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.

Induction of ornithine decarboxylase activity by 4,4'-methylene bis(2-chloroaniline) in the rat.

The effects of the curative extender 4,4'-methylene bis (2-chloraniline) (MOCA), an established experimental carcinogen that exhibits activity in rat liver, on hepatic ornithine decarboxylase (ODC) activity was investigated. Male Sprague-Dawley rats were injected i.p. with 75 mg/kg MOCA and killed 6, 12, 18, 24, 42 and 48 h later. Stimulation with MOCA of liver cytosolic ODC was first evident at 6 h, peaked at 12 h and returned to control levels by 42 h. The liver enzyme was refractory to stimulation by a second treatment of MOCA within the dosing intervals examined. The magnitude of stimulation of the enzyme by this aromatic amine was dependent on dose and route of administration.

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'-Methylene-bis(2-chloroaniline) (MOCA): comparison of macromolecular adduct formation after oral or dermal administration in the rat.

The macromolecular binding of 4,4'-methylenebis(2-chloroaniline) (MOCA), a suspect human carcinogen, was studied in the adult male Sprague-Dawley rat after both oral and dermal administration. Rats were euthanized 1, 3, 7, 10, 14, and 29 days after a single 281 mumol/kg body wt dose of [14C]MOCA (oral, 213 muCi/kg; dermal, 904 muCi/kg). DNA from various tissues and hemoglobin were isolated for determination of the time course of MOCA macromolecular binding. After oral administration adduct formation was rapid with maximum levels appearing at 24 hr. The 24-hr covalent binding associated with the globin was 7.84 pmol/mg globin (t1/2 = 14.3 days). More extensive 24-hr covalent binding was detected for liver DNA with 49.11 pmol/mg DNA (t1/2 = 11.1 days). After dermal administration of MOCA the major portion of the dose, 86.2%, remained at the application site throughout the study. For these rats the 24-hr covalent binding determined for liver DNA was 0.38 pmol/mg DNA (t1/2 = 15.6 days). Although lower levels were detected after dermal application, similar stability of MOCA-DNA adducts indicates that quantification of such MOCA adducts may be useful for the long-term industrial biomonitoring of MOCA exposure and for the evaluation of human DNA-MOCA adduct formation, a lesion thought to be associated with the production of cancer.

Testicular effects of bis(2-methoxyethyl) ether in the adult male rat.

The onset of testicular pathology in the rat and possible recovery over an 8-week period were evaluated after the administration of up to 20 daily oral doses of bis(2-methoxyethyl) ether (diglyme) at 5.1 mmol/kg bw (684 mg/kg bw). Primary and secondary spermatocyte degeneration and spermatidic giant cells were observed after six to eight treatments. In addition, the testes-to-body weight ratio was significantly reduced by the tenth day of treatment and continued to be depressed eight weeks after discontinuation of the treatment. Testicular LDH-X activity, a pachytene spermatocyte marker enzyme, was significantly decreased in animals by the eighteenth day of treatment with diglyme.

The role of enzyme induction on metabolite formation of bis(2-methoxyethyl) ether in the rat.

The effect of enzyme induction on the metabolism of the reproductive toxicant bis (2-methoxyethyl) ether (diglyme) was studied in male Sprague-Dawley rats. Rats were given either daily doses of diglyme at 5.1 mmol/kg body wt. by gavage or 0.1% (w/v) phenobarbital (PB) in the drinking water for 22 consecutive days. In one study, a significant reduction in the hexobarbital sleeping time was determined for rats pretreated with diglyme or PB in comparison with that determined for naive rats. In a second study, naive and pretreated rats given single oral doses of 14C-diglyme at 5.1 mmol/kg body wt. showed similar urinary 14C excretion patterns. Urinary metabolites were separated and quantified by hplc to evaluate the influence of pretreatment with either diglyme or PB on the 14C-diglyme urinary metabolite profile. The amount of (2-methoxyethoxy) acetic acid, the principal metabolite, was similar for rats given no pretreatment and for rats pretreated with either diglyme or PB. However, both pretreatments resulted in significant increases in the formation of methoxyacetic acid, a recognized reproductive toxicant.

