CYP330A1 and CYP4C39 enzymes in the shore crab Carcinus maenas: sequence and expression regulation by ecdysteroids and xenobiotics

Cytochrome P450 enzymes (CYP enzymes) catalyse important metabolic reactions of exogenous and endogenous substrates, including steroid hormones. Here, we report the first two CYP sequences from the shore crab, Carcinus maenas. Two complete cDNAs isolated from crab hepatopancreas encode CYP enzymes named CYP330A1, the first member of a new family, and CYP4C39. CYP330A1 is closest related to members of the CYP2 family (37.3% identical to mouse CYP2J6) and CYP4C39 is most identical to crayfish CYP4C15 (59.5%). CYP330A1 gene expression was induced in hepatopancreas of male green intermoult crabs by ecdysone and ponasterone A, but also by benzo(a)pyrene and phenobarbital. CYP330A1 induction was not observed in red crabs. The present results indicate that the CYP330A1 enzyme may be involved in ecdysteroid metabolism, presumably catabolism, and in the detoxification of environmental pollutants. Ecdysteroids or xenobiotics did not affect CYP4C39 gene expression. The fact that both ecdysteroids and xenobiotics affect CYP330A1 gene expression indicates that mutual interactions between chemical exposures and endocrine functions may exist in the shore crab.     

Glucocorticoid-dependent induction of HMG-CoA reductase and malic enzyme gene expression by polychlorinated biphenyls in rat hepatocytes

Administration of xenobiotics to rats results in hypercholesterolemia and in the induction of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase and malic enzyme. To investigate the mechanism of the induction of the enzymes by xenobiotics, the effects of xenobiotics on gene expressions for HMG-CoA reductase, malic enzyme, and cytochrome P-450 in rat liver and in cultured hepatocyte were investigated. The treatment of rats with polychlorinated biphenyls (PCB) as a xenobiotic induced mRNAs for HMG-CoA reductase and malic enzyme as well as CYP2B1/2 (cytochrome P-450b/e). Other xenobiotics, 1,1,1-trichloro-2,2-bis(p-chlorophenyl)ethane (DDT), and chloretone, also increased HMG-CoA reductase mRNA. In an investigation of diurnal rhythm of mRNA for HMG-CoA reductase, the induction by PCB was observed in a dark period. Induced expressions of HMG-CoA reductase gene and malic enzyme gene by PCB were observed in primary cultured rat hepatocytes and showed that the action of PCB on the gene expression relating to lipid metabolism was directed on hepatocytes. The induction was observed only in hepatocytes cultured on Engelbreth-Holm-Swarm sarcoma basement membrane gel (EHS-gel), not on type I collagen, which is usually used for monolayer culture of hepatocytes. The induction of CYP2B1/2 gene expression also was observed only in the cells cultured on EHS-gel. The induction of HMG-CoA reductase and malic enzyme by PCB required dexamethasone. However, the addition of dexamethasone per se to medium containing insulin did not show an inductive effect on levels of mRNA for HMG-CoA reductase and malic enzyme. From the data of diurnal variation and hepatocyte culture experiment, HMG-CoA reductase and malic enzyme are considered to be induced by PCB through the so-called "permissive effect" of glucocorticoid.     

Co-planar 3,3',4,4',5-pentachlorinated biphenyl and non-co-planar 2,2',4,6,6'-pentachlorinated biphenyl differentially induce recruitment of oestrogen receptor alpha to aryl hydrocarbon receptor target genes

In the present study we examined the ability of 3,3',4,4',5-pentachlorinated biphenyl [PCB126 (polychlorinated biphenyl 126)], a prototypical AHR (aryl hydrocarbon receptor) agonist, and 2,2',4,6,6'-PCB (PCB104), which does not activate AHR, to induce the recruitment of ERalpha (oestrogen receptor alpha) to CYP1A1 (cytochrome P4501A1 gene) and CYP1B1 promoters in T-47D human breast cancer cells and other cell lines. PCB126 treatment strongly induced CYP1A1 and CYP1B1 mRNA expression that was unaffected by co-treatment with E2 (17beta-oestradiol). PCB104 failed to induce changes in either CYP1A1 or CYP1B1 expression levels. ChIP (chromatin immunoprecipitation) assays show that PCB126, but not PCB104, increased the promoter occupancy by ERalpha to CYP1A1 and CYP1B1 promoters. Co-treatment with PCB126+E2 significantly enhanced the promoter occupancy of ERalpha at CYP1A1, whereas co-treatment with PCB126+4-hydroxytamoxifen or ICI182,780 did not. Competitive binding studies revealed that neither PCB126 nor PCB104 bound to ERalpha. HEK-293 cells (human embryonic kidney-293 cells) stably transfected with ERalpha showed significantly higher PCB126-induced CYP1A1 expression compared with empty vector controls, whereas no increase was observed in cells stably transfected with ERalpha lacking its N-terminal AF1 (activation function-1) domain (ERalphaDeltaAF1). Despite no increase in AHR-mediated gene expression, ChIP assays revealed that ERalphaDeltaAF1 was present at CYP1A1 and CYP1B1 promoters. HC11 mouse mammary cells stably expressing shRNA (small-hairpin RNA) against ERalpha showed an 8-fold reduction in PCB126-dependent Cyp1a1 expression. Our results provide further evidence that AHR agonists induce ERalpha promoter occupancy at AHR target genes through indirect activation of ERalpha, and support a role for ERalpha in AHR transactivation.     

