Covalent binding of 1,2-dihaloalkanes to DNA and stability of the major DNA adduct, S-[2-(N7-guanyl)ethyl]glutathione.

The major DNA adduct formed from the carcinogen ethylene dibromide (1,2-dibromoethane, EDB) is S-[2-(N7-guanyl)ethyl]glutathione, resulting from the reaction of guanyl residues with the half-mustard S-(2-bromoethyl)glutathione, which is generated by glutathione S-transferase-catalyzed conjugation of EDB with glutathione. The half-life of the alkylating species [putative S-(2-bromoethyl)glutathione or the derived episulfonium ion] was estimated to be less than 10 s. However, the stability was enough for approximately half of the alkylating metabolites to leave isolated rat hepatocytes before reacting with nucleic acids. Treatment of isolated rat hepatocytes with diethylmaleate decreased covalent binding of EDB to DNA, but treatment with 1-phenylimidazole did not, consistent with the view that conjugative metabolism is of greater importance than oxidation with regard to DNA binding. When EDB was administered to rats in vivo, only one major adduct, S-[2-(N7-guanyl)ethyl]glutathione, was formed in liver or kidney. S-[2-(N7-Guanyl)ethyl]glutathione was found in liver and kidney DNA of rats treated with 1,2-dichloroethane, but other adducts were also present. The gamma-glutamyl transpeptidase inhibitor AT-125 [L-(alpha-(5S)-alpha-amino-S-chloro-4,5-dihydro-5-isoxazoleacetic acid] did not affect the level of EDB bound to DNA by glutathione-fortified rat kidney homogenates or bound to liver or kidney DNA in vivo. The in vitro half-life of S-[2-(N7-guanyl)ethyl]glutathione in calf thymus DNA was 150 h; the half-life of the adduct in rat liver, kidney, stomach, and lung was between 70 and 100 h. Isolated S-[2-(N7-guanyl)ethyl]glutathione did not react with DNA to form new adducts. These results provide a further basis for understanding the carcinogenic action of 1,2-dihaloethanes.

Selectivity of rat and human glutathione S-transferases in activation of ethylene dibromide by glutathione conjugation and DNA binding and induction of unscheduled DNA synthesis in human hepatocytes.

The major DNA adduct formed by the carcinogen ethylene dibromide (EDB) is S-[2-(N7-guanyl)ethyl]glutathione. This adduct results from the glutathione S-transferase (GST)-catalyzed conjugation of EDB with glutathione (GSH), which generates an episulfonium ion capable of reacting with cellular nucleophiles. Purified rat and human GST enzymes were compared for their ability to conjugate EDB with GSH and displayed high selectivity. Of the six forms of rat GST tested, conjugation was catalyzed by the alpha class enzyme 2-2 and, to a lesser extent, by the mu class enzyme 3-3. Of the three classes of cytosolic human GST, EDB conjugation was catalyzed by the alpha class enzymes. Three dimers of the human alpha class (alpha x-alpha x, alpha x-alpha y, and alpha y-alpha y) were separated by chromatofocusing. The alpha x-alpha x preparation demonstrated the highest specific activity. Rat microsomal GST had negligible activity for the conjugation of EDB with GSH. The levels of EDB-DNA adducts formed in rat and human hepatocytes were compared. DNA was isolated from both rat and human hepatocytes incubated with 0.5 mM EDB, and the level of DNA adduct formation in the human samples was about 40% of that in the rat hepatocytes. EDB concentration-dependent unscheduled DNA synthesis was demonstrated in isolated human hepatocytes. Concurrent treatment of the hepatocytes with diethylmaleate to deplete intracellular GSH inhibited EDB-induced unscheduled DNA synthesis. These results indicate that EDB alkylates DNA in human hepatocytes and that enzymatic repair of adducts may occur. The results of experiments done in rat and human systems using both purified GST enzymes and intact hepatocytes imply that the genotoxic pathway of EDB metabolism in rats and humans is similar.

