Hypothesis Papers
The heme synthesis and degradation pathways: role in oxidant sensitivity: Heme oxygenase has both pro- and antioxidant properties

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Abstract

The heme biosynthetic and catabolic pathways generate pro- and antioxidant compounds, and consequently, influence cellular sensitivity to oxidants. Heme precursors (δ-aminolevulinic acid, porphyrins) generate reactive oxygen species (ROS), from autoxidation and photochemical reactions, respectively. Heme, an essential iron chelate, serves in respiration, oxygen transport, detoxification, and signal transduction processes. The potential toxicity of heme and hemoproteins points to a critical role for heme degradation in cellular metabolism. The heme oxygenases (HOs) provide this function and participate in cellular defense. This hypothesis emerges from the observation that the activation of HO-1 is an ubiquitous cellular response to oxidative stress. The reaction products of HO activity, biliverdin, and its subsequent metabolite bilirubin, have antioxidant properties. Furthermore, iron released from HO activity stimulates ferritin synthesis, which ultimately provides an iron detoxification mechanism that may account for long-term cytoprotection observed after HO induction. However, such models have overlooked potential pro-oxidant consequences of HO activity. The HO reaction releases iron, which could be involved in deleterious reactions that compete with iron reutilization and sequestration pathways. Indeed, the induction of HO activity may have both pro- and antioxidant sequelae depending on cellular redox potential, and the metabolic fate of the heme iron.

Introduction

The heme biosynthetic and degradation pathways affect cellular oxidant metabolism because both are closely linked with iron cycling. Under certain conditions, heme biosynthesis generates reactive oxygen species (ROS) by two general mechanisms: (i) metal catalyzed aerobic oxidation of the porphyrin precursor δ-aminolevulinic acid (ALA) and (ii) the photochemical reactions of protoporphyrin IX (PPIX) and other porphyrins [1]. The heme molecule itself, although serving as a component of many essential enzymatic activities, represents a potentially harmful iron chelate, which may promote deleterious cellular processes such as oxidative membrane damage [2], [3], [4].

The rate-determining step in heme breakdown, the microsomal enzyme heme oxygenase-1 (HO-1) has emerged as a central component of the mammalian stress response, since the corresponding gene can be induced by multiple chemical and physical stimuli [5], [6], [7], [8], [9]. Keyse and Tyrrell [10] demonstrated the identity of HO-1 with the 32-kDa mammalian stress protein inducible by hydrogen peroxide (H2O2) and ultraviolet A (UVA: 320–380 nm) radiation. The transcriptional regulation of the HO-1 gene by oxidants, as well as the ubiquitous occurrence of the response in mammalian tissues, led to an hypothesis that HO-1 provided a cellular defense mechanism during oxidative stress [10], [11], [12], [13], [14]. Furthermore, the potential of oxidants to induce the HO-1 gene increases under diminishing cellular concentrations of reduced glutathione (GSH), an important intracellular antioxidant compound [15], [16].

Cellular adaptation to adverse environmental conditions, a multifactorial process, occurs in association with increases in the expression of distinct stress protein genes. In the classical example of the stress response, acute cellular hyperthermia induces the synthesis of the heat shock proteins [HSPs, Mr: 28, 58 (hsp60 family), 70–73 (hsp70 family), 90, and 100–110 kDa] [17], [18]. The induction of HSPs correlates with acquired cellular thermotolerance [19]. The heat shock paradigm has led to speculation that HO-1 (32 kDa), though structurally and functionally unrelated to the HSPs, contributes to the development of acquired cellular resistance to the various agents which trigger its synthesis [9].

The heme oxygenase (HO) system consists of three isozymes, the inducible form HO-1, a constitutively expressed form HO-2, and a newly cloned and identified species, HO-3 [20], [21]. HO metabolic activity converts heme to biliverdin IXα, carbon monoxide (CO), and ferrous iron [22]. Previously, a dual antioxidant function of HO has been proposed: (i) the prevention of free heme from participating in pro-oxidant reactions [10] and (ii) the generation of the bile pigments biliverdin and bilirubin, which possess in vitro antioxidant properties [23]. Furthermore, the CO derived from the HO reaction has attracted much recent attention as a potential regulator of neural processes and vascular tone [24], [25], [26]. Roles of CO in organismic adaptation (to ischemia-reperfusion situations) have been hypothesized based on its vasodilatory action; however, the cellular function(s) of CO are not clear [27].

HO may participate in a coupled cellular protection mechanism in which the iron-storage protein, ferritin, ultimately provides the protection by sequestering and oxidizing the iron released by the HO-catalyzed breakdown of heme [28]. HO activity influences the de novo synthesis of ferritin apoprotein [29], [30]. Before its sequestration by ferritin, the released iron may be available for the catalysis of deleterious oxidation reactions, and promote membrane damage. Our recent experimental findings support an hypothesis that HO potentially behaves as a pro-oxidant [31]. We have monitored cellular sensitivity to oxidative challenge (H2O2 and UVA radiation) in HO-2 overexpressing cell lines and their corresponding parent lines, which differ in endogenous HO activity. We have observed quantitative differences in cellular sensitivity to oxidative membrane damage during active heme metabolism. Our studies indicate that under certain conditions heme metabolism is not necessarily an antioxidative process.

This article: (i) examines the conditions whereby heme, its metabolic precursors, and catabolites may produce cellular pro- or antioxidant states; (ii) traces the fate of iron through the heme synthesis and degradation pathways, and identifies points where this cycle may lead to toxic rather than beneficial outcomes for the cell; (iii) considers the step of heme iron liberation by HO activity as a critical event that may lead to both pro- and antioxidant effects; and (iv) presents a hypothesis describing the early and late consequences of HO activity.

