Gold Nanoparticle Toxicity in Mice and Rats: Species Differences.

Nanotoxicity studies are greatly needed to advance nanomedical technologies into clinical practice. We assessed the toxic effects of a single intravenous exposure to commercially available gold nanoparticles (GNPs) in mice and rats. Fifteen-nm GNPs were purchased and independently characterized. Animals were exposed to either 1,000 mg GNPs/kg body weight (GNP group) or phosphate-buffered saline. Subsets of animals were euthanized and samples collected at 1, 7, 14, 21, and 28 days postexposure. Independent characterization demonstrated that the physicochemical properties of the purchased GNPs were in good agreement with the information provided by the supplier. Mice exposed to GNPs developed granulomas in the liver and transiently increased serum levels of the pro-inflammatory cytokine interleukin-18. No such alterations were found in rats. While there was no fatality in mice post-GNP exposure, a number of the rats died within hours of GNP administration. Differences in GNP biodistribution and excretion were also detected between the two species, with rats having a higher relative accumulation of GNPs in spleen and greater fecal excretion. In conclusion, GNPs have the ability to incite a robust macrophage response in mice, and there are important species-specific differences in their biodistribution, excretion, and potential for toxicity.

Mitigating or exacerbating effects of maternal-fetal programming of female mice through the food choice environment.

Humans live, eat, and become overweight/obese in complex surroundings where there are many available food choices. Prenatal exposure to poor food choices predisposes offspring to increased negative health risks, including obesity. Many animal experiments have analyzed intergenerational body weight parameters in an environment without food choices, which may not be directly translatable to the human food environment. In this study, offspring from mothers with a defined high-fat diet (HFD) or low-fat diet (LFD) were arbitrarily assigned to either an exclusively LFD or HFD or to a diet where they have a choice between LFD and HFD (choice diet). Offspring displayed negative outcomes of increased body weight, body fat, serum leptin, and blood glucose levels when given the choice diet compared with offspring on the LFD. Conversely, improved energy expenditure was found for offspring given the choice diet compared with offspring from HFD dams given LFD. In addition, maternal diet-specific influences on offspring metabolic parameters were identified, especially in offspring from HFD dams, including positive outcomes of reduced leptin in LFD offspring, reduced corticosterone and cholesterol levels in HFD offspring, and increased exercise levels in choice offspring, as well as the negative outcome of increased calorie intake in LFD offspring from HFD dams. This defined model can now be used as the basis for future studies to characterize the cycle of inter- and intragenerational obesity and whether more realistic diet environments, especially those including choice, can mitigate phenotype.

Prevalence and characterization of methicillin-resistant Staphylococcus aureus isolates from healthy university student athletes.

BACKGROUND: The prevalence of methicillin-resistant Staphylococcus aureus (MRSA) has been increasing in the general population, and there is concern that close or physical contact, such as in professional and collegiate sports, may increase spread of MRSA. We sought to determine the prevalence of MRSA colonization of male and female athletes from 9 different sports at a major, Division I University during a 12-week period, and determine the USA and SCCmec type from select isolates. METHODS: Swabs for culture of MRSA were obtained from nasal, axillary, and inguinal sites from healthy, asymptomatic student athletes and support staff each week for 12 weeks. Select MRSA isolates were typed by pulsed field gel electrophoresis (PFGE), and the genes encoding for MecA, cassette chromosome recombinase (Ccr), and several toxins were determined by multiplex polymerase chain reaction (PCR). Discrepant results were clarified by multi-locus sequence typing (MLST) and spa typing. RESULTS: Thirty-five percent (78/223) of test subjects were positive for MRSA during the study period, resulting in isolation of 139 MRSA isolates. However, 47% (37/78) of MRSA-positive participants carried MRSA in axillary or inguinal sites, but not in the anterior nares. There was significant correlation between MRSA carriage and participation in wrestling (76%, 19/25; adjusted odds ratio 29.7, 95% CI 5.8-151.5) and baseball (44%, 17/39; adjusted odds ratio 4.4, 95% CI 1.1- 17.4), compared with a staff prevalence of 18.1% (4/22), but other factors were not examined. Multiplex PCR analysis indicated that of the 32 isolates examined 26 could be typed, and all of these carried the SCCmec type IV cassette. PFGE typing identified USA types 300, 400, 500, 700, and 800. However, one isolate was not a known USA type, but was identified as a novel ST951 by MLST, and as spa type t216. Of the strains typed from the same individual, there was consistency, but also variation and alternation of the SCCmec and spa types isolated from individual subjects. Various staphylococcal toxin genes were identified in 31 of the 32 isolates analyzed. CONCLUSIONS: Colonization by MRSA was greater in some student athletes than the average carriage rate for the general population, and only 53% of MRSA carriers were identified by nasal cultures. Carriage of MRSA clones on the same individual and transmission to contacts could vary over time, indicating colonization can be a dynamic process that may be difficult to control.

