Radiotherapy treatment of human inflammatory diseases and conditions: Optimal dose.

During the early part of the past century, hundreds of clinical studies involving more than 37,000 patients were conducted that showed radiotherapy (RT) to be a successful and safe alternative to drug therapy for the treatment of many diverse inflammatory conditions and diseases (e.g. tendonitis, bursitis, arthritis, and serious inflammatory lung conditions). Data from these studies were collected and analyzed with the intent of estimating an optimal dosing range for RT that would induce an efficacious treatment response. RT was reported to be frequently effective after only a single treatment, with a rapid (within 24 h) and often long-lasting (from months to years) relief from symptoms. Over a two-decade span from the 1920s to the 1940s, the therapeutic responses to a single RT treatment consistently improved as the dosing for multiple ailments decreased over time to between 30 roentgen (r) and 100 r. These findings are significant and in agreement with a number of contemporary reports from Germany where RT has been commonly and successfully employed in treating ailments with an inflammatory origin. A proposed mechanism by which RT mitigates inflammation and facilitates healing is via the polarization of macrophages to an anti-inflammatory or M2 phenotype.

Products of ozonized arachidonic acid potentiate the formation of DNA single strand breaks in cultured human lung cells.

In this study we examined the potential for environmental levels of ozone (03) to degrade arachidonic acid (AA), a polyunsaturated fatty acid abundantly present in the lung, into products that can produce DNA single strand breaks (ssb) in cultured human lung cells. Human lung fibroblasts were incubated with 60 microM AA that had been previously exposed to and degraded by 0.4 ppm 03 (1 hr.) Incubation of the cells with 03-exposed AA (but not with vehicle alone) for 1 hr at 4 degrees C and 37 degrees C produced 555 and 245 rad-equivalents of DNA ssb, respectively, as determined by the DNA alkaline elution technique. These breaks were completely eliminated when the ozonized AA solution was incubated with catalase prior to cell treatment, indicating that h202 was solely responsible for damaging DNA. Superoxide dismutase bovine serum albumin, or heat-inactivated catalase showed little, if any, inhibitory activity. The H202 content of the ozonized AA (31 +/- 4 microM) could account for only about 40% of the observed breaks. Potentiation of the H202-induced DNA ssb persisted after removal of the carbonyl substances by chromatographic procedures, suggesting that the non-carbonyl component of ozonized AA was the responsible component for inducing augmentation of the observed increases in DNA ssb. Ozonized AA also induced DNA ssb in cultures of the human bronchial epithelial cell line BEAS-2B. Again, these breaks were shown to exceed levels that could be attributed to the presence of H202 alone. These results indicate that products of ozonized AA can interact to potentiate DNA ssb in human lung cells.

Breakage and binding of DNA by reaction products of hypochlorous acid with aniline, 1-naphthylamine, or 1-naphthol.

Hypochlorous acid (HOCl) is a chemically reactive oxidant and a potent microbicidal agent that is synthesized in phagosomes of inflammatory neutrophils and released into extracellular spaces. Besides reducing pathogenicity by reacting with phagocytized infectious agents, HOCl may damage tissues and yield toxic products upon reaction with various other molecules, including xenobiotics. As model xenobiotics, the substituted aryl compounds aniline, 1-naphthylamine, and 1-naphthol (1-NOH) were investigated herein for their potential to react with HOCl and the transformed into genotoxic products. The compounds were first exposed to HOCl (25-150 microM) in phosphate buffer and afterward used to treat human fibroblasts or purified DNA. DNA single-strand breaks in cells and the binding of HOCl-reacted 1-[14C]NOH to purified DNA were assessed by DNA alkaline elution and scintillation spectrometry, respectively. It was found that neither HOCl nor compounds alone could break cellular DNA. But HOCl-reacted compounds produced up to 400 rad equivalents of DNA breaks. HOCl reaction products of aniline and the model bicyclic aryl compounds differed in their DNA-breaking characteristics. HOCl-reacted 1-[14C]NOH was stable and bound to DNA at up to 124 pmol/mg DNA. Sodium thiosulfate, glutathione, and taurine inhibited the transformation reactions; but only the former two blocked binding of HOCl-reacted 1-NOH to DNA. Ultraviolet spectra showed that HOCl reacted rapidly (less than 1 min) and equally well with 1-NOH at pH 7.2 or at an intraphagosomal pH of 5.0. Reaction concentrations of HOCl in this study were 2- to 11-fold lower than levels generated in vitro by stimulated neutrophils. These results show that certain aryl compounds can react readily with approximated physiological levels of HOCl (-OCl) to form relatively long-lived products that bind DNA and are genotoxic to human cells.

