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PLACENTAL TOXICOLOGY Edited by B.V. Rama Sastry, D.S¢., Ph.D. Professor of Pharmacology Professor of Anesthesiology School of Medicine Vanderbilt University Medical Center Nashville. Tennessee CRC Press Boca Raton Ann Arbor London Tokyo 0 O~

CONTENTS I. Techniques: In Vitro Perfusion of Human Placenta ...................................................... I Hemmt.,, St'Im¢ider .-klcohol. Placental Function. and Fetal Gro~.,.th ........................................................... George I. Henderson and Steven Schenker 3. Smoking. Placental Function. and Fetal Growth ......................................................... 45 B. V. Rama Sastry attd t 7ctoria E. Janson -1.. Opioid Addiction. Placental Function. and Feta~ Growth ...........................................83 B.V. Rama Sast~3. 5. Opioid Receptors in Placenta and Their Functional Role ......................................... 107 Mahmoud S. Ahmed and Bojana Cemerikic 6. Cocaine Addiction. Placental Function. and Fetal Growth ....................................... 133 B. ~." R,tma Sastry 7. Placental Biogenic Amines and Their Transporters .................................................. 161 Vadivel Ganapatt~y and Frederick H. Leibach 8. Renin-Angiotensin System in Placenta ...................................................................... 175 Alan M. Poisner and Gregory J. Downing 9. Placental Enzymes: Cytbchrome P450s and Their Significance ............................... 197 :14o111 R. Juchau I0. Placental Metabolism or" Xenobtottcs .......................................................................... l.~ Raymond D. Harbison. Christopher J. Borgerr. and Christopher M. Tea)" II. HIV and the Placenta: Mechanisms of Transfer. Drug Therapy. and Drug "i'ox,~:,ty .................................................................................................... ... 239 Rodney I.Y. 14o, Conrad .14. Pereira. and Jashvant D. Unadkat 12. Environmental Agents and Placental Toxicity: Anticholinesterases and Other Insecticides ................................................................................................ 257 Ramesh C. Gupta Index .............................. : .............................................................................................. ~9 ~x

Chapter 3 SMOKING. PLACE.NTAL FU.NC'I'I()N. AND FETAL (;ROWTH B. V. Rama Sastry and Victnria E..|anson [I. Ill. iV. VII. CONTENTS Ao introduction .................................................................................................... ............. Smoking. Birth Weight. and Fetal Growth .......................................................... 46 Tobacco Smoke Components ............................................................................... I. Tobacco Alkaloids ........................................................................................... at) 2. Nitro,amines .................................................................................................... . 50 3. Tcbaccb Ga.~es ........................................... .. ..................................................... 5 .t. Metals .................................................................................................... ........... 52 Metabolism of Nicotine and its Placental Transfer .................................................... 52 A. Elimination and Metabolism of Nicotine ............................................................. 52 B. Cotinine Plasma Levels: Qualitative Marker of Smoking ................................... S# C. Placental Transfer or" Nicotine and its Metabolites ............................................. 55 D. The Human Placenta as a Metabolic Organ in Smokers ..................................... 56 .Maternal Smoking. Fetal Tobacco Syndrome. and R.eproducti~e To,cicit5 of Nicotine .................................................................................................... ............... 57 Maternal Smoking and Placental Function ................................................................. A. Mammal Smoking and Blood Flow in the lntervillous Spac, ............................ 38 B. Maternal Smoking and Depression of Amino Acid Uptake by Placenta ........... 60 C. Maternal Smoking and t.he Regulatory. Mechanisms for Amino Acid Upr,.kt b.~ Plactntal Trophobl~t Cells ................................................................ 60 I. The Gammaglutamyl Cycle ............................................................................. 2. Placental Acetvlcholine and Regulation .of Amino Au . Transport ............... 6_ 3. Phospholipid N=Methylation in Placental Pl~.sma Membrane and Amino Acid Transport ..................................................................................... -; Ox~d"-tix e E;:er~': Sour~¢.~ .rod P~a~ental Amino Acid T:';:n,port .................. :.'.-~ D. NIatema! Smoking and Transfer of Amino Acids from the Trophoblast to the Umbilical Circulation " ' E. Maternal Smoking and Function of Endothelial Cells in Human Placental Vasculature ............................................................................................ (55 Maternal Tobucco Smoking and Covalent DNA Adduct~ in the Human Placenta .................................................................................................... ....... ~ .Alterations m Amino Acid and Protein .Metabolism in Growth-Retarded Babies as Indicated by Blood Analysis ...................................................................... 67 Scope of Future Investigations ................................................................................... 70 A. Placental Compensator3." and Genetic F'a.ctors ............... : ...................................... -0 B. Genetic Factors in the Placental Toxicity of Tobacco Alkaloids ........................ "71

Plttt'ent~d C. Placental .Metabolism o( Tobacco Alkztlt_+~.~ ....................................................... 7 13. Influence of Environmental l=actor,~ and Other Abused on Placental Toxicity of Tobacco Smoke Compot+ents ....................................... 7 A,:k:;,,~ led,__,mc nt.-, .................................................................................................... ............. 72 References .................................................................................................... ......................... 73 I. INTRODUCTION Scientific literature published during the past century, indicates that cigarette smoking during pregnancy has significant adverse effects on the development of the fetus and the health and development of the newborn baby. The hazardous effects of smoking during pregnancy include possible abortion, fetal death, fetal growth retardation, and ,~hort- or )ong-ten'n developmental deficits in the infants. Concern about the exposure of pregnant women to tobacco dust and smoke has been expressed since 1868.. Abortion was frequent among women workers in .tobacco factories in Europe. There was also a high. rate of mortaliw among the infants of females in tobacco- feinted occupations. In 1935. Sorting and Wallace-" obse~,ed that cigarette smoking by preg- nant women increased fetal heart rate. and they speculated that this was probably due to transl~)lacental transfer of nicotine into fetal circulation. Campbell:.-" obsetwed that a woman who smoked heavily during pregnancy ~,as likely to have more difficulty during the course of pregnancy, parturition, and lactation than a nonsmoker. In 19~,0. lEssenberg and collabora- tors'* investigated the influence of nicotine and ~obacco smoke on pregnant albino rats. They found that the young of nicotine-treated as well as tobacco-smoke-exposed rats were under- weight compared to those of control untreated rats. These studies raised important questions about the effects of tobacco smoke and nicotine on intrauterine fetal growth retardation ~ IUGR). In 1957. Simpson* reported that babies of women smokers had significantly lower birth weight than those of nonsmokers. Simpson's studies were confirmed by several others in',-oi','mg more than half a million bir',hs. These studies have been discussed in detail in reports of the U.S. Surgeon'General.''~ In general, babies of p~gnant women smokers are 200 g lighter than babies born to comparable pregnant women who are nonsmokers. The mecha- nisms of smoking-induced IUGR and the role of placental function in IUGR have yet to Le established. A. SMOKING. BIRTH WEIGHT. AND FETAL GROWTH it: '~mok:,ng ~tudies. the birth ~,eights of babies in groups of smoking and nonsmoking women are expressed as mean birth weights. In some studies, the percentages of babies (of smoking and nonsmoking mothers) who at birth weighed less than a specified weight, namely 2500 g. were compared. According to the available evidence, maternal smoking during ~regnancy exe.,'t,s a retarding influence on fe',al grn~ th wh{ch i,~ manifested by decrease~;, high ~eight of the ;,nfant. The evidence that maternal smoking causes reduced birth weight is indicated by :he following:--`) I. Results are consistent in studies from many different countries, cultures, races, and geographical settings. 2. The relationship between smoking and reduced birth weight is independent of all other factors which affect birth weight, such as race. maternal size. socioeconomic status, and sex of the infant.

Smoking and Plat'et~tal Fum.'timl 47 3. This relationship is independent of gestationa[ age."' ~ [n other words, the infants smoking mothe~ are small For date r~ther than preterm t Figure I~. 4. There is a dose-response relationship between the degree of maternal smoking and fetal growth retardation.I"- The more a woman smokes during pregnancy, the greater the reduction in birth weight. This dose-response relationship is also independent of the gestational age of the fetus,I: 5. If a woman gives up smoking during pregnancy, her chance of deti~'ering a normal- weight baby is similar to that of a nonsmoker.=: Since the low birth weight associated with maternal smoking occurs at all gestationai ages and is not due ~o a significant reduction in mean gestation, it mus~ be due to a reduction in the rate of fetal growth. The pattern of fetal growth retardation ~ssociated with maternal smoking is a decrease in all dimensions." This indicates a common cause for the development of all organs of the fetus. The disparity in growth and development between the children of smokers and those of nonsmokers disappears by the age or" l I. indicating that maternal smoking produces reversibic intrauterine fetal _m'owth retardation." One of the requirements for the growth and maturation or" all organs of th~ fetus is availability, or" essential amino acids and other nutrients. The fetus is dependent on the placental transfer of amino acids and other nutrients from maternal blood to fetal circulation. The requirement of essential amino acids for fetal growth is met only when these nutrients am available in t'eta! blood in requisite proportions and enter the fetal cells. Several factors will influence the transfer of nutrients from maternal blood into fetal circulation: (1) rate of maternal blood flow through the inter~iilous space. ~2~ pO: in 130 120 100 Non-Smokers Smokers 37 40 43 37 43 Weeks of Gestation FIGURE 1. Relationships between human infant birth weishts in ounces and gestation in weeks in nonsmokers and ~mokers. Smokers used less th~n one pack (20 cigarettes) per day. Gestat|on is expressed in completed weeks. Each vertical bar in.dicates gestation in weeks from ~7 to .t4 weeks. At each gcs~ational week. the menn birth weight of smokers" bab=es =s lower than ~nt of nonsmokers" babies. The dnt= in the figur~ :tin from a report by Butler and A.Iberman" involving 17.000 births in Great Brt~in.

4~ Placental T~.rtcnh~:4y 155 150 ~ 145 140 125 A B Non-Smokers Light-Smokers 120 37 40 43 37 ~0 43 C Heavy Smokers 37 40 43 Weeks of Gestation li~t smokm (~0 ci~y). ~d iC~ ~d ~v~ smoke~ (>~0 ci~uesldayL A¢ ~¢h smo~ng in¢~d PR ~d hmvy smo~ng i~m~d PR ~m ~ li~t smoking PR wh~ ~mp~ no~mo~'s P~ ~¢ ~ m ~e fi~ ~ from = s~y by Wing~ ¢~ ~.~= involving 7~ maternal blood. (3) uptake of amino acids and other nutrients by the p[acental trophobiast, (4) transfer of amino acids f~'om lxophoblast to umbilical circulation, and (5) rate of blood flow through the umbilical a~erial-venous system. Once amino acids and other basic nutrients enter the fetal circulation, their utilization by fetal tissues is dependent on (I) pO,. in the fetal blood and (2) uptake of amino acids by the fetal cells. Components of tobacco smoke may interfere with one or more steps in these processes and. therefore, utilization of essential amino acids and nutrients by the fetus. Placental weights ate either increased or less affected than birth weights by maternal smoking. As previously discussed, birth weights of infants axe affected by maternal smoking, and the deg~'~ of reduction in birth weight is related to the number of cigarettes smoked. Therefore. the ratio of placental weight to birth weight, or the placental ratio, tends to be larger for smokers than for nonsmokers fFigure 2).:'.~-~ The placenta] ratio increases with an increase in the degree of smoking, and this increase has been obce,,'ved at all ,.ze~ta;ional pe~ods." These observations indicate that a large or normal-size placenta tends to support the nutritional" transport requirements of a small fetus. Even then the effects of smoking on the growth of the fetus ate not overcome, and fetal gTowth is retarded in smokers. These observations suggest that smoking and tobacco components affect the functional efficiency of the placenta more than its size. B. TOBACCO SMOKE COMPONENTS The raw materials in a cigarette provide only a prelude to what happens when it is smoked. A lit cigarette generates more than 2000 known compounds by a number of processes responsive to temperature profiles. The lit cigarette has a steep temperature gradient. 880°C to 40°C, which can be demarcated into three reaction zones:=as (I) high temperature zone (900 to 600°C). which has 8% (v/v) hydrogen, 15% (v/v) carbon monoxide, and insignificant o 03 o~ o

49 TABLE i Ma.j.r Components in Tobucco Smoke Alkaloids .%;icotm= n. 1-2..~ mLz Tobacco g~es CO 16.2 mg 17.18 CO: H~dm~=n c~nid= Pul~nuc~¢ar aromatic h~dn~a~)n Mc~ Cu i qO n~ :1 Cd Hg 4 ng 21 Ni ~0 • ,Mainstream ~mok¢ or panicula¢e..,dcig:m=tt¢. amounts of oxygen: (2) the oxygen-depleter pyrolysis-distillation zone (600 to 100°C): and ~3) the low temperature zone (<100°C3. which contains about 12% oxygen. Mainstream smoke is formed within these zones by hydrogenation, pyrolysis, oxidation, decarboxylation, dehy- dration, chemical condensation, distillation, and sublimation. The exit temperature of the mainstream smoke is about 25 to 50°C depending on butt length. Sidesu'eam smoke is produced during smoldering of the cigarette at the peak temperatures inside the glowing cone (about 800=C/. The composition of mainstream and sidestream smoke varies depending on the type of tobacco, filler mamrials, packing density, additives, moisture, and the filters. Of all the tobacco smoke constituent.s, only a limited number of compounds have been investigated for human o~ad animal toxiciw,. The placental transt'cr and toxiciw, of any of these have been completely studied. For the purposes of this chapter, tobacco smoke components are divided into five groups: (1) tobacco alkaloids and their metaboiitcs. [2) nitrosamincs. [3~ tobacco gases. I J,) metals, and ~5~ ~oxic hydrocarbons (Tables [ and 2). I. Tobacco Alkaloids The smoking of a cigarette satisfies a .~moker's ph.~ siolo,='ical and p,,ycholo,='ical needs, and nicotine is generally regarded as the principal alkaloid responsible for the pharmacodynamic effects. There arc several other alkaloids in tobacco and tobacco smoke. Nicotine in tobacco smoke particulates is about 100 to 2~00 .Lt~cigarette. while all other minor alkaloids amount to 61 ¢o 199 g~cigarette (Table I. Figure 3). All of the other alkaloids have been compared to L-nicotine in several pharmacological systems.:-" All of them were less active than nicotine. the closest being anabasine, which is 18 to 7.5% as active as nicotine in different pharmaco- logical models.:* However. in these studies, optically pure nicotine was compared with racemic forms of other alkaloids for their pharmacological activities. Racemic anabasine is about 75% as active as L-nicotine in releasing catecholamines from rat adrenal glands.:= Optically pure anabasine may be as active as L-nicotine in releasing adrenal catechotamines. Them is no intbrmation on the chronic effects of other tobacco alkaloids in man or animals. More experimental work on other tobacco alkaloids is necessary, to determine their contribu- tion to the effects and toxicity or" tobacco smoke.

