Hepatopancreas Microsomes Research Articles

Differences in cytochrome P450 enzyme activities between fish and crustacea: Relationship with the bioaccumulation patterns of polychlorobiphenyls (PCBs)

Variations in cytochrome P450 enzyme (CYPs) distribution and function between animal groups could result in differential metabolism and elimination kinetics for certain contaminants. Although a number of studies have suggested that differences in polychlorobiphenyl (PCB) accumulation profiles between crustacea and fish might result from differential CYP patterns, the relationship between PCB bioaccumulation and CYP capacities has not been demonstrated in these organisms. In the present study we investigated the hepatic microsomal catalytic activities in three deep-sea fish species, Alepocephalus rostratus (Alepocephalidae), Coelorinchus mediterraneus (Macrouridae), and Lepidion lepidion (Moridae), and the decapod crustacean Aristeus antennatus (Decapoda), using six fluorescent CYP-mediated substrates, namely ER (7-ethoxyresorufin), PR (7-pentoxyresorufin), BR (7-benzyloxyresorufin), CEC (3-cyano-7-ethoxycoumarin), DBF (dibenzylfluorescein) and BFC (7-benzyloxy-4-trifluoromethylcoumarin). Furthermore, we related the metabolic activities to the accumulation patterns of 41 PCB congeners in the muscle of these organisms. The results indicated a marked difference in the presence and activities of CYP isoforms between fish and the crustacean A. antennatus. Liver microsomes of the three selected fish species were capable of metabolizing all six CYP-mediated substrates and enzymes were identified as primarily belonging to CYP1A and CYP3A subfamilies. In contrast, hepatopancreas microsomes from A. antennatus only showed activity for PR and DBF substrates, generally related to mammalian CYP2-like enzymes. Furthermore, a direct relationship between metabolic activities and PCB accumulation profiles could be established. Results revealed that A. antennatus accumulated significantly higher proportions of PCBs 28, 52, 118, 138, 158 and 169 than fish, which is in accordance with the previously observed lack of CYP1A-like biotransformation capacities. Moreover, A. antennatus exhibited lower levels of PCBs 87, 149, 153, 170, 180, 183, 194 and 206 indicating that this crustacean is able to metabolize congeners considered mammalian CYP2B inducers. Hence, the present findings highlight the role of CYP-mediated metabolism in the congener-specific accumulation of PCBs in aquatic organisms and stress the need to further investigate quantitative and qualitative differences in xenobiotic metabolism among animal groups.

Macrobrachium borellii Hepatopancreas Contains a Mitochondrial Glycerol‐3‐Phosphate Acyltransferase Which Initiates Triacylglycerol Biosynthesis

Mammals express four isoforms of glycerol-3-phosphate acyltransferase (GPAT). The mitochondrial isoform GPAT1 may have been the acyltransferase that appeared first in evolution. The hepatopancreas of the crustacean Macrobrachium borellii has a high capacity for triacylglycerol (TAG) biosynthesis and storage. In order to understand the mechanism of glycerolipid biosynthesis in M. borellii, we investigated its hepatopancreas GPAT activity. In hepatopancreas mitochondria, we identified a GPAT activity with characteristics similar to those of mammalian GPAT1. The activity was resistant to inactivation by SH-reactive N-ethylmaleimide, it was activated by polymyxin-B, and its preferred substrate was palmitoyl-CoA. The reaction products were similar to those of mammalian GPAT1. A 70-kDa protein band immunoreacted with an anti-rat liver GPAT1 antibody. Surprisingly, we did not detect high GPAT specific activity in hepatopancreas microsomes. GPAT activity in microsomes was consistent with mitochondrial contamination, and its properties were similar to those of the mitochondrial activity. In microsomes, TAG synthesis was not dependent on the presence of glycerol-3 phosphate as a substrate, and the addition of monoacylglycerol as a substrate increased TAG synthesis 2-fold. We conclude that in M. borellii the de novo triacylglycerol biosynthetic pathway can be completed in the mitochondria. In contrast, TAG synthesis in the ER may function via the monoacylglycerol pathway.

