A Sensitive Sensor Cell Line for the Detection of Oxidative Stress Responses in Cultured Human Keratinocytes

Abstract

In the progress of allergic and irritant contact dermatitis, chemicals that cause the generation of reactive oxygen species trigger a heat shock response in keratinocytes. In this study, an optical sensor cell line based on cultured human keratinocytes (HaCaT cells) expressing green fluorescent protein (GFP) under the control of the stress-inducible HSP70B' promoter were constructed. Exposure of HaCaT sensor cells to 25 μM cadmium, a model substance for oxidative stress induction, provoked a 1.7-fold increase in total glutathione and a ∼300-fold induction of transcript level of the gene coding for heat shock protein HSP70B'. An extract of Arnica montana flowers resulted in a strong induction of the HSP70B' gene and a pronounced decrease of total glutathione in keratinocytes. The HSP70B' promoter-based sensor cells conveniently detected cadmium-induced stress using GFP fluorescence as read-out with a limit of detection of 6 μM cadmium. In addition the sensor cells responded to exposure of cells to A. montana extract with induction of GFP fluorescence. Thus, the HaCaT sensor cells provide a means for the automated detection of the compromised redox status of keratinocytes as an early indicator of the development of human skin disorders and could be applied for the prediction of skin irritation in more complex in vitro 3D human skin models and in the development of micro-total analysis systems (μTAS) that may be utilized in dermatology, toxicology, pharmacology and drug screenings.

Identification of HSP70-inducing activity in Arnica montana extract and purification and characterization of HSP70-inducers

“…Thus, we selected A. montana extract and tried to identify active molecules within this extract that had HSP70-inducing ability. Induction of HSP70 by A. montana extract in HaCaT keratinocytes was also reported recently [47]. We identified eight sesquiterpene lactones in fractions that showed HSP70-inducing activity (AM-1-8).…”

“…Thus, we consider that the anti-inflammatory activity of A. montana extract is likely to be mediated by an HSP70-independent mechanism. It should be noted that induction of HSP70 by helenalin in HaCaT keratinocytes was reported recently [47].…”

Medaka (Oryzias latipes) Embryo as a Model for the Screening of Compounds That Counteract the Damage Induced by Ultraviolet and High-Energy Visible Light

“…Considering the previous results, medaka embryos could be an alternative economic platform for screening of compounds capable of counteracting the harmful effects of UV radiation and HEV light. To evaluate this possibility, we compared the effect of three well-known antioxidant compounds in medaka embryos and HaCat culture, as this last model is a typical platform used for compound screening [ 38 , 39 , 40 ]. The selected antioxidant compounds were vitamin C (ascorbic acid), vitamin A (retinoic acid), and resveratrol [ 41 , 42 , 43 , 44 , 45 ].…”

Biomimetic Receptors and Sensors

“…Keratinocyte cells in epidermis act as a barrier against environmental threat such as pathogens, or chemical and physical influences, respectively [ 12 ]. In case of pathogen invasion, keratinocytes will generate mediators, which attract leukocytes to this site and defend the body against invaders.…”