Christian Khalil

Toxicity assessment of industrial chemicals and airborne contaminants: Transition from in vivo to in vitro test methods : A review

Exposure to occupational and environmental contaminants is a major contributor to human health problems. Inhalation of gases, vapors, aerosols, and mixtures of these can cause a wide range of adverse health effects, ranging from simple irritation to systemic diseases. Despite significant achievements in the risk assessment of chemicals, the toxicological database, particularly for industrial chemicals, remains limited. Considering there are approximately 80,000 chemicals in commerce, and an extremely large number of chemical mixtures, in vivo testing of this large number is unachievable from both economical and practical perspectives. While in vitro methods are capable of rapidly providing toxicity information, regulatory agencies in general are still cautious about the replacement of whole-animal methods with new in vitro techniques. Although studying the toxic effects of inhaled chemicals is a complex subject, recent studies demonstrate that in vitro methods may have significant potential for assessing the toxicity of airborne contaminants. In this review, current toxicity test methods for risk evaluation of industrial chemicals and airborne contaminants are presented. To evaluate the potential applications of in vitro methods for studying respiratory toxicity, more recent models developed for toxicity testing of airborne contaminants are discussed.

A novel in vitro exposure technique for toxicity testing of selected volatile organic compounds

Exposure to vapours of volatile chemicals is a major occupational and environmental health concern. Toxicity testing of volatile organic compounds (VOCs) has always faced significant technological problems due to their high volatility and/or low solubility. The aim of this study was to develop a practical and reproducible in vitro exposure technique for toxicity testing of VOCs. Standard test atmospheres of xylene and toluene were generated in glass chambers using a static method. Human cells including: A549-lung derived cell lines, HepG2-liver derived cell lines and skin fibroblasts, were grown in porous membranes and exposed to various airborne concentrations of selected VOCs directly at the air/liquid interface for 1 h at 37 degrees C. Cytotoxicity of test chemicals was investigated using the MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) and NRU (neutral red uptake) assays following 24 h incubation. Airborne IC(50) (50% inhibitory concentration) values were determined using dose response curves for xylene (IC(50)=5350+/- 328 ppm, NRU; IC(50)=5750+/- 433 ppm, MTS in skin fibroblast) and toluene (IC(50)=0 500+/- 527 ppm, NRU; IC(50)=11,200 +/- 1,044 ppm, MTS in skin fibroblast). Our findings suggest that static direct exposure at the air/liquid interface is a practical and reproducible technique for toxicity testing of VOCs. Further, this technique can be used for inhalational and dermal toxicity studies of volatile chemicals in vitro as the exposure pattern in vivo is closely simulated by this method.

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.

Purified c-phycoerythrin: safety studies in rats and protective role against permanganate-mediated fibroblast-DNA damage†

We have evaluated in vitro cytotoxicity of cyanobacterial phycoerythrin (C‐PE) on three human cell lines by cell proliferation and neutral red uptake assays. No toxic effects of C‐PE were observed to any of the cell lines tested. The protective role of purified C‐PE to potassium permanganate‐mediated human fibroblast‐DNA damage was assessed by comet assay at 0 (control), 10 and 20 µg C‐PE ml−1 doses in pre‐, simultaneous and post‐mutagen exposure conditions. Significant DNA damage was detected only in post‐mutagen exposure conditions. Our findings confirmed that the C‐PE is non‐toxic and provides protection against permanganate‐mediated DNA damage. The preliminary acute (2000 mg C‐PE kg−1 body weight, b.w.) and 90 day sub‐chronic (0, 5, 15 and 25 mg C‐PE kg−1 b.w./day) oral toxicity studies of purified C‐PE in male albino rats showed no mortality or treatment‐related major clinical signs, and all the doses of C‐PE were well tolerated. The no observed adverse effect level and no observed effect level were found to be 15 and 5 mg C‐PE kg−1 b.w./day respectively. Copyright

