The effect of a color tattoo on the local skin redox regulatory network: an N-of-1 study

Abstract Biomedical aspects of tattooing have been extensively discussed in literature, however pathophysiological effects of tattoo inks in the human body are still unexplored. Oxidative stress is considered responsible for the adverse effects of tattooing, however no experimental evidence for tattoo ink-related oxidative stress in the human body currently exists. The aim was to examine the effect of a blue tattoo on skin redox regulatory network (RRN) parameters in a single human subject. Skin surface oxidation-reduction potential (ORP) was analyzed with a PH60F flat probe. Interstitial and intracellular fluid enriched capillary blood from the tattoo and the control area was extracted and analyzed with I2/KI-stabilized microORP, nitrocellulose redox permanganometry (NRP), carbonato-cobaltate (III) formation-derived H2O2 dissociation rate assay, 1,2,3-trihydroxybenzene autoxidation assay, thiobarbituric reactive substances (TBARS) assay and 5,5,’-dithio-bis-(2-nitrobenzoic acid) (DTNB)-based determination of free thiol content in low molecular weight and protein precipitate fractions. Surface ORP analysis revealed a greater antioxidant capacity of tattooed skin in comparison with the control (CTR). Capillary blood analysis confirmed greater reductive capacity in the tattoo sample both by microORP (−4.33 mV vs CTR) and NRP (+10.8%). Hydrogen peroxide dissociation rate (+11.8%), and protein sulfhydryl content (+8.5%) were increased, and lipid peroxidation (−15%) was reduced in the tattoo sample in comparison with the CTR. In this N-of-1 study, RRN of tattooed skin was shifted toward a more reductive state with all parameters indicating reduced levels of oxidative stress in comparison with nontattooed skin. The local antioxidant effect of copper(II) phthalocyanine provides one possible explanation of the observed effects.


Introduction
Tattooing, a practice of inserting ink that contains insoluble pigments into the dermis for esthetic reasons, is becoming increasingly popular especially among young people. Medical consequences of tattooing have been thoroughly discussed in the literature, however mostly in the context of hygiene, the transmission of infectious diseases, or allergic reactions. On the other hand, the pathophysiological consequences of tattooing are still largely unknown. A cursory look at the chemical constituents of tattoo inks reveals a great chemical variability with an equally enormous list of possible biological effects of different chemicals and their combinations. The composition and toxicological effects of tattoo inks have been reviewed in detail in the literature and interested readers are pointed to informative articles by Arl et al. [1] and Laux et al. [2]. The main components of tattoo inks are usually vehicles (e.g. water and glycerine), additives (e.g. surfactants and polymers), and pigments (organic or inorganic compounds). Different metals are also often present either as part of the pigment (e.g. titanium, barium, aluminum, and copper) or contaminants (e.g. arsenic, cadmium, chromium, cobalt, lead, or nickel) [1][2][3][4][5]. Black and colored inks differ greatly in their chemical composition. Black ink often contains iron(II) oxide (FeO) or carbon black. In contrast, red ink was often based on cadmium selenide (CdSe) also known as cadmium red, green ink chromium(III) oxide (Cr 2 O 3 ), and blue cobalt(II) aluminate (CoAl 2 O 4 ) [6]. Modern colored inks have a different chemical constitution. Red and yellow tattoo inks contain azo pigments, and phthalocyanines are usually found in different shades of blue and green [7]. The significant chemical variability of tattoo inks is probably responsible for different biological effects. For example, one in vitro study on cellular viability and protein synthesis in human fibroblasts exposed to different tattoo inks reported that there was no toxic effect of commonly used black ink Strong Black, however, red ink Biolip 27 significantly reduced viability and decreased expression of procollagen a1 type I protein [8]. One other study tested 19 different black tattoo inks and found significant variability in both their chemical constitution and the potential to reduce the mitochondrial activity of human keratinocytes in vitro [9]. Apart from great chemical variability and complexity of commonly used tattoo inks, another challenge for understanding the pathophysiological effects of tattooing arises from the fact that pharmacology of tattooing has never been extensively studied in humans in vivo, except in the context of tattoo-induced side effects that are well documented in the form of case reports but offer limited insight into underlying pharmacodynamics. Biological differences of animals, cells, and humans are well known in pharmacology where many drugs display promising effects in vitro and laboratory animals but fail to induce biological effects in humans, or even worse detrimental effects manifest for the first time in human trials due to significant biochemical, physiological and structural differences. However, majority of studies on the toxic effects of tattoo inks were done in different cell lines in vitro, and papers describing the biological effects of tattoos in humans are extremely scarce. Moreover, not a single study I am aware of examined the effect of tattoo ink on oxidative stress in humans.
Oxidative stress is a biological phenomenon that emerges as a result of a disbalance between generation and removal of reactive molecules often referred to as reactive oxygen species (ROS) [10]. The most common cellular ROS are superoxide and hydroxyl radicals, hydrogen peroxide, and singlet oxygen that are generated as by-products of metabolism [10]. When present at low levels, ROS are involved in the regulation of cellular signaling pathways regulating proliferation, differentiation, and stress response. For example, it has been reported that low levels of ROS stimulate phosphorylation of the protein kinase C, mitogen-activated protein kinases, and phosphoinositide 3-kinase, protein kinase B, and JUN N-terminal kinase [11]. However, once the production of ROS overpowers cellular detoxification capacities, excess radicals damage lipids, proteins, and nucleic acids that lead to loss of cell function and structure. Redox disbalance that results in oxidative stress has been recognized as an important etiopathogenetic factor in many human diseases such as Alzheimer's [12] and Parkinson's disease [13], cardiovascular diseases [14], and cancer [15]. Oxidative stress also plays a major role in the maintenance of skin homeostasis where cellular detoxification mechanisms have to neutralize ROS generated from both internal processes (e.g. mitochondrial electron transport chain) and external stimuli (e.g. ultraviolet radiation and pollutants) [16]. The importance of skin oxidative stress is the most pronounced in inflammatory conditions of the skin where redox homeostasis has been recognized as a pathogenetic factor and an important pharmacological target [17].
A lucky coincidence that I am actively involved in redox regulation research and prone to self-experimentation in combination with the fact that I was equipped with adequate curiosity and a blue tattoo on my left forearm motivated me to conduct an N-of-1 study on myself to try to understand how the presence of chemical constituents of a color tattoo affects local skin redox regulatory network (RRN).

