title: Cytotoxicity and molecular effects of biocidal disinfectants (quaternary ammonia, glutaraldehyde, poly(hexamethylene biguanide) hydrochloride PHMB) and their mixtures in vitro and in zebrafish eleuthero-embryos.
abstract: Frequently used biocidal disinfectants, including quaternary ammonium compounds (QAC), glutaraldehyde and poly(hexamethylene biguanide) hydrochloride (PHMB), occur in the aquatic environment but their potential effects in fish are poorly known, in particular when occurring as mixtures. To investigate their joint activity, we assessed the cytotoxicity of three QACs (BAC, barquat and benzalkonium chloride), glutaraldehyde andPHMB by the MTT assay individually, followed by assessing binary and ternary mixtures in zebrafish liver cells (ZFL) and human liver cells (Huh7). We also analysed molecular effects by quantitative PCR in vitro and in zebrafish eleuthero-embryos employing a targeted gene expression approach. QACs displayed strong cytotoxicity in both cell lines with EC50values in the low μg/ml range, while glutaraldehyde and PHMB were less cytotoxic. Most of the binary and both ternary mixtures showed synergistic activity at all equi-effective concentrations. A mixture containing all five compounds mixed at their no observed effect concentrations showed strong cytotoxicity, suggesting a synergistic interaction. Additionally, we determined transcriptional alterations of target genes related to endoplasmatic reticulum (ER) stress, general stress, inflammatory action and apoptosis. Induction of ER stress genes occurred at non-cytotoxic concentrations of barquat, glutaraldehyde and BAC in ZFL cells. Barquat and BAC induced tumor necrosis factor alpha (tnf-α). Similar transcriptional alterations were found in vivo upon exposure of zebrafish eleuthero-embryos for 120h. Glutaraldehyde led to induction of ER stress genes and tnf-α, while BAC additionally induced genes indicative of apoptosis, which was also the case with benzalkonium chloride at the highest concentration. We demonstrated strong cytotoxicity of QACs, and synergistic activity of binary, ternary and quintuple mixtures. Barquat and BAC let to induction of ER stress and inflammation in vitro, and BAC and glutaraldehyde at non-toxic concentrations in vivo, while benzalkonium chloride induced expression of tnf-α only.
authors.affiliation: University of Applied Sciences and Arts Northwestern Switzerland, Gründenstrasse 40, CH-4132 Muttenz, Switzerland.
authors.forename: Verena
authors.initials: V
authors.lastname: Christen
authors.affiliation: University of Applied Sciences and Arts Northwestern Switzerland, Gründenstrasse 40, CH-4132 Muttenz, Switzerland.
authors.forename: Susanne
authors.initials: S
authors.lastname: Faltermann
authors.affiliation: University of Applied Sciences and Arts Northwestern Switzerland, Gründenstrasse 40, CH-4132 Muttenz, Switzerland.
authors.forename: Nadja Rebecca
authors.initials: NR
authors.lastname: Brun
authors.affiliation: Federal Office for the Environment (FOEN), Air Pollution Control and Chemicals Division, Biocides and Plant Protection Products Section, 3003 Berne, Switzerland.
authors.forename: Petra Y
authors.initials: PY
authors.lastname: Kunz
authors.affiliation: University of Applied Sciences and Arts Northwestern Switzerland, Gründenstrasse 40, CH-4132 Muttenz, Switzerland; Swiss Federal Institute of Technology Zürich, Department of Environmental System Sciences, CH-8092 Zürich, Switzerland. Electronic address: karl.fent@bluewin.ch.
authors.forename: Karl
authors.initials: K
authors.lastname: Fent
mesh_terms: D000641:Ammonia / Q000633:toxicity; D000818:Animals; D001645:Biguanides / Q000633:toxicity; D002460:Cell Line; D004202:Disinfectants / Q000633:toxicity*; D004625:Embryo, Nonmammalian / Q000187:drug effects*; D005976:Glutaral / Q000633:toxicity; D006801:Humans; D018675:Toxicity Tests; D015027:Zebrafish
chemical_list: D001645:Biguanides; D004202:Disinfectants; C031233:polihexanide; D000641:Ammonia; D005976:Glutaral
keywords: Biocidal disinfectants; Cytotoxicity; ER stress; Inflammation; Mixture activity; Synergism