Degenerated human articular cartilage at autopsy represents preclinical osteoarthritic cartilage: comparison with clinically defined osteoarthritic cartilage

To investigate whether macroscopically fibrillated human articular knee cartilage observed at autopsy can be considered an early, preclinical phase of osteoarthritis (OA). Histological and biochemical characteristics of 3 types of articular knee cartilage were compared: macroscopically degenerated knee cartilage obtained at autopsy (6 donors) from donors without clinical history of OA, normal healthy knee cartilage obtained at autopsy (6 donors), and OA cartilage obtained during joint replacement surgery from patients (n = 6) with clinically defined OA of the knee. From the same donors synovial tissue and synovial fluid were obtained and analyzed for features of inflammation. Histological changes of OA were comparable for degenerated and OA cartilage and significantly different from normal cartilage. Content and synthesis of proteoglycans showed intermediate levels for degenerated tissue compared to normal and OA cartilage. Analysis of synovial tissue revealed a low, mild, and moderate degree of inflammation for joints with normal, degenerated, and OA cartilage, respectively. The same sequence was found for metalloproteinase activity in synovial fluid. In general, all changes observed in OA joints were, to a lesser extent, observed in the joints with degenerated cartilage and were significantly different from joints with normal cartilage. We conclude that cartilage degeneration observed at autopsy can be considered a preclinical phase of OA, suitable for studying the process of cartilage degeneration in O

Cytochrome P450 2B1-mediated one-electron reduction of adriamycin: a study with rat liver microsomes and purified enzymes

The role of cytochrome P450 (CYP) in the one-electron reductive bioactivation of Adriamycin (ADR) (doxorubicin) was investigated in subcellular fractions of the rat liver. The rate of one-electron reduction of ADR to its semiquinone free radical (ADRSQ), measured by ESR, was 5-fold greater with phenobarbital (PB)-induced (PB microsomes) than with beta-naphthoflavone (beta NF)-induced (beta NF microsomes) rat liver microsomes under anaerobic conditions. ADRSQ formation was inhibited by SK&F 525-A and metyrapone (MP) in PB microsomes but was not significantly inhibited in beta NF microsomes. Under aerobic conditions, the formation of ADRSQ from ADR was diminished in microsomal incubations and concomitant reduction of molecular oxygen occurred instead. Whereas ADR-induced H2O2 formation in PB microsomes was strongly inhibited by SK&F 525-A and MP, only a slight inhibition was observed with 2-ethylnylnaphthalene and 1-ethynylpyrene in beta NF microsomes. In addition, MP produced strong inhibition of ADR-stimulated lipid peroxidation in PB microsomes, compared with beta NF microsomes. The idea that CYP2B1 was involved in the one-electron reduction of ADR in PB microsomes and in reconstituted systems of purified CYP2B1 and purified NADPH-CYP reductase (RED) under anaerobic conditions could be concluded from inhibition studies using SK&F 525-A and antibodies (KO1) against CYP2B enzymes. Moreover, it was calculated from reconstitution experiments using varying amounts of purified CYP2B1 and purified RED that the contribution of CYP2B1 to the one-electron reduction of ADR was similar to that of RED alone

The cytotoxicity of mitomycin C and adriamycin in genetically engineered V79 cell lines and freshly isolated rat hepatocytes.

The objective of the present study was to investigate the cytotoxicity of Adriamycin (ADR) and mitomycin C (MMC) in tumor and non-tumor cells with respect to the role of cytochrome P450 (P450). Therefore, genetically engineered V79 Chinese hamster fibroblasts expressing only single enzymes of P450 were used. SD1 and XEM2 cells expressed rat P450IIB1 and P450IA1, respectively, whereas the V79 parental cells contained no detectable P450 levels. The cytotoxicity of ADR and MMC in the V79 cell system was compared with that in freshly isolated hepatocytes from phenobarbital (PB-hepatocytes)- and β-naphthoflavone (βNF-hepatocytes)-induced rats. Following 24 h of exposure to ADR equal cytotoxicity was observed in V79, SD1 and XEM2 cells. Addition of metyrapone (MP, an inhibitor of P450IIB1) and α-naphthoflavone (αNF, an inhibitor of P450IA1) had no effect on the ADR-induced cytotoxicity in SD1 and XEM2 cells, respectively. Likewise, MMC was equitoxic in V79 and SD1 cells. Co-incubation of SD1 cells with MP did not alter MMC-induced cytotoxicity. MMC, however, showed a decreased cytotoxicity in XEM2 cells when compared to the parental V79 cells. Unexpectedly, the cytotoxicity of MMC in XEM2 cells was increased by aNF to the same level as observed in the parental V79 cells. In contrast to V79- and V79-derived cells, in freshly isolated hepatocytes from PB or βSNF-induced rats, MMC was cytotoxic (measured as lactate dehydrogenase leakage) within 3 h of incubation. ADR, however, was only cytotoxic to the hepatocytes when intracellular glutathione was first depleted by diethylmaleate. The MMC- and ADR-induced cytotoxicity was found to be more pronounced in PB-hepatocytes than in βNF-hepatocytes. Contrary to the findings in the V79-derived cells, MP afforded complete protection against both MMC- and ADR-induced cytotoxicity in PB-hepatocytes, whereas aNF only partially inhibited the cytotoxicity of MMC in βNF-hepatocytes. In conclusion, we have demonstrated that PB-inducible P450s play a role in the cytotoxicity of both MMC and ADR in freshly isolated PB-hepatocytes but that P450IIB1 does not in genetically reconstituted SD1 cells. P450IA1, however, decreased the cytotoxicity of MMC in the XEM2 cells. The ADR-induced cytotoxicity, which was observed in XEM2 cells, was not mediated by P450IA1. The present study underscores the complexity in the comparison of ADR- and MMC-induced cytotoxicities in normal and tumor cells. © 1995