UV-B Exposure Increases the Activity of Indoleamine 2, 3-Dioxygenase (Ido) and Alters the Levels of Tryptophan Metabolites in Indian Ground Squirrel (Funambulus Palmarum) Lens

The ocular lens of the ground squirrel is an excellent model for studies of eye light interactions that may apply to the human system. UV-B radiation induced damage to the eye lens is believed to be mediated through ROS. Antioxidant systems, both enzymatic and nonenzymatic aid in preventing the damage to the lens. The activity of indoleamine 2,3dioxygenase (IDO), a first rate limiting enzyme of tryptophan catabolism, has been studied in the lenses of Indian ground squirrel exposed to UV-B radiation. Further, antioxidants and oxidative stress markers have also been investigated to assess the oxidative status. Our investigation revealed an increased indoleamine 2,3-dioxygenase activity and tryptophan metabolites suggesting their protective role in UV stress. The study indicates that UV-B irradiation for 12 h and 24 h (300 nm; 100 μw/cm2) of lens samples led to a marginal decrease in GSH and increase in carbonyls and MDA levels reflecting the role of indoleamine 2,3-dioxygenase in protecting the lens tissue. UV-B Exposure Increases the Activity of Indoleamine 2, 3-Dioxygenase (Ido) and Alters the Levels of Tryptophan Metabolites in Indian Ground Squirrel (Funambulus Palmarum) Lens Nagalaxmi V1, Praveen Kumar M1, Sashidhar RB2 and Turlapati Naga Raju1* 1Department of Zoology, Osmania University, Hyderabad, India 2Department of Biochemistry, Osmania University, Hyderabad, India *Corresponding author: Dr. Turlapati NagaRaju, Department of Zoology, Physiology Division, University College of Science, Osmania University, Hyderabad, 500 007, India, Tel: 040-42212242; E-mail: rajutn57@gmail.com Received October 12, 2015; Accepted December 11, 2015; Published December 21, 2015 Citation: Nagalaxmi V, Praveen Kumar M, Sashidhar RB, Naga Raju T (2015) UV-B Exposure Increases the Activity of Indoleamine 2, 3-Dioxygenase (Ido) and Alters the Levels of Tryptophan Metabolites in Indian Ground Squirrel (Funambulus Copyright: © 2015 Nagalaxmi V, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.


Introduction
UV-B radiation (280-320 nm) is absorbed predominantly by the outer structures of the eye, particularly the cornea, with relatively little radiation reaching the lens and retina [1,2]. Under natural conditions these rays are fltered partially by the ozone layer, but some still reach the earth's surface. Near-ultraviolet (UV) radiation represents around 25% of the solar spectrum and approximately 10 mW/cm 2 (300-400 nm) can impinge upon the eye from direct sunlight. Light in the near-UV range at this irradiance level is capable of photochemically damaging mammalian cells [3]. Te eye thus does receive UV-B radiation, which is greatly attenuated by the time it gets past the cornea and aqueous humor before reaching the lens [4]. Yet even this amount of UV-B, incident for extended periods of time, can cause chemical and biochemical damage to the constituents of the lens causing cataract [5][6][7]. Ultraviolet-B (UV-B) and ultraviolet-C (UV-C) irradiation induce the oxidation of tryptophan [8]. Te oxidation of tryptophan catalyzed by indoleamine 2, 3-dioxygenase (E.C 1.13.11.52) results in oxidative end products within the kynurenine pathway [9]. Tey are also produced, albeit, to a lesser extent, by enzymeindependent UV mediated oxidation of tryptophan [10]. Earlier observations from our laboratory with WNIN rats showed changes in indoleamine 2, 3-dioxygenase expression during cataractogenesis [11]. Tryptophan oxidation is enhanced when IDO is activated by interferon-γ or by superoxide anion. A series of enzymatic reactions convert kynurenine to anthranilic acid (AA), 3-hydroxykynurenine (3HK) and 3-hydroxyanthranilic acid (3HAA) [12,13]. Subsequently, 3HK is glycosylated to form 3-hydroxykynurenine O-glucoside (3HKG). Van Heyningen [12] frst reported the identifcation of the lens pigment in the grey squirrel (Sciurus carolinensis leucotis) as N-acetyl-3-hydroxykynurenine and this was confrmed at a later date using more advanced techniques [3]. Major UV flters (N-acetyl 3-hydroxykynurenine and N-acetyl-kynurenine) in the lens of the thirteen lined ground squirrel (S. tridecemlineatus) have been identifed and quantifed [7]. Te lenses of both the grey squirrel and ground squirrel contain N-acetyl-3-hydroxykynurenine as the major UV flter; however, N-acetyl-kynurenine appears to be unique to the ground squirrel. For the present investigation we have chosen Indian 3-lined ground squirrel which is found naturally in southern India. Tese kynurenine metabolites may also act as UV flters and reduce chromatic aberration to sharpen the retinal image. Further, they may also minimize or reduce photo-oxidative damage that may result from prolonged exposure of the lens and retina to the high energy wavelengths of light.
Te lens has an efcient UV protection system that acts to ameliorate the efects of photo-oxidation. In the present study, we have measured the activity of indoleamine 2,3-dioxygenase and quantifed the tryptophan metabolites in Indian ground squirrel (Funambulus palmarum) lens samples which were subjected to UV-B irradiation. Our experimental investigation indicated that exposure to UV-B light leads to an increase in indoleamine 2,3-dioxygenase activity, resulting in an increase in tryptophan metabolites (kynurenine and 3-hydroxykynurenine), which act as UV flters. Further, irradiation led to an increased lipid peroxidation and protein carbonyls and a decrease in GSH suggesting an oxidative insult.  acetate, catalase, kynurenine, 3-hydroxykynurenine, L-tryptophan,  2-thiobarbituric acid (TBA), 1,1,3,3-tetraethoxy propane (TEP),  ortho-pthalaldehyde  (OPT),  reduced  glutathione  (GSH), 2,4-dinitrophenylhydrazine, diethylenetriaminepentaacetic acid (DETAPA), pyrogallol and bovine serum albumin (BSA) were obtained from Sigma Chem.co. All other chemicals and reagents used were of analytical grade.

