Wendy M. Purcell

Peroxynitrite Mediates Nitric Oxide–Induced Blood–Brain Barrier Damage

Using the in vitro blood–brain barrier (BBB) model ECV304/C6, which consists of cocultures of human umbilical vein endothelial-like cells (ECV304) and rat glioma cells (C6), the role of peroxynitrite (OONO−) in nitric oxide (NO·)-mediated BBB disruption was evaluated. Endothelial cell cultures were exposed to NO· gas, in the presence or absence of the OONO− blocker FeTPPS. Separate exposure to NO· and OONO− resulted in endothelial cell cytotoxicity and a decline in barrier integrity. Unfortunately, FeTPPS induced significant detrimental effects on model BBB integrity at a concentration of 300 μM and above. At 250 μM (the highest concentration usable), FeTPPS displayed a trend toward prevention of NO· elicited perturbation of barrier integrity. Dichlorofluorescein diacetate is oxidized to fluorescent dichlorofluorescein by OONO− but only marginally by NO· or O2·−. We observed large and rapid increases in fluorescence in ECV304 preloaded cells following NO· exposure, which were blocked by FeTPPS. Furthermore, using an antinitrotyrosine antibody we detected the nitration of endothelial cell proteins following NO· exposure and conclude that NO·-mediated BBB dysfunction is predominantly elicited by OONO− and not NO·. Proposed mechanisms of NO·-mediated OONO− elicited barrier dysfunction and damage are discussed.

Characterisation of some cytotoxic endpoints using rat liver and HepG2 spheroids as in vitro models and their application in hepatotoxicity studies. I. Glucose metabolism and enzyme release as cytotoxic markers

Cytotoxicity endpoints, spontaneous glucose secretion/consumption and LDH and gamma-GT release, were characterised in rat liver and HepG2 spheroids as in vitro models for toxicology studies. Preprepared rat liver spheroids and HepG2 spheroids cultured in a six-well plate format were exposed to varying concentrations of galactosamine, propranolol, diclofenac, and paracetamol. All four model toxins significantly affected glucose secretion, which agreed well with LDH and/or gamma-GT release in rat liver spheroids. These toxins also significantly increased LDH and/or gamma-GT release in HepG2 spheroids. Whereas glucose consumption in HepG2 spheroids did not show conclusive results, LDH activities in both types of spheroids were similar and their levels were relatively high. Accordingly, the level of LDH leakage in both types of spheroids was much higher than gamma-GT after exposure to the toxins. In contrast, gamma-GT activity in HepG2 spheroids was sixfold higher than that in rat liver spheroids. This study revealed that galactosamine interfered with the gamma-GT assay and paracetamol interfered with the LDH assay. It demonstrated, for the first time, that glucose secretion by liver spheroids can be used as a functional indicator of cytotoxicity. Test compounds may interfere with enzymatic assays as indicated by LDH and gamma-GT release in this study. Combining functional parameters together with two or more indicators of enzyme releases can provide a reliable cytotoxicity evaluation. Liver and HepG2 spheroids as in vitro models showed good predictions in chemical-induced hepatic cytotoxicity.

Nociceptin/orphanin FQ modulates human T cell function in vitro

Although nociceptin/orphanin FQ (N/OFQ) and its receptor (ORL-1) are widely distributed throughout the immune system, its role has yet to be elucidated. This study shows that N/OFQ (10(-14)-10(-12) M) modulates T cell activation by up-regulating activation marker expression, e.g. CD28, leading to enhanced proliferation and modulation of TNFalpha secretion. However, on re-stimulated T cells N/OFQ causes inhibition of proliferation, which could be linked with N/OFQ up-regulating CTLA-4 expression. We have also shown that some of these effects are partly prostaglandin-dependent and that N/OFQ induces prostaglandin synthesis. This report suggests that N/OFQ could exert a key modulatory role in human T cell functions.

