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Insights & Perspectives hn again Think

Antibiotic use and abuse: A threat to mitochondria and chloroplasts with impact on research, health, and environment

Xu Wang1)†, Dongryeol Ryu1)†, Riekelt H. Houtkooper2)* and Johan Auwerx1)*

Recently, several studies have demonstrated that , the Introduction most intensively used in livestock and that are also widely applied in biomedical research, interrupt mitochondrial proteostasis and physiology in animals Mitochondria and chloroplasts are ranging from round worms, fruit flies, and mice to human cell lines. Importantly, unique and subcellular organelles that a plant chloroplasts, like their mitochondria, are also under certain conditions have evolved from endosymbiotic - proteobacteria and cyanobacteria-like vulnerable to these and other antibiotics that are leached into our environment. prokaryotes, respectively (Fig. 1A) [1, 2]. Together these endosymbiotic organelles are not only essential for cellular and This endosymbiotic origin also makes organismal homeostasis stricto sensu, but also have an important role to play in theseorganellesvulnerabletoantibiotics. the sustainability of our ecosystem as they maintain the delicate balance Mitochondria and chloroplasts retained between autotrophs and heterotrophs, which fix and utilize energy, respec- multiple copies of their own circular DNA (mtDNA and cpDNA), a vestige of the tively. Therefore, stricter policies on usage are absolutely required as bacterial DNA, which encodes for only a their use in research confounds experimental outcomes, and their uncontrolled few polypeptides, tRNAs and rRNAs [1, 3, applications in medicine and agriculture pose a significant threat to a balanced 4]. Furthermore, both mitochondria and ecosystem and the well-being of these endosymbionts that are essential to chloroplasts have bacterial-type ribo- sustain health. somes that are distinct from the 80S ribosomes in the cytoplasm; for instance, Keywords: all chloroplasts contain 70S ribosomes, .antibiotics; chloroplasts; ; environmental pollution; mitochondria; whereas animal mitochondria have 55– 60S ribosomes and plant mitochondria mitochondrial unfolded protein response; have 70–80S ribosomes, depending on the species [5, 6]. Mitochondria are biochemical hubs contributing to a diversity of cellular events such as energy homeostasis, calcium homeostasis, thermogenesis, DOI 10.1002/bies.201500071 steroidogenesis, detoxification, inflam- mation, oxidative stress, cell death, and 1) Laboratory of Integrative and Systems Abbreviations: Physiology, Ecole Polytechnique Fed erale de cpDNA, chloroplast DNA; ETC, electron transport so on [7–12]. One of the major and well- Lausanne, Lausanne, Switzerland chain; mtDNA, mitochondrial DNA; nDNA, nuclear characterized roles of mitochondria is 2) Laboratory Genetic Metabolic Diseases, DNA; ROS, reactive oxygen species; UPRmt, oxidative phosphorylation, the harvest- Academic Medical Center, Amsterdam, The mitochondrial unfolded protein response. ing of energy contained in nutrients into Netherlands † These authors contributed equally to this work. adenosine triphosphate (ATP), the major *Corresponding authors: energy currency molecule of life. The Riekelt Houtkooper majority of the mitochondrial proteins are E-mail: [email protected] Johan Auwerx encoded by nuclear DNA (nDNA) and E-mail: admin.auwerx@epfl.ch transcribed by the general transcriptional

Bioessays 37: 1045–1053, ß 2015 The Authors. Bioessays published by WILEY Periodicals, Inc. This is an www.bioessays-journal.com 1045 open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. X. Wang et al. Insights & Perspectives.....

reduced nicotinamide adenine dinucleo- tide phosphate (NADPH) through the ETC and photophosphorylation on the thylakoid membrane. Then in the dark reaction also known as the Calvin cycle, the energy is transferred into sugars following carbon fixation in the stroma. Photosynthesis is sensitive to environ- mental perturbations, which may inhibit plant growth and influence crop yield,

