Phenols, Quinolines, Indoles, Benzene, and 2-Cyclopenten-1-ones are Oviductal Toxicants in Cigarette Smoke
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《毒物学科学杂志》
Graduate Program in Environmental Toxicology and Department of Cell Biology and Neuroscience, University of California Riverside, Riverside, California 92521
ABSTRACT
Previously, we showed that pyridines and pyrazines in cigarette smoke inhibit oviductal functioning in vitro in nanomolar and picomolar doses. The purpose of this study was to determine the lowest observable adverse effect levels (LOAELs) of phenols, quinolines, indoles, benzene, and 2-cyclopenten-1-ones found in mainstream smoke solutions on ciliary beat frequency, oocyte pickup rate, and infundibular smooth muscle contraction using the hamster oviduct. After solid phase extraction, mainstream smoke solution fractions were tested in the oviductal assays. The active fractions were analyzed using gas chromatography-mass spectrometry to identify individual chemicals. Using this approach, benzene, eleven phenolic, two indole, two quinoline, and two 2-cyclopenten-1-one derivatives were identified in the active fractions. Commercially available authentic standards of the identified compounds were tested in dose-response studies on hamster oviducts. The LOAELs were determined for each compound using the ciliary beat frequency, oocyte pickup rate, and infundibular smooth muscle contraction rate assays. Indole, the compound with the highest potency, showed inhibition of ciliary beat frequency (10–13 M), oocyte pickup rate (10–14 M), and infundibular smooth muscle contraction rate (10–15 M) in femtomolar doses. All of the other compounds tested, except phenol, which only showed inhibition at millimolar concentrations, were inhibitory in picomolar, nanomolar, or micromolar concentrations. Derivitization of phenol increased its toxicity in the oviductal assays, especially when a methyl or ethyl group was substituted on the fourth position. The indoles, quinolines, and four phenolic compounds had both high potencies and efficacies in the oviductal assays.
Key Words: cigarette smoke; oviduct; oocyte pickup; phenols.
INTRODUCTION
Numerous epidemiological studies have linked cigarette smoking with reproductive problems in women. For example, an association has been observed between women who smoke and increased incidences of ectopic pregnancy, tubal infertility, and spontaneous abortion (Castles et al., 1999; Mueller and Ciervo, 1998; Saraiya et al., 1998). These problems could be due to an effect on the oviduct, and indeed, recent in vivo and in vitro studies have shown an effect on oviductal functioning by cigarette smoke (DiCarlantonio and Talbot, 1999; Knoll et al., 1995; Knoll and Talbot, 1998; Magers et al., 1995; Riveles et al., 2003). Factors such as cigarette smoke that impair oviductal functioning can diminish or prevent fertility (Florek and Marszalek, 1999; Shiverick and Salafia, 1999).
Inhalation of mainstream (MS) or sidestream (SS) smoke by hamsters, at serum cotinine levels that were within the ranges found in active and passive human smokers, adversely affected the ultrastructure of the oviductal epithelium (Magers et al., 1995). Decreases in the ratio of ciliated to secretory cells in the ampulla were also observed in these hamsters (Magers et al., 1995). In another study on hamsters, inhalation of MS and SS smoke slowed embryo transport, and 75% of the embryos were retained in the oviduct (DiCarlantonio and Talbot, 1999). Moreover, oviductal contractions were inhibited differentially in the ampulla and isthmus in vivo after smoke exposure, suggesting that embryo transport rates were slowed by smooth muscle contraction (DiCarlantonio and Talbot, 1999). Studies have also shown that cigarette smoke exposure slowed the rate of smooth muscle contraction in humans (Neri and Eckerling, 1969) and in rabbits (Ruckebusch, 1975) after in vivo exposure.
In vitro studies have also shown that MS and SS smoke solutions adversely affect proper functioning of the oviduct. MS and SS smoke solutions made with University of Kentucky 2R1 research cigarettes inhibited oocyte pickup rate in a dose-dependent manner in explants of hamster oviducts in vitro (Knoll et al., 1995; Knoll and Talbot, 1998). Ciliary beat frequency decreased in MS smoke solutions; however, SS produced no change or stimulated ciliary beating (Knoll et al., 1995; Knoll and Talbot, 1998). The gas and particulate phases of the smoke solutions were also tested in the oocyte pickup rate assay. Oocyte pickup rate was more sensitive to the gas than the particulate phase of MS and SS smoke solutions (Knoll and Talbot, 1998).
Several studies have been done to identify the toxicants in cigarette smoke that affect the oviduct. For example, oral administration of nicotine inhibited oviductal contractions in Rhesus monkeys (Neri and Marcus, 1972). Intravenous administration of nicotine delayed rat embryo cleavage, reduced embryo cell number, and reduced oviductal blood flow (Mitchell and Hammer, 1985). In another study, other individual smoke components (potassium cyanide, acrolein, phenol, acetaldehyde, and formaldehyde), inhibited ciliary beat frequency in the hamster oviduct in vitro; however, only cyanide was present in cigarette smoke solutions in sufficiently high concentrations to account for the effects seen in vitro (Talbot et al., 1998).
As a result of earlier epidemiological and model systems studies, our lab developed an assay using the hamster infundibulum in vitro to study oviductal functioning and we have been determining the compounds in smoke that affect oocyte pickup rate, ciliary beat frequency, and infundibular smooth muscle contraction rate. Pyridine compounds, which are added directly to tobacco and are generated indirectly by combustion processes associated with burning of the cigarette, have been identified in MS and SS smoke solutions and adversely affect hamster oviductal functioning in vitro (Riveles et al., 2003). Twelve pyridine compounds, including nicotine, were tested on oviductal functioning in vitro, measuring ciliary beat frequency, oocyte pickup rate, and oviductal smooth muscle contraction rate (Riveles et al., 2003). Pyridine derivatives with single ethyl or methyl substitutions (2-ethylpyridine, 3-ethylpyridine, 2-methylpyrdine, and 4-methylpyridine) were inhibitory at picomolar doses for all three parameters measured (Riveles et al., 2003). Nornicotine and -nicotyrine, which are similar in structure to nicotine, were inhibitory in nanomolar doses in the bioassays, while nicotine was only inhibitory at 10–2 M.
In the second study of the series, pyrazine compounds, which are additives in food, tobacco, and cosmetics, and are combustion products of tobacco, were identified in both MS and SS smoke solutions and tested on the hamster oviduct in vitro (Riveles et al., 2004). Six of the seven pyrazine compounds inhibited oviductal functioning at picomolar or nanomolar doses in vitro. Several pyrazine compounds with single ethyl or methyl substitutions (2-ethylpyrazine and 2-methylpyrazine), as well as unsubstituted pyrazine, inhibited ciliary beat frequency, oocyte pickup rate, and smooth muscle contraction rate at picomolar doses (Riveles et al., 2004).
The purpose of this study was to test the hypothesis that there are additional classes of components identified in MS and SS smoke solutions that adversely affect ciliary beat frequency, oocyte pickup rate, and infundibular smooth muscle contraction rate. To test this hypothesis, benzene, indole, 5-methylindole, quinoline, isoquinoline, 2-cyclopenten-1-one, 3-methyl-2-cyclopenten-1-one, and eleven phenolic compounds were individually tested in dose-response experiments on the hamster oviduct in vitro, measuring ciliary beat frequency, oocyte pickup rate, and smooth muscle contraction rate. Our data show that several compounds were inhibitory in as low as femtomolar or picomolar doses.
MATERIALS AND METHODS
Animals, media, and authentic standards.
Female golden hamsters (Mesocricetus auratus) purchased from Harlan (San Diego, CA) were maintained on a 14L:10D cycle in a room at 26°C. Food was administered ad libitum. Hamsters were cycled daily by checking for a vaginal discharge, which occurs on day 1 of their estrous cycle. Female golden hamsters were induced to superovulate by intraperitoneal injection with 25 International Units (IU) of pregnant mare's serum gonadotropin (PMSG) (CalBiochem, La Jolla, CA.) at 10 A.M. on day 1 of their estrous cycle, followed by 25 IU of human chorionic gonadotropin (hCG) (Sigma Chemical Co., St. Louis, MO) on day 3 of the estrous cycle. Hamsters were sacrificed with CO2 on day 4, approximately 12 h after the hCG injection, and oviducts and ovaries were removed and separated. The two ovaries were isolated in Earle's Balanced Salt Solution supplemented with sodium bicarbonate, HEPES, and 0.1% BSA at pH = 7.4 (EBSS-H). The expanded follicles were poked with a dissecting needle to release the oocyte cumulus complexes (OCCs). Approximately 7–15 mature expanded OCCs were recovered from each ovary. The two infundibula were dissected from the oviducts and placed into perfusion chambers containing EBSS-H. The ampullas were left attached to the infundibula and placed into holding pipettes so the infundibula remained stationary throughout the experiment. EBSS-H was used as the control solution for all experiments. The protocol for the use of hamsters in this study was approved by the campus animal use committee.
2-Cyclopenten-1-one (98%), 2,4-dimethylphenol (98%), 2-ethylphenol (99%), 4-ethylphenol (99%), indole (98%), 2-methoxyphenol (98%), p-methoxyphenol (99%), 3-methyl-2-cyclopenten-1-one(97%), 5-methylindole (99%), 2-methylphenol (99+%), 4-methylphenol (99%), and quinoline (98%) were purchased from Aldrich Chemical Company (Milwaukee, WI). 2,6-Dimethoxyphenol (96%) and isoquinoline were purchased from City Chemical LLC (West Haven, CT.). Hydroquinone (99%) was purchased from VWR (Brisbane, CA). Benzene (99%) and phenol (99%) were purchased from Fisher Scientific (Pittsburgh, PA).
Preparation of smoke solutions.
Mainstream (MS) smoke solutions were made in EBSS-H using 2R1 research-grade cigarettes (University of Kentucky, Louisville, KY) that were smoked on a puffer box built at the University of Kentucky (Knoll et al., 1995; Knoll and Talbot, 1998). This method has been described and published previously in detail (Knoll et al., 1995; Knoll and Talbot, 1998; Riveles et al., 2003). MS smoke solutions were made from 60 puffs of MS smoke pushed through 10 ml of EBSS-H, which equals 6 puff equivalents.
