UNIVERSITY OF CALGARY

The Dose-Kinetic Response of Cigarette Smoke and Nicotine in Pregnant Rats

by

Jabeen Sadrudin Ali Hussein

A THESIS

SUBMllTED TO THE FACULTY OF GRADUATE STUDIES

IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF

MASTER OF SCIENCE

DEPARTMENT OF MEDICAL SCIENCE

CALGARY, ALBERTA

NOVEMBER, 2000

O Jabeen Sadrudin Ali Hussein 2000

National Library 1+1 ofCamda Bibliothèque nationale du Canada

Acquisitions and Acquisitians et Bibliographie Services seniices bibliographiques

395 Wellington Street 395. rue WeWngion Ottawa ON K1A ON4 OaawaON K l A W canada CaMda

The author has granted a non- L'auteur a accordé une licence non exclusive licence allowing the exchisive permettant à la National Library of Canada to Bibliothèque nationale du Canada de reproduce, loan, distnbute or seii reproduire, prêter, distribuer ou copies of this thesis in microform, vendre des copies de cette thèse sous paper or electronic formats. la fome de microfiche/fïim, de

reproduction sur papier ou sur format électronique.

The author retains ownership of the L'auteur conserve la propriété du copyright in this thesis. Neither the droit d'auteur qui protège cette thèse. thesis nor substantial extracts fiom d Ni Ia thèse ni des extraits substantiels may be printed or otherwise de celle-ci ne doivent être imprimes reproduced without the author's ou autrement reproduits sans son permission. autorisation.

ABST RACT

Smoking during pregnancy has adverse effects on the perinate including

spontaneous abortion, low birth weight and increased mortality. Better animal

models may help determine the mechanisms of these effects and ultirnately

reasons why smoking is hamiful in humans. In Our studies, plasma nicotine

concentrations, embryo implantation and corticosterone concentrations were

determined in pregnant rats exposed to cigarette smoke via inhalation and to

nicotine via infusion. In both cigarette smoke and nicotine studies. plasma

nicotine concentrations decreased with increasing exposure Iikely due to enzyme

induction and the number of pups per litter remained unaffected. The dosage of 6

mglkglday of nicotine produced nicotine concentrations that were several-fold

higher than those observed in moderate smokers, while 2 mglkglday of nicotine

produced concentrations comparable to those in moderate smokers. ln addition,

corticosterone concentrations in the nicotine group decreased with increasing

exposure while concentrations remained high in the inhalation group. With

nicotine infusion. pup number and weights were not significanüy different from

saline-infused pups and thus nicotine may not be the constituent in cigarette

smoke responsible for adverse effects.

ACKNOWLEDGEMENTS

I would first of al1 like to thank my mentor, supervisor, and friend Dr. Shabih U.

Hasan, for giving me the opportunity to gain such valuable experience in his

laboratory. I would have been Iost without his guidance, support and his constant

strive to make me a better investigator. I will never be able to express my

gratitude for al1 that he has done for me.

I would also like to express my sincere gratitude and appreciation to my

committee members, Dr. Quentin Pittrnan and Dr. John Tyberg for accepting me

as their student and agreeing to be on my supervisory committee. I would also

like to extend my appreciation to Dr. Sheldon Roth for being on my examining

committee.

I would also like to thank my family, my father and sister Salima for their

inestimable support throughout my Master's program. Their unconditional love

and caring has meant more to me than words can describe and has helped me

through my most trying times. I would also like to remember my mother who is no

longer with me. but who I know has been there with me every step of the way.

How much your love has helped me. you will never know. I would also like to

thank the love of my Me Faheem, for always being there through so many

agonizing experiments and long nights. 1 will always be indebted to you for this. I

love al1 of you very much.

I would also like to give thanks to my colleagues: Svetlana Farkas, SaIim Lalani,

Dr. Luxmi Gahlot, Dr. Ather Bano, Yolanda MacKinnon, Xiolan Zhou, and Anita

Rigaux. You al1 were such a joy to work with. Never have I been so fortunate to

work with such a diverse and wonderful group of people. The last two and a half

years just would not have been the same without you.

1 would also like to thank Doreen, Marion and Joanne for al1 of their support,

especially in the last months leading up to my defense. You are al1 wonderful!

Finally, 1 would like to thank al1 of my friends for being there for me to lean on

when things were tough for me, Your love and support was so very appreciated!

To my parents Sadru and Zohora

and my sister Salima

TABLE OF CONTENTS

APPROVAL PAGE.. .............................................................................. ii

... ABSTRACT.. ........................................................................................ III

ACKNOWLEDGEMENTS. ...................................................................... .iv

DEDICATION.. ..................................................................................... vi

TABLE OF CONTENTS.. ..................................................................... .vii

LIST OF TABLES ........................................................-...--................... xv

LIST OF FIGURES .............................................................................. xvi

***

LIST OF ABBREVIATIONS. .......... ,. .....................-............................. WIII

CHAPTER ONE: INTRODUCTION .... ...,. ................................................. 1

1 .1 CtGARElTE SMOKE CONSTITUENTS .......................... 2

................................................................ 1.2 NICOTINE 3

............................................... 1.2.1. Nicotine Structure 3

............................................. 1.2.2. Nicotine Receptors 3

....................................... 1.2.3. Mechanisms of Action ..4

......................... 1.2.4.1. Absorption and Distribution 6

1.2.4.2 Distribution of Nicotine in Body Tissues ......... 8

......... 1.2.4.3. Elirnination (Pathways of Metabolisrn).. 9

1.2.4.4. Factors Influencing the Rate of Nicotine

............................................ Metabolism 11

vii

1.2.5. Regulation of Nicotine lntake During Cigarette

......................................................... Smoking 11

........ 1.3 PATHOPHYSIOLOGICAL EFFECTS OF SMOKING 13

.............. . 1.3.1 Effects on Reproduction and Pregnancy -13

1.3.2.1. Effects on Birth Weight: Human Studies ....... 14

1.3.2.2. Effects on Birth Weight: Animal Studies ....... 15

1.3.3.1. Effects on Fetal Organ Weights: Human

............................... Studies

1.3.3.2. Effects on Fetal Organ Weights: Animal

............................................... Studies -17

.......................... 1.3.4. Effects on Carboxyhemoglobin -19

........................................ 1.3.5. Effects on Hematocrit 20

................................. 1.3.6. Effects on Glucocorticoids -22

1.4. ANIMAL MODELS ADMINETERING CIGARETTE SMOKE

AND NICOTINE ....................................................... 23

1.4.1. Animal Models of Cigarette Smoke Inhalation ......... 23

? .4.2. Recent Studies in our Laboratory ......................... 25

1.4.3. Animal Models of Nicotine Infusion ....................... 27

1.5. RATIONALE ............................................................. 29

1 S.1. Cigarette Smoke Exposure .................... ...... .... 29

1.5.2. Osmotic Minipump Nicotine Infusion ..................... 30

1.6 SPECIFIC AIMS ....................................................... 30

CHAPTER TWO: MATERIALS and METHODS: CIGARETTE SMOKE

EXPOSURE STUD IES .......................................................................... 32

2.1. ANIMALS ................................................................ 32

.................................... 2.2. SMOKE WPOSURE SYSTEM 33

......................... ........ . 2.2.1 General Description ... -33

................................. 2.3. RESEARCH CIGARETTES (2R1) 37

.................................. 2.4. EXPERIMENTAL PROTOCOLS -38

............ .......... 2.4.1. Plasma Nicotine Concentrations , -38

2.4.2. Fetal Weights and Litter Size .............................. 40

2.4.3. Carboxyhemoglobin and Hematocrit ..................... 40

............................ 2.4.4. Corticosterone Concentrations 41

2.5. CARDIAC PUNCTURE PROCEDURE ........................... 41

2.6. DETERMINATION OF PLASMA NICOTINE AND

CORTICOSTERONE CONCENTRATIONS .................... 42

2.6.1. Gas-Chromatography Mass-Spectrometry Method .. 42

.............. .......... 2.6.2. Radioimmunoassay Method ... -44

CHAPTER THREE: MATERIALS and METHODS: NICOTINE INFUSION

.......................................................................... .............. STUDIES .. 45

3.1. OSMOTIC MINIPUMPS ............................................. 45

3.2. EXPERIMENTAL PROTOCOLS .................................. 47

........................... . 3.2.1 Plasma Nicotine Concentrations 47

3.2.2. Hematocrit ........................................................ 49

............................. 3.2.3. Corticosterone Concentrations 50

........................................... CHAPTER FOUR: STATlSTlCAL ANALYSES 51

CHAPTER FIVE: RESULTS IN CIGARETTE SMOKE HPOSED RATS .......... 52

5.1. PLASMA NICOTINE CONCENTRATIONS (nglml) ........... 52

5.1.1. Plasma NIC Concentrations in Pregnant Rats after

Exposure to 1000 ml of CS From Day 2 of Gestation

Compared with Rats Exposed to Room Air ............ 52

5.1.2. Plasma NIC Concentrations in Pregnant and Non-

Pregnant Rats Exposed to 1000 ml of CS .............. 52

5.2. CARBOXYHEMOGLOBIN AND HEMATOCRIT IN

............................................... PREGNANT RATS (%) 53

5.2.1. COHb (Oh) in CS and Air Exposed (Sham) Pregnant

............................................................... Rats 53

5.2.2. Hematocrit (Oh) in CS and Air Exposed (Sham)

.............................................. Pregnant Rats - 5 4

5.3. PLASMA CORTICOSTERONE CONCENTRATIONS

..................................................................... ( pgldl) 54

5.3.1. CORT Concentrations (pgldl) in CS and Air Exposed

(Sham) Pregnant Rats ..................................... 54

X

5.4. MATERNAL AND FETAL OUTCOME ........................... 54

5.4.1. Matemal Weights in CS and Air Exposed (Sham)

Pregnant Rats.. ............................................. 3 4

5.4.2. Pup Weights in CS and Air Exposed (Sham) Rat

Litters.. ........................................................... 55

5.4.3. Pup Numbers per litter in CS and Air Exposed (Sham)

........................................... Rat Litters 55

5.4.4. Placental and Fetal Organ Weights in CS and Air

Exposed (Sham) Pregnant Rats at 22 Days of

..................................................... Gestation.. .55

5.4.5. Placental and Fetal Organ Weights in Control

Pregnant Rats Compared with Air Exposed (Sham)

Pregnant Rats at 22 Days of Gestation ................. 56

5.4.6. Placental and Fetal Organ Weights in Control

Pregnant Rats Compared with CS Exposed Pregnant

Rats at 22 Days of Gestation .............................. 56

CHAPTER SIX: RESULTS IN NICOTINE EXPOSED RATS .......................... 68

6.1. PLASMA NICOTINE CONCENTRATIONS.. .............. .... .68

6.1 -1. Plasma NIC Concentrations in Pregnant Rats

Exposed to NIC via OMPs at Dosages of 2 and 6

mglkglday at Exact Weights .............................. .68

6.1.2. Plasma NIC Concentrations in Pregnant Rats

Exposed to NIC via OMPs at Dosages of 2 and 6

mglkglday at Proiected Weights .................. ........ 68

HEMATOCRIT (Oh) ....... ............................................. 69

6.2.1. Hernatocrit in Rats Exposed to NIC and Saline via

OMPs at Various Dosages ................................. 69

PLASMA CORTICOSTERONE CONCENTRATIONS

( pgldl ) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .69

6.3.1. Corticosterone Concentrations in NIC Exposed

(6rnglkglday a l Proiected Weight) and Saline

Exposed Pregnant Rats .................................... 69

MATERNAL AND FETAL OUTCOME ..... .... . ... ............... 70

6.4.1. Maternal Weights in NIC lnfused and Saline Infused

Pregnant Rats ................................................. 70

6.4.2. Pup Weights in NIC Infused and Saline lnfused Rat

Litters.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .70

6.4.3. Pup Numbers in NIC lnfused and Saline Infused Rat

Litters.. . . . . . . . . . . Litterç....,............................ Litterç....,............................ Litterç....,............................ Litterç....,............................ Litterç....,............................ Litterç....,............................ Litterç....,............................ Litterç....,............................ Litterç....,............................ Litterç....,............................ Litterç....,............................ Litterç....,............................ Litterç....,............................ Litterç....,............................ Litterç....,............................ Litterç....,............................ Litterç....,............................ Litterç....,............................ . . . . . . . . . . . . . . . . . . . -. . -70 6.4.4. Placental and Fetal Organ Weights in Saline and NIC

lnkrsed Rats at 21 Days of Gestation. .... .... .. . . . ..... 71

CHAPTER SEVEN: DISCUSSION .....+...............-.-...-............---.----.. ......... 81

7.1. GENEWL ..... .. . ... .... . .. .. .... . ... .. .. ... ................ .... .. ...... 81

7.2. PLASMA NICOTINE CONCENTRATIONS ...... .... ........ 83

xii

7.3. CARBOXYHEMOGLOBIN. HEMATOCRIT AND

................................................ CORTICOSTERONE 87

7.3.1. Carboxyhemoglobin ......................................... 87

..................................................... 7.3.2. Hematocnt 88

................ 7.3.3. Plasma Corticosterone Concentrations 90

7.3.3.1. Effects of Cardiac Puncture and

........................................... Anesthesia -90

7.3.3.2. Effects of Hormonal Changes during

............................................. Pregnancy 92

7.3.3.3. Effects of Frequent Handling ..................... 92

.... 7.3.3.4. Effects of Nicotine and Cigarette Smoking 93

7.4. MATERNAL WEIGHTS ............................................. -94

7.5. FETAL VARIABLES - BIRTH WEIGHTS, LITTER SIZES,

and ORGAN WEIGHTS .............................................. 95

7.5.1 . Birth Weights ................................................. 95

....................................................... 7.5.2. Litter Size 96

7.5.3. Organ Weights ................................................. 97

............................. CHAPTER EIGHT: SIGNIFICANCE AND RELEVANCE 101

CHAPTER NINE: LIMITATIONS OF THE EXPERIMENTAL DESIGN ............ 102

.................... CHAPTER TEN: CONCLUSIONS .................................. .. 103

xiii

CHAPTER ELEVEN: FUTURE DIRECTIONS ............ ........... ... ..... . .. .--....... 105

CHAPTER TWELVE: BIBLIOGRAPHY. .... .............+ .. ... . . . ....... . ..... . . .. .. --. --A06

xiv

LIST OF TABLES

Table 1.

Table 2.

Table 3.

Table 4.

Table 5.

Table 6.

Table 7.

Table 8.

Table 9.

Fetal Organ Weights in Control and Smoke Exposed Groups ...... 18

Summary of CS Experimental Design ..................................... 39

Summary of OMP Experimental Design .................................. 48

The Placental and Fetal Organ Weights in Cigarette Smoke and

Air Exposed (Sham) Pregnant Rats at 22 Days of Gestation.. . . . ..65

The Placental and Fetal Organ Weights in Control Pregnant Rats

Compared with Air Exposed (Sham) Pregnant Rats at 22 Days of

Gestation.. . . . . . . . . . . . . . . . . . . . . . . , . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . -66

The Placental and Fetal Organ Weights in Control Pregnant Rats

Compared with Cigarette Smoke Exposed Pregnant Rats at 22

Days of Gestation ................... ............................................ 67

The Placental and Fetal Organ Weights in Saline and Nicotine (2

mglkglday) Exposed Rats at 21 Days of Gestation ..................... 79

The Placenta1 and Fetal Organ Weights in Saline and Nicotine (6

mglkglday) Exposed Rats at 21 Days of Gestation .................... 80

The Placental and Fetal Organ Weights in Control Pregnant Rats

Compared with Sham (Air) Exposed Pregnant Rats at 22 Days of

Gestation (subsequent studies) ..... . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ..-.. 1 O0

LIST OF FIGURES

..................................................... Figure 2.2.2. Smoke Exposure System 36

Figure 3.1 .l. Cross-section of OMP showing design, components and

..................................................................... mechanism of operation.. -46

Figure 5.1 .l. Plasma NIC Concentrations (mean i SD) in Pregnant Rats after

Exposure to 1000 ml (1 0 ml puff volume X 10 puffs X 10 cigaretteslday) of CS

and Room Air (sham) at Various Gestational Ages ...................................... 57

Figure 5.1.2. Plasma NtC Concentrations in Pregnant and Non-Pregnant Rats

.................................................................. Exposed to 1000 ml of CS.. -58

Figure 5.2.1. Carboxyhemoglobin (COHb) (%) (mean I SD) in CS Exposed and

Air Exposed (Sham) Pregnant Rats .......................................................... 59

Figure 5.2.2. Hematocrit (%) (rnean r SD) in CS Exposed and Air Exposed

(Sham) Pregnant Rats .......................................................................... 60

Figure 5.3.1. Corticosterone Concentrations (mean t SD) in CS Exposed and Air

Exposed (Sham) Pregnant Rats ............................................................... 61

Figure 5.4.1. Materna1 Weights of CS Exposed and Air Exposed (Sham)

Pregnant Rats ..................................................................................... 62

Figure 5.4.2. Pup Weights (mean r SD) from CS Exposed and Air Exposed

(Sharn) Rat Litters ................................................................................ 63

Figure 5.4.3. Pup Numbers per Litter (mean i SD) from CS Exposed and Air

Exposed (Sharn) Rat Litters ................. ... ............................................ 64

xvi

Figure 6.1.1. Plasma NIC Concentrations (mean I SD) in Pregnant Rats

Exposed to NIC via OMP's at Dosages of 2 and 6 mglkglday at Exact

........ Weights.. ............................ .. ..72

figure 6.1.2. Plasma NIC Concentrations (mean + SD) in Pregnant Rats

Exposed to NIC via OMP's at Dosages of 2 and 6 mglkglday at Projected

Weights.. ............................................................................................ 73

Figure 6.2.1. Hematocrit (mean t SD) in Rats Exposed to NIC and Saline via

OMP's at Various Dosages ............................... -74

Figure 6.3.1. Corticosterone Concentrations (mean 2 SD) in NIC Exposed (6

mglkgiday at Projected Weight) and Saline Exposed Pregnant Rats.. ............. 75

Figure 6.4.1. Maternal Weights of NIC lnfused and Saline Inkised Pregnant

Rats.. ................................................................................................ 76

Figure 6.4.2. Pup Weights (mean 2 SD) from NIC lnfused and Saline lnfused Rat

Litters.. .............................................................................................. -77

Figure 6.4.3. Pup Numbers (mean I SD) from NIC lnfused and Saline Infused

Rat Litters.. ........................... .,.. .......................................................... 78

xvii

LIST OF ABBREVIATIONS

Abbreviation

ACTH

CO

CO2

COHb

CORT

CS

DA

ETS

GC-MS

GEE

HPA

IGF

MS

NE

NIC

OMPs

SIDS

SS

Definition

Adrenocorticotropic hormone

Carbon monoxide

Carbon dioxide

Carboxyhemoglobin

Corticosterone

Cigarette smoke

Dopamine

Environmental tobacco smoke

Gas chromatography- mass spectrometry

Generalized estimating equations

Hypothalamo-pituitary- adrenal axis

Insulin-like growth factors

Mainstream smoke

Norepinephrine

Nicotine

Osmotic minipumps

Sudden infant death syndrome

Sidestream smoke

Page Number of First Appearance

xviii

1 .O. INTRODUCTION

Approximatefy 20% of pregnant women smoke during pregnancy (9).

nu me roi!^ studies in recent years have demonstrated that smoking during

pregnancy is detrimental to the developing fetus. Matemal smoking during

pregnancy produces adverse effects on the fetoplacental unit including

spontaneous abortion, abruptio placentae, placenta previa, prematurity, low birth

weight, increased mortality, increased ainnray thickness and reduced lung

function. Cigarette smoke (CS) exposure may also lead to skull deformation and

congenital defects such as polycystic kidneys in the fetus (7-

9; 19;21;24;27;37:41;44;46:60;76;81;86). Furthemore, materna1 smoking during

pregnancy also increases the risk for Sudden Infant Death Syndrome (SIDS) in a

dose-dependent manner (37).

