Evaluation of a procedure for the simultaneous quantification of 4-ketocyclophosphamide, cyclophosphamide, and Ifosfamide in human urine.

An accurate and precise analysis procedure is presented for the detection and quantification of cyclophosphamide (CP), 4-ketocyclophosphamide (4-keto-CP), a primary metabolite of CP, and ifosfamide (IF) in human urine. CP and IF are common antineoplastic drugs used for the treatment of many types of cancer. Workers in the healthcare field, including nurses and pharmacists who interact with or prepare prescriptions for patients, have potential low-level exposure to the parent drugs; therefore, an analysis procedure is needed. The main focus of this procedure is the quantitation of 4-keto-CP because it is a primary metabolite of CP exposure and stable under physiological conditions. Sample preparation consists of liquid-liquid extraction of urine with ethyl acetate, and the analysis consists of reversed-phase high-performance liquid chromatography coupled with tandem mass spectrometry for detection of the analytes. Accuracy and precision of this procedure is demonstrated by means of recovery experiments. Recoveries are between 97-105% of theory for the three target analytes at various concentrations (25, 50, 100, and 375 ng/mL for 4-keto-CP; 1, 2, 4, and 15 ng/mL for CP and IF) with relative standard deviations of 8.4% or less. The limit of detection for this procedure is 1 ng/mL for 4-keto-CP, 0.1 ng/mL for CP, and 0.05 ng/mL for IF in urine.

Development of an HPLC-MS procedure for the quantification of N-acetyl-S-(n-propyl)-l-cysteine, the major urinary metabolite of 1-bromopropane in human urine.

An analytical procedure was developed for the detection and quantification of N-acetyl-S-(n-propyl)-l-cysteine (n-propylmercapturic acid, AcPrCys), a metabolite and biomarker for exposure to 1-bromopropane (1-BP). 1-BP is used as an industrial solvent and exposure is a health concern for industrial workers due to its toxicity. It has been associated with neurological disorders in both animals and humans. Urine sample preparation for the determination of AcPrCys consisted of solid phase extraction (SPE). Urine samples on preconditioned SPE (C18) columns were washed with 40% methanol/60% water solution prior to elution with acetone. Quantification was by means of a liquid chromatograph (LC) equipped with a mass spectrometer (MS) using an Aqua 3 microm C18 300A column and [d(7)]-AcPrCys was used as internal standard. Electrospray ionization (ESI) was used with the MS operated in the negative ion mode and selected ion monitoring (SIM) at m/z 204 for AcPrCys and m/z 211 for [d(7)]-AcPrCys. Demonstrated recovery of urine samples fortified at multiple levels (0.625-10 microg/ml) varied between 96 and 103% of theory with relative standard deviations (RSD) of 6.4% or less. The limit of detection (LOD) for the procedure was approximately 0.01 microg/ml AcPrCys in urine. These data will be discussed as well as other factors of the development of this test procedure.

Development of a headspace gas chromatographic test for the quantification of 1- and 2-bromopropane in human urine.

A test procedure was developed for the detection and quantification of 1- and 2-bromopropane in human urine. 1-Bromopropane (1-BP) is a commonly used industrial solvent, and 2-bromopropane (2-BP) is often found as an impurity component in industrial grade 1-BP. Both compounds are a health concern for exposed workers due to their chronic toxicity. Bromopropanes have been associated with neurological disorders in both animals and humans. Sample preparation consisted of diluting urine with water and fortification with 1-bromobutane (1-BB), which was used as an internal standard; then each sample was sealed in a headspace vial. A static-headspace sampler (Teledyne-Tekmar Model 7000) was used to heat each sample at 75 degrees C for a 35-min equilibrium time. Quantification was by means of a gas chromatograph (GC) equipped with an electron capture detector (ECD) and a dimethylpolysiloxane (DB-1) capillary column. A recovery study using fortified urine samples at multiple concentrations (0.5-8 microg/ml) demonstrated full recovery; 104-121% recovery was obtained. Precision ranged from 5 to 17% for the 15-20 spiked samples used at each concentration, which were analyzed over multiple experimental trial days. The limit of detection (LOD) for this test procedure was approximately 2 ng/ml 1-BP and 7 ng/ml 2-BP in urine. A recovery study of 1- and 2-BP from fortified urine stored in vials appropriate for field collection was also completed. These results and other factors of the development and validation of this test procedure will be discussed.

