Bright-field Microscopy Research Articles

Comparison of bull sperm morphology evaluation methods under field conditions

Sperm morphological assessment is a critical component in bull breeding soundness evaluations. Although sperm morphology is an important parameter to identify subfertile from fertile bulls, the evaluation can be biased because sperm staining methods used under field conditions may not be exact due to various factors, including artifacts. Objective was to compare 2 sperm morphological evaluation methods. Prebreeding season ejaculates of 1,216 Angus cross bulls collected via electroejaculation were evaluated. For each bull, an unstained (UNS) and an eosin-nigrosin stained (ENS) semen smear were viewed under a phase contrast microscope and a brightfield microscope with an oil immersion lens, both at 1,000 × magnification. Normal and percentage of abnormal sperm were identified by counting 200 sperm. Inter-rater agreements between 2 clinicians for the percentage of abnormal sperm determination and its categories were very good (ENS method, r = 0.84 – 0.96; UNS method, r = 0.76 – 0.96; p < 0.01). No differences (p > 0.1) were observed for abnormal sperm percentage determination and its categories between 2 methods. Correlation was very good between 2 methods for total abnormal sperm percentage determination (r = 0.91; p < 0.01) and its categories (r = 0.84 – 0.96; p < 0.05). Additionally, 60 ejaculates were evaluated by triple stain (TS), ENS, and UNS methods. Agreements between TS (percentage of sperm with damaged membrane) and ENS (percentage of abnormal sperm) and between TS and UNS methods were moderate (r = 0.58; p < 0.05) and fair (r = 0.43; p < 0.05), respectively. Based on our findings, either technique can be used for bull sperm morphological evaluation under field conditions. Considering the ease of semen smear preparation, the UNS method can be a viable alternative to the ENS method.

Myenteric Plexus Immune Cell Infiltrations and Neurotransmitter Expression in Crohn's Disease and Ulcerative Colitis.

Pain is a cardinal symptom in inflammatory bowel disease [IBD]. An important structure in the transduction of pain signalling is the myenteric plexus [MP]. Nevertheless, IBD-associated infiltration of the MP by immune cells lacks in-depth characterisation. Herein, we decipher intra- and periganglionic immune cell infiltrations in Crohn´s disease [CD] and ulcerative colitis [UC] and provide a comparison with murine models of colitis. Full wall specimens of surgical colon resections served to examine immune cell populations by either conventional immuno-histochemistry or immunofluorescence followed by either bright field or confocal microscopy. Results were compared with equivalent examinations in various murine models of intestinal inflammation. Whereas the MP morphology was not significantly altered in IBD, we identified intraganglionic IBD-specific B cell- and monocyte-dominant cell infiltrations in CD. In contrast, UC-MPs were infiltrated by CD8+ T cells and revealed a higher extent of ganglionic cell apoptosis. With regard to the murine models of intestinal inflammation, the chronic dextran sulphate sodium [DSS]-induced colitis model reflected CD [and to a lesser extent UC] best, as it also showed increased monocytic infiltration as well as a modest B cell and CD8+ T cell infiltration. In CD, MPs were infiltrated by B cells and monocytes. In UC, mostly CD8+ cytotoxic T cells were found. The chronic DSS-induced colitis in the mouse model reflected best the MP-immune cell infiltrations representative for IBD.

Heth longquani sp. n.—(Rhigonematomorpha: Hethidae)—obligate commensalistic nematode of camballoid millipede (Spirostreptida: Cambalopsidae) from Viet Nam

A new nematode species, Heth longquani sp. n. (Hethidae, Ransonematoidea, Rhigonematomorpha, Rhabditida) is described from the camballoid millipede, Chonecambala crassicauda from Thanh Hóa province, Viet Nam. The morphological features of this new species were studied with brightfield and scanning electron microscopies. Partial sequences of LSU rDNA and Cox1 mtDNA are provided. The phylogeny of the nematodes of the genus Heth Cobb, 1898 is discussed.

