Disruption of normal embryonic angiogenesis by direct exposure of mainstream whole smoke solutions of commercial cigarettes

Environmental Toxicology and Pharmacology 20 (2005) 188–198

Disruption of normal embryonic angiogenesis by direct exposure of mainstream whole smoke solutions of commercial cigarettes Sohail Ejaza , Kim Bum Seokb , Lim Chae Woongc,∗ a

Biosafety Research Institute, Chonbuk National University, Jeonju, 561-756, South Korea Department of Pathobiology, College of Veterinary Medicine, University of Tennessee, USA Department of Pathology, College of Veterinary Medicine, Chonbuk National University, Jeonju, 561-756, South Korea b

c

Received 10 October 2004; accepted 13 December 2004 Available online 8 February 2005

Abstract Angiogenesis is activated in the female reproductive system during embryogenesis and embryo implantation. Smoking during pregnancy has been linked to interfere with normal process of angiogenesis resulting an increased incidence of ectopic pregnancy, spontaneous abortion, preterm delivery and sudden infant death syndrome. Chorioallantoic membrane (CAM) assay was used as an alternative in vivo approach to evaluate the toxicological effects of different mainstream whole smoke solutions (MSWSS) of commercial cigarettes on embryonic angiogenesis. Seventy 5-day-old CAMs, divided in seven groups were exposed to MSWSS with different nicotine concentration: 0.2 mg (group B), 0.3 mg (group C), 0.5 mg (group D), 0.6 mg (group E), 0.7 mg (group F) and 1 mg (group G). All smoke solutions caused varying levels of disruption on the normal process of angiogenesis and have shown to adversely affect the diameters of blood vessels, capillary plexus formation and organization of the fibrillar materials of CAMs. Abbot curve, angular spectrum and 3D surface roughness of CAMs were also measured for precise quantification of angiogenesis. Moderate to dramatic changes were observed in all treated groups with a very highly significant (P < 0.001) disruption observed on CAMs of group G. No significant change was observed in different groups treated with pure nicotine. Current observations demonstrated that MSWSS of different commercial cigarettes have toxic effects on the process of angiogenesis and smoking during pregnancy may lead to an increased risk of spontaneous abortion and preterm delivery. © 2005 Elsevier B.V. All rights reserved. Keywords: Angiogenesis; MSWSS; Nicotine; CAM; Cigarette

1. Introduction Angiogenesis is the sprouting of new blood vessels from pre-existing capillaries and requires the multiplication of endothelial cells, their migration, remodeling of the extracellular matrix, tube formation and recruitment of surrounding structures to maintain the newly formed blood vessels (Ramsden, 2000). Angiogenesis is activated in the female reproductive system during ovulation, corpus luteum formation, embryogenesis, and embryo implantation (Folkman, 1995). Vascularization of the human embryo takes place very early in pregnancy (second week post conception) and is ini∗

Corresponding author. Tel.: +82 63 270 3788; fax: +82 63 270 3780. E-mail address: [email protected] (L.C. Woong).

1382-6689/$ – see front matter © 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.etap.2004.12.058

tiated in the extraembryonic areas. Angiogenesis is vital for embryonic development, and disruption of this process can be a powerful mechanism of abortion or teratogenesis (Meegdes et al., 1988). Cigarette smoking has a detrimental impact on smokers with respect to health issues such as addiction, lung cancers, and cardiovascular problems (Bartal, 2001). One of the most unfortunate facts about smoking is the double jeopardy of maternal smoking during pregnancy because it affects not only the health of the mother, but also the developing fetus. Currently, the spectrum of the damaging effects of smoking prior to or during pregnancy includes decreased success rate of becoming pregnant or carrying a baby to term (Economides and Braithwaite, 1994), an increased number of births by cesarean section and decrease of postpartum breastfeeding

