Impacts of Bacteriostatic and Bactericidal Antibiotics on the Mitochondria of the Age-Related Macular Degeneration Cybrid Cell Lines

We assessed the potential negative effects of bacteriostatic and bactericidal antibiotics on the AMD cybrid cell lines (K, U and J haplogroups). AMD cybrid cells were created and cultured in 96-well plates and treated with tetracycline (TETRA) and ciprofloxacin (CPFX) for 24 h. Reactive oxygen species (ROS) levels, mitochondrial membrane potential (ΔψM), cellular metabolism and ratio of apoptotic cells were measured using H2DCFDA, JC1, MTT and flow cytometry assays, respectively. Expression of genes of antioxidant enzymes, and pro-inflammatory and pro-apoptotic pathways were evaluated by quantitative real-time PCR (qRT-PCR). Higher ROS levels were found in U haplogroup cybrids when treated with CPFX 60 µg/mL concentrations, lower ΔψM of all haplogroups by CPFX 120 µg/mL, diminished cellular metabolism in all cybrids with CPFX 120 µg/mL, and higher ratio of dead cells in K and J cybrids. CPFX 120 µg/mL induced overexpression of IL-33, CASP-3 and CASP-9 in all cybrids, upregulation of TGF-β1 and SOD2 in U and J cybrids, respectively, along with decreased expression of IL-6 in J cybrids. TETRA 120 µg/mL induced decreased ROS levels in U and J cybrids, increased cellular metabolism of treated U cybrids, higher ratio of dead cells in K and J cybrids and declined ΔψM via all TETRA concentrations in all haplogroups. TETRA 120 µg/mL caused upregulation of IL-6 and CASP-3 genes in all cybrids, higher CASP-7 gene expression in K and U cybrids and downregulation of the SOD3 gene in K and U cybrids. Clinically relevant dosages of ciprofloxacin and tetracycline have potential adverse impacts on AMD cybrids possessing K, J and U mtDNA haplogroups in vitro.


Introduction
The introduction of antibiotics in the 20th century is considered an astounding breakthrough in medicine [1]. Antibiotics cure a wide range of infectious diseases, and they facilitate the success of modern medical procedures such as organ transplantations, surgeries and cancer therapy regarding prevention of opportunistic infections [2]. However, over time, the growing pattern of antimicrobial resistance (AMR) and some remarkable adverse effects resulting from administration or misusage of antibiotics have attracted attention [3]. Fluoroquinolones are antibiotics frequently used in medical and surgical fields, such as ophthalmology. These bactericidal agents (e.g., norfloxacin, lomefloxacin, ciprofloxacin and ofloxacin) have broad-spectrum antibacterial activity, and can be administered via topical, intravitreal and systemic routes [4]. Their principal mechanism of action increased risk of age-related macular degeneration [33]. Kenney at al. demonstrated that mtDNA haplogroups have key roles in numerous molecular pathways associated with AMD progression such as energy production, cell growth and signaling [34].
In this study, our aim is to assess the potential damaging effects of CPFX and TETRA on the cellular health and mitochondria of AMD cybrid cell lines.

Ethic Statement
This study involving human subjects was performed according to stated principals in Declaration of Helsinki. After obtaining the informed written consent, the Institutional Review Board of the University of California, Irvine approved the research (UCI IRB #2003-3131).

Cybrids Creation
The process of creation of trans-mitochondrial cybrids is described in previous studies [33]. After collecting 10 mL of peripheral blood using tubes with sodium citrate, DNA extraction kits (Puregene, Qiagen, Valencia, CA, USA) and the Nanodrop 1000 (Thermo Scientific, Wilmington, DE, USA) were used for DNA isolation and quantification, respectively. Then, a series of centrifuge steps were performed for platelet isolation followed by using Tris buffer saline (TBS) for platelet suspension. ARPE-19 cell lines, purchased from ATCC (Manassa, VA, USA), demonstrated similar functional and structural characteristics to in vivo RPE cells [35]. The ARPE-19 were exposed to low-dose ethidium bromide and serially passaged until they were deficient in mtDNA (Rho0) [36]. As described in previous studies, cybrids were created by fusion of Rho0 ARPE-19 cells with platelets using polyethylene glycol [32].

