Paula D Johnson

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• Runyan, R. B., Thorne, P. A., Selmin, O. I., Runyan, R. B., Johnson, P. D., Caldwell, P. T., & Boitano, S. (2008).

  Trichloroethylene disrupts cardiac gene expression and calcium homeostasis in rat myocytes.

  . Toxicological sciences : an official journal of the Society of Toxicology, 104(1), 135-43. doi:10.1093/toxsci/kfn078
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  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.
• Runyan, R. B., Thorne, P. A., Selmin, O. I., Runyan, R. B., Johnson, P. D., & Caldwell, P. T. (2005).

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

  . Cell biology and toxicology, 21(2), 83-95. doi:10.1007/s10565-005-0124-3
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  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.
• Thorne, P. A., Selmin, O. I., Romagnolo, D. F., Johnson, P. D., & Blachere, F. M. (2005).

  Transcriptional activation of the membrane-bound progesterone receptor (mPR) by dioxin, in endocrine-responsive tissues.

  . Molecular reproduction and development, 70(2), 166-74. doi:10.1002/mrd.20090
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  We originally identified the membrane-bound progesterone receptor (mPR) using a screening for genes differentially expressed in liver of rats exposed to dioxin. Recent findings have suggested a role for the mPR in sperm cells, ovary, and brain; however, its mechanisms of action are largely unknown. In this study, we examined the expression pattern of the mPR in liver of rats exposed to dioxin and identified possible mechanisms of its regulation. We observed that mPR expression was induced by dioxin, but was also dependent on the hormonal responsiveness of the tissue. In particular, in male, but not female liver, dioxin induced the expression of the mPR. However, in control, untreated female liver the level of mPR transcript was higher than in control males. Moreover, in breast cancer cells MCF-7 dioxin induced mPR expression. Promoter studies using the luciferase assay indicated that a fragment of approximately 350 bp of the mPR promoter was able to induce luciferase activity in the presence of dioxin, suggesting that the presumptive XREs sites contained in this mPR promoter region are responsive to dioxin. Analysis of mPR protein level confirmed the results observed at the RNA level, both in rat liver and MCF-7 cells. Taken together, these observations suggest the existence of a novel cross-talk between steroid and aromatic hydrocarbon receptors (AhR), and underline the importance of the mPR as a mediator of physiologic effects of the sex hormones.
• Mays, M. Z., Johnson, P. D., Goldberg, S. J., & Dawson, B. V. (2004).

