How Cigarette Smoke Impairs Reproduction and Development

Overview
What are Mainstream and Sidestream Smoke?
In Vivo Effects of Mainstream and Sidestream Smoke Inhalation on Female Reproductive Organs
Conclusions from the in vivo studies
In Vitro Effects of Smokeon the Oviduct
Mainstream and Sidestream Smoke Inhibit Growth and Angiogenesis
Identification of Novel Toxicants in Cigarette Smoke
Bottom Line
OVERVIEW:

Our lab began investigating the effect of cigarette smoke on the female reproductive tract in 1992. This study grew out of previous literature, largely epidemiological, that showed a correlation between cigarette smoking and adverse effects on reproduction in women. We have conducted a number of experiments, both in vivo and in vitro to determine which reproductive organs are targets of cigarette smoke, what specific effects smoke has on reproductive organs, and what chemicals in smoke produce these effects (publications). Our studies have included both mainstream and sidestream smoke.

We have identified chemicals in smoke that may have therapeutic value in blocking unwanted growth of cells, and we have patented their possible use for treatment of diseases requiring tissue growth and/or angiogenesis. We are currently evaluating their in vitro and in vivo effects on mammalian cells. We are interested in collaborating with clinicians or biotech companies that would be interested in helping us evaluate the efficacy of these chemicals in treatment of cancer and other growth requiring diseases. Please contact us if you are interested in pursuing a possible collaboration or in licensing these chemicals for further studies (talbot@citrus.ucr.edu).


WHAT ARE MAINSTREAM AND SIDESTREAM SMOKE?


Mainstream smoke is the smoke that an active smoker inhales each time they take a puff on a cigarette. Humphrey Bogart is actively smoking in the figure above.




Sidestream smoke is the smoke that burns off the end of a cigarette. It is the major component of environmental tobacco smoke (ETS) and it is the main type of smoke inhaled by passive smokers.
IN VIVO EFFECTS OF MAINSTREAM AND SIDESTREAM SMOKE INHALATION ON FEMALE REPRODUCTIVE ORGANS:

In our first study (Magers et al., 1995), we evaluated the effects of mainstream and sidestream smoke inhalation on the ovaries, oviducts, and uteri using an analytical smoking machine that exposed hamsters to nose only inhalation of either mainstream or sidestream smoke. Our smoking machine which is diagramed below allows simultaneous exposure of 8 females to mainstream smoke and 8 females to sidestream smoke. In our first experiment, hamsters inhaled cigarette smoke for 30 days prior to pregnancy and for the first 7 days of pregnancy. We then evaluated the effects of smoke inhalation on various aspects of their reproductive tracts. The doses used in this experiment were similar to those received by active and passive human smokers. Inhalation of mainstream and sidestream cigarette smoke produced adverse effects in the ovaries, oviducts, and uteri.


FIGURE: (Top) Schematic diagram of our analytical smoking machine. Mainstream smoke (red tubing) is generated by a puffer box and is diluted with air before being delivered to hamsters via a Delrin nose cone. Sidestream smoke (blue tubing) is collected in a glass tube as it burns off the end of the cigarette and is delivered to another group of hamsters. Used smoke (yellow tubes) is drawn away from the smoking machine by a small rotary pump. Up to eight mainstream and eight sidestream animals can be exposed simultaneously. Details of the method used for exposure can be found in Magers et al., (1995).


FIGURE: A schematic diagram of a female reproductive tract (one side) showing the ovary, oviduct, and uterus. Notice that the oviduct is divided into three anatomical regions, the infundibulum, ampulla, and isthmus.
Ovaries: The corpora lutea on ovaries of smokers were affected by smoke exposure. Corpora lutea are endocrine organs that function mainly in producing the hormones estrogen and progesterone, which are important in maintaining a pregnancy. Ovaries from females exposed to mainstream smoke had significantly fewer corporal lutea than controls or sidestream smokers.

Moreover, the corpora lutea of females exposed to mainstream or to sidestream smoke were not as red in color as those from smokers ( figure below, left). To determine if the pink color was related to a decrease in vasculature, histological sections of corpora lutea were examined and the area occupied by blood vessels was computed. The corpora lutea from smokers had less vascular area than those from control groups (figure below).


