In Utero Exposure to 1R4F Reference Cigarette Smoke: Evaluation of Developmental Toxicity

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

* Philip Morris USA Research Center, 4201 Commerce Road, Richmond, Virginia 23261, and {dagger} IIT Research Institute, 10 W. 35th Street, Chicago, Illinois 60616

Received February 6, 2003; accepted May 14, 2003


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The potential developmental effects of 1R4F reference cigarette smoke were examined using Sprague-Dawley rats exposed for 2 h/day, 7 days/week, by nose-only inhalation at target mainstream smoke concentrations of 150, 300, and 600 mg/m3 total particulate matter (TPM). Males were exposed 4 weeks prior to and during mating, with females exposed 2 weeks prior to mating and during mating, and through gestation day (GD) 20. Sham controls received filtered air to simulate nose-only exposure, while cage controls were maintained untreated. Smoke exposure was confirmed through biomarker evaluation (parental: carboxyhemoglobin, nicotine, and cotinine; fetal: nicotine and cotinine). Characteristic cigarette smoke-related histopathologic changes including nasal epithelial hyperplasia and squamous metaplasia and pigmented macrophages in the lung were observed in all exposed parental groups. Maternal toxicity during gestation was indicated at smoke concentrations of 300 and 600 mg TPM/m3, where corrected total body weight gain was significantly (p <= 0.05) decreased compared to sham controls. Food consumption was unaffected. Mating performance was unaffected by exposure: 71–75% pregnant in sham or cage control groups compared to 66–76% pregnant in smoke-exposed groups. Nicotine and cotinine were identified in the blood of fetuses from smoke-exposed dams on GD 20. Resorption rates, litter size, and sex ratio were equivalent among the groups. Fetal body weights at GD 20 were significantly reduced in the group exposed to 300 or 600 mg TPM/m3 compared to sham exposed controls. Developmental abnormalities were rare and sporadic in nature and the absence of a clear smoke concentration relationship suggested they were not related to cigarette smoke inhalation. Skeletal examinations revealed delayed ossification (supra occipital and sternebrae) in fetuses from dams exposed to 300 or 600 mg TPM/m3 smoke. High concentrations of 1R4F cigarette smoke were not teratogenic. The methodology used for this study was able to detect a decrease in fetal birthweight and this approach may be a useful tool for cigarette evaluation.

Key Words: 1R4F cigarette smoke; nose-only inhalation; fetal weight; Sprague-Dawley; in utero; developmental toxicity.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The 1964 Surgeon General’s report stated that: "Women who smoke cigarettes during pregnancy tend to have babies of lower birthweight" (U.S. Public Health Service, 1964Go). Low birthweight has been extensively investigated since 1964, and it is now clear that there is a direct relationship between the number of cigarettes smoked, the carbon monoxide level, or the tar yield of the cigarettes and the relative risk for low birthweight. However, despite numerous investigations, the mechanism for the effects remains elusive. In a later Surgeon General’s report entitled "Women and Smoking," it was further concluded that there was an increased risk of stillbirths among women who smoked during pregnancy (U.S. Public Health Service, 2001Go). Most epidemiology studies have found no association between cigarette smoking and the overall risk of birth defects (U.S. Public Health Service, 2001Go).

Laboratory animal studies have produced mixed results concerning an association between cigarette smoke and fetal effects. In an early study, Essenberg et al. (1940)Go used a whole body apparatus to expose rats from the time of mating through weaning to the smoke of about a pack of cigarettes a day. The smoke was crudely generated by pumping a rubber bulb, which drew smoke into a 6-l exposure jar. Two-thirds of all of the young were underweight at birth. In another whole body inhalation study, rats were exposed to the smoke of 30 cigarettes over 7–11 min for up to four smoking cycles per day (Reznik and Marguard, 1980Go). A reduction in birthweight was observed. Reckzeh et al. (1975)Go used nose-only exposure to study the effects of diluted mainstream smoke from 30 cigarettes for 9–10 min twice per day. One group of female rats was exposed after mating, with a second female group exposed before and after mating and males exposed before mating. While maternal body weight gain was reduced by smoke exposure compared to controls, no significant exposure effects were found for litter weights, litter sizes, length of fetuses, number of implantation sites, or the incidence of resorptions. No malformations were found, and there was no effect on the offspring of the males exposed to smoke before mating. Bertolini et al. (1982)Go studied the reproductive effects of unknown commercial cigarettes in rats by using whole body exposure to smoke for 15 min/day during gestation. The offspring were allowed to deliver normally and were followed for 60 days. Carboxyhemoglobin (COHb) levels in similarly exposed rats reached 16.25% at the end of the exposure period. Dam weight gain was significantly reduced, but there was no effect on pup weight or any other reproductive parameter and no gross malformations were observed.

