Impact of structural and metabolic variations on the toxicity and carcinogenicity of hydroxy- and alkoxy-substituted allyl- and propenylbenzenes.

The metabolic fate of a compound is determined by numerous factors including its chemical structure. Although the metabolic options for a variety of functional groups are well understood and can often provide a rationale for the comparison of toxicity based on structural analogy, at times quite minor structural variations may have major consequences for metabolic outcomes and toxicity. In this perspective, the effects of structural variations on metabolic outcomes is detailed for a group of related hydroxy- and alkoxy-substituted allyl- and propenylbenzenes. These classes of compounds are naturally occurring constituents of a variety of botanical-based food items. The classes vary from one another by the presence or absence of alkylation of their para-hydroxyl substituents and/or the position of the double bond in the alkyl side chain. We provide an overview of how these subtle structural variations alter the metabolism of these important food-borne compounds, ultimately influencing their toxicity, particularly their DNA reactivity and carcinogenic potential. The data reveal that detailed knowledge of the consequences of subtle structural variations for metabolism is essential for adequate comparison of structurally related chemicals. Taken together, it is concluded that predictions in toxicological risk assessment should not be performed on the basis of structural analogy only but should include an analogy of metabolic pathways across compounds and species.

Antioxidant intake, GSTM1 polymorphism and pulmonary function in healthy young adults.

Dietary antioxidants may protect lung tissue against reactive oxygen species-induced injury, adverse respiratory effects and reduced pulmonary function. Genetic variability in antioxidant enzymes also determines response to oxidative stress in the lung. The current authors evaluated whether lung function levels are associated with dietary intake of antioxidants and the glutathione S-transferase M1 (GSTM1) polymorphism. The current study cohort consisted of healthy, nonsmoking freshmen students who were lifetime residents in the Los Angeles or the San Francisco Bay areas (CA, USA). Participants completed comprehensive residential history, health history and food frequency questionnaires. Blood for genotyping was collected and forced expiratory volume measurements were obtained. Dietary vitamin C, magnesium and daily fruit servings were associated positively with forced expiratory volume in one second in males and with maximum mid-expiratory flow, forced expiratory flow after 75% of expelled volume, and the ratio of maximum mid-expiratory flow to forced vital capacity in females. In multivariable regression, vitamin C (or fruit for male students) and magnesium showed a consistent, positive association with lung function. Among healthy female adolescents, dietary intake of vitamin C is associated with increased levels of lung function. The current study does not support a role for the glutathione S-transferase M1-null genotype as an independent risk factor for decrements in lung function.

Absence of direct delivery for single transmembrane apical proteins or their "Secretory" forms in polarized hepatic cells.

The absence of a direct route to the apical plasma membrane (PM) for single transmembrane domain (TMD) proteins in polarized hepatic cells has been inferred but never directly demonstrated. The genes encoding three pairs of apical PM proteins, whose extracellular domains are targeted exclusively to the apical milieu in Madin-Darby canine kidney cells, were packaged into recombinant adenovirus and delivered to WIF-B cells in vitro and liver hepatocytes in vivo. By immunofluorescence and pulse-chase metabolic labeling, we found that the soluble constructs were overwhelmingly secreted into the basolateral milieu, which in vivo is the blood and in vitro is the culture medium. The full-length proteins were first delivered to the basolateral surface but then concentrated in the apical PM. Our results imply that hepatic cells lack trans-Golgi network (TGN)-based machinery for directly sorting single transmembrane domain apical proteins and raise interesting questions about current models of PM protein sorting in polarized and nonpolarized cells.

Role of endothelial cell extracellular signal-regulated kinase1/2 in urokinase-type plasminogen activator upregulation and in vitro angiogenesis by fibroblast growth factor-2.

