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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. Reddys 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

ABSTRACT The object of the present study was to investigate the effect (s] of UV-B irradiation on the functional integrity, metabolic and detoxifying capacity of the isolated goat hepatocytes. Isolated goat hepatocytes were subjected to UV-B irradiation invitro for 0, 250, 500, 1250, 2500 and 7500 Joules/m2 which corresponds to the irradiation time of 0,1,2,5,10 and 30 minutes. Cells were then analyzed for Viability (Trypan blue exclusion test [TBE], 3-[4,5-dimethylthiozol-2yl]-2,5-diphenyltetrazoloum bromide [MTT] assay, Membrane integrity (Lactate dehydrogenase [LDH] leakage, Lipid peroxidation) Detoxification (Ureagenesis, Cytochrome P450 activity [CYP450, Diazepam metabolism] and Glutathione-S-Transferase [GST] activity. The results show that there was no difference in functional, metabolic as well as detoxifying parameters of the hepatocytes when irradiated from 0-1250 Joules/m2, whereas a significant alteration was appreciable in the parameters such as LDH leakage, lipid peroxidation, and CYP450 activity when irradiated beyond 1250 Joules/m2. Our present findings suggest that the biologically compatible and feasible dose of UV-B irradiation for xenotransplantation appears to be 1250 Joules/m2.

KEYWORDS Goat, Hepatocytes, Ultraviolet B irradiation, viability, detoxification. transplantation,

Xenogeneic

hepatocyte

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 [1,2]. However, the major problem associated with XHT is the predominance of immune rejection due to transplantation across the species barrier. Studies invitro and invivo have demonstrated the beneficial effects of UV-B irradiation that abrogates the allograft rejection of transplanted hepatocytes [3,4]. UV-B irradiation has shown to be biologically compatible and it spares the functions of specialized cells unlike UV-C, which renders damage to the haemopoietic stem cells [5]. Further, hepatocytes subjected to UV-B irradiation have elicited changes in their cell membranes, cell surface antigens, cell-to-cell contact and suppression in delayed hypersensitivity [6,7]. These observations implicate a clinical significance for UV-B irradiation in transplantation biology. 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. Literature survey shows that there are few reports available correlating the efficacy of UV-B irradiation and hepatocyte transplantation in higher animals [8]. In the present study, we have used the outbred adult goat as the donor animal as it fulfills the criteria of an optimum donor species such as domestication, less expensive, having big litters, gentle, easy to feed and grows rapidly [9,10]. There are no reports in the literature demonstrating the use of goat hepatocytes for transplantation in the management of acute liver failure. The aim of the present investigation was to study the invitro the effect(s) of UV-B irradiation at 250, 500, 1250, 2500 and 7,500 J/m2 on goat hepatocytes. Hepatocyte functions following UV-B irradiation have been evaluated by measuring the parameters such as viability, membrane integrity and detoxification.

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, Ministry of Social Justice and Empowerment, Government of India. The outbred adult goats used for the study were examined by a veterinarian to monitor their status of health. Hepatocytes were isolated from the liver using the collagenase perfusion method as

4 described by Vijayalakshmi et al. 2003[11]. Morphology of the isolated hepatocytes was


checked by Haemtoxylin and Eosin (H&E) staining. Hepatocytes at a concentration of 0.1x10 6 cells/ml was subjected to cytospin (Shandon Cytospin, Shandon Southern Products Ltd, Chesire, UK) at 1000 rpm x 10 min. The samples were prepared and immediately fixed in ethanol and acetone (1: 1) at R.T. After drying the slides, samples were stained with Haematoxylin and Eosin ( H &E).

The isolated hepatocytes were suspended in Hanks medium at a concentration of 4x106/ml between 22-25oC. Cells were subjected to UV-B irradiation at doses 250, 500, 1250, 2500 and 7500 J/m2 which corresponds to 0,1,2,5,10,30 minutes, using the UV lamp (UVM-57, Ultraviolet Products Limited, USA) and radiometer assembly (UVX-31, Ultraviolet Products Limited, USA) (12).

Viability: Viability of the hepatocytes before and after UV-B irradiation was determined by TBE test and MTT assay [13,14]. For TBE, cell suspension was diluted with Trypan blue (0.4%) in the ratio of 1:1 and counted in a haemocytometer under the microscope. For MTT assay, the cells (4x106) were incubated with MTT (1mg/ml, Sigma Chemical Co) at 37oC for 2 hours. Hepatocytes were then sedimented and the formazan formed was solubilized by adding isopropanol. The purplish blue colour of the supernatant is the amount of formazan formed, which was measured at 540nm and expressed as M formazan/106 cells. Membrane integrity: Membrane integrity was determined by LDH leakage and Lipid peroxidation. For LDH leakage, 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.Merk India Ltd). LDH activity in pellet was determined after solubilizing the pellet with 200 l of solubilizing solution containing 0.15% triton-X100, 0.1% BSA, 0.9% NaCl. 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. Lipid peroxidation was measured by malondialdehyde (MDA) formation by the

5 method of Ohkawa et al. 1979 (15). MDA was estimated in the hepatocyte suspension by ultilizing its property to react with 2-thiobarbituric acid (TBA), a pink coloured product formed was measured at 532nm.