Development of a gas chromatographic test for the quantification of the biomarker 3-bromopropionic acid in human urine.

An accurate and precise method was developed for the detection and quantification of 3-bromopropionic acid (3-BPA), a metabolite and biomarker for exposure to 1-bromopropane (1-BP). 1-BP is used as an industrial solvent and exposure is a health concern for industrial workers due to its toxicity. It has been associated with neurological disorders in both animals and humans. Urine sample preparation for the determination of 3-BPA consisted of liquid-liquid extraction (LLE) with ethyl acetate and silylation with N-methyl-N-[tert-butyldimethylsilyl]trifluoroacetamide (MTBSTFA). Quantification was by means of a gas chromatograph (GC) equipped with a mass selective detector (MSD) using a dimethylpolysiloxane (HP-1) capillary column and 3-chloropropionic acid was used as an internal standard in the procedure. Demonstrated accuracy and precision during this method's validation was good; recovery varied between 93 and 98% with relative standard deviations (R.D.S.) of 5.7% or less. The limit of detection (LOD) for the procedure was approximately 0.01microg/ml 3-BPA in urine. These data and other factors of the development and validation of this test method will be discussed.

Procedure for the quantification of the biomarker (2-methoxyethoxy)acetic acid in human urine samples.

An accurate and precise procedure was developed for the detection and quantification of (2-methoxyethoxy)acetic acid (MEAA), a metabolite and biomarker for human exposure to 2-(2-methoxyethoxy)ethanol. The compound 2-(2-methoxyethoxy)ethanol has a wide array of industrial applications including its use as an additive in military jet fuel. Exposure to 2-(2-methoxyethoxy)ethanol is a health concern owing to its toxicity which includes developmental and teratogenic properties. Sample preparation consisted of liquid-liquid extraction (LLE) and esterification of MEAA to produce the ethyl ester. Measurement was by a gas chromatograph (GC) equipped with a mass selective detector (MSD) using a HP-1 capillary column. Recovery studies of spiked blank urine demonstrated good accuracy and precision; recovery varied between 95 and 103% with relative standard deviations of 8.6% and less. The limit of detection (LOD) for this procedure was found to range from 0.02 to 0.08 microg/ml equivalent levels of MEAA in urine. These data and other aspects of the validation of this procedure will be discussed.

DNA strand breaks, oxidative damage, and 1-OH pyrene in roofers with coal-tar pitch dust and/or asphalt fume exposure.

OBJECTIVE: To determine the potential for asphalt fume exposure to increase DNA damage, we conducted a cross-sectional study of roofers involved in the application of roofing asphalt. METHODS: DNA strand breaks and the ratio of 8-hydroxydeoxyguanosine (8-OHdG) to 2-deoxyguanosine (dG) were measured in peripheral blood leukocytes of roofers. In addition, urinary excretion of 8-OHdG and 8-epi-prostaglandin F2alpha (8-epi-PGF) was also measured. The study population consisted of 26 roofers exposed to roofing asphalt and 15 construction workers not exposed to asphalt during the past 5 years. A subset of asphalt roofers (n = 19) was exposed to coal-tar pitch dust (coal tar) during removal of existing roofs prior to applying hot asphalt. Personal air monitoring was performed for one work-week to measure exposure to total particulates, benzene-soluble fraction of total particulates, and polycyclic aromatic compounds (PACs). Urinary 1-OH-pyrene levels were measured as an internal biomarker of PAC exposure. RESULTS: Full-shift breathing zone measurements for total particulates, benzene-solubles and PACs were significantly higher for coal-tar exposed workers than for roofers not exposed to coal tar. Similarly, urinary 1-OH-pyrene levels were higher in coal-tar exposed roofers than roofers not exposed to coal tar. Total particulates or benzene-soluble fractions were not associated with urinary 1-OH-pyrene, but PAC exposure was highly correlated with urinary 1-OH-pyrene. When stratified by 1-OH-pyrene excretion, DNA strand breaks increased in a dose-dependent manner, and leukocyte 8-OHdG/dG decreased in a dose-dependent manner. Significant changes in DNA damage appeared to be linked to PACs from coal-tar exposure, although asphalt fume alone was associated with a small but significant increase in urinary 1-OH-pyrene and DNA strand breaks. CONCLUSIONS: Results are consistent with previous reports that asphalt or coal-tar exposure can cause DNA damage. Urinary 8-epi-PGF remained relatively constant during the week for virtually all subjects, regardless of exposure indicating that neither asphalt nor coal-tar exposure induces an overt oxidative stress. A small, but statistically significant increase in 8OHdG was evident in end-of-week urine samples compared with start-of-week urine samples in roofers exposed to coal-tar. The increase in urinary 8OHdG coupled with the decrease in leukocyte 8-OHdG/dG, suggests that coal-tar exposure induces protective or repair mechanisms that result in reduced levels of steady-state oxidative-DNA damage.

