Melatonin protects bovine cerebral endothelial cells from hyperoxia-induced DNA damage and death

Neuroscience Letters 229 (1997) 193–197

Melatonin protects bovine cerebral endothelial cells from hyperoxia-induced DNA damage and death Arif Y. Shaikh, Jian Xu, Yingji Wu, Lucy He, Chung Y. Hsu* Department of Neurology, Box 8111, Washington University School of Medicine, 660 S. Euclid Ave., St. Louis, MO 63110, USA Received 16 December 1996; received in revised form 11 April 1997; accepted 17 April 1997

Abstract Hyperoxia leads to excessive formation of reactive oxygen species (ROS). ROS cause damage to many cellular components, including DNA. Exposure of bovine cerebral endothelial cells to 95 or 100% oxygen resulted in an increase in DNA fragmentation, the appearance of DNA ladders, and cell death with morphological features suggestive of apoptosis. Melatonin, an antioxidant, reduced hyperoxia-induced DNA fragmentation and cell death in a dose-dependent manner. Results from the present study support the contention that ROS play a major role in DNA damage and apoptotic death. Melatonin is an effective agent in reducing ROS-mediated DNA fragmentation and death in bovine cerebral endothelial cells.  1997 Elsevier Science Ireland Ltd. Keywords: Apoptosis; Antioxidant; Brain; Free radicals; Oxygen

Reactive oxygen species (ROS) damage many cellular components, including proteins, lipids, and DNA [17]. ROS have been detected following cerebral ischemia reperfusion [7,11,31] and are implicated in the pathogenesis of ischemic brain injury [4,20]. ROS produce DNA lesions such as base modifications, single-strand breaks, doublestrand breaks, and the crosslinking of bases [12]. DNA damage in the brain can be repaired, although the repair may be error-prone with low fidelity [8,26] leading to mutations, genomic instability, and cell death through apoptotic mechanisms [17]. Apoptosis features cell shrinkage, chromatin condensation, and endonuclease cleavage of DNA into multiples of 180 base pairs [14]. Cerebral endothelial cells (CECs) play a major role in maintaining brain homeostasis, including cerebral blood flow and blood–brain barrier function. We explored the effect of ROS generation on bovine CECs in vitro and studied melatonin’s actions. Melatonin is a pineal hormone which was only recently discovered to be an efficient free radical scavenger [27]. Melatonin reduces apoptosis in neurons [3,10], thymocytes [23], and ischemia-reperfusion injury in cheek pouch endothelial cells [2,15]. In the present * Corresponding author. Tel.: +1 314 3629461; fax: +1 314 3629462; e-mail: [email protected]

study, we noted that melatonin is a potent agent in protecting bovine CECs from oxidative DNA damage and cell death. Bovine CECs were prepared as described [1,5,6] with modification. Briefly, bovine brains from freshly slaughtered adult animals were immediately placed in ice-cold Hank’s balanced salt solution (HBSS) (Gibco BRL, Grand Island, NY) with antibiotics. After removing the meninges and superficial blood vessels, the gray matter was disrupted in a loose Dounce homogenizer and the homogenates sequentially filtered through 300 mm and 80 mm nylon meshes. Collagenase B (4 mg/ml) (Boehringer Mannheim, Indianapolis, IN) was added to the microvessel fraction for 2 h to dissociate brain tissue. The lipid fraction was separated by adding 25% bovine serum albumin (BSA). The pellets were further digested with collagenase/dispase (1 mg/ml) (Boehringer Mannheim, Indianapolis, IN) for 8 h, suspended in HBSS, loaded onto 40% Percoll, and centrifuged at 1400 × g for 15 min. The second band containing microvessels was collected and washed before plating onto tissue culture dishes precoated with collagen. Bovine CECs migrating from vessels were pooled to form a culture of proliferating endothelial cells that were maintained in medium containing 10% fetal calf serum (FCS), heparin (0.5 mg/ml), and endothelial growth supplements (75 mg/ml)

0304-3940/97/$17.00  1997 Elsevier Science Ireland Ltd. All rights reserved PII S0304-3940 (97 )0 0307-8

