Sexual Dimorphism of Skeletal Muscle in a Mouse Model of Breast Cancer: A Functional and Molecular Analysis.

Breast cancer incidence in men is statistically rare; however, given the lack of screening in males, more advanced stages at initial diagnosis result in lower 5-year survival rates for men with breast cancer compared to women. A sexual dimorphism, with respect to the effect of tumor growth on cachexia incidence and severity, has also been reported across cancer types. The purpose of this study was to examine the sexual dimorphism of breast cancer as it pertains to skeletal muscle function and molecular composition. Using female and male transgenic PyMT mice, we tested the hypothesis that the isometric contractile properties and molecular composition of skeletal muscle would be differentially affected by breast tumors. PyMT tumor-bearing mice of each sex, corresponding to maximal tumor burden, were compared to their respective controls. RNA sequencing of skeletal muscle revealed different pathway alterations that were exclusive to each sex. Further, differentially expressed genes and pathways were substantially more abundant in female tumor mice, with only minimal dysregulation in male tumor mice, each compared to their respective controls. These differences in the transcriptome were mirrored in isometric contractile properties, with greater tumor-induced dysfunction in females than male mice, as well as muscle wasting. Collectively, these data support the concept of sexually dimorphic responses to cancer in skeletal muscle and suggest that these responses may be associated with the clinical differences in breast cancer between the sexes. The identified sex-dependent pathways within the muscle of male and female mice provide a framework to evaluate therapeutic strategies targeting tumor-associated skeletal muscle alterations.

Sexual Dimorphism of Skeletal Muscle in a Mouse Model of Breast Cancer: A Functional and Molecular Analysis.

Breast cancer incidence in men is statistically rare; however, given the lack of screening in males, more advanced stages at initial diagnosis results in lower 5-year survival rates for men with breast cancer compared to women. A sexual dimorphism, with respect to the effect of tumor growth on cachexia incidence and severity, has also been reported across cancer types. The purpose of this study was to examine the sexual dimorphism of breast cancer as it pertains to skeletal muscle function and molecular composition. Using female and male transgenic PyMT mice, we tested the hypothesis that isometric contractile properties and molecular composition of skeletal muscle would be differentially affected by breast tumors. PyMT tumor-bearing mice of each sex, corresponding to maximal tumor burden, were compared to their respective controls. RNA-sequencing of skeletal muscle revealed different pathway alterations that were exclusive to each sex. Further, differentially expressed genes and pathways were substantially more abundant in female tumor mice, with only minimal dysregulation in male tumor mice, each compared to their respective controls. These differences in the transcriptome were mirrored in isometric contractile properties, with greater tumor-induced dysfunction in females than male mice, as well as muscle wasting. Collectively, these data support the concept of sexually dimorphic responses to cancer in skeletal muscle and suggest these responses may be associated with the clinical differences in breast cancer between the sexes. The identified sex-dependent pathways within muscle of male and female mice provide a framework to evaluate therapeutic strategies targeting tumor-associated skeletal muscle alterations.

Associations of Adiposity and Diet Quality with Serum Ceramides in Middle-Aged Adults with Cardiovascular Risk Factors.

Rates of adverse cardiovascular events have increased among middle-aged adults. Elevated ceramides have been proposed as a risk factor for cardiovascular events. Diet quality and weight status are inversely associated with several traditional risk factors; however, the relationship to ceramides is less clear. This study aimed to determine associations of adiposity and diet quality with circulating ceramides in middle-aged adults (n = 96). Diet quality was estimated using the Healthy Eating Index 2015 (HEI-2015). Serum ceramide concentrations were determined by liquid chromatography-mass spectrometry. A ceramide risk score was determined based on ceramides C16:0, C18:0, and C24:1 and their ratios to C24:0. Participants who were classified as at 'moderate risk' compared to 'lower-risk' based on a ceramide risk score had significantly higher body mass index (BMI) values, as well as higher rates of elevated fibrinogen levels, metabolic syndrome, and former smoking status. BMI was positively associated with the ceramide C18:0 (R2 = 0.31, p < 0.0001), the ratio between C18:0/C24:0 ceramides (R2 = 0.30, p < 0.0001), and the ceramide risk score (R2 = 0.11, p < 0.009). Total HEI-2015 scores (R2 = 0.42, p = 0.02), higher intakes of vegetables (R2 = 0.44, p = 0.02) and whole grains (R2 = 0.43, p = 0.03), and lower intakes of saturated fats (R2 = 0.43, p = 0.04) and added sugar (R2 = 0.44, p = 0.01) were associated with lower C22:0 values. These findings suggest that circulating ceramides are more strongly related to adiposity than overall diet quality. Studies are needed to determine if improvements in weight status result in lower ceramides and ceramide risk scores.

