Polycyclic aromatic hydrocarbons (PAHs) and dioxins are ubiquitous environmental pollutants and activate the aryl hydrocarbon receptor (AhR), a ligand-activated transcription factor. It has been reported that testosterone represses 2,3,7,8-tetrachlorodibenzo-p-dioxin-induced transcription of the cytochrome P450 (CYP) 1A1 gene in LNCaP cells. In this study, we investigated the mechanism for the repression of 3-methylcholanthrene (3MC)-induced transcription of AhR-regulated genes, CYP1A1, CYP1A2, CYP1B1, and AhR repressor (AhRR), by 5alpha-dihydroteststerone (DHT) in LNCaP and T47D cells, which are androgen receptor (AR)- and AhR-positive. Real-time PCR analysis showed that DHT repressed 3MC-induced mRNA expression of the CYP1 family and AhRR genes. DHT repressed 3MC-induced luciferase activity in an AhR response element-driven luciferase reporter assay in LNCaP and T47D cells. The inhibitory effect of DHT was abolished by knockdown of AR protein with siRNA. The protein levels of AhR and AhR nuclear translocator (Arnt), the AhR-dimerizing partner, were not affected by DHT. Co-immunoprecipitation assay showed that DHT significantly facilitated the complex formation between AR and AhR in 3MC-treated cells. These results suggest that complex formation between activated AR and AhR plays an important role in the suppression of 3MC-induced transcription of CYP1 family genes by DHT.

Organic solvents, such as dimethylsulfoxide (DMSO) and methanol are widely used as vehicles to solubilise lipophilic test compounds in toxicity testing. However, the effects of such solvents upon innate detoxification processes in aquatic organisms are poorly understood. This study assessed the effect of solvent exposure upon cytochrome P450 (CYP)-mediated xenobiotic metabolism in Daphnia magna and zebrafish larvae (4d post fertilisation). Adult D. magna were demonstrated to have a low, but detectable, metabolism of ethoxyresorufin in vivo and this activity was not modulated by pre-exposure to DMSO or methanol (24 h, up to 0.1% and 0.05% v/v, respectively). In contrast, the metabolism of ethoxyresorufin in zebrafish larvae was significantly reduced by both solvents (0.1% and 0.05% v/v, respectively) after 24 h of exposure. In zebrafish, these observed decreases in activity towards ethoxyresorufin were accompanied by decreased expression of a variety of genes coding for drug metabolising enzymes (corresponding to CYP1, CYP2, CYP3 and UDP-glucuronyl transferase [UGT] family enzymes), measured by quantitative PCR. Reduction of gene expression and CYP1 enzyme activities by methanol (0.05% v/v) in zebrafish larvae was partially reversed by co-exposure with Aroclor 1254 (100 μg L(-1)). Overall this study suggests that relatively low concentrations of organic solvents can impact upon the biotransformation of certain xenobiotics in zebrafish larvae, and that this warrants consideration when assessing compounds for metabolism and toxicity in this species.

The AhR was initially identified as a ligand-activated transcription factor mediating effects of chlorinated dioxins and polycyclic aromatic hydrocarbons on cytochrome P450 1 (CYP1) expression. Recently, evidence supporting involvement of the AhR in cell-cycle regulation and tumorigenesis has been presented. To further define the roles of the AhR in cancer, we investigated the effects of AhR expression on cell proliferation, migration, invasion, and tumorigenesis of MCF-7 human breast cancer cells. In these studies, the properties of MCF-7 cells were compared with those of two MCF-7-derived sublines: AH(R100) , which express minimal AhR, and AhR(exp) , which overexpress AhR. Quantitative PCR, Western immunoblots, 17β-estradiol (E2 ) metabolism assays, and ethoxyresorufin O-deethylase assays showed the lack of AhR expression and AhR-regulated CYP1 expression in AH(R100) cells, and enhanced AhR and CYP1 expression in AhR(exp) cells. In the presence of 1 nM E2 , rates of cell proliferation of the three cell lines showed an inverse correlation with the levels of AhR mRNA. In comparison with MCF-7 and AhR(exp) cells, AH(R100) cells produced more colonies in soft agar and showed enhanced migration and invasion in chamber assays with E2 as the chemoattractant. Despite the lack of significant AhR expression, AH(R100) cells retained the ability to form tumors in severe combined immunodeficient mice when supplemented with E2 , producing mean tumor volumes comparable to those observed with MCF-7 cells. These studies indicate that, while CYP1 expression and inducibility are highly dependent on AhR expression, the proliferation, invasion, migration, anchorage-independent growth, and estrogen-stimulated tumor formation of MCF-7 cells do not require the AhR.

