Characteristics of cytochrome P450-dependent metabolism in the liver of the wild raccoon, Procyon lotor.

Wildlife is exposed to a wide range of xenobiotics in the natural environment. In order to appropriately assess xenobiotic-induced toxicity in wildlife, it is necessary to understand metabolic capacities. Carnivores, in general, have low metabolic abilities, making them vulnerable to a variety of chemicals. Raccoons (Procyon lotor) in the wild have been found to have high levels of xenobiotics. However, little is known about the metabolic capacity of the cytochrome P450 (CYP) enzymes in this species. Thus, this study used liver samples to investigate the characteristics of CYP enzymes in wild raccoons. In 22 wild raccoons, CYP concentrations in hepatic microsomes were examined. To better understand the properties of CYP-dependent metabolism, in vitro metabolic activity studies were performed using ethoxyresorufin, pentoxyresorufin and testosterone as substrates. In addition, three raccoons were fed commercial dog food in the laboratory for one week, and the effects on CYP-dependent metabolism were investigated. In comparison to other mammalian species, raccoons had very low concentrations of CYP in their livers. In an in vitro enzymatic analysis, raccoons' ethoxyresorufin O-deethylase (EROD) and pentoxyresorufin O-depentylase (PROD) metabolic capacities were less than one-fifth and one-tenth of rats', respectively. These results indicate the possible high risk in raccoons if exposed to high levels of environmental xenobiotics because of their poor CYP activity. In this study, the features of CYP-dependent metabolism in wild raccoons are described for the first time.

Role of Insulin-like Growth Factor-1 in the Central Regulation of Feeding Behavior in Chicks.

Insulin-like growth factor-1 (IGF-1) is a key regulator of muscle development and metabolism in chickens. Recently, we have demonstrated that intracerebroventricular administration of IGF-1 significantly decreased food intake in broiler chicks. However, the molecular mechanisms underlying the IGF-1-induced anorexia and the anorexigenic effect of IGF-1 in different strains of commercial chicks have not been investigated. Neuropeptide Y (NPY, a hypothalamic orexigenic neuropeptide), agouti-related protein (AgRP, a hypothalamic orexigenic neuropeptide), and proopiomelanocortin (POMC, the precursor of hypothalamic anorexigenic neuropeptides) play important roles in the regulation of food intake in both mammals and chickens. Evidence shows that several cell signaling pathways in the hypothalamus are involved in regulating the feeding behavior of mammals. In the present study, we first investigated the effects of IGF-1 on the expression of appetite-regulating neuropeptides and phosphorylation of signaling molecules in the hypothalamus of broiler chicks. Intracerebroventricular administration of IGF-1 significantly increased the mRNA levels of POMC, whereas the mRNA levels of NPY and AgRP were not significantly altered. IGF-1 also significantly induced the phosphorylation of v-Akt murine thymoma viral oncogene homolog 1 (AKT) in the hypothalamus of chicks, but did not influence the phosphorylation of forkhead box O1, S6 protein, AMP-activated protein kinase, and extracellular signal-regulated kinase 1/2. We also compared the effect of IGF-1 on food intake in broiler chicks (a hyperphagic strain of chickens) and layer chicks. Results demonstrated that the threshold of IGF-1-induced anorexia in broiler chicks was higher than that in layer chicks. Our observations suggest that hypothalamic POMC and AKT may be involved in the IGF-1-induced anorexigenic pathway and that high threshold of IGF-1-induced anorexia in broiler chicks might be one of the causes of hyperphagia in broiler chicks. Overall, it appears that IGF-1 plays important roles in the central regulation of feeding behavior in chicks.

Hypothalamic Akt-mediated signaling regulates food intake in chicks.

