Sumi Nagase

COMPARISON OF THE EFFECTS OF VARIOUS FINE PARTICLES ON IgE ANTIBODY PRODUCTION IN MICE INHALING JAPANESE CEDAR POLLEN ALLERGENS

The adjuvant effects of various fine particles [Kanto loam dust, fly ash, carbon black, diesel exhaust particles (DEP), and aluminum hydroxide (alum)] on immunoglobulin E (IgE) antibody production in female BDF1 mice were examined. In experiment 1, animals both received 25 micrograms of each particle intranasally and were exposed to aerosolized Japanese cedar pollen allergens (JCPA) for 30 min/d at 1-wk intervals for the first 8 wk. This was followed by exposure for 30 min every 3 wk for the next 9 wk. As parameters of allergic rhinitis, measurements were made of JCPA-specific IgE and IgG antibody titers, the protein-adsorbing capacity of each type of particle, and nasal rubbing movements. The increases in anti-JCPA IgE and IgG antibody production in mice treated with aerosolized JCPA plus respective particles were significantly greater than that found with aerosolized JCPA alone. This was associated with no marked differences in the other allergic rhinitis parameters. In experiment 2, after the administration of particles as in experiment 1, about 160,000 grains of Japanese cedar pollen (JCP, native dry pollen) were dropped onto the tip of the nose of mice twice a week for 16 wk. Six weeks after the first immunization, the anti-JCPA IgE antibody titers of groups treated with the respective particles were greater than 1:20, whereas those of mice treated with JCP alone were 1:10. No significant differences in the anti-JCPA IgE and IgG antibody productions, nasal rubbing counts, or histopathological changes were observed after 18 wk. These results suggested the nature of the particles, their capacity to adsorb antigens, and/or their size may not be related to enhancement of IgG antibody production nor symptoms of allergic rhinitis. However, IgE antibody production seemed to occur earlier in mice treated with particles than in mice immunized with allergens alone.

Effect of albumin on hepatic uptake of warfarin in normal and analbuminemic mutant rats: Analysis by multiple indicator dilution method

Multiple indicator dilution studies of warfarin uptake were carried out on isolated perfused rat livers in the presence and absence of bovine serum albumin (BSA) in the perfusate using normal rats and Nagase analbuminemic rats (NAR). A distributed model was fitted to the dilution data and estimates of influx, efflux, and sequestration rate constants were obtained. In both groups of rats, the intrinsic clearance for unidirectional hepatic uptake (CLinl,influx) of warfarin in the presence of 1.6g/dl BSA was approximately 37–45% of that in the absence of BSA, while the unbound fraction of warfarin with 1.6 g/dl BSA in the perfusate was only 4.2% of that in the absence of BSA. Thus the degree of BSA-induced reduction of the value of CLinl,influx and that of the unbound fraction are different. From these observations, it was found that the hepatic uptake of warfarin is not driven solely by the unbound concentration of warfarin, supporting the recent concepts of albumin-mediated transport for tightly albumin bound ligands as reported by Ockner et al. In addition, the fact that the same hepatic uptake mechanism of warfarin was also observed in NAR suggested that the hepatic uptake of warfarin may not necessarily require a special albumin receptor on the hepatocyte surface.

Regulation of Redox States of Plasma Proteins by Metabolism and Transport of Glutathione and Related Compounds

