Antioxidant health effects of aged garlic extract.

Oxidative modification of DNA, proteins and lipids by reactive oxygen species (ROS) plays a role in aging and disease, including cardiovascular, neurodegenerative and inflammatory diseases and cancer. Extracts of fresh garlic that are aged over a prolonged period to produce aged garlic extract (AGE) contain antioxidant phytochemicals that prevent oxidant damage. These include unique water-soluble organosulfur compounds, lipid-soluble organosulfur components and flavonoids, notably allixin and selenium. Long-term extraction of garlic (up to 20 mo) ages the extract, creating antioxidant properties by modifying unstable molecules with antioxidant activity, such as allicin, and increasing stable and highly bioavailable water-soluble organosulfur compounds, such as S-allylcysteine and S-allylmercaptocysteine. AGE exerts antioxidant action by scavenging ROS, enhancing the cellular antioxidant enzymes superoxide dismutase, catalase and glutathione peroxidase, and increasing glutathione in the cells. AGE inhibits lipid peroxidation, reducing ischemic/reperfusion damage and inhibiting oxidative modification of LDL, thus protecting endothelial cells from the injury by the oxidized molecules, which contributes to atherosclerosis. AGE inhibits the activation of the oxidant-induced transcription factor, nuclear factor (NF)-kappa B, which has clinical significance in human immunodeficiency virus gene expression and atherogenesis. AGE protects DNA against free radical--mediated damage and mutations, inhibits multistep carcinogenesis and defends against ionizing radiation and UV-induced damage, including protection against some forms of UV-induced immunosuppression. AGE may have a role in protecting against loss of brain function in aging and possess other antiaging effects, as suggested by its ability to increase cognitive functions, memory and longevity in a senescence-accelerated mouse model. AGE has been shown to protect against the cardiotoxic effects of doxorubicin, an antineoplastic agent used in cancer therapy and against liver toxicity caused by carbon tetrachloride (an industrial chemical) and acetaminophen, an analgesic. Substantial experimental evidence shows the ability of AGE to protect against oxidant-induced disease, acute damage from aging, radiation and chemical exposure, and long-term toxic damage. Although additional observations are warranted in humans, compelling evidence supports the beneficial health effects attributed to AGE, i.e., reducing the risk of cardiovascular disease, stroke, cancer and aging, including the oxidant-mediated brain cell damage that is implicated in Alzheimer's disease.

Cyclin D1 induced apoptosis maintains the integrity of the G1/S checkpoint following ionizing radiation irradiation.

Cell cycle "checkpoints" help to ensure the integrity of normal cellular functions prior to replicative DNA synthesis and/or cell division. Cell kinetic abnormalities, particularly arrests at the G1/S and G2/M cell cycle checkpoints, are induced following exposure to ionizing radiation in vitro. Following irradiation, cellular signaling pathways may lead to G1 arrest and/or apoptosis at the G1/S cell cycle transition point. Transfection of cyclin D1, a G1/S cyclin, into a rat embryo cells (REC) results in cellular populations that overexpress cyclin D1, are transformed morphologically, demonstrate an increased incidence of apoptosis, and are tumorigenic in immune-deficient mice. Despite such phenotypic changes, transfected cell populations maintain the integrity of the G1 checkpoint following ionizing radiation. The transfected cells overexpressing Cyclin D1 have a statistically significant increase in the incidence of apoptosis as compared to parental REC strains or mock-transfected REC. The work provides further evidence of Cyclin D1 playing a critical role in maintaining the integrity of the G1/S checkpoint, via the activation of apoptotic pathways following exposure to ionizing radiation in vitro.

Transfection of rat embryo cells with mutant p53 increases the intrinsic radiation resistance.

Dominant oncogenic sequences have been shown to modulate the intrinsic radiation sensitivity of cells of both human and murine tumor cell lines. Whether transfection with candidate tumor-suppressor genes can modulate intrinsic radiation sensitivity is unknown. The data presented here demonstrate that transfection of rat embryo cells with a mutant p53 allele can increase the intrinsic radiation resistance of cells in vitro. First, transfection with mutant p53 resulted in transformed cellular morphology. Second, the transfected clone and the corresponding pooled population of transfected clones were more resistant to ionizing radiation in vitro. Last, analyses of the parameters of cell kinetics suggested that the radiobiological effects were unlikely to be due to altered parameters of cell kinetics at the time of irradiation, suggesting that mutant p53 altered the intrinsic radiation resistance of transfected cells by a more direct mechanism. Further experimentation will be necessary to develop a mechanistic approach for the study of these alterations.

