[Show abstract][Hide abstract] ABSTRACT: The spirochete periplasmic flagellum has many unique attributes. One unusual characteristic is the flagellar hook. This structure serves as a universal joint coupling rotation of the membrane-bound motor to the flagellar filament. The hook is comprised of about 120 FlgE monomers, and in most bacteria these structures readily dissociate to monomers (∼ 50 kDa) when treated with heat and detergent. However, in spirochetes the FlgE monomers form a large mass of over 250 kDa [referred to as a high molecular weight complex (HMWC)] that is stable to these and other denaturing conditions. In this communication, we examined specific aspects with respect to the formation and structure of this complex. We found that the Lyme disease spirochete Borrelia burgdorferi synthesized the HMWC throughout the in vitro growth cycle, and also in vivo when implanted in dialysis membrane chambers in rats. The HMWC was stable to formic acid, which supports the concept that the stability of the HMWC is dependent on covalent cross-linking of individual FlgE subunits. Mass spectrometry analysis of the HMWC from both wild type periplasmic flagella and polyhooks from a newly constructed ΔfliK mutant indicated that other proteins besides FlgE were not covalently joined to the complex, and that FlgE was the sole component of the complex. In addition, mass spectrometry analysis also indicated that the HMWC was composed of a polymer of the FlgE protein with both the N- and C-terminal regions remaining intact. These initial studies set the stage for a detailed characterization of the HMWC. Covalent cross-linking of FlgE with the accompanying formation of the HMWC we propose strengthens the hook structure for optimal spirochete motility.
PLoS ONE 05/2014; 9(5):e98338. DOI:10.1371/journal.pone.0098338 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Spirochete motility is enigmatic: It differs from the motility of most other bacteria in that the entire bacterium is involved in translocation in the absence of external appendages. Using the Lyme disease spirochete Borrelia burgdorferi (Bb) as a model system, we explore the current research on spirochete motility and chemotaxis. Bb has periplasmic flagella (PFs) subterminally attached to each end of the protoplasmic cell cylinder, and surrounding the cell is an outer membrane. These internal helix-shaped PFs allow the spirochete to swim by generating backward-moving waves by rotation. Exciting advances using cryoelectron tomography are presented with respect to in situ analysis of cell, PF, and motor structure. In addition, advances in the dynamics of motility, chemotaxis, gene regulation, and the role of motility and chemotaxis in the life cycle of Bb are summarized. The results indicate that the motility paradigms of flagellated bacteria do not apply to these unique bacteria.
[Show abstract][Hide abstract] ABSTRACT: Spirochetes have a unique cell structure: These bacteria have internal periplasmic flagella subterminally attached at each cell end. How spirochetes coordinate the rotation of the periplasmic flagella for chemotaxis is poorly understood. In other bacteria, modulation of flagellar rotation is essential for chemotaxis, and phosphorylation-dephosphorylation of the response regulator CheY plays a key role in regulating this rotary motion. The genome of the Lyme disease spirochete Borrelia burgdorferi contains multiple homologues of chemotaxis genes, including three copies of cheY, referred to as cheY1, cheY2, and cheY3. To investigate the function of these genes, we targeted them separately or in combination by allelic exchange mutagenesis. Whereas wild-type cells ran, paused (flexed), and reversed, cells of all single, double, and triple mutants that contained an inactivated cheY3 gene constantly ran. Capillary tube chemotaxis assays indicated that only those strains with a mutation in cheY3 were deficient in chemotaxis, and cheY3 complementation restored chemotactic ability. In vitro phosphorylation assays indicated that CheY3 was more efficiently phosphorylated by CheA2 than by CheA1, and the CheY3-P intermediate generated was considerably more stable than the CheY-P proteins found in most other bacteria. The results point toward CheY3 being the key response regulator essential for chemotaxis in B. burgdorferi. In addition, the stability of CheY3-P may be critical for coordination of the rotation of the periplasmic flagella.
