Hum. Reprod. Advance Access published October 27, 2006

Human Reproduction Page 1 of 10

doi:10.1093/humrep/del380

Smoke from traditional commercial, harm reduction and research brand cigarettes impairs oviductal functioning in hamsters (Mesocricetus auratus) in vitro K.Riveles1, V.Tran2, R.Roza2, D.Kwan2 and P.Talbot1,2,3 1

Graduate Program in Environmental Toxicology and 2Department of Cell Biology and Neuroscience, University of California Riverside, Riverside, CA, USA

3

To whom correspondence should be addressed at: Department of Cell Biology and Neuroscience, University of California Riverside, Riverside, CA 92521, USA. E-mail: [email protected]

BACKGROUND: Cigarette smoke from 2R1 research brand cigarettes and specific toxicants in smoke inhibit oviductal functioning. Our purpose was to test the hypothesis that smoke from commercial cigarettes, including harm reduction cigarettes, inhibits oviductal functioning and to measure the concentration of previously identified toxicants in smoke from research and commercial cigarettes. METHODS: Mainstream (MS) and sidestream (SS) smoke solutions from two research, six traditional commercial and three harm reduction brands were tested in vitro using an oviductal assay that measures ciliary beat frequency, oocyte retrieval rate and smooth muscle contraction. RESULTS: Generally, smoke from each brand of cigarette was inhibitory in the three oviductal bioassays. SS, the major component of environmental tobacco smoke, was usually more inhibitory than MS, the smoke inhaled by active smokers. Nine cigarette toxicants, previously shown to be highly inhibitory in the oviductal bioassays, were quantified in MS and SS. 4Methylpyridine, which was inhibitory by itself in picomolar doses, was present in the highest concentration in MS and SS solutions from all brands tested. In general, toxicant concentrations were higher in SS than in MS solutions. CONCLUSIONS: These data show that commercial brands of cigarettes, including harm reduction cigarettes, contain toxicants that inhibit biological processes in the oviduct and could affect reproductive outcomes. Key words: oviduct/cigarette smoke/cilia/oocyte/smooth muscle contraction

Introduction The oviduct plays important roles in mammalian reproduction (Harper, 1994; Puikkinen, 1995; Talbot and Riveles, 2005). The infundibular portion of the oviduct is responsible for picking up the cumulus–oocyte complex following ovulation (Mahi-Brown and Yanagimachi, 1983; Talbot et al., 1999, 2003) while the ampulla and isthmus provide suitable microenvironments for fertilization and preimplantation development (Hunter, 1994; Puikkinen, 1995; Buhi et al., 1997; Menezo and Guerin, 1997; Killian, 2004). Once fertilization occurs in the ampulla, the embryo is transported through the remainder of the oviduct to the uterus with the aid of smooth muscle contraction (Hafez, 1973; Blandau and Verdugo, 1976; Harper, 1994; Croxatto, 2002; Talbot and Riveles, 2005). Correct timing of preimplantation embryo transport is essential for successful implantation to occur (Coutinho et al., 1976; Cummings and Perreault, 1990), and factors that impair oviductal functioning can diminish or prevent fertility (Phipps et al., 1987; Chow et al., 1988; Stergachis et al., 1991; Puikkinen, 1995; Florek and Marszalek, 1999; Shiverick and Salafia, 1999).

Both mainstream (MS) and sidestream (SS) cigarette smoke target the oviduct and impair its functioning in vivo (Magers et al., 1995; DiCarlantonio and Talbot, 1999; Talbot and Riveles, 2005). For example, inhalation of MS or SS smoke from University of Kentucky 2R1 research cigarettes retarded embryo transport in the hamster oviduct by slowing muscle contraction (DiCarlantonio and Talbot, 1999) and causing blebbing of the oviductal epithelium at doses that produced serum cotinine levels within the ranges found in active and passive smokers (Magers et al., 1995). Inhalation of cigarette smoke also altered the rate of smooth muscle contraction in humans and rabbits (Neri and Eckerling, 1969; Ruckebusch, 1975). Other in vivo studies have shown that nicotine, a major component of cigarette smoke, slowed oviductal contraction in the Rhesus monkey (Neri and Marcus, 1972) and decreased blood flow to the oviduct of rats (Mitchell and Hammer, 1985). In vitro studies have further demonstrated that the oviduct is a target of MS and SS cigarette smoke. In mammals, the oocyte and its surrounding cumulus cells (cumulus–oocyte complex) are ovulated into the peritoneal or bursal cavity, ‘picked up’ by cilia on the infundibulum of the oviduct and rapidly transported

© The Author 2006. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: [email protected]

