Betty Herndon

Role of anthocyanin‐enriched purple‐fleshed sweet potato p40 in colorectal cancer prevention

SCOPE Anthocyanins, the natural pigments in plant foods, have been associated with cancer prevention. However, the content of anthocyanins in staple foods is typically low and the mechanisms by which they exert anticancer activity is not yet fully defined. METHODS AND RESULTS We selected an anthocyanin-enriched purple-fleshed sweet potato clone, P40, and investigated its potential anticancer effect in both in vitro cell culture and in vivo animal model. In addition to a high level of total phenolics and antioxidant capacity, P40 possesses a high content of anthocyanins at 7.5 mg/g dry matter. Treatment of human colonic SW480 cancer cells with P40 anthocyanin extracts at 0-40 μM of peonidin-3-glucoside equivalent resulted in a dose-dependent decrease in cell number due to cytostatic arrest of cell cycle at G1 phase but not cytotoxicity. Furthermore, dietary P40 at 10-30% significantly suppressed azoxymethane-induced formation of aberrant crypt foci in the colons of CF-1 mice in conjunction with, at least in part, a lesser proliferative PCNA and a greater apoptotic caspase-3 expression in the colon mucosal epithelial cells. CONCLUSION These observations, coupled with both in vitro and in vivo studies reported here, suggest anthocyanin-enriched sweet potato P40 may protect against colorectal cancer by inducing cell-cycle arrest, antiproliferative, and apoptotic mechanisms.

Pleurodesis by autologous blood, doxycycline, and talc in a rabbit model.

BACKGROUND Management of recurrent spontaneous pneumothorax or symptomatic pleural effusion often uses thoracoscopic pleurodesis, about which many questions remain. Both effectiveness and toxicity of agents currently used for pleurodesis were evaluated in a rabbit model. METHODS Agents administered were autologous blood 1 mL/kg, talc slurry (70 mg x mL(-1) x kg(-1)), and doxycycline 10 mg/mL, given through a chest tube to 30 rabbits. Controls had only chest tubes inserted. At 30 days surfaces were graded by gross observation and histologic examination. Blood and lung tissue from all animals were analyzed for enzymes and blood chemistries. RESULTS Gross observations showed mediastinal thickening and adhesions with doxycycline, and threadlike adhesions with talc. Autologous blood was only slightly more effective than a chest tube alone. Talc significantly increased angiotensin converting enzyme activity in serum, whereas doxycycline changed liver function enzymes and produced tissue toxicity. CONCLUSIONS Doxycycline produced effective pleurodesis but yielded remarkably severe local effects. The distant sequelae of talc and doxycycline pleurodesis-histologic changes in the contralateral lung and serum enzyme elevations-suggests undesirable systemic effects for the commonly used agents, and autologous blood exhibited no significant pleurodesis, short-term. The search for the ideal agent for chemical pleurodesis continues.

Effects of Dietary Calorie Restriction or Exercise on the PI3K and Ras Signaling Pathways in the Skin of Mice

Weight control by exercise and dietary calorie restriction (DCR) has been associated with reduced cancer risk, but the underlying mechanisms are not well understood. This study was designed to compare the effects of weight loss by increasing physical activity or decreasing calorie intake on tumor promoter-induced Ras-MAPK and PI3K-Akt pathways. SENCAR mice were randomly assigned to one of the following five groups: ad libitum-fed sedentary control, ad libitum-fed exercise (AL+Exe), exercise but pair-fed at the amount as controls (PF+Exe), 20% DCR, and 20% DCR plus exercise (DCR+Exe). After 10 weeks, body weight and body fat significantly decreased in the groups of DCR, DCR+Exe, and PF+Exe when compared with the controls. AL+Exe did not induce weight loss due to, at least in part, increased food intake. Plasma IGF-1 levels reduced significantly in DCR and DCR+Exe but not PF+Exe. The protein H-Ras and activated Ras-GTP significantly decreased in TPA-induced skin tissues of DCR-fed mice but not exercised mice. PI3K protein, phosphoserine Akt, and p42/p44-MAPK were reduced, however, in both DCR and PF+Exe groups. Immunohistochemistry demonstrated that the significantly reduced H-Ras occurred in subcutaneous fat cells, while the reduced PI3K and PCNA took place only in the epidermis. Plasma leptin decreased in PF+Exe, DCR, and DCR+Exe, while the caspase-3 activity increased in DCR+Exe only. Genomic microarray analysis further indicated that the expression of 34 genes relevant to PI3K and 31 genes to the MAPK pathway were significantly regulated by either DCR or PF+Exe treatments. The reduced PI3K in PF+Exe mice was partially reversed by IGF-1 treatment. The overall results of this study demonstrated that DCR abrogated both Ras and PI3K signaling, which might inhibit TPA-induced proliferation and anti-apoptosis. Selective inhibition of PI3K by PF+Exe but not AL+Exe seems more attributable to the magnitude of the caloric deficit and/or body fat loss than diet versus exercise comparison.

