The case for thyroid disruption in early life stage exposures to thiram in zebrafish (Danio rerio).

Thiram, a pesticide in the dithiocarbamate chemical family, is widely used to prevent fungal disease in seeds and crops. Its off-site movement to surface waters occurs and may place aquatic organisms at potential harm. Zebrafish embryos were used for investigation of acute (1 h) thiram exposure (0.001-10 µM) at various developmental stages. Survival decreased at 1 µM and 10 µM and hatching was delayed at 0.1 µM and 1 µM. Notochord curvatures were seen at 0.1 and 1 µM thiram when exposure was initiated at 2 and at 10 hpf. Similar notochord curvatures followed exposure to the known TPO inhibitor, methimazole (MMI). Changes were absent in embryos exposed at later stages, i.e., 12 hpf. In embryos exposed to 0.1 or 1 µM at 10 hpf, levels of the thyroid enzyme, Deiodinase 3, increased by 12 hpf. Thyroid peroxide (TPO), important in T4 synthesis, decreased by 48 hpf in embryos exposed to 1 µM at 10 hpf. Thiram toxicity was stage-dependent and early life stage exposure may be responsible for adverse effects seen later. These effects may be due to impacts on the thyroid via regulation of specific thyroid genes including TPO and Deiodinase 3.

Characterization of feline cytochrome P450 2B6.

1. Little is known about drug metabolism in carnivores. Although the domestic cat (Felis catus) is an obligate carnivore and is the most common companion animal, usage and dosage of many drugs are determined according to information obtained from humans and dogs. We determined the complete cDNA sequence of CYP2B6 from the feline lung. 2. Feline CYP2B6 consists of 494 deduced amino acids, showing highest identity with the dog CYP2B ortholog, followed by those of horse, pig, primate and human. 3. Feline CYP2B6 transcripts were expressed predominantly in the lung and slightly in the small intestine but not in the liver without significant sex-dependent differences. Western blot analysis with an anti-human CYP2B6 antibody confirmed the presence of CYP2B protein in the lung but not in the liver. 4. Feline CYP2B6 proteins heterologously expressed in Escherichia coli metabolized several substrates specific to human CYP2B6, including 7-ethoxy-4-(trifluoromethyl) coumarin (EFC). The metabolic activity was strongly inhibited by medetomidine and atipamezole, potent inhibitors of canine CYP2B11 (now officially CYP2B6) as well as by ticlopidine and sertraline, inhibitors selective to human CYP2B6. 5. The results suggest that feline CYP2B6 is a functional CYP2B ortholog that plays a role in the local defense mechanism in the cat respiratory system and intestine.

Accumulation properties of inorganic mercury and organic mercury in the red-crowned crane Grus japonensis in east Hokkaido, Japan.

The red-crowned (Japanese) crane Grus japonensis is native to east Hokkaido, Japan, in contrast to the East Asia mainland. Previously, we reported that red-crowned cranes in Hokkaido were highly contaminated with mercury in the 1990s and that the contamination rapidly decreased to a moderate level in the 2000s. In the present study, we determined levels of organic mercury (O-Hg) in the liver and kidney of cranes in east Hokkaido in comparison with levels of total mercury (T-Hg). T-Hg levels in the kidneys were higher than those in the livers in adults but not in subadults and juveniles; however, the reverse was the case for O-Hg even for adults. The ratio of O-Hg to T-Hg in both the liver and kidney decreased as T-Hg increased in the three developmental stages. While the ratios of O-Hg to T-Hg in the liver and kidney of adults were significantly lower than those of juveniles, the ratios were similar for adults and juveniles in a lower range of T-Hg. The ratio of selenium (Se) to T-Hg decreased as T-Hg increased in both the liver and kidney, irrespective of stages. Mercury burdens in feathers were about 59% and 67% of the total body burdens for juveniles and adults, respectively. Furthermore, ratios of carbon and nitrogen stable isotopes to T-Hg varied greatly, with no relation to mercury level in the liver. The results suggest slow accumulation of inorganic mercury in the kidney of red-crowned cranes in east Hokkaido, Japan.

