In Utero and Lactation Exposure of Rats to 1R4F Reference Cigarette Mainstream Smoke: Effect on Prenatal and Postnatal Development

C. L. Gaworski*,1, E. L. Carmines*, A. S. Faqi{dagger},2 and N. Rajendran{dagger}

* Philip Morris USA, Richmond, Virginia 23224 {dagger} IIT Research Institute, Chicago, Illinois 60616

Received November 20, 2003; accepted January 16, 2004


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Childhood cognitive and behavioral deficits have been reported in children born to mothers who smoked during pregnancy (Institute of Medicine, 2001Go). To investigate these potential responses in an animal model, reproductive and neurotoxicity evaluations based on the U.S. FDA guidelines were used to examine the offspring of male and female Sprague-Dawley rats exposed 2 h/day, 7 days/week by nose-only inhalation to whole mainstream smoke total particulate matter (TPM). Concentrations of 150, 300, or 600 mg/m3 were used (males: 4 weeks prior to and during mating; and females: 2 weeks prior to mating, during mating, and through weaning at postnatal day 21). Sham air controls receiving filtered air and cage controls were also maintained. F1 rats were weighed, identified by gender, examined for clinical signs of toxicity, and evaluated for neurobehavioral effects through postnatal day 65. Parental exposure was evidenced by smoke concentration-related increases in blood carboxyhemoglobin, nicotine, and cotinine and by characteristic cigarette smoke-related rodent respiratory tract histopathology. Also, nicotine and cotinine were found in F1 blood through the lactation period. Maternal toxicity occurred at concentrations of 300 and 600 mg TPM/m3, where total body weight gain during gestation was significantly (p <= 0.05) decreased compared to sham controls. While smoke concentration-related decreases in F1 birth weight and growth were evident (600 mg TPM/m3, significantly different from sham at all time points), no adverse effects on developmental landmarks, including age at vaginal patency or preputial separation, motor activity, acoustic startle response or learning, and memory, were observed in the F1 generation. This study confirmed that maternal exposure to high levels of mainstream cigarette smoke during gestation and lactation reduces birth weight and retards growth in the rat neonate; however, the developmental and neurobehavioral testing methodologies employed did not appear to be sensitive for an evaluation of neonatal behavioral effects following parental smoke exposure.

Key Words: IRAF mainstream cigarette smoke; developmental toxicity; in utero exposure.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The Surgeon General has concluded that cigarette smoking is a risk factor for human reproductive effects in both parents and infants (U.S. DHHS, 1988Go, 1990Go). Specifically, the Surgeon General indicates that smoking during pregnancy may lead to an increased risk of ectopic pregnancy, spontaneous abortion, abruptio placentae, placenta previa, and preterm delivery (U.S. DHHS, 2001Go). Smoking among males has been suggested as a risk factor for erectile dysfunction (Forsberg et al., 1979Go), decreased libido (Ochsner, 1971Go), and decreased sperm count (Raboch and Mellan, 1975Go). Furthermore, the Surgeon General has reported that women who smoke during pregnancy have a decreased risk for preclampsia, but the risks for perinatal mortality—both stillbirth and neonatal deaths—and sudden infant death syndrome are increased among the offspring of women who smoke during pregnancy (U.S. DHHS, 2001Go).

A consistent association between smoking during pregnancy and reduced average birth weight, with the risk of having a low-birth-weight baby (under 2500 g) increased 53% in light smokers (< 1 pack/day) and 130% in heavy smokers (>=1 pack/day) compared with nonsmokers, has been reported (Meyer et al., 1976Go). Follow-up studies on children born to mothers who smoked during pregnancy have indicated growth retardation at 5 years (Wingred and Schoen, 1974Go) and 7 years (Goldstein, 1972Go) of age. The relationship between cognitive and mental development in children born to mothers who smoked during pregnancy is less clear. The 1979 Surgeon General’s report indicated that the data suggest unfavorable effects of smoking during pregnancy on the child’s long-term growth, intellectual development, and behavioral characteristics (U.S. DHHS, 1979Go). The Royal College of Physicians (1992)Go cited a study showing a "strong correlation between smoke exposure during pregnancy and lower academic achievement even after allowing for social class."

Along with the risk for reduced birth weight, decreases in the infant’s cognitive and motor development and abilities have been suggested in infants born to parents who smoked (Institute of Medicine, 2001Go). However, as acknowledged by the Surgeon General’s report, these changes are often difficult to study because of the vast complexity of possible antecedent and confounding variables (U.S. DHHS, 1979Go). Furthermore, the authors of studies investigating an association between smoking during pregnancy and antisocial behavior, violent arrests, delinquency hyperactivity, and mental retardation generally note that some confounding factors may have been missed. Although trends were suggested by the data, evidence for causation was usually insufficient (Brennan et al., 1999Go; Drews et al., 1996Go; Rantakallio et al., 1992Go; Roeleveld et al., 1993Go; Weitzman et al., 2002Go).

Although several embryo/fetal toxicity studies on mainstream cigarette smoke exposure can be found (Essenberg et al., 1940Go; Magers et al., 1995Go; Peterson et al., 1981Go; Reckzeh et al., 1975Go; Reznik and Marquard, 1980Go; Schoeneck, 1941Go; Seller and Bnait, 1995Go; Wagner et al., 1972Go), studies on postnatal developmental parameters in animal models are rare. Bertolini et al. (1982)Go described a study where the offspring of rats exposed to commercial cigarettes during pregnancy were trained for avoidance conditioning when 60 days old. The number of rats satisfying the criterion of learning at the end of conditioning was similar in all groups; however, the rate of avoidance acquisition was significantly greater in the offspring from dams exposed to mainstream cigarette smoke. Characteristics of the exposure were not described.

A controlled laboratory study investigated the potential for mainstream cigarette smoke exposure to produce reproductive effects (Carmines et al., 2003Go). The objective of that study was to develop a nose-only smoke inhalation design in a laboratory animal (rat) using well-defined smoke generation and analysis techniques. Parental exposure to total particulate matter (TPM), 300 or 600 mg TPM/m3 1R4F reference mainstream cigarette smoke for two 1-h periods each day (females: 2 weeks prior to mating and through gestation; males: 4 weeks prior to mating), resulted in a statistically significant reduction of fetal body weight compared to the fetal body weights obtained with a sham control group exposed to air. Exposure to high levels of smoke produced signs of maternal toxicity (reduced body weight gain) and blood levels of ~250 ng/ml nicotine, ~250 ng/ml cotinine, and ~35% carboxyhemoglobin (COHb), which were in excess of those levels typically found in human smokers. (COHb levels in smokers reportedly average 4%, usually in the range of 3–8% [1–2% in nonsmokers], with smoker blood nicotine ranging from 10–50 ng/ml and blood cotinine ranging from 250–300 ng/ml [WHO, 2003Go]). Still, the exposure design was considered suitable for studying the potential for other smoke-attributed fetal effects including delayed development. A sensitive animal model investigating potential developmental effects could be useful for a comprehensive evaluation of cigarette smoke toxicity.

