Occupational and tobacco exposure to aromatic amines (AAs) including 4-aminobiphenyl (4-ABP) and 2-naphthylamine (2-NA) are associated with bladder cancer (BC) risk. Several epidemiological studies have also reported a possible role for structurally related heterocyclic aromatic amines (HAAs) formed in tobacco smoke or cooked meats with BC risk. We had screened for DNA adducts of 4-ABP, 2-NA, and several prominent HAAs formed in tobacco smoke or grilled meats including 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), 2-amino-3,8-dimethylmidazo[4,5-f]quinoxaline (MeIQx), and 2-amino-9H-pyrido[2,3-b]indole (AαC) in the bladder DNA of BC patients, using liquid chromatography/mass spectrometry. We detected DNA adducts of 4-ABP, but not adducts of the other carcinogens. In this study, we have examined the capacity of RT4 cells, an epithelial human bladder cell line, to bioactivate AAs and HAAs to DNA damaging agents, which may contribute to BC. 4-ABP and AαC formed DNA adducts, but DNA adducts of 2-NA, PhIP, and MeIQx were not detected. 4-ABP DNA adducts were formed at tenfold higher levels than AαC adducts. Pretreatment of RT4 cells with α-naphthoflavone (1-10 µM), a specific cytochrome P450 1 (CYP1) inhibitor, decreased AαC adduct formation by 50% but did not affect the level of 4-ABP adducts. However, cell pretreatment with 8-methoxypsoralen (0.1-1 µM), a potent inhibitor of CYP2A, resulted in a 90% decrease of 4-ABP DNA adducts levels. These data signify that CYP2A and CYP1A isoforms expressed in the target urothelium bioactivate 4-ABP and AαC, respectively, and may be a critical feature of aromatic amine-induced urinary bladder carcinogenesis. The bioactivation of other tobacco and environmental AAs by bladder CYPs and their ensuing bladder DNA damage warrants further study.

Antifungal azoles are widely used in medicine, agriculture, and material protection and several antifungal azoles have been found in environmental samples. Although these compounds were designed to inhibit fungal enzymes such as lanosterol-14-demethylase (cytochrome P450 (CYP) 51), it is well established that the inhibitory actions of azoles are not specific for fungal CYP isozymes. We refined a gill filament assay to determine the inhibition of <em>CYP1</em>, measured as reduced 7-ethoxyresorufin-O-deethylase (EROD) activity, in rainbow trout (Oncorhynchus mykiss) gill tissue ex vivo. The advantage of this method is that both induction and inhibition of EROD are performed ex vivo. Among thirteen azoles studied, the five that caused the strongest inhibition of gill EROD activity at a concentration of 5 μM were selected for concentration-response assessment. These compounds (bifonazole, clotrimazole, imazalil, miconazole, and prochloraz) showed IC50 values ranging from 0.1 to 1.5 μM. <em>CYP1</em>9 (aromatase) inhibition was measured using microsomes from rainbow trout brains. Concentration-response curves for <em>CYP1</em>9 inhibition were determined for letrozole, bifonazole, clotrimazole, imazalil, miconazole and prochloraz, which gave IC50 values ranging from 0.02 to 3.3 μM. It was further found that mixtures of the five most potent azoles reduced both <em>CYP1</em> and 19 catalytic activity in an additive fashion (IC50 = 0.7 μM and 0.6 μM, in the respective assay). Bifonazole (IC50 = 0.1 μM) is not previously known to inhibit <em>CYP1</em> activity. The additive inhibition of <em>CYP1</em> and <em>CYP1</em>9 catalytic activity is an important finding of the present study. We conclude that this additive action of azoles could mediate adverse impacts on CYP regulated physiological functions in environmentally exposed fish.