Because of these adverse effects of CS, a suitable animal model would be

desirable to elucidate the mechanisms for the detrimental effects seen in fetal

and neonatal development. The rat is a good animal model because these

animals are small, easy to handle, less expensive than larger mammals, have a

short period of gestation (21 - 23 days of gestation) and a great deal of

physiological information on them is available in the literature.

Previously animal models were designed in order to study the effects of

chronic inhalation of CS or nicotine (NIC) infusion via osmotic minipumps

2 (OMPs). However, the objectives of my project were to establish a model of

inhalation that provides a more suitable animal model for smoking related fiealth

by closely approximating human active smoking. My project was also designed

to detemiine the effects of continuous NIC infusion on plasma NIC and

corticosterone (CORT) concentrations and its effect on the size of the litter and

weight of the pups.

1 .l. CIGARElTE SMOKE CONSTITUENTS

Environmental tobacco smoke (ETS) contains over 4,000 agents. At least

40 of these constituents have been identified as carcinogens (48). The smoke

components have been divided into gas and particulate phases. The gaseous

phase consists of carbon monoxide (CO), carbon dioxide (CO2), nitnc oxides,

ammonia, nitrosamines, hydrogen cyanide, nitrites, voiatile hydrocarbons,

alcohols, aldehydes and ketones whereas the particulate phase consists of NIC,

water and tar. The tar in turn consists of polycyclic aromatic hydrocarbons,

rnetallic ions and radioactive compounds (48). Cyanide has been proposed to

affect the abiiity of the rnother to store vitamin 8-12, which is a vitamin with the

function of controlling pernicious anemia, During pregnancy, levels of vitamin B-

12 are lower in women who smoke than in nonsmokers.

The effects of NIC and CO have been studied extensively in pregnant

women and animak and most studies speculate that both substances are

3 potential causes of impaired fetal growth (64). NIC reduces uteroplacental blood

ffow, increases catecholamine levels, and rnay reduce uptake of nutrients by the

placenta. In addition, NIC passes rapidly into the fetal circulation. CO, as

mentioned, interferes with oxygen carrying capability by reducing both the

oxygen-carrying capacity of the blood and its ability to release oxygen to the

tissues.

1.2. NICOTINE

Nicotine structure

NIC is a tertiary amine that contains a pyridine and pyrolidine ring (4;7;45).

Tobacco contains pure levorotatory S-NIC, while tobacco smoke also contains

the R-isorner in quantities up to 10 percent of the total NIC present (7;85). S-NIC

binds stereoselectively to nicotinic cholinergic receptors while R-NIC is a weak

agonist at cholinergic receptors (85).

1.2.2. Nicotine Receptors

Nicotinic cholinergic receptors are found in the brain, autonomic ganglia,

and the neurornuscular junction. Most relevant to NIC addiction are the neuronal

nicotinic acetylcholine receptors (7). These receptors are found throughout the

brain, with the greatest number of binding sites in the cortex, thalamus, and

4 interpeduncular nucleus (45). Different subtypes of nicotinic cholinergic

receptors have varying chemical conductances for sodium and calcium and differ

in their sensitivity to a number of nicotinic agonists. Thus these receptors result in

correspondingly different pharmacological actions. The diversity in nicotinic

cholinergic receptors rnay explain the multiple effeds of NIC in humans and may

present targets for specific nicotinic agonist or antagonist therapies.

W h e ~ NIC binds to NIC receptors, altostenc changes occur such that

there are several different functional states (4;45). These include the resting

state, an activated state (channel open), and two desensitized states (channel

closed). An increase in the number of NIC receptors has been observed both in

experirnental anirnals during NIC treatment and in human smokers at autopsy.

NIC facilitates the release of various neurotransmitters with different

effects. These include dopamine (DA), norepinephrine (NE), acetylcholine,

serotonin, vasopressin, and beta-endorphin (5). With repetitive exposures to NIC,

neural adaptation and tolerance develop, resulting in diminished neurotransmitter

release. Such tolemnce underlies the development of physical dependence to

NIC, which conhbutes to NIC addiction,

Mechanisms of Action

NIC in small doses results in peripheral effects via stimulation of autonomic

5 ganglia (8;85) and of peripheral sensory receptors in the heart and lungs (72).

Activation of these receptors results in various autonornic reflex responses,

causing tachycardia, increased cardiac output and increased arterial pressure,

reduction of gastrointestinal motility and sweating (72). Central nervous system-

mediated sympathetic stimulation can also occur via activation of peripheral

chemoreceptors, direct effect on the brain stem, andlor effects on the ventral

hom of the spinal cord.

In addition, blood flow to various vascular beds is adversely affected as

blood vessels in the skin are constricted while skeletal muscle blood vessels are

dilated (8;85). The coronary blood vessels also appear to be constricted by NIC,

as seen by the decrease in coronary blood ffow when heart rate is increased.

Some in vitro studies, have also suggested that NIC reduces vascular synthesis

of prostacyclin, an endothelia1 cell-derived local vasodilator and antiplatelet

hormone. Reduced prostacyclin synthesis could interfere with blood perfusion.

NIC also has metabofic effects on body weight and serum Iipids (8;85).

Smokers on average weigh 4 kg less than nonsmokers which is maintained by

an increase in metabolic rate, along with appetite suppression which is observed

via the absence of a compensatory increase in caloric intake that would be

expected when metabolic rate increases. In addition, NIC (via release of

catecholamines) induces tipolysis and reieases free fatty acids into the plasma

6 and also substantially enhances free fatty acid turnover, which could cause liptd

abnormalities.

NIC also has endocrine effects in that it increases adrenocorticotropic

hormone and cortisol release (8;85). Excessive cortisol release coufd have

effects on mood and could contribute to osteoporosis by affecting bone mineral

density (25).

A study by Marit;! (1993) has also shown that adult lung ascorbic acid

content was reduced by 76% after exposure to NIC (52). Ascorbic acid

accumulates in the fiuid lining the air spaces where it appears to act as an

extracellular antioxidant. protecting the lung against inhaled oxidants. Ascorbic

acid is also involved in carbohydrate metabolism.

1.2.4.1. Absorption and Distribution

NIC is distilled from buming tobacco and is carried proximally on tar

droplets which are inhaled into the pulrnonary venous circulation via the

pulmonary capillaries (7;11;29). After reaching the small airways and alveoli of

the lung, NIC is rapidly absorbed independent of the pH of the smoke. The rapid

absorption of NIC from CS through the lung is presumably due to the large

surface area of the alveoli and srnall airways. In addition, NIC dissolves into fiuid

of pH that is in the hurnan physiobgic range, which facilitates transfer across ceil

7 membranes. After leaving the pulmonary circulation, NIC will enter the left side

of the heart. Arterial blood perfuses tissues, which takes up a variable amount of

NIC before blood reaches a venous sampling site. (4;7;45). Thirty to 60 minutes

after intravenous administration of NIC to rats, concentrations that are 2 to 15

times higher than those of plasma occur in the adrenals, liver, brain, lung, heart,

intestinal wall, spleen, thymus, skeletal muscle and kidney (15).

NIC absorption is dependent upon body pH, meaning that the extent of

NIC absorption varies markedly in different organs and regions of the body

according to differences in the pH of their mucosal or membrane lining (41 ). NIC

is Iipid soluble and readily permeates cell membranes of the lungs, skin,

gastrointestinal tract, buccal and nasal mucosae, and also the renal tubule.

Pulmonary NIC absorption occurs at a rapid phase similar to that after

intravenous administration.

The actual arnount of NIC absorbed by the smoker during the smoking

process depends upon several factors incfuding the type of tobacco, the NIC and

moisture content of the tobacco, the shape and configuration of smoke particles,

pH of body fluids and surfaces coming into contact with smoke and the duration

of contact between srnoke and mucous membranes (41).

1.2.4.2. Distribution of Nicotine in Body Tissues

After NIC is absorbed rapidly from CS, it enters the arterial circulation and

is rapidly distributed to the body tissues (85). NIC levels then decrease, due to

uptake by the peripheral tissues and later, to elimination of NIC from the body.

NIC takes 10 - 15 s to enter the brain. Arteriovenous differences during cigarette

smoking are substantial, with arterial levels exceeding venous levels six - to -

tenfold (85). At any given time, the concentration of a drug in venous blood

reflects the concentration of the drug in arterial blood less the net amount

extracted during its passage through the body tissues (29). It is the arterial

concentration that best represents the concentration that drives the

pharrnacodynamic effects. Peak arterial plasma NIC concentrations of up to 93

nglml were observed while venous concentrations remained low (8;29).

In both human and animal studies, NIC crosses the placenta freely and

has been found in amniotic fluid and the umbilical-cord blood of neonates (7).

The amniotic fluid provides a reservoir for continuous delivery of NIC to the fetus,

even when materna1 levels are low (1 1). Pregnant smokers expose the fetus to a

substantial amount of NIC and its metabolites such that in primates, the fetus had

up to 15 times higher levels of NIC than in the mother (71:84).

1.2.4.3. Elimination (Pathways of Metabolism)

NIC is rapidly and extensively metabolized, primarily in the liver, but also

to a small extent in the kidneys and lungs (7). The level of renal excretion is

dependent upon urinary pH (norrnally between 4.5 and 8.0 (31)) and urine flow

which accounts for approximately 2 to 35% of total elimination. At urinary pH

above 7, NIC is readily reabsorbed through the renal tubule, and only 2% of NIC

is recovered unchanged in urine (41). When urine is more acidic (pH less than S),

however, as much as 23% of NIC can be recovered as the unmetabolized

alkaloid. Saliva also constitutes an important route for NIC excretion. Less

important routes of NIC elimination incfude sweat, breast milk, bile, gastric juice

and feces.

The half-life of NIC varies from 1 to 2 hours, although there is

considerable variability among peopk (range of 1 - 4 hours) (7). However, in a

manner consistent with a half-life of Wo hours, NIC accumulates over six to eight

hours of regular smoking and persists overnight, even as the smoker sleeps.

Thus smoking r~sults not oniy in intermittent exposure to NIC, but in exposure

that lasts 24 hours a day. There is also a very iong terminal half-life, 20 hours or

more, presumably reflecting slow release of NIC from body tissues. It is apparent

that the decay curve of NIC is made up of two components; an initial rapid phase,

which is probably due to uptake of NIC from the blood into various tissues, and a

slower phase, which represents metabolism and excretion of NIC (1 1).

10 NIC's primary metabolite is cotinine (11). Other metabolites of NIC

include nomicotine and nicotine-N-oxide. Cotinine is fonned in a two step

process, the first of which involves oxidation of the five position of the pyrolidine

ring in a cytochrome P-450 rnediated process, to NIC-* l'(5')-imminium ion

(1 1;85). The latter is then metabolized by a cytoplasmic aldehyde oxidase to

cotinine. Cotinine is ideai in surveys and treatment studies as a marker of NIC

intake because of its long half-life (16 to 20 hours). Cotinine itself is also

extensively metabolized, with only about 17% excreted unchanged in the urine.

Several metabolites of cotinine have been reported, including trans-3'-

hydroxycotinine, 5'-hydroxycotinine, cotinine-N-oxide, and cotinine methonium

ion. The most abundant metabolite in the urine is 3'-hydroxycotinine which could

also prove to be a useful indicator of NIC exposure. Cotinine is present in the

blood of smokers in much higher concentrations than NIC. Cotinine blood levels

average about 250 - 300 nglml in groups of cigarette smokers. After cessation of

smoking, levels decline in a log-linear fashion with a half-life averaging 20 hours.

Because of the long half-life, there is much less fluctuation in cotinine

concentrations throughout the day than in NIC concentrations. As expected,

there is a gradua1 rise in cotinine levels during the day, peaking at the end of

smoking, and persisting in high concentrations ovemight. Mean urine cotinine

concentrations are hig her in active smokers (> 10 cigaretteslday, 1 1 O0 nglml)

than in passive smokers (9.2 nglmt) and nonsmokers (6.0 nglml) (87). At levels

to which smokers are generally exposed, cotinine appears to exert little, if any,

direct pharmacological effect.

11 1.2.4.4. Factors lnfiuencing the Rate of Nicotine Metabolism

Smoking status is only one of the numerous host factors that can alter,

both individually and through dynamic interactions with each other, the

disposition of NIC (71). A recent study of serum cotinine concentrations in

healthy Black and White smokers 18 to 30 years old dernonstrated much higher

mean concentrations in the former than the latter. Multiple genetic and

environmental causes were considered for this racial difference in NIC

metabolism. It has also been reported that men metabolize NIC faster than

women and that metabolism decreases with age. Other reports have concluded

that cigarette smoking accelerated NIC metabolism. In addition, inductive and

inhibitory effects on NIC metabolism may be exerted by NIC itself and other

chemicals, such as phenobarbitol and fi-naphthoflavone. Dietary habits, such as

the undefined association between NIC kinetics and caffeine consumption have

also been speculated to affect NIC metabolism. Disease may also have adverse

effects on NIC disposition, especially diseases impairing hepatic blood fow. In

addition, since NIC metabolism is dependent upon hepatic blood flow, such

factors as posture will affect NIC metaboikm (71).

Regulation of Nicotine lntake During Cigarette Smoking

Depending on the tar and NIC yields of cigarettes, smokers tend to alter

their smoking pattern in order to regulate NIC intake (8;11). Thus, when the NIC

12 yield is high they tend to smoke less, and when it is low they compensate by

smoking more. Smokers may also consciously or unconsciously control their NIC

intake by altering the number of cigarettes smoked, puff size, puff velocity, puff

rates, nurnber of puffs per cigarette, proximity of puffs to tip, proportion of puff

inhaled, and depth and duration of inhalation.

In one study, smokers took a 35 ml, two second puff once a minute to a

butt length of 20 mm. However, there was no guarantee that they would smoke

the cigarettes in the same way irrespective of NIC yield (75).

In a study by Benowitz et al., (1984), NIC intake was measured per

cigarette and found to be 1.04 i 0.36 mg (9). This study found that NIC intake

per cigarette was consistently less than machine yield, and that smokers of

nonfiltered cigarettes. which have higher machine yields than filtered cigarettes,

seemed to smoke their cigarettes less intensively than smokers of filtered

cigarettes.

ln a similar study, when a puff of CS was inhaled, the amount of NIC

absorbed was the equivalent of a venous injection of 0.1 mg (64). Up to two

milligrams of NIC was absorbed frorn the smoke of a single cigarette.

13 Another study showed that 50 - 90% of the NIC in smoke was

absorbed by the smoker and NIC levels in plasma could be detected shortly after

smoking (23).

1.3. PATHOPHYSIOLOGICAL EFFECTS OF SMOKING

1.3.1. Effects on Reproduction and Pregnancy

Reproductive processes in both humans and animals have been affected

by CS exposure. In laboratory animals, smoking adversely influences several

reproductive processes that are essential for pregnancy to occur, such as

ovulation, tuba1 transport, pre-implantation development, and implantation (3;34).

There is also a causal relationship between smoking and reduced fecundity (the

biological ability to become pregnant and maintain pregnancy), as pointed out in

both animal and human studies. However, human studies have shown that if

women stop smoking, fecundity levels will retum to normal (3).

In humans, menstrual cyclicity was also reported to differ between

smokers and non-smokers, and several epidemiological studies have found that

smokers experience menopause at earlier ages than non-smokers do. Smoking

can also influence estrogen metabolism as human smokers metabolize more of

their estradiol to catechol fonns (3).

1.3.2.1. Effects on Birth Weight: Human Studies

Much attention has been placed on birth weight because of the fact that it

is a relatively accurate index of prenatal development and it provides a constant

predictor of prenatal or neonatal mortality (1). Maternal cigarette smoking in

humans during pregnancy is associated with low birth weight babies

(1;14;34;86). It has been found in a wide variety of study designs and

epidemiological contexts, that maternal smoking and low birth weight exhibit a

dose-response relationship, such that as the number of cigarettes consumed

increases, birth weight will decrease (1;33). In addition, birth weights of babies

born to smokers who did not smoke during pregnancy resernbled those of non-

smokers more than those of smokers (1). This indicates that it is smoking and not

any physiological characteristic of the srnoker herself causing the growth

restriction. In addition, infants that are prenatally exposed to CS tend to have

abnorrnally low height and weight postnatally (1).

Maternal weight may also have a significant impact on the birth weight of

babies as seen in a study designed to investigate the effects of smoking to term

on materna1 weight gain and on the fetus after delivery (30). Of 238 pregnant

women who were studied, 115 smoked at least 5 cigarettes weekly and 123

stopped smoking prenatally. When women stopped smoking prenatally, they had

a greater increase in weight (16.6 kg) as compared to women who smoked until

term (1 3.2 kg). This greater increase in maternal weight gain was associated with

15 a 3.1 times more frequent delivery of babies weighing over 4000 g. Other

studies have found similar results (70). ln addition, as maternal weight gain

decreased, so did bieh weights. Thus perhaps low birth weights in babies may

not be due to CS exposure, but to maternal weight gain.

