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Effect of ultraviolet B (302nm) irradiation on viability, metabolic and detoxification functions of goat hepatocytes-In vitro study

Naseem Begum Shakeel1, Vijayalakshmi Venkateshan2, Parveen1, Adarsh K Capoor1, , Mohammed Aejaz Habeeb1, Ansar Ali Khan3, Syed Muzeeb3, NVS Rao Mamidi3, Aleem Ahmed Khan1, Chittoor Mohammed Habibullah1
1. Owaisi Hospital and Research Centre, Deccan College of Medical Sciences, Kanchanbagh, Hyderabad-500 058. India. Tel/Fax: 091-040-4342954. 2. National Institute of Nutrition, Tarnaka, Hyderabad. Tel: 27008921. 3. Dr. Reddy’s Research Foundation, R&D Centre, Bollaram Road, Miyapur, Hyderabad. Tel: 3045439.

Correspondence:

Dr. Vijalakshmi Venkateshan Assistant Director National Institute of Nutrition, Tarnaka, Hyderabad. 500 007 Email: v_venkateshan@hotmail.com naseembegum@hotmail.com

2. lipid peroxidation. metabolic and detoxifying capacity of the isolated goat hepatocytes.5-diphenyltetrazoloum bromide [MTT] assay. The results show that there was no difference in functional. 3-[4.2 ABSTRACT The object of the present study was to investigate the effect (s] of UV-B irradiation on the functional integrity. Our present findings suggest that the biologically compatible and feasible dose of UV-B irradiation for xenotransplantation appears to be 1250 Joules/m2. viability.1. 250. and CYP450 activity when irradiated beyond 1250 Joules/m2. Cells were then analyzed for Viability (Trypan blue exclusion test [TBE].5. Cytochrome P450 activity [CYP450.5-dimethylthiozol-2yl]-2. 2500 and 7500 Joules/m2 which corresponds to the irradiation time of 0. Xenogeneic hepatocyte . metabolic as well as detoxifying parameters of the hepatocytes when irradiated from 0-1250 Joules/m2. Hepatocytes. Diazepam metabolism] and Glutathione-S-Transferase [GST] activity. whereas a significant alteration was appreciable in the parameters such as LDH leakage. Membrane integrity (Lactate dehydrogenase [LDH] leakage. 1250. 500. transplantation. detoxification.10 and 30 minutes. Ultraviolet B irradiation. Lipid peroxidation) Detoxification (Ureagenesis. KEYWORDS Goat. Isolated goat hepatocytes were subjected to UV-B irradiation invitro for 0.

2500 and 7. membrane integrity and detoxification. These observations implicate a clinical significance for UV-B irradiation in transplantation biology. 500.10]. the major problem associated with XHT is the predominance of immune rejection due to transplantation across the species barrier. MATERIALS AND METHODS: The use of animals for experimentation was approved by the Committee for the Purpose of Control and Supervision of Experiments on Animals. Studies invitro and invivo have demonstrated the beneficial effects of UV-B irradiation that abrogates the allograft rejection of transplanted hepatocytes [3. having big litters. cell-to-cell contact and suppression in delayed hypersensitivity [6. cell surface antigens.7]. The aim of the present investigation was to study the invitro the effect(s) of UV-B irradiation at 250. we have used the outbred adult goat as the donor animal as it fulfills the criteria of an optimum donor species such as domestication. gentle. Literature survey shows that there are few reports available correlating the efficacy of UV-B irradiation and hepatocyte transplantation in higher animals [8]. 1250.2]. UV-B irradiation has shown to be biologically compatible and it spares the functions of specialized cells unlike UV-C. Ministry of Social Justice and Empowerment. Hepatocytes were isolated from the liver using the collagenase perfusion method as . Government of India.4]. easy to feed and grows rapidly [9. hepatocytes subjected to UV-B irradiation have elicited changes in their cell membranes. There are no reports in the literature demonstrating the use of goat hepatocytes for transplantation in the management of acute liver failure. The outbred adult goats used for the study were examined by a veterinarian to monitor their status of health.500 J/m2 on goat hepatocytes. However. less expensive.3 INTRODUCTION: Xenogeneic hepatocyte transplantation (XHT) is emerging as a therapeutic potential and an alternative to orthotopic and auxillary liver transplantation in the treatment of acute liver failure [1. In the present study. Further. which renders damage to the haemopoietic stem cells [5]. Nevertheless these investigations have been carried out in small animal models such as rodents and studies in larger animals merit advantage as a therapeutic potential prior to the clinical transplantation in man. Hepatocyte functions following UV-B irradiation have been evaluated by measuring the parameters such as viability.

