Effects of microsomal enzyme inducers on glutathione S-transferase isoenzymes in livers of rats and hamsters.

The effects of microsomal enzyme inducers on glutathione S-transferase (GST) isoenzymes were studied in livers of rats and hamsters using three hypolipidemic drugs of the peroxisome proliferator type and the two model substances phenobarbital (PB) and 3-methylcholanthrene (MC). The effects were investigated by immunoblot analysis of the various GST subunits using polyclonal antibodies directed to rat subunits 1-4. In untreated animals the subunit composition was different, with hamsters having a much higher content of class mu isoenzymes. Administration of all three hypolipidemic drugs reduced the protein concentration of both alpha and mu class GSTs in rats but reduced only class mu subunits in hamsters. This reduction was in good agreement with the decreased activity observed with the broad-spectrum substrate 1-chloro-2,4-dinitrobenzene (CDNB) in both species. As expected, PB and MC increased GST activity together with the concentration of subunits 1 and 3 in rats. In hamsters, PB significantly increased subunit 1 and slightly reduced subunits 3 and 4, although this decrease was not significant. Total GST, measured with CDNB, was reduced by 17%. In contrast, MC slightly decreased subunit 1 and markedly raised subunits 3 and 4, resulting in a net increase in total GST activity. All drugs increased relative liver weight, microsomal protein concentration and total P450 in both species; in contrast, total cytosolic proteins were raised by all drugs in rats but not in hamsters, except for MC. The results obtained in these two species show that GST activity is not always increased by microsomal enzyme inducers. The response may depend in part on isoenzyme profile, and varies with the subunit considered.

Impairment of hepatic glutathione S-transferase activity as a cause of reduced biliary sulfobromophthalein excretion in clofibrate-treated rats.

Administration of clofibrate reduced the maximal excretion rate of bile sulfobromophthalein (BSP) in rats but left that of phenol-3,6-dibromophthalein (DBSP) unchanged. This decrease in liver transport of BSP was due to reduced bile excretion of conjugated BSP. Hepatic uptake and storage of this dye were not impaired. Liver glutathione S-transferase activity in vitro, measured with BSP, 1,2-dichloro-4-nitrobenzene (DCNB) or 1-chloro-2, 4-dinitrobenzene (CDNB) was significantly reduced. This alteration in liver conjugating activity was probably not related to a modification of the hepatic GSH pool, since the GSH level was unchanged or only increased slightly after clofibrate treatment. Detection of this inhibition required at least two daily doses of clofibrate. Inhibition was dose-related and lasted for several days after cessation of the drug. In clofibrate-treated rats, Lineweaver-Burk plots showed a reduced Vmax for both the BSP and GSH substrates. These results suggest that clofibrate decreases hepatobiliary transport of BSP by lowering glutathione S-transferase activity in the liver.

Inhibition of liver glutathione S-transferase activity in rats by hypolipidemic drugs related or unrelated to clofibrate.

The effects of in vivo administration of six hypolipidemic drugs on rat liver glutathione S-transferase activity were compared. This activity was measured with sulfobromophthalein (BSP), 1,2-dichloro-4-nitrobenzene (DCNB) or 1-chloro-2,4-dinitrobenzene (CDNB) as substrate. Except for the nicotinic acid derivative ethanolamine oxiniacate, all the compounds tested significantly reduced it, whether or not they were related to clofibrate. The hepatic glutathione concentration either remained unchanged or only increased slightly after treatment with the various drugs. When measured, the maximal excretion rate of bile BSP dropped significantly, but not that of phenol-3,6-dibromophthalein (DBSP). Hepatic dye uptake and storage were not impaired. These results show that hypolipidemic drugs of the peroxisome proliferator type inhibit rat liver glutathione S-transferase activity and may reduce transport of anions conjugated with glutathione before excretion.

Deficient induction of sulfobromophthalein conjugating activity by phenobarbital in hamster liver.

Administration of phenobarbital, a known inducer of glutathione S-transferase activity in rat liver, failed to stimulate sulfobromophthalein (BSP) conjugation by liver cytosol in hamsters. The latter displayed poor ability to conjugate this substrate, despite very high glutathione-conjugating activity with the broad-spectrum substrate 1-chloro-2,4-dinitrobenzene (CDNB). Of the six substrates tested, in this species, 1,2-epoxy-3-(4-nitrophenoxy)propane (ENPP) was the only one whose conjugation was greatly enhanced by phenobarbital (+172%). Nevertheless, hamsters proved as responsive to phenobarbital induction as rats, since it increased their relative liver weight and microsomal enzyme activity. The deficient induction of liver BSP-conjugating activity observed with phenobarbital is consistent with the finding that it did not affect the hepatic transport of this substrate in hamsters.

Bilirubin content and 4-nitrophenol glucuronosyltransferase activity in Gunn rat liver.

