Free Rad. Res., Vol. 35, pp. 215-231 Reprints available di~y from the publisher Photocopying permitted by license only © 2001 OPA (Overseas Publishers Association) N.V. Published by license under the Harwood Academic Publishers imprint, part of Gordon and Breach Publishing, a member of the Taylor & Francis Group. Invited Review The Mechanisms for Nitration and Nitrotyrosine Formation in vitro and in vivo: Impact of Diet CERI OLDREIVE and CATHERINE RICE-EVANS* Wolfson Centre for Age-Related Diseases, Guy's, King's and St. Thomas School of Biomedical Sciences, King's College London, London SE1 9RT Accepted by Prof. V. Darley-Usmar (Received 2 January 2001) The detection of 3-nitro-L-tyrosine residues associated with many disease states, including gastric cancer, has implicated a role for peroxynitrite in vivo, and thus endogenously produced nitric oxide and superoxide. Additionally, dietary nitrate has been suggested to be involved in the pathogenesis of gastric cancer through a mechanism involving reduction to nitrite and sub- sequent formation of potentially mutagenic nitroso- compounds. Studies have now demonstrated that a multitude of reactive nitrogen species other than per- oxynitrite are capable of producing nitrotyrosine. Thus, we have reviewed the evidence that dietary nitrate, amongst other reactive nitrogen species, may contribute to the body burden of nitrotyrosine. Keywords: Nitrotyrosine, nitrate, nitrite, peroxynitrite, nitric oxide, nitration, reactive nitrogen species Abbreviations: Nitrotyrosine, 3-nitro-L-tyrosine; RNS, reactive nitrogen species; BSA, bovine serum albumin; LDL, low density lipoprotein; MCP-1, monocyte chemoat~actant protein; EGC, epigaliocatechin; ECG, epicatechin gallate; EGCG, epigaliocatechin gallate; HNO2, nitrous acid; C1-NO2, nitryl chloride; SIN-l, 3-morpholino-syndominine; NO~, nitrogen dioxide; NO-, Angeli's salt; NO2BF4~ nitryl salt; iNOS, inducible nitric oxide synthase; NFL, low molecular weight neurofilament; MnSOD, managanese superoxide dismutase; SERCA, sarcoplasmic reticulum calcium ATPase; ALS, amyotrophic lateral sclerosis; SP-A, surfactant protein A; i.v., intravenous; NHPA, 3-nitro-4-hydroxyphenylacetic acid; NHPL, 3-nitro-4-hydroxyphenyllactic acid; NO{, nitrite; NO;, nitrate; N203, dinitl-ogen trioxide; NO', nitric oxide; HOC1, hypochlorous acid; EDRF, endothelium derived relaxing factor; NOS, nitric oxide synthase; eNOS, endothelial nitric oxide synthase; nNos, neuronal nitric oxide synthase; HbO2, oxyhaemoglobin; Hb 3+, methaemoglobin; NH3, ammonia; O~-, superoxide; XOD, xanthine oxidase; M~, macrophage; N, nitrogen; DNA, deoxyribonucleic acid; RNA, ribonucleic acid; HRPT, hypoxanthine-guanine phosphoribosyl transferase; DEN, diethylrtitrosamine; DEA, diethylamine INTRODUCTION The presence of 3-nitro-L-tyrosine (Figure 1) has often been considered to be a "fingerprint" of peroxynitrite formation in vivo. However, it * Corresponding author. Tel.: +44-207-848 6141. Fax: +44-207-848 6143. E-mail: catherine.rice-evans@kcl.ac.uk. 215
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`216 C. OLDREIVE AND C. RICE-EVANS COOH ~IOH "~'NH2 NH2 OH Tyrosine Nitrotyrosine FIGURE 1 The structures of tyrosine and its nitrated product, 3-nitro-L-tyrosine. has been demonstrated that, at physiological pH, a multitude of reactive nitrogen species (RNS) that may occur in vivo can nitrate both free and protein-bound tyrosine on the ortho-position in vitro (Table I). In addition, nitration of tyrosine residues by tetranitromethane has been used to elucidate the role of specific tyrosine residues in protein function. I24"25] Inhibition