Sex-specific impact of maternal obesity on fetal placental macrophages and cord blood triglycerides

IntroductionMaternal obesity is associated with increased risk of offspring obesity and cardiometabolic disease. Altered fetoplacental immune programming is a potential candidate mechanism. Differences in fetal placental macrophages, or Hofbauer cells (HBCs), have been observed in maternal obesity, and lipid metabolism is a key function of resident macrophages that may be deranged in inflammation/immune activation. We sought to test the following hypotheses: 1) maternal obesity is associated with altered HBC density and phenotype in the term placenta and 2) obesity-associated HBC changes are associated with altered placental lipid transport to the fetus. The impact of fetal sex was evaluated in all experiments. MethodsWe quantified the density and morphology of CD163-and CD68-positive HBCs in placental villi in 34 full-term pregnancies undergoing cesarean delivery (N = 15, maternal BMI ≥30 kg/m2; N = 19, BMI <30 kg/m2). Antibody-positive cells in terminal villi were detected and cell size and circularity analyzed using a semi-automated method for thresholding of bright-field microscopy images (ImageJ). Placental expression of lipid transporter genes was quantified using RTqPCR, and cord plasma triglycerides (TGs) were profiled using modified Wahlefeld method. The impact of maternal obesity and fetal sex on HBC features, lipid transporters, and cord TGs were evaluated by two-way ANOVA. Spearman correlations of cord TGs, HBC metrics and gene expression levels were calculated. ResultsMaternal obesity was associated with significantly increased density of HBCs, with male placentas most affected (fetal sex by maternal obesity interaction p = 0.04). CD163+ HBCs were larger and rounder in obesity-exposed male placentas. Sexually dimorphic expression of placental FATP4, FATP6, FABPPM, AMPKB1 and AMPKG and cord TGs was noted in maternal obesity, such that levels were higher in males and lower in females relative to sex-matched controls. Cord TGs were positively correlated with HBC density and FATP1 expression. DiscussionMaternal obesity is associated with sex-specific alterations in HBC density and placental lipid transporter expression, which may impact umbilical cord blood TG levels and offspring cardiometabolic programming.

Direct imaging of local atomic structures in zeolite using optimum bright-field scanning transmission electron microscopy.

Zeolites are used in industries as catalysts, ion exchangers, and molecular sieves because of their unique porous atomic structures. However, direct observation of zeolitic local atomic structures via electron microscopy is difficult owing to low electron irradiation resistance. Subsequently, their fundamental structure-property relationships remain unclear. A low-electron-dose imaging technique, optimum bright-field scanning transmission electron microscopy (OBF STEM), has recently been developed. It reconstructs images with a high signal-to-noise ratio and a dose efficiency approximately two orders of magnitude higher than that of conventional methods. Here, we performed low-dose atomic-resolution OBF STEM observations of two types of zeolite, effectively visualizing all atomic sites in their frameworks. In addition, we visualized the complex local atomic structure of the twin boundaries in a faujasite (FAU)-type zeolite and Na+ ions with low occupancy in eight-membered rings in a Na-Linde Type A (LTA) zeolite. The results of this study facilitate the characterization of local atomic structures in many electron beam-sensitive materials.

