Transgenic Zebrafish Expressing Rat Cytochrome P450 2E1 (CYP2E1): Augmentation of Acetaminophen-Induced Toxicity in the Liver and Retina.

Metabolic activation is the primary cause of chemical toxicity including hepatotoxicity. Cytochrome P450 2E (CYP2E) is involved in this process for many hepatotoxicants, including acetaminophen (APAP), one of the most common analgesics and antipyretics. Although the zebrafish is now used as a model for toxicology and toxicity tests, the CYP2E homologue in zebrafish has not been identified yet. In this study, we prepared transgenic zebrafish embryos/larvae expressing rat CYP2E1 and enhanced green fluorescent protein (EGFP) using a ß-actin promoter. Rat CYP2E1 activity was confirmed by the fluorescence of 7-hydroxycoumarin (7-HC), a metabolite of 7-methoxycoumarin that was specific for CYP2 in transgenic larvae with EGFP fluorescence (EGFP [+]) but not in transgenic larvae without EGFP fluorescence (EGFP [-]). APAP (2.5 mM) caused reduction in the size of the retina in EGFP [+] larvae but not in EGFP [-] larvae, while APAP similarly reduced pigmentation in both larvae. APAP at even 1 mM reduced the liver size in EGFP [+] larvae but not in EGFP [-] larvae. APAP-induced reduction of liver size was inhibited by N-acetylcysteine. These results suggest that rat CYP2E1 is involved in some APAP-induced toxicological endpoints in the retina and liver but not in melanogenesis of the developing zebrafish.

Cytochrome P450 2J Genes Are Expressed in Dogs, Cats, and Pigs, and Encode Functional Drug-Metabolizing Enzymes.

Cytochrome P450s (P450s) have been identified and analyzed in dogs and pigs, species that are often used in preclinical drug studies. Moreover, P450s are clinically important for drug therapy not only in humans, but also in species under veterinary care, including dogs and cats. In the present study, seven P450s homologous to human CYP2J2, namely, dog CYP2J2; cat CYP2J2; and pig CYP2J33, CYP2J35, CYP2J91, and CYP2J93, were newly identified and characterized, along with pig CYP2J34 previously identified. The cDNAs of these CYP2Js contain open reading frames of 502 amino acids, except for CYP2J35 (498 amino acids), and share high sequence identity (77%-80%) with human CYP2J2. Phylogenetic analysis revealed that dog and cat CYP2J2 were closely related, whereas pig CYP2Js formed a cluster. All seven CYP2J genes contain nine coding exons and are located in corresponding genomic regions, with the pig CYP2J genes forming a gene cluster. These CYP2J2 mRNAs were predominantly expressed in the small intestine with additional expression in the kidney and brain for dog CYP2J2 and pig CYP2J91 mRNAs, respectively. All seven CYP2Js metabolized human CYP2J2 substrates terfenadine, ebastine, and astemizole, indicating that they are functional enzymes. Dog CYP2J2 and pig CYP2J34 and CYP2J35 efficiently catalyzed ebastine primary hydroxylation and secondary carebastine formation at low substrate concentrations, just as human CYP2J2 does. Velocity-versus-substate plots exhibited sigmoidal relationships for dog CYP2J2, cat CYP2J2, and pig CYP2J33, indicating allosteric interactions. These results suggest that dog, cat, and pig CYP2Js have similar functional characteristics to human CYP2J2, with slight differences in ebastine and astemizole oxidations. SIGNIFICANCE STATEMENT: Dog CYP2J2; cat CYP2J2; and pig CYP2J33, CYP2J34, CYP2J35, CYP2J91, and CYP2J93, homologous to human CYP2J2, were identified and characterized by sequence, phylogenetic, and genomic structure analyses. Intestinal expression patterns of CYP2J mRNAs were characteristic in dogs, cats, and pigs. Dog, cat, and pig CYP2Js likely play roles as drug-metabolizing enzymes in the small intestine, similar to human CYP2J2.

Pheasant motilin, its distribution and gastrointestinal contractility-stimulating action in the pheasant.

