Alpha(α)Klotho, a soluble transmembrane protein, facilitates calcium-phosphorus homeostasis through feedback between bone and kidney and is a potential systemic biomarker for bone-kidney health during spaceflight. We determined if: (1) plasma αKlotho was reduced after both spaceflight aboard the ISS and hindlimb unloading (HU); and (2) deficiency could be reversed with exercise. Both spaceflight and HU lowered circulating plasma αKlotho: plasma αKlotho recovered with exercise after HU.

Reduced knee weight-bearing from prescription or sedentary lifestyles are associated with cartilage degradation; effects on the meniscus are unclear. Rodents exposed to spaceflight or hind limb unloading (HLU) represent unique opportunities to evaluate this question. This study evaluated arthritic changes in the medial knee compartment that bears the highest loads across the knee after actual and simulated spaceflight, and recovery with subsequent full weight-bearing. Cartilage and meniscal degradation in mice were measured via microCT, histology, and proteomics and/or biochemically after: (1) ~ 35 days on the International Space Station (ISS); (2) 13-days aboard the Space Shuttle Atlantis; or (3) 30 days of HLU, followed by a 49-day weight-bearing readaptation with/without exercise. Cartilage degradation post-ISS and HLU occurred at similar spatial locations, the tibial-femoral cartilage-cartilage contact point, with meniscal volume decline. Cartilage and meniscal glycosaminoglycan content were decreased in unloaded mice, with elevated catabolic enzymes (e.g., matrix metalloproteinases), and elevated oxidative stress and catabolic molecular pathway responses in menisci. After the 13-day Shuttle flight, meniscal degradation was observed. During readaptation, recovery of cartilage volume and thickness occurred with exercise. Reduced weight-bearing from either spaceflight or HLU induced an arthritic phenotype in cartilage and menisci, and exercise promoted recovery.

Reduced weight bearing, and to a lesser extent radiation, during spaceflight have been shown as potential hazards to astronaut joint health. These hazards combined effect to the knee and hip joints are not well defined, particularly with low-dose exposure to radiation. In this study, we examined the individual and combined effects of varying low-dose radiation (≤1 Gy) and reduced weight bearing on the cartilage of the knee and hip joints. C57BL/6J mice (n = 80) were either tail suspended via hindlimb unloading (HLU) or remained full-weight bearing (ground). On day 6, each group was divided and irradiated with 0 Gy (sham), 0.1 Gy, 0.5 Gy or 1.0 Gy (n = 10/group), yielding eight groups: ground-sham; ground-0.1 Gy; ground-0.5 Gy; ground-1.0 Gy; HLU-sham; HLU-0.1 Gy; HLU-0.5 Gy; and HLU-1.0 Gy. On day 30, the hindlimbs, hip cartilage and serum were collected from the mice. Significant differences were identified statistically between treatment groups and the ground-sham control group, but no significant differences were observed between HLU and/or radiation groups. Contrast-enhanced micro-computed tomography (microCECT) demonstrated decrease in volume and thickness at the weight-bearing femoral-tibial cartilage-cartilage contact point in all treatment groups compared to ground-sham. Lower collagen was observed in all groups compared to ground-sham. Circulating serum cartilage oligomeric matrix protein (sCOMP), a biomarker for ongoing cartilage degradation, was increased in all of the irradiated groups compared to ground-sham, regardless of unloading. Mass spectrometry of the cartilage lining the femoral head and subsequent Ingenuity Pathway Analysis (IPA) identified a decrease in cartilage compositional proteins indicative of osteoarthritis. Our findings demonstrate that both individually and combined, HLU and exposure to spaceflight relevant radiation doses lead to cartilage degradation of the knee and hip with expression of an arthritic phenotype. Moreover, early administration of low-dose irradiation (0.1, 0.5 or 1.0 Gy) causes an active catabolic response in cartilage 24 days postirradiation. Further research is warranted with a focus on the prevention of cartilage degradation from long-term periods of reduced weight bearing and spaceflight-relevant low doses and qualities of radiation.