nitrotyrosine formation by RNS has been demonstrated in vitro by many compounds including dietary phenolics, such as the catechine polyphenols I3I and to a lesser extent hydroxycinnamates [21 (Table I). In vivo, nitrotyrosine formation has been prevented in humans by supplementation with ascorbic acid (1 g, twice daily) I261 and in a guinea pig model of ileitis by genistein. [27] The formation of nitrotyrosine has been implicated in many inflammatory conditions, neurodegenerative diseases and cancer due to elevated levels observed both in humans (Table II) and in animal models of gut inflamma- tion [27"3°] and skin cancer I6°1 amongst others. However, there are some conflicting studies in which nitrotyrosine levels do not appear to change in disease. [61'621 The nitrotyrosine detected in vivo appeared to be localised in a variety of cells: epithelial cells, [26"31'33'571 polymor- phonudear leukocytes, [261 macrophages, [29-31"5°] smooth muscle cells, [29"31] extracellular matrix, [26'57] neurons [4°'42"48'49] and microglia. [42~511 Within cells it is often located close to inducible nitric oxide synthase (ilNJOS). [29'30'51'54] Nitric oxide itself is unable to nitrate tyrosine, I6'63~s] thus indicating the involvement of higher oxides of nitrogen. Several purified proteins (Table I) have been nitrated in vitro, and some nitrated proteins have been identified in cell studies. These include prostacyclin synthase from rat mesangial ceils treated with peroxynitrite or SIN-l, [661 the focal adhesion protein p130 cas in human neuroblasto- mas SH-SY5Y cells treated with SIN-1 [67] and multiple nitrated proteins in heart homogenates treated with peroxynitrite, or a mixture of mye- loperoxidase, nitrite and hydrogen peroxide. I681 At present, however, nitrotyrosine residues have only been isolated from human serum albu- mirl, [7] human low density lipoprotein, I7'91 low molecular weight neurofilament (NFL), I621 manganese superoxide dismutase (MnSOD), [69] rabbit fl-VLDL apoproteins I7°l and murine sarcoplasmic reticulum calcium-ATPase 2a (SERCA2a). I651 Nitration of tyrosine residues in proteins in- duces the change of tyrosine into a negatively charged hydrophilic nitrotyrosine moiety that may alter enzyme activity. This could have dele- terious effects due to mitochondrial respirat- ory dysfunction, [711 impaired lung function following inactivation of surfactant protein A (SP-A), [17] disrupted microtubule organisation that leads to altered cell morphology and epithe- lial-barrier dysfunction, [721 reduced antioxidant defence due to diminished activity of superoxide dismutase, [12"64'131 impaired cellular function due to reduced GTP binding, [lsl inactivation of tyro- sine kinases resulting in decreased phosphoryla- tion [73] and thus affecting signalling cascades and inhibited neurofilament assembly weakens cell structure [74] and may reduce motor neuron survi- val. However, nitration of tyrosine may play a protective role by attenuating the thrombotic properties of tissue factor, [is] elimination of for- eign bodies engulfed by phagocytes, [751 thymo- cyte negative-selection via apoptosis [761 and targeting proteins for degradation and elimina- tion. [731 The majority of proteins appear to be unable to incorporate free nitrotyrosine upon their assembly, [72'771 the exception being a-tubu- lin that subsequently cannot function correctly. [721
