Environmental metabolite, 1,2-diacetylbenzene, produces cytotoxicity through ROS generation in HUVEC cells

Organic solvents are ubiquitous in industrial and household surroundings, and thus individuals are easily exposed. 1,2-Diethylbenzene (DEB) is one of organic solvents contained in gasoline or jet fuels. DEB is absorbed by dermal or inhalation routes, metabolized by cytochrome P-450 in the liver, and ultimately affects mammalian functions. 1,2-Diacetylbenzene (1,2-DAB), which is a putative metabolite of 1,2-DEB, resulted in neuropathological effects on rodent central and peripheral nervous systems. To elucidate the possibility of 1,2-DAB effects on the vascular system, studies were undertaken to examine whether 1,2-DAB induces endothelial cytotoxicity through reactive oxygen species (ROS) generation. Incubation of human umbilical vein endothelial cells (HUVEC) with lower concentrations (4 or 8 microM) of 1,2-DAB induced inhibition of cellular growth and at higher amounts (16 or 32 microM) produced apoptosis. Endothelial cells cultured with 1,2-DAB also showed increased intracellular ROS production and morphological alterations indicative of senescence. Pretreatment with the well-known antioxidant glutathione or N-acetylcysteine (NAC) reduced cytotoxicity induced by 1,2-DAB. Taken together, the results provide evidence that cytotoxicity induced by 1,2-DAB in endothelial cells may be mediated by ROS generation.     

Possible involvement of 1,2-diacetylbenzene in diethylbenzene-induced neuropathy in rats

The role of 1,2-diacetylbenzene (1,2-DAB) in the peripheral nerve toxicity of 1,2-diethylbenzene (1,2-DEB) was investigated in rats. Gas chromatography-mass spectrometry identified 1,2-DAB in the urine samples of rats given 165 mg kg-1 1,2-DEB orally on four consecutive days. 1,2-DAB shared not only the ability of 1,2-DEB to cause bluish discoloration of skin, internal organs and urine, but unlike 1,2-DEB it turned hair blue at the site of intraperitoneal injection. Intraperitoneal administration of 10 mg kg-1 and 20 mg kg-1 1,2-DAB to groups of 12 rats, 4 days a week for 11 and 6 weeks, caused a dose- and time-dependent decrease in mean sensory and motor conduction velocities. Recovery in a 5-week post-exposure period was gradual but consistent. The effect of 1,2-DAB on the amplitude of the sensory action potential was ambiguous. The findings support the hypothesis that the formation of 1,2-diacetylbenzene derivatives contributes to the neurotoxicity of 1,2-DEB.     

Alteration of brainstem auditory evoked potentials in diethylbenzene and diacetylbenzene-treated rats.

Male Sprague-Dawley rats were treated either with 1,2-diethylbenzene (1,2-DEB) or its putative active metabolite, 1,2-diacetylbenzene (1,2-DAB). Experimental rats and appropriate controls were examined electrophysiologically for brainstem auditory evoked potentials (BAEP). Oral administration of 1,2-DEB (75 or 100 mg kg-1 once a day, 4 days a week, for 8 weeks) and intraperitoneal injection of 1,2-DAB (10 or 15 mg kg-1 once a day, 4 days a week, for 8 weeks) produced time- and dose-dependent increases in the peak latencies of all BAEP components as well as in interpeak (I-V) differences, and a decrease in the amplitudes of all the components. The absolute and interpeak latencies recovered partially during an 8-week (1,2-DEB) or a 10-week (1,2-DAB) recovery period, whereas there were long-lasting decreases in peak amplitudes.     

