ANNALS OF CLINICAL AND LABORATORY SCIENCE, Vol. 27, No. 5 Copyright © 1997, Institute for Clinical Science, Inc.

Efficacy of Taurine Based Compounds as Hydroxyl Radical Scavengers in Silica Induced Peroxidation* XIANGLIN SHI, Ph.D.,f DANIEL C. FLYNN, Ph.D.,* DALE W. PORTER, Ph.D.,f STEPHEN S. LEONARD, B.S.,t VAL VALLLYATHAN, Ph.D.,f and VINCE CASTRANOVA, Ph.D.f fPathology and Physiology Research Branch, Health Effects Laboratory Division, National Institute for Occupational Safety and Health, Morgantown, WV 26505 and $Department of Microbiology and Immunology, West Virginia University, Morgantown, WV 26506

ABSTRACT While it is widely believed that taurine may play an important role in protecting cells against toxic injury by functioning as an antioxidant, there is a lack of evidence to support this hypothesis. In this study, electron spin resonance (ESR) was used to investigate the reaction of taurine and hypotaurine with hydroxyl radicals (OH). The Fenton reaction (Fe(II) + H 20 2 —» Fe(III) + OH + OH“) and the Cr(V)-mediated Fenton-like reaction (Cr(V) + H 20 2 —> Cr(VI) + OH + OH“) were used as sources of OH radicals. The results show that hypotaurine but not taurine effectively scav­ enges OH radicals with a reaction rate constant of k = 1.6 x 1010 M“xs_1. That is comparable with other efficient OH radical scavengers. The effect of taurine and hypotaurine on silica-induced lipid peroxidation was evaluated using linoleic acid as a model lipid. Hypotaurine, but not taurine, caused a significant inhibition of silica- induced lipid peroxidation. The results show that hypotaurine is an excellent anti­ oxidant and appears to have the potential for being a therapeutic agent against silica-induced lung injury.