The developing fetus is highly sensitive to the carcinogenic effects of dietary and environmental carcinogens in cigarette smoke, charbroiled foods and air pollution following transplacental exposure. Oxidative metabolism of toxicants by both maternal and fetal tissue causes DNA damage which can ultimately result in the initiation of childhood cancers. Due to the latency of cancer formation, in utero exposure to carcinogens may even predispose the individual to cancers developing later in life. The goal of this study is to determine whether a proprietary muscadine grape extract (MGE) effectively reduces lung tumor formation in the fetus following transplacental exposure to a polycyclic hydrocarbon. The muscadine grape (Vitis rotundifolia) has a distinct phytochemical composition compared to other grape varieties and limited reports indicate that the extract has potential anti-tumor activity. BALB/c male mice were mated to C57BL/6 female mice to serve as a model for in utero exposure to environmental toxicants. The pregnant mice were treated on the 17th day of gestation by intraperitoneal injection with a 45 mg/kg dose of 3-methylcholanthrene dissolved in olive oil, to induce lung tumors in the fetus. An injection of the olive oil vehicle (0.5 mL/0.35 kg) served as the control. Day 0 was considered as the first day when the vaginal plug was detected. After weaning, cohorts of mice received drinking water alone or water with MGE (approximately 1.0 mg phenolics/25 g mouse) and were sacrificed at 12 months of age. Although all of the mice developed lung tumors, MGE significantly reduced tumor burden in female mice by approximately 48% as compared to the tumor tissue found in control mice drinking regular water (12.4 ± 1.9 g versus 6.5 ± 1.2 g, n = 26-32, p = 0.014). In addition, tumor multiplicity was higher in control mice as compared to MGE-treated animals (6.8 ± 0.4 observed tumors versus 4.2 ± 0.5 observed tumors, n = 26-32, p = 0.0001). Tumor tissue sections from mice administered MGE had a significant decrease in proliferating cell nuclear antigen as compared to tumors from control animals (22.4 ± 1.8 versus 14.2 ± 1.3, p = 0.0009), suggesting that in part MGE reduces tumor burden by decreasing tumor cell proliferation. Lung tumor sections from female mice drinking regular water or MGE were incubated with an antibody to the endothelial cell marker, CD34, and vessels were identified by a combination of morphology and positive CD34 immunoreactivity. MGE significantly reduced blood vessel density in lung tumor tissue as compared to female mice drinking regular water (20.5 ± 2.2 versus 11.3 ± 1.3, p = 0.0012), suggesting that the grape extract inhibits angiogenesis. These results indicate that MGE reduces both tumor proliferation and angiogenesis, leading to a decrease in tumor burden and multiplicity. Thus, MGE may represent a novel nutraceutical for the prevention of lung tumors induced by in utero exposure to environmental toxicants. Citation Format: Brooke E. Porter, Joseph E. Moore, Mark S. Miller, E. Ann Tallant, Patricia E. Gallagher. Muscadine grape extract reduces lung carcinogenesis in female mice exposed to 3-methylcholanthrene in utero. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4563. doi:10.1158/1538-7445.AM2015-4563

Data from the National Lung Screening Trial suggested that annual computed tomography (CT) screening of at-risk patients decreases lung cancer mortality by 20%. We assessed the effects of low-dose CT radiation in mice exposed to 4-(methylnitrosoamino)-1-(3-pyridyl)-1-butanone (NNK) to mimic the effects of annual CT screening in heavy smokers and ex-smokers. A/J mice were treated at 8 weeks with NNK followed 1 week later by 4 weekly doses of 0, 10, 30 or 50 mGy of whole-body CT and euthanized 8 months later. Irradiated mice exhibited significant 1.8- to 2-fold increases in tumor multiplicity in males (16.1 ± 0.8 versus 9.1 ± 1.5 tumors per mouse; P < 0.0001) and females (21.6 ± 0.8 versus 10.5 ± 1.4 tumors per mouse; P < 0.0001), respectively, compared with unirradiated mice with no dose effect observed; female mice exhibited higher sensitivity to radiation exposure than did males (P < 0.0001). Similar results were obtained when tumor area was determined. To assess if the deleterious effects of radiation could be prevented by antioxidants, female mice were fed a diet containing 0.7% N-acetylcysteine (NAC) starting 3 days prior to the first CT exposure and continuing for a total of 5 weeks. NAC prevented CT induced increases in tumor multiplicity (10.5 ± 1.2 versus 20.7 ± 1.5 tumors per mouse; P < 0.0001) back to levels seen in NNK/unirradiated mice (10.5 ± 1.2). Our data suggest that exposure of sensitive populations to CT radiation increases the risk of tumorigenesis, and that antioxidants may prevent the long-term carcinogenic effects of low-dose radiation exposure. This would allow annual screening with CT while preventing the potential long-term toxicity of radiation exposure.

