Primary human hepatocytes (PHHs) are commonly used for in vitro studies of drug-induced liver injury. However, when cultured as 2D monolayers, PHH lose crucial hepatic functions within hours. This dedifferentiation can be ameliorated when PHHs are cultured in sandwich configuration (2Dsw), particularly when cultures are regularly re-overlaid with extracellular matrix, or as 3D spheroids. In this study, the 6 participating laboratories evaluated the robustness of these 2 model systems made from cryopreserved PHH from the same donors considering both inter-donor and inter-laboratory variability and compared their suitability for use in repeated-dose toxicity studies using 5 different hepatotoxins with different toxicity mechanisms. We found that expression levels of proteins involved in drug absorption, distribution, metabolism, and excretion, as well as catalytic activities of 5 different CYPs, were significantly higher in 3D spheroid cultures, potentially affecting the exposure of the cells to drugs and their metabolites. Furthermore, global proteomic analyses revealed that PHH in 3D spheroid configuration were temporally stable whereas proteomes from the same donors in 2Dsw cultures showed substantial alterations in protein expression patterns over the 14 days in culture. Overall, spheroid cultures were more sensitive to the hepatotoxic compounds investigated, particularly upon long-term exposures, across testing sites with little inter-laboratory or inter-donor variability. The data presented here suggest that repeated-dosing regimens improve the predictivity of in vitro toxicity assays, and that PHH spheroids provide a sensitive and robust system for long-term mechanistic studies of drug-induced hepatotoxicity, whereas the 2Dsw system has a more dedifferentiated phenotype and lower sensitivity to detect hepatotoxicity.

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Ah.wocf: Clodronate. etidronate and pamidronate are bisphosphonates introduced in the treatment of hypercalcaemia and osteoporosis. Interestingly, they also inhibit development of experimental atherosclerosis and affect smooth muscle tone of isolated rat tail artery. We have studied in vitro whether these hydrophilic compounds 1) accumulate in the wall of the human artery, 2) influence human arterial tone, and 3) interfere with the vascular action of L-type Ca2+ antagonists. Human internal mammary artery rings were incubated with ''C-labelled bisphosphonates. After a 2-hr incubation, the ratios of artery-to-incubate concentrations with 4 and 40 pmolil of clodronate were, respectively, 3.020.5 (mean?S.E.M.) and 1.320.2, with 4 and 40 pmol/l of etidronate 7.4+-0.9 and 3.2?0.4, and with 0.4 and 4 pmol/l of pamidronate 4.7+0.7 and 3.920.8. Both tested bisphosphonates, clodronate and pamidronate, reduced the arterial contractile force induced by a-adrenergic stimulation with noradrenaline and membrane depolarization with high concentration of KCI. Clodronate also decreased the arterial contraction induced by cumulative addition of Ca2+ with KCI as the agonist, and had an additive inhibitory effect on this response with the L-type Ca2+-channel blocker nifedipine. The results demostrate that I ) bisphosphonates accumulate markedly in human artery, 2) clodronate and pamidronate reduce human arterial contactile force to a-adrenergic and depolarizing stimuli, and 3) as shown with clodronate, bisphosphonates may exert an additive inhibitory effect on human arterial contractions with an L-type Ca2+-channel blocker.

Objective: To study, if there are differences in the fatty acid composition of low-density lipoprotein (LDL) in people eating three different long-standing habitual diets: vegetarian, high ®sh intake, or high saturated fat (milk fat) diet as a control group, and to study if these differences in¯uence the oxidation susceptibility of LDL. Design: Cross-sectional study using blood samples and a validated dietary frequency questionnaire with illustrations. Setting: Helsinki University Central Hospital, Finland. Subjects: The effect of three different types of long-standing diets of different fatty acid content (a strict vegetarian diet, n 11; a high ®sh intake diet, n 9; and a high saturated fat (milk fat) diet, controls, n 7) on the serum and LDL fatty acid content, and on the susceptibility of LDL to oxidation in vitro, was studied in healthy normocholesterolemic volunteers who had been on these diets for years. Oxidation of LDL was carried out by using CuSO 4 as a pro-oxidant. Results: There were no statistically signi®cant differences in the serum lipids or lipoproteins, though the vegetarian group exhibited lowest mean values of total, high-density lipoprotein (HDL) and LDL cholesterol levels. Both the serum and LDL eicosapentaenoic, docosapentaenoic and docosahexaenoic acid proportions were highest in the ®sh and lowest in the vegetarian groups. Linoleic acid was highest among the vegetarians. In the ®sh group, the vitamin A concentration in serum was higher than in vegetarians and controls and b-carotene lower than in controls, but in a-tocopherol, or lycopene concentrations there were no statistically signi®cant differences. The lag phase of LDL oxidation was shortest (116 min) in the ®sh group and longest (165 min) in the vegetarian group, and the control group was between them (129 min). The mean oxidation percentage after 2.5 h of copper-induced oxidation was highest (44%) in the ®sh group and lowest (22%) in the vegetarian group and intermediate (31%) in the control group. Conclusion: Long-term dietary habits predict the fatty acid composition of serum and LDL, and in¯uence the susceptibility of LDL to oxidation. In the ®sh group with the highest content of omega-3 fatty acids in LDL, the oxidation susceptibility of LDL was highest. In the vegetarian group with less omega-3 fatty acids in LDL, the LDL was more resistant to oxidation.

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