Optimisation of cell-based assays for medium throughput screening of oxidative stress.

Identification and development of cell-based assays adapted to medium throughput screening requirements is important when screening chemicals for their potential toxic properties. We describe here rapid fluorometer-based and spectrophotometer-based microplate assays which allow for the evaluation of oxidative stress in hepatocyte cell cultures by measurement of three markers: production of hydroperoxide assessed by the DCFH-DA probe, cellular antioxidant status by measurement of reduced glutathione and glutathione peroxidase activity and cytotoxicity and mitochondrial damage by evaluation of the mitochondrial transmembrane potential with rhodamine 123 fluorescent dye. The assays described here are rapid, simple and inexpensive, which are desirable when setting up screening assays.

Tumorigenic potential of carbaryl in the heterozygous p53 knockout mouse model.

The heterozygous p53 knockout mouse model was used to assess whether vascular tumors noted in a 2-year carcinogenicity study in CD-1 mice with carbaryl were induced through a genotoxic mechanism. This knockout mouse model was selected for carbaryl because of the high sensitivity of this model to genotoxic events and its low spontaneous incidence of tumors until 9-12 months of age. Carbaryl was administered continuously via the diet to groups of 20 male heterozygous p53 knockout mice at concentrations of 0, 10, 30, 100, 300, 1000 and 4000 ppm for 180 days.

Evaluation of the effect of culture configuration on morphology, survival time, antioxidant status and metabolic capacities of cultured rat hepatocytes.

We evaluated the antioxidant status, namely cellular lipid peroxidation, by measuring thiobarbituric acid reactive substances (TBARS), cellular reduced glutathione (GSH) content, glutathione reductase (GSSG-R), glutathione transferase (GST), glutathione peroxidase (GSH-Px) and catalase activities in rat liver, hepatocytes immediately after isolation and in two-dimensional (2D) culture (on non-coated or collagen-coated dishes, as collagen-collagen or collagen-Matrigel sandwich cultures) or three-dimensional (3D) culture on Matrigel-coated dishes. Microsomal cytochrome P450 (CYP)- and UDP-glucuronosyl transferase (UGT)- dependent activities were also assessed in rat livers and hepatocyte cultures. The overall antioxidant status of rat hepatocytes immediately after isolation was not significantly different from that of rat livers.

Spontaneous nitric oxide in hepatocyte monolayers and inhibition of compound-induced apoptosis.

Primary cultures of hepatocytes are a widely used in vitro model for biochemical research. Following isolation, hepatocytes produce large amounts of nitric oxide (NO), which is known to have both pro- and anti-apoptotic effects in hepatocytes in vivo and in vitro. Previous work has not determined the effect of these increased levels of NO on the response of hepatocytes to apoptotic stimuli.

Effects of model inducers on thyroxine UDP-glucuronosyl-transferase activity in vitro in rat and mouse hepatocyte cultures.

Thyroxine (T(4))-UDP-glucuronosyltransferase (UGT) activity was measured directly in cultured male Sprague-Dawley rat and OF-1 mouse hepatocyte monolayers. The activity of T(4)-UGT (pmol/min/g liver) in vitro in hepatocyte cultures was, after 24 hr in culture, equivalent to that previously measured in vivo in rat and mouse liver microsomes (Viollon-Abadie et al., 1999).

The p53 heterozygous knockout mouse as a model for chemical carcinogenesis in vascular tissue.

Heterozygous p53 knockout mice were investigated as a potential model for vascular tumor carcinogenesis. Groups of 20 male mice were exposed by gavage for 6 months to the vascular carcinogen urethane at 1, 10, or 100 mg/kg body weight/day. Wild-type and heterozygous p53 knockout control groups were exposed by gavage to the vehicle alone.