The present neonate development study was designed to deliver mainstream smoke under well-controlled and defined exposure conditions using a standard reference cigarette type that represents a medium tar yield commercial cigarette. Male and female rats were exposed prior to mating and during the gestation and lactation periods to increase the probability of producing an effect. Preweaning development and growth as well as neurobehavior were assessed in the F1 generation rats through postnatal day 65. Protocol strategies were based the U.S. Food and Drug Administration guidelines for conducting reproductive and neurotoxicity evaluations (U.S. FDA, 2000Go). Smoke exposures were conducted by a nose-only technique to reduce potential exposure to smoke components deposited on the fur and subsequent ingestion of smoke particulates during preening. Smoke constituent absorption was demonstrated using maternal and fetal blood biomarkers, while respiratory tract histopathology was performed on the parental rats to demonstrate the degree of toxicity.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Study design.
Table 1 summarizes the study design. The rats were exposed by nose-only inhalation at target mainstream smoke concentrations of 150, 300, or 600 mg TPM/m3 for 2 h/day (1 h smoke/30 min air/1 h smoke) using Canon-style exposure chambers with plastic restraint tubes matching their body size (CH Technologies, Westwood, NJ). Males were exposed 4 weeks prior to and during mating and females were exposed for 2 weeks prior to mating and during gestation and lactation. Because it was considered necessary to maintain the dams with the litters immediately after birth, the daily exposure regimen was interrupted at parturition day until lactation day 4 and then continued through lactation day 21. Rats in the sham control group were exposed to filtered air on the nose-only exposure system, while cage controls remained in transport cages during the exposure periods. Separate groups of sentinel animals undergoing exposure and mating were included in each group and used for smoke biomarker determination. As appropriate, developmental and behavioral end points through the lactation period were assessed using the litter as the experimental unit. Smoke concentrations and exposure time periods were based on previous experience with nose-only mainstream cigarette smoke exposure in rats and International Congress on Harmonization reproductive toxicity study guidelines, which suggest exposure prior to mating and during the gestation period to increase the probability of producing an effect (ICH, 1993Go). Animals were acclimated to the mainstream cigarette smoke and animal holders during the first 3 days of the premating exposure period using a reduced schedule (30 min, 1 h, and 1.5 h on exposure days 1, 2, and 3, respectively). The first full set of two 1-h exposures began on day 4. Because of the high levels of carbon monoxide (CO) produced in the smoke, the daily exposure period was broken into two individual 1-h smoke exposures separated by a 30-min air exposure period. The position of the rats on the nose-only exposure system was changed on a weekly basis according to a rotation scheme.


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TABLE 1 Study Design Used to Examine the Effect of Mainstream 1R4F Cigarette Smoke on Development

 
Cigarettes and smoke generation.
The 1R4F reference cigarettes were obtained from the Tobacco and Health Institute at the University of Kentucky (Diana and Vaught, 1990Go). All cigarettes were conditioned according to ISO 3402 (International Organization for Standardization, 1991aGo) before being smoked. Smoke was generated on an automatic 30-port carrousel smoking machine designed for continuous smoke generation over several hours (modified, type SM85; Reininghaus and Hackenberg, 1977Go). The machine operates in basic conformity with ISO 3308 (International Organization for Standardization, 1991bGo) standard smoking protocol. The key parameters were a puff volume of 35 ml, a puff duration of 2 s, and a puff frequency of 1 puff per cigarette every 60 s. The carrousel rotated in steps at a speed of 1 puff/2 s, resulting in a constant stream of mainstream smoke. Some minor deviations from the ISO standard, such as rectangular puff profile, free (open-end) smoking, and air velocity, were necessary for technical reasons (Vanscheeuwijck et al., 2002Go). At the puffing port, cigarettes were lit with a halogen spot lamp; butts were ejected at a mean butt length of 35 mm. Near the smoking position, the continuously generated smoke was diluted with filtered conditioned fresh air at a rate of 1:150 to 1:170 (vol:vol) depending on the desired yields of TPM.

Animals and animal care.
Care and use of the animals was in conformity with the American Association for Laboratory Animal Science Policy on the Humane Care and Use of Laboratory Animals (AAALAC, 1991Go). Sprague-Dawley rats were obtained from Taconic Farms (Germantown, NY). Male rats ranged from 9 to 10 weeks of age (256–310 g) and female rats ranged from 7 to 8 weeks of age (145–204 g) upon receipt. Male and female rats were double-housed (same sex) upon arrival and were held in quarantine for approximately 2 weeks prior to exposure initiation, during which time they were observed at least once daily for mortality and morbidity. After randomization by body weight, each parental animal was single-housed (except during mating when a male rat and a female rat were paired overnight). Animals were housed in plastic shoebox cages (Lab Products, Maywood, NJ) with corncob bedding (Anderson, Maumee, OH) during nonexposure periods. The animal rooms were maintained at 20–24°C and 20–54% relative humidity. Rats were provided with Certified Rodent Meal 5002 (PMI Nutrition International, Inc., Brentwood, MO) and City of Chicago municipal tap water ad libitum except during inhalation exposures.

Analytical characterization of the test atmospheres.
After the smoke adaptation period, TPM concentrations were determined at least twice daily (once per hour) in each chamber using a gravimetric filter-collection method. The sampling system consisted of a preweighed filter connected to a constant flow vacuum pump. Smoke particles were collected on preweighed 47-mm fiberglass filter disks (Pall Corp., Ann Arbor, MI) from inhalation exposure ports at flow rates matching the smoke test atmosphere delivery rate. The aerosol mass collected on the filter was weighed and a dry gas meter was used to measure the corresponding sample volume. The weight to volume ratio was determined to provide the TPM concentration in the test atmosphere. Nicotine concentration was determined twice per group per exposure week by trapping in a sulfuric acid-impregnated silica gel tube (Extrelut NT, Merck, Darmstadt, Germany) and eluting from the tube with an n-butyl acetate solution. The amount of nicotine collected was determined by gas chromatography using a GC 6890 series (Agilent Technologies, Wilmington, DE) equipped with nitrogen phosphorus detector and an HP-5 column (30 m long x 0.32 mm diameter). Results of the two analyses were then averaged. CO concentrations were monitored in each exposure chamber continuously with a dedicated infrared gas analyzer (Model ZRH-1, California Measurements, Inc., Sierra Madre, CA) by drawing filtered smoke through the gas analyzer. The gas analyzers were calibrated with gas standards and the calibration was checked prior to the initiation of daily exposure. The aerosol particle size distribution was determined during exposure weeks 4 and 9 in each chamber using a Quartz Crystal Microbalance (California Measurements, Inc., Sierra Madre, CA).