  Trichloroethylene: Johnson et al.’s Response

  . Environmental Health Perspectives, 112(11). doi:10.1289/ehp.112-1247490
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  We share Hardin et al.’s belief that any apparent conflict of interest should be reported. We note that Brent provided testimony for the defense in TCE litigation, notably for the same case in which Goldberg (based on his extensive epidemiologic and laboratory research on the effects of TCE) acted as an expert witness for the plaintiff. We did not report Goldberg’s experience acting as an expert witness because the point of expert witness is to provide unbiased, factual explanations of extant data. We believe this does not constitute a conflict of interest; we have included a caveat about extrapolating data to humans in our publications. To our knowledge none of our data have been used inappropriately. The work published in 1993 (Dawson et al.) and in 2003 (Johnson et al.) was actually performed during a much shorter period of time. Many extraneous factors contributed to the late publication of the 2003 paper. Data from our previous work was included in the more recent paper because we needed “boundary values” between or below which we were looking for a threshold or a critical level. This was a long-term study, and it would have been an inappropriate use of animals to repeat the earlier animal studies for those groups. We should have stated more clearly that we were using the groups already studied to prevent repetition and to conserve animal resources, as recommended by the Animal Welfare Act (1990); however, we did refer to our previous paper. Our 2003 publication contained new data as well as previously published data. We welcome this opportunity to clarify our method. Our alleged reclassification of defects in our Table 2 (Johnson et al. 2003) merely reflects careful reevaluation by the cardiologist and minor updates in terminology that mirror current clinical usage to clarify the nature of a defect (e.g., great vessel defect vs. the more specific aortic hypoplasia; L-transposition vs. abnormal looping, etc.). There are other minor numerical differences in the tables (Table 2, Johnson et al. 2003, and Tables 1 and 3, Dawson et al. 1993), not remarked upon by Hardin et al., which derive from the more extensive statistical analysis in the later paper. In an apparent typographic error, we failed to report a pulmonary valve defect for the 1.5 ppm TCE in the 2003 paper. This should have been included in Table 2; however, it would not have changed the number of hearts with defects. Again, because this was a long-term continuous project, we did use all of the controls together in a cumulative manner. We used the larger sample size with data collected over a long period because it increases the generalizeability of our data, demonstrating clearly the background rate and the variability around rate estimates. Control values were consistent throughout our studies. The larger sample size did increase statistical power somewhat in our most recent paper (Johnson et al. 2003), again without inappropriate use of further valuable animal resources. It should be noted that the increase in statistical power is small compared to the increase generated by the effect sizes and the increase in the number of dose groups—data that can only be generated in a long-term project. Our statistical analysis was simple and conventional. Hardin et al. are incorrect in stating that the differences at the 1.5-ppm dose were statistically significant in our recent paper (Johnson et al. 2003). The p-values were reported in Figures 1 and 2 of our paper as 0.14 and 0.08, respectively, values not conventionally seen as statistically significant. Different levels of statistical significance used in each of the studies for each of the groups were carefully listed in the tables and figures and explained in the text. There are many references in the scientific literature about effects of halogenated hydrocarbons on development. We included only a few of these in our articles. We are a multidisciplinary team and have studied both TCE and its major metabolites, often basing some of our work on the findings of others in the field without duplicating the work of others. We have consulted with other prominent researchers in the field from time to time in establishing our experimental design or in interpreting our results. We have found only heart defects associated with these compounds, despite looking for other effects. This work has been consistent with the original epidemiological studies on which our laboratory work was based. We have been funded by government and other nonbiased agencies requiring competitive grant application and accountability. We have presented our results as peer-reviewed published articles in excellent journals. Our work has all been carried out at The University of Arizona. A major strength of our studies was microdissection of each heart by investigators fully versed in the pathology of congenital cardiac malformations as well as noncardiac anatomy. We fully agree with Hardin et al. that studies in this area “have potential for important health and public policy implications, so it is particularly important for the scientific and regulatory communities to have confidence in the conduct and reporting of those studies.” We believe that our studies have been rationally planned, are statistically and scientifically sound, and are of value for this purpose. We welcome this opportunity for postpublication discussion of results.
• Mays, M. Z., Johnson, P. D., Goldberg, S. J., & Dawson, B. V. (2003).

  Threshold of trichloroethylene contamination in maternal drinking waters affecting fetal heart development in the rat.

  . Environmental health perspectives, 111(3), 289-92. doi:10.1289/ehp.5125
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  Halogenated hydrocarbons such as trichloroethylene (TCE) are among the most common water supply contaminants in the United States and abroad. Epidemiologic studies have found an association but not a cause-and-effect relation between halogenated hydrocarbon contamination and increased incidence of congenital cardiac malformations or other defective birth outcomes. Avian and rat studies demonstrated statistically significant increases in the number of congenital cardiac malformations in those treated with high doses of TCE, either via intrauterine pump or in maternal drinking water, compared with controls. This study attempts to determine if there is a threshold dose exposure to TCE above which the developing heart is more likely to be affected. Sprague-Dawley rats were randomly placed in test groups and exposed to various concentrations of TCE (2.5 ppb, 250 ppb, 1.5 ppm, 1,100 ppm) in drinking water or distilled water (control group) throughout pregnancy. The percentage of abnormal hearts in the treated groups ranged from 0 to 10.48%, with controls having 2.1% abnormal hearts, and the number of litters with fetuses with abnormal hearts ranged from 0 to 66.7%, and the control percentage was 16.4%. The data from this study indicate not only that there is a statistically significant probability overall of a dose response to increasing levels of TCE exposure, but also that this trend begins to manifest at relatively low levels of exposure (i.e., < 250 ppb). Maternal rats exposed to more than this level of TCE during pregnancy showed an associated increased incidence of cardiac malformations in their developing rat fetuses.
• Runyan, R. B., Selmin, O. I., Runyan, R. B., Johnson, P. D., & Collier, J. M. (2003).

  Trichloroethylene effects on gene expression during cardiac development.

