Skin cancer prevention is important and sunscreen use has been recommended as a cost-effective preventative, especially in countries with a high-sunlight environment [1
]. It has been estimated that a majority of cancers could potentially be reduced by regular sunscreen use. It included around 9.3% of squamous cell skin carcinomas and 14% melanomas that may be prevented if UV exposure was reduced. On the other hand, the effect of the sunscreen concerning the prevention of basal cells carcinomas’ incidence was not seen [1
]. The studies showing effect mostly used sunscreens with organic filters, not sunscreens with nanoparticles [2
]. However, some studies have reported potentially unfavourable effects of sunscreens, since they can extend the duration of sunbathing and increase the risk of skin malignancies [3
]. Other studies point to a high reactivity of nanoparticles and their potential to produce reactive oxygen species (ROS), alter the skin structure [7
], and/or penetrate the skin [9
Ultraviolet (UV) radiation, encompassing UVA (wavelengths 320–400 nm), UVB (280–320 nm), and UVC (200–280 nm), including UV-emitting tanning devices, may cause each of the three main types of skin cancer: basal cell carcinoma, squamous cell carcinoma, and melanoma [10
While UVA and UVB rays are transmitted through the atmosphere, all UVC and some UVB rays are absorbed by the Earth’s ozone layer. Thus, most of the UV rays we encounter are UVA (95%).
UVA penetrates deeply into the dermal layer of the skin, promoting skin aging, wrinkles, and in large doses, cancer [11
]. Only 10%–30% of UVB reaches the surface, but its biological effects are larger. UVB radiation only enters the epidermis and triggers erythema, pigmentation, and optical aging, however, it can cause chemical changes in DNA, increasing the risk of skin cancer [12
]. On the other hand, UVB is also known to play positive roles, including supporting the synthesis of vitamin D in the human body [13
Similar to the sun, tanning beds typically emit 95% UVA and 5% UVB [11
There are several complex mechanisms by which non-melanoma and melanoma skin cancers can be attributed to DNA damage caused by UV radiation exposure (photocarcinogenesis), involving the interplay between various biochemical processes [14
]. UV can generate ROS, such as hydroxyl radicals (OH·), hydrogen peroxide (H2
), and superoxide anions (O2−
), leading to oxidative stress, which is considered a key mechanism of cell and DNA damage. It was recently shown that the use of antioxidants, such as ascorbic acid and rutin, appears beneficial following UV irradiation [15
The formation of a marker of the peroxidation of lipids, such as 8-iso
-prostaglandin F2α (8-isoprostane) by free-radical lipid peroxidation of arachidonic acid, has been detected in human skin in response to UVB radiation [16
]. Another lipid peroxidation product, malondialdehyde (MDA), can accumulate in human skin and MDA-protein adducts have also been found in skin cancer. UV radiation induces mutation of nucleotides that are highly susceptible to these free radical injuries [13
]. For instance, guanine oxidation leads to the formation of 8-hydroxy-2-deoxyguanine (8-OHdG), and changes in subsequent base pairing can cause mutations, as has been found in skin cancer.
Sunscreens should be effective in blocking both the UVA and UVB spectrum. Unique properties of nanoparticles have led to an increase in their use in various applications, including medicine [2
], however, there are also concerns regarding the safety of nanoTiO2
, not solely in sunscreens.
One of the most frequently used nanoparticles is titania (TiO2
), which has the ability to reflect, scatter, and absorb UV radiation [18
], however this results in photocatalysis, releasing ROS capable of altering DNA [11
]. Additionally, oxidative damage and genotoxicity without UV radiation have been found in vivo [19
Based on animal studies, TiO2
was reclassified in 2010 by the International Agency for Research on Cancer as a group 2B carcinogen, i.e., possibly carcinogenic to humans [21
]. In the experimental studies, nanoTiO2
toxicity was higher than that of bulk TiO2
due to the higher reactivity of the nanoparticles, since they have a highly active surface area (hundreds m2
/g). This is due to three main mechanisms: (1) ROS production following the induction of electron-hole pairs, (2) damage of the cell membranes due to lipid peroxidation by the attachment of nanoparticles to cells via electrostatic forces as a result of their large specific surface area, and (3) TiO2
nanoparticle attachment to intracellular organelles and macromolecules following cell membrane damage [20
can be found in three crystalline structures that differ in their potential permeation, i.e., anatase, rutile, and brookite, in addition to an amorphous phase. Rutile is more desirable for use in sunscreens, since it is less photoreactive [11
]. Both the anatase and rutile forms, including anatase/rutile combinations, can generate ROS [22
] and perturb the structure of the stratum corneum [8
There are concerns regarding the potential penetration of nanoTiO2
particles from sunscreens into viable skin, and subsequent systemic absorption, however the conclusions are ambiguous, since these data are very limited [23
