The Amazon catfish genus Pterygoplichthys (Loricariidae, Siluriformes) is closely related to the loricariid genus Hypostomus, in which at least two species lack detectable ethoxyresorufin-O-deethylase (EROD) activity, typically catalyzed by cytochrome P450 1 (CYP1) enzymes. Pterygoplichthys sp. liver microsomes also lacked EROD, as well as activity with other substituted resorufins, but aryl hydrocarbon receptor agonists induced hepatic CYP1A mRNA and protein suggesting structural/functional differences in Pterygoplichthys CYP1s from those in other vertebrates. Comparing the sequences of CYP1As of Pterygoplichthys sp. and of two phylogenetically related siluriform species that do catalyze EROD (Ancistrus sp., Loricariidae and Corydoras sp., Callichthyidae) showed that these three proteins share amino acids at 17 positions that are not shared by any fish in a set of 24 other species. Pterygoplichthys and Ancistrus (the loricariids) have an additional 22 amino acid substitutions in common that are not shared by Corydoras or by other fish species. Pterygoplichthys has six exclusive amino acid substitutions. Molecular docking and dynamics simulations indicate that Pterygoplichthys CYP1A has a weak affinity for ER, which binds infrequently in a productive orientation, and in a less stable conformation than in CYP1As of species that catalyze EROD. ER also binds with the carbonyl moiety proximal to the heme iron. Pterygoplichthys CYP1A has amino acid substitutions that reduce the frequency of correctly oriented ER in the AS preventing the detection of EROD activity. The results indicate that loricariid CYP1As may have a peculiar substrate selectivity that differs from CYP1As of most vertebrate.

Cytochrome P450 family 1 (CYP1) includes four subfamilies of enzymes: CYP1A, CYP1B, CYP1C, and CYP1D. In many vertebrates, CYP1A, 1B, and 1C expression is induced by agonists of the aryl hydrocarbon receptor, including toxic contaminants such as chlorinated dioxins, coplanar chlorinated biphenyls, and polynuclear aromatic hydrocarbons. Assessed at the level of mRNA, protein, or enzyme activity, CYP1s (especially CYP1As) represent potent and popular biomarkers of contaminant exposure in aquatic vertebrates. Alkylated resorufins are synthetic substrates used to detect, quantify, and describe catalytic activities of cytochrome P450s. The ability to oxidize specific resorufin-based substrates can distinguish the catalytic activities of individual CYP1s. Xenopus laevis, the African clawed frog, is the most widely employed amphibian model in aquatic toxicology, yet the number, inducibility, and activities of CYP1s have not been systematically characterized in this species. Here we report the cloning of cDNAs encoding two new CYP1 family members, X. laevis CYP1B and CYP1C, along with an integrated assessment of the induction of alkyloxyuresorufin-O-dealkylase (AROD) activities and mRNA expression of four known X. laevis CYP1s: CYP1A6, CYP1A7, CYP1B, and CYP1C. Using XLK-WG, an X. laevis kidney epithelial cell line, we determined that EROD (ethoxyresorufin substrate) and MROD (methoxyresorufin) were both induced 3000- to 5000-fold following 2,3,7,8 tetrachlorodibenzo-p-dioxin (TCDD) exposure up to 250 nM, while BROD (benzyloxyresorufin) and PROD (pentyloxyresorufin) activity was not detectable regardless of TCDD treatment. TCDD induced CYP1A6 and CYP1A7 mRNAs by 2-3 orders of magnitude, while CYP1B and CYP1C were unchanged. The more potent AHR agonist, FICZ (6-formylindolo[3,2-b]carbazole), induced CYP1B up to 10-fold at concentrations between 0.1 and 250 nM, while CYP1C induction was less than 3-fold. CYP1B mRNA showed the highest constitutive mRNA expression, 5- to 75-fold greater than the other CYP1 transcripts. Taken together, these results suggest that CYP1A6 and CYP1A7 perform the bulk of EROD and MROD activities we observed in these cells. The ability of each X. laevis CYP1 to catalyze oxidation of individual resorufin substrates remains to be determined. Correlating CYP1 mRNA and induced AROD activity is a significant step toward clarifying the biochemical meaning of these biomarkers and the roles of CYP1 enzymes in X. laevis. The cell culture approach represents an important complement to the long standing use of frog embryos and tadpoles in toxicological studies, providing a well suited model system for determining the molecular mechanisms underlying the regulation of these important biomarkers of contaminant exposure.

