Differential expression of two cinnamate 4-hydroxylase genes in 'Valencia' orange (Citrus sinensis Osbeck).

Two different full-length cDNAs for cinnamate 4-hydroxylase (C4H1 and C4H2) were isolated from Citrus sinensis Osbeck cv. Valencia libraries. C4H1 (1708 bp) and C4H2 (1871 bp) share only 65% identity on nucleotide and 66% identity on the amino acid level, respectively. C4H1 is most homologous to a cinnamate 4-hydroxylase sequence from French bean (Phaseolus vulgaris), but codes for a unique N-terminus. C4H2 shows highest similarity to a poplar (Populus kitakamiensis) sequence, but also shows a unique N-terminus. The two genes are expressed differentially in orange flavedo, C4H2 is constitutive, C4H1 is wound-induced. In competitive RT-PCR, the mRNA for both genes in wounded and untreated tissue was quantified. C4H1 is strongly wound-inducible from 'not detectable' to about 35 fg mRNA per 50 ng total RNA 8 h after wounding. The first detectable C4H1 mRNA was found 4 h after wounding. After reaching peak levels 4 h later the levels slightly declined, but stayed elevated until the end of the experiment (48 h). C4H2 is expressed 3-10 times higher than wound-induced C4H1 even in the control sample; wounding transiently increases the level of expression another 2-3 times. The existence of different N-termini and their effects on the possible role of both genes in phenylpropanoid pathways is discussed.

Bioflavonoids: selective substrates and inhibitors for cytochrome P450 CYP1A and CYP1B1.

Interactions of six naturally occurring flavonoids (acacetin, diosmetin, eriodictyol, hesperetin, homoeriodictyol, and naringenin) with human cytochrome P450 (CYP1) enzymes were studied. The flavones acacetin and diosmetin were potent inhibitors of ethoxyresorufin O-dealkylase (EROD) activity of CYP1A and CYP1B1. Hydroxy and/or methoxy substitutions at the 3' and 4' positions in the flavonoid structures were the major factors involved in conveying selectivity for the different cytochrome P450 enzymes. Eriodictyol, homoeriodictyol and naringenin were very poor inhibitors of human CYP1A EROD activity (IC(50)4 microM). Hesperetin and homoeriodictyol selectively inhibited human CYP1A1 and CYP1B1. Only homoeriodictyol selectively inhibited human CYP1B1 (IC(50) 0.24 microM). Hesperetin was O-demethylated by both human CYP1A1 and 1B1 to eriodictyol, which was then further metabolized by the same enzymes. Hesperetin was not metabolized by human CYP1A2 or CYP3A4.

cDNA cloning and expression of a class II acidic chitinase from sweet orange.

A citrus cDNA encoding a class II acidic chitinase was isolated from a nonembryogenic cell line of sweet orange using the tobacco cDNA clone PROB3. Northern blot analysis revealed that the corresponding mRNA is expressed in young, green bark but not in leaves, roots, or flavedo.

Characterization of seven basic endochitinases isolated from cell cultures of Citrus sinensis (L.).

Seven endochitinases (EC 3.2.1.14) (relative molecular masses 23,000-28,000 and isoelectric points 10.3-10.4) were purified from nonembryogenic Citrus sinensis L. Osbeck cv. Valencia callus tissue. The basic chitinase/lysozyme from this tissue (BCLVC) exhibited lysozyme, chitinase and chitosanase activities and was determined to be a class III chitinase. While BCLVC acted as a lysozyme at pH 4.5 and low ionic strength (0.03) it acted as a chitinase/chitosanase at high ionic strengths (0.2) with a pH optimum of ca. 5. The lysozyme activity of BCLVC was inhibited by histamine, imidazole, histidine and the N-acetyl-D-glucosamine oligosaccharide (GlcNAc)3. The basic chitinase from cv. Valencia callus, BCVC-2, had an N-terminal amino acid sequence similar to tomato and tobacco AP24 proteins. The sequences of the other five chitinases were N-terminal blocked. Whereas BCLVC was capable of hydrolyzing 13.8-100% acetylated chitosans and (GlcNAc)4-6 oligosaccharides, BCVC-2 hydrolyzed only 100% acetylated chitosan, and the remaining enzymes expressed varying degrees of hydrolytic capabilities. Experiments with (GlcNAc)2-6 suggest that BCLVC hydrolysis occurs in largely tetrasaccharide units whereas hydrolysis by the other chitinases occurs in disaccharide units. Cross-reactivities of the purified proteins with antibodies for a potato leaf chitinase (AbPLC), BCLVC, BCVC-3, and tomato AP24 indicate that these are separate and distinct proteins.