Expression of multidrug resistance associated protein 5 (MRP5) on cornea and its role in drug efflux.

PURPOSE The purpose of this manuscript is to investigate the presence of nucleoside/nucleotide efflux transporter in cornea and to evaluate the role in ocular drug efflux. METHODS RT-PCR, immunoprecipitation followed by Western blot analysis and immunostaining were employed to establish molecular presence of multidrug resistance associated protein 5 (MRP5) on cornea. Corneal efflux by MRP5 was studied with bis(POM)-PMEA and acyclovir using rabbit and human corneal epithelial cells along with MRP5 over expressing cells (MDCKII-MRP5). Ex vivo studies using excised rabbit cornea and in vivo ocular microdialysis in male New Zealand white rabbits were used to further evaluate the role of MRP5 in conferring ocular drug resistance. RESULTS RT-PCR confirms the expression of MRP5 in both rabbit and human corneal epithelial cells along with MDCKII-MRP5 cells. Immunoprecipitation followed by Western blot analysis using a rat (M511-54) monoclonal antibody that reacts with human epitope confirms the expression of MRP5 protein in human corneal epithelial cells and MDCKII-MRP5 cells. Immunostaining performed on human cornea indicates the localization of this efflux pump on both epithelium and endothelium. Efflux studies reveal that depletion of ATP decreased PMEA efflux significantly. MRP5 inhibitors also diminished PMEA and acyclovir efflux. However, depletion of glutathione did not alter efflux. MDR1 and MRP2 did not contribute to PMEA efflux. However, MRP2 is involved in acyclovir efflux while MDR1 do not participate in this process. TLC/autoradiography suggested the conversion of bis(POM)-PMEA to PMEA in rabbit and human corneal epithelial cells. Two well known antiglaucoma drugs, bimatoprost and latanoprost were rapidly effluxed by MRP5. Ex vivo study on intact rabbit corneas demonstrated accumulation of PMEA in cornea in the presence of ATP-depleting medium. In vivo ocular pharmacokinetics also revealed a significant increase in maximum aqueous humor concentration (C(max)) and area under the aqueous humor time curve (AUC) of acyclovir in the presence of MK-571, a specific MRP inhibitor. CONCLUSIONS Taken together immunolocalization on human cornea, in vitro efflux in human, rabbit corneal and MRP5 over expressing cells, ex vivo and in vivo studies in intact rabbit cornea suggest that MRP5 on cornea can significantly lower the permeability of antiviral and glaucoma drugs. These findings may be valuable in developing formulation strategies to optimize ocular bioavailability of topically administered ocular agents.

Bronchiolitis Obliterans Induced by Intratracheal Papaverine: A Novel Animal Model

Constrictive bronchiolitis obliterans (CBO), a highly fatal syndrome seen following toxicant exposure and lung transplantation, is in need of mechanistic study. This report creates an animal model of toxicant induced CBO, validates its pathology and suggests a physiologic mechanism for its origin. Papaverine, the alkaloid in Sauropus plants and responsible for human toxicant-induced CBO, was used to create the rat model. A mini-osmotic pump delivered papaverine intratracheally for up to 28 days (0.25 µL/hr, totaling 6.4 mg). Bronchoalveolar lavage (BAL) was measured. Lung tissue was evaluated for signs of CBO (H&E and Trichrome staining). Cytokines deregulated in human CBO were also measured (TGF-β and eNOS). Peribronchial inflammation, extensive denudation and destruction of bronchial mucosa, as well as increased peribronchial collagen (all classic signs of CBO) were observed as early as 7 days in papaverine treated animals, with more extensive damage after 28 days. Significant elevations of TGF-β and eNOS were seen in lung homogenates. Our toxicant induced model of CBO via a novel delivery method of intratracheal papaverine accurately reproduces pathology and cytokine profiles of human CBO. Papaverine has potential for producing CBO by multiple routes. This model introduces a vehicle for both understanding and development of innovative treatment for CBO.

