Pyrazine derivatives in cigarette smoke inhibit hamster oviductal functioning

Female golden hamsters (Mesocricetus auratus) purchased from Harlan Sprague Dawley (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 [27]. Female golden hamsters were induced to ovulate by intraperitoneal injection with 25 IU of pregnant mare's serum gonadotropin (PMSG) (CalBiochem, La Jolla, CA.) at 10 AM 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 their estrous cycle, and used twelve hours later on day 4 for all experiments. One animal was injected and used on any given day for an experiment. Unfertilized follicular oocytes were collected by a method previously described [16]. The protocol for the use of hamsters in this study was approved by the campus animal use committee.

Oocyte pickup rate, ciliary beat frequency, and infundibular smooth muscle contraction assays were done in 1x Earle's Balanced Salt Solution (EBSS-H) supplemented with sodium bicarbonate, HEPES, and 0.1% BSA [23]. The pH was adjusted to 7.4 with NaOH. This solution was used for dissection, incubation, and as the control solution for all experiments. Pyrazine (99+ %), 2-methylpyrazine (99+ %), 2,5-dimethylpyrazine (98%), 2,6-dimethylpyrazine (98%), 2,3,5-trimethylpyrazine (99%), 2-ethylpyrazine (98%), and 2-methoxy-3-methylpyrazine (99%) were purchased from Aldrich Chemical Company (Milwaukee, WI).

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 [23, 25]. Diagrams showing the arrangement of the puffer box and vacuum pumps have been published previously [25]. The MS was adjusted to a pH of 7.4 and 0.1% BSA was added. MS smoke solutions were made from 60 puffs of MS smoke pushed through 10 ml of EBSS-H, which is equal to 6 puffs/ml and is defined as 6 puff equivalents per millilitre [16, 23, 25].

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. Cartridges which were screened for their ability to bind inhibitory chemicals included a variety of non-polar, polar, and anion and cation exchange cartridges: 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 previously [16].

The C8, PH, and CN cartridges retained most of the inhibitory activity in MS smoke solutions as determined in the oviductal assays. To identify the components in aqueous smoke solutions that inhibit oviductal functioning, MS smoke solutions were analyzed with GC-MS after solid phase extraction on a C8, PH, or CN cartridge and elution with methanol. Two microliters of each sample was injected into a Hewlett Packard 5890 GC interfaced to an HP-5971A MSD quadrupole mass selective detector with a Zebron ZB1701 cyanopropyl phenyl column 30 m × 0.32 mm with 1 micron phase thickness from Phenomenex (Torrance, CA.) as described previously [16]. Identifications of compounds were made using the mass spectrometry data matched to mass spectral library entries. Compound identities were confirmed using purified standards and matching both mass spectra and retention times.

For this assay, one hamster was injected with 25 IU of PMSG on day 1 of the estrous cycle, followed by 25 IU of hCG on day 3. On the day of the experiment, approximately 12 hours after the injection of hCG, the hamster was sacrificed using CO₂, and two oviducts and ovaries were removed and separated. The two ovaries were isolated in EBSS-H with 0.1% BSA, and 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. OCCs were transferred to a fresh Petri dish and placed in EBSS-H with 0.1% BSA. Two infundibula were dissected from the oviducts leaving the ampullas attached to function as handles. The infundibula were placed in perfusion chambers containing EBSS-H with 0.1% BSA, and the ampullas were placed into the holding pipettes so the infundibula remained stationary throughout the experiment. Oocyte pick-up rate was measured using hamster oviduct explants in an in vitro assay developed in our lab [4, 16, 25, 26]. Using a Wild 5A stereoscopic microscope, an OCC was placed onto the infundibulum, and the length of time for the OCC to traverse a defined path was recorded. This defined path was used for both the control and treatment groups. A total of six to ten measurements were made and the means ± standard deviations were calculated for each treatment group. A single experiment was defined as the treatment of a single infundibulum with the control medium (EBSS-H with 0.1% BSA), and a single dose of the test chemical diluted in EBSS-H with 0.1% BSA.

Ciliary beat frequency was measured using hamster oviductal explants in an in vitro assay developed in our lab [23, 25, 26]. A video image capture system was used to measure ciliary beat frequency as described previously [16]. Infundibula were placed on a Wild M5A stereoscopic microscope, and a video image was captured using an Hitachi KP-D50 color digital camera and viewed on a Dell Optiplex GXA computer. Images of the beating cilia were recorded onto 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) were measured and then converted into beats per second. A single experiment was defined as the treatment of a single infundibulum with the control medium (EBSS-H with 0.1% BSA) and a single dose of the test chemical diluted in EBSS-H with 0.1% BSA. Ten measurements were made for each infundibulum for each treatment group. Recordings were made for 60 to 150 seconds per region, which ensured an adequate amount of data for analysis. The same region was recorded for both the control and treated groups.