Hydantoin bioisosteres. In vivo active spiro hydroxy acetic acid aldose reductase inhibitors.

The hypothesis that clinical side effects of the aldose reductase inhibitor (ARI) sorbinil were related to its hydantoin ring led to a bioisosteric analysis and replacement of the hydantoin by a spiro hydroxy acetic acid moiety as in 40. These hydroxy acids, compared to hydantoins, showed a similar potency increase on chroman 2-methyl substitution, a similar orthogonal relationship of acidic to aromatic moieties, and similar ARI enantioselectivity. In this series the six-membered spiro hydroxy acetic acid anion array is a bioisostere for a spiro hydantoin anion and leads to ARIs with excellent in vivo activity. In vitro and in vivo activity was improved over 40 by chroman cis 2-methylation as in 4 and by aromatic 6,7-halogen substitution. Compounds with the best acute in vivo activity in rats were compared for chronic in vivo activity. The highest tissue levels and best chronic in vivo activities were found in the racemic 6,7-dichloro and 6-fluoro-7-chloro analogues 18 and 23. ARI activity was enantioselective for 58 and 60, the 2R,4R-enantiomers of 18 and 23. 7-Chloro-6-fluoro-cis-4-hydroxy-2(R)-methyl-chroman-4-acetic acid (60) was selected for phase 1 clinical trials and did not exhibit sorbinil-like hypersensitivity side effects.

Activation of dihaloalkanes by glutathione conjugation and formation of DNA adducts.

Ethylene dibromide (1,2-dibromoethane, EDB) can be activated to electrophilic species by either oxidative metabolism or conjugation with glutathione. Although conjugation is generally a route of detoxication, in this case it leads to genetic damage. The major DNA adduct has been identified as S-[2-(N7-guanyl)ethyl]glutathione, which is believed to arise via half-mustard and episulfonium ion intermediates. The adduct has a half-life of about 70 to 100 hr and does not appear to migrate to other DNA sites. Glutathione-dependent DNA damage by EDB was also demonstrated in human hepatocyte preparations. The possible relevance of this DNA adduct to genetic damage is discussed.

Pharmacokinetics of the aldose reductase inhibitor, zopolrestat, in humans.

The pharmacokinetics of zopolrestat, an aldose reductase inhibitor that may be useful for the treatment of complications of diabetes, have been investigated using oral doses ranging from 50 to 1200 mg administered to healthy male volunteers. In a single-dose study, Cmax, AUC(0-48), and urinary elimination of zopolrestat increased linearly with increasing dose. The amount of zopolrestat excreted unchanged in the urine within 48 hours ranged from 34 to 45% of the administered dose. Renal clearance ranged from 2.6 to 5.6 mL/min, and appeared to decrease as the dose was increased. In a 2-week multiple dose study, the mean steady-state minimum and maximum plasma concentrations, Cmin and Cmax, were 91.5 and 196 micrograms/mL for subjects administered 800 mg/day, and 131 and 281 micrograms/mL for subjects administered 1200 mg/day. Steady-state AUC(0-24) was also dose proportional. The mean steady state half life of about 30.3 hours was consistent with the observed 2.2-fold accumulation in plasma. Apparent oral clearance (Clpo) was 5.2 mL/min, and apparent volume of distribution (Vdss/F) was 12 L. Mean renal clearance was 2.2 mL/min, and approximately 45% of the administered dose was excreted into the urine at steady state. There was no effect of food consumption during dosing on the extent of absorption of zopolrestat. In in vitro studies, extensive, concentration-dependent binding of zopolrestat to plasma proteins was observed. These data indicate that once-daily dosing of zopolrestat will provide suitable exposure in the treatment of diabetic complications.

Induction of fluconazole metabolism by rifampin: in vivo study in humans.

