Effects of Arsenic on Cytokeratins in Foetal Stem Cells

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Arsenic is an environmental contaminant and potential human carcinogen. Foetal stem cell are key target in transplacental carcinogenesis. CK10 expresion increased after transplacental treatment. CK14 expression declined after transplacental treatment. But in invitro treatment CK1 expression increased many fold. CK13 expresion increased upto 4 folds and CK 15 expression increased upto 7 folds. Invitro arsenic treatment can slow down epidermal stem cell exit into differentiation pathway.



Inorganic arsenic is naturally occuring drinking water contaminant and potential carcinogen, affecting millions of people worldwide. (IARC,2004; NRC 2001). Inorganic arsenic is generally present in arsenite or arsenate form and is very readily found in contaminated ground waters. Skin is the major target site for arsenic and it is considered a human skin carcinogen (Straif et al, 2009). At very high exposure, it may cause squamous cell carcinoma (IARC,2004). Skin is most sensitive tissue to chronic arsenic exposure. In human chronic exposure can lead to hyperpigmentation, hyperkeratosis, basal cell carcinoma and squamous cell carcinoma (IARC,2004 ; NRC2001). Arsenic also has transplacental activity (Smith et al 2006). It is readily transferred from mother to foetus via the placenta. Foetal stem cell are supposed to be prime target in transplacental activity (Anderson et al, 2000).

Cyto keratins are major structural proteins of epidermis (Coulombe and Omary, 2002). Expression of keratins is strongly linked to tissue specific and differentiation specific manner. Cytokeratin expressing cells found in epidermis are called keratinocyte. They are derived from a pool of multipotent stem cell found in the epidermis. Basal cell layer and hair follicle bulges are the stem cell niches. Cytokeratin are intermediate filament and are major structural proteins. There aberrant expression can be linked to various diseases. Alteration in expression of cytokeratin can lead to skin pathological conditions, such as, hyperkeratosis or skin cancers (Tseng et al, 1968). There is a strong relation between cytokeratin expression and stem cells. cytokeratin 5, 14 and 15 are all epidermal markers for stem cell.(Gerdes and Yuspa,2005; Lyle et al., 1998; Liu et al., 2003). Cytokeratin 10 are markers for cell differentiation and are found expressed in differentiated cell than in stem cells.

Arsenic treatment causes alteration in expression of cytokeratin in stem cells. Some studies report that invitro treatment of stem cell, can slow down the exit of epidermal stem cell into differentiation pathways, thus disturbing the population dynamics (Patterson et al, 2005, 2007). The present study involves ex vivo treatment of animal with sodium arsenite and hence can lead to different results.

The present study has been conducted to see the expression of cytokeratin in stem cells following exposure to arsenic in vivo, ex vivo or in vitro.


Cytokeratin or Keratins

Keratins are intermediate filament proteins, specific for epithelia, that are expressed in tissue specific and differentiation specific manner. (Moll et. al.,1982).

Keratins are specific for epithelial tissue.( Coulombe and Omary, 2002). They are divided into two subtypes : Type I is of lower molecular weight and acidic (K9-K28) and type II is of higher molecular weight and basic in nature (K1–K8 and K71–K74). . (Schweizer et al., 2006). They are divide into acidic and basic type on the basis of properties like molecular weight and isoelectric point (Hesse et al., 2004). Cytokeratins are obligatory heteropolymers and one of each type is assembled in 1:1 ratio for proper filament formation. (Coulombe and Omary, 2002; Herrmann and Aebi, 2004).

Keratins perform several functions some of which are that they modulate processes like apoptosis (Gilbert et al 2004), osmolarity (Toivola et al 2004) and protein synthesis (Kim et al 2006). They regulate these processes by modulating signaling pathways. K14 modulate TNFα signaling/apoptosis (Inada et al.,2001). K10 modulate PI-3K pathway/ cell proliferation (Paramio et al., 2001) , TNFα signaling/ apoptosis (Chen et al., 2006) and Notch signalling pathway/ differentiation( Santos et al .,2005). K17 regulate TNFα signalling/apoptosis (Tong et al., 2006) and mTOR pathway/ protein synthesis (Kim et al., 2006).K18 regulate cell cycle (Kuno et al 2002). K6/16 regulate Cell proliferation (Paladini et al., 1998). K8/18 regulate Fas signalling/apoptosis (Gilbert et al .,2001).

Keratin 10 is type I keratin and expressed by KRT10 gene. (Schweizer et al 2006). These are expressed in suprabasal layers of keratinized stratified epithelia, foot sole epidermis, anal canal epithelium. They are markers of cellular differentiation. (Magin et al 2005) and many squamous cell carcinoma which have been obtained from non keratinizing stratified epithelia. (Vaidya et al 1989; Vigneshwaran et al 1989). It is expressed in combination with K1. K1 is expressed in Epidermis and foot sole epidermis. Like K10 it is also a marker for cell differentiation. (Moll et al.,1982).Many squamous cell carcinoma obtained from non-keratinizing stratified epithelia also express this pair of keratin (Vaidya et al 1989; Vigneshwaran et al 1989).