  . Birth defects research. Part A, Clinical and molecular teratology, 67(7), 488-95. doi:10.1002/bdra.10073
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  Halogenated hydrocarbon exposure is associated with changes in gene expression in adult and embryonic tissue. Our study was undertaken to identify differentially expressed mRNA transcripts in embryonic hearts from Sprague-Dawley rats exposed to trichloroethylene (TCE) or potential bio-transformation products dichloroethylene (DCE) and trichloroacetic acid (TCAA)..cDNA subtractive hybridization was used to selectively amplify expressed mRNA obtained from control or halogenated hydrocarbon exposed rat embryos. The doses used were 1100 and 110 ppm (8300 and 830 microM) TCE, 110 and 11 ppm (1100 and 110 microM) DCE, and 27.3 and 2.75 mg/ml (100 and 10 mM) TCAA. Control animals were given distilled drinking water throughout the period of experiments..Sequencing of over 100 clones derived from halogenated hydrocarbon exposed groups resulted in identification of numerous differentially regulated gene sequences. Up-regulated transcripts identified include genes associated with stress response (Hsp 70) and homeostasis (several ribosomal proteins). Down-regulated transcripts include extracellular matrix components (GPI-p137 and vimentin) and Ca(2+) responsive proteins (Serca-2 Ca(2+)-ATPase and beta-catenin). Two possible markers for fetal TCE exposure were identified: Serca-2 Ca(2+)-ATPase and GPI-p137, a GPI-linked protein of unknown function. Differential regulation of expression of both markers by TCE was confirmed by dot blot analysis and semi-quantitative RT-PCR with levels of TCE exposure between 100 and 250 ppb (0.76 and 1.9 microM) sufficient to decrease expression..Sequences down-regulated with TCE exposure appear to be those associated with cellular housekeeping, cell adhesion, and developmental processes, while TCE exposure up-regulates expression of numerous stress response and homeostatic genes.
• Johnson, P. D., & Besselsen, D. G. (2002).

  Practical aspects of experimental design in animal research.

  . ILAR journal, 43(4), 202-6. doi:10.1093/ilar.43.4.202
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  A brief overview is presented of the key steps involved in designing a research animal experiment, with reference to resources that specifically address each topic of discussion in more detail. After an idea for a research project is conceived, a thorough review of the literature and consultation with experts in that field are pursued to refine the problem statement and to assimilate background information that is necessary for the experimental design phase. A null and an alternate hypothesis that address the problem statement are then formulated, and only then is the specific design of the experiment developed. Likely the most critical step in designing animal experiments is the identification of the most appropriate animal model to address the experimental question being asked. Other practical considerations include defining the necessary control groups, randomly assigning animals to control/treatment groups, determining the number of animals needed per group, evaluating the logistics of the actual performance of the animal experiments, and identifying the most appropriate statistical analyses and potential collaborators experienced in the area of study. All of these factors are critical to designing an experiment that will generate scientifically valid and reproducible data, which should be considered the ultimate goal of any scientific investigation.
• Warren, D. A., Sudberry, G. L., Macmahon, K. L., Latendresse, J. R., Johnson, P. D., Graeter, L. J., Goodyear, C. D., Fisher, J. W., Eggers, J. S., & Channel, S. R. (2001).

  Trichloroethylene, trichloroacetic acid, and dichloroacetic acid: do they affect fetal rat heart development?

  . International journal of toxicology, 20(5), 257-67. doi:10.1080/109158101753252992
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  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 litters 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.
• Johnson, P. D., Goldberg, S. J., & Dawson, B. V. (1998).

  A review: trichloroethylene metabolites: potential cardiac teratogens.

  . Environmental health perspectives, 106 Suppl 4(suppl 4), 995-9. doi:10.1289/ehp.98106s4995
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  This review is a a series of the authors' studies designed to test the hypothesis that administration of trichloroethylene (TCE), dichloroethylene (DCE), their metabolites, and related compounds are responsible for fetal cardiac teratogenesis when given to pregnant rats during organogenesis. Identification of teratogenic compounds will allow more accurate assessment of environmental contaminants and public health risks. Epidemiologic studies and previous teratogenic studies using chick embryos and fetal rats have reported an increased number of congenital cardiac defects when exposed to TCE or DCE during fetal development. Metabolites of TCE and DCE studied in the drinking-water exposure study include trichloroacetic acid TCAA), monochloroacetic acid, trichloroethanol, carboxymethylcysteine, trichloroacetaldehyde, dichloroacetaldehyde, and dichlorovinyl cysteine. Varying doses of each were given in drinking water to pregnant rats during the period of fetal heart development. Rats receiving 2730 ppm TCAA in drinking water were the only metabolite group demonstrating a significant increase in the number of cardiac defects in fetuses on a per-litter basis (p = 0.0004 Wilcoxon test and p =0.0015 exact permutation test). Maternal and fetal variables showed no statistically significant differences between treated and untreated groups. When treated with TCAA the increased cardiac defects, as compared to controls, do not preclude the involvement of other metabolites as cardiac teratogens, but indicates TCAA as a specific cardiac teratogen. Further studies of drinking-water exposure and potential mechanisms of action on the developing heart are proceeding.
• Johnson, P. D., Goldberg, S. J., & Dawson, B. V. (1998).