Solid-phase extraction and gas-chromatography-mass spectrometry (GC-MS) of smoke solutions.
Bond Elut solid phase extraction (SPE) cartridges, 3 cc, 500 g capacity (Phenomenex, Torrance, CA) were used to fractionate smoke solutions and concentrate chemicals that inhibit oviductal functioning. A variety of nonpolar, polar, and anion and cation exchange cartridges were used: NH2, 2OH, CN, CBA, SCX, SAX, C18, C8, C2, CH, SI, and PH. The protocol used to screen the cartridges for their ability to bind oviductal toxicants in smoke solutions has been described in detail previously (Riveles et al., 2003). MS smoke solutions were passed through the cartridges, and the individual fractions and corresponding cartridge eluates were collected and tested in the oviductal bioassays measuring ciliary beat frequency, oocyte pickup rate, and smooth muscle contraction rate (Riveles et al., 2003).
Four cartridges (C8, CN, PH, and NH2) were particularly effective at retaining the oviductal toxicants, and their eluates were characterized using GC-MS. A Hewlett Packard 5890 GC interfaced to an HP-5971A MSD quadrupole mass selective detector with a Zebron ZB1701 cyanopropyl phenyl column 30 m x 0.32 mm with 1-μm phase thickness from Phenomenex (Torrance, CA.) was used as described previously in detail (Riveles et al., 2003). The eluate sample (2 μl) was injected directly into the GC, and compounds were identified using the mass spectrometry data matched to mass spectral library entries. Compound identities were confirmed using authentic standards and by matching both mass spectra and retention times.
Oviductal assays: ciliary beat frequency, oocyte pickup rate, and infundibular smooth muscle contraction rate.
Ciliary beat frequency, oocyte pickup rate, and smooth muscle contraction rate were measured using hamster oviduct explants in vitro in a bioassay developed in our lab and described previously in detail (Riveles et al., 2003). A single experiment was defined as the treatment of one infundibulum with the control medium (EBSS-H with 0.1% BSA) and a single dose of the test chemical diluted in EBSS-H for a 5-min exposure period. The mean ± standard deviation of six to ten measurements was made on an infundibulum for each treatment group. For each parameter, a region of the infundibulum was selected, and measurements for both the control and treated groups were performed in the same region.
Each parameter was measured using the same experimental setup with a Wild 5A stereoscopic microscope and a video image capture system as described previously (Riveles et al., 2003). Ciliary beat frequency was measured using the video capture system and expressed as number of beats per second. Oocyte pickup rate was performed by manually placing an oocyte cumulus complex (OCC) onto the infundibulum and measuring the speed at which the OCC traversed a defined path expressed as microns per second. Infundibular smooth muscle contractions were also measured using the video image capture system. The distance of the contractions was measured directly on the computer monitor using a ruler as described previously (Riveles et al., 2003). Frequency was measured as the number of times the infundibulum contracted per minute. Smooth muscle contraction rate was calculated and expressed as microns per minute.
Determination of LOAELs and efficacies.
To determine LOAELs for each compound tested in each bioassay, a preliminary screen was done using one infundibulum per dose group (10–16–10–1 M). For each infundibulum, 6–10 control, treatment, and recovery measurements were taken, and the means were compared using a one-way ANOVA. The lowest significant inhibitory dose provided an estimate of the LOAELs for each test compound in each assay. Since the estimated LOAELs were based on only one infundibulum per dose, additional experiments were performed at the estimated NOAEL and LOAEL doses to increase the sample size to n = 4 infundibula for all of the tested compounds in each bioassay.
Using the initial dose-response curves, the efficacy values were estimated for each test compound in each assay. Efficacies were determined by finding the point on the curve that reached a maximum percentage of inhibition and then leveled off over two or more consecutive doses. These efficacy values are estimations based on a limited dose selection and a sample size of only one infundibulum. A maximum percentage of inhibition was not reached in all cases based on the doses selected, in which case efficacies were indicated as greater than or equal to the highest percentage of inhibition observed for that compound in that assay at the highest dose tested.
Statistical analysis.
The statistical significance of the results was evaluated in the oocyte pickup rate, ciliary beat frequency, and infundibular muscle contraction rate assays using a one sample t-test to compare the control (n = 4 infundibula) set to 100% to the mean at the estimated LOAEL dose (n = 4 infundibula) for each test compound in each bioassay (Fig. 7) with Instat (Graphpad, San Diego, CA). Means were considered to be significantly different for p < 0.05.
RESULTS
Phenols, Indoles, Quinolines, Benzene, and 2-Cyclopenten-1-Ones Identified in MS Smoke Solutions
A preliminary screen of 12 solid phase extraction cartridges revealed that four of the cartridges (C8, CN, PH, and NH2) retained chemicals in MS smoke solutions that were inhibitory in the ciliary beat frequency, oocyte pickup rate, and smooth muscle contraction rate assays. Phenols, indoles, quinolines, benzene, and 2-cyclopenten-1-ones were subsequently identified in the eluates of these four cartridges using GC-MS (Table 1). Commercially available authentic standards of the identified compounds from Table 1 were purchased and tested in dose-response studies on the oviduct to determine their lowest observable adverse effect levels (LOAELs) in the oviductal bioassays.
Dose-Response, LOAELs, and Estimated Efficacies of the Phenolic Compounds in the Oviductal Bioassays
Phenol and 10 phenolic derivatives (2-methylphenol, 4-methylphenol, 2-ethylphenol, 4-ethylphenol, 2,4-dimethylphenol, 2,5-dimethylphenol, 2-methoxyphenol, 4-methoxyphenol, 2,6-dimethoxyphenol, and hydroquinone) were tested in dose-response studies on the hamster oviduct based on a 5-min exposure in the ciliary beat frequency, oocyte pickup rate, and smooth muscle contraction rate assays. For each phenolic compound tested, an initial screen of doses, ranging from 10–13 M to 10–1 M was performed for a single infundibulum per dose group. For the parent compound, phenol, the dose range was extended to 10 M. Dose-response curves for the ciliary beat frequency, oocyte pickup rate, and smooth muscle contraction rate assays were created based on these data (Figs. 1–3 ). LOAELs were estimated from the dose-response curves and then confirmed using an increased sample size (n = 4 infundibula) for each of the bioassays. The confirmed LOAELs for the phenolic compounds (n = 4) are summarized in Table 2. Figure 7 has the mean percent inhibition and standard deviation at each LOAEL for each of the bioassays. The estimated efficacies were determined from the initial dose-response curves as explained in the Materials and Methods.
For the ciliary beat frequency assay, the dose-response curves were similar for most of the phenolic compounds, except for 2-ethylphenol and phenol, which were inhibitory at higher doses (Fig. 1). It is interesting that all of the phenolic derivatives were more potent than phenol itself. Additional infundibula were tested at the estimated LOAEL doses for confirmation. The LOAELs for the phenolic compounds ranged from 10–12 M to 10–1 M (Table 2). 4-Methylphenol (10–12 M), 4-ethylphenol (10–11 M), 2,6-dimethoxyphenol (10–10 M), and hydroquinone (10–10 M) inhibited ciliary beat frequency at picomolar doses. 2-Methylphenol (10–9 M), 2,4-dimethylphenol (10–9 M), 2-methoxyphenol (10–8 M), 2,5-dimethylphenol (10–8 M), and 4-methoxyphenol (10–7 M) inhibited ciliary beat frequency at nanomolar doses. 2-Ethylphenol (10–5 M) inhibited ciliary beat frequency at micromolar doses. Phenol had the highest LOAEL (10–1 M). The substitution of an ethyl or methyl group in the fourth position on the phenol moiety compared to the substitution on the second position increased its potency by a million-fold and one-thousand-fold, respectively.
For the oocyte pickup rate assay, the dose-response curves were similar for six of the phenolic compounds (2-methylphenol, 2,4-dimethylphenol, 4-ethylphenol, 2-methoxyphenol, 2,6-dimethoxyphenol, and hydroquinone) (Fig. 2). The dose-response curves for 2-ethylphenol, 2,5-dimethylphenol, and 4-methoxyphenol showed inhibition at higher doses. All of the phenolic derivatives were more potent than phenol itself, which had a LOAEL for oocyte pickup rate (10–2 M), similar to the ciliary beat frequency assay (10–1 M). Additional infundibula were tested at the estimated LOAEL doses for confirmation. The LOAELs for the phenolic compounds in the oocyte pickup rate assay ranged from 10–12 M to 10–2 M (Table 2). 4-Ethylphenol, (10–12 M), 2-methylphenol (10–11 M), 4-methylphenol (10–11 M), 2,6-dimethoxyphenol (10–11 M), 2,4-dimethylphenol (10–10 M), 2-methoxyphenol (10–10 M), and hydroquinone (10–10 M) inhibited oocyte pickup rate in picomolar doses. 2-Ethylphenol (10–8 M), 4-methoxyphenol (10–8 M), and 2,5-dimethylphenol (10–7 M) inhibited oocyte pickup rate at nanomolar doses. Phenol had the highest LOAEL (10–2 M) (Table 2).
For the smooth muscle contraction rate assay, all of the derivatives were more potent than phenol (10–2 M) (Fig. 3). The dose-response curves were similar for most of the phenolic compounds, except for 2-methylphenol, 4-methylphenol, and phenol (Fig. 3). The dose-response curves for 2-ethylphenol (10–7 M), 4-methoxyphenol (10–7 M), and 2,5-dimethylphenol (10–6 M) had LOAELs at higher doses in this assay, as was the case in the oocyte pickup rate assay (10–8, 10–8 M, and 10–7, respectively). Additional infundibula were tested at the estimated LOAEL doses for confirmation. For the smooth muscle contraction rate assay, the LOAELs for the phenolic compounds ranged from 10–12 M to 10–2 M (Fig. 3). 4-Ethylphenol (10–12 M), 2-methylphenol (10–11 M), 4-methylphenol (10–11 M), and hydroquinone (10–10 M) inhibited smooth muscle contraction rate at picomolar doses. 2,4-Dimethylphenol (10–9 M), 2,6-dimethoxyphenol (10–9 M), 2-methoxyphenol (10–8 M), 2-ethylphenol (10–7 M), and 4-methoxyphenol (10–7 M) inhibited smooth muscle contraction rate at nanomolar doses. 2,5-Dimethylphenol (10–6 M) inhibited smooth muscle contraction rate at micromolar doses. The LOAEL for phenol was the highest at millimolar doses (10–2 M) (Fig. 3).