194

A.Y. Shaikh et al. / Neuroscience Letters 229 (1997) 193–197

(Sigma, St. Louis, MO). Purity of bovine CECs was more than 95% based on immunocytochemical detection of the expression of Factor VIII and vimentin and in the absence of the expression of fibronectin, a-actinin [29], and glial fibrillary acidic protein [1]. Bovine CECs expressing functional bradykinin receptors [30] were plated on 100 mm dishes or 24-well plates, maintained in Dulbecco’s modified Eagle’s medium (DMEM) with 10% FCS, and grown to confluence. When suitable for study, bovine CECs were maintained in DMEM containing 0.5% FCS. Bovine CECs were subjected to hyperoxia by placing culture dishes in an incubation chamber (Billups-Rothenberg, Del Mar, CA) with 100% O2 delivered at 2 l/min through a flow regulator for 8 h at room temperature (24°C). Another set of CECs which served as a normoxic control were placed in the normal atmosphere at the same room temperature for the same length of time. In another series of experiments, hyperoxia was carried out by exposing bovine CECs to 95% O2 and 5% CO2 for 8 h at 37°C. Control CECs were exposed to 95% air and 5% CO2 for the same length of time at 37°C. Results were similar under these two conditions. Certain cells were treated with melatonin (0.04–5 mM) (Sigma, St. Louis, MO) immediately prior to hyperoxia. Bovine CECs were maintained in 100 mm culture dishes with DMEM containing 0.5% FCS. After treating the cells with either hyperoxia or normoxia, DNA was extracted as previously described [24] with modification. Briefly, cells were lysed in lysis buffer (0.01 M Tris–HCl, pH 8.0, 0.1 M EDTA, 0.5% SDS). Following lysis, RNase A was added to a final concentration of 10 U/ml, and samples were incubated at 37°C for 2 h. DNA was extracted twice with phenol/chloroform/amyl alcohol (26:25:1). The total DNA

Fig. 1. Analysis of DNA fragmentation by agarose gel electrophoresis. Lane 1 represents the normoxic bovine CECs, in which no degradation of DNA is visible. Lane 2 represents hyperoxic bovine CECs and displays the stepladder-like pattern associated with the internucleosomal cleavage of DNA occurring in multiples of approximately 180 base pairs. Molecular weight markers are shown on the left. Representative of two experiments.

Fig. 2. Quantification of DNA fragmentation in bovine CECs by ELISA. Hyperoxia (95% O2 / 5% CO2) at 37°C increased levels of DNA fragments in bovine CECs as compared to normoxic samples. Treatment with melatonin immediately prior to hyperoxia caused a dose-dependent reduction in DNA fragmentation with significant difference from hyperoxia samples noted at 0.2, 1, and 5 mM (P , 0.05). Results are from quadruplets of two separate experiments.

contained in the aqueous phase was precipitated with ethanol. The DNA pellet obtained was washed twice with 70% ethanol and resuspended in TE buffer (0.01 M Tris–HCl, 0.001 M EDTA, pH 8.0). Aliquots (10–15 mg of DNA) were loaded onto a 1.5% agarose gel. Following electrophoresis and staining with ethidium bromide, the gel was visualized under ultraviolet light and photographs were taken. A cell death detection ELISA kit (Boehringer Mannheim, Indianapolis, IN) was used to quantitatively determine levels of histone-associated DNA fragments, including mono- and oligonucleosomes. The assay measures levels of mono- and oligonucleosomes in cell lysates [16]. Bovine CECs were maintained in 24-well clusters with DMEM containing 0.5% FCS. After treating the cells with hyperoxia (with and without melatonin) and normoxia, samples were processed as described [16] and detailed in the protocol provided with the ELISA kit. Materials used in the in situ nick labeling of DNA were obtained from Boehringer Mannheim in Indianapolis, IN. Biotin high prime non-radioactive nucleic acid labeling mixture was used to identify individual cells undergoing apoptosis. The biotin high prime reaction mixture contains the Klenow polymerase (1 U/ml), biotin-16-dUTP (0.35 mM), dATP (1 mM), dCTP (1 mM), dGTP (1 mM), and dTTP (0.65 mM). Biotinylated nucleotides were added to DNA by the Klenow polymerase, which catalyzes the addition of deoxyribonucleoside triphosphate to the 3′-OH end of DNA. The biotin-labeled DNA is detected by streptavidin conjugated to alkaline phosphatase (AP), which catalyzes a color reaction with 5-bromo-4-chloro-3-indolyl-phosphate (BCIP) and 4-nitro blue tetrazolium chloride (NBT), resulting in the deep blue staining of nuclei of apoptotic cells. Bovine CECs were maintained in 24-well clusters on cover slips in DMEM containing 0.5% FCS. After treating the cells with hyperoxia (with and without melatonin) and normoxia, DNA in samples were end labeled as described above using a protocol provided by the manufacturer of the nick labeling kit. After treating cells with hyperoxia (with and without

