Bactericidal Antibiotics Induce Mitochondrial Dysfunction and Oxidative Damage in Mammalian Cells

Abstract

Prolonged antibiotic treatment can lead to detrimental side effects in patients, including ototoxicity, nephrotoxicity, and tendinopathy, yet the mechanisms underlying the effects of antibiotics in mammalian systems remain unclear. It has been suggested that bactericidal antibiotics induce the formation of toxic reactive oxygen species (ROS) in bacteria. We show that clinically relevant doses of bactericidal antibiotics-quinolones, aminoglycosides, and β-lactams-cause mitochondrial dysfunction and ROS overproduction in mammalian cells. We demonstrate that these bactericidal antibiotic-induced effects lead to oxidative damage to DNA, proteins, and membrane lipids. Mice treated with bactericidal antibiotics exhibited elevated oxidative stress markers in the blood, oxidative tissue damage, and up-regulated expression of key genes involved in antioxidant defense mechanisms, which points to the potential physiological relevance of these antibiotic effects. The deleterious effects of bactericidal antibiotics were alleviated in cell culture and in mice by the administration of the antioxidant N-acetyl-l-cysteine or prevented by preferential use of bacteriostatic antibiotics. This work highlights the role of antibiotics in the production of oxidative tissue damage in mammalian cells and presents strategies to mitigate or prevent the resulting damage, with the goal of improving the safety of antibiotic treatment in people.

Full text

DOI: 10.1126/scitranslmed.3006055
, 192ra85 (2013);5 Sci Transl Med
et al.Sameer Kalghatgi
Damage in Mammalian Cells
Bactericidal Antibiotics Induce Mitochondrial Dysfunction and Oxidative
Editor's Summary
made about oxidative damage to mammalian tissues.
important to confirm this antibiotic effect in humans, with a broader range of antibiotics, before any conclusions can be
can also be prevented by taking antioxidants or by switching to bacteriostatic antibiotics. Nevertheless, it will be
suggest that not only does this damage occur with long-term use of antibiotics, but itet al.These studies by Kalghatgi
-acetyl-l-cysteine (NAC) without disrupting the bacteria-killing properties of the antibiotics.Npowerful antioxidant
in blood tests, tissue analysis, and gene expression studies. This ROS-mediated damage could be reversed by the
Mice treated with clinically relevant doses of bactericidal antibiotics similarly showed signs of oxidative damage
transport chain, which would lead to a buildup of ROS.
showed that bactericidal antibiotics disrupted the mitochondrial electronet al.shed light on the mechanism, Kalghatgi
DNA, protein, and lipid damage in vitro. A bacteriostatic antibiotic, tetracycline, had no effect on ROS production. To
dose- and time-dependent increases in intracellular ROS in various human cell lines. Such increases in ROS led to
induced−−-lactam), and kanamycin (an aminoglycoside)βciprofloxacin (a fluoroquinolone), ampicillin (a −−antibiotics
bactericidaltriggering mitochondrial release of reactive oxygen species (ROS). Indeed, in culture, three representative
antibiotics damage mammalian tissues by−−but not bacteriostatic−−The authors hypothesized that bactericidal
regimens.
antibiotics may also cause damage to mammalian cells and thus pose problems for patients on long-term antibiotic
Antibiotics hurt only bacteria, right? According to a new study from Kalghatgi and colleagues, certain types of
Antibiotics Affect Mitochondria in Mammalian Cells
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ANTIBIOTICS
Bactericidal Antibiotics Induce Mitochondrial
Dysfunction and Oxidative Damage in
Mammalian Cells
Sameer Kalghatgi,
1
* Catherine S. Spina,
1,2,3
* James C. Costello,
1
Marc Liesa,
3
J. Ruben Morones-Ramirez,
1
Shimyn Slomovic,
1
Anthony Molina,
3,4
Orian S. Shirihai,
3
James J. Collins
1,2,3†
Prolonged antibiotic treatment can lead to detrimental side effects in patients, including ototoxicity, nephro-
toxicity, and tendinopathy, yet the mecha nisms underlying the effects of antibiotics in mammalian systems
remain unclear. It has been suggested that bactericidal antibiotics induce the formation of toxic reactive oxy-
gen species (ROS) in bacteria. We show that clinically relevant doses of bactericidal antibiotics—quinolones,
aminoglycosi des, and b-lactams—cause mitochondrial dysfunction and ROS overproduction in mammalian
cells. We demonstrate that these bactericidal antibiotic–induced effects lead to oxidative damage to DNA, pro-
teins, and membrane lipids. Mice treated with bactericidal antibiotics exhibited elevated oxidative stress markers
in the blood, oxidative tissue damage, and up-regulated expression of key genes involved in antioxidant de-
fense mechanisms, which points to the potential physiological relevance of these antibiotic effects. The deleterious
effects of bactericidal antibiotics were alleviated in cell culture and in mice by the administration of the antioxidant
N-acetyl-
L-cysteine or prevented by preferential use of bacteriostatic antibiotics. This work highlights the role of
antibiotics in the production of oxidative tissue damage in mammalian cells and presents strategies to mitigate
or prevent the resulting damage, with the goal of improving the safety of antibiotic treatment in people.
INTRODUCTION
Antibiotics have led to an extraordinary decrease in morbidity and
mortality associated with bacterial infections. Yet, despite the great
benefits, antibiotic use has been linked to various adverse side effects,
including ototoxicity (1), nephrotoxici ty (2), and tendinopathy (3). Al-
though antibiotic targets and modes of action have been widely studied
and well characterized in bacteria, the mechanistic effects of common-
ly prescribed antibiotics on mammalian cells remain unclear. Recently,
it has been demonstrated that major classes of bactericidal antibiotics,
irrespective of their drug-target interactions, induce a common oxida-
tive damage cellular death pathway in bacteria, leading to the produc-
tion of lethal reactive oxygen species (ROS) (4–12) via disruption of
the tricarboxylic acid (TCA) cycle and electron transport chain (ETC)
(4, 6). The role of ROS in antibiotic-induced bacterial killing is cur-
rently a matter of debate (13, 14) and the subject of intense experime n-
tal investigation in our laboratory and other laboratories; however,
the techniques critiqued in (13, 14) were not used in the present study,
which focuses on mammalian systems.
Bactericidal and bacteriostatic antibiotics have been shown to target
mitochondrial components (15–20). In mammalian cells, the mito-
chondrial ETC is a major source of ROS during normal metabolism
because of leakage of electrons (21). Given the proposed bacterial or-
igin of mitochondria (22), we hypothesized that bactericidal antibiot-
ics commonly disrupt mitochondrial function in mammalian cells,
leading to oxidative stress and oxidative damage. Previous work has
shown that mammalian cells can be damaged by antibiotic treatment,
but these results were shown at concentrations considerably higher
than those applied clinically. At thesehighconcentrations, select anti-
biotics inhibited cell growth and metabolic activity, in addition to im-
pairing mitochondrial function in vitro (23, 24).
Here, we focused on characterizing the mechanist ic effects of clini-
cally relevant levels of bactericidal antibiotics on mammalian cells,
both in vitro and in vivo. We showed that bactericidal antibiotics—
quinolones, aminoglycosides, and b-lactams—caused mitochondrial
dysfunction and ROS overproduction in mammalian cells, ultimately
leading to the accumulation of oxidative tissue damage. We found that
these deleterious effects could be alleviated by administration of the
Food and Drug Administration (FDA)–approved antioxidant, N-acetyl-
L-cysteine (NAC), or prevented by preferential use of bacteriostatic
antibiotics. These results reflect two therapeutic strategies to combat
the adverse side effects of long-term antibiotic treatment.
