P21-03 Effects of individual and combined exposure to parabens on neutrophil oxidative burst

Parabens, a group of alkyl esters of 4-hydroxybenzoic acid, are widely used as antimicrobial preservatives in cosmetics, pharmaceuticals, and food products [1] . However, increasing evidence suggests that parabens act as endocrine-disrupting chemicals with the potential to induce inflammatory responses [2] . While the effects of individual parabens have been described, little is known about their combined impact, despite daily exposure typically occurring through mixtures. Given that neutrophils are among the first immune cells to respond to xenobiotics, this study aimed to investigate the effects of parabens, both individually and in combination, on human neutrophils. Isolated human neutrophils were exposed to methylparaben (MeP), ethylparaben (EtP), propylparaben (PrP), butylparaben (BuP), and to a mixture designed combining these parabens at their EC01, to assess their impact on oxidative burst and cell viability. Reactive species (RS) production by neutrophils was evaluated using the fluorescent probe dihydrorhodamine (DHR) 123, while inhibitors of oxidative burst-related enzymes were employed to elucidate the mechanisms underlying RS production. To predict the combined effects, two additive models – concentration addition (CA) and independent action (IA) – were applied based on DHR 123 assay data. Overall, all parabens induced neutrophils’ oxidative burst, with MeP leading to the highest RS production, involving phospholipase C activation, followed by protein kinase C activation, and ultimately resulting in the activation of NADPH oxidase and myeloperoxidase. Notably, when combined at concentrations that elicited individual effects that did not differ from controls, these parabens acted additively to elicit significant effects. None of the parabens, either individually or in combination, induced apoptosis or necrosis at the tested concentrations. This study provides novel insights into the effects of paraben exposure on human neutrophils. Given their widespread use, these findings support that future studies should not neglect the potential health implications of mixture effects, as additive toxicity may arise even at low individual exposures. Funding: This work received financial support from the PT national funds (FCT/MECI, Fundação para a Ciência e Tecnologia and Ministério da Educação, Ciência e Inovação) through the project UID/50006 –Laboratório Associado para a Química Verde – Tecnologias e Processos Limpos. Acknowledgements: This work was also supported by the FCT/MCTES through the Strategic Projects LA/P/0008/2020 (DOI: 10.54499/LA/P/0008/2020) and UIDP/50006/2020 (DOI: 10.54499/UIDP/50006/2020. AR thanks FCT and ESF (European Social Fund) for her PhD grant ref. 2023.00361.BD. MF acknowledges her CEEC contract- 2020.04126. CEECIND/CP1596/CT0006 and to LAQV-REQUIMTE for her contract under the ref. LA/P/0008/2020:DOI 10.54499/LA/P/0008/2020.