Evaluation of in vitro cytotoxic and genotoxic effects of the 3-(3,4-dihydroxyphenyl)-8-hydroxycoumarin.

A wide variety of natural and synthetic coumarins present therapeutic potential. Therefore, the assessment of their safety for humans is essential. 3-(3,4-Dihydroxyphenyl)-8-hydroxycoumarin is a coumarin derivative with antioxidant properties, among other biological activities. The aim of this study is to evaluate the cytotoxic and genotoxic potential of this molecule on peripheral blood mononuclear cells (PBMC) and human hepatocellular carcinoma cells (HepG2/C3A). The results obtained for the cytotoxicity assays, evaluated by the trypan blue staining assay, using concentrations between 0.1 and 20 µg/mL, showed that there is no decrease in cell viability for both cell lines. The MTT assay showed a significant decrease in the viability of HepG2/C3A cells at the highest concentrations tested, after 48 h, for all the tested concentrations, after 72 h of exposure. Regarding the genotoxic assays, the data obtained by the comet assay and the micronucleus test, up to the tested concentration of 10 µg/mL, do not show significant DNA damage and/or chromosomal mutations, for both cell lines. However, at the highest tested concentration of 20 µg/mL, a small but significant genotoxic effect was observed in PBMC. In view of the observed results, it can be concluded that the 3-(3,4-dihydroxyphenyl)-8-hydroxycoumarin, up to a concentration of 10 µg/mL, does not present genotoxic effects in human cells with and without liver enzymes metabolism. Additional studies with higher concentrations of this molecule need to be performed to address its complete biosafety.

Intestinal epithelial damage due to herbal compounds - an in vitro study.

Intestinal epithelial injury from herbal products has rarely been reported, despite the gut being the first point of contact for oral preparations. These products often consist of multiple herbs, thereby potentially exposing consumers to higher levels of reactive phytochemicals than predicted due to pharmacokinetic interactions. The phytochemical coumarin, found in many herbal products, may be taken in combination with herbal medicines containing astragalosides and atractylenolides, purported cytochrome P450 (CYP) modulators. As herbal use increases, the need to predict interactions in multiple at-risk organ systems is becoming critical. Hence, to determine whether certain herbal preparations containing coumarin may cause damage to the intestinal epithelium, Caco2 cells were exposed to common phytochemicals. Coumarin, astragaloside IV (AST-IV) or atractylenolide I (ATR-I) solutions were exposed to Caco2 cultures in increasing concentrations, individually or combined. Coumarin produced a significant concentration-dependant fall in cell viability that was potentiated when CYP enzymes were induced with rifampicin and incubated with CYP3A4 inhibitor econazole, suggesting a role for other CYP enzymes generating toxic metabolites. ATR-I alone produced no toxicity in uninduced cells but showed significant toxicity in rifampicin-induced cells. ATR-I had no effect on coumarin-induced toxicity. AST-IV was nontoxic alone but produced significant toxicity when combined with nontoxic concentrations of coumarin. The combination of coumarin, ATR-I and AST-IV was significantly toxic, but no synergistic interaction was seen. This investigation was conducted to determine the likelihood for intestinal-based interactions, with the results demonstrating coumarin is potentially toxic to intestinal epithelium, and combinations with other phytochemicals can potentiate this toxicity.

Natural Coumarin Shows Toxicity to Spodoptera litura by Inhibiting Detoxification Enzymes and Glycometabolism.

Coumarin and its derivatives are plant-derived compounds that exhibit potent insecticidal properties. In this study, we found that natural coumarin significantly inhibited the growth and development of Spodoptera litura larvae through toxicological assay. By transcriptomic sequencing, 80 and 45 differentially expressed genes (DEGs) related to detoxification were identified from 0 to 24 h and 24 to 48 h in S. litura after coumarin treatment, respectively. Enzyme activity analysis showed that CYP450 and acetylcholinesterase (AChE) activities significantly decreased at 48 h after coumarin treatment, while glutathione S-transferases (GST) activity increased at 24 h. Silencing of SlCYP324A16 gene by RNA interference significantly increased S. litura larval mortality and decreased individual weight after treatment with coumarin. Additionally, the expression levels of DEGs involved in glycolysis and tricarboxylic acid (TCA) cycle were inhibited at 24 h after coumarin treatment, while their expression levels were upregulated at 48 h. Furthermore, metabonomics analysis identified 391 differential metabolites involved in purine metabolism, amino acid metabolism, and TCA cycle from 0 to 24 h after treated with coumarin and 352 differential metabolites associated with ATP-binding cassette (ABC) transporters and amino acid metabolism. These results provide an in-depth understanding of the toxicological mechanism of coumarin on S. litura.

