Into the world of steroids

Abstract

Evolution of steroids such as sex hormones and ecdysteroids occurred independently in the animal and plant kingdoms. Plants use phytoecdysteroids (PEs) to control defense interactions with some predators; furthermore, PEs can exert beneficial influence on many aspects of mammalian metabolism. Endocrine disrupting compounds such as the estrogen agonist bisphenol A (BPA) are widespread in the environment, posing a potential hormonal risk to animals and plants. Adverse BPA effects on reproductive development and function are coupled with other toxic effects. BPA bioremediation techniques could be developed by exploiting some tolerant plant species.

Introduction

Life is simultaneously extraordinarily homogeneous and diverse. Mammalian sex hormones have quite important and specific roles in animals, and ecdysteroids are essential insect hormones that control moulting and metamorphosis. Vascular plants also produce these steroidal compounds, that are far less prominent in plant physiology than brassinosteroids, a group of essential plant hormones. This mini-review will briefly survey the plant steroid groups shared with animals, reflecting on their convergent biochemical evolution that may be analogous to substantial rearrangements of the same music composition.

Phytoecdysteroids: Plants Produce Insect Hormones

A wide range of vascular plant species, if not all, produce phytoecdysteroids (PEs) as secondary metabolites. PEs are a family of over 300 plant steroids, analogous to the arthropod steroid hormones ecdysteroids, which have essential regulatory roles in all stages of insect development and reproduction, controlling many biochemical and physiological processes. Indeed, many ecdysteroids are present in both animals and plants. The most frequent PE is 20E, which is also the major inducer of moulting and metamorphosis in arthropods.^1,2

PE biosynthesis.

PEs are derived from phytosterols with a number of chemical modifications, including addition of multiple hydroxyl groups.² There is little information on the PE biosynthetic pathways in plants. Cytochrome P450s (CYPs) are a class of membrane-bound proteins localized in the mitochondria and endoplasmic reticulum. CYPs are key enzymes involved in hydroxylation/oxidation reaction in mammalian, insect and plant steroid biosynthesis.³ In several plant species, C20-hydroxylase is a characteristic microsomal CYP enzyme, especially in spinach. Also C2-hydroxylase of spinach is only microsomal. Overall, there are significant differences in plant and insect ecdysteroid biosynthesis based on enzyme sub-cellular localization, reaction sequences, and the various biosynthetic steps. These data support the hypothesis of independent pathway origins in the two kingdoms.³

A role (or two) for PEs in plants.

All known PEs have agonists activity in the insect ecdysteroid receptor complex.⁴ Feeding experiments demonstrated the endocrine-disrupting activity of dietary ecdysteroids on phytophagous insects and nematodes, whereby some insects avoided feeding on ecdysteroid-containing food.⁵ Due to the specific PE interference in the ecdysteroid signal transduction in insects, PEs appear to have a clear ecological role in plant-insect interactions in deterring invertebrate predators.² Hence, PEs are good candidates for the development of an environmentally safe approach to crop protection.⁵ The huge array of plant secondary compounds offers also offer insect steroid antagonists such as cucurbitacins and cucurbitanes.⁶ Brassinosteroids also can have weak ecdysteroid antagonistic biological activity on insects.⁵

Secondly, PEs may perform a direct physiological function in plants. For example, Arabidopsis thaliana seedlings and wheat coleoptiles showed growth and enzyme activity responses to exogenous ecdysterone (20E), possibly through interaction with receptors of 24-epibrassinolide and GA₃, which have similar effects on some growth and metabolic processes.⁷

PEs are not plant hormones.

Plants may accumulate high levels of PEs, even higher than insects, unlike the very low concentrations of plant hormones. Paradoxically, advances in insect endocrinology knowledge is due to the abundance of plant ecdysteroids.¹ However, most crop plant species are ecdysteroid-negative, except for spinach and other Chenopodiaceae such as beets and quinoa.² The biosynthetic pathways are thought to be downregulated in many species. Thus, elevation of PE levels could be achieved by altering regulation of existing genes without genetic engineering.⁴ Overall, the uneven distribution in vascular plants, the huge levels found in some species, and the chemical traits do not support a phytohormonal function of PEs.^1,5