Purified c-phycoerythrin

We have evaluated in vitro cytotoxicity of cyanobacterial phycoerythrin (C‐PE) on three human cell lines by cell proliferation and neutral red uptake assays. No toxic effects of C‐PE were observed to any of the cell lines tested. The protective role of purified C‐PE to potassium permanganate‐mediated human fibroblast‐DNA damage was assessed by comet assay at 0 (control), 10 and 20 µg C‐PE ml−1 doses in pre‐, simultaneous and post‐mutagen exposure conditions. Significant DNA damage was detected only in post‐mutagen exposure conditions. Our findings confirmed that the C‐PE is non‐toxic and provides protection against permanganate‐mediated DNA damage. The preliminary acute (2000 mg C‐PE kg−1 body weight, b.w.) and 90 day sub‐chronic (0, 5, 15 and 25 mg C‐PE kg−1 b.w./day) oral toxicity studies of purified C‐PE in male albino rats showed no mortality or treatment‐related major clinical signs, and all the doses of C‐PE were well tolerated. The no observed adverse effect level and no observed effect level were found to be 15 and 5 mg C‐PE kg−1 b.w./day respectively. Copyright

Characterization of cytotoxicity of airborne particulates from urban areas of Lahore

A number of species (organic and inorganic) in airborne particulates cause the toxicity to living being. The potential of in vitro test methods were explored for toxicity assessment of trace toxic elements (inorganic species) present in ambient air on human being (lungs). A year long sampling of airborne particles (PM2.5) was carried (April 2008 to March 2009) in Lahore, Pakistan. A total of thirty nine samples were collected on 47 mm Zefluor Teflon filter membranes and each was analysed to characterize for the elements: Sb, As, Be, Cd, Cr, Co, Pb, Mn, Hg using ICP-MS in water extract and total acid digestate. The samples cytotoxicity was also established using lung derived cells and MTS colorimetric assays. This generated dose response curves and IC50 values for the elemental mixtures identified on the Teflon filter membrane. The results indicated that even at low concentrations airborne elemental mixtures displayed an additive toxic effect.

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

Graphical abstract

Human skin explants an in vitro approach for assessing UVB induced damage

Lifestyle changes involving frequent outdoor activities are contributing to higher exposure to harmful ultraviolet light (UVB). The acute effects of UVB irradiation on human skin was evaluated in this study using freshly excised human skin from elective surgery subjected to UVB doses (0-3.76 J/cm2). The assessment of UVB induced cellular and skin damages was undertaken at two time points immediately and 24 h post exposure using in vitro, and immunohistochemical staining techniques. The results indicated no significant loss of skin integrity or significant acute mitochondrial cellular damages in UVB exposed skin sections as measured by the MTS cytotoxicity assay. The other key markers of damage showed significant extracellular LDH membrane leakages and upregulation of inflammatory cytokines such as IL-1β. Skin integrity analysis was also undertaken using H&E, HLADR, and anti-cytokeratin antibodies. The results showed significant epidermal changes, basal cell activation and Langerhans cells depletion. The research proved the usefulness of freshly excised human skin explant model in measuring UVB damage. Furthermore, freshly excised human skin maintains the natural layering and therefore does not pose the same challenges faced by commercially available reconstructed skin in terms of higher costs and accurate mimicking of all the complex interactions observed in human skin.

Toxicity Of volatile organic compounds (VOCs) mixtures using human derived cells

Assessing the effects of contaminants is an issue of high priority for governmental safety health and environmental agencies around the world. The general conservative consensus is that chemicals in mixtures interact by concentration addition. However, previous studies also report that concentration addition of mixture components does not always reflect the overall toxicity of a mixture. Volatile organic compounds (VOCs) such as Benzene, Toluene, Xylene and Formaldehyde (BTXF) belong to the air pollutants found in urban and indoor environments. They could trigger acute and chronic adverse health effects like allergy, respiratory and cardiovascular diseases. The volatile nature of these compounds poses additional problems in assessing individual volatile chemical toxicity let alone mixtures of these chemicals. Our research aims at establishing the true toxic effects of VOC exposure in vitro using a static direct exposure glass-chamber method. This was achieved by assessing and comparing individual and interactive effects of VOCs in exposed human epithelial lung (A549) and liver cells (HepG2) using the MTS cytotoxicity assay to assess cell viability upon VOC insult. The study results clearly indicated the limitation of the concentration addition method used in assessing volatile mixtures cytotoxicity and the need to develop new techniques for rapid and accurate mixture toxicity determination. The study may have implications for regulatory risk assessment of environmental volatile organic chemicals.