Skip Navigation


Carcinogenesis Advance Access originally published online on February 12, 2006
Carcinogenesis 2006 27(6):1146-1152; doi:10.1093/carcin/bgi353
This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow All Versions of this Article:
27/6/1146    most recent
bgi353v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrow Search for citing articles in:
ISI Web of Science (4)
Right arrowRequest Permissions
Google Scholar
Right arrow Articles by Steinetz, B. G.
Right arrow Articles by Bosland, M. C.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Steinetz, B. G.
Right arrow Articles by Bosland, M. C.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?

© The Author 2006. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

The parity-related protection against breast cancer is compromised by cigarette smoke during rat pregnancy: observations on tumorigenesis and immunological defenses of the neonate

Bernard G. Steinetz *, Terry Gordon, Salamia Lasano, Lori Horton, Sheung Pui Ng, Judith T. Zelikoff, Arthur Nadas and Maarten C. Bosland

Department of Environmental Medicine, Department of Statistics and Department of Urology, New York University School of Medicine, Tuxedo, NY and New York, NY

* To whom correspondence should be addressed. Tel: +1 845 731 3517; Fax: +1 845 351 4510; Email: Steinetz{at}env.med.nyu.edu


   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 References
 
Early pregnancy is a powerful negative risk factor for breast cancer (BCa) in women. Pregnancy also protects rats against induction of BCa by carcinogens such as N-methyl-N-nitrosourea (MNU), making the parous rat a useful model for studying this phenomenon. Smoking during early pregnancy may lead to an increased risk of BCa in later life, possibly attributable to carcinogens in cigarette smoke (CS), or to reversal of the parity-related protection against BCa. To investigate these possibilities, 50-day-old timed first-pregnancy rats were exposed to standardized mainstream CS (particle concentration = 50 mg/m3) or to filtered air (FA) 4 h/day, Day 2–20 of gestation. Age-matched virgin rats were similarly exposed to CS or FA. At age 100 days, the CS or FA-exposed, parous and virgin rats were injected s.c. with MNU (50 mg/kg body wt), or with MNU vehicle. Mammary tumors (MTs) first appeared in virgin rats 9 weeks post-MNU injection. While no MTs were detected in FA-exposed parous rats until 18 weeks post-MNU, MTs appeared in the CS-exposed parous rats as early as 10 wks (P < 0.02). As no MTs developed in CS-exposed rats not injected with MNU, CS did not act as a direct mammary carcinogen. Serum prolactin concentration on Day 19 of pregnancy in CS-exposed dams was reduced by 50% compared with FA-exposed dams (P < 0.005). CS exposure during a pregnancy may thus ‘deprotect’ rats, enhancing their vulnerability to MNU-induced BCa. Prenatal CS exposure had no detectable effect on the immune responses of the pups examined at 3, 8 or 19 weeks of age. However, prolactin concentration in stomach contents (milk) of 3-day-old pups suckled by CS-exposed dams was decreased when compared with that of FA-exposed dams (P < 0.032). As milk-borne prolactin modulates development of the central nervous and immune systems of neonatal rats, CS exposure of the dams could adversely affect later maturation of these systems by reducing milk prolactin.

Abbreviations: BCa, breast cancer; CS, cigarette smoke; FA, filtered air; MNU, N-methyl-N-nitrosourea; MTs, mammary tumors; NK, natural killer; PCBS, phosphate-citrate-buffered saline; PGE2, prostaglandin E2; RIA, radioimmunoassays.


   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 References
 
Breast cancer (BCa) is a leading cause of cancer deaths in women (1). Risks include geographic area of residence, diet and lifestyle, genetic influences, age at menarche and menopause, and both endogenous and environmental estrogens (c.f. ref. 1 for review). However, in most cases there are no obvious causes of BCa (1), supporting the view that a variety of environmental carcinogens play a major role in initiation of the disease (2). There is one clearly negative risk factor for BCa: the incidence of breast tumors in women who carry a pregnancy to term prior to age 20 is only about one-third to one-half that of nulliparous women or women who have children in their later years (1,3,4). Thus, early pregnancy and childbirth may confer a lifelong refractoriness to BCa, but the protective action of a successful pregnancy is lost after the early reproductive years. The mechanism(s) responsible for these phenomena remain a mystery, although they have been widely studied in both women and laboratory animals (1,3,4).

