Reducing Antimicrobial Toxicity: Time to Move from Mice to Men in a Clinical Trial
ABSTRACT & COMMENTARY
Richard R. Watkins, MD, MS, FACP
Division of Infectious Diseases, Akron General Medical Center, Akron, OH; Associate Professor of Internal Medicine, Northeast Ohio Medical University, Rootstown, OH
Dr. Watkins reports no financial relationships in this field of study.
SYNOPSIS: Clinically relevant doses of bactericidal antibiotics (quinolones, aminoglycosides and β-lactams) were shown to cause mitochondrial dysfunction and overproduction of reactive oxygen species in mammalian cells in vitro and in mice, leading to oxidative tissue damage. The antioxidant acetylcysteine prevented these effects.
SOURCE: Kalghatgi S, et al. Bactericidal antibiotics induce mitochondrial dysfunction and oxidative damage in mammalian cells. Sci Transl Med 2013; 5:192ra85.
It is quite an understatement to say that antibiotics have been tremendously valuable for treating bacterial infections. Indeed, untold millions of humans and animals have benefited since widespread antimicrobial use began in the 1940s. Yet, clinicians are acutely aware of the downsides of these miracle drugs including adverse side effects, some of which have serious consequences for the recipient (i.e., nephrotoxicity, ototoxicity, tendinopathy). These toxicities and others tend to happen more with long-term rather than short-term antibiotic therapy. Efforts to understand the mechanisms of antibiotic toxicity and their prevention are the subject of ongoing experimental investigation. An emerging theory of how bactericidal antibiotics work is through the induction of a common cellular death pathway in bacteria that leads to the production of lethal reactive oxygen species (ROS).1
One of the innocent bystanders that can be affected by ROS is the mitochondria within mammalian cells. This consequence is not surprising since according to the endosymbiotic theory, mitochondria likely originated from free-living, aerobic bacteria. Moreover, it is known that high concentrations of antibiotics (greater than what is used clinically) inhibit mammalian cell growth and metabolism, although the mechanistic pathways by which this occurs remain obscure. Kalghatgi and colleagues sought to elucidate the effects of clinically relevant concentrations of bactericidal antibiotics on mammalian cells, both in vitro and in vivo.
First, the investigators exposed a number of human cell lines to three different classes of bactericidal antibiotics: ciprofloxacin (fluoroquinolone), ampicillin (β-lactam), and kanamycin (aminoglycoside). They also exposed the cells to tetracycline to compare the effects of a bacteriostatic agent. All three bactericidal antibiotics induced a dose- and time-dependent increase in intracellular ROS production that damaged cellular DNA and protein and induced mitochondrial dysfunction, but tetracycline did not. Further experiments determined the mitochondrial electron transport chain as the major source for intracellular ROS and that the bactericidal but not bacteriostatic antibiotics reduced the overall respiratory capacity of the cell.
Next, the investigators tested whether this widespread cellular damage could be alleviated through the use of an antioxidant. They used N-acetyl-L-cysteine (NAC), an FDA-approved antioxidant used in clinical practice and a known buffer for extraneous intracellular ROS. Mammalian cells pretreated with NAC had reduced bactericidal-induced ROS levels and restored mitochondrial membrane potential compared to untreated cells. One concern with this approach is the potential to reduce the bacterial killing efficacy of the antibiotic, since bactericidal-induced ROS formation is a key mechanism for causing bacterial cell death. To address this concern a urinary tract infection model was constructed using Escherichia coli transurethrally introduced into the bladder of mice. Mice given ciprofloxacin compared to ciprofloxacin plus NAC had similar levels of bactericidal killing, which suggests that NAC does not interfere with antibacterial activity of ciprofloxacin. Serum glutathione levels were tested in the mice at 2 weeks and 16 weeks as a proxy measurement for ROS production. At 2 weeks mice treated with NAC plus the bactericidal antibiotics had lower glutathione levels compared to mice that did not receive NAC. At 16 weeks only ciprofloxacin caused a significant increase in ROS. Additional experiments using human epithelial cells yielded similar findings whereby NAC reduced bactericidal antibiotic-induced oxidative damage.
Through a series of well-conduced experiments, Kalghatgi and colleagues have provided evidence to support a novel mechanism of how bactericidal antibiotics can generate deleterious side effects. The three major classes of bactericidal antibiotics- quinolones, aminoglycosides, and β-lactams- cause ROS production that results in damage to mammalian DNA, proteins, and lipids. These data suggest a basis for potential therapeutic strategies that could reduce the toxicities associated with bactericidal antibiotics. One example is the co-administration of an intracellular antioxidant like NAC, which the investigators found reduced ROS levels and oxidative damage while not impacting the bacterial killing capacity of the antibiotics. The results from this study might be especially applicable to certain patients with a genetic predisposition towards developing a mitochondrial dysfunction disease. This group would in theory have an increased risk for toxicities from bactericidal agents. It remains to be elucidated whether monitoring specific blood markers (e.g. glutathione) would be beneficial.
The study had several limitations. First, many of the experiments were conducted in mice and the literature abounds with examples in which experimental data derived from animal models are different after testing in human subjects. Second, the hypothesis that all antibiotics kill by producing ROS, while biologically plausible, is controversial and other studies do not support it.2 Third, the author’s suggestion for reducing the risk of ROS and oxidative damage by using bacteriostatic rather than bactericidal antibiotics, especially for prolonged courses, needs to be taken with caution. The prevailing belief among many infectious disease experts is that bactericidal antibiotics are often preferred to bacteriostatic agents largely because of differences in pharmacokinetic and pharmacodynamics properties. Moreover, there are many examples in clinical practice (i.e. infective endocarditis, bacteremia, sepsis) where rapid bacterial killing is the goal.
Overall this paper advances our understanding of the mechanisms of bacterial action in mammalian cells in several ways. Whether antioxidants are helpful in preventing toxicities from bactericidal antibiotics in humans remains to be elucidated and a clinical trial seems warranted.
- Kohanski MA, et al. A common mechanism of cellular death induced by bactericidal antibiotics. Cell 2007; 130:797-810.
- Keren I, et al. Killing by bactericidal antibiotics does not depend on reactive oxygen species. Science 2013; 339:1213-1216.