3.1. l-Ergothioneine Increases the Reduced Glutathione (GSH) Level
The tripeptide glutathione (GSH) is the most abundant intracellular non-protein thiol. It is present predominantly in a reduced form (GSH), which is the biologically active form. Over the years, a great deal of information has been gathered on the role of GSH in maintaining the intracellular reduction–oxidation (redox) environment including antioxidant defense via direct interaction with reactive oxygen/nitrogen species (ROS/RNS) or via activities of detoxification enzymes like GSH peroxidases and GSH-
S-transferases [
16]. In our study we tested the effect of
l-ergothioneine on the reduced glutathione (GSH) level in the KB cell line using the GSH/GSSG-412 colorimetric assay. Our studies showed that
l-ergothioneine at the 20 µM concentration (chosen according to our previous studies [
17]) increased the reduced glutathione (GSH) level both in control and UVB irradiated KB cells (
Table 2). Changes between GSH level in control compared with GSH level after treatment with
l-ergothioneine, both in cells exposed and unexposed to UVB, were statistically significant. The UVB light was chosen for the irradiation of KB cells since it is the light of the UVB spectrum that is for the most part absorbed by the epidermis
in vivo [
18]. UVB may also damage epidermal cells both directly and indirectly by inducing the formation of reactive oxygen species,
i.e., the superoxide anion [
19]. Thus, it was deemed necessary to study the antioxidative effect of EGT on UVB irradiated epidermis.
Table 2.
Quantitative determination of glutathione (GSH) level in KB cells.
Table 2.
Quantitative determination of glutathione (GSH) level in KB cells.
KB cells | µmol GSH/µg protein |
---|
−UVB | +UVB (300 J/m2) |
---|
control | 6.07 ± 0.15 | 3.03 ± 0.02 |
20 µM l-ergothioneine | 15.37 ± 0.13 | 4.65 ± 0.06 |
Our results are in line with numerous studies demonstrating that
l-ergothioneine is an integral component of the cellular antioxidant defense system. It scavenges hydroxyl and peroxynitrite radicals as well as the superoxide anion radical and singlet oxygen.
In vitro tests have also demonstrated that EGT also has strong copper chelating ability—it potentially inhibits tyrosinase activity [
20,
21,
22].
Moreover,
in vitro tests have demonstrated that in cultured fibroblasts EGT suppressed the tumor necrosis factor α (TNF α) up-regulation induced by UVB irradiation. In addition in fibroblasts exposed to UVA, EGT suppressed the expression of matrix metalloproteinase 1 (MMP 1) protein [
23]. EGT also inhibited nuclear factor kβ (NF kβ) activation and limited the transcriptional activation of the gene for interleukin 8 (IL-8)—a chemotaxin which might account for neutrophil recruitment [
24].
It has been demonstrated that EGT is a more powerful antioxidant than either coenzyme Q(10) or idebenone due to its relatively greater efficiency in directly scavenging free radicals and in protecting cells from UV-induced ROS [
25].
Recently, a high specific transporter for EGT (ETT) was identified in mammalian tissues, which explains the high tissue levels of EGT and implies a physiological role. Depletion of ETT leads to augmented oxidative stress and cell death. ETT is highly concentrated in the plasma membrane and mitochondria [
11].
3.2. l-Rrgothioneine Shows Protective Properties against the Induction of a Photoaging-Associated mtDNA “Common Deletion”
One of the best described mt deletions, the 4977 bp “common deletion”, is known to be involved in photoaging of the skin. Intraindividual comparison studies have revealed that the “common deletion” is increased up to 10-fold in photoaged skin, as compared with sun-protected skin of the same individuals [
7]. The objective of our study was to evaluate the effect of
l-ergothioneine on the occurrence of mtDNA “common deletion” in UV-irradiated human primary fibroblasts obtained from various donors. For the experiments on dermal cells only UVA irradiation was performed, since the great majority of UVB rays is absorbed by the epidermis and hence never reaches the dermal level [
18]. UVA is known to cause mainly indirect damage to cellular structures and nucleic acids by inducing the formation of radical oxygen and nitrogen species but may also be responsible for single- and double-strands breaks in DNA [
19]. Thus, exposure to UVA may be one of the main causes of mtDNA mutations including mtDNA deletions [
26]. In our experiments, in fibroblasts which were not exposed to UVA we did not observe the “common deletion” after DNA isolation nor during 3 weeks of culture (
Figure 2, lanes 9, 11 and 13). When we incubated fibroblasts with
l-ergothioneine we also observed no “common deletion” PCR product (
Figure 3, lanes 9, 11 and 13). To summarize this part of our study: first of all, cultured fibroblasts isolated from various donors did not initially show the “common deletion”. Secondly,
l-ergothioneine, itself, had no influence on the presence of the “common deletion”.