2-Methoxyethanol metabolism, embryonic distribution, and macromolecular adduct formation in the rat: the effect of radiofrequency radiation-induced hyperthermia.

Exposure of pregnant rats to the solvent 2-methoxyethanol (2ME) and radiofrequency (RF) radiation results in greater than additive fetal malformations (Nelson, B.K., Conover, D.L., Brightwell, W.S., Shaw, P.B., Werren, D.W., Edwards, R.M., Lary, J.M., 1991. Marked increase in the teratogenicity of the combined administration of the industrial solvent 2-methoxyethanol and radiofrequency radiation in rats. Teratology 43, 621-34; Nelson, B.K., Conover, D.L., Shaw, P.B., Werren, D.W., Edwards, R.M., Hoberman, A.M., 1994. Interactive developmental toxicity of radiofrequency radiation and 2-methoxyethanol in rats. Teratology 50, 275-93). The current study evaluated the metabolism of 14C-labeled 2ME and the distribution of methoxyacetic acid (MAA) in maternal and embryonic tissues of pregnant Sprague-Dawley rats either exposed to 10 MHz RF radiation or sham conditions. Additionally, adduct formation for both plasma and embryonic protein was tested as a possible biomarker for the observed 2ME/RF teratogenicity. Rats were administered [ethanol-1,2-(14)C]-2ME (150 mg/kg, 161 microCi/rat average) by gavage on gestation day 13 immediately before RF radiation sufficient to elevate body temperature to 42 degrees C for 30 min. Concurrent sham- and RF-exposed rats were sacrificed at 3, 6, 24 or 48 h for harvest of maternal blood, urine, embryos and extra-embryonic fluid. Tissues were either digested for determination of radioactivity or deproteinized with TCA and analyzed by HPLC for quantification of 2ME metabolites. Results show the presence of 2ME and seven metabolites, with the major metabolite, MAA, peaking at 6 h in the tissues tested. MAA, the proximal teratogen, was detectable in maternal serum, urine, embryo and extraembryonic fluid 48 h after dosing. Clearance of total body 14C was significantly reduced for the RF-exposed animals (P<0.05) for the 24-48 h period, but MAA values for serum, embryos and extraembryonic fluid were similar for both sham- and RF-exposed rats. Additionally, no difference was noted for 2ME metabolite profiles in urine or tissue for sham- or RF-exposed rats, thus eliminating an effect of RF radiation on MAA production as a possible explanation for the reported RF-2ME synergism. Subsequently, serum and embryo protein-bound adducts were evaluated by analysis of covalently bound radioactivity. Serum protein binding was significantly higher for sham than RF rats at 3- and 6-h - highest for sham rats at 6 h (519+/-95 microg as parent 2ME/g of protein) whereas RF serum values were highest at 24 h (266+/-79 microg/g protein). Embryonic protein binding was significantly higher for sham rats at 6 h, but binding was highest for both groups at 24 h (sham=229+/-71 microg/g, RF=185+/-48 microg/g). Formation of protein adducts after 2ME is thought to be related to levels of methoxyacetaldehyde, a reactive intermediate in the formation of MAA. These results suggest that no direct relationship exists for covalent binding in the embryo which would explain RF-2ME synergistic malformations. In comparison with urinary metabolites, the relatively slow elimination of adducted serum 2ME indicates that analysis of protein-bound concentrations could be a potential tool for long- term biomonitoring of worker exposure.