FIGURE: Corpora lutea on the ovary of a smoker (left) and of a control (right). Corpora lutea are much redder on the control ovary. Ovaries form mainstream smokers also had fewer corpora lutea.


FIGURE: Tracings of blood vessels in the apex of corpora lutea from control and smoke exposed females. The amount of vasculature is significantly reduced in the smoke exposed females. Similar results were obtained for females that inhaled sidestream smoke.
Uterus: The uterus is the site of blastocyst implantation and subsequent development of the embryo and fetus. The uterine horns of smokers were more taut than those from controls. The number of implantation sites was not affected by smoke inhalation, but the spacing of implantation sites was not normal in smokers. Significantly more implantation sites touched each other in the smoked exposed group than in the controls, indicating that spacing at implantation had been affected by smoke inhalation.
Oviduct: The mammalian oviduct has three anatomical parts: the infundibulum, ampulla and isthmus (figure at right). The infundibulum functions in rapidly picking up the oocyte cumulus complex and transporting it to the ampulla where fertilization occurs. The zygote and preimplantation embryo then spend several days traveling through the isthmus. Four to five days after fertilization the preimplantation embryo arrives in the uterus where implantation takes place on day 5-6. The exterior surface of the infundibular part of the oviduct is covered predominantly with ciliated cells and a smaller number of secretory cells (figure below, left). Oviducts from hamsters exposed to mainstream or sidestream cigarette smoke had numerous blebs on the ciliated cells of the infundibulum, indicating that damage had occurred to these cells (figure below, right). In addition, the percentage of ciliated cells relative to secretory cells decreased in smokers.


FIGURE: Electron micrograph of cilia (arrow) on the exterior surface of the infundibulum of the oviduct. The cilia beat in the direction of the ostium (opening of the oviduct) and propel the oocyte cumulus complex into the oviduct's lumen. The arrowhead points to microvilli on the surface of some secretory cells.


FIGURE: Electron micrograph showing blebs (arrowhead) on ciliated cells on the surface of an oviduct from a hamster exposed to sidestream smoke.
Reversal Experiment:

To determine if the above described effects on the reproductive tract were reversible, females were exposed to mainstream or sidestream smoke for 30 days, then were allowed to breathe fresh air for 30 days before mating. No significant differences were found in the number of pink vs. red corpora lutea, in the strechability of the uterine horns, or in the number of touching of implantation sites in the uterus in the reversal experiment indicating that these effects were reversible if smoking stopped before pregnancy began.

A follow-up study was done to examine the effect of smoking on oviductal functioning more closely (DiCarlantonio and Talbot, 1999). Hamsters were exposed on the smoking machine to doses of mainstream or sidestream cigarette smoke similar to those received by human smokers. Transport of embryos through the oviduct was retarded in female hamsters at doses that produced serum cotinine levels similar to those found in humans who smoke actively or passively during pregnancy. In addition, hamster oviductal smooth muscle contraction rate decreased significantly during a single exposure to either mainstream or sidestream (figure below) smoke.


FIGURE: Oviductal smooth muscle cell contraction slows in vivo after inhalation of sidestream smoke. Contraction rate partially recovers when smoking stops. Similar results were obtained with mainstream smoke.
Conclusions from the in vivo studies:

Inhalation of mainstream or sidestream cigarette smoke at levels comparable to what active and passive human smokers receive can adversely affect the ovary, oviduct, and uterus of hamsters. Vascular area is significantly reduced in the corpora lutea of smokers. Muscle contraction is slowed in the oviduct, and this is correlated with retardation of embryo transport in the ampulla and isthmus. Implantation sites are not spaced normally in uterine horns of smokers, suggesting muscle contraction in the uterus is also affected by smoking.
IN VITRO STUDIES ON SMOKE EXPOSURE AND THE OVIDUCT:

In vitro assays to study oviductal functioning: We have developed an in vitro assay using the infundibulum that allows four parameters of oviductal functioning to be measured in the same infundibular preparation (Huang et al., 1997; Knoll and Talbot, 1998; Lam et al., 2000; Riveles et al., 2003). These parameters are ciliary beat frequency, smooth muscle contraction rate, oocyte pick-up rate, and adhesion of the oocyte cumulus complex to the infundibulum. To perform any of these assays, an infundibulum is mounted in a holding pipette and placed in culture medium in a Petri dish so that it can be observed with a dissecting microscope and manipulated by the observer. The figures below show a schematic view of the assay, a microscope with infundibulum mounted in a Petri dish, and the actual appearance of a mounted infundibulum as viewed through the microscope and ready for the assay. You can also view a video of the oocyte cumulus complex being picked-up by the oviduct.


