The Society for Integrative and Comparative Biology
Testosterone-Fatty Acid Esterification: A Unique Target for the Endocrine Toxicity of Tributyltin to Gastropods1
1 Department of Environmental and Molecular Toxicology, North Carolina State University, Raleigh, North Carolina 27695-7633
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Over the past thirty years, a global occurrence of sexual aberration has occurred whereby females among populations of prosobranch snails exhibit male sex characteristics. This condition, called imposex, has been causally associated with exposure to the biocide tributyltin. Tributyltin-exposed, imposex snails typically have elevated levels of testosterone which have led to the postulate that this endocrine dysfunction is responsible for imposex. This overview describes recent evidence that supports this postulate. Gastropods maintain circulating testosterone levels and administration of testosterone to females or castrates stimulates male sex differentiation in several snail species. Studies in the mud snail (Ilyanassa obsoleta) have shown that gastropods utilize a unique strategy for regulating free testosterone levels. Excess testosterone is converted to fatty acid esters by the action of a testosterone-inducible, high capacity/low affinity enzyme, acyl-CoA:testosterone acyl transferase, and stored within the organisms. Free testosterone levels are regulated during the reproductive cycle apparently due to changes in esterification/desterification suggesting that testosterone functions in the reproductive cycle of the organisms. Testosterone esterification provides a unique target in the testosterone regulatory machinery of snails that is altered by tributyltin. Indeed, imposex and free testosterone levels were elevated in field collected snails containing high tin levels, while testosterone-fatty acid ester pools were reduced in these organisms. These observations indicate that tributyltin elevates free testosterone by reducing the retention of testosterone as fatty acid-esters. This endocrine effect of tributyltin may be responsible for imposex.
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INTRODUCTION |
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SYNOPSISINTRODUCTIONTESTOSTERONE AS A MASCULINIZING...PUTATIVE TARGETS OF TRIBUTYLTIN...CONCLUSIONReferences |
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Over the past thirty years, a global occurrence of sexual aberration has occurred whereby females among populations of gonochoristic prosobranch snails exhibit male sex characteristics. This condition is most typically associated with the development of a penis by female snails (Matthiessen and Gibbs, 1999
; Blaber, 1970) although more severe cases can involve the development of a vas deferns or a convolution of the oviduct that resembles the seminal vesicles of males (Jenner, 1979; Smith, 1971). This condition has been termed imposex (Smith, 1971) and has been causally associated with exposure to the biocide tributyltin (Smith, 1981a, b, c; Bryan et al., 1986, 1987). Tributyltin has been used extensively as an antifoulant in marine paints. Tributyltin levels are commonly measured in the marine environment at levels sufficient to cause imposex (summarized in LeBlanc and Bain, 1997).
The development of imposex by neogastropods is a highly specific response to exposure to organotins. In addition to tributyltin, triphenyltin also can cause imposex though with significantly lower potency (Schulte-Oehlmann et al., 2000). Exposure of snails to other environmental contaminants including bisphenol A, octylphenol, (Oehlmann et al., 2000), vinclozolin (Tillmann et al., 2001), and 4-nonylphenol (unpublished observations) have failed to induce imposex. This apparent high degree of specificity implies that the organotins target a specific site (i.e., receptor, enzyme, etc.) that is a critical component to the sex differentiation pathway of neogastropods. Unfortunately, the endocrinology of sex differentiation among neogastropods is poorly understood. Male sex differentiation occurs seasonally which implies that environmental factors (i.e., photoperiod, temperature, etc.) stimulate a neuro-endocrine pathway leading to the development of male sex characteristics. Various hormones have been evaluated for their ability to participate in this neuroendocrine signaling cascade. Two hormones have emerged as candidate participants– APGWamide and testosterone.
APGWamide is a neuropeptide secreted by the anteromedial region of the right cerebral ganglion, the right pedal ganglion, the copulatory duct, and penial complex of male mollusks (deLange and van Minnen, 1998; Fan et al., 1997). APGWamide contributes to the activity of the penis retractor muscle and muscles involved in ejaculation (Li et al., 1992) as well as male mating behavior (Koene et al., 2000). APGWamide was reported to stimulate penis growth in the female mud snails (Ilyanassa obsoleta) (Oberdorster and McClellan-Green, 2000). This observation, coupled with the fact that APGWamide is secreted by the cerebral ganglia and the pedal ganglia provide compelling evidence for APGWamide being the penultimate neuroendocrine hormone of prosobranch gastropods that responds to the environmental stimuli responsible for male sex differentiation.
