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Warren Heideman

Publications and source records attributed to Warren Heideman.

At least 19 recordsLinked to original sources
PubMed2006

Blocking expression of AHR2 and ARNT1 in zebrafish larvae protects against cardiac toxicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin.

The zebrafish (Danio rerio) has become an attractive vertebrate model for studying developmental processes, and is emerging as a model system for studying the mechanisms by which xenobiotic compounds perturb normal development. Embryos treated with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) shortly after fertilization exhibit a range of adverse effects on the heart: an early reduction in cardiac myocyte number, followed by a change in heart looping and morphology, with an apparent compaction of the ventricle and overall decrease in heart size. These changes are accompanied by impaired cardiac function including a decrease in cardiac output and eventually irreversible ventricular standstill. The mechanisms involved in mediating effects of TCDD on the heart remain unknown. However, it is widely accepted that aryl hydrocarbon receptor (AHR) activation mediates endpoints of TCDD toxicity in vertebrates. In zebrafish, there are multiple forms of AHR and AHR nuclear translocator protein (ARNT) raising the question about whether different endpoints of TCDD toxicity are mediated by different components of the AHR/ARNT pathway. To address this question we used morpholino oligonucleotide technology to specifically block the expression of zfAHR2, zfARNT1, zfARNT2, and zfCYP1A, and assessed the previously described effects of TCDD on heart morphology, size, and function in the developing morphants. We report that blocking zfAHR2 and zfARNT1 expression provided protection against the TCDD-mediated alteration in heart morphology, reduced cardiac myocyte number, decreased cardiac output and ventricular standstill in zebrafish larvae, while the zfarnt2 and zfcyp1a morpholinos did not block the TCDD-induced cardiac toxicity.

Animals↗
PubMed2006

Aryl hydrocarbon receptor activation produces heart-specific transcriptional and toxic responses in developing zebrafish.

Proper regulation of the aryl hydrocarbon receptor (AHR), a ligand-activated transcription factor, is required for normal vertebrate cardiovascular development. AHR hyperactivation by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) during zebrafish (Danio rerio) development results in altered heart morphology and function, culminating in death. To identify genes that may cause cardiac toxicity, we analyzed the transcriptional response to TCDD in zebrafish hearts. Zebrafish larvae were exposed to TCDD for 1 h at 72 h after fertilization (hpf), and the hearts were extracted for microarray analysis at 1, 2, 4, and 12 h after exposure (73, 74, 76, and 84 h postfertilization). The remaining body tissue was also collected at each time for comparison. TCDD rapidly induced expression in 42 genes within 1 to2hof exposure. These genes function in xenobiotic metabolism, proliferation, heart contractility, and pathways that regulate heart development. Furthermore, these expression changes preceded signs of cardiovascular toxicity, characterized by decreased stroke volume, peripheral blood flow, and a halt in heart growth. This identifies strong candidates for important AHR target genes. It is noteworthy that the TCDD-induced transcriptional response in the hearts of zebrafish larvae was substantially different from that induced in the rest of the body tissues. One of the biggest differences included a cluster of genes that were down-regulated 12 h after exposure in heart tissue, but not in the body samples. More than 70% of the transcripts in this heart-specific cluster promote cellular growth and proliferation. Thus, the developing heart stands out as being responsive to TCDD at both the level of toxicity and gene expression.

Animals↗
PubMed2006

Understanding dioxin developmental toxicity using the zebrafish model.

Zebrafish (Danio rerio) have advantages over mammals as an animal model for investigating developmental toxicity. 2,3,7,8-Tetrachlorodibenzo-p-dioxin (dioxin, TCDD), a persistent global contaminant, is the most comprehensively studied developmental toxicant in zebrafish. The hallmark responses of TCDD developmental toxicity manifested in zebrafish larvae include edema, anemia, hemorrhage, and ischemia associated with arrested growth and development. Heart and vasculature development and function are severely impaired, and jaw malformations occur secondary to inhibited chondrogenesis. The swim bladder fails to inflate, and the switch from embryonic to adult erythropoiesis is blocked. This profile of developmental toxicity responses, commonly referred to as "blue sac syndrome" because the edematous yolk sac appears blue, is observed in the larval form of all freshwater fish species exposed to TCDD at the embryonic stage of development. Components of the aryl hydrocarbon receptor/aryl hydrocarbon receptor nuclear translocator (AHR/ARNT) signaling pathway in zebrafish have been identified and functionally characterized. Their role in mediating TCDD toxicity has been determined using morpholinos to specifically knockdown the translation of zfAHR1, zfAHR2, zfARNT1, and zfARNT2 mRNAs, respectively, and a line of zfARNT2 null mutant zebrafish has provided further insight. These studies have shown that zfAHR2 and zfARNT1 mediate TCDD developmental toxicity. In addition, the growing use of molecular and genomic tools for research on zebrafish have led to advances in our understanding of the mechanism of TCDD developmental toxicity at the molecular level, including the recent finding that toxicity is not mediated by increased cytochrome P4501A (zfCYP1A) expression.