Intermoult duration affects the susceptibility of shore crabs Carcinus maenas (L.) to pyrene and their ability to metabolise it

We have studied pyrene (PYR) toxicity and the ability to metabolise and eliminate PYR in two colour forms of shore crabs Carcinus maenas (Linnaeus, 1758). In addition, we analysed differences in the expression of expressed sequence tags (ESTs) encoding specific cytochrome P450s (CYPs) by quantitative realtime PCR. Green and red intermoult crabs are considered to represent different adaptational life stages, allocating energy into growth (green) and reproduction (red), respectively. PYR injection resulted in significantly higher mortality in red crabs than in green crabs during a 51 days period. PYR is an ideal model PAH compound as only 1 phase I metabolite, 1-hydroxypyrene (1-HP) is formed, which is further conjugated to form various phase II metabolites. In this study, 1-HP was detected only after deconjugation of total PYR derived metabolites indicating that PYR hydroxylation (putatively CYP catalysed) conceivably is the rate-limiting step in PYR metabolism. Investigation of the accumulation of PYR and 1-HP (after deconjugation), in different tissues revealed a significantly higher accumulation of PYR in muscle and epidermis of red crabs compared to green crabs. Consistent with this observation, green crabs had significantly higher levels of 1-HP in the hepatopancreas than red crabs. This indicates that a larger portion of the injected PYR was metabolised into 1-HP in green crabs compared to red crabs. 1-HP was mainly detected in the hepatopancreas confirming its major role in the biotransformation of lipophilic compounds. CYP enzymes typically mediate the phase I hydroxylations of lipohilic contaminants such as PYR. In agreement with the higher rate of conversion of PYR into 1-HP in green compared to red crabs, increased abundance of several CYP transcripts was observed in green crabs. Furthermore, in vitro pyrene hydroxylase assays revealed significantly higher NADPH-depedent pyrene hydroxylase activity in hepatopancreas microsomes of green crabs (18.4 ρmol min −1 mg −1 protein) compared to red crabs (8 ρmol min −1 mg −1 protein). The present study demonstrates that the susceptibility of shore crabs to PYR and their ability to metabolise it is life stage dependent, conceivably due to life stage related differences in the expression of certain CYP genes, suggesting a mechanistic explanation of the observed life stage differences in PYR toxicity.

Reductive Dechlorination of p, p'-DDT Mediated by Hemoproteins in the Hepatopancreas and Blood of Goldfish, Carassius auratus

The in vitro metabolism of p, p'-DDT (p, p'-dichlorodiphenyltrichloroethane), an important environmental pollutant, was examined in fish, focusing on reductive dechlorination. When p, p'-DDT was incubated with hepatopancreas microsomes or the blood of goldfish, Carassius auratus, in the presence of both a reduced pyridine nucleotide and FMN, a dechlorinated metabolite, p, p'-DDD (p, p'-dichlorodiphenyldichloroethane) was formed under anaerobic conditions. These reductase activities were inhibited by carbon monoxide. Although the microsomes or blood was boiled, the dechlorinating activity was not abolished. Hemoglobin and hematin exhibited the reductase activity toward p, p'-DDT with NADH and FMN. The activity of hematin was also exhibited with FMNH2. The reductive dechlorination appears to proceed nonenzymatically by the reduced flavin, catalyzed by the heme group of hemoproteins.

Interconversion between Fenthion and Fenthion Sulfoxide in Goldfish, Carassius auratus

The in vitro metabolism of fenthion, an insecticide, was examined in fish, focusing on the interconversion between fenthion and its sulfoxide (fenthion sulfoxide). When fenthion was incubated with hepatopancreas microsomes of goldfish, Carassius auratus, in the presence of NADPH, the oxidized metabolite, fenthion sulfoxide was formed, but not fenthion sulfone. The oxidase activity was inhibited by SKF 525-A, and partly by α-naphthylthiourea. In contrast, fenthion sulfoxide was reduced back to fenthion by the hepatopancreas cytosol of goldfish in the presence of 2-hydroxypyrimidine, an electron donor for aldehyde oxidase. The activity was markedly inhibited by menadione, an inhibitor for aldehyde oxidase. The interconversion appears to be mediated mainly by the cytochrome P450 system and aldehyde oxidase.

