The purpose of this paper is to review the development of the mammalian kidney and to assess the influence that various perinatal manipulations may have on the developmental process either morphologically or functionally. Immature kidneys in general have less functional capacity than adult kidneys and a low rate of glomerular filtration, perhaps related to renal blood flow, which appears to limit the disposition of a fluid or solute load. Tubular reabsorption is also limited leading to the urinary loss of glucose, amino acids, bicarbonate and phosphate. Although the relatively low function of the immature kidney is a normal part of development, its capacity to respond under conditions of stress may be less adequate than in adults. An additional concern is that a variety of perinatal manipulations, such as the incidental or accidental ingestion of a chemical, may lead to varying degrees of altered morphogenesis or functional development of the kidney. Chemical induced renal anomalies may be of several types, but in typical teratology experiments hydronephrosis may be the most frequent observation. The functional consequences of these renal malformations may be lethal or inconsequential or while an animal may be able to survive and develop normally in the presence of a renal malformation, it is possible that a stressful situation would unmask a functional malformation which could compromise survival. Thus, some renal abnormalities may be subtle enough to go unnoticed without experimental tests. Without such tests it is impossible to evaluate the effect of functional alterations on successful adaptation. (+info)
Endocrine biomarkers of early fetal loss in cynomolgus macaques (Macaca fascicularis) following exposure to dioxin.
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This study examines the endocrine alterations associated with early fetal loss (EFL) induced by an environmental toxin, TCDD (2,3,7, 8-tetrachlorodibenzo-p-dioxin), in the cynomolgus macaque, a well-documented reproductive/developmental model for humans. Females were administered single doses of 1, 2, and 4 microgram/kg TCDD (n = 4 per dose group) on gestational day (GD) 12. Urinary estrogen metabolites (estrone conjugates) were monitored to establish the day of ovulation, and serum hormones (estradiol, progesterone, chorionic gonadotropin, relaxin) were measured to assess ovarian and placental endocrine status before and after treatment. EFL occurred between GDs 22 and 32 in 10 of the 12 animals treated with TCDD. The primary endocrine alterations associated with TCDD treatment were significant decreases in serum estradiol and bioactive chorionic gonadotropin concentrations (p < 0.02). Less pronounced decreases in serum progesterone (p = 0.10) and relaxin (p < 0.08) also followed TCDD treatment. In contrast, immunoreactive chorionic gonadotropin concentrations were not reduced by TCDD exposure at any level, indicating that TCDD targets specific components of the chorionic gonadotropin synthesis machinery within the trophoblast to alter the functional capacity of the hormone. These data demonstrate the value of endocrine biomarkers in identifying a toxic exposure to primate pregnancy many days before direct signs of reproductive toxicity were apparent. The increased EFL that occurred after exposure to TCDD might reflect a toxic response initially mediated via endocrine imbalance, leading to placental insufficiency, compromised embryonic circulation, and subsequent EFL. (+info)
2,3,7,8-Tetrachlorodibenzo-p-dioxin alters cardiovascular and craniofacial development and function in sac fry of rainbow trout (Oncorhynchus mykiss).
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Hallmark signs of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) toxicity in rainbow trout sac fry, are yolk sac edema, hemorrhage, craniofacial malformation, and growth retardation culminating in mortality. Our objective was to determine the role of cardiovascular dysfunction in the development of this toxicity. An embryotoxic TCDD dose (385 pg/g egg) caused a progressive reduction in blood flow in rainbow trout sac fry manifested first and most dramatically in the 1st and 2nd branchial arches and vessels perfusing the lower jaw. Blood flow was reduced later in the infraorbital artery and occipital vein of the head as well as segmental vessels and caudal vein of the trunk. Reduced perfusion occurred last in gill branchial arteries involved with oxygen uptake and the subintestinal vein and vitelline vein involved with nutrient uptake. Although heart rate throughout sac fry development was not affected, heart size at 50 days post-fertilization (dpf) was reduced far more than body weight or length, suggesting that the progressive circulatory failure caused by TCDD is associated with reduced cardiac output. Craniofacial development was arrested near hatch, giving rise to craniofacial malformations in which the jaws and anterior nasal structures were underdeveloped. Unlike the medaka embryo, in which TCDD causes apoptosis in the medial yolk vein, endothelial cell death was not observed in rainbow trout sac fry. These findings suggest a primary role for arrested heart development and reduced perfusion of tissues with blood in the early-life stage toxicity of TCDD in trout. (+info)
AhR, ARNT, and CYP1A1 mRNA quantitation in cultured human embryonic palates exposed to TCDD and comparison with mouse palate in vivo and in culture.
