Experimental Biology and Medicine

Dietary behavior has been identified as one of the most important modifiable determinants of cancer risk. Which personalized modifications are needed remains an area of considerable controversy. Part of this uncertainty may arise from interactions among dietary bioactive compounds and/or food combinations. These interactions may either enhance or negate the response to specific foods. Evidence suggests that the cancer-protective effects of an individual’s diet may reflect the combined effects of various vitamins, minerals, and other bioactive components such as flavonoids, isothiocyanates, and/or allium compounds rather than from the effect of a single ingredient. A better understanding of physiologically important interactions is needed to determine the merit of combining foods for maximum efficacy for cancer prevention. Furthermore, the response is complicated, since multiple cellular processes associated with carcinogenesis can be modified simultaneously, including sites such as drug metabolism, DNA repair, cell proliferation, apoptosis, inflammation, differentiation, and angiogenesis. Current evidence suggests that bioactive food components can typically influence more than one process. It is essential to have a better understanding of how the response relates to exposures and credentialing which process is most involved in bringing about a change in tumor incidence and/or tumor behavior. Credentialing is being defined as a determination of which cellular process(es) and which bioactive food components are most important for bringing about a phenotypic change. Additional attention is needed to determine the critical intake of dietary components, their duration, and when they should be provided to optimize the desired physiological response. Further research is also needed on the molecular targets for bioactive components and whether genetic and epigenetic events dictate the direction and magnitude of the response.

Ghrelin and adiponectin are recently discovered peptides that are both associated with energy homeostasis and insulin action. In addition, circulating levels of both peptides are altered in obese populations and are associated with poor health. Moreover, expression of ghrelin and adiponectin returns to normal levels following weight loss in obese patients. Because exercise training improves the health status of obese individuals and is associated with reduction of body weight, there is interest in the effects of exercise on adiponectin and ghrelin and whether these peptides may provide better understanding of how exercise improves health. Ghrelin levels do not increase in response to acute running and cycling in humans, and therefore ghrelin does not appear to regulate growth hormone (GH) release during exercise. There is some evidence that ghrelin levels are suppressed following resistance exercise of moderate intensity and are lower with higher GH concentrations during aerobic exercise. It has been suggested that negative feedback from elevated GH produces the reductions, but why these responses have not been consistently found in other studies and whether postexercise reduction in ghrelin affects appetite warrants further investigation. There are a few studies (but not all) that suggest long-term chronic exercise produces increases in ghrelin levels when weight loss is produced. Ghrelin levels are much higher in amenorrheic athletes than in ovulating exercisers or in female exercisers with a luteal phase defect, suggesting an association with reproductive function. Adiponectin concentrations do not change in response to moderate and strenuous running or low- and moderate- intensity cycling. Most studies have revealed that chronic exercise that improves fitness levels, increases insulin sensitivity, and reduces body weight, will increase resting adiponectin levels. However, it does not appear that changes in insulin sensitivity brought about by moderate exercise training are attributable to adiponectin.

Systemic lupus erythematosus (SLE) is a complex systemic autoimmune disorder of unknown etiology. However, the definitive mechanisms remain obscure. Recently, transglutaminase 2 (TG2) was implicated in the pathogenesis of SLE. Cystamine, which inactivates TG2 activity by forming a mixed disulfide, may interfere with and inhibit other thiol-dependent enzymes such as caspases. To investigate the effects of cystamine in SLE pathogenesis, this in vivo study assessed the serum and macrophage response after administration of cystamine to NZB/W F₁ mice. The experimental results demonstrated for the first time a significant reduction in TG2 and matrix metalloproteinase (MMP)-9 activity; tissue inhibitor of metalloproteinases (TIMP)-1, TIMP-2, TG2, tumor necrosis factor alpha, and tumor growth factor beta mRNA expression; and anticardiolipin autoantibodies (aCL) in NZB/W F₁ mice following cystamine administration. It strongly suggests the therapeutic potential of cystamine in SLE.