The effects of rifampin on the pharmacokinetics of fluconazole were analyzed in an open-label, placebo-controlled, parallel study. Sixteen healthy male volunteers, randomized into two groups, received 200 mg of oral fluconazole on days 1 and 22. On days 8 through 27, group I received oral rifampin, 600 mg/d, and group II received placebo. Fluconazole in serum was analyzed by HPLC. On days 1 and 22, respectively, the AUC (micrograms.hr/mL) (mean +/- SD) was 160.5 +/- 19.5 and 124 +/- 22.2 in group I, 152 +/- 25 and 152.8 +/- 33.9 in group II; the Kel (hr-1) was .0211 +/- .0030 and .0264 +/- .0040 in group I, .0219 +/- .0036 and .0216 +/- .0053 in group II. Cmax and Tmax did not change significantly in either group. Urinary 6 beta-hydroxycortisol/cortisol increased from 3.47 +/- 1.04 to 15.2 +/- 5.07 in group I, but was unchanged (3.54 +/- 1.33-4.26 +/- 2.36) in group II on days 1 and 22, respectively. The findings in this study indicate that rifampin induces the metabolism of fluconazole.

Pharmacokinetics of the acyl coenzyme A:cholesterol acyl transferase inhibitor CP-105,191 in dogs--the effect of food and sesame oil on systemic exposure following oral dosing.

Inhibition of acyl coenzyme A:cholesterol acyl transferase (ACAT) decreases total plasma cholesterol in animals and may be an effective therapy for atherosclerosis in man. The pharmacokinetics of CP-105,191, a potent inhibitor of ACAT, were explored in fed and fasted dogs. Following oral administration of drug, mean apparent plasma half-life ranged from 9 to 16 h. Systemic availability of CP-105,191, as determined by AUC(0-infinity), was approximately 3-4-fold higher in fed dogs than in fasted dogs when 50 mg doses were administered as aqueous suspensions. Tmax was achieved more rapidly and Cmax was lower in fasted dogs. When 50 mg doses, partially dissolved in 20 mL sesame oil, were administered to fed dogs, the availability of CP-105,191 increased by another factor of 2. A 12.5 mg dose of CP-105,191, completely dissolved in sesame oil, was administered to fed and fasted dogs. Plasma AUC's were similar for fed and fasted dogs following the 12.5 mg dose, indicating that the increased availability of drug when administered with food is related to the presence of lipid.

Pharmacokinetics of tiqueside (beta-tigogenin cellobioside) in dogs, rats, rabbits, and monkeys.

Tiqueside (CP-88,818, beta-tigogenin cellobioside) is an effective cholesterol absorption inhibitor that may be useful in the treatment of hypercholesteremia. We have investigated the pharmacokinetics of tiqueside in dogs, rats, rabbits, and monkeys. In dogs, the volume of distribution (Vdss) was 2.11 L/kg, clearance was 0.58 mL/min-kg, and half-life was 45 h following a 1.4 mg/kg intravenous dose. Absolute bioavailability in fed dogs decreased from 6.7% for a 30 mg/kg dose to 1.7% for a 375 mg/kg dose. The oral bioavailability at a dose of 375 mg/kg was approximately 4-fold lower in fasted dogs than fed dogs. AUC-(0-24) for doses up to 2000 mg/kg were only slightly greater than AUC-(0-24) for a 375 mg/kg dose. In rats dosed intravenously at 8.0 mg/kg, Vdss was 3.52 L/kg, clearance was 14.6 mL/min-kg, and half-life was 3.6 h. Estimated bioavailability for rats dosed in feed at 250-2000 mg/kg/day was less than 0.5%. In rabbits dosed at 4.0 mg/kg i.v., Vdss was 2.95 L/kg, clearance was 0.59 mL/min-kg, and half-life was 61 h. Bioavailability for rabbits dosed in feed at 62.5 or 125 mg/kg/day was approximately 7%. Systemic exposure in rhesus monkeys after oral dosing was lower than that for dogs and rabbits. Thus, low systemic exposure to tiqueside following oral administration has been demonstrated in several animal species.

