title: Paradoxical cytotoxicity of tert-butylhydroquinone in vitro: What kills the untreated cells?
abstract: At high concentrations, tert-butylhydroquinone (tBHQ), a phenolic antioxidant frequently used as a food preservative, exerts cytotoxic effects, which are closely linked to its ability to form reactive oxygen species as a consequence of redox cycling processes. Here we describe that treatment of murine 3T3 cells with 300 μg/ml of tBHQ in 96-well culture plates induces the death of untreated cells in neighboring wells on the same plate. The mechanisms underlying that effect were investigated. Death of the seemingly untreated neighboring cells was caused by the more toxic and volatile tBHQ oxidation product tert-butyl-p-benzoquinone (tBQ) present at up to 3 μg/ml in the untreated neighboring wells. tBQ was formed from tBHQ in a non-enzymatic process involving copper ions and oxygen. The unexpected perturbation of cytotoxicity testing following treatment with tBHQ by its volatile metabolite tBQ shows that not only metabolic processes but also non-enzymatic mechanisms have to be considered as important parameters for in vitro assays. Furthermore, our data show that even cells several wells away from the treated wells do not necessarily constitute proper "untreated" controls when cells are treated with the frequently used compound tBHQ. This might lead to an underestimation of the effects observed on the Nrf2 signaling pathway, where tBHQ is frequently used as an inductor in vitro.
authors.affiliation: Department of Toxicology, Institute of Experimental and Clinical Pharmacology and Toxicology, University of Tübingen, Wilhelmstr. 56, 72074 Tübingen, Germany. albert.braeuning@uni-tuebingen.de
authors.forename: Albert
authors.initials: A
authors.lastname: Braeuning
authors.forename: Silvia
authors.initials: S
authors.lastname: Vetter
authors.forename: Silvia
authors.initials: S
authors.lastname: Orsetti
authors.forename: Michael
authors.initials: M
authors.lastname: Schwarz
mesh_terms: D016475:3T3 Cells; D000818:Animals; D000975:Antioxidants / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology*; D016227:Benzoquinones / Q000032:analysis / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D002470:Cell Survival / Q000187:drug effects; D002614:Chelating Agents / Q000737:chemistry; D003300:Copper / Q000737:chemistry; D005347:Fibroblasts / Q000187:drug effects / Q000378:metabolism; D005520:Food Preservatives / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology*; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects; D005982:Glutathione Transferase / Q000235:genetics / Q000378:metabolism; D006873:Hydroquinones / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology*; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism; D051379:Mice; D009994:Osmolar Concentration; D010084:Oxidation-Reduction; D012333:RNA, Messenger / Q000378:metabolism; D015203:Reproducibility of Results; D013997:Time Factors; D014835:Volatilization
chemical_list: D000975:Antioxidants; D016227:Benzoquinones; D002614:Chelating Agents; D005520:Food Preservatives; D006873:Hydroquinones; D007527:Isoenzymes; D012333:RNA, Messenger; D003300:Copper; C018855:2-tert-butylhydroquinone; D005982:Glutathione Transferase; C119995:glutathione S-transferase alpha; C051290:2-tert-butyl-4-quinone