Hello, and welcome to the Bio-Rad Antibodies podcast.
I am Annalise Barnette, Technical Writer within the antibody team. Today I will be talking about bromodexoyuridine, also known as BrdU, a neurogenesis lab classic with a dark twist. The story includes three sections focusing on the history of BrdU as a neurogenesis research tool, latest scientific findings, and experimental control tips aimed at assisting you with your BrdU labeling experiments.
Did you know that BrdU played a major role in revolutionizing neurogenesis research? It is even today a popular reagent used by neuroscientists. To understand its rise and how it came to be so commonplace in neurogenesis research, we need to go back in time to the 1960s.
Up until the mid-1960s, scientists believed that neurogenesis, which is the process of developing new neurons, only occurred in early human development. Essentially, the working model was that new neurons could not be generated in the adult brain. In 1965, biologists Joseph Altman and Gopal Das of the Massachusetts Institute of Technology provided the first evidence that this hypothesis might be incorrect in a seminal study published in the Journal of Comparative Neurology. Using adult rat models, they demonstrated the synthesis of new neurons in the hippocampus using radioactively labeled tritiated thymidine. This radiolabeled thymidine is incorporated during DNA replication instead of thymidine in the S phase of cell growth. Autoradiography is then used to detect the proliferating cells. However, while Altman and Das conclusively showed that adult neurogenesis does occur in rats, scientists were still unsure about the situation in humans.
Another method or chemical, other than radiolabeled thymidine would be more suitable to answer this question in humans due to the general complications associated with handling radiolabeled substances and the 3-12 week duration for developing autoradiographs.
Fast forward to the 1990s when scientists began using BrdU, a non-radioactive thymidine analog, to label newly synthesized DNA. BrdU can be supplemented in cell growth media, and can be administered in vivo through different methods such as orally via the drinking water as well as through intraperitoneal injection*1 and 2. Similar to radioactively labeled thymidine, BrdU is incorporated into newly synthesized DNA instead of the DNA base thymine during replication. The structure of BrdU differs from thymidine in that a bromine atom replaces the methyl group at carbon number 5*3 and *4. The incorporated BrdU can in turn be detected with antibodies that specifically bind to BrdU but not to thymidine thereby allowing the identification of proliferating cells*5.
At this time in the clinic, BrdU was already well established in cancer research to estimate the rate of tumor growth in cancer patients. Patients were administered BrdU via the blood stream, where it would incorporate into growing tumors*6. A tumor biopsy is then taken, and anti-BrdU antibodies are used to detect cells that have incorporated BrdU into their DNA. The ratio of BrdU positive cells to total tumor cells measured over time provided an estimate of how fast the tumor is growing. Professor Fred Gage, a prominent neuroscientist at Salk Research Institute for Biological Studies was aware of this application of BrdU in cancer detection, and reasoned that BrdU would also be incorporated into the brain cells of patients administered the drug.
He hypothesized that BrdU would be incorporated into new but not old brain cells, and could therefore be used to birthdate individual neurons and ultimately determine whether neurogenesis occurs in the adult human brain.
To test this hypothesis, Professor Gage and a research team including physician scientists at the Sahlgrenska University Hospital in Sweden acquired post mortem brain samples from adult cancer patients who were previously administered BrdU.
Using immunofluorescence labeling of BrdU and neuronal markers such as calbindin and neuron specific enolase, they determined that neurogenesis did in fact occur in adults.
This finding revolutionized the neurogenesis research field, and it has been described as one of the greatest scientific discoveries of the 20th century. The finding was published in the Nature Medicine journal in 1998 and has been cited an impressive 5797 times*7.
This highlights why I have introduced BrdU as a lab classic. The thymidine analog truly played a significant role in defining adult neurogenesis, and has made significant contributions to advancing neurogenesis research.
However, like many research tools and assays, BrdU incorporation has side effects and limitations. For example, although the acute toxic effects of BrdU on humans appears to be limited, its incorporation may lead to subacute and chronic effects such as anemia, skin lesions, and thrombocytopenia as well as induce birth defects and genetic mutations in exposed pregnant women.
Interestingly, the off-target effects of BrdU have been known since the 1970s. In 1977, Professor Barry Goz published a study titled “The effects of incorporation of 5-halogenated deoxyuridines into the DNA of eukaryotic cells” in the journal Pharmacological Reviews. The paper reported that BrdU and similar halogenated thymidine analogs can induce structural changes in the conformation of DNA, which could have harmful effects such as altering cell cycle progression.
Despite these findings, early studies on adult neurogenesis did not consider these possible side effects of BrdU.
Perhaps due to advances in neural stem cell research, the impact of BrdU in various cell types and research models re-emerged as an area of concern by the 2000s.
Recent studies by the research group of Professor Ludwig Aigner and lead researcher Bernadette Lehner at the Paracelsus Medical University Salzburg in Austria show that BrdU negatively impacts cell proliferation as well as the differentiation of adult neural progenitor cells.
Another study by Leonid Schneider and Fabrizio d’Adda di Fagagna of the IFOM foundation FIRC Institute of Molecular Oncology in Milan published in 2012 further highlighted unwanted side effects of BrdU incorporation. The study reported that BrdU exposure resulted in loss of global DNA methylation in neural stem cells.