Think again such as nutrient deficiency [26–29], high light intensity [30], and diverse biotic and abiotic stresses [31, 32]. In addition, impaired chloroplast translation can also significantly decrease chlorophyll con- tent and efficiency of photosynthesis [5]. Antibiotics are antimicrobial organic substances that are produced from natural microorganisms or through industrial synthesis [33]. Since the intro- duction of antibiotics for the treatment and prevention of bacterial infection Figure 1. Conceptual figure illustrating the general idea showing the targeted effect of about 7 decades ago, a wide variety of antibiotics. A: Antibiotics induce bacterial cell death meanwhile also interrupting the function antibiotics have been used in human of endosymbiotic organelles, mitochondria, and chloroplast, and generating a signal turning on the unfolded protein response pathways in eukaryotic cells. B: A schematic figure medicine as well as in agriculture for showing the targeted effect of tetracyclines on the Tet-On/Tet-Off system in nucleus and preventing or treating animal and plant their adverse collateral effects on mitochondrial translation. Other antibiotics, such as the bacterial infections [34]. In addition, , also have similar effects on the mitochondria. Using antibiotics that impair antibiotics are also used as feed additives mitochondrial translation can induce a mitonuclear protein imbalance and lead to the for animals (mammals, birds, and fishes) mt mitochondrial unfolded protein response (UPR ) pathway. to promote their growth [33]. During the production and various application of such massive amount of antibiotics, they machinery into mRNAs that are trans- dynamics (fission/fusion), mitophagy, are released and can affect the environ- ported into cytosol where they are and the mitochondrial unfolded protein ment. Whereas the public and scientific translated into proteins by the cytoplas- response (UPRmt) [18–22]. Chronic over- community has been mostly focusing on mic ribosomes. These nuclear encoded load of these quality control pathways is the influence of overuse or misuse of and mitochondria-targeted proteins are a major cause for mitochondrial dysfunc- antibiotics on human health, there have imported in a co-translation manner, tion and contributes to the pathogenesis been relatively few studies on the impact folded, and assembled together with of mitochondrial diseases, ranging from of antibiotics on ecosystems, especially mtDNA-encoded polypeptides that are rare inherited mitochondrial diseases toa on the kingdom Plantae. Here, we translated by the specific translation palette of common age-related disorders summarize recent achievements show- machinery that resides in mitochondria that include metabolic diseases, neuro- ing adverse effects of antibiotics on [13–15]. Assembly of complexes and degeneration, and cancer [8, 9, 23]. mitochondria and chloroplast function, supercomplexes of the mitochondrial Chloroplasts are located in the cyto- which are off-site targets of several electron transport chain (ETC) hence plasm of plant leaf cells as well as in antibiotics. The ultimate goal of this requires a stoichiometric match between micro- and macroalgae. There are three review is to inform the reader about why nDNA-encoded and mtDNA-encoded key subcompartments in the chloro- judicious antibiotic usage is required to polypeptides [16]. An imbalance in the plasts, including the chloroplast enve- protect our health and the ecosystem. ratio between nuclear- and mitochon- lope, a double membrane, the stroma, drial-encoded proteins, termed the mito- and the thylakoid membranes, which nuclear protein imbalance, inflicts a form thylakoid vesicles [24]. Chloroplasts Elevated antibiotic proteotoxic and metabolic stress on the fulfill many essential functions in plant production increases mitochondria [17]. The exposure of cells, such as carbon assimilation mitochondria to many stressors of a through photosynthesis, nitrogen and potential of environmental proteotoxic, energetic, osmotic, and sulfur metabolism, and biosynthesis of release oxidative nature explains the existence amino acids, chlorophyll, and fatty of multiple quality control mechanisms acids [24]. Photosynthesis consist of light Due to the successful application of and adaptive pathways to maintain reactions and dark reactions [25]. In light antibiotics in human medicine and espe- proper mitochondrial function, such as reaction, the energy of light is trans- cially in agriculture, antibiotic produc- mitochondrial biogenesis, mitochondrial formed into chemical energy in ATP and tion has increased massively recently.

1046 Bioessays 37: 1045–1053, ß 2015 The Authors. Bioessays published by WILEY Periodicals, Inc...... Insights & Perspectives X. Wang et al.

