Jun-Lan Wang

Purification and characterization of hepatic steroid hydroxylases from untreated rainbow trout

Purification of cytochrome P450 from liver microsomes of untreated juvenile male rainbow trout yielded five fractions designated LMC1 to LMC5. All fractions, except LMC4 and LMC5, appeared homogeneous on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and showed minimum molecular weights of 50,000 (LMC1), 54,000 (LMC2), 56,000 (LMC3), 58,000 (LMC4), and 59,000 (LMC5). Specific contents ranged from 2.8 (LMC3) to 14.9 (LMC5) nmol heme/mg protein. The catalytic activity of LMC1, LMC2, and LMC5 toward various substrates was examined. LMC2 exhibited the highest estradiol 2-hydroxylase activity and progesterone 16 alpha-hydroxylase activity. LMC2 also was most active in the metabolic activation of aflatoxin B1 (AFB1). In contrast, LMC5 was most active in catalyzing the 6 beta- and 16 beta-hydroxylation of testosterone and the 6 beta-hydroxylation of progesterone. LMC1 showed the highest lauric acid hydroxylase activity. The three isozymes tested had low activity (for LMC2 and LMC5) or no activity (for LMC1) toward benzphetamine or benzo[a]pyrene. Polyclonal antibodies to all five isozymes were raised in rabbits and the antibodies were used to examine the contribution of the P450s to microsomal enzyme activities. The results of microsomal enzyme inhibition studies with polyclonal antibodies showed that anti-LMC2 IgG significantly inhibited the oxidative metabolism of testosterone, lauric acid, AFB1, and benzphetamine. Anti-LMC5 IgG inhibited the oxidation of progesterone, estradiol, benzo[a]pyrene, and benzphetamine. Anti-LMC1 IgG slightly inhibited the microsomal hydroxylation of lauric acid. Anti-LMC3 and anti-LMC4 IgG did not inhibit any of the measured microsomal enzyme activities. These findings suggest that individual constitutive isozymes of trout cytochrome P450 have well-defined contributions to the microsomal metabolism of steroids, fatty acids, and xenobiotics.

Comparison of rainbow trout and mammalian cytochrome P450 enzymes: Evidence for structural similarity between trout P450 LMC5 and human P450111A4

Studies were undertaken to determine the immunochemical relationship between constitutive trout cytochrome P450s and mammalian cytochrome P450IIIA enzymes. Polyclonal antibodies (IgG) generated against trout P450 LMC5 reacted strongly with P450IIIA1 in dexamethasone-induced rat liver microsomes and with P450IIIA4 in human liver microsomes in immunoblots. In contrast, rabbit anti-P450 LMC1 IgG did not recognize these proteins in rat and human liver microsomes. Reciprocal immunoblots using anti-rat P450IIIA1 showed that this antibody does not recognize trout P450 LMC1 or LMC5. However, anti-human P450IIIA4 IgG was found to cross react strongly with P450 LMC1 and LMC5. Progesterone 6 beta-hydroxylase activity of trout liver microsomes, a reaction catalyzed by P450 LMC5, was markedly inhibited by anti-P450IIIA4 and by gestodene, a mechanism-based inactivator of P450IIIA4. These results provide evidence for a close structural similarity between trout P450 LMC5 and human P450IIIA4.

Flavin-containing monooxygenase: a major detoxifying enzyme for the pyrrolizidine alkaloid senecionine in guinea pig tissues.

Evidence based on optimal pH, thermal stability, and enzyme inhibition data suggests that the NADPH-dependent microsomal N-oxidation of the pyrrolizidine alkaloid senecionine is carried out largely by flavin-containing monooxygenase in guinea pig liver, lung, and kidney. In contrast, the hepatic microsomal conversion of senecionine to the pyrrole metabolite (+/-)-6,7-dihydro-7-hydroxy-1-hydroxymethyl-5H-pyrrolizine (DHP) is catalyzed largely by cytochrome P450. However, the rate of senecionine N-oxide formation (detoxication) far exceeded the rate of DHP formation (activation) in guinea pig liver microsomes over a range of pHs (pH 6.8 to 9.8). In guinea pig lung and kidney microsomes, N-oxide was the major metabolite formed from senecionine with little or no production of DHP. The high rate of detoxication coupled with the low level of activation of senecionine in liver, lung, and kidney may help explain the apparent resistance of the guinea pig to intoxication by senecionine and other pyrrolizidine alkaloids.