Figure 2.
Representative PCR analysis of the presence of the “common deletion” in a culture of human fibroblasts incubated with and without UVA exposure, without the presence of l-ergothioneine. M, molecular weight marker; C, control PCR reaction, confirming the presence of mtDNA; 1, fibroblasts after 1 week with UVA exposure; 3, fibroblasts after 2 weeks with UVA exposure; 5, fibroblasts after 3 weeks with UVA exposure; 7, positive control—DNA from a patient with a previously confirmed presence of “common deletion”; 9, fibroblasts after 1 week without UVA exposure; 11, fibroblasts after 2 weeks without UVA exposure; 13 fibroblasts after 3 weeks without UVA exposure.
Figure 2.
Representative PCR analysis of the presence of the “common deletion” in a culture of human fibroblasts incubated with and without UVA exposure, without the presence of l-ergothioneine. M, molecular weight marker; C, control PCR reaction, confirming the presence of mtDNA; 1, fibroblasts after 1 week with UVA exposure; 3, fibroblasts after 2 weeks with UVA exposure; 5, fibroblasts after 3 weeks with UVA exposure; 7, positive control—DNA from a patient with a previously confirmed presence of “common deletion”; 9, fibroblasts after 1 week without UVA exposure; 11, fibroblasts after 2 weeks without UVA exposure; 13 fibroblasts after 3 weeks without UVA exposure.
We were able to induce the “common deletion” using repetitive UVA irradiation in cultured human fibroblasts derived from two donors (
Figure 2, lanes 1, 3 and 5) suggesting that there is an individual susceptibility to the induction of CD, at least in our
in vitro model. Due to the fact that our “common deletion” analysis was performed on fibroblasts isolated from four donors, this requires further studies. When fibroblasts were irradiated and co-incubated with 20 µM
l-ergothioneine we did not observe the “common deletion” even after 3 weeks with UVA exposure, suggesting that
l-ergothioneine may prevent the formation of this particular damage in mtDNA (
Figure 3, lanes 1, 3 and 5). Our results are in accordance with the previous studies where repetitive exposure of keratinocytes, fibroblasts or human skin to UVA at physiological doses was found to induce mutations of mtDNA [
7,
27,
28,
29]. Interestingly, it has been shown that in fibroblasts the level of CD is decreasing with prolonged culture, which can be counteracted with uridine supplementation of culture media [
6,
7,
30]. For this reason, uridine supplementation was employed during all CD analyses in this study.
Figure 3.
Representative PCR analysis of the presence of the “common deletion” in a culture of human fibroblasts incubated with and without UVA exposure, in the presence of 20 µM l-ergothioneine. M, molecular weight marker; C, control PCR reaction, confirming the presence of mtDNA; 1, fibroblasts after 1 week with UVA exposure; 3, fibroblasts after 2 weeks with UVA exposure; 5, fibroblasts after 3 weeks with UVA exposure; 7, positive control—DNA from a patient with a previously confirmed presence of “common deletion”; 9, fibroblasts after 1 week without UVA exposure; 11, fibroblasts after 2 weeks without UVA exposure; 13, fibroblasts after 3 weeks without UVA exposure.
Figure 3.
Representative PCR analysis of the presence of the “common deletion” in a culture of human fibroblasts incubated with and without UVA exposure, in the presence of 20 µM l-ergothioneine. M, molecular weight marker; C, control PCR reaction, confirming the presence of mtDNA; 1, fibroblasts after 1 week with UVA exposure; 3, fibroblasts after 2 weeks with UVA exposure; 5, fibroblasts after 3 weeks with UVA exposure; 7, positive control—DNA from a patient with a previously confirmed presence of “common deletion”; 9, fibroblasts after 1 week without UVA exposure; 11, fibroblasts after 2 weeks without UVA exposure; 13, fibroblasts after 3 weeks without UVA exposure.
Protection against the induction of a photoaging-associated mtDNA mutation has been also previously shown for two other widely applied cosmetic actives—β-carotene and genistein [
31,
32].
Our results demonstrated that l-ergothioneine may protect skin cells against some UV-induced damage; in particular against the occurrence of mitDNA “common deletion”. Further studies are required to assess the full-spectrum of l-ergothioneine’s protective activity. We conclude that l-ergothioneine, as an active compound of cosmetic products, shows considerable promise as an effective agent against the aging process. On the other hand, we have shown that in cosmetology, the detection of mtDNA deletions could be a useful tool to assess the effect of active ingredients on UV-irradiated human skin cells.