Reactive Oxygen Species : The Universal I<illers?

FABADJ. Pharm. Sci., 24, 171-179, 1999

BlLlMSEL TARAMALAR/ SClENTIFIC REVIEWS

Reactive Oxygen Species : The Universal I<illers?

Diana IVANOVA*, Tatyana YANKOVA•^0, Ganka BEKYAROVA**

Reactive Oxygen Species: The Universal Killers?

Summary : Free radicals are highly reactive species character- ised by an unpaired electron in their outer orbital. Oxygen- derived free radicals appear in the organism during in physio- logical and pathological processes. On the other hand they par- ticipate in development of many diseases, including rheumatoid arthritis, hemorrhagia, and AIDS in human and animals. There is alsa evidence that reactive oxygen species (ROS) play a sig- nificant role in the pathogenesis ofplants. The biological effects of these ROS controlled by antioxidant mechanisms. Over- whelming production of free radicals or deficiencies in the anti- oxidant defences may lead to pathological process.

The production and the activity of ROS as un universal phe- nornenon far plant and anirnals as phylogenetically distant organisrns and their role in the development of diseases as factors of pathological processes and irnmune response are discussed in the present paper.

Key words: reactive oxygen species, antioxidant de/ense, pathological processes

Received Revised Accepted

15.06.1999 15.09.1999 15.09.1999

What are the reactive oxygen species and how are they formed?

The terms ⁰activated oxygen", "reactive" or "acti.vat- ed oxygen species", "reactive oxygen intermediates", define various short-living, interconvertible highly reactive oxygen species, which appear as a resul! of electron excitation or redox processes. The par- ticular reactive oxygen species (ROS) - superoxide anion, hydroxyl and hydroperoxyl radicals, singlet oxygen and the more stable hydrogen peroxide -

Reaktif Oksijen Türleri : Genel Öldürücüler?

Özet : Serbest radikaller dış orbitalinde eşlenmemiş elek- tron içeren, oldukça reaktif türlerdir. Oksijen türevli serbest radikaller organizmadaki fizyolojik ve patolojik re- aksiyonlarda ortaya çıkarlar. Diğer taraftan insan ve hay- vanlarda, romatoid artrit, hemoraji ve AIDS gibi birçok has-

talığın gelişimine katılırlar. Reaktif oksijen türleri (ROT) bitkilerin patojenezinde de önemli rol oynarlar. ROT'nin bi- yolojik etkileri antioksidan mekanivnalarla kontrol edilir.

Serbest radikallerin aşırı üretimi veya antioksidan sa- vunmadaki yetersizlikler patolojik ilerlemeye neden olabilir.

Bu derlemede filogenetik olarak farklı olan bitki ve hay- vanlar için evrensel bir olay olarak ROT'nin üretimi ve ak- tivitesi, patolojik işlemlerin ve immün ^yanıtın faktörleri ola- rak hastalıkların gelişmesindeki rolleri tartışılmaktadır.

Anahtar kelimeler: Reaktif oksijen türleri, Antioksidan savunma, Patolojik ilerleme

and the by-products generated from the oxygen me- tabolism (Fig. 1), although they differ in their re- activity and half-life, are not usually considered sep- arately in biological systems because of their in- stability and easy conversion, and the technical dif- ficulties concerning their detection (1-3). Reactive molecules such as hydrogen peroxide and singlet oxygen are not free radicals but are certainly capable of causing damage and being strong oxidizing agents !hey are often discussed on equal terms with the oxygen free radicals. When H₂0₂ reacts with the

* Department of Medical Chemistry and Biochemistry, University of Medicine - Vama, 9002, Bulgaria.

**

°

lvanova, Yankova, Bekyarova

ıo2 ^singlet

oxygen

H202 OH'

--c;J

--

--

ıt ıt ıt

o ·-

superoxide anion-radical

hydroperoxyl oxyle anion

ani on radical

tı

02-

NO' nitric

oxide

--

Figure 1. Schematic diagram illustrating ROS: oxygen radicals and nonradicals that easily convert one into another and are active as oxidizing agents (based on 2, with permission).

superoxide anion radical (02-) it forms the extreme- ly destructive hydroxyl radicals (OH•) that attack the cellular content.

Formation and utilization of ROS lake place in parallel in biological systerns of different level of organization, i.e. even in oıganisms distant in evolutionary aspects, microorganisrns, plants, anirnals and rnan. Production of ROS depends on changes in environment and the on- togenetic slage of development of the organism. What is more, it may change in accordance to the particular physiological slate of a given organism. However it is not completely clear yet in which reactions, when and where in the organism ROS appear. Are ROS com- ponents of the normal melabolism or is their presence generally related to stress and various pathological slates? Which are the target cells of their action, foreign or those of the oıganism? Which are the detailed mech- anisrns of their interference into the cell melabolism?

