Rapid assessment of the toxicity of oil sands process-affected waters using fish cell lines

Rapid and reliable toxicity assessment of oil sands process-affected waters (OSPW) is needed to support oil sands reclamation projects. Conventional toxicity tests using whole animals are relatively slow, costly, and often subjective, while at the same time requiring the sacrifice of test organisms as is the case with lethal dosage/concentration assays. A nonlethal alternative, using fish cell lines, has been developed for its potential use in supporting oil sands reclamation planning and to help predict the viability of aquatic reclamation models such as end-pit lakes. This study employed six fish cell lines (WF-2, GFSk-S1, RTL-W1, RTgill-W1, FHML, FHMT) in 24 h viability assays for rapid fluorometric assessment of cellular integrity and functionality. Forty-nine test water samples collected from the surface of oil sands developments in the Athabasca Oil Sands deposit, north of Fort McMurray, Alberta, Canada, were evaluated in blind. Small subsample volumes (8 ml) were mixed with 2 ml of 5× concentrated exposure media and used for direct cell exposures. All cell line responses in terms of viability as measured by Alamar blue assay, correlated well with the naphthenic acids (NA) content in the samples (R ² between 0.4519 and 0.6171; p?<?0.0001) when data comparisons were performed after the bioassays. NA or total acid-extractable organics group has been shown to be responsible for most of the acute toxicity of OSPW and our results further corroborate this. The multifish cell line bioassay provides a strong degree of reproducibility among tested cell lines and good relative sensitivity of the cell line bioassay as compared to available in vivo data that could lead to cost effective, high-throughput screening assays.     

In vitro and in vivo toxicity evaluation of the freshwater cyanobacterium Heteroleiblenia kuetzingii

Cyanobacteria are prokaryotic organisms characterized by their ability to produce secondary metabolites with different biological activities. The aim of this work was to evaluate the in vitro and in vivo toxicity of the cosmopolitan freshwater cyanobacterium H. kuetzingii. An extract from H. kuetzingii and cyanobacterial growth media were assessed for presence of intracellular and extracellular toxins by in vitro tests using primary cell cultures from mouse kidney and fibroblasts, cell lines A549 and 3T3, a fish cell line RTgill-W1 as well as by a traditional in vivo mouse bioassay. The presence of toxicity was compared with the ELISA and HPLC data for corresponding cyanotoxins. In vitro tests showed pronounced cytotoxicity of the cyanobacterium extract and growth medium in which H. kuetzingii released potential extracellular toxic compounds as the mammalian cells were significantly more sensitive to exposure compared to the fish cells. Histopathological analyses of the liver and kidneys of treated mice showed pathological changes such as leukocyte infiltration and necrosis, changes in the proximal and distal convoluted tubules, lack of differentiation of Bowman’s space, enlarged Bowman’s capsules and massive hemorrhages. ELISA and HPLC analyses confirmed the presence of saxitoxins and microcystins at low concentrations. In addition, the histological analyses suggest that H. kuetzingii produces other, yet unknown toxic metabolites. Monitoring efforts are therefore required to evaluate the potential hazard for the freshwater aquatic systems and possible public health implications associated with this cyanobacterium.     

Oxidative Stress and Nano-Toxicity Induced by TiO2 and ZnO on WAG Cell Line

Metallic nanoparticles are widely used in cosmetics, food products and textile industry. These particles are known to cause respiratory toxicity and epithelial inflammation. They are eventually released to aquatic environment necessitating toxicity studies in cells from respiratory organs of aquatic organisms. Hence, we have developed and characterized a new cell line, WAG, from gill tissue of Wallago attu for toxicity assessment of TiO₂ and ZnO nanoparticles. The efficacy of the cell line as an in vitro system for nanoparticles toxicity studies was established using electron microscopy, cytotoxicity assays, genotoxicity assays and oxidative stress biomarkers. Results obtained with MTT assay, neutral red uptake assay and lactate dehydrogenase assay showed acute toxicity to WAG cells with IC₅₀ values of 25.29±0.12, 34.99±0.09 and 35.06±0.09 mg/l for TiO₂ and 5.716±0.1, 3.160±0.1 and 5.57±0.12 mg/l for ZnO treatment respectively. The physicochemical properties and size distribution of nanoparticles were characterized using electron microscopy with integrated energy dispersive X-ray spectroscopy and Zetasizer. Dose dependent increase in DNA damage, lipid peroxidation and protein carbonylation along with a significant decrease in activity of Superoxide Dismutase, Catalase, total Glutathione levels and total antioxidant capacity with increasing concentration of exposed nanoparticles indicated that the cells were under oxidative stress. The study established WAG cell line as an in vitro system to study toxicity mechanisms of nanoparticles on aquatic organisms.     

