Epidemiological evidence suggests a link between meat consumption and prostate cancer. In this study, benign prostatic hyperplasia tissues, obtained by transurethral resection or radical retropubic prostatectomy from UK-resident individuals (n = 18), were examined for CYP1 expression and for their ability, in short-term organ culture, to metabolically activate carcinogens found in cooked meat. Semi-quantitative RT-PCR analysis of CYP1 expression detected CYP1A2 mRNA transcripts in the prostates of four individuals, as well as mRNA transcripts from CYP1A1 and CYP1B1. The compounds tested for metabolic activation were 2-amino-1-methyl-6-phenylimidazo[4, 5-b]pyridine (PhIP; 500 microM, n = 9) and its metabolite N:-hydroxy PhIP (20 microM, n = 8), 2-amino-3-methylimidazo[4,5-f]quinoline (IQ; 500 microM, n = 6) and benzo[a]pyrene (B[a]P; 50 microM, n = 5). After incubation (PFMR medium, 22 h, 37 degrees C), DNA was isolated from tissue fragments and DNA adducts were detected and quantified by (32)P-postlabelling analysis. DNA adduct formation was detected in all samples incubated with PhIP (mean, adducts per 10(8) nucleotides), N:-hydroxy-PhIP (2736/10(8)) or B[a]P (1/10(8)). IQ-DNA adducts were detected in 5/6 tissues (mean, 1/10(8)). The CYP1 inhibitor alpha-naphthoflavone (10 microM) reduced B[a]P-DNA adduct formation in tissues from two individuals by 96 and 64%, respectively. This pilot study shows that human prostate tissue can metabolically activate 'cooked meat' carcinogens, a process that could contribute to prostate cancer development.

Pomegranate juice derived ellagitannins and their intestinal bacterial metabolites, urolithins, inhibited TCDD-induced CYP1-mediated EROD activity in vitro with IC(50) values ranging from 56.7 microM for urolithin A to 74.8 microM for urolithin C. These compounds exhibited dose- and time-dependent decreases in cell proliferation and clonogenic efficiency of HT-29 cells. Inhibition of cell proliferation was mediated through cell cycle arrest in the G(0)/G(1) and G(2)/M stages of the cell cycle followed by induction of apoptosis. These results indicate that the ellagitannins and urolithins released in the colon upon consumption of pomegranate juice in considerable amounts could potentially curtail the risk of colon cancer development, by inhibiting cell proliferation and inducing apoptosis.

The CYP1A family of cytochrome P450s (CYPs), comprising CYP1A1, CYP1A2, and CYP1B1, plays a role in bioactivation of several procarcinogens to carcinogenic derivatives, and also in detoxification of several xenobiotic compounds. Resveratrol (3,4,5-trihydroxystelbine) is a naturally occurring compound that has been shown in a number of studies to inhibit the induction of CYP1A1 and CYP1B1 by dioxin (2,3,7,8-tetrachloro-dibenzo-p-dioxin), but the mechanism(s) of resveratrol inhibition is controversial. In the current study, 100nM dioxin treatment for 24, 48, and 72 h induced CYP1A1, CYP1A2, and CYP1B1 mRNA levels in the human breast cancer cell line MCF-7, and CYP1A1 and CYP1A2 mRNA levels in the human hepatocellular carcinoma cell line, HepG2. Simultaneous treatment with 10 microM resveratrol significantly inhibited dioxin-induced mRNA expression levels of these genes in both cell lines. Our studies are novel in that we used the chromatin immunoprecipitation assay to assay dioxin-induced recruitment of the aryl hydrocarbon receptor (AHR), and aryl hydrocarbon nuclear translocator (ARNT) to the enhancer regions and recruitment of RNA polymerase II to the promoter regions, of the CYP1A1 and CYP1B1 genes in their natural chromosomal settings. These recruitments were significantly inhibited in cells cotreated with resveratrol. Our studies thus indicate that resveratrol inhibits dioxin induction of the CYP1 family members either by directly or indirectly inhibiting the recruitment of the transcription factors AHR and ARNT to the xenobiotic response elements of the corresponding genes. The reduced transcriptional factor binding at their enhancers then results in reduced pol II recruitment at the promoters of these genes.