Immunological characterization of constitutive isozymes of cytochrome P-450 from rainbow trout. Evidence for homology with phenobarbital-induced rat P-450s

Immunoglobulin G fractions (IgGs), isolated from rabbits immunized against hepatic cytochrome P-450 isozymes were used to investigate the immunochemical homology among trout P-450s and between trout and rat P-450s. The antigens used for immunization were five constitutive trout P-450s (LMC1 to LMC5), one beta-naphthoflavone (BNF)-inducible trout P-450 (LM4b), and one phenobarbital-induced rat P4500IIB1 (PB-B). In the enzyme-linked immunosorbent assay (ELISA), strong cross-reactivity was observed between anti-LMC2 IgG and P-450 LMC1, and between anti-LMC3 IgG and P-450 LMC4. There was little or no cross-reactivity of anti-LMC5 IgG with other trout P-450s. Trout P-450 LM4b was not recognized by any of the antibodies against constitutive trout P-450s. Antibodies to P-450 LMC1 and P450 LMC2 cross-reacted strongly with rat P450IIB1 and with proteins of PB-induced rat liver microsomes. Rat P450IA1 (BNF-B) did not cross-react with anti-LMC1 or anti-LMC2 IgG. These cross-reactions were essentially confirmed by immunoblot (Western blot) analysis. Western blots of PB-induced rat liver microsomes probed with anti LMC1 revealed two major immunoreactive proteins in the P-450 region, one of which co-migrated with rat P450IIB1. P450IIB1 itself cross-reacted strongly with anti-LMC1 IgG. In control rats, a single protein band cross-reacted poorly with anti-LMC1 IgG. Antibodies to LMC1 and LMC2 did not cross-react with rat P450IA1 in Western blots. The antigenic epitopes in rat P450IIB1 recognized by anti-LMC1 IgG and anti-LMC2 IgG are probably not located at or near the active site of the enzyme since these antibodies did not inhibit benzphetamine N-demethylase activity of P450IIB1 or of PB-induced rat liver microsomes. In general, our results demonstrate: (1) the presence of a significant homology between LMC1 and LMC2, and between constitutive trout P-450 (LMC1) and PB-induced rat P-450 (P450IIB1); and (2) distant homology between constitutive trout P-450s and constitutive rat P-450s or BNF-induced rat P-450s.

Regiospecificity in the hydroxylation of lauric acid by rainbow trout hepatic cytochrome P450 isozymes

The catalytic activity of two hepatic cytochrome P450 isozymes from untreated rainbow trout towards lauric acid was investigated. In a reconstituted system, cytochrome P450 LMC1 and P450 LMC2 were found to catalyze exclusively the omega- and (omega-1)-hydroxylation of lauric acid, respectively. Microsomal enzyme inhibition studies with polyclonal antibodies raised against the individual P450 isozymes showed that P450 LMC1 and LMC2, respectively, accounted for most if not all the omega- and (omega-1)-lauric acid hydroxylase activity of trout liver microsomes. The polyclonal antibodies were highly specific in that they only inhibited the enzyme activity of the P450 used as the immunogen. These results illustrate that as in mammals, omega- and (omega-1)-hydroxylation of lauric acid by trout liver microsomes can be carried out separately by distinct isozymes of cytochrome P450.

Comparative effects of the polychlorinated biphenyl mixture, aroclor 1242, on porphyrin and xenobiotic metabolism in kidney of japanese quail and rat

1. Aroclor 1242 (500 mg/kg, p.o.) produced a marked increase in porphyrin content of quail kidney (1800-fold), and of rat kidney but to a lesser extent (6-fold). 2. delta-Aminolevulinic acid synthetase activity was increased 12-fold in quail kidney but was unchanged in rat kidney following Aroclor 1242 treatment. 3. Uroporphyrinogen decarboxylase activity was significantly inhibited in quail kidney but not in rat kidney. 4. Renal ethoxyresorufin O-deethylase activity was induced in rat and quail whereas renal ethoxycoumarin O-deethylase and glutathione S-transferase activities were induced only in rats by Aroclor 1242.

Inhibition of dehydrogenase enzymes by hexachlorophene

Abstract Low concentrations of the antibacterial agent hexachlorophene (HCP) inhibited a number of pyridine nucleotide-linked dehydrogenase enzymes, including bovine liver glutamate dehydrogenase (GDH), beef heart malate dehydrogenase (MDH), torula yeast glucose 6-phosphate dehydrogenase (G-6-P-D), horse liver alcohol dehydrogenase (ADH), pig heart isocitrate dehydrogenase (ICD), and beef heart lactate dehydrogenase (LDH). Initial velocity studies at appropriate enzyme concentrations gave I 50 values for the dehydrogenases which ranged between 1.6 μM for GDH and 105 μM for ICD and LDH. More detailed kinetic analyses of G-6-P-D, ICD and GDH showed that inhibition by HCP in most cases was of the noncompetitive type. Calculations made from the kinetic data gave apparent K i values with G-6-P-D, of 59 μM for NADP + and of 38 μM for glucose 6-phosphate; with ICD, of 1.0 μM for NADP + and of 25 μM for isocitrate; and, with GDH, of 2.0 μM for NADH, of 7.4 μM for α-ketoglutarate, and of 2.3 μM for ammonium acetate.