Male adolescent exposure to endocrine-disrupting pesticides: vinclozolin exposure in peripubertal rabbits.

Adolescence is a time of dramatic neuroendocrine changes that are required for sexual maturation. Hormonal mimicking or inhibiting chemicals can cause significant impairment during this critical period. Vinclozolin (Vin) has been shown to be an anti-androgen affecting male offspring in rats in utero, and its mechanism of action may be mediated by inhibition of androgenic receptor action. The majority of teenagers working on farms are male, and therefore a systemic fungicide, vinclozolin, was selected for study. The rabbit has proved to be an excellent species for modelling reproductive toxicant effects in the male and was selected as the test species. The peripubertal phase for the rabbit was determined to be between the 3rd and 4th months. A 2-month dosing period was therefore initiated at 3 months of age and carried through to the 4th month. Vin was administered by dermal application (100 mg kg(-1) in 100 microl of dimethylsulphoxide) daily. Body weights were determined weekly. The rabbits were then held until fully mature (6 months of age). Semen was collected and evaluated from sexually mature males on a weekly schedule for 5 weeks to maximize sperm output. An automated solid phase extraction procedure for monitoring exposures through isolation and quantification of Vin and its metabolic products was developed. Increased plasma levels of Vin and M2 were found throughout the experimental period. The exposed rabbits had a smaller weight gain during pubertal growth (approaching significance; P=0.059). At maturity, the accessory sex glands of the exposed animals weighed less than those of the controls (P=0.016). Surprisingly, the pooled sperm count of the exposed animals was significantly higher (P=0.017) than that of the unexposed animals. The anti-androgenic effects of Vin may have blocked the negative feedback mechanism of testosterone on the hypothalamus or pituitary gland, allowing for an increase in gonadotrophin release, and consequently increasing sperm production at puberty.

Mutagenicity of N-OH-MOCA (4-amino-4'-hydroxylamino-bis-3,3'-dichlorodiphenylmethane) and PBQ (2-phenyl-1,4-benzoquinone) in human lymphoblastoid cells.

The genotoxic potential of two occupationally significant chemicals, 4,4'-methylene-bis-2-chloroaniline (MOCA) and 2-phenyl-1,4-benzoquinone (PBQ), was explored by monitoring the induction of mutations at the HPRT locus of AHH-1 human lymphoblastoid cells. Exposure of AHH-1 cells to the putative carcinogenic metabolite of MOCA, N-OH-MOCA, induced a 6-fold increase in mutant frequency and resulted in base pair substitutions primarily at A:T base pairs. In contrast, exposure to PBQ did not result in an increased mutant frequency although this compound was significantly more cytotoxic than N-OH-MOCA at equimolar doses. The induction of mutations at A:T sites by N-OH-MOCA is consistent with the type of DNA damage known to be produced by MOCA and provides a specific marker of genotoxic damage for exposed populations.

Neoplastic transformation and DNA-binding of 4,4'-methylenebis(2-chloroaniline) in SV40-immortalized human uroepithelial cell lines.

