The purpose of this position paper is to present a critical analysis of the challenges and limitations of the most widely used fluorescent probes for detecting and measuring reactive oxygen and nitrogen species. Where feasible, we have made recommendations for the use of alternate probes and appropriate analytical techniques that measure the specific products formed from the reactions between fluorescent probes and reactive oxygen and nitrogen species. We have proposed guidelines that will help present and future researchers with regard to the optimal use of selected fluorescent probes and interpretation of results.

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Heme oxygenase-1 (HO-1), the rate-limiting enzyme in heme degradation, is an integral membrane protein of the smooth endoplasmic reticulum. However, we detected an HO-1 immunoreactive signal in the nucleus of cultured cells after exposure to hypoxia and heme or heme/hemopexin. Under these conditions, a faster migrating HO-1 immunoreactive band was enriched in nuclear extracts, suggesting that HO-1 was cleaved to allow nuclear entry. This was confirmed by the absence of immunoreactive signal with an antibody against the C terminus and the lack of a C-terminal sequence by gas chromatographymass spectrometry. Incubation with leptomycin B prior to hypoxia abolished nuclear HO-1 and the faster migrating band on Western analysis, suggesting that this process was facilitated by CRM1. Furthermore, preincubation with a cysteine protease inhibitor prevented nuclear entry of green fluorescent proteinlabeled HO-1, demonstrating that protease-mediated C-terminal cleavage was also necessary for nuclear transport of HO-1. Nuclear localization was also associated with reduction of HO activity. HO-1 protein, whether it was enzymatically active or not, mediated activation of oxidant-responsive transcription factors, including activator protein-1. Nevertheless, nuclear HO-1 protected cells against hydrogen peroxide-mediated injury equally as well as cytoplasmic HO-1. We speculate that nuclear localization of HO-1 protein may serve to up-regulate genes that promote cytoprotection against oxidative stress.Heme oxygenase (HO) 3 catalyzes the degradation of heme and the formation of biliverdin and carbon monoxide. It is highly inducible in response to various stimuli, including oxidative stress, heavy metals, UV radiation, and inflammation (1-4). Cytoprotective roles for HO have been demonstrated in many models; however, the mechanisms by which this occurs are still under intensive study. Many have speculated that either heme catabolites, such as biliverdin, or its derivative, bilirubin, and carbon monoxide or the degradation of the pro-oxidant heme results in cytoprotection against oxidative stress (5-7). Nevertheless, all of the by-products of the HO reaction, despite being potentially cytoprotective, are also cytotoxic. Bilirubin is a potent neurotoxin (8), as is carbon monoxide (9). Furthermore, the HO reaction releases iron, which could interact with cellular oxidants to generate the hydroxyl radical (10). Transfection with an inactive HO-1 mutant protein results in cytoprotection against chemically induced oxidative stress (11). Because this effect of the mutant HO-1 could not be attributable to changes in heme catabolites, it alludes to a role for the HO-1 protein itself. Furthermore, the inactive form of HO-1 increased catalase and glutathione content (11). This suggests that the HO-1 protein itself may play a role in cellular signaling. If this were true, HO-1 would need to migrate to the nucleus or produce nuclear changes that affect transcription. There are several examples of cytoplasmic enzymes serving in nuclear functi...

Control of gene expression often involves an interwoven set of regulatory processes. As information regarding regulatory pathways may be lost in ex vivo analyses, we used bioluminescence to monitor gene expression in living mammals. Viral promoters fused to firefly luciferase as transgenes in mice allowed external monitoring of gene expression both superficially and in deep tissues. In vivo bioluminescence was detectable using either intensified or cooled charge-coupled device cameras, and could be detected following both topical and systemic delivery of substrate. In vivo control of the promoter from the human immunodeficiency virus was demonstrated. As a model for DNA-based therapies and vaccines, in vivo transfection of a luciferase expression vector (SV-40 promoter and enhancer controlling expression) was detected. We conclude that gene regulation, DNA delivery and expression can now be noninvasively monitored in living mammals using a luciferase reporter. Thus, real-time, noninvasive study of gene expression in living animal models for human development and disease is possible.

