Induction of anoikis by sodium arsenite in rat hepatoma FGC4 cells: comparison with cadmium chloride and implications for assessment of regulation of heat shock protein 70.

CONTEXT: Arsenic, a toxic metalloid with major health concerns, elicits upregulation of heat shock protein 70 (HSP70) in rat hepatoma FGC4 cells, together with evidence of detachment of viable cells from the growth substratum. OBJECTIVE: To determine if this cell detachment was linked to anoikis, and the impact of this on measurement of HSP70 expression. MATERIALS AND METHODS: FGC4 cells were exposed to sodium arsenite, and detached and attached cells were taken for assessment of cell viability, activation of procaspase-3, and expression of HSP70. RESULTS: Exposure to sodium arsenite led to loss of viable cells from the substratum, associated with apoptosis in detached, but not attached, cells. Upregulation of HSP70 of a similar magnitude was demonstrated in both cell populations. Exposure of cells to cadmium chloride, a toxic metal, also of major environmental concern and believed to act by an oxidative stress mechanism, produced very little release of viable cells from the culture substratum, was not associated with apoptosis, but did elicit a modest upregulation of HSP70 in both cell populations. DISCUSSION: Exposure of FGC4 cells to sodium arsenite elicits anoikis, a form of anchorage-dependent apoptosis, and assessment of the level of HSP70 upregulation in such cells should take account of the detached cell population. Further, the data suggest that this phenomenon is selective to sodium arsenite, rather than to another toxic element that shares a similar mechanism of toxicity.

A double-blind atropine trial for active learning of autonomic function.

Here, we describe a human physiology laboratory class measuring changes in autonomic function over time in response to atropine. Students use themselves as subjects, generating ownership and self-interest in the learning as well as directly experiencing the active link between physiology and pharmacology in people. The class is designed to concomitantly convey the importance of bias in experimentation by adopting a double-blind placebo-controlled approach. We have used this class effectively in various forms with ∼600 students receiving atropine over the last 16 yr. This class has received favorable feedback from staff and students of medicine, pharmacy, and neuroscience, and we recommend it for such undergraduates. The learning objectives that students are expected to achieve are to be able to 1) know the ethical, safety, and hygiene requirements for using human volunteers as subjects; 2) implement and explain a double-blind placebo-controlled trial; 3) design, agree, and execute a protocol for making (and accurately recording) precise reproducible measurements of pulse rate, pupil diameter, and salivary flow; 4) evaluate the importance of predose periods and measurement consistency to detect effects (including any reversibility) after an intervention; 5) experience direct cause-and-effect relationships integrating physiology with pharmacology in people; 6) calculate appropriate summary statistics to describe the data and determine the data's statistical significance; 7) recognize normal variability both within and between subjects in baseline physiological parameters and also recognize normal variability in response to pharmacological treatment; 8) infer the distribution and role of muscarinic receptors in the autonomic nervous system with respect to the heart, eye, and mouth; 9) identify and explain the clinical significance of differences in effect due to the route and formulation of atropine; 10) produce and deliver a concise oral presentation of experimental findings; and 11) produce a written report in the form of a short scientific research article. The results of a typical study are presented, which demonstrate that the administration of atropine by a subcutaneous injection elicited a significant increase in pulse rate and pupil diameter and a significant decrease in salivary flow, whereas administration of atropine in an oral liquid elicited significant effects on pulse rate and salivary flow, and an oral solid format elicited a significant alteration in salivary flow alone. More detailed analysis of the salivary flow data demonstrated clear differences between the routes of administration and formulation in the onset and magnitude of action of atropine.

Recombinant expression of aryl hydrocarbon receptor for quantitative ligand-binding analysis.

Recombinant expression of the aryl hydrocarbon receptor (AhR) yields small amounts of ligand-binding-competent AhR. Therefore, Spodoptera frugiperda (Sf9) cells and baculovirus have been evaluated for high-level and functional expression of AhR. Rat and human AhR were expressed as soluble protein in significant amounts. Expression of ligand-binding-competent AhR was sensitive to the protein concentration of Sf9 extract, and coexpression of the chaperone p23 failed to affect the yield of functional ligand-binding AhR. The expression system yielded high levels of functional protein, with the ligand-binding capacity (Bmax) typically 20-fold higher than that obtained with rat liver cytosol. Quantitative estimates of the ligand-binding affinity of human and rat AhR were obtained; the Kd for recombinant rat AhR was indistinguishable from that of native rat AhR, thereby validating the expression system as a faithful model for native AhR. The human AhR bound TCDD with significantly lower affinity than the rat AhR. These findings demonstrate high-level expression of ligand-binding-competent AhR, and sufficient AhR for quantitative analysis of ligand binding.

A reassessment of the neurotoxicity of pyrethroid insecticides.

