* Reproductive Toxicology Division and
** Experimental Toxicology Division, National Health and Environmental Effects Research Laboratory, U.S. Environmental Protection Agency, Research Triangle Park, North Carolina 27711;
DuPont Merck Pharmaceutical, Newark, Delaware; and
Mantech Environmental Sciences, Research Triangle Park, North Carolina
Received May 23, 2000; accepted September 22, 2000
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ABSTRACT |
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Key Words: mammalian developmental toxicity; BBDR modeling; 5-fluorouracil; thymidylate synthetase inhibition; dNTP imbalance; limb dysmorphogenesis.
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INTRODUCTION |
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Biologically based dose-response modeling is an emerging approach to reducing the uncertainties inherent in the risk-assessment process. The concept, depicted in Figure 1, is to incorporate the necessary biological information into a model that captures the sequence of key events involved in disruption of homeostasis, with mathematical terms that describe the linkage of these biological processes, thereby improving the ability for low-dose and cross-species extrapolation. Ideally, physiologically based pharmacokinetic (PBPK) data (pharmacokinetic linkage) should be applied for the dose component of the model, to facilitate direct comparisons of exposure levels between tissues and across species. Pertinent biomarkers based on mechanisms of toxicity (pharmacodynamic linkage) should be evaluated for the response component to enhance the sensitivity of detection for adverse biological outcomes (O'Flaherty, 1997
). Although this concept is appealing, the actual practice of model construction proves to be daunting. For developmental toxicity, at least a dozen or so general categories of mechanisms have been identified to elicit various untoward cellular responses, leading to tissue damage and adverse pregnancy outcomes (Wilson, 1973
). This list of toxic mechanisms will undoubtedly be expanded as our understanding of developmental biology is advanced. It would also be naive to assume that a chemical would act through only a single mechanism. From a toxicologist's standpoint, one must ask which and how many mechanism(s) should be embraced for modeling?
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Applying these principles, we have previously described a BBDR model comprising the biochemical and cellular sequelae of 5-fluorouracil (5-FU) exposure during embryonic development, with specific reference to the drug-induced digital defects (Shuey et al., 1994b). 5-FU is a chemotherapeutic agent and a teratogen in a number of laboratory animal species, including rat, mouse, chick, hamster, and monkey (Dagg, 1960
; Karnofsky et al., 1958
; Shah and MacKay, 1978
; Shuey et al., 1994b
; Wilson, 1971
; Wilson et al., 1969
). Clinically, a single case of multiple congenital anomalies in a fetus exposed to 5-FU during the first trimester of pregnancy was reported by Stephens et al. (1980), and the lesions described were generally consistent with those seen in the animal models. 5-FU acts by at least 3 known mechanisms (reviews: Parker and Cheng, 1990; Pinedo and Peters, 1988; Valeriote and Santelli, 1984): first, inhibition of thymidylate synthetase, the enzyme that catalyzes the conversion of uridylate to thymidylate (Danenberg and Lockshin, 1981
); second, incorporation of FUTP (as substitute for UTP) into RNA, producing miscoding in transcription and leading to translation of nonfunctional proteins (Dolnick and Pink, 1985
; Glazer and Lloyd, 1982
; Lenz et al., 1994
), and third, incorporation of FdUTP into DNA, requiring excessive repairs and resulting in damage due to fragmentation (Kufe et al., 1983
; Parker et al., 1987
; Schuetz et al., 1984
). In addition to these 3 mechanisms, drug-induced fetal anemia could also contribute to 5-FU developmental toxicity (Shuey et al., 1994c
; Zucker et al., 1995
). A composite scheme of putative mechanisms for 5-FU developmental toxicity is depicted in Figure 2
. In a previous paper, Shuey et al. (1994b) constructed a BBDR model primarily for the 5-FU-induced hind-limb dysmorphogenesis. However, 5-FU produces a gamut of anatomical abnormalities and defects, each possessing a slightly different dose-response profile. Thus, instead of providing a model for each of the malformations, the question remains as to whether a BBDR model can be extended to the whole-embryo level in order to capture the embryotoxicity of 5-FU at large, rather than specific insults of one anatomical structure.