Benzo[a]pyrene (BaP) is a prototypical polycyclic aromatic hydrocarbon (PAH); this ubiquitous environmental carcinogenic agent is found in tobacco smoke, charcoal-grilled foods, and PAH-contaminated surfaces of roofs, playgrounds, and highways. Cytochrome P450 1 wild-type, Cyp1a2(-/-), Cyp1b1(-/-), or Cyp1a2/1b1(-/-) knockouts, and mice with Cyp1a1 expression deleted in hepatocytes can ingest large oral BaP doses (125 mg/kg/d) without apparent toxicity. Cyp1a1(-/-) and Cyp1a1/1a2(-/-) knockouts and mice with Cyp1a1 expression deleted in gastrointestinal (GI) tract epithelial cells develop immunotoxicity and die within 32 days, indicating that GI tract inducible CYP1A1 is absolutely required for detoxication of oral BaP. Cyp1a1/1b1(-/-) and Cyp1a1/1a2/1b1(-/-) mice are rescued from immunosuppression and early death due to absent metabolic activation of BaP by CYP1B1 in immune cells. Ten-fold lower oral BaP doses result in adenocarcinoma of the proximal small intestine (PSI) in Cyp1a1(-/-) mice; Cyp1a1/1b1(-/-) double-knockout mice show no PSI cancer but develop squamous cell carcinoma of the preputial gland duct (PGD). BaP-metabolizing CYP1B1 in the PSI and CYP3A59 in the PGD are the most likely candidates to participate in tumor initiation in the epithelial cells of these two tissues; oncogenes and tumor-suppressor genes upregulated and downregulated during tumorigenesis are completely different between these tissues. This "oral BaP Cyp1" mouse paradigm represents a powerful teaching tool, showing that gene-environment interactions depend on route-of-administration: the same oral, but not intraperitoneal, BaP exposure leads to dramatic differences in target-organ toxicity and tumor type as a function of dose and Cyp1 genotype.

The overexpression of human cytochrome P450 CYP1B1 has been observed in a wide variety of malignant tumours, but the protein is undetectable in normal tissues. A number of cytochrome P450 enzymes are known to metabolise a variety of anticancer drugs, and the consequence of cytochrome P450 metabolism is usually detoxification of the drug, although bioactivation occurs in some cases. In this study, a Chinese hamster ovary cell line expressing human cytochrome P450 CYP1B1 was used to evaluate the cytotoxic profile of several anticancer drugs (docetaxel, paclitaxel, cyclophosphamide, doxorubicin, 5-fluorouracil, cisplatin, and carboplatin) commonly used clinically in the treatment of cancer. The MTT (3-[4,5-dimethylthiazol-2yl]-2,5-diphenyltetrazolium bromide) assay was used to determine the levels of cytotoxicity. The key finding of this study was that on exposure to docetaxel, a significant decrease in sensitivity towards the cytotoxic effects of docetaxel was observed in the cell line expressing CYP1B1 compared to the parental cell line (P = 0.03). Moreover, this difference in cytotoxicity was reversed by co-incubation of the cells with both docetaxel and the cytochrome P450 CYP1 inhibitor alpha-naphthoflavone. This study is the first to indicate that the presence of CYP1B1 in cells decreases their sensitivity to the cytotoxic effects of a specific anticancer drug.

The phytopathogenic basidiomycetous fungus Ustilago maydis secretes large amounts of the glycolipid biosurfactant ustilagic acid (UA). UA consists of 15,16-dihydroxypalmitic or 2,15,16-trihydroxypalmitic acid, which is O-glycosidically linked to cellobiose at its terminal hydroxyl group. In addition, the cellobiose moiety is acetylated and acylated with a short-chain hydroxy fatty acid. We have identified a 58 kb spanning gene cluster that contains 12 open reading frames coding for most, if not all, enzymes needed for UA biosynthesis. Using a combination of genetic and mass spectrometric analysis we were able to assign functional roles to three of the proteins encoded by the gene cluster. This allowed us to propose a biosynthesis route for UA. The Ahd1 protein belongs to the family of non-haem diiron reductases and is required for alpha-hydroxylation of palmitic acid. Two P450 monooxygenases, Cyp1 and Cyp2, catalyse terminal and subterminal hydroxylation of palmitic acid. We could demonstrate that infection of tomato leaves by the plant pathogenic fungus Botrytis cinerea is prevented by co-inoculation with wild-type U. maydis sporidia. U. maydis mutants defective in UA biosynthesis were unable to inhibit B. cinerea infection indicating that UA secretion is critical for antagonistic activity.