A gated-7T magnetic resonance imaging (MRI) application is described that can accurately and efficiently measure the size of in vivo mouse lung tumors from ∼0.1 mm3 to >4 mm3. This MRI approach fills a void in radiation research because the technique can be used to noninvasively measure the growth rate of lung tumors in large numbers of mice that have been irradiated with low doses (<50 mGy) without the additional radiation exposure associated with planar X ray, CT or PET imaging. High quality, high resolution, reproducible images of the mouse thorax were obtained in ∼20 min using: (1) a Bruker 7T micro-MRI scanner equipped with a 60 mm inner diameter gradient insert capable of generating a maximum gradient of 1000 mT/m; (2) a 35 mm inner diameter quadrature radiofrequency volume coil; and (3) an electrocardiogram and respiratory gated Fast Low Angle Shot (FLASH) pulse sequence. The images had an in-plane image resolution of 98 μm and a 0.5 mm slice thickness. Tumor diameter measured by MRI was highly correlated (R2 = 0.97) with the tumor diameter measured by electronic calipers. Data generated with an initiation/promotion mouse model of lung carcinogenesis and this MRI technique demonstrated that mice exposed to 4 weekly fractions of 10, 30 or 50 mGy of CT radiation had the same lung tumor growth rate as that measured in sham-irradiated mice. In summary, this high-field, double-gated MRI approach is an efficient way of quantitatively tracking lung tumor development and progression after exposure to low doses of ionizing radiation.

The p53 tumor suppressor gene plays an essential role in tumorigenesis, and the chromosomal region maintenance 1 (CRM1) has been suggested to export p53 protein from the nucleus to the cytoplasm. The objectives of the present study were to evaluate p53 expression and subcellular localization as well as CRM1 expression using immunohistochemistry in our established bitransgenic mouse lung tumor model. In this model, expression of the mutant human Ki-rasG12C allele was regulated in a doxycycline (DOX)-inducible, lung-specific manner. Following treatment with curcumin, we found that although overall p53 expression levels were not significantly changed among the three groups, lung lesions in mice treated with DOX alone had the highest proportion of N>C (nucleus predominant) p53 staining (46±7%), followed by lung lesions in mice co-treated with DOX and curcumin (31±12%) and controls (17±4%). CRM1 expression was dramatically inhibited in lung lesions in mice treated with DOX (0±0) as compared to controls (90±17, P=0.001), and could be partially reversed after curcumin treatment (47±21, P=0.028, DOX vs. DOX+curcumin). Collectively, the results from this study demonstrated that p53 accumulated in the nucleus in lung lesions in mice expressing the mutant Ki-rasG12C transgene as a result of down-regulation of CRM1. Furthermore, these alterations could be partially reversed by curcumin treatment. p53 subcellular localization resulting from CRM1 alterations may play an important role in lung tumorigenesis.

A >20-fold increase in X-ray computed tomography (CT) use during the last 30 years has caused considerable concern because of the potential carcinogenic risk from these CT exposures. Estimating the carcinogenic risk from high-energy, single high-dose exposures obtained from atomic bomb survivors and extrapolating these data to multiple low-energy, low-dose CT exposures using the Linear No-Threshold (LNT) model may not give an accurate assessment of actual cancer risk. Recently, the National Lung Cancer Screening Trial (NLST) reported that annual CT scans of current and former heavy smokers reduced lung cancer mortality by 20%, highlighting the need to better define the carcinogenic risk associated with these annual CT screening exposures. In this study, we used the bitransgenic CCSP-rtTA/Ki-ras mouse model that conditionally expresses the human mutant Ki-rasG12C gene in a doxycycline-inducible and lung-specific manner to measure the carcinogenic risk of exposure to multiple whole-body CT doses that approximate the annual NLST screening protocol. Irradiated mice expressing the Ki-rasG12C gene in their lungs had a significant (P = 0.01) 43% increase in the number of tumors/mouse (24.1 ± 1.9) compared to unirradiated mice (16.8 ± 1.3). Irradiated females had significantly (P < 0.005) more excess tumors than irradiated males. No tumor size difference or dose response was observed over the total dose range of 80–160 mGy for either sex. Irradiated bitransgenic mice that did not express the Ki-rasG12C gene had a low tumor incidence (≤0.1/mouse) that was not affected by exposure to CT radiation. These results suggest that (i) estimating the carcinogenic risk of multiple CT exposures from high-dose carcinogenesis data using the LNT model may be inappropriate for current and former smokers and (ii) any increased carcinogenic risk after exposure to fractionated low-dose CT-radiation may be restricted to only those individuals expressing cancer susceptibility genes in their tissues at the time of exposure.