Cytochrome P450-dependent metabolism of PCB52 in the nematode Caenorhabditis elegans

There are 75 full length cytochrome P450 (CYP) genes known in the genome of the nematode Caenorhabditis elegans. The individual biological functions of the vast majority are mostly as yet unknown. Here the impact of cytochrome P450 isoforms on the metabolism of PCB52, an ortho-substituted, non-coplanar 2,2′,5,5′-tetrachlorbiphenyl, as a model PCB of these worldwide distributed pollutants is investigated. Organic extracts, isolated from treated worms and analyzed by GC/MS, contained two obvious PCB52-derived products which have been identified as C3-, C4- and/or C6-hydroxy-PCB52. Moreover, these hydroxylase reactions strictly required the functional expression of the NADPH-dependent cytochrome P450 reductase (CPR) encoding emb-8 gene, which was recently shown to be essential also for several other cytochrome P450-dependent enzymatic reactions. Multiple and subsequent single RNAi-gene silencing experiments, as well as the use of cyp-mutant strains, identified members of the CYP-14A subfamily and CYP-34A6 as the major isoforms contributing to PCB52 metabolism in C. elegans. In the gene-silenced worms and mutants, the reduction in formation of hydroxylated products ranged from 55% to 78%. These results demonstrate for the first time that C. elegans shares with mammals the capacity to produce CYP-dependent PCB metabolites and may thus facilitate future studies on biotransformation.     

Cytochrome P450 1A, 1B, and 1C mRNA induction patterns in three-spined stickleback exposed to a transient and a persistent inducer

Cytochrome P450 1 (CYP1) mRNA induction patterns in three-spined stickleback (Gasterosteus aculeatus) were explored for use in environmental monitoring of aryl hydrocarbon receptor (AHR) agonists. The cDNAs of stickleback CYP1A, CYP1B1, CYP1C1, and CYP1C2 were cloned and their basal and induced expression patterns were determined in the brain, gill, liver and kidney. Also, their induction time courses were compared after waterborne exposure to a transient (indigo) or a persistent (3,3',4,4',5-pentacholorbiphenyl PCB 126) AHR agonist. The cloned stickleback CYP1s exhibited a high amino acid sequence identity compared with their zebrafish orthologs and their constitutive tissue distribution patterns largely agreed with those reported in other species. PCB 126 (100 nM) induced different CYP1 expression patterns in the four tissues, suggesting tissue-specific regulation. Both indigo (1 nM) and PCB 126 (10 nM) induced a strong CYP1 expression in gills. However, while PCB 126 gave rise to a high and persistent induction in gills and liver, induction by indigo was transient in both organs. The number of putative dioxin response elements found in each CYP1 gene promoter roughly reflected the induction levels of the genes. The high responsiveness of CYP1A, CYP1B1, and CYP1C1 observed in several organs suggests that three-spined stickleback is suitable for monitoring of pollution with AHR agonists.     

