Detection and Modulation of Nitric Oxide in an In Vitro ...
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ABSTRACT Asthma is an inflammatory disease of the lungs and airways that presents challenges for management in diverse populations in the United States. These challenges reflect the conclusion that asthma is not caused by a single agent and is not infectious, but rather represents a disregulated host response to a number of inflammatory stimuli. Moreover, unlike many acute diseases, asthma is marked by a pattern of recurrent exacerbations, which may have relatively benign early signs and symptoms but may then progress to life-threatening manifestations. An optimal strategy for management of asthmatic patients would benefit from a simple means of monitoring the early signs of lung inflammation that mark an asthma attack. Our team has been developing a tool for monitoring a well-recognized biomarker of lung inflammation that employs a portable sensor for nitric oxide, a gas in exhaled breath that is produced at levels that are correlated with severity of asthma 1 . The device is intended to provide a readout of nitri c oxide that can be recorded in a home setting by non-medically trained people and can be easily transmitted remotely to professionals for rapid adjustment of therapeutic interventions for the asthmatic patient. This capacity to evaluate the extent of lun g inflammation remotely on a regular ongoing basis is ideal for low cost management strategies that incorporate lifestyle adjustments, including use of accepted dietary supplements, without adding to the burden faced by health care delivery teams and facilities. We report here on preliminary steps to evaluate the use of one dietary ingredient, curcumin, a component of the Indian spice turmeric, that has been reported to ameliorate the histopathology of lung inflammation in animal models, to inhibit the production of NO in cultured human cells, and to lower the levels of inflammatory biomarkers in mouse models of asthma 2-5 . Individuals with a history of asthma who consume this spice regularly could be periodically monitored with our nitric oxide detector to see if their suceptibility to asthmatic attacks has been reduced before their lung inflammation becomes so severe as to require medical intervention. Our detector should alo be a useful tool in evaluating derivatives of curcumin with improved bioavailability for future use in management of asthma. CURCUMIN AND ITS DERIVATIVES Curcumin (diferuloylmethane) is a low molecular weight molecule that has multiple anti - inflammatory activities, including inhibition of a large list of destructive proteolytic enzymes from mammalian and bacterial sources and down-regulation of intracellular inflammatory cascades. As a component of the spice turmeric, it has been consumed for centuries in large quantities without toxic side effects 6 . The extremely hydrophobic nature of curcumin may be related to its biolgoical activities, but presents a challenge for oral administration. The laboratory of our colleague, Dr. Francis Johnson, in the Department of Chemistry at Stony Brook, has been developing derivatives of curcumin that are more water-soluble than the parent compound and therefore have greater bioavailability without loss of the inhibitory activity characteristic of curcumin itself 7 . One of these chemically modified curcumins, CMC 2.24, is being examined as a possible candidate for control of a variety of inflammatory conditions. NO IS A BIOMARKER OF INFLAMMATION IN HUMAN LUNG AND AIRWAY CELL LINES We have been using the human lung carcinoma-derived cell line A549 as a source of nitric oxide (NO) because these cells generate levels of NO as well as its products, nitrite and nitrate ions, spontaneously formed in aqueous environments, that reflect the cells’ response to pro-inflammatory stimuli. This serves as a human cell-based model of NO by airway epithelium in inflammatory diseases of the lung, including asthma 9 . It is especially straightforward to collect aliquots of supernatant medium from stimulated or unstimulated A549 cultures for measurement of nitrate and nitrite ions by first eliminating high molecular weight components from the culture medium by ultrafiltration and then assaying the filtrates with a two-step sequence of