Various environmental contaminants are known agonists for the aryl hydrocarbon receptor (AHR), which is highly conserved across vertebrate species. Due to gene duplication events before and after the divergence of ray- and lobe-finned fishes, many teleosts have multiple AHR isoforms. The zebrafish (Danio rerio) has three identified AHRs: AHR1A and AHR1B, the roles of which are not yet well elucidated, and AHR2, which has been shown to mediate the toxicity of various anthropogenic compounds including dioxins, polychlorinated biphenyls (PCBs), and polycyclic aromatic hydrocarbons (PAHs). In this study, we sought to explore the role of the two AHR1 isoforms in PAH- and PCB-induced toxicity in zebrafish embryos utilizing morpholino gene knockdown of the AHR isoforms. Knockdown of AHR1B did not affect the toxicity of PAH mixtures or PCB-126, whereas knockdown of AHR1A exacerbated the cardiac toxicity caused by PAH mixtures and PCB-126. Knockdown of AHR1A did not impact the mRNA expression of CYP1A, CYP1B1, and CYP1C1 in exposed embryos, but it did result in increased CYP1 activity in exposed embryos. As has been shown previously, knockdown of AHR2 resulted in protection from PAH- and PCB-induced cardiac deformities and prevented CYP1 enzyme activity in exposed embryos. Co-knockdown of AHR1A and AHR2 resulted in an intermediate response compared to knockdown of AHR1A and AHR2 individually; co-knockdown did not exacerbate nor protect from PAH-induced deformities and embryos exhibited an intermediate CYP1 enzyme activity response. In contrast, co-knockdown of AHR1A and AHR2 did protect from PCB-126-induced deformities. These results suggest that AHR1A is not a nonfunctional receptor as previously thought and may play a role in the normal physiology of zebrafish during development and/or the toxicity of environmental contaminants in early life stages.

The antibacterial personal care product triclosan is discharged in municipal waste, and converted in part by bacteria in sewage sludge and soil to its more lipid-soluble methyl ether, methyl triclosan. Triclosan and methyl triclosan have been detected in water, sediment, fish and invertebrates near sewage treatment facilities. Understanding the biotransformation of methyl triclosan and triclosan in a model food fish, the channel catfish, will be of value in assessing the likelihood that these compounds will bioaccumulate in exposed fish, and therefore potentially pass up the food chain. We hypothesize that cytochrome P450 will catalyze the O-demethylation of methyl triclosan to yield triclosan, which is likely to undergo glucuronidation or sulfonation of the phenolic hydroxyl group. Conversion of methyl triclosan to triclosan was measured by LC/MS/MS following aerobic incubation of varying concentrations of methyl triclosan with NADPH and hepatic and intestinal microsomes from untreated, 3-methylcholanthrene-treated (10 mg/kg, i.p.) or PCB-126-treated (0.1 mg/kg, i.p.) channel catfish (n=4 per treatment group). The K(m) values for methyl triclosan were similar for untreated, 3-methylcholanthrene-treated and PCB-126-treated catfish liver microsomes, ranging from 80 to 250 μM. V(max) values for O-demethylation ranged from 30 to 150 pmol/min/mg protein, with no significant differences between controls, PCB-126-treated or 3-methylcholanthrene-treated fish, suggesting that methyl triclosan O-demethylation was not a CYP1-catalyzed reaction. Methyl triclosan O-demethylation activities in intestinal microsomes were similar to or lower than those found with liver microsomes. The calculated rate of O-demethylation of methyl triclosan in catfish liver at 1 μM, a concentration reported in exposed fish, and 21°C, an early summer water temperature, is 0.10 pmol/min/mg protein. This slow rate of metabolism suggests that upon continued exposure, methyl triclosan may bioaccumulate in the channel catfish. Triclosan itself, however, was readily glucuronidated by hepatic and intestinal microsomes and sulfonated by hepatic and intestinal cytosol. Triclosan glucuronidation followed Michaelis-Menten kinetics when rates were measured across a concentration range of 5-1000 μM, whereas triclosan sulfonation exhibited substrate inhibition at concentrations above 10-20 μM in both intestinal and hepatic cytosol. Based on the enzyme kinetic constants measured in hepatic and intestinal fractions at 21°C, triclosan at 1 μM could be glucuronidated at rates of 23 and 3.2 pmol/min/mg protein respectively in liver and intestine, and sulfonated at rates of 277 (liver) and 938 (intestine) pmol/min/mg protein. These rates are much higher than the rates of demethylation of methyl triclosan, and suggest that triclosan would be rapidly cleared and unlikely to bioaccumulate in catfish tissues.

