The adverse effect of gentamicin on cell metabolism in three cultured mammary cell lines: “Are cell culture data skewed?”

Abstract

Many cells are cultured in media that contains an antibiotic to prevent bacterial contamination. Mycoplasma and other bacterial contamination is a serious problem for those involved in cell culture. Antibiotics in the media helps prevent this contamination and make life easier for the investigators; as performing cell culture experiments in antibiotic free media is difficult and requires vigorous sterile technique. There are many reports of antibiotics causing mitochondrial damage. In this study, we tested the effect of gentamicin in culture media on human mammary epithelial MCF-12A and breast cancer MCF-7 and MDA-MB-231 cell lines by real time PCR, immunofluorescent microscopy, lactate assay, DNA damage assay. We found that the addition of gentamicin in media upregulated the gene expression of hypoxia inducer factor 1 alpha (HIF1a), glycolytic enzymes and glucose transporters, compared to the cells cultured in gentamicin free media. Gentamicin also increased the lactate production and inhibited mitochondrial membrane potential of the cell lines. Furthermore, the antibiotics in media induced mitochondrial reactive oxygen species causing DNA damage. We found an increase of 8-hydroxy-2’-deoxyguanosine a product of DNA oxidative damage in the media of MCF-12A, MCF-7 and MDA-MB-231 cell lines. These results showed that normal epithelial and breast cancer cells cultured in the media with gentamicin had increased HIF1a, aerobic glycolysis and DNA oxidative damage. If we use these unhealthy cells in the experiment, all data will be different, compared to cells grown in gentamicin free media. We have studied the detrimental effects of three antibiotics on mitochondrial function in the untransformed MCF-12A human mammary cell line and two human mammary cancer cell lines, MCF-7 and MB-MDA-231. The metabolic changes in all cell lines were dramatically different between those in antibiotic free media versus antibiotic containing media. There was a marked difference in gene expression of glycolytic enzymes, reactive oxygen species production and effects on membrane potential. Ironically, our first studies were done in media containing gentamicin, and repeated studies were done in gentamicin free media. The results were very different. The purpose of this report is to emphasize that metabolic cell culture data may be inaccurate because experiments were performed in cell culture media containing antibiotics. We will present evidence to support this theory.

Neurodegeneration and Mitochondria Organelle Transplantation: “A Technology That Proof of Principle Suggest Is Ready for Prime Time”

“…I predict had they harvested mitochondria from cells not exposed to antibiotic cell culture media, results would have been even better. The damage to mitochondria caused by antibiotics and how the effect of antibiotics skews cell culture data has recently been published by us in PLOS one [23]. Therefore, for accurate cell culture data and healthier mitochondria for MOT antibiotics should be avoided.…”

Mitochondrial Toxicity of Azithromycin Results in Aerobic Glycolysis and DNA Damage of Human Mammary Epithelia and Fibroblasts

“…Many cell lines are incubated in media containing antibiotics, such as gentamicin, penicillin or streptomycin, to prevent bacterial contamination. We found that gentamicin addition in routine culture media caused mitochondrial toxicity, increased mitochondrial ROS and changed cell metabolism [36]. There are many papers in the literature on the effects of antibiotics on metabolism of cell lines.…”

Biophysical characterization of melanoma cell phenotype markers during metastatic progression

“…Some antibiotics may also present a significant influence on cell metabolism by inducing mitochondrial dysfunction and oxidative damage in mammalian cells. Taking this into consideration, cell culture studies on metabolism should probably be done in an antibiotic free media (Elliott and Jiang 2019 ). An example is the class of aminoglycoside antibiotics, which consists mainly of gentamicin and geneticin.…”

“…They show diverse usability against prokaryotic and eukaryotic cells, especially due to the fact that cell response depends on its metabolism (Sobiepanek et al 2020c ). The addition of gentamicin in cell culture media inhibits: the mitochondrial membrane potential gradient in the cells, the upregulation of HIF1a gene expression, the glycolytic enzymes and glucose transporters as well as the increase of lactate production, induction of mitochondrial superoxide and the increase in the DNA oxidative damage in the cells (Elliott and Jiang 2019 ). The addition of geneticin (G418) may cause the change of glucose flux from glycolysis towards either the TCA cycle or other biosynthetic pathways in various cell lines like baby hamster kidney (BHK) and Chinese hamster ovary (CHO) cells (Yallop and Svendsen 2001 ; Yallop et al 2003 ).…”