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THE STUDY AND EXPLORATION ABOUT
NANOTOXlCITY OF OXIDE NANOPARTICLES ON
ESCHERICHIA COLI AND EXIGUOBACTERIUM
SIBIRICUM

presented by

Yu Yang

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5/08 K:IProj/Aoc&Pres/ClRClDaIeDue.indd

 

THE STUDY AND EXPLORATION ABOUT NANOTOXICITY OF OXIDE
NANOPARTICLES ON ESCHERICHIA COLI AND EXIGUOBACTERIUM
' SIBIRICUM

By

Yu Yang

A DISSERTATION
Submitted to
Michigan State University

In partial fulfillments of requirements
for the degree of

DOCTOR OF PHILOSOPHY

Crop and Soil Science

2009

 

 

 

ABSTRACT
THE STUDY AND EXPLORATION ABOUT NANOTOXICITY OF OXIDE
NANOPARTICLES ON ESCHERICHIA COLI AND EXIGUOBACTERIUM
SIBIRICUM
By

Yu Yang

Nanoparticles (NPS) have been used in a variety of areas, including cosmetics,
pigments, water purification, environmental remediation and drug delivery. However,
their toxicological and environmental effects have not been adequately investigated. I
found that ZnO and TiOz NPS reduced cell viability of Escherichia coli K12 (Gram
negative) and Exiguobacterium sibiricum strain 255-15 (Gram positive) in a
dose-dependent manner, while SiOz and Fe3O4 NPS did not in a 12 h exposure. Among
the test NPS, ZnO NPS were more toxic to E. coli than to E. sibiricum, while TiOz NPS
were more toxic to E. sibiricum than to E. coli. The smallest ZnO or TiOz NPS, 5-10 mn,
were more toxic than the larger particles to both bacteria. Zn2+ dissolved from ZnO NPS
in both LB and TSB broth, reached saturation at 100-110 ppm. The release of Zn2+ ion
played a more important role for ZnO NP toxicity to E. coli than E. sibiricum, due to the
greater sensitivity of E. coli to Zn”, while both Zn2+ and ZnO NPS showed the toxicity to
E. sibiricum.

Si02 and TiOz NPS were internalized in E. sibiricum but not in E. coli cells as
observed by transmission electron microscopy-dispersive spectroscopy, while ZnO NPS

were found internal to both of them. No particle larger than 150 nm was found

intracellularly. NPS that can be internalized by microbes may possess the stronger toxicity
than those not internalized, which is consistent with my Observations.

ZnO and TiOz NPS induced production of reactive oxygen species (ROS) in E. coli
K12 and E. sibiricum in a dose-dependent manner. Both material-effect and size-effect
were evidenced in ROS induction studies in that the capability for ROS induction
increased generally from SiOz to TiOz to ZnO, and that the smaller TiOz NPS induced
more ROS than the larger ones. More ROS was induced by SiOz NPS in E. sibiricum than
in E. coli, while ZnO NPS induced more ROS in E. coli than in E. sibiricum, which is
consistent with the viability results. Both TiOz NPS and ZnO NPs caused significant DNA
damage in lymphocytes as measured by the comet assay, and TiOz NPS were more
genotoxic than ZnO NPS. The latter is probably due to the Solubility or smaller size of the
ZnO NPS.

Using microarray technology, 1 found that SiOz, HQ», and ZnO NPS influenced gene
expression of E. coli and E. sibiricum in a variety of function (COG) categories. Genes in
the “transport” category were most affected by all treatments. When genes for amino acid
and carbohydrate transport were repressed, genes for metal iron transport were induced.
Other important categories with altered expression due to NPS were “carbohydrate
metabolism”, “lipid metabolism”, “amino acid metabolism” and “DNA repair”. The latter
may be caused by ROS damage to the membrane, amino acids in proteins, and DNA. NPs
also led to up-regulation of stress-related genes and down-regulation of cellular

respiration, also evidence for ROS induction.

Copyright by
Yu Yang
2009

To my parents Zhaolan Liang and Xiaoping Yang

ACKNOWLEGEMENTS

I would like to express my deepest gratitude to my advisors, Drs. James Tiedje and
Syed Hashsham, for their continuous guidance, mentoring, and support through my Ph.D
study. I learn a lot by their examples and I believe their infectious enthusiasm and passion
about science will always inspire me in my future research. I so appreciate their generous
encouragement and confidence, especially when I struggled with my research and almost
lost confidence in myself. I can not imagine, without their constructive advice and
comments, how I can finish this work.

I would also like to extend my great appreciation to my other guidance committee
members: Drs. Terence Marsh and Stephen Boyd, for their valuable comments and
suggestions on this work. Thanks to all the members of Tiedje’s lab and Hashsham’s lab,
for their invaluable discussions and kind help during the past five years, who make this
period of time more colorful and give me so many precious memories.

I would like to thank Drs. Alicia Pastor and Xudong Fan for their constant assistance
and advice about transmission electron microscopy and sample preparation. I also want to
say thanks to Dr. Alok K. Pandey for his valuable patience and assistance about comet
assay and Mr. Yang-lyang Pan for his generous help about research supports.

I can never thank my parents, Zhaolan Liang and Xiaoping Yang enough. Without
their love, support, motivation and encouragement over these years, I would have never
achieved my dream. NO matter what happens, they always trust me and have confidence

in me. No matter when I feel weak inside and turn to them, they always open arm and

vi

give me their generous love. I hope I can make them proud of me.

vii

TABLE OF CONTENTS

LIST OF TABLES ............................................................................................................................ x
LIST OF FIGURES ......................................................................................................................... xi
ABBREVIATIONS ........................................................................................................................ xii
CHAPTER 1. ................................................................................................................................... I
INTRODUCTION: TOXICITY OF NANOMATERIALS TO MICROORGANISMS
Abstract ............................................................................................................................... 2
Introduction ......................................................................................................................... 3
Classes ofNMs and their toxicity ....................................................................................... 5
Mechanisms of NP toxicity ............................................................................................... 11
Conclusion ........................................................................................................................ 15
References ......................................................................................................................... 20
CHAPTER 2 .................................................................................................................................. 34

TOXICITY OF OXIDE NANOPARTICLES ON ESCHERICHIA COLI AND
EXIGUOBACTERIUM SIBIRICUM

Abstract ............................................................................................................................. 35
Introduction ....................................................................................................................... 36
Materials and methods ...................................................................................................... 37
Results ............................................................................................................................... 39
Discussion ......................................................................................................................... 44
References ......................................................................................................................... 59
CHAPTER 3. ................................................................................................................................. 65

INDUCTION OF INTRACELLULAR REACTIVE OXYGEN SPECIES BY NANOPARTICLES
AND MEASUREMENT OF GENOTOXICITY BY COMET ASSAY

Abstract ............................................................................................................................. 66
Introduction ....................................................................................................................... 67
Materials and methods ...................................................................................................... 68
Results and discussion ...................................................................................................... 71
References ......................................................................................................................... 82
CHAPTER 4. ................................................................................................................................. 89

INFLUENCES OF OXIDE NANOPARTICLES ON GENE EXPRESSION OF ESCHERICHIA
COLIKIZ
Abstract ............................................................................................................................. 90
Introduction ....................................................................................................................... 91

viii

Materials and methods ...................................................................................................... 92

Results ............................................................................................................................... 98
Discussion ....................................................................................................................... I 13
References ....................................................................................................................... 120
CHAPTER 5 ................................................................................................................................ 133

COMPARATIVE ANALYSIS OF DIF F ERENTIALLY EXPRESSED GENES
EXIGUOBACTERIUM SIBIRICUM FOLLOWING EXPOSURE TO OXIDE NPS

Abstract ........................................................................................................................... I 34
Introduction ..................................................................................................................... 135
Materials and methods .................................................................................................... 136
Results ............................................................................................................................. 1 4 I
Discussion ....................................................................................................................... 155
References ....................................................................................................................... I 63

‘ix

LIST OF TABLES

Table 1.1 Examples of NMs showing antibacterial activity ............................................. 18
Table 2.1 The concentration of Zn2+ ion in LB or TSB medium from ZnO NPS ......... 53
Table 2.2 The toxicity of ZnClz to E. coli and E. sibiricum ............................................. 53

Table 3.1 Measurement of tail length, tail DNA and olive tail moment by TiOz NPS in
comet assay ................................................................................................................................. 79

Table 3.2 Measurement of tail length, tail DNA and olive tail moment by ZnO NPS in
comet assay ................................................................................................................................. 80

Table 4.1 primer sequence for target genes for validation .............................................. 118

LIST OF FIGURES

Figure 1.1 Images of nanomaterials ....................................................................................... 17
Figure 2.1 The effect Of ZnO NPs on E. coli and E. sibiricum ........................................ 49
Figure 2.2 The effect of TiOz NPs on E. coli and E. sibiricum ........................................ 50
Figure 2.3 The toxicity of iron oxide NPs to E. coli and E. sibiricum ........................... 51
Figure 2.4 The influence Of SiOz NPs on E. coli and E. sibiricum ................................. 52

Figure 2.5 The toxicity of TiOz NP suspension and soluble fraction to E. coli and E.
sibiricum ...................................................................................................................................... 54

Figure 2.6 The toxicity of ZnO NP suspension and soluble fraction to E. coli and E.

sibiricum ...................................................................................................................................... 55
Figure 2.7 TEM and EDS images of SiOz NPs in E. sibiricum ....................................... 56
Figure 2.8 TEM and EDS images of TiOz NPs in E. sibiricum ....................................... 57
Figure 2.9 Internalization of ZnO NPs in E. coli and E. sibiricum ................................. 58
Figure 3.1 ROS induction by TiOz and ZnO NPs in E. coli and E sibiricum ............... 77
Figure 3.2 Human lymphocytes treated with TiOz NPs for 6 h exposure ...................... 81

Figure 4.1 The functional categories of the gene profiles in SiOz, TiOz and ZnO NP
for E. coli ................................................................................................................................... 119

Figure 5.1 The upregulated and downregulated gene profiles in three different
treatments .................................................................................................................................. 160

Figure 5.2 The functional categories of the gene profiles in SiOz, TiOz and ZnO NP
for E. sibiricum ........................................................................................................................ 161

Figure 5.3 The functional categories of the shared gene profile among SiOz, TiOz and
ZnO NP exposure .................................................................................................................... 162

xi

Nanoparticles

Nanomaterials

Carbon nunotubes

Quantum dots

Reactive oxygen species
Transmission electron microscopy
Energy dispersive spectrometry
Luria-Bertani

Tryptic soy broth

Tryptic soy agar

2’, 7’-dichlorofuorescin diacetate
Polymerase chain reaction
ATP-binding cassette
Phosphotransferase system
Coenzyme A

Tricarboxylic acid

ABBREVIATIONS

NPs
NMs
CNTs
QDs
ROS
TEM
EDS
LB
TSB
TSA
DCFH-DA
PCR
ABC
PTS
COA

TCA

xii

CHAPTER 1

INTRODUCTION: TOXICITY OF NAN OMATERIALS TO
MICROORGANISMS

Abstract

The unique and advanced functions of nanomaterials (NMs) make them broadly
useful and powerful for research, industrial development and many products. Their use,
however, also brings uncertainties regarding their bio-safety and environmental impacts.
The goal of this study is to introduce the main types of W3 and review their
environmental adverse effect, with a particular emphasis on microorganisms. I also
collate published papers regarding the antibacterial activity of different NMs, and
describe several possible mechanisms of NM toxicity. Production of reactive oxygen
species (ROS) is the most favored explanation for NP toxicity. ROS can attack DNA,
amino acids in proteins and lipids in membranes, causing extensive damage to cells.

Direct contact between NMs and microorganisms is also necessary for NP toxicity.

Introduction

Nanotechnology, a broad interdisciplinary field revolutionizing research
development and new product and process, is one of the most rapidly growing fields. In
nanotechnology and nanoscience, NMs, nanotools and nanodevices are the important
segments, and the global nanotech market is estimated to grow to $25.2 billion by 2011
(75). The Royal Society stated that “Nanotechnologies are the design, characterization,
production and application of structures, devices and systems by controlling shape and
size at nanometer scale”, when there have also been a few definitions offered by other
organizations (25). Most of these definitions refer only to the size range or new
functionalities, but neglect risks and uncertainties which also need to be considered and
resolved. The development of nanotechnology and nanoscience requires
multidisciplinary integration of physics, chemistry, biology, and engineering, and mainly

consists of nano-medicine, nanO-fabrication, nano-metrology and nano-materials (59).

Manufactured NMs are defined by the US. National Nanotechnology Initiative as
materials that have at least one dimension in the 1 to 100 nm range (88). Recently more
and more NMs have been synthesized and applied in various fields, such as
environmental remediation, drug delivery, cosmetics, electronics, energetics and
photonics (3). For instance, 2954 kg of fullerenes were synthesized world-wide in 2003
(10). In 2005, more than ten billion dollars was spent internationally on nanotechnology

development, and it is estimated that by 2015 the global market for nanotechnology and

NMs will be around $1 trillion (35). Because of wide and abundant applications, NMs
may enter environment during their life cycle and end up as environmental contaminants
Of water, air and soil (93). The concerns about the toxicological and environmental
impacts of NMs have been increasing rapidly, and the research conducted about safety of
NMs remains focused on the issues related to occupational health and human safety. The
concerns about environmental safety of NMs need attention; these materials are too small
to detect and track in environment. The entrance of NMs to environment can be divided
into intentional releases and unintentional releases, such as atmospheric emission and

liquid waste streams.

Nanoparticles (NPs), one of the most useful subsets of NMs, are widely applied in
water purification, catalysis, pigments, food, cosmetics, and lubrication (3, 31, 38, 86,
116). NPs have natively existed in nature, while the exposure to the NPs may have some
adverse effect on the environmental microorganisms(81). The mechanisms of NP toxicity
are not yet clear, and the studies associated on their potential influence on public health
and environments are inadequate. In this chapter, I focus on the toxicity Of NPS to
microorganisms. Understanding NP toxicity to microorganism is important to evaluate
the risk of NPs to environment, since microorganisms play a fundamental role of the food
chain as the lowest level and are responsive for much of biogeochemical cycles.
Moreover, they grow rapidly and are sensitive to some toxicants, which make them as a

good test model for NP toxicity. These studies will supply helpful information for

regulatory policy concerning the manufacture, application and disposition of NPs.
Classes of NMs and their toxicity

NMs have always existed in‘the environment, from both natural and anthropogenic
sources. The natural NMs include clays and particulate organic matter in soil, volcanic
ash and forest-fire smoke, and, colloid and ocean spray in aquatic systems. The
engineered NMs possess many unique characteristics, which are attributable to their
small size, large surface area. chemical composition, surface structure, solubility, shape,

and aggregation. The main types Of manufactured NMS include:
Fullerene

The manufacture of carbon-base fullerenes, spherical molecules comprising 60
carbon atoms, opened the gate of the nanotechnology era (58). Its configuration is like a
football with 20 hexagons and 12 pentagons and its diameter is about 1 nm (Figure
l.1(a)). Underivatized fullerenes are extremely insoluble in water, and subject to
precipitation and aggregation (4, 54). Fullerene has been applied in polymer and
structural composites, electromagnetic shielding, electron field emitters, super capacitors
and batteries, due to their high conductivity, high surface area and unique electronic

properties (3, 74).

F ullerenes are redox active and can generate ROS, which may cause damage to DNA,

membranes and amino acids (20, 100). Fullerenes have been observed to alter the
membrane lipid composition, phase transition temperature and membrane fluidity in
Pseudomonas putida and Bacillus subtilis (100). The antibacterial activity of fullerene on
Escherichia coli and B. subtilis has been reported in literature, as a decreased aerobic
respiration rate (28, 63). Surface-modified fullerene can generate singlet oxygen, causing
lipid peroxidation and exhibiting antibacterial activities (48, 64, 67). Carboxyfullerene
caused the inhibition on Gram positive species by damage to cell membranes, i. e.
Staphylococcus spp., Streptococcus spp., Enterococcus faecalis, but not for Gram
negative E. coli, Pseudomonas aeroginosa, Salmonella typhmuriumi and Klebsiella
pneumoniae (108, 109). However, toxicity of a carboxy-fullerene derivative to E. coli

was detected in a dose-dependent manner (107).

Some other fullerene derivatives were also studied for their antibacterial activities.
For instance, one positively charged fullerene derivative inhibited the growth of
Mycobacterium tuberculosis, and alkylated fullerene derivatives could inhibit the growth
of E. faecalis, S. aureus, S. epidermidis, and Enterococcus hirae (11, 67). Their toxicity
to E. coli was explained as respiratory chain inhibition and hydrogen peroxide production
(68, 69). Moreover, fullerene derivatives had a mutagenic effect on S. typhimurium (6). It

was also reported that fullerene can alter the microbial community in soil (106).

Nanotubes and nanowires

Carbon nanotubes (CNTS), first observed by Sumio Iijima in 1991, are a particular
form of fullerenes, including single-walled carbon nanotubes (SWCNTS) and
multi-walled carbon nanotubes (MWCNTS) (Figure 1.1(b)) (44). Both SWCNBs and
MWCNTS show antibacterial activity: direct contact between SWCNBs and bacteria can
cause membrane damage and lead to cell death (49, 85). Dr Elimelech’s group published
a serial of papers about CNT toxicity. They provided the direct evidence that both
SWCNTS and SWCNTS exhibited strong antimicrobial activity on E. coli, and SWCNBs
were more toxic to bacteria than MWCNTs (50, 51). They also reported that these CNBS
could regulate gene expression of E. coli and induce stress-related genes, and that the
size of CNT could influence the antibacterial effects (49). Meanwhile, the surface coating
is also important for CNT antimicrobial activity (85). Nanowires are ultrafine wires or
linear arrays of dots formed by self assembly, and used as interconnectors in
nanoelectronic devices (25). There are not many studies of nanowire toxicity, and

perhaps they are less likely to be toxic due to their long dimension.
Nanoparticles

One important subset of NMs are NPs, which are particles with a diameter less than
100 nm. The materials of NPs include metal, metal oxides and silica. The metal oxide
NPs can be consisted of individual oxides, such as ZnO, T102, CeOz and CrOz, and

binary oxides, such as BaTi03 and LiCOOz.

NPS of Silicon oxide (SiOz), Titanium oxide (TiOz) and Zinc oxide (ZnO) are the
popular ones among the oxide NPs. SiOz is used as an additive in food, drugs, cosmetics
and printer toners (Figure l.1(a)). It is also a good semiconductor and an electrical
insulator. TiOz is used as a photocatalyst for disinfection of bacteria, and a number of the
papers explored this subject (9, 14, 66, 72, 94, 96, 104). It is also employed as a pigment
in paints and a sunscreen product in cosmetics. ZnO is widely used in pigments,
semiconductors, and cosmetics. It was estimated that globally 1000 tons of NPs were

used in sunscreen products in 2004 (10).

There have been a ntunber of reports on the toxicity Of SiOz, TiOz and ZnO NPS.
SiOz NPs are the least toxic among these three NPs. Their antibacterial activity on E. coli
and B. subtilis was reported while some studies indicated that they were not toxic (1).
Due to the wide application of TiOz NPs, their toxicity on microorganisms has been well
studied. The TiOz NP photocatalytic inactivation of E. coli, S. aureus and E. faecalis was
reported, and scientists also demonstrated antibacterial activity to Streptococcus spp.,
Enterococcus spp., Vibrio fischeri, Serratia marcescens, Bacillus megaterium and some
fungi (29, 36, 76, 94, 95, 118). N-doped TiOz NPs and Ag-coated TiOz NPs also showed
inhibition of E. coli and Micrococcus lylae, respectively (60, 61, 123). Matsunaga et al.
reported that TiOz can oxidize COA and cause cell death (72, 73). Moreover, the cell
membrane may be oxidized prior to the oxidation of COA, and a partial decomposition of

outer membrane is followed by disordering of the cytoplasmic membrane, leading to cell

death (19, 76, 104). Later, Saito et al. found rapid leakage of K+ ion and slow release of
proteins and RNA. They explained that the cell death was due to a significant disorder in
cell permeability (7, 96). The toxicity of Ti02 NPs can be influenced by NP size and light
intensity (2, 39). ZnO NPs are probably the most toxic among these three NPs. They
inhibited the growth of E. coli, S. aureus, V. fishceri, B. subtilis, S. typhimurium and
Streptococcus agalactiae (43, 83, 92, 122). The release Of Zn2+ ion from ZnO NPs, direct
contact between ZnO NPs and membranes, and ROS induction all contributed to their
antibacterial activity. A NP size-effect was also demonstrated in ZnO toxicity studies,
and internalization of ZnO was found in E. coli, S. agalactiae, Exiguobacterium

sibiricum and S. aureus (Figurel.1 (b)) (12, 43, 120).