RESULTS
Bactericidal antibiotics induce oxidative stress and damage
in mammalian cells
We first examined whether clinically relevant doses of antibiotics in-
duce the formation of ROS in mammalian cells. Here, clinically rele-
vant doses are defined by peak serum levels (25). We exposed a human
mammary epithelial cell line, MCF10A, to representative bactericidal
antibiotics from three different classes: ciprofloxacin (a fluoroquinolone),
ampicillin (a b-lactam), and kanamycin (an aminoglycoside). All three
bactericidal antibiotics induced a dose- and time-dependent increase
1
Howard Hughes Medical Institute, Department of Biomedical Engineering and Cen ter of
Synthetic Biology, Boston University, Boston, MA 02215, USA.
2
Wyss Institute for Bio-
logically In spired Engineering, Harvard University, Boston, MA 02215, USA.
3
Department of
Medicine, Boston University School of Medicine, Boston, MA 02118, USA.
4
Department of
Internal Medicine, Section on Gerontology and Geriatric Medicine, Wake Forest University
School of Medicine, Winston-Salem, NC 27105, USA.
*These authors contributed equally to this work.
†Corresponding author. E-mail: jcollins@bu.edu
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in intracellular ROS production (Fig. 1A and fig. S1), but a bacterio-
static antibiotic (tetracycline) did not lead to a significant increase in
ROS production (Fig. 1A). Notably, bactericidal antibiotic–induced
ROS was not elevated in mammary epithelial cells as a result of an
increased presence of dead or dying cells in vitro (fig. S2). To establish
that the observed bactericidal antibiotic–induced oxidative stress was
not cell line–specific, we tested several additional cell types, including
primary human aortic endothelial cells (PAEC), primary human mam-
mary epithelial cells (HMEC), human gut epithelial cells (CACO-2),
and normal human diploid skin fibroblasts (NHDF). In all instances,
we found the same pattern of significantly elevated ROS levels induced
by bactericidal antibiotics (fig. S3), with little effect seen after bacterio-
static antibiotic treatment.
Superoxide, a reactive oxygen by-product generated by leakage of
electrons from the ETC to oxygen, is a precursor to many other forms
of ROS, one instance being the enzymatic conversion into hydrogen
peroxide (H
2
O
2
) through superoxide dismutase (26). We measured
mitochondrial superoxide production and extracellular H
2
O
2
release
in mammalian (human) MCF10A cells and found that all three bac-
tericidal antibiotics induced a dose- and time-dependent increase in
both mitochondrial superoxide (Fig. 1B and fig. S4) and H
2
O
2
(Fig.
1C and fig. S5). Bacteriostatic antibiotic treatment did not lead to a
significant increase in superoxide (Fig. 1B) or H
2
O
2
production (Fig. 1C).
ROS can directly interact with cellular components resulting in
DNA, protein, and lipid damage. To characterize DNA damage in-
duced by bactericidal antibiotics in mammalian cells, we used indirect
immunofluorescence and Western blot anal-
ysis to measure g-H2AX, a core histone
protein that is phosphorylated in response
to DNA damage. We observed a persist-
ent and significant increase in g-H2AX
in MCF10A cells exposed to bactericidal
antibioticsafter6and96hoursoftreatment
compared with untreated cells (Fig. 1D and
fig. S6). Additionally, we quantified the
presence of 8-hydroxy-2 ′-deoxyguanosine
(8-OHdG), an oxidized DNA by-product.
Similar to g -H2AX, we found significant-
ly elevated levels of 8-OHdG after 6 and
96 hours of bactericidal antibiotic treat-
ment (Fig. 1E) but negligible change in
response to tetracycline compared with un-
treated controls.
To further investigate the accumulation
of cellular oxidative damage, we measured
levels of protein carbonyls, a modifica-
tion of proteins resulting from oxidative
damage, and levels of malondialdehyde
(MDA), an end product of lipid peroxi-
dation. We found significantly elevated lev-
els of protein carbonylation (Fig. 1F) and
lipid peroxidation (Fig. 1G) in MCF10A
cells after 96 hours of bactericidal antibi-
otic treatment. Consistent with the gen-
eral ROS, mitochondrial superoxide, and
H
2
O
2
results, bacteriostatic antibiotics
had little effect on protein carbonylation
or lipid peroxidation (Fig. 1, F and G).
Bactericidal antibiotics induce
mitochondrial dysfunction
In bacteria, the common mechanism of
killing by bactericidal antibiotics advances
the finding that toxic ROS are generated
via the disruption of the TCA cycle and
ETC (4). Bacteriostatic antibiotics do not
stimulate ROS production in bacteria, sug-
gesting that bactericidal antibiotics uniquely
affect major sources of ROS. In mamma-
lian cells, mitochondria are major sources
of intracellular ROS; therefore, we tested
6 h
Cell count
96 h
A
400
200
0
CM-H DCFDA
2
B
Untreated CiproAmp Kan
-Tubulin
-H2AX
6 h
96 h
Cell count
400
200
0
1234
Fluor intenstiy (A.U.)
010101010
6 h
96 h
-H2AX
-H2AX
D
AmpicillinCiprofloxacin Kanamycin Tetracycline
C
FE
96 h6 h
8-OHdG Lipid peroxidation
96 h6 h
G
1234
Fluor intenstiy (A.U.)
010101010
CM-H DCFDA
2
-Tubulin
-H2AX
2
3
5
1
0
4
Untreated
Protein carbonylation
96 h6 h
*
0.3
0.4
0.6
0.5
5
7
9
8
10
Carbonyls (nmol/mg)
MDA (nmol)
0
300
200
100
96 h
6 h
ROS
Change over
control (%)
96 h6 h
Hydrogen peroxide
0
300
200
100
Change over
control (%)
0
50
100
Mitochondrial superoxide
6 h 96 h
Change over
control (%)
8-OHdG/mg DNA (ng)
6
0.0
0.4
0.8
1.2
1.6
Ratio
( -H2AX/ -tubulin)
*
**
*
*
***
***
***
***
******
***
***
***
***
***
***
***
***
***
***
***
***
***
***
***
***
***
***
**
***
***
***
***
***
***
Fig. 1. Bactericidal antibiotics cause oxidative damage to mammalian cells. ROS and oxidative
damage were measured in human mammary epithelial cells (MCF10A) after 6 and 96 hours of treatment
with bactericidal [ciprofloxacin (10 mg/ml), ampicillin (20 mg/ml), and kanamycin (25 mg/ml)] or bacterio-
static [tetracycline (10 m g/ml)] antibiotics compared to untreated cells. (A) ROS was q uantified using
CM-H
2
DCFDA by flow cytometry (histograms on left) and a microplate spectrophotometer (bar graphs on
right). (B) Mitochondrial superoxide was measured using MitoSox Red. (C) Hydrogen peroxide release was
quantified by measuring Amplex Red fluorescence. (D) Antibiotic-induced DNA damage was evaluated by
Western blot analysis to measure the abundance of the phosphorylated histone protein, g-H2AX, com-
pared to a-tubulin serving as the loading control (quantifi catio n in the bar graph). (E) Additionally, oxida-
tive DNA damage was assessed by measuring the abundance of 8-OHdG. (F) Oxidative protein damage,
proteincarbonylation,wasdetectedusinganenzyme-linked immunosorbent assay (ELISA). (G)Lipidper-
oxidation was evaluated by measuring MDA. All bar graphs display means ± SEM (n ≥ 3). Comparisons
between treatments and untreated controls were made using a Student’s t test (*P <0.5,**P <0.01,***P <
0.001).
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the hypothesis that bactericidal antibiotics uniquely disrupt the func-
tion of the mitochondrial ETC, leading to the observed overpro-
duction of ROS and oxidative damage (Fig. 1).