Bio-distribution study of 1,2-diethylbenzene and its main metabolites by whole-body autoradiography and tissue homogenates

The bio-distribution of the neurotoxic 1,2-diethylbenzene (1,2-DEB) was studied in male Sprague-Dawley rats after intravenous administration of [(14)C] 1,2-DEB (1 mg kg(-1)). The highest concentrations of [(14)C] non-volatile metabolites, determined by whole-body auto-radiography, were in the nasal cavity, ethmoid turbinates and in kidney. Whatever the time after dosing, the [(14)C] concentrations in the cerebrum, cerebellum, spinal cord and lung were lower than those in the blood. In contrast, after killing of batch of administered rats, the [(14)C] concentrations in the brain homogenates were higher than in plasma for 5-15 min. In addition, the [(14)C] concentrations in the lung were higher than in the plasma for 24 h post-dose. Moreover, the concentrations of unchanged 1,2-DEB and one of its metabolites, 1-(2'-ethylphenyl)ethanol (1,2-EPE) in the brain, were higher than in the plasma until 1 h post-dose. The concentrations of 1,2-DEB in the blood cells were tenfold higher than in the plasma. The clearance of unchanged 1,2-DEB in the whole blood and in the blood cells was 6.4 and 3.9 ml min(-1), respectively. The apparent half-life of unchanged 1,2-DEB in plasma is very fast (5 min) which suggests a quick distribution and/or metabolism in liver and/or other tissues such as the lung. In conclusion, unchanged 1,2-DEB has a high affinity for the brain and blood cells and its concentrations in plasma and brain decreased rapidly with time.     

Diethylbenzene-induced sensorimotor neuropathy in rats

The commercial isomer mixture of diethylbenzene (DEB mixture), 1,2-diethylbenzene (1,2-DEB), 1,3-diethylbenzene (1,3-DEB) and 1,4-diethylbenzene (1,4-DEB) were administered orally to male Sprague-Dawley rats. The experimental rats and the appropriate controls were examined electrophysiologically for motor and sensory conduction velocities (MCV and SCV), and for the amplitude of the sensory action potential (ASAP) of the tail nerve, at weekly or bi-weekly intervals. Oral administration of DEB mixture (750 or 500 mg kg-1, once daily, 5 days per week for 10 weeks) and 1,2-DEB (100 mg kg-1, once daily, 4 days per week for 8 weeks) produced a time-dependent decrease in MCV, SCV and ASAP. Rats treated with DEB mixture and 1,2-DEB exhibited a blue discoloration of tissues and urine. No changes in MCV, SCV and ASAP developed in rats administered orally with 1,3-DEP and 1,4-DEB (500 mg kg-1, once daily, 5 days per week for 8 weeks). The results indicate that 1,2-DEB is the isomer responsible for neurotoxicity and suggest that a metabolic pathway giving rise to coloured compounds is involved in the neurotoxicity of DEB.     

1,2:3,4-Diepoxybutane in blood of male B6C3F1 mice and male Sprague-Dawley rats exposed to 1,3-butadiene

The important industrial chemical 1,3-butadiene (BD; CAS Registry Number: 106-99-0) is a potent carcinogen in B6C3F1 mice and a weak one in Sprague-Dawley rats. This difference is mainly attributed to the species-specific burden by the metabolically formed 1,2:3,4-diepoxybutane (DEB). However, only limited data exist on the DEB blood burden of rodents at BD concentrations below 100 ppm. Considering this, DEB concentrations were determined in the blood of mice and rats immediately after 6 h exposures to various constant concentrations of BD of between about 1 and 1200 ppm. Immediately after its collection, blood was injected into a vial that contained perdeuterated DEB (DEB-D₆) as internal standard. Plasma samples were prepared and treated with sodium diethyldithiocarbamate that derivatized metabolically produced DEB and DEB-D₆ to their bis(dithiocarbamoyl) esters, which were then analyzed by high performance liquid chromatography coupled with an electrospray ionization tandem mass spectrometer. DEB concentrations in blood versus BD exposure concentrations in air could be described by one-phase exponential association functions. Herewith calculated (±)-DEB concentrations in blood increased in mice from 5.4 nmol/l at 1 ppm BD to 1860 nmol/l at 1250 ppm BD and in rats from 1.2 nmol/l at 1 ppm BD to 92 nmol/l at 200 ppm BD, at which exposure concentration 91% of the calculated DEB plateau concentration in rat blood was reached. This information on the species-specific blood burden by the highly mutagenic DEB helps to explain why the carcinogenic potency of BD in rats is low compared to that in mice.     

