methoxykynuramine and its precursor melatonin ... - The FASEB Journal

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Novel rhythms of N1-acetyl-N2-formyl-5methoxykynuramine and its precursor melatonin in water hyacinth: importance for phytoremediation Dun-Xian Tan,*,1 Lucien C. Manchester,†,1 Paolo Di Mascio,‡,2 Glaucia R. Martinez,‡ Fernanda M. Prado,‡ and Russel J. Reiter*,3 *Department of Cellular Structural Biology, The University of Texas, Health Science Center, San Antonio, Texas, USA; †Department of Biological Sciences, St Mary’s University, San Antonio, Texas, USA; and ‡Departamento de Bioquı´mica, Instituto de Quı´mica, Universidade de Saõ Paulo, Saõ Paulo, SP, Brazil N1-acetyl-N2-formyl-5-methoxykynuramine (AMFK) is a major metabolite of melatonin in mammals. To investigate whether AFMK exists in plants, an aquatic plant, water hyacinth, was used. To achieve this, LC/MS/MS with a deuterated standard was employed. AFMK was identified in any plant for the first time. Both it and its precursor, melatonin, were rhythmic with peaks during the late light phase. These novel rhythms indicate that these molecules do not serve as the chemical signal of darkness as in animals but may relate to processes of photosynthesis or photoprotection. These possibilities are supported by higher production of melatonin and AFMK in plants grown in sunlight (10,000 –15,000 ␮W/cm2) compared to those grown under artificial light (400 – 450 ␮W/ cm2). Melatonin and AFMK, as potent free radical scavengers, may assist plants in coping with harsh environmental insults, including soil and water pollutants. High levels of melatonin and AFMK in water hyacinth may explain why this plant more easily tolerates environmental pollutants, including toxic chemicals and heavy metals and is successfully used in phytoremediation. These novel findings could lead to improvements in the phytoremediative capacity of plants by either stimulating endogenous melatonin synthesis or by adding melatonin to water/soil in which they are grown.—Dun-Xian Tan, Lucien C. Manchester, Paolo Di Mascio, Glaucia R. Martinez, Fernanda M. Prado, and Russel J. Reiter. Novel rhythms of N1-acetylN2-formyl-5-methoxykynuramine and its precursor melatonin in water hyacinth: importance for phytoremediation. FASEB J. 21, 1724 –1729 (2007) ABSTRACT

Key Words: plant 䡠 AFMK 䡠 circadian rhythm 䡠 antioxidant

ronmental conditions (1); it is referred to as the chemical expression of darkness (2). A decade ago, melatonin was first identified in the unicellular organism, a marine alga, the dinoflagellate Lingulodinium polyedrum [Gonyaulax polyedra] (3); subsequently, it was also found in vegetables, seeds, numerous herbs, and recently in grapes (4 – 8). Generally, plants have higher melatonin levels than those normally present in the blood of animals (9). Considering the antioxidative activity of melatonin (10, 11) and its presence in plant products that humans consume, melatonin is also classified as an antioxidant vitamin (12). High levels of melatonin in plants not only benefit the organisms that consume them but also protect plants from oxidative damage induced by harsh environments such as UV irradiation (4), extremely cold or hot weather (13) and spontaneous lipid peroxidation, which normally occurs in lipid-rich seeds (6). It is speculated that plants that contain high levels of melatonin may tolerate environmental pollutants well and thus may have utility in phytoremediation (14). The water hyacinth [Eichhornia crassipes (Mart) Solms] is an aquatic plant widely distributed in tropical and subtropical regions, and it survives in highly polluted environments (15). Studies have shown that the hyacinth tolerates contamination by the phosphorus pesticide ethion (16), the heavy metal mercury (17), and carcinogenic arsenic (18), and it has been used to remove these pollutants and serve in phytoremediation for wastewater generated from industrial and agricultural sources (19 –21). It was our interest to determine whether this pollutant-tolerant plant contains the antioxidant melatonin and its oxidative metabolite, N11

