Comparative studies of apoptosis in Xenopus laevis and mouse thymoma cell lines.

With the use of in vitro methods and cell lines, functional aspects of apoptosis in the Xenopus laevis B3/B7 and mouse EL4 thymoma cell lines are revealed. Moreover, by using information gleaned from digital imaging and immunocytochemistry, changes in locations of key proteins implicated in apoptotic anti-cancer responses, e.g. p53 and Mdm2, are shown. Suggestions are offered as to what these results might mean with respect to the evolutionary conservation of the function and structure of these two molecules and to cancer resistance in amphibians. Finally, studies are described on resveratrol as an anti-cancer therapeutic reagent in the two thymoma cell lines and in normal X. laevis thymocytes.

Resistance to cancer in amphibians: a role for apoptosis?

The rarity of spontaneous cancer in amphibians, and the difficulty of inducing cancer in these lower vertebrates, suggest that they possess an effective system for resistance to the development of cancer. The first part of this narrative presents evidence for cancer resistance in amphibians, and then a variety of studies designed to help understand the physiological basis for this resistance are reviewed. Here, our emphasis is on evidence with regard to the role that apoptosis might play.

An evaluation of the chick cardiomyocyte micromass system for identification of teratogens in a blind trial.

The chick micromass culture system has advantages over the validated rat system - ready availability and non-culling of the donor parent - but needs to give comparable results. This study confirmed comparability and the ability to extend the system to cover cardiac effects. It was also compared with the validated embryonic stem cell cardiomyocyte model. A teratogen and paired non-teratogen with known in vivo effects were used. Differential effects were measured via changes in cell protein content, cell viability (resazurin reduction and neutral red uptake), and cell contractility. Results showed that teratogens [L-ethionine, 5-fluorouracil and sulphisoxazole] could be distinguished from non-teratogens [DL-methionine, 6-methyluracil and sulphanilamide respectively]. Dichloroacetone and dichloropropanol affected embryonic stem cells but not the micromass; dichloropropanol had a greater effect than dichloroacetone. This approach revealed differential effects on contractility independent of effects on activity/viability, whilst the total cell protein remained unchanged. We suggest that pre-validation of this system should be examined.

The effects of terahertz radiation on human keratinocyte primary cultures and neural cell cultures.

Terahertz (THz) frequencies are found in a previously underexploited region of the radiation spectrum. This non-ionising energy is now being employed in medical imaging, so the possibility of adverse effects on human skin was evaluated. Primary cultures of normal human keratinocytes (NHKs) express adhesion molecules that comprise part of the natural barrier function of the skin. The effects of exogenous agents on this barrier function can be measured. The ND7/23 cell line, which displays the characteristics of sensory neurones, can proliferate in the undifferentiated state, but can be induced to differentiate and develop neurite-like projections. Previous studies with NHK and neural cell cultures produced no evidence of the inability of these cells to differentiate and form a barrier following THz exposure. The cells were exposed to 0.14THz radiation for times varying from 10 minutes to 24 hours. For each 80-nanosecond pulse, the cells were exposed to a peak power of between 24 and 62mW/cm(2), i.e. a total energy at peak power of 345J, or 86J at average power over 24 hours. No changes in cell activity occurred, as monitored with the resazurin reduction assay, or with the barrier function of the human corneal cells, as measured with the fluorescein leakage assay. The monitoring of differentiation by using an assay for cornified envelope formation, revealed no adverse effects. Glutathione (GSH) and heat shock protein 70 levels were examined before and after differentiation, to determine the degree of the stress response, with the effects of UVB radiation as a control. UVB induced a stress response, as did heat shock treatment at 43 degrees C, whilst 0.15THz radiation, even after 24 hours of exposure, did not. Repeated exposure to THz radiation at this level, also resulted in no detectable adverse reactions.

Cancer resistance in amphibians.