Introduction characterized in the cat. 1,2 L-cysteine is the Taurine (2-aminoethane sulfonic acid) is Prf*ursor- and ^ steic acildl and hypotaurine ubiquitous amino acid present in most mam- af ke>' intermediates. In addition to its various malian tissues, especially in those exposed to Physiol°gical r°Ies> Murine is believed to be an elevated levels of pre-oxidants. The pathway antioxidant and to protect cells against toxic for the synthesis of taurine is relatively well injur>' Í,n" nS that from P * 0* ? Com- pounds. ■ ’ ■ ■ It may be noted that hypotau­ rine is a precursor of taurine synthesis.4,5,6 Its * Send reprint requests to: Xianglin Shi, Ph.D. or Vince chemical structure (figure 1), specially the Castranova, Ph.D Pathology and Physiology Research _SQ - makes ^ molecu]e a particu. branch, National Institute tor Occupational Sarety and , , i 1 rni • Health, 1095 Willowdale Hoad, Mail Stop L2015, Mor- l&rly good reducing agent. Thus, hypotaurine is gantown, w v 26505. likely to be a good antioxidant. 365 0091-7370/97/0900-0365 $02.50 © Institute for Clinical Science, Inc. 366 SHI, PORTER, LEONARD, VALLYATHAN, AND CASTRANOVA nh2+ - ch2- ch2- so3 rate constant of taurine or hypotaurine with hydroxyl radical ( OH). This radical is chosen Taurine because it is the most reactive and toxic free radical among ROS. The possible inhibitory effect of taurine and hypotaurine on silica- nh2+- ch2- ch2- so2- induced lipid peroxidation has also been inves­ Hypotaurine tigated. The major questions to be answered F ig u r e 1. The chemical structure of taurine and in this study are as follows: (1) Are taurine hypotaurine. and hypotaurine good antioxidants? What are reaction rate constants? (2) How do It is known that the pulmonary system is taurine and hypotaurine compare with other most susceptible to oxidative stress owing to its well established antioxidants? (3) Do tau­ exposure to the highest oxidation tension and rine and hypotaurine inhibit silica-induced toxic foreign substances. Alveolar macro­ lipid peroxidation? phages phagocytize inhaled particles and gen­ erate reactive oxygen species (ROS) as means Materials and Methods of antimicrobial defense.8 This process plays an important role in the mechanism leading to M aterials oxidative lung injury. Although further studies Iron(II) chloride (FeCl2),* hydrogen per­ are needed to investigate the protective effect oxide (H 2 0 2),* potassium dichromate of taurine against cell damage, recent reports (K2 Cr2 0 7),* and 5,5-dimethyl-l-pyrroline indicate that taurine may function as an anti­ N-oxide (DMPO)* as well as nicotinamide- oxidant in the lung. Banks et al9,10 have adenine-dinucleotide phosphate [reduced reported that there is a Na-coupled transport form] (NADPH),t glutathione,! cis-9-cis-12- pathway for the uptake of taurine by alveolar octadecadienoic acid (linoleic acid),f taurine,f macrophages that results in an inhibition of hypotaurine,f and sodium formatef were pur­ oxidative cell injury caused by ozone exposure. chased from chemical companies. Chelex-100 A correlation has been found between taurine chelating resin i was also purchased. Phos­ content of alveolar macrophages, type I and phate buffer (pH 7.4) was treated with Chelex- type II cells, and their susceptibility to oxi­ 1 0 0 to remove putative metal ion contami­ dant injury.3 nants. The DMPO solutions were purified It may be noted that epidemiologic and using activated charcoal until free radical pathologic studies have established that inha­ impurities disappeared as verified by ESR lation of silica incites the development of acute spectroscopy. Crystalline silica (Min-U-Sil)§ and chronic pulmonary silicosis.11 In addition, was obtained and fractionated to <5|x using a increasing evidence from epidemiologic and Donaldson particle classifier. X-ray energy animal experimental studies has implicated spectrometric measurements show that 98.7 silica as a potential carcinogen.12 Although the percent of the particles were pure crystal­ mechanism of silica-induced lung injury line silica. remains to be elucidated, it is believed that one event of primary importance is the pertur­ E lectro n Spin R eson a nce M easurements bation of cell membrane due to silica- Electron spin resonance (ESR) spin trap­ mediated free radical reactions. 