The Ratio of Live, Apoptotic and Dead Cells (Flow Cytometry)
To measure the ratio of live and apoptotic cells affected by exogenous 120 µg/mL CPFX and TETRA treatments, cells were cultured in 6-well plates (50,000/well) and incubated for 24 h. After 48 h treatment period with antibiotics, 50 µL propidium iodide (PI, representing dead cells) and YO-PRO-1 (an early marker for apoptosis) stains were added to seeded cells. Cells were incubated for 30 min on ice followed by flow cytometry analyses (Flow Cytometer, Novocyte 3000; Catalog# 4355488, San Diego, CA, USA). The flow cytometry values were measured using 488 nm excitation with green fluorescence emission for YO-PRO ® -1 dye (i.e., 530/30 bandpass) and red fluorescence emission for propidium iodide (i.e., 610/20 bandpass). This technique measures the physical and chemical characteristics of the cells such as the proportion of live, apoptotic and dead cells in untreated, vehicle-control and antibiotic-treated (CPFX and TETRA) groups. To accomplish standard compensation, single-colored stained cells were used. To assure a statistically significant determination of a sample volume, 10,000 bead events were collected per each sample.

Isolation of RNA and cDNA Amplification
Cultured cybrids in 6-well plates were treated with 120 µg/mL CPFX and TETRA. After 48 h incubation period, RNA was isolated from the cell lysis using the RNeasy Mini-Extraction kit (Puregene, Qiagen, Valencia, CA, USA). Nano Drop 1000 (Thermo Scientific, Wilmington, DE, USA) was used for RNA quantification. Then, each isolated RNA was reverse transcribed to complementary DNA (cDNA) by using Superscript IV VILO Master Mix with the DNase Enzyme (ThermoFisher, Waltham, MA, USA).

Quantitative Real Time Polymerase Chain Reaction (qRT-PCR)
Isolation of total RNA was performed from cultured CPFX-exposed (n = 3), TETRAexposed (n = 3), associated vehicle-control (HCl and TETRA) (n = 3) and untreated (n = 3) cells. Table 2 demonstrates all target primers, which are predesigned via Qiagen Quan-tiTect Primer Assays or KiCqStart SYBR ®® Green primers (Sigma-Aldrich, Burlington, MA, USA). PowerUp SYBR Green Master Mix (ThermoFisher) on a ThermoFisher Quant Studio 3 Real-Time PCR System was used for performing qRT-PCR, which evaluated the relative expression level of various genes associated with pro-inflammatory (IL-6, IL-33 and TGF-β1), pro-apoptotic (CASP-3, CASP-7 and CASP-9) and antioxidant enzyme (SOD2, SOD3 and GPX3) pathways genes. In this study, the housekeeping gene was HPRT1, which is an enzyme recycling inosine and guanine in the purine salvage pathway and is considered a stable endogenous control gene when investigating alterations in gene expression. Therefore, as a consistent endogenous control, HPRT1 primer was used as the reference gene for reaching a standard expression level for all primers [37]. The ∆∆ Ct method was used for analyzing the data, in which ∆ Ct = [Ct (threshold value) of the target gene] − [Ct for HPRT1], and ∆∆ Ct = ∆ Ct of the treatment condition − ∆ Ct of the untreated condition. When comparing the treated conditions to untreated conditions, the fold changes were measured as: fold change = 2 −∆∆Ct . Triplicate formats of antibiotic treated cells (CPFX and TETRA) compared to untreated and vehicle-control (HCl and Meth) samples were analyzed.