The tumorigenic transformation of certain occupationally significant chemicals, such as N-hydroxy-4-4'-methylenebis[2-chloroaniline] (N-OH-MOCA), N-hydroxy-ortho-toluidine (N-OH-OT), 2-phenyl-1,4-benzoquinone (PBQ) and N-hydroxy-4-aminobiphenyl (N-OH-ABP) were tested in vitro using the well established SV40-immortalized human uroepithelial cell line SV-HUC.PC. SV-HUC cells were exposed in vitro to varying concentrations of N-OH-MOCA, N-OH-OT, N-OH-ABP and PBQ that caused approximately 25% and 75% cytotoxicity. The carcinogen treated cells were propagated in culture for about six weeks and subsequently injected subcutaneously into athymic nude mice. Two of the fourteen different groups of SV-HUC.PC treated with different concentrations of N-OH-MOCA, and one of the three groups exposed to N-OH-ABP, formed carcinomas in athymic nude mice. 32P-postlabeling analyses of DNA isolated from SV-HUC.PC after exposure to N-OH-MOCA revealed one major and one minor adduct. The major adduct has been identified as the N-(deoxyadenosin-3',5'-bisphospho-8-yl)-4-amino-3-chlorob enz yl alcohol (pdAp-ACBA) and the minor adduct as N-(deoxyadenosin-3',5'-bisphospho-8-yl)-4-amino-3-chlorot oluene (pdApACT). Furthermore, SV-HUC.PC cytosols catalyzed the binding of N-OH-MOCA to DNA, in the presence of acetyl-CoA, to yield similar adducts. The same adducts were also formed by chemical interaction of N-OH-MOCA with calf thymus DNA, suggesting that the aryl nitrenium ion may be the ultimate reactive species responsible for DNA binding. The tumorigenic activity of N-OH-MOCA in this highly relevant in vitro transformation model, coupled with the findings that SV-HUC.PC cells formed DNA-adducts in vitro and contained enzyme systems that activated N-OH-MOCA to reactive electrophilic species that bound to DNA, strongly suggest that MOCA could be a human bladder carcinogen. These findings are consistent with the International Agency for Research on Cancer's classification of MOCA as a probable human carcinogen.

Determination of 4,4'-methylene-bis(2-chloroaniline)-DNA adduct formation in rat liver and human uroepithelial cells by the 32P postlabeling assay.

The probable human carcinogen 4,4'-methylene-bis(2-chloroaniline) (MOCA) was utilized to develop biomarkers of exposure to occupational carcinogens. The 32P postlabeling assay, utilizing the nuclease P1 enhancement procedure, was used to evaluate MOCA-DNA adduct formation in target tissues. Male Sprague-Dawley rats were treated with different dosing regimens of MOCA, and DNA was isolated from the liver. Additionally, a human uroepithelial cell (HUC) line was treated with N-hydroxy-MOCA for 24 hr, cells were harvested, and DNA was isolated. DNA was analyzed for MOCA-DNA adduct formation by the 32P postlabeling assay. Five MOCA adducts were detected in rat liver DNA. Adduct A, which corresponded to N-(deoxyadenosin-8-yl)-4-amino-3-chlorobenzyl alcohol, was the major adduct in rat liver DNA appearing in all treatment groups. Levels of adduct A were higher when MOCA was administered by ip injection versus oral gavage. Phenobarbital pretreatment increased the amount of adduct A approximately 12-fold. The pathway leading to the formation of adduct A in DNA from HUC appeared to be saturated at the concentrations used: 2.5, 5, and 10 microM. However, an additional adduct (E) was observed at the 10 microM treatment level only. A major DNA adduct was detected in the target tissue of rats and target human cells for MOCA-induced carcinogenesis, thus making it useful as a biomarker of exposure. Other DNA adducts were also observed with the different doses and routes of exposure investigated.

Alterations of histone phosphorylation in rat spleen cells after treatment with the aromatic amine, 4,4'-methylene-bis(2-chloroaniline).

Alterations of the phosphorylation pattern of histones by the carcinogen, 4,4'-methylene-bis(2-chloroaniline) (MOCA) were investigated using rodent spleen cells. Spleen cells were isolated from Sprague-Dawley rats and treated with either 5, 10, 25, or 50 microM MOCA or acetone vehicle controls for 1, 2, 4, or 8 hours. Cells were incubated with 32P-phosphoric acid, and histones from these cells were fractionated utilizing two-dimensional polyacrylamide gel electrophoresis. Marked stimulation of histone phosphorylation was observed with the 10 microM MOCA treatment. A transient decrease in histone phosphorylation was observed at the 1 and 2 hour time points followed by a marked stimulation at 4 hours.

