The skeletal effects of spaceflight in growing rats: tissue-specific alterations in mRNA levels for TGF-beta.

Dynamic weight bearing is important for normal growth and maintenance of the skeleton in humans and laboratory animals. Transforming growth factor-beta (TGF-beta) has been implicated as having autocrine and paracrine actions in bone. The purpose of this study was to examine mRNA levels of TGF-beta in skeletal tissues of growing male rats following skeletal unweighting during an 11-day spaceflight. Animals were sacrificed 5-8 h after the skeleton was reloaded. Spaceflight resulted in decreases in cortical bone area and periosteal bone formation, but no change in medullary area and endocortical bone formation. In addition, spaceflight had no effect on longitudinal bone growth. TGF-beta was reduced relative to the ground controls in the hindlimb periosteum, but was not significantly altered in the growth zone of the tibial metaphysis. Similarly, mRNA levels for type I collagen were reduced in the periosteum, but not in the metaphysis of flight animals. The results suggest a potential role of TGF-beta as an intermediate in the signal transduction pathway for mechanical loading. Further, they indicate skeletal tissue compartment-dependent changes in mRNA levels for TGF-beta following weightlessness.

Differential effects of estrogen metabolites on bone and reproductive tissues of ovariectomized rats.

The effects of 17 beta-estradiol and the important estrogen metabolites, 2-hydroxyestrone (2-OHE1) and 16 alpha-hydroxyestrone (16 alpha-OHE1) on bone, mammary gland, and uterine histology, and on blood cholesterol were investigated in ovariectomized growing rats. Rats were treated with 200 micrograms/kg of body weight/day of each of the test compounds for 3 weeks. Ovariectomy resulted in uterine and mammary gland atrophy, increased body weight, bone turnover and tibia growth, and hypercholesterolemia. 17 beta-estradiol treatment prevented these changes, with the exception that this high dose of estrogen did not prevent hypercholesterolemia. 2-OHE1 had no effect on any of the measurements. 16 alpha-OHE1 resulted in bone measurements that did not differ from the 17 beta-estradiol-treated rats and prevented the increase in serum cholesterol. In contrast, 16 alpha-OHE1 resulted in increases in uterine weight, uterine epithelial cell height, and mammary gland cell proliferation that were significantly less than the 17 beta-estradiol treatment. These findings demonstrate that 16 alpha-hydroxylation of estrone results in tissue-selective estrogen agonistic activity, whereas 2-hydroxylation resulted in no measured activity. Furthermore, they suggest that factors that modulate the synthesis of these metabolites could selectively influence estrogen target tissues.

Estrogen does not increase bone formation in growing rats.

The purpose of these experiments was to test the hypothesis that estrogen has an anabolic effect on cancellous bone formation in long bones of growing rats. In the present studies, estrogen decreased measurements related to cancellous bone formation, including osteoblast number, fluorochrome-labeled bone perimeter, and incorporation of [3H]proline into osteoid. Steady state mRNA levels for bone matrix proteins and mineral apposition rate were unchanged or reduced. The [3H]thymidine labeling index for osteoblasts was very low and was reduced by estrogen treatment. Finally, estrogen decreased the rate of removal of tetracycline previously incorporated into cancellous bone as well as the growth-dependent decrease in cancellous bone area, indicating that hormone treatment reduced net bone resorption. In summary, no evidence was secured to support the hypothesis that estrogen has an anabolic action on bone formation; the results obtained in these studies consistently demonstrated a pronounced inhibitory action of the hormone on bone turnover.

The effects of orbital spaceflight on bone histomorphometry and messenger ribonucleic acid levels for bone matrix proteins and skeletal signaling peptides in ovariectomized growing rats.