Cytochrome P450 specificities of alkoxyresorufin O-dealkylation in human and rat liver.

The O-dealkylations of ethoxyresorufin and pentoxyresorufin are widely used activity probes for measuring the cytochrome P450 forms, CYP1A1 and CYP2B1, respectively, and their induction by xenobiotics, but there is confusion in the literature about which P450 forms are detected in human and rat liver microsomes by these and homologous alkoxyresorufins. High performance liquid chromatographic analysis confirmed that O-dealkylation to resorufin was the sole or predominant route of metabolism for both short-chain and long-chain alkoxyresorufins and benzyloxyresorufin by rat liver microsomes. The purified 3-methylcholanthrene (3MC)-induced rat P450 forms, CYP1A1 and CYP1A2, and a possible variant form, CYP1A1*, showed substrate selectivities for propoxyresorufin, methoxyresorufin and ethoxyresorufin, respectively. Purified phenobarbitone (PB)-induced CYP2B1 was selective for benzyloxyresorufin and pentoxyresorufin. Purified constitutive CYP2C6 was much less active than CYP2B1 or the CYP1A forms but showed distinctive selectivity for benzyloxy-, propoxy- and butoxyresorufin. CYP1A2 and CYP2C6 metabolised n-propoxy- and n-butoxyresorufin much more rapidly (8-23-fold) than iso-propoxy- and iso-butoxyresorufin, whereas CYP1A1 and CYP2B1 showed only small differences (2-5-fold) between the n- and iso-homologues and CYP1A1* and CYP2B2 did not discriminate between them. The results show that ratios between different alkoxyresorufin O-dealkylation (AROD) activities can be more useful than absolute values of single activities for identifying P450 forms. Anti-P450 antibody and furafylline inhibition of rat liver microsomal AROD confirmed that ethoxyresorufin was a selective probe for CYP1A1 in 3MC-induced and isosafrole (ISF)-induced microsomes and that pentoxy- and benzyloxyresorufins both selectively measured CYP2B1 in PB-induced and ISF-induced microsomes. Ethoxyresorufin was not a selective probe for CYP1A in liver microsomes from untreated or PB-induced rats, however, where it was metabolised mainly by CYP2C6 and CYP2B1, respectively. Pentoxyresorufin and benzyloxyresorufin were metabolised by several different P450 forms in non-induced rat liver microsomes but mainly by the CYP1A subfamily in 3MC-induced microsomes and by CYP2B1 in PB- and ISF-induced microsomes. Although with purified rat P450s methoxyresorufin appeared not effectively to discriminate CYP1A2 from CYP1A1, CYP1A1* or CYP2C6, furafylline inhibition indicated that methoxyresorufin was a selective measure of CYP1A2 in uninduced and 3MC-induced rat liver microsomes but not in ISF- or PB-induced microsomes. In human liver microsomes, antibody inhibition and furafylline inhibition showed that ethoxyresorufin and methoxyresorufin were metabolised mainly by CYP1A2, whilst benzyloxyresorufin metabolism was due mainly to the CYP3A subfamily but also involved CYP1A2 and CYP2A6.(ABSTRACT TRUNCATED AT 400 WORDS)

Identification and characterization of acidic hydrolases with chitinase and chitosanase activities from sweet orange callus tissue.