Lipid peroxidation was measured by malondialdehyde (MDA) formation by the .1% BSA.15% triton-X100. The percentage of extracellular LDH activity (LDH leakage) was calculated as the ratio of LDH activity in the supernatant to the total activity in the supernatant and cell pellet x100.5. samples were stained with Haematoxylin and Eosin ( H &E). UK) at 1000 rpm x 10 min. using the UV lamp (UVM-57. For LDH leakage. USA) and radiometer assembly (UVX-31. LDH activity in pellet was determined after solubilizing the pellet with 200 µl of solubilizing solution containing 0. Shandon Southern Products Ltd. Morphology of the isolated hepatocytes was checked by Haemtoxylin and Eosin (H&E) staining. The samples were prepared and immediately fixed in ethanol and acetone (1: 1) at R. For TBE. Chesire.Merk India Ltd). Sigma Chemical Co) at 37oC for 2 hours.4 described by Vijayalakshmi et al. hepatocyte suspension was centrifuged at 700xg for 10 minutes and LDH activity was determined in the pellet and in the supernatant using the LDH kit (E. The isolated hepatocytes were suspended in Hank’s medium at a concentration of 4x106/ml between 22-25oC.10. Cells were subjected to UV-B irradiation at doses 250. Viability: Viability of the hepatocytes before and after UV-B irradiation was determined by TBE test and MTT assay [13. Ultraviolet Products Limited. 0. 2500 and 7500 J/m2 which corresponds to 0. For MTT assay. 1250. Ultraviolet Products Limited. Membrane integrity: Membrane integrity was determined by LDH leakage and Lipid peroxidation. which was measured at 540nm and expressed as µM formazan/106 cells. Hepatocytes were then sedimented and the formazan formed was solubilized by adding isopropanol. 0.2.T.4%) in the ratio of 1:1 and counted in a haemocytometer under the microscope. After drying the slides. the cells (4x106) were incubated with MTT (1mg/ml. The purplish blue colour of the supernatant is the amount of formazan formed. cell suspension was diluted with Trypan blue (0.14]. USA) (12).1x10 6 cells/ml was subjected to cytospin (Shandon Cytospin.9% NaCl. Hepatocytes at a concentration of 0. 500.1. 2003[11].30 minutes.

1b). The activity of the detoxifying enzyme.D of eight independent experiments. viability as assessed by TBE and MTT assay was not affected upto 2. With increase in linear dosage of UV-B. GST was assayed using 1-chloro2. a pink coloured product formed was measured at 532nm. STATISTICS: The data presented here are the mean ± S.05. MDA was estimated in the hepatocyte suspension by ultilizing its property to react with 2-thiobarbituric acid (TBA). hepatocytes were suspended in 10mM ammonium chloride and incubated for a period of 1 hour at 37oC.4dinitrobenzene(CDNB) as substrate (17).001) compared to control hepatocytes (Fig 1a.500 J/m2 but there was a significant decrease at 7. The supernatant was determined for the amount of urea formed at 530nm using the urea Kit (E. RESULTS: Light Microscopy: H and E staining of the isolated hepatocytes reveals an intact cell membrane with cytoplasm and nuclei. . The measurement of CYP450 activity was based on the metabolism of diazepam as substrate by HPLC method.5 method of Ohkawa et al.).Merck. The values have been expressed as mM urea/106 cells. Level of significance was considered as p<0.. India Ltd. 1979 (15).500 J/m2 (**p<0. The cytosolic fraction was monitored for measuring the increase in absorbance of CDNB-GSH conjugate at 340nm and the activity expressed as µM CDNB-GSH conjugate/min/mg protein. Detoxification: For the measurement of urea. One-way analysis of variance has been used to compare the mean values between groups with post-hoc test. Viability: TBE test demonstrated a viability of 90-95% for control (non-irradiated) hepatocytes. Monitoring the eluent for metabolites (Oxazepam and Desmethyl diazepam) and drug (Diazepam) using a PDA detector operating at 247nm shows the amount of diazepam metabolized which is directly related to CYP450 activity (16).

