* Curriculum in Toxicology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 275997270; and
Reproductive Toxicology Division, National Health and Environmental Effects Research Laboratory, United States Environmental Protection Agency, Research Triangle Park, North Carolina 27711
Received April 6, 1999; accepted June 1, 1999
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key Words: disinfection byproducts; haloacetic acids; embryotoxicity; whole-embryo culture; protein kinase C; apoptosis; cell cycle.
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
One potential mechanism by which the HAs may perturb development is inhibition of protein kinases. Dichloroacetate in particular has been shown to inhibit various protein kinases, including pyruvate dehydrogenase kinase (Crabb et al., 1981), succinyl-CoA synthetase (Yang and Smith, 1983
), and protein kinase A and MAP kinase (Smith et al., 1997
). Also, it has recently been demonstrated that various HAs inhibit purified human protein kinase C (PKC) and PKC isolated from neurulation-stage mouse embryos (Ward et al., 1997
). Protein kinases, especially PKC, are important regulators of many different cellular events, including apoptosis (Leszczynski, 1996
) and regulation of the cell cycle (Fishman et al., 1998
). Given the importance of these molecular processes in development (Elstein et al., 1993a
; Knudsen, 1997
), it is intuitive that perturbation of PKC activity could result in dysregulation of critical events during development, to produce embryotoxicity.
The objective of this study was to describe the cellular pathogenesis of HA embryotoxicity in the neurulation-stage mouse. Whole-embryo culture was chosen as the experimental approach for this investigation because of the necessity to eliminate maternal influences (e.g., maternal toxicity or pharmacokinetics) in the study design, while retaining the structural and functional integrity of the embryo. Because of the potential role of HA-induced perturbations in PKC activity as a mechanism for the observed dysmorphogenicity, Bis I, a specific PKC inhibitor with previously defined embryotoxic effects (Toullec et al., 1991; Ward et al., 1998
) was employed in this investigation as a comparator to the HAs. Also, as a positive control, the pathogenesis of staurosporine, a potent but non-specific protein kinase inhibitor known to interact with the cell cycle (Bruno et al., 1992
) and with apoptosis (Falcieri et al., 1993
; Rajotte et al., 1992
), was investigated. Finally, Bis V, a structural analog of Bis I that possesses neither PKC-inhibitory activity (Davis et al., 1992
) nor substantial embryotoxicity (Ward et al., 1998
) was employed in this investigation as a negative control.
![]() |
MATERIALS AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Animals.
CD-1 mice were purchased from Charles River Laboratories (Raleigh, NC). Mice were housed in a temperature- and humidity-controlled vivarium using a 14:10-h light:dark cycle with lights out at 23:00. Purina rodent chow and filtered tap water were available ad libitum. Females were housed 35 per cage for 2 weeks before being included in the breeding colony. Males were individually housed. From 08:00 to 10:00 A.M., 3 females were placed in a cage containing a male mouse. At the end of this time, females were removed and checked for a vaginal plug. Presence of a vaginal plug indicated that mating had occurred and was designated gestational day (gd) 0. On gd 8, pregnant females were killed by cervical dislocation and the uterus was removed from the dam. Early somite-stage conceptuses (36 somites) were prepared for culture as described previously (Sadler, 1979). Briefly, the maternal connective tissue and myometrium were removed, leaving the conceptus surrounded by decidua. The decidua was removed, along with the parietal yolk sac and Reichert's membrane, leaving the conceptus intact.
Whole embryo culture.
Conceptuses were cultured in 30-mL serum bottles in culture medium consisting of 75% immediately centrifuged, heat-inactivated rat serum and 25% Tyrode's buffer and test compound. Up to 5 embryos were cultured per bottle in 4 mL of medium; embryos from a given litter were randomly distributed among treatments and doses. Culture bottles were gassed using a mixture of 5% O2, 5% CO2, and 90% N2, and sealed with a silicon stopper. Bottles were placed onto a rotating wheel (30 rpm) in a 38°C incubator. For extended culture periods, cultures were re-gassed after 20 to 21 h of culture, using 20% O2, 5% CO2, and 75% N2.