1.3.2.2. Effects on Birth Weight: Animal Studies

When Nelson et al., (1999) exposed pregnant Wistar rats to 1, 2, 3, or 4

cigaretteslday during either the first, second or third week of gestation, there was

a dose-dependent reduction in birth weights of pups whose mothers had been

exposed to CS, similar to that seen in humans (28). They also found that body

length was significantly reduced.

In a study by Bassi et al., (1984). birth weights of pups b o n to smoking

dams (4.60 i 0.16 g) were significantly lower than birtti weights of the pups born

to control unexposed dams (5.85 5 0.1 3 g) (6).

Younoszai et al., (1969) exposed pregnant Holtzman albino rats to CS

from three different types of cigarettes: (1 ) tobacco cigarettes each containing 15

mg of NIC, (2) lettuce leaf cigarettes, and (3) lettuce leaf cigarettes to which 15

mg of NIC had been added (90). Fetuses of ail of the smoke exposed rats had

lower birth weights as cornpared to conh l unexposed animals. However,

16 animals exposed only to tobacco smoke had the most severely affected birth

weights.

In a study by Reznik (1 980), pregnant Sprague-Dawley rats were exposed

to the smoke of different research cigarettes with varying amounts of NIC,

condensate and CO (73). The birth weights and lengths of fetuses were reduced

in a dose-dependent fashion, meaning that the higher the dosage of CS given,

the lower the birth weights were.

Leichter (1 995) exposed pregnant Sprague-Dawley rats (21 0-240 g) to the

CS of commercial cigarettes (1.4 mg N1C) from day 1 to 21 of gestation (43). CS

exposed pups were significantfy smaller compared to control and pair-fed groups.

However, this difference was no longer present at one and two weeks

postnatally. Thus, this study concluded that fetal growth restriction caused by CS

exposure did not persist after birth, contradictory to the results observed in

human studies.

1.3.3.1. Effects on Fetal Organ Weights: Human Studies

Epidemiological studies in humans have shown that the placenta is

disproportionately large at delivery in smokers (68). The peripheral villous tree

which is the major component of the placental wéight, adapts to hypoxic stress,

such as high altitude or matemal anemia. The data suggests an adaptive

17 response of the fetal capiliary bed within the placental villi of smokers. The

study predicted that this response would increase the surface area for diffusional

exchange and thus compensate for the impaired oxygen transport caused by the

CO that displaces oxygen from hemoglobin in both matemal and fetal blood. The

sample size in this study was quite small (n = 4 smokers, and n = 4 non-

smokers) and thus with a larger sample size, these results may or may not differ.

Numerous studies have also shown that there is an increased placenta

coefficient (ratio between placenta weight and birth weight) in smokers. These

studies attribute the increased coefficient to a decrease in birth weight while

placental weight remains the same. This differs from the observation that the

hypoxia caused by smoking could result in placental hypertrophy, which

compensates for such fetal hypoxia (1).

1.3.3.2. Effects on Fetal Organ Weights: Animal Studies

In a study by Bassi etal., (1984), fetal brain, lungs, Iiver and placenta

were weighed and examined for any gross malformations at day 21 of gestation

in both control and smoke exposed groups (Table 1) (6).

18 Table 1. Fetal Organ Weights in Control and Smoke Exposed Groups

! l

Organ 1 Controi ! Smoke Exposed i N 1 l I I

i Brain (mg) 1

I 1 1 18425.4' 1 221 t 9.1 5 - 7 1 I i !

I l I I

: Liver (mg) 491 i 20 346 2 19' I I 5 - 7 i 1

! ! I 1 I

5 - 7 Fetal Weight (g) 1 5.85 i 0.13 4.60 5 0.16' 1 1

i

1 I ! 1 I

I l 1

'Significantly different from control, p < 0.05.

Bassi JA et al., Pediatric Research:18(2), pp. 727-i29, 1984 (6).

I j I I

Fetal heart and lung weights were measured in a study by Mortola et al.,

j Lungs (mg)

(1990) which focused on stunted body growth in the chronically hypoxic newborn

155 F 6.7 5 - 7 I

animai (55). In the control group, fetal rat lung and heart weights were 0.046 and

0.017 g, respectively, at postnatal day 7. In this study, severe hypoxia (10% O*)

1 108 2 8.0' l I j I

Placenta (mg) / 604 c_ 46 522 i 63 5 - 7 I l i I 1

caused growth restriction along with hypoplasia in these hivo organs.

Histological changes in the Iungs, liver, stomach, kidneys and intestinal

tissues have also been observed (27). The Iung tissues of smoke exposed pups

shawed evidence of greaUy enhanced apoptosis, mesenchymal changes and

branchial muscle hyperplasia. Liver tissues were also observed to exhibit striking

abnomalities in hernatopoiesis and bile-duct cell proliferation. In addition,

19 immature glomeruli of the kidney and hypoplasia of the intestinal villi were

common among newborns of smoke exposed mothers compared with controls.

Effects on Carboxyhemoglobin

CO is a colorless, odorless and tasteless gas that is produced by

incomplete combustion of hydrocarbons and is found commonly in the extemal

environment and is a major component of CS (48;49). Maternal smoking during

pregnancy may induce chronic fetal hypoxia which may be due to impaired

oxygen transport capacity because of increased carboxyhemoglobin (COHb) in

the mother and the fetus. CO binds to hemoglobin with a 200 times greater

affinity than oxygen and foms COHb. By cornpeting with oxygen for hemoglobin,

CO decreases the capacity of the blood to transport oxygen. Therefore, CO

readily displaces oxygen from hemoglobin even in small concentrations and thus

interferes with tissue oxygenation.

Values of COHb in normal non-smoking pregnant women have been

reported to be 0.4 - 2.6% (48;49). Several factors influence COHb percentages

in the pregnant individual including the individual's endogenous CO production,

rate of CO excretion, affinity of maternal hemoglobin for CO, the concentration of

exogenous CO in inspired air, fetal endogenous production of CO and also the

rate of exchange of CO across the placenta. When a pregnant woman smokes.

maternal COHb concentrations range fiom 2-0 - 8.3% with concentrations of up

20 to 14% reported in one study (48;49). Mean COHb in the fetuses of smoking

humans ranged from 2.4 - 7.6% with levels of up to 9.8%. An increase in fetaI

COHb to 9% is equivalent to a 41% reduction in blood flow or hemoglobin

concentration (48).

COHb has also been measured in pregnant rats that have been exposed

to CS. In studies by Bassi et al., (1984), percent CO saturation which was

between 9 and 20% was used as an index of the amount of CS inhaled by

animals to make sure that al1 animals inhaled similar amounts of CS (6). In a

study by Nelson et al., (1999) pregnant rats were exposed to sidestream smoke

(SS) (smoke emitted from the end of a burning cigarette, also known as second

hand smoke) and COHb increased from 1.2% in controls to 6% (exposure to 1

cigarette) and 12.2% (exposure to 4 cigarettes) (28).

1.3.5. Effects on Hematocrit

Elevated hematocrit has been reported in smoking mothers and their

babies (54). Hematocrit is the fraction of blood that is composed of red biood

cells (31). Normal hematocnt values have been reported as being between 30

and 36% in humans (13;31). In adults a positive correlation exists between

hematocrit and CS consumption, that is the more cigarettes that are consumed,

the higher the hematocrit (22;36;54). The high hematocrit in cigarette smokers

may be caused by elevated blood levels of CO, which limits tissue oxygen

21 canying capacity and stimulates eryttiropoiesis (36). ln addition, smoking in

adults may induce polycythemia and thus decrease the plasma volume (22). In

pregnant wornen, a hematocrit higher than 38 and a high erythrocyte count (2

4.51~1) was associated with low birth weight (c 2,500 g), preterrn birth (< 37

weeks) and pregnancy induced hypertension (39).

Smoking is thought to produce hypoxic conditions in the fetus (54). This in

turn wiil stimulate fetal erythropoiesis, which causes an increase in red cell

production and ultimately an increase in tissue oxygenation. Hematocrit in infants

of smoking and non-smoking mothers after one day of birth, was 49 - 7S0I0 and

48 - 67%. respectively (p < 0.05). Another study by D'Souza et al., (1978) found

that babies born to smoking mothers had a higher mean capillary blood

hematocrit during the first 48 hours of life (22). Similar studies by Bulterys, et al..

(1990) found increased values of hematocrit in the cord blood of infants of

smoking mothers which was likely to be caused by induction of fetal

erythropoiesis (1 3).

Conflicting results have been observed when hematocrit was measured in

both pregnant and non-pregnant rats at various tirne periods (42). In non-

pregnant rats, hematocrit was 42.1 c 1 .l%, which was significantly higher than

hematocrit in pregnant rats. Hematocrit in pregnant rats during gestation (day 2

to day 19) was 39.2 I 2.0, while in the perinatal period (gestational day 18 - 20

22 to postnatal day 2) hematocrit dropped significantly to 35.3 + 2.6. In the

postnatal period (postnatal day 1 - 9) hematocrit was 39.4 5 1.6.

1.3.6. Effects on Glucocorticoids

The rate of adrenocortical secretion of glucocorticoids is increased

moderately throughout the duration of pregnancy (31). It is possible that the

glucocorticoids help mobilize amino acids directly from mother's tissues, which

can then be utilized in fetal tissue formation. In addition, several studies have

observed increased cortisol, and 17-hydroxycorticosteroid in humans aller

cigarette smoking (1 2; 16;17;53). In addition, elevated CORT concentrations have

been found in both rats and dogs after NIC injections (16;53). This increase in

plasma glucocorticoid levels is thought to be mediated through the release of

adrenocorticotropic hormone (ACTH) from the anterior pituitary gland

(1 2;%;17;37;53).

In addition, exposure to any kind of physical or psychological stress has

been associated with a rise in plasma glucocorticoids which does adapt with

prolonged exposure to the stressor (16)- In a study by Henry et al., (1994) (34),

plasma CORT concentrations were significantly elevated in prenatally stressed

rats at 3 and 21 days of gestation after exposure to postnatal stress. At 90 days

of age, prenatally stressed rats showed a longer duration of CORT secretion

after exposure to stress. Thus the findings suggest that prenatal stress produces

long term changes in the hypothaiamo-pituitary-adrenai

offspring .

23 (HPA) axis in the

Upon comparing our two rnodels of CS and NIC exposure, both models

may elicit a CORT response due to some type of stress. In the CS model, there

is frequent handling of animals, effect of CS exposure, cardiac puncture, effect of

anesthesia itself, and the actual injection of the anesthesia. In the OMP model,

the animals are handled much less frequently; however. stress from surgery, the

weight of the OMP mounted un the back of the animal, cardiac puncture, effect of

anesthesia itself, and the injection of the anesthesia still exist. These factors of

stress in both models make CORT concentrations a desirable variable to be

measured.

1.4. ANIMAL MODELS ADMlNlSTERlNG CIGARETTE SMOKE AND

NICOTINE

Several investigators have established models for CS and NIC infusion in

animals.

Animal ModeIs of Cigarette Smoke Inhalation

Younoszai et al., (1 969) exposed pregnant Holtzman albino rats to CS by

24 placing rats in a chamber five times daily for 4 minutes from day 3 to 22 of

gestation (90). Three types of cigarettes were used in these studies: (1) tobacco

cigarettes each containing 15 mg of NIC, (2) lettuce leaf cigarettes, and (3)

lettuce leaf cigarettes to which 15 mg of NIC had been added.

In a study by Reznik (1980), pregnant Sprague-Dawley rats were exposed

to the smoke of different research cigarettes with varying amounts of NIC,

condensate and CO (73). Smoke exposures were done through the use of a

smoking machine (Hamburg Il). In one exposure, ten animals received the

smoke of 30 cigarettes (puff volume 35 ml; puff duration 2 sec; puff frequency

Ilmin) in a chamber for 7 - 11 minutes depending on the type of cigarette.

Bassi et al., (1984) also conducted CS chamber studies in which pregnant

Sprague-Dawley rats were exposed to CS from day 5 to 20 of gestation using the

P and I Walton Exposure Machine (6). Animals in three groups (control, CS

exposed and pair-fed) were exposed in cycles of 7 minutes, 16 times a day, to

the puff of three lighted cigarettes pushed into the exposure chamber. In a typical

1 minute cycle of operation, each puff of smoke was held in the chamber for 30 s,

and the chamber was diluted with fresh air for the next 30 seconds.

Leichter (1 995) exposed pregnant Sprague-Dawley rats (21 0-240 g) to CS

using a chamber (43). Two rats were pIaced inside the Plexiglass chamber with

openings where the commercial cigarettes (1.4 mg NIC) were placed for a 2 hour

25 period each day from day 1 to 21 of gestation. Each animal was exposed to

the smoke of 10 cigarettes over the short two hour period.

The CS studies described above had some limitations that are worthy of

attention. The first is that these studies used chambers for the administration of

CS. This does not accurately simulate human smoking. The chamber studies are

limited, because not only does this method allow NIC absorption through the

skin, but if any constituents are caught in animal's skin. these rnay be ingested

when animals are cleaning themselves. ln addition, with chamber systems

smoke distribution may not be uniform and could depend on the dimensions of

the chamber and the position of the animal.

The second limitation of these studies is that none of the above studies

rneasured plasma NIC concentrations at any time during the pregnancy. These

concentrations are important if one is to compare rat CS studies with human CS

studies.

Recent Studies in our Laboratory

In Our laboratory, a CS exposure made1 was recently established and it

showed that rats exposed to 1000 ml of CS from day 6 to day 21 of gestation

produced plasma NIC concentrations that were similar to those seen in moderate

smokers (day 1 of exposure = 65.6 2 9.5 ngiml and day 21 of exposure = 22.9 I

26 5.9 nglml). Thus this mode1 circumvented the limitations of prior studies in that

plasma NIC concentrations were measured, puff volume was known and

dosages were comparable to those observed in moderate smokers. However,

these exposures began on gestational day 6, after embryo implantation, which

occurs on day 5. Although implantation was measured in these studies, it is

unclear whether implantation would be affected if CS exposure began prior to

embryo implantation. Therefore, the studies in this thesis intended to begin

exposures on gestational day 2, so that the embryo implantation effects could be

observed when pup numbers, weights, and organs are measured. In addition,

since these exposures have a longer duration than those in previous

expenments, it will be interesting to observe whether plasma NIC concentrations

decrease further with prolonged penods of smoke exposure.

Previous studies in Our laboratory along with the experiments described in

this thesis differ from that of the previous studies in that they are nose only

exposures. This type of exposure system has h o main advantages which are

that rats are exposed to CS in a manner which closely parallels human smoking

and that this type of system minimizes the amount of CS particles depositing in

animal's fur. Our studies using this system also expose anirnals to srnaller doses

of CS giving plasma NIC concentrations similar to those seen in moderate

smokers. In addition, instead of exposing animals to al1 of the cigarettes in a

short tirne period, in our study design. cigarettes are given at regular intervals

throughout the day, sirnilar to human srnokers.

1.4.3. Animal Models of Nicotine Infusion

. - Mumn et al., (1987) examined different methods of administering NIC to

rats. Through these studies they observed that drinking water containing NIC

was unpalatable to the rats (57). As well, oral NIC administration interfered with

nutrient absorption. In addition, subcutaneous injections of NIC produced sudden

spike concentrations of NIC in the plasma and presumably also in the fetus.

Although the acute effects of NIC can be mimicked by iniection of NIC, the spikes

of NIC can cause hypoxia in the fetus due to constriction of the placental

vasculature. Thus OMPs were found to be the best model in which to administer

NIC and are used widely as a method of NIC delivery (1;2;4;5;22;24;38-

41;43;57;60;77:80:81;82). No significant effects on food or water intake or on

weight gain were observed, especially after using a short-term anesthetic for

pump imptantation. As well, since the anirnals were not handled frequently,

stress levels were postulated to be lower than with other methods of NIC

administration. Although OMP infusion of NIC does not accurately simulate the

actions of a hurnan smoker, this method was stiIl found to be the best way to

administer NIC compared with previous methods.

Mumn et al., (1 987) administered 1.5 mg/kg, 4.5 mglkg, and 13.5 mglkg of

NIC in pregnant Sprague-Dawley rats on day two of gestation at exact weights

(-250 g) (Section 3.2. a.) (57). Matemal plasma NIC concentrations were

measured two days after delivery from tmnk blood. Plasma NIC concentrations

28 were 23.3, 66.6 and 180.0 nglml for the 1.5, 4.5 and 13.0 mglkglday dosages

of NIC, respectively. No fetal measurements were obtained in this study.

In a study by Schuen et al., (1997) OMPs delivered 12 mglkglday of NIC

bitartrate throughout gestation and one week postnatally in pregnant rats based

on projected weight (Section 3.2. a.) (77). Matemal blood was withdrawn from

the tail vein for NIC assay at some point during the pregnancy as well as

following delivery. Materna1 plasma NIC concentrations were 134 i 42 nglml

during gestation, and 70 + 13 nglml after gestation. There was a significant

difference between pup weights of the NIC exposed and the saline exposed rats.

In addition, no significant difference in litter size was obsewed between the two

groups.

Another study by Bamford et al., (1999) implanted pregnant rats with

OMPs delivering 6 mglkglday ai projected weights of NIC or saline for 4 weeks

on day 3 of gestation (4). There was a consistent and statistical reduction of

postnatal pup weights when comparing the NIC exposed group and the saline

group.

Navarro et al., (1988) impfanted pregnant rats with OMPs administering a

dosage of 6 mglkglday of NIC at projected weight on day 4 of gestation. NIC

infusion did not cause matemal death but did slow weight gain for the first 3 to 4

days of treatment, after which normal weight gain was resumed (61).

In subsequent studies by Slotkin and colleagues (1987, 1989, 1993, and

1995), both 2 and 6 mglkglday of NIC was admisiistered in pregnant rats at

projected weights on day 4 or 5 of gestation (60:79;80;81).