1,2-diacetylbenzene, the neurotoxic metabolite of a chromogenic aromatic solvent, induces proximal axonopathy.

Several widely used aromatic hydrocarbon solvents reportedly induce blue-green discoloration of tissues and urine in animals and humans. The chomophore has been proposed to result from a ninhydrin-like reaction with amino groups in proteins. The present study examines the neurotoxic property of 1,2-diacetylbenzene (1,2-DAB), the active metabolite of the chromogenic and neurotoxic aromatic solvent 1,2-diethylbenzene. Rats treated with 1,2-DAB, but not with the nonchromogenic isomer 1,3-DAB or with ninhydrin developed blue discoloration of internal organs, including the brain and spinal cord. Only 1,2-DAB induced limb weakness associated with nerve fiber changes, which were most prominent in spinal cord and spinal roots. Changes began with the formation of proximal, neurofilament-filled axonal swellings of the type seen after treatment with 3,4-dimethyl-2,5-hexanedione, a potent derivative of the active metabolite of the neurotoxic aliphatic hydrocarbon solvents n-hexane and methyl n-butyl ketone. These compounds are metabolized to a gamma-diketone that forms pyrroles with target proteins, such as neurofilament proteins. A comparable mechanism is considered for 1,2-DAB, an aromatic gamma-diketone.     

Diethylbenzene inhalation-induced electrophysiological deficits in peripheral nerves and changes in brainstem auditory evoked potentials in rats.

Motor and sensory conduction velocities (MCV and SCV), amplitude of the sensory action potential (ASAP) of the tail nerve and parameters of brainstem auditory evoked potentials (BAEP) were studied in male Sprague-Dawley rats after prolonged inhalation exposure to a commercial isomer mixture of diethylbenzene (DEB mixture) containing 6% 1,2-DEB. The MCV, SCV and ASAP were studied in one control group (10 rats) and three groups of 12 rats exposed to 500, 700 or 900 ppm DEB mixture for 6 h daily, 5 days per week, for 18 weeks. Rats used for recording BAEP (one control group and two other groups of 15 rats) were exposed to 600 and 800 ppm DEB mixture. The exposure time was the same. Rats exposed to DEB mixture exhibited a time- and concentration-dependent decrease in MCV, SCV and ASAP and a time- and concentration-dependent increase of both the peak latencies of all BAEP components and the interpeak (I-V) differences.     

Cytotoxicity of 1,2-diacetylbenzene in human neuroblastoma SHSY5Y cells is mediated by oxidative stress

Environmental substances or metabolites induce neuronal damage through oxidative stress. Environmental organic solvent metabolite, 1,2-diacetylbenzene (1,2-DAB), treated rats develop limb weakness with neuropathological damage in both the central and peripheral nervous systems. In this experiment, we examined the relevance of 1,2-DAB-induced toxicity to increased oxidative stress using human dopaminergic neuroblastoma SHSY5Y cells. 1,2-DAB (4, 16, and 32 microM) disrupted cytoskeletal integrity and caused morphological changes. 1,2-DAB significantly decreased cell viability and induced cell cycle arrest in the G(1) phase in a concentration-dependent manner. At higher concentration, it produced apoptosis. Pre-treatment of cells with the antioxidants, GSH or N-acetylcysteine (NAC), effectively blocked 1,2-DAB-mediated cytotoxicity including cell viability, and morphological changes. These results therefore suggest that oxidative stress is involved in environmental metabolite 1,2-DAB-mediated neurotoxicity and that antioxidant treatment can effectively protect the nervous system from environmental hazards.     

Assessment of the developmental toxicity and placental transfer of 1,2-diethylbenzene in rats.

