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Human cytochromes P450 1A1 (CYP1A1) and P450 1B1 (CYP1B1) catalyze the metabolic activation of a number of procarcinogens and the hydroxylation of 17β-estradiol (E2) at the C-2 and C-4 positions, respectively. The aromatic hydrocarbon receptor (AhR) agonist 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) has a marked effect on estrogen metabolism in MCF-7 breast-tumor cells by induction of these two enzymes. To investigate whether induction of CYP1A1 and CYP1B1 by AhR agonists and the associated increase in E2 metabolism are common to all breast epithelial cells and breast-tumor cells, we determined the effects of TCDD on E2 metabolism, and CYP1A1 and CYP1B1 mRNA levels in a series of non-tumor-derived breast epithelial (184A1 and MCF-10A) and breast-tumor (MCF-7, T-47D, ZR-75-1, BT-20, MDA-MB-157, MDA-MB-231 and MDA-MB-436) cell lines. In 184A1 cells, which did not express detectable estrogen receptor (ER) αmRNA, CYP1A1 mRNA and activity were induced by TCDD, and enhanced E2 metabolism in TCDD-treated cells was predominantly E2 2-hydroxylation. In MCF-10A, MCF-7, T-47D, ZR-75-1 and BT-20 cells, which expressed varying levels of ERα mRNA, both CYP1A1 and CYP1B1 mRNA levels and rates of both E2 2- and 4-hydroxylation were highly elevated following exposure to TCDD. In MDA-MB-157, MDA-MB-231 and MDA-MB-436 cells, which did not express detectable ERα mRNA and generally displayed fibroblastic or mesenchymal rather than epithelial morphology, CYP1B1 induction was favored, and the rate of E2 4-hydroxylation exceeded that of 2-hydroxylation in TCDD-treated cells. These results show that breast epithelial cells and tumor cells vary widely with regard to AhR-mediated CYP1A1 and CYP1B1 induction, suggesting that factors in addition to the AhR regulate CYP1A1 and CYP1B1 gene expression. In these cell lines, significant CYP1A1 inducibility was restricted to cultures dismaying epithelial morphology, whereas CYP1B1 inducibility was observed in cells of both epithelial and mesenchymal morphology. [ABSTRACT FROM PUBLISHER]

Cyanide (CN = HCN + CN(-)) is a renowned poison and neurotoxicant that is prevalent throughout the environment. Despite a plethora of studies conducted over the last half century, relatively little is known of its potential to cause adverse health outcomes at sublethal exposures. CN exposure is normally determined from blood, but because CN is rapidly metabolized and cleared from this compartment (t(1/2) < 1 h), it is common for several half-lives to have passed before blood samples are drawn for analysis. This variable, coupled with a very narrow toxic index and metabolic diversity within the human population, has rendered accurate assessment of CN exposure, and consequently any predictions of possible adverse health outcomes, highly problematic. Prior studies by us showed the potential of Cys-SCN adducts within human serum albumin (HSA) to act as retrospective surrogates of CN exposure. Here, we report the discovery of a stable, SCN adduct at Cys(567) formed by the reaction of CN with the C-terminal Cys(558)Cys(567) disulfide bond of HSA. Treatment of HSA purified from human serum with base in guanidine hydrochloride releases a readily detectable, uniquely modified, C-terminal-19-mer peptide from Cys(567)-SCN moieties in all the samples examined thus far. Inclusion of a HSA-Cys(567)-S(13)C(15)N labeled internal standard permits quantitation of the Cys(567)-SCN adduct by LC-MS/MS in selective reaction monitoring (SRM) of the surrogate peptide with high sensitivity and good precision. Reaction of CN in vitro with the Cys(558)Cys(567) disulfide bond in HSA is specific, rapid, and concentration dependent within a putative, physiologically relevant range. Data from various human sera demonstrate the potential usefulness of this adduct as a biomarker of CN exposure.

Arsenite, an environmental cocontaminant of polycyclic aromatic hydrocarbons (PAHs), diminishes the PAH-mediated upregulation of human CYP1A1, the enzyme that bioactivates PAHs to carcinogenic metabolites. Mechanistically, while transcriptional downregulation contributes to these effects, a role for posttranslational regulation has been implicated but not proven. We hypothesize that arsenite induces heme oxygenase-1 (HO-1), which catabolizes CYP1A1 heme or cellular heme pools, thereby downregulating CYP1A1. Arsenite (5 microM), in HepG2 cells, induced HO-1 mRNA 7.4-fold over the 48 h observation period, and it upregulated HO-1 protein expression. Arsenite decreased the induction of CYP1A1 by a PAH, benzo[k]fluoranthene (BKF), by 50%; and transfection of HepG2 cells with siRNA targeting the human HO-1 gene, reduced the arsenite downregulation of BKF-induced CYP1A1 from 54% to 27%, relative to untransfected cells. Reconstituted HO-1 did not significantly catabolize CYP1A1 heme in vitro. Together these findings demonstrate that a posttranslational mechanism involving decreases in the cellular heme pool by arsenite-induced HO-1 may contribute to arsenite-mediated downregulation of CYP1A1.

Cyanide (CN) is a ubiquitous environmental toxicant. The measurement of CN in whole blood is a common exposure assay, but values are error prone because of CN's rapid metabolism and clearance (t1/2 < 1 h) from this compartment. This study was undertaken to determine whether CN forms covalent adduct(s) with plasma proteins that could serve as stable biomarker(s) and potential surrogate(s) of exposure. When added to human blood, plasma, or serum, CN formed covalent adducts with immunoglobulin G (IgG) and serum albumin (HSA) in the plasma fraction. Covalent adducts were not detected in the cellular, primarily erythrocyte, fraction. With human, mouse, and rabbit IgGs, the reaction with CN occurred at intra- and/or interchain disulfide linkages in the heavy and light chains. Digestion of CN-treated HSA with trypsin or the endoproteinase Lys-C at basic pH produced tautomeric 2-iminothiazoline-4-carboxylyl/2-aminothiazolidine-4-carboxylyl (itcCys) N-terminal peptides exclusively, consistent with prior model peptide/protein studies showing that under basic conditions internal S-cyanylated-Cys residues cyclize with concomitant release of the upstream peptide. The most readily detectable reaction of CN with purified HSA was at Cys34, the only Cys of the 35 present not connected as internal cystines. Because CN does not react with free sulfhydryl groups, it is probable that S-cyanylation at Cys34 occurs at those residues that carry GSH, Cys, or other small molecules as mixed disulfides. Relatively less detectable, modified Cys residues were also identified at positions 53, 124, 392, 477, and 487. When 14CN was added to human serum or whole blood at concentrations spanning a putative nontoxic to lethal range, stable adduct formation with HSA occurred in a linear, concentration-dependent reaction that was complete within 2 h. These attributes of the reaction, coupled with a plasma compartment location, suggest that quantitation of CN bound to HSA would provide a much more reliable assessment of exposure than does measurement of CN in blood.

