Department of Cell Biology and Neuroscience, 135 Spieth Hall, University of California, Riverside, California 92521-0146
Received March 15, 2002; accepted May 17, 2002
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ABSTRACT |
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Key Words: pyridine; pyridine derivative; 3-ethylpyridine; growth; chick chorioallantoic membrane; cigarette smoke; passive smoking; active smoking.
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INTRODUCTION |
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Several lines of in vivo evidence indicate that components in cigarette smoke can inhibit growth. We recently showed that mainstream whole, sidestream whole, and sidestream gas phase smoke solutions significantly inhibited growth of the chick chorioallantoic membrane (CAM) in a dose-dependent manner and that sidestream whole and sidestream gas solutions were more inhibitory than the mainstream whole solutions (Melkonian et al., 2000, 2002a
). It was deduced from these data that growth inhibitory chemicals in sidestream whole smoke partition mainly with the gas phase, while in mainstream smoke inhibitory chemicals partition mainly with the particulate phase. Inhibition of growth was shown by BrdU labeling to be due to inhibition of cell division (Melkonian et al., 2002a
). Several other processes in smokers are linked to inhibition of cell proliferation. In the rat, cigarette smoke inhibited gastric ulcer healing by inhibiting cell proliferation (Ma et al., 2000
). Slower healing of wounds has also been observed clinically in smokers (Silverstein, 1992
), and could be due to an inhibition of cell proliferation by chemicals in smoke. In addition, incomplete development in the placentas of human smokers has been correlated with a decrease in the number of cytotrophoblasts in the S phase of the cell cycle (Genbacev et al., 2000
).
Based on prior work, we hypothesized that cigarette smoke contains chemicals that can inhibit growth of the CAM. Our current research goal was to identify the chemicals in cigarette smoke solutions that inhibit tissue growth. To accomplish this goal, we first screened a number of solid phase extraction cartridges to identify one that retained almost 100% of the CAM growth inhibitory activity. The eluate from this cartridge was subsequently analyzed by gas chromatography-mass spectrometry (GC-MS) and 12 pyridine derivatives were identified as the main class of compounds in the cartridge eluate. The pyridine identities were then confirmed using purified standards matched by mass spectrum and retention time. In the present study, the lowest observed adverse effect level (LOAEL) and no observed adverse affect level (NOAEL) were determined for each purified pyridine derivative using the day 5 CAM growth assay. The majority of the pyridine derivatives and pyridine itself had LOAELs and NOAELs in the micromolar range. However, several chemicals were identified that had LOAELs 10,000 to 10 million times lower than pyridine. A correlation was observed between the functional group substitutions and the relative toxicity of each pyridine derivative. At least one of the chemicals that inhibited growth in the picomolar range is an approved additive in consumer products.
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MATERIALS AND METHODS |
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Smoke solutions.
Smoke solutions were made fresh in EBSS-H using 2R1 research grade cigarettes (University of Kentucky, Louisville, KY) as described in detail previously (Knoll and Talbot, 1998). Sidestream (SS) whole smoke solutions were made by collecting the smoke that was produced at the burning end of a cigarette during 30 puffs of MS smoke and pushing it through 10 ml of EBSS-H. The gas phase of SS (SSG) smoke was made by passing whole SS smoke through a Cambridge filter prior to bubbling it into EBSS-H (Knoll and Talbot, 1998
). Smoke solutions and control medium (EBSS-H) applied to CAMs were passed through a 0.22 µm Acrodisk filter (Fisher Scientific, Tustin, CA) and handled using sterile technique. The pH of each solution was adjusted to 7.4, and solutions were added to CAMs immediately after preparation.
Solid phase extraction of smoke solutions.
Solid phase extraction (SPE) cartridges were used to preclean smoke solutions and concentrate chemicals that inhibit growth. All SPE cartridges were Bond Elut cartridges 3 cc with 500 g capacity purchased from Phenomenex, Torrance, CA. Cartridges that were screened for their ability to bind inhibitory chemicals included NH2, 2OH, CN, CBA, SCX, SAX, C18, C8, C2, CH, SI, and PH. This range of cartridges was chosen with the expectation that one or several of them would be effective at retaining the growth inhibitory chemicals in cigarette smoke.
