Climate change: Why higher education matters?

Higher education (HE) matters to the global struggle to combat climate change. Research builds knowledge and informs climate solutions. Educational programmes and courses upskill current and future leaders and professionals to tackle the systems change and the transformation needed to improve society. Through their outreach and civic engagement work, HE helps people understand and address the climate change impacts, notably on under-resourced or marginalised people. By raising awareness of the problem and supporting capacity and capability building, HE encourages changes in attitudes and behaviours, focusing on adaptive change in preparing people to face the challenges of a changing climate. However, HE has yet to fully articulate its contribution towards climate change challenges, which means that organisational structures, curricula and research programmes do not reflect the interdisciplinary nature of the climate crisis. This paper describes the role of HE in supporting education and research efforts on climate change and outlines areas where further action is urgently needed. The study adds to the empirical research on HE's role in combating climate change and the role of cooperation in maximising the global effort to cope with a changing climate.

Higher education and the COVID-19 pandemic: navigating disruption using the sustainable development goals.

The COVID-19 health crisis has caused profound social and economic disruptions. Affecting everyone, its impact is not equal. Exacerbating deep social fissures and long-standing systemic inequalities, the pandemic reveals the fragilities and inequities in global higher education. Accelerating underlying trends and triggering seismic shifts in the sector, collective efforts over a period of weeks delivered massive change in a sector not known for being fleet-of-foot. As we envisage the 'next normal' for higher education, problems with the 'old normal' may be fixed and some recent innovations carried forward. Representing a period of punctuated equilibrium, COVID-19 could lead to transformation of the sector towards greater equity and impact across teaching/learning, research/innovation, community service/engagement and the staff/students' experience. As we ask, "When will HE open again?", and "When we do, what will it be like?" we may also find ourselves considering "Will some institutions open at all?" Seeking to navigate these volatile, uncertain, complex and ambiguous times-so-called VUCA conditions-we propose that the SDGs are positioned as a lens to re-imagine higher education in mounting an antifragility response. We examine whether a new paradigm is forming that could be more sustainable as budgets, priorities and institutional archetypes are challenged fundamentally in line with delivery against the SDGs.

Spheroid Size Does not Impact Metabolism of the ß-blocker Propranolol in 3D Intestinal Fish Model.

Compared to two-dimensional (2D) cell culture, cellular aggregates or spheroids (3D) offer a more appropriate alternative in vitro system where individual cell-cell communication and micro-environment more closely represent the in vivo organ; yet we understand little of the physiological conditions at this scale. The relationship between spheroid size and oxygen microenvironment, an important factor influencing the metabolic capacity of cells, was first established using the fish intestine derived RTgutGC cell line. Subsequently, pharmaceutical metabolism (Propranolol), as determined by high performance liquid chromatography, in this intestinal model was examined as a function of spheroid size. Co-efficient of variation between spheroid size was below 12% using the gyratory platform method, with the least variation observed in the highest cell seeding density. The viable, high oxygen micro-environment of the outer rim of the spheroid, as determined by electron paramagnetic resonance (EPR) oximetry, decreased over time, and the hypoxic zone increased as a function of spheroid size. Despite a trend of higher metabolism in smaller spheroids, the formation of micro-environments (quiescent, hypoxic or anoxic) did not significantly affect metabolism or function of an environmentally relevant pharmaceutical in this spheroid model.

Application of the rainbow trout derived intestinal cell line (RTgutGC) for ecotoxicological studies: molecular and cellular responses following exposure to copper.

There is an acknowledged need for in vitro fish intestinal model to help understand dietary exposure to chemicals in the aquatic environment. The presence and use of such models is however largely restrictive due to technical difficulties in the culturing of enterocytes in general and the availability of appropriate established cell lines in particular. In this study, the rainbow trout (Oncorhynchus mykiss) intestinal derived cell line (RTgutGC) was used as a surrogate for the "gut sac" method. To facilitate comparison, RTgutGC cells were grown as monolayers (double-seeded) on permeable Transwell supports leading to a two-compartment intestinal model consisting of polarised epithelium. This two-compartment model divides the system into an upper apical (lumen) and a lower basolateral (portal blood) compartment. In our studies, these cells stained weakly for mucosubstances, expressed the tight junction protein ZO-1 in addition to E-cadherin and revealed the presence of polarised epithelium in addition to microvilli protrusions. The cells also revealed a comparable transepithelial electrical resistance (TEER) to the in vivo situation. Importantly, the cell line tolerated apical saline (1:1 ratio) thus mimicking the intact organ to allow assessment of uptake of compounds across the intestine. Following an exposure over 72 h, our study demonstrated that the RTgutGC cell line under sub-lethal concentrations of copper sulphate (Cu) and modified saline solutions demonstrated uptake of the metal with saturation levels comparable to short term ex situ gut sac preparations. Gene expression analysis revealed no significant influence of pH or time on mRNA expression levels of key stress related genes (i.e. CYP3A, GST, mtA, Pgp and SOD) in the Transwell model. However, significant positive correlations were found between all genes investigated suggesting a co-operative relationship amongst the genes studied. When the outlined characteristics of the cell line are combined with the division of compartments, the RTgutGC double seeded model represents a potential animal replacement model for ecotoxicological studies. Overall, this model could be used to study the effects and predict aquatic gastrointestinal permeability of metals and other environmentally relevant contaminants in a cost effective and high throughput manner.