A calorimetric method to assess endogenous metabolism and its application to the study of bovine sperm.

Calorimetry was used to assess the importance of endogenous metabolism towards total ATP synthesis in bovine sperm in the presence of extracellular glucose. Sperm were incubated in the calorimeter with D-[U-14C]glucose without or with electron transport inhibitors, rotenone and antimycin A. Steady-state heat production during the incubations was measured for 30 min, the incubations were terminated, and the cell suspensions removed for analysis of radioactive glucose and its metabolic end-products. Heat production (mean +/- S.E.) associated with the metabolism of glucose was calculated, from enthalpies of formation of glucose and its end-products, as -412 +/- 34 mJ/h/10(8) cells in control incubations and -263 +/- 18 mJ/h/10(8) cells in the incubations with electron transport inhibitors. Measured heat production was -455 +/- 36 and =263 +/- 17 mJ/h/10(8) cells, respectively. Thus, heat production by endogenous pathways, the difference between measured total heat production and calculated exogenous heat production, was -43 +/- 14 mJ/h/10(8) cells for control cells and about -6 mJ/h/10(8) cells for inhibited cells. The ratio of heat produced per mol of ATP synthesized is similar for all ATP-producing pathways. Therefore, about 10% of total ATP synthesis in control cells and less than 2% in inhibited cells is provided by endogenous pathways when extracellular glucose is present.

Comparative tissue distribution and excretion of [1-14C]acrylamide in beagle dogs and miniature pigs.

Male beagle dogs and miniature pigs were given acrylamide in the diet for 3-4 wk at a dosage of 1 mg/kg/day. They were then given [1-14C]acrylamide as a single oral dose of 1 mg/kg. The animals were killed 6 hr or 1, 2, 4 or 14 days after administration of the radioactive compound and tissues were analysed for radioactivity. The radiolabelled material was distributed to a major extent in muscle tissue in both species (31-35% of the dose at 6 hr and 5-7% at 14 days). Although the nervous system is the primary target for acrylamide monomer toxicity, less than 1% of the administered 14C was found in the brain in both species. No neurotoxic signs were evident during the exposure period at the dosage used. Analysis of discrete areas of the brain for radioactivity revealed that the levels of penetration of [1-14C]acrylamide in brain paralleled the vascularization pattern of the tissues. Approximately 60% of the administered radiolabel was excreted in the urine in both species and smaller amounts were excreted in the faeces. However, recovery in the faeces was higher in pigs (c. 25%) than in dogs (c. 7%) and this and the considerably higher levels demonstrated in the gastro-intestinal tract of the pigs indicated that the absorption of acrylamide was more rapid and more extensive in dogs than in pigs.

Glutathione-mediated binding of dibromoalkanes to DNA: specificity of rat glutathione-S-transferases and dibromoalkane structure.

1,2-Dibromo-[1,2-14C]ethane was bound irreversibly to DNA when glutathione S-transferase or rat liver cytosolic components were added to incubations of calf thymus DNA and glutathione at 37 degrees C. There was no DNA binding of 1,2-dibromoethane when glutathione was absent or in incubations of DNA with microsomal proteins with or without NADPH, thus supporting the proposal that the major route of DNA binding by 1,2-dibromoethane occurs via conjugation to glutathione. In vitro binding of 1,2-dibromoethane occurred most effectively when the YaYc (or 'B') isozyme of glutathione S-transferase was included in incubations of DNA with 1,2-dibromoethane and glutathione. Other dihaloalkanes were incubated with DNA in the presence of glutathione S-transferase and [35S]glutathione. Of these, only 1,2-dibromo-3-chloropropane and tris-(2,3-dibromopropyl)-phosphate led to significant DNA binding of [35S]glutathione. 1,2-Dibromo-3-chloro-[1,3-14C]propane was bound to DNA when glutathione and glutathione S-transferase were present. However, even higher 1,2-dibromo-3-chloropropane binding to DNA occurred when cytosol or microsomes were included in incubations without glutathione. When glutathione was added to incubations containing cytosol and 1,2-dibromo-3-chloropropane, total DNA binding was decreased. Thus, the actual amount of DNA binding by dihaloethanes in vivo may be the result of a complicated balance among the opposing roles of glutathione conjugation in detoxicating and activating processes.