Journal of bacteriology 07/2011; 193(13):3332-41. DOI:10.1128/JB.00362-11 · 2.81 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: HD-GYP domain cyclic dimeric GMP (c-di-GMP) phosphodiesterases are implicated in motility and virulence in bacteria. Borrelia burgdorferi possesses a single set of c-di-GMP-metabolizing enzymes, including a putative HD-GYP domain protein, BB0374. Recently, we
characterized the EAL domain phosphodiesterase PdeA. A mutation in pdeA resulted in cells that were defective in motility and virulence. Here we demonstrate that BB0374/PdeB specifically hydrolyzed
c-di-GMP with a Km of 2.9 nM, confirming that it is a functional phosphodiesterase. Furthermore, by measuring phosphodiesterase enzyme activity
in extracts from cells containing the pdeA pdeB double mutant, we demonstrate that no additional phosphodiesterases are present in B. burgdorferi. pdeB single mutant cells exhibit significantly increased flexing, indicating a role for c-di-GMP in motility. Constructing and
analyzing a pilZ pdeB double mutant suggests that PilZ likely interacts with chemotaxis signaling. While virulence in needle-inoculated C3H/HeN
mice did not appear to be altered significantly in pdeB mutant cells, these cells exhibited a reduced ability to survive in Ixodes scapularis ticks. Consequently, those ticks were unable to transmit the infection to naïve mice. All of these phenotypes were restored
when the mutant was complemented. Identification of this role of pdeB increases our understanding of the c-di-GMP signaling network in motility regulation and the life cycle of B. burgdorferi.
Infection and immunity 06/2011; 79(8):3273-83. DOI:10.1128/IAI.05153-11 · 3.73 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The cyclic-dimeric-GMP (c-di-GMP)-binding protein PilZ has been implicated in bacterial motility and pathogenesis. Although
BB0733 (PlzA), the only PilZ domain-containing protein in Borrelia burgdorferi, was reported to bind c-di-GMP, neither its role in motility or virulence nor it's affinity for c-di-GMP has been reported.
We determined that PlzA specifically binds c-di-GMP with high affinity (dissociation constant [Kd], 1.25 μM), consistent with Kd values reported for c-di-GMP-binding proteins from other bacteria. Inactivation of the monocistronically transcribed plzA resulted in an opaque/solid colony morphology, whereas the wild-type colonies were translucent. While the swimming pattern
of mutant cells appeared normal, on swarm plates, mutant cells exhibited a significantly reduced swarm diameter, demonstrating
a role of plzA in motility. Furthermore, the plzA mutant cells were significantly less infectious in experimental mice (as determined by 50% infectious dose [ID50]) relative to wild-type spirochetes. The mutant also had survival rates in fed ticks lower than those of the wild type. Consequently,
plzA mutant cells failed to complete the mouse-tick-mouse infection cycle, indicating plzA is essential for the enzootic life cycle of B. burgdorferi. All of these defects were corrected when the mutant was complemented in cis. We propose that failure of plzA mutant cells to infect mice was due to altered motility; however, the possibility that an unidentified factor(s) contributed
to interruption of the B. burgdorferi enzootic life cycle cannot yet be excluded.
Infection and immunity 02/2011; 79(5):1815-25. DOI:10.1128/IAI.00075-11 · 3.73 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Scuba divers typically rinse equipment in communal tanks. Studies show these tanks are contaminated with bacteria, but the types of bacteria have not been studied. We sought to identify bacteria in rinse tanks at a dive facility at San Pedro, Belize, to determine the origin of the bacteria and determine whether the bacteria represented potential threats to human health. The identity of bacteria was investigated using reverse line blot (RLB) assays based on 28 different rDNA probes designed to detect known pathogens of sepsis, as well as by sequencing 23S rDNA from isolates and performing VITEK identification of several isolates. Based on the identities of bacteria in divers' rinse tanks, many likely originate from the ocean, and others likely originate from the divers themselves. None of the bacteria identified would be considered overt human pathogens. However, some of the bacteria found in the tanks are known to be associated with unsanitary conditions and can cause opportunistic infections, which may pose health problems to some individuals. Rinsing scuba equipment in communal tanks has the potential to transmit disease among some divers. Equipment, especially regulators and masks, should be rinsed/cleaned individually and not be placed in communal tanks.
