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The Lancet.
Volume 358, Number 9296 01 December 2001
Effects of dose, sex, and long-term abstention from use on toxic
effects of MDMA (ecstasy) on brain serotonin neurons
Liesbeth Reneman, Jan Booij, Kora de Bruin, Johannes B Reitsma,
Frederik A de Wolff, W Boudewijn Gunning, Gerard J den Heeten, Wim van
den Brink
Departments of Nuclear Medicine (L Reneman MD, J Booij MD, K de
Bruin), Clinical Epidemiology and Biostatistics (J B Reitsma MD),
Human Toxicology (F A de Wolff PhD), Child and Adolescent Psychiatry
(W B Gunning MD), Radiology (G J den Heeten MD), and Psychiatry (W van
den Brink MD), Academic Medical Center, Meibergdreef 9, 1105 AZ
Amsterdam, Netherlands; and Toxicology Laboratory, Leiden University
Medical Center, Leiden, Netherlands (F A de Wolff)
=========================================================================
Summary
Background 3,4-methylenedioxymethamphetamine (MDMA or ecstasy) is a
popular recreational drug that has been shown to damage brain
serotonin neurons in high doses. However, effects of moderate MDMA use
on serotonin neurons have not been studied, and sex differences and
the long-term effects of MDMA use on serotonin neurons have not been
identified. We investigated the effects of moderate and heavy MDMA
use, sex differences, and long-term effects of MDMA use on serotonin
neurons in different brain regions.
Methods By means of flyers posted in "rave" venues in Amsterdam, the
Netherlands, we recruited 15 moderate MDMA users, 23 heavy MDMA users,
16 ex-MDMA users who had stopped using MDMA for more than 1 year, and
15 controls who claimed never to have used MDMA. We studied the
effects of MDMA on brain serotonin neurons using
123iodine-2ß-carbomethoxy-3ß-(4-iodophenyl) tropane ([123I]ß-CIT)--a
radioligand that binds with high affinity to serotonin transporters.
Density of binding (expressed as a ratio of region-of-interest binding
over binding in the cerebellum) was calculated by
single-photon-emission computed tomography (SPECT).
Findings We saw significant effects of group and group by sex (p=0*041
and p=0*022, respectively) on overall [123I]ß-CIT binding ratios. In
heavy MDMA users, significant decreases in overall binding ratios were
seen in women (p99*0%) and high specific activity (>185
MBq/nmol). Potassium iodide was used to block thyroid uptake of free
radioactive iodide.
SPECT studies were done with a brain-dedicated SPECT system
(Strichman Medical Equipment 810X, Medfield, MA, USA). This
12-detector single-slice scanner has a full width-at-half-maximum
resolution of about 7*5 mm. Transaxial slices parallel to and upward
in 5 mm steps from the orbitomeatal line to the vertex were acquired
after positioning of the individual in the camera with a fixed light
source oriented along the orbitomeatal line. Each image consisted of
about 15 slices (in a 64x64 matrix) with a 3-min scanning time per
slice. The energy window was set at 135-190 keV. Scanning started 4 h
after intravenous injection of about 140 MBq [123I]ß-CIT--a time when
specific binding to serotonin transporters is at a maximum.18
Reconstruction and attenuation correction of all images were done as
described previously.19
Density (mean counts per pixel) of the different regions of interest
(ROIs) were calculated by an investigator unaware of the participant's
history. We did the ROI analysis with a standard template constructed
manually from coregistered magnetic resonance images in four control
individuals. The template, including ROIs for the thalamus, frontal
cortex, temporal cortex, parieto-occipital cortex, and occipital
cortex was placed on three consecutive SPECT slices that showed best
visualisation of the striatum (typically 30 mm above the orbitomeatal
line). An additional template was constructed with an ROI for the
midbrain and cerebellum. Binding in the cerebellum, which was assumed
to be free from serotonin transporters, was used as a reference for
background radioactivity (non-specific binding and free ligand). The
ratios of [123I]ß-CIT binding were calculated by division of ROI
binding by binding in the cerebellum.
=============================================================================
Discussion
The results of our study suggest that MDMA use could lead to
decreases in the density of brain serotonin transporters, and that men
and women differ in their susceptibility to the neurotoxic effects of
MDMA. We found that, in women, use of MDMA is associated with
dose-related decreases in densities of brain serotonin transporters.