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MATERIALS AND METHODS |
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Biochemical assays.
Four embryos were removed randomly from each dam, placed individually in 1 ml of ice-cold 125-mM Tris buffer containing 150 mM KCl (pH 7.5) and homogenized immediately by polytron. In addition, maternal spleens were dissected, weighed, and homogenized in 20 volumes of Tris buffer. A 0.2-ml aliquot of the tissue homogenate was removed for DNA and protein determination; total protein was determined in duplicate 25 µl aliquots of tissue homogenate by the dye-binding method of Bradford (1976), and DNA content was measured in duplicate 5 µl samples using a modified method of LaBarca and Paigen (1980) as described previously by Abbott and Pratt (1988). The remainder of the homogenate was sedimented for 30 min at 32,000 x g, and duplicate 50 µl aliquots of the supernatant solution were withdrawn for the thymidylate-synthetase assay, using a modified method of Roberts (1966) that measured the release of tritiated water from enzyme reaction substrate deoxyuridine 5'-monophosphate, after a methylation step to produce thymidylate. The incubation mixture contained 50 µl of reagent containing 0.86 mM tetrahydrofolate, 75 mM mercatpoethanol, 235 mM tris, 0.43% formaldehyde (pH 7.5), 25 µl of 1 M NaF, 10 µl of 2.8 mM deoxyuridine 5'-monophosphate (dUMP), 65 µl of 1 M Tris (pH 7.5) and 0.4 µCi of deoxy[3H]uridine 5'-monophosphate (specific activity 1619 Ci/mmol., Amersham). Samples were incubated at 37°C for 1 h and pooled supernatant samples, incubated at 0°C for 1 h, served as assay blanks. Reaction was terminated on ice and by addition of 100 µl of 40% trichloroacetic acid. 50 µl of dUMP (5 mg/ml) was then added to each sample, followed by 1.8 ml of activated charcoal (100 mg/ml). The mixture was sedimented at 500 x g for 15 min, and 0.5 ml of the supernatant was withdrawn and counted by liquid scintillation spectrometry. Enzyme activity is expressed as nmols 3H2O released/mg DNA/h.
For pharmacokinetic studies, 2 blocks of 3 to 4 pregnant rats from the 4-h breeding scheme were given 10, 20, or 40 mg/kg of 5-FU on GD 14. For one block of rats, blood samples were collected from the tail vein at 5, 15, 25, and 30 min after injection; for another block, samples were collected at 10, 20, 45, and 60 min. The serum fraction was separated from whole blood and stored frozen at 80°C until analysis. Samples were thawed and extracted by the method of Barberi-Heyob et al. (1992). In brief, 500 µl of serum was added to a Chem-Elut column (Analytichem International, Harbor City, CA) for 5 min, followed by elution with 3 aliquots of ethyl-acetate-methanol (95:5, v/v). The eluent was concentrated by evaporation under nitrogen and reconstituted with 200 µl of water. The extracted samples were analyzed for 5-FU by a modified method of Schwartz et al. (1992). 5-FU was separated from its metabolites with an adsorbosphere 5 µm C18 column (250 mm x 4.6 mm, Alltech, Deerfield, IL) isocratically using a mobile phase of 5 mM tetrabutylammonium hydrogen sulfate and 5 mM potassium phosphate (pH 7), and detected by UV spectrophotometry at 268 nm. Recovery of 5-FU under these conditions was typically greater than 95%. Results were expressed as µg of 5-FU/ml serum. In an attempt to measure the 5-FU content in whole embryos, 2 embyros were pooled and deproteinized in 0.5 ml of 0.3 M perchloric acid, and the supernatant was analyzed for 5-FU by HPLC, as described above. However, an interfering peak co-eluted with 5-FU that was also detected in saline-treated controls, thereby precluding accurate assessment of 5-FU content in the embryonic tissue.