Glutathione is one of the most abundant naturally occurring thiols in living organisms and is synthesized in its reduced from (GSH). GSH has been known to play a fundamental role in cellular events in different cells and tissues, including protection of organisms against oxidative stress. The two peptide linkages of GSH are sequentially degraded by γ-glutamyltransferase and peptidases that hydrolyze the cysteinylglycine bond; all these enzymes are localized on the outer surface of cell membranes. The turnover of GSH in animals can be understood on the basis of the following three factors: (1) synthesis of GSH occurs exclusively intracellularly, while its degradation occurs predominantly extracellularly; (2) plasma membranes of many tissues and cells have secretory transport systems for GSH and its derivatives; (3) levels of the transferase, a key enzyme for GSH degradation, differ from one tissue to another. Thus, GSH released from tissues with low transferase activity (such as the liver) must be transferred for its rapid turnover to tissues with high enzyme activity (such as the kidney). Further studies on the states of thiol compounds transported via the circulation should be relevant to the understanding of the full scope and physiological significance of the interorgan cooperation of GSH metabolism. Many enzymes and proteins have free SH and disulfide groups within molecules. Function, stability, and in vivo fate of these macromolecules could be affected significantly by their redox state. Although cells and tissues have enzymic defense mechanisms against oxidative stress, the mechanism by which the homeostasis of the redox state of extracellular compartments (such as plasma, urine, bile, etc.) is maintained remains obscure. Plasma mercaptoalbumin (M-Alb) has 17 disulfide bonds and one free cysteinyl residue (Cys-34). This free thiol group can form mixed disulfides with low-molecular weight compounds, such as GSH and cysteine, to generate nonmercaptoalbumin (NM-Alb). Thus, when titrated by several different thiol reagents, less than 1 mole of free SH group (0.4–0.7) was usually detected per mole albumin. The ratio of M-Alb to NM-Alb in plasma samples varies significantly from one sample to another. Many plasma proteins in nonalbumin fractions also formed mixed disulfides with GSH and cysteine. The extent of mixed disulfide formation and the ratio of M-Alb to NM-Alb appeared to change markedly, depending on the redox state of the organisms. The present paper describes the mode of interorgan metabolism and transport of GSH and related compounds, the mechanism by which the redox state of albumin and other plasma proteins is controlled, and their biological significance in healthy and diseased conditions in normal and analbuminemic mutant rats.

Turnover of serum proteins in rats with analbuminemia

Summary In the sera of a strain of rats with analbuminemia, the albumin concentration is less than a thousandth of that in normal rats. However, the total amount of serum proteins in the analbuminemic rats is almost the same as that in normal rats and no spots of additional proteins were observed on two dimensional gel electrophoresis. The half life of serum proteins as a whole in analbuminemic rats injected with [ 3 H]-leucine was 3.0 days, being similar to that of 2.9 days in normal rats. The serum of analbuminemic rats gave 3 distinct peaks of protein and radioactivity (P 1 to P 3 ), while those of normal rats gave an additional peak of albumin (P 4 ) on polyacrylamide gel electrophoresis. The half lives of P 1 , P 2 and P 3 were 4.2, 2.4 and 2.1 days, respectively in analbuminemic rats and 2.5, 1.7 and 2.3 days, respectively in normal rats. The concentrations of P 1 , P 2 and P 3 were 23.1, 19.9 and 20.9 mg/ml, respectively in analbuminemic rats and 4.4, 10.7 and 11.0 mg/ml, respectively in normal rats. The half lives of [ 3 H]-labeled rat albumin injected into the blood of analbuminemic and normal rats were 8.0 and 3.5 days, respectively.

Strain Differences in Susceptibility to 2-Acetylaminofluorene and Phenobarbital Promotion of Rat Hepatocarcinogenesis in a Medium-term Assay System: Quantitation of Glutathione S-Transferase P-positive Foci Development

Strain differences in susceptibility to promotion by the liver carcinogens 2‐acetylaminofluorene (2‐AAF) and phenobarbital (PB) were examined in the medium‐term bioassay system initially developed in our laboratory using male F344 rats as the test animal and glutathione S‐transferase placental form (GST‐P)‐positive foci as the lesion end‐point. Numbers and areas per cm2 of induced GST‐P‐positive hepatocellular foci were compared in LEW, F344, NAR, SD, WBN, SHR, Wistar and ODS rats initiated with diethylnitrosamine (DEN) and subjected to partial hepatectomy during subsequent administration of 2‐AAF or PB. LEW, SD, WBN, and F344 rats were most susceptible to hepatopromotion by both compounds, with a hundred fold increase in lesion area being observed for 2‐AAF in the LEW case. NAR and SHR strains demonstrated an intermediate response, while Wistar and, in particular, the related ODS rats demonstrated very low susceptibilities. The obvious strain differences could be expressed in terms of comparative indices of promoting effects of 2‐AAF and PB as well as DEN itself regarding each of the 8 strains tested. The use of F344 rats for the bioassay model was validated by the relatively high sensitivity to both DEN and 2‐AAF initiation as well as second‐stage promotion stimulus exhibited.

