Toxicological Sciences 56, 437-438 (2000)
Copyright © 2000 by the Society of Toxicology
Letters to the Editor |
Halogenated Solvents Industry Alliance 2001 L Street NW, Suite 506A Washington, DC 20036
To the Editor:
Boyer et al. (2000) report effects of trichloroethylene (TCE) on an in vitro chick atrioventricular canal culture as a method for investigating the induction of cardiac defects. Unfortunately the authors have overlooked factors that should always be considered when employing in vitro techniques. One such factor is the physical chemistry of the test agent in the incubation medium; another is the relationship between the in vitro conditions and those found in whole animal experiments or during human exposures.
The solubility of TCE in water is 1.366 g/l, or 1,366 ppm at 25°C (McNeill, 1979
). Solubility will be slightly higher at the temperature of incubation, but the conclusions of the analysis that follows would be unaffected. The highest level used in the study was 250 ppm. Even allowing for slight modifications to solubility arising from components in the incubation medium, the level of saturation used was very high. In terms of thermodynamic activity, a level of 1400 ppm would be the equivalent of bathing the in vitro system in undiluted TCE; 250 ppm is proportionately lower in activity, but nevertheless represents an extraordinarily high dose. Under these conditions, effects would be expected, and it is surprising that the cultures fared as well as they did.
When the question is asked, "How do the conditions in vitro relate to the whole animal or human situation?" it would be possible to employ PBPK modeling to provide a sophisticated analysis for lower concentrations.
However, on simple physical chemical grounds, it would take inhalation of an atmosphere of approximately 180,000 ppm to achieve an internal level of 250 ppm in aqueous body fluids at equilibrium. Even the lowest concentration tested in vitro, 50 ppm, is the equivalent of an intolerably high in vivo dose.
The authors consider that little or no effect would have occurred at TCE concentrations below 50 ppm in the incubation medium. This evidence suggests that it is highly unlikely that TCE would have any influence on cardiac development through the mechanisms investigated at dose levels that could be tolerated by animals in vivo or at levels of exposure experienced by humans.
REFERENCES
Boyer, A. S., Finch, W. T., and Runyan, R. B. (2000). Trichloroethylene inhibits development of embryonic heart valve precursors in vitro. Toxicol. Sci. 53, 109–117.
McNeill, W. C., Jr. (1979). Trichloroethylene. In Kirk-Othmer Encyclopedia of Chemical Technology, 3rd ed. (M. Grayson and P. Eckroth, Eds.), Vol. 5, pp. 745–753. John Wiley & Sons Inc., New York.
Dept. of Cell Biology and Anatomy University of Arizona P.O. Box 245044 1501 N. Campbell Ave.,LSN 462 Tucson, AZ 85724-5044
To the Editor:
Dr. Dugard raises several issues in his letter that can be addressed. First, the concentrations of TCE we used in our study were in the low millimolar range and are comparable to levels used by other investigators studying solvents and anesthetics in vitro. They are also comparable to doses utilized in previous in vivo studies of TCE. Our manuscript described the empirical addition of TCE to an in vitro collagen gel culture system where we tested an initial dose of 250 ppm and then decreased levels down to 50 ppm. At this lowest dose reported, the measured effect was still significant but produced only a modest loss of mesenchymal cells. In considering the physical chemistry of the organic solvent, it is unlikely that the actual concentration remained at the added level. Our dose-response data reported the concentration delivered to the culture, not the final effective concentration. In the culture dishes we used, TCE would be expected to partition into the head space above the medium and diffuse into the incubator atmosphere. Further, it would also potentially adhere to the plastic of the culture dish and bind to the type I collagen gel. All of these factors would lower the actual concentration. Thus, TCE exposure in the explant cultures varied unavoidably over time and were likely only at the levels indicated after initial addition and at the time when the medium was replaced at 24 h. In terms of the viability of the cultures under the test conditions, we point to the fact that the myocardial portion of the explant was beating vigorously throughout the test culture period, mesenchymal cell expression of type I collagen was unperturbed, and that cell migration was indistinguishable from untreated controls.
While in vitro models can be interpreted on many levels, the suggested extrapolation of our data to an unusually large human airway exposure and specific blood levels both seem inappropriate. There have been reports to suggest a relationship between exposure to TCE and an increased incidence of heart defects. These data are reportedly linked to environmental levels of water exposure, not to large and unusual exposures to volatile TCE, nor is it clear that human blood levels of TCE would be an appropriate comparison to that found in our culture medium. In circulating blood, the major portion of TCE would be sequestered or metabolized in various tissues. Fisher and colleagues (1998) found considerably higher levels of TCE in liver (6-fold) and fat tissue (64-fold) than seen in blood. We know of no specific data for the heart, but a level of 50 ppm in the blood would likely reflect a considerably greater exposure for cardiac tissue. Our culture conditions provided a finite amount of TCE and limited cell sources for TCE metabolism.
Extrapolation of in vitro sensitivity levels towards TCE by avian heart cells to human embryonic exposures is beyond our intent. Several investigators feel that there are more proximate teratogens produced by TCE such as trichloroacetic acid. If true, the relative metabolic activity of avian cardiac endothelial and myocardial cells may be considerably different than human maternal, placental, or embryonic tissues. The exposure levels utilized in our cultures are specific to both the tissue culture conditions and the source of cells. While the data suggest that TCE exposures are of concern, one cannot determine from these studies whether specific levels of exposure would be teratogenic in other animal or human populations or even in chick embryos in vivo. Rather, the point of our contribution is that if TCE is a teratogen, it makes sense to explore the effects of TCE on molecular and cellular events critical to normal heart development. Our data show that moderate levels of TCE produce specific effects on a subset of molecules and cells in embryonic chick heart tissue. Further studies will be needed to determine whether these are direct or indirect effects and to identify sensitivities in various species.
REFERENCE
Fisher, J. W., Mahle, D., and Abbas, R. (1998). A human physiologically based pharmacokinetic model for trichloroethylene and its metabolites, trichloroacetic acid and free trichloroethanol. Toxicol. Appl. Pharmacol. 152, 339–359.[Web of Science][Medline]
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