5O TABLE 2 Nitros-~mines in Tobacco and Tobacco Smoke" rig/cigarette" ~;itrns-~mine Tf~ha¢¢o sme~ke smoke 680 9.4 30O • Summanzad from data r~poncd by Brunnemann and Hoffmann.= Amounts vary type of cigm'elte and length of cigareRe (70--g5 gm long). Higher limit is estimated and not determined. Average values from Hecht et al."J bIicodne Dehydronicotine Nicotyrine Cot/nine Nor-cot/n/he Myosamin¢ Bipyridyl Ambasine Anatabine FIGURE 3. Common tobacco alkaloids in tobacco smoke. 2. Nitrosamines The nitrosamines in tobacco smoke can be divided into two ,m'oups: (l) volatile nitro- samines, including N-nitrosodimethylamine (NDMA), N-nitroethylmethylamine (N]~MA). N- nitrosodiethylarnine (NDF_A), and N-nia'osopyrrolidine (NPYR): and (2) tobacco-specific nitrosamines, including N-nitrosonomicotin¢ (NNN), 4-(N-methyl-N-nitrosamino)-4-(3- pyridyl)- 1-butanal (NNA), and 4-(N-methyl-N-nitrosamino)- l-(3-pyridyl)- l-butanone (NNK). Both volatile and tobacco-specific nitrosamines (Figure ,t) have been identified in mainstream smoke (Table 2). Volatile nitrosamines have also been identified in sidestream smoke of cigarctms and cigars, and their levels in sidest~am smoke am at least t0 times higher than in mainstream smoke. During I h in a smoke-filled indoor environment, one may inhale volatile nitrosamines in quantities equal to those in the mainstream smoke of 0.5 to 30 cigarettes. If the mother smokes, volatile nitrosarnines of sidestmara smoke may b~ harrnfi~l to the fetus and the nursing baby. However. i~ has yet to be determined whether volatile nitrosamines play a role in cancer induction in the lung. oral cavity., placenta, or other tobacco-related cancer sites.

/%., Several tobacco-re!areal nitrosamines have also been identified in :obacco smok.~ (Figure ¢. Table Z~. Of all .:.~e :obacco-s.~ific ,~itros-,.'n. ines. ~ NNN;. '-t-(N-me~yI-N-nitrosamino~- ~.- • 3-py.,mdy~)- i -:.'-ut:av:n¢ : ?iNK . .':-..'-.::r.~;:~pig~=.q.:_- . NP*.P). and N-~fitrosoanab~me :NAB) ~ ccnddered m _be tumomgenic or .':a~ireg~.~ic in exaedmenml animals.'--" NNK and NPIP "-= stronger :arc!nogens :hun NNN ann .";AS. 3. Tobacco Gases In addition to mcodne '--".d "'~,-. tobacco or a!_,g.a~=¢ smoke contains a dozen gases ~,Tabie of which carbon mono'dde, hydrege.-, cTanide, and nitrogen ex,de.~ ,a mixture of NO.. and NO., genervAly mea~ur.*d as NC:; a~ c: pharm,.,ucoiog;.cal significance.'-'-:° Each of these gases causes dssue hyg'oxla :hrough ~.ifferent mechanisms. The af.e.'ni~- of CO for hemog!obin is about .~C0 times that of ex'.:,='en.:" ~e:e.,'or=. CO displaces O: ~rom :ombinatien and forms carboxyhemoglobin .~ COHb). which is not capabie of respiratory, function. Carbon monoxide may also inhibit ~-.e ~-iac.*nta! "'t'acilitated oxygen :ransfer ~ystem'" ,*FOTS; and the placental cn.-bonic anhydrase.:".:° which r,-sults in an inc.-ease of the pCO:. The O: uptake by placenta .'armor be aw.dic:~" " . • ~, simv/e di,'guston or" O:. Therefore. a r'acilimted oxygen transfer system was proposed for placenta. This is a carv.e." system which does not require adenosine 5"-~.:'il:he~hate ).~,TP~.. It is irh~bited by CO. Nitrogen oxides oxidize me fe.-.ous ::vn of hemogiobin -:o :he fa."ric s:ate, r'orming methemogiobin IMHb. ~e.":ic hemoglobin), which is not capable of carrying ,:xygen. Cya- nides combine with me:hemoglobin..-'e,r:.-..ing cyanme:nemog!obin *.%tHb-CN:. ,vhich also ,:annct can:y oxygen.* F'ar:,ne:. cyanides :eversibly inhibit oxidative enzymes such as cytochrome oxidase ~,cy,'echrome ..~ 3) and dep.dve placental tissue of necessary oxygen. of these effects of gases in :obacco smoke are expected to cause, tissue hypoxia, which ~lepresses ceil function.

52 4. Metals About 30 metal.~, o( which special interest has be~n focussed on Ni. Cd. As. Ca. and Hg. have been identified ~n tobacco ~m~k¢ (Table I~. ~¢s¢ are present m [oh~cco pa~icula[es and are inhibitors of sulfahyd~'l ¢nzym¢~. All tbrms of Ni (m~tal and sanou. s~[~) are c=~no~en[cJ~.,~: Dudn~ ~mokin~. 10 ~o 20% o£ the Ni ~n the tobacco (~0 n~/ cig~ette) is t~nsfe~ed into mainstream smoke and is present in the gas phase ~ nickel carbonyl. Ni(COL. a carcinogen. [n mainstream smoke. 7 to 90 ng of Cd are present per cigarette.'s A heavy ~moker retains about 1.5 gg of C~ per day and ma~ accumulate up to 0.5 m~ through inhalation. Dependin~ on the smoking pa~tems of a smoker. 7 to I $% or" ~he total arsenic in cigarette tobacco * 12 ci~amtte~ is tmnsfe~ed imo the respiratory tract, and abou~ 50% or" this is deposited in the tissues.~ Due to ~eir cumu{ative natu~, all o~ ~ese m~mls ma~ accumulate in the placenta of a pmgn~t smoker ~d affect i~ Junction. II. METABOLISM OF NICOTINE AND ITS PLACENTAL TRANSFER Nicotine, the primary alkaloid of tobacco, has been extensively studied for its distribution. metabolism, and elimination both in animals-~`~ and man."z~: After its absorption into the blood (pH 7.4). about 69% of nicotine is ionized and 31% is nonionized. Less than 5% of the absorbed nicotine binds to plasma proteins.'-* Nicotine is distributed extensively to body tissues, with a steady-state volume of" distribution averaging 180 1 (2.5 times the body weight in kilograms). When nicotine concentrations have fully equilibrated, the amount of nicotine in the body tissues is 2.6 times the amount predicted by the product of the blood concentration and body weight. The. pattern of tissue uptake has been examined in tissues of rabbits by measuring concentrations of nicotine in various dssues after infusion of nicotine to a steady state. Spleen. liver, lungs, and brain have high concentrations of nicotine, whereas adipose tissue has a r~latively low concentration. After rapid intravenous (i.v.) injection, concentrations ot" nicotine decline rapidly because of its uptake by tissues. After i.v. injection, concentrations in arterial blood, lung. and brain are high, while concentrations in. tissues such as muscle and adipose (major storage tissues at steady state) are low. The uptake into the brain is rapid, occurring within 1 or 2 rain. and blood levels fail because of peripheral tlssde uptake for 20 or 30 rain after administration. Thereafter. blood concentrations decline slowly depending on rates ot" elimination and dismbution out of storage tissues. Nicotine is also secre~d into saliva."~ It is also identified in the freshly shampooed hair of smokers and of nonsmokers environmentally exposed to tobacco smoke,a-~ Nicotine is also secreted into breast milk. breast fluid of nonlactating women.-~.:: and cervical mucous.:" A. ELIMINATION AND METABOLISM OF NICOTINE Nicotine is metabolized primarily in the liver but also secondarily, to a small extent, in the lung."~ Renal excretion of unchanged nicotine depends on urinary pH and urine flow and may range from 2 to 35%. but typically accounts tbr 5 to 10% of total ellruination.:''-'~ The main metabolites of nicotine are eotinine, nicotine N'-oxide. and nomieotine (Figure 5). Codnine is formed in the liver in a two-step process: (I) oxidation or" position 5 or" the pyrrolidine ring in a cytochrome P450-mediated process to nicotine-.Av'r,iminium ionS: and I2) metabolism of iminium ion by a cytoplasmic aldehyde oxidase to cotinine2s Pan of the nicotine iminium ion (or intermediate carbinolamine) is convened stepwise into 7-3-pyridyl oxobutyric acid.

Nicodne-].'-N-oxide H $(-)-Nicotine / v Nor-nicotine Nicotine glucuronide N-Methylnicotine Cotinine /-~ 3- Pyridyl--~,-methylamino)- C.arbinolamine butyraldehyde • ~,-(3-Pyridyl--z-me~hylamino)- butyric acid Nicodne ~ninium ion -y-3-Pyridyl- oxobutyric acid Cotinine is also extensively metabolized, with only about 13 to 17% excreted unchanged in ~he urine?~-~ Sever~[ rnet~bo[kes of cotinine have been reported, including trans-3"- hydroxycotinineJ~" 5"-hydroxycodnine.~ cotinine N-oxide.~ and cotinine methonium ion~ (Figure 6). Little is known abou~ ~he quantitative importance or" these metabo[ites. Hydroxycotinine appeoxs to be a major memboliteY"~s wkh urinary, concen~ons exceeding codnine by ~wo- to ~hmefo[d. Codnine N-oxide {s R minor membo[{~e {n humans, accounting Oxidative degra~don of the py~o[idinc dng fo~s "/-3-pyddy[ oxobu~dc acid nicotine an~o[ codnine, which ~s ~nher converted ~mo 3-pyddyI acetic acid (F~gum 7). compound h~ been [demificd qu~imdve[y in hum~ urine.~ Benowi[z and his co[labomzo~ have me~umd qumdtadve ~pec~ of n~codne metabolic profiles in man. L'~na~" excretion profiles of nicotine and ci~ht of i~s metaboli[cs have b~en dete~incd"~ (Figure 8). According to ~eir studies, t I) a high pc~enmgc (avenging 88%) of a systemic dose of nicotine can be accounted for by me~ufing nicotine ~d i~ metaboIites: ~2) the pattern of metabolism is similar when nicotine is inhaled or abso~ed t~nsde~alIy: t3) there is considerable intc~ndividual vadability in the pattern of metabolism, but the pattern is consistent for an individuak and (4) within individuals, the ext~nt of conjugation of nicotine ~d cotinine is highly co.elated, but neither is co.elated wi~ the extent of conjugation of Y= hyd~xycotinine. ~is suggests that similar enzymes ~ involved in the conjugation of nicodne and codnine and that a different enzyme may ~ involved in the conjugation of

5-I Cotinine-N-oxide N-Mcttaylcotinine ~ CH~ J CH~ O t Hydroxycotinine Cotinine OH Nnr-eotinine N-metlaylbutyramide ~ Cotit~e -t-3-Pyridyl- glueutoa2de oxobutyri¢ acid FIGI.~'RI~ 6. Metal0olic scheme foe, the conversion of cotinine, a major metabolite of nicotine, into hydroxy¢otinin¢, .V-me~.vleotinin,. ¢otinine N.oxide. nort:otinine, and -p.3-pyridyl oxobutyrie acid. Cotinine and its glucuronide. 3- hydroxyeotmine, its glueuronide, and ¢otinine N-oxide are d~teeted in the urine after sysmmi¢ administration of nicotine."t 3"-hydroxycotinine. 3"-Hydroxycotinine. cotinine, and nicotine account for 47%, 30%, and 15%. respectively, of the dose of nicotine in urine as free alkaloids or glucuronides. The levels of their glucuronides in urine are higher than those of their free bases. Cotinine N-oxide, nicotine N-oxide, and nomicotine are only minor metabolites in human urine and account for only 9% of the dose of nicotine. There are no studies to indicate whether this metabolic pattern remains the same or is altered during pregnancy. B. COTINI'NE PLASMA LEV'ELS: QUALITATIVE MARKER OF SMOKING The elimination half-life of nicotine after nicodne administration in man to a steady state is about 2 h?~.e'v This half-life of nicodne is useful in p~dieting its accumulation rate in the body with repetitive doses as well as the time course of its decline after cessation of dosing. .assuming a half-life of 2 h. one would predict nicotine to accumulate over 6 to 8 h (3 to 4 half- lives) of regular smoking and to persist at significant nicotine levels for 6 to 8 h after cessation of smoking. Peaks and troughs follow the use of each cigarette, but after smoking a number of cigarettes trough levels rise and the influence of peak levels becomes less important. However. a more reliable marker whose levels are maintained constant is necessary, to determine moderate use of cigarettes or other forms of tobacco. Cotinine levels are of special interest as qualitative markers and quantitative indicators of nicotine intake. Cotinine is present in the blood of smokers in much higher concentrations than

and Placental Func'tmn -~-3-Pyridyl- o×obutyric acid 3-Pyridytacetic acid FIGURE 7. Metabolic scheme t'or the conversion of .pj-pyridyl oxobutyric formed from nicotine and/or codnin+ mto .~-p.vrid¥! ~c~ic a~id (3-PAA). 3-P.~A is quafi~a~i~eiy de~ccd in urine of exl:~rnen~.al animals:' nicotine. Cotinine blood levels average about .~0 to 300 ng/ml in cigarette smokers.6~-~s After cessation of smoking, levels decline with a half-life averaging 18 to 20 h (range I I to 37 h). However, because of its tong half-tile, there is much tess fluctuation in cotinine concentrations than in nicotine concentrations throughout the day. C. PLACENTAL TRANSI~R OF NICOTINE AND ITS M~TABOLITES Three types of experimental approaches indicate that nicotine crosses the placenta: (l) securing nicotine or cotinine in umbilical blood after a mother uses tobacco preparations. (2) monitoring the effects of nicotine on the fetus, and (3) measuring placental transfer of nicotine in isolated human placental cotyledon. Nicotine has been found in amniotic fluid and umbili- cal cord blood of neonates after maternal tobacco use/~-es Nicotine infusion in pregnant sheep increases uterine vascular resistance and reduces uterine blood flow. These effects appear to be mediated by catecholamine release..°,~o Both cigarette ~moking and nicotine gum chewing increaqe fetal heart rate during the qecond mme~ter in humans, consistent with sympathetic activation.'= During the third trimester in humans, cigarette smoking or nicotine gum chewing decreases fetal heart rate and reduces fetal breathing movements, both of which may be signs of fetal hypoxia:~-7~ or nicotine- or catecholamine-induced release or" opioid peptides (enkephalins) in fetal brain:=.vs There are no detailed in ~.itro studies on the placental transfer of nicotine or its metabolites in placental cotyledon. In a preliminary, study. Sastry et al.:~ reported the transport of nicotine

i 56 Nornico~Jne Placental Toxicology ~ Nicotine 10 20 30 Percent of Go tmlne N-oxlcle Nicotine N-oxide 3-Hydroxycotinine (HC) glucuronide ' 3-HC ~Cotinine glucuronide ~ Cotinine Nicotine glucuromde 40 50 60 Systemic Dose of Nicotine in isolated perfused cotyledon of normal human term placent~ The perfusion technique is classical and. has be~n validated by several investigators.~-~ The placental cotyledon was perfused with aerated (21%. O:. 5% CO,.) IC,~bs-Ringer bicarbonate buffer (pH 7.4. 37"C~ containing 2% albumin on both the maternal (2,30 ml. 15 ml/min, 0.6 in Hg) and fetal (93 ml. 1.75 ml/min. 1.75 in Hg) sides in a closed recirculadng system. Nicotine (2 rag) was added to the maternal perfusate, and perfusate samples (I ml) were collected from both sides at regular intervals and analyzed for nicotine content by high performance liquid chromatogra- phy (HPLC~.~° In about 40 rain. tS.6q~ of the nicotine added to the maternal perfusate was transferred to the fetal pwrfusate, and the matwmal/fetal concentration redo reached 1,0: this was maintained for the next 60 rain. These results show rapid placenta[ transfer of nicotine. consistent with its high lipid solubility. D. THE HUMAN PLACENTA ~ A METABOLIC ORGAN IN SMOKERS The function of placenta as a metabolic organ is of interest in several aspects: (l) metabo- lism of nicotine and other tobacco alkaloids and components of tobacco smoke into toxic components like nitrosamines, (2) induction of placental enzymes, and (3) conversion of polyaromatic hydrocarbons (PAHs) into mutagenic metaboLites. Most of the enzymes for biowansformations present in the Liver are also present in the placenta. Induction compounds affecting liver enzymes also affect the inducible placental metabolic pathways.8t This subject has been discussed in detail in several reviews and articles.~Z-~s Glucuronidadon, sulfation, and glutathione conjugation as well as reactions dependent on cytochrome P-t-50, epoxide hydrase, catechol-O-mcthyltransferase, and monoamine oxidase