In adult rabbits, the CYP1A1 and CYP1A2 genes are expressed constitutively. Exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) leads to elevations in both CYP1A1 and CYP1A2 gene products (S. T. Okino et al., 1985, Proc. Natl. Acad. Sci. USA 82, 5310-5314). In this report, we have characterized the rabbit CYP1A1 and CYP1A2 genes, and analyzed the pattern of expression of these genes in neonatal animals following exposure to TCDD. Genomic clones encoding the entire rabbit CYP1A1 and CYP1A2 genes were characterized. Restriction enzyme analysis and partial DNA sequence analysis identified the seven exons for the CYP1A1 and CYP1A2 genes. Primer extension analysis using mRNA from TCDD-treated neonatal rabbits helped confirm the start of transcription for the CYP1A genes. The length of the noncoding first exon of the CYP1A1 gene was 74 bases, compared to 90 and 88 bases for the human and rodent CYP1A1 genes. The length of the noncoding CYP1A2 gene first exon was 53 bases, similar to its counterpart in human and rodents. DNA sequence analysis of the 5' regulatory regions and comparison to the rodent and human CYP1 genes demonstrated that the rabbit CYP1A1 and CYP1A2 genes were most similar to their human orthologs. The 5' region of the CYP1A1 gene contained several consensus dioxin (Ah)-receptor responsive elements (XREs), while no functional XRE sequences were identified in the CYP1A2 gene. When expression of the two genes were monitored, a small amount of constitutive P4501A1 mRNA was detected in neonatal rabbits from the ages of 1 to 17 days, while P4501A2 mRNA levels could not be observed until 8-12 days postpartum. In response to TCDD treatment, P4501A1 mRNA levels were inducible at all neonatal time points, while P4501A2 mRNA levels could not be induced until the animals were 3-5 days postpartum. While the dioxin Ah-receptor most likely plays a major role in the induction of these genes by TCDD, early expression of the CYP1A1 and CYP1A2 genes is differentially regulated in a developmental fashion.

Differences in drug metabolism among individuals are caused by numerous factors: differences in production and stability of mRNA of xenobiotic metabolizing enzymes, differences in the rate of enzyme synthesis and degradation, or enzyme inhibition. One of the most important reasons is genetic polymorphism of cytochrome P450 genes or cytochrome P450 regulatory factors. Nuclear receptors play great role in the regulation of these genes. The presence of the ligand induces the nuclear receptor to bind to the dimerisation partner and as a hetero/homodimer it can activate the DNA responsive element. In addition, several co-activators, co-repressors and other factors can modulate the effect of nuclear receptors. Hepatic levels of cytochrome P450 enzymes are reduced in multiple models of inflammation or infection. Cytochrome P450 enzymes of four families (CYP1-CYP4) are known to be involved in xenobiotic metabolism. Their genetic polymorphism and regulation are discussed in this review.

CYP1 determines the expression of several genes whose transcription is heme-dependent in yeast. It exerts regulatory functions even in the absence of heme, usually considered to be its effector. It mediates both positive and negative effects, depending on the target gene and on the redox state of the cell. In the presence of heme, it binds through a cysteine-rich domain in which a histidine residue occupies the position of the sixth and essential cysteine of the otherwise classical zinc cluster DNA-binding domain exemplified by GAL4. We constructed specific missense mutations in the potential CYP1 zinc cluster domain by site-directed mutagenesis and looked for regulatory effects of the mutated proteins under specific physiological conditions. We show that CYP1 does belong to the zinc cluster regulatory family since a sixth essential cysteine residue is indeed present, albeit at a modified position when compared to the consensus sequence. We also show that the amino acid preceding the first cysteine residue of the DNA-binding domain critically affects the efficiency of regulation both in the presence and in the absence of heme: mutations known to affect DNA binding under heme-sufficient conditions also affect regulation under heme-deficient conditions. We therefore surmise that regulation under heme-deficient conditions is dependent upon DNA binding.