Cloning, sequencing and expression of the major rainbow trout constitutive cytochrome P450 (CYP2K1). Identification of a new cytochrome P450 gene subfamily

Abstract A full-length cDNA has been cloned and sequenced for the major constitutive rainbow trout liver cytochrome P450 that had earlier been designated as cytochrome P450 LMC2. This cDNA clone encodes for a protein of 504 amino acids with a calculated M r of 56,795. While the cDNA nucleotides for trout P450 LMC2 shared a 49 and 47% homology, respectively, with the two rainbow trout P450s previously sequenced (hepatic P4501A1 and an ovarian 17-hydroxylase), it showed a 54–56% nucleotide identity with several human or other mammalian P450 forms from family 2. From amino acid sequence comparisons, the trout P450 LMC2 has been assigned to a new cytochrome P450 gene family designated P4502K1 (CYP2K1). Northern blots of total RNA isolated from sexually mature male and female rainbow trout examined with a cDNA probe (2K1,7c), indicated that mRNA levels were substantially higher in the trunk kidney of male trout compared to that seen in females. Multiple 2K1,7c hybridizable bands at about 2.8 kb and 1.6–1.9 kb were found in the trunk kidney preparations, with the 2.8 kb band predominating. A major 1.9 kb mRNA band, detectable with the 2K1,7c probe, was present at similar concentrations in livers of both sexes.

Studies on the porphyrinogenic action of 1,2,4-trichlorobenzene in birds.

The porphyrinogenic action of 1,2,4-trichlorobenzene (TCB) was examined in 17-day-old embryos, day-old chicks, 18-day-old chickens and adult Japanese quail. The quail was found to be the most sensitive species towards TCB induced porphyria whereas the chick embryo was totally non-responsive. The liver porphyrins of Japanese quail were increased in a dose-dependent manner 1 day after TCB. Elevation in porphyrin levels in quail was associated with comparable increases in delta-aminolevulinic acid synthetase (ALA-S) activity 1 day after TCB treatment. In contrast, ferrochelatase activity was found to be unchanged 1 day after TCB. Multiple administration of TCB produced only a slight increase in liver porphyrin levels and ALA-S activity in quail. However, there was a marked induction in ferrochelatase activity suggesting increased porphyrin turnover. Liver glutathione and glutathione S-transferase activity were also significantly increased following repeated administration of TCB in quail, which could indicate an enhancement of detoxication of reactive metabolites of TCB. Thus, it is suggested that the inability of low multiple doses of TCB to cause porphyria in Japanese quail may be related to the low responsiveness of ALA-S but high inducibility of ferrochelatase liver GSH and glutathione S-transferase.

The effects of a luteinizing hormone-releasing hormone analogue on cytochrome P450 in rainbow trout

Profound sex-related differences in P450 levels and P450 activities in rainbow trout trunk kidney occur during the time of spawning. Since spawning in salmonids can be induced by certain analogues of luteinizing hormone-releasing hormone (LHRH), the role of the analogue, [D-Ala6, Pro9-NHEt]-LHRH (LHRH-A) in the expression of selected P450 enzymes in trunk kidney of 2-year old rainbow trout was evaluated. LHRH-A, 5 μg/ kg, injected i.p. at days 0 and 3, reduced or abolished the expression of P450 LMC2 (P4502K1) (a catalyst for aflatoxin B1 bioactivation and lauric acid hydroxylation) in trunk kidney of female, but not male trout, at days 10, 17, and 24. However, the expression of P4502K1 in liver of male and female trout was unchanged after LHRH-A treatment. P450 LMC1 (another catalyst for lauric acid hydroxylation) in trunk kidney and liver of both males and females was not affected by LHRH-A. No P4502K1 was detected in head kidney of control and LHRH-A-treated trout of both sexes. This study provides evidence that the expression of P4502K1 protein in rainbow trout may be affected by synthetic peptide hormones such as LHRH-A. Additional studies are being carried out to determine whether the LHRH analogue affects P4502K1 expression at the level of transcription.