Acidic chitinases (EC 3.2.1.14) were isolated and characterized from 4-week-old nonembryogenic Citrus sinensis L. Osbeck cv 'Valencia' callus tissue. The enzymes were purified using size exclusion, anion exchange, and chromatofocusing HPLC techniques. Eleven isoforms were isolated with M(r)s between 26,000 and 37,400. Eight of the isoforms were purified to homogeneity, and all but one cross-reacted with a polyclonal antibody raised against a basic class I potato leaf chitinase. The isoelectric points (determined by chromatofocusing) were from pH 4.5 to 5.4. All hydrolases degraded chitin and four were capable of hydrolyzing solubilized shrimp shell chitosan suggesting they may be chitosanases (EC 3.2.1.99). Apparent chitosanase activity generally decreased with decreasing acetylation of the chitosan (i.e. from 20% to 0% acetylation). The chitinases and chitinases/chitosanases are predominantly endochitinases. Chitosanase activity was optimal at pH 5 while the pH optimum for chitinase activity ranged between pH 3.5 and 5.5. The chitinases and chitinases/chitosanases were stable up to 60 degrees C and showed their highest enzyme activity at that temperature. N-terminal sequences were obtained on three of the isoforms. One of the isoforms was identified as a class II chitinase and the other two as class III chitinases.

A comparative study of constitutive and induced alkoxyresorufin O-dealkylation and individual cytochrome P450 forms in cynomolgus monkey (Macaca fascicularis), human, mouse, rat and hamster liver microsomes.

The expression of constitutive and inducible cytochrome P450 forms was measured in cynomolgus monkey liver and compared with man, rat, mouse and hamster. Four alkoxyresorufin O-dealkylation (AROD) activities widely used as indicators of P450 induction were measured: methoxyresorufin O-demethylation (MROD), ethoxyresorufin O-deethylation (EROD), pentoxyresorufin O-dealkylation (PROD) and benzyloxyresorufin O-dealkylation (BROD). In monkeys there were no sex-differences in untreated, phenobarbitone (PB)- or beta-naphthoflavone (BNF)-treated animals in AROD activities, or in individual P450 proteins detected by immunoblotting. Basal MROD and EROD activities varied by less than 7-fold between the five species, but the comparative pattern of basal MROD, EROD, PROD and BROD activities (the "MEPB profile") was very species-specific, with monkeys being similar to rats but different from man, mouse and hamster. The induction of AROD activities by PB and BNF was also highly species-specific. Monkeys expressed constitutive proteins immunorelated to the CYP1A, CYP2A, CYP2B, CYP2C and CYP3A sub-families (human CYP2A6 cross-reacted with the anti-rat CYP2B1 antibodies used, and so CYP2A and CYP2B forms could not be separately identified in the monkey). Single constitutive immunoblot bands were identified in monkey for CYP1A (54 kDa), CYP2A/CYP2B (51 kDa) and CYP3A (51 kDa), respectively, but two strong (51 and 52 kDa) plus two weak (49 and 49.5 kDa) bands were shown for CYP2C. Human liver expressed CYP1A2 (54 kDa), CYP2A6 (51 kDa), CYP3A4 (50.5 kDa) and three CYP2C9-immunorelated protein bands (48, 50 and 54 kDa). In monkeys BNF induced the 54 kDa CYP1A protein and CYP1A-dependent MROD, EROD and PROD activities (18-, 15- and 6-fold increases in activity, respectively), whereas PB strongly induced the 51 kDa CYP2A/CYP2B protein but did not induce PROD activity. PB also induced non-constitutive CYP2A/CYP2B protein bands at 49 and 52 kDa in some monkeys. BROD activity was induced less that four-fold by either PB or BNF in monkeys. In conclusion, cynomolgus monkeys expressed a range of constitutive CYP1A, CYP2A or CYP2B, CYP2C and CYP3A proteins similar to man, and a range of AROD monooxygenase reaction rates similar to both man and rat, but the basal MEPB profile of AROD activities in monkeys was more similar to rat than to man. MROD and EROD were good measures of CYP1A induction by polycyclic aromatic hydrocarbons in cynomolgus monkeys, but neither PROD nor BROD were indices of CYP2B induction by PB.

