Neutral Red Cytotoxicity Assay Research Articles

Impact of face masks weathering on the mussels Mytilus galloprovincialis

The COVID-19 pandemic has triggered an unprecedented need for single-use face masks, leading to an alarming increase in plastic waste globally. Consequently, the improper disposal of face masks has added to the existing burden of plastic pollution in the oceans. However, the complete environmental and marine ecotoxicological impact remains unclear. This study aims to investigate the ecotoxicological impact caused by the weathering of disposable face masks (DFMs) in the marine environment on mussels Mytilus galloprovincialis (M. galloprovincialis ) by assessing biochemical, cytotoxic, and genotoxic effects. The mask leachate was analysed for the presence of nano and microplastics. Furthermore, the leachate was used in in vivo and in vitro toxicity bioassays to assess its impacts on M. galloprovincialis . The in vivo exposure of M. galloprovincialis to face mask leachate for 14 days induced a significant increase in catalase (CAT) activity in mussel gills, although not enough to prevent oxidative damage to cell membranes. DNA damage was also registered in mussel haemocytes after in vivo exposure to mask leachate. The in vitro Neutral Red (NR) cytotoxicity assay indicated that leachate concentrations ≤ 0.5 g/L-1 pose a significant risk to the health of mussel haemocytes, which seems a reliable tool for the cytotoxicity impact assessment of face masks in the marine environment. Therefore, the leachate obtained from face masks in seawater causes oxidative stress, oxidative damage, cytotoxicity, and genotoxicity in M. galloprovincialis , indicating that the plastic burden generated by DFMs in the ocean and its subsequent weathering represents a ubiquitous and invisible threat to the marine biota.

In-vitro cytotoxicity effects and bioactive constituents of chloroform extract of Vernonia glaberrima Welw. ex O. Hoffm (Asteraceae)

Vernonia glaberrima plant is used traditionally in the treatment of cancer, diabetes and malaria in North central Nigeria. This study was designed to evaluate the cytotoxic effect of chloroform extract of Vernonia glaberrima and isolate some bioactive constituents. Neutral red cytotoxicity assay was performed using breast cancer (MCF7), cervical cancer (HeLa) and Liver cancer (HepG2) cells upon exposure to predetermined concentrations of the chloroform extract. The cell viability was determined colorimetrically after 72 hours of incubation. The extract was fractionated using silica gel column chromatography and preparative TLC. Isolated compounds were characterized using FTIR and NMR spectroscopy. The extract inhibited 50% cell proliferation of MCF7, HeLa and HepG2 cells at an IC50 value of 14.02 ± 0.03, 15.78. ± 0.04, and 16.77± 0.1 μg/mL, respectively. Two compounds namely, lupeol (1) and betulinic acid (2) were isolated from the extract. The two isolated compounds have been previously reported in literature to possess anticancer activities. Hence, the findings from this study demonstrate the potential anticancer properties of chloroform extract of Vernonia glaberrima.

In Vivo and In Vitro Toxicity Testing of Cyanobacterial Toxins: A Mini-Review.

Harmful cyanobacterial blooms are increasing and becoming a worldwide concern as many bloom-forming cyanobacterial species can produce toxic metabolites named cyanotoxins. These include microcystins, saxitoxins, anatoxins, nodularins, and cylindrospermopsins, which can adversely affect humans, animals, and the environment. Different methods to assess these classes of compounds in vitro and in vivo include biological, biochemical, molecular, and physicochemical techniques. Furthermore, toxic effects not attributable to known cyanotoxins can be observed when assessing bloom material. In order to determine exposures to cyanotoxins and to monitor compliance with drinking and bathing water guidelines, it is necessary to have reliable and effective methods for the analysis of these compounds. Many relatively simple low-cost methods can be employed to rapidly evaluate the potential hazard. The main objective of this mini-review is to describe the assessment of toxic cyanobacterial samples using in vitro and in vivo bioassays. Newly emerging cyanotoxins, the toxicity of analogs, or the interaction of cyanobacteria and cyanotoxins with other toxicants, among others, still requires bioassay assessment. This review focuses on some biological and biochemical assays (MTT assay, Immunohistochemistry, Micronucleus Assay, Artemia salina assay, Daphnia magna test, Radionuclide recovery, Neutral red cytotoxicity and Comet assay, Enzyme-Linked Immunosorbent Assay (ELISA), Annexin V-FITC assay and Protein Phosphatase Inhibition Assay (PPIA)) for the detection and measurement of cyanotoxins including microcystins, cylindrospermopsins, anatoxin-a, saxitoxins, and nodularins. Although most bioassay analyses often confirm the presence of cyanotoxins at low concentrations, such bioassays can be used to determine whether some strains or blooms of cyanobacteria may produce other, as yet unknown toxic metabolites. This review also aims to identify research needs and data gaps concerning the toxicity assessment of cyanobacteria.