Melatonin was once considered to be exclusively a molecule primarily synthesized by the pineal gland of vertebrates. Melatonin regulates seasonal reproductive function by transducing annual changes of the lightdark environment into a chemical message that synchronizes their physiology with the appropriate envi1724

These authors contributed equally to this work. Present address: Departamento de Bioquı´mica e Biologia Molecular, Setor de Cieˆncias Biolo´gicas, Universidade Federal do Parana´, 81531–990, Curitiba, PR, Brazil. 3 Correspondence: Department of Cellular Structural Biology, The University of Texas Health Science Center, 7703 Floyd Curl Dr., San Antonio, TX, 78229, USA. E-mail: [email protected] doi: 10.1096/fj.06-7745com 2

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acetyl-N2-formyl-5-methoxykynuramine (AFMK), and whether melatonin and AFMK exhibit circadian rhythms in the water hyacinth.

MATERIALS AND METHODS Melatonin (100% chromatographically pure) was a gift from the Helsinn Chemical Co. (Biasca, Switzerland); AFMK, deuterated melatonin (D3-melatonin); and deuterated AFMK (D3-AFMK) were made available by the San Antonio Laboratory (San Antonio, TX, USA). Other chemicals and reagents were purchased from Sigma (St. Louis, MO, USA). Determination of the production of melatonin and AFMK in cultured water hyacinth. Water hyacinths were collected from the local pond in San Antonio, TX, USA during May. The selected plants were roughly 15 cm in height and 10 cm wide with 8 –10 leaves. The plants were divided into two groups with 6 plants per group. The hyacinths were cultured in glass containers (12⫻10⫻8 cm) with 400 ml pond water. The hyacinths were exposed to constant light (400 – 450 ␮W/cm2) at room temperature (22⫾0.5°C). One group served as a control, and another group was treated with tryptophan at a concentration of 5 ␮M in the culture water. Every 3 days, water consumption was measured, and the water remaining was replaced by 400 ml fresh pond water with or without tryptophan. After 15 days of culture, the leaves of the hyacinths were harvested and stored at – 80°C for subsequent melatonin and AFMK analyses. Examination of whether water hyacinths absorb exogenously provided melatonin The experimental conditions were as above. Two plants served as controls and two were treated with 5 ␮M melatonin in the culture media. Every 3 days, water consumption was measured, and the water remaining was replaced by 400 ml of fresh pond water with or without melatonin. The experiment lasted for 7 days, at which time leaves were collected and stored at – 80°C. Tests of the circadian rhythm of melatonin and AFMK in water hyacinths This study was carried out on May 20, 2004, at a natural pond near San Antonio, TX, USA. It was a sunny day with temperature of 29.5–31.5°C during the day and 24.5–26.5°C at night. The light intensity of the sun at noon was (10,000 –15,000 ␮W/cm2). The sample collections were started at 16:00 h and then every 4 h; 4 samples of leaves and flowers were collected at each time point during one 24-h period. During darkness, no artificial light was used for the sample collection. The samples were stored at – 80°C for future melatonin and AFMK analyses. Melatonin and AFMK extraction from water hyacinths One gram of each leaf was pulverized in 10 ml phosphate buffer (50 mM, pH 7.4) using mortar and pestle and then sonicated for 20 min. The mixtures were centrifuged at 3,000 g for 15 min. The supernatants were mixed with 10 ml chloroform and horizontally shaken for 5 min. The organic phase was collected and evaporated under vacuum. The residue was redissolved with 200 ␮l methanol, vor-