Animals↗
PubMed2006

The function and properties of the Azf1 transcriptional regulator change with growth conditions in Saccharomyces cerevisiae.

Azf1 activates CLN3 transcription in Saccharomyces cerevisiae cells growing in glucose. Paradoxically, other studies have shown Azf1 to be almost undetectable in glucose-grown cells. Microarray experiments showed that Azf1 activates nonoverlapping gene sets in different carbon sources: in glucose, Azf1 activates carbon and energy metabolism genes, and in glycerol-lactate, Azf1 activates genes needed for cell wall maintenance. Consistent with the decreased expression of cell wall maintenance genes observed with azf1Delta mutants, we observed a marked growth defect in the azf1Delta cells at 37 degrees C in nonfermentable medium. Cell wall integrity assays, such as sensitivity to calcofluor white, sodium dodecyl sulfate, or caffeine, confirmed cell wall defects in azf1Delta mutants in nonfermentable medium. Gel shift experiments show that Azf1 binds to DNA elements with the sequence AAAAGAAA (A4GA3), a motif enriched in the promoters of Azf1-sensitive genes and predicted by whole-genome studies. This suggests that many of the Azf1-dependent transcripts may be regulated directly by Azf1 binding. We found that the levels of Azf1 protein in glucose-grown cells were comparable to Azf1 levels in cells grown in glycerol-lactate; however, this could only be demonstrated with a cell extraction procedure that minimizes proteolysis. Glucose produces conditions that destabilize the Azf1 protein, a finding that may reflect a glucose-induced change in Azf1 tertiary or quaternary structure.

Base Sequence↗
PubMed2005

Identification of zebrafish ARNT1 homologs: 2,3,7,8-tetrachlorodibenzo-p-dioxin toxicity in the developing zebrafish requires ARNT1.

To use the zebrafish (Danio rerio) as a model to study 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) developmental toxicity, it is essential to know which proteins are involved in mediating toxicity. Previous work has identified zfAHR2 as the receptor that binds TCDD mediating downstream responses. Although zfARNT2b can form a functional heterodimer with zfAHR2 in vitro, zfarnt2 null mutants show no protection against endpoints of TCDD developmental toxicity, demonstrating that zfARNT2b cannot be the physiological dimerization partner for zfAHR2 mediating responses to TCDD in zebrafish embryos. The purpose of the current study was to identify an alternate dimerization partner(s) for zfAHR2 that may function to mediate TCDD developmental toxicity. By searching zebrafish genomic sequence and using the polymerase chain reaction-based rapid amplification of cDNA ends technique, three forms of cDNA that seem to be alternate mRNA splice variants of a zebrafish homolog of ARNT1 were detected. Analysis of the zfARNT1 proteins in vitro demonstrates that the two longest forms of zfARNT1, zfARNT1b and zfARNT1c, can form functional heterodimers with zfAHR2. However, the shortest form, zfARNT1a, seems to be nonfunctional with zfAHR2 in vitro. To determine whether a zfARNT1 protein functions with zfAHR2 in vivo, a morpholino targeted against the 5' end of zfARNT1 (zfarnt1-MO) was used. Injection of the zfarnt1-MO before TCDD treatment significantly decreases the induction of zfCYP1A mRNA and protein. In addition, zfarnt1 morphants show complete protection against TCDD-induced pericardial edema and show partial protection against reduced blood flow and craniofacial malformations caused by TCDD, demonstrating the role of zfARNT1 proteins in mediating these responses.

Amino Acid Sequence↗
PubMed2005

Zebrafish as a model vertebrate for investigating chemical toxicity.

