ToxSci Advance Access originally published online on June 21, 2006
Toxicological Sciences 2006 93(1):82-95; doi:10.1093/toxsci/kfl047
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Cigarette Smoke Toxicants Alter Growth and Survival of Cultured Mammalian Cells
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* Graduate Program in Cell, Molecular, and Developmental Biology; and Department of Cell Biology and Neuroscience, University of California, Riverside, Riverside, California 92521
1 To whom correspondence should be addressed at 2320 Spieth Hall, Department of Cell Biology and Neuroscience, University of California, Riverside, 900 University Avenue, Riverside, CA. Fax: (951) 827-4286. E-mail: talbot{at}ucr.edu.
Received April 27, 2006; accepted June 14, 2006
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Our purpose was to determine the effects of six cigarette toxicants (pyridine, nicotine, 2-ethylpyridine, 3-ethylpyridine, p-cresol, and pyrazine) on three types of cultured mammalian cells (human umbilical vein endothelial cells [HUVECs], human microvascular endothelial cells [HMVECs], and NIH 3T3 cells) using a cell proliferation/survival assay. Synchronized cells were cultured in proliferation or survival medium containing various doses (10–18M–10–2M) of the tested chemicals. After 48 h, cells were counted using a hemacytometer. The no observable adverse effect level (NOAEL), lowest observable adverse effect level (LOAEL), and the efficacy were determined for each compound in the cell proliferation and survival assays. Pyridine and p-cresol did not show significant effects with any cell types, except at high doses. Derivitization of the pyridine ring altered its potency, especially when an ethyl group or nitrogen was added. In survival medium, nicotine stimulated proliferation of all three cell types at doses found in smoker's serum (10–8M–10–7M). For HUVEC and HMVEC, 2-ethylpyridine, 3-ethylpyridine, and pyrazine inhibited proliferation in proliferation medium and induced cell death in survival medium at attomolar and femtomolar doses. All chemicals, except pyridine and pyrazine, stimulated NIH 3T3 cell proliferation at low doses and induced cell death at high doses. LOAELs and efficacies revealed that endothelial cells from a developing organ (umbilical cord) were more sensitive to these chemicals than endothelial cells from an adult organ (lung). 3-Ethylpyridine and pyrazine, which induced cell death at low doses, are added to consumer products and should be subjected to further toxicological testing.
Key Words: cigarette smoke; cell growth; cell death; pyrazines; pyridines; phenols.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The gas and particulate phases of cigarette smoke contain over 4000 chemicals (EPA, 1992
). Most studies on cigarette smoke and its components have focused on nicotine, polycyclic aromatic hydrocarbons, tobacco-specific nitrosamines, carbon monoxide, tar, and 10–15 other compounds, including phenol, acrolein, acetaldehyde, hydrogen cyanide, and formaldehyde (detailed references in Talbot and Riveles, 2005). However, the majority of the chemicals in cigarette smoke have not been examined for toxicity.
Our previous studies using the chick chorioallantoic membrane (CAM) showed that whole mainstream smoke, whole sidestream smoke, and the gas phase of sidestream smoke significantly impaired CAM growth, cell proliferation, blood vessel development, and vessel migration (Melkonian et al., 2000, 2002). Using solid phase extraction cartridges and gas chromatography–mass spectrometry (GC–MS), 12 pyridine (Ji et al., 2002), 10 pyrazine (Melkonian et al., 2003), and 11 phenolic (Melkonian and Talbot, unpublished data) derivatives were identified in the active fraction of sidestream gas phase smoke solutions and were subsequently found to adversely affect CAM growth at very low doses.