Establishment and characterization of permanent cell line from gill tissue of Labeo rohita (Hamilton) and its application in gene expression and toxicology

Rohu gill cell line (LRG) was established from gill tissue of Indian major carp (Labeo rohita), a freshwater fish cultivated in India. The cell line was maintained in Leibovitz's L-15 supplemented with 10 % foetal bovine serum (FBS). This cell line has been sub-cultured more than 85 passages over a period of 2 years. The LRG cell line consists of both epithelial and fibroblastic-like cells. The cells were able to grow at a wide range of temperatures from 22 to 32 °C, the optimum temperature being 28 °C. The growth rate of gill cells increased as the FBS proportion increased from 2 to 20 % at 28 °C. The plating efficiency was also high (34.37 %). The viability of the LRG cell line was 70–80 % after 6 months of storage in liquid nitrogen. The karyotype analysis revealed a diploid count of 50 chromosomes. The gill cells of rohu were successfully transfected with pEGFP-N1. Amplification of mitochondrial Cox1 gene using primers specific to L. rohita confirmed the origin of this cell line from L. rohita. The cytotoxicity of malathion was assessed in LRG cell line using multiple endpoints such as 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, Neutral Red assay, Alamar Blue assay and Coomassie Blue protein assay. Acute toxicity assay on fish was conducted by exposing L. rohita for 96 h to malathion under static conditions. Statistical analysis revealed good correlation with r ²?=?0.946–0.990 for all combinations between endpoints employed. Linear correlations between each in vitro effective concentration 50 and the in vivo lethal concentration 50 data were highly significant.     

水环境中工程纳米颗粒物的生态毒理学机理及理想模式生物的筛选

随着纳米技术产业的高速发展,大量工程纳米颗粒物(Engineering nano-particles,ENPs)被排放到自然水环境中,因此对其进行生态毒性及环境风险的研究尤为迫切。综述了ENPs在水环境中的毒理学机理及理想模式生物筛选的研究进展。目前的研究表明ENPs的毒性作用机制主要包括两方面:一是影响细胞信号通路,二是氧化应激造成基因表达的变化。此外,光催化活性、细胞表面附着、溶解特性、表面特征、赋存形态、溶剂效应及与其他环境污染物的协同作用也是可能的毒性作用机理。模式生物的筛选与确定在纳米生态毒理学研究中极为重要。鱼类作为水环境中普遍存在的脊椎动物,群落庞大,其具有行为端点敏感性高、且在生物毒性实验中存在明显的量效关系等特征,被认为是研究ENPs生态毒理学最适合的水生模式生物。研究表明针对在ENPs影响下的未成年鱼类的行为特征研究比传统的胚胎发育及致死率研究更为有效。无脊椎动物和浮游植物同样在各种水环境中普遍存在,对环境污染物极为敏感,且对有害物质具有显著的富集放大效应,因此作为模式生物也具有一定的优势。     

Changes in the selected hematological parameters and gill Na/K ATPase activity of juvenile crucian carp Carassius auratus during elevated ammonia exposure and the post-exposure recovery

The increase in concentration of ammonia in lake water during the degradation of algal blooms may last for several weeks and thus cause chronic toxicity to aquatic organisms. The purpose of this study was to assess the chronic toxicity of ammonia on the selected hematological parameters and gill Na⁺/K⁺ ATPase activity of juvenile crucian carp Carassius auratus during elevated ammonia exposure and the post-exposure recovery. Juvenile crucian carp were exposed in different ammonia solutions for 45 days and then immediately transferred to pristine freshwater to initiate a 15-day recovery period. Results showed sub-lethal ammonia significantly deters growth and a 15-day recovery period was not sufficient for the fish to compensate for the loss of growth. The fish exhibited a continuous decrease in red blood cell (RBC), the total hemoglobin (Hb), and gill Na⁺/K⁺ ATPase activity as the concentration of NH₃-N increased. After the 15-day recovery period, RBC, Hb, and gill Na⁺/K⁺ ATPase activity had recovered to similar levels as the controls.     

