Perspective on this Article from Early Changes in Gene Expression Induced by Tobacco Smoke: Evidence for the Importance of Estrogen within Lung Tissue

ObjectivesWe hypothesized that short chain fatty acid (SCFA) production by oral pathogens is suppressed by exposure to cigarette smoke extract (CSE).BackgroundTobacco smoking is a major risk factor for plaque‐induced periodontal diseases. Despite increased disease susceptibility, overt oral inflammation is suppressed in smokers, presenting a diagnostic conundrum. Bacterial‐derived SCFAs can penetrate into oral tissues where they influence multiple components of immune and healing responses. Indeed, the SCFA burden has been correlated with the inflammatory condition of the gingiva. However, the influence of cigarette consumption on SCFA production is unknown.MethodsGC/MS was employed to monitor the production of several SCFAs (propionic acid, isobutyric acid, butyric acid, and isovaleric acid) by representative anaerobic oral pathogens (Filifactor alocis 35896, Fusobacterium nucleatum 25586, Porphyromonas gingivalis 33277) that were exposed, or not, to a physiologically relevant dose of CSE (2000 ng/ml nicotine equivalents) generated from 3R4F reference cigarettes.ResultsThe growth of all three bacterial species was unaffected by CSE. The capacity to produce SCFAs by these bacteria was highly varied. F alocis produced the highest concentration of a specific SCFA (butyrate); P gingivalis provided the most robust overall SCFA signal, while F alocis and F nucleatum did not release detectable levels of isobutyrate or isovalerate. As P gingivalis 33277 was the broadest SCFA producer, three low‐passage clinical isolates (10208C, 5607, and 10512) were also examined. Compared to unconditioned microbes, reduced SCFA release was apparent in CSE‐exposed low‐passage clinical isolates of P gingivalis which reached significance for one of the three isolates (propionic, isobutyric, butyric, and isovaleric acids, all P < 0.05).ConclusionsThere is high disparity in the SCFA profiles of variant chronic periodontitis‐associated bacteria, while CSE exposure reduces SCFA production by a specific clinical strain of P gingivalis. If the latter phenomenon occurs in vivo, a reduced SCFA burden may help explain the reduced vascular response to dental plaque in tobacco smokers.

: The twenty-first century warfighter will encounter the challenge of the potential use of toxic industrial chemicals (TICs) as chemical warfare agents. Large quantities of TICs are manufactured, stored, transported and used throughout the world. Many of these TICs are highly toxic and can rapidly affect exposed individuals causing severe injury and even deaths. Because TICs are stored and transported under relatively less secure conditions, these materials are easier for terrorists to inflict significant casualties against not only military but also civilian populations. In the event of a chemical attack, it will be vital to determine those individuals whose level of exposure would be anticipated to result in significant injury and possible death from those receiving only minimal or no exposure. The purpose of this project is to define the chemical signatures of the TICs acrylonitrile and acrolein in human blood, and potentially to devise a rapid, high throughput screening technology to enable examination of large groups of individuals following a known or suspected exposure. Studies have been directed toward defining the patterns of chemical reactivity of blood components with these TICs in order to use these adducts as biomarkers of chemical exposure. More recently experiments have focused on inventorying the tissue protein targets of acrylonitrile in order to gain insight into its mechanism of toxicity so that countermeasures (antidotes) may be developed.

