Effects of in vitro brevetoxin exposure on apoptosis and cellular metabolism in a leukemic T cell line (Jurkat)

Harmful algal blooms (HABs) of the toxic dinoflagellate, Karenia brevis, produce red tide toxins, or brevetoxins. Significant health effects associated with red tide toxin exposure have been reported in sea life and in humans, with brevetoxins documented within immune cells from many species. The objective of this research was to investigate potential immunotoxic effects of brevetoxins using a leukemic T cell line (Jurkat) as an in vitro model system. Viability, cell proliferation, and apoptosis assays were conducted using brevetoxin congeners PbTx-2, PbTx-3, and PbTx-6. The effects of in vitro brevetoxin exposure on cell viability and cellular metabolism or proliferation were determined using trypan blue and MTT (1-(4,5-dimethylthiazol-2-yl)-3,5-diphenylformazan), respectively. Using MTT, cellular metabolic activity was decreased in Jurkat cells exposed to 5 – 10 μg/ml PbTx-2 or PbTx-6. After 3 h, no significant effects on cell viability were observed with any toxin congener in concentrations up to 10 μg/ml. Viability decreased dramatically after 24 h in cells treated with PbTx-2 or -6. Apoptosis, as measured by caspase-3 activity, was significantly increased in cells exposed to PbTx-2 or PbTx-6. In summary, brevetoxin congeners varied in effects on Jurkat cells, with PbTx-2 and PbTx-6 eliciting greater cellular effects compared to PbTx-3.     

Neurotoxic Shellfish Poisoning

Neurotoxic shellfish poisoning (NSP) is caused by consumption of molluscan shellfish contaminated with brevetoxins primarily produced by the dinoflagellate, Karenia brevis. Blooms of K. brevis, called Florida red tide, occur frequently along the Gulf of Mexico. Many shellfish beds in the US (and other nations) are routinely monitored for presence of K. brevis and other brevetoxin-producing organisms. As a result, few NSP cases are reported annually from the US. However, infrequent larger outbreaks do occur. Cases are usually associated with recreationally-harvested shellfish collected during or post red tide blooms. Brevetoxins are neurotoxins which activate voltage-sensitive sodium channels causing sodium influx and nerve membrane depolarization. No fatalities have been reported, but hospitalizations occur. NSP involves a cluster of gastrointestinal and neurological symptoms: nausea and vomiting, paresthesias of the mouth, lips and tongue as well as distal paresthesias, ataxia, slurred speech and dizziness. Neurological symptoms can progress to partial paralysis; respiratory distress has been recorded. Recent research has implicated new species of harmful algal bloom organisms which produce brevetoxins, identified additional marine species which accumulate brevetoxins, and has provided additional information on the toxicity and analysis of brevetoxins. A review of the known epidemiology and recommendations for improved NSP prevention are presented.     

Development of a Rapid Throughput Assay for Identification of hNav1.7 Antagonist Using Unique Efficacious Sodium Channel Agonist,Antillatoxin

Voltage-gated sodium channels (VGSCs) are responsible for the generation of the action potential. Among nine classified VGSC subtypes (Na_v1.1–Na_v1.9), Na_v1.7 is primarily expressed in the sensory neurons, contributing to the nociception transmission. Therefore Na_v1.7 becomes a promising target for analgesic drug development. In this study, we compared the influence of an array of VGSC agonists including veratridine, BmK NT1, brevetoxin-2, deltamethrin and antillatoxin (ATX) on membrane depolarization which was detected by Fluorescence Imaging Plate Reader (FLIPR) membrane potential (FMP) blue dye. In HEK-293 cells heterologously expressing hNa_v1.7 α-subunit, ATX produced a robust membrane depolarization with an EC₅₀ value of 7.8 ± 2.9 nM whereas veratridine, BmK NT1, and deltamethrin produced marginal response. Brevetoxin-2 was without effect on membrane potential change. The ATX response was completely inhibited by tetrodotoxin suggesting that the ATX response was solely derived from hNa_v1.7 activation, which was consistent with the results where ATX produced a negligible response in null HEK-293 cells. Six VGSC antagonists including lidocaine, lamotrigine, phenytoin, carbamazepine, riluzole, and 2-amino-6-trifluoromethylthiobenzothiazole all concentration-dependently inhibited ATX response with IC₅₀ values comparable to that reported from patch-clamp experiments. Considered together, we demonstrate that ATX is a unique efficacious hNa_v1.7 activator which offers a useful probe to develop a rapid throughput screening assay to identify hNa_v1.7 antagonists.     