Transfection efficiency and cytotoxicity of cationic liposomes in primary cultures of rainbow trout (Oncorhynchus mykiss) gill cells

Immunisation of fish by immersion has been applied for inactivated, whole cell bacterins, where the gill epithelial cells are considered as one of the prime uptake sites. Antigen entry is a critical factor for delivery of vaccine antigens through the immersion route, also for DNA vaccines, and delivery systems like cationic liposomes may enhance uptake. In this study, the aim was to examine the efficiency of cationic liposomes as a means to transfect primary cultures of rainbow trout gill cells with plasmids encoding viral or reporter proteins. Furthermore, the effects of the concentration and composition of liposomes/lipoplex on the viability of the cells were evaluated. Transfection of the gill cells was possible with both plasmids following transfection with lipoplexes of a neutral charge. Low concentrations and neutral/negatively charged formulations were favourable with respect to the toxicity of the formulations. Given that the mucous barrier covering the gills is overcome, this system might be useful for the priming of the local immunity in the fish gills.     

Transfection efficiency and cytotoxicity of cationic liposomes in primary cultures of rainbow trout (Oncorhynchus mykiss) gill cells

Immunisation of fish by immersion has been applied for inactivated, whole cell bacterins, where the gill epithelial cells are considered as one of the prime uptake sites. Antigen entry is a critical factor for delivery of vaccine antigens through the immersion route, also for DNA vaccines, and delivery systems like cationic liposomes may enhance uptake. In this study, the aim was to examine the efficiency of cationic liposomes as a means to transfect primary cultures of rainbow trout gill cells with plasmids encoding viral or reporter proteins. Furthermore, the effects of the concentration and composition of liposomes/lipoplex on the viability of the cells were evaluated. Transfection of the gill cells was possible with both plasmids following transfection with lipoplexes of a neutral charge. Low concentrations and neutral/negatively charged formulations were favourable with respect to the toxicity of the formulations. Given that the mucous barrier covering the gills is overcome, this system might be useful for the priming of the local immunity in the fish gills.     

A mechanism for acute aluminium toxicity in fish

Aluminium is acutely toxic to fish in acid waters. The gill is the principal target organ and death is due to a combination of ionoregulatory, osmoregulatory and respiratory dysfunction. The toxic mechanism has hitherto received little direct consideration and is unknown. In this paper the mechanism of acute aluminium toxicity is approached from a chemical perspective. Symptomatic evidence of toxicity is taken from the literature and combined with our own research to elucidate a biochemically sound model to describe a possible mechanism of acute aluminium toxicity in fish. The proposed model delineates the chemical conditions immediately adjacent to the gill surface and emphasizes their importance in aluminium's toxic mode of action. The mechanism is shown to be bipartite. Aluminium binding to functional groups both apically located at the gill surface and intracellularly located within lamellar epithelial cells disrupts the barrier properties of the gill epithelium. The concomitant iono- and osmoregulatory dysfunction results in accelerated cell necrosis, sloughing and death of the fish. The mechanism of epithelial cell death is proposed as a general mechanism of aluminium-induced accelerated cell death.     

Effects of deoxynivalenol (DON) and its microbial biotransformation product deepoxy-deoxynivalenol (DOM-1) on a trout,pig, mouse,and human cell line

Deoxynivalenol (DON), a trichothecene produced by various Fusarium species, is one of the most prevalent food- and feed-associated mycotoxins. The effects of DON and deepoxy-deoxynivalenol (DOM-1) were assessed in five different cell lines from different tissues and species starting from the first line of defense, the trout gill (RTgill-W1) and pig intestinal cells (IPEC-1 and IPEC-J2) over immune cells, as second line of defense (mouse macrophages RAW 264.7) to human liver cells (HepG2). Viability was assessed with a WST-1 assay, except for RTgill-W1, where a neutral red (NR) and sulforhodamine B (SRB) assay was performed. Additionally, more sensitive parameters, such as interleukin-, nitric oxide (NO)-, and albumin-release were determined. Viability was affected by DON at concentrations starting at 10 μmol/L (RTgill-W1), 0.9 μmol/L (IPEC-1), 3.5 μmol/L (IPEC-J2), and 0.9 μmol/L (HepG2), whereas DOM-1 did not have such an effect. Additionally, NO was decreased (0.84 μmol/L DON), whereas interleukin (IL)-6 was increased (0.42 μmol/L DON) in lipopolysaccharide (LPS)-stimulated DON-, but not DOM-1-treated RAW cells. Tumor necrosis factor (TNF)-α release, however, was not affected. Interestingly, albumin secretion of HepG2 cells was decreased by both DON and DOM-1 but at a much higher concentration for DOM-1 (228 versus 0.9 μmol/L for DON). 98.9% of DOM-1 was retrieved by liquid chromatography tandem mass spectrometry at the end of the experiment, proving its stability. In this study, IL-6 was the most sensitive parameter, followed by NO and albumin release and viability for HepG2 and IPEC-1.     