Statistical Analyses
GraphPad Prism (Version 5.0, GraphPad Software, Inc., San Diego, CA, USA) was used for statistical analyses. One-way ANOVA Tukey test was performed when comparing the differences among untreated, vehicle-control (HCl and Meth) and antibiotic-treated (CPFX and TETRA) cells. The p value of less than 0.05 was considered statistically significant. Three replicates were allocated for each condition in this study and the associated data analyzed in triplicate. Table 2. Information of the genes associated with inflammatory, apoptotic and antioxidant enzymes pathways in AMD cybrid cells.

Symbol
Gene Name GenBank Accession No.

Sigma Primer Sequences
Or Qiagen Gene Globe ID

CASP-3
Caspase 3, apoptosis-related cysteine peptidase NM_004346 NM_032991 QT00023947 Encodes protein as a cysteine-aspartic acid protease that plays a central role in the execution phase of cell apoptosis.

QT00003549
This gene encodes a member of the cysteine-aspartic acid protease (caspase) family. Sequential activation of caspases plays a central role in the execution phase of cell apoptosis.

CASP-9
Caspase 9, apoptosis-related cysteine peptidase NM_001229 NM_032996 QT00036267 Encodes a member of the cysteine-aspartic acid protease (caspase) family, which is involved in the execution phase of cell apoptosis.

IL-6
This gene encodes a cytokine that functions in inflammation and the maturation of B cells. In addition, the encoded protein has been shown to be an endogenous pyrogen capable of inducing fever in people with autoimmune diseases or infections.

IL-33
Interleukin 33 The protein encoded by this gene is a cytokine that binds to the IL1RL1/ST2 receptor. The encoded protein is involved in the maturation of Th2 cells and the activation of mast cells, basophils, eosinophils and natural killer cells.

TGF-β1
Transforming growth factor beta-1-like This gene is a polypeptide member of the transforming growth factor beta superfamily of cytokines. It is a secreted protein that performs many cellular functions, including the control of cell growth, cell proliferation, cell differentiation and apoptosis.

Superoxide dismutase 1 NM_000454 QT01671551
This gene is a member of the iron/manganese superoxide dismutase family. The protein encoded by this gene is a soluble cytoplasmic protein, acting as a homodimer to convert naturally occurring but harmful superoxide radicals to molecular oxygen and hydrogen peroxide.

Symbol
Gene Name GenBank Accession No.

Sigma Primer Sequences
Or This gene encodes a mitochondrial protein that forms a homotetramer and binds one manganese ion per subunit. This protein binds to the superoxide byproducts of oxidative phosphorylation and converts them to hydrogen peroxide and diatomic oxygen.

Superoxide dismutase 3 NM-003102 QT01664327
This gene encodes a member of the superoxide dismutase (SOD) protein family, which catalyzes the conversion of superoxide radicals into hydrogen peroxide and oxygen, effective in protection of the brain, lungs and other tissues from oxidative stress.

GPX3
Glutathione peroxidase 3 NM_002084 FH1-5 -GCAACCAATTTGGAAAACAG-3 RH1-5 -CTCAAAGAGCTGGAAATTAGG-3 The protein encoded by this gene belongs to the glutathione peroxidase family, members of which catalyze the reduction of organic hydroperoxides and hydrogen peroxide (H 2 O 2 ) by glutathione, and thereby protect cells against oxidative damage. Several isozymes of this gene family exist in vertebrates, which vary in cellular location and substrate specificity.

HPRT1
Hypoxanthine Phosphoribosyl The protein encoded by this gene is a transferase, which catalyzes conversion of hypoxanthine to inosine monophosphate and guanine to guanosine monophosphate via transfer of the 5-phosphoribosyl group from 5-phosphoribosyl 1-pyrophosphate. This enzyme plays a central role in the generation of purine nucleotides through the purine salvage pathway.