Deoxyribonucleotide (dNTP) determination in whole embryos was also performed with pregnant rats from the 4-h breeding scheme. On GD 14, 6 to 8 rats were given either 10, 20, or 40 mg/kg 5-FU, and animals were sacrificed at 0.5, 1, 2, 4, 8, or 12 h after injection. Four embryos were removed randomly from each dam, weighed, homogenized (sonication) individually, and deproteinized in 300 µl of 0.3 M perchloric acid and stored frozen at 80°C until analysis by an HPLC method described previously (Mole et al., 1998). Duplicate samples were measured from each dam to ensure reproducibility, and data were expressed as pmol of dNTP/mg embryonic tissue.
To evaluate the extent of 5-FU incorporation into nucleic acid, 0.51 mCi/kg of 5-FU-63H (specific activity 20 Ci/mmol, Sigma) was isotopically diluted to 20 or 40 mg/kg and given to pregnant rats on GD 14. Rats were sacrificed by asphyxiation 8 h later, and embryos were removed. Two embryos were pooled for each sample, and duplicate samples from each dam were assayed. Embryos were homogenized (polytron) in 3.5 ml of 4 M guanidine isothiocyanate buffer. A 0.75-ml aliquot of homogenate was removed and counted by liquid scintillation spectrometry. Homogenate (2.4 ml) was then loaded onto 1.7 ml of CsCl2 buffer (0.96 g/ml) for overnight centrifugation at 100,000 x g. The DNA fraction from the gradient was collected, precipitated, and washed with 6 ml of 70% ethanol, and the resultant pellet was dissolved in 1 ml of water. The remaining CsCl2 buffer was removed and the RNA pellet was dissolved in 0.5 ml of water. Aliquots of DNA and RNA extract were removed, and the nucleic acid concentrations of each sample was determined by spectrophotometry at 260 nm. The remaining fractions were counted by liquid scintillation spectrometry, and incorporation of 5-FU into nucleic acid was calculated as follows:
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Statistical analysis.
Data were computed with a linear model, treating each experimental day as a random effect (PROC MIXED, SAS, 1990), and expressed as means and 95% confidence intervals. Litter means were used for analysis to eliminate the variability among embryos within each dam. The cell size of each time and dose point were not balanced due to experimental limitations; typical intra- and inter-block variabilities of each measurement were reflected by the 95% confidence interval estimation in the control groups. Because of the heterogeneity of variance and to facilitate the expression of contrasts as ratios to the corresponding control means, values were subject to log-transformation before statistical evaluation, and the resulting contrasts were back-transformed for graphing. Pharmacokinetic data were analyzed as mixed-effect linear models using "lme" in S+ version 3.4 (Mathsoft, Seattle, WA), treating results from the same dam as repeated measures. Curves were fitted using the method of nonlinear mixed-effect models of Lindstrom and Bates (1990).
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RESULTS |
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To compare the effects of 5-FU between embryonic and maternal tissues, TS activity in the spleen of the pregnant rats were evaluated. TS activity in maternal spleens ranging from 1.6 to 3.9 nmol/mg DNA/h (Table 7), was substantially lower than that in the embryo. As seen in the embryonic tissues, 5-FU produced dose-dependent enzyme inhibition at doses as low as 1 mg/kg, although prominent effects were generally observed at and above 20 mg/kg.
