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research-article |
RTI International,4 Research Triangle Park, North Carolina 27709
National Institute of Environmental Health Sciences,5 Research Triangle Park, North Carolina 27709
ABSTRACT
1-Bromopropane (1-BrP) induces dose- and time-dependent reproductive organ toxicity and reduced sperm motility in rodents. The contribution of cytochrome P4502E1 (CYP2E1) to both 1-BrP metabolism and the induction of male reproductive toxicity was investigated using wild-type (WT) and Cyp2e1–/– mice. In gas uptake inhalation studies, the elimination half-life of [1,2,3-13C]-1-BrP was longer in Cyp2e1–/– mice relative to WT (3.2 vs. 1.3 h). Urinary metabolites were identified by 13C nuclear magnetic resonance. The mercapturic acid of 1-bromo-2-hydroxypropane (2OHBrP) was the major urinary metabolite in WT mice, and products of conjugation of 1-BrP with glutathione (GSH) were insignificant. The ratio of GSH conjugation to 2-hydroxylation increased 5-fold in Cyp2e1–/– mice relative to WT. After 1-BrP exposure, hepatic GSH was decreased by 76% in WT mice vs. 47% in Cyp2e1–/– mice. Despite a 170% increase in 1-BrP exposure in Cyp2e1–/– vs. WT mice, sperm motility in exposed Cyp2e1–/– mice did not change relative to unexposed matched controls. This suggests that metabolites produced through CYP2E1-mediated oxidation may be responsible for 1-BrP-induced sperm toxicity. Both 1-BrP and 2OHBrP inhibited the motility of sperm obtained from WT mice in vitro. However, only 2OHBrP reduced the motility of sperm obtained from Cyp2e1–/– mice in vitro, suggesting that conversion of parent compound to 2OHBrP within the spermatozoa may contribute, at least in part, to reduced motility. Overall, these data suggest that metabolism of 1-BrP is mediated in part by CYP2E1, and activation of 1BrP via this enzyme may contribute to the male reproductive toxicity of this chemical.
epididymis, male reproductive tract, sperm, sperm motility and transport, toxicology
The solvent 1-bromopropane (n-propyl bromide [1-BrP]) is used in spray adhesives, fats, waxes, and resins, and as a synthetic intermediate. Annual production, which currently exceeds 1 million pounds in the United States, will likely increase following the adoption of 1-BrP as a replacement for halogenated ozone-depleting solvents [1]. The acute toxicity of 1-BrP is relatively low by single inhalation exposure. The 4-h LC50 (lethal concentration, 50%) is 7000 parts per million (ppm) in rats, and the oral/i.p. LD50 (lethal dose, 50%) exceeds 2 g/kg in rats and mice [2]. On repeat inhalation, 1-BrP was found to be both a male rodent reproductive toxicant and a neurotoxicant [2, 3]. In workers exposed to 1-BrP, reports of neurological findings [4] and DNA damage [5] have resulted in an increased interest in understanding the metabolic and cellular mechanisms of 1-BrP-induced toxicity.
The majority of 1-BrP toxicity work has been conducted in the rat. In a two-generation reproductive toxicity study [6], groups of rats were exposed to 1-BrP at 0, 100, 250, 500, or 750 ppm by inhalation for 6 h/day, 7 days/wk for at least 70 days prior to mating. In males, histological effects in the reproductive organs were minimal, and testicular sperm counts were not significantly altered by treatment with 1-BrP. Cauda epididymal sperm numbers were significantly reduced by 1-BrP at 750 ppm, and the percentage of motile sperm was significantly reduced at 500 (72% vs. 87% in controls) and 750 ppm (53% vs. 87% in controls). The F1 generation was exposed to 1-BrP for a similar period at 100, 250, and 500 ppm beginning on Postnatal Day 21, and male reproductive toxicity was assessed. Male reproductive organs were histologically normal, but the percentage of motile sperm was reduced in the F1 males in the 250 and 500 ppm groups.
Ichihara et al. [3] exposed male rats to 1-BrP at 0, 200, 400, or 800 ppm for 8 h/day for 12 wk and then examined the testicular effects, including sperm motility and morphology and testicular histology. Significant reductions in organ weights from exposed males were noted for seminal vesicles (
200 ppm) and epididymides (800 ppm). Histopathological changes were observed in the epididymides, prostate, and seminal vesicles of the 800 ppm group. Sperm quality also was affected, as observed by significant dose-related reductions in sperm count and motility. At 800 ppm, a significant increase in epididymal sperm with abnormal heads or no tails was observed. In these studies the mechanism of adverse effects on sperm motility was not elucidated.
Analysis of published sperm motility data across a number of studies suggested that the effect of 1-BrP on rat sperm motility is concentration dependent and apparently independent of duration of daily exposure [3, 6]. However, 12 wk of inhalation exposure to high levels of 1-BrP did cause structural changes in the spermatozoa [3].