Strain Differences in N-Butyl-N-(4-hydroxybutyl)nitrosamine Bladder Carcinogenesis in Rats

Differences in susceptibility of the urinary bladder epithelium to N‐butyl‐N‐(4‐hydroxybutyl)‐nitrosamine (BBN) in various strains were examined. In experiment 1, 5 strains of male rats were given 0.025% BBN in the drinking water for 8 weeks followed by drinking water without BBN for 32 weeks. Analbuminemic rats (NAR) and ACI rats had high incidences of urinary bladder lesions (papillary or nodular hyperplasia, papilloma and carcinoma), F344 and Wistar rats had low incidences, and Sprague‐Dawley (SD) rats showed an intermediate incidence. Carcinoma area was largest in NAR rats followed in decreasing order by SD, ACI and F344 rats. The extent of tumor invasion was higher in NAR and ACI rats than in SD rats. In experiment 2, the 5 strains of male rats were administered 0.025% BBN in the drinking water. Some rats from each group were killed after each of weeks 4 and 8. The urinary bladder of ACI and NAR rats given BBN had the most marked lesions observed by scanning electron microscopy, with less marked changes in SD rats. F344 and Wistar rats showed the weakest response. Cytochrome P‐450 content of the liver in ACI rats treated with BBN for 4 weeks was significantly higher than those of the controls. Cytochrome P‐450 and Cytochrome b5 contents of the control and BBN‐treated rats were significantly higher in ACI and SD rats than in Wistar, F344 or NAR rats. These results indicate that there are strain differences in the urinary bladder response to BBN.

Hypertriacylglycerolemia and adipose tissue lipoprotein lipase activity in the Nagase analbumineric rat

In Nagase analbuminemic rats, serum triacylglycerol levels were significantly elevated. This abnormality was accompanied by decreased adipose tissue fat stores, and both were more marked in female than in male rats. Parametrial adipose tissue lipoprotein lipase activity was determined in normally fed female rats. When expressed per mg protein, the activity in analbuminemic rats was only 35% of that in control rats. The activity in analbuminemic rats, however, could be increased as in control rats by refeeding starved animals with a fat-free and carbohydrate-rich diet, and the peak values recorded were the same with the two groups. Treatment of animals with streptozotocin lowered adipose tissue lipoprotein lipase activity in both groups to similar levels. These results suggest that hypertriacylglycerolemia associated with analbuminemia may be caused, at least in part, by altered hormonal control of adipose tissue lipoprotein lipase activity.

Identification of F344 rat hepatocytes transplanted within the liver of congenic analbuminemic rats by the polymerase chain reaction.

Hepatocytes isolated from F344 rats were transplanted into the liver of congenic albumin-deficient rats (Nagases analbuminemic rats [NAR]) by infusion into the mesenteric vein. Both albumin-positive hepatocytes in the liver and the serum albumin level increased proportionally to the number of the infused F344 hepatocytes in the recipients. However, there was no such increase in the control rats which had received transplantation of NAR hepatocytes. After the polymerase chain reaction (PCR)-mediated analysis of cDNA and genomic DNA of the recipient livers, the implantation of the F344 hepatocytes was confirmed by the increase in normal albumin mRNA and the presence of 7 bp which are missing in the NAR albumin gene. Although treatment of NAR with the 2-acetylaminofluorene diet turned albumin-negative hepatocytes to positive ones, the sequence of the normal albumin gene could not be identified in the NAR liver. This study demonstrates that the hepatocytes infused into the portal vein are readily organized into the host liver parenchyma and continue to produce albumin.