Permethrin Induction of Multiple Cytochrome P450 Genes in Insecticide Resistant Mosquitoes,Culex quinquefasciatus

The expression of some insect P450 genes can be induced by both exogenous and endogenous compounds and there is evidence to suggest that multiple constitutively overexpressed P450 genes are co-responsible for the development of resistance to permethrin in resistant mosquitoes. This study characterized the permethrin induction profiles of P450 genes known to be constitutively overexpressed in resistant mosquitoes, Culex quinquefasciatus. The gene expression in 7 of the 19 P450 genes CYP325K3v1, CYP4D42v2, CYP9J45, (CYP) CPIJ000926, CYP325G4, CYP4C38, CYP4H40 in the HAmCqG8 strain, increased more than 2-fold after exposure to permethrin at an LC50 concentration (10 ppm) compared to their acetone treated counterpart; no significant differences in the expression of these P450 genes in susceptible S-Lab mosquitoes were observed after permethrin treatment. Eleven of the fourteen P450 genes overexpressed in the MAmCqG6 strain, CYP9M10, CYP6Z12, CYP9J33, CYP9J43, CYP9J34, CYP306A1, CYP6Z15, CYP9J45, CYPPAL1, CYP4C52v1, CYP9J39, were also induced more than doubled after exposure to an LC50 (0.7 ppm) dose of permethrin. No significant induction in P450 gene expression was observed in the susceptible S-Lab mosquitoes after permethrin treatment except for CYP6Z15 and CYP9J39, suggesting that permethrin induction of these two P450 genes are common to both susceptible and resistant mosquitoes while the induction of the others are specific to insecticide resistant mosquitoes. These results demonstrate that multiple P450 genes are co-up-regulated in insecticide resistant mosquitoes through both constitutive overexpression and induction mechanisms, providing additional support for their involvement in the detoxification of insecticides and the development of insecticide resistance.     

Estrogen-mediated suppression of cytochrome P4501A (CYP1A) expression in rainbow trout hepatocytes: role of estrogen receptor.

Hepatic CYP1A expression in fish can be modulated by the female sex hormone, 17beta-estradiol (E2), however neither the mechanism of E2 suppression of CYP1A nor the capacity for hormonal regulation to overcome CYP1A induction by xenobiotics are known. The present study investigates for the first time in fish if the estrogen receptor (ER) is involved in the suppressive action of E2 on CYP1A gene expression. The study further examines, if the E2 effect is able to overcome xenobiotic induction of CYP1A. As experimental model, in vitro cultures of rainbow trout, Oncorhynchus mykiss, hepatocytes were used. The effect of E2 on CYP1A was assessed by measuring the CYP1A-associated 7-ethoxyresorufin-O-deethylase (EROD) enzyme activity, and CYP1A mRNA contents. E2 at non-cytotoxic concentrations caused a significant time- and concentration-dependent decline of basal but not of induced hepatic EROD activities. The inhibitory action of E2 on basal CYP1A was also evident at the mRNA level. The presence of the ER antagonist tamoxifen abolished the inhibitory action of E2 on CYP1A expression. The results from these in vitro experiments provide evidence (a) that the ER is involved in the suppressive action of E2 on CYP1A, and (b) that E2 inhibitory action does not overcome xenobiotic induction of CYP1A.     

Oxidation of xenobiotics by plant microsomes, a reconstituted cytochrome P450 system and peroxidase: a comparative study

The microsomal fraction from tulip bulbs (Tulipa fosteriana, L.) contains cytochrome P450 (CYP3, EC 1.14.14.1) and peroxidase (EC 1.11.1.7.) enzymes catalyzing the NADPH--and hydrogen peroxide--dependent oxidation of the xenobiotic substrates, N-nitrosodimethylamine (NDMA), N-nitrosomethylaniline (NMA), aminopyrine and 1-phenylazo 2-hydroxynaphthalene (Sudan I), respectively. Oxidation of these model xenobiotics has also been assessed in a reconstituted electron-transport chain with a partially purified CYP fraction, phospholipid and isolated tulip NADPH:CYP reductase (EC 1.6.2.4.). Peroxidase isolated from tulip bulbs (isoenzyme C) oxidizes these xenobiotics, too. Values of kinetic parameters (Km, Vmax), requirements for cofactors (NADPH, hydrogen peroxide), the effect of inhibitors and identification of products formed from the xenobiotics by the microsomal fraction, partially purified CYP and peroxidase C were determined. These data were used to estimate the participation of the CYP preparation and peroxidase C in oxidation of two out of the four studied xenobiotics (NMA, Sudan I) in tulip microsomes. Using such detailed study, we found that the CYP-dependent enzyme system is responsible for the oxidation of these xenobiotics in the microsomal fraction of tulip bulbs. The results demonstrate the progress in resolving the role of plant CYP and peroxidase enzymes in oxidation of xenobiotics.     

Lack of CYP1A responsiveness in species inhabiting chronically contaminated habitats: Two varieties of resistance?