Biochemical and functional changes of rat liver spheroids during spheroid formation and maintenance in culture: I. morphological maturation and kinetic changes of energy metabolism, albumin synthesis, and activities of some enzymes

In the process of isolated single liver cells coming together to form three‐dimensional spheroids, cells undergo dramatic environmental changes. How liver cells respond to these changes has not been well studied before. This study characterized the functional and biochemical changes during liver spheroid formation and maintenance. Spheroids were prepared in 6‐well plates from freshly isolated liver cells from male Sprague rats by a gyrotatory‐mediated method. Morphological formation, and functional and biochemical parameters of liver spheroids were evaluated over a period of 21 days in culture. Liver spheroid formation was divided into two stages, immature (1–5 days) and mature (>5 days), according to their size and shape, and changes in their functionality. Galactose and pyruvate consumption was maintained at a relatively stable level throughout the period of observation. However, glucose secretion and cellular GPT and GOT activities were higher in immature spheroids, decreased upto day 5 and remained stable thereafter. Cellular γ‐glutamyltransferase (γ‐GT) and lactate dehydrogenase (LDH) activities were initially undetectable or low and increased as spheroids matured. Albumin secretion decreased rapidly within the first 2 days and increased as spheroids matured. It is concluded that cells undergo functional and biochemical changes during spheroid formation following isolation of liver cells from intact tissue. Functionality and biochemical properties recovered and were maintained in mature spheroids. A relatively stable period (6–15 days) of functionality in mature spheroids was identified and is recommended for applications of the model.

Characterisation of some cytotoxic endpoints using rat liver and HepG2 spheroids as in vitro models and their application in hepatotoxicity studies. II. Spheroid cell spreading inhibition as a new cytotoxic marker

Cells in liver spheroids and Hep G2 spheroids transferred from gyrotatory culture conditions and maintained in normal static culture conditions will spread out at the edges. Based on this observation, we developed a new test called the Spheroid Cell Spreading Inhibition Test (SCSIT) to screen hepatic cytotoxicity of xenobiotics and determine the spheroid cell spreading inhibition concentration (SCSIC) of test chemicals. Four model hepatoxicants, D-galactosamine, propranolol, diclofenac, and paracetamol, were studied with SCSIT in both rat liver and HepG2 spheroids. Both liver and HepG2 spheroids were prepared under gyrotatory culture conditions and used at 6 days in vitro. The results showed that all four hepatotoxicants tested inhibited cell spreading in liver spheroids (D-galactosamine at 20 mM, propranolol at 125 microM, diclofenac at 500 microM, and paracetamol at 25 mM) and HepG2 spheroids (D-galactosamine at 16 mM, propranolol at 125 microM, diclofenac at 500 microM, and paracetamol at 25 mM). The SCSIT results agreed with the conventional cytotoxic indicators, release of LDH and/or gamma-GT and the inhibition of glucose secretion from rat liver spheroids. In conclusion, this study, for the first time, described the biological characteristics of liver and HepG2 spheroid cell spreading and demonstrates its application in hepatic cytotoxicity studies. This method may be used in testing in vitro acute toxicity, comparing relative cytotoxicity and generating reference concentrations for subsequent studies. Therefore, SCSIT could be a useful tool for screening hepatotoxicity relevant to preclinical lead optimization and compound library screening.

Biochemical and functional changes of rat liver spheroids during spheroid formation and maintenance in culture: II. nitric oxide synthesis and related changes