Mapping highly complicated disulfide linkages and free thiols via liquid chromatography-tandem mass spectrometry (LC-MS²) is challenging because of the difficulties in optimizing sample preparation to acquire critical MS data and detecting mispairings. Herein, we report a highly efficient and comprehensive workflow using an on-line UV-induced precolumn reduction tandem mass spectrometry (UV-LC-MS²) coupled with two-stage data analysis and spiked control. UV-LC-MS² features a gradient run of acetonitrile containing a tunable percentage of photoinitiators (acetone/alcohol) that drives the sample to the MS through a UV-flow cell and reverse phase column to separate UV-induced products for subsequent fragmentation via low energy collision-induced dissociation. This allowed the alkylated thiol-containing and UV-reduced cysteine-containing peptides to be identified by a nontargeted database search. Expected or unexpected disulfide/thiol mapping was then carried out based on the search results, and data were derived from partially reduced species by photochemical reaction. Complete assignments of native and scrambled disulfide linkages of insulin, α-lactalbumin, and bovine serum albumin (BSA) as well as the free C34-BSA were demonstrated using none or single enzyme digestion. This workflow was applied to characterize unknown disulfide/thiol patterns of the recombinant cyclophilin 1 monomer (rTvCyP1 mono) from the human pathogen Trichomonas vaginalis. α-Lactalbumin was judiciously chosen as a spiked control to minimize mispairings due to sample preparation. rTvCyP1 was determined to contain a high percentage of thiol (>80%). The rest of rTvCyP1 mono were identified to contain two disulfide/thiol patterns, of which C41-C169 linkage was confirmed to exist as C53-C181 in rTvCyP2, a homologue of rTvCyP1. This platform identifies heterogeneous protein disulfide/thiol patterns in a de-novo fashion with artifact control, opening up an opportunity to characterize crude proteins for many applications.

In Trichomonas vaginalis (T. vaginalis), cyclophilins play a vital role in dislodging Myb proteins from the membrane compartment and leading them to nuclear translocation. We previously reported that TvCyP1 cyclophilin from T. vaginalis forms a dimer and plays an essential role in moving the Myb1 transcription factor toward the nucleus. In comparison, TvCyP2 containing an extended segment at the N-terminus (N-terminal segment) formed a monomer and showed a different role in regulating protein trafficking. Four X-ray structures of TvCyP2 were determined under various conditions, all showing the N-terminal segment interacting with the active site of a neighboring TvCyP2, an unusual interaction. NMR study revealed that this particular interaction exists in solution as well and also the N-terminal segment seems to interact with the membrane. In vivo study of TvCyP2 and TvCyP2-∆N (TvCyP2 without the N-terminal segment) indicated that both proteins have different subcellular localization. Together, the structural and functional characteristics at the N-terminal segment offer valuable information for insights into the mechanism of how TvCyP2 regulates protein trafficking, which may be applied in drug development to prevent pathogenesis and disease progression in T. vaginalis infection.

Keywords: Cyclophilin; Trichomonas vaginalis; cytoadherence; peptidyl-prolyl isomerase; protein trafficking; trichomoniasis.

Estrogen hydroxylases (EHs) are cytochrome P450 Family 1 (Cyp1, Clan 2) proteins involved in estrogen hydroxylations at 2-, 4- or 16- carbon positions to form catecholestrogens. EHs are encoded by CYP1A1, CYP1A2 and CYP1B1 in mammals. In the catfish Heteropneustes fossilis, cyp1a1 and cyp1b1 cDNAs were cloned and characterized from liver and ovary. The cyp1a1 cDNA is 2071 bp long and codes for a 518 amino acids (aa) long protein. The cloned cyp1b1 cDNA is 1927 bp long and codes for a 509 residue protein. The deduced proteins clustered distinctly into teleost Cyp1a1 and Cyp1b1 clades, distinct from the tetrapod clusters and featured common function domains and homology with other teleost proteins. In the qPCR assay, the transcripts were the most abundant in the liver, followed by brain and ovary, and moderate in gill, kidney and muscle. Evidence was presented to show the involvement of the genes in reproduction. Expression of brain and ovarian transcripts showed significant seasonal variations with the highest abundance in the spawning phase. In situ hybridization showed the transcripts in the follicular layer (theca and granulosa) of the ovarian follicles. Periovulatory changes in the expression cyp1a1 and cyp1b1 were obtained during final oocyte maturation (FOM) and ovulation induced by human chorionic gonadotropin (hCG), both in vivo and in vitro, and by 2-hydroxyestradiol-17β (catecholestrogen) in vitro. In the brain, the transcript levels increased with time but in the ovary, the increase was maximal at 16 h and decreased at 24 h. The periovulatory activation of the cyp1 genes was reported in this study and discussed on the basis of complex regulation of FOM and ovulation.

