Long-term Xenopus laevis tadpole -heart-organ-culture: Physiological changes in cholinergic and adrenergic sensitivities of tadpole heart with thyroxine-treatment.

The present study clarified changes in physiological sensitivities of cultured Nieuwkoop and Faber stage 57 Xenopus laevis tadpole-organ-heart exposed to thyroxine (T4) using acetylcholine (ACh), norepinephrine (NE) and atropine. For preliminary life span and the chemical tests, 60% minimum essential medium (MEM), two types of modified Hank's balanced salt-solution-culture-media (MHBSS-CM) I and II containing relatively lower concentrations of amino acids and collagen were prepared. In preliminary lifespan-test of cultured tadpole hearts, the hearts maintained in 60% MEM was 50 days on average, whereas that of the tadpole-hearts in MHBSS-CMs was extended by 109 days on average, showing superior effectiveness of MHBSS-CMs. 4 min-stimulation by 5 × 10-9 M T4 tended to increase the tadpole heartbeat. 10-9 M ACh decreased the tadpole heartbeat. Frog-heart at 2-4 weeks after metamorphosis completion and tadpole heart treated with 5 × 10-10 M T4 for 45 h also responded to 10-9 M ACh, and low-resting hearts were restored to the control level with the competitive muscarinic antagonist 10-8 M atropine, whereas excessive exposure of 10-5 M atropine to T4-treated tadpole heart did not increase heartbeat in spite of the increased frog heartbeat over the control. 10-14 -10-12 M NE increase the tadpole heartbeat in a concentration-dependent manner, however, 10-12 M NE did not act to stimulate adrenergic receptors on both T4-treated tadpole- and the frog-hearts. These results suggest that T4 induces the desensitization of atropine-sensitive muscarinic and adrenergic receptors in organ-cultured tadpole-heart.

Amiodarone bioconcentration and suppression of metamorphosis in Xenopus.

Trace concentrations of a number of pharmaceutically active compounds have been detected in the aquatic environment in many countries, where they are thought to have the potential to exert adverse effects on non-target organisms. Amiodarone (AMD) is one such high-risk compound commonly used in general hospitals. AMD is known to alter normal thyroid hormone (TH) function, although little information is available regarding the specific mechanism by which this disruption occurs. Anuran tadpole metamorphosis is a TH-controlled developmental process and has proven to be useful as a screening tool for environmental pollutants suspected of disrupting TH functions. In the present study, our objective was to clarify the effects of AMD on Xenopus metamorphosis as well as to assess the bioconcentration of this pharmaceutical in the liver. We found that AMD suppressed spontaneous metamorphosis, including tail regression and hindlimb elongation in pro-metamorphic stage tadpoles, which is controlled by endogenous circulating TH, indicating that AMD is a TH antagonist. In transgenic X. laevis tadpoles carrying plasmid DNA containing TH-responsive element (TRE) and a 5'-upstream promoter region of the TH receptor (TR) ßA1 gene linked to a green fluorescent protein (EGFP) gene, triiodothyronine (T3) exposure induced a strong EGFP expression in the hind limbs, whereas the addition of AMD to T3 suppressed EGFP expression, suggesting that this drug interferes with the binding of T3 to TR, leading to the inhibition of TR-mediated gene expression. We also found AMD to be highly bioconcentrated in the liver of pro-metamorphic X. tropicalis tadpoles, and we monitored hepatic accumulation of this drug using mass spectrometry imaging (MSI). Our findings suggest that AMD imposes potential risk to aquatic wildlife by disrupting TH homeostasis, with further possibility of accumulating in organisms higher up in the food chain.

Developmental changes in drug-metabolizing enzyme expression during metamorphosis of Xenopus tropicalis.

