Background
Holi is a traditional Hindu festival originated in India. Lasting for several days in spring and celebrating the victory of good over evil it is a very colourful event. Differences in people’s caste, age, sex and status seem to be abrogated by throwing Gulal, a special coloured powder, at each other to make everybody and everything look equal. About 3 years ago, the traditional Indian festival was transferred to Europe and adapted to a commercial party event. Meanwhile it takes place in many different cities all over the world: people listen to music, dance and throw coloured powder. This so called Holi powder/Holi colour/Gulal powder or Micro Confetti is distributed by different companies. The quality and the amount of information given on the outer packaging about the ingredients of the particular Holi colour are mostly very poor. So far, there is no European or US regulation about Holi colours and it is not clear whether they should be categorised for instance as cosmetic products or general consumer products. Although many manufacturers claim that their products are harmless to health and environment, organisers of Holi festivals often recommend wearing eye and respiratory protection (like goggles and face masks) and advise people with respiratory disorders not to participate. There are several reports of adverse health effects probably caused by the use of Holi powder: various forms of cutaneous diseases [
1] as well as ocular irritations [
1,
2] and a case of periorbital necrotizing fasciitis [
3] have been described after exposure to Holi colours. In addition, high total suspended particulate concentrations occurred during Holi events [
4] that may also cause or aggravate adverse health effects like respiratory irritations.
To assess possible hazards to human health, we ordered four different Holi powder products of three different distributors via the internet and analysed them regarding particle size and their potential to induce pro-inflammatory responses in cell culture systems. Cell-based in vitro assays have been used widely to imitate in vivo situations [
5,
6]. For example the whole blood assay was used to describe pro-inflammatory activity of dust sample extracts [
7]. Moreover, we tested the cytotoxic potential of corn starch and the different Holi colours, analysed the endotoxin and mould content of corn starch and the Holi colours and screened for corn starch-/Holi colour-leukocyte interactions in vitro.
Methods
Holi colours
Four different Holi colour products were ordered via a renowned internet retailer. General information like producing country, ingredients or indications relevant for impacts on health either given on the internet or on the outer packaging of the product are shown in Table
1. As corn starch is often used as carrier substance for Holi colours, commercially available corn starch (RUF Lebensmittelwerk KG, Quakenbrück, Germany) designated as food ingredient was purchased to serve as a control.
Table 1
Available information on the different Holi colours, either communicated with the product itself or online
Shade/ characterisation of colour | black/ Holi colour, perfumed natural colour, Holi powder Gulal | pink/ Holi powder (Holi gulal Holi colour), used at Holi Festivals | orange/ “Effect colour powder”, optically like Holi powder/ Holi colours | green/ Holi colour, Gulal festival |
Produced in | India | Germany | n.s. | n.s. |
MSDS available | n.s. | yes | n.s. | n.s. |
EU Regulation | n.s. | according to Art. 19 EU regulation of cosmetic products | n.s. | n.s. |
Ingredients | natural colour, plant based | corn starch, water, hydrated silica, sodium chloride, sodium sulphate, E127 | n.s. | flour powder, dyed |
Recommended application | Holi open air festivals, creative areas | Holi festivals, for throwing up into the air, sprinkle powder onto hands and throw up into air (away from body) for adult use only | for decor use or throwing up into the air | for decor use or throwing up into the air |
Warning | environmental friendly, mild on skin, nontoxic | don’t use with a history of asthma and allergies, avoid contact with mucus membranes, wear mouth, nose and eye protection, not edible | don’t use at festivals or on skin, not classified as cosmetic product | don’t use at festivals or on skin, not classified as cosmetic product |
Electric field cell counting system (CASY®) of corn starch and Holi particles
We used a CASY® cell counter (Schärfe System GmbH, CASY Cell Counter + Analyser System, Model TTC) with a 60 μm measuring capillary to determine particle size and number of (i) BD Cytometer Setup and Tracking beads of 2 and 3 μm diameter (Becton Dickinson, Lot: 22680), (ii) blank calibration particles (BD Biosciences, Lot: 63100) of 6.0–6.4 μm diameter, (iii) corn starch and (iiii) four different Holi colours. Respective particles were suspended in PBS (Phosphate buffered saline) (Biochrom, Berlin, Germany) and diluted with CASY® ton (La Roche Diagnostics GmbH, Basel, Switzerland) according to the manufacturer’s instructions for measuring. Particle count was evaluated in a size range between 0.7 and 30 μm.
