Dataset Information

A preparation of murine liver fragments for in vitro studies: liver preparation for toxicological studies.

ABSTRACT:

Background

The aim of this study was to develop liver tissue preparation suitable for investigating toxins. Hepatocyte respiration, ATP content, urea synthesis, caspase activity and morphology were measured as a function of in vitro incubation time. Mice were anesthetized by sevoflurane inhalation. Small liver fragments were then rapidly excised and incubated at 37°C in Krebs-Henseleit buffer (continuously gassed with 95% O2: 5% CO2) for up to 6 h. Phosphorescence O2 analyzer was used to determine the rate of cellular mitochondrial O2 consumption (kc, μM O2 min-1 mg-1). Cellular ATP was measured using the luciferin/luciferase system. The caspase-3 substrate N-acetyl-asp-glu-val-asp-7-amino-4-methylcoumarin (Ac-DEVD-AMC) was used to monitor intracellular caspase activity; cleaved AMC moieties (reflecting caspase activity) were separated on HPLC and detected by fluorescence.

Findings

Respiration was inhibited by cyanide, confirming the oxidation occurred in the respiratory chain. The values of kc (mean ± SD) for 0≤ t ≤6 h were 0.15 ± 0.02 μM O2 min-1 mg-1 (n = 18, coefficient of variation, CV = 13%), ATP content 131 ± 69 pmol mg-1 (1≤ t ≤6 h, n = 16, CV = 53%), synthesized urea 0.134 ± 0.017 mg/dL mg-1 in 50 min (0≤ t ≤6 h, n = 14, CV = 13%), and AMC peak area 62,540 ± 26,227 arbitrary units mg-1 (1≤ t ≤6 h, n = 3, CV = 42%). Hepatocyte morphology and organelles were reasonably persevered.

Conclusions

The described liver tissue preparation demonstrates stable hepatocyte structure, ultrastructure and biomarkers for up to 6 h, permitting in vitro studies.

SUBMITTER: Alfazari AS

PROVIDER: S-EPMC3598918 | biostudies-literature |

REPOSITORIES: biostudies-literature

ACCESS DATA

Similar Datasets

Project description:Brazil has the highest biodiversity of native stingless bees in the world. However, Brazilian regulations are based on protocols standardized by the Organization for Economic Cooperation and Development (OECD), which uses Apis mellifera as a model organism. The safety of the use of an exotic species as a substitute for a native species is a problem that concerns members of the academy and the government agencies responsible for studies of this nature in the neotropical regions where there are occurrences of stingless bee species. Regarding the exposure of larvae to pesticides, several indicators suggest that the same rearing method for A. mellifera cannot be applied to stingless bees, mainly because of their different feeding systems. Thus, it is necessary to establish an in vitro rearing method for native social bees. We developed a larval rearing method for the stingless bee species Melipona scutellaris and evaluated parameters such as the defecation rate, pupation, emergence, mortality and morphometry of the newly emerged workers. The control was represented by the morphometry of individuals that emerged from natural combs (in vivo). In addition, we determined the average lethal concentration (LC50) of the insecticide dimethoate, the standard active ingredient used for the validation of toxicity tests. Procedures conducted prior to the in vitro bioassays allowed us to obtain the actual dimensions of the rearing cells for making acrylic plates for use in establishing how much each larva consumes during its development, that is, determining how much larval food should be placed in every artificial cell. Tests performed with M. scutellaris indicated an average of 80.2% emergence of individuals relative to the larvae, 92.61% relative to the pupae and a mean of 7.42% larval mortality. The mean of the intertegular distance, head width and wing asymmetry parameters were not significantly different between individuals from the in vitro and in vivo rearing methods. The LC50 value determined was 27.48 ng dimethoate / μL diet. The method described for M. scutellaris showed development rates above OECD standards, which requires at least 75% emergence, and produced newly emerged workers with similar dimensions to those produced under natural conditions; thus these results enable their use as a rearing protocol for this species (or genus) and, consequently, their use in toxicity tests. The results produced with M. scutellaris are the first steps for a proposed toxicity test protocol for stingless bee larvae that can be standardized and included as a protocol in the OECD.