Sensitivity of (1)H NMR analysis of rat urine in relation to toxicometabonomics. Part I: dose-dependent toxic effects of bromobenzene and paracetamol.

(1)H nuclear magnetic resonance (NMR) spectroscopy of rat urine in combination with pattern recognition analysis was evaluated for early noninvasive detection of toxicity of investigational chemical entities. Bromobenzene (B) and paracetamol (P) were administered at five single oral dosages between 2 and 500 mg/kg and between 6 and 1800 mg/kg, respectively. The sensitivity of the proposed method to detect changes in the NMR spectra 24 and 48 h after single dosing was compared with histopathology and biochemical parameters in plasma and urine. Both B and P applied at the highest dosages induced liver necrosis and markedly increased aspartate aminotransferase (AST) and alanine aminotransferase (ALT) plasma levels. At dosages of 125 mg/kg B and 450 mg/kg P, liver necrosis and changes in AST and ALT were less pronounced, while at lower dose levels these effects could not be detected. Changes in kidney pathology or standard urine biochemistry were not observed at any of these dosages. Evaluation of the total NMR dataset showed 80 signals to be sensitive for B and P dosing. Principal component analysis on the reduced dataset revealed that NMR spectra were significantly different at dosages above 8 mg/kg (B) and 110 mg/kg (P) at both sampling times. This implies a 4- to 16-fold increased sensitivity of NMR versus histopathology and clinical chemistry in recognizing early events of liver toxicity.

Comparison of tissue distribution and metabolism of 1,2- and 1,4-dibromobenzenes in female rats.

The distribution, excretion and metabolism of 1,4-dibromobenzene (1,4-DBB) and 1,2-dibromobenzene (1,2-DBB), following a single intraperitoneal administration to female Wistar rats, were investigated using radiotracer 3H and GC-MS technique. The maximum level of 3H after 1,4-DBB administration was detected in all examined rat tissues between 4 and 24 h foltowing the injection. The highest concentrations of 3H were found in fat tissue, muscles, adrenal glands and sciatic nerve. About 50% of administered dose was still retained in the rat 72 h after injection. For 1,2-DBB, the highest level of 3H was in the liver, kidneys and fat tissue 4 and 8 h after administration. Three days after injection, less than 2% of the given dose was retained in the rat body. Urine turned out to be the main route of 3H excretion following the injection of both compounds (30% and 82%, after 1,4-DBB and 1,2-DBB, respectively), and about 4% of the given dose was excreted in feces. In urine of rats the following substances were identified (in sequence 1,4-dBB and 1,2-dBB): (1) unchanged parent compounds (5 and 11%); (2) dibromophenols (84 and 73%); (3) dibromothiophenols (5 and 10%) and (4) monobromophenols (1.9 and 0.7%). This study suggests that 1,2-DBB is characterized by a relatively high turnover rate, whereas 1,4-DBB shows a tendency for long-term retention in the body.

The disposition and metabolism of 1,3-dibromobenzene in the rat.

The distribution, excretion and metabolism of 1,3-dibromobenzene following a single i.p. administration to rats 100 or 300 mg/kg was investigated using radiotracer [3H] and GC-MS technique. After 72 hours about 74 to 90% were excreted in urine. The highest radioactivity was observed in the liver, kidneys and fat tissue. Later on a steady decline of radioactivity was apparent in all investigated tissues except for blood cells and the sciatic nerve, where constant levels were noted. In urine the following substances were identified and quantified by GC peak areas: unchanged 1,3-DBB (18%), dibromophenols (34%), dibromothiophenols (28%), dibromothioanisole (1.8%), bromophenol (5.5%), bromohydroxythiophenols (5%), and bromohydroxythioanisole (7.5%).

Metabolites of ebrotidine, a new H2-receptor antagonist, in human urine.

Ebrotidine is a new H2-receptor antagonist which exhibits a remarkable ability for gastric mucosal protection. A preliminary metabolic pathway for this compound was proposed and the hypothetic metabolites were synthesized. The presence of ebrotidine and its metabolites ebrotidine S-oxide and 4-bromobenzenesulfonamide in human urine has been confirmed by HPLC separation and spectroscopic characterization of the collected fractions by FT-IR and 1H NMR. Ebrotidine S,S-dioxide has been identified by HPLC using diode-array detection.

Effects of model traumatic injury on hepatic drug metabolism in the rat. VI. Major detoxification/toxification pathways.