Use of 7-alkoxyphenoxazones, 7-alkoxycoumarins and 7-alkoxyquinolines as fluorescent substrates for rainbow trout hepatic microsomes after treatment with various inducers.

Various fluorescent substrates have been used as specific indicators of induction or activity of different cytochrome P450 isozymes in both fish and mammalian species. In an attempt to identify additional definitive fluorescent substrates for use in fish, we examined a series of 7-alkoxyphenoxazones, 7-alkoxycoumarins and 7-alkoxyquinolines as substrates in O-dealkylation assays with hepatic microsomes from rainbow trout (Oncorhynchus mykiss). Microsomes were prepared after 48 hr of treatment with beta-naphthoflavone (beta-NF), pregnenolone-16 alpha-carbonitrile (PCN), phenobarbital (PB), isosafrole (ISF), or dexamethasone (DEX). Total P450 spectra were obtained, and spectral binding studies were performed. Microsomal O-dealkylation rates were greater after ISF treatment than after beta-NF treatment for 7-methoxy-, 7-ethoxy-, 7-propoxy- and 7-benzyloxyphenoxazones but not for 7-butoxyphenoxazone. DEX treatment resulted in a significant elevation of pentoxyphenoxazone metabolism (about a 144-fold increase) compared with microsomes induced by beta-NF (11-fold) and ISF (37-fold). The rates of dealkylation of the alkoxyphenoxazones by ISF-treated microsomes occurred in the following order: methoxy > ethoxy > propoxy > benzxyloxy > butoxy > pentoxy. When beta-NF-treated microsomes were used, the 7-alkoxyphenoxazones were metabolized as follows: methoxy > ethoxy > propoxy > butoxy > benzyloxy = pentoxy, while the order of metabolism of the 7-alkoxycoumarins was: ethoxy >> butoxy > propoxy = methoxy > benzyloxy > pentoxy. None of the other treatments significantly increased the rate of metabolism of any of the alkoxycoumarins. Treatment with beta-NF did not significantly elevate the rate of metabolism of any of the alkoxyquinolines. DEX treatment produced significant elevations in the rate of metabolism of benzyloxy-, ethoxy-, and butoxy- = pentoxy- = propoxyquinoline, in that order. ISF treatment significantly elevated the rate of metabolism of benzyloxy-, methoxy- and butoxyquinoline, in that order. These results suggest that some of these new fluorescent substrates can be used to characterize induction of rainbow trout hepatic microsomal monooxygenase activity by ISF and DEX, in addition to the commonly used ethoxyphenoxazone and ethoxycoumarin for the characterization of induction by beta-NF or other 3-methylcholanthrene-type P450 inducers. Distinction between ISF-type and beta-NF-type inducers in rainbow trout hepatic microsomes may best be made using 7-methoxycoumarin as a substrate. Distinction between ISF-type and DEX-type inducers and between beta-NF-type and DEX-type inducers may best be made using 7-methoxyphenoxazone as a substrate.(ABSTRACT TRUNCATED AT 400 WORDS)

Conformers of acetylcholinesterase: a mechanism of allosteric control.