1 S.1. Cigarette Smoke Exposure

lnvestigators have established models of CS exposure and NIC infusion in

animals. CS inhalation models expose rats to CS using chambers. This mode of

CS exposure does not ensure that CS will be uniformly distributed to animals. In

addition, CS can be absorbed through the skin and CS constituents may be

deposited on fur and ingested by the animal during the cleaning process.

Although CS constituents are afso deposited on human skin and may be

absorbed, smoke is also camed away into the atrnosphere while in the rat, most

of the smoke remains in the chamber. Our CS model will circumvent this problem

and will provide a model that more cioseiy simulates human smoking. This model

will also address the issue of embryo implantation and whether this is affected by

earlier smoke exposure (gestational day 2).

Osmotic Minipump Nicotine infusion

Other investigators have chosen to administer only NIC in animals to

observe whether this constituent is responsible for some of the adverse effects of

smoking. NIC has been adrninistered orally. via intravenous injections and NIC

has also been infused via OMPs. Of the models used in NIC administration,

OMPs were found to be the best method of NIC infusion. However, when

investigators used this infusion model, most of ttiern did not measure plasma NIC

concentrations and fetal parameters such as fetal birth weights, litter sizes, and

fetal organ weights. With our model of NIC infusion, using OMPs these variables

will be addressed.

1.6. SPEClFlC AlMS

Specific Aim 1:

Establish a CS inhalation model to investigate the changes in matemal NIC

concentrations at various days of gestation after exposure to CS from day h o to

22 of pregnancy.

Specific Airn 2:

lnvestigate the effects of CS exposure starting during the preimplantation period

on pregnancy outcome (Le. number and size of pups) to test the hypothesis that

the CS exposed animais will have decreased pup numbers and bitth weights.

Specific Airn 3:

Establish a NIC infusion mode1 to deterrnine the maternai plasma N1C

concentrations at various pregnancy intervals when NIC is administered via

OMPs.

Specific Aim 4:

lnvestigate the effect of CS and NIC exposure on maternal COHb and maternal

hematocrit along the gestational age, to test the hypothesis that COHb will

increase with CS exposure, and that hematocrit will be higher as the pregnancy

continues in both groups.

Specific Aim 5:

lnvestigate the effects of chronic prenatal administration of CS and continuous

infusion of NIC on matemal plasma CORT concentrations in rats throughout the

pregnancy.

32 2.0 MATERIALS and METHODS: ClGARElTE SMOKE EXPOSURE

Time-dated pregnant Sprague-Dawley rats (CS group - 49: sham group -

8) weighing 220 - 270 g were exposed to 1000 ml of mainstream smoke (MS)

(10 ml puff volume x 10 cigarettesiday x 10 puffs per cigarette) or roorn air

respectively using the Smoke Exposure System (Tobacco Research Council,

University of Kentucky). A separate group of control rats (n = 9) that were not

exposed to CS or air was also used. The rats were mated at the Biosciences

Department, University of Calgary and pregnancy was confirmed by the

formation of a vaginal mucus plug. The first day after mating was considered to

be day 1 of the pregnancy. The rats were housed individually in breeding cages

in the Biohazard area of the Heritage Medical Building, University of Calgary.

Temperature in the facility was maintained at 20°C with a 12 hour light to dark

cycle. Humidity in the room was kept between 40% and 60%. Food (rat chow,

Lab Diet 5001 ) and tap water was provided ad libitum except during exposures.

All experimental procedures were carried out in accordance with the

"Guide to the Care and Use of Expenmental Animals" provided by the Canadian

Council on Animal Care and with the approval of the Animal Care Committee at

the University of Calgary.

2.2 SMOKE EXPOSURE SYSTEM

The Smoke Exposure System (Figure 2.2.2.) has the capability of

providing al1 three modes of exposures (i.e. MS only, SS, or MS + SS) plus Sham

(room air) exposures. MS is the smoke inhaled directly in the lungs from a

buming cigarette while SS is the smoke emitted from the end of a buming

cigarette. The system was developed at the Tobacco Research Institute,

University of Kentucky for nose-only exposure of small rodents (rats, mice and

guinea pigs) to MS andlor SS from the same cigarette and is designed so that al1

anirnals receive modest and similar quantities of smoke simultaneously.

2.2.1. General Description

The smoke exposure system has three main components: smoke

generation, smoke distribution and the animal exposure units.

Smoke Generation: The three main components of this unit are the puffer, the

pump and the SS collection chamber. The puff volume can be adjusted to any

desired amount by adjusting the Cam on the puffer or Sy calibration of the

airlsmoke flow through the main vacuum Iine, waste line and purnp's head line.

Under standard conditions, one puff is produced every minute and lasts

approximately 2.4 seconds. The smoke generated during the puff is transferred

34 to the smoke distribution units. While MS is generated intemittently, SS is

generated continuously.

Smoke Distribution: The smoke distribution unit distributes both MS and SS to

the exposure blocks. The MS distribution unit consists of one pump, two pump

heads (catalogue no. 7015) and a recycle diluter. Generated MS gets diluted to

50% before being distributed to the animals. It takes about 15-30 seconds for the

smoke to clear the recycle diluter. The SS distribution unit has one pump and

one purnp head (catalogue no. 7015). The desired amount of generated SS c m

be distributed to the animals.

Animal Exposure Units: The smoke exposure portion has two exposure blocks

MS and SS and it is designed to expose 8 rats to MS andlor 8 rats to SS smoke

simultaneously from the same cigarette. In the case when only one exposure

block is used. the unused exposure ports are closed using rubber stoppes.

The system consists of additional parts including a vacuum source, a flow

meter used for airfiow calibrations, and filters CF1, CF2, and CF3 which are

placed in given locations and changed three times daily.

Aifflow and PufF Volume Calibrations

A vacuum swrce with two vacuum control valves was required to operate

35 the system. A flow meter was provided for calibration of airfiow through the

lines. Air fiow through the pump heads Iine was adjusted by connecting the flow

meter to the suction end of the pump head tubing, and adjusting the speed dial

on the pump to obtain the desired flow. Airfiow through the main vacuum line and

waste line was calibrated by connecting one line at a time to the flow meter. The

desired flow was achieved by opening the vacuum control valves until the

desired airflow was achieved. For example, in order to generate a 40 ml puff

volume in MS exposures, the airfiow through the main vacuum line was 40

mllsec, 16.7 mllsec through pump heads line and 16.7 mllsec through the waste

line. It is important to note that the flow meter was always disconnected during

the exposure sessions.

Operation and Maintenance

Before each exposure was started, the MS pump was warmed up for at

least 10 minutes and operated in the reverse position. Calibration of the srnoke

exposure system was done every day before and after the first and last cigarette

respectively and a control run (without animals) was performed to monitor the

machine's performance. In addition, the pump head tubing was changed daily

and other machine parts and connectors were cleaned with 70% ethanol. Before

the exposures began, animals were placed in wire mesh restrainers and loaded

onto the machine. In sharn exposures, the MS pump was replaced by the sham

pump with the speed and volume similar to the MS pump.

PPimc<mr P U S VOLUME - 4 ml WiT DURATION - 2 4 ur 7015 PUMP HEAD R O W - 16.7 mllwrr CF3 FILTER FLOW- 16.7 rnUsa=

Figure 2.2.2. Smoke Exposure System

2.3. RESEARCH CIGARETTES (2Rl)

Research cigarettes (2R1) with a resistance to draw 8.94 cmHnO, 660

static burn sed40 mm, 85 cm long, 25 mm circumference, paper porosity of 47.6

sed50 cc and containing 2.46 mg of NIC were used to match the commercial

brands. The reference research cigarettes were purchased from the Tobacco

and Health Research lnstitute where they were in cold storage (3.3% and 50-

60% humidity). One day prior to shipment, the cigarettes were removed from cold

storage and placed in a humidity-controlled room (23.8% and 60% relative

humidity) for equilibration,

The reference cigarettes were placed in a plastic airtight bag and stored in

a standard laboratory refrigerator untif used for research. This procedure was

suitable to hold the proper moisture Ievel in the cigarettes and is recommended

by the Tobacco and Health Research Institute. ln addition, before the cigarettes

were used for research they were exposed to room temperature for

approximately 15 minutes. Each puff lasted 2.4 seconds at a puff volume of 10

ml. The system was calibrated every day by using a flow meter (Tobacco

Research Council. University of Kentucky) mode1 number: 150ECA2AEAl A) in

order to achieve a given puff volume.

2.4. EXPERIMENTAL PROTOCOLS

2.4.1. Plasma Nicotine Concentrations

Establish a CS inhalation model to investigate the changes in materna1 NIC

concentrations at various days of gestation after exposure to CS from day

two to 22 of pregnancy.

Time-dated pregnant Sprague-Dawley rats (200 - 270 g) were exposed to

CS (Group 1, CS) or air (Group 2, sham) via the Smoke Exposure System

(Tobacco Research Council, University of Kentucky) from day 2 until 22 of

gestation.

Eight rats were exposed at the same tirne, as this is the maximum

capacity that the Exposure System can accommodate at any given time.

Group 1 CS: These rats were exposed to 1000 ml of MS. Animals were weighed

every other day in the morning after the first exposure.

Group 2 Sham: Eight time-dated Sprague-Dawley rats (250-270 g) were

attached to the Smoke Exposure System and exposed to air in the same manner

as the rats in Group 1. They were exposed to air via a 'sham pump" from the 2nd

39 day of gestation to the 22nd day of gestation. The purpose of this was to have

a group of rats that had been handled and restrained but not been exposed to

CS.

For both groups, on days 2, 8, 15, and 22 of pregnancy, 5 ml of maternal

blood was withdrawn via cardiac puncture under general anesthesia (sodium

pentobarbital; 40 mglkg - Animal Care Committee, Standard Operating

Procedure; A7 method #2) (Table 1).

For the quantitation of NIC in serum, 3-5 ml of maternal blood was

collected in heparinized glass tubes and centrikiged at approximately 1860 rpm

for 12 minutes at 4°C. Plasma was subsequently collected and stored at -70°C.

Plasma çamples were analyzed thereafter using the Gas Chromatography-Mass

Spectrometry (GC-MS) method (Section 2.5) (Toxicology Lab, Health Sciences

Center) to obtain plasma NIC concentrations.

Table 2. Summary of CS Experimental Design

Group Dosage Number of Rats Sacrificed on Designated

Day of Exposure

Day 1 Day 7 Day 14 Day 21

1 CS (1000 ml) 9 9 8 7

2 Sham (air) 1 1 1 5

Fetal Weights and Litter Sire

lnvestigate the effects of CS exposure starting during the preimplantation

period on pregnancy outcome (Le. number and sire of pups) to test the

hypothesis that the CS exposed animals will have decreased pup numbers

and birth weights.

In groups 1 and 2 after matemal blood was withdrawn on the 22" day of

gestation, pups from both the left and right uterine horns were removed, weighed

and counted. In addition, fetal organs at gestational day 22 were dissected,

weighed and snap frozen for future studies. Fetal organs were also extracted

from a control group of animals for comparison.

2.4.3. Carboxyhemoglobin and Hematocrit

lnvestigate the effect of CS exposure on materna1 COHb (54) and matemal

hematocrit along the gestational age, to test the hypothesis that COHb (%)

and hematocrit will be higher in the CS exposed animals as compared with

the sham group.

After completion of exposures on day 2, 15, and 22 of pregnancy, 5 ml of

matemal blood was withdrawn as mentioned previously and from this a 1 ml

aliquot of blood was obtained to measure COHb (%) and hematocrit at various

4 1 tirne points of gestation. The blood gas analyzer used to measure COHb was

suitable for rat blood analysis and the temperature of the blood gas analyzer was

modified to 37OC (IL 1301: Instrumentation Laboratory System, 482 Co-

Oximeter). In addition, hematocrit samples were spun using a microhematocrit

centrifuge (DamonllEC Division) for 5 minutes after which the hematocrit value

was measured.

2.4.4. Corticosterone Concentrations

lnvestigate the effects of chronic prenatal administration of CS on maternal

plasma CORT concentrations in rats throughout the pregnancy.

It is known that restraint stress of the mother increases maternal CORT,

which can cross the placenta1 barrier and affect the fetal growth and adrenal

function. Since the CS model requires hourly daytime handling of rats, restraint

and chronic prenatal CS administration will likely cause stress in the animal and

thus it was important to measure CORT concentrations. For the quantitation of

CORT in plasma, samples were collected as previously rnentioned (Section 2.4.

4.) and analyzed using the radioimmunoassay rnethod.

2.5. CARDIAC PUNCTURE PROCEDURE

BIood samples for NIC, CORT assays, COHb (%), and hematocrit were

42 cotIected by cardiac puncture using a 23 gauge; 3 needle attached to a 5 ml

heparinized syringe. Prior to cardiac punctures, each pregnant animai was

anesthetized using sodium pentobarbital (40 mglkg). Once the animal no longer

demonstrated a paw withdrawal reflex, cardiac punctures were perfomed.

Usually, three to five ml of matemal blood was withdrawn. Because of NIC's

short half-life in rats (60 minutes) al1 cardiac punctures were performed within 15

minutes following the last cigarette. Blood samples were collected in heparinized

glass tubes and centrifuged at 1860 rpm for 12 minutes at 4OC. Plasma was then

collected and stored at - 70% until plasma NIC and CORT assay were

conducted.

2.6. DETERMINATION OF PLASMA NICOTINE AND CORTICOSTERONE

CONCENTRATIONS

2.6.1. Gas-Chromatography Mass-Spectrornetry Method

Plasma NIC concentrations were determined by the GC-MS method at the

Center for Toxicology, University of Calgary. These analyses involved a nurnber

of steps.

Step 1: Before any extraction of NIC occurred, an internai standard,

deuterium NIC, was added. Deuterium NIC has properties similar to NIC.

43 Step 2: NIC was extracted from the biological matrix (water or plasma)

and placed in organic solvent. The organic layer was taken into a tube and put

through a concentrating process (drying) to get a 10 - 20 x concentration.

Step 3: One pl of concentrate was injected into the gas chromatograph

(Hewlett Packard GC 6890 System).

Step 4: Inside the gas chromatograph is a 20 m column made of fused

silica and helium gas, which pushes the sample through the column. The organic

solvent interacts with the column and the stronger the interaction, the slower the

molecules will proceed down the column. The time that the compound peaked or

came out onto the chmmatograph is called the retention time. Usually the

retention time is very specific to each compound with an accuracy of 0.02 min.

The retention time for NIC is 4.78 min and the chance of another compound

having the sarne retention time is marginai.

Step 5: Once the sample came out of the gas chrornatograph, it went into

the mass selective detector (Hewlett Packard 5973, USA) which can give the

"fingerprintn of a compound. The mass spectrometer gave two signals of NIC and

deuterium NIC and also produced a calibration curve (5-500 nglml). A

cornparison of these two signais produced the plasma NIC concentrations. it is

also important to note that a quality control was run alongside the sarnple with

the precision of 1 % for each sample analyzed.

Radioimmunoassay Method

Rat plasma CORT concentrations were measured by the

radioimmunoassay method using a procedure developed by the Endocrine

Sciences' laboratory (Calabasas Hills, California).

After nonpolar solvent extraction, duplicate aliquots of sample extract were

transferred to assay tubes, dried and incubated for three hours with an antibody

made to a CORT-3-oxime-albumin conjugate. Recovery of the hormone was

monitored with 3H-CORT. Assay reliability was checked using one water blank,

three control pools and a 1000 PQ accuracy standard. Ammonium sulfate was

added after incubation, to separate antibody-bound and free steroids. Free

CORT was quantified by liquid scintillation counting. The amount of CORT in

each sample was calculated from a curve derived from purified CORT standards

ranging O - 500 PQ. Sensitivity of the method was 1 pg/dl when employing the

usual 0.01 ml aliquot of senim.

3.0 MATERIALS and METHODS: NICOTINE INFUSION STUDIES

OMPs are miniature pumps that continuously deliver test agents at

controlled rates into various species of experimental animals (Figure 3.1 -1 .).

When these pumps are implanted subcutaneously, they serve as a constant

source of prolonged drug deiivery. These pumps offer an alternative to repeated

intravenous or intrapentoneal injections, which minimizes animal handling and

stress. The pumps are composed of three layers: the drug reservoir, the osmotic

sleeve, and the rate controlling, semipermeable membrane. There is also an

additional component, the flow moderator (21-gauge stainless steel tube with a

plastic end-cap) which is inserted into the body of the pump after filling with NIC.

It is also important to note that the pump should be inserted with this delivery

portlflow moderator side first. This model 2ML4 pump contains approximately 2

ml of solution and has an effective pumping life of at least 28 days (Figure

3.1 -1 .).

Delivery portal

- Removable cap

- Flange

- Flow moderator

- Impermeable reservoir wall

- Osmotic agent

- Semi permeable membrane

+ Aqueous environment

, Reservoir

Figure 3.1 .l. Cross-section of OMP showing design, components and

mechanism of operation

3.2. EXPERIMENTAL PROTOCOLS

3.2.1. Plasma Nicotine Concentrations

Establish a NIC infusion model to determine the materna1 plasma NIC

concentrations at various pregnancy intervals when NIC is administered

via OMPs.

To establish the dose of NIC in OMPs needed to achieve a plasma NIC

concentration of 30-60 nglml, pregnant Sprague-Dawley rats (220-270 g) (NIC

group - 85; saline group - 27) were lightly anesthetized on day 2 of gestation

using halothane and oxygen and a two to five cm incision was made in the

interscapular region. An OMP containing a prepared solution of a known

concentration of NIC bitartrate (dosages of 2 or 6 mglkglday) or saline was

implanted port side first (Alzet, 2ML4 flow rate = 2.5 pllhr) (Figure 3.1.1 .). The

incision was closed with 4-5 sutures of 3.0 vicryl. The rats recovered a few

minutes after the surgery was completed and one day was given to allow for

priming of the pump.

The dams were separated into five groups each receiving a different

dosage of NIC or vehicle, as shown below. NIC dosages of 2 mglkglday and 6

mglkglday were calculated for two groups at the exact weight of the dams on the

day of surgery, and for two groups at 350 g, the projected weight at the end of

48 pregnancy. Exact weight refers to the weight of the animal at the time of pump

implantation while projected weight refers to the estirnated weight of the animal

at term (350 g), as used in previous studies.

