Isolation of a novel hepatic CYP2-related protein from channel catfish, Ictalurus punctatus

Channel catfish (Ictalurus punctatus) have been previously shown to express two major cytochrome P450 (CYP) protein bands that are cross-reactive with anti-CYP2K1 (rainbow trout, Oncorhynchus mykiss) antibodies on Western blots. These proteins appear to be the major constitutive CYPs in channel catfish and show distinct sex- and age-specific variations in expression. Because I. punctatus is an important agricultural and ecological commodity, and because it displays a high degree of resistance to the toxic effects of many pesticides, the molecular and catalytic characteristics of its biotransformation systems are of interest to those in areas of environmental science and aquaculture research. Using a chromatographic method similar to that employed in the purification of other fish CYP2 enzymes, a single CYP2-related protein (CM-HA3) was isolated from channel catfish hepatic microsomes. The isolated protein displays a relative molecular mass of approximately 47 kDa, and a CO-reduced difference spectrum _max of 449.6 nm. The sequence of 15 residues at the amino-terminal of CM-HA3 is 27% identical to both CYP2K1 and CYP2M1 isoforms of rainbow trout. Correlational analysis was employed to characterize potential substrates for this isoform, but no significant relationship was observed with E₂ hydroxylation, testosterone hydroxylation, or 7-ethoxycoumarin O-deethylase activities. These data indicate that CM-HA3 is a CYP2 family protein, with as yet uncharacterized substrate specificities.     

Species differences and effects of soft coral extracts from <Emphasis Type="Italic">Sinnularia maximus</Emphasis> on the expression of cytochrome P4501A and 2N in butterflyfishes (<Emphasis Type="Italic">Chaetodon</Emphasis> spp.)

Cytochrome P450 (CYP) has been shown to confer resistance in numerous terrestrial insects that consume potentially toxic secondary metabolites in plants, but fewer studies have examined the role of critical biotransformation enzymes in allowing marine organisms to consume chemically defended foods. This study examined the expression of CYP1A and CYP2N mRNAs in several butterflyfish species, which can feed on numerous chemically defended soft and hard corals. In addition, the effect of an extract from a soft coral (Sinnularia maxima) on expression of hepatic CYP1A and CYP2 mRNAs was also examined. Fish were fed extracts on days 1, 3 and 5, and expression was examined on day 5. Phylogenetic analyses of the CYP1A cDNA from 12 species of butterflyfish (DNA, amino acid) indicate well-separated groupings according to their feeding strategies. The non-coralline feeding Chaetodon xanthurus exhibited a 7-fold higher basal expression of CYP2N8 relative to the other species studied. Although induction of CYP2N7 expression was observed in C. punctatofasciatus, CYP1A and CYP2N was largely unaffected or diminished by extract treatment in the other species of butterflyfish. These results indicated groupings of feeding strategy with CYP1A phylogeny in Chaetodon, but generally unaltered expression of CYP1A and CYP2N following dietary treatment with an extract from a chemically defended soft coral suggesting an inconclusive role of these isoforms in the detoxification of chemicals in these extracts.     

Impacts of hypersaline water on the biotransformation and toxicity of fenthion on rainbow trout (Oncorhynchus mykiss), striped bass (Morone saxatilis X Morone chrysops) and tilapia (Oreochromis mossambicus)

Hypersaline water derived from agricultural practices primarily in arid landscapes can impact fisheries in streams receiving run-off from fields. Previous studies have indicated significant elevation in the toxicity of thio-ether pesticides by hypersaline water in certain species of euryhaline fish due to enhanced formation of sulfoxide metabolites which may be more toxic than the parent compounds. The objectives of this study were to evaluate the effects of salinity on the toxicity of the thio-ether organophosphate pesticide, fenthion in three species of euryhaline fish: rainbow trout (Oncorhynchus mykiss), striped bass (Morone saxatilis X Morone chrysops), tilapia (Oreochromis mossambicus) and determine whether a relationship was observed between toxicity and enantioselective sulfoxidation. Stereochemical formation and total sulfoxide formation did not mirror acute toxicity in the three species exposed in fresh or hypersaline conditions. Mortality of striped bass and rainbow trout due to fenthion exposure was enhanced by hypersaline treatments and a trend towards increased toxicity was observed in tilapia. In liver microsomes of rainbow trout, inhibition of cytochrome P450 (CYP) caused a respective 7- and 3-fold increase in sulfoxide formation in liver microsomes fresh and saltwater fish, and a significant increase in the formation of the (+)R-sulfoxide. CYP inhibition also caused a significant elevation of the (+)R-sulfoxide in freshwater striped bass, but not in hypersaline-treated bass. The results indicated contribution by CYP in the sulfoxidation of fenthion as well as the formation of other metabolites in all three species. In summary, hypersaline conditions impacted fenthion toxicity under conditions that appeared to be independent of fenthion sulfoxide formation indicating a much more complex mechanism of action for compounds with phosphorothioate and thio-ether structural features potentially involving multiple oxidative pathways.     

