Bietti's crystalline corneoretinal dystrophy (BCD) is a recessive degenerative eye disease caused by germline mutations in the CYP4V2 gene. More than 80% of mutant alleles consist of three mutations, that is, two splice-site alterations and one missense mutation, c.992C>A, which translates to p.H331P. In the present study, we analyzed the expression of CYP4 family members in human tissues and conducted functional studies with the wild-type and p.H331P enzymes, to elucidate the link between CYP4V2 activity and BCD. Expression analysis of 17 CYP1 to CYP4 genes showed CYP4V2 to be a major cytochrome P450 in ARPE-19 cells (a human cell line spontaneously generated from normal human retinal pigmented epithelium) and the only detectable CYP4 transcript. Immunohistochemical analyses demonstrated that CYP4V2 protein was present in epithelial cells of the retina and cornea and the enzyme was localized to endoplasmic reticulum. Recombinant reconstituted CYP4V2 protein metabolized eicosapentaenoic acid and docosahexaenoic acid (an important constituent of the retina) to their respective ω-hydroxylated products at rates similar to those observed with purified CYP4F2, which is an established hepatic polyunsaturated fatty acid (PUFA) hydroxylase. The disease-associated p.H331P variant was undetectable in Western blot analyses of HepG2 cells stably transduced with lentiviral expression vectors. Finally, overexpression of functional CYP4V2 in HepG2 cells altered lipid homeostasis. We demonstrated that CYP4V2 protein is expressed at high levels in ocular target tissues of BCD, that the enzyme is metabolically active toward PUFAs, and that the functional deficit among patients with BCD who carry the H331P variant is most likely a consequence of the instability of the mutant protein.

The dimorphic basidiomycete Ustilago maydis produces large amounts of surface-active compounds under conditions of nitrogen starvation. These biosurfactants consist of derivatives of two classes of amphipathic glycolipids. Ustilagic acids are cellobiose lipids in which the disaccharide is O-glycosidically linked to 15,16-dihydroxyhexadecanoic acid. Ustilipids are mannosylerythritol lipids derived from acylated beta-d-mannopyranosyl-d-erythritol. Whereas the chemical structure of these biosurfactants has been determined, the genetic basis for their biosynthesis and regulation is largely unknown. Here we report the first identification of two genes, emt1 and cyp1, that are essential for the production of fungal extracellular glycolipids. emt1 is required for mannosylerythritol lipid production and codes for a protein with similarity to prokaryotic glycosyltransferases involved in the biosynthesis of macrolide antibiotics. We suggest that Emt1 catalyzes the synthesis of mannosyl-d-erythritol by transfer of GDP-mannose. Deletion of the gene cyp1 resulted in complete loss of ustilagic acid production. Cyp1 encodes a cytochrome P450 monooxygenase which is highly related to a family of plant fatty acid hydroxylases. Therefore we assume that Cyp1 is directly involved in the biosynthesis of the unusual 15,16-dihydroxyhexadecanoic acid. We could show that mannosylerythritol lipid production is responsible for hemolytic activity on blood agar, whereas ustilagic acid secretion is required for long-range pheromone recognition. The mutants described here allow for the first time a genetic analysis of glycolipid production in fungi.

Compound lipophilicity is of key importance to P450 binding affinity and enzyme selectivity. Here, lipophilicity is discussed with reference to the human drug-metabolizing P450 enzymes of families CYP1, CYP2 and CYP3. From an extensive compilation of log P values for P450 substrates, and by analysis of relationships between partitioning energy and substrate-binding free energy, the relevance of lipophilicity and other factors pertaining to P450 binding affinity is explained, leading to the formulation of lipophilicity relationships within substrates of each human P450 enzyme involved in drug metabolism. Furthermore, log P values for P450 substrates appear to represent markers for enzyme selectivity. Together with the important roles of hydrogen bonding and pi-pi stacking interaction energies, the desolvation of the P450 active site makes a major contribution to the overall substrate-binding energy and, consequently, a good agreement with experimental information is reported based on this analysis.