texed, and centrifuged at 5,000 g for 10 min; 180 ␮l supernatant was collected and dried under vacuum. The residues were stored at – 80°C for future analyses. To estimate the recovery rates of melatonin, D3-melatonin was added to some samples and passed through the same extraction procedure. To minimize melatonin decay, all procedures were performed under dim light. Melatonin and AFMK analyses using LC/ESI/MS-MS A liquid chromatograph/tandem mass spectrometry (LC/ ESI/MS-MS) analysis in the positive mode was carried out on a Quatro II mass spectrometer (Micromass, Altrincham, UK). The cone voltage and the source temperature were adjusted to 25 V and 100°C, respectively. The flow rate of drying and nebulizing gases were optimized at 300 and 15 Lh-1. The capillary potential and RF lens were set to 3.5 and 0.5 kV, respectively. For collision-induced dissociation of compounds, the collision energy of the mass spectrometer was set at 15 eV and the argon pressure at 2.1 ⫻ 103 mbar. The HPLC system consisted of two LC-10ADvp pumps and a SCL-10A system (Shimadzu, Kyoto, Japan). An analytic Phenomenex Mercury MS column (4⫻20 mm, 5-␮m particle size) connected to a precolumn was used to separate the compounds at a flow rate of 0.2 ml/min. Two linear gradients were used: A) 20 – 40% acetonitrile in water for 20 min and 40 to 20% for an additional 5 min, and B) 40 to 10% acetonitrile in water for 10 min, and 10 to 40% for additional 5 min. The data were processed using the Mass Lynx NT data system, version 3.2 (Micromass, Altrincham, UK). The detection of melatonin, D3-melatonin, AFMK, and D3-AFMK were performed in the Multiple Reaction Monitoring (MRM) mode. The m/z transition from 233 to 174 (melatonin), 236 to 174 (D3-melatonin), 265 to 178 (AFMK), and 268 to 178 (D3-AFMK) were chosen for MRM detection experiments as shown in Fig. 2A, B, using gradient A and B, respectively. Statistics Data are expressed as means ⫾ sem. Two-sample assuming equal variances t test was used for statistical analysis. A P ⬍ 0.05 was considered to be statistically significant.

RESULTS Using a LC/ESI/MS-MS method and D3-melatonin as a reference standard, melatonin was identified in water hyacinths (Fig. 1A). Melatonin levels in the water hyacinth are unexpectedly high, especially when they were grown under sunlight. The levels of melatonin in leaves of hyacinth under sunlight (10,000 –15,000 ␮W/ cm2) are 48 ⫾ 14.3 ng/g (0800 –1200) and under the artificial night (400 – 450 ␮W/cm2) are 2.9 ⫾ 0.64 ng/g (0800 –1200), respectively. In addition to melatonin, AFMK, a metabolite of melatonin in animals, is also for the first time identified in this plant (Fig. 1). To test whether the water hyacinth has the capacity to synthesize melatonin, some plants were provided tryptophan, a precursor of melatonin in the media in which the plants were grown. Melatonin levels in the plants treated with tryptophan were significantly higher than those in control plants (Fig. 2). In addition to its synthesis, whether exogenous melatonin can be extracted from the growth medium by

NOVEL RHYTHMS OF N1-ACETYL-N2-FORMYL-5-METHOXYKYNURAMINE

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Figure 1. Detection of melatonin, D3-melatonin, AFMK and D3-AFMK by HPLC/ESI/MS-MS in the multiple reaction monitoring (MRM) mode using linear gradient A (A) and B (B). Conditions were as described in the Materials and Methods.

this plant was also examined. When melatonin at a concentration of 5 ␮M was provided in the growth media, the melatonin levels in the leaves of hyacinth were dramatically elevated when compared to the plants, which grew in the media without melatonin (Fig. 3). The results show that the plants also absorb exogenous melatonin via the roots. In addition, we observed that melatonin levels in water hyacinth exhibited an obvious novel diurnal rhythm under natural environmental conditions. The unique rhythm of melatonin in the water hyacinth is noticeably different from that of animals in that its peak occurs late in the light phase of the light-dark cycle, i.e., near sunset. The plant melatonin peak appeared around 20:00 h with a level of 306 ⫾ 63.4 ng/g vs. 4 ⫾ 0.8 ng/g at its lowest levels at 0800 (Fig. 4A). AFMK, also exhibits a rhythm

similar to that of melatonin. Its peak levels were 20 ⫾ 1.7 ng/g at 20:00 h vs. its minimal levels of 2.0 ⫾ 0.5 ng/g at 08:00 h (Fig. 4B). When the levels of melatonin and AFMK in the leaves of water hyacinths were compared to their levels in flowers at the same time (08:00), it shows that the levels of both melatonin and AFMK are much higher in flowers than they are in leaves (Fig. 5).