CYP2S1 is a recently discovered member of the cytochrome P450 (CYP) gene superfamily. Interestingly, even though the DNA sequence identifies it as the sole member of the new CYP2S family, CYP2S1 exhibits many features typical to CYP1 family members, e.g. dioxin-inducibility mediated by the aryl hydrocarbon receptor (AHR) and the aryl hydrocarbon receptor nuclear translocator (ARNT). In addition, CYP2S1 metabolises some aromatic hydrocarbons as well as cellular substances. These characteristics, together with a wide extrahepatic tissue distribution, suggest that CYP2S1 may have an important role in both exogenous and endogenous metabolism. This is the first study characterising CYP2S1 alleles and naming them with the recommended CYP allele nomenclature. We used denaturing gradient gel electrophoresis (DGGE) and direct sequencing to investigate genetic variation of CYP2S1 in 100 male Finnish Caucasians. Those exons in which variation was found were examined in subsequent 100 subjects. The coding region of all of the nine exons, as well as a 449 bp fragment of the proximal promoter region, was analysed. This systematic investigation revealed eight single nucleotide polymorphisms (SNPs), which comprise nine different variant alleles (haplotypes), in addition to the wild-type allele. Seven of the SNPs occurred in the protein-coding areas and one in the proximal 3' untranslated region (3'UTR). Two of these sequence variations (10347C>> T and 13106C>> T) result in non-conservative amino acid substitutions, i.e. Arg380Cys and Pro466Leu, respectively. The respective allelic variants, CYP2S1*2 ([10347C>> T]) and CYP2S1*3 (13106C>> T; 13255A>> G]), occurred in our study population at frequencies of 0.50 and 3.75%, respectively. The most common of the variant alleles was CYP2S1*1H (23.8%), harbouring a 13255A>> G substitution located in the 3'UTR.

The level of mRNA for cytochromes P450 (CYP1A1, CYP1A2, and CYP1B1) and CYP1 regulatory proteins (heat shock protein, aryl hydrocarbon receptor, aryl hydrocarbon receptor repressor, and aryl hydrocarbon receptor nuclear translocator) was measured in the liver of rats after cold stress (4 degrees C). The CYP1A1 mRNA level increased and remained high for 10 days after 5-day cold exposure. The level of mRNA for CYP1A2, heat shock protein, and aryl hydrocarbon receptor nuclear translocator decreased by the 10th day. The level of mRNA for CYP1B1, aryl hydrocarbon receptor, and aryl hydrocarbon receptor nuclear translocator remained unchanged over this period.

The current studies investigate whether synergistic or antagonistic interactions in the upregulation of CYP1 activity occur in binary mixtures of polycyclic aromatic hydrocarbons (PAHs) involving benzo[a]pyrene and five other structurally diverse PAHs of varying carcinogenic activity. Precision-cut rat liver slices were incubated with benzo[a]pyrene alone or in combination with a range of concentrations of a second PAH, and ethoxyresorufin O-deethylase, CYP1A1 and CYP1B1 mRNA levels determined. Concurrent incubation of benzo[a]pyrene with either dibenzo[a,h]anthracene or fluoranthene in liver slices led to a synergistic interaction, at least at low concentrations, in that ethoxyresorufin O-deethylase activity was statistically higher than the added effects when the slices were incubated with the individual compounds. In contrast, benzo[b]fluoranthene and, at high doses only, dibenzo[a,l]pyrene gave rise to antagonism, whereas 1-methylphenanthrene had no effect at all concentrations studied. When CYP1A1 mRNA levels were monitored, benzo[b]fluoranthene gave rise to an antagonistic response when incubated with benzo[a]pyrene, whereas all other compounds displayed synergism, with 1-methylphenathrene being the least effective. A similar picture emerged when CYP1B1 mRNA levels were determined, though the effects were less pronounced. In conclusion, it has been demonstrated that the benzo[a]pyrene-mediated upregulation of CYP1, at the mRNA and activity levels, is synergistically and antagonistically modulated by other PAHs. © 2016 Wiley Periodicals, Inc. Environ Toxicol 32: 764-775, 2017.

Polychlorinated diphenyl sulfides (PCDPSs) are a group of environmental pollutants for which limited toxicological information is available. This study tested the hypothesis that PCDPSs could activate the mammalian aryl hydrocarbon receptor (AhR) mediated toxicity pathways. Eighteen PCDPSs were tested in the H4IIE-luc transactivation assay, with 13/18 causing concentration-dependent AhR activation. Potencies of several congeners were similar to those of mono-ortho substituted polychlorinated biphenyls. A RNA sequencing (RNA-seq)-based transcriptomic analysis was performed on H4IIE cells treated with two PCDPS congeners, 2,2',3,3',4,5,6-hepta-CDPS, and 2,4,4',5-tetra-CDPS. Results of RNA-seq revealed a remarkable modulation on a relatively short gene list by exposure to the tested concentrations of PCDPSs, among which, Cyp1 responded with the greatest fold up-regulation. Both the identities of the modulated transcripts and the associated pathways were consistent with targets and pathways known to be modulated by other types of AhR agonists and there was little evidence for significant off-target effects within the cellular context of the H4IIE bioassay. The results suggest AhR activation as a toxicologically relevant mode of action for PCDPSs suggests the utility of AhR-related toxicity pathways for predicting potential hazards associated with PCDPS exposure in mammals and potentially other vertebrates.

