Influence of a cysteine prodrug, L-2-oxothiazolidine-4-carboxylic acid, on the urinary elimination of mercapturic acids of ethylene oxide, dibromoethane, and acrylonitrile: a dose-effect study

Metabolic disposition of ethylene oxide, dibromoethane, and acrylonitrile in rats after acute exposure was studied by examining the relationship between dose and urinary metabolites, and by establishing the influence of a glutathione precursor, L-2-oxothiazolidine-4-carboxylic acid (OTCA), on the above relationship. Respective urinary metabolites, hydroxyethylmercapturic acid, cyanoethylmercapturic acid, thiocyanate, and ethylene glycol, were quantified to estimate the extent to which each compound was metabolized. The animals were given either ethylene oxide (0.34, 0.68, or 1.36 mmol/kg), dibromoethane (0.2, 0.4, or 0.6 mmol/kg), or acrylonitrile (0.10, 0.38, or 0.76 mmol/kg). Urine samples were collected at 24 h. The metabolic biotransformation of all three chemicals to their respective mercapturic acids was strongly indicative of saturable metabolism. Administration of OCTA (4-5 mmol/kg) enhanced gluthathione availability and increased excretion of urinary mercapturic acids at the higher doses of the chemicals. This study indicates that OTCA increases the capacity for detoxification via the glutathione pathway thereby partially correcting the nonlinearity between the administered dose of ethylene oxide, dibromoethane, and acrylonitrile and the amount of certain urinary metabolites.     

The contribution of bromobenzene to our current understanding of chemically-induced toxicities

The metabolism and toxicity of bromobenzene has been investigated for well over one hundred years. The urinary excretion of mercapturic acids was first reported in 1879, in animals treated with bromobenzene. Bromobenzene has since proven to be a valuable tool in efforts to unravel the complexities involved in chemical- induced toxicities. For example, the importance of metabolic activation via the cytochrome(s) P-450; the role of glutathione in the detoxification of reactive metabolites; and the toxicological significance of covalent binding, enzyme inactivation and lipid peroxidation have all been illustrated in studies with bromobenzene. Thus, many of the principles involved in chemical-induced toxicity have been exemplified in studies with bromobenzene. These studies have provided substantial insight into the role of chemically reactive metabolites in the genesis of xenobiotic-mediated cytotoxicity.     

Assessment of hepatoprotective and nephroprotective potential of withaferin A on bromobenzene-induced injury in Swiss albino mice: possible involvement of mitochondrial dysfunction and inflammation

Bromobenzene is a well-known environmental toxin which causes liver and kidney damage through CYP450-mediated bio-activation to generate reactive metabolites and, consequently, oxidative stress. The present study aimed to evaluate the possible protective role of withaferin A against bromobenzene-induced liver and kidney damage in mice. Withaferin A (10 mg/kg) was administered orally to the mice for 8 days before intragastric intubation of bromobenzene (10 mmol/kg). As results of this experiment, the levels of liver and kidney functional markers, lipid peroxidation, and cytokines (TNF-α and IL-1β) presented an increase and there was a decrease in anti-oxidant activity in the bromobenzene-treated group of mice. Pre-treatment with withaferin A not only significantly decreased the levels of liver and kidney functional markers and cytokines but also reduced oxidative stress, as evidenced by improved anti-oxidant status. In addition, the mitochondrial dysfunction shown through the decrease in the activities of mitochondrial enzymes and imbalance in the Bax/Bcl-2 expression in the livers and kidneys of bromobenzene-treated mice was effectively prevented by pre-administration of withaferin A. These results validated our conviction that bromobenzene caused liver and kidney damage via mitochondrial pathway and withaferin A provided significant protection against it. Thus, withaferin A may have possible usage in clinical liver and kidney diseases in which oxidative stress and mitochondrial dysfunction may be existent.     

