Oxidative stress and inflammation response after nanoparticle exposure: differences between human lung cell monocultures and an advanced three-dimensional model of the human epithelial airways

Combustion-derived and manufactured nanoparticles (NPs) are known to provoke oxidative stress and inflammatory responses in human lung cells; therefore, they play an important role during the development of adverse health effects. As the lungs are composed of more than 40 different cell types, it is of particular interest to perform toxicological studies with co-cultures systems, rather than with monocultures of only one cell type, to gain a better understanding of complex cellular reactions upon exposure to toxic substances. Monocultures of A549 human epithelial lung cells, human monocyte-derived macrophages and monocyte-derived dendritic cells (MDDCs) as well as triple cell co-cultures consisting of all three cell types were exposed to combustion-derived NPs (diesel exhaust particles) and to manufactured NPs (titanium dioxide and single-walled carbon nanotubes). The penetration of particles into cells was analysed by transmission electron microscopy. The amount of intracellular reactive oxygen species (ROS), the total antioxidant capacity (TAC) and the production of tumour necrosis factor (TNF)-alpha and interleukin (IL)-8 were quantified. The results of the monocultures were summed with an adjustment for the number of each single cell type in the triple cell co-culture. All three particle types were found in all cell and culture types. The production of ROS was induced by all particle types in all cell cultures except in monocultures of MDDCs. The TAC and the (pro-)inflammatory reactions were not statistically significantly increased by particle exposure in any of the cell cultures. Interestingly, in the triple cell co-cultures, the TAC and IL-8 concentrations were lower and the TNF-alpha concentrations were higher than the expected values calculated from the monocultures. The interplay of different lung cell types seems to substantially modulate the oxidative stress and the inflammatory responses after NP exposure.

Paracrine factors secreted by endothelial progenitor cells prevent oxidative stress-induced apoptosis of mature endothelial cells

Endothelial progenitor cells (EPC) play a fundamental role in tissue regeneration and vascular repair. Current research suggests that EPC are more resistant to oxidative stress as compared to differentiated endothelial cells. Here we hypothesized that EPC not only possess the ability to protect themselves against oxidative stress but also confer this protection upon differentiated endothelial cells by release of paracrine factors. To test this hypothesis, HUVEC incubated with conditioned medium obtained from early EPC cultures (EPC-CM) were exposed to H2O2 to assess the accumulation of intracellular ROS, extent of apoptosis and endothelial cell functionality. Under oxidative stress conditions HUVEC treated with EPC-CM exhibited substantially lower levels of intracellular oxidative stress (0.2+/-0.02 vs. 0.4+/-0.03 relative fluorescence units, p<0.05) compared to control medium. Moreover, the incubation with EPC-CM elevated the expression level of antioxidant enzymes in HUVEC (catalase: 2.6+/-0.4; copper/zinc superoxide dismutase (Cu/ZnSOD): 1.6+/-0.1; manganese superoxide dismutase (MnSOD): 1.4+/-0.1-fold increase compared to control, all p<0.05). Furthermore, EPC-CM had the distinct potential to reverse the functional impairment of HUVEC as measured by their capability to form tubular structures in vitro. Finally, incubation of HUVEC with EPC-CM resulted in a significant reduction of apoptosis (0.34+/-0.01 vs. 1.52+/-0.12 relative fluorescence units, p<0.01) accompanied by an increased expression ratio of the anti/pro-apoptotic factors Bcl-2/Bax to 2.9+/-0.7-fold (compared to control, p<0.05). Most importantly, neutralization of selected cytokines such as VEGF, HGF, IL-8 and MMP-9 did not significantly reverse the cyto-protective effect of EPC-CM (p>0.05), suggesting that soluble factors secreted by EPC, possibly via broad synergistic actions, exert strong cyto-protective properties on differentiated endothelium through modulation of intracellular antioxidant defensive mechanisms and pro-survival signals.