Animals
Adult three lined ground squirrels (Funambulus palmarum), weighing approximately 150 g, were obtained and animal care protocols were in accordance with and approved by Institutional Animal Ethics Committee (383/01/CPCSEA). Animals were sacrifced by Co2 asphyxiation and lenses were dissected by posterior approach.

Irradiation
Intact lenses were suspended in RPMI-1640 medium, containing 10 mM HEPES bufer pH 7.2 and 0.01% sodium azide (1 ml/lens) in the quartz cuvctte by employing a perplex sheet. Lenses were irradiated at 300 nm at room temperature (22 o C) for diferent time periods (up to 24 h), placed in the cuvette in such a way that the anterior portion faces the light source. Lenses kept in the dark under similar conditions served as control. Te source of UV light was a 150 W xenon high pressure lamp attached to a double monochromator [14]. Te distance between the sample holder (cuvette) and exit slit of the monochromator was 6 cm. Te intensity of the incident light falling on the sample was found to be 100 µW cm, as measured with an ultraviolet meter (Blak-Ray, UVP Inc, Model J-225). At the end of the time period, the lenses of 12 h and 24 h corresponding to control and irradiated samples were taken, homogenized and centrifuged at 14,000×g for 20 min at 4 o C for indoleamine 2,3-dioxygenase, SOD and protein carbonyls. Total lens homogenate (10%) was used to determine the levels of MDA, GSH and tryptophan metabolites.

Preparation of the lens homogenate
Four to six lenses have been pooled, weighed and 10% (w/v) homogenate was prepared in 50 mM sodium phosphate bufer pH 7.4 for all assays. Lenses used for the study are categorized as follows: C12 h (Control 12 hours), C24 h (Control 24 hours), E12 h (Experimental 12 hours), E24 h (Experimental 24 hours).

Protein estimation
Protein (total, soluble and insoluble) was estimated according to Lowry [14] using BSA as reference standard. For total protein estimation the lens homogenate (10%) was prepared in 50 mM sodium phosphate bufer pH 7.4. Total homogenate was centrifuged at 14,000×g for 20 min at 4 o C. Te bufer soluble supernatant was used for estimation of soluble protein and the residue was dissolved in TNEN bufer pH 8.0 [Tris bufer 25 mM containing Nacl (0.1 M), EDTA (0.5 mM), and sodium azide (0.01%)] and used for the estimation of insoluble protein.