One earlier study confirmed that nanoTiO2
absorption occurred from sunscreens that were used twice daily for 9–31 days in 13 patients scheduled for skin surgery [25
]. In subsequent histological examination, TiO2
was detected in both the epidermis and dermis. In another study in two volunteers, TiO2
nanoparticles were found in viable cells of the epidermis following sunscreen application six times a day for seven consecutive days. However, these studies did not ascertain whether the particles were translocated into the systemic circulation [18
To date, several molecular epidemiological studies in workers exposed to different nanoparticles during handling engineered nanomaterials suggest health impairment [26
]. Elevated pro-inflammatory markers, antioxidant enzymes, cardiovascular markers [27
], pro-inflammatory leukotrienes (LTs), and tumour necrosis factor [29
] in the circulation, markers of oxidative stress in the exhaled breath condensate (EBC) and/or circulation, and impaired lung functions have been observed [32
EBC is a liquid, collected during tidal breathing, which is composed mainly of water (99.9%) and contains only a small proportion of water-soluble and insoluble compounds, presumably originating from the airway lining fluid in the form of aerosolised particles generated during the re-opening of distal airways [38
Unlike the non-invasive marker of eosinophilic airway inflammation, fractional exhaled nitric oxide (FeNO) [39
], EBC is not considered a standardised biological specimen. Its analysis is used in research but not yet in clinical practice, since the percentage of condensed liquid of the exhaled volume is not constant for each collection process. Nevertheless, it enables the non-invasive collection and detection of a broad spectrum of biomarkers and particles from the airways and lungs [38
Oxidative alteration of lipids occurs in vivo during aging and in certain health disorders. It includes stable 8-isoprostane and unstable indicators of oxidative stress in cells, such as lipid peroxides forming reactive compounds, i.e., MDA, 4-hydroxy-trans
-hexenal (HHE), and 4-hydroxy-trans
-nonenal (HNE), which can covalently bind to DNA and proteins, and are therefore considered genotoxic and cytotoxic [40
]. The stable products formed from peroxynitrite (ONOO-) and hypochlorous acid (HClO) with tyrosine residues in proteins are 3-nitrotyrosine (3-NOTyr) and 3-chlorotyrosine (3-ClTyr) respectively, both of which are related to neutrophilic inflammation and have been found in patients with interstitial respiratory disorders, in addition to o-tyrosine (o-Tyr) [40
]. Finally, 8-OHdG and 8-hydroxyguanosine (8-OHG), formed by the oxidation of guanine in DNA and 5-hydroxymethyl uracil (5-OHMeU) in RNA respectively, reflect oxidative damage to nucleic acids [40
Several biomarkers of inflammation have been successfully measured in EBC using highly sensitive techniques, such as liquid chromatography mass spectrometry (LC/MS). LTB4 activates leukocytes and induces chronic neutrophilic inflammation. Cysteinyl LTs (LTC4, LTD4, and LTE4) increase vascular permeability and induce airway smooth-muscle contraction [41
]. Proinflammatory LTs have been found in workers exposed to nanoTiO2
for an entire shift, but also in office workers with only an approximate 15-min of daily exposure [29
Although the use of sunscreen containing nanoTiO2 is extensive, a limited number of human studies have been conducted to assess the response triggered by the nanoparticles and the uptake of nanoTiO2 by human skin. In the present pilot study, biological fluids taken non-invasively, such as EBC, blood, and urine, were used to study the effects of sunscreen in humans. We aimed to test:
potentially deleterious effects of commercially available nanoTiO2 sunscreen by measuring markers of oxidative stress and/or inflammation in plasma, urine, and EBC;
beneficial preventive effects of commercial nanoTiO2 sunscreen in limiting/blocking oxidative stress and inflammatory markers in plasma, urine, and EBC, triggered by UV radiation from a commercial tanning bed;
potential absorption of titania and TiO2 nanoparticles by their presence in biological fluids—plasma and urine—prior to, during, and one week after sunscreen use;
potential inhalational absorption of titania and TiO2 nanoparticles (as powder contamination from the dry sunscreen on skin) by analysing the EBC in the collected samples;
spirometry and FeNO to evaluate potential respiratory disorders.
There were five key findings of the present study. Firstly, a potentially damaging effect of sunscreen was not found. We did not see elevation in the markers of oxidative stress and inflammation in any sample—plasma, urine, or EBC—after using sunscreen for three consecutive days or one week after their thorough removal from the skin. This indicates that even a longer usage of sunscreen than is common, i.e., without washing off the sunscreen each evening, was not detrimental.
There was an elevation in a few biomarkers in the EBC in study A, as compared with pre-test levels, however, due to the higher variability of the results and technical challenges of EBC collection and analysis, and since EBC is not yet considered a routine clinical method, we do not want to overestimate their significance.