Previously, pheasant motilin was identified as a 22-amino acid peptide with a sequence of FVPFFTQSDI QKMQEKERIK GQ. In the present study, the distribution of pheasant motilin mRNA was determined and compared with that of ghrelin, a motilin-related peptide. The effects of pheasant motilin on the cognate gastrointestinal (GI) muscle strips were also examined in an in vitro contraction study. The expression of pheasant motilin mRNA was highest in the small intestine (duodenum, jejunum and ileum), moderate in the colon and very low in the brain, lung, heart, pancreas, esophagus, proventriculus, gizzard and caecum, and this distribution was in contrast with that of ghrelin mRNA. Pheasant motilin caused contraction of the cognate GI tract in a region-dependent manner, similar to chicken motilin. The contraction in the small intestine was large and was not affected by atropine. In contrast, contraction in the proventriculus was small and was decreased by atropine. The crop and colon were insensitive to pheasant motilin. Neither GM109 nor MA2029, mammalian motilin receptor antagonists inhibited the contractions of pheasant motilin. Erythromycin was ineffective in the pheasant ileum, although it caused contraction of the rabbit duodenum. These results indicate that pheasant motilin caused contraction through an action on smooth muscles in the small intestine and an action on enteric cholinergic nerves in the proventriculus. This high responsiveness of the small intestine suggests that motilin is a regulator of small intestinal motility in avians, and the characteristic of the motilin receptor in the pheasant might be different from that in mammals, as is that in chickens.

Motilin- and ghrelin-induced contractions in isolated gastrointestinal strips from three species of frogs.

Ghrelin (GHRL) and motilin (MLN), gut peptides isolated from the mucosa of the stomach and duodenum, respectively, stimulate gastrointestinal (GI) motility in mammals and birds. However, the functions of MLN and GHRL in amphibian GI tracts have not been examined in detail. To clarify the regulation of GI motility by the two peptides, the effects of human MLN and rat GHRL on contractility of isolated GI strips from three species of frogs, the black-spotted pond frog (pond frog; Pelophylax nigromaculata), bullfrog (Lithobates catesbeiana) and Western clawed frog (Xenopus; Xenopus tropicalis), were examined in in vitro experiments. The GI tract of each frog was divided into the stomach, upper intestine, middle intestine and lower intestine. Human MLN caused contractions of the stomach in the pond frog and upper intestine in the bullfrog and Xenopus, but other GI regions were insensitive to human MLN. Erythromycin did not cause contraction of the upper intestine of the bullfrog and Xenopus. Rat GHRL did not cause contraction of the stomach and small intestines in the pond frog and bullfrog, but it caused a concentration-dependent contraction in the stomach and upper intestine of Xenopus, while des-acyl rat GHRL did not cause any contraction of them. In conclusion, human MLN caused the contraction of the stomach or upper intestine in the three species of frogs, but GHRL was effective only in the stomach and upper intestine of Xenopus. On the basis of these data, MLN but not GHRL causes the GI region-dependent contractions in the frogs.

Identification of pheasant ghrelin and motilin and their actions on contractility of the isolated gastrointestinal tract.

Motilin and ghrelin were identified in the pheasant by molecular cloning, and the actions of both peptides on the contractility of gastrointestinal (GI) strips were examined in vitro. Molecular cloning indicated that the deduced amino acid sequences of the pheasant motilin and ghrelin were a 22-amino acid peptide, FVPFFTQSDIQKMQEKERIKGQ, and a 26-amino acid peptide, GSSFLSPAYKNIQQQKDTRKPTGRLH, respectively. In in vitro studies using pheasant GI strips, chicken motilin caused contraction of the proventriculus and small intestine, whereas the crop and colon were insensitive. Human motilin, but not erythromycin, caused contraction of small intestine. Chicken motilin-induced contractions in the proventriculus and ileum were not inhibited by a mammalian motilin receptor antagonist, GM109. Neither atropine (a cholinergic receptor antagonist) nor tetrodotoxin (a neuron blocker) inhibited the responses of chicken motilin in the ileum but both drugs decreased the responses to motilin in the proventriculus, suggesting that the contractile mechanisms of motilin in the proventriculus was neurogenic, different from that of the small intestine (myogenic). On the other hand, chicken and quail ghrelin did not cause contraction in any regions of pheasant GI tract. Since interaction of ghrelin and motilin has been reported in the house musk shrew, interaction of two peptides was examined. The chicken motilin-induced contractions were not modified by ghrelin, and ghrelin also did not cause any contraction under the presence of motilin, suggesting the absence of interaction in both peptides. In conclusion, both the motilin system and ghrelin system are present in the pheasant. Regulation of GI motility by motilin might be common in avian species. However, absence of ghrelin actions in any GI regions suggests the avian species-related difference in regulation of GI contractility by ghrelin.