reactions with the Greiss Reagents. The mixture of nitrate and nitrite ions in the filtrates is first converted to al l nitrite with nitrate reductase and the nitrite is then reacted with sulfanilamide and naphthyl ethylenediamine to form an intensely colored azo compound. The color from the azo compound is linearly proportional to the amounts of nitrite and nitrate in the filtrates 10 . ONGOING EXPERIMENTS We are now culturing the A549 cells in chambers from which the headspace above the medium can be collected in Douglas bags for measurement of NO using our breathalyzer. These bags have already been evaluated for preservation and retention of NO. Similar Douglas bags have been used for collection of breath samples from human patients being treated for inflammatory diseases of the lung, including asthma. Detection and Modulation of Nitric Oxide in an In Vitro Asthma Model CONVERSION OF NO TO NITRITE AND NITRATE IN WATER THE REACTIONS OF INDUCIBLE NO SYNTHASE (iNOS) DIXON PLOTS FOR INHIBITION OF THERMOLYSIN BY CURCUMIN AND CMC 2.24 CMC 2.24 CURCUMIN REFERENCES 1. Smith, AD, Cowan, JO, Brassert, KP, Herbison, KP, Taylor, DR (2005) Use of exhaled nitric oxide measurements to guide treatment in chronic asthma. N Engl J Med 352:2163- 2173. 2. Moon, DO, Kim, MO, Lee, HJ, Choi, YH, Park, YM, Heo, MS, Kim, GY (2008) Curcumin attenuates ovalbumin-induced airway inflammation by regulating nitric oxide. Biochem Biophys Res Comm 375:275-279. 3. Karaman, M, Firinci, F, Cilaker, S, Uysal, P, Tugyan, K, Yilmaz, O, Uzuner, N, Karaman, O. (2012) Anti-inflammatory effects of curcumin in a murine model of chroni c asthma. Allergol Immunopathol 40:210-214. 4. Wang, W, Zhu, R, Zie, Q, Li, A, Xiao, Y, Li, K, Liu H, Cui, D, Chen, Y, Wang, S (2012) Enhanced bioavailability and efficiency of curcumin for the treatment of asthma by its formulation in solid lipid nanoparticles. Internat J Nanomed 7:3667-3677. 5. Jang, DJ, Kim, ST, Oh, E, Lee, K. (2014) Enhanced oral bioavailability and antiasthmatic efficacy of curcumin using redispersible dry emulsion. Bio-Med Mat Engin 24:917-930. 6. Gupta, SC, Prasad, S, Kim, JH, Patchva, S, Webb, LK, Priyadarsini, IK, Aggarwal, BB (2011) Multitargeting by curcumin as revealed by molecular interaction studies. Nat Prod Rep 28:1937-1955. 7. Zhang, Y, Gu, Y, Lee, HM, Hambardjieva, E, Crankova, K, Golub, LM, Johnson, F (2012) Design, synthesis and biological activity of new polyphenolic inhibitors of matrix metalloproteinases: a focus on chemically-modified curcumins. Curr Med Chem 19:4348- 4356. 8. Antonelli, AC, Zhang, Y, Golub, LM, Johnson, F, Simon, SR (2013) Inhibition of anthrax lethal factor by curcumin and chemically modified curcumin derivatives. J Enz Inhib Med Chem early online 1-7. 9. Jiang, J., and S.C. George (2011) Modeling gas phase nitric oxide release in lung epithelial cells. Nitric Oxide 25:275-281. 10. Nims, RW, Derbyshire, JF, Saavedra, JE, Christodoulou, D, Hanbauer,I, Cox, GW, Grisham, MB, Laval, JA, Krishna, MC, Wink, DA. (1995) Colorimetric methods for the determination of nitric oxide concentration in neutral aqueous solutions. Methods 7:48 - 54. 11. Curcumin (Turmeric) (2014) Natural Medicines Comprehensive Database, Therapeutic Research Faculty, 3120 W March Lane, Stockton, CA 95219. Supported by NSF Award IIS-1231767, SHB:Type I (EXP): Personalized Asthma Monitor Detecting Nitric Oxide in Breath, P.I. Gouma, Principal Investigator, S.R. Simon and M. Stanacevic, Co-PIs RECENT RESULTS We have shown that production of nitrate and nitrite ions derived from NO released by A549 cells can be increased by culturing the cells in the presence of a classic pro - inflammatory stimulus, bacterial lipopolysaccharide (LPS, endotoxin). When the cells are cultured in the presence of LPS and 20 µM curcumin, the levels of nitrate and nitrite ions are significantly lowered without any evidence of cytotoxicity. These results support the previous observations of Moon et al. 2 that curcumin inhibits formation of nitrite and nitrate ions from A549 cells stimulated with gamma-interferon, another known pro-inflammatory stimulus. Yimei Miao a , Joanna Ghobrial a , Virghinya Ridwan a , Junmei Seki a , Sanford Simon a,b , Milutin Stanacevic c , Pelagia-Irene Gouma d Departments of Biochemistry a , Pathology b , Electrical Engineering c , and