All our prior studies on growth with these chemicals were done using the chick model. The purpose of this study was to test the hypothesis that the cigarette toxicants that inhibited CAM growth would inhibit proliferation and survival of cultured mammalian cells. The chemicals that were chosen for this study included pyridine, nicotine, 2- and 3-ethylpyridine, pyrazine, and p-cresol. Pyridine had a very high LOAEL (lowest observable adverse effect level) in the CAM bioassay (Melkonian et al., 2002) and was hypothesized to produce little or no effect on cultured mammalian cells. Nicotine, a pyridine derivative, was previously shown to promote in vitro DNA synthesis and cell proliferation in several types of mammalian endothelial cells (Heeschen et al., 2001; Villablanca, 1998) and to stimulate angiogenesis, tumor growth, and atherosclerosis in vivo (Heeschen et al., 2001). The use of nicotine served two purposes: (1) to ensure that our cell proliferation/survival assay would work properly and give us results similar to those obtained in other laboratories and (2) to extend the testing of nicotine to human microvascular endothelial cells (HMVECs) and NIH 3T3 cells. 2-Ethylpyridine, 3-ethylpyridine, p-cresol, and pyrazine were chosen because they were the most potent chemicals in their respective groups when tested in the CAM bioassay (Ji et al., 2002; Melkonian et al., 2003). Pyridine, 3-ethylpyridine, pyrazine, and p-cresol were also of interest because they are included on the FEMA GRAS list (Flavor and Extract Manufacturers' Association—Generally Regarded As Safe) and the FDA EAFUS list (Everything Added to Food in the United States) (Talbot and Riveles, 2005).
The cell types used were human umbilical vein endothelial cells (HUVECs), HMVECs from the lung, and NIH 3T3 cells. Endothelial cells were chosen because in our previous CAM experiments, cigarette toxicants adversely affected blood vessel development (Melkonian et al., 2003). HUVEC and HMVEC were used for two reasons. First, previous studies indicated that endothelial cells from different types of blood vessels had distinct gene expression profiles (Chi et al., 2003; Ho et al., 2003), suggesting that they could react differently to the test chemicals. Secondly, HUVEC and HMVEC allowed comparison of endothelial cells from a developing (umbilical cord) and adult tissue (lung). NIH 3T3 cells, which were derived from mouse embryonic fibroblasts, allowed comparison of endothelial cells to a cell line that proliferates rapidly and continuously in culture under monolayer conditions.
The results of our study demonstrate that smoking adversely affects human endothelial cells and further show that not all cells respond in the same way to cigarette toxicants.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Chemicals and tissue culture supplies.
Pyridine, nicotine (free base), 2-ethylpyridine, 3-ethylpyridine, p-cresol, and pyrazine were purchased from Sigma–Aldrich (St Louis, MO), and the identity and purity of each chemical were confirmed using HPLC and/or GC–MS. Endothelial Basal Medium (EBM-2), EGM-2 MV Singlequot, and Reagent Pack (trypsin/EDTA, trypsin neutralizing solution [TNS], and HEPES buffer saline [HBSS]) were purchased from Clonetics (BioWhittaker, Walkersville, MD). Dulbecco's Modified Eagle Medium (DMEM), 2mM glutamine, and penicillin/streptomycin were purchased from Sigma–Aldrich. Fetal calf serum (FCS) was purchased from Gibco (Invitrogen, Carlsbad, CA). Tissue culture plates were from Falcon (Becton Dickinson Labware, Franklin Lakes, NJ). Tissue culture flasks (T-25 and T-75) were from Nunc (Fisher Scientific, Indiana, PA). The Cell Counting Kit-8 was purchased from Dojindo (Dojindo Molecular Technologies, Inc, Gaithersburg, MD).
Cell culture.
HUVECs and HMVECs were purchased from Clonetics (BioWhittaker), and passages 6–10 were used for all experiments. NIH 3T3 cells, originally from American Type Culture Collection (ATCC), were a gift from Dr Manuela Martins-Green (University of California, Riverside, CA). All three cell types were expanded; frozen overnight at – 80°C in solution containing 80% basal medium, 10% FCS, and 10% DMSO; and then stored in liquid nitrogen for future usage.
To prepare cells for experiments, endothelial cells and NIH 3T3 cells were cultured (5000 cells/cm2) in monolayers in either T-25 (25 cm2) or T-75 (75 cm2) tissue culture flasks. Endothelial cells were expanded in proliferating medium (EBM-2 BulletKit for HUVEC and EGM-2-MV BulletKit for HMVEC), and NIH 3T3 cells were expanded in 87% DMEM, 2% 2mM glutamine, 10% FCS, and 1% penicillin/streptomycin medium (DMEM proliferation medium) in a humidified 37°C incubator with an atmosphere of 95% air and 5% CO2. For both endothelial cells and NIH 3T3 cells, the medium was changed every 2 days, at which time the cells were examined with an inverted microscope (Diavert Inverted Microscope—Leitz, Wetzlar, Rockleigh, NJ). When cell confluence reached 85%, subculturing was done according to the protocol from Clonetics.
