Toxicological and pathophysiological roles of reactive oxygen and nitrogen species

Abstract

'Oxidative and Nitrative Stress in Toxicology and Disease' was the subject of a symposium held at the EUROTOX meeting in Dresden 15th September 2009. Reactive oxygen (ROS) and reactive nitrogen species (RNS) produced during tissue pathogenesis and in response to viral or chemical toxicants, induce a complex series of downstream adaptive and reparative events driven by the associated oxidative and nitrative stress. As highlighted by all the speakers, ROS and RNS can promote diverse biological responses associated with a spectrum of disorders including neurodegenerative/neuropsychiatric and cardiovascular diseases. Similar pathways are implicated during the process of liver and skin carcinogenesis. Mechanistically, reactive oxygen and nitrogen species drive sustained cell proliferation, cell death including both apoptosis and necrosis, formation of nuclear and mitochondrial DNA mutations, and in some cases stimulation of a pro-angiogenic environment. Here we illustrate the pivotal role played by oxidative and nitrative stress in cell death, inflammation and pain and its consequences for toxicology and disease pathogenesis. Examples are presented from five different perspectives ranging from in vitro model systems through to in vivo animal model systems and clinical outcomes.

Figure 1:. Inflammation and the role of ROS and RNS in tissue damage

Inflammation begins with a reaction to an irritant or infection that is characterized by movement of fluid and white blood cells into extravascular tissue. This is followed by tissue repair and regeneration and involves cell proliferation. Associated with these processes are the release of free radicals, such as reactive oxide species (ROS) and reactive nitrogen species (RNS). This can activate a process called lipid peroxidation and the arachidonic-acid cascade, with the production of cell-proliferation-stimulating eicosanoids. Also, DNA-damaging agents, such as malondialdehyde (MDA) and 4-hydroxynonenol (4-HNE), are byproducts of the arachidonic-acid cascade. The free radicals can also damage DNA and modify the structure and function of cancer-related proteins. OH•, hydroxyl radical; O₂^-, superoxide; NO, nitric oxide; ONOO^-, peroxynitrite; N₂O₃, nitrous anhydride.

Fig 2:. Liver damage, inflammation and cancer: lessons from PPARα

A schematic depicting the pathway from toxicant exposure to inflammation and ultimately to tumor development. Inflammation can be triggered by toxicant exposure or by viral infection; if this inflammation persists it can progress to the development of tumors. Similalry, exposure of rodent liver to peroxisome proliferators causes activation of PPARα and hepatic growth changes ultimately leading to hepatocellular carcinoma. Cytokines such as TNFα and survival signalling molecules such as NFκB are implicated in both processes.

Figure 3:

A summary diagram of the oxidative stress insults applied to primary cultures of postnatal cerebellar granule neurons (Smith et al., 2003), the mechanistic and signalling pathways investigated, and the neuroprotective strategies employed to attenuate neuronal damage and loss (see text for details of the individual studies).

Figure 4:. Inhibition of NF-κB prevents protein nitration in dopaminergic neurons in vivo.

C57Bl/6 mice were treated with MPTP (4×10 mg/Kg, 2 hr intervals) in the presence or absence of DIM-C-pPhtBu (DIM-C) (4 daily doses by intragastric gavage, 50 mg/Kg) and sacrificed after 7 days. Frozen sections were stained by immunofluorescence for typrosine hydroxylase (green), 3-nitrotyrosine (red; an indicator of peroxynitrite formation), and nuclear morphology (DAPI; blue). (a - d) - Control; (e - h) MPTP; (i - l) MPTP + DIM-C-pPhtBu. Yellow staining the merged images in d, h, and l indicates co-localization.

Figure 5:

Schematic of the roles of reactive oxygen species (ROS), reactive nitrogen species (RNS) oxidative DNA damage, pro-inflammatory cytokines, and vascular endothelial growth factor (VEGF) in multi-stage skin carcinogenesis.

Figure 6:

Schematic diagram of doxorubicin-induced cardiotoxicity: role of superoxide, NO, and peroxynitrite. Doxorubicin initially increases mitochondrial superoxide and, consequently, the generation of other ROS (e.g., H₂O₂) in cardiomyocytes and/or endothelial cells by redox cycling. Increased doxorubicin-induced ROS generation in cardiomyocytes triggers the activation of the transcription factor NF-kB, leading to enhanced NOS2 expression and NO generation. NO reacts with superoxide to form peroxynitrite both in the cytosol and mitochondria, which, in turn, induces cell damage via lipid peroxidation, inactivation of enzymes and other proteins by oxidation and nitration, and activation of stress signaling pathways (e.g., MAPK), MMPs, and PARP-1, among others. In the mitochondria, peroxynitrite, in concert with other ROS/RNS, impairs various key mitochondrial enzymes, leading to more sustained intracellular ROS generation (persistent even after doxorubicin already metabolized), triggering further activation of transcription factor(s) and NOS2 expression, resulting in the amplification of oxidative/nitrosative stress. In the mitochondria, peroxynitrite also triggers the release of proapoptotic factors (e.g., Cytochrome C and apoptosis-inducing factor) mediating caspase-dependent and -independent cell death pathways, which are also pivotal in doxorubicin-induced cardiotoxicity. Peroxynitrite, in concert with other oxidants, also causes strand breaks in DNA, activating the nuclear enzyme PARP-1. Once excessive oxidative and nitrosative stress-induced DNA damage occurs, overactivated PARP initiates an energy-consuming cycle by transferring ADP-ribose units from NAD⁺ to nuclear proteins, resulting in the rapid depletion of intracellular NAD⁺ and ATP pools, slowing the rate of glycolysis and mitochondrial respiration, eventually leading to cellular dysfunction and death, mostly by necrosis. Overactivated PARP may also facilitate the expression of a variety of inflammatory genes leading to increased inflammation (PARP-1 is a known co-activator of NF-kB) and associated oxidative stress, thus facilitating the progression of cardiovascular dysfunction and heart failure. PARG, poly(ADP-ribose) glycohydrolase (Reproduced with permission from Mukhopadhyay et al., 2009).