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DISCUSSION |
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While ample evidence has indicated that all 4 putative mechanisms depicted in Figure 2 are likely to contribute to the expression of 5-FU developmental toxicity, we chose only one of these for the construction of a BBDR model in accordance with the model assumptions. Although direct incorporation of the 5-FU metabolite (FUTP) into RNA has been shown in a variety of cell culture systems, concentrations of 5-FU found to be associated with cytotoxicity were typically high, in the mM range, resulting in approximately 12% substitution for the natural substrate UTP (Dolnick and Pink, 1985
; Glazer and Lloyd, 1982
). Doses employed in our in vivo study were comparatively lower, embryonic exposure being estimated at the µmol/kg range. Indeed, the extent of 5-FU tracer incorporation into embryonic RNA in our study was considerably lower, with substitution for UTP being estimated at only 0.2%. Although transcriptional miscoding leading to production of nonfunctional proteins was hypothesized with direct incorporation of FUTP into RNA, recent findings have brought this hypothesis into doubt. Takimoto and co-workers (1993) transcribed mRNA with 100% substitution with 5-FU but did not note any abnormality with the translation product. Schuetz et al. (1984) also could not correlate incorporation of 5-FU into RNA with toxicity. Furthermore, although the 2-D electrophoresis experiment in our study provided only a relatively crude screen for protein abnormalities, the paucity of changes noted between controls and 5-FU-exposed embryos also did not support any significant aberrant production of proteins by the drug treatment. Thus, while the mechanisms and impact of RNA-directed actions of 5-FU involved in the overall expression of cytotoxicity remains to be addressed, the contribution of this pathway to embryotoxicity in the current study was likely nominal. On the other hand, the extent of erroneous incorporation of FdUTP into embryonic DNA noted in the present study (300700 fmol/µg) was comparable to that observed in CF-1 mouse bone-marrow cells where cytotoxicity was associated (Scheutz et al., 1984). However, the net effect of erroneous DNA incorporation in the whole embryo is difficult to ascertain because it is dependent on the capacity and effectiveness of the DNA repair machinery. Excessive DNA damage due to saturation of these repair mechanisms has been linked to fragmentation of newly synthesized DNA and cell death (apoptosis) (Curtin et al., 1991
; Golos and Malec, 1989
; Holliday, 1985
; Snyder, 1988
). Although 5-FU-induced apoptosis has been reported recently in several tumor cells (Guchelaar et al., 1998
; Inada et al., 1997
; Koshiji et al., 1997
; Okamoto et al., 1996
) and rat tissues (Sakaguchi et al., 1994
), the extent of excessive cell death in embryonic tissues caused by DNA damage remains to be evaluated carefully. 5-FU-induced fetal anemia observed in previous studies (Shuey et al., 1994c
; Zucker et al., 1995
) was likely a secondary effect arising from fetal hepatotoxicity because the fetal liver serves as a primary source of newly synthesized erythrocytes at this stage of development. Indeed, Shuey and co-workers (1994a) demonstrated that early biochemical events that occurred rapidly within 3 h of 5-FU administration were sufficient to produce growth retardation and dysmorphogenesis. Thus, although physiological deficiency such as fetal anemia is of significant note to the overall picture of 5-FU developmental toxicity, it should probably be modeled as an adjuvant to the primary pathway of TS inhibition and nucleotide pool imbalance.
Inhibition of TS and the attendant dNTP imbalance in the whole embryos provided sensitive endpoints to monitor the dose-response effects of 5-FU, and the temporal profiles of changes afforded a concentration x time (c x t) estimation for mathematical modeling. Indeed, while 5-FU inhibited embryonic TS in a dose-dependent fashion, the manner in which enzyme activities recovered perhaps provided a more significant clue to the drug's toxic potentials. Thus, the combination of duration as well as magnitude of enzyme inhibition might confer the overall toxicity. Mathematical modeling of these data (in the accompanying paper) may shed light on this issue. It is also heartening to note that the dose-response profile of TS inhibition in the maternal spleen (a tissue undergoing rapid cell replication) resembled that in the embryos, suggesting that the enzyme was equally sensitive to 5-FU regardless of tissue or cell type, thus lending credence to extrapolation based on this mechanistic step.