Structure and function of CinD (YtjD) of Lactococcus lactis, a copper-induced nitroreductase involved in defense against oxidative stress

In Lactococcus lactis IL1403, 14 genes are under the control of the copper-inducible CopR repressor. This so-called CopR regulon encompasses the CopR regulator, two putative CPx-type copper ATPases, a copper chaperone, and 10 additional genes of unknown function. We addressed here the function of one of these genes, ytjD, which we renamed cinD (copper-induced nitroreductase). Copper, cadmium, and silver induced cinD in vivo, as shown by real-time quantitative PCR. A knockout mutant of cinD was more sensitive to oxidative stress exerted by 4-nitroquinoline-N-oxide and copper. Purified CinD is a flavoprotein and reduced 2,6-dichlorophenolindophenol and 4-nitroquinoline-N-oxide with k(cat) values of 27 and 11 s(-1), respectively, using NADH as a reductant. CinD also exhibited significant catalase activity in vitro. The X-ray structure of CinD was resolved at 1.35 A and resembles those of other nitroreductases. CinD is thus a nitroreductase which can protect L. lactis against oxidative stress that could be exerted by nitroaromatic compounds and copper.

Sperm of colourful males are better protected against oxidative stress

Sperm cells are highly vulnerable to free radicals, and sperm quality and male fertility are critically affected by oxidative stress. Recently, sexual ornaments, particularly carotenoid-based colourful traits, have been proposed to depend on a male's capacity to resist oxidative stress, and thus to signal sperm quality. We conducted an experimental test of this hypothesis on great tits Parus major, in which adults are sexually dichromatic in carotenoid-based breast plumage. We report the first evidence that ornaments and sperm quality may be linked through oxidative stress. When experimentally subjected to oxidative stress resulting from increased workload, less colourful males suffered a greater reduction in sperm motility and swimming ability, and increased levels of sperm lipid peroxidation compared to more colourful males. Moreover, the level of sperm lipid peroxidation was negatively correlated with sperm quality. Finally, carotenoid supplementation increased sperm quality of less colourful males, suggesting that pale males are deficient in carotenoid antioxidants.

Effect of sibling competition and male carotenoid supply on offspring condition and oxidative stress

Early developmental conditions have major implications for an individual's fitness. In species where offspring are born simultaneously, the level of sibling competition for food access is intense. In birds, high sibling competition may subject nestlings to decreased growth rate as a result of limited food and increased levels of oxidative stress through high metabolic activity induced by begging behaviors. We manipulated the level of sibling competition in a natural population of great tits and assessed the consequences for nestling body condition and resistance to oxidative stress. In a full factorial design, we both augmented brood size to increase sibling competition and supplemented the male parents with physiological doses of carotenoids thereby doubling the natural carotenoid intake, aiming at increasing the males' investment in current reproduction and thereby decreasing sibling competition. Nestling body mass was reduced by the brood enlargement and enhanced by the carotenoid supplementation of fathers. Nestling resistance to oxidative stress, measured as total antioxidant defenses in whole blood, was not influenced by the treatments. Because nestlings experience high metabolic activities, an absence of an effect of sibling competition on free radicals production seems unlikely. Nestling body mass decreased and resistance to oxidative stress tended to increase with initial brood size, and hence these correlational effects suggest a trade-off between morphological growth and development of the antioxidant system. However, the result of the experimental treatment did not support this trade-off hypothesis. Alternatively, it suggests that nestling developed compensatory mechanisms that were not detected by our antioxidant capacity measure.

Risk factors and mechanisms of hepatocarcinogenesis with special emphasis on alcohol and oxidative stress

Hepatocellular cancer is the fifth most frequent cancer in men and the eighth in women worldwide. Established risk factors are chronic hepatitis B and C infection, chronic heavy alcohol consumption, obesity and type 2 diabetes, tobacco use, use of oral contraceptives, and aflatoxin-contaminated food. Almost 90% of all hepatocellular carcinomas develop in cirrhotic livers. In Western countries, attributable risks are highest for cirrhosis due to chronic alcohol abuse and viral hepatitis B and C infection. Among those with alcoholic cirrhosis, the annual incidence of hepatocellular cancer is 1-2%. An important mechanism implicated in alcohol-related hepatocarcinogenesis is oxidative stress from alcohol metabolism, inflammation, and increased iron storage. Ethanol-induced cytochrome P-450 2E1 produces various reactive oxygen species, leading to the formation of lipid peroxides such as 4-hydroxy-nonenal. Furthermore, alcohol impairs the antioxidant defense system, resulting in mitochondrial damage and apoptosis. Chronic alcohol exposure elicits hepatocyte hyperregeneration due to the activation of survival factors and interference with retinoid metabolism. Direct DNA damage results from acetaldehyde, which can bind to DNA, inhibit DNA repair systems, and lead to the formation of carcinogenic exocyclic DNA etheno adducts. Finally, chronic alcohol abuse interferes with methyl group transfer and may thereby alter gene expression.