A 14-day orbital spaceflight was performed using ovariectomized Fisher 344 rats to determine the combined effects of estrogen deficiency and near weightlessness on tibia radial bone growth and cancellous bone turnover. Twelve ovariectomized rats with established cancellous osteopenia were flown aboard the space shuttle Columbia (STS-62). Thirty ovariectomized rats were housed on earth as ground controls: 12 in animal enclosure modules, 12 in vivarium cages, and 6 killed the day of launch for baseline measurements. An additional 18 ovary-intact rats were housed in vivarium cages as ground controls: 8 rats were killed as baseline controls and the remaining 10 rats were killed 14 days later. Ovariectomy increased periosteal bone formation at the tibia-fibula synostosis; cancellous bone resorption and formation in the secondary spongiosa of the proximal tibial metaphysis; and messenger RNA (mRNA) levels for the prepro-alpha2(1) subunit of type 1 collagen, osteocalcin, transforming growth factor-beta, and insulin-like growth factor I in the contralateral proximal tibial metaphysis and for the collagen subunit in periosteum pooled from tibiae and femora and decreased cancellous bone area. Compared to ovariectomized weight-bearing rats, the flight group experienced decreases in periosteal bone formation, collagen subunit mRNA levels, and cancellous bone area. The flight rats had a small decrease in the cancellous mineral apposition rate, but no change in the calculated bone formation rate. Also, spaceflight had no effect on cancellous osteoblast and osteoclast perimeters or on mRNA levels for bone matrix proteins and signaling peptides. On the other hand, spaceflight resulted in an increase in bone resorption, as ascertained from the diminished retention of a preflight fluorochrome label. This latter finding suggests that osteoclast activity was increased. In a follow-up ground-based experiment, unilateral sciatic neurotomy of ovariectomized rats resulted in cancellous bone loss in the unloaded limb in excess of that induced by gonadal hormone deficiency. This additional bone loss was arrested by estrogen replacement. We conclude from these studies that estrogen alters the expression of signaling peptides believed to mediate skeletal adaptation to changes in mechanical usage and likewise modifies the skeletal response to mechanical unloading.

Effects of spaceflight and simulated weightlessness on longitudinal bone growth.

Indirect measurements have suggested that spaceflight impairs bone elongation in rats. To test this possibility, our laboratory measured, by the fluorochrome labeling technique, bone elongation that occurred during a spaceflight experiment. The longitudinal growth rate (LGR) in the tibia of rats in spaceflight experiments (Physiological Space Experiments 1, 3, and 4 and Physiological-Anatomical Rodent Experiment 3) and in two models of skeletal unloading (hind-limb elevation and unilateral sciatic neurotomy) were calculated. The effects of an 11 day spaceflight on gene expression of cartilage matrix proteins in rat growth plates were also determined by northern analysis and are reported for the first time in this study. Measurements of longitudinal growth indicate that skeletal unloading generally did not affect LGR, regardless of age, strain, gender, duration of unloading, or method of unloading. There was, however, one exception with 34% suppression in LGR detected in slow-growing, ovariectomized rats skeletally unloaded for 8 days by hind-limb elevation. This detection of reduced LGR by hind-limb elevation is consistent with changes in steady-state mRNA levels for type II collagen (-33%) and for aggrecan (-53%) that were detected in rats unloaded by an 11 day spaceflight. The changes detected in gene expression raise concern that spaceflight may result in changes in the composition of extracellular matrix, which could have a negative impact on conversion of growth-plate cartilage into normal cancellous bone by endochondral ossification.

Tissue-selective effects of continuous release of 2-hydroxyestrone and 16alpha-hydroxyestrone on bone, uterus and mammary gland in ovariectomized growing rats.

2-Hydroxyestrone (2-OHE(1)) and 16alpha-hydroxyestrone (16alpha-OHE(1)) have been reported to be risk factors for negative bone balance and breast cancer, respectively. The roles of these two metabolites of estrone as estrogen agonists or antagonists with respect to estrogen target tissues, or both, are poorly defined. The purpose of this study was to characterize metabolite and tissue-specific differences between the actions of hydroxylated estrones on selected reproductive and non-reproductive estrogen target tissues in growing rats. First, the effects of ovariectomy were determined. Ovariectomy had the expected effects, including increases in all dynamic bone measurements at the proximal tibial epiphysis, without induction of bone loss. Second, ovariectomized growing rats were continuously treated for 3 weeks with 2-OHE(1), 16alpha-OHE(1), 17beta-estradiol (E(2)), a combination of E(2) and 2-OHE(1) (E(2)+2-OHE(1)), or a combination of E(2) and 16alpha-OHE(1) (E(2)+16alpha-OHE(1)), using controlled release subcutaneous implanted pellets containing 5 mg 2-OHE(1), 5 mg 16alpha-OHE(1), 0.05 mg E(2) or placebo. E(2) reduced body weight gain and radial and longitudinal bone growth as well as indices of cancellous bone turnover, and increased serum cholesterol, uterine wet weight and epithelial cell height, and proliferative cell nuclear antigen labeling in mammary gland. The hydroxylated estrones did not alter uterine wet weight and 16alpha-OHE(1) antagonized the E(2)-stimulated increase in epithelial cell height. 2-OHE(1) had no effect on cortical bone, whereas 16alpha-OHE(1) was an estrogen agonist with respect to all cortical bone measurements. 16alpha-OHE(1) also behaved as an estrogen agonist with respect to serum cholesterol and cancellous bone measurements. 2-OHE(1) had no effect on most E(2)-regulated indices of cancellous bone growth and turnover, but was a weak estrogen agonist with respect to mineral apposition rate and bone formation rate. Neither estrogen metabolite influenced body weight gain. Third, weanling rats were treated for 1 week with vehicle, E(2) (200 microg/kg per day) or 16alpha-OHE(1) (30, 100, 300, 1000 and 3000 microg/kg per day) to confirm uterotropic effects of daily subcutaneous (s.c.) administration of 16alpha-OHE(1). 16alpha-OHE(1) increased uterine weight in a dose-response manner to values that did not differ from rats treated with E(2). We conclude that the estrogen metabolites 2-OHE(1) and 16alpha-OHE(1) have target tissue-specific biological activities which differ from one another as well as from E(2). These findings add further support to the concept that there are several classes of estrogens with distinct biological activities. Furthermore, differences in the route of administration could influence the tissue specificity of estrogen metabolites.

