Maternal Obesity during Pregnancy is Associated with Lower Cortical Thickness in the Neonate Brain.

BACKGROUND AND PURPOSE: Recent studies have suggested that maternal obesity during pregnancy is associated with differences in neurodevelopmental outcomes in children. In this study, we aimed to investigate the relationships between maternal obesity during pregnancy and neonatal brain cortical development. MATERIALS AND METHODS: Forty-four healthy women (28 normal-weight, 16 obese) were prospectively recruited at <10 weeks' gestation, and their healthy full-term neonates (23 boys, 21 girls) underwent brain MR imaging. All pregnant women had their body composition (fat mass percentage) measured at ∼12 weeks of pregnancy. All neonates were scanned at ∼2 weeks of age during natural sleep without sedation, and their 3D T1-weighted images were postprocessed by the new iBEAT2.0 software. Brain MR imaging segmentation and cortical surface reconstruction and parcellation were completed using age-appropriate templates. Mean cortical thickness for 34 regions in each brain hemisphere defined by the UNC Neonatal Cortical Surface Atlas was measured, compared between groups, and correlated with maternal body fat mass percentage, controlled for neonate sex and race, postmenstrual age at MR imaging, maternal age at pregnancy, and the maternal intelligence quotient and education. RESULTS: Neonates born to obese mothers showed significantly lower (P ≤ .05, false discovery rate-corrected) cortical thickness in the left pars opercularis gyrus, left pars triangularis gyrus, and left rostral middle frontal gyrus. Mean cortical thickness in these frontal lobe regions negatively correlated (R = -0.34, P = .04; R = -0.50, P = .001; and R = -0.42, P = .01; respectively) with the maternal body fat mass percentage measured at early pregnancy. CONCLUSIONS: Maternal obesity during pregnancy is associated with lower neonate brain cortical thickness in several frontal lobe regions important for language and executive functions.

Brain Cortical Structure and Executive Function in Children May Be Influenced by Parental Choices of Infant Diets.

BACKGROUND AND PURPOSE: While it is known that breastfeeding promotes healthy brain development in children, the potential effects of formulas substantially differing in composition (ie, milk-based versus soy-based) during infancy on brain development are unclear. MATERIALS AND METHODS: Seventy-one 8-year-old children who were predominantly breastfed, milk formula fed, or soy formula fed during infancy were recruited for an MR imaging examination of the brain and a Behavior Rating Inventory of Executive Function assessment (completed via a questionnaire to the parents). Brain cortical features measured from MR imaging such as cortical thickness and surface area were extracted and compared among groups and correlated with Behavior Rating Inventory of Executive Function test scores. RESULTS: Clusters in the frontal and occipital lobes showed significant differences (cluster-wise P ≤ .05, corrected for multiple comparisons) in cortical thickness or surface area among the 3 diet groups. The effects were more prominent for boys, particularly for comparison of the milk formula fed versus soy formula fed boys. Assessments of executive function and behavior showed significantly lower Behavior Rating Inventory of Executive Function test scores in soy formula fed versus milk formula fed groups, which were mostly attributed to differences in boys. There were no differences between milk formula fed and breastfed groups for either sex. Mean cortical thickness for several of the clusters in the brain showing infant diet-associated effects significantly correlated with Behavior Rating Inventory of Executive Function scores. CONCLUSIONS: Choices of infant diets (ie, breastfed, milk formula fed, soy formula fed) may have long-term and sex-specific effects on the cortical development and executive function and behavior of children's brains.

Cesarean Delivery Impacts Infant Brain Development.