Zebrafish (Danio rerio) has been a prominent model vertebrate in a variety of biological disciplines. Substantial information gathered from developmental and genetic research, together with near-completion of the zebrafish genome project, has placed zebrafish in an attractive position for use as a toxicological model. Although still in its infancy, there is a clear potential for zebrafish to provide valuable new insights into chemical toxicity, drug discovery, and human disease using recent advances in forward and reverse genetic techniques coupled with large-scale, high-throughput screening. Here we present an overview of the rapidly increasing use of zebrafish in toxicology. Advantages of the zebrafish both in identifying endpoints of toxicity and in elucidating mechanisms of toxicity are highlighted.

Animals↗
PubMed2005

Heart malformation is an early response to TCDD in embryonic zebrafish.

The zebrafish (Danio rerio) has become an attractive vertebrate model for studying developmental processes, and is emerging as a model system for studying the mechanisms by which toxic compounds perturb normal development. When exposed to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) shortly after fertilization, zebrafish embryos exhibit pericardial edema and reduced blood flow by 72 h post fertilization (hpf). To better understand the progression of dioxin toxicity in zebrafish, we have examined the effects of TCDD on heart development. At 72 hpf, TCDD-treated embryos exhibited altered looping, with the atria positioned distinctly posterior to the ventricles, contrary to the looping of control hearts, where the two chambers lied side by side. Moreover, the ventricles in dioxin-exposed hearts became more compact, and the atria elongated in comparison to controls. These defects are not secondary to pericardial edema because they were observed when edema formation was suppressed with osmotic support. In addition to morphological changes, TCDD produced functional deficits in the developing hearts, including blood regurgitation and a striking ventricular standstill that became prevalent by 120 hpf. We also assessed the effect of TCDD on the heart size using stereological measurements, which demonstrated significant reduction in heart tissue volume at 72 hpf. Perhaps our most significant finding was a decrease in the total number of cardiomyocytes in TCDD-exposed embryos by 48 hpf, one day prior to observable effects on peripheral blood flow. We conclude that the developing heart is an important target for TCDD in zebrafish.

Abnormalities, Drug-Induced↗
PubMed2005

Zebrafish and cardiac toxicology.

Model systems are a mainstay in toxicological research. Zebrafish are rapidly becoming an important model organism for studying vertebrate development. The advantages of zebrafish: short reproductive cycle, production of numerous transparent, synchronously developing embryos, low cost, and standardization make zebrafish an attractive model for toxicologists as well. The use of these fish to study heart development has moved forward very rapidly, laying the groundwork for studying the effects of chemicals on cardiac development and function. Here we describe approaches that can be used to study cardiac toxicity in developing zebrafish, focusing on examples where zebrafish embryos have been especially useful in understanding the impact of specific toxicants on heart development and function.

Animals↗
PubMed2004

ARNT2 is not required for TCDD developmental toxicity in zebrafish.

ZfAHR2 has been identified as the receptor that is essential for mediating the developmental toxicity caused by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in zebrafish. One form of zfARNT2, zfARNT2b, forms a functional heterodimer with zfAHR2 that specifically recognizes XREs in gel shift experiments and induces XRE-driven transcription in COS-7 cells treated with TCDD. However, it has not been demonstrated that zfARNT2b acts as the physiological dimerization partner for zfAHR2 to mediate TCDD toxicity in developing zebrafish. An antisense morpholino targeted against zfARNT2 (zfarnt2-MO) along with a line of mutant zebrafish lacking expression of the zfarnt2 gene have been used to test the hypothesis that zfARNT2 mediates the developmental toxicity of TCDD. Injection of the zfarnt2-MO decreased expression of the zfARNT2 protein but did not provide any protection against the formation of pericardial edema at 72 hpf. In addition, in TCDD dose response studies the zfarnt2(-/-) embryos showed no protection against three endpoints of TCDD toxicity observed at 96 hpf: pericardial edema, reduced trunk blood flow, and shortened lower jaw. Finally, immunostaining results at 96 hpf demonstrate that the zfarnt2(-/-) embryos show a similar pattern of TCDD-induced zfCYP1A expression as WT embryos. These results demonstrate that zfARNT2 is not essential for mediating TCDD developmental toxicity in zebrafish and suggest that alternate dimerization partner(s) exist for zfAHR2 in vivo.

Abnormalities, Drug-Induced↗
PubMed2004

Water permeability and TCDD-induced edema in zebrafish early-life stages.