Mutagenicity and metabolism of dimethyl phthalate and its binding to epidermal and hepatic macromolecules.

As an active ingredient in insect repellents, dimethyl phthalate (DMP) had previously been shown to produce chromosomal aberrations in the livers of rats following subchronic application of the phthalate to skin. When we tested DMP in the Ames mutagenesis assay, it produced in bacterial tester strain TA100 (but not TA98) a dose-related mutagenic response that was abolished by NAD- and NADP-independent metabolism associated with rat liver microsomal preparations (S9). In a host-mediated mutagenesis assay, rats were injected ip with DMP (2 g/kg body weight); urine was collected for 24 h, extracted, and analyzed for mutagenic activity and phthalic acid-containing derivatives. The extracted urine was not mutagenic to TA100 and contained an equivalent of 1.96 mg phthalate/ml urine. More than 97% of the phthalic acid-containing derivatives present in the extracted urine consisted of the nonmutagenic metabolite of DMP, monomethyl phthalate (MMP). In vitro experiments showed that rat liver homogenates hydrolyzed 93% of carbonyl-labeled 14C-DMP (7.7 mM) to MMP in 2 h and bound 0.07 nmol of [14C]phthalate/mg liver macromolecules. By contrast, rat epidermal homogenates metabolized only 5% and bound 38-fold higher levels of carbonyl-labeled 14C-DMP (2.66 nmol/mg of macromolecules), with no detectable binding to nucleic acids. Compared to epidermis and plasma, liver had a fivefold higher rate of DMP monoesterase activity (1240 nmol/h/mg protein), which, when inhibited by 67%, resulted in a 4.4-fold increase in phthalate-bound hepatic macromolecules (0.31 vs. 0.07 nmol of carbonyl-labeled 14C-DMP/mg macromolecules). In addition to MMP, formaldehyde was produced during the metabolism of DMP by liver. When ethanol was used to inhibit the oxidation of DMP-derived methanol by hepatic homogenates, there resulted a 74% reduction in the accumulation of formaldehyde and similar reductions of 71 and 73% in the binding of methyl-labeled 14C-DMP to nucleic acids and macromolecules. (Methyl-labeled, unlike carbonyl-labeled, 14C-DMP yields a 14C-labeled methanol when hydrolyzed.) These results indicate that the DMP diester is a weak bacterial mutagen, which binds to epidermal and hepatic macromolecules other than nucleic acids, and that although the rapid hepatic metabolism of DMP to its monoester (MMP) and methanol affords protection against higher levels of phthalate binding as well as against DMP-induced bacterial mutagenesis, it also oxidizes DMP-derived methanol to formaldehyde, a metabolite that binds macromolecules, including nucleic acids.

Human alveolar and peritoneal macrophages mediate fungistasis independently of L-arginine oxidation to nitrite or nitrate.

Human alveolar macrophages (HAM) from 28 normal volunteers were found to inhibit replication of Cryptococcus neoformans. Conditions under which fungistasis occurred were different than those required for mouse peritoneal macrophage-mediated fungistasis. Inhibition of fungal replication by mouse peritoneal macrophages (MPM) requires that the macrophages are activated and that the cocultures of C. neoformans and macrophages be done in the presence of serum, L-arginine, and endotoxin. During MPM-mediated fungistasis and tumor cell killing, L-arginine is oxidized to NO2-, NO3-, and L-citrulline. In addition, MPM have arginase activity that converts L-arginine to L-ornithine and urea. HAM-mediated fungistasis was similar to that mediated by MPM in terms of the serum requirement, but HAM did not require L-arginine or endotoxin. HAM did not produce NO2- or NO3- detectable by colorimetric and bioassay, nor did HAM produce L-citrulline or L-ornithine from 14C-radiolabeled L-arginine as detectable by reverse-phase ion-pairing HPLC of macrophage-C. neoformans coculture supernatants. HAM had no detectable arginase activity, hence there was no evidence for L-arginine nitrogen metabolism in HAM. HAM-mediated fungistasis was not enhanced by endotoxin or by recombinant human interferon-gamma (rHIFN-gamma). The combination of endotoxin and rHIFN-gamma inhibited the fungistatic effect of HAM. Human peritoneal macrophages (HPM) from women undergoing laparoscopy were tested for fungistasis and L-arginine nitrogen oxidation. Partial inhibition of cryptococcal replication occurred; however, there was no evidence of L-arginine metabolism to NO2- or NO3-.(ABSTRACT TRUNCATED AT 250 WORDS)

Induction of DNA damage in cultured human lung cells by tobacco smoke arylamines exposed to ambient levels of ozone.

