In JoVE (1)

Other Publications (15)

Articles by Courtney Sulentic in JoVE

Other articles by Courtney Sulentic on PubMed

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) Alters the Regulation and Posttranslational Modification of P27kip1 in Lipopolysaccharide-activated B Cells

Toxicological Sciences : an Official Journal of the Society of Toxicology. Oct, 2003  |  Pubmed ID: 12883080

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) alters B-cell differentiation, as evidenced by a marked decrease in immunoglobulin M (IgM) secretion and in the number of antibody-forming cells (AFC) induced by antigenic stimulation. The objective of the present studies was to evaluate the effect of TCDD on the level of p27kip1, a cyclin-dependent kinase inhibitor that is a critical regulator of cellular differentiation. In the well-characterized B-cell line, CH12.LX, a modest decrease in p27kip1 was observed during the initial 24-h post-LPS (lipopolysaccharide) activation, which then gradually increased above background at 48 and 72 h. Conversely, in the presence of TCDD, p27kip1 was not induced and remained unchanged from LPS unstimulated cells throughout the entire 72-h period post-LPS activation. In addition, Western blotting revealed that TCDD treatment altered the profile of p27kip1 migration as compared to the LPS-activated control. Time-of-addition studies demonstrated that the greatest sensitivity of p27kip1 to TCDD treatment occurred within the initial 24-h post-LPS activation. Interestingly, LPS-induced Ig kappa light chain and IgM secretion also exhibited the greatest period of sensitivity (i.e., inhibition) to TCDD during the first 24-h post-LPS activation. In addition, TCDD markedly suppressed the LPS-induced differentiation of CH12.LX cells into IgM secreting AFC, with a modest but cumulative effect on cell proliferation over a 72-h period. Collectively, these findings show that TCDD altered the cellular concentration and posttranslational modification of p27kip1 in this activated B-cell line model, which occurred concomitantly with altered B-cell differentiation and suggests that cyclin-dependent kinase inhibitors may be an important intracellular target in TCDD-mediated inhibition of B-cell differentiation.

2,3,7,8-tetrachlorodibenzo-p-dioxin, an Exogenous Modulator of the 3'alpha Immunoglobulin Heavy Chain Enhancer in the CH12.LX Mouse Cell Line

The Journal of Pharmacology and Experimental Therapeutics. Apr, 2004  |  Pubmed ID: 14718603

Transcriptional regulation of the Ig heavy chain gene involves several regulatory elements, including the 3'alpha enhancer, which is composed of four distinct regulatory domains. DNA binding sites for several transcription factors, including B cell-specific activator protein, nuclear factor for immunoglobulin kappa chain in B cells, and octamer have been identified within the 3'alpha enhancer domains and are believed to be important in regulating 3'alpha enhancer activity. We have identified an additional DNA binding motif, the dioxin-responsive element (DRE), which can contribute to 3'alpha enhancer regulation. 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD), a known disrupter of B cell differentiation (i.e., decreased plasma cell formation, inhibition of micro heavy chain expression, and suppression of IgM secretion), induces binding of the aryl hydrocarbon receptor (AhR) nuclear complex to DREs. TCDD also induces AhR binding to the hypersensitive (hs)4 domain of the 3'alpha enhancer. Interestingly, TCDD enhances LPS-induced activation of the hs4 domain but profoundly inhibits LPS-induced activation of the complete 3'alpha enhancer. Furthermore, site-directed mutational analysis demonstrated that a DRE and kappaB element in the hs4 domain is modulated by TCDD in lipopolysaccharide-activated B cells. We propose that the AhR is a novel transcriptional regulator of the 3'alpha enhancer, which can mediate, at least in part, the effects of TCDD on the 3'alpha enhancer and its domains, putatively contributing to a marked suppression of IgM production.