Using the initial dose-response curves, the efficacies for phenol and several of its derivatives were estimated as described in the Materials and Methods. The estimated efficacies of phenol in the ciliary beat frequency, oocyte pickup rate, and smooth muscle contraction rate assays were 22%, 68%, and 100%, respectively (Figs. 1–3). The chemicals having estimated efficacies in the ciliary beat frequency assay that were lower than phenol were 2,4-dimethylphenol (7%) and 4-methylphenol (16%) (Fig. 1). 2-Methylphenol (25%), 2,5-dimethylphenol (28%), 2-ethylphenol (32%), and hydroquinone (37%) had estimated efficacies in the ciliary beat frequency assay that were higher than phenol (Fig. 1). The efficacies for 2-methoxyphenol (9%), 4-methoxyphenol (11%), 2,6-dimethoxyphenol (14%), and 4-ethylphenol (19%) were estimated based on the initial dose-response curves (Fig. 1).
For the oocyte pickup rate assay, from the dose-response curve, the efficacy for phenol was 68% (Fig. 2). The estimated efficacy of 4-methylphenol (6%) was lower than for phenol in the oocyte pickup rate assay (Fig. 2). The efficacies for 4-ethylphenol (41%), 2-methoxyphenol (41%), 2-methylphenol (42%), 4-methoxyphenol (50%), 2,6-dimethoxyphenol (52%), hydroquinone (63%), 2,4-dimethylphenol (64%), 2,5-dimethylphenol (67%), and 2-ethylphenol (75%) were estimated for the oocyte pickup rate assay based on the dose-response curves (Fig. 2). The efficacies in the oocyte pickup rate assay were higher than for the ciliary beat frequency assay in most cases.
In the smooth muscle contraction rate assay, the estimated efficacy of phenol was 100% (Fig. 3). The estimated efficacies of 4-methylphenol (6%) and 2,6-dimethoxyphenol (66%) were lower than phenol in the smooth muscle contraction rate assay (Fig. 3). 4-Ethylphenol (100%) and 4-methoxyphenol (100%) had efficacies similar to phenol (Fig. 3). The efficacies for 2-methylphenol (16%), 2-methoxyphenol (30%), 2-ethylphenol (39%), 2,4-dimethylphenol (65%), and hydroquinone (71%) were estimated in the smooth muscle contraction rate assay based on the initial dose-response curves (Fig. 3).
Dose-Response, LOAELs, and Estimated Efficacies of Indoles, Quinolines, Benzene, and 2-Cyclopenten-1-ones in the Oviductal Bioassays
An initial screen of doses ranging from 10–16 M to 10–4 M was used to test indole, 5-methylindole, quinoline, isoquinoline, benzene, 2-cyclopenten-1-one, and 3-methyl-2-cylcopenten-1-one in the three bioassays with a single infundibulum per dose group. Dose-response curves for the ciliary beat frequency, oocyte pickup rate, and smooth muscle contraction rate assays were created based on these data (Figs. 4–6). LOAELs were estimated from the dose-response curves and then confirmed using an increased sample size (n = 4 infundibula). The confirmed LOAELs for these compounds (n = 4) are summarized in Table 3. Figure 7 has the mean percent inhibition and standard deviation at each LOAEL for each of the bioassays.
For the ciliary beat frequency assay, the dose-response curves were similar for the indoles and quinolines (Fig. 4). Benzene, 2-cyclopenten-1-one, and 3-methyl-2-cyclopenten-1-one showed inhibition at higher doses and were the least potent in this group of chemicals (Fig. 4). Additional infundibula were tested to confirm the LOAELs for each compound in the ciliary beat frequency assay. The LOAELs ranged from 10–13 M to 10–8 M (Table 3). Indole (10–13 M), quinoline (10–13 M), isoquinoline (10–12 M), and 5-methylindole (10–10 M) inhibited ciliary beat frequency at femtomolar or picomolar doses. Benzene (10–8 M) and 2-cyclopenten-1-one (10–8 M) inhibited ciliary beat frequency at nanomolar doses. The LOAEL for 3-methyl-2-cyclopenten-1-one in the ciliary beat frequency was not determined with the dose range used (Fig. 4).
For the oocyte pickup rate assay, the dose-response curve for indole showed the highest potency of all the chemicals tested (Fig. 5). The dose-response curves for 5-methylindole, quinoline, and isoquinoline in the oocyte pickup rate assay were similar to the ciliary beat frequency assay. Benzene and 3-methyl-2-cyclopenten-1-one were the least potent in this assay as was the case in the ciliary beat frequency assay (Fig. 5). Additional infundibula were tested to confirm the LOAELs for each compound in the oocyte pickup rate assay (Fig. 7). The LOAELs for this group of compounds ranged from 10–14 M to 10–7 M (Table 3). Indole (10–14 M), isoquinoline (10–13 M), quinoline (10–11 M), 5-methylindole (10–11 M), and 3-methyl-2-cyclopenten-1-one (10–10 M) inhibited oocyte pickup rate at femtomolar or picomolar doses. Oocyte pickup rate was inhibited by 2-cyclopenten-1-one (10–9 M) and benzene (10–7 M) at nanomolar doses.
For the smooth muscle contraction rate assay, indole showed the highest potency of all the chemicals tested, as was the case for oocyte pickup rate (Fig. 6). The dose-response curves for 5-methylindole, quinoline, isoquinoline 2-cyclopenten-1-one, and 3-methyl-2-cyclopenten-1-one were similar for the smooth muscle contraction rate assay. The dose-response curve for benzene showed little potency in this assay, as was the case for the ciliary beat frequency and oocyte pickup rate assays (Fig. 6). Additional infundibula were tested to confirm the LOAELs for each compound in the smooth muscle contraction rate assay (Table 3). Indole (10–15 M) and isoquinoline (10–13 M) inhibited smooth muscle contraction rate at femtomolar doses. Quinoline (10–11 M), 5-methylindole (10–10 M), and 3-methyl-2-cyclopenten-1-one (10–10 M) inhibited smooth muscle contraction rate at picomolar doses. Smooth muscle contraction rate was inhibited by 2-cyclopenten-1-one (10–9 M) at nanomolar doses and by benzene (10–6 M) at micromolar doses (Fig. 6).
Estimated Efficacies for Indoles, Quinolines, 2-Cyclopenten-1-ones, and Benzene
Using the initial dose-response curves, the efficacies for the indoles, quinolines, 2-cyclopenten-1-ones, and benzene were estimated as described in Materials and Methods. For the ciliary beat frequency assay, the efficacies for indole (25%) and 5-methylindole (15%) could not be compared (Fig. 4). The efficacy of isoquinoline in the ciliary beat frequency assay was 27%; however, the efficacy of quinoline could only be predicted to be 22% (Fig. 4). The efficacies for benzene (56%), 2-cyclopenten-1-one (6%), and 3-methyl-2-cyclopenten-1-one (2%) were estimated based on the initial dose-response curves for the ciliary beat frequency assay (Fig. 4). For the oocyte pickup rate assay, 5-methylindole (73%) had a higher estimated efficacy than indole (52%) (Fig. 5). The estimated efficacies of quinoline (70%) and isoquinoline (61%) were high for oocyte pickup rate (Fig. 5). 3-Methyl-2-cyclopenten-1-one (20%) had a lower efficacy in the oocyte pickup rate assay than 2-cyclopenten-1-one (66%) (Fig. 5). The efficacy of benzene in the oocyte pickup rate assay was predicted to be 52% (Fig. 5). The exact efficacies in the smooth muscle contraction rate assay could not be determined based on the initial dose-response curves (Fig. 6). The efficacies could only be estimated for: quinoline (75%), 2-cyclopenten-1-one (61%), isoquinoline (60%), indole (55%), 3-methyl-2-cyclopenten-1-one (45%), 5-methylindole (39%), and benzene (26%) (Fig. 6).
DISCUSSION
The purpose of this study was to identify additional compounds in cigarette smoke that inhibit oviductal functioning. Phenol, 10 phenolic derivatives, indole, 5-methylindole, quinoline, isoquinoline, benzene, 2-cyclopenten-1-one, and 3-methyl-2-cyclopenten-1-one were identified in fractions of mainstream smoke solutions that inhibited oviductal functioning. The hierarchy of potency of the individual chemicals tested in this study and their LOAELs in the ciliary beat frequency, oocyte pickup rate, and infundibular smooth muscle contraction rate assays are summarized in Table 4. Several of these smoke toxicants were highly effective at femtomolar or picomolar doses in all three oviductal assays (indole, isoquinoline, 4-ethylphenol, quinoline, 4-methylphenol, 5-methylindole, and hydroquinone) (Table 4).
Previous studies have shown inhibition of oviductal functioning in picomolar or nanomolar ranges by pyridines (Riveles et al., 2003) and pyrazines (Riveles et al., 2004). While pyridine itself was not very potent in the oviductal assays, addition of a single methyl or ethyl group increased its potency greater than a 100-million-fold. Within the pyrazine group, addition of single ethyl and methyl groups had minor effects on its potency, in most cases. As was the case for the pyridines, substitution of an ethyl or methyl group to the ring increased the potency of the phenolic derivatives. In the case of the phenols, this increase was clearly correlated with the position of the substitution.