Current thoughts on maternal nutrition and fetal programming of the metabolic syndrome.

Chronic diseases such as type 2 diabetes and cardiovascular disease are the leading cause of death and disability worldwide. Although the metabolic syndrome has been defined in various ways, the ultimate importance of recognizing this combination of disorders is that it helps identify individuals at high risk for both type 2 diabetes and cardiovascular disease. Evidence from observational and experimental studies links adverse exposures in early life, particularly relating to nutrition, to chronic disease susceptibility in adulthood. Such studies provide the foundation and framework for the relatively new field of developmental origins of health and disease (DOHaD). Although great strides have been made in identifying the putative concepts and mechanisms relating specific exposures in early life to the risk of developing chronic diseases in adulthood, a complete picture remains obscure. To date, the main focus of the field has been on perinatal undernutrition and specific nutrient deficiencies; however, the current global health crisis of overweight and obesity demands that perinatal overnutrition and specific nutrient excesses be examined. This paper assembles current thoughts on the concepts and mechanisms behind the DOHaD as they relate to maternal nutrition, and highlights specific contributions made by macro- and micronutrients.

Production of a type 2 maternal diabetes rodent model using the combination of high-fat diet and moderate dose of streptozocin.

INTRODUCTION: Pregnancy may be complicated by maternal diabetes. The following experiments were performed in an attempt to produce mouse models of insulin-resistant maternal diabetes. METHODS: CD1 females received 200 mg/kg streptozocin (STZ) to model insulin-dependent diabetes (T1 group). Another group of females (T2 group) was put on a HFD 4 weeks before receiving 100 mg/kg STZ. After 4 additional weeks of HFD, hyperglycemic females were separated and bred. In another experiement, CD1 females were fed a HFD for 4 weeks before receiving an intravenous (GDM1 group) or intraperitoneal (GDM2 group) injection of 100 mg/kg STZ. Females from GDM2 group were bred at the same day of the STZ injection. Females from GDM1 group were bred 4 weeks after the STZ injection. RESULTS AND CONCLUSION: About 25% of the females from T2 group became hyperglycemic after 4 weeks of the injection of STZ. Fifty percent of the females from GDM1 group reached hyperglycemic levels greater than 250 mg/dl during pregnancy. The combination of HFD and moderate STZ in CD1 mice therefore produced hyperglycemic females; however numbers of these mice were somewhat low.

Late-gestation ventricular myocardial reduction in fetuses of hyperglycemic CD1 mice is associated with increased apoptosis.

