Gerard J. Mulder

[11] Isolated perfused rat liver technique

Publisher Summary The function of the liver in the metabolism of xenobiotics can be studied with a spectrum of liver preparations ranging from the intact organ in vivo, through perfusion systems, liver slices, isolated hepatocytes, homogenates, and membrane fractions, to purified enzymes.. The use of the isolated perfused liver is also attractive because of the intact architecture of the organ, the possibilities of controlling blood and bile flow, and the ease of the manipulation of the composition of the perfusion medium. Advantages include the large number of perfusate samples that may be collected from and compared with the intact animal and the smaller number of interactions with endogenous compounds. In addition, it is possible to use the concentrations of the substrates and metabolic inhibitors that would not be tolerated in vivo because of their toxicity. Compared with isolated hepatocyte preparations, the perfusion system does not require damaging treatment with Ca 2+ -free solutions and digesting enzymes and the normal functional polarity of cells and their localization in the liver lobule is maintained.

Dose-dependent shifts in the sulfation and glucuronidation of phenolic compounds in the rat in vivo and in isolated hepatocytes: The role of saturation of phenolsulfotransferase

Abstract The role of enzyme-kinetic parameters of sulfotransferase and UDP-glucuronyltransferase in the balance between sulfation and glucuronidation of various phenolic substrates was studied in the rat in vivo after i.v. administration and in isolated hepatocytes. A pronounced shift from sulfation to glucuronidation was observed in vivo upon increasing the dose of two phenols, harmol and phenol. Similar shifts were found when these compounds were incubated with isolated hepatocytes. However, the shift from sulfation to glucuronidation was small when 4-chlorophenol, or absent when 4- t -butylphenol were given in vivo . Such shifts were also absent when 4-chlorophenol and 4- t -butylphenol were incubated at increasing concentrations with isolated hepatocytes. The in vivo results with the various phenols were very similar to the conjugation patterns found in isolated hepatocytes. This suggests that these conjugations in hepatocytes are regulated by similar factors as in the intact animal. In isolated hepatocytes at most 16 per cent of the available pool of inorganic sulfate was consumed during the incubation. Since Cheng and Levy have shown [ J. biol. Chem. 255 , 2637 (1980)] that uptake of inorganic sulfate by hepatocytes is very rapid, the present results suggest that the limitation of sulfation of harmol and phenol at increasing dose was caused by saturation of the overall sulfation process by the acceptor substrate, rather than by depletion of inorganic sulfate.

UDP glucuronyltransferase and phenolsulfotransferase in vivo and in vitro: Conjugation of harmol and harmalol

Abstract Glucuronidation and sulfation of the phenolic — OH group of harmol and harmalol by the rat in vivo , and by rat liver subcellular fractions in vitro have been studied. In vivo harmol was extensively excreted (59% of the dose in 3 hr) in bile and urine, mostly as harmol-sulfate (70%) but also as harmol-glucuronide (30%). Harmalol was also excreted in bile and urine (50% of the dose in 3 hr), mostly as harmalol-glucuronide (70%), with only a trace of harmalol-sulfate present (less than 3%). In vitro kinetic parameters of conjugating activities towards both substrates were determined. Harmol and harmalol were glucuronidated by UDP glucuronyltransferase at comparable rates. Phenolsulfotransferase converted harmol readily to its sulfate-conjugate, whereas harmalol was a very poor substrate of this enzyme. Thus, the excretory pattern of harmol and harmalol can be explained by different rates of conjugation of these substrates by UDP glucuronyltransferase and phenolsulfotransferase, as found in vitro .

Absorption, serum levels and urinary excretion of inorganic sulfate after oral administration of sodium sulfate in the conscious rat

The absorption of inorganic sulfate after ingestion was investigated in rats. After oral administration of Na235SO4, 35S radioactivity was measurable in plasma already after 15 min and its plasma concentration reached a peak after about 1.5--2 h. The 35S-radioactivity excreted in urine during 24 h after ingestion of Na235SO4 together with varying amounts of unlabelled Na2SO4 (0.25--5.0 mmol Na2SO4 per rat) indicated an almost complete absorption of inorganic sulfate from the gastrointestinal tract. Determination of the inorganic sulfate concentration in rat serum 2 h after oral administration of 5.0 mmol Na2SO4 revealed a three-fold increase in serum sulfate concentration. The data suggest a rapid and almost complete absorption of inorganic sulfate after oral administration in the rat. Its importance in relation to the sulfate availability for sulfate conjugation of drugs is discussed.

