Effects of Taurine on Nitric Oxide and 3-Nitrotyrosine Levels in Spleen During Endotoxemia
- First Online:
- Cite this article as:
- Bircan, F.S., Balabanli, B., Turkozkan, N. et al. Neurochem Res (2011) 36: 1978. doi:10.1007/s11064-011-0521-3
- 138 Views
Taurine (2-aminoethanesulfonic acid) is a free sulfur-containing β-amino acid which has antioxidant, antiinflammatory and detoxificant properties. In the present study, the role of endotoxemia on peroxynitrite formation via 3-nitrotyrosine (3-NT) detection, and the possible antioxidant effect of taurine in lipopolysaccharide (LPS)-treated guinea pigs were aimed. 40 adult male guinea pigs were divided into four groups; control, endotoxemia, taurine and taurine+endotoxemia. Animals were administered taurine (300 mg/kg), LPS (4 mg/kg) or taurine plus LPS intraperitoneally. After 6 h of incubation, when highest blood levels of taurine and endotoxin were attained, the animals were sacrificed and spleen samples were collected. The amounts of 3-nitrotyrosine and taurine were measured by HPLC, and reactive nitrogen oxide species (NOx) which are stable end products of nitric oxide was measured spectrophotometrically in spleen tissues. LPS administration significantly decreased the concentration of taurine whilst increased levels of 3-NT and NOx compared with control group. It was determined that taurine treatment decreased the levels of 3-nitrotyrosine and NOx in taurine+endotoxemia group. The group in which taurine was administered alone, contradiction to well-known antioxidant effect, taurine caused elevated concentration of 3-NT and NOx. This data suggest that taurine protects spleen against oxidative damage in endotoxemic conditions. However, the effect of taurine is different when it is administered alone. In conclusion, taurine may act as an antioxidant during endotoxemia, and as a prooxidant in healthy subjects at this dose.
Lipopolysaccharide (LPS) is a glycolipid component of the cell wall of gram-negative bacteria, and the administration of LPS can stimulate the development of the systemic inflammatory response syndrome . The main cellular targets for LPS are endothelial cells lining the vasculature and macrophages which found on high concentrations in the spleen [2,3]. Macrophages and endothelial cells are two major biological sources of nitric oxide (NO). It has been shown that released NO plays an important role in the development of LPS-mediated endotoxin shock . Induction of NO synthesis during inflammatory processes represents a defence mechanism against invading microorganisms, but excessive formation of NO has also been implicated in host tissue injury . Excessive NO rapidly reacts with superoxide anion (O2·−) to form peroxynitrite (ONOO−) that is far more toxic and reactive than its precursors. ONOO− is a potent oxidant and nitrating agent which causes modifications in numerous biomolecules including proteins, lipids, thiols, sulfhydryl groups, DNA bases, and preferentially nitrates tyrosine residues of protein or non-protein origins. Because the direct measurement of ONOO− in vivo is difficult, nitration of the amino acid tyrosine, to produce 3-nitrotyrosine (3-NT), is reportedly a reliable index of damage left by ONOO− in various inflammed tissues [6–8]. In addition, nitrite (NO2−) and nitrate (NO3−), major oxidation products derived from NO, are useful markers to evaluate NO production [9,10].
Taurine is a sulfur-containing β-amino acid that that is found in milimolar concentrations in most mammalian tissues and plasma. Metabolic actions of taurine include: bile acid conjugation , detoxification [12,13], osmoregulation , immunomodulation , membrane stabilization , calcium homeostasis maintenance , growth modulation , glycolysis and glycogenesis stimulation . Moreover, it has been shown in many in vitro and in vivo studies to have cytoprotective effects, and these actions are often attributed to an antioxidant mechanism [20,21]. Hovewer, many biological functions of taurine rely upon its intracellular concentration, which is determined for a given cell by its capacity to synthesize and to transport it from the extracellular medium .
In the present study, we investigated that the effect of taurine on the NO-mediated damage in spleen during endotoxemia. In addition, we aimed to determine taurine content of spleen tissues under this conditions.
LPS (0111:B4 from Escherischiacoli) and taurine were purchased from Sigma–Aldrich (St. Louis, MO, USA). All other chemicals used were of the highest analytical grade, and purchased from Merck (Darmstadt, Germany) or Sigma–Aldrich (St. Louis, MO, USA).
