Publisher Summary This chapter discusses the role of free radicals and catalytic metal ions in human disease. The importance of transition metal ions in mediating oxidant damage naturally leads to the question as to what forms of such ions might be available to catalyze radical reactions in vivo . The chapter discusses the metabolism of transition metals, such as iron and copper. It also discusses the chelation therapy that is an approach to site-specific antioxidant protection. The detection and measurement of lipid peroxidation is the evidence most frequently cited to support the involvement of free radical reactions in toxicology and in human disease. A wide range of techniques is available to measure the rate of this process, but none is applicable to all circumstances. The two most popular are the measurement of diene conjugation and the thiobarbituric acid (TBA) test, but they are both subject to pitfalls, especially when applied to human samples. The chapter also discusses the essential principles of the peroxidation process. When discussing lipid peroxidation, it is essential to use clear terminology for the sequence of events involved; an imprecise use of terms such as initiation has caused considerable confusion in the literature. In a completely peroxide-free lipid system, first chain initiation of a peroxidation sequence in a membrane or polyunsaturated fatty acid refers to the attack of any species that has sufficient reactivity to abstract a hydrogen atom from a methylene group.

Role of free radicals and catalytic metal ions in human disease: an overview.

Flavonoids are nearly ubiquitous in plants and are recognized as the pigments responsible for the colors of leaves, especially in autumn. They are rich in seeds, citrus fruits, olive oil, tea, and red wine. They are low molecular weight compounds composed of a three-ring structure with various substitutions. This basic structure is shared by tocopherols (vitamin E). Flavonoids can be subdivided according to the presence of an oxy group at position 4, a double bond between carbon atoms 2 and 3, or a hydroxyl group in position 3 of the C (middle) ring. These characteristics appear to also be required for best activity, especially antioxidant and antiproliferative, in the systems studied. The particular hydroxylation pattern of the B ring of the flavonoles increases their activities, especially in inhibition of mast cell secretion. Certain plants and spices containing flavonoids have been used for thousands of years in traditional Eastern medicine. In spite of the voluminous literature available, however, Western medicine has not yet used flavonoids therapeutically, even though their safety record is exceptional. Suggestions are made where such possibilities may be worth pursuing.

The Effects of Plant Flavonoids on Mammalian Cells:Implications for Inflammation, Heart Disease, and Cancer

If carcinogenesis occurs by somatic evolution, then common components of the cancer phenotype result from active selection and must, therefore, confer a significant growth advantage. A near-universal property of primary and metastatic cancers is upregulation of glycolysis, resulting in increased glucose consumption, which can be observed with clinical tumour imaging. We propose that persistent metabolism of glucose to lactate even in aerobic conditions is an adaptation to intermittent hypoxia in pre-malignant lesions. However, upregulation of glycolysis leads to microenvironmental acidosis requiring evolution to phenotypes resistant to acid-induced cell toxicity. Subsequent cell populations with upregulated glycolysis and acid resistance have a powerful growth advantage, which promotes unconstrained proliferation and invasion.

Why do cancers have high aerobic glycolysis

Phenolics are broadly distributed in the plant kingdom and are the most abundant secondary metabolites of plants. Plant polyphenols have drawn increasing attention due to their potent antioxidant properties and their marked effects in the prevention of various oxidative stress associated diseases such as cancer. In the last few years, the identification and development of phenolic compounds or extracts from different plants has become a major area of health- and medical-related research. This review provides an updated and comprehensive overview on phenolic extraction, purification, analysis and quantification as well as their antioxidant properties. Furthermore, the anticancer effects of phenolics in-vitro and in-vivo animal models are viewed, including recent human intervention studies. Finally, possible mechanisms of action involving antioxidant and pro-oxidant activity as well as interference with cellular functions are discussed.

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Plant phenolics: extraction, analysis and their antioxidant and anticancer properties.

The relationship between antioxidant activity and antimutagenicity of various tea extracts (green tea, pouchong tea, oolong tea, and black tea) was investigated. All tea extracts exhibited markedly antioxidant activity and reducing power, especially oolong tea, which inhibited 73.6% peroxidation of linoleic acid. Tea extracts exhibited a 65-75% scavenging effect on superoxide at a dose of 1 mg and 30 - 60% scavenging effect on hydrogen peroxide at a dose of 400 microgram. They scavenged 100% hydroxyl radical at a dosage of 4 mg except the black tea. Tea extracts also showed 50 - 70% scavenging effect on alpha, alpha-diphenyl-beta-picrylhydrazyl radical. The antioxidant activity and the scavenging effects on active oxygen decreased in the order semifermented tea > nonfermented tea > fermented tea. Tea extracts showed strong antimutagenic action against five indirect mutagens, i.e., AFB1, Trp-P-1, Glu-P-1, B[a]P, and IQ, especially oolong and pouchong teas. The antioxidant effect of tea extracts was well correlated to their antimutagenicity in some cases but varied with the mutagen and antioxidative properties.