Comparative metabolism of bis(2-methoxyethyl)ether in isolated rat hepatocytes and in the intact rat: effects of ethanol on in vitro metabolism.

The metabolism of the reproductive and developmental toxicant bis(2-methoxyethyl)ether (diglyme) was studied in isolated rat hepatocytes and in the intact rat. Male Sprague-Dawley rats (190-220 g) were used in both studies. Hepatocytes, isolated by a two-step in situ collagenase perfusion of the liver, were cultured as monolayers and incubated with [14C]diglyme at 1, 10, 30, and 50 microM for up to 48 h. For the in vivo study, rats were given single oral doses of [14C]diglyme at 5.1 mmol/kg body wt, and urine was collected for up to 96 h. Radioactive compounds in the culture medium or in the urine were separated by high performance liquid chromatography and quantified with an in-line radioactivity monitor. Metabolites were identified by comparison of their chromatographic retention times and their mass spectra with those of authentic compounds. The principal metabolite from hepatocytes and in the urine was (2-methoxyethoxy)acetic acid (MEAA). This metabolite accounted for approximately 36% of the radioactivity in the 48-h culture medium and about 67% of the administered dose in the 48-h urine. Other prominent metabolites common to both systems included 2-(2-methoxyethoxy)ethanol, methoxyacetic acid (MAA), 2-methoxyethanol, and diglycolic acid. The diglyme metabolite profiles from urine and from hepatocytes were qualitatively similar, demonstrating that, in the rat, hepatocytes serve as a good model system for predicting the urinary metabolites of diglyme. Moreover, MEAA was shown to be the metabolite best suited for use as a short-term biological marker of exposure to diglyme.(ABSTRACT TRUNCATED AT 250 WORDS)

Biological monitoring for occupational exposures to o-toluidine and aniline.

Epidemiological evidence that occupational exposure to o-toluidine and aniline is associated with an increased risk of bladder cancer led to efforts to identify biomarkers of workplace exposures to these aromatic amines. For the determination of o-toluidine and aniline in worker urine specimens, a method using high performance liquid chromatography (HPLC) followed by electrochemical detection was developed. The limits of detection were 0.6 microgram/l and 1.4 micrograms/l for o-toluidine and aniline, respectively. Recovery of o-toluidine and aniline from spiked urine averaged 86% and 93%, respectively, over a range of 4-100 micrograms/l. Reproducibility in the range 2-100 micrograms/l for analyses of split field samples was 13% (average RSD) for o-toluidine and 16% (average RSD) for aniline. Application of this method to pre- and post-shift samples collected from potentially exposed and unexposed workers indicated elevated concentrations of o-toluidine and aniline in urine from exposed workers. To develop methods for biomarkers of internal dose, o-toluidine binding to the blood proteins hemoglobin and albumin was investigated utilizing in-vivo (rodent) and in-vitro (hemoglobin and albumin) studies. Base-hydrolyzable protein adducts were analyzed by HPLC (fluorescence) and/or GC/electron capture (EC). The methods were compared for sample preparation requirements, selectivity and sensitivity. While the GC/EC method was more sensitive than HPLC, the presence of interfering peaks limited the utility of this approach. Results from these studies suggested that the HPLC method could be useful for determination of o-toluidine exposures in individuals acutely or chronically exposed to high levels.

ortho-toluidine blood protein adducts: HPLC analysis with fluorescence detection after a single dose in the adult male rat.

