Bioelectrochemistry 126 (2019) 163–171

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Bioelectrochemistryjournal homepage: www.elsevier.com/locate/bioelechem

Cytochrome P450 genes play central roles in transcriptional responseby keratinocytes to a high-voltage alternating current electric ﬁeld
Masayo Aoki ⁎, Noriko M. Matsumoto, Yuri Okubo, Rei Ogawa
Department of Plastic, Reconstructive, and Aesthetic Surgery, Nippon Medical School, 1-1-5 Sendagi, Bunkyo-ku, Tokyo 113-8602, Japana r t i c l ei n f oArticle history:
Received 6 September 2018
Received in revised form 29 November 2018
Accepted 30 November 2018
Available online 11 December 2018
Keywords:
Electric ﬁeld
Keratinocytes
Cytochrome P450
Wound healing
Anti-aging

a b s t r a c t
The endogenous electric ﬁeld (EF) of skin wounds plays an important role in the biological processes that underlie wound healing. Treatments that modulate wound-EFs promote healing. However, the mechanism(s) that
underlie this effect remain unclear. Agilent-based microarrays were used to determine the transcriptomes of
the keratinocyte line HaCaT, normal human dermal ﬁbroblasts, and the human dermal endothelial cell line
HMEC-1 before and after high-voltage alternating current (AC)-EF (14,000 V, 90 Hz) treatment. Thekeratinocytes had the most genes whose transcription was altered by EF. They included the cytochrome P450(CYP) genes CYP1A1 and CYP1B1, HMOX1, EREG, DUSP5, and SLC7A11 (all upregulated), and DOCK8, ABCC6,and CYP26A1 (all downregulated). As shown by transcriptional-network analysis, all three CYP genes played central roles in the EF-induced changes in keratinocyte transcriptome. To the best of our knowledge, this is the ﬁrst
study that demonstrates that CYP genes play a key role in the transcriptional responses of human keratinocytes toEF treatment. Further investigations into the effects of EF on wound healing, aging, and regenerative medicine arelikely to yield promising results.
© 2018 Elsevier B.V. All rights reserved.

1. Introduction
Electric ﬁeld (EF) therapy has evolved as a traditional complementary and alternative medicine in Japan, and was approved for therapeutic use in Japan by the Ministry of Health, Labor and Welfare in 1972. It isbelieved to modulate the nervous, endocrine, and other health systems
and is used to treat headaches, shoulder stiffness, chronic constipation,and insomnia, among many other diseases [1]. Several studies haveshown formally that EF has therapeutic effects. Thus, high-voltage static
EF reduces the subjective pain in active rheumatoid arthritis [1].
Moreover, when a non-healing bone defect in rats was subjected to a
100 Hz pulsing electromagnetic ﬁeld, the defective bone exhibited increased alkaline phosphatase activity, calcium uptake, and bridging
bone production [2].
Currently, the mainstream EF therapy in Japan is a high-voltage alternating EF of 9000–14,000 V and 50–90 Hz. The EFs used in these
therapies do not have a deleterious effect on human health [3]. However, the precise molecular mechanisms that underlie the therapeutic
effects of EF remain unclear. Electroporation and electrofusion, achieved
via pulsed EF, are particularly important in cell biology and biotechnology [4]. However, even in the case of electroporation, which iscommonly used to deliver molecules such as drugs, proteins, and DNA
into cells, the mechanism remains unclear [5]. EF pulses induce neural⁎ Corresponding author.
E-mail address: masayok@nms.ac.jp (M. Aoki).

https://doi.org/10.1016/j.bioelechem.2018.11.014
1567-5394/© 2018 Elsevier B.V. All rights reserved.

electrostimulation, Ca2+ signaling activation, and tissue ablation [6].
Low frequency EF induces heat-inducible heat shock protein 70(HSP70) expression in rat primary ﬁbroblasts [7].
Multiple lines of evidence suggest that EF therapy may promotewound healing. In particular, the human body bears an endogenous
EF: while it plays important physiological roles in the electrical activation of the nervous system and muscles, it also appears to participate
in the dynamic and well-ordered biological processes that underlie
wound healing and scarring [8,9]. For example, Yan et al. showed that
endogenous EF plays a key role in the re-epithelialization of wounds[9]. The putative beneﬁcial role of EF in wound healing is also supported
by Graebert et al., who showed that when the ischemic skin wounds inpreclinical in vivo model rats were subjected to exogenous electricalstimulation, they exhibited improved wound healing [10].
The mechanisms that underlie the beneﬁcial effects of EF on woundhealing remain unclear. However, multiple in vitro and in vivo studies
show that exogenous EFs shape the activities of the cells that play key
roles in wound healing. Wound healing is a dynamic and complex process that consists of several overlapping phases, namely, the hemostasis,inﬂammatory, proliferative, and remodeling phases [11]. Many cell
types are involved in these phases, including keratinocytes, endothelialcells, and ﬁbroblasts [12–14]. Dubey et al. showed that exogenous pulse
EF stimulation affects the metabolism of and multiple biochemicalmechanisms in both keratinocytes and endothelial cells [15]. Moreover,electrical stimulation enhances epithelial cell proliferation [16], promotes dermal ﬁbroblast activity, increases the differentiation of dermal