Material and methods
Tattoo A tattoo used in the research was made by Bo Mademoiselle in Tattoo and Piercing studio Memories in Zagreb (Croatia), and was 7 months old at the time of sampling. Intenze TM inks (Intenze, USA) were used for tattooing. Although I am not aware of the exact proportions of different shades of blue inks used for the tattoo, analysis of safety data sheets of all shades of blue available from the company website reveals copper(II) phthalocyanine (C.I.74160; CAS 147-14-8; EC 205-685-1) is present in all blue inks. The tattoo was not exposed directly to the sun before the experiment and was covered with long sleeves outdoors. During the first month following tattooing, the tattoo was treated by Bepanthen Plus (Bayer, Germany) creme (50 mg/g dexpanthenol, 5 mg/g chlorhexidinum), however, no creme was applied in the period of 6 months before the experiment.

Skin oxidation-reduction potential
The skin was thoroughly cleaned with soap, water, and distilled water and dried. Surface skin oxidation-reduction potential (ORP) measurements were conducted with PH60F (Apera Instruments, Germany) with a flat end to ensure optimal contact of the electrode with the skin, and the instrument was calibrated and cleaned before measurements. A tattoo area was divided into 12 equal parts as shown in Figure 1(B). Measurements were designed in a way to maximally reduce experimental error. Every area was measured once, followed by the corresponding control area on the forearm Figure 1. A blue tattoo redox regulatory network analysis. A) A photograph of the tattoo on the left forearm with the area used for prick-sampling for the tattoo sample indicated with a blue triangle, and the area used for prick-sampling of the control sample indicated with a red circle. B) Enlarged photograph of the tattoo with 12 sampling areas used for ORP measurements illustrated with blue squares. The same anatomical area on the other forearm was used for control measurements. C) Results of the ORP analysis with potentials from the tattooed forearm depicted with blue circles, and potentials from the corresponding areas on the right forearm presented with red circles. An illustration of the tattoo is overlaid onto the graph. Mean values of three technical replicates are illustrated. D) Control measurements of ORP to control for possible ipsilateral-contralateral differences in reductive potential. Two control areas are illustrated -an area above the fifth rib in the anterior axillary line (5th RIB AAL) and a clavicular (anterior) part of the deltoid muscle (SHOULDER). Mean values of three technical replicates are presented. E) Redox regulatory network subsystem analysis in the sample obtained from the tattooed and control area by the prick-sampling method as illustrated in Figure 1 without a tattoo (1-tattoo, 1-control, 2-tattoo, 2-control, … ) and everything was repeated three times. A mean value of three measurements was used even though variations were minimal. To exclude possible unilateral differences due to other physiological reasons additional body parts were chosen as controls for unilateral variation -skin above the 5th rib in the anterior axillary line (5th RIB AAL) and clavicular (anterior) part of the deltoid muscle (SHOULDER). Skin pH was analyzed with PH60F as an additional control for the ORP measurements.