In recently reported experiments from this laboratory we used 13C nuclear magnetic resonance (13C-NMR) techniques to identify urinary metabolites of rats and mice exposed to 1-BrP via inhalation [7]. Qualitatively, both rats and mice produce the same metabolites, which include: N-acetyl-S-propylcysteine, N-acetyl-3-(propylsulfinyl)alanine, N-acetyl-S-(2-hydroxypropyl)cysteine, 1-bromo-2-hydroxypropane-O-glucuronide, N-acetyl-S-(2-oxopropyl)cysteine, and N-acetyl-3-[(2-oxopropyl)sulfinyl]alanine. These metabolites are formed following oxidation of 1-BrP to 1-bromo-2-propanol and bromoacetone, and following subsequent glutathione (GSH) conjugation with either of these compounds. In rats the proportion of 1-BrP metabolized via oxidation relative to pathways dependent on direct GSH conjugation is inversely proportional to dose. In mice, however, oxidative metabolism is dose independent [7]. In B6C3F1 mice, the major 1-BrP metabolites found in urine were N-acetyl-S-propylcysteine, N-acetyl-S-(2-hydroxypropyl)cysteine, and 1-bromo-2-hydroxypropane-O-glucuronide [7].
Cytochromes P450 (CYPs) are the major oxidative enzymes system implicated in 1-BrP metabolism [7]. Recently, we reported that following intravenous or inhalation exposure to a mixture of 14C- and 13C-labeled 1-BrP to rats pretreated with the universal potent CYP inhibitor 1-aminobenzotriazole, the production of potentially reactive oxidative metabolites was nearly completely eliminated [7]. Parent 1-BrP and most of its oxidative metabolites appear to be readily conjugated with GSH, with subsequent loss of bromine [7, 8]. Exposure to 1-BrP has been shown to deplete GSH in rat brain [9] in a dose-dependent manner, independent of duration of exposure. Recently, Lee et al. [10] demonstrated that hepatic levels of GSH are lowered after single oral doses of 1-BrP to mice for up to 24 h after administration at doses over 150 mg/kg.
In the present studies we used Cyp2e1–/– and wild-type (WT) mice to determine the contribution of cytochrome P4502E1 (CYP2E1) to the kinetics of elimination of [1,2,3-13C]-1-BrP following inhalation exposure. The metabolite content of urine from these animals was determined using 13C-NMR techniques. Hepatic GSH levels also were determined in these same animals. Finally, experiments were conducted to assess the relationships between 1-BrP oxidative metabolism at doses known to cause male reproductive toxicity and inhibition of sperm motility.
The [1,2,3-13C]-1-BrP, >98% pure with 99% 13C enrichment, was obtained from Cambridge Isotope Laboratories (Andover, MA) and gave a 1H-decoupled 13C-NMR spectrum in deuterium oxide (D2O) consisting of a doublet at 12.3 ppm, a doublet of doublets at 25.9 ppm, and a doublet at 37.4 ppm. Multiplets associated with 2-BrP were negligible. Neat 1-BrP (99% pure) was purchased from Aldrich Chemical Company (Milwaukee, WI). The identities of test articles were confirmed by 1H-NMR and 13C-NMR and gas chromatography/mass spectrometry. Acetonitrile, acetone, methanol, sodium hydroxide, and water were purchased from Fisher Scientific (Fair Lawn, NJ). Saline (0.9%) was purchased from The Butler Company (Columbus, OH). Alkamuls EL-620/L was purchased from Rhodia (Cranbury, NJ). Trifluoroacetic acid was purchased from J. T. Baker (Phillipsburg, NJ). All other chemicals were purchased from Sigma-Aldrich (St. Louis, MO).
All animal experiments were conducted under protocols approved by the Institutional Animal Care and Use Committee of RTI International. Adult Cyp2e1–/– (KO) and WT mice were developed at the National Cancer Institute [11] and were rederived and bred at Charles River Inc. (Wilmington, MA). J1 embryonic stem cells generated from 129/Sv mice were used to generate the CYP2E1 mutant null mice [11]. Chimeric males were crossed with C57BL/6N females once to generate heterozygous mutant mice as the F1 hybrid [11]. Homozygous mutant mice for CYP2E1 were generated from an intercross of F1 mice, then maintained at Charles River Laboratories (Wilmington, MA) by intercrossing homozygous mice. WT littermates obtained from the F1 intercross also were maintained by intercrossing at Charles River Laboratories to serve as age-matched, strain-matched, WT mice. Animals (3–4 mo old) were individually housed in polycarbonate cages and supplied certified Purina Rodent Chow (5002) and tap water ad libitum. Room temperature was maintained at 17°C–26°C and relative humidity at 30%–70%. Light/dark cycle was kept at 12L:12D.