Assay of Indoleamine 2, 3 -dioxygenase activity
Lens homogenate was centrifuged at 14,000×g, 4°C, for 15 min and supernatant was used for the indoleamine 2,3-dioxygenase assay. Te assay was carried out according to the method of Mailankot with minor modifcations. Te standard assay medium (200 µl) contained 0.5 M potassium phosphate bufer (20 µl, pH 6.5) at a fnal concentration of 50 mM, 0.2 M ascorbic acid (20 µl neutralized with 1M NaOH solution) at a fnal concentration of 20 mM, 0.5 mM methylene blue (4 µl) at a fnal concentration of 10 M, 5 mg/ml catalase (4 µl) at a fnal concentration of 100 g/ml, 2 mM L-tryptophan (20 µl) at a fnal concentration of 200 mM, MilliQ water (112 µl) and enzyme source (total soluble protein 1 mg equivalent protein). Te reaction mixture was incubated at 37°C for 1 h and 40 µl of 30% (v/v) TCA was added to the assay mixture. Te mixture was incubated at 60°C for 15 min to hydrolyze N-formyl kynurenine to kynurenine. It was then centrifuged at 14,000×g, 4°C, 15 min and the amount of kynurenine and 3-hydroxykynurenine formed were quantifed by method outlined above. Indoleamine 2,3-dioxygenase activity was expressed as nanomoles of both kynurenine and 3-hydroxykynurenine formed per milligram protein per minute.

Quantifcation of tryptophan metabolites by HPLC
To 100 µl of lens homogenate (control and UV irradiated samples) 0.3 ml of 100% (v/v) ethanol was added and placed at room temperature for 1 h and then centrifuged (14,000×g, 15 min). Te supernatant was removed, kept at -20°C, whereas the pellet was re extracted twice with 0.3 ml of 80% ethanol. Te supernatant was observed to be pale yellow in colour. Te supernatants were pooled and dried in a speedvac concentrator (Eppendorf AG · 22331 Hamburg, Germany). Kynurenines were quantifed by the method described by Hains et al. [6]. Metabolites have been estimated by reverse phase HPLC. Te column was equilibrated in 0.1% (v/v) TFA, at a fow rate of 0.7 ml/min. Samples were resuspended in 100 µl of 0.1% (v/v) TFA, centrifuged for 10 min at 12,000×g and loaded on to the column. UV flters (N-acetyl-3-hydroxykynurenine, N-acetyl-kynurenine, kynurenine and 3-hydroxykynurenine) were eluted with the following gradient; 0-5 min 0% bufer B (0.08% (v/v) TFA, 80% (v/v) acetonitrile, water), 5-50 min 0-50% B, 50-60 min 50-0% B. Te eluent was monitored using UV detector at 360 nm. In a similar way, reference kynurenine and 3-hydroxykynurenine were used to construct standard curves.

Synthesis of α-N-acetyl-kynurenine and α-N-acetyl-3hydroxykynurenine
Kynurenine (2 mM in 800 mM sodium phosphate bufer pH 7.5) was taken in a reaction chamber at 37°C and to this 8 mM acetic anhydride was added drop wise. Te reaction was allowed for 1 hr with continuous and gentle magnetic stirring under subdued light and N-acetyl-kynurenine were found to be unaltered in both 12 h and 24 h exposures. Te α-amino groups of both Kynurenine and 3-hydroxykynurenine can act as acceptors for the acetyl group suggesting an efcient lenticular mechanism for acetylation of the α-amino group of these metabolites. Acetylation of the α-amino group will prevent the deamination of the tryptophan derived UV flters and this perhaps represents an efcient mechanism for stabilizing UV flters in the squirrel lens (Figure 4).

Protein content
Tere was no net change in the soluble and insoluble protein content (Table 3) in both control and UV-B (12 h and 24 h) exposed samples.

Antioxidants
In oxidative stress, the parameters commonly considered are glutathione (GSH), superoxide dismutase (SOD), malondialdehyde conditions. Te fnal reaction mixture contained 1 mM kynurenine in 400 mM sodium phosphate bufer pH 7.5 with 4 mM acetic anhydride. Te products were separated by HPLC as described above and the major components were analyzed by mass spectrometry.

Statistical Analysis
Te diferences between the control and UV irradiated samples were analyzed using simple t-test and the diferences were considered signifcant if p<0.05. All data is expressed as mean ± SD.