Inter-organ metabolism and transport of a cysteine-S-conjugate of xenobiotics in normal and mutant analbuminemic rats

Biosynthesis of N-acetylcysteine S-conjugates of toxic electrophiles, mercapturic acids, occurs via inter-organ metabolism and transport in which liver, small intestine and kidney play an important role. Since a mercapturic acid is a hydrophobic organic anion and strongly binds to plasma albumin in vitro, the ligand-albumin interaction may affect the metabolic fate of this final metabolite in vivo. To investigate the role of the circulating albumin in detoxication and elimination of a toxic electrophile, urinary occurrence of the final metabolite was determined in normal and mutant Nagase analbuminemic rats (NAR) after administration of S-benzylcysteine, a model compound of cysteine conjugates. S-Benzylcysteine intravenously administered was excreted rapidly into urine as its N-acetyl derivative in both animal groups. However, the urinary recovery of this mercapturic acid was significantly lower in NAR than in normal animals. The lower urinary recovery in NAR was due to a rapid and random distribution of the unbound metabolite in the circulation to extrarenal tissues. In contrast, no significant difference in the urinary recovery of the final metabolite was observed between the two animal groups if S-benzylcysteine was given orally. Kinetic analysis revealed that the major part of the orally administered S-benzylcysteine was transferred to the liver and acetylated predominantly in this organ in both animal groups; the mercapturic acid which was synthesized in the liver can be transferred to the kidney and excreted into urine even in the absence of the circulating albumin. These results indicate that albumin is important for a final elimination of a mercapturic acid when animals were extraorally challenged with a large dose of toxic electrophiles by which the rate of biosynthesis and the plasma level of the amphipathic metabolites were increased.

Recovery from changes in the blood and nasal cavity and/or lungs of rats caused by exposure to methanol-fueled engine exhaust

One group of male, pathogen-free, Fischer 344 rats was exposed to about 17-fold diluted exhaust generated by an M85 methanol-fueled engine (methanol with 15% gasoline) without catalyst for 8 h, and then the rates of recovery from the resulting increased levels of plasma formaldehyde and carboxyhemoglobin in their erythrocytes were measured. The carboxyhemoglobin level in the erythrocytes was restored within 4 h, whereas the plasma formaldehyde level was still elevated after 4 h but was restored to the normal level within 8 h. No methanol or formic acid was detected in the plasma. Another group of rats was exposed to the same dilution of exhaust for 8 h/d for 7 d, and then the recovery from histopathological damage of the nasal cavity and lungs was also examined. Hyperplasia/squamous metaplasia and erosion of the respiratory epithelium lining the nasoturbinate, maxilloturbinate, or nasal septum, and infiltration of neutrophils into the submucosa at level 1 (level of the posterior edge of the upper incisor teeth) were observed immediately after the exposure period. Lesions of the respiratory epithelium at level 2 (incisive papilla) were less than those at level 1. Slight lesions at levels 1 or 2 were still noticed 1 wk after exposure, but not 4 wk after exposure. Just after exposure, decreases of Clara cells in the terminal bronchiolus and of cilia in the bronchial/bronchiolar epithelium were also observed. Moreover, focal hypertrophy of alveolar walls and increase of macrophages were observed in parts adjacent to respiratory bronchiolus. One week after the exposure period, these changes were no longer seen. These results indicate that changes in the blood and in the nasal cavity and lungs caused by methanol-fueled engine exhaust are reversible. However, complete recovery from damage of the nasal cavity caused by 7-d exposure to the exhaust takes 4 wk, and recovery from elevated plasma formaldehyde and erythrocyte carboxyhemoglobin levels caused by a single 8-h exposure takes 4-8 h.