Erythrosin B: From Food Dye to Multifunctional Biological Tool

Erythrosin B, also called as eosin B, is a xanthene dye and has been used as a dye in fluorescence lifetime imaging. It belongs to the family of fluorescein dyes.Erythrosin B is an artificial food dye that induces hyperkinesis when swallowed by susceptible children.It plays a major role in inhibiting dopamine uptake.

Application of a non-hazardous vital dye for cell counting

EB, also known as erythrosine or Red No. 3, is primarily used for food coloring. Although Erythrosin B has already been introduced as a vital dye, it is not widely used to count viable cells manually or with automated instruments. Because biosafety is a growing concern, the use of EB with automated instruments is an option to lessen consumption of TB. Here we report, for the first time, the use of Erythrosin B for vital staining of three cell lines for automatic counting and demonstrate that the use of safer alternative EB avoids the toxic side effect of trypan blue exposure on mammalian cells. Various vital dyes, including trypan blue (TB), methylene blue, erythrosin B (EB), nigrosine, eosin, safranin, propidium iodide, and 7-aminoactinomycin D, have been introduced to count viable cells. Among these, TB is widely used for viable cell counting with bright-field optics. In addition, most of automated cell counters without fluorescence optics are optimized for viable cell counting based on TB dye exclusion.[1]

Recent advances in automated cell counters enable us to count cells more easily with consistency. However, the wide use of the traditional vital dye trypan blue (TB) raises environmental and health concerns due to its potential teratogenic effects. To avoid this chemical hazard, it is of importance to introduce an alternative non-hazardous vital dye that is compatible with automated cell counters. Erythrosin B is a vital dye that is impermeable to biological membranes and is used as a food additive. Similarly to TB, EB stains only nonviable cells with disintegrated membranes. However, Erythrosin B is less popular than TB and is seldom used with automated cell counters. We found that cell counting accuracy with EB was comparable to that with TB. Erythrosin B was found to be an effective dye for accurate counting of cells with different viabilities across three different automated cell counters. In contrast to TB, EB was less toxic to cultured HL-60 cells during the cell counting process. These results indicate that replacing TB with EB for use with automated cell counters will significantly reduce the hazardous risk while producing comparable results.

Erythrosin B as a potent and broad-spectrum orthosteric inhibitor

Many flaviviruses, such as Zika virus (ZIKV), Dengue virus (DENV1-4) and yellow fever virus (YFV), are significant human pathogens. Infection with ZIKV, an emerging mosquito-borne flavivirus, is associated with increased risk of microcephaly in newborns and Guillain-Barré syndrome and other complications in adults. Currently, specific therapy does not exist for any flavivirus infections. In this study, we found that erythrosin B, an FDA-approved food additive, is a potent inhibitor for flaviviruses, including ZIKV and DENV2. Erythrosin B was found to inhibit the DENV2 and ZIKV NS2B-NS3 proteases with IC50 in low micromolar range, via a non-competitive mechanism. Erythrosin B can significantly reduce titers of representative flaviviruses, DENV2, ZIKV, YFV, JEV, and WNV, with micromolar potency and with excellent cytotoxicity profile. Erythrosin B can also inhibit ZIKV replication in ZIKV-relevant human placental and neural progenitor cells. As a pregnancy category B food additive, erythrosin B may represent a promising and easily developed therapy for management of infections by ZIKV and other flaviviruses.[2]

In this study, we identified erythrosin B as a candidate orthosteric inhibitor blocking interactions between flavivirus NS2B and NS3. Erythrosin B, also known as Red No. 3, erythrosine, or food additive E127, is an organoiodine compound primarily used in food coloring in many countries. In this study, we show for the first time that EB not only has novel anti-flaviviral activities but also has novel mechanism of action. Erythrosin B has been used as a dye in foods, drugs and cosmetics since its approval by the US Department of Agriculture in 1907. Erythrosin B is a substance within the FDA pregnancy category B that has failed to demonstrate a risk to the fetus in animal reproduction studies, although there are no adequate and well-controlled studies in pregnant women. Our studies showed that erythrosin B is a potent ZIKV and DENV2 inhibitor with a novel mechanism by inhibition of interactions between viral protease components NS2B and NS3. As a category B substance, erythrosin B has low acute oral toxicity, and does not appear to cause reproductive or developmental toxicity.