Theoretical considerations on two equations for estimating the extent of absorption after oral administration of drugs.

PURPOSE: The amount of drug absorbed into portal blood after oral dosing (Dp.o,g) has been estimated using Fick's principle (Q-method), i.e., Dp.o,g = Qh x (AUCp.o,g--AUCp.o,c), where Qh is the portal blood flow rate, and AUCp.o,g and AUCp.o,c are the areas under the concentration-time curves of portal vein and systemic blood after oral dosing, respectively. However, this method may underestimate Dp.o,g, when the drug is subject to systemic intestinal elimination. An alternate equation (CL-method; Dp.o,g = CLs x AUCp.o,g) is described using a simple pharmacokinetic model, to estimate Dp.o,g in the presence of systemic intestinal elimination, where CLs is systemic clearance. METHODS: The model is composed of central, intestine and liver compartments, assuming that drug is eliminated by intestinal and/or hepatic pathways only. A comparison of both methods for estimating Dp.o,g was made using computer-simulation or experimental data of phenacetin from the literature. RESULTS: The simulation study demonstrated that the Q-method underestimated Dp.o,g in the presence of significant intrinsic intestinal clearance, compared to the CL-method. The similar results were observed using the experimental data of phenacetin. CONCLUSIONS: The CL-method can provide a better estimate of Dp.o,g, while the Q-method may underestimate Dp.o,g, when there is significant systemic intestinal elimination of drugs after oral administration. In addition, useful information for understanding the relationship between the extent of absorption and the first-pass effect by intestine and/or liver after oral dosing of drugs can be obtained from the present approach.

Changes in metabolism of ram sperm associated with epididymal transit or induced by exogenous carnitine.

Differences in the metabolism of testicular and cauda epididymal sperm have been demonstrated for several species, but the region(s) of the ram epididymis in which changes in metabolism occur is not known. In these experiments respirometric and radioisotopic methods were used to monitor metabolic activity of ram sperm isolated from six regions of the epididymis. Effects of exogenous carnitine on metabolism of the sperm also were studied. Sperm from the caput and corpus epididymidis had similar rates of glucose and oxygen consumption and of lactate and CO2 production. However, the magnitude of each parameter was severalfold higher for cauda sperm. In addition, cauda sperm produced 25 times more acetate than caput or corpus sperm. The amount of energy derived from utilization of endogenous substrates was similar for sperm from all regions of the epididymis. Exogenous D,L-carnitine (5 mM) had no effect on the metabolism of caput or corpus sperm. However, when cauda sperm were incubated with carnitine, they consumed less glucose and produced less lactate and more acetate; thus they produced the same amount of ATP from less glucose than did control aliquots incubated without exogenous carnitine. Since the rate of ATP synthesis was equivalent for both incubations, we believe this change in metabolism reflects an increased efficiency of glucose utilization. This increased efficiency may be vital for sperm motility.

Endogenous metabolism by sperm in response to altered cellular ATP requirements.

A quantitative description of the relative importance of endogenous metabolism to overall ATP production has not been established for mammalian cells. We report herein results of experiments using sperm (selected because of their simple metabolic potential and absence of biosynthetic pathways) and calorimetry (chosen because it serves as a general monitor of metabolism) to assess the importance of endogenous metabolism to total ATP synthesis under several incubation conditions. In experiments in which sperm were incubated at different temperatures (20 degrees C, 25 degrees C, 30 degrees C or 35 degrees C) and with different substrates (glucose, fructose, lactate, or beta-hydroxybutyrate), endogenous metabolism occurred at a constant rate regardless of the rate of ATP turnover in the cells or the nature of the exogenous substrate available to them. Sperm incubated at 35 degrees C with glycolyzable substrates synthesized more ATP (9 mumol ATP . h-1/10(8) cells) than did sperm incubated with the nonglycolyzable substrate, lactate (6.2 mumol ATP . h-1/10(8) cells). To investigate this substrate-related difference in the rate of ATP synthesis, the motility of sperm incubated at 35 degrees C with glucose or with lactate was determined. The velocities of the sperm incubated with either substrate were identical, indicating that the rates of ATP consumption for support of motility were identical. Most of the additional ATP synthesized by cells with glycolyzable substrates was consumed in the process of substrate cycling of the metabolic intermediates of glucose.