Stimulation of PBMCs
Blood was withdrawn under authorised supervision from six healthy donors who had given their informed consent. PBMCs were isolated from lithium heparin anti-coagulated blood by Ficoll-Paque™ Plus (GE Healthcare Bio-Sciences AB, Uppsala, Sweden) density gradient centrifugation and resuspended in VLE RPMI 1640 (Very Low Endotoxin) media (Biochrom AG, Berlin, Germany) supplemented with 10 % (v/v) FCS (Fetal Calf Serum), 2 mM L-glutamine (Sigma Aldrich, Chemie GmbH, München, Germany) and 1 % (v/v) penicillin/streptomycin (Biochrom AG, Berlin, Germany). 500 μl of a suspension of PBMCs (2 × 106 cells/ml) were incubated in sterile 24-well plates (Multiwell™, Falcon®, Becton Dickinson Labware, NJ, USA) for 4 h at 37 °C and 5 % CO2 with corn starch and the four different Holi colours (see above), respectively, each at 1.5 × 106 particles/ml. The utilised concentration of the Holi colours and corn starch arose from dose-effect experiments that we performed with ambient dust (data not shown). Cells treated with PBS only (7.5 % v/v) served as negative control. Cells treated with LPS (Lipopolysaccharide) (Enzo Life Sciences, NY, USA) at 100 ng/ml served as positive control. Samples were centrifuged for five minutes at 300 g and supernatants were transferred to fresh cell culture plates and frozen at −20 °C until further analysed.
Stimulation of whole blood
Blood was withdrawn under authorised supervision from six healthy donors who had given their informed consent. 100 μl of a PBS suspension containing corn starch, the four different Holi colours or LPS respectively was mixed with 1 ml of a 0.9 % NaCl solution (Fresenius Kabi Deutschland GmbH, Bad Homburg, Germany). Subsequently 100 μl of lithium heparin anti-coagulated whole blood was added, samples were mixed carefully and stimulated in sterile 1.5 ml safe-lock tubes (Biopur, Eppendorf AG, Hamburg, Germany) overnight at 37 °C and 5 % CO2. The final concentration of corn starch and the four different Holi colours was 6.8 × 105 particles/ml and for LPS it was 45.45 ng/ml. PBS only served as negative control. Samples were then mixed carefully and centrifuged for 5 min at 300 g and supernatants were transferred to fresh Eppendorf tubes and frozen at −20 °C until further analysed.
Analysis of cytokine production by ELISA
The concentration of the cytokines TNF-α, IL-6 and IL-1β in the supernatant was determined using commercially available ELISA kits (R&D Systems, Minneapolis, USA) following the manufacturer’s instructions. PBMC samples were blocked with 300 μl 1 % BSA (Bovine Serum Albumin) (Sigma Aldrich, St. Louis, USA) in PBS and whole blood with 300 μl 3 % BSA in PBS.
ELISA data are presented as mean ± SD (standard deviation) values from six independent experiments. Statistical analysis was performed using SPSS software version 18 (SPSS Inc., Chicago, USA). The Wilcoxon matched pairs signed-ranked test was used to test for group differences. P-values < 0.05 (two-tailed) were considered as significant.
Analysis of endotoxin content of samples by Limulus Amebocyte Lysate test (LAL test)
The endotoxin content of an unstimulated control, Holi colours 1–4, corn starch and LPS was determined using a commercially available Limulus Amebocyte Lysate test kit (Pierce® LAL Chromogenic Endotoxin Quantitation Kit, Pierce Biotechnology, Rockford, USA) as described by the manufacturer. Corn starch and Holi colour samples were applied in duplicate at 1.5 × 106 particles/ml, LPS at 100 ng/ml.