This study presents a reliable method for performing reverse transcription quantitative realtime PCR (RT-qPCR) to measure gene expression in the whitefly Bemisia tabaci (Asia I) (Gennadius) (Hemiptera: Aleyrodidae), utilising suitable reference genes for data normalisation. We identified orthologs of commonly used reference genes (actin (ACT), cyclophilin 1 (CYP1), elongation factor 1α (EF1A), glyceraldehyde 3-phosphate dehydrogenase (GAPDH), ribosomal protein L13a (RPL13A), and α-tubulin (TUB1A)), measured the levels of their transcripts by RT-qPCR during development and in response to thermal stress, and evaluated their suitability as endogenous controls using geNorm, BestKeeper, and NormFinder programs. Overall, TUB1A, RPL13A, and CYP1 were the most stable reference genes during B. tabaci development, and TUB1A, GAPDH, and RPL13A were the most stable reference genes in the context of thermal stress. An analysis of the effects of reference gene choice on the transcript profile of a developmentally-regulated gene encoding vitellogenin demonstrated the importance of selecting the correct endogenous controls for RT-qPCR studies. We propose the use of TUB1A, RPL13A, and CYP1 as endogenous controls for transcript profiling studies of B. tabaci development, whereas the combination of TUB1A, GAPDH, and RPL13A should be employed for studies into thermal stress. The data pre- sented here will assist future transcript profiling studies in whiteflies.

Dopaminergic systems regulate the release of several hormones including growth hormone (GH), thyroid hormones, insulin, glucocorticoids and prolactin (PRL) that play significant roles in the regulation of various Cytochrome P450 (CYP) enzymes. The present study investigated the role of dopamine D2-receptor-linked pathways in the regulation of CYP1A1, CYP1A2 and CYP1B1 that belong to a battery of genes controlled by the Aryl Hydrocarbon Receptor (AhR) and play a crucial role in the metabolism and toxicity of numerous environmental toxicants. Inhibition of dopamine D2-receptors with sulpiride (SULP) significantly repressed the constitutive and benzo[a]pyrene (B[a]P)-induced CYP1A1, CYP1A2 and CYP1B expression in the rat liver. The expression of AhR, heat shock protein 90 (HSP90) and AhR nuclear translocator (ARNT) was suppressed by SULP in B[a]P-treated livers, whereas the AhRR expression was increased by the drug suggesting that the SULP-mediated repression of the CYP1 inducibility is due to inactivation of the AhR regulatory system. At signal transduction level, the D2-mediated down-regulation of constitutive CYP1A1/2 and CYP1B1 expression appears to be mediated by activation of the insulin/PI3K/AKT pathway. PRL-linked pathways exerting a negative control on various CYPs, and inactivation of the glucocorticoid-linked pathways that positively control the AhR-regulated CYP1 genes, may also participate in the SULP-mediated repression of both, the constitutive and induced CYP1 expression. The present findings indicate that drugs acting as D2-dopamine receptor antagonists can modify several hormone systems that regulate the expression of CYP1A1, CYP1A2 and CYP1B1, and may affect the toxicity and carcinogenicity outcome of numerous toxicants and pre-carcinogenic substances. Therefore, these drugs could be considered as a part of the strategy to reduce the risk of exposure to environmental pollutants and pre-carcinogens.

The principal objective was to ascertain whether precision-cut tissue slices can be used to evaluate the potential of chemicals to induce CYP1, epoxide hydrolase and glutathione S-transferase activities, all being important enzymes involved in the metabolism of polycyclic aromatic hydrocarbons. Precision-cut rat liver and lung slices were incubated with a range of benzo[a]pyrene concentrations for various time periods. A rise in the O-deethylation of ethoxyresorufin was seen in both liver and lung slices exposed to benzo[a]pyrene, which was accompanied by increased CYP1A apoprotein levels. Pulmonary CYP1B1 apoprotein levels and hepatic mRNA levels were similarly enhanced. Elevated epoxide hydrolase and glutathione S-transferase activities were also observed in liver slices following incubation for 24h; similarly, a rise in apoprotein levels of both enzymes was evident, peak levels occurring at the same time point. When mRNA levels were monitored, a rise in the levels of both enzymes was seen as early as 4h after incubation, but maximum levels were attained at 24 h. In lung slices, induction of epoxide hydrolase by benzo[a]pyrene was observed after a 24-h incubation, and at a concentration of 1 microM; a rise in apoprotein levels was seen at this time point. Glutathione S-transferase activity was not inducible in lung slices by benzo[a]pyrene but a modest increase was observed in hepatic slices. Collectively, these studies confirmed CYP1A induction in rat liver slices and established that CYP1B1 expression, and epoxide hydrolase and glutathione S-transferase activities are inducible in precision-cut tissue slices.