The powerful protective effects of early pregnancy may be compromised by environmental factors as suggested by the observation in a case–control study that smoking during a first pregnancy was associated with a 3–4-fold increase in BCa risk (5). Reports of effects of cigarette smoking on mammary cancer incidence in women are inconsistent. Numerous studies have failed to find a strong association between smoking and BCa in women with no familial history of the disease. Although cigarette smoking may increase BCa incidence in women with high familial risk (6), smoking may actually be a strong negative risk factor in BRCA1 and BRCA2 carriers (7). These findings present a challenge to researchers. Thus, cigarette smoking could either act directly to initiate tumorigenesis in the young, undifferentiated mammary gland, or perhaps act indirectly to prevent or reverse the factors involved in the protective action of parity against BCa. Elucidation of the mechanisms whereby cigarette smoking ‘deprotects’ primiparous women could provide new and important clues to the understanding of the nature of the protective action of parity against BCa and might identify a previously unrecognized property of tobacco smoke.

To our knowledge, the effect of cigarette smoking during a first pregnancy on parity-induced mammary cancer resistance has not been studied previously in animal models. N-methyl-N-nitrosourea (MNU) is a DNA-methylating agent that induces estrogen-dependent mammary carcinomas in a high proportion of Sprague–Dawley rats in 2–6 months (4,8–10). A prior pregnancy or even a pregnancy occurring soon after MNU injection protects Sprague–Dawley rats against mammary tumor development (8–10). The latter observation suggests that pre-neoplastic cells present prior to pregnancy are either destroyed or altered during pregnancy by hormone-induced differentiation of the glands (11). Thus, pregnancy may somehow prevent not only initiation of BCa but also tumor progression. Because the Sprague–Dawley rat is known to be sensitive to many of the carcinogens (e.g., benzo[a]pyrene, nitromethane and benzene) present in tobacco smoke (12), it offers a suitable animal model for examining the effects of cigarette smoking on the protective action of parity against MNU-induced mammary cancer.

It has been well documented in women and in rodent models that cigarette smoking during pregnancy can exert harmful effects on the unborn (c.f. ref. 13 for review). Some of these untoward effects relate to suppression of the immune system, which in turn can lead to an increased sensitivity to toxic, immunomodulatory and carcinogenic substances (14–16). Accordingly, in the present study, several parameters of the immune systems of the pups were measured at various time intervals after birth following in utero exposure to cigarette smoke (CS) or filtered air (FA). It was of further interest to investigate the possible effects of CS exposure on the transmission of milk-borne hormonal growth factors from dam to pup during lactation, as some of these are known to modulate development of the immune system in rats (17,18).

The present investigation was undertaken specifically to test the hypotheses that cigarette smoking negates the protective action of parity against mammary cancer and may also have deleterious effects on the immune systems of the neonates.


   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 References
 
These experiments were approved by the NYU School of Medicine Institutional Animal Care and Use Committee (IACUC).

Experimental protocol and animals
Seventy timed pregnant (mated when 50 ± 2 days old) and 70 age-matched virgin female Sprague–Dawley rats were purchased from Charles River Breeding Laboratories (Wilmington, MA) and shipped to the NYU Institute of Environmental Medicine on the second (±1) day of gestation for the mated rats. The rats were individually housed on corncob bedding in solid bottom plastic cages and fed Purina Rat Chow and water ad libitum. The light–dark schedule was as follows: lights on, 6 a.m.–8 p.m.; lights out, 8 p.m.–6 a.m. The rats were then divided into the experimental groups indicated in Table I:


View this table:
[in this window]
[in a new window]
  		Table I. Treatment groups

 
Virgin rats (Groups 1 and 2 and Control groups C1 and C2)
Groups 1 (25 rats) and C1 (10 rats) were placed in a whole-body exposure chamber and exposed only to FA for 4 h/day for 19 days (age 52–70 days). Groups 2 (25 rats) and C2 (10 rats) were placed in a similar chamber and exposed to mainstream CS for 4 h/day for 19 days (ages 52–70 days). Groups 1 and 2 were then injected i.v. with 50 mg MNU in 6.67 ml phosphate-citrate-buffered saline (PCBS)/kg body wt on Day 100 (±2 days), whereas Control Groups C1 and C2 received only the PCBS vehicle at age 100 ± 2 days.

Pregnant rats (Groups 3 and 4 and Control groups C3 and C4)
Groups 3 (25 rats) and C3 (10 rats) were exposed only to FA for 4 h/day for 19 days (Day 2–20 of pregnancy). Groups 4 (25 rats) and C4 (10 rats) were exposed to CS for 4 h/day for 19 days (Day 2–20 of pregnancy). Groups 3 and 4 were then injected i.v. with 50 mg MNU in 6.67 ml PCBS/kg body wt on Day 100 ( ± 2 days) as described above, whereas Control Groups C3 and C4 received only the PCBS vehicle on the same schedule.

In each of Groups 3, 4, C3 and C4, 1.5 ml blood samples were drawn from the tail vein under restraint on Day 8, 12 and 19 of gestation using an i.v. cannula, and the serum was harvested and frozen for hormone assays.

