Relationship between the lipophilicity of gallic acid n-alquil esters' derivatives and both myeloperoxidase activity and HOCl scavenging

The gallic acid and several n-alkyl gallates, with the same number of hydroxyl substituents, varying only in the side carbonic chain length, with respective lipophilicity defined through the C log P, were studied. It evidenced the structure-activity relationship of the myeloperoxidase activity inhibition and the hypochlorous acid scavenger property, as well as its low toxicity in rat hepatic tissue. The gallates with C log P below 3.0 (compounds 2-7) were more active against the enzyme activity, what means that the addition of 1-6 carbons (C log P between 0.92 and 2.92) at the side chain increased approximately 50% the gallic acid effect. However, a relationship between the HOCl scavenging capability and the lipophilicity was not observed. With these results it is possible to suggest that the gallates protect the HOCl targets through two mechanisms: inhibiting its production by the enzyme and scavenging the reactive specie.     

Further immunochemical and biocatalytic characterization of CYP1A1 from feral leaping mullet liver (Liza saliens) microsomes

CYP1A is known to play important roles in the metabolism, detoxification and bioactivation of carcinogens and other xenobiotics in animals including fish. In our laboratory, CYP1A1 was obtained in a highly purified form with a specific content of 15-17 nmol P450 per mg protein from liver microsomes of feral fish, leaping mullet (Liza saliens). Purified mullet CYP1A1 showed a very high substrate specificities for 7-ethoxyresorufin and 7-methoxyresorufin in a reconstituted system containing purified fish P450 reductase and lipid. In addition, effects of each individual components of the reconstituted system, i.e., CYP1A1 and P450 reductase on 7-methoxyresorufin O-demethylase (MROD) activity were studied. 7-ethoxyresorufin O-deethylase (EROD) activity was strongly inhibited by alpha-naphthoflavone (ANF). At 0.5 and 2.5 microM. ANF inhibited EROD activity by 90 and 98%, respectively. Mullet CYP1A1 did not catalyze monooxygenations of other substrates such as aniline, ethylmorphine, N-nitrosodimethylamine and p-nitrophenol. Antibodies produced against CYP1A1 orthologues in fish such as trout and scup showed strong cross-reactivity with the purified mullet CYP1A1. In addition, anti-L. saliens liver CYP1A1 produced in our laboratory inhibited both the EROD and MROD activities catalyzed by L. saliens liver microsomes but stronger inhibition was observed with EROD activity. On the other hand, anti-mullet CYP1A1 antibodies showed very weak cross-reactivity with two proteins (presumably CYP1A1 and CYP1A2) in 3MC-treated rat liver microsomes. Moreover, 3MC-treated rat liver microsomal EROD activity was weakly inhibited by the anti-L. saliens liver CYP1A1. These results strongly suggested that the purified mullet CYP1A1 is structurally, functionally and immunochemically similar to the CYP1A1 homologues purified from other teleost species but functionally and immunochemically distinct from mammalian CYP1A1.     

Cytochrome P450 1A1 (CYP1A1) in blood lymphocytes evidence for catalytic activity and mRNA expression.

Studies initiated to characterise the catalytic activity and expression of CYP1A1 in rat blood lymphocytes revealed significant activity of 7-ethoxyresorufin-O-deethylase (EROD) in rat blood lymphocytes. Pretreatment with 3-methylcholanthrene (MC) and beta-naphthoflavone (NF) resulted in significant induction in the activity of lymphocyte EROD suggesting that like the liver enzyme, EROD activity in lymphocytes is inducible and is mediated by the MC inducible isoenzymes of P450. The increase in the activity of EROD was associated with a significant increase in the apparent Vmax and affinity of the substrate towards EROD. That this increase in the activity of EROD could be primarily due to the increase in the expression of CYP1A1 isoenzymes was demonstrated by RT-PCR and western immunoblotting studies indicating an increase in the expression of CYP1A1 in blood lymphocytes after MC pretreatment. Significant inhibition in the EROD activity of MC induced lymphocyte by anti-CYP1A1/1A2 and alpha-naphthoflavone further provided evidence that the CYP1A1/1A2 isoenzymes are involved in the activity of EROD in blood lymphocytes. The data indicating similarities in the regulation of CYP1A1 in blood lymphocytes with the liver isoenzyme suggests that factors which may affect expression of CYP1A1 in liver may also affect expression in blood lymphocytes and that blood lymphocytes could be used as a surrogates for studying hepatic expression of the xenobiotic metabolising enzymes.     