Mice (n = 4 per experiment) were exposed to [1,2,3-13C]-1-BrP at 800 ppm in a gas uptake chamber [7]. During exposure the concentration of 1-BrP within the chamber was monitored with a Hewlett Packard 5890 gas chromatograph equipped with a flame ionization detector (GC/FID) and a 1.8 m stainless steel GC column packed with 0.2% Carbowax 1500 on 80/100 Carbopack C (Supelco, Altoona, PA). The GC oven was maintained isothermally at 150°C. After a standard curve was prepared, a known amount of 1-BrP was introduced into the empty gas uptake chamber to achieve the desired initial exposure concentration. 1-BrP was allowed to equilibrate, and then its concentration was monitored for 1 h to estimate 1-BrP loss from chamber atmosphere. If the loss rate was below 4% per h, then the 1-BrP gas uptake inhalation experiment was performed. After determining the 1-BrP loss rate, the system was prepared to perform the animal gas uptake inhalation experiment. Approximately 110 g of lithium hydroxide and 4 g of citric acid were added to their respective traps. Four mice then were placed in the chamber and allowed to acclimate within the system for 30 min. After the acclimation period, sufficient [1,2,3-13C]-1-BrP was introduced into the gas uptake chamber to produce the desired atmospheric concentration of 800 ppm. The concentration of 1-BrP in the chamber atmosphere was then monitored for the subsequent 6 h. During the course of each experiment, chamber temperature, relative humidity, and oxygen levels also were monitored. Following the 6-h exposure, animals were killed using CO2/O2 mixture. Urine (collected from the chamber plus urinary bladder at termination) was collected for analysis by 13C-NMR spectroscopy. Livers were collected and frozen at –70°C for measurement of GSH content, and sperm were collected from cauda epididymes for motility analysis.
Animals were removed from the inhalation chamber and killed with CO2.
Seminal vesicles, testes, ventral prostate, and one epididymis, sectioned into initial segment, caput, corpus, and cauda segments, were collected and immediately frozen in liquid nitrogen. Sperm samples were collected from the distal cauda of the remaining epididymis and used for manual and computer-assisted sperm analysis (CASA). All of the above was accomplished within 5 min of removal from the exposure chamber. The epididymis was trimmed free of fat and clamped at the corpus-cauda junction, then severed on the corpus side of the clamp and blotted. While still clamped, the cauda epididymis was pierced with a scalpel blade to tease out approximately three tubules, and the sperm were allowed to diffuse into the medium without the epididymis touching the medium. Medium 199 modified with 0.5% BSA was used for the motility determinations. The sperm were allowed to disperse for approximately 5 min in 3 ml media in a covered Petri dish at 37°C. Then, 0.1 ml of this preparation was added to 1.0 ml medium 199 plus BSA in a vial that was maintained at 37°C. Sperm were placed on a flat microscope slide by capillary action.
For manual evaluations, a phase contrast microscope was used with a hemacytometer and a manual counter. Multiple sperm motility determinations were made over designated time periods. The sperm were diluted to a consistent concentration of approximately 10 x 106/ml with about 3 ml medium 199 + 0.5% BSA, placed in a capped test tube, and maintained at a constant temperature of 34°C. Motility was determined again at this point and was recorded as the initial motility. Motility readings were verified with an HTM-IVOS (Hamilton-Thorne Research, Beverly, MA) equipped with version 12.1c of the Toxicology software (Hamilton-Thorne Research). Microslides (100 µm) were used for motility determinations with IVOS settings of a frame rate of 60 Hz, minimum duration of tracking of 0.5 sec, and progressive minimum average-path velocity (VAP) of 50.0 µm/sec. Approximately 3000 cauda epididymal sperm were analyzed for each treatment group, and over 300 sperm were analyzed per animal. The percentages of motile and progressively motile sperm and the following kinematic parameters were determined by CASA: VAP, curvilinear velocity (VCL), straight-line velocity (VSL), amplitude of lateral head displacement, beat cross frequency, linearity, and straightness.
For in vitro experiments, caudal sperm from unexposed WT and Cyp2e1–/– mice were harvested, diluted, and prepared as described above. The sperm were diluted to a consistent concentration of approximately 10 x 106/ml with about 3 ml medium 199 + 0.5% BSA, placed in a capped test tube, and maintained at a constant temperature of 34°C. Incubations with either 1-BrP or 1-bromo-2-hydroxypropane (50 µM) were initiated by the addition of media containing the test article to the sperm preparations, and the caps were tightly sealed to prevent loss of test article. For each experiment, one tube of each type of sperm was left without added test chemical and served as the control. The sperm preparations then were held at the same temperature for 2 h, and the motility was determined using a phase contrast microscope with a hemacytometer and a manual counter. All determinations were made by the same experienced individual.
Hepatic levels of reduced GSH were measured after derivatization with O-phthalaldehyde using the method of Akerboom and Sies [12]. Assay mixtures contained 950 µl assay buffer (0.1 M potassium phosphate, pH 8.0, and 0.005 M EDTA), 10 µl of 9000-g supernatant, and 50 µl o-phthalaldehyde. Fluorescence intensity was measured at an excitation wavelength of 350 nm and an emission wavelength of 420 nm. A standard curve was prepared using authentic standards of reduced GSH.