BACKGROUND AND PURPOSE: The cesarean delivery rate has increased globally in the past few decades. Neurodevelopmental outcomes associated with cesarean delivery are still unclear. This study investigated whether cesarean delivery has any effect on the brain development of offspring. MATERIALS AND METHODS: A total of 306 healthy children were studied retrospectively. We included 3 cohorts: 2-week-old neonates (cohort 1, n = 32/11 for vaginal delivery/cesarean delivery) and 8-year-old children (cohort 2, n = 37/23 for vaginal delivery/cesarean delivery) studied at Arkansas Children's Hospital, and a longitudinal cohort of 3-month to 5-year-old children (cohort 3, n = 164/39 for vaginal delivery/cesarean delivery) studied independently at Brown University. Diffusion tensor imaging, myelin water fraction imaging, voxel-based morphometry, and/or resting-state fMRI data were analyzed to evaluate white matter integrity, myelination, gray matter volume, and/or functional connectivity, respectively. RESULTS: While not all MR imaging techniques were shared across the institutions/cohorts, post hoc analyses showed similar results of potential effects of cesarean delivery. The cesarean delivery group in cohort 1 showed significantly lower white matter development in widespread brain regions and significantly lower functional connectivity in the brain default mode network, controlled for a number of potential confounders. No group differences were found in cohort 2 in white matter integrity or gray matter volume. Cohort 3 had significantly different trajectories of white matter myelination between groups, with those born by cesarean delivery having reduced myelin in infancy but normalizing with age. CONCLUSIONS: Cesarean delivery may influence infant brain development. The impact may be transient because similar effects were not observed in older children. Further prospective and longitudinal studies may be needed to confirm these novel findings.

Gestational Age at Birth and Brain White Matter Development in Term-Born Infants and Children.

BACKGROUND AND PURPOSE: Studies on infants and children born preterm have shown that adequate gestational length is critical for brain white matter development. Less is known regarding how variations in gestational age at birth in term infants and children affect white matter development, which was evaluated in this study. MATERIALS AND METHODS: Using DTI tract-based spatial statistics methods, we evaluated white matter microstructures in 2 groups of term-born (≥37 weeks of gestation) healthy subjects: 2-week-old infants (n = 44) and 8-year-old children (n = 63). DTI parameters including fractional anisotropy, mean diffusivity, axial diffusivity, and radial diffusivity were calculated by voxelwise and ROI methods and were correlated with gestational age at birth, with potential confounding factors such as postnatal age and sex controlled. RESULTS: Fractional anisotropy values, which are markers for white matter microstructural integrity, positively correlated (P < .05, corrected) with gestational age at birth in most major white matter tracts/regions for the term infants. Mean diffusivity values, which are measures of water diffusivities in the brain, and axial and radial diffusivity values, which are markers for axonal growth and myelination, respectively, negatively correlated (P < .05, corrected) with gestational age at birth in all major white matter tracts/regions excluding the body and splenium of the corpus callosum for the term infants. No significant correlations with gestational age were observed for any tracts/regions for the term-born 8-year-old children. CONCLUSIONS: Our results indicate that longer gestation during the normal term period is associated with significantly greater infant white matter development (as reflected by higher fractional anisotropy and lower mean diffusivity, axial diffusivity, and radial diffusivity values); however, similar associations were not observable in later childhood.

Differences in brain functional connectivity at resting state in neonates born to healthy obese or normal-weight mothers.

Recent studies have shown associations between maternal obesity at pre- or early pregnancy and long-term neurodevelopment in children, suggesting in utero effects of maternal obesity on offspring brain development. In this study, we examined whether brain functional connectivity to the prefrontal lobe network is different in newborns from normal-weight or obese mothers. Thirty-four full-term healthy infants from uncomplicated pregnancies were included, with 18 born to normal-weight and 16 born to obese mothers. Two weeks after delivery, the infants underwent an magnetic resonance imaging (MRI) examination during natural sleep, which included structural imaging and resting-state functional MRI (fMRI) scans. Independent component analysis was used to identify the prefrontal lobe network, and dual regression was used to compare functional connectivity between groups. Infants born to normal-weight mothers had higher recruiting (P<0.05, corrected) of dorsal anterior cingulate cortex regions to the prefrontal network after adjusting for maternal intelligence quotient, gestational weight gain and infant postmenstrual age, gender, birth weight/length, head circumference and neonatal diet. The functional connectivity strength in dorsal anterior cingulate cortex negatively correlated (P<0.05) with maternal fat mass percentage measured at early pregnancy. This preliminary study indicates that exposure to maternal obesity in utero may be associated with changes in resting-state functional connectivity in the newborn offspring's brain.

Voxel-Based Morphometry and fMRI Revealed Differences in Brain Gray Matter in Breastfed and Milk Formula-Fed Children.