FIGURE: Schematic diagram of the oviductal assay set-up showing an infundibulum mounted in a holding pipette in a Petri dish. The assay can be done with or without oil depending on the experimental design. A second pipette can be used to add or remove medium.


FIGURE: Microscope with dish and oviduct mounted.


FIGURE: Actual preparation of a hamster infundibulum (white) and oocyte cumulus complex (stained blue) as it would appear through the microscope. The oocyte cumulus complex is gliding over the oviduct's surface toward the opening in the oviduct (right end). Click on image to watch a movie of this oocyte cumulus complex being picked up. Notice how the oocyte cumulus complex adheres to the surface of the oviduct during pick-up.
The outer surface of the infundibulum is covered with cilia that beat in the direction of the ostium or opening of the oviduct. Oocyte cumulus complexes adhere to the cilia by their extracellular matrix, and the beating of the cilia causes the oocyte cumulus complex to glide over the surface of the infundibulum and eventually through the ostium. Smooth muscles in the wall of the oviduct also contract and may aid in transport of the oocyte/preimplantation embryo through the oviduct. With our in vitro assay, we are able to measure ciliary beat frequency, the strength of adhesion between the oocyte cumulus complex and infundibulum, oocyte cumulus complex pick-up rate, and muscle contraction rate.


FIGURE: Scanning electron micrograph showing a hamster infundibulum with an oocyte cumulus complex (colorized blue) entering the ostium or opening of the oviduct. The oocyte cumulus complex is actually wider that the opening of the oviduct in hamsters and must undergo a churning processes that compresses the extracellular matrix in the complex so that it can pass through the ostium.
Effect of Smoke Exposure on Ciliary Beat Frequency and Oocyte Cumulus Complex Pick-up Rate:

The oviductal assay was used to examine the effect of mainstream and sidestream smoke solutions on ciliary beat frequency and oocyte pick-up rate (Knoll and Talbot, 1998). Smoke solutions were made using a puffer box (figure below) to draw cigarette smoke through a physiological solution. Both mainstream and sidestream smoke solutions were studied. In some cases, solutions of just the gas phase or just the particulate phase of cigarette smoke were used.


FIGURE: Puffer box and pump set-up for making mainstream smoke solutions. To make sidestream solutions, smoke is collected from the burning end of the cigarette and drawn through the physiological solution.
Graphs showing main findings:

Mainstream smoke decreased ciliary beat frequency and oocyte pick-up rate in a dose dependent manner. Ciliary beat frequency recovered during washout, while pick-up rate did not. Sidestream smoke slightly stimulated ciliary beat frequency, while oocyte pick-up rate decreased. These data are important as they show both types of smoke solution impair pick-up of the oocyte independently of their effect on beat frequency (see figure below). Pick-up could be impaired by a decrease in beat frequency in mainstream solutions, but clearly pick-up is impaired when beating returns to normal in mainstream solutions. Moreover, in sidestream solutions ciliary beat frequency is stimulated above control levels, but pick-up rate decreases.


FIGURE: Sidestream smoke inhibited oocyte pickup rate (OPR) even when ciliary beat frequency (CBF) increased. These data demonstrate that sidestream smoke produces its effect on oocyte pick-up rate by affecting a parameter other than ciliary beat frequency.
Do smoke solutions affect adhesion of the oocyte cumulus complex to infundibula?

Since adhesion is necessary for successful pick-up of the oocyte cumulus complex by the infundibulum, we have examined the effect of smoke solutions on adhesion using the infundibular assay. Details of the method for measuring adhesion have been published previously (Lam et al, 2000). The effect of various types of smoke solution on adhesion of the oocyte cumulus complex to the infundibulum was measured using our adhesion assay. Data that are currently in preparation for publication indicate that pretreatment of either the infundibulum or oocyte cumulus complex with mainstream or sidestream smoke increased adhesion and correlated with a decrease of oocyte pick-up rate. This effect occurs even when ciliary beat frequency is stimulated.