The catechol estrogen, 4-hydroxyestrone, has tissue-specific estrogen actions.

Recent data indicate that the catechol estrogen, 2-hydroxyestrone (2-OHE(1)), has no effect on any target tissue including bone, whereas 16 alpha-hydroxyestrone (16 alpha-OHE(1)) exerts tissue-selective estrogen agonist activity. The effect of the catechol estrogen, 4-hydroxyestrone (4-OHE(1)), putatively associated with tumorigenesis, has not been studied in the skeleton. The purpose of this study was to assess the effect of 4-OHE(1) on tibia, uterine and mammary gland histology and blood cholesterol in ovariectomized (OVX'd) growing rats. Ten-week-old female Sprague-Dawley rats were injected subcutaneously with 200 microg/kg BW per day with 4-OHE(1), 17 beta-estradiol (E(2)) or vehicle for three weeks. OVX resulted in uterine atrophy, increased body weight, radial bone growth and cancellous bone turnover, and hypercholesterolemia. E(2) prevented these changes with the expected exception that the subcutaneous infusion of this high dose of estrogen did not prevent the hypercholesterolemia. 4-OHE(1) prevented the increase in blood cholesterol and the increase in body weight. 4-OHE(1) appeared to have partial estrogen activity in the uterus; uterine weight and epithelial cell height were significantly greater than the OVX rats but significantly less (twofold) than the E(2) animals. Analysis of variance indicated that 4-OHE(1) slightly decreased the periosteal mineral apposition rate (P<0.05) compared with vehicle-treated rats but had no effect on double-labeled perimeter or bone formation rate. Similarly, 4-OHE(1) was a partial estrogen agonist on cancellous bone turnover. The data suggest that the catechol estrogen, 4-OHE(1), unlike 2-OHE(1), has estrogen activity. Furthermore, the profile of activity differs from that of 16 alpha-OHE(1). Our results suggest that estrogen metabolites may selectively influence estrogen-target tissues and, concomitantly, modulate estrogen-associated disease risk.

A comparison of the oxygen drift in downhill vs. level running.

This investigation explored the recent theory that muscle damage causes the drift in oxygen consumption (VO2) during low-intensity downhill running. Seven subjects participated in a maximal VO2 (VO2max) test and three submaximal bouts [one level (Level) and two downhill runs (Down 1, Down 2) at 40% peak VO2]. Two downhill runs (30 min at -10% grade) were performed to vary the extent of muscle damage. Creatine kinase (CK) increased more after Down 1 (61%) than after Down 2 (11%), as did soreness ratings, indicating reduced muscle damage during Down 2. Significantly greater increases in VO2 over time were noted for Down 1 (15.6%) and Down 2 (14.7%) than for Level (1.2%). Heart rate increased 8 beats/min for Level but 29 and 25 beats/min for Down 1 and Down 2, respectively. Expired ventilation increased more for Down 1 (20.5%) and Down 2 (24%) than for Level (3.5%). Rectal temperature increased approximately 0.8 degree C for all bouts. Because the magnitude of the drift was similar in the two downhill bouts, the findings suggest that muscle damage does not cause the drift in VO2 during low-intensity downhill running.

