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Reactive oxygen species not only modulate important signal transduction pathways, but also induce DNA damage and cytotoxicity in keratinocytes. Hydrogen peroxide and organic peroxides are particularly important as these chemicals are widely used in dermally applied cosmetics and pharmaceuticals, and also represent endogenous metabolic intermediates. Lipid peroxidation is of fundamental interest in the cellular response to peroxides, as lipids are extremely sensitive to oxidation and lipid-based signaling systems have been implicated in a number of cellular processes, including apoptosis. Oxidation of specific phospholipid classes was measured in normal human epidermal keratinocytes exposed to cumene hydroperoxide after metabolic incorporation of the fluorescent oxidation-sensitive fatty acid, cis-parinaric acid, using a fluorescence high-performance liquid chromatography assay. In addition, lipid oxidation was correlated with changes in membrane phospholipid asymmetry and other markers of apoptosis. Although cumene hydroperoxide produced significant oxidation of cis-parinaric acid in all phospholipid classes, one phospholipid, phosphatidylserine, appeared to be preferentially oxidized above all other species. Using fluorescamine derivatization and annexin V binding it was observed that specific oxidation of phosphatidylserine was accompanied by phosphatidylserine translocation from the inner to the outer plasma membrane surface where it may serve as a recognition signal for interaction with phagocytic macrophages. These effects occurred much earlier than any detectable changes in other apoptotic markers such as caspase-3 activation, DNA fragmentation, or changes in nuclear morphology. Thus, normal human epidermal keratinocytes undergo profound lipid oxidation with preference for phosphatidylserine followed by phosphatidylserine externalization upon exposure to cumene hydroperoxide. It is therefore likely that normal human epidermal keratinocytes exposed to similar oxidative stress in vivo would under go phosphatidylserine oxidation/translocation. This would make them targets for macrophage recognition and phagocytosis, and thus limit their potential to invoke inflammation or give rise to neoplastic transformations.

Carbon nanotubes (CNT), with their applications in industry and medicine, may lead to new risks to human health. CNT induce a robust pulmonary inflammation and oxidative stress in rodents. Realistic exposures to CNT may occur in conjunction with other pathogenic impacts (microbial infections) and trigger enhanced responses. We evaluated interactions between pharyngeal aspiration of single-walled CNT (SWCNT) and bacterial pulmonary infection of C57BL/6 mice with Listeria monocytogenes (LM). Mice were given SWCNT (0, 10, and 40 mug/mouse) and 3 days later were exposed to LM (10(3) bacteria/mouse). Sequential exposure to SWCNT/LM amplified lung inflammation and collagen formation. Despite this robust inflammatory response, SWCNT pre-exposure significantly decreased the pulmonary clearance of LM-exposed mice measured 3 to 7 days after microbial infection versus PBS/LM-treated mice. Decreased bacterial clearance in SWCNT-pre-exposed mice was associated with decreased phagocytosis of bacteria by macrophages and a decrease in nitric oxide production by these phagocytes. Pre-incubation of naïve alveolar macrophages with SWCNT in vitro also resulted in decreased nitric oxide generation and suppressed phagocytizing activity toward LM. Failure of SWCNT-exposed mice to clear LM led to a continued elevation in nearly all major chemokines and acute phase cytokines into the later course of infection. In SWCNT/LM-exposed mice, bronchoalveolar lavage neutrophils, alveolar macrophages, and lymphocytes, as well as lactate dehydrogenase level, were increased compared with mice exposed to SWCNT or LM alone. In conclusion, enhanced acute inflammation and pulmonary injury with delayed bacterial clearance after SWCNT exposure may lead to increased susceptibility to lung infection in exposed populations.

A variety of phenolic compounds are utilized for industrial production of phenol-formaldehyde resins, paints, lacquers, cosmetics, and pharmaceuticals. Skin exposure to industrial phenolics is known to cause skin rash, dermal inflammation, contact dermatitis, leucoderma, and cancer promotion. The biochemical mechanisms of cytotoxicity of phenolic compounds are not well understood. We hypothesized that enzymatic one-electron oxidation of phenolic compounds resulting in the generation of phenoxyl radicals may be an important contributor to the cytotoxic effects. Phenoxyl radicals are readily reduced by thiols, ascorbate, and other intracellular reductants (e.g., NADH, NADPH) regenerating the parent phenolic compound. Hence, phenolic compounds may undergo enzymatically driven redox-cycling thus causing oxidative stress. To test the hypothesis, we analyzed endogenous thiols, lipid peroxidation, and total antioxidant reserves in normal human keratinocytes exposed to phenol. Using a newly developed cis-parinaric acid-based procedure to assay site-specific oxidative stress in membrane phospholipids, we found that phenol at subtoxic concentrations (50 μM) caused oxidation of phosphatidylcholine and phosphatidylethanolamine (but not of phosphatidylserine) in keratinocytes. Phenol did not induce peroxidation of phospholipids in liposomes prepared from keratinocyte lipids labeled by cis-parinaric acid. Measurements with ThioGlo-1 showed that phenol depleted glutathione but did not produce thiyl radicals as evidenced by our high-performance liquid chromatography measurements of GS·-5,5-dimethyl1pyrroline N-oxide nitrone. Additionally, phenol caused a significant decrease of protein SH groups. Luminol-enhanced chemiluminescence assay demonstrated a significant decrease in total antioxidant reserves of keratinocytes exposed to phenol. Incubation of ascorbate-preloaded keratinocytes with phenol produced an electron paramagnetic resonance-detectable signal of ascorbate radicals, suggesting that redox-cycling of one-electron oxidation products of phenol, its phenoxyl radicals, is involved in the oxidative effects. As no cytotoxicity was observed in keratinocytes exposed to 50 μM or 500 μM phenol, we conclude that phenol at subtoxic concentrations causes significant oxidative stress.