Conclusions from in vitro studies with the oviduct:

Both mainstream and sidestream smoke solutions inhibit oocyte cumulus complex pick-up independently of their effect on ciliary beat frequency. This inhibition appears to be due mainly to the smoke solutions ability to increase adhesion between the oocyte cumulus complex and the infundibulum and thereby retard the rate at which the complex travels over the surface of the infundibulum. Failure of the oocyte cumulus complex to be picked up by the oviduct could affect fertility or result in ectopic pregnancy, which numerous epidemiological studies have shown is more frequent among smokers.


MAINSTREAM AND SIDESTREAM SMOKE INHIBIT GROWTH AND ANGIOGENESIS:

Since the vasculature of corpora lutea decreased in female hamsters that inhaled smoke (Magers et al., 1995), we have examined the effects of cigarette smoke on growth and angiogenesis using an extraembryonic membrane found in chicken eggs called the chick chorioallantoic membrane (CAM). The CAM provides a rapid, relatively inexpensive method to assay growth of extraembryonic tissues and to evaluate angiogenesis. As an additional bonus, growth of embryos can be evaluated in the same preparations.


Smoke solutions perturb vessel development and affect the extracellular matrix in day 7- day 11 CAMs.

Our first experiments were done using older (day 7 to day 11) CAMs. When smoke solutions were placed on CAMs 7 days after fertilization then examined on day 11, the pattern of the CAM vasculature was disturbed (Melkonian et al., 2000). The vessels normally branch in a dendritic fashion. In smoke treated CAMs, branching was aberrant and many vessels were long and thin without many branches, as shown in the figure below.


FIGURE: A control CAM (A)and a CAM treated with mainstream smoke solution (B). The vascular pattern has been disrupted by smoke treatment. Vessels in the control show the normal branching pattern for day 11 CAMs, while vessels in the treated CAM appear aberrant. Sidestream smoke solutions produced similar effects on the CAM vasculature.
This effect on the vascular pattern was accompanied by changes in the extracellular matrix, as shown in the western blots below. The levels of both collagen I and collagen III increased dramatically in both mainstream and sidestream smoke treated CAMs. Fibronectin, another extracellular matrix molecule, increased in CAMs treated with sidestream smoke but not in CAMs treated with mainstream smoke, as shown in the western blot below.


FIGURE: A western blot probed to demonstrate collagens I and III in control CAMs and CAMs treated with either mainstream (MSW) or sidestream (SSW) smoke. The levels of both collagen I and collagen III increased in both smoke treatments.


FIGURE: A western blot probed to demonstrate fibronectin which has increased in CAMs treated with sidestream smoke (SSW), but not in CAMs treated with mainstream (MSW) smoke solution.
The increase in collagen can also be visualized in scanning electron micrographs (figure below). In control CAMs, relatively few collagen fibrils were present in the mesodermal layer, while CAMs treated with mainstream or sidestream smoke solution had an extensive tangle of collagen fibrils.

Smoke treatment also caused the loss of hyaluronan from beneath the ectoderm of day 7 day 11 CAMs (figure below). Hyaluronan is a large sugar polymer that plays important roles in embryonic development. Hyaluronan is nomally present in the extracellular matrix beneath the ectoderm in day 11 CAMs. When CAMs were treated with mainstream or sidestream smoke solution, hyaluronan was either much less abundant or completely lacking from this region, as shown in the fluorescent images. Hyaluronan has been labeled with a probe that binds specifically to it.


FIGURE: Scanning electron micrographs of sections through control and mainstream treated CAMs to demonstrate collagen fibrils. (A) Section through a control CAM. (B) Section through a CAM treated with mainstream smoke solution. Smoke treatment has increased the number of extracellular collagen fibrils (arrows).