While the effect of 5-FU on de novo synthesis of dNTP mediated by TS inhibition is well established, the influences of the drug on the nucleotide salvage pathways have received less attention. For instance, thymidine kinase, an enzyme involved in maintaining the level of dTTP, was reported to be inhibited by 5-FU as well (Nord and Martin, 1991; Tsukamoto and Kojo, 1991
). Thus, the profiles of individual dNTPs in the whole embryo would provide a clearer and more definite picture of the 5-FU effects. Results from our present study are consistent with a previous report where embryonic dNTP was determined at a single time point after 5-fluoro-deoxyuridine treatment (Ritter et al., 1980
). Corresponding to TS inhibition, there was an abrupt fall in the concentration of dTTP, reaching a nadir of over 50% deficit at 2 h after drug administration. Two points are noteworthy from these data. Little change in dTTP was seen with the 10 mg/kg group despite effective TS inhibition (although the extent was less than the higher-dose groups), and the time course of recovery was faster than that of TS, suggesting a significant participation of nucleotide salvage and feedback control mechanisms (review: Lau, 1997). Corresponding to the intracellular nucleotide feedback regulation, dGTP levels fell along with dTTP, while the negative feedback control of dCTP by dTTP was repressed, leading to a dramatic rise of dCTP. It is interesting to note that while dTTP is most proximal to the 5-FU action (TS inhibition), the magnitude and duration of changes in dGTP and dCTP were greater than those in dTTP. Embryonic dATP, on the other hand, was not affected significantly by 5-FU. Because little information is available to discern the relative importance of specific dNTPs in DNA synthesis and cell replication, a direct biochemical correlation of changes in an individual dNTP to embryotoxicity is not readily attainable. Nonetheless, Yoshioka and co-workers (1985, 1987) reported that cytotoxicity of fluorodeoxyuridine in mouse mammary tumor cells correlated with dNTP pool imbalance and fragmentation of parental DNA (double-strand breaks). These researchers noted that inhibition of protein synthesis prevented DNA fragmentation and attenuated the cytotoxicity, and therefore hypothesized that dNTP imbalance induced an endonuclease that destroyed the DNA and resulted in cell death. Indeed, they were able to isolate a DNA endonuclease in extracts from the cells exposed to fluorodeoxyuridine but not in the untreated cells. Interestingly, Hirota et al. (1989) also reported DNA double-strand breaks and cell death associated with dNTP imbalance caused by another agent, 2-chlorodeoxyadenosine. Thus, activation of an endonuclease by dNTP pool imbalance may be a general mechanism for a number of agents to induce apoptosis.
Total DNA and protein contents in the whole embryo appeared to be less sensitive indicators of 5-FU toxicity. Notable changes were obtained only with the high doses. However, it is worth noting that reductions of these parameters were persistent and correlated well with the deficits of fetus weights in these dose groups. Because 5-FU should affect only the dividing cells within the embryo and for a relatively short duration, the lack of dramatic changes in total macromolecule contents may not be too surprising. A more accurate measurement to indicate the 5-FU effect was, perhaps, biosynthesis of DNA and protein. However, because thymidine kinase is known to be altered by 5-FU (Bagrij et al., 1993; Tsukamoto and Kojo, 1991
), results from the conventional method of labeled thymidine uptake and incorporation into DNA would be confounded. Indeed, preliminary studies in our laboratory indicated a small increase of labeled thymidine incorporated into DNA shortly after 5-FU administration (data not shown), perhaps reflecting an acute response of the salvage pathway rather than the rate of de novo DNA synthesis. Another method of assessing DNA synthesis using BrdU was limited because of the 5-FU-induced TS inhibition.
In summary, pharmacokinetic and pharmacodynamic information of a chemical, as well as the biochemical alterations leading to the breakdown of homeostasis (toxicodynamics) within a developing organism (embryo), can be evaluated and used for the construction of a BBDR model of developmental toxicity. Indeed, data derived from the maternal serum concentrations of 5-FU, the TS activities, and the dNTP levels in the embryos, as well as the fetus weights reported in this study, were selected for the construction of a mathematical model in the accompanying paper by Setzer et al. (2001) that would describe the developmental toxicity of this chemotherapeutic agent. Although 5-FU was used as a prototypic agent in this study, the correlation of molecular and cellular changes with toxicity should reflect an inherent property of the developing organism (embryo) that can be verified and extended to other potential developmental toxicants.
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ACKNOWLEDGMENTS |
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NOTES |
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1 To whom correspondence should be addressed at Mail Drop 67, U.S. Environmental Protection Agency, Research Triangle Park, NC 27711. Fax: (919) 541-4017. E-mail: lau.christopher{at}epa.gov.
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