Arsenic-Induced Carcinogenesis and Immune Dysregulation.

Arsenic, a metal ubiquitously distributed in the environment, remains an important global health threat. Drinking arsenic-contaminated water is the major route of human exposure. Exposure to arsenic contributes to several malignancies, in the integumentary, respiratory, hepatobiliary, and urinary systems. Cutaneous lesions are important manifestations after long-term arsenic exposure. Arsenical skin cancers usually herald the development of other internal cancers, making the arsenic-induced skin carcinogenesis a good model to investigate the progression of chemical carcinogenesis. In fact, only a portion of arsenic-exposed humans eventually develop malignancies, likely attributed to the arsenic-impaired immunity in susceptible individuals. Currently, the exact pathophysiology of arsenic-induced carcinogenesis remains elusive, although increased reactive oxidative species, aberrant immune regulations, and chromosome abnormalities with uncontrolled cell growth might be involved. This review discusses how arsenic induces carcinogenesis, and how the dysregulated innate and adaptive immunities in systemic circulation and in the target organs contribute to arsenic carcinogenesis. These findings offer evidence for illustrating the mechanism of arsenic-related immune dysregulation in the progression of carcinogenesis, and this may help explain the nature of multiple and recurrent clinical lesions in arsenic-induced skin cancers.

Treatment of Arsenic-Induced Bowen's Disease With Topical 5-Fluorouracil

Here, we present a case of arsenic-induced Bowen's disease treated with a regimen consisting of topical 5-fluouracil and oral nicotinamide. The use of this therapy modality resulted in near complete resolution of all of the patient's lesions except for those on her palms, soles, and scalp. Excellent wound care and treatment adherence were major factors contributing to the success of this treatment option. Our results ultimately provide an alternative approach to treating multiple arsenical keratoses in patients who are limited to a drug plan involving 5-FU and oral nicotinamide and who are able to be rigorously compliant with application of medication and wound care. J Drugs Dermatol. 2019;18(5):477-479.

Multiple Bowen's diseases and basal cell carcinomas in a patient with acute promyelocytic leukemia treated with arsenic trioxide: A case report and effective treatment with photodynamic therapy.

It is very rare that multiple Bowen's disease (BD) and basal cell carcinoma (BCC) lesions develop in a single patient. Routine therapy for the multiple lesions is not satisfactory. The present authors report on a patient who had developed multiple BD and BCC lesions for 12 years after arsenic trioxide treatment for his acute promyelocytic leukemia 20 years before. The patient with multiple lesions was successfully treated with photodynamic therapy.

Immunological dysfunction in chronic arsenic exposure: From subclinical condition to skin cancer.

Exposure to arsenic is a global health issue. Long-term arsenic exposure may associate with various cancers and many other pathological effects. Over 100 million people worldwide are exposed to arsenic particularly in countries such as Bangladesh, Chile, China, India, Mexico, Taiwan and the USA. Drinking of water contaminated with arsenic is the major route of human exposure. Skin lesions are considered to be the most common adverse effects associated with chronic arsenic exposure. Skin lesions usually develop with the latency period spanning more than 20 years from first exposure. Arsenic-induced Bowen's disease, the most frequently encountered carcinoma in situ resulting from chronic arsenic exposure, is characterized by multiple and recrudescent lesions. Long-term arsenic exposure results in impaired immunity in susceptible individuals. In the prenatal stage, enhanced placental inflammatory responses and reduced placental T cells by arsenic may result in decreased thymic size and functions in newborns. In childhood, arsenic exposure may reduce peripheral CD4+ cells and interleukin-2 secretion which leads to susceptibility to opportunistic infections. There was an impairment of macrophage function and oxidative DNA damage of peripheral polymorphonuclear leukocytes in adults with skin lesions. In arsenic-induced Bowen's disease lesions, a decrease in the number and functions of Langerhans cells and, in parallel, a selective CD4+ cell apoptosis was noticed. These findings provide scientific evidence for understanding the phenomenon of arsenic-induced immune escape in the early stage of carcinogenesis and a reasonable explanation for multiple and recrudescent arsenic cancers in the skin.

ΔNp63 promotes abnormal epidermal proliferation in arsenical skin cancers.