NONSS'IOKING MOTIIER villus SMOKING MO'rlIER Vaso~onszricdon of Umbilical Vcsscls Vasoconstricdo~ of Umbilica] V©sscls

6O Placental Tm-icolo~y TABLE 3 Influence of Nicotine on the Uptake of c~.Aminoisobutyric Acid ((~-A[B) by Human Placental Villi~ Treazment K~ Y,,., (n) (nmol/I) (nmoUmin/g) Irreversible component Control 114) "~ "~- ..-) _ 0.7 69 _ 8 Nicotine (8) 0.79 - 0.42 .0 _ 9 Adapted from Bamwell and Sast~') E~ch value is a mean * SE from the number (n) of placentas subjected to Ireatment with nicotine, which lowered both the Michaelis-Memen constant (K,J and maximum velo~i~ (V.m) for a-AIB 07 B. MATERNAL SMOKING AND DEPRESSION OF AMINO ACID UPTAKE BY PLACENTA The fetus is dependent on placental transfer of amino acids from maternal to fetal circu- lation for its requirements of amino acids.In The placental transfer of amino acids is a two- step process: (1) active uptake of amino acids by placental trophoblast cells from the mother's blood, and (2) passive diffusion of amino acids from placental trophoblast ceils into umbilical venous blood. The first step is critical, and its efficiency may be depressed under placental hypoxic conditions induced by maternal smoking. Nicotine and several components of to- bacco smoke (carbon monoxide, cyanides, and nitrites) reduce active uptake of amino acids by isolated human placental villi,u~.~-~-') Exposure of human placental villi to nicotine inhibits the upt~. e of ct-aminoisobutyric acid (ct-AIB). a nonmetabolizable amin6 acid. and decreases both the maximum velocity (V,,~) and the Michaelis-Menten constant (K,,) for uptake of ~-AIB'~9 (Table 3). The inhibition is neither competitive, noncompetitive, nor uncompetitive, indicating the complex nature of the inhibition of placental AIB uptake by nicodne (Figure 10). Part of the inhibition is not reversible by washing the placental tissue. which may be of significance in chronic smoking (Table 3). Concentrations of several essential amino acids (val. met. lieu. leu. tyr, pbe. his) and nonessential amino acids (asp. glu. gly. ala. arg) in the placental villi of nonsmoking mothers a,re about 30 to 50% higher than those of smokers.''--'~:~ Concentrations of thr and phe are about 14 to I5% higher in the placental villi of nonsmokers than in those of smokers. Maternal smoking decreases the uptake of amino acids by the placenta and the net transfer of amino acids from maternal to fetal blood. Therefore. fetal undernutrition for amino acids may partially explain fetal intrauterine growth retardation in tobacco smokers. C. MATERNAL SMOKING AND THE REGULATORY MECHANISMS FOR AMINO ACID UPTAKE BY PLACENTAL TROPHOBLAST CELLS c~-AIB, a nonmetabolizable amino acid. has been used as a model amino acid to study the mechanisms of amino acid uptake by placental tissue. Several regulatory mechanisms have been proposed for the cellular uptake of amino acids in the placenta: (1) the gam,maglutamyi cycle.~-'#.t~ (2) placental acetylcholine release and amino acid transport coupling.~'~-~v.~-'°.~-'o (3) phospholipid N-methylation in the plasma membraneJ~ and (4) oxidative energy sources. 12~1.129 1. The Gammaglutamyl Cycle The gammaglutamyl (GG) cycle has been postulated by Meister and Anderson~-'~ as a mechanism for amino acid transport. A hypothetical model links the GG cycle to amino acid

III i Sraoking and Pht¢'ental Function A o' ¢o " ~o ao ' ~:o ~ Nicotine (raM) 61 B ~oo! 5oo o 400 o • • 300 I00 0 -5 0 5 10 15 2o 25 1/Concentration (AIB) FIGIJ'RI~ 10. Inhibition of the uptake of ct-aminoisobu .tyric acid (a-AIB) by the human placental villus. Data ate summarized from Barnwell end Sastry.I1' (..~) Concentration*response curve for the uptake of a-aminoisobutyric acid in the presence of nicotine. "i'he ordinate represent~ the percent of control UO ratio II: inrracellular concenn'ation: O: extracellular concenmation~ for the uptake of Ct-AIB (uptake when no drug is present). No nicotine was present in the baths during preincubation. Exposure time = 2 h. Points ate means _. SE from a minimum of t0 values. The control UO ratio was 6.8 = 0.J. IB) T.vpical double reciprocal plots for the uptake of ct-AIB in the presence and absence of nicotine. Concenmation is expressed as IJJnol/l. velocity as rate of uptake in IJ.mol/g/min. The straight lines were established with linear re~,ression analysis by the method of least squares. In neither case did these lines cross on the abscissa or the ordinate. In the presence of ~ min/I. Both K~ and V,~, decreased in the presence of nicotine. (Control: V,,~ = 12 x I0"~ tool/g/rain. I~ ,, 0.26 x .10-~ tool/l: 5 rru~f, nicotine: V,,.u = 8.J, x 10-" mol/.g/min. K,, = 0.23 x 10-~ tool/l). Slopes of V,~, and K,, for ct-AIB uptake in the presence and absence of nicotine suggest that the inhibition cannot be classified as simple competitive. noncompetitive or uncomp~-.titiv¢. The results from one placenta ~re presented and -,re representative ot" eight placentas studied tsee Table I transport systems, y-Glutamyl transpeptidase tGGTP) plays a key r~le in the GG cycle. It is bound in the outside bilayer of the plasma membrane. Glutathione (glutamylcysteinylglycin~) • is continuously secreted onto the cell surt'aee, where GGTP transfers a glutamyl, group to an i incoming molecule of an amino acid. The products, glutamyl-amino acid and cysteinylglycine, are reabsorbed into the ceil. Most amino acid molecules are absorbed by independent transport systems. The number of these carrier molecules may be inversely related to the degree of

Pl¢~¢'ental Toxicology :lh,+~,.,T'.i. ;i • .i' :~;tmm;.~glul;.Ull.+, I-imfinu acid. Durra,=, ~arva~on oI" lh¢ cells I~r amino acids with low I¢veN nf extr:lccllul~lr amin+, :u'ids ~. m,,r~ mm,p~+rt cagier mtflc~ulcs arc induc+d and mco~oratcd into th¢ plasma membrane. Thu~. ~amm:~gluramyl-amino acids may s¢~'¢ as tran~mcmhran¢ or environmental signals t~r ~he uprak¢ or" amino acids by cells.~ Cells can use cbe GG cycle tbr efficient recov¢~ of cysmin¢. Human cmphoblas~ic microvilli contain high levels o~ GG~)c Maternal smokin~ du~n8 pregnancy decreases the activi=y and of GGTP in the micmvillous pl~ma mcmb~n¢~:"=~: ~T~bl¢ 4). ~is d¢cre~¢s the and abso~fion o). ~amma~luramyl-amino acids, si~nallin~ the cell m induce ~h¢ ~ynchcsis of more ~mino ~cid tran~po~ c~¢~ ~nd ~o ins~ them ~nto ~h~ plasm~ membrane. ~ere ~s an increase in ~h¢ uptake of ~-AIB. m~inly duc =o an increase in V~. in washed placenral villi from .chronic smokers (Table 4). ~e induction or" placent~[ ~-AIB tr~nspo~ ~hes durin~ m~¢emal ~mokin8 can be pa~i=lly explained by depression o[ ~e GG cycle. However, in ~be pmsenc~ of tobacco smoke component, the inc~¢d ca~¢r sysmms do nm fully compensate ~or ~be dep~ssion of the GG cycle. ~e effects of mammal smoking on componcn~ o~ ~be GG cycle ocher ~ GG~ ~¢ no~ known. 2. Placental Acetylcholine and Regulation of Amino Acid Transport The chemical transmitter function of acetylcho[ine LA.Ch) in nervous tissue is well estab- lished. Human placenta is not innervated. The evolutionary appearance of ACh preceded that of the nervous system. The ACh system is fully developed in the placenta during maturation and development of the placenta and the fetus:-~ It is synthesized in the syncytiou'ophoblast and is released into both the maternal and fetal circulations of the placenta. There are several studies which indicate a coupling link between ACh release and active uptake of amino acids from maternal blood. Atropine causes a reduction in the uptake of cc-AIB by isolated placental villous==~ or placental fragmentsJ~.~ Four different types of choline acetyltransferase (ChA) inhibitors depress the uptake of a-AIB by placental villus,n:.=:~-~-~s There is a positive corre- lation between the inhibition of ChA, ACh synthesis by ChA. and the depression of o~-AIB uph~k¢. P-xr~acellular Ca** must be present for placental release of ACh. The uptake of ce-AIB by the placental villus is depressed by 90% in Ca--free medium:-~ Inhibition of ChA decreases the synthesis of ACh and i~ release as well as a-AIB uptake in placental villi or Placentas from women with preeclampsia have higher levels of ACh than control placentas. but the outpm of ACh from preeclamptic placentas is si~mificantly reduced,tz~-*'~ The trans- port of a-AIB from the maternaJ side to the fetal side is reduced in preeclamptic peffused placenta. :'== These observations suggest tha~ feral intrauterine growth rer.~rdation in preeclamptic women can be partially explained by reduced placental ACh release and reduced amino acid transport. Clinical observations give further evidence )'or a )'unctional link between ACh and ~lacentai • ".r:.m<fer ,,f ::2:ino ;:cid.-. Hi.='h cunccnti'atioa.,, of nicotine ~gcrcase placental ACh releaseY') Maternal smoking depresses placental transfer of amino acids:~'~.0pioids decrease ACh release from placenta and depress amino acid uptake.~'-)~:-~-~ Cocaine decreases placental ACh release as well as amino acid uptake?~ Tobacco smoking and addiction to opioids or cocaine cause intrauterine growth retardation. All these observations suggest a link between ACh release and amino acid transport in placenta. There are only preliminary reports on the effects of chronic maternal smoking on the placental ACh system)~-' Maternal smoking decreases placental ChA activity (Table 4). This decrease may be due to decreased synthesis of ChA protein or inhibition of ChA. an enzyme containing -SH groups, by Cd, Ca, and Ni in tobacco smoke. Further investigations are necessary to completely evaluate the relationships among the placental AC'h system, maternal smoking, and placental amino acid transport.

Smukin:¢ and Pl~tcc, ntal t"um'ti,n TABLE 4 .Maternal Smoking and Changes in the Regulator) of Amino Acid Trunsport in Placenta" ?*lean ± .',;EM• p value t'~)r Par'amele~ Nunsmokers I.~i0 Smoker~ I,M..~ ~1~ vs. ~,1., Plasma membrane. GGTP V~ of GGTP ~nmollmg pro[c~min~ 47.39 ~ 7.~7 15.50 : 2.73 <0.o2 Pl~ memb~e. PMT PMT I ~fmol/mg p~tcin/min) 23.17 ~ I.~ 13.~) ~ 1.99 <0.02 Micmviscosi~y ~cP~ ~8 ~ 14 513 ~ 26 <O.t)2 Placental ~A ~A ,nmol ~Ch h~nncdlm~ pn~c~n/m~n 3.2 • ~-AIB upz~ ~w~shed placental viilih [~ mzio 9..SO ~ 0.4 15.30 = o.~ <0.05 K~ ~ff) 0.~ ~ 0.41 1.45 ~ 0.62 <0.05 V~, mmol/~min} 30.7 ~ ~3 • Emzh ~'aiu¢ is t~r 6-9 nonsmok~t~ or 4-.5 smokers: smukcrs u,~d 20 =,g c~garcffes/day. All va|u~s arc summarized from Sasl~" and Horst.~.~° Horsl and Sa.slryY~ Sa.slr}..,~' and Jan,,am et al.'.~ GGTP. ~ammaglutarn.~ l ~ranspepfida.,~: PMT. phospholipid N-melhyllranslcr'a.s¢: ChA. choline ac¢lyhran.~eras¢: ¢t-AIB. ¢z-aminoisobu~ync acid: I and E. in|ra- and ~xlrac~llular conccn~ratiuns of o¢-A[B: K,,..Michacli.,,-Mcmen conslan! for ~h¢ uptake of c[-AIB by placemal villi: V ..,,. maximum velocity for ~h¢ uptake of ~-A[B by isolated placental vdli. 3. Phospholipid N-Methylation in Placental Plasma Membrane and Amino Acid Transport Enzymatic methylations, in the presence of 5-adenosy[-|.-methionine [SAM) as a methyl donor, play a significant role in several cellular functions. During the past decade, three of these SAM-mediated methylations .have received special attention:'z:a'=-'~-~ (I) stepwise con- version of membrane phosphatidylethanolamine (PE) to phosphatidyl-N-methylethanolamine ~PME}. phosphatidyl-..VaV-dimethylethanolamine ~PMME~. and phosphatidylcholine (PC) by t~o phospholipid .\'-methyltransferases ~PMT [ and [I) in ~eve.-'al tissues: 12~ formation of protein carboxymethylesters (PCME) by protein carboxymethylase ~PCM) in several cell systems: and ~3) methylation of endogenous fatty acids by fatty acid carboxymethylases (FACM') in several tissues. These methylations in placental human trophoblast have been reviewed by Bamwell and Sastry?:: Enzymatic phospholipid N-methylation is known to occur in placental tissues. Further. it has been demonstrated that increasing the intracellular levels of 5-adenosyl-~- homocysteine (SAH), an inhibitor of phospholipid N-methylation, inhibits the uptake of c¢-AIB by the human placental villus.~= SAH is not a selective inhibitor of any specific enzymatic methylation: it inhibits all SAM-mediated enzymatic methylations. A question arises as to which one of the SAM-mediated enzymatic methylations plays a significant in the uptake ofcc-AIB by the placental villus. Amino acid uptake systems occur in the plasma m~mbrane. Barnwell and Sastry~-'v have analyzed the human placental villus for the above three groups of enzymes involved in SAM-mediated methylations.~'-~ According to these investigations, f I ) the relative distribution of PMT enzymes and amino acid carrier systems ~PCM and FACM) in the plasma membrane and placental villus homogenates and depression of (z-AIB uptake by inhibition of PMT enzymes indicate that plasma membrane phospholipid .V-methylation plays a more significant role than other types of methylations in the uptake of amino acids by the human trophoblast. tl

