Partial characterization of the human CYP1A1 negatively acting transcription factor and mutational analysis of its cognate DNA recognition sequence.

Previous studies in our laboratory identified a negative regulatory domain in the 5'-flanking region of the human CYP1A1 gene containing two negative regulatory elements (NRE). Characterization of one of these elements revealed three nuclear protein binding regions: a 21-bp palindrome with a point of symmetry at -784 and two guanine- and cytosine-rich elements that flank the palindrome. Functional studies suggested the palindrome is critical for transcriptional repression, whereas the guanine- and cytosine-rich sequences play a secondary role. In this study, the interaction between nuclear proteins and the CYP1A1 NRE was further defined. Electrophoretic mobility shift assays (EMSA) indicated that the NRE -784 palindrome alone, but not the guanine- and cytosine-rich sequences minus the palindrome, was capable of specific nuclear protein binding. Competitive cotransfection experiments confirmed this observation in intact cells. Specific residues important for DNA-protein interactions were identified by site-directed mutagenesis and competitive EMSA. The loss of specific protein binding was also correlated with the loss of negative regulatory activity in a transient-expression assay. Finally, competitive EMSA was performed with consensus oligonucleotides for known transcription factors. An NF-Y consensus sequence efficiently competed with the NRE probe for specific nuclear protein binding. EMSA supershift analyses indicate that a protein immunologically related to NF-YB is part of the specific nuclear protein complex binding the human CYP1A1 NRE. These studies have refined our understanding of the sequences critical for the transcriptional repression of human CYP1A1. To our knowledge, this is also the first report implicating a member of the NF-Y transcription factor family in negative gene regulation.

Altered regulation of the cytochrome P4501A1 gene: novel inducer-independent gene expression in pulmonary carcinoma cell lines.

The cytochrome P450 (CYP) systems catalyze the metabolic transformation of a wide variety of xenobiotics including procarcinogens present in cigarette smoke condensate as well as atmospheric pollutants. The CYP1A1 isoenzyme is of particular interest because it has been implicated as a risk factor in the etiology of lung cancer in heavy cigarette smokers. The identification and expression of the structural CYP1A1 gene in either normal human lung or lung cancer cells has not been reported. Because of its potential significance in human lung cancer, we investigated the expression of the CYP1A1 structural gene in 24 established human lung cancer cell lines including 15 non-small cell (eight adenocarcinomas, three large cell undifferentiated carcinomas, two bronchioloalveolar cell carcinomas, and two squamous cell carcinomas) and nine small cell lung carcinomas. CYP1A1 mRNA was detected in 14 of 15 (93%) of the non-small cell lung carcinoma cell lines examined following 24-hour treatment with benz[a]anthracene (BA) and in nine of 15 (60%) of the non-small cell lines cultured without an inducer in the medium. When the small cell lung cancer lines were evaluated for CYP1A1 gene expression, two of nine (22%) expressed detectable CYP1A1 mRNA in both BA-induced cell cultures and constitutive (control) cultures. A positive correlation was noted between BA-induced CYP1A1 mRNA levels and the corresponding aryl hydrocarbon hydroxylase activity expressed as absolute BA-induced enzyme activity (r = 0.74; P less than .01; n = 24), which further demonstrated that CYP1A1 mRNA expression reflects CYP1A1 enzyme activity in the individual cell lines. These observations represent the first known demonstration of constitutive (non-induced) CYP1A1 gene expression in human cells and suggest altered regulation of the CYP1A1 gene in selected lung cancer cell lines. These human pulmonary carcinoma cell lines, which have documented regulatory defects, could be useful for further identification of the mechanisms associated with CYP1A1 gene regulation.

The role of P(i) in glycolytic inhibition of calcium ion uptake by ELD ascites tumor cells.

