Oxidative Stress and Opioids

In recent years, research has shown the involvement of free radicals in the development of the pain that accompanies many pathological conditions. In the treatment of acute and chronic pain, the most effective therapies are natural and synthetic opioid alkaloids. Their metabolism in itself may contribute to the formation of free radicals and thus affect body system load and the perception of pain. Long-term treatment with opioids is a tool of choice for the treatment of medium and severe pain. Opioids stimulate the effect of endogenous opioids, endorphins, by binding to multiple subtypes of opioid receptors (μ,κ,σ) in spinal, supraspinal and peripheral tissues. Morphine is a typical, natural opioid analgesic utilised in practice in the treatment of severe chronic pain. In addition, similar effects can be expected from semisynthetic opioids such as oxycodone and hydromorphone. However, during treatment with opioids some adverse effects can appear regardless of whether treatment is short-term or long-term. One potentially serious side effect is the induction of oxidative stress. The purpose of this present work is to determine the main sources of reactive oxygen and nitrogen in the development of infl ammatory and neuropathic pain, and the manner in which metabolism of morphine contributes to oxidative stress alone. Review Article Oxidative Stress and Opioids Janka Vašková1*, Ladislav Kočan2 and Ladislav Vaško1 1Department of Medical and Clinical Biochemistry, Faculty of Medicine, Pavol Jozef Šafárik University in Košice, Trieda SNP 1, 040 66 Košice, Slovak Republic 2Clinic of Anaesthesiology and Intensive Care Medicine, East Slovak Institute of Cardiovascular Disease, Ondavská 8, 040 11 Košice, Slovak Republic Dates: Received: 13 December, 2016; Accepted: 27 December, 2016; Published: 30 December, 2016 *Corresponding author: Dr. Janka Vašková, PhD, Associate professor, Department of Medical and Clinical Biochemistry, Faculty of Medicine, Pavol Jozef Šafárik University in Košice, Trieda SNP 1, 040 66 Košice, Slovak Republic, E-mail:


Introduction
Chronic pain is currently one of the major problems and, as such, has lost its defensive physiological importance. Its intensity, duration, and nature do not match the extent of tissue damage. It distorts the psyche of the individual, behaviour, quality of life, undermines the family, social life and, ultimately, presents a socio-economic burden to society. This problem has been so severe, extensive and complex that it required the creation of a new department, known as algesiology. Chronic pain requires a multidisciplinary approach. Examples include non-pharmacological methods -physical therapy, physical therapy and rehabilitation, acupuncture, psychotherapy,

Oxidative stress and pain
Reactive oxygen species (ROS) play an important role in the pathogenesis of disease states [1,2]. Disease-state is characterized by altered metabolic conditions of cells, increased production of ROS, which themselves act as mediators, and other Citation: Vašková  mediators of infl ammation mediating pain expression. ROS are signifi cantly involved in the formation and maintenance of neuropathic pain, which was confi rmed by the alleviation of neuropathic pain following administration of non-toxic doses of free oxygen radicals' scavengers [3]. Free radicals are atoms or molecules containing one or more unpaired electron. However, several reactive intermediates are formed from oxygen, not all of them radicals. Therefore, radical and non-radical derivatives of oxygen, including the excited state, and other oxidizing agents of which are easily formed radicals are collectively referred to as the reactive oxygen species (ROS). Due to the many physiological functions in organisms, ROS are normally produced continuously in controllable and uncontrollable processes. Organelles, enzymes and molecules subject to auto-oxidation are all endogenous sources of ROS.
The most productive regulated sources include the activities of nitric oxide synthase (NOS), enzymes of NADPH-enzyme family (NOX), lipoxygenase and cyclooxygenase (LOX and COX) as well as the non-adjustable processes of the respiratory chain in mitochondria: xanthine oxidase (XO), autooxidation of dopamine, and photosensitization [4]. Among these are a few fundamental sources directly associated with disease states.
Interconnection between ROS, ATP production in mitochondria and pain: Firstly, we can mention the mitochondrial respiratory chain. Following oxidation of the substrates in the Krebs cycle come electron carriers NADH and FADH 2 oxidised at complexes I and II. Reduction equivalents of the released electrons are then transferred through the respiratory chain complex III, and IV by the prosthetic groups arranged according to increasing redox potential to the terminal electron acceptor, which is oxygen. Because of the structure of an oxygen atom, 4 electrons should be simultaneously transferred to the oxygen molecule to allow its reduction to H 2 O. The introduction of one electron to an oxygen atom causes its univalent reduction and formation of reactive intermediates. The biggest producers of ROS are complex I and III. Maintaining effi cient electron transport to O 2 is a potential mechanism for control of the production of ROS.