64 Placemal Toxicology Increasing the intracellular level of SAH blocks both phospholipid N-methylation and amino acid uptake by the placental villus under the same conditions. The blockade of amino acid transport is not complete. When phospholipid :V-methylation is blocked more than 60%. amino acid uptb, ke decreases by only about 40%. There might be a basal level of amino acid uptake which is not regulated by phospholipid N-methylation. High SAH levels inhibit PCM and FACM in addition to phospholipid N-methylation. The distribution of PMT L PMT II, PCM, and FACM as well as the amino acid carrier systems (e.g.. GGTP) indicate that phospholipid .V-methylation is possibly more intimately involved in the amino acid transport across the plasma membrane of the trophoblast than the other two enzymatic methylations. Phospholipid N-methyhransfemses contain active -SH ~roups. The cadmium ions in tobacco smoke inhibit PMTs. The PMT I activity~'~.~" of placental plasma membranes decreases in cigarette smokers~) (Table 4). However. it has yet to be evaluated whether the decrease in PMT activity, is due to a decrease in the enzyme protein or the inhibition of the enzyme by tobacco smoke components. 4. Oxidative Energy Sources and Placental Amino Acid Transport Oxygen consumption by placental slices from smoking mothers decreased in proportion to CoHb concentrations in maternal blood.~ The rate of oxygen consumption in placental slices from nonsmoking mothers ( 1.9 p,l/mgjh) is about 30% higher than that in placental slices from smoking mothers ( 1.3 gl/mg/h) at an 8% maternal CoHb concentration. These observations indicate that the energy-dependent processes of placental cells may be depressed and that the formation and concentrations of cellular ATP may be affected by hypoxia. All of the above mechanisms for the regulation of amino acid transpor~ are ATP dependent. Three ATP molecules are required for operation of the GG cycle and the uptake of one molecule of garnrnaglutamyl-amino acid. Another ATP molecule is required to form one molecule of acetyl.,coenzyrne, a substrat¢ for ChA to form one molecule of ACh. Still another ATP molecule is required to form one molecule of SAM. Three molecules of SAM are required to form one molecule of phosphatidylcholine by phospholipid methylation. Therefore. all pos- tulated regulatory, mechanisms of amino acid u'ansport in the placenta may be indirectly affected by maternal smoking. The effects of maternal smoking on placental ATP levels have ye~ to be determined. D. MATERNAL SMOKING AND TRANSFER OF AMINO ACIDS FROM THE TROPHOBLAST TO THE UMBILICAL CIRCULATION The placental trophoblasr cell membrane not only pumps amino acids into the cell but also effects a net transfer across the cells into fetal interstitial fluid and thus into fetal circulation. Most essential amino acids are actively taken up by the placenta from the maternal circulation and diffuse out into the fetal plasma. Both of these processes are influenced by maternal smoking. The former is affected by placental hypoxia and the influence of components of tobacco smoke on amino acid transpor~ systems and their regulation. The latter is influenced by both hypoxia and release of biogenic amines (catecholamines and 5-hydroxytryptamine [~- HT]) which cause constriction of.the umbilical vasculature. The rate of oxygen consumption by placental slices from smoking mothers decreases in proportion to CoHb levels in maternal blood,m Therefore, the energy-dependent processes of placental cells may b~ depressed by hypoxia. These processes include actively transporting amino acids, vitamins, and other substances. Smoking induces a certain amount of vascular damage of placental vessels. Changes in umbilical arteries, villous arterioles, and villous capillaries, have been observed in placentas from smoking mothers. These changes include a broadening of the basement membrane, increased collagen content, decreased vascularization, and pronounced intimal edema.*~:-m Depending on the degree of edema, the vascular diam- eters will decrease and hinder blood flow in the umbilical arterial-venous system.

~5 Placental blood flow facilitates diffusion, but .,,ubstances that cause vasoconstriction of placental blood ves.~ls interfere with transpl~centai transport. A good example of a subst~nc~ that decreases placental bl~od t]nw and ~;lU~C~ ~r:xnxplacental transpo~ ir~ularil~cx ~, 5-I IT. 5-HT decreases perfusion tlow through the umhilicnpl:,cental vasculalure in the human placenta.*.~ The I~tal blood circulatio~ or" 5-HT increases ~tbre high. which may prevent loss of blood througi, the placenta durin~ birth.~ A ~ingle injection of 5-HT in mice causes rapid death of t~tuses ~ith little eft~t on the mother.*'~ Some investi~ato~ attributed fetal death or congenital mal~ations caused by 5-HT to a decrease in placental blood flow and transt~r fimction.~',:'~ In view of the potent actions of 5-HT on uterine and placental vasculatum, the substance has been implicated in a number of physiological and pathophysiological conditions related to pm~nancy and uterine hemodynamics, includin~ pmeel~psia, a~ion, and p~ufition.*~*~* Became eff~em on placental blood flow can be deleterious to the t~tus, drugs used in human p~gnancy have to be scrutinized t~r their effects on placental blood flow. Maternal tobacco ~mokin~ can advemely affect placental blood flow. An acute decrease in inte~illous bio~ flow of appmximamly 15 to 20% h~ ~en obsemed in healthy women after they smoke a eig~tte. However. the bi~ flow mmms to nodal within 15 rain a~er t~nishing ~e cigarette.*"~ ~icotine is known to mle~ $-HT from nemo~ tissues. Nico~ne enmm the fetal cimulation from the maternal bl~d of tobacco smokem. Released HT decmmes feral blood flow ~mugh the placenta ~d contributes to t~tal hypoxia and ~mwth intonation in babies of tobacco smokem. Nicotine may also have a di~ct effect on placental bl~d vessels.~ Pe~usion of isolated hum~ placent~ h, ritro through the umbilical a~e~ h~s shown that the placental vmculature is sensitive m a v~ety of compounds with vasoactive prope~ies. ~is p~edum provides use~hl info~ation on the ~sion flow and overall resistance of the system. However. it d~s not distinguish ~e sites of vmcul~ resistance or pe~it the qu~tit~cation of drag actions. Strips of umbilical ~efies and veins have ~en examined for their sensitivity to d~gs. but these studies reflect only the sensitivity of cord vessels per se ~d not that of ~e placent~ v~culatum. Different regions of the placental vmcular tree exhibit different sensitivities to vasoaetiv~ peptides.*~ ~e small a~efies or amefioles of the placental vmculamm am the most probable s~ms of plaeen~ vmculatum resistance. ~e pa~s of the human placental vaseulamm ~ umbilici, chofionic, and villous stem a~eries ~ have ~en studied to dete~ine the sensitivities or" resis~ce of vessels to 5-~ and nicotine.*~ HelicMly cut st~ps of vessels were ~ed in these studies. Intruded vascular resistance is indicated by increased tension or contraction of the vascular strips. 5-HT is potent for causing eon~ctions of umbilical, chofionic, and villous stem a~efial s~ps (EC~: 84.47. and 80 riM. mspeetivelyL Nicotine induces contractions in umbilical, ehofionie, and villous ~tem a~efial strips ~EC~,,: 2.4. 3.3. and 2.4 raM. res~tively~. All of the above obse~'ations indicam that hypoxia and bioactive compounds released by nicotine decmme umbilical blood flow and thereby decrease the rote of diffusion of amino acids and other nutrients from the tmphoblmt cells into cetal ci~ulation. E..~IATERNAL SMOKING AND FUNCTION OF ENDOTHELIAL CELLS IN HUMAN PLACENTAL VASCULATURE ACh is released from the trophoblast not only into maternal blood*"-" but also into the fetal circulation.I('' Intravascular injection or infusion of ACh causes vasodilation in experimental animals. Therefore. it is tempting to speculate that endogenously released ACh ¢82.4 n~min/ ,, tissue at 37.5=C) causes vasodilation of the placental vasculature and decreases the resis- : "1 ~ lance to fetal blood flow. ~ placental vasculature. The effects of exogenously injected ACh on the peffused human

66 placenta have been studied by several investigators.17"-==: In such experiments endogenous ACh is released in considerable amounts into the perfusion medium~ and the placental vascu]ature is already dilated, making it difficult to further demonstrate the vasodi[atory effects of exogenously injected ACh. Therefi)re, investigators have reported that there it no eft'oct for cxo,=,cnou.,, ACh and thal weak vasodilauon or vastx:onstriction is enhanced by physostigmine and abolished by atropine. Exogenou.~ly admini.~lered ACh (0.05 to 13 caused vasodilation in 27.% of pert'used human term placentas, vasoconstriction in I 1%. and had no effect in 56%.E:" The vasoconstrictive response, normally observed with high concen- trations of ACh. may be due to the release of vasoactive substances (e.g.. catechol~mines. HTI. or it may have causes thal are as yet unexplained. In vin-¢~ preparations of the placental vasculamre may be more suitable tbr these studies than perfused whole placentas. Furchgottev-~ and Furchgo~t et al.t~ have reported that the presence of intact endothelial cell.~ is necessary for the relaxation effect of ACh on isolated blood vessels. According to their invesdsadons, (I) muscadnic receptors that am activated by ACh to release endothelium- derived relaxing factor (EDRF) am present on endothelial cells and (2) EDRF mediates the ACh-induced relaxation of vascular smooth muscle. Electron microscopy of placentas of smokers revealed swollen and irregular endothelial cells and cytoplasmic prors'usions, or blebs, on their surfaces. Transmission electron microscopy disclosed degenerative changes. including endothelial swelling and subendothelial edema. At[ of these changes indicam damaged endothelial cells and possible deficits in ACh-induced relaxation of the placental vasculature of smokers. V. MATERNAL TOBACCO SMOKING AND COVALENT DNA ADDUCTS IN THE HUMAN PLACENTA One of the largest risk factors in the induction of lung cancer in humans is exposure to cigarette smoke. While there is no specific evidence implicating any particular constituent of ~ tobacco smoke as the major factor, polynuclear aromatic compounds and tobacco-specific :~nitrosaraines are carcinogenic in several species. Human lung and placenta have the similar 'function of gaseous exchange, and placenta is a readily accessible tissue. Therefore. placentas of smokers will provide information as to whether tobacco smoke components induce DNA damage and are responsible for carcinogenesis in humans. In humans and animals, chemical carcinogenesis is a multistep process comprised of discrete events occurring over a considerable portion of the life span. The initial and/or first event (initiation) involves DNA dar-.',ge, as indicated by the ability of a large number of chemical carcinogens to form covalent addition products (adducts) with DNA. Altemately. the presence of adducts in tissue DNA samples from an individual indicates that that individual has been exposed to carcinogens. The concentrations of DNA adducts in the tissues of an individual probably represent a "'steady state" between the formation of new adducts and the loss of old ones through cell turnover and DNA repair. In investigations involving DNA adducts. DNA is isolated from tissues and hydrolyzed to form mononucleoddes. These nucleotides are then labelled with radioactive ATP and analyzed by thin-layer chromatogra- phy ([~'-P]-postlabelling assay) and/or other methods. DNA adducts have been qtudied in the placentas of smokers and nonsmokers by the [~:PI- postlabelling assay.~s-~'v Several modified nucleotides, one of which (adduct [) is strongly related to maternal smoking (Figure I l). have been detected in placentas of smokers. The chemical identification of adduct I has yet to be accomplished. There are nicotine binding sites and nicotinic receptors in placenta. [t has not yet been demonstrated whether human placenta itself converts nicotine into its corresponding nitro- samines. When tobacco and/or the nicotine-specific nitrosamine 4-(N-methyI-N-nitrosamino)- l-(3-pyridyl)-l-butanone (NNK. Figure ,¢) was administered by injection (200 mg/kg, i.p.) to ,o

-- 6 - "~ 18C 8 120 [7"~ .--'_ ,~ ~,~ . ~ 5 - ~ 150 ~ 5 e ~- ~ 120 ~ 4 80 O 0 30 1 20 NS S NS S NS S NS S ~S. nonsmok¢~ S. smoker. ~is figure is ba~ on ~m m~med by Eye.on. ¢o [7 values. Smokin~ signific~dy i~ed the intensity of adduct I I A ). ~e ~cnmge of bl~ ~xyh=mogl~in IC). ~d pl~ma thi~yan:[¢ ID). lncm~cs in B. C, ~d D idemi~v subj~ ~ smoke~. ~¢ chemi~i nature of adduct I h~ yet to ~ dete~ined. Other adduc~ not mla¢~ to smoking am also ~s=n¢ in the pi~en~ o~ smokc~. ~e bl~ samples w¢~ co[lcct=d at a~uc 32 weeks o( pm~n~cy. l~-day p~ant h~st¢~, NNK w~ detected in amniotic fluid (3~.07 ~ 7.~2 nmol/mD. placenta (0.72 • 0AS nmol/mg protein), and feral lung (0.39 suggest thac ~K crosses the placental ba~¢r in p~gnant hamster. When ¢~plan~ w~r~ cultured in the p~senc~ o~ [m¢chyl-~H]-~K. ¢~plant DNA w~ mezhylated at the 6-O- and 7-N-guanine sites. Chromosomal a~tions we~ also obse~ed in epithelial cells established from lung cxplmt out~wths. All of these obse~adons suggest ~at NNK foxed in the mammal liver crosses the placental ba~er and is convened into genotoxic inte~ediams in pregn~t h~sm~. It is not known if NNK crosses the human placental b~cr ~d me~ylates placcn~ DNA to fo~ ~ adduct in p~nant smokc~. Alternately, NNK may ~ foxed in placenta ~d subsequently placental DNA. ' VI. ALTERATIONS IN AMINO ACID AND PROTEIN &IETABOLISM IN GRO~VTH-RET.ARDED B,ABIES AS INDICATED BY BLOOD ANALYSIS ~ere a~ no studies compmng ~e blood concen~tions of amino acids in babies who are in.uterine ~ow~ ~ded due to mammal smoking ~d in babies of nonsmoking mothers. However. ~e~ m some studies on alterations in blood chemist~ of neonates who ~e growth ret~ded for unexplained ~ons. ~ese Mterations ~flect t~tal deprivation of food ~d oxygen resulting in a cata~li¢ state,t~ Chronic oxygen deprivation may be indicated by higher e~ropoietin ~d hemoglobin concentrations ~d a higher e~th~cyte count at bi~.~°'~ ~¢re is a rapid incre~e in t~e fat~ acids ~d glycerol atier birth in the growth- ~tarded neonate. ~is indicates that these neonates have a greater ~li~ce on lipid catabolism " th~ on glycoiysis,ta: Elevated levels of urea md ~monia may reflect an unusual ~li~ce on protein substrates in energy metabolism.)~z)~ ~e ~tio of nonessentiM to essential ~ino acids at birth is higher than in either no~ally ~own gestationai or weight ~e~. which may indicate t~ml protein calorie malnu~tion,ts~ Ch~ges in nonessential/essential amino acid