Cytochromes P450 in crustacea

Since the last review of this topic, further insight has been gained into the presence and functions of cytochrome P450 proteins in the hepatopancreas and other organs of aquatic crustacean species, although progress has been slow relative to the advances in other species. Recent studies with several lobster, shrimp, crab and crayfish species suggest that cytochromes P450 in the 2 and 3 families are the most abundant forms in hepatopancreas microsomes. Substrates normally metabolized by CYP2 and CYP3 family members are monooxygenated more rapidly by crustacea than substrates normally metabolized by CYP1 family enzymes, e.g. erythromycin, testosterone and aminopyrine are much more rapidly monooxygenated than ethoxyresorufin. Some progress has been made in cloning and sequencing crustacean P450 forms. CYP2L1 and CYP2L2 cDNA sequences have been cloned from spiny lobster hepatopancreas libraries, and there was evidence for at least two more cytochromes P450 in spiny lobster hepatopancreas. An area of continued interest, but of no consensus or general findings, relates to the presence and inducibility of CYP1 family members in crustacea. Some studies indicate weak induction of total cytochrome P450 and increased turnover of substrates normally associated with CYP1, while others show no effect of the classic inducers that act at the Ah receptor in vertebrates. A few studies of the roles of cytochromes P450 in the biosynthesis and degradation of steroids, including ecdysteroids, have been published. Further studies are needed to understand the regulation and normal function of the crustacean cytochromes P450.

Cytochrome P450 system in the hepatopancreas of the red swamp crayfish Procambarus clarkii: a field study

Cytochrome P450 monooxygenase system and acetylcholinesterase (AChE) activity were studied in the red swamp crayfish Procambarus clarkii sampled from the Ebro Delta before and after spraying of the area with fenitrothion. Analysis of hepatopancreas microsomes revealed an increase of 7-ethoxyresorufin O-deethylase (EROD) activity in organisms sampled after spraying. AChE activity was significantly inhibited in the same specimens, corroborating that these organisms were exposed to fenitrothion, and the usefulness of AChE as a marker of pesticide poisoning in P. clarkii. Crayfish from a reference site were analyzed and the results compared with those obtained in organisms from the Ebro Delta. The ability of P. clarkii to metabolize the organophosphorus pesticide fenitrothion was determined in vitro by using microsomal fractions isolated from hepatopancreas. The results pointed out the existence of an oxidative metabolism, which was inhibited by different P450 inhibitors (clotrimazole, α-naphthoflavone and N-benzylimidazole) and not affected by methimazole.

Isolation of CYP2L2 and two other cytochrome P450 sequences from a spiny lobster, Panulirus argus, hepatopancreas cDNA library

Cytochrome P450 monooxygenases constitute an enzyme superfamily, with at least 74 known families. Members of CYP families 1–4 are important in the phase 1 metabolism of lipophilic xenobiotics, such as those found in contaminated marine environments. Previous studies (James et al. Arch. Biochem. Biophys. (1996) 329, 31–38) showed that a major form of P450 in spiny lobster, Panulirus argus, hepatopancreas was CYP2L1, a new sub-family, and that there was evidence for other P450 forms in hepatopancreas. We now report the sequence of a second member of this subfamily, named CYP2L2, present in P. Argus hepatopancreas. The deduced amino acid sequences of CYP2L1 and CYP2L2 share 54.7% sequence identity and an additional 13.6% of the sequences show conservative substitutions. Analysis of the sequences of CYP2L1, CYP2L2 and other representative CYP2 family members (from rat and mouse sub-families 2A, 2B, 2D and 2E) showed that the crustacean sequences clustered together. In addition to CYP2L2, cDNA clones of 66 to 117 base pairs from the 5′ coding region of two more P450 isoforms were isolated from the spiny lobster cDNA library. The deduced amino acid sequence of one of these additional cDNA clones was identical to the first 22 amino acids of the N-terminal sequence of a P450 protein previously isolated from hepatopancreas microsomes. These studies confirm earlier biochemical evidence that the hepatopancreas contains multiple forms of cytochrome P450.