Quantifying the influence of heating and resting on the bitumen microstructure

The investigation of the bitumen microstructure can provide an alternative approach to characterise the material. However, various factors prior to its analysis need to be considered, before being able to come to a meaningful conclusion. This involves the identification of factors like heating time and temperature as well as resting time and how it can affect the resulting microstructure. Furthermore, the need for quantifying the microstructure is sought after. Thus, the goal of this study is to address these parameters and quantify their respective influence via optical inverse dark- and brightfield microscopy, subsequent particle analysis of the darkfield images and partial link to infrared spectroscopy. The results show that optical microscopy in combination with particle analysis from dark field images can be used for quantification of the microstructure and even differentiate binders from different crude oil sources. However, small binder quantities, typically used for microscopy, can oxidize rapidly. Thus, a heating time of 30 – 60 s at a temperature of 150 °C was deemed sufficient for these unmodified binders. In addition to that the microstructure becomes stable after 30 min of resting, making binder characterisation via microscopy much more convenient. For imaging analysis an overall area covered by bee structure enables universal characterisation and comparison, while providing good differentiation between binder A (10%), B (2%) and C (16%). These findings should set the basis for future research, where microscopy can be used to identify and tackle important questions related to changes caused by ageing, modification with polymers, additives, filler or rejuvenators.

The role of microbe-microplastic associations in marine Nematode feeding behaviors

Fauna across many taxa and trophic levels have been shown to consume microplastics (MPs) in experiments, providing evidence that supports field-based gut content assessments. Multiple explanations exist regarding why fauna consume MPs, one of which posits that microbial growth on MPs may facilitate faunal ingestion. However, laboratory assessments on the reasons why MPs are consumed remain limited. Here, we assessed if the presence of microbes on MPs altered marine nematode feeding behaviors across current and potential future concentrations of MPs in a local system. We used a microcosm experiment in which field-collected sediment was spiked with bacterially treated or untreated fluorescent plastic microbeads (1.0–5.0 μm) in concentrations of 102, 104, and 106 per microcosm, representing local and potential future concentrations of MPs. Ingestion by the dominant interstitial fauna was investigated after 0, 3, and 7 days using bright field microscopy. Nematodes were the only fauna across microcosms that consumed MPs, but this consumption was variable and there were no apparent trends across exposure time, bacterial treatment, or MP concentration. There were also no genera- or feeding-type-specific trends in the number of MPs consumed, though four of the top five nematode genera that consumed MPs were pollution-tolerant genera. Our study demonstrates that microbe-MP associations do not drive marine nematodes to eat MPs, especially at local field concentrations. While there were no trends across any of the nematode genera in our study, we recognize that unrealistic MP concentrations in other studies may provide alternative explanations for nematode consumption of MPs.

Real-time morphological detection of label-free submicron-sized plastics using flow-channeled differential interference contrast microscopy

Owing to the surge in plastic waste generated during the COVID-19 pandemic, concerns regarding microplastic pollution in aqueous environments are increasing. Since microplastics (MPs) are broken down into submicron (< 1 µm) and nanoscale plastics, their real-time morphological detection in water is necessary. However, the decrease in the scattering cross-section of MPs in aqueous media precludes morphological detection by bright-field microscopy. To address this problem, we propose and demonstrate a differential interference contrast (DIC) system that incorporates a magnification-enhancing system to detect MPs in aqueous samples. To detect MPs in both the stationary and mobile phases, a microfluidic chip was designed, taking into consideration the imaging depth of focus and flow resistance. MPs of various sizes flowing in deionized, tap, and pond water at varying speeds were observed under Static and Flow conditions. Successful real-time morphological detection and quantification of polystyrene beads down to 200 nm at a constant flow rate in water were achieved. Thus, the proposed novel method can significantly reduce analysis time and improve the size-detection limit. The proposed DIC microscopy system can be coupled with Raman or infrared spectroscopy in future studies for chemical composition analysis. Environmental implicationMicroplastics (MPs), particularly submicron plastics < 1-µm, can pose a risk to human health and aquatic ecosystems. Existing methods for detecting MPs in the aqueous phase have several limitations, including the use of expensive instruments and prolonged and labor-intensive procedures. Our results clearly demonstrated that a new low-cost flow-channeled differential interference contrast microscopy enables the real-time morphological detection and quantification of MPs down to 200 nm under flowing conditions without sample labeling. Consequently, our proposed rapid method for accurate quantitative measurements can serve as a valuable reference for detecting submicron plastics in water samples.

Evaluating the utility of brightfield image data for mechanism of action prediction.