In vitro growth of microsporidia Anncaliia algerae in cell lines from warm water fish

Anncaliia algerae is an aquatic microsporidium that most commonly infects mosquitoes but can be grown on the rabbit kidney cell line, RK-13. Spores were purified from RK-13 cultures and added to cell lines from warm water fish and from an insect. The cell lines were GFSK-S1 and GFB3C-W1 from goldfish skin and brain respectively, ZEB2J from zebrafish embryos, FHMT-W1 from fathead minnow testis, and Sf9 from ovaries of a fall armyworm moth. All cultures were maintained at 27°C. Infection was judged to have taken place by the appearance of sporonts and/or spores in cells and occurred in all cell lines. Spores were also isolated from ZEB2J cultures and used to successfully infect new cultures of ZEB2J, RK-13 and Sf9. These results suggest that cells of a wide range of vertebrates support A. algerae growth in vitro and fish cells can produce spores infectious to cells of mammals, fish, and insects.     

Trangenic fish as models in environmental toxicology

Historically, fish have played significant roles in assessing potential risks associated with exposure to chemical contamination in aquatic environments. Considering the contributions of transgenic rodent models to biomedicine, it is reasoned that the development of transgenic fish could enhance the role of fish in environmental toxicology. Application of transgenic fish in environmental studies remains at an early stage, but recent introduction of new models and methods demonstrates progress. Rapid advances are most evident in the area of in vivo mutagenesis using fish carrying transgenes that serve as recoverable mutational targets. These models highlight many advantages afforded by fish as models and illustrate important issues that apply broadly to transgenic fish in environmental toxicology. Development of fish models carrying identical transgenes to those found in rodents is beneficial and has revealed that numerous aspects of in vivo mutagenesis are similar between the two classes of vertebrates. Researchers have revealed that fish exhibit frequencies of spontaneous mutations similar to rodents and respond to mutagen exposure consistent with known mutagenic mechanisms. Results have demonstrated the feasibility of in vivo mutation analyses using transgenic fish and have illustrated their potential value as a comparative animal model. Challenges to development and application of transgenic fish relate to the needs for improved efficiencies in transgenic technology and in aspects of fish husbandry and use. By taking advantage of the valuable and unique attributes of fish as test organisms, it is anticipated that transgenic fish will make significant contributions to studies of environmentally induced diseases.     

Biochemical and hematological parameters and histological alterations in fish Cyprinus carpio L. as biomarkers for water pollution with chlorpyrifos

The possible effects of the chlorpyrifos (CPF) on the fish Cyprinus carpio L. exposed to sub-lethal concentrations (52, 79, and 158 µg/L) of CPF for 6 weeks were evaluated, and an analysis was made of their hematological parameters, biochemical parameters, and histology of various organs such as liver, gill, and muscle. At three concentrations, packed cell volume (PCV), mean corpuscular hemoglobin, Red blood cells (RBC) Hb, T3, T4, and total protein were significantly decreased in fish treated with CPF, whereas the parameters White blood cells (WBC), creatinine, Glutamic Oxaloacetic Transaminase (GOT) and Glutamic Pyruvic Transaminase (GPT) presented a significant increase at the two higher concentrations. However, the sub-chronic exposure to CPF resulted in histological lesions and caused clear damage to liver, muscles, and gill tissues of the fish Cyprinus carpio. Thus, we may conclude that the altered biochemical and hematological parameters can be used as efficient biomarkers in monitoring the toxicity of CPF in aquatic organisms. At the same time, histopathology proves as a reliable and easy tool for toxicological studies.     