For evaluation of staurosporine-induced dysmorphogenesis, staurosporine was dissolved in DMSO and added to the culture medium at various concentrations ranging from 0.005 to 0.1 µM (N = 6 to 9 embryos per staurosporine concentration). In no instance did the DMSO content in the culture system exceed 20 µM; this concentration of DMSO produces no adverse effects in rat or mouse whole-embryo culture (Kitchin and Ebron, 1984; Ward et al., 1998
). Appropriate control embryos were incubated with identical concentrations of DMSO to the staurosporine-treated embryos (N = 9). At the end of 24 to 26 h of culture, embryos were evaluated for dysmorphogenesis according to previously defined criteria (Sadler and Warner, 1984
), including an evaluation of craniofacial structures, as well as heart and somite morphology. In embryos lacking a detectable heartbeat, the embryo was classified as dead and excluded from morphological evaluations.
Effect of test compounds on cell cycle regulation.
To evaluate whether the test compounds altered the cell cycle in the neurulation-stage embryo, flow cytometry was used to assess the percentage of cells that were in different stages of the cell cycle (i.e., G0/G1, S, or G2/M). Embryos were cultured for 6 (gd 8.25), 12 (gd 8.5), 18 (gd 8.75), or 24 h (gd 9) in the presence or absence of DCA (11000 µM), DBA (300 µM), BCA (300 µM), Bis I (10 µM), or Bis V (10 µM), or for 8 (gd 8.33), 18 (gd 8.75), or 24 h (gd 9) in the presence of staurosporine (0.1 µM). The test agents were assessed independently. Each agent was dissolved in deionized water (with the exception of staurosporine and Bis V, which were dissolved in DMSO) and added to the culture medium to give the desired concentration. Concentrations of each test compound used were selected in order to produce 100% embryotoxicity with no consequent embryolethality, except for Bis V, which was used at the same concentrations as Bis I to facilitate a direct comparison (Hunter et al., 1996; Ward et al., 1998
). At the end of the culture period, embryos were removed from the culture system and the visceral yolk sac was removed.
Embryos were either used whole or were dissected into 4 segments: head (including both pharyngeal arches and the otic anlagen), heart, midpiece (from the otic anlagen to approximately the midpoint of the body), and hindpiece, and appropriate segments from 6 to 12 embryos were pooled for analysis. Cell cycle analysis was conducted using a FACSCalibur flow cytometer (Becton-Dickinson, San Jose, CA) essentially as described by Elstein and Zucker (1997). Briefly, immediately upon collection, embryo segments were placed in 1 mL of phosphate-buffered saline containing 10% DMSO and 10% fetal bovine serum. Samples were cooled at 4°C for approximately 30 min, then stored overnight at 20°C before final storage at 80°C until analysis. On the day of analysis, embryos were thawed at 37°C and all excess buffer was removed. Each sample was triturated with 20 strokes of a fine-tipped plastic pipet in 0.5 mL of lysing buffer (phosphate-buffered saline (pH 7.4) containing 0.2% NP-40 and 0.5 mg/mL RNAse A). Samples were allowed to incubate at room temperature for 30 min, and then were placed on ice, where they were kept for the remainder of analysis. At the end of the incubation period, 25 µL of propidium iodide (1 mg/mL) was added to each 0.5-mL sample; samples were vortexed immediately after addition of stain. Red fluorescence of 10,000 nuclei was detected using 488-nm excitation, a 630-nm Schott barrier filter, and doublet discrimination. The percentage of cells in each phase of the cell cycle was calculated using the ModFit 2.0 LT software package (Becton-Dickinson, San Jose, CA).
Effect of test compounds on embryonic apoptosis.