BACKGROUND AND PURPOSE: Infant diets may have significant impact on brain development in children. The aim of this study was to evaluate brain gray matter structure and function in 8-year-old children who were predominantly breastfed or fed cow's milk formula as infants. MATERIALS AND METHODS: Forty-two healthy children (breastfed: n = 22, 10 boys and 12 girls; cow's milk formula: n = 20, 10 boys and 10 girls) were studied by using structural MR imaging (3D T1-weighted imaging) and blood oxygen level-dependent fMRI (while performing tasks involving visual perception and language functions). They were also administered standardized tests evaluating intelligence (Reynolds Intellectual Assessment Scales) and language skills (Clinical Evaluation of Language Fundamentals). RESULTS: Total brain gray matter volume did not differ between the breastfed and cow's milk formula groups. However, breastfed children had significantly higher (P < .05, corrected) regional gray matter volume measured by voxel-based morphometry in the left inferior temporal lobe and left superior parietal lobe compared with cow's milk formula-fed children. Breastfed children showed significantly more brain activation in the right frontal and left/right temporal lobes on fMRI when processing the perception task and in the left temporal/occipital lobe when processing the visual language task than cow's milk formula-fed children. The imaging findings were associated with significantly better performance for breastfed than cow's milk formula-fed children on both tasks. CONCLUSIONS: Our findings indicated greater regional gray matter development and better regional gray matter function in breastfed than cow's milk formula-fed children at 8 years of age and suggested that infant diets may have long-term influences on brain development in children.

RNA-seq analysis of the rat placentation site reveals maternal obesity-associated changes in placental and offspring thyroid hormone signaling.

INTRODUCTION: In animal models, maternal obesity (OB) leads to augmented risk of offspring OB. While placental function is influenced by maternal habitus, the effect of maternal obesity on the interacting zones of the placenta [the labyrinth (LZ), junctional (JZ) and metrial gland (MG)] remains unknown. METHODS: Using a rat maternal obesity model, we conducted transcriptomic profiling of the utero-placental compartments and fetal liver (FL) at dpc 18.5, in conjunction with analyses of mRNA expression of key thyroid hormone (TH) signaling genes in the placenta, fetus and weanling offspring. RESULTS AND DISCUSSION: Gene expression analysis of placenta and offspring revealed that each utero-placental compartment responds distinctly to maternal OB with changes in inflammatory signaling, lipid metabolism and hormone stimulus being the predominant effects. OB-induced alterations in 17 genes were confirmed by qPCR, including reductions in thyrotropin-releasing hormone (Trh) in JZ. We further characterized mRNA and protein expression of TH signaling regulators including deiodinases (Dio), TH receptors (Tr), and downstream targets (uncoupling proteins (Ucp)). A concerted down-regulation of multiple facets of thyroid hormone signaling in the JZ and FL was observed. JZ expression of thyroid hormone signaling components Trh, Dio2, Trα, and Ucp2 were negatively associated with maternal leptin. mRNA expression of TRH, TRß and UCP1 were also decreased in term placenta from OB women. Finally, our studies identified persistent impairments in expression of TH related genes in tissues from offspring of obese dams. CONCLUSIONS: The role of lower placental thyroid expression is worthy of further study as a possible pathway that leads to low energy metabolism and obesity in animals born to obese mothers.

Maternal obesity is associated with a lipotoxic placental environment.

Maternal obesity is associated with placental lipotoxicity, oxidative stress, and inflammation, where MAPK activity may play a central role. Accordingly, we have previously shown that placenta from obese women have increased activation of MAPK-JNK. Here, we performed RNA-sequencing on term placenta from twenty-two subjects who were dichotomized based on pre-pregnancy BMI into lean (BMI 19-24 kg/m(2); n = 12) and obese groups (BMI, 32-43 kg/m(2); n = 12). RNA-seq revealed 288 genes to be significantly different in placenta from obese women by ≥ 1.4-fold. GO analysis identified genes related to lipid metabolism, angiogenesis, hormone activity, and cytokine activity to be altered in placenta from obese women. Indicative of a lipotoxic environment, increased placental lipid and CIDEA protein were associated with decreased AMPK and increased activation of NF-κB (p65) in placenta from obese women. Furthermore, we observed a 25% decrease in total antioxidant capacity and increased nuclear FOXO4 localization in placenta from obese women that was significantly associated with JNK activation, suggesting that maternal obesity may also be associated with increased oxidative stress in placenta. Maternal obesity was also associated with decreased HIF-1α protein expression, suggesting a potential link between increased inflammation/oxidative stress and decreased angiogenic factors. Together, these findings indicate that maternal obesity leads to a lipotoxic placental environment that is associated with decreased regulators of angiogenesis and increased markers of inflammation and oxidative stress.