The central anorexigenic mechanism seems to be similar in mammals and chicks, because the appetite-suppressive action of a number of peptide hormones is similar in both species. Accumulating evidence in mammals has revealed that hypothalamic Akt-mediated signaling factors (for instance, mTOR and FOXO1) are significantly involved in the regulation of food intake. However, the role of hypothalamic Akt in feeding regulation is yet to be determined in chickens. In this study, we showed that pAkt (Thr308)/Akt, pFOXO1/FOXO1, and pS6 levels were significantly increased in the hypothalami of chicks refed 1 h after a 24 h-fast in correlation to increases in the plasma concentrations of insulin, one of the activators of the Akt-mediated signaling pathways. In addition, central administration of insulin increased the phosphorylation of Akt, FOXO1, and S6 in chicken hypothalami. Furthermore, intracerebroventricular injections of both phosphoinositide 3-kinase inhibitor LY294002 and mTOR inhibitor rapamyacin enhanced the food intake of chicks. These findings suggest that hypothalamic Akt-mediated signaling pathways contribute to the regulation of food intake in chicks.

Effects of Fasting and Refeeding on the mRNA levels of Insulin-like Growth Factor-binding Proteins in Chick Liver and Brain.

The physiological functions of insulin-like growth factor-binding proteins (IGFBPs) in mammals have been evaluated in several studies. However, the physiological roles of IGFBPs in chickens have not yet been elucidated. In this study, we examined the effects of short-term (6 h) fasting and refeeding on the mRNA levels of IGFBPs in chick liver and brain. Eighteen 8-day-old chicks were weighed and allocated to three groups on the basis of body weight, and subjected to ad libitum feeding, 6 h of fasting, or 6 h of fasting followed by 6 h of refeeding. After the chicks were euthanized by decapitation, the liver and brain were excised, and the brain was dissected into six segments (telencephalon, optic lobes, cerebellum, rostral part of the brainstem, middle part of the brainstem, and caudal part of the brainstem). IGFBP mRNA levels were determined by qRT-PCR. Fasting significantly increased the mRNA levels of IGFBP-1 and -2 in the chick liver, and these changes were reversed by 6 h of refeeding. The mRNA levels of IGFBP-3 in the middle part of the brainstem and IGFBP-5 in the optic lobes were decreased by 6 h of fasting and were not reversed after 6 h of refeeding. These findings suggest that IGFBP-1 and -2 in the liver, IGFBP-3 in the middle part of the brainstem, and IGFBP-5 in the optic lobes may play physiological roles in response to short-term changes in the nutritional status of chicks.

Central and peripheral administrations of insulin-like growth factor-1 suppress food intake in chicks.

A number of studies have been made on the physiological actions of insulin-like growth factor-1 (IGF-1) in mammals and birds. In mammals, the effects of central administration of IGF-1 on food intake have been examined. For example, intracerebroventricular administration of IGF-1 significantly decreased food intake in diabetic rats, but not in sheep and nondiabetic rats. The chicken is known to be a hyperglycemic animal. Like satiety hormones, plasma IGF-1 levels are elevated postprandially in chickens. In this study, we hypothesized that IGF-1 is involved in the regulation of food intake in chickens. Intracerebroventricular administration of IGF-1 significantly suppressed food intake in chicks in a dose dependent manner. Both the mRNAs of IGF-1 and its receptor were expressed throughout the brain. However, the mRNA levels of IGF-1 were not influenced by fasting and refeeding in all regions of the brain. On the other hand, 6h of fasting significantly suppressed mRNA expression of hepatic IGF-1, and this effect was significantly reversed by 6h of refeeding. Furthermore, intravascular administration of IGF-1 significantly suppressed food intake in chicks. These findings suggest that IGF-1 may function as a satiety hormone in chickens.

Contamination status and possibility of toxic effects of co-planar polychlorinated biphenyls, polycyclic aromatic hydrocarbons, and dichlorodiphenyltrichloroethane in large japanese field mouse (Apodemus speciosus) collected from Hokkaido and Aomori.