DISCUSSION The water hyacinth readily tolerates environmental pollutants (15) and has been successfully used in phytoremediation for wastewater generated from industrial and agricultural sources (19 –21). In this study,

Figure 2. Melatonin and AFMK levels in the cultured water hyacinths with or without tryptophan treatment. Data are expressed as means ⫾ sem of three plants; Con, control; ** P ⬍ 0.05.

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Figure 5. Comparison of the melatonin levels in leaves and flowers of naturally growing water hyacinths in the local pond. The data are expressed as means ⫾ sem of 4 plants; *P ⬍ 0.05.

Figure 3. The HPLC chromatograms of the extracts from leaves of cultured water hyacinths with or without melatonin treatment. Top: An HPLC spectrum of a plant without melatonin treatment. Bottom: An HPLC spectrum of a plant treated with melatonin (5 ␮M) for 7 days. Mel, melatonin.

the potent antioxidants, melatonin and AFMK, were found in this plant. This is the first evidence for the presence of AFMK in any plant. AFMK can be formed from melatonin via several means, including enzymatic,

Figure 4. The circadian rhythms of melatonin (A) and AFMK (B) in naturally growing water hyacinths in a pond in San Antonio, TX, USA. The data are expressed as means ⫾ sem of 4 plants at each time point; dark bar indicates the daily dark period.

pseudoenzymatic and photocatalytic processes and via numerous free radical reactions (22). Interactions of melatonin with several reactive species, including hydrogen peroxide (H2O2), superoxide anion, singlet oxygen, peroxynitrite, and hypochlorous acid generate AFMK as one of the major metabolites (23). Recently, it was found that cytochrome c can also oxidize melatonin to form AFMK (24). In a unicellular organism (dinoflagellate), AFMK is generated when this organism is exposed to UV or to short-wavelength visible light (25). Because relatively high levels of H2O2 and singlet oxygen are produced during photosynthesis of plants, melatonin as a unique antioxidant may defend plants against toxic reactive oxygen and reactive nitrogen species; as a result, AFMK is generated. AFMK is an active biogenic amine in animals. It functions as an antioxidant to protect cells from oxidative damage (23) and can also selectively down-regulate the expression of the proinflammatory gene for cyclooxygenase 2 (26, 27). However, because this is the first report to show the presence of AFMK in plants, its functions in plants have obviously never been examined and deserve further investigation. When tryptophan, a precursor of melatonin, was provided to water hyacinths, melatonin production was elevated significantly. This finding is similar to previously published data in a land plant, St. John’s wort (Hypericum perforatum L) (28). The current data indirectly prove that the water hyacinth has the necessary enzymatic machinery for melatonin biosynthesis. In addition to its synthesis, exogenously provided melatonin can also be rapidly absorbed by water hyacinths. In another photoautotroph, Gonyaulax, exogenously added melatonin is reportedly also rapidly taken up (29, 30). The marked uptake of melatonin by water hyacinth could make this plant even more useful in phytoremediation since artificially supplemented melatonin, because it is a powerful antioxidant, would elevate their tolerance to pollutants and remove additional contaminants; as a result, the cleansing activity of polluted water would be improved. It is well documented that melatonin production exhibits a circadian rhythm in the pineal gland of animals; however, whether it is rhythmic in plants has been sparingly investigated. Wolf et al. (31) reported a