Enniatin B1-induced lysosomal membrane permeabilization in mouse embryonic fibroblasts

The mycotoxin enniatin B1 (ENN B1) is widely present in grain-based feed and food products. In the present study, we have investigated how this lipophilic and ionophoric molecule can affect the lysosomal stability and chaperone-mediated autophagy (CMA) in wild-type (WT) and in lysosome-associated membrane proteins (LAMP)-1/2 double-deficient (DD) mouse embryonic fibroblasts (MEF). The cell viability and lysosomal pH were assessed using the Neutral Red (NR) cytotoxicity assay and the LysoSensor® Yellow/Blue DND-160, respectively. Changes in the expression of the CMA-related components LAMP-2 and the chaperones heat shock cognate (hsc) 70 and heat shock protein (hsp) 90 were determined in cytosolic extracts by immunoblotting. In the NR assay, LAMP-1/2 DD MEF cells were significantly less sensitive to ENN B1 than WT MEF cells after 24h exposure to ENN B1 at levels of 2.5-10μmol/L. Exposure to ENN B1 at concentrations below the half maximal effective concentration (EC50) (1.5-1.7μmol/L) increased the lysosomal pH in WT MEF, but not in LAMP-1/2 DD cells, suggesting that lysosomal LAMP-2 is an early target of ENN B1-induced lysosomal alkalization and cytotoxicity in MEF cells. Additionally, cytosolic hsp90 and LAMP-2 levels slightly increased after exposure for 4h, indicating lysosomal membrane permeabilization (LMP). In summary, it appeared that ENN B1 can destabilize the LAMP-2 complex in the lysosomal membrane at concentrations close to the EC50, resulting in the alkalinization of lysosomes, partial LMP, and thereby leakage of CMA-associated components into the cytosol.

GENETIC EFFECT OF MIGRATORY COMPOUNDS FROM BOTTLED NATURAL DRINKING WATER STORED UNDER DIRECT SUNLIGHT

The drinking water is one of the important routes for human exposure to contaminants through releasing of antimony (Sb) and polymers from polyethylene terephthalate (PET) plastic. The aim of this study was to investigate the effect of sunlight on chemical compounds migration into PET-bottled water and studying the cytotoxicity of di-butyl phthalate (DBP) and di-octyl phthalate (DOP) by SMART in Drosophila melanogaster. Four random water bottle samples produced by various companies as: A (PET, clear, 0.6L), B (PET, clear, 1.5L), C (PET, blue, 1.5L) and D (polycarbonate PC, blue, 19L) for studying the effect of direct sunlight exposure on migration of antimony and phthalates. Experiment was carried out in the presence of sunlight (7 h daily) for 210 day. The migrated compounds profile (µg/L) that detected sample (A) before storage were only two compounds formaldehyde (FA) and acetaldehyde (AA) out of nine compounds. During storage under sunlight, four compounds (Sb), bis-phenol A (BPA), dimethyl phthalate (DMP) and diethyl phthalate (DEP) were increased till 30th day then did not affect till the end of storage. Other detected compounds were continuously progressed till the end of storage with different rates. The highest rate was appeared in case of FA, it was 222 fold followed by DOP compound (173 fold) as well as DBP (75.3 fold) and finally the AA compound with 17.9 fold. Regarding to another sample B, only 3 compounds (BPA, DMP and DBP) were not detected at zero time. The AA compound was detected with the highest concentration (0.9µg/L) and the lowest one was DOP (0.007µg/L). Only four compounds were detected before storage named Sb, DOP, FA and AA, the lowest level (0.006µg/L) was noticed in DOP, while the moderate level was recorded in FA (0.03µg/L) and Sb (0.08µg/L). Other detected compounds; i.e. DBP, DOP, AA and FA were consequently increased with higher levels. This study investigated genotoxic effects of (DBP) and (DOP) at 30µg/ml concentration using the somatic mutation and recombination test (SMART). The cytotoxicity of the tested phthalate compounds was also assessed at five different concentrations 0.5, 1, 5, 10 and 20µg/ml in two types of human cell lines; liver cancer (HepG2), colon cancer (HCT-116) using neutral red cytotoxicity assay. All of tested compounds significantly showed high levels of tumor induction and frequency compared to the negative control in SMART assay. It was also reduced the viability of the HepG2 cell lines cells using different concentrations and the highest cytotoxic effect. While, on HCT-116 showed no cytotoxic effect.

