Establishment of a model of acetaminophen-induced hepatotoxicity in different weekly-aged ICR mice.

Acetaminophen (APAP), a widely used analgesic and antipyretic drug, has the potential to cause lethal hepatotoxicity. Mice are widely used for developing murine models of APAP-induced hepatotoxicity, and many researchers have used these models for APAP-related studies including the fields of biology, pharmacology and toxicology. Although drug-induced hepatotoxicity is dependent on a number of factors (species, gender and age), very few studies have investigated the effect of aging on APAP hepatotoxicity. In this study, we evaluated the effect of age on APAP-induced hepatotoxicity in different weekly-aged mice to establish a model of APAP-induced hepatotoxicity that is an accurate reflection of general experimental conditions. Male ICR mice 4, 6, 8, 10 and 12 weeks old were given APAP intraperitoneally, and mortality, hepatic damage and the plasma concentration of APAP metabolites were evaluated. It was found that younger male ICR mice were relatively resistant to hepatotoxicity induced by intraperitoneal APAP administration. In addition, the APAP-glucuronide concentration in plasma remained essentially the same among the differently-aged mice, while APAP-sulfate levels were dramatically decreased in an age-dependent manner. Thus, it is recommended that mice of the same ages be used in studies related to APAP-induced hepatotoxixity. These results provide evidence in support of not only the age-related changes in susceptibility to APAP-derived hepatotoxicity in mice but also in developing mouse models for APAP-related studies.

Arginine 485 of human serum albumin interacts with the benzophenone moiety of ketoprofen in the binding pocket of subdomain III A and III B.

Arylpropionic acid nonsteroidal anti-inflammatory drusg (NSAIDs) primarily bind to subdomain III A (site II) of human serum albumin (HSA). Ketoprofen (KP), an arylpropionic acid that contains a photoreactive benzophenone moiety, was used to photolabel the binding region of site II. LC/Q-TOF mass spectrometry determination revealed that R485 was the amino acid residue that formed covalent adduct with the benzophenone moiety of KP. Point mutation of arginine 485 to alanine showed a slight decrease in the overall binding percentage of KP when compared to that of native HSA. The induced circular dichroism spectral data of KP with both R485A and native albumin confirmed the photolabeling findings. Interestingly, an increase in the extent of [14C]KP covalent adduct formation with the 11.6 kDa peptide derived from subdomain IIB-IIIA was observed for R485A. In contrast, mutation of arginine 410 caused a significant reduction of binding percentage, confirming the importance of this residue in high affinity binding of arylpropionic acid derivatives. This may indicate that while KP's carboxylate interacts electrostatically with arginine 410, the benzophenone moiety may have swung away from helix 6 in the absence of arginine 485. In this study, photolabeling of native and mutants albumins, R485A and R410C with [14C]KP confirmed that R485 involved in the non-electrostatic interaction with the benzophenone moiety of KP, but not vital to hold KP in the binding pocket of subdomain IIIA.

Genogroup distribution of F-specific coliphages in wastewater and river water in the Kofu basin in Japan.

AIMS: To determine the genogroup distribution of F-specific coliphages in aquatic environments using the plaque isolation procedure combined with genogroup-specific real-time PCR. METHODS AND RESULTS: Thirty water samples were collected from a wastewater treatment plant and a river in the Kofu basin in Japan on fine weather days. F-specific coliphages were detected in all tested samples, 187 (82%) of 227 phage plaques isolated were classified into one of the 4 F-specific RNA (F-RNA) coliphage genogroups and 24 (11%) plaques were F-specific DNA coliphages. Human genogroups II and III F-RNA coliphages were more abundant in raw sewage than animal genogroups I and IV, excluding one sample that was suspected to be heavily contaminated with sporadic heavy animal faeces. The secondary-treated sewage samples were highly contaminated with genogroup I F-RNA coliphages, probably because of different behaviours among the coliphage genogroups during wastewater treatment. The river water samples were expected to be mainly contaminated with human faeces, independent of rainfall effects. CONCLUSIONS: A wide range of F-specific coliphage genogroups were successfully identified in wastewater and river water samples. SIGNIFICANCE AND IMPACT OF THE STUDY: Our results clearly show the usefulness of the genogroup-specific real-time PCR for determining the genogroups of F-specific coliphages present in aquatic environments.