Using repetitive 8-min exposures in mice and variable exposure duration during discrete periods of gestation, Wagner et al. (1972)Go found that while maternal body weight gain was significantly reduced during gestation there was no effect on fetal weights. In another study in mice, after nose-only exposure to the smoke of six coded branded cigarettes for 10 min per day, a reduction in fetal body weight and delayed development as indicated by a reduction in the number of skeletal ossification centers was observed (Seller and Bnait, 1995Go). Mice were also used in another whole-body exposure using the smoke of one and one-half University of Kentucky reference cigarettes (specific type unknown) from gestation days (GD) 6–17 (Peterson et al., 1981Go). There was no effect on fetal mortality, weight, or length. No smoke related malformations were observed, nor was there any adverse effect on fetal weight. Magers et al. (1995)Go exposed hamsters to the smoke of one or two University of Kentucky 2R1 reference cigarettes twice per day via whole body inhalation for 30 days prior to mating. On GD 7, the reproductive organs were removed and evaluated. The authors concluded that smoke decreased the number of and vascular area of corpora lutea, induced blebbing of the ciliated/secretory cells in the ampulla, decreased stretched uterine length, and increased the number of crowded implantation sites.

In a unique rabbit study, the smoke of one cigarette was blown into the nostril of a doe each day for 4 weeks prior to mating, during pregnancy, and lactation (Schoeneck, 1941Go). The does were "smoked" daily throughout pregnancy and lactation. The average birthweight of the offspring was reduced by 17%, and there was an increase in stillbirths and death rate of the offspring. It was concluded that under the conditions of this test, cigarette smoke produced a deleterious effect on the offspring.

While human data show a consistent association between maternal smoking and risk for low birthweight and small for gestational age births (Royal College of Physicians, 1992Go; U.S. Public Health Service, 2001Go), the information presently available from animal studies does not provide sufficient exposure characterization to further investigate the association between smoke exposure and potential fetal effects. Specifics lacking include a description of the cigarette tested, smoke yield of the cigarette, chemical characterization of the smoke atmosphere, and biomarker evaluation as a measure of smoke absorption. Additionally, in most cases it is not evident that a maximum tolerated dose was obtained. This study was intended to provide a design where products or tobacco ingredients may be evaluated for potential maternal, paternal, and fetal effects of cigarette smoke under well-controlled and defined exposure conditions. The goal was to investigate whole cigarette smoke rather than individual smoke components such as nicotine, carbon monoxide, and aldehydes, although these parameters were used for smoke exposure characterization. Using a standard reference cigarette type that represents a medium tar yield commercial cigarette, male and female rats were exposed according to International Congress on Harmonization reproductive toxicity study guidelines prior to mating and during the gestation period to increase the probability of producing an effect (ICH, 1993Go). Smoke exposures were conducted by 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, and respiratory tract and placental histopathology was performed on the dams to demonstrate the degree of toxicity.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Study design.
Table 1Go summarizes the study design. Males were exposed by nose-only inhalation to diluted mainstream cigarette smoke at target total particulate matter (TPM) concentrations of 150, 300, or 600 mg TPM/m3 for 2 h (1 h smoke/30 min air/1 h smoke) per day for four weeks prior to and during mating. Females were exposed under the same conditions for 2 weeks prior to mating, during mating, and through GD 20 when cesarean sections were performed and the fetuses evaluated. Rats in the sham control group were exposed to filtered air, while cage controls remained in their cages and had access to food and water. Separate groups of sentinel animals undergoing exposure and mating were included in each group and used for smoke biomarker determination. Exposure concentrations and time periods were based on previous experience with nose-only cigarette smoke exposures.


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TABLE 1 Study Design Used to Examine the Effect of Cigarette Smoke on Fetal 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 standard 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 (Reininghaus and Hackenberg, 1977Go, modified, type: SM85). 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 per 2 seconds, 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, and were not considered to substantially affect the smoke composition. 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 1:60 to 1:170 (volume:volume) 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 9–10 weeks, while female rats ranged from 7–8 weeks in age upon receipt. Male and female rats were double-housed (same sex) upon arrival and were held in quarantine for approximately two weeks prior to exposure initiation, during which time they were observed at least once daily for mortality and moribundity. 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.

Exposure.
The rats were nose-only exposed in Canon-style exposure chambers using plastic restraint tubes matching their body size (CH Technologies, Westwood, NJ). The daily routine was a 1-h exposure, followed by one-half h smoke free period, followed by another 1-h exposure. Animals were acclimated to the cigarette smoke and animal holders during the first three days of the pre-mating exposure period using a reduced schedule (30 min, 1 h, and 1–1/2 h on exposure days 1, 2, and 3, respectively). The first full 2-h exposure began on day 4. The position of the rats on the nose-only exposure system was changed on a weekly basis according to a rotation scheme.