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into the ampulla where fertilization occurs (Talbot and Riveles, 2005). Our laboratory has developed an in vitro technique in which multiple bioassays can be performed on a single oviductal preparation. This technique allows quantitative measurements to be made on oocyte retrieval rate, ciliary beat frequency, adhesion between the cumulus–oocyte complex and infundibular cilia, and infundibular muscle contraction rate (Huang et al., 1997; Knoll and Talbot, 1998; Lam et al., 2000; Riveles et al., 2003, 2005; Gieseke and Talbot, 2005; Talbot and Riveles, 2005). MS smoke solutions made from the 2R1 research cigarettes inhibited ciliary beat frequency of hamster oviducts in vitro, while the corresponding SS smoke solutions stimulated ciliary beat frequency when albumin was present (Knoll et al., 1993, 1995; DiCarlantonio et al., 1995; Riveles et al., 2003). Both MS and SS smoke solutions made from 2R1 research cigarettes inhibited oocyte retrieval rate, adhesion of cumulus complexes to cilia and infundibular muscle contraction of hamster oviducts in a dose-dependent manner during acute in vitro exposure (Knoll and Talbot, 1998; Talbot et al., 1999; Riveles et al., 2003; Gieseke and Talbot, 2005). Although in vitro analyses have repeatedly shown that MS and SS smoke solutions from 2R1 cigarettes adversely affect oviductal functioning, the smoke from commercial brands of cigarettes has not been tested using the hamster oviductal bioassays. The first purpose of this study was to test the hypothesis that MS and SS smoke solutions from research, traditional commercial and harm reduction cigarettes inhibit oviductal functioning similarly to 2R1 research cigarettes in the oviductal bioassays. The brands tested were unfiltered and filtered research cigarettes (University of Kentucky 2R1 and 1R4F), a light and regular traditional commercial cigarette (Marlboro light and Marlboro red), a filtered and unfiltered traditional commercial cigarette (Camel filtered and Camel unfiltered), a mentholated commercial cigarette (Kool), which was tested with and without its filter, a regular and light harm reduction cigarette (Omni and Omni light) and a second harm reduction brand (Advance). We had previously identified about 40 chemicals in smoke from 2R1 cigarettes that were inhibitory in the oviductal bioassays, and many of these chemicals were active at very low doses (Riveles et al., 2003, 2004, 2005). The second purpose of this study was to quantify some of these known oviductal toxicants in solutions of MS and SS smoke made from research, traditional commercial and harm reduction cigarettes. The toxicants that were chosen for quantification were the ones that had been shown previously to be highly potent in the oviductal bioassay. Filtered, light and harm reduction brands were hypothesized to remove or reduce levels of these chemicals and thereby enhance their safety. Materials and methods Research and commercial cigarettes Two research cigarettes (2R1s and 1R4Fs) were purchased from the University of Kentucky. 2R1s are high-tar unfiltered cigarettes (tar and nicotine per cigarette = 37 mg and 2.5 mg, respectively), whereas 1R4Fs are lower in tar and filtered (tar and nicotine per cigarette = 9 mg and 0.8 mg, respectively). Commercial brand cigarettes were

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purchased locally and included Marlboro (red pack filter cigarettes, tar = 15 mg, nicotine = 1.1 mg and batch = V137Z22-C4) and Marlboro lights (filter cigarettes, tar = 10 mg, nicotine = 0.8 mg and batch = N141Y2-S1) from Philip Morris Inc. (Richmond, VA, USA); Camel filtered (tar = 17 mg and nicotine = 1.2 mg) from RJ Reynolds Tobacco Co. (Winston-Salem, NC, USA); Camel unfiltered (tar = 26 mg and nicotine - 1.7 mg) from RJ Reynolds Tobacco Co.; Advance Premium Lights 100s (tar = 10 mg and nicotine = 0.8 mg) from Brown & Williamson Tobacco (Louisville, KY, USA) and Omni 100s (tar = 15 mg and nicotine = 1 mg) and Omni light 100s (tar = 12 mg and nicotine = 0.8 mg) from Vector Tobacco Ltd. (Durham, NC, USA). Preparation of smoke solutions Smoke solutions were prepared using a University of Kentucky analytical smoking machine as described in detail previously (Knoll et al., 1995; Knoll and Talbot, 1998). MS smoke solutions, which contained chemicals inhaled by active smokers, were produced using six cigarettes with 10 puffs per cigarette for 60 puffs of smoke passed through 10 ml of a 1× solution of Earle’s balanced salt solution (EBSS-H), which was made fresh daily from a 10× stock solution. Sodium bicarbonate, HEPES and 1% bovine serum albumin (BSA) were added, and the pH was adjusted to 7.4 with NaOH. SS smoke solutions, which contained the chemical inhaled by passive smokers, were collected from three cigarettes for 30 puffs also passed through 10 ml of 1× EBSS-H. Puff equivalents (p.e.) are defined as the number of puffs of cigarette smoke drawn through 1 ml of aqueous solution. In the bioassays, MS smoke solutions were used at 6 p.e. (60 puffs per 10 ml), and SS solutions were used at 3 p.e. (30 puffs per 10 ml). For the extraction assays, the concentrations of smoke solution used were 12 p.e. for MS (60 puffs through 5 ml) and 6 p.e. for SS (30 puffs through 5 ml). Animals, media, oviduct and cumulus–oocyte complex preparation The biological in vitro assays used female golden hamsters (Mesocricetus auratus) purchased from Harlan (San Diego, CA, USA). Females were maintained on a 14:10 light : dark cycle (6 AM to 8 PM light) in a room at 26°C, and food was administered ad libitum. Hamsters were induced to superovulate by i.p. injection of 25 IU of equine CG (CalBiochem, La Jolla, CA, USA) at 10 AM on day 1 of their estrous cycle, followed by 25 IU of HCG (Sigma Chemical Co., St. Louis, MO, USA) on day 3 of the estrous cycle. Cumulus–oocyte complexes from the ampullas of oviducts and infundibula (from different females), which were used in the in vitro bioassays, were collected 12 h after the hCG injection. The care and use of animals in this study was approved by the campus Animal Care Committee. The control culture medium was a 1× solution of EBSS-H, which was made fresh daily from a 10× stock solution. Sodium bicarbonate, HEPES and 1% BSA were added, and the pH was adjusted to 7.4 with NaOH. This solution was used for dissection, for incubation and as the control solution for all experiments. Infundibula were separated from the ampulla and placed in perfusion chambers. Measurements of ciliary beat frequency, oocyte retrieval rate and muscle contraction rate were first made in the control medium (EBSS-H) and again on the same infundibular preparation after a 5-min exposure to MS or SS smoke solution. Ciliary beat frequency, oocyte retrieval rate and infundibular smooth muscle contraction bioassays MS and SS smoke solutions were tested on at least four infundibula in each experiment. Each infundibulum was exposed for 5 min to 1 ml of smoke solution, and then 6 to 10 measurements were made for each assay (ciliary beat frequency, oocyte retrieval rate and smooth muscle contraction rate). Assays were completed within 3 h of removal of the