From 2000 to 2010, human consumption to the data in 2009 [37]. More critically, trying to make antibiotics only available of antibiotics increased by 36%, primarily China produces and consumes the most onmedical orveterinaryprescription[39]. in developing countries [35]. According to antibiotics of all countries with an However, in 2012 there were still 8 hn again Think Food and Drug Administration (FDA) estimated 162 million kg of antibiotics million kg of antibiotics delivered to reports [36], in 2011, 3.3 million kg of being sold in China in 2013, of which animals in EU countries [40] (Fig. 2A). antibiotics were sold for human use and almost half was used in animal feed [38] The particular antibiotics used vary 13.6 million kg were sold for animal use (Fig. 2A). It has been accepted that in different countries. For instance in in the USA, indicating that 80% of overuse or misuse of antibiotics may China, fluoroquinolones and b-lactams antibiotics were destined for agriculture promote the development of antibiotic- are among the most commonly used applications (Fig. 2A). By 2013, the resistant bacteria [33]. To solve this antibiotics, while tetracyclines are also amount of antibiotics used in food- problem, the European Union (EU) has widely used for both humans and producing animals increased to 14.8 already forbidden the use of antibiotics to animals (Fig. 2B) [38]. In the USA and million kg, increasing by 17% compared promote animal growth from 2006, and is EU, tetracyclines are the most com- monly used antibiotics in animals, accounting for 40% of total antibiotics use (Fig. 2B) [36, 40]. Following the rising demand for animal protein, stockbreeding and aquaculture are developing rapidly, therefore, an unprecedented increase in the amount of veterinary antibiotics used is foreseen. According to an assessment of antibiotic consumption in livestock around the world using Bayesian statistical models, between 2010 and 2030, the global consumption of antimicrobials will increase by 67% in 2030 [41]. For countries such as Brazil, Russia, India, China, and South Africa (BRICS), the increase will be 99%, indicating that the double amount of antibiotics may be consumed in 2030 [41]. If such a prediction comes true, it will create even more challenges to control the release of antibiotics into the environment.

Tetracyclines, lost in translation

Tetracyclines are broad-spectrum poly- ketide antibiotics discovered from the genus of actinobacteria and they are acting by inhibiting bacterial protein synthesis (for detail see below). In the late 1940s, chlorte- tracycline and were identified, and soon after tetracycline Figure 2. Usages of antibiotics in China, USA, and EU. A: Uses of antibiotics in human and doxycycline were synthesized medicine, animals, and crop production. Data are from sales figures, which may not exactly [42–44]. Before the wide awareness of reflect consumption. Usage data in crops in China were estimated from validamycin use, antibiotic resistance in medicine, tetra- which consists of the majority of antibiotics consumption in China. A zoomed-in chart shows cyclines were the most common class of antibiotics usage for crop production in the USA, which can hardly be seen in the main antibiotics used worldwide to treat graph. Application of antibiotics in crop production is banned in the EU. B: Antibiotics most infectious diseases and they are now frequently used in humans and animals. The data on the antibiotics used in China are still an available choice to manage selected from those antibiotics that are frequently detected in the environment. Some antibiotics with relatively high use are not included due to their low detection frequencies [38]. certain diseases, such as acne, chlamy- Although not in the top, a substantial amount of tetracyclines were sold in China, as dia infections, and Lyme disease [42]. indicated. While their use in medicine has

Bioessays 37: 1045–1053, ß 2015 The Authors. Bioessays published by WILEY Periodicals, Inc. 1047 X. Wang et al. Insights & Perspectives.....

reduced, tetracyclines are still the the proteobacterial origin of mitochon- similarity of ribosomal machinery most commonly used antibiotic class dria [53]. Tetracyclines are potent inhib- between bacteria and mitochondria, it in veterinary medicine (sales: 2,943 tons itors of mitochondrial translation in rat is not surprising that, besides tetracy- in EU, 2012 [40] and 6,515 tons in US, heart and liver (IC50 ¼ 2.1 mM) [54]. Along clines, also other antibiotics that target 2013 [37]). This wide use is explained with this, several studies reported that bacterial protein synthesis can affect because tetracyclines are relatively tetracyclines reduce cell proliferation in mitochondrial protein synthesis (Fig. 1, cheap and can be applied in the diet various human cell lines and cause many and Table 1). Antibiotics of the families of farm animals at therapeutic levels to adverse effects in thymocytes and HepG2 of the , amphenicols, treat disease or at a subtherapeutic dose cells [55, 56]. Very recently, we have , , oxazolidi- to improve animal growth rates [45]. demonstrated that doxycycline disturbed nones, – all known as