Coronary artery disease, nitric oxide and oxidative stress: the "Yin-Yang" effect--a Chinese concept for a worldwide pandemic

Prevention of coronary artery disease (CAD) and reduction of its mortality and morbidity remains a major public health challenge throughout the "Western world". Recent evidence supports the concept that the impairment of endothelial function, a hallmark of insulin resistance states, is an upstream event in the pathophysiology of insulin resistance and its main corollaries: atherosclerosis and myocardial infarction. Atherosclerosis is currently thought to be the consequence of a subtle imbalance between pro- and anti-oxidants that produces favourable conditions for lesion progression towards acute thrombotic complications and clinical events. Over the last decade, a remarkable burst of evidence has accumulated, offering the new perspective that bioavailable nitric oxide (NO) plays a pivotal role throughout the CAD-spectrum, from its genesis to the outcome after acute events. Vascular NO is a critical modulator of coronary blood flow by inhibiting smooth muscle contraction and platelet aggregation. It also acts in angiogenesis and cytoprotection. Defective endothelial nitric oxide synthase (eNOS) driven NO synthesis causes development of major cardiovascular risk factors (insulin resistance, arterial hypertension and dyslipidaemia) in mice, and characterises CAD-prone insulin-resistant humans. On the other hand, stimulation of inducible nitric oxide synthase (iNOS) and NO overproduction causes metabolic insulin resistance and characterises atherosclerosis, heart failure and cardiogenic shock in humans, suggesting a "Yin-Yang" effect of NO in the cardiovascular homeostasis. Here, we will present a concise overview of the evidence for this novel concept, providing the conceptual framework for developing a potential therapeutic strategy to prevent and treat CAD.

Neuregulin-1 beta attenuates doxorubicin-induced alterations of excitation-contraction coupling and reduces oxidative stress in adult rat cardiomyocytes

Treatment of metastatic breast cancer with doxorubicin (Doxo) in combination with trastuzumab, an antibody targeting the ErbB2 receptor, results in an increased incidence of heart failure. Doxo therapy induces reactive oxygen species (ROS) and alterations of calcium homeostasis. Therefore, we hypothesized that neuregulin-1 beta (NRG), a ligand of the cardiac ErbB receptors, reduces Doxo-induced alterations of EC coupling by triggering antioxidant mechanisms. Adult rat ventricular cardiomyocytes (ARVM) were isolated and treated for 18-48 h. SERCA protein was analyzed by Western blot, EC coupling parameters by fura-2 and video edge detection, gene expression by RT-PCR, and ROS by DCF-fluorescence microscopy. At clinically relevant doses Doxo reduced cardiomyocytes contractility, SERCA protein and SR calcium content. NRG, similarly as the antioxidant N-acetylcystein (NAC), did not affect EC coupling alone, but protected against Doxo-induced damage. NRG and Doxo showed an opposite modulation of glutathione reductase gene expression. NRG, similarly as NAC, reduced peroxide- or Doxo-induced oxidative stress. Specific inhibitors showed, that the antioxidant action of NRG depended on signaling via the ErbB2 receptor and on the Akt- and not on the MAPK-pathway. Therefore, NRG attenuates Doxo-induced alterations of EC coupling and reduces oxidative stress in ARVM. Inhibition of the ErbB2/NRG signaling pathway by trastuzumab in patients concomitantly treated with Doxo might prevent beneficial effects of NRG in the myocardium.

Vitamin C deficiency in weanling guinea pigs: differential expression of oxidative stress and DNA repair in liver and brain

Neonates are particularly susceptible to malnutrition due to their limited reserves of micronutrients and their rapid growth. In the present study, we examined the effect of vitamin C deficiency on markers of oxidative stress in plasma, liver and brain of weanling guinea pigs. Vitamin C deficiency caused rapid and significant depletion of ascorbate (P < 0.001), tocopherols (P < 0.001) and glutathione (P < 0.001), and a decrease in superoxide dismutase activity (P = 0.005) in the liver, while protein oxidation was significantly increased (P = 0.011). No changes in lipid oxidation or oxidatively damaged DNA were observed in this tissue. In the brain, the pattern was markedly different. Of the measured antioxidants, only ascorbate was significantly depleted (P < 0.001), but in contrast to the liver, ascorbate oxidation (P = 0.034), lipid oxidation (P < 0.001), DNA oxidation (P = 0.13) and DNA incision repair (P = 0.014) were all increased, while protein oxidation decreased (P = 0.003). The results show that the selective preservation of brain ascorbate and induction of DNA repair in vitamin C-deficient weanling guinea pigs is not sufficient to prevent oxidative damage. Vitamin C deficiency may therefore be particularly adverse during the neonatal period.