Keywords: Catecholestrogens; Cyp1 family genes; Estrogen hydroxylation; Periovulatory changes; Stinging catfish; hCG.

Zebrafish are used widely in biomedical, toxicological, and developmental research, but information on their xenobiotic metabolism is limited. Here, we characterized the expression of 14 xenobiotic cytochrome P450 (CYP) subtypes in whole embryos and larvae of zebrafish (4 to 144 h post-fertilization (hpf)) and the metabolic activities of several representative human CYP substrates. The 14 CYPs showed various changes in expression patterns during development. Many CYP transcripts abruptly increased at about 96 hpf, when the hepatic outgrowth progresses; however, the expression of some <i><em>cyp1</em></i>s (<i>1b1</i>, <i>1c1</i>, <i>1c2</i>, <i>1d1</i>) and <i>cyp2r1</i> peaked at 48 or 72 hpf, before full liver development. Whole-mount in situ hybridization revealed <i>cyp2y3</i>, <i>2r1</i>, and <i>3a65</i> transcripts in larvae at 55 hpf after exposure to rifampicin, phenobarbital, or 2,3,7,8-tetrachlorodibenzo-<i>p</i>-dioxin from 30 hpf onward. Marked conversions of diclofenac to 4'-hydroxydiclofenac and 5-hydroxydiclofenac, and of caffeine to 1,7-dimethylxanthine, were detected as early as 24 or 50 hpf. The rate of metabolism to 4'-hydroxydiclofenac was more marked at 48 and 72 hpf than at 120 hpf, after the liver had become almost fully developed. These findings reveal the expression of various CYPs involved in chemical metabolism in developing zebrafish, even before full liver development.

Keywords: cytochrome P450; developing zebrafish; drug metabolism.

Cytochromes P-450 are a superfamily of hemoproteins which represent the main pathway for drug and chemical oxidation. This superfamily is divided into families, subfamilies and/or single enzymes. The majority of P-450s involved in drug metabolism appear to belong to three distinct families termed CYP1, CYP2 and CYP3. Numerous invasive and non-invasive methodologies have been developed to study these enzymes. Their activities are modulated by genetic and nongenetic factors as well as pathological conditions. In this work, the significance of genetic and nongenetic control of P-450s activities in normal subjects is described. Thereafter, the impact of P-450s on the apparition of liver diseases and the effects of liver disease on P-450s activities is emphasized. In conclusion, future perspectives on this field are presented.

Cytochrome P450 family 1 (CYP1) is involved in polycyclic aromatic hydrocarbons (PAHs) biotransformation. PAHs can induce CYP1 protein expression and enzyme activity, the latter being usually quantified as 7-ethoxyresorufin O-deethylase activity (EROD). The aim of this study was to characterize EROD activity in the bivalve mollusk Crassostrea brasiliana. EROD activity was evaluated in cytosolic and microsomal fractions of gills, digestive gland and mantle of C. brasiliana. No EROD activity was detected in mantle, but it was present in microsomal fraction of gills and digestive gland with NADPH as coenzyme. Optima temperature and pH for EROD assay were 30°C and 7.4, respectively. EROD apparent Km (Kmapp) was 4.32μM for gills and 5.56μM for digestive gland. EROD Vmax was 337.3fmol·min-1·mg of protein-1 in gills and 297.7fmol·min-1·mg of protein-1 in digestive gland. Compared to other bivalves, a higher Kmapp and a lower Vmax was found in oyster which may suggest that oyster CYP1-like enzyme has lower affinity for substrate 7-ethoxyresorufin (7-ER) than those species. CYP1 inhibitor ellipticine (ELP) inhibited EROD activity in all tested concentrations in both tissues. The higher ELP concentration, 100μM, inhibited 78% of EROD activity in gills and 47% in digestive gland. The CYP1 inhibitors α-naphthoflavone and furafylline did not inhibited EROD activity in microsomes of both tissues. In conclusion, EROD activity can be used to determine CYP1-like activity in oysters and possibly a CYP1A1/A2-like enzyme is responsible for this catalysis.