Rate control in acetylcholinesterase (AChE) involves a single anionic site whose anionic center controls rate-related biochemical and conformational changes in the E (free enzyme) and EA (acylated enzyme) conformers. Change in conformer structure and biochemistry affect binding, acylation, and hydrolysis. It is significant that the anionic-esteratic intersite distance is not altered during conformer change as E is converted to EA. In this enzyme system, cationic acetylcholine and anionic AChE are true structural, functional, and biochemical counterparts. The anionic center in the E conformer lies at the bottom of a sterically restricted, hydrophobic cleft < 8 A wide at the top and > 3 A wide at the bottom, while the anionic center in the EA conformer is relatively open. It is characterized by a decrease in the relative binding of hydrophobic cations and by an ability to bind large organic cations. Binding of acetylcholine, H+, or organic cations at the anionic site controls k2(acylation) in the E conformer and k3(hydrolysis) in the EA conformer. Acetylcholine binding forms the ES complex in which the cation maximizes k2. In the EAS complex, the cation reduces k3 and provides allosteric control. Anionic site structure and biochemistry and the effect of pH on k2 and k3 differentiates AChE from butyrylcholinesterase. This comprehensive study of kinetic and thermodynamic processes in AChE was made possible by the synthesis and/or use of families of over 30 cationic and acylation probes of known stereochemistry. They act as rulers of the E and EA conformers of AChE and provide comparative data on kinetic-based and thermodynamic-based constants. Cationic inhibitors affect decarbamylation rates in AChE and provide an additional set of comparative data related to the mechanism of substrate hydrolysis by AChE. Acridine araphanes are unique neural receptor and cholinergic enzyme probes. Their parallel plane and coplanar conformations are related to bridge length. Two parallel plane acridine araphanes are pure uncompetitive inhibitors of AChE. Scatchard plots of the binding of methylacridinium and 9-aminoacridine with the E conformer and 9-aminoacridine with the EA conformer indicate binding at a single anionic site. No ternary complex (EII or EAII) from two-site binding was detected. In AChE, nonspecific, low-level binding at surface ionic and hydrophobic areas is ubiquitous. Binding affinity differences greater than two orders of magnitude distinguish binding at the anionic site from low level binding at surface moieties. Surface binding provides environmental and stability changes in the enzyme but does not modify the fundamental biochemistry of the E and EA conformers.

7-Alkoxyquinoline O-dealkylation by microsomes from human liver and placenta.

1. The O-dealkylation of seven 7-alkoxyquinoline derivatives by human hepatic and placental microsomes and the effect of maternal cigarette smoking on placental 7-alkoxyquinoline metabolism was studied. 2. None of several monoclonal antibodies to isoenzymes of cytochrome P450 had a clear effect on metabolism of the compounds by liver microsomes. 3. Maternal cigarette smoking induced the O-dealkylation of all of the 7-alkoxyquinoline derivatives, being greatest for 7-butoxy- and 7-benzyloxyquinoline. 4. Placental 7-alkoxyquinoline metabolism induced by smoking was partially inhibited by the monoclonal antibody 1-7-1 raised against 3-methylcholanthrene-induced rat liver P450. 5. None of the 7-alkoxyquinoline O-dealkylations could be assigned specifically to any known P450 isoenzyme in human liver or placenta.

Quantitative fluorometric analysis of plant and microbial chitosanases.

A quantitative fluorometric assay for chitosanase activity in bacterial and plant tissues was developed. The assay can be conducted with either finely milled preparations of chitosan in suspension or dissolved chitosan; activity is based on measurements of glucosamine (GlcN) or oligomers of GlcN. GlcN is detected fluorometrically after reaction with fluorescamine with detection in the nanomole range. Fluorescence measurements of chitosanase activity and radioassay of chitinase in commercial preparations of chitinase from Streptomyces griseus revealed that both activities were present. Specific activities for the S. griseus chitosanase using suspended and soluble chitosans were respectively 1.24 and 6.4 mumol GlcN.min-1.mg protein-1. Specific activity of the S. griseus chitinase was 0.98 mumol GlcN.min-1.mg protein-1. Sweet orange callus tissue was tested for chitosanase and chitinase activity. It was necessary to remove small amine-containing molecules from the callus preparations before chitosanase activity could be assayed. The specific activity for chitinase and chitosanase in desalted extracts of nonembryogenic Valencia sweet orange callus tissue was determined to be 18.6 and 89.4 nmol GlcN.min-1.mg protein-1, respectively.