A peptide corresponding to an efficient peroxisomal targeting sequence, the carboxy terminal 12 amino acids of PMP20 from Candida boidinii, was employed as an affinity ligand to search for a peroxisomal targeting receptor. Two proteins from yeast extracts with apparent molecular masses of 20 and 80 kDa were detected by chemical cross-linking to radioiodinated peptide. Both proteins were present in cytosolic supernatants. The 20-kDa species did not cross-link to a control peptide with reversed sequence, whereas the 80-kDa protein cross-linked to both peptides. The cross-linking assay was used to purify the 20-kDa protein from Saccharomyces cerevisiae. Partial protein sequencing identified this protein as cyclophilin, the product of the CYP1 gene. This protein, a peptidyl-prolyl cis-trans isomerase, is the yeast homologue of the protein that mediates the immunosuppressant effects of the drug cyclosporin A (CsA). Cross-linking of peptide to cyclophilin was inhibited by CsA. The cross-linking of cyclophilin to the PMP20-derived peptide was unanticipated because the peptide contains no prolines. The CYP1-encoded protein was not required to target proteins to peroxisomes because this organelle appeared to be assembled normally in a CYP1-disrupted strain. Furthermore, the final three amino acids of the peptide, which are critical for peroxisomal sorting, were not required for cross-linking to cyclophilin. We conclude that either cyclophilin is playing a nonessential facilitating role in peroxisomal targeting or that the interaction of the targeting peptide to cyclophilin is mimicking an interaction with an unidentified substrate or effector of cyclophilin.

Cross-talk between the aryl hydrocarbon receptor (AhR) pathway and the typical stress response is thought to be an important signal transduction in response to nutrient-stress conditions, such as glucose deprivation in liver cells. In the present study, we demonstrate that reduction of glucose concentration in the medium of HepG2 cells, a human hepatocellular carcinoma cell line, induces the CYP1 family and Nrf2. RNAi for AhR abolishes the induction of expression of CYP1 and Nrf2. These inductions are accompanied by the translocation of AhR into the nucleus in response to low-glucose conditions. Endogenous compounds are recruited as AhR ligands to induce various gene expressions, and our present results suggest that an endogenous AhR ligand is produced under low-glucose conditions and that the role of AhR as a transcription factor is related to the low-glucose response. The recommended glucose concentration (4.5 g/L) in the medium for culture of HepG2 was used as the high-glucose concentration in this study. We adopted 1.0 g/L as the low-glucose condition for elucidation of mechanisms of the stress response. These results will be useful to understand the relationship between drug-metabolizing enzymes and mechanisms of the anti-stress response of tumor cells, and will also be useful for investigating preventive remedies against tumor angiogenesis.

In this work, we sought to understand how glycolipid production and the availability of nutrients could explain the ecology of Pseudozyma flocculosa and its biocontrol activity. For this purpose, we compared the development of P. flocculosa to that of a close relative, the plant pathogen Ustilago maydis, under different environmental conditions. This approach was further supported by measuring the expression of cyp1, a pivotal gene in the synthesis of unique antifungal cellobiose lipids of both fungi. On healthy cucumber and tomato plants, the expression of cyp1 remained unchanged over time in P. flocculosa and was undetected in U. maydis. At the same time, green fluorescent protein (GFP) strains of both fungi showed only limited green fluorescence on control leaves. On powdery mildew-infected cucumber leaves, P. flocculosa induced a complete collapse of the pathogen colonies, but glycolipid production, as studied by cyp1 expression, was still comparable to that of controls. In complete contrast, cyp1 was upregulated nine times when P. flocculosa was applied to Botrytis cinerea-infected leaves, but the biocontrol fungus did not develop very well on the pathogen. Analysis of the possible nutrients that could stimulate the growth of P. flocculosa on powdery mildew structures revealed that the complex Zn/Mn played a key role in the interaction. Other related fungi such as U. maydis do not appear to have the same nutritional requirements and hence lack the ability to colonize powdery mildews. Whether production of antifungal glycolipids contributes to the release of nutrients from powdery mildew colonies is unclear, but the specificity of the biocontrol activity of P. flocculosa toward Erysiphales does appear to be more complex than simple antibiosis.