Within the large genus of Silene, more than 100 of the 700 species produce PEs, and 26 species have a high PE content; this has no counterpart among plants. About 40 PEs were first isolated from various Silene species.⁸ PE fluctuations showed qualitative and quantitative changes during plant development, with higher levels located in aerial parts during periods of intensive growth.⁹ In both annual and perennial species of Silene, the highest content (20E, up to 34%) was associated with the reproductive portions, although they contribute a small percentage of the total body mass.⁹ This is in line with the “optimal δϵϕϵνσϵ” theory, where high PE levels are usually found in tissues of high value for plant fitness (reproductive tissues and apical tips in annuals; roots in perennials). PE biosynthesis and transport in developing spinach had a continuous redistribution within the growing plant between ‘source’ older leaves and young, developing ‘sinks’ leaves.⁵ Spinach PE synthesis showed direct negative feedback of the major PE, 20E.⁵

Biomedical applications of PEs.

PE accumulation from plant species have been used in folk medicine.¹⁰ Plant cell culture techniques to produce PEs have been developed, and PE-based preparations are available on the market.^8,11 PEs exert many beneficial effects on important mammalian physiological functions without known side-effects.^1,2,12 Pharmacological effects include enhancement of metabolism, tonic and anti-depressive as well as anti-diabetic properties, and use as anti-osteoporosis, hypocholesterolemic, hepato- and nephroprotective and anti-tumor agents.^2,13 However, the PE mode of action on mammal cells is quite different from that in lower animals since PEs do not interact with vertebrate nuclear steroid receptor systems. Most of the ecdysteroid action in insects is mediated via nuclear receptors and only some by membrane-bound receptors.²

Plants Produce Mammalian Sex Hormones

Steroidal sex hormones are reported in many plants. In particular, androsterone and progesterone occur in more than 80% of the plant species investigated, while testosterone and estrogens (estrone and 17β-estradiol) occur in 70 and 50%, respectively. Their occurrence in various vegetative and reproductive tissues and organs, changes during development and enzymes for biosynthesis and conversion have been described in many plants.¹⁴ There are also reports on the effects of exogenous steroid hormones on plant cell division, embryo growth, root and shoot growth and flower development.¹⁴ Their involvement in plant growth and development cannot be denied, although steroidal sex hormones, like PEs, are not considered as phytohormones.

Animal cells respond to steroids using nuclear receptor molecules. After binding of a specific ligand, the structural conformation is changed and the receptor is transferred into the nucleus, binding to corresponding DNA responsive elements and triggering gene expression.¹⁵ The presence of estrogen-binding proteins that share structural similarity to the well characterized mammalian estrogen receptor α-subtype has been reported in plants, namely in Solanum glaucophyllum and Lycopersicon esculentum.¹⁶ Using western blot analysis with highly specific monoclonal antibodies against different domains of the α-receptor, various organs of the two species were shown to contain a protein of about 67 kDa, which coincides with the molecular weight of the mammalian receptor. The approximately 67 kDa protein was most concentrated in the nuclear fraction, while two additional lower weight reactive bands were localized in the cytosol and microsomal fractions. All these bands were able to bind 17β-estradiol in ligand blot assays.¹⁶

Environmental Pollution can Destroy the Steroid Hormonal Balance in Animals and Plants: A Domino Effect

A large number of environmental pollutants can interfere with signalling pathways of nuclear receptors, including those for steroid hormones.¹⁵ The best known are the environmental estrogens, which mimic estradiol and bind to estrogen receptors. These agonistic molecules belong to the large group of ‘endocrine disrupting compounds’ (EDCs). Bisphenol A (BPA) is a well-known estrogen-like EDC used as a monomer in the production of polycarbonate and epoxy resin compounds found in several consumer products. There are multiple sources, even common objects, releasing BPA into the environment, including foodstuff packed in plastics.¹⁷ BPA contamination is spread everywhere (air, soil and water systems), routinely exposing living beings to biologically active levels of the compound. Indeed, BPA experienced quite a lot of media coverage in recent years for its significant impact on wildlife. For instance, environmentally relevant BPA doses can induce sex reversion in crocodiles.¹⁸