BACKGROUND: Previous work in our laboratory showed reduced myocardium and dilated ventricular chambers in gestation day (GD) 17 hearts that were collected from hyperglycemic CD1 mouse dams. Pre-breeding maternal immune stimulation, using Freund's complete adjuvant (FCA), diminished the severity of these fetal heart lesions. The following experiments were performed to detect possible changes in fetal heart apoptotic cell death, under hyperglycemic conditions and with or without maternal immune stimulation. METHODS: Female CD1 mice were injected with 200 mg/kg of streptozocin (STZ) to induce insulin-dependent diabetes mellitus. Half of these mice received prior FCA injection. Fetal hearts were collected on GD 17 and myocardial apoptotic cells were quantified using flow cytometry. A panel of apoptosis regulatory genes (Bcl2, p53, Casp3, Casp9, PkCe) was then examined in the fetal myocardium using RT-PCR. RESULTS: Early apoptotic cells and late apoptotic/necrotic cells were significantly increased in fetal hearts from STZ or STZ+FCA dams. Pre-treatment with FCA reduced late apoptotic/necrotic cells to control level, suggesting some cell death protection was rendered by FCA. Paradoxically in the face of such increased cell death, the expression of pro-apoptotic genes Casp3 and Casp9 was decreased by diabetes, while the anti-apoptotic gene Bcl2 was increased. CONCLUSIONS: Maternal hyperglycemia causes dys-regulated apoptosis of fetal myocardial cells. Such effect may be prevented by maternal immune stimulation.

Gestational high saturated fat diet alters C57BL/6 mouse perinatal skeletal formation.

BACKGROUND: Our present work joins growing evidence that gestational environment (maternal nutrition, health, and chemical exposures) strongly influences prenatal development (www.thebarkertheory.org). The present study suggests that maternal consumption of a diet high in saturated fats (HFD), which approximates the macronutrient content of fast food, impairs perinatal skeletal development. METHODS: In this study, administration of HFD (32% saturated fat) for one month prior to conception and throughout gestation in C57BL/6J mice was associated with a marked reduction in late-gestation fetal skeletal developmental delay that included shorter long bone lengths, decreased average bone mineral density (ABMD; 20%), lower total bone volume (TBV; 45%), and shorter crown-to-rump length (C-R; 12%), as compared to controls. RESULTS: A putative mechanism linking prenatal HFD to dysregulated fetal osteogenesis is HFD-induced oxidative stress (OS), which has been shown in our laboratory to cause placental labyrinthine vascular damage and impaired fetal signaling pathways associated with osteogenesis (Liang et al., unpublished data). CONCLUSIONS: The theory of HFD-associated, OS-mediated placental damage and skeletal pathogenesis was supported by demonstrating a protective effect of the dietary antioxidant quercetin (Q) against HFD-associated fetal skeletal developmental delay. Improved understanding of the role of HFD and elevated OS in fetal skeletal development will help to more completely elucidate the importance of the prenatal environment to fetal formation, and will be applied to better understand the contribution of the fetal environment to long-term risk of adult-onset disease.

Intrauterine exposure to high saturated fat diet elevates risk of adult-onset chronic diseases in C57BL/6 mice.

BACKGROUND: The developmental environment is thought to determine, in part, lifelong metabolic parameters and risk of adult disease. Effects of maternal malnutrition on fetal growth have been studied extensively, and the role of poor prenatal diet in elevating lifelong risk of cardiovascular and metabolic disease has been well characterized (www.thebarkertheory.com). However, the contribution of gestational high saturated fat diet (HFD) to adult-onset metabolic disease and skeletal dysfunction has only recently been recognized, and as such is incompletely understood. METHODS: The present study evaluates the pathophysiologic mechanisms linking gestational HFD (approximating the macronutrient content of fast food) and elevated oxidative stress (OS) to adult-onset skeletal, cardiovascular, and metabolic dysfunction. RESULTS: Results of this study demonstrate that adult offspring of dams fed HFD during pregnancy exhibited adult hyperglycemia, insulin resistance, obesity, and hypertension, despite being fed healthy standard rodent chow throughout postnatal life. These offspring also showed significantly lower femoral epiphyseal average bone mineral density (ABMD) at 6 months of age, and dysregulation of distal femoral trabecular architecture at 12 months of age, characteristic of osteoporosis. Incidence of these adult-onset adverse skeletal and metabolic effects was reduced by supplementing the pregnant dam with the antioxidant (quercetin, Q) during pregnancy. CONCLUSIONS: Collectively, these data suggest that offspring of dams who consume a diet rich in saturated fats during pregnancy are at increased risk of adult-onset chronic disease. Additionally, these chronic diseases were determined to be in-part OS-mediated, and preventable by increasing a prenatal dietary antioxidant; this knowledge offers both a putative mechanism of disease pathogenesis and suggests a potential preventive strategy.