Prick sampling and sample preparation
To explore local skin RRN a first drop of capillary blood following skin puncture was used as it is enriched with intracellular and interstitial fluid [18]. A capillary blood sample was taken from two locations on the tattooed forearm as shown in Figure 1(A). The skin was cleaned as described previously, and a lancing device was used to obtain a local capillary blood sample with a new lancet used each time. One microliter of the blood was taken from each site with a micropipette and dissolved in 39 ll of double-distilled water (ddH 2 O) validated for 0.055 lS/cm. Samples were vortexed thoroughly and stored at À20 C.

Sample oxidation-reduction potential
In vitro sample reductive capacity was measured by means of I 2 /KI -redox-coupled microORP to ensure redox system stabilization as described previously [19]. In short, 0.1 M I 2 and 0.4 M KI solutions were used as the redox pair electrode solution in the experiment. Samples (2 ll) were mixed with 5 ll of redox solution and left for 1 h in the dark at 25 C. After the incubation period, samples were measured in three time points with redox microsensor system ORP-146S (Shelf scientific, Lazar Research Laboratories, Inc., Los Angeles, USA) composed of platinum sensing element with Ag/ AgCl reference (KCl was used as a filling solution). Readings were obtained by 6230 N Microprocessor meter (Jenco Instruments, San Diego, USA). System accuracy was ±0.5 mV, and the upper bioanalytical method variability was determined to be 2 mV (Supplement). Readings were taken as mV differences between the control and the tattoo sample.

Nitrocellulose redox permanganometry (NRP)
The total reductive capacity of the sample was additionally checked with NRP to control for reductive specificity and overcome possible bias due to a redox pair used for ORP stabilization. A Standard NRP protocol was used [20]. Briefly, 1 ll of each sample was placed on a nitrocellulose membrane (Amersham Protran 0.45; GE Healthcare Life Sciences, USA), left to dry out, and developed in KMnO 4 solution (0.2 g KMnO 4 in 20 ml ddH 2 O). Excess reagent was removed under flowing dH 2 O, and MnO 2 precipitate was visualized by scanning. The membrane was analyzed in Fiji (NIH, USA) by means of GelAnalyzer plugin for integrated density quantification.

Hydrogen peroxide dissociation rate
Hydrogen peroxide dissociation rate indicating the activity of peroxidases was done by multi-point H 2 O 2 quantification derived from carbonato-cobaltate(III) complex ([Co(CO 3 ) 3 ]Co) spectrophotometric assessment [21]. In short, samples were analyzed multiple times until concentration with corresponding activities was inside the quantification range with 2 ll of samples used for the preparation of serial dilution samples. Once optimal dilution was determined, samples (8 ll of the final working solution corresponding to 1:200 dilution of the whole blood sample) were incubated with 40 ll 10mM H 2 O 2 dissolved in phosphate-buffered saline for 60 s, and the reaction was stopped by adding 100 ll of cobalt(II) hexametaphosphate working solution. The absorbance of the carbonato-cobaltate complex was determined at 450 nm with Infinite F200 PRO multimodal microplate reader (Tecan, Switzerland). The computed coefficient of variation (CV) of the hydrogen peroxide dissociation rate measurement was 6.5% (Supplement).