A previously validated small mammal trauma model, hindlimb ischemia secondary to infrarenal aortic ligation in the rat, was utilized to investigate the effects of traumatic injury on two of the major hepatic enzymes of detoxification, glutathione S-transferase and epoxide hydrolase. Hepatic cytosolic glutathione S-transferase activity toward a variety of substrates showed a 26-34% decrease at 24 hr after model injury. Hepatic microsomal epoxide hydrolase activity toward 1,2-epoxy-3-(p-nitrophenoxy)propane was diminished by 53% after model trauma. Both enzymatic activities toward styrene oxide were similarly depressed. The toxicological sequelae of these derangements were illustrated by administering a dose of styrene oxide (300 mg/kg, ip) which was below the threshold dose (350 mg/kg, ip) necessary to produce hepatotoxicity in control animals. Model trauma dramatically enhanced the hepatotoxic effects of the subthreshold dose, as well as the covalent binding of labeled styrene oxide to liver proteins. These findings indicate that traumatic injury renders the animal more susceptible to agents which are detoxified by glutathione S-transferase and epoxide hydrolase. Conversely, model trauma provided almost complete protection from the hepatotoxic effects of a standard dose (200 mg/kg, ip) of bromobenzene. This protection appeared to derive from a post-traumatic alteration of cytochrome P-450 subpopulations that decreased the formation of the potentially toxic 3,4-epoxide metabolite, despite an increase in the cytochrome P-448-mediated generation of the nontoxic 2,3-epoxide. For bromobenzene, the change in cytochrome P-450-mediated activation appeared quantitatively more significant in overall toxicity than the post-traumatic depression of detoxification pathways described above, leading to decreased toxicity in vivo. For other compounds, the combination of post-traumatic influences on cytochrome P-450/P-448 activity and epoxide hydrolase/glutathione S-transferase activities could lead to markedly enhanced toxicity.

Metabolism of bromobenzene. Analytical chemical and structural problems associated with studies of the metabolism of a model aromatic compound.

Our studies of bromobenzene metabolism have shown that the 3,4-oxide is metabolized to 3- and 4-bromophenol through an extended glutathione pathway. The mechanism of sulfur elimination from a dihydrobromobenzene metabolite is not known, although it is known that the aromatization reaction will occur in a 9000-g supernatant fraction of rat liver. The hepatotoxic and nephrotoxic metabolites of bromobenzene are most likely bromthiocatechols and a bromothiopyrogallol, respectively.

Effect of a cysteine prodrug (L-2-oxothiazolidine-4-carboxylic acid) on the metabolism and toxicity of bromobenzene: a repeated exposure study.

The relationship between dose, toxicity, and metabolism of bromobenzene and the use of urinary metabolite excretion as an index of internal exposure to the reactive electrophilic intermediate bromobenzene 3,4-epoxide after repeated treatments with bromobenzene in presence or absence of L-2-oxothiazolidine-4-carboxylic acid (OTCA) were evaluated in mice. Repeated treatments with bromobenzene doses of 0.5, 0.75, and 1.0 mmol/kg ip twice a day for 18 d produced a marked reduction in 24-h urinary excretion of bromophenylmercapturic acid and p-bromophenol; this was accompanied by increases in plasma transaminases. Treatment with OTCA (1.0-3.0 mmol/kg) prevented toxicity and enhanced the 24-h urinary excretion of various bromobenzene metabolites by approximately 30-75, 104-145, and 164-269% for bromobenzene doses of 0.5, 0.75, and 1.0 mmol/kg, respectively. The effect of OTCA was further characterized by investigating the metabolism of bromobenzene given as a challenge dose of 4.0 mmol/kg to mice pretreated with bromobenzene and OTCA for 18 d. Pretreatment with bromobenzene reduced the 0- to 6-h urinary excretion of all metabolites after the challenge dose; this effect was virtually reversed by OTCA. It is concluded that repeated bromobenzene administration reduces its own detoxification to mercapturic acid and phenolic metabolites and elicits toxicity. This phenomenon is reversed after OTCA administration. This study further provides evidence that internal exposure to the reactive electrophilic intermediate bromobenzene 3,4-epoxide could be assessed more accurately by summing the urinary excretion of bromophenylmercapturic acid and p-bromophenol after OTCA treatment.