A comprehensive analysis of the human placenta transcriptome.

As the conduit for nutrients and growth signals, the placenta is critical to establishing an environment sufficient for fetal growth and development. To better understand the mechanisms regulating placental development and gene expression, we characterized the transcriptome of term placenta from 20 healthy women with uncomplicated pregnancies using RNA-seq. To identify genes that were highly expressed and unique to the placenta we compared placental RNA-seq data to data from 7 other tissues (adipose, breast, hear, kidney, liver, lung, and smooth muscle) and identified several genes novel to placental biology (QSOX1, DLG5, and SEMA7A). Semi-quantitative RT-PCR confirmed the RNA-seq results and immunohistochemistry indicated these proteins were highly expressed in the placental syncytium. Additionally, we mined our RNA-seq data to map the relative expression of key developmental gene families (Fox, Sox, Gata, Tead, and Wnt) within the placenta. We identified FOXO4, GATA3, and WNT7A to be amongst the highest expressed members of these families. Overall, these findings provide a new reference for understanding of placental transcriptome and can aid in the identification of novel pathways regulating placenta physiology that may be dysregulated in placental disease.

Dietary fat source alters hepatic gene expression profile and determines the type of liver pathology in rats overfed via total enteral nutrition.

To determine if dietary fat composition affects the progression of nonalcoholic fatty liver disease (NAFLD), we overfed male Sprague-Dawley rats low (5%) or high (70%) fat diets with different fat sources: olive oil (OO), corn oil (CO), or echium oil (EO), with total enteral nutrition (TEN) for 21 days. Overfeeding of the 5% CO or 5% EO diets resulted in less steatosis than 5% OO (P < 0.05). Affymetrix array analysis revealed significant differences in hepatic gene expression signatures associated with greater fatty acid synthesis, ChREBP, and SREBP-1c signaling and increased fatty acid transport (P < 0.05) in the 5% OO compared with 5% CO group. The OO groups had macrosteatosis, but no evidence of oxidative stress or necrosis. The 70% CO and 70% EO groups had a mixture of micro- and macrosteatosis or only microsteatosis, respectively; increased oxidative stress; and increased necrotic injury relative to their respective 5% groups (P < 0.05). Oxidative stress and necrosis correlated with increasing peroxidizability of the accumulated triglycerides. Affymetrix array analysis comparing the 70% OO and 70% CO groups revealed increased antioxidant pathways and lower expression of genes linked to inflammation and fibrosis in the 70% OO group. A second study in which 70% OO diet was overfed for 50 days produced no evidence of progression of injury beyond simple steatosis. These data suggest that dietary fat type strongly influences the progression of NAFLD and that a Mediterranean diet high in olive oil may reduce the risk of NAFLD progressing to nonalcoholic steatohepatitis.

QMR: validation of an infant and children body composition instrument using piglets against chemical analysis.

OBJECTIVE: This study was undertaken to validate the first quantitative nuclear magnetic resonance (QMR) instrument designed and built to assess body composition in children from birth to adulthood (up to 50 kg). DESIGN: A total of 50 pigs weighing between 3.0 and 49.1 kg were studied. Each piglet's body composition was assessed by quantitative nuclear magnetic resonance (QMR, EchoMRI-AH small), whole-body chemical carcass analysis for lipid and water content, and dual-energy X-ray absorptiometry (DXA, Hologic QDR 4500, using infant or adult whole-body scan acquisition programs where appropriate). Twenty-five piglets (3.1-47.2 kg) were randomly selected to calibrate the QMR instrument. The remaining 25 piglets (3.0-49.1 kg) were used to validate the instrument. RESULTS: The precision of QMR to estimate fat mass (FM), fat-free mass (FFM) and total body water (TBW) for five consecutive scans was excellent (1.3, 0.9 and 0.9%, respectively). QMR measures of FM were highly and significantly correlated with chemical carcass analyses and DXA measures (r(2)=0.99 and r(2)=0.98, respectively). QMR and DXA FFM results were highly correlated (R(2)=0.99, P<0.01). TBW measures were strongly correlated between QMR and carcass analyses (R(2)=0.99, P<0.01). QMR overestimated FM by 2% and DXA measures (using the infant and adult scan programs) overestimated FM by 15% on average. CONCLUSION: QMR provides precise and accurate measures of FM, FFM and TBW in piglets weighing up to 50 kg. As the piglet is considered to be an excellent model of human development, these data suggest that QMR should provide the opportunity to acquire valuable body composition data in longitudinal studies in children, which is not possible or practical with other commercially available instrumentation.