Superoxide dismutase
Superoxide dismutase activity was assessed by 1,2,3-trihydroxybenzene autooxidation inhibition determined from quantification of absorbance increment at 325 nm for 300 s [22,23]. Briefly, 10 ll of the sample was added to 1000 ll of 0.05 M Tris-HCl and 1 mM Na 2 EDTA (pH 8,2) and vortexed in the 1.5 ml reaction tube. 15 ll of 60 mM 1,2,3-trihydroxybenzene dissolved in 1 mM HCl was added and the sample was briefly vortexed again and pipetted into the spectrophotometric cuvette. The absorbance difference was analyzed and compared to the increment of the standard sample. CamSpec M350 DoubleBeam UV-Visible Spectrophotometer (Cambridge, UK) was used in the experiment. The estimated CV of the method was 1.7% (Supplement).

Thiobarbituric reactive substances assay (TBARS)
TBARS assay was used for the assessment of lipid peroxidation [23]. In short, 10 ll of the sample was mixed with 190 ll of ddH 2 O and 400 ll of TBARS reagent (0,375% thiobarbituric acid; 15% trichloroacetic acid). Samples were incubated at 95 C for 30 min in perforated 1.5 ml reaction tubes and placed under tap water to cool down afterward. Once the sample was cool, 600 ll of n-butanol was added to the mixture to extract malondialdehyde-thiobarbituric acid (MDA-TBA) chromogen. The absorbance of the butanol fraction was analyzed at 532 nm and the amount of TBARS was estimated based on the molar extinction coefficient of 1,56 Â 10 5 M À1 cm À1 . CamSpec M350 DoubleBeam UV-Visible Spectrophotometer (Cambridge, UK) was used in the experiment. The estimated CV of the method was 1.9% (Supplement).

Protein sulfhydryl content and low molecular weight thiol determination
Protein thiols and low molecular weight thiols (LMWT) were determined by reacting the samples with 5,5 0dithio-bis(2-nitrobenzoic acid) (DTNB) with subsequent quantification of 5-thio-2-nitrobenzoic acid (TNB) [23,24]. Briefly, samples (10 ll) were mixed with 15 ll of ddH 2 O, and 25 ll of sulfosalicylic acid (4% w/v) was added to the mixture. Samples were left on ice in dark for 1 h and spun at 10 000 rotations per min (RPM) for 10 min afterward. The supernatant (45 lL) was reacted with 45 ll of DTNB reagent (4 mg/ml in 5% sodium citrate) for analysis of LMWT, and the remaining solution was removed from the tubes by gentle tapping and 45 ll was reacted with the protein precipitate. After the reaction, samples were placed in a transparent 96 well plate and analyzed with F200 PRO multimodal microplate reader (Tecan, Switzerland) at 405 nm. Protein precipitate was analyzed with the addition of potassium phosphate buffer (0.1 M, pH 7.4) to increase the volume. The estimated CV of the method was 3.8% (Supplement).

Skin surface ORP
Analysis of skin surface ORP revealed that tattooed skin had increased reductive potential in comparison to the surface of the control skin area. In other words, slightly lower ORP values suggest that tattooed skin was less oxidative. This finding was at first counterintuitive as the literature suggests increased oxidative stress in tattooed skin, however, out of 72 consecutive measurements (36 of the 12 tattoo areas of interest and 36 of the 12 control areas as explained in the Methods section) 54 measurements showed that tattooed skin was stronger antioxidant, 12 measurements suggested it had the same reductive potential as the control skin, and only 6 measurements suggested tattooed skin was slightly more oxidative. Measurement areas are presented in Figure 1(B) and the mean results of all areas are shown in Figure 1(C). Control measurements are presented in Figure 1(D). Skin pH measurements with the same instrument indicated no difference between tattooed and nontattooed skin.