Carcinogenic and Cytotoxic Effect of Some Food Additives on Drosophila melanogaster and Human Cell Lines

Some food additives that are commonly used by humans were recently proved to be mutagenic. It is of significant importance to evaluate their genotoxic effects, since they are frequently consumed by humans in their daily meals. In this proposal, we investigated the effects of sodium sulphite, boric acid, and benzoic acid on human cell lines; liver cancer (HepG2), colon cancer (HCT-116), lung cancer (A-459), and normal lung (Wi38) and cells were evaluated using neutral red cytotoxicity assay and assessed using the somatic mutation and recombination test (SMART). These compounds at 100mM concentrations induced tumor induction and increased the frequency compared to a negative control in SMART assay. Also, they reduced the viability of the four examined cell lines cells using different concentrations (75, 150, 300 and 600µg/ml). Boric acid had the highest toxic effect while benzoic had the lowest on the examined cells. The toxicity effect of the tested food additives was higher on normal lung human cells than on lung cancer cells, therefore, these food additives may act as carcinogenic agents

UVB damage onset and progression 24h post exposure in human-derived skin cells.

The focus of this research was on UVB radiation (280-320nm) responsible for cellular changes in skin of acute and chronically exposed individuals. This study investigated the acute cellular damages triggered by UVB exposure of cultured human fibroblasts and keratinocyte cells immediately and 24h post exposure in order to understand damage onset and progression. The study evaluated a number of cellular parameters including mitochondria, lysosomes, cell membrane, DNA damages as well as pro and anti-apoptotic protein expression levels. Cellular organelle damages were assessed by a battery of in vitro toxicological assays using MTS and Neutral red cytotoxicity assays. Cell membrane damages were also assessed by measuring lactate dehydrogenase (LDH) enzyme leakage from UVB exposed cells. Lastly DNA damages was assessed using the comet assay while protein expression was evaluated using Western Blot. In this study we reported in all our assay systems (MTS, NR and LDH) that cellular damages were UVB dose dependent with damages amplified 24h post exposure. Our results also indicated that incubation of exposed cells for a period of 24h increased the sensitivity of the assay systems used. The increased sensitivity in detecting early cytotoxic damages was manifested though organelle damage measurement at very low doses which were not manifested immediately post exposure. The data also indicated that HaCaT cells were most sensitive in detecting UVB triggered damages immediately and 24h post exposure using the MTS assay. We also established upregulation and downregulation of various apoptotic proteins at various time points post exposure. The presented data clearly indicated the need for a comprehensive assessment of UVB damages 4 and 24h post exposure due to the different assay sensitivities in addition to various signaling mechanisms activated at different time points post exposure.

Oak and chestnut wood extracts enhance the intestinal gut barrier function in a porcine in vitro cell model

The protective effect of oak wood and chestnut wood extracts on the gut barrier function of a porcine intestinal epithelial cell line (IPEC-J2) was investigated. Tannins, polyphenolic compounds, are likely to improve the impaired epithelial barrier function by inhibiting the disruptive effect of TNF-α [Ren et al., 2012]. Therefore, extracts from oak and chestnut wood were prepared and tested in an in vitro cell culture model, using non-transformed, differentiated IPEC-J2. Cells were seeded in non-coated Transwell® membrane inserts (1.12 cm2; 0.4 µm pore size) for 7 days, allowing the formation of tight junctions (TJs) and the build-up of a representative intestinal layer, which was observed by an increase of transepithelial electrical resistance (TEER). Cells were pre-treated with a previously selected non-toxic concentration of the respective extracts. On day 8, a stressor was additionally added to the pre-treated cells and to the control. TEER was measured after 24, 48, and 72h. Following the final TEER measurement, a neutral red cytotoxicity assay was performed.

