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.