Two different theories of migraine aura exist: In the vascular theory of Wolff, intracerebral vasoconstriction causes migraine aura via energy deficiency, whereas in the neuronal theory of Leão and Morison, spreading depression (SD) initiates the aura. Recently, it has been shown that the cerebrovascular constrictor endothelin-1 (ET-1) elicits SD when applied to the cortical surface, a finding that could provide a bridge between the vascular and the neuronal theories of migraine aura. Several arguments support the notion that ET-1–induced SD results from local vasoconstriction, but definite proof is missing. If ET-1 induces SD via vasoconstriction/ischemia, then neuronal damage is likely to occur, contrasting with the fact that SD in the otherwise normal cortex is not associated with any lesion. To test this hypothesis, we have performed a comprehensive histologic study of the effects of ET-1 when applied topically to the cerebral cortex of halothane-anesthetized rats. Our assessment included histologic stainings and immunohistochemistry for glial fibrillary acidic protein, heat shock protein 70, and transferase dUTP nick-end labeling assay. During ET-1 application, we recorded (i) subarachnoid direct current (DC) electroencephalogram, (ii) local cerebral blood flow by laser-Doppler flowmetry, and (iii) changes of oxyhemoglobin and deoxyhemoglobin by spectroscopy. At an ET-1 concentration of 1 μM, at which only 6 of 12 animals generated SD, a microarea with selective neuronal death was found only in those animals demonstrating SD. In another five selected animals, which had not shown SD in response to ET-1, SD was triggered at a second cranial window by KCl and propagated from there to the window exposed to ET-1. This treatment also resulted in a microarea of neuronal damage. In contrast, SD invading from outside did not induce neuronal damage in the absence of ET-1 (n = 4) or in the presence of ET-1 if ET-1 was coapplied with BQ-123, an ET_A receptor antagonist (n = 4). In conclusion, SD in presence of ET-1 induced a microarea of selective neuronal necrosis no matter where the SD originated. This effect of ET-1 appears to be mediated by the ET_A receptor.

Endothelin-1 (ET-1) has been implicated in many neurological diseases, including subarachnoid hemorrhage (SAH) and cerebral ischemia. ET-1 is also proved to deteriorate the ischemia-reperfusion injury in many organs. Our previous studies demonstrated that the endothelin-converting enzyme (ECE) inhibitor, CGS 26303, possessed beneficial effects for the treatment of SAH and transient middle cerebral artery occlusion. In this study, we investigated the neuroprotective effect of CGS 26303 on the locomotor function and mRNA expression of heme-oxygenase-1 (HO-1) in rats subjected to a 15-min spinal cord ischemia. The results showed that pretreatment with CGS 26303 significantly preserved the locomotor function and decreased the paraplegia rate at Days 1 and 3 as compared with a saline-treated group. Furthermore, rats pretreated with CGS 26303 had a significant increase in the levels of HO-1 mRNA expression at Day 3 when compared with animals pretreated with saline after spinal cord ischemia and the sham operation group. These results suggest that CGS 26303 may have a promising neuroprotective effect in the spinal cord after ischemia-reperfusion injury, and beneficial result may be due to an adaptive mechanism involved by HO-1 overexpression.

Antithrombin, the most potent anticoagulant in vivo, displays a significant conformational flexibility. The native five-stranded anticoagulant form transforms under different conditions or mutations to inactive six-stranded conformations: latent or polymer. However, the function, potential deleterious effects, and clearance of these forms are not completely known. The dimerization of latent antithrombin with a native molecule has been suggested to have thrombotic potential. We have assessed the potential thrombogenicity of high amounts of latent and polymeric antithrombin by experiments performed in mice and human plasma. Moreover, we have analyzed the clearance of ¹²⁵I-labeled native, latent, polymer, and thrombin-complexed antithrombins in rat, as well as the clearance of latent antithrombin from plasma of patients treated with commercial concentrates. Our results show that high plasma levels of latent or polymeric antithrombin do not interfere with the anticoagulant function of native antithrombin. Moreover, we confirm that all monomeric forms of antithrombin have similar turnover. Finally, we show that polymers have the longest half-life of all conformers, being in circulation for prolonged periods of time. In conclusion, our data support that latent and polymeric antithrombin would not likely have a thrombotic effect, thus dispelling doubts about the potential harmful effect of latent antithrombin present in commercial concentrates for therapeutic use. Moreover, the suggested antiangiogenic role of latent antithrombin, together with its stability in plasma and its negligible thrombogenicity raises the possibility of its use as a new antiangiogenic drug.