At parturition, the size of each litter and birth weight of each pup was recorded. Three pups from each litter were euthanized (by CO2 inhalation) on Day 2 or 3 of lactation and the stomach contents (milk) were removed and frozen for hormone assays. The remaining pups were left with the dams for the remainder of the lactation period and then utilized as described in Table II.


View this table:
[in this window]
[in a new window]
  		Table II. Experiments on immune system of pups

 
Smoke generation and exposure of rats
Mainstream CS was generated with a cigarette-smoking machine (CH Technologies) at the NYU inhalation Core facility. The CS machine is housed in a 2 m3 generation box operated under slightly negative pressure (0.1 inches of water). Tobacco and Health Research Institute's 1R1 non-filtered cigarettes were used to generate the exposure atmosphere. The reference cigarettes were stored as suggested at 4°C and acclimated to room temperature and ~60% room humidity prior to burning. The number of cigarettes smoked at a time and the chamber flow rates were adjusted to achieve a target concentration of 50 (±10) mg/m3. Particle concentration was monitored continuously with a real-time aerosol monitor (RAM-1, MIE) and by taking gravimetric filter samples on an hourly basis. The smoke exposure of the rats (50 mg particulate matter/m3/4 h/day/200 g rat) was approximately equivalent to an individual smoking 2.7 packs of cigarettes per day.

Rat Groups 2, 4, C2 and C4 were exposed to smoke for 4 h/day for Days 2–20 of pregnancy [either during pregnancy (Groups 4 and C4) or corresponding virgin ages 52–70 days (Groups 2 and C2)]. Groups 1, 3, C1 and C3 were exposed only to FA as a control for effects of the daily confinement.

Injections of the carcinogen, MNU or saline vehicle
When the rats (virgin and parous) were 100 ± 2 days old and in proestrus/estrus (the time of maximum sensitivity of breast cells to MNU), each was sedated with a s.c. injection of Ketamine (Aveco, Fort Dodge, IO) and Xylazine (Mobay Shawnee, KS) in a mixture of 10 mg Ketamine/3 mg Xylazine/kg body wt. The sedated rats then received a single injection of either MNU (NCI Chemical Carcinogen Reference Standard Repository, Midwest Research Institute, Kansas City, MO) at a dose of 50 mg/6.67 ml/kg body wt in 0.1 M phosphate-citrate buffer (pH 4.8), diluted 1 : 14 with physiological saline (PCBS) (Groups 1–4) or 6.67 ml/kg body wt PCBS vehicle alone (Groups C1–C4) in the tail vein.

Mammary biopsies
Using Ketamine–Xylazine anesthesia (described above) a biopsy of a cervical mammary gland was taken from half of the rats in each group on Day 140 (40 ± 2 days after MNU injection) and fixed in 10% neutral buffered formalin (NBF). These samples were to be utilized for possible detection of CS and/or MNU-induced changes in estrogen and prostaglandin E2 (PGE2) receptors and p53 expression.

Histology and immunohistochemistry
At necropsy, tissues were fixed in 10% NBF embedded in paraffin, sectioned at 4 µm, and stained with H&E unless otherwise specified. Tumors were classified according to criteria established by the 1987 Hanover, Germany Consensus Conference on rat and human breast neoplasms as described by Russo et al. (4) Visualization of estrogen receptors, PGE2 receptors and p53 expression in the mammary biopsies was carried out by immunohistochemistry.

Hormone assays
Serum samples collected during exposure of pregnant rats to CS or FA were assayed for estradiol-17ß, progesterone, insulin, prolactin, relaxin and corticosterone to determine whether CS exposure had affected the concentrations of hormones known to play a role in mammary gland development. Because of the limited amount of serum available from each pregnant rat, the serum samples were diluted 1 : 5 with assay buffer to permit duplicate determinations. Specific radioimmunoassays (RIAs) were used to determine each of the following hormones:

  1. Serum estradiol-17ß: specific RIA using Kit no. 07-138015 supplied by ICN, Costa Mesa, CA.
  2. Serum progesterone: specific RIA using Kit no. 07-17015 supplied by ICN.
  3. Serum corticosterone: specific RIA using Kit no. 07-12013 supplied by ICN.
  4. Serum insulin concentrations were determined using a specific rat insulin RIA kit provided by LINCO, St Charles, MO.
  5. Serum prolactin was assayed using a specific rat prolactin RIA kit provided by Albert F. Parlow, Scientific Director of the NIDDK National Hormone and Peptide Program.
  6. Serum relaxin was measured by a specific rat relaxin RIA (19), the reagents for which were provided by the innovator, Dr O.D. Sherwood.