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.

Limited expression of functional cytochrome p450 2c subtypes in the liver and small intestine of domestic cats.

1. Compared to information for herbivores and omnivores, knowledge on xenobiotic metabolism in carnivores is limited. The cytochrome P450 2C (CYP2C) subfamily is recognized as one of the most important CYP groups in human and dog. We identified and characterized CYP2C isoforms and variants in cat, which is an obligate carnivore. 2. Quantitative RT-PCR and immunoblot analyses were carried out to evaluate the expression of CYP2C in the liver and small intestine. A functional CYP2C isoform was heterologously expressed in yeast microsomes to determine the enzymatic activity. 3. Cat had two CYP2C genes, 21 and 41, in the genome; however, CYP2C21P was a pseudogene that had many stop codons. Three splicing variants of CYP2C41 were identified (v1-v3), but only one of them (v1) showed a complete deduced amino acid sequence as CYP2C protein. Transcripts of feline CYP2C41v1 were detected but the amounts were negligible or very small in the liver and small intestine. Immunoreactivity to an antihuman CYP2C antibody was confirmed in the recombinant feline CYP2C41v1 but not in the feline liver. 4. Recombinant feline CYP2C41v1 metabolized several substrates, including dibenzylfluorescein that is specific to human CYP2C. 5. The results suggest a limited role of functional CYP2C isoforms in xenobiotic metabolism in cat.

Usefulness of zebrafish in evaluating drug-induced teratogenicity in cardiovascular system.

To confirm the usefulness of zebrafish for evaluating the teratogenic potential of drug candidates, the effect of O-ethylhydroxylamine hydrochloride (OHY), which induces mutagenesis by methylation, was evaluated in teratogenicity studies in rats and zebrafish. In the rat teratogenicity study, OHY-induced cardiovascular malformations such as increased abnormal vascular structures and ventricular septal defects. In the teratogenicity study using zebrafish-injected microspheres and green fluorescent protein-expressing Tg zebrafish (flk1:EGFP), OHY exposure was associated with the loss or malformation of the mandibular arch, opercular artery, and fourth branchial arch. These results suggested that OHY-induced external malformations in zebrafish eleutheroembryos adequately reflect OHY's teratogenicity in rat fetuses. Moreover, the zebrafish teratogenicity study incorporating vascular morphological examinations, including those of blood vessels in the heart, head and trunk, is an easy and reliable screening method to detect potential drug-induced teratogenicity and phenotypic characteristics.

Contamination Status of Seven Elements in Hooded Cranes Wintering in South-West Kyushu, Japan: Comparison with Red-Crowned Cranes in Hokkaido, Japan.

The hooded crane is designated as an endangered species. The cranes breed primarily in wetlands in southeast Russia and China in summer. Most of the hooded crane population winters in the Izumi plain in Japan. It is difficult to know the contamination status of their habitat because of their vast breeding area. We determined the levels of Cd, Pb, As, (total) Hg, Se, Zn, and Cu in the liver, kidney, and muscle of hooded cranes that were found dead in Izumi in the periods 2003-2006 and 2014-2015 compared with the levels in red-crowned cranes in Hokkaido, Japan, as the only cranes in which these elements had been studied extensively. There were no notable differences between levels of the seven elements in the two periods. Overall, tissue levels of the elements examined in hooded cranes were comparable to those in red-crowned cranes except for Hg and Se. Tissue levels of Hg and Se were clearly lower in hooded cranes than in red-crowned cranes that were found dead from 2000. One lead poisoning case was confirmed. The results suggest that Hooded cranes wintering in Izumi are not extensively contaminated with the seven elements examined.