To evaluate whether the test compounds altered patterns of embryonic cell death, fluorescence microscopy was employed. Embryos (N = 6 to 12 per treatment group) were cultured for 24 h in the presence or absence of DCA, DBA, BCA, Bis I, or Bis V as described for cell cycle analysis above. After 24 h of culture, embryos were removed from the culture system and the visceral yolk sac was removed. For a qualitative determination of apoptosis, the acidophilic dye LysoTracker Red (LT) was employed. LT consists of a fluorophore moiety linked to a weak base that is only partially protonated at neutral pH; it is freely permeant to cell membranes and preferentially accumulates in acidic environments, such as with lysosomes (Haugland, 1996). Because one hallmark of cells undergoing apoptosis is cytoplasmic acidification (Lincz, 1998
; Meisenholder et al., 1996
), and also because cells neighboring regions of apoptosis may display enlargement/activation of lysosomes as they phagocytose apoptotic bodies, LT staining has been used as a marker of apoptosis (Hunter et al., 1997
; Lau et al., 1998
; Zucker et al., 1997
). The LT staining procedure was similar to that described by Zucker and co-workers (1998). Essentially, after removal of the yolk sac, whole embryos were shaken and incubated in 5 µM LT for 30 min at 37°C and then fixed in 2% paraformaldehyde in phosphate buffered saline at 4°C for 12 h. Following fixation, embryos were stored at 4°C in a tightly sealed container until analysis. Embryos were visualized using a Leica fluorescence microscope (Leica MZ12 stereofluorescence microscope, 100-W Hg bulb) with a green fluorescence filter module (546/10 nm excitation filter; dichromatic beam splitter, 565 nm; Barrier filters OG 590 nm) and photographed with a Dage MTI CCD-300T-RC camera. A Scion video capture card was used in a Macintosh 7100 computer. Images were averaged for 16 frames before capture. Adobe Photoshop 5.0.2 was used for creating the figures of embryo images. Figures were printed on a Codonics 1600 printer.
Statistical analysis.
The percentage of cells in each phase of the cell cycle, in embryos exposed at different times to each agent, was compared to time-matched control embryos using either a 1-way analysis of variance (ANOVA) with a Bonferroni correction for multiple comparisons or a 2-tailed Student's t-test, as noted below. The level of statistical significance of differences was predetermined as p 0.05 for all comparisons.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
|
|
|
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
In contrast, however, flow cytometry did reveal substantial accumulation of sub-G1 events in whole-embryo homogenates treated with the HAs. Because of the characteristic intranucleosomal DNA cleavage that occurs during apoptosis, sub-G1 peaks may be used as a flow cytometric marker of apoptosis (Hanon et al., 1996; McCarthy and Evan, 1998
). While DBA was not a potent inducer of sub-G1 events, both BCA and DCA did produce an increased incidence of sub-G1 peaks in whole-embryo homogenates. For BCA, this induction appeared to exclude the heart region of the embryo; this result was confirmed using LysoTracker staining and fluorescence microscopy. Because the predominant morphologic effect of the HAs in whole-embryo culture is induction of neural tube defects, these data are suggestive of a mechanistic linkage between induction of apoptosis and HA embryotoxicity. These data also are consistent with previous investigations demonstrating that the heart of the neurulation-stage mouse embryo is relatively resistant to the induction of apoptosis by various developmental toxicants (Mirkes and Little, 1998
).
Although the effects of DCA were generally similar to those observed with BCA (i.e., induction of apoptosis with no concomitant change in the cell cycle), some differences were observed between DCA and the other test agents. DCA pathogenesis was characterized by an apparent subtle pattern shift in the time course of the cell-cycle data; percentage of cells in S phase rose and fell across time differently after treatment with DCA than with the other toxicants. This effect was even more pronounced in the region-specific data; DCA appeared to induce a shift in the population of cells towards S phase in the heart, and towards G1 in the head region. Furthermore, DCA induced cardiac apoptosis in addition to apoptosis in the head region. Several possibilities exist which may explain these data. First, it is important to note that in general, DCA is a weaker embryotoxicant than many of the other HAs. The benchmark concentration (the calculated lower 95% confidence interval for production of 5% incidence of neural tube defects) for DCA toxicity is about 2500 µM, compared to about 150 µM for DBA (Hunter et al., 1996). Similarly, to obtain a test cohort of embryos in which 100% embryotoxicity was produced, DCA had to be administered at 11 mM, as opposed to 300 µM for both BCA and DBA. This large difference in benchmark concentrations alone is suggestive of a potentially different mechanism of action for DCA. Second, it is possible that decreased intracellular pH resulting from the relatively large concentration of DCA employed may have produced a general effect on the embryo not present with the other test compounds. Although the pH of the embryo culture medium does not decrease significantly with inclusion of 11 mM DCA (Hunter et al., 1996
), the intracellular embryonic pH is unknown, and may have played a role in the effects observed in the present study. Finally, DCA is a well-characterized in vivo developmental cardiotoxicant in the rat (Epstein et al., 1992
; Smith et al., 1992
); the developmental cardiotoxicity of other HAs has not been fully characterized.
