Introduction
Oxidative stress is defined as an excessive load of Reactive Oxygen Species (ROS) which cause reversible or persistent damage on a cellular or systemic level. However, oxidative stress is dose dependent [
1]: high oxygen levels can cause severe damage while low levels of ROS can be beneficial to the organism, resulting in an extended life span [
2,
3]. Such biphasic responses to a potentially harmful compound are commonly named hormesis, a concept that was initially postulated by [
4] and which was shown to have significant impact on aging with a variety of stressors described [
3,
5‐
10]. Adaptive response processes may explain how increased ROS formation culminates in the promotion of life span [
2,
11,
12]. Yet, it is not fully elucidated however, which molecular sensors become directly activated by ROS. In yeast, inhibition of Target of Rapamycin (TOR) extends chronological life span by increasing mitochondrial ROS (mROS) [
13]. In
C. elegans, glucose restriction increases mROS to increase life span [
14,
15]. A redox-dependent hormetic response can also regulate the life span of Drosophila [
16] and a correlation between increased mROS and prolonged life span was observed in mice [
17]. These data can be explained by the hypothesis that a mild increase of ROS and other stressors might lead to a secondary increase in stress defense, culminating in reduced net stress levels and possibly extended life span [
14,
18‐
24]. Currently however, we cannot exclude alternative hypotheses explaining low level ROS induced hormesis. Low mROS levels might also extend life span in humans.
In vivo data regarding regulation of life span of humans are scarce. Instead, replicative senescence of human cells
in vitro has been studied as a surrogate for the human life span. In cellular senescence, cells, though metabolically active, stop dividing after a finite number of cell divisions (called the “Hayflick limit”) [
25]. Cellular senescence contributes to aging via accumulation of senescent cells in various tissues and organs during life; senescent cells have been hypothesized to disrupt tissue structure and function due to the components they secrete. In primates, the percentage of senescent skin fibroblasts increases with age
in vivo [
26] while senescent cell deletion delays aging-associated disorders in mice [
27]. Senescent cells contribute to the decline in tissue integrity and function, rendering the human body susceptible to a number of age-related diseases [
28,
29]. These results indicate that cellular senescence is causally implicated in generating age-related phenotypes and that removal of senescent cells can prevent or delay tissue dysfunction and extend health span, linking cellular to tissue and organismal aging. Cellular senescence can be induced by several mechanisms, in most cases involving oxidative or oncogenic stress [
30]. Human diploid fibroblasts display an increase in replicative life span under hypoxia [
31]. Hypoxia increases cellular ROS levels which were found to be required for the increase of the replicative life span of human fibroblast cells [
32]. However, a brief exposure to hyperbaric oxygen or juglone (a compound that generates ROS) can increase life span in
C. elegans [
33]. Rotenone interferes with the electron transport chain in mitochondria, producing increased levels of intracellular ROS due to inhibition of electron transfer from complex I to ubiquinone [
34,
35]. Therefore, rotenone can be applied to mimic a physiological increase of ROS as a trigger for cellular aging [
36]. Rotenone is a color- and odorless chemical with a broad spectrum of use as an insecticide [
37], pesticide [
38] and piscicide [
39]. Rotenone has been extensively used in age related studies revealing cell line and experimental model specific responses [
35,
36,
40‐
46]. Rotenone induced ROS increase can accelerate telomere shortening and can cause DNA damage, followed by a robust DNA damage response and senescence [
47‐
50]. In addition to aging, mitochondrial dysfunction can result in a number of chronic conditions in humans, including Alzheimer’s disease [
51], diabetes [
52] and obesity [
53]. However, low dose rotenone revealed a lifespan extending capability in
C. elegans [
40].
In this study we investigated the effect of rotenone as a stressor in primary human fibroblasts. We assessed the transcriptomes of primary human fibroblast strains in the presence and absence of mild doses of rotenone during their transition into senescence. We studied the effects of rotenone in MRC-5 fibroblasts derived from male embryonic lung [
54], human foreskin fibroblasts (HFF) derived from foreskin of 10 year old donors [
55,
56] and WI-38 fibroblasts derived from female embryonic lung [
57,
58]. Our data show that the concept of hormesis also applies to
in vitro aging of primary human fibroblasts.