Recently there have been a few reports about the antimicrobial activity of some other
NPS, and on the interaction between NPS and microorganisms (12, 15, 29, 89, 90, 103).
CuO NPs inhibited the growth of V. fischeri, and the growth of E. coli was found to be
inhibited by N-doped ZrOz, CeOz, Ag/A1203 and Fe203 NPS (5, 15, 80, 105). Both CaO
and MgO NPs demonstrated antibacterial activity to B. subtilis, E. coli, S. aureus, S.

typhimurium and B. megaterium (41, 42, 65, 97, 98).

Silver NPs are one of the most promising metal NPs for future applications. They are
antibacterial, antimicrobial and antifungal (24). Silver NPS can also interact with DNA,
and interrupt RNA replication and microbial reproduction (79, 110).The toxicity of Ag

NPS to E. coli was reported by Sondi and Salopek-Sondi, and Ag NPS also inhibited the

growth of Salmonella typhi, P. aeruginosa, and Vibrio cholerae (79, 90, 102). The
toxicity of gold NPs was demonstrated to depend on surface coating. Cationic gold NPs
are moderately toxic and anionic gold NPs are quite nontoxic (30, 117). Organic
compound NPs also possess antibacterial activity, such as the growth inhibition of K.
pneumonia, S. aureus, and Streptococcus mutans by polyethylenimine NPs and

Ag-loaded polystyrene NPs (8, 89).

Quantum dots (QDs).

Quantum dots, which are small assemblies of metal, metal oxide or semiconductor
materials in size of 2-10 nm, were first created in the early 19803 (25). Quite a few QDs
contain some noble or transition metals, such as CdSe, InAs and ZnS. The components
for QD shell or core can be quite different (18, 52). The application of QDs covers
biomedical imaging, drug delivery and electronics industry (34). They can be coated by
some organic molecules, which can influence their characteristics and toxicity (40). QDs

can induce ROS and nick supercoiled DNA to cause genotoxicity (32, 46).

Other NMs

There are some other types of NMS, such as dendrimers and biopolymers. Few
toxicity studies have been conducted on these NMS, perhaps due to their limiting

applications.

10

 

Mechanisms of NP toxicity

There have been a few hypothesized mechanisms Of NP toxicity. such as disruption
of cell membranes, induction of ROS, dissolution of hazardous constituents and
interruption Of energy production (54. 84). NPs may cause toxicity to bacteria via one or

more Of these mechanisms.

ROS induction

Induction of ROS by NPs is currently the most favored explanation for the toxicity
of NPs. In 1997, Rahman et al. found the increasing induction of ROS by nano-sized
Ti02 in human and rat alveolar macrophages (91). That Ti02 NPs were toxic to some
cells and caused oxidative stress in different cell types, including bacteria and mammal

cells (2, 33, 62,101, 113-115).

The chemical reactions by which Ti02 NPs induce ROS are showed below (16, 17,
123). In the first equation, a hole (e')/electron (h+) in the valence band and conduction
band is induced by light (k<390 nm) and Ti02 NPs. The e' is absorbed onto the surface of
a TiO2 NP and available for electron transfer, which can reduce 02 to 02' (reaction 2),
and produce H202 by further reducing 02' (reaction 3). The OH radicals can be produced
by the reaction of superoxide with H202 (reaction 4) and reduction of H202 by e'
(reaction 5). The OH radicals can also be formed by the reactions of H20 or OH" with h+

and electron abstraction from absorbed oxidizable species by h+ (reaction 6 and 7). H202

11

can be produced by either a reductive pathway (reaction 3) or an oxidative pathway

(reaction 8), while it can be decomposed into OH radicals by light (reaction 9).

TiO2 + hv ——> TiO2 (e‘ + h*) (1)

6" + 02 —> 02' (2)
02' + 2H“ + e' —+ H202 (3)
H202 + 02' —> °0H + OH' + 02 (4)

e' + H202 —->°0H + OH' (5)

h” + OH' ——>°OH (6)

h+ + H2O —+ °OH + IT“ ’ (7)
2h+ + 2H2O —+ 2H“ + H202 (8) hv
+ H202 —» 2°0H (9)

An interfacial charge transfer has been reported to occur between Ti02 NPs and E.
coli membrane (82). Reaction 10 and 11 exhibit the surface charge trapping on Ti02 NPs.
During the process of interfacial charge transfer, h+ reacts with peptidoglycan (PGN) and
lipo-polysaccharides (LPS) on microbial cell wall membranes, such as E. coli (reaction
12). Meanwhile, h+ can be scavenged by PE, LPS or E. coli during surface oxidation

(reaction 13).

e' + Ti(IV)0-H —+ Ti(III)0-H' (10)
h+ +Ti(IV)0-H -2 Ti (Iv)O--H+ (11)
hI+RH—>R°+H+ (12)
Ti(1v)O--H++RH—»OH+R- +H+ (13)

12

ROS can be transferred through membranes, and oxidize fatty acids of phospholipids
and cause lipid peroxidation. This process can change membrane permeability and
fluidity, and make bacteria more vulnerable to osmotic stress and interrupt nutrient
uptake (13). ROS can also oxidize and damage DNA to cause genotoxicity. For instance,
there have been a few reports about the DNA lesions caused by ROS induction by Ti02
NPs (22, 33, 37, 57, 70, 111-113). Moreover. ROS can oxidize amino acids in proteins,
and especially attack sulfur-iron assembly, which can release Fe ions, induce Fenton
chemistry and lead to more production of ROS (45). The induction Of ROS by ZnO NPs
was also hypothesized as an explanation for their toxicity, although the mechanisms for
ZnO NP toxicity are not yet well understood (99). ZnO NP toxicity may be caused by the
binding of the NPs to the bacterial surface due to electronic forces (103, 122). The
internalization of NPs is not required for ROS induction. ROS can be transferred into
bacteria via direct contact between NPs and their membranes, although the lifetime for

ROS is very short (10'9 second).

Release of toxic components from NPs

Hazardous components can be released by some NMs. Silver NPs are a good
example, as release of silver ions has been hypothesized as one of the mechanisms for
toxicity of silver NPs (79). That the interaction between silver ions and thiol groups in
proteins can inactivate some enzymes (71). Moreover, silver ions can also attack

membranes and prevent DNA replication (27). ZnO NPs can release Zn2+ ion, which is

13

antibacterial (121). Kirchner et al. reported that QDs (CdSe/ZnS) released toxic Cd”,
which increased their toxicity (53). Meanwhile, both free Cd and Se from QDS was found
inside S. aureus before QDs was internalized, which confirmed the release of

components from QDS (56).

Damage to membrane integrity

Membrane integrity is important for microorganisms to maintain cell activity, such
as transport and energy transduction, and to protect cells from potential damage. Some
NMs can attack bacterial membranes directly. Zn0 NPs were internalized into E. coli
cells (12). E. coli and E. sibiricum cells with broken membrane and leaking cytoplasm
were found when they were exposed to ZnO NPs in our studies. QDs were also found to
enter bacterial cells (55). However, there is no clear explanation for how NP
internalization occurs, since NPs are still too big for channels or transporters to transport
through membranes. Moreover, Ag NPs can also interact with sulphur-containing amino
acids in membranes, prohibiting transport through membranes and causing cell death
(102, 119). The embedding of silicon NPs and fullerene in membranes was also reported
(47, 107). ROS can disrupt membranes too, via oxidizing lipids or proteins in membrane.
Cell membrane damage by direct contact of CNTs is proposed as the main mechanism

for CNT cytotoxicity.

Damage to nucleic acid

14

Some NMs have characteristics of interacting with DNA, and they can be used to
label or cleave DNA. For example, QDs have been used for imaging applications via
tagging DNA, and they were also found to nick DNA (21, 23, 32). ROS induced by NPs

can damage DNA, such as by Ti02 and ZnO NPs (22).

Conclusion

The NM toxicity studies require multidisciplinary collaboration of physicist, chemist,
microbiologist and other experts. On one hand, when microbiologists focus on
antibacterial activity of NMS, they may neglect the characterization of NPs. However, it
is essential to characterize the physical or physieochemieal properties of NMs before any
toxicity studies, since their features are likely critical to any toxicity. Therefore, the

measurement Of the NP properties should be the first step for studying their toxicity.

The main characteristic Of NPs is their size. which may extend down to individual
atoms or molecules, and much smaller than cells. The small size can influence other
physicochemical properties of materials and increases the Opportunity of uptake and
interaction with or within bacteria. Surface area is another important factor in toxic
effects. Normally when the size decreases, the surface area increases and there are more
reactive groups. The surface group is dependent on the chemical composition and surface
structure of NPS, such as electronic properties and coating. which determines the

solubility of NPS. hydrophobic or hydrophilic. lipophobic or lipophilic, or active or

15

passive. The other main properties of NPs that may affect their toxicity include
agglomeration state, shape, crystal structure, chemical composition, surface chemistry,
surface charge and porosity (88). Standard and characterized NPS are critical for NP

toxicity studies, as well are standardized and validated test methods.

0n the other hand, physicists and chemists should use more relevant biological
methods to explore toxicity study at molecular level. Simple viability tests and growth
inhibition are not very helpful to answer the questions about mechanisms of NP toxicity.
Use of genetics and proteomics can supply more details about bacterial reaction to NPs,
and reveal increasing complex that can be useful in refining experiments for exploration

of NP fate, bioavailability and effect.

There are still many unexplored areas for NP toxicity studies. For instance, there are
few anaerobic studies, although it was found that fullerenes have no significant effect on
anaerobic microbial communities (87). How microbial communities react to NMs still
remains unknown, and it is very difficult to estimate the consequences of wide
environmental impact during the exposure to NMs. Finally, it is critical to improve our

understanding of the interaction between NPs and microorganisms.

16

w . .,
.v. .2 . . u v .mw . «w...

I.

a

 

Figure 1.] images of NPs. (a) TEM image of a fullerene (28).(b) TEM image of a SWCMT

(49). (C) TEM image of 8102 NPs. (b) TEM image of internalized ZnO NPs in E. sibiricum.

17

Table 1.1 Examples of NMs showing antibacterial activity

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

NMs Microorganism Reference
Fullerenes E. coli, B. subtilis, and P. putida (94), (64), (63). (28),
(26),
fullerene E. coli, S. typhimurium, and M. tubercolosis (6), (11), (68), (69)
derivatives
carboxyfullerene E. coli, S. pyogenes, Staphylococcus spp., (107), (109), (108)
Streptococcus spp., E. faecalis, P.
aeruginosa, S. typhi, and K. pneumoniae
SW and MWNTs E. coli, M lysodeikticus, and S. aureus (85), (49), (51), (50)
Carbon Nanohom E. coli (77)
Carbon NPs K. pneumoniae (78)
QDS E. coli and B. subtilis (55)
Gold NPs E. coli (30), (117)
CuO NPS V. fischeri (80), (36)
Si02 E. coli and B. subilis (I)
TiO2 E. coli, M. luteus, B. subtilis, Streptococci spp., S. (95), (94), (9), (116),
marcescens, Enterococcus spp., Saccharomyces (76), (118), (29),
Serevisiae, S. aureus, E. faecalis, and V. fischeri (104), (l), (16), (7),
(96), (72), (39), (124),
(36)
N-doped Ti02 E. coli (60), (61)
A g-coated TiO2 M lylae (123)
Silver E. coli, P. aeruginosa, V. cholera, and s. typhi (102), (79), (90)
N-doped Zr02 E. coli (61)
ZnO S. agalactiae, S. aureus, V. fischeri, B. subilis, and (98), (92), (l), (122),
S. typhimurium (36), (97), (83), (120),
(43), (80)
MgO B. subtilis, E. coli, S. aureus, B. megaterium, (98), (42), (41), (103),
and S. typhimurium (97), (65)
CaO B. subtilis, E. coli, S. typhimurium, and S. aureus (98), (97)
Ce02 E. coli (105)
Ag/Al203 E. coli (15)
Fe203 E. coli (5)
Silica, silica/iron E. coli (117)
oxide
Polyethylenimine Streptococcus mutans (8)
NPs

 

 

18

 

Table 1.1 cont

 

Ag-loaded
polystyrene NPs

 

 

K. pneumonia and S. aureus

 

(89)

 

19

 

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33

CHAPTER 2

TOXICITY OF OXIDE NANOPARTICLES ON ESCHERICHIA C 0L1 AND
EXI G U OBA C TERI UM SIBIRIC UM

34

Abstract

NPs are being used in a variety of areas, including cosmetics, pigments, water
purification, environmental remediation and drug delivery. However, their toxicological
and environmental effects have not been. thoroughly investigated. In our studies, zinc
oxide (ZnO) and titanium oxide (Ti02) NPs were found to decrease cell viability of
Escherichia coli strain K12 (Gram-negative) and Exiguobacterium sibiricum strain
255-15 (Gram-positive) in a dose-dependent manner, while silicon oxide (Si02) and iron
oxide (Fe3O4) NPs did not inhibit the microbial growth after a 12 h exposure. Five
nanometer Ti02 NPs were more toxic to both E. coli and E. sibiricum than 150 nm Ti02
NPs, which confirmed that size is one of the most important factors for NP toxicity. ZnO
NPs displayed more toxicity to E. coli compared to E. sibiricum, while Ti02 NPs were
more toxic to E. sibiricum than to E. coli. Zn2+ ion dissolved from ZnO NPs in both LB
and TSB medium, and reached saturation of approximately 100- 110 ppm. E. coli was
more vulnerable to the toxicity of Zn2+ than E. sibiricum. Transmission electron
microscopy (TEM) and energy dispersive spectroscopy (EDS), showed that Si02 and
Ti02 NPs were internalized by E. sibiricum but not by E. coli, and ZnO NPs were found
in both. We concluded that size and chemical composition play a key role in NP toxicity,
and that solubility of metal ions from NPs could also attribute to the toxicity of NPs.
These results also show that different bacteria have different tolerance and internalization

ofNPs.

35

Introduction

NMs are defined by the US. National Nanotechnology Initiative as materials that
have at least one dimension in the 1 to 100 nm range (34). Nanotechnology has become
one of the rapidly developing fields with the potential to revolutionize medical science,
biology and engineering. Meanwhile, the concerns about the toxic and environmental
impacts of exposure to these NMs have been increasing and starting to attract public and
governmental attention. The critical risk assessment issues about NMs include toxicology,
exposure assessment, environmental and biological fate, persistence, sustainability,
transportation and transformation. Moreover, a number of studies suggest that NMs
might cause damage, such as lung cancer, silicosis and some autoimmune diseases (9, 10,
13,20, 37, 42).

NPs, the type most important in NMs, are applied in environmental remediation,
catalysis, drug delivery, cosmetics, water purification and lubrication (2, 14, 18, 33, 49).
$02, Ti02 and ZnO NPs were the main NPs test in this study. Si02 is used as an additive
in food, drugs, cosmetics and printer toners, as well as it is a good semiconductor and an
electrical insulator. Ti02 is used‘as a photocatalyst for disinfection of bacteria and a
number of the papers explored the mechanism (3, 7, 27, 29, 38, 40, 44). It is also
employed as a pigment in paints and as a sunscreen product for cosmetics. ZnO is widely
used for application as pigments, semiconductors, and as a sunscreen. It was estimated
that globally 1000 tons of NPs were used for sunscreen products during 2003 and 2004

and more than 2500 tons in 2008 (4). Because of their numerous applications, NPs may

36

enter the environment during their life cycle and end up as environmental contaminants
in water, air or soil. However, very few studies on NPs about their environmental and
toxicological effects on the microorganisms have been conducted, and their mechanism
of toxicity is still unknown. The main properties of NPs that may affect the toxicity
include size distribution, agglomeration state, shape, crystal structure, chemical
composition, surface area, surface chemistry, surface charge and porosity (34).

We evaluated the effects of Si02, Fe3O4, Ti02 and ZnO NPs on E. coli K12 and E.
sibiricum representative Gram—negative and Gram-positive bacteria, and explored if the
size and composition of these NPs influenced the degree of toxicity. Moreover, we
investigated the dissolution of ZnO NPs, and compared the toxicity of Zn2+ ion with that
of the NP suspension. TEM and EDS were employed to study the internalization of NPs

and the morphological changes of these bacteria after the exposure to NPs.

Materials and Methods
Reagents and bacterial culture. E. coli K12 was grown aerobically in
Luria-Bertani (LB) liquid broth at 37 °C and 200 rpm for 12 h. E. sibiricum was cultured
in tryptic soy broth (TSB) medium at 30 °C and 150 rpm for 12 h. Solid medium for E.
coli or E. sibiricum was as above with 1.5% agar (Difco). The bacteria were in stationary
phase when the NPs were added, as well as after 12 h exposure. All the cultures with NPs
were grown under the same conditions.

Preparation of NP suspensions. Si02 (10 nm and 1 pm), Fe3O4 (50 nm and 1 um),

37

Ti02 (5 nm, 150 nm and 5 mm) and ZnO (60 nm and 1 mm) were purchased from
Sigma-Aldrich (St. Louis, MO). Sixty namometer Si02 was synthesized by reverse
microemulsion (45, 50). NPs stock solutions of 100 g/L were prepared in deionized and
distilled water.

Assessment of NP toxicity to bacteria. E. coli K12 and E. sibiricum were cultured
as above for 12 h with NPs at 0, 10, 50, 100, 500, 1000, 2000, and 5000 ppm. Cultures
were then diluted and spread onto LB or trypic soy agar (TSA) plates, and colonies were
counted after growth over-night. All the cultures were grown in triplicate.

Evaluation of Zn2+ toxicity in ZnO NP medium. Different concentrations of 60 nm
and 1 pm ZnO NPs were mixed in TSB and LB for 12 h (50 ppm, 200 ppm, 1000 ppm
and 5000 ppm). After 20 min of centrifugation at 20,000Xg, the supernatants were
collected, diluted and measured with atomic absorption spectrometry. Zinc standard
solution was purchased from Fischer Scientific Co. (Pittsburgh, PA) and diluted to
construct a standard curve for Zn2+ concentration. E. coli K12 and E. sibiricum were
cultured in triplicate in each of three concentrations of ZnClz (0.5 mM, 1.0 mM and 2.0
mM). Culture viability was measured by plate counts and all the samples were prepared
in triplicate.

Toxicity of NPs versus soluble metals from NPs. LB or TSB medium containing
possible metal impurities from TiOz or ZnO NPs was prepared by making suspension of
NPs with concentrations of 1000, 2000, and 5000 ppm for 10 nm, 150 nm and 5 pm Ti02

NPs, and, 100, 200, 500, 1000, 2000, and 5000 ppm for 60 nm ZnO NPs. After leaving

38

the LB suspension at 30 °C and TSB suspension at 37 °C for 12 h, the NPs were removed
by centrifuging the suspension at 20,000Xg for 20 min. The supernatant was collected
and used to culture E. coli and E. sibiricum respectively for 12 h, and the microbial
growth was evaluated by plate counting method.

Morphological studies

E. coli K12 and E. sibiricum with 1000 ppm of NPs (60 nm Si02, 5 nm Ti02 and 60
nm ZnO) were cultured in LB or TSB medium overnight at 30 0C or 37 0C respectively.
After culturing, the mixture of cells and NPs in each sample was collected by
centrifugation at 12,000 X g for 30 min. These samples were fixed with 2.5%
glutaraldehyde and 2.5% paraformaldehyde in 0.1 M cacodylate buffer and then 1%
osmium tetroxide in cacodylate buffer, dehydrated in serial acetone buffer (from 30% to
100%), and infiltrated with acetone: Poly/Bed 812 buffer for TEM. The samples were cut
by MTX ultramicrotome into 70-100 nm thick sections. All the sections were collected
on copper grids, and a JEOL 2200F S Transmission Electron Microscope was used for the

EDS-TEM detection.