Cell proliferation and survival assay.
To assess the effects of the test chemicals on endothelial cells and NIH 3T3 cells, two different media were used, one to assess the effect of the chemicals on dividing cells (proliferation medium) and one to assess the effect of the chemicals on cells that survived but did not divide (survival medium). The proliferation medium was serum and growth factor rich (2% FCS plus growth factors in EBM-2 BulletKit for HUVEC, 5% FCS plus growth factors in EGM-2-MV BulletKit for HMVEC, and 10% FCS in DMEM proliferation medium for NIH 3T3 cells), while the survival medium was serum and growth factor poor (1% FCS in EBM-2 basal medium for HUVEC and HMVEC and 1% FCS in DMEM survival medium for NIH 3T3 cells). The doubling time for HUVEC and HMVEC in proliferation medium was 18–24 h, while NIH 3T3 cells doubled every 16–24 h.
To determine if a test chemical affected cell proliferation and/or survival, cells were synchronized using the survival medium 24 h prior to an experiment. Then the appropriate amount of HBSS, trypsin, and TNS (according to the protocol from Clonetics) was added to either the T-25 or T-75 tissue culture flask, and the detached cells were centrifuged at 400 x g for 7 min. Proliferation medium was used to break the cell pellet, and a small portion of the cell suspension was then added to trypan blue for both live and dead cell counting (according to the protocol from Clonetics) using a hemacytometer (Hausser Scientific, Horsham, PA).
After cell counting, 3 ml of proliferation or survival medium containing varying concentrations of the test chemicals was added to 35-mm tissue culture dishes ("Easy Grip" Tissue Culture Dishes—Falcon—Becton Dickinson Labware). A total of 35,000 cells were then added to the dishes immediately. For each test chemical, duplicated dishes were prepared for each experiment. Doses that were tested ranged from 10–18M to 10–2M (7.9 x 10–20 to 7.9 x 1.08 x 10–3 g/ml).
After 48 h of incubation, cells were detached from the dishes using trypsin (Reagent Pack), and the attached cells or total cells (attached and suspended) were counted using a hemacytometer. To count suspended cells, supernatants from the dishes were collected, and then 3 ml of HBSS was added to the dishes for 5 min at room temperature to wash out proteins that might inhibit the function of trypsin and to recover additional suspended cells. The 3 ml of wash HBSS was added to the "supernatant." The centrifuge tubes were then spun at 400 x g for 7 min, and 100 µl of proliferation medium was used to break the pellet. A portion of the pellet was added to trypan blue to distinguish live and dead cells, and the live cell count was done using a hemacytometer. To count the adherent cells, 3 ml of trypsin was used for detachment. During the lifting process, the tissue culture dishes were put on a lab shaker (Rocking platform—VWR Scientific, Brisbank, California) for 10 min to ensure even lifting. After 10 min, each dish was examined with an inverted microscope to confirm detachment of all cells. Then the solution containing the cells and trypsin was aspirated into a centrifuge tube, and 3 ml of TNS was added to the dishes to remove any left over adherent cells. The TNS was aspirated into the same tube containing the trypsin solution to neutralize trypsin. These tubes were then centrifuged at 400 x g for 7 min, and the number of live cells was determined using a hemacytometer.
Validation of the counting method.
To ensure that the treatment of the cells by trypsin and centrifugation needed for hemacytometer counting did not affect the actual number of cells being counted, a water-soluble tetrazolium salt (WST-8)–based colorimetric microplate assay (Cell Counting Kit-8, Dojindo Molecular Technologies, Inc) was used to validate the hemacytometer protocol for cell counting. The cell count data obtained with the tetrazolium kit were compared to data previously obtained using the hemacytometer. HUVECs treated with 3-ethylpyridine in survival medium for 48 h were used for this purpose, and the resulting cell counts did not differ significantly from the hemacytometer data (data not shown).
Coefficient of variation.
To determine the coefficient of variation for cell counting using the hemacytometer, 35,000 HUVECs were cultured for 48 h in proliferation medium in two 35-mm tissue culture dishes. This experiment was done three times on three separate days. Cells from each dish were counted five times. The cell count from each dish on a given day was used to calculate the intradish coefficient of variation, and the readings generated between the two dishes in the three experiments on different days were used to obtain the interdish coefficient of variation. The intradish and interdish coefficient of variation were 4.5% and 5.0%, respectively.