68 Plucentul T,xicoh~gy ratios are detectable in t'~tal urine and consequently in amniotic fluid, which may permit an early prenatal diagnosis of intrauterine growth retardationJ'~" Although'the above concept is speculative, it is very exciting)s':-t"~* Growth hormone levels are normal to high in growth- retarded babies, suggesting that this hormone deficit is not a cause of fetal growth retarda- tion,:"' Growth retardation due to unknown etiologies is ct~aracterized by (I) chronic O, dcprivation. ~2) retianc¢ on lipid calaboli.~m. (3~ excessive reliance on protein substrates for energy metabolism, and (~) protein calorie malnutrition ~Table :5 L At high altitudes, variation in arterial oxygenation during pregnancy is related to birth weight of the offspring. Thus. maternal O: transport and hypoxia may be responsible for fetal growth retardation."'~'~'-' Maternal smoking further enhances growth retardation by two- to threefold at high altitudes. ~'~ Besides fetal hypoxia, there are other causes of fetal growth retardation during maternal smoking. These include (l) decreased blood flow through uterine spiral arteries (release of catecholamines in maternal circulation): (2) lowered blood flow through umbilical circulation (vasoconstriction by S-HT and intimal edema): (3) malnutrition due to decreased uptake of essential amino acids by placenta, placental transfer, and fetal utilization (inhibition by tobacco smoke components and hypoxia): and ~4) release of opiate pepddes in the fetal brain a~Id co~l~¢qu~tlt r~,,,plt'atory depression ~t'eedback effect due to release or" neurotransmitter. ACh. catecholamines, and S-HT by nicotine) (Table 6). Various maternal, placental, and fetal factors which are influenced by maternal smoking are summarized in Figure 9. Three of the consequences of maternal smoking are depression of placental uptake, placental transfer, and fetal-utilization of essential amino acids. In these processes, l~iacental uptake of neutral amino acids is affected more than uptake of basic amino acids.~ ~' In other words, the amino acid pools are disturbed in the fetus, and this effect may contribute to fetal malnutrition and retarded growth. TABLE 5 Biochemical Status and Chemical Findings in Intrauterine Gro~h-Retarded Babies Contrasted to Those of Gestationai Peers No. Biochemical status Chronic O., deprivation and con.c~-quent changes Reliance on lipid catabolism ~ not glycolysis F_'ccessive reliance on protein subs/rates for energy metabolism Protein-calorie malnutrition Hurrnone~ 6 Body water Parameter Change ReL E~thropoietin Higher Hematoctit Higher Hemoglobin Higher RBC count Higher Free fatty acids Higher Glycerol Higher Urea Higher Ammonia Higher Amino acids: glycin~, alanine. cystine, tyrosine Nones,~ntial to es~ntial amino F~ng+r nail N Growth hormone Conisol Extmcellular water tntracellular water Plasma volume Higher Higher Normal or higher Higher Higher Higher Higher t?) Finne=,m Lugo and Cassady='* Humt:mn. ¢t Humbert et Robertsoa et ai.v~ Melichar and WolP~: Rubalmlli ¢t ai.~u Rubaltelli and Young and Prtnton='~' Mestyan ctal.t"* Lindbiad et Lindblad eta|.t~ Me,~tyan et Younoszai et al. Lockard et aL:'~.~m Humbert and Godint'~' Haymond et Haymond et aL2m Brans and Ca.ssady~''~

Smokm.'4 aml Plcwental Fmwtitm 69 TABLE 6 Maternal Smoking and Alterations in Biochemical Parameters Materna! Ptacental Parameter Change• Re£ Bh~l CoHh Higher Lt~ngo:": i E. NEI Higher Quigley et al.~'' Y-HT Amino actd~ utilized "? Uterine bl~ flow Lowe~ G~is~ and Gobble:" Dyer ~d Goughz'~ ~i~ht Hi~her Wilson ~" Inte~'illo~ blo~ flow Lowe~ Adam~ns ~d Myers:'" O: consumption Lower T~aka~. Vj~ular ~ma~e Higher A~mu.~,cn and K.ieltl~en;" A~ ~te;t~ Lo~er Olub~ewo and Birth weight Lower U.S. ~p~ment of Health ~d Human See'ices" Umbilical bl~ flow Low~ S~sl~ and Owens CoHbHigher Youn~i ~d Hawonh~': He~t~m Higher Younoszai ~d Hawonh~ Br~ing m~ ements Lower Manning et al. He~ rote Hi~her M~ning et ai. ~-~t ? HeWet ReI~ of opiate '? Tayeb and ~tW ~tid~ in CNS~ Tayeb and S~tW CoHb = earboxyhemoglobin: E = epinephrine: NE = norepinephrine: 5-HT = 5-hydroxyt~.ptamine: ACh ~ acetylcholine: CNS = central ne~,ous system. Contrasted to nonsmoking mother. Due to constriction of spiral art. cries by E and NE. Effects of nicotine, cyanide, and nitrogen oxides. Due to constriction of umbilical arteries by 5-HT. -" Althouo~h there ~ been no direct demonstration, nicotine increases release of 5-H~ from ~me CNS sites. Nicotine releases ACh from central sites: accumulation of excess ACh at synaptic sites may trigger release of .-nke~halin> ,.,,'hich may Iov.-er breathing movements. There is some evidence that smoking disturbs the nitrogen balance in man. Cigarette smoking reduces t ,ryptophan utilization in humans. This reduction is associated with relative impairment of protein utilization which can be reversed in part by an increased protein-calor.ie intake,tg-~ Cessation of cigarette smoking is frequently associated with significant weight gain. while resumption of smoking is associated with .weight loss. According to the studies of Barnweil and Sastry.tt9 nicotine interferes with different transport systems in human placentas to varying degrees. Its inhibition of the uptake of typical neutral amino acids by the placental villous is higher than that of the uptake of basic and acidic amino acids. Even at the level of placental transfer, amino acid pools are disturbed. This suggests that e~sential amino acids are not entering the fetal circulation in the proper proportions to be utilized for protein synthesis and ti.~sue gro~ th. The pla.,,ma concentrations of several essential and nonessential amino acids in nonpregnant and pregnant women smokers increase when compared to nonsmoking and nonpregnant women. The concentrations of several amino acids in umbilical plasma of smokers are higher than the corresponding concentrations in the umbilical plasma of non- smokers. Different amino acids are affected to varying degrees. These observations indicate that not only smoking women but also their fetuses h~ tltt, t7,~ experience negative nitrogen balance.

7O Placental VII. SCOPE OF FUTURE INVESTIGATIONS There are many gaps in our knowledge of the toxicities of tobacc~ ,~moke components on the placenta and their molecular mechanisms. Placental toxicity ~f drug,~ and envir~mn~eni:d chemical.,, has received less attention than the corresponding toxicities to the fetus and the mother. This is justifiable in that the placenta is discarded at the termination of pregnancy. However. proper function of the fully formed placenta during the fetal period (2 to 9 months) is critical to the fetus for nutrient transport and as a protective barrier against environmental insults. Human placenta is an easily accessible tissue, and molecular mechanisms of cellular toxicity of environmental chemicals in placenta may provide clues about their toxicity in other tissues. Areas in which progress can be made include (1) genetic factors in fetal growth retardation induced by tobacco smoking. (2) genetic factors causing placental toxicity of tobacco smoke components, (3) placental metabolism of nicotine and other tobacco alkaloids in placenta, and (4) influence of other environmental chemicals and other abused drugs on the placental toxicity of tobacco smoke components. A. PLACENTAL COMPENSATORY AND GENETIC FACTORS One well-documented effect of maternal smoking during pregnancy is fetal growth retar- dation. When percentage distributions by birth weight of babies of nonsmoking mothers were plotted, a normal distribution curve was obtained with a mean birth weight of about 7.75 lb.'~.~*' A similar distribution curve for babies of mothers who smoked one pack of cigarettes daily during pregnancy indicated that birth weights were normally distributed with a mean birth weight of 6.75. There is considerable overlap between these two birth weight curves, indicat- ing that some babies of smokers escape itt utero growth retardation. While this overlap can be partially explained by good nutrition during pregnancy, it raises the question of genetic differences in the population ~ certain individuals are more susceptible to the ill effects of smoking whereas others are more capable of developing physiological compensation or adaptation to the ill effects of smoking. Some smoking expectant mothers who attend smoking cessation clinics ask the question why the birth weights of babies of smokers are normal. An answer based on genetic differences may persuade the smoker not to take a risk and to quit smoking during pregnancy. Smoking increases the concentrations of several primary, amino acids in the plasma of nonpregnant women, pregnant women, and umbilical arterial blood,t'-~-~:-:t~-'~-" This means that the utilization of amino acids by fetal as well as adult tissues decreases during smoking. The impact of nonudlization of amino acids may be greater for the fetus, which is developing and growing, than it is for adults. Increased plasma concentrations of amino acids may not be harmful to the majority of people. However, individuals who are genetically predisposed to certain amino acid disorders must be identified. This becomes more important for pregnant women. For example, women who are predisposed to metabolic disorders of phenylalanine resulting in phenylketonuda should be identified during prenatal care. Otherwise. damage may be done to the fetus due to increases in the steady-state plasma concentrations of phenylalanine. This is important be- cause more and more information is becoming available to indicate that there is a genetic component in certain tobacco-related diseases (e.g.. lung and colon cancer). This means that certain population groups may be more susceptible to tobacco-related diseases than others. .According to discussions in previous sections, smoking depresses amino acid uptake by the trophoblast and transfer of amino acids to the: placental circulation, and it releases vasoactive biogenic amines that decrease placental blood flow. Compensation for this insult takes three forms: I l ~ an increase in the size of the placenta'to sustain a smaller fetus. (2) an increase in .i

71 the number of amino acid carriers in the plasma membrane of the ¢rophohlast to increase amino acid uptake, and (.3) a decrease in the membrane fluidity of placental blood vessels ~o that they are less rcsponhiv¢ to vasoconstriction by biogtnic amines.'~''-, ~: All of these changc~ improve the placental transport of amino acid~, and ~l~i~ cfl~ct is unma~kcd in the absence of tobacco smoke compounds (e.g.. washed placenta of a smoker). Studies on the influence of nicotine, nicotine mtmbolites, and other tobacco components on different genetic strains of pregnant experimental animals and their placentas are needed to explain why certain babies ¢~ca~ in u~ero grog'th retardation and why some babies are competent ¢o induct physiologi- cal compensation for inadequate amino acid wanspon in their placemas. B. GENETIC FACTORS IN THE PLACENTAL TOXICI'~Y OF TOBACCO ALKALOIDS Genetic factors influence the responses of inbred mouse strains to acute doses of nicotine and their tolerance to the effects of nicotine after chronic treatment. These differences in responses to nicotine among mouse strains cannot be explained by differences in metabolism ,~t" aicotin,: or differences in nicotinic receptor numbers.::,~ Other unknown genetic differences in various mouse strains have yet to be investigated. Investigations on the influence of nicotine and other tobacco alkaloids on placental function of pregnant rodent strains are needed. According to studies on male twins, there are moderate genetic influences on lifetime smoking effects,zt'~ Three components of smoking behavior tnever having smoked, currently smoking, and quitting) are moderately influenced by genetic factors. Knowledge of the aspects of behavior in which genetic influence is greatest may help us to identify children at risk of smoking by virvae of their parents" smoking behavior. However. there are no reported studies on biochemical indices such as nicotine metabolism or characteristics of nicotinic receptors. which are traits closer to the gene products responsible for nicotine addiction in humans. There are both muscarinic and nicotinic receptors in placenta. Investigations of these receptors and their gene products in placentas from smokers and nonsmokers may provide clues for the treatment of nicotine addiction in women and their children. C. PLACENTAL METABOLISM OF TOBACCO ALKALOIDS Drug metabolism studies in placenta deserve more attention. Drugs and chemicals may undergo alterations not only in the maternal liver and tissues but also in the placenta before they reach the fetus. The t~tus may be more sensitive to certain drug metabolites than is the mother. Many drugs may induce not o~[y Jr.:,..'-metabolizing enzymes in the placenta but also enzyme systems involved in the function of the placenta. Drug metabolites deleterious to ptacental function are hazardous to the fetus. DNA adducts occur in placentas of smokers. A question arises as to whether nicotine is convened into NNK ¢ Figure 4~ in the placenta itself. NNK is capable of methylating DNA and inducing carcinogenesis. Nicotine is a pharmacological agent and is useful as a medication for certain clinical states. Anabasine and anatabine are two other alkaloids which occur in tobacco in addition to nicotine ~Figure 3L They are not metabolically derived from nicotine and are useful as biochemical markers during nicotine replacement therapy.:t-~ The metabolism of these two alkaloids by the liver and placenta has yet to be investigated. D. INFLUENCE OF ENVIRONMENTAL FACTORS AND OTHER ABUSED DRUGS ON PLACENTAL TOXICITY OF TOBACCO SMOKE COMPONENTS Many opiate, cocaine, and alcohol addicts smoke tobacco preparations. For example. 80 to 90% of alcoholics smoke.:~" ~kccording to Sastr2d and Ov,'ens?~ alcohol influences the