Effect of resistance exercise training on cortical and cancellous bone in mature male rats.

The effect of resistance training on tibial cancellous and cortical bone was evaluated in rats by using static histomorphometry and Northern analysis. Five-month-old male Sprague-Dawley rats were randomly assigned to exercise (Ex; n = 8) or control (Con; n = 4) groups. Animals were operantly conditioned to press two levers, facilitating full extension and flexion of the hindlimbs ("squats"), while wearing an unweighted vest. After an 8-wk familiarization period, Ex animals performed 3 sessions/wk for 17-19 sessions with progressively increased amounts of weight applied to the vest. Con rats completed the same exercise protocol without applied resistance. No difference in cross-sectional, medullary, or cortical bone area was observed between Ex and Con rats in the tibial diaphysis. In contrast, the cancellous bone area in the proximal tibial metaphysis was significantly larger in trained rats. Trabecular number, trabecular thickness, and the percentage of cancellous bone covered by osteoid were significantly greater in the Ex animals compared with Con animals. In addition, steady-state mRNA levels for osteocalcin for the Ex group were 456% those expressed in the Con group. The data demonstrate that resistance training increases cancellous bone area in sexually mature male rats and suggest that it does so, in part, by stimulating bone formation.

Estrogen has rapid tissue-specific effects on rat bone.

The decrease in cancellous bone formation after estrogen treatment is generally thought to be coupled with a prior decrease in bone resorption. To test the possibility that estrogen has rapid tissue-specific actions on bone metabolism, we determined the time course (1-32 h) effects of diethylstilbestrol on steady-state mRNA levels for immediate-response genes, extracellular matrix proteins, and signaling peptides in the proximal tibial metaphysis and uterus by using Northern blot and RNase protection assays. The regulation of signaling peptides by estrogen, although tissue specific, followed a similar time course in bone and uterus. The observed rapid decreases in expression of insulin-like growth factor I, a growth factor associated with bone formation; decreases in mRNA levels for bone matrix proteins; evidence for reduced bone matrix synthesis; failure to detect rapid increases in mRNA levels for signaling peptides implicated in mediating the inhibitory effects of estrogen on bone resorption (interleukin-1 and -6) as well as other cytokines that can increase bone resorption; and the comparatively long duration of the bone remodeling cycle in rats indicate that estrogen can decrease bone formation by a mechanism that does not require a prior reduction in bone resorption.

Moderate exercise training slows mammary tumour growth in adolescent rats.

Adolescence and young adulthood may be critical windows in establishing risk for breast cancer development in humans. Epidemiological data suggest that exercise during this life stage is associated with decreased breast cancer risk yet few experimental studies to elucidate the mechanism have been performed. The purpose of these studies was to evaluate the effects of moderate exercise training on mammary tumour development in adolescent rats using the 1-methyl 1-nitrosourea (MNU) chemical carcinogen model. Exercise (EX) consisted of moderate-intensity treadmill running 30 min/day, 5 days a week. A total of 274 animals were used: 94 in study 1 and 180 in study 2. Animals were injected with MNU (50 and 25 mg/kg body weight in studies 1 and 2, respectively) at 21 days of age and began training at 28 days of age. Groups of animals (n=10-30 depending on the study and time point) were sacrificed every 2 weeks for 8 weeks to evaluate tumour development. No difference in median tumour-free survival time was observed in the EX versus sham-exercise (SHAM), nor were there any differences in multiplicity at either a high or moderate dose of MNU. Latency to first tumour palpated was increased in both studies by 3-4 days. Consistent across both studies, tumour weights were less and the growth rates of the tumours, defined as tumour weight divided by the number of days elapsed since the tumour was first palpated, were reduced in the EX group. The data suggest that latency is increased and tumour growth is retarded in response to moderate exercise training.

The effect of diurnal and menstrual cyclicity and menopausal status on estrogen metabolites: implications for disease-risk assessment.