Berberine is a pharmacologically active alkaloid present in widely used medicinal plants, such as Coptis chinensis (Huang-Lian). The hormone estradiol is oxidized by cytochrome P450 (CYP) 1B1 to primarily form the genotoxic metabolite 4-hydroxyestradiol, whereas CYP1A1 and CYP1A2 predominantly generate 2-hydroxyestradiol. To illustrate the effect of berberine on the regioselective oxidation of estradiol, effects of berberine and its metabolites on CYP1 activities were studied. Among CYP1s, CYP1B1.1, 1.3 (L432V), and 1.4 (N453S)-catalyzed 4-hydroxylation were preferentially inhibited by berberine. Differing from the competitive inhibition of CYP1B1.1 and 1.3, N453S substitution in CYP1B1 allowed a non-competitive or mixed-type pattern. An N228T in CYP1B1 highly decreased its activity and preference to 4-hydroxylation. A reverse mutation of T223N in CYP1A2 retained its 2-hydroxylation preference, but enhanced its inhibition susceptibility to berberine. Compared with berberine, metabolites demethyleneberberine and thalifendine caused weaker inhibition of CYP1A1 and CYP1B1 activities. Unexpectedly, thalifendine was more potent than berberine in the inhibition of CYP1A2, in which case an enhanced interaction through polar hydrogen-π bond was predicted from the docking analysis. These results demonstrate that berberine preferentially inhibits the estradiol 4-hydroxylation activity of CYP1B1 variants, suggesting that 4-hydroxyestradiol-mediated toxicity might be reduced by berberine, especially in tissues/tumors highly expressing CYP1B1.

Breast cancer is the most common malignancy among women worldwide. In addition to reproductive factors, environmental factors such as nutrition and xenobiotic exposure have a role in the etiology of this malignancy. A stimulating and a potentially protective effect on experimental breast cancer has been previously described for high corn oil and high extra-virgin olive oil diets, respectively. This work investigates the effect of these lipids on the metabolism of 7,12-dimethylbenz(a)anthracene (DMBA), a polycyclic aromatic hydrocarbon that can initiate carcinogenesis and its consequences in an experimental rat breast cancer model. The PUFA n-6-enriched diet increased expression of Phase I enzymes prior to DMBA administration and raised the activity of CYP1s in the hours immediately after induction, while reducing the activity of Phase II enzymes, mainly NQO1. The levels of reactive metabolites measured in plasma by GC-MS and DMBA-DNA adducts in the mammary gland of the animals fed the high corn oil diet were also higher than in the other groups. On the other hand, the high extra-virgin olive oil diet and the control low-fat diet exhibited better coordinated Phase I and Phase II activity, with a lower production of reactive metabolites and less DNA damage in the mammary gland. The concordance between these effects and the different efficacy of the carcinogenesis process due to the dietary treatment suggest that lipids may differently modify mammary gland susceptibility or resistance to cancer initiation over the exposure to environmental carcinogens.

Dietary lipids influence the initiation of DMBA-induced mammary cancer through the modulation of liver xenobiotic metabolism, formation of reactive metabolites and subsequent DNA damage in the target tissue.

There is currently no reproducible model of the painful and lithogenic disease, chronic pancreatitis. Its biphasic evolution, from acinar cell hyperplasia and hyperactivity toward effacement of enzyme as well as bicarbonate secretory parenchyma, would be rationalized if it was linked to induction of cytochrome P450 mono-oxygenases (CYP): the increased oxidant load from long-term CYP induction eventually erodes micronutrient antioxidant defenses to injure cells. This philosophy would also rationalize the reported hepatobiliary aberrations associated with the human disease, including increases in free radical oxidation products in bile. Accordingly, pancreatic and biliary secretions were studied in Syrian golden hamsters that were reared for 6 mo on low or high (16% corn oil) fat diets that were supplemented with a prototype inducer of CYP2 (200 ppm phenobarbitone) or CYP1 (100 ppm beta naphthoflavone) enzyme families, with or without a putative enzyme inhibitor (400 ppm cimetidine). The drugs did not alter the reduction in flow rate or bicarbonate concentration of pancreatic juice caused by the high fat diet alone, but, in contrast, evoked pancreatic protein hypersecretion in a number of animals. beta naphthoflavone, but not phenobarbitone, augmented the output of biliary lipid peroxidation products irrespective of dietary fat content, and cimetidine cotreatment with either inducer did the same. We conclude: (1) that drug modifiers of CYP magnify the deleterious pancreatobiliary effects of corn oil-enriched diets and draw them closer to those found in human chronic pancreatitis; (2) that these functional derangements are accompanied by pancreatic lipoatrophy; and (3) that long-term CYP induction does not, of its own, cause fibrosis or the ductal abnormalities that generally accompany loss of pancreatic acinar cells in the human disease and, also in contrast, the changes that are caused appear to be painless.

