Smoking and reproduction: The oviduct as a target of cigarette smoke

The oviduct is an exquisitely designed organ that functions in picking-up ovulated oocytes, transporting gametes in opposite directions to the site of fertilization, providing a suitable environment for fertilization and early development, and transporting preimplantation embryos to the uterus. A variety of biological processes can be studied in oviducts making them an excellent model for toxicological studies. This review considers the role of the oviduct in oocyte pick-up and embryo transport and the evidence that chemicals in both mainstream and sidestream cigarette smoke impair these oviductal functions. Epidemiological data have repeatedly shown that women who smoke are at increased risk for a variety of reproductive problems, including ectopic pregnancy, delay to conception, and infertility. In vivo and in vitro studies indicate the oviduct is targeted by smoke components in a manner that could explain some of the epidemiological data. Comparisons between the toxicity of smoke from different types of cigarettes, including harm reduction cigarettes, are discussed, and the chemicals in smoke that impair oviductal functioning are reviewed.     

TLR4 protein contributes to cigarette smoke-induced matrix metalloproteinase-1 (MMP-1) expression in chronic obstructive pulmonary disease

Cigarette smoke is the major risk factor associated with the development of chronic obstructive pulmonary disease and alters expression of proteolytic enzymes that contribute to disease pathology. Previously, we reported that smoke exposure leads to the induction of matrix metalloproteinase-1 (MMP-1) through the activation of ERK1/2, which is critical to the development of emphysema. To date, the upstream signaling pathway by which cigarette smoke induces MMP-1 expression has been undefined. This study demonstrates that cigarette smoke mediates MMP-1 expression via activation of the TLR4 signaling cascade. In vitro cell culture studies demonstrated that cigarette smoke-induced MMP-1 was regulated by TLR4 via MyD88/IRAK1. Blockade of TLR4 or inhibition of IRAK1 prevented cigarette smoke induction of MMP-1. Mice exposed to acute levels of cigarette smoke exhibited increased TLR4 expression. To further confirm the in vivo relevance of this signaling pathway, rabbits exposed to acute cigarette smoke were found to have elevated TLR4 signaling and subsequent MMP-1 expression. Additionally, lungs from smokers exhibited elevated TLR4 and MMP-1 levels. Therefore, our data indicate that TLR4 signaling, through MyD88 and IRAK1, plays a predominant role in MMP-1 induction by cigarette smoke. The identification of the TLR4 pathway as a regulator of smoke-induced protease production presents a series of novel targets for future therapy in chronic obstructive pulmonary disease.     

Human urine mutagenicity study comparing cigarettes which burn or only heat tobacco

Cigarette smokers have been reported to void urine which is more mutagenic, as measured in the Ames bacterial mutation assay, than urine voided by non-smokers. Condensate from the mainstream smoke of a cigarette which heats, but does not burn tobacco (test cigarette) showed no evidence of mutagenicity in a battery of in vitro genotoxicity assays under conditions in which condensate from the mainstream smoke of cigarettes that burn tobacco was mutagenic. The objective of this study was to determine whether the absence of mutagenic activity observed in the in vitro assays would be reflected in the urine of smokers of the test cigarette. 72 subjects (31 smokers and 41 non-smokers) were enrolled in a 6-week study, with the smokers randomly divided into 2 groups. The study was designed as a double crossover, with each smoker smoking both test (tobacco-heating) and reference (tobacco-burning) cigarettes. This design allowed each smoker to serve as his or her own control while at the same time allowing comparisons between groups of non-smokers and smokers of both test and reference cigarettes. 24-h urine samples were collected twice a week and concentrated using XAD-2 resin. Urine concentrates were tested in Ames bacterial strains TA98 and TA100, with and without metabolic activation and with and without beta-glucuronidase/aryl sulfatase. Individuals who smoked the test cigarette voided urine which was significantly less mutagenic than that voided when they smoked reference cigarettes. The mutagenicity of urine from smokers who smoked the test cigarette and non-smokers did not differ under any of the assay conditions used in this study.     

Revealing the hidden mechanisms of smoke-induced fallopian tubal implantation

Ectopic pregnancy (EP) is an enigmatic reproductive disorder. Although tubal EP is difficult to predict, several hypotheses about its etiology have been proposed. In retrospective case-control studies, smoking is associated with an increased rate of EPs in the fallopian tube. Studies of experimental animals in vivo and human fallopian tubal tissues in vitro have suggested mechanisms of fallopian tubal damage and dysfunction induced by nicotine and other smoking-related chemicals that may explain this association. However, the pathogenesis of smoking-induced modulation of implantation leading to tubal EP is largely unknown. Because cigarette/tobacco smoke adversely affects the success of intrauterine implantation, there is a great need to determine how embryo implantation occurs in the fallopian tube in female smokers of reproductive age.     