FIGURE: CAMs labeled to show presence of hyaluronan. (A) A control CAM showing a region (red) enriched in hyaluronan beneath the ectoderm of a day 11 CAM. (C) A CAM exposed to sidestream smoke showing the hyaluronan is absent beneath the ectoderm of treated CAMs on day 11.
This study demonstrated that in day 7 to day 11 CAMs, both mainstream and sidestream smoke solutions perturbed vascular development, apparently through an effect on the extracellular matrix. The main effect observed was on the pattern of the blood vessels that formed in the CAM. This alteration in blood vessel pattern formation correlated with changes in the amounts of four extracellular matrix molecules collagen I and II, fibronectin, and hyaluronan.
Smoke solutions impair growth and vascular development of younger CAMs :

Younger CAMS (day 5- day 6), as shown below, were subsequently tested with smoke solutions as this assay is more rapid and growth and vascular development can be studied simultaneously (Melkonian et al., 2002). Growth between day 5 and day 6 after fertilization can easily be quantified by measuring CAM area, which normally increases about 20 fold during this 24 hour window.


FIGURE: A chick chorioallantoic membrane (CAM) surrounding a young chick embryo. The CAM contains numerous developing blood vessels.


FIGURE: Microscopic view of a CAM showing the blood vessels as they appear in a normal CAM 6 days after fertilization.
Mainstream and sidestream cigarette smoke solutions both inhibited growth of young (day 5- day 6) CAMs in a dose dependent manner, as shown in the figure below for mainstream smoke (Melkonian et al., 2002). Interestingly, sidestream smoke was more inhibitory than mainstream smoke in this assay.


FIGURE: CAM growth, as measured by CAM area, was significantly inhibited in a dose dependent manner by whole (MSW) or gas (MSG) phase mainstream smoke. MSW smoke was more inhibitory than the gas phase. Doses are shown as cigarette puff equivalents (PE). For MSW, 6 PE corresponds to the concentration of dissolved chemicals in smoke solution from 6 puffs of MSW smoke. Control = CAMs that were treated like all other groups except that no solutions were applied to the CAMs. EBSS is a physiological solution used to dissolve the smoke components. It serves as a control. Addition of EBSS to CAMs did not effect growth when compared to the control receiving no solutions.
Smoke treatment also affected aspects of angiogenesis in young CAMs. For example, a well-developed capillary plexus is present beneath the ectoderm of day 6 CAMs. This plexus forms in part by migration of mesodermal blood vessels to the ectoderm, where they differentiate into the capillary plexus. Both mainstream and sidestream smoke solutions inhibited plexus formation by inhibiting migration of mesodermal vessels to the ectoderm (figure below). In addition to this effect on the capillary plexus, smoke solutions also resulted in formation of blood vessels with smaller diameters than in the controls and caused the formation of abnormal blood vessels patterns and branching, as had been seen in the older CAMs treated with smoke solutions.


FIGURE: In control CAMs (A), the capillary plexus was well developed beneath the ectoderm (arrowheads). In CAMs treated with mainstream (B) or sidestream smoke (C), little capillary plexus formed; however, many mesodermal vessels (arrows) were still present, indicating these vessels had not migrated to the ectoderm to form the plexus.
IDENTIFICATION OF NOVEL TOXICANTS IN CIGARETTE SMOKE:

The preceding work demonstrated that cigarette smoke solutions impair a variety of processes including beating of oviductal cilia, contraction of oviductal muscle, pick-up of the oocyte cumulus complex by the oviduct, growth of developing tissues, and vascular development. Our next goal was to identify the toxicants in smoke that caused inhibition of these processes (Ji et al., 2002; Riveles et al., 2003). Since cigarette smoke contains over 4,000 chemicals, this was a challenging task. Our strategy was to use solid phase extraction cartridges to pull out families of chemicals from smoke. Each cartridge was eluted to remove bound chemicals, and the eluates were tested in screens using either the CAM or oviductal assay to determine which cartridges retained inhibitory activity. When high levels of inhibitory activity were recovered from a cartridge, the chemicals in that eluate were identified using gas chromatography and mass spectrometry. Purified standards of the identified compounds were then purchased and used in dose response studies to verify that a particular chemical was inhibitory and to determine its potency and efficacy in each assay. Several classes of compounds, including pyridine derivatives, were identified in inhibitory eluates in both the oviductal and CAM screen.