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due in part to a lack of milk production (Habek et al., 2002; Leston et al., 2002). Moreover, smoking during pregnancy has been linked to an increased incidence of sudden infant death syndrome (DiFranza and Lew, 1995), a reduction in birthweight (Ernst et al., 2001), and disruptions in the functions of the central nervous system (Xu et al., 2001) on the affected offspring. Studies on animals have suggested that tobacco compounds can affect fertility from gametogenesis to implantation (Mattison, 1982). The explanatory mechanisms for the effect of smoking on future reproductive potential are uncertain and controversial. Women who were exposed to smoking in intrauterine but not in adult life presented a reduction in fertility in comparison to those who had no direct exposure to tobacco (Jensen et al., 1998). On the other hand, studies have also indicated that smoking reduces fertility only during exposure; when women quit smoking, their fertility is restored to normal (Bolumar et al., 1996). Smoking during gestation causes serious, well-known effects on intrauterine growth of babies. There is a greater risk for prematurity and low birthweight in pregnant women who smoke during the third trimester; this risk increases proportionally to the number of cigarettes smoked (Mainous and Heuston, 1994). Women who smoked during the second and/or third trimester presented the same risk than those who smoked from beginning to end of pregnancy. Thus, it is possible that smoking has a greater effect on decreasing fetal growth during the third trimester (Kirkland et al., 2000) by reduction in uterine and placental perfusion and a consequent poor fetal oxygenation and nutrition. Therefore, it is undisputed that smoking can interfere the normal process of angiogenesis, which is a vital process to maintain pregnancy and development of the fetus. In order to evaluate the toxic effects of smoking on health, series of research grade cigarettes has been developed jointly by the US National Cancer Institute, the Agriculture Research Service of the US Department of Agriculture, and the University of Kentucky Tobacco and Health Research Institute (Lexington, Kentucky) to serve as reference cigarettes for experimental purposes (Steele et al., 1995). The intention in developing these cigarettes was to provide standard reference cigarettes for use in comparative chemical and biological studies of cigarette smoke. Since, different tobacco blends, ingredients, cigarette design, papers, filters, flavoring ingredients and methods of manufacturing of cigarettes influence the efficiency of combustion of cigarettes and thus modify the quantity and/or the quality of delivering different toxic chemical compounds in smoke (Chepiga et al., 2000; Lodovici et al., 2004; Seeman et al., 2003; Wayne and Connolly, 2002). A great deal of information has already been published regarding toxicological evaluation of research grade cigarettes but no study has attempted to concurrently evaluate the toxicological effect of different commercially available cigarettes on angiogenesis. The present study was therefore undertaken to screen the effects of mainstream whole smoke solution (MSWSS) of

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different brands of commercially available cigarettes on embryonic angiogenesis. The chorioallantoic membrane (CAM) of the developing chick embryo was selected as a complimentary in vivo assay system. The CAM is a densely vascularized and rapidly growing extra-embryonic membrane that has been used for many years to investigate the effect of a variety of substances on the formation of new blood vessels (Folkman, 1974) and to study the mechanisms of tumor cell invasion (Kim et al., 1998). CAM assay allows continuous visualization of the implant area while providing a rapid, simple and low-cost screening of tissue reactions to different toxic materials. Since mammalian placenta is an evolutionary homolog of the chick chorioallantoic membrane and CAM–yolk interactions are analogous to that of the placenta and maternal blood (Eckstein et al., 1997), CAM assay can be a good alternative in vivo approach to evaluate the toxicological effects of different MSWSS of commercially available cigarettes on embryonic angiogenesis.

2. Materials and methods 2.1. Preparation of chorioallantoic membranes Fertilized eggs of the commercial white Leghorn, obtained form local hatchery (Harim, Iksan), were incubated after cleaning at 37 ◦ C and 85–90% relative humidity throughout the experiment. On day 3 of incubation, 2.5 ml of albumin was aspirated through a hole made with a sterile 21G cannula, to allow the CAM in a way accessible to treatments. The hole was sealed with sterile parafilm tape (American National Can). At day 4, a square window (1.5 cm2 ) was opened in the shell. The shell membrane was then sealed along the longitudinal axis at the apex pole by using sterile parafilm tape. The eggs were then incubated until day 5, when test materials were placed on the developing CAMs and the response of different tested materials on angiogenesis was quantified after 24 h. We have used CAMs at day 5 of incubation, as extensive capillary formation takes place during 5–6 days (Melkonian et al., 2002b). Hence the effect of different tested materials on angiogenesis and capillary plexus formation can be evaluated during this period, as well. 2.2. Preparation and administration of the solutions Smoke solutions were prepared from six different brands of commercially available cigarettes (KT & G, Korea) by using an analytical smoking device (Knoll and Talbot, 1998) containing 10 mL of distilled water (DW). MSWSS were prepared by passing the puffs of mainstream smoke through DW (Knoll et al., 1995) and pH was adjusted to 7.4. Nicotine levels of different MSWSS (Table 1) were evaluated as described previously (Tyrpien et al., 2003). In the similar way, six nicotine solutions, respective to nicotine concentration evaluated in each commercial cigarette, were prepared by serially diluting nicotine (Sigma) in DW. All solutions as well as control

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Table 1 Experimental groups with respective treatments

medium (DW) applied to the CAMs were passed through 0.22 ␮m Acrodisc filter (Whatman) and handled using sterile technique. On day 5 of incubation, 200 ␮l of all solutions were applied to CAMs immediately after preparation.