Changes of mercury contamination in red-crowned cranes, Grus japonensis, in East Hokkaido, Japan.

Red-crowned cranes (Grus japonensis) are native to eastern Hokkaido (island population), in contrast to the mainland, which migrates between the Amur River basin and eastern China-Korea peninsula. During the 1990s we found that Red-crowned cranes in Hokkaido were highly contaminated with mercury: however, the source was unknown. We investigated the time trend of mercury contamination in Red-crowned cranes. Total mercury levels in the livers and kidneys from cranes dead in the 2000s were lower than those dead in the 1990s. Feather is a major pathway of mercury excretion for many bird species and is used as an indicator of blood mercury level during feather growth. As internal organs from the specimens collected before 1988 were not available, we analyzed the flight feather shavings from stuffed Red-crowned cranes dead in 1959-1987 and found that the mercury level of feathers from cranes dead in the 1960s and 1970s was not more than those from the cranes dead in the 2000s. These results suggest that mercury contamination in Red-crowned cranes in Hokkaido decreased temporally during the 1990s-2000s. This indicates the possible occurrence of some mercury pollution in Red-crowned cranes' habitat in this region in the 1990s or before.

Role of zebrafish cytochrome P450 CYP1C genes in the reduced mesencephalic vein blood flow caused by activation of AHR2.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) causes various signs of toxicity in early life stages of vertebrates through activation of the aryl hydrocarbon receptor (AHR). We previously reported a sensitive and useful endpoint of TCDD developmental toxicity in zebrafish, namely a decrease in blood flow in the dorsal midbrain, but downstream genes involved in the effect are not known. The present study addressed the role of zebrafish cytochrome P450 1C (CYP1C) genes in association with a decrease in mesencephalic vein (MsV) blood flow. The CYP1C subfamily was recently discovered in fish and includes the paralogues CYP1C1 and CYP1C2, both of which are induced via AHR2 in zebrafish embryos. We used morpholino antisense oligonucleotides (MO or morpholino) to block initiation of translation of the target genes. TCDD-induced mRNA expression of CYP1Cs and a decrease in MsV blood flow were both blocked by gene knockdown of AHR2. Gene knockdown of CYP1C1 by two different morpholinos and CYP1C2 by two different morpholinos, but not by their 5 nucleotide-mismatch controls, was effective in blocking reduced MsV blood flow caused by TCDD. The same CYP1C-MOs prevented reduction of blood flow in the MsV caused by ß-naphthoflavone (BNF), representing another class of AHR agonists. Whole-mount in situ hybridization revealed that mRNA expression of CYP1C1 and CYP1C2 was induced by TCDD most strongly in branchiogenic primordia and pectoral fin buds. In situ hybridization using head transverse sections showed that TCDD increased the expression of both CYP1Cs in endothelial cells of blood vessels, including the MsV. These results indicate a potential role of CYP1C1 and CYP1C2 in the local circulation failure induced by AHR2 activation in the dorsal midbrain of the zebrafish embryo.

Molecular cloning and expression of cytochrome P450 2D6 in the livers of domestic cats.

Cytochrome P450 2D (CYP2D) is one of the major metabolizing enzymes for drugs in humans and other mammals. Although domestic cats (Felis catus) are the most common companion animal in many countries, the properties of their drug-metabolizing system are largely unknown. We determined the complete cDNA sequence of the CYP2D open reading frame (1,500 bp) with both 5'- and 3'-untranslated regions in domestic cats. Feline CYP2D consists of 500 deduced amino acids and shows high homology with CYP2Ds of primates and rodents, particularly with the canine CYP2D homologue, and was designated as CYP2D6 based on its genomic structure. CYP2D6 transcripts were expressed predominantly in the liver and to a much lesser in the testis, suggesting the role of xenobiotic metabolism in the liver.