Results
Antibacterial activity of ZnO and Ti02 NPs. E. coli and E. sibiricum was exposed
to two different sizes of ZnO NPs for 12 h to evaluate their effect on microbial growth.
As illustrated in Figure 2.1, the toxicity of ZnO NPs to E. coli and E. sibiricum rose with

the increasing doses. ZnO NPs significantly inhibited the growth of E. coli when the

39

concentration was higher than 100 ppm (Figure 2.1(a)). The 60 nm ZnO NPs were more
toxic to E. coli than the 1 pm ones. In the case of E. sibiricum, a gradual increase in
growth inhibition was observed with increasing doses of ZnO NPs, while E. sibiricum
showed more tolerance to ZnO NPs (Figure 2.1(b)). In addition, the 60 nm ZnO NPs
displayed less toxicity to E. sibiricum than the 1 pm NPs, which was different from the
size-effect of ZnO NPs on E. coli.

Similar to the results with ZnO NPs, all three Ti02 NPs showed a gradual increase in
toxicity to both E. coli and E. sibiricum as the doses increased (Figure 2.2). Moreover, E.
sibiricum was more vulnerable to Ti02 NPs than E. coli, which was contrary to the
toxicity of ZnO NPs to E. coli and E. sibiricum. In the case of E. coli, both 5 nm and 150
nm NPs significantly inhibited growth of E. coli at 2000 and. 5000 ppm, with 5 nm NPs
being more toxic than the 150 nm ones (Figure 2.2(a)). The 5 pm Ti02 NPs did not
display any toxicity even at 5000 ppm. As illustrated in Figure 2.2(b), the growth of E.
sibiricum was inhibited significantly by all the three different size Ti02 NPs, even for 5
pm NPs when their concentrations were higher than 1000 ppm. The 5 nm NPs strongly
inhibited the growth of E. sibiricum at concentrations above 500 ppm while the 150 nm
TiOz NPs were intermediate in their inhibition of the growth.

Zn2+ toxicity in ZnO NP solution. Zn2+ ion concentration in LB or TSB solution of
ZnO NPs was measured with atomic absorption spectrometry, and the results
demonstrated that the concentrations of Zn2+ ion rose with the increasing ZnO NP

concentrations (Table 2.1). The concentrations of Zn2+ ion dissolved from the same

40

concentration of 60 nm and 1 pm ZnO NPs were similar, which suggested that the size of
NPs did not influence the dissolution of ZnO NPs much. Additionally, there was no
significant difference in Zn2+ ion concentrations between the same concentration of ZnO
NPs in LB and TSB broth. In the ZnO NP suspension of 50 ppm, the concentration of
Zn2+ ion was approximately 40 ppm in either TSB or LB broth, which meant that most of
ZnO NPs were dissolved in the solution. The concentration of Zn2+ was saturated at 100-
110 ppm in TSB or LB medium, which occurred when the concentration of NPs was
higher than 200 ppm.

In Table 2.2, the viability 0f E. coli or E. sibiricum decreased with increasing
concentrations of ZnClz in broth, with E. coli being more vulnerable of the two strains.
The growth of these two strains to Zn2+ ions suggested that the most of overall ZnO NP
toxicity came from the dissolved Zn2+. The ZnO NPs, however, still played an important
role in the toxicity for the solutions containing high concentrations of ZnO NPs, since the
concentration of Zn2+ reached the saturation of 110 ppm.

Toxicity of NPs versus soluble metals from NPs. The toxicity of Ti02 or ZnO NPs
was compared to that of possibly released metal ion Ti4+ or Zn2+ from the same
concentration of suspension. Since no toxicity in the Ti02 NP suspension lower than 1000
ppm was found for either E. coli or E. sibiricum, only the suspensions > 1000 ppm were
tested. The soluble fraction of 10 nm, 150 nm and 5 pm Ti02 NPs showed little effect on
the grth of E. coli (Figure 2.5(a)). The reason for this is probably that the Ti02 NPs is

highly insoluble, so the concentration of Ti“ ion is not high enough to cause damage to E.

41

coli. Another reason may be that membrane of E. coli is not that permeable to Ti02 NPs
compared to E. sibiricum. In the case of E. sibiricum (Figure 2.5(b)), the biggest
difference between the suspension and the soluble metal was seen at 5 um Ti02 NPs: no
toxicity was found in soluble fractions while the viability of E. sibiricum in suspension of
2000 ppm and 5000 ppm 5 pm Ti02 NPs was accordingly 74 % and 20 %.

Compared to Ti02 NPs, the soluble fraction in ZnO NP suspension played a more
important role in the microbial growth. For E. coli, the suspension of ZnO NPs was 10 to
40 fold more toxic than the soluble fraction, when the ZnO NP concentrations ranged
from 100 to 200 ppm (Figure 2.6(a)). Furthermore, there was no toxicological difference
on the microbial grth between the suspension and the soluble fraction at the
concentration of ZnO NPs higher than 500 ppm. Compared to E. coli, E. sibiricum was
less vulnerable to Zn2+ ion or ZnO NPs. Contrary to the results with E. coli, there was
almost no significant toxic difference on the growth of E. sibiricm between the ZnO NP
suspension and the soluble fraction, when the ZnO NP concentration is no higher than
1000 ppm (Figure 2.6(b)). At high concentrations of ZnO NPs (2000 and 5000 ppm),
however, the soluble fraction was 3 to 6 fold less toxic than the whole suspension.

It is well known that low concentration of Zn2+ is safe or even beneficial for the
bacteria, by providing stability to the membrane for example. For E. coli, the
concentration of Zn2+ was unsaturated and not that high in solutions containing low
concentrations of ZnO NPs, so ZnO NPs in the suspension could raise the toxicity and

played a predominant role on the inhibition of the microbial growth. Furthermore, when

42

the concentration of Zn2+ reached saturation at the high concentrations of ZnO NP
suspension, the soluble fraction dominated the toxicological effects. Compared to E. coli,
E. sibiricum was more tolerant to the toxicity of Zn2+. That is why the increasing
concentrations of ZnO NPs in the suspension still could enhance the inhibition to the
growth of E. sibiricum when the concentration of Zn2+ was saturated.

Morphological studies. TEM was applied to visualize the morphological changes in
E. coli and E. sibiricum exposed to 1000 ppm NPs in LB or TSB medium for 12 h. EDS
was performed for the validation of NPs, because osmium tetroxide, used to fix lipid in
the membrane, gave some confusing black spots inside cells. The 60 nm 810; NPs were
found in E. sibiricum not in E. coli, which meant that it was easier for Si02 NPs to go
through the membrane of E. sibiricum than that of E. coli (Figure 2.7(a)). The EDS
results revealed that Silicon element appeared along the cross-section line and there was
an increase in the distribution of both silicon and oxygen elements along the line, which
confirmed the internalization of Si02 NPs (Figure 2.7(b) and (c)). Furthermore, the width
of the silicon peak was similar to the 60 nm size of NPs.

Similar to the results with ZnO NPs, 5 nm Ti02 NPs was found inside E. sibiricum
cells but none in E. coli (Figure 2.8(a)). This is consistent with the toxicological results
that Ti02 NPs showed more toxic to E. sibiricum than to E. coli. The EDS results
confirmed the presence of elemental titanium along the cross-section line that
corresponds on the thin section samples. The width of the titanium peak was similar to

the size of the NPs in the cell (Figure 2.8(b)).

43

Sixty nanometer ZnO NPs were found inside both E. coli and E.sibiricum cells
(Figure 2.9(a) and (c)), which suggested that the ZnO could pass the both of membranes.
Lots of dead cells with broken membranes and leaking cytoplasm were also found under
TEM. The EDS data confirmed that the region along the cross-section line contained
elemental zinc. The distribution of zinc displayed a peak whose width was similar in size
to ZnO NPs (Figure 2.9(b) and ((1) dark spot). Together these data validated that the dark

spot is a ZnO NP. Similar internalization results was published by Brayner et al. (5).

Discussion

The chemical composition of NPs is one of the most important factors determining
biological response, and causes their toxicity. As illustrated in Figure 2.1 and 2.2, ZnO
NPs and Ti02 NPs were toxic to both E. coli and E. sibiricum, while Si02 and Fe3O4
NPs were not generally. Only in the case of E. sibiricum, did 500 ppm 10 nm Si02 NPs
inhibit growth, and then only after the exposure time was increased to 48 h. The
composition of NPs also influences the possible mechanisms of NP toxicity. For
instance, ZnO NPs are slightly soluble (5.4mg/L), and the released Zn2+ ion plays a key
role in ZnO NP toxicity. In the case of Ti02 NPs, the ROS leads to oxidation of
membrane and proteins, and hinders nutrient uptake and respiration, resulting cell death.
Recently several papers show the toxicity of Si02 NPs to E. coli, B. subtilis and human
cells (1, 21 , 24), and some groups reported that no cytotoxicity was found for iron oxide

NPs, similar to our results (15, 16, 19, 23, 36). All these data demonstrate that chemical

44

composition is one of the physicochemical properties important to NP toxicity.

Size is also an important factor to NP toxicity. As size decreases, both surface area
and surface reactive groups increase, which may enhance the NP toxicity (32, 35). We
found that the smaller ZnO or Ti02 NPs were more toxic to E. coli than larger NPs
(Figure 21(3) and 2.2(a)), similar to previous findings (19, 51). There were some
conflicts in the size-effect with E. sibiricum, since 1 um ZnO NPs were more toxic than
60 nm ZnO NPs (Figure 2.1(b)), and 5 um Ti02 NPs were more toxic to E. sibiricum
than 150 nm Ti02 NPs (Figure 2.2(b)).

Aggregation of NPs in the medium may be one reason for these conflicts. High
concentrations of salts and proteins in the medium can cause the aggregation of NPs,
which increases the size the cell experienced (26). For instance, Long et al. found that
commercial grade, nano-size Ti02 NPs aggregated in Hank’s basic salt solution and
Dulbecco’s modified eagle’s medium, and the hydrodynamic diameter of the TiOz NPs
increased to 2368i163 nm at 120 ppm in their studies (25). Although there is no clear
consensus about the upper size limit for the NP toxicity, it was estimated that it might be
between 65 nm and 200 nm (11). Additionally, there are other factors of NPs which can
influence NP toxicity, such as shape and surface modification (41). So it is important for
toxicology and exposure assessment of NPs to determine and characterize these
parameters.

Gram-positive or Gram-negative species may show different tolerance to the toxicity

of the same NPs. For example, Ti02 NPs were more toxic to E. sibiricum than to E. coli

45

 

 

(Figure 2.2). Some groups reported that the Ti02 NPs should be more toxic to Gram
negative than Gram positive species, since Gram positive species have thicker cell walls
and can form spores (49). E. sibiricum is non-spore-forming, which may be one of the
reasons why Ti02 NPs are more toxic to E. sibiricum. We also found that ZnO NPs were
more toxic to E. coli compared to E. sibiricum. Adams et al. (1) found that ZnO NPs
were the most toxic ones of three NMs they tested, and were more toxic to B. subtilis,
Gram positive, than to E. coli, Gram negative. Other external factors, such as the initial
concentration of bacteria, the physiological state of the bacteria, light intensity, pH and
02, also can influence the toxicity of NPs (38).

The dissolution of NPs was hypothesized as one of the mechanisms by which NPs
interact with cells (32). The dissolution can be influenced by the coating of NPs, pH of
solution and other factors (12, 52). Ti02 is highly insoluble (0.15 mg/L) while ZnO is
slightly soluble (5.4 mg/L) (6). Even if Ti4+ is dissolved into solution, the reaction below
usually happens:

Ti4+ + 2HZO —+ Tio2 + 4H+

Ti2+ is much less stable than Ti“, and it is probably oxidized by 02 to form Ti“. The
dissolution of ZnO NPs was previously reported, with the suggestion that toxicity might
depend on Zn2+ ion in NP solution (6, 12, 17, 22). The dissolution of ZnO NPs may
worsen the adverse effects on aquatic biota and cause more ecotoxicity (39). We did not
find that the dissolution of ZnO NPs was size-dependent, although it had been reported

that Zn2+ release rate of ZnO NPs was faster than that of ZnO micro-particles (52). Zinc

46

ions play an important role in cellular process, mostly as an enzyme cofactor (47). It has
been reported that high concentrations of Zn2+ ion are harmful for cells by inhibiting
some enzyme activity or affecting ATP (8, 28, 48). The viability results demonstrated that
Zn2+ was more toxic to E. coli than E. sibiricum. According to the genome sequences of
E. coli and E. sibiricum in the database of National Center for Biotechnology
Information, there are more proteins annotated for zinc transport, efflux, or uptake in E.
coli than in E. sibiricum, which may make E. coli more vulnerable to Zn2+ toxicity.
Intemalization of NPs can be influenced by size and chemical composition of NPs
and by types of microorganisms. For instance, 5 nm Ti02 NPs were found in E. sibiricum
rather than in E. coli, which suggests that it is easier for Ti02 NPs to go through the
membrane of E. sibiricum than that of E. coli. This could be one of the reasons why Ti02
NPs were more toxic to E. sibiricum than E. coli, since the internalized NPs may cause
direct damage and induce serious toxicity. Ti02 NPs can decompose and peroxidate
membranes, which is the explanation for the killing effect by Ti02 photocatalysis (27, 40,
44). In the ZnO NP samples, many dead cells with damaged membranes and leaking
intercellular contents were noted, also observed in a previous study (5). Furthermore, we
investigated the morphology of E. coli and E.sibiricum exposed to 50 nm Fe3O4 NPs and
5 pm Ti02 particles by TEM, and found that both E. coli and E. sibiricum could
internalize Fe3O4 NPs, although Fe304 NPs were non-toxic. No 5 pm Ti02 particles were
found inside these microorganisms. The largest Ti02 particles found inside the cells were

around 150 nm. Other NPs, such as CeOz and silver, were previously found internalized

47

in E. coli or incorporated into the membrane, as well as interfacial charge transfer and the
interaction between the Ti02 NPs and membrane of E. coli (30, 43, 46). There is no clear
explanation, however, about the mechanisms of internalization. The contact between NPs
and bacteria was enhanced by the repulsive force generated between negatively charged
bacterial cells and positively charged NPs (31).

In conclusion, we demonstrated that ZnO and Ti02 NPs were toxic to E. coli and E.
sibiricum in a dose-dependent manner, while SiOz and Fe3O4 NPs did not inhibit their
growth during a 12 h exposure. Among these NPs, ZnO NPs were the most toxic ones for
either of these two microorganisms. ZnO was more toxic to E. coli than to E. sibiricum
with the toxicity mostly due to the Zn2+ ion dissolved from the NP, while TiOz was more
toxic to E. sibiricum than to E. coli. By TEM-EDS, we found Si02 and Ti02 NPs
internalized only in E. sibiricum not in E. coli, while ZnO NPs appeared inside both of

these bacteria.

48

 

 

 

 

 

 

 

 

 

1000 -
- 60 nm
100 1 um
" ii u:
g
C E
e\: 10 J I *Itbl 11*
go 5 l u
3 1 , "' _ a
8 i an: a E
11 u
1* ** 13* *1!
L ‘ a It ~— slut:
‘ - ** *t **
01 l i i ..
1". :5 a 'i if": E 5: is
0.01 " :91 l‘; i“ 3 g iii A 3:

control 50 100 200 300 400 500 1000 2000 5000
Nanoparticles concentration (ppm)

 

 

 

 

 

 

 

 

 

 

 

 

 

#9533-

b
1000 -
1 um
- 60 nm
100 " r: " at it:
to, B a an at
g; 11 1:33 i it 1
V ,, .' .' . E u: an
3:. .- :2 ii . ;
9 1o - l. g iii a: .. E g
31': I' l :51} ”'- link
8 ii a s ' .
Z 1 F 1‘ E a
1 - it -. 1:. ' l . “ g n
3"; ‘51 i t. E t

 

 

5’fo /

0.1 - a
control 50 100 200 300 400 500 1000 2000 5000
Nanoparticle concentration (ppm)

Figure 2.1 (a) The effect of ZnO NPs on E. coli. (b) The effect of ZnO NPs on E. sibiricumi. *p<0.05
versus control cells;*"'p<0.005 versus control.

49

140 -

120 -

100 a

80+

60-

Cell Growth (%)

40‘

20a

 

120 a

100 -

80-

60-

Cell Growth (%)

4o-

20'

 

1." ' -w.lmmme ' :-

control

 

3
,l

 

WHJWW

control

 

 

 

32'

5O

j_l
if
t!

 

50

 

-5nm

 

 

 

 

 

 

 

 

 

 

- 150 nm
- 5 um
i
a ! ‘.‘ *
l i *-
-' p . an:
: ‘ fl }
1 f a
l l " i
ii I .~
i: “ ‘
l ~
1 t
100 500 1 000 2000 5000
TiO2 concentration (ppm)
b
— 5 nm
T - 150 nm
1‘ - 5 um
i at:
l *
. if 1 . t
l a , ii ,
t. l ,i ,‘ ‘7'
l i ll . .
l; f .. -
100 500 1000 2000 5000

Ti02 concentration (ppm)

Figure 2.2 (a) The effect of Ti02 NPs on E. coli. (b) The effect of Ti02 NPs on E. sibiricum. *p<0.05
versus control cells;**p<0.005 versus control.

50

- 60nm
140 m1um

Cell Growth (%)

is

 

 

 

20‘

 

 

 

 

 

 

 

 

 

  

control 50 100

Fe304 concentration (ppm)

140 - - 60 nm

1 l l

60-

Cell growth (%)

4o — - it .
5 in .+ .

20‘

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Drag: ‘

control 50 100 500 1 000 2000 5000

Fe304 concentration (ppm)
Figure 2.3 (a) the toxicity of 60 nm and 1 pm iron oxide NPs to E coli, (b) the toxicity of 60 nm and 1 pm
iron oxide NPs on E. sibiricum.

51

 

 

 

 

 

 

   

 

 

   

 

 

 

 

 

 

 

 

 

 

 

 

 

 

140 ‘ - 10 nm
_ 1 um
120 -
100 ‘ .‘133
{a {3
o z
.c 80 ‘ g
E
9 ‘_
2’ 60 - ; 5
8 5' 2:1
40 ‘ ~ 3:
20 - 1;
0113'"; § Fifi 2‘5».
control 50ppm 100ppm 500ppm 1000ppm 2000ppm 5000ppm
nanoparticles concentration (ppm)
b
140 - - NPs1O nm
Es: NPs1 um
120 -
100 J E El .5
E 80 d " -.;
2 f"- I
52 6O 4 '. -.-' L. t
35 I .
40 ‘ :' fir %
l. .‘ '
20 ‘ a

 

 

 

control 50ppm 100ppm 500ppm 1000ppm 2000ppm 5000ppm

Nanoparticle Concentration (ppm)

Figure 2.4 (a) the influence of Si02 NPs (10 nm and 1 pm) on the growth of E. coli, (b) the influence of
Si02 NPs (10 nm and 1 pm) on the growth of E. sibiricum.

52

Table 2.1 The concentration of Zn2+ ion dissolved in LB or TSB from various concentrations of ZnO NPs

 

 

 

ZnO NPs Zn2+ Conc in LB (ppm) Zn2+ Come in TSB (ppm)
Cone

60 nm 1pm 60 nm 1pm
(ppm)
0 1.2:t0.0 1.2:t0.0 0.7:i:0.0 0.7:t0.0
50 38.0:t2.8 37.0:t:1.4 40.0i0.0 38.5:h2.1
200 90.0:t0.0 85.0:t7.1 110.0i0.0 100.0:t0.0
1000 100.0:t0.0 100.0i0.0 10502157.] 100.0i0.0
5000 115.0i7.1 100.0101) 110.0i0.0 100.0100

 

Table 2.2 Viability of E. coli and E. sibiricum in various concentrations of Zan solution

 

 

 

ZnClz Conc Viability (%)

(ppm) E. coli E. sibiricum
Control 100.0i10.3 100.0i2.0
32.5 87.3i10.4 113.9i4.6
65 68.2:t4.2 77.3i7.8
130 <0.01 16.4i3.9

 

53

 

 

 

 

 

 

 

 

a
+ 5 nm Ti02 solution only
—0— 5 nm Ti02 NPs and solution
140 1 + 150 nm Ti02 solution only
—A— 150 nm Ti02 NPs and solution
+ 5 um Ti02 solution only
120 ' —U—- 5 um Ti02 NPs and solution
g 100 -
g
'5
to
'5 80 -
60 d
40 l I I l

 

control 1 000 2000 5000
TiO2 NP concentration (ppm)

 

+ 5 nm Ti02 solution only
—0— 5 nm Ti02 NPs and solution
+ 150 nm Ti02 solution only
—A— 150 nm Ti02 NPs and solution
140 ' + 5 um Ti02 solution only
—t:l— 5 urn Ti02 NPs and solution

 

 

120 a

100 - . «b»

\.