The cynomolgus monkey is used to study drug metabolism because of its evolutionary closeness to humans. Despite their importance, regional distribution of cytochrome P450 (CYP) enzymes including CYP3As in the liver and small intestine, the major sites of drug metabolism, has not been fully investigated in cynomolgus monkeys. In this study, we measured mRNA expression levels of 14 CYPs in the CYP1, 2, and 3 subfamilies, including orthologs of human CYP3A4 and CYP3A5, in the liver and small intestine of cynomolgus monkeys. Expression levels of each CYP mRNA in various regions of the liver were quantified and comparisons were made between the right lobe, quadrate lobe, left medial lobe, left lateral lobe, and caudate lobe and with four different sections of the right lobe. In the small intestine, the same mRNAs were measured in the duodenum and six different sections from the proximal jejunum to the distal ileum. Expression levels of the CYP mRNAs were not substantially different between liver samples, but varied between the different sections of the small intestine, including CYP3A4. These results suggest that analysis of distinct sections is required for a better understanding of cynomolgus monkey CYPs in the small intestine.

We have previously reported very high concentrations of pharmaceuticals in the effluent from a treatment plant receiving wastewater from about 90 bulk drug manufacturers near Hyderabad, India. The main objective of the present study was to examine how high dilutions of this effluent affect mRNA expression of cytochrome P450 (CYP) 1 family genes and ethoxyresorufin O-deethylase (EROD) activity in exposed wildlife, using the three-spined stickleback (Gasterosteus aculeatus) as a model. In gill filaments exposed to diluted effluent ex vivo, EROD activity was strongly inhibited in a concentration-dependent manner. In a subsequent in vivo study, groups of fish were exposed (24h) to three concentrations of effluent, 0.8%, 1.6% or 3.2%. In this experiment, EROD in gills was induced 27-, 52- or 60-fold, respectively. Accordingly, CYP1A mRNA was markedly up-regulated in gill, liver and brain of fish exposed to all three effluent concentrations. Expression of mRNA for CYP1B1 and CYP1C1 was induced in gills at all concentrations while effects on these genes in liver and brain were weak or absent. The results of a time course study suggested that most CYP1-inducing substances in the effluent were readily metabolised or excreted, because the induced EROD activity and mRNA expression decreased when the fish were transferred to clean water. Considering that CYP1 enzymes play important roles in biotransformation of endogenous and foreign compounds, the observed dual effect of the effluent on CYP1 catalytic activity and mRNA expression suggests that multiple physiological functions could be affected in exposed wildlife.

The presence of mRNA of individual members of the CYP1 gene family in normal and neoplastic kidney has been investigated by RTPCR. CYP1B1 mRNA was consistently expressed in both normal and neoplastic kidney while CYP1A1 was present in the majority of normal and neoplastic whereas CYP1A2 was infrequently expressed. Expression of the Ah receptor and Arnt which are involved in the transcriptional activation of the CYP1 genes was also studied. The Ah receptor mRNA and Arnt mRNA were consistently expressed both in kidney tumours and normal kidney. These results indicate differential expression of individual members of the CYP1 gene family in normal and neoplastic kidney and suggest that several mechanisms including the Ah receptor complex could be involved in their regulation.

The cytochrome P4501 (CYP1) gene family comprises four subfamilies in fish: CYP1A, CYP1B, CYP1C, and CYP1D. Only two CYP1 genes, CYP1A1 and CYP1A3, are so far known in rainbow trout (Oncorhynchus mykiss). The present study aimed to identify other CYP1 subfamily genes in rainbow trout, to establish methods for quantitative mRNA expression analysis of these genes, and to determine their basal and induced mRNA expression in gills and liver. Another goal was to examine their mRNA expression in environmentally exposed fish. We cloned four new transcripts, denoted rbCYP1B1, rbCYP1C1, rbCYP1C2, and rbCYP1C3. Levels of these and the previously known rbCYP1A transcripts were determined by real-time PCR in unexposed fish, fish exposed to the potent aryl hydrocarbon receptor (AhR) agonist 3,3',4,4',5-pentachlorobiphenyl (PCB126), and fish caged in various waters in the Uppsala region (Sweden). The mRNA expression patterns observed in unexposed rainbow trout (basal levels) were markedly similar to those reported for orthologous genes in other species. All six transcripts were induced by PCB126 in gills and liver, suggesting all genes to be AhR regulated. The caged fish showed clear rbCYP1 induction in gills at all monitoring sites (up to 70-fold the basal level), whereas the liver responses were weak; induction (up to 5-fold) was recorded only at the Uppsala municipal sewage treatment plant outlet. Gill filament EROD activity was induced at all caging sites. Most interestingly, the rbCYP1 gene response patterns in gills differed among caging sites and among subfamilies. The EROD induction seemed to only reflect induction of rbCYP1A transcription. Response patterns of multiple CYP1 genes in gills and liver could provide an improved monitoring strategy. Such patterns could be used to characterize complex mixtures of AhR agonists and antagonists in aquatic environments.

