Generation of 'humanized' hCYP1A1_1A2_Cyp1a1/1a2(-/-) mouse line

Human/rodent CYP1A1 and CYP1A2 orthologs are well known to exhibit species-specific differences in substrate preferences and rates of metabolism. This lab previously characterized a BAC-transgenic mouse carrying the human CYP1A1_CYP1A2 locus; in this line, human dioxin-inducible CYP1A1 and basal vs dioxin-inducible CYP1A2 have been shown to be expressed normally (with regard to mRNAs, proteins and three enzyme activities) in every one of nine mouse tissues studied. The mouse Cyp1a1 and Cyp1a2 genes are oriented head-to-head and share a bidirectional promoter region of 13,954 bp. Using Cre recombinase and loxP sites inserted 3' of the stop codons of both genes, we show here a successful interchromosomal excision of 26,173 bp that ablated both genes on the same allele. The Cyp1a1/1a2(-) double-knockout allele was bred with the "humanized" line; the final product is the hCYP1A1_1A2_Cyp1a1/1a2(-/-) line on a theoretically >99.8% C57BL/6J genetic background-having both human genes replacing the mouse orthologs. This line will be valuable for human risk assessment studies involving any environmental toxicant or drug that is a substrate for CYP1A1 or CYP1A2.     

Cytochrome P450 1A1 (CYP1A1) Catalyzes Lipid Peroxidation of Oleic Acid-Induced HepG2 Cells

Nonalcoholic fatty liver disease (NAFLD) is a chronic hepatic disease associated with excessive accumulation of lipids in hepatocytes. As the disease progresses, oxidative stress plays a pivotal role in the development of hepatic lipid peroxidation. Cytochrome P450 1A1 (CYP1A1), a subtype of the cytochrome P450 family, has been shown to be a vital modulator in production of reactive oxygen species. However, the exact role of CYP1A1 in NAFLD is still unclear. The aim of this study was to investigate the effects of CYP1A1 on lipid peroxidation in oleic acid (OA)-treated human hepatoma cells (HepG2). We found that the expression of CYP1A1 is elevated in OA-stimulated HepG2 cells. The results of siRNA transfection analysis indicated that CYP1A1-siRNA inhibited the lipid peroxidation in OA-treated HepG2 cells. Additionally, compared with siRNA-transfected and benzo[a]pyrene (BaP)-OA-induced HepG2 cells, overexpression of CYP1A1 by BaP further accelerated the lipid peroxidation in OA-treated HepG2 cells. These observations reveal a regulatory role of CYP1A1 in liver lipid peroxidation and imply CYP1A1 as a potential therapeutic target.     

Genotype frequencies of polymorphic GSTM1, GSTT1, and cytochrome P450 CYP1A1 in Mexicans

The genotype frequencies of three metabolic polymorphisms were determined in a sample of a typical community in central Mexico. CYP1A1*3, GSTM1, and GSTT1 polymorphisms were studied in 150 donors born in Mexico and with Mexican ascendants; with respect to ethnicity the subjects can be considered Mestizos. PCR reactions were used to amplify specific fragments of the selected genes from genomic DNA. An unexpected 56.7% frequency of the CYP1A1*3 allele (which depends on the presence of a Val residue in the 462 position of the enzyme, instead of Ile) was found, the highest described for open populations of different ethnic origins (i.e., Caucasian, Asian, African, or African American). The GSTM1 null genotype was found with a frequency of 42.6%, which is not different from other ethnicities, whereas the GSTT1 null genotype had a frequency of 9.3%, one of the lowest described for any ethnic group but comparable to the frequency found in India (9.7%). The frequency of the combined genotype CYP1A1*3/*3 and the GSTM1 null allele is one of the highest observed to date (or perhaps the highest): 13.7% among all the ethnicities studied, including Caucasians and Asians, whereas the combination of CYP1A1*3/*3 with the GSTT1 null allele reached only 2.8%. The GSTM1 null allele combined with the GSTT1 null allele, on the other hand, has one of the lowest frequencies described, 4.24%, comparable to the frequencies found in African Americans and Indians. Finally, the combined CYP1A1*3/*3, GSTM1 null allele, and GSTT1 null allele genotype could not be found in the sample studied; it is assumed that the frequency of carriers of these combined genotypes is less than 1%. CYP1A1*3 and CYP1A1*2 polymorphisms were also evaluated in 50 residents in a community of northern Mexico; the CYP1A1*3 frequency was 54%, similar to that found in the other community studied, and the CYP1A1*2 frequency was 40%, which is high compared to Caucasians and Asians but comparable to the frequency found in Japanese and lower than the frequency found in Mapuche Indians. Haplotype frequencies for these CYP1A1 polymorphisms were estimated, and a linkage disequilibrium value (D) of 0.137 was calculated.     