Think again In addition to their medical and mitochondrial proteostasis through the inhibitors of bacterial protein synthesis – veterinary applications, tetracyclines induction of an imbalance between also block mitochondrial polypeptide are also used as tool compounds in mitochondrial and nuclear protein pro- synthesis, often without a parallel biomedical research to control the tran- duction, aka the mitonuclear protein effect on the cytoplasmic ribosome. scriptional regulator (Tet-On/Tet-Off imbalance [17]. This effect was observed Conversely, the antibiotic cyclohexi- system), to inhibit matrix metallopro- in human embryonic kidney (HEK) 293 mide, which is an antifungal agent, teases and to label bone remodeling and HeLa cells as well as in mouse does not inhibit bacterial and mito- [46–50]. Among those applications, the hepatoma Hepa1-6 and hypothalamic chondrial protein synthesis but prevents Tet-On/Tet-Off system is accounting for GT1-7 cell lines, and was present even eukaryotic cytoplasmic polypeptide most of the research use of the tetracy- at low concentrations (0.5 mg/mL) [17, synthesis [58]. clines. In Tet-Off systems, the trans- 57] (Fig. 1). In mouse and human cells, Aminoglycosides are a group of activator (tTA) protein, which is a fusion the induction of mitonuclear protein antibiotics that include , dibe- protein of the tetracycline repressor imbalance was accompanied by major kacin, , kanamycin, neomy- (TetR) of E. coli and the trans-activating changes in mitochondrial function (e.g. cins, , and . domain of VP16 of Herpes Simplex oxygen consumption rate), mitochon- Aminoglycosides induce the misreading Virus, can be used to express genes drial dynamics (e.g. induced fragmented and premature termination of mRNA placed under the control of a tetracy- mitochondria), as well as marked repres- translation through perturbing peptide cline-response element (TRE). When sion of 10% of nuclear genes [57]. elongation at the bacterial 30S riboso- tetracycline or a tetracycline derivative Moreover, doxycycline impaired devel- mal subunit [59]. Several of the side such as doxycycline binds to tTA opment and mitochondrial function in effects of the aminoglycosides, such as protein, the tTA protein is released from the nematode C. elegans and the fruit fly kidney injury, ototoxicity, and vestibu- the TRE and shuts down transcription. D. melanogaster [57]. Similarly, C57BL/6J lar toxicity, are hallmarks of mitochon- The Tet-On system is basically operating mice that drank water containing dox- drial toxicity; especially, the ototoxicity in the opposite fashion to the Tet-Off ycycline (50 or 500 mg/kg/day) for has been associated with mitochondrial system and upon tetracycline binding to 14 days displayed a similar mitonuclear ribosomal dysfunction [60]. Chloram- the rTA protein it allows it to interact imbalance and mitochondrial dysfunc- phenicol, a member of the with the TRE and for transcriptional tion. Furthermore, energy expenditure class that was isolated from Streptomy- activation to occur [51]. Although the wasreducedindoxycycline-treatedmice, ces venezuelae, binds to the 23S rRNA Tet-On/Tet-Off system is exquisitely compared to the controls receiving of the 50S ribosomal subunit and flexible to study gene function without amoxicillin, which does not prevent prevents bacterial protein elongation apparent developmental defects in vivo bacterial/mitochondrial translation but by overlapping with the binding site and in vitro, several studies have rather targets bacterial wall synthesis. at the A-site [52, 61]. Likewise, chlor- warned about the potential detrimental Also in plants, 25 mg/L doxycycline amphenicol and shut and confounding effects of the use of severely repressed growth of Arabidopsis down mitochondrial translation [62, tetracyclines (Fig. 1B). seedlings, significantly decreased oxy- 63]. In mammalian cells treated with Tetracyclines occupy the A-site of the gen consumption, and reduced mito- either or thiampheni- bacterial 30S ribosomal subunit and chondrial translation, indicative of col, mtDNA-encoded proteins (e.g. inhibit bacterial polypeptide synthesis repressed mitochondrial function. In MT-ND1, MT-CO1, and MT-CO2) were by sterically blocking the recruitment of summary, doxycycline altered mitochon- dramatically reduced while nDNA- the aminoacyl-tRNA to the bacterial drial function in immortalized mamma- encoded respiratory gene transcripts ribosome [43, 44, 52]. Ribosomes are lian cell lines, worms, fruit flies, mice, (e.g. ATP5A1, COX5A, and COX8A) [62] biological machines composed of RNAs and across kingdoms in plants [57]. and proteins (e.g. ATP5A) were and proteins that are responsible for increased [unpublished results of protein synthesis and that are conserved the authors]. Finally, the amphenicol- across kingdoms. Many antibiotics that induced mitonuclear imbalance are clinically approved and widely used Other antibiotics that between nDNA- and mtDNA-encoded in research, such as the tetracyclines, impact on mitochondria cellular respiratory proteins induces also have powerful inhibitory effects on the mitochondrial unfolded protein mitochondrial ribosomes and protein Given the body of evidence for the response, typified by the induction of synthesis, which is not surprising given endosymbiotic theory [53], and the HSP60, Mortalin, LONP1, and CLPP