Undersea & hyperbaric medicine: journal of the Undersea and Hyperbaric Medical Society, Inc 07/2010; 37(4):233-40. · 0.77 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The genome of Borrelia burgdorferi encodes a set of genes putatively involved in cyclic-dimeric guanosine monophosphate (cyclic-di-GMP) metabolism. Although BB0419 was shown to be a diguanylate cyclase, the extent to which bb0419 or any of the putative cyclic-di-GMP metabolizing genes impact B. burgdorferi motility and pathogenesis has not yet been reported. Here we identify and characterize a phosphodiesterase (BB0363). BB0363 specifically hydrolyzed cyclic-di-GMP with a K(m) of 0.054 microM, confirming it is a functional cyclic-di-GMP phosphodiesterase. A targeted mutation in bb0363 was constructed using a newly developed promoterless antibiotic cassette that does not affect downstream gene expression. The mutant cells exhibited an altered swimming pattern, indicating a function for cyclic-di-GMP in regulating B. burgdorferi motility. Furthermore, the bb0363 mutant cells were not infectious in mice, demonstrating an important role for cyclic-di-GMP in B. burgdorferi infection. The mutant cells were able to survive within Ixodes scapularis ticks after a blood meal from naïve mice; however, ticks infected with the mutant cells were not able to infect naïve mice. Both motility and infection phenotypes were restored upon genetic complementation. These results reveal an important connection between cyclic-di-GMP, B. burgdorferi motility and Lyme disease pathogenesis. A mechanism by which cyclic-di-GMP influences motility and infection is proposed.
[Show abstract][Hide abstract] ABSTRACT: Lyme disease is a common vector-borne disease caused by the spirochete Borrelia burgdorferi (Bb), which manifests as systemic and targeted tissue inflammation. Both in vitro and in vivo studies have shown that Bb-induced inflammation is primarily host-mediated, via cytokine or chemokine production that promotes leukocyte adhesion/migration. Whether Bb produces mediators that can directly alter the vascular permeability in vivo has not been investigated. The objective of the present study was to investigate if Bb produces a mediator(s) that can directly activate endothelial cells resulting in increases in permeability in intact microvessels in the absence of blood cells.
The effects of cell-free, spent culture medium from virulent (B31-A3) and avirulent (B31-A) B. burgdorferi on microvessel permeability and endothelial calcium concentration, [Ca(2+)](i), were examined in individually perfused rat mesenteric venules. Microvessel permeability was determined by measuring hydraulic conductivity (Lp). Endothelial [Ca(2+)](i), a necessary signal initiating hyperpermeability, was measured in Fura-2 loaded microvessels. B31-A3 spent medium caused a rapid and transient increase in Lp and endothelial [Ca(2+)](i). Within 2-5 min, the mean peak Lp increased to 5.6+/-0.9 times the control, and endothelial [Ca(2+)](i) increased from 113+/-11 nM to a mean peak value of 324+/-35 nM. In contrast, neither endothelial [Ca(2+)](i) nor Lp was altered by B31-A spent medium.
A mediator(s) produced by virulent Bb under culture conditions directly activates endothelial cells, resulting in increases in microvessel permeability. Most importantly, the production of this mediator is associated with Bb virulence and is likely produced by one or more of the 8 plasmid(s) missing from strain B31-A.