A.Y. Shaikh et al. / Neuroscience Letters 229 (1997) 193–197

melatonin) and normoxia, LDH activity was assayed in the culture medium. The spectrophotometric assay was performed at l = 340 nm using Noll’s method [19]. Data are expressed as means and standard error of mean. Comparisons among experimental and control variables were made using one-way analysis of variance (ANOVA) followed by a post-hoc Tukey test. A P value less than 0.05 is considered significant. DNA ladders characteristic of apoptosis were noted on agarose gel electrophoresis in DNA from hyperoxic, but not normoxic, bovine CECs (Fig. 1). Hyperoxia for 8 h was found to induce a significant increase in levels of DNA fragments, more than 7-fold of the normoxic cells, suggest-

Fig. 3. Visualization of apoptotic cells by the nick-labeling method. Nuclei with DNA fragmentation are stained dark blue in original color photos. Figures shown here are black and white photos reproduced from the original color photos. Positive labeling of damaged nuclei, which are dark blue in the original figure, are black with image enhancement. Negative labeling of normal nuclei, which are pink in the original figure, are gray with image enhancement. Original color photos are available upon request. Panel A shows normoxia treated bovine CECs with little if any indication of DNA damage or apoptosis. Panel B shows that hyperoxia led to the appearance of nuclei with positive nick-labeling in black. Panel C shows the reduction of cell injury caused by melatonin (1 mM) pretreatment. Very few nuclei with positive nick-labeling were noted. Representative of three experiments. Magnification: 200× for all panels.

195

Fig. 4. The extent of LDH release from bovine CECs. LDH levels in normoxia group were 36.4 ± 2.1 units/well (n = 12). Hyperoxia (95% O2 / 5% CO2) caused significant increase in LDH release. Melatonin (0.04–5 mM) caused a dose-dependent decrease in LDH release with significant difference from hyperoxia samples noted at 0.2, 1, and 5 mM (P , 0.05). Results are from quadruplets of two separate experiments.

ing DNA cleavage into mono- and oligonucleosomes (Fig. 2). Melatonin, a potent antioxidant, proved to be highly efficient in protecting bovine CECs from DNA damage, reducing DNA fragmentation in a dose-dependent manner. Results were similar whether cells were treated with 100% O2 without 5% CO2 at room temperature (data not shown) or with 95% O2 with 5% CO2 at 37°C (Fig. 2). In situ nick labeling also provided evidence of hyperoxia-induced DNA damage in bovine CECs. Melatonin reduced the intensity of DNA end labeling in CECs exposed to hyperoxia (Fig. 3). Lactate dehydrogenase (LDH), which is released from cells during cell death, was measured to assess the extent of cell death and protective effects of melatonin (Fig. 4). Results indicate that hyperoxia caused significant cell death. LDH content was low and comparable to basal level in the normoxia treated samples (LDH levels: 36.4 ± 2.1 units/well, n = 12). Hyperoxic bovine CECs released significant amounts of LDH, greater than 5-fold of the normoxic cells. Melatonin again demonstrated a protective role, reducing LDH release in a dose-dependent manner. LDH results were similar whether cells were treated with 100% O2 without 5% CO2 at room temperature (data not shown) or with 95% O2 and 5% CO2 at 37°C (Fig. 4). In this study, we found that hyperoxia induced DNA damage in bovine CECs as reflected by DNA laddering on gel electrophoresis, increased DNA cleavage into monoand oligonucleosomes, and enhanced intensity in DNA nick labeling. These features are suggestive of cell death by apoptosis. To our knowledge, this is the first study demonstrating that hyperoxia causes CEC death with features suggestive of apoptosis. This has serious implications because hyperoxia is clinically utilized to treat lung dysfunction, decompression sickness and other disorders. Our studies raise the possibility that CECs may be a target for hyperoxia induced cytotoxicity. Melatonin is a highly efficient free radical scavenger and reduced DNA damage induced in bovine CECs by hyperoxia. Melatonin, N-acetyl-5-methoxytryptamine, is phylogenetically a very old molecule that is derived from tryptophan. It is produced in the pineal gland and is mainly