ROS Levels
First, we compared the effect of antibiotics on the oxidative stress in the cells of different cybrid haplogroups. In terms of intracellular ROS levels, the different mtDNA haplogroups cybrids demonstrated varying results. After treatment with 60 µg/mL CPFX, U cybrids showed increased ROS levels (p < 0.05) (Figure 1b). In contrast, CPFX-treated J cybrids showed decreased ROS levels caused by CPFX 120 µg/mL (p < 0.05), (Figure 1c). Similarly, TETRA 120 µg/mL decreased ROS levels (p < 0.001) in J cybrids (Figure 1c). Furthermore, neither of the antibiotics affected the ROS levels in treated K haplogroups (Figure 1a). These results indicate that higher dosages of these antibiotics could have impacts on the ROS levels in U and J haplogroups. (h) CPFX 120 µg/mL reduced cellular metabolism of U cybrids but hig with TETRA 120 µg/mL; (i) All CPFX treatments and TETRA 30 µg/mL declined cellular me of J cybrids. (* p < 0.05, ** p < 0.01, *** p < 0.0001).

Ratio of Apoptosis and Dead Cells (Flowcytometry)
We investigated whether apoptotic cell death contributes to the negative impact of antibiotics on the different haplogroup cybrids. In K cybrids, treatment with 120 µg/mL of CPFX induced 100% value dead cells (p < 0.0001), versus 9.53% dead cells induced by vehicle-control treatment (Figure 2a,d). The TETRA-treated cells showed 18.66% (p < 0.05) dead cells versus 15.98% for the vehicle-control treated cells (Figure 2g,j). In U cybrids, there was no significant alteration of the ratio of dead cells with neither CPFX (17.81%) (Figure 2b,e) and TETRA (25.12%) (Figure 2h,k) versus HCl (15.64%) and Meth (23.61%)-exposed cells, respectively. Compared to vehicle-control-exposed cells, the J cybrids treated with CPFX 120 µg/mL showed a substantial increase in relative dead cells (90.52% versus 25.40%) (p < 0.05) (Figure 2c,f). The TETRA-treated cells also showed an increase in dead cells (16.36% versus 6.47%) (p < 0.05) compared to vehicle-control treated cells (Figure 2i,l). Therefore, CPFX and TETRA significantly increase the proportion of apoptotic and finally dead cells.

Alterations of Pro-Inflammatory, Pro-Apoptotic and Antioxidant Enzymes Genes
Next, we evaluated the effects of the antibiotics on the different cybrids at the gene level. treated with CPFX 120 µg/mL showed a substantial increase in relative dead cells ( versus 25.40%) (p < 0.05) (Figure 2c,f). The TETRA-treated cells also showed an incr dead cells (16.36% versus 6.47%) (p < 0.05) compared to vehicle-control treated cel ure 2i,l). Therefore, CPFX and TETRA significantly increase the proportion of ap and finally dead cells.

K Cybrids
Inflammation genes: Performing qRT-PCR, in all experiments, the value of 1 is assigned to the vehicle-control samples. In terms of the pro-inflammatory gene pathway in K cybrids, the relative gene expression of IL-33 (p < 0.0001) (Figure 3b) increased significantly with CPFX treatment. There was higher expression of IL-6 gene (p < 0.0001) (Figure 3a) with TETRA treatment compared to vehicle-control samples. However, TETRA treatment showed no effect on the IL-33 (Figure 3b) and TGF-β1 gene expression levels (Figure 3c) compared to the vehicle-control group. Additionally, there was no alteration of TGF-β1 gene after CPFX treatment (Figure 3c). K cybrids, the relative gene expression of IL-33 (p < 0.0001) (Figure 3b) increased significantly with CPFX treatment. There was higher expression of IL-6 gene (p < 0.0001) ( Figure  3a) with TETRA treatment compared to vehicle-control samples. However, TETRA treatment showed no effect on the IL-33 (Figure 3b) and TGF-β1 gene expression levels ( Figure  3c) compared to the vehicle-control group. Additionally, there was no alteration of TGF-β1 gene after CPFX treatment (Figure 3c).  Antioxidant genes: The CPFX-treated K cybrids showed no significant impacts on the expression levels of SOD1 (Figure 5a), SOD2 (Figure 5b), SOD3 (Figure 5c) and GPX3 (Figure 5d) genes. With TETRA treatment, there were lower expression levels of SOD3 (p < 0.01) (Figure 5c) and GPX3 (p < 0.05) (Figure 5d) genes in K cybrids versus vehicle-controlexposed samples.