Section snippets

The biochemistry of heme synthesis

In mammals, the mitochondrial enzyme δ-aminolevulinate synthase (ALAS)(E.C. 2.3.1.37) provides the rate limiting step in the heme biosynthetic pathway (Fig. 1). ALAS exists in two forms, an erythroid specific isozyme (eALAS), and a nonerythroid isozyme common to the liver and other tissues (ALAS1) [32], [33], [34], [35]. Natural regulation of the (hepatic) heme synthesis pathway occurs by end-product inhibition of the synthesis and mitochondrial translocation of ALAS [36], [37]. The

Heme: a catalyst of essential cellular functions

The heme molecule promotes most biological oxidation processes and, thus, performs vital functions in cellular and whole body homeostasis. Heme serves as a prosthesis for proteins involved in oxygen transport, mitochondrial respiration, drug metabolism, steroid biosynthesis, cellular antioxidant defenses, and signal transduction processes [5]. Erythroid cells synthesize heme exclusively for incorporation into hemoglobin. In muscle, myoglobin predominates as the principle hemoprotein used for

Route of heme degradation

The majority of heme in mammals is utilized for the oxygen transport protein hemoglobin. The fate of this hemoglobin heme is well defined: it undergoes synthesis in erythrocytes and ultimate degradation in the reticuloendothelial system, and accounts for 80% of BR production in man [92], [93]. Red blood cell hemoglobin in intact, but senescing erythrocytes, undergoes degradation in the reticuloendothelial system of the liver, kidney, and especially the spleen, where HO activity is highest [94].

HO as heme removal mechanism in response to hemoprotein degradation

In discussing HO as a heme detoxification mechanism at the cellular level, several general principles should be considered: (i) newly synthesized cellular heme that is destined for incorporation into the hemoprotein pool exists transiently in a “free” pool; (ii) heme released during the natural or pharmacological induction of hemoprotein turnover must also exist transiently in a “free” pool, before its ultimate degradation by HO; (iii) The pre- and post-hemoprotein “free” heme pool occurs in

In vivo models of HO as cytoprotectant

New information of the functional roles of HO-1 and HO-2 arises from recent in vivo studies [169], [170], [171], [172]. Adenoviral mediated gene transfer of HO-1 into rat lung protected the rat against lung apoptosis and inflamation during hyperoxia [169]. HO-1 homozygous knockout mice HO-1(−/−) had low serum iron anemia, yet accumulated nonheme iron in the kidney and liver. The authors concluded that iron recycling by HO-1 is critical in maintaining blood iron levels [170]. The exact mechanism

Carbon monoxide: is there a significance in cellular signaling or cellular protection?

The carbon monoxide (CO) released directly from heme during HO activity may function as a soluble second messenger molecule in a fashion similar to the free radical gas, NOradical dot [24], [25], [26], [173], [174]. Both CO and NOradical dot bind to ferrous heme iron to form hexa- and penta-coordinate complexes, respectively [173]. Both NOradical dot and CO have demonstrated inhibitory effects on most known hemoproteins including the NOS enzymes and the heme-HO complex that generate them [175], [176], [177]. Thus, NOradical dot and CO

An equilibrium model for the functional consequences of HO activity

In considering the experimental evidence described herein, we propose the following equilibrium model for the functional consequences of HO activity with respect to cellular systems (Fig. 6). Cells subjected to metabolic stress resulting in the increased production of ROS may sustain protein modification, rendering the proteins more susceptible to proteolytic degradation. Such a phenomenon may result in a transient increase in the “free” heme pool as a result of increased turnover of

Conclusions

Intracellular iron cycling plays a critical role in how cells cope with the generation of ROS. In this article we have stressed the role of the heme oxygenases as regulators of intracellular iron homeostasis, and imply that HO enzyme function has both indirect antioxidative and potential pro-oxidant consequences, depending on the magnitude of induction. Furthermore, on a temporal basis, active heme metabolism may increase apparent oxidant sensitivity in the short-term by creating a transient

Acknowledgements

R. M. Tyrrell was supported by core grants from the International Association for Cancer Research (UK) and the Department of Health (UK), contract no. 121/6378, with additional support from the League Against Cancer of Central Switzerland, the Neuchateloise League Against Cancer, and the Swiss National Science Foundation. We would like to thank the Swiss Institute for Experimental Cancer Research (ISREC) for use of the facilities for part of the experimental work described. We would also like

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    1

    Stefan Ryter received his Ph.D. from the Institute for Toxicology, University of Southern California (Los Angeles, CA, USA), and spent a postdoctoral period at the Swiss Institute for Experimental Cancer Research (ISREC) in Epalinges, Switzerland. Dr. Ryter is currently a faculty member at Southern Illinois University School of Medicine, in Springfield, IL, USA.

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    Professor Rex M. Tyrrell received his Ph.D. from the University of Bath (UK), and spent a postdoctoral period at Argonne National Labs (Chicago, IL, USA) in the 1970s. After a period as a staff scientist in a Medical Research Council unit in London (UK) and several years at the Biophysics Institute of the Federal University of Rio de Janeiro (Brazil), Professor Tyrrell returned to head the Physical Carcinogenesis Unit, Swiss Institute for Experimental Cancer Research, ISREC, in Epalinges, Switzerland, for 15 years. He has recently been appointed to a chair at the Department of Pharmacy and Pharmacology, University of Bath.

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