Induction of Human UDP-glucuronosyltransferase 1A1 by Cortisol-GR

During the course of the study of UGT1A1 induction by bilirubin, we could not detect the induction of the reporter gene (−3174/+14) of human UGT1A1 in HepG2 by bilirubin (Mol. Biol. Rep. 31: 151–158 (2004)). In this report, we show the finding of the induction of the reporter gene of UGT1A1 by cortisol at 1 μM, a major natural cortico-steroid, with human glucocorticoid receptor (GR). RU486 of a typical GR antagonist at 10 μM inhibited the induction by cortisol from 5.9- to 1.8-fold. This result indicates that the induction by cortisol-GR is dependence on ligand-binding. This induction is caused by the UGT reporter gene itself, from the results of noinduction with control vector pGL2 (equal to pGV-C) in the presence of cortisol-GR. We confirmed that the induction of the reporter gene by cortisol is dependent on the position of proximal element (−97/−53) of UGT1A1. From this result, we concluded that the increase of corticosteroid in neonates must induce the elevation of UGT1A1 after birth and prevent jaundice. With the study of induction by corisol, we studied the influence of co-expression of PXR (pregnenolone xenobiotic receptor) with the UGT1A1 reporter gene and we could not find the induction of UGT1A1 expression in the presence of dexamethasone, rifampicin, or pregnenolone 16α-carbonitrile of the PXR ligands. These results suggest that the induction of UGT1A1 expression by GR is not mediated by PXR, unlike the induction of CYP3A4 through PXR.     

Antagonism between Cry1Ac1 and Cyt1A1 toxins of bacillus thuringiensis

Most strains of the insecticidal bacterium Bacillus thuringiensis have a combination of different protoxins in their parasporal crystals. Some of the combinations clearly interact synergistically, like the toxins present in B. thuringiensis subsp. israelensis. In this paper we describe a novel joint activity of toxins from different strains of B. thuringiensis. In vitro bioassays in which we used pure, trypsin-activated Cry1Ac1 proteins from B. thuringiensis subsp. kurstaki, Cyt1A1 from B. thuringiensis subsp. israelensis, and Trichoplusia ni BTI-Tn5B1-4 cells revealed contrasting susceptibility characteristics. The 50% lethal concentrations (LC50s) were estimated to be 4,967 of Cry1Ac1 per ml of medium and 11.69 ng of Cyt1A1 per ml of medium. When mixtures of these toxins in different proportions were assayed, eight different LC50s were obtained. All of these LC50s were significantly higher than the expected LC50s of the mixtures. In addition, a series of bioassays were performed with late first-instar larvae of the cabbage looper and pure Cry1Ac1 and Cyt1A1 crystals, as well as two different combinations of the two toxins. The estimated mean LC50 of Cry1Ac1 was 2.46 ng/cm2 of diet, while Cyt1A1 crystals exhibited no toxicity, even at very high concentrations. The estimated mean LC50s of Cry1Ac1 crystals were 15.69 and 19.05 ng per cm2 of diet when these crystals were mixed with 100 and 1,000 ng of Cyt1A1 crystals per cm2 of diet, respectively. These results indicate that there is clear antagonism between the two toxins both in vitro and in vivo. Other joint-action analyses corroborated these results. Although this is the second report of antagonism between B. thuringiensis toxins, our evidence is the first evidence of antagonism between toxins from different subspecies of B. thuringiensis (B. thuringiensis subsp. kurstaki and B. thuringiensis subsp. israelensis) detected both in vivo and in vitro. Some possible explanations for this relationship are discussed.     