Table 3. Summary of OMP Experimental Design

Group Dosage Number of Rats Sacrificed on Designated

Day 1

2 mglkglday at exact wt 4

2 mglkglday at projected wt 4

6 mglkglday at exact wt 4

6 mglkglday at projected wt 4

Vehicle or Sham (saline) 2

Day of Exposure

Day 21

6

6

6

6

2

Following surgery, rats were housed individually with free access to food

and water. The rats were weighed every other day of gestation. Three to five ml

of matemal blood was obtained by cardiac puncture on days 4 and 21 of

gestation (Section 2.5). Thereafter, the rats were sacrificed using sodium

pentobarbitone (40 mglkg - in accordance with the Canadian Council on Animal

Care Guidelines). For the quantitation of NIC in serum, the materna1 blood was

collected in an anticoagulant free tube and centrifuged at 1860 rpm for 12

minutes at 4°C. Serum was then collected and stored at -70°C for assays at a

later date. The GC-MS Method (Toxicology Lab, Health Sciences Center) was

used :O detect NIC (Section 2.6.1 .),

For dams sacrificed at term (day 21), a rnidline cesarean section was

performed exposing both the left and right uterine horns. Pups and placentas

from each side were counted, removed and weighed. Brain, heart, lungs, liver,

and kidneys were dissected from the proximal, middle and distal pups and were

also weighed.

3.2.2. Hematocrit

lnvestigate the effect of NIC exposure on materna1 hematocrit along the

gestational age, to test the hypothesis that hematocrit levels would be

hig her as the pregnancy continues.

OMPs containing a prepared solution of a known concentration of NIC

bitartrate or saline were implanted as described previously. On various days of

gestation (4 and 21), three to five ml of matemal blood was obtained as

mentioned previously. Hematocrit levels were rneasured using methods that

have already been described (please refer to pages 40 - 41).

Corticosterone Concentrations

lnvestigate the effects of chronic prenatal administration of continuous

infusion of NIC on materna1 plasma CORT concentrations in rats

throughout the pregnancy.

The OMP model has been cited as causing minimal stress in the animal,

however, no parameters of stress in pregnant rats are available in the literature.

Thus, it would be important to investigate CORT as an indicator of stress in this

group. For the quantitation of CORT in plasma, samples were collected and

analyzed using the radioimmunoassay method (Section 2.6.2.).

4.0. STATISTICAL ANALYSES

Plasma concentrations of NIC and CORT, as well as blood COHb (Oh) and

hematocrit levels in CS, sham, NIC and saline treated animais were compared

within each and behveen the groups using analysis of variance. To test the

differences in materna1 weight gain in both the CS and NIC studies throughout

the gestation, a method for repeated measures with the Generalized Estimating

Equations (GEE) analysis was used. Pup weights and pup numbers in the CS

exposed group were analyzed using the Student's t-test, whereas pup waights

and numbers in the NIC and saline groups were examined using analysis of

variance.

In certain instances, additional tests were perforrned using statistical

models, which are analogs of the analysis of variance based on regression

analysis. As an adjustment for multiple comparisons, Bonferroni corrections were

used when considering the pairwise comparisons.

All results are presented as mean i SD, and a p-value of < 0.05 is

considered to be statistically significant.

5.0 RESULTS IN CIGARETTE SMOKE EXPOSED RATS

5.1. PLASMA NICOTINE CONCENTRATIONS (nglml)

5.1 .l. Plasma NIC Concentrations in Pregnant Rats after Exposure to 1000

ml of CS From Day 2 of Gestation Compared with Rats Exposed to

Room Air

Exposure to CS on day 2 of gestation led to concentrations that were 45.4

i 7.5 nglml (Figure 5.1.1.). Plasma NIC concentrations decreased with

continuous exposures on day 7 foliowed by a signifiant rebound on day 14. At

day 21, plasma NIC concentrations once again significantly decreased.

Significant differences were also observed in plasma NIC concentrations at day 7

of exposure, compared to concentrations at day 1 and 14. In addition, plasma

NIC concentrations at day 1 of exposure were significantly higher than at day 21

of exposure.

5.1.2. Plasma NIC Concentrations in Pregnant and Non-Pregnant Rats

Exposed to 1000 ml of CS

Pregnant rats that were exposed to CS from the second day of pregnancy

showed a steady decline of NIC concentrations as the pregnancy progressed. In

non-pregnant animals, the same decline of plasma NIC concentrations was

53 ubserved throughout gestation. It is important to note that at day 1 of

exposure, it was impossible to distinguish between pregnant and non-pregnant

rats and thus concentrations at day 1 are the same for both groups (Figure

5.1.2.). The concentrations at day 21 of exposure in the pregnant group were

19.2 .c 5.2 ng/ml compared to 12.0 + 6.7 nglml in non-pregnant animals. There

was a significant difference in plasma NIC concentrations in animals between

day 1 and 21 of exposure in both the pregnant and non-pregnant groups. In

addition, no detectable differences were observed between pregnant and non-

pregnant animals at day 14 or 21.

5.2. CARBOXYHEMOGLOBIN AND HEMATOCRIT IN PREGNANT RATS (%)

5.2.1. COHb (%) in CS and Air Exposed (Sham) Pregnant Rats

In the sham group, hematocrit did not change at various intervals during

the pregnancy (Figure 5.2.1 .). After exposure to CS, COHb (%) was significantly

higher on day 1 of exposure as compared with day 14 and 21. In addition,

significant differences occurred between the CS and sham COHb (%) at days 1

and 14 of exposure.

54 5.2.2. Hematocrit (%) in CS and Air Exposed (Sham) Pregnant Rats

Hematocrit remained unchanged throughout the gestation in the CS group

(Figure 5.2.2). However, in the sham group, hematocrit was significantly higher at

day 14 as compared to day 21 of exposure. Furthemore, no significant

differences were observed between the CS and sham groups at any of the

exposure days.

5.3. PLASMA CORTICOSTERONE CONCENTRATIONS (pgldl)

5.3.1. CORT Concentrations (pgldl) in CS and Air Exposed (Sham) Pregnant

Rats

CORT concentrations showed no significant differences between the CS

or sham group as the pregnancy pfogressed (Figure 5.3.1 .).

5.4. MATERNAL AND FETAL OUTCOME

5.4.1. Maternai Weights in CS and Air Exposed (Sham) Pregnant Rats

No significant differences in matemal weight gain were seen between

the CS and sham groups (Figure 5.4.1 .).

5.4.2. Pup Weights in CS and Air Exposed (Sham) Rat Litters

Pup weights on day 22 in the CS and sham groups were not significantly

different (Figure 5.4.2.).

5.4.3. Pup Nurnbers per litter in CS and Air Exposed (Sham) Rat Litters

Pup number on day 22 in the CS and sham groups was not significantly

different (Figure 5.4.3.).

5.4.4. Placental and Fetal Organ Weights in CS and Air Exposed (Sham)

Pregnant Rats at 22 Days of Gestation

The placental and various fetal organ weights are given in Table 4. The

number of pups used for each organ is also included in the table. Significant

differences were observed in the brain, heart, liver, lungs and kidneys of the CS

exposed group when compared with the sham group. Similarly, the ratio of

organs to body weight in the CS group were significantly decreased when

compared to the sham group. Fetal kidneys, heart and brain were the most

severely affected (23% 28% and 4q0/0 reduction in organ weight, respectively,

compared with the sham group) whereas fetal lungs and liver were reduced to

51% and 60% respectively. Placental weights were similar between both groups.

56 5.4.5. Placenta1 and Fetal Organ Weights in Control Pregnant Rats

Compared with Air Exposed (Sham) Pregnant Rats at 22 Days of

Gestation

The placenta1 and various fetal organs in the control and sham group are

compared in Table 5. Placental organ weights and al1 fetal organ weights are

significantly different between the two groups. In addition, organ to body weight

ratios are significantly different upon comparison of these two groups.

5.4.6. Placental and Fetal Organ Weights in Control Pregnant Rats

Compared with CS Exposed Pregnant Rats at 22 Days of Gestation

The placental and various fetal organs in the control and CS group are

compared in Table 6. The placenta, brain, liver and kidney raw weights were

significantly different between the two groups. However, the heart and lungs

were similar in weight. When comparing the organ to body weight ratios of these

two groups. significant differences were seen in the brain and kidney ratios onfy.

1 (2) 7(8) 14(15) 2 1 (22) Day of Exposure (Day of Gestation)

Figure 5.1.1 . Plasma NIC Concentrations (mean f SD) in

Pregnant Rats after Exposure to 1000 ml (1 0 ml

puff volume X 10 puffs X 10 cigaretteslday) of CS

and Room Air (sham) at Various Gestational

Aqes (n = 3 - 5 animals per rime interval (CS))

'p c 0.01 at day 1 of exposure to CS as compared to

day 7, and 21 of exposure to CS.

'p = 0.008 at day 7 of exposure to CS as compared to

day 14 of exposure to CS.

'p c 0.09 at day 14 of exposure to CS as compared to

day 21 of exposure to CS.

Figure 5.1.2.

Day of Exposure

H Pregnant

Plasma NIC Concentrations in Pregnant and

Non-Pregnant Rats Exposed to 1000 ml of CS {n = 3 - 5

animals per time intewal J

*p < 0.04 at day 1 of exposure to CS as compared to day 21 of

exposure to CS in pregnant and non pregnant animals.

Day 1 represents both pregnant and non pregnant animais

because on day 1 of pregnancy it is impossible to distinguish

between the two groups.

7

6

5 . h

à? Y

Il 4

8 O 3

2

1

O

Figure 5.2.1.

U2) M15) wa Day of Exposure (Day of Gestation)

Carboxyhemoglobin (COHb) (%) (mean f SD) in

CS Exposed and Air Exposed (Sham) Pregnant

Rats

{n = 9 - 17 animals per time interval (CS) and 3 - 8 animals per tirne interval (Sham))

'p = 0.004 at 1 day of exposure to CS as compared to al1 other

exposure days in both CS and air exposed (sham) groups.

'p c 0.004 at 14 days of exposure to CS as compared to al1

other exposure days in air exposed (sham) groups.

Day of Exposure (Day of Gestation)

Figure 5.2.2. Hematocrit (%) (mean f SD) in CS Exposed and Air Exposed (Sham)

Pregnant Rats

{n = 2 - 15 animals per time interval (CS) and 4 - 8 animals per time

interval (sham))

'p = 0.013 at day 14 of exposure to air as compared to day 21

of exposure to air (sham).

l(L3 7@

Day of Exposure (Day of Gestation)

Figure 5.3.1. Corticosterone Concentrations (mean f SD) in CS

Exposed and Air Exposed (Sham) Pregnant Rats

{n = 2 - 4 animals per fime interval (CS) and 3 animals

per time interval (sham))

Day of Gestation

Figure 5.4.1. Materna1 Weights (mean + SD) in CS Exposed and

Air Exposed (Sham) Pregnant Rats

{n = 8 animals per time interval (CS) and 5 animals per

tirne interval (sham)}

Although we suspected that there were differences in

maternai weights between the CS and sham groups, the

GEE test found no significant differences between the two

groups.

CS Sham

Exposure Group

Figure 5.4.2. Pup Weigtits (mean f SD) in CS Exposed and

Air Exposed (Sham) Rat Litters

{n = 32 animals (CS) and 35

animais per time interval (sham))

CS Sham

Exposure Group

Figure 5.4.3. Pup Numbers per Litter (mean f SD) in CS Exposed and

Air Exposed (Sham) Rat Litters

{n = 4 litters (CS and sham) and each litter between 4 - 12

P U P S ~

65 Table 4. The Placental and Fetal Organ Weights in Cigarette Srnoke

and Air Exposed (Sham) Pregnant Rats at 22 Days of

Gestation.

Placenta

Placenta1

Body weight

ratio

Brain

BrainlBody

weight ratio

Heart

HeartBody

weight ratio

Lungs

LungIBody

weight ratio

Liver

LiverIBod y

weight ratio

Kidneys

KidneylBody

weight ratio

Sham

0.90 i 0.1 7

0.22 i 0.05

n=35

- - - - - - - - --

Cigarette Smoke

11 000 mllday) 0.76 i 0.24

0.16 i 0.05'

n = 32

3 A11 organs were weighed in grams (g).

p value*

0.196

0.001

' Significant differences are seen when comparing the sham and cigarette

smoke exposed average organ weights.

66 Table 5. The Placental and Fetal Organ Weights in Control Pregnant

Rats Compared with Air Exposed (Sham) Pregnant Rats at

22 Days of Gestation.

OrganO

Placenta

Placenta1

Body weight

ratio

Brain

Brain/Body

weight ratio

Heart

HeartIBody

weight ratio

Lungs

LungIBody

weight ratio

Liver

LiverlBody

weight ratio

Kidneys

Kidney/Body

weight ratio

Control

0.54 I 0.09

0.15 i 0.03

n = 128

Sham

0.90 I 0.1 7'

0.22 k 0.05'

n = 35

a All organs were weighed in grams (g).

- - - -- -

P value'

0.001

0.001

' Significant differences are seen when comparing the control and sham

average organ weights and organ to body weight ratios.

67 Table 6. The Placental and Fetal Organ Weights in Control Pregnant

Rats Compared with Cigarette Smoke Exposed Pregnant Rats at

22 Oays of Gestation.

organe

Flacenta

Placental

Body weight

ratio

Brain

BrainlBody

weight ratio

Heart

HeartlBody

weight ratio

Lungs

LunglBody

weight ratio

Liver

LiverIBod y

weight ratio

Kidneys

KidneylBody

weight ratio

Control

0.54 2 0.09

0.15 t 0.03

n = 128

- - - - - - - - -

Cigarette Smoke

(1 000 mllday) 0.76 + 0.24* 0.16 k 0.05

n = 32

O. 16 2 0.03'

0.03 I 0.01'

n = 21

0.07 I 0.14

0.02 + 0.03 n =21

0.19 2 0.20

0.04 t 0.05

n = 21

0.35 i 0.16'

0.07 I 0.04

n = 21

0.07 I 0.03*

0.02 I 0.03*

n =21

a All organs were weighed in grams (g).

- -- - -

p value*

0.001

0.139

0.031

0.007

0.108

0.156

O. 186

0.633

0.014

0.700

0.001

0.051

Significant differences are seen when comparing the controt and cigarette

smoke exposed average organ weights and organ to body weight ratios.

6.0 RESULTS IN NICOTINE EXPOSED RATS

6.1. PLASMA NICOTINE CONCENTRATIONS

6.1 .l. Plasma NIC Concentrations in Pregnant Rats Exposed to NIC via

OMPs at Dosages of 2 and 6 mglkglday at Exact Weights

Plasma NIC concentrations decreased significantly frorn day 1 to day 18

of exposure, 181.9 r 45.3 to 85.9 r 20.0 nglml in the 6 mglkglday group (Figure

6.1.1.). No significant differences were observed between days 1 and 18 of

exposure in the 2 mglkglday group, There were however, significant differences

in plasma NIC concentrations between the two dosage groups at both days 1

and 18 of exposure.

6.1.2. Plasma NIC Concentrations in Pregnant Rats Exposed to NIC via

OMPs at Dosages of 2 and 6 mglkglday at Projected Weights

In the 6 mglkglday dosage group plasma NIC concentrations decreased

from day 1 to day 18 of exposure, 173.9 2 19.9 to 114.8 2 24.1 nglml (Figure

6.1.2.). No significant differences were observed between days 1 and 18 of

exposure in the 2 mglkglday group. There were however, significant differences

in plasma NIC concentrations between the two dosage groups at both days 1

and 18 of exposure. Furthermore, plasma NIC concentrations were dependent

6 9 upon the dosage only (either 2 or 6 mglkglday) and were not dependent upon

whether animals were in the exact or projected weight groups.

6.2.1. Hematocrit in Rats Exposed to NIC and Saline via OMPs at Various

Dosages

No significant differences were observed in hematocrit in the 6 mglkglday

group at exact and projected weights between day 1 and day 18 of exposure

(Figure 6.2.1 .). Similarly, no significant differences were observed in the 2

mglkglday group at exact and projected weights or in the saline group at either

exposure day. The only signifiant difference that existed was between the 2

rnglkglday group at exact weights on day 1 of exposure and the saline group on

day 18 of exposure.

6.3. PLASMA CORTICOSTERONE CONCENTRATIONS (pgldl)

6.3.1. Corticosterone Concentrations in NIC Exposed (6 mglkglday at

Proiected Weight) and Saline Exposed Pregnant Rats

Plasma CORT concentrations significanffy decreased from day 1 to day

18 of exposure in the NIC group (Figure 6.3.1.). No significant differences were

70 observed from day 1 to 18 of exposure in the satine group. In addition, no

significant differences were observed between the NIC group and the saline

group at either exposure day.

6.4. MATERNAL AND FETAL OUTCOME

6.4.1. Materna1 Weights in NIC lnfused and Saline lnfused Pregnant Rats

Matemal weights in al1 of the dosage groups and the saline group

increased as the pregnancy progressed (Figure 6.4.1 .). No significant differences

were seen between any of the dosage groups and the saline group.

6.4.2. Pup Weights in NIC lnfused and Saline lnfused Rat Litters

Pup weights of al1 of the pups in the NIC group and the saline infused

group were similar (Figure 6.4.2.).

6.4.3. Pup Numbers in NIC lnfused and Saline lnfused Rat Litters

Pup numbers per Iitter of al1 of the pups in the NIC group and the saline

infused group were similar (Figure 6.4.3.).

7 1 6.4.4. Placental and Fetal Organ Weights in Saline and NIC lnfused Rats

at 21 Days of Gestation

No significant difference was obsenred between fetal organ weights in the

saline group and fetal organ weights in any of the dosage groups (Tables 7 and

8). In addition, there were no significant effects on organ growth in any of the

dosage groups when compared with the saline group.

Day of Exposure (Day of Gestation)

Figure 6.1.1. Plasma NIC Concentrations (mean i SD) in Pregnant Rats

Exposed to NIC via OMP's at Dosages of 2 and 6 mglkglday at

Exact Weig hts

{n = 4 - 5 animals per time interval; 6 and 2 mgj

*p = 0.001 at day 1 of exposure vs day 18 of exposure to 6

mglkglday of NIC.

'p = 0.001 at 1 day of exposure to 2 mg/kg/day of NIC as compared

to day t of exposure to 6 rngikgf day of NIC.

'p = 0.003 at day 18 of exposure to 6 mglkgiday of NIC as

compared to 2 rnglkgiday of NIC.