Immune Modulating Brevetoxins: Monocyte Cytotoxicity,Apoptosis, and Activation of M1/M2 Response Elements Is Dependent on Reactive Groups

Brevetoxins are a suite of marine neurotoxins that activate voltage-gated sodium channels (VGSCs) in cell membranes, with toxicity occurring from persistent activation of the channel at high doses. Lower doses, in contrast, have been shown to elicit neuroregeneration. Brevetoxins have thus been proposed as a novel treatment for patients after stroke, when neuron regrowth and repair is critical to recovery. However, findings from environmental exposures indicate that brevetoxins may cause inflammation, thus, there is concern for brevetoxins as a stroke therapy given the potential for neuroinflammation. In this study, we examined the inflammatory properties of several brevetoxin analogs, including those that do and do not bind strongly to VGSCs, as binding has classically indicated toxicity. We found that several analogs are toxic to monocytes, while others are not, and the degree of toxicity is not directly related to VGSC binding. Rather, results indicate that brevetoxins containing aldehyde groups were more likely to cause immunotoxicity, regardless of binding affinity to the VGSC. Our results demonstrate that different brevetoxin family members can elicit a spectrum of apoptosis and necrosis by multiple possible mechanisms of action in monocytes. As such, care should be taken in treating “brevetoxins” as a uniform group, particularly in stroke therapy research.     

Cyclodextrin Formulation of the Marine Natural Product Pseudopterosin A Uncovers Optimal Pharmacodynamics in Proliferation Studies of Human Umbilical Vein Endothelial Cells

Pseudopterosin A (PsA) treatment of growth factor depleted human umbilical vein endothelial cell (HUVEC) cultures formulated in hydroxypropyl-β-cyclodextrin (HPβCD) for 42 h unexpectedly produced a 25% increase in cell proliferation (EC₅₀ = 1.34 × 10⁻⁸ M). Analysis of dose response curves revealed pseudo-first order saturation kinetics, and the uncoupling of cytotoxicity from cell proliferation, thereby resulting in a widening of the therapeutic index. The formulation of PsA into HPβCD produced a 200-fold increase in potency over a DMSO formulation; we propose this could result from a constrained presentation of PsA to the receptor, which would limit non-specific binding. These results support the hypothesis that the non-specific receptor binding of PsA when formulated in DMSO has ostensibly masked prior estimates of specific activity, potency, and mechanism. Collectively, these results suggest that the formulation of PsA and compounds of similar chemical properties in HPβCD could result in significant pharmacological findings that may otherwise be obscured when using solvents such as DMSO.     

Structure Activity Relationship of Brevenal Hydrazide Derivatives

Brevenal is a ladder frame polyether produced by the dinoflagellate Karenia brevis. This organism is also responsible for the production of the neurotoxic compounds known as brevetoxins. Ingestion or inhalation of the brevetoxins leads to adverse effects such as gastrointestinal maladies and bronchoconstriction. Brevenal shows antagonistic behavior to the brevetoxins and shows beneficial attributes when administered alone. For example, in an asthmatic sheep model, brevenal has been shown to increase tracheal mucosal velocity, an attribute which has led to its development as a potential treatment for Cystic Fibrosis. The mechanism of action of brevenal is poorly understood and the exact binding site has not been elucidated. In an attempt to further understand the mechanism of action of brevenal and potentially develop a second generation drug candidate, a series of brevenal derivatives were prepared through modification of the aldehyde moiety. These derivatives include aliphatic, aromatic and heteroaromatic hydrazide derivatives. The brevenal derivatives were tested using in vitro synaptosome binding assays to determine the ability of the compounds to displace brevetoxin and brevenal from their native receptors. A sheep inhalation model was used to determine if instillation of the brevenal derivatives resulted in bronchoconstriction. Only small modifications were tolerated, with larger moieties leading to loss of affinity for the brevenal receptor and bronchoconstriction in the sheep model.     