Time-resolved quantitative phosphoproteomics reveals cellular responses induced by caffeine and coumarin.

Protein phosphorylation is a critical way that cells respond to external signals and environmental stresses. However, the patterns of cellular response to chemicals at different times were largely unknown. Here, we used quantitative phosphoproteomics to analyze the cellular response of kinases and signaling pathways, as well as pattern change of phosphorylated substrates in HepG2 cells that were exposed to caffeine and coumarin for 10 min and 24 h. Comparing the 10 min and 24 h groups, 33 kinases were co-responded and 32 signaling pathways were co-enriched in caffeine treated samples, while 48 kinases and 34 signaling pathways were co-identified in coumarin treated samples. Instead, the percentage of co-identified phosphorylated substrates only accounted for 4.31% and 9.57% between 10 min and 24 h in caffeine and coumarin treated samples, respectively. The results showed that specific chemical exposure led to a bunch of the same kinases and signaling pathways changed in HepG2 cells, while the phosphorylated substrates were different. In addition, it was found that insulin signaling pathway was significantly enriched by both the caffeine and coumarin treatment. The pattern changes in phosphorylation of protein substrates, kinases and signaling pathways with varied chemicals and different time course shed light on the potential mechanism of cellular responses to endless chemical stimulation.

Comparing toxicity of galbanic acid, auraptene and umbelliprenin on adult T-cell leukaemia-lymphoma in normoxia and hypoxia.

Natural coumarins are valuable agents that induce anticancer effects and/or enhance sensitivity to therapeutic modalities. Galbanic acid (GBA), auraptene (AUR) and umbelliprenin (UMB) are coumarins derived from Ferula species with various pharmaceutical activities. The aim of the current research was to compare toxic effects of GBA, AUR, and UMB on human lymphoma cells in normoxia and hypoxia. In this regard, GBA and AUR were extracted from the roots of F. szowitsiana and UMB was derived from the roots of F. persica, all by thin-layer chromatography. MT-2 cells were treated with each agent for 3 consequent periods, while exposed to different O2 contents (21% and 2%). By the end of each treatment, the viability of MT-2 cells was determined by resazurin dye-based colorimetric assay. Obtained results revealed that low doses of GBA (10 and 20 µM) induced significant (p < 0.0001) toxic effects in hypoxia. However, similar toxicity was observed when cells were treated with 40 µM AUR in normoxia and hypoxia. Notably, UMB was the only coumarin that exerted cytotoxic effects in all time points (48, 72 and 96 h) in normoxia and hypoxia, although its concentration was highest (80 µM). In conclusion, this is the first report indicating GBA was the most toxic coumarin against ATL cells in hypoxia, AUR induced similar effects in normoxia and hypoxia, and low toxicity of UMB was stable during the time and different O2 contents. Future studies on other ATL cell lines are recommended to better evaluate the toxic effects of GBA, AUR and UMB in vitro.

Implications for herbal polypharmacy: coumarin-induced hepatotoxicity increased through common herbal phytochemicals astragaloside IV and atractylenolide I.

Hepatotoxicity is a well-known adverse effect of many substances, with toxicity often resulting from interactions of drugs with other drug-like substances. With the increased availability of complementary and alternative medicines, including herbal medicines, the likelihood of adverse interactions between drugs and drug-like substances in herbs increases. However, the impact of potential herb-herb interactions is little understood. To assess the potential of two cytochrome P450 enzyme modulating phytochemicals common to many herbal medicines, atractylenolide I (ATR-I) and astragaloside IV (AST-IV), to interact with coumarin, another phytochemical common in many foods, a hepatocyte function model with a liver carcinoma cell line, HepG2, was exposed to these agents. To determine the effects of cytochrome P450 modulation by these phytochemicals certain cells were induced with rifampicin to induce cytochrome P450. Increasing concentrations of ATR-I combined with a fixed, nontoxic concentration of coumarin (200 µM), demonstrated significant additive interactions. 300 µM ATR-I produced a 31% reduction in cell viability (p < 0.01) with coumarin in rifampicin uninduced cells. In rifampicin-induced cells, ATR-I (100-300 µM) produced a significant reduction in cell viability (p < 0.01) with coumarin (200 µM). AST-IV with fixed coumarin (200 µM) showed 27% toxicity at 300 µM AST-IV in rifampicin uninduced cells (p < 0.05) and 30% toxicity in rifampicin induced cells (p < 0.05). However, when fixed coumarin and AST-IV were combined with increasing concentrations of ATR-I no further significant increase in toxicity was observed (p > 0.05). These results demonstrate the potential toxic interactive capabilities of common traditional Chinese herbal medicine phytochemicals and underline the potential importance of coumarin-mediated toxicity.

