Chronic obstructive pulmonary disease (COPD), which comprises emphysema and chronic bronchitis resulting from prolonged exposure to cigarette smoke (CS), is a major public health burden with no effective treatment. Emphysema is also associated with pulmonary hypertension, which can progress to right ventricular failure, an important cause of morbidity and mortality among patients with COPD. Nuclear erythroid 2 p45 related factor-2 (Nrf2) is a redox-sensitive transcription factor that up-regulates a battery of antioxidative genes and cytoprotective enzymes that constitute the defense against oxidative stress. Recently, it has been shown that patients with advanced COPD have a decline in expression of the Nrf2 pathway in lungs, suggesting that loss of this antioxidative protective response is a key factor in the pathophysiological progression of emphysema. Furthermore, genetic disruption of Nrf2 in mice causes early-onset and severe emphysema. The present study evaluated whether the strategy of activation of Nrf2 and its downstream network of cytoprotective genes with a small molecule would attenuate CS-induced oxidative stress and emphysema. Nrf2 ؉/؉ and Nrf2 ؊/؊ mice were fed a diet containing the potent Nrf2 activator, 1-[2-cyano-3-,12-dioxooleana-1,9(11)-dien-28-oyl]imidazole (CDDO-Im), while being exposed to CS for 6 months. CDDO-Im significantly reduced lung oxidative stress, alveolar cell apoptosis, alveolar destruction, and pulmonary hypertension in Nrf2 ؉/؉ mice caused by chronic exposure to CS. This protection from CS-induced emphysema depended on Nrf2, as Nrf2 ؊/؊ mice failed to show significant reduction in alveolar cell apoptosis and alveolar destruction after treatment with CDDO-Im. These results suggest that targeting the Nrf2 pathway during the etiopathogenesis of emphysema may represent an important approach for prophylaxis against COPD. chronic obstructive pulmonary disease ͉ oxidative stress

Nuclear factor-erythroid 2 p45-related factor 2 (Nrf2) is the primary transcription factor protecting cells from oxidative stress by regulating cytoprotective genes, including the antioxidant glutathione (GSH) pathway. GSH maintains cellular redox status and affects redox signaling, cell proliferation, and death. GSH homeostasis is regulated by de novo synthesis as well as GSH redox state; previous studies have demonstrated that Nrf2 regulates GSH homeostasis by affecting de novo synthesis. We report that Nrf2 modulates the GSH redox state by regulating glutathione reductase (GSR). In response to oxidants, lungs and embryonic fibroblasts (MEFs) from Nrf2-deficient (Nrf2−/−) mice showed lower levels of GSR mRNA, protein, and enzyme activity relative to wild type (Nrf2+/+). Nrf2−/− MEFs exhibited greater accumulation of glutathione disulfide and cytotoxicity compared to Nrf2+/+ MEFs in response to t-butylhydroquinone, which was rescued by restoring GSR. Microinjection of glutathione disulfide induced greater apoptosis in Nrf2−/− MEFs compared to Nrf2+/+ MEFs. In silico promoter analysis of the GSR gene revealed three putative antioxidant-response elements (ARE1, −44; ARE2, −813; ARE3, −1041). Reporter analysis, site-directed mutagenesis, and chromatin immunoprecipitation assays demonstrated binding of Nrf2 to two AREs distal to the transcription start site. Overall, Nrf2 is critical for maintaining the GSH redox state via transcriptional regulation of GSR and protecting cells against oxidative stress.

Exposure to cigarette smoke (CS) is the primary factor associated with the development of chronic obstructive pulmonary disease (COPD). CS increases the level of oxidants in the lungs, resulting in a depletion of antioxidants, which promotes oxidative stress and the destruction of alveolar tissue. In response to CS, pulmonary epithelial cells counteract increased levels of oxidants by activating Nrf2-dependent pathways to augment the expression of detoxification and antioxidant enzymes, thereby protecting the lung from injury. We hypothesize that increasing the pathways activated by Nrf2 will afford protection against CS-induced lung damage. To this end we have developed a novel mouse model in which the cytosolic inhibitor of Nrf2, Keap1, is genetically deleted in Clara cells, which predominate in the upper airways in mice. Deletion of Keap1 in Clara cells resulted in increased expression of Nrf2-dependent genes, such as Nqo1 and Gclm, as determined by microarray analysis and quantitative PCR. Deletion of Keap1 in airway epithelium decreased Keap1 protein levels and significantly increased the total level of glutathione in the lungs. Increased Nrf2 activation protected Clara cells against oxidative stress ex vivo and attenuated oxidative stress and CS-induced inflammation in vivo. Expression of KEAP1 was also decreased in human epithelial cells through siRNA transfection, which increased the expression of Nrf2-dependent genes and attenuated oxidative stress. In conclusion, activating Nrf2 pathways in tissue-specific Keap1 knockout mice represents an important genetic approach against oxidant-induced lung damage.

It was postulated that thalidomide causes birth defects by being metabolized to a toxic electrophilic intermediate. This hypothesis was tested by using an in vitro assay in which drug toxicity to human lymphocytes was assessed in the presence of a hepatic microsomal drug metabolizing system. Maternal hepatic microsomes from pregnant rabbits mediated the production of a metabolite that was toxic to lymphocytes. Toxicity was enhanced by inhibitors of epoxide hydrolase (EC 3.3.2.3) and abolished by adding the purified enzyme to the incubation medium. The metabolite thus appears to be an arene oxide, consistent with the previously reported isolation of phenolic metabolites of thalidomide from the urine of treated animals. Two teratogenic analogs of thalidomide (phthalimidophthalimide and phthalimidinoglutarimide) were also toxic in the system; two nonteratogenic analogs (phthalimide and hexahydrothalidomide) were not toxic, even in the presence of epoxide hydrolase inhibitors. The toxic metabolite of thalidomide was not produced by rat liver microsomes (the rat is not sensitive to thalidomide teratogenesis) but was produced by hepatic preparations from maternal rabbits, and rabbit, monkey, and human (all sensitive species) fetuses. A toxic arene oxide therefore may be involved in the teratogenicity of thalidomide.Thalidomide was identified as a human teratogen 20 years ago (1-3). Compared to other teratogens, thalidomide's selective toxicity in the embryo, particularly for the developing limbs, and its relative lack oftoxicity in the adult is striking (4). Despite intensive investigation, however, the mechanism of the fetal toxicity of the drug remains unknown.An interesting early observation was that rats were resistant to the teratogenic effects of thalidomide but rabbits and monkeys were sensitive (5-7). Differences in species susceptibility could result from differences in biotransformation of the compound. It was noted that rabbit liver homogenates enhanced the rate of disappearance of the drug from incubation mixtures whereas rat liver homogenates did not (8). Similarly, in vivo, more thalidomide metabolites were bound to liver macromolecules in the rabbit than in the rat, suggesting that a metabolite might interact covalently with macromolecules important in morphogenesis. Furthermore, after thalidomide treatment, 4-and 5-hydroxylated metabolites of thalidomide were recovered from the urine of rabbits but not from rats (9).The presence ofphenolic derivatives ofthalidomide suggests that the drug might undergo oxidative metabolism via an arene oxide intermediate. Arene oxides have been implicated as mutagens, cytotoxins, and teratogens (10-12). Thus, birth defects caused by the anticonvulsant phenytoin may result from an arene oxide metabolite which covalently binds to critical structures in the developing fetus (12). Therefore, we have attempted to look for a possible toxic arene oxide metabolite ofthalidomide. We have used an in vitro assay system in which human lymphocytes are the target of metabol...