Evaluation of the skin sensitization potential of chemicals in THP-1/keratinocyte co-cultures

Abstract

Many attempts have been made to develop in vitro sensitization tests that employ dendritic cells (DCs), DC-like cell lines or keratinocytes. The aim of the present investigation was to establish a co-culture of THP-1 cells and keratinocytes for evaluation of skin sensitization potential of chemicals. Co-cultures were constructed by THP-1 cells cultured in lower compartments and keratinocytes cultured in upper compartments of cell culture inserts. After 24 h exposure to sensitizers (2, 4-dinitrochlorobenzene, p-phenylenediamine, formaldehyde, nickel sulfate, isoeugenol and eugenol) and non-sensitizers (sodium lauryl sulfate, benzalkonium chloride and lactic acid), the expression of CD86 and CD54 on THP-1 cells were evaluated by flow cytometry, and cell viabilities were determined. The sensitizers induced the augmentation of CD86 and CD54 expression, but the non-sensitizers had no significant effect. Compared with mono-cultures of THP-1 cells, the augmentation of CD86 and CD54 could be detected even at a non-toxic concentration of sensitizers in THP-1 cell/keratinocyte co-cultures. Moreover, isoeugenol was distinguished as a sensitizer in co-cultures, but failed to be identified in mono-cultures. These results revealed that the co-cultures of THP-1 cells and keratinocytes were successfully established and suitable for identifying sensitizers using CD86 and CD54 expression as identification markers.

In vitro detection of chemical allergens: an optimized assay using mouse bone marrow‐derived dendritic cells

“…This conclusion has since been challenged, on the basis of misinterpretation of these clinical tests owing to the strong irritant potential of this substance . BC is therefore commonly used in studies as an irritant non‐sensitizer . However, recent studies have shown that the BC could, in fact, be a contact sensitizer .…”

The role of transcription factor Nrf2 in skin cells metabolism

“…Xenobiotics strongly sensitizing skin such as formaldehyde, eugenol, or dinitrochlorobenzene elicit the skin’s defences through Nrf2 activation. As a result of covalent links between these compounds and the cysteine residues in Keap1, Nrf2 dissociates from Nrf2-Keap1-Cul3 complex and consequently begins the ARE-dependent gene expression [ 10 , 78 ]. Many chemopreventive phytochemicals are known to activate Nrf2 either by oxidative or covalent modification of its cytosolic repressor—Keap1 or by phosphorylation of Nrf2 [ 15 ].…”

The Overt and Hidden Use of Animal-Derived Products in Alternative Methods for Skin Sensitisation: A Systematic Review

“…The use of animal-derived trypsin was clearly described in only one of the 50 methods to assess DC response, in which it was used to dissociate keratinocytes from foreskin. 112 As the trypsin manufacturer (Invitrogen Corp., now ThermoFisher Scientific) was mentioned in this case, it was possible to identify its porcine origin. Whenever trypsin was used for the isolation of cells from epidermal human or animal skin, 50,51 or for subculturing primary human keratinocytes or keratinocyte lineages in co-culture with DCs, 111,112 most of the analysed methods did not state the source or the manufacturer.…”

“…For example, one experimental study involved the use of bovine pituitary extract, BSA, goat serum, dried skimmed milk and antibodies. 141 In some cases, the origin of the products was unclear: for instance, Cao et al 112 mentioned the use of a human keratinocyte growth supplement, which, after checking the manufacturer’s website, was found to include animal-derived bovine pituitary extract and transferrin.…”