Two human bone marrow stromal cell lines, HS5 and HS27a, co-cultured with myeloid cells, have frequently been used in studies of cross talk between cells in the bone marrow microenvironment and hematopoietic cells. Altered expression of proteins is typically associated with cell–cell signal transduction and regulation of cellular functions. Many studies have focused on key proteins that contribute to functional differences in cell co-culture models, but global quantitative proteome analysis of HS5 and HS27a has not been performed. We employed the stable isotope labeling by amino acids in cell culture (SILAC) method using two stable isotopes each of arginine and lysine to label proteins in the two cell lines. Labeled proteins were analyzed by 2-D ultrahigh-resolution liquid chromatography– LTQ/Orbitrap mass spectrometry. Among 4,213 unique identified and annotated proteins in the cell lines, 1,462 were detected in two independent experiments. Of these, 69 exhibited significant upregulation and 48 significant downregulation (>95% confidence) in HS27a relative to HS5 cells. Gene ontology term and pathway analysis indicated that the differentially regulated proteins were involved in cellular movement, cell-to-cell signaling and interaction, and hematologic system development and function. A total of 55 items were identified in both genomic and proteomic databases. Quantitative reverse transcription polymerase chain reaction and Western blotting were performed on 7 proteins randomly selected from 28 differentially expressed proteins that were identified in both databases and were involved in the top networks/pathways. We observed a decrease in apoptosis in co-cultured KG1a cells when integrin αV was inhibited in HS27a cells, which suggested the functional role of integrin αV in the co-culture system. The integrated genomic/proteomic approach described here, and the identified proteins, will provide a useful basis for further elucidation of molecular mechanisms in the bone marrow microenvironment and for ongoing studies of cross talk among stromal cells and myeloma cells in co-culture systems.

Abstract Lung cancer is the leading cause of cancer deaths in the United States, surpassing breast cancer as the primary cause of cancer-related mortality in women. The goal of the present study was to identify early molecular changes in the lung induced by exposure to tobacco smoke and thus identify potential targets for chemoprevention. Female A/J mice were exposed to either tobacco smoke or HEPA-filtered air via a whole-body exposure chamber (6 h/d, 5 d/wk for 3, 8, and 20 weeks). Gene expression profiles of lung tissue from control and smoke-exposed animals were established using a 15K cDNA microarray. Cytochrome P450 1b1, a phase I enzyme involved in both the metabolism of xenobiotics and the 4-hydroxylation of 17β-estradiol (E2), was modulated to the greatest extent following smoke exposure. A panel of 10 genes were found to be differentially expressed in control and smoke-exposed lung tissues at 3, 8, and 20 weeks (P < 0.001). The interaction network of these differentially expressed genes revealed new pathways modulated by short-term smoke exposure, including estrogen metabolism. In addition, E2 was detected within murine lung tissue by gas chromatography-coupled mass spectrometry and immunohistochemistry. Identification of the early molecular events that contribute to lung tumor formation is anticipated to lead to the development of promising targeted chemopreventive therapies. In conclusion, the presence of E2 within lung tissue when combined with the modulation of cytochrome P450 1b1 and other estrogen metabolism genes by tobacco smoke provides novel insight into a possible role for estrogens in lung cancer. Cancer Prev Res; 3(6); 707–17. ©2010 AACR.

Previous studies on the metabolic activation of polycyclic aromatic hydrocarbons have shown conclusively that activation to reactive electrophilic intermediates occurs via metabolism of the unsubstituted aromatic hydrocarbon to electrophilic diol-epoxide intermediates. These reactive electrophilic derivatives undergo reaction with various biological nucleophilic centers to form covalent modifications, which can be assessed by a variety of analytical techniques. While the formation of these adducts with various cellular nucleophiles has been clearly established, the relationships between relative rates of formation of various adducts in biological systems has not been significantly investigated. In the present study, we investigated the pharmacokinetics of reaction of the ultimate carcinogenic metabolite of benzo(a)pyrene, (±)-anti-7,8-dihydroxy-9α,10α -epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE), with both male C57BL/6 mouse red cell hemoglobin and human red cells, in vitro. Comparative pharmacokinetic studies using both mouse and human hemoglobin were carried out by incubation of the packed red cells together with solutions of (±)-anti-7,8-dihydroxy-9α,10α -epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene. Similar rate constants for the binding of the epoxide to hemoglobin in both mouse and human were found, suggesting a similarity of amino acids that were adducted in both species. Overall binding of (±)-anti-7,8-dihydroxy-9α,10α -epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene to mouse hemoglobin was found to be slightly higher than that found using human hemoglobin, suggesting the potential of a facilitation of the binding of the epoxide with mouse protein or an additional reactive amino acid to which the epoxide was bound. In parallel studies, mice were injected intraperitoneally with benzo(a)pyrene to compare the kinetics of binding of this carcinogen to hemoglobin, in vivo. Blood samples were obtained at specific intervals, and the kinetics of binding to hemoglobin characterized. These results suggested that the binding to hemoglobin in the in vivo studies was slightly lower than that compared to the in vitro assessments. However the differences in adduct levels strongly suggests that other metabolic pathways are involved in the metabolic activation of benzo(a)pyrene as well as other related carcinogenic compounds.