1(4) 18(21)

Day of Exposure (Day of Pregnancy)

Figure 6.1.2. Plasma NIC Concentrations (mean f SD) in Pregnant

Rats Exposed to NIC via OMP's at Dosages of 2 and 6

mglkglday at Proiected Weights

{n = 4 - 5 animals per time intemal; 6 and 2 mg]

'p = 0.018 at day 1 compared to day 18 of exposure to 6

mglkglday of NIC.

'p = 0.001 at day 1 of exposure to 2 mglkglday of NIC as

compared tu day 1 of exposure to 6 mglkglday of NIC.

'p = 0.001 at day A8 of exposure to 6 mglkglday of NIC as

compared to 2 mglkgtday of NE.

0 2 mglkglday projected 1 2 mglkglday exact

1 (4) 18 (21)

Day of Exposure (Day of Gestation)

Figure 6.2.1. Hematocrit (mean f SD) in Rats Exposed to NIC and

Saline via OMP's at Various Dosages

{n = 3 - 6 animais per time interval (6 mg and 2 mg

exact and projected, and saline))

'p = 0.045 at 1 day of exposure to 2 mglkglday (exact) of NIC as

compared to 18 days of exposure to saline

Day of Exposure (Day of Gestation)

Figure 6.3.1. Corticosterone Concentrations (mean f SD) in NIC

Exposed (6 mglkglday at Projected Weight) and Saline

Exposed Pregnant Rats

{n = 3 animals per time interval (6 mg) and 3 - 4 animals

per time interval (saline)}

*p < 0.04 at day 1 compared to day 18 of exposure of

exposure to 6 mglkglday of NIC.

Figure 6.4.1.

6 8 10 12 14 16 18 Day of Gestation

Matemal Weights in NIC lnfused and Saline lnfused Pregnant

Rats

{n = 5 - 8 animals per time interval (6 mg and 2 mg exact and

projected, and saline)}

Dosage group

Figure 6.4.2. Pup Weights (mean f SD) in NIC lnfused and Saline lnfused

Rat Litters

{n = 75 - 313 animals (6 mg and 2 mg exact and projected, and

saline))

1 6 mglkgiday projected

Q 6 mglkgiday exact

O 2 mglkgiday projeded

00 2 rng/kg/day exact

O Saline - . . - . . -. . . . . - - - - . - - .

Dosage Group

Figure 6.4.3. Pup Numbers (mean f SD) in NIC lnfused and Saline

lnfused Rat Litters

{n = 4 - 6 litters with 13 - 21 pups per lifter (6 mg and 2

mg exact and projected, and saline))

79 Table 7. The Placental and Fetai Organ Weights in Saline and Nicotine

(2 mglkglday) Exposed Rats at 21 Oays of Gestation.

Placenta

PlacentalBody

weight ratio

O rgan'

Liver

LiverlBod y

weight ratio

Kidneys

KidneyiBody

weight ratio

Saline

Brain

BrainIBody

weight ratio

Heart

HeartlBody

weight ratio

Lungs

LungIBody

weight ratio

B Ali organs were weighed in grams tg).

No significant differences were observed between any of the groups.

2 mglkglday at

Exact Weig ht

0.18 I 0.09

0.05 k 0.03

n=23

2 mglkglday at

Piojected Weight

0.1 7 i 0.05

0.05 I 0.08

n=36

0.16 t 0.05

0.04 k 0.01

n = 24 0.05 k 0.03

0.01 10.01

nt24

0.05 + 0.08 0.02 + 0.02

n = 23

0.06 I 0.06

0.02 * 0.02

n = 36

0.18 t 0.15

0.04 I 0.03

n = 23

0.18 + 0.08 0.05 f 0.01

n = 36

O. 15 t 0.03

0.04 k 0.01

n = 24

80 Table 8. The Placental and Fetal Organ Weights in Saline and Nicotine

(6 mglkglday) Exposed Rats at 21 Oays of Gestation.

organe

Placenta

PlacentaIBody

weight ratio

Brain

BrainIBody

weight ratio

Saline

0.61 i 0.1 5

0.16 * 0.04 n = 63

0.18 + 0.09 0.05 c 0.03

n =23

Heart

HeartlBody

weight ratio

Lungs

LunglBody

weight ratio

Liver

LiverlBody

weight ratio

Kidneys

KidneyjBody

weight ratio

6 mglkglday at

Exact Weig ht

0.60 k 0.16

0.A 5 k 0.05

n = 71

0.05 i 0.08

0.02 + 0.02

n =23

0.18 10.15

0.04 k 0.03

n = 23 0.29 I 0.12

0.07 i 0.03

n = 23

0.07 * 0.09

0.02 2 0.03

n = 23

No significant differences were obsenred between any of the groups.

6 mglkglday at

Projected Weig ht

0.64 k 0.12

0.16 k 0.04

n = 75

0 1 !

AI1 organs were weighed in grams (g).

7.0. DISCUSSION

7.1. GENERAL

The present study was performed to establish h o new models of CS and

NIC exposure. One may question the necessity of establishing two different

models of CS and NIC exposure. However, we did this because we wanted to

determine whether NIC was the agent in CS which was responsible for growth

restriction or whether it was constituents other than NIC which act together or

individually in causing these effects.

The main difference between this study and the study performed

previously in our laboratory is that in our study CS exposures began on day 2 of

gestation in order to elucidate the effects on the fetoplacental unit since

implantation occurs on day 5 of gestation. The unique aspect of our studies is the

comparative analysis of CS and NIC exposures starting on day 2 of gestation.

We provide new information on the effects of prenatal CS and NIC exposures on

plasma NIC and CORT concentrations. Iitter size, birth weights, and organ

weights at various gestational ages.

From the CS inhalation studies, we observed that NIC concentrations

decreased with increasing days of gestational age and exposures. The studies

also detemined that CS exposure that took place phor to embryo implantation

had no significant effect on fetal birth weights or litter sizes at tem, when

82 compared with animals exposed to room air. Fetal organ weights in the CS

and sham groups were significantly different, with the sham organs being much

larger. The NIC and saline groups had fetal organ weights that were statistically

similar. In addition, fetal organ weights reported in the literature were similar to

organ weights in the CS, NIC and saline groups and different from the sham

group organ weights. We also observed that COHb (%) in the sham group

remained unchanged throughout the pregnancy while COHb (%) in the CS group

was higher on day 1 of exposure but decreased by day 21 of exposure. We also

found that, in both the CS and sham groups, hematocrit did not change during

the early and mid portions of gestation with a significant decrease at the end of

gestation in the sham group.

In the OMP studies, plasma NIC concentrations decreased as the

pregnancy advanced. This pattern of NIC concentrations is similar to the one

seen in the CS studies. NIC concentrations in the 6 mglkglday dosage groups

were significantly higher at term than concentrations in the 2 mglkglday dosage

groups. In addition, no significant differences in fetal birth weights or Iitter sizes

were observed between any of the NIC groups and the saline group. Because no

significant differences were seen in birth weights and litter sizes between NIC

and saline groups, we tentatively concluded that NIC is not the agent in CS that

is responsible for growth restriction. We also observed that hematocrit was not

significantly different over the exposure period, except for a significant difference

between the 2 mglkglday group (at exact weights) at day 1 of exposure and the

saline group at day 21 of exposure.

Using plasma CORT as our indicator of stress in the inhalation and

infusion models, it appears that the NIC infusion mode1 with OMPs may cause

less stress in the animal because CORT concentrations decreased as the

pregnancy progressed. In the CS group, GORT concentrations remained higher

than control levels throughout the entire duration of pregnancy. However, before

drawing any unequivocal conclusions, further studies are needed to study the

variables that interact with CS and NIC, thiis influencing the CORT

concentrations.

7.2. PLASMA NICOTINE CONCENTRATIONS

In a number of previous studies, CS has been administered via whole

body or nose only methods. CS studies have been performed in humans as well

as in animals. Benowitz et al., (1984) reported plasma NIC concentrations in

moderate smokers to be between 30 and 50 nglml (9). Another study by Isaac

and Rand (1971) found plasma NIC concentrations to be between 12 - 44 nglml

in male and female smokers who smoked between 5 and 14 cigarettes per day

(35). In addition, pregnant women who smoked one cigarette had concentrations

between 14 - 41 nglml (1). The large variation in concentrations reported in the

literature is most likely due to differences in smoking practices as mentioned

eariier.

84 A number of animal exposure models have been utilized to administer

CS in animals via chamber inhalation systems using cigarettes with high NIC

content (74;90), large CS doses (6;73) and in some cases with unknown puff

volumes (6;90j. However, a very Iimited number of these studies have

investigated plasma NIC concentrations in rats at any point during the

pregnancy. A study by Rotenberg and Adir (1983) measured plasma NIC

concentrations in male rats after chamber inhalation to CS with cigarettes

containing 9.5 mg of NIC per cigarette (74). Concentrations at the end of 15 days

of CS exposure were 242 nglml.

The present study has found that exposure to 1000 ml of CS from day 2 of

gestation produces plasma NIC concentrations sirnilar to the values obtained by

Benowitz (9) and Isaac and Rand (35). Plasma NIC concentrations in my study

ranged from 45.4 nglml at day 1 of exposure to 19.2 ng/ml at the end of the

exposure period (day 21 of gestation), concentrations that mimic those seen in

human smokers.

Plasma NIC concentrations in CS exposed animals showed an interesting

pattern at various gestational ages. At day 7 of exposure, plasma NIC

concentrations decreased significantly and then rose at day 14 of exposure

(Figure 5.1.1.). VVe speculate that the decrease at day 7 may be due to

cytochrome P-450 enzyme induction by NIC, which increases metabolism in the

liver, lungs, kidneys and brain leading to a decrease in NIC concentrations

(1 0;32;65). The xenobiotic-rnetabolizing P-450 exists in multiple forms, each with

85 its own distinct substrate specificity and many of these forms are induced by

exposure to exogenous agents (65). Although metabolites that are produced are

inactive, reactive or toxic metabolites rnay be forrned. Also, there may be an

endogenous inhibitor present that becomes active at day 14, which inhibits the

enzyme system, and causes plasma NIC to increase as seen in in vitro studies

with cotinine (38). It is also possible at day 14, that there are other constituents of

CS, such as CO which are causing injury to the enzyme system leading to

increased plasma NIC concentrations (10). In addition, although there is no

evidence to support this, it is plausible that metabolic acidosis develops due to

hypoxia at day 7 to cause NIC concentrations to decrease. The rnetabolic

acidosis may be caused by repeated hypoxic episodes through exposure to CS.

This would then cause increased secretion of hydrogen ions and thus more

unchanged NIC would be excreted into the urine causing a decrease in NIC

concentrations. Plasma NIC concentrations also decrease significantly at day 21

as compared to concentrations at day 14. This decrease may be caused by

hemodilution due to the large increase in blood volume that takes place during

the latter part of pregnancy. Increasing delivery of NIC to the many fetal

compartments in the rat via the placenta and the increasing size of these

compartrnents rnay also cause NIC concentrations to decrease. Placental

enzymes may also be induced to increase metabolism, causing concentrations to

decrease.

Our data comparing the plasma NIC concentrations in pregnant and non-

pregnant rats show that concentrations decrease significantly in both groups at

86 term, and thus that the decrease is not dependent on pregnancy. In addition,

there were no significant differences between the two groups, which is opposite

to what was reported previously (89). In the pregnant animals, the factors

described above, the hernodilution factor, fetal compartments, and placental

enzyme induction, are still present. However, in non-pregnant animals, these

factors are not present and thus the decrease in concentrations is likely due to

increased enzyme induction in both animal groups.

Previous studies using OMPs have seldom measured plasma NIC

concentrations during the pregnancy and if NIC concentrations were measured,

blood samples were taken either h o days after delivery (57) or at mid-gestation

(77). In the current study, when OMPs were used to administer NIC, plasma NIC

concentrations were significantfy higher at 6 mglkglday at both exact and

projected weights as compared with 2 rnglkglday. However, the dosage of 2

mglkglday gave concentrations similar to those seen in moderate smokers.

In the OMP model, plasma NIC concentrations also decreased with

advancing pregnancy, similar to the CS group. This is most likely due to

pregnancy-induced physiologicaI or hemodynamic changes in the body

(hemodilution, development of fetal compartrnents and placental enzyme

induction) as stated above. Further studies need to be performed in order to

investigate whether the sigrnoidal plasma NIC concentrations curve seen in the

CS studies would also occur in the OMP rnodel.

87 One may question the utility of measuring plasma NIC concentrations

when using OMPs because in my studies there did not appear to be any effect

on fetal variables. However, it is important to know these concentrations in

studies because even though NIC may not cause any g ros malformations, it

may be causing damage at receptor sites or in growth factors. Thus the OMP

studies that were performed previoudy with higher dosages (6 mglkglday) should

be interpreted with caution. In addition, in order to compare rat CS and NIC

studies to human studies, plasma NIC concentrations should be in a comparable

range (Le. if studying moderate smokers, plasma NIC concentrations should be

between 30 and 60 nglml). In addition, although the OMP model is an easier

procedure to perform in cornparison with the CS studies, it does not mimic

smoking in humans like the CS studies.

7.3. CARBOXYHEMOGLOBIN, HEMATOCRIT AND CORTICOSTERONE

7.3.1. Carboxyhemoglobin

In studies by Bassi et al., (1984) (6) and Nelson (1999) (63), percent CO

saturation was taken as an index of the arnount of CS inhaled by animals to

make sure that al1 aaimals inhaled similar amounts of CS, usually between 9 and

2C%. Our experiments showed that after exposure to 1000 ml of CS from day 2

of gestation, matemal COHb (%) values ranged from 5.0% at day 2 to 2.5% at

the end of gestation. Literature has given COHb (%) in pregnant smoking

mothers as being between 2.0 and 8.3% and reaching peaks of 14% in some

88 instances (48;49). Our results are comparable to these results but are on the

low end and this is most likely due to the low puff volume (10 ml) and CS dose

given to our animals.

Fetal hemoglobin binds to CO with greater affinity than adult hemoglobin

(31;48;49). As a result, the fetus will accumulate CO, and also experience an

increase in COHb ( O h ) . Fetal COHb (%) levels have been found to be

approximately two times higher than in the mother (59). In addition, the half-life of

CO is three times longer in the fetal than maternal circulation, 7 and 2.5 hours,

respectively (48;49;59). However, we were unable to measure fetal COHb (%)

because of the difficulty in obtaining blood samples from pups at 21 days of

gestation. An increase in COHb (%), whether fetal or maternal, will shift the

oxygen saturation curve to the left. This shift will tend to decrease the normal

partial pressure gradient for oxygen from matemal to fetal blood across the

placenta. This impairs placenta1 oxygen exchange.

7.3.2. Hematocrit

High hematocrït values compensate for the decrease in oxygen carrying

capacity of the blood due to increasing COHb (90). In addition, in hypoxic

situations, the red blood celf count decreases and erythropoietin is released from

the kidneys to offset the decreased ce11 count. An increase in red blood cell

production follows and may attenuate hypoxia. In most studies, maternal

hematocrït values above 38 are thought to be associated with an increased risk

89 of unfavorable outcomes, such as spontaneous abortion, hypertension and low

birth weight (28). In our CS group, hematocrit remained constant, behveen 40.8

and 42.3%. In the sham group, hematocrit also remained constant but dropped

significantly between day 14 and 21 of exposure. As mentioned, during

pregnancy a 30 - 40% increase in blood volume close to terrn occurs due to the

extra release of hormones such as aldosterone and estrogens, which induce

increased water retention (31). Such an increase in blood volume causes the

bone marrow to increase red cell production to compensate. However, it must be

noted that a 30 - 40% increase in red blood ceil production does not occur which

causes a relative decrease in hematocnt during the last third of pregnancy in the

sham group.

In our NIC infusion studies, there were no signifiant differences in

hematocnt levels between the two dosage groups of 2 and 6 mgikg/day.

However. hematocrit was slightly lower in the NIC groups than in the CS groups

especially at the end of gestation. Thus hematocrit did not remain high in NIC

infused rats throughout the pregnancy and did not support our hypothesis. It is

plausible that since only NIC is being infused into animais, there should be no

change in hematocrit. Even though NIC is a placental artery vasoconstrictor and

may thus cause fetal hypoxia, stimulating erythropoietin, it was still not enough to

cause hematocrit to increase and other compensatory mechanisms might have

occu rred .

Plasma Corticosterone Concentrations

In both the CS and sham groups, CORT was elevated throughout the

pregnancy. In the OMP studies, CORT in both the 6 mglkglday group at

projected weights and the saline group was elevated above control levels;

however, by the end of the pregnancy there was a significant decrease in CORT

concentrations.

These elevated CORT concentrations imply that rats were exposed to a

certain amount of stress. There are a number of factors that could cause this

increase in stress including cardiac puncture, anesthesia, hormonal changes

during pregnancy, frequent handling of animais and CS components, including

NIC (l5;l8;l9;37:83).

7.3.3.1. Effects of Cardiac Puncture and Anesthesia

Carlberg et al., 1996 (18) performed a study to develop a blood collection

protocol for fetal rats and to examine its effect on the HPA axis in mothers and

fetuses. They found that materna1 CORT at the end of the blood sampling

procedure was higher than at the beginning of the procedure in six of the eight

rats. However, upon fetal blood collection they found that fetal CORT

concentrations were not affected by the blood sarnpling procedure. The blood

sampling procedure used in this study was cardiac puncture, similar to our

studies, however the anesthetic used in their study was halothane compared to

91 sodium pentobarbitol used in Our studies. Because matemal CORT

concentrations were shown to increase after the blood sarnpling procedure (20 -

39 minutes), one may suggest the possibility that CORT concentrations in Our

studies also remained high because of the blood sampling procedures. However,

although CORT in the CS studies remained high throughout gestation, CORT

concentrations in the OMP studies decreased at the end of gestation. In both

studies, the same blood sampling procedure was used and thus it is unlikely mat

the blood sampling procedure influenced CORT concentrations in our studies.

Stern and Voogt reported a rise in CORT concentrations 30 minutes after

a two minute ether exposure in cycling as compared to lactating rats 24 hours

after deiivery (83). It is also known that the peak secretion of ACTH occurs

between 2 and 5 minutes after the onset of ether. In our studies, approximately

15 minutes after animals were anesthetized, blood samples were obtained.