Contamination levels of coplanar polychlorinated biphenyls (Co-PCBs), polycyclic aromatic hydrocarbons (PAHs), and dichlorodiphenyltrichloroethane (DDTs) were measured in the entire body of the large Japanese field mouse (Apodemus speciosus) collected from Hokkaido (Ishikari and Rankoshi) and Aomori prefecture (Takko) in Japan. Higher concentrations of PCBs including Co-PCBs, were observed in the mice collected from Ishikari than those from Rankoshi. The concentration of PAHs in the soil from Ishikari was also higher than that in the other sampling sites. The findings suggest that Ishikari is the most polluted area, probably because of human activities, depending on the population distribution. However, the observed contaminant levels were extremely lower compared to those in previous studies. The ratio of testis weight to body weight (TW/BW) was the lowest in the mice collected from Ishikari, which is the area contaminated with PAHs and p,p'-dichlorodiphenyldichloroethylene (DDE). However, the serum testosterone levels of mice from the Ishikari area were higher than those from the non-contaminated other areas although no significant differences. Previous studies have shown that a low-level exposure to dioxin related compounds (DRCs) disturbances in sexual function, resulting in the production of testosterone. This study showed that POPs exposure is one of the possibility of the high testosterone concentration in the mice of the Ishikari area in addition to a cause of biological and environmental factors such as habitat density, age, temperatures and/or food riches.

Clofibric acid induces hepatic CYP 2B1/2 via constitutive androstane receptor not via peroxisome proliferator activated receptor alpha in rat.

Peroxisome proliferator activated receptor α (PPARα) ligands, fibrates used to control hyperlipidemia. We demonstrated CYP2B induction by clofibric acid (CFA) however, the mechanism was not clear. In this study, HepG2 cells transfected with expression plasmid of mouse constitutive androstane receptor (CAR) or PPARα were treated with CFA, phenobarbital (PB) or TCPOBOP. Luciferase assays showed that CFA increased CYP2B1 transcription to the same level as PB, or TCPOBOP in HepG2 transfected with mouse CAR But failed to induce it in PPARα transfected cells. CYP2B expressions were increased with PB or CFA in Wistar female rats (having normal levels of CAR) but not in Wistar Kyoto female rats (having low levels of CAR). The induction of CYP2B by PB or CFA was comparable to nuclear CAR levels. CAR nuclear translocation was induced by CFA in both rat strains. This indicates that fibrates can activate CAR and that fibrates-insulin sensitization effect may occur through CAR, while hypolipidemic effect may operate through PPARα.

Stereoselective synthesis of P-modified α-glycosyl phosphates by the oxazaphospholidine approach.

α-Glycosyl phosphate derivatives are widely known as constituents of biomolecules. To date, several types of non-natural α-glycosyl phosphates including "P-modified analogs" have been synthesized to investigate their characteristics. Herein a new approach to the stereoselective modification of the intersugar phosphorus atom in α-glycosyl phosphates by use of the oxazaphospholidine method is presented. Via this approach, the dimers of α-glycosyl phosphorothioates and α-glycosyl boranophosphates were obtained efficiently and stereoselectively.

Repeated treatment with furazolidone induces multiple cytochrome p450-related activities in chicken liver, but not in rat liver.

The nitrofuran antimicrobial agent, furazolidone (FZ), is still used in veterinary medicine in some countries in the Middle and Far Eastern countries. The present study aimed to investigate the effects of successive bolus doses of FZ and its metabolite 3-amino-2-oxazolidinone (AOZ) on cytochrome P450 (CYP)-related activities in the livers of rats and chickens. Female Wistar rats and white Leghorn chickens were orally administered FZ once a day for 4 consecutive days. FZ-treated chickens showed an increase in multiple CYP-related activities, however, rats treated with FZ did not show these changes. In chickens, treatment with FZ also induced production of microsomal CYP2C6-like apoprotein. The present study demonstrated that FZ caused a multiple-type induction of CYP-related activities in chickens, but not in rats.

A novel mutation in VKORC1 and its effect on enzymatic activity in Japanese warfarin-resistant rats.

