www.elsevier.com/locate/biochemsyseco

Available online at www.sciencedirect.com

Biochemical Systematics and Ecology 36 (2008) 71e73

Acetanilide as the only constituent in skin secretion of Xenohylatruncata Izecksohn, 1959 (1998) and its biological significance

M�ario Geraldo de Carvalho a,*, Ronald Bastos Freire b, Felipe Baggio Vianna b,Marli Terezinha Frana Cornelius a

a Departamento de Quımica, ICE, UFRRJ, BR 465 Km. 07, 23890-000 Seropedica, RJ, Brazilb Departamento de Biologia Animal, IB, UFRRJ, BR 465 Km. 07, 23890-000 Seropedica, RJ, Brazil

Received 9 February 2007; accepted 27 May 2007

Keywords: Xenohyla truncata; Hylidae; Acetanilide

1. Subject and source

Xenohyla truncata Izecksohn, 1959, is one of the two species of the genus Xenohyla I., 1998 bufonid treefrogs,order Anura, Hylinae subfamily of the Hylidae family (Izecksohn, 1998; Faivovich et al., 2005; Frost et al., 2006;Frost, 2007). Members of this family are 3 cm in length and are distributed throughout North and South America,with one complex species across part of Europe, Africa, and Asia (Bevins and Zasloff, 1990). In Brazil, X. truncatais endemically distributed on scrubby vegetation restricted to the coppices bordering the seashore of Rio de JaneiroState (Napoli, 2001). This is the only treefrog species known to eat fruit (Da Silva et al., 1989; Santos et al., 2004; DaSilva and de Britto-Pereira, 2006). It is not known whether the chemical composition, such as alkaloids, of the fruitseaten by this bufonid treefrog influences the chemical constituents of its skin exhalation. In order to study the skinexhalations, two individuals were collected in the wet season (July 2004) in the Rio de Janeiro’s Maric�a lagoon at22�590 SE latitude; 44�050W longitude. A voucher specimen (number EI-053) was deposited at the Vertebrate Collec-tion, Zoology Section, Animal Biology Department, Universidade Federal Rural do Rio de Janeiro. The treefrogs werehandled with care and caged in a shock-chamber to induce the skin exhalation. The electrically elicited secretion wascollected and we observed that the identified chemical constituent is different from those commonly in the fruit eatenby the treefrog.

2. Previous work

No previous work has been reported on the chemistry of the skin secretion of this treefrog genus.

* Corresponding author. Tel.: þ55 2126820721; fax: þ55 2126822807.

E-mail address: mgeraldo@ufrrj.br (M.G. de Carvalho).

0305-1978/$ - see front matter � 2007 Elsevier Ltd. All rights reserved.

doi:10.1016/j.bse.2007.05.016

72 M.G. de Carvalho et al. / Biochemical Systematics and Ecology 36 (2008) 71e73

3. Present study

The X. truncata skin exhalation was induced through 1 min application of a continuous current of 1.0 V (1.0 A) toeach animal in a 3.0� 15 cm shock-chamber previously added with 3.0 ml of sterile distillated water. A whitecrystalline substance (acetanilide) was obtained by filtration through a Buchner funnel under vacuum and dried inan Abder Haldem pistol system. This procedure gave 0.09 g exhalation per gram of treefrog body weight, correspond-ing to a final yield of 2.2 g. Because a crystalline substance was obtained, we believed the main constituent might bea single substance. Its melting point (112e114 �C) was determined on Kofler’s block adapted to a microscope, withoutcorrection values. Afterwards, it was analyzed by silica gel w/UV254, aluminum backed, TLC plates (Sorbent) usingCHCl3/MeOH (9:1) as eluent. The substance spot was visualized by UV (254 and 366), ceric sulfate, aluminumchloride and by Dragendorff reagent. IR spectra were carried out on Perkin Elmer 1600/2605 FT-IR spectrophotom-eter using KBr disks. NMR spectra were recorded on Bruker (200 MHz for 1H and 50.3 MHz for 13C) dissolving10.0 mg/ml and 40 mg/ml, respectively, using CDCl3 as solvent and TMS as internal standard. Electron ionizationmass spectra (EIMS) were taken with gas chromatography coupled to a mass spectrometry (GC/MS) on Varian Saturn2000 (2.0 mg/ml in CHCl3). Although fruits of Erythroxylum ovalifolium eaten by the X. truncata treefrogs are knownto contain alkaloids, the Dragendorff reaction did not confirm the presence of such chemical group of substances in theexudates from the frogs’ skin. The IR spectra showed bands at 3294 cm�1 (nNeH), 1663 cm�1 (nC]O), 1601, 1550,1495 cm�1 (attributed to aromatic ring), compatible with aromatic amides. The 1H NMR spectrum gave rise to signalsat dH: 7.49 (d, J¼ 8 Hz, 2H, H-2,6), 7.29 (t, J¼ 8 Hz, 2H, H-3,5) and 7.08 (t, J¼ 8 Hz, 1H, H-4), 2.15 (s, 3H, H-8) andsinglet at dH 7.61 (change with D2O, NeH). The 13C NMR (BBD) spectral analysis led to identify the signal at dCH

119.9 (C-2,6), 128.9 (C-3,5), 124.4 (C-4), dCH324.4 (C-8) and dC at 168.7 (C-7) and 137.9 (C-1). These data led us to

identify the compound in the skin emission from X. truncata to be N-phenyl-acetamide. The mass spectrum revealedpeaks at m/z (%): 135 (90, Mþ

�), 93 (100, C6H7Nþ

�) and 66 (25, 93�HCN) corroborating the N-phenyl-acetamide (1)

identification.