A large number of chemicals are routinely detected in aquatic environments, and these chemicals may adversely affect aquatic organisms. Accurate risk assessment requires understanding drug-metabolizing systems in aquatic organisms because metabolism of these chemicals is a critical determinant of chemical bioaccumulation and related toxicity. In this study, we evaluated mRNA expression levels of nuclear receptors and drug-metabolizing enzymes as well as cytochrome P450 (CYP) activities in pro-metamorphic tadpoles, froglets, and adult frogs to determine how drug-metabolizing systems are altered at different life stages. We found that drug-metabolizing systems in tadpoles were entirely immature, and therefore, tadpoles appeared to be more susceptible to chemicals compared with metamorphosed frogs. On the other hand, cyp1a mRNA expression and CYP1A-like activity were higher in tadpoles. We found that thyroid hormone (TH), which increases during metamorphosis, induced CYP1A-like activity. Because endogenous TH concentration is significantly increased during metamorphosis, endogenous TH would induce CYP1A-like activity in tadpoles.

Acetyl-L-carnitine suppresses thyroid hormone-induced and spontaneous anuran tadpole tail shortening.

Mitochondrial membrane permeability transition (MPT) plays a crucial role in apoptotic tail shortening during anuran metamorphosis. L-carnitine is known to shuttle free fatty acids (FFAs) from the cytosol into mitochondria matrix for ß-oxidation and energy production, and in a previous study we found that treatment with L-carnitine suppresses 3, 3', 5-triiodothyronine (T3 ) and FFA-induced MPT by reducing the level of FFAs. In the present study we focus on acetyl-L-carnitine, which is also involved in fatty acid oxidation, to determine its effect on T3 -induced tail regression in Rana rugosa tadpoles and spontaneous tail regression in Xenopus laevis tadpoles. The ladder-like DNA profile and increases in caspase-3 and caspase-9 indicative of apoptosis in the tails of T3 -treated tadpoles were found to be suppressed by the addition of acetyl-L-carnitine. Likewise, acetyl-L-carnitine was found to inhibit thyroid hormone regulated spontaneous metamorphosis in X. laevis tadpoles, accompanied by decreases in caspase and phospholipase A2 activity, as well as non-ladder-like DNA profiles. These findings support our previous conclusion that elevated levels of FFAs initiate MPT and activate the signaling pathway controlling apoptotic cell death in tadpole tails during anuran metamorphosis.

Phenolic antioxidant 2,6-di-tert-butyl-p-cresol (vitamin E synthetic analogue) does not inhibit 1,1'-dimetyl-4,4'-bipyridium dichloride (paraquat)-induced structural chromosomal damage in cultured leukocytes of the dark-spotted-frog Pelophylax (Rana) nigromaculatus.

Pro-oxidative effect of phenolic antioxidant (vitamin E) in combination with the initiators on human low-density lipoprotein is known. Recently I reported that oxidative stress induced by vitamin E in combination with the herbicide paraquat enhances structural chromosomal damage in cultured anuran leukocytes. In the present study, the phenolic antioxidant vitamin E-synthetic-analogue 2,6-di-tert-butyl-p-cresol (BHT) in combination with paraquat was found to enhance structural chromosomal damage in cultured Pelophylax (Rana) nigromaculatus leukocytes more than paraquat only and paraquat plus nicotinamido adenine dinucleotido phosphate served as positive control, although BHT only had no effect on induction of structural chromosomal damage. Paraquat plus BHT-enhanced structural chromosomal damage was inhibited by combination of the superoxide dismutase mimic Mn(III)tetrakis(1-methyl-4-pyridyl)porphyrin and the hydrogen peroxide scavenger catalase. In test based on reduction of paraquat cation, BHT was found to reduce paraquat cation chemically to paraquat monocation radical. These results suggest that BHT functions in chemically donating electron to paraquat and thereby induces an acute accumulation of reactive oxygen species, resulting in increase in chromosomal damage.

Dl-α-tocopherol enhances the herbicide 1,1'-dimetyl-4,4'-bipyridium dichloride (paraquat, PQ) genotoxicity in cultured anuran leukocytes.