In contrast to previous investigations at 25 degrees C, glucose was shown to support 45Ca2+ uptake at 37 degrees C in intact ELD ascites tumor cells. Intact ascites tumor cells in vitro accumulated up to 5.0 micromol of 45Ca2+ per g cells dry wt. within 20 min. In the presence of 10.0 mM glucose, intracellular P(i) levels fell from 40.0 micromol x g(-1) cells dry wt. to 20.0 micromol x g(-1) cells dry wt. in 5 min. Intracellular P(i) levels were maintained by 20.0 mM extracellular Tris-P(i). 45Ca2+ uptake was inhibited in P(i)-depleted cells, even though the metabolic rate (as measured by Q(lactate)) and energy state (as measured by ATP levels) were at acceptable levels. Evidence has been presented suggesting that previous reports of glucose inhibition of calcium uptake can be attributed to a competition for available intracellular P(i) between glycolytic processes and the mitochondrial calcium uptake mechanism.

Oligonucleotide design and optimized protocol for site-directed mutagenesis.

We have optimized conditions for the successful execution of site-directed mutagenesis (SDM) in systems that utilize mutagenic oligonucleotide primers to direct the synthesis of mutant plasmids. In this report, we describe strategies for the design of single-strand DNA templates for SDM, mutagenic oligonucleotide primers, as well as conditions for the annealing, synthesis and propagation of mutant plasmids. The primary focus of the report details the technical aspects of computer-assisted mutagenic oligonucleotide design. Important features include oligonucleotide length, number of matched bases flanking the point(s) of mutation(s), melting temperature, internal stability of the 5' and 3' ends, hairpin and dimer formation, and potential false-priming sites. Largely through a retrospective analysis of our successes and failures, we describe features of the mutagenic oligonucleotide primer that appear critical in this mutagenesis system. Specific examples of efficient and inefficient oligonucleotides are discussed. These characteristics and guidelines should be applicable for SDM of a broad range of target sequences of varying composition, complexity and secondary structure.

Large-scale purification of plasmid DNA by anion-exchange high-performance liquid chromatography.

Numerous methods have previously been reported for the final steps in the large-scale purification of plasmid DNA. Although gel permeation and reverse-phase high-performance liquid chromatography have been utilized for this procedure in the past, the limited capacity of these systems often necessitated multiple rounds of chromatography, especially with the high copy number plasmids commonly in use today. In this paper, the use of the high-capacity, high-resolution Protein-Pak DEAE 8HR column is presented for the large-scale isolation of highly purified plasmid DNA from crude E. coli cell lysates. Up to 5 mg of plasmid DNA have been purified in a single 50-minute chromatography run. The purified DNA demonstrated excellent biological activity as demonstrated by restriction endonuclease digestion, E. coli transformation and DNA-mediated gene transfection of eukaryotic cells.

Induction of cytochrome P450 1A1 gene expression, oxidative stress, and genotoxicity by carbaryl and thiabendazole in transfected human HepG2 and lymphoblastoid cells.

Carbaryl and thiabendazole, two widely used pesticides, have been shown to induce cytochrome P450 1A1 (CYP1A1) expression, but neither compound is capable of displacing [3H] 2,3,7,8-tetrachlorodibenzo-P-dioxin from its aryl hydrocarbon receptor binding site. In the present study, we investigated the transcriptional regulation of CYP1A1 as well as other genes in various human hepatoma HepG2 cell lines stably transfected with the chloramphenicol acetyl transferase (CAT) reporter gene and cloned under the control of each of 14 promoters or response elements from relevant stress genes. Carbaryl and thiabendazole were found to activate CYP1A1 at the level of transcription, as demonstrated by the dose-dependent increase in reporter CAT and CYP1A1 mRNAs. Moreover, this effect appeared to be mediated via the xenobiotic responsive element (XRE), because both pesticides specifically activated various fusion constructs containing XRE sequences (CYP1A, glutathione S-transferase, and XRE). Carbaryl and to a lesser extent thiabendazole also activated other stress genes such as c-fos and NF-kappaBRE, HSP70 and GRP78, and GADD153 at a transcriptional level. These data suggest that these molecules induce early alert genes, including those known to be sensitive to oxidative stress. This led us to examine the genotoxic effect of carbaryl and thiabendazole by an in vitro DNA repair solid-phase assay. Both compounds provoked a strong DNA-damaging activity in the human lymphoblastoid cell line that constitutively expresses human CYP1A1 cDNA, but not in the parental line, indicating that CYP1A1 is chiefly implicated in carbaryl and thiabendazole genotoxicity. This effect was confirmed on HepG2 cells. These observations support the notion that intracellular signals leading to CYP1A1 induction, oxidative stress, and genotoxicity are intimately related.