Hemoglobin (Hb) and albumin (Alb) adducts of the suspect human carcinogen ortho-toluidine (OT) were quantified in blood samples collected from rats after a single i.p. injection. Mild alkaline hydrolysis of Hb adducted with [14C]OT followed by extraction with ethyl acetate resulted in recovery of 63% of the bound radioactivity. HPLC analysis revealed a single radiolabeled peak which was identified as OT by GC-MS. In subsequent experiments Hb and Alb adduct levels were determined by HPLC analysis of this cleavage product using fluorescence detection. 4-Ethylaniline was used as internal standard. The detection limit for OT was approximately 450 pg/injection or 5 pmol/mg Hb. Mean adduct levels for Hb increased rapidly over the first 4 hr with the highest (ng/mg Hb +/- SD) 3.7 +/- 0.5 detected 24 hr after OT administration at 50 mg/kg body wt. In contrast, adduct levels for pooled Alb samples increased from 0.7 ng/mg Alb at 2 hr to 2.5 ng/mg Alb at 4 hr, but were not detectable 24 hr after dosing. Hb adducts showed a linear relationship for OT doses of 10, 20, 40, 50, and 100 mg/kg body wt. The Hb adduct t1/2 (11 days) was determined after a single 100 mg/kg OT dose. Hb adduct levels were quantifiable (1.3 +/- 0.2 ng/mg Hb) by HPLC/fluorescence 28 days after 100 mg/kg OT. Although Hb and Alb adducts differ in stability, a ratio of such OT adducts may be useful in long-term industrial biomonitoring for evaluation of OT exposure.

Binding characteristics of ortho-toluidine to rat hemoglobin and albumin.

The binding characteristics of [14C]ortho-toluidine (OT), a suspect human carcinogen, were investigated in male Sprague-Dawley rats. Rats were administered [14C]OT i.p. at 10, 20, 40, 50, or 100 mg/kg body weight, then sacrificed at 2, 4, 8, 18, 24, 48, or 72 h, or 7, 14, or 28 days. Hemoglobin (Hb) and albumin (Alb) were isolated from blood, and OT binding was determined by liquid scintillation counting. For Alb, peak binding occurred at 50 mg/kg at the 4-h time point (15.6 ng OT/mg Alb), while for Hb peak binding was observed at 24 h at the 100 mg/kg dose (23.0 +/- 5.1 ng OT/mg Hb). OT-Alb binding was not linear; however, OT-Hb binding appeared to increase linearly in a dose-dependent manner. Biological half-lives of OT bound to Alb or Hb were observed to be 2.6 and 12.3 days, respectively, after rats were administered a single dose of [14C]OT and sacrificed after 4 h to 28 days. The effect of route of administration on OT-Hb adduct formation was investigated, and approximately a two-fold increase in radioactivity bound to Hb was observed after i.p. administration of 100 mg/kg [14C]OT versus oral intubation. Additional studies were carried out to investigate the effect of microsomal enzyme induction. An increase in OT-Hb binding was seen in rats pretreated with phenobarbital compared to rats pretreated with beta-naphthoflavone or without pretreatment; however, this increase was not statistically significant. These results suggest that OT-Hb and OT-Alb adduct formation may be a valuable biomarker for assessing workplace exposure.

Bis(2-methoxyethyl) ether: metabolism and embryonic disposition of a developmental toxicant in the pregnant CD-1 mouse.

An embryotoxic oral dose of bis(2-methoxyethyl) ether (DGDME), 3.73 mmol/kg body wt (500 mg/kg), administered on the 11th day of gestation to pregnant CD-1 mice was metabolized predominantly by O-demethylation to 2-(2-methoxyethoxy)ethanol with subsequent oxidation to (2-methoxyethoxy)acetic acid. Urinary excretion of this metabolite over 48 hr amounted to 63 +/- 2% of the dose. A smaller percentage of the administered dose was metabolized at the central ether linkage to produce 2-methoxyethanol, which was further metabolized by alcohol dehydrogenase to methoxyacetic acid. Urinary excretion of methoxyacetic acid, a potent developmental toxicant, amounted to 28 +/- 1% of the administered dose by 48 hr and was the second most prominent urinary metabolite. Unchanged DGDME and methoxyacetic acid were detected in the embryonic tissues from these animals, and embryos harvested after the initial 6-hr period showed detectable amounts of only methoxyacetic acid. The average amount of methoxyacetic acid per embryo was calculated to be 1.5 +/- 1.0 mumol (5.9 mmol/kg body wt) at the 6-hr termination time. This finding suggests that the reported teratogenic effects of DGDME are due to methoxyacetic acid formed, either in the fetus or by hepatic metabolism in the dam with subsequent distribution to the embryonic tissue. These results suggest that such developmental toxicity may occur with structurally similar aprotic ethylene glycol ethers in which metabolic O-dearylation would yield 2-methoxy-ethanol.