Biomonitoring.
To measure smoke exposure, biomonitoring was conducted on the sentinel animals and their offspring. Within 5 min of being removed from the exposure chamber, male and female parental sentinels were anesthetized with 70% CO2 /30% air and bled from the retro-orbital sinus for determination of COHb, nicotine, and cotinine. Blood was collected from the parental males at weeks 1 and 3 prior to mating and at termination (1 week after mating), from the females at week 1 and at termination after weaning, and from the pups at weaning (postnatal day 21) and at termination (postnatal day 65). Serum nicotine and cotinine levels were determined using the radioimmunoassay previously described (VanVunakis et al., 1987Go). COHb was determined using an IL-482 CO-Oximeter (Instrumentation Laboratory Company, Lexington, MA).

In-life and post-mortem parental evaluation.
The rats were observed twice daily during the exposure period for mortality and morbidity. Animals on study received weekly clinical observations concurrent with body weight and food consumption measurements. Male rats were single-housed in shoebox cages at least 24 h prior to introduction of the females for mating. Prior to mating, vaginal smears were collected on 4 consecutive days to ensure cyclicity and determine the state of the estrus cycle. During the mating period, one male and one female were housed together overnight and the next morning female rats determined to have mated (sperm-positive vaginal smears) were removed and single-housed. In most cases, a male was allowed to mate with only one female. A limited number of multiple matings (three cases) was not considered to have adversely affected the study, since male reproductive evaluation was not a measurement parameter in the study. The mating was continued for 10 days and terminated after sufficient numbers of sperm-positive females were obtained for the core study and sentinel groups. Unmated and nonpregnant females were euthanized by CO2 asphyxiation at the end of the mating period without necropsy. The day a sperm-positive smear was observed was considered gestation day 0. Parental animals were euthanized by CO2 asphyxiation (females after weaning and males 1 week after mating), and 10 males and 10 females were randomly selected from each group for organ weight measurements (males and females: lungs, trachea, and larynx weighed as a unit; females: ovaries and uterus).

The respiratory tract was examined microscopically as follows: larynx (three sections consisting of base of epiglottis, arytenoid projections, and vocal folds), nose (four rostral to caudal sections [Young, 1981Go]), lung (longitudinal and cross-sections [Dungworth et al., 1976Go; Lamb and Reid, 1969Go]), and trachea (longitudinal sections including bronchial bifurcation and bronchial lymph nodes). Lungs were inflated via the trachea and nasal passages were flushed with formalin to ensure fixation. All tissues collected were preserved in 10% neutral-buffered formalin and stained with hematoxylin and eosin. Additional sections of the lung, trachea, and nose were stained with periodic acid-schiff stain/alcian blue for goblet cell enumeration.

F1 generation evaluation.
Each pup was identified by gender at birth or soon thereafter. Physical examinations and body weight measurements were performed on lactation days 0, 4, 7, 14, and 21. The litters were randomly culled to four males and four females on lactation day 4. On lactation day 11, one male and one female pup per litter were randomly selected, weighed, and necropsied for brain collection, weighing, and histopathology (hematoxylin and eosin stains; three sections consisting of medulla/pons, cerebrum, and cerebellum). All pups were assessed for developmental landmarks through postnatal day 21. On postnatal day 17, 10 pups/sex/group were randomly selected for motor activity testing, with an additional 20 pups/sex/group randomly selected on postnatal day 21 for acoustic startle response and learning and memory testing. Animals not selected by postnatal day 21 for neurological testing were euthanized without necropsy. Animals selected for neurobehavioral testing were housed individually, weighed, and observed weekly. Age of vaginal opening or preputial separation was recorded. Aerial righting was performed three consecutive times for each rat on postnatal day 30–32 by holding the rat upside down (i.e., limbs upward) and dropping it from approximately 30 cm high. Normal rats, which righted themselves in midair and landed on their feet, were scored with a plus, and abnormal rats, landing on their sides or backs, were scored with a minus to indicate a negative/failed response.

Within each study group, the 30 pups/sex selected for neurobehavioral testing were allocated to one of three subgroups consisting of 10 pups/sex (limit of 1 pup/sex/litter). The pups were tested for neurobehavior including motor activity (postnatal days 17 and 61), acoustic startle response (postnatal days 22 and 61), and learning and memory (postnatal days 21 and 61). The same pups were used for testing at each of the respective time points. Motor activity was measured using an Open Field Motor Activity System (San Diego Instruments, San Diego, CA). The photo beam activity system was composed of hardware and software that enabled the monitoring and the real-time counting of as many as 480 photo beam channels. The test was performed at room temperature with normal illumination. Two enclosures were used and for each animal the motor activity was run for 10 min (two consecutive 5-min trials) after 1 min of acclimation; the numbers of central, peripheral, rear, and total beams interrupted were recorded.

Acoustic startle response was measured with an Animal Acoustic Startle System (Coulbourn Instruments, Inc., Allentown, PA). The acoustic startle system consisted of a large chamber with four startle response platforms. The platforms were equipped to measure the movement of the animal in response to a sound burst. All platforms were calibrated prior to testing. Animals were placed in platforms and allowed to acclimate for 1 min. Each animal was exposed to 50-ms bursts of white noise at 80 and 120 dB, with 20 trials each and with 5-s intertrial intervals. The peak amplitude of each startle movement and the interval between the acoustic stimulus and the peak amplitude was recorded.

Learning and memory tests were run in a room with normal illumination and using a computer-controlled, two-compartment shuttle box (Gemini Passive Avoidance System, San Diego Instruments). The animals were placed in the right compartment of the chamber (light off), which was equipped with the house light, cue light, and a tone. The testing sequence consisted of a 1-min acclimation period, after which the light turned on and the gate separating the right from the left chamber opened automatically. Rats that entered the dark compartment broke an infrared beam, which caused an automatic closure of the gate. After the gate was closed a scrambled shock of 0.5 mA for 2 s was delivered to the cage floor. A maximum of five testing sessions were conducted where the animal received foot shocks repeatedly until they reached the stop criterion (defined as remaining in the lighted compartment for two consecutive 180-s trials). The program recorded the latency (in seconds) from the beginning of the stimulus. If the animal remained passive for the trial length (180 s), the conditional stimulus ceased, the gate closed, and the program recorded "Max" as the latency. Animals that failed to remain passive for two consecutive of the five testing sessions were reported as nonpassive animals.

On postnatal day 65, 10 pups/sex/group were selected for necropsy and organ weight measurements (adrenals, brain, heart, kidneys, liver, lung, spleen, thymus, testes, and ovaries). Final body weights were used for all body weight-relative calculations.