Molecular mechanisms in cancer induction and prevention.

Chemical and physical carcinogens, present in our environment and encountered in a variety of occupations, produce damage to DNA. X-rays produced direct ionizations and indirect hydroxyl radical attack. UV light in the short wavelength is specifically absorbed by unsaturated bonds in DNA, RNA, and proteins. There are a number of genetic sites that are specifically affected by environmental agents, and an increased sensitivity is found in certain genetic diseases. The development of a fully malignant tumor involves the activation or altered expression of oncogenes or the inactivation of tumor-suppressor genes that control normal cellular development. Mutations in the p53 tumor-suppressor gene are common in diverse types of cancer and could perhaps provide clues to the etiology of some cancers and to the effect of various environmental and occupational carcinogens in cancer development. The fact that environmental factors are involved to a great extent in cancer suggest that cancer may be preventable. Experimental as well as epidemiological data indicate that a variety of nutritional factors can act as anticarcinogens and inhibit the process of cancer development and reduce cancer risk. The interaction of cells with a number of environmental and occupational genotoxic substances such as X-rays, UV light, and a variety of chemicals including ozone results in an enhanced generation of free oxygen radicals and in modified pro-oxidant states. A number of nutritional factors such as vitamins A, C, E, beta-carotene, and micronutrients such as selenium act as antioxidants and anticarcinogens. Certain hormones such as thyroid hormones enhance oxidative processes and act as a co-transforming factor in carcinogenesis.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of omega 3 and omega 6 fatty acids on transformation of cultured cells by irradiation and transfection.

Mouse embryo fibroblasts (C3H 10T1/2) were exposed to 4 Gy of gamma-rays. The cells yielded 5-8 transformed foci per 10(4) surviving cells. Addition of 100 microM of either eicosapentaenoate or docosahexaenoate to the tissue culture medium reduced the number of transformed foci to 0-1.4. C3H 10T1/2 and NIH 3T3 cells were transfected with plasmid T24 containing the Harvey ras oncogene. C3H 10T1/2 cells yielded 0.85-1.1 foci/ng DNA, while NIH 3T3 cells yielded 0.12-0.14 foci/ng DNA. Foci formation was suppressed 65% in C3H 10T1/2 cells and 93% in NIH 3T3 cells when 100 microM eicosapentaenoate was present in the culture medium. Docosahexaenoate had a similar but somewhat weaker effect. Addition of arachidonate to the medium had little or no effect. Cells grown in the presence of added eicosapentaenoate or docosahexaenoate produced much less prostaglandin E when challenged with calcium ionophore A23187. This is a reflection of changes in arachidonate production or utilization that occur during transformation which are suppressed by the added omega 3 fatty acids. Addition of eicosapentaenoate or docosahexaenoate to the culture medium resulted in extensive remodeling of the molecular species of the four major phospholipid classes that were examined. In its simplest form, omega 3-fatty acid-containing species substantially replaced omega 6-fatty acid-containing species. However, many more subtle changes occurred, and the different phospholipids responded differently to different polyunsaturated fatty acids. A feature of C3H 10T1/2 cells was their preferential accumulation of molecular species of 22-carbon fatty acids such as docosapentaenoate (22:5 omega 3) and docosatetraenoate (22:4 omega 6) in preference to eicosapentaenoate (20:5 omega 3) and eicosapentaenoate (arachidonate, 20:4 omega 6). It is proposed that the protective effect of eicosapentaenoate and docosahexaenoate arises out of the changes in the composition of the fatty acids that are released from one or more phospholipids by the action of phospholipases. The changes consist of a reduced release of arachidonate, the normal substrate of cyclooxygenase and lipoxygenases, and a greatly increased release of eicosapentaenoate and docosahexaenoate, which inhibit one or more of these enzymes, or form oxygenated products which are not as active as the arachidonate-derived products. Other mechanisms are also considered.

Role of transfection and clonal selection in mediating radioresistance.