Role of the cyclooxygenase 2-thromboxane pathway in 2,3,7,8-tetrachlorodibenzo-p-dioxin-induced decrease in mesencephalic vein blood flow in the zebrafish embryo.

Previously, we reported that 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) evoked developmental toxicity required activation of aryl hydrocarbon receptor type 2 (AHR2), using zebrafish embryos. However, the downstream molecular targets of AHR2 activation are largely unknown and are the focus of the present investigation. TCDD induces cyclooxygenase 2 (COX2), a rate-limiting enzyme for prostaglandin synthesis in certain cells. In the present study, we investigated the role of the COX2-thromboxane pathway in causing a specific endpoint of TCDD developmental toxicity in the zebrafish embryo, namely, a decrease in regional blood flow in the dorsal midbrain. It was found that the TCDD-induced reduction in mesencephalic vein blood flow was markedly inhibited by selective COX2 inhibitors, NS-398 and SC-236, and by a general COX inhibitor, indomethacin, but not by a selective COX1 inhibitor, SC-560. Gene knock-down of COX2 by two different types of morpholino antisense oligonucleotides, but not by their negative homologs, also protected the zebrafish embryos from mesencephalic vein circulation failure caused by TCDD. This inhibitory effect of TCDD on regional blood flow in the dorsal midbrain was also blocked by selective antagonists of the thromboxane receptor (TP). Treatment of control zebrafish embryos with a TP agonist also caused a reduction in mesencephalic vein blood flow and it too was blocked by a TP antagonist, without any effect on trunk circulation. Finally, gene knock-down of thromboxane A synthase 1 (TBXS) with morpholinos but not by the morpholinos' negative homologs provided significant protection against TCDD-induced mesencephalic circulation failure. Taken together, these results point to a role of the prostanoid synthesis pathway via COX2-TBXS-TP in the local circulation failure induced by TCDD in the dorsal midbrain of the zebrafish embryo.

Lead exposure induces pycnosis and enucleation of peripheral erythrocytes in the domestic fowl.

Lead (Pb) shot were administered orally to young chickens to determine the effects on red blood cells (RBCs). The concentrations of Pb in the blood of young chickens rose rapidly after Pb administration and were maintained at high levels for several days. The number of RBCs with pycnotic nuclei, reticulocytes and enucleated RBCs increased concurrently. Pycnotic nuclei were surrounded by enlarged nucleolemmal cisternae, which sometimes opened to the extracellular space. Gel electrophoresis showed that the presence of pycnotic nuclei was not associated with DNA fragmentation typical of apoptosis. It was concluded that exposure to lead shot changes nuclear morphology in the peripheral blood of domestic fowl.

Functional disorder of the retina in manganese-deficient Japanese quail revealed by electroretinography using a contact lens electrode with built-in light source.

Manganese deficiency results in neurological and skeletal defects, together with ultrastructural disarrangement of the retina in rats. Wild birds show a range of Mn concentrations in their tissues, including the liver, raising the possibility of Mn-related disorders in the wild. Electroretinography (ERG) provides a useful noninvasive approach to evaluate visual function. This method is especially useful in birds, as objective analysis of them is very difficult, while they have well-developed vision. In this study, we carried out a convenient and reliable ERG recording using a contact lens electrode with a built-in light source (LED electrode) of Japanese quail (Coturnix coturnix japonica) fed a Mn-deficient diet. After 10 min light adaptation, single-flash and flicker cone responses were reproducibly recorded to cause an intensity-dependent increase in amplitude of both a-wave and b-wave in single-flash ERG. Mn-deficient feeding markedly decreased the Mn concentration in the liver by almost half in 3 to 6 weeks, followed by body weight loss in 13 to 15 weeks. Implicit time of a-wave and b-wave cone response by single-flash stimulation was significantly delayed in quail with a Mn depletion from 3 to 6 weeks. Every cone response of the Mn-deprived quail had a tendency to decrease amplitude. The ultrastructure of cone photoreceptor cells was disorganized by Mn deficiency, including changes in outer segment discs of photoreceptor cells. These results suggest the essential role of Mn in the integrity of the retinal function of birds.