Cytochrome P4501A1-inhibitory action of antimutagenic anthraquinones in medicinal plants and the structure-activity relationship

We have earlier found that flavones and flavonols in vegetables specifically inhibited one of the carcinogenesis-related enzymes, cytochrome P450 (CYP) 1A1, and subsequently suppressed the mutagenicity of food-derived carcinogens. In this study, we explored other candidates for the enzyme inhibitor in Chinese medicinal plants. Some of them were antimutagenic toward 3-amino-1-methyl-5H-pyrido[4,3-b]indole (Trp-P-2). For example, Rheum officinale contained large amounts of anthraquinones as the active compounds, 3.4 mg of emodin, 2.1 mg of chrysophanol and 1.8 mg of rhein in 10 g of dry matter. Anthraquinones showed similar IC50 values for antimutagenicity against Trp-P-2 to those for inhibition of the N-hydroxylation activity of CYP1A1 toward Trp-P-2, indicating that the antimutagenicity was attributable to CYP inhibition. The structure-activity relationships were then examined with 14 commercial chemicals, and it was found that the interaction with an enzyme required three rings and an oxygen group in the side ring. This characteristic is similar to that of flavones and flavonols.     

Comparative CYP1A1 and CYP1B1 substrate and inhibitor profile of dietary flavonoids

CYP1A1 and CYP1B1 are two extrahepatic enzymes that have been implicated in carcinogenesis and cancer progression. Selective inhibition of CYP1A1 and CYP1B1 by dietary constituents, notably the class of flavonoids, is a widely accepted paradigm that supports the concept of dietary chemoprevention. In parallel, recent studies have documented the ability of CYP1 enzymes to selectively metabolize dietary flavonoids to conversion products that inhibit cancer cell proliferation. In the present study we have examined the inhibition of CYP1A1 and CYP1B1-catalyzed EROD activity by 14 different flavonoids containing methoxy- and hydroxyl-group substitutions as well as the metabolism of the monomethoxylated CYP1-flavonoid inhibitor acacetin and the poly-methoxylated flavone eupatorin-5-methyl ether by recombinant CYP1A1 and CYP1B1. The most potent inhibitors of CYP1-EROD activity were the methoxylated flavones acacetin, diosmetin, eupatorin and the di-hydroxylated flavone chrysin, indicating that the 4'-OCH(3) group at the B ring and the 5,7-dihydroxy motif at the A ring play a prominent role in EROD inhibition. Potent inhibition of CYP1B1 EROD activity was also obtained for the poly-hydroxylated flavonols quercetin and myricetin. HPLC metabolism of acacetin by CYP1A1 and CYP1B1 revealed the formation of the structurally similar flavone apigenin by demethylation at the 4'-position of the B ring, whereas the flavone eupatorin-5-methyl ether was metabolized to an as yet unidentified metabolite assigned E(5)M1. Eupatorin-5-methyl ether demonstrated a submicromolar IC(50) in the CYP1-expressing cancer cell line MDA-MB 468, while it was considerably inactive in the normal cell line MCF-10A. Homology modeling in conjunction with molecular docking calculations were employed in an effort to rationalize the activity of these flavonoids based on their CYP1-binding mode. Taken together the data suggest that dietary flavonoids exhibit three distinct modes of action with regard to cancer prevention, based on their hydroxyl and methoxy decoration: (1) inhibitors of CYP1 enzymatic activity, (2) CYP1 substrates and (3) substrates and inhibitors of CYP1 enzymes.     