Skin capillary blood RRN
As surface skin measurements suggested tattooed skin was "less oxidative," I decided to obtain interstitial and intracellular fluid enriched capillary blood samples from tattooed and control skin to assess oxidative stress parameters that should reflect the redox status of the surrounding biological environment. Here, I analyzed ORP in vitro with a redox micro measurement system as described in the Methods section and obtained similar results as tattoo samples were again a few mV more reductive in comparison with the control samples (Figure 1(E)). Furthermore, samples were analyzed by NRP, a novel method characterized by great accuracy and precision for the measurement of reductive capacity in tiny volumes of biological samples [20]. Nitrocellulose redox permanganometry confirmed both surface measurements and in vitro ORP and indicated the increased antioxidant capacity of the tattooed skin (Figure 1(F)). As both methods used for overall redox balance analysis indicated that a small antioxidative shift was present in the tattoo samples (Table 1), I decided to analyze specific subsystems involved in cellular redox regulation to see what mechanisms were responsible for the observed effect. As shown in Figure  1(G), the hydrogen peroxide dissociation rate was increased in tattoo samples by 11.8% indicating greater activity of catalase and other peroxidases in the inked skin. Superoxide dismutase activity was also measured, however, the 0.7% increment observed was lower than the CV of the method (1.7%) and was thus considered inconclusive (Table 1, Supplement). Lipid peroxidation measurement was the most surprising as tattoo samples had 15% less harmful electrophile aldehyde peroxidation end products as determined by the TBARS assay (Figure 1(H)). Protein sulfhydryl content was also slightly increased in tattoo samples (þ8.5%; Figure 1(I)) indicating fewer proteins were oxidized by environmental redox homeostatic perturbations. Low molecular weight thiols were also measured, however, the observed difference was smaller than the CV of the method and was considered inconclusive (Table 1, Supplement). Percentage changes might be misleading, and should be carefully considered in terms of the biological information they convey, however, taken all together, RRN data indicated reduced oxidative stress in the tattoo sample (Table 1).