Neutral red cytotoxicity assays for assessing in vivo carbon nanotube ecotoxicity in mussels — Comparing microscope and microplate methods

The purpose of the present study was to compare two neutral red retention methods, the more established but very labour-intensive microscope method (NRR) against the more recently developed microplate method (NRU). The intention was to explore whether the sample volume throughput could be increased and potential operator bias avoided. Mussels Mytilus sp. were exposed in vivo to 50, 250 and 500μgL−1 single (SWCNTs) or multi-walled carbon nanotubes (MWCNTs). Using the NRR method, SWCNTs and MWCNTs caused concentration dependent decreases in neutral red retention time. However, a concentration dependent decrease in optical density was not observed using the NRU method. We conclude that the NRU method is not sensitive enough to assess carbon nanotube ecotoxicity in vivo in environmentally relevant media, and recommend using the NRR method.

Cytotoxicity and mutagenicity of sidestream cigarette smoke particulate matter of different particle sizes.

Sidestream cigarette smoke, the major component of environmental tobacco smoke, is a complex and reactive aerosol. The particulate matter (PM) in sidestream smoke is one of the carriers of chemical constituents. However, particle size-dependent toxicological effects of PM are poorly understood. In this study, we evaluated the relationship between the PM size and in vitro cytotoxicity and mutagenicity of sidestream cigarette smoke. A NanoMoudi-II(™) 125A was used to collect PM samples ranging from 10 nm to 10 μm. The in vitro toxicity of PM was evaluated using a neutral red cytotoxicity assay and Salmonella mutagenicity assay. The results showed that the cytotoxicity and mutagenicity of PM larger than 1 μm was significantly lower than PM sized 10 nm-1 μm. Furthermore, there was a noticeable trend that the smaller the size of the PM of sidestream cigarette smoke, the greater the toxicity. This study suggests that the toxicity of PM in sidestream cigarette smoke is size-dependent.

Changes in the activity of antioxidant barrier after treatment of K562 and CCRF-CEM cell lines with doxorubicin-transferrin conjugate.

Doxorubicin (DOX), one of the oldest member of the anthracycline antibiotics, has been administered for over 50 years to patients with leukemias and solid tumors. However, the high unspecified DOX toxicity, related to reactive oxygen species (ROS), affects its limitation in clinical application. Therefore we proposed the usage of human transferrin as a doxorubicin carrier in order to improve the quality of doxorubicin application in conventional chemotherapy. In this study we continue our investigations related to the mechanism of the toxicity of doxorubicin-transferrin (DOX-TRF) conjugate in human leukemia cells. Consequently, we are now concentrating on the influence of this compound on the antioxidative system in K562 and CCRF-CEM cell lines (chronic erythromyeloblastoid leukemia and acute lymphoblastic leukemia cells, respectively). We carried out a neutral red cytotoxicity assay, reduced (GSH) and total (GSH+GSSG) glutathione content, alterations in the activity of catalase and enzymes responsible for maintaining glutathione in reduced form. Exposure of leukemia cells to the investigated anticancer agents caused a time-dependent depletion of intracellular GSH, accompanied by an increase of catalase activity. Moreover, analysis of GSH-related enzymes showed a significant increase in the activities of thioredoxin reductase and glutathione peroxidase after DOX-TRF application. In contrast, glutathione reductase activity was reduced by conjugate treatment to 50%. Significant differences between the pro-oxidative actions of the investigated anticancer compounds were observed in RT-PCR experiments, which confirmed that changes in the activity of catalase and GSH-related enzymes are strictly correlated with their gene transcription changes.

Comparison of in vitro toxicity of mainstream cigarette smoke particulate matter from nano- to micro-size

In order to investigate the interrelationship between particulate matter (PM) size and in vitro toxicological effects of mainstream cigarette smoke, PM sized between 10nm and 10μm in mainstream cigarette smoke was sampled and divided into six stages. The in vitro cytotoxicity, genotoxicity and cell inhibition effects of PM were assessed by the neutral red cytotoxicity assay, Salmonella mutagenicity assay, micronucleus test and flow cytometry analysis, respectively. The results showed that all test samples were cytotoxic in the neutral red cytotoxicity assay. The IC50 values in the small-sized groups were significantly lower than those in the large-sized groups. Most test samples were mutagenic in the Salmonella mutagenicity assay (TA98 with S9 and TA100 with S9) and increased the frequency of micronucleated cells. Most PM disturbed the normal progression of the cell cycle, resulting in the accumulation of cells in the G0/G1 phase and the induction of apoptosis. In these tests, PM of a large size induced less toxicity compared with PM of a small size. These findings suggest that most PM samples induced toxicity in vitro, and PM of a small size was more toxic than PM of a large size.