CT and gross pathology are comparable methods for detecting some degenerative sacroiliac joint lesions in dogs.

Degenerative sacroiliac joint disease is a cause of lumbosacral pain in dogs; however, published information on cross-sectional imaging characteristics is limited. Objectives of this retrospective, secondary analysis, methods-comparison study were to test hypotheses that CT lesions reported in humans with degenerative sacroiliac joint disease are also present in dogs, and that CT is comparable to gross pathology for detecting these lesions. Matched CT and gross pathology slice images of 30 sacroiliac joints were retrieved from a previous prospective, canine cadaver study. A veterinary radiologist interpreted randomized CT images for each joint based on previously published CT characteristics of lesions in humans with degenerative sacroiliac joint disease. A veterinary pathologist independently interpreted randomized gross pathology images using the same criteria. All joints had at least one CT lesion consistent with degenerative sacroiliac joint disease. A new CT lesion was also identified and termed "subarticular cleft." The CT and gross pathology methods agreed for detecting joints with subchondral sclerosis, subchondral erosion, and intra-articular ankylosis lesions (P > .05, McNemar's test), but disagreed for detection of joints with subchondral cyst, para-articular ankylosis, and subarticular cleft lesions (P ≤ .05). Using gross pathology as the reference standard, CT had 100% sensitivity for detection of subarticular cleft and subchondral cyst lesions, with 56% and 22% specificity, respectively. Para-articular ankylosis lesions were detected by CT but not by gross pathology. Findings supported the hypothesis that CT lesions reported in humans with degenerative sacroiliac joint disease are also present in dogs, and partially supported the hypothesis that CT is comparable to gross pathology for detecting joints with these lesions.

Nutrition Risk is Associated with Leukocyte Telomere Length in Middle-Aged Men and Women with at Least One Risk Factor for Cardiovascular Disease.

Poor diet quality has been associated with several age-related chronic conditions, but its relationship to telomere length, a biological marker of cellular aging, is unclear. The purpose of this cross-sectional study was to determine whether overall diet quality was associated with relative leukocyte telomere length (rLTL) in a sample (n = 96) of nonsmoking middle-aged adults in Appalachia with at least one risk factor for cardiovascular disease. Diet quality was assessed using the Healthy Eating Index (HEI-2015), the alternate Mediterranean diet score (aMed), and the Dietary Screening Tool (DST). Peripheral rLTL was measured by quantitative real-time polymerase chain reaction. The associations between potentially confounding sociodemographic, lifestyle and health-related factors and the first and fourth rLTL quartile groups were examined using Chi-square or Fisher's Exact tests or logistic regression. The relationships between diet quality index scores and rLTL as a continuous variable were analyzed using simple linear regression and multivariate linear models, analogous to linear covariance analyses. The rLTL ranged from 0.46 to 1.49 (mean ± SEM was 1.02 ± 0.18). Smoking history, income level, and cardiovascular health (Life's Simple 7) were associated with the lowest and highest quartiles of rLTL and were used as covariates. In adjusted and unadjusted models, participants considered "at nutrition risk" by the DST were more likely to have shorter rLTL than those "not at risk or at potential risk" (p = 0.004). However, there was no evidence that adherence to the 2015⁻2020 Dietary Guidelines for Americans or to a Mediterranean diet was associated with rLTL in this sample. Intervention studies are needed to determine if improving the diet quality of those at nutrition risk results in reduced telomere attrition over time.

Educational intervention improves fruit and vegetable intake in young adults with metabolic syndrome components.