Developmental regulation of flavin-containing monooxygenase (FMO) isoforms 1 and 2 in pregnant rabbit.

Mammalian flavin-containing monooxygenase (FMO, EC 1.14.13.8) metabolizes a vast number of structurally diverse xenobiotics containing a soft-nucleophile, typically a nitrogen or sulfur. FMO is not inducible by the classical cytochrome P450 (CYP) inducers, such as phenobarbital, polycyclic aromatic hydrocarbons, ethanol or macrolide antibiotics. Evidence does exist from a number of laboratories, however, for developmental and hormonal regulation of FMO. Our laboratory has confirmed previous observations of enhanced FMO activity during mid- and late-gestation in maternal rabbit lung and have demonstrated that this response is due to increased protein and catalytic activity associated with FMO2. The time course of expression of FMO2 during mid- and late-gestation correlates to plasma peaks of progesterone or cortisol. FMO2 also peaks at parturition in maternal kidney, coincident with plasma cortisol levels. FMO2 is induced by s.c. administration of either progesterone or dexamethasone in lung, or by dexamethasone in kidney. Correlation of plasma progesterone or cortisol levels during gestation and postpartum support a role for progesterone, but not cortisol in regulation of FMO2 in maternal rabbit lung. The levels of FMO1 also appear to be increased during mid- and late-gestation in liver. FMO1 in liver may also be regulated during gestation by progesterone or glucocorticoids as administration of these steroids enhanced FMO1 mRNA levels 4-fold.

Differential expression and activity of flavin-containing monooxygenases in euryhaline and stenohaline flatfishes indicates potential osmoregulatory role.

N,N-Dimethylaniline (DMA) N-oxidase activity indicative of flavin-containing monooxygenase (FMO) was examined in four tissues (liver, gill, muscle, and kidney) of the flounder (Platichthys flesus). Gill microsomes had the highest levels of activity (456 +/- 343 pmol/min/mg), while kidney (121 +/- 109) and liver (67 +/- 26) had levels just above detection. A single faint band of a 56 kD protein was observed in liver and gill microsomes following Western blot analyses with polyclonal antibodies to FMO 1. DMA N-oxidase activity in gill and liver directly correlated with the expression of the 56 kD protein recognized by polyclonal antibodies against FMO form 1. Likewise a mRNA band of approximately 2.5 kilobases was higher in gill than a 3.0 kb band in liver following hydridization with an FMO 1 cDNA probe. Gill and liver microsomal DMA N-oxidase from the euryhaline P. flesus was compared with that of the stenohaline turbot (Scophthalmus maximus). DMA oxidase activity, FMO protein and mRNA were significantly greater in the gill of P. flesus, while S. maximus had higher levels of enzyme activity in the liver, but also significant levels in gill. Comparison of the enzymatic properties of the P. flesus gill and S. maximus liver enzymes indicated dramatic differences in Km between gill and liver, but were both inhibited by equimolar concentrations of trimethylamine (TMA). Gill microsomal activity in each species was unaffected by the mammalian FMO 2 substrate (competitive inhibitor) n-octylamine. Differential expression of FMO in tissues from stenohaline and euryhaline fish suggests a functional relationship between FMO and osmoregulation.

Developmental expression of drug metabolizing enzymes: impact on disposition in neonates and young children.

Profound changes in drug metabolizing enzyme expression occurs during development that impacts drug efficacy and the risk of adverse events in the neonate and young child. A review of our current knowledge suggests individual hepatic drug metabolizing enzymes can be categorized into one of three classes based on developmental trajectories. The time frame for the perinatal changes observed for both Class 1 and Class 3 enzymes varies considerably between different enzymes. However, for a given enzyme, significant interindividual variation is observed in the timing of the perinatal changes, creating windows of hypervariability. Genetic variation clearly impacts drug disposition in children. However, developmental factors can dominate pharmacogenetic factors. Thus, a major challenge in applying pharmacogenomics to improve pediatric drug safety is determining at what age functional genetic variants identified in adults become a major determinant of expression in children. Developmental and genetic data on drug metabolizing enzyme ontogeny, as well as age-dependent changes in other physiological factors impacting drug disposition, can be integrated into physiologically-based pharmacokinetic models. Such models have proven useful in predicting the range of expected metabolic capacities at a given age.