We measured the inhibitory effects of bactericidal antibiotics on
individual, immunocaptured ETC complexes I to V. Bactericidal anti-
biotics inhibited complex I activity by 16 to 25% and complex III ac-
tivity by 30 to 40% compared to untreated samples (Fig. 2A), whereas
bacteriostatic antibiotics and negative controls exhibited only 5 to 10%
inhibition(fig.S7).Inaddition,weobservedavarieddegreeofinhi-
bition of complexes II and IV across the tested bactericidal antibiotics,
with complex V showing less change in activity compared to com-
plexes I to IV. These data indicate that bactericidal antibiotics inhibit
mitochondrial ETC complexes, in particular complexes I and III, which
have been identified as major sources of ROS formation (27).
Because mito chondri al energy metabolism is tightl y linked to or-
ganelle function, disruption of theETCshouldleadtoadecreasein
mitochondrial membrane potential (DY
m
), adenosine triphosphate
(ATP) levels, and overall metabolic activity. Indeed, bactericidal anti-
biotic treatment led to a correspondin g and significant decrease in all
three metabolic functions after 96 hours of treatment (Fig. 2, B to D),
further supporting the hypothesis that bactericidal antibiotics induce
mitochondrial dysfunction.
If bactericidal antibiotics induce ROS by impairing the function of
the ETC, then cells devoid of a functional ETC should show little ROS
formation after antibiotic treatment. Mammalian cells devoid of mito-
chondrial DNA (mtDNA), but retaining their nuclear genomes, lack
functional ETC complexes; these cells are referred to as r
0
cells. By
selectively eliminating mtDNA and providing nutrient supplementa-
tion, we generated an MCF10A r
0
cell line (fig. S8A). Bactericidal an-
tibiotic treatments of r
0
cells showed no difference in ROS production
when compared to untreated cells (fig. S8B). This was in sharp con-
trast to the large, significant increase in ROS production stimulated by
bactericidal antibiotic treatment in normal MCF10A cells (Fig. 1 and
fig. S8B). Furthermore, we found relatively high levels of nuclear DNA
damage (g-H2AX) in normal cells after bactericidal antibiotic treat-
ments, whereas the level of DNA damage in r
0
cells was similar to
untreated controls (Fig. 2E). These results suggest that the mitochon-
drial ETC is a major source of bactericidal antibiotic–induced intra-
cellular ROS.
The homeostatic balance between mitochondrial fission and fusion
can be altered in response to oxidative stress (28). Disruption of this
balance can lead to changes in mitochondrial morphology and func-
tion, resulting in the generation of ROS (28). Using live-cell imaging of
primary human mammary epithelial cells, we measured the morpho-
logical changes in mitochondria and found short, swollen, fragmented
mitochondria (smaller aspect ratio) with highly reduced branching
(smaller form factor) in bactericidal antibiotic–tr eated cells compared
to long, tubular (larger aspect ratio), and extensively branched (larger
form factor) mitochondria in untreated cells (Fig. 3). These results
suggest that bactericidal antibiotics shift the balance toward a profis-
sion state. Consistent with our data in Fig. 2, a profission state can lead
to a loss of membrane potential, loss of metabolic activity, and an
overall increase in oxidative stress (28).
Thus far, we have characterized isolated processes involved in
mitochondri al respiration. To directly test the mitochondrial respira-
tory capacity of antibiotic-treated cells, we measured changes in the
oxygen consumption rate (OCR) of intact MCF10A cells using the
Seahorse XF24 flux analyzer. Compared to untreated cells, bactericidal
antibiotic–treated cells exhibited a significant reduction in both basal
respiration and maximal respiratory capacity after 6 hours of treat-
ment (Fig. 4A), which was further reduced after 96 hours of treatment
(Fig. 4B). In contrast, cells treated for 6 and 96 hours with tetracycline
showed little change in respiratory capacity compared to untreated
cells (Fig. 4). These data demonstrate that bactericidal, but not bacterio-
static, antibiotics impair the function of mitochondria and, consequent-
ly, the overall respiratory capacity of the cell.
A
ATP levels
Ampicillin
Ciprofloxacin
Kanamycin
C
–60
Mitochondrial complex inhibition
–40
–20
0
D
Untreated
-H2AX
-Tubulin
0 0 0 0
NNNN
Cipro Amp Kan
E
6 h
96 h
B
-H2AX
-Tubulin
6 h 96 h
Decrease in
complex activity (%)
IC
50
Tetracycline
*
6 h 96 h
Normal
cells (N)
cells
0
0.8
0.6
0.4
1.0
6 h 96 h
Mitochondrial potential Metabolic activity
6 h 96 h
2.05
1.35
1.00
2.40
1.70
Untreated
*
*
Cipro Amp KanCtrl
Flour. (A475-660nm)
Ratio
(TMRE/MitoTracker)
15
25
35
45
ATP ( mol)
Cipro Amp KanCtrl
0.0
0.5
1.0
1.5
2.0
Ratio
( -H2AX/ -tubulin)
( -H2AX/ -tubulin)
0.0
0.5
1.0
Ratio
***
**
**
**
**
**
**
**
***
***
***
***
***
***
**
***
***
***
***
***
***
**
**
*
Fig. 2. Bactericidal antibiotics induce mitochondrial dysfunction. (A)
The effects of bactericidal [ciprofloxacin (10 mg/ml), ampicillin (20 mg/ml),
and kanamycin (25 mg/ml)] or bacteriostatic [tetracycline (10 mg/ml)] anti-
biotics on the function of individual, isolated ETC protein complexes were
measured. Bars represent the change in activity of individual antibiotic-
treated complexes compared to untreated complexes. The dashed gray
line represents the maximum inhibition (10%) seen across several indepen-
dent negative controls. The solid gray line represents the IC
50
(median inhib-
itory concentration) of positive control drugs shown to inhibit specific target
complexes (see fig. S7 for all positive and negative control results). (B)Mito-
chondrial membrane potential was quantified using tetramethylrhodamine
ester (TMRE) and MitoTracker Green. (C) ATP levels were measured using a
luciferin/luciferase assay. (D) Metabolic activity was measured using a color-
imetric tetrazolium dye (XTT). (E) DNA damage in normal (N) and mtDNA-
depleted (r
0
) MCF10A cells was measured by Western blot to evaluate the
ratio of g-H2AX compared to the loading control, a-tubulin. Quantification
is shown below the blots. All bar graphs display means ± SEM (n ≥ 3). Com-
parisons between treatments and untreated controls were made using a
Student’s t test (*P <0.5,**P <0.01,***P <0.001).
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Antibiotic-induced oxidative damage is rescued by an antioxidant
Characterization of bactericidal antibiotic effects on mammalian cells
points to widespread cellular oxidative damage. Therefore, we explored
the possibility of using an antioxidant to alleviate these deleterious
effects. We chose to test the ability of NAC to alleviate bactericidal
antibiotic–induced oxidative damage in mammalian cells. NAC was
selected because it is an FDA-approved antioxidant that is well tolerated
bypatientsandcommonlyusedtobufferextraneousintracellularROS
in mammalian systems (29, 30). MCF10A cells were pretreated with NAC
for 2 hours, followed by bactericidal antibiotic treatment for 6 and
96 hours. NAC pretreatment reduced bactericidal antibiotic–induced
ROS levels (Fig. 5A and fig. S9) and restored mitochondrial membrane
potential to levels seen in untreated cells after 96 hours of antibiotic treat-
ment (Fig. 5B). Furthermore, NAC restored basal respiration and
maximal respiratory capacity of bactericidal antibiotic–treated cells to
near-normal levels (Fig. 5C and figs. S10 and S11) and alleviated bacte-
ricidal antibiotic–induced DNA damage (g-H2AX) (fig. S12), 8-OHdG
formation (Fig. 5D), protein carbonylation (Fig. 5E), and lipid peroxida-
tion (Fig. 5F) after only 6 hours of treatment. Cells treated with NAC
alone showed no significant effects from untreated cells (Student’s t test).