Tetrapod cytochrome P4501 family (CYP1A1, CYP1A2 and CYP1B1) enzymes are most active in hydroxylating a variety of environmental contaminants including polyaromatic hydrocarbons (PAH), planar polychlorinated biphenyls and arylamines and thus play a pivotal role in the toxicology of these compounds. Mammalian CYP1A1 and CYP1A2 genes appear to have diverged after the evolutionary emergence of mammals, whereas fish species apparently possess only one CYP1A family gene, and fish CYP1A enzymes exhibit properties of both of the mammalian isoforms. We have isolated a further CYP1 family gene from a marine flatfish (plaice; Pleuronectes platessa), which, on the basis of exon organisation and sequence similarity, can be assigned as a piscine CYP1B. Its deduced amino acid sequence shows the closest (54%) identity to mammalian CYP1B1 proteins and, on the basis of molecular modeling studies, shows a high degree of positional and structural conservation of the substrate contacting amino acid residues in its putative active site when compared to other CYP1 enzymes. Phylogenetic analysis of fish and mammalian CYP1 family sequences indicates that the plaice CYP1B and mammalian CYP1B1 genes share a common ancestry. Plaice CYP1B has a more restricted tissue expression profile than the previously isolated plaice CYP1A, only being detectable, by Northern blotting, in gill tissue. In contrast to CYP1A, which shows extensive PAH-dependent induction in a variety of tissues, plaice CYP1B appears unresponsive to treatment with a prototypical PAH-type inducer, beta-naphthoflavone (BNF).

Exposure of precision-cut rat liver slices to six structurally diverse polycyclic aromatic hydrocarbons, namely benzo[a]pyrene, benzo[b]fluoranthene, dibenzo[a,h]anthracene, dibenzo[a,l]pyrene, fluoranthene and 1-methylphenanthrene, led to induction of ethoxyresorufin O-deethylase, CYP1A apoprotein and CYP1A1 mRNA levels, but to a markedly different extent. In liver slices, constitutive CYP1A1 mRNA levels were higher, as well as being markedly more inducible by PAHs, compared with CYP1B1, a similar profile to that observed in human liver slices following exposure to the PAHs. Increase in ethoxyresorufin O-deethylase and in CYP1A1 apoprotein levels was also observed when precision-cut rat lung slices were incubated with the same PAHs, the order of induction potency being similar to that observed in liver slices. Under the same conditions of exposure, CYP1B1 apoprotein levels were elevated in the lung. Up-regulation of CYP1A1 by the six PAHs correlated with their affinity for the Ah receptor, determined using the chemical-activated luciferase expression (CALUX) assay. It may be concluded that (a) precision-cut liver and lung slices may be used to assess the CYP1 induction potential of chemicals at the activity, apoprotein and mRNA levels; (b) rat is a promising surrogate animal for human in studies to evaluate CYP1 induction potential; (c) CYP1A1 is far more inducible than CYP1B1 in both rat liver and lung; (d) CYP1 up-regulation by PAHs is related to their affinity for the Ah receptor, and finally (e) computer analysis revealed that the ratio of molecular length/width is an important determinant of CYP1 induction potency among equiplanar PAHs.