It has been proposed that the ratio of two estrogen metabolites, 2-hydroxyestrone (2-OHE1) and 16alpha-hydroxyestrone (16alpha-OHE1), may represent a marker to predict a woman's risk for developing breast cancer and other estrogen-related disease. The present studies evaluated the potential confounders of type of sample, diurnal rhythm, menstrual cycle phase, and menopausal status on the ratio of 2/16alpha-OHE1 using an urine-based monoclonal antibody enzyme immunoassay. Two initial studies to compare a 24-h urine collection with a first-morning void and to evaluate diurnal variation were performed. Subsequently, urine samples were collected every other day for 2 months from five premenopausal subjects to assess the impact of the menstrual cycle. Spot urine samples were then obtained from a total of 67 pre, peri-, early post-, and late post-menopausal women to assess the effect of menopausal status. No significant difference in the ratio of 2/16alpha-OHE1 was found between a 24-h and first-morning void or over a 24-h period. No significant difference in the mean ratio of 2/16alpha-OHE1 was found with the menstrual phase. Intra-individual variability was observed in the ratio of 2/16alpha-OHE1, which was attributable to small fluctuations in the small denominator, 16alpha-OHE1. No difference in the ratio of 2/16alpha-OHE1 was observed in groups of women of different menopausal status. The data suggest that a first-morning void is representative of a 24-h collection and that the 2/16alpha-OHE1 ratio is constant throughout a 24-h period. Moreover, menstrual phase and menopausal status do not appear to significantly influence the ratio of 2/16alpha-OHE1.

Estrogen receptor mRNA is expressed in vivo in rat calvarial periosteum.

Estrogen deficiency is well recognized as a cause of bone loss in rats and humans. Likewise, treatment with estrogen results in prevention of this loss. Initially, this effect was thought to be indirectly mediated but, more recently, estrogen receptors (ER) have been reported in osteosarcoma cells and primary cultures originating from surgical waste, suggesting a direct effect of this steroid hormone. Detection of ER in skeletal tissues, however, has remained elusive. The purpose of this investigation was to establish the efficacy of the highly sensitive reverse-transcription polymerase chain reaction (RT-PCR) technique to detect ER in a well defined skeletal tissue (calvarial periosteum) that is responsive to the hormone. Primers were made specific to rat ER sequences. Total RNA was extracted from rat uterus, liver, spleen, and the periosteum using an organic solvent method. cDNA was synthesized from 2 micrograms total RNA. cDNA corresponding to 40 ng total RNA/sample produced intense PCR products for ER. In descending order of intensity were uterus, liver, bone, and spleen. Importantly, a similar time-course for estrogen-induced down-regulation of steady-state mRNA levels for alkaline phosphatase and osteonectin was observed in calvarial periosteum and tissues known to express estrogen receptors. These data provide in vivo evidence of ER mRNA in bone and suggest that at least some of estrogen's action on bone is directly modulated.

Alterations in oxygen consumption during and between bouts of level and downhill running.

Since the etiology of the drift in VO2 during downhill running is unclear, this study was designed to assess the contribution of heart rate (HR), ventilation (VE), blood lactate, rectal temperature (RT), muscle damage, and several variables that have not previously been included in VO2 drift research: muscle temperature (MT), and stride rate (SR) and length (SL), to the drift in VO2. Six subjects participated in a 45-min level run (LEVEL) and two 45-min downhill runs (DOWN1 and DOWN2) at 50% VO2max. Although VO2 increased significantly over time for all bouts, the magnitude [4.3% (LEVEL), 5.4% (DOWN1), and 8.1% (DOWN2)] did not differ between bouts (P > 0.05). VO2 was significantly lower during DOWN2 than during LEVEL and DOWN1 (P < 0.05). MT increased during the three bouts (P < 0.05) but the change over time was not different between bouts. SR and SL did not change over time within each bout nor between the two downhill runs. Muscle damage, as indicated by serum creatine kinase levels and perceived soreness, was less following LEVEL and DOWN2 than DOWN1 (P < 0.05). HR and RT increased over time (P < 0.05) but did not differ between bouts. VE and blood lactate did not differ over time or between bouts. VO2 drift during the three bouts paralleled changes in RT, MT, and HR but appears unrelated to muscle damage or biomechanical factors.

Exercise and Serum Androgens in Women.

In brief: Little information exists about the role of androgens in women relative to exercise, particularly resistance exercise. This study examines (1) the effect of a ten-week hydraulic resistance exercise program on serum androgen levels, strength, and lean body weight and (2) the relationship of serum androgen levels to strength and lean body weight before and after the ten-week program. Thirteen experimental and five control subjects, all college women, participated. Training did not result in significant increases in androgen hormones, although there were significant gains in strength. In addition, no significant correlations were observed between testosterone, androstenedione, or sex hormone-binding globulin and pretraining and delta values of strength or lean body weight (r <.49).