Antioxidant potential, cytotoxic activity and total phenolic content of Alpinia pahangensis rhizomes

BackgroundAlpinia pahangensis, a wild ginger distributed in the lowlands of Pahang, Malaysia, is used by the locals to treat flatulence. In this study, the antioxidant and cytotoxic activities of the crude aqueous methanol and fractionated extracts of Alpinia pahangensis against five different cancer and one normal cell lines were investigated. The total phenolic content of each extract and its fractions were also quantified. This is the first report on the antioxidant and cytotoxic activities of Alpinia pahangensis extract.MethodsIn the current study, the crude methanol and fractionated extract of the rhizomes of Alpinia pahangensis were investigated for their antioxidant activity using four different assays namely, the DPPH scavenging activity, superoxide anion scavenging, β-carotene bleaching and reducing power assays whilst their phenolic contents were measured by the Folin-Ciocalteu’s method.In vitro neutral red cytotoxicity assay was employed to evaluate the cytotoxic activity against five different cancer cell lines, colon cancer (HCT 116 and HT-29), cervical cancer (Ca Ski), breast cancer (MCF7) and lung cancer (A549) cell lines, and one normal cell line (MRC-5). The extract that showed high cytotoxic activity was further investigated for its chemical constituents by GC-MS (gas chromatography–mass spectrometry) analysis.ResultsThe ethyl acetate fraction showed the strongest DPPH radical scavenging (0.35 ± 0.094 mg/ml) and SOD activities (51.77 ± 4.9%) whilst the methanol extract showed the highest reducing power and also the strongest antioxidant activity in the β-carotene bleaching assays in comparison to other fractions. The highest phenolic content was found in the ethyl acetate fraction, followed by the crude methanol extract, hexane and water fractions. The results showed a positive correlation between total phenolic content with DPPH radical scavenging capacities and SOD activities. The hexane fraction showed potent cytotoxic effect against KB, Ca Ski and HCT 116 cell lines with IC50 of 5.8 ± 0.1 and 9.1 ± 2.0 ug/ml, respectively. The major components of hexane fraction analysed by GC-MS analysis were mostly methyl esters.ConclusionsThe current study suggests that the methanol extract and ethyl acetate fraction of A. pahangensis is a potential source of natural antioxidant for protective as well as prevention of life-threatening diseases. The hexane fraction of A. pahangensis may have the potential to be developed into therapeutic option for treating cancer.

Selective Cytotoxicity of a Grape Seed Proanthocyanidin Extract to Human Oral Carcinoma HSC-2 Cells

Grape seed extract (GSE), a nutraceutical rich in polyphenol proanthocyanidins, was evaluated for its toxicity to human oral cells. Using the neutral red cytotoxicity assay, human squamous carcinoma (HSC-2) cells were shown to be more sensitive to GSE than were gingival fibroblasts. Polyphenols undergo auto-oxidation reactions in cell culture medium, to generate reactive oxygen species, in particular, hydrogen peroxide. Using the FOX assay to quantify hydrogen peroxide, GSE, when added to cell culture medium, generated hydrogen peroxide, albeit at relatively low levels. No hydrogen peroxide was detected in the presence of catalase, which catalyzes the decomposition of hydrogen peroxide, and minor levels were detected in the presence of superoxide dismutase, which stabilizes polyphenols. Superoxide free radical, detected with nitroblue tetrazolium, was identified in GSE-amended medium. Focusing on carcinoma HSC-2 cells, the 24-hr toxicity of GSE was unaffected by the hydrogen peroxide scavengers, catalase and pyruvate, indicating that hydrogen peroxide played no role in toxicity. For HSC-2 cells cotreated with GSE and D,L-buthionine-[S,R]-sulfoximine, a depleter of intracellular glutathione, no potentiation of toxicity occurred. Over the 24 hr toxicity range, GSE did not affect the level of intracellular glutathione; however, some depletion occurred, but only at elevated GSE concentrations. GSE toxicity, apparently, was independent of oxidative stress. The 24-hr cytotoxicity of GSE to HSC-2 cells was potentiated in the presence of SOD, indicating that the proanthocyanidins per se, rather than their auto-oxidation products, accounted for toxicity. To confirm the instability of GSE in cell culture medium with a concomitant lowering of potency, studies compared medium freshly-amended with GSE to “spent” medium, i.e., GSE-amended medium left in the incubator for 24 hr prior to usage. The potency of GSE to HSC-2 cells was significantly lowered following incubated in “spent” medium for 24 hours.