XTT test
100 μl HepG2 cells (c = 2.5 × 105 cells/ml, Leibniz Institut, DSMZ, Braunschweig, Germany) were disseminated in RPMI 1640 media (Sigma-Aldrich Chemie GmbH, München, Germany) supplemented with 10 % (v/v) FCS (Sigma-Aldrich Chemie GmbH, München, Germany) in a 96 well plate (Cellstar, Greiner Bio One International GmbH, Leipzig, Germany) and incubated over night at 37 °C and 5 % CO2 for adherence. Then media was discarded and replaced by 100 μl RPMI 1640 supplemented with 1 % (v/v) FCS. Subsequently 100 μl of a single Holi colour or corn starch suspension in PBS were added at a final concentration of 1.7 × 106, 2.5 × 106, 5 × 106 and 1 × 107 particles/ml, each in quadruplicate. After 4 h of incubation cells were washed with PBS and supplemented with 100 μl of fresh RPMI 1640 containing 1 % (v/v) FCS. Then 50 μl of XTT reagent was added according to the manufacturer’s instructions (Cell Proliferation Kit II (XTT), product number 11465015001, Sigma-Aldrich Chemie GmbH, München, Germany). After 3 h of incubation at 37 °C and 5 % CO2 samples were mixed thoroughly and measured on a microplate reader (Anthos Zenyth 200RT, Biochrom, Cambridge, UK) at 490 nm (reference wavelength 690 nm). Vitality of cells was calculated in percent in relation to an unstimulated control sample.
Propidium iodide test
500 μl Jurkat cells (c = 1 × 106 cells/ml, Leibniz Institut, DSMZ, Braunschweig, Germany) in RPMI 1640 media supplemented with 10 % (v/v) FCS were disseminated in a 24 well plate (Cellstar, Greiner Bio One International GmbH, Leipzig, Germany). Then 500 μl of single Holi colour or corn starch suspension in PBS were added to reach a final concentration of 1.7 × 106, 2.5 × 106, 5 × 106 and 1 × 107 particles/ml, respectively. Samples were incubated for 4 h and for 20 h at 37 °C and 5 % CO2. After washing with 1 ml PBS cells were suspended in 1 ml fresh media without FCS and phenol red (RPMI 1640, Sigma-Aldrich Chemie GmbH, München, Germany), incubated for 15 min with 8 μg/ml propidium iodide (Sigma-Aldrich Chemie GmbH, München, Germany) in the dark at room temperature and analysed by Flow Cytometry (FACS Calibur, BD Biosciences). Vitality of cells was calculated in percent in relation to an unstimulated control sample.
Light Microscopy of corn starch and Holi colours together with human leukocytes
Blood was withdrawn under authorised supervision from two healthy donors who had given their informed consent. Cells were treated as described for the stimulation of PBMCs and stimulation of whole blood. After the 4 h, respectively overnight incubation, 20 μl of the cell-corn starch or cell-Holi colour mixture was pipetted onto an object slide, covered with a cover slip and immediately analysed under a light microscope (Axio Vert A1 with Axiovision Rel.4.8.2 software, Zeiss, Jena, Germany). Some of the whole blood samples were also stained with Syto 9 (Life Technologies, USA,) for 10 min as described by the manufacturer and analysed under the microscope in the green fluorescence channel.
Phagoburst Assay
Blood was withdrawn under authorised supervision from four healthy donors who had given their informed consent. Phagoburst™ reagent Kit (Glycotope Biotechnology, Germany) was used to measure the oxidative burst activity of monocytes and granulocytes in human whole blood as described by the manufacturer except for the incubation time at 37 °C which was reduced to overall 12 min instead of 20 min due to a reduced cell viability observed at the longer incubation time. Additionally to the reagents in the kit, corn starch and Holi colour 1 were incubated with the heparinised human whole blood. The concentrations of corn starch and Holi colour 1 corresponded to the E.coli (Escherichia coli) concentration of 2 × 109 particles/ml. Samples remaining on ice served as negative control.