Curcumin exhibits anti-inflammatory and antitumor activity and is being tested in clinical trials as a chemopreventive agent for colon cancer. Curcumin's chemopreventive activity was tested in a transgenic mouse model of lung cancer that expresses the human Ki-ras(G12C) allele in a doxycycline (DOX) inducible and lung-specific manner. The effects of curcumin were compared with the lung tumor promoter, butylated hydroxytoluene (BHT), and the lung cancer chemopreventive agent, sulindac. Treatment of DOX-induced mice with dietary curcumin increased tumor multiplicity (36.3 +/- 0.9 versus 24.3 +/- 0.2) and progression to later stage lesions, results which were similar to animals that were co-treated with DOX/BHT. Microscopic examination showed that the percentage of lung lesions that were adenomas and adenocarcinomas increased to 66% in DOX/BHT, 66% in DOX/curcumin and 49% in DOX/BHT/curcumin-treated groups relative to DOX only treated mice (19%). Immunohistochemical analysis also showed increased evidence of inflammation in DOX/BHT, DOX/curcumin and DOX/BHT/curcumin mice relative to DOX only treated mice. In contrast, co-treatment of DOX/BHT mice with 200 p.p.m. [DOSAGE ERROR CORRECTED] of sulindac inhibited the progression of lung lesions and reduced the inflammation. Lung tissue from DOX/curcumin-treated mice demonstrated a significant increase (33%; P = 0.01) in oxidative damage, as assessed by the levels of carbonyl protein formation, relative to DOX-treated control mice after 1 week on the curcumin diet. These results suggest that curcumin may exhibit organ-specific effects to enhance reactive oxygen species formation in the damaged lung epithelium of smokers and ex-smokers. Ongoing clinical trials thus may need to exclude smokers and ex-smokers in chemopreventive trials of curcumin.

Studies in cell culture have suggested that the level of RAS expression can influence the transformation of cells and the signaling pathways stimulated by mutant RAS expression. However, the levels of RAS expression in vivo appear to be subject to feedback regulation, limiting the total amount of RAS protein that can be expressed. We utilized a bitransgenic mouse lung tumor model that expressed the human Ki-ras(G12C) allele in a tetracycline-inducible, lung-specific manner. Treatment for 12 months with 500 mu g/ml of doxycycline (DOX) allowed for maximal expression of the human Ki-ras(G12c) allele in the lung, and resulted in the development of focal hyperplasia and adenomas. We determined if different levels of mutant RAS expression Would influence the phenotype of the lung lesions. Treatment with 25, 100 and 500 mu g/ml of DOX resulted in close-dependent increases in transgene expression and tumor multiplicity. Microscopic analysis of the lungs of mice treated with the 25 mu g/ml dose of DOX revealed infrequent foci of hyperplasia, whereas mice treated with the 100 and 500 mu g/ml doses exhibited numerous hyperplastic foci and also adenomas. Immunohistochemical and RNA analysis of the downstream effector pathways demonstrated that different levels of mutant RAS transgene expression resulted in differences in the expression and/or phosphorylation of specific signaling molecules. Our results suggest that the molecular alterations driving tumorigenesis may differ at different levels of mutant Ki-ras(G12C) expression, and this should be taken into consideration when inducible transgene systems are utilized to promote tumorigenesis in mouse models. (c) 2008 Elsevier Inc. All rights reserved.

