Lack of bufadienolides in the skin secretion of red bellied toads, Melanophryniscus spp. (Anura, Bufonidae), from Uruguay

Comparative Biochemistry and Physiology, Part C 144 (2007) 398 – 402 www.elsevier.com/locate/cbpc

Lack of bufadienolides in the skin secretion of red bellied toads, Melanophryniscus spp. (Anura, Bufonidae), from Uruguay Dietrich Mebs a,⁎, Moritz G. Wagner a , Werner Pogoda a , Raul Maneyro b , Axel Kwet c , Gerold Kauert a a b

Institute of Forensic Toxicology, Zentrum der Rechtsmedizin, University of Frankfurt, Kennedyallee 104, D-60596 Frankfurt, Germany Sección Zoología Vertebrados, Facultad de Ciencias, Iguá 4225, CP: 11400, Montevideo, Uruguay and Museu de Ciência e Tecnologia and Faculdade de Biociências, Pontifica Universidade Católica do Rio Grande do Sul, Brazil c Staatliches Museum für Naturkunde, Zoologie, Rosenstein 1, D-70191 Stuttgart, Germany Received 19 July 2006; received in revised form 23 November 2006; accepted 23 November 2006 Available online 29 November 2006

Abstract The South-American red bellied toads (Melanophryniscus spp.) belonging to the Bufonidae family contain toxic alkaloids in their skin, predominantly of the pumiliotoxin group. Whole animal methanolic extracts of individual specimens of three species (Melanophryniscus atroluteus, M. devincenzii, and M. montevidensis) were analyzed for the presence of toad specific bufadienolides and indolalkylamines (serotonin derivatives) by HPLC-electrospray (ESI)-MS-TOF. No bufadienolides, but few bufotenines, mainly dehydrobufotenine, were detected in the extracts in variable amounts. The concentration of the dehydrobufotenine in the extracts seems to be species specific. Whereas M. atroluteus and M. montevidensis contain very low or trace amounts, M. devincenzii specimens exhibit high concentrations of this indolalkylamine. In comparison, analysis of extracts from Bufo arenarum (Uruguay) and from B. bufo (Germany) confirmed the presence of bufadienolides as well as of bufotenine derivatives. Tadpoles of both species exhibited a different pattern: extracts from B. arenarum tadpoles contained only dehydrobufotenine, but those from B. bufo tadpoles bufotoxin and two alkylamines. Melanophryniscus toads appear not to be able to compensate the high variability of toxic skin alkaloids by producing defensive bufadienolides. © 2006 Elsevier Inc. All rights reserved. Keywords: Red bellied toad; Melanophryniscus; Skin secretion; Bufadienolides; Dehydrobufotenine; Indolalkylamines; Pumiliotoxin

1. Introduction Red bellied toads of the genus Melanophryniscus, which are distributed in the southern parts of South-America (Kwet et al., 2005), contain toxic alkaloids in their skin (Daly et al., 1984; Garraffo et al., 1993; Mebs et al., 2005). In whole animal extracts of specimens from Uruguay (Melanophryniscus montevidensis) alkaloids of the pumiliotoxin (PTX) group and indolizidines were identified. Among them PTX 251D was the predominant compound. The PTX-content of the various toad populations varied considerably. Whereas very high levels of PTX 251D were detected in toads from a certain area, those from other areas exhibited low or undetectable levels of this alkaloid (Mebs et al., 2005). ⁎ Corresponding author. Tel.: +49 69 6301 7418; fax: +49 69 6301 5882. E-mail address: [email protected] (D. Mebs). 1532-0456/$ - see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.cbpc.2006.11.009

The genus Melanophryniscus belongs to the anuran family Bufonidae, toads sensu strictu. Bufonids are characterized by the presence of bufadienolides (bufogenins) and indolalkylamines in their skin secretion (Deulofeu and Ruveda, 1971; Krenn and Kopp, 1998; Steyn and van Heerden, 1998). Bufadienolides are polyhydroxy C24 steroids exhibiting a wide variety of genins and existing in free or conjugated entities. These compounds exert cardiotonic activity by inhibiting the Na+, K+-ATPase thereby increasing the contractile force of the heart muscle. Moreover, some bufadienolides possess antitumor activity (Kamano et al., 2002), insect antifeedant (Steyn and van Heerden, 1998), insecticidal (Supratman et al., 2001), and antimicrobial properties (Taniguchi and Kubo, 1993; Cunha Filho et al., 2005). Other compounds in the skin secretion of toads are indolalklyamines, e.g. bufotenines. They represent derivatives of serotonin and occur as dehydrobufotenine, O-methylbufotenine, bufotenidine,