In VitroMorphological Assessment of Apoptosis Induced by Antiproliferative Constituents from the Rhizomes ofCurcuma zedoaria

Bioassay-guided isolation of the active hexane fractions of Curcuma zedoaria led to the identification of five pure compounds, namely, curzerenone (1), neocurdione (2), curdione (3), alismol (4), and zederone (5) and a mixture of sterols, namely, campesterol (6), stigmasterol (7), and β-sitosterol (8). Alismol has never been reported to be present in Curcuma zedoaria. All isolated compounds except (3) were evaluated for their cytotoxic activity against MCF-7, Ca Ski, and HCT-116 cancer cell lines and noncancer human fibroblast cell line (MRC-5) using neutral red cytotoxicity assay. Curzerenone and alismol significantly inhibited cell proliferation in human cancer cell lines MCF-7, Ca Ski, and HCT-116 in a dose-dependent manner. Cytological observations by an inverted phase contrast microscope and Hoechst 33342/PI dual-staining assay showed typical apoptotic morphology of cancer cells upon treatment with curzerenone and alismol. Both compounds induce apoptosis through the activation of caspase-3. It can thus be suggested that curzerenone and alismol are modulated by apoptosis via caspase-3 signalling pathway. The findings of the present study support the use of Curcuma zedoaria rhizomes in traditional medicine for the treatment of cancer-related diseases. Thus, two naturally occurring sesquiterpenoids, curzerenone and alismol, hold great promise for use in chemopreventive and chemotherapeutic strategies.

Comparison of Primary Human Fibroblasts and Keratinocytes with Immortalized Cell Lines Regarding their Sensitivity to Sodium Dodecyl Sulfate in a Neutral Red Uptake Cytotoxicity Assay

Cell lines present a valuable tool for in vitro assessment of skin damage caused by application of cosmeticals or pharmaceuticals. They form a reproducible test system under controllable test conditions and, in many cases, can be used as alternatives to animal testing in order to assess the compatibility of drugs or cosmetics and human skin. Yet, it can not necessarily be assumed that the behavior of cultured cells, when treated with different substances, is exactly consistent with the behavior of cells being part of a live organism. Becoming immortal, cells exhibit changes in genotype and/or phenotype, possibly resulting in modified reactions to external influences. Therefore, to obtain results close to in vivo studies, it seems apparent to use primary cells for testing that have not yet undergone any modifications. To compare the properties of primary fibroblasts (Normal Human Dermal Fibroblasts, NHDF) and primary keratinocytes (Normal Human Epidermal Keratinocytes, NHEK) with those of immortal cell lines (3T3 (ACC 173) Swiss albino mouse fibroblasts and HaCaT (human, adult, low calcium, high temperature, human adult skin keratinocytes) cells), their sensitivities in cytotoxicity assays have been assessed. While both fibroblast cell cultures showed similar sensitivities towards sodium dodecyl sulfate (SDS), primary keratinocytes died at SDS concentrations about three times lower than the immortal HaCaT cells.

In vitro study on the effects of iron sucrose, ferric gluconate and iron dextran on redox-active iron and oxidative stress

Concerns exist that administration of intravenous (i.v.) iron preparations is associated with oxidative stress. Therefore iron sucrose (CAS 8047-67-4), ferric gluconate (CAS 34098-81-1) and iron dextran (CAS 9004-66-4) were assessed for redox-active iron by a dichlorofluorescein assay and for intracellular reactive oxygen species (ROS) generation and cytotoxicity in HepG2 cells. Examining each i.v. iron preparation at its maximum concentration achieved following clinically frequently used doses in a 70 kg individual in in vitro experiments, redox-active iron was highest with ferric gluconate, followed by iron dextran and iron sucrose. Interestingly, when the i.v. iron preparations were diluted in human serum instead of buffer, redox-active iron was highest with iron dextran, followed by iron sucrose, and practically disappeared with ferric gluconate. ROS production in HepG2 cells was increased by all i.v. iron preparations. However, in the neutral red cytotoxicity assay all i.v. iron preparations were non-toxic. In conclusion, ferric gluconate showed the highest increase in intracellular ROS-production in HepG2 cells and the highest amount of redox-active iron in buffer in the in vitro assays. In contrast to the other i.v. iron preparations, redox-active iron from ferric gluconate was rendered completely redox-inactive by serum, indicating that redox-active iron in the various preparations has different chemical properties.