FIGURE: Schematic of the solid phase extraction cartridges used to identify toxicants in smoke solutions that inhibited oviductal functioning and CAM development. A smoke solution was passed through a cartridge, and bound chemicals were eluted off with methanol, dried down, resuspended in culture medium, and tested in the oviductal or CAM assays. Fractions that showed high degrees of inhibition in the oviductal or CAM assay were subjected to gas chromatography and mass spectrometry to identify the chemicals in the eluate.
Purified standards of the pyridine derivatives were purchased and evaluated in dose response experiments using both assays. The graph below shows the results obtained with 3-ethylpyridine in the CAM growth assay (Ji et al., 2002). 3-Ethylpyridine inhibited CAM growth at picomolar doses and was 10 million times more potent than the parent compound, pyridine!


FIGURE: Picomolar doses of 3-ethylpyridine significantly inhibited growth of day 5 CAMs. CN = control with no treatment. EBSS = control treated with the culture medium used to dissolve 3-ethylpyridine. The remaining bars show various doses of 3-ethypyridine that were tested. Significant inhibition was seen at doses as low as 5 x 10-12 M (yellow arrow)!


FIGURE: The chemical structure of 3-ethylpyridine, one of the chemicals in smoke that was highly inhibitory in both the CAM and oviductal assays.
The table below summarizes data obtained when growth was evaluated following treatment with 12 different pyridine derivatives using the CAM assay (Ji et al., 2002). Single ethyl or methyl substitutions to the pyridine greatly increased the toxicity of the tested compounds. 2-and 3-ethylpyridine were inhibitory in the CAM growth assay at picomolar doses (LOAEL= lowest observed adverse effect level). Similar results were obtained when these chemicals were tested in the oviductal assay (Riveles et al., 2003). Methyl and ethyl pyridines strongly inhibited ciliary beat frequency, oocyte pick-up rate, and smooth muscle cell contraction at very low doses in oviducts in vitro as shown in the figure below.


FIGURE: This table shows the LOAELs relative to pyridine, the parent compound, for each pyridine derivative tested for its effect on CAM growth. The table also shows the structure of each compound. Most pyridines have LOAELs similar to pyridine. However, single methyl and ethyl substitutions to the pyridine ring greatly increased its toxicity in this assay.


FIGURE: This figure shows the LOAEL doses of 3-ethylpyridine in the oocyte pickup rate, ciliary beat frequency, and muscle contraction assay. All assays were done using the hamster infundibulum... In each assay, the LOAEL was in the picomolar range.
3-Ethylpyridine, which was highly inhibitory in the oviductal and CAM assays, is on the FEMA GRAS list (GRAS = generally regarded as safe) and the FDA EAFUS list (everything added to food in the United States) and is added to consumer products including cigarettes, food, and cosmetics. Our data demonstrate that very low doses of this chemical can be highly inhibitory in a variety of unrelated assays that measure very different cell processes. Clearly, further toxicological testing should be done on this and related compounds.

Images below are examples of consumer products, in addition to cigarettes that contain 3-ethypyridine.

CANDY


COSMETICS


BAKED GOODS


BEER


HAMBURGERS
BOTTOM LINE:

Our studies show that the female reproductive tract and developing tissues are targeted by both mainstream and sidestream cigarette smoke and that perturbations occur in vivo in smokers in the ovary (corpus luteum), oviduct, and uterus.

In vitro, chemicals in smoke impaired oviductal functioning by altering ciliary beat frequency, inhibiting muscle contraction, increasing adhesion between the OCC and oviduct, and inhibiting oocyte pick-up rate. In the CAM assay, chemicals in smoke inhibited growth of the CAM and perturbed angiogenesis. The effect on angiogenesis appeared to be at least in part through alterations in the extracellular matrix including collagen I and collagen III, fibronectin, and hyaluronan.

Pyridine derivatives have been identified as one of the major classes of chemicals in smoke that impair oviductal functioning, CAM growth, and angiogenesis, and some pyridine derivatives were found to be inhibitory at picomolar doses. At least one of these chemicals, 3-ethylpyridine, which was toxic at picomolar doses, has previously been considered safe and is often added to consumer products, including tobacco. Clearly additional studies are needed on this compound.


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Talbot Central : Home | About PI | Staff | Research | Publications | Movies | Lab Meetings | Protocols | Congrats | Festivities | Conferences | Alumni