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To understand the detoxification and bioactivation mechanisms for organic contaminants, it is essential to identify the cytochrome P450 (CYP) complement. Therefore, this study aimed to clone a partial cDNA sequence of the novel CYP1D1 gene from the fish Oreochromis niloticus and examine whether intraperitoneal injection of benzo-a-pyrene (BaP), a potent AHR agonist, is capable of inducing CYP1D1 mRNA expression in different tilapia fish tissues. The cloned nucleotide sequence consisted of 713 bp representing a portion of the tilapia CYP1D1 cDNA ORF, encoding 237 amino acids. Amino acid sequence comparison of O. niloticus CYP1D1 with the sequences of CYP1D1 from other species showed that this gene shared the highest identity of 81% with Fundulus heteroclitus CYP1D1. Furthermore, analysis of the percent identities shared by the deduced amino acid sequence of O. niloticus CYP1D1 with the sequences of CYP1 from other species revealed that the highest identities were shared with fish CYP1As. Real-time PCR results revealed that the highest expression level of CYP1D1 mRNA was found in muscles, followed by gills, liver, and intestine, while there was no detectable expression recorded in bile acid. These results indicate that tilapia CYP1D1 plays an important role in the metabolism of xenobiotics, expanding our knowledge regarding the diversity of CYP1 genes in this important model fish species.

We evaluated the metabolism of several herbicides and progesterone by two P450 proteins (CYP1A9 and CYP1C1) from Japanese eel (Anguilla japonica). Expression vectors harboring CYP1A9 and CYP1C1 sequences were introduced into Escherichia coli. E. coli membrane fractions were incubated with each substrate, and the metabolites were analyzed. CYP1A9 and CYP1C1 deethylated 7-ethoxycoumarin and phenacetin, and demethylated chlorotoluron, diuron, and linuron. CYP1C1 specifically hydroxlyated progesterone at the 6β and 16α positions. Five amino acids of CYP1A9 related to substrate binding were selected for mutation analyses [CYP1A9(F128A), CYP1A9(F229A), CYP1A9(F263A), CYP1A9(V387A), and CYP1A9(I391A)]. Two variants, CYP1A9(F229A) and CYP1A9(F128A), changed the ratio of 16α hydroxyprogesterone to 6β hydroxyprogesterone. Among all the variants, CYP1A9(F263A) showed the highest activity towards substrates used. CYP1A9(V387A) and CYP1A9(I391A) showed higher activities than that of CYP1A9 toward progesterone. The substrate specificity of CYP1A9 may be altered by replacing an amino acid related to substrate binding.

In this study, the localization of fluorescent protein (FP) was characterized in the muscles of four species and two subspecies of eels Anguilla anguilla, A. australis, A. bicolor bicolor (b.), A. bicolor pacifica (p.) and A. mossambica in addition to the previously reported A. japonica. The open reading frame of each eel FP was 417 bp encoding 139 amino acid residues. The deduced amino acid sequences among the four species and two subspecies exhibited 91.4–100% identity, and belonged to the fatty-acid-binding protein (FABP) family. The gene structure of eel FPs in A. japonica, A. anguilla, A. australis, A. bicolor b., A. bicolor p. and A. mossambica have four exons and three introns, and were common to that of FABP family. The apo eel FPs expressed by Escherichia coli with recombinant eel FP genes were analysed for the fluorescent properties in the presence of bilirubin. The excitation and emission spectra of holo eel FPs had the maximum wavelengths of 490–496 and 527–530 nm, respectively. The holo eel FPs indicated that the fluorescent intensities were stronger in A. japonica and A. bicolor than in A. mossambica, A. australis and A. anguilla. The comparison of amino acid sequences revealed two common substitutions in A. mossambica, A. australis and A. anguilla with weak fluorescent intensity.

