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Interviews with two foremost researchers into neurotoxicity who hold opposing views

Dr. Ricaurte believes that MDMA is highly neurotoxic which may result in long term problems for ecstasy users, while Dr. O'Callaghan believes it is not, even when taken in large amounts. Both researchers are established and respected in their fields. Dr. Ricaurte has carried out a series of studies at one of the world's top medical research centres, Johns Hopkins; while Dr. O'Callaghan is senior researcher for the US Environmental Protection Agency. Both interviews were conducted face-to-face in October 1995. References are included in Ecstasy Reconsidered.

Interview with George Ricaurte

on his research study using humans into ecstasy neurotoxicity in which I was a volunteer

What is the specific aim of this study?

The issue as to whether or not MDMA damages serotonin neurons in the human brain is still unresolved. There is some preliminary indication that the serotonin neurotoxicity that was produced in animals may also occur in humans, but we still don't know that for sure. The purpose of the ongoing study is two fold. Firstly, can we obtain further indications as to whether MDMA's serotonin neurotoxic potential generalises to humans. To do that we are going to look not only as CSF and 5HIA as markers of neurotoxicity, but we are also looking at a different neuroendocrine measures of serotonin function in living humans. That's the MCCPT test. Secondly, we are asking the simple question: "If there is any suggestion of serotonin toxicity in humans, is it associated with any functional change?", so along those lines we look for changes in pain perception or sensitivity, changes in sleep, the cognitive functions that are evaluated by the computerised test batteries, and the various behavioural spheres that are assessed with a series of questionnaires. Those are the major things within the human study. Apart from that, we are also trying to look for evidence of possible toxicity in humans with a ligand that selectively labels a serotonin neuron in the human brain.

So its like an extension of the squirrel monkey comparison looking for functional damage?

That's exactly right. I still strongly believe that in humans we are forced to use indirect measures of serotonin brain functions. What we need to do is to gauge serotonin function in a number of different ways, and hopefully collect data that is consistent and points to the same conclusion. If it doesn't, that's obviously important and raises questions about the validity of the methods we are using.

How was damage seen in squirrel monkeys?

We look for specific markers for serotonin neurons, and simply determine their presence or absence. We do this long after the drug has been discontinued and we inferred that if all the markers for neurons are missing for a long time after we discontinued the drug, then it seams reasonable to conclude that the drug has produced a loss of the axons that pertain to the serotonin nerve cell.

Can you not look for the axons visually, or are they too small?

We can look directly at the brain tissue, postmortem, in animals. I think one of the things to do in the future is to look at the brain tissue of people who have died, from whatever cause, who have been MDMA users. That's how we have come to learn about Parkinson's disease. There is no reason why the same strategy should not be used with MDMA users.

What do you believe is the reason that you did not come across functional effects of MDMA in your previous study?

We actually did find some functional differences. As a group, the individuals that had prior exposure to MDMA scored lower on hostility and impulsively. In the sleep studies, the MDMA group had less stage 2 time. We didn't note any negative functional consequences. Its a very difficult thing to tap into functional difference. That is a very imperfect science. One of the promising things is that by carefully examining such a group of subjects we may get some direct insight into the functional role. As long as we don't have a clear idea of what it is that serotonin does in the human brain, it is difficult to set up a testing strategy.

Is the damage direct, like that of a bullet, or is it more of a distortion?

I think the latter. Typically the neurotoxic effects of MDMA are extraordinarily selective. That's why MDMA is such a useful tool in research. You can see how, for experimental purposes, MDMA could be such a useful tool. Because damage is generally restricted to just the axon and axon terminals, there is a potential for regrowth. In monkeys, regrowth occurs, but the regrowth is abnormal, i.e. different to that observed in the control. One of the reasons why regrowth can take place is that the MDMA lesion, in contrast to a knife wound, doesn't form a scar, leaving potential to regrow. In monkeys this leads to a rewiring of the serotonin system, and at this point in time we have no idea what the rewiring does to the function of the animal. Those are things that have yet to be addressed.

A commonly expressed concern about MDMA use is that damage may not show up now in tests, but may do so in the future. Can you see any reason why that may be the case?

There are theoretical reasons, at least three that I can think of. If one uses the dopamine system as an example, we know that if you deplete something over 90% of an individual's dopamine, that individual becomes Parkinsonian. We also know that, unless you deplete dopamine by at least 90% behaviour looks entirely normal. We also know from the dopamine system that there are toxins, some related to drugs of abuse, that can damage the system; we also know that there appears to be a decline of dopamine with age; we also know that dopamine neurons can be damaged by various disease processes.