Warfarin is a rodenticide commonly used worldwide. It inhibits coagulation of blood by inhibiting vitamin K 2,3-epoxide reductase (VKOR) activity. An inadequate supply of vitamin K blocks the production of prothrombin and causes hemorrhage. Recently, warfarin-resistant brown rats (Rattus norvegicus) were found around the Aomori area of Japan. There is no significant difference in the metabolic activity of warfarin in sensitive and resistant brown rats. To clarify the mechanism underlying warfarin resistance, we cloned the VKORC1 gene from rats and identified a novel substitution of arginine to proline at position 33 of the VKORC1 amino acid sequence. Then, we determined the differences in kinetics of VKOR activity between warfarin-resistant and sensitive rats. Hepatic microsomal VKOR-dependent activity was measured over a range of vitamin K epoxide concentrations from 6.25 to 150 µM. The Vmax values of resistant rats (0.0029 ± 0.020 nmol/min/mg) were about one tenth of those of sensitive rats (0.29 ± 0.12 nmol/min/mg). The Km values of resistant rats (47 ± 32 µM) were similar to those of sensitive rats (59 ± 18 µM). Warfarin-sensitive rats exhibited enzyme efficiencies (Vmax/Km) which were ten-fold greater than those observed in resistant rats. It may mean that VKOR activity of warfarin-resistant Aomori rats is almost lost, because their enzymatic efficiencies are very low even without warfarin. Further studies are needed to clarify how these rats can survive with a markedly reduced VKOR activity and how they simultaneously exhibit warfarin resistance.

Synthesis of oligo(α-D-glycosyl phosphate) derivatives by a phosphoramidite method via boranophosphate intermediates.

An efficient method for the synthesis of short oligo(α-D-glycosyl boranophosphate) derivatives by using an α-D-glycosyl phosphoramidite as a monomer unit was developed. The synthesis of oligomers was carried out by repeating a cycle consisting of the condensation of the monomer unit with a terminal hydroxy group of carbohydrates, boronation of the resultant phosphite intermediates, and terminal deprotection. The phosphoramidite monomer unit was synthesized from the corresponding glycosyl iodide and methyl N,N-diisopropylphosphonamidate in a highly α-selective manner. Di- and tri(α-D-glycosyl boranophosphate) derivatives obtained by the synthetic cycle were converted into the corresponding H-phosphonate diester derivatives, which were then used to synthesize di- and tri(α-D-glycosyl phosphate) derivatives including a fragment of Leishmania glycocalyx lipophosphoglycans.

Sodium 2-propenyl thiosulfate derived from garlic induces phase II detoxification enzymes in rat hepatoma H4IIE cells.

There is evidence that onions and garlic protect against cancer in humans. It has been suggested that this effect is partly due to the organosulfur compounds in Allium vegetables and that these substances act through induction of phase II detoxification enzymes. Here, we hypothesized that alk(en)yl thiosulfates, sodium n-propyl thiosulfate (NPTS), and sodium 2-propenyl thiosulfate (2PTS), which were identified in onions and garlic, respectively, may induce phase II enzymes. Therefore, rat hepatoma cells (H4IIE) were cultured with 1 to 100 micromol/L of NPTS or 2PTS for 48 hours at 37 degrees C; and the activities and messenger RNA (mRNA) expression levels of phase II enzymes in H4IIE cells were investigated. The effects of diallyl trisulfide and tert-butylhydroquinone, known as phase II inducers, were also examined as positive controls and compared with the responses of NPTS and 2PTS. Quinone reductase (QR) activity and mRNA expression levels of QR and epoxide hydrolase 1 were significantly increased by 2PTS (P < .05-.005). In particular, QR activity was increased at a relatively low concentration of 2PTS (10 micromol/L). However, glutathione S-transferase activity and mRNA expression levels of glutathione S-transferase A5 and uridine diphosphate glucuronosyl transferase 1A1 were not changed by 2PTS. In contrast, NPTS did not affect the activities and mRNA expression levels of these phase II enzymes. These results show that 2PTS can induce phase II enzymes, and its inductive effect is comparable or superior to that of diallyl trisulfide and tert-butylhydroquinone.

The CYP1D subfamily of genes in mammals and other vertebrates.