7-Alkoxyquinolines: new fluorescent substrates for cytochrome P450 monooxygenases.

A series of 7-alkoxyquinolines was synthesized and tested as substrates with hepatic microsomes prepared from male Wistar rats. Microsomal O-dealkylation rates and kinetic constants were determined for the 7-alkoxyquinolines with microsomes from control, 3-methylcholanthrene (MC)-pretreated, and phenobarbitone (PB)-pretreated rats. Structure-activity relationship studies indicated that the 7-benzyloxyquinoline was the most rapidly metabolized substrate for control microsomes and those from PB-pretreated rats, whereas the 7-ethoxy- and 7-propoxyquinolines were O-dealkylated more rapidly by microsomes of MC-pretreated animals. Differences in activities occurred in Vmax and apparent Km values; however, there does not appear to be a correlation between these two values for the different quinoline substrates. Apparent Km and Vmax values for the 7-alkoxyquinolines were: control microsomes, Km = 71-773 microM, Vmax = 0.37-8.4 nmol 7-quinolinol/min/mg protein; MC microsomes, Km = 0.5-14 microM, Vmax = 0.29-2.7 nmol 7-quinolinol/min/mg protein; PB microsomes, Km = 2.8-46 microM, Vmax = 0.9-12 nmol 7-quinolinol/min/mg protein. All of the quinoline substrates gave Type I binding spectra with control and MC microsomes. With PB microsomes, Type I. Reverse Type I, and a mixture of the two types of binding spectra were observed. Comparisons of the structure-activity relationships, levels of induction, and kinetic constants were made with 7-alkoxycoumarin and 7-alkoxyphenoxazone analogs. In addition, three new coumarin substrates (7-pentoxy-, 7-hexoxy-, and 7-benzyloxycoumarin) are described.

Purification of phospholipase D from citrus callus tissue.

Phospholipase D in extracts of soluble proteins from callus cultures derived from cotyledons of Citrus sinensis (L.) Osbeck is activated by Ca2+ and anionic detergents and has a pH optimum of 6.5. The enzyme was purified 703-fold over the crude protein extract with a yield of 15% by ammonium sulfate precipitation, ion exchange chromatography, gel filtration, hydrophobic interaction chromatography, and preparative acrylamide gel electrophoresis. Preparative electrophoresis was carried out using conventional slab gel equipment and electroelution of the sliced gel. Analytical sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified phospholipase revealed two bands of the same staining intensity running at 94.2K and 90.5K.

The effects of cigarette smoke compared to 3-methylcholanthrene and phenobarbitone on alkoxyresorufin metabolism by lung and liver microsomes from rats.

The rates of metabolism of phenoxazone and a homologous series of its ethers (alkoxyresorufins) by liver and lung microsomes of rats exposed to cigarette smoke were compared with the metabolism in rats pretreated with 3-methylcholanthrene (3MC) or phenobarbitone (PB). The rate of resorufin production was dependent on the length of the ether side chain. Liver and lung microsomes from control rats differed in their activity profiles (rate of resorufin production plotted against side-chain length), showing highest activity with ethoxy- and benzyloxyresorufin respectively. 3MC and PB selectively induced hepatic microsomal resorufin production with only certain of the substrates and the two agents differed in their selectivity, inducing most greatly with ethoxy- and benzyloxyresorufin respectively. Pulmonary microsomal resorufin production was induced by 3MC with a substrate selectivity similar to that shown for liver, but PB suppressed pulmonary metabolism with all the substrates. A single, short exposure to cigarette smoke induced ethoxyresorufin O-deethylase activity transiently in liver and lung microsomes. Three consecutive daily short exposures to cigarette smoke caused a weak 3MC-like induction of liver microsomal alkoxyresorufin metabolism, but the effect on lung microsomes was like weak 3MC and PB inductions combined. It is concluded that cigarette smoke induces selected cytochrome P-450-linked alkoxyresorufin O-dealkylase activities to a similar extent in both lung and liver and that the effects of cigarette smoke are characteristic of both 3MC-type and non-3MC-type inducers.