Soy protein isolate reduces hepatosteatosis in yellow Avy/a mice without altering coat color phenotype.

Agouti (A(vy)/a) mice fed an AIN-93G diet containing the soy isoflavone genistein (GEN) prior to and during pregnancy were reported to shift coat color and body composition phenotypes from obese-yellow towards lean pseudoagouti, suggesting epigenetic programming. Human consumption of purified GEN is rare and soy protein is the primary source of GEN. Virgin a/a female and A(vy)/a male mice were fed AIN-93G diets made with casein (CAS) or soy protein isolate (SPI) (the same approximate GEN levels as in the above mentioned study) for 2 wks prior to mating. A(vy)/a offspring were weaned to the same diets and studied at age 75 d. Coat color distribution did not differ among diets, but SPI-fed, obese A(vy)/a offspring had lower hepatosteatosis (P < 0.05) and increased (P < 0.05) expression of CYP4a 14, a PPARalpha-regulated gene compared to CAS controls. Similarly, weanling male Sprague-Dawley (SD) rats fed SPI had elevated hepatic Acyl Co-A Oxidase (ACO) mRNA levels and increased in vitro binding of PPARalpha to the PPRE promoter response element. In another hepatosteatosis model, adult SD rats fed a high fat/cholesterol diet, SPI reduced (P < 0.05) steatosis. Thus, 1) consumption of diets made with SPI partially protected against hepatosteatosis in yellow mice and in SD rats, and this may involve induction of PPARalpha-regulated genes; and 2) the lifetime (in utero, neonatal and adult) exposure to dietary soy protein did not result in a shift in coat color phenotype of A(vy)/a mice. These findings, when compared with those of previously published studies of A(vy)/a mice, lead us to conclude that: 1) the effects of purified GEN differ from those of SPI when GEN equivalents are closely matched; 2) SPI does not epigenetically regulate the agouti locus to shift the coat color phenotype in the same fashion as GEN alone; and 3) SPI may be beneficial in management of non-alcoholic fatty liver disease.

Effects of chronic ethanol on hepatic and renal CYP2C11 in the male rat: interactions with the Janus-kinase 2-signal transducer and activators of transcription proteins 5b pathway.

Chronic alcohol intake in male rats results in: 1) demasculinization of the GH pulse pattern; 2) reduced serum testosterone concentrations; and 3) decreased expression hepatic CYP2C11. Hepatic CYP2C11 expression is regulated by the male pattern of GH through the Janus-kinase/signal transducer and activators of transcription proteins (JAK/STAT) signal transduction pathway in the male rat. Renal CYP2C11 is regulated by testosterone, not GH. The involvement of the JAK/STAT5b signal transduction pathway in renal CYP2C11 signaling has not been studied. We tested the hypothesis that ethanol reduces CYP2C11 levels by interfering with the JAK/STAT5b pathway. Using a total enteral nutrition (TEN) model to feed rats a well-balanced diet, we have studied the effects of chronic ethanol intake (21 d) on hepatic and renal JAK/STAT pathway of adult male rats (8-10/group). We found decreased hepatic and renal expression of CYP2C11 in ethanol-fed rats with concomitant decreases in STAT5b and phospho-STAT5b, decreased in vitro hepatic STAT5b binding to a CYP2C11 promoter element and no effects on hepatic GHR levels. Ethanol caused tissue specific effects in phospho-JAK2 and JAK2, with increased levels in the liver, but decreased JAK2 expression in the kidney. We conclude that ethanol suppression of CYP2C11 expression is clearly associated with reductions in STAT5b levels, but not necessarily in reductions of JAK2 levels. The mechanisms underlying ethanol-induced suppression of STAT5b is yet to be determined, as is the question of whether this is secondary to hormonal effects or a direct ethanol effect.