Toll-like receptor genetic variants are associated with Gram-negative infections in VLBW infants.

OBJECTIVE: To test the hypothesis that single-nucleotide polymorphisms (SNPs) in Toll-like receptor (TLR) genes alter susceptibility to bacterial infections and modulate white blood cell (WBC) counts during infections in very low birth weight (VLBW) infants (birth weight <1500 g). STUDY DESIGN: VLBW infants recruited in a multicenter study were genotyped for nine functional TLR SNPs and associations between SNPs and infection rates examined. WBC counts obtained during infections were compared among infants with and without SNPs. RESULT: In our cohort (n=408), 90 infants developed bacterial infections. Presence of TLR4 (rs4986790 and rs4986791) variants were associated with Gram-negative (G-ve) infections. Female infants heterozygous for the X-linked IRAK1 (rs1059703) SNP had less G-ve infections. In regression models controlling for confounders, the TLR4 (rs4986790) SNP was associated with increased G-ve infections. The TLR5 (rs5744105) variant was associated with elevated WBC counts during infections. CONCLUSION: TLR genetic variants can contribute to increased risk of bacterial infections and altered immune responses in VLBW infants.

Modeling interchild differences in pharmacokinetics on the basis of subject-specific data on physiology and hepatic CYP2E1 levels: a case study with toluene.

The objective of the present study was to evaluate the magnitude of interindividual variability in the internal dose of toluene in children of various age groups, on the basis of subject-specific hepatic CYP2E1 content and physiology. The methodology involved the use of a previously validated physiologically based pharmacokinetic (PBPK) model, in which the intrinsic clearance for hepatic metabolism (CL(int)) was expressed in terms of the CYP2E1 content. The adult toluene PBPK model, with enzyme content-normalized CL(int), facilitated the calculation of child-specific CL(int) based on knowledge of hepatic CYP2E1 protein levels. The child-specific physiological parameters, except liver volume, were computed with knowledge of age and body weight, whereas physicochemical parameters for toluene were kept age-invariant based on available data. The actual individual-specific liver volume (autopsy data) was also included in the model. The resulting model was used to simulate the blood concentration profiles in children exposed by inhalation, to 1 ppm toluene for 24 h. For this exposure scenario, the area under the venous blood concentration vs. time curve (AUC) ranged from 0.30 to 1.01 microg/ml x h in neonates with low CYP2E1 concentration (<3.69 pmol/mg protein). The simulations indicated that neonates with higher levels of CYP2E1 (4.33 to 55.93 pmol/mg protein) as well as older children would have lower AUC (0.16 to 0.43 microg/ml x h). The latter values were closer to those simulated for adults. Similar results were also obtained for 7 h exposure to 17 ppm toluene, a scenario previously evaluated in human volunteers. The interindividual variability factor for each subgroup of children and adults, calculated as the ratio of the 95th and 50th percentile values of AUC, was within a factor of 2. The 95th percentile value of the low metabolizing neonate group, however, was greater than the mean adult AUC by a factor of 3.9. This study demonstrates the feasibility of incorporating subject-specific data on hepatic CYP2E1 content and physiology within PBPK models for evaluating the age, interchild and population variability of internal dose for use in risk assessment of inhaled volatile organics.

In vitro binding and functional studies comparing the human CYP1A1 negative regulatory element with the orthologous sequences from rodent genes.

