Biochemical and Biological Effects of Organotins

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The legislation on organotin-based antifouling paints, including the International Convention on the Control of Harmful Anti-fouling Systems on Ships (AFS Convention) is here summarized. Concentrations of TBT and TPhT in the marine environment and toxicities of TBT and TPhT to mainly marine organisms are also overviewed, as is the relevant legislation. As one of the most typical toxicological effects of TBT and TPhT, imposex in gastropod mollusks is reviewed. Imposex is a superimposition of male-type genitalia (penis and vas deferens) in females and is considered an irreversible pseudohermaphroditic condition. It is typically induced by very low concentrations (~1 ng/L) of TBT, TPhT, or both. Reproductive failure occurs in the severe stages of imposex, either because of oviduct blockage by the formation of vasa deferentia or because of ovarian disorder (spermatogenesis as well as suppressed production of matured oocytes), and eventually results in population decline or mass extinction. Globally, approximately 200 species of mesogastropods and neogastropods are affected by imposex. Imposex among gastropods has been recognized as a clear manifestation of endocrine disruption. Five main hypotheses of the mechanisms by which organotins induce imposex in gastropods have been proposed: (1) an increase in androgen (e.g., testosterone) levels due to TBT-mediated inhibition of aromatase; (2) TBT-mediated inhibition of the excretion of androgen sulfate conjugates; (3) TBT interference in the release of penis morphogenetic/retrogressive factor from the pedal/cerebropleural ganglia; (4) an increase in the level of alanine-proline-glycine-tryptophan (APGW)amide neuropeptide in response to TBT; and (5) involvement of the retinoid X receptor (RXR), a nuclear receptor. Each hypothesis is critically reviewed.

OTC are characterized by the presence of at least one covalent C–Sn bond. The compounds contain tetravalent {Sn} centre and are classified as mono-, di-, tri- and tetraorganotins, depending on the number of alkyl (R) or aryl (Ar) moieties. The counter anions are usually Cl–, F–, O2–, OH–, –COO– or –S2–. The nature of the anionic group has only secondary importance. The organotin(IV) compounds display biological activity in aquatic environments. Most of the compounds are very toxic, even at low concentration. The biological activity is essentially determined by the number and nature of the organic groups bound to the central {Sn} atom, and by the structure of compounds. The [R3Sn(IV)]+ and [Ar3Sn(IV)]+ derivatives exert powerful toxic action on the central nervous system. Within the series of [R3Sn(IV)]+ compounds, the lower homologues (Me, Et) are the most toxic and the toxicity decreases progressively from propyl (Pr) to octyl (Oc), the latter not being toxic at all. The structure is varied between regular or distorted tetrahedral Th, trigonal-bipyramidal Tbp and octahedral Oh. Some of the complexes display antitumor activity. The mechanism of biological (including antitumor) action is still unclear.

Organotins are widely distributed environmental contaminants with potent toxicity phenotypes and established endocrine disrupting properties in diverse species and ecosystems. Of particular interest has been the recent discovery of direct high affinity interactions between trialkyltins, such as tributyltin chloride (TBT), and the family of nuclear hormone receptors (NRs) which function as ligand dependent transcription factors for a diverse set of lipophilic endocrine hormones in vertebrates and invertebrates. Perturbed NR activity can provide a direct molecular link between environmental organotin exposure and their endocrine disrupting effects. The ability of organotins to activate NRs is surprising since they share neither structural characteristics with the endogenous steroidal or lipophilic hormone ligands nor classical modes of binding. Instead, crystallographic and mass spectroscopic data for the retinoid X receptor alpha (RXRα) shows a covalent interaction between TBT or TPhT and a critical receptor cysteine residue (C432) at the entrance to the ligand binding pocket. Therefore, covalent interactions between organotins and reactive cysteines residues, as also observed with stannin and predicted for aromatase, appears to be an emerging theme for modifying protein function in eliciting organotin toxicity. The stoichiometric modification of nuclear receptor activity, especially of RXRs that heterodimerize with a broad subset of NRs, has profound implications for assessment of organotin toxicity in organismal physiology particularly with respect to invertebrate imposex phenotypes and vertebrate metabolic integration of energy homeostasis. It is likely that additional nuclear receptors and proteins will be identified that are sensitive to organotin covalent modification.

