Marie A. Amoruso

Stimulation of human polymorphonuclear leukocyte superoxide anion radical production by tumor promoters

Comparison was made of the ability of the potent tumor promoter phorbol myristate acetate (PMA), as well as less active PMA analogs and non-phorbol ester tumor promoters, to stimulate superoxide anion radical (O-.2) production by human polymorphonuclear leukocytes (PMN). The rate of O-.2 production was found to correlate with the tumor-promoting activity of the phorbol esters as opposed to their inflammatory activity. Mezerein and telocidin B were slightly better stimulators of O-.2 production than PMA. Acetic acid was inactive. These data are discussed in terms of a possible role for O-.2 and other reactive oxygen species in tumor promotion.

Retinoid inhibition of superoxide anion radical production by human polymorphonuclear leukocytes stimulated with tumor promoters

Abstract All-trans retinol, retinyl acetate and retinoic acid were found to be effective inhibitors of O 2 − • polymorphonuclear leukocytes stimulated with the tumor promoter phorbol myristate acetate. Retinol similarly inhibited cells stimulated with mezerein or teleocidin B. No-effect concentrations of the protease inhibitor antipain potentiated the inhibitory effect of low levels of retinol. Higher concentrations of retinol and antipain resulted in an additive or less than additive inhibitory effect.

Enhancement of rat and human phagocyte superoxide anion radical production by cadmium in vitro

The mechanism by which cadmium produced oxidizing effects in vivo is unknown. We show that cadmium enhances the production of superoxide anion radical (O-(2) .), a reactive oxygen species, in digitonin-stimulated phagocytes from man and rat. Cadmium concentrations ranging from 3.6 X 10(-2)M to 3.6 X 10(-4)M inhibited O-(2) . production in rat alveolar macrophages or human granulocytes. However, when activated in the presence of 3.6 X 10(-5)M cadmium, the production of O-(2) . was increased by a factor of 2.11 +/- 0.25 above control levels in human granulocytes and 3.6 +/- 0.62 above control levels in rat alveolar macrophages. This effect by levels of cadmium within the range of those occurring during in vivo toxicity might provide an explanation for the oxidizing effects of this metal ion.

Degradation of soluble collagen by ozone or hydroxyl radicals

Collagen exposed to ozone or hydroxyl radicals was degraded in a time‐ and dose‐dependent manner. This degradation was inhibited by free radical scavengers. Furthermore, lower levels of these oxidants did not degrade the molecule, but caused it to become susceptible to proteolytic degradation. We suggest an alternative mechanism by which oxygen‐derived free radicals participate in the destruction of extracellular matrix observed during acute lung injury by oxidant gas, in addition to the commonly accepted proteinase‐antiproteinase theory of lung injury.

Decreased superoxide anion radical production by rat alveolar macrophages following inhalation of ozone or nitrogen dioxide.

Abstract In vivo exposure of rats to ozone or nitrogen dioxide results in a dose-dependent decrease in superoxide anion radical production (O 2 − ·) by alveolar macrophages isolated from the exposed animals. When alveolar macrophages from ozone-exposed animals were stimulated with phorbol myristate acetate (PMA, a non-phagocytic stimulus of O 2 − · production) the decrease in O 2 − · production ranged from 85.9% of control at 3.2 ppm-hrs ozone to 7% of control at 10.5 ppm-hrs. In a similar fashion, O 2 − · production by PMA-stimulated macrophages from NO 2 -exposed rates ranged from 78% of control at 18.3 ppm-hrs NO 2 down to 14.5% of control at 51 ppm-hrs. Since the viability of the alveolar macrophages obtained from ozone or nitrogen dioxide-exposed animals was 88% or better in all cases as judged by both Trypan blue exclusion and lactate dehydrogenase release, the decreased ability of these cells to produce superoxide anion radical cannot be attributed to a pollutant effect on cell viability. This diminution in superoxide anion radical production by alveolar macrophages from the pollutant-exposed animals might account, in part, for the ability of these 2 air pollutants to potentiate bacterial infections in laboratory animals.

Muconaldehyde, A Potential Toxic Intermediate of Benzene Metabolism

The metabolite of benzene that is responsible for its hematological toxicity is unknown. Benzene is of course the parent aromatic hydrocarbon and mush attention has been focussed on classical pathways of aromatic hydrocarbon metabolism in the search for toxic benzene metabolites. Elegant studies by a number of groups, including work presented at this symposium by Snyder, Irons, and Tunek, have evaluated metabolites such as benzene oxide, catechol, phenol, hydroquinone and their derivatives (See reviews by Snyder et al, 1977; Laskin and Goldstein, 1977). While there are some interesting clues concerning potentially toxic intermediates, and much important information has been obtained, the metabolic pathway and agent(s) responsible for the hematological toxicity of benzene remains unidentified.