The solid phase extraction cartridges were activated with 50 ml of HPLC grade methanol (Fisher Scientific, Tustin, CA), and then rinsed with 200 ml of deionized water. A column control was collected to assure that the methanol had been rinsed from the cartridge and that any toxic effect observed was solely due to the smoke solution. The smoke solution was run through the cartridge and the filtrate was collected and tested in the CAM assay. The cartridge was then either eluted immediately or frozen at 20°C for later elution. Solid phase extraction cartridges were eluted with 1 ml of HPLC grade methanol. The eluate was collected and used in the GC-MS analysis and the biological assays. For the biological assays, the methanol was evaporated under nitrogen, and the residue was resuspended in EBSS-H. A methanol control was used to assure that any observed activity was not due to residual methanol in the sample, and in no case did the methanol control affect growth.
GC-MS.
A preliminary screen of solid phase extraction cartridges had shown that the C2 cartridge retained most of the inhibitory activity in the CAM growth assay. To identify the components in aqueous smoke solutions that inhibit growth, mainstream and sidestream smoke solutions were analyzed with GC-MS after solid phase extraction on a C2 column. The equipment used was a Hewlett Packard 5890 GC interfaced to an HP-5971A MSD (quadropole mass selective detector) with a Zebron ZB1701 cyanopropyl phenyl column 30 m x 0.32 mm and with a 1 µm phase thickness (Phenomenex, Torrance, CA.). Helium was used as the carrier gas. The mass range examined was 40 to 350 with a threshold of 1500 with a sampling of 2.3 scans per second. The temperature program was an initial temperature of 45°C for 1 min with an increase of 10°C per min to a final temperature of 280°C after 10 min. The total run time was 34.5 min. Two µl of the eluate sample were injected directly into the GC using a Hamilton gas-tight syringe. Identifications of compounds were made using the mass spectrometry data matched to library entries. Compound identities were confirmed using purified standards matched by mass spectrum and retention time.
CAM assay.
CAM growth was evaluated by measuring CAM area using the CAM assay as modified by Melkonian et al.(2002b). Because the CAM undergoes very rapid growth, increasing in size 20-fold between days 5 and 6 of development (Melkonian et al., 2002b), it can be used as a screening assay to quickly identify chemicals that inhibit tissue growth (Melkonian et al., 2002a
). The CAM assay is both economical and rapid. Since it is an in vivo assay, it may provide more meaningful data than in vitro screens using cultured cells. Fertilized chicken eggs were purchased from Hy-Line International (Lakeview, CA), and then incubated at 37°C at 8590% relative humidity throughout the experiment. Albumin (2.53.0 cc) was removed on the fourth day after fertilization to drop the embryo away from the shell. Windows were then made in shells, and sealed with transparent tape to allow subsequent access to the CAM for experimental treatment. Two hundred µl of either EBSS-H or different concentrations of pure chemicals were added to the surface of each CAM at 1000 h on the fifth day after fertilization. A sham control group in which the window was opened and resealed was also included in each experiment. In all cases, the following 5 concentrations of test reagents were used, 5 x 10-3 M, 5 x 10-5 M, 5 x 10-7 M, 5 x 10-9 M, and 5 x 10-11 M. For 2-ethylpyridine and 3-ethylpyridine additional concentrations of 5 x 10-12 M, 5 x 10-13 M, 5 x 10-14 M, and 5 x 10-15 M were used. For nicotine and nornicotine, concentrations of 5 x 10-4 M, 5 x 10-6 M, 5 x 10-8 M, and 5 x 10-10 M were also used. Development was terminated at 1000 h on the sixth day after fertilization by fixing the embryo and the CAM with 3% glutaraldehyde in 0.1M cacodylate buffer (pH 7.4) for 2 h at room temperature. A small volume of fixative was also injected under the CAM. The CAMs were later dissected from eggs, postfixed in the same fixative for 24 h at room temperature, then thoroughly rinsed in PBS.
Quantification and imaging of CAMs.