PLoS ONE 02/2008; 3(12):e4101. DOI:10.1371/journal.pone.0004101 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Measuring the chemotactic response of Borrelia burgdorferi, the bacterial species that causes Lyme disease, is relatively more difficult than measuring that of other bacteria. Because these spirochetes have long generation times, enumerating cells that swim up a capillary tube containing an attractant by using colony counts is impractical. Furthermore, direct counts with a Petroff-Hausser chamber is problematic, as this method has a low throughput and necessitates a high cell density; the latter can lead to misinterpretation of results when assaying for specific attractants. Only rabbit serum and tick saliva have been reported to be chemoattractants for B. burgdorferi. These complex biological mixtures are limited in their utility for studying chemotaxis on a molecular level. Here we present a modified capillary tube chemotaxis assay for B. burgdorferi that enumerates cells by flow cytometry. Initial studies identified N-acetylglucosamine as a chemoattractant. The assay was then optimized with respect to cell concentration, incubation time, motility buffer composition, and growth phase. Besides N-acetylglucosamine, glucosamine, glucosamine dimers (chitosan), glutamate, and glucose also elicited significant chemoattractant responses, although the response obtained with glucose was weak and variable. Serine and glycine were nonchemotactic. To further validate and to exploit the use of this assay, a previously described nonchemotactic cheA2 mutant was shown to be nonchemotactic by this assay; it also regained the wild-type phenotype when complemented in trans. This is the first report that identifies specific chemical attractants for B. burgdorferi and the use of flow cytometry for spirochete enumeration. The method should also be useful for assaying chemotaxis for other slow-growing prokaryotic species and in specific environments in nature.
[Show abstract][Hide abstract] ABSTRACT: Constructing mutants by targeted gene inactivation is more difficult in the Lyme disease organism, Borrelia burgdorferi, than in many other species of bacteria. The B. burgdorferi genome is fragmented, with a large linear genome and 21 linear and circular plasmids. Some of these small linear and circular plasmids are often lost during laboratory propagation, and the loss of specific plasmids can have a significant impact on virulence. In addition to the unusual structure of the B. burgdorferi genome, the presence of an active restriction-modification system impedes genetic transformation. Furthermore, B. burgdorferi is relatively slow growing, with a 7- to 12-h generation time, requiring weeks to obtain single colonies. The beginning part of this chapter details the procedure in targeting specific B. burgdorferi genes by allelic exchange mutagenesis. Our laboratory is especially interested in constructing and analyzing B. burgdorferi chemotaxis and motility mutants. Characterization of these mutants with respect to chemotaxis and swimming behavior is more difficult than for many other bacterial species. We have developed swarm plate and modified capillary tube assays for assessing chemotaxis. In the modified capillary tube chemotaxis assay, flow cytometry is used to rapidly enumerate cells that accumulate in the capillary tubes containing attractants. To assess the swimming behavior and velocity of B. burgdorferi wild-type and mutant cells, we use a commercially available cell tracker referred to as "Volocity." The latter part of this chapter presents protocols for performing swarm plate and modified capillary tube assays, as well as cell motion analysis. It should be possible to adapt these procedures to study other spirochete species, as well as other species of bacteria, especially those that have long generation times.
Methods in Enzymology 02/2007; 422:421-37. DOI:10.1016/S0076-6879(06)22021-4 · 2.09 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Borrelia burgdorferi has a complex chemotaxis signal transduction system with multiple chemotaxis gene homologs similar to those found in Escherichia coli and Bacillus subtilis. The B. burgdorferi genome sequence encodes two cheA, three cheY, three cheW, two cheB, two cheR, but no cheZ genes. Instead of cheZ, B. burgdorferi contains a different CheY-P phosphatase, referred to as cheX. The multiple B. burgdorferi histidine kinases (CheA1 and CheA2) and response regulators (CheY1, CheY2, and CheY3) possess all the domains and functional residues found in E. coli CheA and CheY, respectively. Understanding protein phosphorylation is critical to unraveling many biological processes, including chemotaxis signal transduction, motility, growth control, metabolism, and disease processes. E. coli, Salmonella enterica serovar Typhimurium, and B. subtilis chemotaxis systems have been studied extensively, providing models to understand chemotaxis signaling in the Lyme disease spirochete B. burgdorferi. Both genetic approaches and biochemical analyses are essential in understanding its complex two-component chemotaxis systems. Specifically, gene inactivation studies assess the importance of specific genes in chemotaxis and motility under certain conditions. Furthermore, biochemical approaches help determine the following in vitro reactions: (1) the extent that the histidine kinases, CheA1 and CheA2, are autophosphorylated using ATP; (2) the transfer of phosphate from CheA1-P and CheA2-P to each CheY species; and (3) the dephosphorylation of each CheY-P species by CheX. We hypothesize that characterizing protein phosphorylation in the B. burgdorferi two-component chemotaxis system will facilitate understanding of how the periplasmic flagellar bundles located near each end of B. burgdorferi cells are coordinately regulated for chemotaxis. During chemotaxis, these bacteria run, pause (stop/flex), and reverse (run again). This chapter describes protocols for assessing B. burgdorferi CheA autophosphorylation, transfer of phosphate from CheA-P to CheY, and CheY-P dephosphorylation.