Expression of CYP2L1 in the yeast Pichia pastoris, and determination of catalytic activity with progesterone and testosterone

Cytochrome P450 (P450) enzymes are widely distributed throughout the plant and animal kingdoms. In previous work, two forms of P450 partially purified from spiny lobster, Panulirus argus, hepatopancreas microsomes were shown to metabolize several substrates, including testosterone and progesterone at the 16α, 6β and 21 positions f James (1990) Archives of Biochemistry and Biophysics 282, 8–17]. We have recently cloned and sequenced a major form of P450, CYP2L1, from hepatopancreas cDNA [James et al. (1996) Archives of Biochemistry and Biophysics 329, 31–38]. CYP2L1 is one of at least four P450s detected in a cDNA library constructed from spiny lobster hepatopancreas. The spiny lobster CYP2L1 cDNA was introduced into the methylotrophic yeast Pichia pastoris and the crustacean CYP2L1 was expressed in microsomes of the methanol-induced yeast. On the basis of immunoblot analysis and ability to catalyse the 16α-hydroxylation of testosterone and progesterone, the catalytically active, heterologously expressed spiny lobster CYP2L1 appears to be very similar to the major spiny lobster hepatopancreas P450 previously purified in this laboratory. This is the first report of heterologous expression of a crustacean P450.

Induction of cytochrome P450 3A and heat shock protein by tributyltin in blue crab, Callinectes sapidus

Tributyltin (TBT), a toxic contaminant in aquatic environments, breaks down cytochrome P450 enzymes (P450s) in vitro. To determine in vivo effects of long-term TBT-exposure in aquatic invertebrates, respiration rates, heat shock protein induction, microsomal cytochrome P450 levels and metabolism of [ 14C]testosterone were examined in the hepatopancreas of blue crabs fed TBT. Four groups of crabs were fed fish injected with ethanol or 2, 4 or 8 μg TBT Cl dissolved in ethanol/g meat; doses of 0, 125, 250, or 500 μg kg −1 respectively. Crabs were fed TBT-treated meat every other day for 16 days. Respiration rates were significantly decreased after TBT exposure. Hepatopancreas microsomes and cytosol were prepared 24 h after the last feeding. Heat shock protein was induced at the two highest TBT exposure concentrations. Total P450 levels were similar in all samples; however, protein cross-reacting with anti-scup CYP 3A, an isozyme responsible for 6 β-hydroxylation of testosterone in vertebrates, increased at the 250 μg and 500 μg TBT Cl kg −1 dose. EROD activity showed that CYP 1A was not elevated. Hydroxylation of [ 14C]testosterone at the 6 β, 6 α, 7 α, and 16 α positions by hepatopancreas microsomes increased significantly in crabs exposed to 250 μg TBT Cl kg −1. The lack of increase in [ 14C]testosterone hydroxylation at the 500 μg kg −1 dosage may result from TBT Cl's disruption of P450 activity at high concentrations. Incubation of control microsomes with TBT significantly reduced spectrally quantified P450 as well as P450 activity after 2 h, showing that TBT Cl interferes with crab microsomal P450 proteins. Immunoinhibition of testosterone 6 β-hydroxylation was accomplished by using scup CYP 3A antibody, showing that the antibody specifically binds to a protein with 6 β-hydroxylase activity. These results indicate that blue crabs are stressed by TBT-exposure, and upregulate P450 isozymes possibly to aid in metabolism and elimination of TBT.

Cross-reactivity of an antibody to spiny lobster P450 2l with microsomes from other species

The gene for a new form of P450, CYP2L, was recently sequenced in this laboratory, using a primer designed from the N-terminal amino acid sequence of a P450 partially purified from spiny lobster hepatopancreas microsomes. The partially purified (75% pure) P450 was used to immunize rabbits. Serum containing the rabbit anti-CYP2L antibody was demonstrated to possess good cross-reactivity on Western blots with microsomal preparations from hepatopancreas of the spiny lobster. In Western blot studies with microsomal preparations from other aquatic invertebrates, the horseshoe crab, the blue crab, the slipper lobster, the American lobster and the brown shrimp, the slipper lobster microsomal preparations showed strong cross-reactivity with the spiny lobster antibody and the American lobster weak cross-reactivity. Hepatic microsomes from all of the vertebrate species studied showed cross-reactivity with the anti-CYP2L antibody. The fish species studied were killifish, clear-nose skate, snook and catfish. Strong cross-reactivity was found with killifish and catfish, and weak cross-reactivity was found with skate and snook in the 45–66 kDa region. Cross-reactivity was also found with microsomes from male control and phenobarbital-induced rats. These studies suggest that epitopes in CYP2L are also present in other invertebrate and vertebrate species.