Combining an antioxidant with a bactericidal antibiotic carries the
potential risk of reducing the bacterial killing efficacy of the antibiotic,
given that bactericidal antibiotic–induced ROS formation facilitates
bacterial killing (4). Tested in bacterial cultures, we found that the con-
centration of NAC used to rescue antibiotic-induced oxidative damage
in mammalian cells did not decrease the bacterial killing efficacy of
bactericidal antibiotics (fig. S13). We extended this study to test the
antimicrobial efficacy of the antibiotic-NAC combination in a mouse
urinary tract infection (UTI) model. Escherichia coli were transurethrally
introduced into the bladder of mice. Twenty-four hours after estab-
lishing infection, mice were treated with ciprofloxacin (50 mg/ml),
NAC (10 mM), and ciprofloxacin + NAC or untreated (vehicle only).
Ciprofloxacin + NAC and ciprofloxacin-only treatments showed simi-
lar levels of bacterial killing (fig. S14), suggesting that NAC does not
interfere with the antimicrobial activity of clinically relevant doses of
bactericidal antibiotics. Given that other antioxidants, such as glutathione,
have been shown to reduce the bacterial killing efficacy of bactericidal
antibiotics (8), further work is needed to characterize the effects of
NAC on both Gram-negative and Gram-positive bacteria.
Oxidative damage in vivo caused by bactericidal antibiotics
can be rescued
To determine the in vivo relevanc e of bactericidal antibiotic–induced
ROS production and mitochondrial dysfunction, we investigated oxidative
Short Long Rounded Branched
Form factor
24 h
Aspect ratio
24 h
Mitochondria (%)
AmpicillinCiprofloxacin KanamycinUntreated
100
75
50
25
0
Untreated
24 h
Ciprofloxacin
24 h
10 m
100
75
50
25
0
Mitochondria (%)
10 m
Fig. 3. Mitochondria in bactericidal antibiotic–treated cells show an
abnormal, profission state. Mitochondrial morphology was measured
in primary human mammary epithelial cells using TMRE and MitoTracker
Green. Untreated samples (left image) show mitochondria with normal
morphology, which are long and highly branched. Ciprofloxacin-treated
cells (10 mg/ml) (right image) had abnormally short and truncated mitochon-
dria. The percentages of mitochondria with a short versus long aspect ratio
and rounded versus branched form factor were measured for untreated and
bactericidal antibiotic–treated cells. Data are means ± SEM (n ≥ 5).
Kanamycin
Ampicillin
Background correction
Tetracycline
Untreated
Ciprofloxacin
0 1835537188
Time (min)
1353
971
590
208
–174
1735
OCR (pmol/min)
i ii iii
0 18355371
Time (min)
88
1174
846
519
191
–136
1501
OCR (pmol/min)
i ii iii
6 h
Basal Maximal
96 h
Basal Maximal
B
A
Respiratory capacity
Respiratory capacity
OCR (pmol/min)
0
350
700
1050
1400
OCR (pmol/min)
0
350
700
1050
1400
***
***
***
***
***
***
***
***
***
***
***
***
Fig. 4. Bactericidal antibiotics decrease mitochondrial basal respiration
and maximal respiratory capacity.(A and B) OCR was measured in
MCF10A cells after 6 hours (A) and 96 hours (B) of bactericidal [ciprofloxacin
(10 mg/ml), ampicillin (20 mg/ml), and kanamycin (25 mg/ml)] or bacteriostatic
[tetracycline (10 mg/ml)] antibiotic treatment. Cells were treated with anti-
biotics followed by the Seahorse OCR protocol including treatment with
three mitochondrial ETC complex inhibitors: (i) olig omycin, (ii) carbonyl cy-
anide p-trifluoromethoxyphenylhydrazone, and (iii) antimycin A. OCR mea-
sured before (i) represents basal respiration, whereas OCR measured
between (ii) and (iii) represents the maximal respiratory capacity. Represent-
ative Seahorse OCR plots are shown, and the bar graphs are means ± SD
for n = 3. Comparisons between treatments and untreated controls were
made using a Student’s t test (***P <0.001).
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stress markers and accumulation of oxidative damage in tissues of mice
treated with antibiotics. Mice received clinically relevant doses of anti-
biotics [ciprofloxacin (12.5 mg/kg per day), ampicillin (28.5 mg/kg per
day), and kanamycin (15 mg/kg per day)] in their drinking water (n ≥
3 animals per treatment group). At 2 and 16 weeks, peripheral blood
was drawn from the same animals to measure intracellular ROS, lipid
peroxidation, and glutathione levels. A reduction in glutathione is a
proxy measure for ROS production because it is an intracellular scav-
enger of ROS and a key component of the enzymatic antioxidant sys-
tem (31).
Bactericidal antibi otic–treated mice showed elevated lipid peroxi-
dation in their peripheral blood; these incr eases were statistically sig-
nificant after 16 weeks of treatment (Fig. 6). The treated mice also
exhibited reduced glutathione levels after 2 weeks of antibiotic treat-
ment, which was significantly decreased after 16 weeks of treatment
(Fig. 6). However, only ciprofloxacin led to a significant increase in
ROS in treated animals after 16 weeks.
To further characterize the in vivo oxidative stress response, we
evaluated the transcriptio nal changes in key genes invol ved in antiox-
idant defense mechanisms. Mammary glands were extracted from
mice treated with bactericidal antibiotics in the presence and absence
of NAC or the bacteriostatic antibiotic tetracycline. From these tissues,
the expression of the antioxidant defense genes—Sod1, Sod2, Gpx1,
and Foxo3a—was measured by quantitative polymerase chain reaction
(qPCR). After 2 weeks of treatment, these genes showed more than a
2-fold increase in expression in tissues extracted from bactericidal
antibiotic–treated mice, which were further elevated to ~10-fold change
after 16 weeks of treatment (fig. S15A). These genes were not up-regulated
0
4
6
2
0
4
6
2
0.15
0.35
0.45
0.25
0.15
0.35
0.45
0.25
Lipid peroxidationProtein carbonylation
DE
8-OHdG
F
C
NAC
Untreated
Ciprofloxacin
Cipro + NAC
Background
correction
0 1937567593
Time (min)
923
665
408
150
–107
1180
OCR (pmol/min)
i ii iii
96 h
0 1835537188
Time (min)
i ii iii
6 h
1174
846
519
191
–136
OCR (pmol/min)
1501
A
6 h 96 h
B
96 h 6 h 6 h 96 h 6 h 96 h
0.85
0.90
0.95
1.00
Mitochondrial superoxide
0.85
0.90
0.95
1.00
Mitochondrial potential
UntreatedAmpicillin Amp + NACCiprofloxacin Cipro + NAC Kanamycin Kan + NAC
NAC
Untreated Ampicillin Amp + NACCiprofloxacin Cipro + NAC Kanamycin Kan + NAC
NAC
Ratio (TMRE/MitoTracker)
Ratio (TMRE/MitoTracker)
Carbonyls (nmol/mg)
Carbonyls (nmol/mg)
6 h
0
10
20
30
40
50
Change over control (%)
96 h
0
20
40
60
80
100
Change over control (%)
*
8-OHdG/mg DNA (ng)
8-OHdG/mg DNA (ng)
4
7
8
5
MDA (nmol)
6
9
4
7
8
5
MDA (nmol)
6
9
** **
**
**
**
**
***
***
***
***
***
***
***
***
***
***
***
***
Fig. 5. NAC rescues bactericidal antibiotic–induced oxidative damage
in vitro. MCF10A cells were incubated with and without NAC (10 mM) for
2 hours, followed by treatment with ciprofloxacin (10 mg/ml), ampicillin
(20 mg/ml), kanamycin (25 mg/ml), or tetracycline (10 mg/ml) for 6 and
96 hour s. (A) Mitochondrial superoxide was measured using MitoSox
Red. (B) Mitochondrial membrane potential was quantified using TMRE
and MitoTracker Green. (C) OCR was measured using the Seahorse XF24
flux analyzer, and a representative diagram testing the ciprofloxacin + NAC
is shown (see figs. S10 and S11 for ampicillin and kanamycin). (D)Oxidative
DNA damage was assessed by quantification of 8-OHdG. (E) Protein carbonyls
were quantified to evaluate oxidative protein damage. (F) Lipid peroxidation
was measured by quantifying MDA. Data are means ± SEM (n ≥ 3). Compar-
isons between treatment + NAC and treatments were made using a Student’s
t test (*P <0.5,**P <0.01,***P <0.001).