Detoxification: For the measurement of urea, hepatocytes were suspended in 10mM ammonium chloride and incubated for a period of 1 hour at 37oC. The supernatant was determined for the amount of urea formed at 530nm using the urea Kit (E.Merck, India Ltd.). The values have been expressed as mM urea/106 cells. The measurement of CYP450 activity was based on the metabolism of diazepam as substrate by HPLC method. 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). The activity of the detoxifying enzyme, GST was assayed using 1-chloro2,4dinitrobenzene(CDNB) as substrate (17). The cytosolic fraction was monitored for measuring the increase in absorbance of CDNB-GSH conjugate at 340nm and the activity expressed as M CDNB-GSH conjugate/min/mg protein. STATISTICS: The data presented here are the mean S.D of eight independent experiments. One-way analysis of variance has been used to compare the mean values between groups with post-hoc test. Level of significance was considered as p<0.05. RESULTS: Light Microscopy: H and E staining of the isolated hepatocytes reveals an intact cell membrane with cytoplasm and nuclei..
Viability: TBE test demonstrated a viability of 90-95% for control (non-irradiated)

hepatocytes. With increase in linear dosage of UV-B, viability as assessed by TBE and MTT assay was not affected upto 2,500 J/m2 but there was a significant decrease at 7,500 J/m2 (**p<0.001) compared to control hepatocytes (Fig 1a,1b).

6 Membrane integrity as assessed by LDH leakage and lipidperoxiation was comparable to non-irradiated hepatocytes upto 1250 J/m2. However, there was a significant damage at 2500 J/m2 and 7500 J/m2 (*p<0.05, **p<0.001). Detoxifying functions such as Ureagenesis, CYP450 and GST activity showed comparable results upto 1250 J/m2 and a significant decrease at 2,500, 7,500 J/m2 (*p<0.05,**p<0.001). DISCUSSION Hepatocyte Transplantation (HT) appears to be a promising alternative to liver transplantation. Studies have been carried out to assess the efficacy of HT in acute liver failure in animal models and have shown promising results [1]. Investigators have also demonstrated improvement in survival in fulminant hepatic failure patients with allogeneic hepatocyte transplantation [18,19]. Shortage of human donors is the major limitation for clinical transplantation, which has renewed interest in the use of xenogeneic cells for transplantation. Goats appear to be attractive candidates as donor animals for xenotransplantation. They have shown promising results in xenobiotic oxidative metabolism and they have also been used as recipients in xenotransplantation studies [20,21]. Among the strategies to prevent immune rejection, 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. 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 [1,3,4]. 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. So, an attempt has been made to understand the viability, membrane integrity and detoxification of goat hepatocytes as a function of UV-B dose response. 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

7 when transplanted in patients with acute liver failure. The dosage of UV-B irradiation used was 250, 500, 1250, 2500 and 7,500 J/m2. A high dose of 7,500 J/m2 was used in the experiment as a positive control to check the deleterious effect of Ultraviolet B irradiation.

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.. Many investigators have used MTT assay as a marker of cellular viability [22,23]. In compliance with our observations on goat hepatocytes, in vitro irradiated rat hepatocytes (200-1000 J/m2) and fetal, porcine islets (300-1800 J/m2) elicited a similar non-toxic response in the functional integrity of the cell [24]. LDH leakage and lipid peroxidation have been used as markers of the cellular viability and integrity of the hepatocytes [25]. The effect of UV-B irradiation on the membrane integrity was measured by lipid peroxidation as shown by accumulation of malondialdehyde [26]. 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. However membrane integrity showed a significant decrease at 2500 and 7,500 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. In the present study, we have also investigated the xenobiotic metabolizing function of goat hepatocytes on exposure to UV-B irradiation at different doses of irradiation. CYP3A4 is the most abundant CYP450 isoform in human liver and responsible for detoxification (Phase I metabolism) of most of the drugs. Endogenous benzodiazepine like substances are known to involve in mediation and pathogenesis of the neuronal inhibition observed in hepatic encephalopathy. Hence, the detoxification of diazepam (Benzodiazepine) by CYP2C19 and CYP3A4 isoforms of CYP450 familiy is of particular importance in hepatic transplantation studies. Our results showed that CYP450 activity was not altered upto a UV-B dose of 1250 Joules/m2. Glutathione S-transferases are a group of multifunctional proteins involved in the detoxification of a wide spectrum of compounds. The major role of GSTs is to increase the detoxification of exogenous xenobiotics and their metabolites and

8 endogenous toxic compounds via the phase II reaction of detoxification pathyway. Investigators have measured GST activity in hepatic detoxification studies. GST activity has been studied in adult rat and adult human hepatocytes in primary, pure and mixed monolayer culture to compare the conjugation pathways in drug metabolism. As GST is an important detoxifying enzyme, 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. 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. 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. 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.