Fluorescence staining techniques, such as Cell Painting, together with fluorescence microscopy have proven invaluable for visualizing and quantifying the effects that drugs and other perturbations have on cultured cells. However, fluorescence microscopy is expensive, time-consuming, labor-intensive, and the stains applied can be cytotoxic, interfering with the activity under study. The simplest form of microscopy, brightfield microscopy, lacks these downsides, but the images produced have low contrast and the cellular compartments are difficult to discern. Nevertheless, by harnessing deep learning, these brightfield images may still be sufficient for various predictive purposes. In this study, we compared the predictive performance of models trained on fluorescence images to those trained on brightfield images for predicting the mechanism of action (MoA) of different drugs. We also extracted CellProfiler features from the fluorescence images and used them to benchmark the performance. Overall, we found comparable and largely correlated predictive performance for the two imaging modalities. This is promising for future studies of MoAs in time-lapse experiments for which using fluorescence images is problematic. Explorations based on explainable AI techniques also provided valuable insights regarding compounds that were better predicted by one modality over the other.

Quantitative Analyses for Early Tempo-spatial Patterning of Differentiated Human Induced Pluripotent Stem Cells on Micropatterns using Time-lapse Bright-field Microscopy Images.

Three germ layer formation on micropatterns are extremely useful for quantitative analysis of hiPSC (human induced pluripotent stem cells) pluripotency. Spatial patterns of stem cells differentiated on the micropatterns will be formed from about 24 hours after differentiation induction and usually quantitated near 48 hours. To delineate the germ layer formation process, temporal changes in spatial patterning of germ layers should be analyzed by noninvasive microscopy. This study proposed a series of image processing methods combined with a U-net automatic segmentation to segment differentiated hiPSCs captured by bright-field microscopy. High segmentation accuracy (83.3%) for the test bright-field images compared with their concurrent Hoechst images (85%) was achieved. Tempo-spatial patterning and formation process of germ layers on the micropatterns can be visualized and quantified by segmenting time-lapse bright-field microscopy images using our method.

Analyses of the effects of fiber diameter, fiber fibrillation, and fines content on the pore structure and capillary flow using laboratory sheets of regenerated fibers

Abstract The aim of this work is to investigate the influence of fiber fibrillation and fines on the pore structure of well-defined regenerated fiber sheets as well as the water flow through the sheet. For this purpose, sheets were produced with refined, fibrillated fibers only, with unfibrillated fibers and fines, as well as with fibrillated fibers and fines. Next, the samples were analyzed by brightfield and fluorescence microscopy, mercury porosimetry, and an ascending test. Both the fibrils and the added fines reach into the pores between the fibers or are deposited there. As a result, pore size decreases and capillary flow slows down. The two effects overlap when the fiber surface is fibrillated and fines are present. Sheets with thicker fibers form a pore structure with larger pores in between the fibers. However, such a change in pore size has no significant influence on the flow of water through the sheet in the performed ascending tests. It is shown that a statistical model with the parameters fibrillation and fines content can be used to describe the ascending rate nearly as well as the Lucas–Washburn equation. Consequently, the equation could be improved by the addition of further fiber and sheet properties.

The Role of Trypan Blue as a Conventional and Fluorescent Dye for the Diagnosis of Superficial Mycoses by Potassium Hydroxide (KOH) Testing.

Direct microscopic examination of samples using potassium hydroxide (KOH) is a fast, simple, and inexpensive method to confirm clinical suspicion of superficial mycosis. However, the lack of color contrast in this test makes it difficult to separate any fungal structures from artifacts. The sensitivity of the KOH mount technique may be enhanced using both fluorochromes and conventional stains that highlight the fungal structures when observed under fluorescence microscopy and bright-field, respectively. Here we study the possibility of using Trypan Blue (TB), an azo dye which is often used as a live/dead marker, in the diagnosis of superficial mycoses by KOH testing. TB at 0.01% displayed a fluorescent staining pattern similar to that of Calcofluor White (CFW), the conventional cell wall fluorophore. Furthermore, by adjusting the TB concentration to 0.1-0.3%, in addition to maintaining the fluorescent staining pattern, the fungal elements were stained in blue under bright-field microscopy. Thus, we demonstrate for the first time that TB has the unique property as a fungal stain that can be used in both bright-field and fluorescence microscopy for diagnosis of superficial mycoses by direct microscopic examination.