So one can see the scenario that if an individual sustained partial insult during early life, damaging 50% of the dopamine neurons, that individual will be fine as long as he doesn't suffer some other insult but might develop symptoms in conditions that others with normal reserve would not. The same may apply to serotonin systems. One could make the case that an individual that has the normal complement of brain serotonin has a normal risk of developing brain disorders, but the individual who has lower serotonin would be at increased risk of developing diseases associated with depletion of brain serotonin.

Would this also be true for fenfluramine and fluoxetine [Prozac]?

It would certainly apply to fenfluramine. Drugs like fluoxetine actually enhance serotonin transmission. The fundamental difference is that the effect of MDMA is long lasting and related to actual destruction of the nerve axons, whereas fluoxetine just alters the metabolism. When you stop taking fluoxetine, serotonin returns to its normal level. What's unique about drugs like MDMA is that they actually prune the axon field.

How does MDMA compare with fenfluramine in the way they cause damage?

As best I can tell, they are identical. If anything, fenfluramine might be a little more potent as a serotonin toxin.

How about a study on fenfluramine users and the onset of Parkinson's disease?

Given that serotonin is more implicated in things like mood regulation, as opposed to movement which is primarily affected in Parkinson's disease, a nice study would be to see if people exposed to fenfluramine are at a higher risk of developing neuropsychiatric disorders which have been linked, albeit indirectly, to serotonin dysfunction: such as depression, anxiety, sleep disturbances, memory function. Those would be some of the areas I would begin to look at in the fenfluramine cohort to see if humans exposed to high doses of fenfluramine show any evidence of long lasting serotonin dysfunction.

Might that mean the doses taken would not be high enough to show effects?

The issue of possible serotonin toxicity secondary to fenfluramine use is, I think, a very, very important one. In every experimental animal tested to date, the dose of fenfluramine required to reduce food intake is virtually identical to the dose that produces serotonin toxicity, or only slightly lower. When humans use fenfluramine, they are told to take it 2 or 3 times a day for long periods. For d-fenfluramine which is presently being evaluated in the US, the manufacturers recommendation are life long use. The suggestion put forth is that obesity should be regarded like diabetes: the obese person may need to take fenfluramine daily for life. When you consider that the use is chronic and the dose is very close to toxic level, then I think you have cause to question the safety of its use.

Going back to present study, how long before you will get preliminary results, and are you involved with other studies?

Its a five year study, and I expect it will be at least a year before we get preliminarily results. The human studies are complemented by animal studies, some directed to consequences, others to mechanisms of toxicity. We are particularly interested in the aberrant reinnervation in monkeys. Without the knowledge we have obtained with monkeys, one might have predicted that in the wake of MDMA use we might expect a blunted response to neuroendocrine challenge with specific pharmacological probes. Now I'm not so sure that hypothesis is correct. Perhaps we should be looking for augmented responses if indeed the regions of the brain that subserve the neuroendocrine response in the long term are hyperinnervated as opposed to hypoinnervated. The animal studies serve an important role in helping direct us as to the proper way to asses humans, and how to appropriately interpret the data. There is a constant interplay in this study between the two.

Does the neuron degradation you have observed with MDMA also occur with alcohol or aging or in other ways?

It can occur within some diseases, but whether serotonin declines with age has not been determined. The same toxicity occurs with a limited number of other drugs that are close chemical cousins, such as MDA, but not amphetamine sulphate or anti depressants.

Just how harmful is MDMA in comparison to other drugs or alcohol?

Not to be alarmed, but I think that if you compare MDMA to other psychoactive drugs, it is unique precisely because it has the potential to damage serotonin systems at doses close to that taken to produce the required effect. You would have to drink far more than social drinking to produce such damage. So if you consider the problem from that perspective, these drugs have a far higher risk associated with their use.

Interview with Dr. James O'Callaghan

I understand that you and your colleagues at the U.S. Environmental Protection Agency were awarded over $750,000 by the National Institute on Drug Abuse to provide a reliable assessment of the potential neurotoxic effects of MDMA and other drugs of abuse. Why is it so difficult to determine if a drug is neurotoxic?

The overall problem is that the brain is exceedingly complex. This complexity is reflected in all of the different types and sub types of neurons and glia, the cells that compose the brain. Because there are all these diverse types of cells it is not surprising that there are diverse and unpredictable targets of damage that result from exposure to neurotoxic agents. You can't predict what compound will hit what, because you don't know what would make a given cell type vulnerable to damage, and you don't know anything about the chemical to predict which cell is going to be targeted. You can't do this by computer modelling ; you can't look at the molecule and say "Gee, I know that's going to damage this particular brain cell".