Secretoglobin 1A1 and 1A1A Differentially Regulate Neutrophil Reactive Oxygen Species Production,Phagocytosis and Extracellular Trap Formation

Secretoglobin family 1A member 1 (SCGB 1A1) is a small protein mainly secreted by mucosal epithelial cells of the lungs and uterus. SCGB 1A1, also known as club (Clara) cell secretory protein, represents a major constituent of airway surface fluid. The protein has anti-inflammatory properties, and its concentration is reduced in equine recurrent airway obstruction (RAO) and human asthma. RAO is characterized by reversible airway obstruction, bronchoconstriction and neutrophilic inflammation. Direct effects of SCGB 1A1 on neutrophil functions are unknown. We have recently identified that the SCGB1A1 gene is triplicated in equids and gives rise to two distinct proteins. In this study we produced the endogenously expressed forms of SCGBs (SCGB 1A1 and 1A1A) as recombinant proteins, and analyzed their effects on reactive oxygen species production, phagocytosis, chemotaxis and neutrophil extracellular trap (NET) formation ex vivo. We further evaluated whether NETs are present in vivo in control and inflamed lungs. Our data show that SCGB 1A1A but not SCGB 1A1 increase neutrophil oxidative burst and phagocytosis; and that both proteins markedly reduce neutrophil chemotaxis. SCGB 1A1A reduced chemotaxis significantly more than SCGB 1A1. NET formation was significantly reduced in a time- and concentration-dependent manner by SCGB 1A1 and 1A1A. SCGB mRNA in bronchial biopsies, and protein concentration in bronchoalveolar lavage fluid, was lower in horses with RAO. NETs were present in bronchoalveolar lavage fluid from horses with exacerbated RAO, but not in fluid from horses with RAO in remission or in challenged healthy horses. These findings indicate that SCGB 1A1 and 1A1A have overlapping and diverging functions. Considering disparities in the relative abundance of SCGB 1A1 and 1A1A in airway secretions of animals with RAO suggests that these functional differences may contribute to the pathogenesis of RAO and other neutrophilic inflammatory lung diseases.     

Autocrine activation of adenosine A1 receptors blocks D1A but not D1B dopamine receptor desensitization

Adenosine is known to modulate dopamine responses in several brain areas. Here, we show that tonic activation of adenosine receptors is able to impede desensitization of D1 dopamine receptors. As measured by cAMP accumulation in transfected COS-7 cells, long-term exposure to dopamine agonists promoted desensitization of D1B receptor but not that of D1A receptor. The inability of D1A receptor to desensitize was a result of the adenosine present in culture medium acting through activation of adenosine A1 receptors. Cell incubation with either adenosine deaminase, CGS-15943, a generic adenosine receptor antagonist, or the A1 antagonist DPCPX restored the long-term desensitization time-course of D1A receptors. In Ltk cells stably expressing A1 adenosine receptors and D1A dopamine receptors, pre-treatment of cells with R(-)-PIA, a full A1 receptor agonist, did not significantly inhibit the acute increase in cAMP levels induced by D1 receptor agonists, but blocked desensitization of D1A receptors. However, simultaneous activation of A1 and D1A receptors promoted a delayed D1A receptor desensitization. This suggests that functional interaction between A1 and D1A receptors may depend on the activation kinetics of components regulating D1 receptor responses, acting differentially on D1A and D1B receptors.     