Animals and Study Protocol
This study was carried out in accordance with the regulations of Animal Experimentation Ethics Committee at Gazi University. 40 adult male Dunkin Hartley guinea pigs weighing 500–600 g were obtained from Laboratory Animal Husbandry and Experimental Research Center at Gazi University. They were provided with standard rat chow and tap water ad libitum until the experiments. Guinea pigs were randomly divided into four groups at 10 animals each; control, endotoxemia, taurine and taurine+endotoxemia. Group 1 served as control group and control group was intraperitoneally (i.p.) injected 0.9% NaCl. Group 2 served as endotoxemia group and endotoxemia was induced by a single i.p. injection of LPS (4 mg/kg b.w.) . Group 3 served as taurine group and taurine was administered i.p. at a single dose of 300 mg/kg b.w. Group 4 served as taurine+endotoxemia group and was administered consecutively i.p. taurine and LPS. After 6 h of incubation, when highest blood levels of taurine and endotoxin were attained, all animals were anesthetized with ketamine (60 mg/kg b.w.) and xylazine (10 mg/kg b.w.) intramuscularly, and sacrificed. The spleens were removed and immediately frozen in liquid nitrogen, and then stored at –80°C until assay.
The dose of taurine was selected based upon previous study that reported that this dosage scheme can produce marked antioxidant and antiinflammatory effects in endotoxemia model . LPS and taurine were prepared daily, dissolved in non-pyrogenic sterile saline, and warmed to body temperature (approximately 37°C) before the injections.
Measurement of Tyrosine Nitration
Tissue homogenates were prepared according to the method described by Kamisaki et al. . Briefly, 0.5 g spleen tissue was homogenized in 50 mM potassium phosphate buffer (pH 7.2) and then centrifuged for 5 min at 1.000 × g. Following the acid hydrolysation of the precipitate with 6 M HCl, it was evaporated under nitrogen gas. After 1 ml distilled water was added, 10 μl of the sample was auto-injected a Microtech Scientific C18 analytical column (particle size 5 μm, 50 × 1 mm). All samples were analysed by HPLC with electrochemical detector (ECD) using the method described by Maruyama et al. . Mobil phase was 50 mM H3PO4, 50 mM citric acid, 40 mg/L EDTA, 100 mg/L octane sulfonic acid and 5% methanol (v/v) (pH 3.1 with KOH). The flow rate was 0.05 ml/min. ECD was set at –850/600 mV. Tissue 3-NT content was calculated from a 3-NT standard curve (3.125, 6.25, 12.5, 25 and 50 μmol/L) and expressed as μmol/g tissue.
Measurement of NOx Levels
The NOx values are given by the sum of nitrite and nitrate, which are the stable end products of NO. Tissue NOx levels were measured spectrophotometrically by Griess method, according to Green et al. . Sodium nitrite and nitrate solutions (1, 10, 50, 100 μmol/L) were used as standards, and tissue NOx content was expressed as μmol/g tissue.
Measurement of Taurine Levels
Measurement of taurine was accomplished by HPLC, using the method described by McMahon et al. . Tissue homogenate preparation was made according to Nusetti et al. . In brief, 100 mg spleen sample in 0.1 N perchloric acid was homogenized and then centrifuged for 10 min at 6.000 × g and 4°C. 10 μl supernatant was treated with 150 μl of acetonitrile, and centrifuged at 3.500 × g for 10 min, and 50 μl of 10 mM borate buffer (pH 9.2) were added to the supernatant solution. This was followed by the addition of a 50 μl aliquot of 5 mM fluorescamine in acetonitrile for derivatisation, and the mixture was immediately vortexed. A 20 μl sample was on to reversed-phase HPLC system using a bondclone C18 analytical column (particle size 10 μm, 300 × 3.9 mm). The mobile phase was tetrahydrofuran/acetonitrile/phosphate buffer (15 mM, pH 3.5) (4:24:72, v/v/v) and the flow rate was 1 ml/min. The taurine derivatives were detected at a wavelength of 385 nm. Tissue taurine concentrations were calculated from a taurine standard curve (2.5, 5, 10 and 20 μg/ml) and expressed as μg/g tissue.