At least 6 CAMs were used in each control and experimental group. The area of each CAM was measured by placing the CAMs in PBS in a Petri dish under a Wild-M5A dissecting microscope (Max ERB Instrument Co., Burbank, CA), then measuring the longest and shortest length of each CAM with a ruler to the precision of 0.5 mm. The data were recorded into an Excel spreadsheet. CAM area was calculated using the formula:
Area = (1/2 A) x (1/2 B) x () where A = longest length, B = longest width,
= 3.14.
Means and SD were then calculated for each control and treatment group. The treated group means were used to compare with control means to determine the effect of each chemical on CAM growth (the LOAEL, the NOAEL, and the maximum percentage of inhibition for each chemical were determined).
BrdU labeling.
EBSS-H or 3-ethylpyridine were placed on CAMs between 11001200 h on day 5 after fertilization. At 8 h after the start of an experiment, control and treated CAMs were exposed to 100 µl of BrdU (10 mM) for 55 min; they were then transferred to a 20°C freezer for 23 min and fixed at 20°C in 70% ethanol containing 50 mM glycine buffer, pH 2.0 for 1.5 h. CAMs were cut out of eggs and washed in PBS and dehydrated in an ethanol series, and further processed in a Hypercenter II Tissue Processing System (Shandon Inc., Pittsburgh, PA), then embedded in paraffin using a Sakura Tissue-TE (Shandon Inc., Pittsburgh, PA). Sections (5 µm) were cut on an S20 Spencer microtome (Labequip, American Optical Corp., Ontario, Canada), and placed on Superfrost/plus glass slides (Fisher Scientific, Tustin, CA). Sections were deparaffinized with 3 changes of Hemo-De for 15 min each, washed in 100% ethanol 2 times for 5 min each, then rehydrated to PBS. Sections were incubated in 1N HCl for 1 h at 40°C, washed in PBS for 1015 min, then incubated in 1% sodium borohydride (Sigma, St. Louis, MO) for 10 min, and washed again in PBS. Sections were incubated in anti-BrdU IgG (1/100 dilution in PBS containing 1% BSA) for 2 h at room temperature. Slides were washed in PBS containing 0.1% BSA for 4560 min, then incubated in secondary antibody (Alexa antimouse IgG, 1/100 dilution) for 1 h at room temperature. Slides were washed with PBS plus 0.1% BSA for 45 min, and cover slips were mounted with Vectashield. BrdU and antibody controls were performed on both control and treated CAMs. Controls consisted of CAMs that were not exposed to BrdU and CAMs exposed to secondary antibody only. Slides were examined using a Zeiss photomicroscope with epifluorescence. Images were recorded using a Spot digital camera (SP401-115, Diagnostic Instruments, Sterling Heights, MI) and processed and montaged using Adobe Photoshop (San Jose, CA). The number of labeled cells/7.8 mm2 was counted in CAMs exposed to EBSS-H or to smoke solutions and was significantly less in both smoke treated groups than in the EBSS-H control.
Statistical analyses.
Group means of CAM area were compared by ANOVA. When significance was found (p < 0.05), Dunnetts post hoc test was used to identify significantly affected groups. Dunnetts test compares treated group means to the sham control group. Analyses were done using GraphPad (Instat, San Diego, CA). Data were checked to determine if they satisfied the assumptions of ANOVA. The assumptions tested involved determining if the SDs of all groups were equal and if the data were sampled from a Gaussian distribution. If the assumptions were not satisfied, the Kruskal-Wallis nonparametric test was used followed by Dunns post hoc test.
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RESULTS |
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Using the LOAEL of the pyridine ring as a comparative reference, the 12 pyridine derivatives identified in sidestream smoke solution were then tested for their effects in the CAM assay. 4,4-Bipyridine, which has two pyridine rings dimerized at the 4,4 position, increased the LOAEL to 5 x 10-4 M (7.81 x 10-2 mg/ml; Fig. 2B).