I have seen 'before and after' photographs of the brains of monkeys used as evidence of neurotoxicity by Ricaurte. The 'after MDMA' slide shows far less lines. Is this a fair visual representation of damage caused by MDMA?

NO. This is the water in the pipe issue, not evidence of damage to the pipe. Serotonin immunostaining data does not indicate that neurodegeneration occurred. It is very subjective; in the absence of quantitative data (i.e. levels of 5-HT) to support the photomicrographs, it is difficult if not impossible to claim that what you see is what you get in a representative sense. In other words, if you see one (selected) picture this tells you very little. My point is underlined by the fact that the quantitative data does not support the "representative" photomicrographs in the paper you refer to.

The recently published article by Ricaurte and colleagues [J. Neurosci. 15: 5476-5485, 1995] describes the effects of MDMA on various indices thought to reflect the integrity of serotonergic innervation in the brain. Both the rat and squirrel monkey were examined in this study. The conclusions reached by the authors are that the long term effects of MDMA cause a reorganization of serotonin innervation of the brain, effects which are repeatedly referred to as evidence of "serotonin neurotoxicity." While the actions of MDMA in the rat and primate brain at the high dosages employed in the study (at least 25 times the human dosage) may in fact reflect a toxic effect of this drug on serotonin pathways, other interpretations of the data are possible.

For example, what Ricaurte and others have shown is, at high dosages, MDMA causes prolonged decreases in brain serotonin. This effect, per se, cannot be equated to destruction of serotonergin axons (i.e. bonafide serotonin neurotoxicity) because assessments of serotonin are only indicative of the presence of this transmitter in neurons, not the actual neuronal structures themselves.

In other words, MDMA can decrease the level of serotonin without necessarily destroying serotonergic axons. An analogy would be draining water from a pipe without destroying the plumbing. The prolonged decreases in serotonin seen after high dosages of MDMA have not been associated with the classic evidence of neuronal destruction such as astrogliosis [O'Callaghan and Miller, 1993] or silver degeneration staining [Commins et al., 1987; Jensen et al., 1993]. Moreover, retrograde transport studies, which could be used to assess whether serotonergic axons are still functional even when depleted of serotonin, have yet to be performed following exposure of rats or primates to MDMA.

How, then, do you find damage to the nervous system?

Historically, and to this day, the gold standard for determining whether something damages the nervous system is based on morphology. This means that you rely on histological staining to determine if you see loss of a given cell type or change in staining patterns of specific groups of cells. However, in the brain that doesn't work so well.

Why is that?

The problem is that there are so many myriads of cell types that compose the brain with different structural DNA/RNA make ups that you can't tell one from another, they look kind of the same, but they aren't the same, they perform very different functions, based on their specific molecular components. Obvious damage can be seen if a sufficiently large numbers of cells are lost and such findings would certainly constitute a neurotoxic effect because, once lost, these cells will not likely regenerate. Unfortunately, classical staining procedures (i.e. ones that will tell you if a brain cell is lost) will not allow you to determine if there has been damage to nerve terminals, nerve axons, or damage to a small population of cells. You can't see it even with a systematic survey of the entire brain using classical techniques.

Why is that, can't you get sufficient magnification?

If you could magnify the whole brain so that you could survey every cell type, it would take you two millenniums to sample everything because there are so many different cell types. Sampling at that level just cannot be done, so you have to rely on other ways of detecting damage or, alternatively, you have to start with a hint as to where the target is. The standard techniques only allow you to see a very small percentage of the total population of cells in the brain. Even if those staining techniques only missed one cell out of ten, that might be a very important cell in terms of brain functioning.

So, if the damage can't be assessed visually, how can you measure it?

I have gone into the literature and looked at what is known in terms of general responses of the nervous system to damage, and I include trauma-induced, disease-induced, stroke-induced damage besides chemical-induced damage that is of issue with respect to MDMA. When you get damage to any part of the brain and spinal cord the damaged area responds by an enlargement of a brain cell known as an astrocyte, a star-shaped glial cell. Astrocyte hypertrophy (enlargement) is called gliosis (the response of astrocytes to injury of neurons or glia to injury). This response recently has been associated with a very specific protein, GFAP. Increases in GFAP can be detected by a specific antibody and this is easy to see in sections of brain tissue when the increase in astrocyte size is on the order of 2 to 3-fold.

Is this a classical test, or one developed in your lab?

GFAP staining of tissue sections in not a classical test, but it is one that has gained wide acceptance for assessing, qualitatively, the presence of brain damage. I have extended this analysis to a different level by developing an assay for this GFAP, so that you can actually quantify the degree of increase in this protein in a given brain area. Its analogous to a blood test for the antibody to HIV; its a test for the presence of increased levels of GFAP.

