Project ID Assignment: HTS-3: High Throughput Screening to Determine the Mechanistic Toxicology
of Engineered Nanomaterials in Bacteria
Hilary Godwin, Patricia Holden, Angela Ivask, Chitrada Kaweeteerawat
Abstract:
This project aims to demonstrate that high-throughput screening (HTS) of nanoparticles (NPs) can be
performed in bacteria as models for environmentally relevant organisms and that these experiments
can be used to predict the hazards that different NPs pose to environmental systems. Because
bacteria are critical sentinel species and form a biological foundation for ecosystems, demonstration
of effective use of HTS for nanotoxicology in bacterial systems is a high priority for the UC CEIN. In
this project, we are capitalizing on the ground work that has been laid in bacterial nano highthroughput screening in UC CEIN in performing the following experiments: (i) screening of a broad
range of ENMs using a HT growth assay with E. coli cells, (ii) screening of a broad range of ENMs using
HT sub-lethal assays for toxicological endpoints in E. coli and (iii) systematically studying which gene
pathways are involved in toxicological responses to highly toxic NPs using a genome-wide collection
of E. coli gene deletion strains.
To date, we have identified optimal suspension conditions (using DLS) and screened the toxicity of a
sets of: (i) nine different TiO2 nanomaterials including a series of Fe-doped TiO2, (ii) 24 metal oxide
nanomaterials, (iii) five different Ag nanomaterials, and (iv) four polystyrene nanomaterials. These
ENM libraries were selected to complement the studies in mammalian cells as well as in vivo studies.
Our major findings to date are that:
(i)
Under the conditions studied, the library of TiO2 nanomaterials, including Fe-doped TiO2,
exhibited toxicity only after UV illumination conditions; the antibacterial effects of this library
of ENMs decreased with increasing Fe doping as a result of decreased hydroxyl radicals
production. This work is complementary to phototoxicity studies performed on these
materials using mammalian cells under the Project ENM-4.
(ii) Eight out of 24 metal oxide nanomaterials exhibited toxicity in the E. coli parent strain; these
results are complementary to those obtained using mammalian cell lines and will be used to
develop nano-SAR models for metal oxide nanomaterials (in collaboration with H. Zhang,
Nel´s group).
(iii) All Ag nanomaterials showed toxic effects that differed according to the materials' primary
size and surface coating. To elucidate whether mechanistic differences underlie the observed
differences in toxicity, these Ag materials will be analyzed in an HT assay using 4000 E. coli
mutant strains (see below).
(iv) The set of 4 polystyrene materials were not toxic except for smallest sized (60 nm primary
size) cationic (amino charged) material (PS-NH2). This highly toxic PS-NH2 nanomaterial has
also proven highly toxic in the study of mammalian cells, which constitutes the basis of why
this library was chosen to develop and validate the HT assay with bacterial gene deletion
mutants (see below).
We are currently in the process of developing HTS platforms to complement the bacterial growth
inhibition assays with sub-lethal assays. These assays are being developed premised on existing
protocols from Holden ´s group, which include assays for membrane integrity & permeability, ROS
generation, and electron transport. In addition, to systematically study which gene pathways are
involved in the toxicity of certain nanomaterials, we have developed a genome-wide HTS assay
exploiting 4000 E. coli mutant single gene mutant strains. In the last year, we have optimized the test
procedure for HTS with bacterial single gene deletion mutants using a toxic standard reference
nanomaterial, PS-NH2 and used this methodology to demonstrate that the two primary mechanisms
of toxicity for this material are generation of ROS and destabilization of the outer membrane. We are
currently applying this methodology to investigate the mechanisms of toxicity of Ag nanomaterials in
E. coli and to determine whether the differential levels of toxicity observed for Ag nanomaterials with
different sizes, shapes, and surface coatings reflect different mechanisms of toxicity. Based on the
results obtained from our genome-wide HTS toxicity screens, and earlier data from the literature, we
are currently developing a database of E. coli single gene mutants that are more sensitive or resistant
towards ENMs. This set of strains should serve as an important tool for defining the relationship
between physicochemical properties and mechanisms of toxicity in nanomaterials.
The impact of this work from a theme-wide perspective is that we are poised to compare bacterial
HTS results to those obtained in HTS studies in mammalian cell lines and zebrafish. What is already
clear is that not all particles that are toxic to organisms in one kingdom, class or phylum are
necessarily toxic to all others, which reinforces the importance of the holistic approach taken by the
UC CEIN in studying nanotoxicology in a broad range of platforms and organisms. At the same time,
we have been able to demonstrate that the “positive control NM” (60 nm PS-NH2 NM) which is used
across the UC CEIN is not only toxic across all organisms studied to date, but that at least one of the
mechanisms of toxicity for this NM (ROS generation) is conserved across all of the organisms studied
to date – including ones from different kingdoms. Going forward, our hope is that the sub-lethal
assays in bacteria which we are transitioning to high-throughput will provide a rapid way to prioritize
testing for not only bacteria, but other environmentally-relevant organisms.