13 This cell ping15,16 was used to detect short-lived free damage releases certain lytic enzymes that radical intermediates. This technique involves cause additional cell injury.14 The protection against silica-induced membrane damage * Aldrich, Milwaukee, WI. appears to be a key step to attenuate or pre­ t Sigma, St. Louis, MO. vent silica-induced lung injury. $ Bio-Rad Laboratories, Richmond, CA. The present study investigates the reaction § U.S. Silica Products, Berkeley Springs, WV. EFFICACY OF TAURINE BASED COMPOUNDS AS HYDROXYL ION SCAVENGERS 367 the addition-type reaction of a short-lived radi­ reacted similarly to obtain a calibration curve, cal with a diamagnetic compound (spin trap) to which was used to calculate the amount form a relatively long-lived free radical prod­ of TRA reactive substances produced by uct, the so-called spin adduct, which can be silica samples. studied by conventional ESR. The intensity of The concentrations provided in figure leg­ the spin adduct signal corresponds to the ends are final concentrations. All the experi­ amount of short-lived radicals trapped, and the ments were carried out in air at room tempera­ hyperfine splittings of the spin adduct are gen­ ture except those specifically indicated. erally characteristic of the original, short-lived, trapped radical. All ESR measurements were Results made using a Varian E4 spectrometer and a flat cell assembly. Hyperfine splittings were Scavenging of 'OH Ry Hypotaurine measured (to 0.1 G) directly from magnetic field separations using potassium tetraperoxo- The Fenton reaction (Fe(II) + H 2 0 2 —» chromate (K 3 Cr08) and 1,1-diphenyl- Fe(III) + 'OH + OH-) was used as a source of 2-picrylhydrazyl (DPPH) as standards. The "OH radicals. As shown in figure 2a, an aque­ relative radical concentration was estimated ous solution containing Fe(II), H 20 2, and a by multiplying half of the peak height by spin trap (DMPO) in a phosphate buffer solu­ (AHp p )2 (where AHpp represents peak-to-peak tion (pH 7.4), generated a 1:2:2:1 quartet with width). Reactants were mixed in test tubes in hyperfine splittings of aN = aH = 14.9 G. Based a total final volume of 0.50 ml. The reaction on these splitting constants, the 1 :2 :2 :1 quartet mixture was then transferred to a flat cell for was assigned to a DMPO/'OH adduct, as an ESR measurement. indirect evidence for "OH generation.17 As may be noted from figure 2b, 0.125 mM hypo­ L ipid P eroxidation M easurements taurine effectively reduced the intensity of the DMPO/'OH spin adduct signal (note the dif­ Lipid peroxidation of the model polyunsatu- ference of the spectrometer setting). Increas­ red lipid, linoleic acid, by silica was measured ing the hypotaurine concentration further by monitoring the thiobarbituric acid (TRA) decreased the intensity of DMPO/'OH signal reactive substances formed in the reaction (figures 2c, 2d, 2e, and 2f). At a concentration mixture. A typical reaction mixture contained of 20 mM, hypotaurine completely abolished 10 mg/ml quartz particles and 100 (xl of 0.9 M the DMPO/'OH spin adduct signal (figure 2g). linoleic acid emulsion in a total volume of 1 .0 The control, which has an equivalent volume ml hydroxyethylpiperazine ethanesulphonic of hypotaurine vehicle without hypotaurine, acid (HEPES) buffered medium containing did not alter the spectral intensity (data not 140 mM NaCl, 5 mM KCl, and 10 mM shown), demonstrating that the apparent HEPES (pH 7.4). The mixture was incubated effects of hypotaurine were not due to its for 1 hour in a shaking water bath at 37°C. The vehicle. In contrast, at concentrations as high reaction was terminated by the addition of as 20 mM, taurine did not significantly alter 0.625 ml of 40 percent trichloroacetic acid and the DMPO/'OH spin adduct signal intensity 0.3 ml 5 N hydrochloric acid. Vials were vor- (figure 2 h), demonstrating that taurine is not a texed for 10 seconds and 0.625 ml 2 percent "OH radical scavenger. thiobarbituric acid was added and mixed again. In order to examine whether or not the The mixture was then heated for 20 minutes at decreased intensity was related to the reaction 95° to 100°C. The tubes were cooled and cen­ of hypotaurine with the DMPO/'OH spin trifuged for 1 0 minutes at 600 g and the absor­ adduct, measurements were made on solutions bance of the supernatant was measured at 585 in which Fe(II) was reacted with H 20 2 in the nm. Malondialdehyde standards were pre­ presence of DMPO first, and then hypotaurine pared from 1,1,3,3-tetramethoxypropane and was added 30 seconds later. The intensity of 368 SHI, PORTER, LEONARD, VALLYATHAN, AND CASTRANOVA the DMPO/OH spin adduct signal (data not shown) was essentially the same as that in fig­ ure 2a without hypotaurine. This result shows Gain x 1/2 that the decrease in spectral intensity observed in the reaction of Fe(II) with H 20 2 in the pres­ ence of hypotaurine was not related to the reaction of hypotaurine with the DMPO/OH adduct but to the scavenging of OH radicals. Additional evidence for the scavenging of 'OH by hypotaurine was obtained by using a different source of OH, namely, reaction of Cr(V) with H 20 2, i.e, Cr(V) + H 20 2 Cr(VI) + 'OH + OH “ .18 The Cr(V) was produced by one-electron reduction of Cr(VI) by glutathi­ one reductase in the presence of NADPH .10 In figure 3a is shown the 1:2:2:1 quartet of the DMPO/OH adduct obtained, as an evidence of OH formation. Upon addition of hypotau­ rine, the intensity of the 1 :2 :2 :1 quartet signal decreased, demonstrating the OH scavenging