Sulforaphane (SFN) is an isothiocyanate found in cruciferous vegetables such as broccoli. This anticarcinogen was first identified as a potent inducer of Phase 2 enzymes, but evidence is mounting that SFN acts through other cancer chemopreventive mechanisms. We recently reported on a novel mechanism of chemoprotection by SFN in human colon cancer cells and prostate epithelial cells, namely the inhibition of histone deacetylase (HDAC). In the present investigation, we sought to test whether SFN also might inhibit HDAC activity in vivo. When consumed in the diet at an average daily dose of 7.5 μmol per animal for 21 days, SFN suppressed the growth of human PC-3 prostate cancer cells by 40% in male nude mice. There was a significant decrease in HDAC activity in the xenografts, as well as in the prostates and mononuclear blood cells (MBC), of mice treated with SFN, compared to controls. There also was a trend towards increased global histone acetylation in the xenografts, prostates, and MBC. In human subjects, a single dose of 68 g BroccoSprouts inhibited HDAC activity significantly in peripheral blood mononuclear cells (PBMC) 3 and 6 hrs following consumption. These findings provide evidence that one mechanism through which SFN acts as a cancer chemopreventive agent in vivo is through the inhibition of HDAC activity. Moreover, the data suggest that HDAC activity in PBMC may be used as a biomarker for assessing exposure to novel dietary HDAC inhibitors in human subjects.

Numerous polyphenolic compounds have been found to inhibit adhesion and migration of leukocytes to sites of inflammation that are partly regulated by the expression of cell adhesion molecules (CAM) such as vascular cell adhesion molecule-1 (VCAM-1), E-selectin, and platelet endothelial cell adhesion molecule-1 (PECAM-1). Licorice root extracts have been used in traditional Chinese, Tibetan, and Indian medicine for the treatment of pulmonary diseases and inflammatory processes. Expression of CAM proteins was examined in human umbilical vein endothelial cells (HUVEC) treated with a licorice component (isoliquiritigenin, 18β-glycyrrhetinic acid, glycyrrhizin, formononetin, or ononin) and exposed to TNF-α. The involvement of NF-κB in the transcriptional control of CAM proteins was assessed by degradation of IκBα and nuclear translocation of NF-κB using Western blotting techniques and immunocytochemical staining. At nontoxic ≥10 μM, isoliquiritigenin blocked the induction of VCAM-1 and E-selectin on activated HUVEC and markedly interfered with THP-1 monocyte adhesion to TNF-α-activated endothelial cells. Isoliquiritigenin abolished TNF-α-induced mRNA accumulation of VCAM-1 and E-selectin. Additionally, immunocytochemical staining revealed that isoliquiritigenin attenuated PECAM-1 expression induced by TNF-α. In contrast, other components recognized in licorice, 18β-glycyrrhetinic acid, glycyrrhizin, formononetin, and ononin did not down-regulate the expression of VCAM-1 and/or PECAM-1 activated by TNF-α, implying that these components are inactive in modulating adhesion of leukocytes to stimulated endothelial cells. Isoliquiritigenin downregulated CAM proteins in TNF-α-activated HUVEC at the transcriptional levels by blocking degradation of IκBα and nuclear translocation of NF-κB. These results demonstrate that the induction blockade of VCAM-1 and E-selectin by isoliquiritigenin was directly mediated by its interference with the CAM mRNA transcription through NF-κB-dependent mechanisms under inflammatory conditions.

Mixed isomers of conjugated linoleic acid (CLA) have been shown to have variable effects on bone formation and resorption in animals. The variable effects of CLA on bone physiology may be due to the different isomers present in common commercial preparations of CLA, and the effects of the predominant individual isomers (9cis,11trans and 10trans,12cis CLA) are not clear. The objective of this study was to determine the effects of individual and mixed isomers of CLA on mineralized bone nodule formation and alkaline phosphatase (ALP) activity in vitro using long-term cultures of SaOS-2 cells. Mineralized bone nodules were stained using the von Kossa method, and ALP activity in cell lysates was measured as a marker of early osteoblast differentiation. The 9cis,11trans isomer increased the number (~4- to 11-fold) and size (~2- to 5-fold) of mineralized bone nodules from 25 to 100 μM, but the 10trans,12cis isomer did not. The increase in mineralized bone nodule formation by 9cis,11trans CLA was accompanied by a variable increase in ALP activity. These results show that the 9cis,11trans isomer of CLA increases the formation of mineralized bone nodules using bone cells of human origin, and provide evidence for isomer-specific effects of CLA on bone health.