Regulation of this situation is particularly important in sites of infl ammation and triggered immune response. There is, in fact, a signifi cant shift in metabolic activity resulting from a profound recruitment of infl ammatory cells (neutrophils and monocytes) and high proliferation rates among lymphocyte populations. The resultant shifts in energy supply and demand (e.g., glucose, oxygen, ATP) can result in metabolic acidosis and diminished delivery and/or availability of oxygen. This can lead to hypoxia extensive enough to trigger transcriptional and translation changes in tissue phenotype [5]. One possible regulatory mechanism is the effect of hexokinases I and II of the ADP:ATP complex (voltage-dependent anion channel). Part of ATP is exported immediately for glucose phosphorylation from mitochondria through the effect of hexokinases. This then enters glycolysis on the mitochondrial surface to form ADP in reverse. Mitochondrial creatine kinase works similarly to the continuous formation of ADP so that the respiratory chain complexes work constantly dragging electrons to complex IV, keeping ATP synthase active and membrane potential low. Inducibility of proton leakage is the major mechanism of maintaining membrane potential and thus controlling the release of ROS. There is therefore a slight loss of energy (as heat) from the formation of ATP, which can prevent excessive supply of mitochondria by electron/reducing equivalents for the respiratory chain complexes and to minimize the likelihood of interaction of electrons with O 2 and ROS formation. Voltagedependent anion channels and uncoupling protein UCP1 -5 (family of mitochondrial anion carriers) permit the escape of protons. However, the ability to catalyse the release of protons which reduce production of ROS was detected only in UCP2 and UCP3. Activation or inhibition of UCPs is controlled by allosteric regulators, which include fatty acids, nucleotides and retinoic acid. Some derivatives of ROS such as O 2 •and 4-hydroxynonenal are able to activate UCP2 mediated release of protons. So the overproduction of ROS itself in mitochondria leads to a feedback regulation [6].
In response to oxidative stress and infl ammatory conditions, however, the formation and use of energy is important, particularly for phagocytic cells of the immune system that control the resolution of transient infl ammatory pain [7]. Cells of myeloid lineages derive their energy almost exclusively from glycolysis, whereas lymphocytes predominantly use oxidative phosphorylation [8,9]. As opposed to lymphocytes that proliferate within tissues, myeloid cells, such as polymorphonuclear leukocytes (neutrophils), macrophages, and dendritic cells, are recruited to sites of infl ammation during immune responses. In transit, these cells expend tremendous amounts of energy. Cell migration requires large amounts of actin turnover, which is particularly demanding on ATP [10]. Energy demands of phagocyting cells increase at sites of infl ammation. Predominantly using glycolysis for obtaining energy allows them to function at low oxygen concentration and even anoxic conditions associated with deep infl ammation. To cover increased energy demands, a glycerol-3-phosphate shuttle is used [9]. Lymphocytes (B and T) use glucose, lipids, and amino acids as energy sources in oxidative phosphorylation. During proliferation, lymphocytes become more glucose dependent, resulting in a nearly 20-fold increase in glucose uptake. This is accomplished by high expression of GLUT-1 [11] tightly controlled by hypoxia-inducible factor, and lactate production can increase 40-fold [12]. In naive T-cells the nutrient uptake is controlled through cytokines (IL-7 and IL-4) [13].
Produced ATP plays an important role in extracellular signalling reactions in infl ammation and immune responses.
Extracellular ATP (or ADP) can directly bind to cell surface purinergic receptors (P2-type) or can be metabolized to adenosine at the cell surface, where it is made available to bind and activate adenosine receptor(s) (P1-type) [5].
The data available would tend to suggest a role for COX-1 in acute nociception, with inducible COX-2 mechanisms becoming prevalent in more pathological conditions [17]. The synthesis thereof is induced by cytokines and oxidative stress. COX-1 is inducible under infl ammatory conditions in the kidney [18], and COX-2 is constitutively expressed in CNS, kidney and blood vessels [19]. As the major inducible isoform, COX-2 is strongly upregulated during infl ammation by the products of tissue damage and infl ammation such as pro-infl ammatory cytokines (IL-1beta, IL-15, TNF-), and lipopolysaccharides. Indeed, IL-1beta is thought to be one of the major inducers of COX-2 in the CNS under infl ammatory conditions [16]. Induction of COX-2 via the MAPK pathway has also been suggested, together with the involvement of peroxynitrite [17]. Interestingly, NO at higher concentrations can suppress induction of COX-2 [20] as can glucocorticoids in the spinal cord [21].