196

A.Y. Shaikh et al. / Neuroscience Letters 229 (1997) 193–197

known for its ability to regulate the function of the endocrine and circadian systems [21]. Melatonin acts differently from many other antioxidants. It is capable of trapping one or more hydroxyl radicals to form a stable metabolite, which does not participate in redox cycling [13]. This unique scavenging mechanism of melatonin makes it less toxic to DNA and more suitable for protection of DNA damage than many other free radical scavengers. Not surprisingly, melatonin is the most potent hydroxyl radical scavenger discovered to date [27] with broad cytoprotective effects [2,3,10, 22,23]. Membranes are permeable to melatonin due to its small size and high lipophilicity [21]. Melatonin is also widely distributed in cells and has actions in the membrane, cytosol, and nucleus [27]. The tendency for melatonin to be present in the nucleus provides an effective means of on-site protection against DNA damage [28]. Our results suggest that melatonin might play a role as a possible endogenous antioxidant that may protect CECs. Nocturnal melatonin excretion is lower in patients with ischemic stroke [9]. Rats deficient in melatonin sustained greater brain damage after stroke [18]. Thus, reduction in melatonin levels may be detrimental in cerebrovascular diseases. Further studies on the mechanism of action of melatonin in preventing CEC injury may broaden our insight into the development of new effective therapeutic strategies aiming at ROS-mediated cytotoxicity [25].

[8]

[9]

[10]

[11]

[12] [13]

[14]

[15]

[16]

We thank Dr. Tom Lin and Dr. Laura Dugan for advice on free radical chemistry and biology, Dr. Mark Goldberg for image analysis, and Ms. Kelly Treat for editorial assistance. This study is supported in part by an Office of Naval Research grant (N00014-95-1-0582) and an NINDS grant (NS28995). Support for Arif Y. Shaikh to participate in this research was provided by a grant to Washington University from the Howard Hughes Medical Institute through the Undergraduate Biological Sciences Education Program. [1] Abbott, N.J., Hughes, C.A.W., Revest, P.A. and Greenwood, J., Development and characterization of a rat brain capillary endothelial culture: towards an in vitro blood-brain barrier, J. Cell Sci., 103 (1992) 23–37. [2] Bertuglia, S., Marchiafava, P.L. and Colantuoni, A., Melatonin prevents ischemia reperfusion injury in hamster cheek pouch microcirculation, Cardiovasc. Res., 31 (1996) 947–952. [3] Cagnoli, C.M., Atabay, C., Kharlamova, E. and Manev, H., Melatonin protects neurons from singlet oxygen-induced apoptosis, J. Pineal Res., 18 (1995) 222–226. [4] Chan, P.H., Role of oxidants in ischemic brain damage, Stroke, 27 (1996) 1124–1128. [5] DeBault, L.E., Henriquez, E., Hart, M.N. and Cancilla, P.A., Cerebral microvessels and derived cells in tissue culture. II. Establishment, identification and preliminary characterization of endothelial cell line, In vitro, 17 (1981) 480–494. [6] DeBault, L.E., Kahn, L.E., Fromomes, S.P. and Cancilla, P.A., Cerebral microvessels and derived cells in tissue culture: isolation and preliminary characterization, In vitro, 15 (1979) 473–487. [7] Dugan, L.L., Lin, T.S., He, Y.Y., Hsu, C.Y. and Choi, D.W., Detection of free radicals by microdialysis/spin trapping EPR following

[17]

[18]

[19] [20] [21] [22]

[23]

[24]

[25]

[26]