Heavy metal contamination status of Japanese cranes (Grus japonensis) in east Hokkaido, Japan--extensive mercury pollution.

Japanese cranes (Grus japonensis) of eastern Hokkaido, Japan, and migrants between the Amur River basin and the eastern China-Korea Peninsula, live around fresh and brackish wetlands. Only a few thousand cranes are confirmed to exist in the world, so they are under threat of extinction. To understand the adverse effects of metal accumulation, we measured concentrations of three heavy metals in the liver, kidney, and muscle of 93 Japanese cranes from Hokkaido. The cranes were classified into six categories according to their sex and three life stages. Cadmium and mercury (Hg: total mercury) showed age-dependent but not sex-dependent accumulation in the liver and kidney. Twenty cranes showed 30 microg/g or higher levels of Hg in dry tissue and five adult cranes had more than 100 microg/g in their livers or kidneys. Cadmium concentrations were generally lower in all samples. Two adult cranes showed extremely high lead levels of more than 30 microg/g in their livers, suggesting lead poisoning. These results have highlighted the widespread and high levels of Hg pollution in Japanese cranes in Hokkaido, Japan.

Impairment of lower jaw growth in developing zebrafish exposed to 2,3,7,8-tetrachlorodibenzo-p-dioxin and reduced hedgehog expression.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) has been shown to cause a multitude of detrimental effects to developing zebrafish (Danio rerio). Previously, we demonstrated that jaw growth was impaired by TCDD exposure, but the exact mechanism underlying these malformations remained unknown. In the present study, we investigated the involvement of hedgehog genes and their downstream signaling in TCDD-mediated jaw malformation. We demonstrate that the developing lower jaw expresses sonic hedgehog a (shha), sonic hedgehog b (shhb) and their receptors, patched1 (ptc1) and patched2 (ptc2), as well as the downstream transcription factors, gli1 and gli2a. Loss of Hh signaling in mutants (sonic you) and larvae treated with a Hh inhibitor (cyclopamine), resulted in similar effects as those caused by TCDD. Moreover, TCDD exposure caused downregulation of shha and shhb in a manner dependent on aryl hydrocarbon receptor 2 (ahr2). Although this suggested an involvement of Hh signaling in TCDD-mediated impairment of jaw growth, we did not observe downregulation of ptc1 and ptc2, receptors dependent on Hh signaling. Furthermore, while the overall occurrence of apoptosis in the developing jaw was minimal, it was significantly increased in larvae treated with cyclopamine. In contrast, both TCDD and cyclopamine markedly reduced immunoreactivity against phosphorylated histone 3, a cell proliferation marker in the developing jaw. Taken together, our data suggest that Ahr2-mediated downregulation of Hh signaling, leading to a failure of cell proliferation, contributes to TCDD induced inhibition of lower jaw growth. TCDD may impair jaw growth through other pathway(s) in addition to Hh signaling.

Retinal neuronal cell is a toxicological target of tributyltin in developing zebrafish.

Organotins are among the most common marine pollutants in the world, as they are widely used as antifouling paint on ships and fishing nets. It has been reported that organotin preferentially accumulates in the central nervous system, especially in the retinal neurons of marine organisms including fish. In this study, we investigated the effects of waterborne tributyltin (TBT) on early-stage developing zebrafish (Danio rerio). Below the lethal concentrations, TBT specifically increased the number of apoptotic cells in the retina as well as some cells near trigeminal neurons, detected by terminal transferase-mediated nick-end-labeling staining. Apoptosis peaked at 60 hpf and decreased by 72 hpf, which was associated with macrophage accumulation. Furthermore, the effect of TBT was markedly inhibited by antioxidants, ascorbic acid or trolox. These results suggest that TBT preferentially induces apoptosis in the retinal neuron of developing zebrafish. Oxidative stress may be involved in this toxicological response.