Discussion
Taken together, the results of this experiment strongly suggest that oxidative stress was reduced, rather than increased in the tattooed skin in this particular case. I find this extremely interesting as I originally expected to see the exact opposite. As briefly mentioned in the introduction, biological phenomena underlying tattooinduced changes are still largely unexplored. Nevertheless, oxidative stress has been proposed as the most important pathophysiological mediator responsible for tattoo-related health complaints such as swelling, itching, and redness following solar radiation exposure in the famous "Beach study" [25]. Interestingly, the association of reactive oxygen species (ROS) and tattoo ink in humans has so far only been supported by indirect findings from in vitro toxicological studies [26,27] and theoretical assumptions based on the fact that mechanisms of photoactivation underlying the biological effects of photodynamic therapy were also shown for some chemicals that are often present in tattoo inks such as polycyclic aromatic hydrocarbons [25,28]. Nevertheless, PubMed search for "tattoo" AND "oxidative stress" and "tattoo" AND "ROS" only yields 7 and 8 hits respectively, and none of the results provide any evidence for direct oxidative stress effects of tattooing in humans. Consequently, this N-of-1 study, although limited by its design, provides the first reported evidence of redox system-related effects of tattoo ink in the human body. Interestingly, the results seem to contradict the hypothesis that tattoo ink would induce oxidative stress in humans. One possible explanation of this paradoxical finding is related to the chemical variability of color tattoo inks briefly discussed in the introduction. A tattoo in this particular experiment was blue (Figure 1(A)) and most of the modern blue tattoo inks are based on copper(II) phthalocyanine (CuPC) also known as Phthalocyanine Blue BN, a bright crystalline synthetic blue pigment first prepared by accident in 1927 [29] and extensively studied in industrial context due to its interesting physicochemical properties. For example, CuPC is a wellsuited material for organic solar cells [30] and it has been suggested for data storage in quantum computing [31]. Considering its low toxicity (e.g. FDA approval for use in infant furniture and toys, and contact lenses [6,32]) and compelling properties, CuPC might even turn out to be interesting in the growing field of biocompatible organic bioelectronics [33]. Although biological effects of CuPC are yet to be elucidated, one especially interesting finding relevant for this paper is related to its strong antioxidant activity explored for the first time in 2012 by Amaral et al. where they showed that phthalocyanines were able to both reduce and prevent lipid peroxidation in a mouse brain, kidney, and liver [34]. Moreover, CuPC (alongside manganese(II) phthalocyanine was found to be especially protective in this study as it also exerted strong antioxidant properties in H 2 O 2 , Fe 2þ, and H 2 O 2 þFe 2þ -induced oxidative stress in deoxyribose degradation assay [34]. Based on these results authors suggest that CuPC might act by inhibiting the generation of free radicals or inhibiting their actions against lipids. This is in concordance with the results of this study as overall RRN of the tattoo was shifted toward antioxidative values (Figure 1(C-F)), and the protective effect was especially pronounced for The usual interpretation of the oxidative-stress-related changes of redox regulatory network subsystems is presented in column "Oxidative stress" with changes indicative of reduced oxidative stress in a tattoo sample marked with an arrow facing downward (#), and changes indicative of increased oxidative stress marked with an arrow facing upward ("). Changes that were smaller than the estimated coefficient of variation of the method used were considered inconclusive (marked with a "?" and highlighted in red). Ã Detailed calculation of CV and validations experiments are provided in the Supplement. ÃÃ The greatest electrode drift for the I 2 /KI-redox-stabilized oxidation-reduction potential was estimated to be 2 mV (described in the Supplement).
lipid peroxidation (Figure 1(H), Table 1). Authors also suggest that phthalocyanines might exert their antioxidant properties by directly degrading hydroperoxides such as H 2 O 2 . This is very interesting as the second most pronounced change of RRN subsystems observed in this study was related to sample H 2 O 2 dissociation rate (þ11.8% vs CTR). I originally assumed that the effect might be related to modulation of endogenous peroxidases, enzymes that play one of the most important roles in cellular defence against oxidative stress, however, it is possible that the change in the H 2 O 2 dissociation rate was also mediated by CuPC present in the tattoo sample ( Figure 2). Even though CuPC is the most prominent chemical constituent of blue tattoo inks, other chemicals or possibly even contaminants might be responsible for the observed effect and further research is needed to answer the intriguing question of how tattooing might shift local skin redox balance toward antioxidative values and reduce lipid peroxidation. Another possibility is the hormetic effect of tattoo ink as small quantities of harmful oxidative substances might trigger hyper compensation and induce a net increase of the total antioxidant capacity [35]. This interesting phenomenon has been observed for 3,5,4 0 -trihydroxy-trans-stilbene (resveratrol), a wellknown wine polyphenol with myriad health benefits exerted through potentiation of the antioxidant systems [36]. Apart from the exact mechanism that might be responsible for the apparent paradoxical reductive potential of the tattoo, numerous other questions remain to be answered. For example, as the experiment was conducted during the spring, and the tattoo has not been exposed to the sun for several hours prior to testing, it would be interesting to see whether the observed effect would also be present after it was exposed to UV light as it has been shown that polycyclic aromatic hydrocarbons and other components of tattoo ink can generate ROS upon exposure to solar radiation [9], and sun-induced tattoo-related complaints are a common finding among inked people. Considering the fact that CuPC is popular in material sciences due to its photovoltaic effect, the ability to generate electric current upon exposure to light, it is possible that the same effect in the human body is responsible for the oxidative shift of the RRN upon exposure to solar radiation and possibly related to unpleasant sensations such as itching. However, the author has never experienced this so far. Another interesting question is related to the fact that due to the apparent paucity of data, the explanation of the observed effects with the color of the tattoo is highly hypothetical, and it is possible that other tattoos might also demonstrate antioxidant properties.

Limitations of the study
The study presented here is obviously limited by its nature because it is an N-of-1 study. Although numerous control procedures were introduced to maximally reduce experimental error (e.g. technical replicates, multiple measure points, carefully chosen prick-sampling anatomical site, scrupulous setup of biochemical experiments), none of the precautionary settings can overcome a limitation that the presented results reflect a single person, a single time-point and a single tattoo. It would also be fair to point out that the author of the paper was at the same time the only subject in the experiments so this should also be taken into account. However, this didn't affect the experiment or writing of this manuscript in any way.

Conclusion
In conclusion, this N-of-1 study provides the first evidence of the effect of a tattoo on the human redox regulatory network and oxidative stress parameters. Furthermore, it provides preliminary evidence that there is a possibility of antioxidative, in contrast to the expected pro-oxidative changes following the administration of tattoo ink into the skin, and raises the question of different biological effects that might be mediated by a variety of chemical constituents of colored inks.

Data availability statement
All data generated by this research has been presented in the manuscript. Raw data can be obtained from the author's GitHub account: https://github.com/janhomolak or directly from the author. Supplementary material can be obtained from https://github.com/janhomolak/colortattoo/blob/master/Supplement_tattoo_Homolak.docx or directly from the author.