Cadmium (Cd) is an environmental pollutant of global concern with a 10-30-year biological half-life in humans. Accumulating evidence suggests that the lung is one of the major target organs of inhaled Cd compounds. Our previous report demonstrated that 100 microM Cd induces MRC-5 cells, normal human lung fibroblasts, to undergo caspase-independent apoptosis mediated by mitochondrial membrane depolarization and translocation of apoptosis-inducing factor (AIF) from mitochondria into the nucleus. Here, using benzyloxycarbonyl-Val-Ala-Asp-(ome) fluoromethyl ketone (Z-VAD.fmk) as a tool, we further demonstrated that Cd could induce caspase-independent apoptosis at concentrations varied from 25 to 150 microM, which was modulated by reactive oxygen species (ROS) scavengers, such as N-acetylcysteine (NAC), mannitol, and tiron, indicating that ROS play a crucial role in the apoptogenic activity of Cd. Consistent with this notion, the intracellular hydrogen peroxide (H2O2) was 2.9-fold elevated after 3 h of Cd treatment and diminished rapidly within 1 h as detected by flow cytometry with 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA) staining. Using inhibitors of the mitochondrial electron transport chain (ETC) (oligomycin A and rotenone for complex I and V, respectively) and mitochondrial permeability transition pore (MPTP) (cyclosporin A and aristolochic acid), we coincidently found the ROS production, mitochondrial membrane depolarization, and apoptotic content were almost completely or partially abolished. As revealed by confocal microscopy staining with chloromethyl-X-rosamine (CMXRos) and an anti-AIF antibody, the collapse of mitochondrial membrane potential induced by Cd (3 h-treatment) was a prelude to the translocation of caspase-independent pro-apoptotic factor, AIF, into the nucleus (after 4 h of Cd treatment). In summary, this study demonstrated that, in MRC-5 fibroblasts, Cd induced caspase-independent apoptosis through a mitochondria-ROS pathway. More importantly, we provide several lines of evidence supporting a role of mitochondrial ETC and MPTP in the regulation of caspase-independent cell death triggered by Cd.

Cadmium, a well-known environmental hazard, has caused serious health problems in humans and animals. Accumulating evidence suggests the cadmium toxicity is mediated by oxidative stress-induced cell death. However, the molecular signaling underlying cadmium-induced apoptosis remains unclear. In this study, we demonstrate here that cadmium induced mixed types of cell death including primary apoptosis (early apoptosis), secondary necrosis (late apoptosis), and necrosis in normal human lung cells, MRC-5, as revealed by chromatin condensation, phosphatidylserine (PS) externalization, and hypodiploid DNA content. The total apoptotic cells reached a plateau of around 40.0% after 24 h exposure of 100 microM cadmium. Pretreatment with Z-Val-Ala-Asp-fluoromethylketone (Z-VAD-fmk), a broad spectrum of caspase inhibitor, could not rescue apoptotic cells from cadmium toxicity. Coincidently, we failed to detect the activation of pro-caspase-3 and cleavage of PARP by immunoblot, which implies the apoptogenic activity of cadmium in MRC-5 cells is caspase-independent. JC-1 staining also indicated that mitochondrial depolarization is a prelude to cadmium-induced apoptosis, which was accompanied by a translocation of caspase-independent pro-apoptotic factor apoptosis-inducing factor (AIF) into the nucleus as revealed by the immunofluorescence assay. In summary, this study demonstrated for the first time that cadmium induced a caspase-independent apoptotic pathway through mitochondria-mediated AIF translocation into the nucleus.

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Intrauterine growth restriction (IUGR) is associated with reduced lung function during infancy and throughout adulthood. We investigated the effects of maternal undernutrition (50% rations of the control food intake) during the last week of gestation on the pulmonary surfactant system and lung morphometry in postnatal rats. IUGR rats exhibited a significantly lower body weight, lower lung weight, lower lung/body weight ratio, lower lung volume, and lower lung volume/body weight ratio on some postnatal days. IUGR rats had a significantly lower lung saturated phosphatidylcholine and lower plasma corticosterone levels on postnatal d 1 only, and values were comparable between control and IUGR rats in the ensuing weeks. Lung surfactant protein (SP)-A, SP-B, SP-C, and SP-D mRNA expressions were similar between control and IUGR rats. Volume fractions of the alveolar airspace were significantly lower in IUGR rats on postnatal d 7, 14, and 42. Alveolar surface areas were significantly lower in IUGR rats during the study period. The alveolar surface area/body weight ratio reached a peak on postnatal d 7, and values were significantly lower in IUGR rats on postnatal d 1, 14, 28, and 42. We conclude that maternal undernutrition during late gestation decreases lung surfactant lipid levels in the immediate postnatal period and alters the development of lung structure during the postnatal period. Alteration of lung surfactant and structure may be important in the pathogenesis of impaired pulmonary function in IUGR infants and children.

We hypothesized that lung inflammation and parenchymal apoptosis in ventilator-induced lung injury (VILI) are related to ANG II and assessed the ability of the angiotensin-converting enzyme inhibitor captopril to attenuate VILI in rats. Adult male Sprague-Dawley rats were randomized to receive two ventilation strategies for 2 h: 1) tidal volume of 40 ml/kg, respiratory rate of 25 breaths/min, and inspiratory O2 fraction of 0.21 [high-volume, 0 positive end-expiratory pressure (HVZP) group] and 2) injection of captopril (100 mg/kg ip) 30 min before HVZP ventilation (HVZP+CAP group). Another group, which did not receive ventilation, served as the control. Mean arterial pressure was significantly lower in the HVZP+CAP group than in the HVZP group at 2 h of ventilation. Total protein levels were significantly higher in bronchoalveolar lavage fluid (BALF) recovered from HVZP-ventilated rats than from controls. BALF macrophage inflammatory protein-2 and lung ANG II were significantly higher in the HVZP group than in the control and HVZP+CAP groups. Lung ANG II levels correlated positively with BALF protein and macrophage inflammatory protein-2. The number of apoptotic airway and alveolar wall cells was significantly higher in the HVZP and HVZP+CAP groups than in the control group and significantly lower in the HVZP+CAP group than in the HVZP group. These results suggest that the efficiency of captopril to attenuate VILI is related to reduction of inflammatory cytokines and inhibition of apoptosis and indicate that VILI is partly mediated by the local angiotensin system.

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Isovitexin, isolated from rice hull of Oryza sativa, has been characterized as a potent antioxidant. Its antioxidant activity, determined on the basis of inhibition of lipid peroxidation by the Fenton reaction, was comparable with that of alpha-tocopherol, a well-established antioxidant. Isovitexin was able to reduce the amount of hydrogen peroxide production induced by lipopolysaccharide (LPS) in mouse macrophage RAW264.7 cells. In this study, we assessed its effects on the production of tumor necrosis factor alpha (TNF-alpha), prostaglandin E2 (PGE2), and the expression of cyclooxygenase-2 (COX-2) in LPS-activated RAW 264.7 macrophages. Isovitexin inhibited the release of TNF-alpha, a proinflammatory cytokine, upon LPS activation with a 50% inhibitory concentration (IC50) of 78.6 microM. Isovitexin markedly reduced LPS-stimulated PGE2 production in a concentration-dependent manner, with an IC50 of 80.0 microM. The expression of COX-2 was also inhibited by isovitexin treatment. Our results suggest that suppression of ROS-mediated COX-2 expression by isovitexin is beneficial in reducing inflammation and carcinogenesis.