The LOAELS for phenol and its derivatives ranged from 10–12 M to 10–1 M. Four phenolic compounds (2-methylphenol, 4-ethylphenol, 2,6-dimethoxyphenol, and hydroquinone) had both high potencies and high efficacies in all three oviductal assays. 2,4-Dimethylphenol was potent in picomolar doses in all three assays, but only had high efficacies for the ooycte pickup rate and smooth muscle contraction rate assays. Four phenolic derivatives (2-ethylphenol, 2,5-dimethylphenol, 2-methoxyphenol, and 4-methoxyphenol) had high efficacies, but medium or low potencies.
Addition of either a methyl or ethyl group to the two or four position of phenol increased its potency in the oviductal assays. In general, the four substitution resulted in higher potency than the two. For the oocyte pickup rate and smooth muscle contraction rate assays, substitution of an ethyl group in the four position increased potency more than a two or four methyl substitution. Substitution of a methyl group in the two position or an ethyl group in the two or four positions was also highly efficacious in all three assays. 4-Methylphenol was very potent in all three oviductal assays, but had very low efficacies. The parent compound, phenol, was the least potent chemical in the oviductal assays with the highest LOAELs (10–2 to 10–1 M). Phenol was very effective in the oocyte pickup rate and smooth muscle contraction rate assays, but only at very high doses. In general, both substitution and position of the substitution influenced the degree of toxicity and efficacy in the bioassays. The data in this study show that several phenolic compounds are highly effective and inhibitory of oviductal functioning at very low doses.
Although the mechanism of action of phenols on the oviduct is not known, phenolic compounds studied previously exerted toxicity through the formation of phenoxy-free radicals (Smith et al., 2002). High concentrations of phenoxy radicals at the oviduct could attack biochemical processes such as production of ATP, cAMP, Ca2+, or nitric oxide (NO), which are important in oviductal muscle contraction and ciliary beat frequency (Cometti et al., 2003; Lansley et al., 1992; Lindblom and Hamberger, 1980). The biochemical affected by the phenoxy radical would depend on its interactions within the cell or on its surface (Selassie et al., 1998). The hydrophobicity of the phenoxy free radicals would enable them to penetrate into the hydrophobic region of the membranes (Selassie et al., 1998). This could facilitate interaction with polyunsaturated fatty acids and initiate free radical chain reactions (Selassie et al., 1998). In our study, one possible mechanism by which the phenolic compounds exerted toxicity on the oviduct may have been through the formation of phenoxy radicals. The free radicals produced and circulated to the oviduct may interfere with the production of biochemicals necessary for adhesion of the oocyte to the tips of the cilia on the infundibular surface. These free radicals could also inhibit oviductal functioning by slowing or stopping smooth muscle contraction by interfering with ATP or NO production. The oxidative damage produced by phenoxy free radicals could also result in cell death or mutation (Selassie et al., 1998).
Previous studies have evaluated the reproductive toxicity of phenol and its derivatives (Bian and Wang, 2004; Bruce et al., 1987). Phenol itself is rapidly absorbed and distributed to all tissues in animals and humans after oral, dermal, or inhalation exposure and has a half-life of approximately 3.5 h in humans (Bruce et al., 1987). Oral doses of phenol in rats showed dose-related fetal toxicity with a decrease in average fetal body weight per litter (Bruce et al., 1987; Narotsky and Kavlock, 1995). Reduction of growth (decreased somite number) and development (tail bud abnormalities) were observed in embryo cultures in vitro after exposure to phenol and its para-substituted cogeners (Oglesby et al., 1992). In vivo exposure to phenol decreased weight gain in pregnant rats (Kavlock, 1990). A limited number of studies have shown that phenol and its derivatives have reproductive effects (Bian and Wang, 2004) and our study clearly shows that certain phenolic compounds interfere with proper oviductal functioning in vitro in as low as picomolar and nanomolar doses.
Phenolic compounds have been measured in cigarettes (Chen and Moldoveanu, 2003; Czogala and Wardas, 1998; Forehand et al., 2000; Nanni et al., 1990; Rustemeir et al., 2002; White et al., 1990). The concentration of phenol and its derivatives in cigarette smoke varies significantly among different brands of cigarettes (Nanni et al., 1990). For example, in most cases, commercial brands (3.6–17 μg/cigarette) had higher concentrations of phenol than 1R4F research cigarettes (7 μg/cigarette) or ultra-low-tar cigarettes (0.26 μg/cigarette) (Nanni et al., 1990). However, the concentrations of phenol vary among different research brands, for example, 1R3 cigarettes (55.4 μg/cigarette) have more phenol than 1R4F cigarettes (6.9 μg/cigarette) (Forehand et al., 2000). In our study, phenol was identified in the MS smoke solutions from 2R1 cigarettes and in the active fractions. Phenol was the least potent chemical tested in this study with LOAELs in the millimolar range; however, it was very effective at these high doses. Phenol (5.7 μg/ml) and 4-methylphenol (1.78 μg/ml) have been detected in the urine of smokers (Orejuela and Silva, 2002). Taken together, the data suggest that phenol, if present in cigarette smoke in high enough concentration, could enter the circulatory system and inhibit oviductal functioning, especially oocyte pickup rate and smooth muscle contraction rate, effectively.
In addition to phenol, the concentration of hydroquinone has been measured in 1R3 research cigarettes (70.5 μg/cigarette) (Forehand et al., 2000) and MS smoke from a nonfilter cigarette (110–300 μg/cigarette) (U.S. EPA, 1992). In the current study, hydroquinone was highly potent and efficacious in all three oviductal assays. The concentration of hydroquinone in MS smoke could be sufficient to account for the effects we observed in vitro on hamster oviductal functioning.
Humans are also exposed to phenols through foods (Bruce et al., 1987). 2-Methoxyphenol, also known as guaiacol, is an additive in cigarettes (http://www.rjrt.com/TI/Ticig_ingred_summary.asp, Cigarette Ingredients, 2004), a Food and Drug Administration (FDA) approved food additive, and is on the Flavor and Extract Manufacturer's Association (FEMA) generally regarded as safe (GRAS) list. It is also found in celery, cocoa, coffee, rum, soybean, tea, tomato, whiskey, wine, and used in ice cream, baked goods, meat, chewing gum, and dairy products (http://www.bbc.co.uk/worldservice/sci_tech/features/health/tobaccotrial/inacigarette599.htm, What's in a Cigarette, 2004). In our study, 2-methoxyphenol inhibited oviductal functioning in nanomolar and picomolar doses and was highly efficacious in all three assays. 4-Ethylphenol and 2,6-dimethoxyphenol are also on the FEMA GRAS list and are found and used in a variety of consumer products. In the current study, 4-ethylphenol and 2,6-dimethoxyphenol had both high efficacies and high potencies in all three oviductal assays.
The LOAELs for the indole, quinoline, 2-cyclopenten-1-one compounds, and benzene ranged from 10–15 M to 10–6 M. Four compounds (indole, 5-methylindole, quinoline, and isoquinoline) had both high potencies (femtomolar and picomolar doses) and high efficacies in all three oviductal assays. Benzene had a high efficacy and medium potency (nanomolar doses) for ciliary beat frequency and oocyte pickup rate, but a lower potency (micromolar doses) for the smooth muscle contraction rate assay. 2-Cyclopenten-1-one and 3-methyl-2-cyclopenten-1-one were highly potent for the oocyte pickup rate and contraction rate assays, but not for the ciliary beat frequency assay, and they had varying efficacies.
Two of the most potent and efficacious cigarette smoke components tested in this study were indole and 5-methylindole. Although reproductive studies were not found on these two compounds, another type of indole, indole-3-acetic acid (I3C), is a negative regulator of estrogen (Auborn et al., 2003) and has been shown to be teratogenic in mice and rats at 500 mg/kg/day resulting in decreased weight gain (John et al., 1979). I3C is an endocrine disrupter that can block ovulation in adult rats at 0.5 to 1.5 g/kg/day and cause decreases in body and ovarian weight gain at 1.5 g/kg/day (Gao et al., 2002). In the current study, ciliary beat frequency, oocyte pickup rate, and oviductal smooth muscle contraction were inhibited by indole in femtomolar doses and 5-methylindole in picomolar doses in vitro. The results from our study combined with previous studies suggest that indoles are effectors that can adversely affect several reproductive processes including oviductal functioning.
Although information is available on phenols and certain indoles, the reproductive effects of quinoline and isoquinoline are unknown. In our study, both quinoline and isoquinoline inhibited oviductal functioning and were highly efficacious at very low doses. Quinoline has been measured in the MS smoke from 1R4F (0.30 μg/cigarette) and 2R4F (0.23 μg/cigarette) research cigarettes (Chen and Moldoveanu, 2003); however smoke condensate from 1R4F cigarettes had lower levels of quinoline (0.19 μg/cigarette) (White et al., 1990). Higher concentrations of quinoline were reported for a nonfiltered cigarette (0.5–2 μg/cigarette) (U.S. EPA, 1992). In our study, quinoline inhibited oviductal functioning at femtomolar or picomolar concentrations. Therefore, even small concentrations of quinoline in cigarettes may adversely affect reproductive processes.
In the current study, benzene was inhibitory of oviductal functioning at nanomolar or micromolar doses in the oviductal assays and had efficacies greater than 50% for two of the assays. Smokers inhale greater concentrations of benzene (2 mg/day) than nonsmokers (0.2 mg/day) as measured by breath levels (Wallace et al., 1987). Commercial cigarettes contain, on average, more benzene (50 μg/cigarette) (Darrall et al., 1998) than 1R4F (44.33 μg/cigarette) and 2R4F (43.39 μg/cigarette) research cigarettes (Chen and Moldoveanu, 2003). Occupational benzene exposure during pregnancy has been associated with an increased incidence of spontaneous abortion (Marinova, 1978), which can occur as a result of diminished oviductal functioning. Taken together, these results suggest that benzene contributes to the inhibition of oviductal functioning observed in hamster oviducts in vitro.
In summary, our study has shown that indole, 5-methylindole, quinoline, isoquinoline, 2-methylphenol, 4-ethylphenol, 2,6-dimethoxyphenol, and hydroquinone inhibit oviductal functioning of hamsters in vitro at very low doses and high efficacies. Based on the LOAELs determined in our study, it is likely that the doses of these compounds
ACKNOWLEDGMENTS
This work was funded by grants 10RT-0239 and 13RT-0068 from the Tobacco-Related Disease Research Program, California.
