Effect of methyl p-hydroxybenzoate on the culture of mammalian cell

Several chemicals, such as methyl p-hydroxybenzoate (MHB), have been widely used as preservatives in the water baths of CO₂ incubators used for mammalian cell culture, and they are not considered to produce any biological effects. However, no detailed analyses of the effects of these compounds on cultured cells have been reported. In this study, we thus examined whether MHB in the incubator water bath affects cell viability or genome-wide gene expression in mouse embryonic stem cells under control conditions [using only dimethyl sulfoxide (DMSO) in the culture medium] and under chemical-treated conditions using benzene and chloroform; conditions that simulate a cell-based toxicity assay. We found that (i) MHB significantly altered cell growth rate, and (ii) MHB affected gene expression levels related to pathways that modulate cell growth and basic molecular processes, not only under control conditions but also the chemical-treated conditions. Furthermore, Gene Ontology term analyses revealed that the effects of MHB cannot be accounted for by subtracting the gene expression pattern in the control conditions from that in the chemical-treated conditions. Thus, we suggest that the use of MHB or other preservatives in a CO₂ incubator water bath is reconsidered in terms of potential confounding effects on cultured cells.