All urine collected was centrifuged for 10 min at ~1000 x g. Aliquots of urine supernatant then were analyzed by one- and two-dimensional 13C-NMR as described previously [7].
Briefly, urine samples for NMR analysis were prepared by mixing 750 µl pooled urine and 250 µl D2O. 1H-decoupled 13C-NMR spectra were obtained at room temperature using a Varian Unity Inova 500-MHz NMR spectrometer equipped with a Varian 5-mm pulse field gradient (PFG) switchable probe for 13C-NMR. Typically, 20 000 transients were acquired with a 30° pulse width and a 1.5-sec relaxation delay. The spectra were acquired as 32 000 complex data points and were zero filled to 64 000, multiplied by a 1-Hz Lorentzian function and Fourier transformed to provide the spectrum. Chemical shift data were expressed in ppm and referenced to urea (162.5 ppm). An additional spectrum was acquired with a 30-sec relaxation delay and integrated to provide an estimate of the quantitative contribution of metabolites within the profile. Known volumes of mouse urine samples were fortified with known amounts of tetraethylammonium acetate standard and D2O. The samples then were subjected to proton-decoupled 13C-NMR analysis with nuclear overhouser effect suppression. The spectra of the fortified samples were integrated to determine absolute amounts of carbon from each major metabolite relative to the standard.
Spectra from the two-dimensional 13C Incredible Natural Abundance Double Quantum Transfer Experiment (INADEQUATE) were obtained using the standard Varian "inadqt" pulse sequence with a 7.5-sec interpulse delay. The experiment was optimized for 13C-13C coupling constants of 40 Hz. Predicted carbon shift values for proposed metabolites were determined with ACD/CNMR Predictor, version 7.03 (Advanced Chemistry Development Inc., Toronto, ON, Canada).
Concentrations of 1-BrP within the inhalation chamber were analyzed using noncompartmental models using WinNonlin Software (Pharsight Software, Palo Alto, CA).
In cases where a test of statistical significance was necessary, Student t-test was used [13], and values were considered statistically significant at P 
0.05.
Differential 1-BrP Exposure in WT vs. Cyp2e1–/–Mice
WT and Cyp2e1–/– mice were exposed to 13C-1-BrP (800 ppm) over 6-h exposure periods, and the profile of 13C-1-BrP concentrations inside the gas uptake chamber was determined (Fig. 1). Both WT and Cyp2e1–/– mice removed 13C-1-BrP from the chamber. In control experiments it was determined that 1-BrP losses due to adsorption onto chamber surfaces and skin and hair were negligible (data not shown), so the chamber elimination rates were representative of the rates of uptake and clearance of 1-BrP by the mice [14]. During the equilibration phase, the decline of 1-BrP from the chamber was similar between groups. However, after equilibrium (approximately 1 h), removal of 1-BrP from the chamber atmosphere was at a significantly slower rate for Cyp2e1–/– mice than for the corresponding WTs (Fig. 1 and Table 1). Kinetic parameters were calculated from chamber concentrations of 1-BrP (Table 1). The chamber elimination half-life of Cyp2e1–/– mice was ~3.2 h vs. ~1.3 h for WT mice. Cyp2e1–/– mice had a lower chamber clearance than the WT (~1.6 vs. ~2.7 ml/h) and a higher chamber area under the curve (AUC) (~11.6 vs. ~6.9 mg h–1 l–1). Chamber mean residence time for Cyp2e1–/– mice was ~4.1 h vs. ~1.6 h for WT mice.
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Differential Metabolism in WT vs. Cyp2e1–/– Mice
Urine samples collected from the animals in the inhalation kinetics/sperm motility experiments were analyzed to determine potential differences in 1-BrP urinary metabolites between WT and Cyp2e1–/– mice. Use of pooled, unconcentrated urine from mice in the 13C-NMR experiments allowed characterization of metabolites without loss of metabolites due to volatilization, adsorption, or chemical decomposition. NMR spectral data generated in these studies were compared to those previously published [7] to confirm final assignments.
The proton-decoupled 13C-NMR spectra of control, WT, and Cyp2e1–/– mouse urine collected following 6-h exposure to 800 ppm [1,2,3-13C]-1-BrP are shown in Figure 2. The carbons of endogenous compounds in urine gave rise to signals, which appear as singlets because of a low incidence of adjacent 13C nuclei. The spectrum of control urine (Fig. 2a) shows an intense singlet at 162.5 ppm (assigned to urea) and singlets of lower intensity due to sugars, hippurate, citrate, and creatine [15]. The 13C-NMR spectrum of urine from WT and Cyp2e1–/– mice exposed to [1,2,3-13C]-1-BrP (Fig. 2, b and c) contain multiplets due to 13C-13C coupling between the adjacent labeled carbons of each metabolite. Chemical shifts associated with the parent compound [1,2,3-13C]-1-BrP, 12.4 ppm (d), 25.9 ppm (d,d) and 37.4 ppm (d), were not detected.