Liver cells isolated from intact tissue can reaggregate to form three‐dimensional, multicellular spheroids in vitro. During this process, cells undergo a histological and environmental change. How cells respond biochemically to this change has not been studied in detail previously. We have investigated some biochemical changes in rat liver cells during the formation and maintenance of spheroids. Liver cells were isolated from male Sprague rats and spheroids cultured by a gyrotatory‐mediated method. Liver cells were shown to respond to the isolation procedure and the formation of spheroids triggered histological environmental changes that increased arginine uptake, nitric oxide (NO) and urea syntheses, as well as raised levels of GSH, GSSG, glutamic acid and aspartic acid secretion within the first couple of days after cell isolation. Levels were maintained at a relatively stable level in the mature spheroids (>5 days) over the 3 week period of observation. P450 1A1 activity was lost in the first 2 days and gradually recovered thereafter. This study, for the first time, shows that liver cells after isolation and during spheroid formation actively uptake arginine and increase NO and urea syntheses. A high level of NO is likely to play an important role in modulating a series of biochemical changes in liver cells. It is considered that liver cells actively respond to the ‘challenge’ induced by the isolation procedure and subsequent histological environmental changes, and biochemical modulation and instability result. The stable cell–cell contacts and histological environment in mature spheroids permit and support functional recovery and maintenance in vitro. This period of stability permits the use of spheroids in toxicity studies to establish acute and chronic paradigms.

Optimizing the enzymatic determination of galactose in the culture medium of rat liver and HepG2 cell spheroids.

Enzymatic determination of b-galactose by b-galactose dehydrogenase has been a useful method to determine b-galactose in biological substances [1,2] since the enzyme was identified and purified from Pseudomonas saccharophila by Doudoroff et al. [3] in the 1950s. This method was based on the b-galactose dehydrogenase catalysis of the oxidation of galactose in the presence of oxidized b-nicotinamide adenine dinucleotide (NAD) to produce galactonolactone and the reduced form of bnicotinamide adenine dinucleotide (NADH) which can be detected at 340 nm. The production of NADH is proportional to the concentration of galactose [1–3]. In this study, we optimized the enzymatic method for galactose determination and investigated galactose metabolism in liver spheroids, HepG2 spheroids, and HepG2 cells cultured in monolayer. To determine an optimal pH range, the conditions of 1mg/ml b-galactose, 4U/ml b-galactose dehydrogenase, and 16mM b-nicotinamide adenine dinucleotide in the pH 5, 6, 7, 8, 9, and 10 buffers (Na2HPO4=KH2PO4 buffer, 200mM) were tested. The assays including all following assays were performed in a 96-well format and absorbance was read on a microplate reader (Multiscan RC, Labsystems). After 50min incubation at 37 C, the reactions in pH 7–10 buffers (Na2HPO4=KH2PO4 buffer, 200mM) were completed, whereas the reactions in these buffers at pH 5 and 6 slowly increased. The best results were obtained between pH 8 and 9. This indicates that b-galactose dehydrogenase works well in an alkaline condition. pH 8 buffer was used in the following assays in this study. At the concentrations of 1 and 2U/ml b-galactose dehydrogenase in the conditions of 1mg/ml galactose and 16mM NAD, the reactions were completed at 70 and 90min, respectively. The maximum absorbance of both concentrations was 20–25% lower than that in 4U/ml b-galactose dehydrogenase. A suitable ratio of substrate concentration and enzyme concentration is essential for an optimal enzymatic reaction. Based on this result, 4U/ml b-galactose dehydrogenase was utilized for all the following experiments. Fig. 1 shows the effect of NAD concentration on catalysis of b-galactose dehydrogenase. As shown in Fig. 1, the concentration of NAD modified the enzymatic reaction rate. A higher concentration of NAD produced a higher OD value after the completion of the reaction although all three concentrations of NAD tested were high enough to receive hydrogen from 1mg/ml galactose in the defined method. Therefore, a concentration of NAD between 12 and 16mM is recommended. The concentration of 16mM was used for the following experiments. The catalytic kinetics and changes of OD readings are shown in Fig. 2A. The lower the substrate concentration, the sooner the reaction was completed as indicated by OD values. At 50min and the concentration of 1mg/ ml galactose or lower, the absorbance no longer increased with time, indicating completion of the reaction. In the higher concentrations of galactose, the absorbance slowly increased until 110min. Once the absorbance reached the peak value, it decreased slowly and steadily. The average decrease of OD values was only 0.44% every 10min and a total 3.54% decrease was detected 80min after peak time point. This indicates that the end product, NADH, is stable enough to allow a flexible assay. The linearity of galactose concentrations versus OD values after 60min reaction is shown in Fig. 2B. When the OD value was lower than 1.8 (equivalent to 1mg/ml galactose or 5.5mM NADH), the OD values were linear Analytical Biochemistry 311 (2002) 179–181