A new assay for measuring infundibular muscle contraction was developed in our lab after observing consistent infundibular contractions that slowed or ceased after treatment with MS smoke solutions [16]. A single experiment was defined as the treatment of a single infundibulum with the control medium (EBSS-H with 0.1% BSA) and a single dose of the test chemical diluted in EBSS-H with 0.1% BSA. A perfusion chamber containing the infundibulum was placed on a Wild M5A stereoscopic microscope, and a video image was captured using an Hitachi KP-D50 color digital camera and viewed with a Dell Optiplex GXA computer and an NEC Multisync 5FG monitor. Infundibular contractions were observed and measured directly on the computer monitor. As the infundibulum contracted, the distance of the contraction was measured in centimeters and then converted to micrometers as described previously [16]. For each experiment, distance and frequency were measured six times for a single infundibulum per treatment group (the control medium EBSS-H with 0.1% BSA and a single dose of the test chemical diluted in EBSS-H with 0.1% BSA). 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 both the control and treatment measurements. For the infundibular smooth muscle contraction assay, the same region was observed and the same direction of contraction was used for both the control and treatment groups.

To determine LOAELs for each pyrazine derivative in each bioassay, two levels of analysis were performed. First, a preliminary screen of the pyrazine compounds was done using a single infundibulum per dose group (10^-13–10^-5 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 pyrazine derivative in each assay.

Since the estimated LOAELs were based on only one infundibulum per dose, additional experiments were performed at the estimated LOAEL doses to increase the sample size to n = 4 infundibula for each pyrazine derivative in each bioassay. The statistical significance of these results was evaluated using Student's t-Test to compare the mean of the control (n = 4 infundibula) to the mean at the estimated LOAEL dose (n = 4 infundibula) for each pyrazine derivative in each bioassay (Figs. 1,2,3,4,5,6,7). MS Excel 2002 (Microsoft Corp, Redmond, WA.) was used for all statistical analysis. Means were considered to be significantly different for p < 0.05.

To analyze the efficacies of each pyrazine derivative relative to the parent compound pyrazine, each derivative was treated as a separate experiment, and statistical analyses were performed by comparing the percent inhibition of the derivative (n = 4 infundibula) to the percent inhibition of pyrazine (n = 4 infundibula) at the LOAEL doses using Student's t-Test. These statistics are reported in Table 2.

When MS cigarette smoke solution was passed through twelve solid phase extraction cartridges, the CN, PH, and C8 cartridges were the most effective at retaining the compounds responsible for the inhibition of ciliary beat frequency, oocyte pickup rate, and infundibular smooth muscle contraction [16]. Pyrazine derivatives were identified by gas chromatography-mass spectrometry (GC-MS) as a predominant class of compounds that were retained by these cartridges, and the specific pyrazines in the active fractions of MS smoke solutions are shown for each cartridge in Table 1. 2-Methylpyrazine was the most abundant in the cartridge eluates. Commercially available standards of the identified pyrazines (2-methylpyrazine, 2,5-dimethylpyrazine, 2,6-dimethylpyrazine, 2,3,5-trimethylpyrazine, 2-ethylpyrazine, and 2-methoxy-3-methylpyrazine) were purchased and tested in dose-response studies on the oviduct to determine their effect on ciliary beat frequency, oocyte pickup rate, and infundibular muscle contraction. In addition, pyrazine, which was not found in the cartridge eluates, was also purchased and tested to compare the parent compound to derivatives with methyl, ethyl, or methoxy substitutions on the pyrazine ring. The results from the dose-response experiments are reported for each compound in Figures 1,2,3,4,5,6,7 and are summarized in Table 2.

Pyrazine inhibited ciliary beat frequency and oocyte pickup rate at picomolar doses and infundibular smooth muscle contraction at nanomolar doses when compared to the control, which was treated with the vehicle (EBSS-H with 0.1% BSA) alone (Fig. 1). Significant inhibition of ciliary beat frequency, oocyte pickup rate, and infundibular smooth muscle contraction occurred at 10^-12 M, 10^-11 M, and 10^-9 M, respectively, which were the lowest observable adverse effect levels (LOAELs) (Fig. 1, Table 2). The efficacy for all three oviductal assays was determined using the percent inhibition at the LOAEL. The efficacies in the ciliary beat frequency, oocyte pickup rate, and infundibular smooth muscle contraction assays were 25%, 29%, and 40%, respectively (Table 2).

