Passivated-electrode insulator-based dielectrophoretic separation of heterogeneous cell mixtures.

Rapid and accurate purification of various heterogeneous mixtures is a critical step for a multitude of molecular, chemical, and biological applications. Dielectrophoresis has shown to be a promising technique for particle separation due to its exploitation of the intrinsic electrical properties, simple fabrication, and low cost. Here, we present a geometrically novel dielectrophoretic channel design which utilizes an array of localized electric fields to separate a variety of unique particle mixtures into distinct populations. This label-free device incorporates multiple winding rows with several nonuniform structures on to sidewalls to produce high electric field gradients, enabling high locally generated dielectrophoretic forces. A balance between dielectrophoretic forces and Stokes' drag is used to effectively isolate each particle population. Mixtures of polystyrene beads (500 nm and 2 µm), breast cancer cells spiked in whole blood, and for the first time, neuron and satellite glial cells were used to study the separation capabilities of the design. We found that our device was able to rapidly separate unique particle populations with over 90% separation yields for each investigated mixture. The unique architecture of the device uses passivated-electrode insulator-based dielectrophoresis in an innovative microfluidic device to separate a variety of heterogeneous mixture without particle saturation in the channel.

Scalable nanolaminated SERS multiwell cell culture assay.

This paper presents a new cell culture platform enabling label-free surface-enhanced Raman spectroscopy (SERS) analysis of biological samples. The platform integrates a multilayered metal-insulator-metal nanolaminated SERS substrate and polydimethylsiloxane (PDMS) multiwells for the simultaneous analysis of cultured cells. Multiple cell lines, including breast normal and cancer cells and prostate cancer cells, were used to validate the applicability of this unique platform. The cell lines were cultured in different wells. The Raman spectra of over 100 cells from each cell line were collected and analyzed after 12 h of introducing the cells to the assay. The unique Raman spectra of each cell line yielded biomarkers for identifying cancerous and normal cells. A kernel-based machine learning algorithm was used to extract the high-dimensional variables from the Raman spectra. Specifically, the nonnegative garrote on a kernel machine classifier is a hybrid approach with a mixed nonparametric model that considers the nonlinear relationships between the higher-dimension variables. The breast cancer cell lines and normal breast epithelial cells were distinguished with an accuracy close to 90%. The prediction rate between breast cancer cells and prostate cancer cells reached 94%. Four blind test groups were used to evaluate the prediction power of the SERS spectra. The peak intensities at the selected Raman shifts of the testing groups were selected and compared with the training groups used in the machine learning algorithm. The blind testing groups were correctly predicted 100% of the time, demonstrating the applicability of the multiwell SERS array for analyzing cell populations for cancer research.

Biophysical phenotyping of cells via impedance spectroscopy in parallel cyclic deformability channels.

This paper describes a new microfluidic biosensor with capabilities of studying single cell biophysical properties. The chip contains four parallel sensing channels, where each channel includes two constriction regions separated by a relaxation region. All channels share a pair of electrodes to record the electrical impedance. Single cell impedance magnitudes and phases at different frequencies were obtained. The deformation and transition time information of cells passing through two sequential constriction regions were gained from the time points on impedance magnitude variations. Constriction channels separated by relaxation regions have been proven to improve the sensitivity of distinguishing single cells. The relaxation region between two sequential constriction channels provides extra time stamps that can be identified in the impedance plots. The new chip allows simultaneous measurement of the biophysical attributes of multiple cells in different channels, thereby increasing the overall throughput of the chip. Using the biomechanical parameters represented by the time stamps in the impedance results, breast cancer cells (MDA-MB-231) and the normal epithelial cells (MCF-10A) could be distinguished by 85%. The prediction accuracy at the single-cell level reached 97% when both biomechanical and bioelectrical parameters were utilized. While the new label-free assay has been tested to distinguish between normal and cancer cells, its application can be extended to include cell-drug interactions and circulating tumor cell detection in blood.

Kernel-Based Microfluidic Constriction Assay for Tumor Sample Identification.