Multiple forms of cytochrome P-450 related to forms induced marginally by phenobarbital. Differences in structure and in the metabolism of alkoxyresorufins.

The properties of five structurally related forms of cytochrome P-450 (PB1a, PB1b, PB2a, PB2b and PB2d) isolated from rats treated with phenobarbital have been compared with two forms isolated previously now termed 'PB1c' and 'PB2c'. These enzymes were characterized by their marginal inducibility by phenobarbital and are clearly distinguishable from the major phenobarbital-inducible proteins. PB1a and PB1b differed in Mr (52,700 and 52,900), absorption spectra and papain-proteolysis fragments. However, they had identical N-terminal sequences. PB2a, PB2b and PB2d had apparent Mr values of 52,900, 52,900 and 50,800. PB2a and PB2b had different N-terminal sequences and, after digestion with papain, gave different papain-proteolysis fragments. The N-terminal sequence of PB2b was similar to, but not identical with, that of pregnenolone-16 alpha-carbonitrile-inducible P-450 species, and PB2b was the protein most closely related to PB2c. The extent of immunocross-reactivity among the forms was stronger within, than between, the PB1 and PB2 groups. Even structurally similar forms were functionally diverse, exhibiting large differences in metabolic specificity in the dealkylation of a series of alkoxyresorufins.

Major differences between lung, skin and liver in the microsomal metabolism of homologous series of resorufin and coumarin ethers.

Phenoxazone and a homologous series of its ethers (methoxy to octoxy plus benzyloxy), and coumarin and a series of its ethers (methoxy to propoxy), were metabolized by liver, lung and skin microsomes of normal adult female BALB/c mice. For each series of substrates, and with each tissue, clear structure-activity relationships were seen, relating metabolic activity to the length of the ether side-chain. With the coumarin series of substrates the structure-activity relationships were almost identical in the three tissues, with liver more active than lung and lung more active than skin. Liver, lung and skin microsomes each showed very different structure-activity relationships, however, for metabolism of the phenoxazone series of substrates. Benzyloxyphenoxazone was metabolized almost twice as fast in lung as in liver, but for the other phenoxazone substrates the activities were much greater in liver than in lung or skin. Liver, lung and skin microsomal propoxy- and benzyloxyphenoxazone dealkylase activities differed in their sensitivities to inhibition by metyrapone and alpha-naphthoflavone. The structure-activity relationship and inhibitor data for the phenoxazone substrates are consistent with a view that mouse lung and sking cyt. P-450 are predominantly similar to phenobarbitone-induced and 3-methylcholanthrene-induced forms of hepatic cyt. P-450 respectively. The results also show that the pattern of microsomal metabolism of xenobiotics in lung and skin cannot be reliably predicted from that in liver.

Alkoxyresorufin O-dealkylases: association with the murine Ah locus.

The hepatic microsomal dealkylation of a series of alkoxyresorufins and the oxidation of phenoxazone to resorufin were investigated in C57BL/6 and DBA/2 mice of both sexes. In both strains of mice and in both sexes the dealkylation rate decreased with increasing length of the alkyl chain. With all alkoxyresorufins the dealkylation rates were higher in the C57BL mice than the DBA mice, whereas the rate of phenoxazone hydroxylation was higher in the latter. In the C57BL mice, and to a lesser extent in the DBA mice, females were more efficient in dealkylating the resorufin ethers. Treatment with 3-methylcholanthrene (3MC) enhanced the rates of dealkylation of all alkoxyresorufins in the C57BL mice but not in the DBA mice, the extent of stimulation being highest for the propoxy- and butoxyresorufins and least for pentoxy-, heptoxy- and benzyloxyresorufins. The same treatment had no effect on the oxidation of phenoxazone in either strain of mice. It is concluded that the dealkylation of alkoxyresorufins, not the oxidation of phenoxazone, is associated with the murine Ah locus.