Materials Sciences d Stony Brook University, New York 11794 DISCUSSION At this early stage in development, we now have a prototype of a portable breathalyzer that can measure levels of NO in exhaled breath samples and can transmit the data to a remote location. Such a device is well suited to observing the exacerbations of airway inflammation that are the hallmark of asthma in human patients. We have already demonstrated that human lung epithelial cells in culture can be induced to produce NO in response to inflammatory stimuli and we have recently shown that the active ingredient of a widely used nontoxic Indian spice can diminish the production of NO and its products, nitrite and nitrate ions, from the cultured cells. The Natural Medicines Comprehensive Database compiled by the National Institutes of Health classifies turmeric and its active ingredient, curcumin, as safe for human consumption and apparently effective for use i n a variety of inflammatory conditions. 11 . Future studies that incorporate nutritional supplementation with curcumin or preferably, its more bioavailable derivatives, in asthmatic patients should be considered in which severity of disease and response to dietary management may be correlated with levels of NO released in exhaled breath and detected with our NO breathalyzer. INHIBITION OF TARGETED ENZYMES BY CURCUMIN AND CMC 2.24 Curcumin and CMC 2.24 are both inhibitors of a representative metalloproteinase, thermolysin, that is a very close homolog of the human enzyme Angiotensin Converting Enzyme, a key regulator of blood pressure. The pattern of inhibition by both curcumin and CMC 2.24 indicates binding at multiple sites on the targeted enzymes to achieve IC50 values in the micromolar range without toxicity. This pattern of inhibition is similar to that observed for other bacterial and mammalian enzymes that contribute to inflammatory tissue damage 8 .
Transcript of Detection and Modulation of Nitric Oxide in an In Vitro ...
ABSTRACT Asthma is an inflammatory disease of the lungs and airways that presents challenges for management in diverse populations in the United States. These challenges reflect the conclusion that asthma is not caused by a single agent and is not infectious, but rather represents a disregulated host response to a number of inflammatory stimuli. Moreover, unlike many acute diseases, asthma is marked by a pattern of recurrent exacerbations, which may have relatively benign early signs and symptoms but may then progress to life-threatening manifestations. An optimal strategy for management of asthmatic patients would benefit from a simple means of monitoring the early signs of lung inflammation that mark an asthma attack. Our team has been developing a tool for monitoring a well-recognized biomarker of lung inflammation that employs a portable sensor for nitric oxide, a gas in exhaled breath that is produced at levels that are correlated with severity of asthma1. The device is intended to provide a readout of nitric oxide that can be recorded in a home setting by non-medically trained people and can be easily transmitted remotely to professionals for rapid adjustment of therapeutic interventions for the asthmatic patient. This capacity to evaluate the extent of lung inflammation remotely on a regular ongoing basis is ideal for low cost management strategies that incorporate lifestyle adjustments, including use of accepted dietary supplements, without adding to the burden faced by health care delivery teams and facilities. We report here on preliminary steps to evaluate the use of one dietary ingredient, curcumin, a component of the Indian spice turmeric, that has been reported to ameliorate the histopathology of lung inflammation in animal models, to inhibit the production of NO in cultured human cells, and to lower the levels of inflammatory biomarkers in mouse models of asthma2-5. Individuals with a history of asthma who consume this spice regularly could be periodically monitored with our nitric oxide detector to see if their suceptibility to asthmatic attacks has been reduced before their lung inflammation becomes so severe as to require medical intervention. Our detector should alo be a useful tool in evaluating derivatives of curcumin with improved bioavailability for future use in management of asthma.