The mechanism of nociception induction is attributed to the mediation of diverse activities through PGE 2 binding to P (1-4) receptors coupled to different signal transduction pathways. While the P1 receptor is coupled to intracellular Ca 2+ mobilisation, P2 and P4 are coupled to the stimulation of adenylate cyclase. The P2 receptor is also implicated in pain. Harvey et al. [22] hypothesise that activation of the P2 receptor inhibits, by phosphorylation, the glycine receptor (GlyR3), which thus mediates spinal hyperexcitability. The role of P3 and P4 receptors in pain remains to be more clearly explained, as EP3 receptor knockout mice do show reduction in pain behaviours [23]. Conversely, activation of the P4 receptor appears to have an inhibitory function; P4 receptor agonists mediate an inhibition in hyperalgesic scores in an infl ammatory pain model [24]. PGE 2 is also able to increase the sensitivity of certain sodium channels, notably those resistant to the effects of tetrodotoxin (TTX). In addition, PGE 2 can modulate the activity of other ion channels, and e.g. inhibits a Ca 2+ -dependent K+ current, which mediates slow afterhyperpolarisation [17].
In contrast to COX, LOX isoforms also oxidise other lipids, including membrane phospholipids [25]. Oxidized lipids lose their stereospecifi city. The oxidation products can include isoprostanes, cholesteryl hydroperoxides, hydroperoxides, epoxides, hydroxycholesterol derivatives, and reactive aldehydes, which enhance the conditions of oxidative stress.
Moreover, arachidonic acid can undergo non-enzymatic (free-radical mediated) oxidation to generate prostaglandin-like products [26]. Oxidatively damaged lipoproteins that can cross-react with antibodies were generated via PGE2 and PGF2 [27]. This fi nding adds to the speculation that oxidativelymodifi ed lipoproteins can mimic the effects of prostaglandin.
In parallel reactions with the particles formed in other systems and from NOS activity, the oxidants are formed, and either will form other reactive intermediates in reaction with uric acid, causing oxidative tissue damage [32]. The released NO performs two functions in the brain, both as an atypical neurotransmitter and in cell defence, due to its cytotoxic properties. In the peripheral nervous system, NO is a major neurotransmitter in the non-adrenergic and noncholinergic neurons (mediating smooth muscle relaxation, e.g., in the gastrointestinal tract).Excessive production under pathological conditions may not only be involved in tissue damage, but can directly activate sensitive fi bres in the CNS followed by the release of Calcitonin gene-related peptide (CGRP), thereby extending the burning pain [3,33]. In the absence of haem, NOS function as monomers [35].
However, monomers of all isoforms are incapable of binding tetrahydrobiopterine (BH4) without haem and do not catalyse the production of NO [35].
In the absence of BH4, nNOS and eNOS may form stable dimers which catalyse the simultaneous production of NO and O 2 •- [36,37], immediately resulting in ONO 2 release. NOS only works as NO synthase at very high (up to 1 uM) concentrations of BH4 [38]. Under conditions of oxidative stress, however, it can very easily become scarce, as it acts as an effective radical scavenger. Its availability therefore depends on the suffi ciency of ascorbate and glutathione [39]. It has been shown that uncoupled nNOS reaction is relevant in induced neuronal toxicity [41]. On the other hand, uncoupled iNOS is often present in a state where there is a lack of L-arginine, such as in wound repair. The eNOS uncoupled reaction is often due to lack of BH4 but not L-arginine [42].

Opioid receptors
Based on their affi nity and intrinsic activity with the tract: application of an opioid antagonist methylnaltrexone, which does not cross the blood brain barrier, or use of naloxone, which is metabolized by "fi rst-pass" metabolism in the liver, is an example of a combined preparation oxycodone/naloxone) [44]. Also disorders of parenchymatous organs especially the liver in relation to bottlenecks caused by biliary excretion of biliary tract spasm are known. Skin problems caused by the effects of opioids include urticaria, dermatitis, and pruritus.