Methods in Enzymology 02/2007; 422:438-47. DOI:10.1016/S0076-6879(06)22022-6 · 2.09 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Steroid hormones are known to affect the humoral immune response to a variety of antigens. However, the mechanisms regulating these effects are poorly understood. The immunotoxic chemical propanil and estrogen have similar effects on the immune system including augmentation of humoral immune responses. Propanil enhances the number of phosphorylcholine (PC)-specific IgG2b, IgG3, and IgM antibody-secreting cells (ASCs) in the spleen four- to sixfold 7 days after vaccination of female C57BL/6 mice with heat-killed Streptococcus pneumoniae. Several experiments were performed to test the hypothesis that propanil increases the response via an estrogenic pathway. Ovariectomy abrogated the effect of propanil on the PC-specific ASC response. Both in vitro and in vivo assays indicate that propanil does not bind either estrogen receptor (ER) alpha or beta. Exogenous estradiol administration in ovariectomized mice failed to restore the effect of propanil on the PC response. Treatment of female mice with a pure ER antagonist, ICI 182,780, or the progesterone antagonist RU486 did not inhibit the increase in ASCs. These data suggest that estrogen and progesterone do not regulate the effect of propanil. However, complete inhibition of steroid synthesis with the gonadotropin-releasing hormone (GnRH) antagonist antide abrogated the increased response in propanil-treated mice, indicating a necessary role for steroid synthesis. Experiments in male mice demonstrated that propanil increased the number of ASCs comparable to female mice. However, orchiectomy did not inhibit this effect, suggesting that androgens do not regulate the amplification of the humoral response. These data suggest a novel role for the ovarian hormones in the regulation of the PC-specific antibody response.
[Show abstract][Hide abstract] ABSTRACT: Motility and chemotaxis are believed to be important in the pathogenesis of Lyme disease caused by the spirochete Borrelia burgdorferi. Controlling the phosphorylation state of CheY, a response regulator protein, is essential for regulating bacterial chemotaxis
and motility. Rapid dephosphorylation of phosphorylated CheY (CheY-P) is crucial for cells to respond to environmental changes.
CheY-P dephosphorylation is accomplished by one or more phosphatases in different species, including CheZ, CheC, CheX, FliY,
and/or FliY/N. Only a cheX phosphatase homolog has been identified in the B. burgdorferi genome. However, a role for cheX in chemotaxis has not been established in any bacterial species. Inactivating B. burgdorferi cheX by inserting a flgB-kan cassette resulted in cells (cheX mutant cells) with a distinct motility phenotype. While wild-type cells ran, paused (stopped or flexed), and reversed, the
cheX mutant cells continuously flexed and were not able to run or reverse. Furthermore, swarm plate and capillary tube chemotaxis
assays demonstrated that cheX mutant cells were deficient in chemotaxis. Wild-type chemotaxis and motility were restored when cheX mutant cells were complemented with a shuttle vector expressing CheX. Furthermore, CheX dephosphorylated CheY3-P in vitro
and eluted as a homodimer in gel filtration chromatography. These findings demonstrated that B. burgdorferi CheX is a CheY-P phosphatase that is essential for chemotaxis and motility, which is consistent with CheX being the only
CheY-P phosphatase in the B. burgdorferi chemotaxis signal transduction pathway.