Experimental protocol for neonatal rat pups
Following parturition, the pups were utilized according to the experimental design outlined in Table II:

RIA of hormones in milk obtained from the stomachs of suckling pups
The stomach contents of each pup were diluted 1 : 1 with HOH and centrifuged to sediment the solids. Rat prolactin and rat relaxin in the aqueous phase of the pup stomach milk extracts were assayed using the specific RIAs described for serum hormones under the section Hormone assays. Dilution curves of the immunoactive relaxin and prolactin in milk were parallel to the rat standards in the respective RIAs.

Natural killer (NK) cell activity
NK cell activity was determined using a modification of the protocol initially described by Djeu (20). Briefly, recovered splenocytes were resuspended in RPMI 1640 media (40 ml) and incubated for 1 h at 37°C. Non-attached cells were recovered and resuspended in RPMI to a final concentration of 2 x 107 viable cells/ml. At the same time, 5 x 106 cultured YAC1 mouse lymphoma cells (ATCC, Manassas, VA) were incubated (at 37°C for 1 h) with 200 µCi 51Cr and then diluted (in RPMI) to a final concentration of 1 x 105 viable cells/ml. Splenocytes and 51Cr-labeled YAC1 cells (each at 0.1 ml) were then incubated (at 37°C in 5% CO2) for 4 h in individual wells of a round-bottom microtiter plate and cytotoxicity calculated as the % cytotoxicity = [(ER-SR)/(TR-SR)] x 100, where ER, SR and TR represent experimental release, spontaneous release and total releasable counts, respectively.

Lymphocyte proliferation assay
Single cell suspensions of splenic lymphocytes were resuspended to 2 x 106 cells/ml in RPMI 1640 (containing 5% FBS, 1% non-essential amino acids, 1% sodium pyruvate solution, 0.2% gentamycin, 1% penicillin-streptomycin and 0.1% L-glutamine). Aliquots (100 µl) of cells were added in triplicate to each well of a 96-welled microtiter plate along with an additional 100 µl of medium, Con A (concanavalin A, 20 µg/ml) or LPS (lipopolysaccharide, 20 µg/ml) to permit assessment of spontaneous and mitogen-induced proliferative activity. The plate was then incubated for 48 h (at 37°C with 5% CO2) before 20 µl of MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) was added to each well. Following another 4 h incubation, 50 µl of SDS (sodium dodecyl sulfate) was added to each well. The absorbance in all wells was determined at 600 nm on a microtiter plate-reader (Bio-Tek Instruments, Winooski, VT) following overnight incubation (at 37°C with 5% CO2). The stimulation ratio was calculated on the basis of the absorbance measured in response to a mitogenic stimulus relative to that in wells receiving medium alone.

Cytokine assays
Concentrations of interleukin-1ß (IL-1ß) and tumor necrosis factor-{alpha} (TNF-{alpha}) in pup serum samples were determined by enzyme-linked immunosorbent assays (ELISAs) using commercially available kits purchased from R&D Systems, Minneapolis, MN.

Statistical analyses
The data are presented in tables or graphs as mean values ± standard errors or ± standard deviations as specified in the legends. Differences in continuous variables were statistically evaluated after transformation of the data to their natural logarithms (ln) using the two-tailed Student's t-Test, or when standard deviations were statistically different, using the alternate Welch t-Test or Mann–Whitney Test. Differences in mammary tumor incidence in response to MNU between CS- and FA-treatment groups were statistically evaluated by modeling the time-to-first-tumor distribution in CS-exposed animals as a negative exponential distribution. It was then shown that according to this fitted distribution, the FA-exposed animals time-to-first-tumor is improbably (P < 0.02) large. A ‘P-value’ of <0.05 was considered statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 References
 
No significant differences were observed in body weight gain of dams exposed to CS as compared with FA dams during pregnancy (Table III). Litter size and neonatal pup weights of CS- and FA-exposed dams did not differ significantly (Table III).


View this table:
[in this window]
[in a new window]
  		Table III. Body weights and litter size and weight in rats exposed to CS or FA during gestation

 
There were no significant differences between groups in the concentrations of estradiol-17ß, progesterone, corticosterone, growth hormone, relaxin or insulin measured in the serum samples obtained during pregnancy (Table IV). However, there was a highly significant (P < 0.005) decrease in serum immunoactive prolactin concentration on Day 19 of pregnancy in the rats exposed to CS as compared with FA controls, whereas on Day 8 serum prolactin levels were slightly higher in FA controls than in CS-exposed dams (Figure 1).