Results
Te present study is an attempt to understand the role of indoleamine 2,3-dioxygenase and antioxidants under UV-B stress. Indoleamine 2,3-dioxygenase enzyme was assayed by the method described [9] with minor changes. Enzyme activity was expressed as the quantity of both kynurenine and 3-hydroxykynurenine (Table  1) formed and standard graphs were plotted. Te retention times for 3-hydroxykynurenine and kynurenine are 11 and 21 min respectively. Signifcant (p<0.05) increase in 3-hydroxykynurenine and marginal increase in kynurenine (Figure 1) was observed when lens homogenate was exposed to UV-B irradiation for 12 h in relation to control and 24 h UV exposure (Figure 2) led to a signifcant (p<0.05) increase in both kynurenine and 3-hydroxykynurenine.

Content of kynurenines in control and UV-B irradiated groups
A typical HPLC profle of the UV flters in control and experimental (12 h and 24 h) of the ground squirrel lens homogenate is depicted in (Figure 3). All the four metabolites viz.,Kynurenine, 3-hydroxykynurenine, N-acetyl-3-hydroxykynurenine and N-acetylkynurenine in squirrel lens were quantifed (Table 2) by HPLC. Mass spectrometry was carried out to confrm their conversion and mass values were in agreement with those reported in literature [7] several major peaks have been observed that absorbed at 360 nm. Our data confrms the conversion of 3-hydroxykynurenine to N-acetyl-3-hydroxyKynurenine and a clear cut wavelength shif was also observed. As a result of conversion, the maximum absorption of N-acetyl-3-hydroxyKynurenine was recorded at 300 nm. A marginal increase in both 3-hydroxykynurenine and kynurenine for 12 h exposure and signifcant (p<0.05) increase in kynurenine for 24 h exposure was observed. However, the levels of N-acetyl-3-hydroxykynurenine      (Table 3). SOD activity marginally increased when the lens sample was exposed to UV light for 12 h. However, exposure to UV-B for a longer time (24 h) resulted in marginal inhibition of SOD activity (Table 3) in relation to the 12 h exposure and the activity had reverted to the control level.

Oxidative stress markers TBARS and protein carbonyls content
A marginal rise in MDA levels was observed upon 12 h exposure and 24 h UV exposure showed signifcant (p<0.05) increase (Table 4). Protein carbonyls (12 h and 24 h UV exposures) were signifcantly (p<0.05) higher (Table 4) in irradiated groups than their corresponding control groups.