The diene monomers, 1,3-butadiene, chloroprene, and isoprene, respectively, differ only in substitution of a hydrogen, a chlorine, or a methyl group at the second of the four unsaturated carbon atoms in these linear molecules. Literature reviewed in the preceding sections indicates that these chemicals have important uses in synthesis of polymers, which offer significant benefits within modern society. Additionally, studies document that these monomers can increase the tumor formation rate in various organs of rats and mice during chronic cancer bioassays. The extent of tumor formation versus animal exposure to these monomers varies significantly across species, as well among strains within species. These studies approach, but do not resolve, important questions of human risk from inhalation exposure. Each of these diene monomers can be activated to electrophilic epoxide metabolites through microsomal oxidation reactions in mammals. These epoxide metabolites are genotoxic through reactions with nucleic acids. Some of these reactions cause mutations and subsequent cancers, as noted in animal experiments. Significant differences exist among the compounds, particularly in the extent of formation of highly mutagenic diepoxide metabolites, when animals are exposed. These metabolites are detoxified through hydrolysis by epoxide hydrolase enzymes and through conjugation with glutathione with the aid of glutathione S-transferase. Different strains and species perform these reactions with varying efficacy. Mice produce these electrophilic epoxides more rapidly and appear to have less adequate detoxification mechanisms than rats or humans. The weight of evidence from many studies suggests that the balance of activation versus detoxification offers explanation of differing sensitivities of animals to these carcinogenic actions. Other aspects, including molecular biology of the many processes that lead through specific mutations to cancer, are yet to be understood. Melnick and Sills (2001) compared the carcinogenic potentials of these three dienes, along with that of ethylene oxide, which also acts through an epoxide intermediate. From the number of tissue sites where experimental animal tumors were detected, butadiene offers greatest potential for carcinogenicity of these dienes. Chloroprene and then isoprene appear to follow in this order. Comparisons among these chemicals based on responses to external exposures are complicated by differences among studies and of species and tissue susceptibilities. Physiologically based pharmacokinetic models offer promise to overcome these impediments to interpretation. Mechanistic studies at the molecular level offer promise for understanding the relationships among electrophilic metabolites and vital genetic components. Significant improvements in minimization of industrial worker exposures to carcinogenic chemicals have been accomplished after realization that vinyl chloride caused hepatic angiosarcoma in polymer production workers (Creech and Johnson 1974; Falk et al. 1974). Efforts continue to minimize disease, particularly cancer, from exposures to chemicals such as these dienes. Industry has responded to significant challenges that affect the health of workers through efforts that minimize plant exposures and by sponsorship of research, including animal and epidemiological studies. Governmental agencies provide oversight and have developed facilities that accomplish studies of continuing scientific excellence. These entities grapple with differences in perspective, objectives, and interpretation as synthesis of knowledge develops through mutual work. A major challenge remains, however, in assessment of significance of environmental human exposures to these dienes. Such exposure levels are orders of magnitude less than exposures studied in experimental or epidemiological settings, but exposures may persist much longer and may involve unknown but potentially significant sensitivities in the general population. New paradigms likely will be needed for toxicological evaluation of these human exposures, which are ongoing but as yet are not interpreted.