Enhancement of dissolution and bioavailability of flurbiprofen by low molecular weight chitosans.

The dissolution behavior and absorption of flurbiprofen (FP) following oral administration from:three types of chitosans (LM chitosans), with different molecular weights and degree of acetylation, have been studied in comparison with those of the drug alone. The solubility of FP increased with concentrations of LM chitosan, especially in the case of C-III, with the highest degree of deacetylation degree among the three chitosans. This indicates that amino groups of LM chitosan play an important role in its interaction with FP. Moreover, spectroscopic studies, including NMR data, indicate that the binding involves interactions between the carboxyl group of FP and the amino group of the chitosans. The dissolution rates of FP for a C-III kneaded mixture were enhanced with increasing amounts of C-III. The oral absorption of FP from a C-III kneaded mixture was improved to a significant extent, compared to FP alone. These results suggest that FP from LM chitosan kneaded mixture increases the dissolution rate and improves the bioavailability of the drug by the formation of a water-soluble complex.

Renal clearance of glycolaldehyde- and methylglyoxal-modified proteins in mice is mediated by mesangial cells through a class A scavenger receptor (SR-A).

AIMS/HYPOTHESIS: Glomerular mesangial expansion is a characteristic feature of diabetic nephropathy, and the accumulation of AGE in the mesangial lesion has been implicated as one of its potential causes. However, the route for the AGE accumulation in mesangial lesions in diabetic patients is poorly established. METHODS: Glycolaldehyde-modified BSA (GA-BSA) and methylglyoxal-modified BSA (MG-BSA) were prepared as model AGE proteins, and their in vivo plasma clearance was examined in mice, and renal uptake by in vitro studies with isolated renal mesangial cells. RESULTS: Both (111)In-GA-BSA and (111)In-MG-BSA were rapidly cleared from the circulation mainly by both the liver and kidney. Immunohistochemical studies with an anti-GA-BSA antibody demonstrated that intravenously injected GA-BSA accumulated in mesangial cells, suggesting that such cells play an important role in the renal clearance of circulating AGE proteins. Binding experiments at 4 degrees C using mesangial cells isolated from mice showed that (125)I-GA-BSA and (125)I-MG-BSA exhibited specific and saturable binding. Upon incubation at 37 degrees C, (125)I-GA-BSA and (125)I-MG-BSA underwent endocytic degradation by these cells. The binding of the ligands to these cells was inhibited by several ligands for scavenger receptors. The endocytic degradation of GA-BSA by mesangial cells from class A scavenger receptor (SR-A) knock-out mice was reduced by 80% when compared with that of wild-type cells. The glomerular accumulation of GA-BSA after its intravenous administration was attenuated in SR-A knock-out mice, as evidenced by immunohistochemical observations. CONCLUSIONS/INTERPRETATION: These results raise the possibility that circulating AGE-modified proteins are subjected to renal clearance by mesangial cells, mainly via SR-A. This pathway may contribute to the pathogenesis of AGE-induced diabetic nephropathy.

Differential hormonal regulation of carbonyl reductase activities in liver and kidney microsomes of rat.

1. In the male rat, hepatic microsomal carbonyl reductase (CR) activity decreased by testectomy (Tx) was restored to the control level by the treatment with testosterone propionate (TP), even though the enzyme activity decreased by hypophysectomy (Hx) was not increased by the treatment with TP. On the other hand, renal microsomal CR activities decreased by Tx and Hx were markedly increased by the treatment with TP. 2. The treatment with TP had no effect on the CR activity in liver microsomes of the ovariectomized or hypophysectomized female rat. On the other hand, the CR activities in kidney microsomes of the ovariectomized and hypophysectomized female rat were significantly increased by the treatment with TP. 3. The results indicate that in rat programmed by neonatal androgens, the hepatic microsomal CR activity is regulated indirectly by androgens through the hypothalamus-pituitary system, whereas the hormonal regulation of the renal microsomal CR activity is not via the pituitary. We conclude that the regulatory mechanism of the CR activity in liver microsomes is distinguishable from that in kidney microsomes.