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ABSTRACT
Previously, we showed that pyridines and pyrazines in cigarette smoke inhibit oviductal functioning in vitro in nanomolar and picomolar doses. The purpose of this study was to determine the lowest observable adverse effect levels (LOAELs) of phenols, quinolines, indoles, benzene, and 2-cyclopenten-1-ones found in mainstream smoke solutions on ciliary beat frequency, oocyte pickup rate, and infundibular smooth muscle contraction using the hamster oviduct. After solid phase extraction, mainstream smoke solution fractions were tested in the oviductal assays. The active fractions were analyzed using gas chromatography-mass spectrometry to identify individual chemicals. Using this approach, benzene, eleven phenolic, two indole, two quinoline, and two 2-cyclopenten-1-one derivatives were identified in the active fractions. Commercially available authentic standards of the identified compounds were tested in dose-response studies on hamster oviducts. The LOAELs were determined for each compound using the ciliary beat frequency, oocyte pickup rate, and infundibular smooth muscle contraction rate assays. Indole, the compound with the highest potency, showed inhibition of ciliary beat frequency (10–13 M), oocyte pickup rate (10–14 M), and infundibular smooth muscle contraction rate (10–15 M) in femtomolar doses. All of the other compounds tested, except phenol, which only showed inhibition at millimolar concentrations, were inhibitory in picomolar, nanomolar, or micromolar concentrations. Derivitization of phenol increased its toxicity in the oviductal assays, especially when a methyl or ethyl group was substituted on the fourth position. The indoles, quinolines, and four phenolic compounds had both high potencies and efficacies in the oviductal assays.
Key Words: cigarette smoke; oviduct; oocyte pickup; phenols.
INTRODUCTION
Numerous epidemiological studies have linked cigarette smoking with reproductive problems in women. For example, an association has been observed between women who smoke and increased incidences of ectopic pregnancy, tubal infertility, and spontaneous abortion (Castles et al., 1999; Mueller and Ciervo, 1998; Saraiya et al., 1998). These problems could be due to an effect on the oviduct, and indeed, recent in vivo and in vitro studies have shown an effect on oviductal functioning by cigarette smoke (DiCarlantonio and Talbot, 1999; Knoll et al., 1995; Knoll and Talbot, 1998; Magers et al., 1995; Riveles et al., 2003). Factors such as cigarette smoke that impair oviductal functioning can diminish or prevent fertility (Florek and Marszalek, 1999; Shiverick and Salafia, 1999).
Inhalation of mainstream (MS) or sidestream (SS) smoke by hamsters, at serum cotinine levels that were within the ranges found in active and passive human smokers, adversely affected the ultrastructure of the oviductal epithelium (Magers et al., 1995). Decreases in the ratio of ciliated to secretory cells in the ampulla were also observed in these hamsters (Magers et al., 1995). In another study on hamsters, inhalation of MS and SS smoke slowed embryo transport, and 75% of the embryos were retained in the oviduct (DiCarlantonio and Talbot, 1999). Moreover, oviductal contractions were inhibited differentially in the ampulla and isthmus in vivo after smoke exposure, suggesting that embryo transport rates were slowed by smooth muscle contraction (DiCarlantonio and Talbot, 1999). Studies have also shown that cigarette smoke exposure slowed the rate of smooth muscle contraction in humans (Neri and Eckerling, 1969) and in rabbits (Ruckebusch, 1975) after in vivo exposure.
In vitro studies have also shown that MS and SS smoke solutions adversely affect proper functioning of the oviduct. MS and SS smoke solutions made with University of Kentucky 2R1 research cigarettes inhibited oocyte pickup rate in a dose-dependent manner in explants of hamster oviducts in vitro (Knoll et al., 1995; Knoll and Talbot, 1998). Ciliary beat frequency decreased in MS smoke solutions; however, SS produced no change or stimulated ciliary beating (Knoll et al., 1995; Knoll and Talbot, 1998). The gas and particulate phases of the smoke solutions were also tested in the oocyte pickup rate assay. Oocyte pickup rate was more sensitive to the gas than the particulate phase of MS and SS smoke solutions (Knoll and Talbot, 1998).
Several studies have been done to identify the toxicants in cigarette smoke that affect the oviduct. For example, oral administration of nicotine inhibited oviductal contractions in Rhesus monkeys (Neri and Marcus, 1972). Intravenous administration of nicotine delayed rat embryo cleavage, reduced embryo cell number, and reduced oviductal blood flow (Mitchell and Hammer, 1985). In another study, other individual smoke components (potassium cyanide, acrolein, phenol, acetaldehyde, and formaldehyde), inhibited ciliary beat frequency in the hamster oviduct in vitro; however, only cyanide was present in cigarette smoke solutions in sufficiently high concentrations to account for the effects seen in vitro (Talbot et al., 1998).
As a result of earlier epidemiological and model systems studies, our lab developed an assay using the hamster infundibulum in vitro to study oviductal functioning and we have been determining the compounds in smoke that affect oocyte pickup rate, ciliary beat frequency, and infundibular smooth muscle contraction rate. Pyridine compounds, which are added directly to tobacco and are generated indirectly by combustion processes associated with burning of the cigarette, have been identified in MS and SS smoke solutions and adversely affect hamster oviductal functioning in vitro (Riveles et al., 2003). Twelve pyridine compounds, including nicotine, were tested on oviductal functioning in vitro, measuring ciliary beat frequency, oocyte pickup rate, and oviductal smooth muscle contraction rate (Riveles et al., 2003). Pyridine derivatives with single ethyl or methyl substitutions (2-ethylpyridine, 3-ethylpyridine, 2-methylpyrdine, and 4-methylpyridine) were inhibitory at picomolar doses for all three parameters measured (Riveles et al., 2003). Nornicotine and -nicotyrine, which are similar in structure to nicotine, were inhibitory in nanomolar doses in the bioassays, while nicotine was only inhibitory at 10–2 M.
In the second study of the series, pyrazine compounds, which are additives in food, tobacco, and cosmetics, and are combustion products of tobacco, were identified in both MS and SS smoke solutions and tested on the hamster oviduct in vitro (Riveles et al., 2004). Six of the seven pyrazine compounds inhibited oviductal functioning at picomolar or nanomolar doses in vitro. Several pyrazine compounds with single ethyl or methyl substitutions (2-ethylpyrazine and 2-methylpyrazine), as well as unsubstituted pyrazine, inhibited ciliary beat frequency, oocyte pickup rate, and smooth muscle contraction rate at picomolar doses (Riveles et al., 2004).
The purpose of this study was to test the hypothesis that there are additional classes of components identified in MS and SS smoke solutions that adversely affect ciliary beat frequency, oocyte pickup rate, and infundibular smooth muscle contraction rate. To test this hypothesis, benzene, indole, 5-methylindole, quinoline, isoquinoline, 2-cyclopenten-1-one, 3-methyl-2-cyclopenten-1-one, and eleven phenolic compounds were individually tested in dose-response experiments on the hamster oviduct in vitro, measuring ciliary beat frequency, oocyte pickup rate, and smooth muscle contraction rate. Our data show that several compounds were inhibitory in as low as femtomolar or picomolar doses.
MATERIALS AND METHODS
Animals, media, and authentic standards.
Female golden hamsters (Mesocricetus auratus) purchased from Harlan (San Diego, CA) were maintained on a 14L:10D cycle in a room at 26°C. Food was administered ad libitum. Hamsters were cycled daily by checking for a vaginal discharge, which occurs on day 1 of their estrous cycle. Female golden hamsters were induced to superovulate by intraperitoneal injection with 25 International Units (IU) of pregnant mare's serum gonadotropin (PMSG) (CalBiochem, La Jolla, CA.) at 10 A.M. on day 1 of their estrous cycle, followed by 25 IU of human chorionic gonadotropin (hCG) (Sigma Chemical Co., St. Louis, MO) on day 3 of the estrous cycle. Hamsters were sacrificed with CO2 on day 4, approximately 12 h after the hCG injection, and oviducts and ovaries were removed and separated. The two ovaries were isolated in Earle's Balanced Salt Solution supplemented with sodium bicarbonate, HEPES, and 0.1% BSA at pH = 7.4 (EBSS-H). The expanded follicles were poked with a dissecting needle to release the oocyte cumulus complexes (OCCs). Approximately 7–15 mature expanded OCCs were recovered from each ovary. The two infundibula were dissected from the oviducts and placed into perfusion chambers containing EBSS-H. The ampullas were left attached to the infundibula and placed into holding pipettes so the infundibula remained stationary throughout the experiment. EBSS-H was used as the control solution for all experiments. The protocol for the use of hamsters in this study was approved by the campus animal use committee.
2-Cyclopenten-1-one (98%), 2,4-dimethylphenol (98%), 2-ethylphenol (99%), 4-ethylphenol (99%), indole (98%), 2-methoxyphenol (98%), p-methoxyphenol (99%), 3-methyl-2-cyclopenten-1-one(97%), 5-methylindole (99%), 2-methylphenol (99+%), 4-methylphenol (99%), and quinoline (98%) were purchased from Aldrich Chemical Company (Milwaukee, WI). 2,6-Dimethoxyphenol (96%) and isoquinoline were purchased from City Chemical LLC (West Haven, CT.). Hydroquinone (99%) was purchased from VWR (Brisbane, CA). Benzene (99%) and phenol (99%) were purchased from Fisher Scientific (Pittsburgh, PA).
Preparation of smoke solutions.
Mainstream (MS) smoke solutions were made in EBSS-H using 2R1 research-grade cigarettes (University of Kentucky, Louisville, KY) that were smoked on a puffer box built at the University of Kentucky (Knoll et al., 1995; Knoll and Talbot, 1998). This method has been described and published previously in detail (Knoll et al., 1995; Knoll and Talbot, 1998; Riveles et al., 2003). MS smoke solutions were made from 60 puffs of MS smoke pushed through 10 ml of EBSS-H, which equals 6 puff equivalents.