In previous studies, we identified a 21 bp palindrome (-794 to -774) located within the negative regulatory element of the human CYP1A1 gene consisting of an 8 bp inverted repeat and 5 bp spacer. This element specifically binds protein(s) present in HepG2 nuclear extract preparations and is capable of down-regulating heterologous promoters and enhancers in transient expression assays. Conserved guanine/cytosine-rich regions which flank the palindrome also were implicated in this activity. In the present study, we examined similar regions from the rat (-881 to -746) and mouse (-822 to -683) CYP1A1 genes for their ability to bind nuclear protein and down-regulate heterologous promoters and enhancers. These rodent DNA fragments contain the conserved guanine/cytosine-rich sequences, as well as half-sites similar to those found in the human CYP1A1 palindrome. However, each half-site is separated by approximately 40 bp. DNase I footprint analyses revealed the presence of rat and mouse nuclear proteins which gave a similar protection pattern as that observed with nuclear proteins from the human cell line, HepG2. Electrophoretic mobility shift assays with the human negative regulatory element demonstrated the formation of specific DNA-protein complexes with rat and mouse nuclear protein(s). Interestingly, two specific DNA-protein complexes were observed with rodent extracts as compared to the single specific complex seen with human extract. Specific binding was not observed with either the orthologous rat or mouse fragments using human or rodent extracts. In transient expression assays, the rat and mouse fragments were unable to down-regulate enhancer/promoter activity. This absence of negative regulatory activity occurred whether transfections were performed in human, rat or mouse hepatoma cell lines. The human negative regulatory element, which was previously shown to down-regulate heterologous enhancers/promoters approximately 70% in human cells, did not exhibit this activity in rodent cell lines. UV cross-linking and southwestern blot analyses indicated a high degree of similarity between human and rodent NRE binding proteins, although some differences also were apparent. The possible implications of these findings with regards to species differences in the regulation of CYP1A1 expression are discussed.

Identification of multiple rabbit flavin-containing monooxygenase form 1 (FMO1) gene promoters and observation of tissue-specific DNase I hypersensitive sites.

We have cloned and partially characterized the rabbit FMO1 gene as a first approach to understanding mechanisms controlling its tissue-specific expression. The isolated clones contain 14 kb of 5' flanking information and approximately 30 kb of the structural gene, but do not include the 3'-end. Two upstream exons were defined, both encoding 5' leader information. The first exon, termed exon 0, contains information not previously reported. The second exon, termed exon 1, contains information previously reported for the rabbit FMO1 cDNA. Protein coding information begins seven nucleotides from the start of exon 2. A single transcription start site was localized in exon 1, while a cluster of sites were defined in exon 0, consistent with two alternative promoters. Transcripts initiating in exon 0 do not contain exon 1 information due to alternative processing and represent the major FMO1 mRNA. Neither promoter contains a TATA box or GC islands, although the exon 1 promoter does share some sequence identity with initiator-type elements. Homologous sequences to several known transcription factor binding sites were found in the upstream region of the FMO1 promoters. Both promoters were active in directing luciferase expression when transiently transfected into human HepG2 cells, although the data are consistent with both requiring upstream enhancer sequences. Consistent with this observation, DNase I hypersensitive sites were mapped to a 600-bp region immediately upstream of exon 0 using liver nuclei. No such sites were detected with nuclei from lung. Differential DNA methylation also was not observed between these two tissues.

Further characterization of the major and minor rabbit FMO1 promoters and identification of both positive and negative distal regulatory elements.

Previously, two promoters were identified for the rabbit FMO1 gene: a major, upstream promoter (P0) that initiates transcription from exon 0 and a second, minor promoter (P1) located approximately 200 bp downstream and initiating transcription from exon 1. Transcription initiation from the P0 promoter results in elimination of the exon 1 leader sequence from the mature transcript. In this report, we further define the major promoter and identify several positive and negative upstream regulatory domains employing deletion analysis and transient expression in HepG2 cells. Of interest, P0 and P1 were equally active in these assays. A 49-bp fragment spanning position -41 to +8 was found essential for the activity of P0 and also capable of basal transcriptional activity. Interestingly, this same 49-bp region was found necessary for P1 activity. Upstream of P0, three positive regulatory regions (positions -348 to -176, -757 to -584, and -1196 to -829) and two negative regulatory regions (positions -2120 to -1724 and 829 to -757) were identified using deletion mutants. Both P0 and P1 share the most proximate, positive regulatory domain but were regulated differentially by more distal 5' sequences. In addition to the upstream regulatory sequences, a potent negatively acting element was observed within intron 1. Using DNA fragments representing the most potent positive (position -348 to -176) and negative (position -829 to -757) regulatory sequences as probes, we demonstrate the formation of multiple specific DNA/protein complexes with protein factor(s) present in HepG2 nuclear extract.