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INADEQUATE spectroscopy was used to correlate carbon signals in the complex mixture, allowing determination of connectivity between carbon signals that arose from each [1,2,3-13C]-1-BrP-derived metabolite. Inspection of the INADEQUATE spectrum of WT mouse urine collected following inhalation exposure suggests at least two major three-carbon systems (Fig. 3), whereas that of Cyp2e1–/– mouse urine suggested at least three three-carbon systems (Fig. 4). This information and the chemical shifts, peak multiplicity, and carbon-carbon coupling constants (Jcc) from the one-dimensional (1D) spectra were used to assign structural types for metabolites derived from [1,2,3-13C]-1-BrP.
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N-acetyl-S-(2-hydroxypropyl)cysteine. A major three-carbon system in the INADEQUATE spectra of WT and Cyp2e1–/– mouse urine following [1,2,3-13C]-1-BrP inhalation exposure (Fig. 4) had carbon peaks aligned along the same double-quantum frequency (y-axis) between 39.8 ppm and 66.5 ppm and between 66.5 and 21.0 ppm. The doublets at 39.8 ppm (Jcc = 39 Hz) and 21.0 ppm with long-range coupling (Jcc = 39, 6.9 Hz) and the doublet of doublets at 66.5 ppm (Jcc = 39, 39 Hz) observed within the 1D (Fig. 3) spectra further support their connectivity. Based on comparison with a synthetic standard and previously published spectral data, the structure was definitively assigned as N-acetyl-S-(2-hydroxypropyl)cysteine [7]. Multiplets associated with N-acetyl-S-(2-hydroxypropyl)cysteine were the predominant signals in the urine of WT mice, but were less predominant in the urine of Cyp2e1–/–.
1-Bromo-2-propanol-glucuronide. The second three-carbon system had chemical shifts with connectivity between 36.5 ppm and 74.5 ppm and between 74.5 ppm and 19.2 ppm. The doublets at 36.5 ppm (Jcc = 41 Hz) and 19.2 ppm (Jcc = 40 Hz) and the doublet of doublets at 74.5 ppm (Jcc= 40, 40 Hz) observed within the 1D spectra further support their connectivity. These data are consistent with the spectral data of 1-bromo-2-propanol-glucuronide [7]. The intensity of multiplets associated with 1-bromo-2-propanol-glucuronide was significantly lower than the signals associated with N-acetyl-S-(2-hydroxypropyl)cysteine in the urine of WT and Cyp2e1–/– mice.
N-acetyl-S-propylcysteine. The third three-carbon system had chemical shifts with connectivity between 33 ppm and 22 ppm and between 22 ppm and 12 ppm. The 1D spectrum of urine for both WT and Cyp2e1–/– mice (Fig. 3, b and c) contained two doublets at 33.4 ppm (Jcc = 34 Hz) and 12 ppm (Jcc = 34 Hz) and a doublet of doublets at 22.9 ppm (Jcc = 34, 34 Hz). Based on comparison with a synthetic standard and spectral data reported previously, the structure was definitively assigned as N-acetyl-S-propylcysteine [7]. The intensity of these resonances is relatively minor in the spectra of urine from the WT mice and was significantly higher in the spectra of urine from the Cyp2e1–/– mice.
Inspection of the 1D spectra shows several small signals that are suggestive of additional 1-BrP metabolites but do not appear in the INADEQUATE spectra due to low signal-to-noise ratio. Inspection of the 1D spectra shows evidence of connectivity between doublets present at 53.2 ppm (Jcc = 34 Hz) and 12.0 ppm (Jcc = 35 Hz) and a doublet of doublets at 15.5 ppm (Jcc = 35, 34 Hz). Based on comparisons between the measured chemical shifts and those previously published [7] for plausible metabolites the structure was assigned as N-acetyl-3-(propylsulfinyl)alanine.
An additional three-carbon system was observed with doublets at 41.9 ppm (Jcc = 40 Hz) and 27.7 ppm (Jcc = 41 Hz) and a doublet of doublets at 209.3 ppm (Jcc = 39, 39 Hz) observed within the 1D spectra. Comparisons between the measured chemical shifts and those previously published [7] for plausible metabolites suggest a pattern consistent with the assignment of N-acetyl-S-(2-oxopropyl)cysteine.
Another three-carbon system is shown by inspection of the 1D spectra with doublets at 82.8 ppm (Jcc = 71 Hz) and 20.6 ppm (Jcc = 43 Hz) and a doublet of doublets at 151.5 ppm (Jcc = 72, 43 Hz). Comparisons between the measured chemical shifts and those previously published [7] support assignment of the structure as R-S(O)-CH=C(OH)CH3, the enol form of N-acetyl-3-[(2-oxopropyl)sulfinyl]alanine.