The FRUVEDomics study investigates the effect of a diet intervention focused on increasing fruit and vegetable intake on the gut microbiome and cardiovascular health of young adults with/at risk for metabolic syndrome (MetS). It was hypothesized that the recommended diet would result in metabolic and gut microbiome changes. The 9-week dietary intervention adhered to the US Department of Agriculture Dietary Guidelines for Americans and focused on increasing fruit and vegetable intake to equal half of the diet. Seventeen eligible young adults with/or at high risk of MetS consented and completed preintervention and postintervention measurements, including anthropometric, body composition, cardiovascular, complete blood lipid panel, and collection of stool sample for microbial analysis. Participants attended weekly consultations to assess food logs, food receipts, and adherence to the diet. Following intention-to-treat guidelines, all 17 individuals were included in the dietary, clinical, and anthropometric analysis. Fruit and vegetable intake increased from 1.6 to 3.4 cups of fruits and vegetables (P < .001) daily. Total fiber (P = .02) and insoluble fiber (P < .0001) also increased. Clinical laboratory changes included an increase in sodium (P = .0006) and low-density lipoprotein cholesterol (P = .04). In the fecal microbiome, Erysipelotrichaceae (phylum Firmicutes) decreased (log2 fold change: -1.78, P = .01) and Caulobacteraceae (phylum Proteobacteria) increased (log2 fold change = 1.07, P = .01). Implementing a free-living 9-week diet, with intensive education and accountability, gave young adults at high risk for/or diagnosed with MetS the knowledge, skills, and feedback to improve diet. To yield greater impact, a longer diet intervention may be needed in this population.

Long-term Immunotoxic Effects of Oral Prenatal and Neonatal Atrazine Exposure.

Atrazine and its metabolites are present at high concentrations in many water supplies in agro-intensive areas. Because residents in these areas drink water from sources fed from these contaminated supplies, we investigated the long-term immunotoxicity of combined prenatal and neonatal (perinatal) exposure to atrazine via drinking water, on the immune system in mice. At 6 months of age, upon immunization with heat-killed Streptococcus pneumoniae, the serum IgG antibody response against the T independent antigen phosphorylcholine was significantly higher in male, but not female, atrazine-exposed mice as compared with that in untreated controls. No alterations were present in all offspring in the serum antibody response against the T-dependent antigen pneumococcal surface protein A (PspA). ELISpot analysis showed only a small, insignificant reduction in PspA-specific IgG producing splenocytes in atrazine-treated male offspring. Interestingly, upon ex vivo stimulation with anti-CD3 and anti-CD28 antibodies, significant decreases in interleukin (IL)-2, tumor necrosis factor-α, interferon-γ, and IL-17A and a decreasing trend in IL-10 were observed in splenocytes from atrazine-exposed male, but not female mice. Analysis of thymic and splenic cell populations showed no effects of atrazine exposure in either sex. This is the first time that long-term changes in the immune response were observed after a perinatal exposure to atrazine and it demonstrates that these early life exposures can result in permanent changes to the immune system as well as a male bias in these effects.

Suppression of arthritis-induced bone erosion by a CRAC channel antagonist.

OBJECTIVE: We have shown in vitro and in vivo that osteoclast maturation requires calcium-release activated calcium (CRAC) channels. In inflammatory arthritis, osteoclasts mediate severe and debilitating bone erosion. In the current study, we assess the value of CRAC channels as a therapeutic target to suppress bone erosion in acute inflammatory arthritis. METHODS: Collagen-induced arthritis (CIA) was induced in mice. The CRAC channel inhibitor 3,4-dichloropropionaniline (DCPA) and a placebo was administered 1 day prior to collagen II booster to induce arthritis. Effects on swelling, inflammatory cell invasion in joints, serum cytokines and bone erosion were measured. RESULTS: Assays, by blinded observers, of arthritis severity showed that DCPA, 21 mg/kg/day, suppressed arthritis development over 3 weeks. Bone and cartilage damage in sections of animal feet was reduced approximately 50%; overall swelling of joints was reduced by a similar amount. Effects on bone density by µCT showed clear separation in DCPA-treated CIA animals from CIA without treatment, while differences between controls without CIA and CIA treated with DCPA differed by small amounts and in most cases were not statistically different. Response was not related to anticollagen titres. There were no adverse effects in the treated group on animal weight or activity, consistent with low toxicity. The effect was maximal 12-17 days after collagen booster, during the rapid appearance of arthritis in untreated CIA. At 20 days after treatment (day 40), differences in arthritis score were reduced and tumour necrosis factor α, interleukin (IL)-1, or IL-6 in the serum of the animals were similar in treated and untreated animals. CONCLUSIONS: DCPA, a novel inhibitor of CRAC channels, suppresses bone erosion associated with acute arthritis in mice and might represent a new treatment modality for acute arthrits.