We previously demonstrated that co-exposing pre-steatotic hepatocytes to benzo[a]pyrene (B[a]P), a carcinogenic environmental pollutant, and ethanol, favored cell death. Here, the intracellular mechanisms underlying this toxicity were studied. Steatotic WIF-B9 hepatocytes, obtained by a 48h-supplementation with fatty acids, were then exposed to B[a]P/ethanol (10 nM/5 mM, respectively) for 5 days. Nitric oxide (NO) was demonstrated to be a pivotal player in the cell death caused by the co-exposure in steatotic hepatocytes. Indeed, by scavenging NO, CPTIO treatment of co-exposed steatotic cells prevented not only the increase in DNA damage and cell death, but also the decrease in the activity of CYP1, major cytochrome P450s of B[a]P metabolism. This would then lead to an elevation of B[a]P levels, thus possibly suggesting a long-lasting stimulation of the transcription factor AhR. Besides, as NO can react with superoxide anion to produce peroxynitrite, a highly oxidative compound, the use of FeTPPS to inhibit its formation indicated its participation in DNA damage and cell death, further highlighting the important role of NO. Finally, a possible key role for AhR was pointed out by using its antagonist, CH-223191. Indeed it prevented the elevation of ADH activity, known to participate to the ethanol production of ROS, notably superoxide anion. The transcription factor, NFκB, known to be activated by ROS, was shown to be involved in the increase in iNOS expression. Altogether, these data strongly suggested cooperative mechanistic interactions between B[a]P via AhR and ethanol via ROS production, to favor cell death in the context of prior steatosis.

Heterocyclic aromatic amines (HAA) are formed in cooked meat, poultry and fish but also arise in tobacco smoke and exhaust gases. HAA are potential human carcinogens, which require metabolic activation to exert their genotoxicity. Human tissues can bioactivate HAA to produce reactive intermediates that bind to DNA. HAA DNA adduct formation occurs in human hepatocytes; however, the potential of HAA to form DNA adducts has not been investigated in human T lymphocytes. In this study, we investigated the ability of human T lymphocytes activated with PMA/Ionomycin or CD3/CD28 to express functional CYP1 activity and bioactivate three major HAA: 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx), and 2-amino-9H-pyrido[2,3-b]indole (AαC) to form DNA adducts. Adducts were measured by ultraperformance liquid chromatography-electrospray ionization/multistage scan mass spectrometry. The highest level of DNA adducts occurred for AαC (16 adducts per 109 nucleotides), followed by PhIP (9 adducts per 109 nucleotides). In contrast, DNA adducts formed from MeIQx and the structurally related aromatic amine 4-aminobiphenyl, a known human carcinogen, were below the limit of detection (< 3 adducts per 109 nucleotides). Moreover, we demonstrate that AαC is a potent inducer of CYP1A1 and CYP1B1 activity through a transcriptional mechanism involving the AhR pathway. Overall, our results highlight the capacity of activated human T lymphocytes to more efficiently bioactivate AαC to form DNA adducts than other prominent HAA or 4-ABP. Environ. Mol. Mutagen. 57:656-667, 2016. © 2016 Wiley Periodicals, Inc.

To investigate the role of detoxification-related liver genes in amnesic shellfish poisoning toxin metabolism, red sea bream Pagrus major were exposed to domoic acid (DA, 2mugg(-1) wet weight) for 24h. Hepatic mRNA expression levels of AHR, ARNT, CYP1 and GSTs were determined by semi-quantitative RT-PCR. The cytosolic factors aryl hydrocarbon receptor (AHR) and aryl hydrocarbon receptor nuclear translocator (ARNT) mRNA levels of DA exposure group were substantially enhanced by 113.3% and 90.9%, respectively. Consistent with this result, the phase I xenobiotic metabolizing enzyme (XME) cytochrome P-450 1A (CYP1A) was significantly induced. In contrast, the transcriptions of three major phase II XME glutathione S-transferases as well as heat shock protein 70 were not significantly affected by DA exposure. These results suggest a possible role of CYP1A after DA exposure in the toxin metabolism of marine fish, possibly through the AHR/ARNT signaling pathway.

