Bioactivation of the hepatocarcinogen N-hydroxy-2-acetylaminofluorene by sulfation in the rat liver changes during the cell cycle.

Sulfation activity towards N-hydroxy-2-acetylaminofluorene and 4-nitrophenol was determined in male rat liver cytosol at several time points after partial hepatectomy corresponding to G1-, S-, and M-phase. N-hydroxy-2-acetylaminofluorene sulfation activity decreased by 80% when hepatocytes entered the G1-phase. This lower activity was maintained during the S-phase and M-phase, but was restored when hepatocytes entered the G0-phase again. Sulfation activity towards 4-nitrophenol did not alter after hepatectomy. Various other cytosolic enzyme activities were determined after hepatectomy to investigate the specificity of the decrease in sulfation activity. Lactate dehydrogenase and glucose-6-phosphate dehydrogenase activities were increased in the S- and M-phase by maximally 80% and 60%, respectively. Glutathione-S-transferase and glutamate-pyruvate transaminase activity did not alter during the cell cycle. These results indicate that sulfation of N-hydroxy-2-acetylaminofluorene in hepatocytes may depend on the phase of the cell cycle. The relevance of the finding is discussed in relation to the resistance of proliferating (pre)neoplastic hepatocytes to the toxic and mitoinhibitory effects of N-hydroxy-2-acetylaminofluorene.     

Effect of metabolic control on urinary excretion and plasma levels of catecholamines in diabetics.

Urinary excretion and plasma levels of catecholamines were determined in 20 normal and 39 diabetic subjects to evaluate the sympathetic activity. Diabetic patients were divided into 4 groups according to the metabolic control. Sympathetic activity showed no differences between normal and subjects with chemical diabetes (group I, n = 5). In insulin-treated diabetics in good metabolic control (group II, n = 11) only urinary excretion of free norepinephrine was significantly higher than normals (p less than .05). In insulin-treated diabetics in poor metabolic control (group III, n = 16) urinary excretion and plasma levels of norepinephrine showed a marked increase over groups I and II (p less than .001). In insulin-treated diabetics with ketosis (group IV, n = 7) urinary excretion and plasma levels of both norepinephrine and epinephrine showed the highest values (p less than .001 and less than .1). Finally, in groups III and IV, after achieving improved metabolic control, a significant decrease of urinary excretion and plasma levels of catecholamines was observed. The results confirm that there is an increased rate of catecholamine release in poorly controlled diabeties and suggest a close correlation between sympathetic activity and metabolic derangement in diabetes.     

Taurolithocholate-induced inhibition of biliary lipid and protein excretion in the rat.

Taurolithocholate (TLC), a natural bile salt, induces selective impairment on canalicular membrane of the hepatocyte, which seems to be a major determinant of its cholestatic effect in experimental animals. In order to extend existing studies about the effects of TLC on bile secretion, we examined in TLC-treated rats the biliary excretion of compounds that are transported to canalicular membrane via vesicles, such as lipids and proteins. The single intravenous injection of TLC (3 mumol/100 g body wt.) inhibited transiently the biliary bile salt excretion, while the biliary excretion of lipids (i.e., cholesterol and phospholipids) and proteins remained inhibited even though the biliary excretion and composition of bile salts were normalized. Under such a condition, TLC also inhibited the transcellular vesicular pathway to the exogenous protein horseradish peroxidase entry into bile, without altering the paracellular biliary access of the protein. The hepatic uptake of horseradish peroxidase was unaffected by TLC-treatment. The results indicate that TLC can inhibit the biliary excretion of compounds that reach the canaliculus via a vesicular pathway, such as lipids and proteins, by a mechanism not related to a defective bile salt excretion. Possible explanations for these findings are discussed.     

Decreased calcium and magnesium urinary excretion during prostaglandin synthesis inhibition in the rat

The effect of endogenous renal prostaglandins on calcium and magnesium reabsorption was investigated. Renal tubular handling of calcium and magnesium was studied by clearance methods in anesthetized Sprague-Dawley and Brattleboro rats, either intact or thyroparathyroidectomized (ATPTX), before and during prostaglandin synthesis inhibition by meclofenamate, indomethacin, or piroxicam infusion. These three inhibitors had similar effects on calcium and magnesium excretion: A significant decrease in absolute and fractional excretions of both cations was observed in intact Sprague-Dawley rats, and in ATPTX rats of both strains, but not in intact Brattleboro rats. These results suggest an inhibitory effect of prostaglandins on vasopressin-, glucagon-, but not PTH-mediated calcium and magnesium reabsorption. This effect is likely to occur in the thick ascending limb of Henle, which is both a target site for these polypeptidic hormones, and a segment where the bulk of calcium and magnesium is reabsorbed.     