1048 Bioessays 37: 1045–1053, ß 2015 The Authors. Bioessays published by WILEY Periodicals, Inc...... Insights & Perspectives X. Wang et al.

Table 1. Antibiotics affecting bacterial protein synthesis and human health

Class Name Target Reported side effects References hn again Think Aminoglycosides Amikacin, , Peptide elongation at the Kidney injury, ototoxicity, [60] Gentamicin, Kanamycin, bacterial 30S ribosomal and vestibular toxicity Neomycins, Streptomycin, subunit Tobramycin Amphenicols Chloramphenicol, Protein elongation by Aplastic anemia, bone [62, 63] Thiamphenicol overlapping with the marrow suppression, binding site at the A-site neurotoxicity of 50S ribosomal subunit Macrolides , Carbomycin A, Peptide-bond formation Myopathy, QT prolongation, [65] , and ribosomal nausea translocation Oxazolidinones , , Peptide-bond formation by Nausea, bone marrow [54, 61] , , blocking tRNA binding at suppression, lactic the A-site of 50S ribosome acidosis Streptogramins , Quinupristin/ Protein elongation at the Nausea, myalgia, arthralgia [68] dalfopristin, A- and P-sites of 50S ribosome Tetracyclines Doxycycline, , Polypeptide synthesis by Phototoxicity, secondary [53–56] , , sterically blocking the intracranial hypertension, , Tetracycline recruitment of the teeth discoloration, aminoacyl-tRNA at the steatosis, liver toxicity A-site of the bacterial 30S ribosomal subunit

References are reporting an effect on mitochondria, except for [68] which refers to chloroplasts.