View this table:
[in this window]
[in a new window]
  		Table IV. Serum hormones in rats exposed to CS or FA from Days 2–20 of pregnancy

 

Figure 1
View larger version (17K):
[in this window]
[in a new window]
  		Fig. 1. Immunoactive prolactin concentration in serum of CS- or FA-exposed rats on Days 8, 12 and 19 of pregnancy.

 
Following injection of MNU at 100 days of age, the rats were observed five times per week for the appearance of mammary (and other) tumors, and the pattern that emerged is shown in Figure 2. At 22 weeks post-MNU, 77% of the CS-exposed AMV rats exhibited mammary tumors (MTs), whereas the incidence for the FA-exposed controls was only 62% (Figure 2 and Table V).


Figure 2
View larger version (17K):
[in this window]
[in a new window]
  		Fig. 2. Mammary tumor progression following MNU injection in primiparous and virgin rats previously exposed to CS or FA. Sprague–Dawley rats were bred at 50 days of age and exposed to either CS or FA in a chamber 4 h/day Days 2–20 of pregnancy. Age-matched virgin rats were exposed to CS or FA according to the same schedule. At Day 100 the rats were injected with MNU or vehicle. The graph illustrates the appearance of MTs in the MNU-injected groups (the vehicle-injected controls did not develop tumors).

 

View this table:
[in this window]
[in a new window]
  		Table V. Mammary gland tumorigenesis in virgin and primiparous rats injected with MNU

 
Tumors were first detected 10 weeks after MNU injection in the CS-exposed animals, but not until 18 weeks in the FA-exposed dams (Figure 2). This difference in time to first tumor appearance between FA- and CS-exposed parous rats was significant (P = 0.02). At 22 weeks after MNU injection, 23% of the CS-exposed parous rats had palpable MTs, compared with 8% of the FA-exposed controls. There were no significant differences between CS- and FA-exposed virgin rats regarding total numbers of tumors per group or per rat at 22 weeks post-MNU (Table V).

Histochemical studies of the mammary biopsies taken 40 days after injection of MNU did not reveal any differences between CS- and FA-exposed parous or virgin rats for within-tissue distribution and number of positively staining cells, or for cellular staining intensity of estrogen receptor-alpha, PGE2 receptor and p53 expression in epithelial cells of ducts and alveoli of mammary tissue (data not shown). The biopsies were not adequate to determine histological difference in branching patterns between the four groups of animals.

The MTs observed at necropsy at age 240 days were predominantly invasive compact tubular carcinomas (non-invasive compact tubular carcinomas, cribriform-comedocarcinomas and tubulopapillary carcinomas were also observed, but were less frequent). The spectrum of specific tumor types and the histological appearance of the various tumor types were similar in the mammary glands of virgins and in dams exposed to CS or FA, and there were no differences in cervical, thoracic, abdominal and inguinal location of these tumors (data not shown).

The experiments performed on the pups are summarized in Table VI and Figure 3. Body weight and relative weights of the thymus and spleen in pups whose mothers had been exposed to CS or FA during pregnancy were comparable at all age groups studied (3, 8 and 19 weeks of age; Figure 3).


View this table:
[in this window]
[in a new window]
  		Table VI. Lack of effects of prenatal CS exposure on selected immune parameters of rat pupsa

 

Figure 3
View larger version (16K):
[in this window]
[in a new window]
  		Fig. 3. Effects of prenatal exposure to CS on (A) body, (B) spleen and (C) thymus weights of the offspring at 3, 8 and 19 weeks of age. There are four pups per group.

 
Although age- and sex-related variations in immune function parameters were observed in the prenatally exposed offspring, no differences were detected that could be ascribed to CS exposure. The parameters examined included NK cytotoxicity, lymphocyte proliferation and serum concentrations of IL-1ß and TNF-{alpha} in samples obtained from pups born to CS- or FA-exposed dams at 3, 8 or 19 weeks post-partum (Table VI). However, a significant decrease (P = 0.032) was observed in the concentration of immunoactive prolactin in the milk of CS- versus FA-exposed dams (Figure 4) that was recovered from the stomachs of pups euthanized on Day 2–3 of lactation. No differences in relaxin immunoactivity were detected in the same samples (data not shown).


Figure 4
View larger version (33K):
[in this window]
[in a new window]
  		Fig. 4. Immunoactive prolactin concentration in milk recovered from stomachs of suckling pups on Day 3 of lactation. The stomach contents were extracted as outlined in the text.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
References
 
Exposure of female rats to CS (50 mg PM/m3 for 4 h/day for 20 days) during a first pregnancy partially reversed the protective action of parity against MNU-induced BCa by shortening tumor latency. Exposure to CS also tended to increase the tumor response in virgin rats, which were considerably more sensitive to MNU than parous rats. These results corroborate the observation in the human case–control study by Innes and Byers (5), supporting the notion that mainstream CS negatively impacts the protection of parity against BCa. Present findings also suggest that CS may enhance—possibly acting as a weak tumor promoter—the BCa-inducing action of MNU in the virgin rats, although the difference in tumor incidence or latency failed to reach statistical significance. In the absence of MNU, CS-exposed rats did not develop MTs, indicating that CS, at the concentration used for these studies, was not by itself carcinogenic.

