ABSTRACT
Currently, there is no test system, whether in vitro or in vivo, capable of examining all endpoints required for genotoxicity evaluation used in pre-clinical drug safety assessment. The objective of this study was to develop a model which could assess all the required endpoints and possesses robust human metabolic activity, that could be used in a streamlined, animal-free manner. Liver-on-chip (LOC) models have intrinsic human metabolic activity that mimics the in vivo environment, making it a preferred test system. For our assay, the LOC was assembled using primary human hepatocytes or HepaRG cells, in a MPS-T12 plate, maintained under microfluidic flow conditions using the PhysioMimix® Microphysiological System (MPS), and co-cultured with human lymphoblastoid (TK6) cells in transwells. This system allows for interaction between two compartments and for the analysis of three different genotoxic endpoints, i.e. DNA strand breaks (comet assay) in hepatocytes, chromosome loss or damage (micronucleus assay) and mutation (Duplex Sequencing) in TK6 cells. Both compartments were treated at 0, 24 and 45â¯h with two direct genotoxicants: methyl methanesulfonate (MMS) and ethyl methanesulfonate (EMS), and two genotoxicants requiring metabolic activation: benzo[a]pyrene (B[a]P) and cyclophosphamide (CP). Assessment of cytochrome activity, RNA expression, albumin, urea and lactate dehydrogenase production, demonstrated functional metabolic capacities. Genotoxicity responses were observed for all endpoints with MMS and EMS. Increases in the micronucleus and mutations (MF) frequencies were also observed with CP, and %Tail DNA with B[a]P, indicating the metabolic competency of the test system. CP did not exhibit an increase in the %Tail DNA, which is in line with in vivo data. However, B[a]P did not exhibit an increase in the % micronucleus and MF, which might require an optimization of the test system. In conclusion, this proof-of-principle experiment suggests that LOC-MPS technology is a promising tool for in vitro hazard identification genotoxicants.
Subject(s)
Hepatocytes , Micronucleus Tests , Mutagenicity Tests , Mutagens , Humans , Hepatocytes/drug effects , Hepatocytes/metabolism , Mutagens/toxicity , Micronucleus Tests/methods , Mutagenicity Tests/methods , Liver/drug effects , Liver/metabolism , Lab-On-A-Chip Devices , DNA Damage/drug effects , Comet Assay/methods , Cyclophosphamide/toxicity , Methyl Methanesulfonate/toxicity , Cell Line , Benzo(a)pyrene/toxicity , Coculture Techniques , Ethyl Methanesulfonate/toxicity , Mutation/drug effectsABSTRACT
The present study investigated the postnatal establishment of the laminar pattern of acetylcholinesterase (AChE) activity in the visual cortex (Oc1) of normal and neonatally hemidecorticate rates. Rat pups received a hemidecortication on post-natal day (PND) 3 and sacrificed at three day intervals starting at PND-6 through PND-24. Laminar patterns of AChE activity in Oc1 are described qualitatively and quantitatively using optical densitometry. The postnatal development of the laminar distribution of AChE activity is similar in normal and hemidecorticate rats. In both cases, AChE activity is intense in layer I, in the deep layer III as well as in layer IV. This pattern is first detected at the end of the first postnatal week. AChE activity reaches a peak intensity during the third postnatal week and gradually declines to adult levels during the fourth postnatal week. Hemidecortication has no significant effect on the intensity of AChE activity measured in the visual cortex. Neonatal hemidecortication does not affect AChE activity levels, structure of AChE neurites or the laminar distribution pattern, nor does it affect the time course of the establishment of this pattern in Oc1 of the remaining cortex. These data do not support the hypothesis that massive cortical lesions in rats result in an increase in contralateral cholinesterase activity nor do they suggest terminal sprouting of basal forebrain projections to the visual cortex.