Cytochrome P450 (CYP) enzymes constitute a multigene family of many endogenous and xenobiotic substances. The CYP1 family is of particular interest in environmental toxicology because its members are dominant in the metabolism of polycyclic aromatic hydrocarbons (PAHs), polychlorinated biphenyls (PCBs), and aryl amines. A new cDNA of the CYP1C subfamily encoding CYP1C1 was isolated from Japanese eel (Anguilla japonica) liver after intraperitoneal injection with βnaphthoflavone (BNF). The full-length cDNA obtained (3508 bp) contained a 5' noncoding region of 355 bp, an open reading frame of 1581 bp coding for 526 amino acids, a stop codon, and a 3' noncoding region of 1572 bp. The predicted molecular weight of the protein was approximately 59.33 kDa. The deduced amino acid sequence of Japanese eel CYP1C1 had the lower similarity of 70% with that of killifish CYP1C1 while the higher similarity (79 and 81%) was observed with that of rainbow trout CYP1C2 and -1C1 sequences respectively. It exhibited similarities of 71% with that of Indian medaka CYP1C1 and zebrafish CYP1C2. Also the similarity of 74% was registered with the sequence of three-spined stickleback fish CYP1C1, 1C2 and carp CYP1C2. It showed similarity of 77% with that of Nile tilapia CYP1C1, scup CYP1C1 and scup CYP1C2. The phylogenetic tree showed the newly identified Japanese eel CYP1C1 sequence to be clustered with rainbow trout CYP1C1 and -1C2. Japanese eel CYP1C1 was aligned with the CYP1 sequences and has been deposited in the Gen Bank / EMBL data bank with the accession number AY444748. Quantitative real-time polymerase chain reaction (QRT-PCR) analysis of liver, kidney, intestine and gills revealed a distinct induced expression in all organs studied (283.33, 579.35, 20.96 and 3642.32 respectively).

The CYP1 family, one of the gene families of the CYP superfamily, has four subfamilies deposited in the GenBank/EMBL so far; CYP1A, CYP1B, CYP1C, and the newly identified CYP1D. The metabolic activation and elimination of polyaromatic hydrocarbons, polychlorinated biphenyls, and aryl amines from fish body is largely mediated by the CYP enzymes. A new cDNA of the CYP1B subfamily encoding CYP1B1 was isolated from Japanese eel liver after a single intraperitoneal injection of β-naphthoflavone (BNF). The full-length cDNA obtained was 2985 bp and contained a 5' noncoding region of 294 bp, an open reading frame of 1626 bp coding for 541 amino acids and a stop codon and a 3' noncoding region of 1065 bp. The predicted molecular weight of the protein was approximately 61.27 kDa. The deduced amino acid sequence of Japanese eel CYP1B1 showed 62% similarity to three-spined stickleback CYP1B1 and zebrafish CYP1B1. It exhibited similarities of 66% with that of killifish, Indian medaka and our previously reported carp CYP1B1 and -1B2 while the higher similarities (67 and 69%) of the deduced amino acids was observed with that of Nile tilapia CYP1B1 and rainbow trout respectively. The percent identities of Japanese eel CYP1B1 cDNA showed similarities with those of the reported CYP1Bs of mammals of 57, 57, and 56% for human, rat, and mouse CYP1B1, respectively. Japanese eel CYP1B1 was aligned with the CYP1 sequences and has been deposited in the GenBank/EMBL data bank with the accession number AY518340. The phylogenetic tree constructed using the previously reported CYP1B sequences of mammals and fish suggested the closer relationship of the newly identified Japanese eel CYP1B1 to rainbow trout CYP1B1. QRT-PCR analysis of liver, kidney, gills and intestine revealed a distinct induced expression in liver, kidney and gills (71.93, 3.87 and 539.56 respectively) while the constitutive expression (0.062) was observed in intestine.