Arsenic is known to perturb epidermal homeostasis and induce abnormal keratinocyte proliferation, leading to skin carcinogenesis. P63 and its isoforms are essential to regulate epidermal homeostasis. This study aimed to investigate the role of p63 isoforms in abnormal epidermal proliferation induced by arsenic. Using arsenic-induced Bowen's disease (As-BD; an intraepidermal carcinoma) as a disease model, we found that in As-BD, the expression of proliferating basal keratinocytes marker cytokeratin 14 (CK14) and N-terminal truncated p63 isoform (ΔNp63; proliferation regulator) was increased, however, that of the differentiation marker cytokeratin 10 (CK10) and full-length p63 isoform (TAp63; differentiation regulator) was decreased in squamous cells as compared with healthy subjects. These observations were recapitulated in the arsenic-treated skin equivalents (SEs). The SEs showed that arsenic increased epidermal thickness, induced abnormal proliferation, and increased ΔNp63 expression in squamous cells as compared with the control. Treatment of cultured normal human epidermal keratinocytes (HKCs) with arsenic increased CK14 and △Np63 expressions, but decreased TAp63 and CK10 expressions. Furthermore, knockdown of ΔNp63 by RNA interference abrogated arsenic-induced CK14 expression and recovered the reduction of TAp63 and CK10 caused by arsenic. These findings indicated that ΔNp63 is a pivotal regulator in the abnormal cell proliferation in arsenical cancers.

Cyclin D1 promoter -56 and -54bp CpG un-methylation predicts invasive progression in arsenic-induced Bowen's disease.

BACKGROUND: Patients with arsenic-induced Bowen's disease (As-BD) are at risk of developing invasive cancers in the skin, lung, and urinary bladder. However, a longitudinal follow-up study on the association between As-BD and invasive cancers is still lacking. OBJECTIVES: This study aims to investigate the underlying molecular mechanisms of this malignant progression in the skin and internal organs. METHODS: This is a biopsy-based follow-up study. We tested the DNA histograms, Cyclin D1 (CCND1) protein expression and CCND1 promoter DNA methylation in 40 pathologically confirmed specimens from As-BD patients to correlate with individual's invasive cancer occurrence in the 5-year follow-up. RESULTS: Flow cytometric DNA histogram analysis of skin specimens showed aneuploid (n=15), G2/M arrest (n=22), and normal (n=3) DNA histograms. No patients with normal DNA histograms developed invasive cancers, whereas 13 developed invasive cancers in the aneuploid group and 2 developed invasive cancers in the G2/M arrest group. The aneuploid group showed a high risk of invasive cancer development. In all assessed aneuploid specimens, the CCND1 promoter hypomethylation was observed. Statistically, percentage of un-methylation more than 55.85% among 17 detected CpG sites showed extremely high predictive power in the occurrence of invasive arsenical cancers. Furthermore, the un-methylation at -56 and -54bp CpG sites was statistically significantly associated with invasive arsenical cancer development (p=1.29×10-5). CONCLUSIONS: As-BD lesions showing an aneuploid DNA histogram had a high risk of invasive cancer development. Un-methyaltion at -56 and -54bp CpG in the CCND1 promoter serves as a predictor for invasive progression in As-BD patients.

Presence of the Merkel cell polyomavirus in Merkel cell carcinoma combined with squamous cell carcinoma in a patient with chronic arsenism.

We present a case of Merkel cell carcinoma (MCC) coincident with squamous cell carcinoma (SCC) on the breast of a woman with chronic arsenism. This case demonstrates the distinct association of chronic arsenism with two different primary cutaneous carcinomas. Merkel cell polyomavirus (MCPyV) was identified in the lesional skin of the MCC but not in that of the SCC, suggesting there are different interactions of MCPyV in the pathogenesis of SCC and MCC related to arsenic. Physicians need to be vigilant in the occurrence of both SCC and MCC in patients with chronic arsenism. To our knowledge, this is the first study to show the presence of MCPyV in the MCC but not the SCC portion of an arsenic-induced tumour.

Role of mitochondria, ROS, and DNA damage in arsenic induced carcinogenesis.

The International Agency for Research on Cancer (IARC) declared arsenic a class I carcinogen. Arsenic exposure induces several forms of human cancers, including cancers of skin, lung, liver, and urinary bladder. The majority of the arsenic-induced cancers occur in skin. Among these, the most common is Bowen's disease, characterized by epidermal hyperplasia, full layer epidermal dysplasia, leading to intraepidermal carcinoma as well as apoptosis, and moderate dermal infiltrates, which require the participation of mitochondria. The exact mechanism underlying arsenic induced carcinogenesis remains unclear, although increased reactive oxidative stresses, leading to chromosome abnormalities and uncontrolled growth, and aberrant immune regulations might be involved. Here, we highlight how increased mitochondrial biogenesis and oxidative stress lead to mitochondrial DNA damage and mutation in arsenic induced cancers. We also provide therapeutic rationale for targeting mitochondria in the treatment of arsenic induced cancers.