Metal working fluids (MWFs) are widely used in industry for metal cutting, drilling, shaping, lubricating, and milling. Potential for dermal exposure to MWFs exists for a large number of men and women via aerosols and splashing during the machining operations. It has been reported earlier that occupational exposure to MWFs causes allergic and irritant contact dermatitis. Previously, we showed that dermal exposure of female and male B6C3F1 mice to 5% MWFs for 3 months resulted in accumulation of mast cells and elevation of histamine in the skin. Topical exposure to MWF also resulted in elevated oxidative stress in the liver of both sexes and the testes in males. The goal of this study was to evaluate the interaction between oxidative stress in the skin and topical application of MWF. Oxidative stress in skin of B6C3F1 mice of both sexes was generated by intradermal injection of the hydrogen peroxide (H2O2) -producing enzyme, glucose oxidase with polyethylene glycol (GOD+PEG). In mice given GOD+PEG, topical treatment with MWF (200 l, 30%, for 1, 3, or 7 days) resulted in a mixed inflammatory cell response, accumulation of peroxidative products, and reduction of GSH content in the skin. Such changes were not observed with MWF treatment alone. These data indicate that oxidative stress can enhance dermal inflammation caused by occupational exposure to MWF.

Over 10 million workers in the United States are exposed to metal working fluids (MWFs) through dermal contact and/or inhalation of aerosolized fluids. The objective of this study was to elucidate the response of skin to dermal exposure to MWFs. Four-to six-week-old B6C3F1 mice of both sexes were divided into eight groups (n=5/group) and exposed to 200 μ1 of 0%, 5% (pH 7 and 9.7) and 100% (pH 10.4), unused MWFs/H2O by topical application to the unshaven back (cervical to sacral region), twice a week for 6 weeks. Skin-mast cell number in females of most treated groups and of two male groups (100% and 5%, pH 7) were significantly higher than the control groups. Eventhough both males and females (treated with 100% MWF/H2O) showed an increase in the skin-histamine levels (38% and 41%, respectively), this increase was significant in females only (ANOVA, P≤0.05). Dermal exposure to 100% MWFs increased liver weight significantly in both sexes. Ulcers and associated inflammation were seen in the skin of mice treated with 100% unused MWFs and sacrificed at 6 weeks, but not in the recovery groups. Hypertrophy of the sebaceous gland epithelium is present in all mice treated for 6 weeks and sacrificed immediately. However, only the mice treated with 100% MWF retained the hypertrophy of this epithelium after a 6-week recovery period. In conclusion, dermal exposure to unused semi-synthetic MWF penetrates the normal skin, induces mast cell accumulation in the skin, produces hypertrophy of the sebaceous glands, and may affect females more than males.

Zymbal glands were excised bilaterally from the ear ducts of female Sprague-Dawley rats (three/group), minced into approximately four fragments per gland, and transferred into a microtiter plate containing 1.5 mL per well of Waymouth's tissue culture medium supplemented with fetal calf serum, hydrocortisone, insulin, and gentamicin. After addition of a test compound or solvent vehicle, plates were incubated for 6, 24, 48, or 96 hr at 37°C in a humidified atmosphere of 5% CO2in air. Tissue in culture for 6 hr was histologically indistinguishable from the freshly excised tissue, while that in culture for 24, 48, and 96 hr showed a progressive deterioration often with necrosis and/or squamous metaplasia. More pronounced deterioration was noted in samples treated with 750 or 1500 μg/mL of benzene. Using a nuclease P1-enhanced32P-postlabeling assay, aromatic DNA adducts were detected in cultured Zymbal glands exposed for 48 hr to benzene and its derivatives, as well as to 7,12-dimethylbenzanthracene (DMBA) and 2-acetylaminofluorene (AAF). Benzene produced very low levels of adducts (0.5 adducts per 109nucleotides), whereas its congeners produced relatively high levels of adducts (50-2000 lesions per 109nucleotides), which decreased in the order benzoquinone > hydroquinone > phenol > benzenetriol > catechol. Each adduct profile overall was characteristic for the compound studied, suggesting the formation of compound-specific electrophiles. AAF and DMBA adducts were identical to those formed in vivo in animals. Our results show that the Zymbal glands are capable of metabolizing different carcinogens to DNA-reactive intermediates, a process that may be causally associated with tumor formation in vivo in this organ.

Rats, guinea pigs and monkeys were exposed by inhalation (6 hr/day, 5 days/week) for up to 22 months to a 13 mg/ m3concentration of PVC dust. Autopsies on rats and guinea pigs were performed after 12 months of exposure and on monkeys after 22 months of exposure. Lung function tests were performed on monkeys after 9, 14 and 22 months of exposure. Aggregates of alveolar macrophages containing PVC particles were found in the lungs of all animals. These aggregates were more numerous in the monkey lungs. No fibrosis or significant cellular infiltrates were present in or near these cellular aggregates. No significant effects on pulmonary function could be demonstrated in the monkeys exposed to PVC. Under the conditions of this experiment, inhaled PVC produced a benign pneumoconiosis.