It may be that the observed induction of apoptosis in the heart in whole-embryo culture (as determined by the increase in sub-G1 events) is a more sensitive indicator of eventual developmental cardiotoxicity than simple gross morphological examination alone at this developmental stage, and that the observed increase in apoptosis is a precursor to later dysmorphogenesis. Further research is required to more thoroughly characterize the differences between DCA-induced pathogenesis and that induced by other HAs. Interestingly, the observed induction of embryonic apoptosis by DCA contrasts with work by Snyder and co-workers (1995), who demonstrated that DCA (0.5 and 5 g/L in drinking water) significantly decreased hepatocellular apoptosis in vivo. These data indicate that DCA may interact with the whole embryo by a different mechanism than that acting in the liver; alternatively, the lower dosage of DCA administered in vivo may underlie this discrepancy. Further research will be necessary to clarify these findings.
In this investigation, DCA and BCA increased apoptosis with no concomitant alteration in cell cycle pattern. The complex and sometimes contradictory interaction between induction of apoptosis and changes in the cell cycle has been investigated previously (King and Cidlowski, 1998). Some toxicants have been demonstrated to produce both changes in apoptosis and alterations in the cell cycle, while other toxicants may affect either endpoint. For example, zinc deficiency and ethanol appear to induce embryonic apoptosis with no parallel change in the cell cycle (Cartwright et al., 1998
; Rogers et al., 1995
); the hypolipidemic drug nafenopin perturbs the cell cycle but not apoptosis (Elcock et al., 1998
); and methyl tertiary butyl ether and 5-aza-2'-deoxycytidine appear to affect both endpoints simultaneously (Rogers et al., 1994
; Vojdani et al., 1997
). Furthermore, Park and co-workers (1997) have demonstrated that cell-cycle blockers such as deferoxamine and mimosine actually suppress camptothecin-induced apoptosis in vitro. The present data, demonstrating an effect of DCA and BCA on apoptosis with no change in the cell cycle, lend further support to the hypothesis that cell cycle alterations are not necessary precursors to apoptosis. This hypothesis, by extension, would suggest that both apoptosis and the cell cycle warrant investigation as cellular markers of toxicity, since it is not obvious a priori whether a toxicant will induce changes in the cell cycle, apoptosis, both, or neither.
Previously, the embryotoxicity of staurosporine has been characterized in rat, but not mouse, embryos (Fujinaga et al., 1994). In the present investigation in mouse embryos, the predominant dysmorphogenic effects of staurosporine were observed in the pharyngeal arch, eye, and heart. These data differed from the previously described effects of staurosporine in rat embryos. Mesencephalic vesicles, observed in staurosporine-treated rat embryos, were not observed in staurosporine-treated mouse embryos in the present study. Also, the concentration required to attain approximately 100% incidence of embryotoxicity was greater in the rat than in the mouse. However, this apparent difference in sensitivity may be due in part to differences in experimental design. Fujinaga and co-workers (1994) exposed egg cylinder-stage rat conceptuses for 4 h to staurosporine at concentrations
0.1 µM, since exposure for the full 50-h culture period was embryolethal. In the present investigation, developmentally older neurulation-stage mouse embryos were exposed to staurosporine at concentrations
0.1 µM for the entire 24- to 26-h culture period.
From a biochemical standpoint, the staurosporine data from the present investigation are consistent with data from the literature. Staurosporine is well-characterized as an agent that both induces apoptosis and alters the cell cycle distribution in a variety of cell types and under many different conditions (Courage et al., 1996; Qiao et al., 1996
; Stokke et al., 1997
; Swe and Sit, 1997
). The observations in the present study are consistent with these previous findings. In particular, staurosporine is associated with a G1 blockade (Orr et al., 1998
; Wang and Xue, 1997
), a finding also consistent with the increase in the percentage of embryonic cells in G1 in the present investigation.