Extracts from Black Carrot Tissue Culture as Potent Anticancer Agents

Black carrots contain anthocyanins possessing enhanced physiological activities. Explants of young black carrot shoots were cultured in Murashige and Skoog (MS) medium for callus initiation and were transferred to new MS medium supplemented with four different combinations of 2,4-dichlorophenoxyacetic acid and kinetin. Subsequently, the lyophilized calli and black carrot harvested from fields were subjected to ultrasound extraction with ethanol at a ratio of 1:15 (w:v). Obtained extracts were applied to various human cancer cell lines including MCF-7 SK-BR-3 and MDA-MB-231 (human breast adenocarcinomas), HT-29 (human colon adenocarcinoma), PC-3 (human prostate adenocarcinoma), Neuro 2A (Musmusculus neuroblastoma) cancer cell lines and VERO (African green monkey kidney) normal cell line by MTT assay. The highest cytotoxic activity was achieved against Neuro-2A cell lines exhibiting viability of 38–46 % at 6.25 μg/ml concentration for all calli and natural extracts. However, a significantly high IC₅₀ value of 170.13 μg/ml was attained in normal cell line VERO indicating that its natural counterpart is an ideal candidate for treatment of brain cancer without causing negative effects to normal healthy cells.     

Comparative Analysis of the Cytotoxic Effects of Okadaic Acid-Group Toxins on Human Intestinal Cell Lines

The phycotoxin, okadaic acid (OA) and dinophysistoxin 1 and 2 (DTX-1 and -2) are protein phosphatase PP2A and PP1 inhibitors involved in diarrhetic shellfish poisoning (DSP). Data on the toxicity of the OA-group toxins show some differences with respect to the in vivo acute toxicity between the toxin members. In order to investigate whether OA and congeners DTX-1 and -2 may induce different mechanisms of action during acute toxicity on the human intestine, we compared their toxicological effects in two in vitro intestinal cell models: the colorectal adenocarcinoma cell line, Caco-2, and the intestinal muco-secreting cell line, HT29-MTX. Using a high content analysis approach, we evaluated various cytotoxicity parameters, including apoptosis (caspase-3 activation), DNA damage (phosphorylation of histone H2AX), inflammation (translocation of NF-κB) and cell proliferation (Ki-67 production). Investigation of the kinetics of the cellular responses demonstrated that the three toxins induced a pro-inflammatory response followed by cell cycle disruption in both cell lines, leading to apoptosis. Our results demonstrate that the three toxins induce similar effects, as no major differences in the cytotoxic responses could be detected. However DTX-1 induced cytotoxic effects at five-fold lower concentrations than for OA and DTX-2.     

Synthesis and Cytotoxicity Evaluation of Spirocyclic Bromotyrosine Clavatadine C Analogs

Marine-originated spirocyclic bromotyrosines are considered as promising scaffolds for new anticancer drugs. In a continuation of our research to develop potent and more selective anticancer compounds, we synthesized a library of 32 spirocyclic clavatadine analogs by replacing the agmatine, i.e., 4-(aminobutyl)guanidine, side chain with different substituents. These compounds were tested for cytotoxicity against skin cancer using the human melanoma cell line (A-375) and normal human skin fibroblast cell line (Hs27). The highest cytotoxicity against the A-375 cell line was observed for dichloro compound 18 (CC₅₀ 0.4 ± 0.3 µM, selectivity index (SI) 2). The variation of selectivity ranged from SI 0.4 to reach 2.4 for the pyridin-2-yl derivative 29 and hydrazide analog of 2-picoline 37. The structure–activity relationships of the compounds in respect to cytotoxicity and selectivity toward cancer cell lines are discussed.     

Evaluation of Macroalgae Sulfated Polysaccharides on the Leishmania (L.) amazonensis Promastigote

The sulfated polysaccharides from Solieria filiformis (Sf), Botryocladia occidentalis (Bo), Caulerpa racemosa (Cr) and Gracilaria caudata (Gc) were extracted and extensively purified. These compounds were then subjected to in vitro assays to evaluate the inhibition of these polysaccharides on the growth of Leishmania (L.) amazonensis promastigotes. Under the same assay conditions, only three of the four sulfated polysaccharides were active against L. amazonensis, and the polysaccharide purified from Cr was the most potent (EC₅₀ value: 34.5 μg/mL). The polysaccharides derived from Bo and Sf demonstrated moderate anti-leishmanial activity (EC₅₀ values of 63.7 μg/mL and 137.4 μg/mL). In addition, we also performed in vitro cytotoxic assays toward peritoneal macrophages and J774 macrophages. For the in vitro cytotoxicity assay employing J774 cells, all of the sulfated polysaccharides decreased cell survival, with CC₅₀ values of 27.3 μg/mL, 49.3 μg/mL, 73.2 μg/mL, and 99.8 μg/mL for Bo, Cr, Gc, and Sf, respectively. However, none of the sulfated polysaccharides reduced the cell growth rate of the peritoneal macrophages. These results suggest that macroalgae contain compounds with various chemical properties that can control specific pathogens. According to our results, the assayed sulfated polysaccharides were able to modulate the growth rate and cell survival of Leishmania (L.) amazonensis promastigotes in in vitro assays, and these effects involved the interaction of the sulfated polysaccharides on the cell membrane of the parasites.     