Cr(V) g = 1.9792

hJ! Gain x 1/2

15G 15G i------1 -► H ' ♦ h ' F ig u r e F ig u r e 2. Electron spin resonance (ESR) signals from 3. Electron spin resonance (ESR) signals from 5,5-dimethyl-l-pyrroline N-oxide (DMPO/OH) adducts 5,5-dimethyl-l-pyrroline N-oxide (DMPOVOH adducts obtained from the reaction of fe(II) with H20 2 (a) ESR obtained from the reaction of Cr(V) with H20 2. (a) ESR spectrum recorded 1 minute after mixing 1.0 mM FeCl2, spectrum recorded 1 minute after mixing 0.75 mM 1.0 mM H20 2, and 1.0 mM DMPO in a pH 7.4 phosphate K2Cr207, 0.75 mM, nicotinamide-adenine-dinucleotide buffer solution, (b) Same as (a) but with 0.125 mM hypo­ phosphate [reduced form] (NADPH), 10 units/ml gluta­ taurine added, (c) Same as (a) but with 0.25 mM hypo­ thione reductase, 0.75 mM H20 2, and 1.0 mM DMPO in taurine added, (d) Same as (a) but with 0.5 mM hypotau­ a pH 7.4 phosphate buffer solution, (b) Same as (a) but rine added, (e) Same as (a) but with 1.0 mM hypotaurine with 0.5 mM hypotaurine added, (c) Same as (a) but with added, (f) Same as (a) but with 2.0 mM hypotaurine 20 mM taurine added. The spectrometer settings were: added, (g) Same as (a) but with 20 mM hypotaurine added, receiver gain, 5.0 x 104, modulation amplitude, 0.5 G; (h) Same as (a) but with 20 mM taurine added. The spec­ magnetic field, 3500 ± 100 G; scan time, 4 minutes. trometer settings were: receiver gain, 5.0 x 103 (for a and h), 1.0 x 104 (for b to g); modulation amplitude, 0.5 G; magnetic field, 3500 ± 100 G; scan time, 4 minutes. EFFICACY OF TAURINE BASED COMPOUNDS AS HYDROXYL ION SCAVENGERS 369 activity of hypotaurine (figure 3b). In contrast, the addition of hypotaurine. This spectrum is taurine did not exhibit any significant inhibi­ essentially identical to that in figure 4a, indi­ tory effect (figure 3c). cating that hypotaurine did not significantly The possibility was checked that hypotau­ react with Cr(V)-NADPH complex. rine may have reacted with metal to block the As mentioned in the introduction, silica par­ 'OH radical generation. As shown in figure 4a, ticles may cause cellular damage via 'OH radi­ the reduction of Cr(VI) by glutathione reduc­ cal generation. It is possible that hypotaurine tase in the presence of NADPH generated a may protect the cell from OH-mediated dam- spectrum centered at g = 1.9792 with five prin­ age by silica particles. Reaction of silica par­ cipal components having 0.84 G spacing. The g ticles with aqueous medium in the presence of value is typical of that of a Cr(V) complex, and DMPO generates a DMPO/'OH spin adduct the splittings are characteristic of the superhy- (data not shown) as reported earlier.20 The perfine interaction of nearby hydrogens. Thus, addition of 1 mM of hypotaurine decreased this spectrum was assigned to a Cr(V)- the intensity of the DMSO/'OH spin adduct NADPH complex.18 In figure 4b is shown the signal by about 40 percent (data not shown), spectrum obtained by the reaction of Cr(VI) indicating that hypotaurine is able to scavenge with glutathione reductase and NADPH with the OH radical generated by silica reactions.

C alculation o f R eaction R ate C onstant o f H ypotaurine w ith 'O H To verify that the decrease of DMPO/’OH is due to the OH radical scavenging effect of hypotaturine and not due to any other mechanism, spin trapping competition reac­ tions were carried out using formate as another OH radical scavenger. It is known that the reaction of formate with OH gener­ ates formate-derived (carbon-centered) radi­ cals with a reaction rate of kf = 3.9 x 109 M _1 sec-1 (Equation [l ] ) . 21'22 kf •OH + HCOO-----> H20 + -COO“ [1] kf = 3.9 x 109 M“ 1 sec^ 1 The newly generated formate-derived radicals will react with DMPO to produce DMPO/ COO” adduct. The reaction rate of DMPO with 'OH is kd = 2.1 x 109 M_1 sec-1 (Equa­ I------1 tion [2]) (Morehouse and Mason, 1988.) ♦ H kd Figure 4. Effect of hypotaurine on the generation of •OH + DMPO-----> DMPO OH [2] Cr(V). (a) Electron spin resonance (ESR) spectrum 9 “ 1 “ 1 recorded 1 minute after mixing 0.75 mM K2Cr20 7, 0.75 kd = 2.1 x 10 M sec mM nicotinamide-adenine-dinucleotide phosphate [reduced form] (NADPH), and 10 units/ml glutathione Because the kf and kd are comparable, with an reductase in a pH 7.4 phosphate buffer solution, (b) Same excess amount of formate, the OH will pre­ as (a) but with 0.5 mM hypotaurine added. The spectrom­ eter settings were: receiver gain, 5.0 x 104; modulation dominately react with formate to generate amplitude, 0.2 G; magnetic field, 3500 ± 25 G; scan time, COO". In this case, the spin adducts obtained 4 minutes. are mostly DMPO/ COO-. As shown in figure 370 SHI, PORTER, LEONARD, VALLYATHAN, AND CASTRANOVA