(see [17] and unpublished results of the demonstrated, inhibitory actions of vir- which is not so surprising given the authors) (Fig. 1). Also in C. elegans, giniamycin on protein synthesis in endosymbiotic nature of these organ- chloramphenicol induced a mitonuclear chloroplasts were reported [68]. elles (Fig. 1B). Future studies should imbalance, activated the UPRmt and In addition to antibiotics directly define the mechanisms how these anti- reduced mitochondrial respiration [17]. targeting the mitochondrial ribosome, biotics achieve these mitochondrial Erythromycin was the first of the several studies demonstrated that a effects (mitochondrial translation, oxi- antibiotics discovered in 1952. number of antibiotics, including quino- dative stress, NADH depletion, or other Macrolides including azithromycin, car- lones (i.e. ciprofloxacin), b-lactams (i.e. mechanisms), potentially leading to bomycin A, clarithromycin, and eryth- ampicillin), and aminoglycosides (i.e. the development of new antibiotics that romycin bind within the exit tunnel of kanamycin), induce oxidative stress via are safer antimicrobials and cleaner the bacterial ribosome and perturb the depletion of the primary reducing research tools and which leave organ- peptide-bond formation and ribosomal equivalent NADH in bacterial, as well elle function intact. translocation [52, 64], and consequently as, in mammalian cells [69–71]. The fact also have an inhibitory action on mito- that highly deleterious hydroxyl radi- chondrial protein synthesis [65]. Oxazo- cals and the NADþ/NADH ratio was also lidinones are a class of antimicrobial increased after the addition of anti- Antibiotics reach plants agents that prevent peptide-bond for- biotics to wild-type E. coli, led to the through multiple pathways mation by blocking tRNA binding at the hypothesis that an oxidative damage- A-site of the bacterial 50S ribosome [52]. induced cell death pathway could There are mainly three ways for the The adverse effects of oxazolidinones underpin the bactericidal effects of the environmental release of antibiotics: (i) reflect their deleterious action on mito- antibiotics [71]. Interestingly, in 6 to after therapeutic use in human and chondria, and include hyperlactemia, 8-week-old wild-type female FVB/NJ veterinary medicine; (ii) after agricul- metabolic acidosis, and peripheral mice, a 16-week treatment of ciprofloxacin tural use, i.e. for growth promotion in neuropathy [54, 66]. Pristinamycin, (12.5 mg/kg/day), ampicillin (28.5 mg/kg/ stockbreeding and aquaculture, and quinupristin/dalfopristin, and virgin- day), or kanamycin (15 mg/kg/day) also therapeutic and preventive use in plant iamycin are of the family of the induced oxidative stress in blood and production; and (iii) non-intentional streptogramins isolated from Strepto- mammary gland [69]. Evaluating the release from industrial production [33, myces pristinaespiralis, and inhibit effect of antibiotics on NADþ and NADH 72, 73] (Fig. 3). According to the bacterial protein synthesis by occupy- levels and mitochondrial function, hence, statistical data from China, USA, and ing on the A- and P-sites of 50S warrants future investigation. EU, most of the antibiotics are used in ribosome [52, 67]. While the effect of This short literature review actually humans and farm animals (Fig. 2A). streptogramins on mitochondrial trans- underscores that several antibiotic Previous reports showed that due to lation and function are not yet clearly classes affect mitochondrial activity, incomplete absorption, 30–90% of

Bioessays 37: 1045–1053, ß 2015 The Authors. Bioessays published by WILEY Periodicals, Inc. 1049 X. Wang et al. Insights & Perspectives.....

improve plant growth [75, 88–90] prob- ably due to “hormesis,” an adaptive and mostly beneficial response to low levels of toxins. However, the beneficial range of antibiotics is usually quite narrow, for example, 0.5–5 mg/L of tetracycline can stimulate cell mitotic division and growth of wheat seedlings, while con- centrations higher than 5 mg/L cause adverse effects on wheat growth [75]. Think again Figure 3. Sources and pathways of how antibiotics are released into the environment. Antibiotics reach the environment through multiple ways, the main pathways beginning from human and agricultural use are highlighted. The thickness of the arrows reflects the relative Plant chloroplasts and importance of the pathways. mitochondria are vulnerable to antibiotics