Glucose inhibition of the induction of CYP2E1 mRNA expression by ethanol in FGC-4 cells.

1. Rats fed intragastrically with ethanol-containing diets made with low levels of carbohydrates have greater CYP2E1 induction than rats fed similar diets made with high carbohydrate levels. 2. FGC-4 rat hepatoma cells were used to test the hypothesis that carbohydrates could down-regulate ethanol-induced CYP2E1 induction. 3. FGC-4 cells grown in a glucose-free media and treated with 1-100 mM ethanol for 24 h exhibited a dose-dependent increase (p < 0.05) in CYP2E1, with maximum mRNA steady-state (3.8-fold) or protein (3.1-fold) levels measured at 30 or 100 mM ethanol, respectively. 4. In cells treated with 30 mM ethanol, a glucose concentration-dependent inhibition (p < 0.05) of CYP2E1 mRNA was observed between 2.5 and 10 mM glucose. 5. Induction by 30 mM ethanol of CYP2E1 protein was reduced in cells co-treated with 1 mM or greater glucose concentration and complete inhibition was measured with 5 mM glucose co-treatment. 6. These data demonstrate that under culture conditions of extremely low carbohydrate concentrations: (1) ethanol treatment of FGC-4 cells results in elevated steady-state levels of CYP2E1 mRNA and protein; and (2) glucose inhibits this increase. 7. It is concluded that glucose can negatively regulate CYP2E1 expression and could at least partially explain the greater induction of hepatic CYP2E1 in rats fed low carbohydrate ethanol-containing diets compared with high carbohydrate diets at the same ethanol level.

Estrogenic and antiproliferative properties of soy sapogenols in human breast cancer cells in vitro.

Two soy sapogenols, soyasapogenol A (SA) and soyasapogenol B (SB) were tested for their estrogenic activities in estrogen responsive MCF-7 or estrogen-insensitive MDA-MB-231 (MDA) human breast cancer cells. SB and SA had differential actives on cell proliferation with 10 microM SB being growth inhibitory to MDA cells with no significant effect at any concentration on MCF-7 cells. SA also inhibited MDA cell proliferation at 10 micro, but at this same dose stimulated a 2.5-fold increase in MCF-7 proliferation. SA (0.1-10 microM) induced pS2 mRNA levels and the induction was blocked by co-treatment of cells with the anti-estrogen ICI 182,780. SA also induced the formation of an ER-ERE DNA complex measured by electrophoretic mobility shift assay. In summary, these results show that soyasapogenol A is estrogenic, whereas soyasapogenol B is growth inhibitory.

Soy and whey proteins downregulate DMBA-induced liver and mammary gland CYP1 expression in female rats.

One possible mechanism by which diet may reduce cancer risk is through enhancement of metabolic systems that prevent activation of carcinogens or accelerate carcinogen inactivation. We studied the effects of diet and 7,12-dimethylbenz-(a)anthracene (DMBA) on hepatic and mammary gland CYP1A1, CYP1A2 and CYP1B1 enzymes in female Sprague-Dawley rats. Diets (AIN-93G) were fed from conception to adulthood, and DMBA was given by oral gavage at age 48-50 d. The protein sources of diets were casein (CAS), soy protein isolate (SPI) or whey protein hydrolysate (WPH). The DMBA-induced hepatic ethoxyresorufin-O-deethylase and methoxyresorufin-O-demethylase activities and CYP1A1 protein and mRNA expression were lower (P < 0.05) in SPI-fed rats compared with those fed casein. Differences in mammary gland CYP1 expression were also observed with decreased DMBA induction (P < 0.05) of all three CYP1 proteins and mRNAs in rats fed either SPI or WPH compared with those fed CAS. Most notable were the decreased constitutive and DMBA-induced mammary gland expression of CYP1B1 protein of 93 and 96%, respectively, in the SPI-fed rats relative to the CAS-fed controls. The diet-induced changes in CYP1 enzyme expression were consistent with changes in the AhR and ARNT transcription factors that regulate them. Decreased (P < 0.05) mammary constitutive AhR and ARNT proteins were measured in SPI-fed rats. There was also a 100% increase in constitutive AhR protein in the WPH-fed rats that paralleled a 100% increase in constitutive CYP1B1 protein in the mammary gland. These results demonstrate the importance of diet in regulation of phase I metabolism in liver and mammary gland, and suggest a potential mechanism by which soy or whey proteins reduce DMBA-induced mammary tumor incidence.