 

 

 

 

 

 

:3? A“
:5 so - ..
.0
.9
> 40 a
20 “ 6 I
0 .. :
control 1000 ' 2000 5000

TiO2 NPs concentration (ppm)

Figure 2.5 (a) The toxicity of Ti02 NP suspension and soluble fraction on E. coli. (b) The toxicity of Ti02
NP suspension and soluble fraction on E. sibiricum.

54

 

 

 

 

 

 

 

 

 

 

 

 

 

1000 -
100 g + 60 nm ZnO solution only
—0- 60 nm ZnO NPs and solution
g 10 -
g
B
.9
> 1 r
0.1 d
0.01 r . r r r r f
control 100 200 500 1000 2000 5000
ZnO NP concentration (ppm)
b
1000 -
+ 60 nm ZnO solution only
—0— 60 nm ZNO NPs and solution
100 4
E
g 10 .
.o
.3!
>
1 ..
0.1 I I I T l l I

control 100 200 500 1000 2000 5000
ZnO NP concentration (ppm)

Figure 2.6 (a) The toxicity of ZnO NP suspension and soluble fraction on E. coli. (b) The toxicity of ZnO
NP suspension and soluble fraction on E. sibiricum.

55

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58

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64

CHAPTER 3

INDUCTION OF INTRACELLULAR REACTIVE OXYGEN SPECIES BY
NANOPARTICLES AND THE MEASUREMENT OF GENOTOXICITY BY
COMET ASSAY

65

Abstract

With the rapid development of nanotechnology, various NMs have been applied in
diverse areas, including cosmetics, water purification, material science, electrical devices
and drug delivery. The biosafety of NPs has begun to attract attention with regard to their
toxicological and environmental effects. I found that zinc oxide (ZnO) and titanium
oxide (Ti02) NPs induced reactive oxygen species (ROS) in Escherichia coli strain K12
(Gram-negative) and Exiguobacterium sibiricum strain 255-15 (Gram-positive) in a
dose-dependent manner. A 12 h exposure to silicon oxide (Si02) NPs induced ROS in E.
sibiricum, but not in E. coli except for 5000 ppm Si02 NPs. There were more ROS
induced by Ti02 NPs and Si02 NPs in E. sibiricum than in E. coli, while ZnO NPs
induced more ROS in E. coli than in E. sibiricum, which is consistent with our viability
results. The 5 nm Ti02 NPs caused more ROS induction than 150 nm Ti02 NPs, which
confirmed that size is one of the important factors in NP toxicity. Both Ti02 NPs and ZnO
NPs displayed genotoxicity as measured by comet assay, and Ti02 NPs were more
genotoxic than ZnO NPs. During 3 h exposure, 10 ppm Ti02 and 1 ppm ZnO NPs caused

significant DNA damage in lymphocytes.

66

Introduction

The development in NMs and nanotechnology offers various new opportunities in
diverse fields, such as electronics, medicines, environmental remediation and food. In
2005, more than $10 billion were spent on the nanotechnology development
internationally (21). However, the environmental exposure to the NPs may have some
adverse effect on microorganisms, although NPs have existed in the nature for eons (31).
Few studies about the influence of NP on public health and environment are available,
and there is no clear understanding of the mechanisms of NP toxicity.

Recently there have been a few reports on the antimicrobial activity of NPs and
interaction between NPs and microorganisms (7, 8, 18, 34, 35, 42). The induction of
ROS by NPs is considered as one of the possible mechanisms for NP toxicity. In 1997,
Rahman et al. found the increasing induction of ROS by nano-sized Ti02 in human and
rat alveolar macrophages (36). Meanwhile, Ti02 NPs were toxic and caused oxidative
stress in different cell types, including bacteria and mammal cells (2, 20, 26, 39, 50-52).
The induction of ROS by ZnO NPs has also been hypothesized as an explanation for
their toxicity, though the mechanisms for their toxicity are not yet well established (3 8).

NPs may cause genotoxicity to different cells, since the ROS can enter cells and
oxidize membranes, and further damage the DNA, lipids and proteins. There have been a
few papers reporting DNA lesions caused by ROS induction from TiOz NPs (16, 20, 24,
27, 50). The alkaline comet assay is a well-established method to detect and measure

DNA damage, since it is sensitive, rapid, flexible, cheap and easy to apply (45-47). This

67

 

technology has been used in different fields, including radiation biology, genotoxicity,
clinical research, and genetic ecotoxicology (3, 12, 14).

In this study, we investigated the ROS induction by Si02, Ti02 and ZnO NPs in E.
coli and E. sibiricum. I also suggested a possible mechanism of ROS induction by Ti02
NPs. The comet assay was performed to evaluate the genotoxicity of Ti02 and ZnO NPs,

and Olive tail moment, tail DNA and tail length were measured to quantify DNA damage

in lymphocytes.

Materials and Methods

Reagents and preparation of NP suspensions. RPMI-l640 medium and 2’, 7’-
dichlorofluorescin diacetate (DCFH-DA) was obtained from Invitrogen (Carlsbad, CA).
Normal melting agarose (NMA), low melting point agarose (LMPA), Histopaque,
Phosphate Buttered Saline (PBS), Si02 (10 nm), Ti02 (5 nm and 150 nm) and ZnO (60
nm) were purchased from Sigma-Aldrich (St. Louis, MO). One hundred g/L NPs stock
solutions were prepared in distilled and deionized water.

Bacterial culture and cell culture. E. coli K12 was grown aerobically in
Luria-Bertani (LB) liquid broth at 37 °C and 200 rpm for 12 h. E. sibiricum was cultured
in TSB medium at 30 °C and 150 rpm for 12 h. Solid medium for E. coli or E. sibiricum
was as above with 1.5% agar (Difco) added. All the cultures with NPS were grown under
the same conditions.

Intracellular ROS measurement. The intracellular ROS was measured by using

68

DCFH-DA as described previously (25, 49). Ten mM of DCFH-DA stock solution in
DMSO was diluted with PBS buffer into 20 M of working solution. E. coli K12 or E.
sibiricum was exposed to different concentrations of NPs in 2 ml of liquid media for 24 h.
Cells were collected by centrifugation in 1.8 ml of liquid medium, washed twice with
water and then incubated in 1.8 ml of DCFH-DA working solution at 37 °C or 30 °C for
1 h. Samples were washed twice with 1X PBS, and their fluorescence was measured at
485-nm excitation and 520-nm emission with SpectraMax M2 spectrometer (Molecular
Devices, Sunnyvale, CA). Blank samples (only microorganisms) and control samples
(only NPs) were treated in the same way. Viability was measured by plate counting. All
the data are the mean of triplicate samples.

Alkaline comet assay. Fresh Heparinized venous blood was used for separation of
lymphocytes. Blood (100 pl) was diluted with RPMI-164O medium (1 ml) and the
dilution was layered over 200 pl Histopaque buffer. After centrifuged at 800><g for 20
min, the buffy coat from the medium/Histopaque interface was collected. After washed
once with RPMI-1640 and centrifuged at 250><g for 10 min, the pellet was resuspended
in 1 ml RPMI-1640. The lymphocyte suspensions were exposed to various
concentrations of Ti02 NPs or ZnO NPs (1 ppb, 10 ppb, 100 ppb, 1 ppm, 10 ppm, and
100 ppm) for 3 h or 6 h at 37 °C, described by Tice et al. (46). The positive control
groups received 2 mM ethyl methanesulphonate (EMS) for 3 h exposure or 1 mM for 6 h
exposure. After the treatment, the lymphocytes were centrifuged at 500x g for 5 min and

resuspended in 110 pl PBS solution. The Trypan blue dye viability test was applied

69

before and after the NP exposure, and the viability was essential for all the experiments.
The lymphocyte suspension (100 pl) was mixed with 1% LMPA (100 pl) at 37 °C, and
80 pl of mixture was added onto one of the duplicate microscope slides (coated with 1%
NMP and dried in advance), and covered with a cover-slip. After 5 min solidification on
ice, the cover-slip was slid off, and 0.5% LMA (90 ul) was spread as a third layer with a
similar procedure. After the removal of the cover-slip, the slides were dipped into freshly
made, cold-lysing solution (2.5 M NaCl, 100 mM EDTA and 10 mM Tris (pH 10), with
1% Triton X-100 added just before use) for a minimum of 2 h at 4 °C. All the slides were
closely placed in a horizontal electrophoresis box containing freshly-prepared cold
electrophoresis buffer (1 mM EDTA and 300 mM NaOH, pH >13) for 20 min to allow
for unwinding of the DNA, and subsequently electrophoresis was performed at 24 V and
300 mA for 30 min. After that, the slides were washed three times (5 min each) with
neutralization buffer (400 mM Tris, pH 7.5) and stained with 1 ug/ml ethidium bromide
(80 ul) for 5 min. The slides were then soaked in cold distilled water for 5 min, covered
with cover-slips, and then stored at 4 0C in a humidified slide box (no more than 2 days).
The observation was made at 400><g magnification with a Komet 3.1 Image Analysis
System (Kinetic Imaging, Liverpool, U.K.), and the DNA damage parameters, including
olive tail moment, tail DNA and tail length, were measured (17). One hundred total cells
on duplicate slides were analyzed in each of three independent experiments. The data
were analyzed with one-way analysis of variance (ANOVA), and p < 0.05 was

considered significant compared with the respective controls.

70

Results and Discussion

Cellular oxidative stress level. DCFH-DA can react with ROS and produce
fluorescence. Intracellular oxidative stress increased as NP concentrations increased
during the 24 h exposure (Figure 3.1). Si02 NPs slightly induced ROS in E. coli only at
5000 ppm in Figure 3.1(a). For Ti02 NPs, ROS was induced when the concentrations
were higher than 100 ppm, which meant that Ti02 NPs induced more ROS in E. coli than
Si02 NPs. Furthermore, 5 nm Ti02 caused more oxidative damage in E. coli than 100 nm
Ti02, which is consistent with the size-effect found in our viability studies. In the case of
E. sibiricum, the ROS increased When the concentrations of Si02 NPs were higher than
500 ppm, and ROS was induced in E. sibiricum at Ti02 NP concentrations above 100
ppm (Figure 3.1(b)). Similar to the results with E. coli, 5 nm Ti02 NPs induced more
ROS in E. sibiricum than the 150 nm NPs. Hence, Si02 and Ti02 NPs induced more ROS
in E. sibiricum than in E. coli, which might be one of the reasons why the growth of E.
sibiricum was more inhibited by the same NPs than E. coli in our previous studies.

ZnO NPs were the most toxic of these three test NPs, and they caused the most
serious inhibition on the microbial growth and induced the most ROS in both E. coli and
E. sibiricum. As illustrated in Figure 3.1(c), the ROS in both E. coli and E. sibiricum
began to increase at 50 ppm of ZnO NPs. ZnO NPs induced more ROS in E. coli than in
E. sibiricum, which was consistent with our viability test.

Our ROS induction data also proved the size-dependent effect of NPs, and were

71

consistent with our previous viability test. The 5 nm Ti02 NPs induced more ROS in
either E. sibiricum or E. coli than the same concentration of 150 nm Ti02 NPs. Other
groups also have reported the size effect on ROS induction by ZnO NPs or silver NPs (11,
33). The ROS formed on the surface of Ti02 NPs was also reported to depend on particle
size, aggregation of particles and other factors (1). Since the lifetime of hydroxyl radicals
is extremely short (10'9 s), the direct contact between NPs and membranes seems
essential for an effect of NPs on bacteria (19, 22, 44). Although Gram-negative bacteria
usually have thinner cell walls than Gram positive bacteria, it seems that there was no
significant difference in their membrane permeability to ROS.

Photocatalytic Ti02 chemistry. The reactions below describe the possible
mechanisms of ROS induced by Ti02 NPs (9, 10, 53). In the first equation, a hole
(e')/electron (W) in the valence band and conduction band is generated and induced by
light 0t<390 nm) and Ti02 NPs. The e' is absorbed onto the surface of Ti02 NPs and
available for electron transfer, which can reduce 02 to Oz' (reaction 2), and produce H202
by further reducing Oz' (reaction 3). The OH radicals can be produced by the reaction of
superoxide with H202 (reaction 4) and reduction of H202 by e" (reaction 5). The OH
radicals can also be formed by the reactions of H20 or OH’ with h+ and electron
abstraction from absorbed oxidizable species by W (reaction 6 and 7). H202 can be
produced by either a reductive pathway (reaction 3) or an oxidative pathway (reaction 8),
while it can be decomposed into OH radicals by induction of light (reaction 9).

Tio2 + hv —+ no2 (e' + 17*) (1)

72

e' + 02 —+ 02' (2)

02' + 2H+ + e' -—> H202 (3)
H202 + 02' —> °OH + OH' + 02 (4)
e' + H202 —->°OH + OH' (5)
h+ + 011' —+°OH (6)
h+ + H20 —+ °OH + H* (7)
2h* + 21120 —> 2H+ + 11202 (8)
hv + H202 -> 2'OH (9)

An interfacial charge transfer occurred between T102 NPs and the membrane of E.
coli (32). The reaction 10 and 11 exhibit the surface charge trapping on T102 NPs. During
the process of interfacial charge transfer, h+ reacts with peptidoglycan (PGN) and
lipo-polysaccharides (LPS) on microbial cell wall membranes, such as E. coli (reaction
12). As well, h+ can be scavenged by PE, LPS or E. coli during surface oxidation

(reaction 13).

e' + Ti(IV)O-H —+ Ti(III)O-H' (10)
h++Ti(IV)O-H—-Ti (IV)o--H+ (11)
h++RH—>R'+H+ (12)
Ti(IV)O--H+ + RH —-» OH + R- + H" (13)

ROS induction can result in other intracellular damage and disorder of cellular
activities. Matsunaga et al. reported that TiOz could oxidize CoA and cause cell death (28,

29). The cell membrane may be oxidized prior to the oxidation of CoA (13, 30, 43). Saito

73

et al. found rapid leakage of K+ ions and slow release of proteins and RNA, and they
explained that the cell death was due to a significant disorder in cell permeability (5, 37).

Genotoxicity of Ti02 and ZnO NPs. Exposure to Ti02 and ZnO NPs caused
significant DNA damage in lymphocytes. Lymphocytes treated with different
concentration of T102 NPs for 6 h, and the clear DNA tails, appeared at concentrations
greater than 1 ppm. As shown in Table 3.1, the tail length, DNA tail and Olive tail
moment increased with the increasing Ti02 NP concentration for both 3 h and 6 h
exposures, which meant that DNA damage aggravated as the NP concentration increased.
The lymphocytes displayed significant DNA damage in 3 h exposure when the Ti02 NP
concentrations were greater than 1 ppm. During the 6 h exposure, the tail length, the tail
DNA and the Olive tail moment demonstrated significant difference at concentrations
above 100 ppb, compared to the negative control. There is no significant difference
between 3 h and 6 h exposures.

In the case of ZnO NP treatment, the enhancement of tail length, DNA tail and olive
tail moment was demonstrated as the concentration of ZnO NPs increased. The
concentrations of ZnO NPs, at which significant DNA damage started, were 10 ppm for 3
h exposure and 1 ppm for 6 h exposure. These results were ten times higher than those
for Ti02 NPs. There is no clear explanation about genotoxicity caused by NPs, although
one of the hypotheses is ROS induction (8, 16, 19, 20, 35, 40, 43, 48).

The results suggest that Ti02 NPs caused more genotoxicity than ZnO NPs,

consistent with other reports (23). The explanations are:

74

(a) Dissolution of ZnO NPs. Our previous studies have shown that ZnO dissolves in
LB and TSA medium to form Zn2+ ion, which is not genotoxic. Actually low
concentrations of Zn2+ ion can stabilize membrane structures and prevent ROS
production (6, 41). The saturation concentration of Zn2+ ion in the solution of ZnO NPs is
approximately 100-110 ppm. The dissolution of ZnO NPs may reduce the genotoxicity of
ZnO NPs.

(b) Size difference between T102 NPs and ZnO NPs. The size of Ti02 NPs was 5 nm,
while the size of ZnO NPs was 60 nm. For the same mass, Ti02 NPs provide more
surface area and hence supply more surface reactive sites than ZnO NPs. There may be
more Ti02 NPs absorbed to the lymphocyte membrane than ZnO NPs due to their smaller
size. Moreover, it is probably easier for the much smaller Ti02 NPs to enter the
membranes of lymphocytes than for the larger ZnO NPs.

Induction of ROS has been hypothesized as one of possible mechanisms for
genotoxicity. As shown in Figure 3.1, ZnO NPs induced much more ROS than Ti02 NPs.
When the concentrations of NPs were lower than 100 ppm, however, there was not much
difference between the ROS induced by ZnO NPs and by TiOz NPs, especially in E.
sibiricum. Fullerenes can cause DNA damage, and it is much more genotoxic than ZnO
NPs and Ti02 NPs (15). The reason may be that fullerenes can disperse more evenly in
solution and not aggregate as easily as ZnO NPs and Ti02 NPs. In addition, Silica NPs
was reported to caused no genotoxicity by the comet assay (4).

In conclusion, the ROS induction by NPs was found in both E. coli and E. sibiricum.

75

SiOz NPs could induce ROS in E. sibiricum when the concentration was greater than 500
ppm, while they did not induce ROS in E. coli except at 5000 ppm. TiOz NPs induced
more ROS in E. sibiricum than in E. coli, which is consistent with our previous viability
tests. The 5 nm Ti02 NPs induced more ROS than 150 nm Ti02 NPs in either E.
sibiricum or E. coli. According to the measurement of Olive tail moment, tail DNA and
tail length in comet assay, 1 ppm ZnO NPs in 6 h exposure and 10 ppm ZnO NPs in 3 h
exposure caused significant DNA damage in lymphocytes. The 100 ppb Ti02 NPs for 6 h
exposure and 1 ppm Ti02 NPs for 3 h exposure damaged DNA significantly, which

demonstrated that Ti02 NPs are more genotoxic than ZnO NPs for lymphocytes.

76

 

 

 

 

 

 

 

 

 

 

 

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77

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Figure 3.2. Human lymphocytes treated with Ti02 showing genotoxicity (magnification 400): (a, b,
c, d, e, and f) nuclei from Ti02-treated 6 h (1 ppb, 10 ppb, 100 ppb, 1 ppm, 10 ppm and 100 ppm)
human lymphocytes showing DNA damage; (g) nucleus from an untreated human lymphocyte
(negative control); (h) nucleus from an ethyl methanesulfonate (2 mM; positive control) treated

human lymphocyte showing DNA damage.

81

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Warheit, D. B., T. R. Webb, C. M. Sayes, V. L. Colvin, and K. L. Reed. 2006.
Pulmonary instillation studies with nanoscale Ti02 rods and dots in rats: Toxicity

is not dependent upon particle size and surface area. Toxicological Sciences
91:227-236.

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53.

Zhang, L. Z., J. C. Yu, H. Y. Yip, Q. Li, K. W. Kwong, A. W. Xu, and P. K.
Wong. 2003. Ambient light reduction strategy to synthesize silver nanoparticles
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Langmuir 19: 10372-10380.

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CHAPTER 4

THE INFLUENCES OF OXIDE NANOPARTICLES ON GENE EXPRESSION OF
ESCHERICHLA COL! K12

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Abstract

Si02, Ti02, and ZnO NPs are used in various fields as support or catalysts. Our
previous toxicity data demonstrated their antibacterial activities. In this study, we
explored their influence on the gene expression of E. coli, which generally increased
from Si02 to Ti02 to ZnO NPs. In Si02 and ZnO NPs treatments, the number of repressed
genes was higher than that of the induced ones, while more genes were induced than the
repressed in Ti02 exposure. The transporters for amino acid and carbohydrate were
down-regulated, while the transporter for iron or a few other metal ions were
up-regulated. Moreover, stress-related genes and DNA repair genes were generally
induced. This may be due to a number of reasons including ROS induction, damage to
membrane, proteins or DNA, alteration of membrane permeability and fluidity, attack to
iron-sulfur cluster and nick on DNA. It was also demonstrated that ZnO NPs influenced
microbial respiration. The microarray data demonstrated that NPs regulated the genes in
different categories, and E. coli adjusted their metabolic pathways and defense

mechanisms during the exposure to NPS.