Several systemic and cellular markers of 3'-azido-3'-dideoxythymidine (AZT) metabolism and AZT incorporation into nuclear DNA were measured in cord blood from uninfected infants born to HIV-1-infected mothers receiving prepartum therapies based on AZT or AZT in combination with 2',3'-dideoxy-3'-thiacytidine (3TC). In addition, the relationships among these pharmacological end points, levels of AZT-DNA incorporation, and the previously reported mutagenic responses in these infants were evaluated. AZT- and 3TC-specific radioimmunoassays (RIAs), or HPLC coupled with AZT-RIA, were used to measure plasma levels of AZT and the AZT-glucuronide, and cellular levels of AZT, phosphorylated AZT, and DNA incorporation of AZT or 3TC in cord blood mononuclear cells from treated infants compared with unexposed controls born to HIV-uninfected mothers. Fewer infants had detectable AZT-DNA incorporation levels in the group exposed to AZT (71%; n = 7) compared with those receiving AZT-3TC (100%; n = 21), and the mean AZT-DNA incorporation for AZT-exposed infants (14.6 +/- 6.3 AZT/10(6) nucleotides) was significantly lower than that in AZT-3TC exposed infants (51.6 +/- 10.2 AZT/10(6) nucleotides; P = 0.028). Low levels of 3TC-DNA incorporation found in a few AZT-3TC-exposed newborns correlated with AZT-DNA incorporation values in the same samples. Among the metabolites studied, there were positive correlations between levels of AZT-diphosphate and AZT-triphosphate, and AZT-triphosphate and AZT-DNA incorporation, in nucleoside analog-exposed infants. Levels of AZT-DNA incorporation, however, did not correlate well with the reported frequencies of somatic mutations in the same population of nucleoside analog-treated children. While these data support the continued use of AZT-based therapies during pregnancy, infants receiving prepartum AZT should be monitored long-term for adverse health effects., ((c) 2006 Wiley-Liss, Inc.)

Gender-specific estrogen receptor alpha (ERalpha) expression may plausibly influence lung carcinogenesis in females. Initial genome-wide microarray studies confirmed that carcinogen metabolism genes (CYP1A1, CYP1B1) were those most responsive to cigarette smoke extract (CSE) in normal bronchial epithelial (NHBE) cells. These two genes encoding phase I bioactivating enzymes and the GSTP1 gene encoding a phase II deactivating enzyme were then tested for induction by ERalpha. NHBE cells (native ERalpha-) were transfected with wild-type ERalpha-adenoviral constructs, and then exposed to CSE, 17beta-estradiol (E2), and/or the ERalpha inhibitor, ICI 182,780. The expression levels of CYP1A1, CYP1B1, and GSTP1 were then determined by RNA-specific quantitative RT-PCR and immunoassay. ERalpha increased the basal expression of CYP1B1 4.04-fold (P < 0.01) at the mRNA level and 6.5-fold at the protein level. ERalpha also increased the CSE-induced mRNA expression of CYP1B1 2.26-fold (P < 0.01), but not the protein expression. ERalpha did not alter the CYP1A1 mRNA levels, but did increase protein expression 2.0-fold (P < 0.01) on CSE exposure, and 6.2-fold (P < 0.01) upon E2 exposure. These effects could be inhibited by ICI 182,780. ERalpha did not alter the expression of GSTP1. Chromatin immunoprecipitation assay (ChIP) assay confirmed ERalpha binding to CYP1B1 promoter near the transcription start site. These results suggest that ERalpha regulates the CYP1B1 expression at a transcriptional level, and CYP1A1 expression at a translational level. These data raise the possibility that inter-gender differences in expression of ERalpha that are known to exist in human lung may contribute to inter-individual expression differences in CYP1A1 and CYP1B1, and to differences in carcinogen metabolism and mutation., (Copyright 2005 Wiley-Liss, Inc)

In the described studies, we have developed a variant of the MCF-7 cell line, M-ERd3/g8, for analysis of 17-beta-estradiol (E2)-action without direct DNA interaction by E2-receptors. M-ERd3/g8 cells principally express the estrogen receptor alpha (ER) form ERDelta3 which, due to skipping of exon 3, lacks the second zinc finger of ER that is required for direct DNA interaction. This was achieved by introduction of siRNA targeting exon 3 to a Tamoxifen-selected MCF-7 variant, TMX 2-11, expressing approximately equal amount of full-length ER and ERDelta3 proteins. M-ERd3/g8 cells exhibited a normal nuclear ER localization, and ERDelta3 expression levels were similar to those for full-length ER protein in MCF-7 cells. Ser 118 phosphorylation of the ERDelta3 was triggered by E2 treatment. The expression of several well characterized E2-responsive markers was strongly modified in the M-ERd3/g8 cells. The E2-induction of progesterone receptor (PR) and HEM45 mRNAs was abolished. The effect on pS2 mRNA expression was complex: the pS2 mRNA levels fell approximately 50-fold in control M-ERd3/g8 cells. There was E2-induction of pS2-expression but with an altered temporal pattern. This was blocked by inhibitors of the p42/44 mitogen activated protein (MAP) kinase and inositol triphosphate (PI3) kinase pathways suggesting a role for cytoplasmic signaling pathways. Gene array analysis and real-time polymerase chain reaction (PCR) studies identified several genes whose expressions were induced in E2-treated M-ERd3/g8 cells. These included A-Myb, a homolog to the avian myoblastosis virus oncogene, carbonic anhydrase XII (CAXII), chemokine ligand 12 (CXCL-12), early growth response 3 (EGR 3), fibrinogen B beta (FibBbeta), along with serine protease 23 (SPUVE). The responses fell into several temporal patterns. A-Myb, CAXII, CXCL-12 and EGR 3 were E2-induced within 2 h. The expression of CXCL-12 and EGR 3 was persistent to 24 h, while that of A-Myb and CAXII was not persistent in M-ERd3/g8 cells. FibBbeta and SPUVE expression was not induced until times later than 6 h. Expression of none of the genes was elevated prior to 2 h, but the utilization of a 24 h time point for the gene array analysis may have eliminated the most transiently responsive genes. Immediate early 3 (IE3) was down-regulated by E2 in the M-ERd3/g8 cells but was transiently up-regulated during the 2-6 h period in MCF-7 cells. Basal levels of several of the genes were strongly reduced in M-ERd3/g8, compared to MCF-7. The studies suggest that M-ERd3/g8 cells provide a new model for studies of E2-action without direct ER binding to DNA and where E2-action must be via alternate pathways.

Polycyclic aromatic hydrocarbons (PAHs) and heavy metals are often environmental cocontaminants that could interact to alter PAH carcinogenicity. The heavy metal, arsenite, and the PAH, benzo[k]fluoranthene, were used as prototypes to investigate, in human HepG2 cells, mechanisms whereby the bioactivation of benzo[k]fluoranthene by human CYP1A1 could be diminished by arsenite-mediated decreases in CYP1A1 induction by benzo[k]fluoranthene. To determine whether arsenite down-regulates CYP1A1 transcription, quantitative real-time reverse transcriptase-polymerase chain reaction assays and luciferase reporter gene expression assays were used with HepG2 cells treated with benzo[k]fluoranthene and arsenite, separately and as a mixture. Benzo[k]fluoranthene (0.5 microM) and arsenite (5 microM) markedly decreased benzo[k]fluoranthene-mediated induction of CYP1A1 mRNA by 45%. Plasmids containing the CYP1A1 promoter region (pHu-1A1-FL) were induced 7.4-fold over vehicle by benzo[k]fluoranthene (0.5 microM), whereas arsenite (1, 2.5, or 5 microM) decreased reporter gene expression by 46%, 45%, and 61%, respectively. The plasmid, pHu-1A1-Delta100-FL, lacked xenobiotic response element (XRE) sites at -1061 and -981 and showed greater responsiveness relative to pHu-1A1-FL, by 1.7-fold. Benzo[k]fluoranthene (0.5 microM) and arsenite (1, 2.5, or 5 microM) decreased reporter gene expression by 0%, 27%, and 39%, respectively, relative to expression levels produced by benzo[k]fluoranthene alone. Arsenite is stable for at least 48 h in the HepG2 cell medium with respect to its ability to diminish CYP1A1 benzo[k]fluoranthene induction. Arsenite did not affect benzo[k]fluoranthene induction directly through XRE sites, nor did it affect the stability of CYP1A1 mRNA. Thus, arsenite affects the transcriptional regulation of the benzo[k]fluoranthene-mediated induction of CYP1A1 and could diminish PAH carcinogenicity by decreasing bioactivation by CYP1A1.