Pyrazine compounds with single methyl (2-methylpyrazine) or single ethyl (2-ethylpyrazine) substitutions had LOAELs in the picomolar range for all three bioassays (Figs. 2, 3). For 2-methylpyrazine, the LOAELs were 10^-12 M, 10^-11 M, and 10^-9 M in the ciliary beat frequency, oocyte pickup rate, and infundibular smooth muscle contraction assays, respectively (Fig. 2, Table 2). For 2-ethylpyrazine, the LOAELs were 10^-12 M, 10^-11 M, and 10^-12 M in the ciliary beat frequency, oocyte pickup rate, and infundibular smooth muscle contraction rate assays, respectively (Fig. 3, Table 2). While the LOAELs for ciliary beat frequency and oocyte pickup rate did not change relative to pyrazine for these two derivatives, the LOAELs in the smooth muscle contraction assay decreased 100 and 1000-fold, respectively for 2-methylpyrazine and 2-ethylpyrazine (Figs. 2, 3, Table 2).

In most cases, single methyl and single ethyl substitutions on pyrazine altered the efficacies when compared to pyrazine alone. For both 2-methyl and 2-ethylpyrazine, the efficacies for the ciliary beat frequency assay were significantly lower than for pyrazine alone, while efficacies for these two compounds significantly increased relative to pyrazine in the oocyte pickup rate assay (Table 2). In the infundibular smooth muscle contraction assay, the single ethyl substitution did not have a significant effect on efficacy, while the single methyl substitution significantly increased efficacy compared to pyrazine (Table 2).

In general, the dimethylpyrazines were less potent than the single ethyl or methyl substituted pyrazines or pyrazine alone. 2,5-Dimethylpyrazine had LOAELs equivalent to pyrazine in the oocyte pickup rate (10^-11 M) and infundibular smooth muscle contraction rate (10^-9 M) assays; however, its LOAEL in the ciliary beat frequency assay (10^-8 M) was 10,000 times greater than that of pyrazine (10^-12 M) (Fig. 4, Table 2). In all cases, 2,5-dimethylpyrazine was more potent than 2,6-dimethylpyrazine, which inhibited ciliary beat frequency, oocyte pickup rate, and infundibular smooth muscle contraction with LOAELs of 10^-6 M, 10^-9 M, and 10^-7 M, respectively (Fig. 5, Table 2).

2,5-Dimethylpyrazine had the highest efficacy of all the pyrazine compounds in the ciliary beat frequency assay (34%), however it was not statistically different from pyrazine. 2,5-Dimethylpyrazine (9.9%) and 2,6-dimethylpyrazine (3.5%) had significantly lower efficacies in the oocyte pickup rate assay compared to pyrazine. 2,6-Dimethylpyrazine was the least potent of all the compounds tested, however, its efficacy in the infundibular smooth muscle contraction assay (85%) was significantly higher than pyrazine (Table 2).

For 2,3,5-trimethylpyrazine, the LOAELs were 10^-9 M, 10^-10 M, and 10^-9 M in the ciliary beat frequency, oocyte pickup rate, and infundibular smooth muscle contraction assays, respectively (Fig. 6, Table 2). For the ciliary beat frequency assay, the LOAEL was lower than the dimethyl pyrazines, but not as low as the ethyl or methyl substituted pyrazines or pyrazine alone. For the oocyte pickup rate assay, the LOAEL was lower than 2,6-dimethylpyrazine, but higher than all the other pyrazines. For the infundibular smooth muscle contraction assay, the LOAEL was the same as that for pyrazine and 2,5-dimethylpyrazine, but higher than the ethyl or methyl substituted pyrazines.

For 2-methoxy-3-methylpyrazine the LOAELs were 10^-9 M, 10^-12 M, and 10^-12 M, respectively, for the ciliary beat frequency, oocyte pickup rate, and infundibular muscle contraction assays (Fig. 7, Table 2). In the oocyte pickup rate and infundibular smooth muscle contraction rate assays the LOAELs were the lowest (10^-12 M) compared to all the other pyrazines tested. For the ciliary beat frequency assay, the LOAEL was the same as for the trimethyl substituted pyrazine (10^-9 M).

The efficacies for 2,3,5-trimethylpyrazine (10%) and 2-methoxy-3-methylpyrazine (13%) for oocyte pickup rate were significantly lower than for pyrazine alone (Table 2). The efficacy of 2,3,5-trimethylpyrazine (58%) for the infundibular smooth muscle contraction rate assay was significantly higher than for pyrazine. 2-Methoxy-3-methylpyrazine had the lowest efficacy (17%) in the infundibular smooth muscle contraction assay and was significantly lower than pyrazine, although its LOAEL is among the lowest (Table 2). The efficacies for 2,3,5-trimethylpyrazine and 2-methoxy-3-methylpyrazine for the ciliary beat frequency assay were not significantly different than for pyrazine (Table 2).