Three mutations, each of which causes overproduction of iso-2-cytochrome c, were characterized biochemically. Two, CYP3-4 and CYP3-15, were previously shown to be cis-dominant and map to the CYC7 locus which encodes the iso-2 protein, while the third, cyp1-16, maps to an unliked locus. All three mutations caused dramatically increased levels of transcription of the CYC7 gene, and the CYC7 mRNA in mutant cells was found to be the same size as that in wild type cells. The CYP3-4 mutation was found to be caused by the integration of a transposable element, Tyl, 269 base pairs 5' to the coding sequences. The CYP3-15 mutation was also found to alter the DNA, probably through a deletion or inversion with one endpoint 285 base pairs upstream from the coding sequence. The CYC7 gene in both wild type and mutant cells was not subject to catabolite repression.

The CYP1 (HAP1) gene of Saccharomyces cerevisiae is known to activate a number of target genes in response to the presence of heme. Several features of the protein, deduced from the sequence of the gene, suggest that CYP1 is a general sensor of the redox state of the cell. To investigate further the function of CYP1, we analysed its effects on the transcription of two genes, HEM13 and 14DM, which are preferentially expressed in anaerobiosis. HEM13 encodes coproporphyrinogen oxidase which catalyses the sixth enzymatic step in the heme biosynthetic pathway and 14DM encodes lanosterol-14-demethylase which is involved in sterol biosynthesis and is a member of the cytochrome P450 family. Isogenic CYP1+ and cyp1 degree deleted strains, either heme-sufficient or heme-deficient (HEM1 disrupted), were grown in aerobic or anaerobic conditions, and transcripts of HEM13 and 14DM were analysed on Northern blots. The results show that in anaerobic and in heme-deficient cells, CYP1 activates the transcription of HEM13 and inhibits that of 14DM. Opposite effects of CYP1 are observed in aerobic, heme-sufficient cells. We concluded that: (i) CYP1 is an efficient activator especially in heme-depleted cells; (ii) CYP1 exerts both positive and negative regulatory effects; (iii) the nature of the regulatory function of CYP1 depends on the target gene; and (iv) for a given gene, the presence or absence of heme or oxygen reverses the sense of CYP1-dependent regulation.

Cytochrome P450 (CYP) enzymes are members of the hemoprotein superfamily, and are involved in the mono-oxygenation reactions of a wide range of endogenous and exogenous compounds in mammals and plants. Characterization of CYP genes in fish has been carried out intensively over the last 20 years. In Japanese pufferfish (Takifugu rubripes), 54 genes encoding P450s have been identified. Across all species of fish, 137 genes encoding P450s have been identified. These genes are classified into 18 CYP families: namely, <em>CYP1</em>, CYP2, CYP3, CYP4, CYP5, CYP7, CYP8, <em>CYP1</em>1, <em>CYP1</em>7, <em>CYP1</em>9, CYP20, CYP21, CYP24, CYP26, CYP27, CYP39, CYP46 and CYP51.We pinpointed eight CYP families: namely, <em>CYP1</em>, CYP2, CYP3, CYP4, <em>CYP1</em>1, <em>CYP1</em>7, <em>CYP1</em>9 and CYP26 in this review because these CYP families are studied in detail. Studies of fish P450s have provided insights into the regulation of P450 genes by environmental stresses including water pollution. In this review, we present an overview of the CYP families in fish.

A potato cysteine protease (cyp) cDNA expressed at an early stage of an incompatible interaction with Phytophthora infestans was isolated. Both the nucleotide and deduced amino acid sequences are highly homologous to those of a tomato cysteine protease, CYP1. Striking protein similarity to all known cathepsins in animals, particularly cathepsin K, was also observed. However, unlike cathepsins, a granulin binding domain is located near the carboxyl terminus of the putative CYP protein. In animals, granulins bind to receptors in the plasma membrane and signal cell growth and division. A ribonuclease protection assay demonstrated that the cyp gene is tightly regulated and is induced 15 h post inoculation with P. infestans in potato leaves either with high field resistance or in which a resistance (R) gene is activated. We conclude that a common signaling pathway is activated in each form of resistance.