Such a reduction was also seen in men, but this finding was not
significant. The use of MDMA in quantities regarded as moderate does
not lead to significant reductions in densities of serotonin
transporters. Lastly, our data suggest that MDMA-induced decreases in
serotonin transporters could be reversible in female MDMA users, but
we cannot rule out the possibility that they might be long-lasting or
only partly reversible in the parieto-occipital cortex and occipital
cortex.
Decreases in densities of serotonin transporters are thought to be a
consequence of MDMA-induced brain serotonin neurotoxicity, since
similar reductions have been documented in rodents and primates with
MDMA-induced serotonin injury.1-3,5,10,20,21 We found that heavy MDMA
use seems to be associated with global decreases in serotonin
transporters. In MDMA-treated monkeys, the most severe serotonin
depletion was found in the occipital cortex.5 However, additional
studies and converging lines of evidence are needed to delineate
better the neurotoxic potential of moderate MDMA use in human beings.
In our study, women were more susceptible than men to MDMA
dose-related decreases in [123I]ß-CIT-labelled serotonin transporters.
Sex differences in the effects of MDMA exposure on radioligand binding
to serotonin transporters have not been previously published. Semple
and colleagues6 only included male MDMA users in their study, and saw
decreases in serotonin-transporter densities in posterior cortical
areas. McCann and co-workers4 found global decreases in the densities
of these transporters when investigating male and female MDMA users.
Contrary to the findings by Semple, we did not observe significant
reductions in serotonin transporter densities in male MDMA users.
Discrepancies between Semple's study and ours could be partly due to
the fact that participants in our study abstained from psychoactive
drugs (including MDMA) for at least 3 weeks, whereas they avoided such
drugs for only 1 week in the study by Semple.
In our study, the observed sex difference might have been due to
various things. First, inaccurate self-reporting of MDMA use might
have biased the results. However, the positive correlation between
reported MDMA exposure and densities of serotonin transporters in
women suggests accurate self-reporting of MDMA use, so there seems to
be no reason why men's self-reporting would not be accurate. Second,
women, who on average have a lower bodyweight than men, might have
been exposed to higher doses of MDMA on a mg/kg basis. However, on a
tablet/kg basis, men had a higher exposure to MDMA than women (table
2). Third, sample size could have contributed to the apparent absence
of neurotoxic effects in male MDMA users. However, a retrospective
power analysis showed that, to detect with 80% power a significant
reduction in overall [123I]ß-CIT binding in heavy MDMA users, seven
women and 393 men were required in each group. Another potential
explanation for the observed sex differences could be related to age.
Age is known to affect serotonin-transporter densities,18 so we
analysed our results with age as a potential confounder. We did not
see a significant effect of age on [123I]ß-CIT binding ratios, and
addition of age to the model did not change the overall effect of
group on specific to non-specific [123I]ß-CIT-labelled serotonin
transporters. We therefore conclude that, after accounting for the
differences in age, sex had an independent effect on [123I]ß-CIT
binding ratios.
There is other evidence that the consequences and mechanisms of MDMA
use are not identical in men and women. In line with our observations,
McCann and co-workers observed greater reductions in 5
hydroxyindoleacetic acid concentrations in the cerebrospinal fluid of
female MDMA users than in male users.9 Furthermore, Liechti and
co-workers reported more pronounced subjective responses to MDMA in
women than in men.8 These observations support our finding that women
could be more susceptible than men to the effects of MDMA; the
consequences and mechanisms of action of other drugs are not identical
in men and women.22,23 The cause of these sex differences is unknown,
but they could be related to differences in innate hormonal
profiles,24 volume and morphology of certain brain structures,25 or
monoaminergic neurotransmission. Future studies in larger experimental
groups are needed to investigate further sex differences in the
effects of MDMA exposure.