It is often postulated that the cytoprotective nature of heme oxygenase (HO‐1) explains the inducible nature of this enzyme. However, the mechanisms by which protection occurs are not verified by systematic evaluation of the physiological effects of HO. To explain how induction of HO‐1 results in protection against oxygen toxicity, hamster fibroblasts (HA‐1) were stably transfected with a tetracycline response plasmid containing the full‐length rat HO‐1 cDNA construct to allow for regulation of gene expression by varying concentrations of doxycycline (Dox). Transfected cells were exposed to hyperoxia (95% O2/5% CO2) for 24 h and several markers of oxidative injury were measured. With varying concentrations of Dox, HO activity was regulated between 3‐ and 17‐fold. Despite cytoprotection with low (less than fivefold) HO activity, high levels of HO‐1 expression (greater than 15‐fold) were associated with significant oxygen cytotoxicity. Levels of non‐heme reactive iron correlated with cellular injury in hyperoxia whereas lower levels of heme were associated with cytoprotection. Cellular levels of cyclic GMP and bilirubin were not significantly altered by modification of HO activity, precluding a substantial role for activation of guanylate cyclase by carbon monoxide or for accumulation of bile pigments in the physiological consequences of HO‐1 overexpression. Inhibition of HO activity or chelation of cellular iron prior to hyperoxic exposure decreased reactive iron levels in the samples and significantly reduced oxygen toxicity. We conclude that there is a beneficial threshold of HO‐1 overexpression related to the accumulation of reactive iron released in the degradation of heme. Therefore, despite the ready induction of HO‐1 in oxidant stress, accumulation of reactive iron formed makes it unlikely that exaggerated expression of HO‐1 is a cytoprotective response.—Suttner, D. M., Dennery, P. A. Reversal of HO‐1 related cytoprotection with increased expression is due to reactive iron. FASEB J. 13, 1800–1809 (1999)

Oxygen radicals, or reactive oxygen species (ROS) act as primary or secondary messengers to promote cell growth or death. Many instances demonstrate an important direct role of ROS in development because redox status regulates key transcription factors that influence cell signaling pathways involved in proliferation, differentiation, and apoptosis. Therefore, oxidative stress can alter many important reactions that affect embryonic development both positively and negatively. During particular periods in development, the embryo is more or less susceptible to oxidative stress, and teratogens, which can modify redox status, such as thalidomide, phenytoin, and ethanol, will disrupt fetal development. Various events in pregnancy such as diabetes also alter the redox state. Fortunately, antioxidants can obviate these effects through modification of gene expression, transcription factor signaling, and cell cycle alterations. A better understanding of ROS-mediated reactions and their impact on embryonic development is important to ensure optimal outcomes.

Free radicals and oxidants are now implicated in physiological responses and in several diseases. Given the wide range of expertise of free radical researchers, application of the greater understanding of chemistry has not been uniformly applied to biological studies. We suggest that some widely used methodologies and terminologies hamper progress and need to be addressed. We make the case for abandonment and judicious use of several methods and terms and suggest practical and viable alternatives. These changes are suggested in four areas: use of fluorescent dyes to identify and quantify reactive species, methods for measurement of lipid peroxidation in complex biological systems, claims of antioxidants as radical scavengers, and use of the terms for reactive species.

Background: A 28-kDa HO-1 isoform is induced by oxidative stress and cancer and accumulates in the nucleus. Results: Nuclear HO-1 interacts with Nrf2 and alters expression of its target genes. Conclusion: HO-1 modulates Nrf2 function. Significance: Exploiting the synergistic benefits of the HO-1⅐Nrf2 protein complex is important for developing therapeutic strategies against oxidative stress or cancer.