The effect of cigarette smoke on fertilization and pre-implantation development: assessment using animal models, clinical data, and stem cells

Numerous studies have repeatedly shown that women who smoke experience problems establishing and maintaining pregnancies, and recent work has further demonstrated that the in utero effects of smoke may not be manifested until months or even years after birth. The purpose of this review is to examine the recent literature dealing with the effects of cigarette smoke on the earliest stages of human prenatal development. Studies in this area have included the use of animal models, patients undergoing in vitro fertilization, and embryonic stem cell models. Events leading to fertilization, such as cumulus expansion, hyperactivation of sperm motility, and oocyte pick-up by the oviduct are all impaired by smoke exposure in animal models. Steps crucial to fertilization such as the acrosome reaction and sperm binding to the zona pellucida are likewise inhibited by cigarette smoke. Preimplantation embryos and stem cells that model embryos show a number of adverse responses to smoke exposure, including poor adhesion to extracellular matrices, diminished survival and proliferation, and increased apoptosis. The current literature demonstrates that the earliest stages of prenatal development are sensitive to smoke exposure and indicates that pregnant women should be advised not to smoke during this time.     

ADAMTS proteases in fertility

The reproductive organs are unique among adult organs in that they must undergo continual tissue remodelling as a key aspect of their normal function. The processes for persistent maturation and release of new gametes, as well as fertilisation, implantation, placentation, gestation and parturition involve cyclic development and regression of tissues that must continually regenerate to support fertility. The ADAMTS family of proteases has been shown to contribute to many aspects of the tissue morphogenesis required for development and function of each of the reproductive organs. Dysregulation or functional changes in ADAMTS family proteases have been associated with reproductive disorders such as polycystic ovarian syndrome (PCOS) and premature ovarian failure (POF). Likewise, proteolytic substrates of ADAMTS enzymes have also been linked to reproductive function. New insight into the roles of ADAMTS proteases has yielded a deeper understanding of the molecular mechanisms behind fertility with clinical potential to generate therapeutic targets to resolve infertility, develop biomarkers that predict dysfunction of the reproductive organs and potentially offer targets for development of non-hormonal male and female contraceptives.     

Cigarette smoke increases progesterone receptor and homeobox A10 expression in human endometrium and endometrial cells: a potential role in the decreased prevalence of endometrial pathology in smokers

Cigarette smoking has long been tied to a multitude of poor health outcomes; however, in reproductive biology, smoking has shown several unintuitive findings. Smoking is associated with significantly decreased rates of endometriosis and endometrial cancer. Here, we show that treatment with cigarette smoke extract leads to increased mRNA and protein expression of homeobox A10 (HOXA10) and progesterone receptor (PGR) as well as more rapid decidualization of endometrial stromal cells in vitro. In vivo, mice exposed to cigarette smoke similarly showed increased expression of HOXA10 and PGR in the endometrium. Both HOXA10 and PGR drive endometrial differentiation and are suppressed in endometrial tumors and in endometriosis. The increased expression found upon exposure to cigarette smoke may provide a protective effect, mediating the decreased incidence of endometrial disease among smokers. This mechanism contrasts with the accepted paradigm that the effects of smoking on the uterus are secondary to ovarian alterations rather than direct effects on endometrium as demonstrated here.     

The effect of exposure to nicotine, carbon monoxide, cigarette smoke or cigarette smoke condensate on the mutagenicity of rat urine

Cigarette smokers have been reported to void urine which is more mutagenic than that voided by non-smokers, but the specific urinary mutagen(s) have not been identified. Since mechanistic studies are best performed in animal models, the objective of this study was to determine if a model to study the role of cigarette smoke and its components in urinary mutagenicity could be developed in rats. XAD-2 resin was used to concentrate the urine and the microsuspension modification of the Ames test used to quantify mutagenicity. Nicotine administered by intraperitoneal injection at 0.8 mg/kg (the maximum tolerated dose) or inhalation of carbon monoxide for 14 days at the maximum tolerated dose (1800 ppm, resulting in 68% carboxyhemoglobin) did not increase urinary mutagenicity. Cigarette smoke condensate (CSC) prepared by electrostatic precipitation of mainstream smoke increased urinary mutagenicity at doses of 100 and 200 mg/kg when administered acutely by either i.p. injection or gavage, verifying that the assay system was capable of detecting cigarette smoke-related mutagens in the urine. However, cigarette smoke administered by the appropriate route of exposure, nose-only inhalation, for 1, 7, 14 or 90 days (1 h per day) did not increase urinary mutagenicity. The smoke concentration administered was at or near the maximum tolerated dose as evidenced by carboxyhemoglobin concentrations of approximately 50%, and of 10% or more weight loss in exposed animals. Thus, although cigarette smoke condensate is mutagenic in vitro and mutagenic urine was observed when rats were given high doses of CSC by inappropriate routes of administration, acute or subchronic inhalation exposure to the maximum tolerated dose of whole cigarette smoke did not increase urinary mutagenicity in rats. These results indicate that the rat may be an inappropriate model to study urinary mutagenicity following the inhalation of tobacco smoke.     