Analysis of mould content
100 μl of each Holi colour and corn starch suspension (c = 1.5 × 106 particles/ml) was inoculated under sterile conditions on malt extract agar (MEA, OXOID GmbH, Wesel, Germany) and Dichloran 18 % Glycerol agar (DG18, heipha Dr. Müller GmbH, Eppelheim, Germany). The agar plates were incubated at 25 °C and 36 °C for 10 days, respectively. The micro-morphology of selected colonies was analysed by optical microscopy (Axioskop 40 FL, Carl Zeiss MicroImaging GmbH, Göttingen, Germany) at 200× and 1000× magnification and subsequently photographed with a digital camera (Canon PowerShot Gs, Canon Deutschland, Krefeld, Germany).
Discussion
Holi colours can potentially be harmful to human health: they contain a considerable amount of particles with an aerodynamic diameter smaller than 10 μm and at least in vitro they show a close association with human leukocytes, a pro-inflammatory potential, they can have cytotoxic effects in higher concentration and can induce an oxidative burst in human granulocytes and monocytes.
Extensive use of Holi powder at Holi festivals will result in a considerable increase in PM10 concentrations in ambient air. Adverse health effects due to long-term exposure to high PM10 concentrations are widely known: increasing concentrations of particulate matter are related to a higher cardiovascular and respiratory morbidity and mortality [
9‐
13]. Also, there is evidence for short-term effects of air pollutants: increased PM10 concentrations were also associated with an increase in daily mortality [
14,
15]. In addition, a positive association of dust storms with mild asthma manifestations in children, as indicated by medication purchases, could be shown recently [
16].
In this study the particle size of the examined Holi colours differed (Fig.
1). While Holi colours 2 and 3 consisted of about 80 % of particles smaller than 10 μm in diameter - with the majority of particles being even smaller than 5 μm (Fig.
1d–
e) -, the particle size of Holi colours 1 (Fig.
1c) and 4 (Fig.
1f) ranged between 0.7 and 20 μm with a PM10 content of 67 and 43 %, respectively.
The size resolution in the CASY® analysis confirmed approximately the average particle size of corn starch (Fig.
1b) stated in the literature: 15 μm in diameter [
17]. But except for Holi colour 4 the other colours contained a greater number of smaller particles. Thus, the corn starch used for Holi colour production might be somehow modified during the colour production process or mixed with other substances. For the Holi colours 1, 3 and 4 it is also possible that they comprise another carrier substance, e.g. rice flour or other anticaking agents.
It is difficult to compare the specific exposure at/or in the vicinity of Holi festivals with the “normal” ambient PM10 exposure i.e. caused by road traffic or combustion processes. The composition of the particulate matter seems to have a great influence on the biological effects as i.e. assessed by cytokine induction [
18]. Also epidemiological data show that not only the particle size but furthermore the chemical composition accounts for harmful health effects [
19,
20].
In this study, the four different Holi colours induced diverging amounts of pro-inflammatory cytokines as shown in the cell culture experiments (Fig.
2). Differences in the proportion of particle size alone cannot account for the different levels of pro-inflammatory potential: although Holi colours 2 and 3 had nearly the same amount of particles >0.7 μm and <10 μm (about 80 %), they induced significantly diverging amounts of TNF-α, IL-6 and IL-1β.
In general as compared to PBMCs, which were stimulated for 4 h, (Fig.
2a) we found a decrease in TNF-α production and an increase in IL-1β production in the whole blood experiments (Fig.
2b), where cells were incubated with the respective substances overnight. This corresponds well with the kinetics of release of TNF-α and IL-1β in whole blood and PBMCs: TNF-α reaches maximal levels within 6 h of stimulation, whereas IL-1β reaches maximal levels between 12 and 16 h [
21,
22].
The release of the pro-inflammatory cytokines TNF-α, IL-6 and IL-1β by human cells stimulated in vitro with particulate matter PM10 or nanoparticles has been shown in various studies [
18,
23‐
27]. The fact that exposure to swine dust in vivo also leads to an increase of TNF-α, IL-6 and IL-1β in peripheral blood of healthy volunteers [
28] suggests, that these cytokines might also be able to mediate this kind of inflammatory response in vivo.