Pharmaceutical Metabolism in Fish: Using a 3-D Hepatic In Vitro Model to Assess Clearance.

At high internal doses, pharmaceuticals have the potential for inducing biological/pharmacological effects in fish. One particular concern for the environment is their potential to bioaccumulate and reach pharmacological levels; the study of these implications for environmental risk assessment has therefore gained increasing attention. To avoid unnecessary testing on animals, in vitro methods for assessment of xenobiotic metabolism could aid in the ecotoxicological evaluation. Here we report the use of a 3-D in vitro liver organoid culture system (spheroids) derived from rainbow trout to measure the metabolism of seven pharmaceuticals using a substrate depletion assay. Of the pharmaceuticals tested, propranolol, diclofenac and phenylbutazone were metabolised by trout liver spheroids; atenolol, metoprolol, diazepam and carbamazepine were not. Substrate depletion kinetics data was used to estimate intrinsic hepatic clearance by this spheroid model, which was similar for diclofenac and approximately 5 fold higher for propranolol when compared to trout liver microsomal fraction (S9) data. These results suggest that liver spheroids could be used as a relevant and metabolically competent in vitro model with which to measure the biotransformation of pharmaceuticals in fish; and propranolol acts as a reproducible positive control.

Correction: Direct Measurements of Oxygen Gradients in Spheroid Culture System Using Electron Parametric Resonance Oximetry.

[This corrects the article DOI: 10.1371/journal.pone.0149492.].

Direct Measurements of Oxygen Gradients in Spheroid Culture System Using Electron Parametric Resonance Oximetry.

Advanced in vitro culture from tissues of different origin includes three-dimensional (3D) organoid micro structures that may mimic conditions in vivo. One example of simple 3D culture is spheroids; ball shaped structures typically used as liver and tumour models. Oxygen is critically important in physiological processes, but is difficult to quantify in 3D culture: and the question arises, how small does a spheroid have to be to have minimal micro-environment formation? This question is of particular importance in the growing field of 3D based models for toxicological assessment. Here, we describe a simple non-invasive approach modified for the quantitative measurement and subsequent evaluation of oxygen gradients in spheroids developed from a non-malignant fish cell line (i.e. RTG-2 cells) using Electron Paramagnetic Resonance (EPR) oximetry. Sonication of the paramagnetic probe Lithium phthalocyanine (LiPc) allows for incorporation of probe particulates into spheroid during its formation. Spectra signal strength after incorporation of probe into spheroid indicated that a volume of 20 µl of probe (stock solution: 0.10 mg/mL) is sufficient to provide a strong spectra across a range of spheroid sizes. The addition of non-toxic probes (that do not produce or consume oxygen) report on oxygen diffusion throughout the spheroid as a function of size. We provide evidence supporting the use of this model over a range of initial cell seeding densities and spheroid sizes with the production of oxygen distribution as a function of these parameters. In our spheroid model, lower cell seeding densities (∼2,500 cells/spheroid) and absolute size (118±32 µm) allow control of factors such as pre-existing stresses (e.g. ∼ 2% normoxic/hypoxic interface) for more accurate measurement of treatment response. The applied methodology provides an elegant, widely applicable approach to directly characterize spheroid (and other organoid) cultures in biomedical and toxicological research.

In-vitro maintenance and functionality of primary renal tubules and their application in the study of relative renal toxicity of nephrotoxic drugs.