Human bronchial epithelial cell transcriptome: gene expression changes following acute exposure to whole cigarette smoke in vitro

Cigarette smoke is a complex mixture of more than 4,000 constituents. Its effects on cell biology are poorly understood, partly because whole smoke exposure in vitro is technically challenging. To investigate the effects of smoke on cell signaling and function, a three-dimensional air-liquid interface model of tracheobronchial epithelium, grown from primary human lung epithelial cells, was exposed to air or whole mainstream cigarette smoke for 1 h in a purpose-designed chamber. Gene expression profiles were then determined at 1, 6, and 24 h postexposure using Affymetrix HGU133-2 Plus microarrays. Cells from three different donors were used in the study, and the experiment was performed in triplicate for each donor. Genes significantly regulated by smoke, compared with the air control, in all experiments were determined. Genes exhibiting differential expression were assigned to functional categories and mapped to signaling pathways. Effects were observed on many cellular processes including xenobiotic metabolism, oxidant/antioxidant balance, and DNA damage and repair. Notably, there was marked downregulation of the transforming growth factor-beta pathway, which has not been previously reported. This study provides important data on the acute effects of whole cigarette smoke on mucociliary epithelium and may be used to gain a greater understanding of smoke toxicity.     

The cytotoxic effect of volatile organic compounds of the gas phase of cigarette smoke on lung epithelial cells

Health effects of cigarette smoke (CS) in humans are well known from both clinical and epidemiological studies. However, the mechanism behind CS toxicity and carcinogenicity remains mainly unknown. Recent studies have pointed to the major importance of the gas phase of CS in generating its cytotoxic effects. In the current study, an exposure system capable of introducing the gas phase of mainstream cigarette smoke deprived of its volatile organic constituents (VOCs) was used to study the role of the nonorganic components of the gas phase on the cytotoxicity of smoke to monolayer cultures of mouse lung epithelial cells. Cell viability was measured by Wst-1 and the lactate dehydrogenase (LDH) assays. In cells treated with increasing doses of mainstream cigarette smoke gas phase (one to nine puffs), a dose-dependent increase in cytotoxicity was observed (one puff, 95% viability; nine puffs, 40% viability). Cell viability of cultures exposed to gas phase with only the nonorganic components was found to be equivalent to control, unexposed cultures, indicating that removal of VOCs resulted in almost eliminating the cytotoxic ability of the gas phase of CS. Furthermore, the removal of VOCs seems to reduce the effects of protein tyrosine nitration mediated through the gas phase constituents. The results obtained suggest the important and decisive role of VOCs in inducing cytotoxic effects.     

Gene transfer to the mammalian reproductive tract

This review summarizes the results of research on gene transfer to the mammalian genital tract. Gene transfer experiments have been developed during the last 2 decades and have been applied using in vitro, ex vivo and in vivo procedures. (i) In vitro methods have been applied to the uterine epithelial cells with the principal purpose of analysing some pathological change occurring in the uterus. In the male tract, epididymal cell lines have been used to evaluate the expression of particular genes and the function of specific proteins. (ii) Ex vivo methods have been applied to both the uterus and the vas deferens in humans, and good transgene expression has been recorded. (iii) In vivo gene transfer in the female tract has been employed in the uterus and oviduct using gene injections or electroporation methods. The glandular epithelium of both organs can be transfected efficiently, and transfection efficiency depends on the hormonal stage of the animal. The best expression occurred during pseudopregnancy and meta-estrus periods, when high progesterone and low estradiol concentrations occur. In the male tract, in vivo methods have been applied to mouse vas deferens and epididymis. In both organs, patches of epithelial regions appeared to express the transgenes. Furthermore, the secretions of both organs were also modified using gene constructions that led to the expression of some secretory proteins. In summary, gene modifications in the epithelium of the mammalian reproductive tract have been successful employing different technologies. Further improvements in transfection efficiency would help provide new insights into the physiology of these reproductive organs. Furthermore, the use of these methods could also be used to modify the fertility of mammals.     