(4/1351)
2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is developmentally toxic in many species and induces cleft palate in the C57BL/6N mouse embryo. Palatogenesis in mouse and human embryos involves homologous processes at the morphological, cellular, and molecular levels. In organ culture, mouse and human palates respond similarly to TCDD. The present study quantitates the expression of AhR, ARNT, and CYP1A1 mRNA in human embryonic palates in organ culture. Palatal tissues were exposed to 1 x 10(-10), 1 x 10(-9), or 1 x 10(-8) M TCDD or control medium and sampled at 0, 2, 4, and 6 hours for quantitative RT-PCR using a synthetic RNA internal standard. Similar measurements of CYP1A1 gene expression were collected for mouse palates cultured in this model. In human palates, AhR expression correlated with ARNT and CYP1A1 mRNA expression. TCDD induction of CYP1A1 was time- and concentration-dependent. The expression of these genes presented a uniform and continuous distribution across the group of embryos, with no subset of either high or low expressors/responders. The ratio of AhR to ARNT was approximately 4:1. AhR mRNA increased during the culture period in both treated and control subjects; however, ARNT expression was relatively constant. TCDD did not alter either AhR or ARNT expression in a consistent dose- or time-related manner. Comparison of human and mouse data showed a high correlation across species for the induction of CYP1A1. Human embryos expressed approximately 350 times less AhR mRNA than the mouse, and in earlier studies it was shown that human palates required 200 times more TCDD to produce the same effects. When the morphological, cellular, and molecular responses to TCDD between mouse and human are compared, it seems highly unlikely that human embryos could be exposed to sufficient TCDD to achieve changes in palatal differentiation that would lead to cleft palate. (+info)
RT-PCR quantification of AHR, ARNT, GR, and CYP1A1 mRNA in craniofacial tissues of embryonic mice exposed to 2,3,7,8-tetrachlorodibenzo-p-dioxin and hydrocortisone.
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C57BL/6N mouse embryos exposed to hydrocortisone (HC) or 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) develop cleft palate. An interaction between these agents produces clefts at doses which alone are not teratogenic. The glucocorticoid receptor (GR) and dioxin receptor (AhR) mediated these responses and their gene expression was altered by TCDD and/or HC in palates examined on gestation day (GD) 14 by Northern blot analysis and in situ hybridization. The present study quantifies AhR, AhR nuclear translocator (ARNT), and GR mRNA at 4, 12, 24, and 48 h after exposure (time 0 = dose administration at 8 A.M. on gestation day 12) on GD12 to TCDD (24 micrograms/kg), HC (100 mg/kg) or HC (25 mg/kg) + TCDD (3 micrograms/kg). The induction of CYP1A1 mRNA was also quantified at 2, 4, 6, 12, 24, and 48 h for control and TCDD-exposed samples. Total RNA was prepared from midfacial tissue of 4-6 embryos/litter at each time and dose. An RNA internal standard (IS) for each gene was synthesized, which included the gene's primer sequences separated by a pUC19 plasmid sequence. Reverse transcription-polymerase chain reaction (RT-PCR) was performed on total RNA + IS using a range of 5-7 IS concentrations across a constant level of total RNA. PCR products were separated in gels (mRNA and IS-amplified sequences differed by 30-50 bases), ethidium bromide-stained, imaged (Hamamatsu Photonics Systems, Bridgewater, NJ), and quantified with NIH Image. CYP1A1 mRNA was significantly induced in the TCDD-exposed samples at all time points examined (p = 0.005 at 2 h and 0.001 after 2 h). During palatal shelf outgrowth on GD12, AhR mRNA levels increased significantly and this was not affected by treatment with TCDD or HC + TCDD. A significant increase in GR was detected at 24 h (p < 0.05) and this was unaffected by any of the exposures. Expression of ARNT increased at 12 h (p < 0.001); however, treatment with HC or HC + TCDD blocked this increase (p < 0.05). At 24 h, the TCDD-treated embryos had significantly lower ARNT mRNA compared with controls (p < 0.001). The relative overall expression level of the genes was AhR > ARNT > GR. Within individuals, expression of AhR and/or ARNT was highly correlated with GR level. In conclusion, CYP1A1 mRNA was expressed in developing craniofacial tissue and was highly induced by TCDD exposure. AhR, ARNT, and GR mRNA are upregulated in early palatogenesis, although not on the same schedule. The TCDD-induced decrease in ARNT at 24 h after dosing and the HC and HC + TCDD-induced delay in upregulation of ARNT may affect the dynamics of heterodimer formation between AhR and ARNT. The changes in ARNT mRNA level could also affect availability of this transcriptional regulator to interact with other potential partners, and these effects, separately or in combination, may be involved in disruption of normal embryonic development. (+info)
Amelioration of TCDD-induced teratogenesis in aryl hydrocarbon receptor (AhR)-null mice.