Sprague-Dawley rats were administered 1,2-diethylbenzene (1,2-DEB) by gavage on gestational days (GD) 6 through 20 at dose levels of 0 (corn oil), 5, 15, 25 or 35 mg/kg. The dams were euthanized on GD21 and the offspring were weighed and examined for external, visceral and skeletal alterations. Maternal toxicity, indicated by significant decreases in body weight gain and food consumption, was observed at doses of 15 mg/kg and above. Developmental toxicity, expressed as significantly reduced foetal body weights, was seen at doses of 15 mg/kg and higher. There was no evidence of embryolethal or teratogenic effects at any dose tested. The placental transfer of 1,2-DEB was examined after a single oral dose of 25 mg [14C]1,2-DEB/kg on GD18. Maternal and foetal tissues were collected at intervals from 1 to 48 hours. Placental and foetal tissues accounted for less than 0.35% of the administered dose. Levels of radiocarbon in foetuses were lower than those in maternal plasma and placenta at all time points. Analysis performed at 1, 2 and 4 hours indicated that ethyl acetate extractable (acidic) metabolites were predominant in the maternal plasma while n-hexane extractable (neutral) compounds represented the major part of radioactivity in the placenta and foetus. In conclusion, this study demonstrated that 1,2-DEB causes mild foetotoxicity at maternal toxic doses and that the exposure of the developing rat foetus to 1,2-DEB and/or metabolites after maternal administration of 1,2-DEB in late gestation is small.     

Toxicokinetics of 1,2-diethylbenzene in male Sprague-Dawley rats-part 1: excretion and metabolism of [(14)C]1,2-diethylbenzene.

The excretion and metabolism of neurotoxic 1,2-diethylbenzene (1, 2-DEB) was studied in male Sprague-Dawley rats after i.v. (1 mg/kg) or oral (1 or 100 mg/kg) administration of 1,2-diethyl[U-(14)C]benzene ([(14)C]1,2-DEB). Whatever the treatment, radioactivity was mainly excreted in urine (65-76% of the dose) and to a lower extent in feces (15-23% of the dose), or via exhaled air (3-5% of the dose). However, experiments with rats fitted with a biliary cannula demonstrated that about 52 to 64% of the administered doses (1 or 100 mg/kg) were initially excreted in bile. Biliary metabolites were extensively reabsorbed from the gut and ultimately excreted in urine after several enterohepatic circulations. Insignificant amounts of unchanged 1,2-DEB were recovered in the different excreta (urine, bile, and feces). As reported previously, presence of 1-(2'-ethylphenyl)ethanol (EPE) was confirmed in urine and demonstrated in bile and feces. The two main [(14)C]1,2-DEB metabolites accounted for 57 to 79% of urinary and biliary radioactivity, respectively. Beta-Glucuronidase hydrolysis and electron impact mass spectra results strongly supported their glucuronide structure. Additionally, these two main metabolites were thought to be the glucuronide conjugates of the two potential enantiomers of EPE. The results indicate that the main initial conversion step of the primary metabolic pathway of 1,2-DEB appears to be the hydroxylation of the alpha-carbon atom of the side chain. The presence of two glucuronide conjugates of EPE in the urine in a ratio different from one suggests that the metabolic conversion of 1, 2-DEB is under stereochemical control.     

Toxicokinetics and metabolism of 1,2-diethylbenzene in male Sprague Dawley rats--part 2: evidence for in vitro and in vivo stereoselectivity of 1,2-diethylbenzene metabolism.