A high-throughput multiconstriction microfluidic channels device can distinguish human breast cancer cell lines (MDA-MB-231, HCC-1806, MCF-7) from immortalized breast cells (MCF-10A) with a confidence level of ∼81-85% at a rate of 50-70 cells/min based on velocity increment differences through multiconstriction channels aligned in series. The results are likely related to the deformability differences between nonmalignant and malignant breast cells. The data were analyzed by the methods/algorithms of Ridge, nonnegative garrote on kernel machine (NGK), and Lasso using high-dimensional variables, including the cell sizes, velocities, and velocity increments. In kernel learning based methods, the prediction values of 10-fold cross-validations are used to represent the difference between two groups of data, where a value of 100% indicates the two groups are completely distinct and identifiable. The prediction value is used to represent the difference between two groups using the established algorithm classifier from high-dimensional variables. These methods were applied to heterogeneous cell populations prepared using primary tumor and adjacent normal tissue obtained from two patients. Primary breast cancer cells were distinguished from patient-matched adjacent normal cells with a prediction ratio of 70.07%-75.96% by the NGK method. Thus, this high-throughput multiconstriction microfluidic device together with the kernel learning method can be used to perturb and analyze the biomechanical status of cells obtained from small primary tumor biopsy samples. The resultant biomechanical velocity signatures identify malignancy and provide a new marker for evaluation in risk assessment.

Entrapment of Prostate Cancer Circulating Tumor Cells with a Sequential Size-Based Microfluidic Chip.

Circulating tumor cells (CTCs) are broadly accepted as an indicator for early cancer diagnosis and disease severity. However, there is currently no reliable method available to capture and enumerate all CTCs as most systems require either an initial CTC isolation or antibody-based capture for CTC enumeration. Many size-based CTC detection and isolation microfluidic platforms have been presented in the past few years. Here we describe a new size-based, multiple-row cancer cell entrapment device that captured LNCaP-C4-2 prostate cancer cells with >95% efficiency when in spiked mouse whole blood at ∼50 cells/mL. The capture ratio and capture limit on each row was optimized and it was determined that trapping chambers with five or six rows of micro constriction channels were needed to attain a capture ratio >95%. The device was operated under a constant pressure mode at the inlet for blood samples which created a uniform pressure differential across all the microchannels in this array. When the cancer cells deformed in the constriction channel, the blood flow temporarily slowed down. Once inside the trapping chamber, the cancer cells recovered their original shape after the deformation created by their passage through the constriction channel. The CTCs reached the cavity region of the trapping chamber, such that the blood flow in the constriction channel resumed. On the basis of this principle, the CTCs will be captured by this high-throughput entrapment chip (CTC-HTECH), thus confirming the potential for our CTC-HTECH to be used for early stage CTC enrichment and entrapment for clinical diagnosis using liquid biopsies.

Microfluidic Iterative Mechanical Characteristics (iMECH) Analyzer for Single-Cell Metastatic Identification.

This study describes the development of a microfluidic biosensor called the iterative mechanical characteristics (iMECH) analyzer which enables label-free biomechanical profiling of individual cells for distinction between metastatic and non-metastatic human mammary cell lines. Previous results have demonstrated that pulsed mechanical nanoindentation can modulate the biomechanics of cells resulting in distinctly different biomechanical responses in metastatic and non-metastatic cell lines. The iMECH analyzer aims to move this concept into a microfluidic, clinically more relevant platform. The iMECH analyzer directs a cyclic deformation regimen by pulling cells through a test channel comprised of narrow deformation channels and interspersed with wider relaxation regions which together simulate a dynamic microenvironment. The results of the iMECH analysis of human breast cell lines revealed that cyclic deformations produce a resistance in non-metastatic 184A1 and MCF10A cells as determined by a drop in their average velocity in the iterative deformation channels after each relaxation. In contrast, metastatic MDA-MB-231 and MDA-MB-468 cells exhibit a loss of resistance as measured by a velocity raise after each relaxation. These distinctive modulatory mechanical responses of normal-like non-metastatic and metastatic cancer breast cells to the pulsed indentations paradigm provide a unique bio-signature. The iMECH analyzer represents a diagnostic microchip advance for discriminating metastatic cancer at the single-cell level.

Single-Cell Mechanical Characteristics Analyzed by Multiconstriction Microfluidic Channels.

A microfluidic device composed of variable numbers of multiconstriction channels is reported in this paper to differentiate a human breast cancer cell line, MDA-MB-231, and a nontumorigenic human breast cell line, MCF-10A. Differences between their mechanical properties were assessed by comparing the effect of single or multiple relaxations on their velocity profiles which is a novel measure of their deformation ability. Videos of the cells were recorded via a microscope using a smartphone, and imported to a tracking software to gain the position information on the cells. Our results indicated that a multiconstriction channel design with five deformation (50 µm in length, 10 µm in width, and 8 µm in height) separated by four relaxation (50 µm in length, 40 µm in width, and 30 µm in height) regions was superior to a single deformation design in differentiating MDA-MB-231 and MCF-10A cells. Velocity profile criteria can achieve a differentiation accuracy around 95% for both MDA-MB-231 and MCF-10A cells.