The toxicity of the intermediates and products of oxy- gen metabolism poses a conslant threat to the cell or- ganelles, such as mitochondria, nuclei, chloroplasts, per- oxisomes or the cell membrane structures, but is it right to always give them the role of "cell killers"?

ROS appear in photoreactions, asa result of interactions

with protons, transient metals, other radicals and single electron transfer. At present it is stili not possible to give a detailed description of ROS production within the cell.

In some cases enzymes such as superoxide dismulase (SOD) known for their antioxidant activity in the ab- sence of peroxidase appear as sources of H 20 2 (4). it seerns feasible, however, to group the huge body of dala that has accurnulated on model systerns, on the ha- sis of the localization of ROS production. A great num- ber of systerns located at different cell compartments or extracellularly, in pa;allel to their other functions, ap- pear to be sources of ROS (fable 1) (4-8). Obviously, the bioproduction of ROS is not an evolutionary new phe- nomenon and is typical for phylogenetically distant or- ganisrns, prokaryotes and eucaryotes; bacteria, fungi, plants, animals and man. ROS can be produced both in the cell and in the extracellular space. They are derived from normal physiological and melabolic processes that are essential to the organism. Their in vivo generation is

· known to depend on the presence of oxygen and its concentration, the availability of transient metals and the level of reductants and antioxidants. However, the exact nature and even sites for production have yet to be eslablished and it is stili not clear whether the mech- anism of their action in various organisrns and cells is as universal as their production is.

FABAD J. Pharm. Sci .. 24, 171-179, 1999

•

Table 1. Normal produetion of RüS extraeellularly and in the cells of different organisms

Plants Animals, man

1. Cytosol: by enzymes and in nonenzymie redox 1. Cytosol: eiızyme activities (xanthine oxidase, systems with the participation of transition met- other oxidases) and in nonenzymie redox sys-

als tems with the partieipation of transient metals

2. Mitoehondria: respiratory ehain, superoxide dis- 2. Mitoehondria: respiratory ehain, superoxide dis-

rnutase mutase

3. Endoplasmie retieulum: eytoehrome e, li- 3. Endoplasmie retieulum: eytoehrome b5, ey- poxigenase, NAD(P) oxidase tochrome P, ""

4. Peroxisomes, glyoxisomes: glyeolate oxidase, 4. Peroxisornes: peroxidase, aerobic de- xanthine oxidase, urate oxidase, cytochrome b_5, hydrogenases

eytoehrome e reductase, NADPH oxidase, su- peroxide dismutase

5. Chloroplasts: photosensibilization of pigments, 5. Melanosomes: synthesis of melanins photooxidation of water, oxyıı;enases

6. Nuclear membranes: ? ^. 6. Nuclear membranes: eytoehrome b5, eytoehrome P.oo

7. Plasmalemma: NAD(P)H oxidase, peroxidase, li- 7. Plasmalemma: NADPH oxidase, Nü synthase,

poxigenase lipoxigenase

Cell wall: peroxidase oxidation of polyphenols, lignifieation

8. Extraeellularly, in vaeuoles ete.: as by-produets 8. Extraeellularly: in the thyroglobuline - T _3, T ₄ of ethylene produetion, processes of auto- produetion

oxidation (polyphenols, aglieones, legh- emoglobin) or photosensibilization (hyperieine, alkaloids ete.)

Microorganisms: in photoreaetions with the participation of pigments, toxins ( cercosporine, ela- dochromes ete.).

Involvement of ROS in physiological processes There are few <lata directly demonstrating how RüS interfere and partieipate in the metabolism:

1. it is eonsidered that they may play the role of en- dogenous seeondary messengers in the signal trans- duetion and neurotransmission (4, 9-16):

* Skeletal muscle myoeytes produee oxygen rad- icals, nitrie oxide, and a variety of redox-aetive de- rivatives that modulate muscle function under physiologieal eonditions. In unfatigued muscle RüS and Nü exert opposing effeets on excitation - eon- traction eoupling.

* Nü appears to be important in neural bron- ehodilator and vasodilator meehanisms, in the regu- lation of airway and pulmonary blood flow.

* Nü and Cü may modulate enzyme aetivities: they

are activators of soluble guanylyl eyclase, thus regu- lating, via the inerease in eyclie guanosine 3'5'- monophosphate, a great variety of proeesses in tar- get eells.