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in the tissues of tetracycline-treated mice. Tissues from mice treated
with a combination of bactericidal antibiotic + NAC or NAC alone
showed decreased expression of the tested genes compared to antibi-
otic treatment (fig. S15B). These data support our in vitro results, dem-
on strating that NAC rescued bactericidal antibiotic–induc ed oxidati ve
damage in MCF10A human epithelial cells (Fig. 5).
To evaluate bactericidal antibiotic–induced oxidative stress at the
tissue level, we measured the accumulation of oxidative tissue damage
directly in mammary glands. Quantification of protein carbonylation
(Fig. 7A) and lipid peroxidation (Fig. 7B) revealed that bactericidal
antibiotic–induced oxidative damage was significantly mitigated by co-
administration of NAC. We also found no significant difference be-
tween tetracycline-treated and untreated mice. To further characterize
these effects, we measured oxidative damage of proteins (nitration) caused
by peroxynitrite, the product of the reaction between superoxide and
nitric oxide. After 16 weeks of bactericidal antibiotic treatment, with
and without NAC, as well as the bacteriostatic antibiotic tetracy cline,
we evaluated the number and size of anti-nitrotyrosine–stained foci in
mouse mammary glands. Animals treated with bactericidal antibiotics
showed increased protein nitration compared to untreated mice, whereas
mice treated with tetracycline showed no change (Fig. 7C). The observed
increase in protein damage caused by bactericidal antibiotic–treated
mice was rescued by co-administration of NAC. Immunohistochemical
analysis revealed that the damaged proteins in bactericidal antibiotic–
treated mice tended to localize to the cytoplasm of mammary ductal
epithelial cells (Fig. 7D). However, damaged proteins in NAC-treated
mice, in the presence or absence of bactericidal antibiotics, were more
likely to be found in the connective tissue, localizing to the nuclei of
adipocytes and stromal cells (Fig. 7D).
DISCUSSION
The emergence of drug-resistant bacterial strains has been one un-
intended consequence of the ubiquitous and frequent use of antibiot-
ics in medicine and food production. With the belief that antibiotics
specifically target bacteria, the consequences of how they interact with
mammalian cells have largely been overlooked, despite instances of
known adverse effects, including ototoxicity (1), nephrotoxicity (2), and
tendinopathy (3). Gaining a deeper mechanistic understanding of the
effects of commonly prescribed antibiotics in mammalian systems, par-
ticularly for extended periods of treatment, is critical for achieving a
clear safety profile for these drugs.
According to the endosymbiotic theory, mitochondria originated
from free-living, aerobic bacteria (22). It is likely then that antibiotics
target mitochondria and mitochondrial components, similar to their
action in bacteria. Indeed, previous studies in mammalian systems
have revealed parallel antibiotic-target interactions in mitochondria
(15 –19, 32). It has been shown, for instance, that aminoglycosides tar-
get both bacterial (33) and mitochondrial ribosomes (16), quinolones
target bacterial gyrases (34) and mtDNA topoisomerases (15), and
b-lactams inhibit bacterial cell wall synthesis (35)andmitochondrial
carnitine/acylcarnitine transporters (19). The work presented here ad-
vances our understanding of antibiotic action in mammalian systems
in two distinct and important ways. First, we show that, regardless of
their molecular targets, three major classes of bactericidal antibiotics—
quinolones, aminoglycosides, and b-lactams—induce ROS production
in mammalian cells, leading to DNA,protein,andlipiddamage.Sec-
ond, we demonstrate that these deleterious effects are produced by
clinically relevant doses of bactericidal antibiotics, both in cell culture
and in mice. These findings are analogous to our previous work in
bacteria, in which we showed that clinically relevant doses of bacteri-
cidal antibiotics induce a common oxidative damage pathway (4).
Identifying bactericidal antibiotics as a cause of ROS overpro-
duction and mitochondrial dysfunctioninmammaliancellsprovides
a basis for developing therapeutic strategies that could help allevia te
adverse side effects associated with antibiotics. For instance, by co-
administering an intracellular antioxidant, in this case NAC, we showed
that ROS levels and oxidative damage induced by bactericidal antibio-
tics could be abrogated while having little effect on the bacterial killing
efficacy of the antibiotics. Additionally, we showed that bacteriostatic
antibiotics, such as tetracycline, did not contribute to the overpro-
duction of harmful ROS in mammalian cells. When appropriate for
patient health, substituting a bacteriostatic antibiotic for a bactericidal
antibiotic could be a simple treatment strategy aimed at preventing
cellular oxidative damage.
To establish the link between bactericidal antibiotics and ROS pro-
duction, we have limited this study to antibiotic-treated human cell
lines and mice. It will be important to extend our investigation to hu-
man subjects to confirm our findings, prove their relevance to humans,
and maximize the translational value of our work. Epidemiologic
studies could provide valuable insight into the clinical implications
of antibiotic-induced oxidative damage and define some of the risks
associated with bactericidal antibiotic exposure. Another limitation to
consider is the set of antibiotics tested. Although we present results on
16 weeks
2 weeks
Blood
Glutathione
Change over control (%)
0
–25
–50
–75
Cipro
Amp Kan
ROS
0
50
Change over control (%)
25
75
Cipro Amp Kan
100
Lipid peroxidation
Change over control (%)
Cipro Amp Kan
50
25
0
*
*
*
*
*
*
*
*
*
*
Fig. 6. Bactericidal antibiotics induce oxidative damage in mice.Oxi-
dative stress markers were measuredinblooddrawnfromwild-typemice
treated with ciprofloxacin (12.5 mg/kg per day), ampicillin (28.5 mg/kg per
day),orkanamycin(15mg/kgperday)for2or16weeks.Dataaremeans±
SEM (n ≥ 3 animals per treatment group). Comparisons between treat-
ments and untreated controls were made using a Student’s t test (*P <0.5,
**P <0.01).
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three major classes of bactericidal anti-
biotics, interrogation across a broader range
of antibiotic classes is warranted before
conclusions can be drawn about the gen-
eral category of bactericidal antibiotics.
Last, it has been shown that both bacte-
ricidal and bacteriostatic antibiotics target
mitochondrial components (17–19, 23, 24),
yet we observed a marked differenti al ef-
fect between bactericidal and bacteriostatic
antibiotic treatments on cellular oxidative
damage. Disentangling these differences
in antibiotic action will be essential to gain
a full understanding of how antibiotics
interact with mammalian systems.
In humans, it is likely that the oxidative
stress and related oxidative cellular dam-
age induced by bactericidal antibiotics un-
derlie many of the adverse side effects
associated with these antibiotics (1–3). In
particular, patients with compromised anti-
oxidant defense systems or those genetically
disposed to developing a mitochondrial
dysfunction disease (36)mightbeatgreater
risk from bactericidal antibiotic treatments.