Avian whole‐brain spheroid cultures: applications in pesticide toxicity

The adult hen is the species of choice when assessing the potential of organophosphate compounds (OPs) to evoke OP-induced delayed neuropathy (OPIDN). OPIDN has been shown to depend on the irreversible covalent binding of certain OPs to the enzyme neuropathy target esterase (NTE). Levels of this enzyme in human brain are similar to those in avian species. We have previously shown that the hen embryo whole brain spheroids are histotypically similar to adult brain and express measurable levels of acetylcholine esterase (AChE) and NTE, the key enzymes in mechanisms of OP toxicity. n n n nSingle-cell suspensions were prepared from meninges-free 7 day in ovo hen brains. Cells were grown in serum-free media and incubated on an orbital shaker at 37u2009°C in 5% carbon dioxide humidified air. Spheroids can be maintained long-term in culture and allow the effects of repeated dose and recovery studies to be conducted. Hen embryo brain spheroids were exposed to varying concentrations of tri-o-cresyl phosphate, leptophos and its metabolite leptophos oxon at day 14 in vitro and the effects on the activity of AChE and NTE measured. Whilst current legislation regarding toxicity testing of OPs requires the use of animals, our in vitro model provides a potential pre-screen for novel chemical entities and commercial OP mixture variants. Such an approach should refine and reduce the number of animals used in development of new materials. n n n n© 2000 Society of Chemical Industry

Application of the high-performance liquid chromatographic method for separation, purification and characterisation of p-bromophenylacetylurea and its metabolites.

This study presents a HPLC method for the separation and purification of p-bromophenylacetylurea (BPAU) and its metabolites. The method effectively separated and purified BPAU and its metabolites. Three metabolites of BPAU, M1, M2 and M3 were characterised by mass spectroscopy and nuclear magnetic resonance. They are named as N-hydroxy-p-bromophenylacetylurea, 4-(4-bromophenyl)-3-oxapyrrolidine-2,5-dione and N-methyl-p-bromophenylacetylurea, respectively. The major metabolic pathways of BPAU were proposed. The establishment of the HPLC method and characterisation of BPAU metabolites make it possible for further pharmacokinetic studies to explore the mechanism of BPAU-induced delayed neuropathy.

Absorption, distribution, metabolism and excretion of p-bromophenylacetylurea in the female rat

1. This study has investigated absorption, distribution, metabolism and excretion of p-bromophenylacetylurea (BPAU) in the F344 female rat. BPAU and its metabolites were determined by HPLC. 2. Following a single p.o. dose of 150u2009mg/kg BPAU, the absorbed fraction of dosed BPAU was 65.9% and its half-life in the blood was 9.4 h. The relative distribution of BPAU (tissue/serum ratio) at 6 h (peak time point) after a single i.p. dose of 150u2009mg/kg BPAU was spinal cord (4.6 +/- 0.2) > liver (3.7 +/- 0.1) > brain (2.9 +/- 0.1) (mean +/- SD, n = 5), and they were significantly different from each other (p<0.05). BPAU in spinal cord reached the highest level. 3. Absorbed BPAU was metabolized in vivo into three major metabolites. N-hydroxy- p-bromophenylacetylurea (M1) was a dominant metabolite in tissues, whereas 4-(4-bromophenyl)-3-oxapyrrolidine-2,5-dione (M2) reached a high concentration in blood. N-methyl-p-bromophenylacetylurea (M3) was mainly found in the urine. All three metabolites were excreted via the urine and together accounted for 87% of absorbed BPAU. 4. This study provides a basic understanding of BPAU absorption, distribution, metabolism and elimination in rat.