In this study, there were no significant effects of smoke exposure on serum concentrations of immunoactive estradiol-17ß, progesterone, corticosterone, growth hormone, insulin or relaxin in the pregnant rats (compared with the FA-exposed controls). However, there was a highly significant (P < 0.005) decrease in serum immunoactive prolactin on Day 19 of pregnancy in the CS-exposed animals. As prolactin is a likely factor in BCa susceptibility (21), this finding requires further study. Whether this decrease represents an actual change in secretion, or is due to proteolytic cleavage of the prolactin molecule (22) or to increases in receptor binding requires clarification. It is generally assumed that increases in prolactin secretion are associated with increased susceptibility to BCa in both female rodents and women (21). Thus, the decrease in serum prolactin appears contrary to the observed increase in mammary tumor response, suggesting that factors other than hormones are part of the mechanism that underlies the increased mammary gland sensitivity to later injection of MNU. However, other factors may modulate the actions of prolactin on the mammary glands. Evidence is accumulating that supports the view that local production (autocrine) of prolactin by mammary tissue may be more important than its hypophyseal secretion (endocrine) in breast tumor growth (21,23,24). There is a family of isoforms of the prolactin receptor that mediate the effects of prolactin in human (24) and rat (25) mammary tissue. Importantly, these prolactin isoforms are located in the stroma as well as the epithelium of the glands (26); their distribution and occupancy in this rat model requires determination. Such studies may provide an explanation for the apparently conflicting effects of CS on serum prolactin and mammary cancer induction by MNU. It is also possible that differences in serum prolactin at other time points during pregnancy than were measured in this study are more important than the difference at Day 19 that was observed. This possibility is supported by the observation of non-significantly higher serum prolactin levels on Day 8 of pregnancy in CS-exposed dams compared with air controls, which is in line with the hypothesis that prolactin confers increased BCa risk in rats.

The histochemical studies evaluating p53 expression, as well as estrogen and PGE2 receptors in the mammary biopsies obtained from the CS-exposed and control rats 40 days post-MNU injection (prior to visible tumor appearance) failed to reveal any notable differences between the groups. Whether selection of different time periods for biopsy would have detected CS- or MNU-related effects on these parameters is unknown. The possible induction of p53 mutations by MNU also warrants investigation (27,28).

It has been well documented in women and in rodent models that cigarette smoking during pregnancy can exert harmful effects on the unborn (13). Some of these untoward effects relate to suppression of the immune system, which in turn can lead to an increased sensitivity to toxic, immunomodulatory and carcinogenic substances (14–16). Thus, effects of CS exposure during pregnancy on the transmission of milk-borne hormonal growth factors from dam to pup during lactation and on some indicators of immune function in the prenatally exposed pups were investigated. Hormonal analysis revealed a significant decrease (P < 0.032) in immunoactive prolactin concentration in the stomach contents of pups suckled by CS-exposed dams as compared with that of those exposed only to FA. As milk-borne prolactin plays a role in the normal development of the immune system (17,18) and also the CNS (29,30) in neonatal rats, the results suggest that CS exposure of the dams could indirectly impair maturation of these functions in the young by further reducing prolactin secretion into the milk. However, at the concentration of smoke employed, no significant changes were detected in pup thymus or spleen weights or in the particular immune functions selected for examination in this study. The lack of CS-induced effects on the pup's immune response is in contrast to those immune alterations observed in mice exposed prenatally to an even lower dose of CS (31). Other more sensitive immune end-points such as antibody-forming cell numbers and/or immune cell-surface markers should therefore be evaluated in immunological studies utilizing rats.

Finally, it should be mentioned that the concentration of CS particulates used in the present studies (50 mg PM/m3/200 g rat/4 h/day) was calculated to be roughly equivalent to an individual smoking 2.7 packs of cigarettes per day. This is a relevant exposure scenario for humans classified as ‘heavy smokers’. However, the CS particulate dose used is below that shown in other toxicological studies to reduce maternal and neonatal body weight in rodents (see refs 32–35).

In conclusion, the authors suggest that one or more components of CS may specifically negate or reverse the action of the unknown gestational factor that protects against BCa. Elucidation of the mechanisms involved in these phenomena will be important in the eventual identification of the gestational BCa protective factor itself.