5a, reaction of Fe(II) with H 20 2 in the pres­ -d[-OH]/dt = kd[DMPO][-OH] ence of DMPO and 0.05 M formate indeed + kh[-OH][hypotaurine] generates DMPO/COO“ as a major spin [4] adduct signal. If hypotaurine blocks the OH generation, the intensity of DMPO/'COO“ d[DMPO/-OH]/dt = kd[DMPO][-OH] should significantly decrease. As shown in fig­ [5] ure 5b, addition of hypotaurine did not signifi­ Dividing equation [4] by equation [5], one cantly affect the intensity of DMPO/'COO“ obtains equation [6 ]. adduct. Thus, the results show that hypotau­ rine is capable of scavenging the "OH radicals -d[-OH]/dt kh[hypotaurine] and does not significantly block its generation. d[DMPO/-OH]/dt + kd[DMPO] [ ] To determine the reaction rate constant of hypotaurine with "OH radicals, kinetic studies At a saturating level of DMPO and in the were carried out according to methods absence of hypotaurine, the rate of "OH spin reported earlier for the reaction of ethanol trapping is equal to the rate of OH generation, d[’OH]/dt. If V and v represent the rate of with "OH.22,23 The reaction steps may be writ­ ten as: "OH spin trapping in the absence and in the presence of hypotaurine, respectively, one kd obtains equation [7]. •OH + DMPO-----» DMPO • OH [2] V/v = 1 + kh[hypotaurine]/kd[DMPO] [7] k, = 2.1x!0 9 M“ 1 sec“ 1 kh •OH + hypotaurine-----> product [3] kh[hypotaurine] kd[DMPO] [8]

In figure 6 is shown the inhibition of "OH by hypotaurine. The data were plotted according to equation [8 ]. A straight line is obtained with a slope of 7.6, which is the ratio of kh/kd (figure 6 ). Using the value of kd = 2.1 x 109 M “ 1 sec“ 1 for the "OH trapping by DMPO ,21 the value of kh is calculated as follows: kh = 7.6 kd = 7.6 x 2 .1 x 10y M" sec 1 ____ = 1.6 x lO10 M It should be noted that the rate constant kh as calculated may not be very accurate. For example, this method did not include the decay of the spin adduct after its formation.24 -► H However, it does provide a convenient method F igure 5. Effect of hypotaurine on the 5,5-dimethyl- for obtaining a relative value. 1-pyrroline N-oxide (DMPO)/COO adduct. (a) Electron spin resonance (ESR) spectrum recorded 1 minute after mixing 1.0 mM DMPO, 1.0 mM FeCl2, 1.0 mM H2Oa, In h ib itio n o f Silica -in d u c e d and 50 mM sodium formate in a pH 7.4 phosphate buffer L ipid P eroxidation solution, (b) Same as (a) but with 1.0 mM hypotaurine added. The spectrometer settings were: receiver gain, 2.5 x 104; modulation amplitude, 0.5 G; magnetic field, 3500 To measure the enhanced adverse biologic ± 100 G; scan time, 4 minutes. effect of oxidant generation, silica was evalu- EFFICACY OF TAURINE BASED COMPOUNDS AS HYDROXYL ION SCAVENGERS 371 caused lipid peroxidation as measured by for­ mation of thiobarburic acid reactive sub­ stances. Hypotaurine exhibited a significant dose-dependent inhibition (figure 7). In con­ trast, 2 0 mM taurine failed to inhibit silica- induced lipid peroxidation in vitro (figure 7). Discussion Using ESR spin trapping with DMSO as a spin trap, the present study shows that hypo­ taurine and not taurine is an efficient Oil radical scavenger. The reaction rate constant is 1.6 x 1010 M _1 sec-1. Hypotaurine is compa­ rable with other well established antioxidants, such as ascorbate (1.2 x 1010 M “ 1 sec-1), reduced glutathione (GSH) (1.5 x 1010 M _1 (hypotaurine)/(DMPO) sec-1) and cysteine (1.3 x 1010 M -1 sec-1)