CDNA and Protein Sequence of a Major Form of P450, CYP2L, in the Hepatopancreas of the Spiny Lobster,Panulirus argus

A P450 fraction was previously isolated from spiny lobster hepatopancreas microsomes and shown in reconstitution experiments to be efficient in catalyzing the monooxygenation of benzphetamine, aminopyrine, benzo(a)pyrene, progesterone, and testosterone. In this study, N-terminal sequence information up to residue 39 was obtained from this P450 and used to design degenerate primers for screening a cDNA library constructed from hepatopancreas mRNA. Clones were obtained that contained part of the coding region of a P450 protein. Further exact primers were designed that permitted the isolation of clones containing coding information for other parts of the P450 sequence, as well as a clone that coded for the complete P450 protein sequence. The open reading frame of the complete coding region corresponded to a protein of 492 amino acids. The deduced amino acid sequence of this P450 was about 36% similar to individual mammalian P450s in the 2 family and did not show strong matches with other proteins in the data base. Based on sequence and the previously determined function, this spiny lobster P450 was assigned by the P450 nomenclature committee to a new P450 subfamily, CYP2L. This is the first description of a P450 primary sequence from a marine crustacean species and the first assignment of an invertebrate P450 into the 2 family.

Isolation of cytochrome P450 from hepatopancreas microsomes of the spiny lobster, Panulirus argus, and determination of catalytic activity with NADPH cytochrome P450 reductase from vertebrate liver

One major form of cytochrome P450 has been isolated from the hepatopancreas of untreated spiny lobsters, Panulirus argus. This form, termed here D1, was purified to a specific content of 12.1 ± 1.8 nmol/mg protein. Two minor forms, termed D2 and D3 were partially purified to 4.6 ± 1.6 and 2.3 ± 0.2 nmol P450/mg protein, respectively. No NADPH-cytochrome P450 reductase activity was detected in spiny lobster hepatopancreas microsomes and no purification of spiny lobster reductase was achieved in this study. Very low NADPH-cytochrome c reductase activity was found in hepatopancreas microsomes and also in cytosol. Indirect evidence suggested that proteolysis of spiny lobster P450 reductase during the preparation of hepatopancreas microsomes may in part account for the lack of detectable monooxygenase activity in hepatopancreas microsomes. The catalytic activities of the D1 or D2 forms of spiny lobster P450 were measured by mixing D1 or D2 with NADPH-cytochrome P450 reductase isolated from pig or rat liver microsomes. D2 was very efficient in demethylating benzphetamine, with a turnover number of 122 per minute, and D1 was an efficient catalyst of progesterone 16 α-hydroxylation, with a turnover number of 43 per minute. Other good substrates for D1 and D2 forms were aminopyrine, testosterone, benzo(a)pyrene, and 7-ethoxycoumarin. Little activity was found with methyl-, ethyl-, pentyl-, or benzyl-phenoxazone ethers as substrates. The profile of metabolites formed by D1 or D2 with benzo(a)pyrene as substrate were more similar to those formed with uninduced rat liver microsomes than to those formed by liver microsomes from uninduced flatfish species.

Palmitoyl-CoA ligase activity in hepatopancreas and gill microsomes of the freshwater shrimp Macrobrachium borellii

1. 1. The kinetic parameters of microsomal palmitoyl-CoA ligase from gills and hepatopancreas of Macrobrachium borellii were investigated. 2. 2. The optimal temperature for the enzymatic activity was 37°C in gill microsomes and 30°C in hepatopancreas microsomes. 3. 3. The maximal affinity of the enzyme for its substrate was higher in gill microsomes (apparent K m = 12.59 μ M ) compared with hepatopancreas microsomes (apparent K m = 49.74 μ M ). 4. 4. A lower lipid mobility in gill microsomes than in hepatopancreas microsomes was found. It was attributed to a high phosphatidyl ethanolamine content in gill membranes. This could also explain the differences in the optimal temperatures for the activities of the enzyme from both organs.