   Acknowledgments
 
The reagents for the rat relaxin radioimmunoassay were generously provided by Dr O.D. Sherwood of the University of Illinois. The rat prolactin RIA kit was kindly provided by Dr Albert F. Parlow, Scientific Director of the NIDDK National Hormone and Peptide Program. Research described in this article was supported by the External Research Program of Philip Morris USA and Philip Morris International, and in part by NIEHS Center Grant P30 ES000260 and NCI Center Grant P30 CA016087.

Conflict of Interest Statement: None declared.


   References
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

  1. Lippman,M.E. (1998) Endocrine responsive cancer. In: Wilson,J.D., Foster,D.W., Kronenberg,H.M. and Larson,P.R. (eds) Williams Textbook of Endocrinology, 9th edn, WB Saunders, Philadelphia and London, pp. 1675–1692
  2. Coyle,Y.M. (2004) The effect of environment on breast cancer risk. Breast Cancer Res. Treat., 84, 273–288.
  3. Russo,J., Calaf,G., Sohi,N., Tahin,Q., Zhang,P.L., Alvarado,M.E., Estrada,S. and Russo,I.H. (1993) Critical steps in breast carcinogenesis. Ann. NY Acad. Sci., 698, 1–20.
  4. Russo,J., Gusterson,B.A., Rogers,A.E., Russo,I.H., Wellings,S.R. and van Zwieten,M.J. (1990) Biology of disease. Comparative study of human and rat mammary tumorigenesis. Lab. Invest., 62, 1–32.
  5. Innes,K.E. and Byers,T.E. (2001) Smoking during pregnancy and breast cancer risk in very young women (United States). Cancer Causes Control, 12, 179–185.
  6. Couch,F.J., Cerhan,J.R., Vierkant,R.A., Grabrick,D.M., Therneau,T.M., Pankratz,V.S., Hartmann,L.C., Olson,J.E., Vachon,C.M. and Sellers,T.A. (2001) Cigarette smoking increases risk for breast cancer in high risk breast cancer families. Cancer Epidemiol Biomarkers Prev., 10, 327–332.
  7. Brunet,J.S., Ghadirian,P., Rebbeck,T.R. et al. (1998) The effect of smoking on breast cancer incidence in BRCA1 and BRCA2 carriers. J. Natl Cancer Inst., 90, 761–765.
  8. Sukumar,S., McKenzie,K. and Chen,Y. (1995) Animal models for breast cancer. Mutat. Res., 333, 37–44.
  9. Moon,R.C. (1969) Relationship between previous reproductive history and chemically induced mammary cancer in rats. Int. J. Cancer, 4, 312–317.
  10. Thordarson,G., Jin,E., Guzman,R.C., Swanson,S.M., Nandi,S. and Talamantes,F. (1995) Refractoriness to mammary tumorigenesis in parous rats: is it caused by persistent changes in the hormonal environment or permanent biochemical alterations in the mammary epithelia? Carcinogenesis, 16, 2847–2853.
  11. Grubbs,C.J., Hill,D.L., McDonough,K.C. and Peckham,J.C. (1983) N-nitroso-N-methylurea-induced mammary carcinogenesis: effects of pregnancy on preneoplastic cells. J. Natl Cancer Inst., 71, 625–628.
  12. Hecht,S.S. (2002) Tobacco smoke carcinogens and breast cancer. Environ. Mol. Mutagen., 39, 119–126.
  13. Haustein,K.O. (1999) Cigarette smoking, nicotine and pregnancy. Int. J. Clin. Pharmacol. Ther., 37, 417–427.
  14. Braun,K.M., Cornish,T., Valm,A., Cundiff,J., Pauly,J.L. and Fan,S. (1998) Immunotoxicology of cigarette smoke condensates: suppression of macrophage responsiveness to interferon gamma. Toxicol. Appl. Pharmacol., 149, 136–143.
  15. Byrd,R.S. and Howard,C.R. (1995) Children's passive and prenatal exposure to cigarette smoke. Pediatr. Ann., 24, 640–645.
  16. Cederqvist,L.L., Eddey,G., Abdel-Latif,N. and Litwin,S.D. (1984) The effect of smoking during pregnancy on cord blood and maternal serum immunoglobulin levels. Am. J. Obstet. Gynecol., 148, 1123–1126.
  17. Grove,D.S., Bour,B., Kacsoh,B. and Mastro,A.M. (1991) Effect of neonatal milk-prolactin deprivation on the ontogeny of the immune system of the rat. Endocr. Regul., 25, 111–119.
  18. Kacsoh,B., Toth,B.E., Avery,L.M., Yamamuro,Y. and Grosvenor,C. (1991) Molecular heterogeneity of prolactin in lactating rats and their pups: biological and immunological activities in pituitary gland, serum and milk. Endocr. Regul., 25, 98–110.