Pyrrole Substitutions on Pyridine Produced a Bimodal Effect on CAM Growth
Three of the pyridine derivatives identified in C2 eluates of sidestream smoke had pyrrole substitutions (Fig. 3). 3-(1-Methylpyrrol-2-yl)pyridine (ß-nicotyrine), 3-(1-pyrrolidin-2-yl)pyridine (nornicotine), and 1-methyl-2-(3-pyridyl)pyrrolidine (nicotine) were tested on day 5 CAMs and produced a bimodal effect in which CAM growth decreased at high concentrations and increased at low concentrations. The LOAELs for inhibiting growth for ß-nicotyrine, nornicotine, and nicotine were 5 x 10-7 M (7.90 x 10-5 mg/ml), 5 x 10-5 M (7.40 x 10-3 mg/ml), and 5 x 10-4 M (8.10 x 10-2 mg/ml), respectively. Unlike all other pyridine derivatives tested, a significant stimulatory effect was observed on growth for nornicotine and nicotine at 5 x 10-11 M. There was also a slight stimulation for ß-nicotyrine, but this was not significantly different from the controls.
Single Linear Substitutions to Pyridine Inhibited CAM Growth at Nano and Picomolar Concentrations
Two of the pyridine derivatives identified in the C2 eluate had single methyl substitutions. Treatment of CAMs with either 2-methylpyridine or 3-methylpyridine significantly decreased CAM growth in a dose-dependent manner (Figs. 4A and 4B). Both 2-methylpyridine and 3-methylpyridine were effective at doses as low as 5 x 10-9 M (4.66 x 10-7 mg/ml), which is 10,000 times lower that the LOAEL of pyridine.
In contrast to pyridines with single methyl substitutions, the LOAELs for 2,3-dimethylpyridine and 2,4-dimethylpyridine were 5 x 10-5 M (5.36 x 10-3 mg/ml) and the LOAEL for 2,4,6-trimethylpyridine was 5 x 10-5 M (6.10 x 10-3 mg/ml; Fig. 5). Thus LOAELs for the double and triple methyl substituted derivatives were the same as for pyridine.
Two of the compounds in the C2 eluate had single ethyl substitutions. In the CAM growth assay, substitution of a single ethyl group at either position 2 or 3 of the pyridine ring decreased the LOAEL to at least 5 x 10-11 M, a concentration that was ineffective for all other chemicals tested (Figs. 6A and 6B). Since all doses were highly inhibitory in this set of experiments, a second set of experiments was performed over the concentration range of 5 x 10-9 M to 5 x 10-14 M to define the LOAEL (Figs. 6C and 6D
). For both 2-ethylpyridine and 3-ethylpyridine, the LOAEL was 5 x 10-12 M (5.36 x 10-10 mg/ml), which is 10 million times lower than the LOAEL of pyridine.
One compound identified in the C2 eluate, 3-ethenylpyridine, is neither commercially available nor readily synthesized, therefore, its isomer, 4-ethenylpyridine, which is commonly used as its substitute, was tested in the CAM growth assay (Fig. 7). The LOAEL for 4-ethenylpyridine was 5 x 10-5 M (5.26 x 10-3 mg/ml), which was equivalent to that of the pyridine. Addition of a double bond to the ethyl functional group created an ethenyl substitution and completely reversed the highly inhibitory effect of 3-ethylpyridine.
3-Ethylpyridine Inhibited CAM Growth by Inhibiting Cell Proliferation
To determine if inhibition of CAM growth correlated with inhibition of cell proliferation, BrdU labeling was done on CAMs treated with 10-8 M 3-ethylpyridine for 8 h (Fig. 8). 3-Ethylpyridine was chosen for this assay as it was one of the chemicals that inhibited growth at picomolar doses. Treated CAMs had significantly (p < 0.001) fewer BrdU labeled nuclei than the controls (control = 16.6 ± 3.3 labeled nuclei/7.8 mm2; 10-8 M 3-ethylpyridine = 7.25 ± 2.5 labeled nuclei/7.8 mm2), indicating that DNA synthesis had been inhibited by 3-ethlypyridine.
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DISCUSSION |
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BrdU labeling indicated that 3-ethylpyridine blocked CAM growth by inhibiting cell division. Both the number of labeled nuclei and the intensity of the fluorescence in labeled nuclei was decreased by treatment with 3-ethylpyridine, which is not generally regarded as a toxicant and has not previously been reported to inhibit cell growth at picomolar doses. It is not yet known how 3-ethylpyridine affected cell division but inhibition by such low doses suggests that it acts through a receptor. Interestingly, our data also show a slight but significant stimulation of CAM growth at very low concentrations (5 x 10 -11 M) of nornicotine and nicotine. This is consistent with the previous finding that 10-11 to 10-8 M nicotine stimulates DNA synthesis and proliferation in vascular endothelial cells (Heeschen et al., 2001; Villablanca, 1998
). Nicotine induced stimulation of endothelial cell division can be inhibited by hexamethonium indicating it acts through a nicotinic receptor (Villablanca, 1998
). However, the effects of nicotine on cultured cells appear to be complex as others have reported that nicotine inhibited proliferation of cultured human umbilical vein endothelial cells at 10-9 M (Albaugh et al., 2001
).