Sulfated Polysaccharides from Seaweed Strandings as Renewable Source for Potential Antivirals against Herpes simplex Virus 1

Herpes simplex virus 1 (HSV-1) remains a prominent health concern widespread all over the world. The increasing genital infections by HSV-1 that might facilitate acquisition and transmission of HIV-1, the cumulative evidence that HSV-1 promotes neurodegenerative disorders, and the emergence of drug resistance signify the need for new antiviral agents. In this study, the in vitro anti-herpetic activity of sulfated polysaccharides (SPs) extracted by enzyme or hot water from seaweeds collected in France and Mexico from stranding events, were evaluated. The anti-herpetic activity evaluation of the semi-refined-polysaccharides (sr-SPs) and different ion exchange purified fractions showed a wide range of antiviral activity. Among them, the sr-SPs from the Rhodophyta Halymenia floresii showed stronger activity EC₅₀ 0.68 μg/mL with SI 1470, without cytotoxicity. Further, the antiviral activity of the sr-SPs evaluated at different treatment schemes showed a high EC₅₀ of 0.38 μg/mL during the viral adsorption assays when the polysaccharide and the virus were added simultaneously, whilst the protection on Vero cell during the post-infection assay was effective up to 1 h. The chemical composition, FTIR and ¹H NMR spectroscopic, and molecular weights of the sr-SPs from H. floresii were determined and discussed based on the anti-herpetic activity. The potential utilization of seaweed stranding as a source of antiviral compounds is addressed.     

Griffithsin and Carrageenan Combination Results in Antiviral Synergy against SARS-CoV-1 and 2 in a Pseudoviral Model

Over 182 million confirmed cases of COVID-19 and more than 4 million deaths have been reported to date around the world. It is essential to identify broad-spectrum antiviral agents that may prevent or treat infections by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) but also by other coronaviruses that may jump the species barrier in the future. We evaluated the antiviral selectivity of griffithsin and sulfated and non-sulfated polysaccharides against SARS-CoV-1 and SARS-CoV-2 using a cytotoxicity assay and a cell-based pseudoviral model. The half-maximal cytotoxic concentration (CC₅₀) and half-maximal effective concentration (EC₅₀) were determined for each compound, using a dose-response-inhibition analysis on GraphPad Prism v9.0.2 software (San Diego, CA, USA). The therapeutic index (TI = CC₅₀/EC₅₀) was calculated for each compound. The potential synergistic, additive, or antagonistic effect of different compound combinations was determined by CalcuSyn v1 software (Biosoft, Cambridge, UK), which estimated the combination index (CI) values. Iota and lambda carrageenan showed the most potent antiviral activity (EC₅₀ between 3.2 and 7.5 µg/mL). Carrageenan and griffithsin combinations exhibited synergistic activity (EC₅₀ between 0.2 and 3.8 µg/mL; combination index <1), including against recent SARS-CoV-2 mutations. The griffithsin and carrageenan combination is a promising candidate to prevent or treat infections by SARS-CoV-1 and SARS-CoV-2.     