Cyp1p (Hap1p) activates, among others, the two structural genes, CYC1 and CYP3 (CYC7) which encode isocytochromes c in Saccharomyces cerevisiae. This activation is believed to occur through the binding of the protein to the dissimilar upstream activation sequences (UASs), UAS1 and UAS', present upstream of CYC1 and CYP3, respectively. In this paper, we describe a novel promoter mutation, CYP3-5, which results from a 39-bp deletion located about 160 bp upstream of the well-characterized CYP3 UAS. This deletion includes a sequence identical to the 3' moiety of the CYC1 UAS1. Strikingly, a sequence identical to the 5' part of the CYC1 UAS1 is also present 60 bp downstream of the 3' half in the wild-type gene, suggesting that a spatial organization of the promoter might lead to the reconstitution in vivo of an active UAS1-like sequence. Interestingly, we find that in the presence of the CYP3-5 mutation, which disrupts this potential UAS1, the CYP-UAS' complex is importantly diminished and the transcription of CYP3 is insensitive to the wild-type CYP1-activating protein.

Disinfection by-products (DBPs) are generated during chlorination of drinking water. Previous studies demonstrate that DBPs are cytotoxic, genotoxic and associated with an increased risk of human cancer. However, the molecular basis of DBPs-induced toxic effects remains unclear. Here, we chlorinated samples of algal-derived organic matter (AOM) and sediment organic matter (SOM) from a local drinking water reservoir. Chemical properties, toxicities and transcriptomic profiles of human Caco-2 cell exposed to AOM and SOM were compared before and after chlorination. We analyzed chlorination-caused distinct gene expression patterns between AOM and SOM, and identified a set of 22 differentially expressed genes under chlorination of AOM that are different from chlorinated SOM. Consequent network analysis indicates that differential CYP1A1, CYP1B1, ID1 and ID2 are common targets of the upstream regulators predicted in the AOM group, but not the SOM group. Through experimental validation and data integration from previous reports related to DBPs or environmental stressors, we found that CYP1A1 and CYP1B1 are specifically up-regulated after chlorinating AOM. Our study demonstrates that the two CYP1 genes likely act as novel biomarkers of AOM derived DBPs, and this would be helpful for testing drinking water DBPs toxicity and further monitoring drinking water safety.

The hepatic cytochrome P450 enzymes (CYP450) play a prominent part in the metabolism of drugs, toxicants and some endogenous compounds. After a brief recall of their biochemical properties, the recent nomenclature is proposed. They are members of a large superfamily with various functions; the specialization of 3 families, CYP1, CYP2, CYP3 towards the metabolism of foreign and artificial compounds gives way to some evolutive considerations. Three main features are characteristic: these enzymes are poorly selective as a matter of substrates; some display genetic polymorphism; some are highly inducible. The mechanism of induction is detailed for CYP1A1. The consequences in pharmacotherapy are deduced: the CYP450 are the main cause of the variability in pharmacokinetics, of drug interactions and of drug adverse events of type II. Their implication in carcinogenesis, although controversial, deserves attention and requires further studies.