Correlation of ghrelin concentration and ghrelin, ghrelin-O-acetyltransferase (GOAT) and growth hormone secretagogue receptor 1a mRNAs expression in the proventriculus and brain of the growing chicken.

To determine mechanisms for age-related decrease of GHS-R1a expression in the chicken proventriculus, changes in mRNA expression of ghrelin and ghrelin-O-acetyltransferase (GOAT) as well as ghrelin concentrations in the proventriculus and plasma were examined in growing chickens. Changes in expression levels of ghrelin, GOAT and GHS-R1a mRNAs were also examined in different brain regions (pituitary, hypothalamus, thalamus, cerebellum, cerebral cortex, olfactory bulb, midbrain and medulla oblongata). Ghrelin concentrations in the proventriculus and plasma increased with aging and reached plateaus at 30-50 days after hatching. High level of ghrelin mRNA decreased at 3 days after hatching, and it became stable at half of the initial level. Expression levels of GHS-R1a and GOAT decreased 3 or 5 days after hatching and became stable at low levels. Significant negative correlations were found between plasma ghrelin and mRNA levels of GOAT and GHS-R1a. Expression levels of ghrelin mRNA were different in the brain regions, but a significant change was not seen with aging. GHS-R1a expression was detected in all brain regions, and age-dependent changes were observed in the pituitary and cerebellum. Different from the proventriculus, the expression of GOAT in the brain increased or did not change with aging. These results suggest that decreased GHS-R1a and GOAT mRNA expression in the proventriculus is due to endogenous ghrelin-induced down-regulation. Expression levels of ghrelin, GOAT and GHS-R1a in the brain were independently regulated from that in the proventriculus, and age-related and region-dependent regulation pattern suggests a local effect of ghrelin system in chicken brain.

Role of aryl hydrocarbon receptor in mesencephalic circulation failure and apoptosis in zebrafish embryos exposed to 2,3,7,8-tetrachlorodibenzo-p-dioxin.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is a persistent and potent developmental toxicant in various animals, with developing fish being the most sensitive organisms. Although the expression of aryl hydrocarbon receptor (AHR) as well as the partner molecule, AHR nuclear translocator (ARNT) in the brain has been reported, the effect of TCDD on the brain remains to be clarified in detail. Previously, we reported local circulation failure and apoptosis in dorsal midbrain caused by TCDD in developing zebrafish. In the present experiments, we investigated the effects of morpholino antisense oligos against aryl hydrocarbon receptor 2 (zfAHR2) (AHR2-MO) on toxicological endpoints caused by TCDD in developing zebrafish. AHR2-MO but not its negative homologue (4mis-AHR2-MO) improved TCDD-evoked circulation failure in mesencephalic vein and reduced the occurrence of apoptosis in dorsal midbrain, with concomitant inhibition of CYP1A induction in vascular endothelium. Injection of bovine serum albumin (BSA) into the general circulation, followed by immunohistochemistry with anti-BSA, showed that TCDD raised vascular permeability to albumin in dorsal midbrain, which was blocked by AHR2-MO and N-acetlycystein. In the absence of TCDD, development of embryos injected with AHR2-MO appeared normal at least until 60 h after fertilization. It is concluded that AHR2 activation in the vascular endothelium of the zebrafish embryo midbrain is involved in the mesencephalic circulation failure and apoptosis elicited by TCDD. This is the further evidence that vascular endothelium is the target of TCDD in relation to local circulation failure and apoptosis in dorsal midbrain.

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.