The purpose of this study was to determine whether the hepatic content of bilirubin could influence liver 4-nitrophenolglucuronosyltransferase (4-NP-GT) in the Gunn rat. In animals fed on a 45% lipid diet, compared with rats fed on a normal lipid diet (3%), the bilirubin content of the hepatic microsomal fraction decreased and the bilirubin/protein ratio was reduced. 4-NP-GT activities were comparable in both groups. Administration of clofibrate to Gunn rats greatly enhanced the bilirubin content of liver microsomal fraction. Since this treatment raised the microsomal protein content, the bilirubin/protein ratio was not modified. No significant change in 4-NP-GT was noted. After bilirubin perfusion in Gunn and Wistar rats, no change was observed in hepatic monooxygenase activities or in 4-NP-GT, although the bilirubin/protein ratio was dramatically increased in the microsomal fraction. From these results the low activity of liver 4-NP-GT in Gunn rats does not seem directly related to the hepatic content of bilirubin.

Free fatty-acid uptake by isolated rat hepatocytes.

In isolated rat hepatocytes, the rate of palmitic acid binding and uptake is directly related to the concentration of free fatty acid (FFA) in the medium. After their entry into the cell, FFA are immediately incorporated into cellular phospholipids and triglycerides and no accumulation of free fatty acids can be demonstrated inside the cell. The rate of free fatty-acid uptake remains unchanged after incubation in a 2 mM KCN containing medium, indicating that in the range of fatty-acid concentrations used in this study, this phenomenon does not require energy.

Long-term maintenance of taurocholate uptake by adult rat hepatocytes co-cultured with a liver epithelial cell line.

Taurocholate (TC) uptake by adult rat hepatocytes co-cultured with other rat liver epithelial cells (RLEC) was studied comparatively to hepatocytes in primary culture. Cells were cultured on Petri dishes for desired times prior to measuring their ability to transport TC. TC uptake was linear for 150 sec in both culture conditions. In hepatocytes cultured alone, the initial rate of TC uptake at an extracellular concentration of 100 microM was 0.19 +/- 0.02 nmole per min per 10(6) cells after 48 hr of culture and decreased by 75% after 4 to 6 days. In hepatocytes co-cultured with RLEC, the rate of uptake at 48 hr (0.31 +/- 0.01 nmole per min per 10(6) cells) was significantly higher than in hepatocytes cultured alone (p less than 0.01); in addition, TC uptake remained stable at an average rate of 0.17 +/- 0.01 nmole per min per 10(6) cells for up to 56 days. No detectable uptake was found in RLEC cultured alone. TC uptake exhibited both saturable (Vmax = 0.30 +/- 0.03 nmole per min per 10(6) cells and Km = 42.6 +/- 4.4 microM) and nonsaturable components. These kinetic parameters were similar to those previously reported in isolated hepatocytes and in short-term cultured hepatocytes. TC uptake exhibited sodium dependence and was significantly reduced when extracellular sodium was replaced by lithium and sucrose, or in the presence of 1 mM ouabain. After 18 days of co-culture, TC uptake had qualitatively the same characteristics as at 48 hr, with a saturable and a nonsaturable component.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of clofibrate on the enzyme activity of rat liver plasma membranes.

The activity of 3 plasma membranes marker enzymes (5'-nucleotidase, Mg++-ATPase and alkaline phosphodiesterase-I) was determined in plasma membranes isolated from liver of control and of clofibrate-treated rats. A complete indentity of plasma membranes enzyme activity in the 2 groups of experimental animals was observed for the 3 enzymes studied.

Breast milk jaundice: in vitro inhibition of rat liver bilirubin-uridine diphosphate glucuronyltransferase activity and Z protein-bromosulfophthalein binding by human breast milk.

Twenty-four samples of breast milk from nine mothers of infants suffering from breast milk jaundice were studied. Eight samples of milk from mothers of nonjaundiced infants, along with five formula milks enriched with polyunsaturated fatty acids, served as controls. Milks from mothers with jaundiced infants had no inhibitory effect when assayed immediately after thawing. However, after these milk samples were stores at 4 degrees, they strongly inhibited bilirubin conjugation (80.3% inhibition of uridine diphosphate glucuronyltransferase (UDPGT) activity) and bromosulfophthalein (BSP) binding to cytoplasmic Z protein (dye binding inhibited 82.1%). There was no effect on BSP binding to Y protein (see Table 1). Heating the milk to 56 degrees modified the results in the following manner; when the milk was heated immediately after thawing, no inhibitory effect was seen, even after storage for 96 hr. On the other hand, when the milk was first stored at 96 hr and then heated, it had the same inhibitory effects as the milks which were stored without heating. The present study shows that pathologic breast milk will inhibit BSP-Z protein binding only when stored under conditions that also cause the appearance of the capacity to inhibit bilirubin conjugation in vitro, as well as causing the liberation of nonesterified fatty acids. Thus, the appearance of this inhibitory capacity in vitro seems linked to the lipolytic activity particular to pathologic milks.