A common response to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) exposure in teleost embryos is blue-sac disease, characterized by pericardial and yolk-sac edema. The cellular and extracellular fluids of freshwater fish are hyperosmotic compared to the surrounding water. In order to be in osmotic balance, freshwater fish must maintain a barrier to minimize water entry and excrete excess water that passes the barrier. We hypothesized that edema observed in TCDD-exposed zebrafish was caused by a failure of a barrier to incoming water. As a test of this hypothesis, we removed the osmotic gradient that drives water entry by increasing the osmolarity of the surrounding water with mannitol. Abolishing the osmotic gradient between the interior body fluids and the water environment of the developing zebrafish significantly reduced both pericardial and yolk-sac edema. When added after edema formation had already started, mannitol only partially reversed pre-existing edema. An alternate hypothesis is that TCDD impairs water excretion, allowing water to accumulate as edema fluid. However, we were unable to demonstrate an alteration in kidney function: expression of early markers for kidney development appeared normal, and we did not observe TCDD-induced changes in kidney filtration. An alteration in the overall shape of the kidney was observed, but this may be a consequence of compression by edema. In conclusion, TCDD exposure may inhibit the function of a permeability barrier to water, which is critical for maintaining osmotic balance in early development.

Aging↗
PubMed2004

Agonist-induced internalization of leukotriene B(4) receptor 1 requires G-protein-coupled receptor kinase 2 but not arrestins.

The leukotriene B(4) (LTB(4)) receptor (BLT1) becomes desensitized upon repeated agonist stimulation. Little is known, however, about BLT1 internalization, which follows desensitization in most G-protein-coupled receptors (GPCR). In the current study, transiently expressed BLT1 readily internalized, after LTB(4) stimulation, in RBL-2H3 cells that express high levels of endogenous GPCR kinase 2 (GRK2) but did not in COS-7 or human embryonic kidney (HEK) 293 cells, which do not overexpress GRK. The internalization of BLT1 could be blocked in RBL-2H3 cells by coexpressing dominant-negative (DN) GRK2 K220R and could be promoted in HEK293 cells by coexpressing wild-type (WT) GRK2. Coexpression of WT or DN nonvisual arrestins had no effect on BLT1 internalization. Moreover, upon stimulation with LTB(4), BLT1 did not induce arrestin-green fluorescence protein redistribution in either cell type, even in the presence of overexpressed GRK2. Coimmunoprecipitation experiments confirmed that BLT1 could associate with GRK2 but not with arrestins. A C-tail-truncated mutant of BLT1 lost the capacity to internalize and associate with GRK2 upon exposure to LTB(4), suggesting that the C-tail was required for receptor internalization and association with GRK2. Taken together, our results indicate that the C terminus of BLT1 plays a pivotal role in receptor internalization and GRK2 association. Moreover, ligand-induced BLT1 internalization is dependent on GRK2 but independent of arrestins. This may allow differential, cell-type-specific signaling in response to LTB(4), depending on GRK expression levels.

Animals↗
PubMed2003

Interactions between 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and hypoxia signaling pathways in zebrafish: hypoxia decreases responses to TCDD in zebrafish embryos.

The aryl hydrocarbon receptor (AHR) interacts with the aryl hydrocarbon receptor nuclear translocator (ARNT) to form a heterodimer that binds to promoters in target genes to alter transcription in response to xenobiotics such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). The ARNT protein also forms heterodimers with other proteins such as HIF-1alpha and HIF-2alpha to alter gene expression in response to low oxygen conditions. Because ARNT is shared between multiple signaling pathways it is possible that activation of one ARNT-requiring pathway could inhibit the activation of other pathways that depend on ARNT. One hypothesis to explain TCDD toxicity in early life stage fish is that TCDD activation of zfAHR2 sequesters zfARNT2 away from the hypoxia signaling pathway. To test this hypothesis we measured the ability of TCDD to prevent induction of heme oxygenase by hypoxia (40% saturation), as well as the ability of hypoxia to increase the sensitivity of zebrafish to the effects of TCDD during the first week of life. As a further test of the model we examined mutant zebrafish that lack zfARNT2 for phenotypes that resemble the effects of TCDD exposure. Our results demonstrate that sequestration of zfARNT2 is not causing TCDD toxicity. TCDD did not inhibit hypoxia induction of heme oxygenase, hypoxia and TCDD exposures were not additive in causing developmental toxicity, and mutant embryos that lack zfARNT2 do not develop defects mimicking TCDD toxicity. However, our results demonstrate some level of cross talk between the two pathways in the zebrafish embryo. Hypoxia decreased TCDD induction of zfCYP1A mRNA, and decreased the potency of TCDD in causing edema. It is not clear whether this is mediated through competition for zfARNT2, or through other mechanisms.