Prevention of the hepatotoxic action of N-hydroxy-2-acetylaminofluorene in the rat by inhibition of NO-sulfation by pentachlorophenol

The extent of the hepatotoxic action of $\text{N}$-hydroxy-2-acetylaminofluorene in the rat was determined by following changes in histochemistry, and the activities of glutamate-oxaloacetate transaminase (EC 2.6.1.1) and glutamate-pyruvate transaminase (EC 2.6.1.2) in serum. Administration of $\text{N}$-hydroxy-2-acetylaminofluorene (120 μmol/kg i.v.) cased a periportal (zone I) necrosis which was accompanied by a large increase in glutamate-oxaloacetate transaminase and glutamate-pyruvate transaminase activity in serum. Treatment of rats with pentachlorophenol and 2, 6-dichloro-4-nitrophenol, known inhibitors of NO-sulfation, 45 min before the administration of $\text{N}$-hydroxy-2-acetylaminofluorene, completely prevented the hepatotoxic effects of this carcinogenic hydroxamic acid. Therefore, it is concluded that NO-sulfation is responsible for the hepatotoxic action of $\text{N}$-hydroxy-2-acetylaminofluorene.     

Relation between glutathione redox changes and biliary excretion of taurocholate in perfused rat liver

The influence of the intracellular glutathione status on bile acid excretion was studied in the perfused rat liver. Perturbation of the thiol redox state by short term additions of diamide (100 microM) or hydrogen peroxide (250 microM) or t-butyl hydroperoxide (250 microM) led to a reversible inhibition of biliary taurocholate release without affecting hepatic uptake; inhibition amounted to 45% for diamide and 90% for the hydroperoxides. Concomitantly, the bile acid accumulated intracellularly. Bile flow increased from 1.3 to 2.0 microliters X min-1 X g liver-1 upon infusion of taurocholate (10 microM); the latter value was suppressed to 1.2 microliters X min-1 X g liver-1 by the addition of t-butyl hydroperoxide (250 microM). Similarly, the hepatic disposition of another bile constituent, bilirubin, was suppressed by 70% upon addition of hydrogen peroxide. While the addition of hydrogen peroxide inhibited also the endogenous release of bile acids almost completely, endogenous bile flow was much less affected, decreasing from 1.3 to 1.0 microliters X min-1 X g liver-1. Measurement of [14C]erythritol clearance showed bile/perfusate ratios of about unity both in the absence and presence of hydrogen peroxide, suggesting canalicular origin of the bile under both conditions. In livers from Se-deficient rats low in Se-GSH peroxidase (less than 5% of controls), hydrogen peroxide inhibited taurocholate transport substantially less, providing evidence for the involvement of glutathione in mediating the inhibition observed in normal livers. The percentage inhibition of taurocholate release and intracellular glutathione disulfide (GSSG) content were closely correlated. The addition of t-butyl hydroperoxide caused a several-fold increase of biliary GSSG release, whereas biliary GSH release was even decreased. The results establish a role of glutathione in canalicular taurocholate disposition.     

Species variations in the biliary and urinary excretion of arsenate, arsenite and their metabolites

In most mammalian species, inorganic arsenicals are extensively biotransformed and excreted both in unchanged form and as metabolites. In the bile of rats receiving arsenate (AsV) or arsenite (AsIII) we have identified monomethylarsonous acid (MMAsIII), purportedly the most toxic metabolite of inorganic arsenic. As rats are not commonly accepted for studying arsenic metabolism, we carried out a comparative investigation on the excretion of AsV, AsIII and their metabolites in five animal species in order to determine whether they also form MMAsIII from AsV and AsIII. Anaesthetised bile duct-cannulated rats, mice, hamsters, rabbits, and guinea pigs were injected with AsV or AsIII (50 micromol/kg, i.v.) and their bile and urine was collected for 2 h. Arsenic in bile and urine was speciated by HPLC-hydride generation-atomic fluorescence spectrometry and the excretion rates of AsV, AsIII, monomethylarsonic acid (MMAsV), MMAsIII and dimethylarsinic acid (DMAsV) were quantified. All species injected with AsV excreted arsenic preferentially into urine, whereas all animals receiving AsIII, except rabbits, delivered more arsenic into bile than urine. Bile contained almost exclusively trivalent arsenic (i.e. AsIII and/or MMAsIII), whereas AsV, AsIII and DMAsV appeared in urine. Except for guinea pigs, which do not methylate arsenic, the other species formed MMAsIII and excreted it into bile. Having excreted as much as 8% of the dose of AsIII or AsV in 2 h as MMAsIII, rats were by far the most efficient producers of this supertoxic metabolite. Thus, although the rat is not a good model for studying long-term arsenic disposition, this species appears especially valuable in studies on AsIII methyltransferase and in vivo formation of MMAsIII.     

