Tuesday, 1 May 2018

Achatina Snail: An invasive species in Hong Kong 2. My fruitless hunt for steroid hormones

In my last post, I mentioned that the accidentally introduced giant snail, Achatina, was very common in the gardens of the University of Hong Kong. The mid-1960s were the heyday of comparative endocrinology. Having established the presence of the same or closely-related steroid hormones from the adrenal glands and reproductive organs in all the vertebrates, the question was whether these steroids were present and working as hormones in the invertebrates. With such a plentiful supply of an invertebrate, in this case a mollusc, outside the lab door, I had the animals.

A well-established method to see which steroids vertebrate endocrine glands synthesise was to incubate the tissue with a radioactively-labelled compound in the early part of the biosynthetic pathway and see what was produced. I used the paper chromatography separation systems made famous by the late Ian Elcock Bush (1928-1986) in his book, The Chromatography of Steroids, published in 1961. We had a good set up for descending paper chromatography in Hong Kong and although newer thin-layer techniques were also in use in the lab, the Bush systems still had a lot to offer, especially for a quick look-see. The chromatography tanks were in a small back room of the old medical school building and Hong Kong being Hong Kong there was no need to heat the tanks. The smell of warm solvents greeted you at the door. It was a glue-sniffer’s paradise if an out and out fire hazard. The dangers inherent of inhaling industrial quantities of benzene fumes were also in the future.

Shandon Panglas chromatography tanks
are now museum pieces or, like this one,
for sale on eBay. This one lacks the trough
at the top for the mobile phase.
To cut a long story short, I could find no known adrenal steroids in the incubation fluids from several possible organs of the snail. Although we were not that interested in reproductive steroids I had run known a number of oestrogens and androgens standards as well. Again, nothing radioactive from the incubates ran in the chromatography systems with them. There were however, very small quantities of a few compounds that just might have been steroids of some sort. However, they were beyond my capacity to determine what they were. Indeed there seemed to be no chemical techniques available at the time that would have enabled me to do so.

The pilot trial on the snail of what after all was a cast-in-the-dark fishing expedition was the end of the line for my only foray into invertebrate life. Other, more productive lines beckoned.

I have though always wondered what those radioactive spots on a paper chromatogram were. Had I thrown the baby out with the bath water? Yesterday, though, I was gratified and breathed a 52-years later sigh of relief when I read of a similar early study on a different mollusc. Alexander Scott wrote in his first review of 2013 (see below):

In some experiments within this study, the gonads and hepatopancreas of this animal [Atlantic Sea Scallop, Placopecten magellanicus] converted up to 70% of added radiolabeled Pregnenolone and 17-hydroxyprogesterone into several metabolites. However, David Idler, who was probably the greatest steroid chemist of his time…was unable to identify a single one of these (apart from a trace of androstenedione)!

Those people working on fish endocrinology, incidentally, were scared of what David Richard Idler (1923-1996), then of the Fisheries Research Board of Canada, would have to say about their work. He was certain to spot any flaws in claims of chemical identification of steroids. He blew a fair number of claims—and their authors—out of the water.

Some time after my quick look-see I saw that several papers had been published suggesting that sex steroids but not had been found in molluscs and that there was some evidence for some possible involvement in hormonal signalling. However, I had seen nothing to indicate a major rĂ´le for steroid hormones. In order to become less ignorant on this matter I discovered two reviews by Alexander Scott which gathered all the information of steroids in molluscs and subjected the evidence to a pretty devastating analysis.

Historically, there was a problem with proving that a substance found in, or synthesised by, an organism was a particular steroid. Chemical techniques were simply not then available. There was considerable dissent on what was acceptable evidence of chemical identity. Those with a background in steroid chemistry, like David Idler, had the whip hand. Some would write a paper saying that they had identified the compound produced as hormone x because they had run it in two chromatographic systems along with authentic x; others insisted on using more systems, sometimes changing chemically the structure of the putative and known steroid. For studies on steroid synthesis from radioactive precursors, there was a gold standard: recrystallisation of the separated radiolabelled product with added pure compound to constant specific radioactivity but a lot of early work stopped short of this final step and even with its use there were traps for the unwary.

Looking back at the literature on vertebrate steroid hormones an awful lot of poor work was published. However, because evidence of identity was difficult to get, there was also bad luck. I remember being told of one case where radiolabelled product and pure sample ran together in 10 chromatographic systems; in the 11th they separated showing they were not the same.