72 Placental Toxicoh~gy responsiveness of the placental vasculature to the effects of 5-HT and nicotine. In their investigations, helically cut strips of( I ) umbilical. (2) chorionic, and (3) villnus stem arrerie~ were examined tbr their sensitivity to ~-HT. nicotine, and ethyl alcohol (ETOH). In these preparations, increased vascular resistance is indicated by increased tension or contraction of the vascular strips. These experiments gave the following results: 1. 5-HT was very. potent for causing contractions of umbilical, chorionic, and villous stem arterial strips (EC,,: 84. "47. and 80 nM. respectively. 2. Nicotine induced contractions in umbilical, chorionic, and .villous stem arterial strips tEC:,: 2.4. 3.3. and 2.4 rml4. respectively). 3. ETOH (0.5 to 2.0%, v/v) had differential effects on umbilical and chorionie arterial segments. Its effect on umbilical arterial strips was minimal contractions or flacidity. 5- HT was less effective in umbilical arterial strips pretreated with ETOH. The contraction height induced by 5-HT was reduced by about 50% when the umbilical ,strips were pretreated with ETOH. However, El'OH potentiated the contm, ctile responses of umbili- cal strips to nicotine. 4. El'OH. by itself, caused contractions of chorionic and villous-stem arterial strips and potentiated the effects of 5-HT and nicotine by about 115 to 160% depending on the concentration of El'OH and the nature of the strip. It also enhanced the effects of KCI on these strips. These observations suggest that Ca-" movements required for nicotine-induced contractions may be facilitated in all vessels, whereas Ca- movements from its sources for 5-HT-induced contractions are partially hindered in umbilical arteries. The significance of these observations has yet to be investigated, Susceptibility to alcoholism and alcohol abuse exist for genetic reasons.-~lr-'ts Although 80 to 90% of alcoholics am also heavy smokers, there has been no progress in investigating the genetic basis of the joint use of alcohol and tobacco. Although there have been some studies on the deleterious effects of certain tobacco smoke components on embryonic development in the early stages of pregnancy, before the placenta is fully tbrmed,s.9.'~z there have been only a few studies on the molecular mechanisms of these deleterious effects. The question arises whether certain tobacco alkaloids interfere with the proper dew:opment of the placenta itself. The hydatidiform mole contains a negligible amount of choline acet'yltransferase (C'hA). the en .z3mae that catalyzes the synthesis of acetyl- choline (ACh). ACh seems to play a role in maturation of the human placenta and serves as a growth t'actor. Drugs that inhibit ChA may interfere with placental development.t:~ Placenta is also a source of several -_rowTh fac~or~.:~* However. there is no infomaation about ~i~ether nicotine and other alkaloids interfere with the function of growth factors in the placenta and/ or fetus. Thus, many gaps in our knowledge of the placental toxicology of tobacco alkaloids. environmental chemicals, and drugs have yet to be filled. ACKNOWLEDGMENTS Parts of the work of the authors cited in this review were supported by The Council for Tobacco Research, U.S.A., Inc.: The Study Center for Anesthesia Toxicology. Vanderbilt University. Nashville, TN: The United States Department of Health and Human Services; The National Institutes of Health: and The National Institute on Drug Abuse.

Star,kin,., attd Placental Functi.tt 73 REFERENCES I. Ballantyne..].W.. Manual of ant~:natal path~h~,=.'y and hy,._'lcnc, i1~ The Fct,~. William ~re~,n md ~inburgh. 1~02. 272. 2. S,mta~. L.W. and Wallace. R.F.. The effect ofci~aretl¢ %rooking during p~gnancy u~m the fetal h~a~ rote. Am. J, Ohstet, G~w,',d.. 29. 77. 1935. 3. Campbell. A,M.. ~e effect of excessive ciga~t/e smoking on maIamai h~ailh. Am. J, Ohster. Gyncc.I.. 31. 5{}2. 1936. 4. Campbell. A.M.. Exc~,i~ ¢i~a~t~e ~mokm~ in w~men and ~s ~ft~t u~n ~heir repr~lucti~'e ~,ffici~n~'~..1. Mi¢'h. Mc'd. S~c.. 34. 146. 1935. 5. ~enherg. J.M. Schwind..I.V.. and Pallas. A.R.. ~e effect of nicn~ine and cigarette ~moke ~n p~gnan~ femal~ albino m~ ~d their offsprings. J. ~b. Ctin. Mcd.. 25. 708. 6. Simp~n. WJ. A p~limina~ report on cig~Ite smokin~ and ~h~ mcid~n¢~ ~' prcmaturi~., bn. ,I Oh~t,'t. G~ecoL. 73. 808. 19~7. 7. U.S. ~blic Health Se~ice. ~e Health Con~quences of Smoking. A Rein of the Surgeon Gene~l. DHEW ~blicafion No. ~HSM~ 71-75 i3. U.5. D~pa~ment of Health. Education. ~d W~lt~re. W~hing~on. 1971. 387. 2. ['.S. Public Heahh S~n'ice. Pregnancy and infan~ heahh, in Smoking and Heahh. DHEW Publication iPHS) 7%5~. Chapter 8. U.S. Dep~mem of Health. Education. ~d W¢lt~e. W~hing~on. D.C.. 1979, 8-3. 9. U.S. ~p~menl of Health ~d Human S~'ic~. Toxicity of nicotine, in ~e Health Con~quences of Smoking: Nicotine A~dicfion. Ap~ndix B. U.S. ~p~m~n~ of H~alth ~d Hum~ S~,ices. W~shington. D.C.. 1988. ~89. Macmahon, B. Alpen. M. and Saiber, E~. [nt~l weight ~d ~n~al smoking, habits. Am. J. Epidemi¢~l.. 82. 247. 19~. Butl~. N.R. and Albe~n, E.D. Perinatal Prt~hlems. The 5et'ond Re~ t~" the 195,~ British Pt, rinutal M¢~alio" Su~'ey. E. & S. Livingstone. Lt&. London. Iq69. 36. Meyer, M.B. How d~ mmemal smoking aff~t binh weight ~d mate~ weight gain? Evidence f~m the On~o Pefinatal Moffality Study. Am. J. Obstet. Gynet'ol.. 131. 888. 1978. Ye~s~imy, J. Mother's cig~tte smoking and su~'ival of inlet. Am. J. Ohstet. G3~tctwl.. 88. 505. Wi~n, ~W. ~e eff~t of smokin[ in p~cy on the placent~ c~fficiem. N, Z Med. J.. 74. 384. 197 I. Winger~ J. Ch~stianson, R. Lovitt. W.V. and Sch~n. ~. Pl~ental ratio in white ~d bl~k women: ~tion to smoking ~d anemi~ Am. J. Ohstet. G)met~f.. 124. 671.1~76. Osdene. T~. Reaction m~h~isms in the burning ci$~tte, in ~e R~ent Chennst~ ~tf ~atural I~luding Tob~co. ~n~ NJ.. ~. ~din~ of the ~ond Philip Mo~s Soiree Sym~sium. Richmond. Virgini~ ~to~r 30. 1975. Philip Mo~s. Inc.. 1~6. 42. K~th, C.H. and T~h, P.G. Md~m~t of the total smoke i~uing ~m a burning ci$~ffe. T.~t-ct~ Sci.. 9. 61. 1965. Gori. G.B. Approache~ to the reduclion of total p~iculate matter ~MI in ciga~t~e smoke, in Preceeding~ of the ~i~ World Cont~nce on Smoking a~'" H~lth. New Yo~. June 2-5. 1~75. Vol. I. Modi~'ing the Risk tbr the Smoker. Wynder. ~.L.. Hoffman. D.. and Gofi. G.B.. ~.. DH~W Publication No. tNIH) 7~ 1221. U.S. ~p~ent of Health. ~ducation. ~d Welf~e. Public Health Se~i~. National Institutes of Health. Nation~ C~cer Institute. 1976. 451. Hoffmann, D. and Wynder. ~L. ldentifiziemn$ und R~ucktion von Respimtions~inogenen t Identifi- ¢a~on ~d ~duetion of c~inogens in the ~spi~to~" envi~nment). Zent~lbl. Bakte~ol.. Parasirenkd.. I&~ktionskr. Hyg.. Abt. I Orig. Reihe B. 166. 113. 1978. Hoff~nn. D. Schmelt~ I. H~ht, S.S.. and Wynder, ~ ~emic~ studies on mb~co smoke. XXXIX. On the identification of ~inogens. tumor p~mot~, and c~inogens in tobacco smoke, in ~eedings of the ~ird World Confe~nce on Smoking ~d Health. New Yo~. June 2-5. 1975. Vol. 1. M~itying the Risk tbr the Smoker. Wynder. E.L.. Hoffm~n. D.. and Gofi. G.B.. E~.. DHEW ~blication No. INIH~ 7~ I~1. U.S. Dep~menI of Health. Education. ~d Welt~. Public Health Se~ice. National Institutes of Health. National Cancer Institute. 1976. l~. Nnrman. V. An ove~'iew of the vapor ph~¢. semivolatile ~d non~olattle ¢om~nents of cig~ette ,moke. Rec. Adv. T.lau'tv~ 5ci.. 3. 28. I977. B~nnemann. K.D. and Hoffmann. D.. ~emical ~tudi~ on tob~co ~moke. LIX. Analysis of volatile nit~samines in tobacco smoke ~d ~llut~ ind~r ~vimnmen~. in ~vimnmental Asp~ts of N-Nitroso Compounds. Walker. E.A.. C~tegn~. M.. Gficiute. L.. and Lyle. R.~.. ~.. International Agency Resea~h on Cancer. Lyon. 1978. 343. 10. 11. 12. 13. 14. 15. 16. 17. Ig. 19. 20. 21.

74

75 52. Peterson. L.A. Tre','t~r. A. and Casta=noli. N, Jr. Stete~v~h~:n'nca| ,tudi¢,. t~n ih~ c}'t~hr~n~ 53. Hihherd. A.R, and (;or~d. ,I.W., Enzymolo~' of lh~ metallic path~aF Ir~m nl~()lln¢ to ~ouns~l~ ~ Eur. J. Dru~ Mcrab. P/u~rma¢',km.. S. 15 I. 1983. 54. McKennis. H. Jr, Turnl)ull. L.B, and Bowmn. ~R- N-Mcthyla¢~on of nicufin< and ¢o¢inioe m t'i~',. Bi¢~L Chem.. 238. 719. 1963. 55. Bowman. E.R. and McKennis. N, Jr.. S=udi~ on t~ mcta~)li>m t)f(-~<o~=nin¢ in the humun.J. E.~p, Tlu'r.. 135.3(~6. 1~62. ~6. Shuigin. A.T, Jacob. P. Ill. Bcnot~it~ N.~ and Lau. D, ~¢ idcmifica[ion and quant=¢a¢=vc analy,i~ cotinine-N~xid¢ in human urine. J. Chrnmar,~r. Bi, m~ed..4pplic.. 423.3h5, I9~7. 57. Neu~th. G.B.. Diincer. M.. O~h. D., and Pein, F.G. ;~n.~-3"-H~,l~¢)x}~,¢=mn¢ .~ a m;m~ m¢¢,=b~iitc 58. Jacob. P, Ill. Shuigin. A.T, and Benowit¢ N.~ Syn~is of (3"R~'S~-rrans-3"-hyd~xycotinin¢. a major meta~iite of nicotine. Metallic fo~ation oe'3.hydmxycotinine in humans is highly ~ter~sel~tive..l. Mcd. Chem.. 33. 1888. 1990. 59. McKenn~ H. Jr, Schwa~ S.~ and Bow~n. ~R, Alternate mutes in Ihe metallic degradation of the pym~lidin~ rm~ ,~[ nicotine. J 8i,,i. Chron.. 239. 3~). I~. ~. A~itage. A.K, Doilew. C.T.. G~ C.F, Ho~man. T,H. Lewi~ P,I. and Tu~er, D.M. Ab~o~tion ~d u~¢~bott~m ot m~ne Irom ci$~x~. Br. Med. J.. 4. 313. 1975. 61. Feye~ben¢ C. ln~ ~M, and R~eil, M~. Ni~t~e ph~oki~tics ~d its application to intake f~m smoking. Br. J. Clin. Pl~a~at'~t., 19. 239. 198~. 62. ~ngon~ JJ. Gjikm H.B.. and Van Vu~k~ H. Nicotine ~d its m~a~lites. Radioimmuno~ssays for nicotine ~d co~inine. 8im'hemi.t~'. 12. 50~. 1973. 63. Zeiden~, P. Jaffe, J.H. ~nzler. M, Le~'i~ M~, ~ngone. JJ, and Van Vunakb. H.. Nicotine: co~inine levels in bl~ du~ng cession of smokin~ C~pr. P.~'hiatD'. 18. ~3, 1977. ~. Haley, NJ, Axei~d. C.M. and ~itom ~A_ V~i~ion of ~l~n~ smoking ~havio~ hi,heroical ~lym of cotinin¢ ~nd thi~y~me. ~. J. Public H~lth. 73. 12~. 1983. 65. Benowi~ N.~ Hall. S.M. Hernia. R.I, J~b, P, IIl. Jon~ R.T.. and Osman. A.-L, Smokers of yield ci$=e~ do not ~mume I~s nicmi~..V. Engl. J. Mt'd.. 3~. 139. 1983. ~. Van Vu~k~ H, ~ngone, JJ, and Mil~ky, ~ Nicotine ~d cotinine in the amniotic fluid of smoke~ in ~¢ ~ond ~m~ter of p~gn~, Am. J. Obxtet. Gynecol.. 120, ~. 197~. 67. Hibbe~. A.R. O'Connor. V, and Go~. J.W, ~t~tion of nicotine, nicotine- I'-N-oxide and cotinine in maternal and f~ ~y fluid, in 8ioh~t-al O.viduucm t~f.~'itro~en. Go~. J.W., Ed.. Elsevier:No~h- Holl~d. Amster~m. [9~7. 353. 68. Luc~ W, Hahn R. Steldinger. ~ ~d Nam H, Ni~tine ~d colinine ~ two ph~cologically active subst~c~ ~ p~e~ for th~ s~n on fet~s ~d ~i~ of mothe~ who smoke. J. Perinat. Med.. I O. 107. 1982. ag. R~nick. R. Brin~ G.W. and Wiik~ M. Ca~holamme-m~ia~ed ~eduution m uterine bl~ t]t~w atter nicotine infusion in the p~nant e~'¢. J. Clin. I,n'e~r.. 63. 113. 70. Ay~mi~i. J. D~idedo. D. and Tobi~ M, Eff~t of nicotine sult~te in the hemodynamics and acid b~e b~c¢ of chronically instmmem~ p~gn~t sh~, De~ PharmacaL Tht, r.. 3. 205. 1981. 71. Lehtovi~a. P_ Fo~s. M. ~u~mo. I, and ~dniemi. V., Acute cff~ts of nicotine on fetal he~ 72. Genn~er. G.. Mural. K. and B~n~mark. B.. ~latemal ,mokin~ am[ fe~a[ breathing .~¢ment~. Ohste[. @~ec~d.. l~. 801. l~75. 73. Manning. F.A. and Feyerabend. C. Cigml~e smoking ~d fetal b~athing movements. Br. J. Obstet. Gynaecol.. 83. 262. 1976. 74. S~try. B.V.R. and Tayeb. O~. Regulation of ~e~icholine ~le~e ia the mouse ce~b~m b~, enkephalin and substance P. Ad~'. Biosci.. 38. 165. 1982. 75. Ta~eb. O~. and S~try. B.V.R_ Rele~ of ~u~nue P. acetylchoiine ~d methionine enkephalin f~m mouse ce~b~l slices, in Suhstauce P and .Veurokhm~s. HeaD'. J.L.. Couture. R.. Cuetlo. A.C.. Pelletier. G.. Quimn. R.. and R~goli. D.. ~.. Spfinger-Vertag. Ne~ York. 19S7. 350. 76. S~try. B.V.R.. Chance. M.B. G~dijn-W~i. T.A.W. and Johnson. R.F.. ~e human placental ~ransfer of nict,tine. Ph~trmeu't~lt~,i~'t. 35. I¢8. TT. Schneider, H.. Panigel. M.. and Dancis. J. T~sf~ acmes the ~ffu~dd human placenta of antipyfine. ,~ium and leucine. Am. J. Ol~n.t, G:.m.~,,I.. 114. 822. 7~. B~nd~..L31. Tavolini, N.. Potter, BJ, Sarkozi. L, Shepard. M.D, and Berk. P.D, A n~ t~hnique for human placental cotyl~on ~ffu~ion: appli~tion to ~udies ~f the fetomatcmal trundler gluco~, inulin and antipynn~. Am. ]. Oh.~tet. Gym'ct~l.. 146. 8~.

