Paula D. Johnson

Cardiac teratogenesis of halogenated hydrocarbon—Contaminated drinking water☆

OBJECTIVES The purpose of this study was to test the hypothesis that administration of trichloroethylene and dichloroethylene to pregnant rats during organogenesis would produce a significant fetal cardiac teratogenic effect. It was also hypothesized that administration of these compounds only before pregnancy would not be associated with fetal cardiac teratogenesis. BACKGROUND Epidemiologic observations demonstrated an increased number of congenital cardiac defects in children whose mother resided in an area with drinking water contaminated by trichloroethylene and dichloroethylene. A prior provocative intrauterine exposure study in rats established a positive link between these contaminants and an increased number of fetal hearts with congenital cardiac defects. METHODS Sprague-Dawley rats were given pure tap drinking water (control subjects) or water contaminated with high or low dose of trichloroethylene or dichloroethylene (experimental groups) during prepregnancy only, prepregnancy and pregnancy or during pregnancy alone. RESULTS A total of 2,045 fetuses were examined. Trichloroethylene or dichloroethylene delivered exclusively in the period before pregnancy caused no increase in congenital cardiac malformations over the control level. Compared with the control group, rats exposed to these agents both before and during pregnancy, had a significantly greater number of fetuses with cogenital cardiac malformations. Trichloroethylene (high dose only) administered only during pregnancy produced a significant increase in cardiac defects. Other fetal variables, including noncardiac congenital abnormalities, showed no significant difference between control and treated groups. CONCLUSIONS Trichloroethylene and dichloroethylene administered during organogenesis are cardiac, but not general, teratogens. The data indicate that these agents administered in drinking water to pregnant rats caused an increased number of congenital cardiac defects in rat fetuses.

Cardiac teratogenicity of trichloroethylene metabolites

OBJECTIVES The hypothesis of this study was that metabolites of trichloroethylene (TCE), dichloroethylene (DCE) and related compounds were responsible for fetal cardiac teratogenic effects seen when TCE or DCE is consumed by pregnant rats during organogenesis. Identification of teratogenic metabolites would allow more accurate assessment of environmental contaminants and public health risks from contaminated water or possibly municipal water supplies which, when chlorinated, may produce these potentially dangerous chemicals. BACKGROUND Human epidemiologic studies and previous teratogenic studies using chick embryos and fetal rats have shown an increased incidence of congenital cardiac lesions in animals exposed to TCE and DCE. METHODS Metabolites and compounds studied in drinking water exposure included: trichloroacetic acid (TCAA), monochloroacetic acid (MCAA), trichloroethanol (TCEth), carboxy methylcystine (CMC), trichloroacetaldehyde (TCAld), dichloroacetaldehyde (DCAld), and dichlorovinyl cystine (DCVC). Compounds were administered to pregnant rats during fetal heart development. RESULTS Fetuses of rats receiving 2,730 ppm TCAA in drinking water were the only group that demonstrated a significant increase in cardiac defects (10.53%) compared with controls (2.15%) on a per fetus basis (p = 0.0001, Fischers exact test), and a per litter basis (p = 0.0004, Wilcoxon and p = 0.0015, exact permutation tests). Trichloroacetic acid also demonstrated an increased number of implantation and resorption sites (p < 0.05) over controls. Other maternal and fetal variables showed no statistically significant differences between treated and untreated groups. CONCLUSIONS Of the metabolites tested, only TCAA appeared to be a specific cardiac teratogen in the fetus when imbibed by the maternal rat.