As a result of BPA exposure, general hormone imbalances have been well documented in animal systems, including humans, with alterations in levels of all sex hormones and also expression of estrogen receptors. In addition to disorders in reproductive function and sex-related behavior, adverse effects on the brain were also shown, suggesting that BPA can contribute in developing psychological dependence on drugs.¹⁹ Furthermore, disruption of the endocrine environment can alter metabolism, and susceptibility to cardiovascular diseases may increase in humans.²⁰ While estrogens are almost exclusively produced in females, estrogen receptors, however, are located in numerous tissues of both sexes. Therefore, the abnormal presence of estrogen-like molecules in the environment can also cause a huge array of negative effects in males.¹⁵ In animals, BPA responses independent of estrogen receptors include modulation of enzyme levels or activity involved in estradiol biosynthesis or catabolism.¹⁵

In invertebrates, BPA-induced endocrine disruption was described in the soil organism, Porcellio scaber.²¹ Ecdysteroid levels were altered, whereby 20E increased to 170% of controls in the lowest concentration tested (10 mg BPA kg⁻¹ soil). The ecdysteroid upregulation, in turn, was associated with reproductive toxic effects such as reduction of time taken to first moult, alteration of the sex-ratio in favor of females, and increased abortions. Consequently, compounds like BPA might have serious impact on isopod population dynamics, which may eventually lead to population declines.²¹ Interestingly, in Drosophila melanogaster, BPA is able to compete with ecdysteroids for the ligand binding site on the receptor complex.²² In the freshwater midge, Chironomus riparius, BPA was shown to upregulate levels of ecdysone receptor.²³

In higher plants, available information mainly concern species able to absorb, bioaccumulate and metabolize BPA in cell cultures, whereas little attention was given to the whole plant system, except for description of BPA toxicity and clastogenic effects on seedlings of a number of crop species.²⁴ There are no studies examining BPA effect on the hormonal milieu of the vegetative plant body. In contrast, BPA impact on the abundance of steroid hormones was quantified for the first time in a plant system using the male gametophyte of kiwifruit.²⁵ Dramatic imbalances of both androgen and estrogen levels occurred during germination in the presence of BPA, accompanied by severe impairment of tube emergence and elongation and increased expression of stress-related proteins. The hypothesis of an association between hormone imbalance and pollen tube growth inhibition is currently largely speculative, but is supported by the experimental finding that exogenous hormones can seriously alter in vitro pollen performance.²⁵ On the other hand, hormone levels are surely important for normal development of plant male reproductive structures. In vitro maturation of the so called ‘minimal pollen’ of tobacco presumably lacked essential components normally provided by the mother plant. In such poorly germinating pollen, exogenously supplied steroid hormones recovered good tube emergence and growth.²⁶ This further supports an active, though undefined, involvement of steroidal sex hormones in plant development and growth processes.

Plants Can be Used for BPA Remediation

Biological techniques to remediate BPA contamination could rely on the ability of some plant species to tolerate, accumulate or metabolize BPA. For instance, several Salvia cultivars grown in hydroculture showed a high BPA-eliminating ability, and 74 to 100% of BPA (50 µM) was eliminated after 3 days.²⁷ Dracoena sanderiana tolerates BPA levels up to 80 µM. Plants cultivated in hydroponic systems with an initial BPA concentration of 20 µM had approximately 50% BPA uptake, which was accumulated in the roots and stems. Thus, Dracoena sanderiana is a potential candidate for the phytoremediation of BPA contaminated wastewater or industrial leachate.²⁸ Moreover, photodegradation is another way to destroy BPA in aqueous solutions using the green alga Chlorella vulgaris.²⁹ BPA biotransformation in cultured Eucalyptus perriniana plant cells accumulated the compound as glycosides devoid of estrogenic activity. This procedure using enzymatic reactions could be at the basis of phytoremediation of diphenyl EDC compounds.³⁰

Conclusion and Perspectives

Steroids such as sex hormones and ecdysteroids occur in plants and animals. Therefore, both plants and animals are at risk of endocrine disruption and various metabolic disorders due to widespread environmental pollutants such as BPA that are able to interfere agonistically with hormone receptors. Biotechniques for amending BPA contamination appear to be feasible using some plant species capable of BPA accumulation and transformation.