Up-regulation by clarithromycin of alpha(1)-acid glycoprotein expression in liver and primary cultured hepatocytes.

alpha(1)-Acid glycoprotein (AGP) is the major transport protein for cationic drugs, endogenous ligands, and some anionic drugs in plasma. Hepatic synthesis and secretion of AGP are altered during acute inflammation as well as by a number of drugs. This alteration could influence the binding of drugs and its biological function. Macrolide antibiotics are widely used in the treatment of a variety of infectious diseases. The effects of macrolide antibiotics have been studied with respect to rat AGP expression in vivo. After the individual administration of six macrolides to rats, with the exception of oleandomycin, five increased AGP levels in serum. Of these five, clarithromycin (CAM) was the most potent inducer of AGP, which reached a maximum level between 3 to 7 days after administration. CAM increased the steady-state level of AGP mRNA in liver as well as protein level in serum in a dose-dependent manner. In addition, CAM increased AGP mRNA levels in primary cultured hepatocytes. In the luciferase promoter assay, CAM potentiated dexamethasone-increased promoter activity of the AGP gene, which contained the glucocorticoid response element, in cultured rat hepatocytes, although CAM itself had no effect on its activity. The effect of CAM and dexamethasone was diminished by glucocorticoid response element deletion or mutation or by adding the antiglucocorticoid, RU486. Further, in the mouse mammary tumor virus (MMTV) promoter containing functional glucocorticoid response element, CAM potentiated dexamethasone-increased promoter activity. In the adrenalectomized rats, CAM did not increase AGP levels in serum. These findings suggest that CAM may cause transcriptional induction of AGP, at least in part, via a glucocorticoid-mediated mechanism.

Hormonal regulation of male-specific 20beta-hydroxysteroid dehydrogenase with carbonyl reductase-like activity present in kidney microsomes of rats.

Progesterone, 17alpha-hydroxyprogesterone, cortisone and cortisol, which are C(21)-steroids with a ketone group at the 20-position, potently inhibited the activity of enzyme acetohexamide reductase (AHR) responsible for the reductive metabolism of acetohexamide in kidney microsomes of male rats. Furthermore, progesterone was a competitive inhibitor of AHR. In the case of progesterone usage as the substrate, 20beta-hydroxysteroid dehydrogenase (20beta-HSD) activity was much higher than 20alpha-hydroxysteroid dehydrogenase (20alpha-HSD) activity in kidney microsomes of male rats. These results indicate that AHR present in kidney microsomes of male rats, functions as 20beta-HSD with carbonyl reductase-like activity. In male rats, both testectomy and hypophysectomy decreased the renal microsomal 20beta-HSD activity, but the decreased enzyme activities were increased by the treatment with testosterone propionate (TP). We propose the possibility that TP treatment regulates the renal microsomal 20beta-HSD activity by acting directly on the kidney of male rats. This is supported from the fact that when TP was given to ovariectomized and hypophysectomized female rats, the male-specific 20beta-HSD activity was detected in their kidney microsomes.

Purification and characterization of L-2,3-butanediol dehydrogenase of Brevibacterium saccharolyticum C-1012 expressed in Escherichia coli.

The L-2,3-butanediol dehydrogenase produced in E. coli JM109/pLBD2-CTC was purified by 5 steps. The molecular mass of this enzyme was estimated at 110 kDa and the subunit was measured to be 30 kDa. The L-BDH had some differences from the BDHs from other sources in substrate specificity, pI value, pH stability, effects of divalent cations, and organic acids.

Stability of a cisplatin-chondroitin sulfate A complex in plasma and kidney in terms of protein binding.