001). UV-B irradiation in the range of 280-320nm has emerged as a simple and promising technique of immunosuppression and its therapeutic efficacy has been demonstrated in transplantation biology. They have shown promising results in xenobiotic oxidative metabolism and they have also been used as recipients in xenotransplantation studies [20. an attempt has been made to understand the viability. It been shown to elicit profound immunomodulatory effect by minimizing the rejection signals resulting in the achievement of allograft survival with or without the need for short-term immunosuppression [1.4]. DISCUSSION Hepatocyte Transplantation (HT) appears to be a promising alternative to liver transplantation.500 J/m2 (*p<0. **p<0.001). CYP450 and GST activity showed comparable results upto 1250 J/m2 and a significant decrease at 2. The present study has been undertaken to assess the optimal UV-B irradiation dose on goat hepatocytes that may be used in invivo transplantation in an appropriate acute liver failure model.3. This is of utmost importance as the primary requirement of cells used for therapy is the preservation of the viability and metabolic functions so that they prevent/decrease hepatic encephalopathy . membrane integrity and detoxification of goat hepatocytes as a function of UV-B dose response.21]. Among the strategies to prevent immune rejection. Investigators have also demonstrated improvement in survival in fulminant hepatic failure patients with allogeneic hepatocyte transplantation [18. Studies have been carried out to assess the efficacy of HT in acute liver failure in animal models and have shown promising results [1]. which has renewed interest in the use of xenogeneic cells for transplantation.**p<0. Goats appear to be attractive candidates as donor animals for xenotransplantation. So. Shortage of human donors is the major limitation for clinical transplantation. However.6 Membrane integrity as assessed by LDH leakage and lipidperoxiation was comparable to non-irradiated hepatocytes upto 1250 J/m2.19].500.05.05. there was a significant damage at 2500 J/m2 and 7500 J/m2 (*p<0. Detoxifying functions such as Ureagenesis. 7.

Glutathione S-transferases are a group of multifunctional proteins involved in the detoxification of a wide spectrum of compounds. Many investigators have used MTT assay as a marker of cellular viability [22. The major role of GSTs is to increase the detoxification of exogenous xenobiotics and their metabolites and . LDH leakage and lipid peroxidation have been used as markers of the cellular viability and integrity of the hepatocytes [25]. in vitro irradiated rat hepatocytes (200-1000 J/m2) and fetal. In the present study.500 J/m2 was used in the experiment as a positive control to check the deleterious effect of Ultraviolet B irradiation.500 J/m2. The rate of formation of urea that reflects the key hepatocyte function showed a similar response between the irradiated and the non-irradiated cells not showing any significant difference upto a UV-B dose of 1250 Joules/m2. The effect of UV-B irradiation on the membrane integrity was measured by lipid peroxidation as shown by accumulation of malondialdehyde [26]. 1250. Endogenous benzodiazepine like substances are known to involve in mediation and pathogenesis of the neuronal inhibition observed in hepatic encephalopathy. A high dose of 7.500 J/m2. CYP3A4 is the most abundant CYP450 isoform in human liver and responsible for detoxification (Phase I metabolism) of most of the drugs. porcine islets (300-1800 J/m2) elicited a similar non-toxic response in the functional integrity of the cell [24]. 500. Our results revealed no damage to the hepatocyte / hepatocyte membranes upto UV-B irradiation dose of 1250 J/m2 compared to the non-irradiated hepatocytes as assessed by LDH leakage and lipid peroxidation. the detoxification of diazepam (Benzodiazepine) by CYP2C19 and CYP3A4 isoforms of CYP450 familiy is of particular importance in hepatic transplantation studies. In compliance with our observations on goat hepatocytes. Our results showed that CYP450 activity was not altered upto a UV-B dose of 1250 Joules/m2. we have also investigated the xenobiotic metabolizing function of goat hepatocytes on exposure to UV-B irradiation at different doses of irradiation. However membrane integrity showed a significant decrease at 2500 and 7. 2500 and 7. Hence. The dosage of UV-B irradiation used was 250. The present data on the isolated goat hepatocytes showed that the viability of the cells was retained upto 2500 J/m2 with a significant decrease beyond it..7 when transplanted in patients with acute liver failure.23].