The CYP1 gene has previously been identified as coding for a positive trans active factor that activates the expression of CYC1 and CYP3, which are the structural genes for isol1- and iso2-cytochrome c. Two phenotypically distinct classes of CYP1 mutations can be obtained indicating that CYC1 and CYP3 are differentially regulated by the product of CYP1. The HAP1 gene codes for a product which has previously been proved to be necessary for the expression of the heme dependent CYC1-UAS1 cis regulatory sequence. In this article, we show by complementation and recombination that CYP1 and HAP1 are the same gene, moreover we identify hap1-1 as an iso2-cytochrome c underproducer mutation of the CYP1 gene.

Cyclophilins are peptidyl-prolyl cis-trans isomerases (PPIases) which have been implicated in intracellular protein folding, transport and assembly. Cyclophilins are also known as the intracellular receptors for the immunosuppressive drug cyclosporin A (CsA). The most common type of cyclophilins are the 18 kDa cytosolic proteins containing only the highly conserved core domain for PPIase and CsA binding activities. The wis2+ gene of the fission yeast Schizosaccharomyces pombe was isolated as a multicopy suppressor of wee1-50 cdc25-22 win1-1, a triple mutant strain which exhibits a cell cycle defect phenotype. Sequence analysis of wis2+ reveals that it encodes a 40 kDa cyclophilin-like protein, homologous to the mammalian cyclophilin 40. The 18 kDa cyclophilin domain (CyP-18) of wis2 is followed by a C-terminal region of 188 amino acids. The C-terminal region of wis2 is essential for suppression of the triple mutant defect. Furthermore this region of the protein is able to confer suppression activity on the 18 kDa S.pombe cyclophilin, cyp1, since a hybrid protein consisting of an 18 kDa S.pombe cyclophilin (cyp1) fused to the C-terminus of wis2 shows suppression activity. We also demonstrate that the level of wis2+ mRNA increases 10- to 20-fold upon heat shock of S.pombe cells suggesting a role for wis2+ in the heat-shock response.

The basic-helix/loop/helix per-Arnt-sim (bHLH/PAS) family comprises many transcription factors, found throughout all three kingdoms of life; bHLH/PAS members "sense" innumerable intracellular and extracellular "signals" - including endogenous compounds, foreign chemicals, gas molecules, redox potential, photons (light), gravity, heat, and osmotic pressure. These signals then initiate downstream signaling pathways involved in responding to that signal. The term "PAS", abbreviation for "per-Arnt-sim" was first coined in 1991. Although the mouse Arnt gene was not identified until 1991, evidence of its co-transcriptional binding partner, aryl hydrocarbon receptor (AHR), was first reported in 1974 as a "sensor" of foreign chemicals, up-regulating cytochrome P450 family 1 (CYP1) and other enzyme activities that usually metabolize the signaling chemical. Within a few years, AHR was proposed also to participate in inflammation. The mouse [Ah] locus was shown (1973-1989) to be relevant to chemical carcinogenesis, mutagenesis, toxicity and teratogenesis, the mouse Ahr gene was cloned in 1992, and the first Ahr(-/-) knockout mouse line was reported in 1995. After thousands of studies from the early 1970s to present day, we now realize that AHR participates in dozens of signaling pathways involved in critical-life processes, affecting virtually every organ and cell-type in the animal, including many invertebrates.