Cardiac teratogenesis of trichloroethylene and dichloroethylene in a mammalian model

Recent epidemiologic studies have demonstrated a greater than expected number of pediatric patients with congenital heart disease in areas where drinking water was contaminated by halogenated aliphatic hydrocarbons. Trichloroethylene, trichloroethane and dichlorethylene were the principal contaminants in the groundwater. A previous study of chick embryos demonstrated that when injected into the air sacs of fertilized eggs trichloroethylene produced more than three times the number of cardiac defects that are found in control embryos. This mammalian study demonstrates similar effects of trichloroethylene and dichloroethylene when applied under provocative circumstances (that is, solutions delivered through a catheter into the gravid uterus from an intraperitoneal osmotic pump) to the developing rat fetus in utero during the period of organ differentiation and development. Furthermore, the effect is dose dependent for both agents. Although only a very small number of congenital heart anomalies (3%) were found in the control group, 9% and 12.5% were found in the lower dose trichloroethylene and dichloroethylene groups and 14% and 21% in the higher dose groups, respectively (p less than 0.05). A variety of cardiac defects were found. Dichloroethylene appears to be at least as great a cardiac teratogen as trichloroethylene even though it was administered at a 10-fold lower concentration. These agents appear to be specific cardiac teratogens because only a single noncardiac anomaly was found. This study in a rat model demonstrates a dose-dependent relation between fetal exposure to trichloroethylene and dichloroethylene in utero during the period of organogenesis and the appearance of a variety of congenital cardiac defects.

Trichloroethylene, Trichloroacetic Acid, and Dichloroacetic Acid: Do They Affect Fetal Rat Heart Development?

Trichloroethylene (TCE), trichloroacetic acid (TCA), and dichloroacetic acid (DCA) are commonly found as groundwater contaminants in many regions of the United States. Cardiac birth defects in children have been associated with TCE, and laboratory studies with rodents report an increased incidence of fetal cardiac malformations resulting from maternal exposures to TCE, TCA, and DCA. The objective of this study was to orally treat pregnant CDR(CD) Sprague-Dawley rats with large bolus doses of either TCE (500 mg/kg), TCA (300 mg/kg), or DCA (300 mg/kg) once per day on days 6 through 15 of gestation to determine the effectiveness of these materials to induce cardiac defects in the fetus. All-trans retinoic acid (RA) dissolved in soybean oil was used as a positive control. Soybean oil is commonly used as a dosing vehicle for RA teratology studies and was also used in this study as a dosing vehicle for TCE. Water was used as the dosing vehicle for TCA and DCA. Fetal hearts were examined on gestation day (GD) 21 by an initial in situ, cardiovascular stereomicroscope examination, and then followed by a microscopic dissection and examination of the formalin-fixed heart. The doses selected for TCA and DCA resulted in a modest decrease in maternal weight gain during gestation (3% to 8%). The fetal weights on GD 21 in the TCA and DCA treatment groups were decreased 8% and 9%, respectively, compared to the water control group and 21% in the RA treatment group compared to soybean oil control group. The heart malformation incidence for fetuses from the TCE-, TCA-, and DCA-treated dams did not differ from control values on a per fetus or per litter basis. The rate of heart malformations, on a per fetus basis, ranged from 3% to 5% for TCE, TCA, and DCA treatment groups compared to 6.5% and 2.9% for soybean oil and water control groups. The RA treatment group was significantly higher with 33% of the fetuses displaying heart defects. For TCE, TCA, and DCA treatment groups 42% to 60% of the litters contained at least one fetus with a heart malformation, compared to 52% and 37% of the Utters in the soybean oil and water control groups. For the RA treatment group, 11 of 12 litters contained at least one fetus with a heart malformation. Further research is needed to quantify the spontaneous rates of heart defects for vehicle control rats and to explain the disparity between findings in the present study and other reported findings on the fetal cardiac teratogenicity of TCE, TCA, and DCA.