Morphometrics of gills during growth and development of the air-breathing habit in Colisa fasciatus (Bloch and Schneider)

Gill area and other component parameters of Colisa fasciatus during early life were measured for fish larvae divided into two groups (a) exclusively aquatic and (b) bimodal breathers. Statistical analyses of the data in relation to body size yielded two significantly different straight lines (one for aquatic and other for bimodal breathers) for each parameter. Morphological examinations of gill arches indicated that an increase in the gill area was brought about mainly by an increase in the filament length. The higher slope value (2.41) of gill area in the aquatic phase than that in the bimodal phase ( b = 0.80) is suggestive of a higher weight-specific metabolism in the younger larvae. A heterogenous growth pattern during early ontogenesis of the fish results in intraspecific variation in the gill dimensions which might have been influenced by the acquisition of the air-breathing mechanism in the post-larval stage, besides ecological factors.     

Natural Mineral Particles Are Cytotoxic to Rainbow Trout Gill Epithelial Cells In Vitro

Worldwide increases in fluvial fine sediment are a threat to aquatic animal health. Fluvial fine sediment is always a mixture of particles whose mineralogical composition differs depending on the sediment source and catchment area geology. Nonetheless, whether particle impact in aquatic organisms differs between mineral species remains to be investigated. This study applied an in vitro approach to evaluate cytotoxicity and uptake of four common fluvial mineral particles (quartz, feldspar, mica, and kaolin; concentrations: 10, 50, 250 mg L⁻¹) in the rainbow trout epithelial gill cell line RTgill-W1. Cells were exposed for 24, 48, 72, and 96 h. Cytotoxicity assays for cell membrane integrity (propidium iodide assay), oxidative stress (H₂DCF-DA assay), and metabolic activity (MTT assay) were applied. These assays were complemented with cell counts and transmission electron microscopy. Regardless of mineral species, particles ≤2 µm in diameter were taken up by the cells, suggesting that particles of all mineral species came into contact and interacted with the cells. Not all particles, however, caused strong cytotoxicity: Among all assays the tectosilicates quartz and feldspar caused sporadic maximum changes of 0.8–1.2-fold compared to controls. In contrast, cytotoxicity of the clay particles was distinctly stronger and even differed between the two particle types: mica induced concentration-dependent increases in free radicals, with consistent 1.6–1.8-fold-changes at the 250 mg L⁻¹ concentration, and a dilated endoplasmic reticulum. Kaolin caused concentration-dependent increases in cell membrane damage, with consistent 1.3–1.6-fold increases at the 250 mg L⁻¹ concentration. All effects occurred in the presence or absence of 10% fetal bovine serum. Cell numbers per se were marginally affected. Results indicate that (i.) natural mineral particles can be cytotoxic to gill epithelial cells, (ii.) their cytotoxic potential differs between mineral species, with clay particles being more cytotoxic, and (iii.) some clays might induce effects comparable to engineered nanoparticles.     

A fish out of water: gill and skin remodeling promotes osmo- and ionoregulation in the mangrove killifish Kryptolebias marmoratus

The euryhaline, amphibious mangrove killifish Kryptolebias marmoratus is known to survive weeks out of water in moist environments. We tested the hypothesis that the skin is a site of osmo- and ionoregulation in K. marmoratus. We predicted that under terrestrial conditions, gill and skin remodeling would result in an enhanced role for skin and a diminished role for the gills in osmo- and ionoregulation. Fish were exposed to water-either freshwater (FW, 1‰) or hypersaline water (saltwater [SW], 45‰)-or air over a moist surface of FW or SW for 9 d and then recovered in water. When fish were emersed for 9 d, (22)Na and (3)H-H(2)O were exchanged across the cutaneous surface. Homeostasis of whole-body Cl(-) and water levels but not of Na(+) levels was maintained over 9 d in air. In air-exposed fish, there was a significant increase in the size of skin ionocytes (in SW), a decrease in the number of skin mucous cells (in SW), and an increase in the gill interlamellar cell mass relative to those of fish in water. Gill ionocytes were mostly embedded away from the external surface in air-exposed fish, but the number and size of ionocytes increased (in FW). Interestingly, skin ionocytes formed distinct clusters of 20-30 cells. The estimated number of ionocytes over the whole skin surface was comparable to that in the gills. Overall, the findings support the hypothesis that the skin is a site of osmo- and ionoregulation in K. marmoratus in aquatic and terrestrial environments. Reversible cellular and morphological changes to the skin and gills during air exposure probably enhanced the cutaneous contribution to ion and water balance.     