Found in tobacco smoke, fossil fuel and other organic combustion products, 7H-dibenzo[c,g]carbazole (DBC) is a potent mouse lung carcinogen and potential human carcinogen. Although the first hydroxylation is critical for determining activation versus detoxication, the enzymes responsible for site-specific hydroxylation of DBC are not known. We found that DBC-DNA adduct levels are significantly higher in aromatic hydrocarbon receptor null Ahr(-/-) mice, suggesting that the induction of Aromatic hydrocarbon receptor (AHR)-regulated genes, such as those in the CYP1 family, decrease DBC genotoxicity. Using knockout mice for Cyp1a1, Cyp1a2 and Cyp1b1, we showed that the major CYP1 enzymes that metabolize DBC are CYP1A1 in beta-naphthoflavone (BNF)-induced liver, CYP1A2 in non-induced liver, CYP1B1 and CYP1A1 in induced lung and none in non-induced lung. DBC metabolism by the human CYP1 enzymes was examined in vitro using Supersomestrade mark. Each mouse CYP1, as well as each human CYP1, has a unique DBC metabolite profile. Comparison of the metabolite profile in BNF-induced mice suggested that CYP1A1 primarily generates 1-OH, 2-OH and (5 + 6)-OH-DBC, whereas CYP1A2 generates primarily (5 + 6)-OH-DBC and CYP1B1 primarily generates 4-OH-DBC. This was similar to that observed in the human CYP1 enzymes. Most importantly, lung CYP1B1 is associated with forming 4-OH-DBC, the most potent metabolite leading to DBC-DNA adducts. These studies suggest that for non-pulmonary routes of exposure (i.e. skin, gastric, i.p.), low hepatic expression of CYP1A2 and CYP1A1, together with high expression levels of lung CYP1B1 and CYP1A1, may define a phenotype for high susceptibility to carcinogens such as DBC.

A major development in fishery science has been the Fulton's condition factor (CF) as a reliable physiological index of fish growth and health status (Fulton 1902). As a general rule, CF-value greater than 1 (>1) should be regarded as an indicator for good growth and health. Therefore, exposure of fish to contaminants in the environment will be expected to produce a reduction in scope for growth, since energy for growth will be allocated to overcome stressful conditions. In the present study, we hypothesized that tilapia species from Ogun River (Nigeria) are experiencing severe contaminant-induced obesogen effects leading to high CF (≥ 2) in fish with pathological alterations. The environmental obesogen hypothesis has related the interaction between environmental pollutants and PPAR isoform activation In this respect, peroxisome proliferator-activated receptors (PPARs) and biotransformation responses in relation to contaminant burden were investigated in a total of 1074 specimens of Tilapias species (Tilapia guineensis, Sarotherodon galileaus and Oreochromis niloticus) collected from three areas with different degrees of anthropogenic contamination and from a putative control site along the Ogun River. Liver mRNA expression of cytochrome cyp1 isoforms (cyp1a, 1b and 1c) and PPAR isoforms (ppar-α, β and γ) were analyzed using validated real-time PCR. Fish were also analyzed for CF and muscle contaminant burden (aliphatic and polycyclic aromatic hydrocarbons, organochlorine pesticides, and polychlorinated biphenyls). A significant increase in mRNA expression of cyp1- and ppar isoforms was observed in fish from polluted areas, and these results paralleled data on PCBs and PAHs tissue concentrations. Further, cyp1 isoforms showed clear sex-related differences, with higher mRNA expression in male fish than in females. Principal component analysis revealed a relationship between cyp1 isoforms, ppar-α, β, PCBs and PAHs and these interactions may suggest a crosstalk between AhR- and PPARs mediated pathways on metabolic and energetic processes. The PCA biplot also highlighted a positive relationship between ppar-γ, body weight, total length and PAHs. The CF for fish from all the sites was ≥ 2 indicating that this parameter may not be a reliable index for evaluating fish growth and health condition, especially in wild fish population exposed to complex cocktails of environmental pollutants.

The tryptophan derivative formylindolo[3,2-b]carbazole (FICZ) binds with high ligand affinity to the aryl hydrocarbon receptor (AHR) and is readily degraded by AHR-regulated cytochrome P450 family 1 (CYP1) enzymes. Whether in vivo exposure to FICZ can result in toxic effects has not been examined and the main objective of this study was to determine if FICZ is embryotoxic in birds. We examined toxicity and CYP1 mRNA induction of FICZ in embryos from chicken (Gallus domesticus) and Japanese quail (Coturnix japonica) exposed to FICZ (2-200μgkg(-1)) by yolk and air sac injections. FICZ caused liver toxicity, embryo mortality, and CYP1A4 and CYP1A5 induction in both species with similar potency. This is in stark contrast to the very large difference in sensitivity of these species to halogenated AHR agonists. We also exposed chicken embryos to a low dose of FICZ (4μgkg(-1)) in combination with a CYP inhibitor, ketoconazole (KCZ). The mixture of FICZ and KCZ was lethal while FICZ alone had no effect at 4μgkg(-1). Furthermore, mixed exposure to FICZ and KCZ caused stronger and more long-lasting hepatic CYP1A4 induction than exposure to each compound alone. These findings indicate reduced biotransformation of FICZ by co-treatment with KCZ as a cause for the enhanced effects although additive AHR activation is also possible. To conclude, FICZ is toxic to bird embryos and it seems reasonable that the toxicity by FICZ involves AHR activation. However, the molecular targets and biological events leading to hepatic damage and mortality are unknown.