Thus, rapid detection (for example, via mea-
surements from peripheral blood) and mit-
igation of these adverse effects could have
important implications for patient care.
Our data suggest that oxidative damage
markers can be measured in the blood, but
further work is needed to determine the
efficacy of such a test in humans. Once de-
tected, mitigation strategies such as co-
administration with an antioxidant or treat-
ment with bacteriostatic antibiotics could
be used. It will be intriguing to explore these
possibilities with appropriate clinical trials,
with the goal of developing effective anti-
bacterial therapies with minimal adverse
side effects.
MATERIALS AND METHODS
Study design
The objective of our work was to investi-
gate the effects of clinically relevant doses
of bactericidal and bacteriostatic antibiot-
ics on mammalian systems in vitro and
in vivo. A human mammary epithelial cell
line (MCF10A) served as our in vitro mod-
el, whereas 8-week-old female wild-type
mice (FVB) were used to study the effect
of antibiotics in vivo. To understand the
putative role of antibiotic-induced ROS
in mammalian cells and accumulation of
oxidative damage, we used an antioxidant
A
NAC
Tetracycline
Ampicillin
Amp + NAC
Kanamycin
Kan + NAC
Protein carbonylation Lipid peroxidation Protein nitration
BC
D
Vehicle control NAC Tetracycline
Ciprofloxacin Ampicillin Kanamycin
Kan + NACAmp + NACCipro + NAC
Protein nitration
1.5
2.5
3.5
4.5
MD
A
(nmol)
0.10
0.20
0.30
0.40
Carbonyls (nmol/mg)
Ciprofloxacin
Cipro + NAC
Untreated (basic H 0)
Untreated (H 0)
2
2
0.0
0.4
0.8
1.2
Tissue stained for nY (%)
50 m
*
***
**
**
**
**
*
**
***
Basic H O
2
H O
2
Basic H O
2
H O
2
Basic H O
2
H O
2
Fig. 7. Oxidative damage induced in mouse mammary gland tissue by bactericidal antibiotics is
rescued by an antioxidant. Mouse mammary glands were harvested 16 weeks after being treated with
bactericidal antibiotics [ciprofloxacin (12.5 mg/kg per day), ampicillin (28.5 mg/kg per day), and kanamycin
(15 mg/kg per day)], with and without NAC (1.5 g/kg per day), or a bacteriostatic antibiotic [tetracycline
(27 mg/kg per day)]. Ciprofloxacin requires basic water (pH 8.0) to dissolve; thus, the antibiotic treatments
in each plot have been grouped according to their control treatments: basic H
2
O (pH 8) for ciprofloxacin,
and H
2
O for ampicillin, kanamycin, and tetracycline. (A and B) Protein carbonylation and lipid peroxidation
were measured in mouse mammary gland tissue collected from treated mice. (C) Mammary tissue was
stained with an anti-nitrotyrosine (nY) antibody to measure oxidat ive protein damage (nitration). Quanti-
fication of the protein nitration was defined as the percentage of total tissue area that was stained with
the anti-nitrotyrosine antibody. Data are means ± SEM (n ≥ 3 animals per treatment group). Comparisons
between treatment + NAC and treatments were made using a Student’s t test (*P <0.5,**P <0.01,***P <
0.001). (D) Representative immunohistochemical images that were quantified in (C). Red arrows indicate
protein damage foci (nitration) to ductal epithelial cells; black arrows point to protein damage to connec-
tive tissue cells (adipocytes and stromal cells).
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(NAC) alone or in combination with the bactericidal antibiotics. In vitro,
ROS was measured with fluorescent indicators [CM-H
2
DCFDA, Amplex
Red and horseradish peroxidase (HRP), MitoSox Red], and oxidative
damage to DNA (8-OHdG), proteins (carbonylation), and lipids (per-
oxidation) was quantified, whereas the functional implications were
assessed by evaluation of mitochondrial respiration (oxygen consump-
tion). In vivo, oxidative stress was evaluated in the peripheral blood of
treated mice with fluorescent indicators (H
2
DCFDA, fluor-DHPE, DCF),
whereas oxidative damage to mammary gland tissue (protein carbon-
ylation, lipid peroxidation, protein nitration) was assessed by immuno-
histochemistry and qPCR quantification of key antioxidant genes. Mice
were purchased from the vendor and randomly assigned to treatment
groups. Antibiotics were prepared and administered in a nonblinded
fashion, both in vitro and in vivo. Experiments inc lu de d th re e [F i gs . 1
(C, D, F, and G), 2 (A, B, and E), 4, 5B, and 6 and figs. S3, S5 to S7, S9
to S11, S13, and S15], four [Fig. 1 (A and B), 5 (A and D to F), and 8
(AtoC)andfigs.S1,S2,S4,andS14],five(Figs.1Eand3),six(Fig.2,
C and D), or eight (fig. S8) replicates per group.
Cell culture and antibiotic treatments
Human mammary epithelial cells, MCF10A [American Type Culture
Collection (ATCC)], were maintained in Dulbecco’s modified Eagle’s
medium and Ham’s F12 50/50 Mix (Cellgro, Mediatech) supple-
mented with 5% horse serum, epidermal growth factor (100 mg/ml),
hydrocortisone (1 mg/ml), cholera toxin (1 mg/ml), insulin (10 mg/ml),
and 5 ml of penicillin (10,000 U/ml) and streptomycin (10,000 mg/ml).
Hereafter, this medium will be referred to as “complete medium.” For
antibiotic treatments, complete medium was used without the addition
of penicillin/streptomycin. We refer to this medium as “treatment me-
dium.” All supplements were purchased from Sigma-Aldrich.
Before antibiotic treatments, cells were washed with phosphate-
buffered saline (PBS), detached with 0.25% trypsin-EDT A (Life Tech-
nologies), and seeded in 6-, 12-, 24-, or 96-well plates (Corning) in
treatment medium. After 24 hours, peak serum levels (25) of anti-
biotics were added: ciprofloxacin (10 mg/ml), ampicillin (20 mg/ml),
kanamycin (25 mg/ml), tetracycline (10 mg/ml), and spectinomycin
(100 mg/ml) (Fisher Scientific). Cells were plated at roughly equal num-
bers (near confluence) at each measurement time point. NAC (10 mM;
Sigma-Aldrich)wasusedasanintracellular ROS scavenger. For anti-
biotic treatments in conjunction with NAC, cells were incubated in
treatment medium plus NAC (pH adjusted to 7.4) for 2 hours and
then washed with PBS before the addition of antibiotics. The growth
conditions for the additional cell lines tested are described in the Sup-
plementary Materials and Methods.
MCF10A r
0
cell line
MCF10A rho-zero (r
0
) cells are MCF10A cells that have been cultured
to eliminate mtDNA. Following the protocol from King and Attardi
(37), MCF10A cells were incubated in complete medium supple-
mented with
D-glucose (4.5 g/liter), ethidium bromide (50 ng/ml), uridine
(50 mg/ml), and pyruvate (1 mM) for 6 weeks. After complete elimi-
nation of mtDNA (as verified through PCR described below), cells
were maintained in complete medium supplemented with
D-glucose
(4.5 g/liter), uridine (50 mg/ml), and pyruvate (1 mM). A set of MCF10A
control cells was grown alongside the MCF10A r
0
cells maintained in
complete medium supplemented with
D-glucos e (4.5 g/liter), uridine
(50 mg/ml), and pyruvate (1 mM). The verification of r
0
is described
in the Supplementary Materials and Methods.