Micro-CT evaluation of murine fetal skeletal development yields greater morphometric precision over traditional clear-staining methods.

Traditional techniques for quantification of murine fetal skeletal development (gross measurements, clear-staining) are severely limited by specimen processing, soft tissue presence, diffuse staining, and unclear landmarks between which to make measurements. Nondestructive microcomputed tomography (micro-CT) imaging is a versatile, well-documented tool traditionally used to generate high-resolution 3-D images and quantify microarchitectural parameters of trabecular bone. Although previously described as a tool for phenotyping fetal murine specimens, micro-CT has not previously been used to directly measure individual fetal skeletal structures. Imaging murine fetal skeletons using micro-CT enables the researcher to nondestructively quantify fetal skeletal development parameters including limb length, total bone volume, and average bone mineral density, as well as identify skeletal malformations. Micro-CT measurement of fetal limb lengths correlates well with traditional clear-staining methods (83.98% agreement), decreases variability in measurements (average standard errors: 6.28% for micro-CT and 10.82% for clear-staining), decreases data acquisition time by eliminating the need for tissue processing, and preserves the intact fixed fetus for further analysis. Use of the rigorous micro-CT technique to generate 3-D images for digital measurement enables isolation of skeletal structures based on degree of mineralization (local radiodensity), eliminating the complications of blurred stain boundaries and soft tissue inclusion that accompany clear-staining and gross measurement techniques. Microcomputed tomography provides a facile, accurate, and nondestructive method for determining the developmental state of the fetal skeleton using not only limb lengths and identification of malformations, but total skeletal bone volume and average skeletal mineral density as well.

Mid-gestation exposure of C57BL/6 mice to 2,3,7,8-tetrachlorodibenzo-p-dioxin causes postnatal morphologic changes in the spleen and liver.

Pregnant C57BL/6 mice were exposed to 5 microg/kg 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) or vehicle by oral gavage between gestation days (GDs) 11 and 13. The thymus, spleen, and liver of the pups were examined histologically, and cell surface antigen expression was assessed on postnatal days (PNDs) 1, 14, 25, and 46. In addition to the expected decrease in thymic weight on PND 1, TCDD caused an increase in splenic weight on PND 14 and in hepatic weight on PNDs 14 and 25. The apoptotic index was increased and the corticomedullary border poorly defined in thymuses of TCDD-exposed mice on PND 1, but not at later endpoints. T lymphocytes were increased and B lymphocytes decreased in spleens of the TCDD-exposed mice on PND 46. TCDD-exposed mice had a nearly significant (p =.051) decrease in the number of splenic germinal centers on PND 46. Foci of extramedullary hematopoiesis (EMH) were increased in number in the livers of TCDD-exposed mice on PND 14, suggesting possible increased production of immune cells of unknown phenotype and function in this organ. These results suggest that late-gestation thymic architectural changes caused by TCDD resolve shortly after birth: however, abnormalities in other immunologically important areas may appear later in postnatal life.

2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) or diethylstilbestrol (DES) cause similar hematopoietic hypocellularity and hepatocellular changes in murine fetal liver, but differentially affect gene expression.

TCDD and DES have immunotoxic effects, including selective diminution of T lymphocyte progenitors in the fetal liver. The histologic presentation of fetal liver after exposure to either chemical has not been described. Similarly, limited information exists regarding mechanisms by which TCDD or DES may alter fetal hematopoiesis. Treatment of pregnant C57BL/6 mice with either 10 micro g/kg/day TCDD or 48 micro g/kg/day DES on gestation days (gd) 14 and 16 led to increased fetal liver weight on gd 18. Moderate anisocytosis and anisokaryosis with increased cytoplasmic and nuclear sizes, and increased cytoplasmic basophilia were present within hepatocytes after TCDD or DES. Both chemicals also decreased the presence of hematopoietic cells, however megakaryocyte numbers were unaffected. In contrast to these similar outcomes, real time quantitative PCR using a preliminary panel of 4 genes suggested that the chemicals act through different gene targets. TCDD increased c-jun gene expression in fetal liver, and decreased p53 without alteration in bcl-2 expression, indicating possible pro-proliferative and antiapoptotic effects. DES decreased c-jun and bcl-2, without altering p53, suggesting a shift away from proliferation. Both agents decreased PKCalpha expression, which may suggest shared decreased phosphorylation of substrates required for normal cell cycle progression.