Organisms chronically exposed to organic pollutants such as polychlorinated biphenyls (PCBs) can develop resistance to these chemicals, a condition associated with reduced inducibility of the biomarker enzyme cytochrome P450 1A (CYP1A). This study addresses the CYP1A response of members of the families Ictaluridae and Centrarchidae, two fish families found throughout much of the United States. We measured CYP1A expression, PCB body burdens, and conducted CYP1A challenge experiments in species from these families residing in the Town Branch/Mud River system (Logan County, KY, USA), a stream system historically contaminated with high levels of PCBs. Despite PCB concentrations in muscle tissue typically associated with elevated CYP1A (16.7 to 75.2μgPCB/g wet edible flesh), resident fish in the contaminated Town Branch/Mud River sites (yellow bullhead [Ameiurus natalis], green sunfish [Lepomis cyanellus], and spotted bass [Micropterus punctulatus]) had hepatic CYP1A activity levels similar to, rather than higher than, those in reference fish, suggesting reduced sensitivity to CYP1A induction. Lack of CYP1A expression following direct contaminant exposure has often been associated with resistance to those contaminants. To determine if CYP1A in resident populations was resistant to induction by PCBs, we exposed resident fish to a single, intraperitoneal injection with a potent CYP1A inducer, 3,4,3',4'-tetrachlorobiphenyl (PCB 77). PCB 77 treatment significantly induced hepatic CYP1A activity and protein in yellow bullhead from reference, but not contaminated, sites and had no effect on CYP1A in green sunfish from either site. The low CYP1A expression levels in resident fish with elevated PCB body burdens, together with the failure of PCB injection to induce CYP1A in certain populations, indicate an acclimatory CYP1A response in yellow bullheads and likely an inherently resistant CYP1A in green sunfish. This work demonstrates for the first time acclimation of CYP1A to PCBs in a species within the family Ictaluridae and provides further support for our previous work indicating an apparent inherent lack of CYP1A sensitivity to chlorinated inducers in Centrarchids. These traits may explain, at least in part, the common association of these families with degraded habitats and indicate Lepomis members are likely to be excellent candidates for exploring the mechanistic basis of 'inherent' CYP1A resistance. This study also underlines to the need for thorough characterization of the CYP1A responsivity of a population and/or species prior to using CYP1A as a reliable biomonitoring tool.     

Effects of the novel anthelmintic emodepside on the locomotion, egg-laying behaviour and development of Caenorhabditis elegans

Emodepside, a cyclooctadepsipeptide, is a broad-spectrum anthelmintic previously shown to paralyse body wall muscle and pharyngeal muscle in the model nematode Caenorhabditis elegans. We demonstrate that wild-type C. elegans L4 are less sensitive than adults to emodepside in two independent assays of locomotor behaviour: body bend generation on agar (adult IC(50) 3.7 nM, L4 IC(50) 13.4 nM) and thrashing behaviour in liquid (thrashing behaviour as a % of controls after 1h in 10 microM emodepside: adults 16%, L4 worms 48%). We also show that continuous exposure of wild-type C. elegans to emodepside throughout the life-cycle from egg onwards, slows worm development, an effect that is emodepside concentration-dependent. The rate of worm-hatching from eggs on agar plates containing emodepside was not significantly different from controls, suggesting that it is development post-hatching rather than hatching itself that is affected by the drug. Emodepside also inhibits wild-type C. elegans egg-laying, with acute exposure to the drug at 500 nM resulting in an almost total inhibition within the first hour. However, the rate of egg production was not inhibited and therefore emodepside-treated worms became bloated with eggs, eventually rupturing. This suggests that the effect of emodepside on reproduction is not due to an inhibition of egg production but rather a paralytic effect on the egg-laying muscles. These results, when coupled with previous research, suggest that emodepside interferes with signalling at the neuromuscular junction on the body-wall muscles (Willson et al., 2003), pharynx (Willson et al., 2004) and egg-laying muscles and thus inhibits three important physiological functions: locomotion, feeding and reproduction.     

Inhibition of human drug metabolizing cytochrome P450 enzymes by plant isoquinoline alkaloids