J Cybrids
Inflammation genes: The CPFX-treated J cybrids showed upregulation of IL-33 (p < 0.01) (Figure 3h) but lower expression levels of IL-6 (p < 0.0001) (Figure 3g) along with no effects on TGF-β1 (p = 0.013) (Figure 3i) genes. The TETRA-treated J cybrids showed an Figure 5. Impacts of CPFX and TETRA on genomic expression of antioxidant enzymes in K, U and J cybrids. In K cybrids, (a,b) no significant change in SOD1 and SOD2 by either treatment, (c,d) downregulation of SOD3 and GPX3 by TETRA. In U cybrids, (e,h) no significant change in SOD1 and GPX3 by either treatment, (f) higher expression of SOD2 and (g) lower expression of SOD3 via TETRA. In J cybrids, (i) no significant change in SOD1 by either treatment, (j) upregulation of SOD2 via CPFX and TETRA, (k) higher expression of SOD3 by CPFX, and (l) overexpression of GPX3 by CPFX. (* p < 0.05, ** p < 0.01, *** p < 0.0001).
These results demonstrate that CPFX and TETRA treatments affect the relative expression levels of various genes associated with inflammatory, apoptotic and antioxidant enzymes. Figure 6 summarizes the expression levels of genes associated with apoptotic, inflammatory and antioxidant enzymes after CPFX and TETRA treatments. GPX3 (p < 0.05) (Figure 5l) genes but no alteration of expression level of SOD1 (Figu  and SOD3 genes (Figure 5k).
These results demonstrate that CPFX and TETRA treatments affect the relativ pression levels of various genes associated with inflammatory, apoptotic and antiox enzymes. Figure 6 summarizes the expression levels of genes associated with apop inflammatory and antioxidant enzymes after CPFX and TETRA treatments.

Discussion
Fluoroquinolones and tetracyclines have a wide range of antibacterial activitie previous studies have demonstrated possible ocular toxicities as a result of fluoroq lone administration. In Canada, Etminan et al. conducted a case-control study on 98 patients with ophthalmic disease, who were followed between 2000 and 2007. Th ported a higher risk of retinal detachment in patients who used oral fluoroquino (3.3% of cases) compared to the control group (0.6%) [38]. Another group concluded the hazard of uveitis-associated systemic illnesses was higher in individuals treated fluoroquinolones in the outpatient setting [39]. These findings suggest that the mito

Discussion
Fluoroquinolones and tetracyclines have a wide range of antibacterial activities but previous studies have demonstrated possible ocular toxicities as a result of fluoroquinolone administration. In Canada, Etminan et al. conducted a case-control study on 989,591 patients with ophthalmic disease, who were followed between 2000 and 2007. They reported a higher risk of retinal detachment in patients who used oral fluoroquinolones (3.3% of cases) compared to the control group (0.6%) [38]. Another group concluded that the hazard of uveitis-associated systemic illnesses was higher in individuals treated with fluoroquinolones in the outpatient setting [39]. These findings suggest that the mitochondrial sensitivities to these antibiotics may play a role in the side effects seen in some patients. The present study investigates the effects of CPFX and TETRA on cybrid cell lines that possess different mtDNA haplogroup patterns.