F ig u r e 6 . Scavenging of OH radical by hypotaurine. (table I). The OH radicals were produced by the reaction of 1.0 It thus appears that hypothaurine is a direct mM FeCl2 with 1.0 mM H20 2 in the presence of 1.0 mM antioxidant. An antioxidant is defined as any 5,5-dimethyl-l-pyrroline N-oxide (DMPO). The data were plotted according to V/v - 1 = k^hypotaurineJ/kjIDMPO], chemical or biological species which reacts as explained in the text. efficiently with an oxidant, specially ROS, to prevent oxidizable target from damage by this oxidant. It appears that hypotaurine may ated for the potential to induce lipid peroxida­ inhibit OH radical-induced cellular damage tion in a simulated biologic model using lin- predominantly via scavenging OH radical and oleic acid. As shown in figure 7, silica particles not via attenuating its generation as supported by competition reactions using excess formate. 2.00 - |------TABLE I 1.75 - Reaction Rate Constants of Some 1.50 - Antioxidants with OH Radical

Antioxidant k (M~1sec-1) Reference

Ascorbate 1.3 x 1010 a Glutathione 1.5x10'° a Cysteine 1.5 x 1010 a Azide 1.1 x 1010 a Lipoic acid 1.9 x 1010 b Hypotaurine 1.6x1010 c