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`NITROTYROSINE FORMATION AND DIETARY NITRATE/NITRITE TABLE I Reactive nitrogen species involved in tyrosine nitration in vitro 217 Nitrating species Substrate Inhibitors Peroxynitrite (OONO-) Nitrous acid (HNO2) Nitryl chloride (C1-NO2) produced from: nitrite + HOCI 3-Morpholino- syndominine (SIN-l): produces both O~- and NO" SpermineNONOate produces NO" Oxidation products of nitrite produced by: peroxide + H202 + NaNO2 Nitrogen dioxide (NO~) Angeli's salt (NO-) Nitryl salt (NO2BF4): produces NO + Tyrosine ]1-51 Bovine serum albumin (BSA) t6--8] Human serum albumin [71 t--~v\[7 910] Low density lipo~rotein ~LtaL) ' ' al-antiproteinase [7] Angiotensin 11 I111 Fibrinogen [71 Manganese superoxide dismutase tm'131 Monocyte chemoattractant Protein-1 (MCP-1) 041 Pepsinogen [7] Rac2[15l Ribonucleotide reductase 06] Surfactant protein A [17] Tissue factor 081 Tyrosine D9,2°1 BSAI8,191 Angiotensin I/011 BSA I8] LDL [m] Tyrosine [41 Bovine serum albumin [8] Surfactant protein A [21] Tyrosine [4] BSA [sl Surfactant protein A [21] Tyrosine [22] BSA [22] Tyrosine [1,231 BSA [23] Bovine eye lens c~-crystaUin [23] BSA [81 Tyrosine [22] Ascorbyl palmitate, [1] ascorbic acid, [5] caffeic acid, [2] catechin, TM chlorogenic acid, t2! m-coumaric acid, [2! o-coumaric acid, [21 p-coumaric acid, TM epicatechin, TM EGG, TM ECG, TM ferulic acid, TM gallic acid, TM EGCG, TM glutathione, Is] c~-tocopherol, TM trolox, 12"3] uric acid Isl Ascorbate, [6] uric acid, [6] sulfhydryls [61 Catechin, [2°] epicatechirt, [2°] EGC,[ 2°] ECG,[ 2°] EGCG,I 2°] ferulic acid, [2°] rulin [2°] Urate 121] Proteins capable of removing the nitro-group from nitrotyrosine in proteins, in the absence of protein degradation, have been isolated from canine prostate I781 and and there appears to b ted proteins from rat
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`218 C. OLDREIVE AND C. RICE-EVANS TABLE II Evidence of in vivo formation of nitrotyrosine, nitrate and nitrite in human disease. A selection of the diseases and the biological samples in which nitric oxide has been implicated due to the detection of elevated levels of nitrotyrosine, nitrate and nitrite Disease Site of elevated nitrotyrosine Site of elevated nitrate/nitrite Inflammation Atherosderosis Atherosclerotic lesions I9'2s'291 Atherosclerotic plaques 1~'311 Gastritis Gastric antral biopsy 1261 Neurodegenerative Gastric fundus biopsy [311 Ulcer margins 1311 Colonic mucosa |33'~1 Colonic mucosa I341 Plasma is61 Duodenal biopsy i371 Plasma 1391 Hippocampal tissue 14°-421 Cerebrospinal fluid I41"44] Cerebrospinal fluid [451 Spinal cord i48"49I Central nervous system tissue [5°1 Brain tissue [51! Subtantia nigra I4°1 Gastric ulcer Ulcerative colitis Crohn's disease Celiac disease Septic shock Alzheimer'sdisease Amyotrophic lateral sclerosis (ALS) Multiple sclerosis Parkinson'sdisease Cancer Colon cancer Tumour tissue I54"5sl Gastric cancer Gastric biopsy Is71 Gastric juice/3°1 Gastric juice 132J Urine [351 Urine [351 Plasma ~361 UrineI381 Plasma 1391 Cerebrospinal fluid 1431 Cerebrospinal fluid [46"471 Serum (461 Cerebrospinal fluid tS°l Cerebral spinal fluid 1521 NeutrophiIs [531 Plasma 1561 Serum 158] Gastric juice I591 effect of nitrotyrosine. However, nitrotyrosine can be transported into cells I721 and absorbed from the diet by rats. Isll Intravenous (i.v.) admin- istration of nitrotyrosine in rats inhibited the vasoconstrictive and hernodynamic responses to angiotensin II [82] and attenuated the hemody- namic responses produced by a- and fl-adreno- ceptor agonists (norepinephrine, epinephrine, phenylephrine and isoproterenol). [s3J It has also been demonstrated that nitrotyrosine adminis- tered by i.v. injection to rats has a half-life in the plasma of 1.67 hours Ia4l and an oral dose (100 ~g) was converted