This study planned to isolation and characterization of AKR1A1 cDNA from Bap injected nile tilapia (Oreochromis niloticus), comparison of its characteristic structures with those of other species, characterization of AKR1A1 gene and promoter, and investigation of AKR1A1 mRNA expression in various organs of Bap injected tilapia. The cDNA was 1172 bp long which includes an open reading frame of 975 bp encoding a 324 amino acids protein and a stop codon. The sequence showed 3' and 5' non-coding regions of 179 and 18 bp. The amino acid sequence of O. niloticus AKR1A1 shows similarities of 60, 60, 60.6, 61.2 62.2, and 57.8% with mouse AKR1A1, Norway rat AKR1A1, zebrafish AKR1A1, African clawed frog AKR1A1, human, and yellow perch AKR1A1, respectively. Nucleotide sequence investigation of AKR1A1 gene and 5'-flanking region showed that the structural gene and the 5'-flanking region were approximately 2975 bp and 4006 bp in length, respectively. The protein-coding region contained eight exons, and one additional upstream exon. Real-time polymerase chain reaction (PCR) results showed that the highest level of AKR1A1 expression was found in bile (108.7), followed by kidney (77.9), muscles (37.3), and liver (24.7). mRNA levels of AKR1A1 were almost negligible in gills (0.6) while no detectable (ND) constitutive expression was detected in gut. In conclusion, our results concluded that tilapia AKR1A1 is inducible by BaP and have a significant function in the metabolism of xenobiotics and, therefore, may used as biomarker in fish.

Through the use of a number of bioconversion experiments we demonstrated that P450 proteins (CYP1A9 and CYP1C1) from Japanese eel ( Anguilla japonica ) metabolized a number of herbicides and the drug phenacetin. We performed bioconversion experiments in which substrates were added directly to incubation medium. The resulting metabolites were extracted and analyzed by high-performance liquid chromatography. Proteins CYP1A9 and CYP1C1 metabolized 50 nmol of the drug phenacetin to yield 12.1 and 1.1 nmol of product (acetaminophen), respectively. Further incubation of CYP1A9 with 50 nmol of the herbicides chlorotoluron, diuron, linuron, simazine, or atrazine yielded 16.5, 18.5, 7.3, 1.6, or 0.8 nmol of product, respectively. CYP1C1 also metabolized linuron, diuron, and simazine yield 5.4, 4.6, or 0.7 nmol of product, respectively. Next, polyclonal antibody was isolated by immunizing with two conjugated-peptides (amino acid residues 272–290 and 294–310) of CYP1A9. This antibody did not recognize human CYP1A2 or CYP1C1. Western blotting using the antibody revealed one band in the livers of Japanese eel and tilapia ( Oreochromis niloticus ). Theses results suggest that CYP1A9 and CYP1C1 metabolize herbicides, and that CYP1A9 is an useful biomarker of contamination when detected with this antibody.

Green fluorescent protein (GFP) from eel (Anguilla japonica) muscle (eelGFP) is unique in the vertebrates and requires bilirubin as a ligand to emit fluorescence. This study was performed to clarify the physiological function of the unique GFP. Investigation of susceptibility to oxidative stress was carried out using three types of cell lines including jellyfish (Aequorea coerulescens) GFP (jfGFP)-, or eel GFP (eelGFP)-expressing HEK293 cells, and control vector-transfected HEK293 cells. Binding of eelGFP to bilirubin was confirmed by the observation of green fluorescence in HEK293-eelGFP cells. The growth rate was compared with the three types of cells in the presence or absence of phenol red which possessed antioxidant activity. The growth rates of HEK293-CV and HEK293-jfGFP under phenol red-free conditions were reduced to 52 and 31% of those under phenol red. Under the phenol red-free condition, HEK293-eelGFP had a growth rate of approximately 70% of the phenol red-containing condition. The eelGFP-expressing cells were approximately 2-fold resistant to oxidative stress such as H2O2 exposure. The fluorescence intensity partially decreased or disappeared after exposure to H2O2, and heterogeneous intensity of fluorescence was also observed in isolated eel skeletal muscle cells. These results suggested eelGFP, but not jfGFP, coupled with bilirubin provided the antioxidant activity to the cells as compared to non-bound free bilirubin.

SUMMARY: A freshness index for fish muscle (K-value) was measured for six species of fish using freshness testing paper (FTP) and column chromatography. The correlation coefficient between these K-values determined by both methods was 0.917, although K-values of reddish muscle of big-eyed tuna, flying fish and common horse mackerel were evaluated lower by FTP than by column chromatography. The changes of K-values which were determined at regular intervals by FTP indicated that the decrease in freshness of amberjack and big-eyed tuna muscles was due to a first-order reaction. The kinetic parameters for the freshness-lowering, activation energy and frequency factor were obtained from Arrnhenius’ plots of K-values at the storage temperatures between –1 and +20°C, and then it was clarified that the quality of amberjack and big-eyed tuna with the different history of storage temperature should be predictable on the basis of these kinetic parameters.