In a previous study, it was shown that the neurotoxic compound 1,2-diethylbenzene (1,2-DEB) is mainly hydroxylated in the alkyl chain to give 1-(2'-ethylphenyl)ethanol (1,2-EPE) and excreted in urine of rats as two glucuronide compounds (GA1 and GA2). Some findings have suggested that the two enantiomers of 1,2-EPE are formed in vivo. In the present study, a chiral high-performance liquid chromatography method was developed to separate the two enantiomers of 1,2-EPE from a synthesized racemic mixture. Absolute configuration of both enantiomers was determined after esterification with (R)-(+)-alpha-methoxy-alpha-(trifluoromethyl)phenylacetic acid and analysis of their (1)H NMR spectra in CCl(4) added with Eu (fod)(3). The two main urinary metabolites, GA1 and GA2, from [(14)C]1,2-DEB-treated Sprague-Dawley rats (80 mg/kg, i.p.) were identified, after hydrolysis with beta-glucuronidase from Escherichia coli, as (R) and (S) glucuronide conjugates of 1,2-EPE, respectively. In vitro hydroxylation of 1,2-DEB and glucuroconjugation of 1,2-EPE were under stereoselective control in S9 fraction or microsomes from male Sprague-Dawley rat liver. The V(max) and K(m) constants for (R)1,2-EPE enantiomer formation determined in S9 fraction were greater than those for the (S) enantiomer. In the plasma of bile duct-cannulated rats, the ratio was 1.2 +/- 0.02 over the 1- to 4-h period after oral administration of [(14)C]1,2-DEB (100 mg/kg). In contrast, the glucuroconjugation rate of (S)1,2-DEB enantiomer was 4 times that of (R)1,2-EPE glucuroconjugation. A similar ratio of (R) to (S)1,2-EPE glucuronide conjugates was obtained in the plasma of bile duct-cannulated rats.     

Increased formic acid excretion and the development of kidney toxicity in rats following chronic dosing with trichloroethanol,a major metabolite of trichloroethylene

The chronic toxicity of trichloroethanol, a major metabolite of trichloroethylene, has been assessed in male Fischer rats (60 per group) given trichloroethanol in drinking water at concentrations of 0, 0.5 and 1.0 g/l for 52 weeks. The rats excreted large amounts of formic acid in urine reaching a maximum after 12 weeks ( approximately 65 mg/24 h at 1 g/l) and thereafter declining to reach an apparent steady state at 40 weeks (15-20 mg/24 h). Urine from treated rats was more acidic throughout the study and urinary methylmalonic acid and plasma N-methyltetrahydrofolate concentrations were increased, indicating an acidosis, vitamin B12 deficiency and impaired folate metabolism, respectively. The rats treated with trichloroethanol developed kidney damage over the duration of the study which was characterised by increased urinary NAG activity, protein excretion (from 4 weeks), increased basophilia, protein accumulation and tubular damage (from 12 to 40 weeks), increased cell replication (at week 28) and evidence in some rats of focal proliferation of abnormal tubules at 52 weeks. It was concluded that trichloroethanol, the major metabolite of trichloroethylene, induced nephrotoxicity in rats as a result of formic acid excretion and acidosis.     

In vitro and in vivo nephrotoxicity of the L and D isomers of S-(1,2-dichlorovinyl)-cysteine

The toxicity of the optical isomers S-(1,2-dichlorovinyl)-L-cysteine (L-DCVC) and S-(1,2-dichlorovinyl)-D-cysteine (D-DCVC) was investigated in vivo and in vitro. In vitro studies, utilizing a rabbit renal cortical slice system, demonstrated toxicity due to both forms with the L-form being more toxic. Dose- and time-dependent decreases in intracellular K+ and LDH were observed. Both compounds produced an initial S3 lesion, L-DCVC at 10(-5) M (12 h), D-DCVC at 10(-4) M (8 h), followed by a lesion encompassing all proximal tubules. In vivo studies demonstrated elevated blood urea nitrogen values at 24 and 48 h with 25 mg/kg of either isomer. Histopathology indicated both D and L-DCVC produced a straight proximal tubular lesion by 48 h, the lesion produced by L-DCVC being more severe. The D and L isomers of DCVC were both shown to be toxic, the toxicity assessed in vitro corresponded well with the toxicity in vivo.     

Absorption, metabolism and excretion of safrole in the rat and man.

The metabolic disposition of different doses of [14C] safrole were studied in rat and man. In both species, small amounts of orally administered safrole were absorbed rapidly and then excreted almost entirely within 24 h in the urine. In the rat, when the dose was raised from 0.6 to 750 mg/kg, a marked decrease in the rate of elimination occurred as only 25% of the dose was excreted in the urine in 24 h. Furthermore, at the high dose level, plasma and tissue concentrations of both unchanged safrole and its metabolites remained elevated for 48 h probably indicating impairment of the degradation/excretion pathways. The main urinary metabolite in both species was 1,2-dihydroxy-4-allylbenzene which was excreted in a conjugated form. Small amounts of eugenol or its isomer 1-methoxy-2-hydroxy-4-allylbenzene were also detected in rat and man. 1'-Hydroxysafrole, a proximate carcinogen of safrole, and 3'-hydroxyisosafrole were detected as conjugates in the urine of the rat. However, in these investigations we were unable to demonstrate the presence of the latter metabolites in man.     