CAMs were prepared by fixing them with 2% glutaraldehyde solution, both on top and underneath the CAM, and then processed for scanning electron microscope (SEM) as described previously (Magers et al., 1995). Briefly, CAMs were cut out of the eggs, washed in cacodylate buffer, and postfixed in 1% osmium tetroxide for 30–60 min. Samples were washed in deionized water, 3–5 min each, followed by dehydration in an ascending ethanol series from 30 to 100%. To ensure complete removal of water from the tissue, five changes of 100% ethanol were performed. CAM samples were critically point dried, mounted on aluminum pedestals, and further processed by using standard electron microscopy techniques. Amount of the fibrillar material and capillary plexus were carefully screened to quantify in-depth changes in CAMs by application of MSWSS.

2.3. Evaluation of angiogenesis

2.5. Statistics

To ensure an objective 3D measurement of angiogenesis on CAMs, serial images with respective x, y and z dimensions were recorded on day 6 of incubation, by using DSC-F717 CyberShot (Sony-Japan) camera at 30 frames/s with shutter speed of 1/2000 s. In order to improve image contrast, a 30gauge needle was used to inject skim milk (10% wt/vol in water) into the CAMs. After image acquisition, all images were imported to scan probing image processing software (IBM-Denmark) that works on specific algorithm (Garnaes et al., 1998; Jørgensen et al., 1998). Respective x, y and z dimensions of each image were loaded to quantify different parameters of angiogenesis. In order to evaluate the interim change in angiogenesis, the diameter of secondary and tertiary blood vessels was measured by using calibration and measurement command. Abbot curve (graphical presentation of the height of blood vessels on CAM), angular spectrum (graphical presentation of the angular distribution of blood vessels on CAM) and 3D surface roughness of CAMs were also measured for precise quantification of angiogenesis on the surface of CAMs. Thus, blood vessels of micrometer and/or nanometer scale were also evaluated for holistic quantification of angiogenesis. After imaging, CAMs were carefully cut, fixed in 10% formaldehyde solution and processed for histological examination. The tissues were then embedded in paraffin wax, sectioned at 3 ␮m thickness, mounted on slides and stained with haematoxylin-eosin (H&E) for routine light microscopy. The slides were investigated for subtle changes in CAMs matrix and capillary plexus formation. Histological sections were digitized with a spot camera and numbers of capillary plexus that had formed immediately beneath the ectoderm were calculated.

One-way analysis of variance (ANOVA) was performed to evaluate different parameters between control and treated samples; statistical significance was set at P < 0.05. Post hoc Student’s t-test was also performed when significance was found P < 0.05 (Bradley and Sebelski, 2000).

Group

Number of samples

Nicotine (mg)

A (control) B (MSWSS 1) C (MSWSS 2) D (MSWSS 3) E (MSWSS 4) F (MSWSS 5) G (MSWSS 6)

10 10 10 10 10 10 10

NA 0.2 0.3 0.5 0.6 0.7 1

3. Results 3.1. Evaluation of angiogenesis Progressive decrease in the diameter of secondary blood vessels was observed in all MSWSS treated groups and the decline was relevant to the increase in amount of nicotine in that solution. A significant decrease (P < 0.05) in diameter of secondary blood vessels was observed in group D, while highly significant difference (P < 0.01) was recorded in groups E and F. Excessive decrease (P < 0.001) in the diameter of secondary blood vessels was observed in group G (Fig. 1). A non-significant decrease in the diameter of secondary blood vessels, with fluctuation, was observed in nicotine treated groups. For more accuracy, diameter of the tertiary blood vessels of all groups was also calculated. A significant decrease (P < 0.05) in diameter of the tertiary blood vessels

2.4. Scanning electron microscopy Topographical and ultrastructural evaluation of CAMs reactions to different MSWSS was performed using scanning electron microscope (Jeol, JSM-5900). The samples from

Fig. 1. Graphical outline representing comparative measurements of the diameter of secondary blood vessels. Note the highly significant difference among groups E, F and G.