The human cytochrome P450 (CYP) enzymes play a major role in the metabolism of endobiotics and numerous xenobiotics including drugs. Therefore it is the standard procedure to test new drug candidates for interactions with CYP enzymes during the preclinical development phase. The purpose of this study was to determine in vitro CYP inhibition potencies of a set of isoquinoline alkaloids to gain insight into interactions of novel chemical structures with CYP enzymes. These alkaloids (n = 36) consist of compounds isolated from the Papaveraceae family (n = 20), synthetic analogs (n = 15), and one commercial compound. Their inhibitory activity was determined towards all principal human drug metabolizing CYP enzymes: 1A2, 2A6, 2B6, 2C8, 2C9, 2C19, 2D6 and 3A4. All alkaloids were assayed in vitro in a 96-well plate format using pro-fluorescent probe substrates and recombinant human CYP enzymes. Many of these alkaloids inhibited the CYP3A4 form, with 30/36 alkaloids inhibiting CYP3A4 with at least moderate potency (IC₅₀ < 10 μM) and 15/36 inhibiting CYP3A4 potently (IC₅₀ < 1 μM). Among them corydine, parfumine and 8-methyl-2,3,10,11-tetraethoxyberbine were potent and selective inhibitors for CYP3A4. CYP2D6 was inhibited with at least moderate potency by 26/34 alkaloids. CYP2C19 was inhibited by 15/36 alkaloids at least moderate potently, whereas CYP1A2, CYP2B6, CYP2C8, and CYP2C9 were inhibited to a lesser degree. CYP2A6 was not significantly inhibited by any of the alkaloids. The results provide initial structure-activity information about the interaction of isoquinoline alkaloids with major human xenobiotic-metabolizing CYP enzymes, and illustrate potential novel structures as CYP form-selective inhibitors.     

N-1H-benzimidazol-5-ylbenzenesulfonamide derivatives as potent hPXR agonists

The Human Pregnane X Receptor (hPXR) is a nuclear receptor that regulates the expression of phase I and phase II drug-metabolizing enzymes, as well as that of drug transporters. Because this receptor plays a critical role in protecting tissues from potentially toxic endo- and xenobiotics, highly active agonists could represent novel therapeutic tools in treating several human diseases. Using an in vitro screening reporter system that allow to characterize hPXR activators and a first step of chemical modifications of an original agonist ligand (C2BA-4, 1-(2-chlorophenyl)-N-[1-(1-phenylethyl)-1H-benzimidazol-5-yl]methanesulfonamide), we identified compounds with a N-1H-benzimidazol-5-ylbenzenesulfonamide scaffold as a potent family of hPXR agonists. Further chemical modifications allowed us to identify enhanced activators, notably N-(1-benzyl-1H-benzimidazol-5-yl)-2,3,4,5,6-pentamethylbenzenesulfonamide (6n) with an EC(50) value in the subnanomolar range. Accordingly to their potent EC(50), these compounds induced an efficient protection of hPXR against proteolytic digestion by trypsin even at very low ligand concentrations and were able to induce the expression of the main target genes of hPXR, CYP3A4 and CYP2B6, in primary cultures of human hepatocytes.     

Temporal kinetics and concentration-response relationships for induction of CYP1A, CYP2B, and CYP3A in primary cultures of beagle dog hepatocytes

Compared to other species, little information is available on the xenobiotic-induced regulation of cytochrome P450 enzymes in the beagle dog. Dogs are widely used in the pharmaceutical industry for many study types, including those that will impact decisions on compound progression. The purpose of this study was (1) to determine the temporal kinetics of drug-induced changes in canine CYP1A, CYP2B, and CYP3A mRNA and enzymatic activity, and (2) to characterize concentration-response relationships for CYP1A2, CYP2B11, and CYP3A12 using primary cultures of canine hepatocytes treated with beta-naphthoflavone (BNF), phenobarbital (PB), and rifampin (RIF), respectively. CYP1A1 and CYP1A2 mRNA exhibited maximal expression (12,700-fold and 206-fold, respectively) after 36 h of treatment with BNF. PB treatment, but not RIF treatment, caused maximal induction of CYP2B11 mRNA (149-fold) after 48 h of treatment. CYP3A12 and CYP3A26 mRNA levels were increased maximally after 72 h of treatment with PB and RIF (CYP3A12, 35-fold and 18-fold, and CYP3A26, 72-fold and 22-fold with PB and RIF treatment, respectively). Concentration-response relationships for BNF induced 7-ethoxyresorufin O-dealkylation (EROD) (EC(50) = 7.8 +/- 4.2 microM), PB induced 7-benzyloxyresorufin O-dealkylation (BROD) (EC(50) = 123 +/- 30 microM), and PB and RIF induced testosterone 6beta-hydroxylation (EC(50) = 132 +/- 28 microM and 0.98 +/- 0.16 microM) resembled the relationship for human CYP induction compared to that of rodent. Interestingly, RIF had no effect on CYP2B11 expression, which represents a species difference overlooked in previous investigations. Overall, the induction of dog CYP1A, CYP2B, and CYP3A exhibits characteristics that are intermediate to those of rodent and human.