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.

Studies on nonoxynol-9. III. Effect on fibroblasts and spermatozoa.

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.

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

Cell 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.

A quantitative microassay for in-vitro toxicity testing of biomaterials.

A quantitative tissue culture method is described for toxicity testing of biomaterials. The material to be evaluated, or in the case of insoluble materials--an extract of the material, is incubated on a monolayer of fibroblasts in 24 well culture plates. The activity of the fibroblasts is ascertained by measuring the rate of mitosis (3H-thymidine incorporation), the synthesis of glycosaminoglycans (35SO4= incorporation) or production of total protein (3H-tryptophan incorporation). By proper choice of label, the effect of the biomaterial on cellular proliferation or production of macromolecules or both may be assessed. Materials with a cytotoxic effect cause inhibition of incorporation of radioactive label in the above assays. The cell monolayers can also be inspected microscopically for evidence of cytotoxicity. The method is quantitative, simple, reproducible, rapid, and suitable for screening of a large number of samples.

Complementation of predigested proteins: a nutritionally and economically advantageous method of by-products utilization.

Proteins were isolated from four animal by-products: bovine skin trimmings, sheep wool, whole bovine blood and inedible chicken carcass. These waste protein sources were digested and solubilized with nonspecific bacterial proteases. Commercial casein and isolated soybean protein were treated similarly. Partially digested proteins were characterized by some physical measurements, lipid content and complete amino acid composition. The protein quality of individual proteins as well as their combinations was tested on the growth of Tetrahymena pyriformis. We found that the nutritional value of high quality proteins (casein, soy) could be significantly enhanced by mixing with low nutritional value protein(s). At the same time, the cost of the protein mixture was substantially reduced. We suggest that complementation of proteins isolated from animal by-products is a nutritionally and economically advantageous method.

Pulmonary bioactivation of 1,1-dichloroethylene is associated with CYP2E1 levels in A/J, CD-1, and C57BL/6 mice.

1,1-Dichloroethylene (DCE) elicits lung cytotoxicity and selectively targets Clara cells of bronchioles. The toxic effects are ascribed to CYP2E1-mediated formation of reactive intermediates including the DCE epoxide. Here we tested the hypothesis that differential CYP2E1 levels in the lungs of A/J, CD-1, and C57BL/6 mice lead to differences in the extents of DCE bioactivation and lung damage. Our results showed that lung CYP2E1 levels differed significantly in the three murine strains, and followed the rank order A/J > CD-1 > C57BL/6. Covalent binding of [(14)C]DCE to lung proteins in A/J mice was significantly higher than in either CD-1 or C57BL/6 mice. HPLC analysis of lung cytosol from DCE-treated mice showed that 2-S-glutathionyl acetate, a glutathione (GSH) conjugate derived from the epoxide (conjugate [C]), was the major metabolite formed. Levels of [C] detected in cytosol from A/J and CD-1 mice were significantly higher than in C57BL/6 mice. Immunohistochemical staining for [C] was pronounced in the lungs of A/J mice, was lower in CD-1 mice, and was lowest in C57BL/6 mice. Levels of GSH were similar in the lungs of all untreated mice. However, significant reduction in GSH was found in DCE-treated mice, with decreases comparable in all three strains. Bronchiolar Clara cell damage was more severe in A/J and CD-1 mice than in C57BL/6 mice. These results showed differences in CYP2E1 levels in the lungs of A/J, CD-1, and C57BL/6 mice that correlated with the extent to which the DCE epoxide is formed as well as with the severity of lung cytotoxicity.

Protection from 1,1-dichloroethylene-induced Clara cell injury by diallyl sulfone, a derivative of garlic.

