Boban Markovic

The sensitivity and specificity of the MTS tetrazolium assay for detecting the in vitro cytotoxicity of 20 chemicals using human cell lines.

A number of studies reported that the MTS in vitro cytotoxicity assay is a convenient method for assessing cell viability. The main features found with this assay are its ease of use, accuracy and rapid indication of toxicity. It might well be a useful tool in human health risk assessment if it can be shown that this assay also has an acceptable sensitivity and specificity. This is of interest particularly when exposure to unknown chemical substances requires the rapid detection and evaluation of toxic effects. In this study, the cytotoxicity of 20 chemicals selected from the MEIC priority list was determined with the MTS assay. Since it could be shown that interactions between detection reagents and test chemicals might influence the results of this assay, preliminary experiments were carried out such that artifactual results due to test chemical interference could be excluded from this study. IC50 (50% inhibitory concentration) were established for each test chemical in two human cell lines (F1-73 and HeLa) and later compared with published toxicity data of the same chemicals established with in vitro and in vivo toxicological test systems. Direct comparisons of the data showed a generally lower sensitivity of the MTS assay, which is influenced by biological test organisms, cell type and exposure time. In terms of the specificity of the MTS assay, the results showed a good correlation between data obtained with the MTS assay and published data. The lowest correlation was found when the MTS assay was compared with in vivo studies, however, this finding corresponds well with other published in vitro-in vivo correlations. The highest correlation was found when the MTS assay was compared with test systems using human cell lines or exposure times of 3-24 h. Since the sensitivity of the MTS assay might be increased using different cell types or by extended incubation, this assay is found to provide ideal features of a good measurement system that might also be used for on site toxicological assessments.

In vitro Cytotoxicity of Australian Tea Tree Oil using Human Cell Lines

Abstract Cytotoxicity of Australian tea tree oil (oil of Melaleuca alternifolia) and its major oxygenated monoterpenes: terpinen-4-ol, 1,8-cineole and α-terpineol were investigated using the MTS [(3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium)] assay at two exposure times: 4 and 24 h on five different human cell lines. These cell lines included: Hep G2, a heptaocellular carcinomic human cell line; HeLa, an epithelioid carcinomic cell line; MOLT-4, a human lymphoblastic leukaemic T-cell line; K-562, a human chronic myelogenous leukaemia cell line; and CTVR-1, an early B-cell line from the bone marrow cells of a patient with acute myeloid leukaemia. The overall rating for cytotoxicity of tea tree oil and its components was α-terpineol>tea tree oil>terpinen-4-ol> 1,8-cineole and with comparison with the controls used mercuric chloride>tea tree oil>aspirin. Antimicrobial activity (MICs) displayed a similar pattern where α-terpineol>terpinen-4-ol>tea tree oil> 1,8-cin...

A novel technique for quantitative detection of mRNA expression in human bone derived cells cultured on biomaterials

A nonisotopic and quantitative in situ hybridization technique was adapted to investigate the effect of biomaterials on the cellular expression of mRNA from human bone derived cells (HBD cells). HBD cells were cultured for 24 or 48 h on tissue culture plastic, alumina, and ion modified alumina. Osteocalcin, osteopontin, alkaline phosphatase, type I collagen alpha 1, and type I collagen alpha 2 mRNAs were quantified. Protein expression for collagen types I, III, and V, and for anti-human macrophages CD68 (DAKO-CD68, KP1) and CD68 (PG-M1), and anti-human myeloid/histiocyte antigen (DAKO-MAC 387) were determined immunohistochemically using monoclonal antibodies. At 24 and 48 h, levels of mRNA for alkaline phosphatase and osteonectin were greater than mRNA levels for osteopontin, osteocalcin, collagen type I alpha 1, and collagen type I alpha 2 for cells grown on the three substrata. However, at 48 h mRNA levels for alkaline phosphatase and osteonectin were significantly higher on the modified ceramic substrata relative to the native alumina. HBD cells appear to express CD68-KP1 when cultured for 24 h. The techniques provide a sensitive and reproducible assay to evaluate gene and protein expression of cells grown on different substrata.