In a summary, our results demonstrate that flaviviral proteases can be targeted at an orthosteric site and provide a starting point for further development of orthosteric inhibitors; and we found a potent inhibitor, erythrosin B, which has excellent safety profile. Future in vivo efficacy and preclinical studies of erythrosin B will be essential to evaluate the pharmacokinetics, bioavailability, in vivo efficacy, dosage, and potential benefits and risks for patients infected with flaviviruses, including ZIKV.

Erythrosin B: A Versatile Colorimetric and Fluorescent Vital Dye for Bacteria

Rapidly assaying cell viability for diverse bacteria species is not always straightforward. In eukaryotes, cell viability is often determined using colorimetric dyes; however, such dyes have not been identified for bacteria. We screened different dyes and found that erythrosin B (EB), a visibly red dye with fluorescent properties, functions as a vital dye for many Gram-positive and -negative bacteria. EB worked at a similar concentration for all bacteria studied and incubations were as short as 5 min. Given EB's spectral properties, diverse experimental approaches are possible to rapidly visualize and/or quantitate dead bacterial cells in a population. As the first broadly applicable colorimetric viability dye for bacteria, Erythrosin B provides a cost-effective alternative for researchers in academia and industry. In eukaryotes, EB is known to function as a membrane-exclusion vital dye such that dead cells with compromised plasma membranes are dye positive. Despite certain advantages over other traditional colorimetric membrane-exclusion dyes, the application of Erythrosin B as a vital dye in eukaryotes is not as widespread as other dyes with a comparable mechanism of action (e.g., trypan blue).[3]

Erythrosin B is the first broadly applicable colorimetric vital dye for assaying viability in both Gram-positive and -negative bacteria. EB's spectral properties allow for its use in diverse experimental approaches with low-, medium- and high-throughput assays. This versatile bacterial vital dye could significantly reduce the cost and time associated with conducting viability studies on diverse bacterial species as well as experiments involving multiple species. Optimization of dye concentrations for different species is likely unnecessary, as this is an individual dye that works well at a single concentration. Unlike existing methods, EB's colorimetric properties allow for rapid, straightforward live/dead determination by bright-field microscopy with a single dye. Importantly, its sensitivity allows for low rates of death to be accurately quantified. Erythrosin B has diverse commercial and academic applications such as studies screening for new antimicrobial compounds and determining the concentration of these needed for bactericidal effects. EB staining may be particularly useful for field studies looking at diverse microbiomes, in field hospitals in the developing world where equipment and resources are limited, and in studying or identifying pathogenic bacteria that have entered a viable but nonculturable state. Last, while this study examined Erythrosin B 's utility as an individual dye, future studies may identify important uses for it in bacterial viability studies as part of dye mixtures.

References

[1]Kim SI, Kim HJ, Lee HJ, Lee K, Hong D, Lim H, Cho K, Jung N, Yi YW. Application of a non-hazardous vital dye for cell counting with automated cell counters. Anal Biochem. 2016 Jan 1;492:8-12.

[2]Li Z, Sakamuru S, Huang R, Brecher M, Koetzner CA, Zhang J, Chen H, Qin CF, Zhang QY, Zhou J, Kramer LD, Xia M, Li H. Erythrosin B is a potent and broad-spectrum orthosteric inhibitor of the flavivirus NS2B-NS3 protease. Antiviral Res. 2018 Feb;150:217-225.

[3]Franke JD, Braverman AL, Cunningham AM, Eberhard EE, Perry GA. Erythrosin B: a versatile colorimetric and fluorescent vital dye for bacteria. Biotechniques. 2020 Jan;68(1):7-13.