CORT was significantly elevated at day 1 of exposure, but this was not seen at

day 18 of exposure. Cohen et al., (1983) (19) observed no differences in CORT

in ether-anesthetized rats cornpared to decapitated rats. These results may be

oxplained by the fact that blood samples were taken in ether-anesthetized rats in

less than 2 minutes. Other studies reported that CORT concentrations do not

change within this period of time but peak at 15 minutes under ether anesthesia,

sirnilar to our results at day 1 of exposure.

7.3.3.2. Effects of Hormonal Changes during Pregnancy

When pregnant animals were exposed to stresses such as immobilization

or ether inhalation, there were increases in plasma concentrations of ACTH and

some adrenocortical products, including glucocorticoids and androstenedione, in

both mothers and fetuses (18). In Carlberg's study (18), plasma CORT

concentrations were measured in pregnant rats at term after halothane

anesthesia and cardiac punctures. Mean matemal CORT concentrations were

23.9 pgldl as compared with 13.8 pgldl previously reported in non-pregnant

Sprague-Dawley rats sampled in the same manner, with the same anesthetic, at

the same time of day. Our CORT concentrations (20 = 6 pgldl) in control rats

were also comparable to these studies. Pechinot and Cohen (1983) reported

similar values after cervical dislocation and blood collection by intra-cardiac

puncture (66).

7.3.3.3. Effects of Frequent Handling

An increase in plasma CORT concentrations after continuous NIC infusion

has been reported (15). One reason for this could be the actual effects of NIC or

the effects of the surgical procedure. In addition, previous studies indicate mat

adult offspring of dams handled or stressed during gestation, or adult animals

handled during the early postnatal period, can show altered HPA responses to

stress as well as a more rapid recovery of CORT following temination of stress

93 in the Iatter group (69). Upon comparing our two models of CS and NIC

exposure, it is apparent that both models elicit a CORT response due to some

type of stress. In the CS model, there is frequent handling of animals, effect of

CS exposure, effect of anesthesia itself, and the actual injection of the

anesthesia. In the OMP model, the animals are handled much less frequently;

however, stress from surgery, the size and weight of the OMP mounted on the

back of the animal, effect of anesthesia itself, and the administration of the

anesthesia still exist.

7.3.3.4, Effects of Nicotine and Cigarette Smoking

Kershbaum (1968) (37) reported a rise of 58% in plasma 11-

hydroxycorticosteroid in Sprague-Dawley rats 30 minutes after NIC

administration. In addition, human data showed an increase in piasma 11-

hydroxy-corticosteroid by 27-77% after heavy smoking cornpared to the normal

diurnal fall during control observation with no smoking. However, concentrations

of 1 l-hydroxycorticosteroid tended to decrease with some fluctuations during the

remainder of exposures. A possible explanation for this decrease could be the

development of tolerance to chronic NIC or CS administration in both rats and

humans.

Our CS studies do not support the hypothesis that plasma CORT

concentrations would decrease as the pregnancy progressed. Plasma CORT

concentrations were consistently 2 to 3 fold higher in both groups throughout the

94 pregnancy as cornpared to the controls. This inconsistency in CORT may be

due to the frequency of handling that the animals in the CS group were subjected

to. In Our NIC infusion studies, CORT concentrations were higher than control

levels at day 1 of infusion. There was, however, a significant decrease in plasma

CORT concentrations as the pregnancy progressed. This is consistent with the

human and rat data presented, showing the development of tolerance to chronic

NIC administration. As mentioned, CORT concentrations may also be influenced

by the interaction of anesthesia, the injection itself, and CS and NIC components.

However, before any conclusions can be made as to which model is more

stressful for the animal, these variables need to be isolated in the experiments to

determine which factor or factors is causing the effects seen in CORT

concentrations.

7.4. MATERNAL WEIGHTS

In a study by Leichter (1995), dams exposed to CS had significantly

attenuated weight gain at day 21 of gestation compared with unexposed control

dams (43). However in this study, matemal food intake was monitored and dams

in the CS group had lower food intake. In Our studies, food intake was not

monitored. Similar results were found in the study by Younoszai (1969). as mean

materna1 weight gain in animals exposed to CS was decreased in comparison to

the unexposed controls (90). In both the CS inhalation studies and the NIC

infusion studies, matemal weights of the expenmental groups were not

significantly different from matemal weights in the sham and saline groups.

95 It is possible that no difFerences in maternal weight gain were seen

between the CS and sham groups in our studies, because of the fact that lower

dosages of CS were given to animals, as compared to the studies mentioned

above. In the NIC studies, maternal weight gain in al1 of the NIC dosage groups

was similar to the saline group. When comparing the CS and NIC materna!

weight gain figures (Figure 5.4.1 .and Figure 6.4.1 ., respective!^), the OMP group

had a higher rate of matemal weight gain than the CS group.

7.5. FETAL VARIABLES - BIRTH WEIGHTS, LITTER SIZES, and ORGAN

WEIGHTS

Birth Weights

Smoking during pregnancy in humans results in offspring with an average

birth weight that is 150 - 400 g less than that of non-smoking mothers (6). In

studies utilizing animal models, growth restriction and birth weight deficits have

been observed in a dose-dependent manner (6;20;43;62;63;73;89;90). However,

increased birth weights have also been observed in babies of smoking mothers

(88). In my study, birth weights of pups in the CS exposed group were nct

significantly different from birth weights in the sharn group. In the NIC infusion

study, birth weights were not significantly different between any of the NIC

dosage groups and the saline group. A study by Schuen et al., (1997) using

OMPs to administer NIC observed a decreased birth weight in pups that had

prenatal NIC exposure (77). However, in this study a dosage of 12 rng/kg/day

96 was administered to rats at a rate of 70 pllday as compared to my studies

administering either 2 or 6 mglkglday at a rate of 60 pllday. This higher dosage

of NIC may have caused a decrease in pup weights. A study administering NIC

(0.05 mg and 0.10 mg) in the food of pregnant Sprague-Dawley rats, observed

no significant differences in pup birth weight between the NIC group and the

control group (56). Our studies concur with their results despite the fact that the

methodology was slightly different. It has been shown that maternal weight has a

direct impact on birth weight and since we did not observe any differences in

birth weights, this may be due to the fact that matemal weights were not

significantly different (30;70). Despite the large amounr of research in both

humans and animals, that has clearly concluded that birth weights are reduced

after prenatal exposure to CS which cannot be disregarded, it is possible that

pup birth weights are more dependent on maternal weight gain patterns than on

exposure to whole CS or its constituents.

7.5.2. Litter Size

In both the CS and NIC infusion studies, litter sire was not affected by

exposure to CS and NIC respectively. Previous studies administering both CS

and NIC in the literature have similar findings (6;43;77).

Upon examination of the organ weights in the CS exposed and sham

groups, the CS group had significantly lower fetal organ weights, excluding the

placenta, compared with the sham group. ln the NIC and saline groups, no

significant differences were observed. Upon comparing al1 of the data, al! of the

groups had reiatively similar values, with the exception of the sham group. In the

sham group, organ weight values were two to three times larger than in any other

group. Even upon comparing values in the control group to the sham group,

significant differences were found (Table 5). And although sorne control values

were different frorn CS values, they are not as different as with the sharn group

(Table 6). After perforrning a thorough literature search, we found that several

different researchers had investigated fetal organ weights in rat pups

(26;47;50;51;55;58;67;78).

Some studies reported values that were consistent with Our CS, NIC,

saline and control data, while others were very different from Our values. For

instance studies by Bassi et al., (1984) (26), had Iiver (0.49 r 0.02 g) and brain

(0.22 2 0.01 g) weights that were significantly higher than the liver and brain

weights in our CS (Table 4), control (Tables 5 and 61, NIC and saline groups

(Tables 7 and 8). However, iung (0.16 2 0.01 g) weights found by Bassi were

comparable with our experimental groups: CS (0.19 I 0.20 g), 2 mglkglday NIC

at exact weights (0.1 8 2 0.08 g), 2 mg/kg/day of NIC at projected weights (0.1 5 +

98 0.03 g), 6 mglkglday of NIC at exact weights (0.15 s 0.03 g), 6 mg/kg/day at

projected 0.16 I 0.03 g) and saline groups (0.18 +_ 0.15 g).

Two studies conducted by Lueder et al., (1 992, 1995) (50;51) measured

al1 major fetal organ weights in rat pups [fetal heart: 0.0179 I 0.0011 g, liver:

0.253 i 0.00003 g, lungs: 0.124 I 0.0002 g, brain: 0.162 I 0.0002 g, and

kidneys: 0.0275 I 0.0006 g]. These results are comparable to the values in our

experimental groups and our control group [fetal heart: 0.02 c_ 0.01 g, liver: 0.26 +_

0.05 g, lungs: 0.13 I 0.02 g, brain: 0.14 2 0.01 g, and kidneys: 0.030 I 0.01 g].

Several other studies measured various fetal organs such as lungs (58;67)

and liver (78) and these studies also found results comparable to our CS, NIC,

saline and control group data. However, there were some discrepancies in the

literature. For instance a study by Mortola et al., (1990) (55), measured heart

(0.016 r 0.001 g) and lung (0.047 I 0.004 g) weights in pups on postnatal day

7. These values at postnatal day 7 were lower than our values and values of

other investigators at 19 - 22 days of gestation. In this study, before organs were

weighed, portions of each organ were cut for DNA analysis. It is possible that this

may have caused organ weights to be lower. Another study conducted by

Lohninger et al., (1996) (47) measured fetal lung weights at day 19 of gestation

and found lung (55 mg) weights that were significantly smaller than lung weights

from our CS, NIC and saline data as well as Iiterature values. ln this study, mean

birth weight in the control group was 2.7 g while in Our studies and the Iiterature,

99 mean birth weight varied between 3.70 and 5.85 g. This could have caused

lung weights to be lower in these animals.

Despite these discrepancies, the majority of data found in the literature is

similar to our CS, NIC, saline and control groups. However, no data was found to

be comparable with our sham group data. I also compared my sham data with a

similar study performed in our laboratory previously, in which animals were

exposed to air and organ weights were measured. Once again, sham organ

weights in my study were much larger than organ weights in the other sham

study. This leads us to suspect that this data may be faulty due to either error in

weight measurement or organ extraction. In addition, after cornparison of control

fetal organ weights with the sham organ weights, it was apparent that sham

organ weights were significantly different. Upon comparing the control organs

with the CS exposed organs significant differences were found, however, the

weights were more similar. This again leads us to the conclusion that we cannot

assume that the sham fetal organ weights are correct.

The only way to make any concrete conclusions from my data is to repeat

the CS and sham exposure studies and measure fetal organ weights to

detemine if the results in the cuvent data set are reproducible. We performed a

subsequent set of experirnents consisting of control animals and sham (air-

exposed) animals and the results are shown in Table 9 below.

1 O0 Table 9. The Placenta1 and Fetal Organ Weights in Control Pregnant

Rats Compared with Sham (Air) Exposed Pregnant Rats at 22

Days of Gestation (subsequent studies)

1 1 I I ..

Placenta , 0.54 2 0.09 0.51 I 0.07 1

1 l FetaI Tissue (g) j Control

1 1 I 1 n = 128 I n = 95 I

1 Lungs I 0.14 + 0.10 0.13 i_ 0.02 I

S ham

n = 38 1 n =40 I I Liver 0.26 ç 0.05 1 0.27 2 0.07 1 1 I n = 48 1 n =40 , , Heart 0.02 I 0.01 l

1 0.02 i 0.004 ~ 1

l n =48 1 n =35 l

Kidneys 0.03 1 0.01 0.03 ? 0.01 l l

Brain

Pup Weights 3.70 i. 0.40 1

3.55 r 0.88 i

n = 128 n = 97 1

Litter Sixe 1611 1 15.1 IL 2-5 I I n = 9 dams , n = 7 dams I

In the control and sham groups, a comparable litter size is observed. In

addition, there are no significant differences observed in organ weights between

these two groups and the organ weights are similar to those seen in the NIC and

saline groups. This confirrns my belief that the original sham data may not

suitabiy represent valid experimental conditions. Therefore, the only conclusions

that can be drawn are with the NIC and saline groups. Because no significant

differences were observed in organ weights between these two groups and

the data is comparable to the literature, it is reasonable to conclude that

perhaps NIC is not the agent in CS responsible for fetal growth restriction.

8.0 SlGNlFlCANCE AND RELEVANCE

In the present study, we exposed animals to a known amount of CS, air,

NIC and saline frorn the second day of gestation to elucidate the changes in

plasma NIC, CORT, Iitter size, and birth weights. Plasma NIC concentrations

followed a sigmoidal pattern and at terni were similar to concentrations found in

moderate smokers. This was also the first study of its kind to measure plasma

NIC concentrations after 6 and 2 mglkglday NIC infusion via OMPs. The dosage

of 6 mglkglday produces plasma NIC concentrations that are higher than those

observed in moderate smokers. Thus future physiological studies should use the

2 mglkglday dosage which produces plasma NIC concentrations sirnilar to those

seen in moderate smokers, instead of the higher dose of NIC.

CORT concentrations remained high throughout gestation in the CS and

sham groups but decreased at terrn in the NIC and saline group. Thus, it is

possible that the OMP mode1 presents a tess stressful situation for the animals

than the CS model.

We observed no significant differences in litter size or birth weights

between the CS and sharn groups. There were also no observed differences in

terrns of fetal birth weights and litter size between rats exposed to saline and rats

exposed to NIC at either dosage which suggests that NIC may not be the

constituent in CS responsible for fetai growtti restriction.

9.0 LIM1TATlONS OF THE DPERIMENTAL DESIGN

1) In each experiment. we used different animals because upon obtaining

blood samples, anirnals were unable to survive for extended periods of tirne, as

these were terminal experirnents. We did atternpt to insert a catheter into the

carotid artery, but because of the frequency of handling of the rats it was difficult

to keep the lines open and intact during the entire duration of the pregnancy and

smoke exposure.

2) Rats were exposed to a certain amount of stress due to continuous

handling, exposures to CS, surgeries and infusion of NIC, as shown by increased

plasma CORT concentrations. However, similar observations have been reported

after IV injections or continuous infusions of NIC.

3) Some caution needs to be used when interpreting data using the Smoke

Exposure System, because of the stress factor. In order to confidently reproduce

data in human studies, there needs to be a way to regulate the stress caused in

animals when using this system.

The experiments from this study show that, when exposures began on day

2 of gestation, there was no significant impact on embryo implantation, as seen

by similar pup weights and litter sizes in both the CS and sham groups. In

addition, when CS exposures began eariier, plasma NIC concentrations

decreased with increasing exposure to CS, as seen in previous studies in our

laboratory when exposures began on day 6 of gestation.

These studies also showed that plasma NIC concentrations in OMP

studies were significantly higher than those found in moderate srnoketç with the

dosage of 6 mglkglday; a dosage of 2 rnglkglday provided plasma NIC

concentrations that were sirnilar to those found in moderate smokers. Thus,

previous studies that have used the dosage of 6 rnglkglday shoutd be interpreted

with caution. Future studies that are perforrned using OMPs should use a dosage

comparable to 2 mglkglday, to obtain plasma NIC concentrations similar to those

observed in rnoderate smokers.

Because no differences in maternal weight gain and birth weights were

observed in either the CS or NIC groups, it is possible that birtti weight is more

dependent upon maternal weight gain than any other factor and in tum matemal

weight gain is dependent upon CS exposure.

1 O4 Because no significant differences in organ weights were found in the

NIC and saline groups, it is possible that NIC is not the agent in CS that is

responsible for causing fetal growth restriction. In terms of the CS and sham

data, no conclusions can be made because the sham experiments were

apparently unreliable.

In the CS group, COHb (%) was high on day 1 of exposure but decreased

at day 21 of exposure. Hematocnt remained high throughout the pregnancy in

the CS group, which was expected because of exposure to CO. In addition,

hematocrit in the NIC group remained at normal IeveIs because dams were not

exposed to CO.

11.0 FUTURE DIRECTIONS

Before any further physiological work can begin, the CS and sham

inhalation studies, especially the fetal organ weights, need to be performed again

in order to confirm the results.

After the results are confirmed, the models of CS exposure and NIC

exposure established in my study will be used in future studies performed in the

laboratory to investigate the mechanisms of adverse effects on cardiorespiratory

control during postnatal maturation and its implication in SIDS. Briefly, the effects

of smoking on breathing patterns and metabolic rate in newboms during hypoxic

and hypercapnic challenges, on carotid body DA release and on insulin-like

growth factors (IGF) I and II. IGF binding proteins 1-6, and hypoxia-inducible

genes in the placenta and fetal body organs will be investigated.

1. Abel, E. L. Smoking during pregnancy: a review of effects on growth and development of offspring. Human Biology 52: 593-625, 1980.

2. Armitage, A. K., C. T. Dollery, C. F. George, T. H. Houseman, P. J. Lewis, and D. M. Turner. Absorption and metabolism of nicotine from cigarettes. Br. Med. J. 4: 31 3-3 1 6, 1 975.

3. Baird, D. Evidence for reduced fecundity in femate smokers. In Poswillo, D. and E. Alberman, eds., The Effects of Smoking on the Fetus, Neonate and Child. Oxford, Oxford University Press. 1992, 5-22.

4. Bamford, O. S. and J. L. Carroll. Dynamic ventilatory responses in rats: normal development and effects of prenatal nicotine exposure. Respir. Physiol. 1 17: 29-40, 1999.

5. Bamford, O. S., J. N. Schuen, and J. L. Carroll. Effect of nicotine exposure on postnatal ventilatory responses to hypoxia and hypercapnia. Respir. Physiol. 1 06: 1 -1 1 , 1 996.

6. Bassi, J. A., P. Rosso, A. C. Moessinger, W. A. Blanc, and L. S. James. Fetal growth retardation due to materna1 tobacco smoke exposure in the rat. Pediatr. Res. 18: 127-1 30, 1984.

7. Benowitz, N. L. Pharmacologie aspects of cigarette smoking and nicotine addiction. The New England Journal of Medicine 31 9: 131 8-1 330, 1988.

8. Benowitz, N. L. Acute bioiogical effects of nicotine and its metabolites. In Clarke, P. B. S., M. Quik, F. Adikofer, and K. Thurau, eds., Effects of Nicotine on Biological Systems II. Basel, Birkhauser Berlag. 1996, 9-16.