Cytochrome P450s (CYPs) are the main catalytic enzymes for metabolism by a variety of endogenous and exogenous substrates in mammals, fish, insects, etc. We evaluated the application of a multidrug cocktail on changes in CYP1, CYP2, and CYP3 activity in Turbot Scophthalmus maximus. The probe drugs were a combination of caffeine (5 mg/kg body weight), dapsone (5 mg/kg), and chlorzoxazone (10 mg/kg). After a single intraperitoneal injection of the cocktail, the concentration of all three probe drugs in the plasma increased quickly to a peak and then decreased gradually over 24 h. Pharmacokinetic profiles of the three probe drugs were determined using a noncompartmental analysis, and the typical parameters were calculated. In the assay for CYP induction, pretreatment with rifampicin significantly reduced the typical pharmacokinetic metrics for caffeine and chlorzoxazone, but not dapsone, indicating that the activity of CYP1 and CYP2 in turbot were induced by rifampicin.

Extra-hepatic metabolism of xenobiotics by epithelial tissues has evolved as a self-defence mechanism but has potential to contribute to the local activation of carcinogens. Bladder epithelium (urothelium) is bathed in excreted urinary toxicants and pro-carcinogens. This study reveals how differentiation affects cytochrome P450 (CYP) activity and the role of NADPH:P450 oxidoreductase (POR). CYP1A1 and CYP1B1 transcripts were inducible in normal human urothelial (NHU) cells maintained in both undifferentiated and functional barrier-forming differentiated states in vitro. However, ethoxyresorufin O-deethylation (EROD) activity, the generation of reactive BaP metabolites and BaP-DNA adducts, were predominantly detected in differentiated NHU cell cultures. This gain-of-function was attributable to the expression of POR, an essential electron donor for all CYPs, which was significantly upregulated as part of urothelial differentiation. Immunohistology of muscle-invasive bladder cancer (MIBC) revealed significant overall suppression of POR expression. Stratification of MIBC biopsies into "luminal" and "basal" groups, based on GATA3 and cytokeratin 5/6 labeling, showed POR over-expression by a subgroup of the differentiated luminal tumors. In bladder cancer cell lines, CYP1-activity was undetectable/low in basal PORlo T24 and SCaBER cells and higher in the luminal POR over-expressing RT4 and RT112 cells than in differentiated NHU cells, indicating that CYP-function is related to differentiation status in bladder cancers. This study establishes POR as a predictive biomarker of metabolic potential. This has implications in bladder carcinogenesis for the hepatic versus local activation of carcinogens and as a functional predictor of the potential for MIBC to respond to prodrug therapies.

High-throughput in vitro reporter gene assays are increasingly applied to assess the potency of chemicals to alter specific cellular signaling pathways. Genetically modified reporter gene cell lines provide stable readouts of the activation of cellular receptors or transcription factors of interest, but such reporter gene assays have been criticized for not capturing cellular metabolism. We characterized the metabolic activity of the widely applied AREc32 (human breast cancer MCF-7), ARE-bla (human liver cancer HepG2), and GR-bla (human embryonic kidney HEK293) reporter gene cells in absence and in presence of benzo(a)pyrene (BaP), an AhR ligand known to upregulate cytochrome P450 in vitro and in vivo. We combined fluorescence microscopy with chemical analysis, real-time PCR, and EROD activity measurements to track temporal changes in BaP and its metabolites in the cells and surrounding medium over time in relation to the expression and activity of metabolic enzymes. Decreasing BaP concentrations and formation of metabolites agreed with the high basal CYP1 activity of ARE-bla and the strong CYP1A1 mRNA induction in AREc32, whereas BaP concentrations were constant in GR-bla, in which neither metabolites nor CYP1 induction were detected. The study emphasizes that differences in sensitivity between reporter gene assays may be caused not only by different reporter constructs but also by a varying biotransformation rate of the evaluated parent chemical. The basal metabolic capacity of reporter gene cells in absence of chemicals is not a clear indication because we demonstrated that the metabolic activity can be upregulated by AhR ligands during the assay. The combination of methods presented here is suitable to characterize the metabolic activity of cells in vitro and can improve the interpretation of in vitro reporter gene effect data and extrapolation to in vivo human exposure.