While spontaneous tumours may occasionally develop in inbred and isogenic strains of Xenopus laevis, the South African clawed toad, they are extremely rare in natural and laboratory populations. Only two amphibian neoplasms, the renal adenocarcinoma of Rana pipiens and the lymphosarcoma of Xenopus laevis, have been extensively explored. Amphibians are resistant to the development of neoplasia, even following exposure to "direct-acting" chemical carcinogens such as N-methyl-N-nitrosourea, that are highly lymphotoxic, thus diminishing immune reactivity. Regenerative capacity in adults, and a dramatic metamorphosis which remodels much of the larval body to produce the adult form, are unique to amphibian vertebrates, and the control mechanisms involved may protect against cancer. For example, naturally rising corticosteroid titres during metamorphosis will impair some T-cell functions, and the removal of T-regulatory (suppressor) functions inhibits the induction of altered-self tolerance. Altered-self tolerance is not as effectively induced in adult Xenopus laevis as in mammals, so cancer cells with new antigenicity are more likely be rejected in amphibians. Amphibian immunocytes tend to undergo apoptosis readily in vitro, and, unlike mammalian immunocytes, undergo apoptosis without entering the cell cycle. Cells not in the cell cycle that die from nuclear damage (apoptosis), will have no opportunity to express genetic instability leading to cell transformation. We suggest that all these factors, rather than any one of them, may reduce susceptibility to cancer in amphibians.

An evaluation of the effects of photoactivation of bithionol, amiodarone and chlorpromazine on human keratinocytes in vitro.

Human skin is a continual target for chemical toxicity, due to its constant exposure to xenobiotics. The skin possesses a number of protective antioxidant systems, including glutathione and enzymic pathways, which are capable of neutralising reactive oxygen species (ROS). In combination with certain chemicals, the presence of ROS might augment the levels of toxicity, due to photoactivation of the chemical or, alternatively, due to an oxidatively-stressed state in the skin which existed prior to exposure to the chemical. Bithionol is a phototoxic anti-parasitic compound. The mechanism of its toxicity and the possible methods of protection from its damaging effects have been explored. The capacity of keratinocytes to protect themselves from bithionol and other phototoxic chemicals has been investigated. In addition, the potential of endogenous antioxidants, such as vitamin C and E, to afford protection to the cells, has been evaluated. The intracellular glutathione stores of HaCaT keratinocytes were reduced following treatment with biothionol. Following photoactivation, both bithionol and chlorpromazine had similar effects, which suggests that glutathione is important in the detoxification pathway of these chemicals. This was confirmed by means of the visual identification of fluorescently-labelled glutathione. Endogenous antioxidants were unable to protect the HaCaT keratinocytes from bithionol toxicity or chlorpromazine phototoxicity. Amiodarone was shown to have no effect on cellular glutathione levels, which suggests that an alternative mechanism of detoxification was occurring in this case. This was supported by evidence of the protection of HaCaT cells from amiodarone phototoxicity via endogenous antioxidants. Thus, it appears that amiodarone toxicity is dependent on the levels of non-gluathione antioxidants present, whilst bithionol and chlorpromazine detoxification relies on the glutathione antioxidant system. This type of approach could indicate the likely mechanisms of phototoxicity of chemicals in vitro, with relevance to potential effects in vivo.

Development of an innervated model of human skin.

Neuronal cell responses and interactions with the epithelial and fibroblastic cells of the skin are a key factor in the production in vivo of the irritation/inflammatory response. Currently, few in vitro models are available that contain dermal, epidermal and the relevant neuronal components. The primary objective of this study was to produce and maintain a 3-D in vitro model of human skin containing these elements. The relevant neuronal component was supplied by adding sensory neurons derived from the dorsal root ganglion (DRG). Since adult neuronal cells do not grow significantly in vivo or in vitro, and since it is very difficult to obtain such cells from humans, it was necessary to employ embryonic rat DRG cells. The ultimate purpose of this model is to improve prediction of the in vivo skin irritancy potential of chemicals and formulations, without the need to use animal models. In addition, this approach has also been applied to the in vitro human eye and bronchial 3-D models being developed in the FRAME Alternatives Laboratory.