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rhythm of melatonin in a short-day flowering plant, Chenopodium rubrum, and the rhythm resembled that found in animals, that is, high values during darkness and low levels during the day. In another study, Van Tassel et al. (32) found that melatonin levels in the tomato did not exhibit day-night changes. In the current study, novel melatonin and AFMK peaks were revealed, and they occurred near sunset. It has been reported that melatonin production in plants is not inhibited by exposure to light, such as occurs in the pineal gland of animals, and in plants, its synthesis is actually stimulated by increasing intensities of light (28). Afreen et al. (33) showed that UV-B exposure promotes melatonin synthesis in the roots of Glycyrrhiza uralensis. They speculated that the elevated melatonin production under the UV-B is an adaptive reaction of the plants to tolerate the UV irradiation. The highest level of melatonin in the water hyacinth during the late light phase may represent the accumulated melatonin, which is synthesized during daily sunlight exposure. In accordance with the novel diurnal rhythms, we also found that melatonin levels in cultured water hyacinths are significantly lower than those of plants grown under natural sunlight. The intensity of sunlight (10,000 –15,000 ␮W/cm2) is much greater than that of artificial light (400 – 450 ␮W/cm2) used in the current study. Considering the novel 24-h rhythms of melatonin and AFMK and the phenomenon that the light intensity promoted melatonin production in an aquatic plant indicate that melatonin and AFMK do not serve as chemical signals of darkness as they do in animals. Rather, they seem to be more related to the process of photosynthesis or possibly to photoprotection from UV light exposure. During the process of photosynthesis, large quantities of reactive oxygen species (ROS) are generated, and relatively high levels of antioxidants, including melatonin and AFMK, would be beneficial to the plant. In reference to photoprotection, with increasing exposure to light during the photophase, plastidial photoprotection is diminished, including the impairment of the violaxanthin cycle. Additionally, the lightharvesting complexes and photosystems are progressively damaged so that excessive superoxide is formed, and the resulting H2O2 is, in aquatic photoautotrophs, measurably released into the water. The increases in melatonin and AFMK at the end of photophase may, thus, reflect the necessity of photoprotection when other mechanisms become less efficient. The photoprotective effect of melatonin against UV exposure in algae and higher plants has been already suggested (4, 34, 35). This suggestion is supported by an observation of Conti et al. (36), who showed that alpine and Mediterranean plants exposed to high UV in their natural habitat contain much more melatonin than the same species living under lower UV exposure. The protective effects of melatonin against environmental pollutants have been intensively studied in animals, but not in plants. Melatonin administration improves the survival of mice which have been treated 1728

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with mercury (37) and protects against organ damage caused by mercury in rats (38). Melatonin also prevents DNA damage induced by chromium (39) and inhibits organ injury caused by cadmium in hamsters (40). The protective effects of melatonin against these environmental pollutants are attributed to its antioxidant capacity (41, 42). The physiology of melatonin in plants has been sparingly investigated. It is suggested that melatonin may function as an auxin to promote plant growth (43). Recently, Arnao and Hernandez-Ruiz (44) proved that melatonin indeed stimulates plant growth and its action and levels in different parts in the etiolated hypocotyls of Lupinus albus L are very similar to those of auxin. Another potential physiological function of melatonin in plants is speculated to be as an antioxidant to protect them against oxidative stress (45). Plant oxidative stress can also be initiated by contaminants in the environment and during the process of photosynthesis. Harsh environmental changes, for example cold temperature, elevate plant melatonin production (13). Melatonin supplementation attenuates cold-induced apoptosis in carrot root cell suspensions (46). In addition to melatonin, its oxidative metabolite, AFMK, also exhibits antioxidative activity. The ability of melatonin and its metabolites to function as free radical scavengers has been referred to as the antioxidant cascade of melatonin’s interactions with free radicals (14). The rather high levels of melatonin, as well as AFMK, in the water hyacinth indicate that this plant has an elevated antioxidant capacity that may explain their high tolerance to pollutants, which are initiators of oxidative stress. Further research will focus on whether other plants, which are normally used in phytoremediation, also contain high levels of melatonin and AFMK. This work was partially supported by the Brazilian research funding institutions Fundaçaõ de Amparo a` Pesquisa do Estado de Saõ Paulo, Conselho Nacional de Pesquisa, Instituto do Mileˆnio: Redoxoma, and The John Simon Memorial Guggenheim Foundation (P.D.M. Fellow).

REFERENCES 1. 2. 3. 4.

5.

6.