The present study reports the in vitro studies with furafylline and troleandomycin (TAO) as specific inhibitors of activities 7-methoxyresorufin-O-demethylase (MROD) and nifedipine oxidase, catalyzed by cytochrome P450 1 A2 (CYP1 A2) and 3A4 human enzymes, respectively, in hepatic microsomes of quail, duck, turkey and chicken. The results suggest that in chicken and quail the MROD activity is carried out by orthologs CYP1 A4 and 1 A5, meanwhile in duck and turkey by a CYP1 A5 ortholog. The nifedipine oxidase activity is carried out by orthologs of the CYP3A family in the four bird species. The use of furafylline and TAO significantly decreased these activities (P < 0.05) and suggested that the biotransformation of resorufin methyl ether (RME) may be related to more than one avian ortholog.

OBJECTIVE

Recent findings demonstrate that nuclear receptor - aryl hydrocarbon receptor (AhR) may play an important role in the pathogenesis of Sjögren's syndrome (SS) via involvement in Epstein-Barr virus reactivation. In that study a reporter system was used. Therefore, it was decided to define AhR expression in human salivary cell line (HSY) and its functional regulators.

METHODS

The expression and functional regulation of AhR was studied in HSY cells. The cells were incubated with dioxin (TCDD) - AhR model inducer, IL-1 and TNF-α. qRT-PCR was applied to assess the expression of AHR, AHRR (AhR repressor), ARNT (AhR nuclear translocator) as well as AhR dependent genes: CYP1A1 and CYP1B1. Enzymatic activity of CYP1A1 and CYP1B1 was evaluated using luciferin-labelled CYPs substrate.

RESULTS

In general, dioxin did not significantly influence the expression of AHR and ARNT, but reduced AHRR level. AhR dependent gene expression, i.e. CYP1A1 and CYP1B1 increased gradually with TCDD incubation time. TNF-α significantly induced AHR along with CYP1A1 and CYP1B1 expression. IL-1β did not affect AHR expression, and had minimal effects on CYP1 mRNA levels. Exposure of HSY cells to TCDD resulted in time-dependent induction of CYP1A1 and CYP1B1 enzymatic activity.

CONCLUSIONS

This study documents functional expression of AhR in HSY as well as induction of AhR and its dependent genes by TNF-α.

It is hypothesized that polychlorinated diphenyl sulfides (PCDPSs) induce lethal toxicity in zebrafish which is mediated by aryl hydrocarbon receptor 2 (Ahr2) activation. In this study an assay was developed based on in vivo exposure of wild-type and Tg(cyp1a:gfp) transgenic zebrafish embryos/larvae to PCDPS congeners (i.e., six dichloro- to heptachloro-diphenyl sulfides) coupled with a zebrafish Ahr2-luciferase reporter gene (LRG) expression. Waterborne PCDPSs were found to be accumulated in zebrafish larvae, and exposure to PCDPSs led to a significant increase in mortality and cyp1s mRNA expression. Furthermore, treatment with PCDPSs caused a significant induction of Ahr2-LRG activity in COS-7 cells, and extremely significant correlations were observed between the in vivo median lethal concentrations and the levels of cyp1s mRNA expression and Ahr2 activation. Molecular dynamics simulations indicated the interaction between dioxins/dioxin-like compounds (DLCs) and six key amino acid residues in the ligand-binding domain of Ahr2 probably determined the susceptibility to dioxins/DLCs in zebrafish. These results strongly support the hypothesis that early life-stage mortality of zebrafish is initiated and mediated by Ahr2 activation.

Despite numerous studies on the toxicities of planar polycyclic aromatic hydrocarbons (PAHs), very little is known about the toxicological profiles of non-planar PAHs. In the present study, the cytotoxicity of corannulene (COR), a typical bowl-shaped PAH with a myriad of applications in the area of material chemistry, and benzo[a]pyrene (BaP), a typical planar PAH with similar molecular weight, were systematically compared in various cell lines. Compared with BaP, exposure to COR resulted in less cytotoxic responses in both human (HepG2) and murine (Hepa1-6) hepatoma cells, which was characterized with a slower cellular accumulation as well as a weaker induction of cytochrome P450 1 (CYP1/Cyp1) isozymes. Knockdown of aryl hydrocarbon receptor (AhR) by siRNA attenuated the inductive effect of COR on CYP1A/Cyp1a mRNA levels in these two cell lines. Further analysis revealed that derivatization greatly influenced the cytotoxicity of COR, which was positively correlated with their binding affinities to the AhR, as demonstrated by in silico molecular docking. Overall, these results suggest that AhR appears to be involved in the cytotoxic responses of COR and its derivatives, providing a fundamental understanding of the biological effects of bowl-like PAHs.