Antioxidant activity of various tea extracts in relation to their antimutagenicity

An antioxidant fraction of Chinese green tea (green tea antioxidant; GTA), containing several catechins, has been previously shown to inhibit 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced tumor promotion in mouse skin. In the present study, GTA was shown to have antioxidative activity toward hydrogen peroxide (H2O2) and the superoxide radical (O2-). GTA also prevented oxygen radical and H2O2-induced cytotoxicity and inhibition of intercellular communication in cultured B6C3F1 mouse hepatocytes and human keratinocytes (NHEK cells). GTA (0.05-50 micrograms/ml) prevented the killing of hepatocytes (measured by lactate dehydrogenase release) by paraquat (1-10 mM) and glucose oxidase (0.8-40 micrograms/ml) in a concentration-dependent fashion. GTA (50 micrograms/ml) also prevented the inhibition of hepatocyte intercellular communication by paraquat (5 mM), glucose oxidase (0.8 micrograms/ml), and phenobarbital (500 micrograms/ml). In addition, GTA (50 micrograms/ml) prevented the inhibition of intercellular communication in human keratinocytes by TPA (100 ng/ml). Cytotoxicity and inhibition of intercellular communication, two possible mechanisms by which tumor promoters may produce their promoting effects were therefore prevented by GTA. The inhibition of these two effects of pro-oxidant compounds may suggest a mechanism by which GTA inhibits tumor promotion in vivo.

Prevention of cytotoxicity and inhibition of intercellular communication by antioxidant catechins isolated from Chinese green tea

To explain the complex carcinogenic process by which a single normal cell in human beings can be converted to an invasive and metastatic cancer cell, a number of experimental findings, epidemiological observations and their associated hypothesis/theories have been integrated in this review. All cancers have been generally viewed as the result of a disruption of the homeostatic regulation of a cell's ability to respond appropriately to extra-cellular signals of the body which trigger intra-cellular signal transducting mechanisms which modulate gap junctional intercellular communication between the cells within a tissue. Normal homeostatic control of these three forms of cell communication determines whether: (a) the cell remains quiescent (Go); (b) enters into the cell proliferation phase; (c) is induced to differentiate; (d) is committed to apoptose; or (e) if it is already differentiated, it can adaptively respond. During the evolution from single cell organisms to multicellular organisms, new cellular/biological functions appeared, namely, the control of cell proliferation ("contact inhibition"), the appearance of the process of differentiation from committed stem cells of the various tissues and the need for programmed cell death or apoptosis. Interestingly, cancer cells have been characterized as cells: (a) having been derived from a stem-like cell; (b) without their ability to control cell growth or without the ability to contact inhibit; (c) which can not terminally differentiate under normal conditions; and (d) having altered ability to apoptosis under normal conditions. During that evolutionary transition from the single cell organism to the multicellular organism, many new genes appeared to accompany these new cellular functions. One of these new genes was the gene coding for a membrane associated protein channel (the gap junction) which between coupled cells, allowed the passive transfer on ions and small molecular weight molecules. A family of over a dozen of these highly evolutionarily-conserved genes (the connexin genes) coded for the connexin proteins. A hexameric unit of these connexins in one cell (a connexon) couples with a corresponding connexon in a contiguous cell to join the cytoplasms. This serves to synchronize either the metabolic or electrotonic functions of cells within a tissue. Most normal cells within solid tissues have functional gap junctional intercellular communication (GJIC) (exceptions are free-standing cells such as red blood cells, neutrophils, and several, if not all, the stem cells). On the other hand, the cancer cells of solid tissues appear to have either dysfunctional homologous or heterologous GJIC. Therefore, among the many differences between a cancer cell and its normal parental cell, the carcinogenic process involves the transition from a normal, GJIC-competent cell to one that is defective in GJIC. The review examines how GJIC can be either transiently or stably modulated by endogenous or exogenesis chemicals or by oncogenes and tumor suppressor genes at the transcriptional, translational, or posttranslational levels. It also uses the gap junction as the biological structure to facilitate cellular/tissue homeostasis to be the integrator for the "stem cell" theory, "disease of differentiation theory", "initiation/promotion/progression" concepts, nature and nurture concept of carcinogenesis, the mutation/ epigenetic theories of carcinogenesis, and the oncogene/ tumor suppressor gene theories of carcinogenesis. From this background, implications to cancer prevention and cancer therapy are generated.

https://www.fbsserver.org/1998/v3/d/trosko/trosko.pdf

Cell-cell communication in carcinogenesis.