Metabolic activation and hepatotoxicity. Toxicity of bromobenzene in hepatocytes isolated from phenobarbital-and diethylmaleate-treated rats.

Hepatocytes freshly isolated from diethylmaleate-treated rats exhibited a markedly decreased concentration of reduced glutathione (GSH) which increased to the level present in hepatocytes from nontreated rats upon incubation in a complete medium. When bromobenzene was present in the medium, however, this increase in GSH concentration upon incubation was reversed and a further decrease occurred that resulted in GSH depletion and cell death. This was prevented by metyrapone, an inhibitor of the cytochrome P-450-linked metabolism of bromobenzene. Bromobenzene metabolism in hepatocytes was accompanied by a fraction of metabolites covalently binding to cellular proteins. The size of this fraction, relative to the amount of total metabolites, was increased in hepatocytes isolated from diethylmaleate-treated rats and in hepatocytes from phenobarbital-treated rats incubated with bromobenzene in the presence of 1,2-epoxy-3,3,3-trichloropropane, an inhibitor of microsomal epoxide hydrase which, however, also acted as a GSH-depleting agent. In addition, the metabolism of bromobenzene by hepatocytes was associated with a marked decrease in various coenzyme levels, including coenzyme A, NAD(H), and NADP(H). Cysteine and cysteamine inhibited the formation of protein-bound metabolites of bromobenzene in microsomes, but did not prevent bromobenzene toxicity in hepatocytes when added at higher concentrations to the incubation medium (containing 0.4 mm cysteine). Methionine, on the other hand, did not cause a significant effect on bromobenzene metabolism in microsomes and prevented toxicity in hepatocytes, presumably by stimulating GSH synthesis and thereby decreasing the amount of reactive metabolites available for interaction with other cellular nucleophiles. It is concluded that, in contrast to hepatocytes with normal levels of GSH, hepatocytes from diethylmaleate-treated rats were sensitive to bromobenzene toxicity under our incubation conditions. In this system, bromobenzene metabolism led to GSH depletion and was associated with a progressive decrease in coenzyme A and nicotinamide nucleotide levels and a moderate increase in the formation of metabolites covalently bound to protein. Methionine was a potent protective agent which probably acted by enhanced GSH synthesis via the formation of cystathionine.     

Methapyrilene hepatotoxicity is associated with increased hepatic glutathione, the formation of glucuronide conjugates, and enterohepatic recirculation

The mechanisms by which acute administration of methapyrilene, an H(1)-receptor antihistamine causes periportal necrosis to rats are unknown. This study investigated the role of the hepato-biliary system in methapyrilene hepatotoxicity following daily administration of 150 mg/kg per day over 3 consecutive days. Biliary metabolites of methapyrilene were tentatively identified. In male Han Wistar rats administration of methapyrilene significantly increased hepatic reduced glutathione (GSH) to 140% of control levels 24 h following the last dose. There were no significant changes in the activities of glutathione-related enzymes, glutathione peroxidase (GPx) and reductase (GSH), glutathione S-transferase (GST), and gamma-glutamyl cysteine synthetase (gamma-GCS) over 3 days of methapyrilene administration. Methapyrilene treatment resulted in no significant increase in excretion of biliary oxidized glutathione (GSSG), a sensitive marker of oxidative stress in vivo, following the third dose. [3H]Methapyrilene-derived radioactivity was detected in bile, to a greater extent than in feces, indicating that methapyrilene and/or metabolites underwent enterohepatic recirculation. Cannulation and exteriorization of the bile duct (to interrupt enterohepatic recirculation) afforded some protection against the hepatotoxicity, assessed by clinical chemistry and histopathology. Liquid chromatography-mass spectrometry (LC-MS) analysis of bile indicated the presence of unmetabolized methapyrilene, methapyrilene O-glucuronide and desmethyl methapyrilene O-glucuronide. These data demonstrate that acute methapyrilene hepatotoxicity in vivo is not a consequence of GSH depletion, or oxidative stress, but that enterohepatic recirculation of biliary metabolites may be important. Progressive exposure to non-oxidizing, reactive metabolic intermediates may be responsible for hepatotoxicity.     