Ozone (O3) is a powerful oxidizing component of air pollution that may react with other air pollutants before or after inhalation. Because ozonized compounds can be mutagenic to bacteria, we examined whether ambient O3 levels can transform tobacco smoke arylamines into products that are genotoxic to human lung cells. To test this possibility, aqueous solutions of 1-naphthylamine (1-NA) were first exposed to air or O3 in the absence of cells and then used to treat cultured human lung cells, i.e., the diploid fibroblasts CCD-18Lu and the transformed type II epithelial cells A549. DNA single-strand breaks were assayed by DNA alkaline elution. Neither air-exposed 1-NA nor O3-exposed buffer or water were DNA-damaging. However, exposure of 1-NA (15 microM) to O3 (0.1 ppm; 1 h) produced 400 rad equivalents of DNA breaks in either cell type. Although maximal induction of DNA breaks depended upon arylamine concentration, the rates at which DNA-damaging products were formed (activated) and subsequently deactivated depended upon O3 concentration. O3-activated 1-NA was stable for at least 4 h and could damage cellular DNA at 4 degrees C. During ozonization, hydroperoxides were formed at levels equivalent to between 2 and 20 microM of hydrogen peroxide and were eliminated by treatment with catalase. However, failure of catalase and superoxide dismutase to block formation of DNA breaks indicated that neither hydrogen peroxide nor superoxide anions were involved in breaking DNA.(ABSTRACT TRUNCATED AT 250 WORDS)

Ozone-induced inflammation in the lower airways of human subjects.

Although ozone (O3) has been shown to induce inflammation in the lungs of animals, very little is known about its inflammatory effects on humans. In this study, 11 healthy nonsmoking men, 18 to 35 yr of age (mean, 25.4 +/- 3.5), were exposed once to 0.4 ppm O3 and once to filtered air for 2 h with intermittent exercise. Eighteen hours later, bronchoalveolar lavage (BAL) was performed and the cells and fluid were analyzed for various indicators of inflammation. There was an 8.2-fold increase in the percentage of polymorphonuclear leukocytes (PMN) in the total cell population, and a small but significant decrease in the percentage of macrophages after exposure to O3. Immunoreactive neutrophil elastase often associated with inflammation and lung damage increased by 3.8-fold in the fluid while its activity increased 20.6-fold in the lavaged cells. A 2-fold increase in the levels of protein, albumin, and IgG suggested increased vascular permeability of the lung. Several biochemical markers that could act as chemotactic or regulatory factors in an inflammatory response were examined in the BAL fluid (BALF). The level of complement fragment C3 alpha was increased by 1.7-fold. The chemotactic leukotriene B4 was unchanged while prostaglandin E2 increased 2-fold. In contrast, three enzyme systems of phagocytes with potentially damaging effects on tissues and microbes, namely, NADPH-oxidase and the lysosomal enzymes acid phosphatase and beta-glucuronidase, were increased neither in the lavaged fluid nor cells. In addition, the amounts of fibrogenic-related molecules were assessed in BALF.(ABSTRACT TRUNCATED AT 250 WORDS)

Molecular mechanisms involved in T cell activation. III. The role of extracellular calcium in antigen-induced lymphokine production and interleukin 2-induced proliferation of cloned cytotoxic T lymphocytes.

The role that extracellular calcium plays in activating resting cloned cytotoxic T lymphocytes (CTL) to proliferate and to produce lymphokines was examined. In these cells, stimulation with interleukin 2 (IL-2) induced a proliferative response without a concomitant production of macrophage-activating factor (MAF), whereas stimulation with antigen or lectin (in the absence of IL-2) induced MAF production but not proliferation. In the case of IL-2-induced proliferation, extracellular calcium was required to initiate proliferation as well as to prevent cellular arrest later in the G2 + M phase of the cell cycle. In MAF production extracellular calcium was required both to activate the phosphatidylinositol signal-transducing mechanism and to mobilize intracellular calcium in antigen- or lectin-stimulated cytotoxic T lymphocytes. Further, extracellular calcium was required for only 8 of the 18 hr of stimulation time which was needed to achieve maximal MAF production, indicating that both calcium-dependent and -independent events exist in the signal pathway. Additional experiments with calcium ionophores and activators of protein kinase C indicated that although both intracellular calcium mobilization and de novo protein phosphorylation are involved in MAF production, an optimal increase in the level of intracellular calcium by itself is insufficient to induce the production of this lymphokine.