Solid-phase extraction and gas-chromatography-mass spectrometry (GC-MS) of smoke solutions.
Bond Elut solid phase extraction (SPE) cartridges, 3 cc, 500 g capacity (Phenomenex, Torrance, CA) were used to fractionate smoke solutions and concentrate chemicals that inhibit oviductal functioning. A variety of nonpolar, polar, and anion and cation exchange cartridges were used: NH2, 2OH, CN, CBA, SCX, SAX, C18, C8, C2, CH, SI, and PH. The protocol used to screen the cartridges for their ability to bind oviductal toxicants in smoke solutions has been described in detail previously (Riveles et al., 2003). MS smoke solutions were passed through the cartridges, and the individual fractions and corresponding cartridge eluates were collected and tested in the oviductal bioassays measuring ciliary beat frequency, oocyte pickup rate, and smooth muscle contraction rate (Riveles et al., 2003).
Four cartridges (C8, CN, PH, and NH2) were particularly effective at retaining the oviductal toxicants, and their eluates were characterized using GC-MS. A Hewlett Packard 5890 GC interfaced to an HP-5971A MSD quadrupole mass selective detector with a Zebron ZB1701 cyanopropyl phenyl column 30 m x 0.32 mm with 1-μm phase thickness from Phenomenex (Torrance, CA.) was used as described previously in detail (Riveles et al., 2003). The eluate sample (2 μl) was injected directly into the GC, and compounds were identified using the mass spectrometry data matched to mass spectral library entries. Compound identities were confirmed using authentic standards and by matching both mass spectra and retention times.
Oviductal assays: ciliary beat frequency, oocyte pickup rate, and infundibular smooth muscle contraction rate.
Ciliary beat frequency, oocyte pickup rate, and smooth muscle contraction rate were measured using hamster oviduct explants in vitro in a bioassay developed in our lab and described previously in detail (Riveles et al., 2003). A single experiment was defined as the treatment of one infundibulum with the control medium (EBSS-H with 0.1% BSA) and a single dose of the test chemical diluted in EBSS-H for a 5-min exposure period. The mean ± standard deviation of six to ten measurements was made on an infundibulum for each treatment group. For each parameter, a region of the infundibulum was selected, and measurements for both the control and treated groups were performed in the same region.
Each parameter was measured using the same experimental setup with a Wild 5A stereoscopic microscope and a video image capture system as described previously (Riveles et al., 2003). Ciliary beat frequency was measured using the video capture system and expressed as number of beats per second. Oocyte pickup rate was performed by manually placing an oocyte cumulus complex (OCC) onto the infundibulum and measuring the speed at which the OCC traversed a defined path expressed as microns per second. Infundibular smooth muscle contractions were also measured using the video image capture system. The distance of the contractions was measured directly on the computer monitor using a ruler as described previously (Riveles et al., 2003). Frequency was measured as the number of times the infundibulum contracted per minute. Smooth muscle contraction rate was calculated and expressed as microns per minute.
Determination of LOAELs and efficacies.
To determine LOAELs for each compound tested in each bioassay, a preliminary screen was done using one infundibulum per dose group (10–16–10–1 M). For each infundibulum, 6–10 control, treatment, and recovery measurements were taken, and the means were compared using a one-way ANOVA. The lowest significant inhibitory dose provided an estimate of the LOAELs for each test compound in each assay. Since the estimated LOAELs were based on only one infundibulum per dose, additional experiments were performed at the estimated NOAEL and LOAEL doses to increase the sample size to n = 4 infundibula for all of the tested compounds in each bioassay.
Using the initial dose-response curves, the efficacy values were estimated for each test compound in each assay. Efficacies were determined by finding the point on the curve that reached a maximum percentage of inhibition and then leveled off over two or more consecutive doses. These efficacy values are estimations based on a limited dose selection and a sample size of only one infundibulum. A maximum percentage of inhibition was not reached in all cases based on the doses selected, in which case efficacies were indicated as greater than or equal to the highest percentage of inhibition observed for that compound in that assay at the highest dose tested.
Statistical analysis.
The statistical significance of the results was evaluated in the oocyte pickup rate, ciliary beat frequency, and infundibular muscle contraction rate assays using a one sample t-test to compare the control (n = 4 infundibula) set to 100% to the mean at the estimated LOAEL dose (n = 4 infundibula) for each test compound in each bioassay (Fig. 7) with Instat (Graphpad, San Diego, CA). Means were considered to be significantly different for p < 0.05.
RESULTS
Phenols, Indoles, Quinolines, Benzene, and 2-Cyclopenten-1-Ones Identified in MS Smoke Solutions
A preliminary screen of 12 solid phase extraction cartridges revealed that four of the cartridges (C8, CN, PH, and NH2) retained chemicals in MS smoke solutions that were inhibitory in the ciliary beat frequency, oocyte pickup rate, and smooth muscle contraction rate assays. Phenols, indoles, quinolines, benzene, and 2-cyclopenten-1-ones were subsequently identified in the eluates of these four cartridges using GC-MS (Table 1). Commercially available authentic standards of the identified compounds from Table 1 were purchased and tested in dose-response studies on the oviduct to determine their lowest observable adverse effect levels (LOAELs) in the oviductal bioassays.
Dose-Response, LOAELs, and Estimated Efficacies of the Phenolic Compounds in the Oviductal Bioassays
Phenol and 10 phenolic derivatives (2-methylphenol, 4-methylphenol, 2-ethylphenol, 4-ethylphenol, 2,4-dimethylphenol, 2,5-dimethylphenol, 2-methoxyphenol, 4-methoxyphenol, 2,6-dimethoxyphenol, and hydroquinone) were tested in dose-response studies on the hamster oviduct based on a 5-min exposure in the ciliary beat frequency, oocyte pickup rate, and smooth muscle contraction rate assays. For each phenolic compound tested, an initial screen of doses, ranging from 10–13 M to 10–1 M was performed for a single infundibulum per dose group. For the parent compound, phenol, the dose range was extended to 10 M. Dose-response curves for the ciliary beat frequency, oocyte pickup rate, and smooth muscle contraction rate assays were created based on these data (Figs. 1–3 ). LOAELs were estimated from the dose-response curves and then confirmed using an increased sample size (n = 4 infundibula) for each of the bioassays. The confirmed LOAELs for the phenolic compounds (n = 4) are summarized in Table 2. Figure 7 has the mean percent inhibition and standard deviation at each LOAEL for each of the bioassays. The estimated efficacies were determined from the initial dose-response curves as explained in the Materials and Methods.
For the ciliary beat frequency assay, the dose-response curves were similar for most of the phenolic compounds, except for 2-ethylphenol and phenol, which were inhibitory at higher doses (Fig. 1). It is interesting that all of the phenolic derivatives were more potent than phenol itself. Additional infundibula were tested at the estimated LOAEL doses for confirmation. The LOAELs for the phenolic compounds ranged from 10–12 M to 10–1 M (Table 2). 4-Methylphenol (10–12 M), 4-ethylphenol (10–11 M), 2,6-dimethoxyphenol (10–10 M), and hydroquinone (10–10 M) inhibited ciliary beat frequency at picomolar doses. 2-Methylphenol (10–9 M), 2,4-dimethylphenol (10–9 M), 2-methoxyphenol (10–8 M), 2,5-dimethylphenol (10–8 M), and 4-methoxyphenol (10–7 M) inhibited ciliary beat frequency at nanomolar doses. 2-Ethylphenol (10–5 M) inhibited ciliary beat frequency at micromolar doses. Phenol had the highest LOAEL (10–1 M). The substitution of an ethyl or methyl group in the fourth position on the phenol moiety compared to the substitution on the second position increased its potency by a million-fold and one-thousand-fold, respectively.
For the oocyte pickup rate assay, the dose-response curves were similar for six of the phenolic compounds (2-methylphenol, 2,4-dimethylphenol, 4-ethylphenol, 2-methoxyphenol, 2,6-dimethoxyphenol, and hydroquinone) (Fig. 2). The dose-response curves for 2-ethylphenol, 2,5-dimethylphenol, and 4-methoxyphenol showed inhibition at higher doses. All of the phenolic derivatives were more potent than phenol itself, which had a LOAEL for oocyte pickup rate (10–2 M), similar to the ciliary beat frequency assay (10–1 M). Additional infundibula were tested at the estimated LOAEL doses for confirmation. The LOAELs for the phenolic compounds in the oocyte pickup rate assay ranged from 10–12 M to 10–2 M (Table 2). 4-Ethylphenol, (10–12 M), 2-methylphenol (10–11 M), 4-methylphenol (10–11 M), 2,6-dimethoxyphenol (10–11 M), 2,4-dimethylphenol (10–10 M), 2-methoxyphenol (10–10 M), and hydroquinone (10–10 M) inhibited oocyte pickup rate in picomolar doses. 2-Ethylphenol (10–8 M), 4-methoxyphenol (10–8 M), and 2,5-dimethylphenol (10–7 M) inhibited oocyte pickup rate at nanomolar doses. Phenol had the highest LOAEL (10–2 M) (Table 2).
For the smooth muscle contraction rate assay, all of the derivatives were more potent than phenol (10–2 M) (Fig. 3). The dose-response curves were similar for most of the phenolic compounds, except for 2-methylphenol, 4-methylphenol, and phenol (Fig. 3). The dose-response curves for 2-ethylphenol (10–7 M), 4-methoxyphenol (10–7 M), and 2,5-dimethylphenol (10–6 M) had LOAELs at higher doses in this assay, as was the case in the oocyte pickup rate assay (10–8, 10–8 M, and 10–7, respectively). Additional infundibula were tested at the estimated LOAEL doses for confirmation. For the smooth muscle contraction rate assay, the LOAELs for the phenolic compounds ranged from 10–12 M to 10–2 M (Fig. 3). 4-Ethylphenol (10–12 M), 2-methylphenol (10–11 M), 4-methylphenol (10–11 M), and hydroquinone (10–10 M) inhibited smooth muscle contraction rate at picomolar doses. 2,4-Dimethylphenol (10–9 M), 2,6-dimethoxyphenol (10–9 M), 2-methoxyphenol (10–8 M), 2-ethylphenol (10–7 M), and 4-methoxyphenol (10–7 M) inhibited smooth muscle contraction rate at nanomolar doses. 2,5-Dimethylphenol (10–6 M) inhibited smooth muscle contraction rate at micromolar doses. The LOAEL for phenol was the highest at millimolar doses (10–2 M) (Fig. 3).