20mM 10mM 5mM 1mM 0.75mM 0.5mM 20mM Control »Dorfman LM, Adams GE. National Reference Hypotaurine Taurine Data Service, National Bureau of Standards. No. F ig u r e 7. Silica-induced lipid peroxidation and its 46:1-56. inhibition by hypotaurine. Incubation mixture contained bMatsugo S, Yan LJ, Trischler HJ, et al. 10 mg/ml silica particles and different concentrations of Biochem Biophys Res Commun 1995;208:161-7. hypotaurine or taurine as indicated. Data presented are the means of ±SD of a minimum of five sets of experi­ °Shi X, Flynn DC, Porter DW, Leonard SS, ments in duplicate. Other experimental conditions are Vallyathan V, Castranova V. Ann Clin Lab Sei described in the section of Materials and Methods. 1997; 27:365-74. 372 SHI, PORTER, LEONARD, VALLYATHAN, AND CASTRANOVA It may be noted that taurine has been ing alternatives may exist. (1) Taurine may pro­ reported to be an antioxidant, specially in the tect cells through an alternative pathway. For lung. It is known that activation of alveolar example, it may enhance the ability of cells to macrophages results in release of oxygen free repair oxidant-induced damage. In fact, type II radicals.27,28 Pulmonary cells are normally pro­ cells, which contain taurine at a concentration tected against the toxic effects of radicals by a as high as 14.4 mM, are believed to repair well balanced battery of extracellular and oxidant injury to the alveolar epithelium by intracellular defense systems, including super­ replacing injured type I cells.3 (2) Taurine may oxide dismutase, catalase, glutathione peroxi­ modulate the cellular level of oxidants by dase, ascorbate, and vitamin E .8 However, modulation of nitric oxide (NO )32 and cal­ increased production of oxygen free radicals cium33 concentrations. (3) Since hypotaurine and their metabolites may occur in occupa­ is an excellent antioxidant and is a precursor of tional exposures to foreign particles as a result taurine synthesis, a high taurine concentration of repeated processes of phagocytosis, frus­ may be associated with a high concentration of trated phagocytosis, or because the inhaled hypotaurine. It is possible that some protective particles generate reactive oxygen free radicals effect of taurine, in fact, is due to the presence or contain other sites of redox potential. The of hypotaurine. For example, a significant enhanced generation of oxygen free radicals as elevation of intracellular level of taurine upon a result of the phagocytosis of quartz particles exposure of alveolar macrophages to ozone may impair or overwhelm the cellular defense may be due to the enhanced generation of tau­ system, thereby resulting in cell injury. It has rine from the reaction of hypotaurine with been shown that alveolar pneumocytes exhibit ozone or its associated reactive intermediates. different sensitivities to oxidant injury. Type II The results obtained from the present study cells are most resistant;29 alveolar macro­ also demonstrate that silica particles react with phages are moderately resistant,30 type I cells linoleic acid to induce lipid peroxidation and are the most susceptible to oxidant damage.29 that hypotaurine significantly inhibits this A correlation exists between intracellular reaction. It may be noted that when quartz is taurine and resistance to oxidant injury, i.e., fractured in air, silicon-based radicals are gen­ alveolar macrophages and type II cells accu­ erated on the surface of quarts.34,35,36 These mulate taurine and are relatively resistant to radicals subsequently react with atmospheric oxidant injury while type I cells contain low oxygen and water to generate H 2 0 2 OH, and levels of taurine and are more susceptible to O^ 2°,36 j n aJfijj-jQn iron and other transition oxidant damage.3 Taurine levels in alveolar metals that may be present as trace impurities pneumocytes isolated from rats were reported on the silica can surface catalyze the genera­ to be: alveolar macrophages, 4.27 ± 0.41 mM; tion of OH radicals from H20 2. The free radi­ alveolar type II epithelial cells, 14.40 ± 0.95; cals (Si’ and SiO ) on the surface of quartz and alveolar type I epithelial cells, 0.14 ± 0.67 particles and associated oxygenated reactive mM .3 Taurine has been shown to exhibit a pro­ species could perturb the cell membrane and tective effect against oxidant injury induced by initiate lipid peroxidation.13 The result would ozone and result in a significant elevation of be the release of reactive oxygenated species the intracellular content of taurine.10 (H 20 2, 0 2~, OH, R’, and RO ). These reactive In contrast to these reports, the present oxygenated species would further react with study shows that taurine is not an effective the cell membrane, leading to an additional OH radical scavenger and did not significantly release of these species. This process has been protect against silica-induced lipid peroxida­ suggested as a primary step in the pathogen­ tion in vitro. These results brings into question esis of silicosis. 13 The inhibition of silica- the widely believed antioxidant property of induced reactions by hypotaurine may attenu­ taurine. To explain the functioning as an ate or prevent silica-induced lipid peroxidation apparent direct antioxidant in vivo, the follow­ and protect the cell membrane from silica- EFFICACY OF TAURINE BASED COMPOUNDS AS HYDROXYL ION SCAVENGERS 373 induced oxidative damage. It may be noted 10. Banks MA, Porter DW, Martin WG, et al. Taurine uptake by isolated alveolar macrophages and type II that oxidative stress is considered to be respon­ cells. J Nutr Biochem 1991;2:308-13. sible for a variety of diseases, in addition to 11. Silicosis and Silica Disease Committee. Disease asso­ silicosis. Dietary supplementation of antioxi­ ciate with exposure to silica and silicosis. Arch Pathol Lab Med 1988;112:673-720. dants, such as ascorbate, has been shown to 12. IARC. Silica and Some Silicates. In: IARC Mono­ protect against lung oxidant injury. The anti­ graphs on the Evaluation of Carcinogenic Rick of oxidant function of hypotaurine may suggest Chemicals to Humans. Lyon: International Agency for Research on Cancer. 1987;42:39-143. that dietary hypotaurine supplement may pro­ 13. Shi X, Dalai NS, Hu X. et al. The chemical properties tect against lung oxidant injury in general. of silica particle surface in relation to silica-cell inter­ In conclusion, the results obtained from the actions. J Toxicol Environ Health 1989;27:434-54. 14. Reiser KM, Last JA. Silicosis and fibrogenesis: fact present study show that taurine is not an effi­ and artifact. Toxicology 1979;13:15-72. cient OH radical scavenger. It does not sig­ 15. Janzen EG, Blackburn BJ. Detection and identifica­ nificantly protect against lipid peroxidation tion of short-lived free radicals by an electron spin resonance trapping technique. J Am Chem Soc 1968; induced by silica-mediated free radical reac­ 90:5909-10. tion. Thus, it is not a good antioxidant. In con­ 16. Mottley C, Mason RP. Nitroxide radical adducts in trast, hypotaurine is an efficient OH radical biology: chemistry, applications, and pitfalls. Biol Magn Reson 1989;8:489-546. scavenger with reaction rate constant of k = 1 .6 17. Buettner GR. ESR parameters of spin adducts. Free x 10loM-1 sec 1, which is comparable to sev­ Radical Biol Med 1987;3:259-303. eral well established antioxidants, such as 18. Shi X, Dalai NS. On the hydroxyl radical formation in the reaction between hydrogen peroxide and biologi­ ascorbate, glutathione and cysteine. The cally generated chromium (V) species. Arch Biochem results also indicate that hypotaurine may have Biophys 1990;277:342-50. the potential for being a therapeutic agent 19. Shi X, Dalai NS. 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