AVAILABILITY OF INORGANIC SULFATE AS A RATE LIMITING FACTOR IN THE SULFATE CONJUGATION OR XENOBIOTICS IN THE RAT - SULFATION AND GLUCURONIDATION OF PHENOL

Abstract Phenol is converted by the rat in vivo into the sulphate and glucuronide conjugates. When the intravenously administered dose of 14 C-labelled phenol was increased from 13 to 266 μmol/kg the percentage glucuronidated increased from 28 to 60 per cent of the dose; the percentage sulphated decreased from 72 to 40 per cent. In rats with intact kidneys the conjugates were almost completely excreted in the urine: when the kidneys were ligated phenylglucuronide was excreted in bile to a high extent, but biliary excretion of phenylsulphate was still very low. Using 35 S-labelled sodium sulphate, incorporation of [ 35 S]-sulphate into phenylsulphate could be observed; phenol enhanced the disappearance of [ 35 S]-sulphate from plasma. No significant depletion of inorganic sulphate was found when a high dose of unlabelled phenol (266 μmol/kg) was injected. The literature data on sulphate depletion by substrates of phenolsulphotransferase are critically reviewed; from our data is concluded that the relative decrease of phenol sulphation at high phenol doses is not due to sulphate depletion.

PREVENTION OF THE HEPATOTOXIC ACTION OF N-HYDROXY-2-ACETYLAMINOFLUORENE IN THE RAT BY INHIBITION OF N-O-SULFATION BY PENTACHLOROPHENOL

Abstract The extent of the hepatotoxic action of 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 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 Nue5f8O-sulfation, 45 min before the administration of N -hydroxy-2-acetylaminofluorene, completely prevented the hepatotoxic effects of this carcinogenic hydroxamic acid. Therefore, it is concluded that Nue5f8O-sulfation is responsible for the hepatotoxic action of N -hydroxy-2-acetylaminofluorene.

Use of pentachlorophenol as long-term inhibitor of sulfation of phenols and hydroxamic acids in the rat in vivo

Inhibition of sulfation of the phenolic compound harmol (7-hydroxy-1-methyl-9H-pyrido[3,4-b]indole) by pentachlorophenol (PCP) was studied in the Wistar rat: PCP was administered in various ways to find a convenient method for long-term inhibition of sulfation. High doses of PCP or sodium pentachlorophenolate (NaPCP) in the diet (350 ppm) or NaPCP in the drinking water (1.4 mM) of Wistar rats for one week inhibited the sulfation of harmol by 30-45%. The plasma concentration of PCP in rats with NaPCP (1.4 mM) in their drinking water was highest (270 microM) in the period that the animals were kept in the dark and consumed food and water. This is explained by a rapid elimination: the elimination of PCP from plasma, after intravenous administration, showed a biphasic disappearance curve with half-lives of 2.17 and 7.24 hrs, respectively. This is much faster than in Sprague-Dawley rats. A log-linear correlation was found between the plasma concentration of pentachlorophenol and the inhibition of harmol sulfation. Although administration of NaPCP to rats in their drinking water inhibited the sulfation of harmol only by 45%, it inhibited the sulfation of the carcinogenic arylhydroxamic acid N-hydroxy-2-acetylaminofluorene by 70-75%.