The impact of sphingosine kinase inhibitor-loaded nanoparticles on bioelectrical and biomechanical properties of cancer cells.

Cancer progression and physiological changes within the cells are accompanied by alterations in the biophysical properties. Therefore, the cell biophysical properties can serve as promising markers for cancer detection and physiological activities. To aid in the investigation of the biophysical markers of cells, a microfluidic chip has been developed which consists of a constriction channel and embedded microelectrodes. Single-cell impedance magnitudes at four frequencies and entry and travel times are measured simultaneously during their transit through the constriction channel. This microchip provides a high-throughput, label-free, automated assay to identify biophysical signatures of malignant cells and monitor the therapeutic efficacy of drugs. Here, we monitored the dynamic cellular biophysical properties in response to sphingosine kinase inhibitors (SphKIs), and compared the effectiveness of drug delivery using poly lactic-co-glycolic acid (PLGA) nanoparticles (NPs) loaded with SphKIs versus conventional delivery. Cells treated with SphKIs showed significantly higher impedance magnitudes at all four frequencies. The bioelectrical parameters extracted using a model also revealed that the highly aggressive breast cells treated with SphKIs shifted electrically towards that of a less malignant phenotype; SphKI-treated cells exhibited an increase in cell-channel interface resistance and a significant decrease in specific membrane capacitance. Furthermore, SphKI-treated cells became slightly more deformable as measured by a decrease in their channel entry and travel times. We observed no significant difference in the bioelectrical changes produced by SphKI delivered conventionally or with NPs. However, NPs-packaged delivery of SphKI decreased the cell deformability. In summary, this study showed that while the bioelectrical properties of the cells were dominantly affected by SphKIs, the biomechanical properties were mainly changed by the NPs.

Single cell metastatic phenotyping using pulsed nanomechanical indentations.

The existing approach to characterize cell biomechanical properties typically utilizes switch-like models of mechanotransduction in which cell responses are analyzed in response to a single nanomechanical indentation or a transient pulsed stress. Although this approach provides effective descriptors at population-level, at a single-cell-level, there are significant overlaps in the biomechanical descriptors of non-metastatic and metastatic cells which precludes the use of biomechanical markers for single cell metastatic phenotyping. This study presents a new promising marker for biosensing metastatic and non-metastatic cells at a single-cell-level using the effects of a dynamic microenvironment on the biomechanical properties of cells. Two non-metastatic and two metastatic epithelial breast cell lines are subjected to a pulsed stresses regimen exerted by atomic force microscopy. The force-time data obtained for the cells revealed that the non-metastatic cells increase their resistance against deformation and become more stiffened when subjected to a series of nanomechanical indentations. On the other hand, metastatic cells become slightly softened when their mechanical microenvironment is subjected to a similar dynamical changes. This distinct behavior of the non-metastatic and metastatic cells to the pulsed stresses paradigm provided a signature for single-cell-level metastatic phenotyping with a high confidence level of ∼95%.

Congenital infection of mice with Toxoplasma gondii induces minimal change in behavior and no change in neurotransmitter concentrations.

We examined the effect of maternal Toxoplasma gondii infection on behavior and the neurotransmitter concentrations of congenitally infected CD-1 mice at 4 and 8 wk of age when latent tissue cysts would be present in their brains. Because of sex-associated behavioral changes that develop during aging, infected female mice were compared with control females and infected male mice were compared with control males. Only the short memory behavior (distance between goal box and first hole investigated) of male mice congenitally infected with T. gondii was significantly different (P < 0.05) from that of uninfected control males at both 4 and 8 wk by using the Barnes maze test. The other parameters examined in the latter test, i.e., functional observational battery tests, virtual cliff, visual placement, and activity tests, were not significantly different (P > 0.05) at 4 and 8 wk. Concentrations of neurotransmitters and their metabolites (dopamine; 3,4-dihydroxyphenylacetic acid; homovanillic acid; norepinephrine; epinephrine; 3-methoxy-4-hydroxyphenylglycol; serotonin; and 5-hydroxyindoleacetic acid) in the frontal cortex and striatum were not different (P > 0.05) between infected and control mice at 8 wk of age. The exact mechanism for the observed effect on short-term memory in male mice is not known, and further investigation may help elucidate the molecular mechanisms associated with the proposed link between behavioral changes and T. gondii infection in animals. We were not able, however, to confirm the widely held belief that changes in neurotransmitters result from chronic T. gondii infection of the brain.