Aortic and ventricular dilation and myocardial reduction in gestation day 17 ICR mouse fetuses of diabetic mothers.

BACKGROUND: Maternal diabetes mellitus is associated with increased fetal teratogenesis, including cardiovascular defects. Information regarding cardiovascular changes in late-gestation fetal mice, related to maternal hyperglycemia, is not present in the literature. METHODS: Late-gestation fetal heart and great vessel morphology were analyzed in fetuses from control and diabetic mice. Female ICR mice were injected with streptozocin (200 mg/kg IP) prior to mating to induce diabetes (n = 8). Nonhyperglycemic females were used as controls (n = 8). At day 17 of gestation, females were euthanized and one fetus was arbitrarily selected per litter to analyze the heart and great vessels. Six additional fetuses from different litters, showing external malformations (spina bifida and/or exencephaly), were also evaluated from the diabetic group. Fetal thoraxes were processed using routine histopathologic techniques, and 7-mum transversal sections were stained with hematoxylin-eosin. Digital images of sections were made and analyzed using NIH Image J software to compare regional cardiac development. Student's t tests for means were performed to determine differences between groups (p < .05). RESULTS: Maternal hyperglycemia caused a dilation of late-gestation fetal ventricular chambers, a reduction of total ventricular myocardial area, and an increase in transversal ascending thoracic aortic area. Three of six fetuses that displayed external malformations showed an overt cardiac defect, beyond the ventricular and myocardial changes. CONCLUSIONS: Maternal hyperglycemia altered morphology of the late-gestation fetal mouse heart. Postnatal persistence or consequences of late-gestation heart chamber dilation and myocardial reduction are not yet known.

Maternal treatment with a high dose of CpG ODN during gestation alters fetal craniofacial and distal limb development in C57BL/6 mice.

Synthetic oligodeoxynucleotides (ODN) containing CpG motifs, characteristic of bacterial DNA, are currently being evaluated as vaccine adjuvants for inducing protective immunity. Recently, there is increasing pressure to vaccinate pregnant women against maternally transmitted diseases including AIDS and tetanus, as well as against potential bio-weapons such as anthrax. CpG vaccines are effective because they trigger transient increases in T(H)1 cytokine production. Recent literature suggests, however, that a shift toward a T(H)1 cytokine profile during pregnancy may increase the risk of fetal morphologic defects. On this basis, we hypothesized that exposure to CpG motifs during pregnancy could result in T(H)1 inflammation leading to adverse effects on fetal development. To address this hypothesis, pregnant C57BL/6 mice were injected with CpG ODN (0-300 microg/dam) and maternal and fetal outcomes were determined. Injection of dams with the highest dose of CpG ODN resulted in markedly increased fetal resorptions and craniofacial/limb defects, while lower doses had little, if any effects. Histological examination of placentas revealed cellular necrosis with mixed inflammation and calcification in the spongiotrophoblast layer and dysregulation of labyrinthine vascular development. Concomitant elevations in maternal serum cytokine levels were observed including interleukin (IL)-2, IL-10 and IL-12. Treatment with 300 microg of non-CpG ODN did not cause any adverse effects. The 300 microg dose of CpG ODN used in the present study is 30-fold higher than the highest dose that has been administered to humans during clinical trials. These results suggest that the induction of T(H)1 cytokines during pregnancy by CpG motifs may potentially increase the risk of fetal loss and morphologic defects in mice, at least at high doses, and support the need for further investigation of teratogenesis that may result from exposure to vaccine adjuvants designed to produce T(H)1 cytokine profile shifts.

Reduction in diabetes-induced craniofacial defects by maternal immune stimulation.