Statistical procedures.
Means and standard deviations were calculated for all measured parameters. Since the cage control group was not subjected to potential stress factors from the nose-only exposure, statistical analyses included only the sham and smoke-exposed groups. Body weights, body weight gains, food consumption and organ weight data, and goblet cell counts for lung and trachea; litter survival; developmental landmarks; and neurotoxicity data were analyzed by analysis of variance (ANOVA) followed, where appropriate, by Dunnett’s test using SYSTAT software, versions 5.0 and 8.0 (Systat Software Inc., Richmond, CA). Comparison of litter (pup) body weight data was analyzed by an analysis of variance (ANOVA); the litter was the unit of observation. In the presence of significant main effects, all post hoc comparisons between the mainstream cigarette smoke-treated and sham control were conducted using Dunnett’s test. A minimum significance level of p = 0.05 was used for all comparisons.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Exposure
The measured daily mean TPM exposure concentrations for the entire exposure period were 151, 295, and 590 mg TPM/m3, all within 5% of the 150, 300, and 600 mg TPM/m3 target levels. Daily mean smoke concentration variations fell within 10% of the target smoke concentrations, and there was no overlap of the different exposure groups. Table 2 shows the particle size distribution and nicotine and CO concentrations. Measurements of smoke particle size and distribution indicated similar mass median aerodynamic diameters and geometric standard deviations in all smoke-exposed groups, while CO and nicotine levels were proportional to the smoke concentrations. These results demonstrate that the smoke was generated consistently and that the smoke particulates were respirable based on particle size measurements.


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TABLE 2 1R4F Smoke Atmosphere TPM, Nicotine, Particle Size Distribution, and CO Concentrations

 
Biomonitoring
Exposure to smoke resulted in smoke concentration-related increases in serum nicotine and cotinine in both males and females (Table 3). Consistent with the relatively short half-life of nicotine in the rat (1.3 h; Kyerematen et al., 1988Go), there did not appear to be any bioaccumulation over the course of the exposure series. COHb was also increased in a smoke concentration-related manner, with treated parental animals reaching levels of between 32 and 38% in the 600 mg TPM/m3 group. The elimination half-life of COHb in rats is about 42 min at the 600 ppm exposure level (Ayres et al., 1989Go), and there did not appear to be any bioaccumulation of COHb since the levels did not increase during the course of the study.


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TABLE 3 Parental Rat Nicotine, Cotinine, and COHb Biomonitoring

 
Blood from the pups was collected at weaning and at study termination (postnatal day 65). At weaning, nicotine was detectable in the blood of pups from dams being exposed to either 300 or 600 mg TPM/m3 (Table 4). Nicotine was not detectable in the pups from mothers exposed to 150 mg TPM/m3. Pup blood cotinine appeared to increase in relation to maternal smoke exposure and was detected in the blood of pups from all smoke-exposed groups. Since the F1 generation was not directly exposed to smoke, nicotine/cotinine appeared to be transferred via lactation to the pups. Nicotine transfer by milk has been previously reported (Dahlstrom et al., 1990Go). Not unexpectedly, the COHb levels in the blood of the pups from smoke-exposed dams were comparable to the sham control group levels at weaning. Carbon monoxide has not been reported to transfer via lactation. Analysis of F1 generation blood at study termination, ~40 days after weaning, indicated that the nicotine and cotinine levels were below the quantitation levels.


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TABLE 4 F1 Generation Nicotine, Cotinine, and COHb Biomonitoring

 
Paternal Effects
There were no deaths associated with smoke exposure. Diarrhea, salivation, and red material around the eyes and nose were occasionally noted in all nose-only tube-constrained animals and were considered to be signs of tubing stress. All groups of smoke-exposed male rats had significantly lower body weight gains compared to the sham control group (Table 5). The substantial difference between the sham control and cage control groups suggests that nose-only exposure may produce some stress-related effect on body weight. Although the data are not shown, closer examination of the data indicated that most of the body weight gain reduction was due to initial effects noted during the first week of exposure. Food consumption did not appear to be affected by exposure (Table 5). Mating performance, as measured by the number of sperm-positive dams, was slightly lower in the group of rats exposed to 600 mg TPM/m3 compared to the sham control. However, the pregnancy rate was not inconsistent with our previous investigation of smoke (Carmines et al., 2003Go) and was not considered to be an adverse effect of smoke exposure.


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TABLE 5 Summary of Observations in Male Parental Rats Exposed to Mainstream 1R4F Cigarette Smoke

 
Lung weight ratios (relative to body weights) tended to increase with smoke exposure, but the effect was not statistically significant compared to the sham control group (Table 5). Examination of the respiratory tract tissues indicated the presence of tissue changes consistent with those noted in previous nose-only inhalation studies using mainstream cigarette smoke (Vanscheeuwijck et al., 2002Go). These changes included inflammation, epithelial degeneration and atrophy, epithelial hyperplasia, and squamous metaplasia of the nose; inflammation, epithelial hyperplasia, and squamous metaplasia of the larynx: and diffuse pigmented macrophages in the lung (selected data are shown in Table 5). The incidence and severity of the changes indicated a clear smoke-related response, with resolution of the smoke exposure concentration evident in most cases.

Maternal Effects
With the exception of the time between parturition and lactation day 4, the female rats were exposed to smoke for 2 weeks prior to mating through weaning. Because of the nature of the nose-only inhalation exposure where females would have to be removed from the newborn litter, the smoke-exposure period was interrupted for a few days immediately after birth to allow for maternal/offspring bonding. No maternal animals died during the smoke-exposure period, but one female in the cage control group died during parturition.

Clinical observations in the females were the same as those described previously for the males. While the group mean body weight gains of female smoke-exposed rats (premating) were about 20% less than the sham control group mean body weight gains, the differences were not statistically significant (p > 0.05, Table 6). However, during the gestation period, exposure to smoke at concentrations of 300 or 600 mg TPM/m3 significantly reduced maternal body weight gains compared to the sham controls (Table 6). Exposure to 150 mg TPM/m3 had no effect on body weight gain. Reduced fetal body weight may account for some of these body weight effects. During the lactation period, body weight gains in smoke-exposed rats were unaffected by smoke exposure (Table 6). Food consumption measurements obtained during the premating, gestation, and lactation periods did not indicate any significant differences between sham and smoke-exposed groups (Table 6). Female mating and fertility indices were unaffected by smoke exposure. About 40% of the females in the 600 mg TPM/m3 group had a slightly prolonged gestation period, resulting in an average length of gestation increase by approximately 1 day for the group. However, because of the difficulty in specifically establishing the exact time of birth, the impact of a delay in delivery by a day is unknown.