Reaction products of the carcinogen N-hydroxy-4-acetylamino-4'-fluorobiphenyl with DNA in liver and kidney of the rat

The binding of AABP4'F and ABP4'F residues to rat liver and kidney DNA in vivo was studied at different periods of time after administration of N-[G-3H]hydroxy-AABP4'F at dose levels of 5 and 25 mg/kg body weight. DNA preparations from both organs were hydrolyzed enzymatically at pH 8--9 with mixtures of DNAase, snake venom phosphodiesterase and alkaline phosphatase from Escherichia coli. The enzymatic digests were analysed by Sephadex LH-20 chromatography using synthetic N-([G-14C] deoxyguanosin-8-yl)-AABP4'F as marker. Elution with 30% ethanol gave three major peaks of tritium activity. The first peak consisted largely of N-(deoxyguanosin-8-yl)-ABP4'F decomposition products, which were not further characterized. The second product has similar chromatographical and chemical properties as 3-(deoxyguanosin-N2-yl)-AAF; and was also persistent in liver as well as in kidneys. The third peak of tritium activity co-chromatographed with the marker compound N-([G-14C] deoxyguanosin-8-yl)-AABP4'F. Kinetic studies revealed that the latter product was removed rapidly from liver and kidney DNA at equal rates (t1/2 = 2 days). Approximately 80% of the total radioactivity bound to DNA consisted of deacetylated material, which was removed at a much slower rate (t1/2 = 10 days) in both organs. An initial rapid removal of all products in kidney during the first 7 days (t1/2 = 3.3 days) at dose levels of 25 mg/kg is probably due to toxic effects on the kidneys, because this phenomenon was not observed at dose levels of 5 mg/kg. The synthetic ester N-OSO3K-AABP4'F was at least twice as reactive towards L-methionine and guanosine as compared to the corresponding AABP derivative, but had 40% of the reactivity of N-acetoxy-AAF under similar conditions. The new compounds 3-methylmercapto-4-acetylamino-4'-fluorobiphenyl and N-(deoxyguanosin-8-yl)-4-acetylamino-4'-fluorobiphenyl have been characterized by means of their NMR and mass spectra. Attempts to devise an unambiguous synthesis for 3-(deoxyguanosin-N2-yl)arylamides have been unsuccessful.     

Formation of electrophilic N-acetoxyarylamines in cytosols from rat mammary gland and other tissues by trans-acetylation from the carcinogen N-hydroxy-4-acetylaminobiphenyl

The 105 000 × g supernatant fractions of various rat tissues catalyze the transfer of the N-acetyl group of certain carcinogenic aromatic acethydroxamic acids to the O atom of aromatic hydroxylamines. The resulting N-acetoxyhydroxylamines are strongly electrophilic and have been detected and analyzed through their reaction with N-acetylmethionine to yield methylmercaptoaminoarenes.Of the rat tissues studied the liver had the highest activity; kidney and small intestinal mucosa were about 15–20% as active. The transacetylase activities of these tissues were similar with respect to their ability to use either N-hydroxy-2-acetylaminofluorene (N-hydroxy-AAF or N-hydroxy-4-acetylaminobiphenyl (N-hydroxy-AABP) as acetyl donors, their stability on storage at 2–3°C, and their elution patterns from Sephadex G-100 columns. Low transacetylase activity was found in spleen and muscle.Mammary tissue from 16–21 day pregnant rats had 20% of the transacetylase activity of rat liver when N-hydroxy-AABP was used as acetyl donor and N-hydroxy-4-aminobiphenyl (N-hydroxy-ABP) was the acetyl acceptor. This enzyme system from mammary tissue did not utilize the fluorene derivatives as either acetyl donor or acetyl acceptor, was much more labile than the liver, kidney, or intestinal mucosa systems, and had a pH optimum at 7.5, as compared to pH 6.8 for liver. The mammary tissue system was similar to the hepatic system in being inhibited by sulfhydryl reagents; it required a source of reduced pyridine nucleotides for maximum activity.     

Depression of biliary glutathione excretion by chronic ethanol feeding in the rat

The effects of chronic alcohol feeding on biliary glutathione excretion were studied in rats pair fed diets containing either ethanol (36% of total energy) or isocaloric carbohydrate for 4-6 weeks. An exteriorized biliary-duodenal fistula was established and total glutathione (GSH) and oxidized glutathione (GSSG) were measured. A significant decrease was observed in rats fed alcohol chronically compared to their pair fed controls in the biliary excretion of GSH (55.7 +/- 37.0 vs 243.1 +/- 29.0 micrograms/ml bile, p less than 0.025) as well as biliary GSSG (12.5 +/- 5.0 vs 49.9 +/- 8.0 micrograms/ml bile, p less than 0.05) and in bile flow (23.1 +/- 1.6 vs 29.2 +/- 1.3 micrograms/min, p less than 0.05). An acute dose of ethanol tended to exaggerate the decrease on biliary GSH and GSSG in the two groups of animals. The depression in biliary GSH could not be attributed to decreased GSH synthesis since S35-L-methionine incorporation into hepatic and biliary GSH was unchanged or even increased after chronic ethanol feeding.     