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Introduction

Nanotechnology, defined by The Royal Society, is “the design, characterization,
production and application of structures, devices and systems by controlling shape and
size at nanometer scale” (29). The growth in nanotechnology offers numerous
technological advances and industrial development opportunities, and it is estimated that
the global market for nanotechnology grow to about $1 trillion by 2015. NMs, one
proportion of nanotechnology, possess enhanced and unique mechanical, optical and
electrical characteristics, which broaden their application in diverse fields, such as
environmental remediation, cosmetics, medicine and catalysis. Although the number of
studies addressing to the toxicity of NPs is increasing, there are many unknowns about
the environmental impacts of NPs and the mechanisms of NP toxicity (70, 81).

The toxicity of NPs to microorganisms is an important concern for evaluating the
environmental impact of NPs. Microorganisms play a fundamental role in the
environment, and supply key environmental services in maintaining healthy ecosystems.
Studying toxicity of NPs on microorganisms is beneficial for us to evaluate the
environmental impacts of NPs and explore the nanotoxicology on animals and human.
Possible explanations for NP toxicity include induction of ROS, disruption of membrane,
releases of hazardous constituents, and damage to DNA (52).

$02, Ti02, and ZnO NPs are popular NPs, and used for cosmetics, food, painting
and electrical devices. Several papers have been published about their antibacterial

activities (3, 7, 32, 33, 80). In this study, E. coli K12 is chosen as the test species to

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explore gene expression influenced by Si02, Ti02, and ZnO NPs. E. coli K12,
Gram-negative, facultative anaerobic and non-spore forming, is one of the most studied
bacteria in the field of microbiology. Microarray technique was applied to explore the
gene expression of E. coli in the NP exposure. Microarray data were validated by
quantitative PCR for a few target genes. We categorized the influenced genes, and also
analyzed and explored the pathways and the transcriptional units that are regulated by

NPs.

Materials and Methods

Reagents and preparation of NP suspensions. Random hexamer primers, 100 mM
nucleotide set, RNaseOUT, salmon sperm DNA, superscriptase 11 RT, formamide, 10%
sodium dodecyl sulfate, 20x Saline-Sodium Citrate (SSC), and 0.5 M
ethylenediaminetetraacetic acid (EDTA) were obtained from Invitrogen Co. (Carlsbad,
CA). Bovine serum albumin (BSA), dimethyl sulfoxide, sodium hydroxide, 1 M
hydrochloric acid and isopropanol were purchased from Sigma-Aldrich Chemical Co. (St.
Louis, MO). RNAlater, SlideHyb buffer and aminoallyl—modified dUTP were obtained
from Ambion Inc. (Austin, TX). L'ysozyme, DNase I, Tris-Cl EDTA (TE) buffer, RNeasy
mini kit, Quiaquick PCR purification kit and RNA protect bacteria reagents were
purchased from Qiagen Co (Valencia, CA). Si02 (10 nm), Ti02 (5 nm) and ZnO (60 nm)
were purchased from Sigma-Aldrich (St. Louis, MO). One hundred g/L NPs stock

solutions were prepared in deionized and distilled water. Phosphate wash buffer (100 ml)

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for cDNA purification was made of 0.5 m1 K2HPO4 (1 M, pH 8.5), 84.25 ml ethanol and
15.25 ml H20 and phosphate elution buffer (100 ml) contains 0.4 ml KzHPO4 (l M, pH
8.5) and 99.6 ml H20. All solutions were autoclaved and stored at room temperature

Cell growth, harvest and RNA extraction. From the freezer stock of E. coli streak
plates were prepared with LB medium and grown at 37 °C overnight. One colony on the
plate was transferred to the tubes with 5 ml LB broth. The NP stock solution was added
to each tube with 10 ml LB broth to result in final NP concentrations of 1000 ppm for
Si02 NPs, 1000 ppm for Ti02 NPs and 50 ppm for ZnO NPs. The samples were
inoculated and incubated until mid-log growth phase of E. coli (O.5<OD600<1).
According to the protocol of RNAprotect bacteria reagent, 1 ml culture and 2 ml
RNAprotect were mixed by vortexing for 5 s and incubated for 5 min at room
temperature. The pellet was collected by centrifuging for 10 min at 5000xg, and
supernatant was decanted. The 200 pl lysozyme in TE buffer (1 mg/ml) was added to the
pellet, mixed by vortexing for 10 s, and incubated at room temperature for 5 min on a
shaker. Buffer RLT (700 pl) in RNeasy mini kit was applied and vortexed vigorously.
After 2 min centrifugation at maximum speed and removal of the NP sediment, the
supernatant was mixed with 500 pl ethanol by shaking vigorously. The lysate was
transferred to RNeasy Mini spin column as per the procedure described in RNeasy mini
kit, and centrifuged for 14 s at >8000x g. DNase digestion was applied to the column by
buffer RWl washing, DNase I solution washing (10 pl) and incubating, and then buffer

RWl re-washing. The RNA was purified as per the protocol of RNeasy mini kit and the

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final concentration of RNA was measured by Nanodrop from Therrno Scientific Co.
(Wilmington, DE). RNA from control samples was processed in a similar way as NP
samples. All experimental and control samples were prepared in triplicate.

cDNA synthesis and dye-labeling. For each sample, 5 pg of total RNA was used to
synthesize cDNA and two RNA populations of each sample were needed for
dye-swapping. RNA mixture contained 5 pg RNA, 5 p1 random hexamer primers (3 mM),
Rnase-free water in the final volume of 16.8 pl. The RNA mixture was incubated at 70°C
for 10 min and then cooled on ice for another 10 min. The RT-PCR reaction had a 30 pl
volume and contained 6 pl 5x first strand synthesis buffer, 3 pl DTT (0.1 M), 1 pl
RNaseOUT, 1.2 pl 25x dNTPs (7.5 mM aa-dUTP, 5 mM dTTP, 12.5 mM dCTP, 12.5 mM
dGTP and 12.5 mM dATP), 2 pl Superscript 11 RT and 16.8 pl RNA mixture. The
RT-PCR reaction was incubated at 42°C overnight, and then 10 p1 NaOH (1 M) and 10 pl
EDTA (0.5 M) were added to the reaction. After incubating at 65°C for 15 min, 10 pl HCl
was added to neutralize the pH. The cDNA was purified according to the protocol of
Qiagen purification kit with someimodification. The cDNA mixture was mixed with 300
pl PB buffer, and transferred to a Quiaquick column. After 1 min centrifuging at 17900x
g, the collection tube was emptied and the column was washed with 750 pl phosphate
wash buffer twice and centrifuged for 1 min. After 1 min additional centrifuging, the
column was transferred to a new tube, and then eluted twice by incubating the column
with 30 pl phosphate elution buffer for 1 min and centrifuging for 1 min at maximum

speed. The cDNA sample was dried using speedvac from Global Medical Instrumentation

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Inc. (Ramsey, MN). The aa-dUTP cDNA was resuspended in 4.5 p1 sodium carbonate
(0.1 M, pH 9.0) and mixed with 4.5 p1 Cy5 or Cy3. After incubating 2 h at room
temperature in the dark, 35 pl NaOAc (100 mM, pH 5.2) was added and the dye-coupled
cDNA' was purified as described in the protocol of Qiaquick purification kit. The
concentrations of cDNA and dye were measured by Nanodrop from Thermo Scientific Co.
(Wilmington, DE).

Hybridization. The glass-slide arrays were cross-linked at 6000 pJ UV and water
bath was pre-warrned to 42°C. The slides were placed in pre-hybridization buffer
containing 5x SSC, 0.1% SDS and 0.1 mg/ml BSA at 42°C for 90 min. After washed
twice with 0.1x SSC for 5 min and twice with water for 0.5 min, the slides were dried by
centrifuging at 1600 rpm for 3 min and transferred to dry and clean tubes covered with
foil. The cover-slips were washed with 2% SDS, water and ethanol, followed by
additional washing twice with water. Then, the cover-slips were dried with filtered
pressured air and placed in clean tubes covered with foil. The labeled DNA of the control
sample was re-suspended in 4 pl EDTA, and heated at 95°C for 30 s, and then spun down.
The other differently labeled DNA from NP-treated sample was suspended in the same 4
pl EDTA, which was heated at 95°C for 5 min and spun down. The dry slide-array was
placed in a clean and dry hybridization chamber and coverslip was placed on the slide.
The 40 p1 SlideI—lyb buffer, which was preheated to 68°C, was added to the sample and
mixed by pipetting (preventing bubbles). All the hybridization solution was loaded on the

slide, and 25 pl water was added in each well in the hybridization chamber. The chamber

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was placed in water-bath at 42°C for 18 h. For each NP treatment, six hybridizations from
three biological replicates and two technical replicates (dye-swapping) were performed.

Washing, scanning and data analysis. After 18 h, the chamber was removed from
waterbath and dried with paper towel before open. Wash Buffer 1, Wash Buffer 2 and
slide staining jars were prewarmed at 37 °C. The slide was taken out with forceps,
washed once with Wash Buffer 1 (2x SSC and 0.5% SDS) to remove the coverslip on
belly dancer for 5 min, then twice with Wash Buffer 2 (0.1x SSC and 0.1% SDS) on belly
dancer for 5 min, and twice with Wash Buffer 3 (0.1x SSC) for 2.5 min. The slide was
dried by centrifuging at 1800 rpm for 3 min and transferred to dry tubes.

Axon Genepix 4000B laser scanner was used for slide scanning and Genepix 3.0
software was used to produce a raw data set. The scanner was turned on and warmed up
for 15 min while the computer was off. The slide was placed face-down, and PMT and
power % were adjusted to optimal by verifying histogram. The resolution to 5 pixels per
pm and seaming area was selected. Then the interested area was scanned and the image
was saved. GENESPRING 6.0 software (Silicon Genetics, Redwood City, CA) was used
for data analysis. The data was normalized both per chip and per gene (Lowess method),
and dye-swapping. Only the spots with more than 55% of pixel greater than background
plus 2x standard deviation in either Cy5 or Cy3 channel were used for analysis. Genes
that showed a statistically significant change in gene expression (p>0.05) and a greater
than or equal to 2-fold change in magnitude were regarded as significant.

Validation of micoarray data by quantitative PCR. For control samples and

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ZnO treated samples, 5 pg of total RNA was used to synthesize cDNA. RNA mixture
contained 5 pgRNA, 5 pl random hexamer primers (3 mM), Rnase-free water in the final
volume of 16.8 pl. The RNA mixture was incubated at 70°C for 10 min and then cooled
on ice for another 10 min. The reverse transcriptase PCR reaction had a 30 pl volume and
contained 6 pl 5x first strand synthesis buffer, 3 pl DTT (0.1 M), 1 pl RNaseOUT, 1.2 pl
25x dNTPs (12.5 mM dTTP, 12.5 mM dCTP, 12.5 mM dGTP and 12.5 mM dATP), 2 pl
Superscript 11 RT and 16.8 pl RNA mixture. The RT-PCR reaction was incubated at 42°C
overnight, and then 10 pl NaOH (1 M) and 10 pl EDTA (0.5 M) were added to the
reaction. After incubating at 65 °C for 15 min, ten pl HCl was added to neutralize the pH.
cDNA was purified according to the protocol of Qiagen purification kit with some
modification. The cDNA mixture was mixed with 300 pl PB buffer, and transferred to a
Quiaquick column. After 1 min centrifuging at 17900x g, the collection tube was emptied
and the column was washed by 750 pl phosphate wash buffer twice and centrifuged for 1
min as above. After 1 min additional centrifuging, the column was transferred to a new
tube, and then eluted twice by incubating the column with 30pl phosphate elution buffer
for 1 min and centrifuging for 1 min at maximum speed. For quantitative PCR, the
forward primers and the reverse primers were designed for ten target genes and one
reference gene (Table 4.1). The real time PCR reaction had a final volume of 15 pl and
contained 7.5 pl PCR green master mix, 0.9 p1 primer mixture (5 pM for forward primer
and reverse primer respectively), 5.6 pl PCR water and 1 pl DNA template. Real time

PCR was performed on ABI Prism 7900HT Sequence Detection System and the cycling

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parameters were 50 °C for 2 min, 95 °C for 10 min, and 40 cycles of 95 °C for 15 sec and
60 °C for 1 min. The dissociation curves were run for product identification. Real time
PCR was done on ABI Prism 7900HT Sequence Detection System from Applied
Biosystems (Foster City, CA). Comparative CT Method (AACT) was applied for
quantitative PCR analysis. The gene expression fold is equal to 2AACT and AACFACT test

sample —ACT control sample.

Results

Global gene expression profiles under NP exposure. In the treatments of Si02 and
ZnO NPs, a greater number (279 for ZnO and 52 for Si02) of genes were repressed than
the induced (197 for ZnO and 2 for Si02). For Ti02 NPs, there were more up-regulated
genes (28 genes) compared to the number of repressed genes (11 genes). The genetic
response to 100 ppm ZnO NPs was with more than 8.0% of the genome expressing
differentially whereas only approximately 1% of genome showed differential expression
for both 1000 ppm Ti02 NPs and 1000 ppm Si02 NPs respectively. In our previous
studies, E. coli K12 was more tolerant to TiOz and SiOz NPs, but more vulnerable to ZnO
NPs, compared to E. sibiricum. During the 1000 ppm Ti02 NPs exposure, there were 289
down-regulated genes and 197 up-regulated genes in E. sibiricum, while 1] induced
genes and 28 repressed genes were found in E. coli at the same concentration of Ti02

NPs. Not many genes were common among these three different treatments. According to

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the l way-ANOVA analysis, there were 499 genes expressing differently in these three

NP treatments.

Gene expression profile following ZnO treatment. In the ZnO treatment, globally
the expression fold of 5,969 genes ranges from 0.02 to 12.8], while there were 279
down-regulated genes and 197 up-regulate genes. In the treatment of ZnO NPs, there

were more repressed and induced genes than in the treatment of Si02 or Ti02 NPs.

The up-regulated genes in ZnO treatment. The categories “transportation”
(19.7%), “transcription” (12.6%) and “translation” (10.1%) were the biggest ones for the
up-regulated genes in ZnO treatment. The transporters genes included those for
carbohydrate, amino acid, ion, proton, and heme, such as gene glpE rha]? tsp, and nikD
(9, 79, 91, 97). Induction of some transporter genes was the response to Zn2+ in ZnO NP
suspension. For instance, the induced gene zntA (3.8 fold), coding zinc, cobalt and lead
efflux system, was found to be activated by an increased concentration of Zn(II) and
Cd(II), showing greater induction by Cd than Zn (85). Gene znuA, coding subunit of ABC
transporter, was also induced by Zn2+ ions (2.1 fold) (100). Moreover, gene erA (3.5
fold), coding glucitol/sorbitol-specific enzyme IIC component of PTS, was up-regulated,
and its expression was reported to be elevated under basic conditions and addition of Zn
to culture grown in minimal medium (58, 65, 87). The aerobic/anaerobic conditions in
ZnO NP suspension also affected the expression of a few transporter genes. For example,

gene ulaA, coding L-ascorbate transporting phosphotransferase system, was up-regulated

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(2.6 fold), which is required for the ability to utilize L-ascorbate as the carbon source
under anaerobic growth conditions (105). It was also found that induced gene nark (4.6
fold), coding Nark MFS nitrate/nitrite antiporter in anaerobic nitrate respiration, was
regulated by nitrate and oxygen (53). Moreover, a threonine sugar transport protein (STP)
transporter, coded by gene tch, was induced (2.1 fold), and its expression was reported
under anaerobic conditions (86).

The aerobic/anaerobic conditions in ZnO suspension also influenced the expression
of some other genes, especially for those involved in both anaerobic and aerobic
respiration. Both cyoB and cyoD were up-regulated (2.6 and 2.1 fold respectively),
coding subunits of cytochrome b0 terminal oxidase in aerobic respiration. The expression
of gene aldA was also induced (3.0 fold), which codes subunit of aldehyde
dehydrogenase A. Aldehyde dehydrogenase A was expected to be only present under
aerobic conditions and be regulated by catabolite repression, because previous work
showed that it was repressed under anaerobic conditions and induced by the carbon
source (30, 75). Meanwhile, gene sdhC was also up-regulated (2.4 fold), which codes
succinate dehydrogenase membrane protein in aerobic respiration, as a large subunit of
cytochrome b556. Gene ndh is another example, and it codes NADqubiquinone
oxidoreductase II in aerobic respiratory chain (2.0 fold) (69, 73). Genes involved in
anaerobic respiration were also induced, such as genes narG and narH (2.3 and 2.1 fold),
coding subunits of nitrate reductase A in anaerobic respiration. Nitrate reductase A

functions as an electron acceptor and benefits proton motive force (11, 93). These

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suggested that both anaerobic and aerobic respiration is important for survival in ZnO NP
solution.

In the category “transcription”, Induction of some genes were due to the stress
caused by ZnO NPs. TreR transcriptional repressor, coded by gene treR, was induced by
2.4 fold, and treR can de-repress operons related to carbohydrate transport and catabolism
under osmotic stress (44). The induced gene yiiA (2.9 fold), coding CpxR transcriptional
dual regulator, can sense a variety of stress and activate some stress-combative genes,
such as those involved in drug efflux (42). In the presence of zinc, gene zraS (2.5 fold)
was induced, responding to environmental signals. Meanwhile, gene asr (3.3 fold) was
also up-regulated, which responds to zinc and plays a role in survival under acid
conditions (58, 84). Furthermore, induced gene thA (2.2 fold) codes protein involved in
stress resistance, as well as phoH, responding to stress, such as starvation (2.1 fold) (28,
65, 104). All of these demonstrated that E. coli responded to stress resulting from ZnO
NP by inducing some gene expression.

Genes about cell structure and division were also induced. For example, gene ydaD
(2.3 fold) implicated in cell division and cycling was up-regulated, whose
over-expression inhibits cell division and leads to morphological defects (21). Gene ych
was also induced, which involves in flagellar synthesis, swarming, and cell elongation
(26). Moreover, induced gene mukB, coding cell division protein, involves in
chromosome segregation and condensation, and also fimctions in cell division and cycle.

The induction of these genes is probably due to the slow growing rate of bacteria, caused

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by ZnO NPs. The categories about metabolism and biosynthesis of carbohydrate, lipid,
amino acid or nucleotide were also important in the ZnO NP treatment. For instance,
gene sucB and sucD were up-regulated, functioning in tricarboxylic acid cycle. The
changes in these categories are probably due to the ROS induction by ZnO NPs, since

ROS can oxidize amino acid and DNA, and also interrupt nutrient uptake.

Since ZnO NPs can cause genotoxicity, the genes related to DNA repair and RNA
modification were found to be up-regulated. Up-regulation of gene dpiA can induce some
genes involving citrate fermentation and SOS response, or repress some transcriptional
regulator. The subunit of DNA gyrase, coded by induced gene gyrB, involves in DNA
modification and replication, as well as transcription. Meanwhile, gene recC, involved in
DNA repair and essential for recombination and conjugation as well as repair of
double-strand breaks, was also up-regulated (22). Moreover, gene mch which codes for
subunit of S-methylcytosine restriction system and defends cell against foreign DNA, was
induced. Genes involving in detoxification was also up-regulated. For instance, induction
of gene dsdA, coding D-serine deaminase, serves in a catabolic function and
detoxification, since D-serine is still a good nitrogen source and moderate carbon source.
Moreover, gene frmA, coding formaldehyde dehydrogenase, was also induced, which can

detoxify exogenous formaldehyde and be induced by formaldehyde (3 8, 40).

Not only single genes, quite a few operons in different categories was also found to

be induced by ZnO NPs, for example, the transcriptional unit rplNXE-rpsNH—rpIFR

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-rpsE-rme-rplO-sec Y -rpmJ and rsz-rplCD WB-rpsS-rplV-rpsC-rpIP-rme-rpsQ.
These two big operons encode a serial of SOS and 308 ribosomal subunit proteins. The
transcription unit rpsLG-fusA-tqu is another induced operon in the category “translation”,
containing genes tqu (elongation factor Tu), fusA (elongation factor G) and rpsG (3OS
ribosomal subunit protein S7), and it was estimated that elongation factor Tu was the
most abundant protein in E. coli and maximally expressed during stress response (68).
Furthermore, both of the transcriptional unit ngABC and glpT Q were induced, coding
glycerol-3-phosphate dehydrogenase under anaerobic conditions and glycerol-3-P MFS
transporter respectively, which are important for utilization of glycerol (99). The
intracellular level of glycerol-3-phosphate is important for biosynthesis of phospholipids.
Genes narG (a subunit of nitrate reductase A) and narH ([3 subunit of nitrate reductase A),
in the operon narGHJI, was up-regulated, constituting catalytic dimmer of nitrate

reductase A in anaerobic respiration.