Because of their lipophilicity, organotins may become embedded at different levels in lipid bilayer. The nature of the interaction between organotin compounds and membranes has been investigated by different authors using lipid vesicles formed by phosphatidylcholines (the major component of eukaryotic membranes). It has been reported that tributyltin chloride (TBTC) and tributyltin acetate (TBTA) localize differently within the hydrophobic region of dipalmitoil phosphatidylcholine perturbing its thermotropic structural properties. Organotin compounds do not affect the macroscopic bilayer organization of phospholipids which remain lamellar but do affect the grade of hydration of its carbonyl moiety. The effect of trisubstituted organotins (TBT and triphenyltin) on membrane permeability using phospholipid model membranes has been also reported. The results indicate that triorganotin compounds are able to transport organic anions across phospholipid bilayers by exchange diffusion with chloride. The effects of cholesterol on reducing membrane permeability were also well established. The interaction of organotin compounds with biological (real) membranes has been mainly investigated using erythrocytes as membrane target. The ability of organotin compounds to cause hemolysis of human and other erythrocyte species has been described to be very different depending on species and cell aging.

Evidence for the ability of organotins to act as endocrine-disrupting chemicals (EDCs) has been accumulating over the past couple of decades. One of the most consistently cited examples of environmental endocrine disruption in wildlife is the occurrence of male sex characteristics on female neogastropods, termed imposex, as a result of exposure to tributyltin (TBT). Extensive efforts have been made to elucidate the mechanism by which TBT induces imposex. A hypothesis regarding the elevation of testosterone by TBT has been widely touted as a result of the association between exposure to TBT and increased free testosterone titers in imposexed females. The hypothesis specifically states that TBT elevates testosterone which then initiates some unknown biochemical signaling pathway with the ultimate outcome being the development of imposex. Recently, some organotins, including TBT, were shown to be high-affinity ligands of vertebrate retinoid X-receptors (RXRs). This finding has resulted in the development and testing of the latest hypothesis for TBT-induced imposex: TBT causes imposex by disrupting retinoid signaling, i.e., retinoic acid acting via the RXR. Studies suggesting that RXR signaling may be important in the development of the reproductive tract in neogastropods provide additional support for the involvement of retinoid signaling in the development of imposex. Future research regarding the mechanism of TBT-induced imposex should focus on identifying pathways downstream of RXR binding that are involved in reproductive tract development in neogastropods.

Lipid homeostasis is essential for the maintenance of the organism energy balance while enabling the cells to perform vital functions. In mammals, improper control of these metabolic pathways can result in serious health problems, such as obesity, increased risk of coronary artery diseases, diabetes and related problems, such as hypertension and lipidemia. The nuclear receptor peroxisome proliferator-activated receptorγ (PPARγ), together with its heterodimeric partner retinoid X receptor (RXR), is a master regulator of adipocyte differentiation, being involved in the regulation of food intake, metabolic efficiency and energy storage. Triorganotins such as tributyltin (TBT) and triphenyltin (TPhT) are high affinity ligands of both RXRs and PPARγ. In line with these findings, the use of the 3T3-L1 cell system, a well characterized model for adipogenesis, demonstrated that organotins stimulate 3T3-L1 cells differentiation and the expression of adipocyte marker genes. In vivo exposure of neonate mice to TBT significantly elevates lipid accumulation in several tissues, thus supporting the hypothesis that chronic lifetime human exposure to organotins may increase the risk of obesity. While PPAR has not been described outside deuterostomes, RXR is ubiquitous within metazoans. Thus, the taxonomic scope for organotin-mediated lipid homeostasis disruption may be wider than initially anticipated. This is further supported by the observation of increased lipid accumulation in amphibians and teleost fish and changes to the lipid profile in molluscs after organotin exposure. Therefore, we review here the current evidence on the molecular and biochemical mechanisms for organotin-mediated lipid homeostasis disruption within several metazoan phyla.