Our data also suggest reversibility of MDMA-induced changes in brain
serotonin transporters in most of the brain regions of female MDMA
users. A partly reversible neurotoxic action of MDMA on the human
brain has been described by Gerra and co-workers,26 when studying
neuroendocrine responses after D-fenfluramine administration. Studies
in primates show that MDMA-induced changes in serotonin terminal
markers persist for longer than 7 years in the neocortex, particularly
the pyriform and visual cortex, whereas brain regions proximal to the
rostral raphe nuclei showed evidence of complete recovery. The
distance of the serotonin terminal field to the rostral raphe nuclei
influences recovery of serotonin axons after MDMA injury.10 Clearly,
to establish whether the presently observed changes in serotonin
transporter densities in MDMA users are reversible, a prospective
study design would be needed. However, since studies of MDMA in people
are subject to ethical and methodological constraints, such a study
would be difficult to do. Therefore, future studies in individuals
with known MDMA-induced neurotoxicity need to be done to allow
definite conclusions on reversibility or permanence of MDMA-induced
changes in the human brain.
Decreased binding of the radioligand is assumed to reflect a decrease
in the density of serotonin transporters and thus axonal loss, but
several factors, such as allosteric changes in the binding unit of the
protein, could also result in decreased binding. Nevertheless, central
serotonin concentrations are also reduced after MDMA treatment, so
this possibility is unlikely to be important.27 Furthermore,
correlative anatomical studies indicate that loss of presynaptic
serotonin transporters in MDMA-treated animals is directly related to
damage of serotonin axons and axon terminals.20
Several potential limitations of the current study should be
mentioned. First, given the affinity of [123I]ß-CIT for both serotonin
and dopamine transporters, our findings cannot be definitively
ascribed to changes in the densities of serotonin transporters: the
midbrain, thalamus, and cortex also contain dopamine transporters.
However, displacement studies in animals have shown that binding of
ß-CIT is predominantly associated with serotonin transporters in these
brain regions.14 Furthermore, since we did not see differences in
striatal [123I]ß-CIT binding ratios (obtained 24 h after injection of
the radiotracer) between heavy MDMA users and controls, we conclude
that the findings probably reflect differences in densities of
serotonin and not dopamine transporters. Second, as with all
retrospective studies, there is a possibility that pre-existing
differences between MDMA users and controls underlie differences in
serotonin-transporter densities--eg, people with low densities might
be predisposed to use MDMA. Future studies taking the recently
described functional polymorphism in the promoter for the serotonin
transporter gene into account,28 could be of interest. Third, since
[123I]ß-CIT binding ratios are reduced in drug-free patients with
depression,29 we cannot rule out that the present findings are related
to this disease. However, we saw no significant effects of current
depression on [123I]ß-CIT binding, exclusion of depressed individuals
from our statistical analysis did not affect the main outcomes of the
present study (data not shown), and addition of current depression to
the model did not change the overall effect of group on [123I]ß-CIT
binding. Consequently, we conclude that the observed reduction in
serotonin transporter densities is unlikely to be due to this
potential confounding variable. Fourth, observed decreases in the
density of brain [123I]ß-CIT-labelled serotonin transporters are
unlikely to be due to direct pharmacological effects of MDMA or other
drugs, since MDMA-using participants reported that they had abstained
from use of MDMA or other psychoactive drugs for at least 3 weeks
before the study. Urine was screened to detect concealed recent MDMA
use, but other than self-report, we were not able to ensure long-term
abstention from MDMA. However, a recent survey in the Netherlands
investigated the validity of the drug-history questionnaire that was
used in this study. In 93% of the cases (n=594), the reported use of
MDMA was in agreement with the drug-urine test.30 In future studies,
analysis of hair samples would be a useful way to ascertain previous
use of MDMA.
Theoretically, the presently observed changes in [123I]ß-CIT binding
to serotonin transporters could be due to drugs other than MDMA, since
MDMA users in our study had more experience with other recreational
drugs (mainly amphetamine and cocaine) than controls. However, since
none of these drugs is a known serotonin neurotoxin in human beings,
our findings are unlikely to be attributed to substances other than
MDMA. An important part of the present study is that we minimised the
potential confounding effects of psychosocial differences and use of
other drugs between users and controls by studying participants from
the same subculture. This situation differs from those of most
previous studies, in which controls came from a university or general
population.
In summary, our data indicate that heavy use of MDMA could be
associated with neurotoxic effects on serotonin neurons in
serotonin-rich brain regions. Our results indicate that women could be
more susceptible than men to the neurotoxic effects of MDMA, and that
MDMA-induced neurotoxic changes in brain regions of female ex-MDMA
users might be reversible.
------------------
And the Mission is the Mouse...