Engineering a Biomimetic In Vitro Model of Bruch's Membrane Using Hagfish Slime Intermediate Filament Proteins.

Bruch's membrane resides in the subretinal tissue and regulates the flow of nutrients and waste between the retinal pigment epithelial (RPE) and vascular layers of the eye. With age, Bruch's membrane becomes thicker, stiffer, and less permeable, which impedes its function as a boundary layer in the subretina. These changes contribute to pathologies such as age-related macular degeneration (AMD). To better understand how aging in Bruch's membrane affects surrounding tissues and to determine the relationship between aging and disease, an in vitro model of Bruch's membrane is needed. An accurate model of Bruch's membrane must be a proteinaceous, semipermeable, and nonporous biomaterial with similar mechanical properties to in vivo conditions. Additionally, this model must support RPE cell growth. While models of subretinal tissue exist, they typically differ from in vivo Bruch's membrane in one or more of these properties. This study evaluates the capability of membranes created from recombinant hagfish intermediate filament (rHIF) proteins to accurately replicate Bruch's membrane in an in vitro model of the subretinal tissue. The physical characteristics of these rHIF membranes were evaluated using mechanical testing, permeability assays, brightfield microscopy, and scanning electron microscopy. The capacity of the membranes to support RPE cell culture was determined using brightfield and fluorescent microscopy, as well as immunocytochemical staining. This study demonstrates that rHIF protein membranes are an appropriate biomaterial to accurately mimic both healthy and aged Bruch's membrane for in vitro modeling of the subretinal tissue.

Staphylococcus aureus Biofilm Destabilization by Tween-80 and Lung Surfactants to Overcome Biofilm-Imposed Drug Resistance.

This study is aimed to evaluating the potential of tween-80 and artificial lung surfactant (ALS) to destabilize S. aureus biofilm. The biofilm destabilization was studied by crystal violet staining, bright field microscopy, and scanning electron microscopy (SEM). During the study, S. aureus biofilm was exposed with tween-80 along various concentrations (1%, 0.1%, and 0.05%) or LS (lung surfactant) at (2.5%, 5%, and 15%) for 2 hrs. It was observed that 0.1% of tween-80 destabilized 63.83 ± 4.35% and 15% ALS 77 ± 1.7% biofilm in comparison to without treatment. The combination of tween-80 and ALS was used and showed a synergistic effect to destabilize 83.4 ± 1.46% biofilm. These results showed the potential of tween-80 and ALS as biofilm disruptors, which further needs to explore in an in-vivo animal model to access the actual potential of biofilm disruption in natural conditions. This study could play a pivotal role to overcome the problem of antibiotic resistance imposed due to biofilm formation to combat antibiotic resistance imposed by bacteria.

Near infrared laser irradiation on single multicellular spheroids

Light is being widely used in biomedicine due to its non-invasive nature, with application in imaging techniques and as a therapeutic agent. However, several aspects of its effect on irradiated tissues still leads to discussion in the scientific community. This particularly relates to novel biological models, as is the case of 3D multicellular spheroids, which are rising as an intermediate model between in vitro monolayer cultures and small animals. The applications of these spherical cell aggregates are diverse and include tissue reconstruction, drug testing or cancer studies, to cite some. To address the effect of light on these models, we use spheroids formed by MCF-7 (adenocarcinoma) or by U-87 MG (glioblastoma) cells. After their growth, they have been irradiated individually with focused laser radiation in the near-infrared (808 nm and 1450 nm), which provokes size changes in the spheroid. Time-lapse imaging in a brightfield microscope allows to define a reduction parameter, which informs about the extent of the size change. This parameter is correlated with cell viability studies; thus, we can set a safe range of reduction in which spheroids are not damaged by irradiation, and a threshold that should be avoided to keep cell mortality low. This correlation can be used as preliminary and visual information on the survival of cells during optical experiments with 3D spheroids.

