Radiation and chemical reactions that give rise to free radicals cause the formation of highly cytotoxic base propenals, degradation products of DNA. Human glutathione transferases (GSTs; RX:glutathione R-transferase, EC 2.5.1.18) of classes Alpha, Mu, and Pi were shown to promote the conjugation of glutathione with base propenals and related alkenes. GST P1-i was particularly active in catalyzing the reactions with the propenal derivatives, and adenine propenal was the substrate giving the highest activity. The catalytic efficiency of GST P1-i with adenine propenal (kat/K. = 7.7 x 105 M-1's-) is the highest so far reported with any substrate for this enzyme. In general, GST Al-i and GST Mi-i, in contrast to GST P1-i, were more active with 4-hydroxyalkenals (products of lipid peroxidation) than with base propenals. The adduct resulting from the Michael addition of glutathione to the alkene function of one of the base propenals (adenine propenal) was identified by mass spectrometry. At the cellular level, GST P1-i was shown to provide protection against a,4-unsaturated aldehydes. GST Pi-i added to the culture medium of HeLa cells augmented the protective effect of glutathione against the toxicity of adenine propenal and thymine propenal. No protective effect ofthe enzyme was observed in the presence of the competitive inhibitor S-hexylglutathione. GST Pi-i introduced into Hep G2 cells by electroporation was similarly found to increase their resistance to acrolein. The results show that glutathione transferases may play an important role in cellular detoxication of electrophilic a,.-unsaturated carbonyl compounds produced by radical reactions, lipid peroxidation, ionizing radiation, and drug metabolism.

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various uterine responses in female Sprague-Dawley and Failure of Chloro-S-triazine-Derived Compounds to Induce Es-Fischer 344 rats (Eldridge et al., 1994;Stevens et al., 1994; trogen Receptor-Mediated Responses in Vivo and in Vitro. CON- Tennant et al., 1994a,b;Wetzel et al., 1994). These studies NOR, K., HOWELL, J., CHEN, I., LIU, H., BERHANE, K., SCIAR-found that atrazine and simazine may inhibit 17b-estradiol tremely high doses. For example, chronic feeding of high The potential estrogenic activities of atrazine and simazine were dietary levels of atrazine increased the number of days in investigated in vivo using the immature female Sprague-Dawley estrus and hastened the onset but not the incidence of mamrat uterus and in vitro using the estrogen-responsive MCF-7 hu-mary tumors in female Sprague-Dawley rats. In contrast, man breast cancer cell line and the estrogen-dependent recombi-these changes were not observed in female Fischer 344 rats; nant yeast strain PL3. Animals that were dosed with 50, 150, or these animals exhibited a significantly lower incidence of 300 mg/kg of atrazine or simazine alone for 3 consecutive days spontaneous age-dependent mammary tumors than observed did not exhibit any significant increases in uterine wet weight while in Sprague-Dawley rats. There is also evidence suggesting decreases in cytosolic progesterone receptor (PR) binding levels that these chloro-S-triazines decrease rat uterine estrogen and uterine peroxidase activity were observed. 17b-estradiol (E2)-receptor (ER) binding and uterine wet weights. Moreover, induced increases in uterine wet weight were not significantly affected by cotreatment with either chemical; however, some dose-rats that were treated with E2 plus simazine or atrazine expeindependent decreases in E2-induced cytosolic PR binding and rienced a decrease in E2-induced progesterone receptor (PR) uterine peroxidase activity were observed. In vitro, atrazine and binding and DNA synthesis (Tennant et al., 1994b). Taken simazine did not affect basal or E2-induced MCF-7 cell prolifera-together, these results suggest that atrazine and simazine tion or the formation of nuclear PR-DNA complexes as deter-may possess estrogenic and/or antiestrogenic activities that mined by gel electrophoretic mobility shift assays. In addition, are mediated by the ER. these chloro-S-triazines did not display agonist activity or antago-Recently, Tennant and co-workers reported that atrazine nize E2-induced luciferase activity in MCF-7 cells transiently and simazine did not effectively displace [ 3 H]E2 from the transfected with a Gal4-human estrogen receptor chimera (Gal4-rat uterine cytosolic ER (Tennant et al., 1994b). Data from HEGO) and a Gal4-regulated luciferase reporter gene (17m5-GLuc). Moreover, the estrogen-dependent PL3 yeast strain was not competitive displacement assays indicate that k i values for capable of growth on minimal media supplemented with atrazine these chloro-S-triazines ranged from 10 to 100 mM and that or simazine in place of E2. Collectively, these ...

BackgroundPersistant inflammatory responses to infectious agents and other components in organic dust underlie lung injury and development of respiratory diseases. Organic dust components responsible for eliciting inflammation and the mechanisms by which they cause lung inflammation are not fully understood. We studied the mechanisms by which protease activities in poultry dust extracts and intracellular oxidant stress induce inflammatory gene expression in A549 and Beas2B lung epithelial cells.MethodsThe effects of dust extracts on inflammatory gene expression were analyzed by quantitative polymerase chain reaction (qPCR), enzyme linked immunosorbent (ELISA) and western blot assays. Oxidant stress was probed by dihydroethidium (DHE) labeling, and immunostaining for 4-hydroxynonenal (4-HNE). Effects on interleukin-8 (IL-8) promoter regulation were determined by transient transfection assay.ResultsDust extracts contained trypsin and elastase activities, and activated protease activated receptor (PAR)-1 and -2. Serine protease inhibitors and PAR-1 or PAR-2 knockdown suppressed inflammatory gene induction. Dust extract induction of IL-8 gene expression was associated with increased DHE-fluorescence and 4-HNE staining, and antioxidants suppressed inflammatory gene induction. Protease inhibitors and antioxidants suppressed protein kinase C and NF-κB activation and induction of IL-8 promoter activity in cells exposed to dust extract.ConclusionsOur studies demonstrate that proteases and intracellular oxidants control organic dust induction of inflammatory gene expression in lung epithelial cells. Targeting proteases and oxidant stress may serve as novel approaches for the treatment of organic dust induced lung diseases. This is the first report on the involvement of oxidant stress in the induction of inflammatory gene expression by organic dust.Electronic supplementary materialThe online version of this article (doi:10.1186/s12931-016-0455-z) contains supplementary material, which is available to authorized users.

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