Commercial cigarette smoke impairs the oviduct

oviduct from the animal. Ciliary beat frequency was measured using a video image capture system described previously in detail (Knoll et al., 1995; Knoll and Talbot, 1998; Talbot et al., 1998; Riveles et al., 2003, 2004, 2005). Infundibula were placed on a Wild M5A stereoscopic microscope, and a video image was captured using a Hitachi KP-D50 colour digital camera and viewed on a Dell Optiplex GXA computer. Images of beating cilia were recorded on a Super VHS videotape using a Sony Hi-Fi SVHS recorder. To measure ciliary beat frequency, the videotape was played back frame-by-frame. Video images were recorded at 30 frames per second. Each frame represented a fraction of a cycle; the completion of a cycle was equivalent to one beat. The number of frames per cycle (or frames per beat) was measured and then converted into beats per second. Each infundibulum was treated as a single experiment. Ten measurements were made for each infundibulum for each treatment group (control and smoke), and the same region was used for all treatment groups. Recordings were made for 60–150 s per region, which ensured an adequate amount of data for analysis. Oocyte retrieval rate was measured using hamster oviductal explants as described previously (Huang et al., 1997; Knoll and Talbot, 1998; Talbot et al., 1998; Riveles et al., 2003). Oocyte retrieval rate is defined as the length of time needed for the cumulus– oocyte complex to traverse a defined path to the ostium (Huang et al., 1997; Knoll and Talbot, 1998; Talbot et al., 1999). The perfusion chambers containing the infundibular preparations were viewed using a Nikon SMZ-10 microscope containing an ocular micrometer. For each experiment, 6 to 10 measurements were made before (control) and after smoke treatment, and the means ± SDs were calculated for each treatment group. For the infundibular muscle contraction assay, a perfusion chamber containing the infundibulum was placed on a Wild M5A stereoscopic microscope, and a live video image was viewed using a Hitachi KPD50 colour digital camera interfaced to a Dell Optiplex GXA computer with an NEC Multisync 5FG monitor. Infundibular contractions were observed and measured directly on the computer monitor as described previously (Riveles et al., 2003). For each experiment, distance and frequency were measured six times for a single infundibulum per treatment group. The perfusion chamber was equipped with a pipette that held the ampulla of the oviduct in place facilitating the ability to return to the same region for all the measurements. For the smooth muscle contraction rate assay, the same region was observed and the same direction of contraction was used for all the treatment groups. Statistical analysis The statistical significance of the results for the in vitro bioassays was evaluated using a one sample t-test to compare the control (n ≥ 4 infundibula), which was set to 100%, with the mean percent of inhibition (n ≥ 4 infundibula) for each MS or SS smoke solution in the ciliary beat frequency, oocyte retrieval rate or smooth muscle contraction rate assay. Instat (Graphpad, San Diego, CA, USA) was used for all statistical analyses. Means were considered to be significantly different for P < 0.05. Cigarette extraction protocol MS and SS smoke solutions were extracted using dichloromethane (DCM). 4-Vinylpyridine (50 mg/ml) (Sigma-Aldrich, Milwaukee, Wisconsin, USA) was diluted in DCM and added to each smoke solution for use as an internal standard. The final concentration of 4vinylpyridine in the smoke solutions was 1 mg/ml. Three millilitres of smoke solution was extracted three times with 3 ml of DCM using a separatory funnel. After the final extraction, the sample was concentrated to 1 ml under nitrogen. To measure the internal standard using

gas chromatography-mass spectrometry (GC-MS), the smoke samples were diluted 100-fold. By using the authentic standard, we created a calibration curve and calculated the response factor for 4-vinylpyridine. The extraction efficiencies based on recovery of 4-vinylpyridine for the MS smoke solutions were 63% for 2R1, 53% for 1R4F, 59% for Marlboro red, 60% for Marlboro light, 61% for Camel filtered, 80% for Camel unfiltered, 32% for Kool, 62% for Advance, 81% for Omni and 66% for Omni light. The extraction efficiencies for the SS smoke solutions were 88% for 2R1, 76% for 1R4F, 59% for Marlboro red, 63% for Marlboro light, 74% for Camel filtered, 75% for Camel unfiltered, 43% for Kool, 63% for Advance, 60% for Omni and 69% for Omni light. Only samples with extraction efficiencies greater than 50% were reported. Therefore, concentrations in MS and SS Kool and one of the SS Omni cigarette smoke solutions were not determined because of low recoveries. Three samples from each type of smoke solution were injected twice and the mean and SD calculated. When concentrations for two independent samples of the same smoke type were determined, there was good agreement. Each sample was also injected at least two times, and there was good agreement in those values. The means and SDs were based on all the injections for samples with extraction efficiencies above 50%. GC-MS GC-MS was used to identify and quantify specific chemicals in MS and SS cigarette smoke solutions. A Hewlett Packard 5890 GC interfaced to a HP-5971A MSD quadrupole mass selective detector with a Zebron ZB1701 cyanopropyl phenyl column 30 m × 0.32 mm with 1 μm phase thickness from Phenomenex (Torrance, CA, USA) was used. The method parameters have been described previously in detail (Riveles et al., 2003). Samples were injected directly into the GC using a Hamilton gas-tight syringe. Identifications of compounds were made using the mass spectrometry data matched to mass spectral library entries. Compound identities were confirmed using authentic standards matching both mass spectra and retention times. Data analysis The data from the GC-MS were collected and analysed using HPCHEM software G1043C version C.02.00© Hewlett-Packard 1989–1993. Authentic standards were injected in serial dilutions into the GC-MS, and the area counts were recorded and used to calculate response factors for each chemical to be quantified. Quantification of the chemicals in the smoke solution samples was based on the area counts recorded for each peak from the chromatogram. Area counts were multiplied by corresponding response factors for each chemical. After adjusting for injection and dilution volumes, concentrations were calculated in nanograms per microlitre. Authentic standards Authentic standards of each chemical quantified were purchased and used to confirm the identities of compounds discovered in the extracts of MS and SS smoke solutions and to create standard curves for quantification using three serial dilutions (5, 10 and 50 μg/ml). 2Ethylpyridine, 4-methylpyridine, 4-vinylpyridine, 2-ethylpyrazine, 2methylpyrazine, 4-ethylphenol, 4-methylphenol, quinoline and indole were purchased from Sigma-Aldrich.

