3.4 Treatment with arsenite upregulates the level of β-endorphin
in mice
After a 6-month treatment with
different concentrations of arsenite in drinking water (0, 5, 15 mg/L),
the level of β-endorphin in mice increased in a dose-response manner
(Figure 4). The difference of β-endorphin was significant between the
control group and the 15 mg/L arsenite-treated group (P =0.001).
3.5 Naloxone relieves
pruritus in arsenic-exposed
participants
We initially planned to recruit 200 patients but finally enrolled 126
eligible participants. After random allocation, 64 were assigned to
placebo control and 62 were assigned to treatment group (supplementary
Figure S1). A total of 40 patients dropped out in week 1 (the treatment
period) for various reasons, including lack of efficacy (n=35), adverse
events (dizziness, n=2; cold, n=2), and worry of side effects (n=1).
Four patients dropped out in week 2 (the withdrawl period) due to no
efficacy. The baseline characteristics were comparable between the
groups (supplementary Table S4). The changes in itch NRS are displayed
in Figure 5. Compared to the control group, the NRS showed a significant
reduction in the treatment group in week 2
(β Group×Time=–0.98, P =0.040) based on a
mixed effect model (supplementary Table S5).
DISCUSSION
We performed a series of studies to evaluate the association of arsenic
exposure with pruritus: (1) a cross-sectional study to uncover the
positive association of hair arsenic with the intensity of pruritus in
arsenic-exposed residents; (2) a Mendelian randomization study to
confirm the causal relationship between genetic susceptibility to
arsenic exposure and chronic pruritus in the UK Biobank participants;
(3) a case-control study to identify β-endorphin as a serum biomarker
for arsenic-related pruritus; (4) an animal study to validate that
chronic arsenic exposure upregulates the expression of β-endorphin; and
(5) a randomized controlled trail to test the efficacy of naloxone on
pruritus in patients with arseniasis.
During the field survey, many participants reported unrelieved itch
during the dermatologic examination. Common causes for itch including
dermatologic, systemic, neuropathic, and psychogenic factors were
evaluated by physical examination as well as biochemical tests, and it
was not likely to explain such a high prevalence of pruritus in the
residents. Thus, we suspected the effect of arsenic exposure. The
mechanisms underlying pruritus are quite complex. The itch signal is
transmitted mainly by small, itch-selective C fibers originating in the
skin. Histamine-triggered neurons and nonhistaminergic neurons may be
involved. They form a synapse with secondary neurons that cross over to
the contralateral spinothalamic tract and ascend to multiple brain areas
involved in sensation, evaluative processes, emotion, reward, and
memory17. In the peripheral nervous system, T-type
calcium channels are prominent in C fibers. It was reported that
peripheral T-type calcium channels are involved in histamine-dependent
or histamine-independent itch processing. Pre-locally blocking T-type
calcium channels in the peripheral afferents of skins yielded an
inhibition in acute itch or pain behaviors10 18. On
the other hand, it has been reported that arsenic exposure could cause
calcium influx in guinea pigs, human neuroblastoma SY-5Y, and embryonic
kidney cells HEK 29319 20. Steady-state calcium
increases, transient calcium elevations, and calcium spikes were
observed, indicating that both L-type and T-type voltage gated channels
might be involved. However, direct evidences from in vivo andin vitro models are needed to support the hypothesis.
Arsenic in drinking water is predominantly inorganic arsenic. In human
body, inorganic arsenic is first methylated into MMA, which has high
cytotoxicity and genotoxicity21 22. MMA is then
methylated to DMA in liver, which excretes through the kidney together
with MMA and inorganic arsenic23. Epidemiologic
studies reported that a higher MMA% and a lower DMA% in urine are
associated with increased risks of bladder, breast, lung, and skin
cancers, as well as skin lesions24-26. Our MR analysis
consistently showed that a genetically higher MMA% and lower DMA% were
associated with an increased risk of chronic pruritus in a large
external sample. This indicates that chronic pruritus may be
attributable to unobserved environmental pollutants, even in regions
under a low level of exposure.
There is increasing evidence that neuropeptides such as substance P,
calcitonin gene-related protein (CGRP) or β-endorphin are involved in
the pathogenesis of itch27. β-endorphin is an
endogenous agonist for μ-opioid receptor, which are both present in
keratinocytes and free nerve endings28. Chronic
inflammatory skin disorders, such as atopic dermatitis and psoriasis
have common features of increased β-endorphin expression and peptidergic
nerve fibers29 30. Furthermore, μ-opioid receptors
antagonists (MORA) such as naloxone, naltrexone, and nalmefene are known
to suppress pruritus in patients with chronic urticaria, atopic
dermatitis, and psoriasis31. Our clinical trial
provides evidence that naloxone may relieve pruritus in arsenic-exposed
participants, which is deducible based on previous studies.
One possible mechanism underlying the pathogenesis of arsenic-related
pruritus is that arsenic increases the expression of
microRNA-2132 which promotes the secretion of
interleukin-1033, regulating the synthesis of
β-endorphin34. Chronic arsenic exposure leads to
peripheral neuropathy
characterized by symptoms like numbness, weakness, pain as well as
paraesthesia in stocking and glove distributions35.
During the questionnaire interview, many participants reported symptoms
of numbness, itch, and pain which might be caused by the peripheral
neuropathy. Nerve conduction velocity test and electromyography test
would be applied in further studies to identify the arsenic-related
neuropathy.
Our findings are highly relevant to public health and clinical practice.
First, if arsenic is confirmed to cause pruritus by more studies, the
diagnosis criteria of endemic arsenicosis may need revisions. Second,
for those receiving As2O3 therapy or
occupationally exposed to arsenic, pruritus should be noticed as an
adverse drug reaction or a symptom of arsenic poisoning. Third, when
dealing with patients with pruritus of unknown reason in clinical
settings, a history of exposure, including the place of residence,
occupational exposure, and the use of traditional medications that
contain arsenic, should be inquired, in addition to the detection for
hair arsenic. However, hair arsenic only reflects recent exposure (2 to
4 weeks). A national survey in 1996 showed that the hair arsenic in
Chinese residents in non-pollution regions ranged from 0.004 to 9.999
μg/g36.
In our study, 83% of the participants had hair arsenic <1
μg/g, and 99% had hair arsenic <10 μg/g. This indicates that,
patients with pruritus did not necessarily present elevated hair arsenic
level.
Our study systematically investigated the association of arsenic with
pruritus for the first time. It is possible that itch is a neglected but
important symptom in correlation with arsenic exposure. Second, a series
of methods were applied to test the hypothesis. We first observed the
association in a cross-sectional study, and then verified the causal
relationship using the MR analysis. We further performed a case-control
study to identify the biomarker for itch, and validated this in mice and
human.
The study also has limitations. A primary limitation is the lack of data
on external exposure. The level of previous exposure could not be
obtained owing to the lack of historical data, especially at the
individual level. However, we measured the internal level of arsenic
exposure in hair, serum, and urine samples, and consistently observed
the association based on multiple methods. Second, the underlying
mechanism has not been fully investigated, and more in vivo and in vitro
experiments are warranted. Nevertheless, we clearly identified a genetic
correlation between arsenic susceptibility and chronic pruritus in an
external sample, providing new insights for mechanism studies in the
future.
CONCLUSIONS
Our data suggest that chronic arsenic exposure is associated with
pruritus, and β-endorphin may mediate this association.
The treatment with naloxone relieves
the intensity of pruritus in patients with arseniasis. However, the
mechanism underlying arsenic-related itch is not clear yet.