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Fig. 2. Graphical outline showing similar highly significant changes in the diameter of tertiary blood vessels. Note the highly significant difference among groups E, F and G.

was observed in group D, while highly significant reduction (P < 0.01) was recorded in groups E and F. A very highly significant difference (P < 0.001) was observed in diameter of the tertiary blood vessels of group G (Fig. 2). It imitates that diameters of the secondary and tertiary blood vessels are interdependent and decrease in diameter of the secondary blood vessels may contribute in decreasing the diameter of the tertiary blood vessels. A trivial decline in diameter of the tertiary blood vessels, with fluctuation, was observed in all groups treated with different concentration of nicotine. Abbot curve, angular spectrum and 3D surface roughness were also calculated for holistic quantification of an-

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giogenesis. The maximum height of abbott curve observed on normal CAM was 22 ␮m with maximum bearing surface (Fig. 3A). Considerable changes in the height of abbot curve were recorded on CAMs of groups E, F, and G with abbott curve values of 20, 18 and 10 ␮m, respectively (Fig. 3B–D). It imitates a substantial decline in the height of blood vessels on CAMs of groups E, F, and G. Angular distribution of the blood vessels on CAMs was also quantified by evaluating angular spectrum. It was observed that blood vessels on CAM of control egg were uniformly distributed and covered the maximum area ranged from 12,000–16,000 points (Fig. 4A). An impede in angular distribution was observed by application of MSWSS in groups E, F, and G. Different values of angular distribution observed in groups E, F, and G were 400, <300 and <200 points, respectively (Fig. 4B–D). Different other imperative parameters of 3D surface roughness were also quantified including Sa, average roughness of surface, which was significantly higher in control group; Sq, a dispersion parameter defined as the root mean square value of the surface within the sampling area. Sq is a very general and widely used parameter for evaluating surface roughness. Sq values of normal CAM were 3457 ± 205 nm, while it was 3072 ± 312, 2944 ± 842, 2534 ± 358, 1274 ± 214, 424 ± 79 and 2.74 ± 0.32 nm in groups B, C, D, E, F and G, respectively (Table 2). Sa and

Fig. 3. Graphical outline of abbott curve measurements on CAMs of different groups. Note the impediment in height among different treated groups. (A) Control: 22 ␮m, (B) MSWSS 4: 20 ␮m, (C) MSWSS 5: 18 ␮m, (D) MSWSS 6: 10 ␮m.

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Fig. 4. Graphical representation of angular spectrum of blood vessels on CAMs of different groups. Note the impediment in angular spread and distribution of blood vessels. (A) Control: 12,000–16,000 points, (B) MSWSS 4: 400 points, (C) MSWSS 5: <300 points, (D) MSWSS 6: <200 points.

Sq values reveal a significantly higher neovascularization on CAM of control egg. Sy, a parameter that quantify the number of lowest valleys on the bearing surface and Sz, an extreme parameter used to evaluate the average value of the absolute heights of the five highest peaks and the depths of the five deepest pits or valleys within the sampling area. Sy and Sz values for normal CAMs were significantly higher than treated CAMs. Ssk is the measure of asymmetry of surface deviations about the mean plane. This parameter can effectively be used to describe the shape of the topography and height distribution. For a Gaussian surface, which has a symmetrical shape for the surface height distribution, the skewness is zero. For an asymmetric distribution of surface heights, the skewness may be negative if the distribution has a longer tail at the lower side of the mean plane or positive if the distribution

has a longer tail at the upper side of the mean plane. This parameter can give some indication of the existence of “spiky” features (Anselme et al., 2000). The results demonstrate that surface height of the blood vessels on CAMs of control egg was having more symmetry than treated CAMs. Sci is the ratio of the void volume of the unit sampling area at the core zone over the root mean square deviation. This vital parameter of 3D surface roughness is used for determination of fluid retention in that surface (Rudzitis et al., 1998) and larger Sci value designates good fluid retention. A marked decrease in Sci values of all treated CAMs was observed, while higher Sci value of control CAM supports ample fluid retention in blood vessels due to better neovascularization (Table 2). Histological evaluation of CAMs revealed that normal CAM was approximately 200 ␮m thick and composed of a

Table 2 Different parameters of 3D surface roughness of CAMs in different groups Parameters