Analytical characterization of the test atmospheres.
TPM concentrations were determined at least twice daily (once per h) in each chamber using a gravimetric filter-collection method. The sampling train consisted of a pre-weighed filter connected to a constant flow vacuum pump. Smoke particles were collected on pre-weighed 47-mm fiberglass filter disks (Pall Corporation, 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 (Etrelut® NT, Merck, Darmstadt, Germany) and eluted 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, Willmington, DE) equipped with nitrogen phosphorus detector and, HP-5 column 30 m long x 0.32 mm diameter. CO concentrations were monitored in each exposure chamber continuously with a dedicated infrared gas analyzer (California Analytical Instruments, Model ZRH-1) 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 determine smoke exposure, biomonitoring was conducted on the sentinel animals and their pups. Within 5 min after being removed from the exposure chamber (to prevent dissociation of COHb) male and female sentinels were anesthetized with 70% CO2/30% air, and bled from the retro-orbital sinus for determination of COHb, nicotine, and cotinine. Blood from fetal sentinels was collected following decapitation and pooled on a litter basis for nicotine and cotinine determination on GD 20. COHb was analyzed immediately after collection using an IL-482 CO-Oximeter, while the serum for nicotine and cotinine determination was stored at -70°C until all samples could be analyzed by radioimmunoassay methods as described by VanVunakis et al. (1987)Go.

In-life and postmortem parental evaluation.
The rats were observed twice daily during the exposure period for mortality and morbidity. Animals on study received clinical observations concurrent with body weight and food consumption measurements (weekly until sacrifice for male rats and on GD 0, 3, 6, 9, 12, 15, 18, and 20 for female rats) and prior to scheduled termination. 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 four consecutive days to ensure cyclicity of female rats 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. The day a sperm-positive smear was collected was considered GD 0. Dams were euthanized by CO2 asphyxiation on GD 20, and cesarean sections conducted. The uterus was removed from the dam, trimmed free of excess adherent tissue and weighed, with the ovaries, prior to removal of the fetuses. After weighing, the corpora lutea were counted and recorded for the left and right ovaries. Each uterine horn was inspected for resorption and fetal deaths. Resorptions were counted and classified as an early resorption (placenta only), late resorption (placenta and attached fetal parts), early death (fetus weighing less than 0.8 g), or late death (fetus weighing more than 0.8 g). Unmated and nonpregnant females were euthanized by CO2 asphyxiation at the end of the mating period without necropsy.

Male rats were euthanized by CO2 asphyxiation and necropsied approximately one week after mating. The left testis, left epididymis, prostate (both lobes), and seminal vesicles were trimmed of excess fat, collected and weighed from the first 10 males of each group at terminal necropsy, placed in 10% neutral buffered formalin or Bouin’s solution (testes), stained with hematoxylin and eosin, and examined for histopathological changes. The right testis, right epididymis, right cauda, parenchyma, and vas deferens were collected from 15 males/group, trimmed of excess fat and weighed during reproductive function assessment. Sperm count, motility, and morphology were determined.

The larynx (three sections: base of epiglotis, arytenoid projections, and vocal folds), nose (four sections: according to Young, 1981Go), lung (longitudinal and cross sections: according to Lamb and Reid, 1969Go, and Dungworth et al., 1976Go), trachea (longitudinal sections including bronchial bifurcation and bronchial lymph nodes) and placenta were collected for histopathology from 7–10 randomly selected females per group on GD 20; the lungs, trachea, and larynx were weighed as a unit. Lungs were inflated via the trachea and nasal passages were flushed with formalin to insure 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 levels 1 and 2 were stained with periodic acid-schiff stain/alcian blue for goblet cell enumeration.

Fetal evaluation.
Each live fetus was counted and received a gross external morphologic examination. Live fetuses from each litter were weighed and randomized to receive either cephalic and visceral evaluation (~50%) or skeletal evaluation (~50%). Visceral examinations were performed by a modified method of Staples (Staples, 1974Go). Cephalic evaluations were conducted using a modified Wilson’s Razor Blade Sectioning Technique (Wilson, 1965Go). All fetuses designated for skeletal examinations were eviscerated, skinned, fixed in ethyl alcohol, stained with alcian blue and alizarin red.

