Holi colours can potentially be harmful to human health: they contain a considerable amount of particles with an aerodynamic diameter smaller than 10 ?m 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 .
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 ?m in diameter – with the majority of particles being even smaller than 5 ?m (Fig. 1d–e) -, the particle size of Holi colours 1 (Fig. 1c) and 4 (Fig. 1f) ranged between 0.7 and 20 ?m 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 ?m in diameter . 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 . 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 ?m and 10 ?m (about 80 %), they induced significantly diverging amounts of TNF-?, IL-6 and IL-1?.
In general as compared to PBMCs, which were stimulated for 4 h, (Fig. 2a) we found a decrease in TNF-? production and an increase in IL-1? 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-? and IL-1? in whole blood and PBMCs: TNF-? reaches maximal levels within 6 h of stimulation, whereas IL-1? reaches maximal levels between 12 and 16 h [21, 22].
The release of the pro-inflammatory cytokines TNF-?, IL-6 and IL-1? 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-?, IL-6 and IL-1? in peripheral blood of healthy volunteers  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 . 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 . 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 .
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 . As Kawakami et al.  and Wykoff et al.  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.