Members of the cytochrome P450 family 1 (CYP1s) are involved in the detoxification and bioactivation of numerous environmental pollutants and phytochemicals such as polycyclic aromatic hydrocarbons (PAHs), aromatic amines, and flavonoids. The vertebrate CYP1 gene comprises four subfamilies: CYP1A, CYP1B, CYP1C, and CYP1D. Recently, the CYP1D gene was identified in fish, and subsequently in the platypus. These findings indicate the possibility that all vertebrates have a functional CYP1D subfamily. However, there is no information on the mammalian CYP1D gene. In this study we investigated the genomic location of CYP1D genes in mammals and other vertebrates in silico. We also performed phylogenetic analysis and calculated the identities and similarities of CYP1D sequences. The data from synteny and phylogenetic analyses of CYP1D genes demonstrated the evolutionary history of the CYP1 gene family. The results suggested that CYP1D became a nonfunctional pseudogene in human and bovine species; however, several other mammals possess functional CYP1D genes. The promoter regions of CYP1D genes were also examined. Unlike other CYP1 isoforms, few xenobiotic responsive element (XRE)-like sequences were found upstream of the CYP1D genes. Analysis of mammalian CYP1Ds also provided new insight into the relationship between CYP1 genes and the aryl hydrocarbon receptor.

1,2-Trans-selective synthesis of glycosyl boranophosphates and their utility as building blocks for the synthesis of phosphodiester-linked disaccharides.

A highly 1,2-trans-selective synthesis of glycosyl boranophosphate derivatives by glycosylation of dimethyl boranophosphate with glycosyl iodides was developed. A study on the reaction mechanism indicated that the stereoselectivity of the reactions is controlled by neighboring group participation. The resultant glycosyl boranophosphate triesters were converted into the corresponding boranophosphate diesters and condensed with appropriately protected monosaccharides to give disaccharides linked with an anomeric boranophosphate linkage. Furthermore, the disaccharides worked as precursors of the corresponding phosphodiester-linked disaccharides. The whole synthesis of boranophosphate-linked disaccharides and their conversion to the phosphodiester-linked disaccharides were accomplished in good yields without loss of stereopurity at the anomeric position, indicating that the method is useful to synthesize diastereopure glycosyl phosphate-containing biomolecules.

Cytochrome P450 1A-dependent activities in deer, cattle and horses.

The objective of this study was to investigate and characterize the metabolic activities of CYP1A in deer, cattle and horses in comparison to those of rats using ethoxyresorufin O-deethylation (EROD) and methoxyresorufin O-demethylation (MROD) assays. We performed an inhibition study for these activities using anti-rat CYP1A1 antibody and identified that these activities were due to the CYP1A subfamily. Interspecies differences in the CYP1A-dependent activities were highly observed in this study. In particular, we found that the horse had the highest EROD and MROD activities among the examined animal species. In the kinetic analysis, the horses showed the highest Vmax and catalytic efficiency (Vmax/Km), followed by the cattle, deer and rats.

Expression and sequence of CYP1A1 in the camel.

In this study, we determined a partial sequence of CYP1A1 in the camel and its phylogenetic position. The deduced amino acid sequence of camel CYP1A1 showed the highest identity 94% with those of sheep and cattle CYP1A1. In a phylogenetic analysis, the camel CYP1A1 isoform was located beside sheep and cattle CYP1A1. When we studied the distribution of camel CYP1A1 mRNA in different tissues, we found that this isoform was expressed in all tissues except the hump. Interestingly, the lungs of all the camels and tongues of two of the three animals showed high expressions of CYP1A1 mRNA, and this may indicate exposure to ligands of aryl hydrocarbon receptor such as environmental pollutants or flavonoids.

Diazepam metabolism in the kidneys of male and female rats of various strains.