Genetic and pharmacologic manipulation of oxidative stress after neonatal hypoxia-ischemia

Oxidative stress is a critical component of the injury response to hypoxia-ischemia (HI) in the neonatal brain, and this response is unique and at times paradoxical to that seen in the mature brain. Previously, we showed that copper-zinc superoxide-dismutase (SOD1) over-expression is not beneficial to the neonatal mouse brain with HI injury, unlike the adult brain with ischemic injury. However, glutathione peroxidase 1 (GPx1) over-expression is protective to the neonatal mouse brain with HI injury. To further test the hypothesis that an adequate supply of GPx is critical to protection from HI injury, we crossed SOD1 over-expressing mice (hSOD-tg) with GPx1 over-expressing mice (hGPx-tg). Resulting litters contained wild-type (wt), hGPx-tg, hSOD-tg and hybrid hGPx-tg/hSOD-tg pups, which were subjected to HI at P7. Confirming previous results, the hGPx-tg mice had reduced injury compared to both Wt and hSOD-tg littermates. Neonatal mice over-expressing both GPx1 and SOD1 also had less injury compared to wt or hSOD-tg alone. A result of oxidative stress after neonatal HI is a decrease in the concentration of reduced (i.e. antioxidant-active) glutathione (GSH). In this study, we tested the effect of systemic administration of alpha-lipoic acid on levels of GSH in the cortex after HI. Although GSH levels were restored by 24h after HI, injury was not reduced compared to vehicle-treated mice. We also tested two other pharmacological approaches to reducing oxidative stress in hSOD-tg and wild-type littermates. Both the specific inhibitor of neuronal nitric oxide synthase, 7-nitroindazole (7NI), and the spin-trapping agent alpha-phenyl-tert-butyl-nitrone (PBN) did not reduce HI injury, however. Taken together, these results imply that H2O2 is a critical component of neonatal HI injury, and GPx1 plays an important role in the defense against this H2O2 and is thereby neuroprotective.

Oxidative stress and damage in liver, but not in brain, of Fischer 344 rats subjected to dietary iron supplementation with lipid-soluble [(3,5,5-trimethylhexanoyl)ferrocene]

Accumulation of iron probably predisposes the aging brain to progressive neuronal loss. We examined various markers of oxidative stress and damage in the brain and liver of 3- and 24-month-old rats following supplementation with the lipophilic iron derivative [(3,5,5-trimethylhexanoyl)ferrocene] (TMHF), which is capable of crossing the blood-brain barrier. At both ages, iron concentration increased markedly in the liver but failed to increase in the brain. In the liver of TMHF-treated young rats, levels of alpha- and gamma-tocopherols and glutathione (GSH) were also higher. In contrast, the brain displayed unaltered levels of the tocopherols and GSH. Malondialdehyde (MDA) level was also higher in the cerebrospinal fluid (CSF) and the liver but not in the brain. In old rats, the absence of an increase in iron concentration in the brain was reflected by unaltered concentrations of GSH, tocopherols, and MDA as compared to that in untreated rats. In the aging liver, concentrations of GSH and MDA increased with TMHF treatment. Morphological studies revealed unaltered levels of iron, ferritin, heme oxygenase-1 (HO-1), nitrotyrosine (NT), or MDA in the brains of both young and old rats treated with TMHF. In contrast, TMHF treatment increased the level of HO-1 in Kupffer cells, NT in hepatic endothelial cells, and MDA and ferritin in hepatocytes. Although these results demonstrated an increase in the biochemical markers of oxidative stress and damage in response to increasing concentrations of iron in the liver, they also demonstrated that the brain is well protected against dietary iron overload by using iron in a lipid-soluble formulation.