Holi colours contain PM10 and can induce pro-inflammatory responses

Abstract

Background: At Holi festivals, originally celebrated in India but more recently all over the world, people throw coloured powder (Holi powder, Holi colour, Gulal powder) at each other. Adverse health effects, i.e. skin and ocular irritations as well as respiratory problems may be the consequences. The aim of this study was to uncover some of the underlying mechanisms.

Methods: We analysed four different Holi colours regarding particle size using an Electric field cell counting system. In addition, we incubated native human cells with different Holi colours and determined their potential to induce a pro-inflammatory response by quantifying the resulting cytokine production by means of ELISA (Enzyme Linked Immunosorbent Assay) and the resulting leukocyte oxidative burst by flow cytometric analysis. Moreover, we performed the XTT (2,3-Bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide) and Propidium iodide cytotoxicity tests and we measured the endotoxin content of the Holi colour samples by means of the Limulus Amebocyte Lysate test (LAL test).

Results: We show here that all tested Holi colours consist to more than 40 % of particles with an aerodynamic diameter smaller than 10 μm, so called PM10 particles (PM, particulate matter). Two of the analysed Holi powders contained even more than 75 % of PM10 particles. Furthermore we demonstrate in cell culture experiments that Holi colours can induce the production of the pro-inflammatory cytokines TNF-α (Tumor necrosis factor-α), IL-6 (Interleukine-6) and IL-1β (Interleukine-1β). Three out of the four analysed colours induced a significantly higher cytokine response in human PBMCs (Peripheral Blood Mononuclear Cells) and whole blood than corn starch, which is often used as carrier substance for Holi colours. Moreover we show that corn starch and two Holi colours contain endotoxin and that certain Holi colours display concentration dependent cytotoxic effects in higher concentration. Furthermore we reveal that in principle Holi colours and corn starch are able to generate an oxidative burst in human granulocytes and monocytes. In Holi colour 1 we detected a fungal contamination.

Conclusions: Some of the observed unwanted health effects of Holi colours might be explained by the high content of PM10 particles in conjunction with the possible induction of a pro-inflammatory response and an oxidative leukocyte burst.

Keywords: Holi colours; IL-1β; IL-6; Oxidative burst; PM10; Pro-inflammatory response; TNF-α.

Fig. 1

Particle size distribution of beads, corn starch and Holi colours. Illustrated is the particle size distribution of commercially available beads of 2 µm, 3 µm and 6-6.4 µm (a), corn starch (b) and 4 different Holi colours (c-f). The gate _P0.7-10µm indicates the percentage of particles bigger than the detection limit of 0.7 µm but smaller than 10 µm in diameter

Fig. 2

Induction of pro-inflammatory cytokines by corn starch and Holi colours. Shown is the production of TNF-α, IL-6 and IL-1β induced by corn starch and Holi colours 1-4 in (a) PBMCs and (b) whole blood. PBS served as negative and LPS as positive control. Each bar represents the mean value of six independent experiments representing 6 different probands. The standard deviation is depicted by the error bars. # significant (p < 0.05) when compared to unstimulated cells. * significant (p < 0.05) when compared to unstimulated cells and cells treated with corn starch

Fig. 3

Endotoxin content of corn starch and Holi colours 1–4. The endotoxin content of corn starch and Holi colours 1–4 was determined by LAL test. The resulting endotoxin amount was measured in Endotoxin Units per ml (EU/ml). Samples were applied in the same concentration as used for stimulation of PBMCs (corn starch and Holi colours 1–4: c = 1.5 × 10⁶ particles/ml, LPS: 100 ng/ml)

Fig. 4

Light Microscopic images of human PBMCs together with Holi colours or corn starch. a–e shows 200 fold magnifications of native preparations of human PBMCs incubated for 4 h together with corn starch (a), Holi colour 1 (b), Holi colour 2 (c), Holi colour 3 (d) and Holi colour 4 (e). Cell sizes are indicated in black and particle sizes in red and the respective radiuses are stated. (Note that very small particles could not be indexed.) f displays a 400 fold magnification of cells incubated overnight with Holi colour 3, then stained with the nucleic acid dye Syto 9 and analysed in the green fluorescence channel

Fig. 5

Induction of leukocyte oxidative burst by corn starch and Holi colour1. Exemplified is the illustration of the percentage of granulocytes (a-c) and monocytes (d-f) which produce reactive oxidants in one proband after a and d: incubation with corn starch at 0 °C (negative control); b and e: incubation with corn starch at 37 °C; c and f: incubation with Holi colour 1 at 37 °C. The gate P_FITC+ displays the particles considered FITC positive and respective percentages are given

Fig. 6

Holi colour 1 contains mould. a exhibits a photograph of a DG18 agar plate after inoculation with Holi colour 1 and incubation for 10 days at 25 °C. b–d displays microscopic images of different isolates of mould fungi (b Aspergillus sp., c unidentified Zygomycete species, d unidentified Paecilomyces/Bossychlamus species) found in Holi colour 1 (Magnification: 200×)