Efficacies of various antimicrobial agents in treatment of Staphylococcus aureus abscesses and correlation with in vitro tests of antimicrobial activity and neutrophil killing.

A rabbit perforated-capsule model was utilized to study antimicrobial efficacy in treating 2-week-old Staphylococcus aureus abscesses. Animals received either ciprofloxacin (30 mg/kg), cefazolin (100 mg/kg), or ciprofloxacin (30 mg/kg) plus rifampin (20 mg/kg) every 8 h for 8 days or no antibiotic. Antibiotic levels within the abscess exceeded the MIC for the test organism. At the end of treatment, ciprofloxacin was no more effective than the control, animals receiving cefazolin had a mean log10 fall of 2.41 CFU/ml, and animals receiving ciprofloxacin plus rifampin had a mean log10 reduction of 5.06 CFU/ml (P = less than 0.01). Six days after completion of therapy, all abscesses in animals receiving ciprofloxacin plus rifampin were culture negative. Surviving organisms in animals receiving ciprofloxacin or rifampin did not develop resistance to the treatment antibiotics. In vitro time-kill curves performed with logarithmic- and stationary-phase organisms in broth, serum, and abscess fluid supernatants did not correlate with the in vivo results. Neutrophil killing studies of S. aureus pretreated with antibiotics revealed greater killing of organisms pretreated with ciprofloxacin plus rifampin than of those pretreated with cefazolin or ciprofloxacin alone. In conclusion, ciprofloxacin plus rifampin was effective therapy in this staphylococcal abscess model, compared with the moderate efficacy of cefazolin and no effect observed with ciprofloxacin alone. Enhanced neutrophil killing of S. aureus pretreated with antibiotics may be an important mechanism by which bacteria are killed in suppurative infections.

Fat Embolism: Evolution of Histopathological Changes in the Rat Lung

The pathophysiology of Fat Embolism Syndrome (FES) is poorly understood and subject to some controversy. Evaluation of the evolution of histological changes in the lungs of patients with FES is impractical. The current theories of FES were established through acute clinical observations and acute animal experiments, but sequential changes in the histology of lungs over a prolonged period have not been made. The progressive effects of fat embolization of the lungs were examined in a rat model over a period of 11 days. Triolein, a major bone marrow fat, was administered to conscious Sprague‐Dawley rats via the caudal vein. Rats were euthanized at 24, 48, 96 h, and 11 days, but some died within a few hours. Histomorphometric evaluations of lung tissue were made, including stains for fat, collagen, and smooth muscle actin. Arterial and arteriolar patency decreased progressively up to 96 h, but returned toward normal after 11 days. A striking finding was the very early presence of inflammation and fibrosis after only several hours, persisting up to 11 days. The results of this study provide evidence of both very early and prolonged changes due to fat embolization.

Weight Loss via Exercise with Controlled Dietary Intake May Affect Phospholipid Profile for Cancer Prevention in Murine Skin Tissues

Exercise has been linked to a reduced cancer risk in animal models. However, the underlying mechanisms are unclear. This study assessed the effect of exercise with dietary consideration on the phospholipid profile in 12-O-tetradecanoylphorbol-13-acetate (TPA)–induced mouse skin tissues. CD-1 mice were randomly assigned to one of the three groups: ad libitum–fed sedentary control; ad libitum–fed treadmill exercise at 13.4 m/min for 60 min/d, 5 d/wk (Ex+AL); and treadmill-exercised but pair-fed with the same amount as the control (Ex+PF). After 14 weeks, Ex+PF but not Ex+AL mice showed ∼25% decrease in both body weight and body fat when compared with the controls. Of the total 338 phospholipids determined by electrospray ionization–tandem mass spectrometry, 57 were significantly changed, and 25 species could distinguish effects of exercise and diet treatments in a stepwise discriminant analysis. A 36% to 75% decrease of phosphatidylinositol (PI) levels in Ex+PF mice occurred along with a significant reduction of PI 3-kinase in TPA-induced skin epidermis, as measured by both Western blotting and immunohistochemistry. In addition, ∼2-fold increase of the long-chain polyunsaturated fatty acids, docosahexaenoic and docosapentaenoic acids, in phosphatidylcholines, phosphatidylethanolamines, and lysophosphatidylethanolamines was observed in the Ex+PF group. Microarray analysis indicated that the expression of fatty acid elongase-1 increased. Taken together, these data indicate that exercise with controlled dietary intake, but not exercise alone, significantly reduced body weight and body fat as well as modified the phospholipid profile, which may contribute to cancer prevention by reducing TPA-induced PI 3-kinase and by enhancing ω-3 fatty acid elongation. Cancer Prev Res; 3(4); 466–77

