Trichloroethylene exposure during cardiac valvuloseptal morphogenesis alters cushion formation and cardiac hemodynamics in the avian embryo

Abstract

It is controversial whether trichloroethylene (TCE) is a cardiac teratogen. We exposed chick embryos to 0, 0.4, 8, or 400 ppb TCE/egg during the period of cardiac valvuloseptal morphogenesis (2-3.3 days' incubation) . Embryo survival, valvuloseptal cellularity, and cardiac hemodynamics were evaluated at times thereafter. TCE at 8 and 400 ppb/egg reduced embryo survival to day 6.25 incubation by 40-50%. At day 4.25, increased proliferation and hypercellularity were observed within the atrioventricular and outflow tract primordia after 8 and 400 ppb TCE. Doppler ultrasound revealed that the dorsal aortic and atrioventricular blood flows were reduced by 23% and 30%, respectively, after exposure to 8 ppb TCE. Equimolar trichloroacetic acid (TCA) was more potent than TCE with respect to increasing mortality and causing valvuloseptal hypercellularity. These results independently confirm that TCE disrupts cardiac development of the chick embryo and identifies valvuloseptal development as a period of sensitivity. The hypercellular valvuloseptal profile is consistent with valvuloseptal heart defects associated with TCE exposure. This is the first report that TCA is a cardioteratogen for the chick and the first report that TCE exposure depresses cardiac function. Valvuloseptal hypercellularity may narrow the cardiac orifices, which reduces blood flow through the heart, thereby compromising cardiac output and contributing to increased mortality. The altered valvuloseptal formation and reduced hemodynamics seen here are consistent with such an outcome. Notably, these effects were observed at a TCE exposure (8 ppb) that is only slightly higher than the U.S. Environmental Protection Agency maximum containment level for drinking water (5 ppb) .

Figure 1

TCE treatment protocol. TCE was administered four times during cardiac cushion formation, at stages HH13, HH15, HH17, and HH20. Embryos were assessed at HH24 or HH30.

Figure 2

Effects of TCE on chick embryo survival at (A) HH24 and (B) HH30. Values shown are mean percent survival (± SE) for embryos exposed to TCE during cardiac cushion formation. Each panel represents the mean of five experiments; numbers within bars indicate the total number of embryos. *Significantly different from 0 nmol treatment (p < 0.01); p-values are given above bars.

Figure 3

Effects of TCE on cardiac cushion proliferation and cellularity in HH24 chick embryos. Embryos were exposed to TCE during cushion development. Percentage of BrdU-labeled cushion mesenchyme in (A) OFT cushions and (B) AVC cushions. Total mesenchymal cellularity in the (C) OFT cushions and (D) AVC cushions. Values shown are mean ± SE. Experiments were performed in triplicate; the total number of embryos evaluated was 8 for 0 nmol, 7 for 0.2 nmol, 10 for 4 nmol, and 9 for 200 nmol. *Significantly different from 0 nmol treatment; p-values are given above bars.

Figure 4

Effects of TCE and its metabolites on embryo survival and cushion development. Embryos were treated with PBS or with 4 nmol/egg of TCE, TCA, or TCOH during cushion development. (A) Mean embryo survival ± SE at HH30 (n = 3 experiments; values represent 32 embryos for PBS, 46 for TCE, 41 for TCOH, and 34 for TCA). (B and C) Mean cardiac cushion proliferative index ± SE in OFT (B) and AVC (C), assessed at HH24 using BrdU incorporation. (D and E) Mean cushion cellularity of the OFT (D) and AVC (E). Experiments in B–E were performed in duplicate and represent 7 embryos for PBS, 7 for TCE, 5 for TCOH, and 7 for TCA. *Significantly different from PBS treatment; p-values are given above bars.

Figure 5

Effects of TCE on cardiovascular function at HH24. Embryos were treated with PBS or 4 nmol TCE during cushion development. Heart rate (A) and mean dorsal aortic and atrioventricular (both passive and active components for the atrioventricular) blood flow (B) for PBS- and TCE-exposed embryos; values are mean ± SE for 8 PBS or 11 TCE embryos. (C–F) Representative dorsal aortic (C and D) and atrioventricular (E and F) velocity analog waveforms for a PBS-treated (C and E) and a TCE-treated (D and F) embryo; TCE exposure reduced both the dorsal aortic and active atrioventricular velocities. (G and H) Ratio of passive to active atrioventricular blood flow (G) and stroke volume index (H) for PBS- and TCE-exposed embryos. *Significantly different from PBS treatment; p-values are given above bars.

Figure 6

Possible consequences of TCE exposure to cardiac development and function.