Endotoxin is a potent inducer of various pro-inflammatory cytokines [
8]. To elucidate the role of endotoxin in the cytokine induction of the Holi colours, we measured the amount of endotoxin in the different Holi samples by means of the LAL test. We did not detect any endotoxin in Holi colours 1 and 2, but discovered endotoxin in corn starch as well as in Holi colours 3 and 4, albeit these amounts of endotoxin are much below the endotoxin level measured in LPS (Fig.
3). But since already very low amounts of endotoxin in the picogramme range (which represent Endotoxin units of 1 EU/ml or even below according to the manufacturer’s product guide of the LAL test used here) can be responsible for considerable cytokine secretions the observed cytokine induction of Holi colours 3 and 4 might be caused by endotoxin. (We performed dose response tests for endotoxin levels and cytokines of interest. The results will be subject of a separate publication.) Nevertheless as Holi colour 1 contains no endotoxin and Holi colours 3 and 4 contained lesser endotoxin than LPS but induced a stronger cytokine release, we argue that also other factors than endotoxin might drive the cytokine answer.
Hansen et al. have shown that the potency of endotoxin from different Gram-negative bacteria in the LAL-assay is not closely correlated with the potency of the endotoxin to induce IL-8 (Interleukine-8) secretion from pulmonary epithelial cells [
29]. This observation might be true for other pro-inflammatory cytokines as well. Hence the results of the LAL-test do not necessarily have to correlate with the cell culture data. This would also explain why corn starch, which contained endotoxin, only induces a very low cytokine release.
The XTT test and Propidium iodide test showed possible cytotoxic effects of Holi colours 2 and 3 with increasing Holi colour concentration, while corn starch and Holi colours 1 and 4 did not display any cytotoxicity thus underlying the heterogeneity of Holi colours. Since the observed cytotoxicity occurs only at higher concentrations or is rather less pronounced in the lowest concentration tested, respectively, their relevance to health regarding a real Holi festival situation would need further investigation.
Although fluorescence microscopy (Fig.
4f) showed a close particle-cell-association the nature of this association remains unclear: are the particles bound to the cell surface and if so through what kind of receptor or are the particles phagocytosed by the cell? Due to the impossibility of quenching the autofluorescent signal of the Holi colours in the flow cytometric analysis an exact discrimination of particles bound to the cell surface and particles phagocytosed by the cell was not feasible and we were not able to show an internalisation of Holi particles directly (data not shown). But as corn starch and to a lesser extent also Holi colour 1 induced an oxidative burst in human granulocytes and monocytes (Fig.
5), one can speculate that these cells might engulf the particles. However, the limitation remains, that the Phagoburst assay is an in vitro setup that is very different from the situation in vivo regarding i.e. time of exposure and particle concentration. Still, phagocytosis and processing of particulate matter by the macrophages in the lung is an established phenomenon which leads to diverse immunological responses and cytokine release [
30].
Aspergillus spp. as well as other filamentous fungi could be detected in Holi colour 1 (Fig.
6). Moulds in general pose a risk to human health, can be involved in disorders of the respiratory tract and may contribute to the manifestation of asthma and allergies [
31]. As Kawakami et al. [
32] and Wykoff et al. [
33] reported,
Aspergillus and
Paecilomyces species can play a role in ocular infections with soft contact lens use as one predisposing factor. In the context of numerous ocular irritations reported by medical services on Holi festivals (Becker et al.,
submitted) this detection of fungi may be of clinical relevance. Deeper insights in the microbiological contamination profile of Holi colours might be of interest for future investigations.
Not much is known about the ingredients of the Holi colours (see Table
1). Even the carrier substance often remains unknown. As the composition (carrier substance, colour pigments, anticaking agents, etc.) varies from colour to colour, also the observed in vitro effects differ. Similarly, underlying mechanisms as well as the generation of possible health effects might be diverse and dependent on the specific colour. However, these various modes of action might also work in an additive or synergistic fashion as i.e. endotoxin, fungal contamination and leukocyte oxidative bursts might potentially lead to the observed induction of pro-inflammatory cytokines. Further experiments are needed to elucidate the distinct underlying mechanisms in more detail.