Using the initial dose-response curves, the efficacies for phenol and several of its derivatives were estimated as described in the Materials and Methods. The estimated efficacies of phenol in the ciliary beat frequency, oocyte pickup rate, and smooth muscle contraction rate assays were 22%, 68%, and 100%, respectively (Figs. 1–3). The chemicals having estimated efficacies in the ciliary beat frequency assay that were lower than phenol were 2,4-dimethylphenol (7%) and 4-methylphenol (16%) (Fig. 1). 2-Methylphenol (25%), 2,5-dimethylphenol (28%), 2-ethylphenol (32%), and hydroquinone (37%) had estimated efficacies in the ciliary beat frequency assay that were higher than phenol (Fig. 1). The efficacies for 2-methoxyphenol (9%), 4-methoxyphenol (11%), 2,6-dimethoxyphenol (14%), and 4-ethylphenol (19%) were estimated based on the initial dose-response curves (Fig. 1).
For the oocyte pickup rate assay, from the dose-response curve, the efficacy for phenol was 68% (Fig. 2). The estimated efficacy of 4-methylphenol (6%) was lower than for phenol in the oocyte pickup rate assay (Fig. 2). The efficacies for 4-ethylphenol (41%), 2-methoxyphenol (41%), 2-methylphenol (42%), 4-methoxyphenol (50%), 2,6-dimethoxyphenol (52%), hydroquinone (63%), 2,4-dimethylphenol (64%), 2,5-dimethylphenol (67%), and 2-ethylphenol (75%) were estimated for the oocyte pickup rate assay based on the dose-response curves (Fig. 2). The efficacies in the oocyte pickup rate assay were higher than for the ciliary beat frequency assay in most cases.
In the smooth muscle contraction rate assay, the estimated efficacy of phenol was 100% (Fig. 3). The estimated efficacies of 4-methylphenol (6%) and 2,6-dimethoxyphenol (66%) were lower than phenol in the smooth muscle contraction rate assay (Fig. 3). 4-Ethylphenol (100%) and 4-methoxyphenol (100%) had efficacies similar to phenol (Fig. 3). The efficacies for 2-methylphenol (16%), 2-methoxyphenol (30%), 2-ethylphenol (39%), 2,4-dimethylphenol (65%), and hydroquinone (71%) were estimated in the smooth muscle contraction rate assay based on the initial dose-response curves (Fig. 3).
Dose-Response, LOAELs, and Estimated Efficacies of Indoles, Quinolines, Benzene, and 2-Cyclopenten-1-ones in the Oviductal Bioassays
An initial screen of doses ranging from 10–16 M to 10–4 M was used to test indole, 5-methylindole, quinoline, isoquinoline, benzene, 2-cyclopenten-1-one, and 3-methyl-2-cylcopenten-1-one in the three bioassays with a single infundibulum per dose group. Dose-response curves for the ciliary beat frequency, oocyte pickup rate, and smooth muscle contraction rate assays were created based on these data (Figs. 4–6). LOAELs were estimated from the dose-response curves and then confirmed using an increased sample size (n = 4 infundibula). The confirmed LOAELs for these compounds (n = 4) are summarized in Table 3. Figure 7 has the mean percent inhibition and standard deviation at each LOAEL for each of the bioassays.
For the ciliary beat frequency assay, the dose-response curves were similar for the indoles and quinolines (Fig. 4). Benzene, 2-cyclopenten-1-one, and 3-methyl-2-cyclopenten-1-one showed inhibition at higher doses and were the least potent in this group of chemicals (Fig. 4). Additional infundibula were tested to confirm the LOAELs for each compound in the ciliary beat frequency assay. The LOAELs ranged from 10–13 M to 10–8 M (Table 3). Indole (10–13 M), quinoline (10–13 M), isoquinoline (10–12 M), and 5-methylindole (10–10 M) inhibited ciliary beat frequency at femtomolar or picomolar doses. Benzene (10–8 M) and 2-cyclopenten-1-one (10–8 M) inhibited ciliary beat frequency at nanomolar doses. The LOAEL for 3-methyl-2-cyclopenten-1-one in the ciliary beat frequency was not determined with the dose range used (Fig. 4).
For the oocyte pickup rate assay, the dose-response curve for indole showed the highest potency of all the chemicals tested (Fig. 5). The dose-response curves for 5-methylindole, quinoline, and isoquinoline in the oocyte pickup rate assay were similar to the ciliary beat frequency assay. Benzene and 3-methyl-2-cyclopenten-1-one were the least potent in this assay as was the case in the ciliary beat frequency assay (Fig. 5). Additional infundibula were tested to confirm the LOAELs for each compound in the oocyte pickup rate assay (Fig. 7). The LOAELs for this group of compounds ranged from 10–14 M to 10–7 M (Table 3). Indole (10–14 M), isoquinoline (10–13 M), quinoline (10–11 M), 5-methylindole (10–11 M), and 3-methyl-2-cyclopenten-1-one (10–10 M) inhibited oocyte pickup rate at femtomolar or picomolar doses. Oocyte pickup rate was inhibited by 2-cyclopenten-1-one (10–9 M) and benzene (10–7 M) at nanomolar doses.
For the smooth muscle contraction rate assay, indole showed the highest potency of all the chemicals tested, as was the case for oocyte pickup rate (Fig. 6). The dose-response curves for 5-methylindole, quinoline, isoquinoline 2-cyclopenten-1-one, and 3-methyl-2-cyclopenten-1-one were similar for the smooth muscle contraction rate assay. The dose-response curve for benzene showed little potency in this assay, as was the case for the ciliary beat frequency and oocyte pickup rate assays (Fig. 6). Additional infundibula were tested to confirm the LOAELs for each compound in the smooth muscle contraction rate assay (Table 3). Indole (10–15 M) and isoquinoline (10–13 M) inhibited smooth muscle contraction rate at femtomolar doses. Quinoline (10–11 M), 5-methylindole (10–10 M), and 3-methyl-2-cyclopenten-1-one (10–10 M) inhibited smooth muscle contraction rate at picomolar doses. Smooth muscle contraction rate was inhibited by 2-cyclopenten-1-one (10–9 M) at nanomolar doses and by benzene (10–6 M) at micromolar doses (Fig. 6).
Estimated Efficacies for Indoles, Quinolines, 2-Cyclopenten-1-ones, and Benzene
Using the initial dose-response curves, the efficacies for the indoles, quinolines, 2-cyclopenten-1-ones, and benzene were estimated as described in Materials and Methods. For the ciliary beat frequency assay, the efficacies for indole (25%) and 5-methylindole (15%) could not be compared (Fig. 4). The efficacy of isoquinoline in the ciliary beat frequency assay was 27%; however, the efficacy of quinoline could only be predicted to be 22% (Fig. 4). The efficacies for benzene (56%), 2-cyclopenten-1-one (6%), and 3-methyl-2-cyclopenten-1-one (2%) were estimated based on the initial dose-response curves for the ciliary beat frequency assay (Fig. 4). For the oocyte pickup rate assay, 5-methylindole (73%) had a higher estimated efficacy than indole (52%) (Fig. 5). The estimated efficacies of quinoline (70%) and isoquinoline (61%) were high for oocyte pickup rate (Fig. 5). 3-Methyl-2-cyclopenten-1-one (20%) had a lower efficacy in the oocyte pickup rate assay than 2-cyclopenten-1-one (66%) (Fig. 5). The efficacy of benzene in the oocyte pickup rate assay was predicted to be 52% (Fig. 5). The exact efficacies in the smooth muscle contraction rate assay could not be determined based on the initial dose-response curves (Fig. 6). The efficacies could only be estimated for: quinoline (75%), 2-cyclopenten-1-one (61%), isoquinoline (60%), indole (55%), 3-methyl-2-cyclopenten-1-one (45%), 5-methylindole (39%), and benzene (26%) (Fig. 6).
DISCUSSION
The purpose of this study was to identify additional compounds in cigarette smoke that inhibit oviductal functioning. Phenol, 10 phenolic derivatives, indole, 5-methylindole, quinoline, isoquinoline, benzene, 2-cyclopenten-1-one, and 3-methyl-2-cyclopenten-1-one were identified in fractions of mainstream smoke solutions that inhibited oviductal functioning. The hierarchy of potency of the individual chemicals tested in this study and their LOAELs in the ciliary beat frequency, oocyte pickup rate, and infundibular smooth muscle contraction rate assays are summarized in Table 4. Several of these smoke toxicants were highly effective at femtomolar or picomolar doses in all three oviductal assays (indole, isoquinoline, 4-ethylphenol, quinoline, 4-methylphenol, 5-methylindole, and hydroquinone) (Table 4).
Previous studies have shown inhibition of oviductal functioning in picomolar or nanomolar ranges by pyridines (Riveles et al., 2003) and pyrazines (Riveles et al., 2004). While pyridine itself was not very potent in the oviductal assays, addition of a single methyl or ethyl group increased its potency greater than a 100-million-fold. Within the pyrazine group, addition of single ethyl and methyl groups had minor effects on its potency, in most cases. As was the case for the pyridines, substitution of an ethyl or methyl group to the ring increased the potency of the phenolic derivatives. In the case of the phenols, this increase was clearly correlated with the position of the substitution.
The LOAELS for phenol and its derivatives ranged from 10–12 M to 10–1 M. Four phenolic compounds (2-methylphenol, 4-ethylphenol, 2,6-dimethoxyphenol, and hydroquinone) had both high potencies and high efficacies in all three oviductal assays. 2,4-Dimethylphenol was potent in picomolar doses in all three assays, but only had high efficacies for the ooycte pickup rate and smooth muscle contraction rate assays. Four phenolic derivatives (2-ethylphenol, 2,5-dimethylphenol, 2-methoxyphenol, and 4-methoxyphenol) had high efficacies, but medium or low potencies.