Functional characterization of the human CYP1A1 negative regulatory element: modulation of Ah receptor mediated transcriptional activity.

The mechanisms that underly the regulation of human CYP1A1 have merited considerable attention because of their association both with toxic outcomes and the etiology of several cancers. Previous work conducted in this laboratory has identified a negative regulatory element (NRE) in the 5' region of this gene that appeared to modulate CYP1A1 transcriptional activity. This NRE is present in two functional copies, a high affinity 21-bp palindrome centered at position -784, and an additional element found within a GC-rich region between position -728 and -558. In this report, the regulatory function of the NREs in the context of the CYP1A1 promoter was evaluated. This was accomplished by substituting mutated elements for the corresponding wild-type element in a vector that contained human CYP1A1 sequences positions -1140 to +59 directing the transcription of the chloramphenicol acetyltransferase reporter gene. Expression vectors containing specific mutations in each or both NREs were characterized. We show that eliminating the binding of the CYP1A1 repressor protein to one or both repressor motifs results in a significant 2- to 3-fold increase in the inducibility of CYP1A1 promoter activity. Although mutation of both sites appeared to result in an increase in inducibility over that observed with only one site mutated, the effect was not additive. Such aberrant transcriptional activity correlates with the highly inducible aryl hydrocarbon hydroxylase phenotype that is a reported marker for individuals predisposed to lung cancer. Mutation of the NRE, or more likely, the cognate repressor protein(s), may provide a genetic basis for this phenotype.

Regulation of flavin-containing monooxygenase 1 expression by ying yang 1 and hepatic nuclear factors 1 and 4.

The flavin-containing monooxygenases (FMOs) are important for the oxidation of a variety of environmental toxicants, natural products, and therapeutics. Consisting of six family members (FMO1-5), these enzymes exhibit distinct but broad and overlapping substrate specificity and are expressed in a highly tissue- and species-selective manner. Corresponding to previously identified regulatory domains, a YY1 binding site was identified at the major rabbit FMO1 promoter, position -8 to -2, two overlapping HNF1alpha sites, position -132 to -105, and two HNF4alpha sites, position -467 to -454 and -195 to -182. Cotransfection studies with HNF1alpha and HNF4alpha expression vectors demonstrated a major role for each of these factors in enhancing FMO1 promoter activity. In contrast, YY1 was shown by site-directed mutagenesis to be dispensable for basal promoter activity but suppressed the ability of the upstream domains to enhance transcription. Finally, comparisons between rabbit and human FMO1 demonstrated conservation of each of these regulatory elements. With the exception of the most distal HNF4alpha site, each of the orthologous human sequences also was able to compete with rabbit FMO1 cis-elements for specific protein binding. These data are consistent with these same elements being important for regulating human FMO1 developmental- and tissue-specific expression.

Production of growth factors related to fibroblast growth factor and platelet-derived growth factor by human embryonal carcinoma cells.

Previous studies have demonstrated that mouse embryonal carcinoma (EC) cells produce at least two growth factors: one related to platelet-derived growth factor (PDGF) and another related to basic fibroblast growth factor (FGFb). Since human EC cell lines are being used with increased frequency, the current study examined whether human EC cells produce growth factors, in particular those produced by mouse EC cells. In this study, it was determined that the human EC cell line NT2/D1 produces a heat-labile heparin-binding growth factor that behaves like FGF in a bioassay. Three additional criteria suggest that this factor is closely related or identical to FGFb. The factor from NT2/D1 EC cells, bovine FGFb and FGFb produced by the human hepatoma cell line SK-HEP-1 elute from heparin at similar salt concentrations. The factor produced by NT2/D1 EC cells exhibits a thermal stability curve that is nearly identical to those for bovine FGFb and FGFb from SK-HEP-1 cells. Lastly, NT2/D1 and SK-HEP-1 cells express transcripts of the same size that hybridize with a cDNA probe for human FGFb. In the course of these studies it was determined that NT2/D1 EC cells also express several transcripts that hybridize with a cDNA probe for the human PDGF A-chain. Thus, our findings suggest that the pattern of growth factor production by human and mouse EC cells is evolutionarily conserved.