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Fig. 1. Identification of dehydrobufotenine by HPLC-electrospray (ESI)-MS-TOF in methanolic extracts from Melanophryniscus species, corresponding to the theoretical accurate mass of 203.118438, and its two isotopes.

bufoviridine and bufothionine in skin secretions of toads as well as of several frogs occasionally in high concentrations (Daly et al., 1987). Bufotenines, which are also found in various plants (Schultes and Hofmann, 1980) elicit hallucinogenic sensations in humans (Fabing and Hawkins, 1956). In the present study, whole body extracts from individual specimens of red bellied toads, Melanophryniscus atroluteus, M. devincenzii, and M. montevidensis were analyzed for their indolalkylamine and bufadienolide content using HPLC-ESIMS-TOF methodology. 2. Methods and materials 2.1. Collection of animals Specimens of M. montevidensis (36) were collected in various locations in Uruguay as described previously (Mebs et al., 2005). Specimens of M. atroluteus (28) and M. devincenzii (16) were collected at Parque Gran Bretaña in April 2005 and Cuchilla Negra in September 2005, respectively, in both cases in Departamento Rivera (Northern Uruguay). Specimens of Bufo arenarum (3 tadpoles, 6 froglets, 3 adults) were collected in Cabo Polonio, Departamento Rocha, Uruguay, in October 2003. Bufo bufo specimens (24 tadpoles, 5 adults) were from Germany (ponds near Frankfurt, March 2003). 2.2. Analysis of skin extracts Methanol (70%) extracts obtained from each animal were analyzed for the presence of alkaloids by gas chromatography/ mass spectrometry (GC/MS) as described previously (Mebs et al., 2005). Analysis of bufadienolides and indolalkylamines was performed by HPLC-ESI-MS-TOF. The HPLC-system

(1100 series; Agilent Technologies, Waldbronn, Germany) was equipped with an autosampler and a Zorbax™ SB C-18 column (2.1 × 50 mm, 1.8 μm particle size; Agilent Technologies) connected to a time-of-flight mass spectrometer (TOF; Agilent Technologies). Fractionation of the extracts dissolved in 200 μl 50% acetonitrile with 0.1% formic acid was carried out at 50 °C using a linear gradient of 0.1% formic acid as solvent A and acetonitrile as solvent B over 15 min. The LC-MS-TOF interface was an electrospray ion source (ESI) equipped with a dual-sprayer mechanism that allows constant injection of reference substances (purine at 121.05873 Da and HP-921 at 922.009798 Da) for mass shift correction in every spectrum acquired, thus providing mass accuracies in the range of ± 3 ppm. ESI source and the MS-TOF parameters were set according to the recommendations of the supplier except for nebulizer pressure (Pg = 50 psi), capillary voltage (4000 V) and drying gas flow (12 L/min at 350 °C). The fragmentor voltage was set to 180 V. Data analysis was performed using Analyst QS 1.1 software (Applied Biosystems/MDS Sciex, Concord, Canada). For quantitative analysis of bufotenine derivatives, tryptamine (Sigma–Aldrich, St. Louis, USA) was used as standard compound, sensitivity was in a range of 1–2 ng.

Table 1 Concentration of dehydrobufotenine in whole body extracts of Melanophryniscus species (tryptamine equivalents, μg per toad), determined by HPLC-electrospray (ESI)-TOF using tryptamine as standard

M. atroluteus (12/16) M. devincenzii (16/16) M. montevidensis (8/36)

Mean

(Min.–max. values)

14.1 513.3 b3

(0.7–66.0) (93.1–1016.6)

In brackets: number of positive /no. of total extracts.