The European Water Framework Directive aims to achieve a good ecological and chemical status in surface waters until 2015. Sediment toxicology plays a major role in this intention as sediments can act as a secondary source of pollution. In order to fulfill this legal obligation, there is an urgent need to develop whole-sediment exposure protocols, since sediment contact assays represent the most realistic scenario to simulate in situ exposure conditions. Therefore, in the present study, a vertebrate sediment contact assay to determine aryl hydrocarbon receptor (AhR)-mediated activity of particle-bound pollutants was developed. Furthermore, the activity and the expression of the CYP1 family in early life stages of zebrafish after exposure to freeze-dried sediment samples were investigated. In order to validate the developed protocol, effects of β-naphthoflavone and three selected sediment on zebrafish embryos were investigated. Results documented clearly AhR-mediated toxicity after exposure to β-naphthoflavone (β-NF) and to the sediment from the Vering canal. Upregulation of mRNA levels was observed for all investigated sediment samples. The highest levels of all investigated cyp genes (cyp1a, cyp1b1, cyp1c1, and cyp1c2) were recorded after exposure to the sediment sample of the Vering canal. In conclusion, the newly developed sediment contact assay can be recommended for the investigation of dioxin-like activities of single substances and the bioavailable fraction of complex environmental samples. Moreover, the exposure of whole zebrafish embryos to native (freeze-dried) sediment samples represents a highly realistic and ecologically relevant exposure scenario.

The oxidative metabolism of coumarin via several human cytochrome P450 (CYP) enzymes from families CYP1, CYP2 and CYP3 is rationalized in terms of molecular modelling studies carried out on the key interactions with various amino acid residues in the relevant active sites. The findings from modelling by homology with the CYP2C5 crystallographic template are in agreement with the known metabolism of coumarin in human P450s from the CYP1, CYP2 and CYP3 families, which has been published recently, and with independently reported information from site-directed mutagenesis studies.

Candida albicans is a human pathogenic fungus which can undergo a morphological transition from yeast to hyphae in response to a variety of environmental stimuli. We analyzed a C. albicans Asc1 (Absence of growth Suppressor of Cyp1) protein which is entirely composed of seven repeats of the WD domain, and is conserved from fungi to metazoan. Deleting the ASC1 in C. albicans led to a profound defect in hyphal development under hypha-inducing conditions examined. Furthermore, deletion of the ASC1 attenuated virulence of C. albicans in a mouse model of systemic infection. These data strongly suggested that the conserved WD-repeat protein Asc1 is required for morphogenesis and pathogenesis of C. albicans.

Alcohol consumption and tobacco smoking are major causes of head and neck cancers, and regional differences point to the importance of research into gene-environment interactions. Much interest has been focused on polymorphisms of CYP1A1 and of GSTM1 and GSTT1, but a number of studies have not demonstrated significant effects. This has mostly been ascribed to small sample sizes. In general, the impact of polymorphisms of metabolic enzymes appears inconsistent, with some reports of weak-to-moderate associations, and with others of no elevation of risks. The classical cytochrome P450 isoenzyme considered for metabolic activation of polycyclic aromatic hydrocarbons (PAH) is CYP1A1. A new member of the CYP1 family, CYP1B1, was cloned in 1994, currently representing the only member of the CYP1B subfamily. A number of single nucleotide polymorphisms of the CYP1B1 gene have been reported. The amino acid substitutions Val432Leu ( CYP1B1*3) and Asn453Ser ( CYP1B1*4), located in the heme binding domain of CYP1B1, appear as likely candidates to be linked with biological effects. CYP1B1 activates a wide range of PAH, aromatic and heterocyclic amines. Very recently, the CYP1B1 codon 432 polymorphism ( CYP1B1*3) has been identified as a susceptibility factor in smoking-related head-and-neck squamous cell cancer. The impact of this polymorphic variant of CYP1B1 on cancer risk was also reflected by an association with the frequency of somatic mutations of the p53 gene. Combined genotype analysis of CYP1B1 and the glutathione transferases GSTM1 or GSTT1 has pointed to interactive effects. This provides new molecular evidence that tobacco smoke-specific compounds relevant to head and neck carcinogenesis are metabolically activated through CYP1B1 and is consistent with a major pathogenetic relevance of PAH as ingredients of tobacco smoke.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is a widespread environmental contaminant, that has been linked with a variety of deleterious effects on human health, including increased cancer rates and reproductive anomalies. The detrimental effects of TCDD are mediated via the aryl hydrocarbon receptor (AhR), a transcription factor that regulates the expression of the carcinogen-activating enzymes cytochromes P-450 (CYP) 1A1, 1A2, and 1B1. In the present study, we examined the ability of synthetic derivatives of salicylic acid to affect TCDD-stimulated AhR-mediated signal transduction in human hepatoma HepG2 cells. Salicylamide (SAL), an analgesic drug, caused a potent and long-lasting inhibition of TCDD-induced CYP enzyme activity. Acetylsalicylic acid (aspirin) and the naturally occurring phytochemical salicylic acid had no effect on CYP activity. SAL inhibited the increase in CYP1A1, -1A2, and -1B1 mRNA levels that occurs on exposure to TCDD. TCDD-induced transcription of these genes was also inhibited by SAL, but not by aspirin or salicylic acid, as demonstrated by luciferase reporter assays. The transcription of the CYP1 family of genes is regulated by the interaction of TCDD-activated AhR with the xenobiotic-responsive element present in the promoter regions of these genes. As shown by electrophoretic mobility shift assay, SAL completely blocked the binding of TCDD-activated AhR to the xenobiotic responsive element. Also, SAL substantially blocked the binding of TCDD to the cytosolic AhR. These results demonstrate that SAL, a commonly used analgesic, is a potent inhibitor of AhR-mediated signal transduction, and may be an effective agent in the prevention of TCDD-associated disease.

