Efficient hepatic uptake and concentrative biliary excretion of a mercapturic acid.

The role of the liver in the disposition of circulating mercapturic acids was examined in anesthetized rats and in the isolated perfused rat liver using S-2,4-dinitrophenyl-N-acetylcysteine (DNP-NAC) as the model compound. When DNP-NAC was infused into the jugular vein (150 or 600 nmol over 60 min) it was rapidly and nearly quantitatively excreted as DNP-NAC into bile (42-36% of the dose) and urine (48-62% of dose). Some minor metabolites were detected in bile (<4%), with the major metabolite coeluting on HPLC with the DNP conjugate of glutathione (DNP-SG). Isolated rat livers perfused single pass with 3 microM DNP-NAC removed 72 +/- 9% of this mercapturic acid from perfusate. This rapid DNP-NAC uptake was unaffected by sodium omission, or by L-cysteine, L-glutamate, L-cystine, or N-acetylated amino acids, but was decreased by inhibitors of hepatic sinusoidal organic anion transporters (oatp), indicating that DNP-NAC is a substrate for these transporters. The DNP-NAC removed from perfusate was promptly excreted into bile, eliciting a dose-dependent choleresis. DNP-NAC itself constituted approximately 75% of the total dose recovered in bile, reaching a concentration of 9 mM when livers were perfused in a recirculating mode with an initial DNP-NAC concentration of 250 microM. Other biliary metabolites included DNP-SG, DNP-cysteinylglycine, and DNP-cysteine. DNP-SG was likely formed by a spontaneous retro-Michael reaction between glutathione and DNP-NAC. Subsequent degradation of DNP-SG by biliary gamma-glutamyltranspeptidase and dipeptidase activities accounts for the cysteinylglycine and cysteine conjugates, respectively. These findings indicate the presence of efficient hepatic mechanisms for sinusoidal uptake and biliary excretion of circulating mercapturic acids in rat liver and demonstrate that the liver plays a role in their whole body elimination.

Potentiation of cisplatin activity by the bioreductive agent tirapazamine.

PURPOSE: The chemosensitizing potential of the benzotriazine-N-oxide tirapazamine was determined in rodent mammary tumor cells grown as solid tumors. MATERIALS AND METHODS: C3H/HeJ mice bearing i.m. transplanted 16C mammary carcinomas were treated with varying doses of either cisplatin alone or cisplatin in combination with a 0.27 mmol/kg dose of tirapazamine. Tumor response to single agent or combination therapy was assessed using an in situ tumor growth delay assay. Normal tissue toxicity resulting from the tirapazamine, cisplatin, or tirapazamine plus cisplatin was determined by measuring bone marrow stem cell (CFU-GM) toxicities and blood urea nitrogen (BUN) levels. RESULTS: Tirapazamine itself had no measurable effect on the growth of this tumor. However, when administered from 3 h before to simultaneously with a single dose of cisplatin, the resultant tumor growth delay was significantly increased as compared to that seen with cisplatin alone. The administration of tirapazamine 3 h prior to a range of doses of cisplatin was found to result in a dose modifying factor (DMF) of approximately 1.7 in tumor response compared to cisplatin alone. Tirapazamine did demonstrate some hematologic toxicity on its own but it did not potentiate the toxicity of cisplatin when the two agents were administered in combination. BUN analysis showed that tirapazamine had little effect on BUN levels but did suppress the BUN values of mice treated with the combination of tirapazamine and 15 mg/kg cisplatin as compared to cisplatin alone. CONCLUSIONS: The present findings suggest that the addition of tirapazamine to cisplatin therapy may lead to a therapeutic benefit.

Glutathione conjugation and conversion to mercapturic acids can occur as an intrahepatic process.

By catalyzing the reaction of electrophilic compounds with the sulfhydryl group of glutathione, the glutathione S-transferases play physiologically important roles in the detoxication of potential alkylating agents. The glutathione S-conjugates thus formed are transported out of cells for further metabolism by gamma-glutamyltransferase and dipeptidases, ectoproteins that catalyze the sequential removal of the glutamyl and glycyl moieties, respectively. These ectoproteins are not found in all cells, but are localized predominantly to the apical surface of epithelial tissues. The resulting cysteine S-conjugates can be reabsorbed by specific cell types, and acetylated on the amino group of the cysteinyl residue by intracellular N-acetyl-transferases, to form the corresponding mercapturic acids (N-acetylcysteine S-conjugates). Mercapturic acids are then released into the circulation and delivered to the kidney for excretion in urine, or they may undergo further metabolism. Mercapturic acid biosynthesis is generally considered to be an interorgan process, with the liver serving as the major site of glutathione conjugation, and the kidney as the primary site for conversion of glutathione conjugates to cysteine conjugates. Cysteine conjugates formed in the kidney appear to be transported back to the liver for acetylation. This interorgan model of mercapturic acid synthesis is based largely on the interorgan distribution of the enzymes involved in their formation, and in particular of the enzyme gamma-glutamyltransferase. Rats have relatively low hepatic and high renal activities of gamma-glutamyltransferase, the only protein known to initiate the breakdown of glutathione S-conjugates. The low gamma-glutamyltransferase activity in rat liver limits the hepatic degradation of glutathione S-conjugates, particularly after large doses of xenobiotic. In contrast, hepatic gamma-glutamyltransferase is significantly higher in species such as rabbit, guinea pig, and dog, and as a consequence, nearly all of the glutathione and glutathione S-conjugates released by liver cells of these species is degraded within the liver. Recent studies demonstrate that glutathione S-conjugates synthesized within hepatocytes are secreted preferentially across the canalicular membrane into bile, and are broken down within biliary spaces to form cysteine S-conjugates. The latter are then reabsorbed by the liver, N-acetylated to form mercapturic acids, and reexcreted into bile, completing an intrahepatic pathway for mercapturic acid biosynthesis. The contribution of this intrahepatic pathway to overall mercapturate formation is dependent on dose of the electrophile, route of exposure, and the physicochemical properties of the glutathione S-conjugate formed, as well as the tissue distribution and activity of gamma-glutamyltransferase.(ABSTRACT TRUNCATED AT 400 WORDS)