Furocoumarins from grapefruit juice and their effect on human CYP 3A4 and CYP 1B1 isoenzymes

Bioactive compounds present in grapefruit juice are known to increase the bioavailability of certain medications by acting as potent CYP 3A4 inhibitors. An efficient technique has been developed for isolation and purification of three furocoumarins. The isolated compounds have been tested for the inhibition of human CYP 1B1 isoform using specific substrates. Grapefruit juice was extracted with ethyl acetate (EtOAc) and the dried extract was loaded onto silica gel column chromatography. Further, column fractions were subjected to preparative HPLC to obtain three compounds. The purity of these compounds was analyzed by HPLC and structures were determined by NMR studies. The identified compounds, bergamottin, 6',7'-dihydroxybergamottin (DHB), and paradisin-A, were tested for their inhibitory effects on hydroxylase and O-dealkylase activities of human cytochrome P450 isoenzymes CYP 3A4 and CYP 1B1. Paradisin-A was found to be a potent CYP 3A4 inhibitor with an IC50 of 1.2 microM followed by DHB and bergamottin. All three compounds showed a substantial inhibitory effect on CYP 3A4 below 10 microM. Inhibitory effects on CYP 1B1 exhibited a greater variation due to the specificity of substrates. Paradisin A showed an IC50 of 3.56+/-0.12 microM for the ethoxy resorufin O-dealkylase (EROD) activity and 33.56+/-0.72 microM for the benzyloxy resorufin (BROD). DHB and bergamottin showed considerable variations for EROD and BROD activities with an IC50 of 7.17 microM and 13.86 microM, respectively.     

Evaluation of mutagenic and antimutagenic activities of alpha-bisabolol in the Salmonella/microsome assay

alpha-Bisabolol (BISA) is a sesquiterpene alcohol found in the oils of chamomile (Matricaria chamomilla) and other plants. BISA has been widely used in dermatological and cosmetic formulations. This study was undertaken to investigate the mutagenicity and antimutagenicity of BISA in the Salmonella/microsome assay. Mutagenicity of BISA was evaluated with TA100, TA98, TA97a and TA1535 Salmonella typhimurium strains, without and with addition of S9 mixture. No increase in the number of his+ revertant colonies over the negative (solvent) control values was observed with any of the four tester strains. In the antimutagenicity assays, BISA was tested up to the highest nontoxic dose (i.e. 50 and 150 microg/plate, with and without S9 mix, respectively) against direct-acting (sodium azide, SA; 4-nitroquinoline-N-oxide, 4-NQNO; 2-nitrofluorene, 2-NF; and nitro-o-phenylenediamine, NPD) as well as indirect-acting (cyclophosphamide, CP; benzo[a]pyrene, B[a]P; aflatoxin B1, AFB1; 2-aminoanthracene, 2-AA; and 2-aminofluorene, 2-AF) mutagens. BISA did not alter mutagenic activity of SA and of NPD, and showed only a weak inhibitory effect on the mutagenicity induced by 4-NQNO and 2-NF. The mutagenic effects of AFB1, CP, B[a]P, 2-AA and 2-AF, on the other hand, were all markedly and dose-dependently reduced by BISA. It was also found that BISA inhibited pentoxyresorufin-o-depentylase (PROD, IC50 2.76 microM) and ethoxyresorufin-o-deethylase (EROD, 33.67 microM), which are markers for cytochromes CYP2B1 and 1A1 in rat liver microsomes. Since CYP2B1 converts AFB1 and CP into mutagenic metabolites, and CYP1A1 activates B[a]P, 2-AA and 2-AF, results suggest that BISA-induced antimutagenicity could be mediated by an inhibitory effect on the metabolic activation of these promutagens.     

Modulation of porcine cytochrome P450 enzyme activities by surgical castration and immunocastration