Statistical procedures.
Means and SDs 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 were analyzed by ANOVA followed, where appropriate, by Dunnett’s test using SYSTAT (SPSS, Inc., Chicago, IL, version 5.0 and version 8.0). Comparison of litter (fetal) body weight data was analyzed by an ANOVA; the litter was the unit of observation. For viability data, a one-factor (i.e., treatment group) ANOVA was used for mean total males and females per litter, mean corpora lutea, mean total, live and non-live (resorptions and deaths) implants, mean percent live and non-live implants, and mean percent preimplantation loss. Cephalic and skeletal data were analyzed [when the incidence (absolute number of fetuses affected) in the cigarette smoke-treated groups was higher (i.e., greater than 3) than controls] by Chi-square followed, where appropriate, by Fisher Exact test. In the presence of significant main effects, all post hoc comparisons between the 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 152, 292, and 599 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 2Go shows the particle size distribution, nicotine, and CO concentrations. Measurement 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 Smoke Atmosphere TPM (mg TPM/m3), Nicotine (mg/m3), Particle Size Distribution, and CO (ppm) Concentrations
 
Biomonitoring
Blood was collected from the sentinel animals and from selected pups at cesarean section to assess exposure (Table 3Go). Exposure to smoke resulted in a concentration-related increase in serum nicotine and cotinine in the parental animals. There did not appear to be any bioaccumulation as the concentrations did not increase with time. This is to be expected, since the elimination half-life of nicotine in rats is 1.3 h (Kyerematen et al., 1988Go).


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TABLE 3 Biomonitoring for Nicotine, Cotinine, and COHb in Parental Rats and Fetuses
 
COHb was increased in a smoke concentration-related manner, with all treated parental animals reaching a maximum of about 35% in the 600 mg TPM/m3 group. There did not appear to be any bioaccumulation of COHb since the levels did not increase with exposure duration. This is also not unexpected since the elimination half-life of COHb in rats is about 42 min at the 600 ppm exposure level (Ayres et al., 1989Go).

Blood collected after decapitation of selected sentinel pups at cesarean section was pooled on a litter basis for analysis. While this represents an inexact collection technique, it is clear from the nicotine and cotinine results (Table 3Go) that the pups were exposed in utero to some of the constituents of smoke. Because of the time delay necessary for cesarean sectioning and the elimination half-life of COHb, it was not considered feasible to meaningfully measure fetal COHb levels.

Paternal Effects
Males were exposed to smoke for four weeks prior to and during mating. There were no deaths associated with exposure; however, occasional diarrhea, salivation, and red material around the eyes and nose were noted in the nose-only exposed animals (sham and smoke-exposed), while cage control animals generally did not exhibit these signs. The signs were therefore considered indicative of nose-only tubing stress unrelated to smoke exposure. Body weight gain was reduced in the smoke-exposed animals when compared to the sham control (Table 4Go) and the sham control was reduced compared to the cage control. This depression in body weight in the smoke-exposed animals was mostly due to a reduction in body weight gain during the first week of exposure (data not shown). Food consumption was not affected by exposure (Table 4Go).


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TABLE 4 Survival, Body Weight, Food Consumption, Reproductive Function, and Relative Organ Weights of Male Rats Exposed to Cigarette Smoke
 
At the end of the mating period, the males were terminated and reproductive function and organ weights determined. There were no effects on sperm count, motility, nor morphology, and sexual organ weights were unaffected (Table 4Go). There was a statistically significant decrease in the relative seminal vesicle weight of the low and mid-smoke exposure groups. Small seminal vesicles were observed at necropsy in 3/10, 1/10, and 1/10 males from the 150, 300, and 600 mg TPM/m3 smoke exposure groups. The sham and cage control group animals exhibited normal seminal vesicles. A small prostate was observed in one 300 mg TPM/m3 male and a white focus on the prostate was observed in one 150 mg TPM/m3 male. A small epididymis was observed in one 300 mg TPM/m3 male. Red pigmentation of the fat adjacent to the left epididymis was observed in a 300 mg TPM/m3 male. No significant histopathological changes were found in the epididymis, testes, seminal vesicle, or prostate. A reduced size of the accessory sex organs was observed in smoke-exposed groups, but the findings were not consistent within groups and were not influenced by smoke exposure concentration. Additionally, several of the seminal vesicle findings were recorded at trimming of tissues and may have been influenced by loss of contents at necropsy. Since the effects were sporadic and not correlated with histopathology, they were considered to be not treatment related.

Maternal Effects
Females were exposed to smoke for two weeks prior to mating and during gestation. No animals died during the study, but one female in the 150 mg TPM/m3 group was terminated in a moribund state (causes unrelated to exposure) prior to mating. The clinical observations in the females were similar to those seen in males (diarrhea, salivation, and red material around the eyes and nose-only exposed animals). All of the females gained approximately the same amount of weight and consumed the same amount of food during the pre-mating period (Table 5Go and Fig. 1Go). There was a clear difference in the body weight gain between the restrained rats and the untreated cage controls during gestation. The 150 and 300 mg TPM/m3 treated rats appeared to gain about the same amount of weight during the gestation period. The 600 mg TPM/m3 dams gained less weight between GD 15 to 20, with statistical significance being reach at day 20. Food consumption was not affected (Table 5Go). Female mating and fertility indices were not affected by exposure to smoke (Table 5Go). The mean uterine weight was significantly reduced in the 600 mg TPM/m3 (Fig. 2Go). When the GD 20 terminal body weight was corrected for the uterine weight, there was a significant difference in the GD 20 body weight and body weight gain in the 300 and 600 mg TPM/m3 dams. Relative lung weight was not affected by smoke exposure.