Does motilin peptide regulate gastrointestinal motility of zebrafish? An in vitro study using isolated intestinal strips.

Motilin (MOT), a 22-amino-acid peptide hormone produced in the duodenal mucosa, stimulates gastrointestinal motility in mammals and birds, and it is a mediator of interdigestive motor complexes. Recently, expression of MOT-like peptide (MOTLP) and its receptor mRNAs was identified in zebrafish. The aim of the present study was to determine whether the zebrafish MOTLP (zfMOTLP, HIAFFSPKEMRELREKE) affects zebrafish gastrointestinal motility, with comparison to the effect of human MOT, in which five amino acids are identical to zfMOTLP at positions 5, 9, 15, 16, and 17. zfMOTLP caused small contractions of the rabbit duodenum and chicken ileum but, the sensitivity was about 3000-times lower than that of human MOT. zfMOTLP-induced contraction in the rabbit duodenum was decreased by pretreatment of the MOT receptor antagonist GM109, indicating that zfMOTLP could bind to the MOT receptor. zfMOTLP (3-100nM) increased the intracellular Ca2+ concentration in zfMOT receptor-expressing HEK293 cells, but human MOT did not cause responses even at 100nM. In in vitro study using isolated zebrafish gastrointestinal strips, zfMOTLP caused only small contractions even at high doses (1-10µM). zfMOT receptor mRNA is detected in the gastrointestinal tract and brain to almost the same extent, and the expression level (40-70 copies/100ng total RNA) is much lower than that in the chicken gastrointestinal tract. These results suggest that the MOTLP/MOT receptor system is present in zebrafish, but its physiological role for regulation of gastrointestinal motility might be not significant due to the weak contractile activity and low expression level of the receptor.

Functional expression and comparative characterization of four feline P450 cytochromes using fluorescent substrates.

1. Cytochrome P450s (CYP) are a major group of metabolizing enzymes for xenobiotics in humans and other mammals. The properties of CYP isoforms in the domestic cat, an obligate carnivore, are largely unknown at present. In this study, we studied relative expression in tissues and enzymatic properties of nine significant feline CYP isoforms. 2. CYP2E2 transcript was most abundant in the feline liver, followed by CYP2A13 and 2E1. Transcripts of CYP3A131, 1A2 and 1A1 were also present in the liver, while CYP2D6 and 3A132 were only slightly expressed. CYP3A131 was a major transcript in the small intestine. 3. Four major CYP isoforms in the feline liver and small intestine (CYP1A2, CYP2A13, CYP2E2 and CYP3A131) were heterologously expressed in Escherichia coli to generate functional monooxygenase systems. We carried out screenings of 17 test compounds known to be inhibitors of CYP isoforms in other mammals as well as two anticancer drugs to assess the activity modulation of feline CYP isoforms using fluorogenic substrates. These CYP isoforms showed similar selectivity to counterparts in other mammals against inhibitors as a whole but with many exceptions. 4. The present study suggests the usefulness of the feline CYP recombinant system to obtain chemical affinity information and possible drug interactions in CYP metabolism of domestic cats.

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.

The chicken is an interesting animal for study of the functional role of ghrelin in the gastrointestinal tract.

Ghrelin has been identified in vertebrates from fish to mammals, and it has multiple biological activities including gastrointestinal (GI) motor-stimulating action. In some non-mammalian vertebrates, we examined the effects of ghrelin on contractility of the isolated GI tract as well as the mRNA expression of growth hormone secretagogue-receptor 1a (GHS-R1a) to determine whether the motor-stimulating action of ghrelin is common in vertebrates. The expression level of GHS-R1a mRNA differed depending on the species and on the GI region (stomach, small intestine, and colon). GI region-dependent expression of GHS-R1a mRNA was remarkable in chickens, and the expression levels changed depending on age. In a functional study, ghrelin did not cause contraction of unstimulated GI strips in fish (goldfish and rainbow trout) or amphibians (bullfrog and Japanese fire belly newts) even using their homologous ghrelin. In avian species, ghrelin caused contraction of the unstimulated GI tract of the chicken but not of the Japanese quail, and the responses to ghrelin in the chicken GI tract decreased with aging. Our in vitro studies show that the motor-stimulating action of ghrelin is not conserved across vertebrates and that the chicken is a unique animal species for evaluation of the GI-stimulating action of ghrelin of different age.