In contrast to the staurosporine data, the lack of effect of Bis I on the cell cycle or apoptosis in the present study was less anticipated. Effects of Bis I on the modulation of apoptosis and the cell cycle have been demonstrated in a variety of systems. These include G1-phase arrest in C6 glioma cells (O'Brien and Regan, 1998) and prevention of cerebellar granule cell apoptosis (Villalba, 1998
). These data, along with the present findings, suggest that Bis I may interact with embryonic cells in whole-embryo culture differently from those previously studied in in vitro systems, or that some of the in vitro effects mediated by Bis I are compensated for by the developing organism. It has been previously demonstrated that staurosporine, but not Bis I, induces apoptosis in isolated rat thymocytes, indicating that PKC may not be involved in the production of apoptosis in the developing embryo by staurosporine (Harkin et al., 1998
). One further potential mechanism for the apparent lack of effect of Bis I on embryonic apoptosis or the cell cycle could be the PKC isozyme distribution in the neurulation-stage mouse embryo. Previously, it has been reported that atypical (i.e., calcium/phospholipid-independent) PKC isoforms predominate in the mouse embryo at this developmental stage (Ward et al., 1997
). Bis I possesses less affinity for the atypical isoforms of PKC than for the classical isozymes (Gescher, 1998
); this isozyme specificity may underlie the lack of effect of Bis I on these endpoints in the mouse embryo.
In summary, this investigation has generated data indicating that the embryotoxicity of certain HAs may be mediated, in part, by modulation of apoptosis, without concomitant cell cycle perturbations. Furthermore, the dysmorphogenic effects of staurosporine, a broad-spectrum protein kinase inhibitor, were evaluated in the mouse embryo; the staurosporine-treated embryos served as positive controls for induction of apoptosis and changes in the cell cycle distribution. Finally, the effects of Bis I, a specific PKC inhibitor, were investigated; neither alterations in apoptosis nor effects in the cell cycle could be invoked to explain the observed dysmorphogenesis induced by this agent. Future research will be directed towards a more thorough investigation of the role of various signal transduction pathways in the neurulation-stage embryo, with a particular emphasis on elucidating the mechanism(s) by which Bis I may exert its toxic effect. Specific future experiments may include a determination of the contribution of the different isozymes of PKC in the neurulation-stage embryo, as well as evaluation of the role of other important signal transduction mechanisms, such as MAP kinase, in neurulation (Ward et al., 1997). Finally, efforts also will be focused on a direct determination of the ability of the HAs, and perhaps other common environmental toxicants, to inhibit isozymes of PKC and/or other critical kinases involved in embryonic signal transduction.
![]() |
ACKNOWLEDGMENTS |
---|
![]() |
NOTES |
---|
1 Present address: Drug Metabolism and Pharmacokinetics, SmithKline Beecham Pharmaceuticals, 709 Swedeland Road, UW 2720, King of Prussia, PA 19406.
2 To whom correspondence should be addressed at Reproductive Toxicology Division, MD-67, National Health and Environmental Effects Research Laboratory, U.S. Environmental Protection Agency, Research Triangle Park, NC 27711. Fax: (919) 541-4017. E-mail: hunter.sid{at}epamail.epa.gov.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Bruno, S., Ardelt, B., Skierski, J. S., Traganos, F., and Darzynkiewicz, Z. (1992). Different effects of staurosporine, an inhibitor of protein kinases, on the cell cycle and chromatin structures of normal and leukemic lymphocytes. Cancer Res. 52, 470473.[Abstract]
Bull, R. J. (1992). Toxicology of drinking water disinfection. In Environmental Toxicants: Human Exposures and Their Health Effects (M. Lippmann, Ed.), pp. 184230. Van Nostrand Reinhold, New York.
