Association between the levels of biogenic amines and superoxide anion production in brain regions of rats after subchronic exposure to TCDD.

The effects of TCDD on the distribution of biogenic amines and production of superoxide anion (SA) in different brain regions of rats have been studied after subchronic exposure. Groups of females Sprague-Dawley rats were administered daily dose of 46ng TCDD/(kgday) (treated groups), or the vehicle used to dissolve TCDD (control group), for 90 days. The rats were sacrificed at the end of the exposure period and their brains were dissected into different regions including, hippocampus (H), cerebral cortex (Cc), cerebellum (C), and brain stem (Bs). The levels of different biogenic amines and some of their metabolites, including, norepinephrine (NE), dopamine (DA), 3,4-dihydroxy phenyl acetic acid (DOPAC), 4-hydroxy-3-methoxy-phenyl acetic acid (HVA), 5-hydroxy tryptamine (5-HT), and 5-hydroxy indole 3-acetic acid (5-HIAA), were determined in those brain regions, using a high performance liquid chromatography (HPLC) system with an electrochemical detector. SA production was also determined in those regions, using the cytochrome c reduction method. Results of analyses indicate significant increases in the levels of DA, NE and DOPAC in H, NE and HVA in Cc, NE and DA in Bs and NE in C. SA production was significantly increased in H and Cc, but not in Bs or C. The results also indicated strong correlations between DA and DOPAC, and SA and NE in all of the brain regions, and also between SA and 5-HT/HIAA in H and Cc. These results may indicate the contribution of biogenic amines, especially NE and 5-HT/HIAA to SA overproduction in some brain regions and may also indicate the potential of long term neurotoxic effects of those biogenic amines, in response to subchronic exposure to TCDD.

Anti-insulin receptor autoantibodies are not required for type 2 diabetes pathogenesis in NZL/Lt mice, a New Zealand obese (NZO)-derived mouse strain.

The New Zealand obese (NZO) mouse strain shares with the related New Zealand black (NZB) strain a number of immunophenotypic traits. Among these is a high proportion of B-1 B lymphocytes, a subset associated with autoantibody production. Approximately 50% of NZO/HlLt males develop a chronic insulin-resistant type 2 diabetes syndrome associated with 2 unusual features: the presence of B lymphocyte-enriched peri-insular infiltrates and the development of anti-insulin receptor autoantibodies (AIRAs). To establish the potential pathogenic contributions of B lymphocytes and AIRAs in this model, a disrupted immunoglobulin heavy chain gene (Igh-6) congenic on the NZB/BlJ background was backcrossed 4 generations into the NZO/HlLt background and was then intercrossed to produce mice that initially segregated for wild-type versus the mutant Igh-6 allele and thus permitted comparison of syndrome development. A new flow cytometric assay (AIRA binding to transfected Chinese hamster ovary cells stably expressing mouse insulin receptor) showed IgM and IgG subclass AIRAs in serum from Igh-6 intact males, but not in Igh-6null male serum. However, the absence of B lymphocytes and antibodies distinguishing mutant from wild-type males failed to significantly affect diabetes-free survival. The Igh-6null males gained weight less rapidly than wild-type males, probably accounting for a retardation, but not prevention, of hyperglycemia. Thus, AIRA and the B-lymphocyte component of the peri-insulitis in chronic diabetics were not essential either to development of insulin resistance or to eventual pancreatic beta cell failure and loss. A new substrain, designated NZL, was generated by inbreeding Igh-6 wild-type segregants. Currently at the F10 generation, NZL mice exhibit the same juvenile-onset obesity as NZO/HlLt males, but develop type 2 diabetes at a higher frequency (> 80%). Also, unlike NZO/HlLt mice that are difficult to breed, the NZL/Lt strain breeds well and thus offers clear advantages to obesity/diabetes researchers.

Application of a convective-dispersion model to predict in vivo hepatic clearance from in vitro measurements utilizing cryopreserved human hepatocytes.

Growing interest in the prediction of in vivo pharmacokinetic data from purely in vitro data has grown into a process known as the in vitro-in vivo correlation (IVIC). IVIC can be used to determine the viability of new chemical entities in the early drug development phases, leading to a reduction of resource spending by many large pharmaceutical companies. Here, a convective-dispersion model was developed to predict the total hepatic clearance of six drugs using pharmacokinetic data obtained from in vitro metabolism studies in which the drug disappearance from suspensions of human cryopreserved hepatocytes was measured. Predicted in vivo hepatic clearances estimated by the convective-dispersion model were ultimately compared to the actual clearance values and to in vivo hepatic clearances that were scaled based on the well-stirred model. Finally, sensitivity studies were performed to determine the dependence of hepatic clearance on a number of physiological model parameters. Results reaffirmed that low clearance drugs exhibit rate-limited metabolism, and their hepatic clearances are thus independent of blood flow characteristics, whereas drugs with relatively higher clearance values show a more pronounced dependence on the flood flow properties of dispersion and convection. Absent a priori knowledge about the flow-dependent properties of a drug's clearance, the convective dispersion model applied to disappearance data acquired from cryopreserved human hepatocytes is likely to provide satisfactory estimates of hepatic drug clearance.

Microplate screening of the differential effects of test agents on Hoechst 33342, rhodamine 123, and rhodamine 6G accumulation in breast cancer cells that overexpress P-glycoprotein.

A microplate screening method has been developed to evaluate the effects of test agents on the accumulation of the fluorescent P-glycoprotein (Pgp) substrates Hoechst 33342, rhodamine 123, and rhodamine 6G in multidrug-resistant (MDR) breast cancer cells that overexpress Pgp. All three substrates exhibit substantially higher accumulation in MCF7 non-MDR cells versus NCI/ADR-RES MDR cells, while incubation with 50 microM reserpine significantly reduces or eliminates these differences. Rhodamine 123 shows the lowest substrate accumulation efficiency in non-MDR cells relative to the substrate incubation level. The effects of several chemosensitizing agents and a series of paclitaxel analogs on the accumulation of each fluorescent substrate suggest that there are distinct differences in the substrate interaction profiles exhibited by these different agents. The described methods may be useful in Pgp-related research in the areas of cancer MDR, oral drug absorption, the blood-brain barrier, renal/hepatic transport processes, and drug-drug interactions.