Establishment of Salmonella strain expressing catalytically active human UDP-glucuronosyltransferase 1A1 (UGT1A1)

Human uridinediphosphate-glucuronosyltransferase 1A1 (UGT1A1) was expressed in Salmonella typhimurium TA1535 cells by transfection of the cells with plasmids carrying the UGT1A1 cDNA. UGT1A1 cDNA was isolated by a polymerase chain reaction from human liver total RNA and was inserted into the pSE420 plasmid, linked to the trc promoter and terminator. The plasmid thus constructed was introduced into Salmonella TA1535 cells. The expression of human UGT1A1 protein was confirmed by Western blot analysis. The maximal expression was observed at 24 h after the addition of isopropyl-beta-D-thiogalactopyranoside, an inducer. However, the bilirubin conjugation activity of the membrane fraction from the Salmonella cells was not detectable. When a beta-glucuronidase inhibitor such as saccharic acid 1,4-lactone, glycyrrhizin or 1-naphtyl-beta-D-glucuronide was added to the reaction mixture, the bilirubin conjugation activity of the human UGT1A1 was detected. When geniposide was added to the reaction mixture, the bilirubin conjugation activity of UGT1A1 was not seen. Taking these results into account, the established Salmonella strain possesses the beta-glucuronidase activity. Since the beta-glucuronidase activity of the Salmonella was lower than that of E. coli, it was concluded that Salmonella seemed to be a good host to express UGT protein. This is the first study to demonstrate the establishment of a bacterial strain expressing native human UGT protein showing catalytic activity.     

细胞色素P450 1A1和1B1靶向抗肿瘤前药研究进展

细胞色素P450（CYP）能催化各种内源性及外源性化合物的代谢,与多种肿瘤发生有关。其中CYP1A1参与多种前致癌物和致突变物的代谢活化,CYP1B1被认为在许多人癌细胞中特异性表达,参与药物的氧化代谢和前药的活化。CYP1A1和181已成为靶向抗肿瘤前药研究的新靶点。相继有大量相关研究报道,本文就近年来文献报道的CYP1A1和1B1靶向抗肿瘤前药研究进展。     

Characterization of human CYP1A1/1A2 induction by DNA microarray and alpha-naphthoflavone

DNA microarrays and real time PCR were used to analyze the mechanism of gene induction by CYP1A1 inducers, beta-naphthoflavone, and omeprazole, in the human hepatocellular carcinoma HepG2 cells. Reproducible and significant inductions were observed in a limited number of genes including CYP1A1 and CYP1A2. Genes induced by omeprazole included several protein tyrosine kinase targets. This result confirmed that omeprazole could modulate gene expressions through protein tyrosine kinase-mediated pathway. Induction ratios were considerably different from CYP1A1 and CYP1A2 (>10-fold) to other induced genes (<5-fold). alpha-Naphthoflavone, which is known as an antagonist to 2,3,7,8-tetrachlorodibenzo-p-dioxin, inhibited the inductions of heme oxygenase 1, glutamate-cysteine ligase (modifier unit), and thioredoxin reductase by beta-naphthoflavone but not those of CYP1A1 and CYP1A2. It unexpectedly enhanced the beta-naphthoflavone-mediated CYP1A1 and CYP1A2 induction. These results suggest that the CYP1A1 and CYP1A2 genes, which share their 5(') enhancer regions, are regulated differently from the other genes.     

Human cytosolic sulfotransferase SULT1A1

Sulfonation is an important conjugation reaction required for a range of biological processes including phase II metabolism, whereby sulfo-conjugation renders a compound more hydrophilic to aid its excretion. The major enzyme responsible for xenobiotic sulfonation is the widely expressed cytosolic sulfotransferase SULT1A1. The SULT1A1 crystal structure has provided insights into this enzyme's substrate specificity and catalytic function, including its role in the sulfonation of endogenous substrates such as oestrogens. Contrary to its metabolic role, SULT1A1 can also bioactivate compounds; it is known to sulfonate pro-carcinogens such as hydroxymethyl polycyclic aromatic hydrocarbons leading to highly reactive intermediates capable of forming DNA adducts, potentially resulting in mutagenesis. Given the role of SULT1A1 in these diverse functions and the discovery of allelic variants with differing catalytic activities, this enzyme has been the focus of numerous polymorphic studies investigating the link between inter-individual SULT1A1 variance and the etiology of a variety of cancers.     