CURCUMIN AND ITS DERIVATIVES Curcumin (diferuloylmethane) is a low molecular weight molecule that has multiple anti-inflammatory activities, including inhibition of a large list of destructive proteolytic enzymes from mammalian and bacterial sources and down-regulation of intracellular inflammatory cascades. As a component of the spice turmeric, it has been consumed for centuries in large quantities without toxic side effects6. The extremely hydrophobic nature of curcumin may be related to its biolgoical activities, but presents a challenge for oral administration. The laboratory of our colleague, Dr. Francis Johnson, in the Department of Chemistry at Stony Brook, has been developing derivatives of curcumin that are more water-soluble than the parent compound and therefore have greater bioavailability without loss of the inhibitory activity characteristic of curcumin itself7. One of these chemically modified curcumins, CMC 2.24, is being examined as a possible candidate for control of a variety of inflammatory conditions.
NO IS A BIOMARKER OF INFLAMMATION IN HUMAN LUNG AND AIRWAY CELL LINES
We have been using the human lung carcinoma-derived cell line A549 as a source of nitric oxide (NO) because these cells generate levels of NO as well as its products, nitrite and nitrate ions, spontaneously formed in aqueous environments, that reflect the cells’ response to pro-inflammatory stimuli. This serves as a human cell-based model of NO by airway epithelium in inflammatory diseases of the lung, including asthma9.
It is especially straightforward to collect aliquots of supernatant medium from stimulated or unstimulated A549 cultures for measurement of nitrate and nitrite ions by first eliminating high molecular weight components from the culture medium by ultrafiltration and then assaying the filtrates with a two-step sequence of reactions with the Greiss Reagents. The mixture of nitrate and nitrite ions in the filtrates is first converted to all nitrite with nitrate reductase and the nitrite is then reacted with sulfanilamide and naphthyl ethylenediamine to form an intensely colored azo compound. The color from the azo compound is linearly proportional to the amounts of nitrite and nitrate in the filtrates10.
ONGOING EXPERIMENTS We are now culturing the A549 cells in chambers from which the headspace above the medium can be collected in Douglas bags for measurement of NO using our breathalyzer. These bags have already been evaluated for preservation and retention of NO. Similar Douglas bags have been used for collection of breath samples from human patients being treated for inflammatory diseases of the lung, including asthma.
Detection and Modulation of Nitric Oxide in an In Vitro Asthma Model
CONVERSION OF NO TO NITRITE AND NITRATE IN WATER
THE REACTIONS OF INDUCIBLE NO SYNTHASE (iNOS)
DIXON PLOTS FOR INHIBITION OF THERMOLYSIN BY CURCUMIN AND CMC 2.24
CMC 2.24
CURCUMIN
REFERENCES
1. Smith, AD, Cowan, JO, Brassert, KP, Herbison, KP, Taylor, DR (2005) Use of exhaled nitric oxide measurements to guide treatment in chronic asthma. N Engl J Med 352:2163-2173. 2. Moon, DO, Kim, MO, Lee, HJ, Choi, YH, Park, YM, Heo, MS, Kim, GY (2008) Curcumin attenuates ovalbumin-induced airway inflammation by regulating nitric oxide. Biochem Biophys Res Comm 375:275-279. 3. Karaman, M, Firinci, F, Cilaker, S, Uysal, P, Tugyan, K, Yilmaz, O, Uzuner, N, Karaman, O. (2012) Anti-inflammatory effects of curcumin in a murine model of chronic asthma. Allergol Immunopathol 40:210-214. 4. Wang, W, Zhu, R, Zie, Q, Li, A, Xiao, Y, Li, K, Liu H, Cui, D, Chen, Y, Wang, S (2012) Enhanced bioavailability and efficiency of curcumin for the treatment of asthma by its formulation in solid lipid nanoparticles. Internat J Nanomed 7:3667-3677. 5. Jang, DJ, Kim, ST, Oh, E, Lee, K. (2014) Enhanced oral bioavailability and antiasthmatic efficacy of curcumin using redispersible dry emulsion. Bio-Med Mat Engin 24:917-930. 