Keratin 14 is also type I keratin intermediate filament and expressed by KRT14 gene. (Schweizer et al 2006). They are expressed predominantly in basal layer of epidermis (Magin et al 2005). They are also expressed in outer root sheath of hair follicle, sebaceous gland, anal canal epithelium. It is expressed along with K5. It’s main function is to provide structural support to basal layer of stratified epithelia. Mutation in either of the pair leads to development of a disease epidermolysis bullosa.( Coulombe et al ., 2009). K14 is marker for epidermal stem cell in skin. (Metallo et al., 2010 ; Bickenbach 2005). It is expressed with K5. k5 is expressed in epidermis, foot sole epidermis,outer root sheath of hair follicle,sebaceous gland, cornea, but predominantly in basal cell layer (Magin et al 2005) . It is also stem cell marker for skin.

In stratified epithelia, the basal layer cells are highly proliferating and express keratin 5/14. As they move to upper layer they get more differentiated and express keratin 1/10. K14 is thus marker for epidermal stem cells while K10 is marker for cell differentiation.

Keratin 6/16 are marker of cell proliferation.(Moll et al.,1982). They are expressed in suprabasal cells and stratified epithelia having hyperproliferation or abnormal differentiation, such as psoriasis and cancer. (McGowan and Coulombe 1998).

K8 are expressed in colon, small intestine, hepatocytes and gall bladder epithelium. K8 has role in cell transformation/ differentiation (Moll et al., 1982; Magin et al, 2005).

K17 is expressed in outer root sheath of hair follicles, amnion and trachelal epithelium, mammary gland ducts. It regulates growth and proteins synthesis. (Magin et al., 2005; Kim et al.,2006).

K18 is expressed in Hepatocytes, colon, small intestine (mucosa), gall bladder epithelium, excrine sweat glands. It has role in transformation and differentiation. (Moll et al., 1982; Magin et al., 2005).

K15 is an epidermal stem cell marker. and so is K19. It is expressed in bulge region of human hair follicles (Abbas et al.,2009;Braun et al.,2003) outer root sheath of the follicle of human hair, the epidermal basal cell layer and eccrine glands (Kloepper et al.,2008). CK15 expression in the basal cell layers of the hair follicle, which is mitotically active tells that CK15 might have a role in regulating keratinocytes differentiation.(Abbas et al.,2009).

K19 is also an epidermal stem cell marker. It was earlier thought to occur only in bulge region of hair follicle, but now it has been found in the layer tmst of the outer root sheath in the human hair follicle bulge, the outer root sheath near and far to the bulge.(Abbas et al.,2009). In vivo and in vitro studies have lead us to infer that CK19 might be important in the deciding of stem cells to differentiation and an epidermal cell fate. (Morris.,2004).

Stem cells

Zygote and each cell of morula stage of embryo is totipotent, that is, it is capable of generating entire organism. Blastocyst is pluripotent, in which the cells of inner cell mass can form ectoderm, endoderm ad mesoderm. These are embryonic stem cell. But the stem cell found in adult tissue, among the differentiated cell, is only multipotent. It is adult stem cell. They can renew themselves and differentiate to produce cell of tissue in which it is found. This will enable organism to repair the the tissue, which is critical for survival.( Wobur and Boheler 2005).

Stem cell differ from non stem cells in two ways. Stem cell has unlimited capacity for self renewal, which enable them to undergo continuous cell division throughout life of an organism. One of the most distinguishing features of stem cells is their slow-cycling nature. They divide infrequently and have a long cell cycle.

Stem cell can undergo asymmetric cell division to produce two daughter cells with different differentiation capacities. One is the stem cell (undifferentiataed) while the other enters irreversible differentiation pathway. The one which can enter differentiation pathway is called progenitor cell or transit-amplifying cell.

These cells have finite self-renewal capacity and are committed to differentiation following few cell divisions. Transit-amplifying cell never regain stem cell characteristics. (Hall and watt, 1989).

Skin is composed of inner dermis of mesodermal origin and outer epidermis of ectodermal origin. Epidermis is stratified squamous epithelia composed primarily of keratinocytes. These are keratin expressing cells and are generated from multipotent stem cell in skin. These stem cell are located in basal cell layer and hair follicular bulge area.(Poojan and kumar, 2010). In tissues

stem cell are located in specialized area called stem cell “niche”, which consist of supporting cell and extracellular factor which are compatible for maintaining stem cell characters. In skin basal cell layer and hair follicle bulge area are the niche.


fig 2.1 Compartmentalization of growth and differentiation in the epidermis.