Animals↗
PubMed2003

ACE2 is required for daughter cell-specific G1 delay in Saccharomyces cerevisiae.

Saccharomyces cerevisiae cells reproduce by budding to yield a mother cell and a smaller daughter cell. Although both mother and daughter begin G1 simultaneously, the mother cell progresses through G1 more rapidly. Daughter cell G1 delay has long been thought to be due to a requirement for attaining a certain critical cell size before passing the commitment point in the cell cycle known as START. We present an alternative model in which the daughter cell-specific Ace2 transcription factor delays G1 in daughter cells. Deletion of ACE2 produces daughter cells that proceed through G1 at the same rate as mother cells, whereas a mutant Ace2 protein that is not restricted to daughter cells delays G1 equally in both mothers and daughters. The differential in G1 length between mothers and daughters requires the Cln3 G1 cyclin, and CLN3-GFP reporter expression is reduced in daughters in an ACE2-dependent manner. Specific daughter delay elements in the CLN3 promoter are required for normal daughter G1 delay, and these elements bind to an unidentified 127-kDa protein. This DNA-binding activity is enhanced by deletion of ACE2. These results support a model in which daughter cell G1 delay is determined not by cell size but by an intrinsic property of the daughter cell generated by asymmetric cell division.

Base Sequence↗
PubMed2003

Aryl hydrocarbon receptor 2 mediates 2,3,7,8-tetrachlorodibenzo-p-dioxin developmental toxicity in zebrafish.

In order to use the zebrafish as a model vertebrate to investigate the developmental toxicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), it is essential to know whether one or both forms of the zebrafish aryl hydrocarbon receptor (AHR), zfAHR1 or zfAHR2, mediate toxicity. To determine the role of zfAHR2, an antisense morpholino approach was used to knock down translation of the protein. No effect of the zfahr2 morpholino (zfahr2-MO) was seen on normal development in embryos not treated with TCDD. Injection of embryos at the 1-2 cell stage with zfahr2-MO decreased TCDD-induced transcription of zfCYP1A mRNA until 96 h post fertilization (hpf), and immuno-histochemical detection of zfCYP1A protein in embryos at 72 hpf revealed a dramatic decrease in expression. The zfahr2-MO completely protected embryos from TCDD-induced edema and anemia and provided protection against TCDD-induced reductions in peripheral blood flow initially; however, a slight reduction in blood flow was observed at later times when the morpholino was no longer effective. Due to persistence of TCDD and decreasing effectiveness of the zfahr2-MO over time, the morpholino provided only transient protection against TCDD-induced inhibition of chondrogenesis of the lower jaw, and no protection against an effect of TCDD that was initiated late in development, blockade of swimbladder inflation. The zfahr2-MO did not protect embryos from TCDD-induced mortality but did produce a 48 h delay in its onset. Endpoints of TCDD developmental toxicity manifested in zfahr2 morphants at late stages of development, beyond 144 hpf, were clearly different from TCDD-exposed embryos injected with a control morpholino. Most strikingly, zfahr2 morphants exposed to TCDD never developed edema. Taken together, these results demonstrate that zfAHR2 mediates several endpoints of TCDD developmental toxicity in zebrafish.

Animals↗
PubMed2003

Glucose regulation of Saccharomyces cerevisiae cell cycle genes.