Journal of Bacteriology 01/2006; 187(23):7963-9. DOI:10.1128/JB.187.23.7963-7969.2005 · 2.81 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: While some studies report that estradiol (E2) activates extracellular-signal regulated kinase (Erk1/2) in MCF-7 breast cancer cells, others report E2 does not activate this signaling pathway. This study attempted to resolve the conflicting reports by investigating experimental variables that could impact Erk1/2 activation using a high through-put assay that quantitatively assessed Erk1/2 phosphorylation. Variables tested included: cell staging and dosing regimes with and without charcoal-stripped serum, different MCF-7 cell sublines and culture densities and several E2 formulations and solvents. Levels of phosphorylated Erk1/2 were normalized to cellular protein rather than to total Erk1/2 protein because an antibody purported to recognize total Erk1/2 preferentially reacted with non-phosphorylated Erk1/2, potentially exaggerating the apparent level of Erk1/2 activation. Dosing MCF-7 cells with E2 containing small amounts of stripped serum induced Erk1/2 phosphorylation; however, this induction was largely attributed to serum factors. E2 administered in serum-free medium did not significantly alter Erk1/2 phosphorylation under any condition tested; immunocytochemistry corroborated this conclusion. While phosphatase inhibitors generally increased Erk1/2 phosphorylation, they did not impact E2-altered Erk1/2 phosphorylation. It remains important to resolve the basis of conflicting reports regarding E2-induced Erk1/2 activation due to the potential importance of this pathway on breast cancer and other processes.
The Journal of Steroid Biochemistry and Molecular Biology 10/2005; 96(5):375-85. DOI:10.1016/j.jsbmb.2005.05.006 · 3.63 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The purpose of this study was to determine the potential relationship between imiquimod and C/EBPbeta by investigating the extent to which imiquimod could alter C/EBPbeta binding activity to known sequences of the HPV-16 NCR, which could lead to the repression of HPV-16 E6/E7 oncogene expression, possibly impacting a major mechanism by which HPV causes cellular transformation.
The effect of imiquimod treatment on C/EBPbeta binding activity to its consensus sequence as well as to two specific regions of the HPV-16 NCR was determined by electromobility shift assay (EMSA) in CaSki cervical cancer cells. HPV-16 E6/E7 gene expression was evaluated by RNase protection assay (RPA) in CaSki and in W12-E cells treated with imiquimod. In addition, C/EBPbeta mRNA expression and protein production in response to imiquimod were evaluated by reverse transcription polymerase chain reaction (RT-PCR) and Western blotting, respectively, in the cervical cancer cell lines, CaSki, HeLa, and C33A.
C/EBPbeta binding activity, mRNA expression, and protein production remained unchanged with imiquimod treatment. Initially, HPV-16 E6/E7 expression appeared to be increased with imiquimod treatment in CaSki cells, but this effect was not reproducible. HPV-16 E6/E7 expression was not reproducibly altered with imiquimod treatment in W12-E cells.
While these results indicate that imiquimod does not alter C/EBPbeta binding activity, nor does it appear to decrease HPV-16 E6/E7 oncogene expression in vitro, it remains possible that imiquimod may have utility in treating cervical dysplasia or cervical cancer via another mechanism.