Results Biological effects of smoke solutions from research brand cigarettes MS and SS smoke solutions made from the University of Kentucky research brand cigarettes (2R1 and 1R4F) were 3

K.Riveles et al.

tested in the oviductal bioassays measuring ciliary beat frequency, oocyte retrieval rate and infundibular smooth muscle contraction rate after a 5-min exposure period (Figure 1A–C). MS smoke solutions from both 2R1 and 1R4F research brand cigarettes significantly inhibited ciliary beat frequency (87 ± 10% and 77 ± 12% of control, respectively) (Figure 1A). SS

Ciliary Beat Frequency

160

Mainstream Sidestream

140

Percent of Control

120

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100

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60 40 20 0

A Univ. KY 2R1

Univ. KY 1R4F

100 90

Oocyte Retrieval Rate

smoke solutions from 1R4F cigarettes also significantly inhibited ciliary beat frequency (73 ± 10% of control). However, SS smoke solutions from 2R1 cigarettes (Figure 1A) did not inhibit ciliary beat frequency (134 ± 12% of control), in agreement with earlier studies (Knoll et al., 1995; Riveles et al., 2003). All the MS and SS smoke solutions from the 2R1 and 1R4F cigarettes inhibited oocyte retrieval rate (Figure 1B). MS smoke solutions from 2R1 and 1R4F cigarettes significantly inhibited oocyte retrieval rate (41 ± 5% and 43 ± 14% of control, respectively) (Figure 1B). SS smoke solutions from 2R1 and 1R4F cigarettes also inhibited oocyte retrieval rate (17 ± 9% and 0% of control, respectively) (Figure 1B). For both cigarette types, SS smoke solutions were more inhibitory than the MS solutions, and the SS 1R4F smoke solutions completely stopped oocyte retrieval. In the infundibular smooth muscle contraction rate assay, MS smoke solutions from 2R1 and 1R4F research cigarettes significantly inhibited smooth muscle contraction rate by a similar percentage (49 ± 9% and 52 ± 20% of control, respectively) (Figure 1C). The SS smoke solutions from Camel filtered, Kool, and Kool (filter removed) inhibited smooth muscle contraction rate (0.2–23% of control) to a greater extent than their corresponding MS solutions, except for MS smoke from Marlboro light, which appeared to stimulate contraction.

Percent of Control

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Biological effects of smoke solutions from traditional commercial cigarettes

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Univ. KY 2R1

Univ. KY 1R4F

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*** Univ. KY 2R1

*** Univ. KY 1R4F

Figure 1. The effect of mainstream (MS) and sidestream (SS) smoke solutions from research brand cigarettes on oviductal functioning. Hamster oviducts were treated with MS (6 puff equivalents/ml) or SS (3 puff equivalents/ml) smoke solutions from 2R1 and 1R4F research brand cigarettes, and ciliary beat frequency (A), oocyte retrieval rate (B) and infundibular smooth muscle contraction rate (C) were measured. Each point is the mean ± SD of at least four infundibula calculated as the percentage of control. Statistical significance was determined using a one-sample t-test (*P < 0.05, **P < 0.01 and ***P < 0.001).

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Five traditional cigarette brands (Marlboro red, Marlboro light, Camel filtered, Camel unfiltered and Kool) were tested in the oviductal assays (Figure 2A–C). MS smoke solutions from all traditional brand cigarettes significantly inhibited ciliary beat frequency (Figure 2A). The percentages of the control (70–87%) for the traditional brands were similar to the percentages of the control for the research brands (77–87%) (Figure 1A). When the filter was removed from Kool cigarettes before making MS smoke solutions, inhibition was similar to inhibition observed using filter-intact Kool cigarettes (Figure 2A). All SS smoke solutions made from traditional cigarette brands, except for Marlboro light, significantly inhibited ciliary beat frequency (57–86% of control) (Figure 2A). SS smoke solutions from both Marlboro red and Camel filtered cigarettes inhibited ciliary beat frequency more than MS smoke solutions from the same cigarettes. All the MS smoke solutions made from traditional cigarette brands significantly inhibited oocyte retrieval rate (Figure 2B) at percentages of the control (31–57%) similar to the research brand cigarettes (41–43%) (Figure 1B). The MS smoke solutions made from both filter-intact and filter-removed Kool cigarettes inhibited oocyte pickup rate, and removing the filter greatly increased inhibition (Figure 2B). All the SS smoke solutions from traditional cigarette brands significantly inhibited oocyte retrieval rate (6–28% of control) (Figure 2B). Moreover, the SS smoke solutions inhibited oocyte retrieval rate more than their corresponding MS solutions for each type of traditional cigarette (Figure 2B). In the infundibular smooth muscle contraction rate assay, all the MS smoke solutions made from traditional cigarette brands, except Marlboro light (124% of control), significantly

Commercial cigarette smoke impairs the oviduct

Ciliary Beat Frequency

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Mainstream Sidestream

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Marlboro light

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Kool (Filter Removed)

Figure 2. The effect of mainstream (MS) and sidestream (SS) smoke solutions from traditional brand cigarettes on oviductal functioning. Hamster oviducts were treated with MS (6 puff equivalents/ml) or SS (3 puff equivalents/ml) smoke solutions from traditional brand cigarettes, and ciliary beat frequency (A), oocyte retrieval rate (B) and infundibular smooth muscle contraction rate (C) were measured. All the MS and SS smoke solutions from traditional cigarettes significantly inhibited ciliary beat frequency, except SS Marlboro light (A). MS and SS smoke solutions from traditional brand cigarettes significantly inhibited oocyte retrieval rate (B). MS and SS smoke solutions from traditional brand cigarettes significantly inhibited smooth muscle contraction, except MS Marlboro light (C) Each point is the mean ± SD of at least four oviducts and was calculated as the percentage of control. Statistical significance was determined using a one-sample t-test (*P < 0.05, **P < 0.01 and ***P < 0.001).