Subtle Structural Changes of Dyes Lead to Distinctly Different Fluorescent Behaviors in Cellular Context: The Role of G-Quadruplex DNA Interaction Using Coumarin-Quinazolinone Conjugates as a Case Study.

Fluorogenic organic materials have gained tremendous attention due to their unique properties. However, only a few of them are suitable for bioimaging. Their different behaviors in organic and cellular environments hinder their application in bioimaging. Thus understanding the photoluminescent behaviors of organic materials in a cellular context is particularly important for their rational design. Herein, we describe two coumarin-quinazolinone conjugates: CQ and MeCQ. The high structure similarity makes them possess similar physical and photophysical properties, including bright fluorescence ascribed to the monomer forms in organic solvents and aggregation-caused quenching (ACQ) effect due to self-assembly aggregation in aqueous solution. However, they behave quite differently in cellular context: that is, CQ exhibits bright fluorescence in living cells, while the fluorescence of MeCQ is almost undetectable. The different performance between CQ and MeCQ in living cells is attributed to their different scenario in G-quadruplex (G4) DNA interaction. CQ selectively binds with G4 DNA to recover its fluorescence via aggregation-disaggregation switching in living cells, while MeCQ remained in the aggregate form due to its poor interplay with G4 DNA. Furthermore, CQ is applied as a two-photon fluorescent dye, and its photoswitchable fluorescence capability is exploited for super-resolution imaging of the specific mitochondrial structure in living cells via the STORM technique.

A hypothetical skin sensitisation next generation risk assessment for coumarin in cosmetic products.

Next generation Risk Assessment (NGRA) is an exposure-led, hypothesis-driven approach which integrates new approach methodologies (NAMs) to assure safety without generating animal data. This hypothetical skin allergy risk assessment of two consumer products - face cream containing 0.1% coumarin and deodorant containing 1% coumarin - demonstrates the application of our skin allergy NGRA framework which incorporates our Skin Allergy Risk Assessment (SARA) Model. SARA uses Bayesian statistics to provide a human relevant point of departure and risk metric for a given chemical exposure based upon input data that can include both NAMs and historical in vivo studies. Regardless of whether NAM or in vivo inputs were used, the model predicted that the face cream and deodorant exposures were low and high risk respectively. Using only NAM data resulted in a minor underestimation of risk relative to in vivo. Coumarin is a predicted pro-hapten and consequently, when applying this mechanistic understanding to the selection of NAMs the discordance in relative risk could be minimized. This case study demonstrates how integrating a computational model and generating bespoke NAM data in a weight of evidence framework can build confidence in safety decision making.

Preclinical safety assessment of the crude extract from Sida rhombifolia L. aerial parts in experimental models of acute and repeated-dose 28 days toxicity in rats.

Sida rhombifolia (Malvaceae) is popularly used as a treatment for several pathological conditions; however, there is a lack of studies that identify its compounds and that evaluate comprehensively the safety of its consumption. Therefore, the aim of this study was to determinate the phytochemical constitution of the crude extract of Sida rhombifolia (CESR), and its safety in models of acute and repeated doses (28 days) toxicity. The tested dose for the model of acute toxicity was 2000 mg/kg doses for the repeated dose model were 150, 300 e 600 mg/kg. Hematological, biochemical, histopathological and oxidative markers were investigated. HPLC-DAD-MS analysis evidenced the presence of caffeic acid, coumarin, and rutin. In the acute toxicity model the only altered parameters were tissue ROS, and AST and BUN in serum. As for the repeated dose experiment both hematological and biochemical markers remained within the values of reference for the species. Obtained results demonstrate that the CESR did not present significant toxic effects when administrated orally to male and female rats in acute and repeated doses.

Natural Dibenzo-α-Pyrones: Friends or Foes?