Discussion
Oxidants are important intracellular signaling molecules, with mROS levels notifying the cell of a changing extracellular environment. Redox-dependent signals induce transcriptional changes in the nucleus leading to cellular decisions including differentiation, growth, cell death and senescence [
98,
99]. A particular stressor that is incompatible with cell viability might induce larger quantities of mROS, which non-specifically produce cell damage and subsequent cell death, while another moderate stressor might induce smaller quantities of mROS. Relatively minor damage, induced by intracellular stresses including metabolic perturbations and genomic instability, increases ROS levels, predominantly (although not exclusively) from the mitochondria. Low mROS levels promote adaptation to the stressor and consequently promote cell survival [
2,
9] since ROS are not simply a chemical inducing damage but also induce signaling pathways. Thus, the release of oxidants from the mitochondria, or other sources, can provoke a secondary protective response [
3,
100]. This phenomenon, termed hormesis (or mitohormesis), posits that low ROS levels can induce cellular defense mechanisms, resulting in health span-promoting effects, while higher ROS levels can cause cellular and systemic damage, culminating in increased mortality [
101]. Thus, ROS production and subsequent induction of ROS defense can be essential contributors to longevity.
Here, we induced increasing cellular ROS levels by addition of an external stressor and detected a hormetic effect in human cell strains. We investigated the effect of a range of concentrations of rotenone on the growth of primary human fibroblast strains from different tissue origin (MRC-5, WI-38 and HFF) maintained in culture in triplicates. Supplementing with 0.1 μM rotenone revealed a delay in senescence induction in MRC-5 and HFF (male from different tissue; Figs.
1b and
2b) but not in WI-38 fibroblast strains (female from same tissue as MRC-5; Additional file
2: Figure S1B). This rotenone concentration did not or only to a minor extent affects the cumulative PDs in these three fibroblast strains. To a great degree, cells are reported to keep their tissue-specific phenotype in culture [
102]. Interestingly, here we found a similar response for two cell strains (MRC-5 and HFF) from different tissue but major differences between MRC-5 and WI-38 strains, both derived from human lung (though from different genders). A difference between these two cell strains in response to mild stress had been observed by us before: an increase in oxygen levels from 3 % to 20 % induced senescence and a shorter life span in MRC-5 but not in WI-38 cell strains [
58], WI-38 cells are thus less sensitive to higher external oxygen levels. Here, in response to rotenone treatment, we confirmed the different properties of these two cell strains. An individual variation in the hormetic response was also observed in the resistance to type 2 diabetes mellitus in humans [
103].
Concentrations of rotenone higher than 0.1 μM resulted in apoptosis of the fibroblast strains. Thus, low dose rotenone induced a hormetic effect [
104]. The hormetic effect was evident however only in young (low PD) but not in older (higher PDs) cells (Fig.
1c). Possibly, at mid and high PDs, the amount of ROS in the fibroblasts has already increased with age to a value above the hormetic level. The increase of ROS in fibroblasts with age may result in the impairment of mitochondrial membrane potential [
105]. In addition, MRC-5 fibroblasts at PD 50 already showed accelerated levels of other typical mediators of senescence including p16, p21, and γH2AX, while these markers are not expressed in MRC-5 at PD 30 [
58,
106,
107]. Thus, at higher PDs (PD > 50), the senescence-induced feed-back loop of ROS generation may override any potential hormetic effect of rotenone [
108].
The effect of rotenone treatment has previously been investigated in other cell lines and experimental model systems. In MCF-7 cells, 0–20 μM rotenone induced apoptosis in a dose dependent manner [
42], consistent with our findings. Seven days of treatment with 0.2 μM rotenone induced senescence in fibroblasts from skin biopsies derived from healthy humans [
36] while, similar to our results, the higher concentration of 1 μM after 3 days of treatment resulted in apoptosis [
36,
41]. While 0.1 μM rotenone delayed senescence in young PD MRC-5 fibroblasts in our study, the same concentration resulted in depolarization of the mitochondrial membrane potential in skin fibroblasts derived from healthy humans [
43]. In muscle derived C2C12 cells, treatment with 0.005 μM rotenone for 48 h was able to induce lipotoxicity [
35]. However, 0.2 μM and 0.4 μM rotenone treatment were the highest tolerable concentrations for mtDNA mutations in HCT116 cells and immortalized mouse embryonic fibroblasts, respectively [
44]. In rat skeletal and heart mitochondria, 10 μM rotenone treatments significantly increased H
2O
2 production [
45]. Investigation of rotenone as a stressor in
C. elegans revealed a dose dependent effect on cell survival. 5 μM rotenone resulted in death of the organism [
46], whereas 0.1 μM rotenone resulted in an extended lifespan and improved stress resistance in
C. elegans [
40], effects similar to those observed here for MRC-5 and HFF fibroblasts.
