In vitro induction of apoptosis, necrosis and genotoxicity by cosmetic preservatives: application of flow cytometry as a complementary analysis by NRU

Abstract

Preservatives are used in cosmetics to prevent microbial contamination; however, some preservatives are not free of allergenic and cytotoxic potential. Allergenicity and cytotoxicity potential values are major aspects of preservative safety, which determine limitations and maximum concentration dose in a cosmetic product. The purpose of this study was to investigate and compare the in vitro apoptosis, necrosis and genotoxicity-inducing potential of five different types of preservatives: Phenoxyethanol (PE), Propylparaben (PP), Methylparaben (MP), Benzyl Alcohol (BA) and Ethylhexyl Glycerine (EG). In vitro experiments were carried out on human dermal fibroblasts by a quantitative flow cytometry method, using specific cell markers (Annexin V, Propidium Iodide and H2AX). We compared the resulting cell viability by means of neutral red uptake (NRU) and established the IC(50) . Our results showed that PE, PP, MP and BA have similar cytotoxic mechanisms (high apoptosis and necrosis levels only at the test concentration of 1%), whereas EG showed only an apoptosis pathway. For genotoxicity, both parabens yielded the highest values. Results obtained by flow cytometry for necrosis were comparable to those produced by NRU; however, NRU does not distinguish apoptosis from necrosis. We propose that flow cytometry is a more sophisticated methodology for understanding the cytotoxic mechanisms of cosmetic preservatives and can be used to complement the NRU.

Parabens can enable hallmarks and characteristics of cancer in human breast epithelial cells: a review of the literature with reference to new exposure data and regulatory status

“…In this way, apoptosis serves as a natural prevention for cancer, and factors that can inhibit apoptotic mechanisms have the potential to enhance risk of cancer development. Parabens have been shown to induce cell death by apoptosis in several cell types in vitro, including in human skin keratinocytes (Handa et al, 2006;Ishiwatari et al, 2007), in human dermal fibroblasts (Carvalho et al, 2012), in human hepatoma HepG2 cells (Khanal et al, 2012) and in rat pheochromocytoma (adrenal) PC12 cells (Egawa et al, 2012). However, all these studies were carried out at high doses of parabens in the 100 μM or above range, which are well above any concentrations measureable in human breast tissue (Barr et al, 2012).…”

In vitro cytotoxicity of chemical preservatives on human fibroblast cells

“…Other studies confirmed the low toxicity of parabens as well, including that of Soni, Carabin, and Burdock (2005) who showed that methyl and propylparaben at the concentrations of 0.25% and 0.05%, respectively, did not induce hemolysis in human and rabbit erythrocytes. In contrast, Carvalho et al (2012) evaluated the potential of some preservatives for the induction of apoptosis, necrosis, and genotoxicity against HDF cells (human fibroblasts) following the 24hours of exposure. Their results showed that methyl and propylparaben have increased genotoxic potential.…”

DNA damage signaling assessed in individual cells in relation to the cell cycle phase and induction of apoptosis

“…Utility of the γH2AX assay has been also tested in the assessment of genotoxic properties of agents used to manufacture plastics and epoxy resins such as bisphenol A and bisphenol F, 167 ubiquitous pollutants such as polycyclic aromatic hydrocarbons, 168 the non-ionic surfactans nonylphenol polyethoxylates that are widely used for industrial and household purposes, 169 potential genotoxins in cosmetic preservatives, 170 or several different pharmaceuticals frequently detected in freshwater environment. 171 …”