Expression of the Ah receptor-regulated cytochrome P4501B1 (CYP1B1) gene was studied in human adult and fetal tissues and cells in culture by reverse transcriptase-coupled polymerase chain reaction (RT-PCR). In adults, CYP1B1 mRNA was detected in liver, lymphocytes, cells of bronchoalveolar lavage samples and uterine endometrium, but not in lung. The level of expression was very low in adult liver and only three out of six fetal livers expressed CYP1B1. Extrahepatic fetal tissues, especially brains and kidneys, expressed high levels of CYP1B1. CYP1B1 mRNA was constitutively detected at a low level in first trimester and full-term placental samples. A competitive RT-PCR assay was developed to assess the regulation of CYP1B1. CYP1B1 mRNA was not induced in placenta by maternal cigarette smoking. Inducibility of CYP1B1 in cells in culture by the Ah receptor ligand 2,3,7,8-tetrachlorodibenzo-p-dioxin was studied in primary fibroblasts and chorion carcinoma cell line JEG-3 having different CYP1A1 induction properties. Inducibility of CYP1B1 was found to be regulated independently from CYP1A1. In JEG-3 cells CYP1A1 mRNA was induced up to 9000-fold, while the expression of CYP1B1 was not affected. Expression of Ah receptor and Ah receptor nuclear translocator (regulators of the CYP1 family) was determined in human placenta and in the JEG-3 cell line. Expression of these transcription factors was found neither to be co-regulated nor affected by Ah receptor ligands. This study provides evidence that in addition to the Ah receptor complex, other cell-specific factors modulate the response of CYP1B1 and CYP1A1 to Ah receptor ligands.

Expression of the Saccharomyces cerevisiae gene encoding cytochrome b2 (EC 1.2.2.3), CYB2, was investigated by direct analysis of mRNA transcripts and by measurement of the expression of lacZ fused to the CYB2 control regions. These studies indicated that regulation of the CYB2 gene is subject to several metabolic controls at the transcriptional level: inhibition due to glucose fermentation, induction by lactate, and inhibition in anaerobiosis or in absence of heme biosynthesis. Furthermore, we have shown that the CYB2 promoter contains one cis negative regulatory region and two heme-dependent positive regions, one of which is controlled by the transcriptional regulator CYP1 (HAP1) which is involved in the modulation of the expression of several oxygen-regulated genes. The CYP1 (HAP1)-binding sequence was located by gel retardation and DNase I footprinting experiments and compared with the binding sequences previously characterized in detail (UAS1CYC1, UAS'CYP3 (CYC7), and UASCTT1).

We report here that Hap1p (originally named Cyp1p) has an essential function in anaerobic or heme-deficient growth. Analysis of intragenic revertants shows that this function depends on the amino acid preceding the first cysteine residue of the DNA-binding domain of Hap1p. Selection of recessive extragenic suppressors of a hap1-hem1- strain allowed the identification, cloning, and molecular analysis of ASC1 (Cyp1 Absence of growth Supressor). The sequence of ASC1 reveals that its ORF is interrupted by an intron that shelters the U24 snoRNA. Deletion of the intron, inactivation of the ORF, and molecular localization of the mutations show unambiguously that it is the protein and not the snoRNA that is involved in the suppressor phenotype. ASC1, which is constitutively transcribed, encodes an abundant, cytoplasmically localized 35-kD protein that belongs to the WD repeat family, which is found in a large variety of eucaryotic organisms. Polysome profile analysis supports the involvement of this protein in translation. We propose that the absence of functional Asc1p allows the growth of hap1-hem1- cells by reducing the efficiency of translation. Based on sequence comparisons, we discuss the possibility that the protein intervenes in a kinase-dependent signal transduction pathway involved in this last function.