A new member of the cytochrome P450 superfamily, CYP2S1, has recently been identified in human and mouse. In this paper, we review the data currently available for CYP2S1. The human CYP2S1 gene is located in chromosome 19q13.2 within a cluster including CYP2 family members CYP2A6, CYP2A13, CYP2B6, and CYP2F1. These genes also show the highest homology to the human CYP2S1. The gene has recently been found to harbor genetic polymorphism. CYP2S1 is inducible by dioxin, the induction being mediated by the Aryl Hydrocarbon Receptor (AHR) and Aryl Hydrocarbon Nuclear Translocator (ARNT) in a manner typical for CYP1 family members. In line with this, CYP2S1 has been shown to be inducible by coal tar, an abundant source of PAHs, and it was recently reported to metabolize naphthalene. This points to the involvement of CYP2S1 in the metabolism of toxic and carcinogenic compounds, similar to other dioxin-inducible CYPs. CYP2S1 is expressed in epithelial cells of a wide variety of extrahepatic tissues. The highest expression levels have been observed in the epithelial tissues frequently exposed to xenobiotics, e.g., the respiratory, gastrointestinal, and urinary tracts, and in the skin. The observed ubiquitous tissue distribution, as well as the expression of CYP2S1 throughout embryogenesis suggest that CYP2S1 is likely to metabolize important endogenous substrates; thus far, retinoic acid has been identified. In conclusion, CYP2S1 exhibits many features of interest for human health and thus warrants further investigation.

It has been difficult to study the regulation of cytochrome P4501A2 (CYP1A2) because expression of this enzyme is reported to be limited or absent in cell culture. We found that CYP1A2 can be induced significantly by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), 3-methylcholanthrene (MC), or benz[a]anthracene in the human colon carcinoma cell line LS180. TCDD and MC each caused a dramatic elevation of CYP1A2 mRNA, as assessed by reverse transcription-polymerase chain reaction or by northern blot analysis. TCDD also increased immunoreactive CYP1A2 protein and the activity of phenacetin-O-deethylase, a diagnostic catalytic marker for CYP1A2. The induction of CYP1A2 at all levels (mRNA, protein, catalytic activity) was concentration- and time-dependent: the EC50 for mRNA induction by TCDD = 0.5 nM, and by MC = 1.4 microM. Inducible CYP1A2 mRNA also was detected at lower levels in two other human cell lines, the hepatoma cell line HepG2 and the breast carcinoma cell line MCF-7. CYP1A1 and CYP1B1, additional CYP1 enzymes regulated by the aryl hydrocarbon receptor (AHR), also were inducible by TCDD and MC in LS180 cells; their concentration-dependent induction was highly correlated with induction of CYP1A2 at mRNA, protein, and catalytic levels. CYP1B1 was constitutively expressed and inducible in the LS180, MCF-7, and HepG2 cell lines as well as in the human choriocarcinoma cell line JEG-3 and the squamous cell carcinoma line A431. CYP1A2 was neither constitutively expressed nor inducible in A431 or JEG-3 cells. The expression of mRNAs encoding the regulators of CYP1 enzymes-the AHR and its heterodimerization partner, the ARNT (AH receptor nuclear translocator) protein-was not altered by treatment with TCDD or MC. However, the cytosolic content of AHR protein and ARNT protein was depleted substantially following treatment with TCDD. The LS180 cell line should constitute a good model for further mechanistic studies on AHR-regulated CYP1A2 expression.

Enzymes in the cytochrome P450 1 family oxidize many common environmental toxicants. We identified a new CYP1, termed CYP1D1, in zebrafish. Phylogenetically, CYP1D1 is paralogous to CYP1A and the two share 45% amino acid identity and similar gene structure. In adult zebrafish, CYP1D1 is most highly expressed in liver and is relatively highly expressed in brain. CYP1D1 transcript levels were higher at 9h post-fertilization than at later developmental times. Treatment of zebrafish with potent aryl hydrocarbon receptor (AHR) agonists (3,3',4,4',5-pentachlorobiphenyl or 2,3,7,8-tetrachlorodibenzo-p-dioxin) did not induce CYP1D1 transcript expression. Morpholino oligonucleotide knockdown of AHR2, which mediates induction of other CYP1s, did not affect CYP1D1 expression. Zebrafish CYP1D1 heterologously expressed in yeast exhibited ethoxyresorufin- and methoxyresorufin-O-dealkylase activities. Antibodies against a CYP1D1 peptide specifically detected a single electrophoretically-resolved protein band in zebrafish liver microsomes, distinct from CYP1A. CYP1D1 in zebrafish is a CYP1A-like gene that could have metabolic functions targeting endogenous compounds.