* There is evidenee for direet oxidative modifieation of growth-signal transduction proteins sueh as re- eeptors, protein kinases, protein phosphatases and transeription factors by H₂ü₂ or ü_2-, or altemative- ly, H₂ü₂ may modulate the redox state and activity of these important signal transduetion proteins through ehanges in eellular levels of redueed and oxidized glutathione.

* Some proteins may be "marked" for proteolysis by oxidative steps, including interaetion with RüS (16).

* in the eourse of the RüS-mediated oxidation of fatty aeids in planı eells a number. of physiologieally aetive eompounds appear: growth aetivators and in- hibitors, regulators of metabolie pathways, sub-

Ivaııova, Yankova, Bekyarova

stances possessing a bacteriostatic and/ or fungista- tic activity ete.

2. ROS appear during biosynthesis of sorne bio- logically active substances (2, 6, 10):

* They are involved in the biosynthesis of rnelanin in the rnelanocytes.

* Extracellularly - in the thyroglobulin - T 3, T ₄ pro- duction

* in plants ROS appear as a consequence of photo- sensibilization of different biornolecules, shown to occur under physiological conditions in connection with their allelopathic and detergent properties and a role of phytoalexins, in the process of photosyn- thesis.

3. ROS are the factors responsible far the invasion of sorne pathogenic rnicroorganisrns (e.g. necrotrophic fungi) into the host cells (2).

Probably this is not ali that ROS do in a normal healthy organisrn. Much is stili unknown and we of- ten only have inforrnation on what kind of pro-

cesses ROS appear in but not what the rneaning of this is. Maybe we shall have other irnportant aspects of ROS physiological activities described in the near future.

What ^is an antioxidant systern and how does it func- tion in aerobic organisrns?

Every aerobic organisrn with a rnetabolisrn that in- volves the production of such deleterious species as ROS is supplied with a systern far self-protection against their harrnful effects (2). A disturbance in ^fa- vour of the production of ROS and disfavouring their inactivation, known as an "oxidative stress", potentially leads to darnage. Antioxidant defense strategies of the celi and the organisrn are organized at rnultiple levels and include prevention, inter- ception, repair and adaptive responses. Ali those forrns of protection are realized in biology and there are rnany exarnples exploring the different strategies of antioxidant defense in different biological sys- terns (2, 17-28) (Table 2). Generally, ^it is considered that the diversity and localization of antioxidants ("any substance that, when present at low concentra- tions cornparable to that of an oxidizable substrate, Table 2. Antioxidant defense strategies ata cellular or organisrnal !eve! (rnodified frorn 17).

Level of antioxidant delense Examples

Prevention: protection against or decreasing !he rate ol formation of ROS (2. 19- - group protection by enzymes that reduce oxidative stress (catalase in f. ^cofı)

21) - prevenlion of cells against incident radiation (specialized pigments: melanins.

caralinoids)

- control of the level of ROS in lhe cells and body fluids with the participatıon of dilferent enzymes (e.g. glutathione -S-transferases)

- compartmentalization of processes and enzyme activities, inc!uding three- dimensional structure of enzymes (e.g, cytochrome oxidase does not release superoxide or other radicals, even though it contains iron and copper ions) - control of potential radical-generating reactions by metal-binding proteins (fer-

ritin, transferrin, ceruloplasmin)

- control of enzyme activities: NADPH oxidase, NO synthase ete.

lnterception: interceplion of damaging species, once formed (18. 22-24) - nonenzymic {ascorbate, a-locopherol, ~-caroteiıe, glutathione, urate, bi\irubin.

o/cop ene. tlavonoids, ete.)

- enzymatic ( superoxide dismutases, glutathione peroxidases, catalase): an- cillary enzymes (conjugation enzymes, enzymes maintaining glutathione levels and NADPH supply; systems lor export of oxidized glutalhione and glutathıone-

S-conjugates) Repair: repair or eliminalion of macromolecules already damaged by ROS (25) - ONA repair systems

- oxidized protein turnover

- oxidized phospholipid tumover

Adaptation: adaptive responses at cellular ar organismal level to oxidative stress - adaptation to lethal effect of oxidants by induced expression of protective slress

(26-28) genes under the contro! of regulons (e.g. oxyR. soxR) in procaryotes

- production of stress proteins (e.g. heme oxygenase) in response to oxidative stress and adaptive responses at the level of gene regulation (e.g. antioxidant responsive element present in the genes of enzymes related to antioxidant de- lense)

FABAD J. Pharm. Sci., 24, 171-179, 1999

significantly delays or inhibits oxidation of that sub- strate") {29) match these of prooxidants (any com- pound, whether of xenobiotic origin or an inter- mediate of the metabolism or enzyme activity that may exert oxidant activity or may be activated to generate oxidizing agents within the organism or the celi) in the healthy organism.