MCF10A and MDA-MB-231 human breast basal epithelial cell co-culture in silicon micro-arrays.

We developed istotropically etched silicon chip micro-arrays for co-culture of metastatic human breast cancer (MDA-MB-231) and non-tumorigenic human breast (MCF10A) cells. The micro-arrays were fabricated using a single-mask, single-etch step process. Each chip contained a 16×16 array of cavities 140 µm wide by 60 µm deep separated by planar silicon surfaces. Cells occupied 97-100% of the etched cavities. The cavities were enriched three-fold in MDA-MB-231 cells relative to the seeding ratio of, MDA-MB-231(1): MCF10A(10) cells. Micro co-cultures comprised of both MCF10A and MDA-MB-231 cells formed in 26% of cavities and contained 2-10 cells per cavity. Heterotropic cell interactions were seen in co-culture, and sites of these interactions were enriched with vinculin spikes. A selective morphological response to the histone deacetylase inhibitor (HDI), SAHA (suberoylanilide hydroxamic acid), occurred in MDA-MB-231 cells which was quantified by significant increases in cell length and cell area on flat surfaces and in the number of stretched cells inside the etched cavities. The morphology of MCF10A cells was unaltered in response to SAHA. Real time imaging showed the formation of highly dynamic and randomly orienting cytoplasmic extensions in MDA-MB-231 cells 1h after adding SAHA; this is the first report of a rapid, morphological response in breast tumor cells to a histone deacetylase inhibitor. The findings demonstrate the utility of etched silicon micro-arrays for the propagation of human breast cell co-cultures and the application of HDI as a potential marker to distinguish metastatic breast cancer cells in a background of normal breast cell types.

Evaluation of the influence of growth medium composition on cell elasticity.

Recently, there has been an increasing interest in using the biomechanical properties of cells as biomarkers to discriminate between normal and cancerous cells. However, few investigators have considered the influence of the growth medium composition when evaluating the biomechanical properties of the normal and diseased cells. In this study, we investigated the variation in Young's modulus of non-malignant MCF10A and malignant MDA-MB-231 breast cells seeded in five different growth media under controlled experimental conditions. The average Young's modulus of MDA-MB-231 cells was significantly lower (p<0.0001) than the mean Young's modulus of MCF10A cells when compared in identical medium compositions. However, we found that growth medium composition affected the elasticity of MCF10A and MDA-MB-231 cells. The average Young's modulus of both cell lines decreased by 10-18% when the serum was reduced from 10% to 5% and upon addition of epidermal growth factor (EGF, 20 ng/ml) to the medium. Though these elasticity changes might have some biological impact, none was statistically significant. However, the elasticity of MCF10A was significantly more responsive than MDA-MB-231 cells to the medium composition supplemented with EGF, cholera toxin (CT), insulin (INS) and hydrocortisone (HC), which are recommended for routine cultivation of MCF10A cells (M5). MCF10A cells were significantly softer (p<0.002) when grown in medium M5 compared to a standard MDA-MB-231 medium (M1). The investigation of the effects of culture medium composition on the elastic properties of cells highlights the need to take these effects into consideration when interpreting elasticity measurements in cells grown in different media.

Actions of the anti-cancer drug suberoylanilide hydroxamic acid (SAHA) on human breast cancer cytoarchitecture in silicon microstructures.

Micro- and nanotechnologies are increasingly being applied in cancer research. Here we report the effects of an experimental breast cancer agent, SAHA, on the cytoarchitecture and adherence of MDA-MB-231 metastatic human breast cancer cells on flat silicon surfaces and in three dimensional (3-D) isotropic silicon microstructures. The 3-D silicon microstructure were fabricated using a single mask and single etch step process to yield arrays of star- and circular-shaped microchambers 151-168 microm in diameter and 53-68 microm deep. There was a marked expansion of the microtubule network, an increase in mean cell area and mean cell length in response to SAHA. SAHA also decreased the nuclear-to-cytoplasmic area (N/C). Atomic force microscopy (AFM) showed there was no change in cellular elasticity over the nuclear region in response to SAHA. The alterations in cytoarchitecture produced by SAHA were associated with changes in the mode of adhesion of the cells in silicon microstructures. In contrast to control cells which conformed to the microstructures, SAHA caused cells to stretch and attach to the microstructures through actin-rich cell extensions. We conclude that isotropically etched silicon microstructures comprise microenvironments that discriminate metastatic mammary cancer cells in which cytoskeletal elements reorganized in response to the anti-cancer agent SAHA.