Representational difference analysis was used to isolate cDNAs corresponding to 2,3,7,8-tetrachlorodibenzo-p-dioxin (dioxin)-inducible genes from mouse Hepa-1 cells. One cDNA encoded a novel cytochrome P450. The human homolog was also isolated and later proved to be human CYP2S1. The induction of mouse CYP2S1 mRNA by dioxin represents a primary response and required the aryl hydrocarbon receptor and aryl hydrocarbon receptor nuclear translocator proteins. The induction of CYP2S1 also occurred in mouse liver and lung, with the highest expression found in lung. CYP2S1 was also inducible in a human lung epithelial cell line. The dioxin-inducibility of CY2S1 is exceptional, because all previously well-characterized cases of the induction of cytochromes P450 by dioxin involve members of the CYP1 family.

Previously, it was shown that the CYP1(HAP1) gene product mediates the transcription of several oxygen-regulated genes through a metabolic co-effector, heme, in the yeast Saccharomyces cerevisiae. This study investigates the overproduction of the CYP1 protein when the CYP1(HAP1) gene is placed under the control of the GAL10-CYC1 hybrid promoter (either at the locus of the CYP1(HAP1) gene or cloned in a high-copy-number plasmid). In these conditions, the CYP1 protein is detected by Western blot analysis and has a molecular mass in agreement with the open reading frame sequence. Band-shift experiments show that the CYP1(HAP1) protein is able to interact specifically with its target sequences in vitro without addition of hemin, and forms a large complex with one or several unidentified factors denoted as X. Addition of hemin allows the formation of a new complex which has a lower molecular mass. The internal deletion of the seven repeated amino acid sequences containing the KCPVDH motif in the CYP1(HAP1) protein modifies the heme responsiveness phenomenon observed in vitro in the band-shift experiments and in vivo in the transcription of the CYB2, CYC1, CYP3(CYC7) and ERG11 genes. On the basis of these data, we propose a new model for heme-induced activation of the CYP1 protein.

Nitropolycyclic aromatic hydrocarbons (NPAHs) are found in diesel exhaust and ambient air. NPAHs as well as polycyclic aromatic hydrocarbons (PAHs) are known to have mutagenicity, carcinogenicity, and endocrine-disruptive effects. In the present study, the inducibility of the human cytochrome P450-1 (CYP1) family by NPAHs was compared with those produced by their parent PAHs and some reductive metabolites, amino-PAHs. Furthermore, to investigate the differences in the inducibility of the CYP1 family in human tissues, various human tissue-derived cell lines, namely HepG2 (hepatocellular carcinoma), ACHN (renal carcinoma), A549 (lung carcinoma), MCF-7 (breast carcinoma), LS-180 (colon carcinoma), HT-1197 (bladder carcinoma), HeLa (cervix of uterus adenocarcinoma), OMC-3 (ovarian carcinoma), and NEC14 (testis embryonal carcinoma), were treated with NPAHs, PAHs, or amino-PAHs. The mRNA levels of CYP1A1, CYP1A2, and CYP1B1 were determined with reverse transcription-polymerase chain reaction (RT-PCR). The cell lines were classified into two groups: CYP1 inducible cell lines, comprising HepG2, MCF-7, LS-180, and OMC-3 cells, and CYP1 non-inducible cell lines, comprising ACHN, A549, HT-1197, HeLa, and NEC14 cells. In inducible cell lines, the induction profile of chemical specificity was similar for CYP1A1, CYP1A2, and CYP1B1, although the extent of induction differed among the cell lines and for the CYP isoforms. Pyrene, 1-nitropyrene, 1-aminopyrene, 1,3-, 1,6-, and 1,8-dinitropyrenes slightly induced CYP1 mRNAs, but 1,3-dinitropyrene produced a 6-fold induction of CYP1A1 mRNA in MCF-7 cells. 2-Nitrofluoranthene and 3-nitrofluoranthene exhibited stronger inducibility than fluoranthene in the inducible cell lines. 6-Nitrochrysene induced CYP1 mRNAs to the same extent or more potently than chrysene. The induction potencies of 6-nitrobenzo[ a]pyrene and 7-nitrobenz[ a]anthracene were weaker than those of their parents benzo[ a]pyrene and benz[ a]anthracene, respectively. This study demonstrated that NPAHs as well as PAHs induced human CYP1A1, CYP1A2, and CYP1B1 in a chemical-, CYP isoform-, and cell-specific manner. Furthermore, the cell-specific induction of the CYP1 family was not related to the expression levels of aryl hydrocarbon receptor, aryl hydrocarbon nuclear translocator, or estrogen receptors alpha and beta.