focal cerebral ischemia-reperfusion and a cautionary note on the stability of 5,5-dimethyl-1-pyrroline N-oxide (DMPO), Free Radical Res., 23 (1995) 27–32. Feig, D.I. and Loeb, L.A., Mechanisms of mutation by oxidative DNA damage: Reduced fidelity of mammalian DNA polymerase b, Biochemistry, 32 (1993) 4466–4473. Fiorina, P., Lattuada, G., Ponari, O., Silvestrini, C. and Dall’Aglio, P., Impaired nocturnal melatonin excretion and changes of immunological status in ischemic stroke patients, Lancet, 347 (1996) 692– 693. Giusti, P., Gusella, M., Lipartiti, M., Milani, D., Zhu, W., Vicini, S. and Manev, H., Melatonin protects primary cultures of cerebellar granule neurons from kainate but not from N-methyl-D-aspartate excitotoxicity, Exp. Neurol., 131 (1995) 39–46. Globus, M.Y., Busto, R., Lin, B., Schnippering, H. and Ginsberg, M.D., Detection of free radical activity during transient global ischemia and recirculation: effects of intraischemic brain temperature modulation, J. Neurochem., 65 (1995) 1250–1256. Halliwell, B. and Gutteridge, J.M.C., Free Radicals in Biology and Medicine, 2nd edn., Clarendon Press, Oxford, 1989. Hardeland, R., Reiter, R.J., Poeggeler, B. and Tan, D., The significance of the metabolism of the neurohormone melatonin: antioxidative protection and formation of bioactive substances, Neurosci. Biobehav. Rev., 17 (1993) 347–357. Johnson, E.M., Greenlund, L.J.S., Akins, P.T. and Hsu, C.Y., Neuronal apoptosis: current understanding of molecular mechanisms and potential role in ischemic brain injury, J. Neurotrauma, 12 (1995) 843–852. Jornot, L. and Junod, A.F., Variable glutathione levels and expression of antioxidant enzymes in human endothelial cells, Am. J. Physiol., 264 (1993) 482–489. Leist, M., Gantner, F., Bohlinger, I., Germann, P.G., Tiegs, G. and Wendel, A., Murine hepatocyte apoptosis induced in vitro and in vivo by TNF-alpha requires transcriptional arrest, J. Immunol., 153 (1994) 1778–1788. Liu, P.K., Hsu, C.Y., Dizdaroglu, M., Floyd, R.A., Kow, Y.W., Karakaya, A., Rabow, L.E. and Cui, J.K., Damage, repair, mutagenesis in nuclear gene after mouse forebrain ischemia and reperfusion, J. Neurosci., 16 (1996) 6795–6806. Manev, H., Uz, T., Kharlamov, A. and Joo, J.Y., Increased brain damage after stroke or excitotoxic seizures in melatonin-deficient rats, FASEB J., 10 (1996) 1546–1551. Noll, F., In: H.U. Bergmeyer (Ed.), Methods of Enzymatic Analysis, Vol. VI, Deerfield Beach, FL, 1984, pp. 582–588. Phillis, J.W., A ‘radical’ view of cerebral ischemic injury, Prog. Neurobiol., 42 (1994) 441–448. Reiter, R.J., Melatonin: that ubiquitously acting pineal hormone, News Physiol. Sci., 6 (1991) 223–227. Reiter, R.J., Melchiorri, D., Sewerynek, E., Poeggeler, B., BarlowWalden, L., Chuang, J., Ortiz, G.G. and Acuna-Castroviejo, D., A review of the evidence supporting melatonin’s role as an antioxidant, J. Pineal Res., 18 (1995) 1–11. Sainz, R.M., Mayo, J.C., Uria, H., Kotler, M., Antolin, I., Rodriguez, C. and Menendez, A.P., The pineal neurohormone melatonin prevents in vivo and in vitro apoptosis in thymocytes, J. Pineal Res., 19 (1995) 178–188. Sambrook, J., Fritsch, E.F. and Maniatis, T., Molecular Cloning: A Laboratory Manual, 2nd edn., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989, 6.3–6.19. Sanders, S.P., Zweier, J.L., Kuppusamy, P., Harrison, S.J., Bassett, D.J.P., Gabrielson, E.W. and Sylvester, J.T., Hyperoxic sheep pulmonary microvascular endothelial cells generate free radicals via mitochondrial electron transport, J. Clin. Invest., 91 (1993) 46–52. Sobol, R.W., Horton, J.K., Kuhn, R., Gu, H., Singhal, R.K., Prasad, R., Rajewsky, K. and Wilson, S.H., Requirement of mammalian DNA polymerase-beta in base-excision repair, Nature, 379 (1996) 183–186.

A.Y. Shaikh et al. / Neuroscience Letters 229 (1997) 193–197 [27] Tan, D., Chen, L., Poeggeler, B., Manchester, L.C. and Reiter, R.J., Melatonin: a potent, endogenous hydroxyl radical scavenger, Endocr. J., 1 (1993) 57–60. [28] Tan, D., Reiter, R., Chen, L., Poeggeler, B., Manchester, L.C. and Barlow-Walden, L., Both physiological and pharmacological levels of melatonin reduce DNA adduct formation induced by the carcinogen safrole, Carcinogenesis, 15 (1994) 215–218. [29] Tsukada, T., Tippens, D., Gordon, D., Ross, R. and Gown, A.M., HHF35, a muscle-action specific monoclonal antibody. Immunocy-

197

tochemical and biochemical characterization, Am. J. Pathol., 126 (1987) 51–60. [30] Xu, J., Qu, Z.X., Moore, S., Hsu, C.Y. and Hogan, E.L., Receptor linked hydrolysis of phosphoinositide and production of prostacyclin in cerebral endothelial cells, J. Neurochem., 58 (1992) 1930–1935. [31] Zini, I., Tomasi, A., Grimaldi, R., Vannini, V. and Agnati, LF., Detection of free radicals during brain ischemia and reperfusion by spin trapping and microdialysis, Neurosci. Lett. 138:2 (1992) 279– 282.