PIP: 24 samples of breastmilk from 9 mothers of infants suffering from breastmilk jaundice were studied. 8 samples of milk from mothers of nonjaundiced infants, along with 5 formula milks enriched with polyunsaturated fatty acids, served as controls. Milks from mothers with jaundiced infants had no inhibitory effect when assayed immediately after thawing. However, after these milk samples were stored at 4 degrees, they strongly inhibited bilirubin conjugation (80.3% inhibition of uridine diphosphate glucuronyltransferase activity) and (BSP) bromosulfophthalein binding to cytoplasmic Z protein (dye binding inhibited 82.1%). There was no effect on BSP binding to Y protein (intable). Heating the milk to 56 degrees modified the results in the following manner; when the milk was heated immediately after thawing, no inhibitory effect was seen, even after storage for 96 hours. On the other hand, when the milk was 1st stored for 96 hours and then heated, it had the same inhibitory effects as the milks which were stored without heating. The present study shows that pathologic breast milk will inhibit BSP-Z protein binding only when stored under conditions that also cause the appearance of the capacity to inhibit bilirubin conjugation in vitro, as well as causing the liberation of nonesterified fatty acids. Thus, the appearance of this inhibitory capacity in vitro seems linked to the lipolytic activity particular to pathologic milks.

Bilirubin UDP-glucuronyltransferase activity of wistar rat kidney.

Wistar rat kidneys have been shown to possess a bilirubin glucuronyltransferase (BGT) activity capable of conjugating about 3/5 of the total pool of unconjugated bilirubin within 48 h of being grafted to Gunn rat hosts. Bilirubin conjugated by the kidney is taken up by the liver and excreted in the bile. Except when the bile duct is ligated, no conjugated bilirubin appears in the plasma or urine. Renal BGT activity is about 1/20th of the hepatic activity on a weight basis in Wistar rats. The Gunn rat's hyperbilirubinemia probably causes an induction of the renal enzyme since its activity doubles in 48 h.

Effects of pregnancy on the toxicity and metabolism of acetaminophen in mice.

Although acetaminophen is widely used in pregnant women, the effects of pregnancy on its hepatotoxicity remain unknown. We assessed these effects in pregnant mice (17-18 days of gestation). The hepatotoxicity of acetaminophen (300-400 mg X kg-1 i.p.) was increased markedly in pregnant mice, as judged by increased serum glutamic-pyruvic transaminase activity, higher incidence of liver necrosis and greater mortality. In vitro, acetaminophen sulfotransferase activity was increased by 47% in pregnant mice, but acetaminophen glucuronosyltransferase activity was decreased by 54%; the metabolic activation of acetaminophen to covalently bound metabolites was unchanged. Glutathione S-transferase activities were decreased slightly. In vivo, after administration of acetaminophen (300 mg X kg-1 i.p.), the 24-hr urinary excretion of the sulfate conjugate was increased (from 12% of the recovered dose in nonpregnant mice to 21% in pregnant mice), that of the glucuronide was decreased (from 61 to 52%), whereas those of the cysteine and mercapturic acid conjugates and that of acetaminophen were unchanged. Finally, the plasma clearance and the apparent volume of distribution of acetaminophen (both expressed per body weight) remained unchanged. Similarly, in vivo covalent binding to hepatic proteins 4 hr after administration of acetaminophen (300 and 400 mg X kg-1 i.p.) remained unchanged as were in vivo indexes of lipid peroxidation. In contrast, liver glutathione concentration, albeit initially normal, fell to much lower levels after administration of acetaminophen (200-400 mg X kg-1 i.p.) or diethylmaleate (0.5 ml X kg-1 i.p.) in pregnant mice, and recovered more slowly thereafter.(ABSTRACT TRUNCATED AT 250 WORDS)

[Clofibrate for the treatment of hyperbilirubinemia in neonates born at term: a double blind controlled study (author's transl)]. / Traitement curatif de l'ictère du nouveau-né à terme par le clofibrate. Essai thérapeutique contrôlé en double aveugle.

A double blind controlled study of the therapeutic effect of clofibrate, an inductor of bilirubin glucuronyl transferase, was performed in neonates born at term and presenting with physiologic jaundice. 47 children were treated with a single oral dose of clofibrate. 46 control children were given corn oil alone. Results show that mean plasma bilirubin levels are significantly lower in the treated group as compared with the control group, from the 16th hour of treatment, if there is no ABO incompatibility. Clofibrate treatment also resulted in a shorter duration of jaundice and a restricted use of phototherapy. No undesirable side-effect was observed.