Judith B. Ulreich

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 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.

Increased fibronectin production by cell lines from hypertrophic scar and keloid.

Primary cell lines of fibroblasts from 8 tissues were established--three from hypertrophic scars (HS), one keloid (K) and four from the normal uninvolved dermis adjacent to each lesion. The objective was to quantify and compare all eight cell lines on the basis of fibronectin (FN) produced per cell and per total protein (PR). Two hypertrophic scars and their adjacent skin cell lines were evaluated by the ELISA method for FN and a micro Lowry assay for PR. The scar lines showed statistically significant increases in the amount of FN/cell compared to the cell lines from their adjacent normal dermis. The third hypertrophic scar and the keloid with their adjacent skin cell lines were assayed for FN and PR by radioimmunoprecipitation. Subconfluent cells were metabolically labeled with 35S-methionine for 20 hours. Harvested media and cell monolayers were assayed for radioactivity incorporated into FN and PR. The percentage of FN/PR was significantly higher in media for HS and K compared to the adjacent normal skin lines in the three passages tested. These results support our previous immunofluorescence studies and demonstrate that a fibroblast-type cell line from a hypertrophic scar or keloid produces more FN/PR over time than the normal fibroblast-type cell line from adjacent uninvolved dermis.

A microassay for lysyl oxidase activity

Abstract Pinnell and Martins [(1968) Proc. Nat. Acad. Sci. USA61, 708–716] standard assay for lysyl oxidase is modified to determine activity in small samples. Data collected by both methods are comparable, and the microassay has the advantages of being economical and rapid.

Tissue factors influence fibroblast activity in neuromuscular diseases

Abstract Connective tissue proliferation is common in a number of neuromuscular disorders. We tested the hypothesis that one or more factors which may affect fibroblast activity are present in muscle biopsy extracts from patients with neuromuscular disorders characterized by variable degrees of fibrosis of the affected muscles. Thirty-two muscle biopsies from patients and normal controls were examined. Prolyl hydroxylase activity was stimulated in fibroblasts exposed in vitro to dystrophic muscle biopsy extracts compared with controls. Thymidine incorporation into DNA was inhibited. As prolyl hydroxylase activity is indicative of the capacity of fibrogenic cells to manufacture competent structural macromolecules, we conclude that the muscle biopsies of dystrophic patients contain one or more factors which promote the production of structural macromolecules. Because of the nature of the biopsied material, these factors may arise from muscle cells, fibroblasts, connective tissue, or other tissue components.

Altered activity in cultured cells caused by contaminants in tubes widely used for blood collection and serum preparation

SummaryCell culture has been recognized as an extremely sensitive system for measuring the toxicity of various materials. A study was done to determine whether the type of tube used to collect blood or store human serum might affect results in experiments requiring blood drawn into such tubes. In order to test tubes for contaminants that might alter cellular activity, a variety of commercially available tubes used for collection of blood and storage of serum were shaken while containing culture medium with fetal bovine serum. The medium was then applied to 3T3 fibroblasts in culture. Measuring incorporation of tritiated thymidine into DNA in log phase cells as an index of cellular proliferation, it was found that medium containing serum preincubated in tubes routinely used for blood collection could be extremely toxic. The same types of tube were also used to prepare human serum. When serum from some of the tubes was applied to 3T3 fibroblasts, a stimulatory effect was observed, perhaps caused by selective adsorption of inhibitory components of the blood or serum by various tubes. It is, therefore, crucial in a properly controlled experiment using serum in vitro to collect blood in tubes that exert no toxic or stimulatory effects in the assay or, at least, to be consistent in one’s choice of tube. None of the tubes used for storage of serum showed significant effects in our assay.

Directionally Guided Angiogenesis Within Extruded Type I Collagen Filaments In Vivo

Abstract Two types of crosslinked extruded type I collagen filaments were developed and characterized for implantation into canine knee joints to achieve anterior cruciate ligament (ACL) regeneration. Type A filaments were crosslinked with glutaraldehyde. Type B filaments were crosslinked with glyceraldehyde and treated with heparin and basic fibroblast growth factor (bFGF). Microscopic evaluation of the filaments revealed a gradient in the degree of crosslinking, with the surface of the filaments more highly crosslinked than the interior. The heterogeneous nature of the crosslinking was demonstrated by complementary methods using bright-field and polarized light microscopy imaging of differentially stained histological sections. In vivo, in the canine model of ACL replacement, the interior of the Type A filaments was degraded or resorbed well before the surface, thus creating collagen tubes through which new capillary channels developed. This phenomenon was not observed for the Type B collagen filaments that were degraded or resorbed from the periphery of each filament. The in vivo behavior of individual collagen filament types is correlated with the fabrication methods. The results suggest a novel method with which to achieve directionally guided angiogenesis (The J Histotechnol 29:267, 2006). Submitted October 1, 2006; accepted with revisions November 2, 2006

Studies on Nonoxynol-9. III. Effect on Fibroblasts and Spermatozoa**Supported in part by Subcontract PARFR P50 from the Program for Applied Research on Fertility Regulation, Northwestern University, under Contract AID/DSPE-C-0035.

Seven nonionic detergents of the Igepal CO series differing in molecular size and including nonoxynol-9 and a representative anionic (SDS) and cationic (Cepacol) detergent were tested as to their relative cytotoxicity. The biologic effects of these detergents on human W1--38 fibroblasts (DNA and glycosaminoglycans synthesis) and on the motility of human spermatozoa were studied. The relative order of cytotoxicities for both fibroblasts and spermatozoa was cationic greater than nonionic greater than anionic. The concentration of nonoxynol-9-inhibiting fibroblast activity was approximately 30 times less than the amount needed to immobilize spermatozoa. It is concluded that CO-630, used as a source of nonoxynol-9, is the most effective polymer to inhibit spermatozoa and also fibroblasts.