1987 4. The present data on the effect(s) of UV-B irradiation on goat hepatocytes suggests that optimal dose of ultraviolet B irradiation on goat hepatocytes is 1250 Joules/m2. 1997 pp312 – 324 2. UV-B irradiation provides an important tool to study cell/cell and donor/host interactions and necessitates that every model requires a diligent determination of an effective nontoxic and biologically compatible dose-response study. Japan Karger lander systems. we have measured the activity of GST in goat hepatocytes on increasing doses of UV-B irradiation and found that GST was also not altered with UVB irradiation observed upto 1250 J/m2. REFERENCES 1. Fox IJ and Chowdhury JR: Hepatocyte transplantation.19: 989-991. Price J B. Vijayalakshmi V. Transplant Proc. The present observations is significant as UV-B irradiation appears to be biologically compatible and is a promising tool for XHT using the large animal models. Investigators have measured GST activity in hepatic detoxification studies. 4:7. pure and mixed monolayer culture to compare the conjugation pathways in drug metabolism. Hardy M A: Reversal of liver failure in rats by ultraviolet irradiated hepatocyte transplantation.). Masauuki Sawa. Kawai Y. 200 3. As GST is an important detoxifying enzyme. Vijayalakshmi A. GST activity has been studied in adult rat and adult human hepatocytes in primary. (eds. Habibullah C M: An overview of Hepatocyte Transplantation – Fulminant hepatic failure. Rao MN .M: Differential responses of UV-B irradiation on the viability and intracellular . Naseem B S. in: Michio Mito. American Journal of Transplantation. Hepatocyte Transplantation.8 endogenous toxic compounds via the phase II reaction of detoxification pathyway. Nandini R Habibullah C.

Capoor AK. Tanabe S. 19:10291035. Sci. pig and human fetuses. Extention goat handbook. 59 (12):1660-1665. Cooperative Extension Service. Institute of Laboratory Animal Resources. Khan AA.9 calcium influx in goat hepatocytes-in vitro effect. 80:87-93. 1993 8. Vet. Journal of Gastroenterology and Hepatology. Ace. Molecular and Cellular Biochemistry. Immunol. ILAR News. Med. 1994 vol 36 pp21-29 10. 55:853-858. Transplantation. Clinical studies and Biological effects. University of Deaware.M: Comparison of Biochemical and Cytotoxic functions of hepatocytes from goat. 1984 7. Vijayalakshmi V. Taura Y. Newark. Naseem B. Miyamoto M. 2004 5. J. et al: The functional and immunomodulatory effects of ultraviolet light on fetal porcine pancreas. et al: Suppression of delayed type hypersensitivity (DTH) responses on xenografts by pretreatment with ultraviolet irradiated hepatocytes. Farris Jr EH: Farm animals in biomedical research part 2. Immunology Today. Fulton K L. (eds). in: George F W. Hall A: The pygmy. Kenmouchi T. Rev. Clarke S M. Benhamou P Y. Alnaqdy AA. Haenlein and Donald L. Lincicome P P. Tanka M.2004 . The goat as a model for biomedical research and teaching. 1995 9. 1984 pp1-4 11. 266:1616-166. Kripke M L: Immunological unresponsiveness induced by ultraviolet irradiation. Pamphilon DH.1991 6. 12:119123. Deaware. Habibullah C. Wallington TB: Immunomodulation by ultraviolet light.

Karrer F. Syed IH. Transplantation. Ramesh and Srinivas NR:Open acces generic method for continuous determination of major human CYP P450 probe substrates/metabolites and application in drug metabolism studies. Ohishi N. Lahiri S. 1994 19. Rao MNVS. (ed. Habibullah CM. et al: Human fetal hepatocyte transplantation in patients with fulminant hepatic failure. 58: 951-952. Biju B. Anal Biochem. Seglen P O: Preparation of isolated rat liver cells. Ansar AK. Bilir MB. Ohkawa H.15:149-152. Yagi N: Assay of lipid peroxides in animal tissues by thiobarbituric acid reaction. 249: 7130-7139. Qamer Q. et al: Effect of UV-B (302nm) irradiation on isolated rat hepatocytes. van’t Klooster G.2003 17. Mossmann T: Rapid colorimetric assay for cellular growth and survival. Anfossi P. 1976 vol 13 pp29-83 14.10 12. 65:55-63. 6(1): 32-40. 1979 16. Habibullah C M. Chem. Application to proliferation and cytotoxicity assays. Guinette D. Academic Press New York. Liver Transplant.95: 351-358. Methods in Cell Biology. J. 1995 13. 20(5): 449-460. Ayesha Q. 1974 18. Vet Res Commun. in: Priscott DM. Habig WH. 1996 . J. Liver. et al: Hepatocyte transplantation in acute liver failure.Immunol Meth. Biol. 2000 20. Pabst MJ and Jakoby WB: Glutathione-S-Transferases: The first enzymatic step in mercapturic acid formation. 1983 15. Montesissa C. et al: The use of cultured hepatocytes from goats and cattle to investigate xenobiotic metabolism.). Mujeeb M. Xenobiotica 33:1233-1245.