Data analysis.
Cell proliferation and survival data were plotted using Microsoft Excel. Results were expressed as means ± SDs for each group from experiments repeated at least three times (n = 3) in duplicate. Data involving comparisons of experimental treatment groups against a control group were first analyzed by a one-way ANOVA. When significance was found, Dunnett's post hoc test was used to determine which treatment groups were significantly different from the control. In all analyses, data were checked for homogeneity of variances using Bartlett's test. Significance was determined at the p < 0.05 levels.
NOAELs, LOAELs, and efficacies.
To quantify and compare the experimental data (Figs. 1–6), no observable adverse effect level (NOAEL), LOAEL, and efficacies were determined for each experiment (Table 1, A–F). NOAEL is defined as the highest dose at which there was no statistically significant change between the exposed and control groups. LOAEL is defined as the lowest exposure level at which there was a statistically significant change between the exposed group and its control group. Efficacies were determined by finding the dose that reached a maximum percentage of stimulation or inhibition. The percentage increase or decrease between this maximum point and the control was interpreted to be the efficacy. When a maximum percentage of either stimulation or inhibition was not reached, efficacies were indicated as greater than or equal to the highest percentage of either stimulation or inhibition observed for that compound at the highest dose tested.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Experimental Design
To determine the effects of the six cigarette toxicants (pyridine, nicotine, 2-ethylpyridine, 3-ethylpyridne, p-cresol, and pyrazine) on HUVECs, HMVECs, and NIH 3T3 cells, 35,000 synchronized cells were treated with various doses of each test chemical in either proliferation or survival medium for 48 h, and then the effect on cell number was determined (Figs. 1–6
). Data were analyzed to determine the NOAELs, LOAELs, and efficacies as described in Materials and Methods (Table 1, A–F). When there was not sufficient data to establish the LOAEL or efficacy, these values were estimated and are indicated by 
or 
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Cells in Control Medium
In proliferation medium, the number of cells in the control group increased in all experiments from 35,000 cells at plating to between 129,533 and 191, 792 cells by 48 h (Figs. 1–6). This increase is equivalent to about 2–2.5 doublings. In survival medium, all cell types survived, but did not divide, for the 48-h interval in all experiments.
Effect of Pyridine on Proliferation and Survival
Human umbilical vein endothelial cells.
Based on our prior work with CAMs, we hypothesized that pyridine would not have a strong effect on cell growth. In proliferation medium, pyridine produced no effect on total cell number in any of the treated groups (Fig. 1A and Table 1, A). Likewise in survival medium, cells were not very responsive to pyridine. Pyridine significantly inhibited HUVEC survival only at high doses with the LOAEL = 10–4M (7.9 x 10–6 g/ml; efficacy = 
38%), probably due to cytotoxicity. A small, but apparently significant, increase (10%) in total cell number was observed at 10–6M (7.9 x 10–8 g/ml). The data from both media agreed with our previous observation that pyridine did not have a strong effect on CAM growth (Ji et al., 2002).
Human microvascular endothelial cells.
There was no significant effect on total cell number in any of the pyridine-treated groups in either medium, again in agreement with our hypothesis that pyridine would not have a strong effect on cell growth (Fig. 1B and Table 1, A).
NIH 3T3 cells.
In proliferation medium, there was no significant effect on total cell number in any of the pyridine-treated groups (Fig. 1C and Table 1, A). In survival medium, pyridine did not affect total cell number, except at the highest dose (10–4M = 7.9 x 10–6 g/ml), which produced a modest but significant decrease in cell number.
Effect of Nicotine on Proliferation and Survival
Human umbilical vein endothelial cells.
Nicotine, one of the most studied components in cigarette smoke, is a pyridine derivative. In proliferation medium, 10–10M–10–8M nicotine significantly stimulated HUVEC proliferation with a LOAEL at 10–10M (1.6 x 10–11 g/ml; efficacy = 
13%), except at the highest dose of 10–4M (1.6 x 10–5 g/ml), which was inhibitory (14%) (Fig. 2A and Table 1, B).
In survival medium, there was an interesting and significant stimulation of proliferation at femtomolar and picomolar doses with the LOAEL 10–14M (1.6 x 10–15 g/ml; efficacy = 86%). These data were consistent with previous observations on HUVEC (Heeschen et al., 2001; Hill et al., 1983; Villablanca, 1998), demonstrating that our culture conditions produced results similar to those obtained in other laboratories.