Mechanism of cyclosporin A-induced immunosuppression. Cyclosporin A inhibits receptor-mediated and non-receptor-mediated lymphokine production as well as interleukin-2-induced proliferation in cloned T lymphocytes.

The effects of cyclosporin A (CyA) on the activation processes of cloned murine cytotoxic T lymphocytes (CTL) have been examined. With the use of Day 7 resting cloned CTL it was possible to separate the functions of lymphokine production (macrophage-activating factor, MAF) and interleukin 2 (IL-2)-induced proliferation of these cells. The effect of CyA on each of these activities was analyzed independently. CyA was found to inhibit both receptor-mediated MAF production in response to stimulation with antigen and lectin and MAF production in response to non-receptor-mediated stimulation (by anti-Thy-1 antibodies, ionophore, and phorbol ester). Further, CyA was observed to inhibit the re-entry of these resting CTL into the cell cycle upon stimulation with IL-2. The effect of CyA on MAF production did not appear to be due to inhibition of the signal-transducing mechanism involved in this process (i.e., inositol lipid hydrolysis, calcium mobilization, and protein phosphorylation). The action of CyA on the IL-2-induced proliferation was not due to inhibition of IL-2 receptor expression or the binding of IL-2 to its receptor. Thus, CyA appeared to mediate its suppressive effects on MAF production and IL-2-induced proliferation through an action on some later step(s) in the signal pathways of these activities.

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%.

Transmembrane signalling via the T11-dependent pathway of human T cell activation. Evidence for the involvement of 1,2-diacylglycerol and inositol phosphates.

It has previously been shown that some anti-T11 monoclonal antibodies, when used in combination, can activate the human T cell line Jurkat to produce interleukin 2. In this study, we investigate the mechanism by which perturbation of different epitopes of T11 molecules induces activation in Jurkat cells. We show that this activation is initiated by a T11-mediated increase in the concentration of free cytoplasmic calcium ions ([Ca2+]i). The initial increment in [Ca2+]i can occur when extracellular Ca2+ is depleted by EGTA, indicating that Ca2+ from intracellular stores is mobilized. As an early response to extracellular signals provokes a rapid breakdown of a class of lipid known collectively as the phosphoinositides, we measured the levels of phosphatidylinositol bisphosphate (PIP2) which is hydrolyzed to generate inositol triphosphates (IP3), the putative mobilizer of Ca2+ from internal stores and 1,2-diacylglycerol (DAG), the physiological activator of protein kinase C. Monoclonal antibodies directed either against different epitopes of T11 molecules or the T3-Ti antigen receptor complex provoke a rapid breakdown of PIP2, the parental product from which IP3 and DAG derive. In addition antibodies to either the T11 molecules or T3-Ti antigen receptor complex induce marked elevations in IP3, other inositol phosphate compounds and DAG. Taken together, these data indicate that, during T cell activation, due to the perturbation of T11 molecules or T3-Ti antigen receptor complex, membrane phosphoinositides are specifically hydrolyzed. This hydrolysis of phosphoinositides generates two putative second messengers such as IP3 and DAG, which mobilizes Ca2+ from intracellular stores and stimulates protein phosphorylation, respectively.

Molecular mechanisms involved in T cell activation. II. The phosphatidylinositol signal-transducing mechanism mediates antigen-induced lymphokine production but not interleukin 2-induced proliferation in cloned cytotoxic T lymphocytes.