INTRODUCTION: The renal tubules play important roles in substance re-absorption from primary urine of the kidney, drug metabolism and gluconeogenesis in fasting and are vulnerable targets of nephrotoxic chemicals. Therefore, an appropriate functional model of renal tubules would enable the study of their functionality and chemical-induced toxicity. We have developed a method to maintain primary renal tubules and sustain their biochemical functionality in culture for an extended period of time. METHODS: Primary rat renal tubules were isolated from male rat kidneys by collagenase perfusion and the tubules maintained in culture as a suspension by a gyratory culture method. RESULTS: The tubule fragments gradually formed renal tubule aggregates within 6days and were maintained in culture for up to 12days without apparent morphological changes. Biochemical functions including glucose release, galactose uptake and pyruvate uptake were retained for the observed period of 12days after isolation. The aggregates showed significant cytochrome P450 1A1 activity recovery from day 6 after isolation and this was maintained thereafter during the 12-day period of in-vitro culture. A new toxicity test termed the cell spreading inhibition test (CSIT) of renal tubule aggregates was developed to study the effect of toxicants on cell spreading/growth and determine the minimum concentration of each toxicant that caused cell spreading inhibition (CSI-C). The CSI-Cs of selected nephrotoxic drugs were determined as chlorpromazine (60µM), cisplatin (200µM), diclofenac (800µM), valproic acid (10mM), and gentamycin (30mM). DISCUSSION: The gyratory method of primary renal tubule aggregate culture can retain tubular cell functions such as glucose release, galactose uptake and allow cytochrome P450 1A1 activity to recover, which are essential for an in-vitro model. Therefore, renal tubule aggregates can be used as a model for studies of biochemical functions of renal tubules and relative renal toxicity of nephrotoxic agents.

Towards a more representative in vitro method for fish ecotoxicology: morphological and biochemical characterisation of three-dimensional spheroidal hepatocytes.

The use of fish primary cells and cell lines offer an in vitro alternative for assessment of chemical toxicity and the evaluation of environmental samples in ecotoxicology. However, their uses are not without limitations such as short culture periods and loss of functionality, particularly with primary tissue. While three-dimensional (spheroid) technology is now established for in vitro mammalian toxicity studies, to date it has not been considered for environmental applications in a model aquatic species. In this study we report development of a reproducible six-well plate, gyratory-mediated method for rainbow trout (Oncorhynchus mykiss) hepatocyte spheroid culture and compare their functional and biochemical status with two-dimensional (2D) monolayer hepatocytes. Primary liver spheroid formation was divided into two stages, immature (1-5 days) and mature (≥6 days) according to size, shape and changes in functional and biochemical parameters (protein, glucose, albumin and lactate dehydrogenase). Mature spheroids retained the morphological characteristics (smooth outer surface, tight cell-cell contacts) previously described for mammalian spheroids as demonstrated by light and scanning electron microscopy. Glucose production and albumin synthesis were significantly higher in mature spheroids when compared to conventional 2D monolayer cultures (P < 0.01) and increased as spheroids matured (P < 0.01). Basal lactate dehydrogenase (LDH) leakage significantly decreased during spheroid formation and was significantly lower than 2D cultures (P < 0.01). It is therefore suggested that mature spheroids can maintain a high degree of functional, biochemical and morphological status over-time in culture that is superior to conventional 2D models and can provide realistic organotypic responses in vitro. Trout spheroids that take ~6-8 days to reach maturity would be suitable for use in acute toxicological tests and since it is possible to culture individual spheroids for over a month, there is potential for this work to lead towards in vitro bioaccumulation alternatives and to conduct high throughput screens of chronic exposure. This is an important step forward for developing alternative in vitro tools in future fish ecotoxicological studies.

Nociceptin-induced modulation of human T cell function.

There is an accumulating evidence for the immunoregulatory role of the neuropeptide, nociceptin/orphanin FQ (N/OFQ) however its role on T cell function requires elucidation. This study has demonstrated an inhibitory role for N/OFQ on SEB-activated T cell function. N/OFQ decreases T cell proliferation, which is abrogated when the costimulatory receptors CD80 and CD86 are blocked. In addition, evidence suggests that the immunoregulatory cytokines TGF-beta, IFN-gamma and nitric oxide (NO) are involved in the N/OFQ effect. N/OFQ also, through involvement of IFN and NO, induces the expression of the immunosuppressive modulator indoleamine 2,3-dioxygenase (IDO), suggesting a central role for IDO in the N/OFQ effect on T cell proliferation. The data presented in this report indicate a multi-faceted mechanism of action used by N/OFQ to modulate T cell function.

Functional three-dimensional HepG2 aggregate cultures generated from an ultrasound trap: comparison with HepG2 spheroids.