Reiter, R. J. (1991) Melatonin: the chemical expression of darkness. Mol. Cell. Endocrinol. 79, C153–C158 Reiter, R. J. (1991) Pineal melatonin: cell biology of its synthesis and of its physiological interactions. Endocr. Rev. 12, 151–180 Poeggeler, B., Balzer, I., Hardeland, R., and Lerchl, A. (1991) Pineal hormone melatonin oscillates also in the dinoflagellate Gonyaulax polyedra. Naturwissenschaffen 78, 268 –269 Dubbels, R., Reiter, R. J., Klenke, E., Goebel, A., Schnakenberg, E., Ehlers, C., Schiwara, H. W., and Schloot, W. (1995) Melatonin in edible plants identified by radioimmunoassay and by high performance liquid chromatography-mass spectrometry. J. Pineal. Res. 18, 28 –31 Hattori, A., Migitaka, H., Iigo, M., Itoh, M., Yamamoto, K., Ohtani-Kaneko, R., Hara, M., Suzuki, T., and Reiter, R. J. (1995) Identification of melatonin in plants and its effects on plasma melatonin levels and binding to melatonin receptors in vertebrates. Biochem. Mol. Biol. Int. 35, 627– 634 Manchester, L. C., Tan, D. X., Reiter, R. J., Park, W., Monis, K., and Qi, W. (2000) High levels of melatonin in the seeds of

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TAN ET AL.

7. 8. 9. 10. 11.

12.

13.

14.

15.

16. 17.

18. 19.

20. 21. 22. 23.

24. 25.

26.

27.