Volume 358, Number 9296 01 December 2001
Effects of dose, sex, and long-term abstention from use on toxic
effects of MDMA (ecstasy) on brain serotonin neurons
Liesbeth Reneman, Jan Booij, Kora de Bruin, Johannes B Reitsma,
Frederik A de Wolff, W Boudewijn Gunning, Gerard J den Heeten, Wim van
den Brink
Departments of Nuclear Medicine (L Reneman MD, J Booij MD, K de
Bruin), Clinical Epidemiology and Biostatistics (J B Reitsma MD),
Human Toxicology (F A de Wolff PhD), Child and Adolescent Psychiatry
(W B Gunning MD), Radiology (G J den Heeten MD), and Psychiatry (W van
den Brink MD), Academic Medical Center, Meibergdreef 9, 1105 AZ
Amsterdam, Netherlands; and Toxicology Laboratory, Leiden University
Medical Center, Leiden, Netherlands (F A de Wolff)
=========================================================================
Summary
Background 3,4-methylenedioxymethamphetamine (MDMA or ecstasy) is a
popular recreational drug that has been shown to damage brain
serotonin neurons in high doses. However, effects of moderate MDMA use
on serotonin neurons have not been studied, and sex differences and
the long-term effects of MDMA use on serotonin neurons have not been
identified. We investigated the effects of moderate and heavy MDMA
use, sex differences, and long-term effects of MDMA use on serotonin
neurons in different brain regions.
Methods By means of flyers posted in "rave" venues in Amsterdam, the
Netherlands, we recruited 15 moderate MDMA users, 23 heavy MDMA users,
16 ex-MDMA users who had stopped using MDMA for more than 1 year, and
15 controls who claimed never to have used MDMA. We studied the
effects of MDMA on brain serotonin neurons using
123iodine-2ß-carbomethoxy-3ß-(4-iodophenyl) tropane ([123I]ß-CIT)--a
radioligand that binds with high affinity to serotonin transporters.
Density of binding (expressed as a ratio of region-of-interest binding
over binding in the cerebellum) was calculated by
single-photon-emission computed tomography (SPECT).
Findings We saw significant effects of group and group by sex (p=0*041
and p=0*022, respectively) on overall [123I]ß-CIT binding ratios. In
heavy MDMA users, significant decreases in overall binding ratios were
seen in women (p99*0%) and high specific activity (>185
MBq/nmol). Potassium iodide was used to block thyroid uptake of free
radioactive iodide.
SPECT studies were done with a brain-dedicated SPECT system
(Strichman Medical Equipment 810X, Medfield, MA, USA). This
12-detector single-slice scanner has a full width-at-half-maximum
resolution of about 7*5 mm. Transaxial slices parallel to and upward
in 5 mm steps from the orbitomeatal line to the vertex were acquired
after positioning of the individual in the camera with a fixed light
source oriented along the orbitomeatal line. Each image consisted of
about 15 slices (in a 64x64 matrix) with a 3-min scanning time per
slice. The energy window was set at 135-190 keV. Scanning started 4 h
after intravenous injection of about 140 MBq [123I]ß-CIT--a time when
specific binding to serotonin transporters is at a maximum.18
Reconstruction and attenuation correction of all images were done as
described previously.19
Density (mean counts per pixel) of the different regions of interest
(ROIs) were calculated by an investigator unaware of the participant's
history. We did the ROI analysis with a standard template constructed
manually from coregistered magnetic resonance images in four control
individuals. The template, including ROIs for the thalamus, frontal
cortex, temporal cortex, parieto-occipital cortex, and occipital
cortex was placed on three consecutive SPECT slices that showed best
visualisation of the striatum (typically 30 mm above the orbitomeatal
line). An additional template was constructed with an ROI for the
midbrain and cerebellum. Binding in the cerebellum, which was assumed
to be free from serotonin transporters, was used as a reference for
background radioactivity (non-specific binding and free ligand). The
ratios of [123I]ß-CIT binding were calculated by division of ROI
binding by binding in the cerebellum.
=============================================================================
Discussion
The results of our study suggest that MDMA use could lead to
decreases in the density of brain serotonin transporters, and that men
and women differ in their susceptibility to the neurotoxic effects of
MDMA. We found that, in women, use of MDMA is associated with
dose-related decreases in densities of brain serotonin transporters.