Downstream signaling triggered by the binding of fibroblast growth factor-2 (FGF2) to its tyrosine-kinase receptors involves the activation of mitogen-activated protein kinase kinase (MEK) with consequent phosphorylation of extracellular signal-regulated kinases (ERKs). Here we demonstrate that FGF2 induces ERK1/2 activation in bovine aortic endothelial (BAE) cells and that the continuous presence of the growth factor is required for sustained ERK1/2 phosphorylation. This is prevented by the MEK inhibitors PD 098059 and U0126, which also inhibit FGF2-mediated upregulation of urokinase-type plasminogen activator (uPA) and in vitro formation of capillary-like structures in three-dimensional type I collagen gel. Various FGF2 mutants originated by deletion or substitution of basic amino acid residues in the amino terminus or in the carboxyl terminus of FGF2 retained the capacity to induce a long-lasting activation of ERK1/2 in BAE cells. Among them, K128Q/R129Q-FGF2 was also able to stimulate uPA production and morphogenesis whereas R129Q/K134Q-FGF2 caused uPA upregulation only. In contrast, K27, 30Q/R31Q-FGF2, K128Q/K138Q-FGF2 and R118,129Q/K119,128Q-FGF2 exerted a significant uPA-inducing and morphogenic activity in an ERK1/2-dependent manner only in the presence of heparin. Furthermore, no uPA upregulation and morphogenesis was observed in BAE cells treated with the deletion mutant (delta)27-32-FGF2 even in the presence of soluble heparin. Thus, mutational analysis of FGF2 dissociates the capacity of the growth factor to induce a persistent activation of ERK1/2 from its ability to stimulate uPA upregulation and/or in vitro angiogenesis. In conclusion, the data indicate that ERK1/2 phosphorylation is a key step in the signal transduction pathway switched on by FGF2 in endothelial cells. Nevertheless, a sustained ERK1/2 activation is not sufficient to trigger uPA upregulation and morphogenesis. FGF2 mutants may represent useful tools to dissect the signal transduction pathway(s) mediating the complex response elicited by an angiogenic stimulus in endothelial cells.

Basic fibroblast growth factor-induced angiogenic phenotype in mouse endothelium. A study of aortic and microvascular endothelial cell lines.

The mouse is the most commonly used species for in vivo studies on angiogenesis related to tumor development. Yet, to the best of our knowledge, very few reports on the in vitro interaction of the angiogenic basic fibroblast growth factor (bFGF) with mouse endothelial cells are available. Three mouse endothelial cell lines originated from aorta (MAECs), brain capillaries (MBECs), and heart capillaries (MHECs) were characterized for endothelial phenotypic markers, in vivo tumorigenic activity, and the capacity to respond in vitro to bFGF. These cells express angiotensin-converting enzyme, acetylated LDL receptor, constitutive endothelial nitric oxide synthase, and vascular cell adhesion molecule-1 and bind Griffonia simplicifolia-I lectin. When injected subcutaneously in nude mice, MAECs induced the appearance of slow-growing vascular lesions reminiscent of epithelioid hemangioendothelioma, whereas MBEC xenografts grew rapidly, showing Kaposi's sarcoma-like morphological features. No lesions were induced by injection of MHECs. MAECs, MBECs, and MHECs expressed both low-affinity heparan sulfate bFGF-binding sites and high-affinity tyrosine kinase receptors (FGFRs) on their surfaces. In particular, MAECs expressed FGFR-2/bek mRNA, whereas microvascular MBECs and MHECs expressed FGFR-1/flg mRNA. Accordingly, bFGF induced a mitogenic response and the phosphorylation of extracellular signal-regulated kinase-2 in all the cell lines. In contrast, upregulation of urokinase-type plasminogen activator expression was observed in bFGF-treated microvascular MBECs and MHECs but not in MAECs. Also, bFGF-treated MBECs and MHECs but not MAECs invaded a three-dimensional fibrin gel and formed hollow, capillary-like structures. The relevance of the modifications of the fibrinolytic balance of mouse microvascular endothelium in bFGF-induced angiogenesis was validated in vivo by a gelatin-sponge assay in which the plasmin inhibitors tranexamic acid and epsilon-aminocaproic acid given to mice in the drinking water inhibited neovascularization induced by the growth factor. In conclusion, differences in response to bFGF exist between large-vessel MAECs and microvascular MBECs and MHECs. Both in vitro and in vivo data point to a role of the profibrinolytic phenotype induced by bFGF in microvascular endothelial cells during mouse angiogenesis. Our observations make these endothelial cell lines suitable for further studies on mouse endothelium during angiogenesis and in angioproliferative diseases.