Under pathological conditions that delicate balance may be shifted towards the oxidative side with the end result being uncontrolled and potentially lethal damage.

Involvement of ROS in pathological processes There is evidence of an increase in the intracellular and, in some cases, extracellular concentrations of ROS as a consequence either of an altered redox status or of activation of metabolite pathways re- Table 3. ROS production in the course of pathologies

sulting in an enhanced production of different ROS.

ROS production increases occasionally in response to exposure to ionizing radiation, UV light (2), en- vironmental pollution {32, 33, 36), cigarette smoke {35), hyperoxia ete. {Table 3. !) (30-33, 36, 37), dur- ing aging (12), excess exercise (37, 38) and also as a part of an inflammatory reaction (24, 39-41) (Table 3.

il). Furthermore, it is known that, being mediators of inflammation, ROS may even modulate the im- mune response. They have been incriminated in the mechanisms of different pathological processes and over 100 human and animal diseases {Table 3. il), such as atherosclerosis ( 42-44), hypertension ( 45), reperfusion injury (46-48), shock (49-52), retinopathy {53), AIDS (54, 55), diabetes (56, 57), cancer (35, 36, 58, 59), rheumatoid arthritis (60), Parkinson's dis- ease (61, 62), schizophrenia {63, 64) and many others (9, 29, 65-70). Also, although the neutrophil has been

!. ln pathological processes caused by exogenous factors of abiotic nature

Plants Man

- Disbalanced mineral nutrition, excess of transient met- - lonizing radiation, UY light

als inclusive - Transient metals

- NaCl salinity

-

- ^Anoxia

-

-

-

- Mechanical injury

-

- Air pollutants:

so,,

oxiacetylnitrates - ^Others

- Application of herbicides, fungicides ete.

- Thermal stress

-

!!. In pathological processes caused by endogenous factors or by pathogenic organisms

Plants _Man

- Senescence and aging - Excess exercises

- Irnmune reactions: resistance (necrosis)

-

- Infection caused by:

-

-viruses - Apoptosis (?). Necrosis

-bacteria

-

-fungi - Atherosclerosis

-insects - Hypertension

(reactions of susceptibility)

-

- Others reanimation complications ete.)

- Diseases as:

-Septic shock -Retinopathia -AIDS -Diabetes -Can cer

-Rheumatoid arthritis -He~atitis

-Par inson's disease

-

Ivanova/ Yankova/ Bekyarova

the prototypical source of ROS, multiple other cells have been identified as capable of producing ROS in an organism subjected to stresses of abiotic nature or in the course of disease processes caused by en- dogenous factors or pathogens. There is dala show- ing that ROS can activate programmed celi death, although it is not yet clear whether they are actually necessary far the signaling of apoptosis (4, 11, 71- 73).

The production of ROS, particularly H₂0_2, appears to be an ubiqutous defense mechanism of eu- caryotes. This oxidative burst differs from the pro- duction of free radicals !hat occurs as a result of side reactions of metabolism and electron leakage, in that it is deliberately induced as a defense mech- anism far killing and destruction. Under pathogenic conditions such as environmental stress, or fol-

· 1awing plan! - pathogen interactions, ROS are pro- duced by plant cells (74, 75). The oxidative burst ex- hibited by phagocytes is lethal to the engu!fed pathogen but not ta !he celi. In contrast, successful resistance to pathogens in plants is very often man- ifested as hypersensitive celi death of a small group of plan! cells at the site of the interaction with the microbial pathogen. ^It is thought !hat ROS have a complex role in this response: ROS act as direct tox- ins; ta a great extent ROS are responsible far the hy- persensitive reaction at the site of penetration fal- lowed by the immobilization of the pathogen and its subsequent exposure to a battery of defensive molecules (phytoalexines, hydrolytic enzymes ete.);

ROS are involved in the construction of barriers by cross-linking of extracellular components. There- fare, the resistance of the cultivar or the species to a given pathogen is ta some extent in correlation with the induced ROS production in the plan!.