GST isoforms have been extensively studied in adult tissues but little is known about the composition and levels of these enzymes in fetal tissues. As part of our ongoing studies to determine the potential role of metabolic enzymes in mediating the differential susceptibility of different strains of mice to lung tumorigenesis following in utero exposure to 3-methylcholanthrene (MC), we screened for GST enzyme activity and for expression of the individual GSTalpha, pi, mu, and theta isoforms in murine fetal lung and liver tissues isolated from the parental strains and F1 crosses between C57BL/6 (B6) and BALB/c (C) mice. Using 1-chloro-2,4-dinitrobenzene (CDNB) as a substrate, we found that treatment with MC had no effect on the levels of GST enzyme activity in either the fetal lung or liver in either of the two parental strains or their F1 crosses. Low levels of expression of each of the four enzymes were detected by Western blotting in both fetal lung and liver tissues in all four strains. A statistically significant 3.5-fold induction was observed only for GSTmu in the fetal lung of the parental strain of BALB/c mice 48 h after exposure to MC. None of the other enzymes showed any significant differences in the levels of expression following exposure to MC. Although strain-specific differences in the expression of the GSTs that were independent of MC treatment were observed, they could not account for the differences previously observed in either the Ki-ras mutational spectrum or lung tumor incidence in the different strains of mice. Similar results were obtained when the maternal metabolism of MC was assayed in liver microsomal preparations. The results are consistent with previous studies showing low levels and poor inducibility of phase II enzymes during gestation, and demonstrate for the first time that all four of the major GST enzymes are expressed in fetal tissues. While the high inducibility of activating enzymes, such as Cyp1a1, and low, uninducible levels of phase II conjugating enzymes probably account for the high susceptibility of the fetus to transplacentally induced tumor formation, the results also suggest that factors other than metabolism may account for the strain-specific differences in susceptibility to carcinogen-mediated lung tumor induction following in utero exposure to chemical carcinogens.

Fetal mice are more sensitive to chemical carcinogens than are adults. We previously demonstrated that resistant offspring of a DBA/2 x (C57BL/6 x DBA2) backcross exhibited a high incidence of lung tumors 12-13 mo after transplacental exposure to 3-methylcholanthrene (MC). We compared the effects of in utero treatment with MC on lung tumor incidence in the offspring of intermediately susceptible BALB/c (C), resistant C57BL/6 (B6), and reciprocal crosses between these strains. Pregnant mice were treated with 45 mg/kg of MC on day 17 of gestation and tumor incidence, multiplicity, and the Ki-ras mutational spectrum determined in the offspring 12-18 mo after birth. Tumor incidences in C mice and reciprocal crosses were 86% and 100%, respectively, while B6 mice demonstrated resistance to tumorigenesis, with a tumor incidence of 11%. Tumor multiplicities in C, B6C, CB6, and B6 mice were 3.3 +/- 3.2, 5.8 +/- 3.2, 5.0 +/- 2.7, and <0.1, respectively. Ki-ras mutations, which occurred chiefly in the K(s) allele (96%), were found in 79-81% of reciprocally crossed F1 mice, 64% of C mice, and 50% of B6 mice, with the Val(12), Asp(12), and Arg(13) mutations associated with more aggressive tumors. A subset of these mice was used to demonstrate the utility of computer tomography (CT) for the visualization and measurement of lung tumors in the submillimeter range in vivo. Based on known genetic differences in murine strains for lung cancer, our results suggest the presence of a previously unidentified genetic factor(s) which appears to specifically influence lung tumorigenesis following exposure to carcinogens during fetal development.