Control

Sa (nm) Sq (nm) Sy (nm) Sz (nm) Ssk Sci

2692 3457 22503 17453 0.00968 1.91

Group B ± ± ± ± ± ±

115 205 850 1102 0.0002 0.04

2373 3072 19332 15993 0.0195 1.85

± ± ± ± ± ±

Group C 253 312 495 842 0.002 0.03

22435 2944 17432 13458 0.0298 1.71

± ± ± ± ± ±

Group D 352 842 445 945 0.002 0.04

18312 2534 15322 11235 0.32 1.63

± ± ± ± ± ±

Group E 626 358 421 256 0.03 0.03

9312 1274 7322 6512 0.39 1.45

± ± ± ± ± ±

415 214 812 317 0.02 0.04

Group F 2692 424 2444 2170 0.56 1.38

± ± ± ± ± ±

318 79 315 359 0.04 0.03

Group G 2.07 2.74 20.0 15.1 0.67 1.32

± ± ± ± ± ±

0.08 0.32 0.8 1.1 0.04 0.03

Sa: average roughness; Sq: root mean square deviation; Sy: lowest valley; Sz: maximum height of the surface; Ssk: skewness of the surface; Sci: core fluid retention.

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multilayer epithelium at the air interface (ectoderm), a loose stroma, and a 1-cell layer-thick inner epithelium at the interface with the allantoic sac (endoderm). It was observed that new capillary plexus vessels adhered directly to the basal lamina beneath the ectoderm as they formed the plexus. Mesenchymal cells also attached to the surface of the plexus endothelial cells, but initial contact to the basal lamina was via the endothelial cells. In histological sections stained with H&E, the ectoderm and endoderm of control and treated CAMs were almost similar. In control group, extensive capillary plexus formation (Fig. 5A) and migration of blood vessels towards ectoderm was observed. Application of MSWSS1 caused decrease in number and size of capillary plexus formation (Fig. 5B). Rudimentary capillary plexus formation along with very slow migration of blood vessels towards ectoderm of CAMs were observed in group E (Fig. 5C). Most drastic changes were observed in group G, containing less capillary plexus with scanty blood vessels, few fibroblasts, and appeared to have less extracellular matrix than the mesoderm of control CAMs (Fig. 5D). The numbers of capillary plexus formed near ectoderm of CAMs were calculated and very highly significant decrease (P < 0.001) in capillary plexus formation was observed in CAMs of group G (Fig. 6). 3.2. Scanning electron microscopy of capillary plexus formation SEM of the normal CAMs revealed well-developed capillary plexus formation. The lumen was well caverned and surrounded by maximum blood vessels in cuff like manners (Fig. 7A). Deterioration in normal architecture of capillary plexus was observed by application of MSWSS on CAMs. The alteration in architecture ranged form dense meshwork of capillary plexus to complete abrasion of normal cuff like pattern. The relevant doses of pure nicotine did not discernibly affect pattern of capillary plexus formation. Structural alterations of capillary plexus were frequently observed in groups C (Fig. 7B) and D (Fig. 7C) with incomplete junction of blood vessels to form a plexus. Extensive changes were observed in group F where lumens of capillary plexus were almost obliterated (Fig. 7D). Dramatic changes with shallow lumen and disrupted capillary plexus and were observed in group G (Fig. 7E).

Fig. 5. Light micrographs showing variable capillary plexus formation in CAMs of different treatment groups (magnification: ×20). (A) Control: arrows pointing numerous capillary plexuses formed along the ectoderm. Blood vessel of considerable lumen size is seen. (B) MSWSS 1: small sized capillary plexus can be seen with migration of blood vessels towards the ectoderm. (C) MSWSS 4: formation of scanty capillary plexus with retarded migration of blood vessel towards the ectoderm. (D) MSWSS 6: distorted CAM matrix with sparse capillary plexus formation beneath the ectoderm.

3.3. Scanning electron microscopy of structural alterations In order to evaluate the structural alteration, SEM was performed on cut edges of control and treated CAMs. The fibrillar elements of the mesoderm were far more abundant in CAMs treated with MSWSS than CAMs of control eggs (Fig. 8A). Abnormal branching morphogenesis was accompanied by alteration in extracellular matrix. Along with this, disorganization and condensation of mesodermal fibrils were

Fig. 6. Graphical outline of capillary plexus formation in CAMs of different groups. Note the highly significant difference among groups E, F and G.