BACKGROUND: Maternal diabetes can induce a number of developmental abnormalities in laboratory animals and humans, including facial deformities and defects in neural tube closure. The incidence of birth defects in newborns of diabetic women is approximately 3-5 times higher than among non-diabetics. In mice, non-specific activation of the maternal immune system can reduce fetal abnormalities caused by diverse etiologies, including diabetes induced neural tube defects. This study was conducted to determine whether non-specific maternal immune stimulation could reduce diabetes-induced craniofacial defects as well. METHODS: Maternal immune function was stimulated before streptozocin (STZ) treatment by maternal footpad injection with Freund's complete adjuvant (FCA), maternal intraperitoneal (i.p.) injection with granulocyte-macrophage colony-stimulating factor (GM-CSF), or maternal i.p. injection with interferon-gamma (IFNgamma). Streptozocin (200 mg/kg i.p.) was used to induce hyperglycemia (26-35 mmol blood glucose) in female ICR mice before breeding. Fetuses from 12-18 litters per treatment group, were collected at Day 17 of gestation. RESULTS: Craniofacial defects were observed in fetuses from all hyperglycemic groups. The incidence of defects was significantly decreased in fetuses from dams immune stimulated with IFNgamma or GM-CSF. The most common defects were reduced maxillary and mandibular lengths. Both were prevented by maternal stimulation with GM-CSF. CONCLUSION: Maternal immune stimulation reduced the incidence of diabetic craniofacial embryopathy. The mechanisms for these protective effects are unknown but may involve maternal or fetal production of cytokines or growth factors that protect the fetus from the dysregulatory effects of hyperglycemia.

Immunotoxic effects of cis-urocanic acid exposure in C57BL/6N and C3H/HeN mice.

Exposure to ultraviolet radiation results in increased levels of intradermal cis-urocanic acid (cUCA) and alters cutaneous immunity by interfering with processing and presentation of antigen by Langerhans cells. Reports on effects of systemic immunotoxicity with 30 day cUCA exposure in laboratory rodents include thymic atrophy, thymic hypocellularity and decreased T-cell-mediated immunity; however, immune effects of single exposure or 5 day cUCA administration, which may better mimic human exposures, are poorly defined. The present study initially evaluated immune effects of single, 5 day, and 4 week cUCA exposure in C57BL/6N mice. Single administration of intradermal cUCA resulted in decreased splenocyte phagocytosis that persisted for 30 days after cUCA exposure. Five day consecutive cUCA exposure decreased numbers of phenotypically mature CD4(+)CD8(-) and CD4(-)CD8(+) (single positive) thymocytes, increased CD4(+)CD8(+) (double positive) immature thymocytes and increased splenocyte proliferation. Prolonged cUCA exposure (4 weeks) caused profound thymic hypocellularity and splenic hypercellularity and increased splenic macrophage chemiluminescence. Because of this apparent sensitivity of C57BL/6N mice to cUCA, thymic hypocellularity was compared between C57BL/6N and C3H/HeN mice dosed with cUCA, and was found to be more pronounced in the C57BL/6N strain. These results are an extension of previous conclusions on immune modulation caused by cUCA in the spleen and thymus. Further, the observed variation in sensitivity between the mouse strains is consistent with known genetic susceptibility of these strains to the immunomodulatory effects of exposure to sunlight.

A comparison of the cytologic and histologic features of meningiomas in four dogs.

The cytologic and histologic features of 2 intracranial and 2 spinal (extramedullary cervical) canine meningiomas were compared. Cerebrospinal fluid analysis in 2 cases revealed mild, mixed cell pleocytosis, primarily composed of small lymphocytes and monocytoid cells, with a moderate increase in total protein concentration. Cytologic features suggestive of meningioma included cells with both epithelial and mesenchymal characteristics and a tendency towards cell clustering. Tumor location also was useful in making a diagnosis. The 4 meningiomas differed histologically from one another, and included angioblastic, psammomatous, meningotheliomatous, and microcystic anaplastic types, which conformed to a classification scheme for human meningiomas. The classification scheme could not be applied to cytologic specimens.