Palmoplantar keratoses and Bowen's disease in a Vietnam veteran: Could Agent Blue be implicated?

Agent Blue was an arsenical herbicide used extensively in the Vietnam War. Arsenic is one of the known causes of acquired palmoplantar keratoderma (PPK). The most common manifestation of arsenic exposure in susceptible individuals is bilateral palmoplantar hyperkeratosis. We report a 67-year-old man with no known prior exposure to arsenic in the USA or family history of PPK who developed multiple squamous cell carcinoma in situ (SCCIS) and palmoplantar hyperkeratotic lesions beginning 23 years after service in Vietnam. The SCCIS were located on the trunk and extremities in both sun-exposed and non-sun-exposed sites and his palmoplantar lesions were diagnosed concurrently with his SCCIS. He has continued to develop SCCIS since his first visit to our clinic 25 years ago.

Cutaneous malignant and premalignant conditions caused by chronic arsenicosis from contaminated ground water consumption: a profile of patients from eastern India.

Natural arsenic pollution is a major global health problem. The two worst affected areas e Bangladesh and West Bengal, India. Arsenic is a well-documented human carcinogen that affects many organs including the skin. The authors sought to find out the clinical patterns of different malignant and premalignant conditions associated with chronic arsenicosis from drinking contaminated ground water in a group of patients from eastern India. This was a clinical observational study. Patients with chronic arsenicoses with suspected cutaneous malignancies for whom dermatology service was sought were enrolled in the study. A total of 24 patients (male to female ratio, 11:1; age range, 32-71 years; mean age, 52.2 years) were evaluated. Squamous cell carcinoma (SCC) was the commonest malignancies in our series, seen in 10 (41.7%) patients. This was followed by Bowen's disease (9 [37.5%]) and basal cell carcinoma (8 [33.3%]). Three patients (12.5%) had > 1 type of cutaneous malignancies. Multicentric lesions were seen in 3 cases. The most common site of involvement was the chest (8 [33.3%]). No statistically significant correlation was found between number of lesions and arsenic content in the hairs and nails of the patients.

Involvement of mtDNA damage elicited by oxidative stress in the arsenical skin cancers.

Arsenic causes several human cancers. Arsenic-induced Bowen's disease (As-BD), the most common arsenical cancer, is characterized by increased proliferation, dysplasia, and individual cell apoptosis, all of which involve mitochondria. We reported that arsenic causes aberrant keratinocyte proliferation through mtTFA-mediated mitochondrial biogenesis in As-BD. Increasing mitochondrial biogenesis causes cells to undergo oxidative stress. However, how arsenic induces oxidative stress and causes mtDNA damage in arsenical cancers remains largely unknown. Using tissues from As-BD patients and arsenic-treated keratinocytes, we determined the oxidative stress, antioxidant enzymes, DNA-repair enzymes, and 8-hydroxy-2'-deoxyguanosine (8-OHdG) level in mtDNA by immunofluorescence, real-time PCR, and western blot. The results showed that oxidative stress was enhanced in both As-BD and arsenic-treated keratinocytes. Antioxidant enzymes including manganese-superoxide anion and copper/zinc-superoxide anion and DNA-repair enzymes were upregulated concomitantly in tissues and cells. In arsenic-treated keratinocytes, increased mitochondrial oxidative stress and the 8-OHdG level in mtDNA were attenuated by pretreatment with ascorbic acid, a potent antioxidant. Further, we found several somatic mutations in the ND4, ND5, and ND6 genes of mtDNA in lesional but not in perilesional skin from As-BD patients. Taken together, the results suggest that oxidative damage and mutations to mtDNA might be involved in the arsenical skin cancers in the context of mitochondrial biogenesis.

Use of arsenic-induced palmoplantar hyperkeratosis and skin cancers to predict risk of subsequent internal malignancy.