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TABLE 6 Summary of Observations in Female Parental Rats Exposed to Mainstream 1R4F Cigarette Smoke

 
Organ weights recorded in females indicated significantly greater body weight-relative lung weights in animals exposed to 600 mg TPM/m3. Ovary and uterus weights were comparable for all groups. Relative lung weight was increased by smoke exposure to 600 mg TPM/m3. The respiratory tract histopathological observations in the females were similar in distribution and severity to those described for males. They included inflammation, epithelial degeneration and atrophy, epithelial hyperplasia, and squamous metaplasia of the nose; inflammation, epithelial hyperplasia, and squamous metaplasia of the larynx; and diffuse pigmented macrophages in the lung (data not shown).

F1 Generation Effects
The number of pups/litter in dams exposed to smoke at 600 mg TPM/m3 was slightly greater than the numbers born to sham control dams, although the difference was not statistically significant (Table 7). A greater number of pups died immediately after birth (day 0) in the litters from the 600 mg TPM/m3 group compared to the sham control group, thus providing a significantly lower live birth index for this smoke-exposure group (Table 7). Closer examination of the data indicated that the mortality was reasonably consistent across the litters in this smoke-exposure group (~1 pup/litter). Although the day 4 viability indexes for the litters from dams exposed to smoke at 150 or 600 mg TPM/m3 were lower than the sham control group index, no statistically significant difference was indicated (p > 0.05) and the survival rate was considered similar across all groups. To reduce the effect of litter size variability, the litters were culled at postnatal day 4 to contain an even distribution of 4 males and 4 females. No pups died through weaning when the groups were further culled to 15/sex/group and maintained for follow-up neurotoxicity evaluations; however, at the time of postweaning, two deaths (one male and one female) occurred in the 600 mg TPM/m3 smoke-exposure group.


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TABLE 7 F1 Generation Survival and Growth Data

 
Parental smoke exposure resulted in significantly lower pup birth weight at all smoke concentrations (Table 7). Continued observations through postnatal day 63 indicated that the pups born to females exposed to smoke at 600 mg TPM/m3 never attained the body weights of the sham controls. However, body weight effects were less persistent in the groups exposed to lower concentrations of smoke. Despite the initial birth weight differences between smoke-exposed (150 and 300 mg TPM/m3) and sham control groups, by postnatal day 63 only the female pups from the 300 mg TPM/m3 group remained significantly different from the sham group. By postnatal day 28, both sexes of pups from females exposed to 150 mg TPM/m3 smoke were statistically equivalent to the sham group. Although cage controls were not included in the statistical evaluations, comparison of the birth weight and growth data for the sham-exposed and cage control groups may suggest some minimal influence on pup growth during the lactation period due to maternal stress from nose-only tubing.

F1 generation development, as measured by the time to reach specific landmarks, was not significantly affected by parental smoke exposure (Table 8). All pups displayed aerial righting reflex by postnatal day 30–32, with no difference among the groups (Table 8). Motor activity measurements at postnatal day 17 suggested greater activity among the pups in the litters from dams exposed to smoke at 600 mg TPM/m3 (Table 8). However, this may be somewhat misleading since the response of the sham control group appeared to be quite low compared to the response demonstrated by the cage control group. No differences in motor activity were noted at postnatal day 61. Learning and memory trials did not reveal any differences between the pups from smoke-exposed and sham control dams (Table 8). Acoustic startle response measurements indicated a relatively small, although statistically significant, difference in the peak grams displaced in response to 80 dB in some litters from dams exposed to smoke compared to the sham control group; statistical significance was not indicated at 120 dB level (Table 9).


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TABLE 8 F1 Generation Developmental and Neurobehavioral Assessment Results

 

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TABLE 9 F1 Generation Acoustic Startle Response Assessment Results

 
Overall, the inconsistent response between the two analysis periods and the relatively small magnitude of difference with high variability suggests that the findings were by chance and not biologically relevant. The latency periods to reach the peak response were not affected by maternal smoke exposure. Although the sample size was quite limited, histopathological examination of the pup brain at postnatal day 11 did not indicate any smoke-related effects. While brain weights relative to body weights were increased in litters from smoke-exposed dams, the effect was not statistically significant in all groups, lacked a clear smoke concentration-related response, and probably reflected the decreased body weight more than actual effect on brain weight (Table 7). Other relative organ weights analyzed (adrenal, heart, kidney, liver, lung, spleen, testis, and thymus) did not reveal any smoke-related effects (data not shown).


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The potential relationship between smoking during pregnancy and the various aspects of the child’s cognitive and mental development has been assessed in several epidemiologic investigations. Using data from the National Child Development Survey in Britain, Butler and Goldstein (1973)Go analyzed a birth cohort of 17,000 children born in 1958 finding that, at age 7, the children of mothers who smoked during pregnancy had an average 4-month deficit in reading attainment. Additional analysis at 11 years indicated a deficit of 3 months in general ability, 4 months for reading, and 5 months for mathematics. Adjustment for mother’s height, age, social class, number of other children in the household, and gender substantially diminished the relationship. The authors also noted that the observed differences associated with smoking during pregnancy were relatively small compared to other factors, such as social class and number of other children. In a follow-up of the same study, Fogelman (1980)Go reported that smoking during pregnancy was related to measurements of the child’s reading and mathematics attainment. However, the author also noted that the deficit in attainment for children of smokers was relatively small compared to those associated with other factors, such as social class and family size. The author could not establish a causal link because of the potential for residual confounding.

Naeye and Peters (1984)Go examined a subset of data from the Collaborative Perinatal Study in the United States and found that smoking during pregnancy was associated with a slight decrease in children's educational attainment, spelling scores being lower by about 3%, and reading scores being lower by 4%. More frequent short attention spans and hyperactivity in these children were also reported. The authors performed a further analysis comparing sibling pairs where the mother had smoked in one but not the other pregnancy. In total, 578 such sibling pairs were available for analysis and the comparison suggested similar associations with the outcomes measured. Also, Olds et al. (1994)Go reported on a long-term prospective study based on 133 first births from women in semirural New York State. Even after controlling for social class, maternal education, maternal IQ, qualities of caregiving, and conditions in the home environment, children born to women who smoked more than 10 cigarettes per day during pregnancy had a significant difference of 4.35 IQ points at 3 and 4 years of age. However, no association at 1 and 2 years of age was evident. In contrast, a report by Niemela and Jarvenpaa (1996)Go stated there was no effect of maternal smoking on cognitive test performance at 5 months for breast-fed children.

Other studies have looked at associations between smoking during pregnancy and delinquency (Rantakallio et al., 1992Go), antisocial behavior, anxiety and depression, headstrong behavior, hyperactivity, peer conflict and immaturity (Weitzman et al., 2002Go), violent arrests (Brennan et al., 1999Go), attention deficit hyperactivity disorder (Milberger et al., 1996Go), and mental retardation (Drews et al., 1996Go; Roeleveld et al., 1993Go). Many of these studies have demonstrated some association with maternal smoking, but the strength of the association was usually reduced when confounding factors were considered. The authors noted that some confounding factors may have been missed in the studies and sample sizes were small. Thus, although trends were suggested in the data, evidence for causation was generally insufficient. While no specific mechanism for the behavioral observations in humans has been established, several have been hypothesized including fetal hypoxia (Naeye and Peters, 1984Go; Weitzman et al., 2002Go), placental transfer of smoke constituents (Drews et al., 1996Go; Olds et al., 1994Go; Weitzman et al., 2002Go), and nicotine action on the fetal brain (Frydman, 1996Go; Milberger et al., 1996Go).