Leached from various sources, all the organotin compounds have an impact on natural aquatic environments. In both freshwater and seawater ecosystems, they are dangerous in that they can have deleterious effects on biocenoses already at low concentrations. All these compounds are known to be toxic at relatively low levels, not only for aquatic invertebrates, but also for fish and laboratory mammals. Moving easily along the trophic chains, organotins are also rapidly bioaccumulated in the tissues of non-target organisms living in the water-sediment interface, causing severe, long-term toxic effects on local epifauna, with repercussions on biodiversity and human health. Among toxic effects, genotoxicity and immunotoxicity are the most important affecting the capacity for survival of animals. Genotoxicity appearing in the form of chromosomal aberrations, increasing in frequency of micronuclei and induction of cytogenetic damage has recently been reported in mammals, fish and aquatic invertebrates. Organotins interfere selectively with the immune system of vertebrates, causing atrophy of the thymic cortex and lymphoid tissues with a consequent leucopoenia. Short-term in vitro exposures of haemocytes of various vertebrate and invertebrate organisms reveal inhibition of phagocytosis, cytolysis and/or apoptosis of leucocytes after inhibition of chemotaxis and respiratory burst, with resulting depression of cell-mediated immune responses. These immunosuppressive effects are dose- and time-dependent, and vary according to the number and type of organic moiety present. Both Ca2+-dependent and Ca2+-independent mechanisms of action have been proposed. They are linked and synergistic in triggering the cascade of secondary events that lead to toxic action.

In vitro toxicity, molecular study of the toxicity mechanisms, which should account for the observed in vivo effects of toxic compounds, is a complicate issue. The study of organotin compounds is particularly complicate, since organotins, being chemically versatile, are able to bind covalently and non-covalently to many biomolecules; also organotins are soluble in lipophilic environments and can affect a wide variety of biological functions. Therefore, it is difficult to identify the biological mechanism and the molecular target inhibited by the lowest effective dose. In the interactions of organotin compounds with mitochondria, the problem is “simplified” since many organotins cause in vivo acute toxicity. Being mitochondria the energy source of the cell, mitochondrial impairment induces cell damage; therefore, in many cases, mitochondria are the molecular target responsible for the in vivo acute toxicity. On these bases, ATP synthesis mechanism in mitochondria should be carefully analysed, in order to individuate the step(s) inhibited by the toxic compounds. Many organotin effects on the mitochondrial functions are correlated with the ATP synthesis inhibition. In isolated mitochondria, the (alkyl)3-Sn- compounds inhibit all the steps involved in the ATP synthesis mechanism, but experiments performed in isolated cells suggest that the mitochondrial ATP synthesis inhibition is probably related to the opening of the membrane permeability transition pore. In addition, also triphenyltin opens the permeability transition pore and triggers apoptosis. Other putative and proven mechanisms of organotin action in mitochondria are considered in detail.

Enzymes, non-enzymatic proteins and other organic molecules are vital components in living cells. Their respective function depends on specific spatial configurations which are linked to intracellular conditions. Any fluctuation of these conditions, beyond certain threshold values, such as a disruption of ionic regulatory mechanisms, can lead to the destabilisation of a finely balanced intracellular dynamic physiological equilibrium or homeostasis. Hydromineral homeostasis in aquatic organisms is maintained by a complex endocrine controlled array of specialised cross-membrane ion transport systems and the regulation of membrane water permeability. Depending on how aquatic organisms maintain hydromineral homeostasis, they can be roughly divided into two groups: osmoconformers and osmoregulators; the former are mostly invertebrates with high water permeability, the latter include some invertebrates and most fish species, whose permeable external epithelia are usually restricted to the gills. Other important organs involved in hydromineral regulation include the intestine and the various phyla-specific organisational types of renal systems. Environmental concentrations of organotin compounds, such as tributyltin and triphenyltin, have been shown to interfere with the maintenance of hydromineral homeostasis by inhibiting ATPases and affecting membrane permeability for water. The present chapter reviews the impact of organotin exposure on fresh- and seawater organisms of various phyla by examining the histophathological, physiological and molecular interactions of organotin compounds with relevant enzymes, membranes, the endocrine system, and the consequential ramifications for individuals, populations and community structure in aquatic ecosystems.