Human microvascular endothelial cells.
In proliferation medium, nicotine significantly inhibited HMVEC proliferation at 10–4M (1.6 x 10–5 g/ml; efficacy =14%) (Fig. 2B and Table 1, B). In contrast, in survival medium, nicotine significantly stimulated HMVEC proliferation at all tested doses with the LOAEL 10–14M (1.6 x 10–15 g/ml; efficacy = 24%).
NIH 3T3 cells.
High doses (10–6M–10–4M = 1.6 x 10–7–1.6 x 10–5 g/ml) of nicotine significantly inhibited NIH 3T3 cell proliferation in proliferation medium (efficacy =14%) and decreased cell number in survival medium (efficacy = 27%) (Fig. 2C and Table 1, B). In contrast, in survival medium, low doses of nicotine (10–14M–10–8M) significantly stimulated NIH 3T3 cell proliferation with the LOAEL 10–14M (1.6 x 10–15 g/ml; efficacy = 17%).
Effect of 2-Ethylpyridine and 3-Ethylpyridine on Proliferation and Survival
Human umbilical vein endothelial cells.
2- and 3-Ethylpyridine were the two most potent pyridines in the CAM growth assay and were hypothesized to inhibit the growth of cultured mammalian cells (Figs. 3A and 4A and Table 1, C and D). In proliferation medium, both 2- and 3-ethylpyridine significantly inhibited cell proliferation; however, 2-ethylpyridine was 100 million times more potent (LOAEL was 10–14M = 1.1 x 10–15 g/ml) than 3-ethylpyridine (LOAEL was 10–6M = 1.1 x 10–7 g/ml).
In survival medium, both 2- and 3-ethylpyridine decreased total cell number at all doses tested (LOAELs 10–16M = 1.1 x 10–17 g/ml), and efficacies were similar for both chemicals (71% for 2-ethylpyridine, 75% for 3-ethylpyridine). The loss of more than a third of the cells at attomolar (10–16M = 1.1 x 10–17 g/ml) doses supported our hypothesis that these two chemicals would be highly potent in the cell culture assay.
Human microvascular endothelial cells.
In proliferation medium, 2-ethylpyridine (10–8M–10–2M) and 3-ethylpyridine (10–4M–10–2M) significantly inhibited HMVEC growth in a dose-dependent manner (efficacy = 38% for 2-ethylpyridine, and 24% for 3-ethylpyridine) (Figs. 3B and 4B and Table 1, C and D). However, 2-ethylpyridine (LOAEL was 10–8M = 1.1 x 10–9 g/ml) was 10,000 times more potent than 3-ethylpyridine (LOAEL was 10–4M; 1.1 x 10–5 g/ml). The inhibitory effect of both chemicals, as measured by the LOAELs, was not as great as for HUVEC.
In survival medium, both 2- and 3-ethylpyridine decreased the number of HMVEC in a dose-dependent manner between 10–12M–10–2M. For both chemicals, the LOAELs were the same (10–12M = 1.1 x 10–13 g/ml) and the efficacies were similar (efficacy = 63% for 2-ethylpyridine and 67% for 3-ethylpyridine).
NIH 3T3 cells.
In contrast to the endothelial cell lines, in proliferation medium, 2-ethylpyridine (LOAEL was 10–12M = 1.1 x 10–13 g/ml; efficacy = 22%) and 3-ethylpyridine (LOAEL 10–14M; efficacy = 25%) significantly stimulated NIH 3T3 cell proliferation (Figs. 3C and 4C and Table 1, C and D). For both chemicals, inhibition of proliferation was only observed at the highest dose (10–4M = 1.1 x 10–5 g/ml).
When NIH 3T3 cells were incubated with 2- or 3-ethlypyridine in survival medium, the total number of cells increased significantly between 10–14M and10–10M. For both chemicals, the LOAELs were the same ( 10–14M or 1.1 x 10–15 g/ml) and the efficacies were similar (22% for 2-ethylpyridine, 24% for 3-ethylpyridine). In contrast, at high doses (10–6M and 10–4M), both 2- and 3-ethylpyridine significantly decreased cell number, with the same potency (LOAELs were 10–6M = 1.1 x 10–7 g/ml).