2,3,7,8-Tetrachlorodibenzo-p-dioxin toxicity in the zebrafish embryo: altered regional blood flow and impaired lower jaw development.

The effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) exposure on regional red blood cell (RBC) perfusion rate, as an index of blood flow, and lower jaw development were investigated quantitatively in zebrafish embryos (Danio rerio) during early development. As revealed by observation of live embryos and alcian-blue staining, TCDD retarded lower jaw development in a concentration-dependent manner with only a minor inhibitory effect on total body length. Both inhibitory effects were significant as early as 60 h postfertilization (hpf), at which time the area of goosecoid (gsc) mRNA expression was clearly reduced in the lower jaw. To examine effects of TCDD on RBC perfusion rate, time-lapse recording was performed using a digital video camera attached to a light microscope. TCDD did not show marked effects on RBC perfusion rate until 72 hpf, when vessel-specific effects emerged. TCDD severely inhibited RBC perfusion rate in intersegmental arteries of the trunk, but only modestly and slightly inhibited RBC perfusion rate in certain vessels of the head such as the central arteries and optic vein. Conversely, at both 72 and 84 hpf, TCDD significantly increased RBC perfusion rate in the hypobranchial artery branching to the lower jaw primordia, and then reduced it at 96 hpf. RBC perfusion rate in all vessels examined in TCDD-exposed embryos was inhibited at 96 hpf. The zebrafish aryl hydrocarbon receptor 2 (zfAhR2) mRNA was strongly expressed in the lower jaw primordia at 48 hpf, and expression of this transcript was augmented by TCDD treatment. Thus, TCDD exposure of the zebrafish embryo has a disruptive effect on local circulation and lower jaw cartilage growth. Initially, TCDD may act directly on the lower jaw primordia to impair lower jaw development. Reductions in hypobranchial RBC perfusion rate occurred well after the initial retardation in lower jaw development had become apparent, and may contribute further to the effect.

Zebrafish as a novel experimental model for developmental toxicology.

It is widely believed that embryos and infants during development are highly sensitive to chemicals that cause serious damage to growth. However, knowledge on the mechanisms of developmental toxicity is scarce. One reason for this is limited convenient model system other than organ cultures using rodents to study the various aspects of developmental toxicology. Cultured cells are not always adequate for this purpose, since events in morphogenesis are processed through interactions with other tissues. We focused on zebrafish embryo (Danio rerio), one of the most important organisms in developmental biology. Saturation mutagenesis, applied to drosophila and nematode to define the functions of genes, has been carried out in zebrafish but almost no other vertebrate, and several thousand lines are available due to the rapid growth and transparent body of this embryo. Enhanced databases for the genome and ESTs are available at websites with abundant genetic and biological background. By targeted gene knock-down with morpholino-modified antisense oligonucleotieds (morpholinos), the translation of a specific protein can be transiently blocked for several days. Many reporter systems in vivo have been established mainly as GFP-transgenic fish for environmental chemicals. Although several excellent studies have been performed with zebrafish embryos on the effects of chemicals, the developmental toxicology of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) has been most extensively studied to date. We have found that TCDD induces apoptosis in dorsal midbrain with a concomitant decrease in local blood flow, using developing zebrafish. TCDD seems to produce oxidative stress through CYP1A induction in vascular endothelium, resulting in local circulation failure and apoptosis in the dorsal midbrain. In addition to applications in toxicology, an experimental system with zebrafish embryos could help to clarify the mechanism of congenital anomaly, which arises from genetic mutation.

cDNA cloning and expressions of cytochrome P450 1A in zebrafish embryos.