Rap1A regulates generation of T regulatory cells via LFA-1-dependent and LFA-1-independent mechanisms

The small GTPase Rap1A has a critical role in regulating cell-matrix and cell-cell adhesion. In T lymphocytes, Rap1A mediates LFA-1 activation and LFA-1-mediated adhesion. LFA-1 reduces the threshold of TCR signals for low affinity ligands. Previously, we determined that mice expressing constitutively active Rap1A on T cells have increased frequency of CD103⁺ T regulatory cells (Treg). We hypothesized that Rap1A-GTP might affect the differentiation of Treg by regulating LFA-1 activation. Using Foxp3-GFP-KI, LFA-1-KO and Rap1A-GTP-Tg mice we determined that Rap1A has an active role in the development of thymic Treg but LFA-1 is not mandatory for this function. Rap1A is also involved in the generation of peripheral Treg and this effect is mediated via LFA-1-dependent and LFA-1-independent mechanisms. Identification of the signaling pathways via which Rap1-GTP contributes to the differentiation of Treg will provide new insights to the function of Rap1A and to designing targeted approaches for generation of Treg for therapeutic applications.     

cDNA cloning and characterization of feline CYP1A1 and CYP1A2

Deficiency of drug glucuronidation in the cat is one of the major reasons why this animal is highly sensitive to the side effects of drugs. The characterization of cytochrome P450 isoforms belonging to the CYP1A subfamily, which exhibit important drug oxidation activities such as activation of pro-carcinogens, was investigated. Two cDNAs, designated CYP1A-a and CYP1A-b, corresponding to the CYP1A subfamily were obtained from feline liver. CYP1A-a and CYP1A-b cDNAs comprise coding regions of 1554 bp and 1539 bp, and encode predicted amino acid sequences of 517 and 512 residues, respectively. These amino acid sequences contain a heme-binding cysteine and a conserved threonine. The cDNA identities, as well as the predicted amino acid sequences containing six substrate recognition sites, suggest that CYP1A-a and CYP1A-b correspond to CYP1A1 and CYP1A2, respectively. This was confirmed by the kinetic parameters of the arylhydrocarbon hydroxylase and 7-ethoxyresorufin O-deethylase activities of expressed CYPs in yeast AH22 cells and by the tissue distribution of each mRNA. However, theophylline 3-demethylation is believed to be catalyzed by CYP1A1 in cats, based on the high V(max) and low K(m) seen, in contrast to other animals. Because feline CYP1A2 had a higher K(m) for phenacetin O-deethylase activity with acetaminophen, which cannot be conjugated with glucuronic acid due to UDP-glucuronosyltransferase deficiency, it is supposed that the side effects of phenacetin as a result of toxic intermediates are severe and prolonged in cats.     

Stimulation of transcriptional expression of human UDP-glucuronosyltransferase 1A1 by dexamethasone

Human UDP-glucuronosyltransferase (UGT) 1A1 is only enzyme in the conjugation of bilirubin for prevention of hyperbilirubinemia and jaundice. Deletion or mutation of the UGT1A1 gene causes Crigler-Najjar syndrome or Gilbert's syndrome. We previously reported the functional promoter region for expression of UGT1A1 [Hepatology Research 9, 152-163 (1997)]. We investigated the influence of some drugs on the transient transfection assay of the luciferase reporter gene containing the 5'-promoter region -3174/+14 of UGT1A1 in HepG2 cells. Among drugs investigated, dexamethasone was the most effective at the range of concentration of 10-100 microM, whereas stimulation by beta-estradiol was not found. We also could not find stimulation by bilirubin of the endogenous main substrate for UGT1A1. Stimulation by dexamethasone was continued for 48 hr. The luciferase reporter gene containing the 5'-region of -97/+14 was induced by dexamethasone but the gene of the 5'-region -53/+14 was not. The region -97/-53 is essential for induction by dexamethasone. This region contains HNF1 element, therefore, we speculated that dexamethasone directly and/or indirectly stimulates UGT1A1 expression through this HNF1 region in the promoter region of UGT1A1. Thus, we clarified that UGT1A1 was induced by dexamethasone and the key position was the region (-97/-53) in UGT1A1 promoter.     