ROS may initiate pathologic processes

ünce ROS are produced, their concentrations may reach cytotoxic levels and !hey may thus be in- volved in pathologic processes (8, 11, 76-78). They maydamage:

• DNA (base alterations and strand breaks, ox-

idative changes in the deoxyribose residue ( genetic changes ( carcinogenesis);

• proteins ( oxidation of aminoacids, fragmentation, farmation of cross-linkages and aggregation, con- farmational a!terations (proteins become susceptible to proteolytic degradation, affected membrane transporters, channel proteins, receptors and regu- latory proteins, immunomodulators ete.);

• carbohydrate compounds ( e.g. oxidation of mono- saccharides);

* lipids (peroxidation ^-> membrane damage ^-> al- tered fluidity / function relationships, intracellular messaging ete.).

Their presence in the celi is fallowed by the genera- . tion of new activated molecules and radicals which

can now interact with other molecules in chain re- actions, thus being a reason far the collapse of the celi.

However this cytotoxic effect has a double meaning.

First, when generated as a result of the neutrophil activation ⁱⁿ man and animals or ⁱⁿ the processes such as phenol peroxidation or ethylene production in plants, ROS are active against alien to the organ- ism cells and may be considered an element of the immune response. In the reactions of hyper- sensitivity and necrosis ROS are active with regard to the plant's own cells but again this activity may be considered to be a part of the immune response.

And second, when generated, far example, as a product of a microbial pathogen's activity (by its pigments and toxins ), ROS appear rather to be a tool of aggression, than of defense. In such cases, when produced as by-products in disease processes, ROS may cause extra damage to the cells and thus con- tribute to their death, showing themselves to be ^fac~

tors in the pathogenesis of various diseases. In this sense maybe we have the right to cali !hem "!he bad celi killers". But in the sense of their antimicrobial, tumoricidal and immunological activity they rather should be called "!he good", but... nevertheless "celi killers"!

FABAD J. Pharm. Sci., 24, 171-179, 1999

CONCLUSION

It has been shown that ROS may be deleterious and toxic or harmless and even may have a key role in many physiological events. For each of these there is experimental support. Although it is stil! not pos- sible at present to give an estimate of their relative contributions to the overall processes in the celi and to the organism as a whole, the fallowing conclu- sions could be drawn:

l. The ROS production is an universal phenomenon far phylogenetically distant organisms: micro- organisms, plants, anirnals and man.

2. Oxygen activation may potentially occur in ali compartments of the living celi and extracellularly.

3. ROS are necessary far normal celi functioning.

4. In different organisms (plants, animals, man) an increased production of ROS is detected in the course of pathologies caused by factors of different nature (abiotic, endogenous or pathogens).

5. Antioxidant systems organized at multiple levels protect the cells and the organisms from the harm- ful effects of ROS.

6. The mechanisms of celi damage caused by the in- creased production of ROS are similar far planı and animal organisms: ROS oxidize proteins, un- saturated fatty acids, carbohydrates and DNA.

7. At the same time ROS appear to be a means of de- fense via the cytotoxic effect they have on the organ- ism's own altered or alien cells.

We still continue asking ourselves is the appearance of ROS in the organism programmed and which ROS production should be considered normal and which increased?

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"SYMPOSIUM ON LIPID AND SURFACTANT DISPERSED SYSTEMS FUNDAMENTALS, DESIGN, FORMULATION,

PRODUCTION" TOPLANTISININ ARDINDAN

Faculty of Chemistry Lomonosov Moscow State University, APGI (Association de Pharmacie Galenique Industrielle) ve Royal Pharmaceutical Society of Great Britain tarafından ortaklaşa

düzenlenen bu simpozyum, 26-28 Eylül 1999 tarihinde Moskova'da yapılmışhr. Simpozyuma,

yaklaşık 25 ülkeden 150 kadar araşhrmacı katılmışhr.

Bu simpozyumun konusu ve ^amacı, lipid ve surfaktan içeren dispers sistemlerin ^dayandığı

esasları, tasarım, formülasyon ve üretimleri üzerine son yıllarda yapılan araşhrma ve geliştirmeleri

bilimsel ve uluslararası bir platformda birbirine duyurmak ve birlikte ^paylaşmak olarak ifade edilebilir. Dispers sistemler, özellikle kollodial sistemler, miseler sistemler, jel sistemler, ^sıvı kristaller, emülsiyonlar, makroemülsiyonlar, mikroemülsiyonlar, çoklu emülsiyonlar, lipozomlar, niozomlar, mikrokapsüller ve nanokapsüller üzerine 29 sözlü ve 92 poster tebliğin sunulduğu üç gün süren bu simpozyumda en son yenilikler

görüşülmüş, tartışılmış ve paylaşılmıştır.

Doç. Dr. Nurşin GÖNÜL AÜ. Eczacılık Fakültesi

Farınasötik Teknoloji Anabilim Dalı