The present study aimed to evaluate some cytochrome P450 metabolic enzyme activities in hepatic microsomes prepared from entire male pigs (uncastrated pigs), surgically castrated pigs and pigs immunized against gonadotropin-releasing hormone (immunocastrated pigs). The activities of the following enzymes were measured: ethoxyresorufin O-deethylase (EROD, CYP1A1/1A2), methoxyresorufin O-deethylase (MROD, CYP1A2), pentoxyresorufin O-depentylase (PROD, CYP2B), coumarin hydroxylase (COH, CYP2A) and p-nitrophenol hydroxylase (PNPH, CYP2A/2E1). The total cytochrome P450 contents were not affected by either surgical or immunocastration. Hepatic microsomal activities for EROD, PROD, COH and PNPH were lower in entire male pigs compared with surgically castrated and immunocastrated pigs (P < 0.05). Surgically and immunocastrated male pigs were similar with respect to EROD, MROD, PROD and COH activities (P > 0.05), whereas surgically castrated pigs exhibited lower PNPH activity compared with immunocastrated pigs (P = 0.029). The effect of different concentrations of testicular steroids - testosterone, 17β-estradiol, free estrone and androstenone - on enzyme activities was evaluated by in vitro microsomal study. Testosterone at the concentration of 8 pmol/ml inhibited EROD activities and estradiol-17β at the concentration of 1.8 pmol/ml inhibited PROD activities in hepatic microsomes from surgically castrated pigs. The highest concentration of androstenone (7520 pmol/ml) inhibited COH activities, whereas a 42-fold lower concentration of androstenone (180 pmol/ml) stimulated COH activities in surgically castrated pigs. Both free estrone (3.5 pmol/ml) and androstenone (55 pmol/ml) inhibited EROD activities in microsomes from entire male pigs. Stimulation of COH activities by the highest dose of free estrone (18 pmol/ml) was recorded in microsomes from entire male pigs. However, these effects of steroids were not concentration-dependent and the maximum extent did not exceed ±15% variation compared with the controls. There was no inhibition of PNPH activities in the hepatic microsomes from either entire or castrated pigs. In conclusion, we showed that EROD, PROD, COH and PNPH activities were lower in entire male pigs compared with those in surgically and immunocastrated pigs. Direct inhibition by the testicular steroids - testosterone, 17β-estradiol, free estrone and androstenone - was not the primary cause of the reduced enzyme activities.     

Noncompetitive mixed-type inhibition of rainbow trout CYP1A catalytic activity by clotrimazole

Over the past two decades a number of antifungal imidazole derivatives have been approved for use in agricultural. The purpose of this study was to characterize the interaction of a model antifungal imidazole compound with a cytochrome P450 isozyme in a species of fish. Clotrimazole inhibited rainbow trout (Oncorhyncus mykiss) hepatic CYP1A-catalyzed ethoxyresorufin O-deethylase (EROD) activity in vivo and in vitro. Although clotrimazole inhibited EROD activity in vivo, it did not effect CYP1A mRNA levels. Addition of clotrimazole to microsomes produced a type II binding spectrum and clotrimazole was determined to be a noncompetitive mixed-type inhibitor of EROD activity with an IC₅₀ of 190 nM. Since antifungal imidazole compounds may be co-applied with other pesticides, inhibition of cytochrome P450 activity by antifungal imidazole compounds may lead to unexpected toxicological interactions.     

Protein kinase C and CYPlAl induction in rainbow trout (Oncorhynchus mykiss) hepatocyte culture

1. The role of protein kinase C (PKC) in B-naphthoflavone (BNF) induction of CYP1A1 in rainbow trout hepatocytes was investigated.2. Primary cultures of rainbow trout hepatocytes treated with BNF for 24 hr showed an increase in microsomal 7-ethyoxyresorufm-O-deethylase (EROD) activity compared to cells treated with vehicle (DMSO) only.3. Increases in EROD activities were proportional to increased concentrations of BNF from 1 to 10 nM reaching a plateau at higher concentrations (20–100 nM) of BNF.4. Western blot analysis using specific antibody (LM4b) against CYP1A1 showed that changes in microsomal CYP1A1 protein paralleled that of EROD activity.5. The induction of EROD activity by BNF required both protein and RNA synthesis since the process was blocked by both cycloheximide and actinomycin D.6. Pretreatment of hepatocytes with 12-O-tetradecanoyl-phorbol-13-acetate (TPA) led to a dose dependent suppression of BNF-induced EROD activity and CYP1A1 content. TPA alone had no effect on hepatic EROD activity and CYP1A1 protein level.7. Pretreatment with sn-1,2 didecanoylglycerol, a PKC activator, had no effect on BNF-induced EROD activity in these cells.8. Pretreatment of cells with staurosporine, a PKC inhibitor, effectively blocked BNF-induced EROD activity.9. PKC may play a role in the induction of CYP1A1 gene expression in fish liver by BNF.     