Activation of neutrophil collagenase by cathepsin G

Collagenase is secreted from neutrophils as a latent or proenzyme. In an effort to understand the mechanism of collagenase activation in inflammation, human peripheral neutrophils (PMNs) were isolated and incubated with the tumor promotor, phorbol myristate acetate (PMA), which induces the neutrophils to degranulate and secrete proteinases. Neutrophil media were then treated with various activators or inhibitors of collagenase and other proteinases, and the collagenase activity was measured. A serine proteinase secreted from neutrophils, cathepsin G, was found to activate latent collagenase, but it was also found to require activation itself. Both hypochlorous acid (HOCl) and oxidized glutathione (GSSG) were tested for their coilagenase-activating ability and were found to be successful only in the presence of active cathepsin G. A specific cathepsin G inhibitor (0.5 mM Z-Gly-Leu-Phe-CH2Cl) prevented the activation of latent collagenase by HOCl. To confirm these results, purified neutrophil cathepsin G was incubated with a neutrophil proteinase mixture which contained latent collagenase. The collagenase was shown to be activated upon incubation with purified cathepsin G. These results indicate that cathepsin G is a key mediator in neutrophil collagenase activation.

Alteration of erythrocyte membrane fluidity by heavy metal cations.

Fluorescence polarization measurements were carried out on ery throcyte ghosts treated with heavy metal cations. Membranes labelled with the fluorescent lipid probe all-trans 1,6-diphenyl-1,3,5- hexatriene (DPH) had increased DPH polarization (P) values, con sistent with increased membrane lipid viscosity, when treated with 10-3 -10-2 M of the chloride salts of cadmium, zinc or lead for 1 hour at 37° C, but not with calcium. Using cadmium as a prototype heavy metal, additional studies showed that exposure of ghosts to cadmium also increased the native protein fluorescence polariza tion. These increases in P values were observed at temperatures ranging from 15-40° C. These data suggest that interaction of cad mium and other heavy metals with cellular membranes may alter membrane lipid and possibly membrane protein fluidity, which may contribute to abnormal cellular function.

Inhibition by reactive aldehydes of superoxide anion radical production in stimulated human neutrophils

alpha,beta-Unsaturated aldehydes were investigated in vitro for their ability to inhibit superoxide anion radical (O2-.) production in stimulated human polymorphonuclear leukocytes (PMN). The aldehydes investigated were (i) trans-4-hydroxynonenal and malonaldehyde (MDA), two toxic lipid peroxidation products; (ii) acrolein and crotonaldehyde, two air pollutants derived from fossil fuel combustion; (iii) trans,trans-muconaldehyde, a putative hematotoxic benzene metabolite. Preincubation of PMN with reactive aldehydes followed by stimulation with the oxygen burst initiator phorbol myristate acetate (PMA) resulted in a dose-dependent inhibition of O2-. production. The concentration at which 50% inhibition (IC50) was observed was 21 microM for acrolein, 23 microM for trans,trans-muconaldehyde, 27 microM for trans-4-hydroxynonenal and 330 microM for crotonaldehyde. A similar inhibitory effect by these aldehydes was observed in digitonin- and concanavalin A-stimulated PMN. MDA inhibited O2-. production in PMA-stimulated PMN by 100% at 10(-2) M but gave no inhibition at 10(-3) M. The standard aldehyde propionaldehyde did not inhibit O2-. production at 10(-3)-10(-6) M. Preincubation of PMN with acrolein in the presence of cysteine completely protected against the inhibitory effect of this reactive aldehyde. The results indicate that the ability of toxic aldehydes to inhibit O2-. production in stimulated PMN correlates directly with their alkylation potential which is a function of the electrophilicity of the beta carbon.

Estimation of risk of glucose 6-phosphate dehydrogenase-deficient red cells to ozone and nitrogen dioxide

It has been suggested that the more than 1 million black Americans with the A- variant of glucose-6-phosphate dehydrogenase deficiency (G6PD) are at risk for adverse hematologic effects due to inhalation of ambient levels of oxidant gases. To evaluate this hypothesis studies were performed that included direct exposure of human G6PD-deficient red cells, and of mouse strains with different G6PD levels, to the oxidant gases ozone and nitrogen dioxide. Using the oxidant drug phenylhydrazine in part as a point of comparison, conservative extrapolation of the data indicates that exposure to levels of ozone or nitrogen dioxide at least one and probably two orders of magnitude above the LD50 would be required for any hematologic effect to be observed of pertinence to G6PD deficiency. It is concluded that there is no reason to remove or preclude from the workplace black employees with the common A- variant of red cell G6PD deficiency who potentially are exposed to oxidant gases.