1997 22. Xu H. Res. malonaldehyde and related aldehydes. 11:81-128. A study of UV-B irradiation. Shauer RJ.11 21. Tsang A: Xenotransplantation of adult porcine islets in diabetic mice. Song W. Cheung S. 30(80): 509-513.5diphenyltetrazolium bromide (MTT) reduction activity and lactate dehydrogenase. Gundry SR. Tze W J. Am J Clin Med. Tsai SJ. 27 (1):95105. Neurosci. 1999 26. et al: Prolonged discordant cardiac xenograft survival in newborn recipients. Circulation. 21:415-420. Abe K. 1998 25. Zhu XZ. lipid peroxidation. diallyl disulphide on cell viability. 96(2):364-367. Guan HJ.38(4): 325-329.5-dimethylthiazol-2yl)-2. Tai J. 1991 LEGENDS . Hor Metab Res. Zollner H: Chemisty and biochemisty of 4hydoxynonenal. 2000 23. Acta Pharmacol Sin. Free Radic Biol. Matsuki N: Measurement of cellular 3-(4. Esterbauer H. cryopreservation and immunosuppression on graft survival time. et al: Protective effect of bilobalide against nitric oxide-induced neurotoxicity in PC12 cells. Sheu SF. et al: Effect of garlic active principle. Hill AC. 2000 24. Med. glutathione concentration and its related enzyme actitivities in primary rat hepatocytes. Sheen LY.

H&E staining of the isolated goat hepatocytes.05. Fig 2a. 4b Effect of UV-B irradiation on detoxifying functions of goat hepatocytes: Ureagenesis. **p<0.05. .001) compared to control hepatocytes. Fig 4a. 2b Effect of UV-B irradiation on viability of goat hepatocytes as assessed by TBE test and MTT assay. Fig 3a.500 J/m2 (**p<0.500. 3b Effect of UV-B irradiation on membrane integrity as assessed by LDH leakage and lipidperoxiation was comparable to non-irradiated hepatocytes upto 1250 J/m2 with a significant decrease at 2500 J/m2 and 7500 J/m2 (*p<0. 7. viability was not affected upto 2.500 J/m2 (*p<0.001) .500 J/m2 but there was a significant decrease at 7. CYP450 and GST activity showed comparable results upto 1250 J/m2 and a significant decrease at 2. With increase in linear dosage of UV-B.12 Fig 1.001). **p<0.

13 Fig. 1 .

2a T T T ** T TBE (%) 70 60 50 40 0 250 500 1250 2500 16 mM Formazan/ 106 cells 14 12 10 8 6 4 2 7500 UV-B irradiation (J/m2) T Fig. 2b T T T T ** T 0 0 250 500 1250 2500 UV-B irradiation (J/m2) 7500 .14 100 90 80 T T Fig.

15 Fig. 3b UV-B irradiation (J/m2) ** * T T T T T T 0 250 500 1250 2500 7500 UV-B irradiation (J/m2) . 3a 50 45 40 35 30 25 20 15 10 5 0 ** LDH leakage (%) ** T T T T T 0 250 500 1250 2500 6 nmols MDA/mg Protein 5 4 3 2 1 0 7500 Fig.

4a T T T T T ** * T 250 500 1250 2500 7500 UV-B irradiation (J/m2) 100 % CYP450 activity remaining 90 80 70 60 50 40 0 250 Fig. 4b T T T T ** * T 500 1250 2500 7500 UV-B irradiation (J/m2) .16 5 mM urea/ 106 cells 4 3 2 1 0 0 Fig.

4c 80 70 60 50 40 30 20 10 0 T T T T * T ** T 0 250 500 1250 2500 7500 UV-B irradiation (J/m2) .17 n moles CDNB – GSH conjugate/mg/protein Fig.