High-Content Image-Based Single-Cell Phenotypic Analysis for the Testicular Toxicity Prediction Induced by Bisphenol A and Its Analogs Bisphenol S, Bisphenol AF, and Tetrabromobisphenol A in a Three-Dimensional Testicular Cell Co-culture Model.

Emerging data indicate that structural analogs of bisphenol A (BPA) such as bisphenol S (BPS), tetrabromobisphenol A (TBBPA), and bisphenol AF (BPAF) have been introduced into the market as substitutes for BPA. Our previous study compared in vitro testicular toxicity using murine C18-4 spermatogonial cells and found that BPAF and TBBPA exhibited higher spermatogonial toxicities as compared with BPA and BPS. Recently, we developed a novel in vitro three-dimensional (3D) testicular cell co-culture model, enabling the classification of reproductive toxic substances. In this study, we applied the testicular cell co-culture model and employed a high-content image (HCA)-based single-cell analysis to further compare the testicular toxicities of BPA and its analogs. We also developed a machine learning (ML)-based HCA pipeline to examine the complex phenotypic changes associated with testicular toxicities. We found dose- and time-dependent changes in a wide spectrum of adverse endpoints, including nuclear morphology, DNA synthesis, DNA damage, and cytoskeletal structure in a single-cell-based analysis. The co-cultured testicular cells were more sensitive than the C18 spermatogonial cells in response to BPA and its analogs. Unlike conventional population-averaged assays, single-cell-based assays not only showed the levels of the averaged population, but also revealed changes in the sub-population. Machine learning-based phenotypic analysis revealed that treatment of BPA and its analogs resulted in the loss of spatial cytoskeletal structure, and an accumulation of M phase cells in a dose- and time-dependent manner. Furthermore, treatment of BPAF-induced multinucleated cells, which were associated with altered DNA damage response and impaired cellular F-actin filaments. Overall, we demonstrated a new and effective means to evaluate multiple toxic endpoints in the testicular co-culture model through the combination of ML and high-content image-based single-cell analysis. This approach provided an in-depth analysis of the multi-dimensional HCA data and provided an unbiased quantitative analysis of the phenotypes of interest.

Manipulation of Single Cells Using a Ferromagnetic Nanorod Cluster Actuated by Weak AC Magnetic Fields.

A unique noncontact single cell manipulation technique based on the actuation of magnetic nanorods (MNRs) or clusters (MCs) by nonuniform alternating magnetic fields (nuAMFs) is demonstrated. Compared to the actuation of MNRs/MCs by conventional magnetophoresis, the motion of MNRs/MCs actuated by nuAMFs can be tuned by additional parameters including the shape of MNRs/MCs and the frequency of the applied magnetic fields. The manipulation of a single cell by an actuated MNR/MC are divided into five stages, i.e., approaching, pushing, carrying, dragging, and releasing. The interactions between the MNR/MC and the cell in these stages are investigated in detail both experimentally and numerically. Other applications of cell manipulation, such as concentrating cells at target locations and accumulating MNRs/MCs onto a single cell, are also demonstrated. The single cell manipulation system is simple, low-cost, and low-power consumption, and helps advance the state-of-the-art of single-particle manipulation.

From the Cover: An Animal-Free In Vitro Three-Dimensional Testicular Cell Coculture Model for Evaluating Male Reproductive Toxicants.

Primary testicular cell coculture model has been used to evaluate testicular abnormalities during development, and was able to identify the testicular toxicity of phthalates. However, the primary testicular cell coculture model has disadvantages in employing animals for the isolation of testicular cells, and the complicated isolation procedure leads to inconsistent results. We developed an invitro testicular coculture model from rodent testicular cell lines, including spermatogonial cells, Sertoli cells, and Leydig cells with specified cell density and extracellular matrix (ECM) composition. Using comparative high-content analysis of F-actin cytoskeletal structure between the coculture and single cell culture models, we demonstrated a 3D structure of the coculture, which created an invivo-like niche, and maintained and supported germ cells within a 3D environment. We validated this model by discriminating between reproductive toxicants and nontoxicants among 32 compounds in comparison to the single cell culture models. Furthermore, we conducted a comparison between the invitro (IC50) and invivo reproductive toxicity testing (lowest observed adverse effect level on reproductive system). We found the invitro coculture model could classify the tested compounds into 4 clusters, and identify the most toxic reproductive substances, with high concordance, sensitivity, and specificity of 84%, 86.21%, and 100%, respectively. We observed a strong correlation of IC50 between the invitro coculture model and the invivo testing results. Our results suggest that this novel invitro coculture model may be useful for screening testicular toxicants and prioritize chemicals for further assessment in the future.

High-Content Analysis Provides Mechanistic Insights into the Testicular Toxicity of Bisphenol A and Selected Analogues in Mouse Spermatogonial Cells.

Bisphenol A (BPA), an endocrine-disrupting compound, was found to be a testicular toxicant in animal models. Bisphenol S (BPS), bisphenol AF (BPAF), and tetrabromobisphenol A (TBBPA) were recently introduced to the market as alternatives to BPA. However, toxicological data of these compounds in the male reproductive system are still limited so far. This study developed and validated an automated multi-parametric high-content analysis (HCA) using the C18-4 spermatogonial cell line as a model. We applied these validated HCA, including nuclear morphology, DNA content, cell cycle progression, DNA synthesis, cytoskeleton integrity, and DNA damage responses, to characterize and compare the testicular toxicities of BPA and 3 selected commercial available BPA analogues, BPS, BPAF, and TBBPA. HCA revealed BPAF and TBBPA exhibited higher spermatogonial toxicities as compared with BPA and BPS, including dose- and time-dependent alterations in nuclear morphology, cell cycle, DNA damage responses, and perturbation of the cytoskeleton. Our results demonstrated that this specific culture model together with HCA can be utilized for quantitative screening and discriminating of chemical-specific testicular toxicity in spermatogonial cells. It also provides a fast and cost-effective approach for the identification of environmental chemicals that could have detrimental effects on reproduction.