Interactions at a Dioxin Responsive Element (DRE) and an Overlapping KappaB Site Within the Hs4 Domain of the 3'alpha Immunoglobulin Heavy Chain Enhancer

Toxicology. Aug, 2004  |  Pubmed ID: 15212819

Our previous results describing the CH12.LX (AhR-expressing) and BCL-1 (AhR-deficient) B cell lines have supported an AhR/dioxin-responsive element (DRE)-mediated mechanism for TCDD-induced inhibition of micro heavy chain expression and thus of IgM secretion. Transcriptional regulation of the Ig heavy chain genes involves several regulatory elements including the 3'alpha Ig heavy chain enhancer, which is composed of four regulatory domains that span approximately 40 kb. One of these domains, hs4, contains a DRE-like site that overlaps a kappaB motif. We have previously demonstrated TCDD-inducible binding of both the AhR nuclear complex and NF-kappaB/Rel proteins to the DRE and kappaB motifs, respectively, as well as TCDD and LPS-induced transcriptional activity through the hs4 domain. The objective of the present study was to determine if the AhR nuclear complex and NF-kappaB/Rel proteins converge at these two overlapping cis-elements and act cooperatively to influence enhancer activity. To eliminate the potential influence of other transcription factors which bind to the hs4 domain, the approach was to construct a series of luciferase reporters containing a variable heavy chain (VH) promoter and a 42 bp fragment of the 1.4 kb hs4 regulatory domain, that included only the overlapping DRE and kappaB motif or mutations of these motifs for transient transfection experiments in CH12.LX and BCL-1 cells. In the CH12.LX cells, TCDD activated the hs4 fragment; however, co-treatment with LPS led to a marked and synergistic activation as previously observed with the wild type 1.4 kb hs4 domain. Mutation of either or both of the DRE and kappaB motifs diminished the effect of TCDD and LPS on the luciferase reporters possessing the 42 bp portion of hs4, and resembled the effect of these treatments on the promoter alone. In the BCL-1 cells, activity of the hs4 fragment was not induced by TCDD and/or LPS treatment. These results suggest that the AhR nuclear complex and NF-kappaB/Rel proteins converge at the DRE and kappaB motif to influence transcriptional activity of the hs4 enhancer fragment.

Transgenic Expression of Spi-C Impairs B-cell Development and Function by Affecting Genes Associated with BCR Signaling

European Journal of Immunology. Sep, 2008  |  Pubmed ID: 18792411

Spi-C is an Ets family transcription factor closely related to PU.1 and Spi-B. Expression of Spi-C is developmentally regulated in the B-cell lineage, but its function remains unknown. To determine the function of Spi-C in B-cell development, we generated mice expressing a B-cell-specific Spi-C transgene under the control of the IgH intronic enhancer. Spi-C transgenic mice had 50% fewer B cells than wild-type littermates. Flow cytometric analyses showed that splenic transitional B cells and bone marrow pre-B or immature B cells from transgenic mice were dramatically reduced compared with those of wild type. Both nonspecific and Ag-specific serum IgM levels were significantly increased in transgenic mice, while serum IgG levels were significantly decreased compared with wild type. Spi-C transgenic B cells proliferated poorly after stimulation by anti-IgM or anti-CD40 in vitro, although they responded normally to LPS stimulation. Using real-time RT-PCR, we found that several BCR signaling-related mediators were downregulated at pre-B-cell and mature B-cell stages in transgenic mice, while an inhibitor of BCR signaling was upregulated. Taken together, these data indicate that ectopic expression of Spi-C can impair B-cell development and function by affecting genes associated with BCR signaling.

Analysis of Modulation of Immunoglobulin Gene Expression

Current Protocols in Toxicology. May, 2008  |  Pubmed ID: 23045006

Immunoglobulins (Ig) are critical in maintaining host immunity to a variety of pathogens. Regulation of Ig expression is a complex process involving transcriptional regulation of different Ig gene loci by many transcription factors and transcriptional regulatory regions. This complexity suggests many possible molecular targets for immunotoxicants. Therefore, thorough evaluation of chemical-induced modulation of Ig expression may necessitate multiple experimental approaches evaluating: (1) number of B cells secreting antibodies by antibody-forming cell response or plaque assay; (2) concentration of total secreted antibodies by enzyme-linked immunosorbent assay (ELISA); (3) cellular proliferation and viability by cell count measurements, [(3)H] thymidine incorporation, and trypan blue exclusion; (4) Ig mRNA expression by quantitative real-time reverse transcriptase-polymerase chain reaction (RT-PCR); (5) transcriptional activity of specific Ig regulatory regions by reporter gene analysis; and (6) transcription factor binding to specific Ig regulatory regions by electrophoretic mobility shift (EMSA) and chromatin immunoprecipitation (ChIP). These experimental approaches are discussed in the unit, with detailed description of EMSA, EMSA-western analysis, and isolation of nuclear protein.