Assessment of potential behavioral and neurologic effects related to chemical exposure in humans is frequently complicated by the need for retrospective-type analysis where confounding factors may or may not be adequately captured. Therefore, animal models measuring behavioral toxicity have increased in importance. They provide the ability to accurately measure exposure characteristics and allow for more specific determination of the mechanisms responsible for any effect. Coupling these assessment models with defined chemical exposure during the gestational phase may provide a tool for evaluation of potential latent effects that might be passed on through subsequent generations. In the present study, we applied standard behavioral testing methods in an attempt to develop an exposure design suitable for mainstream cigarette smoke evaluation.

Using a controlled nose-only smoke generation system, parental rats were exposed to known levels of mainstream cigarette smoke, which clearly elicited signs of maternal and paternal toxicity at the highest smoke concentration tested. Premating smoke exposure was included in the study design to increase the probability of demonstrating some effect. Parental toxicity was indicated by both body weight decreases in the sires during exposure and in the dams during the gestation phase. Also, smoke exposure produced characteristic smoke concentration-related increases in both respiratory tract histopathological change and blood biomarkers such as nicotine, cotinine, and COHb.

Despite the demonstration of a response that reflects maternal toxicity, the behavioral tests conducted in the offspring of smoke-exposed rats did not display a response that could be considered indicative of a consistent behavioral effect. Given the relatively weak and inconsistent strength of association found in human epidemiologic evidence regarding the relationship between maternal smoke exposure during pregnancy and cognitive and mental development in the child, the absence of an effect on these behavioral measurements in animal studies is perhaps not surprising. These standard animal behavior tests, as currently developed, may lack the required sensitivity to pick up the behavioral associations that have been predicted by some human epidemiologic studies.

In animals, nicotine has been investigated as a possible modulator of sensorimotor reactivity. Nicotine at doses of 6 to 12 mg/kg/day (delivered via minipump) produced a dose-dependent increase in startle amplitude during administration, which subsequently decreased during nicotine dose cessation (Acri, 1994Go; Acri et al., 1991Go). Sensory response to nicotine in the rat may be influenced by several variables. Acri et al. (1995)Go found both an age (70 days > 40 days) and strain (Wistar > Sprague-Dawley > Long-Evans) dependence in acoustic startle response and prepulse inhibition. Faraday et al. (1999)Go found that nicotine reduced both acoustic startle response and prepulse inhibition in Long-Evans rats but enhanced these responses in Sprague-Dawley rats. In another study, direct maternal exposure to nicotine in the mouse via subcutaneous injections resulted in significantly stimulated motor activity in early adulthood of the pups (Ajarem and Ahmad, 1998Go). Popke et al. (1997)Go reported that nicotine delivered during gestation via a minipump reduced the prepulse inhibition of acoustic startle reflex to 98-dB stimulus in female but not male offspring. The differences in the peak acoustic startle response noted between smoke-exposed groups and sham control groups in the present study were relatively small and inconsistent between the measurement periods. A calculation of the theoretical maternal nicotine dose in the 600 mg/m3 smoke-exposure group yields a potential exposure of approximately 1.3 mg/kg/day, representing ~10–20% of the nicotine doses used in the minipump infusion studies where an effect on acoustic startle reflex was demonstrated. Application of greater levels of smoke (and, therefore, nicotine) in whole smoke inhalation studies is limited by the maternal toxicity due to carbon monoxide.

According to data reported by the Centers for Disease Control, approximately 13.6% of women smoked during their pregnancy in 1996 (Centers for Disease Control, 2000Go). Although this frequency dropped to 12.9% by 1998, the number of women who smoke during pregnancy remains relatively high. Epidemiologic investigations have established a relatively consistent association between maternal smoking and the risk of low birth weight and small size for gestational age births in humans, with smoking cessation early in the pregnancy appearing to result in virtually complete disappearance of a birth weight effect (U.S. DHHS, 2001Go). The results of the present study indicate a smoke concentration-related decrease in average birth weight and confirm our earlier observations using this smoke-exposure design (Carmines et al., 2003Go). Our findings were consistent with other reports using less-characterized smoke-exposure situations where birth weight effects were noted (Bertolini et al., 1982Go; Magers et al., 1995Go; Peterson et al., 1981Go; Reckzeh et al., 1975Go; Reznik and Marquard, 1980Go; Schoeneck, 1941Go; Seller and Bnait, 1995Go; Wagner, 1972Go).

In the present study, continued pup body weight measurements during the initial 65 days of life indicated that, with a sufficient level of maternal smoke exposure (600 mg TPM/m3), neonate growth was delayed. However, a threshold for this response was suggested by the fact that, despite lower birth weight in pups from dams exposed to smoke at lower concentrations, catch-up growth was almost immediate (by postnatal day 4). Previously, Leichter (1995)Go reported reduced average birth weight in the offspring of Sprague-Dawley dams exposed to smoke from 10 cigarettes over a 2-h period (actual TPM exposure not quantified) with no fetal growth retardation after birth. In humans, differences in weights of infants of smoking and nonsmoking mothers generally disappear within the first year of age (Day et al., 1992Go, 1994Go; Russell et al., 1968Go). Peacock et al. (1991)Go reported that humans smoking only a low number of low-yield cigarettes had babies of similar mean birth weight to nonsmokers and, thus, postulated a possible threshold effect.

In summary, despite indications of maternal toxicity and reductions in pup body weights that appeared with the mainstream cigarette smoke exposure testing protocol used for this study, the behavioral tests conducted in the offspring of smoke-exposed rats were not responsive and did not display any consistent adverse behavioral effects.


    ACKNOWLEDGMENTS
 
We thank the staffs at Philip Morris USA and IIT Research Institute, Dr. R. L. Morrissey (Pathology Associates, Chicago, IL), D. E. Rodwell (Rodwell & Associates, Lawton, OK), and C. E. Ulrich (Inhalation Toxicology Services, Ashland, OH) for their technical assistance and critical review of the manuscript.


    NOTES
 
Portions of this report were presented at the 42nd Annual Meeting of the Society of Toxicology, March 9–13, 2003, Salt Lake City, Utah.