The cytoskeletal organization of breast carcinoma and fibroblast cells inside three dimensional (3-D) isotropic silicon microstructures.

Studying the cytoskeletal organization as cells interact in their local microenvironment is interest of biological science, tissue engineering and cancer diagnosis applications. Herein, we describe the behavior of cell lines obtained from metastatic breast tumor pleural effusions (MDA-MB-231), normal fibrocystic mammary epithelium (MCF10A), and HS68 normal fibroblasts inside three dimensional (3-D) isotropic silicon microstructures fabricated by a single-mask, single-isotropic-etch process. We report differences in adhesion, mechanism of force balance within the cytoskeleton, and deformability among these cell types inside the 3-D microenvironment. HS68 fibroblasts typically stretched and formed vinculin-rich focal adhesions at anchor sites inside the etched cavities. In contrast, MCF10A and MDA-MB-231 cells adopted the curved surfaces of isotropic microstructures and exhibited more diffuse vinculin cytoplasmic staining in addition to vinculin localized in focal adhesions. The measurement of cells elasticity using atomic force microscopy (AFM) indentation revealed that HS68 cells are significantly stiffer (p < 0.0001) than MCF10A and MDA-MB-231 cells. Upon microtubule disruption with nocodazole, fibroblasts no longer stretched, but adhesion of MCF10A and MDA-MB-231 within the etched features remained unaltered. Our findings are consistent with tensegrity theory. The 3-D microstructures have the potential to probe cytoskeletal-based differences between healthy and diseased cells that can provide biomarkers for diagnostics purposes.

Method to quantify the effect of passivation layer in bio-impedance sensors.

This paper investigates the effect of the passivation layer in a bio-impedance sensor. A sensor with 20 sensing sites has been designed, fabricated using a simple two mask process and tested. We have cultured in-vivo MDA-MB231 mammary cells and recorded the impedance from 1kHz to 1MHz. Processing the recorded data brings to light the drawback of the passivation layer which results in a drop in sensitivity of 13%. Simulation results based on parameters extracted from measurements re-affirm the drop in sensitivity. Thus, the passivation layer needs to be provided a special consideration in future design of the sensor as it can modify the response of the sensor.

Hydroxychloroquine, chloroquine, and all-trans retinoic acid regulate growth, survival, and histone acetylation in breast cancer cells.

The antimalarial drugs chloroquine (CQ) and hydroxychloroquine (HCQ) have potential applications in cancer treatment. The growth of MCF-7 and MDA-MB-231 human breast cancer cells in vitro was inhibited by CQ and HCQ and these cells were more sensitive than nontumorigenic MCF-10A breast epithelial cells. Furthermore, all-trans retinoic acid (ATRA) augmented the anticancer effects of CQ and HCQ as evidenced by significant reductions in Ki67-positive cancer cells and clonogenicity compared with cells treated with CQ or HCQ in the absence of ATRA. As an earlier study suggested that CQ, HCQ, and ATRA are breast cancer cell differentiation agents, these agents were screened in cell-free histone deacetylase (HDAC) and histone acetyltransferase (HAT) assays. ATRA, but not CQ or HCQ, inhibited HDAC activity in HeLa nuclear extracts. Growth inhibitory concentrations of HCQ and ATRA stimulated purified p300/CBP-associated factor, where CBP is the cAMP-response element binding protein, HAT activity. To investigate whether growth inhibitory concentrations of these agents influenced protein acetylation in cells, gel-purified histone H3 and histone H4 were analyzed using mass spectrometry. HCQ alone and HCQ+ATRA treatments altered the acetylation status in the N-terminal lysines of histones H3 and H4 compared with dimethyl sulfoxide (DMSO) controls. The results indicated that HCQ and ATRA regulate protein acetylation events in MCF-7 breast cancer cells, and identify a potential mechanism for their effects on breast cancer cell growth and differentiation.

Cadmium-containing nanoparticles: perspectives on pharmacology and toxicology of quantum dots.