The oxidation of L- and D-cysteine to inorganic sulfate and taurine in the rat

Oral administration of L-cysteine to rats (8 mmol/kg body wt.) caused a rapid increase of the concentration of cystine in serum, from less than 5 micro M in controls to about 200 micro M. Concomitantly, the serum concentration of inorganic sulfate increased, reaching a peak 2 h after L-cysteine administration; this level, twice the control level, was maintained for 4 h. Serum sulfate returned to control concentration 23 h after L-cysteine administration. The urinary excretion of inorganic sulfate during the 24 h after administration increased considerably, and 33% of the dose of L-cysteine was recovered as inorganic sulfate in urine. Consumption of comparable amounts of L-cysteine via the food caused the same increase in urinary excretion of sulfate, but did not increase the concentration of sulfate in serum. After oral administration of D-cysteine (8 mmol/kg body wt.), very high cystine levels were reached in serum (mean concentration about 1500 microM); the sulfate concentration was already maximal 30 min after administration. The increase in urinary excretion of sulfate after D-cysteine was also higher than after L-cysteine administration: 55% of the dose. Possible routes for the rapid degradation of D-cysteine to inorganic sulfate are discussed. The administration of L-cysteine also caused an increase the serum concentration of taurine, while methionine was not influenced. D-Cysteine did not increase the serum concentration of taurine, indicating that it is probably not or only slowly converted to taurine.

An evaluation of methods to decrease the availability of inorganic sulphate for sulphate conjugation in the rat in vivo

Abstract The concentration of inorganic sulphate in serum of the rat (about 0.9 mM) could be lowered in the following three different ways. (1) Oral administration of sodium chloride (8 mmol/kg) decreased serum sulphate within 2 hr to 0.5 mM. Eight hours after administration serum sulphate had returned to the control level. (2) Feeding of a low-protein diet (8 per cent casein, without supplements of sulphur-containing amino acids or inorganic sulphate salts) reduced urinary sulphate excretion in 2 days to 10 per cent of control. Concomitantly, serum sulphate was decreased to half the control level. (3) Paracetamol (1.0 or 1.5 mmol/kg, orally), a substrate of sulphation, reduced serum sulphate within 3 hr to 30 per cent of control. Eight hours after administration the sulphate concentration tended to rise again. Fasting initially increased serum sulphate; after 3 days of fasting still considerable amounts of inorganic sulphate were excreted in urine (50–70 per cent of control). Even after 3 days serum sulphate was not yet significantly decreased below control. Lowering of the serum sulphate concentration results in a decreased availability of inorganic sulphate. Sulphation of a high dose of phenol (266 μmol/kg) was decreased at a serum concentration of sulphate of 0.3 mM, presumably because sulphate was depleted by the high dose of phenol. Feeding the low-protein diet, however, caused no decrease in sulphation at a tracer dose of [ 14 C]-phenol (1.25 μmol/kg), while paracetamol pretreatment did cause a decrease in the fraction of the dose that became sulphated, probably because remaining unconjugated paracetamol competed with phenol for sulphation; the tracer dose of [ 14 C]-phenol did not further deplete sulphate. The findings are discussed in relation to implications for the toxicity of many xenobiotics that are eliminated as sulphate conjugates.

[4] Collection of metabolites in bile and urine from the rat

Publisher Summary The procedure for the collection of metabolites from the rat primarily includes the administration of drugs. Further, for the cannulation of the bile duct, a 2-cm incision is made across the abdomen just below the liver. The overlying liver lobes and gut are gently moved out of the way, and the duodenum is gently pulled. The bile duct is noted as a thin vessel that lies between the hilus, where it leaves the liver, and the point at which it joins the duodenum. The upper part of the bile duct is free and should be used for catheterization at about 1 cm from the liver hilus. In this case, the pancreatic ducts are left intact and mixing of bile with pancreatic secretions is avoided. To collect bile for a prolonged period, the bile is collected external to the animal and to the cage by way of a swivel joint that is connected to polyethylene tubing. An alternative method is to implant a reservoir into the peritoneal cavity of the rat for the collection of bile. To study the role of the liver in the conjugation of xenobiotics, two other operative procedures may be useful. A bypass may be used to temporarily exclude the liver from the circulation. Another useful method is the cannulation of a side branch of the portal vein to both administer compounds and to sample directly at the portal circulation. Additionally, a simple way to collect the metabolites of a compound excreted in urine (and feces) is to place the rats in metabolism cages and collect urine and feces separately.