Estrogen receptor alpha (ER) mRNA is primarily transcribed from two promoters, the two transcripts share identical sequence encoding the same ER protein but differ in upstream regions. The 5' region of the two transcripts contain upstream open reading frames (uORFs) encoding potential peptides of 20 and 18 amino acids. The peptides have five C-terminal residues in common. These studies were undertaken to determine if the uORFs and encoded peptides differentially affected expression of ER. Expression of green fluorescent protein (GFP) reporter constructs containing upstream proximal promoter transcript sequences with the first 18 codons of ER fused to GFP was tested in HeLa cells. The cells expressed reduced levels of GFP as compared to the pEGFP-N1 parent vector; the effect was dependent on the presence of an intact proximal ER transcript uORF. Similar regulation by the uORF was seen in transfected MCF-7, MDA MB-231 and Ishikawa cells. Only protein expression was affected by eliminating the uORF; RNA levels were unchanged. This indicates the mechanism is translational rather than being an effect of the introduced point mutations on either mRNA stability or transcription. Eliminating the uORF did not significantly increase expression from similar distal promoter transcript ER-GFP constructs. However, study of in-frame fusions of GFP to the entire proximal and distal uORFs and to their translational start motifs showed that the translational start region of the distal uORF was inherently better at initiating translation than the AUG environment of the proximal promoter transcript uORF. The data indicate there are regulatory properties suppressing expression from the ER translation start which are specific to the unique regions of the ER proximal promoter transcript and these are likely associated with the proximal transcript uORF peptide product.

Polycyclic aromatic hydrocarbons (PAHs) are common environmental pollutants that have been extensively studied for multiple toxicological endpoints in both laboratory animals and humans. The purpose of this study was to investigate the estrogenicity of PAHs in the human breast cancer cell line MCF-7. We investigated 14 PAHs for their ability to bind either the estrogen receptor (ER) or the aryl hydrocarbon receptor (AhR) and to activate target gene expression. PAHs were tested in a human recombinant estrogen receptor (hrER) competitive binding assay, and in both an estrogen response element (ERE)- and xenobiotic response element (XRE)-mediated reporter gene assay. We used quantitative RT-PCR to examine selected PAHs that showed activity in the ERE reporter gene assay for their ability to upregulate estrogen-responsive genes HEM45, progesterone receptor, and pS2, and the aryl hydrocarbon-responsive CYP1A1 gene. None of the 14 PAHs bound the hrER, but five of the PAHs (anthracene, B[a]A, chrysene, B[b]F, and B[a]P) induced ER-reporter activity. This activity was dependent on the metabolism of PAHs in MCF-7 cells via the AhR pathway, which resulted in the formation of metabolites that bound the ER. None of the five PAHs that induced the ER-reporter were found to upregulate estrogen-responsive genes, yet four of the five PAHs induced AhR-dependent CYP1A1 gene expression. In contrast, a metabolite of B[a]P, 3'OH-B[a]P, and a PCB metabolite, 4'OH-2,4,6-BP, did weakly upregulate all three estrogen-responsive genes. Data from these studies indicate that induction of ER-reporter activity alone does not necessarily parallel endogenous gene transcription, and that the reporter gene assay may detect interactions that are not functional in vivo.

Purpose: The regulation of carcinogen metabolism machinery may involve proximate tobacco smoke exposure, hormonal and other endogenous coregulatory factors, and an individual's underlying genetic responsiveness. The mRNA and protein expression patterns of known carcinogen metabolism genes encoding the aromatic hydrocarbon receptor Ahr; the cytochromes P450 CYP1A1 and CYP1B1; glutathione S-transferases GSTM1, GSTM3, GSTP1, and GSTT1; and NADPH quinone oxidoreductase NQO1 were examined., Experimental Design: Paired tumor and nontumor lung tissue from 45 subjects was subject to a recently devised RNA-specific qualitative reverse transcription-PCR strategy, as well as Western immunoblotting. Tobacco exposure measured by plasma biomarkers nicotine and cotinine, plasma estradiol levels, alpha and beta estrogen receptor (ER) expression in the lung, gender, age, and histological diagnosis were then analyzed using multivariate regression models., Results: In nontumor lung tissue, multivariate models identified several correlates of mRNA expression: (a) CYP1B1 in females (positively: smoke status, P=0.024; ER-beta expression, P=0.024); (b) GSTT1 in females (positively: cotinine, P=0.007; negatively: age, P=0.001; ER-beta expression, P=0.005) and in males (positively: plasma estradiol, P=0.015; ER-beta expression, P=0.025); and (c) NQO1 in females (positively: smoke status, P=0.002) and in males (positively: ER-beta expression, P=0.001). CYP1A1 (mRNA, 9.1%) and GSTM1 (mRNA, 17.5%) are uncommonly expressed in human lung. Confirmation by Western immunoassayed protein is described. The results in nontumor tissue differed from that in tumor tissue., Conclusions: Regulation of carcinogen metabolism genes expressed in human lung seems impacted by hormonal and gender factors, as well as ongoing tobacco exposure. Expression differences between tumor and nontumor tissue in this pathway have both susceptibility and therapeutic implications.

MCF-7 breast tumor cells form multicellular nodules (foci) over a confluent monolayer in an estradiol (E2)-dependent, antiestrogen-sensitive reaction. A cell line cloned from MCF-7 that displays these phenotypes was probed to determine the effects of long term exposure to tamoxifen on the growth of foci, estrogen receptor alpha (ERalpha) status, and gene responsiveness to E2. In one of two experiments, a heterogeneous cell population emerged (TMX2) that over-expressed estrogen receptor alpha wild type mRNA (ERalpha mRNA) (approximately 20-fold) missing exon 3 (ERDelta3 mRNA) and its corresponding protein (ERDelta3P). On a per mRNA to protein basis, ERDelta3P and wild-type ERalpha were equivalently expressed. Return of the TMX2 population to medium without tamoxifen eventually selected for a population that expressed predominately wild-type ERalpha, whereas TMX2 clones over expressing ERDelta3 mRNA and ERDelta3P retained this phenotype in tamoxifen-free media. In both experiments, expression of all ERalpha mRNAs and proteins declined to barely detectable levels during 6-12 months exposure, concomitant with a progressive increase in the ability of the cells to form foci independently of E2 or tamoxifen. Selection for these various populations suggests that tamoxifen can induce and/or support certain cellular changes that lead to altered ERalpha expression, E2-independent cell growth and resistance to antiestrogens.

Estrogen receptor (ER) expression in human lung has been understudied, particularly in light of its potential biological importance in the female lung cancer epidemic. Reverse transcription-polymerase chain reaction was used to probe mRNA expression of wild-type ERalpha and ERbeta and their splice variants in human bronchogenic tumor and adjacent nontumor specimens. In tumor tissue from 13 women and 13 men, ERalpha was expressed in 85% of women versus 15% in men [P=0.001]. ERbeta was expressed equally in tumors from women versus men [92% vs. 69%, P=ns]. Both ERalpha and beta forms were expressed simultaneously in the lung tumors of 77% of women versus 15% of men [P=0.005]. Among adjacent nontumor lung specimens, 31% of the women expressed ERalpha mRNA versus 0% of men [P=0.101], and 39% of women expressed ERbeta mRNA versus 31% of men [P=ns]; only one woman and no men expressed both ERalpha and beta in nontumor tissue. Females expressed ERalpha [P=0.017], ERbeta [P=0.013], and ERalpha+beta [P=0.002] more frequently in tumor versus nontumor tissue, whereas in males expression of ERalpha, beta and both alpha+beta was not clearly different for tumor versus nontumor tissue. In specimens expressing ERalpha mRNA, the transcript lacking exon 7 (delta7) was the major splice variant with varying contributions from the transcripts delta4, delta3+4, delta5 and others unidentified. Alternative splicing of ERbeta mRNA was observed, but not to as great an extent as for ERalpha mRNA. ERalpha promoter usage in tumors varied among individuals. When the ER receptors were co-expressed in tumors, ERalpha was quantitatively more abundant in the majority of cases than ERbeta. Within this small group of 26 patients, no correlation was found between age, smoking history, plasma nicotine, cotinine, estradiol concentrations or histopathologic type with tumor or nontumor estrogen receptor status of any type. However, several positive correlations imply that: (1) ERalpha expression occurs more often in the lungs of women than men; (2) ERbeta is expressed with approximately equal frequency in the lungs of both genders; and (3) tumors display a higher frequency of both receptor types than nontumors in women. We hypothesize that these putative gender-dependent differences in ERalpha and ERbeta expression could contribute unique phenotypic characteristics to lung cancer development or progression in women.