Statistical analysis was performed using SPSS version 16.0 software package program. All values were expressed as mean ± standard deviation. The significance of the data obtained was evaluated using analysis of variance (ANOVA). Differences between means were analyzed using the post ANOVA test (Tukey). All statistical tests were two-tailed, and a P value of 0.05 or less was considered significant.
NOx, 3-NT and taurine levels of control and experimental groups in spleen
NOx (μmol/g tissue)
3-NT (μmol/g tissue)
Taurine (μg/g tissue)
0.613 ± 0.054
0.326 ± 0.053
32.966 ± 6.997
1.279 ± 0.124a
0.612 ± 0.127a
3.649 ± 0.546a
1.432 ± 0.110a,b
0.513 ± 0.102a
85.920 ± 15.627a,b
0.848 ± 0.116a,b,c
0.398 ± 0.091b
141.708 ± 9.912a,b,c
In endotoxemia group, LPS administration increased approximately twofold levels of NOx in spleen tissues compared with control group (P < 0.05). The maximal mean of NOx levels was seen in taurine group (1.432 ± 0.110 μmol/g tissue). As for in the taurine+endotoxemia group, taurine and endotoxin administration together significantly reduced tissue levels of NOx compared to both endotoxemia and taurine groups (p < 0.05).
As seen in Table 1, spleen 3-NT content was 0.326 ± 0.053 μmol/g tissue in control group and 0.612 ± 0.127 μmol/g tissue in endotoxemia group, and the difference between them was statistically significant (P < 0.05). In the taurine+endotoxemia group, taurine and endotoxin administration together significantly decreased tissue 3-NT concentrations compared with endotoxemia group (P < 0.05). Moreover, the lowest 3-NT levels observed were in taurine+endotoxemia group (0.398 ± 0.091 μmol/g tissue).
Endotoxin administration significantly decreased levels of taurine compared with control group (P < 0.05). However, taurine administration elevated spleen levels of taurine, and these differences between control and taurine groups were statistically significant (P < 0.05). In addition, taurine plus endotoxin administration increased levels of taurine more, and the maximal mean of taurine level was seen in the taurine+endotoxemia group (141.708 ± 9.912 μg/g tissue).
The spleen is responsible for nonspecific host defence, and due to its high content of phagocytes and direct connection to the blood stream, the spleen has a important role in the clearance of circulating microorganisms, particles and some large molecules such as LPS . Thus, it has been suggested that spleen can be used to investigate the association between the deleterious effect of endotoxin and NO-related mechanism .
In the present study, we induced endotoxemia by 4 mg/kg i.p. injection of LPS in guinea pigs, and found that spleen tissue NOx levels in the endotoxemia group significantly increased compared with control level. Consistent with our data, Sakemi et al.  reported that spleen and plasma NOx levels approximately increased twofold in the LPS-injected rats. Similarly, several studies showed that NO production increased by activation of inducible nitric oxide synthase (iNOS) mRNA expression in spleen and other principal organs after LPS administration [2,5,31]. We also measured 3-NT levels in spleen tissues by HPLC method to investigate the ONOO−-mediated damage mechanism after endotoxin administration at 6th h, and determined that 3-NT levels increased during endotoxemia. These results indicated that LPS administration resulted in activation of macrophages in the spleen to produce reactive oxygen and nitrogen species [1,32,33]. When released in vivo into biological fluids, NO is autooxidized to NO2− and NO3−. But, autooxidation of NO is relatively slow, and may not have much significance in biological systems. However, NO reacts much faster with other radicals such as O2·−. Because several cell types can generate both NO and O2·− during activation, this reaction is believed to have much biological importance. The reaction product, ONOO−, is a powerful oxidant and cytotoxic species . ONOO− is known to be capable of leading to nitration of tyrosine residues, and nitrotyrosine is a specific biomarker of ONOO− generation [2,5,33]. Bian and Murad  investigated that the distrubution of nitrotyrosine in spleen, lung and liver, all of which are rich in macrophages and endothelial cells in LPS-treated rats, and they showed that the density of some nitrated proteins was significantly enhanced after LPS treatment by immunublotting and immunohistochemical methods.