Crossing the Cyp1a1/1a2(-/-) double-knockout mouse with the Cyp1b1(-/-) single-knockout mouse, we generated the Cyp1a1/1a2/1b1(-/-) triple-knockout mouse. In this triple-knockout mouse, statistically significant phenotypes (with incomplete penetrance) included slower weight gain and greater risk of embryolethality before gestational day 11, hydrocephalus, hermaphroditism, and cystic ovaries. Oral benzo[a]pyrene (BaP) daily for 18 days in the Cyp1a1/1a2(-/-) produced the same degree of marked immunosuppression as seen in the Cyp1a1(-/-) mouse; we believe this reflects the absence of intestinal CYP1A1. Oral BaP-treated Cyp1a1/1a2/1b1(-/-) mice showed the same "rescued" response as that seen in the Cyp1a1/1b1(-/-) mouse; we believe this reflects the absence of CYP1B1 in immune tissues. Urinary metabolite profiles were dramatically different between untreated triple-knockout and wild-type; principal components analysis showed that the shifts in urinary metabolite patterns in oral BaP-treated triple-knockout and wild-type mice were also strikingly different. Liver microarray cDNA differential expression (comparing triple-knockout with wild-type) revealed at least 89 genes up- and 62 genes down-regulated (P-value < or = 0.00086). Gene Ontology "classes of genes" most perturbed in the untreated triple-knockout (compared with wild-type) include lipid, steroid, and cholesterol biosynthesis and metabolism; nucleosome and chromatin assembly; carboxylic and organic acid metabolism; metal-ion binding; and ion homeostasis. In the triple-knockout compared with the wild-type mice, response to zymosan-induced peritonitis was strikingly exaggerated, which may well reflect down-regulation of Socs2 expression. If a single common molecular pathway is responsible for all of these phenotypes, we suggest that functional effects of the loss of all three Cyp1 genes could be explained by perturbations in CYP1-mediated eicosanoid production, catabolism and activities.

Lycoris aurea (L' Hér.) Herb, a perennial grass species, produces a unique variety of pharmacologically active Amaryllidaceae alkaloids. However, the key enzymes and their expression pattern involved in the biosynthesis of Amaryllidaceae alkaloids (especially for galanthamine) are far from being fully understood. Quantitative real-time polymerase chain reaction (qRT-PCR), a commonly used method for quantifying gene expression, requires stable reference genes to normalize its data. In this study, to choose the appropriate reference genes under different experimental conditions, 14 genes including YLS8 (mitosis protein YLS8), CYP2 (Cyclophilin 2), CYP 1 (Cyclophilin 1), TIP41 (TIP41-like protein), EXP2 (Expressed protein 2), PTBP1 (Polypyrimidine tract-binding protein 1), EXP1 (Expressed protein 1), PP2A (Serine/threonine-protein phosphatase 2A), β-TUB (β-tubulin), α-TUB (α-tubulin), EF1-α (Elongation factor 1-α), UBC (Ubiquitin-conjugating enzyme), ACT (Actin) and GAPDH (Glyceraldehyde 3-phosphate dehydrogenase) were selected from the transcriptome datasets of L. aurea. And then, expressions of these genes were assessed by qRT-PCR in various tissues and the roots under different treatments. The expression stability of the 14 candidates was analyzed by three commonly used software programs (geNorm, NormFinder, and BestKeeper), and their results were further integrated into a comprehensive ranking based on the geometric mean. The results show the relatively stable genes for each subset as follows: (1) EXP1 and TIP41 for all samples; (2) UBC and EXP1 for NaCl stress; (3) PTBP1 and EXP1 for heat stress, polyethylene glycol (PEG) stress and ABA treatment; (4) UBC and CYP2 for cold stress; (5) PTBP1 and PP2A for sodium nitroprusside (SNP) treatment; (6) CYP1 and TIP41 for methyl jasmonate (MeJA) treatment; and (7) EXP1 and TIP41 for various tissues. The reliability of these results was further enhanced through comparison between part qRT-PCR result and RNA sequencing (RNA-seq) data. In summary, our results identified appropriate reference genes for qRT-PCR in L. aurea, and will facilitate gene expression studies under these conditions.

Two novel CYP1 genes from teleost fish constituting a new subfamily have been cloned. These paralogous sequences are designated CYP1C1 and CYP1C2. Both genes were initially obtained from untreated scup Stenotomus chrysops tissues by RT-PCR and RACE. Scup CYP1C1 and CYP1C2 code for 524 and 525 amino acids, respectively, and share 80-81% identity at the nucleotide and amino acid levels. Orthologues of CYP1C1 and CYP1C2 were identified in genome databases for other fish species, and both CYP1B1 and CYP1C1 were cloned from zebrafish (Danio rerio). Phylogenetic analysis shows that CYP1Cs and CYP1Bs constitute a sister clade to the CYP1As. Analysis of sequence domains likely to have functional significance suggests that the two CYP1Cs in scup may have catalytic functions and/or substrate specificity that differ from each other and from those of mammalian CYP1Bs or CYP1As. RT-PCR results indicate that CYP1C1 and CYP1C2 are variously expressed in several scup organs.