Differential effects of viral hemorrhagic septicaemia virus (VHSV) genotypes IVa and IVb on gill epithelial and spleen macrophage cell lines from rainbow trout (Oncorhynchus mykiss)

The two most prominent genotypes of viral hemorrhagic septicemia virus (VHSV) are -I in the Northeastern Atlantic region and -IV in North America, but much more is known about the cellular pathogenesis of genotype -I than -IV. VHSV genotype -IV is divided into -IVa from the Northeast Pacific Ocean and -IVb from the Great Lakes and both of which are less virulent to rainbow trout than genotype -I. In this work, infections of VHSV-IVa and -IVb have been studied in two rainbow trout cell lines, RTgill-W1 from the gill epithelium, and RTS11 from spleen macrophages. RTgill-W1 produced infectious progeny of both VHSV-IVa and -IVb. However, VHSV-IVa was more infectious than -IVb toward RTgill-W1: -IVa caused cytopathic effect (CPE) at a lower viral titre, elicited CPE earlier, and yielded higher titres. By contrast, no CPE and no increase in viral titre were observed in RTS11 cultures infected with either genotype. Yet in RTS11 all six VHSV genes were expressed and antiviral genes, Mx2 and Mx3, were up regulated by VHSV-IVb and -IVa. However, replication appeared to terminate at the translational stage as viral N protein, presumably the most abundant of the VSHV proteins, was not detected in either infected RTS11 cultures. In RTgill-W1, Mx2 and Mx3 were up regulated to similar levels by both viral genotypes, while VHSV-IVa induced higher levels of IFN1, IFN2 and LGP2A than VHSV-IVb.     

Effects of manufactured nanomaterials on fishes: a target organ and body systems physiology approach

Manufactured nanomaterials (NM) are already used in consumer products and exposure modelling predicts releases of ng to low μg l(-1) levels of NMs into surface waters. The exposure of aquatic ecosystems, and therefore fishes, to manufactured NMs is inevitable. This review uses a physiological approach to describe the known effects of NMs on the body systems of fishes and to identify the internal target organs, as well as outline aspects of colloid chemistry relevant to fish biology. The acute toxicity data, suggest that the lethal concentration for many NMs is in the mg l(-1) range, and a number of sublethal effects have been reported at concentrations from c. 100 μg to 1 mg l(-1). Exposure to NMs in the water column can cause respiratory toxicity involving altered ventilation, mucus secretion and gill pathology. This may not lead, however, to overt haematological disturbances in the short term. The internal target organs include the liver, spleen and haematopoietic system, kidney, gut and brain; with toxic effects involving oxidative stress, ionoregulatory disturbances and organ pathologies. Some pathology appears to be novel for NMs, such as vascular injury in the brain of rainbow trout Oncorhynchus mykiss with carbon nanotubes. A lack of analytical methods, however, has prevented the reporting of NM concentrations in fish tissues, and the precise uptake mechanisms across the gill or gut are yet to be elucidated. The few dietary exposure studies conducted show no effects on growth or food intake at 10-100 mg kg(-1) inclusions of NMs in the diet of O. mykiss, but there are biochemical disturbances. Early life stages are sensitive to NMs with reports of lethal toxicity and developmental defects. There are many data gaps, however, including how water quality alters physiological responses, effects on immunity and chronic exposure data at environmentally relevant concentrations. Overall, the data so far suggest that the manufactured NMs are not as toxic as some traditional chemicals (e.g. some dissolved metals) and the innovative, responsible, development of nanotechnology should continue, with potential benefits for aquaculture, fisheries and fish health diagnostics.     

Immortalisation of Primary Cells

Knowledge of the target cells is fundamental to maximise efficiency in attempts at immortalisation of specific cell types. It is also important to optimise the primary cell culture system to promote the survival of the target cell population. Other important factors that may influence the success in obtaining immortalised cells include the toxicity and efficiency of the immortalisation procedure. These can be assessed experimentally and if necessary appropriate techniques can be employed to purify the target cells. When cell lines have been established it is vital to assess them at an early stage for desired scientific and practical features as well as determining their stability and life-span. Furthermore, early characterisation of cell line authenticity (e.g. genetic characters, species of origin) and quality control testing will avoid wasted time and resources should contamination with micro-organisms or another cell line occur. Establishing a programme of immortalisation is a serious undertaking that should only be considered when there are no candidate continuous cell lines available. However, new approaches to modify the biology of cells to give extended life-span, whilst retaining the characteristics of differentiated cells in vivo, will hopefully provide valuable new substrates for in vitro toxicology.