It is well known that many burn patients experience psychopathological disorders prior to burn injury. However, it is not known whether individuals that have been exposed to chronic psychological stresses will respond differently than unstressed individuals when challenged by a burn injury. In this study, we assessed whether chronic psychogenic stress prior to burn injury had any significant impact on burn injury-induced alterations in the myeloid compartment in the bone marrow and serum cytokine levels utilizing a well-controlled purely psychogenic stress model (predator exposure). Mice were individually caged and exposed to a Long Evans rat for 1 hr a day on 3 consecutive days prior to a 15% total body surface area flame burn. Four days after burn injury, bone marrow and serum were collected to assess myeloid cells and cytokine levels, respectively. Bone marrow cells were cultured in granulocyte-macrophage colony-stimulating factor (GM-CSF) to assess clonogenic ability. Flow cytometry was also used to characterize the populations of myeloid cells based on Gr-1 and CD11b staining intensity and to determine the expression of the macrophage colony-stimulating factor receptor (M-CSFR). Serum was assayed for IL-6, IL-12p70, MCP-1, and IFN-γ by multiplexed sandwich enzyme-linked immunoabsorbent assay (ELISA). We found that predator exposure prior to burn injury ablated the burn-induced increase in myeloid colony formation and attenuated the burn-induced increases in immature monocytes and immature neutrophils in the bone marrow, as well as MCP-1 levels in the serum. Conversely, psychogenic stress exaggerated the burn-induced increase in the number of M-CSFR-positive cells. This study is the first to show the effects of a pure psychogenic stressor (predator exposure) on burn-induced alterations of the immune system. The clinical ramifications of our findings remain to be elucidated.

Neuropeptide pituitary adenylate cyclase-activating polypeptide (PACAP) is a pleiotropic hormone that is involved in numerous physiologic functions. The present study examines the presence and the functional significance of PACAP and its receptor in the brain and astrocytes of tilapia (Oreochromis mossambicus). This is the first demonstration of the full-length nucleotide sequence of tPACAP gene in tilapia pituitary, brain, and cultured astrocytes. Two cDNA variants of the growth hormone–releasing hormone (GHRH)–PACAP gene were identified in tilapia pituitary, brain, and cultured astrocytes as a result of exon skipping with a long form (271 bp) encoding both tPACAP₃₈ and tGHRH and a short form (166 bp) encoding only tPACAP₃₈. The short form was found to be more abundant in astrocytes. Addition of ovine PACAP₃₈ (1 nM) to cultured astrocytes significantly stimulated the expression of tPACAP₃₈ at 4 hrs, but the effect dropped after 8 hrs of treatment. By contrast, the expression of PACAP type I receptor (PAC₁-R) mRNA in the astrocytes was not responsive to PACAP₃₈ treatment. The tPACAP₃₈ expression also was activated by the cAMP analog, dibutyryl-cAMP, in a dose-dependent manner. Adding high salinity (170 mM NaCl, 500 mOsm/kg osmolarity) to cultured medium substantially increased astroglial tPACAP₃₈ expression over 4 hrs to a level that was maintained for 16 hrs. This observation was not found when mannitol (270 mM) was supplemented as an osmolarity-enhancing agent (500 mOsm/ kg). Taken together, tPACAP expression in tilapia astrocytes was well regulated by exogenous PACAP, cAMP, and salinity and might be involved in the adaptation to high salinity when the fish is in a seawater environment.