6. Gupta, SC, Prasad, S, Kim, JH, Patchva, S, Webb, LK, Priyadarsini, IK, Aggarwal, BB (2011) Multitargeting by curcumin as revealed by molecular interaction studies. Nat Prod Rep 28:1937-1955. 7. Zhang, Y, Gu, Y, Lee, HM, Hambardjieva, E, Crankova, K, Golub, LM, Johnson, F (2012) Design, synthesis and biological activity of new polyphenolic inhibitors of matrix metalloproteinases: a focus on chemically-modified curcumins. Curr Med Chem 19:4348-4356. 8. Antonelli, AC, Zhang, Y, Golub, LM, Johnson, F, Simon, SR (2013) Inhibition of anthrax lethal factor by curcumin and chemically modified curcumin derivatives. J Enz Inhib Med Chem early online 1-7. 9. Jiang, J., and S.C. George (2011) Modeling gas phase nitric oxide release in lung epithelial cells. Nitric Oxide 25:275-281. 10. Nims, RW, Derbyshire, JF, Saavedra, JE, Christodoulou, D, Hanbauer,I, Cox, GW, Grisham, MB, Laval, JA, Krishna, MC, Wink, DA. (1995) Colorimetric methods for the determination of nitric oxide concentration in neutral aqueous solutions. Methods 7:48-54. 11. Curcumin (Turmeric) (2014) Natural Medicines Comprehensive Database, Therapeutic Research Faculty, 3120 W March Lane, Stockton, CA 95219.
Supported by NSF Award IIS-1231767, SHB:Type I (EXP): Personalized Asthma Monitor Detecting Nitric Oxide in Breath, P.I. Gouma, Principal Investigator, S.R. Simon and M. Stanacevic, Co-PIs
RECENT RESULTS We have shown that production of nitrate and nitrite ions derived from NO released by A549 cells can be increased by culturing the cells in the presence of a classic pro-inflammatory stimulus, bacterial lipopolysaccharide (LPS, endotoxin). When the cells are cultured in the presence of LPS and 20 µM curcumin, the levels of nitrate and nitrite ions are significantly lowered without any evidence of cytotoxicity. These results support the previous observations of Moon et al.2 that curcumin inhibits formation of nitrite and nitrate ions from A549 cells stimulated with gamma-interferon, another known pro-inflammatory stimulus.
Yimei Miaoa, Joanna Ghobriala, Virghinya Ridwana, Junmei Sekia, Sanford Simona,b, Milutin Stanacevicc, Pelagia-Irene Goumad
Departments of Biochemistrya, Pathologyb, Electrical Engineeringc, and Materials Sciencesd Stony Brook University, New York 11794
DISCUSSION At this early stage in development, we now have a prototype of a portable breathalyzer that can measure levels of NO in exhaled breath samples and can transmit the data to a remote location. Such a device is well suited to observing the exacerbations of airway inflammation that are the hallmark of asthma in human patients. We have already demonstrated that human lung epithelial cells in culture can be induced to produce NO in response to inflammatory stimuli and we have recently shown that the active ingredient of a widely used nontoxic Indian spice can diminish the production of NO and its products, nitrite and nitrate ions, from the cultured cells. The Natural Medicines Comprehensive Database compiled by the National Institutes of Health classifies turmeric and its active ingredient, curcumin, as safe for human consumption and apparently effective for use in a variety of inflammatory conditions.11. Future studies that incorporate nutritional supplementation with curcumin or preferably, its more bioavailable derivatives, in asthmatic patients should be considered in which severity of disease and response to dietary management may be correlated with levels of NO released in exhaled breath and detected with our NO breathalyzer.
INHIBITION OF TARGETED ENZYMES BY CURCUMIN AND CMC 2.24 Curcumin and CMC 2.24 are both inhibitors of a representative metalloproteinase, thermolysin, that is a very close homolog of the human enzyme Angiotensin Converting Enzyme, a key regulator of blood pressure. The pattern of inhibition by both curcumin and CMC 2.24 indicates binding at multiple sites on the targeted enzymes to achieve IC50 values in the micromolar range without toxicity. This pattern of inhibition is similar to that observed for other bacterial and mammalian enzymes that contribute to inflammatory tissue damage8.