Single capture bright field and off-axis digital holographic microscopy: publisher's note.

This publisher's note contains corrections to Opt. Lett.48, 876 (2023)10.1364/OL.478674.

Lyophilized biopolymeric beads of chitosan-xanthan with edible fungus Laccaria laccata (Scop.) Cooke as forest ectomycorrhizal biofertilizers

Objective: To evaluate whether or not the spores of the edible fungus Laccaria laccata (Scop.) Cooke encapsulated in a lyophilized biopolymeric matrix of chitosan-xanthan can cause ectomycorrhization in Pinus greggii Englem. trees under greenhouse conditions. Methodology: Spores of the edible ectomycorrhizal fungus L. laccata were encapsulated in beads made with the chitosan-xanthan biopolymer. The embedded spores were analyzed using scanning electron microscopy to evaluate possible structural damage. Next, these beads were used as biofertilizers in a greenhouse bioassay using Pinus greggii plants to evaluate their ability to be ectomycorrhized. The bioassay lasted 270 days. Subsequently, stereoscopic and bright field microscopy was used to determine if the roots of the pines had been subjected to an ectomycorrhizal colonization. Additionally, the growth of inoculated plants was evaluated compared to non-inoculated plants, 180 and 270 days after sowing. Results: The spores of L. laccata encapsulated in the biopolymeric matrix formed ectomycorrhizae in the roots of P. greggii. The percentages of ectomycorrhizal colonization in the plants ranged from 80 to 90%, demonstrating that the production of chitosan-xanthan biopolymeric beads can maintain the viability of the spores of the ectomycorrhizal fungus evaluated and extensively colonize the roots of Pinus greggii. Study Limitations/Implications: The biopolymeric matrix beads that contain spores of the fungus L. laccata can induce ectomycorrhization in trees of forest importance. Conclusions: The spores of an edible ectomycorrhizal fungus encapsulated in the chitosan-xanthan biopolymer have potential as a forest biofertilizer, which opens the opportunity to scale its use up to an industrial level.

Colleters, osmophores, and nectaries in the species Ceropegia lenewtonii: a sapromyiophilous stapeliad (Asclepiadoideae, Apocynaceae).

Ceropegia lenewtonii (Plowes) Bruyns (=Huernia keniensis), currently belonging to the Huernia section of the genus Ceropegia, is a stapeliad species distributed in Africa and the Arabian Peninsula; but it is widely cultivated as ornamental in most parts of the world. This species of stapeliad presents "carrion flowers" associated with a sapromyophilous pollination syndrome since the flowers emit an unpleasant odor. In this work, we describe the floral morphology and anatomy of the calyx, corolla, and corona of this species based on bright-field and scanning electron microscope techniques. We detected the presence of diverse floral secretor tissues, and based on different histochemical tests, the principal component of the secreted substance was identified. We interpret the functions of the glands and compare with other related species of stapeliads. Our results indicate that flowers of C. lenewtonii present colleters in sepals, osmophores in corolla, and primary and secondary nectaries in corona. All these floral glands have specific functions that involve the processes of pollination and reproduction of this species, as well as protection and defense mechanisms.

Aqueous spice extracts as alternative antimycotics to control highly drug resistant extensive biofilm forming clinical isolates of Candida albicans.