Developmental effects and health aspects of soy protein isolate, casein, and whey in male and female rats.

Dietary factors other than the traditional nutrients are found in the so-called functional foods. They are becoming increasingly recognized as potentially important for maintaining good health. Soybeans are rich in such factors thought to help prevent certain chronic diseases. Soy protein isolate (SPI) is one of the three major proteins used in infant formulas sold in the United States, with casein (CAS) and whey (WPH) proteins being the others. We have been studying the health effects of these proteins. Safety concerns have developed over the consumption of soy-based infant formula, partly because of the high circulating levels of the total isoflavones (phytoestrogens) during "critical periods of infant development." There is a paucity of data on developmental, physiological, neurophysiological, behavioral, metabolic, or molecular effects of soy phytochemicals in humans, especially during pregnancy and infancy. We have studied the effects of CAS, SPI, and WPH in short-term, long-term, and multigenerational studies in rats. Aside from minor differences in body weight gain profiles, CAS-, SPI- or WPH-fed rats did not differ in development, organ weights, in vitro hepatic metabolism of testosterone (T), or reproductive performance. However, some endocrine-related functions differed between rats fed these proteins. We found that SPI accelerated puberty in female rats (p < .05) and WPH delayed puberty in males and females, as compared with CAS (p < .05). Gender differences were also found in gonadectomy-induced steroid responses. Male rats had normal serum T levels, but female rats fed SPI had reduced serum 17beta-estradiol concentrations and a blunted 17beta-estradiol response to ovariectomy, as compared to rats fed CAS or WHP (p < .05). Female rats fed SPI or WHP or treated with genistein had reduced incidence of chemically induced mammary cancers (p < .05) compared to CAS controls, with WHP reducing tumor incidence by as much as 50%, findings that replicate previous results from our laboratory. Together, these results suggest gender-specific differences in development and certain endocrine responses among rats fed diets composed of a single protein source such as those used in infant formulas. Whether similar developmental effects occur in human infants is unknown, but unlikely because (1) most infants do not consume such diets throughout life as these rats did, and (2) no such effects have been reported in millions of American infants fed infant formula containing these proteins. The long-term health consequence implications of early diet exposure to SPI and WPH, such as reduced breast cancer incidence, are likely to be very positive.

Skeletal effects of developmental lead exposure in rats.

To identify possible direct and indirect mechanisms underlying the effects of lead on skeletal growth, 3 studies were conducted. In the first study, 1 male and 1 female pup/litter (n = 5 litters), were exposed ad libitum to 0, 825, or 2475 ppm lead acetate in the drinking water from gestational day 4 to euthanasia on day 55. Tibial strength was tested by 3-point bending and plasma levels of vitamin D metabolites were measured. A dose-dependent decrease of the load to failure was demonstrated but only in male pups. No differences in plasma levels of vitamin D metabolites were observed. In the second study, conducted to test if hormone treatment would attenuate the lead deficits, male and female pups were exposed to 0 or 2475 ppm lead acetate and then, from 30-60 days of age, received either saline vehicle, L-dopa, testosterone (males only), dihydrotestosterone (DHT, males only), or estradiol (females only). Lead exposure significantly reduced somatic growth, longitudinal bone growth, and bone strength during the pubertal period. Sex steroid replacement did not restore skeletal parameters in lead-exposed rats. L-Dopa increased plasma insulin-like growth factor 1 (IGF(1)) concentrations, rates of bone growth, and bone strength measures in controls while having no effect in lead-exposed pups. The third study was conducted at 100 days of age, when endocrine parameters have been shown to be normalized, to test for effects of lead exposure on bone formation during tibial limb lengthening (distraction osteogenesis, DO). Both DO gap x-ray density and proximal new endosteal bone formation were decreased in the distraction gaps of the lead-treated animals (p < 0.01). In conclusion, lead exposure reduced somatic growth, longitudinal bone growth, and bone strength during the pubertal period, and these effects could not be reversed by a growth hormone (GH) axis stimulator or by sex-appropriate hormones. Finally, lead exposure appears to specifically inhibit osteoblastogenesis in vivo in adult animals.