REFERENCES 1. Habibullah C M: An overview of Hepatocyte Transplantation Fulminant hepatic failure. in: Michio Mito, Masauuki Sawa, (eds.), Hepatocyte Transplantation. Japan Karger lander systems, 1997 pp312 324 2. Fox IJ and Chowdhury JR: Hepatocyte transplantation. American Journal of Transplantation. 4:7, 200 3. Kawai Y, Price J B, Hardy M A: Reversal of liver failure in rats by ultraviolet irradiated hepatocyte transplantation. Transplant Proc.19: 989-991, 1987 4. Vijayalakshmi V, Naseem B S, Rao MN , Vijayalakshmi A, Nandini R Habibullah C.M: Differential responses of UV-B irradiation on the viability and intracellular

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

10 12. Lahiri S, Habibullah C M, Ayesha Q, et al: Effect of UV-B (302nm) irradiation on isolated rat hepatocytes. Liver.15:149-152, 1995 13. Seglen P O: Preparation of isolated rat liver cells. in: Priscott DM, (ed.), Methods in Cell Biology. Academic Press New York, 1976 vol 13 pp29-83 14. Mossmann T: Rapid colorimetric assay for cellular growth and survival; Application to proliferation and cytotoxicity assays. J.Immunol Meth. 65:55-63, 1983 15. Ohkawa H, Ohishi N, Yagi N: Assay of lipid peroxides in animal tissues by thiobarbituric acid reaction. Anal Biochem.95: 351-358, 1979 16. Rao MNVS, Biju B, Ansar AK, Mujeeb M, 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. Xenobiotica 33:1233-1245,2003 17. Habig WH, Pabst MJ and Jakoby WB: Glutathione-S-Transferases: The first enzymatic step in mercapturic acid formation. J. Biol. Chem. 249: 7130-7139, 1974 18. Habibullah CM, Syed IH, Qamer Q, et al: Human fetal hepatocyte transplantation in patients with fulminant hepatic failure. Transplantation. 58: 951-952, 1994 19. Bilir MB, Guinette D, Karrer F, et al: Hepatocyte transplantation in acute liver failure. Liver Transplant. 6(1): 32-40, 2000 20. Montesissa C, Anfossi P, vant Klooster G, et al: The use of cultured hepatocytes from goats and cattle to investigate xenobiotic metabolism. Vet Res Commun. 20(5): 449-460, 1996

11 21. Xu H, Gundry SR, Hill AC, et al: Prolonged discordant cardiac xenograft survival in newborn recipients. Circulation. 96(2):364-367, 1997 22. Song W, Guan HJ, Zhu XZ, et al: Protective effect of bilobalide against nitric oxide-induced neurotoxicity in PC12 cells. Acta Pharmacol Sin. 21:415-420, 2000 23. Abe K, Matsuki N: Measurement of cellular 3-(4,5-dimethylthiazol-2yl)-2,5diphenyltetrazolium bromide (MTT) reduction activity and lactate dehydrogenase. Neurosci. Res.38(4): 325-329, 2000 24. Tze W J, Cheung S, Tai J, Tsang A: Xenotransplantation of adult porcine islets in diabetic mice. A study of UV-B irradiation, cryopreservation and immunosuppression on graft survival time. Hor Metab Res. 30(80): 509-513, 1998 25. Sheen LY, Sheu SF, Tsai SJ, et al: Effect of garlic active principle, diallyl disulphide on cell viability, lipid peroxidation, glutathione concentration and its related enzyme actitivities in primary rat hepatocytes. Am J Clin Med. 27 (1):95105, 1999 26. Esterbauer H, Shauer RJ, Zollner H: Chemisty and biochemisty of 4hydoxynonenal, malonaldehyde and related aldehydes. Free Radic Biol. Med. 11:81-128, 1991

LEGENDS

12 Fig 1. H&E staining of the isolated goat hepatocytes. Fig 2a, 2b Effect of UV-B irradiation on viability of goat hepatocytes as assessed by TBE test and MTT assay. With increase in linear dosage of UV-B, viability was not affected upto 2,500 J/m2 but there was a significant decrease at 7,500 J/m2 (**p<0.001) compared to control hepatocytes. Fig 3a, 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<0.05, **p<0.001).

Fig 4a, 4b Effect of UV-B irradiation on detoxifying functions of goat hepatocytes: Ureagenesis, CYP450 and GST activity showed comparable results upto 1250 J/m2 and a significant decrease at 2,500, 7,500 J/m2 (*p<0.05, **p<0.001) .

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