edible plants: possible function in germ tissue protection. Life. Sci. 67, 3023–3029 Chen, G., Huo, Y., Tan, D. X., Liang, Z., Zhang, W., and Zhang, Y. (2003) Melatonin in Chinese medicinal herbs. Life. Sci. 73, 19 –26 Iriti, M., Rossoni, M., and Faoro, F. (2006) Melatonin content in grapes: myth or panacea? J. Sci. Food. Argic. 67, 833– 838 Reiter. R. J., Tan, D. X., Burkhardt, S., and Manchester, L. C. (2001) Melatonin in plants. Nutr. Rev. 59, 286 –290 Tan, D. X., Chen, L. D., Poeggeler, B., Manchester, L. C., and Reiter, R. J. (1993) Melatonin: a potent, endogenous hydroxyl radical scavenger. Endocr. J. 1, 57– 60 Tan, D. X., Hardeland, R., Manchester, L. C., Poeggeler, B., Lopez-Burillo, S., Mayo, J. C., Sainz, R. M., and Reiter, R. J. (2003) Mechanistic and comparative studies of melatonin and classic antioxidants in terms of their interactions with the ABTS cation radical. J. Pineal. Res. 34, 249 –259 Tan, D. X., Manchester, L. C., Hardeland, R., Lopez-Burillo, S., Mayo, J. C., Sainz, R. M., and Reiter, R. J. (2003) Melatonin: a hormone, a tissue factor, an autocoid, a paracoid, and an antioxidant vitamin. J. Pineal. Res. 34, 75–78 Tan, D. X., Manchester, L. C., Reiter, R. J., Qi, W. B., Karbownik, M., and Calvo, J. R. (2000) Significance of melatonin in antioxidative defense system: reactions and products. Biol. Signals. Recept. 9, 137–159 Tan, D. X., Reiter, R. J., Manchester, L. C., Yan, M. T., El-Sawi, M., Sainz, R. M., Mayo, J. C., Kohen, R., Allegra, M., and Hardeland, R. (2002) Chemical and physical properties and potential mechanisms: melatonin as a broad spectrum antioxidant and free radical scavenger. Curr. Top. Med. Chem. 2, 181–197 Jamil, K., Madhavendra, S. S., Jamil, M. Z., and Rao, P. V. (1987) Studies on water hyacinth as a biological filter for treating contaminants from agricultural wastes and industrial effluents. J. Environ. Sci. Health. B. 22, 103–112 Xia, H., and Ma, X. (2006) Phytoremediation of ethion by water hyacinth (Eichhornia crassipes) from water. Bioresour. Technol. 97, 1050 –1054 Riddle, S. G., Tran, H. H., Dewitt, J. G., and Andrews, J. C. (2002) Field, laboratory, and X-ray absorption spectroscopic studies of mercury accumulation by water hyacinths. Environ. Sci. Technol. 36, 1965–1970 Misbahuddin, M., and Fariduddin, A. (2002) Water hyacinth removes arsenic from arsenic-contaminated drinking water. Arch. Environ. Health. 57, 516 –518 Jayaweera, M. W., and Kasturiarachchi, J. C. (2004) Removal of nitrogen and phosphorus from industrial wastewaters by phytoremediation using water hyacinth (Eichhornia crassipes (Mart.) Solms). Water Sci. Technol. 50, 217–225 Singhal, V., and Rai, J. P. (2003) Biogas production from water hyacinth and channel grass used for phytoremediation of industrial effluents. Bioresour. Technol. 86, 221–225 Trivedy, R. K., and Pattanshetty, S. M. (2002) Treatment of dairy waste by using water hyacinth. Water. Sci. Technol. 45, 329 –334 Hardeland, R. (2005) Antioxidative protection by melatonin: multiplicity of mechanisms from radical detoxification to radical avoidance. Endocrine. 27, 119 –130 Tan, D. X., Manchester, L. C., Burkhardt, S., Sainz, R. M., Mayo, J. C., Kohen, R., Shohami, E., Huo, Y. S., Hardeland, R., and Reiter, R. J. (2001) N1-acetyl-N2-formyl-5-methoxykynuramine, a biogenic amine and melatonin metabolite, functions as a potent antioxidant. FASEB J. 15, 2294 –2296 Semak, I., Naumova, M., Korik, E., Terekhovich, V., Wortsman, J., and Slominski, A. (2005) A novel metabolic pathway of melatonin: oxidation by cytochrome c. Biochemistry. 44, 9300 –9307 Hardeland, R., Ressmeyer, A.-R., Zelosko, V., Burkhardt, S., and Poeggeler, B. (2004) Metabolites of melatonin: Formation and properties of the methoxylated kynuramines AFMK and AMK. In: Recent Advances in Endocrinology and Reproduction: Evolutionary, Biotechnological and Clinical Applications (Haldar, C., and Singh, S.S., eds.), Banaras Hindu Univ., Varanasi, India, pp. 21–38 Cuzzocrea, S., Tan, D. X., Costantino, G., Masoń, E., Caputi, A. P., and Reiter, R. J. (1999) The protective role of endogenous melatonin in carrageenan-induced pleurisy in the rat. FASEB J. 13, 1930 –1938 Mayo, J. C., Sainz, R. M., Tan, D. X., Hardeland, R., Leon, J., Rodriguez, C., and Reiter, R. J. (2005) Anti-inflammatory ac-

28.

29.

30.

31.

32. 33. 34. 35.

36.

37. 38. 39.

40.

41.

42.

43. 44. 45. 46.