Effects of CPFX on AMD Cybrids
Mitochondrial membrane potential and ROS levels after CPFX treatment: Our findings indicate that different cybrid haplogroups respond differently to treatment with CPFX; (i) There is a reduction in ROS levels in J haplogroup after treatment with CPFX 120 µg/mL; (ii) all haplogroup cybrids showed decreased ∆ψM levels and lower cell metabolism with CPFX 120 µg/mL treatment; additionally, (iii) all three haplogroups treated with CPFX 120 µg/mL showed significantly elevated ratios of apoptotic and dead cells.
Other studies have also reported mitochondrial dysfunction and higher ROS levels after antibiotic exposure. Kalghatgi et al. assessed the effects of three bactericidal antibiotics (kanamycin, ciprofloxacin and ampicillin) and one bacteriostatic (tetracycline) on harvested mouse mammary tissues [26]. The bactericidal agents negatively affected mitochondria electron transportation chain (ETC), which led to significantly increased ROS levels and eventually cellular lipid, protein and DNA damage [26]. In another study, application of higher concentrations of fluoroquinolones and tetracycline impaired mitochondrial function in cultured primary human osteoblasts [40]. Similar harmful effects on mitochondria were also shown in animal studies. For example, intraperitoneal injection of fluoroquinolones (alatrofloxacin) caused oxidative stress and mitochondrial injury in a Sod2+/− (heterozygous deficiency in mitochondrial superoxide dismutase, Sod2) mouse model. Compared to wild-type mice, there was a significant reduction in mitochondrial genes and critical enzyme activities in mutant models after injection of 33 mg/kg/day of alatrofloxacin over 28 days. Moreover, the activation of mitochondrial Ca 2+ dependent mechanisms, such as nitric oxide (NOS) production, was more prominent in alatrofloxacin-treated mutant mice [41]. Multiple studies reported substantial structural and functional alterations of mitochondria in AMD subjects, including disorganized membranes, cristae and matrix along with reduction in mitochondria numbers [32,42,43]. Therefore, this harmful role of bactericidal agents could be more prominent in individuals suffering from an impaired antioxidant defense system along with patients with specific age-related disease with damaged mitochondria such as AMD and Parkinson's disease. By identifying eukaryotic mitochondrial dysfunction and over-production of ROS linked to fluoroquinolones, therapeutic approaches are progressing regarding alleviating the harmful side consequences of antibiotics administration [44].
Gene expression studies after CPFX treatment: Exposure with CPFX 120 µg/mL induced (i) overexpression of IL-33 in all treated haplogroups; and (ii) upregulation of CASP-3 and CASP-9 genes in all exposed haplogroups. Prior literature has shown considerable retinal damage due to increased phototoxicity after fluoroquinolone intake. The phototoxicity of five different fluoroquinolones (ciprofloxacin, enoxacin, DR-3355 (the s-isomer of ofloxacin), lomefloxacin and ofloxacin) was demonstrated in culture mouse 3T3 fibroblast cells exposed to ultraviolet-A (UVA) light [45]. Cellular apoptosis has been described as the mechanism associated with the cytotoxic proprieties of fluoroquinolones. Kohanski et al. compared the ROS levels in Escherichia coli produced via three various categories of bactericidal agents including 250 ng/mL norfloxacin, 5 µg/mL kanamycin and 5 µg/mL ampicillin compared to bacteriostatic ones including tetracycline, erythromycin, spectinomycin and chloramphenicol treatments. It was indicated that the bactericidal groups induced higher deleterious production of hydroxyl radicals contributing oxidative damage and cellular death [46]. To our knowledge, this is the first study to measure the effects of CPFX treatment on expression patterns of genes associated with basic pathways.