ROS, H
2
O
2
, and mitochondrial superoxide
To detect ROS, cells plated in 6- or 96-well tissue culture plates were
washed twice with prewarmed PBS and then incubated with 10 mM
CM-H
2
DCFDA (Life Technologies) at 37°C for 45 min. Cells were
washed with PBS to remove excess dye and allowed to recover in
treatment medium at 37°C for 15 min in the dark. Imaging was car-
ried out on a fluorescence-enabled inverted microscope, and quan-
tification was done with a BD FACSAria II flow cytometer (Becton
Dickinson) or SpectraMax M5 microplate reader (Molecular Devices)
at an excitation/emission of 495/525 nm. This protocol was repeated
forallothertestedcelllines.
To detect extracellular hydrogen peroxide, we adapted a protocol
from Panopoulos et al.(38). Cells plated in 96-well tissue culture
plates were washed once with Krebs-Ringer phosphate buffer (KRPG).
KRPG containing 0.1% horse serum (KRPG 0.1%) and clinical levels
of antibiotics were added to the cells in a total volume of 50 ml. Next,
50 ml of prewarmed KRPG containing 100 mM Amplex Red reagent
and horseradish peroxide (0.2 U/ml) (reaction mixture) was added to
each well. Absorbance was measured at 560 nm after the addition of
reaction buffer with a SpectraMax M5 precision microplate reader
(Molecular Devices).
To detect mitochondrial superoxide, cells plated in 6- or 96-well
tissue culture plates were washed twice with prewarmed PBS and then
incubated with 5 mM MitoSox Red at 37°C for 10 min in the dark.
After incubation, cells were washed and resuspended in prewarmed
Hanks’ balanced salt solution (Fisher Scientific). Quantification was
completed with a BD FACSAria II flow cytometer or a SpectraMax
M5 microplate reader at an excitation/emission of 510/580 nm.
Oxidative DNA, protein, and lipid damage
8-OHdG levels were quantified with the OxiSelect Oxidative DNA
Damage ELISA Kit (Cell Biolabs), protein carbonylation was measured
with the Protein Carbonyl ELISA Kit (Enzo Life Sciences), and lipid
peroxidation was measured with the Lipid Peroxidation MDA Assay
Kit (Abcam). All assays were performed according to the manufacturer’s
protocol and are described in the Supplementary Materials and Methods.
Mitochondrial ETC complex activity
Direct inhibition of the activity for each of the five ETC complexes
was measured with the MitoTox Complete OXPHOS Activity Assay
Panel (Abcam), following the manufacturer’s protocol. Each of the five
complexes was captured from isolated bovine heart mitochondria in
their functionally active state with highly specific monoclonal antibo-
dies attached to 96-well microplates. IC
50
values for known inhibitors
of each of the five complexes were used as positive controls: rotenone
(complex I, 17.3 nM), thenoyltrifluoroacetone (complex II, 30 mM),
antimycin A (complex III, 22 nM), KCN (complex IV, 3.2 mM), and
oligomycin (complex V, 8 nM). The same positive controls were tested
on each complex and used as negative controls for off-target complexes
(for example, rotenone was a negative control for complexes II to V).
For each of the complexes treated with antibiotics [ciprofloxacin
(10 mg/ml), ampicillin (20 mg/ml), kanamycin (25 mg/ml), tetracycline
(10 mg/ml), or spectinomycin (100 mg/ml)], activity was determined by
measuring the decrease in absorbance in milli–optical density per mi-
nute at room temperature and at specified wavelengths [340 nm (I
and V), 600 nm (II), and 550 nm (III and IV)] in kinetic mode [every
minute for 2 hours (I) and 1 hour (II, IV, and V) and every 20 s for
5min(III)]withaSpectraMax M5 microplate reader.
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Metabolic activity, mitochondrial potential, and ATP
Metabolic activity was assayed with an XTT cell proliferation kit
(ATCC), cellular ATP was measured with the ATPlite Luminescence
Assay kit (Perkin Elmer), and mitochondrial potential was calculated
with the ratio of TMRE to MitoTracker Green (Life Technologies). All
assays were performed according to the manufacturer’sprotocoland
are described in the Supplementary Materials and Methods.
Mitochondrial morphometry
Primary human mammary epithelial cells (HMEC) were plated in
glass-bottom confocal dishes in treatment medium 24 hours before
addition of antibiotics. Cells were washed once with PBS and treated
with antibiotics for 24 or 96 hours. After treatment, cells were washed
with 1× PBS and incubated in 7 nM TMRE and 10 mM MitoTracker
Green for 30 min. After incubation, cells were returned to fresh treat-
ment medium with only TMRE (7 nM) and immediately imaged on a
Zeiss LSM710 confoc al microscope.
Mitochondrial morphometry was carried out with the particle anal-
ysis function in the image processing software ImageJ [National
Institutes of Health (NIH)] (39), as described in the Supplement ary
Materials and Methods.
Mitochondrial oxygen consumption
OCRs were measured at 37°C with an XF24 extracellular analyzer
(Seahorse Bioscience ). MCF 10A cells were seeded at a density of
40,000 cells per well on XF24 tissue culture plates overnight and then
treated with antibiotics for 6 and 96 hours. Measurements of OCR
were done according to Seahorse Bioscience protocols as described
in the Supplementary Materials and Methods.
Bacterial killing
The growth and survival of untreated exponential-phase wild-type
E. coli (MG1655) were compared to cultures treated with bactericidal
antibiotics alone [ciprofloxacin (5 mg/ml), ampicillin (5 mg/ml), or
kanamycin (5 mg/ml)], 10 mM NAC alone, or a combination of bac-
tericidal antibiotics supplemented with NAC for 3 hours. Cells were
grown overnight at 37°C and 300 rpm in a light-insulated shaker
and then diluted 1:500 in 25-ml LB in a 250-ml flask. Cultures were
growntoanopticaldensity(OD
600
) of about 0.3, as measured with
the SPECTRAFluor Plus spectrophotometer (Tecan). Antibio tics were
added at this point. For bacterial count measurements [colony-forming
units (CFU)/ml], 100 ml of culture was collected 30 min and 1, 2, and
3 hours after addition of antibiotics, washed twice with filtered 1× PBS
(pH 7.2) (Fisher Scientific), and then serially diluted in 1× PBS. Ten
microliters of each dilution was plated onto 120-mm square dishes
(BD Biosciences) containing LB Agar (Fisher Scientific), and plates
were incubated at 37°C overnight. Colonies were counted, and CFU/ml
values were calculated with the following formula:
ð#coloniesÞ10
dilution f actor
ðvolume platedÞ1000
Animal studies and tissue collection
Six- to 8-week-old female FVB/NJ mice (Jackson Laboratory) were ad-
ministered one of the following treatments: ciprofloxacin (12.5 mg/kg
per day), ampicillin (28.5 mg/kg per day), kanamycin (15 mg/kg per day),
with and without NAC (1.5 g/kg per day), tetracycline (13.5 mg/kg per
day),andvehicleonly(basicwater,pH8.0,ordeionizedwater).Solu-
tions were made fresh every 3 days and administered in the drinking
water for mice to feed ad libitum. Doses were achieved on the basis of
an average mouse weight of 20 g and an approximate intake of drink-
ing water of 4 ml per day. After 2 or 16 weeks, the animals were bled
and euthanized. Mammary tissue was collect ed from each mouse, pre-
served in RNAlater (Ambion, Life Technologies) for real-time qPCR
and 10% buffered formalin for immunohistochemistry, and flash-
frozen for protein extraction. The Institutional Animal Care and Use
Committee approved all mouse experiments. Quantitative reverse
transcription PCR of oxidative stress genes is described in the Supple-
mentary Materials and Methods.