Epidermal stem cells residing in specific niches are self renewing, and give rise to transit amplifying which are responsible for proliferative activity in the basal layer. Basal layer keratinocytes express keratin 5 and keratin 14, but as they detach from the basement membrane and migrate into the first suprabasal layer in the spinous layer, they irreversibly exit the cell cycle and switch to CK 1 and CK 10.


Arsenic is a metalloid and an environmental pollutant and multisite human carcinogen.It is common drinking water contaminant. (IARC, 2004 ; NRC 2001).The metalloid arsenic has long

been recognized to be associated with toxicity.The main route of exposure is through contaminated drinking water. Arsenic, besides uniform distribution, often accumulates in hair, nails and skins (Hutton, 1987; Fatur, et al, 2006).Inorganic arsenic is multisite human human carcinogen found in high levels in contaminated drinking water. Inorganic arsenic exist as trivalent (arsenite) and pentavalent (arsenate) from.(Tokar et al, 2011).

Sources of exposure

Maximum arsenic exposure via contaminated drinking water. Ground water is contaminated through high level of aresinc in ground water. Due to use of deep tubewells in ganges delta, arsenic poisoning is a problem here, affecting a large number of people. Also, hydraulic fracturing, a mining technique and other mining activities mobilizes arsenic in ground water and aquifers. It is due to enhanced transportation of methane and resulting change in redox condition and inject fluid containing additional arsenic. According to a study conducted in 2007, over 137 million people residing in more than 70 countries are affected by arsenic poisoning of drinking water. Ground water contamination by arsenic is found in many countries of the world, including USA. (Murcott, S. (2012)

Bioavailability of arsenic

Ingested arsenic is poorly absorbed and eliminated primarily through urine. (IARC, 2004). In small intestine it is absorbed via proton gradient. Both organic and inorganic arsenic are not so well absorbed by skin. After ingestion, inorganic arsenic apperars promptly in circulation. It binds to haemoglobin and within 24 hours it is found mainly n spleen, lungs, kidneys, liver and skin.It accumulates in skin as skin contains abundance of protein with sulphydryl group, with

which it readily reacts. Arsenic can cross placental barrier and is found in cord blood of foetus and mother. (Flora, Pachauri, et al., 2011).

Skin toxicity of Arsenic

Skin is important target site for arsenic carcinogenesis.(IARC 2004, 2009). Skin is most sensitive tissue to chronic arsenic exposure. Chronic exposure to arsenic in human, lead to epidermal lesions, like hyperkeratosis, hyperpigmentation, squamous cell carcinoma and basal cell carcinoma. (IARC 2004, NRC 2001). SCC is the most common cancer in case of cronic exposure to arsenic.(IARC2004, NRC, 2001). Arsenic has transplacental carcinogenic activity in mice( Waalkes et al., 2007) and humans (Smith et al.,2006) . Foetal stem cell are key targets in transplacental activity.(Anderson et al., 2000).

Fetal Stem cell population in skin are key targets in arsenic carcinogenesis,during in utero exposure. (Waalkes et al, 2008). Studies on human skin cancer models in vitro have indicated that slow down epidermal stem cell conversion into differentiated cell, thus disturbing the population dynamics. (Patterson et al,.2005, 2007). So arsenic increases the proportion of epidermal stem cell,in culture, leading to conclusion that this might be target cell population for invivo carcinogenesis. (Patterson et al, 2005, 2007).

According to current studies, human skin cancer models in vitro have indicated that arsenic exposure can slow down epidermal stem cell differentiation, thus distorting the stem cell population dynamics.(15,16). Actually, arsenic increases the proportion of stem cells in culture, leading us to as hypothesis, that these cells might be main target in invivo carcinogenesis.(15,16).If we accept that cancer is a disease by stem cell dysregulation(5), an arsenic induced increase inskin stem cell number invivo might remain quiescent and yet facilitate cancer after some stimulation. This has not been tested directly.

In vitro treatment of arsenic yielded different results. There was alteration in cytokeratin gene expression. CK 1 expression increased upto many fold. CK1 is marker for hyperkeratosis. CK13 increased upto 4 fold, which is a marker for dermal cancer progression. CK 15 upto 7 fold, which is a stem cell marker, indicating increase in population of stem cells like cells.



The present study was carried out on the topic “Cytokeratin expression in stem cell following exposure to arsenic.” The study entails that aberrant cytokeratin expression is seen after exposure to arsenic. CK 10 expression increased after exvivo treatment . CK 10 are markers of cell differentiation. Cells were losing stem cell property and were approaching differentiation. CK 14 expression declined with arsenic exposure exvivo. CK 14 are epidermal stem cell marker. CK 1 expression increased many folds during in vitro arsenic treatment. CK 1 are marker for hyperkeratosis. CK 13 expression increased 4 fold due to in vitro arsenic treatment. CK 13 is marker of cancer progression. CK 15 expression increased 7 fold during in vitro treatment.Arsenic can slow epidermal stem cell entering into differentiation pathway when given a invitro treatment.