Cytochrome P450 1A (CYP1A) is well known for being induced by aromatic hydrocarbons, including 2,3,7,8-tetrachloro-dibenzo-p-dioxin (TCDD). We determined the complete cDNA sequence of a CYP1A open reading frame with both 5'- and 3'-ends in zebrafish (zfCYP1A), a useful model for environmental toxicology. zfCYP1A shows high percentage identity with CYP1As of mammals, domestic fowl and xenopus (51.9-60.4%), as well as the other fish species (63.8-89.2%). As revealed by in situ hybridization and immunohistochemistry, zfCYP1A was scarcely detected in control embryos but was markedly induced by TCDD especially in heart, vascular endothelial cells, intestinal epithelium, pronephros and outer integument in both prehatched and hatched embryos. These expression patterns are consistent with possible involvement of zfCYP1A in TCDD-induced toxicities.

Vertebral formula in red-crowned crane (Grus japonensis) and hooded crane (Grus monacha).

Red-crowned cranes (Grus japonensis) are distributed separately in the east Eurasian Continent (continental population) and in Hokkaido, Japan (island population). The island population is sedentary in eastern Hokkaido and has increased from a very small number of cranes to over 1,300, thus giving rise to the problem of poor genetic diversity. While, Hooded cranes (Grus monacha), which migrate from the east Eurasian Continent and winter mainly in Izumi, Kagoshima Prefecture, Japan, are about eight-time larger than the island population of Red-crowned cranes. We collected whole bodies of these two species, found dead or moribund in eastern Hokkaido and in Izumi, and observed skeletons with focus on vertebral formula. Numbers of cervical vertebrae (Cs), thoracic vertebrae (Ts), vertebrae composing the synsacrum (Sa) and free coccygeal vertebrae (free Cos) in 22 Red-crowned cranes were 17 or 18, 9-11, 13 or 14 and 7 or 8, respectively. Total number of vertebrae was 47, 48 or 49, and the vertebral formula was divided into three types including 9 sub-types. Numbers of Cs, Ts, vertebrae composing the Sa and free Cos in 25 Hooded cranes were 17 or 18, 9 or 10, 12-14 and 6-8, respectively. Total number of vertebrae was 46, 47, 48 or 49, and the vertebral formula was divided into four types including 14 sub-types. Our findings clearly showed various numerical vertebral patterns in both crane species; however, these variations in the vertebral formula may be unrelated to the genetic diversity.

Involvement of COX2-thromboxane pathway in TCDD-induced precardiac edema in developing zebrafish.

The cardiovascular system is one of the most characteristic and important targets for developmental toxicity by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in fish larvae. However, knowledge of the mechanism of TCDD-induced edema after heterodimerization of aryl hydrocarbon receptor type 2 (AHR2) and AHR nuclear translocator type 1 (ARNT1) is still limited. In the present study, microscopic analysis with a high-speed camera revealed that TCDD increased the size of a small cavity between the heart and body wall in early eleutheroembryos, a toxic effect that we designate as precardiac edema. A concentration-response curve for precardiac edema at 2 days post fertilization (dpf) showed close similarity to that for conventional pericardial edema at 3 dpf. Precardiac edema caused by TCDD was reduced by morpholino knockdown of AHR2 and ARNT1, as well as by an antioxidant (ascorbic acid). A selective inhibitor of cyclooxygenase type 2 (COX2), NS398, also markedly inhibited TCDD-induced precardiac edema. A thromboxane receptor (TP) antagonist, ICI-192,605 almost abolished TCDD-induced precardiac edema and this effect was canceled by U46619, a TP agonist, which was not influential in the action of TCDD by itself. Knockdown of COX2b and thromboxane A synthase 1 (TBXS), but not COX2a, strongly reduced TCDD-induced precardiac edema. Knockdown of COX2b was without effect on mesencephalic circulation failure caused by TCDD. The edema by TCDD was also inhibited by knockdown of c-mpl, a thrombopoietin receptor necessary for thromobocyte production. Finally, induction of COX2b, but not COX2a, by TCDD was seen in eleutheroembryos at 3 dpf. These results suggest a role of the COX2b-thromboxane pathway in precardiac edema formation following TCDD exposure in developing zebrafish.