Human estrogen receptor alpha (ER) mRNA is a mixture of wild type and alternatively spliced variants. Many studies have examined the potential of ER mRNA profiles to serve as diagnostic/prognostic cancer biomarkers, but only a few have attempted to correlate ER mRNA profiles with protein expression. Representative ER mRNA pools were reproduced from the cDNAs of MCF-7 cells, a human breast tumor and human uterus and translated in a protease-free environment by reticulocyte lysates to determine relative translation efficiencies between the various ER mRNA transcripts and to facilitate identification of translated proteins. Cell line and tumor extracts were then examined for expression of the ER variant proteins identified in reticulocyte lysate translations. Each of the ER mRNA pools were translated by reticulocyte lysates into two ER proteins with molecular weights of approximately 60 and 52 kD. Western immunoblotting with various C- and N-terminal-directed, anti-ER antibodies and comparison with expressed ER protein standards established that the 52 kD protein (ERDelta7P) was translated from the predominant splice variant mRNA in each pool, which is missing exon 7. The 60 kD protein contained wild type ER sequence minus 61 C-terminal amino acids lost due to an intentional run off truncation. ERDelta7P expression was subsequently demonstrated in MCF-7 cells by Western immunoblotting with the site-directed antibodies. A protein corresponding to ERDelta7P was also detected in other ER positive breast tumor cell lines, and extracts of ER positive breast and uterine tumors. This widespread expression of ERDelta7P in vivo suggests that it may have some biological function. ERDelta7P may also affect immunohistochemical evaluation of ER positivity in tumors depending upon the level of its expression and the antibody used.

The polymerase chain reaction (PCR) has revolutionized molecular biology. Portions of single-copy per cell genes (and cDNAs) prepared from very small tissue or cell samples can be specifically amplified for use in sequence determination, gene identification, and quantitation. Improvements to the method, such as the introduction of genetically engineered, thermostable polymerases, more precise thermocyclers and more efficient reverse transcriptases for mRNA conversion to cDNA, have combined to make RNA-PCR (also called reverse transcriptase, or RT-PCR) and PCR more reproducible and specific. Coupled with the high sensitivity of the reactions, RT-PCR and PCR are increasingly used as quantitative bio-analytical techniques.

Elevated expression of cytochrome P450 1B1 (CYP1B1) and estradiol 4-hydroxylation have been reported to be biomarkers of tumorigenesis in humans. The aromatic hydrocarbon receptor (AhR) regulates expression of human cytochrome P450 1A1 (CYP1A1) and CYP1B1, 17beta-estradiol (E2) 2- and 4-hydroxylases, respectively. There is also evidence that expression of estrogen receptor alpha (ERalpha) potentiates CYP1A1 inducibility in breast cancer cells. To characterize these relationships further, we examined the effects of 12-O-tetradecanoylphorbol-13-acetate (TPA), which downregulates ERalpha, and the high-affinity AhR ligand, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), on the expression of AhR, ERalpha, CYP1A1, and CYP1B1 in MCF-7 human breast cancer cells. Treatment with TPA, which suppressed ERalpha mRNA levels, caused a greater than fourfold elevation of AhR mRNA and protein levels, whereas treatment with TCDD caused a decrease in AhR protein but no change in ERalpha or AhR mRNA levels. In MCF-7 cells treated with TPA prior to treatment with TCDD, the AhR mRNA level was elevated, the ERalpha mRNA level remained suppressed, and the ratio of CYP1B1 to CYP1A1 mRNA was increased compared with treatment with TCDD alone. A corresponding increase in the ratio of the rates of 4- to 2-hydroxylation pathways of E2 metabolism was also observed in response to pretreatment with TPA prior to the addition of TCDD. These results demonstrate differential regulation of the human CYP1A1 and CYP1B1 genes and provide a cellular model to investigate further the mechanisms that may be involved in the elevated expression of CYP1B1 in tumorigenesis.

Relative proportions of the estrogen receptor (ER) alternatively spliced mRNA variants from the proximal (A) and distal (B) promoter pre-mRNA transcripts were measured in normal human uterus, an endometrial tumor, and in T47D, MCF-7, and BT-20 breast tumor cell lines. A single tube RNA-PCR method was developed to determine the proportions of the individual transcripts and a nested, competitive RNA-PCR method to determine the proportions of the alternatively spliced variants. Except for the BT-20 cells, the patterns of splice variants produced from each transcript were very similar. In BT-20 cells no splice variants were detected for the minor (< or = 1%) A promoter transcript, although the B promoter transcript was alternatively spliced similarly to the other samples, with the exon 7 variant as the major mRNA form. These results indicate that the mRNA spliced variant patterns in most tissues and tumors will be essentially unaffected by any changes in the A and B promoter ER mRNA transcript ratios that may occur. At least one exception does exist, however, and only more comprehensive studies can determine whether the BT-20 cells are unique or part of a larger subgroup.

Human cytochromes P450 1A1 (CYP1A1) and P450 1B1 (CYP1B1) catalyze the metabolic activation of a number of procarcinogens and the hydroxylation of 17beta-estradiol (E2) at the C-2 and C-4 positions, respectively. The aromatic hydrocarbon receptor (AhR) agonist 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) has a marked effect on estrogen metabolism in MCF-7 breast-tumor cells by induction of these two enzymes. To investigate whether induction of CYP1A1 and CYP1B1 by AhR agonists and the associated increase in E2 metabolism are common to all breast epithelial cells and breast-tumor cells, we determined the effects of TCDD on E2 metabolism, and CYP1A1 and CYP1B1 mRNA levels in a series of non-tumor-derived breast epithelial (184A1 and MCF-10A) and breast-tumor (MCF-7, T-47D, ZR-75-1, BT-20, MDA-MB-157, MDA-MB-231 and MDA-MB-436) cell lines. In 184A1 cells, which did not express detectable estrogen receptor (ER) alpha mRNA, CYP1A1 mRNA and activity were induced by TCDD, and enhanced E2 metabolism in TCDD-treated cells was predominantly E2 2-hydroxylation. In MCF-10A, MCF-7, T-47D, ZR-75-1 and BT-20 cells, which expressed varying levels of ER alpha mRNA, both CYP1A1 and CYP1B1 mRNA levels and rates of both E2 2- and 4-hydroxylation were highly elevated following exposure to TCDD. In MDA-MB-157, MDA-MB-231 and MDA-MB-436 cells, which did not express detectable ER alpha mRNA and generally displayed fibroblastic or mesenchymal rather than epithelial morphology, CYP1B1 induction was favored, and the rate of E2 4-hydroxylation exceeded that of 2-hydroxylation in TCDD-treated cells. These results show that breast epithelial cells and tumor cells vary widely with regard to AhR-mediated CYP1A1 and CYP1B1 induction, suggesting that factors in addition to the AhR regulate CYP1A1 and CYP1B1 gene expression. In these cell lines, significant CYP1A1 inducibility was restricted to cultures displaying epithelial morphology, whereas CYP1B1 inducibility was observed in cells of both epithelial and mesenchymal morphology.