The phospholipid metabolism of cloned murine cytotoxic T lymphocytes (CTL) was examined under conditions in which the induction of proliferation by interleukin 2 (IL 2) and the stimulated production of lymphokine (macrophage-activating factor (MAF] by concanavalin A (Con A) and specific antigen occurred independently of each other. Activation of the CTL by either of the latter two stimuli resulted in changes in the metabolism of phosphatidylinositol (PI) that were early (less than 2.5 min), specific, and prolonged (6 to 8 hr). These changes were primarily characterized by an increase in phosphatidic acid (PA) and PI, with a decrease in phosphatidylinositol-4,5-bisphosphate. The duration of these phospholipid responses, particularly PA and PI, approximated the minimum time of CTL-stimulus interaction required to produce maximal titers of MAF. No changes were observed in other major classes of phospholipids during 8 hr of continuous stimulation. Stimulation with an irrelevant antigen had no effect on CTL phospholipid metabolism. In contrast to specific antigen or Con A, the T cell growth factor IL 2 failed to elicit specific and early biosynthetic responses from PA and PI. Instead, there were nonspecific biosynthetic responses from all major phospholipid classes (including phosphatidylcholine and phosphatidylethanolamine, as well as PA and PI) which occurred between 1 and 6 hr after IL 2 stimulation. Both 1,2-diacylglycerol (DAG) and inositol phosphates (IP), the hydrolytic products of PI turnover, were produced in response to MAF-inducing stimuli, but neither were detected in response to the proliferative stimulus IL 2. Together, these results indicate that the hydrolysis of PI and the concomitant production of the putative second messengers DAG and IP are involved in signaling the production of lymphokines (MAF) by CTL. On the other hand, the failure of IL 2 to elicit a full-spectrum PI response suggests that signals mediating CTL proliferation may utilize an alternate and still undefined pathway.

Molecular mechanisms involved in T cell activation. I. Evidence for independent signal-transducing pathways in lymphokine production vs proliferation in cloned cytotoxic T lymphocytes.

It is well-established that activated T cells proliferate in response to interleukin 2 (IL 2) and produce various soluble lymphokines such as macrophage-activating factor (MAF) in response to antigen. Prior to investigating the molecular events involved in signaling the initiation of these responses in cloned murine cytotoxic T lymphocytes (CTL), we determined whether these responses could occur independently, and we established for each response the time during which signal transducing mechanisms may function. It was found that this cloned CTL population was in a resting state (G1 phase of cell cycle) 7 days after stimulation with antigen plus IL 2. At this time, the incubation of these resting CTL with IL 2 for 4 to 6 hr resulted in a maximal proliferative response that was not accompanied by the production of MAF. Conversely, the incubation of resting CTL with antigen or lectin (in the absence of IL 2) for at least 8 hr resulted in the maximal production of MAF at 24 hr without inducing a proliferative response. In addition, antigen or lectin, but not IL 2, triggered an immediate (less than 1 min) and sustained (at least 8 hr) mobilization of intracellular calcium. The kinetics of this calcium response paralleled the minimum time (8 hr) that was required for resting CTL to interact with either antigen or lectin in order to produce maximal titers of MAF. These results indicate that proliferation and lymphokine (MAF) production in cloned murine CTL are independent events. In these resting CTL, the signal mechanisms that mediate the production of lymphokines are most likely restricted to the initial 8 hr of stimulation by antigen or lectin and involve the rapid and prolonged mobilization of cytoplasmic calcium. Proliferative signals, however, are probably complete within 4 to 6 hr after stimulation by IL 2 and do not involve readily demonstrable fluxes of cytoplasmic calcium, as determined by the fluorescent calcium probe Quin 2.

Are free radicals involved in tumor promotion?

Previously it was shown that lipophilic analogs of a free-radical scavenger, 2(3)-tert-butyl-4-hydroxyanisole (BHA), inhibit ornithine decarboxylase (ODC) activity which is induced by 12-O-tetradecanoylphorbol-13-acetate (TPA) in mouse epidermis. With regard to this antitumor-promoting effect, eight analogs of BHA (2- and 3-BHA, 2-t-butyl-1, 4-dimethoxybenzene methyl-BHA), t-butylhydroquinone (t-BHQ), p-hydroquinone (HQ), 4-hydroxyanisole, phenol and 2-t-butylphenol) are evaluated herein for their antioxidant capacities for scavenging superoxide anions (O-2), of inhibiting lipid peroxidation and of inhibiting chemiluminescence (CL) in TPA-activated polymorphonuclear leukocytes (PMNs), an event associated with oxy-radical production. None of the analogs reacted with O-2, while 2- and 3-BHA suppressed the formation of O-2 by TPA-activated PMNs. T-BHQ underwent autoxidation in aqueous solution, reducing molecular oxygen and increasing the levels of O-2 that were formed chemically, enzymatically and cellularly. However, all of the phenolic antioxidant analogs of BHA inhibited TPA-stimulated CL in PMNs and ascorbate-initiated lipid peroxidation, while methyl-BHA (a non-antioxidant analog) was inactive. The inhibitory activities of these analogs for lipid peroxidation were related to both their lipophilic and antioxidant properties and corresponded favorably with their inhibitory activities for TPA-induced ODC activities in mouse epidermis. On the other hand, inhibition of the CL response by these antioxidants was independent of their lipophilicity and compared less favorably with their capacities to antagonize phorbol ester-induced ODC activity. These results imply that lipophilic BHA analogs inhibit TPA-induced ODC activity by scavenging free radicals other than O-2. Furthermore, the fact that t-BHQ was the most potent inhibitor of CL, lipid peroxidation and ODC activity and simultaneously reduced molecular oxygen, suggests the possibility that O-2 may act as a precursor to the formation of free radicals which are reactive with t-BHQ and more directly involved in the process of tumor promotion.