Cytochrome P450 CYP1B1 is a recently identified member of the CYP1 P450 family. We have shown that this P450 displays increased expression in several types of human cancer, indicating that CYP1B1 is a potential tumor biomarker. In this study we developed monoclonal antibodies (MAbs) to CYP1B1 that are effective on formalin-fixed, paraffin-embedded tissue sections and investigated the presence of CYP1B1 in a series of primary breast cancers. The MAbs were generated using a synthetic peptide coupled to carrier protein as the immunogen. The MAbs specifically recognized CYP1B1 and did not recognize either CYP1A1 or CYP1A2, related CYP1 forms. The MAbs were tested by immunohistochemistry and were found to be effective on formalin-fixed, paraffin-embedded tissue sections. The majority of breast cancers showed positive immunoreactivity for CYP1B1, and in each case CYP1B1 was specifically localized to tumor cells. The presence of CYP1B1 in breast cancer cells is likely to contribute to their metabolism of estradiol because CYP1B1 is a specific estradiol hydroxylase. (J Histochem Cytochem 47:1457-1464, 1999)

Expression of human cytochrome P450 (CYP) genes in human adult and fetal liver were studied using the reverse transcriptase-polymerase chain reaction (RT-PCR) method. In adult liver mRNA of CYPs 1A1, 1A2, 2A6/2A7, 2B6/2B7, 2C8-19, 2D6, 2E1, 3A3/3A4 and 3A7 were detected while CYPs 2F1 and 4B1 were absent. In fetal liver mRNA of CYPs 2C8, 2D6, 3A3/3A4 and 3A7 were found but all other forms studied were undetectable. The results provide a comprehensive qualitative picture of the expression of CYP genes in families CYP1 through CYP4 in human adult and fetal liver.

Smoking is a major risk factor for osteoporosis and fracture, but the mechanism through which smoke causes bone loss remains unclear. Here, we show that the smoke toxins benzo(a)pyrene (BaP) and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) interact with the aryl hydrocarbon receptor (Ahr) to induce osteoclastic bone resorption through the activation of cytochrome P450 1a/1b (Cyp1) enzymes. BaP and TCDD enhanced osteoclast formation in bone marrow cell cultures and gavage with BaP stimulated bone resorption and osteoclastogenesis in vivo. The osteoclastogenesis triggered by BaP or RANK-L was reduced in Ahr(-/-) cells, consistent with the high bone mass noted in Ahr(-/-) male mice. The receptor activator of NF-κB ligand (RANK-L) also failed to induce the expression of Cyp1 enzymes in Ahr(-/-) cells. Furthermore, the osteoclastogenesis induced by TCDD was lower in Cyp1a1/1a2(-/-) and Cyp1a1/1a2/1b1(-/-) cultures, indicating that Ahr was upstream of the Cyp enzymes. Likewise, the pharmacological inhibition of the Cyp1 enzymes with tetramethylsilane or proadifen reduced osteoclastogenesis. Finally, deletion of the Cyp1a1, Cyp1a2, and Cyp1b1 in triple knockout mice resulted in reduced bone resorption and recapitulated the high bone mass phenotype of Ahr(-/-) mice. Overall, the data identify the Ahr and Cyp1 enzymes not only in the pathophysiology of smoke-induced osteoporosis, but also as potential targets for selective modulation by new therapeutics.

The cytochromes P450 are a large family of haemoproteins which have a major role in the oxidative metabolism of a wide range of xenobiotics and some endogenous compounds. In this study the presence of individual members of the CYP1, CYP2 and CYP3 P450 families has been investigated by reverse transcriptase polymerase chain reaction in different regions of normal human brain consisting of frontal and temporal cortices, mid brain, cerebellum, pons and medulla. All the P450s were identified in specific regions of brain with CYP1A1 and CYP2C being the most frequently expressed forms of P450. Sequencing identified the CYP2C PCR product as CYP2C8. This study indicates that individual P450 mRNAs are present in human brain and are found in specific brain regions. The distribution of individual P450s in different regions of human brain is likely to be highly important in determining the response of the brain to toxic foreign compounds.