The skin reacts to environmental noxae by inducing cytochrome P450 (CYP)-catalyzed reactions via activation of the aryl hydrocarbon receptor (AhR). A drawback of this response is the generation of oxidative stress, which is especially dangerous for postreplicative cells such as dermal fibroblasts, in which damage may accumulate over time. Accordingly, in dermal fibroblasts, CYP1 expression is repressed and it has been proposed that this is due to the AhR repressor (AhRR), which is supposedly overexpressed in fibroblasts as compared with other skin cells. Here, we revisited this "AhRR hypothesis", which has been mainly based on ectopic overexpression studies and correlation analyses of high AhRR gene expression with CYP1A1 repression in certain cell types. In primary human skin fibroblasts (NHDFs) of 25 individuals, we found that (i) the AhRR was expressed only at moderate RNA copy numbers and that, against the common view, (ii) in some fibroblast strains, CYP1A1 mRNA expression could be induced by AhR activators. However, even the highest induction did not translate into measurable CYP1 enzyme activity, and neither basal expression nor mRNA inducibility correlated with AhRR expression. In addition, enhancement of CYP1A1 mRNA expression by trichostatin A, which inhibits AhRR-recruited histone deacetylases at the CYP1A1 promoter, failed to induce measurable CYP1 activity. Finally, AhRR-deficient ((-/-)) mouse embryonic fibroblasts were not induced to biologically relevant CYP1 enzyme activity despite impressive mRNA induction. These data clearly indicate that repressed CYP1 activity in NHDFs is not causally related to AhRR expression, which may serve a different, yet unknown, biological function.

Human CYP1A1, CYP1A2, and CYP1B1 mRNAs were constitutively expressed in MCF-7 (human breast carcinoma) cells and were extensively (6-12-fold) induced by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). In contrast, in HeLa (human cervical adenocarcinoma) cells, CYP1A1 and CYP1B1 were induced by TCDD by up to 2-3-fold but CYP1A2 was not detected even when the cells were treated with TCDD. In the present study, the involvement of histone deacetylation and DNA methylation in the cell-specific inducibility of the human CYP1 family was investigated. The treatment of MCF-7 cells with trichostatin A (TSA), an inhibitor of histone deacetylase, and 5-aza-2'-deoxycitidine (AzaC), an inhibitor of DNA methylase, increased the constitutive expression level of CYP1A1, CYP1A2, and CYP1B1 by 2-3-fold. However, these treatments did not affect the levels of induction by TCDD. In HeLa cells, TSA and AzaC treatment increased the constitutive expression levels of CYP1A1 and CYP1B1. The induction of CYP1A2 was enhanced to a detectable level by TSA and AzaC even when the cells were not exposed to TCDD. Interestingly, pretreatment with TSA and AzaC increased the levels of CYP1A1, CYP1A2, and CYP1B1 induced by TCDD in HeLa cells. Furthermore, it was observed that TSA and AzaC treatment increased the constitutive expression level of AhR by 2-fold only in HeLa cells. To compare the methylation status of the 5'-flanking region of the human CYP1A1 gene including five XREs and the promoter region in MCF-7 and HeLa cells, the bisulfite-modified genes were amplified and sequenced. Since there was no remarkable difference in the methylation status within a -1.4 kb region of the human CYP1A1 gene, the methylation status in the CpG sites that exist in other regions of the human CYP1A1 gene might be involved in the cell-specific induction.