The functional expression of human bone-derived cells grown on rapidly resorbable calcium phosphate ceramics

The use of biodegradable bone substitutes is advantageous for alveolar ridge augmentation, since it avoids second-site surgery for autograft harvesting. This study examines the effect of novel, rapidly resorbable calcium phosphates on the expression of bone-related genes and proteins by human bone-derived cells (HBDC) and compares this behavior to that of tricalciumphosphate (TCP). Test materials were alpha-TCP, and four materials which were created from beta-Rhenanite and its derivatives: R1-beta-Rhenanite (CaNaPO(4)); R1/M2 composed of CaNaPO(4) and MgNaPO(4); R1+SiO(2) composed of CaNaPO(4) and 9% SiO(2) (wt%); and R17-Ca(2)KNa(PO(4))(2). HBDC were grown on the substrata for 3, 5, 7, 14 and 21 days, counted and probed for various mRNAs and proteins (Type I collagen, osteocalcin, osteopontin, osteonectin, alkaline phosphatase and bone sialoprotein). All substrata supported continuous cellular growth for 21 days. At day 21, surfaces of R1+SiO(2) and R17 had the highest number of HBDC. At 14 and 21 days, cells on R1 and on R1+SiO(2) displayed significantly enhanced expression of all osteogenic proteins. Since all novel calcium phosphates supported cellular proliferation together with expression of bone-related proteins at least as much as TCP, these ceramics can be regarded as potential bone substitutes. R1 and R1+SiO(2) had the most effect on osteoblastic differentiation, thus suggesting that these materials may possess a higher potency to enhance osteogenesis than TCP.

Prosthetic particles modify the expression of bone-related proteins by human osteoblastic cells in vitro

Loss of bone near joint prostheses is thought to be caused by activation of recruited osteoclasts by osteolytic mediators induced by wear particles. It is proposed that particles inhibit osteogenesis during bone remodelling causing a reduction in the levels of peri-implant bone. This study explores whether prosthetic particles modulate bone formation by affecting osteoblastic bone-related mRNAs (alkaline phosphatase, pro-collagen Ialpha1, osteopontin, osteonectin, osteocalcin, bone sialoprotein and thrombospondin) or their translated proteins using titanium alloy, commercially pure titanium, and cobalt-chrome particles. The direct effect of the particles revealed no change to the expression of the bone-related mRNAs in human bone-derived cells (HBDC) at the time points investigated; although non-collagenous translated proteins expressed by these HBDC were significantly effected (p<0.05). Different patterns of expression for bone-related proteins were induced by the different particles both directly and indirectly. Inflammatory mediators (interleukin-1beta, tumor necrosis factor alpha, interleukin-6, and prostaglandin E2) had similar effects on HBDC to the media obtained from monocytes incubated with particles. This study shows that prosthetic wear particles can significantly modify the expression of bone-related proteins by osteogenic cells in vitro. These alterations in osteogenic activity at the interface of the implant and bone may be an important factor in the failure of many orthopaedic implants.

In vitro cytotoxicity testing of airborne formaldehyde collected in serum-free culture media

The purpose of this study was to identify a suitable sampling model for on-site toxicity assessment of soluble air contaminants such as formaldehyde, a well known industrial and indoor air contaminant. The in vitro cytotoxicity of formaldehyde, the selected model for soluble air contaminants, was studied using the MTS (tetrazolium salt) assay in two carcinoma cell lines, A549 epithelial lung and HepG2 hepatocarcinoma, and in skin fibroblasts. The cytotoxic effects of airborne formaldehyde were evaluated using test atmospheres in concentrations below 10 ppm (12.3 mg/m3), generated by a dynamic diffusion method and bubbled (0.3 L/min) through serum-free culture media for one or four hours. Human cells were treated with formaldehyde air samples, and cell viability was determined after four hours incubation. In parallel, the concentration of airborne formaldehyde was monitored, using the 3500 NIOSH method. Cell viability of the HepG2 cells exposed to formaldehyde air samples (8.75 ppm-4 h) was reduced to less than 50% (31.69/1.24%). The HepG2 cell lines were found to be more sensitive (IC50=103.799/23.55 mg/L) to formaldehyde than both A549 cell lines (IC50=198.369/9.54 mg/L) and skin fibroblasts (IC50=196.689/36.73 mg/L) (PB/0.01). An average of 96.8% was determined for collection efficiency of formaldehyde in serum-free culture media. The results of this study suggest that absorption of soluble air contaminants, such as formaldehyde, in serum-free culture media can be used as a suitable sampling model for on-site toxicity assessments.