Low level rotenone treatment induced an individual strain specific cellular response. WI-38 cells, found before to be oxygen insensitive [
58], did not show a hormetic effect at all while, to a considerable extent, MRC-5 and HFF displayed cell strain specific most differentially expressed genes and a delayed transition into senescence. By statistical selection we determined the most differentially expressed genes common for both strains (Additional file
3: Table S2). Among these, we identified four genes (
SFRP1,
MMP3,
CCDC68 and
ENPP2) with an expression regulation identifying them as potential candidates for hormesis induction in fibroblasts. Over- and under expression of these genes are envisaged to provide experimental proof for this hypothesis.
Several pathways regulated in different directions due to rotenone treatment compared to transition into senescence were identified. Improved DNA repair capacity and cell cycle progression could well be underlying mechanisms inducing a hormetic effect after low dose rotenone treatment. However, on the DEG as well as on the pathway level, the differential regulation of common genes and pathways were weak compared to that of others in the single cell strains. Thus, the rotenone induced common cellular response is a weak signal, superimposed by individual cell-internal gene expression changes. This is consistent with hormesis being a small effect in general, with the extreme case of WI-38 cells not showing a rotenone induced hormesis at all. This suggests that the observed hormetic phenotype does not result from a specific strong gene or pathway regulation but from weak common cellular processes, probably induced by low dose ROS levels [
3,
101].
A recent microarray study investigated the effect of 0.6 μM rotenone on fibroblasts from skin biopsies derived from healthy young (23–25 year old) and aged (90–91 year old) human subjects [
109], detecting no significantly differentially regulated pathways. This higher rotenone concentration induced apoptosis in the cells studied here. We observed a hormetic effect only in young (low PD) fibroblasts.
0.1 μM rotenone supplementation induced a life span extension in
C. elegans [
40]. As a consequence of the same low dose rotenone treatment, we observed a hormetic effect in two human fibroblast cell strains similar to effects in
C. elegans. We therefore searched for similarities between the significantly differentially regulated pathways on rotenone treatment in
C. elegans and the fibroblast cell strains analyzed here. As in our study, rotenone was supplemented throughout the
C. elegans life span. High-throughput RNA sequencing was conducted at four time points of the
C. elegans life span (after 1, 5, 10 and 20 days), revealing a number of differentially expressed genes (3460, 158, 2 and 18, respectively) compared to untreated
C. elegans worms. From our comparison, we excluded the
C. elegans rotenone data for day one since this may be the immediate organismal response to the addition of a foreign stressor [
110,
111]. The comparison of the common most differentially regulated pathways (
p < 0.05) due to 0.1 μM rotenone treatment in
C. elegans and human MRC-5 and HFF fibroblasts revealed the common up-regulation of ten pathways (“RNA transport”, “Spliceosome”, “DNA replication”, “Nucleotide excision repair”, “Base excision repair”, “Mismatch repair”, “Homologous recombination”, “Pyrimidine metabolism”, “RNA degradation” and “RNA polymerase”). This might indicate that in both systems low dose rotenone could induce similar mechanisms, resulting in the delay of senescence in fibroblasts and the extension of life span in
C. elegans. However, none of the genes belonging to the significantly differentially regulated pathways common for both cell strains and
C. elegans had a log2 fold expression change due to rotenone treatment larger than one in either of the two cell strains. Furthermore, analyzing the genes most differentially expressed due to rotenone treatment in
C. elegans (on days 5 and 10) revealed no common genes compared to either of the fibroblast strains; the genes most significantly differentially regulated in
C. elegans have no human orthologues.
Taken together, we find that on the gene and on the pathway level the dominant cellular response to low level rotenone is mostly cell strain specific while the observed common hormetic effect seems to be based on weaker expression differences. This suggests that hormesis is a rather individual response, consistent with [
103]. Our results obtained for human fibroblast cell strains show that hormesis occurs already on the cellular level and not necessarily requires high-level, like immune or neuronal, regulatory systems for induction. In animals, immune-system-related and neuronal hormetic effects are common [
10,
112,
113] and might add to the hormetic effect induced on the cellular level. Minor stress induced by rotenone or other hormetic agents activates maintenance genes (“vitagenes” [
10]), including DNA repair genes as observed here. Our results could be explained by the hypothesis that minor stress induces an over-shooting stress-response that does more than necessary, in this way slightly delaying senescence induction by counteracting aging effects which are due to the time dependent decay of cellular systems. The dose dependent response of hormetic agents has a broad range of biomedical applications [
114]. This observed effect
in vitro if translated
in vivo might have an impact on longevity in humans.
Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
SM undertook the laboratory work in the laboratory of SD and PH. SM wrote the manuscript together with SD. SP did the bioinformatics analysis under the guidance of RG. MG undertook the high throughput RNA sequencing under the guidance of MP. All authors read the manuscript, studied it critically for its intellectual content and approved the final draft.