Hepatic uptake of intact glutathione S-conjugate, inhibition by organic anions, and sinusoidal catabolism.

To identify potential mechanisms for hepatic removal of circulating glutathione (GSH) conjugates, uptake and metabolism of S-2,4-dinitrophenylglutathione (DNP-SG) were examined in isolated perfused livers from rat and guinea pig. Guinea pig livers perfused with 5 mumol of DNP-SG in a recirculating system (50 microM initial concn) rapidly cleared the conjugate from the perfusate (half time 3.7 min), whereas clearance was considerably slower in rat liver (half time 35 min). Disappearance of DNP-SG from the perfusate was accompanied by a simultaneous appearance of DNP-SG and its metabolites in bile. Addition of acivicin, an inhibitor of gamma-glutamyltransferase (gamma-GT), to the perfusate resulted in a marked decrease in DNP-SG clearance by guinea pig liver but had no effect in rat liver, suggesting that in the guinea pig this process is largely dependent on sinusoidal gamma-GT activity. However, even in the presence of acivicin, rat and guinea pig livers removed nearly one-half of the administered DNP-SG from the recirculating perfusate over 30 min. High concentrations of DNP-SG were found in bile (up to 3.7 mM), indicating that the liver is capable of transporting the intact conjugate from the circulation. When rat livers were perfused with higher concentrations of DNP-SG (100 and 250 microM), biliary excretion of DNP-SG increased dose dependently, with concentrations in bile reaching 10 mM at the higher dose. This was accompanied by a dose-dependent choleresis.(ABSTRACT TRUNCATED AT 250 WORDS)

Intrahepatic conversion of a glutathione conjugate to its mercapturic acid. Metabolism of 1-chloro-2,4-dinitrobenzene in isolated perfused rat and guinea pig livers.

Because of the low hepatic activity of gamma-glutamyl-transferase in the rat, the liver is generally considered to play only a minor role in the degradation of glutathione conjugates, a limiting step in mercapturic acid formation. Recent findings indicate, however, that the liver has a prominent role in glutathione catabolism, particularly in species other than rat. To examine the contributions of liver to mercapturic acid biosynthesis, mercapturate formation was compared in isolated perfused livers from rats and guinea pigs dosed with either 0.3 or 3.0 mumol of 1-chloro-2,4-dinitrobenzene (CDNB). Chemically synthesized glutathione conjugate, mercapturic acid, and intermediary metabolites of CDNB were used as standards in the high performance liquid chromatography analysis of bile and perfusate samples. Biliary excretion accounted for almost all of the recovered metabolites. A marked species difference was observed in the pattern of CDNB metabolism. Rat livers dosed with 0.3 mumol of CDNB excreted 55% of total biliary metabolites as the glutathione conjugate and 8.2% as the mercapturic acid, whereas guinea pig livers excreted only 4.8% as the glutathione conjugate and 47% as the mercapturate. Mercapturic formation was also dose-dependent, with a larger fraction formed at the 0.3- versus the 3.0-mumol dose (8.2 versus 3.7% in the rat; 47 versus 19% in the guinea pig). Hepatic conversion of the glutathione conjugate to the mercapturic acid was markedly inhibited in both species after retrograde intrabiliary infusion of acivicin, an inhibitor of gamma-glutamyltransferase activity. These findings provide direct evidence for intrahepatic biosynthesis of mercapturic acids. Thus, glutathione conjugates synthesized within hepatocytes are secreted into bile and broken down to cysteine conjugates; the latter are then presumably reabsorbed by the liver, N-acetylated to form the mercapturic acid and re-excreted into bile.

Polarity of hepatic glutathione and glutathione S-conjugate efflux, and intraorgan mercapturic acid formation in the skate.