So you have developed a new indirect method for measuring neurotoxicity. But how can you be sure if it gives the correct result?

The general idea is to develop a profile of broad types of known neurotoxic compounds, that is, compounds that are known to damage the nervous system, and see if you can get an increase in GFAP using the immunoassay I have developed. The test shows positive to a broad class of toxicants ­p; from metals to endogenous compounds like bilirubin that accumulate in blue babies at birth; to toxins that poison muscles, to seaweed poisons; to drugs used as tools to damage specific components of the nervous system. Stab wounds to the brain, extremely advanced age, infections to the brain and damage such as induced by Alzheimer's disease would also show positive, because they increase GFAP. Likewise, the test shows negative with a large number of drugs that we have tried at therapeutic doses. These include barbiturates, anti-psychotics, anti-cholinergics, steroids, anti-inflammatories, blockers of monoamine oxidase ­p; even Ketamine under certain circumstances. These agents do not damage the brain and do not increase GFAP.

How does your test compare with traditional methods?

For example, with respect to serotonin containing neurons, the toxicant 5, 7-dihydroxy tryptamine (which selectively damages serotonin nerve terminals) has been found to increase GFAP. Thus, damage to serotonin nerve terminals will cause gliosis and this is very useful for determining that damage has occurred because if the brains of animals treated with 5, 7-dihydroxytryptamine were examined by traditional staining methods, the brain would look normal when, in fact there is underlying damage to serotonin neurons.

So you've applied your test to a number of different known and suspected neurotoxic drugs and chemicals and it always works. How about fenfluramine and MDMA?

Realistically, we don't think fenfluramine, MDMA or even methamphetamine causes any damage in the rodent even at high dosages of these three compounds. The transmitter changes we see are prolonged but they return to control and they are never associated with gliosis (increased GFAP) or evidence of degeneration at the dosages that we have used. In the mouse but not in the rat we have shown clear-cut damage to dopaminergic nerve terminals after very high dosages of methamphetamine, MDA and MDMA. In rats or mice we have not observed damage to serotonin neurons due to methamphetamine, MDA, MDMA or d-fenfluramine, despite the fact that we do see weeks-long decreases in serotonin.

So your test indicates that MDMA is not neurotoxic to serotonin neurons, even at dosages higher than those commonly used in humans. But does this apply at high temperature?

The temperature data is too complicated to go into but I would say that all of the toxic effects of MDA, MDMA and methamphetamine require sustained temperatures above base line levels throughout the dosing regimen. When I refer to toxic here, I'm referring to systemic toxicity (e.g. cardiovascular collapse) that results from malignant hyperthermia. Its the acute toxic effects, not neurotoxicity, that kills you.

Are there any doubts at all about your methods of testing?

A possible argument that could be raised against the GFAP increase as evidence of serotonergic neurotoxicity could be that the serotonin decrease isn't associated with the increase in GFAP. What we have done here is instead of relying on the so-called serotonergic neurotoxic compounds, that is MDMA, d-fenfluramine or even paracloramphetamine, we have given the most widely characterised neurotoxic compound targeting serotonergic neurons which is 5, 7-dihydroxytryptamine. When we administer this compound to rats, the levels of brain serotonin decrease by 50 to 70% while we see 30 to 100% increases in GFAP over control.

What this means is that damage to the serotonergic nervous system leading to prolonged decreases of serotonin give you prolonged increases of GFAP. In other words, damage to serotonin nerve terminals results in gliosis and, therefore, an increase in GFAP. In contrast, when MDMA or d-fenfluramine are administered to rats a 30-70 % decrease in serotonin also is observed but these decreases are not associated with an increase in GFAP. We interpret these findings as evidence for a lack of a neurotoxic effect of these drugs. Instead, we believe that the decrease in serotonin after MDMA and d-fenfluramine can be explained as neuromodulatory/regulatory phenomena, i.e. protein synthesis inhibition as a natural extension of the pharmacological activity of those compounds.

I understand that a recent postmortem study of the brains of chronic methamphetamine users failed to find the expected neuronal damage. Do you think the same might apply to MDMA users?

Yes. It would be very surprising not to find decreases in serotonin in the brains of high-dose MDMA users. Again, as Kish found for methamphetamine abusers, this does not necessarily mean there is damage to nerve terminals.

Both interviews took place face to face in October 1995.
I interviewed Dr O'Callaghan at Rockefeller University where he had the honour of being a Guest Researcher, and Dr Ricaurte at John's Hopkins Medical Institute.

Taken from Ecstasy Reconsidered
©Nicholas Saunders, 1997 index
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