Secretion from rat basophilic leukaemia cells induced by calcium ionophores Effect of pH and metabolic inhibition

Previous experiments on the functional properties of rat basophilic leukaemia cells showed a major anomaly when compared to normal mast cells: though IgE-mediated secretion was dependent on external Ca²⁺ with both types of cells, substantial non-cytotoxic release with ionophore A23187 could be demonstrated with the normal cells but not with the tumour cells. We now show that when the pH of the incubation medium is increased to 8 it is possible to obtain excellent Ca-dependent, non-cytotoxic secretion from tumour basophils with the ionophores A23187 and ionomycin. These results provide further evidence that secretion from the tumour cells occurs via a mechanism similar to that used by normal mast cells and basophils. Experiments with metabolically inhibited tumour cells suggest that their unusual sensitivity to the cytotoxic effects of Ca²⁺ ionophores may be related to their ability to sequester intracellular calcium. Changes in the conditions of cell culture appeared to produce substantial and at least partially reversible changes in responsiveness to IgE-mediated triggering and ionophores.     

Hepatic storage and biliary excretion of rose bengal in the rat

The distribution of intravenously administered rose bengal (RB) depends on its dose. At a low dose (10 mg/kg), RB can be found almost solely in the liver and plasma. However, at higher doses (from 25 up to 200 mg/kg) the amount of RB found in extra-hepatic tissues gradually increases. In this experiment the hepatic transfer maximum of RB amounted to 146 micrograms/kg/min. By increasing the dose from 10 to 200 mg/kg, the hepatic concentration of RB also approached a maximum (1250 micrograms/g). The storage capacity of the liver, however, did not limit the transfer maximum of RB.     

The bile flow and biliary excretion of ursocholate in the rat

Several studies reported that ursodeoxycholate (but not its conjugates), when administered intravenously, increased the biliary bicarbonate concentration in the rat (1–3). At the same time, a complete dissociation between bile flow and the bile salt excretion rate was produced in the second hr of infusion (2). In order to examine whether this property was due to the 7β-hydroxy group in its molecular structure, the choleretic property of ursocholate (3α, 7β, 12α-trihydroxy-5β-cholanoic acid) was investigated in male Wistar rats. Immediately after the start of iv infusion of ursocholate at a rate of 1.2 μmole/min/100 g b. wt., both the bile flow and bile salt excretion rate began to increase. However, unlike with ursodeoxycholate, the bile salt excretion rate continued to be high in the second and third hr of infusion, while the bile flow rate gradually increased. Furthermore, the bicarbonate concentration in the bile fell slightly 10 min after the start of ursocholate infusion. Although the concentration tended to return to the baseline value before the bile salt infusion in the later period of observation, no significant increase in bicarbonate concentration was observed during the whole observation period. These properties were quite similar to those of cholate rather than those of ursodeoxycholate. However, a cholate infusion at the same rate of 1.2 μmole/min/100 g b.wt. caused a cholestasis as early as 20 to 30 min after the start of an infusion. These results suggest that the previously reported properties of ursodeoxycholate (that it causes a complete dissociation between the bile flow and bile salt excretion rate in the second hr and that it increases the biliary bicarbonate concentration) were not due to the 7β-hydroxy group in its steroidal structure, and that the choleretic property of ursocholate is similar to its 7α-hydroxy epimar, cholate. However, the much lower cytotoxicity of ursocholate compared to cholate appears to be due to the 7β-hydroxy group that ursocholate has.     

Effect of probenecid on the urinary excretion of TXB2 and PGE2 in the anaesthetised rat

In the anaesthetised rat, probenecid (33 mg/kg) produced a 50% fall in urinary TXB2 excretion indicating that a component of the TXB2 excreted in the urine is secreted by the proximal tubule. At a higher dose of probenecid (100 mg/kg) this effect was overcome, a relative increase in urinary TXB2 excretion being produced. This may provide evidence for the proximal reabsorption or bi-directional transport of TXB2 in the rat. At 100 mg/kg probenecid also produced an 8-fold increase in urinary PGE2 excretion. Although the bi-directional transport of PGE2 is well known, this is the first time urinary PGE2 excretion rate has been shown to be modified by probenecid. The increase in PGE2 excretion could obscure the assessment of any inhibition by probenecid of proximal PGE2 secretion. It could also provide evidence for the proximal reabsorption of PGE2. However the interpretation of probenecid-induced changes in eicosanoid excretion in terms of modified tubular reabsorption must be treated with caution since urinary eicosanoid excretion could be increased by other properties of probenecid including inhibition of either protein binding or the uptake of eicosanoids into the lung.