Inhibition of estrone sulfatase activity offers the potential for breast cancer prevention therapy by blocking a route to estrogen synthesis. We have investigated the inhibition of this activity by natural flavonoids in a human hepatic microsomal preparation in vitro. The majority of studies were performed with a male liver, but male and female livers exhibited comparable estrone sulfatase activities. The natural flavonoids, quercetin, kaempferol, and naringenin, significantly inhibited estrone sulfatase activity with I50 < 10 microM for the most potent, quercetin. Estrone sulfatase activity in the liver microsomes was biphasic, with a high affinity, low capacity, low concentration activity (Km 14.3 microM, Vmax 0.5 nmol/min/mg protein), probably steroid sulfatase-catalysed, and a low affinity, high capacity, high concentration activity (Km 1.5 mM, Vmax 21.5 nmol/min/mg protein), probably arylsulfatase C or E-catalysed. The former activity was inhibited uncompetitively by quercetin, the latter competitively. Quercetin, a natural dietary constituent, is a potent inhibitor of estrone sulfatase in vitro, and thus has the potential to express antiestrogenic activity in vivo.

The human cytochrome P450 (CYP) 2D subfamily comprises the CYP2D6 gene and four pseudogenes, CYP2D7P1 and 2 and CYP2D8P1 and 2. The CYP2D6 gene product is a prominent drug-metabolizing enzyme, which is probably constitutive and has no known inducing agents. Alternative splicing of the pre-mRNAs of these genes has been detected in human liver and breast tissue. RNA-PCR, competitive RNA-PCR, Southern blotting, cDNA sequencing, and gene-specific PCR have been used to fully characterize the alternatively spliced forms of CYP2D mRNA in human breast tissue in the region of exon 5 to 8. Such alternative splicing could regulate the expression of CYP2D6 protein. A full-length mRNA (exons 5 to 8), and variants c (exon 6 deleted), b' (3' portion of exon 6 deleted), e (3' portion of exon 6 deleted, 3' 57-bp portion of intron 6 included), d (3' 57-bp portion of intron 6 included), and b (intron 6 included) were characterized and quantitated. Variant c was derived from CYP2D6, variants d, e, and b were from CYP2D7P, and variant b' and full-length mRNA were derived from both CYP2D6 and 2D7P. Full-length mRNA was a minor form in human breast tissue where variants b' and c predominated. Human breast tumor MCF-7 cells had CYP2D mRNA splice variant patterns similar to those of human breast tissue, while human liver tumor HepG2 cells had wild-type mRNA predominating. These results suggest that CYP2D6 could be regulated tissue specifically using tissue-specific alternative mRNA splicing.

Allelic variants of the CYP2D6 gene, a member of the cytochrome P450 gene superfamily, have been implicated in susceptibility to lung carcinogenesis. Human breast CYP2D6 and CYP2D7P (from a pseudogene) mRNAs were previously reported to be expressed as a series of splice variants. In this study, the expression of full-length and splice variants of these mRNAs in human lung tissue and tumors are reported for the first time and are compared in order to probe the potential for differential CYP2D6 regulation in lung normal tissue and tumors. The splice variant profiles differed within the same individual, but no consistent differences were detected.

One in ten tobacco smokers develops bronchogenic carcinoma over a lifetime. The study of susceptibility of an individual and a population to lung cancer traditionally has been limited to the study of tobacco smoke dose and family history of cancer. New insights into lung carcinogenesis have made the study of molecular markers of risk possible in human populations in the emerging field of molecular epidemiology. This review summarizes data addressing the relationships of human lung cancer to polymorphisms of phase I procarcinogen-activating and phase II-deactivating enzymes and intermediate biomarkers of DNA mutation, such as DNA adducts, oncogene and tumor suppressor gene mutation, and polymorphisms. These parameters are reviewed as they relate to tobacco smoke exposure, procarcinogen metabolizing polymorphisms, and the presence of lung cancer. Problem areas in biomarker validation, such as cross-sectional data interpretation; tissue source, race, statistical power, and ethical implications are addressed.

Estrogen receptor (ER) mRNA exists as wild-type (full-length) and alternatively spliced variants in cell lines, normal tissues, and tumors. Most of the alternatively spliced variants discovered so far are missing one or more complete exons. RNase protection and RNA-PCR assays used previously to determine the relative concentration of a particular ER spliced-variant mRNA to wild-type mRNA have produced equivocal results because the probes/primers targeted only small regions within the nucleotide sequence. Variant ER mRNAs missing an exon outside the probe/primer region will react as if they were wild-type and any alternatively spliced variants containing a deletion at the probe/primer annealing site(s) will not be detected. A highly sensitive, competitive RNA-PCR assay has been developed that is quantitative with respect to the relative composition of wild-type ER and its alternatively spliced-mRNA forms, and semiquantitative with respect to their concentrations in cells and tissues. Separation and quantitation of the products are rapidly and accurately achieved by, respectively, capillary electrophoresis and laser-induced fluorescence. Wild-type ER mRNA concentration can be measured independently of all the reported exon deletion forms in a single PCR assay. Specific exon deletion forms can be measured by ER cDNA amplification with overlapping primer sets. Results obtained with RNAs isolated from two MCF-7 cell lines, a T-47D cell line, and five breast tumor tissues are presented.

Human cytochrome P4501A1 (CYP1A1) occurs extrahepatically and is polymorphic, the common form having Ile at position 462 and the rare form having Val. The rare allele has been associated with enhanced susceptibility to lung cancer. To resolve its role in cancer we have constructed CYP1A1-Val462 cDNA by site-directed mutagenesis from CYP1A1-Ile462, as confirmed by sequencing and allele-specific PCR. Both alleles were expressed in Escherichia coli, and CYP1A1-Ile462 and -Val462 were purified to electrophoretic homogeneity. The secondary structures of both forms were virtually identical, with high alpha helix content, as assessed by circular dichroism. The P450s stereoselectively and regioselectively catalyzed the metabolism of (R)- and (S)- warfarin, in reconstituted systems, with very similar profiles. Both P450s produced (R)-6- and 8-hydroxy-warfarin with Km values of 0.40 +/- 0.06 and 0.43 +/- 0.05 mM, respectively, and Vmax values of 84.0 +/- 6.8 and 137.7 +/- 8.9 pmol/min/nmol CYP1A1-Val462, respectively, 1.0 +/- 0.1 and 1.0 +/- 0.1 mM, respectively, and 46.7 +/- 2.5 and 80.0 +/- 4.4 pmol/min/nmol CYP1A1-Ile462, respectively. Reconstituted CYP1A1-Val462 catalyzed ethoxyresorufin metabolism at a slightly but significantly higher rate than did CYP1A1-Ile462; Vmax values were 4.4 +/- 0.6 and 3.1 +/- 0.3 nmol/min/nmol CYP1A1, respectively. However, with the carcinogen benzo(a) pyrene as substrate, reconstituted CYP1A1-Ile462 together with epoxide hydrolase produced 7,8- and 9,10-dihydrodiols at comparable rates than did CYP1A1-Val462. Thus, the apparently greater susceptibility of the CYP1A1-Val462 genotype to lung cancer is probably not related to greater extents of carcinogen bioactivation.

