Comparison of different methods of determining cell viability after exposure to cytotoxic compounds

Cell-survival (of DON and L1210 cells) after treatment with cytotoxic compounds was assessed by measuring cloning efficiency, exclusion of trypan blue and erythrosin B, [⁵¹Cr] release, and attachment of DON cells to glass. Cell survival as measured by cloning efficiency did not correlate with survival measured by any of the other methods. We found that the stainability of cells after drug exposure depended on the cell line used. For example, after 3 h exposure to tubercidin although 100% of both DON and L1210 cells were killed (on basis of cloning efficiency), only 11% of DON cells and 68% of L1210 cells were dead as indicated by staining with erythrosin B. The stainability of cells also depended on the particular drug used. For example, after 24 h exposure of L1210 cells to adriamycin and tubercidin (both killed >99% of cells on basis of cloning efficiency) 21% of cells exposed to adriamycin and 99% of cells exposed to tubercidin were stained. The results obtained with several other cytotoxic compounds are discussed.     

Ion-Exchange Chromatography Separates Activities Synthesizing and Degrading Fructose 2,6-Bisphosphate from C3 and C4 Leaves but Not from Rat Liver

Fructose-6-phosphate,2-kinase and fructose-2,6-bisphosphatase were separated on the basis of charge from leaves of C₃ (spinach, lettuce, and pea) and C₄ (sorghum and amaranthus) plants but not from rat liver—a tissue known to contain a bifunctional enzyme with both activities. [2-³²P]Fructose 2,6-bisphosphate binding experiments also suggest that the major forms of these activities reside on different proteins in leaves.     

Continuous high density expression of human beta 2-adrenergic receptors in a mouse cell line previously lacking beta-receptors

The PvuII fragment of human genomic clone LCV-517 which contains the entire coding region of a beta-adrenergic receptor gene was cloned into the SmaI site of the expression vector pMSG. The recombinant DNA was cotransfected with pRSVneo into mouse B-82 cells using the CaPO4 precipitation method. B-82 cells do not possess beta-adrenergic receptors but do contain prostaglandin E1 receptors that stimulate adenylate cyclase. Following transfection, several colonies expressing beta-adrenergic receptors were isolated. Analysis of ligand binding to expressed beta-receptors indicated that the protein encoded by the gene in clone LCV-517 was a beta 2-adrenergic subtype. Human beta 2-adrenergic receptors photoaffinity labeled with [125I]iodocyanopindolol diazirine migrated on sodium dodecyl sulfate-polyacrylamide gels consistent with a molecular mass of 68,000, demonstrating that the receptor is glycosylated to an extent of 25-30% by weight. Addition of isoproterenol to cultures of transfected cells resulted in a 3-4-fold stimulation of adenylate cyclase, an effect similar to that seen in control B-82 cells with prostaglandin E1. These data describe the production of stable murine clonal cell lines expressing human beta 2-adrenergic receptors and illustrate the utility of such lines in the biochemical and pharmacological characterization of receptor proteins.     

Cytotoxicity of the redox cycling compound diquat in isolated hepatocytes: involvement of hydrogen peroxide and transition metals

Diquat is a hepatotoxin whose toxicity in vivo and in vitro is mediated by redox cycling and greatly enhanced by pretreatment with 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU), an inhibitor of glutathione reductase. The mechanism by which redox cycling mediates diquat cytotoxicity is unclear, however. Here, we have attempted to examine the roles of three potential products of redox cycling, namely superoxide anion radical (O2-.), hydrogen peroxide (H2O2), and hydroxyl radical (.OH), in the toxicity of diquat to BCNU-treated isolated hepatocytes. Addition of high concentrations of catalase, but not superoxide dismutase, to the incubations provided some protection against the toxic effect of diquat, but much better protection was observed when catalase was added in combination with the iron chelator desferrioxamine. Addition of desferrioxamine alone also provided considerable protection, whereas the addition of copper ions enhanced diquat cytotoxicity. Taken together, these results indicate that both H2O2 and the transition metals iron and copper could play major roles in the cytotoxicity of diquat. The role of O2-. remains less clear, however, but studies with diethylenetriaminepentaacetic acid indicate that O2-. is unlikely to significantly contribute to the reduction of Fe3+ to Fe2+. The hydroxyl radical or a related species seems the most likely ultimate toxic product of the H2O2/Fe2+ interaction, but hydroxyl radical scavengers afforded only minimal protection.     

Increased efflux rather than oxidation is the mechanism of glutathione depletion by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)

Incubation of isolated hepatocytes in the presence of either the parkinsonian-inducing compound 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) or its putative toxic metabolite 1-methyl-4-phenylpyridinium ion (MPP+) led to a depletion of intracellular reduced glutathione (GSH), which was mostly recovered as glutathione disulfide (GSSG). However, both MPTP- and MPP+-induced glutathione perturbances were relatively unaffected by the prior inhibition of glutathione reductase with 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU), suggesting that intracellular oxidation was not the major mechanism involved in the GSH loss. Inclusion of cystine in the incubation mixtures revealed a time-dependent formation of cysteinyl glutathione (CySSG), indicating that an increased efflux was mostly responsible for the MPTP- and MPP+-induced GSH depletion. Therefore, the measurement of GSSG, which is apparently formed extracellularly, was not associated with oxidative stress.     

The effect of cortisol on thyroid hormone kinetics in the ovine fetus

The mechanism underlying the association of rising concentrations of circulating triiodothyronine (T3) with the prepartum surge in the concentration of cortisol was investigated in 11 fetal sheep. The concentrations and metabolic clearance rates of T3 and thyroxine (T4) were measured prior to and following a continuous intravascular infusion of cortisol (1 mg/h for 84 h). Mean plasma T3 concentrations increased 10-fold following cortisol infusion whereas the concentrations of T4 either remained stable or exhibited a variable decline. Cortisol induced a 5-fold decrease in the metabolic clearance rate of T3 and a 6-fold increase in that of T4. The corresponding mean production rates of T3 and T4 increased significantly although the magnitude of the change varied between fetuses. We conclude that the prepartum rise in plasma T3 concentrations is likely to be a consequence of both a decreased metabolic clearance of T3 and increased peripheral conversion of T4 to T3 caused by rising concentrations of cortisol in fetal plasma.     

The hypogonadotropic state of the prepubertal male rhesus monkey (Macaca mulatta) is not associated with a decrease in hypothalamic gonadotropin-releasing hormone content

Hypothalamic contents of gonadotropin-releasing hormone (GnRH) in neonatally orchidectomized infant, juvenile, and adult monkeys were measured by a radioimmunoassay (RIA) and by an in vivo bioassay that utilized luteinizing hormone (LH) secretion in estrogen- and progesterone-treated ovariectomized rats. The results of the bioassay provided no evidence to suggest that hypothalamic GnRH content in juvenile monkeys (mean = 83 ng/hypothalamus; n = 3) was less than that in infants (mean = 54 ng/hypothalamus; n = 4) and adults (mean = 36 ng/hypothalamus; n = 3). A similar developmental pattern in hypothalamic GnRH content was also observed when the decapeptide was measured by RIA. In striking contrast to the maintenance of hypothalamic GnRH content throughout postnatal development, pituitary gonadotropin contents and serum gonadotropin concentrations were markedly reduced in juvenile monkeys.