暴露三氧化二砷及UVB干擾角質細胞增生和分化之機制探討

Abstract

皮膚主要功能為隔離體內與外在環境，而皮膚障蔽主要是由最外層的角質層所構成。砷廣泛的存在於自然環境中，其主要影響的標的器官包括皮膚。砷又可分為有機砷及無機砷，文獻指出在所有的砷化合物以無機的三價砷最具毒性，因此過去實驗多以三價砷為探討對象。流行病學顯示長期經由飲水暴露砷，會導致皮膚色素沉著、過度角質化並增加罹患癌症之風險，包括皮膚癌、肝癌、肺癌以及膀胱癌。在砷誘導的癌症中，又以皮膚癌最為常見。在流行病學研究中顯示太陽光為另一促使皮膚癌生成之危險因子，尤其是紫外光中的中紫外線(Ultraviolet B, UVB)對於皮膚之致癌性最為顯著。砷與UVB皆會對人體皮膚具有致癌性，且兩者具有共致癌性。根據先前文獻指出在動物實驗中僅長期飲用含砷之水並不會導致皮膚癌生成，其必須要與UVB同時暴露才會增強皮膚致癌性。在一般情況下，砷的暴露途徑會經由食入，吸入或經皮暴露，但至今鮮少研究去探討職業上經皮暴露砷與皮膚癌之關聯性，然而近來有流行病學研究顯示在職業場所上經皮共同暴露砷與UVB會增加罹患皮膚癌之風險。本實驗目的是想要建立經皮接觸砷之方式來探討其與UVB之傷害，並釐清其共致癌性的相關機制。 實驗利用八週大雄性C57BL/6小鼠，在背皮照射100 mJ/cm2 UVB後立即塗抹上0.002% 三氧化二砷，在皮膚組織上發現共同暴露UVB和三氧化二砷顯著性的增加表皮厚度。而為了確定此現象是否是影響了角質細胞的增生與分化，實驗利用免疫螢光染色來確認具有增生功能的基底層(K5)和具有分化功能的上基底層(K10)之變化。結果顯示共同暴露UVB及三氧化二砷在48及72小時，K5和K10之表現顯著增加，這也代表著共同暴露UVB和三氧化二砷會刺激細胞增生並干擾細胞分化。除此之外，細胞增生標記Ki67和DNA損傷標記 gamma-H2Ax 表現之細胞數在共同暴露下48及72小時會協同性的增加。根據過去研究顯示表皮屏障功能被破壞，會誘發細胞激素的產生，因此接下來實驗利用細胞激素蛋白表達微陣列來探討在共同暴露UVB和三氧化二砷後表皮釋出細胞激素之變化。結果顯示UVB和三氧化二砷之暴露會參與IL-1R/TLR之訊息傳遞並影響IL-1beta的分泌。根據上述之結果，IL-1beta為此路徑之重要激素其會正向回饋結合至IL-1R，因此實驗接下來利用IL-1R1-/-小鼠來探討UVB和三氧化二砷協同性傷害是否是經由IL-1R之訊息傳遞所引起。結果顯示和野生型小鼠相比IL-1R-/-小鼠共同暴露UVB和三氧化二砷後，其表皮擁有正常的增生和分化，而Ki67和 gamma-H2Ax 表現之細胞數也顯著減少。綜合以上，本實驗建立了短期經皮接觸三氧化二砷之方式來探討與UVB之傷害之模式，並發現IL-1R之訊息傳遞在此傷害模式扮演著重要角色，而抑制此路徑可以降低UVB與三氧化二砷對表皮的協同性傷害。在未來會進一步探討此動物模式是否在長期下可誘導癌症之生成。

The primary function of the skin is to protect the body from unwanted environmental influences. The main barrier of the skin is the outermost layer of the skin, the stratum corneum. Arsenic existed ubiquitously in the environment in both inorganic and organic forms, and skin is among the major target organs affected by arsenic exposure. The toxicity of trivalent arsenic is greater than other arsenic compounds. From epidemiologic studies, there is strong evidence that exposure to inorganic arsenic is associated with hyperpigmentation, hyperkeratosis in the skin and increased risk of carcinogenesis in various organs, including skin, lung, liver and urinary tract. Among these cancers, skin cancers are the most common. Epidemiologically, exposures to sunlight is shown to be the main cause of skin cancers. It has been shown that ultraviolet B (UVB) irradiation induces skin cancer in animals more effectively than other spectrum of sunlight. Arsenic and UVB, both as human skin carcinogens, may act together as co-carcinogens. Interestingly, arsenic alone does not seem sufficient to induce skin cancer in animals. In mouse skin, it appears that arsenic acts as a co-carcinogen by enhancing UV irradiation-induced skin cancer when it is simultaneously added to drinking water. In general, the primary routes of arsenic entry into the body are via ingestion, inhalation and skin exposure. Up to date, there are few studies examining potential links between arsenic skin exposure at work and skin carcinogenesis. However, recent epidemiological studies showed that workplace coexposure to arsenic and sunlight may increase the risk of skin cancer. This study is aimed at establishing an animal model to investigate the effect of coexposure of skin to arsenic and UVB and to examine the potential Imechenisms of the co-carcinogenicity of UVB and arsenic. The 8week-old male C57BL/6 mice were used for this in vivo model, and 0.002% arsenic trioxide (As2O3, As) was applied immediately onto the skin after exposure to 100 mJ/cm2 UVB. Histopathologically, the epidermis exhibited a dramatic increase in thickness at 48h and 72h following co-exposure to arsenic trioxide and UVB. To evaluate the effects of UVB and arsenic trioxide on the proliferation and differentiation of epidermal keratinocytes, immunofluorescence staining was performed. Histological images of immunofluorescence staining revealed expansion of the proliferating basal layer (K5) and suprabasal layer (K10) after co-exposure to arsenic and UVB at 48h and 72h. This shows that co-exposure to arsenic and UVB may stimulate cell proliferation of the basal layer and disturb the differentiation. In addition, the number of Ki67 and gamma-H2Ax positive cells was also increased at 48h and 72h According to previous studies, epidermal barrier perturbation can induce a cytokine cascade. In this study, a cytokine antibody array was used to evaluate the profiles of the epidermal cytokines released following UVB and arsenic trioxide co-exposure. We revealed increased IL-1beta secretion. We then used mice that were deficient in IL-1R to assess the contribution of IL-1R signaling to the pathological changes. Compared to wild-type mice, IL-1R deficient mice exhibited normal epidermal histology and their Ki67 and gamma-H2Ax cell numbers were significantly reduced.In summary, our study provides a short-term animal model that illustrates the effect of co-exposure to arsenic trioxide and UVB and reveals an important role of IL-1R signaling in the associated pathological changes. The synergistic damage to the epidermis caused by co-exposure to arsenic trioxide and UVB can be reduced by inhibiting IL-1R signaling. In the future, we will examine the potential carcinogenic effect of long-term co-exposure to arsenic trioxide and UVB.