N

1

H

O

1

2

3

4

5

6

7 8

4. Proposed biochemical and ecological significance

In this paper we report that the product discharged under an electrically induced stress from the skin of X. truncatais N-phenyl-acetamide (1). The acetanilide content may be a consequence of the groundwater pollution by farmingactivities (Eykholt and Davenport, 1998). More persistent acetanilide herbicides’ degradates present in groundwaterand surface water (Ujv�ary, 2000) are also detoxified in biological systems by the formation of glutathioneeacetanilideconjugates (Stamper and Tuovinen, 1998; Emtiazi et al., 2001), which might lead acetanilide to accumulate at largeconcentration in the rich poisonous parotid glands and/or miniature glands scattered in the X. truncata skin. Theacetanilide, and its derivatives, may act synergistically with other cutaneous poison to enhance its known insecticide,antimicrobial, anti-inflammatory hypotensive and neuromuscarinic properties (Emtiazi et al., 2001; Farah et al.,2004). In the toads, this secretion generally contains steroids and biogenic amines (Edstrom, 1992). In certain species,the poison contains non-bioactive substances such as glycosaminoglycans and catalase, whose functional significanceis yet unknown (Toledo and Jared, 1995). These differences suggest a high diversity in the phylogenic expression ofenzymes directed to the bioactive content in poisonous treefrogs (Bevins and Zasloff, 1990) and may explain possiblevariations in the poison as well as the evolutionary advantage of those from several treefrogs.

73M.G. de Carvalho et al. / Biochemical Systematics and Ecology 36 (2008) 71e73

Acknowledgements

The authors are grateful to the IBAMA (Instituto Brasileiro do Meio Ambiente) for the permits to Dr Helio Ricardoda Silva, from the Zoology section of the Universidade Federal Rural do Rio de Janeiro for the animal’s capture, and toFAPERJ, CAPES and CNPq for the research fellowships and financial support.

References

Bevins, C.L., Zasloff, M., 1990. Annu. Rev. Biochem. 59, 395.

Da Silva, H.R., de Britto-Pereira, M.C., Caramaschi, U., 1989. Copeia 1989, 785.

Da Silva, H.R., de Britto-Pereira, M.C., 2006. J. Zool. 270, 692.

Edstrom, A., 1992. Venomous and Poisonous Animals. Krieger Publishing Company, Malabar, Florida, pp. 210 (115).

Emtiazi, G., Satarii, M., Mazaherion, U.M., 2001. Water Res. 35, 1219.

Eykholt, G.R., Davenport, D.T., 1998. Environ. Sci. Technol. 32, 1482.

Faivovich, J., Haddad, C.F.B., Garcia, P.C.A., Frost, D.R., Campbell, J.A., Wheeler, W.C., 2005. Bull. Am. Mus. Nat. Hist. 294, 1.

Farah, M.A., Ateeq, B., Ali, M.N., Sabir, R., Ahmad, W., 2004. Chemosphere 55, 257.

Frost, D.R., 1 February, 2007. Amphibian Species of the World: An Online Reference, Version 5.0. American Museum of Natural History, New

York, USA. Electronic Database Accessible at http://research.amnh.org/herpetology/amphibia/index.php.

Frost, D.R., Grant, T., Faivovich, J., Bain, R.H., Haas, A., Haddad, C.F.B., De Sa, R.O., Channing, A., Wilkinson, M., Donnellan, S.C.,

Raxworth, C., Campbell, J.A., Blotto, B.L., Moler, P., Drewes, R.C., Nussbaum, R.A., Lynch, J.D., Green, D.M., Wheeler, W.C., 2006.

Bull. Am. Mus. Nat. Hist. 297, 1.

Izecksohn, E., 1998. Rev. Univ. Rural Ser. Cienc. Vida 18, 47.

Napoli, M.F., 2001. Herpetol. Rev. 32, 36.

Santos, E.M., Almeida, A.V., Vasconcelos, S.D., 2004. Iheringia Ser. Zool. 94, 433.

Stamper, D.M., Tuovinen, O.H., 1998. Crit. Rev. Microbiol. 24 (1), 1.

Toledo, R.C., Jared, C., 1995. Comp. Biochem. Physiol. A Physiol. 111, 1.

Ujv�ary, I., 2000. Pest Manag. Sci. 56, 703.