アナゴの蒲焼きあるいは煮アナゴを製造する際に発生する調味液廃液は工場レベルで以下のような処理により,再利用が可能となった.(1) アナゴ廃液の再利用に障害となる呈味成分の希釈化,夾雑物の混入などは,加熱殺菌,プロテアーゼ処理,濃縮,遠心分離およびSF膜ろ過処理からなる一連の操作によって解決することができた.(2) 細菌数は加熱処理で減少したが,その後の各処理工程で徐々に増加することから,その発育を抑制するために,再生調味液の固形物含量を45%までに濃縮し,さらに食塩を添加することにより商業ベースでの常温流通を可能とした.

To study the effects of urea on the denaturation of myosin B prepared from smooth dogfish, myosin B was kept at 20°C for 10 min with various concentrations (0-3.0M) of urea, and the reduced viscosity was measured as a parameter for structural changes in myosin B. The decrease in the reduced viscosity of myosin B containing urea above 1.0M was similar to that of myosin in the presence of urea, However, the reduced viscosity of F-actin was slightly affected by urea even above 1.0M. The reduced viscosity of myosin B did not recovered to its initial level through the removal of urea at concentrations above 1.0M urea, but that of myosin recovered perfectly through the re-moval of urea at concentrations up to 3.0M urea. In the presence of urea above 1.0M, myosin B irreversibly dissociated into myosin and actin. Therefore, it is concluded that the urea denaturation of myosin B would be attributed to its dissociation into myosin and actin as well as the conforma-tional changes of myosin molecules in the presence of urea above 1.0M. Accordingly, shark myosin B is fairly stable under physiological concentrations of urea below 1.0M.

The quantitative method of ATP in fish muscle was investigated by the measurement of emitted light intensity using luciferase of the American firefly. The peak of the emitted light spectrum was shown at 554nm. The sensitive assay for ATP was improved by increasing the concentration of the enzyme with the presence of bovine serum albumin, DTT, and EDTA. The regression line was obtained between the emitted light intensity and the ATP concentra-tion in the ranges from 1 to 50μM. The bioluminescent reaction was not inhibited by the dilu-tion of neutralized perchlorate as extractive solvent with 100 volumes of distilled water. The ATP contents in the extracts of fish muscle were determined both by bioluminescent reaction and by column chromatography. The values obtained by the two different methods almost agreed. Consequently, the bioluminescent reaction of American firefly luciferase could be used for the rapid determination of the ATP contents in fish muscle.

Cytochrome P450 (CYP) genes, which make up a large gene superfamily, are known to play an important role in drug metabolism. The CYP1 family, one of the gene families of the CYP superfamily, has three subfamilies of genes whose sequences have been deposited in the GenBank/EMBL thus far: CYP1A, CYP1B, and CYP1C. Mammals as well as fish confront numerous foreign chemicals in the environment that may accumulate to toxic levels unless they are metabolized and eliminated by processes largely mediated by CYP enzymes. A new complementary DNA of the CYP1C subfamily encoding CYP1C2 was isolated from the carp liver after a single intraperitoneal injection of beta-napthoflavone (BNF). The full-length cDNA obtained contained a 5' noncoding region of 198 bp, an open reading frame of 1575 bp coding for 524 amino acids and a stop codon, and a 3' noncoding region of 531 bp. The predicted molecular weight of the protein was approximately 59.3 kDa. The amino acid sequence deduced from the carp CYP1C2 sequence showed a similarity of 76.6% with that deduced from our previously reported carp CYP1C1. It exhibited similarities of 77.3, 73.7, and 76.4% with those deduced from scup CYP1C2, scup CYP1C1, and Japanese eel CYP1C1 sequences, respectively. Carp CYP1C2 cDNA showed similarities with reported CYP1Bs of teleosts and mammals, namely, 47.6, 45.3, 45.7, 44.0, and 44.6% for carp, plaice, human, rat, and mouse CYP1B1s, respectively, while it exhibited a similarity of 49.0% with carp CYP1B2. The carp CYP1C2 sequence was aligned with the CYP1 sequences and has been deposited in the GenBank/EMBL data bank with the accession number AY437777. The phylogenetic tree constructed using fish and mammalian CYP1 sequences suggested a closer relationship of CYP1C with CYP1B than with CYP1A. The tree showed possibile existence of CYP1C subfamily genes in mammalian species. Northern blot analysis of the liver, intestines, kidneys, and gills revealed a distinct induced expression only in the kidneys, with no detectable constitutive expression in the other organs studied.

