Margaret E. Fransen

Identification of developmentally toxic drinking water disinfection byproducts and evaluation of data relevant to mode of action

Reactions between chemicals used to disinfect drinking water and compounds present in source waters produce chemical mixtures containing hundreds of disinfection byproducts (DBPs). Although the results have been somewhat inconsistent, some epidemiological studies suggest associations may exist between DBP exposures and adverse developmental outcomes. The potencies of individual DBPs in rodent and rabbit developmental bioassays suggest that no individual DBP can account for the relative risk estimates reported in the positive epidemiologic studies, leading to the hypothesis that these outcomes could result from the toxicity of DBP mixtures. As a first step in a mixtures risk assessment for DBP developmental effects, this paper identifies developmentally toxic DBPs and examines data relevant to the mode of action (MOA) for DBP developmental toxicity. We identified 24 developmentally toxic DBPs and four adverse developmental outcomes associated with human DBP exposures: spontaneous abortion, cardiovascular defects, neural tube defects, and low birth weight infancy. A plausible MOA, involving hormonal disruption of pregnancy, is delineated for spontaneous abortion, which some epidemiologic studies associate with total trihalomethane and bromodichloromethane exposures. The DBP data for the other three outcomes were inadequate to define key MOA steps.

Activin A and transforming growth factor-beta stimulate heart formation in axolotls but not rescue cardiac lethal mutants.

In the Mexican axolotl (salamander), Ambystoma mexicanum, a recessive cardiac lethal mutation causes an incomplete differentiation of the myocardium. Mutant hearts lack organized sarcomeric myofibrils and do not contract throughout their lengths. We have previously shown that RNA purified from normal anterior endoderm or from juvenile heart tissue is able to rescue mutant embryonic hearts in an in vitro organ culture system. Under these conditions as many as 55% of formerly quiescent mutant hearts initiate regular contractions within 48 hours. After earlier reports that transforming growth factor-β1 and, to a lesser extent, platelet-derived growth factor-BB could substitute for anterior endoderm as a promoter of cardiac mesodermal differentiation in normal axolotl embryos, we decided to examine the effect of growth factors in the cardiac mutant axolotl system. In one type of experiment, stage 35 mutant hearts were incubated in activin A, transforming growth factors-β1 or β2, platelet-derived growth factor, or epidermal growth factor, but no rescue of mutant hearts was achieved. Considering the possibility that growth factors would only be effective at earlier stages of development, we tested transforming growth factors-β1 and β5, and activin A on normal and mutant precardiac mesoderm explanted in the absence of endoderm at neurula stage 14. We found that, although these growth factors stimulated heart tube formation in both normal and mutant mesodermal explants, only normal explants contained contractile myocardial tissue. We hypothesize that transforming growth factor-β superfamily peptides initiate a cascade of responses in mesoderm that result in both changes in cell shape (the basis for heart morphogenesis) and terminal myocardial cytodifferentiation. The cardiac lethal mutation appears to be deficient only in the latter process.

Differential expression of C-protein isoforms in the developing heart of normal and cardiac lethal mutant axolotls (Ambystoma mexicanum).

Regulated assembly of contractile proteins into sarcomeric structures, such as A‐ and I‐bands, is still currently being defined. The presence of distinct isoforms of several muscle proteins suggests a possible mechanism by which myocytes regulate assembly during myofibrillo‐genesis. Of several muscle isoforms located within the A‐band, myosin binding proteins (MyBP) are reported to be involved in the regulation and stabilization of thick filaments during sarcomere assembly. The present confocal study characterizes the expression of one of these myosin binding proteins, C‐protein (MyBP‐C) in wild‐type and cardiac lethal mutant embryos of the axolotl, Ambystoma mexicanum. C‐protein isoforms are also detected in distinct temporal patterns in whole‐mounted heart tubes and thoracic skeletal muscles. Confocal analysis of axolotl embryos shows both cardiac and skeletal muscles to regulate the expression of C‐protein isoforms over a specific developmental window. Although the CPROAxslow isoform is present during the initial heartbeat stage, its expression is not retained in the adult heart. C‐protein isoforms are simultaneously expressed in both cardiac and skeletal muscle during embryogenesis.

Immunohistochemical analysis of C-protein isoforms in cardiac and skeletal muscle of the axolotl, Ambystoma mexicanum.

Of the several proteins located within sarcomeric A-bands, C-protein, a myosin binding protein (MyBP) is thought to regulate and stabilize thick filaments during assembly. This paper reports the characterization of C-protein isoforms in juvenile and adult axolotls, Ambystoma mexicanum, by means of immunofluorescent microscopy and Western blot analyses. C-protein and myosin are found specifically within the A-bands, whereas tropomyosin and α-actin are detected in the I-bands of axolotl myofibrils. The MF1 antibody prepared against the fast skeletal muscle isoform of chicken C-protein specifically recognizes a cardiac isoform (Axcard1) in juvenile and adult axolotls but does not label axolotl skeletal muscle. The ALD66 antibody, which reacts with the C-protein slow isoform in chicken, localizes only in skeletal muscle of the axolotl. This slow axolotl isoform (Axslow) displays a heterogeneous distribution in fibers of dorsalis trunci skeletal muscle. The C315 antibody against the chicken C-protein cardiac isoform identifies a second axolotl cardiac isoform (Axcard2), which is present also in axolotl skeletal muscle. No C-protein was detected in smooth muscle of the juvenile and adult axolotl with these antibodies.

Induction of Myofibrillogenesis in Cardiac Mutant Axolotls by RNA from Normal Embryonic Endoderma

Recessive mutant gene c, for “cardiac nonfunction” in the axolotl results in an absence of heart function.’ Skeletal muscle does not appear to be defective. Morphological studies comparing normal and mutant heart development from stage 34 (heart beat stage) through 41 (when mutant embryos die) have been reported? Electron microscopy reveals that normal ventricular heart myocytes contain organized sarcomeric myofibrils at stage 34-35. By stage 41, the normal ventricular myocardium shows trabeculae formation and contains well-differentiated muscle cells. The mutant myocardium does not trabeculate and remains a single cell layer in thickness. Mutant heart ventricular cells contain a few scattered thin (6 nm) and thick ( 15 nm) filaments and occasional Z bodies. Some mutant cells show a partial organization of myofilaments; however, distinct sarcomeric myofibrils are not observed. Mutant cells, instead, show amorphous proteinaceous collections in their peripheral cytoplasm where myofibrils initially organize in normal cells. Humphrey ’ performed heart transplant experiments and showed that mutant hearts transplanted into the heart regions of normal embryos began to beat. In reciprocal transplants, normal into mutant, the normal organs failed to beat. These experiments suggested that gene c might exert its effect by way of abnormal induction or inhibitory processes in the heart region of mutant embryos. It is well-established that anterior endoderm in amphibians is an important heart inductor tissue?-’ Inasmuch as Humphrey’s ’ transplantation experiments were suggestive of abnormal inductive processes in cardiac mutant embryos, we performed experiments to determine whether the cardiac defect could be corrected by culturing mutant (c/c) hearts with normal ( + / + ) anterior endoderm or by medium conditioned by the preculture of normal