Estrogen and its metabolites are believed to play important roles in breast cancer. The influence of genetic polymorphisms in the enzymes responsible for formation and disposition of estrogen on breast cancer risk may shed light on the importance of estrogen metabolites in this disease. However, for such studies to be valid, it is important to correctly identify the enzymes involved in estrogen bioactivation. Therefore, we assessed the human cytochrome P450-dependent oxidation of estrone using substrate concentrations that more closely approximate the maximum expected concentrations in breast tissue. The in vitro metabolism of estrone by recombinant human cytochrome P450 enzymes and human liver microsomes was studied. The formation of estrone metabolites (2-hydroxyestrone, 4-hydroxyestrone, and 16alpha-hydroxyestrone) was monitored by high-performance liquid chromatography. 2-Hydroxyestrone formation was catalyzed predominantly by CYP1A2, CYP1A1, and CYP1B1 enzymes; 4-hydroxyestrone formation was catalyzed predominantly by CYP1B1, CYP1A2, and CYP1A1 enzymes; and 16alpha-hydroxyestrone formation was catalyzed predominantly by CYP2C19, CYP1A1, and CYP3A5. This study confirms the important role of members of the CYP1 family in the 2-hydroxylation and 4-hydroxylation of estrone, but the enzymes identified as responsible for the 16alpha-hydroxylation of estrone are different from those previously identified. The relative importance of these enzymes in vivo would depend on the specific tissue expression of the enzymes. These enzymes are all known to be genetically variant in the human population, and additional studies to assess the role CYP1A2, CYP2C19, and CYP3A5 in breast cancer risk are indicated.

The CYP1A1, CYP1A2, and CYP1B1 enzymes are inducible by benzo[a]pyrene (BaP) and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD); metabolism of BaP by these enzymes leads to electrophilic intermediates and genotoxicity. Throughout the gastrointestinal (GI) tract, we systematically compared basal and inducible levels of the CYP1 mRNAs by Q-PCR, and localized the CYP1 proteins by immunohistochemistry. Cyp1(+/+) wild-type were compared with the Cyp1a1(-/-), Cyp1a2(-/-), and Cyp1b1(-/-) single-knockout and Cyp1a1/1b1(-/-) and Cyp1a2/1b1(-/-) double-knockout mice. Oral BaP was compared with intraperitoneal TCDD. In general, maximal CYP1A1 mRNA levels were 3-10 times greater than CYP1B1, which were 3-10 times greater than CYP1A2 mRNA levels. Highest inducible concentrations of CYP1A1 and CYP1A2 occurred in proximal small intestine, whereas the highest basal and inducible levels of CYP1B1 mRNA occurred in esophagus, forestomach, and glandular stomach. Ablation of either Cyp1a2 or Cyp1b1 gene resulted in a compensatory increase in CYP1A1 mRNA - but only in small intestine. Also in small intestine, although BaP- and TCDD-mediated CYP1A1 inductions were roughly equivalent, oral BaP-mediated CYP1A2 mRNA induction was approximately 40-fold greater than TCDD-mediated CYP1A2 induction. CYP1B1 induction by TCDD in Cyp1(+/+) and Cyp1a2(-/-) mice was 4-5 times higher than that by BaP; however, in Cyp1a1(-/-) animals CYP1B1 induction by TCDD or BaP was approximately equivalent. CYP1A1 and CYP1A2 proteins were generally localized nearer to the lumen than CYP1B1 proteins, in both squamous and glandular epithelial cells. These GI tract data suggest that the inducible CYP1A1 enzyme, both in concentration and in location, might act as a "shield" in detoxifying oral BaP and, hence, protecting the animal.

Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous environmental contaminants often present in aquatic systems as complex mixtures. Embryonic fish are sensitive to the developmental toxicity of some PAHs, but the exact mechanisms involved in this toxicity are still unknown. This study explored the role of the aryl hydrocarbon receptor (AHR) in the oxidative stress response of zebrafish to the embryotoxicity of select PAHs. Embryos were exposed to two PAHs, benzo[k]fluoranthene (BkF; a strong AHR agonist) and fluoranthene (FL; a cytochrome P4501A (CYP1A) inhibitor), alone and in combination. CYP1A, CYP1B1, CYP1C1, and redox-responsive genes glutathione s-transferase pi 2 (GSTp2), glutathione peroxidase 1 (GPx1), the glutamate-cysteine ligase catalytic subunit (GCLc), MnSOD and CuZnSOD mRNA expression was examined. CYP1 activity was measured via an in vivo ethoxyresorufin-O-deethlyase (EROD) activity assay, and the area of the pericardium was measured as an index of cardiotoxicity. BkF or FL alone caused no deformities whereas BkF+FL resulted in extreme pericardial effusion. BkF induced CYP activity above controls and co-exposure with FL inhibited this activity. BkF induced expression of all three CYPs, GSTp2, and GCLc. BkF+FL caused greater than additive induction of the three CYPs, GSTp2, GPx1, and GCLc but had no effect on MnSOD or CuZnSOD. AHR2 knockdown protected against the cardiac deformities caused by BkF+FL and significantly inhibited the induction of the CYPs, GSTp2, GPx1, and GCLc after BkF+FL compared to non-injected controls. These results further show the protective role of AHR2 knockdown against cardiotoxic PAHs and the role of AHR2 as a mediator of redox-responsive gene induction.