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Measurements were replicated twice confirming very low analytical variability of the data. 3. Results 3.1. Analysis of the extracts from Melanophryniscus species In whole body extracts from individual red bellied toads, M. montevidensis, several alkaloids, including pumiliotoxin PTX 251D as a major compound had been identified by gas chromatography/mass spectrometry. In addition remarkable concentrations of hydroquinone were also identified (Mebs et al., 2005). A similar alkaloid and hydroquinone composition was found in extracts of the other Melanophryniscus species, e.g. M. atroluteus (28 specimens) and M. devincenzii (16 specimens) (data not shown). When using the HPLC-ESI-MS-TOF methodology, no bufadienolides (screened for 24 derivatives), neither in free nor in conjugated forms were detected in the extracts of the three Melanophryniscus species. In HPLC elution patterns of the extracts, only minor peaks occurred when recording the absorbance at 300 nm, the absorption maximum of the α-pyrone ring of bufadienolides. A few indolalkylamines only, such as dehydrobufotenine (Fig. 1) and hydroxymethyl–bufotenine were detected in 8 of 36 extracts from M. montevidensis, generally in trace amounts (Table 1). Similar results were obtained by analyzing extracts from M. atroluteus (28 specimens): Dehydrobufotenine and hydroxymethyl–bufotenine were present in 12 extracts. On the other hand, dehydrobufotenine was detected in considerably high concentrations in all extracts from M. devincenzii (16 specimens). 3.2. Analysis of extracts from B. arenarum and B. bufo For comparison analysis of extracts from the toads B. arenarum (Uruguay) and B. bufo (Germany) was performed confirming the presence of bufadienolides as well as indolakylamines (Table 2). In extracts from B. arenarum the two bufadienolides, bufogenin and bufalin, and two indolalkylamines, dehydrobufotenine and bufothionine, were identified, whereas extracts of the European toad B. bufo contained bufotoxin, bufotalin and bufalin in addition to the two Table 2 Bufadienolides and indolalkylamines in extracts from the toads Bufo arenarum and B. bufo, as detected by HPLC-electrospray (ESI)-MS-TOF B. arenarum Tadpoles Bufadienolides Bufogenine B Bufotoxin Bufotalin Bufalin Indolalkylamines Dehydrobufotenine Bufothionine

B. bufo Adults

Tadpoles

X

X X

X

X

X X

X X

Adults

X X X

X X

bufotenine derivatives. It is interesting to note that tadpoles of both species exhibited a different pattern of compounds when compared to that of the corresponding adults: extracts from B. arenarum tadpoles contained only dehydrobufotenine, whereas those from B. bufo contained bufogenine B and bufotoxin as well as two indolalkylamines. 4. Discussion Secretions from skin glands of bufonid anurans render these toads unpalatable to most predators (Hayes, 1989), because of the presence of highly toxic bufadienolides. This observation has also been confirmed for eggs and tadpoles (Licht, 1968). Moreover, these compounds exhibit marked antimicrobial activity protecting the animal's skin from infections by microorganisms (Cunha Filho et al., 2005). Other compounds such as indolalkylamines seem to occur regularly as secondary metabolites in the skin secretion of toads, e.g. bufotenine and its various derivatives (Cei et al., 1972). The small South-American toads of the genus Melanophryniscus contain numerous toxic alkaloids in their skin (Daly et al., 1984; Garraffo et al., 1993; Mebs et al., 2005), which have been suggested to be of dietary origin like in the case of the poisonous frogs belonging to the families Dendrobatidae, Mantellidae and Myobatrachidae (Jones et al., 1999; Daly et al., 1997, 2000, 2002; Saporito et al., 2004; Smith et al., 2002; Takada et al., 2005). In fact, alkaloids such as pyrrolidines, piperidines, indolizidines and pyrrolizidines have been detected in the skin of these frogs as well as in several arthropods which may serve as food. Some ant species (Brachymyrmex and Paratrechina species) have been identified as a potential source of pumiliotoxins (Saporito et al., 2004). Although analysis of 125 samples of various Uruguayan arthropods including ants provided no clues for the origin of pumiliotoxin alkaloids in Melanophryniscus species (Mebs et al., 2005), the high variability in alkaloid levels in skin extracts of these toads supports the assumption that these animals are not able to synthesize these compounds and rely on toxic food sources. Recently, Takada et al. (2005) reported that extracts from mites of the genus Scheloribates revealed the presence of PTX 251D, but also of coccinelline-type alkaloids. Thus, mites may probably serve as a potential source of pumiliotoxins in Melanophryniscus toads. Most members of the family Bufonidae produce various toxic steroidal compounds, e.g. bufogenins or bufadienolides, which are synthesized and secreted by granular glands present in the skin (Neuwirth et al., 1979; Houck and Sever, 1994). Those glands are found all over the body of Melanophryniscus species (Naya et al., 2004; Mebs et al., 2005). It is, therefore, surprising that these toad specific compounds have not been found in whole body extracts of three Melanophryniscus species. The high sensitivity (ng range) and specificity of the analytical methods applied (HPLC-ESI-MS-TOF) for screening a wide range of these compounds was confirmed by the positive identification of these compounds in body extracts from two toad species, e.g. one from Uruguay, B. arenarum, and another from Europe, B. bufo.