Prenatal cadmium exposure produces persistent changes to thymus and spleen cell phenotypic repertoire as well as the acquired immune response.

Cadmium (Cd) is a common environmental contaminant. Adult exposure to Cd alters the immune system, however, there are limited studies on the effects of prenatal exposure to Cd. Pregnant C57Bl/6 mice were exposed to an environmentally relevant dose of CdCl(2) (10 ppm) and the effects on the immune system of the offspring were assessed at 20 weeks of age. Prenatal Cd exposure caused an increase in the percent of CD4(-)CD8(-)CD44(+)CD25(-) (DN1) thymocytes in both sexes and a decrease in the percent of CD4(-)CD8(-)CD44(-)CD25(+) (DN3) thymocytes in females. Females had an increase in the percent of splenic CD4(+) T cells, CD8(+) T cells, and CD45R/B220(+) B cells and a decrease in the percent of NK cells and granulocytes (Gr-1(+)). Males had an increase in the percent of splenic CD4(+) T cells and CD45R/B220(+) B cells and a decrease in the percent of CD8(+) T cells, NK cells, and granulocytes. The percentage of neutrophils and myeloid-derived suppressor cells were reduced in both sexes. The percent of splenic nTreg cells was decreased in all Cd-exposed offspring. Cd-exposed offspring were immunized with a streptococcal vaccine and the antibody response was determined. PC-specific serum antibody titers were decreased in Cd exposed female offspring but increased in the males. PspA-specific serum IgG titers were increased in both females and males compared to control animals. Females had a decrease in PspA-specific serum IgM antibody titers. Females and males had a decrease in the number of splenic anti-PspA antibody-secreting cells when standardized to the number of B cells. These findings demonstrate that very low levels of Cd exposure during gestation can result in long term sex-specific alterations on the immune system of the offspring.

Chronic occupational exposure to arsenic induces carcinogenic gene signaling networks and neoplastic transformation in human lung epithelial cells.

Chronic arsenic exposure remains a human health risk; however a clear mode of action to understand gene signaling-driven arsenic carcinogenesis is currently lacking. This study chronically exposed human lung epithelial BEAS-2B cells to low-dose arsenic trioxide to elucidate cancer promoting gene signaling networks associated with arsenic-transformed (B-As) cells. Following a 6month exposure, exposed cells were assessed for enhanced cell proliferation, colony formation, invasion ability and in vivo tumor formation compared to control cell lines. Collected mRNA was subjected to whole genome expression microarray profiling followed by in silico Ingenuity Pathway Analysis (IPA) to identify lung carcinogenesis modes of action. B-As cells displayed significant increases in proliferation, colony formation and invasion ability compared to BEAS-2B cells. B-As injections into nude mice resulted in development of primary and secondary metastatic tumors. Arsenic exposure resulted in widespread up-regulation of genes associated with mitochondrial metabolism and increased reactive oxygen species protection suggesting mitochondrial dysfunction. Carcinogenic initiation via reactive oxygen species and epigenetic mechanisms was further supported by altered DNA repair, histone, and ROS-sensitive signaling. NF-κB, MAPK and NCOR1 signaling disrupted PPARα/δ-mediated lipid homeostasis. A 'pro-cancer' gene signaling network identified increased survival, proliferation, inflammation, metabolism, anti-apoptosis and mobility signaling. IPA-ranked signaling networks identified altered p21, EF1α, Akt, MAPK, and NF-κB signaling networks promoting genetic disorder, altered cell cycle, cancer and changes in nucleic acid and energy metabolism. In conclusion, transformed B-As cells with their whole genome expression profile provide an in vitro arsenic model for future lung cancer signaling research and data for chronic arsenic exposure risk assessment.

Prenatal cadmium exposure alters postnatal immune cell development and function.