2 Present address: MPI Research, Mattawan, MI 49071-9324. Back

1 To whom correspondence should be addressed at Philip Morris USA, 615 Maury Street, Richmond, VA 23224. Fax: (804) 274-3055. E-mail: charles.l.gaworski{at}pmusa.com


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
AAALAC (1991). American Association for Laboratory Animal Science policy on the humane care and use of laboratory animals. Lab.Animal Sci. 41, 91.

Acri, J. B. (1994). Nicotine modulates effects of stress on acoustic startle reflexes in rats: Dependence on dose, stressor, and initial reactivity. Psychopharmacology (Berl) 116, 255–265.[ISI][Medline]

Acri, J. B., Brown, K. J., Saah, M. I., and Grunberg, N. E. (1995). Strain and age differences in acoustic startle responses and effects of nicotine in rats. Pharmacol. Biochem. Behav. 50, 191–198.[CrossRef][ISI][Medline]

Acri, J. B., Grunberg, N. E., and Morse, D. E. (1991). Effects of nicotine on the acoustic startle reflex amplitude in rats. Psychopharmacology (Berl) 104, 244–248.[ISI][Medline]

Ajarem, J. S., and Ahmad, M. (1998). Prenatal nicotine exposure modifies behavior of mice through early development. Pharmacol. Biochem. Behav. 59, 313–318.[CrossRef][ISI][Medline]

Ayres, P. H., Mosberg, A. T., Burger, G. T., Hayes, A. W., Sagartz, J. W., and Coggins, C. R. (1989). Nose-only exposure of rats to carbon monoxide. Inhal.Toxicol. 1, 349–363.

Bertolini, A., Bernardi, M., and Genedani, S. (1982). Effects of prenatal exposure to cigarette smoke and nicotine on pregnancy, offspring development, and avoidance behavior in rats. Neurobehav. Toxicol. Teratol. 4, 545–548.[ISI][Medline]

Brennan, P. A., Grekin, E. R., and Mednick, S. A. (1999). Maternal smoking during pregnancy and adult male criminal outcomes. Arch. Gen. Psychiatry 56, 215–219.[Abstract/Free Full Text]

Butler, N. R., and Goldstein, H. (1973). Smoking in pregnancy and subsequent child development. Br. Med. J. 4, 573–575.[ISI][Medline]

Carmines, E. L., Gaworski, C. L., Faqi, A. S., and Rajendran, N. (2003). In utero exposure to 1R4F reference cigarette smoke: Evaluation of developmental toxicity. Toxicol. Sci. 75, 134–147.[Abstract/Free Full Text]

Centers for Disease Control (2000). Tobacco use during pregnancy. National Vital Statistics Report 47, 1–12.

Dahlstrom, A., Lundell, B., Curvall, M., and Thapper, L. (1990). Nicotine and cotinine concentrations in the nursing mother and her infant. Acta Paediatr. Scand. 79, 142–147.[ISI][Medline]

Day, N., Cornelius, M., Goldschmidt, L., Richardson, G., Robles, N., and Taylor, P. (1992). The effects of prenatal tobacco and marijuana use on offspring growth from birth through 3 years of age. Neurotoxicol. Teratol. 14, 407–414.[CrossRef][ISI][Medline]

Day, N. L., Richardson, G. A., Geva, D., and Robles, N. (1994). Alcohol, marijuana, and tobacco: Effects of prenatal exposure on offspring growth and morphology at age six. Alcohol Clin. Exp. Res. 18, 786–794.[ISI][Medline]

Diana, J. N., and Vaught, A. (1990). Research Cigarettes. The University of Kentucky, Lexington, KY.

Drews, C. D., Murphy, C. C., Yeargin-Allsopp, M., and Decoufle, P. (1996). The relationship between idiopathic mental retardation and maternal smoking during pregnancy. Pediatrics 97, 547–553.[Abstract]

Dungworth, D. L., Schwartz, L. W., and Tyler, W. S. (1976). Morphological methods for evaluation of pulmonary toxicity in animals. Annu. Rev. Pharmacol. Toxicol. 16, 381–399.[CrossRef][ISI][Medline]

Essenberg, J. M., Schwind, J. V., and Patras, A. R. (1940). The effects of nicotine and cigarette smoke on pregnant female albino rats and their offspring. J. Lab. Clin. Med. 250, 708–717.

Faraday, M. M., O’Donoghue, V. A., and Grunberg, N. E. (1999). Effects of nicotine and stress on startle amplitude and sensory gating depend on rat strain and sex. Pharmacol. Biochem. Behav. 62, 273–284.[CrossRef][ISI][Medline]

Fogelman, K. (1980). Smoking in pregnancy and subsequent development of the child. Child Care Health Dev. 6, 233–249.[ISI][Medline]

Forsberg, L., Gustavii, B., Hojerback, T., and Olsson, A. M. (1979). Impotence, smoking, and ß-blocking drugs. Fertil. Steril. 31, 589–591.[ISI][Medline]

Frydman, M. (1996). The smoking addiction of pregnant women and the consequences on their offspring’s intellectual development. J. Environ. Pathol. Toxicol. Oncol. 15, 169–172.[Medline]

Goldstein, H. (1972). Cigarette smoking and low birth weight babies. Am. J. Obstet. Gynecol. 114, 570–573.[ISI][Medline]

International Conference on Harmonization ICH (1993). Detection of toxicity to reproduction for medicinal products. ICH Secretariat, Geneva, Switzerland. www.ich.org/urlGrpServer.jser?@1D=276&@_TEMPLATE=254.

Institute of Medicine (2001). Clearing the Smoke: Assessing the Science Base for Tobacco Harm Reduction. (K. Stratten, P. Sherry, R. Wallace, and S. Bendurant, Eds.). National Accademy Press, Washington, DC.

International Organization for Standardization ISO (1991a). International Standard ISO 3308. Routine Analytical Cigarette-Smoking Machine: Definitions and Standard Conditions. ISO, Geneva.

International Organization for Standardization ISO (1991b). International Standard ISO 3402. Tobacco and Tobacco Products: Atmosphere for Conditioning and Testing. ISO, Geneva.