into two metabolites; 3-nitro-4- hydroxyphenylacetic acid (NHPA) and 3-nitro- 4-hydroxyphenyllactic acid (NHPL) that were excreted in the urine (44% and 5% of oral dose, respectively), tall These metabolites have been proposed as biomarkers of human exposure to nitrosating agents fall such as nitrate and nitrite. This has been further corroborated as NHPA appears to be excreted in the urine of human volunteers, with a profile similar to that of nitrate, following consumption of a nitrate-rich meal. t8sl Thus, the origin of free nitrotyrosine detected in vivo probably arises from the nitration of free tyrosine, the breakdown of nitrated proteins and dietary intake and absorption. Uncertainty still remains as to whether nitrotyrosine formation initiates the onset of disease or is a consequence of the progression of disease. NITRATE/NITRITE Nitrate (NO3) and nitrite (NO2) are naturally occurring, water soluble anions. The nitrate ion is the conjugate base of the strong acid nitric acid (pKa =-1.37). The nitrite ion is the conjugate base of the weak acid nitrous acid (pKa -- 3.37) which decomposes readily to give water and dinitrogen trioxide (N203) or nitric acid, nitric oxide (NO') and water. Salts of both acids are readily soluble in water with nitrites being much more stable than the acid itself. [861
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`NITROTYROSINE FORMATION AND DIETARY NITRATE/NITRITE 219 I. The Fate of Nitrate and Nitrite in the Human Body The fate of nitrate in the human body is repres- ented schematically in Figure 2. Ingested nitrate first encounters specialised symbiotic nitrate- reducing bacteria, that reside on the dorsal surface of the tongue and which reduce some (5%) of the nitrate to nitrite. [87-91] The nitrite thus formed is then chemically reduced to nitric oxide (NO') under the acidic conditions of the stomach (Figure 3A). [89] Nitrate is absorbed from the sto- mach into the blood stream where it rapidly enters erythrocytes. Is8] Salivary glands concen- trate and actively secrete nitrate from the circu- lation into the oral cavity, [87~s9] 25% of dietary nitrate undergoes enterosalivary recirculation in this way. [87"9°'91] The majority of nitrate (60-70%) is excreted unchanged in the urine within 24 hours of ingestion [85"87"92] and appears to be predominantly tubular. Is81 Maximal urinary excretion occurs 4-6 hours following challenge with potassium nitrate [sTl or as it occurs naturally as part of a meal. [85[ A minor amount is also excreted unchanged in the sweat [87] and faeces (0.1-0.5 %).[87,92] In radiolabel tracer studies in humans using 15N-labeled nitrate, the nitrogen from administered 15NO; was recovered as ammonia and urea in the urine (3%) and faeces (0.2%). [92] The half-life of nitrate in the body has been determined to be approximately 5 hours. [921 After administration of nitrite in meat, urin- ary excretion of nitrate but not nitrite was elevated [93] indicating that nitrite is oxidised during its passage through the body possibly in the stomach (Figure 3A). Oxidation of nitrite to nitrate can also occur via reactions with oxyhaemoglobin, [94] HOC1 [1°'95] and peroxi- dases.[22,961 II. Endogenous Sources Nitrate balance studies conducted in humans consistently conclude that a greater amount of nitrate is excreted than can be accounted for by FIGURE 2 Dietary nitrate- Green leafy vegetables Brassicas Root vegetables I #tt *--- r1111= "- Nitrite formation Olluch 4.-. 6astdclllamb -- Nitrite formation bll Blood I ; Excretion Diagrammatic scheme representing the fate of nitrate following ingestion by humans.