Hyperpigmentation, hyperkeratoses, and Bowen's disease are hallmarks of chronic arsenic exposure. The association between arsenic-induced skin lesions and subsequent internal cancers is examined by using a community-based prospective study. The cohort was enrolled from an arseniasis-endemic area in southwestern Taiwan, where 2,447 residents participated in skin examinations during the late 1980s. The number of participants diagnosed with hyperpigmentation was 673; with hyperkeratosis, 243; and with skin cancer (Bowen's disease or non-melanoma skin cancer), 378. Newly diagnosed internal cancers were ascertained through linkage with National Cancer Registry profiles. Cox regression was performed to estimate hazard ratios with 95% confidence intervals for potential risk predictors. Compared with participants without skin lesions, patients affected with skin cancers had a significantly increased risk of lung cancer (hazard ratio = 4.64, 95% confidence interval: 2.92, 7.38) and urothelial carcinoma (hazard ratio = 2.02, 95% confidence interval: 1.23, 3.30) after adjustment for potential confounders and cumulative arsenic exposure. Hyperkeratosis is significantly associated with an increased lung cancer risk (hazard ratio = 2.76, 95% confidence interval: 1.35, 5.67). A significant interactive effect on lung cancer risk between hyperkeratosis and cigarette smoking was identified, which suggests that patients with hyperkeratosis who have been exposed to arsenic should cease smoking.

Arsenic mobilizes Langerhans cell migration and induces Th1 response in epicutaneous protein sensitization via CCL21: a plausible cause of decreased Langerhans cells in arsenic-induced intraepithelial carcinoma.

Arsenic, still a significant environmental threat in several regions in the world, induces various cancers, including lungs, skin, and bladder. Arsenic-induced Bowen's disease (As-BD) is generally an indolent cutaneous intraepithelial carcinoma in susceptible people. Patients with As-BD have been found to have attenuated contact hypersensitivity. Skin samples collected from these patients have reduced numbers of Langerhans cells (LCs), the major epidermal antigen presenting cells expressing Langerins. This study uses an epicutaneous protein sensitization model to investigate the mechanism through which LCs are decreased in As-BD. It further investigates the possibility that arsenic alters LC migration and polarizes Th responses. To do this, we patch-sensitized Balb/c mice or DT-treated Langerin-DTR mice (conditional depletion of Langerin(+) cells) with OVA or PBS, and fed them water containing 300 ppb arsenic or regular water for 200 µl for five days. Ninety-six hours after OVA sensitization, Langerin(+)EpCAM(+) cells in arsenic-treated WT mice were significantly increased in draining lymph nodes and decreased in epidermis without changes in the dermis. Lymph node cells from arsenic-treated WT mice were found to proliferate more than lymph node cells from control PBS-treated mice after OVA challenge in vitro. They also secreted more IFN-γ and IL-12, but not IL-4, IL-13, or IL-17. However, cell proliferation and the induction of IFN-γ by arsenic were found to be abolished in DT-treated Langerin-DTR mice. The expressions of CCL21 and CXCL12 were also increased in lymph nodes from arsenic-treated WT mice. The administration of a neutralizing antibody against CCL21, but not CXCL12, abolished the increase of LCs in lymph nodes in vivo. The results of this study, the first to study oral arsenic polarization of Th1 responses in epicutaneous protein sensitization through CCL21-mediated LC migration, suggest the chronicity of As-BD without invasion might result from enhanced Th1 responses and altered LC migrations by arsenic.

Aberrant immune responses in arsenical skin cancers.

Arsenic is a well-known human carcinogen. It also impairs immune functions and activation in many aspects. However, only a small portion of arsenic-exposed population develops skin abnormalities, including Bowen's disease and skin cancers. Differential immune activation among the individuals might account for the different susceptibilities. In patients with arsenic-induced Bowen's disease, there is a selective CD4 T-cell apoptosis through tumor necrosis factor-alpha pathway, decrease in macrophage differentiation and phagocytosis, reduced Langerhans cell numbers and dendrites, altered regulatory T-cell distribution, and other immune alterations. Several lines of evidence from mouse and fish studies also confirmed the potent and multifaceted effects of arsenic in the immune system. The molecular bases of immunosuppression by arsenic in lymphocytes may include chromosomal and DNA abnormalities, decreased T-cell receptor activation, and the cellular status of oxidation and methylation. This article also reviews the causative and differential role of selective CD4 cell apoptosis and the carcinogenesis of arsenic-induced Bowen's disease.

Aberrant cell proliferation by enhanced mitochondrial biogenesis via mtTFA in arsenical skin cancers.