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K.Riveles et al.

180

Ciliary Beat Frequency 160

Mainstream Sidestream

140

Percent of Control

inhibited smooth muscle contraction rate (1–63% of control) (Figure 2C). MS smoke solutions from Marlboro red, Camel unfiltered and Kool cigarettes (0.63, 0.57 and 10.48% of control, respectively) were more inhibitory in the smooth muscle contraction rate assay than the research brand cigarettes (41–43% of control) (Figure 2C). MS smoke solutions from Marlboro lights appeared to stimulate smooth muscle contraction although statistical significance was not determined (Figure 2C). The SS smoke solutions from Camel filtered, Kool, and Kool (filter removed) inhibited smooth muscle contraction rate (0.2– 23% of control) to a greater extent than their corresponding MS solutions, except for MS smoke from Marlboro lights, which appeared to stimulate contraction (Figure 2C).

120 100

*

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Biological effects of smoke solutions from harm reduction cigarettes

Quantification of oviductal toxicants in MS and SS smoke solutions of various cigarette brands The concentrations of nine oviductal toxicants (4-methylpyridine, 2-ethylpyridine, 3-ethylpyridine, 2-methylpyrazine, 2-ethylpyrazine, 4-methylphenol, 4-ethylphenol, quinoline and indole) previously identified in MS and SS smoke solutions were determined for the research, traditional and harm reduction cigarettes (Table I). These chemicals were chosen as they had previously been shown to be inhibitory in the oviductal assays at very low doses (Table II) (Riveles et al., 2003, 2004, 6

Advance

Omni

Omni light

100 90

Oocyte Retrieval Rate

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Advance

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Omni light

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Infundibular Smooth Muscle Contraction Rate

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Three harm reduction cigarette brands (Advance, Omni and Omni light) were tested in the oviductal assays (Figure 3A–C). MS smoke solutions from Advance cigarettes, but not from Omni or Omni light, significantly inhibited ciliary beat frequency (83 ± 10% of the control) (Figure 3A), and the level of inhibition was similar to that seen with the research and traditional cigarettes (Figures 1A and 2A). SS smoke solutions from the harm reduction cigarettes either had no effect on ciliary beat frequency (Omni) or appeared to stimulate beat frequency (Advance and Omni light) although statistical significance was not determined. All MS smoke solutions made from harm reduction cigarettes significantly inhibited oocyte retrieval rate (21–57%) (Figure 3B). Levels of inhibition for Omni and Omni light were similar to levels observed with traditional brands, whereas MS solutions from Advance cigarettes (21% of the control) inhibited oocyte retrieval more than any other brand tested. SS smoke solutions from all three brands of harm reduction cigarettes also significantly inhibited oocyte retrieval rate (14–30%) (Figure 3B). For Omni and Omni light, the SS smoke solutions inhibited oocyte retrieval rate more than the MS solutions (Figure 3B). The MS smoke solutions from Advance and Omni harm reduction cigarettes significantly inhibited infundibular smooth muscle contraction rate (66 ± 20% and 60 ± 16% of the control, respectively). In contrast, MS smoke solutions from Omni light (116 ± 23%) appeared to slightly stimulate contraction (Figure 3C). All the SS smoke solutions from harm reduction cigarette brands significantly inhibited contraction rate (4–31%) (Figure 3C). The SS smoke solutions from each brand of harm reduction cigarette inhibited contraction more than their corresponding MS solutions (Figure 3C).

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Figure 3. The effect of mainstream (MS) and sidestream (SS) smoke solutions from harm reduction brand cigarettes on oviductal functioning. Hamster oviducts were treated with MS (6 puff equivalents/ml) or SS (3 puff equivalents/ml) smoke solutions from harm reduction brand cigarettes, and ciliary beat frequency (A), oocyte retrieval rate (B) and infundibular smooth muscle contraction rate (C) were measured. MS Advance smoke solutions significantly inhibited ciliary beat frequency (A). All the MS and SS smoke solutions made from harm reduction cigarettes significantly inhibited oocyte retrieval rate (B). All the MS and SS smoke solutions made from harm reduction cigarettes significantly inhibited smooth muscle contraction rate, except MS Omni light (C). Each point is the mean ± SD of four oviducts and was calculated as the percentage of control. Statistical significance was determined using a onesample t-test (*P < 0.05, **P < 0.01 and ***P < 0.001).

40.60 (16.48) 45.20 (10.43) 0.69 (0.14) 0.72 (0.08) 5.08 (3.27) 6.31 (1.50) 3.74 (0.35) 7.24 (0.98) 0.36 (0.05) 0.37 (0.15) 2.07 (0.50) 1.90 (0.22) 0.35 (0.08) 0.51 (0.20) 0.11 (0.03) 0.52 (0.06) 0.59 (0.19) 0.68 (0.12)

1R4 106.34 (20.85) 151.99 (22.77) 1.11 (0.16) 3.26 (2.12) 9.20 (0.91) 3.26 (2.12) 19.19 (2.69) 21.83 (7.30) 0.239 (0.08) 2.12 (0.71) 5.32 (0.18) 6.02 (1.91) 6.23 (0.10) 8.65 (0.65) 0.61 (0.19) 0.35 (0.24) 1.82 (0.23) 1.23 (0.36)

Marlboro red 80.02 (8.59) 82.78 (14.20) 1.10 (0.14) 1.55 (0.49) 4.71 (1.57) 3.57 (1.94) 10.80 (2.52) 13.75 (2.53) 0.71 (0.04) 3.78 (2.21) 2.12 (0.41) 4.91 (1.47) 0.25 (0.06) 0.68 (0.42) 0.004 (0.001) 0.05 (0.01) 0.47 (0.65) 0.38 (0.31)