Gap-junction channels connect the interiors of adjacent cells and can be arranged into aggregates or plaques consisting of hundreds to thousands of channel particles. The mechanism of channel aggregation into plaques and whether plaques can disaggregate are not known. Many carcinogenic and tumor-promoting chemicals have been identified that inhibit cell-cell gap-junctional coupling. Here, we provide morphological evidence that 18 beta-glycyrrhetinic acid (18 beta-GA), a saponin isolated from licorice root that is an inhibitor of gap-junctional communication, caused the disassembly of gap-junction plaques in WB-F344 rat liver epithelial cells. This effect was dose (5-40 microM) and time dependent (1-4 h treatment). Gap-junction channels in WB-F344 cells are comprised of connexin 43 (Cx43), and the protein is phosphorylated to a species known as Cx43-P2 coincident with the assembly of channels into plaques. Consistent with this, the disassembly of plaques induced by 18 beta-GA was correlated with decreases in Cx43-P2 levels and increases in nonphosphorylated Cx43. Biochemical evidence indicated that these changes in the P2 and NP forms of Cx43 represented 18 beta-GA-induced dephosphorylation of Cx43-P2 and not its degradation or the inhibition of Cx43-NP phosphorylation. Okadaic acid and calyculin A, which are inhibitors of type 1 and type 2A protein phosphatases, prevented the dephosphorylation of Cx43, suggesting that one or both of these phosphatases were involved in Cx43 dephosphorylation. These data indicate that 18 beta-GA causes type 1 or type 2A protein phosphatase-mediated Cx43 dephosphorylation coincident with the disassembly of gap-junction plaques.

Gap‐junction disassembly and connexin 43 dephosphorylation induced by 18β‐glycyrrhetinic acid

The effects of three tumor promoters on gap-junction permeability; connexin 43 and 26 mRNA levels, protein levels, and phosphorylation; and the numbers of gap-junctional membrane plaques were studied in the rat liver epithelial cell line WB-F344 to determine whether changes in these parameters correlated with the inhibition of gap-junction function. 12-O-tetradecanoylphorbol-13-acetate (TPA; 10 ng/mL), dieldrin (10 μg/mL), and heptachlor epoxide (10 μg/mL) inhibited gap-junctional intercellular communication (GJIC) assayed by fluorescent dye transfer by 80–90% after a 5-min exposure and by more than 90% within 1 h. Decreases in steady-state connexin 43 mRNA levels were detected by northern blot analysis within 1 h and paralleled changes in steady-state β-actin mRNA, but these changes did not occur rapidly enough to account for the rapid loss of gap-junction function. A substantial loss in the number of connexin 43 immunostained gap-junctional membrane plaques was detected after a 15-min exposure to all three promoters, but little change had occurred at 5 min. Western blot analyses using connexin 43-specific antibodies showed changes in the degree of connexin 43 phosphorylation for all three tumor promoters. TPA induced the appearance of a fourth connexin 43-im-munoreactive band (P3) and a concomitant decrease in the relative intensity of the unphosphorylated (P0) band within 5 min of treatment. P3, in addition to bands P1 and P2, disappeared after treatment with alkaline phosphatase. In contrast, dieldrin and heptachlor epoxide induced loss of P2 with a concomitant increase in the relative staining intensity of P0 within 1 h of exposure, but no changes were seen after 5 min. Connexin 43 phosphorylation levels recovered in parallel with the recovery of GJIC for all three tumor promoters. Connexin 26 mRNA levels showed little change after a 1-h exposure to the three promoters, but reductions in connexin 26 immunofluorescent staining were observed. These results suggest that (i) TPA-induced hyperphosphorylation of connexin 43 occurred fast enough to account for inhibition of GJIC, (ii) dieldrin and heptachlor epoxide modulated connexin phosphorylation in a manner different from TPA by promoting hypophosphorylation of connexin 43, (iii) redistribution of plasma membrane gap-junctional plaques after treatment with phorbol ester and non-phorbol-ester tumor promoters occurred subsequent to changes in gap-junction permeability, and (iv) changes in connexin mRNA levels could not account for the losses in fluorescent dye coupling induced by these promoters. © 1994 Wiley-Liss, Inc.

Changes in gap-junction permeability, phosphorylation, and number mediated by phorbol ester and non-phorbol-ester tumor promoters in rat liver epithelial cells.

Randall J. Ruch

Papers

Prevention of cytotoxicity and inhibition of intercellular communication by antioxidant catechins isolated from Chinese green tea

Cell-cell communication in carcinogenesis.

Gap‐junction disassembly and connexin 43 dephosphorylation induced by 18β‐glycyrrhetinic acid

Changes in gap-junction permeability, phosphorylation, and number mediated by phorbol ester and non-phorbol-ester tumor promoters in rat liver epithelial cells.

Role of inhibition of intercellular communication in carcinogenesis.