Acute nephrotoxicity induced by N-(3,5-dichlorophenyl)-3-hydroxysuccinamic acid in Fischer 344 rats

N-(3,5-Dichlorophenyl)succinimide (NDPS) induces nephrotoxicity via one or more metabolites which arise from oxidation of the succinimide ring. The purpose of this study was to examine the nephrotoxic potential of N-(3,5-dichlorophenyl)-3-hydroxysuccinamic acid (3-NDHSA), a potential metabolite of NDPS and a positional isomer of N-(3,5-dichlorophenyl)-2-hydroxysuccinamic acid (2-NDHSA), a known nephrotoxic metabolite of NDPS. Male Fischer 344 rats were administered a single intraperitoneal injection of 3-NDHSA (0.2 or 0.4 mmol/kg) or sesame oil (2.5 mmol/kg), and renal function was monitored at 24 and 48 h. Both doses of 3-NDHSA induced diuresis, increased proteinuria, glucosuria and hematuria, elevated blood urea nitrogen (BUN) concentrations and kidney weights, decreased organic ion accumulation by renal cortical slices, and induced proximal tubular necrosis. The characteristics of 3-NDHSA-induced nephrotoxicity were identical to NDPS-induced nephropathy, but were evident at lower doses with 3-NDHSA. These results demonstrate that 3-NDHSA is a nephrotoxicant which might contribute to NDPS-induced nephropathy.     

Species differences in blood profiles, metabolism and excretion of 14C-propofol after intravenous dosing to rat, dog and rabbit.

1. Bolus i.v. doses of 14C-propofol (7-10 mg/kg) to rat, dog and rabbit, or an infusion dose (0.47 mg/kg per min for 6 h) to dog were eliminated primarily in urine (60-95% dose); faecal elimination (13-31%) occurred for rat and dog, but was minimal (less than 2%) for rabbit. 2. After bolus administration, blood 14C concentrations were maximal (8-30 micrograms equiv./ml) at 2-15 min; these declined rapidly during the 0-2 h period and thereafter more slowly. Propofol concentrations were maximal (4-16 micrograms/ml) at 2 min and the profiles were best fitted by a tri-exponential (rat and dog) or bi-exponential (rabbit) equation. Duration of sleep ranged from 5 to 8 min. 3. Infusion of 14C-propofol in dog gave a blood 14C concentration of 117 micrograms equiv./ml at the end of the 6 h infusion period; this declined at a similar rate to that after the bolus dose. Propofol concentration on termination of infusion was 13 micrograms/ml; thereafter, propofol concentrations declined less rapidly than after the bolus dose. Waking occurred about 44 min post-infusion. 4. Propofol was cleared by conjugation of the parent molecule or its quinol metabolite; hydroxylation of an isopropyl group also occurred in rat and rabbit. Biliary excretion leading to enterohepatic recirculation, and in turn increased sulphate conjugation, occurred in rat and dog, but not rabbit, resulting in a marked interspecies variation in drug clearance and metabolite profiles.     

Metabolic and analytical interactions of grapefruit juice and 1,2-benzopyrone (coumarin) in man