Marlboro light 1.43 (0.77) 1.77 (0.28) 0.05 (0.02) 0.14 (0.05) 0.27 (0.15) 0.71 (0.22) 0.24 (0.16) 1.37 (0.43) ND 0.18 (0.04) 0.31 (0.01) 0.41 (0.14) 0.03 (0.02) 0.02 (0.01) 0.003 (0.001) 0.05 (0.002) 0.025 (0.01) 0.04 (0.03)

Camel filtered

9 × 10−9 (9.50 × 10−11) 1.00 × 10−9 (9.35 × 10−12) 1.00 × 10−7 (9.33 × 10−10) 9 × 10−10 (1 × 10−11) 1 × 10−9 (1 × 10−12) 1.08 × 10−9 (1 × 10−11) 1 × 10−10 (1 × 10−12) 1 × 10−9 (1 × 10−11) 1 × 10−13 (1 × 10−15)

37.50 (4.30) 0.35 (0.50) 2.60 (3.50) 1.93 (2.56) 0.19 (0.24) 1.10 (1.42) 0.18 (0.24) 0.11 (0.03) 0.31 (0.40)

9 × 10−9 (9.50 × 10−11) 1.00 × 10−9 (9.35 × 10−12) 1.00 × 10−8 (9.33 × 10−11) 9 × 10−11 (1 × 10−12) 1 × 10−9 (1 × 10−12) 1.08 × 10−10 (1 × 10−12) 1 × 10−9 (1 × 10−11) 1 × 10−11 (1 × 10−13) 1 × 10−11 (1 × 10−13)

9 × 10−9 (9.50 × 10−11) 1.00 × 10−9 (9.35 × 10−12) 1.00 × 10−7 (9.33 × 10−10) 9 × 10−10 (1 × 10−11) 1 × 10−9 (1 × 10−11) 1.08 × 10−9 (1 × 10−11) 1 × 10−10 (1 × 10−12) 1 × 10−9 (1 × 10−11) 1 × 10−12 (1 × 10−14)

86.80 (65.80) 2.70 (4.00) 36.50 (63.60) 28.70 (38.00) 5.46 (8.64) 8.10 (11.22) 2.36 (3.00) 0.21 (0.35) 4.60 (7.68)

TRD

RSH

CBF

ORR

SMC

MS meanb concentration (mg/ml)

a

1.60 (0.90) 0.02 (0.01) 0.63 (0.90) 0.05 (0.06) 0.04 (0.03) 0.10 (0.05) 0.09 (0.13) 0.30 (0.31) 0.02 (0.01)

HR

159.40 (40.82) 127.50 (66.81) 8.39 (1.23) 8.33 (1.42) 131.77 (26.20) 76.55 (1.00) 84.38 (1.39) 94.81 (1.68) 15.44 (3.80) 13.19 (1.38) 24.65 (5.01) 15.01 (6.39) 2.92 (0.61) 2.89 (0.88) ND 0.04 (0.03) 16.05 (1.32) 8.35 (0.76)

Camel unfiltered

LOAEL mg/ml (M)a

CBF, ciliary beat frequency; HR, harm reduction; ORR, oocyte retrieval rate; RSH, research; SMC, smooth muscle contraction; TRD, traditional. Values are from Talbot and Riveles (2005). b Values are means (SDs) of the nine brands shown in Table I.

4-Methylpyridine 2-Ethylpyridine 3-Ethylpyridine 2-Methylpyrazine 2-Ethylpyrazine 4-Methylphenol 4-Ethylphenol Quinoline Indole

Toxicants

Table II. Comparison of toxicant lowest observed adverse effect levels (LOAELS) and toxicant concentrations in smoke solutions

ND, not detected. Concentrations are for MS or SS solutions prepared as described in Materials and methods. Values are means (SDs) of six measurements.

Indole

Quinoline

4-Ethylphenol

4-Methylphenol

2-Ethylpyrazine

2-Methylypyrazine

3-Ethylpyridine

2-Ethylpyridine

34.45 (12.97) 17.84 (1.57) 0.01 (0.002) 1.93 (0.84) 0.12 (0.05) 0.64 (0.28) 0.12 (0.06) 0.18 (0.03) 0.02 (0.01) 0.03 (0.02) 0.06 (0.03) 0.06 (0.01) 0.01 (0.003) 0.02 (0.01) ND 0.08 (0.02) 0.02 (0.005) 0.07 (0.02)

4-Methylpyridine

MS SS MS SS MS SS MS SS MS SS MS SS MS SS MS SS MS SS

2R1

Toxicants [concentration (mg/ml)]

Table I. Concentration of nine toxicants in mainstream (MS) and sidestream (SS) smoke from various types of cigarettes

0.87 (0.07) 5.80 (1.27) 0.02 (0.005) 0.03 (0.02) 0.04 (0.004) 0.37 (0.17) 0.01 (0.001) 0.14 (0.02) 0.06 (0.02) 0.06 (0.003) 0.06 (0.02) 0.04 (0.01) 0.01 (0.006) 0.007 (0.001) ND ND 0.01 (0.002) 0.02 (0.001)

Omni

31.50 (19.30) 1.33 (0.86) 3.50 (4.00) 3.71 (5.00) 0.20 (0.24) 0.98 (1.30) 0.27 (0.35) 0.12 (0.15) 0.38 (0.43)

RSH

91.00 (66.00) 3.32 (3.60) 21.00 (37.00) 33.00 (42.00) 4.81 (5.77) 6.60 (6.12) 3.06 (4.00) 0.04 (0.04) 2.50 (3.93)

TRD

SS meanb concentration (mg/ml)

2.55 (0.18) 8.08 (3.81) 0.01 (0.001) 0.09 (0.05) 1.67 (0.50) 1.70 (0.20) 0.03 (0.01) 0.60 (0.18) ND 0.08 (0.06) 0.08 (0.01) 0.23 (0.05) 0.24 (0.04) 0.66 (0.2) ND 0.06 (0.001) 0.02 (0.01) 0.12 (0.07)

Advance

1.60 (0.87) 0.05 (0.03) 0.74 (0.84) 0.42 (0.25) 0.10 (0.05) 0.10 (0.12) 0.25 (0.36) 0.01 (0.001) 0.06 (0.05)

HR

1.35 (0.08) 5.57 (3.11) 0.02 (0.01) 0.04 (0.02) 0.18 (0.01) 0.15 (0.06) 0.12 (0.02) 0.53 (0.07) 0.02 (0.007) 1.46 (0.12) 0.15 (0.03) 0.02 (0.01) 0.02 (0.004) 0.08 (0.002) 0.01 (0.001) 0.01 (0.002) 0.03 (0.01) 0.04 (0.01)

Omni light

Commercial cigarette smoke impairs the oviduct

7

K.Riveles et al.