Diverse Chemicals Including Aryl Hydrocarbon Receptor Ligands Modulate Transcriptional Activity of the 3'immunoglobulin Heavy Chain Regulatory Region

Toxicology. Jun, 2009  |  Pubmed ID: 19447539

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD), a known disruptor of B-cell differentiation and a ligand for the aryl hydrocarbon receptor (AhR), induces binding of the AhR to dioxin responsive elements (DRE) in sensitive genes. The Ig heavy chain (IgH) gene is a sensitive target of TCDD and may be transcriptionally inhibited by TCDD through inhibition of the 3'IgH transcriptional regulatory region (3'IgHRR). While the 3'IgHRR contains binding sites for several transcription factors, two DRE motifs were also identified which may be responsible for TCDD-induced inhibition of 3'IgHRR activation and may implicate the AhR as an important regulator of IgH expression. The objectives of the present study were to determine if 3'IgHRR modulation is limited to TCDD or if structurally diverse chemicals (AhR ligands and non-AhR ligands) from environmental, industrial, dietary or pharmaceutical origin are also capable of modulating the 3'IgHRR and to verify a correlation between effects on a stable 3'IgHRR reporter and the endogenous IgH protein. Utilizing a CH12.LX mouse B-cell line that stably expresses a 3'IgHRR-regulated transgene, we identified an inhibition of both 3'IgHRR activation and IgH protein expression by the non-dioxin AhR activators indolo(3,2-b)carbazole, primaquine, carbaryl, and omeprazole which followed a rank order potency for AhR activation supporting a role of the AhR in the transcriptional regulation of the 3'IgHRR and IgH expression. However, modulation of the 3'IgHRR and IgH expression was not limited to AhR activators or to suppressive effects. Hydrogen peroxide and terbutaline had an activating effect and benzyl isothiocyanate was inhibitory. These chemicals are not known to influence the AhR signaling pathway but have been previously shown to modulate humoral immunity and/or transcription factors that regulate the 3'IgHRR. Taken together these results implicate the 3'IgHRR as a sensitive immunological target and are the first to identify altered 3'IgHRR activation by a diverse range of chemicals.

The Long Winding Road Toward Understanding the Molecular Mechanisms for B-cell Suppression by 2,3,7,8-tetrachlorodibenzo-p-dioxin

Toxicological Sciences : an Official Journal of the Society of Toxicology. Mar, 2011  |  Pubmed ID: 20952503

Suppression of humoral immune responses by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) was first reported in the mid-1970s. Since this initial observation, much effort has been devoted by many laboratories toward elucidation of the cellular and molecular mechanisms responsible for the profound impairment of humoral immune responses by TCDD, which is characterized by decreased B cell to plasma cell differentiation and suppression of immunoglobulin production. These efforts have led to a significant body of research demonstrating a direct effect of TCDD on B-cell maturation and function as well as a requisite but as yet undefined role of the aryl hydrocarbon receptor (AhR) in these effects. Likewise, a number of molecular targets putatively involved in mediating B-cell dysfunction by TCDD, and other AhR ligands, have been identified. However, our current understanding has primarily relied on findings from mouse models, and the translation of this knowledge to effects on human B cells and humoral immunity in humans is less clear. Therefore, a current challenge is to determine how TCDD and the AhR affect human B cells. Efforts have been made in this direction but continued progress in developing adequate human models is needed. An in-depth discussion of these advances and limitations in elucidating the cellular and molecular mechanisms putatively involved in the suppression of B-cell function by TCDD as well as the implications on human diseases associated in epidemiological studies with exposure to TCDD and dioxin-like compounds is the primary focus of this review.