Double-prodrugs of L-cysteine: differential protection against acetaminophen-induced hepatotoxicity in mice

A series of double-prodrugs of L-cysteine, designed to release L-cysteine in vivo and stimulate the biosynthesis of glutathione (GSH), were synthesized. To evaluate the hepatoprotective effectiveness of these double-prodrugs, male Swiss-Webster mice were administered acetaminophen (ACP) (2.45 mmol/kg (360 mg/kg), intraperitoneally (i.p.)). Prodrug (2.50 mmol/kg, i.p. or 1.25 mmol/kg, i.p., depending on the protocol) was administered 1 h before ACP as a priming dose. A supplementary dose of prodrug (2.5 mmol/kg, i.p. or 1.25 mmol/kg, i.p. depending on the protocol) was administered 0.5 h after ACP. The plasma alanine amino transferase (ALT) values, 24 h after ACP administration were transformed to logs and the 95% and 99% confidence intervals of the log values were plotted and compared for each group. Hepatoprotection was assessed by the degree of attenuation of plasma ALT levels. With these multiple dose schedules, the use of 2% carboxymethylcellulose as vehicle for the prodrugs was found to be detrimental; therefore, the prodrugs were dissolved in dilute aqueous base and the pH adjusted for administration. When a priming dose was given 1 h before ACP followed by a supplementary dose 0.5 h after ACP, only N,S-bis-acetyl-L-cysteine, where both the sulfhydryl and amino groups of L-cysteine were functionalized with the acetyl group, was found to be effective in protecting mice against the hepatotoxic effects of ACP. This suggests that these acetyl groups were rapidly hydrolyzed in vivo to liberate L-cysteine. In contrast, N-acetylation of 2(R,S)-methylthiazolidine-4(R)-carboxylic acid (MTCA) and its 2-n-propyl analog (PTCA), or N-acetylation of 2-oxothiazolidine-4-carboxylic acid (OTCA), reduced the hepatoprotective effects relative to the parent MTCA, PTCA, and OTCA, indicating that the release of L-cysteine in vivo from these N-acetylated thiazolidine prodrugs was metabolically unfavorable. The carbethoxy group, whether functionalized on the sulfhydryl or on the amino group of L-cysteine, or on the secondary amino group of MTCA, appears to be a poor "pro-moiety," since these carbethoxylated double-prodrugs of L-cysteine did not protect mice from ACP-induced hepatotoxicity.     

Induction of hepatic heme oxygenase activity by bromobenzene

Hepatic heme oxygenase, an enzyme which converts heme to carbon monoxide and bile pigment in vitro, is inducible by heme but also by large “toxic” doses of such nonheme substances as hormones, endotoxin, and heavy metal ions. When we gave rats a single hepatotoxic dose of allyl alcohol, ethionine, acetaminophen, furosemide, or endotoxin, hepatic heme oxygenase activity rose modestly (two- to fivefold) after 20 h. In contrast, administration of bromobenzene (5 mmol/kg) induced heme oxygenase in the liver an average of 15-fold after 20 h but was without effect on the enzyme in the kidney or spleen. The change in heme oxygenase was accompanied by a loss in cytochrome P-450 concentration and, in rats labeled with 5-δ-amino[¹⁴C]levulinic acid, an increased rate of degradation of hepatic [¹⁴C]heme to ¹⁴CO. Induction of heme oxygenase by bromobenzene was blocked by cycloheximide, an inhibitor of protein synthesis, but not by actinomycin D, an inhibitor of RNA synthesis. This suggests that bromobenzene stimulates de novo enzyme synthesis at the step of translation. Subtoxic doses of bromobenzene (less than 1 mmol/kg) gave proportionately greater induction of heme oxygenase. Furthermore, induction of the enzyme remained unaffected when bromobenzene hepatotoxicity was blocked by pretreatment of rats with SKF-525A, 3-methylcholanthrene, or cysteine (which supplements liver sulfhydryl content), or when hepatotoxicity was enhanced by pretreatment with phenobarbital or with diethylmaleate (which depletes hepatic glutathione). These data suggest that with induction of heme oxygenase by bromobenzene, neither liver cell necrosis nor alteration in hepatic sulfhydryl metabolism is indispensible. The latter characteristic differs from induction of the enzyme by metal ions in which depletion of sulfhydryl-containing constituents has been thought to be essential. We conclude that bromobenzene is a novel inducer of heme oxygenase activity in the liver, differing from other nonheme substances in potency and specificity for the liver, and in utilizing mechanism(s) which require neither production of hepatotoxicity, depletion of hepatic glutathione, nor sensitivity to actinomycin D.     