To assess the stability of a cisplatin (CDDP) complex prepared with chondroitin sulfate A (CSA) relative to protein binding in the circulation and kidney, a trichloroacetic acid (TCA) precipitation method was developed to measure the protein-unbound species of CDDP and the CDDP-CSA complex in plasma and kidney homogenates. The total and unbound drug concentrations were determined up to 3 h following a 2 mg/kg bolus injection of CDDP or CDDP-CSA complex to rats. The stability against plasma binding was evaluated by a determination of the area under concentration-time curve from time 0 to infinite time (AUC(0-infinity)); the ratio of unbound drug AUC(0-infinity) to total drug AUC(0-infinity) was employed to estimate the availability of the unbound drug in the circulation. The results showed that a competitive reaction to platinum existed between plasma protein and the CDDP-CSA complex, but the complex accounted for more than 60% of the protein-unbound species for a dosage, compared to 30% obtained by an administration of uncomplexed CDDP. The tissue binding kinetics in kidney for CDDP and the CDDP-CSA complex was investigated by the use of homogenates. The binding rate constants of CDDP and CDDP-CSA in a kidney homogenate were 0.0040 min(-1) and 0.0014 min(-1), respectively. The results indicate that the CDDP-CSA complex could effectively retard the binding of CDDP to protein in the kidney. These data provide evidence that endogenous protein is able to compete for platinum from the CDDP-CSA complex, but the complex effectively retarded the protein binding reaction with CDDP in plasma and kidney as compared to native CDDP, which has the potential for reducing the accumulation of CDDP in plasma and kidney.

Interaction mechanism between indoxyl sulfate, a typical uremic toxin bound to site II, and ligands bound to site I of human serum albumin.

PURPOSE: The study was performed for clarifying the mechanism of interaction between indoxyl sulfate (IS), a typical uremic toxin bound to site II, and site I-ligands when bound to human serum albumin (HSA). The effect of the N to B transition on the interactions was also examined. METHODS: Quantitative investigation of the relations between ligands bound to HSA was performed by equilibrium dialysis, and the binding data were analyzed on the basis of a theoretical model for simultaneous binding of two ligands. RESULTS: The high-affinity binding constants for the site I-ligands warfarin (WF) and dansyl-L-asparagine (DNSA) increased with increasing pH, whereas those for the site II-ligands IS and dansylsarcosine (DNSS) were hardly affected by pH. Mutual displacement experiments showed that even though IS binds to site II it influenced binding of DNSA at the azapropazone binding area in site I. By contrast, it is unlikely that IS affects the WF binding area of site I. Furthermore, pH-profiles showed that the interaction between IS and DNSA was very sensitive to the N to B transition: "competitive-like" strong allosteric regulation was observed for binding of the two ligands to the N conformer (pH 6.5), whereas in the B conformation (pH 8.5) binding of these molecules was nearly "independent". CONCLUSIONS: The present data provide useful information for elucidating a potential mechanism of interaction between drugs and endogenous substances including uremic toxins.

Effect of oxidative stress on the structure and function of human serum albumin.

PURPOSE: Human serum albumin (HSA) was mildly oxidized by a metal-catalyzed oxidation system (MCO-HSA), chloramine-T (CT-HSA) or H2O2 (H2O2-HSA), and the effects of these treatments on the structural, drug-binding and esterase-like properties were studied. METHODS: Protein conformation was examined by calorimetric, chromatographic, electrophoretic and spectroscopic techniques. Drug binding was studied by ultrafiltration method, and esterase-like activity was determined using p-nitrophenyl acetate as a substrate. RESULTS: Far-UV and near-UV CD spectra indicated that significant structural changes had occured as the result of treatment with MCO-HSA and CT-HSA but not with H2O2-HSA. However, SDS-PAGE analysis does not provide precise information on gross conformational changes such as fragmentation, cross-linking and SDS-resistant polymerisation. The results of differential scanning calorimetry, the fluorescence of the hydrophobic probe 1,1-bis-4-anilino-naphthalene-5,5-sulfonic acid and the elution time from a hydrophobic HPLC column indicated that MCO-HSA and CT-HSA in particular, have a more open structure and a higher degree of exposure of hydrophobic areas than unoxidized HSA. In all cases, high-affinity binding of warfarin remained unchanged for all the oxidized HSAs. However, high-affinity binding of ketoprofen to CT-HSA and, especially, MCO-HSA was diminished. In addition, the esterase-like activity of these proteins were all decreased to the same low level. CONCLUSIONS: Mild oxidation of HSA has no detectable effect on the binding of drugs to site I in subdomain IIA. In contrast, both the ligand binding property of site II and the esterase-like activity of oxidized HSAs are decreased, most probably due to conformational changes in subdomain IIIA.