Physiologically based pharmacokinetic modeling for 1-bromopropane in F344 rats using gas uptake inhalation experiments.

1-Bromopropane (1-BP) was introduced into the workplace as an alternative to ozone-depleting solvents and increasingly used in manufacturing industry. The potential exposure to 1-BP and the current reports of adverse effects associated with occupational exposure to high levels of 1-BP have increased the need to understand the mechanism of 1-BP toxicity in animal models as a mean of understanding risk in workers. Physiologically based pharmacokinetic (PBPK) model for 1-BP has been developed to examine 2 metabolic pathway assumptions for gas-uptake inhalation study. Based on previous gas-uptake experiments in the Fischer 344 rat, the PBPK model was developed by simulating the 1-BP concentration in a closed chamber. In the model, we tested the hypothesis that metabolism responsibilities were shared by the p450 CYP2E1 and glutathione (GSH) conjugation. The results showed that 2 metabolic pathways adequately simulated 1-BP closed chamber concentration. Furthermore, the above model was tested by simulating the gas-uptake data of the female rats pretreated with 1-aminobenzotrizole, a general P450 suicide inhibitor, or d,l-buthionine (S,R)-sulfoximine, an inhibitor of GSH synthesis, prior to exposure to 800 ppm 1-BP. The comparative investigation on the metabolic pathway of 1-BP through the PBPK modeling in both sexes provides critical information for understanding the role of p450 and GSH in the metabolism of 1-BP and eventually helps to quantitatively extrapolate current animal studies to human.

Regulation of basal lateral membrane mobility and permeability to divalent cations by membrane associated-protein kinase C.

Biological membrane stabilization is essential for maintenance of cellular homeostasis, functionality and appropriate response to various stimuli. Previous studies have showed that accumulation of PKCs in the cell membrane significantly downregulates the membrane fluidity and Ca(2+) influxes through the membranes in activated cells. In addition, membrane-inserted form of PKCs has been found in a variety of resting mammalian cells and tissues. This study is aimed to investigate possible role of the endogenous membrane-associated PKCs in the modulation of basal membrane fluidity. Here, we showed that interfering PKC expression by chronic activation of PKC with phorbol myristate acetate (PMA) or shRNA targeting at PKCα lowered the levels of PKCα in cytosol, peripheral membrane and integral membrane pools, while short-term activation of PKC with PMA induced accumulation of PKCα in the membrane pool accompanied by a dramatic decrease in the cytosol fraction. The lateral membrane mobility increased or decreased in accordance with the abundance alterations in the membrane-associated PKCα by these treatments. In addition, membrane permeability to divalent cations including Ca(2+), Mn(2+) and Ba(2+) were also potentiated or abrogated along with the changes in PKC expression on the plasma membrane. Membrane stabilizer ursodeoxycholate abolished both of the enhanced lateral membrane mobility and permeability to divalent cations due to PKCα deficiency, whereas Gö6983, a PKC antagonist, or Gd(3+) and 2-aminoethyoxydipheyl borne, two Ca(2+) channels blockers, showed no effect, suggesting that this PKC-related regulation is independent of PKC activation or a modulation of specific divalent cation channel. Thus, these data demonstrate that the native membrane-associated PKCα is involved in the maintenance of basal membrane stabilization in resting cells.

Characterization of seventy polymethoxylated flavonoids (PMFs) in the leaves of Murraya paniculata by on-line high-performance liquid chromatography coupled to photodiode array detection and electrospray tandem mass spectrometry.

A sensitive HPLC-DAD-ESI-MS/MS method was established to screen and identify the polymethoxylated flavonoids (PMFs) in the leaves of Murraya paniculata (L.) Jack. 16 PMF standards were first to be analyzed in positive mode by the CID-MS/MS. For polymethoxylated flavones, the fragments of [M+H-n×15](+) produced by loss of one or more methyl radicals from the protonated molecule, as well as [M+H-16](+), [M+H-28](+), [M+H-29](+), [M+H-31](+), [M+H-33](+), [M+H-43](+), [M+H-44](+), [M+H-46](+) and [M+H-61](+) fragment ions were detected, which could be taken as their diagnostic characters. For polymethoxylated flavanones and chalcones, their [M+H](+) ions usually underwent RDA cleavage fragmentation of the C-ring prior to the similar loss of diagnostic fragment ions as polymethoxylated flavones, which could be adopted as a shortcut to distinguish them from ordinary flavones rapidly. For the PMF glycosides, the neutral loss of the similar fragments with polymethoxylated flavones from their [aglycone+H](+) could be adopted as a simple method to screen them out from complex mixture. Based on these characterizations of PMFs and the results of EIC-MS/MS experiment, 70 PMFs including 45 flavones, 17 flavanones or chalcones and 8 PMFs glycosides were screened out from the complex extract of the leaves of M. paniculata. Among them, 16 compounds were unambiguously identified by comparison with reference substances. The results indicated that the developed analysis method could be employed as a rapid, effective technique for structural characterization of PMFs.