Apoptosis, a specific type of programmed cell death, is characterized by cell shrinkage, nucleus condensation and fragmentation, plasma membrane blebbing and final engulfment by neighboring cells or professional phagocytes. The molecular mechanisms of organotin-induced apoptosis, which involve a series of biochemical regulators and molecular interactions, have been extensively studied in different cell types but the apoptotic pathway mechanisms and signaling still remain unexplained in some detail. Apoptosis may be triggered and modulated by caspase-independent, and more frequently by caspase-dependent pathways. Pro-caspase activation is driven by death receptors, and/or by a mitochondrion-mediated mechanism. Although both pathways were described, the mitochondrial mechanism seems to be the most important one in organotin-induced apoptosis. Organotin compounds trigger cytoskeletal modifications and disruption of mitochondrial functions. Generally, the apoptotic pathway induced by organotins starts with their interactions with cellular components leading to perturbation of intracellular Ca2+ homeostasis, the latter especially triggered by endoplasmic reticulum stress, and intracellular Ca2+ concentration increase, cessation of ATP and reactive oxygen species production and loss of mitochondrial membrane potential. These events are followed by cytochrome c release from mitochondria to cytosol, apoptosome formation and final executioner caspase activation. The increase in intracellular Ca2+ level and the consequent mitochondrial cytochrome c release play critical steps in organotin-induced apoptosis. The process not only depends on cell type and sensitivity but also on organotin chemical characteristics and insult intensity. New and promising research on mechanisms of organotin-induced apoptosis is focused on the characterization of organotin interactions with apoptosis-related proteins and regulation of gene expression.

Organotins have been used for a variety of purposes since their discovery and their initial use in the 1920s. However, organotins are described as toxic pollutants introduced into the environment and impacts of organotins on several organisms have been largely documented. Although studies of impacts of organotins on human health are minimal, concerns have been raised regards the entry of organotins in human food chain and its consequences. Therefore, this chapter presents a review of impacts of organotins on humans. The chapter presents major applications of organotins, their entry in the food chain and human exposure via food and other sources and finally effects of organotins on humans.

Most of the biological effects displayed by organotin contaminants, among which trisubsituted species are especially toxic, have increasingly been found to exhibit astonishing analogies in different taxa. While similarities can be perceived from prokaryotes to mammals, different modes and extent of biochemical and biological effects were described in different cells, tissues and species. A broad susceptibility range to organotins emerges from literature. Aquatic biota are mainly affected by organotins as environmental water and sediments act as storage site. Endocrine and lipid homeostasis perturbations span from Mollusks, where first gender changes (imposex) referable to environmental organotin contamination was pointed out, to Mammals, where organotins play the role of environmental obesogens. Organotin immunotoxicity, elicited in various invertebrate Phyla, also affects humans. Inhibition of key membrane-bound enzyme complexes such as Na,K-ATPase and FOF1 complexes, thus affecting hydromineral balance, energy production and related effects, are known to occur in a wide variety of organisms. Mitochondria and all membrane functions apparently represent a preferred target of these lipophilic toxicants. Highly conserved action mechanisms could be involved in the observed effects: organotin binding to nuclear receptors, membrane components and intracellular proteins as well as DNA damage may represent widely shared action modes of these compounds. On the other hand the different response and even the refractoriness to these toxicants shown at different biological levels may mirror biochemical and physiological selectivity of signalling pathways, biomembranes and intracellular protein components.

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