A Comparison of the Inflammatory and Proteolytic Effects of Dung Biomass and Cigarette Smoke Exposure in the Lung

Rationale

Biomass is the energy source for cooking and heating for billions of people worldwide. Despite their prevalent use and their potential impact on global health, the effects of these fuels on lung biology and function remain poorly understood.

Methods

We exposed human small airway epithelial cells and C57BL/6 mice to dung biomass smoke or cigarette smoke to compare how these exposures impacted lung signaling and inflammatory and proteolytic responses that have been linked with disease pathogenesis.

Results

The in vitro exposure and siRNA studies demonstrated that biomass and cigarette smoke activated ERK to up regulate IL-8 and MMP-1 expression in human airway epithelial cells. In contrast to cigarette smoke, biomass also activated p38 and JNK within these lung cells and lowered the expression of tissue inhibitor of matrix metalloproteinase-1 (TIMP-1). Similarly, in the lungs of mice, both biomass and cigarette smoke exposure increased macrophages, activated ERK and p38 and up regulated MMP-9 and MMP-12 expression. The main differences seen in the exposure studies was that mice exposed to biomass exhibited more perivascular inflammation and had higher G-CSF and GM-CSF lavage fluid levels than mice exposed identically to cigarette smoke.

Conclusion

Biomass activates similar pathogenic processes seen in cigarette smoke exposure that are known to result in the disruption of lung structure. These findings provide biological evidence that public health interventions are needed to address the harm associated with the use of this fuel source.     

Pyrazine derivatives in cigarette smoke inhibit hamster oviductal functioning

Background  

Our past studies have shown that cigarette smoke inhibits oviductal functioning in vivo and in vitro. The goals in this study were to identify pyrazine derivatives in cigarette smoke solutions that inhibit ciliary beat frequency, oocyte pickup rate, and infundibular smooth muscle contraction in the hamster oviduct and to determine their lowest observable adverse effect levels (LOAELs) using in vitro bioassays.     

Genotoxicity of tobacco smoke and tobacco smoke condensate: a review

This report reviews the literature on the genotoxicity of mainstream tobacco smoke and cigarette smoke condensate (CSC) published since 1985. CSC is genotoxic in nearly all systems in which it has been tested, with the base/neutral fractions being the most mutagenic. In rodents, cigarette smoke induces sister chromatid exchanges (SCEs) and micronuclei in bone marrow and lung cells. In humans, newborns of smoking mothers have elevated frequencies of HPRT mutants, translocations, and DNA strand breaks. Sperm of smokers have elevated frequencies of aneuploidy, DNA adducts, strand breaks, and oxidative damage. Smoking also produces mutagenic cervical mucus, micronuclei in cervical epithelial cells, and genotoxic amniotic fluid. These data suggest that tobacco smoke may be a human germ-cell mutagen. Tobacco smoke produces mutagenic urine, and it is a human somatic-cell mutagen, producing HPRT mutations, SCEs, microsatellite instability, and DNA damage in a variety of tissues. Of the 11 organ sites at which smoking causes cancer in humans, smoking-associated genotoxic effects have been found in all eight that have been examined thus far: oral/nasal, esophagus, pharynx/larynx, lung, pancreas, myeoloid organs, bladder/ureter, uterine cervix. Lung tumors of smokers contain a high frequency and unique spectrum of TP53 and KRAS mutations, reflective of the PAH (and possibly other) compounds in the smoke. Further studies are needed to clarify the modulation of the genotoxicity of tobacco smoke by various genetic polymorphisms. These data support a model of tobacco smoke carcinogenesis in which the components of tobacco smoke induce mutations that accumulate in a field of tissue that, through selection, drive the carcinogenic process. Most of the data reviewed here are from studies of human smokers. Thus, their relevance to humans cannot be denied, and their explanatory powers not easily dismissed. Tobacco smoke is now the most extreme example of a systemic human mutagen.     