II Imlllll ~11 liB

7'7 lOS. Himme|her--er, l).t~.. P,r¢=wn, |~.W...|r.. and C=~hen. E..N.. Ciga~tlc ~m,~kin~ during p~nuncy ami ~ccn~cncc ~t' ,p~mlanc~=~ ;~ni=m and ~nn~en=¢at ahm~m~ahty, bu .I ~p~h'm~,,I.. 1(1~. 47f). ~. [. 1 I0. Eelsey. J.L, Dwyer. T., Noiford, T.R. a~d Bracken, $I.B., ~at=mal ~mokin~ ;rod tone,total lions..An epidemiological ~¢udy. ~. EpidemhH. C~nm~. Hee/th. 33, IO~. I I I. ~aeTe. R.L, Efl~c=~ o( malcmal cigarette ~moking on lhe t~lus and placenta. Br. ~. Oh.~ct. Gt,,~cu,,I.. ~. 732. I 12. Philipp, ~, Palei~kE, ~.. and Endler. ~1., Effects of ~mokin~ on utemplac=mal blm~ Row. Gwzcc~l. Oh~tct. hfr¢~t.. 17. l~¢~. pla~=m=l t~n~lion. ReproS. Fertil. De~'.. 3. 355. 197~. 114. ~'e~balmy..].. ~¢ relationship of p~nts" cigarette ~mokin~ to outcome o~ pm~n~¢y -- implicaliem~ to the problem of in[¢~n$ cau~don ~mm ob~wed ~s~ialion. Am. ~. Epidemio/.. 93. ~3. 1971. 115. ~leEet, ~.B, Joan, B~, and To=i~ J.A, Pedna¢~ even~ ~ial~ with maternal smokin~ durin~ i lb. Harbison, R.D, Olubadew~ .J, DwivedL C. and S~lr}. B.V.R, Prul~)~d ~)I¢ ol~ pla~¢ztlal syslem in m~ulalion of fetal ~mw~, and ~velopment. in Bus&" end Th¢r~tpel~tic A~pecrs ~ Permutul Phe~c~h~,y. ~o~¢Iti. P.L.. Gamltini. S.. ~d Se~ni. F.. ~s.. Raven P~s. New Yo~. 1~75. ]07. I 17. RowelL P.P. and S=~. B.V.~ ~e influence ofcholine~ic bl~¢ ~ the uptake ol'alp~-aminoisobuty~c ~id by i~latcd human placental villi. Tn.~icol. Appl. Ph~ucnl.. ~5.7q. I Ig. Mo~ F.H. Pl~ental factom condilioning feral nu~d=ion and growth. Am. ]. Cliu, Nu~r.. 3~. 7~. 19~ I. I lq. Ba~well. S.~ and S~I~. B.V.R. Depr~sion of amino acid upl~e in hu~n pl~enlal villu~ by mo~hine ~d nicolin¢. Trnplmhl~st Rcs.. I. I01, 1983. 120. Sast~. B.V.R. Ba~well. S.~ and M~re. R.D. Facto~ aff~lin~ lh¢ uptake of alpha-amino acid~ by human placemal villus: ~elylcholin¢. phospholipid m¢~yladon, Ca--and cymskelelal org~=~¢ion. hlas~ R~s.. I. 81. [983. 12l. S~try. B.V.R_ Amino acid up¢~e by human placenta: ahemtions by nicotine ~d m~ac¢o ~mok¢ nenls .and their implications on t~al ~wth. in PlLx~iolo~ic~l ~d Phu~coh~ical ~mtrt~l ~' .Vcrw~u,t System Det'el~pmenr. Cac~li. F.. Giac~ini. ~. ~ Paoleai. ~., ~.. Elsevier, Amste~m. 19~. 137. 122. S~tO'. B.V.R. Jan~n. V.~ B~hm. F.H. Ahm~ ~l. Kno~ J. and ~hinfeld, J~, ~latemal ~moking dep~s ~ino acid uptake by hum~ placem~ Fed. Proc., 46. 3750, 1987. 123. S~lry. B.V.~ Ja~n, Y.~ Ahm~. M. Kno~ J- and S~Infel~ J~, Ma~e~i ci~tt¢ smoking depresses placental amino acid ~s~ which ~y lower the bi~h weight of inf~. Annu..V.y. Acud. Sci.. 562. 367. 1989. 12g. Melter, A. and Ande~n. ~1.~ Glumlhione. ,4nm~. Rev. Big'hem.. 52. 71 I. 1983. I~. ~yn~ G.M. and ~n~ 3.W. G~-glu~yl~fe~ is ~ involv~ in ~¢ ~lk upt~¢ of amino acids. ~fi~s or g~a~lu~yl-~ino a¢i~ in y~t (Saccharnm~es cer~'is~e). Big'hem. ~.. 218. l g7. t984. 1~6. Rowell. P.P. and S~I~, B,V.~ Hum~ placen~ choline~ic s~stcm: d~sion of the uptake of alpha- aminoi~butydc acid in isolat~ hum~ pl~en=~ villi by choline ~e~l~s~e~¢ inhibitor. ~. Pharm=~c¢~l. ~rp. Ther.. 216. 232. 1981. 127. Ba~well. S.~ and S~t~. B.V.R, S-Adcnozyl~-me=hion~e mediat~ ¢nzy~ti¢ me~yladons in the pl~ma m~mbme of lh¢ hum~ tmphobl~t. Troplmhh~.¢t Res,. 2. o5. Iqg7. 128. Long~ L.D, Yuen. P, and G~se~ Dd, A~¢~bi¢. giyco~¢n~e~ndcnt tms~ of amino acids by placcn~ ~n~r¢ l~mdonl. 243. ~31. 1973. 129. ~liller. R.~ and Bernd~ W.O, ~¢t¢fi~lion of neu~ amino acid ~cumuta¢ion by human placemai slic~. Am. J. PlLvsinl., 2~7. 1~36. 1974. [30. S~I~, B.V.R. and Ho~ ~I.A. Hum~ pl~ental gammaglumm~lt~s~pti~ ~d i~ alterations loba¢¢o smoking, in Placem~l and Endnmerrial Peoteins. Toma~. Y.. Mizumni. S., Nimta, O.. ~d Klopper. .~.. VSP Pm~. U~cht. 1988, 357, 131. Ho~t. ~I.A. and S~I~'. B.~'.R. ~{atemal tobacco smoking ~d aitemlions in amino acid t~s~ in human placema: induction of lmns~ systems. Prn~. Cli~. Bi,~l. Res.. ~. 24~. 132. S~try. B.V.R, Ho~t. ~1,~, and Nau~m. R~, ~la=¢mai ~o~a¢¢o smoking ~d ¢h~gcs in amino acid uptake by hum~ placental villi: induction of uplak¢ syscems, gammaglutamyh~ms~plid~¢ =~d fluidicy. Place~ra. 10. 345. 133. Janson. V.E. Sastry. B.V.R.. and $1outon. S, ~tat¢~l smoking and m~k¢~ or" pl~¢ntal cholin¢rgi¢ system. F.A~B ].. 7. A699. 1993. 13~. S~try. B.V.R.. Pla¢¢mal ac=tyl¢holin¢, m .~l,l¢,¢'tdar Aspects t~'Placemal and Fetal .ilemhr~tc Rice. G.~ and Br¢nn¢ck¢. S.P.. Eds.. CRC Pr~s. B~a ~a¢on. ~. 1993. 157.

78 Phtcental ToA'i¢'olo,~S 135. ~,s~ elsch. F.. Wenger, W,C. and Stedman. D.B.. Acct.', Icht~hne m human tem~ placenta: tissue levels in intact :~a~ment~ after ~nhib~titm in t'ltt'o uf cJlt~line acet~ II~lll~l~r:lse ;aid rel;ilit~l~h~p Io i' 'C)-anlintu~t)butyric acld uptake. Plaeenut. Suppl. 3. 338. 1981. 13~. Levent~. S.M.. Rowell, P.P., and Clark ~ld. ~e eff~t of choli~ acetyttranst~m~ inhibitiun on ace~ylcholine synthesis and ~le~se in ~e~ human placenta. J. Phar~mt~'ol. Ktp, ~her,. 222. 301. 1982. 137. Chen, Y~. Brenn~ke. S.P.. R~mk, TJ. and King. R.G. El'l~ct uf ~2-~nzoylethyl~tfime~hylammonium and ve~micol on acetylcholine ~d prontaglandin ~le~e l~m hu~n placental expla~ts. ReproS. Ferril. Dev.. 3. ~59. 1991. 138. Marry, N.V.A_ Bhamagar. O.P. and Ganguly. A.K.. Placental ~cetylcholine and tox~mi~ of p~gnanc>. Italian .L ~h'd. Rex.. 65. 523. Iq77. 13t~. Mu~. %.~.A. Melville. (;.N.. Wynter. H.H. Wruy. S.R. Rain. N.V~. and Hahn, H.N.V.. human pl~ental ~fftt,ton ~tudie~. ~oliner~ic acti~ i~y in nt:~nal -ubjects and to~aemia of pregnancy. bMia Med. J.. 34. ~7, 1985. 1~. K~hn~ B.~ King, R.G, Waite~ W.A.W, and Gude. N.M. Acetylcholine output ~d amino acid t~fer in nodal ~d p~lamptic human pl~enta. Clin. E.rp. Pharma¢ol. PhysioL. I~ (Suppl. I I~. 1~1. 1q87. I~ I. Olubad¢~'o, J.O. and S~t~', B.V.R. Effects of nicotine on the ~lea~ t~f human placental acet~lcholine and i~ signific~ce on fetal g~wth. Toxi¢oL AppL Pha:w~a~.~,l.. 37. 126. 1976. 1~2. Ahmed. M~. and Ho~K M.A, Opiate ~cepto~ of hum~ pl~enmt villi m~ulate acetylcholine mle~. Lt[t" S¢i.. 39. 535. 1986. 143. Ahmed. M~ Sch~f. T, Zhou, D, and Quarl~ C. ~ppa opioid ~cept~ of hu~ pl~ental villi. l~. Hi~ F. and Ax¢i~. J~ Phospholipid me~ylation and biological signal tr~smi~ion. Scient~. 2~. 1980. 145. Crew~ F. Phospholipid me~ylation ~d membr~e function, in Phospholipids and Cellular Regulation. Kuo. J.F.. ~d.. CRC ~. B~a Raton. ~ 1985. 131. I~. Ba~w¢lk S.~ and S~try. B.V.R.. ~e role of enzymatic phospholipid methylafion on ~ino ~id uptake 147. We~h. F. Wenger. W.C, and Stedman. D.B. ~oline meta~ii~m in plac~m: evide~¢ tbr the bionyn- • ~is of pho~phatidyicholin¢ in micm~mes via the methylation pa~. Placenta. 2. ~I. 1981. 148. S~try. B.V.~ Jan~m V.~ F~n~ P.M. and Merk~ L~ I~ibition of phmpholipid methylation cadmium iota ~d i~ implic~ions on tissue toxicity. J. Am. CoiL T~,xicoL. 3. 169. 19~. 149. S~t~ B.V.~ Ja~m V.~ g~n~ P.M. and Stephan. C.C. Bi~mi~al eff~ts of cadmium: intluence of Cd-- ions on enzy~tic methylatiom~. J. Ant. ~olL T~icol.. 4. ~4. 1985. 150. Ho~k SI.A. and S~try, B.V.R~ Eff~m of matem~ smoking on ph~olipid methylafion in hum~ ~lacenml pl~ membmfles. Pha~acologixt. 31. 138. 1989. tSl. Tana~ M. Studies on the eti~logic~ m~h~isms of t~ developm~t diso~ ca~d by maternal smok~g during p~¢~-. Nippon San~ Ft~jin~ Ga~i ~shi. t7. 1107. 1965. 152. ~m~em I. UIt~cm~ of ~¢ hum~ pimento at te~: ob~ati~s on pl~n~ from new~m child~n of smoking ~d nommoking mo~e~. Acta Obxtet. Gbme~l. Scand.. 56. 119. 1977. 153. ~mmsen. I. and Kjel~n. ~ Infm~ ulfrmt~tu~ of hum~ umbilical ~efi~: o~ations on t~m new~m chil~ of smo~ng ~d nonsmoking mother. Cite. Rex.. 36. 579. 1975. [5~. G autieri. R.F. and Ciuchm. H.P. Eff¢~ of ce~ain drags on ~ffu~d hum~ placent~ I. N~otic ~e~o~onin. ~d relaxin. J. Pharm. 3~'i.. 51.55. 1962. I YS..lon~. J.B. and Rowsell. A.. Fetal 5-hydroxy~,p~amine levels in later p~aney. J. Ohstet. Gynet~l.. 80. 687. 1973. 156. Pou~on. E, Robson. J.M. and Sullivan. F.M. Te~togenic eff~t of 5-hyd~xy~p~ine in mice. S~qence. 157. Robson. J.M. and Sullivan, F,M~ 5-Hydmxyt~pt~ine. placent~ function ~d toxemia of p~gnancy. Prm'. R. Sac'. Med.. 59. 7~. 19~. I YS. Honey. D.P.. Rohmn. J.M. and Sullivan, F.M. M~hanism of inhibito~ a~tion uf 5-h~dmxyt~'ptamine pl~enml funcrion. Am. J. Obstet. Gynecol.. 99. ~0. 1967. 15~. R~bson..1.M. and Sullivan. M. ~ff~t of 5-hydro~y~'ptamine on maintenance of p~gnancy, con~enital abno~aliti~, and the de~elopment t~t" toxemia. Adr. Pharmot'oi.. 6. 187. 1968. I ~). Began. W.. Jr.. Gremlin. R.C. Hey~nn. M.A. and Rudnlph. A.M. Eft~t~ of ph~acologic agent~ on umbilical bl~ fio~ in fetal lambs m merm Bi,L Netmme. 33. ~. 1978. 161. Clark. ~E. MiI~. ~G. Otte. %~ and Sty~ S~. Eff~t of se~tonin on uterine bl~ flow in and nonp~gn~t sheep. ~r~ Sci.. 27. 2655. 1980.