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TABLE 5 Survival, Pregnancy Status, Food Consumption, and Body Weight Data of Female Rats before and during Gestation
 


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FIG. 1. Pre-mating and gestational body weights of female rats exposed to 1R4F cigarette smoke. Data are expressed as the mean ± SD of 34–35 rats/group during pre-mating and 23–25 rats/group during gestation. *Significantly different from the sham control group, p <= 0.05.

 


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FIG. 2. Maternal body weights (BW) and body weight gains (BWG) corrected for uterine weight. Data are expressed as the mean ± SD of 23–25 rats/group. *Significantly different from the sham control group, p <= 0.05.

 
Examination of the respiratory tract tissues from the maternal rats indicated the presence of tissue changes consistent with those noted in previous nose-only inhalation studies with 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 shown in Table 6Go). The incidence and severity of the changes indicated a clear smoke-related response, with resolution of the smoke exposure concentration evident in most cases. Additionally, in the nose, an anterior to posterior progression of lesion incidence and severity was noted with increasing smoke concentration.


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TABLE 6 Summary of the Major Histopathological Changes Observed in Female Rats
 
Gestational data, including corpora lutea, resorptions, implantation sites, live and dead fetuses, and mortality are presented in Table 7Go. Smoke inhalation did not affect these parameters. In summary, while smoke concentration related respiratory tract histopathologic changes were noted in all smoke-exposed groups, corrected body weight and body weight gains were reduced only after exposure to 300 and 600 mg TPM/m3 smoke. The presence of reduced body weight gain in addition to the histopathology of the respiratory tract was considered to be evidence of maternal toxicity.


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TABLE 7 Gestational Data from Rats Exposed to Cigarette Smoke
 
Fetal Effects
Fetuses were removed by cesarean section on GD 20 and evaluated for skeletal and visceral malformations and variations. There was no effect on the ratio of males to females in each litter (Table 7Go). Maternal exposure to cigarette smoke affected fetal body weight. As shown in Figure 3Go, the mean male and female fetal body weight was significantly reduced in the 600 mg TPM/m3 group, while the combined fetal body weight reduction reached statistical significance at 300 and 600 mg TPM/m3. Histopathological evaluation of the placenta did not reveal any changes that could be attributable to treatment (Table 7Go). Comparison of the cage and sham control group results indicated that restraint of the animals in tubes reduced (20%) the dam corrected body weight gain (Table 5Go) but did not adversely affect fetal body weight (Table 7Go). Restraint of the animals had no other effects on pup development.



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FIG. 3. Fetal body weights measured at the time of Cesearean sectioning. Data are expressed as the mean ± SD of 23–25 litters/group. *Significantly different from the sham control group, p <= 0.05.

 
Table 8Go presents the malformations observed in the external, visceral, and skeletal examinations of the fetuses. Only three external and three visceral malformations were observed in this study. No skeletal malformations were found. The very few malformations observed were limited to the three smoke-exposed groups, with no malformations noted in either the sham or cage control groups. However, the malformations were not considered to be smoke-related, since the overall incidences of the individual or total malformations were not statistically significantly different from either control group, there was no smoke concentration-related increase in the incidence of any specific individual or total malformation, and the type of malformations are known to occur in this strain of rat.


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TABLE 8 Malformations in Rat Fetuses following Parental Inhalation Exposure to Cigarette Smoke
 
Table 9Go presents the most frequent anatomical variations observed during the external, visceral, and skeletal examinations of the fetuses. The limited number of external and visceral variations noted in the study did not indicate a smoke-related effect. However, there was a clear smoke concentration-dependent response in the number of skeletal variations noted, with exposure to 300 or 600 mg TPM/m3 smoke producing a significant increases in the number of incomplete or unossifed bones compared to the sham control group.


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TABLE 9 Variations in Rat Fetuses following Parental Inhalation Exposure to Cigarette Smoke
 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
According to data reported by the Centers for Disease Control, in 1996, approximately 13.6% of women smoked during their pregnancy. Although this frequency dropped to 12.9% by 1998, the number of women who smoke during pregnancy remains relatively high (Centers for Disease Control, 2000Go). Additionally, among teenagers smoking during pregnancy increased from 18.8% in 1997 to 19.2% in 1998.

Epidemiological investigations have established a relatively consistent association between maternal smoking and the risk of low birthweight and small for gestational age births in humans; however, it is not totally clear from these studies what impact other environmental factors may play in the observed associations. There does appear to be some agreement however, that whatever effect maternal smoking exerts on fetal development the influence occurs during the later stages of pregnancy. Smoking cessation early in the pregnancy appears to result in virtually complete disappearance of a birthweight effect (U.S. Public Health Service, 2001Go).