Potential applications of electrical impedance techniques in female mammalian reproduction

The measurement of electrical impedance is one of many methods that can be used for monitoring various reproductive events in female mammals. This paper summarizes the key findings in this research area that have been achieved during the past four decades. The electrical impedance in the vagina, vaginal vestibule and vulva during the estrous/menstrual cycle shows significant changes that vary among these reproductive organs, different locations in these organs and mammalian species. The changes of vaginal and vulvar impedance during periestrus are temporarily associated with the preovulatory luteinizing hormone (LH) peak and are significantly correlated with systemic levels of estradiol and progesterone. In humans, significant impedance changes during the menstrual cycle and their close association with the LH peak and ovulation have been found not only in the vagina, but also in the tongue. Findings of a number of studies suggest the possibility of using vaginal, vestibular and vulvar impedance to predict and confirm the fertile period of the estrous/menstrual cycle. There is some evidence that vulvar and cervical impedance may be a reliable indicator of impending parturition. Results in several studies also indicate the possibility of using electrical impedance methods to confirm the existence of ovarian follicular cysts, endometriosis and cervical neoplasia. However, physical and biological factors that may affect the impedance variation in the female reproductive system and tongue are still poorly understood. Their detection allows us to improve our ability to predict more precisely various reproductive processes by impedance techniques. This review also provides some considerations about the origin of impedance changes in the female reproductive system and tongue that may be useful for the systematic development of impedance methods in the field of female mammalian reproduction.     

Functional characterization of the matrix metalloproteinase-1 cigarette smoke-responsive region and association with the lung health study

Background

Prior studies have demonstrated that the distal 1.5 kb of the MMP-1 promoter is fundamental in directing the induction of the MMP-1 gene by cigarette smoke.

Methods

To characterize the genetic variants in the MMP-1 cigarette smoke-responsive element, deep re-sequencing of this element was performed on DNA samples from participants in the Lung Health Study. Furthermore, evidence of Sp1 binding to the MMP-1 promoter was assessed using chromatin immunoprecipitation assays and the influence of cigarette smoke exposure on this interaction was evaluated in cultured human small airway epithelial cells.

Results

Ten polymorphisms (four novel) were detected in the cigarette smoke-responsive element. Chromatin immunoprecipitation assays to assess the protein-DNA interactions at Sp1 sites in the MMP-1 promoter showed increased binding to the Sp1 sites in the cigarette smoke-responsive element in small airway epithelial cells treated with cigarette smoke extract. In contrast, a Sp1 site outside of the element exhibited the opposite effect. None of the polymorphisms were more prevalent in the fast decliners versus the slow decliners (fast decliners = mean −4.14% decline in FEV1% predicted per year vs. decline in FEV1% predicted per year).

Conclusions

Sequencing analyses identified four novel polymorphisms within the cigarette smoke-responsive element of the MMP-1 promoter. This study identifies functional activity within the cigarette smoke-responsive element that is influenced by cigarette smoke and examines this region of the promoter within a small patient population.     

A comparison of the free radical chemistry of tobacco-burning cigarettes and cigarettes that only heat tobacco

Cigarette smoke contains free radicals both in the particulate matter (tar) and in vapor-phase smoke. Vapor-phase smoke decreases the activity of alpha-1-proteinase inhibitor (alpha 1PI) in vitro. The free radical content of the tar and vapor-phase smoke from a cigarette that heats rather than burns tobacco has been compared with data on a standard 1R4F cigarette. No radicals were detected in the tar from the new cigarette and radicals in its vapor-phase smoke are lower by more than 99% relative to the 1R4F standard cigarettes. The vapor-phase smoke from the new cigarette causes essentially no reduction of alpha 1PI activity in vitro. These findings support our previously published mechanisms for the production of radicals in tar and in vapor-phase smoke.     

Polynitroxylated hemoglobin-based oxygen carrier: inhibition of free radical-induced microcirculatory dysfunction.

Reactive oxygen species have been identified as key mediators of leukocyte/endothelial cell interaction under various pathological conditions and diseases such as ischemia/reperfusion injury, inflammation, and after exposure to cigarette smoke. Consequently, antioxidants have been shown to successfully prevent the sequelae of these conditions, ranging from tissue infarction to atherogenesis. In this study we investigated whether, via its established superoxide dismutase-like activity, a novel polynitroxyl hemoglobin-based oxygen carrier (PNH), could affect the stimulation of leukocyte rolling and adhesion to endothelial cells in response to cigarette smoke. Using the dorsal skin fold chamber model for intravital microscopic observation of leukocyte/endothelium and -/platelet interactions in hamsters, we could demonstrate that cigarette smoke exposure elicited in control animals the rolling and adhesion of leukocytes along the endothelium of postcapillary venules and also of arterioles, as well as the formation of leukocyte/platelet aggregates. In contrast to the hemoglobin based oxygen carrier (HBOC) alone, that showed no therapeutic benefit, PNH significantly inhibited these proadhesive processes secondary to cigarette smoke. Also, PNH significantly reduced the formation of leukocyte/platelet aggregates in the blood stream of the cigarette smoke-exposed animals. These effects are not due to changes in microhemodynamic conditions, because wall shear rates remained unchanged in all three groups of animals.