In the urine were detected several resonances with faint intensities but with sufficient detail to suggest structural characteristics of plausible 1-BrP metabolites.
A three-carbon system in 13C spectra generated from urine following inhalation exposure is a doublet of doublets at 74 ppm matched in intensity to a doublet at 35.3 ppm (Jcc = 37 Hz) and a doublet at 64.3 ppm (Jcc = 37 Hz). These chemical shifts are consistent with BrCH2CH(OH)CH2OH, which is supported by calculated chemical shifts of this structure, so these resonances are tentatively assigned to this structure.
Another three-carbon system was shown by inspection of the 1D spectra with doublets at 33.9 ppm and 176.6 (faint) ppm and a doublet of doublets at 66.3 ppm. Comparisons between the measured chemical shifts and those calculated for plausible metabolites support tentative assignment of the structure for bromolactic acid, Br-CH2C(OH)COOH.
A doublet of doublets at 180 ppm (Jcc = 51 Hz) with apparent long-range coupling (Jcc = 6 Hz) was observed. The chemical shift and coupling is consistent with a sp3-sp2-sp2 system containing a carboxylic acid adjacent to a carbonyl within a three-carbon system. This suggests the structure XCH2C(O)COOH, where X = Br, or another group such as a cysteine, cysteine-glycine, or N-acetylcysteine.
Quantitation of Metabolites in Urine
The spectra of the urine samples were integrated to determine absolute amounts of carbon from each major metabolite. The total moles of 1-BrP-derived, 13C-containing metabolites recovered in urine of WT and Cyp2e1–/– mice were equal (~0.05 mmol per 500 µl; Table 2). However, in the urine of 1-BrP-exposed WT mice, the molar ratio of 2-hydroxylated metabolites (N-acetyl-S-(2-hydroxypropyl)cysteine and 1-bromo-2-propanol-glucuronide) relative to N-acetyl-S-propylcysteine is approximately 5-fold (Table 2). When mice lacking CYP2E1 were exposed to 1-BrP, that ratio was essentially 1. The levels of the 2-hydroxylated metabolites were reduced approximately 60% in Cyp2e1–/– mice, whereas the directly conjugated parent metabolite, N-acetyl-S-propylcysteine, increased 190% relative to that measured in the corresponding WT mouse urine. Signals from other metabolites in urine were sufficiently weak such that accurate quantitation was not possible.
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Quantitation of Hepatic GSH in WT vs. Cyp2e1–/– Mice
Levels of reduced GSH were determined in the livers of WT and Cyp2e1–/– mice following a 6-h inhalation exposure to 1-BrP at 800 ppm (n = 3 to 4). There was significant reduction of GSH in the livers of Cyp2e1–/– and WT mice exposed to 1-BrP (Table 3). In WT mice, hepatic GSH levels were lowered by 76% relative to matched unexposed controls (P < 0.005). In Cyp2e1–/– mice, hepatic levels of GSH were lowered by 47% (P < 0.05) relative to matched unexposed controls.
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Sperm Motility in WT vs. Cyp2e1–/– Mice
The motility of caudal spermatozoa was determined in the WT and Cyp2e1–/– mice (n = 8 per genotype) following a 6-h exposure to 1-BrP at 800 ppm. At this exposure WT mice exhibited a 37% reduction in the percentage of motile sperm relative to matched unexposed controls (40% from 63%, P < 0.05; Fig. 5). On the other hand, Cyp2e1–/– mice exposed to similar concentrations of 1-BrP exhibited a slight but insignificant decline in sperm motility (12%) relative to their matched unexposed controls (48% from 57%). Ex vivo, sperm from unexposed WT mice showed a time-dependent decrease in motility when exposed to either 1-BrP (39% from 73%, P < 0.05) or 1-bromo-2-hydroxypropane for 2 h (26% from 73%, P < 0.05; Fig. 6) at 50 µM. Interestingly, motility of sperm from Cyp2e1–/– mice was not significantly reduced by 1-BrP (43% from 57%), but sperm showed a time-dependent decrease in motility when exposed to 1-bromo-2-hydroxypropane (23% from 57%, P < 0.05).
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Early studies demonstrated that 1-BrP causes a decrease in epididymal motile sperm, increased abnormal sperm morphology, and a dose-dependent increase in the number of sperm retained in postspermiation stages [3]. Further, metabolism studies suggested that oxidation via CYPs is the primary pathway of 1-BrP metabolism [7, 8]. However, the specific isozyme responsible for 1-BrP oxidation remains unidentified. Current investigations were undertaken to characterize the enzymatic basis if 1-BrP induced reproductive male toxicity using Cyp2e1–/– and WT mice.