Bronchiolar Clara cell damage ensues after treatment of mice with 1,1-dichloroethylene (DCE). The cytotoxicity is mediated by CYP2E1, a cytochrome P450 isozyme that is highly localized in the Clara cells. Bioactivation of DCE produces the primary metabolites 2,2-dichloroacetaldehyde, which hydrolyzes to the acetal, and DCE epoxide, which reacts with glutathione to form the conjugates 2-(S-glutathionyl) acetyl glutathione [B] and 2-S-glutathionyl acetate [C]. In this study, we investigated the potential of diallyl sulfone (DASO2) to inhibit CYP2E1, to suppress the bioactivation of DCE to reactive intermediates and to abrogate DCE-induced Clara cell cytotoxicity. Our results showed that treatment of mice with DASO2 (100 mg/kg p.o.) produced decreases in CYP2E1-dependent p-nitrophenol hydroxylation that were apparent at 1 h. Enzyme activity plummeted to about 20% of the control by 2 h and remained at this low level from 3 to 8 h. Recovery of activity was evident at 16 h and returned to the control level by 24 h. Immunoreactivity of the CYP2E1 protein was decreased in immunoblots of lung microsomes from DASO2-treated mice. Treatment with DASO2 did not cause any structural alterations in lung tissue; in contrast, treatment with DCE (75 mg/kg) produced Clara cell damage. This lesion was not manifested in mice treated with DASO2 in conjunction with DCE. The lack of cytotoxicity observed in vivo correlated with a reduction of about 45% in the levels of both the acetal and the DCE epoxide-derived conjugates [B] and [C] in vitro. These results demonstrated that DASO2 significantly inhibited the CYP2E1 enzyme, decreased the production of DCE metabolites and protected Clara cells from DCE-induced cytotoxicity.

Cardiac teratogenicity of dichloroethylene in a chick model.

Trichloroethylene (TCE) and dichloroethylene (DCE) are related halogenated aliphatic hydrocarbon industrial solvents that are frequently found as drinking water contaminants. TCE has been implicated as a cardiac teratogen in an epidemiologic study and in a chick model. The purpose of this study was to determine whether DCE was also a cardiac teratogen in the chick embryo. Fertilized White Leghorn chick eggs (n = 418) were inoculated just above the embryo with 30 microL of a test solution on d 3 of incubation. Two control groups were studied: normal saline (n = 96) and the diluent for the DCE, mineral oil (n = 108). DCE was studied at three doses: 5, 20, and 25 microM (n = 76, 62, and 76, respectively). Eggs were coded with a seven-digit number to mask identity. Chicks were terminated on d 18 of incubation, and, after external inspection, hearts and great vessels were dissected macroscopically according to a detailed protocol. Abnormal hearts were reviewed and the diagnosis was agreed upon by three investigators before decoding the seven-digit number and photographing the abnormality. Some embryo death and subsequent tissue autolysis occurred in all groups, but, compared to controls, it was not significantly greater in the treatment group. However, combining all controls and all experimentals, significantly more (p = 0.02) embryonic death occurred in the experimental group. Noncardiac anomalies occurred in 17 embryos and were highest in the saline (four), 5 microM (four), and 20 microM (seven) DCE groups.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

Differential formation of 1,1-dichloroethylene-metabolites in the lungs of adult and weanling male and female mice: correlation with severities of bronchiolar cytotoxicity.

The bronchiolar Clara cell cytotoxicant, 1,1-dichloroethylene (DCE), is selectively metabolized by CYP2E1 to metabolites including 2,2-dichloroacetaldehyde and DCE-epoxide. We have performed comparative studies in the lungs of adult and weanling male and female mice to determine their relative capacities to metabolize DCE. Levels of activities of p-nitrophenol hydroxylase, N-nitrosodimethylamine demethylase and NADPH-cytochrome P450 reductase were all significantly higher in adult female mice than in either adult male or weanling mice of both sexes. The quantities of 2,2-dichloroacetaldehyde (identified as its hydrolysis product, acetal) and the DCE-epoxide (identified as the GSH conjugates, 2-(S-glutathionyl) acetyl glutathione [B] and 2-S-glutathionyl acetate [C]) formed were significantly higher in lung microsomes from adult female mice than in those from either adult male or weanling mice of both sexes. Also, the metabolite levels formed in weanling mice were significantly higher than in adult male mice. The amounts of DCE-metabolites produced correlated with the relative severities of DCE-induced bronchiolar damage. The severities of bronchiolar injury were in the rank order adult female > weanling male and female > adult male mice, and coincided with the rank order of DCE-epoxide formation in these experimental groups of mice. In comparison with adult male and weanling male and female mice, adult female mice expressed highest levels of activities of CYP2E1-selective and reductase enzymes, formed most of the DCE-epoxide and were most susceptible to DCE-induced pneumotoxicity. These findings demonstrated sex-related differences in expression of activating enzymes and DCE metabolism in lung, and only in the adult female vs. female weanling mice were there age-related effects in regard to formation of both DCE-metabolites and cytotoxicity.