We indicated that two P450s (1A9 and 1C1) from Japanese eel (Anguilla japonica) metabolized 7-ethoxycoumarin, 7-ethoxyresorufin, and flavanone. At first, we constructed expression vectors for two types of P450 (1A9 and 1C1). The reduced CO-difference spectra of Escherichia coli cells transformed with these plasmids showed Soret peaks (450 nm) that were typical of P450s. We performed bioconversion experiments in which substrates were added directly to incubation medium. The resulting metabolite(s) were extracted and analyzed by high-performance liquid chromatography and spectrofluorometer. Incubation of 50 nmol 7-ethoxyresorufin with P450 1C1 yielded 0.773 nmol of deethylated product, whereas 50 nmol 7-ethoxycoumarin resulted in 4.76 nmol. P450 1A9 metabolized 50 nmol of 7-ethoxyresorufin and 7-ethoxycoumarin to yield 6.54 and 20.9 nmol of deethylated product, respectively. Incubation of 50 nmol flavanone with P450 1C1 yielded 1.46 nmol and 0.69 nmol of products, whereas 50 nmol flavanone with P450 1A9 resulted in 1.10 nmol. In this system, 4'-hydroxy flavanones were formed by P450 1A9 and P450 1C1. P450 1A9 also metabolized 50 nmol of 17 beta-estradiol to yield 4.25 nmol of product. In this system, 2-hydroxy estradiol was formed by P450 1A9 using 17 beta-estradiol as a substrate. This study is the first to identify the substrates that P450 1C1 and 1A9 metabolize.

Cytochrome P450 (CYP) enzymes constitute a multigene family of many endogenous and xenobiotic substances. The CYP1 family is of particular interest in environmental toxicology because its members are dominant in the metabolism of polycyclic aromatic hydrocarbons (PAHs), polychlorinated biphenyls (PCBs), and aryl amines. A new complementary DNA of the CYP1C subfamily encoding CYP1C1 was isolated from carp liver after intraperitoneal injection of beta-napthoflavone (BNF). The full-length cDNA obtained contained a 5' noncoding region of 244 bp, an open reading frame of 1572 bp coding for 524 amino acids, a stop codon, and a 3' noncoding region of 965 bp. The predicted molecular weight of the protein was approximately 59.3 kDa. The deduced amino acid sequence of this cDNA was 82.1% and 80.2% similar to Japanese eel and scup CYP1C1 sequences, respectively, while it exhibited a similarity of 74.9% with the scup CYP1C2 sequence. The deduced amino acid sequence of carp CYP1C1 showed similarities with those of the reported CYP1B1s of teleosts and mammals of 48.4, 48.8, 48.2, 48.6, 45.3, and 45.5% for carp CYP1B1, carp CYP1B2, plaice CYP1B1, and human, rat, and mouse CYP1B1, respectively. The phylogenetic tree constructed using fish and mammalian CYP1 sequences suggested a closer relationship of the CYP1C subfamily to CYP1B than to CYP1A. The tree showed the possibility of the existence of CYP1C subfamily genes in mammalian species. Northern blot analysis for the liver, intestine, gills, and kidney showed no detectable induced expression but constitutive expression in the gill organs.

