Pathophysiological mechanisms of active oxygen.

Besides being toxic, oxidants can induce pathophysiological effects in mammalian cells. For example they can stimulate rather than inhibit cell growth. Since oxidants are ubiquitous they may represent 'natural' tumour promoters. Our work with xanthine/xanthine-oxidase as an extracellular source of active oxygen (AO) and promotable (clone 41) and non-promotable (clone 30) mouse epidermal cells JB6 allows insights into the mechanism of action of oxidant promoters. We found that AO stimulated the growth only of promotable clone 41 after an initial period of moderate inhibition while it was strongly cytostatic for non-promotable clone 30. Active oxygen induced larger amounts of DNA-strand breaks and poly ADP-ribosylation of chromosomal proteins in non-promotable cells. In addition, AO was capable of inducing the growth- and differentiation-related proto-oncogenes c-fos and c-myc in promotable and non-promotable JB6 cells. We speculate that these genes can exert their functions only in the promotable clone 41 because the general cytostatic effects of AO are moderate. A possible explanation for the differences between these 2 clones was discovered when we compared the constitutive activities, protein concentrations and mRNA levels for the antioxidant enzymes catalase (CAT), Cu,Zn-superoxide dismutase (SOD) and glutathione-peroxidase (GPx). We found that CAT and SOD (but not GPx) levels were 2-3-fold higher in the promotable clone 41. We propose that promotable cells possess a superior antioxidant defence which protects them from excessive cytostatic effects of AO.

Effects of tert-butyl hydroperoxide on promotable and non-promotable JB6 mouse epidermal cells.

Oxidants and agents that induce a cellular prooxidant state can act as carcinogens. We compared the effect of tert-butyl hydroperoxide (Bu-OOH) on DNA strand breakage, poly ADP-ribosylation of chromosomal proteins and the expression of the proto-oncogenes c-fos and c-myc between non-promotable clone 30 and promotable clone 41 of mouse epidermal cells JB6. These pathophysiological effects of oxidants are mechanistically related. Bu--OOH caused more DNA-strand breakage at high concentrations and more extensive poly ADP-ribose accumulation in clone 30 than in clone 41, in reactions which require intracellular free iron. Clone 41 exhibited constitutive c-myc expression while c-fos mRNA was very low in untreated cultures of both clones. Low concentrations of Bu-OOH induced c-myc and more strongly c-fos in clone 41. Both proto-oncogenes were strongly induced in clone 30. Our results allow insights into the mechanisms of action of a typical organic hydroperoxide in JB6 cells. However, they do not uncover the reasons for the differential promotability of the two JB6 clones by oxidants beyond the implication of the constitutive expression of c-myc in promotable clone 41.

Active oxygen induced DNA strand breakage and poly ADP-ribosylation in promotable and non-promotable JB6 mouse epidermal cells.

The evidence is convincing that oxidants and agents which induce a cellular pro-oxidant state can act as carcinogens, in particular as promoters and progressors. Importantly, infiltrated phagocytes represent a source of oxidants in inflamed tissues. We have studied the mechanism of the promotional action of active oxygen (AO) in mouse epidermal cells JB6 by comparing the non-promotable clone 30 to the promotable clone 41. In order to mimick AO released by phagocytes we used xanthine/xanthine oxidase as a source of extracellular superoxide and hydrogen peroxide. We found that AO stimulated the growth only of promotable clone 41 after an initial period of moderate inhibition while it was strongly cytostatic for non-promotable clone 30. Reasons for the higher cytostatic effect of AO on the non-promotable clone 30 were discovered when we measured DNA strand breakage and poly ADP-ribosylation of chromosomal proteins. At equal doses AO induced 4-5 times more DNA breaks in clone 30 in reactions which required iron--and probably also calcium--ions. The higher amount of DNA breakage in clone 30 was reflected in a higher extent of poly ADP-ribosylation. Excessive DNA breakage and poly ADP-ribosylation which causes the depletion of NAD and ATP may be responsible for the strong cytostatic effect of AO in clone 30. We conclude that differential resistance to the cytostatic/cytotoxic effect of AO in part determines the promotability of mouse epidermal cells JB6.

Phorbol ester-induced formation of clastogenic factor from human monocytes.

Phorbol-12-myristate-13-acetate (PMA) acts as a tumor promoter on mouse skin. It induces inflammation and leukocyte-mediated clastogenicity which appears to be related to rapid changes in lipid metabolism. To identify lipids possessing clastogenic and/or tumor-promoting properties, we have characterized the metabolism and release of arachidonic acid (AA) and related lipids during the formation of lipophilic clastogenic factors by PMA-treated human monocytes. In 1 h, [3H]AA-labeled monocytes spontaneously released significant amounts of their total radioactivity (4%) which increased nearly 4-fold (15%) with PMA (30 ng/ml) treatment. Eighty-five per cent of extracellular 3H-label from both control and PMA-treated monocytes was composed of free AA (plus AA-metabolites), while the remaining radioactivity was incorporated in phospholipids and mono- and diacylglycerols. Treated and non-treated cells released essentially the same kind of metabolites but PMA induced a 3- to 4-fold increase in total amounts. The major products consisted of prostaglandins F2 alpha and E2, thromboxane B2, 12-hydroxy-5,8,10-heptadecatrienoic acid and 5-, 11- and 15-hydroxyeicosatetraenoic acids. PMA also induced increases in the levels of three unidentified products. Neither leukotrienes nor 4-hydroxynonenal, a major alkenal degradation product of AA, were found in medium from PMA-treated monocytes. PMA, in contrast to the first-stage tumor promoter calcium ionophore A23187, failed to stimulate the release of platelet activating factor. The increased formation of phorbol ester-induced AA metabolites was proportional to the increase in free extracellular AA. The source of AA from treated and untreated monocytes consisted of cellular phospholipids with phosphatidylcholine and phosphatidylethanolamine accounting for 85%.