[Show abstract][Hide abstract] ABSTRACT: The purpose of this study was to test the hypothesis that acetaminophen would alter an estrogen-regulated process in human cells that express endogenous estrogen receptor alpha and beta (ERalpha and ERbeta). Specifically, the extent to which acetaminophen altered the expression of estrogen-inducible alkaline phosphatase in endometrial adenocarcinoma (Ishikawa) cells and directly interacted with ERbeta and ERalpha was determined. Ishikawa cells were exposed to estradiol and/or to a range of concentrations of acetaminophen for four days, and alkaline phosphatase activity was measured spectrophotometrically. Acetaminophen inhibited both basal and estradiol-induced alkaline phosphatase activity in Ishikawa cells in a concentration-dependent manner. The reduction of Ishikawa cell alkaline phosphatase was not due to direct inhibition of enzyme activity by acetaminophen. Toxic effects of acetaminophen on Ishikawa cells were determined by measuring loss of cellular lactate dehydrogenase to culture medium. High concentrations of acetaminophen (>/=0.5 mM) induced lactate dehydrogenase release from cells and reduced the amount of cellular protein in culture dishes, indicating some acetaminophen-induced reduction of alkaline phosphatase activity might be attributed to toxic effects. However, lower concentrations of acetaminophen significantly reduced alkaline phosphatase activity in the absence of detectable toxicity. Acetaminophen also augmented 4-hydroxy-tamoxifen reduction of alkaline phosphatase activity. Competition binding assays with human ERalpha and ERbeta demonstrated 10(6)-fold molar excess acetaminophen did not directly interact significantly with the ligand-binding domain of either receptor. These studies indicate acetaminophen exerts weak antiestrogenic activity in Ishikawa cells without directly binding ERalpha or ERbeta.
[Show abstract][Hide abstract] ABSTRACT: Studies reported here tested the hypothesis that acetaminophen stimulates proliferation of E2-responsive cells by inducing expression of E2-regulated genes. Ribonuclease protection assays compared the effects of acetaminophen and E2 on expression of selected genes (c-myc, c-fos, cyclin D1, bcl-2, bax, gadd45, mcl-1, p53, p21(CIP1/WAF1), and bcl-xL) in E2-responsive breast cancer (MCF-7) and endometrial adenocarcinoma (Ishikawa) cells as well as in E2-nonresponsive (MDA-MB-231) breast cancer cells. Acetaminophen and E2 increased c-myc RNA levels in MCF-7 cells, consistent with a mitogenic activity of these compounds in MCF-7 cells. However, the magnitude and time course of acetaminophen and E2 induction of c-myc differed. Neither acetaminophen nor E2 induced c-myc in MDA-MB-231 cells, whereas E2, but not acetaminophen, weakly induced c-myc expression in Ishikawa cells. Furthermore, in these 3 cell types, the expression patterns of the other genes differed dramatically in response to acetaminophen and to E2, indicating that acetaminophen does not activate ER as a transcription factor in the same manner as does E2. Additionally, gel shift assays demonstrated that in MCF-7 cells, acetaminophen increased NF-kappaB activity approximately 40% and did not alter AP-1 activity, whereas E2 increased AP-1 activity approximately 50% and did not increase NF-B activity. These studies indicate that acetaminophen effects on gene expression and cell proliferation depend more on cell type/context than on the presence of ER.
[Show abstract][Hide abstract] ABSTRACT: The causes of non-trauma-mediated rhabdomyolysis are not well understood. It has been speculated that ethanol-associated rhabdomyolysis may be attributed to ethanol induction of skeletal muscle cytochrome P450(s), causing drugs such as acetaminophen or cocaine to be metabolized to myotoxic compounds. To examine this possibility, the hypothesis that feeding ethanol induces cytochrome P450 in skeletal muscle was tested. To this end, rats were fed an ethanol-containing diet and skeletal muscle tissue was assessed for induction of CYP2E1 and CYP1A1/2 by immunohistochemical procedures; liver was examined as a positive control tissue. Enzymatic assays and Western blot analyses were also performed on these tissues. In one feeding system, ethanol-containing diets induced CYP1A1/2 in soleus, plantaris, and diaphragm muscles, with immunohistochemical staining predominantly localized to capillaries surrounding myofibers. Antibodies to CYP2E1 did not react with skeletal muscle tissue from animals receiving a control or ethanol-containing diet. However, neither skeletal muscle CYP1A1/2 nor CYP2E1 was induced when ethanol diets were administered by a different feeding system. Ethanol consumption can induce some cytochrome P450 isoforms in skeletal muscle tissue; however, the mechanism of CYP induction is apparently complex and appears to involve factors in addition to ethanol, per se.