Transfected oncogenes have been reported to increase the radioresistance of rodent cells. Whether transfected nononcogenic DNA sequences and subsequent clonal selection can result in radioresistant cell populations is unknown. The present set of experiments describe the in vitro radiosensitivity and tumorigenicity of selected clones of primary rat embryo cells and human glioblastoma cells, after transfection with a neomycin-resistance marker (pSV2neo or pCMVneo) and clonal selection. Radiobiological data comparing the surviving fraction at 2 Gy (SF2) and the mean inactivation dose show the induction of radioresistance in two rat embryo cell clones and one glioblastoma clone, as compared to untransfected cells. Wild-type and transfectant clones were injected into three strains of immune-deficient mice (scid, NIH, and nu/nu) to assay for tumorigenicity and metastatic potential. Only the glioblastoma parent line and its transfectant clones were tumorigenic. None of the cells produced spontaneous or experimentally induced metastases. Flow cytometric analyses indicated that the induction of radioresistance could not be attributed to changes in cell kinetics at the time of irradiation. Our results show that transfection of a neomycin-resistance marker and clonal selection can impart radioresistance on both normal and tumor cells. The work also indicates that altered radiation sensitivity does not necessarily correlate with changes in cell-cycle kinetics at the time of irradiation, tumorigenicity, or altered metastatic potential. Our findings have critical implications for transfection studies investigating determinants of cellular radiosensitivity.

Long-chain (sphingoid) bases inhibit multistage carcinogenesis in mouse C3H/10T1/2 cells treated with radiation and phorbol 12-myristate 13-acetate.

Sphingosine and other long-chain (sphingoid) bases inhibit protein kinase C, the putative cellular receptor for the tumor promoter phorbol 12-myristate 13-acetate (PMA), and exert potent effects on diverse cell functions. We tested the ability of long-chain bases to modulate multistage carcinogenesis in mouse C3H/10T1/2 cells exposed to gamma-rays and PMA. Sphingosine and sphinganine completely blocked the enhancement of radiation-induced transformation by PMA (promotion) and partially suppressed transformation by radiation alone. N-Acetylsphingosine, a ceramide analog, did not inhibit transformation. Sphingosine was rapidly taken up by the cells and metabolized; hence, the long-chain bases were added daily to achieve prolonged inhibition. Long-chain bases inhibited protein kinase C activity in C3H/10T1/2 cells and suppressed the down-regulation of this enzyme by PMA. Our results establish that long-chain bases are highly effective inhibitors of carcinogenesis in this model. Our results also indicate that the suppressive effects may be mediated, in part, by inhibition of protein kinase C. The data suggest that sphingosine and other long-chain bases derived from complex sphingolipids may act as cancer-preventative agents.

Morphological transformation of 10T1/2 mouse embryo cells can be initiated by DNA double-strand breaks alone.

Malignant transformation of mouse fibroblasts was produced by electroporation with restriction enzymes. Similar transformation frequencies were observed with Pstl, Pvull, and Xbal, which cut genomic DNA at similar overall frequencies but have different termini, i.e., a 3' overhang, a blunt end, and a 5' overhang, respectively. The dose-response curve for restriction enzyme transformation shows a marked plateau in frequencies of transformed foci per surviving cell, whereas x-irradiation of the same cells gives a linear dose-response curve. Evidently, transformation can be caused by DNA double-strand breaks alone at a limited number of sites, but the evidence from x rays suggests that other kinds of DNA damage can cause transformation independently.

Free-radical processes in multistage carcinogenesis.

Rodent and human cells in culture, transformed in vitro by radiation or chemicals into malignant cells, afford us the opportunity to probe into early and late events in the neoplastic process at a cellular and molecular level. Transformation can be regarded as an abnormal expression of cellular genes. The initiating agents disrupt the integrity of the genetic apparatus altering DNA in ways that result in the activation of cellular transforming genes (oncogenes) during some stage of the neoplastic process. Events associated with initiation and promotion may overlap to some degree, but in order for them to occur, cellular permissive conditions prevail. Permissive and potentiating factors include free radicals, and thyroid hormone, and inadequate antioxidants. Protective factors which suppress the carcinogenic process include enzymatic and dietary antioxidants. These are constitutive under normal circumstances and can be induced under conditions of oxidative stress produced by a wide range of carcinogens.

Ozone activates transforming genes in vitro and acts as a synergistic co-carcinogen with gamma-rays only if delivered after radiation.

An earlier study indicated that ozone (O3), a major pollutant in our atmosphere, acts as a carcinogen as well as a synergistic co-carcinogen with radiation in cultured hamster embryo cells and in mouse C3H10T1/2 cells. In this investigation we further characterize the oncogenic action of ozone, alone or in combination with radiation, on C3H10T1/2 cells with particular emphasis on transformation produced by different temporal patterns of dose delivery of these two agents and low dose effects. We report that ozone-induced transformation involves the activation of dominant transforming genes, thereby indicating that DNA is a target in ozone induced carcinogenesis. We also report that ozone (5 p.p.m. for 5 min) acts as a synergistic co-carcinogen only if delivered after radiation (4 Gy or gamma-rays); when cells are exposed to ozone prior to radiation no enhanced rates of transformation are observed. Our findings also show that ozone at a low dose of 1 p.p.m. (for 5 min) does not act as a carcinogen but does interact as a co-carcinogen with ionizing radiation. The data indicate that the dose and sequence in which ozone and radiation are delivered have important implications for the putative carcinogenic effects of these two agents, a factor that heretofore has not been recognized.