We have constructed a somatic cell hybrid line, designated 908K1, with a single human der(19) chromosome on a Chinese hamster background by employing conventional as well as microcell-mediated cell fusion techniques. The der(19) chromosome comprises the 19p13.1----q13.2 segment, as well as the distal (Xq24----qter) portion of the X chromosome long arm, and is stably retained by HAT selection. Extensive characterization of this hybrid line and comparison with other somatic cell hybrids has enabled us to regionally assign PGK2 to the distal short arm of chromosome 19 and to narrow down the assignments of CYP1, TGFB, and ERCC1 on 19q. Moreover, a cosmid library has been constructed from this microcell hybrid. By screening this library, as well as a chromosome 19-enriched library obtained elsewhere, 14 single-copy probes have been isolated that map on the 19p13.1----q13.2 segment, and 5 probes were assigned to the distal Xq. It is anticipated that these probes will be useful for the diagnosis of myotonic dystrophy and fra(X) mental retardation.

This review represents a compilation of typical substrates and inhibitors for human cytochrome P450 (CYP) enzymes that are involved in drug metabolism, specifically those from the CYP1, CYP2 and CYP3 families. Relatively recent literature on substrates and inhibitors has been collected and the relevant K(m) and K(i) values, respectively, are tabulated. Furthermore, physicochemical properties in the form of lipophilicity (log P and log D(7.4) values) and acidity/basicity (pK(a) values) are also tabulated for a significant number of substrates, together with some information on inhibitors, although only key inhibitors have been selected as the main focus is on substrates. The collated information indicates that there are certain commonalities between substrates for the same enzyme, especially with respect to their positions of metabolism and likely interactions with the relevant enzyme active site regions. The compilation therefore assists in establishing substrate structure-activity relationships (SSARs) within human drug-metabolizing P450s.

Reference genes are critical for normalization of the gene expression level of target genes. The widely used housekeeping genes may change their expression levels at different tissue under different treatment or stress conditions. Therefore, systematical evaluation on the housekeeping genes is required for gene expression analysis. Up to date, no work was performed to evaluate the housekeeping genes in cotton under stress treatment. In this study, we chose 10 housekeeping genes to systematically assess their expression levels at two different tissues (leaves and roots) under two different abiotic stresses (salt and drought) with three different concentrations. Our results show that there is no best reference gene for all tissues at all stress conditions. The reliable reference gene should be selected based on a specific condition. For example, under salt stress, UBQ7, GAPDH and EF1A8 are better reference genes in leaves; TUA10, UBQ7, CYP1, GAPDH and EF1A8 were better in roots. Under drought stress, UBQ7, EF1A8, TUA10, and GAPDH showed less variety of expression level in leaves and roots. Thus, it is better to identify reliable reference genes first before performing any gene expression analysis. However, using a combination of housekeeping genes as reference gene may provide a new strategy for normalization of gene expression. In this study, we found that combination of four housekeeping genes worked well as reference genes under all the stress conditions.