[Show abstract][Hide abstract] ABSTRACT: Metabolism, DNA adduction, and tumor induction by 7, 12-dimethylbenz(a)anthracene (DMBA) were examined in cultured trout liver cells and in vivo in trout. Modulating CYP1A1 activity indicated this enzyme plays a significant role in metabolizing DMBA to water-soluble compounds in isolated trout liver cells. The major DMBA metabolites identified in trout liver cells were 10-, 11-, 8,9-, and 5,6-DMBA dihydrodiols, and DMBA, 2- or 3- or 4-phenol; 7-OH-methyl-12-methyl-benz(a)anthracene and 12-OH-methyl-7-methyl-benz(a)anthracene were minor metabolites. A very small amount of DMBA-3,4-dihydrodiol was detected, and polar metabolites, which did not migrate with any DMBA metabolite standards, were observed. Incubating trout hepatocytes with DMBA-3, 4-dihydrodiol produced three prominent, nonpolar adducts indistinguishable from those in mouse embryo cells. However, DMBA-DNA adducts, formed in trout in vivo or in trout liver cells exposed to DMBA, were predominantly more polar than those formed in mouse embryo fibroblasts, and levels of DMBA-DNA adducts formed in trout liver cells were not significantly altered by modulating CYP1A1 activity. No significant repair of DMBA-DNA adducts was detected in cultured trout liver cells over a 48-h period, supporting previous studies indicating that fish are less efficient than mammals in repairing polyaromatic hydrocarbon DNA adducts. Compared to animals receiving DMBA alone, beta-naphthoflavone pretreatment in vivo did not affect hepatic CYP1A1, DMBA-DNA adducts, nor hepatic tumor response; but did significantly reduce tumor response in two other target organs. These results collectively indicate that DMBA bioactivation to DNA-binding metabolites in trout liver cells and mouse embryo cells predominantly involve different metabolic pathways to form the DNA-binding intermediates.
[Show abstract][Hide abstract] ABSTRACT: Previous studies have shown that acetaminophen, a common analgesic/antipyretic, induces proliferation of cultured breast cancer cells containing both estrogen and progesterone receptors (ER+/PR+). The main objective of this study was to evaluate the involvement of ERs in this effect. First, the effects of therapeutic acetaminophen concentrations were compared in breast cancer cells with high ERs and in T47Dco cells with lower ERs, to determine if acetaminophen-induced proliferation depends on ER levels. Second, the effects of two antiestrogens (ICI 182,780 and 4'-hydroxytamoxifen) on acetaminophen-induced proliferation were determined in three human breast cancer cell lines: two ER+/PR+ (MCF7, T47D) and one ER-/PR- (MDA-MB-231). Third, ER binding assays were performed in MCF7 cells to determine if acetaminophen competed with estradiol for binding to ERs. Proliferation endpoints monitored included percent cells in the DNA synthesis phase of the cell cycle, 3H-thymidine incorporation into DNA, and cell number. Acetaminophen did not induce DNA synthesis in T47Dco cells, but did in cells with higher ER levels, suggesting high ER levels are necessary for acetaminophen to induce proliferation. Antiestrogens inhibited acetaminophen-induced proliferation in ER+/PR+ cells while no effects were observed in ER-/PR- cells, further supporting ER involvement. However, acetaminophen did not compete with estradiol for binding to ERs in ER+/PR+ cells. Collectively, these data suggest that acetaminophen induces breast cancer cell proliferation via ERs without binding to ERs like estradiol. The second purpose of this study was to determine if acetaminophen is estrogenic/antiestrogenic in vivo (uterotrophic assays). Acetaminophen has no antiestrogenic/estrogenic activity in mice or rats uteri.