Candida albicans form biofilm by associating with biotic and abiotic surfaces. Biofilm formation by C. albicans is relevant and significant as the organisms residing within, gain resistance to conventional antimycotics and are therefore difficult to treat. This study targeted the potential of spice-based antimycotics to control C. albicans biofilms. Ten clinical isolates of C. albicans along with a standard culture MTCC-3017 (ATCC-90028) were screened for their biofilm-forming ability. C. albicans M-207 and C. albicans S-470 were identified as high biofilm formers by point inoculation on Trypticase Soy Agar (TSA) medium as they formed a lawn within 16 h and exhibited resistance to fluconazole and caspofungin at 25 mcg and 8 mcg respectively. Aqueous and organic spice extracts were screened for their antimycotic activity against C. albicans M-207 and S-470 by agar and disc diffusion and a Zone of Inhibition was observed. Minimal Inhibitory Concentration was determined based on growth absorbance and cell viability measurements. The whole aqueous extract of garlic inhibited biofilms of C. albicans M-207, whereas whole aqueous extracts of garlic, clove, and Indian gooseberry were effective in controlling C. albicans S-470 biofilm within 12 h of incubation. The presence of allicin, ellagic acid, and gallic acid as dominant compounds in the aqueous extracts of garlic, clove, and Indian gooseberry respectively was determined by High-Performance Thin Layer Chromatography and Liquid Chromatography-Mass Spectrometry. The morphology of C. albicans biofilm at different growth periods was also determined through bright field microscopy, phase contrast microscopy, and fluorescence microscopy. The results of this study indicated that the alternate approach in controlling high biofilm-forming, multi-drug resistant clinical isolates of C. albicans M-207 and S-470 using whole aqueous extracts of garlic, clove, and Indian gooseberry is a safe, potential, and cost-effective one that can benefit the health care needs with additional effective therapeutics to treat biofilm infections.

#6827 DI(2-ETHYLHEXYL) PHTHALATE INDUCES RENAL PROXIMAL TUBULAR CELLS TO UNDERGO EPITHELIAL-MESENCHYMAL TRANSITION VIA AHR SIGNALING

Abstract Background and Aims Environmental factors account for the majority of identified risks associated with CKD of uncertain etiology (CKDu). Among the various environmental factors, plasticizer di(2-ethylhexyl) phthalate (DEHP) is widely used in modern life. The exposure to DEHP is extensive but mostly underestimated in the general population. The link between DEHP exposure and renal injuries has been scarcely reported but the underlying mechanisms are largely unknown. This study aimed to examine the mechanisms by which DEHP causes renal tubular injuries. Method Human proximal tubular cells (PTC), HK2 cells, were used as a cellular model to test the central hypothesis of this study. Cells were treated with various concentrations of DEHP at different time points. WST-1 assay was used to examine the impacts of DEHP on cell viability. Bright field microscopy and immunofluorescence were used to analyze the morphological evidence of epithelial-mesenchymal transition (EMT). Western blots were used to investigate the changes in the expression of EMT-related proteins and arylhydrocarbon receptor (AhR) signaling. Inhibitors of AhR signaling were employed to investigate the crucial roles of AhR signaling in DEHP-induced EMT in HK2 cells. Results By using WST-1 assay, we found that DEHP did not affect cell viability when the examined concentration was below 50 μM, which resembled the low-level environmental exposures. However, when the concentration was higher than 100 μM, DEHP reduced the cell viability in a dose-dependent manner. In response to DEHP, HK-2 cells changed from cuboid to spindle shape when cultured with DEHP (25 μM). This morphological evidence became evident when the treatment time was longer than 48 hours. At the protein levels, DEHP resulted in down-regulation of e-cadherin and upregulation of vimentin and α-SMA. DEHP also lead to upregulation of AhR. The inhibitor of AhR signaling, CQDP (chloroquine diphosphate) reversed the DEHP-induced EMT, which were evidenced by morphology and by the expression patterns of EMT-related proteins. Conclusion At cellular level, our results suggested that low-level environmental exposures of DEHP led to EMT in renal PTC via AhR signaling.