Effect of glucose-cysteine adduct as a cysteine prodrug in rats

Summary Effect of intraperitoneal administration (5 mmol/kg of body weight) of glucose- cysteine adduct (glc-cys) as a cysteine prodrug in rat tissues was studied. Cysteine levels in liver and kidney increased to 1.08 and 1.98mol per g or ml, respectively, at 2h after the administration. GSH levels did not change substantially. However, when glc-cys was injected to rats treated with diethyl maleate, a GSH-depleting agent, the decreased GSH levels were restored rapidly. The recoveries in liver and kidney were 72% at 1h and 66% at 2h, respectively, after glc-cys administration. Metabolism of glc-cys was assessed by urinary excretion of glc-cys, sulfate and taurine. Average excretion of glc-cys was 2.86mmol/kg/24h after glc-cys administration. Increased excretions of sulfate and taurine were 0.77 and 0.14mmol/kg/24h, respectively. Data show that, although glc-cys excretion was relatively rapid, glc-cys was effectively utilized for GSH synthesis in GSH-depleted tissues.     

Influence of 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor, pravastatin, on corticosteroid metabolism in patients with heterozygous familial hypercholesterolemia.

The present study examined whether hypolipidemic therapy with a potent 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor, pravastatin, influences corticosteroid metabolism in patients with heterozygous familial hypercholesterolemia (FH). Urinary excretion of tetrahydrocortisone, tetrahydrocortisol, 6 beta-hydroxycortisol and free cortisol were determined in 22 patients with heterozygous FH before and after pravastatin administration (10 mg/day for 2 months). Pravastatin induced a statistically significant decrease in serum total cholesterol in patients with heterozygous FH from 6.9 +/- 0.1 to 5.9 +/- 0.1 mmol/l (p less than 0.05). No significant changes were seen in the urinary tetrahydrocortisone, tetrahydrocortisol and free cortisol levels before and after pravastatin therapy. Urinary excretion of 6 beta-hydroxycortisol was significantly (p less than 0.05) increased after pravastatin administration. These results suggest that the hypolipidemic effect of pravastatin in patients with heterozygous FH does not influence the corticosteroid metabolism. The increase in urinary 6 beta-hydroxycortisol may be caused by pravastatin-induced hepatic microsomal 6 beta-hydroxylase induction.     

Protection against acetaminophen-induced hepatotoxicity by L-CySSME and its N-acetyl and ethyl ester derivatives