Effect of p-Cresol on Proliferation and Survival
Human umbilical vein endothelial cells.
In the CAM assay, p-cresol significantly inhibited growth of CAMs at 5 x 10–6 M (1.1 x 10–7 g/ml; Melkonian and Talbot, unpublished data) (Fig. 5A and Table 1, E). In proliferation medium, p-cresol had no significant effect on total cell number in any of the treated groups. However, in survival medium, p-cresol significantly decreased total cell number at moderate to high doses (10–8M–10–4M) (LOAEL was 10–8M = 1.1 x 10–9 g/ml; efficacy = 62%).
Human microvascular endothelial cells.
p-Cresol in proliferation medium had no significant effect on total cell number in any of the treated groups, and in survival medium, it inhibited HMVEC survival only at the highest dose (10–4M = 1.1 x 10–5 g/ml) (Fig. 5B and Table 1, E).
NIH 3T3 cells.
In proliferation medium, p-cresol significantly inhibited NIH 3T3 cell proliferation (12%) only at the highest dose (10–4M = 1.1 x 10–5 g/ml) (Fig. 5C and Table 1, E). In contrast, in survival medium, p-cresol significantly stimulated NIH 3T3 cell proliferation at doses between 10–10M and 10–6M (LOAEL was 10–10M = 1.1 x 10–11 g/ml; efficacy = 45%).
Effect of Pyrazine on Proliferation and Survival
Human umbilical vein endothelial cells.
Pyrazine was the most potent of the pyrazines tested in the CAM assay (Melkonian et al., 2003) and was therefore predicted to have a strong effect on cultured cells. In proliferation medium, pyrazine inhibited HUVEC proliferation in a dose-dependent manner (LOAEL was 10–14M = 8 x 10–16 g/ml; efficacy = 62%) (Fig. 6A and Table 1, F). In survival medium, all doses of pyrazine significantly decreased total cell number in a dose-dependent manner (LOAEL was 10–16M or 10–18 g/ml; efficacy = 62%).
Human microvascular endothelial cells.
In proliferation medium, pyrazine significantly inhibited HMVEC proliferation but only at the highest doses (10–4M–10–2M) (LOAEL was 10–4M = 10–6 g/ml; efficacy 42%) (Fig. 6B and Table 1, F). In survival medium, pyrazine impaired HMVEC survival at doses between 10–8M and 10–2M (LOAEL was 10–8M = 10–10 g/ml; efficacy = 64%).
NIH 3T3 cells.
At high doses (10–6M–10–4M) in proliferation medium, pyrazine significantly inhibited NIH 3T3 cell proliferation (LOAEL was 10–6M = 10–8 g/ml; efficacy 28%) (Fig. 6C and Table 1, F). In survival medium, cell number decreased at doses between 10–10M and 10–4M (LOAEL was 10–10M = 10–12 g/ml; efficacy = 68%).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Six cigarette smoke toxicants that inhibit growth in a chick model (CAM) were shown in this study to affect growth and survival of cultured mammalian cells. Three categories of effects were observed with mammalian cells. First, some chemicals produced different effects on the same cell type (e.g., nicotine both stimulated and inhibited growth of HUVEC depending on the dose). Secondly, structurally similar compounds sometimes had very different potencies when tested on a particular cell type (e.g., in survival medium, pyridine had little effect on HUVEC, but 2- and 3-ethylpyridine caused death of HUVEC at attomolar doses). Finally, the effects of a compound on a particular cell type sometimes differed in proliferation and survival media (e.g., p-cresol significantly inhibited NIH 3T3 cell proliferation in proliferation medium but stimulated NIH 3T3 cell growth in survival medium). In addition to inhibiting CAM growth, most chemicals tested in this study were highly inhibitory at low doses in an in vitro hamster oviductal bioassay that measures ciliary beat frequency, oocyte pickup rate, and smooth muscle contraction (Riveles et al., 2003
, 2004, 2005). Our data from the CAM, oviduct, and cultured mammalian cell assays demonstrate that 2- and 3-ethylpyridine, pyrazine, and p-cresol adversely affect a broad spectrum of biological processes.