  19. Sherwood,O.D. and Crnekovic,V.E. (1979) Development of a homologous radioimmunoassay for rat relaxin. Endocrinology, 104, 893–897.
  20. Djeu,J.Y. (1995) Natural killer activity. In: Burlesson,G.R., Dean,J.H. and Munson,A.E., (eds) Methods in Immunotoxicology. Vol 1, Wiley-Liss, New York, pp. 437–450
  21. Harvey,P.W. (2005) Human prevalence of rodent prolactin-induced non-genotoxic mammary carcinogenesis: prolactin involvement in human breast cancer and significance for toxicological risk assessments. J. Appl. Toxicol., 25, 179–183.
  22. Vick,R.S., Wong,V.L. and Witorsch,R.J. (1987) Biological, immunological and biochemical characterization of cleaved prolactin generated by lactating mammary gland. Biochim. Biophys. Acta., 931, 196–204.
  23. Goffin,V., Bernichtein,S., Touraine,P. and Kelly,P.A. (2005) Development and potential uses of human prolactin receptor antagonists. Endocr. Rev., 26, 400–422.
  24. Clevenger,C.V. (2003) Role of prolactin/prolactin receptor signaling in human breast cancer. Breast Dis., 18, 75–86.
  25. Guillaumot,P. and Cohen,H. (1994) Heterogeneity of the prolactin receptor in the rat mammary gland and liver during various physiological states. J. Endocrinol., 141, 271–278.
  26. Van Horn,K.M., Binart,N., Kelly,P.A. and Talamantes,F. (2001) Prolactin receptor expression in the epithelia and stroma of the rat mammary gland. J. Endocrinol., 171, 85–95.
  27. Hussain,S.P., Harris,C.C., Hainaut,P. and Vahakangas,K. (1999) p53 mutation spectrum and load: the generation of hypotheses linking the exposure of endogenous or exogenous carcinogens to human cancer. Mutat. Res., 16, 23–32.
  28. Hainaut,P. and Vahakangas,K. (1997) p53 as a sensor of carcinogenic exposures: mechanisms of p53 protein induction and lessons from p53 gene mutations. Pathol. Biol., 45, 833–844.
  29. Grosvenor,C.E., Shyr,S.W. and Crowley,W.R. (1986) Effect of neonatal prolactin deficiency on prepubertal tuberoinfundibular and tuberohypophyseal dopaminergic neuronal activity. Endocrinol. Exp., 20, 223–228.
  30. Shyr,S.W., Crowley,W.R. and Grosvenor,C.E. (1986) Effect of neonatal prolactin deficiency on prepubertal tuberoinfundibular and tuberohypophyseal dopaminergic neuronal activity. Endocrinology, 119, 1217–1221.
  31. Ng,S.P., Silvrstone,A.E., Lai,Z.-W. and Zelikoff,J.T. (2006) Effects of prenatal exposure to cigarette smoke on offspring tumor susceptibility and associated immune mechanisms. Toxicol. Sci., 89, 135–144.
  32. Hecht,S.S. (2005) Carcinogenicity studies of inhaled cigarette smoke in laboratory animals: old and new. Carcinogenesis, 26, 1488–1492.
  33. 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.
  34. Witschi,H., Lundgaard,S.M., Rajini,P., Hendrickx,A.G. and Last,J.A. (1994) Effects of exposure to nicotine and to sidestream smoke on pregnancy outcome in rats. Toxicol. Lett., 71, 279–286.
  35. Leichter,J. (1989) Growth of fetuses of rats exposed to ethanol and cigarette smoke during gestation. Growth Dev. Aging, 53, 129–134.
Received October 8, 2005; revised January 7, 2006; accepted January 23, 2006.


Add to CiteULike CiteULike   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us    What's this?


This article has been cited by other articles:


Home page
 Exp. Biol. Med.Home page
S. P. Ng, B. G. Steinetz, S. G. Lasano, and J. T. Zelikoff
Hormonal Changes Accompanying Cigarette Smoke-Induced Preterm Births in a Mouse Model
Experimental Biology and Medicine, September 1, 2006; 231(8): 1403 - 1409.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow All Versions of this Article:
27/6/1146    most recent
bgi353v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrow Search for citing articles in:
ISI Web of Science (4)
Right arrowRequest Permissions
Google Scholar
Right arrow Articles by Steinetz, B. G.
Right arrow Articles by Bosland, M. C.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Steinetz, B. G.
Right arrow Articles by Bosland, M. C.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?