Bull, R. J., Birnbaum, L. S., Cantor, K. P., Rose, J. B., Butterworth, B. E., Pegram, R., and Tuomisto, J. (1995). Water chlorination: Essential process or cancer hazard? Fundam. Appl. Toxicol. 28, 155166.[ISI][Medline]
Cartwright, M. M., Tessmer, L. L., and Smith, S. M. (1998). Ethanol-induced neural crest apoptosis is coincident with their endogenous death, but is mechanistically distinct. Alcohol Clin. Exp. Res. 22, 142149.[ISI][Medline]
Courage, C., Snowden, R., and Gescher, A (1996). Differential effects of stauroporine analogs on cell cycle, growth, and viability in A549 cells. Br. J. Cancer 74, 11991205.[ISI][Medline]
Crabb, D. W., Yount, E. A., and Harris, R. A. (1981). The metabolic effects of dichloroacetate. Metabolism 30, 10241039.[ISI][Medline]
Davis, P. D., Hill, C. H., Lawton, G., Nixon, J. S., Wilkinson, S. E., Hurst, S. A., Keech, E., and Turner, S. E. (1992). Inhibitors of protein kinase C. 1. 2,3-bisarylmaleimides. J. Med. Chem. 35, 177184.[ISI][Medline]
Elcock, F. J., Chipman, J. K., and Roberts, R. A. (1998). The rodent nongenotoxic hepatocarcinogen and peroxisome proliferator nafenopin inhibits intercellular communication in rat but not guinea pig hepatocytes, perturbing S phase but not apoptosis. Arch. Toxicol. 72, 439444.[ISI][Medline]
Elstein, K. H., and Zucker, R. M. (1997). Applications of flow cytometry in developmental and reproductive toxicology. In Molecular and Cellular Methods in Developmental Toxicology (G. P. Daston, Ed.), pp. 195230. CRC Press, Boca Raton.
Elstein, K.H., Zucker, R.M., Andrews, J.E., Ebron-McCoy, M., Shuey, D.L., and Rogers, J.M. (1993b). Effects of developmental stage and tissue type on embryo/fetal DNA distributions and 5-fluorouracil-induced cell-cycle perturbations. Teratology 48, 355363.[ISI][Medline]
Elstein, K. H., Zucker, R. M., Shuey, D. L., Lau, C., Chernoff, N., and Rogers, J. M. (1993a). Utility of the murine erythroleukemic cell (MELC) in assessing mechanisms of action of DNA-active developmental toxicants: Application to 5-fluorouracil. Teratology 48, 7587.[ISI][Medline]
Epstein, D. L., Nolen, G. A., Randall, J. L., Christ, S. A., Read, E. J., Stober, J. A., and Smith, M. K. (1992). Cardiopathic effects of dichloroacetate in the fetal Long-Evans rat. Teratology 46, 225235.[ISI][Medline]
Falcieri, E., Martelli, A. M., Bareggi, R., Cataldi, A., and Cocco, L. (1993). The protein kinase inhibitor staurosporine induces morphological changes typical of apoptosis in MOLT-4 cells without concomitant DNA fragmentation. Biochem. Biophys. Res. Comm. 193, 1925.[ISI][Medline]
Fishman, D. D., Segal, S., and Livneh, E. (1998). The role of protein kinase C in G1 and G2/M phases of the cell cycle. Int. J. Oncol. 12, 181186.[ISI][Medline]
Fujinaga, M., Park, H. W., Shepard, T. H., Mirkes, P. E., and Baden, J. M. (1994). Staurosporine does not prevent adrenergic-induced situs inversus, but causes a unique syndrome of defects in rat embryos grown in culture. Teratology 50, 261274.[ISI][Medline]
Gescher, A. (1998). Analogs of staurosporine: Potential anticancer drugs? Gen. Pharmacol. 31, 721728.[ISI][Medline]
Hanon, E., Vanderplasschen, A., and Pastoret, P.-P. (1996). The use of flow cytometry for concomitant detection of apoptosis and cell cycle analysis. Biochemica 2, 2527.
Harkin, S. T., Cohen, G. M., and Gescher, A. (1998). Modulation of apoptosis in rat thymocytes by analogs of staurosporine: Lack of direct association with inhibition of protein kinase C. Mol. Pharmacol. 54, 663670.
Haugland, R. P. (1996). Handbook of Fluorescent Probes and Research Chemicals, pp. 274275. Molecular Probes, Eugene, OR.
Hunter, E. S., III, Elstein, K. H., and Zucker, R. M. (1997). Studies of the pathogenesis of arsenite-induced dysmorphology. Teratology 55, 43.