Sensitivity of Ascochyta rabiei populations to prothioconazole and thiabendazole

Ascochyta rabiei causes Ascochyta blight, a yield-limiting disease of chickpea (Cicer arietinum) world-wide. In 2007, fungal populations of A. rabiei resistant to the QoI group of fungicides were detected in the Northern Great Plains of the United States. Assays were conducted to determine fungal sensitivity for two alternative fungicidal modes of action. A total of 78 isolates of A. rabiei collected between 1983 and 2007 were screened to determine baseline sensitivity to the demethylation-inhibiting foliar fungicide, prothioconazole, and 100 isolates collected between 1987 and 2007 were screened for sensitivity to the methyl benzimidazole carbamate (MBC) fungicide, thiabendazole. Isolates were tested using an in vitro mycelial growth assay to determine the effective fungicide concentration at which 50% of fungal growth was inhibited (EC₅₀) for each isolate-fungicide combination. Baseline EC₅₀ values of prothioconazole ranged from 0.0526 to 0.2958 μg/ml, with a mean of 0.1783 μg/ml. Isolates of A. rabiei collected from 2007 to 2009 from North Dakota chickpea fields exposed to prothioconazole, were screened for prothioconazole sensitivity using the same assay. Mean EC₅₀ values for these isolates were 0.3544 μg/ml, 0.3746 μg/ml, and 0.7820 μg/ml, respectively. These values represent an approximate 2.0 (2007-2008) and 4.4-fold (2009) decrease in sensitivity from the baseline mean. EC₅₀ values of thiabendazole ranged from 1.192 to 3.819 μg/ml, with a mean of 2.459 μg/ml. No significant decrease in fungicide sensitivity was observed for thiabendazole. To date, no loss of Ascochyta blight control has been observed with the use of either prothioconazole or thiabendazole.     

Occurrence of Lipophilic Marine Toxins in Shellfish from Galicia (NW of Spain) and Synergies among Them

Lipophilic marine toxins pose a serious threat for consumers and an enormous economic problem for shellfish producers. Synergistic interaction among toxins may play an important role in the toxicity of shellfish and consequently in human intoxications. In order to study the toxic profile of molluscs, sampled during toxic episodes occurring in different locations in Galicia in 2014, shellfish were analyzed by liquid chromatography tandem mass spectrometry (LC–MS/MS), the official method for the detection of lipophilic toxins. The performance of this procedure was demonstrated to be fit for purpose and was validated in house following European guidelines. The vast majority of toxins present in shellfish belonged to the okadaic acid (OA) group and some samples from a particular area contained yessotoxin (YTX). Since these toxins occur very often with other lipophilic toxins, we evaluated the potential interactions among them. A human neuroblastoma cell line was used to study the possible synergies of OA with other lipophilic toxins. Results show that combination of OA with dinophysistoxin 2 (DTX2) or YTX enhances the toxicity triggered by OA, decreasing cell viability and cell proliferation, depending on the toxin concentration and incubation time. The effects of other lipophilic toxins as 13-desmethyl Spirolide C were also evaluated in vitro.     

Screening Tests for the Rapid Detection of Diarrhetic Shellfish Toxins in Washington State

The illness of three people due to diarrhetic shellfish poisoning (DSP) following their ingestion of recreationally harvested mussels from Sequim Bay State Park in the summer of 2011, resulted in intensified monitoring for diarrhetic shellfish toxins (DSTs) in Washington State. Rapid testing at remote sites was proposed as a means to provide early warning of DST events in order to protect human health and allow growers to test “pre-harvest” shellfish samples, thereby preventing harvest of toxic product that would later be destroyed or recalled. Tissue homogenates from several shellfish species collected from two sites in Sequim Bay, WA in the summer 2012, as well as other sites throughout Puget Sound, were analyzed using three rapid screening methods: a lateral flow antibody-based test strip (Jellett Rapid Test), an enzyme-linked immunosorbent assay (ELISA) and a protein phosphatase 2A inhibition assay (PP2A). The results were compared to the standard regulatory method of liquid chromatography coupled with tandem mass spectroscopy (LC-MS/MS). The Jellett Rapid Test for DSP gave an unacceptable number of false negatives due to incomplete extraction of DSTs using the manufacturer’s recommended method while the ELISA antibody had low cross-reactivity with dinophysistoxin-1, the major toxin isomer in shellfish from the region. The PP2A test showed the greatest promise as a screening tool for Washington State shellfish harvesters.     