Natural dibenzo-α-pyrones (DAPs) can be viewed from two opposite angles. From one angle, the gastrointestinal metabolites urolithins are regarded as beneficial, while from the other, the emerging mycotoxin alternariol and related fungal metabolites are evaluated critically with regards to potential hazardous effects. Thus, the important question is: can the structural characteristics of DAP subgroups be held responsible for distinct bioactivity patterns? If not, certain toxicological and/or pharmacological aspects of natural DAPs might yet await elucidation. Thus, this review focuses on comparing published data on the two groups of natural DAPs regarding both adverse and beneficial effects on human health. Literature on genotoxic, estrogenic, endocrine-disruptive effects, as well as on the induction of the cellular anti-oxidative defense system, anti-inflammatory properties, the inhibition of kinases, the activation of mitophagy and the induction of autophagy, is gathered and critically reviewed. Indeed, comparing published data suggests similar bioactivity profiles of alternariol and urolithin A. Thus, the current stratification into hazardous Alternaria toxins and healthy urolithins seems debatable. An extrapolation of bioactivities to the other DAP sub-class could serve as a promising base for further research. Conclusively, urolithins should be further evaluated toward high-dose toxicity, while alternariol derivatives could be promising chemicals for the development of therapeutics.

Dihydroisocoumarins and Dihydroisoflavones from the Rhizomes of Dioscorea collettii with Cytotoxic Activity and Structural Revision of 2,2'-Oxybis(1,4-di-tert-butylbenzene).

The investigation of the constituents of the rhizomes of Dioscorea collettii afforded one new dihydroisocoumarin, named (-)-montroumarin (1a), along with five known compounds-montroumarin (1b), 1,1'-oxybis(2,4-di-tert-butylbenzene) (2), (3R)-3'-O-methylviolanone (3a), (3S)-3'-O-methylviolanone (3b), and (RS)-sativanone (4). Their structures were elucidated using extensive spectroscopic methods. To the best of our knowledge, compound 1a is a new enantiomer of compound 1b. The NMR data of compound 2 had been reported but its structure was erroneous. The structure of compound 2 was revised on the basis of a reinterpretation of its NMR data (1D and 2D) and the assignment of the 1H and 13C NMR data was given rightly for the first time. Compounds 3a-4, three dihydroisoflavones, were reported from the Dioscoreaceae family for the first time. The cytotoxic activities of all the compounds were tested against the NCI-H460 cell line. Two dihydroisocoumarins, compounds 1a and 1b, displayed moderate cytotoxic activities, while the other compounds showed no cytotoxicity.

Permeation, stability and acute dermal irritation of miroestrol and deoxymiroestrol from Pueraria candollei var. mirifica crude extract loaded transdermal gels.

In this study, permeation behaviors and chemical stability of miroestrol and deoxymiroestrol from Pueraria candollei var. mirifica (PM), Thai traditional medicine, crude extract containing transdermal gels were firstly evaluated. Three different PM extract containing gels were formulated, including hydroalcoholic and microemulsion gels using carbomer, and silicone gel using silicone elastomer. In vitro permeation through porcine ear skin demonstrated that the flux and 24 h cumulative permeation of miroestrol and deoxymiroestrol were in the order of hydroalcoholic > silicone > microemulsion gels. Hydroalcoholic gel provided the highest partition coefficient from gel onto skin, and thus the skin permeability coefficient. After 24 h permeation, no miroestrol and deoxymiroestrol remained deposited in the skin. Accelerated study using heating-cooling revealed insignificant difference between the remaining percentages of miroestrol and deoxymiroestrol in aqueous and non-aqueous based gels. Long-term stability study showed that miroestrol contents remained constant for 90 d and 30 d under 5 ± 3 °C and 30 ± 2 °C, 75 ± 5%RH, respectively; whereas the percentage of deoxymiroestrol decreased significantly after 30 d storage, irrespective of storage conditions. Acute dermal irritation test on New Zealand White rabbits showed that PM hydroalcoholic gels were non-irritant, with no signs of erythema or oedema.[Figure: see text].

A mitochondria-targeted fluorescent probe for hypochlorite sensing and its application in bioimaging.