Protective effect of Ganoderma lucidum polysaccharide against carbon tetrachloride-induced hepatic damage in precision-cut carp liver slices.

The aim of the present study was to investigate the protective effects of Ganoderma lucidum polysaccharide (GLPS) against carbon tetrachloride (CCl4)-induced hepatotoxicity in vitro in common carp. Precision-cut liver slices (PCLSs), which closely resemble the organ from which they are derived, were employed as an in vitro model system. GLPS (0.1, 0.3, and 0.6 mg/ml) was added to PCLS culture system before the exposure to 12 mM CCl4. The supernatants and slices were collected to detect molecular and biochemical responses to CCl4 and PCLS treatments. The levels of CYP1A, CYP3A, and CYP2E1 were measured by ELISA; the mRNA expressions of TNF-α, IL-1ß, IL-6, and iNOS were determined by RT-PCR; and the relative protein expressions of c-Rel and p65 were analyzed by western blotting. Results showed that GLPS inhibited the elevations of the marker enzymes (GOT, GPT, LDH) and MDA induced by CCl4; it also enhanced the suppressed activity of antioxidant enzymes (SOD, CAT, GSH-Px, T-AOC). The treatment with GLPS resulted in significant downregulation of NF-κB and inflammatory cytokine mRNA levels and significant decreases in the hepatic protein levels of CYP1A, CYP3A, and CYP2E1. These results suggest that GLPS can protect CCl4-induced PCLS injury through inhibiting lipid peroxidation, elevating antioxidant enzyme activity, and suppressing immune inflammatory response.

Effects of ghrelin and motilin on smooth muscle contractility of the isolated gastrointestinal tract from the bullfrog and Japanese fire belly newt.

Ghrelin has been identified in some amphibians and is known to stimulate growth hormone release and food intake as seen in mammals. Ghrelin regulates gastrointestinal motility in mammals and birds. The aim of this study was to determine whether ghrelin affects gastrointestinal smooth muscle contractility in bullfrogs (anuran) and Japanese fire belly newts (urodelian) in vitro. Neither bullfrog ghrelin nor rat ghrelin affected longitudinal smooth muscle contractility of gastrointestinal strips from the bullfrog. Expression of growth hormone secretagogue receptor 1a (GHS-R1a) mRNA was confirmed in the bullfrog gastrointestinal tract, and the expression level in the gastric mucosa was lower than that in the intestinal mucosa. In contrast, some gastrointestinal peptides, including substance P, neurotensin and motilin, and the muscarinic receptor agonist carbachol showed marked contraction, indicating normality of the smooth muscle preparations. Similar results were obtained in another amphibian, the Japanese fire belly newt. Newt ghrelin and rat ghrelin did not cause any contraction in gastrointestinal longitudinal muscle, whereas substance P and carbachol were effective causing contraction. In conclusion, ghrelin does not affect contractility of the gastrointestinal smooth muscle in anuran and urodelian amphibians, similar to results for rainbow trout and goldfish (fish) but different from results for rats and chickens. The results suggest diversity of ghrelin actions on the gastrointestinal tract across animals. This study also showed for the first time that motilin induces gastrointestinal contraction in amphibians.

Identification and functional characterization of novel feline cytochrome P450 2A.

1. Cytochrome P450s are the major metabolizing enzymes for xenobiotics in humans and other mammals. Although the domestic cat Felis catus, an obligate carnivore, is the most common companion animal, the properties of cytochrome P450 subfamilies are largely unknown. 2. We newly identified the feline CYP2A13, which consists of 494 deduced amino acids, showing the highest identity to CYP2As of dogs, followed by those of pigs, cattle and humans. 3. The feline CYP2A13 transcript and protein were expressed almost exclusively in the liver without particular sex-dependent differences. 4. The feline CYP2A13 protein heterogeneously expressed in Escherichia coli showed metabolic activity similar to those of human and canine CYP2As for coumarin, 7-ethoxycoumarin and nicotine. 5. The results indicate the importance of CYP2A13 in systemic metabolism of xenobiotics in cats.