The pyridine derivatives we tested were identified in sidestream cigarette smoke, the major component of environmental tobacco smoke, and most, including 2-ethylpyridine and 3-ethylpyridine, are also present in mainstream smoke (Brunneman et al., 1978), indicating that both active and passive smokers receive exposure to these chemicals. Moreover, the methyl and ethyl substituted pyridines that were most effective in the CAM growth assay are 713 times more abundant in sidestream smoke than in mainstream smoke (Brunneman et al., 1978
). The aggregate concentration of the four most potent pyridine derivatives (2- and 3-methylpyridine and 2- and 3-ethylpyridine) in mainstream and sidestream smoke from one unfiltered cigarette are 46.8 µg/cigarette and 529 µg/cigarette, respectively (Brunneman et al., 1978
). Although the concentrations of these chemicals in smokers have not yet been determined, the effective doses of single methyl and ethyl pyridines are so low that they are likely to be important in human smokers.
The identification of pyridines in sidestream and mainstream cigarette smoke that inhibit growth in nanomolar and picomolar doses could be very important in explaining why human smokers often have difficulty with processes requiring growth of new tissue. It is generally well accepted that smoking impairs wound healing (Frick and Seals, 1994). As an example, in the rat model, gastric ulcers heal much more slowly in animals exposed to cigarette smoke than in unexposed controls (Ma et al., 1999
, 2000
). In addition, reproductive processes that require growth appear to be affected by cigarette smoke (Stillman et al., 1986
). Establishing and maintaining pregnancies require extensive growth in the embryo/fetus, corpus luteum, and the placenta (Findlay, 1986
). Our previous in vivo study showed that inhalation of cigarette smoke by hamsters before and during pregnancy reduced vascular area in their corpora lutea (Magers et al., 1995
). It is also well established that both active (Stillman et al., 1986
) and passive (Martin and Bracken, 1986
) smokers produce fetuses with lower than normal birth weights. This reduction in birth weight is thought to be due to fetal hypoxia caused by nicotine-induced vasoconstriction and decreased placental blood flow and by increased carboxyhemoglobin leading to decreased fetal oxygenation (Tourmaa, 1995). Our current data indicate that 2- and 3-ethylpyridine, which retard growth at picomolar doses, could impair wound healing and contribute to the low birth weights in fetuses delivered by active and passive smokers.
3-Ethylpyridine, which inhibited CAM growth at picomolar doses, is on the FEMA GRAS list of chemicals ("generally regarded as safe"; R. J. Reynolds Tobacco Company) and on the FDA EAFUS list ("everything added to food in the United States"; U.S. FDA). 3-Ethylpyridine is also one of the 599 chemicals reported by tobacco companies to be added to cigarettes to enhance flavor (R. J. Reynolds Tobacco Company). Extracts of the unburned 2R1 research grade cigarettes used to make our smoke solutions did not contain 3-ethylpyridine, thus what we detected was probably produced during combustion. However, several brands of unburned commercial cigarettes did have 3-ethylpyridine, which was presumably added during manufacture to flavor the smoke.
Because it is generally assumed safe and has not been subjected to toxicological testing, 3-ethylpyridine is added to food, cosmetics, and tobacco. Although the amounts added to consumer products are generally not made available to the public, tobacco companies add up to 100 µg/cigarette (R. J. Reynolds Tobacco Company). The doses that are present in humans have not been determined and might be difficult to measure as they are probably low. However, our study shows that very low doses of 3-ethylpyridine have significant inhibitory effects on tissue growth and indicate that further toxicological testing on pyridine derivatives is warranted.
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ACKNOWLEDGMENTS |
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NOTES |
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REFERENCES |
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