2005). This information was collected to compare relative amounts of these oviductal toxicants in the various cigarette brands and to determine whether there is a relationship between concentrations of oviductal toxicants and toxicity in the oviductal assays. When tested individually in a previous study, methyl- and ethyl-substituted pyridines were the most potent pyridine derivatives in the oviductal bioassays, and 4-methylpyridine had lowest observed adverse effect levels (LOAELs) for all three bioassays in the picomolar range (Riveles et al., 2003). Three pyridine derivatives were quantified (4-methylpyridine, 2-ethylpyridine and 3-ethylpyridine). Of the nine chemicals analysed, 4-methylpyridine was present in the highest concentration in both MS and SS smoke solutions from all brands of cigarettes (research, traditional and harm reduction) (Table I). For the MS smoke solutions, the unfiltered Camel cigarettes had the highest concentration of 4-methylpyridine (159.4 mg/ ml). The concentrations of 4-methylpyridine in both MS and SS smoke from the harm reduction brands were less than in the traditional brands. 3-Ethylpyridine was found in all cigarette types and was particularly high (131.77 mg/ml) in unfiltered Camel cigarettes. 3-Ethylpyridine is one of the 599 chemicals added to some cigarettes to enhance flavour (http:// www.ash.org.nz/pdf/Smoking/Tobacco/CigarettesAdditives.pdf). Two pyrazine derivatives, 2-methylpyrazine and 2-ethylpyrazine, were quantified in MS and SS smoke solutions (Table I). When tested individually, 2-methylpyrazine and 2ethylpyrazine were highly potent in the oviductal assays, causing inhibition of ciliary beat frequency, oocyte retrieval rate and infundibular smooth muscle contraction in nanomolar or picomolar doses (Table II). In most brands, 2-methylpyrazine was present in higher concentrations in both MS and SS smoke than 2-ethylpyrazine, and both chemicals were generally present in higher concentrations in SS than in MS smoke solutions within brands. SS smoke from traditional cigarettes (Marlboro red, Marlboro light and Camel unfiltered) had the highest concentrations of 2-methylpyrazine and 2-ethylpyrazine. 2-Methylpyrazine was present in all cigarette types tested, whereas 2-ethylpyrazine was not detected in MS smoke solutions from Camel filtered and Advance cigarettes. MS and SS concentrations of 2-methylpyrazine and 2-ethylpyrazine were considerably higher for the filtered research cigarette (1R4F) than the unfiltered 2R1 cigarettes. In general, the amounts of both pyrazines were lower in the harm reduction cigarettes than in the traditional brands (Table I). Two phenolic derivatives, 4-methylphenol and 4-ethylphenol, were quantified in smoke solutions. Phenolic derivatives with a substitution on the fourth carbon were more potent than the 2-substituted phenols or phenol itself in the oviductal assays (Riveles et al., 2005). In general, the amounts of these two phenols in MS and SS were similar within brands. The relative amounts of 4-methylphenol in MS and SS solutions were higher than for 4-ethylphenol. Marlboro reds had more 4ethylphenol than any other type, including unfiltered Camels (Table I). Quinoline and indole impair oviductal functioning in picomolar or femtomolar doses (Riveles et al., 2005). Quinoline was present in all the MS smoke solutions, except 2R1, Camel 8

unfiltered, Advance and Omni, and in the SS smoke solutions of all cigarette brands, except Omni. The amounts of quinoline in harm reduction cigarettes were in general lower in both MS and SS smoke than in the traditional brands. In most brands, quinoline was present in higher concentration in SS smoke than in MS smoke. Indole was present in all the MS and SS smoke solutions tested (Table I). The relative indole concentrations in both MS and SS smoke solutions were similar within brands, except for Advance SS, which had significantly more indole than the MS solutions. Discussion Previous studies that tested the effect of smoke solutions on oviductal functioning used MS (smoke inhaled by active smokers) and SS (major component of environmental tobacco smoke) smoke solutions from 2R1 research cigarettes, which simulate cigarettes used in the 1950s (Knoll et al., 1993, 1995; Knoll and Talbot, 1998; Lam et al., 2000; Riveles et al., 2003, 2004, 2005). This is the first study to show inhibition of oviductal functioning with MS and SS smoke solutions from 1R4F research cigarettes (a filtered research cigarette similar to many commercial brands smoked today), traditional commercial cigarettes (Marlboro red, Marlboro light, Camel filtered, Camel unfiltered and Kool) and harm reduction cigarettes (Advance, Omni and Omni light). In addition, pyridine, pyrazine, phenol derivatives, quinoline and indole, which were previously shown to be very potent oviductal toxicants (Riveles et al., 2003, 2004, 2005), were quantified in the MS and SS smoke solutions for each cigarette brand that we tested (except Kools). Our quantitative data show that, in general, SS smoke had higher concentrations of toxicants than MS smoke and is in agreement with other published data (Brunnemann and Hoffmann, 1978; White et al., 1990; Chen and Moldoveanu, 2003). This observation correlated well with the oviductal assays that showed, in general, greater inhibition of oviductal functioning by SS than MS smoke solutions. Although each brand of cigarette had its own unique signature with respect to toxicant concentrations, several interesting observations emerge from these data. For the toxicants that we studied, both types of research cigarettes were similar to the traditional commercial brands. Both research cigarettes were inhibitory in all three bioassays, and most toxicants in the 2R1 and 1R4F research cigarettes had values within the range found in the four traditional commercial brands although the research cigarettes often had values at the lower end of the traditional ranges. SS smoke from 1R4Fs completely inhibited oocyte retrieval and infundibular smooth muscle contraction rates and had overall higher concentrations of the toxicants than MS smoke from 1R4Fs or MS and SS smoke from 2R1s. Both MS and SS smoke from all the traditional brands whether filtered, non-filtered, light or mentholated inhibited all the oviductal bioassays, except for MS smoke from Marlboro lights, which appeared to stimulate smooth muscle contraction. In all but three cases, Camel unfiltered cigarettes had the highest concentrations of the chemicals that were quantified. The filtered Camels had reduced levels of all toxicants.