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.

Possible Shifts in the Genetic Diversity of Red-crowned Cranes (Grus japonensis) in Hokkaido, Japan: Indications of Continental Gene Flow.

Red-crowned cranes (Grus japonensis) consist of two distinct groups: the continental population and the island population. The island population, localized in Hokkaido, Japan, exhibits very low genetic diversity due to its rapid recovery from the brink of extinction. Our previous research in 2018 highlighted a possible mating between a male from the continental population, with the Gj5 haplotype, and a female from the island population, with the Gj2 haplotype, at Hitominuma Sawmp shore in northern Hokkaido. The present study attempted to unravel the distribution of their offspring by examining the major histocompatibility complex (MHC) of this mixed breeding pair compared with samples collected from cranes in northern and southeastern Hokkaido between 2008 and 2022. The analysis identified 55 MHC types, including 10 known types in a dataset of 89 crane samples, based on amino acid sequences. A total of 58 MHC types were recognized, based on nucleotide sequences, as there were many cases in which the same amino acid sequence had different nucleotide sequences. The five DNA types of MHC in the Hitominuma Swamp male were predominantly identified in eight cranes from northern Hokkaido and one chick from southeastern Hokkaido. In addition, population genetic analysis, based on insertion/deletion (InDel) polymorphisms, indicates distinct population differentiation between the northern and southeastern regions of Hokkaido. These results suggest that genetic contributions from the continental red-crowned crane population have already been integrated into the Hokkaido populations, with a more pronounced influence in northern Hokkaido.

Augmentation of Pectoral Fin Teratogenicity by Thalidomide in Human Cytochrome P450 3A-Expressing Zebrafish.

The pharmacological and toxicological effects of active metabolites of enzymes including cytochrome P450 (CYP) are important. While it has been believed for a long time that thalidomide causes characteristic limb malformation only in rabbits and primates including humans, the involvement of their CYP3A subtypes (CYP3As) has been suggested. Recently, however, it was reported that zebrafish were sensitive to thalidomide, showing defects of pectoral fins, homologous organs of forelimbs in mammals, as well as other deformities. In this study, we prepared human CYP3A7 (hCYP3A7)-expressing zebrafish (F0) using a transposon system. Thalidomide caused pectoral fin defects and other malformations including pericardial edema in hCYP3A7-expressing embryos/larvae but not in wild-type and hCYP1A1-expressing embryos/larvae. Thalidomide also reduced the expression of fibroblast growth factor 8 in pectoral fin buds in only hCYP3A7-expressing embryos/larvae. The results suggest the involvement of human-type CYP3A in thalidomide teratogenicity.

Analysis of Crop Consumption Using Scatological Samples from the Red-Crowned Crane Grus japonensis in Eastern Hokkaido, Japan.

Total DNA extracts from the intestinal contents of 60 flying red-crowned cranes (juveniles, subadults and adults) found dead in 2006-2021, and the feces of 25 chicks collected in June and July of 2016-2018, were used for PCR reactions with primers specific for 16 crops, followed by high-throughput sequencing. The most predominant crop detected was corn in adult and subadult cranes (61.7%). Other grains (barley, wheat, soybean) (5.0-8.3%) and vegetables (tomatoes, Chinese cabbage, etc.) (1.7-6.7%) were also detected in flying cranes. Surprisingly, some of the detected crops were not grown in the Kushiro and Nemuro regions. There was no significant difference in crop intake status in winter and that in other seasons for most of the crops. Corn (28.0%), soybeans (8.0%), wheat and beet (4.0%) were detected in crane chicks in summer, though the detection rates were generally lower than those in flying cranes. Alfalfa, which is not grown in eastern Hokkaido but is used in some cattle feed, was detected in some cranes. Rice, buckwheat, adzuki beans, common beans, potatoes and carrots were not detected at any life stage, indicating the preferences of red-crowned cranes. The results suggest that red-crowned cranes in Hokkaido are dependent on dairy farmers for their feed supply.