Hydrogen Peroxide Modulates Immunoglobulin Expression by Targeting the 3'Igh Regulatory Region Through an NFκB-dependent Mechanism

Free Radical Research. Jul, 2011  |  Pubmed ID: 21599461

Reactive oxygen species such as hydrogen peroxide (H(2)O(2)) appear to play a role in signal transduction in immune cells and have been shown to be synthesized upon antigen-mediated activation and to facilitate cellular activation in B- and T-cells. However, an effect of H(2)O(2) on B-cell function (i.e. immunoglobulin (Ig) expression) has been less well-characterized. The effects of H(2)O(2) exposure on lymphocytes may be partly mediated by oxidative modulation of the NFκB signal transduction pathway, which also plays a role in Ig heavy chain (Igh) gene expression. Igh transcription in B lymphocytes is an essential step in antibody production and is governed through a complex interaction of several regulatory elements, including the 3'Igh regulatory region (3'IghRR). Utilizing an in vitro mouse B-cell line model, this study demonstrates that exposure to low μM concentrations of H(2)O(2) can enhance 3'IghRR-regulated transcriptional activity and Igh gene expression, while either higher concentrations of H(2)O(2) or the expression of a degradation resistant inhibitory κB (IκBα super-repressor) can abrogate this effect. Furthermore, suppressive H(2)O(2) concentrations increased protein levels of the p50 NFκB sub-unit, IκBα, and an IκBα immunoreactive band which was previously characterized as an IκBα cleavage product exhibiting stronger inhibitory function than native IκBα. Taken together, these observations suggest that exposure of B lymphocytes to H(2)O(2) can alter Igh transcriptional activity and Ig expression in a complex biphasic manner which appears to be mediated by NFκB and altered 3'IghRR activity. These results may have significant implications to disease states previously associated with the 3'IghRR.

2,3,7,8-tetrachlorodibenzo-p-dioxin Induces Transcriptional Activity of the Human Polymorphic Hs1,2 Enhancer of the 3'Igh Regulatory Region

Journal of Immunology (Baltimore, Md. : 1950). Apr, 2012  |  Pubmed ID: 22357631

2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is an environmental toxicant known to inhibit Ab secretion and Ig expression. Inhibition of Ig expression may be partially mediated through repression of the 3'Igh regulatory region (3'IghRR). TCDD inhibits mouse 3'IghRR activation and induces aryl hydrocarbon receptor binding to dioxin response elements within the 3'IghRR enhancers hs1,2 and hs4. The human hs1,2 enhancer (hu-hs1,2) is polymorphic as the result of the presence of one to four invariant sequences (ISs), which have been correlated with several autoimmune diseases. The IS also contains a dioxin response element core motif. Therefore, the objective was to determine whether hu-hs1,2 activity is sensitive to TCDD. Using a mouse B cell line (CH12.LX), we compared the effects of TCDD on mouse hs1,2 versus hu-hs1,2 activity. TCDD inhibited mouse hs1,2 similarly to the mouse 3'IghRR. In contrast, hu-hs1,2 was activated by TCDD, and antagonist studies supported an aryl hydrocarbon receptor-dependent activation, which was replicated in a human B cell line (IM-9). Absence of Pax5 binding sites is a major difference between the human and mouse hs1,2 sequence. Insertion of the high-affinity Pax5 site in hu-hs1,2 markedly blunted reporter activity but did not alter TCDD's effect (i.e., no shift from activation to inhibition). Additionally, deletional analysis demonstrated a significant IS contribution to hu-hs1,2 basal activity, but TCDD-induced activity was not strictly IS number dependent. Taken together, our results suggest that hu-hs1,2 is a significant target of TCDD and support species differences in hs1,2 regulation. Therefore, sensitivity of hu-hs1,2 to chemical-induced modulation may influence the occurrence and/or severity of human diseases associated with hu-hs1,2.

The Toxicology Education Summit: Building the Future of Toxicology Through Education

Toxicological Sciences : an Official Journal of the Society of Toxicology. Jun, 2012  |  Pubmed ID: 22461448

Toxicology and careers in toxicology, as well as many other scientific disciplines, are undergoing rapid and dramatic changes as new discoveries, technologies, and hazards advance at a blinding rate. There are new and ever increasing demands on toxicologists to keep pace with expanding global economies, highly fluid policy debates, and increasingly complex global threats to public health. These demands must be met with new paradigms for multidisciplinary, technologically complex, and collaborative approaches that require advanced and continuing education in toxicology and associated disciplines. This requires paradigm shifts in educational programs that support recruitment, development, and training of the modern toxicologist, as well as continued education and retraining of the midcareer professional to keep pace and sustain careers in industry, government, and academia. The Society of Toxicology convened the Toxicology Educational Summit to discuss the state of toxicology education and to strategically address educational needs and the sustained advancement of toxicology as a profession. The Summit focused on core issues of: building for the future of toxicology through educational programs; defining education and training needs; developing the "Total Toxicologist"; continued training and retraining toxicologists to sustain their careers; and, finally, supporting toxicology education and professional development. This report summarizes the outcomes of the Summit, presents examples of successful programs that advance toxicology education, and concludes with strategies that will insure the future of toxicology through advanced educational initiatives.