Ovarian cancer of surface epithelial origin is an ovulation- and endocrine-related disease. It appears that a cell transformed by genotoxins generated at follicular rupture is propagated during postovulatory wound repair. A consequent steroid hormonal imbalance favoring the mitogenic estrogens is a prospective predisposing factor in ovarian neoplasia. Protection against epithelial ovarian cancer is conferred by progesterone. The objective of this study was to characterize the acute effects of ovulation and steroid hormonal exposure on morphologic responses of surface epithelial cells of mouse ovaries. Follicular development and ovulation were induced in immature animals with equine and human (=Day 0) choriogonadotropins, respectively. On Day 2 (approximately 36 hrs after ovulation), surface epithelial classifications presented in histologic sections were altered from simple (single-layered) squamous and cuboidal toward stratification; this trend was reversed (i.e., reverted to the control status) on Days 4–8. Shifts in the ovarian epithelium from simple to stratified were accentuated following postovulatory (Days 1–8) treatment with estradiol. Surface epithelia of ovaries obtained after 1 week of progesterone administration were exclusively of a simple phenotype. We conclude that the proliferative/procarcinogenic reaction of the ovarian surface epithelium to ovulation is exacerbated by estrogen and counteracted by progesterone.

We reported in a previous work that insulin degradation by insulin-degrading enzyme (IDE) was inhibited by ATP (Exp Biol Med 226:334–341, 2001). Then we studied ATP hydrolysis as a possible mechanism for reversion of this inhibition. ATP hydrolysis was determined by ³²P release after hydrolysis of γ[³²P]ATP. ATP hydrolysis was studied by Sephadex G200 chromatography, immunoprecipitation, and nondissociating gel electrophoresis. Purified recombinant rat IDE and extractive homogenous IDE showed similar ATP hydrolysis. All results showed concordance between insulin degradation and ATP hydrolysis, suggesting that IDE has both functions. In order to define the type of hydrolysis, we studied inhibitors of IDE, phosphohydrolases, and ATPases. Each substance studied had no effect on ATP hydrolysis, except 1 mM orthovanadate, a known inhibitor of ATPases, phosphatases, and insulin degradation. ATP hydrolysis followed a Michaelis-Menten kinetic with Vmax: 570.45 ± 113.08 pmol Pi/hr and apparent Michaelis constant (Km): 63.13 ± 3.48 μM. ATP binding studies strongly suggested an ATP binding site and enzyme kinetics established only one active hydrolytic ATP binding site per IDE molecule. ATP-induced enzyme aggregation changes as observed by electrophoresis mobility in nondissociating conditions and conformational changes on insulin binding as shown by IDE-insulin cross-linking. We conclude that IDEs have ATPase activity and that insulin-binding and degradation are dependent on ATP concentration; however, insulin does not modify the ATPase activity of IDE.

There has been considerable work on the relationships between nutrition and the immune response, particularly on studies that have focused on adaptive responses. There is increasing recognition of the importance of innate immunity in host protection and initiation of cytokine networks. In this study, we examined the effect of select cocoa flavanols and procyanidins on innate responses in vitro. Peripheral blood mono-nuclear cells (PBMCs), as well as purified monocytes and CD4 and CD8 T cells, were isolated from healthy volunteers and cultured in the presence of cocoa flavanol fractions that differ from another by the degree of flavanol polymerization: short-chain flavanol fraction (SCFF), monomers to pentamers; and long-chain flavanol fraction (LCFF), hexamers to decamers. Parallel investigations were also done with highly purified flavanol monomers and procyanidin dimers. The isolated cells were then challenged with lipopolysaccharide (LPS) with quantitation of activation using CD69 and CD83 expression and analysis of secreted tumor necrosis factor (TNF)-α, interleukin (IL)-1β, IL-6, IL-10, and granulocyte macrophage colony-stimulating factor (GM-CSF). The chain length of flavanol fractions had a significant effect on cytokine release from both unstimulated and LPS-stimulated PBMCs. For example, there was a striking increase of LPS-induced synthesis of IL-1β, IL-6, IL-10, and TNF-α in the presence of LCFF. LCFF and SCFF, in the absence of LPS, stimulated the production of GM-CSF. In addition, LCFF and SCFF increased expression of the B cell markers CD69 and CD83. There were also unique differential responses in the mononuclear cell populations studied. We conclude that the oligomers are potent stimulators of both the innate immune system and early events in adaptive immunity.