4,4'-Methylenebis(2-chloroaniline) (MOCA): the effect of multiple oral administration, route, and phenobarbital induction on macromolecular adduct formation in the rat.

The effect of multiple oral administration of MOCA, a suspect human carcinogen, was studied in the adult male rat. As many as 28 consecutive daily doses of [14C]MOCA at 28.1 mumol/kg body wt (5 microCi/day) were administered and rats were euthanized at weekly intervals for 7 weeks. MOCA adduct formation for globin and serum albumin was evaluated by determination of [14C]MOCA covalent binding. The covalent binding associated with globin showed a linear increase over the 28-day exposure period with 342 fmol/mg globin 24 hr after the final dose. More extensive covalent binding was detected for albumin with 443 fmol/mg albumin after the final dose, but increases were not linear. After cessation of dosing, the albumin adduct levels decreased rapidly (t1/2 = 4.6 days) in relation to globin adduct levels (t1/2 = 16.1 days). The MOCA-globin adduct t1/2 is consistent with that determined after a single 281 mumol/kg oral dose of MOCA. Significant differences related to route of administration were detected for 24-hr globin covalent binding with ip greater than po greater than dermal. Distribution of undifferentiated [14C]MOCA was highest in the liver at 24 hr with tissue levels for liver greater than kidney greater than lung greater than spleen greater than testes greater than urinary bladder. Induction of cytochrome P450 enzymes by administration of phenobarbital (100 mg/kg/day/3 days) resulted in a significant (p less than 0.05) increase in MOCA-globin adduct formation detected with 33.5 pmol/mg globin for induced rats versus 13.6 pmol/mg globin for control rats. Although MOCA-globin and albumin adducts show differing stability, quantification of such MOCA adducts may be useful for long-term industrial biomonitoring of MOCA.

Ethylene dichloride: the influence of disulfiram or ethanol on oncogenicity, metabolism, and DNA covalent binding in rats.

Male and female Sprague-Dawley rats were exposed to 50 ppm ethylene dichloride (EDC) for 7 hr/day, 5 days/week, for 2 years by inhalation. Additional rats were exposed to 50 ppm EDC either with 0.05% disulfiram in the diet or with 5% ethanol in the drinking water. Histopathologic lesions related to the combination of inhaled EDC and dietary disulfiram were observed in the liver, mammary, and testicular tissues of rats. This combined exposure resulted in a significant increase in the incidence of intrahepatic bile duct cholangiomas in both male and female rats. Male rats exposed to both EDC and disulfiram also had an increased incidence of subcutaneous fibromas, neoplastic nodules, and interstitial cell tumors in the testes. The female rats exposed to EDC and disulfiram also had a higher incidence of mammary adenocarcinomas. No significant increase in the number of any tumor type was observed in rats exposed to only EDC, disulfiram, or ethanol. Similarly, no significant increase in the number of tumors was observed in rats exposed to inhaled EDC and ethanol in water. At the end of the 2-year period animals from each group were evaluated for EDC metabolism and DNA binding. Blood levels of EDC at the end of a 7-hr exposure period were significantly higher for rats exposed to both EDC and disulfiram than for rats exposed to EDC alone. In addition, the elimination of a single oral dose of radiolabeled EDC was affected. The urinary excretion of 14C from control rats was 47 to 55% of the administered dose with 28 to 30% detected as unchanged EDC in the breath. In disulfiram-treated rats, only 35 to 36% of the administered 14C was eliminated in the urine with 41 to 55% as unchanged EDC in the breath. The urinary metabolite HPLC profile was qualitatively unchanged by long-term EDC, disulfiram, or ethanol treatment, either alone or in combination, and consisted primarily of thiodiglycolic acid, thiodiglycolic acid sulfoxide, and chloroacetic acid.