Renal and urinary disorders may develop and occur as urinary retention and ureteral spasm. Rarely, disorders of the immune system appear, which may lead to anaphylactic shock through the development of hypersensitivity [44].
Opioids have a negative effect on the endocrine system. and related drugs [46].

Oxidative stress and opioids
Several studies show a correlation between the occurrence of oxidative stress and its subsequent complications, in treatment with opioids. The drug most suspected of exhibiting such side effects appears to be morphine. This effect is also likely from the semi-synthetic molecules derived from morphine, altered during degradation in the body to morphine or its metabolites.
Other opioid studies have been suffi ciently undertaken focussed on the excessive production of free radicals.
In principle, the production of reactive oxygen and nitrogen species in connection with long-term use of morphine can proceed in two possible pathways. The fi rst are reactions following the activation of nitric oxide synthase (NOS); with regards this possibility, attention must be paid to the above points. The second pathway is the activation of the enzyme phospholipase A 2 (PLA 2 ). PLA 2 resides in the cytosol, translocates and binds to heparan sulphate proteoglycans on cell membranes in response to an increase in intracellular calcium [47,48]. With the proximity of membrane phospholipids, and prostanoid synthesis enzymes (COX and 5-LOX), arachidonic acid is released to couple COX [49]. PLA 2 is also internalized by neurons and contributes to increased eicosanoid synthesis and infl ammatory pain [50]. However, during transactivation of iNOS involving NFB, released NO interacts with COX-2 and infl uences prostanoid synthesis [51]. In order that that This phenomenon can be explained by reduced nitric oxide concentration, followed by impaired vasodilation, and the subsequent development of atherosclerosis [54]. An increase in the concentration of hydrogen peroxide was observed in the prefrontal cortex and the striatum in rats administered with morphine. Increased concentrations of lipid peroxidation products in the hippocampus and liver were also measured [55].
Increased production of free radicals, especially superoxide, has been shown in tissue macrophages. In contrast, the results of experimental studies of human neuroblastoma cell lines SH-SY5Y have not shown prooxidative effects following morphine administration, nor accelerated the apoptosis of these cells.
This evidence suggests an increased resistance of tumour tissue to the formation of free radicals and subsequent damage [56]. At the same time, it acknowledges the importance of taking into account the specifi c metabolism in different cell types, particularly following injury or during infl ammatory processes accompanied by increased pain sensation. Tissue injury arising from ongoing exposure to high-intensity stimuli leads to a sensation of pain continuing beyond the removal of the originating stimulus. There is, in addition, an enhanced sensitivity to otherwise modestly aversive stimuli applied to the injured tissue (hyperalgesia) [57]. The high production of reactive oxygen and nitrogen species is then also the base for opioid tolerance development. These fi ndings were confi rmed through inhibition of NO and superoxide synthesis [58]. In the case of generated morphine metabolites M-3-G and M-6-G, their biological activity increases [62].
UGTs are expressed in different tissues (including brain, prostate, uterus, placenta, kidney etc.) but the main location of glucuronidation is the liver. UGT genes form on the basis of sequence similarities the two gene families UGT1 and UGT2. Let us return to the more specifi c metabolism of morphine, which is up to 90% (ie usually) metabolised by UGT2B7.
The study of Holthe et al. [68] noted that while the *2 allele Citation: Vašková  For example, in cancers, glucuronic acid is produced more from glycogen or glycogenic amino acids in the liver than from glucose. Therefore, the compounds to be metabolized by UGT intermediates. The mechanism of oxidation of substances is explained by the mechanism of cytochrome P450 [74]. The fact that NOS metabolises and affects the effi ciency and tolerance to morphine has also been suggested by some studies. For example, through the effects of arginine as described by Karami et al., Barghava et al. [75,76], found that the anti-nociceptive effect of morphine as well as its concentration reduced the administration of L-arginine. In contrast, D-arginine administration, did not show this effect. This clearly indicates a signifi cant effect of NOS on the metabolism, the effectiveness of morphine and the generation of reactive species, as NOS has a limited substrate specifi city and is unable to use D-arginine for NO synthesis [74].

Conclusion
The fact that oxidative stress plays a key role in the pathogenesis of many diseases is a generally accepted conclusion chronic pain treated with opioids for severe pain, we are performing a prospective observational multicenter study the results of which will be the subject of experimental study.