A few operons in the category “energy production” were also induced by ZnO NPs.
In the transcriptional unit fdnGHI, fdnG (a subunit of formate dehydrogenase N), fdnH (B
subunit of formate dehydrogenase N) and fan (7 subunit of formate dehydrogenase N)
exhibited induction, and their expression have been found to be induced by nitrate and
anaerobiosis (35, 92). The formate dehydrogenase N is beneficial to generate the proton
motive force and allow generation of the ubiquitous energy carrier ATP by ATP synthase.

One ATP synthase, coded by the transcription unit athEFHAGDC, was induced, proved

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by induction of gene atpC (8 subunit of ATP synthase), atpD ([3 subunit of ATP synthase),
ath (7 subunit of ATP synthase), atpA (a subunit of ATP synthase), atpH (5 subunit of
ATP synthase), and ath (b subunit of ATP synthase), and this ATP synthase can
transport proton and synthesize energy. Moreover, in the transcriptional unit
napFDAGHBC—ccmABCDEFGH, ccmA (subunits of CcmABCDE protoheme IX ABC
transporter), ccmB (subunits of CcmABCDE protoheme IX ABC transporter), and ccmG
(CcmEFGH holocytochrome c synthetase), and napC (cytochrome c protein), napH
(ferredoxin-type protein), napG (ferredoxin-type protein), and napA (large subunit of
periplasmic nitrate reductase), were induced, which were required for electron transfer
from ubiquinol (12, 13, 31). Many genes transporting proton were up-regulated during

ZnO exposure, involved in maintaining the proton motive force and generate energy.

Down-regulated gene profile in the exposure to ZnO NPs.

The categories “transportation” (19.0%) and “hypothetical proteins” (10.8%) had the
highest fraction of down-regulated genes. For instance, the gene manX, manY and manZ
in category “transportation” were repressed, which construct mannose PTS permease and
transport exogenous hexoses (76). The transcriptional unit gatYZABCD was also
down-regulated, coding tagatose-l ,6-biphosphate aldolase (GatYZ),
galactitol-l-l phosphate dehydrogenase (GatD), and galactitol PTS permease (GatABC)
that take up and transport exogenous galactitol. A few genes related to the metabolism of

carbohydrate and amino acid (totally 11.1%) were down-regulated too, maybe due to the

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hindrance of their uptake. Moreover, other categories of genes that were down-regulated
included “transcription” (6.8%), “response to stress” (5.0%), “cell division and motion”
(3.9%), “nucleotide biosynthesis” (3.6%), “DNA repair” (2.9%), RNA modification

(2.9%) and “signal transduction” (2.9%).

The genes related to anaerobic respiration were also found to be down-regulated. On
one hand, some of genes were repressed by the presence of oxygen. For example, the
transcription unit hyaABCDEF was repressed, which codes hydrogenase 1. This enzyme
consists of HyaA (small subunit of hydrogenase 1), HyaB (large subunit of hydrogenase
1), HyaC (b-type cytochrome subunit of hydrogenase 1), HyaD (protein involved in
processing of HyaA and HyaB proteins), HyaE (protein involved in processing of HyaA
and HyaB proteins), HyaF (protein involved in nickel incorporation into hydrogenase 1
proteins), and it is tolerant to oxygen and provide complementary redox properties to the
cell (57). The hya operon was found to be induced under anaerobic conditions and by the
presence of formate, or induced under acidic conditions, but repressed by nitrate (51, 78).
Meanwhile, gene fa’hF, coding formate dehydrogenase, was also down-regulated,'and it
was found to be repressed by nitrate, nitrite and the presence of oxygen (1). On the other
hand, our results also displayed the repression of genes that was reported in anaerobic
conditions. For instance, the repressed gene aldA codes NAD-linked aldehyde
dehydrogenase A, and its repression occurs under anaerobic conditions via ArcA (59).

Under anaerobic conditions, protein ArcA also mediates the repression of L-lactate

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dehydrogenase, coded by gene lldD, which functions in aerobic respiration and
anaerobic nitrate respiration (47). Repressed gene poxB codes pyruvae oxidase, which is
responsible for pyruvate metabolism under aerobic conditions, and its expression
decreases under anaerobic conditions (2, 20). Aerobic/anaerobic conditions in ZnO NP
suspension exhibited down-regulation of some operons. In the transcriptional unit
ynfEFGH-dmsD, gene ynfE (oxidoreductase subunit paralog of DmsA), yan
(oxidoreductase subunit), ynfG (oxidoreductase, Fe-S subunit paralog of DmsB), yan
(DMSO reductase anchor subunit, paralog of DmsC) and dmsD (DMS reductase
maturation protein) were repressed, which can utilize dimethyl sulfoxide as a terminal

oxidant under anaerobic conditions.

The down-regulation of some transcriptional units was also considered as response to
the stress caused by ZnO NPs. The transcriptional unit uxaCA, including uxaA
(D-altronate dehydratase) and uxaC (D-glucuronate isomerase/D-galacturonate isomerase)
was repressed. It was reported that Zn2+ ion could inhibit the enzyme (8). In the
transcriptional unit hdeAB-yhiD, hdeA (inactive form of acid-resistance protein), hdeB
(acid stress chaperone), yhiD (Mg2+ transport ATPase) were also down-regulated, and all
of them involved acid resistance, as well as gene slp (starvation lipoprotein) and hdeD
(acid-resistance membrane protein) (34, 50, 64, 89). The down-regulated transcriptional
unit gadE-mthF also related to low pH, including gene gadE (GadE transcriptional

activator), mth (subunit of MthF-Tolc multidrug efflux transport system) and mth

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(subunit of MthF-Tolc multidrug efflux transport system) (64). Quite a few of
down-regulated stress related genes involved in response to pH.

Even although ZnO NPs can cause genotoxicity, some genes related to DNA repair
and detoxification were still down-regulated, such as gene rqu (Holliday Junction
nuclease). The repressed gene uer, coding DNA-dependent ATPase I and helicase II,
can also protect DNA from degradation. Gene ksgA was downregulated, coding
S-adenosylmethionine-6-N',N'-adenosyl (rRNA) dimethyltransferase, and its expression
is positively regulated by growth rate (74). Meanwhile, some genes functioning in
nucleotide metabolism were also down-regulated. Gene rutC (endoribonuclease), rutD
(hydrolase) and rutE (nitroreductase) in the operon rutABCDEF G were repressed. These
genes function in the pathway for pyrimidine degradation pathway, and the operon is
found under the control of nitrogen regulatory protein (107). The transcriptional unit
xdhABC was also down-regulated, including xdhA, xth, and xdhC, which consist of
xanthine dehydrogenase, and xanthine dehydrogenase plays a role in purine salvage
(101).

Some genes involved in detoxification were repressed by ZnO NPs. For example,
repressed gene yiiM codes a protein involved in base analog detoxification (54).
Moreover, in the transcriptional unit yan-dinJ, coding the Yan-DinJ toxin-antitoxin
system, Yan functions in reducing protein synthesis and inhibiting growth, when DinJ
alleviates that phenotype, acting as the antitoxin (67). Both of these two genes were

down-regulated in the treatment of ZnO NPs. A few genes about cell division, cell motion

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and biofilm formation were also repressed. For example, repressed gene yihA, coding cell
division protein, is essential for normal cell division (6, 23). RNA polemrase sigma F
factor coded by gene fliA was also down-regulated, and it is related to flagellin

production, motility and biofilm formation (10, 60).

Gene expression profiles following Si02 treatment. In SiOz treatment, 5924 genes
ranged in the expression fold between 0.40 and 2.05, which was much narrower than
those in these other two treatments (Fig 5.1). Based on the available information in the
databases of Clusters of Orthologous Groups of proteins (COGS), fifty-two
down-regulated genes in the response to SiOz NPs were grouped into 17 functional
categories, among which “transcription” (11.5%) and “Not in COGS” (30.8%) were the
two of the biggest dominant categories. Other large categories included “Inorganic ion
transport and metabolism” (7.7%), “coenzyme transport and metabolism” (5.8%), “Cell
wall/membrane/envelope biogenesis” (5.8%), “General function” (5.8%), “Function
unknown” (5.8%), “defense mechanisms” (5.8%). However, only 2 genes were

down-regulated in the categories “transcription” and “function unknown”.

“Transportation” is always one of the biggest categories in all the treatments, and for
Si02 NP exposure 10 out of 52 down—regulated genes were in this category, covering
transporters for carbohydrate, amino acid and metal ions, such as gene ych, yeeO, yaaU,
yiaV, and ych (14). Gene ych, functioning as ferrous iron transporter, was repressed

probably due to osmotic up-shift that may be caused by membrane damage from ROS, or

108

anaerobic conditions (48, 95). Meanwhile, gene napC, involving in transportation and
anaerobic respiration, was also repressed. Gene cyoB is another repressed gene involved
in respiration, and it codes cytochrome terminal oxidase subunit 1, a terminal oxidase in
the respiratory chain used under high oxygen growth conditions as a proton pump (77).
Moreover, both genes phnE and ccmB are ABC transporters, functioning as the integral
membrane protein and the membrane protein of type 1 cytochrome c biogenesis system

respectively (77, 94).

“Transcription” is another big category in the treatment of Si02 NPs. The
down-regulated gene ngJ was considered as a positive DNA-binding transcriptional
regulator of transport and utilization of the aromatic B-glucosides arbutin and salicin.
YgeK was repressed as a predicted response regulators of two-component regulatory
systems (62, 103). Gene ascG was also down-regulated, which can repress the expression
of ascF B involving in transport and utilization of the glucosidse suargars arbutin, salicin,

and cellibiose (39).

Exposure to NPs can cause some cellular stress and damage, such as oxidative
stress and genotoxicity by ROS, and they also influence the genes related to stress
condition and DNA repair. For instance, gene yodA, which can bind divalent metal ions
including cadmium, zinc, and nickel and is proposed to be a generalized stress factor, was
repressed (24). Meanwhile, gene yedQ was also found to be down-regulated, which

responds to a number of stress conditions and involve in cellulose biosynthesis (96).

109

Gene xisE was repressed, functioning as DNA recombination, when gene cca was also

down-regulated, involving in RNA repair (56).

There were a few of transcriptional units down-regulated dLuing the exposure to Si02
NPs. The transcriptional unit nhaAR consists of gene nhaA, a sodium ion/proton
antiporter that uses the proton electrochemical gradient to expel sodium ions from the
cytoplasm and functions primarily in the adaptation to high salinity at alkaline pH, and
nhaR, a transcriptional activator. Moreover, nhaA is also believed to be responsible for
adaptation to alkaline pH when sodium is available (49). In the transcriptional unit
hcaEFCBD, hcaE coding a subunit of 3-phenylpropionate dioxygenase, hcaF coding [3
subunit of 3-phenylpropionate dioxygenase, and hcaB coding
3-phenylpropionate-2',3'-dihydrodiol dehydrogenase were repressed, functioning in

utilize aromatic acids as carbon and energy sources (15).

Gene expression profile following Ti02 treatment. Totally 5940 genes ranged in
the expression fold from 0.22 to 7.25. In Figure 5.1, globally 11 down-regulated genes in
the TiOz treatment were grouped into 7 functional categories, including “energy
production and conversion” (27.3%), “coenzyme transport and metabolism” (18.2%),
“general function” (18.2%), “cell motility” (9.1%), “inorganic ion transport and
metabolism” (9.1%), “signal transduction mechanisms” (9.1%) and “not in COGS”
(9.1%). Similarly, twenty-eight up-regulated genes were grouped into 10 functional

categories. Besides “amino acid transport and metabolism” (32.1%) and “not in COGS”

110

(17.9%), most of induced genes belonged to the categories of “carbohydrate transport and
metabolism” (10.7%), “inorganic ion transport and metabolism” (10.7%), “energy

production and conversion” (7.1%), “transcription” (7.1%).

Most of repressed genes in the treatment of TiOz were related to transportation and
cofactor synthesis. Gene yejF was down-regulated, coding an ATP-binding protein of a
ABC transporter that transports peptides through membrane, and its expression was
reported to be regulated by small RNA at the mRNA level (5, 71, 82). The repressed
genes moaA and moaB were in the transcriptional unit moaABCDE, functioning in
Mo-molybdopterin cofactor biosynthetic process (4). The gene deB, coding dimethyl
sulfoxide (DMSO) reductase chain B, was also down-regulated. Moreover, one of DMSO
reductase paralogues was downregulated, including gene ynfE (oxidoreductase subunit
paralog of DmsA), yn/F (oxidoreductase subunit), ynfG (oxidoreductase, Fe-S subunit
paralog of DmsB), and yan (DMSO reductase anchor subunit paralog of DmsC) in the
same operon, which utilize DMSO as a terminal oxidant and function in energy
production and electron transferring (61). Both the DMSO reductase and its paralogue
catalyze in anaerobic electron transport chain, and they contain iron ions and
molybdenum cofactor. Their down-regulation probably relate to the repression of

molybdopterin biosynthesis.

Among up-regulated genes, transportation (32.1%) and amino acid biosynthesis and

catabolism (42.9%) were the biggest categories. For the instance, gene ybaE was

111

up-regulated, functioning as ‘ABC transporter. In the transcriptional unit of
glucitol/sorbitol phosphotransferase system (PTS) permease, genes erA
(glucitol/sorbitol-specific enzyme IIC component of PTS), srlB (glueitol/sorbitol-specific
enzyme IIA component of PTS) and erE (glucitol/sorbitol-Specific enzyme IIB
component of PTS) were up-regulated, which involve in carbohydrate transport via
glycolysis (76). It was reported that GutM and GutR are positive and negative
transcriptional regulators of srl Operon expression respectively, and the operon expression
is also under the control of the cyclic AMP-cyclic AMP receptor protein (CRP) complex
(102). The transcriptional unit of modABC is another induced operon, coding ModA,
ModB and ModC as subunits of molybdate ABC transporter, and their expression was
found to be regulated by a molybdate-dependent repressor protein (36, 37, 100).

For amino acid biosynthesis-and catabolism, the transcriptional unit gltBDF were
induced, coding a glutamate synthase that consists of the large (GltB) and small (GltD)
subunits, and a protein that is implied in the self-operon regulation (GltF). It was reported
that all of these three subunits were induced by exposure to Ti02 NPS, and they involve in
glutamate biosynthesis (18, 19). The activation of this operon could be due to Lrp (global
modulator in response to. leucine levels), IHF (global DNA bending proteins) and GadE
(regulator for the resistance to low pH) (43, 98). The thrLABC operon codes four
enzymes involved in threonine and homoserine biosynthesis, all of which were
up-regulated. Moreover, the tchBCDEFG operon was induced, which codes L-serine

deaminase III (Tch), endoribonuclease (Tch), 2-ketobutyrate formate-lyase/pyruvate

112

formate-lyase (Tch), propionate kinase (Tch), threonine STP transporter (Tch),
catabolic threonine dehydratase (Tch), and Tch transcriptional activator (Tch),
involving amino acid catabolism-transport, and anaerobic respiration (27, 41, 72, 86, 90).
This operon can be induced under anaerobiosis, but it is repressed by glucose (83). The
genes 1'le and nmpC were also up-regulated, relating to amino acid biosynthesis and ion
transporter activity respectively. Gene ach, which functions in two-component signal
transduction system and aerobic respiration, was induced. It involves in amino acid
catabolism, transportation, and anaerobic respiration (27, 41, 90).

Validation of microarray data by quantitative PCR. In Table 5.1, quantitative
PCR for 10 selected genes was used to test the robustness of the microarray hybridization
data, which included two highly repressed genes (yheN and hdeB), two moderately
repressed genes (hyaB and manX), a slightly induced gene (mltC), three slightly induced
genes (atpD, sdhC and mopB), one moderately induced gene (ipr) and another
moderately induced gene with high variations (glpA). Gene ch was used as a reference
gene for comparative CT method; A high correlation (R2=0.95) was obtained between

microarray analysis and quantitative PCR analysis.

Discussion
Aerobic/anaerobic transition in the NP solution. Among the influenced genes,
there were quite a few of them related to aerobic/anaerobic conditions, especially in ZnO

treatment. In ZnO NP treatment, most of repressed genes about aerobic/anaerobic

113

conditions were related to anaerobic respiration, while there were both of genes about
aerobic and anaerobic respiration induced. For example, in the transcriptional unit
appCBA-yccB, genes appC (subunit I of cytochrome bd—II terminal oxidase), appB
(subunit II of cytochrome bd-II terminal oxidase) and appA (acid phosphatase) was
down-regulated, and this operon codes cytochrome bd-II terminal oxidase, which was not
expressed under normal conditions of growth. Meanwhile, gene cyoA, cyoB and cyoD in
transcriptional unit cyoABCDE were induced, which code cytochrome b0 terminal
oxidase used under high oxygen growth conditions. Another example is both aerobic and
anaerobic glycerol-3-phosphate dehydrogenases, functioning in glycerol metabolism and
amino acid transport, as well as providing the proton-motive force for ATP synthesis (55).
The anaerobic glycerol-3-phosphate dehydrogenase consists of GlpA (large subunit),
GlpB (membrane anchor subunit) and GlpC (small subunit), and aerobic one is coded by
gene glpD, both of which was induced by ZnO NPS. Moreover, gene tch coding
catabolic threonine dehydratase was induced by ZnO NPS, and it was reported to be only
expressed during anaerobic growth in the absence of glucose, suggesting the growth
during a transition from aerobic to anaerobic condition. Meanwhile, gene doc was
repressed, coding heme-regulated phosphodiesterase and acting as a direct sensor of
oxygen (25, 66). Gene fucO, coding L-1,2-propanediol oxidoreductase, induced by
growth on L-fucose under both aerobic and anaerobic conditions, but inactivation of the
enzyme under aerobic conditions (16). In TiOz NP suspensions, anaerobic DMSO

reductaSe and its paralogue were down-regulated while the operon tchBCDEF G was

114

induced, which involves in L-serine degradation and anaerobic amino acid metabolism.
The repression of some anaerobic pathways and the induction of some anaerobic/aerobic
pathways suggest that aerobic/anaerobic transition may occur in NP suspension. It was
also reported that fullerene can inhibit E. coli respiration and dioxygen consumption (63).

The function of iron during the exposure to NPS. Iron ions play an important role
for microorganisms. During the exposure to NPS, they can serve as a reducing agent and
repair oxidative damage. For example, gene dps, coding Dps complex that can sequester
iron, was repressed by ZnO NPS, although it can bind to DNA and protect them from
oxidative damage (106). It was reported that protein Dps is very abundant in
stationary-phase and in normal starvation response, which is contrary to our conditions
(45). In the category “transportation”, most of repressed genes related to carbohydrate
and amino acid transport, while 25% of induced transporter genes function in ion
transport. In fact, ROS can attack sulfur-iron cluster , and lead to Fenton chemistry to
generate more ROS (46). Many oxidoreductases, dehydrogenases, hydrogenases,
oxidases, and reductases contain iron-sulfur clusters or bind iron ions, and that is why
their expression was regulated by NPS. In the operon squBCDSE, gene sufB (component
of SufBCD cysteine desulfurase activator complex), squ (component of SufBCD
complex), sujD (component of SufBCD cysteine desulfurase activator complex), sufS
(subunit of selenocysteine lyase), and squ (sulfur acceptor) were repressed. This operon
synthesizes the enzymes in a secondary pathway of iron-sulfur cluster assembly, which is

related to intracellular availability’of iron ions (88). This may be related to oxidation of

115

sulfur atoms by ROS (46).

The bacterial tress-relatedgenes during NP exposures. The stress-related genes
were found in both of down-regulated and up-regulated gene profiles. Some transcription
regulators can sense the stress and influence the expression of other genes. There were
more induced genes about response to stress than repressed ones, such as those coding
starvation proteins, acid resistant proteins and heat shock proteins. Quite a few genes
responding to osmotic stress were up-regulated, maybe due to ROS induction. ROS can
oxidize fatty acids of phospholipids on membrane, and cause lipid peroxidation. This
process can change permeability and fluidity of bacterial membrane, making bacteria

more vulnerable to osmotic stress and interrupt nutrient uptake (17).