Alterations in motility and metabolism associated with sperm interaction with accessory sex gland fluids.

Sperm passing through the male tract interact with accessory sex gland fluids during ejaculation. Cellular metabolism is stimulated by this interaction for unknown reasons. These experiments involved calorimetric measurements [P.B. Inskeep and R.H. Hammerstedt (1983) J. Biochem. Biophys. Methods 7, 199-210] on ejaculated sperm (EJS) and cauda epididymal sperm (CES) from bulls to establish the contribution of individual pathways to total cellular ATP synthesis. Parallel incubations outside the calorimeter yielded samples for oxygen consumption measurements and for motility analysis, the major ATP-consuming reaction of sperm. Energy charge values were identical for incubations of EJS and CES with glucose, thereby establishing that the ratios of rates of ATP synthesis and degradation were equivalent for these cells under this incubation condition. The total rate of ATP synthesis was greater for EJS than for CES (5 vs 13 mumol ATP h-1/10(8) cells) with less than 2 mumol ATP h-1 for each cell type coming from degradation of endogenous reserves. Thus, ejaculation is associated with a large increase in catabolic rate that is satisfied by degradation of extracellular glucose. No difference in percentage of motile sperm was noted, but mean velocity was lower for CES (58 micron s-1) than for EJS (85 micron s-1). A difference in forward motility pattern was observed (wig-wag to flipping). We conclude from these data that interaction with accessory sex gland fluids alters ATP requiring activities of sperm, with one obvious alteration being their motility pattern. The increase in ATP requirement is satisfied by increased degradation of extracellular substrates, but not intracellular reserves, to provide sufficient ATP to satisfy cellular needs.

Tissue distribution and biotransformation of zopolrestat, an aldose reductase inhibitor, in rats.

Zopolrestat (Alond) is a new drug that is being evaluated as an aldose reductase inhibitor for the treatment of diabetic complications. 14C-labeled zopolrestat was orally administered to rats for a tissue distribution study and a bile duct cannulation metabolism study. Tissue samples from the distribution study were analyzed by complete oxidation and liquid scintillation counting. Urine and bile samples from the bile duct cannulation study were analyzed by microbore HPLC, with simultaneous radioactivity monitoring and atmospheric pressure ionization tandem mass spectrometry. The mass balance in the distribution study demonstrated that the greatest exposure (AUC0-infinity) occurred in the liver, followed by the ileum and large intestine. The time of maximal plasma concentrations for nearly all tissues was 4 hr after the dose, and the half-life of radioactivity in most tissues (8-10 hr) was similar to the half-life in plasma. For the bile duct-cannulated rat study, most of the radioactivity was recovered in the bile, indicating that biliary excretion is a major route of elimination of zopolrestat and its metabolites in rats. Numerous oxidative metabolites, as well as phase II conjugates, were identified in the bile and urine samples. Acyl glucuronides of zopolrestat and unchanged drug accounted for >85% of biliary radioactivity, whereas unchanged drug and degradation products of glutathione conjugates were identified as the major urinary metabolites.

Pharmacokinetics of zopolrestat, a carboxylic acid aldose reductase inhibitor, in normal and diabetic rats.