Mouse blood oxidative stress
To measure general oxidative stress, 1 × 10
7
peripheral blood cells were
incubated with 2′,7′-dichlor odihydrofluorescein diacetate (H
2
DCFDA)
(Life Technologies), dissolved in dimethyl sulfoxide (DMSO) (Sigma-
Aldrich), at a final concentration of 0.4 mM at 37°C for 15 min at 5%
CO
2
. Reduced glutathione levels were measured by labeling 2 × 10
7
blood cells with mercury orange (Sigma-Aldrich), dissolved in DMSO,
at a final concentration of 40 mM for 3 min at room temperature. To
measure lipid peroxidation, we washed 2 × 10
7
blood cells with PBS
and labeled with N-(fluorescein-5-thiocarbamoyl)-1,2-dihexadecanoyl-sn-
glycero-3-phosphoethanolamine, triethyl-ammonium salt (fluor-DHPE)
(Life Technologies), dissolved in ethanol, at a final concentration of
50 mM for 1 hour at 37°C in a 5% CO
2
incubator with continuous
agitation. After the incubation period with each dye, blood cells were
washed twice with PBS to remove unbound label, resuspended in PBS,
and analyzed by flow cytometry (FACSFortessa, Becton Dickinson). A
488-nm argon laser beam was used for excitation. Blood cells labeled
with DCF and fluor-DHPE were detected by FL-1 PMT with linear
amplification, whereas mercury orange–labeled red blood cells were
detected by FL-2 PMT with log amplification. For each assay, treated
and untreated unstained cells were used as controls. Instrument cali-
bration and settings were performed with CaliBRITE-3 beads (Becton
Dickinson). The mean fluorescence channel of the entire population was
calculated for DCF, glutathione, and lipid peroxidation by the fluores cence-
activated cell sorting (FACS)–equipped BD FACSDiva software.
UTI mouse model
Eight-week-old C57BL/6 female mice were inoculated with 50 mlof
8% (w/v) mucin solution in sterile saline containing 2 × 10
9
E. coli
(MG1655) cells, via transurethral catheterization into their bladders,
as described previously (40). Briefly, mice were anesthetized with 2
to 4% isoflurane. Urinary catheters (30-gauge ×
1
/
2
-inch hypodermic
needle aseptically covered with polyethylene tubing) were coated in
medical-grade, sterile lubricating jelly. The bladder of the mouse was
gently massaged to expel urine. The lubricated catheter was inserted
into the urethral opening. It was then pushed into the urethra until the
base of the needle reached the urethral opening. Once fully inserted,
50 ml of the inoculum (containing 2 × 10
9
E. coli cells) was inject ed
directly into the bladder.
Infected animals received ciprofloxacin (50 mg/ml), NAC (10 mM),
ciprofloxacin and NAC, or vehicle (PBS) only, via intraperitoneal
delivery 24 hours after inoculation. After treatment, animals were
observed for an additional 24 or 48hours.Attheendoftheexperi-
ment, animals were euthanized by CO
2
asphyxiation followed by cer-
vical dislocation. Bladders were collected in 1 ml of PBS and homogenized
for 30 s for subsequent quantification of bacterial load. For the CFU/
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bladder measurements, the homogenized bladder was serially diluted
in PBS (pH 7.2). A 200-ml portion of each dilution was plated in LB-
agar plates and incubated overnight at 37°C. The colonies were counted,
and CFU/bladder was calculated with the following formula:
5ð#coloniesÞðdilu tion f actorÞ
volume plated
Histology and immunohistochemistry
Six- to 8-week-old female FVB/NJ mice (Jackson Laboratory) were ad-
ministered ciprofloxacin (12.5 mg/kg per day), ampicillin (28.5 mg/kg
per day), kanamycin (15 mg/kg per day), with and without NAC
(1.5 g/kg per day), tetracycline (13.5 mg/kg per day), or vehicle only
(basic or deionized H
2
O). Solutions were made fresh every 3 days and
administered in the drinking water for 16 weeks. Mice were eutha-
nized, and mammary glands were collected and stored in 10% buffered
formalin overnight at 4°C, transferredto70%ethanol,andstoredat
room temperature until sectioning. Tissues were embedded in paraffin
and sectioned for subsequent staining. Mammary gland sections were
stained with anti-nitrotyrosine polyclonal antibody (Millipore), followed
by anti-rabbit immunoglobulin G conjugated to HRP (Vector Labora-
tories). Slides were counterstained with hematoxylin. Image capture and
analysis are described in the Supplementary Materials and Methods.
Statistical analysis
A one-tailed Student’s t test was perform ed on measurements of ox-
idative stress in the blood, UTI model, and for comparisons between
antibiotic-treated groups and groups treated with antibiotic and NAC.
For all other statistical analyses, a two-tailed Student’s t test was per-
formed to compare treatment groups to untreated (control) groups.
We assumed equal variance for all groups.
SUPPLEMENTARY MATERIALS
www.sciencetranslationalmedicine.org/cgi/content/full/5/192/192ra85/DC1
Materials and Methods
Fig. S1. Dose-response and time-course measurements of ROS levels in antibiotic-treated cells.
Fig. S2. Effect of antibiotic treatment on cell viability in vitro.
Fig. S3. ROS production measured across different mammalian cell types treated with antibiotics.
Fig. S4. Dose-response and time-course measurements of mitochondrial superoxide produc-
tion in antibiotic-treated cells.
Fig. S5. Dose-response and time-course measurement of extracellular hydrogen peroxide in
antibiotic-treated cells.
Fig. S6. DNA damage in human mammary epithelial cells treated with antibiotics.
Fig. S7. Effect of antibiotics on the function of isolated ETC complexes.
Fig. S8. ROS production in mammalian cells lacking mtDNA treated with antibiotics.
Fig. S9. NAC reduces bactericidal antibiotic–induced ROS levels.
Fig. S10. OCR in antibiotic- and NAC-treated cells.
Fig. S11. OCR in mammalian cells.
Fig. S12. NAC rescues bactericidal antibiotic–induced DNA damage.
Fig. S13. Bactericidal antibiotic efficacy is unaffected by NAC in vitro.
Fig. S14. Bactericidal antibiotic efficacy is unaffected by NAC when tested in vivo in a murine
UTI model.
Fig. S15. Bactericidal antibiotics induce expression of antioxi dant defense genes in mouse
mammary gland tissue.
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Acknowledgments: We thank E. Cameron and P. Belenky for their help with experimental
design and troubleshooting, A. Saccone for his assistance with experiments, and T. Ferrante
for his assistance with imaging. Funding: This work was supported by the NIH Director’s Pioneer
Award Program and the Howard Hughes Medical Institute. M.L. was the recipient of a post-
doctoral fellowship from Fundacion Ramon Areces and an Evans Center Fellow Award. Author
contributions: S.K., C.S.S., J.C.C., M.L., J.R.M.-R., S.S., A.M., O.S.S., and J.J.C. designed the study,
analyzed the results, and wrote the manuscript. In vitro experiments were performed by S.K.
and S.S. In vivo experiments were performed by S.K., C.S.S., and J.R.M.-R. Seahorse experiments
were completed by S.K. and M.L. Competing interests: The authors declare that they have no
competing interests.
Submitted 1 March 2013
Accepted 3 June 2013
Published 3 July 2013
10.1126/scitranslmed.3006055
Citation: S. Kalghatgi, C. S. Spina, J. C. Costello, M. Liesa, J. R. Morones-Ramirez, S. Slomovic,
A. Molina, O. S. Shirihai, J. J. Collins, Bactericidal antibiotics induce mitochondrial dysfunction
and oxidative damage in mammalian cells. Sci. Transl. Med. 5, 192ra85 (2013).
RESEARCH ARTICLE
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on July 3, 2013stm.sciencemag.orgDownloaded from