A coumarin-diaminomaleonitrile derivative (C-DAN) was designed and prepared. Upon addition of hypochlorite (OCl-), the fluorescence emission peak of C-DAN at 475 nm was gradually enhanced with a rapid response (within 5 s); meanwhile, the color of the solution changed from dark red to bright yellow which can be observed with the naked eye. Furthermore, owing to its advantages including high selectivity, sensitivity, and low toxicity, our C-DAN probe has been applied for detecting OCl- in living cells and zebrafish, indicating it is an ideal tool for biological applications.

Phototoxic risk assessment of dermally-applied chemicals with structural variety based on photoreactivity and skin deposition.

Combined use of photochemical and pharmacokinetic (PK) data for phototoxic risk assessment was previously proposed, and the system provided reliable phototoxic risk predictions of chemicals in same chemical series. This study aimed to verify the feasibility of the screening system for phototoxic risk assessment on dermally-applied chemicals with wide structural diversity, as a first attempt. Photochemical properties of test chemicals, 2-acetonaphthalene, 4'-methylbenzylidene camphor, 6-methylcoumarin, methyl N-methylanthranilate, and sulisobenzone, were evaluated in terms of UV absorption and reactive oxygen species (ROS) generation, and PK profiles of the test chemicals in rat skin were characterized after dermal co-application. All test chemicals showed strong UVA/B absorption with molar extinction coefficients of over 3000 M-1⋅cm-1, and irradiated 2-acetonaphthalene, 6-methylcoumarin, and methyl N-methylanthranilate exhibited significant ROS generation. Dermally-applied 2-acetonaphthalene and 4'-methylbenzylidene camphor indicated high and long-lasting skin deposition compared with the other test chemicals. Based on the photochemical and PK data, 2-acetonaphthalene was predicted to have potent phototoxic risk. The predicted phototoxic risk of the test chemicals by integration of obtained data was mostly consistent with their in vivo phototoxicity observed in rat skin. The screening strategy employing photochemical and PK data would have high prediction capacity and wide applicability for photosafety evaluation of chemicals.

Multi- and transgenerational effects following early-life exposure of zebrafish to permethrin and coumarin 47: Impact on growth, fertility, behavior and lipid metabolism.

Transgenerational effects induced by environmental stressors are a threat to ecosystems and human health. However, there is still limited observation and understanding of the potential of chemicals to influence life outcomes over several generations. In the present study, we investigated the effects of two environmental contaminants, coumarin 47 and permethrin, on exposed zebrafish (F0) and their progeny (F1-F3). Coumarin 47 is commonly found in personal care products and dyes, whereas permethrin is used as a domestic and agricultural pyrethroid insecticide/insect repellent. Zebrafish (F0) were exposed during early development until 28 days post-fertilization and their progeny (F1-F3) were bred unexposed. On one hand, the effects induced by coumarin 47 suggest no multigenerational toxicity. On the other hand, we found that behavior of zebrafish larvae was significantly affected by exposure to permethrin in F1 to F3 generations with some differences depending on the concentration. This suggests persistent alteration of the neural or neuromuscular function. In addition, lipidomic analyses showed that permethrin treatment was partially correlated with lysophosphatidylcholine levels in zebrafish, an important lipid for neurodevelopment. Overall, these results stress out one of the most widely used pyrethroids can trigger long-term, multi- and possibly transgenerational changes in the nervous system of zebrafish. These neurobehavioral changes echo the effects observed under direct exposure to high concentrations of permethrin and therefore call for more research on mechanisms underlying effect inheritance.

Respiratory Toxicity of Azoxystrobin, Pyraclostrobin and Coumoxystrobin on Chlorella vulgaris.

Azoxystrobin (AZ), pyraclostrobin (PYR) and coumoxystrobin (COU) exert negative impacts on Chlorella vulgaris. Thus, in this study, C. vulgaris was used to assess the respiratory toxicity of AZ, PYR and COU by determining the acute toxicity, complex III activity and ATP viability. The 96 h-EC50 values of AZ, PYR and COU for C. vulgaris were 1.85, 2.21 and 1.62 mg/L, respectively. AZ, PYR and COU exerted significant effects on complex III activity and ATP viability after exposure to 0.71, 1.01 and 1.08 mg/L of the fungicides. The binding potentials of AZ, PYR and COU toward ubiquinone were - 10.44, - 9.31 and - 12.98 kcal/mol, respectively, which had adverse effects on amino acids. These results provided new insight into the potential acute respiratory toxicity mechanisms of these strobilurin fungicides in algae.

[Toxicological research and safety consideration of coumarins].