Effect of sodium selenite upon bromobenzene toxicity in rats. II. Metabolism.

The effects of sodium selenite (12.5 or 30 mumol/kg, ip) upon bromobenzene metabolism were examined in male rats treated with selenite at 72 hr prior to bromobenzene exposure (7.5 mmol/kg, ip). The inhibitory nature of selenium treatment upon xenobiotic metabolism and increased production of hepatic thiol suggested that selenite might affect metabolic activation and detoxification of bromobenzene. Selenite treatment lowered in vivo covalent binding of [14C]bromobenzene while the in vitro covalent binding of [14C]bromobenzene in microsomes isolated from selenite-treated rats was unaffected compared to control. When the rate of [14C]bromobenzene decline in whole blood was evaluated in selenite-treated rats over 48 hr after bromobenzene administration, no significant differences were observed when compared to control. Furthermore, values of glutathione conjugates and hydroxylated metabolites of bromobenzene were similar in urine samples collected over 48 hr from selenite and control rats. Mechanistically, reduction of bromobenzene hepatotoxicity by selenite is not mediated through an altered metabolism of bromobenzene, but by alteration of cellular events occurring after metabolic activation.

Influence of selenium on the metabolism of bromobenzene and a possible relationship to its hepatotoxicity.

When male Sprague-Dawley rats were treated with sodium selenite (1 mg/kg, sc) 24 hr prior to or simultaneously with bromobenzene (2.5 mmol/kg, ip) and sacrificed 48 hr after the bromobenzene dose, increased levels of the activities of serum transaminases (serum glutamic-oxaloacetic transaminase (SGOT) and serum glutamic-pyruvic transaminase (SGPT) induced in the bromobenzene-treated rats were significantly reduced in the presence of selenium. However, no such reduction in the transaminases activities were observed when rats were either pretreated with selenite for 48 hr or pretreated with 0.1, 0.2, or 0.5 mg/kg of selenite. Although selenium alone had no effect on the hepatic microsomal drug metabolism, simultaneous treatment of selenite (1 mg/kg) with bromobenzene resulted only an increase in the activity of aniline hydroxylase after 48 hr as compared to that in the bromobenzene-treated group. When rats were given 2.5, 10, and 20 ppm of selenite in drinking water daily for 4 weeks prior to an ip injection of 2.5 mmol/kg of bromobenzene and were sacrificed 48 hr after bromobenzene administration, a reduction in the SGOT activities in all the pretreated groups and a reduction of SGPT activity in 20 ppm selenite-treated group were observed when compared with those in the bromobenzene-treated groups. A dose-dependent increase in hepatic GSH concentrations were observed due to such chronic selenium treatment. Treatment with selenite (1 mg/kg) 24 hr prior to bromobenzene injection (2.5 mmol/kg) increased initially both o and p-bromophenols in the rat urine at 0-7.5 hr without affecting urinary thioethers. On the contrary, the ratio of thioethers to p-bromophenol was significantly higher in both 2.5 and 10 ppm selenite-pretreated (4 weeks) rats as well as a significant increase in the ratio of thioethers to total phenolic metabolites in 10 ppm and an increase close to significant in 2.5 ppm selenite-treated rats were observed initially at 0-7.5 hr urine samples. These results indicate that acute selenium pretreatment under certain conditions, favors increased hydroxylation of the intermediate bromobenzene epoxides, whereas higher detoxification of the epoxides involving hepatic glutathione (GSH)/GSH transferases pathway is more favored due to increased biosynthesis of GSH in certain chronic selenium treated rats.

Excretion of thioethers in urine after exposure to electrophilic chemicals.

Electrophilic agents--a class of chemicals that includes most genotoxic compounds--can be inactivated by reaction with glutathione or other SH-bearing molecules. The conjugates so formed often appear in the urine as mercapturic acids or other thioether products. This paper critically reviews the suitability of the urinary thioether assay as a method for the detection of exposure to electrophilic agents or their precursors. In practice, the greatest value of the thioether assay appears to lie in its signal function. This is demonstrated for cigarette smokers and industrial workers involved in chemical waste incineration. Whenever increased thioether excretion is observed, it is likely to be due to exposure to one or more suspect compounds. However, when the thioether concentration ranges within the limits of the normal value, one must not conclude that there is no, or negligible, exposure. More specific applications of the assay of thio compounds in urine allow development of selective methods that may be useful for biological monitoring.