Proliferation and apoptosis in mammary epithelium during the rat oestrous cycle.

AIM: During each oestrous cycle, the mammary gland is subject to changes in ovarian hormone levels. It responds with limited proliferation, differentiation and regression. To understand the processes resulting in these changes, particularly the regulation of cell death, we examined protein levels in mammary epithelium during the oestrous cycle of the Sprague-Dawley rat. METHODS: Studies of serum hormone levels, vaginal smears, uterine weight and morphology, mammary gland morphology, proliferation and apoptotic indices, and protein levels during the stages of the Sprague-Dawley rat oestrous cycle were used to examine the response of mammary epithelium to the oestrous cycle. RESULTS: Proliferation of mammary epithelium was greater in diestrus and proestrus, while apoptosis was increased in metestrus and diestrus. Growth factor-, hormone- and anchorage-mediated cell survival signalling, indicated by activation of Stat5A, FAK and Akt 1 and expression of anti-apoptotic Bcl-2 family members, was greater in proestrus and reduced in metestrus. In contrast, the levels of pro-apoptotic Bcl-2 family members and proteins associated with apoptosis in mammary epithelium (TGFbeta3, pStat3) were increased during metestrus and diestrus. CONCLUSION: Decreases in growth factor, hormone and cell attachment survival signals corresponded with increased apoptosis during the second half of the oestrous cycle. The protein levels detected during oestrus suggest parallels to apoptosis in mammary involution.

Balance characteristics of persons with osteoporosis.

OBJECTIVE: People affected by osteoporosis are at particular risk for bone fractures caused by falls. Preventive intervention depends on first describing the risk factors for falls present in this population as a group and as individuals. In this preliminary study, balance characteristics of women with and without osteoporosis were measured with computerized dynamic posturography (CDP). DESIGN: A case control design was selected to compare the balance characteristics of each group of patients with osteoporosis. SETTING: Testing was performed in the vestibular assessment area of our multispecialty clinic. SUBJECTS: Patient groups were selected from within our case load. Ten women with osteoporosis were compared with six women with osteoporosis and kyphosis (Cobb angle more than 54 degrees) and with five age-matched normal subjects. INTERVENTIONS: Because this was an observational study, no interventions were used. MAIN OUTCOME MEASURE: Averaged results from all trials of sensory organization tests 5 and 6, with use of sway amplitude and balance strategy scores, were used to compare the performance of each patient group. RESULTS: Both groups with osteoporosis had different balance control strategies than the group without osteoporosis. Specifically, those with osteoporosis had greater use of hip strategies for maintaining balance than did the normal group. Those with kyphosis also had greater postural sway than either of the other two groups. CONCLUSION: Results of this study suggest that there are differences in balance control strategies and sway amplitude between patients with and those without osteoporosis. Further study is recommended in which CDP is used to clarify and confirm these differences. Individual CDP results can be used to optimize habilitative management of these patients.

Effect of exercise on the rat mammary gland: implications for carcinogenesis.

Physical activity has been associated with decreased risk for developing breast cancer yet to date, the mechanism remains unknown. The purpose of this investigation was to evaluate the effects of moderate exercise training on the normal mammary gland in an attempt to identify alterations or differences that might be associated with tumour inhibition. A total of 170 female Sprague-Dawley rats were randomized to baseline (n=10), exercise (EX; n=80), or sham-exercise groups (SHAM; n=80). Treadmill training (20-25 m min-1, 15% grade, 30 min day-1, 5 days week-1) was started at 28 days of age (DOA). Animals were killed at 28, 42, 56, 70 and 84 DOA. Mammary glands were evaluated by histology and immunohistochemistry. Terminal end buds (TEB), structures susceptible to carcinogenesis, were counted. Sexual maturation, estradiol and progesterone, and organ and muscle weights were also evaluated. No differences in growth, sexual maturation, or steroid hormones were observed in response to training. No difference in the number of TEBs was observed at any timepoint between EX and SHAM. Proliferation was significantly increased at 56 DOA and tended to be increased at 42 and 70 DOA in the EX animals whereas cell death was significantly increased at 70 DOA and tended to be increased at 84 DOA in the EX animals. These data suggest no difference in the number of carcinogen-susceptible structures as a result of moderate exercise. The changes in cell proliferation and apoptosis with exercise training suggest altered cell turnover that will necessitate future study particularly with relevance to carcinogenesis.