New model for the study of angiogenesis and antiangiogenesis in the chick embryo chorioallantoic membrane: the gelatin sponge/chorioallantoic membrane assay.

Several methods for the in vivo study of angiogenesis are available, and each angiogenic assay presents distinct advantages and disadvantages. In this study, we present a new method for the quantitation of angiogenesis and antiangiogenesis in the chick embryo chorioallantoic membrane (CAM), based on the implantation of gelatin sponges on the top of growing CAM, on day 8 of incubation. After implantation, the sponges were treated with a stimulator (recombinant human basic fibroblast growth factor, FGF2) or an inhibitor (a rabbit polyclonal anti-FGF2 antibody) of blood vessel formation. Blood vessels growing vertically into the sponge and at the boundary between sponge and surrounding CAM mesenchyme were counted by a morphometric method on day 12. In addition, to assess whether the gelatin sponge is an appropriate vehicle to deliver cultured cells and evaluate their angiogenic potential, mouse aortic endothelial cells were cotransfected with human FGF2 and the Escherichia coli beta-galactosidase (beta-GAL) reporter gene. Stable transfectants were absorbed by the sponge, and evaluation of the angiogenic response was paralleled by beta-GAL staining to visualize implanted cells. This technique may facilitate the discovery and development of agonists or antagonists of angiogenesis.

Basic fibroblast growth factor overexpression in endothelial cells: an autocrine mechanism for angiogenesis and angioproliferative diseases.

Basic fibroblast growth factor (bFGF) is expressed in vascular endothelium during tumor neovascularization and angioproliferative diseases. The ultimate significance of this observation is poorly understood. We have investigated the biological consequences of endothelial cell activation by endogenous bFGF in a mouse aortic endothelial cell line stably transfected with a retroviral expression vector harboring a human bFGF cDNA. Selected clones expressing M(r) 24,000, M(r) 22,000, and/or M(r) 18,000 bFGF isoforms were characterized by a transformed morphology and an increased saturation density. bFGF transfectants showed invasive behavior and sprouting activity in three-dimensional fibrin gels and formed a complex network of branching cord-like structures connecting foci of infiltrating cells when seeded on laminin-rich basement membrane matrix (Matrigel). The invasive and morphogenetic behavior was prevented by anti-bFGF antibody, revealing the autocrine modality of the process. The biological consequences of this autocrine activation were investigated in vivo. bFGF-transfected cells gave rise to highly vascularized lesions resembling Kaposi's sarcoma when injected in nude mice and induced angiogenesis in avascular rabbit cornea. When injected into the allantoic sac of the chick embryo, they caused an increase in vascular density and formation of hemangiomas in the chorioallantoic membrane. In conclusion, bFGF-overexpressing endothelial cells acquired an angiogenic phenotype and recruit quiescent endothelium originating angioproliferative lesions in vivo. These findings demonstrate that bFGF overexpression exerts an autocrine role for endothelial cells and support the notion that tumor neovascularization and angioproliferative diseases can be triggered by stimuli that induce vascular endothelium to produce its own autocrine factor(s).

Suppression of angiogenesis by the antitumor agent titanocene dichloride.

Titanocene dichloride, which is an active antitumor agent against solid but not blood-borne tumors, suppresses angiogenesis and inhibits biosynthesis of collagenous proteins in the in vivo system of the chorioallantoic membrane of the chick embryo. The agent does not affect total protein biosynthesis in the same system. At non-toxic dose regimens titanocene dichloride retards the growth of Walker 256 carcinosarcoma transplants in rats and reduces the number of seeded implants in the mesenteric bed. At concentrations which suppress angiogenesis and inhibit biosynthesis of collagenous proteins, the agent does not affect the viability of Walker 256 carcinosarcoma cells, or the attachment and proliferation of human A549 lung adenocarcinoma or human umbilical vein endothelial cells in culture. It appears that the antitumor activity of titanocene dichloride may be attributed, at least in part, to its ability to suppress angiogenesis.