Testosterone is generally viewed as a vertebrate-type sex steroid. However, mounting evidence suggests that testosterone has a role in molluscan sex differentiation and reproduction. For example, administration of testosterone stimulated the development of a penis and/ or vas deferens in female marine snails, Nucella lapillus, Hinia reticulate, and Ilyanassa obsolete (Bettin et al., 1996; Oberdorster and McClellan-Green, 2000; Spooner et al., 1991). Testosterone administration stimulated spermatogenesis in the land snail Theba pisana (Sakr et al., 1992). In the hermaphroditic snail Helix pomatia, injection of testosterone significantly increased the number of secondary spermatocytes in the gonads (Casaba and Bierbauer, 1979). Testosterone administration to castrated hermaphroditic slugs Euhadra peliomphala led to the development of a head wart, an accessory male sex characteristic (Takeda, 1980). Thus, testosterone may serve as the ultimate sex determinant that is controlled by neuro-endocrine signals (e.g., APGWamide) similar to the hypothalamic/pituitary control of testosterone secretion in vertebrates (discussed in LeBlanc et al., 1999).
Exposure to tributyltin elevates testosterone levels in prosobranch snails (Spooner et al., 1991; Gooding et al., 2003). Increased testosterone levels also have been noted in imposex females sampled from a tin-contaminated location (Gooding et al., 2003). These observations coupled with the demonstration that direct administration of testosterone to prosobranch gastropods causes penis development suggests that imposex results from elevated testosterone levels caused by tributyltin exposure.
Two suppositions must be tested and accepted before this putative causative relationship among tributyltin, testosterone, and imposex can be accepted. First, the supposition must be accepted that testosterone is a male sex differentiating hormone in prosobranch snails. Second, the supposition must be accepted that tributyltin specifically targets some component of the testosterone regulatory machinery causing the aberrant accumulation of this hormone in the snails. In this overview we present recent data that support both suppositions.
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TESTOSTERONE AS A MASCULINIZING HORMONE IN SNAILS |
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Ample evidence exists to conclude that mollusks synthesize and maintain levels of circulating testosterone levels. Testosterone and/or components of its biosynthetic pathway have been measured in bivalve mollusks, opisthobranch gastropods, prosobranch gastropods, pulmonate gastropods, and cephalopods (summarized in Sternberg, 2001
). However, unlike vertebrate species that eliminate testosterone largely through its conversion to polar derivatives (Griffin and Wilson, 1981; Gunderson et al., 2002; Wilson et al., 1999), we have shown that the mud snail converts testosterone largely to apolar fatty acid conjugates that are retained by the organisms (Fig. 1). Fatty acid esterification serves to inactivate and store excess testosterone while maintaining free (unesterified) testosterone at physiologically-appropriate levels (Fig. 2). This pool of esterified testosterone also may serve as a reserve from which testosterone can be drawn in an effort to maintain free testosterone homeostasis (Fig. 3). These conclusions were drawn from experiments in which total testosterone levels in snails were either increased (Fig. 2) or decreased (Fig. 3) and effects on free testosterone and testosterone-fatty acid esters levels were determined.
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Testosterone-fatty acid esterification is catalyzed by a microsomal enzyme acyl coenzyme A (CoA):testosterone acyltransferase (ATAT) that utilizes fatty acid-coenzyme A as the fatty acid donor molecule. The enzyme readily uses palmitoyl CoA and oleoyl CoA as fatty acid donors (Gooding and LeBlanc, 2001
). ATAT is a high capacity (Vmax 
200 pmol/min)/low affinity (Km 6 µM) enzyme (Fig. 4) and its ability to increase conversion of testosterone to fatty acid esters with increasing total testosterone levels (Fig. 2) stems, at least in part, from its high capacity for catalysis. Microsomal ATAT activity also is induced in response to elevated testosterone levels (Fig. 5). This induction suggests that ATAT protein levels are increased in response to elevated testosterone, thought we cannot exclude the possibility that the increased activity is due to increased substrate availability in the form of microsomal membrane-associated testosterone. This novel mode of regulating testosterone levels (esterification/de-esterification) may provide a unique target by which tributyltin can alter the endocrine status of the snails.