Secondly, a beneficial effect of sunscreen application was not seen, since the levels of biomarkers of oxidative stress and inflammation remained unchanged following UV radiation. The sunscreen was applied prior to UV exposure as recommended, and the prescribed dose was used (less than 140 g on average).
On one hand, the sunscreen protected the skin from mild redness as seen in study B. However, on the other hand, the sunscreen did not block/prevent the increase in markers of systemic oxidative stress and inflammation in the test variant C, i.e., sunscreen + UV (conditions for which the use of sunscreen is recommended) as compared with UV only. Both plasma and urine samples reflected a rapid effect in both studies.
With respect to the EBC, the number of positive samples in study C was lower, and the peak elevation of three markers of the oxidation of lipids was postponed to sample 3, i.e., it was seen later, and although there was an increasing trend, significance was not seen in 8-iso, o-Tyr, 3-ClTyr, 3-NOTyr, and LTC4. EBC usually shows a higher variability and may encounter more interfering factors in the respiratory system [38
]. Therefore, the limited number of subjects may have played a role. Although spirometry and FeNO were within the reference values during all test variants and in all samples, subclinical involvement cannot be excluded [38
The length of sunscreen use in the present study was longer than common use by consumers. The difference was that the sunscreen was not washed off in the evenings on days 1, 2, and 3. Nevertheless, the dosage corresponded to the amount recommended by the manufacturer, which was approximately 30 g twice per day per adult and re-application following accidental removal due to swimming or towelling. The total recommended sunscreen amount for three days (180 g) was not exceeded.
Thirdly, there was a measurable titania concentration and the detection of TiO2 particles using TEM in plasma and urine samples in studies A and C, which supports the theory of the skin permeability potential of nanoTiO2.
The sunscreen use duration was three days, and the sunscreen was not washed off at the end of each day, which differs from regular use of the sunscreen. However, the sunscreen dose did not exceed the recommended dose for the population, including children. Similarly, the UV irradiation dose was within the EU limits for commercial tanning beds.
Only nanoparticles up to a diameter of 14 nm were detected in both biological fluids. The average particle size in plasma showed an increase over time from 6.1 nm to 7.8 nm, i.e., larger particles persisted longer. Both measurable titania and nanoparticles were present up to sample 4, i.e., one week after washing the sunscreen off, which excludes contamination from the sunscreen that was thoroughly washed off one week earlier. Of course, urine contamination could not be completely ruled out, however plasma contamination would not occur during standard blood withdrawal.
Fourthly, we could exclude respiratory delivery of nanoTiO2 to the body based on the negative results of both TiO2 and TEM particle analysis of EBC samples.
in the biological samples without sunscreen application also excludes food as a potential source of nanoTiO2.
Potential contamination between skin-hands-mouth cannot be completely excluded, however the absorption from the gastrointestinal tract is very limited [50
], therefore we do not suppose this could bring measurable plasma and urine levels. In addition, we have no explanation as to why it would be more pronounced in females. We would also expect that the nanoparticles, the origin of which would be the skin-hands-mouth contamination, would keep the original size, i.e., of approximately 43 nm, as oral absorption enables absorption of larger particles, with an average that was much higher than in our plasma and urine samples. In human volunteers’ oral exposure of 100-mg dose of TiO2
particles with diameter 50–260 nm appeared in the blood [51
Fifthly, there was an early appearance of TiO2 in both blood and urine under normal conditions of sunscreen use, since titania and TiO2 particles were found 6 h after the first sunscreen use in sample 2 from women. This shows that the continuous use of sunscreen did not affect these results.
This was seen later in men, i.e., in sample 3 collected 42 h later. Since there was no sampling on day 2 and both plasma and urine were actually spot samples and not 24-h collection, it is possible that titania appeared in the samples from men earlier than day 3, when the highest levels were measured in both women and men.
Our study supports the findings by Gulson et al., using sunscreen containing nanoparticles of 68
ZnO, in which the tracer was found in blood and urine samples at the end of the second day when sunscreen was used twice per day [52
]. In their 5-day studies with sunscreen applied to a small area of the skin, such as the mid to upper back, 68
Zn positivity continued to increase for up to 9 days after the end of application [52
In the present study, the concentration of titania in the plasma from women was significantly higher in both studies A and C, although the dose per body surface was not significantly higher than that in men. Titania may have been absorbed by inter- or intra-cellular diffusion, through hair follicles, sweat glands, and skin folds, or a combination. The thinner skin in women relative to men has been suggested as one reason [53
]. Other factors, such as hormone metabolism, hair growth, sweat rate, sebum production, and fat accumulation, should probably be considered [54
]. Here also, the formulation of the sunscreen contained the chelating agent, EDTA, which may potentially bind Ti and promote its absorption [53
The penetration of nanoparticles beyond the stratum corneum suggests that oxidative stress may lead to more serious adverse cellular effects, and potentially cancer [43
]. However, similar to the nanoZnO study, the quantity absorbed was very low.