antibiotic doses given to humans and Antibiotics induce How can antibiotics inhibit plant animals may be released in the urine growth? Many relevant studies focused and feces after [33]. There- phytotoxicity in the on the impact of antibiotics on photo- fore, urban wastewater, biosolids, and environment synthesis and oxidative stress response animal manure contribute most to the in plants [75, 86, 91–94]. The bacterial environmental release of antibiotics. Previous studies showed that terrestrial origins of chloroplasts and mitochon- Among different antibiotic classes, tet- and aquatic plants could take many dria explain why they may be vulner- racyclines are easily dissolved in water kinds of antibiotics up from the polluted able to antibiotics (Figs. 1 and 4). In the and could persist in soil for over 1 year, environment [82, 83]. In general, the moss Physcomitrella patens and the making them the most frequently uptake and effects on plants varies and green algal Closterium, studies showed detected and major antibiotic released depends on the antibiotic and plant that D-, fosfomycin, and b- into the environment [33, 74]. For species, as well as soil and water lactam antibiotics (e.g. ampicillin) inter- example, soil residues of tetracyclines characteristics [82, 84]. In most studies, fered with peptidoglycan biosynthesis have been detected to be up to 307 mg/ antibiotics consistently showed toxic and inhibit chloroplast division, caus- kg in China [75] and 198.7 mg/kg in effects on farm plants, such as rye- ing cell division inhibition and cell Germany [76], while the natural back- grass [85], maize [86], alfalfa, carrot, death [95, 96]. However, chloroplasts ground level of total antibiotics were lettuce [84], cucumber, and rice [87]. in vascular plants have lost the pepti- normally less than 5 mg/kg [33]. Some plants are extremely sensitive to doglycan layer in their envelopes, mak- Since the 1950s, antibiotics have antibiotics; for example, root elongation ing vascular plants insensitive to these been applied to control bacterial infec- in carrot seedlings was reduced by 50% antibiotics [96]. tion in agricultural plants, such as high- at 0.2 mg/L of tetracycline (EC50) [84]. In Chloroplast translation is the main value fruit, vegetable, and ornamental our recent study, 1 mg/L of doxycycline target of many antibiotics because of its plants [34]. In the USA, antibiotics can significantly inhibit root hair similarity to the prokaryotic transla- applied to plants account for only growth in Arabidopsis [57]. More cases tional machinery [97]. The inhibitory 0.26% (36,000 kg) of total agricultural of antibiotic toxicity on plants can be effect of streptogramins (virginiamycin) utilization in 2011, which are mainly found in some recent reviews [33, 82]. on protein synthesis in chloroplasts are confined to use in orchards [77] In some plant species, low concen- already mentioned above [68]. Amino- (Fig. 2A). In China, however, it is tration of antibiotics may oppositely glycoside antibiotics (e.g. streptomycin, estimated that more than 80 million kg of antibiotics, fungicides, and insecticides are produced per year for crops [78], from which validamycin is the most commonly used antibiotics (35 million kg produced per year, Fig. 2A) [79], for the control of sheath blight of rice through inhibiting the trehalase activity in fungal patho- gens [80]. Although validamycin also inhibits trehalase in plants and leads to alterations in carbohydrate allocation, Figure 4. Schematic diagram of the impact of antibiotics on a hypothetical plant cell. For its potential side effects on plant growth simplicity, all factors are presented in the same plant cell. Arrows indicate positive regulations in the environment has not been fully and bars mean negative regulations. ROS, reactive oxygen species; MDA, malondialdehyde, evaluated [81]. is an end product of lipid peroxidation.