Earlier studies have shown that members of the cytochrome P4501 (CYP1) enzyme family are constitutively expressed, and are elevated in the livers of ringed seals (Phoca hispida) and grey seals (Halichoerus grypus) living in the heavily polluted Baltic Sea. In this study, we compared the expression profiles of several additional CYP enzymes in the liver and extrahepatic tissues of Baltic ringed and grey seals with the corresponding CYP expression in seals from relatively unpolluted waters. We used marker enzyme activity levels, diagnostic inhibitors and immunoblot analysis to assess members of the CYP2A, CYP2B, CYP2C, CYP2D, CYP2E and CYP3A sub-families. Coumarin 7-hydroxylation (COH), a marker of CYP2A activity, was high in the liver and the lungs of all the studied seal populations. The presence of a putative CYP2A form in these seals was further supported by the strong inhibition of COH activity by a chemical inhibitor and by an anti-CYP2A5 antibody. However, antibodies to human and rodent CYP2B, CYP2C and CYP2E forms did not recognize any proteins in these seal species. Dextromethorphan O-demethylation (marker for CYP2D activity) and chlorzoxazone 6-hydroxylation (marker for CYP2E activity) were measurable in the livers of all the seals we studied. Both activities were elevated in the Baltic seal populations, showed a strong positive correlation with CYP1A activity and were at least partly inhibited by a typical CYP1A inhibitor, alpha-naphthoflavone. Further studies are needed to determine the presence and characteristics of CYP2D and CYP2E enzymes in ringed and grey seals. Testosterone 6beta-hydroxylation, a CYP3A marker, showed a relatively high level of activity in the livers of both seal species and was potently inhibited by ketoconazole, a CYP3A-selective inhibitor. The putative CYP3A activity showed an opposing geographical trend to that of CYP2D and CYP2E, since it was elevated in the control area. CYP3A protein levels, revealed by immunoblotting, showed a positive correlation with testosterone 6beta-hydroxylation. We conclude tentatively that CYP2A- and CYP3A-like enzymes are expressed in ringed and grey seals, but that CYP2B- and CYP2C-like ones are not. Further information on the individual contaminant profile is needed before any conclusions can be drawn on a possible connection between the varying CYP expressions and the contaminant load.

Phomopsis longicolla T. W. Hobbs (syn. Diaporthe longicolla) is the primary cause of Phomopsis seed decay (PSD) in soybean, Glycine max (L.) Merrill. This disease results in poor seed quality and is one of the most economically important seed diseases in soybean. The objectives of this study were to infer protein-protein interactions (PPI) and to identify conserved global networks and pathogenicity subnetworks in P. longicolla including orthologous pathways for cell signaling and pathogenesis. The interlog method used in the study identified 215,255 unique PPIs among 3,868 proteins. There were 1,414 pathogenicity related genes in P. longicolla identified using the pathogen host interaction (PHI) database. Additionally, 149 plant cell wall degrading enzymes (PCWDE) were detected. The network captured five different classes of carbohydrate degrading enzymes, including the auxiliary activities, carbohydrate esterases, glycoside hydrolases, glycosyl transferases, and carbohydrate binding molecules. From the PPI analysis, novel interacting partners were determined for each of the PCWDE classes. The most predominant class of PCWDE was a group of 60 glycoside hydrolases proteins. The glycoside hydrolase subnetwork was found to be interacting with 1,442 proteins within the network and was among the largest clusters. The orthologous proteins FUS3, HOG, CYP1, SGE1, and the g5566t.1 gene identified in this study could play an important role in pathogenicity. Therefore, the P. longicolla protein interactome (PiPhom) generated in this study can lead to a better understanding of PPIs in soybean pathogens. Furthermore, the PPI may aid in targeting of genes and proteins for further studies of the pathogenicity mechanisms.