This cytogenetic and pharmacological study attempts to clarify genotoxicity-enhancement-effect of dl-α-tocopherol (one form of vitamin E) in combination with the herbicide 1,1'-dimetyl-4,4'-bipyridium dichloride (paraquat, PQ) on cultured anuran leukocytes using the superoxide dismutase-mimic Mn(III)tetrakis(1-methyl-4-pyridyl)porphyrin (Mn(III)TMpyP), the hydrogen peroxide-scavenger catalase and the electron donor nicotinamide adenine dinucleotido phosphate (NADPH). PQ only was found to induce structural chromosomal damage in cultured anuran leukocytes in a dose-dependent manner. PQ plus NADPH, which served as positive control, enhanced the genotoxic effect of PQ. Dl-α-tocopherol only did not induce any structural chromosomal damage in the leukocytes. PQ plus dl-α-tocopherol, however, enhanced the genotoxic effect of PQ. PQ plus Mn(III)TMpyP, PQ plus catalase and PQ plus Mn(III)TMpyP plus catalse suppressed the genotoxic effect of PQ. Furthermore, PQ plus dl-α-tocopherol-enhanced chromosomal damage was also inhibited by Mn(III)TMpyP plus catalase. These results suggest that dl-α-tocopherol in combination with PQ functions as an electron donor to PQ.

Xenopus tropicalis: an ideal experimental animal in amphibia.

Studies using amphibians have contributed to the progress of life science including developmental biology and cell biology for more than one hundred years. Since the 1950s Xenopus laevis in particular has been used by scientists in many fields for experiments, resulting in the development of various techniques such as microsurgery on early embryos, biosynthesis of gene-encoded protein in oocytes by mRNA injection, misexpression experiments by mRNA injection into embryos, gene knockdown studies by injection of morpholino anti-sense oligonucleotide into fertilized eggs, transgenesis by the I-SceI meganuclease method, and so on. In this paper we will introduce Xenopus tropicalis as an alternative experimental animal. It has a shorter generation time and smaller diploid genome, together with whole-genome sequence data. The procedures available for Xenopus laevis can work well with Xenopus tropicalis, and embryos of both species develop at similar rates according to the developmental staging system of Nieuwkoop and Faber. Experimental systems of Xenopus tropicalis will pave the way for a new era of vertebrate genomics and genetics.

Development of metamorphosis assay using Silurana tropicalis for the detection of thyroid system-disrupting chemicals.

The West African clawed frog (Silurana tropicalis), which resembles the South African clawed frog (Xenopus laevis), but is somewhat smaller, has a diploid genome and a shorter generation time. Therefore, S. tropicalis has the potential for use as a new model in ecotoxicology. We demonstrated a S. tropicalis metamorphosis assay based on Xenopus Metamorphosis Assay (XEMA) using 1 microg/L thyroxine (T4) and 75 mg/L propylthiouracil (PTU). Tadpoles at developmental stages 48-50 were exposed to chemicals for 28 days and total body length, developmental stage, and hind limb length were recorded every 7 days. Significant differences in developmental stage and total body length were found for both T4 and PTU after 7-day exposure, which were similar to the results of the XEMA ring-test using the same chemicals. Moreover, in the present study, we measured hind limb length as a new endpoint of thyroid axis. Significant differences in the hind limb length were encountered in both T4 and PTU treatments after 7 days of exposure. These results suggest that S. tropicalis can be used in a XEMA-like protocol to detect agonist and antagonist effects of chemicals on the thyroid system. Hind limb length is also a suitable endpoint in such protocols. A new test protocol detecting both thyroid disruption and reproductive effects of chemicals using S. tropicalis should be established in the near future.

Anti-thyroid hormonal activity of tetrabromobisphenol A, a flame retardant, and related compounds: Affinity to the mammalian thyroid hormone receptor, and effect on tadpole metamorphosis.