The potential for congenital abnormalities resulting from maternal smoking has been the subject of several epidemiological investigations. Malloy et al. (1989)Go found no association of maternal smoking during pregnancy with the occurrence of any congenital malformation or with specific types of malformations in a study of 288,067 live births in Missouri, of which 10,233 had one or more congenital malformation. Werler et al. (1990)Go discussed a possible association between maternal smoking during pregnancy and congenital abnormalities and concluded that the data are conflicting and that the strongest evidence is for oral clefts, although even this is uncertain. Beaty et al. (1997)Go reported an association of maternal smoking with oral clefts in a case-control study which also looked for interaction with a transforming growth factor alpha2 (TGFA2) polymorphism which was suggested as being linked to such disorders. They were unable to confirm the TGFA2 association, nor to find an interaction between the TGFA2 marker and smoking. A case-control study in Finland reported on the potential association of maternal infections, alcohol consumption, and smoking during pregnancy with congenital reduction limb defects (Aro, 1983Go). A weak association of isolated reduction limb defects was reported for smoking, after adjusting for alcohol consumption and maternal age. A similarly designed matched-pair case-control study in Hungary claimed to identify a significantly higher rate of terminal transverse limb defects associated with smoking, although the odds ratio was not significant for smoking in the first trimester (Czeizel et al., 1994Go). Evaluation of the Swedish health registries of 1,109,299 infants born between 1983 and 1993 with known maternal smoking exposure during pregnancy indicated an odds ratio of 1.26 (95% CI 1.06–1.50) for limb reduction (Kallen, 1997aGo). The authors of these studies note that there may be other factors that could bias this result, which were not considered in their analysis. Other epidemiological studies have been conducted on abnormalities including gastroschisis (Haddow et al., 1993Go) and urinary tract infection (Kallen, 1997bGo). Additional risks associated with maternal smoking may include pre-term delivery, perinatal mortality, and spontaneous abortions (Institute of Medicine, 2001Go).

Studies have been conducted using various laboratory animal models to investigate the association between maternal smoke exposure and fetal developmental effects. For the most part, these studies do not capture the smoke exposure conditions in sufficient detail to provide a useful or consistent laboratory model for the study of maternal smoking effects. Using a well-characterized cigarette smoke delivery system and measured biomarkers of exposure, this study investigated the potential for 1R4F cigarette smoke to adversely affect fetal weight and produce congenital abnormalities.

Ideally, exposures in animal models should mimic human exposure conditions. Unfortunately, this is virtually an impossible task in the case of nose-only exposures with pregnant animals. Humans potentially smoke intermittently over an entire day and may experience longer more prolonged expose to carbon monoxide and nicotine than can be accomplished using the two 1-h/day nose-only regimen used in our study. COHb levels in smokers reportedly average 4%, with a usual 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). In humans, the elimination half-life of nicotine is around 2 h, with cotinine displaying a much slower elimination rate of 18–20 h (U.S. Public Health Service, 1988Go). The duration of exposure for published subchronic inhalation studies with smoke using nonpregnant rats ranges from 1 h per day, 5 days/week (Gaworski et al., 1998Go) to 6 h per day for 7 days/week (Vanscheeuwijck et al., 2002Go). Previous work has shown that the exposure-related respiratory tract tissue changes induced by cigarette smoke in rats roughly follows a "concentration x time" relationship (Kaegler et al., 2001Go). Subchronic inhalation studies performed at a smoke concentration of 150 mg TPM/m3 for 6 h per day is therefore equivalent to 450 mg TPM/m3 for 2 h per day. Even at this exposure level, smoke inhalation must be interrupted for 30 min after the first h to allow COHb to dissociate or risk potential CO related mortality. Varsho et al. (2000)Go demonstrated that pregnant rats could be restrained in nose-only exposure tubes up to 6 h without detrimental effects on embryo/fetal development. Because of the expected stress from confinement placed on pregnant rats by tubing constraints during a nose-only exposure and elevated levels of COHb resulting from exposure to the resulting carbon monoxide at high smoke concentrations, it was decided to limit the inhalation exposures to two 1-h periods each day.

Smoke atmosphere characterization indicated very good control of the selected target exposure concentrations. Coupled with the biomarker evidence collected from parental rats during the study, it was apparent that the animals received the intended graded concentration levels of smoke. Furthermore, microscopic evaluation of the selected respiratory tissues indicated changes in the epithelial lining that were consistent with those changes seen in other investigations of cigarette smoke (Vanscheeuwijck et al., 2002Go). Evaluation of blood indicated transfer of maternal nicotine to the fetus during gestation.