Comparisons of Uptake and Depuration of 2-Methylisoborneol in Male, Female, Juvenile, and 3MC-induced Channel Catfish Ictalurus punctatus

The occurrence of off-flavors in farm-raised channel catfish is a major burden to the catfish culture industry, causing millions of dollars worth of market-ready fish to be rejected each year. Off-flavors are believed to be the result of exposure and subsequent accumulation of certain volatile microbial metabolites such as 2-methylisoborneol. In this study, the uptake and elimination of [¹⁴C]-2-methylisoborneol equivalents were examined in juvenile and mature channel catfish. The described method is presented as a simple and inexpensive procedure for comparing treatment effects on off-flavor elimination rates. In addition, the effects of the cytochrome P4501A inducing agent 3-methylcholanthrene on 2-methylisoborneol depuration was studied in juvenile fish. Uptake was determined during a 24–h static exposure to waterborne 2-methylisoborneol (7.6 ± 3.7 μg/L). Uptake equilibrium had been established in all groups by 12 h, with total uptake higher in juvenile fish. Depuration rates were not different between untreated groups (t_½ a = 12.r122.4 h; t_½β= 91.3–143.7 h); however, the depuration half-lives for 3-methylcholanthrene treated juveniles were significantly longer than those of controls.     

2-Methylisoborneol disposition in three strains of catfish: absence of biotransformation

2-Methylisoborneol (MIB) and structurally related terpenoid compounds are responsible for millions of dollars of lost revenue to catfish farmers. In an attempt to determine enzymatic pathways of biotransformation and elimination of MIB, the in vitro metabolism of MIB was examined in the Ulvade strain of channel catfish (Ictalurus punctatus). Although cytochrome P450 (CYP) activities were observed and correlated with expression of specific isoforms (i.e. steroid hydroxylation and CYP3A expression), no metabolites of MIB were observed. To determine whether extrahepatic biotransformation may be occurring the in vivo metabolism and disposition of ¹⁴C-MIB was examined in Uvalde, USDA-103 channel catfish, and a channel catfish X blue catfish (Ictalurus furcatus) hybrid species. Confirming in vitro hepatic studies, no metabolites were observed in plasma from animals treated with an intra-arterial dose of ¹⁴C-MIB. ¹⁴C-MIB elimination was predicted using a two compartment model in each strain of fish. There was no significant difference in terminal half-lives between strains but possible differences in total body clearance and apparent volumes of distribution which may be related to higher lipid content in the hybrids. Results of these studies indicate biotransformation has no involvement in MIB elimination and that other physiological processes may play a more significant role in MIB disposition within Ictalurid fish species.     

Effect of ethanol, clofibric acid and temperature on the uptake and elimination of 2-methylisoborneol in channel catfish (Ictalurus punctatus)

Off-flavor, due to organoleptic compounds such as 2-methylisoborneol (MIB), is the single largest detriment to the harvest, production and profit from the channel catfish aquaculture industry of the Southeastern United States. Methods to increase the metabolism and/or elimination of compounds like MIB would provide a means towards improving catfish rearing practices. Previous studies indicated one or more forms of cytochrome P450 monooxygenase (CYP) may be involved in the biotransformation and elimination of specific organoleptic compounds, such as 2-methylisoborneol (MIB). In order to determine the role of CYP in the elimination of MIB, various compounds that have been shown to modulate cytochrome P450 expression in catfish were administered before and after exposure to ¹⁴C-MIB. Uptake and elimination was monitored in fish over 24 and 48 h, respectively. Pretreatment with clofibric acid (100 mg kg^–1-gavage) which induces a CYP2K-like isoform, and ethanol (1.0% v/v-aqueous) a CYP2 represser, alone and with enhanced temperature (added 10 °C) failed to affect uptake of MIB. Pretreatment with these compounds and conditions also failed to enhance elimination of MIB from channel catfish. However, when fish were treated with 1.0% ethanol after MIB exposure (i.e., during depuration), beta elimination halflives were changed from 144±35 to 71±13 h. in sexually mature animals but unchanged (191±113) in juveniles. The failure of CYP-modulating agents to alter MIB elimination in catfish suggests MIB may not readily undergo Phase I oxidation via CYP. The enhanced elimination of MIB in adults by ethanol warrants further study as to its potential use in aquaculture in purging MIB and related compounds prior to fish processing.     

Effect of urea and low temperature on the expression and activity of flavin-containing monooxygenase in the liver and gill of rainbow trout (Oncorhynchus mykiss)

Little is known about the physiological role of flavin containing monooxygenases (FMOs) in teleost fish. Recent studies have indicated induction during saltwater adaptation in several telost species including rainbow trout. A physiological product of FMOs, trimethylamine N-oxide (TMAO) and urea have also been shown to increase in rainbow trout muscle during saltwater adapatation. TMAO counteracts the denaturing effects of urea. In order to evaluate urea as a possible inducer of FMO expression and activity, adult rainbow trout were infused for 48 h with urea/saline solutions at a loading rate of 8 mmol urea/kg/day. To determine whether low temperature had any effect on FMO expression and activity, one group of animals was infused with urea and exposed to low temperature (2–3 °C) for 48 h. Gill FMO-catalyzed thiourea oxygenase activity was significantly induced by low temperature, with twice the activity observed at low temperature (1.116±0.356 nmol/min/mg) compared to urea infusion at 10 °C (0.585±0.282 nmol/min/mg). Low temperature without urea treatment caused a 50% increase in gill FMO activity. In the liver, urea infusion caused an increase in liver FMO activity (from 0.144±0.053 nmol/min/mg to 0.464±0.237 nmol/min/mg), but was unaffected by co-exposure to low temperature (0.523±219 nmol/min/mg). FMO expression and activity correlated with elevated tissue urea levels, but TMAO concentrations were not related. The interactions between urea, temperature and the tissue-specific induction of FMOs indicate FMOs may contribute to other physiological and cellular processes besides osmoregulation. This revised version was published online in August 2006 with corrections to the Cover Date.