In vitro bioassay for reactive toxicity towards proteins implemented for water quality monitoring

Reactive organic chemicals comprise a large number of compounds with a variety of reactive moieties. While most assays for reactive toxicity focus on DNA damage, reactivity towards proteins can also lead to irreparable damage, but reactivity towards proteins is typically not included in any test battery for water quality assessment. Glutathione (GSH) is a small tripeptide whose cysteine moiety can serve as a model for nucleophilic sites on proteins. GSH is also an important indicator of detoxification processes and the redox status of cells and due to its protective role, depletion of GSH ultimately leads to adverse effects. A bioassay based on genetically modified Escherichia coli strains was used to quantify the specific reactivity towards the protein-like biological nucelophile GSH. The significance of GSH for detoxification was assessed by comparing the growth inhibition induced by reference chemicals or water samples in a GSH-deficient strain to its fully functional parent strain. The GSH deficient strain showed the same sensitivity as the GSH proficient strain to non-reactive and DNA damaging chemicals, but was more sensitive to chemicals that attack cysteine in proteins. The difference in effect concentrations for 50% inhibition of growth assessed as biomass increase (EC50) between the two strains indicates the relevance of GSH conjugation as a detoxification step as well as direct reactivity with cysteine-containing proteins. Seven reference compounds serving as positive and negative controls were investigated. The E. coli strain that lacks GSH was four times more sensitive towards the positive control Sea-Nine, while negative controls benzo[ a] pyrene, 2-aminoanthracene, phenol, t-butylhydroquinone, methyl methane sulfonate and 4-nitroquinoline oxide showed equal effect concentrations in both strains. Water samples collected across an indirect potable reuse scheme representing the complete water cycle from sewage to drinking water in South East Queensland, Australia were used to evaluate the applicability of the E. coli assay for reactive toxicity in water samples. While the EC50 values of the GSH+ strain showed similar trends as in other biological endpoints over the various treatment chains, the specific response indicative of protein damage was only observed in samples that had undergone chlorination as a disinfection process. High natural organic matter or other matrix components disturbed the bioassay so much that we recommend it for future routine testing only in tertiary treated water or drinking water.