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kanamycin, , gentamicin) can reductions in levels of intracellular gene expression, and cell proliferation. enter chloroplast through an iron trans- calcium due to chelation (Fig. 4). In Future research should also define porter (MAR1, multiple antibiotic resist- turn, reduced calcium changes overall cleaner research tools that do not affect hn again Think ance 1), located on chloroplast patterns and levels of protein synthesis the function of organelles, such as membrane; in the chloroplast these and induces toxic effects [102]. There- mitochondria and chloroplast that are antibiotics inhibit chloroplast transla- fore, the mechanisms that antibiotics vital for all aspects of physiology. tion by targeting ribosomal 16S and 23S employ to repress plant growth seem to Global antibiotics production and rRNA [98]. Another study showed that be multiple and not limited to the ones consumption are still increasing year by aminoglycosides (e.g. ), mentioned above. Further studies of the year, and pose a potential threat not lincosamides (e.g. ), and interactions of various antibiotic and only to human health but also to the macrolides (e.g. erythromycin) inhib- plant species will hence be invaluable to delicate homeostasis of our ecosystem. ited translation in the chloroplast with- fully understand the impact of environ- Plants show differential susceptibility out direct effects on cytoplasmic protein mental released antibiotics on plants. to antibiotics, and even in the same synthesis [93]. Lincomycin can also species genotypic differences may repress the transcription rate of some lead to significant divergence in sus- nuclear encoded photosynthesis-related Conclusion and outlooks ceptibility [103, 104], alerting us to take genes, such as Lhcb (Chlorophyll a-b care of their health and to avoid non- binding protein) and RbcS (Ribulose Since 1911 when the first antibiotic, natural selection that may occur in bisphosphate carboxylase small arsphenamine was discovered, antibi- heavily polluted regions. To protect chain) [93], perhaps due to a signal otics took the center stage of human and plants, animals, and humans in our originating from dysfunctional chloro- animal medicine and saved many lives. ecosystem, instead of restricting anti- plasts. Tetracyclines and amphenicols Nowadays antibiotics are not only used biotics use, we may select or design (e.g. chloramphenicol) also repress for medical/veterinary indications, but antibiotics that do not target compo- photosynthesis [75, 86, 91, 99]. also in biomedical research as well as nents of organelles. Therefore, more Although some studies showed that for agricultural applications. They are efforts to study the relationship between tetracyclines at high concentrations these “non-medical,” often uncon- antibiotics and endosymbiotic organ- (500 mM) can quickly inhibit chloro- trolled applications that pose particular elles, such as the mitochondrion and plast translation by binding 16S rRNA threats. chloroplast, are warranted. and blocking the entry of amino-acy- As to the use for research purposes, lated tRNA into the A site of the 70S antibiotics such as penicillin, strepto- The authors report no conflicts of ribosome [97], in our hands chloroplast mycin, and gentamicin are essential to interest. translation is 10-fold less sensitive prevent microbiological contamination to inhibition than mitochondrial trans- in eukaryotic cell culture. Ampicillin, lation [57]. Despite high similarity kanamycin, , hygromycin, Acknowledgements between chloroplast and prokaryotic and geneticin (G418) are the most Work in the RH group is supported by an translational machinery, the exact tar- popular antibiotics for selecting specific ERC Starting grant (no. 638290), and a gets of some antibiotics in the chlor- cells expressing or harboring trans- VENI grant from ZonMw (no. 91613050). oplast remain to be determined. duced genes (typically the antibiotic- JA is the Nestle Chair in Energy Besides these effects on chloro- resistance gene together with the gene- Metabolism and his research is sup- plasts, early work described that tetra- of-interest). In addition, the Tet-On/Tet- ported by EPFL, NIH (R01AG043930), cyclines, as well as chloramphenicol, Off system using tetracyclines allows Krebsforschung Schweiz/Swiss Cancer inhibit translation of proteins encoded the delicate temporal and spatial con- League (KFS-3082-02-2013), Systems X by mtDNA, but not by nDNA [100]. In trol of gene expression avoiding con- (SySX.ch 2013/153), and SNSF (31003A- our recent study [57], plants displayed founding or secondary effects, caused 140780). marked mitonuclear protein imbalance, by chronic overexpression or knock- implying specific inhibition of mito- down of a gene of interest. However, chondrial translation. However, the the use of antibiotics is a double-edged References direct target of tetracyclines in plant sword as exemplified by the induction mitochondria remains unknown. More- of mitochondrial proteotoxic stress by 1. Reyes-Prieto A, Weber AP, Bhattacharya over, lipid peroxidation and reactive doxycycline, which alters not only D. 2007. The origin and establishment of the oxygen species (ROS) were frequently mitochondrial dynamics and function plastid in algae and plants. Annu Rev Genet 41: 147–68. found in plants exposed to tetracy- but also global gene expression patterns 2. Andreux PA, Houtkooper RH, Auwerx J. clines [75, 86, 94]. Since mitochondria in immortalized cell lines, worms, fruit 2013. Pharmacological approaches to are one of the main ROS sources in flies, mice, and plants [56, 57]. To avoid restore mitochondrial function. Nat Rev Drug Discov 12: 465–83. plants [101], it will be interesting to such undesired confounders caused 3. Timmis JN, Ayliffe MA, Huang CY, Martin evaluate in the future whether mito- by organellar – mitochondrial and W. 2004. 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