Purification and characterization of two recombinant human granulocyte colony-stimulating factor glycoforms. Pharmacokinetic and activity studies of single-dose administration in mice.

Two recombinant human granulocyte colony-stimulating factor (rhG-CSF) isoforms were isolated from the medium conditioned by an engineered Chinese hamster ovary (CHO) cell line. The two rhG-CSFs were characterized and were found to differ in the carbohydrate structure attached to Thr-133. The glycoform, referred to as Peak 1, contains the O-linked glycan Neu5Ac(alpha 2-3)Gal(beta 1-3)GalNAc; the Peak 2 glycoform contains the O-linked glycan Neu5Ac(alpha 2-3)Gal(beta 1-3)[Neu5Ac(alpha 2-6)]GalNAc. The two glycoforms displayed a similar biological activity in cultures of a mouse 32D C13 cell line and human bone-marrow myelo-monocytic progenitor cells (CFU-GM). In the latter test both glycoforms displayed a higher activity than nonglycosylated rMet-hG-CSF from Escherichia coli. The pharmacokinetic profile and activity of the two rhG-CSF glycoforms and of a mixture of them (Pool) were investigated in mice treated with a single injection of rhG-CSF at the doses of 125 micrograms and 250 micrograms/kg, given via the intravenous (i.v.) and the subcutaneous (s.c.) route, respectively. The plasma concentration profiles obtained were similar for all three substances and did not show any relevant differences in absorption or elimination. The pharmacokinetic parameters indicate that the three substances have similar area under the curve (AUCs), volumes of distribution, and terminal half-life. Furthermore, our data indicate a high bioavailability of the two different glycoforms of rhG-CSF when given to mice via the s.c. route either singularly or as a mixture. Detectable levels of rhG-CSF persisted for more than 8 h in the i.v. and more than 24 h in the s.c. route of administration. All three substances induced early neutrophilia in mice. All rhG-CSF-treated mice developed a two-four-fold rise in neutrophil counts as early as 4 h after the intravenous and 2 h after the subcutaneous injection. Relatively high levels of neutrophils were maintained for at least 8 and 24 h after i.v. and s.c. administration, respectively.

Light-dependent activation of 7,12-dimethylbenz[a]anthracene to a potent genotoxicant.

7,12-Dimethylbenz[a]anthracene (DMBA), which is widely used in mutagenesis and experimental carcinogenesis, is activated to a mutagen by white fluorescent light. A 40 min exposure to white fluorescent light of Salmonella typhimurium TA98 plates treated with DMBA, in the absence of exogenous metabolism, resulted in an approximately 30-fold increase in the number of histidine revertants. This phenomenon also occurs, with lesser intensity, with other promutagens, such as benzo[a]pyrene or 2-acetylaminofluorene, and in other Salmonella tester strains. Moreover, white fluorescent light is able to activate DMBA to a toxicant for Chinese hamster V79 cells in culture, resulting in very low cell survival. Under these conditions, white fluorescent light-activated DMBA was shown to cause chromosomal aberrations, but not gene mutations, as determined by resistance to thioguanine. This white fluorescent light-dependent activation of DMBA seems to be related to the formation of reactive species, as the addition of vitamin E results in a reduction in the number of histidine revertants induced by white fluorescent light in S. typhimurium TA98.

Effects of ouabain and chloride-free medium on isoosmotic volume control and ultrastructure of hepatocytes in primary culture.

Rat hepatocytes in primary monolayer culture have been studied by a combination of physiological and morphological approaches under conditions affecting ion transport and cell volume. A concentration of ouabain completely inhibiting the coupled transport of Na+ and K+ had little effect on cell volume, as indicated by cell water content, but induced the formation of many vesicles in the cytoplasm. Apparent fusion of vesicles was often observed. By itself, replacement of medium Cl by NO3- had little effect on cell volume or morphology. However, when NO3- replaced Cl- in the presence of ouabain the cells swelled and the numbers and size of vesicles were much reduced. The vesicles accumulating in the presence of ouabain showed a yellow fluorescence after the cells were loaded with acridine orange, implying that the vesicular contents were acidic. Total fluid-phase endocytosis, determined by uptake of Lucifer yellow, was not affected by ouabain or the absence of Cl-. However, ouabain considerably retarded the subsequent release of Lucifer yellow; this suggests that the dye originally taken into endocytotic vesicles became diluted by mixing with contents of ouabain-induced vesicles, an explanation consistent with the vesicle fusion seen by electron microscopy. The Cl-free medium also retarded Lucifer yellow efflux, to the same extent as ouabain, and the effects of the two treatments were not additive. These observations are consistent with the activity in hepatocytes of an ouabain-resistant, Cl(-)-dependent mechanism for cell volume control. It is suggested that this depends on the accumulation of water into acidic vesicles, which is driven by the Cl(-)-coupled activity of the vacuolar ATPases of the organelles, followed by exocytotic expulsion of their contents.

Regulation of cellular water and ionic content in lungs of fetal and adult rats.