Oxyradical production by hepatopancreas microsomes from the red swamp crayfish, Procambarus clarkii

Microsomes catalyze the one electron reduction of many xenobiotics leading to proliferative production of damaging oxyradicals via redox cycling. The oxidation of 2-keto-4-thiomethylbutyric acid (KMBA) to ethylene was used as a means to study the O 2-/H 2O 2-generating oxidase pathway of hepatopancreas microsomes from the red swamp crayfish, Procambarus clarkii. Ethylene was produced by P. clarkii hepatopancreas microsomes in a chelex-treated incubation medium. The reaction was dependent upon NAD(P)H, O 2 and intact microsomes. NADH supported a 2- to 3-fold greater rate of KMBA oxidation by hepatopancreas microsomes than did NADPH, whereas NADPH supported a higher rate of KMBA oxidation than did NADH using rat liver microsomes. Ethylene production was increased approximately 3-fold in the presence of azide, an inhibitor of catalase, suggesting that H 2O 2 served as the precursor of · OH ∗ ∗ ·OH is taken here to include any 3 electron reduction product of O 2 with the oxidizing power of ·OH, e.g. a ferryl species (Aust and Svingen, 1982) production. Likewise, the addition of exogenous catalase in an azide-free system almost completely eliminated ethylene production. Superoxide dismutase was without any significant effect on KMBA oxidation by P. clarkii microsomes. Ethylene production by P. clarkii hepatopancreas microsomes was suppressed by competing hydroxyl radical scavenging agents, i.e. benzoate, mannitol, ethanol, and dimethyl sulfoxide (DMSO). Ferric ethylenediaminetetracetic acid (EDTA) stimulated NAD(P)H-dependent ethylene production in a dose-related manner, thus ferric was reduced by the system. KMBA oxidation was stimulated by EDTA alone and inhibited by the potent ferric-binding chelate, desferrioxamine suggesting the presence of iron in the microsomal system. Rates of · OH generation as measured by formaldehyde production from DMSO were consistent with rates calculated via ethylene production from KMBA. These data show that hepatopancreas microsomes generate potent oxidants during NAD(P)H-dependent microsomal electron transport. The kinetics of oxidant production appears to reflect differential reactivities of ferric-EDTA with NADH- vs. NADPH-dependent reductase(s) which varies considerably between hepatopancreas and rat liver microsomal systems.

Characterization of the microsomal mixed-function oxygenase system of the hepatopancreas and green gland of the red swamp crayfish, Procambarus clarkii

1. The microsomal mixed-function oxygenase (MFO) system from the hepatopancreas and green gland of the red swamp crayfish, Procambarus clarkii has been characterized with respect to the constitution of electron transport proteins and the ability to catalyze the metabolism of xenobiotics. 2. Cytochrome P-450 content of hepatopancreas microsomes was approximately 10-fold higher than that of green gland and comparable to that of rat liver. NADPH-cytochrome c reductase activity in hepatopancreas microsomes was approximately 2% of that found in rat liver microsomes. 3. Green gland microsomes catalyzed higher turnover rates of aminopyrine N-demethylase and benzo[a]pyrene hydroxylase than hepatopancreas microsomes. With hepatopancreas microsomes, organic hydroperoxides supported a greater rate of aminopyrine N-demethylation than did NADPH plus O2. 4. P. clarkii hepatopancreas microsomes generally displayed a lower binding affinity (Ks) for a number of type I and type II ligands than did rat liver microsomes.

Cytochrome P450 monooxygenases in crustaceans.

1. The hepatopancreas is the major site of cytochrome P450-dependent xenobiotic monooxygenation in crustacean species, but the presence of monooxygenase inhibitors in hepatopancreas microsomes and cytosol from many decapod species has impeded in vitro studies. Cytochrome P450 and monooxygenase activities have been reported in other crustacean organs including the antennal gland (green gland) and stomach. 2. NADPH cytochrome c reductase activity is often very low (typically less than 10 nmol cytochrome c reduced/min per mg microsomal protein) in hepatopancreas microsomes from crustacean species. NADPH cytochrome P450 reductase activity has not yet been detected in crustacean hepatopancreas microsomes. 3. The cytochrome P450 present in hepatopancreas of several crab species and the spiny lobster has been resolved into several fractions by chromatography on DEAE-cellulose. One form of cytochrome P450 from spiny lobster has been purified to 12 +/- 2 nmol/mg protein. 4. Reconstitution studies with spiny lobster hepatopancreas P450 have shown that the vertebrate sex steroids, progesterone and testosterone, are excellent substrates, whereas ecdysone--the crustacean molting hormone--is not a substrate. Activity was found with several xenobiotic substrates including benzphetamine, aminopyrine, benzo(a)pyrene, ethyl-, benzyl- and pentyl-phenoxazones and ethoxycoumarin. Highest activities (greater than 50 nmol/min per nmol P450) were found for N-demethylation of benzphetamine and aminopyrine. 5. The ability of agents which induce vertebrate cytochrome P450 to induce cytochrome P450 in crustaceans is still unclear. Some studies indicate that polycyclic aromatic hydrocarbons, but not phenobarbital-type inducers, could induce cytochrome P450 in crustaceans, whereas other studies showed no effect of either inducer type. Crustaceans are not as sensitive as fish to induction of P450 and monooxygenase activity.