In an effort to determine which members of the cytochrome P450 (CYP) superfamily are expressed in human breast tissue and tumors, RNA-polymerase chain reaction studies have been undertaken. Detection of expressed CYP mRNAs identifies those forms of the enzyme that are capable of expression in breast tissue, and provides insight into the potential for in situ xenobiotic and therapeutic drug metabolism. CYP1A1 mRNA was present in (5/11) breast tissues and (6/13) tumors. When normal and tumor tissues were from the same individuals, higher amplification occurred in normal tissues. CYP1B1 mRNA was present in all but one tissue, and CYP2C mRNA forms were present in all of the tissues. CYP3A4 mRNA was present in (8/11) normal breast tissues and (2/13) tumor tissues, and CYP3A5 mRNA was present in (9/11) normal tissues and (2/13) tumor tissues. The expression of the CYP3A mRNA forms was not coincident, suggesting differential regulation. CYP2D6 mRNA was present in (10/11) normal breast tissue and (10/13) tumors. Two splice variants of CYP2D6 mRNA were also detected; one with a 207 bp intron spliced in was detected in all of the normal tissue samples and (11/13) tumors, whereas another (which lacks a 3'-portion of exon 6) was detected in (9/11) normal breast tissues and (7/13) tumors. Thus, examples of each of the xenobiotic-metabolizing CYP1, CYP2, and CYP3 subfamilies were detected in low levels in human normal breast tissue and tumors. The machinery for possible in situ bioactivation of xenobiotics and modification of therapeutic drugs is thus present in human breast tissue.

Two forms of the cytochrome P450 enzyme superfamily, P4501A1 and P4501A2, that are heterogeneously distributed in populations and are induced in response to environmental factors are important because of their capacity to bioactivate procarcinogens. Phenotyping P4501A1 and P4501A2 in individuals will thus provide assessments of those individuals' susceptibility to procarcinogens. The anticoagulant drug warfarin is metabolized by human P4501A1 and P4501A2, and we have characterized this metabolism for the R-warfarin enantiomer as a potential in vivo probe. cDNA-expressed human P4501A1 and P4501A2 are regioselective for R-warfarin 6- and 8-hydroxylation with very similar KM values: 1.4 mM (6-hydroxylation), 1.2 mM (8-hydroxylation), 1.6 mM (6-hydroxylation), and 1.4 mM (8-hydroxylation), respectively, indicating possible binding competition for R-warfarin between the two forms. However, when comparing 6- and 8-hydroxylation, P4501A1 showed weak regioselectivity for 8-hydroxylation, whereas P4501A2 exhibited strong regioselectivity for 6-hydroxylation, with 6-hydroxylation/8-hydroxylation ratios of 0.6 and 5.0, respectively. These findings were confirmed by using microsomes from 2,3,7,8-tetrachlorodibenzo-p-dioxin-treated HepG2 and MCF-7 cells expressing only P4501A1 (ratios of 0.7), and from human hepatic microsomal preparations containing only P4501A2 (average ratios of 4.0). P4501A2 levels in the liver preparations, as assessed by densitometry of immunoblots, correlated with R-warfarin 6-hydroxylation rates (r2 = 0.83) and caffeine 3-demethylation rates (r2 = 0.67), but not with R-warfarin 8-hydroxylation rates. P450s 2A6, 2B6, 2C9, 2D6, 2E1, and 3A4 did not yield either 6- or 8-hydroxy-warfarin from R-warfarin. We conclude that R-warfarin 6-hydroxylation rates are markers for human hepatic P4501A2, whereas ratios of 6-hydroxylation/8-hydroxylation could be used in vitro as a marker for P4501A1.

A capillary electrophoretic (CE) method has been developed for the determination of theophylline and all of its identified and potential metabolites. The method is rapid, resolves all metabolites to baseline, and requires extraction of only some biological fluids. It has been applied to the analysis of theophylline metabolism by hepatic microsomes from rats treated with a variety of inducing agents for different forms of P450 enzymes which metabolize theophylline, and to human urine spiked with theophylline and its metabolites, and concentrated by solid-phase extraction.

Quantitative RNA-polymerase chain reaction (RNA-PCR) is an extremely powerful analytical tool owing to its specificity and high level of sensitivity. Quantitative RNA-PCR is, however, highly labor intensive. No analytical method currently exists that can accurately and rapidly quantitate the small quantities of DNA in RNA-PCR reaction mixtures. We have developed a method using capillary electrophoresis and laser-induced fluorescence to detect YOYO-1 complexes of DNA produced by PCR. RNA-PCR mixtures can be analyzed either directly (without primer and protein removal) or by electrokinetic injection following desalting. Modified competitive and multiplex competitive RNA-PCR assays for glyceraldehyde-3-phosphate dehydrogenase and P4501A1 were tested in a series of mixtures containing equal concentrations, but different proportions, of RNA from untreated (essentially no P4501A1 mRNA) and 2,3,7,8-tetrachlorodibenzo-p-dioxin-treated (high levels of P4501A1 mRNA) HepG2 cells. Twofold differences in concentrations between two P4501A1 mRNA solutions could be detected by competitive RNA-PCR. Glyceraldehyde-3-phosphate dehydrogenase concentrations were constant throughout. Multiplex competitive PCR produced more variable results due to the presence of contaminating peaks, which hindered accurate area integration. These data demonstrate the potential usefulness of capillary electrophoresis in a variety of quantitative PCR applications.

Small intestinal cytochromes P450 (P450s) provide potential first-pass metabolism of ingested xenobiotics. To investigate this system, this study addresses the procedure for elution of enterocytes from male rat small intestine, histological evaluation of the elution procedure, and assessment of the functional microsomal P450s in small intestine of untreated and induced rats, using warfarin metabolism as a probe. Histologically it was demonstrated that villous enterocytes are initially detached in sheets and are subsequently eluted without clear resolution into villous tip, midvillous, and lower villous cells, contrary to previous reports. Crypt cells are eluted after cells from the villus. The following functional P450s were identified, using stereo- and regioselectivity of warfarin metabolism, in small intestinal microsomes: P4502B1 in untreated rats, P4501A1 in beta-naphthoflavone-induced rats, P4502B1 in phenobarbital-induced rats, and P4503A1/2 in pregnenolone-16 alpha-carbonitrile-induced rats. In contrast to hepatic microsomes from untreated or induced rats, P4502C11 and -2C6 were not present or inducible by these inducing agents in rat intestine. Western immunoblots, warfarin assays, and P450 assays all indicated that beta-naphthoflavone induced P4501A1 in small intestinal villous and crypt cells, but in contrast to the liver neither apo-P4501A2 nor functional P4501A2 was induced.

Small intestinal cytochromes P450 (P450) provide the principal, initial source of biotransformation of ingested xenobiotics. The consequences of such biotransformation are detoxification by facilitating excretion, or toxification by bioactivation. P450s occur at highest concentrations in the duodenum, near the pylorus, and at decreasing concentrations distally--being lowest in the ileum. Highest concentrations occur from midvillus to villous tip, with little or none occurring in the crypts of Lieberkuehn. Microsomal P4503A, 2C8-10, and 2D6 forms have been identified in human small intestine, and P450s 2B1, possibly 2B2, 2A1, and 3A1/2 were located in endoplasmic reticulum of rodent small intestine, while P4502B4 has been purified to electrophoretic homogeneity from rabbit intestine. Some evidence indicates a differential distribution of P450 forms along the length of the small intestine and even along the villus. Rat intestinal P450s are inducible by xenobiotics--with phenobarbital (PB) inducing P4502B1, 3-methylcholanthrene (3-MC) inducing P4501A1, and dexamethasone inducing two forms of P4503A. Induction is most effectively achieved by oral administration of the agents, and is rapid--aryl hydrocarbon hydroxylase (AHH) was increased within 1 h of administration of, for example, 3-MC. AHH, 7-ethoxycoumarin O-deethylase (ECOD), and 7-ethoxyresorufin O-deethylase (EROD) have been used most frequently as substrates to characterize intestinal P450s. Dietary factors affect intestinal P450s markedly--iron restriction rapidly decreased intestinal P450 to beneath detectable values; selenium deficiency acted similarly but was less effective; Brussels sprouts increased intestinal AHH activity 9.8-fold, ECOD activity 3.2-fold, and P450 1.9-fold; fried meat and dietary fat significantly increased intestinal EROD activity; a vitamin A-deficient diet increased, and a vitamin A-rich diet decreased intestinal P450 activities; and excess cholesterol in the diet increased intestinal P450 activity. The role of intestinal P450 in toxifying or detoxifying specific xenobiotics has been clearly demonstrated to only a limited extent. However, elevated intestinal P450 levels have been indirectly linked to gastrointestinal cancer. Intestinal metabolism of 2,2,2-trifluoroethanol produces intestinal lesions with consequent systemic bacterial infection.