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For testosterone to function as a masculinizing hormone in snails, a receptor protein must exist in these organisms that functions to relay the signal initiated by the androgen. We are not aware of definitive evidence for the expression of an androgen receptor in mollusks. D'Aniello et al. (1996)
reported the existence of a high affinity (Kd = 0.5 nM) testosterone binding protein in the octopus (Octopus vulgaris). These investigators also reported high affinity (Kd = 1.8 nM) estrogen binding activity in this species (D'Aniello et al., 1996). Since this report, a molluscan estrogen receptor has been identified and characterized (Thornton et al., 2003; Di Cosmo et al., 2002; Stefano et al., 2003). The identification of a molluscan estrogen receptor raises expectations that a receptor protein responsible for the high affinity binding of testosterone ultimately will be identified in these organisms.
Reports of coordinate regulation of testosterone levels with the reproductive cycle of mollusks also suggests a functional role for testosterone in these organisms. In a study of the Florida apple snail (Pomacea paludosa), Sternberg (2001) observed that testosterone levels increased in males commensurate with the onset of egg laying then progressively declined over the reproductive phase of the organisms. Testosterone levels remained low throughout the reproductive phase in females. Testosterone esters were not saponified prior to radioimmunoassay in these experiments, thus testosterone levels reported likely reflect free testosterone (i.e., testosterone:fatty acid esters were not likely detected by the radioimmunoassay). Similarly, we observed that free testosterone levels in male mud snails increased just prior to the onset of egg laying then remained low through the reproductive period only to increase again at the end of the reproductive phase (Fig. 6A). Testosterone levels were typically lower in females although increases also were discerned at the onset and end of egg laying. The seasonal elevations in free testosterone were accompanied by precipitous decreases in testosterone-fatty acid ester pools within the organisms (Fig. 6B). These observations suggest that the surges in free testosterone levels were due to either liberation of free testosterone that was stored as the fatty acid esters or due to the suppression of ATAT activity resulting in the reduced storage of testosterone as the ester.
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It should be noted that the increase in free testosterone levels at the onset of reproduction of the mud snail did not precede the development of male sex characteristics (Table 1). In fact, penis development was evident prior to the surge in free testosterone levels. Thus, the increase in testosterone levels apparently did not stimulate male sex organ development. Among females, the increase in free testosterone levels appeared to co-occur with the development of reproductive organs. Hence, in both sexes, the increase in free testosterone may have been stimulated by some product of the reproductive system. Oberdorster and McClellan-Green (2002)
proposed that steroidal androgens have a greater role in maintaining male accessory sex organs than in initiating their development. The present data are partially consistent with this proposal, although free testosterone levels were not elevated during reproduction when the penis was maintained. Rather than having a primary role in the initiation or maintenance of the penis, perhaps testosterone functions to stimulate rapid, full development of the copulatory organ.
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In the absence of a demonstrated signaling pathway that is stimulated by steroidal androgen, the postulate that testosterone is a functional hormone in some mollusks remains tentative. However, ample circumstantial evidence exists to warrant investigation aimed at identifying a molluscan androgen receptor and associated signaling pathway. Furthermore, the identification of a unique regulator of testosterone levels in the snail— ATAT—suggests a possible target at which tributyltin could specifically disrupt endocrine signaling in these organisms.
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PUTATIVE TARGETS OF TRIBUTYLTIN ACTION ON TESTOSTERONE LEVELS |
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Aromatase inhibition
Having observed that tributyltin elevated testosterone levels in the dogwhelk (Nucella lapillus), Spooner et al. (1991)
proposed that this compound inhibited aromatase (CYP19), the enzyme responsible for the aromatization of testosterone to estradiol. Bettin et al. (1996) provided supporting evidence for the inhibition of aromatase as being the mechanism by which tributyltin elevates testosterone levels. These investigators reported that two aromatase inhibitors, flavone and SH489, also caused imposex in the dogwhelk. The aromatase inhibition hypothesis should be viewed as tentative since estradiol levels were not commensurately decreased with the increase in testosterone observed in tributyltin-treated organisms (Spooner et al., 1991) and the aromatase inhibitors used by Bettin et al. (1996) likely interfered with steroid metabolic enzymes in addition to aromatase (Schrag and Wienkers, 2001; Vietri et al., 2002; Haraguchi et al., 2003).