High-performance liquid chromatography with chemiluminescence detection of penbutolol and its hydroxylated metabolite in rat plasma.

This paper describes a new method of high-performance liquid chromatography with chemiluminescence detection for the analysis of penbutolol (PB) and its main metabolite, 4-hydroxy penbutolol (4-OH PB) in rat plasma. 4-Dimethylaminosulfonyl-7-(N-chloroformylmethyl-N-methyl) amino-2,1,3-benzoxadiazole (DBD-COCl) was used as a fluorogenic labeling reagent. A mixture of hydrogen peroxide and bis[4-nitro-2-(3,6,9-trioxadecyloxycarbonyl)phenyl]oxalate (TDPO) in acetonitrile was used as a post-column chemiluminogenic reagent. The derivatives of PB and 4-OH PB with DBD-COCI were separated by isocratic effluent with 0.01 M imidazole buffer (pH 7.0)-acetonitrile within 10 min. The detection limits of the proposed method for PB and 4-OH PB were 9.9 and 15 fmol on column, respectively. After intravenous administration of PB in rats, its plasma concentration profiles of PB and 4-OH PB were determined by the proposed method. PB was demonstrated to be rapidly metabolized to 4-OH PB at the same rate as cardiac output.

Conformational stability and warfarin-binding properties of human serum albumin studied by recombinant mutants.

Correctly folded recombinant wild-type human serum albumin and the single-residue mutants K199A, W214A, R218H and H242Q were produced with the use of a yeast expression system. The changes in R218H resulted in a pronounced decrease in intrinsic fluorescence. Thermodynamic parameters for thermal denaturation of the present mutants and of five additional mutants have been determined, showing small increases in stability for two mutants (R218H and H242Q) and a larger decrease in stability for one (W214A). In the last of these, denaturation was a heterogeneous process starting at physiological temperature. The high-affinity binding constant for warfarin at pH 7.4 was determined by fluorescence spectroscopy: there was a significant increase in affinity for binding of warfarin to H242Q and K199A and a smaller decrease in affinity for W214A and R218H. The findings show that Trp-214 is not as essential for the high-affinity binding of warfarin as has previously been thought.

FK506 (tacrolimus) increases rat alpha1-acid glycoprotein expression in liver and primary cultured hepatocytes.

FK506 (tacrolimus) (10 mg/kg, s.c., 5 days) increased rat alpha1-acid glycoprotein (AGP) in serum and AGP mRNA in liver. FK506 potentiated the dexamethasone-increased AGP expression in primary cultured hepatocytes. In the luciferase promoter assay, FK506 potentiated the dexamethasone-increased promoter activity of the AGP gene in cultured rat hepatocytes, although FK506 alone had no effect on its activity. The combined effect of FK506 and dexamethasone was diminished by glucocorticoid responsive element (GRE) deletion and mutation or by an anti-glucocorticoid. These results indicated that FK506 causes the transcriptional induction of AGP, at least in part, via a glucocorticoid-mediated mechanism.

Crystallization and preliminary X-ray studies of meso-2,3-butanediol dehydrogenase from Klebsiella pneumoniae IAM1063.

Meso-2,3-butanediol dehydrogenase (meso-BDH) has been crystallized and preliminary X-ray crystallographic characterization of meso-BDH crystals has been performed. Single crystals of meso-BDH were prepared in two forms by the hanging-drop vapour-diffusion method using polyethylene glycol as a precipitant. Form I crystals belong to space group C2, with unit-cell parameters a = 215.5, b = 79.4, c = 134.8 A, beta = 98.22 degrees, and form II crystals belong to space group P2(1), with unit-cell parameters a = 69.16, b = 109.78, c = 127.28 A, beta = 102.29 degrees. The crystals diffracted to 2.0 and 1.7 A resolutions, respectively, using synchrotron radiation.