The metabolism of the clinically utilized, anticoagulant warfarin [4-hydroxy-3-(3-oxo-1-phenylbutyl)-2H-1-benzopyran-2-one] by rat liver microsomes has been investigated. The structure of a new warfarin metabolite [4-hydroxy-3-(3-oxo-1-phenyl-1-butenyl)-2H-1-benzopyran-2-one] (dehydrowarfarin) has been determined by mass spectral comparison with the chemically synthesized compound. The formation of dehydrowarfarin is catalyzed by cytochrome P-450 and is unusual in that the final product is effectively dehydrogenated warfarin.

A highly metastatic line of Lewis lung tumor cells established in fetal bovine serum (10%) was subcultured into normal rodent (mouse or rat) serum or rodent serum made deficient in functional vitamin K-dependent proteins (barium sulfate adsorption or warfarin treatment of animals). Following injection of cells cultured in normal rodent serum into C57BL/6 mice, Factor X activator activity in the primary tumors increased at a near linear rate per gram tumor and attained 5- to 8-fold higher levels than did cells grown in either of the deficient sera. Secondary lung foci were also visible in all mice of the normal-rodent serum groups within 10 days after injection, and by 21 days extensive tumor growth in the lungs had developed. No secondary lung foci were apparent in any mice of the deficient serum groups throughout 21 days of tumor burden. Cells cultured in nonrodent serum (fetal bovine serum) were less proficient than cells grown in normal mouse serum in developing primary tumor Factor X activator activity and producing secondary tumors. Exposure of cells cultured in barium sulfate-treated mouse serum to normal mouse serum for 3 h and 3 weeks prior to injection partially restored primary tumor Factor X activator and metastatic competence. These data strongly suggest that in Lewis lung tumor cells at least one species selective, plasma/serum vitamin K-dependent protein plays a major role in the regulation of metastatic events and demonstrate that there is a positive correlation between primary tumor Factor X activation activity and metastasis.

The development of the rat hepatic microsomal mixed-function oxidase system was investigated by analyzing its components in fetal and young pups of postconceptional age 18-54 days. Cytochrome P-450 and cytochrome b5 concentrations and NADPH-cytochrome c reductase and R-warfarin hydroxylase activities were determined as a function of postconceptional age. The cytochromes and reductase were present and increased with age before birth, and major increases were detected in the first 2 days after birth. The cytochrome P-450 concentration increased again markedly at puberty, whereas reductase activity increased before puberty and reached the adult level at puberty. Metabolism of warfarin to specific metabolites indicated that at 37 days postconception the isozyme cytochrome P-450 UT-C began to predominate. Its domination of the hepatic cytochrome P-450 pool increased with age.

A partially purified, 200S submicrosomal fraction exhibiting thiol-dependent vitamin K1 (vitamin K) and epoxide reductase activities has been isolated by partial solubilization of rat hepatic microsomes with sodium cholate and separation by centrifugation at 105 000 g into a discontinuous sucrose gradient. At pH 7.4, the rates of vitamin K and vitamin K 2,3-epoxide reduction per milligram of 200S fraction protein were equivalent and were 2.5-3.0 times faster than in microsomes. Reduction of vitamin K 2,3-epoxide occurred in a tightly coupled, two-step reaction initially to vitamin K and subsequently to vitamin K hydroquinone (vitamin KH2). Incorporation of glycerol or sucrose and of sodium cholate into reaction mixtures equivalently affected the rates of both vitamin K and vitamin K 2,3-epoxide reduction, but in the case of epoxide metabolism, the ratios of vitamin KH2/vitamin K were much lower, suggesting that the second reaction has been partially uncoupled from the first. A 14 000-17 000-dalton warfarin-sensitive protein (WSP) that participates in vitamin K and vitamin K 2,3-epoxide reduction in the 200S fraction was identified by incorporation of N-[3H]ethylmaleimide ([3H]NEM) into the catalytically active reduced form of one or more attached disulfides. Reduction of WSP with dithiothreitol was required for reaction with [3H]NEM, and the substrates vitamin K and vitamin K 2,3-epoxide and the inhibitor warfarin all effectively blocked the reaction. 2-Mercaptoethanol could not substitute for dithiothreitol.(ABSTRACT TRUNCATED AT 250 WORDS)

Reduction of vitamin K 2,3-epoxide and vitamin K catalyzed by hepatic microsomal enzymes is required for normal, postribosomal, gamma-carboxyglutamate formation in the prothrombin complex Factors II, VII, IX, and X. The R- and S-warfarin enantiomers differentially inhibit (S-warfarin is 2 to 5 times more active) vitamin K function by mechanisms which have not been unambiguously determined. As a step toward determining the physiologically relevant site(s) of warfarin-antivitamin K activity we investigated in Wistar rats the effects of R- and S-warfarin on vitamin K 2,3-epoxide and vitamin K reductase activities and correlated them with effects on plasma concentrations of the Factors II, VII, and X. Based on the results of these studies we conclude that: 1) warfarin inhibition of the vitamin K 2,3-epoxide and vitamin K reductases is essentially irreversible; 2) S-warfarin stereoselectively inhibits both reductases in vivo but not in vitro; 3) the vitamin K reductase which utilizes dithiothreitol as cofactor in vitro is primarily responsible for vitamin K reduction to vitamin K hydroquinone under physiological conditions; 4) warfarin initially inhibits gamma-carboxyglutamate formation by inhibiting simultaneously the vitamin K 2,3-epoxide and vitamin K reductases; and 5) following enantiomer administration there is an apparent lack of correlation between the restoration of the reductase activities and the reinitiation of coagulation factor synthesis.

The effects of thiols and sulfhydryl blocking reagents on the reduction of vitamin K to vitamin K hydroquinone and vitamin K 2,3-epoxide to vitamin K and vitamin K hydroquinone catalyzed by rat hepatic microsomes were investigated to determine the mechanism(s) for these reactions. Both vitamin K and vitamin K 2,3-epoxide reductions were catalyzed more effectively with dithiols than with monothiols as the reductant. The sulfhydryl reagent N-ethylmaleimide (NEM) inhibited vitamin K and vitamin K 2,3-epoxide reduction much more effectively when microsomes were initially treated with dithiothreitol (prereduced). In prereduced microsomes iodoacetamide was approximately half as effective an inhibitor of vitamin K and vitamin K 2,3-epoxide reduction as NEM, but in microsomes not prereduced it was more effective. Iodoacetic acid was ineffective as an inhibitor. Vitamin K or vitamin K 2,3-epoxide added to prereduced microsomes blocked subsequent inhibition by NEM of vitamin K and vitamin K 2,3-epoxide metabolism, respectively. Vitamin K added to prereduced microsomes also blocked inhibition by NEM of vitamin K 2,3-epoxide metabolism, and vitamin K 2,3-epoxide addition blocked inhibition by NEM of vitamin K metabolism. Vitamin K did not diminish the rate of vitamin K 2,3-epoxide metabolism, however, nor did vitamin K 2,3-epoxide diminish the rate of vitamin K metabolism. These data establish that exogenous thiol compounds promote the reduction of at least one protein disulfide which participates in the metabolism of vitamin K and vitamin K 2,3-epoxide. Presumably, the resultant sulfhydryl groups are reoxidized to the disulfide form during the metabolism of either vitamin which protects them from reaction with NEM.