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When using a test system, e.g. inhibition of the uptake of radioactive rubidium ions into red blood cells, for assaying the potency of various amphibian skin extracts to prevent binding of ouabain to Na+, K+-ATPase, Flier et al. (1980) observed high inhibitory activity in an extract from M. moreirae. The active principles were found to be highly polar, but appeared to be devoid of the bufadienolide chromophore, the α-pyrone ring. This confirms findings in the present study in which none or only minor peaks of the bufadienolide chromophore were detected when measuring absorbance of the eluent at 300 nm. It seems that toads of the genus Melanophryniscus are not able to synthesize typical toad bufadienolides. However, the lack of those compounds in the extracts does not rule out the presence of other compounds interfering with the ouabain binding site. Beside bufadienolides toads also produce several indolalkylamines such as bufotenine and its derivatives. These compounds, e.g. dehydrobufotenine and bufothionine were also found in the extracts of the two Bufo species. But in most extracts of Melanophryniscus species, e.g. M. atroluteus and M. montevidensis, dehydrobufotenine and hydroxymethyl–bufotenine were either estimated in low amounts only or were not detectable. However, extracts of M. devincenzii exhibited considerably high concentrations of dehydrobufotenine, up to 1 mg per toad. These results confirm studies of Cei et al. (1972) and Cei (1980), who reported only one indolalkylamine in extracts from Melanophryniscus species (M. moreirae, M. stelzneri), e.g. bufotenine (5 μg per g tissue), but no other compounds such as 5-hydroxytryptamine, N-methyl-5-HT, dehydrobufotenine, bufotenidine or bufothionine. Previously, Cei et al. (1968) had mentioned that in extracts of M. moreirae up to 2200 μg of dehydrobufotenine per g were present. The variability of the dehydrobufotenine content in Melanophryniscus toads appears to be species specific. Whereas some species seem to produce very low amounts or none of this indolalkylamine, others such as M. devincenzii contain rather high amounts. It is tempting to speculate, whether the lack of toxic bufadienolides in Melanophryniscus toads are an adaptation to another defensive strategy: accumulating toxic alkaloids sequestered from dietary sources in the skin instead of investing in expensive toxin synthesis. In this respect it is interesting to note that frogs of the genus Pseudophryne are able to synthesize pseudophrynamine alkaloids beside their ability to accumulate pumiliotoxins from dietary sources. But biosynthesis of pseudophrynamines is reduced when there are high levels of dietary pumiliotoxins present (Smith et al., 2002). However, the lack of toxin synthesis as in the case of Melanophryniscus toads involves the risk that the toxicity of their skin secretion may be low or even negligible when the toxic food source is not available. This is demonstrated by the high variability of alkaloid levels among diverse Melanophryniscus populations, ranging from zero to high levels of the major alkaloid PTX 251D (Mebs et al., 2005). The toads are obviously not able to compensate the low level or even total lack of alkaloid toxicity by producing their own defensive compounds, e.g. bufadienolides. The way out of the dilemma might be the fact that most Melanophryniscus species have a dark body colouration and exhibit cryptic behaviour. The red belly is rarely presented as an

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