CYP1C is the newest member of the CYP1 family of P450s; however, its physiological significance, inducers, and metabolic functions are unknown. Two full-length alleles of Fundulus heteroclitus CYP1C1 complementary DNA were cloned. The 529 amino acid protein shared the highest amino acid identity with Stenotomus chrysops CYP1C1 (81%). To investigate whether the carcinogen benzo[a]pyrene (BaP) was a CYP1C1 inducer, we used real-time PCR to quantitatively measure tissue- and sex-specific expression of both CYP1C1 and CYP1A messenger RNAs (mRNAs) in BaP-exposed adult fish. CYP1C1 mRNA expression was constitutively higher than CYP1A in brain, spleen, eye, and gonad, while CYP1A was higher in gastrointestinal tract (GI), heart, gill, and liver. Kidney had equal but high expression of both CYP1s. There were sex differences in constitutive CYP1 expression in the GI, liver, gill, and eye. BaP exposure caused induction of CYP1C1 expression in female and male heart (31- and 17-fold), gill (seven- and four-fold), and liver (six- and five-fold), respectively. Embryo CYP1 expression was constitutively highest at 2 weeks posthatch, and whole embryos expressed 3- to 15-fold more CYP1C1 mRNA compared to CYP1A. BaP, 10 microg/l for 10 days, caused induction of both genes at 120 and 240 h postfertilization. Our results suggest that teleost CYP1C, in addition to CYP1A, is inducible by BaP, has a broad tissue distribution, and should be further investigated for its role in carcinogen bioactivation.

Five chromosomal genes, CYPI to CYP5 involved in the regulation of the synthesis of iso-1-cytochrome c, iso-2-cytochrome c and cytochrome b2 are described. The function of these genes was studied either by varying the proportion of the mutated and wild type alleles in the cell vy varing the growth conditions, or else by transforming the mutants into sigma-cytoplasmic petites. We have shown a network of genetic interactions which regulate the synthesis of three structurally different proteins : iso-1-cytochrome c, iso-2-cytochrome c and cytochrome b2, by two unlinked genes : CYC1 and CYP1, one of which (CYC1) is the structural gene by iso-1-cytochrome c. Within this network the interactions are proportional to the gene dosage and are either antagonistic or synergistic depending on the allele combination and the protein studied. The mutated alleles cyp1 stimulate the synthesis of iso-2-cytochrome c, inhibit the synthesis of iso-1-cytochrome c, while the cytochrome b2 synthesis is also inhibited but by a combination of cyp1 mutated alleles CYC1 wild type allele. Other loci, CYP2, CYP3, CYP4 and CYP5 were also studied in various allelic combinations. They show some interactions between them or with CYC1 locus but these interactions are different and less pronounced than those involving loci CYP1 and CYC1.

Photooxidized tryptophan (TRP) in tissue culture medium elicits a transient cytochrome P450 (CYP1) induction response in cultured cells. We show here that exposure of TRP to window sunlight (aTRP) greatly increased the potency, efficacy, and duration of CYP1A induction by TRP in primary chick embryo hepatocytes and in vivo. Aqueous TRP exposed to sunlight for 7 days exhibited a 100-fold or greater increase in potency over TRP in medium. The induction response was sustained for at least 48 h and was comparable in efficacy to 2,3,7,8-tetrachlorodibenzo-p-dioxin. In hepatocytes, increases in mRNAs for CYP1A4 and CYP1A5, chick orthologs of mammalian CYP1A1 and 1A2, preceded increases in CYP1A proteins and enzyme activities, 7-ethoxyresorufin deethylase (EROD) for CYP1A4 and arachidonic acid epoxygenation for CYP1A5, consistent with a transcriptional mechanism. Aryl hydrocarbon receptor (AhR) dependence was evidenced by aTRP induction of EROD in wild-type Hepa1c1c7 cells but not in AhR-defective (c35) mutants. Preparations of aTRP were stable for many months at 4 degrees C and were relatively resistant to metabolism by hepatocytes or liver microsomes. Fractionation of aTRP by HPLC analysis coupled with EROD assays showed that aTRP contained multiple photoproducts and CYP1A inducing components, which varied in sensitivity to metabolism by hepatocytes. The previously identified TRP photoproduct, 6-formylindolo[3,2-b]carbazole (FICZ), was one component, but FICZ was not required for CYP1A induction by the aTRP mixture. These findings identify the indoor environment, and window sunlight in particular, as a new source of CYP1A inducers. Further, the evidence that biologically active metabolites of an endogenous substrate, arachidonic acid, are formed by aTRP-induced CYP1A provides a pathway by which TRP photoproducts, like toxic xenobiotics, could have significant physiologic effects.