NAD(P)H-dependent oxidation of the hydroxyl radical scavenging agent 2-keto-4-thiomethylbutyric acid (KMBA) by microsomal fractions from the hepatopancreas of the red swamp crayfish, Procambarus clarkii

A secondary pathway of the microsomal mixed-function oxidase system in which the terminal oxidase is expressed as hydrogen peroxide (H 2O 2) production is well documented in mammalian systems. 1 H 2O 2 is a precursor of hydroxyl radical (·OH), a potent oxidant that has been implicated in numerous pathologies and in the metabolism of various xenobiotic substrates. 2 The ability of ·OH to oxidize 2-keto-4-thiomethylbutyric acid (KMBA) was used as a means to study the ·OH-generating oxidase pathway of P. clarkii hepatopancreas microsomes. Both a post-mitochondrial supernatant (S9) and microsomes catalyzed the oxidation of KMBA in a reaction that was dependent upon NAD(P)H, stimulated by added iron-EDTA and inhibited by superoxide dismutase (SOD), catalase and competing ·OH scavenging agents. The H 2O 2/·OH generating oxidase activity of P. clarkii microsomal fractions was mainly NADH-dependent, which is in contrast to the rat system which is mainly NADPH-dependent. This oxidase activity should be considered in evaluating xenobiotic metabolism by this aquatic invertebrate.

Purification and characterization of a single form of cytochrome P-450 from the spiny crab Maja crispata

1. 1. Microsomes from Maja crispata hepatopancreas contain all the components of the functional mixed function oxidase system: cytochrome P-450 (0.47 nmol/mg), the activity of NADPH cytochrome c reductase (12.25 nmol/mg/min) and benzo[a]pyrene monooxygenase activity (6.58 pmol/mg/min). 2. 2. Solubilization of hepatopancreas microsomes with sodium cholate, and affinity chromatography on omega-amino- n-octyl Sepharose 4B, gave a single cytochrome P-450 peak eluting with 0.2% Emulgen 913. 3. 3. DEAE cellulose chromatography of this cytochrome peak gave rise to a single haemoprotein peak, with apparent monomer M τ = 53,500, as determined by sodium dodecyl sulphate polyacrylamide gel electrophoresis.

Cytochrome P-450-dependent oxidation of progesterone, testosterone, and ecdysone in the spiny lobster, Panulirus argus

Experiments were performed to determine the ability of the cytochrome P-450 present in hepatopancreas microsomes from the spiny lobster, Panulirus argus, to catalyze oxidation of progesterone, testosterone, and ecdysone. Preparations of hepatopancreas microsomes fortified with NADPH cytochrome P-450 reductase from pig liver efficiently catalyzed NADPH-dependent 16α- and 6β-hydroxylation of progesterone and testosterone, and 21-hydroxylation of progesterone. These products were also found if NADPH and NADPH cytochrome P-450 reductase were replaced by cumene hydroperoxide. Cytochrome P-450 purified from hepatopancreas microsomes catalyzed NADPH- and reductase-dependent 16α-hydroxylation of progesterone and testosterone 10 times more rapidly than the original microsomal preparation. While ecdysone was not a substrate for the hepatopancreas microsomal cytochrome P-450, low ecdysone 20-monooxygenase activity was found in hepatopancreas mitochondria. Further studies showed that homogenates of green gland, ovaries, and testes had higher ecdysone monooxygenase activities than hepatopancreas, and that the activity in green gland was localized in mitochondria.