Quantitation of FcγRII mRNA in platelets and megakaryoblastic cell lines by a new method of in situ hybridization

We have developed a highly sensitive and quantitative, non-isotopic method of in situ hybridization in which the level of probe binding to intracellular mRNA is determined using an ELISA based detection method. Highly purified cell preparations or cells from a cultured cell line are centrifuged into 96 well microtiter plates. The cells are fixed with formalin and pre-treated with Triton X-100 and Nonidet P40 before photobiotin labeled cDNA probes are applied. The biotin from the hybridization is detected using multiple applications of streptavidin and biotinylated alkaline phosphatase and then visualized by the p-NPP (p-nitrophenyl phosphate) conversion method. We have determined a number of the optimal parameters in the procedure including the effects of cell numbers per well, development times and standardization of data using ubiquitous beta-actin mRNA and poly-A+ RNA expression as controls. We have used the technique to study the level of expression of FcgR mRNA in platelets and precursors. We found that platelets and megakaryoblastic cell lines only express mRNA for Fc gamma RII. The presence of the Fc gamma RII molecules was confirmed by complementary studies using immunohistochemistry with specific monoclonal antibodies IV.3 and KB61.

Quantitation of soluble and membrane-bound FC7RIIA (CD32A) mRNA in platelets and megakaryoblastic cell line (Meg-01)

Summary .FCγ receptors (FCγRS) are glycoproteins on platelet surface that bind IgG‐containing immune complexes. However, excessive binding of immune complexes leads to platelet activation and thrombosis or increased platelet clearance and thrombocytopenia. In this study, FC7R transcripts in platelets and megakaryoblastic cell line (Meg‐01) were investigated using specifically designed oligonucleotides and a new quantitative in situ hybridization assay. Platelets and Meg‐01 cells were found to express only FCγRII transcripts. Of FCγRIIA mRNA isoforms (FCγRIIA, B and C), FCgMRIIA mRNA predominates in these cells. Platelets and Meg‐01 cells contain both alternative spliced forms of FCγRIIA mRNA, those with and without the transmembrane (TM) exon and both forms were present in near equal amounts. In contrast, FCγRIIA transcript with the TM exon predominates in neutrophils and monocytes, suggesting that the splicing of the TM exon is under lineage‐specific control.

Quantitative aspects of anin situ hybridization procedure for detecting mRNAs in cells using 96-well microplates

The universal quantitation of the DNA hybridization reaction has been a goal sought by many researchers. Part of this search has been the need to develop a rapid, sensitive, easy-to-perform, and quantitative method to measure the abundance of specific mRNAs directly within cells. Conventionally mRNA detection can be done by advanced quantitativein situ hybridization (ISH) using either image analysis or fluorescencein situ hybridization (FISH), or indirectly by extraction of mRNA from cells or tissue and using Northern blot or quantitative polymerase chain reaction (PCR). We examined the quantitative nature of probe binding to intracellular mRNA in a sensitive and easy-to-use nonisotopic method of ISH previously developed in our laboratories. The method is applicable to isolated primary cells or cells in culture. The procedural details are very simple, with cells being centrifuged into 96-well microplates, fixed with formalin, and pretreated with Triton X-100 and Nonidet P-40 before photobiotin-labeled cDNA probes are applied. Biotin from the hybridization of probe to target is detected using multiple applications of streptavidin and biotinylated alkaline phosphatase and visualized by thep-nitrophenyl phosphate conversion method. The quantitative parameters of the ISH procedure were determined by measuring the levels of expression of erythropoietin (EPO) mRNA and its translated protein in transfected COS-7 cells. There is a log-linear relationship between the levels of signal obtained in the ISH reaction in 96-well microplates and the EPO protein levels measured by enzyme-linked immunosorbent assay (ELISA). This demonstrated relationship is important in the standardization and use of these procedures to measure quantitatively mRNAs within cells.

An immunohistological study of anhydrous topical ascorbic acid compositions on ex vivo human skin

Background  Ascorbic acid has numerous essential and beneficial functions in normal and photoaged skin. Ionisation of ascorbic acid in aqueous topical formulations leads to oxidative degradation. Ascorbic acid in an anhydrous vehicle would inherently have greater stability.