Nutrient-limited Saccharomyces cerevisiae cells rapidly resume proliferative growth when transferred into glucose medium. This is preceded by a rapid increase in CLN3, BCK2, and CDC28 mRNAs encoding cell cycle regulatory proteins that promote progress through Start. We have tested the ability of mutations in known glucose signaling pathways to block glucose induction of CLN3, BCK2, and CDC28. We find that loss of the Snf3 and Rgt2 glucose sensors does not block glucose induction, nor does deletion of HXK2, encoding the hexokinase isoenzyme involved in glucose repression signaling. Rapamycin blockade of the Tor nutrient sensing pathway does not block the glucose response. Addition of 2-deoxy glucose to the medium will not substitute for glucose. These results indicate that glucose metabolism generates the signal required for induction of CLN3, BCK2, and CDC28. In support of this conclusion, we find that addition of iodoacetate, an inhibitor of the glyceraldehyde-3-phosphate dehydrogenase step in yeast glycolysis, strongly downregulates the levels CLN3, BCK2, and CDC28 mRNAs. Furthermore, mutations in PFK1 and PFK2, which encode phosphofructokinase isoforms, inhibit glucose induction of CLN3, BCK2, and CDC28. These results indicate a link between the rate of glycolysis and the expression of genes that are critical for passage through G(1).

CDC28 Protein Kinase, S cerevisiae↗
PubMed2002

Identification of a critical amino acid in the aryl hydrocarbon receptor.

Two aryl hydrocarbon receptors (rtAHR2alpha and rtAHR2beta) have been identified in the rainbow trout (Oncorhynchus mykiss). These receptors share 98% amino acid identity, yet their functional properties differ. Both rtAHR2alpha and rtAHR2beta bind 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), dimerize with rainbow trout ARNTb (rtARNTb), and recognize dioxin response elements in vitro. However, in a transient transfection assay the two proteins show differential ability to recognize enhancers, produce transactivation, and respond to TCDD. To identify the sequence differences that confer the functional differences between rtAHR2alpha and rtAHR2beta, we constructed chimeric rtAHRs, in which segments of one receptor form was replaced with the corresponding part from the other isoform. This approach progressively narrowed the region being examined to a single residue, corresponding to position 111 in rtAHR2beta. Altering this residue in rtAHR2beta from the lysine to glutamate found in rtAHR2alpha produced an rtAHR2beta with the properties of rtAHR2alpha. All other known AHRs resemble rtAHR2alpha and carry glutamate at this position, located at the N terminus of the PAS-A domain. We tested the effect of altering this glutamate in the human and zebrafish AHRs to lysine. This lysine substitution produced AHRs with transactivation properties that were similar to rtAHR2beta. These results identify a critical residue in AHR proteins that has an important impact on transactivation, enhancer site recognition, and regulation by ligand.

Amino Acid Sequence↗
PubMed2002

Tissue-specific expression of AHR2, ARNT2, and CYP1A in zebrafish embryos and larvae: effects of developmental stage and 2,3,7,8-tetrachlorodibenzo-p-dioxin exposure.

To better understand the role of the aryl hydrocarbon receptor (AHR) signaling pathway in causing tissue-specific signs of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) toxicity in zebrafish, the temporal and spatial expression of the zebrafish aryl hydrocarbon receptor 2 (zfAHR2), aryl hydrocarbon receptor nuclear translocator 2 (zfARNT2), and an AHR regulated gene, cytochrome P4501A (zfCYP1A), were assessed in larvae exposed to vehicle or TCDD (1.55 nM) from 3-4 h postfertilization (hpf). Coexpression of a transcriptionally active AHR pathway was apparent by the expression of zfCYP1A mRNA and protein in certain larval tissues. zfCYP1A protein was first detected in the skin and vasculature of TCDD-exposed larvae at 36 hpf. Vascular-specific zfCYP1A protein expression continued from 36 to120 hpf at which time it was also detected in the heart, kidney, and liver. zfCYP1A mRNA was observed in TCDD treated larvae as early as 24 hpf in the developing vascular system. Vascular specific zfCYP1A mRNA expression in the head, trunk, and tail by 36 hpf in TCDD-exposed larvae, confirmed immunohistochemical localization. The expression of zfAHR2 and zfARNT2 mRNAs was generally similar in control and TCDD-exposed larvae. Coexpression of zfAHR2, zfARNT2, and zfCYP1A mRNAs was evident in TCDD-exposed larvae by 36 hpf and in the vasculature, heart, and trunk kidney by 48 hpf, well before the first signs of overt developmental toxicity are observed. In addition to their function in response to AHR agonists, zfAHR2 and zfARNT2 may be involved in development and function of the nervous system. zfAHR2 and zfARNT2 were detected in the brain, spinal cord, and sensory organs. However, TCDD-induced zfCYP1A expression was not detected in these tissues. Taken together, these results are consistent with the notion that the cardiovascular system is a primary target of TCDD developmental toxicity in zebrafish.

Animals↗