Ozone and ultraviolet light act as additive cocarcinogens to induce in vitro neoplastic transformation.

Ozone, a major chemical oxidant in our environment, is an environmental air pollutant with putative carcinogenic action. Using in vitro transformation, we report for the first time that ozone (6 ppm for 10 min) acts in additive fashion with ultraviolet light (4 J/m2) to produce enhanced levels of transformation in hamster embryo cells and mouse C3H/10T-1/2 cells as compared to rats induced by each of the agents alone. The results underscore the hazard of ozone as a toxic pollutant which may have putative carcinogenic effects and interact with other environmental carcinogens.

X-ray-induced changes in gene expression in normal and oncogene-transformed rat cell lines.

As an approach to identifying specific cellular markers for the cytotoxic action of x rays in mammalian cells, we used the QUEST system of high-resolution, two-dimensional protein gel electrophoresis and a computerized data base on proteins to score quantitative changes in patterns of protein synthesis. We measured the responses elicited after x irradiation of cells from the normal rat cell line REF52 as well as two oncogene-transformed REF52 cell lines with E1a or E1a plus the mutated c-Harvey-ras T24 (HRAS1 T24) allele. The transformed cell lines differed substantially in the patterns of changes in protein synthesis seen immediately after DNA damage. In addition, we identified a specific subset of growth-regulated cellular polypeptides that are correlated with the observed increase in x-ray-induced cell killing in the transformed cell lines. One of these polypeptides was cyclin (proliferating-cell nuclear antigen), a cell-cycle-specific DNA polymerase delta auxiliary factor. Synthesis of this set of coregulated polypeptides was rapidly suppressed by x irradiation in normal REF52 cells only. The inability of x irradiation to induce suppression of protein synthesis in cells from the transformed cell lines correlated with the increased susceptibility to x-ray-induced cell killing. This finding suggests that the cellular processes that underlie regulation of DNA-damage-induced growth arrest at the level of replicative elongation plays a role in determining the survival of x-irradiated cells.

Basic radiobiology.

Experimental studies of the biological effects of radiation were started soon after the discoveries of x-rays in 1895, but there is still much that is not known. This article includes some research objectives that are essentially pragmatic in nature, intended to support and improve the current practice of radiotherapy, but the central thrust is the understanding of the mechanisms involved in the biological effects of radiation at the cellular and molecular levels. The article was written by a consortium of scientists and suffers inevitably from the drawback that writing styles are inconsistent, and coverage is not uniform. However, it benefits from the enormous advantage that it reflects the accumulated wisdom and judgment of more than a dozen scientists who, in their own areas of expertise, are recognized as being at the cutting edge of radiation research. The niceties of style and syntax are sacrificed in favor of the quality of the science and the maturity of judgment. The study of DNA damage as a mechanism for cell injury in early- and late-responding tissues, as well as a comparison of DNA damage that leads to lethality, as opposed to transformation and mutagenesis, are key items. The study of cell lethality with cells in culture led to the identification of repair, both sublethal and potentially lethal, as well as the dose-rate effect, and has had a considerable impact on radiotherapy. Future studies should focus on understanding the factors that determine radiosensitivity/radioresistance. A variety of approaches are available, including the study of genetically deficient cell lines from cancer-prone individuals. A parallel approach is the application of the techniques of molecular biology to clone the repair genes in mammalian cells, and to understand genetic defects that alter gene regulation, or to regulate biochemical factors in the cell. Substantial progress has been made in developing in vitro assays for mutagenesis, particularly using hybrids of rodent and human cells. Better methods are needed to study the effects of mutation on gene expression, and sensitive systems are needed that can detect low doses of radiation. Assays of oncogenic transformation, the in vitro counterpart of carcinogenesis, have been used to investigate the oncogenic potential of various types of radiation and chemotherapy agents. Key topics in future will include the investigation of supra-additivity between different agents, the identification and characterization of oncogenes that may be activated by radiation, the development of quantitative assays based on human cells, and further studies involving cell-to-cell communication.(ABSTRACT TRUNCATED AT 250 WORDS)