FBG1 Is the Final Arbitrator of A1AT-Z Degradation

Alpha-1 antitrypsin deficiency is the leading cause of childhood liver failure and one of the most common lethal genetic diseases. The disease-causing mutant A1AT-Z fails to fold correctly and accumulates in the endoplasmic reticulum (ER) of the liver, resulting in hepatic fibrosis and hepatocellular carcinoma in a subset of patients. Furthermore, A1AT-Z sequestration in hepatocytes leads to a reduction in A1AT secretion into the serum, causing panacinar emphysema in adults. The purpose of this work was to elucidate the details by which A1AT-Z is degraded in hepatic cell lines. We identified the ubiquitin ligase FBG1, which has been previously shown to degrade proteins by both the ubiquitin proteasome pathway and autophagy, as being key to A1AT-Z degradation. Using chemical and genetic approaches we show that FBG1 degrades A1AT-Z through both the ubiquitin proteasome system and autophagy. Overexpression of FBG1 decreases the half-life of A1AT-Z and knocking down FBG1 in a hepatic cell line, and in mice results in an increase in ATAT. Finally, we show that FBG1 degrades A1AT-Z through a Beclin1-dependent arm of autophagy. In our model, FBG1 acts as a safety ubiquitin ligase, whose function is to re-ubiquitinate ER proteins that have previously undergone de-ubiquitination to ensure they are degraded.     

Comparative CYP1A1 and CYP1B1 substrate and inhibitor profile of dietary flavonoids

CYP1A1 and CYP1B1 are two extrahepatic enzymes that have been implicated in carcinogenesis and cancer progression. Selective inhibition of CYP1A1 and CYP1B1 by dietary constituents, notably the class of flavonoids, is a widely accepted paradigm that supports the concept of dietary chemoprevention. In parallel, recent studies have documented the ability of CYP1 enzymes to selectively metabolize dietary flavonoids to conversion products that inhibit cancer cell proliferation. In the present study we have examined the inhibition of CYP1A1 and CYP1B1-catalyzed EROD activity by 14 different flavonoids containing methoxy- and hydroxyl-group substitutions as well as the metabolism of the monomethoxylated CYP1-flavonoid inhibitor acacetin and the poly-methoxylated flavone eupatorin-5-methyl ether by recombinant CYP1A1 and CYP1B1. The most potent inhibitors of CYP1-EROD activity were the methoxylated flavones acacetin, diosmetin, eupatorin and the di-hydroxylated flavone chrysin, indicating that the 4'-OCH(3) group at the B ring and the 5,7-dihydroxy motif at the A ring play a prominent role in EROD inhibition. Potent inhibition of CYP1B1 EROD activity was also obtained for the poly-hydroxylated flavonols quercetin and myricetin. HPLC metabolism of acacetin by CYP1A1 and CYP1B1 revealed the formation of the structurally similar flavone apigenin by demethylation at the 4'-position of the B ring, whereas the flavone eupatorin-5-methyl ether was metabolized to an as yet unidentified metabolite assigned E(5)M1. Eupatorin-5-methyl ether demonstrated a submicromolar IC(50) in the CYP1-expressing cancer cell line MDA-MB 468, while it was considerably inactive in the normal cell line MCF-10A. Homology modeling in conjunction with molecular docking calculations were employed in an effort to rationalize the activity of these flavonoids based on their CYP1-binding mode. Taken together the data suggest that dietary flavonoids exhibit three distinct modes of action with regard to cancer prevention, based on their hydroxyl and methoxy decoration: (1) inhibitors of CYP1 enzymatic activity, (2) CYP1 substrates and (3) substrates and inhibitors of CYP1 enzymes.