Arsenic-induced Bowen's disease (As-BD), a cutaneous carcinoma in situ, is thought to arise from gene mutation and uncontrolled proliferation. However, how mitochondria regulate the arsenic-induced cell proliferation remains unclear. The aim of this study was to clarify whether arsenic interfered with mitochondrial biogenesis and function, leading to aberrant cell proliferation in As-BD. Skin biopsy samples from patients with As-BD and controls were stained for cytochrome c oxidase (Complex IV), measured for mitochondrial DNA (mtDNA) copy number and the expression levels of mitochondrial biogenesis-related genes, including peroxisome proliferator-activated receptor gamma coactivator-1α (PGC-1α), nuclear respiratory factor 1 (NRF-1), and mitochondrial transcription factor A (mtTFA). The results showed that expression of cytochrome c oxidase, mtTFA, NRF-1, and PGC-1α was increased in As-BD compared with in healthy subjects. Treatment of primary keratinocytes with arsenic at concentrations lower than 1.0 µmol/L induced cell proliferation, along with enhanced mitochondrial biogenesis. Furthermore, we observed that the mitochondrial oxygen consumption rate and intracellular ATP level were increased in arsenic-treated keratinocytes. Blocking of mitochondrial function by oligomycin A (Complex V inhibitor) or knockdown of mtTFA by RNA interference abrogated arsenic-induced cell proliferation without affecting cyclin D1 expression. We concluded that mtTFA up-regulation, augmented mitochondrial biogenesis, and enhanced mitochondrial functions may contribute to arsenic-induced cell proliferation. Targeting mitochondrial biogenesis may help treat arsenical cancers at the stage of cell proliferation.

Differential effects of arsenic on cutaneous and systemic immunity: focusing on CD4+ cell apoptosis in patients with arsenic-induced Bowen's disease.

Bowen's disease (BD), a carcinoma in situ of the skin, has been identified as an early lesion in arsenic carcinogenesis. Patients with arsenic-induced Bowen's disease (As-BD) showed both cutaneous and systemic immune dysfunctions. We set out to evaluate the interactions between keratinocytes and lymphocytes in the context of As-BD carcinogenesis. Our results showed that As-BD lesions demonstrated a significant dermal CD4+ cell, an essential regulator of proper tumor immunity, undergoing apoptosis. In addition, it was found that the As-BD patients have lower percentage of peripheral CD4+ cells as compared with control subjects. However, the CD4+ cells from As-BD patients were less susceptible to arsenic-induced apoptosis, due to reduced tumor necrosis factor receptor 1 expression. Interestingly, arsenic was found to induce Fas expression on CD4+ cells and increase the soluble Fas ligand (sFasL) production from keratinocytes. This sFasL-containing keratinocyte supernatant was able to induce comparable CD4+ cell apoptosis for both patients and controls. Using immunofluorescent staining, increased FasL was observed in keratinocytes of As-BD lesions and Fas was expressed among infiltrating CD4+ cells. Our findings suggested that systemically, the percentage of CD4+ cells was decreased in the peripheral blood of As-BD patients. These residual CD4+ cells were less susceptible to arsenic-induced apoptosis. However, once infiltrated into the As-BD lesions, the selective CD4+ cell apoptosis might be mediated by FasL from keratinocytes. This additional tumor-anti-immune phenomenon present in the cutaneous environment provides a reasonable explanation for frequent occurrence of arsenic cancers in the skin.

Enhanced gastrointestinal motility with orally active ghrelin receptor agonists.

The orexigenic peptide ghrelin has been shown to have prokinetic activity in the gastrointestinal (GI) system of several species, including humans. In this series of experiments, we have evaluated the prokinetic activity of novel, small-molecule ghrelin receptor (GhrR) agonists after parenteral and peroral dosing in mice and rats. Gastric emptying, small intestinal transport, and fecal output were determined after intraperitoneal and intracerebroventricular dosing of GhrR agonists, using ghrelin as a positive control. These same parameters were evaluated after oral gavage dosing of the synthetic agonists. Regardless of dose route, GhrR agonist treatment increased gastric emptying, small intestinal transit, and fecal output. However, fecal output was only increased by GhrR agonist treatment if mice were able to feed during the stimulatory period. Thus, GhrR agonists can stimulate upper GI motility, and the orexigenic action of the compounds can indirectly contribute to prokinetic activity along the entire GI tract. The orexigenic and prokinetic effects of either ghrelin or small-molecule GhrR agonists were selective for the GhrR because they were absent when evaluated in GhrR knockout mice. We next evaluated the efficacy of the synthetic GhrR agonists dosed in a model of opiate-induced bowel dysfunction induced by a single injection of morphine. Oral dosing of a GhrR agonist normalized GI motility in opiate-induced dysmotility. These data demonstrate the potential utility of GhrR agonists for treating gastrointestinal hypomotility disorders.