Some forms of cytochrome P450 (CYP) genes are known to be induced by xenobiotics such as dioxins. Induction of the CYP1A gene is mediated by an aryl hydrocarbon receptor (AHR) which binds to a specific nucleotide sequence called a dioxin-responsive element (DRE) located in the 5' enhancer region of the gene. Functional analysis of the regulatory region of the eel CYP1A gene had shown that a 654-bp region near the basal promoter, containing no DREs but three motifs that resemble estrogen-responsive element (ERE) halfsites, contributes substantially to the induced expression. Considering the importance of non-DRE elements in CYP1A gene induction, we investigated the role of ERE-like motifs using a point mutation technique. The regulatory region of the eel CYP1A gene was identified by creating mutations in all three ERE half-sites simultaneously or individually. The regulatory region constructs were cloned upstream of the luciferase gene in an expression vector which was microinjected into medaka ova. Expression of luciferase as a foreign gene in medaka fry exposed to 3-methylcholanthrene (3-MC)-treated feed was measured by competitive PCR analysis. Mutation in all the three ERE half-sites reduced expression to 10%. Mutation in ERE(-2) or ERE(-3) reduced the expression to 50%, whereas mutation in ERE(-1) did not affect the induction. The two functional half-sites of the eel CYP1A gene are palindromic to each other and appear to constitute the full-ERE.

Cytochrome P450 (CYP) enzymes constitute a multigene family of many endogenous and xenobiotic substances. The CYP1 family is of particular interest in environmental toxicology because its members are dominant in the metabolism of polycyclic aromatic hydrocarbons (PAHs), polychlorinated biphenyls (PCBs), and aryl amines. A new complementary DNA of the CYP1B subfamily encoding CYP1B2 was isolated from carp liver after intraperitoneal injection with 3-methylcholanthrene (3-MC). The obtained full-length cDNA contained a 5'noncoding region of 161 bp, an open reading frame of 1593 bp coding for 530 amino acids and a stop codon, and a 3'noncoding region of 1457 bp. The predicted molecular weight of the protein was approximately 60.2 kDa. The deduced amino acid sequence of this cDNA was 91% similar to that of our previously reported carp CYP1B1; its similarities with those of the reported CYP1B1s of teleosts and mammals were 60.8, 54.4, 50.8, and 51.4% for plaice, human, rat, and mouse, respectively. The phylogenetic tree of fish and mammalian CYP1 sequences constructed by the protein maximum-likelihood method suggested a relatively recent divergence of CYP1B2 from CYP1B1 in the ancestor of carp and closely related species. Despite the structural similarity of CYP1B2 with CYP1B1, which showed induced expression in 3-MC-treated liver, intestine, and gills with marked constitutive expression in gills, CYP1B2 revealed induced expression in gills but not in liver or intestine, and no detectable constitutive expression in the tissues studied.

Cytochrome P450 (CYP) genes, which make up a large gene superfamily, are known to play an important role in drug metabolism. The levels of expression of CYP genes in the tissue of fish inhabiting polluted areas have been used extensively in biomonitoring studies as indicators of dioxin pollution. Complementary DNA of cytochrome CYP1B1 was isolated from carp (Cyprinus carpio) liver 24 h after the injection of 3-methylcholanthrene (3-MC). The full length cDNA obtained contained a 5' noncoding region of 178 bp, an open reading frame of 1593 bp coding for 530 amino acids and a stop codon, and a 3' noncoding region of 1599 bp. The predicted molecular weight of the encoded protein was approximately 60.2 kDa. The deduced amino acid sequence exhibited an identity of 60.6% with reported CYP1B sequences of plaice CYP1B, and of 52.4, 51.4, and 50.3% with human, rat, and mouse CYP1B1s, respectively. It exhibited similarities of 48.4 and 47.3% with scup CYP1C2 and -1C1 sequences. The percent identities with CYP1A sequences showed lower values in the range from 35.3 to 39.5% with mammals and teleosts. The phylogenetic tree of the CYP1 family members constructed by the protein maximum likelihood method indicates that the carp CYP1B1 and plaice CYP1B share a common ancestry with the mammalian CYP1B1s. Carp treated with 3-MC showed expression of CYP1B1 in liver, intestine and gills with distinct induction except for the gills that showed marked constitutive expression. The presence of two successive signals in treated gills at low stringency hybridization may suggest the existence of another CYP1B member that is expressed in the gills of carp.