Maternal smoke exposure decreased body weight gain during gestation in the 600 mg TPM/m3 exposed animals. Additionally, the mean uterine weight was significantly reduced in the 600 mg TPM/m3 group. When the GD 20 terminal body weights were corrected for the uterine weight, there was a significant difference in the GD 20 body weight and body weight gain in dams from both the 300 and 600 mg TPM/m3 smoke exposure groups. Not unexpectedly, COHb levels reached 20 to 40% in the 300 and 600 mg TPM/m3 smoke exposure groups, respectively; placing considerable stress on the parental rat. Additionally, exposure to cigarette smoke produced an array of respiratory tract histopathological changes in the parental rats that were consistent with other studies of cigarette smoke. These findings provide evidence of maternal toxicity at 300 and 600 mg TPM/m3.

There are conflicting published data concerning a possible causal association between congenital malformations and cigarette smoking. The general consensus at present is that maternal smoking does not appear to be a major factor in the induction of congenital malformation (Schardein, 1985Go). No treatment-related malformations were observed in the present study, even at doses that were clearly maternally toxic. This finding is consistent with the other animal studies with cigarette smoke that have failed to produce clear treatment-related malformations. These results and the literature support the observation of a lack of smoking related induction of congenital malformations in humans.

The association of altered male fertility with cigarette smoking is unclear (Institute of Medicine, 2001Go). Studies have shown mixed results with respect to sperm and semen quality and have not supported detrimental effects on male fertility. Exposure to high concentrations of cigarette smoke in the present study did not produce any affect on male reproductive function or organ pathology. This lack of an effect is consistent with the findings of Reckzeh et al. (1975)Go who exposed paternal rats to the smoke of 30 cigarettes twice per day from 45 days prior to mating and found no smoke related effect on skeletal malformations or fertility rate.

In our study, the principal impact of maternal smoke exposure and associated stress on the developing fetus appeared to be limited to a decrease in fetal weight. Reduced ossification at several sites may or may not be a sign of delayed development. There were no effects on the number of corpora lutea, implantation sites, resorptions, or post implantation loss. Previous studies in rats, mice, and rabbits have generally failed to demonstrate any effects on reproductive function with the exception of reduced fetal weight. However, stillbirths in rabbits (Schoeneck, 1941Go), and reduction in the number of corpora lutea in hamsters (Magers et al., 1995Go) have been reported. While the exposure conditions and test cigarettes used in previous studies are not comparable to the current study, the low fetal weight effect remains a reasonably consistent finding. In all cases where there was a reduced maternal body weight gain, there was also reduced fetal weight. The present study also suggests a smoke dose-response relationship. Exposure to 150 mg TPM/m3 did not produce an effect on maternal body weight gain nor on fetal weights. Exposure at 300 and 600 mg TPM/m3 clearly produced these effects.

The specific mechanism for causes for reduced maternal bodyweight gain and reduced fetal weight are unknown. Nicotine is a major constituent of tobacco smoke, and when administered to pregnant mice as a pure material at high levels (25 mg/kg) produced both limb malformations and fetal lethality (Nishimura and Nakai, 1958Go). Lower levels (12 mg/kg) produced no deaths or deformities (Paulson et al., 1989Go). In rats, nicotine causes a decrease in embryo growth, delays implantation, retards parturition, reduces the number of litters and of total young born, and produces an increase in mortality of the young during the nursing period (Becker and King, 1966Go; Becker et al., 1968Go; Essenberg et al., 1940Go; Hammer and Mitchell, 1979Go; Haworth and Ford, 1972Go; Thienes, 1960Go). It has been long known that the pharmacology and toxicology of tobacco smoking and the pharmacology and toxicology of nicotine are not identical and are often not comparable (Silvette et al., 1962Go).

Haworth and Ford (1972)Go pair-fed dams the same amount of food as was consumed by smoke-exposed dams. Smoke exposure retarded the growth of the offspring, but fetal body weight was not affected by food restriction. Younoszai et al. (1969)Go performed a similar experiment with food restriction and cigarette smoke. In this study, food intake was correlated with fetal body weight. In the rats exposed to cigarette smoke, fetal weight was reduced more than expected from the reduction in maternal food intake. Food intake by the dams was unaffected in the present study and would therefore not appear to be a likely cause of reduced fetal weight. Since nicotine stimulates the release of epinephrine, which thereby causes vasoconstriction, chronic reduction in placental blood flow has been postulated as a potential mechanism for low fetal birthweight (Tachi and Aoyama, 1983Go). Trend and Bruce (1989)Go found that increased epinephrine levels do not adversely affect fetal development. Younoszai et al. (1969)Go exposed pregnant rats to the smoke of tobacco cigarettes, lettuce leaf cigarettes, or lettuce leaf cigarettes with nicotine added. The fetuses of all of the smoke-exposed animals were growth retarded. The cigarette smoke-exposed animals were more affected than the lettuce leaf or lettuce leaf and nicotine smoke-exposed animals. The authors concluded that since the all of the rats were exposed to the same amount of carbon monoxide and that the tobacco and lettuce leaf/nicotine smoke-exposed animals got the same amount of nicotine, CO and nicotine are unlikely to be directly responsible for the fetal growth retardation.