Fetal mice are more sensitive to chemical carcinogens than are adults. Previous studies from our laboratory demonstrated differences in the mutational spectrum induced in the Ki-ras gene from lung tumors isolated from [D2 × B6D2F1]F2 mice and Balb/c mice treated in utero with 3-methylcholanthrene (MC). We thus determined if differences in metabolism, adduct formation, or adduct repair influence strain-specific responses to transplacental MC exposure in C57BL/6 (B6), Balb/c (BC), and reciprocal F1 crosses between these two strains of mice. The induction of Cyp1a1 and Cyp1b1 in fetal lung and liver tissue was determined by quantitative fluorescent real-time PCR. MC treatment caused maximal induction of Cyp1a1 and Cyp1b1 RNA 2–8 h after injection in both organs. RNA levels for both genes then declined in both fetal organs, but a small biphasic, secondary increase in Cyp1a1 was observed specifically in the fetal lung 24–48 h after MC exposure in all four strains. Cyp1a1 induction by MC at 4 h was 2–5 times greater in fetal liver (7000- to 16,000-fold) than fetal lung (2000- to 6000-fold). Cyp1b1 induction in both fetal lung and liver was similar and much lower than that observed for Cyp1a1, with induction ratios of 8- to 18-fold in fetal lung and 10- to 20-fold in fetal liver. The overall kinetics and patterns of induction were thus very similar across the four strains of mice. The only significant strain-specific effect appeared to be the relatively poor induction of Cyp1b1 in the parental strain of B6 mice, especially in fetal lung tissue. We also measured the levels of MC adducts and their disappearance from lung tissue by the P32 post-labeling assay on gestation days 18 and 19 and postnatal days 1, 4, 11, and 18. Few differences were seen between the different strains of mice; the parental strain of B6 mice had nominally higher levels of DNA adducts 2 (gestation day 19) and 4 (postnatal day 1) days after injection, although this was not statistically significant. These results indicate that differences in Phase I metabolism of MC and formation of MC-DNA adducts are unlikely to account for the marked differences observed in the Ki-ras mutational spectrum seen in previous studies. Further, the results suggest that other genetic factors may interact with chemical carcinogens in determining individual susceptibility to these agents during development.

Recent research has shown that physically attractive persons are viewed by others, and by themselves, as "better" in many ways than less attractive persons. The trend in research findings is pervasive, and a stereotype has been proposed: "what is beautiful is good." This study explored the relationship between physical attractiveness and emotional adjustment of hospitalized schizophrenic patients. It was hypothesized (1) that judges would consider attractive patients better adjusted than unattractive patients; and (2) that attractive patients would appear healthier or better adjusted than unattractive patients on standard diagnostic measures. The results of multivariate analyses supported the first hypothesis, but consistently failed to support the second. Limitations of the present findings and of the "beautiful is good" stereotype are discussed.

Therapists' expectancies for therapeutic gain in patients reputedly are linked to the actual outcome of treatment for their patients. Theoretical papers have suggested that therapists' expectancies may cause or facilitate patient therapeutic response to treatment. Unfortunately, empirical data related to these notions are greatly lacking. The present study tested the relationship between therapist expectancy and treatment outcome for hospitalized schizophrenic patients. It also tested two opposing hypotheses re the nature of the link between therapist expectancy and outcome (causative vs. predictive). Multiple regression analyses revealed that therapists' expectancies are multi-dimensional, formed partly on the basis of therapists' knowledge of psychopathology in general and partly on their perception of their patients' pretreatment adjustment. The analyses also revealed that therapists' expectancies were associated significantly with treatment outcome; the data supported a predictive, not causative, interpretation of the nature of therapists' expectancies.

It has been hypothesized (a) that patients' expectancies for therapeutic gain are linked to the clinical improvement that the patients realize in treatment; and (b) that patients' expectancies may play a causative role in such improvement. The first hypothesis has received empirical support, but the second has not. This study tested the causativeinterpretation of patients' expectancies and a second interpretation, which states that patients' expectancies predict, but do not cause clinical improvement. The shape of thelink between expectancy and improvement for hospitalized schizophrenics was explored. Based on motivation research it was reasoned that a curvilinear relationship between expectancy and improvement would support a causative interpretation and that a linear relationship would support a predictive interpretation of the nature of expectancy. Multiple regression analyses found a linear relationship between expectancies and objective measures of improvement for the patients, but no evidence of a curvilinear relationship between these measures. The results were interpreted as supporting a predictive interpretation of the expectancies of hospitalized schizophrenic patients.

Therapists' expectations with regard to in-therapy behavior of hospitalized patients were measured and factor analyzed. Five interpretable factors emerged and supported the conclusion that therapists' role expectations for patients complement patients' role expectations for therapists. Mean scores on the factors were independent of therapists' experience and professional training.