The effect of aging on distraction osteogenesis in the rat.

The effect of age on bone formation in the limb lengthening model of distraction osteogenesis (DO) was investigated in two studies using Sprague-Dawley (SD) rats from two colonies at various ages (CAMM: 9 vs 24 months, Harlan: 4 vs 24 months). External fixators were placed on the right tibiae of 30 male SD rats (20 CAMM, 10 Harlan) and mid-diaphyseal osteotomies were performed. Distraction was performed at 0.2 mm bid for 20 days (CAMM) or 14 days (Harlan). The experimental (DO) and control (contra-lateral) tibiae were removed for high-resolution radiography and decalcified histology. Videomicroscopy was used to quantitate radiodensity, histology (matrix type) and relative areas of cell proliferation, which was identified by proliferating cell nuclear antigen (PCNA) immunochemistry. Both studies demonstrated an age-related decrease in the percent mineralized bone (radiodensity) in the distraction gap (CAMM 9 vs 24 months: 68% vs 51%, P < 0.003; Harlan 4 vs 24 months: 95% vs 36%, P < 0.001) and no significant colony or distraction time-specific difference was seen between the two colonies of 24-month-old rats. Histology was performed on the Harlan rats. The DO gaps in the 24-month-old rats demonstrated less endosteal new bone compared to the 4-month-old rats (P < 0.01), but equivalent periosteal new bone. In 4-month-old rats, PCNA-immunostained cells were organized along the primary matrix front (where the first deposition of osteoid occurs) extending across both periosteal and endosteal surfaces. In 24-month-old rats, PCNA+ cells were organized in zones along the periosteal new bone fronts only and irregularly scattered throughout the endosteal gap within a fibrovascular non-ossifying matrix. These results indicate that 24-month-old rats have a relative deficit in endosteal bone formation which may not be related to cell proliferation but rather to cell organization. This model reflects the clinical situation where radiographic findings in older patients demonstrate significant delays in mineralization during DO. We believe this model of DO in aged rats presents unique in vivo opportunities to test hypotheses concerning (1) the effects of aging on bone repair, (2) the effects of pharmacological agents on bone repair in a geriatric setting, and (3) to study the mechanisms underlying DO.

Development of tensile strength during distraction osteogenesis in a rat model.

These studies were designed to determine the reliability of in vitro tensile testing to measure the temporal development of regenerate bone strength in rats during limb lengthening (distraction osteogenesis, DO). External fixators were placed on the right tibiae of 36 virus-free, 400-450 g male Sprague Dawley rats, and osteotomies (n = 33) were performed. Distraction was initiated the following morning (0 day latency) at 0.4 mm/day and continued to day 20. The 8 mm gap was allowed to consolidate for up to 50 days (day 70 postop). Contralateral unoperated and operated (fixator only) controls were included. On days 20, 30, 50 and 70 postop, the rats were anesthetized, and their tibiae were radiographed prior to undergoing sacrifice for histological or tensile analysis. On day 70, an additional group was tested by three-point bending. Radiodensity measurements demonstrated progressive mineralization of the DO gap, and histology confirmed typical intramembranous ossification of collagen bundles oriented parallel to the distraction force. Tensile stiffness increased significantly between days 20 and 30 postop, this increase correlated with initial radiographic and histologic bridging of the DO gap. Energy to failure and ultimate tensile strength increased progressively to day 70. At day 70, the force to failure for three-point bending was 65% of control tibiae. In conclusion, in vitro tensile testing provides a reliable method to test the development of structural integrity during the early stages of DO. Therefore, the biomechanical effects of postulated modulators of bone repair can be measured during early stages (bone formation, bridging, early consolidation) of DO in a rat model.