A series of four 11-alkoxy cyclopenta[a]phenanthren-17-ones, ranging from the methoxy to the butoxy derivative, has been synthesised in order to investigate the effect of the size of the 11-substituent on the mutagenicity and ability of these compounds to induce hepatic CYP1 activity in rats. The latter was monitored by using as diagnostic probes methoxy and ethoxy-resorufin, and immunologically in Western blots employing anti-CYP1A1 antibodies. All four members of the series induced both CYP1A1 and CYP1A2 activities and apoprotein levels, but the methoxy- and ethoxy-CPP-17-ones were clearly the most potent. Of the four isomers, only 11-methoxy-CPP-17-one displaced 3H-TCDD from the cytosolic Ah receptor. Similarly only 11-methoxy-CPP-17-one elicited a positive mutagenic response in the Ames test in the presence of an Aroclor 1254-induced activation system. The relevance of these findings to the carcinogenicity of these compounds in the mouse skin painting model is discussed.

The micronucleus assay in the 3D human reconstructed EpiDerm™ skin model (RSMN) is a promising new assay for evaluating genotoxicity of dermally applied chemicals. To complement the testing of metabolically activated chemicals, such as cyclophosphamide (CPA) and benzo[a]pyrene (B[a]P), we measured phase 1 (ethoxyresorufin O-deethylation (EROD) and testosterone metabolism) and 2 activities (UGTs and GSTs) in non-treated and genotoxin treated EpiDerm™ models in a study design which mimics the RSMN assay. The assay involved a three-dose dosing regimen over 72 h to take into account effects e.g. enzyme induction, which requires longer than the standard 2 dose 48-h assay. These studies demonstrated the presence of basal phase 1 and 2 activities of EpiDerm™ models. With the exception of GST, all of the activities measured did not reproducibly change over time. It was possible to measure enzyme induction using this assay design. EROD activity was significantly induced by B[a]P but not by CPA. CPA and B[a]P had little or no reproducible effects on GST and UGT activities. In conclusion, a number of metabolic enzyme activities were present in the EpiDerm™ skin model and at least the CYP1 family was inducible.

Heat stress (HS) is an important factor for the survival of the marine organism Apostichopus japonicus. Lysine acetylation is a pivotal post-translational modification that modulates diverse physiological processes including heat shock response (HSR). In this study, 4028 lysine acetylation sites in 1439 proteins were identified in A. japonicus by acetylproteome sequencing. A total of 13 motifs were characterized around the acetylated lysine sites. Gene Ontology analysis showed that major acetylated protein groups were involved in "oxidation-reduction process", "ribosome", and "protein binding" terms. Compared to the control group, the acetylation quantitation of 25 and 41 lysine sites changed after 6 and 48 h HS. Notably, lysine acetyltransferase CREB-binding protein (CBP) was identified to have differential acetylation quantitation at multiple lysine sites under HS. Various chaperones, such as caseinolytic peptidase B protein homolog (CLBP), T-complex protein 1 (TCP1), and cyclophilin A (CYP1), showed differential acetylation quantitation after 48 h HS. Additionally, many translation-associated proteins, such as ribosomal proteins, translation initiation factor (IF), and elongation factors (EFs), had differential acetylation quantitation under HS. These proteins represented specific interaction networks. Collectively, our results offer novel insight into the complex HSR in A. japonicus and provide a resource for further mechanistic studies examining the regulation of protein function by lysine acetylation.