M. Aoki et al. / Bioelectrochemistry 126 (2019) 163–171

165

Fig. 1. Photograph of the electric ﬁeld treatment setup that was used to stimulate the cultured cells. The voltage sheet was placed in an insulator box so that it efﬁciently produced anelectric ﬁeld. The cell culture dishes were placed cell-side down onto the sheet so that an electric ﬁeld would be established inside the dish.

the gene expression proﬁles of all three cell types. However, they differed markedly in terms of their sensitivity to EF exposure. Speciﬁcally,
HaCaT cells had the largest number of genes whose expression wassigniﬁcantly changed by EF exposure. Thus, keratinocytes appear to beparticularly sensitive to EF. By contrast, the HMEC-1 cells were relativelyresistant (Fig. 2). NHDF cells had an intermediate number of genes thatwere differentially expressed after EF exposure. The genes that exhibited the most marked up or downregulation are listed on Supplementary Tables 1–3.
The genes whose transcription was signiﬁcantly altered by EF in thethree types were categorized according to their gene ontology (GO) into
three categories (Biological Process, Molecular Function, and CellularComponent). The three cell types differed markedly in terms of the distribution of their EF-upregulated genes in the three GO categories. InHaCaT cells, EF treatment signiﬁcantly upregulated 155 genes in the Biological Process GO category; it also signiﬁcantly upregulated 11 genes
in the Molecular Function GO category. By contrast, in HMEC-1 cells,
EF treatment only signiﬁcantly upregulated nine, three, and two genes
in the Biological Process, Molecular Function, and Cellular Component
GO categories; it also only downregulated seven genes in the BiologicalProcess GO category. In NHDF cells, EF treatment signiﬁcantly upregulated 14 and eight genes in the Biological Process and Molecular Function GO categories, respectively (Fig. 3).

Fig. 4 shows the pathways in each cell type that demonstrated significant enrichment in terms of EF-induced component-gene up or downregulation. HaCaT cells had the largest number of pathways that wereinﬂuenced by EF treatment, followed by NHDF cells. The HMEC-1 cells
had the lowest number of affected pathways. A pathway that was significantly affected by EF in all three cell types was not detected. In HaCaTcells, CYP and T cell receptor signaling pathways were signiﬁcantly inﬂuenced in terms of both up and downregulation.
The validity of the microarray results in the three cell types wasconﬁrmed by quantitative RT-PCR of the genes that exhibited extremely strong responsiveness to EF (deﬁned as fold change N2 orN−2). Thus, the effect of EF on the expression of 15, 3, and 7 genes
in HaCaT, NHDF, and HMEC-1 cells, respectively, was tested by RTPCR. In HaCaT cells, the expression of CYP1A1 and CYP1B1 were signiﬁcantly increased by 20.3- and 5.9-fold, respectively. Moreover,HMOX1, EREG, DUSP5, and SLC7A11 expression was increased by3.8-, 2.6-, 1.4-, and 2.0-fold, respectively, while DOCK8, ABCC6, andCYP26A1 were signiﬁcantly downregulated by 0.34-, 0.70-, and0.44-fold, respectively (Fig. 5).
By contrast, in NHDF, none of the 3 tested genes exhibited signiﬁcant
EF-induced changes on RT-PCR. Furthermore, in HMEC-1 cells, only
MGA expression was conﬁrmed to be slightly upregulated by EF exposure (by 1.2-fold) (Fig. 6).

Fig. 2. Changes in the transcriptomes of the three cell types after high-voltage electric ﬁeld treatment. The Venn diagram shows the number of genes in the HaCaT, normal human dermalﬁbroblast (NHDF), and HMEC-1 cells that were (a) upregulated or (b) downregulated after high-voltage electric ﬁeld treatment, and how many of these genes showed the sametranscriptional changes in the three cell types.