In vitro assessment of inhibition by natural polyphenols of metabolic activation of procarcinogens by human CYP1A1

Previously, inhibitors of CYP1A1 were rated as candidate chemopreventive agents against cancer mainly according to their effects on the 7-ethoxyresorufin O-deethylation (EROD) of diagnostic probe substrates. Surprisingly, several polyphenols including resveratrol, formerly identified as potent inhibitors by the EROD assay, exhibited no or weak inhibition of procarcinogen activation. We compared the effects of 11 representative natural polyphenols, which normally occur in food, on different activities of CYP1A1, namely epoxidation of 7,8-dihydrodiol-benzo[a]pyrene, the terminal step in the activation leading to the ultimate carcinogenic diolepoxides, hydroxylation of benzo[a]pyrene, and EROD. For the first time, a reconstituted system was used for the determination of IC(50) values, consisting of purified enzymes (human CYP1A1 and human NADPH-cytochrome P450 reductase) and dilaurylphosphatidylcholine. The results demonstrate that the inhibitory effects of dietary polyphenols on CYP1A1 activity depend on both the structure of the inhibitor and the type of the reaction and substrate used in the assay. Consequently, a potent EROD inhibition alone is insufficient to count a substance among the chemoprotective agents.     

Induction of cytochrome P450 1A1 and conjugating enzymes in rainbow trout (Oncorhynchus mykiss) liver: A time course study

1. The effects of i.p. injections of isosafrole (ISF) or β-naphthoflavone (β-NF) on the cytochrome P450 (CYP) 1A1 system and conjugating enzymes were investigated in livers from juvenile rainbow trout in a time course study employing catalytic, immunochemical and cDNA probes.2. β-NF treatment resulted in a rapid rise in CYP1A1 mRNA followed by accumulation of P450 1A1 protein and P450 1A1 mediated enzyme activity measured as ethoxyresorufin-O-deethylase (EROD) activity.3. ISF treatment resulted in a comparatively weak induction of CYP1A1 mRNA and P450 1A1 protein levels whilst EROD activity was markedly induced; thus when expressed on the basis of immunoquantified P450 1A1 protein, the specific EROD activity was signficantly higher in ISF than β-NF treated fish.4. In vitro inhibition studies revealed that ISF inhibited EROD activity to a far lesser extent than β-NF.5. Conjugation enzymes represented by phenol UDP-glucuronosyltransferase and glutathione S-transferase (GST) activities, were induced by β-NF, whereas ISF treatment had no effect on these enzyme activities.6. Immunoblotting using antibodies raised against rat GST7-7 showed that a Pi class trout GST enzyme was induced by β-NF treatment.     

Phase I and II liver enzyme activities in juvenile alligators (Alligator mississippiensis) collected from three sites in the Kissimmee-Everglades drainage, Florida (USA)

We examined CYP1A (measured using hepatic EROD and MROD activities) and glutathione-S-transferase (GST) activities in juvenile alligators (Alligator mississippiensis) collected from three sites with varying contamination in the Kissimmee-Everglades drainage in south Florida. We hypothesized that contaminants present in areas with intermediate or higher contaminant concentrations would alter hepatic enzyme activities in juvenile alligators from those sites when compared to hepatic enzyme activity in animals from the area with the least contamination. EROD activity was found to be higher in animals from the site with lower reported levels of contamination relative to those from the site with the highest reported contamination suggesting an inhibition of CYP1A expression or activity. No differences among animals from the three sites were observed for hepatic MROD and GST activities. A significant negative relationship between EROD, MROD, and GST activities and body size was exhibited in alligators from the site with the lowest contamination. No relationship between body size and hepatic enzyme activity was found in animals from the sites with intermediate and higher contamination, suggesting that contaminants present at these sites act to alter this relationship. No correlation was observed in this study between plasma steroid concentrations (estradiol-17 beta or testosterone) and hepatic EROD, MROD, or GST activities.     