Placental complications of smoking are well known in humans (Institute of Medicine, 2001Go). While there is a positive association with placenta previa and placental abruption, the exact mechanism by which maternal smoking causes placental complications is unknown. Placentas of smokers frequently exhibit anatomical and histological changes that suggest hypoxia and underperfusion (Voigt et al., 1990Go). Studies in rats conducted by Barr and Brent (1970)Go and Bruce (1976)Go have shown no effect on fetal development when placental blood flow was altered by unilateral ligation of the uterine artery. These rodent studies suggest that placental vasoconstriction is not a principal cause of the low birthweight. Microscopic evaluation of the placenta of rats in the present study exposed to high concentrations of cigarette smoke did not reveal any adverse changes. Based on this histopathological evidence, it does not appear that a microscopically observable change in the placenta was responsible for the fetal body weight observed in the rats in this study.

Hypoxia due to the CO in smoke is a potential mechanism for low birthweight. CO is able to cross the placenta, and in humans the affinity of fetal hemoglobin for CO is greater than maternal (Hill et al., 1977Go). Astrup et al. (1972)Go have previously demonstrated a direct relationship between CO intoxication and lower birthweight in rabbits. Exposure of laboratory animals to CO has resulted in developmental toxicity in the rat, rabbit, guinea pig, pig, and monkey (Schardein and Keller, 1989Go). In a unique smoke-related investigation, Reznik and Marquard (1980)Go exposed pregnant rats to either whole smoke or the gas phase of smoke. Whole smoke produced a greater effect on fetal birthweight than did comparable dosing with the gas phase suggesting that CO alone is not the cause of low birthweight. Tachi and Aoyama (1983)Go exposed dams to cigarette smoke during the total gestation period and to six-day segments. A separate group of dams was exposed to CO at the same concentration as was found in the smoke. Cigarette smoke reduced fetal weight with the largest effect being produced during the last seven days of gestation. The CO exposed group experienced a reduced fetal weight also, but not to the extent of the smoke-exposed group. The authors concluded that CO exposure was a contributor to the effects of cigarette smoke, but not the sole factor responsible for the adverse effects.

Ultimately, it is possible that the effect of smoke on birthweight is not due to a single component of smoke but to the whole mixture. Cigarette smoke is reported to contain approximately 4000 chemicals (Dube and Green, 1982Go). Teratology studies have been performed on some of the more well-known chemicals including nicotine, carbon monoxide, benzo(a)pyrene, benzene, toluene, styrene, acrolein, and formaldehyde. None of these materials produce a pattern of fetal toxicity similar to cigarette smoke at the levels reported in smokers. Any cigarette smoke constituent studied in isolation may give results quite different from the effects of the mixture of whole smoke and it is critical that one consider smoke as a complex mixture that cannot be easily broken out into individual chemicals with unique effects. Not all women who smoke cigarettes during pregnancy have low birthweight infants. Wang et al. (2002)Go evaluated the metabolic gene polymorphisms in 741 mothers. Maternal CYP1A1 and GSTT1 genotypes modified the association between maternal cigarette smoking and infant birthweight, suggesting an interaction between metabolic genes and cigarette smoking. This work suggests that the relationship between smoking and low birthweight may be even more complex than just exposure to the complex mixture of chemicals in smoke.

In humans, the average birthweight is 2700 g. Infants born weighing less than 2500 g are considered to have low birthweight (Institute of Medicine, 2001Go). This represents a 7.4% decrease. Under the conditions of this study it was possible to detect at a statistically significant decrease in rat fetal weight at 6.6%. The methodologies used in this study may provide a useful model for studying potential smoke-related fetal effects.


    ACKNOWLEDGMENTS
 
The authors thank Drs. Richard Carchman, Hans-Juergen Haussmann, D. E. Rodwell, and C. E. Ulrich for their contributions to the preparation of the manuscript; Dr. R. L. Morrissey (Pathology Associates, Chicago, IL) and the staffs at Philip Morris USA and IIT Research Institute for their excellent technical assistance.

The authors acknowledge that they are employed by Philip Morris USA or are under contract to conduct research for Philip Morris USA, a cigarette company.


    NOTES
 
Portions of this work were presented at the 42nd Annual Meeting of the Teratology Society, June 23–26, 2002, Scottsdale, AZ.

1 To whom correspondence should be addressed. Fax: (804) 274-3055. E-mail: edward.l.carmines{at}pmusa.com. Back


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