Slices of lungs from late-fetal (1 day pre-partum) and adult rats lost K+ and gained Na+, Cl-, water and Ca2+ during pre-incubation at 1 degrees C. These changes were reversed upon restoration to 37 degrees C. The recovery of composition at 37 degrees C was completely dependent on cell respiration in adult slices; by contrast, glycolysis could support partial recovery in the fetal slices. Ouabain completely inhibited K+ reaccumulation at both ages but inhibited net extrusion of water by no more than 50%. Replacement of medium Cl- with NO3- prevented the extrusion of water in the presence of ouabain in adult but not fetal slices. Transmission electron microscopy of type II epithelial cells in slices of both ages showed that ouabain induced the formation of many cytoplasmic vesicles, apparently derived from the Golgi apparatus. Regulation of cell ionic and water content is thus generally similar in late-fetal and adult lung tissue, but there are differences in the source of ATP and in some features of ouabain-resistant volume regulation.

Effects of chlorinated hydrocarbons on cellular volume regulation in slices of rat liver.

The effects of carbon tetrachloride and 1,2-dichloroethane (1,2DCE) on the recovery of slices of rat liver from cellular swelling in vitro were studied. Slices took up water during pre-incubation at 1 degrees C, then cellular volume and ultrastructure were rapidly restored during subsequent incubation at 38 degrees C. Ouabain (2 mm) inhibited water extrusion by less than 50%, while inducing formation of peri-canalicular vesicles, apparently derived from the Golgi apparatus. Neither CCl(4) nor 1,2DCE (up to 10 mm) affected the initial extrusion of water at 38 degrees C in the absence of ouabain, but renewed swelling occurred after 60 min with either agent; this was associated with loss of membrane selectivity and some histological damage. By contrast, 1,2DCE inhibited water extrusion in the presence of ouabain after less intensive exposure, for example with 5 mm-1,2DCE for 60 min or 10 mm for 15-30 min. With ouabain present, 1,2DCE (10 mm) caused marked swelling of the endoplasmic reticulum, reduced the peri-canalicular vesicles seen with ouabain alone and reduced the formation of canalicular microvilli. Both CCl(4) and 1,2DCE inhibited the ATP-dependent accumulation of Cl(-) by isolated vesicles of the Golgi apparatus, The delayed swelling of hepatocytes at high concentrations of 1,2DCE and CCl(4) in the absence of ouabain is probably a non-specific consequence of membrane damage. By contrast, 1,2DCE specifically inhibits the ouabain-resistant extrusion of water, possibly by interfering with a postulated mechanism for the exocytotic expulsion of water.

Effects of monensin on ATP levels and cell functions in rat liver and lung in vitro.

Effects of the proton-alkali cation-exchanging ionophore, monensin, on aspects of cellular metabolism and ionic exchanges have been studied in rat tissues in vitro. Incubation of liver slices at 38 degrees C with 0.1 microM monensin induced time-dependent vesiculation, initially in the Golgi region, reduction of ATP content and of protein synthesis. At 1 microM, monensin also reduced net, active movements of K+, Na+, Cl- and water in liver slices and inhibited state 3 respiration in isolated mitochondria. The respiratory inhibitor, amytal, similarly reduced ATP content and protein synthesis at concentrations lower than those inhibiting ion transport in slices. Low concentrations of monensin (0.1-1.0 microM) had similar effects on ATP and ion transport in slices of adult lung. By contrast, late-fetal liver and lung were much less sensitive to monensin; in these tissues, glycolysis sustained substantial levels of ATP. Monensin also induced vesiculation of the Golgi apparatus in fetal lung cells. It is concluded that by lowering ATP levels, monensin can markedly alter various metabolic activities in those cells which depend primarily on oxidative phosphorylation for their metabolic energy.

Comparison of metabolic effects of carbon tetrachloride and 1,2-dichloroethane added in vitro to slices of rat liver.

Carbon tetrachloride and 1,2-dichloroethane (1,2DCE) were added in vitro to freshly prepared slices of rat liver and the time- and concentration-dependence of their toxic effects on several metabolic parameters determined. With each agent, the most sensitive effect was an increase of malondialdehyde production by a microsomal preparation isolated from the treated slices. The next most sensitive parameter was the inhibition of amino acid incorporation into slice proteins, followed by inhibition of net K(+) accumulation and the induction of early necrotic changes, as indicated by loss of histological staining with azure II. Substantially greater exposures were required to reduce cellular ATP and to initiate entry of Ca(2+). This sequence was similar with both agents, but CCl(4) was the more potent in each case. When added in combinations of submaximally effective concentrations, the two agents produced at least additive inhibitions of protein synthesis and K(+) accumulation. We conclude that metabolic effects in liver slices can be a useful in vitro test for potential toxicity of chlorinated hydrocarbons. Amino acid incorporation and K(+) transport are the most convenient indicator systems, combining considerable sensitivity to relatively low levels of exposure with convenience of measurement.