79 } t~ ptamin, by ~lt h~atn homogemltes. Et," .I Ph,o'ma,'td.. ]3. Io. l t~73 I(~. (;mMman. R.F. ~nd Weiss. (;.B.. Ahemtton of 5-it~dm~tr)ptalnlll¢- "C" ¢II]ux i*~ Itl~Otln¢ In rat brain %]ic¢s. Neurt~pimrmu~'oit~y. 12. u55. 1~73. 16~. He~. F.. Bour~oin. S.. Ham~m. M. Ternaux. J.P.. and (;Iowinskl. J.. Ctmtfo[ of thd r¢lca,¢ o( ,ynth~ized 'H-hydrnxyt~'ptamm¢ by nicotinic and mu~carmtc rec~pto~ in nit il~)thalaml~ ~lic¢~. .$vhmie~h'her~'.~ Arch. Ph~trmar, d.. 29~. 9 [. I ~7~. I~. S~t~'. B.V.R. and ()wins. L.K.. Re,tonal anti dift~rential ,¢nsttivit~ t)t" umbilico-placental va~cttiature Am. J. Oh.*tet. Gynecol.. 133. 629. 1979. 108. Ot~adtwo. J.O. and S~t~. B.V.R. Hum~ piactnml ¢~oiinergi¢ system: stimuiadon-~e~tion . .~. 4. ~. 1973. t~r ml¢~ of acetvlcholin¢ from isolated placental villms. J. Ph~trmucol. ~. Ther.. " 169. Rag~van. K~. and S~tW, P.B. EffccLs of tem~tum on acetylcholine ~ynth~is and mlem~ in p¢#u~d hum~ placentm Indian I. Med. Res.. 38. [718. 1970. 170. van Euler. U~. Action of adrenaline, acetylcholin¢ and other subst~c~ on newe-f~ yes, Is {human placental../ Physical. tLomhm~. 03. ~29. l o3S. 17[. Ellen. R. and Astr0m. A. Pha~acologica[ studies on the ~used human pl~¢nt~ A¢'m Phw'mucol. 172. Ciu~ta. H.P. and (;autieri. R.F. Eff~ts of ce~ain dm~s on ~ human placenta, lit. S} mpathomi- tactics, a~tylcholin¢ and histamine. J. Phurm. 5ci.. 53. [84. 19~. 173. Fu~hgott. R.F. Role tff ¢ndothclium in ms~nses of vmseular smith mu~le. Chx'. Rex.. 53. 537. !7~. Fu~h=ott. R.F. ~wadzki. J.V_ and Chert'. P.D.. Role of ~mdmhelium in the v~ilator response acetylcholin¢, in ¢~s,~ddudon. Valthout¢. P.M. ~d Leu~n. I.. ~.. Raven ~s. Ne~t Yo~. 19~1. 173. Liji~ky. W. Ch6mist~" and meta~lism of N-nitmso compounds ~lat¢d to respirator' t~t ca~mo~¢ne~is. in C#mparadt'e Respirator" D'm-r Cdrt'hu~qene$is. Reznik-Schiiller. H.M.. Ed.. CRC ~ss. Bt~a Raton. [983.95. i76. Ese~m. R.B. Rande~ath. E. Santella. R.3[.. Cefaio. R.C.. Avitts. T.A. and Rande~th. K.. of smoking-~lated covulent DNA adducts in hum~ placenta. S¢'tettt-e. ~ I. ~. [996. 17S. Rtmignok G. Alaoui,lamali. M.~. C~tongua?. A. and Sehuller. H.M.. Meta~limx ~d DNA damage Cant'er Rex.. *~. 567l. i989. 179. B~ Y.W. and C~dy. G. [nt=utenn¢ g~th and maturation in ~tation to feel ~pfivation. in The BMtimom. 1975. 305. I SO. ~nne. P.H. E~ tnm~i¢tin levels in ¢o~ bl~ ~ ~ indicator of in,uterine hy~xia..4cm Paediarr. Stand.. 55. 478. 1966. I Sl. Humb~. J.R. Abebon. H.. Hatha~*'ay. W,E., and Batta~li~. F.C.. Ptfl~c3 themia m ~mall t~r ge,tational 1... Nielichar. V. and Wolf. H.. Ol~ccnn und frei, Fett~ uren im Blntolasma ~t hv~m~ohen 183. RubaitellL F.F. and Peratonen. L. Ammonia nitrogen in ~mall-for-dates n¢~m babi¢~. ~ncet. [. 208. 1909. I $4. Rubaltelli. F.F.. Formentin. P.A. ~nd Taro. L.. Ammonia ntlro~en u~a ~d uric acid blo~ levels in nom~al and byes'strophic new,ms. Bird. Neonate. 13. 129. 1970. 185. 31~tyan. J.. Ftkete. M...la~i. I. 5ulyok. E.. Imhnf. S.. and Solt~ G.Y.. ~e ~>tnatal changes in the ci~ulatin~ f~* amino acid pt~l in the n¢~m~. If. ~e plasma amino ~id ~atio in mwaut¢rin¢ malnulrition tsmall-for~t~ t~ll-te~, p~te~ and twin infants~. Bi,l..Veonute. 14. I~. 1989. I g~. Schulman..I.D.. Queenan..I.K.. and D*mr~. L.. O~mmatographic anal) ~i~ of concentration of amino acid> in amniotic fluid from early, middle and lale ~ri~ of hum~ ge>tatton.. ~m. J. Ohster. Gynev,i.. 116. 243. 1072. I~7. Wha~on. B.A.. Foulds. J.W.. Frazier. I.D.. and Penm~k. C.A. Amniotic fluid total h~d~}p~linc and intmuterine ~th. J. Ohstet. Gynaev*d. Br. Commonw.. 78. 791. 1971.

188. Shah. S.I. Alderman. M.. Queenan..I.K. Br'~se..l. and Winick. M..Nondial.vzabl¢ pept,dc-b~,und hydroxypmhn¢ m human amniot~c fired: ~n ~nd~calor of (c~al g~)wlh. Am. 3. Oh.~eL ~yn£,c'td.. I I ~, ~(). I ~. 1~9. C~sada)'. (;~ Amni~cmcsis. Clin. P~.rtm~t.. I. ~T. I~(I. Humbert..I.R. m~fl (;~tlin. R.W.. Gru~ th [tUHIlt)II~ icvul~ m m~m~oglyeaem~¢ ;lnd hypoglycacm=c maternal arterial ox}gcnalion at hzgh alntudc. J AppL Plty$ie~/. Rc~plr. Era'trim. E.~'ercise Pllystol.. 52. 19~2. 192. Mimre. L.G..lahni=en. D.. R, unds. 5.5.. Re~v~..l.T.. and ¢;rover. R.F.. Mazemal hypcw~nzilati~n help~ precede ~cr=ul ox}genmion durtng )l~gh-al~liud¢ pregnancy. J. :lppl. Pltysu~l. Rdspw. Ei1vtron. E.vurc'L~c Plty~ted.. 52. 690. 1982. Iq3. Albany. A.A- Ort~. L.A. W~in, E.H.. and ~v~tt~ro. D.N_ Ett~cl of cigar~t¢¢ smoking on p~iein amino acid meta~flism..V~m'. Rep. ht¢..5. ~5. 194. Lugu. (;. and C~sady. G, [mrau=¢rin¢ growth ~lardalion: ciimcopaehologic findings in 233 con~uliv¢ inf~ts. Am. J. Obsrer. Gywecol.. [~. 6[5. 1971. 195. Robe~son. A.F. Sprecher. H.W, and Wilcox. J.P, Total lipid fa¢iy acid patterns of umbilical cord blood in inlm-u~¢fine gmwih failure. Biol. Neonate. i~. 28. 1969. 196. Young. M. and P~nton. M.. Ma¢¢mal and t~tal pl~ma amino ~id concentrations during g~m~ion and in mta~ed f~ml growth. J. Oh.s'wt. Gynue¢'e~l. hr. Commtmw.. 76. 333. 1969. Iq7. Lindhlad. B~. Rahimlmfla. R~.. aud Khan. N.. The ~cn¢~u~ pli~ma try= =mno =c=d I~t~ dunng th= tirs¢ hou~ of life. II. In a lower s~io~onomic group of mt~$~ ~a in Ka~hi. West P~isi~. with s~ciai mt~mnc¢ to ~e smaii-for~l~ syn~m¢. A¢~a Paediatr. Stand.. 59. 21. 1970. Younos~i, M.~ ~cie. A. Dilling, L. and Hawo~h. J.C, Urine" hy~xypmline/cmaiinin¢ ralio in noel. te~. pre-m~ ~d Smwih mt~ed in¢~. Arch. Dis. Childh.. ~. ~17. 1969. L~kard, D. P~ R. and C~ady. G. Hngemail nitrogen conten¢ in n~naees. Pediatrics. 49. 618. L~rd, W. B~ Y, Sumne~ J, Dw~k. H. and C=~dy, G., Fingernail nitrogen ~cm=ion in the and new~m. Pediatir. Res.. 7. 176. 1973. Haymond. M. ~rL I. and Paglia~. A. ~f~tiv¢ glucon¢ogenesis in small for geslafional age infants. J. Pediutr.. 83. 153. 1973. Longo. ~D. ~¢ biologic[ elf, s of c~n monoxide on the pregnant woman, fc~ and nc~m infant. Am. J. Ohster. Gb'n¢col.. 129. 69. 19~. Quigley, M.~ Sh~han. K.~ Wilk~ M.M, and Yen. S~.. Effec~ of ma¢¢mul smoking on ci~ulaling cat~holamine levels and t~ml h~ rams. Am. J. Ohster. Gbwecol.. 133. 685. 1979. Gre~. F.C. Jr. and Gobble. F.~ Jr, Et3~cl of syn)paih=tic n=~¢ stimulation on the ui¢fin¢ v~cular ~d. Am. J. Obster. Gynec~l.. 97. ~ 1967. Dyer. D.C. and Gough. ~D.. Comp~ative actions of selected vlsoacliv¢ drags on isola¢¢d human ~¢ri=s. Am. J. Qi~swr. Gynei'td.. I l I. 82t~. 1971. Ada~ons. ~ and Mye~, R.~ Ci~uia=ion m lh= int¢~ illous spat=: obstetrical consid=~tion in fetal dep~vation. ~ The Placenta and d=e Maternal Supply ~ne. Gfilenw~ P.. ~.. Univ¢~ity P~ Bahimom. i97~. I58. 207. Youn~=L M.~ and Hawo~h, J.C~ ~tion of hFdmxypm[in¢ in urine by premam~ ~d no~a[ te~ inf~ and ~hose with inl~ut~fine growth ~r~atlon dunng ch~ fi~t th~ ~ys of 1i¢¢. Pediau'. Rcs.. 2, 17. 1968. ?nq ~lanni~. ~. Wyn-P~h. E.. and ~-ddy. K.. Elf::: .~" .~;.:r:::: ,~;..~.~;;; .,:: :'c:.:. '~r:.::hh;~ m~cmc:=:, nodal pregnancies, hr..lied. J.. 1i5957~. 552. 1975. 200. Tat'eb. O.S. and ~:~try. R.V.R.. ~¢~"7"- '; ...... ; ...... ' ....... :. : .".:.=:~ ")- ' .:.. ::.:;:..:..;= ~:[.=~-::..~ .... and ~ubs~c¢ P rmm mouse c¢~bml slic~s. Fed. Pr~c., ~. 269. 198[. 210. S~try. B.V.R. Mouton. S, Janson. V.E., and Kambam. J.R.. Tobacco smoking by p~anl women: dism~ances in meta~lism of bmnch~ chain ~ino acids and re=a[ gmwlh. Amt. N.Y. Acad. Sci.. 678. 361. 1993. 21 I. S~try. B.V.R. Janson. V.~ Kambam. J.R.. Parris. W.C.V, and Perry. S.M.. Cigam=te smoking inc~as~ plasma amino acid concemmiions in hum~s. F~EB J.. 3. A416. 19~9. 212. S~tD'. B.V.R, Janson. V.~ PerD'. S.3L and Kamba~ J.R.. Cigareit¢ smokin$ increises pl~ma amino acid conc=neralmns in pregnant women. Phannace~logrsr. 31. 178. 1989. 213. Marks. M,J.. Sdtzel..J.A, and Cullins, A.C. Dos=-res~nse analysi~ of nicotine ¢oi~mnc¢ and ~cepmr changes in ¢wo inbred mouse sixths. J. Pltarmaco/. ~rp. Thor.. 239. 358. [986. 214. Carmelll. D., Swan. (;.~ Robinette. D, and Fabsil~ R. Gen=tic influence on ~moking ~ a studF of male ti~=n~. 3. Engl. J. Med.. 327. 829. 1992. 198. 199. 200. 201. 202. 203. 204. 205. 206. O cr~ 4~ O

81 215..laclJbo P.. Bem~,~'it~'~ N.L. Se,,'erson. H.° and Hatsukami. D.. ~nnary. ~xcr~t~on of anah~ine and analabin~: biochemical marK~ tot whacco u~ durm~ ni~olin~ ~plac~mCnl lh~py, CHn. Pluu'm~¢',l. Thor. 55. 132. 21~. K~zlo~ki. L.T., .l¢linck. L.. and Pnp~. M.A.. Ci~ar~t~ ~m~Km~ amon~ alc~holi~: a ~,mmum~ and n~l~t~d p~bl~m. Cun. J. Public' Health. 77, 2()~. 1986. 217. Kozlowski. L.T. R~habilitaling a ~n¢lic ~p~cfiv¢ in the ~tudy of tobacco and alcohol use. Br. J. ,-hMi~'r.. 86. 117. IgUl. 218. Koziowski. L.T. Skinner. W, Kent. C, and Pope. ~I.A. Pro~cts l~r ~mukmg treatment in individuals ~king (~atmen¢ {or alcohol and drag problems. Addict. Behat'.. 14. 273. [989. 219. Hart. V.~M. G~m fa¢lo~ in placental growth and development, in M,l¢,cuh~r Feud Membrane .4ulu~',ids. Rice. G.E. and B~nneck¢. S.P.. E~.. CRC 0