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Fig. 7. Scanning electron micrographs showing disrupted capillary plexus formation in CAMs of different groups (magnification: ×6000). (A) Control: SEM showing a well developed capillary plexus. (B) MSWSS 2: dense meshwork of capillary plexus wall with concavity of central lumen. (C) MSWSS 3: shallow lumen with seemingly compacted wall of capillary plexus. (D) MSWSS 5: obliterated lumen of capillary plexus. (E) MSWSS 6: shallow lumen with distorted capillary plexus wall.

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also observed in treated CAMs (Fig. 8B–D). Dense arrangement representing focal conjoining of fibrillar material was extensively observed in group F treated with MSWSS 5 (Fig. 8C). Most extensive structural deviation was observed

Fig. 8. (Continued ).

in CAMs of group G with distorted and fuzzy appearance of fibrillar material.

4. Discussion

Fig. 8. Scanning electron micrographs showing arrangement of fibrillar material in mesoderm of CAMs of different groups (magnification: ×5000). (A) Control: SEM showing normal arrangement of fibrillar material in mesoderm. (B) MSWSS 3: disorientation of fibrillar material in mesoderm. (C) MSWSS 4: dense arrangement representing focal adjoining of fibrillar materials. (D) MSWSS 5: thickened fibrillar material almost covering all extracellular matrix of CAM.

Toxicology of different MSWSS of commercial cigarettes was investigated on day 5 CAMs. It was observed that almost all smoke solutions caused varying levels of disruption on normal process of angiogenesis and adversely affected the diameters of secondary and tertiary blood vessels. The most consistent findings were diminished height and spread of the blood vessels on CAMs treated with different MSWSS. Progressive deterioration of blood vessels due to the exposure of different MSWSS might result in deprivation of nutrient and substrate supply resulting to the death of embryo. It has been shown that there is a close relationship between embryonic development and the state of vascularization of the chorionic villi and that normal chorionic villous vascularization is essential for the undisturbed development of pregnancy (teVelde et al., 1997). Meegdes and co-workers (Meegdes et al., 1988) described a poor vascular development in cases of intrauterine embryonic death and blighted ova compared to normal villous tissue obtained from legal abortions. The effects of smoking during pregnancy on the fetus have opened a new era on the health consequences of smoking. The fetus is not like a passive smoker who inhales cigarette smoke involuntarily from the environment; rather, the fetus is a highly vulnerable being in a stage of high risk for involvement of growth (Wilcox et al., 1989). It was observed that application of pure nicotine, equivalent to the nicotine concentration in each type of smoke solution, did not affect the process of angiogenesis. Almost similar result was stated by Melkonian et al., suggesting that chemicals other than nicotine play a role in inhibiting cell division, and that in complex mixtures of chemicals containing nicotine, other chemicals dominate over nicotine and inhibit vessel growth (Melkonian et al., 2002a). Pyrazine (chemical

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present in cigarette smoke) and some of its derivatives were reported to inhibit vascular growth and certain other vital processes of angiogenesis (Melkonian et al., 2003). Our results revealed a positive correlation between nicotine concentration in MSWSS and reduction in diameter of secondary and tertiary blood vessels. Hence it might be assumed that nicotine boosts the activities of other chemicals in cigarette smoke that inhibit angiogenesis. Determining the exact efficacy of agents to regulate angiogenesis requires precise quantification of CAM. However, quantification of this assay has been troublesome and often done using subjective scoring methods, leading to difficulties in comparison of results (Vu et al., 1985). In this study, we have used a novel image probing technique for 3D quantification of angiogenesis from images of normal and treated CAMs. One of the main parameters in 3D image analysis is “surface roughness” and its accurate measurement gives the exact values of angiogenesis on that surface (Sohail et al., 2004). Considerable decline in the surface roughness of treated CAMs entail decreased neovascularization on CAMs. Increase amount of heights and pits were observed on CAMs of control eggs, which imitate enhanced angiogenic activity on CAMs of control eggs than treated CAMs. Similarly, asymmetric distribution and diminutive fluid retention in blood vessels of treated CAMs reflect a positive correlation between tobacco smoke solution and retarded angiogenesis. The current study strongly supports the idea that smoke exposure can affect the architecture of blood vessels in tissues actively undergoing angiogenesis and demonstrates that this technique is useful for holistic quantification of angiogenesis. The respiratory exchange in the CAM occurs by means of an extensive capillary plexus that develops initially adjacent to the chorionic ectoderm and later interdigitates between the ectodermal cells of the chorion (Ausprunk et al., 1974; Burton and Palmer, 1989). The CAM capillary plexus provides an excellent model to study capillary formation in vivo. Different extent of capillary plexus formation was adversely affected in treated CAMs by blocking migration of the mesodermal blood vessels to the ectodermal basal lamina. Most treated CAMs that had little plexus also had numerous angiogenic clusters near the ectoderm, indicating that vasculogenesis was also inhibited and contributed to the scarcity of the capillary plexus. Melkonian et al. (2002b) has also demonstrated that mainstream of cigarette smoke contain certain chemicals that inhibit the development of capillary plexus formation. The extracellular matrix plays important roles in regulating many developmental processes, including angiogenesis and branching morphogenesis (Matsui et al., 1996). In the CAM, the extracellular matrix undergoes compositional changes (Rooney and Kumar, 1993) that are known to be important in influencing blood vessel development (Ingber, 1992), although the exact effects of individual matrix components are not yet fully understood. Different preparations of MSWSS produced abnormal pattern formation of the fibrillar material in CAMs, which might be associated with increases in the interstitial collagens (type I and III) and decreases in