Present studies demonstrated that CYP2E1 plays a significant role in the uptake and eliminations kinetics of [13C]-1-BrP in mice exposed at 800 ppm via inhalation, a concentration that had been shown to have significant impact on sperm motility in rats [3]. These experiments comprehensively linked pharmacokinetic and metabolism data with a toxicological effect within the same transgenic animals. Using 13C-NMR techniques, we demonstrated a marked reduction in CYP2E1 oxidative metabolites in the urine of animals used in the gas uptake studies. Finally, in the same animals we demonstrated that a reduction in sperm motility was observed in the WT mice but not in the Cyp2e1–/– mice exposed to 1-BrP, suggesting that the reduction in sperm motility was related to CYP2E1 oxidative metabolites. In vitro sperm incubation experiments showed that both parent 1-BrP and its hydroxylated metabolite, 1-bromo-2-hydroxypropane, caused a time-dependent decrease in motility of sperm isolated from WT mice. However, in the absence of CYP2E1 in the KO animals the effect of parent 1-BrP was not detected, but there was no change on the effect of its metabolite, 1-bromo-2-hydroxypropane. It must be noted that nonsignificant changes in the KOs might approach significance with larger sample sizes.
Earlier work from this laboratory has shown that administration of 1-aminobenzotriazole, a potent general suicide inhibitor of P450, caused a nearly complete elimination of 1-BrP oxidative metabolism and a compensatory shift toward GSH conjugation of 1-BrP in rats [7]. In these experiments the absence of CYP2E1 resulted in reduced systemic clearance of 1-BrP by the Cyp2e1–/– mice, and therefore higher levels remained present in the chambers. Thus, the Cyp2e1–/– mice were exposed to significantly higher levels of the parent compound 1-BrP and concomitantly lower levels of oxidative metabolites. Although CYP2E1 is not the only oxidative enzyme contributing to 1-BrP clearance, it is a predominant isoform.
In Cyp2e1–/– mice, the urinary concentration of the major 1-bromo-2-hydroxypropane-derived metabolite (N-acetyl-S-(2-hydroxypropyl)cysteine was reduced approximately 50% relative to WT mice. 13C-NMR data suggest that the ratio of the products of direct conjugation of 1-BrP with GSH to oxidative 2-hydroxylation increased 5-fold in Cyp2e1–/– relative to WT mice. A similar compensatory shift from oxidative metabolism toward direct GSH conjugation was seen in our earlier work in rats when general CYP inhibitors were administered [7]. This suggests that CYP2E1 contributes significantly to the production of the major oxidative metabolites of 1-BrP. However, production of these metabolites is not entirely CYP2E1 mediated, since the KO mice did not exhibit a complete blockade of the formation of hydroxylated 1-BrP metabolites. Other CYPs also may contribute to the 2-hydroxylation of 1-BrP. The presence of the glucuronide conjugate of 1-bromo-2-propanol in urine and its decrease (approximately 50%) in the urine of Cyp2e1–/– mice suggest that this metabolite is produced directly by oxidation of the parent molecule and that CYP2E1 contributes significantly to its production.
Reduced GSH levels were lowered by 76% in the livers of the WT animals following 6-h inhalation exposure to 1-BrP. These data agree with recent data from Lee et al. [15], which demonstrated that hepatic levels of GSH are lowered by approximately 80% following single oral doses of 1-BrP to mice for up to 24 h after administration at doses of 150-1000 mg/kg. Levels of GSH were lowered in the livers of Cyp2e1–/– mice exposed to 1-BrP via inhalation, but not to the same extent (47% vs. 76%). This suggests that reactive products originating via CYP2E1-mediated oxidation of 1-BrP may contribute to the depletion of GSH following exposure to this chemical. GSH depletion may impact sperm motility by increasing lipid peroxidation products [16, 17]. However, the mechanism of 1-BrP-mediated sperm motility inhibition must be dependent on more than just GSH depletion, since GSH is depleted at molar doses significantly below those that result in reduced sperm motility.
At 800 ppm 1-BrP exposure for 6 h, sperm motility in WT mice was significantly reduced. This suggests that a single high exposure can result in a measurable change in sperm motility in rodents, and this implies direct affects on mature sperm (vs. effects mediated by the testis). Despite a 170% increase in the AUC of parent 1-BrP in the chamber (and, presumably, systemic exposure), sperm motility in Cyp2e1–/– mice did not significantly decrease relative to matched controls. This suggests that the parent compound itself may not directly mediate 1BrP-induced reduction in sperm motility, but that a CYP2E1-mediated oxidation metabolite may be critical to 1-BrP-mediated decreases in sperm motility in vivo. This hypothesis is supported by our concomitant measurement of a 38% reduction in concentration of N-acetyl-S-2-hydroxypropyl-cysteine and a 64% reduction in 2-hydroxy-1-bromopropane-O-glucuronide in the urine of the Cyp2e1–/– relative to WT mice. The epididymides of mice are known to express CYP2E1 [18, 19], and other small halogenated molecules, such as trichloroethylene [20] and halothane [21], have been shown to be activated by this enzyme in the epididymis, resulting in reproductive effects.