The pharmacokinetics of zopolrestat, a carboxylic acid aldose reductase inhibitor, were examined in normal male rats dosed intravenously at 2 mg/kg and in normal and streptozotocin-diabetic male rats after oral administration at 50 mg/kg. After oral dosing, Cmax was 127 micrograms/ml for normal rats and 144 micrograms/ml for diabetic rats. AUC(0-infinity), however, was lower for diabetic rats than for normal rats and plasma half-life was longer in normal rats (8.0 vs 6.6 hr). Half-lives of zopolrestat in nerve, kidney, and lens were longer than plasma half-life and were similar for both diabetic and normal rats. Less than 2% of the dose was excreted in the urine as unchanged zopolrestat during the 48-hr period following dosing by diabetic or normal rats. Protein binding of zopolrestat was less extensive in plasma from diabetic rats than in plasma from normal rats. Similar kinetics were observed in diabetic animals receiving five daily doses of zopolrestat at 50 mg/kg/day. There was no plasma or liver accumulation of zopolrestat at steady state, consistent with the observed half-lives. However, zopolrestat did accumulate in nerve, kidney, and lens to varying degrees during multiple dosing, reflecting the longer half-lives of zopolrestat in these tissues.

Bioavailability, multiple-dose pharmacokinetics, and biotransformation of the aldose reductase inhibitor zopolrestat in dogs.

Zopolrestat (Alond) is a new drug that is being evaluated as an aldose reductase inhibitor for the treatment of diabetic complications. The bioavailability in dogs of a 2 mg/kg oral dose of zopolrestat was 97.2%. In a 1-year, multiple-dose, pharmacokinetic study, systemic exposure increased with increasing dose (50, 100, and 200 mg/kg/day), and there were no consistent changes in exposure with multiple dosing. Renal clearance at 1 year appeared to be higher in males. The magnitude of the potential gender difference in exposure was relatively small and was unlikely to have had a meaningful impact on the pharmacokinetics of zopolrestat in dogs. In studies with bile duct-cannulated dogs, radioactivity from [14C]zopolrestat was primarily eliminated as unchanged drug and acyl glucuronide in the bile and feces (77.3% of the dose) and in urine (18.3% of the dose). The concentrations of acyl glucuronide in urine and feces were approximately 50% of the zopolrestat concentrations. Minor metabolites (each accounting for <1% of the dose) included those resulting from hydroxylation of the phthalazinone ring and glutathione conjugation of the benzothiazole ring.

S-[2-(N7-guanyl)ethyl]glutathione, the major DNA adduct formed from 1,2-dibromoethane.

The reaction of 1,2-dibromoethane and glutathione with DNA in the presence of glutathione S-transferase results in the formation of a single major DNA adduct, which can be released by thermal hydrolysis at neutral pH and separated by octadecylsilyl and propylamino high-performance liquid chromatography. The same DNA adduct is the only major one formed in livers of rats treated with 1,2-dibromo[1,2-14C]ethane. The DNA adduct was identified as S-[2-(N7-guanyl)ethyl]glutathione: (1) The chromatographic behavior was altered by treatment with gamma-glutamyl transpeptidase or Streptomyces griseus protease. (2) The molecular ions observed in positive and negative mode fast atom bombardment mass spectrometry were those expected for the structure when either glycerol or a mixture of dithiothreitol and dithioerythritol was used as the bombardment matrix. (3) The two-dimensional 1H NMR correlated spectroscopy spectrum of the DNA adduct was compared to the spectra of glutathione, oxidized glutathione, and N7-methylguanine and found to be consistent with the assigned structure. No evidence for in vitro or in vivo opening of the guanyl imidazole ring was observed under these conditions. The structure of the adduct supports a pathway involving enzyme-catalyzed conjugation of 1,2-dibromoethane with glutathione, non-enzymatic dehydrohalogenation of the resulting half-mustard to form a cyclic episulfonium ion, and attack of the N7 nitrogen of DNA guanine on the episulfonium ion to generate this major DNA adduct, which may be related to the carcinogenicity of this chemical.