Flunitrazepam, a 7-nitro-1,4-benzodiazepine that is unable to bind to the indole-benzodiazepine site of human serum albumin.

Benzodiazepine (BDZ) is generally thought to bind to site II of human serum albumin (HSA), also known as the indole-BDZ site, which is located at subdomain III A of the molecule. However, differences in the binding characteristics of BDZ drugs with HSA have been reported. The photolabeling profiles of HSA with [(3)H]flunitrazepam (FNZP) in the presence and absence of diazepam (DZP) were shown to be identical, suggesting that each drug primarily binds to different regions. The results of fluorescent probe displacement experiments showed that FNZP failed to decrease the fluorescence of dansylsarcosine to an extent similar to that of DZP. In the photoinhibition experiment, site I and site II ligands failed to inhibit the photoincorporation of [(3)H]FNZP to HSA. In order to evaluate the photolabeling specificity of FNZP, an attempt was made to photolabel alpha(1)-acid glycoprotein (AGP) which also binds BDZ with similar affinity as HSA. The effect of myristate (MYR) and DZP on the FNZP photolabeling of these two major drug binding plasma proteins was examined. Photoincorporation was inhibited when HSA was photolabeled with [(3)H]FNZP in the presence of MYR but not in the presence of DZP. Conversely, DZP inhibited the photolabeling of [(3)H]FNZP to AGP. These results suggest that FNZP interacts with HSA at regions which are not located in the preformed binding pocket of subdomain III A.

Determination of reduced, protein-unbound, and total concentrations of N-acetyl-L-cysteine and L-cysteine in rat plasma by postcolumn ligand substitution high-performance liquid chromatography.

A high-performance liquid chromatographic assay was developed for the quantitative determination of the sulfur-containing amino acids N-acetyl-L-cysteine (NAC) and L-cysteine (Cys) in rat plasma. The thiols were separated by reverse-phase ion-pair chromatography, and the column eluent was continuously mixed with an iodoplatinate-containing solution. The substitution of sulfur of the thiol compound with iodide was quantitatively determined by measuring changes in the absorption at 500 nm. The low-molecular-weight disulfides and mixed disulfide conjugates of thiols with proteins were entirely reduced to the original reduced compounds by dithiothreitol. By reducing these two types of disulfides separately during sample pretreatment, the reduced, protein-unbound, and total thiol concentrations could also be determined. Validation testing was performed, and no problems were encountered. The limit of detection was approximately 20 pmol of thiol on the column. The present method was used to measure the plasma concentrations of NAC and Cys in the rat after a bolus intravenous administration of NAC, focusing on disulfide formation. The binding of NAC to protein through mixed disulfide formation proceeds in a time-dependent and reversible manner. Moreover, this "stable" covalent binding might limit total drug elimination, while the unbound NAC is rapidly eliminated. Consequently, the analytical method described in this study is very useful for the determination of plasma NAC and Cys, including disulfide conjugates derived from them.

[Strain-, sex- and species-related differences of acetohexamide reductase and 20 beta-hydroxysteroid dehydrogenase activities in liver microsomes of experimental animals].

We examined physiological and genetic factors affecting acetohexamide reductase (AHR) and 20 beta-hydroxysteroid dehydrogenase (20 beta-HSD) activities in liver microsomes of experimental animals. Pronounced strain-related differences were found in both activities of AHR and 20 beta-HSD present in liver microsomes of male rats. Among rat strains tested in this study, even though a Wistar-Imamichi (WIM) rat strain was taken to lack AHR activity, it exhibited a significant 20 beta-HSD activity. These findings appeared to be in conflict with our conclusion reported so far, which AHR and 20 beta-HSD present in liver microsomes of male rats are identical enzymes. Thus the reason for this discrepancy was discussed. Furthermore, AHR and 20 beta-HSD activities were little or not observed in liver microsomes of female rats or male experimental animals other than the rat, indicating the existence of sex- and species-related differences in these two enzyme activities.