Commercial cigarette smoke impairs the oviduct

Nevertheless, the smoke passing through the Camel filter retained sufficient concentrations of toxicants to be significantly inhibitory in all three bioassays. Although oviductal toxicants in Marlboro lights were also generally reduced in MS and SS smoke compared to Marlboro reds, concentrations of most toxicants were still relatively high in the Marlboro lights and were in general not as low as in the filtered Camels. Although filtered and ‘light’ cigarettes may be perceived by some smokers as less hazardous than high-yield cigarettes (Shiffman et al., 2001; Hamilton et al., 2004), the results of our study and others (Warner, 2005) do not support this idea. The harm reduction cigarettes that we tested are marketed as having reduced levels of carcinogens. To determine whether the concentrations of oviductal toxicants were lower in harm reduction than in traditional cigarettes, we examined the number of times the concentrations of the toxicants in harm reduction brands fell below the range of concentrations in the traditional brands. For the nine chemicals analysed, oviductal toxicant concentrations in MS and SS smoke from harm reduction cigarettes were below the range of the traditional brands in 70% of our samples. In no case were the concentrations in harm reduction cigarettes above the ranges for the traditional commercial brands. Reduction below the range of traditional cigarettes occurred more often in MS smoke (79.2%) than in SS smoke (61.5%) from harm reduction cigarettes. Comparing across brands, the percentage of toxicants was below the traditional range more often (94%) in Omni cigarettes (MS plus SS combined) than in Omni lights (66.7%) and least often in Advance cigarettes (50%). Importantly, although there was an apparent reduction of some oviductal toxicants in the three harm reduction brands, each brand nevertheless was highly inhibitory in the oocyte retrieval rate and smooth muscle contraction bioassays, indicating that the harm reduction cigarettes retained sufficient amounts of these toxicants to adversely affect oviductal functioning. Moreover, MS smoke from Advance cigarettes inhibited oocyte retrieval rate more than any of the research and traditional brands tested. Although harm reduction cigarettes may be reduced in carcinogens, our data indicate that they still contain high enough levels of toxicants to impair the biological activities of the oviduct and may therefore not be safer than traditional brands for women who are attempting to become pregnant. Three factors lowered the levels of toxicants in the cigarettes tested. First, the inclusion of a filter in Camel cigarettes appeared to greatly reduce the levels of all toxicants in MS smoke. Secondly, harm reduction cigarettes also had, in general, lower levels of the oviductal toxicants than the traditional and research brands. Finally, Marlboro lights, in general, had reduced levels of toxicants when compared to Marlboro reds. However, in no case was the toxicant reduction great enough to eliminate the inhibitory effects of smoke on oviductal functioning. Although toxicants were not quantified in Kool cigarettes, the presence of a filter in Kools did not eliminate their inhibitory activity in the three bioassays. Therefore, our data support the conclusion that reduction of toxicants by inclusion of a filter, by the harm reduction process or by creation of a ‘light’ cigarette may in some cases reduce levels of oviductal toxicants but

does not protect against the inhibitory effects of MS cigarette smoke in the oviductal bioassays. Interestingly, the ability of filters to remove the toxicants was variable. Although the filtered Camels had significantly lower concentrations of all quantified chemicals in MS than their non-filtered counterpart, the filter in the 1R4Fs appeared, in general, not to be effective in removing these chemicals. In all instances, MS smoke from 1R4Fs had higher concentrations of the quantified chemicals than the unfiltered 2R1s. The concentrations of the chemicals measured in smoke solutions were compared with the concentrations previously determined as the LOAELs (Table II). LOAEL doses for each chemical in each bioassay are compared with the mean concentration measured in the three classes of cigarettes. Table II summarizes that each chemical is present in smoke at a high enough concentration to cause inhibition of each oviductal process. Even the harm reduction cigarettes, which had reduced levels of toxicants, had levels that were significantly higher than the LOAEL doses. This comparison supports our experimental observation that all brands of cigarettes were inhibitory in the oocyte retrieval rate and infundibular smooth muscle contraction rate assays. In conclusion, the results of this study show that MS and SS smoke from research, traditional and harm reduction cigarettes inhibit oviductal functioning as assessed with bioassays that measure ciliary beat frequency, oocyte retrieval rate and infundibular smooth muscle contraction. On the basis of comparison of P values, SS smoke was generally more inhibitory than MS smoke for all types of cigarettes and in general had higher levels of the quantified toxicants than MS smoke. Reduction of toxicants by filters, by the harm reduction process or by creation of a ‘light’ cigarette did not reduce toxicant levels sufficiently to prevent inhibition of oviductal functioning. These observations indicate that cigarettes that are perceived by smokers as ‘safe’ may in fact have high enough levels of toxicants to still be dangerous and further show that at least one reproductive organ, the oviduct, could be adversely affected by smoke from commercial cigarettes. Acknowledgements We are very grateful to Dr. Janet Arey who allowed us to use her mass spectrometry instrumentation and also provided helpful advice on the quantification of smoke toxicants. This work was supported by grants 12DT-0012, 13RT0068 and 10RT0239 from the Tobacco Related Disease Research Program and grants from the Academic Senate.

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