Objective: Grapefruit juice is known to inhibit mammalian cytochrome P450 isozymes such as CYP3A4. The aim of this study was to investigate the influence of the juice on the fate of coumarin (1,2-benzopyrone) metabolized by CYP2A6 in man. Its potentially inhibitory effect was examined when low and high amounts of grapefruit juice were taken. Methods: In crossover studies, doses of 10 mg coumarin (Venalot) were given orally to a healthy male volunteer. The drug was taken either with water or with grapefruit juice, at different volumes (300 ml or 4 × 250 ml at intervals of 30 min). Urine samples were collected up to 24 h after dosing. After in vitro hydro-lysis they were analysed fluorimetrically for umbelliferone, the metabolite of coumarin, and cumulative excretion curves were established. HPLC and TLC served to identify fluorescent metabolites from the juice. Results: If coumarin is given in water its excretion is complete after 6 h and 70% of the dose is recovered. Grapefruit juice (300 ml) given simultaneously slightly retards the appearance of the fluorescent metabolite in the urine within the first few hours. The recovery of coumarin remains unaffected. One litre of juice enhances the delay and increases the recovery of coumarin to nearly 100%. Respective controls with grapefruit juice alone lead to remarkable excretions of a fluorescent material identified as conjugated scopoletin, which strongly interferes with the analysis of the coumarin experiment. The precursor of scopoletin is widely present at different concentrations in commercially available grapefruit juices. However, the autoinhibition of the juice is correlated neither to the concentration of naringin nor to that of scopoletin. Conclusion: Only grapefruit juice given at high doses (1 L) retards the appearance of the main metabolite of coumarin administered orally but increases its reco-very. Due to scopoletin formed from the grapefruit juice, experiments especially with coumarin are strongly affected. Received: 27 September 1995/Accepted in revised form: 5 December 1995     

Hydroxyethyl starch. An agent for hypovolaemic schock treatment II. Urinary excretion in normal volunteers following three consecutive daily infusions.

1 Urinary hydroxyethyl starch (HES) concentrations were determined by the anthrone method, in four healthy normal male volunteers following three consecutive daily 500 ml infusions (total 1500 ml), in order to ascertain excretion rates under normal controlled conditions. 2 The HES was excreted at a rate of 2.57, 2.46, and 2.44 g/h during the first hour postinjection, on days 1, 2, and 3, respectively. 3 The rate during the interval 12-24 h postinjection, averaged 0.14, 0.21, and 0.10 g/h, on days 1,2, and 3, respectively. 4 In the intervals 24-72, 72-120, and 120-168 h after the third and final injection, the excretion rate was 0.07, 0.04, and 0.02 g/h, respectively. 5 Renal function, as assessed by serum creatinine and uric acid concentrations and 24 h void volumes, was normal during the entire period of observation. 6 The results indicate that HES is excreted at a similar rate following three consecutive daily infusions without evidence of renal injury.     

Identification of glutathione conjugates and mercapturic acids of 1,2-dibromopropane in female BALB/c mice by liquid chromatography-electrospray ionization tandem mass spectrometry

Based on recent results that 1,2-dibromopropane (1,2-DBP) causes hepatotoxicity and immunotoxicity in female BALB/c mice as well as a reduction of hepatic glutathione levels, the possible formation of glutathione conjugates and mercapturic acids of 1,2-DBP was investigated in vivo in the present studies. The following four metabolites were identified in the liver at 12 h after treatment with 1,2-DBP, by liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI/MS): M1, 2-hydroxypropylglutathione; M2, 2-oxopropylglutathione; M3, N-acetyl-S-(2-hydroxypropyl)-L-cysteine; and M4, N-acetyl-S-(2-oxopropyl)-L-cysteine. Ions of individual conjugates were observed at m/z 366, 364, 222 and 220, respectively. Characteristic product ions at m/z 237, 217, 204 and 202 for the identification of M1, M2, M3 and M4 were observed, respectively. In the sera isolated from the same animals, only mercapturic acids (M3 and M4) were observed by LC-ESI/MS. When female BALB/c mice were treated orally with 1,2-DBP at doses of 150, 300 and 600 mg kg(-1) once for 12 h, the production of glutathione conjugates and mercapturic acids in liver was apparently dose dependent, as were the concentrations of them in sera. When the production of metabolites from 1,2-DBP was investigated in liver following oral treatment with 600 mg kg(-1) 1,2-DBP for 6, 12, 24 and 48 h, metabolite concentrations were greatest at the first time point (6 h). The results explain the authors' previous studies that oral treatment with 1,2-DBP reduces the hepatic content of glutathione.