Kyerematen, G. A., Owens, G. F., Chattopadhyay, B., deBethizy, J. D., and Vesell, E. S. (1988). Sexual dimorphism of nicotine metabolism and distribution in the rat. Studies in vivo and in vitro. Drug Metab. Dispos. 16, 823–828.[Abstract]

Lamb, D., and Reid, L. (1969). Goblet cells increase in rat bronchial epithelium after exposure to cigarette and cigar tobacco smoke. Br. Med. J. 1, 33–35.[ISI][Medline]

Leichter, J. (1995). Decreased birth weight and attainment of postnatal catch-up growth in offspring of rats exposed to cigarette smoke during gestation. Growth Dev. Aging 59, 63–66.[ISI][Medline]

Magers, T., Talbot, P., DiCarlantonio, G., Knoll, M., Demers, D., Tsai, I., and Hoodbhoy, T. (1995). Cigarette smoke inhalation affects the reproductive system of female hamsters. Reprod. Toxicol. 9, 513–525.[CrossRef][ISI][Medline]

Meyer, M. B., Jonas, B. S., and Tonascia, J. A. (1976). Perinatal events associated with maternal smoking during pregnancy. Am. J. Epidemiol. 103(5), 464–476.[Abstract]

Milberger, S., Biederman, J., Faraone, S. V., Chen, L., and Jones, J. (1996). Is maternal smoking during pregnancy a risk factor for attention deficit hyperactivity disorder in children? Am. J. Psychiatry 153, 1138–1142.[Abstract]

Naeye, R. L., and Peters, E. C. (1984). Mental development of children whose mothers smoked during pregnancy. Obstet. Gynecol. 64, 601–607.[Abstract]

Niemela, A., and Jarvenpaa, A. L. (1996). Is breastfeeding beneficial and maternal smoking harmful to the cognitive development of children? Acta Paediatr. 85, 1202–1206.[ISI][Medline]

Ochsner, A. (1971). The health menace of tobacco. Am. Sci. 59, 246–252.[ISI][Medline]

Olds, D. L., Henderson, C. R., Jr., and Tatelbaum, R. (1994). Intellectual impairment in children of women who smoke cigarettes during pregnancy. Pediatrics 93, 221–227.[Abstract]

Peacock, J. L., Bland, J. M., Anderson, H. R., and Brooke, O. G. (1991). Cigarette smoking and birth weight: Type of cigarette smoked and a possible threshold effect. Int. J. Epidemiol. 20, 405–412.[Abstract]

Peterson, K. L., Heninger, R. W., and Seegmiller, R. E. (1981). Fetotoxicity following chronic prenatal treatment of mice with tobacco smoke and ethanol. Bull. Environ. Contam. Toxicol. 26, 813–819.[ISI][Medline]

Popke, E. J., Tizabi, Y., Rahman, M. A., Nespor, S. M., and Grunberg, N. E. (1997). Prenatal exposure to nicotine: Effects on prepulse inhibition and central nicotinic receptors. Pharmacol. Biochem. Behav. 58, 843–849.[CrossRef][ISI][Medline]

Raboch, J., and Mellan, J. (1975). Smoking and fertility. Br. J. Sex. Med. 2, 35–37.

Rantakallio, P., Laara, E., Isohanni, M., and Moilanen, I. (1992). Maternal smoking during pregnancy and delinquency of the offspring: An association without causation? Int. J. Epidemiol. 21, 1106–1113.[Abstract]

Reckzeh, G., Dontewill, W., and Leuschner, F. (1975). Testing of cigarette smoke inhalation for teratogenicity in rats. Toxicology 4, 289–295.[CrossRef][ISI][Medline]

Reininghaus, W., and Hackenberg, U. (1977). Anlage zur langzeit-inhalation mit zigarettenrauch fur kleine labortiere. Medizin. Technik 97, 5–6.

Reznik, G., and Marquard, G. (1980). Effect of cigarette smoke inhalation during pregnancy in Sprague-Dawley rats. J. Environ. Pathol. Toxicol. 4, 141–152.[ISI][Medline]

Roeleveld, N., Zielhuis, G. A., and Gabreels, F. (1993). Mental retardation and parental occupation: A study on the applicability of job exposure matrices. Br. J. Ind. Med. 50, 945–954.[ISI][Medline]

Royal College of Physicians (1992). Smoking and the Young: A Report of the Working Party of the Royal College of Physicians. Royal College of Physicians, London.

Russell, C. S., Taylor, R., and Law, C. E. (1968). Smoking in pregnancy, maternal blood pressure, pregnancy outcome, baby weight and growth, and other related factors. A prospective study. Br. J. Prev. Soc. Med. 22, 119–126.[ISI][Medline]

Schoeneck, F. J. (1941). Cigarette smoking in pregnancy. NY Med. J. 410, 1945–1948.

Seller, M. J., and Bnait, K. S. (1995). Effects of tobacco smoke inhalation on the developing mouse embryo and fetus. Reprod. Toxicol. 9, 449–459.[CrossRef][ISI][Medline]

U.S. Department of Health and Human Services DHHS (1990). The Health Benefits of Smoking Cessation: A Report of the Surgeon General. U.S. Department of Health and Human Services, Centers for Disease Control and Prevention, Rockville, MD.

U.S. Department of Health and Human Services DHHS (1988). The Health Consequences of Smoking. Nicotine Addiction: A Report of the Surgeon General. U.S. Department of Health and Human Services, Centers for Disease Control and Prevention, Rockville, MD.

U.S. Department of Health and Human Services DHHS (1979). Smoking and Health: A Report of the Surgeon General. U.S. Department of Health and Human Services, Centers for Disease Control and Prevention, Rockville, MD.

U.S. Department of Health and Human Services DHHS (2001). Women and Smoking: A Report of the Surgeon General. U.S. Department of Health and Human Services, Centers for Disease Control and Prevention, Rockville, MD.

U.S. Food and Drug Administration FDA (2000). Toxicological Principles for the Safety of Food Ingredients. U.S. Food and Drug Administration, Center for Food Safety and Nutrition, Office of Premarket Approval, http://vm.cfsan.fda.gov/~redbook/red-toca.html, Washington, DC.

Vanscheeuwijck, P. M., Teredesai, A., Terpstra, P. M., Verbeeck, J., Kuhl, P., Gerstenberg, B., Gebel, S., and Carmines, E. L. (2002). Evaluation of the potential effects of ingredients added to cigarettes. Part 4: subchronic inhalation toxicity. Food Chem. Toxicol. 40, 113–131.[CrossRef][ISI][Medline]

VanVunakis, H., Gijka, H., and Langone, J. J. (1987). Environmental Carcinogen. Methods of Analysis and Exposure Measurement. IARC Scientific Publications, Lyon, France.

Wagner, B., Lazar, P., and Chouroulinkov, I. (1972). The effects of cigarette smoke inhalation upon mice during pregnancy. Rev. Eur. Etud. Clin. Biol. 17, 943–948.[ISI][Medline]

Weitzman, M., Byrd, R. S., Aligne, C. A., and Moss, M. (2002). The effects of tobacco exposure on children’s behavioral and cognitive functioning: Implications for clinical and public health policy and future research. Neurotoxicol. Teratol. 24, 397–406.[CrossRef][ISI][Medline]

World Health Organization WHO (2003). IPCS: Environmental Health Criteria Series. Carbon Monoxide. World Health Organization, Geneva.

Wingerd, J., and Schoen, E. J. (1974). Factors influencing length at birth and height at five years. Pediatrics 53, 737–741.[Abstract]

Young, J. T. (1981). Histopathologic examination of the rat nasal cavity. Fundam. Appl. Toxicol. 1, 309–312.[Medline]





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