  Cardiac teratogenicity of trichloroethylene metabolites.

  . Journal of the American College of Cardiology, 32(2), 540-5. doi:10.1016/s0735-1097(98)00232-0
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  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..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..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..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, Fischer's 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..Of the metabolites tested, only TCAA appeared to be a specific cardiac teratogen in the fetus when imbibed by the maternal rat.
• Johnson, P. D., Hruby, V. J., Levine, N., Johnson, P. D., Hruby, V. J., Hadley, M. E., Dorr, R. T., & Dawson, B. V. (1994).

  Coat color darkening in a dog in response to a potent melanotropic peptide.

  . American journal of veterinary research, 55(11), 1593-6.
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  Analogues of a melanocyte-stimulating hormone (alpha-MSH) have been documented to be effective in inducing integumental melanogenesis in several species. These melanotropin analogues are more potent than the natural hormone and have prolonged biological activity, without apparent teratogenic or other toxic effects, at least in rodents. In a pilot study, a cyclic alpha-MSH analogue, Ac-[Nle4, Asp5, D-Phe7, Lys10] alpha-MSH4-10-NH2, was administered SC to a dog at a dose of 1 mg of analogue in 1 ml of 0.9% NaCl for 3 weeks, without noticeable adverse effects. There was gradual and extensive darkening of the coat, which originally was predominantly tan, with tips of black. Initially, the darkening involved face and extremities, then gradually expanded to include the trunk and tail hair. Visual pigmentation peaked approximately 2 months after injections were completed. As new hair growth continued subsequent to the injections, the original tan color appeared at the proximal end of the hair shaft, leaving a dark terminal band on all affected hairs. These observations clearly indicated that follicular melanogenesis can be induced in dogs by treatment with a melanotropic peptide.
• Johnson, P. D., Holloway, H., Ford, C. A., Dorr, R. T., & Dawson, B. V. (1993).

  Administration of melanotropic peptides during gestation in the rodent.

  . Toxicology, 77(1-2), 91-101. doi:10.1016/0300-483x(93)90140-n
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  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.
• Ulreich, J. B., Johnson, P. D., Goldberg, S. J., & Dawson, B. V. (1993).

  Cardiac teratogenesis of halogenated hydrocarbon-contaminated drinking water.

  . Journal of the American College of Cardiology, 21(6), 1466-72. doi:10.1016/0735-1097(93)90325-u
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  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..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..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..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..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.
• Hoyme, H. E., Ulreich, J. B., Johnson, P. D., Hoyme, H. E., Goldberg, S. J., & Dawson, B. V. (1992).

  Cardiac teratogenicity of dichloroethylene in a chick model.

  . Pediatric research, 32(1), 23-6. doi:10.1203/00006450-199207000-00005
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  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)
• Sj, G., Johnson, P. D., & Bv, D. (1992).

  Spontaneous congenital heart malformations in the Sprague-Dawley rat

  . Laboratory Animal Science, 43(2), 183-188.
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  The incidence rate of spontaneous congenital cardiac defects in Sprague Dawley rats has not been extensively studied. The incidence of general congenital defects in these animals has been thoroughly investigated and is very low. We sought to determine the prevalence of congenital heart defects in Sprague Dawley rats. Teratology studies using mammalian models have become increasingly important as the number of known environmental contaminants has increased; thus, it is essential to know the incidence of spontaneous congenital cardiac defects in this most commonly used mammalian model so that statistically appropriate research protocols can be planned. Using a thorough method of evaluating the structure of the heart, 624 Sprague Dawley fetal rat hearts were examined just before parturition. Animals used in this study were control groups for surgically manipulated, hydrocarbon-treated animals in a study by Dawson et al. in 1990. The overall incidence of spontaneous congenital cardiac defects was found to be 2.3%. This is similar to the incidence of congenital cardiac defects in humans and thus enhances the suitability of Sprague Dawley rats as a small mammalian model to study the factors that affect cardiac development.
• Ulreich, J. B., Johnson, P. D., Goldberg, S. J., & Dawson, B. V. (1990).

  Cardiac teratogenesis of trichloroethylene and dichloroethylene in a mammalian model.

  . Journal of the American College of Cardiology, 16(5), 1304-9. doi:10.1016/0735-1097(90)90569-b
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  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.