(6/1351)
The aryl hydrocarbon receptor (AhR) mediates many of the biological effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and transcriptional activation of genes encoding a number of xenobiotic metabolizing enzymes. Prenatal exposure of mice to TCDD causes severe alterations in embryo and fetal development, including hydronephrosis and cleft palate. However, the mechanisms underlying these effects are unclear. In this work, the teratogenicity of TCDD in AhR-null mice was evaluated to determine if this effect is mediated by the AhR. Homozygous wild-type (+/+) or AhR-null (-/-) female mice were mated with males of the same genotype overnight. On gestation day (GD)-10, mice were intubated orally with either corn oil (vehicle control) or 25 micrograms/kg TCDD. Fetuses were examined on GD18 for visceral and skeletal alterations. For non-TCDD-exposed litters, all developmental endpoints were comparable between genotypes, with the exception of a lower incidence of large interfrontal bones in (-/-) mice. For TCDD-exposed litters, (+/+) fetuses had a significantly greater incidence of cleft palate, hydronephrosis, small kidneys, tortuous ureters and greater dilation of the renal pelves and ureters compared to (-/-) fetuses. Interestingly, an increased resorption rate was observed in (-/-) fetuses exposed to TCDD. Results from this work demonstrate that fetal development per se is generally unaffected by the absence of the AhR or that other genes may have compensated for the loss of the AhR. More importantly, these data indicate that the AhR mediates TCDD-induced teratogenicity. Further, since a higher percentage of resorptions was observed in (-/-) litters from TCDD-treated dams, it is possible that AhR-independent mechanisms contribute to TCDD-induced developmental toxicity. (+info)
Cytochrome P450 CYP1B1 determines susceptibility to 7, 12-dimethylbenz[a]anthracene-induced lymphomas.
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CYP1B1-null mice, created by targeted gene disruption in embryonic stem cells, were born at the expected frequency from heterozygous matings with no observable phenotype, thus establishing that CYP1B1 is not required for mouse development. CYP1B1 was not detectable in cultured embryonic fibroblast (EF) or in different tissues, such as lung, of the CYP1B1-null mouse treated with the aryl hydrocarbon receptor agonist 2,3,7,8-tetrachlorodibenzo-p-dioxin whereas the equivalent wild-type EF cells express basal and substantial inducible CYP1B1 and lung expresses inducible CYP1B1. CYP1A1 is induced to far higher levels than CYP1B1 in liver, kidney, and lung in wild-type mice and is induced to a similar extent in CYP1B1-null mice. 7,12-dimethylbenz[a]anthracene (DMBA) was toxic in wild-type EFs that express CYP1B1 but not CYP1A1. These cells effectively metabolized DMBA, consistent with CYP1B1 involvement in producing the procarcinogenic 3,4-dihydrodiol as a major metabolite, whereas CYP1B1-null EF showed no significant metabolism and were resistant to DMBA-mediated toxicity. When wild-type mice were administered high levels of DMBA intragastrically, 70% developed highly malignant lymphomas whereas only 7.5% of CYP1B1-null mice had lymphomas. Skin hyperplasia and tumors were also more frequent in wild-type mice. These results establish that CYP1B1, located exclusively at extrahepatic sites, mediates the carcinogenicity of DMBA. Surprisingly, CYP1A1, which has a high rate of DMBA metabolism in vitro, is not sufficient for this carcinogenesis, which demonstrates the importance of extrahepatic P450s in determining susceptibility to chemical carcinogens and validates the search for associations between P450 expression and cancer risk in humans. (+info)
Quantitative analysis of constitutive and 2,3,7,8-tetrachlorodibenzo-p-dioxin-induced cytochrome P450 1B1 expression in human lymphocytes.
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Exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD or dioxin) results in a broad spectrum of biological responses, including altered metabolism, disruption of normal hormone signaling pathways, reproductive and developmental effects, and cancer. Cytochrome P450 1B1 (CYP1B1) is a dioxin-inducible gene that is active in the formation of 4-hydroxyestradiol, a potentially genotoxic catechol estrogen. Therefore, the analysis of CYP1B1 in humans may be useful in establishing relationships between dioxin exposure and adverse health effects. In this study, we examined the expression of CYP1B1 in human peripheral blood lymphocytes of unexposed individuals using a quantitative reverse transcription-PCR method. Absolute CYP1B1 RNA levels varied more than 30-fold in uncultured mononuclear cells obtained from 10 individuals. In vitro treatment of mitogen-stimulated lymphocytes with TCDD for 1-5 days of culture resulted in a peak induction of CYP1B1 after 3 days. The induction of CYP1B1 RNA levels after 3 days of culture was dose-dependent, exhibited a maximum response above 10 nM TCDD, and varied greatly among different individuals. However, the half maximal dose required for this induction was similar between individuals and comparable to that observed in the MCF-7 and HepG2 human cell lines. These observations indicate that CYP1B1 exhibits variable constitutive expression and is inducible in vitro by TCDD in human lymphocytes and that the magnitude of induction varies within the population. These data define the suitability of CYP1B1 for use as a mechanistically based biomarker in ongoing molecular epidemiological studies of human populations exposed to dioxins and related chemicals that bind the aromatic hydrocarbon receptor. (+info)