The thyroid hormone-disrupting activity of tetrabromobisphenol A (TBBPA), a flame retardant, and related compounds was examined. TBBPA, tetrachlorobisphenol A (TCBPA), tetramethylbisphenol A (TMBPA) and 3,3'-dimethylbisphenol A (DMBPA) markedly inhibited the binding of triiodothyronine (T3; 1 x 10(-10) M) to thyroid hormone receptor in the concentration range of 1 x 10(-7)-1 x 10(-4) M, while bisphenol A and 2,2-diphenylpropane were inactive. TBBPA, TCBPA, TMBPA and DMBPA did not exhibit thyroid hormonal activity in a thyroid hormone-responsive reporter assay using a Chinese hamster ovary cell line (CHO-K1) transfected with thyroid hormone receptor alpha1 or beta1, but TBBPA and TCBPA showed significant anti-thyroid hormone effects on the activity of T3 (1 x 10(-8) M) in the concentration range of 3 x 10(-6) - 5 x 10(-5) M. The thyroid hormone-disrupting activity of TBBPA was also examined in terms of the effect on amphibian metamorphosis stimulated by thyroid hormone. TBBPA in the concentration range of 1 x 10(-8) to 1 x 10(-6) M showed suppressive action on T3 (5 x 10(-8) M)-enhancement of Rana rugosa tadpole tail shortening. These facts suggest that TBBPA, TCBPA, TMBPA and DMBPA can act as thyroid hormone-disrupting agents.

Effects of high gravity on amphibian development.

In order to clarify the possible effects of high gravity environments on eggs and developing embryos, Rana rugosa and Xenopus laevis fertilized eggs and early embryos were raised in 2 G, 5 G, 7 G and 10 G up to the hatched tadpole stage. The results showed that: (1) High gravity significantly retarded the development of eggs and embryos beginning treatment before the blastula stage and induced various abnormalities, including two heads and microcephally suggesting that high gravity is apt to disrupt the animal-vegital axis. On the other hand, embryos beginning treatment after the gastrula stage showed a striking increase in the number of normal-appearing feeding tadpoles. (2) Autopsy revealed that brains, notochords and muscles were reduced in development and differentiation for embryos and tadpoles developed in high gravity. (3) It seems likely that the system for hydrogen peroxide detoxification develops abnormally in high gravity-treated embryos and tadpoles, which probably results in oxidative stress, leading to considerable cell damage.

Cyclosporin A inhibits thyroid hormone-induced shortening of the tadpole tail through membrane permeability transition.

Regression of the tadpole tail through muscule cell apoptosis is one of the most spectacular events in amphibian metamorphosis. Accumulated evidence has shown that mitochondrial membrane permeability transition (MPT) plays a crucial role in apoptosis. Previously we reported that cyclosporin A (CsA) suppressed 3,5,3'-triiodothyronine (T(3))-induced mitochondrial swelling, which was coupled with cytochrome c (Cyt.c) release through MPT [Comp. Biochem. Phys. 130 (2001) 411-418]. To further clarify the mechanism of tadpole metamorphosis, the present study investigates the effect of CsA on T(3) induced tadpole tail shortening. A low concentration of T(3) (5 x 10(-8) M) was found to induce a shortening of stage X Rana rugosa tadpole tails, accompanied by an increase in caspase-3- and -9 like protease activity, as well as an increase in DNA-fragmentation and ladder formation, while CsA was seen to suppress the effects of T(3). The stage X tadpole tail was found to express Bax mRNA and this expression was not affected by T(3) treatment. CsA, on the other hand, proved to have a slightly supressive effection on Bax expression. 20 microM T(3) as well as 50 microM Ca(2+) induced swelling in mitochondria isolated from the liver of R. rugosa resulting in the release of apoptosis related substances, and the released fraction activated cytosolic caspase-3 and -9 in the presence of dATP. This result indicated that Cyt.c might be released from mitochondria by treatment with T(3) through both direct and indirect action of T(3). From these results and other data it was concluded that mitochondrial MPT plays an important role in T(3)-induced apoptosis in the tadpole tail, resulting in tail shortening, and CsA was seen to suppress the effects of T(3).

G and C banding show structural differences between the Z and W chromosomes in the frog Buergeria buergeri.

G and C banding studies were made on cultured blood cells of matured male and female Buergeria (B.) buergeri frogs. The Z is subtelocentric and the W is submetacentric. There is a satellite near the end of the long arm of the Z. Constitutive heterochromatin is seen near the proximal and distal portions of the long arms of both Z and W. A single heavily stained G band is seen in the short arm of Z, while one or two heavily stained G bands are observed in the short arm of W. The Z may have originated through a process, in the course of which parts of W have been translocated to Z.