TCDD (2,3,7,8-tetrachlorodibenzo-p-dioxin) induces cytochromes P450 (CYPs) such as CYP1A1 and CYP1A2 via activation of the aromatic hydrocarbon receptor (AHR). Herein we describe the TCDD-dependent enrichment of CYPs in liver microsomes and mitoplasts from C57BL/6J mice. TCDD-induced accumulation of CYP1A1 and CYP1A2 was observed in microsomes and mitoplasts after treatment with 15 microg TCDD/kg/d for 3d. While microsomal CYP1 proteins peaked at 1 week and diminished thereafter, mitoplast CYP1 proteins persisted 8 weeks at high levels. TCDD also induced microsomal CYP2A5, but not microsomal proteins immunoreactive to CYP2C11, CYP3A2 or CYP4A1 antibodies. Nevertheless, each of these proteins increased in mitoplasts following TCDD exposure. These results suggest that TCDD increases mitochondrial CYP immunoreactive proteins under the transcriptional control of the AHR, as well as CYPs that are not under AHR control. We speculate that such mitochondrial CYPs may be involved in the generation, or mitigation, of the well-known TCDD-inducible oxidative stress response.

Flavonoids are polyphenolic compounds that have attracted the attention of the scientific community as the hallmark molecules responsible for cancer prevention by a plethora of different mechanisms. One of their most important characteristics, responsible for their cancer preventive properties, is their interaction with cytochrome P450 CYP1 enzymes. Flavonoids have traditionally been described as CYP1 inhibitors due to the inhibition of carcinogenic product formation and consequent blockage of the initiation stage of carcinogenesis. However, mounting evidence indicate that flavonoids are also capable of acting as CYP1 substrates, undergoing bioactivation to more antiproliferative agents within cancer cells. In this review, a comprehensive summary of the two models is presented. Structural features responsible for CYP1 inhibition or substrate turnover are discussed and limitations as well as discrepancies between procarcinogen-activating and 7-ethoxyresorufin-inhibition assay systems are further explored in vitro and in vivo. Moreover, a thorough investigation of the substrate specificity of flavonoids for the active site of CYP1 enzymes is undertaken. Finally, issues concerning the bioavailability and metabolic fate of these compounds in vivo are addressed. Ultimately, the mode of flavonoid action, in terms of CYP1 inhibition or CYP1-mediated bioactivation, is dependent on the lipophilicity or hydrophilicity of each compound. The degree of hydroxylation or methoxylation of the A and B rings is the major factor which determines the accessibility to the tumor site, in terms of hepatic and intestinal metabolism, and the introduction of the molecules to the CYP1 active site, respectively.

The aryl hydrocarbon receptor (AhR) is an important mediator of toxic responses after exposure to xenobiotics including 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and dioxin-like polychlorinated biphenyls (PCBs). Activation of AhR responsive genes requires AhR dimerization with the aryl hydrocarbon receptor nuclear translocator (ARNT), a heterodimeric partner also shared by the hypoxia-inducible factor-1α (HIF-1α) protein. TCDD-stimulated AhR transcriptional activity can be influenced by hypoxia; however, it less well known whether hypoxia interferes with AhR transcriptional transactivation in the context of PCB-mediated AhR activation in human cells. Elucidation of this interaction is important in liver hepatocytes which extensively metabolize ingested PCBs and experience varying degrees of oxygen tension during normal physiologic function. This study was designed to assess the effect of hypoxia on AhR transcriptional responses after exposure to 3,3',4,4',5-pentachlorobiphenyl (PCB 126). Exposure to 1% O2 prior to PCB 126 treatment significantly inhibited CYP1A1 mRNA and protein expression in human HepG2 and HaCaT cells. CYP1A1 transcriptional activation was significantly decreased upon PCB 126 stimulation under conditions of hypoxia. Additionally, hypoxia pre-treatment reduced PCB 126 induced AhR binding to CYP1 target gene promoters. Importantly, ARNT overexpression rescued cells from the inhibitory effect of hypoxia on XRE-luciferase reporter activity. Therefore, the mechanism of interference of the signaling crosstalk between the AhR and hypoxia pathways appears to be at least in part dependent on ARNT availability. Our results show that AhR activation and CYP1A1 expression induced by PCB 126 were significantly inhibited by hypoxia and hypoxia might therefore play an important role in PCB metabolism and toxicity.