Hunter, E. S., III, Rogers, E. H., Schmid, J. E., and Richard, A. (1996). Comparative effects of haloacetic acids in whole-embryo culture. Teratology 54, 5764.[ISI][Medline]
King, K. L., and Cidlowski, J. A. (1998). Cell cycle regulation and apoptosis. Annu. Rev. Physiol. 60, 601617.[ISI][Medline]
Kitchin, K. T., and Ebron, M. T. (1984). Further development of rodent whole-embryo culture: Solvent toxicity and water-insoluble compound delivery systems. Toxicology 30, 4557.[ISI][Medline]
Knudsen, T. B. (1997). Cell death. In Drug Toxicity in Embryonic Development I (R. J. Kavlock and G. P. Daston, Eds.), pp. 211244. Springer-Verlag, New York.
Lau, C., Zucker, R. M., Price, O., Rosen, M. B., Setzer, R. W., Rogers, J. M., and Kavlock, R. J. (1998). Detecting cell death pattern in rat limb buds by confocal laser scanning microscopy: Dose-dependent influences of 5-fluorouracil. Teratology 57, 231.
Leszczynski, D. (1996). The role of protein kinase C in regulation of apoptosis: A brief review of the controversy. Cancer J. 9, 308313.[ISI]
Lincz, L. F. (1998). Deciphering the apoptotic pathway: All roads lead to death. Immunol. Cell Biol. 76, 119.[ISI][Medline]
McCarthy, N. J., and Evan, G. I. (1998). Methods for detecting and quantifying apoptosis. Curr. Top. Dev. Biol. 36, 259278.[ISI][Medline]
Meisenholder, G. W., Martin, S. J., Green, D. R., Nordberg, J., Babior, B. M., and Gottlieb, R. A. (1996). Events in apoptosis: Acidification is downstream of protease activation and BCL-2 protection. J. Biol. Chem. 271, 1626016262.
Mirkes, P. E., and Little, S. A. (1998). Teratogen-induced cell death in postimplantation mouse embryos: Differential tissue sensitivity and hallmarks of apoptosis. Cell Death Differ. 5, 592600.[ISI][Medline]
O'Brien, E., and Regan, C. M. (1998). Protein kinase C inhibitors arrest the C6 glioma cell cycle at a mid-G1 phase restriction point: Implications for the antiproliferative action of valproate. Toxicol. In Vitro 12, 914.[ISI]
Orr, M. S., Reinhold, W., Yu, L., Schreiber-Agus, N., and O'Connor, P. M. (1998). An important role for the retinoblastoma protein in staurosporine-induced G1 arrest in murine embryonic fibroblasts. J. Biol. Chem. 273, 38033807.
Park, D. S., Morris, E. J., Greene, L. A., and Geller, H. M. (1997). G1/S cell cycle blockers and inhibitors of cyclin-dependent kinases suppress camptothecin-induced neuronal apoptosis. J. Neurosci. 17, 12561270.
Peeters, M. C. E., Schutte, B., Lenders, M.-H. J. N, Hekking, J. W. M., Drukker, J., and Van Straaten, H. W. M. (1998). Role of differential cell proliferation in the tail bud in aberrant mouse neurulation. Dev. Dyn. 211, 382389.[ISI][Medline]
Qiao, L., Koutsos, M., Tsai, L.-L., Kozoni, V., Guzman, J., Shiff, S. J., and Rigas, B. (1996). Staurosporine inhibits the proliferation, alters the cell cycle distribution, and induces apoptosis in HT-29 human colon adenocarcinoma cells. Cancer Lett. 107, 8389.[ISI][Medline]
Rajotte, D., Haddad, P., Haman, A., Cragoe, E. J., Jr., and Hoang, T. (1992). Role of protein kinase C and the Na+/H+ antiporter in suppression of apoptosis by granulocyte macrophage colony-simulating factor and interleukin-3. J. Biol. Chem. 267, 99809987.