Previously, we have reported drastic strain differences of diazepam metabolism in the livers of a variety of rat strain. In this study, to characterize strain and sex differences of diazepam metabolism in the kidney, renal microsomal diazepam metabolic activities were determined in the Dark Agouti (DA), Sprague-Dawley (SD), Brown Norway (BN) and Wistar (WS) strains of rat. We found that the major pathway of diazepam metabolism in the kidney was diazepam N-demethylation, which is different from that in the liver, 3-hydroxylation. A Dose-course (12.5-200 muM of diazepam) study revealed that the DA and WS male rats had higher diazepam N-demethylation activity than the SD and BN rats. In contrast to the males, a lower activity of diazepam N-demethylation was observed in female BN rats. By Western blot analysis, constitutive protein expressions of cytochrome P450 (CYP) 2C11, which is responsible for diazepam N-demethylation, were detected in the 4 strain in both the male and female rats, and the BN rats had lower expression levels of CYP2C11 protein. However, we did not observe significant differences in the kinetic parameters of diazepam N-demethylation. Our results suggested that there was a strain difference in CYP-dependent diazepam N-demethylation in the rat kidney, which is different from the finding in liver microsomes.

Metabolic alkalosis due to feeding chicks in breeding Adélie penguins Pygoscelis adeliae under natural conditions.

Prolonged abnormal vomiting causes metabolic alkalosis. Many seabirds are known to feed their chicks by regurgitation. We hypothesized that metabolic alkalosis occurs in seabirds even under natural conditions during the breeding season. Adélie penguins Pygoscelis adeliae feed their chicks by regurgitating food for 50-60 d until the chicks fledge. In this study, the concentrations of Cl(-), HCO(3)(-), Na+, K+, pH, and PCO2 in the blood of breeding Adélie penguins were measured throughout the chick-rearing season. The pH of penguin venous blood shifted from 7.54 in the guarding period to 7.47 in the crèche period. Decreasing Cl(-) and increasing HCO(3)(-) blood concentrations in parents were associated with increasing mass of their brood in the guarding period, the early phase of the rearing season, indicating that regurgitating to feed chicks causes loss of gastric acid and results in relative metabolic alkalosis. The inverse trend was observed during the crèche period, the latter phase of the rearing season, when parents spent more time at sea and have fewer opportunities for gastric acid loss. This was assumed to be the recovery phase. These results indicate that regurgitation might cause metabolic alkalosis in breeding Adélie penguins. To our knowledge, this is the first report to indicate that seabirds exhibit metabolic alkalosis due to regurgitation to feed chicks under natural conditions.

Molecular cloning of novel cytochrome P450 1A genes from nine Japanese amphibian species.

Novel cytochrome P450 1A (CYP1A) cDNA fragments were isolated from the liver of nine Japanese amphibian species using reverse transcription polymerase chain reaction (RT-PCR). Degenerate PCR primers were used to amplify 122-bp fragments of CYP1A cDNAs. Construction of a phylogenetic tree revealed that urodele and anuran amphibians formed two branches. Within the anuran species, three branches were formed: 1) Ranidae and Rhacophoridae, 2) Bufo japonicus formosus and Hyla japonica, and 3) Xenopus laevis. The cDNA nucleotide sequence of these CYP1A fragments showed identities ranging 72-98% (all), 72-78% (Anura vs. Urodela), 75 to 98% (Anura), 81% (Urodela), 74-80% (Xenopus laevis vs. nine Japanese amphibians).

Protective effect of Pleurotus cornucopiae mushroom extract on carbon tetrachloride-induced hepatotoxicity.

Pleurotus cornucopiae (PC) mushrooms are found in the field and commonly known in Japan as Tamogidake mushrooms. The present study investigated the protective effects of an aqueous extract of PC on carbon tetrachloride (CCl4)-induced hepatotoxicity and the possible mechanism involved in this protection including cytochrome P450 (CYP) 2E1. Wistar rats were pretreated with aqueous extracts of PC (0, 100, 200, and 400 mg/kg) orally for 8 days prior to the intraperitoneal administration of a single dose of CCl4 (0.5 ml/kg) or corn oil. Pretreatment with PC mushroom extract significantly prevented the increased serum enzyme activities of alanine and aspartate aminotransferases in a dose-dependent manner, and suppressed the expression of CYP2E1. PC mushroom extract also protected hepatocytes from the damage effects of CCl4 as remarked by histological and electromicroscopical findings. It was concluded that repeated daily doses of aqueous extracts of PC mushroom reduced the toxic effects exerted by CCl4 on the liver.