Proc Amer Assoc Cancer Res, Volume 45, 2004 3191 In the following experiments we investigated the binding of selected PAH epoxides (benzo(a)pyrene, benzo(b)fluoranthene, dibenz(a,hanthracene, fluoranthene, benzo(ghi)perylene, and benz(a)anthracene) to hemoglobin in order to determine the kinetics of binding of the PAH epoxide to hemoglobin. The following experiments were carried out using mouse (C57BL/6 male mice) hemoglobin in vitro . Through a series of binding studies as well as known carcinogenicity of the parent PAHs, we correlated extent of reaction of various epoxides with known carcinogenicity of the parent PAH. PAH epoxides arise via metabolism of the parent PAH to epoxides, followed by the addition of water to generate a diol. These reactions are catalyzd by microsomal enzymes as well as epoxide hydrolase. A second epoxidation reaction occurs at an adjacent double bond to yield a diol-epoxide of the parent PAH. Packed red cells (mouse) were resuspended in isotonic saline (2 mls). 100 μl aliquots of RBC suspension were used in reactions (volume brought to 500 μl with saline). PAH epoxide stock solutions were dissolved in tetrahydrofuran (1 mg/ml). 10 μl epoxide PAH stock was added to reactions. Reactions were carried out at 37° for 0,1,2,4,8,10,15,20,45,and 60 minutes. Reactions were stopped by addition of 500 μl water to hydrolyze unreacted PAH epoxide. Red cells were precipitated by centrifugation at 3,000 rpm for 10 minutes. The packed red cells were lyzed using 1 volume cold water and globin precipitated by addition of hemoglobin to acidified (0.1% HCl) acetone. Globin was collected, rinsed with acetone (100%), dried under nitrogen and stored at -20° until analysis. Control reactions were performed by preincubation of the PAH epoxides in saline for 15 minutes prior to addition of the red cell suspension. PAH epoxides were released from the globin by incubation (1 ml) with pronase for 24 hours at 37°. After reaction, released PAH tetrols were extracted using combinations of liquid and solid phase extraction and the tetrols taken to dryness under vacuum. The resulting PAH tetrols were analyzed spectrophotometrically by HPLC using both UV and fluorescence detection. Results from the study indicated that PAH epoxides bound covalently to the protein hemoglobin. The extent of PAH binding to the protein was similar with all epoxides, however there appears to be a correlation between the extent of binding and the overall carcinogenicity of the PAH with the stronger carcinogens having a faster rate of protein adduction than weaker carcinogens. These results suggest that the formation of hemoglobin adducts to PAH carcinogens may serve as reliable biomarkers of exposure as well as carcinogenicity. The limit of sensitivity of the assay by fluorometric detection of the released PAH tetrols is <10 pmols PAH epoxide/ mg protein. This limit of sensitivity suggests that the methods developed may be useful in routine monitoring of PAHs in population studies.

Purpose: To determine the effects of midazolam, 30 ng·mL −1 , on altered perception, mood, and cognition induced by ketamine. Methods: After ketamine was administered to achieve target concentrations of 50, 100, or 150 ng·mL −1 in 11 volunteers, perception, mood, and thought process were assessed by a visual analog scale. Mini-Mental State examination (MMSE) assessed cognition. Boluses of midazolam, 30, 14.5, and 12 µ g·kg −1 , were injected every 30 min to maintain the plasma concentration at 30 ng·mL −1 , which was reached 30 min after each injection. Results: Ketamine produced changes in perception about the body ( P <0.01, 0.001, and 0.001 at 30, 60, and 90 min), surroundings ( P <0.01 and 0.0001 at 60 and 90 min), time ( P <0.002 and 0.0001 at 60 and 90 min), reality ( P <0.001 and 0.0001 at 60 and 90 min), sounds ( P <0.002 at 90 min), and meaning ( P <0.05 at 90 min). Subjects felt less energetic and clearheaded ( P <0.02 and 0.05) during ketamine, midazolam, and their co-administration. Ketamine impaired thought process ( P <0.003 and 0.0001 and 60 and 90 min). Ketamine and midazolam decreased mean total MMSE and recall scores ( P <0.001 for both). Co-administraion reduced the number of subjects with perceptual (body, P <0.01 and 0.001 at 30 and 60 min) and thought process abnormalities. Within the range of observation, co-administration did not affect the changes in mood or recall. Conclusion: Midazolam attenuates ketamine-induced changes in perception and thought process.