Substitutions to the pyridine ring (nicotine, 2- and 3-ethylpyridine) greatly affected cell proliferation and survival. In survival medium, nicotine stimulated all three cell types to divide, even at very low doses (10–14M) (Fig. 2). Similar stimulation in survival conditions has been reported for calf pulmonary artery endothelial cells (Villablanca, 1998), HUVEC, and human coronary artery endothelial cells (Heeschen et al., 2001). Although the nicotine LOAELs for all three cell types in our study in survival medium were the same (10–14M), the efficacies were quite different with HUVEC being the most sensitive (Table 1, B). In a related study, HUVEC also appeared more responsive to nicotine stimulation than human coronary artery endothelial cells under survival conditions (Heeschen et al., 2001). In proliferation medium, nicotine stimulated proliferation of HUVEC at doses found in the plasma of smokers (10–8M–10–7M) (Hill et al., 1983). Interestingly, HMVEC and NIH 3T3 cells were not stimulated with nicotine in proliferation medium, indicating that this effect was specific for certain cells. While the decrease in cell number we observed in both media at high doses was probably due to cytotoxicity, the stimulatory effects at low doses showed that nicotine can act as a potent growth factor to which endothelial cells in a developing organ are particularly responsive.
The other pyridine derivatives (2- and 3-ethylpyridine) were far more potent in the cell culture assays than the parent compound pyridine. However, unlike nicotine, which stimulated cell growth at low doses, 2- and 3-ethylpyridine promoted death of HUVEC and HMVEC at low doses in survival medium (Figs. 3–4). Both 2- and 3-ethylpyridine also inhibited CAM growth (Ji et al., 2002) and adversely affected oviductal functioning (Riveles et al., 2003) at picomolar doses. In survival medium, HUVECs were killed by very low doses of 2- and 3-ethylpyridine, were equally sensitive to both chemicals, and were 10,000 times more sensitive to 2- and 3-ethylpyridine than HMVEC (HUVEC LOAEL = 10–16M; HMVEC LOAEL = 10–12M).
In proliferation medium, both HUVEC and HMVEC were more sensitive to 2-ethylpyridine than to 3-ethylpyridine (e.g., HUVEC LOAEL for 2-ethylpyridine = 10–14M and for 3-ethylpyridine = 10–6M). However, in proliferation medium, HUVEC were 1,000,000 times more sensitive to 2-ethylpyridine than HMVEC (HUVEC LOAEL = 10–14M; HMVEC LOAEL = 10–8M) but were only 100 times more sensitive to 3-ethylpyridine than HMVEC (HUVEC = 10–6M; HMVEC = 10–4M). These results showed that addition of an ethyl group to the pyridine ring creates a derivative that can inhibit growth or kill cultured human endothelial cells at very low doses.
NIH 3T3 cell proliferation was stimulated by both 2- and 3-ethylpyridine at low doses that killed endothelial cells (Figs. 3–4). These results showed that different mammalian cells responded differently to 2- and 3-ethylpyridine and that at low doses, both compounds can kill endothelial cells, while stimulating growth of NIH 3T3 cells.
p-Cresol was one of the most inhibitory phenols tested in the CAM (Melkonian and Talbot, unpublished data) and oviductal bioassays (Riveles et al., 2005) with LOAELs in the picomolar range. However, in the cultured cell assay, p-cresol, like pyridine, produced little effect on HUVEC or HMVEC in either medium. Interestingly, NIH 3T3 cells were stimulated to proliferate by p-cresol in survival medium at doses as low as 10–10M (Fig. 5). These observations showed that p-cresol differentially affected cells from different sources (CAM vs. cultured endothelial cells) to different degrees and that opposite effects can be elicited by this chemical (e.g., CAM growth was inhibited while NIH 3T3 cells were stimulated to grow). p-Cresol has been detected (1.78 µg/ml) in the urine of smokers (Orejuela and Silva, 2002) at a concentration approximately equivalent to 10–5M in our studies. p-Cresol did induce HUVEC death in survival medium at doses of 10–8M–10–6M (Fig. 5), suggesting that its concentration in smokers is high enough to be effective.