Mechanisms of hepatic glutathione and glutathione S-conjugate efflux were investigated in isolated hepatocytes and perfused liver of the little skate (Raja erinacea). Glutathione was released by isolated skate hepatocytes at a rate of 0.12 +/- 0.03 nmol.hr-1.(mg protein)-1. In the perfused liver, glutathione concentrations in bile were high (approximately 0.7 mM) compared to hepatic tissue levels (0.61 +/- 0.11 mumol.g-1). During the first hour of perfusion, the biliary glutathione excretion rate was 3 nmol.hr-1.(g liver)-1, whereas glutathione accumulated in the recirculating perfusate at a rate of only 1.5 nmol.hr-1.(g liver)-1. Release of glutathione by isolated hepatocytes and perfused liver was not affected by the addition of acivicin, an inhibitor of gamma-glutamyltransferase (EC 2.3.2.2), to cell suspension medium or liver perfusate. 1-Chloro-2,4-dinitrobenzene (CDNB) was taken up by isolated hepatocytes, conjugated to glutathione, and released as S-(2,4-dinitrophenyl) (DNP)-glutathione. After infusion of 0.5 mumol CDNB in perfused liver, S-DNP-glutathione was concentrated in bile (0.5 mM) and was associated with choleresis. S-DNP-Conjugates of cysteinylglycine, cysteine and N-acetylcysteine, were also found in bile, suggesting intrahepatic breakdown of S-DNP-glutathione and subsequent acetylation of the resulting cysteine conjugate to form the mercapturic acid, S-DNP-N-acetylcysteine. This mercapturic acid accounted for 31% of the total S-DNP-conjugates collected in bile. In contrast, neither S-DNP-glutathione nor other S-DNP-conjugates were detected in the perfusate (less than 0.5 microM). These findings demonstrate that biliary excretion is the predominant route for efflux of glutathione and a glutathione S-conjugate from skate liver. The results also identify an intrahepatic pathway for mercapturic acid biosynthesis facilitated by biliary glutathione S-conjugate excretion.

Metabolism of dibasic esters by rat nasal mucosal carboxylesterase.

Inhalation exposure of rats to dibasic esters revealed lesions of the nasal olfactory epithelium similar to those observed with other ester solvents. Female rats are more sensitive to these effects than are male rats. It has been proposed that carboxylesterase conversion of inhaled esters within nasal tissues to organic acids may be a critical biochemical step in converting these chemicals to toxic substances. These experiments measured the kinetic parameters Vmax, KM, Ksi, and V/K for the hydrolysis of the dibasic esters in the target nasal tissue, olfactory mucosa, and nontarget tissue, respiratory mucosa. It was determined that under the conditions of these experiments, diacid metabolites are not formed. Esterase activity was inhibited by pretreatment with bis p-nitrophenyl phosphate. Vmax values for the three dibasic esters were 5- to 13-fold greater in olfactory mucosa than respiratory mucosa for male or female rats. V/K values were 4- to 11-fold greater in olfactory mucosa than respiratory mucosa for male or female rats. V/K was similar between male and female olfactory mucosa when dimethyl glutarate was used as the substrate. With dimethyl succinate or dimethyl adipate as the substrate, V/K for female olfactory tissue was 0.5- or 2-fold that of males, respectively. Differences in V/K were mainly due to decreases in KM associated with increasing carbon chain length. Substrate inhibition was observed at dibasic ester concentrations greater than approximately 25 mM, which are unlikely to be achieved in vivo. These results lend further support to the hypothesis that organic acid accumulation in the target tissue, olfactory mucosa, plays a significant role in the pathogenesis of dibasic ester-induced nasal lesions. This mechanism may be applicable to a wide range of inhaled esters.

Glutathione-degrading capacities of liver and kidney in different species.

Although the liver is recognized as a major site of glutathione (GSH) synthesis, it is thought to play only a minor role in GSH catabolism. This is primarily because in the rat, the most commonly used experimental animal, hepatic gamma-glutamyltransferase (gamma-GT) activity is very low, whereas kidney activity is quite high. gamma-GT is the only enzyme known to catalyze the initial step in GSH degradation. The present work compares gamma-GT and dipeptidase activities in liver, kidney, and gallbladder of six mammalian species to assess the importance of hepatobiliary catabolism of GSH, relative to renal degradation. Marked species differences were observed in gamma-GT activities, and in kidney to liver (K/L) ratios for both gamma-GT concentration (milliunits/mg protein) and whole organ activities (total activity per liver or two kidneys). The K/L concentration ratios for gamma-GT activities ranged from 875 in the rat to 15 in the guinea pig. Whole organ gamma-GT ratios were approximately 150 in mouse and rat, and only 2-5 in guinea pig. pig, and macaque. Human K/L ratios calculated from gamma-GT activities reported previously were similar to those of the guinea pig. Species differences were also observed in K/L ratios for dipeptidase activities, though these differences were not as large as those for gamma-GT, gamma-GT and dipeptidase activities were also measured in gallbladders of all species examined (except rat which does not have this organ), and were found to be comparable to those of liver. These results suggest that in species such as the guinea pig and perhaps humans, the liver and biliary tree play a prominent role in GSH turnover. Because of the low hepatic and high renal gamma-GT activities of the rat, and because it does not have a gallbladder, this species may not be the best model for studying the catabolism of GSH and GSH conjugates. Use of the rat model may underestimate the contribution of liver, and overestimate that of kidney, in these degradative processes.