Such a reduction was also seen in men, but this finding was not
significant. The use of MDMA in quantities regarded as moderate does
not lead to significant reductions in densities of serotonin
transporters. Lastly, our data suggest that MDMA-induced decreases in
serotonin transporters could be reversible in female MDMA users, but
we cannot rule out the possibility that they might be long-lasting or
only partly reversible in the parieto-occipital cortex and occipital
cortex.
Decreases in densities of serotonin transporters are thought to be a
consequence of MDMA-induced brain serotonin neurotoxicity, since
similar reductions have been documented in rodents and primates with
MDMA-induced serotonin injury.1-3,5,10,20,21 We found that heavy MDMA
use seems to be associated with global decreases in serotonin
transporters. In MDMA-treated monkeys, the most severe serotonin
depletion was found in the occipital cortex.5 However, additional
studies and converging lines of evidence are needed to delineate
better the neurotoxic potential of moderate MDMA use in human beings.
In our study, women were more susceptible than men to MDMA
dose-related decreases in [123I]ß-CIT-labelled serotonin transporters.
Sex differences in the effects of MDMA exposure on radioligand binding
to serotonin transporters have not been previously published. Semple
and colleagues6 only included male MDMA users in their study, and saw
decreases in serotonin-transporter densities in posterior cortical
areas. McCann and co-workers4 found global decreases in the densities
of these transporters when investigating male and female MDMA users.
Contrary to the findings by Semple, we did not observe significant
reductions in serotonin transporter densities in male MDMA users.
Discrepancies between Semple's study and ours could be partly due to
the fact that participants in our study abstained from psychoactive
drugs (including MDMA) for at least 3 weeks, whereas they avoided such
drugs for only 1 week in the study by Semple.
In our study, the observed sex difference might have been due to
various things. First, inaccurate self-reporting of MDMA use might
have biased the results. However, the positive correlation between
reported MDMA exposure and densities of serotonin transporters in
women suggests accurate self-reporting of MDMA use, so there seems to
be no reason why men's self-reporting would not be accurate. Second,
women, who on average have a lower bodyweight than men, might have
been exposed to higher doses of MDMA on a mg/kg basis. However, on a
tablet/kg basis, men had a higher exposure to MDMA than women (table
2). Third, sample size could have contributed to the apparent absence
of neurotoxic effects in male MDMA users. However, a retrospective
power analysis showed that, to detect with 80% power a significant
reduction in overall [123I]ß-CIT binding in heavy MDMA users, seven
women and 393 men were required in each group. Another potential
explanation for the observed sex differences could be related to age.
Age is known to affect serotonin-transporter densities,18 so we
analysed our results with age as a potential confounder. We did not
see a significant effect of age on [123I]ß-CIT binding ratios, and
addition of age to the model did not change the overall effect of
group on specific to non-specific [123I]ß-CIT-labelled serotonin
transporters. We therefore conclude that, after accounting for the
differences in age, sex had an independent effect on [123I]ß-CIT
binding ratios.
There is other evidence that the consequences and mechanisms of MDMA
use are not identical in men and women. In line with our observations,
McCann and co-workers observed greater reductions in 5
hydroxyindoleacetic acid concentrations in the cerebrospinal fluid of
female MDMA users than in male users.9 Furthermore, Liechti and
co-workers reported more pronounced subjective responses to MDMA in
women than in men.8 These observations support our finding that women
could be more susceptible than men to the effects of MDMA; the
consequences and mechanisms of action of other drugs are not identical
in men and women.22,23 The cause of these sex differences is unknown,
but they could be related to differences in innate hormonal
profiles,24 volume and morphology of certain brain structures,25 or
monoaminergic neurotransmission. Future studies in larger experimental
groups are needed to investigate further sex differences in the
effects of MDMA exposure.