Pyrazine, which has one more nitrogen than pyridine, was highly inhibitory in both the CAM (Melkonian et al., 2003) and oviductal bioassays (Riveles et al., 2004). Pyrazine was the only chemical tested that was inhibitory in both proliferation and survival media for all three cell types. In proliferation medium, HUVECs were far more sensitive than HMVECs or NIH 3T3 cells (Table 1, F). In survival medium, similar results were observed, except that the LOAELs for all three cell types were 100–10,000 times lower than in proliferation medium. A study dealing with synthesis of pyrazine–pyridine biheteroaryl compounds to block vascular endothelial growth factor receptor-2 (VEGFR-2) showed that the second nitrogen in the pyridine ring enhanced binding affinity for VEGFR-2 (Kuo et al., 2005). In our study, pyrazine may have been more effective than pyridine in blocking cell proliferation because the second nitrogen in pyrazine inhibited VEGF binding to its receptor.
With respect to the cell types tested, two interesting trends emerged. First, HUVECs, which come from a developing organ (umbilical cord), were consistently more sensitive to chemical treatment than HMVECs, which come from an adult organ (lung). A previous DNA microarray study on differential gene expression in 53 types of cultured endothelial cells showed that endothelial cells from microvasculature, arteries, and veins each had characteristic patterns of gene expression (Chi et al., 2003; Ho et al., 2003). These expression data indicate that endothelial cells are highly diverse and help to explain our observation that HUVEC and HMVEC responded differently to the same chemical treatments. Secondly, NIH 3T3 cells were stimulated to proliferate in survival medium by all chemicals tested except pyridine and pyrazine, which killed them. This clearly shows that the test chemicals may have profoundly different effects on different cell types. Every cell of the NIH 3T3 line examined by chromosome banding exhibited a unique karyotype that was continuously changing (Rubin, 2005). The differences in karyotype stability between NIH 3T3 cells and human endothelial cells may influence their responses to the test compounds, possibly due to over- or underexpression of genes regulating cell proliferation and/or survival.
Four of the six chemicals tested (pyridine, 3-ethylpyridine, p-cresol, and pyrazine) are on the FEMA-GRAS and FDA EAFUS lists and are added to consumer products including cigarettes and food for flavoring and cosmetics (R.J. Reynolds Tobacco Company, 2002). Two of these chemicals, 3-ethylpyridine and pyrazine, were among the most potent that we tested in the cell culture assay. In a recent reevaluation for FEMA, it was concluded that pyrazines are safe for human consumers (Adams et al., 2002). However, most studies on pyrazine have been done on adult tissues. The significant inhibition of CAM growth and HUVEC cell proliferation and survival at low doses indicate that more work is needed to determine how pyrazine affects developing issues.
Our data document a clear effect of individual cigarette toxicants on mammalian cells in vitro. It will be important in future studies to determine how these chemicals affect cells in vivo, where they would be delivered via smoking in a complex mixture. Since some of the chemicals that we studied exerted their effects at very low doses (e.g., 3-ethylpyridine), it is likely that smokers are exposed to amounts of these chemicals that are above the LOAELs observed in vitro.
In summary, of the six chemicals tested, nicotine, 2- and 3-ethylpyridine, and pyrazine produced the strongest effects on proliferation and survival of cultured mammalian cells. While p-cresol was less potent, it nevertheless showed activity at concentrations found in smokers' urine. HUVECs were in general more responsive to treatment than HMVECs, suggesting that cells from developing tissue are more susceptible to these chemicals than cells from adult tissue. Small modifications to the pyridine ring greatly increased its potency (e.g., 2- and 3-ethylpyridine, pyrazine). Nicotine acted like a growth factor for all three cell types in survival media. In contrast, 2- and 3-ethylpyridine acted like death factors for both types of endothelial cells, while pyrazine killed all three cell types. Some cells responded oppositely to the same chemical (e.g., 2-ethylpyridine killed endothelial cells but stimulated growth of NIH 3T3 cells). While endothelial cells were killed by all chemicals except nicotine, NIH 3T3 cells were stimulated to grow by all chemicals except pyrazine at doses below those that were cytotoxic. These data support prior work showing that cigarette toxicants, in particular 2- and 3-ethylpyridine, p-cresol, and pyrazine, inhibit a broad spectrum of biological processes and should be studied in more detail, especially with respect to developing tissue.
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ACKNOWLEDGMENTS |
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The authors are very grateful for the help, suggestions, and advice of Manuela Martins-Green, PhD., and Christian Lytle, PhD. (University of California, Riverside, Riverside, CA) during the course of this study and to Yuhuan Wang for her help with some of the experiments. This work was funded by grants 10RT-0239 and 13RT-0068 from the Tobacco-Related Disease Research Program, California.
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