This was an important discovery since methylation tends to suppress gene expression. Therefore, BrdU mediated DNA demethylation could initiate unwanted systemic outcomes. The authors also determined that BrdU resulted in the loss of stem cell characteristics through the reduced expression of key stem cell markers such as the transcription factors Sox2 and paired boxed protein 6.
Importantly, however, Schneider and d’Adda di Fagagna also reported that BrdU did not induce significant toxicity in neural stem cells as less than 10% of BrdU treated cells demonstrated apoptotic DNA fragmentation.
Although Schneider and d’Adda di Fagagna did not observe significant toxicity with BrdU in neural stem cells, another study by Pasko Rakic and Alvaro Duque of Yale University School of Medicine demonstrated that BrdU incorporation into the genes of adult Macaque monkey brain cells induced significant toxicity as indicated by a reduction in the number of labeled cells and cell survival compared to incorporation of tritiated thymidine alone.
The difference in the structures of thymidine and BrdU has been proposed as a contributing factor for the opposing effects of BrdU and thymidine. BrdU differs from thymidine with the introduction of the bromine atom in BrdU. It is speculated that the incorporation of bromine into the genome could induce mutations and the phenotypic changes observed in BrdU treated cells in the studies discussed.
These studies highlight the two sides of BrdU, as a lab classic and as a chemical that even when properly dosed can induce off-target effects. They also tell a cautionary tale about how researchers should interpret results from BrdU incorporation experiments and emphasize the importance of including appropriate controls in your experimental design when using BrdU incorporation as a readout for cell proliferation.
To decide on the most suitable experimental controls, here are our top five tips for controlling BrdU labeling experiments
Tip number 1 – It’s all in the solvent
Always include samples treated with the solvent in which you reconstituted the BrdU as a negative control. For example, if you dissolved or diluted the BrdU in ethanol, then your experiment should include samples treated with ethanol only to determine the impact of the solvent.
Tip number 2 – Titrate to succeed
Titrate the BrdU reagent and antibody to determine the optimal concentration for your specific samples as recommended concentrations may not be suitable.
Tip number 3 – Time matters
Similar to titrating your BrdU reagent and antibody, we also recommend that you optimize the BrdU incubation time for your specific samples.
Tip number 4 – Don’t forget about epigenetics
For determining the effect of BrdU on loss of DNA methylation in your samples, the methylation inhibitor drug 5-azacytidine may be used as a positive control. For more information about this topic, please consult Schneider and d’Adda di Fagagna’s 2012 study.
And last but not least, Tip number 5, specifically for studies involving neural stem cells,
– Do a differentiation check
Fetal calf serum can be used as a positive control when determining the impact of BrdU on cell differentiation in neural stem cell studies. This control allows you to monitor differentiation of neural stem cells compared to cells cultured under serum free conditions since exposure to growth factor rich fetal calf serum leads to neural stem cell differentiation into astrocytes.
To go over these tips again, and to find references to the studies mentioned in this podcast, please visit our two part blog series on BrdU in neurogenesis research at bio-rad-antibodies.com/blog/brdu1 and /brdu2.
Also, check out Bio-Rad’s Mouse Anti-BrdU Antibody, clone Bu20a, which has been cited in neurogenesis studies and is suitable for applications such as flow cytometry and immunocytochemistry.
You can learn more about this antibody on our website bio-rad-antibodies.com by typing in the product code MCA2483 in the search bar. If you are interested in BrdU antibody protocols you can go to bio-rad-antibodies.com/protocols to find out more.
That’s it for this BrdU podcast.
If you enjoyed this story and are interested in other neuroscience related posts, feel free to check out our blog post on autophagy and neurodegeneration at bio-rad-antibodies.com/blog/autophagy. Other topics featured in our blog include cancer, immunology and veterinary research, as well as antibody application tips.
Thanks for listening.
End of Podcast: 12:23
*1 Tesfaigzi Y et al. (2004). DNA synthesis and Bcl-2 expression during development of mucous cell metaplasia in airway epithelium of rats exposed to LPS. Am J Physiol Lung Cell Mol Physiol 286, L268-2674.
*2 Moser VC et al. (2004). Neurotoxicity produced by dibromoacetic acid in drinking water of rats. Toxicol Sci 79, 112-122.
*3 Young DW et al. (1969). The crystal and molecular structure of thymidine. Acta Cryst B25, 1423-1432.
*4 Kolb B et al. (1999). Embryonic and postnatal injections of bromodeoxyuridine produce age-dependent morphological and behavioral abnormalities. J Neurosci 19, 2337-2346.
*5 Aten JA et al. (1992). DNA double labelling with IdUrd and CldUrd for spatial and temporal analysis of cell proliferation and DNA replication. Histochem J 24, 251-259.
*6 Waldman FM et al. (1988). Clinical applications of the bromodeoxyuridine/DNA assay. Cytometry Suppl 3, 65-72.
*7 Google Scholar (2017). Search for “Neurogenesis in the adult human hippocampus. Nat Med 4, 1313-1317”, scholar.google.co.uk/scholar?hl=en&q=Neurogenesis+in+the+adult+human+hippocampus.+Nat+Med+4%2C+1313-1317&btnG=&as_sdt=1%2C5&as_sdtp=, accessed June 2, 2017.