Coumarin is an important class of natural organic compounds, which widely exists in a variety of plants and microorganisms. Coumarins have many biological activities and wide clinical applications, such as anti-tumor, anti-HIV, anti-bacterial, anti-inflammatory, anti-oxidation, anti-coagulation, but they have obvious toxic effects in rodents. It was found that the toxicity of coumarins in different animals and organs was significantly different, and high dose oral administration was more likely to produce toxic reactions. Based on the research and analysis of domestic and foreign literatures in recent 60 years, this paper mainly summarized the hepatotoxicity and pulmonary toxicity induced by coumarins, and probed into their possible mechanisms. It was found that the toxicity of coumarins had metabolic differences and species differences. The liver of rats and lungs of mice were more susceptible to coumarins. Toxic reactions occurred mainly in the second metabolic pathway of coumarin metabolism in vivo. In order to put forward safety considerations and evaluate the impact of coumarin on human body, it was found that coumarin is unlikely to produce hepatotoxicity at normal exposure level. It was also suggested that species differences due to different metabolic patterns in model animals should be carefully considered when assessing coumarin toxicity, in order to provide reference for clinical research and rational use of coumarins and improve the rational use of coumarins.

Colocalization Coefficients of a Target-Switchable Fluorescent Probe Can Serve As an Indicator of Mitochondrial Membrane Potential.

The mitochondrial membrane potential (MMP) definitely reflects mitochondrial function. Thus, it is very essential to found a physical parameter as MMP indicator. At present, available parameters are either fluorescent intensity of monochromatic probes such as rhodamine 123 or a ratio of fluorescent intensity at different wavelengths of dual-color dyes such as JC-1, but the inconvenience in practice as well as serious effect of loading concentrations on experimental results limited their application. To address this concern, herein,we found a reliable and easily obtainable colocalization coefficient (CLC) of a fluorescent probe as new MMP indicator and developed a target switchable fluorescent probe (Mito-Lyso) to attain the aim. Because of its intrinsic nature, Mito-Lyso exclusively stains mitochondria with normal MMP and a subsequent decreasing of MMP results in release of some Mito-Lyso. Importantly, the released Mito-Lyso can reversibly transfer between mitochondria and lysosomes. Thus, CLCs of Mito-Lyso and a commercial lysosomal probe (NIR-Lyso) can be MMP-dependent. CLCs gradually increased from 0.20 to 0.8 with the decreasing of MMP and then returned to 0.3 with the recovering of MMP, which better proves that the CLC is a valuable MMP indicator. Furthermore, both the design principle and action mechanism of Mito-Lyso has been explained in detail for the development of this type of probes.

A near-infrared and two-photon dual-mode fluorescent probe for the colorimetric monitoring of SO2in vitro and in vivo.

SO2 has been recently identified as an essential gas messenger followed by NO, CO and H2S. However, abnormal concentrations of SO2 in our bodies can cause many diseases. Thus, the real-time monitoring of SO2 to well define the generation, physiological and pathological functions of SO2 is urgently needed. In this work, we developed a novel SO2 fluorescent probe on the basis of the conjugation of semi-cyanine and coumarin derivate dyes with superior features, such as near-infrared (NIR) and two-photon dual-mode monitoring, a large Stokes shift (175 nm), ultrafast response towards SO2 (within 10 s), high selectivity and photostability. Furthermore, this probe could sense SO2 by dual colorimetric and fluorescence means. In biological imaging, the probe was able to trace exogenous and endogenous SO2 in living cells, mitochondria, E. coli, zebrafish and mice under an NIR and two-photon dual-mode. These results demonstrated that the probe has strong potential for studying the physiological and pathological functions of SO2in vitro and in vivo.

An in cellulo-activated multicolor cell labeling approach used to image dying cell clearance.

Dying cell clearance is critical for myriad biological processes such as tissue homeostasis. We herein report an enzyme-activated fluorescence cell labeling approach and its use for multicolor imaging of dying cell clearance. Diacetylated 4-hydroxymandelic acid (DHA)-conjugated dyes give rise to reactive quinone methides upon deacetylation in live cells, which in turn covalently labels cellular proteins. With partner cells tagged with distinct fluorescence, apoptotic cell clearance by Raw 264.7 macrophages and epithelial HeLa cells was captured by confocal microscopy, showing the potential of DHA-based cell labeling for investigating cell-cell interactions.