CYP2E1-dependent bioactivation of 1,1-dichloroethylene in murine lung: formation of reactive intermediates and glutathione conjugates.

We investigated the cytochrome P450-dependent metabolism of 1,1-dichloroethylene (DCE) in murine lung microsomal incubations. The metabolites were identified as their glutathione conjugates or hydrolyzed products, analyzed by HPLC and quantified with [14C]DCE. We determined the relative quantities of DICE metabolites formed in lung microsomal incubations and compared them to those produced in liver. Furthermore, we used antibody inhibition experiments to investigate the CYP2E1-dependent metabolism of DCE in lung. Our results demonstrated that reactive intermediates were generated from DCE in the lung microsomal incubations. The DCE epoxide (12.6 +/- 1.4 pmol/mg protein/min) was the major metabolite formed and was identified as two glutathione conjugates, 2-(S-glutathionyl) acetyl glutathione and 2-S-glutathionyl acetate. Lower levels of the acetal of 2,2-dichloroacetaldehyde (3.6 +/- 0.25 pmol/mg protein/min) were detected. The ratio of acetal to DCE epoxide was higher in lung (0.30 +/- 0.04) than in liver (0.12 +/- 0.02). Preincubation of microsomes with a CYP2E1-inhibitory monoclonal antibody resulted in a maximum inhibition of 50% in the formation of both the acetal and the glutathione conjugates derived from the DCE epoxide. These data demonstrated that lung CYP2E1 metabolizes DCE to reactive intermediates of which the DCE epoxide is both the major metabolite formed and an efficient scavenger of glutathione, implicating it as an important toxic species mediating DCE-induced lung cytotoxicity.

Reaction of vaginal tissue of rabbits to inserted sponges made of various materials.

Sponges of the same size made of collagen (CS), polyurethane (PU), polyvinylalcohol (PVA) and acetylcellulose (AC) were inserted for 10 days in the vaginas of 22 rabbits. Light and scanning electron microscopy of the vaginal wall and its mucosal lining showed signs of cytotoxicity only with PU and AC while CS and PVA picture did not differ from sham controls. In order to explain the reasons for the toxic effects, all sponges were extracted into aqueous or organic solvent media and analyzed by gas liquid chromatography. Only several minute peaks in organic solvents were found. Extracts of all sponges tested for cytotoxicity in fibroblast cultures showed significant inhibition of H3-thymidine uptake. Nevertheless, extract of collagen sponge was significantly less cytotoxic than the extracts of all other sponges.

Cytochrome P-450-dependent bioactivation of 1,1-dichloroethylene to a reactive epoxide in human lung and liver microsomes.

We investigated the cytochrome P-450-dependent metabolism of 1, 1-dichloroethylene (DCE) by human lung and liver microsomes and compared the results from analogous experiments in mice. Metabolites were identified by HPLC analysis of their glutathione conjugates and/or hydrolyzed products and were detected by using [14C]DCE. The role of human CYP2E1 in the metabolic reactions was examined by comparing p-nitrophenol hydroxylase activities with levels of metabolites formed and by using the CYP2E1-selective inhibitor diallyl sulfone. The major products formed in microsomal incubations containing NADPH were the DCE-epoxide-derived glutathione conjugates 2-(S-glutathionyl)acetyl glutathione and 2-S-glutathionyl acetate. Lower levels of the acetal of 2,2-dichloroacetaldehyde were also detected. In lung samples from eight patients, the amounts of epoxide-derived conjugates formed ranged from 15.6 +/- 4.23 to 34.9 +/- 12.75 pmol/mg protein/min. The levels in murine lung were higher at 40.0 +/- 3.8 pmol/mg protein/min. In liver samples from five patients, conjugate levels ranged from 46.5 +/- 8.3 to 240.0 +/- 10. 5 pmol/mg protein/min, whereas levels in murine liver were 83.0 +/- 6.2 pmol/mg protein/min. Conjugate levels formed in human liver correlated with the relative levels of p-nitrophenol hydroxylase activity present, but this relationship was equivocal in human lung. Diallyl sulfone inhibited the formation of the glutathione conjugates (20-65%) in liver samples from all four patients, whereas only one of five human lung samples exhibited this inhibition (27%). These results demonstrated that the DCE-epoxide is a major metabolite formed by human microsomes and is mediated by CYP2E1 in liver and in some individuals in lung.