Richard, A. M., and Hunter, E. S., III (1996). Quantitative structure-activity relationships for the developmental toxicity of haloacetic acids in mammalian whole-embryo culture. Teratology 53, 352360.[ISI][Medline]
Rogers, J. M., Francis, B. M., Sulik, K. K., Alles, A. J., Massaro, E. J., Zucker, R. M., Elstein, K. H., Rosen, M. B., and Chernoff, N. (1994). Cell death and cell cycle perturbation in the developmental toxicity of the demethylating agent, 5-aza-2'deoxycytidine. Teratology 50, 332339.[ISI][Medline]
Rogers, J. M., Taubeneck, M. W., Daston, G. P., Sulik, K. K., Zucker, R. M., Elstein, K. H., Jankowski, M. A., and Keen, C. L. (1995). Zinc deficiency causes apoptosis but not cell cycle alterations in organogenesis-stage rat embryos: Effect of varying duration of deficiency. Teratology 52, 149159.[ISI][Medline]
Sadler, T. W. (1979). Culture of early somite embryos during organogenesis. J. Embryol. Exp. Morphol. 49, 1725.[ISI][Medline]
Sadler, T. W., and Warner, C. W. (1984). Use of whole-embryo culture for evaluating toxicity and teratogenicity. Pharmacol. Rev. 36, 145S150S.[Medline]
Smith, M. K., Randall, J. L., Read, E. J., and Stober, J. A. (1989). Teratogenic activity of trichloroacetic acid in the rat. Teratology 40, 445451.[ISI][Medline]
Smith, M. K., Randall, J. L., Read, E. J., and Stober, J. A. (1992). Developmental toxicity of dichloroacetate in the rat. Teratology 46, 217223.[ISI][Medline]
Smith, M. K., Thrall, B. D., and Bull, R. J. (1997). Dichloroacetate (DCA) modulates insulin signaling. Toxicologist 36, 233.
Snyder, R. D., Pullman, J., Carter, J. H., Carter, H. W., and DeAngelo, A. B. (1995). In vivo administration of dichloroacetic acid suppresses spontaneous apoptosis in murine hepatocytes. Cancer Res. 55, 37023705.[Abstract]
Stokke, T., Smedshammer, L., Jonassen, T. S., Blomhoff, H. K., Skarstad, K., and Steen, H. B. (1997). Uncoupling of the order of the S and M phases: Effects of staurosporine on human cell cycle kinases. Cell Prolif. 30, 197218.[ISI][Medline]
Swe, M., and Sit, K. H. (1997). Staurosporine induces telophase arrest and apoptosis blocking mitosis exit in human Chang liver cells. Biochem. Biophys. Res. Comm. 236, 594598.[ISI][Medline]
Toullec, D., Pianetti, P., Coste, H., Bellevergue, P., Grand-Perret, T., Ajakane, M., Baudet, V., Boissin, P., Boursier, E., Loriolle, F., Duhamel, L, Charon, D., and Kirilovsky, J. (1991). The bisindolylmaleimide GF 109203X is a potent and selective inhibitor of protein kinase C. J. Biol. Chem. 266, 1577115781.
Villalba, M. (1998). Bisindolylmaleimide prevents cerebellar granule cells apoptosis: A possible role for PKC. Neuroreport 9, 17131717.[ISI][Medline]
Vojdani, A., Mordechai, E., and Brautbar, N. (1997). Abnormal apoptosis and cell cycle progression in humans exposed to methyl tertiary-butyl ether and benzene-contaminating water. Hum. Exp. Toxicol. 16, 485494.[ISI][Medline]
Wang, R.-H., and Xue, S.-B. (1997). Staurosporine blocked normal cells at G1/S boundary. Acta Pharm. Sinica 18, 161165.[ISI]
Ward, K. W., Mundy, W. R., Rogers, E. H., and Hunter, E. S., III (1997). Effect of haloacetic acids on embryonic protein kinase C activity during neurulation. Teratology 55, 52.
Ward, K. W., Rogers, E. H., and Hunter, E. S., III (1998). Dysmorphogenic effects of a specific protein kinase C inhibitor during neurulation. Reprod. Tox. 12, 525534.
Yang, J., and Smith, R. A. (1983). The effect of dichloroacetate on the phosphorylation of mitochondrial proteins. Biochem. Biophys. Res. Comm. 111, 10541058.[ISI][Medline]
Zucker, R. M., Hunter, E. S. III, Haas, A. R., and Rogers, J. M. (1997). Confocal laser scanning microscopy of apoptosis in embryogenesis. Teratology 55, 44.
Zucker, R. M., Hunter, E. S., III, and Rogers, J. M. (1998). Confocal laser scanning microscopy of apoptosis in organogenesis-stage mouse embryos. Cytometry 33, 348354.[ISI][Medline]