BACKGROUND

The natural product eupatorin has been reported to have antiproliferative activity in tumour cell lines, but the exact mechanism is unclear. The cytochromes P450 CYP1B1, CYP1A1, and CYP1A2 have been shown to participate in the activation of various xenobiotics, compounds derived from the diet as well as chemotherapeutic drugs. CYP1B1 and CYP1A1 have also been proposed as targets for cancer chemotherapy for their differential and selective overexpression in tumour cells. In this study, we aimed to identify a possible mechanism of action for the antiproliferative effect of eupatorin, which can be attributed to CYP1 family-mediated metabolism.

METHODS

The study focuses on the antiproliferative action of eupatorin on the human breast carcinoma cell line MDA-MB-468 and on a cell line derived from normal mammary tissue, MCF-10A. The cytotoxicity of the flavone, its effect on the cell cycle of the abovementioned cell lines, and its metabolism by CYP1 family enzymes were examined.

RESULTS

Eupatorin showed a dose-dependent inhibitory effect of cell growth on MDA-MB-468 cells with a submicromolar median inhibition concentration (IC50) whereas the IC50 of this compound in MCF-10A cells was considerably higher. The antiproliferative effect, as measured by EROD (ethoxyresorufin-O-deethylase) assay and Western immunoblotting, was attributed mainly to CYP1A1 expression in MDA-MB-468 cells but not in MCF-10A cells. Moreover, CYP1 family enzymes were shown to metabolise eupatorin in vitro to the flavone cirsiliol and two other unidentified metabolites. Metabolism of eupatorin was also detected in MDA-MB-468 cell cultures, whereas metabolism by MCF-10A cells was negligible. Eupatorin was further shown to arrest the cell cycle of the CYP1-expressing cell line MDA-MB-468 in G2/M phase, whereas no effect was observed in MCF-10A cells, which do not express CYP1 enzymes. The effect of eupatorin on the MDA-MB-468 cell cycle could be reversed by co-application of the CYP1 inhibitor acacetin.

CONCLUSIONS

The flavone eupatorin is selectively activated in breast cancer cells, but not in normal breast cells, due to CYP1 family metabolism. This provides a basis for selectivity which is desired against breast tumour cells. In this sense, eupatorin is shown by this study to be a very promising chemopreventative candidate that should be examined further in an in vivo study.

The aryl hydrocarbon receptor (AhR) is a ligand-activated transcriptional factor that dimerizes with aryl hydrocarbon receptor nuclear translocator (ARNT). This complex binds to xenobiotics response element (XREs), and then starts the expressions of downstream genes including cytochrome P450 (CYP) 1 family members: CYP1A1, CYP1A2 and CYP1B1. Role of CYP1 family is involved in the metabolism of endogenous hormones, xenobiotics and drug. The expression of CYP1 family is regulated by estradiol (E2) or xenobiotics in diverse cancers. In breast cancers expressing estrogen receptors (ERs), level of CYP1B1 is increased by E2 and reversed by an estrogen receptor antagonist, ICI 182,780 or 4-hydrotamoxifen, which indicates that the expression of CYP1 family in downstream region of AhR is regulated by an activation of ERα. In metabolic pathways, E2 is converted into 4-hydroxyestradiol by CYP1B1, which can be converted into mainly estradiol-3,4-quinone, a potential carcinogen, by peroxidase. Increased expression of CYP1 family indicates the possibility of carcinogenesis by exposure of xenobiotics in endometrial and ovarian cancers. Apart from roles of CYP1 family in relation with ER pathway, CYP1 family is over-expressed in ER independent cancers. CYP1A1 exhibits hydroxylase activity in oxidation of arachidonic acid, which has been transformed to 12(R)-hydrxyeicosatetraenoic (HETEs), a potent activator of AhR activity. On the basis of results, phytoestrogens and dexamethasone are provided as cancer therapy regulating the expression of CYP1 family. Thus, this review focuses on the role(s) of CYP1 family in ER-dependent or ER-independent cancers and the potential for cancer therapy to target CYP1 family in these cancers.