A review of in vitro modelling approaches to the identification and modulation of squamous metaplasia in the human tracheobronchial epithelium.

Squamous metaplasia in the tracheobronchial epithelium (TBE) involves the replacement of the normal pseudostratified mucociliary epithelium with a stratified squamous epithelium. Squamous metaplasia is considered to be an adaptive response that protects the lumen from the effects of inhaled airborne pollutants, but which might also feature as a pre-neoplastic lesion preceding squamous cell carcinoma. With the exception of transglutaminase I, involucrin, and cytokeratins 5, 6 and 13, few markers that contribute to the squamous phenotype have been identified in human TBE that can be used in diagnosis or to monitor its development in laboratory investigations, and current models are inadequate to provide statistically meaningful data. Therefore, new predictive markers have been identified, and new techniques established, in epithelial in vitro models capable of expressing squamous characteristics, which will be used to identify hazardous exposures and elucidate the mechanisms by which they induce their effects. A protocol for the quantitative detection of transglutaminase activity has been standardised in keratinocytes, based on the enzymatic incorporation of fluorescein-cadaverine (FC) into bis(gamma-glutamyl) polyamine cross-links. The specificity of this compound as a transglutaminase substrate was demonstrated by using a range of competitive transglutaminase inhibitors, and by modulation of the squamous pathway. FC incorporation was localised to the cell membrane of terminally differentiating cells, and was not visible in basal, proliferating cells. High calcium-containing medium, nicotine and cigarette smoke condensates (CSC) induced an increase in FC incorporation, providing evidence of their role in enhancing the squamous pathway. Analysis by flow cytometry was used to provide a quantitative assessment of a range of optimised squamous differentiation markers, identified in normal human bronchial epithelia and in a bronchial cell line. Transglutaminase I was induced in a time-dependent manner, in post-confluent cells induced to differentiate down the squamous pathway, whereas involucrin was ubiquitously expressed and the levels of cytokeratins 5, 6 and 18 were reduced. The response of these and other differentiation markers to squamous-inducing conditions is being explored.

An evaluation of a novel chick cardiomyocyte micromass culture assay with two teratogens/embryotoxins associated with heart defects.

This study was aimed at determining whether the chick cardiomyocyte micromass (MM) system could be employed to predict the teratogenicity/embryotoxicity of exogenous chemicals. Two documented teratogens/embryotoxins, sodium valproate (the sodium salt of valproic acid; VPA) and all-trans retinoic acid (tRA), were used in the initial phase of the study. White Leghorn 5-day-old embryo hearts were dissociated to produce a cardiomyocyte suspension in Dulbecco's Modified Eagle's Medium. Cultures were incubated at 37 degrees C in 5% CO(2) in air, and observations were made every 24 hours over 5 days, for the detection of beating. Culture viability was assessed by using the resazurin reduction assay for determining culture activity and the kenacid blue assay for determining cell number. It was found that tRA significantly reduced cell activity and beating, whilst not affecting total cell number. VPA up to 500 microM induced no cytotoxicity in the MM cardiomyocyte cultures, whilst all the VPA concentrations tested reduced beating. The results demonstrate the potential of the chick cardiomyocyte MM culture assay to identify teratogens/embryotoxins that alter functionality, which may result in a teratogenic outcome, whilst not causing cytotoxicity (direct embryotoxicity). This could form part of a screen for developmental toxicity related to cardiac function, whilst limb cultures and brain cultures based on the same system could be relevant to teratogenic effects on those tissues.

Phototoxicity and acute toxicity studies conducted by the FRAME Alternatives Laboratory: a brief review.