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`220 C. OLDREIVE AND C. RICE-EVANS A NOt" + H + .4 HNO2 3HNO2 @ H20 + 2NO" + NO3 + H + 2HNO2 ~ H20 + N203 N203 "4 NO" + NO2 B 2NO" + 02 4 2NO2 2NO" + 2NO2 "} 2N203 2N203 + 2H20 ~ 4NO2" + 4H + 2NO2 "} N204 N20, + H20 -~ NO2- + NO3- + 2H + C NO2 + I-I* ~ liNO2 HNO2 + I i + '(--¢ HzNO2 + ~ H20 + NO + 2HNO2 +-¢ HzO + NzO3 HNO-2 + HX ~ H20 + NOX FIGURE 3 The steps involved in the chemical reduction of nitrite to nitric oxide (NO') in the acidic conditions of the human stomach (A); the autoxidation of NO" to nitrite in aqueous media (B); the interconversion of various proposed nitrosating species (bold) in acidic media (C). ingestion, it therefore appears that humans can synthesise 600-2,200 )lmol nitrate per day. [92"93'971 Additionally, ceils appear to contain an NO~-H ÷- cotransporter that has been suggested to elimin- ate the products of NO" metabolism from cells. 1981 The sources of this endogenous production, namely nitric oxide and bacteria resident in the gastrointestinal tract are discussed below. Nitrogen Monoxide Nitrogen monoxide or nitric oxide (NO') is a free radical whose biological role began to emerge in the early 1980s. This small molecule produced by the endothelium, with vasorelaxant proper- ties, was named endothelium derived relaxing factor (EDRF). [99-1°1] Its production and multiple roles have since been extensively investigated. Several isoforms of nitric oxide synthase (NOS) the enzyme responsible for the production of NO" from arginine with the concomitant forma- tion of citrulline have been identified, tl°2-1°sl The main isoforms are inducible NOS (iNOS), constitutive endothelial NOS (eNOS) found in vascular endothelial cells I1°6! and constitutive neuronal NOS (nNOS) found in human brain, I1°71 skeletal muscle [1°71 and both the per- ipheral and central nervous systems. I1°6"1°81 iNOS resides in many different cell types includ- ing endothelial cells, [1°9'11°1 macrophages, tl°9-ml chondrocytes, [1121 neutrophils [26'75] and gastric mucosal cells. II131 Additionally, bacteria such as Helicobacter pylori also contain NOS [114] and nitrite can be reduced to NO" in the ischaemic heart. II15'H61 NO" serves many useful functions such as regulation of blood pressure, 0~7'118t in- hibition of platelet aggregation, II19-1211 inhibition of phagocyte adhesion to the endothelium I12°I and neurotransmission. I122-~241 It is also thought to act as an antioxidant and an anti-atherosclerotic agent due to its ability to induce glutathione synthesis [125] but too much NO" can be toxic.[126,127] In aqueous solutions, NO" autoxidises to nitrite [128'129] (Figure 3B) and addition of NO" to blood results in the formation of both nitrate and nitrite. ~3°1 It is thought that in the vascular system, NO" is rapidly oxidised by reaction with oxyhaemoglobin (HbO2) resulting in the forma- tion of methaemoglobin (Hb 3+) and nitrate (NO3).I13°] NO" also reacts with Hb 3+ forming a complex (Hb-NO) that can hydrolyse to Hb 2+ and nitrite. I1311 That NO" is a source of endogenous nitrite is confirmed by the incorporation of radio- labelled ammonia (15NH3) [132] and arginine (15N- arginine) [1331 into urinary nitrate and nitrite. Peroxynitrite Peroxynitrite (ONOO-) has been implicated in the pathogenesis of many disease states encom- passing cancers and autoimmune, inflammatory,
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`NITROTYROSINE FORMATION AND DIETARY NITRATE/NITRrI'E 221 and neurodegenerative diseases via detection of increased levels of nitrotyrosine, despite evid- ence that peroxynitrite is not the sole species capable of nitrating tyrosine (Table I). Addi- tional evidence that peroxynitrite plays a role in atherosclerosis is its ability to initiate the per- oxidation of low density lipoprotein I1341 that can result in the formation of F2-isoprostanes, lipid oxidation products that may contribute to the atherosclerotic process. [1351 A requirement for the formation of peroxyni- trite from the near diffusion limited reaction between superoxide and nitric oxide (6.7 x 10 9 M -1 s-l) [1361 is that both O~- and NO" are formed in close proximity at high local con- centrations. Thus production is dependent on the rates of production of these two species [4A371 d peroxynitrite is most likely to form near macrophages, [138A391 neutrophils [14°'1411 and en- dothelial cells [142'~43! which produce both O~- and NO'. The function of peroxynitrite is not well estab- lished as it may be an important detoxification mechanism for reactive oxygen species I144'1451 yet it exhibits cytotoxic properties