Three-dimensional culture systems are an ideal in vitro model being capable of sustaining cell functionalities in a manner that resembles the in vivo conditions. In the present study, we developed an ultrasound trap-based technique to rapidly produce HepG2 hepatocarcinoma cell aggregates within 30 min. Enhanced junctional F-actin was observed at the points of cell-cell contact throughout the aggregates. HepG2 aggregates prepared by the ultrasound trap can be maintained in culture on a P-HEMA-coated surface for up to 3 weeks. The cells in these aggregates proliferated during the initial 3 days and cell number was consistent during the following maintenance period. Albumin secretion from these HepG2 aggregates recovered after 3 days of aggregate formation and remained relatively stable for the following 12 days. Cytochrome P450-1A1 activity was significantly enhanced after 6 days with maximal enzyme activity observed between 9 and 18 days. In addition, comparison experiments demonstrated that HepG2 aggregates generated by the ultrasound trap had comparable functional characterizations with HepG2 spheroids formed by a traditional gyrotatory-mediated method. Our results suggest that HepG2 aggregate cultures prepared through the ultrasound trap-based technique may provide a novel approach to produce in vitro models for hepatocyte functional studies.

Energy metabolism and biotransformation as endpoints to pre-screen hepatotoxicity using a liver spheroid model.

The current study investigated liver spheroid culture as an in vitro model to evaluate the endpoints relevant to the status of energy metabolism and biotransformation after exposure to test toxicants. Mature rat liver spheroids were exposed to diclofenac, galactosamine, isoniazid, paracetamol, m-dinitrobenzene (m-DNB) and 3-nitroaniline (3-NA) for 24 h. Pyruvate uptake, galactose biotransformation, lactate release and glucose secretion were evaluated after exposure. The results showed that pyruvate uptake and lactate release by mature liver spheroids in culture were maintained at a relatively stable level. These endpoints, together with glucose secretion and galactose biotransformation, were related to and could reflect the status of energy metabolism and biotransformation in hepatocytes. After exposure, all of the test agents significantly reduced glucose secretion, which was shown to be the most sensitive endpoint of those evaluated. Diclofenac, isoniazid, paracetamol and galactosamine reduced lactate release (P <0.01), but m-DNB increased lactate release (P <0.01). Diclofenac, isoniazid and paracetamol also reduced pyruvate uptake (P <0.01), while galactosamine had little discernible effect. Diclofenac, galactosamine, paracetamol and m-DNB also reduced galactose biotransformation (P <0.01), by contrast, isoniazid did not. The metabolite of m-DNB, 3-NA, which served as a negative control, did not cause significant changes in lactate release, pyruvate uptake or galactose biotransformation. It is concluded that pyruvate uptake, galactose biotransformation, lactate release and glucose secretion can be used as endpoints for evaluating the status of energy metabolism and biotransformation after exposure to test agents using the liver spheroid model to pre-screen hepatotoxicity.

Electrophysiological measurements in three-dimensional in vivo-mimetic organotypic cell cultures: preliminary studies with hen embryo brain spheroids.

Using three-dimensional artificial tissue constructs shown to offer organotypic functionality, hen embryo brain spheroids were used as a novel electrophysiological paradigm. For the first time, single spontaneous action potentials were recorded from spheroids in culture at day 7 in vitro (DIV) using multi-electrode arrays. At DIV14 'bursting behaviour' was observed. Simple stimulation was found to induce an increase in spiking frequency with an effect that ramped up over DIV7-14. By DIV14, the frequency under stimulation was typically over twice that of the corresponding spontaneous spiking. These results indicate strong self-organizing processes in vitro within the neuronal networks of the three-dimensional spheroid cell cultures. The organotypic in vivo-mimetic nature of the spheroid paradigm was confirmed by electron microscopy that revealed an outer layer of glial cells, a glial limitans, while immunostaining for Neurofilament and Glial Fibrilliary Acidic Protein demonstrated neuronal cells with a centralized neuronal and synaptic distribution. Basic biochemical functionality was also determined and Acetylcholinesterase measured, indicating the activity of acetylcholine receptors. Thus the organotypic hen embryo brain spheroid model may offer a new paradigm in which to explore neuronal networks.

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 microM and above. At 250 microM (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.

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.

Accurate dry weight assessment: reducing the incidence of hypertension and cardiac disease in patients on hemodialysis.

Hypertension is a major cause of cardiac disease in patients on hemodialysis (HD) and is most commonly due to hypervolemia. Removal of excess water during HD can successfully normalize blood pressure, but its success depends on an accurate assessment of dry weight. This article reviews the literature concerning hypertension in patients on HD and proposes that increased attention by dialysis staff to assessing dry weight may reduce the incidence of hypertension and cardiac disease in these patients.

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 gamma-glutamyltransferase (gamma-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.

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.

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.

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.