A new metabolite of vitamin K, 2(3)-hydroxy-2,3-dihydro-2-methyl,3-phytyl-1,4-naphthoquinone (hydroxyvitamin K), has been identified as a product of vitamin K epoxide metabolism in hepatic microsomes from warfarin-resistant rats, but not in those derived from normal rats. The structure was determined by comparison of the high performance liquid chromatography retention times, UV, IR, CD, and mass spectra of the unknown with chemically synthesized standards. Alterations in the formation of hydroxyvitamin K occur in parallel with alterations in total vitamin K epoxide conversion with respect to reaction time, extent of reaction, detergent stimulation, and inhibition by warfarin. Thus, hydroxyvitamin K appears to be a product of the warfarin-resistant vitamin K epoxide reductase. It is neither a substrate nor an inhibitor of epoxide reduction. Hydroxyvitamin K is formed from both enantiomers of racemic vitamin K epoxide with little stereoselectivity for the configuration of either the oxirane ring or the phytyl side chain. The reaction is stereospecific; however, the biologically formed (+)-vitamin K epoxide yields exclusively (+)-3-hydroxyvitamin K. Observation of this product is discussed as a key to understanding the normal reaction mechanism of the enzyme.

The effects of cefoxitin and cefotetan on vitamin K metabolism and clotting parameters in five healthy subjects were investigated. No changes in prothrombin time or in the formation of abnormal prothrombin were seen either during or following the cefoxitin or cefotetan phase. However, when phytonadione (10 mg) (vitamin K1) was administered at the completion of each course of antibiotics, formation of vitamin K 2,3-epoxide was observed only during the cefotetan phase. It is probable, therefore, that cefotetan, a cephamycin antibiotic containing the N-methylthiotetrazole side chain, inhibits hepatic vitamin K 2,3-epoxide reductase. While hypoprothrombinemia and formation of abnormal prothrombin were not seen in healthy subjects, the effect of cefotetan on the coagulation status of vitamin K-depleted patients may be adverse.

A quantitative high-pressure liquid chromatographic assay of warfarin and its diastereoisomeric alcohols and 4'-, 6-, 7-, 8- and benzylic hydroxylated metabolites was accomplished using a reversed-phase, microparticle column (muBondapak/C18) employing 1.5% acetic acid (pH 4.7)-acetonitrile (69:31) as solvent. Concentration of these compounds on the column prior to their elution permitted the loading of 0.01-0.5 ml solutions of warfarin and metabolites without consequent alterations in peak heights and resolution. Details of the use of this technique in the study of R and S warfarin metabolism by hepatic mixed function oxidases and in the determination of warfarin and its metabolites in blood are presented.

Microsomes isolated from Lewis lung (LL) primary tumors raised in C57BL/6 mice have been shown to (i) contain a 4-hydroxycoumarin (warfarin)-sensitive cycle of vitamin K metabolism which is at least qualitatively similar to that of liver, and (ii) catalyze the incorporation of NaH14 CO3 into endogenous protein in a vitamin-K hydroquinone-dependent reaction to produce gamma-carboxyglutamate. As in liver microsomes, LL microsomal reduction of vitamin K 2,3-epoxide to vitamin K was greatly enhanced by exogenous dithiols such as dithiothreitol, but under identical conditions the former was 10-fold faster. The R(+) and S(-) warfarin enantiomers were highly and equally effective inhibitors of both the liver and tumor vitamin K 2,3-epoxide reductases-the average I50 against the tumor enzyme was 0.25 microM. Partially purified reductases isolated by centrifugation of sodium-cholate-treated liver and LL tumor microsomes over a discontinuous sucrose gradient were also inhibited by the sulfhydryl reagent N-ethylmaleimide following their reduction by dithiothreitol. Like the activity of the epoxide reductase, that of the gamma-carboxylase was much lower in tumor than in liver microsomes and was only detectable in microsomes isolated from tumor-bearing mice previously administered S(-) warfarin. In view of the reported inhibition of LL tumor metastasis by warfarin and diet-induced vitamin-K deficiency, vitamin-K-dependent proteins may play a role in the spread and/or subsequent growth of LL cells.

Warfarin is a potent inhibitor of vitamin K 2,3-epoxide reduction to vitamin K in vitro and in vivo. Dithiothreitol, an in vitro reductant for the vitamin K 2,3-epoxide reductase, antagonizes inhibition of the reductase by warfarin via mechanisms that have not been determined [Zimmermann, A., & Matschiner, J. T. (1974) Biochem. Pharmacol. 23, 1033-1040]. Experiments with rat hepatic microsomes were undertaken to characterize the interactions that exist between vitamin K 2,3-epoxide, warfarin, and dithiothreitol. Increasing concentrations of dithiothreitol decreased inhibition of the reductase by warfarin. When dithiothreitol was present prior to exposure of the reductase to warfarin, there was less inhibition than when the same concentration of dithiothreitol was present after its exposure to warfarin. Moreover, maximum inhibition of the reductase by warfarin occurred at a much slower rate when dithiothreitol was present initially. Inhibition of the reductase by warfarin was greater when the substrate concentration was 100 microM vitamin K 2,3-epoxide than when it was 10 microM epoxide. On the basis of these data, we conclude that (i) dithiothreitol reduces either directly or indirectly a critical disulfide within the reductase that it reoxidized during reduction of the epoxide substrate, (ii) warfarin and vitamin K 2,3-epoxide are not competitive with respect to one another, and (iii) warfarin binding, which produces inhibition, occurs solely to the disulfide form of the reductase. Once it is bound, warfarin inhibits further reduction of the critical disulfide by dithiothreitol. Dithiothreitol therefore antagonizes warfarin by maintaining the reductase in the reduced state.

Human alpha-thrombin, the thromboplastin activation product of prothrombin with high clotting and esterase activity, was produced from Cohn Fraction III paste. The procedure started with 0.4 to 3.2 kg of frozen paste and was completed in 2 or 3 days. Some 23 g of thrombin were recorded for 65 quantitated preparations made from 11 lots of Fraction III paste. These preparations were obtained at protein concentrations of 3.9 +/- 1.3 mg/ml with a yield of 340 +/- 110 mg/kg of paste, which represented 48 +/- 14% of the clotting potential extracted as prothrombin. They had specific clotting activities of 2.8 +/- 0.4 U.S. (NIH) units/microng of protein and titrated to 88 +/- 8% active with p-nitrophenyl-p'-guanidinobenzoate (NPGB). Those (N - 29) examined by labeling with [14C]diisopropyl phosphorofluoridate (iPr2P-F) and electrophoresing in sodium dodecyl sulfate (SDS)-polyacrylamide gels were found to contain only (N = 4) or predominantly alpha-thrombin (97 +/- 3%) and corresponding amounts of ists degradation product, beta-thrombin (2.6 +/- 3.1%). No plasmin(ogen), prothrombin complex factors (II, VII, IX, IXalpha, X, Xalpha), or prothrombin fragments were detected in representative preparations. As produced in 0.75 M NaCl, pH approximately 6, thrombin was stable for approximately 1 week at 4 degrees and for greater than 1 year at less than or equal to 50 degrees; freeze-dried thrombin stored at 4 degrees for greater than 1 year displayed stable clotting activity and no vial to vial variation, permitting its use for reference purposes. Human thrombin generated by Taipan snake venom activation was compared with that produced by rapid thromboplastin activation: after treatment with [14C]iPr2P-F, greater than 95% of the label in both thrombins migrated at the same rate during electrophoresis in SDS; identical pairs of NH2-terminal residues were released in three consecutive Edman degradation cycles.