Discussion
Risk of cortical cataract mediated by UV light was reported by Heck [24]. Near-UV radiation is known to cause oxidative damage to the lens by a variety of free radical mediated reactions. In the present investigation, characteristic alterations in indoleamine 2,3-dioxygenase activity were observed in 12 h UV-B exposure, which points to the fact that shorter duration of UV-B was found to induce the changes in enzyme activity and other concomitant changes reported herein.
Te present investigation was an attempt to study direct efects of UV-B radiation in squirrel lenses in vitro on indoleamine 2,3-dioxygenase which is involved in many biological processes. Indoleamine 2,3-dioxygenase activity has been expressed by taking into consideration of the levels of both 3-hydroxykynurenine and kynurenine and this is in agreement. In the present study a signifcant (p< 0.05) increase in indoleamine 2,3-dioxygenase activity has been observed in 12 h and 24 h exposure of UV-B radiation. Under normal physiological conditions indoleamine 2,3-dioxygenase activity is reported to be low [20,21]. However, the indoleamine 2,3-dioxygenase activity of the 24h exposure was higher than that of its corresponding control lens sample. Eventhough the 24 h UV exposure control values decreased, the fold diference remains the same for both 12 h and 24 h exposures, indicating an increased indoleamine 2,3-dioxygenase activity under UV-B exposure. Te rise in indoleamine 2,3-dioxygenase has been in agreement with the previous studies [22] which suggest its protective infuence on corneal endothelial cells mediated by UV by reducing rate of apoptosis and lipid peroxidation. It has been previously reported that indoleamine 2,3-dioxygenase is an antioxidant enzyme since it happens to be a direct scavenger of superoxide radicals [23]. Te native state of lens sample continued to be intact and this may be attributed to an increase in indoleamine 2,3-dioxygenase activity and hence not much oxidative damage has been observed during 12 h exposure. Te increase in levels of kynurenines may be linked to an increase in indoleamine 2, 3 dioxygenase activity which cleaves the pyrrole ring of tryptophan and forms formyl kynurenine using free oxy radicals. Tey are also produced, albeit, to a lesser extent, by enzyme independent UV mediated oxidation of tryptophan. Tese kynurenine metabolites may also act as UV flters and reduce chromatic aberration to sharpen the retinal image. Further, they may also minimize or reduce photo-oxidative damage that may result from prolonged exposure of       John had reviewed the activity of IDO in relation to tryptophan metabolism [23]. Tis review clearly indicates that >95% of tryptophan is channelized through kynurenine pathway. Upon UV exposure tryptophan metabolites such as kynurenine, 3-hydroxy kynurenine are also formed and, it can be speculated that based on the previous studies, under UV exposure it is quite logical to infer that almost all the tryptophan would be oxidized through the kynurenine pathway as well as the UV derived tryptophan derivatives due to enhanced IDO activity whose presence was reported by Heck [24]. Tis could perhaps be the reason that no detectable tryptophan was recorded in the present investigation afer UV exposure.
Our observations suggest that N-acetyl-3-hydroxykynurenine is the major UV flter in the ground squirrel lens and besides this, 3-hydroxykynurenine and kynurenine have also been found in reduced amounts. N-acetyl-kynurenine is unique to ground squirrel but its concentration is far less when compared to N-acetyl-3hydroxykynurenine. Our observations revealed a signifcant (p<0.05) elevation in kynurenine for 24 h exposure and other metabolites remain unchanged for both 12 h and 24 h exposures indicating a protective mechanism. Besides this, 3-hydroxykynurenine was also found to show a marginal increase for 12 h exposure ( Figure 5).
Te role of glutathione (GSH), as an essential and primary lenticular antioxidant is well known. Exposure for 12 h showed a marginal fall in GSH content and exposure for 24 h clearly demonstrated a signifcant (p<0.05) depletion of GSH [25,26]. Tis perhaps suggests the generation of oxyradicals in the lens homogenate mediated by UV-B stress and the impaired lens antioxidant status may be correlated to increase in ROS, as evidenced by decrease in GSH content.
SOD plays an important role as an antioxidant protein by reducing the level of intracellular superoxide radical induced by extracellular stimuli such as UV radiation. In the present study, the change in SOD activity suggests the release of superoxide radicals mediated through UV-B exposure. SOD activity marginally increased when the lens sample was exposed to UV for 12 h, suggesting an activation of SOD by scavenging superoxide radical to protect the lens from UV stress. It has been reported that an increase in SOD activity is probably a response to increased ROS generation [26]. However, exposure to UV light for longer duration (24 h) resulted in marginal inhibition of SOD activity in comparison with the 12 h exposure, and the activity reverted to the control level. Tis is in accordance with the earlier fndings which suggest that the longer duration of UV exposure suppresses the activity of SOD.
Protein carbonyl content was found to be signifcantly elevated in lens homogenate exposed to UV-B for 12 h and 24 h exposures in relation to control suggesting UV-B mediated oxidative stress. Protein carbonyls tend to get accumulated on the side chains of proteins as a result of oxidative stress [26]. MDA, a major oxidation product of peroxidized polyunsaturated fatty acids, has been used to determine the degree of lipid peroxidation and as a biological marker of oxidative stress. A marginal rise in lipid peroxidation has been observed in 12 h exposure. Tis marginal rise of MDA may be due to the enhanced activities of antioxidant enzymes which can scavenge excess lipid peroxidation. And, a signifcant (p<0.05) increase was noticed upon 24 h exposure in relation to control. Tese results are in agreement with the observation of [14] and Reddy [13] who reported variations in lipid peroxidation and GSH in rat lens subjected to UV-B radiation.
Our observations have revealed that squirrel lens homogenate exposed to UV-B light has led to an increase in IDO activity and tryptophan metabolites. Among UV radiation blocking compounds, certain aromatic amino acids of proteins, such as tryptophan and its metabolites including kynurenine and 3-hydroxykynurenine play a key role as UV flters in lenticular tissue by absorbing UV radiation between 300 to 400 nm and thus protecting both the lens and the retina from photo-oxidative damage. Keeping all this in view and based on previous fndings we hypothesize that indoleamine 2,3-dioxygenase renders distinct a protective infuence against UV damage in squirrel lens.