Our data also suggest reversibility of MDMA-induced changes in brain
serotonin transporters in most of the brain regions of female MDMA
users. A partly reversible neurotoxic action of MDMA on the human
brain has been described by Gerra and co-workers,26 when studying
neuroendocrine responses after D-fenfluramine administration. Studies
in primates show that MDMA-induced changes in serotonin terminal
markers persist for longer than 7 years in the neocortex, particularly
the pyriform and visual cortex, whereas brain regions proximal to the
rostral raphe nuclei showed evidence of complete recovery. The
distance of the serotonin terminal field to the rostral raphe nuclei
influences recovery of serotonin axons after MDMA injury.10 Clearly,
to establish whether the presently observed changes in serotonin
transporter densities in MDMA users are reversible, a prospective
study design would be needed. However, since studies of MDMA in people
are subject to ethical and methodological constraints, such a study
would be difficult to do. Therefore, future studies in individuals
with known MDMA-induced neurotoxicity need to be done to allow
definite conclusions on reversibility or permanence of MDMA-induced
changes in the human brain.
Decreased binding of the radioligand is assumed to reflect a decrease
in the density of serotonin transporters and thus axonal loss, but
several factors, such as allosteric changes in the binding unit of the
protein, could also result in decreased binding. Nevertheless, central
serotonin concentrations are also reduced after MDMA treatment, so
this possibility is unlikely to be important.27 Furthermore,
correlative anatomical studies indicate that loss of presynaptic
serotonin transporters in MDMA-treated animals is directly related to
damage of serotonin axons and axon terminals.20
Several potential limitations of the current study should be
mentioned. First, given the affinity of [123I]ß-CIT for both serotonin
and dopamine transporters, our findings cannot be definitively
ascribed to changes in the densities of serotonin transporters: the
midbrain, thalamus, and cortex also contain dopamine transporters.
However, displacement studies in animals have shown that binding of
ß-CIT is predominantly associated with serotonin transporters in these
brain regions.14 Furthermore, since we did not see differences in
striatal [123I]ß-CIT binding ratios (obtained 24 h after injection of
the radiotracer) between heavy MDMA users and controls, we conclude
that the findings probably reflect differences in densities of
serotonin and not dopamine transporters. Second, as with all
retrospective studies, there is a possibility that pre-existing
differences between MDMA users and controls underlie differences in
serotonin-transporter densities--eg, people with low densities might
be predisposed to use MDMA. Future studies taking the recently
described functional polymorphism in the promoter for the serotonin
transporter gene into account,28 could be of interest. Third, since
[123I]ß-CIT binding ratios are reduced in drug-free patients with
depression,29 we cannot rule out that the present findings are related
to this disease. However, we saw no significant effects of current
depression on [123I]ß-CIT binding, exclusion of depressed individuals
from our statistical analysis did not affect the main outcomes of the
present study (data not shown), and addition of current depression to
the model did not change the overall effect of group on [123I]ß-CIT
binding. Consequently, we conclude that the observed reduction in
serotonin transporter densities is unlikely to be due to this
potential confounding variable. Fourth, observed decreases in the
density of brain [123I]ß-CIT-labelled serotonin transporters are
unlikely to be due to direct pharmacological effects of MDMA or other
drugs, since MDMA-using participants reported that they had abstained
from use of MDMA or other psychoactive drugs for at least 3 weeks
before the study. Urine was screened to detect concealed recent MDMA
use, but other than self-report, we were not able to ensure long-term
abstention from MDMA. However, a recent survey in the Netherlands
investigated the validity of the drug-history questionnaire that was
used in this study. In 93% of the cases (n=594), the reported use of
MDMA was in agreement with the drug-urine test.30 In future studies,
analysis of hair samples would be a useful way to ascertain previous
use of MDMA.
Theoretically, the presently observed changes in [123I]ß-CIT binding
to serotonin transporters could be due to drugs other than MDMA, since
MDMA users in our study had more experience with other recreational
drugs (mainly amphetamine and cocaine) than controls. However, since
none of these drugs is a known serotonin neurotoxin in human beings,
our findings are unlikely to be attributed to substances other than
MDMA. An important part of the present study is that we minimised the
potential confounding effects of psychosocial differences and use of
other drugs between users and controls by studying participants from
the same subculture. This situation differs from those of most
previous studies, in which controls came from a university or general
population.
In summary, our data indicate that heavy use of MDMA could be
associated with neurotoxic effects on serotonin neurons in
serotonin-rich brain regions. Our results indicate that women could be
more susceptible than men to the neurotoxic effects of MDMA, and that
MDMA-induced neurotoxic changes in brain regions of female ex-MDMA
users might be reversible.
------------------
And the Mission is the Mouse...