itself. [144A461 The conflicting effects are further emphasised by the demonstration that peroxynitrite exerts both damaging and cytoprotective effects towards platelets. I~471 It has also been postulated to be an important microbicidal/bacteriocidal compound. I1481 The reaction of peroxynitrite or its conjugate acid (ONOOH), with a wide variety of biomole- cules results in production of nitrite, t1491 Per- oxynitrite exists in two different conformations cis- and trans-. The trans-conformation forms upon protonation of the cis-form I15°1 and re- arranges to nitrate. I~5°Asll However, the majority (80%) of peroxynitrite exists in the stable cis- conformation in vivo. t~511 III. Intake from Exogenous Sources Nitrogen (N) is required for the synthesis of deoxyribonucleic acid (DNA), ribonucleic acid (RNA) and proteins in both plants and ani- mals. i1521 Humans need to acquire compounds containing nitrogen from food and therefore a minimum quantity of animal or plant protein is needed. It is estimated that one third of human dietary protein now derives from synthetic nitrogen fertilisers via the Nitrogen cycle, t152! A large collaborative study (ECP-INTER- SALT) determined that intake of nitrate in Brit- ain is in the lower tertile of Europe and around the median of the 30 countries studied world- wide. 1153'154] A Total Diet study in 1997 found the daily intake of nitrate by the UK population to be 88 mg, with an upper range of 136 mg. tlssl It has been calculated that vegetarians are exposed to the greatest amount of nitrate (34.3- 163.0 mg/day) in the UK. [1561 It appears that the total dietary intake of nitrate in the UK was lower in 1979 being 61 rng/person (ranging from 24 to 102rag/person) with 75% derived from vegetables, 8 mg from water used to make non- alcoholic drinks and 53 mg from food alone, t1571 This is similar to the amount calculated for the UK population in 1973 (63 mg/person) tlsSl with 50% contributed by vegetables and water and meat contributing 25% each. Higher daily nitrate consumption (215rag/person) from vegetables, fruit and meat in Singapore has been reported, but this differs between the Chinese (250rag/ person) and the Malay and Indian (113mg/ person) populations. I1591 The contribution from water with different nitrate contents was high- lighted in a sample of 50 people from the Netherlands who consumed 145-231 mg nitrate/day with the amount contributed by water varying from 0 to 25 mg in tap water and 34-132mg in well water. I16°~ Daily intake of nitrite in 1979 in the UK was low (0.3-0.9 mg/ person) having been detected only at low con- centrations (< 2.4mg/kg) in cereals, meats and vegetables. [1571 Several factors may account for the large vari- ation in the daily intake including the amount and type of foods consumed by individuals. The levels of nitrate in a plant differ depending
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`222 C. OLDRE1VE AND C. RICE-EVANS on the site within the plant, application of fertiliser,[ 161,1621 season,| ]63,1641 sunshine,[163,1641 maturity at harvest, [1621 storage |162"1651 and cook- ing. |i6L166I Negligible nitrite has been detected in the majority of fruit and vegetables. |8s'1s91 Nitrate levels are also negligible in fruit [s5'1591 but are wide ranging in vegetables of differ- ent types [85'159'163] and within a specific vari- ety. [159"163] Green leafy vegetables consistently have high nitrate contents [85'159"163] and along with water contribute the majority of nitrate in- take by humans. [159l Nitrate and nitrite as their sodium and potassium salts are used as addit- ives (E249-252) in certain foods. |167] They are added to meat products (cured meat, bacon, ham, sausages, pork pies) due to their ability to inhibit the growth of Clostridium botulinum I~57! and although not used in cheese manufactured in the UK, nitrate can be used in cheesemaking in Continental Europe. t1571 IV. Nitrate/Nitrite and Illness/Disease Nitrite can cause a condition called methaemo- globinaemia through its interaction with hae- moglobin so that the blood is less efficient in transporting oxygen. |157! The majority of cases occur in infants and areas where drinking water is obtained from wells with high nitrate content. |168'169| It has also been suggested that exposure of pregnant mothers to high levels of nitrate in drinking water can lead to neural tube defects in their babiesJ 17°1 This reaction with hae- moglobin to form methaemoglobin has been utilised in the use of sodium nitrite as an antidote against cyanide poisoning as methaemoglobin can extract the cyanide anion from cytochrome