Effects of Neutrophils on Cefazolin Activity and Penicillin-Binding Proteins in Staphylococcus aureus Abscesses

ABSTRACT Bacteria survive within abscesses despite antimicrobial therapy, usually necessitating drainage. Our previous work showed that bacterial killing is diminished within the neutrophils of animals with abscesses. To further assess the role of neutrophils in Staphylococcus aureus survival and the poor activities of β-lactams in abscesses, tissue cage abscess-bearing rats were given polymorphonuclear leukocyte (PMN)-depleting antibody prior to and several times following inoculation of the tissue cages with S. aureus. Cefazolin (300 mg/kg of body weight/day) was administered to all animals in appropriately divided doses. After 7 days of antimicrobial therapy, the 17 animals that received anti-PMN serum had significantly fewer abscess neutrophils than the 18 controls and fewer abscess bacteria (5.55 versus 3.79 log10 CFU/ml [P = 0.04]) than the 18 controls. The data were consistent with the premise that cefazolin is more effective in abscesses depleted of neutrophils. To investigate further, S. aureus was incubated with rat peritoneal neutrophils; and bacterial cell membrane proteins were isolated, labeled with biotinylated ampicillin, separated by electrophoresis, blotted onto nitrocellulose, and stained for biotin reactivity. PBP 2 expression was consistently and significantly decreased after a brief, nonkilling PMN exposure. These experiments showed that PMN depletion enhanced the activity of cefazolin in the abscess milieu. Furthermore, altered bacterial cell wall cefazolin targets may be the mechanism by which the PMN diminishes antimicrobial activity, suggesting the importance of the staphylococcus-PMN interaction in the outcome of established infections.

Mitigating effects of captopril and losartan on lung histopathology in a rat model of fat embolism.

BACKGROUND Fat embolization (FE) is an often overlooked and poorly understood complication of skeletal trauma and some orthopedic procedures. Fat embolism can lead to major pulmonary damage associated with fat embolism syndrome (FES). METHODS A model of FE in unanesthetized rats, using intravenous injection of the neutral fat triolein, was used to study the potential therapeutic effect on lung histopathology of altering the production of, or response to, endogenous angiotensin (Ang) II. Either captopril, an Ang I converting enzyme inhibitor, or losartan, an Ang II type 1 receptor blocker, was injected 1 hour after FE by triolein injection. After euthanasia at 48 hours, histopathologic evaluation was used to compare the drug-treated animals with control animals that received only triolein. RESULTS Histology of the lungs of rats treated only with triolein revealed severe, diffuse pathology. Alveolar septa showed severe, diffuse inflammation. Bronchial lumina showed severe mucosal epithelial loss. The media of the pulmonary small arteries and arterioles was thicker, and the lumen patency was reduced 60% to 70%. Trichrome staining confirmed the abundant presence of collagen in the media and adventitia, as well as collagen infiltrating the bronchial musculature. Both captopril and losartan treatments reduced the inflammatory, vasoconstrictor, and profibrotic effects present at 48 hours (p<0.001). With treatment, the vascular lumen remained patent, and the fat droplets were reduced in size and number. There was a reduction in the number of infiltrating leukocytes, macrophages, myofibroblasts, and eosinophils, along with a significant decrease in hemorrhage and collagen deposition (p<0.001). Pathologic changes in bronchial epithelium were also diminished. CONCLUSIONS The results suggest that the use of drugs that act on the renin-Ang system might provide an effective and targeted therapy for fat embolism syndrome.