In the current study C57BL/6J mice were injected intraperitoneally with Hg(2+) in the absence and presence of TCDD. After 6 and 24h the liver was harvested and the expression of Cyps was determined. In vitro, isolated hepatocytes were incubated with TCDD in the presence and absence of Hg(2+). At the in vivo level, Hg(2+) significantly decreased the TCDD-mediated induction of Cyps at 6h while potentiating their levels at 24h. In vitro, Hg(2+) significantly inhibited the TCDD-mediated induction of Cyp1a1 in a concentration- and time-dependent manner. Interestingly, Hg(2+) increased the serum hemoglobin (Hb) levels in mice treated for 24h. Upon treatment of isolated hepatocytes with Hb alone, there was an increase in the AhR-dependent luciferase activity with a subsequent increase in Cyp1a1 protein and catalytic activity levels. Importantly, when hepatocytes were treated for 2h with Hg(2+) in the presence of TCDD, then the medium was replaced with new medium containing Hb, there was potentiation of the TCDD-mediated effect. In addition, Hg(2+) increased heme oxygenase-1 (HO-1) mRNA, which coincided with a decrease in the Cyp1a1 activity level. When the competitive HO-1 inhibitor, tin mesoporphyrin was applied to the hepatocytes there was a partial restoration of Hg(2+)-mediated inhibition of Cyp1a1 activity. In conclusion, we demonstrate for the first time that there is a differential modulation of the TCDD-mediated induction of Cyp1a1 by Hg(2+) in C57BL/6J mice livers and isolated hepatocytes. Moreover, this study implicates Hb as an in vivo specific modulator of Cyp1 family.

BACKGROUND

Although the expression levels of many P450s differ between tumour and corresponding normal tissue, CYP1B1 is one of the few CYP subfamilies which is significantly and consistently overexpressed in tumours. CYP1B1 has been shown to be active within tumours and is capable of metabolising a structurally diverse range of anticancer drugs. Because of this, and its role in the activation of procarcinogens, CYP1B1 is seen as an important target for anticancer drug development.

OBJECTIVE

To synthesise a series of chalcone derivatives based on the chemopreventative agent DMU-135 and investigate their antiproliferative activities in human breast cancer cell lines which express CYP1B1 and CYP1A1.

METHODS

A series of chalcones were synthesised in yields of 43-94% using the Claisen-Schmidt condensation reaction. These were screened using a MTT assay against a panel of breast cancer cell lines which have been characterised for CYP1 expression.

RESULTS

A number of derivatives showed promising antiproliferative activities in human breast cancer cell lines which express CYP1B1 and CYP1A1, while showing significantly lower toxicity towards a non-tumour breast cell line with no CYP expression. Experiments using the CYP1 inhibitors acacetin and α-naphthoflavone provided supporting evidence for the involvement of CYP1 enzymes in the bioactivation of these compounds.

CONCLUSIONS

Chalcones show promise as anticancer agents with evidence suggesting that CYP1 activation of these compounds may be involved.

PAC2 cell line is, along most of the developed zebrafish cell lines, poorly characterized concerning its response to genotoxicants. To define the PAC2 cell line response to different forms of genotoxic stress, we exposed the cells to model genotoxic agents (benzo[a]pyrene, B[a]P, and ethyl methanesulfonate) and subsequently monitored DNA damage and alterations by using the battery of tests, including the Comet assay, quantitative random-amplified polymorphic DNA and amplified fragment length polymorphism. The expression of several DNA repair (xpc, xpd, hr23b, rad51, msh2) and oxidative stress response (sod (Cu/Zn)) genes was monitored as well. To obtain an indication of the PAC2 cell line metabolizing capacity, the expression of genes belonging to cyp1, cyp2 and cyp3 families was assessed upon exposure to B[a]P. Genotoxic responses were observed in all the used methods, and quantitative random-amplified polymorphic DNA and amplified fragment length polymorphism proved to be more sensitive by revealing DNA alterations even when the Comet assay indicated lack of significant damage. The PAC2 cell line demonstrated basal and B[a]P-induced expression of several cyp genes, suggesting its ability to metabolize indirect acting xenobiotics to a certain point. Based on these results, PAC2 cells seem to be sensitive zebrafish in vitro model in the genotoxicity assessment of the direct acting genotoxicant; however, they are less sensitive toward the indirect acting genotoxicant due to their limited metabolizing properties.

Naphthoflavones are synthetic flavonoids containing a conjugated phenyl group attached to A-ring of flavones. Most of their synthetic studies involved the Baker-Venkataraman rearrangement and subsequent cyclization catalyzed by acid. Based on their special structural features, these synthetic flavones exert pronounced influences on the metabolism of various endogenous and exogenous substances as well as the bioactivation of certain procarcinogens. Several mechanisms of these effects have been established, including the potent inhibition on CYP1 and aromatase, allosteric activation of CYP3A4 and/or activation of AhR. Furthermore, they have also been identified as CFTR activators, BCRP inhibitors and/or anticancer agents. All of the findings suggest that these synthetic ones are a series of promising lead compounds in cystic fibrosis therapeutic and anticancer drug discovery. This review primarily focuses on the structure, chemistry and pharmacology of naphthoflavones, while benzothioflavones, benzoflavanones, benzoflavans and benzochalcones as their analogues are also included.