Testosterone:estradiol ratios are typically in the order of 10:1 in mollusks (Morcillo and Porte, 1999; Bettin et al., 1996; Reis-Hendriques et al., 1990; Spooner et al., 1991; Schulte-Oehlmann et al., 1995). It seems unlikely that even the complete inhibition of estradiol synthesis by tributyltin would significantly impact testosterone levels. Measured aromatase activity in the snail Bolinus brandaris constituted less that 1.0% of the total Phase I metabolism of testosterone measured in another snail species Littorina littorea (Morcillo and Porte, 1999) indicating that even the total inhibition of aromatase would have a negligible effect on overall testosterone metabolism. Similarly, we have observed that the conversion of testosterone to estradiol is insignificant when compared to the amount of testosterone converted to fatty-acid esters in the mud snail (unpublished observations). Thus, while aromatase may be susceptible to inhibition by tributyltin in mollusks, it is unlikely that this inhibition is responsible for elevated testosterone levels.
Suppression of testosterone esterification
Upon establishing that testosterone is highly susceptible to fatty esterification by the action of the enzyme ATAT, we hypothesized that tributyltin elevates testosterone by inhibiting ATAT. Indeed, exposure of mud snails to tributyltin reduced testosterone-fatty acid ester levels while elevating free (unesterified) testosterone levels (Gooding et al., 2003). Field collected mud snails from a tributyltin-contaminated site also exhibited elevated free testosterone levels and reduced testosterone ester levels (Fig. 7).
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0.05) difference between sites. Data derived from Gooding et al. (2003)These data supported our hypothesis that tributyltin inhibited ATAT. However, microsomal ATAT activity was not significantly inhibited when incubated with tributyltin at levels comparable to those measured in imposex snails sampled from a tributyltin-contaminated location (Fig. 8). Furthermore, exposure of snails to tributyltin, under a regimen that elevated free testosterone levels, had no effect on ATAT activity associated with microsomes isolated from these organisms, indicating that tributyltin did not lower ATAT protein levels (Gooding et al., 2003
). While current data support the hypothesis that tributyltin elevates free testosterone levels by suppressing the retention of testosterone as fatty acid esters, the precise mechanism by which tributyltin elicits this effect remains an enigma. Experiments are currently underway to determine the mechanism by which free testosterone levels are elevated and testosterone-fatty acid ester pools are depleted seasonally in the mud snail (Fig. 6). These studies may reveal that tributyltin activates this mechanism resulting in aberrant testosterone levels.
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CONCLUSION |
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SYNOPSISINTRODUCTIONTESTOSTERONE AS A MASCULINIZING...PUTATIVE TARGETS OF TRIBUTYLTIN...CONCLUSIONReferences |
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Ample evidence exists to indicate that tributyltin elevates testosterone levels in imposex-susceptible prosobranch gastropods. However as stated initially, two suppositions must be tested and accepted before concluding that tributyltin causes imposex in gastropods by elevating testosterone levels. First, testosterone must be accepted as a male sex differentiating hormone in prosobranch snails. Sufficient corroborative evidence exists to suggest that testosterone is a masculinizing hormone in these organisms. However to date, an androgen signaling pathway in these organisms has not been identified. Second, the supposition must be accepted that tributyltin specifically targets some component of the testosterone regulatory machinery causing the aberrant accumulation of this hormone in the snails. A viable target for this action of tributyltin has been identified. Free testosterone levels are regulated by an esterification process whereby excess free testosterone is conjugated to fatty acids and stored within the tissues of the organisms. Presumably, though not yet demonstrated, free testosterone can be liberated from the ester pool through the action of an esterase when testosterone is required by the organism. Tributyltin disrupts this esterification/desterification process resulting in a reduction in the size of the testosterone-fatty acid pools and a commensurate increase in free testosterone levels. The mechanism through which tributyltin disrupts this balance between free and esterified testosterone has not yet been determined. In conclusion, abundant evidence exists that supports each postulate. However, more research is needed before the causal relationships among tributyltin exposure, testosterone levels, and imposex can be established.
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ACKNOWLEDGMENTS |
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The authors acknowledge the technical support provided by Dr. Harmit S. Ranhotra and Ms. Heather Hoy. This research was supported by grants from the US Environmental Protection Agency (STAR R826129, CT826512010) and the National Science Foundation (IBN 0234676).
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FOOTNOTES |
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1 From the Symposium EcoPhysiology and Conservation: The Contribution of Endocrinology and Immunology presented at the Annual Meeting of the Society for Integrative and Comparative Biology, 5–9 January 2004, at New Orleans, Louisiana.
2 E-mail: ga_leblanc{at}ncsu.edu
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