The pyrethroids are a widely used class of insecticides to which there is significant human exposure. They are however generally regarded as safe to man, and there have been few reports of human fatalities. Their acute toxicity is dominated by pharmacological actions upon the central nervous system (CNS), predominantly mediated by prolongation of the kinetics of voltage-gated sodium channels, although other mechanisms operate. This review summarizes our present understanding of such actions and the pharmacological options to antagonize them. One significant problem is the very clear heterogeneity of pyrethroid sensitivity that is seen across sodium channel subtypes; however, the distribution and function of these across the central nervous system are poorly characterized. The review also provides an overview of recent studies that suggest additional effects of pyrethroids: developmental neurotoxicity, the production of neuronal death, and action mediated via pyrethroid metabolites. The evidence for these is at present equivocal, but all 3 carry important implications for human health.

Antioxidant activity of rosmarinic acid and its principal metabolites in chemical and cellular systems: Importance of physico-chemical characteristics.

Persistent accumulation of reactive oxygen species causes cellular oxidative stress which contributes strongly towards the induction and progression of various diseases. Therapeutic focus has therefore shifted towards the use of antioxidants, with recent interest in those of plant origin. In the current study, rosmarinic acid (RA) and its key metabolites were evaluated in non-cellular and cellular antioxidant assays, using quercetin (Q) as a positive control. The non-cellular assay was performed as scavenging of DPPH radical, whilst the cellular assay was performed as protection from an oxidant stress. Radical-scavenging activity of RA and two of its primary metabolites, CA and DHPLA, were comparable to that of Q, whilst FA was of lower potency and m-CoA was inactive. In the cellular assay, RA and CA were markedly less potent than Q, with DHPLA, FA and m-CoA being inactive, this being true in short-term (5-h) or long-term (20-h) exposure conditions. However, antioxidant potency of Q and methyl rosmarinate, a non-ionisable ester of RA, was similar in the non-cellular and short-term cellular assays. It is proposed that marked ionisation of organic acids such as RA and its metabolites at physiological pH greatly limits their intracellular accumulation, and so attenuates intrinsic antioxidant ability demonstrated in the non-cellular assay. This study demonstrates some of the factors that prevent well-known phytochemicals from progressing further along the drug discovery chain.

Phenotypic anchoring of arsenic and cadmium toxicity in three hepatic-related cell systems reveals compound- and cell-specific selective up-regulation of stress protein expression: implications for fingerprint profiling of cytotoxicity.

Exposure of cells to toxic chemicals is known to up-regulate the expression of a number of stress proteins (SPs), including metallothionein (MT) and members of the heat shock protein (HSP) family, and this response may allow the development of a fingerprint profile to identify mechanisms of toxicity in an in vitro toxicology setting. To test this hypothesis, three hepatic-derived cell culture systems (rat hepatoma FGC4 cell line, rat hepatocytes, human hepatoma HepG2 cell line) were exposed to cadmium (as CdCl2) and arsenic (as NaAsO2), two compounds believed to exert their toxicity through an oxidative stress mechanism, under conditions of phenotypic anchoring defined as minimal and mild toxicity (approximately 5 and 25% reduction in neutral red uptake, respectively). The expression of six SPs--MT, HSP25/27, HSP40, HSP60, HSP70, and HSP90--was then determined by ELISA. Expression of four of these SPs--MT, HSP25/27, HSP40 and HSP70--was up-regulated in at least one experimental condition. However, the patterns of expression of these four SPs varied across the experimental conditions, according to differences in toxicant concentration and/or level of toxicity, cell-type and toxicant itself. This lack of uniformity in response of a focussed set of mechanistically defensible targets suggests that similar problems may emerge when using more global approaches based on genomics and proteomics, in which problems of redundancy in targets and uncertain mechanistic relevance will be greater.

Sensory nerves, neurogenic inflammation and pain: missing components of alternative irritation strategies? A review and a potential strategy.

The eyes and skin are highly innervated by sensory nerves; stimulation of these nerves by irritants may give rise to neurogenic inflammation, leading to sensory irritation and pain. Few in vitro models of neurogenic inflammation have been described in conjunction with alternative skin and eye irritation methods, despite the fact that the sensory innervation of these organs is well-documented. To date, alternative approaches to the Draize skin and eye irritation tests have proved largely successful at classifying severe irritants, but are generally poor at discriminating between agents with mild to moderate irritant potential. We propose that the development of in vitro models for the prediction of sensory stimulation will assist in the re-classification of the irritant potential of agents that are under-predicted by current in vitro strategies. This review describes the range of xenobiotics known to cause inflammation and pain through the stimulation of sensory nerves, as well as the endogenous mediators and receptor types that are involved. In particular, it focuses on the vanilloid receptor, its activators and its regulation, as these receptors function as integrators of responses to numerous noxious stimuli. Cell culture models and ex vivo preparations that have the potential to serve as predictors of sensory irritation are also described. In addition, as readily available sensory neuron cell line models are few in number, stem cell lines (with the capacity to differentiate into sensory neurons) are explored. Finally, a preliminary strategy to enable assessment of whether incorporation of a sensory component will enhance the predictive power of current in vitro eye and skin testing strategies is proposed.

Phytochemicals content, antioxidant activity and acetylcholinesterase inhibition properties of indigenous Garcinia parvifolia fruit.