Styrene trimers migrate from polystyrene food container into foods. We evaluated the estrogenic activity of styrene trimers such as 2,4,6-triphenyl-1-hexene (ST-1), 1a-phenyl-4a-(1′-phenylethyl)tetralin (ST-2), 1a-phenyl-4e-(1′-phenylethyl)tetralin(ST-3), 1e-phenyl-4a-(1′-phenylethyl)tetralin (ST-4), and 1e-phenyl-4e-(1′-phenylethyl)tetralin (ST-5) using the reporter-gene assay with MVLN cells stably expressing the estrogen-stimulated reporter gene, and it was confirmed that ST-1, ST-3, and ST-4 had estrogen-like activity. On the other hand, ST-2 and ST-5 had anti-estrogen-like activity. We examined the estrogenic activity in vivo of ST-1, ST-3, and ST-4. The styrene trimers were administered to pregnant rats, and the effects on the offspring were examined. ST-1, ST-3, or ST-4 (0, 10, 100, 1000 μg/kg body wt/day) were subcutaneously injected into pregnant rats from gestational Day 11 through 17, and the male offspring were sacrificed on postnatal days (PND) 101–103. In the ST-4 treatment groups, the relative anogenital distance on PND 3 was significantly shortened. The relative testis weight was remarkably decreased in all styrene trimer treatment groups. Relative weights of the prostate and epididymides significantly decreased in the ST-4 treatment groups. The relative brain weight was markedly reduced in the ST-3 and ST-4 treatment groups. A significant decrease of the Sertoli cell count was observed in the ST-1 and ST-4 treatment groups. The serum follicle stimulating hormone level was remarkably reduced in all styrene trimer treatment groups. The luteinizing hormone level was significantly decreased and the testosterone level increased in the ST-1 and ST-4 groups. These results suggest that prenatal exposure to estrogenic styrene trimers at low levels obstructed genital organ development, and disrupted the endocrine systems of male rat offspring.

Neonatal cattle and in part neonates of other species have manyfold higher plasma concentrations of nitrite plus nitrate than mature cows and subjects of other species, suggesting an enhanced and needed activation of the nitric oxide (NO) axis at birth. While the biological half-life of NO is short (<1 sec), its functionality can be prolonged, and in many regards more discretely modulated, when it reacts with low-molecular-weight and protein-bound thiols to form S-nitrosothiols (RSNO), from which NO subsequently can be rereleased. We used the calf as a model to test the hypothesis that plasma concentrations of RSNO are elevated at birth in mammals, correlate with ascorbate and urate levels, are selectively generated in critical tissue beds, and are generated in a manner temporally coincident with changes in tissue levels of active NO synthases (NOS). Plasma concentrations of RSNO, ascorbate, and urate were highest immediately after birth (Day 0), dropped >50% on Day 1, and gradually decreased over time, reaching a nadir in mature cattle. Albumin and immunoglobulin G were identified as major plasma RSNO. The presence of S-nitrosocysteine (SNC, a validated marker for S-nitrosylated proteins), inducible NOS (iNOS), and activated endothelial NOS (eNOS phosphorylated at Ser1177) in different tissues was analyzed by immunohistochemistry in another group of similar-aged calves. SNC, iNOS, and phosphorylated eNOS were detected in liver and ileum at the earliest timepoint of sampling (4 hrs after birth), increased between 4 and 24 hrs, and then declined to near-nondetectable levels by 2 weeks of life. Our data show that the neonatal period in the bovine species is characterized by highly elevated and coordinated NO-generating and nitrosylation events, with the ontogenetic changes occurring in iNOS and eNOS contents in key tissues as well as RSNO products and associated antioxidant markers.

In this review, we point out that natural selection does not act to lessen human diseases after the reproductive and caregiving period and that normal levels of iron and copper that may be healthy during the reproductive years appear to be contributing to diseases of aging and possibly the aging process itself. It is clear that oxidant damage contributes to many of the diseases of aging, such as atherosclerosis, Alzheimer’s disease, Parkinson’s diseases, diabetes, diseases of inflammation, diseases of fibrosis, diseases of autoimmunity, and so on. It is equally clear that both iron and copper can contribute to excess production of damaging reactive oxygen species through Fenton chemistry. Here, we examine the evidence that “normal” levels of iron and copper contribute to various diseases of aging.