In vitro experiments were conducted in which spermatozoa isolated from WT and Cyp2e1–/– mice were incubated with either 1-BrP or the major mouse and rat metabolite 1-bromo-2-hydroxypropane. Sperm of WT mice exhibited a statistically significant inhibition of motility as a result of exposure to either compound. However, while sperm isolated from unexposed Cyp2e1–/– mice exhibited 1-bromo-2-hydroxypropane-mediated decrease in motility, these sperm were not sensitive to parent 1-BrP. The blockade of 1-BrP-mediated decrease of sperm motility both ex vivo and in vitro in Cyp2e1–/– mice suggests that CYP2E1 within the spermatozoa mediates the conversion of 1-BrP to 1-bromo-2-hydroxypropane, and this metabolite may be the ultimate sperm toxicant.
The sperm of several species have been shown to metabolize endogenous compounds, such as steroids [22, 23], and xenobiotics, such as 1,6-dichloro-1,6-dideoxy-D-fructose [24]. Functional CYP19 [23, 25] and CYP51 [22] expression in ejaculated sperm has been reported recently. Cotman et al. [22] further demonstrated expression and activity of NADPH CYP reductases within the acrosome of mouse, bull, and ram sperm. Although expression of CYP2E1 in spermatozoa has not been published in the literature, spermatozoa have been demonstrated to oxidize short-chain halogenated alcohols and ketones. The cauda epididymal sperm isolated from mature boars converted 3-bromo-1-hydroxypropanone (BOP) to (S)-3-bromolactaldehyde [26], which is an inhibitor of glyceraldehyde 3-phosphate dehydrogenase. Boar spermatozoa converted (S)-alpha-chlorohydrin and 3-chloro-1-hydroxyacetone to (S)-3-chlorolactaldehyde, which inhibited glycoloysis, and therefore sperm motility [27, 28].
We believe that reduction of sperm motility by 1-BrP may be mediated, at least in part, by disruption of energetic pathways by metabolites derived from the oxidative metabolite 1-bromo-2-hydroxypropane, which originates from its metabolism via CYPs in the rat and mouse (Fig. 7). This report and recent reports from this laboratory [7] have demonstrated that this metabolite undergoes subsequent CYP-mediated oxidation to bromoacetone and 
-bromohydrin, both precursors to metabolic intermediate analogs. Extensive work has demonstrated that -bromohydrin and its chlorinated analog -chlorohydrin are converted in situ by spermatozoa into halolactates, which have been shown to be metabolic inhibitors causing reduced motility in spermatozoa [27–31]. Data from these studies suggest the presence of bromolactate in urine following inhalation exposure. Metabolism of bromoacetone in a manner analogous to acetone [32] would yield either 1-hydroxy-1-bromoacetone, which ultimately yields pyruvate and CO2, or BOP. BOP has been shown to inhibit sperm energetics and motility by conversion to bromolactaldehyde and bromopyruvaldehyde, ultimately yielding bromopyruvate, a metabolic poison [26, 33]. Thus, we suggest that further metabolism of known 1-BrP metabolites to metabolic inhibitors, both in the epididymis and, speculatively, within the spermatozoa themselves, may be the mechanism of 1-BrP-mediated reduction in sperm motility.
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In conclusion, these studies have demonstrated that CYP2E1 plays a significant role in the uptake and elimination kinetics and metabolism of 13C-1-BrP in mice following inhalation exposure. Further, we demonstrated that significant reduction in sperm motility was observed in WT mice but not in Cyp2e1–/– mice following inhalation exposure to 1-BrP, suggesting that the reduction in sperm motility was caused by products of CYP2E1-mediated oxidation of 1-BrP. In vitro sperm incubation experiments showed that both parent 1-BrP and its hydroxylated metabolite, 1-bromo-2-hydroxypropane, caused a time-dependent decrease in motility of sperm isolated from WT mice, whereas only the hydroxylated metabolite caused a decreased motility in sperm from Cyp2e1–/– mice. Collectively, these data suggest that CYP2E1-mediated oxidation of 1-BrP, presumably to 1-bromo-2-hydroxypropane, is a prerequisite for the sperm toxicity of this chemical in male rodents.
ACKNOWLEDGMENTS
The authors would like to express thanks to Ms. Vicki McCall for her help in the preparation of this manuscript.
FOOTNOTES
3Current address: Drug Metabolism and Pharmacokinetics, Cancer and Infection, Discovery Research, AstraZeneca, 35 Gatehouse Rd., Waltham, MA 02451. 
1This research was supported by the Intramural Research Program of the National Institutes of Health/National Institute of Environmental Health Sciences.
Correspondence: 2C. Edwin Garner, Department of Drug Metabolism and Pharmacokinetics, RTI International, Research Triangle Park, NC 27709. FAX: 781 839 4380; e-mail: cegarner{at}rti.org
Received: 23 June 2006.
First decision: 27 August 2006.
Accepted: 23 October 2006.
REFERENCES
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