Pharmacokinetics of Infused Antithrombin Concentrate in Children with Disseminated Intravascular Coagulation Secondary to Sepsis † 794

The metabolism of ethyl carbamate and the localization of its metabolites have been shown to be almost completely inhibited by ethanol in the mouse [Waddell, Marlowe, Pierce: Food Chem. Toxicol.25, 527 (1987); Yamamoto, Pierce, Hurst, Chen, Waddell: Drug Metab. Dispos. 16, 355 (1988)]. The enzyme system catalyzing this metabolism which is inhibited by ethanol now has been further investigated in both in vivo and in vitro studies. There is a direct, highly significant relationship between the extent of metabolism of ethyl carbamate and covalent binding of metabolites to liver protein. Paraoxon, carbaryl, CCl4 ethanol, methimazole, 4-methylpyrazole, diethyl maleate, ethyl N-hydroxycarbamate, and t-butyl carbamate inhibit, to different extents, the metabolism of ethyl carbamate in vivo; SKF-525A, CoCl2, Cacyanamide, chloral hydrate, 2-oxo-4-thiazolidine carboxylic acid, allopurinol, and methyl carbamate do not. Porcine liver esterase, yeast aldehyde dehydrogenase and mouse liver catalase catalyzed the metabolism in vitro; dog or bovine catalase, acid phosphatase, alcohol dehydrogenase, or carbonic anhydrase did not under the conditions tested. Paraoxon, 4-methylpyrazole, carbaryl, and NaF significantly inhibited the hydrolytic activity of mouse liver homogenates toward p-nitrophenyl acetate; ethanol or ethyl carbamate did not. However, each of these, except 4-methylpyrazole, inhibited the metabolism of ethyl carbamate by mouse liver homogenate or porcine liver esterase to about the same extent. Ion exchange chromatography of mouse liver cytosol revealed that the fraction with ability to metabolize ethyl carbamate co-chromatographed almost exactly with the ability to hydrolyze p-nitrophenyl acetate. It is proposed that ethyl carbamate is metabolized in the mouse, at least partially, by esterases; however, metabolism by other enzyme systems cannot be excluded.

Methodology is presented for convenient, reproducible and direct measurement of blood concentrations of ethyl carbamate, an experimental animal carcinogen. Extraction techniques requiring 20 μl of blood and selected ion monitoring using ethyl (13C, 15N)carbamate as internal standard enabled quantification of ethyl carbamate concentrations ranging from 50 ng ml−1 to 100 m̈g ml−1. Coefficients of variation at several representative concentrations averaged less than 4%. The method was used to determine the time course of elimination of ethyl carbamate from mice receiving doses of 125 m̈mol kg−1.

An investigation was made of the suitability of administering nicotine to experimental animals by inclusion in the drinking water. It was found that, after an initial accommodation period of several weeks, nicotine could be administered up to a concentration of 100 μg/ml with no decrease in fluid intake or weight gain compared to control. An analysis of the steady-state plasma levels and distribution of nicotine was made in mice which had received nicotine in the drinking water at a concentration of 60 μg/ml. The average daily dose of nicotine received by these animals was 17.2 mg/kg. The steady-state plasma level of nicotine was 34.4 ng/ml, representing 6% of the total compound present at steady-state as determined by thin-layer chromatography. The distribution of nicotine or metabolite in mice which had received [methyl-14C]-nicotine orally was determined. Wholebody autoradiography, as well as direct tissue counting, demonstrated that nicotine accumulates in a number of areas, particularly the salivary gland, nasal epithelium, uterus, and liver. There was relatively little material in the blood or brain. This investigation indicates that ad libitum oral administration is an acceptable method for maintaining experimental animals on nicotine for long periods of time.