Trichloroethylene Disrupts Cardiac Gene Expression and Calcium Homeostasis in Rat Myocytes

We have been investigating the molecular mechanisms by which trichloroethylene (TCE) might induce cardiac malformations in the embryonic heart. Previous results indicated that TCE disrupted expression of genes encoding proteins involved in regulation of intracellular Ca2+, [Ca2+](i), in cardiac cells, including ryanodine receptor isoform 2 (Ryr2), and sarcoendoplasmatic reticulum Ca2+ ATPase, Serca2a. These observations are important in light of the notion that altered cardiac contractility can produce morphological defects. The hypothesis tested in this study is that the TCE-induced changes in gene expression of Ca2+-associated proteins resulted in altered Ca2+ flux regulation. We used real-time PCR and digital imaging microscopy to characterize effects of various doses of TCE on gene expression and Ca2+ response to vasopressin (VP) in rat cardiac H9c2 myocytes. We observed a reduction in Serca2a and Ryr2 expression at 12 and 48 h after exposure to TCE. In addition, we found significant differences in Ca2+ response to VP in cells treated with TCE doses as low as 10 parts per billion. Taken all together, our data strongly indicate that exposure to TCE disrupts the ability of myocytes to regulate cellular Ca2+ fluxes. Perturbation of calcium signaling alters cardiac cell physiology and signal transduction and may hint to morphogenetic consequences in the context of heart development. These results point to a novel area of TCE biology and, if confirmed in vivo, may help to explain the apparent cardio-specific toxicity of TCE exposure in the rodent embryo.

Effects of trichloroethylene and its metabolite trichloroacetic acid on the expression of vimentin in the rat H9c2 cell line.

Trichloroethylene (TCE) and its metabolite trichloroacetic acid (TCAA) are environmental contaminants with specific toxicity for the embryonic heart. In an effort to identify the cellular pathways disrupted by TCE and TCAA during heart development, we investigated their effects on expression of vimentin, a marker of cardiac differentiation. Previous studies had shown that the level of vimentin transcript was inhibited in rat embryonic heart after maternal exposure to TCE via drinking water. In the same study, maternal exposure to TCAA produced the opposite effect, inducing an increased level of vimentin mRNA. In this study, we selected an in vitro system, the rat cardiac myoblast cell line H9c2, to further characterize the molecular mechanisms used by TCE and TCAA to disrupt normal heart development. In particular, we investigated the effects of both toxicants on vimentin, at both the RNA and protein levels, using dose–response and time course curves. Our experimental findings indicate that vimentin expression is affected by TCE and TCAA in H9c2 cells similarly as in vivo. The work is significant because it provides a suitable in vitro model for studies looking at toxicant effects on myocardiac cells, and it suggests that vimentin is a good marker of TCE exposure in the embryonic heart.

Administration of melanotropic peptides during gestation in the rodent

A potent analogue of alpha-MSH (alpha-melanocyte stimulating hormone, S-alpha-melanotropin), [Nle4,D-Phe7] alpha-MSH, induces darkening of follicular melanocytes when injected or applied topically to the skin of mice [1]. This analogue also results in increased pigmentation when injected subcutaneously (s.c.) in humans. Toxicological studies have been performed on rodent models with administration topically or by intraperitoneal (i.p.) injection. No toxicity was observed and no pathological or significant biochemical changes were found. However there has been some controversy in the literature revolving around whether or not alpha-MSH is trophic for fetal growth and whether the hormone affects fetal adrenal development. These questions have been addressed in this study. All previous studies on the possible reproductive function of alpha-MSH have involved use of the natural hormone only. This is first to demonstrate the effects of the more potent analogue. The rat was used as the animal model to determine if the potent analogue of alpha-MSH affects events in gestation and embryonic fetal development and to determine if the analogue was a developmental toxicant. This study also examines the effect of a melanotropic peptide delivered directly to the conceptus in utero during organogenesis. No changes were found in the parameters examined (sex ratio, weight, morphology or histology, etc.) between treated and control fetuses. There was no evidence of premature parturition or pigmentation changes in the fetuses. The work reported in this study is of relevance if such a melanotropic peptide is to be used in women of childbearing age to induce pigmentation of the skin. Although the present results cannot necessarily be extrapolated to humans, indications are that, in rodents at least, [Nle4,D-Phe7] alpha-MSH and natural alpha-MSH have no adverse effects when administered during gestation and fetal development.