Conclusion

The influence on the gene expression of E. coli increased generally, from Si02 to
TiOz to ZnO NPS. The number of induced and repressed genes was the highest in ZnO
NPS, and for all the three treatment the number of repressed genes is higher than the
repressed ones. ROS induction was evidenced by microarray data, such as the
over-expression of osmotic stress genes that may be due to the alteration of permeability
and fluidity caused by lipid peroxidation, and expression change of iron ion transporter
led by attacking iron-sulfur cluster. Both of them result in the damage from ROS, which
also hinder nutrient uptake and induce DNA pair genes. It was also demonstrated that

ZnO NPS influenced the microbial respiration. The genes related to anaerobic and aerobic

116

respiration was induced, while only genes about anaerobic respiration were repressed.
The microarray data demonstrated that NPS regulated the genes in various categories, and

adjusted the pathways and the metabolisms of E. coli.

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CHAPTER 5

COMPARATIVE ANALYSIS OF DIFFERENTIALLY EXPRESSED GENES IN
EXIGUOBA C TERI UM SIBIRIC UM FOLLOWING EXPOSURE TO OXIDE NPS

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Abstract

NPs are being developed for diverse applications, such as water purification, drug
delivery and electronic manufacturing. However, their environmental impact and
toxicological implications are still unknown. Although numerous studies are now
available about their antibacterial activity, information related to their impact at the level
of gene expression are only beginning to emerge. In this study, microarrays were used to
study the influence of Si02, Ti02, and ZnO NP on gene expression of E. sibiricum. The
toxicity of NPS was consistent with their impact of gene expression. Both the number of
repressed and induced genes generally increased in the order: Si02 to TiOz to ZnO. For
all the three treatments, more genes were down-regulated genes compared to the number
of up-regulated genes. The results demonstrated that they influenced expression of genes
in different functional categories, including transportation, carbohydrate metabolism,
lipid metabolism and DNA repair. Microbial respiration was suppressed during exposure
of NPS, especially for aerobic respiration, as well as TCA cycle, oxidation of pyruvate
and oxidative phosphorylation. Up-regulation of genes related to DNA damage repair and
lipid or fatty acid biosynthesis, indicated that ROS induction in E. sibiricum was a

response to NP exposure.

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Introduction

Nanotechnology is defined as “the understanding and control of matter at dimensions
of roughly 1 to 100 nanometers, where unique phenomena enable novel applications” by
National Nanotechnology Initiative. It is an emerging science that is revolutionizing the
advancements taking place in many industries e.g., electronics, food, cosmetiCs, and
medicines (http://www.nano.gov/html/facts/whatlsNano.htm1). It is estimated that by
2015 the market about nanotechnology will be around $1 trillion (44). Due to the
increasing production and widespread application of diverse NMs, the concerns about the
exposure to human and environmental release are also widespread (19, 59). However,
studies related to the impacts of NMs on environment and human health are limited.
Their small size, large surface area, shape, chemical composition, charge, solubility and
aggregation, increase the difficulty to study and characterize their toxicological
properties.

NPS, the building blocks of NMs, have been used in diverse fields, such as
environmental remediation, paint manufacturing and drug delivery. It has been reported
that NPS had adverse impacts on bacteria and animals (16, 21, 31, 32, 58). However,
there is little explanation about the mechanism of NP toxicity. It is hypothesized that the
toxicity of NPS might be due to ROS induction (8, 44, 46, 49).

DNA microarrays consist of short oligonucleotides or PCR products attached to a
solid substrate. Oligonucleotide microchips containing multiple oligonucleotides are

spotted on the chip surface. Probes targeting an entire microbial genome can be easily

135

represented in a Single array, making it feasible to perform genome-wide analysis. Since
its inception more than a decade ago, DNA microchips have been used widely for gene
expression study (6, 11, 51). DNA samples for analysis are labeled with fluorescent dyes
and hybridized with the Oligonucleotide spots on the chip. The fluorescence pattern is
then recorded by a laser scanner, and then Signals are quantified and analyzed using
dedicated software to handle the amount of data. This technique can be extremely helpful
in increasing our understanding about the effects of NP exposure to microorganisms and
aiding in explaining the possible mechanisms of NP toxicity.

Exiguobact‘erium sibiricum Strain 255-15, one member of the Bacillaceae, is a low
G+C, Gram-positive, rod-shaped and non-spore-forrning psychrotrophic bacterium (53,
i 62). It is wide-spread capable of living over a broad temperature range (-5 °C-40 °C) (54).
The goal of this study was to explore the microbial response to different oxide NPS and
possible mechanisms for their toxicity. More specifically, we wanted to evaluate the gene
expression profiles of E. sibiricum when exposed to Si02, Ti02 and ZnO NPS. Moreover,
we wanted to evaluate the influences of NPS on selected pathways focusing on the ROS

damage response mechanisms in E. sibiricum.

Materials and Methods
Reagents and preparation of NP suspensions. Random hexamer primers, 100 mM
nucleotide set, RNaseOUT, salmon sperm DNA, Superscriptase II RT, formamide, 10%

sodium dodecyl sulfate, 20X Saline-Sodium Citrate (SSC), and 0.5 M

136

ethylenediaminetetraacetic acid (EDTA) were obtained from Invitrogen Co. (Carlsbad,
CA). Bovine serum albumin (BSA), dimethyl sulfoxide, sodium hydroxide, 1 M
hydrochloric acid and isopropanol were purchased from Sigma-Aldrich Chemical Co. (St.
Louis, MO). RNAlater, SlideHyb buffer and aminoallyl-modified dUTP were obtained
from Ambion Inc. (Austin, TX). Lysozyme, DNase I, Tris-Cl EDTA (TE) buffer, RNeasy
mini kit, Quiaquick PCR purification kit and RNA protect bacteria reagents were
obtained from Qiagen Co (Valencia, CA). Si02 (10 nm), Ti02 (5 nm) and ZnO (60 nm)
were purchased from Sigma-Aldrich (St. Louis, MO). The 100 g/L NPs stock solutions
were prepared in deionized and distilled water. Phosphate wash buffer (100 ml) for
cDNA purification was made of 0.5 ml KzHPO4 (1 M, pH 8.5), 84.25 ml ethanol and
15.25 ml H20 and phosphate elution buffer (100 ml) contains 0.4 ml KZHPO4 (l M, pH
8.5) and 99.6 ml H20. All the solutions were autoclaved and stored at room temperature
Cell growth, harvest and RNA extraction. From a freezing stock of E. sibiricum,
streak plates were prepared with TSA medium and grown at 30 °C overnight. One loop
of the plate was transferred to the tubes with 5 ml TSB. The NP stock solution was added
to each tube with 100 ml TSB to result in final NP concentrations of 5000 ppm for Si02
NPs, 1000 ppm for Ti02 NPs and 500 ppm for ZnO NPS. The samples were inoculated
and incubated until mid-log growth phase of E. sibicirum (0.1<OD600<0.4). According to
the protocol of RNAlater, 1 ml of RNAlater was added to 100 ml culture for RNA
extraction, and incubated for 5 min at 30 °C. The cells were collected by centrifuging for

20 min at 5000>< g at 4 °C, and the supernatant was decanted. The cells were resuspended

137

in 1 ml RNAlater, transferred to a microcentrifuge tube and pelleted at SOOOX g at 4 °C
for 10 min. The 100 pl lysozyme in TE buffer (3 mg/ml) was added to the cells, mixed by
vortexing for 10 s, and incubated at room temperature for 30 min on a shaker. Buffer RLT
(350 pl) in RNeasy mini kit was applied and vortexed vigorously for 30 5. After 2 min
centrifiagation at maximum speed and removal of the sediment of NPS, the supernatant
was mixed with 250 p1 ethanol by shaking vigorously (but not centrifuging after shaking).
As described in RNeasy mini kit, the lysate was transferred to RNeasy Mini Spin column,
and centrifuged for 14 s at >8000x g. DNase digestion was applied to the column by
buffer RWl washing, DNase I solution washing (10 pl) incubating, and then buffer RWl
re-washing. The RNA was purified as the protocol of RNeasy mini kit and the final
concentration of RNA was measured by Nanodrop from Thermo Scientific Co.
(Wilmington, DE). RNA from control samples was processed in a Similar way as NP
samples. All experimental and control samples were prepared in triplicate.

cDNA synthesis and dye-labeling. For each sample, 5 pg of total RNA was used to
synthesize cDNA and two RNA populations of each sample were prepared for
dye-swapping. RNA mixture contained 5 pg RNA, 5 pl random hexamer primers (3 mM),
Rnase-free water in the final volume of 16.8 pl. The RNA mixture was incubated at 70
°C for 10 min and then cooled on ice for another 10 min. The RT—PCR reaction had a 30
pl volume and contained 6 pl 5X first strand synthesis buffer, 3 pl DTT (0.1 M), 1 pl
RNaseOUT, 1.2 pl 25x dNTPs (7.5 mM aa-dUTP, 5 mM dTTP, 12.5 mM dCTP, 12.5 mM

dGTP and 12.5 mM dATP), 2 pl Superscript 11 RT and 16.8 pl RNA mixture. The

138

RT-PCR reaction was incubated at 42 °C overnight, and then 10 pl NaOH (1 M) and 10
pl EDTA (0.5 M) were added to the reaction. After incubating at 65 °C for 15 min, 10 pl
HCl was added to neutralize the pH. The cDNA was purified according to the protocol of
Qiagen purification kit with some modification. The cDNA mixture was mixed with 300
pl PB buffer, and transferred to a Quiaquick column. After 1 min centrifuging at 17,900X
g, the collection tube was emptied and the column was washed by 750 pl phosphate wash
buffer twice and centrifuged for 1 min as above. After 1 min additional centrifuging, the
column was transferred to a new tube, and then eluted twice by incubating the column
with 30 pl phosphate elution buffer for 1 min and centrifuging for 1 min at maximum
Speed. The cDNA sample Was dried using Speedvac from Global Medical
Instrumentation Inc. (Ramsey, MN). The aa-dUTP cDNAwaS resuspended in 4.5 pl
sodium carbonate (0.1 M, pH 9.0) and mixed with 4.5 pl Cy5 or Cy3. After incubating 2
h at room temperature in the dark, thirty-five pl NaOAc (100 mM, pH 5.2) was added and
the dye-coupled cDNA was purified as described in the protocol of Qiaquick purification
kit. The concentrations of cDNA and dye were measured by Nanodrop from Thermo
Scientific Co. (Wilmington, DE).

Hybridization. The glass-slide arrays were cross-linked at 6000 p] UV and
waterbath was prewarmed to 46 °C. The slides were placed in prehybridization buffer
containing 50% formamide, 5>< SSC, 0.1% SDS and 0.1 mg/ml BSA at 46 °C for 60 min.
After washing three times with deionized and distilled water and once with isopropanol,

the slides were dried by centrifuging at 1600 rpm for 3 min and transferred to dry and

139

clean tubes covered with foil. The coverslips were washed with RNase-free water and
ethanol, dried by centrifuging 600 rpm and then placed in clean tubes covered with foil.
The heat block was preheated to 95 °C and the hybridization oven to 60 °C. The labeled
DNA of the control sample was resuspended in 35 pl hybridization buffer containing 50%
formamide, 5>< SSC, 0.1% SDS, 0.1 pg/pl salmon sperm DNA. The other differently
labeled DNA from NP-treated sample was suspended in the same hybridization solution,
which was heated at 95 °C for 5 min. The dry slide-array was placed in a clean and dry
hybridization chamber and coverSlip was placed on the slide. The hybridization solution
was loaded on the slide, and 20 pl 5>< SSC was added in each well in the hybridization
chamber. The chamber was placed in waterbath at 46 °C for 16 h. For each NP treatment,
Six hybridizations from three biological replicates and two technical replicates
(dye-swapping) were performed.

Washing, scanning and data analysis. After 16 h, the chamber was removed from
waterbath and dried with paper towel before opening. Wash Buffer l and slide staining
jars were prewarmed at 46 °C. The Slide was taken out with forceps, and the coverslip
was removed by immersing the array in a tube containing Wash Buffer 1 (1>< SSC and
0.1% SDS). The Slide was washed twice with Wash Buffer 1 (l X SSC and 0.1% SDS) for
5 min at 50 °C, then twice with Wash Buffer 2 (O.1>< SSC and 0.1% SDS) for 10 min at
room temperature, and four times with Wash Buffer 3 (0.1>< SSC) for 1 min. The slide
was dried by centrifuging at 1600 rpm for 3 min and transferred to dry tubes.

Axon Genepix 4000B laser scanner was used for Slide scanning and Genepix 3.0

140

software was used to produce a raw data set. The scanner was turned on and warmed up
for 15 min while the computer was off. The slide was placed face-down, and PMT and
power was adjusted to yield optimal signals (as verified by histogram). A 5 pm pixels
resolution setting was chosen for scanning. The area of interest was scanned and the
image was saved. GENESPRING 6.0 software (Silicon Genetics, Redwood City, CA)
was used for data analysis. The data was normalized both per chip and per gene (Lowess
method), and dye-swapping. Only the spots with more than 55% of pixel greater than
background plus 2X standard deviation in either Cy5 or Cy3 channel were used for
analysis. Genes that showed a statistically significant change in gene expression (p>0.05)

and a greater than or equal to 2-fold change in magnitude were regarded as significant.

Results

Global gene expression profiles under NP exposures. For all three NP exposures,
the number of expressed genes exceeded the number of induced genes. The expression
response to 500 ppm ZnO NPS was broader with more than 27% of the genome
expressing differentially. For 1000 ppm Ti02 NPS, 18% of genome showed differential
expression response while for 5000 ppm Si02 NPs, it was 14%. According to our
previous viability data, it could be concluded that the exposure to more toxic NPS caused
more differential expression in E. sibiricum. Among the up-regulated genes, almost 40%
of Ti02-induced genes were also up-regulated in ZnO treatment, while only 15% of

Ti02-induced genes were up-regulated in Si02 treatment (Figure 5.1(a)). Meanwhile,

141

48% of Ti02-repressed genes were down-regulated in ZnO treatment, whereas 27% of
Ti02-repressed genes were down-regulated in Si02 treatment (Figure 5.1(b)). Thus the
response of E. sibiricum to ZnO NP and Ti02 NP exposures was closer, compared to SiOz
NP exposure. A total of 13 genes were induced in all the three treatment, and 38 genes

were repressed by all three different NP exposures in Figure 5.1.

Gene expression profiles following Si02 treatment. For Si02 treatment,
fold-expression for 2539 genes ranged between 0.01 and 11.1. Based on the available
information in the databases of Kyoto Encyclopedia of Genes and Genomes, UniProt and
InterPro, the set of 234 down-regulated genes in the response to Si02 NPS were grouped
into 17 functional categories. Categories “transportation” (24.8%) and “hypothetical”
(20.9%) were the two dominant categories. Others included “biosynthesis” (8.5%),
“carbohydrate metabolism” (7.3%), “amino acid metabolism” (6.4%), “transcription”
(5.6%), “nucleotide metabolism” (3.8%), and “energy metabolism” (3.8%). Similarly,
158 up-regulated genes were grouped into the same 17 functional categories. Besides
“hypothetical” (20.9%) and “transportation” (12.7%), most of induced genes belonged to
the categories of “unknown” (9.5%), “carbohydrate metabolism” (9.5%), “transcription”
(7.0%), “replication and repair” (7.0%), “amino acid metabolism” (6.3%), “translation”
(5.1%), “lipid metabolism” (4.4%), “biosynthesis” (4.4%) and “cellular process” (3.8%).
There were more down-regulated genes than up-regulated ones in categories of

“biosynthesis” and “xenobiotics degradation and metabolism”, which might mean that

142

the bacteria closed some unnecessary pathways to save energy for survival. Meanwhile,
more genes were induced compared to repressed ones in “lipid metabolism”, “replication

and repair” and “signal transduction”, which could be related to the damage to membrane

and DNA by ROS induction.

Response of “transportation” related to genes in Si02 NP exposure.
Transportation and internalization of NPs is an important factor influencing the fate of
NPS. Because NPs are generally much larger than all the transportation channels, the
mechanisms about how they enter the bacterial cells require additional exploration. In all
the expression profiles, “transportation” was always one of the largest groups for both of
induced and repressed genes. During the exposure to SiOz NPS, there were 58
down-regulated genes and 20 up-regulated genes in the “tranSportation” category. Among
the repressed genes, 41.4% belonged to ABC transporters, 6.9% related to Na+
symporters, 5.2% functioned as PTS components, and 13.8% involved in sugar or amino
acid transportation. For the induced genes in “transportation”, 50.0% were

ABC-transporters, 10.0% K+ transporters and 5.0% were involved in PTS system.

ABC transporters are a family of membrane proteins that function in the
ATP-dependent transportation of many compounds across cellular membranes (1, 23).
ABC transporters were found in both induced and repressed genes. The down-regulated
genes mainly involve in transporting sugar, amino acid and peptides, such as ABC-type

sugar transport system permease components and olio-peptide ABC transporter permease.

143

The up-regulated ones are related to ferric and other metal ions, such as ABC-type
Fe3+-hydroxamate transport system components and ferric anguibactin transport system
permease. Zhang et al. also reported over-expression of ABC transporters in mouse cells
by poly(ethylene glycol)-block-polylactide NPs (66). ROS is known to oxidize fatty acids
of phospholipids on membrane and cause lipid peroxidation (2). This process can change
the membrane permeability and fluidity, and interrupt nutrient uptake, which may explain
the response of amino acid and carbohydrate transport genes. Iron ion is another damage

target for ROS, which may change the expression of metal ion ABC-transporter.

Sodium/solute symporters and Potassium (K+) transporters were the other two groups
of transporters influenced by Si02 NPS. Sodium/solute symporters catalyze the uptake
various solutes into the microbial cells (50). Under extreme conditions, bacterial cells can
use Na+ as coupling ions to generate Na+-electrochemical gradients by Na+ pumps and
NaIL/HJr antiporters, and this is important for accumulation of nutrients (25, 26). K+ ion, as
a major intracellular cation, plays a fundamental role for many physiological activities,
such as protein synthesis and enzyme activation (14, 29). Under some conditions, Na+
substitutes functionally for K+ (55). In the Si02 treatment, Na+ symporters were repressed
while K+ transporters were induced. The rapid leakage of K ions in bacterial cells during
NP exposure was reported by Saito et al., and this may be related to the up-regulation of

these K+ transporters influenced by Si02 NPS (56).

144

Category of “carbohydrate metabolism” in Si02 treatment. “Carbohydrate
metabolism” is another important category in the gene expression profile. It involves
various biochemical processes and is helpful to understand how bacteria survive through
the exposure to NPS. There were 17 repressed genes and 14 induced genes in this
category. Among the down-regulated genes, 23.5% of the repressed genes were related to
CoA, 23.5% to pyruvate, and 23.5% to glycosidases or glucosidases. CoA functions as an
acyl group carrier and plays a fundamental role in TCA cycle as well as synthesis and
oxidation of fatty acids. It was reported that NPS could cause oxidation of CoA, and
decrease of CoA may influence the activity of COA-related enzymes (37, 43). CoA is also
involved in the oxidation of pyruvate, which may explain why the genes for pyruvate
dehydrogenases were also down-regulated. The pyruvate dehydrogenase complex
transforms pyruvate into acetyl-CoA, controls pyruvate entry into TCA cycle, and also
participates in glycolysis and glucose oxidation (38). It may be concluded that Si02 NPS
can repress the TCA cycle by oxidizing CoA and down-regulating the pyruvate
dehydrogenases. The Si02 NPs also down-regulated the genes encoding glucosidases,
glucosidase-related glycosidases and l-phosphofructoknase. These enzymes are involved

in glycolysis, generating ATP and producing pyruvate as a part of aerobic respiration.

For up-regulated genes in “carbohydrate metabolism”, there was no clear pattern
among them. However, more than half of them were dehydrogenases, including alcohol

dehydrogenases and aldehyde dehydrogenases, which transfer protons or electrons to

145

acceptors in electron transport chains, usually some coenzymes such as nicotinamide
adenine dinucleotide/ nicotinamide adenine dinucleotide phosphate (NAD/NADP) (60).
It is well known that NAD/NADP participates in generation of ATP, which might explain
how E. sibiricum survives the exposure to Si02 NPs. Most of dehydrogenases contain
sulfur-iron cluster, which is another attacking target for ROS. There were some
coenzyme-dependent enzymes that were up-regulated in this category, such as
flavin-dependent oxidoreductases, which may also adjust the cellular redox conditions

since NPS can induce intracellular ROS.