Cadmium (Cd) is generally found in low concentrations in the environment due to its widespread and continual use, however, its concentration in some foods and cigarette smoke is high. Although evidence demonstrates that adult exposure to Cd causes changes in the immune system, there are limited reports of immunomodulatory effects of prenatal exposure to Cd. This study was designed to investigate the effects of prenatal exposure to Cd on the immune system of the offspring. Pregnant C57Bl/6 mice were exposed to an environmentally relevant dose of CdCl(2) (10ppm) and the effects on the immune system of the offspring were assessed at two time points following birth (2 and 7weeks of age). Thymocyte and splenocyte phenotypes were analyzed by flow cytometry. Prenatal Cd exposure did not affect thymocyte populations at 2 and 7weeks of age. In the spleen, the only significant effect on phenotype was a decrease in the number of macrophages in male offspring at both time points. Analysis of cytokine production by stimulated splenocytes demonstrated that prenatal Cd exposure decreased IL-2 and IL-4 production by cells from female offspring at 2weeks of age. At 7weeks of age, splenocyte IL-2 production was decreased in Cd-exposed males while IFN-γ production was decreased from both male and female Cd-exposed offspring. The ability of the Cd-exposed offspring to respond to immunization with a S. pneumoniae vaccine expressing T-dependent and T-independent streptococcal antigens showed marked increases in the levels of both T-dependent and T-independent serum antibody levels compared to control animals. CD4(+)FoxP3(+)CD25(+) (nTreg) cell percentages were increased in the spleen and thymus in all Cd-exposed offspring except in the female spleen where a decrease was seen. CD8(+)CD223(+) T cells were markedly decreased in the spleens in all offspring at 7weeks of age. These findings suggest that even very low levels of Cd exposure during gestation can result in long term detrimental effects on the immune system of the offspring and these effects are to some extent sex-specific.

Evidence that the arcuate nucleus is an important site of progesterone negative feedback in the ewe.

There is now considerable evidence that dynorphin neurons mediate the negative feedback actions of progesterone to inhibit GnRH and LH pulse frequency, but the specific neurons have yet to be identified. In ewes, dynorphin neurons in the arcuate nucleus (ARC) and preoptic area (POA) are likely candidates based on colocalization with progesterone receptors. These studies tested the hypothesis that progesterone negative feedback occurs in either the ARC or POA by determining whether microimplants of progesterone into either site would inhibit LH pulse frequency (study 1) and whether microimplants of the progesterone receptor antagonist, RU486, would disrupt the inhibitory effects of peripheral progesterone (study 2). Both studies were done in ovariectomized (OVX) and estradiol-treated OVX ewes. In study 1, no inhibitory effects of progesterone were observed during treatment in either area. In study 2, microimplants of RU486 into the ARC disrupted the negative-feedback actions of peripheral progesterone treatments on LH pulse frequency in both OVX and OVX+estradiol ewes. In contrast, microimplants of RU486 into the POA had no effect on the ability of systemic progesterone to inhibit LH pulse frequency. We thus conclude that the ARC is one important site of progesterone-negative feedback in the ewe. These data, which are the first evidence on the neural sites in which progesterone inhibits GnRH pulse frequency in any species, are consistent with the hypothesis that ARC dynorphin neurons mediate this action of progesterone.

Neurokinin B acts via the neurokinin-3 receptor in the retrochiasmatic area to stimulate luteinizing hormone secretion in sheep.

Recent data have demonstrated that mutations in the receptor for neurokinin B (NKB), the NK-3 receptor (NK3R), produce hypogonadotropic hypogonadism in humans. These data, together with reports that NKB expression increases after ovariectomy and in postmenopausal women, have led to the hypothesis that this tachykinin is an important stimulator of GnRH secretion. However, the NK3R agonist, senktide, inhibited LH secretion in rats and mice. In this study, we report that senktide stimulates LH secretion in ewes. A dramatic increase in LH concentrations to levels close to those observed during the preovulatory LH surge was observed after injection of 1 nmol senktide into the third ventricle during the follicular, but not in the luteal, phase. Similar increases in LH secretion occurred after insertion of microimplants containing this agonist into the retrochiasmatic area (RCh) in anestrous or follicular phase ewes. A low-dose microinjection (3 pmol) of senktide into the RCh produced a smaller but significant increase in LH concentrations in anestrous ewes. Moreover, NK3R immunoreactivity was clearly evident in the RCh, although it was not found in A15 dopaminergic cell bodies in this region. These data provide evidence that NKB stimulates LH (and presumably GnRH) secretion in ewes and point to the RCh as one important site of action. Based on these data, and the effects of NK3R mutations in humans, we hypothesize that NKB plays an important stimulatory role in the control of GnRH and LH secretion in nonrodent species.