Inhibition of tumor promotion by a biomimetic superoxide dismutase.

A low molecular weight, lipophilic, copper coordination complex with superoxide dismutase-mimetic activity inhibited biochemical and biological actions of a tumor promoter in mouse epidermis. Such inhibitory effects implicate reactive oxygen species in the tumor promotion process.

Inhibition by 2(3)-tert-butyl-4-hydroxyanisole and other antioxidants of epidermal ornithine decarboxylase activity induced by 12-O-tetradecanoylphorbol-13-acetate.

The relationship between reactive oxygen and/or free radical species and tumor promotion was evaluated by investigating the inhibitory effects of 2(3)-tert-butyl-4-hydroxyanisole (BHA) and other antioxidants on the induction of ornithine decarboxylase (ODC) activity in mouse epidermis by a tumor promoter, 12-O-tetradecanoylphorbol-13-acetate (TPA). Mice maintained on a diet containing 0.75% BHA for 8 days showed a 50% reduction in maximal ODC induction following treatment with TPA when compared to mice fed a control diet. Topical application of BHA (55 mumol) 30 min prior to TPA treatment (17 nmol) elicited an 80% inhibition of promoter-induced ODC activity. BHA was ineffective as an inhibitor when administered either 16 hr before or 2 hr after the promoter. The inhibition by BHA was dose dependent with a dose producing a 50% inhibition of ODC induction of 6 mumol. A structure-activity study with BHA analogues (2-tert-butyl-4-hydroxyanisole, 3-tert-butyl-4-hydroxyanisole, 2-tert-butyl-1,4-dimethoxybenzene,tert-butylhydroquinone, 4-hydroxyanisole, p-hydroquinone, phenol, and 2-tert-butyl-phenol) showed that hydroxyl and tert-butyl substituents were important determinants of inhibitory activity. A spectrum of other antioxidants were also tested. Butylated hydroxytoluene was nearly equipotent to BHA; alpha-tocopherol, propyl gallate, and disulfiram were all less potent, and L-ascorbate was inactive. None of the antioxidants affected basal ODC activity in non-TPA-treated mice. Collectively, these results demonstrate an early and direct inhibition of TPA-induced ODC activity by lipophilic phenolic antioxidants and suggest a role for reactive oxygen and/or free radical species in tumor promotion.

Assessment of the mutagenicity of phthalate esters.

The Ames assay was used to investigate the mutagenicity of several phthalate esters as an approximation of their carcinogenic potential. The ortho diesters, dimethyl phthalate (DMP) and diethyl phthalate (DEP) produced a positive dose-related mutagenic response with Salmonella TA100, but only in the absence of S-9 liver enzymes. Dibutyl, di(2-ethylhexyl), mono(2-ethylhexyl), and butyl benzyl phthalate as well as the dimethyl isophthalate and terephthalates and the trimethyl ester, trimellitate, were not mutagenic with TA100 or TA98 in the presence or absence of S-9. In a host-mediated assay, extracts of 24-hr urines of rats injected IP with DMP (2 g/kg) were not mutagenic to TA100 at levels up to 8 equivalent-ml of urine/plate (representing 30% of their daily urinary output). In vitro studies revealed that S-9 associated esterase hydrolyzed DMP to the monoester and methanol and eliminated its mutagenicity. Whole rat skin was shown to have about 1.5% of the DMP-esterase activity of liver, when compared on a wet weight basis. An in vitro binding study indicated that epidermal macromolecules bound DMP at a severalfold greater rate than hepatic macromolecules. Thus, both the mutagenicity and binding of DMP are inversely related to the metabolism of this compound. These results suggest that skin could be at high risk for a mutagenic/carcinogenic insult.