated with a robust response to treatment (Tennant et al., 2001
). It may be possible that a response could have been seen with amiloride if DMSO had not been used in the vehicle.
For maximal drug exposure, a skin paint solvent should evaporate or be rapidly absorbed after application. This study clearly shows that using a vehicle with incomplete wettability (67% ethanol/33% H2O) does not deliver a full dose of drug to the animal. Greater toxicity was seen with a 3 µg dose of pyrimethamine in 87% ethanol/13% H2O than with a higher dose (6 µg) that was applied in 67% ethanol/33% H2O. These data emphasize that the wettability of a nonstandard vehicle should be confirmed before the study commences.
The conceptual basis of this project lies in the hypothesis that enhancement of the expression of the v-Ha-ras transgene, particularly through stimulation of the transcriptional activity of the -globin promoter directly or through upstream enhancer elements, can be a mechanism by which positive responses are generated in the Tg.AC assay by noncarcinogens. Based on this theory, a reporter assay measuring activation of the
-globin promoter in a permissive cell line was constructed for use in an in vitro screen. However, activation of the
-globin promoter in vitro did not have high correlation with the ability to induce papillomas in Tg.AC mice (Thompson et al., 2000
). Subsequently, through analysis of Tg.AC mice that were nonresponsive to TPA, we observed that the linear positioning of copies of the transgene in a head-to-head palindromic orientation correlated with induction of transgene expression and papilloma formation in response to tumor promoter treatment (Honchel et al., 2001
; Thompson et al., 1998
). Evidence to date suggests that the secondary structure of the transgene locus, the nucleotide sequence of the transgene, and the surrounding insertion site are all involved in the regulation of transgene expression. Therefore, this system would be very difficult to model in an in vitro screen. Based on the transgene structure and sequence, it might be thought that the reporter phenotype of Tg.AC mice, i.e., the generation of skin papillomas upon dermal application of carcinogens or tumor promoters, is simply a direct consequence of induction of transgene expression through regulating the activity of the
-globin promoter. The reality appears to be much more complex and to involve a series of specific genetic and epigenetic events that are incompletely understood at present. Activation of the
-globin promoter may be a necessary component for Tg.AC tumorigenesis but it appears not to be sufficient.
The presence of a nonresponder Tg.AC mouse in our positive control group whose phenotype could not be detected using the standard genotyping protocol emphasizes the importance of monitoring for phenotypic responsiveness among the Tg.AC breeding colony. We are currently investigating whether this mouse carried a deletion in the right hand Line-1 sequence recently associated with loss of responsiveness in Tg.AC mice (Leder et al., 2002). There may be alternative mechanisms in the Tg.AC mouse model besides loss of palindromic transgene sequence or integration site sequence that result in loss of responsiveness and, although these examples may be rare, they require continual vigilance.
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ACKNOWLEDGMENTS |
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NOTES |
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REFERENCES |
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