Garcinia parvifolia belongs to the same family as mangosteen (Garcinia mangostana), which is known locally in Sabah as "asam kandis" or cherry mangosteen. The present study was conducted to determine the phytochemicals content (total phenolic, flavonoid, anthocyanin, and carotenoid content) and antioxidant and acetylcholinesterase inhibition activity of the flesh and peel of G. parvifolia. All samples were freeze-dried and extracted using 80% methanol and distilled water. For the 80% methanol extract, the flesh of G. parvifolia displayed higher phenolic and flavonoid contents than the peel, with values of 7.2 ± 0.3 mg gallic acid equivalent (GAE)/g and 5.9 ± 0.1 mg rutin equivalent (RU)/g, respectively. Anthocyanins were detected in the peel part of G. parvifolia but absent in the flesh. The peel of G. parvifolia displayed higher total carotenoid content as compared to the flesh part with the values of 17.0 ± 0.3 and 3.0 ± 0.0 mg ß-carotene equivalents (BC)/100 g, respectively. The free-radical scavenging, ferric reducing, and acetylcholinesterase inhibition effect of the flesh were higher as compared to the peel in both extracts. These findings suggested that the edible part of G. parvifolia fruit has a potential as a natural source of antioxidant and anti-Alzheimer's agents.

Cellular assessment of the extract of bambangan (Mangifera pajang) as a potential cytoprotective agent for the human hepatocellular HepG2 cell line.

This study was conducted to investigate the potential of bambangan (Mangifera pajang) fruit extracts in the protection against oxidative damage caused by tert-butyl hydroperoxide in the human hepatocellular HepG2 cell line. Proteins which might be involved in the cytoprotective mechanism were investigated using western blotting technique. Quercetin was used as a positive control. The results showed that only the kernel extract of M. pajang and quercetin displayed cytoprotective activity in HepG2 cells, with EC(50) values of 1.2 and 5.3µg/ml, respectively. Expression of quinone reductase, glutathione reductase and methionine sulfoxide reductase A proteins were significantly up-regulated by quercetin, suggesting their involvement in the cytoprotective activity of quercetin. However, expressions of only glutathione reductase and methionine sulfoxide reductase A proteins were significantly up-regulated by the kernel extract, again suggesting their involvement in the cytoprotective activity of bambangan kernel extract. Future study is needed to investigate the involvement of other cytoprotective proteins in the cytoprotection mechanism.

Cytotoxicity, cell cycle arrest, and apoptosis in breast cancer cell lines exposed to an extract of the seed kernel of Mangifera pajang (bambangan).

An extract of Mangifera pajang kernel has been previously found to contain a high content of antioxidant phytochemicals. The present research was conducted to investigate the anticancer potential of this kernel extract. The results showed that the kernel crude extract induced cytotoxicity in MCF-7 (hormone-dependent breast cancer) cells and MDA-MB-231 (non-hormone dependent breast cancer) cells with IC50 values of 23 and 30.5 microg/ml, respectively. The kernel extract induced cell cycle arrest in MCF-7 cells at the sub-G1 (apoptosis) phase of the cell cycle in a time-dependent manner. For MDA-MB-231 cells, the kernel extract induced strong G2-M arrest in cell cycle progression at 24h, resulting in substantial sub-G1 (apoptosis) arrest after 48 and 72 h of incubation. Staining with Annexin V-FITC and propidium iodide revealed that this apoptosis occurred early in both cell types, 36 h for MCF-7 cells and 24 h for MDA-MB-231 cells, with 14.0% and 16.5% of the cells respectively undergoing apoptosis at these times. This apoptosis appeared to be dependent on caspase-2 and -3 in MCF-7 cells, and on caspase-2, -3 and -9 in MDA-MB-231 cells. These findings suggest that M. pajang kernel extract has potential as a potent cytotoxic agent against breast cancer cell lines.

The effect of three-dimensional co-culture of hepatocytes and hepatic stellate cells on key hepatocyte functions in vitro.

In this study, we demonstrate the ability of a three-dimensional co-culture model to preserve some key aspects of differentiated hepatocyte function in vitro. Freshly isolated rat hepatocytes in co-culture with activated stellate cells rapidly aggregate to form well-defined viable spheroids. After 5 days in culture, the spheroids have a complex extracellular matrix support and hepatic ultrastructure including bile canaliculi, tight junctions, desmosomes and lipid storage. Co-culture spheroids have superior cytochrome P450 (CYP450) 3A and 2B function, and increased inducibility of 2B function, relative to a range of hepatocyte monoculture techniques (high-performance liquid chromatography of testosterone metabolites). Increased function in co-culture is supported by greater expression of CYP450 3A23, 1A2, and 2E1 mRNA relative to monoculture (reverse transcriptase quantitative polymerase chain reaction). Also, high hepatocyte growth factor mRNA expression in co-culture suggests a post-traumatic, or possibly regenerative, environment. A preliminary study of human hepatocytes co-cultured with rat stellate cells demonstrated prolonged function of CYP450 3A4, 2C19 and 2C9. This study shows that stellate cells facilitate spheroid formation, influence spheroid architecture, and are an effective method of preserving some aspects of hepatocyte function in the early stage of culture.