oxidase. [171] Nitrate intake has been linked to human can- cers of the stomach due to the effect of nitrite produced from it, but a lot of controversy re- mains. [154'159'172-174] Nitrate intake has also been implicated in non-Hodkgin's lymphoma |1751 and type I diabetes 0761 whilst dietary nitrite may play a role in glioma (brain cancer) |1771 and col- orectal cancer. I1781 The putative role of nitrate in cancer is supported by the finding that nitrate in drinking water can elicit a dose dependent in- crease in hypoxanthine-guanine phosphoribosyl transferase (HPRT) variant frequency in periph- eral lymphocytes (a marker of DNA damage) which is a prerequisite for tumour forma- tion. 116°1 Nitrate itself (117mM) does not appear to mediate any toxic effects in mammalian cells I981 but nitrous acid (HNO2) has been shown to be mutagenic in bacterial systems such as Esch- erichia col| I179"1801 and Salmonella typhimurium 11811 and also in the yeast Saccromyces cerev/s/ae. |182I Administration of sodium nitrite (100-3,000 rng/L) in the drinking water of rats raised their methaemoglobin levels, led to some changes in the liver and spleen, fibrosis in the heart, thin and dilated arteries, and atrophied and dilated bronchi accompanied by lung emphysema. |1831 Whilst the presence of nitrite in the stomach is thought to have adverse health effects due to its ability to form nitroso-compounds which can be carcinogenic, [ls4-18sl it has also been proposed to serve a protective role in augmenting the anti- microbial effects of stomach acid, a possible reason for the recirculation of nitrate into the salivary glands. |189"19°! Levels of nitrate and nitrite in biological fluids are enhanced in several diseases (Table 1I) in which elevated nitrotyro- sine has been detected indicating that either they or their precursors nitric oxide and peroxynitrite play role in the pathogenesis of these diseases. NITROSO-COMPOUND FORMATION Nitration and nitrosation constitute the majority of chemistry associated with reactive nitrogen species. Many different reactive nitrogen species have been proposed to be responsible for the nitration and nitrosation of compounds. Exam- ples include N203, [191-193] NO2, [192] N204, [191"193] amides by nitracidium ion H2NO~, I1941 NOX (where X is a base such as a chloride an- ion), [195'1961 NO~ [951 and NO2-C1, [951 (whereas
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`NITROTYROSINE FORMATION AND DIETARY NITRATE/NITRITE 223 nitric oxide per se is unable to participate directly in nitration and nitrosation reactions [1921). These species can undergo interconversion under acidic conditions [128"194'196] (Figure 3C). Elevated levels of N-nitroso compounds are found in the gastric juice of patients with gastric cancer and its precursor states. Is9] In animal models N-nitro- so-compounds have been shown to be carcino- genic. [la4-~ss] In addition, a nitrosated extract of Japanese fish (sanma hiroki) induced tumours in the forestomach and glandular stomach of rats. [197] Specifically, tyrosine has been identified as one of the precursors of nitroso-compounds in food that exhibit direct acting mutagenicity towards Salmonella strains after treatment with nitrite.[ 198,199] Human urine contains several N-nitrosamino acids [16°'2°°] that could originate either from the diet [1571 or be endogenously produced and which increase in amount after ingestion of nitrate. [2°1] However, there appeared to be no difference in the amount of N-nitroso com- pounds excreted between people consuming different amounts of nitrite in their drinking water I16°! but the total amount of nitrate ingested was not that different. Secondary amines, amino acids and amides react optimally at pH<4, [194'2°21 nitrosation being enhanced by halides [194"195"2°21 and thio- cyanate. [195"2°21 This suggests that the majority of nitrosation by nitrite occurs in the acidic conditions of the stomach. This is probable, especially for tyrosine, as its nitration appears to be highly dependent upon pH with little nitrotyrosine formed at pH > 2.2. I22'81"a51 Addi- tionally, it has been shown that DEN (diethyl- nitrosamine) is formed upon incubation of diethylarnine (DEA) and nitrite in gastric juice from rat, rabbit, cat, dog and humans and is also detected in stomach of cats and rabbits fed DEA and nitrite. [2°3"2°41 Some N-nitroso compounds may arise from the action of certain bacteria that may colonise the stomach during disease states which at elevated pH are capable of catalysing nitrosation reactions I2°51 and activated macro- phages also have the ability to nitrosate amines