9. Benowitz, N. L. and P. Jacob. Daily intake of nicotine during cigarette smoking. Clin. Pharmacol. Ther. 35: 499-504, 1984.

10. Benowitz, N. L. and P. Jacob. Effects of cigarette smoking and carbon monoxide on nicotine and cotinine metabolism. Clinical Pharmacology and Therapeutics 67: 653-659,2000.

11. Benowitz, N. L., H. Porchet, and P. Jacob. Pharmacokinetics, metabolism, and pharmacudynarnics of nicotine. In Wonnacott, S., M. A. H. Russell, and 1. P. Stolerman, eds., Nicotine Psychopharrnacoiogy Molecular, Cellular and Behavioral Aspects. Oxford, Oxford University Press. 1990, 1 12-1 57.

12. Benwell, M. E. M. and O. J. K. Balfour. Effects of chronic nicotine administration on the response and adaptation to stress. Psychophamacology 76: 160-1 62.1982.

I O7 13. Bulterys, M. G., S. Greenland, and J. F. Kraus. Chronic fetal

hypoxia and sudden infant death syndrome: interaction between matemat smoking and low hematocrit dunng pregnancy. Pediatrics 86: 535-540, 1990.

14. Butler, N. R., H. Goldstein, and E. M. Ross. Cigarette smoking in pregnancy: its influence on birt!! weight and pennatal mortality. 6r.Med.J. 2: 127-130, 1972.

15. Caggiula, A. R., L. H. Epstein, S. M. Antelman, S. Saylor, S. Knopf, K. A. Perkins, and R. Stiller. Acute stress or corticosterone administration reduces responsiveness to nicotine: implications for a mechanism of conditioned tolerance. Psychopharmacology 1 1 1 : 499-507, 1993.

16. Cam, G. R. and J. R. Basset. Effect of prolonged exposure to nicotine and stress on the pituitary-adrenocortical response; the possibility of cross-adaptation. Phamacology, Biochemistry & Behaviour 20: 221 -226, 1984.

17. Cam, G. R., J, R. Bassett, and K. O. Cairncross. The action of nicotine on the pituitary-adrenal cortical axis. Arch.lnt.Phannacodyn. 237: 49-66, 1979.

18. Carlberg, K. A,, B. L. Alvin, and A. R. Gwosdow. Exercise during pregnancy and maternai and fetal plasma corticosterone and androstenedione in rats. A m J Physiol. 271 : E896-E902, 1996.

19. Cohen, H., P. Guillaumot, 1. Sabbagh, and J. Bertrand. A new hypoprolactinernic rat strain, prolactin, luteinizing hormone, follicle- stimulating hormone, testosterone and corticosterone levels in males and effects of two anesthetics. Biol. Reprod. 28: 122-1 27, 1983.

20. Collins, M. H., A. C. Moessinger, J. Kleinerrnan, J. Bassi, P. Rosso, A. M. Collins, L. S. James, and W. A. Blanc. Fetal lung hypoplasia associated with maternai smoking: a morphometric analysis. PediatrRes. 19: 408-41 2, 1985.

21. Cooke, R. W. 1. Smoking, intrauterine growth retardation and sudden infant death syndrome. lntemational Journal of Epidemiology 27: 238-241, 1998.

22. D'Souza, S. W., P. M. Black, N. Williams, and R. F. Jennison. Effect of smoking during pregnancy upon the haernatological values of cord blood. British Journal of Obstetrics and Gynaecology 85: 495-499. 1978.

23. Dahlstrom, A., B. Lundell, M. Curvall, and L. Thapper. Nicotine and cotinine concentrations in the nursing mother and her infant. Acta Pediatr-Scand. 79: 142-1 47. 1990.

1 O8 Dahlstrom, A., 8. LundeIl, M. Curvall, and L. Thapper. Nicotine and cotinine concentrations in the nursing mother and her infant. Acta Paediatr.Scand. 79: t42-M?,l99O.

Dennison, E., P. Hindmarsh, C. FaIl, S. Kellingray, O. Baker, D. Phillips, and C. Cooper. Profiles of endogenous circulating cortisol and borie mineral density in healthy elderly men. J.Clin.Endocnnol.Metab, 84: 3058-3063, 1999.

Dixon M., Jackson DM., and Richards I.M. The effects of histamine, acetykholine, and 5-hydroxytryptarnine on lung mechanics and irritant receptors in the dog. J.Physio/.London 287: 393-403, 1979.

Elliot, J., P. Vullerrnin, and P. Robinson. Matemal cigarette smoking is associated with increased inner airway wall thickness in children who die from sudden infant death syndrome. Am J Respir Crit Care Med 158: 802- 806, 1998.

Garn, S. M., S. A. Ridella, A. S. Petzold, and F. Faulkner. Maternai hematologic levels and pregnancy outcornes. Semin.Perinafo1. 5: 155-1 62, 1981.

Gourlay, S. G. and N, L. Benowitr. Arteriovenous difference in plasma concentration of nicotine and catecholamines and related cardiovascular effects after smoking, nicotine nasal spray, and intravenous nicotine. Clin. Phamacol. Ther. 62: 453463, 1995.

Groff, J. Y., Mullen, P. O., Mongoven, M., and Burau, K. Prenatal weight gain patterns and infant birthweight associated with materna1 smoking. Birth 24(4), 234-239. 1997. Ref Type: Abstract

Guyton A.C. and Hall J.E. Textbook of Medical Physiology. Philadelphia, W.B. Saunders Company. 1996,3-1148.

Hakkola, J., O. Pefkonen, M. Pasanen, and H. Raunio. Xenobiotic- Metabolizing Cytochrome P450 Enzymes in the Hurnan Feto-Placental Unit: Role in lntrauterine Toxicity. Critical Reviews in Toxicology 28: 35- 72, 1998.

Haustein, K. O. Cigarette smoking, nicotine and pregnancy. lntemational Journal of Chical Phamacology and Therapeutics 37: 41 7427, 1999.

Henry, C., M. Kabbaj, H. Simon, M. Le Moal, and S. Maccari. Prenatal stress increases the hypothalamo-pituitary-adrenal axis response in young and adult rats. Journal of Neuroendocrinolocrv 6: 341-345. 1994.

1 O9 Isaac, P. F. and M. J. Rand. Cigarette smoking and plasma levels of nicotine. Nature 236: 308-31 0, 1972.

Isager, H. and L. Hagerup. Relationship between cigarette smoking and high packed cell volume and haemoglobin levels. Scandinavian Journal of Haernatology 8: 241-244, 1971.

Kershbaum A, P. O., Bellet S, Hirabayashi M, and Shafiiha H. Effects of smoking and nicotine on adrenocortical secretion. Journal of American Medical Association 203: 275-278, 1968.

Keysell, G. R. O. M. W.The in vitro metabolism of S-(-)-nicotine by liver preparations from pregnant and lactating rats. In Gorrod, J. W. and L. A. Damani, eds., Siological oxidation of nitrogen inorganic molecules. Chichester, England, Ellis Horwood. 1985, 122-127.

Knottnerus, J. A., L. R. Delgado, P. G. Kipschild, G. G. M. Essed, and F. Smits. Haematologic parameters and pregnancy outcorne; a prospective cohort study in the third trimester. Journal of Clinical Epidemiology 43: 46 1 -466, 1 990.

Koren, G. Fetal toxicology of environmental tobacco smoke. Cunent Opinion in Pediatrics 7: 128-1 3 1 , 1995.

Kyerematen, G. A. and Vesell E.S. Metabolism of Nicotine. Dmg Metabolism Reviews 23: 3-41, 1991.

LaBorde, J. B., K. S. Wall, B. Bolon, T. S. Kumpe, R. Patton, Q. Zheng, R. Kodell, and J. F. Young. Haematology and serum chemistry parameters of the pregnant rat. Laboratory Animais 33: 275-277, 1999.

Leichter, J. Decreased birth weigth and attainment of postnatal catch-up growth in offspring of rats exposed to cigarette smoke during gestation. Growth, Development & Aging 59: 63-66, 1 995.

Li, W., 2. Weiyuan, and 2. Yanhui. The study of matemal and fetal plasma catecholarnines levels during pregnancy and delivery. Journal of Perinatal Medicine 27: 195-1 98, 1999.

Lichtensteiger, W., U. Ribary, M. Schlumpf, B. Odematt, and H. R. Widmer. Prenatal adverse effects of nicotine on the developing brain. Progress in Brain Research 73: 137-1 57, 1988.

Lodrup Carlsen, K. C., J. J. K. Jaakkola, P. Nafstad, and K.-H. ~arlsen. In utero exposure to cigarette smoking influences lung function - at birth. European ~es~irator-y ~&imal10: 1 7761 779, 1 997.

110 47. Lohninger, A,, B. Auer, S. Lechner, and H. Salzer. Prenatal effects

of betarnethasone-L-carnitine combinations on fetal rat lung. Journal of Perinatal Medicine 24: 591 -599, 1996.

48. Longo, L. D. Carbon monoxide in the pregnant mother and fetus and its exchange across the placenta. Annals New York Academy of Sciences 174: 313-341, 1970.

49. Longo, L. O. The biological effects of carbon rnonoxide on the pregnant woman, fetus, and newbom infant. Am.J.Obstet.Gyneco1. 129: 69-1 03, 1977.

50. Lueder, F. L., C. A. Buroker, S. Kim, A. S. Flozak, and E. S. Ogata. Differential effects of short and long durations of insulin-induced maternai hypoglycernia upon fetal rat tissue growth and glucose utilization. Pediatr. Res. 32: 436-440, 1992.

51. Lueder, F. L., S. B. Kim, C. A. Buroker, S. A. Bangalore, and E. S. Ogata. Chronic materna1 hypoxia retards fetal growth and increases glucose utilization of select fetal tissues in the rat. Metabolism 44: 532- 537,1995.

52. Maritz, G. S. The influence of matemal nicotine exposure on neonatal lung metabolism. Protective effect of ascorbic acid. Cell 6iol.lnt. 17: 579- 585,1993.

53. Matta, S. G., Y. Fu, J. D. Valentine, and B. M. Sharp. Response of the hypothalamo-pituitary-adrenal axis to nicotine. Psychoneuroendocrinology 23: 103-1 13, 1998.

54. Meberg, A., P. Haga, H. Sande, and 0. P. Foss. Smoking during pregnancy-hematological observations in the newbom. Acta Paediatrica Scandinavica 68: 731-734, 1979.

55. Mortola, J. P., L. J. Xu, and A. M. Lauzon. Body growth, lung and heart weight, and DNA content in newborn rats exposed to different IeveIs of chronic hypoxia. Canadian Journal of Physiology 8 Phamacology 68: 1590-7 594, 1990.

56. Mosier Jr., H. 0. and M. K. Armstrong. Effect of matemal nicotine intake on fetal weight and length in rats. PSEBM 124: 11 35-1 137, 1967.

57. Murrin, L. C., J. R. Ferrer, Z. Wanyun, and N. J. Haley. Nicotine administration to rats: methodological considerations. Life Sciences 40: 1699-1708, 1987.

11 1 58. Nagai, A., K. Sakamoto, and K. Konno. The effect of maternal

exercise on somatic growth and lung developrnent of fetal rats: morphologic and morphometric studies. Pediatr. Pulmonol. 15: 332-338, 1993.

59. Nash, J. E. and T. V. N. Persaud. Embryopathie risks of cigarette smoking. Experimental Pathoiogy 33: 65-73, 1988.

60. Navarro, H. A., F. J. Seidler, J. P. Eylers, F. E. Baker, S. S. Dobbins, S. E. Lappi, and T. A. Slotkin. Effects of prenatal nicotine exposure on development of central and peripheral cholinergic neurotransmitter systems. Evidence for cholinergic trophic influences in developing brain. The Journal of Pharmacology and Expenmental Therapeutics 251 : 894- 899,1989.

61. Navarro, H. A., F. J. Seidler, W. L. Whitmore, and T. A. Slotkin. Prenatal exposure to nicotine via matemal infusions: effects on development of catecholamine systems. J. Phamacol. Exp. Ther. 244: 940- 944,1988.

62. Nelson, E., C. Goubet-Wiemers, Y. Guo, and K. Jodscheit. Maternal passive smoking during pregnancy and foetaf developmental toxicity. Part 2: histological changes. Human and Experhental Toxicology 18: 257-264, 1999.

63. Nelson, E., K. Jodscheit, and Y. Guo. Maternal passive smoking during pregnancy and fetal developmental toxicity. Part 1 : gross morphological effects. Human and Experimental Toxicology 1 8: 252-256, 1 999.

64. Oxforn, H. and P. A. Fried. Smoking for two: cigarettes and pregnancy. New York, Free Press. 1980.

65. Pasanen, M. and 0. Pelkonen. The expression and environmental regulation of P450 enzymes in human placenta. Critical Reviews in Toxicology 24: 21 1 -229. 1 994.

66. Pechinot, D. and A. Cohen. The determination of maternal and foetal rat plasma corticosterone concentration in late pregnancy by competitive protein binding analysis. Journal of Steroid Biochemistry 1 8: 601 -606, 1983.

67. Petit, K. P. and H. C. Nielsen. Chronic in utero beta-blockade alters fetal lung development. Developmental Phamacology & Therapeutics 19: 131 - 140,1992.

68. Pfarrer, C., L. Macara, R. Leiser, and J. Kingdom. Adaptive angiogenesis in placentas of heavy smokers. Lancet 354: 303-303, 1999.

112 69. Poiand, E. R., P. Lutchmansingh, D. Au, M. Edelstein, S.

Lydecker, C. Hsieh, and J. T. McCracken. Exposure to threshold doses of nicotine in utem: neuroendocrine response to restraint stress in adult male offspring. Life Sci. 55: 1567-1 575, 1994.

70. Porozhanova, V., Bozhinova, S., and Popovski, K. The effect of tobacco smoking on matemal weight gain and the neonatal results. Akush Ginekol (Sofiia) 37(3), 10-1 1.1998. Ref Type: Abstract

71. Puizillout, J. J., G. Gaudin-Chazal, and H. Bras. Vagal mechanisms in sleep regulation. Exp.Br& Res. Suppl. 8: 19-38. 1984.

72. Rang, H. P., M. M. Dale, and J. M. Ritter. Drug dependence and drug abuse. Pharmacology. Edinburgh, London, Churchiil Livingstone. 1999, 614-633.

73. Reznik, G. and G. Marquard, Effect of cigarette smoke inhalation during pregnancy in sprague-dawley rats. Journal of Environmental 4: 141 -1 52, 1980.

74. Rotenberg, K. S. and J. Adir. Phannacokinetics of nicotine in rats after multiple-cigarette smoke exposure. Toxicology and Applied Phamacology 69: 1-11, 1983.

75. Russell, M. A. H., C. Wilson, U. A. Patel, C. Feyerabend, and P. V. Cole. Plasma nicotine levels after smoking cigarettes vrith high, medium, and low nicotine yields. 8r.Med.J. 2: 414-416, 1975.

76. Schelischeidt, J., G. Jorch, and J. Menke. Effects of heavy matemal smoking on intrauterine growth patterns in sudden infant death victims and surviving infants. Eur J Pediatr 157: 246-251, 1998.

77. Schuen, J. N., O. S. Barnford, and J. L. Carol!. The Cardiorespiratory Response to Anoxia: Normal Development and the Effect of Nicotine. Respir. Physiol. 1 09: 231 -239, 1 997.

78. Singh, S. P., A. K. Snyder, and G. L. Pullen. Fetal alcohol syndrome: glucose and Iiver metabolism in term fetus and neonate. Alcoholism: Clinical and Expefimental Research 10: 54-58, 1986.

79. Slotkin, T. A., S. E. Lappi, E. C. McCook, B. A. Lorber, and F. J. Seidler. Loss of neonataf hypoxia tolerance after prenatal nicotine exposure: implications for sudden infant death syndrome. Brain Research Bulletin 38: 69-75, 1995.

113 80. Slotkin, T. A., S. E. Lappi, and F. J. Seidler. Impact of fetal nicotine

exposure on developrnent of rat brain regions: critical sensitive periods or efFects of withdrawal? Brain Res.BuH. 31 : 319-328, 1993.

81. Slotkin, T. A., L. Orband-Miller, and K. L. Queen. Devebpment of [%] nicotine binding sites in brain regions of rats exposed to nicotine prenatally via materna1 injections or infusions. J. Pharmacol. Exp. Ther. 242: 232-237, 1987.

82. St.John, W. M. and J. C. Leiter. Matemal nicotine depresses eupneic ventilation of neonatal rats. NeurosciLet. 267: 206-208, 1999.

83. Stern, J. M. and J. L. Voogt. Comparison of plasma corticosterone and protactin levels in cycling and lactating rats. Neuroendocrinology 1 3: 173- l8t, l9?4.

84. Suzuki, K., T. Horiguehi, A. C. Comas-Unutia, E. Mueller-Heubach, H. O. Morishima, and K. Adamsons. Placenta[ transfer and distribution of nicotine in the pregnant rhesus monkey. Arn.J.Obstet.Gynecol. t 19: 253- 262.1974.

85. The Report of the U.S.Environmenta1 Protection Agency. Respiratory health effects of passive smoking: lung cancer and other disorders. Smoking and Tobacco Control. 1993, 248-259.

86. Tuthill, D. P., J. H. Stewart, E. C. Cotes, J. Andrews, and P. H. T. Cartiidge. Maternai cigarette smoking and pregnancy outcome. Paediatric and Perinatal Epidemiology 13: 253, 1999.

87. Wall, M. A., J. Johnson, P. Jacob, and N. L. Benowitz. Cotinine in the serum, safiva, and urine of nonsmokers, passive smokers. and active smokers. American Journal of Public Health 78: 699-701, Z 988.

88. Wheeler-Sherman, J., Sherman, M. P., and Bernert, J. T. Prenatal Nicotine Exposure Lowers Newbom Heart Rate and Limits Variability. Society for P ediatric Research .2000. Ref Type: Abstract

89. Witschi, H. and A. Lundgaard. Effects of exposure to nicotine and sidestream smo ke on pregnancy outcomss in rats. Toxicology Letters 7 1 : 279-286.1994-

90. Younoszai, M. K., J. PeIoso, and J. C. Haworth. Fetal gruwth retardation in rats exposed to cigarette smoke during pregriancy. A r n J Obsfet Gynecol. 1 04: 1 207-1 21 3, 1 969.