We have recently observed that S-(2-hydroxyethylmercapto)-L-cysteine (L-CySSME), the mixed disulfide of L-cysteine and 2-mercaptoethanol, prevented cataracts induced in mice by acetaminophen (ACP) by functioning as a prodrug of L-cysteine and protecting the liver. This prompted the evaluation of the more lipophilic N-acetyl (Ac-CySSME) and ethyl ester (Et-CySSME) derivatives of L-CySSME as pro-prodrug forms, as well as the “D” enantiomer, as hepatoprotective agents. Serum ALT levels were measured at 24 hours after a toxic but nonlethal dose of ACP that insured 48 hour survival of the animals. Since the increases in ALT produced were highly variable (even after log transformation) and complicated the statistical analyses, we calculated confidence intervals for the mean ALT levels for each treatment group. This enabled comparisons to be made of the efficacy of L-CySSME as well as Ac-CySSME and Et-CySSME with other representative prodrugs of L-cysteine, namely, 2(RS)-methylthiazolidine-4(R)-carboxylic acid (MTCA), L-2-oxothiazolidine-4-carboxylic acid (OTCA), and N-acetyl-L-cysteine (NAC), in protecting the liver. It was shown that L-CySSME and MTCA administered intraperitoneally at 2.5 mmol/kg were superior to the other cysteine prodrugs at equimolar doses in protecting mice from hepatotoxicity elicited by a 400 mg/kg (2.65 mmol/kg) dose of ACP given i.p. 30 minutes prior to the prodrugs. The “D” form of CySSME was totally without protective effect. Oral doses of the prodrugs even at 2× the i.p. dose were less effective, although MTCA was the most protective. © 1997 John Wiley & Sons, Inc. J Biochem Toxicol 11: 289–295, 1997.     

The tissue distribution in rats of [195mPt]carboplatin following intravenous,intraperitoneal and oral administration

[^195mPt]carboplatin has been administered intravenously, intraperitoneally and orally to Wistar rats and the tissue distribution, metabolism, and pharmacokinetics of the drug investigated. The urinary and faecal excretion and toxicity following oral [^195mPt]carboplatin administration has also been studied. Virtually identical results have been observed following i.v. and i.p. administration, indicating a rapid absorption of the unaltered compound from the abdominal cavity into the systemic circulation. Thus i.p. administered drug should produce a similar therapeutic response as i.v. administration, but may produce an additional local effect within the peritoneal cavity. Orally administered compound shows a pattern of distribution which is similar to that following parenteral injection for all tissues (except for the increased relative concentration in the stomach tissue), the concentration being lower by a factor of 4–5. However, the overall fraction of the dose retained within the body at 24 h is similar to that following i.v. administration. The toxicity is considerably lower for the orally administered drug compared with i.v. injection. These results clearly show that oral doses could be adjusted to produce a comparable therapeutic effect as i.v. or i.p. doses, and should also result in a higher efficacy against gastric carcinomas than achievable with parenteral administration.     

The cysteine proteinase inhibitor, E-64, reduces proteinuria in an experimental model of glomerulonephritis

Proteinuria is a major manifestation of glomerular disease (glomerulonephritis, GN). We examined the effect of trans-epoxysuccinyl-L-leucylamido-(4-guanidino)butane (E-64), a specific and irreversible cysteine proteinase inhibitor, on urinary protein excretion in a complement- and neutrophil-independent model of antiglomerular basement membrane (GBM) antibody disease. A single injection of rabbit antirat-GBM IgG produced a marked increase in urinary protein excretion 24hr after injection. In two separate studies using different pools of antiGBM IgG, administration of E-64 (5mg every 6h starting 2hr prior to induction of GN) reduced proteinuria (-45 +/- 7%, and -41 +/- 14%, Mean +/- SEM, n = 6; P less than 0.001) in the 24 hour period following induction of the disease. This reduction in urinary protein excretion was accompanied by a marked decrease in the specific activity of the cysteine proteinases cathepsins B and L in glomeruli (B: -97%; L: -84%) and renal cortex (B: -87%; L: -75%) isolated from the same E-64-treated rats compared to same saline-treated controls. These data, combined with the specificity of E-64 for cysteine proteinases, suggest a potential role for cysteine proteinases in the increased GBM permeability and proteinuria in this experimental model of glomerular disease.     