Addition of either a methyl or ethyl group to the two or four position of phenol increased its potency in the oviductal assays. In general, the four substitution resulted in higher potency than the two. For the oocyte pickup rate and smooth muscle contraction rate assays, substitution of an ethyl group in the four position increased potency more than a two or four methyl substitution. Substitution of a methyl group in the two position or an ethyl group in the two or four positions was also highly efficacious in all three assays. 4-Methylphenol was very potent in all three oviductal assays, but had very low efficacies. The parent compound, phenol, was the least potent chemical in the oviductal assays with the highest LOAELs (10–2 to 10–1 M). Phenol was very effective in the oocyte pickup rate and smooth muscle contraction rate assays, but only at very high doses. In general, both substitution and position of the substitution influenced the degree of toxicity and efficacy in the bioassays. The data in this study show that several phenolic compounds are highly effective and inhibitory of oviductal functioning at very low doses.
Although the mechanism of action of phenols on the oviduct is not known, phenolic compounds studied previously exerted toxicity through the formation of phenoxy-free radicals (Smith et al., 2002). High concentrations of phenoxy radicals at the oviduct could attack biochemical processes such as production of ATP, cAMP, Ca2+, or nitric oxide (NO), which are important in oviductal muscle contraction and ciliary beat frequency (Cometti et al., 2003; Lansley et al., 1992; Lindblom and Hamberger, 1980). The biochemical affected by the phenoxy radical would depend on its interactions within the cell or on its surface (Selassie et al., 1998). The hydrophobicity of the phenoxy free radicals would enable them to penetrate into the hydrophobic region of the membranes (Selassie et al., 1998). This could facilitate interaction with polyunsaturated fatty acids and initiate free radical chain reactions (Selassie et al., 1998). In our study, one possible mechanism by which the phenolic compounds exerted toxicity on the oviduct may have been through the formation of phenoxy radicals. The free radicals produced and circulated to the oviduct may interfere with the production of biochemicals necessary for adhesion of the oocyte to the tips of the cilia on the infundibular surface. These free radicals could also inhibit oviductal functioning by slowing or stopping smooth muscle contraction by interfering with ATP or NO production. The oxidative damage produced by phenoxy free radicals could also result in cell death or mutation (Selassie et al., 1998).
Previous studies have evaluated the reproductive toxicity of phenol and its derivatives (Bian and Wang, 2004; Bruce et al., 1987). Phenol itself is rapidly absorbed and distributed to all tissues in animals and humans after oral, dermal, or inhalation exposure and has a half-life of approximately 3.5 h in humans (Bruce et al., 1987). Oral doses of phenol in rats showed dose-related fetal toxicity with a decrease in average fetal body weight per litter (Bruce et al., 1987; Narotsky and Kavlock, 1995). Reduction of growth (decreased somite number) and development (tail bud abnormalities) were observed in embryo cultures in vitro after exposure to phenol and its para-substituted cogeners (Oglesby et al., 1992). In vivo exposure to phenol decreased weight gain in pregnant rats (Kavlock, 1990). A limited number of studies have shown that phenol and its derivatives have reproductive effects (Bian and Wang, 2004) and our study clearly shows that certain phenolic compounds interfere with proper oviductal functioning in vitro in as low as picomolar and nanomolar doses.
Phenolic compounds have been measured in cigarettes (Chen and Moldoveanu, 2003; Czogala and Wardas, 1998; Forehand et al., 2000; Nanni et al., 1990; Rustemeir et al., 2002; White et al., 1990). The concentration of phenol and its derivatives in cigarette smoke varies significantly among different brands of cigarettes (Nanni et al., 1990). For example, in most cases, commercial brands (3.6–17 μg/cigarette) had higher concentrations of phenol than 1R4F research cigarettes (7 μg/cigarette) or ultra-low-tar cigarettes (0.26 μg/cigarette) (Nanni et al., 1990). However, the concentrations of phenol vary among different research brands, for example, 1R3 cigarettes (55.4 μg/cigarette) have more phenol than 1R4F cigarettes (6.9 μg/cigarette) (Forehand et al., 2000). In our study, phenol was identified in the MS smoke solutions from 2R1 cigarettes and in the active fractions. Phenol was the least potent chemical tested in this study with LOAELs in the millimolar range; however, it was very effective at these high doses. Phenol (5.7 μg/ml) and 4-methylphenol (1.78 μg/ml) have been detected in the urine of smokers (Orejuela and Silva, 2002). Taken together, the data suggest that phenol, if present in cigarette smoke in high enough concentration, could enter the circulatory system and inhibit oviductal functioning, especially oocyte pickup rate and smooth muscle contraction rate, effectively.
In addition to phenol, the concentration of hydroquinone has been measured in 1R3 research cigarettes (70.5 μg/cigarette) (Forehand et al., 2000) and MS smoke from a nonfilter cigarette (110–300 μg/cigarette) (U.S. EPA, 1992). In the current study, hydroquinone was highly potent and efficacious in all three oviductal assays. The concentration of hydroquinone in MS smoke could be sufficient to account for the effects we observed in vitro on hamster oviductal functioning.
Humans are also exposed to phenols through foods (Bruce et al., 1987). 2-Methoxyphenol, also known as guaiacol, is an additive in cigarettes (http://www.rjrt.com/TI/Ticig_ingred_summary.asp, Cigarette Ingredients, 2004), a Food and Drug Administration (FDA) approved food additive, and is on the Flavor and Extract Manufacturer's Association (FEMA) generally regarded as safe (GRAS) list. It is also found in celery, cocoa, coffee, rum, soybean, tea, tomato, whiskey, wine, and used in ice cream, baked goods, meat, chewing gum, and dairy products (http://www.bbc.co.uk/worldservice/sci_tech/features/health/tobaccotrial/inacigarette599.htm, What's in a Cigarette, 2004). In our study, 2-methoxyphenol inhibited oviductal functioning in nanomolar and picomolar doses and was highly efficacious in all three assays. 4-Ethylphenol and 2,6-dimethoxyphenol are also on the FEMA GRAS list and are found and used in a variety of consumer products. In the current study, 4-ethylphenol and 2,6-dimethoxyphenol had both high efficacies and high potencies in all three oviductal assays.
The LOAELs for the indole, quinoline, 2-cyclopenten-1-one compounds, and benzene ranged from 10–15 M to 10–6 M. Four compounds (indole, 5-methylindole, quinoline, and isoquinoline) had both high potencies (femtomolar and picomolar doses) and high efficacies in all three oviductal assays. Benzene had a high efficacy and medium potency (nanomolar doses) for ciliary beat frequency and oocyte pickup rate, but a lower potency (micromolar doses) for the smooth muscle contraction rate assay. 2-Cyclopenten-1-one and 3-methyl-2-cyclopenten-1-one were highly potent for the oocyte pickup rate and contraction rate assays, but not for the ciliary beat frequency assay, and they had varying efficacies.
Two of the most potent and efficacious cigarette smoke components tested in this study were indole and 5-methylindole. Although reproductive studies were not found on these two compounds, another type of indole, indole-3-acetic acid (I3C), is a negative regulator of estrogen (Auborn et al., 2003) and has been shown to be teratogenic in mice and rats at 500 mg/kg/day resulting in decreased weight gain (John et al., 1979). I3C is an endocrine disrupter that can block ovulation in adult rats at 0.5 to 1.5 g/kg/day and cause decreases in body and ovarian weight gain at 1.5 g/kg/day (Gao et al., 2002). In the current study, ciliary beat frequency, oocyte pickup rate, and oviductal smooth muscle contraction were inhibited by indole in femtomolar doses and 5-methylindole in picomolar doses in vitro. The results from our study combined with previous studies suggest that indoles are effectors that can adversely affect several reproductive processes including oviductal functioning.
Although information is available on phenols and certain indoles, the reproductive effects of quinoline and isoquinoline are unknown. In our study, both quinoline and isoquinoline inhibited oviductal functioning and were highly efficacious at very low doses. Quinoline has been measured in the MS smoke from 1R4F (0.30 μg/cigarette) and 2R4F (0.23 μg/cigarette) research cigarettes (Chen and Moldoveanu, 2003); however smoke condensate from 1R4F cigarettes had lower levels of quinoline (0.19 μg/cigarette) (White et al., 1990). Higher concentrations of quinoline were reported for a nonfiltered cigarette (0.5–2 μg/cigarette) (U.S. EPA, 1992). In our study, quinoline inhibited oviductal functioning at femtomolar or picomolar concentrations. Therefore, even small concentrations of quinoline in cigarettes may adversely affect reproductive processes.
In the current study, benzene was inhibitory of oviductal functioning at nanomolar or micromolar doses in the oviductal assays and had efficacies greater than 50% for two of the assays. Smokers inhale greater concentrations of benzene (2 mg/day) than nonsmokers (0.2 mg/day) as measured by breath levels (Wallace et al., 1987). Commercial cigarettes contain, on average, more benzene (50 μg/cigarette) (Darrall et al., 1998) than 1R4F (44.33 μg/cigarette) and 2R4F (43.39 μg/cigarette) research cigarettes (Chen and Moldoveanu, 2003). Occupational benzene exposure during pregnancy has been associated with an increased incidence of spontaneous abortion (Marinova, 1978), which can occur as a result of diminished oviductal functioning. Taken together, these results suggest that benzene contributes to the inhibition of oviductal functioning observed in hamster oviducts in vitro.
In summary, our study has shown that indole, 5-methylindole, quinoline, isoquinoline, 2-methylphenol, 4-ethylphenol, 2,6-dimethoxyphenol, and hydroquinone inhibit oviductal functioning of hamsters in vitro at very low doses and high efficacies. Based on the LOAELs determined in our study, it is likely that the doses of these compounds
ACKNOWLEDGMENTS
This work was funded by grants 10RT-0239 and 13RT-0068 from the Tobacco-Related Disease Research Program, California.
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