Alexander Scott, a noted fish endocrinologist, in his two reviews has looked at all the evidence of the presence, formation and possible functions of steroids in molluscs. It is an important topic because of the environmental effects of anthropogenic discharge of hormonal steroids, their metabolites and their chemical analogues. This is how he introduced his first review (references deleted):

The question as to whether vertebrate steroids act as hormones in mollusks is a very important one. Many compounds that behave like vertebrate estrogens are present in the environment (so-called ‘endocrine disrupters’), and if they act in the same way in mollusks as they do in vertebrates then there is genuine cause for concern. The first reports of the existence of vertebrate-type steroids in mollusks appeared in the 1950s. Since then, all but a handful of the 200+ scientific papers and reviews that have been published in this area have a positive message (i.e. they conclude either that mollusks contain vertebrate steroids, are able to biosynthesize them de novo, appear to contain steroid receptor-like binding activity or respond in one way or another when exposed to vertebrate steroids). The sheer ‘weight of evidence’ would seem, on the surface, to make it an ‘open and shut’ case that vertebrate steroids are an important component of molluscan endocrinology. However, if one looks beyond the headline claims (i.e. essentially what is written in the titles and abstracts of many of the papers), a different story emerges – one in which most, if not all, of the positive evidence can be seen to be rather weak (in that the data are open to alternative interpretations). This review paper deals with the strength of the evidence for the presence of steroids in mollusks, for their biosynthesis and for the presence of steroid receptors. Another review paper deals with the strength of evidence for the biological actions of vertebrate steroids on mollusks.

Indeed, in Scott’s detailed analysis the evidence is weak. Steroids found by molluscs may have been taken up and sequestered from the environment, for example. Studies of the sort I did in Hong Kong in 1965-66 if apparently positive can have alternative explanations.

Scott’s second review was concerned with the effects vertebrate sex steroids on molluscs. This is what he concluded:

In assessing the evidence as to whether vertebrate sex steroids (e.g. testosterone, estradiol, progesterone) have hormonal actions in mollusks, ca. 85% of research papers report at least one biological effect; and 18 out of 21 review papers (published between 1970 and 2012) express a positive view. However, just under half of the research studies can be rejected on the grounds that they did not actually test steroids, but compounds or mixtures that were only presumed to behave as steroids (or modulators of steroids) on the basis of their effects in vertebrates (e.g. Bisphenol-A, nonylphenol and sewage treatment effluents). Of the remaining 55 papers, some can be criticized for having no statistical analysis; some for using only a single dose of steroid; others for having irregular dose–response curves; 40 out of the 55 for not replicating the treatments; and 50 out of 55 for having no within-study repetition. Furthermore, most studies had very low effect sizes in comparison to fish-based bioassays for steroids (i.e. they had a very weak ‘signal-to- noise’ ratio). When these facts are combined with the fact that none of the studies were conducted with rigorous randomization or ‘blinding’ procedures (implying the possibility of ‘operator bias’) one must conclude that there is no indisputable bioassay evidence that vertebrate sex steroids have endocrinological or reproductive roles in mollusks. The only observation that has been independently validated is the ability of estradiol to trigger rapid (1–5 min) lysosomal membrane breakdown in hemocytes of Mytilus spp. This is a typical ‘inflammatory’ response, however, and is not proof that estradiol is a hormone – especially when taken in conjunction with the evidence (discussed in a previous review) that mollusks have neither the enzymes necessary to synthesize vertebrate steroids nor nuclear receptors with which to respond to them.

As he points out, there are still a few cases where mollusc tissues appear to have converted known vertebrate hormones into others. Such work needs to be followed up to see if the entire pathway is present or if, fortuitously, an enzyme system in molluscs is doing the job of a specific enzyme in vertebrates.

What is really lacking in all the work is the integrated experimental approach in a single species. The work I first became of that seemed to implicate steroids in the reproductive physiology of a mollusc was that by Herbert Gottfrield and Ralph Dorfman in the Giant Land Slug, Ariolimax californicus and published in a series of papers in the late 1960s. They seemed to have established a steroid-synthesising pathway similar to that in vertebrates that led to the production of androgens by the ovotestis. The work also involved experimental manipulation of possible signalling pathways. Unfortunately, this work does not appear to have been followed up but there appears, prima facie, to be a good experimental animal and an interesting and possibly important phenomenon to investigate.

Herbert Gottfried and Ralph Dorfman (1911-1985) were both then at the Syntex research centre in Palo Alto, California and later at Applied Steroid Laboratories also in Palo Alto. Gottfried had been at Sheffield when I was a student but I do not recall meeting him. He had also been in Hong Kong in 1965 before we arrived introducing the use of gas-liquid chromatography for separating steroids. Ralph Dorfman was famous for his work on steroid hormones—he was elected to the National Academy of Sciences in 1978—including the development of the first oral contraceptives. He was President of Syntex Research. I spoke to him several times in January 1967 at the 3rd Asia and Oceania Congress of Endocrinology in Manila (the first scientific meeting of any sort I had been to) and he invited the Brits and Hongkongers there for drinks in his hotel suite. The bottle of Dimple Haig (named for the shape of the bottle and more popular in the USA and the far east than in Britain) I remember particularly well.

So while it is possible to agree with Scott on the inadequacy of a great deal of past work on steroids in molluscs, there is still the odd twinkle of light to suggest that the role of what might in the circumstances be termed vertebrate-type sex steroids in molluscan physiology can be entirely discounted. Another case of more work—avoiding the many pitfalls highlighted by Scott—being needed.

Scott AP. 2012. Do mollusks use vertebrate sex steroids as reproductive hormones? Part I: Critical appraisal of the evidence for the presence, biosynthesis and uptake of steroids. Steroids 77, 1450-1468.

Scott AP. 2013. Do mollusks use vertebrate sex steroids as reproductive hormones? II. Critical review of the evidence that steroids have biological effects. Steroids 78, 268-281.