Guidelines for in-season nitrogen application for maize (Zea mays L.) based on soil and terrain properties

Nitrogen (N) fertilizers are often applied to maize (Zea mays L.) in excess of economically optimal rates because of the uncertainty of dealing with seasonal and spatial variability. A better understanding of the relationships among field, apparent soil electrical conductivity (EC_a), elevation, slope and seasonal characteristics is therefore essential for performing optimal variable-rate N applications. This study focused on responses during the exponential growth phase, when it is critical that N supply be not limited. Measurements at high spatial resolution allowed to understand the effects of the relationships among N, EC_a, elevation, slope and season on future yield formation. The study was conducted over three years (2005-2007). Mid-season growth responses to applied N were greatest where EC_a levels were high and elevation was low in 2005 and 2007, but not in 2006. Areas with slope ≥1 degree were also more responsive to N rates. Overall best mid-season growth was found in areas of low EC_a, high Elevation and low Slope. However, the best responses to in-season N fertilization were found in areas with opposite properties (high EC_a, low Elevation and high Slope). Indeed, relatively high rates of in-season N were needed to enhance crop growth in areas of high EC_a, low Elevation and high Slope, which are characteristic of unfavourable growth conditions. In counterpart, lower N rates were sufficient for optimal growth in soils at low EC_a high Elevation and low Slope. Also, despite the fact that conditions of high soil variability were specifically selected for the study, the effects and interactions reported for soil NO₃-N content were small. The interaction of EC_a with early seasonal precipitation is likely a key relationship to consider in variable-rate N application: low-lying areas with fine soil texture showed the greatest dependence on weather for optimal N rates. Indeed, the relationships among factors influencing the response to in-season N fertilization were stronger when seasonal conditions were particularly favourable to maize growth. These results are fundamental to the establishment of in-season application rules for spatially variable N algorithms.     

Arbutus unedo L. leaves as source of phytochemicals with bioactive properties

In recent years the strawberry tree (Arbutus unedo L.) is being gradually replaced by other species with higher economic value. With the ultimate goal of selecting superior genotypes, the present work was initiated to study the antioxidant and antimicrobial activities, and total phenolic content in 19 different genotypes of A. unedo leaves from the Trás-os-Montes region of Portugal.The genotype Bragança 1 contains higher total phenolic content (215.0 mg GAE/g_extract) whereas the Vila Boa 4 genotype shows lower total phenolic content (148.0 mg GAE/g_extract). In both methods tested to evaluate the antioxidant activity, Vila Verde and Donai displayed the highest antioxidant capacity (EC₅₀ values of 0.088 and 0.090 mg/mL, respectively, for DPPH; EC₅₀ values of 0.233 and 0.245 mg/mL, respectively, for reducing power assay) while Vila Boa 2 reported the lowest antioxidant potential (EC₅₀ values of 0.142 and 0.378 mg/mL, respectively, in DPPH and reducing power methods). Linear negative correlations were established between the total phenol contents and the EC₅₀ values for both of the antioxidant activity methods tested. Preliminary assays for antimicrobial potential showed that extracts from A. unedo leaves display antibacterial activity, with MIC values of 1 and 5 mg/mL for some Gram-positive and Gram-negative bacteria, respectively. Taken together, the results suggest that A. unedo leaves are a potential source of natural compounds with valuable bioactive properties that could be explored by the pharmaceutical, chemical and food industries.     

Comparative cytotoxicity evaluation of lanthanide nanomaterials on mouse and human cell lines with metabolic and DNA-quantification assays

Lanthanide nanomaterials are considered a less toxic alternative to quantum dots for bioimaging applications. This study evaluated the cytotoxicity of terbium (Tb)-doped gadolinium oxide (Gd(2)O(3)) and dysprosium oxide (Dy(2)O(3)) nanoparticles exposed to human (BEAS-2B) and mouse (L929) cell lines at a concentration range of 200-2000?μg/ml for 48 h. Two assay methods were utilized-WST-8 assay (colorimetric) based on mitochondrial metabolic activity and Pico-Green assay (fluorescence), which measures total DNA content. The authors' data showed that Tb-doped Gd(2)O(3) nanoparticles were consistently more toxic than Tb-doped Dy(2)O(3) nanoparticles. However, exposure to these nanomaterials caused a decrease in proliferation rate for both cell lines rather than a net loss of viable cells after 48 h of exposure. Additionally, there was some degree of discrepancy observed with the two assay methods. For the mouse L929 cell line, the WST-8 assay yielded consistently lower proliferation rates compared to the Pico-Green assay, whereas the opposite trend was observed for the human BEAS-2B cell line. This could arise because of the differential effects of these nanoparticles on the metabolism of L929 and BEAS-2B cells, which in turn may translate to differences in their postexposure proliferation rates. Hence, the Pico-Green assay could have an advantage over the WST-8 assay because it is not skewed by the differential effects of nanomaterials on cellular metabolism.