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High-throughput screening of mitochondrial toxicity

By Mamta Behl
September 2010

Craig Beeson, Ph.D.
"RPTCs cultured under standard conditions in which they are stationary with glucose medium have basal oxygen consumption rates 100-fold lower than those for RPTCs cultured under optimized conditions in which they were shaken with lactate as media," explained Beeson. (Photo courtesy of Steve McCaw)

Scott Auerbach, Ph.D.
Auerbach, above, moderated active discussion on potential upstream energy stress depletion signals which, in turn, may alter the ATP/AMP ratio in the mitochondria. (Photo courtesy of Steve McCaw)

Ray Tice, Ph.D.
Beeson was the latest in a series of speakers Tice has invited to NIEHS whose research has direct bearing on the high-throughput screening initiative. "Given our interest in identifying mitochondrial toxicants, we are very pleased that Dr. Beeson found the time in his busy schedule to present his research findings and meet with key NTP staff," Tice said. (Photo courtesy of Steve McCaw)

The NTP Biomolecular Screening Branch (BSB) hosted a talk by Craig Beeson, Ph.D., July 30 on "High-Throughput Respirometric Assay for Mitochondrial Biogenesis and Toxicity" as part of the NTP High-Throughput Screening Initiative (http://ntp.niehs.nih.gov/?objectid=05F80E15-F1F6-975E-77DDEDBDF3B941CD). Beeson described the results he has obtained using the Seahorse Bioscience XF analyzer for real-time measurement of mitochondrial function in adapted primary cultures of renal proximal tubular cells.

As an associate professor in the Department of Pharmaceutical and Biomedical Sciences and the director of the Metabolomics Core and Drug Design and Synthesis Core at the Medical University of South Carolina, Beeson (http://academicdepartments.musc.edu/psci/faculty/beeson.html) Exit NIEHS focuses on the biochemical networks responsible for the regulation of energy metabolism and cellular proliferation and their potential applications in the areas of predictive toxicology and drug discovery.

Mitochondrial toxicity assays in the Tox21 partnership

"The BSB is evaluating mitochondrial toxicity as a potential toxicity pathway using in vitro studies as part of its high-throughput screening initiative," NTP molecular toxicologist and lecture host Scott Auerbach, Ph.D., said of the branch's decision to invite Beeson to speak at NIEHS.

The NTP, NIH Chemical Genomics Center (NCGC), and U.S. Environmental Protection Agency (EPA), along with their most recent partner, the U.S. Food and Drug Administration (FDA), are striving to advance the state of toxicity testing in the 21st century (http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2721892/) Exit NIEHS through a consortium known as Tox21. The consortium is seeking to identify new mechanisms of chemical activity in cells, effectively prioritize the backlog of untested chemicals for more extensive evaluations, and develop better predictive models of human response to such toxicants as industrial and environmental chemicals and drugs.

Overcoming limitations in current mitochondrial cell-based assays

Although many drugs and chemicals are mitochondrial toxicants, according to Beeson, assessing mitochondrial function has posed a challenge for investigators, because there is no direct high-throughput assay for mitochondrial function. In addition, the current cell-based models of mitochondrial toxicity are inadequate, because immortalized cell lines have lost differentiated function and are highly glycolytic, with minimal aerobic metabolism and altered mitochondrial physiology.

Beeson said that although roughly half of the drugs with FDA black box warnings for hepatotoxicity or cardiotoxicity also have documented mitochondrial effects, the same level of attention has not been given for nephrotoxicity, despite the frequency of loss in renal function due to adverse drug effects and xenobiotic exposure.

To address this issue, Beeson's group adapted primary renal proximal tubular cells (RPTCs) as a model to study mitochondrial loss following oxidative injury. "In contrast to immortalized cells, RPTCs are robust primary cells, which are completely differentiated and polarized with good brush border enzyme activity and sodium-dependent glucose transport," Beeson explained. "The RPTCs are somewhat unique among differentiated tissues in that they have some capacity for repair and regeneration," he added. Beeson described the use of multiple endpoints, such as basal and uncoupled respiration rates, and provided examples of compounds that induce increased uncoupled respiration, confirming that it is a biomarker of mitochondrial biogenesis.

Collaborative efforts for a promising future

Beeson provided examples of loss of respiratory capacity of the mitochondria in degenerative diseases such as retinitis pigmentosa and macular degeneration, as well as the development of novel molecular models, known as pharmacophores, that serve as computational templates for discovering drugs to use in their treatment. "Our strategy involves looking at protection of the uncoupled rate for new molecules in the prevention of these disorders," concluded Beeson.

The lively discussion enabled researchers at the NTP to identify several areas of mutual interest where collaborations might be possible. "One area of immediate interest is in applying Dr. Beeson's mitochondrial toxiphore descriptor model to the Tox21 10K-compound library that will be tested for mitochondrial toxicity in high-throughput mode at the NIH Chemical Genomics Center," said BSB Chief Ray Tice, Ph.D. (http://www.niehs.nih.gov/research/atniehs/labs/bmsb)

Citation: Beeson CC, Beeson GC, Schnellmann RG(http://www.ncbi.nlm.nih.gov/pubmed/20465991) Exit NIEHS. 2010. A high-throughput respirometric assay for mitochondrial biogenesis and toxicity. Anal Biochem 404(1):75-81. Epub ahead of print.

(Mamta Behl, Ph.D., is a research fellow in the NTP Toxicology Branch.)

The Seahorse technology for high-throughput screening

The limitation in the availability of high-throughput real-time assays to assess mitochondrial function poses additional restrictions in the screening of mitochondrial toxicity. The existing high-throughput assays often use alterations in mitochondrial gene expression thereby leading to false negatives and false positives due to lack of correlation between messenger RNA and biogenesis(http://en.wikipedia.org/wiki/Mitochondrial_biogenesis) Exit NIEHS. To circumvent this problem, the Seahorse Biosciences XF(http://www.seahorsebio.com/products/xf-analyzers/how-xf-works.php) Exit NIEHS analyzer was recently introduced. Using this technique, researchers can measure shifts between aerobic respiration and glycolysis as a result of genetic, pharmacologic, or physiologic manipulation simultaneously and non-invasively using a medium- to high-throughput platform.

Beeson's laboratory utilized the Seahorse technology to demonstrate that known toxicants produced concentration-dependent changes in respiratory capacity. "Mitochondrial damage is a phenotypic stress response and is detected prior to cell death, suggesting its role in capturing chronic toxicity," he explained.

According to Auerbach, investigators at NIEHS can access the Seahorse analyzer through one of NTP's contractors.



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