Postabsorption antidotal effects of N-acetylcysteine on acetaminophen-induced hepatotoxicity in the mouse

Male Swiss Webster mice, treated with N-acetylcysteine (NAC, 500 mg/kg po) 1 h following acetaminophen (NAPA, 350 mg/kg po) administration, had control levels of transaminases indicating that NAC protects against NAPA-induced hepatotoxicity by postabsorption antidotal mechanism(s). Hepatic congestion induced by NAPA was reduced by NAC. Significantly higher elimination rate constants (K) for indocyanine green (500 micrograms/kg, iv) in mice treated with NAPA and NAC (K = 0.676 +/- 0.062) than in animals receiving NAPA alone (0.341 +/- 0.105) suggested NAC improved or preserved the hepatic circulation of the compromised liver. This NAC-induced improvement and (or) preservation of hepatic circulation was reflected in biliary and urinary excretion of acetaminophen and its metabolites by a general increase in elimination during the first 6 h (70.2 +/- 2.6 vs. 32.6 +/- 7.1%), and in the repletion of glutathione (GSH) in the liver by a return to control levels more quickly (3 vs. greater than 5 h) following depletion by NAPA. The metabolic consequences of the postabsorption antidotal effect of NAC in the compromised liver was a preferential excretion of sulphydryl-derived metabolites in the 1-4 h bile (GSH conjugate 11.30 +/- 1.25 vs. 7.25 +/- 0.39%) which was subsequently observed in the urine by preferential excretion of glutathione degradation products.     

The metabolic fate of the anti-androgenic agent, oxendolone, in man

After intramuscular administration of 16 beta-ethyl-17 beta-hydroxy-4-4-[4-14C]estren-3-one (14C-oxendolone; 300 mg) to 3 human subjects, excretion of 14C was very slow and incomplete despite a 20-day sample collection period. During this time, means of 37% and 21% of the administered 14C were recovered in urine and faeces, respectively, and if excretion continued at the same rate, approximately 90% of the administered 14C would have been excreted during 5-12 weeks. Peak plasma 14C concentrations were reached at 3-6 days after dosing, when they represented 0.2-1.1 micrograms equiv./ml, and declined very slowly thereafter with a half-life of 5.0-6.6 days. Concentrations of unconjugated drug-related steroids circulating in plasma never exceeded about 0.1 microgram/ml. Mass spectroscopic analysis of isolated urinary and faecal metabolites indicated that the principal routes of biotransformation of oxendolone in man are similar to those of the endogenous androgens-namely, reduction of the 4,5-double bond, further reduction of the saturated 3-ketone to the 3 alpha-hydroxysteroid, and oxidation of the 17 beta-alcohol to the corresponding ketone, followed by conjugation, mainly with glucuronic acid, and excretion in the urine and bile.     

C20 steroids: The metabolism of 3-hydroxy-19-nor[21-14C]pregna-1,3,5(10)-trien-20-one in the rabbit

1. The metabolism of 3-hydroxy-19-norpregna-1,3,5(10)-trien-20-one, a possible product of the aromatization of progesterone or pregnenolone, has been studied. 2. After oral administration of this C(20) steroid as the 21-(14)C-labelled compound to two groups of rabbits, the excretion pattern of metabolites in the urine was examined. 3. At 14 days after administration, 3.3-6.5% of the radioactivity had appeared in the urine, 71-79% in the faeces and approx. 10% remained in the gut. 4. A metabolite, isolated from urine mainly as the unconjugated steroid, was identified as 19-norpregna-1,3,5(10)-triene-3,20alpha-diol and constituted 18.5-22% of the total urinary radioactivity. 5. A minor component of the urinary unconjugated steroids was identified as 19-norpregna-1,3,5(10)-triene-3,17alpha,20alpha-triol. 6. A further 2-7.5% of the total urinary radioactivity, isolated only from the urinary sulphate fraction, was tentatively identified as an 18-oxygenated derivative of the administered steroid.     