The aryl hydrocarbon receptor (AhR), a ligand-activated transcription factor, contributes to carcinogenesis through its role in the regulation of cytochrome P450 1 (CYP1)-catalyzed metabolism of carcinogens. Here, we investigated genetic and epigenetic mechanisms that affect AhR expression. Analyses of the human AHR proximal promoter in MCF-7 human breast cancer cells using luciferase assays and electrophoretic mobility shift assays revealed multiple specificity protein (Sp) 1 binding sequences that are transcriptional activators in vitro. The regulation of AhR expression was evaluated in long-term estrogen exposed (LTEE) MCF-7 cells, which showed increased AhR expression, enhanced CYP1 inducibility, and increased capacity to form DNA adducts when exposed to the dietary carcinogen, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine. The increased AhR expression in LTEE cells was found not to result from increased mRNA stability, differential RNA processing, or decreased DNA methylation. Analysis of the AHR proximal promoter region using chromatin immunoprecipitation confirmed that enhanced expression of AhR in LTEE cells involves changes in histone modifications, notably decreased trimethylation of histone 3, lysine 27. Upon further examination of the GC-rich Sp1-binding region, we confirmed that it contains a polymorphic (GGGGC)(n) repeat. In a population of newborns from New York State, the allele frequency of (GGGGC)(n) was n = 4>> 5 ≫ 6, 2. Circular dichroism spectroscopy revealed the ability of sequences of this GC-rich region to form guanine-quadruplex structures in vitro. These studies revealed multiple levels at which AhR expression may be controlled, and offer additional insights into mechanisms regulating AhR expression that can ultimately impact carcinogenesis.

Rats were exposed to freshly prepared aqueous extracts of green tea (2.5% w/v) as the sole source of drinking water for 4 weeks. Hepatic cytochrome P450 activity was determined using chemical probes, showing selectivity for particular isoforms, and by immunoblot analysis employing polyclonal antibodies. Exposure to green tea gave rise to increases in the O-demethylation of methoxyresorufin and, to a lesser extent, in the dealkylations of ethoxyresorufin and pentoxyresorufin. An increase was also seen in lauric acid hydroxylation but, in contrast, the N-demethylation of erythromycin was inhibited. p-Nitrophenol oxidase activity was unaffected by the same treatment. Immunoblot analysis revealed increases in the apoprotein levels of CYP1A2 and CYP4A1 following treatment with green tea. A significant increase was also noted in the CN(-)-insensitive palmitoyl CoA oxidation and this was paralleled by an increase in the levels of the peroxisomal trifunctional protein determined immunologically. Hepatic S9 and microsomal preparations from tea-treated animals were more effective than controls in activating 2-amino-3-methylimidazol[4,5-f]quinoline and 2-aminoanthracene to mutagens in the Ames test. When N-nitrosopyrrolidine served as the promutagen, tea did not influence its mutagenicity when isolated microsomes comprised the activation system but a significant inhibition was observed when hepatic S9 was used. The above findings are discussed within the context of the established anticarcinogenic and anti-mutagenic properties of green tea.