Effects of TETRA on AMD Cybrids
Mitochondrial membrane potential and ROS levels after TETRA treatment: Our results demonstrated: (i) significantly diminished ROS levels in U and J haplogroup cybrids via TETRA 120 µg/mL; (ii) declining levels of ∆ψM with all TETRA treatment concentrations in all three haplogroups; (iii) diminished cellular metabolism of treated K and J haplogroups cybrids after TETRA 30 µg/mL; and (iv) significant increase in the ratio of dead cells of K and J haplogroups treated with TETRA 120 µg/mL. Similar detrimental effects have been reported by Ding et al. who showed the toxicity of a series of concentrations (0, 45, 60 and 90 mg/L) of two types tetracyclines (doxycycline and chlortetracycline) and four fluoroquinolones (enrofloxacin, norfloxacin, ciprofloxacin and ofloxacin) on wild-type zebrafish. It was suggested that over 7-, 14-and 21-day exposure periods, there was a significant decrease in mitochondria crista along with severe mitochondria swelling in the treated subjects [47]. Another study in agreement with our results investigated the role of doxycycline on the proliferation and metabolism of in vitro breast epithelial lines (MCF12A) [48]. Moreover, the similar risk and mitochondrial-damaging mechanisms are identified as a consequence of topical administration of tetracyclines [49].
Expression studies after TETRA treatment: Our findings showed cybrids treated with TETRA 120 µg/mL had (i) up-regulation of IL-6 and CASP-3 levels in all haplogroups; but (ii) down-regulation of IL-33 and SOD3 genes in K and U haplogroup cybrids. Ahler et al. studied the impacts of doxycycline on alteration of proliferation and metabolism of multiple human cell lines in vitro, such as cervical cancer cells (Hela), lung cancer cells (H157) and prostate cancer cells (LNCaP). There was a widespread alteration of expression of genes associated with the oxidative metabolic pathway along with dominancy of the glycolytic metabolism pathway [50]. They examined expression levels of genes associated with gluconeogenesis (fructose-1,6-bisphosphatase (FBP) and glucose-6-phosphatase (G6PC)) along with regulatory enzymes of the glycolysis pathway (pyruvate kinase (PK), hexokinase (HK) and phosphofructokinase (PFK)). They reported downregulation after TETRA treatment of the G6PC and FBP genes, along with upregulatory effects on PK gene levels [49]. In our study, the principal enzymes related to the antioxidant pathway were affected by treatments. TETRA 120 ug/mL induced downregulation of SOD2 expression in U and J haplogroups along with lower expression of GPX3 in exposed K haplogroups. Noteworthily, Jones et al. reported the importance of the disruptive role of tetracyclines in individuals with mitochondrial defects. The fibroblast cells derived from four categories of patients with defective mitochondrial diseases were exposed to doxycycline (10 µg/mL) or tetracycline (77 µg/mL). There was significant disruption of mitochondrial translation resulting from exposure to these antibiotics [50].

Conclusions
In light of these results, the clinically relevant dosages of bactericidal and bacteriostatic agents showed detrimental effects on treated in vitro AMD cybrid cell lines. The negative association of antibiotics and mitochondria might be important in populations suffering from age-related disorders that are associated with damaged mitochondria, such as AMD, Parkinson's and Alzheimer's diseases. In the future, additional in vivo and clinical trials may need to be conducted to improve our understanding and knowledge regarding the negative contributory roles of these antibiotics on mitochondrial dysfunction.

Strength and Limitations of the Study
The findings of this study might be significant in clinical settings, particularly regarding the approach to treat the patients suffering from specific age-related disease such as AMD, Parkinson's disease and Alzheimer's disease. The only limitation of the study is the smaller cohort of individuals with AMD for three different cybrid cell lines.

Informed Consent Statement:
In terms of various phases of this study including designing, performing and revealing of the results, patients or the public were not involved.

Data Availability Statement:
All data relevant to the study are included in the article.
Conflicts of Interest: B.D.K. is consultant to Alcon, Alimera, Allegro, Allergan, Genentech, Glaukos, GSK, Neurotech, Novagali, Novartis, Ophthotech, Pfizer, Regeneron, Santen, SecondSight, Teva, ThromboGenics. The rest of the authors declare that they have no competing interests. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.