FRAME and the University of Nottingham have been in association for the past 25 years. During this time, the research in the FRAME Alternatives Laboratory (FAL) at the University of Nottingham, which is partly funded by FRAME and also, more recently, by ECVAM, has involved participation in a number of international validation studies. Validation has become a pre-requisite for the regulatory acceptance of in vitro alternative test procedures, and a number of key lessons have been learned from these studies. The directors of validation studies need to ensure that standard operating procedures (SOPs) are fully complied with, and that the equipment used is certified to be of an acceptable standard. Database managers need to be able to check the original data, and to ensure adherence to procedures agreed before the study began. When the validation study is part of an integrated EU Framework Project, such as ACute-Tox, the Workpackage Leader must have the ability to understand and evaluate the data to be presented for inclusion in the study analysis, and to check that it complies with acceptance criteria. The potential to relate observed cellular biochemical changes to morphological endpoints also increases the level of understanding of the relevance and/or limitations of an assay. For example, exposure to a surfactant can induce the temporary loss of adhesion junctions between adjacent epithelial cells, resulting in the loss of barrier integrity and other effects on cell culture activity, which can potentially be restored over time. Unexpected results from the NRU phototoxicity assay with human keratinocytes instead of 3T3 cells, stimulated research into the ability of the in vitro assay, not only to identify phototoxins, but also to identify their possible mechanisms of action and mechanisms underlying the protective capacity within human primary keratinocytes in vitro. The protective effects of UV-filters can also be used to ascertain their effects on the photoactivation of drugs.

Cannabinoid receptor agonists are mitochondrial inhibitors: a unified hypothesis of how cannabinoids modulate mitochondrial function and induce cell death.

Time-lapse microscopy of human lung cancer (H460) cells showed that the endogenous cannabinoid anandamide (AEA), the phyto-cannabinoid Delta-9-tetrahydrocannabinol (THC) and a synthetic cannabinoid HU 210 all caused morphological changes characteristic of apoptosis. Janus green assays of H460 cell viability showed that AEA and THC caused significant increases in OD 595 nm at lower concentrations (10-50 microM) and significant decreases at 100 microM, whilst HU 210 caused significant decreases at all concentrations. In rat heart mitochondria, all three ligands caused significant decreases in oxygen consumption and mitochondrial membrane potential. THC and HU 210 caused significant increases in mitochondrial hydrogen peroxide production, whereas AEA was without significant effect. All three ligands induced biphasic changes in either mitochondrial complex I activity and/or mitochondrial complex II-III activity. These data demonstrate that AEA, THC, and HU 210 are all able to cause changes in integrated mitochondrial function, directly, in the absence of cannabinoid receptors.

Comparison of an animal product free medium and normal growth supplement on the growth and barrier integrity of a human corneal epithelial cell line.

With the development of defined media for general and specific use with cell cultures, and concern over the use of human cells and over potential prion infections associated with growth factor extracts such as bovine pituitary extract, an animal product-free medium has become available. The basic keratinocyte defined medium can be used with a choice of animal product-containing or animal product-free supplements. Human corneal epithelia cell lines were cultured in the media with these two types of supplement, and compared in terms of their growth rates, their capacity to form tight barriers, and calcium regulation of the location of a junction-associated protein, zonula occludins-1 (ZO-1). The growth rates were not different in the two media, as long as the recommended coating was applied to the culture flask for the animal product-free medium. The barrier function was equally effective for confluent cultures seeded at the same densities. A calcium concentration of 100 microM or above resulted in ZO-1 localisation at the cell membrane in either medium. Hence, cultures in the media are comparable, when the coating is employed. Further experiments are being conducted to establish the comparability of responses to chronic treatment with surfactants.

The development of an innervated epithelial barrier model using a human corneal cell line and ND7/23 sensory neurons.