The other important aim of our study was to investigate whether taurine is able to protect spleen tissue against LPS-induced damage. In previous studies, it has been shown that taurine has antioxidant effects through scavenges oxygen and nitrogen free radicals in sepsis models [24, 34]. In the present study, taurine administration significatly reduced spleen NOx and 3-NT levels in taurine+endotoxemia group, and taurine treatment reversed all LPS effect to near control level. We are not able to find any articles about the effect of taurine on spleen NOx and 3-NT levels in endotoxemia. In our previous study, we determined that taurine administration markedly decreased 3-NT concentrations in the hepatocytes of LPS-treated guinea pigs . It is known that taurine regulates inflammatory process through the generation of taurine chloramine (TauCl). TauCl is produced by the reaction between taurine and hypochlorous acid, a microbicidal agent generated by the myeloperoxidase-hydrogen peroxide-halide system, in activated phagocytic cells which found extensively in spleen [36,37]. In in vitro and in vivo studies, it was shown that TauCl inhibits the translation and transcription of iNOS gene, production of O2·−, and down-regulates production of tissue-damaging proinflammatory mediators. Moreover, it was reported that exogenous taurine increases TauCl formation in the cytosol of inflammatory cells [38–41]. These data may be an explanation for decrease in 3-NT and NOx levels in taurine+endotoxemia group. However, as seen in our results, we also observed that there was different effect of taurine if alone and together with LPS. Interestingly, after taurine administration alone, NOx and 3-NT levels increased compared to both control and taurine+endotoxemia groups. Spleen cells have a basal taurine concentrations which are necessary for their physiological functions. Thus, taurine administration may cause cytotoxic effects by accumulating in cells in control animals. Base on these data, it can be suggested that taurine supplementation shows protective effects only under inflammatory conditions due to increased necessity for taurine. However, taurine may act like a prooxidant in healthy subjects.
On the other hand, since many biological functions of taurine rely upon its intracellular concentration, we also measured the levels of taurine in spleen tissues by HPLC. We found that LPS administration significantly decreased levels of taurine compared with control group. In our previous study, we showed that LPS treatment reduced about sevenfold levels of taurine in hepatocytes compared to control level . Similarly, Janssen et al.  reported that the intravenous injection of LPS caused taurine depletion in heart tissues in dogs. In previous studies, it has shown that plasma taurine concentrations are reduced in patients with sepsis, and it has suggested that taurine may has a future role in the management of endotoxemia [43–45]. Decreased taurine levels in endotoxemia may be dependent an increased expenditure and requirement for taurine during these conditions . In this study, we also determined that taurine administration significantly elevated spleen taurine content in both taurine and taurine+endotoxemia groups, a similar finding as previous studies. Parildar et al. [47,48] used a rat model to investigate the influence of chronic taurine treatment on prooxidant-antioxidant balance in several tissues. They reported that taurine levels were significantly increased in the brain, heart and liver tissues of taurine-treated rats. In addition, previously, it was demonstrated that injected [3H] taurine had a rapid uptake by the spleen and liver . The contribution of taurine transport dependens upon the expression level and regulation of a taurine transporter (TauT), but also upon taurine level in extracellular medium. Increased maximal veliocity of taurine transport and increased levels of TauT mRNA have been consistently reported in a great variety of cells exposed to a hypertonic medium [49,50]. In the present study, we found that taurine plus endotoxin administration increased levels of taurine more, and the maximal mean of taurine level was seen in the taurine+endotoxemia group. Bridges et al.  reported that NO regulates the expression and activity of TauT in epithelial cell culture. At taurine+endotoxemia group, highest levels of taurine may be explained by both increased levels of extracellular taurine and stimulation of TauT mRNA expression by NO. These findings suggest that taurine supplementation may be beneficial via increasing tissue taurine content in critical illness such as endotoxemia.
In conclusion, LPS administration significantly elevated NOx and 3-NT levels in spleen tissues. Taurine showed protective effect against LPS-induced oxidative stress, and significantly reduced NO-mediated damage in spleen. However, the effect of taurine is different when it is administered alone, and taurine acts as a prooxidant in healthy subjects. In future studies, various taurine doses and experimental periods (at 0, 6, 12, 18 and 24th hours) may be experienced for understanding of the potential role of taurine in endotoxemia.
This study was supported by Gazi University, Department of Scientific Research Projects Unit. The authors thank TUBITAK (The Scientific and Technological Research Council of Turkey) for the fellowship to F.S.B.