hyaluronic acid in the extracellular matrix. In CAM, interstitial collagen synthesis accompanies angiogenesis, and collagen is thought to provide a substrate for endothelial cell migration. It supports the ideas that collagen metabolism is important in controlling angiogenesis and that either excessive or insufficient amounts of collagen or improper processing of collagen alters normal blood vessel development (Melkonian et al., 2000). A consistent association between smoking during pregnancy and reduced average birth weight, with the risk of having a low-birth-weight baby (under 2500 g) increased 53% in light smokers (<1 pack/day) and 130% in heavy smokers (≥1 pack/day) compared with nonsmokers, has been reported (Meyer et al., 1976). The relationship between cognitive and mental development in children born to mothers who smoked during pregnancy is less clear. The 1979 Surgeon General’s report indicated that the data suggest unfavorable effects of smoking during pregnancy on the child’s long-term growth, intellectual development, and behavioral characteristics (US Department of Health and Human Services DHHS, 1979). The relationship between cognitive and mental development in children born to mothers who smoked during pregnancy is less clear. Along with the risk for reduced birth weight, decreases in the infant’s cognitive and motor development and abilities have been suggested in infants born to parents who smoked (Stratten et al., 2001; Brennan et al., 1999; Drews et al., 1996; Weitzman et al., 2002). Traditionally antismoking interventions aimed at the most preventable cause of cancer (Nutbeam et al., 1993) targeting high-risk groups such as pregnant women (Lowe et al., 1998). Healthy women from socioeconomically deprived classes who traditionally have a higher prevalence of smoking (Hill et al., 1998; Macken et al., 1991) have hitherto escaped this preventive focus. One rationale of cessation programs for pregnant women has been based on the principle that pregnancy is a significant life event and mothers are concerned about the effects of smoking on their babies and themselves (Wakefield et al., 1998; Ziebland and Mathews, 1998). There have been a number of articles published about toxicity of cigarettes using different research grade cigarettes. Research grade cigarettes have specific length, circumference, filter types and chemical composition. As reference cigarettes, they provide a basis for comparing data collected in different laboratories and at different points in time. Many differences do exist among different styles of commercially marketed cigarettes. These differences include the quantity and types of tobacco utilized, the crop of the tobacco utilized; the quantity and types of ingredients utilized; the nontobacco materials utilized (i.e. papers and filters); and the physical design of the cigarette. These differences among cigarettes might result in delivering different chemicals during combustion, thus, inflicting different effects on disruption of angiogenesis than research grade cigarettes (Counts et al., 2004; Ashley et al., 2003; Pauly et al., 2002). Similar findings on blood vessels and capillary plexuses formation were observed in our study. The above observations suggest

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that MSWSS of different commercial cigarettes, which contains the main components of tobacco smoke, is toxic to the normal process of angiogenesis and smoking during pregnancy may lead to an increased risk of spontaneous abortion and preterm delivery. We conclude that since this study was done on commercial cigarettes rather than research grade cigarettes, a more direct impact on studying relationships between cigarette smoking and human health are herein provided. Future work is required to screen the effects of different chemicals and factors in MSWSS that might affect angiogenesis.

Acknowledgement This study was financially supported in part by research grants from Bio-Safety Research Institute, Chonbuk National University in 2004 (CNU-BSRI, No 2004-02).

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