Integrated metabonomic analysis of bromobenzene-induced hepatotoxicity: novel induction of 5-oxoprolinosis

We present here a definitive metabonomic analysis in order to detect novel biomarker and metabolite information, implicating specific putative protein targets in the toxicological mechanism of bromobenzene-induced centrilobular hepatic necrosis. Male Han-Wistar rats were dosed with bromobenzene (1.5 g/kg, n = 25) and blood plasma, urine and liver samples were collected for NMR and magic angle spinning (MAS) NMR spectroscopy at various time-points postdose, with histopathology and clinical pathology performed in parallel. Liver samples were analyzed by 600 MHz 1H MAS NMR techniques and the resultant spectra were correlated to sequential 1H NMR measurements in urine and blood plasma using pattern recognition methods. 1D 1H NMR spectra were data-reduced and analyzed using principal components analysis (PCA) to show the time-dependent biochemical variations induced by bromobenzene toxicity. In addition to a holistic view of the effect of hepatic toxicity on the metabolome, a number of putative protein targets of bromobenzene and its metabolites were identified including those enzymes of the glutathione cycle, exemplified by the presence of a novel biomarker, 5-oxoproline, in liver tissue, blood plasma, and urine. As such, this work establishes the importance of metabonomics technology in resolving the mechanistic complexity of drug toxicity as well as the benefits of frontloading this approach in drug safety evaluation and biomarker discovery.     

Metabolic activation and hepatotoxicity. Effect of cysteine, N-acetylcysteine, and methionine on glutathione biosynthesis and bromobenzene toxicity in isolated rat hepatocytes.

Freshly isolated rat hepatocytes contained a high level (30–40 nmol/10⁶ cells) of reduced glutathione (GSH) which decreased steadily upon incubation in an amino acid containing medium lacking cysteine and methionine. This decrease in GSH level was prevented, and turned into a slight increase, when either cysteine, N-acetylcysteine, or methionine was also present in the medium. The amino acid uptake into hepatocytes was more rapid with cysteine than with methionine. Cystine was not taken up, or taken up very slowly, by the cells and could not be used to prevent the decrease in GSH level which occurred in the absence of cysteine and methionine. The level of GSH in hepatocytes freshly isolated from rats pretreated with diethylmaleate was markedly decreased (to ~5 nmol/10⁶ cells) but increased rapidly upon incubation of the cells in a medium containing amino acids including either cysteine, N-acetylcysteine, or methionine. Again, cysteine was taken up into the cells more rapidly than methionine. The rate of uptake of cysteine was moderately enhanced in hepatocytes with a lowered level of intracellular GSH as compared to cells with normal GSH concentration. Exclusion of glutamate and/or glycine from the medium did not markedly affect the rate of resynthesis of GSH by hepatocytes incubated in the presence of exogenously added cysteine or methionine. Incubation of hepatocytes with bromobenzene in an amino acid-containing medium lacking cysteine and methionine resulted in accelerated cell damage. Addition of either cysteine, N-acetylcysteine, or methionine to the medium caused a decrease in bromobenzene toxicity. The protective effect was dependent, however, on the time of addition of the amino acid to the incubate; e.g., the effect on bromobenzene toxicity was greatly reduced when either cysteine or methionine was added after 1 h of preincubation of the hepatocytes with bromobenzene as compared to addition at zero time. This decrease in protective effect in bromobenzene-exposed cells was related to a similar decrease in the rate of uptake of cysteine and methionine into hepatocytes preincubated with bromobenzene. The rate of uptake, and incorporation into cellular protein, of leucine was also markedly inhibited in hepatocytes preincubated with bromobenzene. In contrast, there was no measurable change in the rate of release of leucine from cellular protein as a result of incubation of hepatocytes with bromobenzene. It is concluded that the presence of cysteine, N-acetylcysteine, or methionine in the medium protects hepatocytes from bromobenzene toxicity by providing intracellular cysteine for GSH biosynthesis and suggested that an inhibitory effect on amino acid uptake may contribute to the cytotoxicity of bromobenzene in hepatocytes.     