Category of “energy metabolism” in Si02 treatment. “Energy metabolism” is
important for understanding how E. sibiricum produces energy and survives in the
exposure to Si02 NPs. Almost 80% of repressed genes in this category were the subunits
of cytochrome c oxidase or cytochrome caa3 oxidase. They function in one of electron
transport chains in oxidative phosphorylation, and this process is important for cellular
metabolism and energy synthesis as a part of aerobic respiration. Many enzymes involved
in oxidative phosphorylation are located on membrane, which is one of the main
attacking targets for ROS. ROS can also be produced during oxidative phosphorylation,

such as free radicals and hydrogen peroxide, which cause some cellular damage.

Among the induced genes in this category, it seems most could serve as antioxidant
preventing the damages from ROS. For example, one gene encoding a catalase which can

catalyze and converse hydrogen peroxide into water and oxygen, was up-regulated (24).

146

Two genes related to cysteine synthesis, which is beneficial for glutathione system in
detoxification metabolism, were also up-regulated (40, 41). This could be another
evidence for ROS damage, since cysteinyl residues in proteins are oxidized at the

presence of ROS (22).

Category of “metabolism of cofactors and vitamins” in $102 treatment. It is well
known that cofactors and vitamins involve many biological processes and own diverse
biochemical functions, such as activation of apoenzymes and regulation of microbial
growth. Some of them function as antioxidant preventing microbes from ROS damages.
Factors related to ROS damage are important in determining the response to NPS. For
SiOz NP treatment, there were 6 repressed genes and 2 induced genes. Among the
down-regulated genes, half were coenzyme F390 synthetafses. Coenzyme F390 system
was first found in Methanobacterium spp., and this system can sense the intracellular
reduction and oxidation potential (61). Coenzyme F390 usually is present when the
cellular redox potential increases. 80 it may function as a signal compound, transferring
information about intracellular redox potential. Two genes encoding ketopantoate
reductase and thiamine monophosphate synthase respectively were repressed. The
ketopantoate reductase is involved in biosynthesis of pantothenate, which is needed to
form CoA and important for carbohydrate metabolism. The thiamine monophosphate

synthase participates in thiamine metabolism, which relates to modification of CoA. It

147

seems that oxidation of CoA by NPs influences the relative pathways to other cofactors

and vitamins.

In the Si02 treatment, a gene encoding isochorismate synthase was up-regulated.
Isochorismate synthase participates in ubiquinone biosynthesis and ubiquinone, also
known as coenzyme Q (CoQ), usually functions as an antioxidant. So the induction of
isochorismate synthase probably benefits the detoxification and protection from oxidative
damages. Another induced gene was related to nitroreductase, which is involved in the

reduction of nitrogen-containing compounds, such as NADH or FMN.

Category of “replication and repair” in Si02 treatment. Since the induction of
ROS by NPs and their genotoxicity have been reported before, “replication and repair”
category may be important in understanding how E. sibiricum protects itself from DNA
damage by NPS. For Si02 NP treatment, there were 5 repressed genes and 11 induced
genes in this category. Thus more DNA repair systems were activated to alleviate DNA
damages. Two bacteriophage integrases and one UV-damage repair protein were
down-regulated by Si02 NPS. It is well known that Short-length UV can induce
prophage-related genes. Therefore it appears that UV-related DNA repair system was
repressed and not applicable for the DNA damage by ROS and NPs. The UV-damage
repair protein was repressed in all the three treatments. It was reported that UV-irradiated
TiOz had been applied as a photocatalyst for bacterial killing, and the suppression of

UV-damage repair protein by NPS could worsen the bacterial survival during the

148

photo-killing (34, 52, 64). The induced genes included transposases, recombinases,
ribonucleases, excisionases and oxidative damage protectants. Almost half of these genes

were related to recombination, which may be important for DNA repair process.

Gene expression profiles following Ti02 treatment. A total of 2681 genes showed
a fold-change in the expression ranging from 0.01 to 17.31. As illustrated in Figure 5.2,
the 289 down-regulated genes in the Ti02 treatment were grouped into 17 functional
categories, including “hypothetical” (25.3%), “transportation” (15.2%), “carbohydrate
metabolism” (8.7%), “amino acid metabolism” (8.7%), “transcription” (7.3%),
“unknown” (5.2%) and “repair and replication” (4.2%). Similarly, 197 up-regulated
genes were grouped into the same 17 functional categories. Besides “transportation”
(26.4%) and “hypothetical” (23.4%), most of induced genes belonged to the categories of
“unknown” (5.6%), “replication and repair” (5.6%), “transcription” (5.1%), “lipid
metabolism” (4.6%), “carbohydrate metabolism” (4.6%), and “nucleotide metabolism”
(4.1%). Usually there were both induced and repressed genes in the same functional
categories, whereas more genes were repressed in the categories of “amino acid

metabolism , carbohydrate metabolism , transcription” and “translation”, which may

mean that E. sibiricum survived exposure to Ti02 NPS by reducing cellular activity.

In “carbohydrate metabolism”, there were 25 down-regulated genes and 9
up-regulated genes. Among the repressed genes, 5 genes were related to CoA and another

5 genes were alcohol or pyruvate dehydrogenases. In the category of “transportation”, a

149

total of 44 genes were down-regulated and 52 genes were up-regulated. The repressed
genes included ABC-transporters (43.2%), PTS components (9.1%), and Na+ or H+
symporters (4.5%), while the up-regulated genes covered ABC-transporters (42.3%),
ferrous related transporters (11.5%), PST components (5.8%), antiporters or sympoters
(5.8%), and K+ or Na+ transporters (3.8%). Similarly to Si02 treatment, Ti02 NPs
repressed a few cytochrome c oxidases and induced some genes encoding
thioredoxin-like proteins or NADPquuinone reductases in the “energy metabolism”
category. It is well known that thioredoxin is another enzyme system functioning as an
antioxidant and it can prevent E. sibiricum from oxidative damage by NP exposure. The
NADPquuinone reductases participates in oxidative stress resistance by acting NADPH
with a quinine as an acceptor (63). In the “replication and repair” category of Ti02
treatment, some UV-related repair genes were down-regulated, such as UV-damage
repair protein and DNA photolyase. Recombinases and recombinational DNA repair
proteins (23.1%) were also repressed by Ti02 NPs. Helicases (18.2%), transposases
(18.2%) and DNA-directed RNA polymerases (18.2%) was among the induced genes by

Ti02 NPS.

Gene expression profiles following ZnO treatment. In the ZnO treatment, globally
the fold-change in expression of 2616 genes ranged from 0.01 to 19.9. There were 402
down-regulated genes and 365 up-regulate genes. ZnO NPs had the largest number of up-

or down-regulated genes (compared to in Si02 and Ti02 treatments). The down-regulated

150

genes can be grouped into 16 functional categories, including “hypothetical” (22.4%),
“transportation” (13.4%), “carbohydrate metabolism” (12.2%), “amino acid metabolism”
(9.5%), “transcription” (8.5%), “unknown” (6.7%) and “repair and replication” (4.0%)
(Figure 6.2). Similarly, the up-regulated genes were grouped into the 17 functional
categories, including “hypothetical” (18.4%), “transportation” (14.8%), “translation”
(11.0%), “replication and repair” (9.3%), “unknown” (5.8%), “nucleotide metabolism”
(5.8%), “amino acid metabolism” (5.8%), “carbohydrate metabolism” (5.2%) and “lipid
metabolism” (4.4%). In ZnO treatment, more genes were repressed in the categories of
“amino acid metabolism”, “carbohydrate metabolism”, and “transcription”, while more
genes were induced in the categories of “replication and repair”, “lipid metabolism” and
“nucleotide metabolism”. It can be concluded that E. sibiricum reduced cellular activity

to save energy and survive the exposure to ZnO NPs, and activated relative pathways to

repair DNA damages by ROS induction.

There were 49 repressed genes and 19 induced genes in “carbohydrate metabolism”.
Similar to the treatment of Si02 and Ti02 NPS, it was found that 5 repressed genes were
related to CoA and another 10 repressed genes were alcohol or pyruvate dehydrogenases.
In the category of “transportation”, there were 54 repressed genes and 54 induced genes.
ABC-transporters (38.9%), PTS components (9.3%), Na+ or K+ transporters (9.3%), and
symporters (5.6%) were found in the repressed transportation genes while there were

ABC-transporters (38.9%), ferrous and ferric transporters (9.3%), antiporters (5 .6%), K+

151

or NaJr transporters (5.6%), and PST components (1.9%) among the induced genes. ZnO
NPS repressed a few cytochrome c oxidases, while they also induced some coenzyme
related oxidoreductases. In ZnO treatment, there were 16 repressed genes and 34 induced
genes in “replication and repair” category. Thus more DNA repair mechanisms were
activated to alleviate the DNA damages from ZnO NPS. Among the down-regulated
genes, helicases (12.5%), DNA-directed RNA polymerases (12.5%), transposases (25%)
and recombinases or recombination proteins (12.5%) contributed the majority. The
up-regulated genes included helicases (8.8%), DNA-directed RNA polymerases (11.8%),
transposases (5.9%) and recombinases or recombination proteins (8.8%), DNA
polymerases (8.8%), nucleases (11.8%), DNA repair proteins (8.8%) and topoisomerases

(5.9%).

Category of “metabolism of cofactors and vitamins” in Ti02 and ZnO
treatments. There were 9 repressed genes and 6 induced genes by Ti02 NPs, while there
were 12 repressed genes and 11 induced genes in the ZnO treatment. Similar to Si02
treatment, coenzyme F390 synthetase was down-regulated, which demonstrated the
changes of intracellular redox potential. Dihydroneopterin aldolase, which participates in
folate biosynthesis, was down-regulated in both Ti02 and ZnO NP treatments. It is well
known that folate is important and necessary for DNA replication and cell division (27).
Glutamyl-tRNA reductase was another repressed gene in both of treatments, which

participates in porphyrin metabolism. Porphyrin is synthesized by reaction of glycine and

152

succinyl-CoA from TCA cycle. It is likely that oxidation of CoA reduced the synthesis of
porphyrin and repressed the activity of glutamyl-tRNA reductase. Porphyrin is contained
in heme, which is one subunit of cytochrome c oxides. In Ti02 treatment,
uroporphyrinogen decarboxylase, also related to heme biosynthesis, was down-regulated
(57). In ZnO treatment, the genes related to metabolism of CoQ and vitamin B6 were
repressed. Vitamin B6 assists in the balancing between Na+ and K+. The repression of

vitamin B6 biosynthesis protein may be related to the rapid leakage of KL

In both TiOz and ZnO NP treatments, thiamine pyrophosphokinase, which catalyzes
the reaction to produce thiamin pyrophosphate, was induced. Glycine oxidase, which
participates in thiamine metabolism, was induced too. Meanwhile, two genes related to
molybdopterin biosynthesis were up-regulated. Molybdopterin usually functions as a
cofactor necessary for sulfite oxidases and nitrate reductases, which are very important in
bacterial electron transfer chains and supply electron acceptors for anaerobic respiration

(28).

Category of “xenobiotics degradation and metabolism” in Si02, Ti02 and ZnO
treatment. Although it is not a large category, it contained some interesting information
about NP exposure, especially among the down-regulated genes. In SiOz treatment, one
gene involved with ethyl tert-butyl ether (ETBE) degradation was down-regulated, which
also happened in the treatments of Ti02 and ZnO NPS. ETBE has been used as a fuel

oxygenate in gasoline and it is toxic and relatively non-degradable (39). Oxidation of

153

ETBE involves electron chain transfer by a cytochrome P450 monooxygenase (3, 13).
The genes related to degradation of phenylacetate or phenylacetic acid were
down-regulated, including some phenylacetate-COA oxygenase subunits that may
produce some TCA cycle intermediates, and phenylacetyl-CoA ligase that activates
phenylacetic acid to phenylacetyl-CoA (12, 45). In SiOz treatment 83.3% repressed genes
were related to phenylacetate or phenylacetic acid, 50.0% in Ti02 treatment and 66.7% in
ZnO treatment. Type I pullulanase and NADH-dependent nitro/flavin oxidoreductase
were induced in both Ti02 and ZnO treatment. Type I pullulanase can specifically cleave
the or-l, 6-glycosidic linkages in pullulan and branched or linear oligosaccharides (33).
NADH-dependent nitro/flavin oxidoreductase is one type of nitroreductases, participating

in reduction of nitrogen containing compounds (9).

Shared down-regulated and up-regulated genes in Si02, Ti02 and ZnO
treatments. As shown in Figure 5.3, there were 13 repressed genes and 38 induced genes
that were common to all the three treatments. The categories “hypothetical” and
“transportation” contained the largest number of common genes for both down-regulated
and up-regulated genes. Among the down-regulated genes, the category “biosynthesis”,
“carbohydrate metabolism” and “xenobiotics degradation and metabolism” were
important. The induced genes in the “transportation” category were associated with ABC
transporters for metal ions, such as ferric or zinc, while the most of repressed genes in the

same category were sugar transporters, including ABC sugar transporters and PTS

154

 

 

components. In “biosynthesis” category, all the repressed genes were related to
polysaccharide synthesis. Among the repressed genes of “carbohydrate metabolism”,
nearly half were COA-related when the other half were pyruvate-related. In the “energy
metabolism” category, the repressed genes mainly function in bacterial electron transfer
chain. All the down-regulated genes in the “metabolism of cofactors and vitamins” were
related to F390 coenzyme, which may be influenced by the changes of intracellular redox
potential, and all the repressed genes in the “xenobiotics degradation and metabolism”
involve in degradation of phenylacetic acid or ETBE. The patterns in these categories

were useful for choosing molecular target candidates of NP toxicity.

Discussion

The microarray data was quite consistent with the studies reported in this dissertation
focusing on viability and ROS. The order for the toxicity of NPs to E. sibiricum was ZnO
> TiOz> Si02, same as ROS induction. The microarray results displayed that the E.
sibiricum exposed to ZnO NPS possessed the most induced and repressed genes and the
expression fold was the widest ranging from 0.01 to 19.9. Ti02 NPs possessed more
influence on gene expression than Si02 NPs. According to 1-way ANOVA analysis, there

were 1,677 genes expressed differently among these three treatments.

The microarray data also indicated that NPS might suppress microbial respiration. A

few genes coding for pyruvate dehydrogenase subunits were repressed by NPs. These

155

 

genes participate in oxidative decarboxylation of pyruvate, which links glycolysis and
TCA cycle. The down-regulation of these genes probably reduced the synthesis of
acetyl-CoA, which is very important for TCA cycle. Meanwhile, some genes encoding
COA-related enzymes were down-regulated during the exposure to NPS. It has been
reported that NPs could oxidize CoA (43). In this study, several cytochrome c oxidases
were repressed by NPs. Cytochrome c oxidases are involved in oxidative phosphorylation,
one of the key pathways for aerobic respiration. The essential enzymes in glycolysis were
not influenced, although the exposure to NPs down-regulated a couple of genes in
glycolysis, not requiring oxygen. It appeared that NPs influenced the aerobic respiration
more than the anaerobic respiration. Similarly, fullerenes or its derivatives have also been
found to inhibit E. coli respiration of glucose, and also hinder dioxygen consumption and
respiratory chain (35-37). During the SiOz treatment, there were a few alcohol and
aldehyde dehydrogenase genes that were up-regulated. These genes facilitate the
conversion between alcohols and aldehydes and play an important role in fermentation.
Quite a few dehydrogenases and Oxidoreductases were also induced by Ti02 NPS. These
included NADPH: quinone reductase and ferredoxin oxidoreductase. For the ZnO NP
exposure, however, some genes encoding important enzymes in the TCA cycle, including
malate dehydrogenase, Succinyl-CoA synthetase beta subunit, isocitrate dehydrogenase
and pyruvate dehydrogenase E2, were induced. Some alcohol and aldehyde

dehydrogenases were repressed in ZnO NP treatment.

156

 

Previously we have demonstrated that ROS was induced in E. sibiricum by these
NPS, and gene expression supported this observation. After exposure to NPS, most of the
repressed ABC transporters were related to sugars, while induced ABC transporters were
mostly related to metal ions, especially for iron ions. Iron is essential for most living
bacteria functioning as a component for some important molecules, such as
ribonucleotide reductase and cytochromes (30, 65). One possible explanation for the
up-regulation of iron transporters is that ROS, induced by NPS, attacks sulfur-iron
clusters and causes release of iron ions. It may also be due to the participation of iron in
some electron transfer reactions since other cellular respiration pathways were suppressed.
Additional evidence is the induction of enzyme in the categories of “lipid metabolism”
and “replication and repair”, Since ROS can cause damage to membrane and DNA.
Although a few enzymes in these two categories were down-regulated, the reason may be
that they participated in the inactive pathway, and furthermore the number of induced
genes was larger than that of the repressed, especially in the ZnO treatment. It was also
observed that the expression of many oxidoreductases was influenced by the NPS, which
might be related to the intracellular changes of redox potential caused by ROS. No

induction of superoxide dismutases was found in E. sibiricum.

The category “transportation” was always the most influenced category in all NP
treatments. ABC transporters were the most influenced class of membrane transport

proteins for various substances by using energy from ATP-binding and hydrolysis (4, 5,

157

 

18, 47). Some Kir transporters were up-regulated. Leakage of K+ occurs in bacteria in
response to exposure to Ti02 NPS, which mayb be the reason for the induction of K+
transporters (56). K+ ion plays an important role in maintaining cellular viability, and the
accumulation of K+ ions influences the rates of cellular growth, respiration and protein
synthesis (10, 17, 20, 42). A few Na+/Solute symporters were found down-regulated while
some Na+/H+ antiporters were up-regulated. Both Na+ ion and H+ ion are critical for cell
bioenergetics and physiology (15, 48). The down-regulation of Na+/solute symporters and
up-regulation of Na‘L/H+ antiporters should be related to H+ or Na+ electrochemical
gradient, which is essential for solute uptake and energy storage. The proton gradient is
important during NP exposure, perhaps due to the suppression of respiration by NPS,

although Na+ ion is particularly used as coupling ions in extreme environments (7, 26).

The categories “translation” and “signal transduction” were also important in all the
three treatment. Most of the influenced genes in the category “translation” coded
ribosomal proteins or enzymes related to tRNA. In the “signal transduction” category, the
influenced genes coded histidine kinases. Not many genes were influenced in the
category “biosynthesis”, and most repressed genes were related to polysaccharide
biosynthesis. In other categories, such as “amino acid metabolism” and “transcription”,

no specific pattern was observed.

In conclusion, the microarray data demonstrated that the three types of NPS tested in

this study influenced the gene expression of E. sibiricum. ZnO NPS were the most toxic

158

 

one. Exposure to ZnO NPS resulted in more up- and down-regulated genes than either
Si02 or TiOz NPS. For all three treatments, the number of down-regulated genes was
higher than the number of up-regulated genes. ROS induced by NPs caused damage to
membrane, protein and DNA. The functional category “transportation” was the largest
for both the induced and repressed genes in these three treatments. These included ABC
transporters, PTS, exporters, and antiporters/symporters. More transporters for
carbohydrate, amino acid and drugs were found in the down-regulated group than in the
up-regulated group, while there were more metal ion transporters induced. For
down-regulated genes, the categories “transcription”, “amino acid metabolism” and
“carbohydrate metabolism” were other important ones, maybe due to the slowdown
activity and growth caused by NP exposure. A number of COA-related genes, which
function in TCA cycle, glycolysis, biosynthesis of fatty acids and amino acid metabolism,
were also repressed. Genes related to DNA repair were induced, maybe due to

genotoxicity caused by NPs. The microarray data demonstrated that NPs influenced the

genes of E. sibiricum in a variety of functional categories.

159

 

 

Upregulated genes Ti02

8102 l 2110

Downregulated genes Ti 02 , b

SiOz
ZnO

Figure 5.1 (a) The up-regulated gene profile in three different treatments. (b) The down-regulated gene
profile in the three treatments.

160

 

400 -

300 1

200 -

Gene numbers

100 -

 

- Amino acid metabolism
- Blosythesis
Carbohydrate metabolism
[:1 Cellular process

- Energy metbolism

- Hypothetical

m Lipid metabolism
Metabolism of cofactors and vitamins
- Nucleotide metabolism
- Protein fate

- Replication and repair
- Signal transduction

- Transcription

- Translation

 

{.259
. '3‘“:
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Treatment

Figure 5.2 The functional categories of the gene profiles in Si02 NP, Ti02 NP and ZnO NP exposures.

161

Shared genes among all the three treatments of Si02, Ti02 and ZnO NPs

- shared downregulated genes
8 - l:l shared upregulated genes

Gene number

 

 

Functional categories

Figure 5.3 The functional categories of the shared genes among Si02 NP, Ti02 NP and ZnO NP
exposures.

162

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