The corneal epithelium is a highly innervated tissue and hence in vitro models that mimic the effects of chemicals or radiation (e.g. ultra violet) on this important barrier should include consideration of the potential role of innervation. A sensory neural cell line, ND7/23, was incorporated into a 2D and 3D model of a corneal epithelium, using a human corneal cell line, and effects on barrier integrity were neither adverse nor stimulatory. In the 3D model the nerve cell bodies were separated from the corneal epithelium, via a porous polycarbonate insert membrane. The ND7/23 cells were induced to form neurites and cease division when cultured in the keratinocyte medium employed for the corneal cells. In the absence of calcium, the epithelial barrier function was lost, shown by enhanced fluorescein leakage and relocation of ZO-1 and E-cadherin from the cell membrane. At 60 microM calcium, and above, the corneal cells formed tight junctions, with peripheral membrane location of ZO-1 and E-cadherin. The presence of the ND7/23 cells did not compromise or enhance the time taken to form these junctions, when monitored at 24-h intervals over 72 h. Both male- and female-derived human corneal cell lines showed a similar tight junction functional response to different medium calcium concentrations in the presence or absence of the ND7/23 cells. Once differentiated in keratinocyte medium, patch-clamped ND7/23 cells were capable of producing a whole-cell current when exposed to low pH (5.4), indicative of the presence of active pH-gated ion channels.

The development of a standardised protocol to measure squamous differentiation in stratified epithelia, by using the fluorescein cadaverine incorporation technique.

Fluorescein cadaverine (FC) incorporation into cornified envelopes during squamous differentiation in stratified epithelia acts as a fluorescent substitute for endogenous transglutaminase substrates that can be visualised and quantified. The FC incorporation technique has been used to evaluate squamous differentiation in keratinocytes cultured in a medium that stimulates differentiation and in response to modulation by chemicals. A Standard Operating Procedure for the measurement of squamous differentiation is required as part of the prevalidation procedure for in vitro assays. In the present study, keratinocytes were isolated from the epidermis of 34 human donors. Cellular metabolic activity (resorufin production), total protein (kenacid blue uptake) and transglutaminase activity (FC incorporation) were measured in 87 and 21 independent runs at 6 and 12 days, respectively. Analysis of the control data showed that the cultures had a mean resorufin production that decreased over 12 days. This was inversely related to FC incorporation, which increased over 12 days. Mean protein concentration was reduced over the 12 days, but not in analyses that used donors for whom both 6-day and 12-day data were available. This information was used to define the normal limits within which the data should fall (mean +/- 1 SD). Data sets falling outside the normal limits performed statistically no differently from the normal responders, in experiments conducted to investigate the effects of chemicals on the modulation of squamous differentiation in keratinocytes. This was demonstrated by using compounds that modify transglutaminase expression and/or activity. All-trans retinoic acid significantly inhibited FC incorporation, but stimulated resorufin production at 1 x 10(-7)M and above. Nicotine significantly up-regulated both FC incorporation and resorufin production at 125 microg/ml. Hence, it was concluded that this robust assay approach, in which keratinocytes from a range of donors respond predictably to the test chemicals employed, did not justify the limitations that would be imposed by setting criteria that eliminated all data lying outside the normal range.

Methoctramine analogues inhibit responses to capsaicin and protons in rat dorsal root ganglion neurons.

We have investigated the possibility that vanilloid receptors have a binding site for polyamines and determined the consequences of binding to such a site. Whole-cell and single-channel patch-clamp recordings were used to investigate the effect of the tetraamine, methoctramine, and 16 of its analogues on capsaicin and proton induced responses of foetal rat dorsal root ganglion neurons. All but two methoctramine analogues inhibited responses to 10 microM capsaicin with IC50 values in the range of 2-70 microM at a holding potential of -100 mV. Inhibition was generally non-competitive and voltage-dependent. Methoctramine at 10 microM reduced the single channel mean open time (>3-fold), but also increased the mean closed time (1.7-fold). Sustained responses to pH 5.4 were antagonized by methoctramine with similar potency to capsaicin responses. Similar data were obtained with adult rat dorsal root ganglion neurons. These data indicate that methoctramine analogues bind to vanilloid receptors to inhibit their function.