A Dioxin Response Element in the Multiple Cloning Site of the PGL3 Luciferase Reporter Influences Transcriptional Activity

Toxicology in Vitro : an International Journal Published in Association with BIBRA. Sep, 2012  |  Pubmed ID: 22652426

Luciferase reporter plasmids (pGL3 backbone, Promega) have been utilized to characterize the transcriptional effects of the environmental contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and other aryl hydrocarbon receptor (AhR) ligands. Following ligand activation, the AhR and its dimerization partner AhR nuclear translocator (ARNT) regulate transcription by binding dioxin response elements (DREs) in regulatory regions of dioxin-sensitive genes. Upon sequencing of our luciferase reporters, we unexpectedly identified a DRE core motif within the multiple cloning site (mcsDRE) of the pGL3 luciferase plasmid backbone in a subset of our reporters. Therefore, the objective of this study was to determine if the mcsDRE inadvertently influences reporter activity. Utilizing deletional analysis we determined that the mcsDRE did significantly alter the transcriptional effect induced by TCDD. Since many chemicals have been shown to interact with the AhR and influence transcription through the DRE, the presence of the mcsDRE in the pGL3 luciferase plasmid may inappropriately influence promoter and enhancer analysis. As such, insertion of regulatory elements into pGL3 reporters should be designed to avoid retaining the mcsDRE core motif (GCGTG) and currently utilized pGL3 reporters should be evaluated for the presence of the mcsDRE.

Gold Nanoparticles Induce Transcriptional Activity of NF-κB in a B-lymphocyte Cell Line

Nanoscale. May, 2013  |  Pubmed ID: 23503581

Gold nanoparticles (Au-NPs) have been designated as superior tools for biological applications owing to their characteristic surface plasmon absorption/scattering and amperometric (electron transfer) properties, in conjunction with low or no immediate toxicity towards biological systems. Many studies have shown the ease of designing application-based tools using Au-NPs but the interaction of this nanosized material with biomolecules in a physiological environment is an area requiring deeper investigation. Immune cells such as lymphocytes circulate through the blood and lymph and therefore are likely cellular components to come in contact with Au-NPs. The main aim of this study was to mechanistically determine the functional impact of Au-NPs on B-lymphocytes. Using a murine B-lymphocyte cell line (CH12.LX), treatment with citrate-stabilized 10 nm Au-NPs induced activation of an NF-κB-regulated luciferase reporter, which correlated with altered B lymphocyte function (i.e. increased antibody expression). TEM imaging demonstrated that Au-NPs can pass through the cellular membrane and therefore could interact with intracellular components of the NF-κB signaling pathway. Based on the inherent property of Au-NPs to bind to -thiol groups and the presence of cysteine residues on the NF-κB signal transduction proteins IκB kinases (IKK), proteins specifically bound to Au-NPs were extracted from CH12.LX cellular lysate exposed to 10 nm Au-NPs. Electrophoresis identified several bands, of which IKKα and IKKβ were immunoreactive. Further evaluation revealed activation of the canonical NF-κB signaling pathway as evidenced by IκBα phosphorylation at serine residues 32 and 36 followed by IκBα degradation and increased nuclear RelA. Additionally, expression of an IκBα super-repressor (resistant to proteasomal degradation) reversed Au-NP-induced NF-κB activation. Altered NF-κB signaling and cellular function in B-lymphocytes suggests a potential for off-target effects with in vivo applications of gold nanomaterials and underscores the need for more studies evaluating the interactions of nanomaterials with biomolecules and cellular components.