Ornithine aminotransferase activity, liver ornithine concentration and acute ammonia intoxication

5-Fluoromethylornithine (5FMOrn) is a specific inactivator of L-ornithine:2-oxoacid aminotransferase (OAT). Inactivation of OAT causes the enhancement of L-ornithine (Orn) concentrations in all tissues. Intraperitoneal or oral administration of 10-50 mg/kg of 5FMOrn per day to albino mice rendered partial protection against lethal intoxication with 26 mmol/kg of ammonium acetate. The protective effect was maximal around 16 h after 5FMOrn administration, at the time when endogenous Orn concentrations were maximal. At this time protection by 5FMOrn against acute ammonia intoxication was comparable to that observed 1 h after the intraperitoneal administration of 10 mmol/kg of L-arginine. Pretreatment with 5FMOrn prevented the enhancement of excessive urinary excretion of orotic acid by ammonia intoxicated mice, and it enhanced urea formation in the liver. These biochemical effects demonstrate that 5FMOrn shifts Orn into the urea cycle, Orn which normally would be transaminated. Since even long-term treatment of mice with 5FMOrn did not reveal toxic effects, this compound may be considered for the treatment of certain conditional deficiencies of Orn or arginine.     

Hispidulin protection against hepatotoxicity induced by bromobenzene in mice

The effects of the natural flavonoid hispidulin (6-methoxy-5, 7, 4′-trihydroxyflavone) on bromobenzene-induced hepatotoxicity in mice were investigated. We found a correlation between liver injury and hepatic lipid peroxidation besides a strong liver glutathione depletion due to the toxicant. Hispidulin at doses between 50 and 150 mg/kg i.p. compared favourably with the reference compound N-acetyl-L-cysteine for inhibition of liver injury and lipid peroxidation. The flavonoid at the highest dose tested was also able to counteract reduced glutathione depletion induced by bromobenzene in starved mice. These hepatoprotective effects can be related to the antioxidant properties of hispidulin.     

Metabolic activation and hepatotoxicity. Metabolism of bromobenzene in isolated hypatocytes.

Rat liver microsomes and isolated rat hepatocytes metabolized bromobenzene to watersoluble and protein-bound metabolites. The latter fraction—which normally accounted for 2–5% of the total products—was slightly increased when 1,2-epoxy-3,3,3-trichloropropane, an inhibitor of microsomal epoxide hydrase, was added to the microsomal incubate. The presence of reduced glutathione (GSH), on the other hand, caused an almost complete inhibition of the formation of protein-bound metabolites from bromobenzene in microsomes. The rates of bromobenzene metabolism were similar in liver microsomes and hepatocytes, and increased severalfold after phenobarbital pretreatment of the rats. Metyrapone and SKF 525-A were inhibitory in both systems. Bromobenzene metabolism in hepatocytes isolated from phenobarbital-treated rats was associated with a rapid and marked decrease in the level of intracellular GSH. When the cells were incubated in a complete medium, however, the decrease in GSH leveled off at about 40% of the original concentration and there was no evidence of any accelerated rate of cell death even when the incubation with bromobenzene was prolonged to 10 h. This was most probably due to resynthesis of GSH by the hepatocytes, which partly compensated for the loss of this thiol associated with bromobenzene metabolism. Accordingly, in a deficient medium (lacking amino acids), the cytotoxic effect of bromobenzene metabolism was pronounced—less than 5% of the zerotime level of GSH and only 25% cell viability remaining after 5 h of incubation. It is concluded that the intracellular level of GSH is of major importance in regard to the cytotoxic effect of bromobenzene metabolism and that hepatocytes incubated in a complete medium are protected against toxicity by their ability to resynthesize this thiol.