tions of melatonin and its metabolites, N1-acetyl-N2-formyl-5methoxykynuramine (AFMK) and N1-acetyl-5-methoxykynuramine (AMK), in macrophages. J. Neuroimmunol. 165, 139 –149 Murch, S. J., Krishna, R. S., and Saxena, P. K. (2000) Tryptophan is a precursor for melatonin and serotonin biosynththesis in in vitro regenerated St. John’s wort (Hypericum perforatum L. cv. Anthos) plants. Plant Cell Rep. 19, 698 –704 Mueller, U., and Hardeland, R. (1999) Transient accumulations of exogenous melatonin indicate binding sites in the dinoflagellate Gonyaulax polyedra. In: Studies on Antioxidants and Their Metabolites (Hardeland, R., ed.), Cuvillier, Goettingen, pp. 140 –147 Mueller, U., Hardeland, R., Fuhrberg, B., and Poeggeler, B. (2000) Accumulation and metabolism of 5-methoxylated indoleamines in the dinoflagellate Gonyaulax polyedra. Eur. J. Cell Biol. 79, Suppl. 50, 98 Wolf, K., Kolar, J., Witters, E., van Dongen, W., van Onckelen, H. C. H., and Machackova, I. (2001) Daily profile of melatonin levels in Chenopodium rubrum L. depends on photoperiod. J. Plant. Physiol. 158, 1491–1493 Van Tassel, D. L., Roberts, N., Lewy, A., and O’Neill, S. D (2001) Melatonin in plant organs. J. Pineal Res. 31, 8 –15 Afreen, F., Zobayed, S. M., and Kozai, T. (2006) Melatonin in Glycyrrhiza uralensis: response of plant roots to spectral quality of light and UV-B radiation. J. Pineal. Res. 41, 108 –115 Balzer, I., and Hardeland, R. (1996) Melatonin in algae and higher plants—Possible new roles as a phytohormone and antioxidant. Bot. Acta 109, 180 –183 Behrmann, G., Fuhrberg, B., Hardeland, R., Uria, H., Poeggeler, B. (1997) Photooxidation of melatonin, 5-methoxytryptamine and 5-methoxytryptophol: aspects of photoprotection by periodically fluctuating molecules? Biometeorology 14, 258 –263 Conti, A., Tettamanti, C., Singaravel, M., Haldar, C., PandiPerumal, R. S., and Maestroni, G. J. M. (2002) Melatonin: An ubiquitous and evolutionary hormone. In: Treatise on Pineal Gland and Melatonin (Haldar C, Singaravel, Maitra SK, eds), Science Publishers, Enfield, NH, pp. 105–143 Kim, C. Y., Nakai, K., Kameo, S., Kurokawa, N., Liu, Z. M., and Satoh, H. (2000) Protective effect of melatonin on methylmercuryInduced mortality in mice. Tohoku. J. Exp. Med. 191, 241–246 Sener, G., Sehirli, A. O., and Ayanoglu-Dulger, G. (2003) Melatonin protects against mercury(II)-induced oxidative tissue damage in rats. Pharmacol. Toxicol. 93, 290 –296 Qi, W., Reiter, R. J., Tan, D. X., Garcia, J. J., Manchester, L. C., Karbownik, M., and Calvo, J. R. (2000) Chromium(III)-induced 8-hydroxydeoxyguanosine in DNA and its reduction by antioxidants: comparative effects of melatonin, ascorbate, and vitamin E. Environ. Health. Perspect. 108, 399 – 402 Karbownik, M., Gitto, E., Lewinski, A., and Reiter, R. J. (2001) Induction of lipid peroxidation in hamster organs by the carcinogen cadmium: melioration by melatonin. Cell Biol. Toxicol. 17, 33– 40 Reiter, R. J., Tan, D. X., Manchester, L. C., and Qi, W. (2001) Biochemical reactivity of melatonin with reactive oxygen and nitrogen species: a review of the evidence. Cell. Biochem. Biophys. 34, 237–256 Tan, D. X., Manchester, L. C., Terron, M. P., Flores, L. J., and Reiter, R. J. (2007) One molecule, many derivatives: A neverending interaction of melatonin with reactive oxygen and nitrogen species? J. Pineal. Res. 42, 28 – 42 Hernandez-Ruiz, J., Cano, A., and Arnao, M. B. (2005) Melatonin acts as a growth-stimulating compound in some monocot species. J. Pineal. Res. 39, 137–142 Arnao, M., and Hernandez-Ruiz, J. (2007) Melatonin promotes adventitious and lateral root regeneration in etiolated hypocotyls of Lupinus albus L. J. Pineal Res. 42, 147–152 Kolar, J., and Machackova, I. (2005) Melatonin in higher plants: occurrence and possible functions. J. Pineal. Res. 39, 333–341 Lei, X. Y., Zhu, R. Y., Zhang, G. Y., and Dai, Y. R. (2004) Attenuation of cold-induced apoptosis by exogenous melatonin in carrot suspension cells: the possible involvement of polyamines. J. Pineal. Res. 36, 126 –131 Received for publication November 9, 2006. Accepted for publication January 4, 2007.

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