The Aryl Hydrocarbon Receptor Regulates an Essential Transcriptional Element in the Immunoglobulin Heavy Chain Gene

Cellular Immunology. May, 2015  |  Pubmed ID: 25749007

Ig heavy chain (Igh) transcription involves several regulatory elements including the 3'Igh regulatory region (3'IghRR). 3'IghRR activity is modulated by several transcription factors, including NF-κB and AP-1 and potentially the aryl hydrocarbon receptor (AhR). The prototypical AhR ligand 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) inhibits antibody secretion and 3'IghRR activity. However, the exact mechanism is unknown and TCDD can modulate NF-κB and AP-1 in an AhR-independent manner. To determine if the AhR is a significant regulator of the 3'IghRR, we utilized a mouse B-cell line that stably expresses a 3'IghRR-regulated transgene and either an AhR antagonist or shRNA targeting the AhR. Disruption of the AhR pathway reversed TCDD-induced suppression of the 3'IghRR-regulated transgene and of endogenous Ig demonstrating a biologically significant effect of the AhR on 3'IghRR activation. Altered human 3'IGHRR activity by AhR ligands, which include dietary, environmental, and pharmaceutical chemicals, may have significant implications to human diseases previously associated with the 3'IGHRR.

The AhR and NF-κB/Rel Proteins Mediate the Inhibitory Effect of 2,3,7,8-Tetrachlorodibenzo-p-Dioxin on the 3' Immunoglobulin Heavy Chain Regulatory Region

Toxicological Sciences : an Official Journal of the Society of Toxicology. Dec, 2015  |  Pubmed ID: 26377645

Transcriptional regulation of the murine immunoglobulin (Ig) heavy chain gene (Igh) involves several regulatory elements including the 3'Igh regulatory region (3'IghRR), which is composed of at least 4 enhancers (hs3A, hs1.2, hs3B, and hs4). The hs1.2 and hs4 enhancers exhibit the greatest transcriptional activity and contain binding sites for several transcription factors including nuclear factor kappaB/Rel (NF-κB/Rel) proteins and the aryl hydrocarbon receptor (AhR). Interestingly, the environmental immunosuppressant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), which potently inhibits antibody secretion, also profoundly inhibits 3'IghRR and hs1.2 enhancer activation induced by the B-lymphocyte activator lipopolysaccharide (LPS), but enhances LPS-induced activation of the hs4 enhancer. Within the hs1.2 and hs4 enhancers, the AhR binding site is in close proximity or overlaps an NF-κB/Rel binding site suggesting a potential reciprocal modulation of the 3'IghRR by AhR and NF-κB/Rel. The objective of the current study was to evaluate the role of NF-κB/Rel and the AhR on the 3'IghRR and its enhancers using the AhR ligand TCDD, the AhR antagonist CH223191, and toll-like receptor agonists LPS, Resiquimod (R848), or cytosine-phosphate-guanine-oligodeoxynucleotides (CpG). Utilizing the CH12.LX B-lymphocyte cell line and variants expressing either a 3'IghRR-regulated transgene reporter or an inducible IκBα (inhibitor kappa B-alpha protein) superrepressor (IκBαAA), we demonstrate an AhR- and NF-κB/Rel-dependent modulation of 3'IghRR and hs4 activity. Additionally, in mouse splenocytes or CH12.LX cells, binding within the hs1.2 and hs4 enhancer of the AhR and the NF-κB/Rel proteins RelA and RelB was differentially altered by the cotreatment of LPS and TCDD. These results suggest that the AhR and NF-κB/Rel protein binding profile within the 3'IghRR mediates the inhibitory effects of TCDD on Ig expression and therefore antibody levels.

The Effect of Shear Flow on Nanoparticle Agglomeration and Deposition in in Vitro Dynamic Flow Models

Nanotoxicology. 2016  |  Pubmed ID: 25961858

Traditional in vitro toxicity experiments typically involve exposure of a mono- or co-culture of cells to nanoparticles (NPs) in static conditions with the assumption of 100% deposition (i.e. dose) of well-dispersed particles. However, cellular dose can be affected by agglomeration and the unique transport kinetics of NPs in biological media. We hypothesize that shear flow can address these issues and achieve more predictable dosage. Here, we compare the behavior of gold NPs with diameters of 5, 10 and 30 nm in static and dynamic in vitro models. We also utilize transport modeling to approximate the shear rate experienced by the cells in dynamic conditions to evaluate physiological relevance. The transport kinetics show that NP behavior is governed by both gravity and diffusion forces in static conditions and only diffusion in dynamic conditions. Our results reveal that dynamic systems are capable of producing a more predictable dose compared to static systems, which has strong implications for improving repeatability in nanotoxicity assessments.

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