Gingko biloba extract and cobalt porphyrin additive to remove harmful components from cigarette smoke and reduce its toxicity

Cigarette filters were modified with a combination of gingko biloba extract and cobalt porphyrin (CGC) to remove harmful components from the cigarette smoke and reduce its toxicity. Smoke analysis results indicated that CGC eliminated up to 32% of benzo[a]pyrene (B[a]P), 52% of N-nitrosonornicotine (NNN), 46% of N-nitrosoanabasine (NAB), 35% of 4-(methylnitrosamine)-1-(3-pyridyl)-1-butanone (NNK), 31% of N-nitrosoanatabine (NAT), 30% of gas-phase free radicals, and 33% of solid-phase free radicals. Biological experiments, including the Ames test, neutral red cytotoxicity assay and chronic toxicity, were conducted for both CGC cigarettes and control cigarettes. Results showed that the toxicity of the CGC cigarettes was lower than those of the control cigarettes. The mechanism by which the CGC components could remove harmful components from cigarette smoke is discussed.

Phytochemical and Cytotoxic Investigations of Curcuma mangga Rhizomes

Investigations on the cytotoxic effects of the crude methanol and fractionated extracts (hexane, ethyl acetate) C. mangga against six human cancer cell lines, namely the hormone-dependent breast cell line (MCF-7), nasopharyngeal epidermoid cell line (KB), lung cell line (A549), cervical cell line (Ca Ski), colon cell lines (HCT 116 and HT-29), and one non-cancer human fibroblast cell line (MRC-5) were conducted using an in-vitro neutral red cytotoxicity assay. The crude methanol and fractionated extracts (hexane and ethyl acetate) displayed good cytotoxic effects against MCF-7, KB, A549, Ca Ski and HT-29 cell lines, but exerted no damage on the MRC-5 line. Chemical investigation from the hexane and ethyl acetate fractions resulted in the isolation of seven pure compounds, namely (E)-labda-8(17),12-dien-15,16-dial (1), (E)-15,16-bisnor-labda-8(17),11-dien-13-on (2), zerumin A (3), β-sitosterol, curcumin, demethoxycurcumin and bis-demethoxycurcumin. Compounds 1 and 3 exhibited high cytotoxic effects against all six selected cancer cell lines, while compounds 2 showed no anti-proliferative activity on the tested cell lines. Compound 1 also demonstrated strong cytotoxicity against the normal cell line MRC-5. This paper reports for the first time the cytotoxic activities of C. mangga extracts on KB, A549, Ca Ski, HT-29 and MRC-5, and the occurrence of compound 2 and 3 in C. mangga.

Extracts of Five Medicinal Herbs Induced Cytotoxicity in Both Hepatoma and Myeloma Cell Lines

The aim of this study is to assess the effect of the crude methanol extracts of five herbs (Pelargonium zonale, Terminalia bellerica, Philodendron selloum, Ulmus pumila and Ulmus parvifolia) on human hepatoma and murine myeloma cell lines. Assessment included in vitro neutral red cytotoxicity assay and cytopathological diagnosis. IC50 values obtained in both cell lines using neutral red assay showed that the plant extracts of both Terminalia bellerica and Philodendron selloum, were the most effective in inducing cytotoxicity in HepG2, IC50 (16.25±0.20 μg/ ml, 17.51±0.70 μg/ml respectively). The result showed that there was no significant difference in the IC50 between the different plants extract in Myeloma cell line. However, in hepatoma cell line, the IC50 of both Terminalia bellerica and Philodendron selloum were significantly lower (p< 0.01) than the IC50 of Ulmus pumila, Ulmus parvifolia and Pelorgonium zonale. The results indicated that the in vitro NR assay of cytotoxic activities induced by plant methanol extracts were supported by the cytopathological changes on the same population of cells. Conclusions: Neutral red cytotoxicity assay is a suitable test for screening anti-cancer potential of natural products materials. The present results showed that Terminalia bellerica and Philodendron selloum methanol extracts induced cytotoxicity on HepG2 cells. The identification of the effect of individual constituents of each plant methanol extract recommended. In vivo study on experimental level is needed to verify the mechanism of action.