We did not see any additional effect of UV irradiation (study C) as compared with sunscreen only (study A) on the mean level of TiO2 and nanoparticles in plasma and urine.
To the best of our knowledge, this is the first study to provide evidence that TiO2 nanoparticles in sunscreen are absorbed through healthy human skin, both exposed and unexposed to UV radiation, and that these particles are detectable in blood and urine.
More data are available concerning the inhalation of nanoTiO2
particles, especially in workers with long-term exposure to TiO2
dust, where particles documented by Raman microspectroscopy of rutile and/or anatase originating from preceding shifts were found in their EBC [55
in EBC reached an average of 24 ng/mL, in contrast to the controls, where it was unmeasurable, similar to the present sunscreen study [55
]. Recently, other methods of nanoparticle detection have been successfully used [56
]. Due to the potential of sunscreen in the spray form to be absorbed by inhalation, such products have been classified as potentially deleterious and are no longer allowed [58
Elevated markers of oxidative stress, including oxidation products of lipids, proteins, and nucleic acids, are associated with aging, metabolic diseases [40
], exposure to carcinogens [60
], and especially cancer [13
], however, the observed effects on oxidative stress and inflammation are unknown. A study in rats exposed to nanoTiO2
by inhalation for four weeks showed a strong inflammatory response in bronchoalveolar lavage peaking on day 3 post-exposure, but the overexpression of genes involved in inflammation was maintained for six months after the end of exposure [63
]. Long-term response was characterised by persistent upregulation of a number of genes, including those involved in oxidative stress, for up to 180 days post-exposure [63
The main limitation of the present pilot study is the relatively low number of exposed subjects, which is a consequence of the high financial demands of such studies [23
]. Only one commercial type of nanoTiO2
sunscreen was used. In addition, this pilot study did not analyse skin samples. Such a study could bring important data, especially in repeated samples. The advantage, on the other hand, is the fact that the participating subjects acted as their own controls and were identical during all three study variants, which eliminates intra-individual variability. Although direct measurement of UV exposure was not possible, the use of UV exposure in the form of a tanning bed with a defined ceiling limit for the intensity and an exact duration enabled achievement of a comparable UV dose for all subjects, not influenced by the weather or location conditions.
Spirometry and FeNO measurements did not show any significant differences and helped to exclude significant respiratory effects and impairments.
Sunscreen alone did not cause an elevation in the vast majority of the biomarkers of oxidative stress and inflammation, however, tanning bed use increased all markers in plasma, urine, and EBC. A preventive effect of sunscreen was not found. Its use prior to UV irradiation suppressed skin redness, however, sunscreen did not prevent the effects of significantly elevated oxidative stress and inflammatory markers, including those of nucleic acid oxidation. Plasma or urine analysis appears preferred to EBC due to the lower potential interference of inhalation.
Titania and nanoTiO2 particles were found in plasma and spot urine samples from women after 6 h of sunscreen use, but in males after 48 h. We show that nanoTiO2 particles can pass through the protective layers of the skin even without UV irradiation, and can be detected in blood and urine up to one week after removal of the sunscreen by bathing and showering. Measurable titania levels and TiO2 nanoparticles prove absorption and exclude potential contamination. No additional effect of the UV irradiation on the absorption of sunscreen was seen.
Positive plasma and urine TiO2 measurements and TEM particle detection during nanoTiO2 sunscreen use confirm that a minor amount is absorbed and slowly eliminated. The amounts were minor, and TiO2 detected in the blood and urine from sunscreen may not necessarily be present in the form of TiO2 nanoparticles.
Importantly, the absence of both titania and TiO2 particles in the EBC by ICP-MS and TEM respectively, excluded the inhalation of TiO2.
The present pilot study doubts the utility and positive effect of nanoTiO2 sunscreen to prevent oxidative stress and inflammation caused by UV irradiation, suggesting that it may not prevent skin cancers.
In addition, it appears that nanoparticles can be absorbed through the skin and pass to the urine, starting 6 h after first exposure, and can remain for up to one week after the sunscreen has been washed off the skin.
Since there is an increasing concern regarding the exposure of pregnant mothers, women, men of fertile age, and notably children to nanoTiO2
], further human studies are needed to explain the impact of the elevated markers used in the present study. Also, our hypothesis that only particles with a diameter size lower than approximately 15 nm may be absorbed through the skin should be verified, as replacing them in the sunscreen with larger particles could potentially prevent absorption.