The field of nanotechnology is rapidly expanding with the development of novel nanopharmaceuticals that have potential for revolutionizing medical treatment. The rapid pace of expansion in this field has exceeded the pace of pharmacological and toxicological research on the effects of nanoparticles in the biological environment. The development of cadmium-containing nanoparticles, known as quantum dots, show great promise for treatment and diagnosis of cancer and targeted drug delivery, due to their size-tunable fluorescence and ease of functionalization for tissue targeting. However, information on pharmacology and toxicology of quantum dots needs much further development, making it difficult to assess the risks associated with this new nanotechnology. Further, nanotechnology poses yet another risk for toxic cadmium, which will now enter the biological realm in nano-form. In this review, we discuss cadmium-containing quantum dots and their physicochemical properties at the nano-scale. We summarize the existing work on pharmacology and toxicology of cadmium-containing quantum dots and discuss perspectives in their utility in disease treatment. Finally, we identify critical gaps in our knowledge of cadmium quantum dot toxicity, and how these gaps need to be assessed to enable quantum dot nanotechnology to transit safely from bench to bedside.

Cytoskeletal role in differential adhesion patterns of normal fibroblasts and breast cancer cells inside silicon microenvironments.

In this paper we studied differential adhesion of normal human fibroblast cells and human breast cancer cells to three dimensional (3-D) isotropic silicon microstructures and investigated whether cell cytoskeleton in healthy and diseased state results in differential adhesion. The 3-D silicon microstructures were formed by a single-mask single-isotropic-etch process. The interaction of these two cell lines with the presented microstructures was studied under static cell culture conditions. The results show that there is not a significant elongation of both cell types attached inside etched microstructures compared to flat surfaces. With respect to adhesion, the cancer cells adopt the curved shape of 3-D microenvironments while fibroblasts stretch to avoid the curved sidewalls. Treatment of fibroblast cells with cytochalasin D changed their adhesion, spreading and morphology and caused them act similar to cancer cells inside the 3-D microstructures. Statistical analysis confirmed that there is a significant alteration (P < 0.001) in fibroblast cell morphology and adhesion property after adding cytochalasin D. Adding cytochalasin D to cancer cells made these cells more rounded while there was not a significant alteration in their adhesion properties. The distinct geometry-dependent cell-surface interactions of fibroblasts and breast cancer cells are attributed to their different cytoskeletal structure; fibroblasts have an organized cytoskeletal structure and less deformable while cancer cells deform easily due to their impaired cytoskeleton. These 3-D silicon microstructures can be used as a tool to investigate cellular activities in a 3-D architecture and compare cytoskeletal properties of various cell lines.

Inhibition of Toxoplasma gondii and Plasmodium falciparum infections in vitro by NSC3852, a redox active antiproliferative and tumor cell differentiation agent.

We searched the National Cancer Institute (NCI) compound library for structures related to the antitumor quinoline NSC3852 (5-nitroso-8-quinolinol) and used a computer algorithm to predict the antiprotozoan activity for each of 13 structures. Half of these compounds inhibited Toxoplasma gondii tachyzoite propagation in human fibroblasts at < or =1 microM. The active compounds comprise a series of low-molecular-weight quinolines bearing nitrogen substituents in the ring-5 position. NSC3852 (EC(50) 80 nM) and NSC74949 (EC(50) 646 nM) were the most potent. NSC3852 also inhibited Plasmodium falciparum growth in human red blood cells (EC(50) 1.3 microM). To investigate the mechanism for NSC3852's anti-T. gondii activity, we used chemiluminescence assays to detect reactive oxygen species (ROS) formation in freshly isolated tachyzoites and in infected host cells; the absence of ROS generation by NSC3852 in these assays indicated NSC3852 does not redox cycle in T. gondii. Inhibitors of enzyme sources of free radicals such as superoxide anion, nitric oxide (NO), and their reaction product peroxynitrite did not interfere with the anti-T. gondii activity of NSC3852. However, inhibition of T. gondii tachyzoite propagation by NSC3852 involved redox reactions because tachyzoites were protected from NSC3852 by inclusion of the cell permeant superoxide dismutase mimetic, MnTMPyP, or N-acetylcysteine in the culture medium. We conclude that the Prediction of Activity Spectra for Substances (PASS) computer program is useful in finding new compounds that inhibit T. gondii tachyzoites in vitro and that NSC3852 is a potent T. gondii inhibitor that acts by indirect generation of oxidative stress in T. gondii.