Induction of CYP1A by carbofuran in primary culture of fish hepatocytes

Carbofuran is a nematicide used in agricultural fields throughout the world. Indiscriminate use of this pesticide poses severe detrimental effects on our ecosystem. We have shown that it induces the CYP1A (cytochrome P4501A) monooxygenase enzyme system in cultured hepatocytes from Indian catfish, Heteropneustes fossilis (Bloch). We have quantified this induction by measuring the activity of the enzyme 7-ethoxyresorufin-O-deethylase (EROD), synthesized from CYP1A1 gene. The induction followed a dose-dependent relationship with carbofuran. The dose-dependent curve of EROD using carbofuran was very much similar with beta-napthoflavone, which is a known inducer of CYP1A1. Coexposure of these compounds to the culture media showed a synergistic effect on the enzyme activity. A blocker of aromatic hydrocarbon receptor, alpha-napthoflavone, blocked carbofuran-induced EROD activity in a dose-dependent manner. All these findings suggest that metabolism of carbofuran might be mediated by the CYP1A monooxygenase system through binding of the aromatic hydrocarbon receptor. We have also studied the superinduction phenomenon, which is a typical characteristic of the CYP1A gene in our system.     

Differential expression, induction, and stability of CYP1A4 and CYP1A5 mRNA in chicken and herring gull embryo hepatocytes

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) induces cytochrome P4501A (CYP1A) catalyzed ethoxyresorufin-O-deethylase (EROD) activity in chickens and other avian species. To investigate mechanisms underlying the effectiveness of EROD activity as a biomarker for exposure to dioxin-like compounds in avian models, we characterized inter-species differences in isoform-specific CYP1A mRNA expression, induction, and stability in chickens (Gallus gallus domesticus) and herring gulls (Larus argentatus). Exposure to 100 nM TCDD significantly increased CYP1A4 and CYP1A5 mRNA expression in chicken and herring gull embryo hepatocyte cultures. Chicken CYP1A4 and CYP1A5 were induced 61-fold and 25-fold respectively. The herring gull isoforms were induced 2.2- and 4.3-fold respectively. In both species, the isoform that was preferentially induced exhibited lower constitutive expression. Half-lives of chicken CYP1A4, chicken CYP1A5, and herring gull CYP1A5 mRNA ranged from 5.0 to 7.0 h in cultured hepatocytes. The half-life of herring gull CYP1A4 mRNA was 2.5 h. Our findings indicate that expression, induction, and stability of CYP1A4 and CYP1A5 mRNA are differentially regulated in chickens and herring gulls. In particular, CYP1A4 is preferentially induced in chickens, while CYP1A5 is preferentially induced in herring gulls. We propose that CYP1A5 mRNA expression may be a sensitive biomarker of exposure to dioxin-like compounds in some avian species.     

Inhibition of CYP450 1A and 3A by berberine in crucian carp Carassius auratus gibelio

Berberine has long been considered as an antibiotic candidate in aquaculture. However, studies regarding its effects on drug-metabolizing enzymes in fish are still limited. In the present study, the effects of berberine on cytochrome P4501A (CYP1A) and CYP3A in crucian carp were investigated. Injection of different concentrations of berberine (0, 5, 25, 50, and 100 mg/kg) inhibited the CYP1A mRNA expression, thereby inhibiting further the catalytic activity of CYP1A-related ethoxyresorufin-O-deethylase (EROD). Furthermore, both CYP1A expression and EROD activity were further inhibited with increasing berberine concentrations. In addition, the CYP3A expressions at both the mRNA and the protein levels were downregulated by higher berberine concentrations. The catalytic activity of CYP3A-related erythromycin N-demethylase (ERND) was also inhibited by berberine at a dose of no less than 25 mg/kg. Moreover, at the berberine concentration exceeding 25 mg/kg, the inhibition of CYP3A expression and ERND activity increased with increasing berberine concentrations. In vitro experiments were also performed. When berberine was pre-incubated with the crucian carp liver microsomes, it competitively inhibited the corresponding EROD activity with the IC₅₀ of 11.7 μM. However, the ERND activity was slightly inhibited by berberine with the IC₅₀ of 206.4 μM. These results suggest that, in crucian carp, berberine may be a potent inhibitor to CYP1A, whereas the CYP3A inhibition needs a higher concentration of berberine.