It was recently shown that the pleiotropic response to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in chick embryo liver includes the induction of cytochrome P450-mediated arachidonic acid epoxygenation, as well as 7-ethoxyresorufin deethylation (EROD) and aryl hydrocarbon hydroxylation (AHH). The TCDD-induced arachidonic acid metabolism in avian liver microsomes is catalyzed by a 55 kDa P450, TCDDAA, whereas the TCDD-induced AHH and EROD are catalyzed by a different 54.5 kDa P450, TCDDAHH. In this study, we investigated the distribution and inducibility of TCDDAA and TCDDAHH in hepatocytes and nonparenchymal cells. Sonicates of freshly isolated hepatocytes from embryos treated with solvent alone (control) metabolized [14C]arachidonic acid principally to a single metabolite, omega-OH arachidonic acid. Treatment with TCDD increased total arachidonic acid metabolism 2.9-fold and epoxygenase products [epoxyeicosatrienoic acids (EETs) and EET-diols] 36-fold. After treatment, EETs and EET-diols constituted 59% of the total metabolites. EROD in hepatocyte sonicates was increased 32-fold by TCDD treatment. The same pattern of arachidonate metabolites and degree of increase in arachidonate metabolism and EROD by TCDD treatment was observed in the hepatocyte sonicates and liver microsomes. TCDD treatment increased arachidonic acid metabolism and EROD activity 3.6- and 50-fold, respectively, in the nonparenchymal cells.(ABSTRACT TRUNCATED AT 250 WORDS)

In order to contribute to a comprehensive understanding of the regulating mechanisms of the aryl-hydrocarbon-receptor (AHR) in zebrafish embryos, we aimed to elucidate the interaction of proteins taking part in this signaling pathway during early development of the zebrafish (Danio rerio) after chemical exposure. We managed to illustrate initial transcription processes of the implemented proteins after exposure to two environmentally relevant chemicals: polychlorinated biphenyl 126 (PCB126) and β-Naphthoflavone (BNF). Using qPCR, we quantified mRNA every 4 h until 118 h post fertilization and found the expression of biotransformation enzymes (cyp1 family) and the repressor of the AHR (ahr-r) to be dependent on the duration of chemical exposure and the biodegradability of the compounds. PCB126 induced persistently increased amounts of transcripts as it is not metabolized, whereas activation by BNF was limited to the initial period of exposure. We did not find a clear relation between the amount of transcripts and activity of the induced CYP-proteins, so posttranscriptional mechanisms are likely to regulate biotransformation of BNF. With regard to zebrafish embryos and their application in risk assessment of hazardous chemicals, our examination of the AHR pathway especially supports the relevance of the time point or period of exposure that is used for bioanalytical investigations and consideration of chemical properties determining biodegradability.

In fish there are four cytochrome P450 (CYP1) subfamilies: CYP1A, CYP1B, CYP1C, and CYP1D. Here we cloned Poecilia vivipara CYP1A, with an inferred amino acid sequence 91% identical to CYP1A from the killifish Fundulus heteroclitus, another member of the Cypriniformes, and an important model in ecotoxicology. In addition, we examined the expression of CYP1A, CYP1B1, and CYP1C1 by qPCR in liver, gill, and intestine of adult P. vivipara injected with 3-methylcholanthrene (3-MC) or held in clean water (control group) for 24h. All three tissues examined showed basal expression of the three CYP1 genes. CYP1A was most strongly expressed in the liver, while CYP1B1, and CYP1C1 were most strongly expressed in the gill and intestine respectively. 3-MC induced CYP1A, CYP1B1, and CYP1C1 significantly (20-120-fold) in the three organs, consistent with the regulation of CYP1A, CYP1B1 and CYP1C1 via the aryl hydrocarbon receptor. Validation of CYP1 gene biomarkers in fish collected from a contaminated urban mangrove environment was confirmed with significant induction of CYP1A and CYP1C1 in gills (10-15-fold) and CYP1B1 in liver (23-fold), relative to fish from a control site. The responsiveness of these CYP1 genes indicates P. vivipara is suitable as a model for environmental toxicology studies and environmental assessment in Brazil.