in vitro. I2°61 Dietary phenolics appear to inhibit nitrosation in vivo [207'208I and nitration of tyrosine in vitro. I85! Nitration and nitrosation reactions can occur under acidic conditions such as that of the stomach with such dietary agents. Paradoxically, however, C-nitrosophenols formed from pheno- lic compounds can act as catalysts for the reac- tion between amines and nitrite in mildly acidic media, t2°9! This is possibly the mechanism by which chlorogenic acid [21°1 and gallic acid [211] catalyse nitrosation of amines but we have seen no evidence for this as, in our systems, phenolic compounds inhibited rather than catalysed the nitration of tyrosine by nitrous acid. Is51 Nitrosa- tion of the flavonoid constituents of food can also result in compounds that are mutagenic in Salmonella Typhimurium TA100. [212I CONCLUSIONS In conclusion, nitrotyrosine detected in vivo may arise from peroxynitrite or nitryl chloride. How- ever, little attention has been paid to sources such as dietary nitrate in the acidic environment of the stomach. The evidence implicating dietary nitrate/nitrite in human disease has been incon- clusive with the exception of nitrite and methae- moglobinaemia (blue baby syndrome) Thus, the impact of diet-derived reactive nitrogen species on the potential formation of nitrotyrosine in vivo and its subsequent incorporation into pro- teins has been little explored. The formation of nitrotyrosine either in the stomach or during inflammation is probably dependent upon a multitude of factors such as the pH, bacterial colonisation, catalytic factors (thiocyanate), inhi- bitory factors (phenolics), competing substrates and the inflammatory mediators (HOC1, perox- idases) produced. The contribution of nitrotyro- sine from the diet needs to be assessed, along with its general pharmacology in humans, namely, absorption, incorporation into proteins,
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`224 C. OLDREWE AND C. RICE-EVANS toxicity and excretion. Finally, the formation of nitrated proteins in vivo whether via their inter- action with reactive nitrogen species or as a con- sequence of the incorporation of nitrotyrosine into proteins remains to be elucidated, as well as the precise role, if any, of nitrotyrosine in the pathogenesis of human disease. Acknowledgments We acknowledge financial support from the Food Standards Agency (UK). References [1 ] K. Kikugawa, K. Hiramoto, S. Tomiyama and K. Nakauchi (1999) Effect of fi--carotene on the transformation of tyr- osIne by nitrogen dioxide and peroxynitrous acid. Free Radical Research, 30, 37-43. [2] A. Pannala, R. Razaq, B. HalliweU, S. Singh and C.A. Rice- Evans (1998) Inhibition of peroxynitrite dependent tyro- sine nitration by hydroxycinnamates: Nitration or electron donation? Free Radical Biology and Medicine, 24, 594-606. [3] A. Pannala, C.A. Rice-Evans, B. HaUiweU and S. Singh (1997) Inhibition of peroxynitrite-mediated tyrosine ni- tration by catechin polyphenols. Biochemical and Biophy- sical Research Communications, 232, 164-168. [4] S. Pfeiffer and B. Mayer (1998) Lack of tyrosine nitration by peroxynitrite generated at physiological pH. The Jour- nal of Biological Chemistry, 273, 27280-27285. [5] M. Whiteman and B. Halliwell (1996) Protection against peroxynitrite-dependent tyrosine nitration and al-anti- proteinase Inactivation by ascorbic acid: A comparison with other biological antioxidants. Free Radical Research, 25, 275-283. [6] A. Gow, D. Duran and S.R. Thorn, H. Ischiropoulos (1996) Carbon dioxide enhancement of peroxynitrite- mediated protein tyrosine nitration. Archives of Biochem- istry and Biophysics, 333, 42-48. [7] J. Khan, D.M. Brennan, N. Bradley, B.R. Gao, R. Bruck- doffer and M. Jacobs (1998) 3-nitrotyrosine in the pro- teins of human plasma determined by an ELISA method. Biochemical Journal, 330, 795-801. [8] H. Ohshima, I. Celan, L. Chazotte, B. PignateUi and H.F. Mower (1999) Analysis of 3-nitrotyrosine in biological fluids and protein hydrolyzates by high-performance liquid chromatography using a postseparation, on-line reduction column and electrochemical detection: Results with various nitrating agents. Nitric Oxide-Biology and Chemistry, 3, 132-141. [9] C. Leeuwenburgh, M.M. Hardy, S.L. Hazen, P. Wagner, S. Oh-ishi, U.P. Steinbrecher and J.W. Heinecke (1997) Reactive nitrogen intermediates promote low density lipoprotein oxidation in human atherosclerotic intima. Journal of Biological Chemistry, 272, 1433-1436. [10] O.M. Panasenko, K. Briviba, L.-O. Klotz and H. Sies (1997) Oxidative modification and nitration of human low-density lipo