Metabolism of bis(2-methoxyethyl) ether in the adult male rat: evaluation of the principal metabolite as a testicular toxicant.

The metabolism of the reproductive toxicant bis(2-methoxyethyl) ether was studied in male Sprague-Dawley rats, and the principal metabolite (2-methoxyethoxy)acetic acid and its metabolic precursor 2-(2-methoxyethoxy)ethanol were evaluated separately as testicular toxicants. For the metabolism study, rats were given single po doses of [1,2-ethylene-14C]bis(2-methoxyethyl) ether at 5.1 or 0.051 mmol/kg body wt. Within 96 hr, approximately 86 to 90% of the radioactivity was excreted in the urine. Urinary metabolites were separated by high-performance liquid chromatography and isolated for characterization by gas chromatography-mass spectrometry. The principal urinary metabolite, accounting for 67.9 +/- 3.3% of the administered high dose and 70.3 +/- 1.3% of the low dose, was identified as (2-methoxyethoxy)acetic acid. A second metabolite, representing 6.2 +/- 0.8% of the high dose and 5.8 +/- 0.8% of the low dose, was identified as methoxyacetic acid, a previously recognized testicular toxicant. In the toxicity study, (2-methoxyethoxy)acetic acid and 2-(2-methoxyethoxy)ethanol were administered to rats at 5.1 mmol/kg body wt by gavage as single daily doses for as many as 20 consecutive days. The testes of rats killed 24 hr after the administration of even numbered doses showed no gross or microscopic abnormalities. These results are in contrast to the previously reported testicular atrophy evoked after as few as 8 daily doses of the parent compound, bis(2-methoxyethyl) ether, tested under the same experimental conditions. Thus, the testicular toxicity reported for bis(2-methoxyethyl) ether could be explained by the presence of a minor metabolite, methoxyacetic acid.

Teratogenicity of n-propanol and isopropanol administered at high inhalation concentrations to rats.

As part of a teratological evaluation of several alcohols, 10,000, 7000 and 3500 ppm n-propanol or isopropanol were administered by inhalation to groups of 15 pregnant Sprague-Dawley rats for 7 hr/day on gestation days 1-19. The dams were killed on day 20. Half of the foetuses were examined for skeletal defects and the others for visceral defects using the Wilson technique. The highest concentration of n-propanol produced only minimal maternal toxicity, as indicated by observation and by measurement of weight gain and feed and water intake. In contrast, the same concentration of isopropanol produced narcosis in the dams, retarded body-weight gain and reduced the feed intake. At 7000 ppm isopropanol, body-weight gain was retarded but there were no other observable effects in the dams. Following exposure to 10,000 ppm of either alcohol, there were significant (P less than or equal to 0.05) increases in resorptions and decreases in foetal weights compared with the control groups. Foetal weights were also reduced significantly following exposure to 7000 ppm of either alcohol and to 3500 ppm isopropanol. Significantly more litters had malformations following exposure to 10,000 or 7000 ppm of either alcohol, but these effects were seen only in the presence of maternal toxicity. At 3500 ppm, no detectable teratogenic effects were produced by either solvent.

4,4'-methylene-bis-ortho-chloro-aniline (MBOCA): absorption and excretion after skin application and gavage.

4,4'-methylene-bis-ortho-chloro-aniline ( MBOCA ) is an aromatic amine and industrial chemical that has been shown to cause cancer of several different organs in rats and mice and bladder cancer in dogs. The purpose of this study was to determine the efficacy of using urinary concentrations of MBOCA as a means for evaluating extent of exposure. Male Sprague-Dawley rats were given MBOCA and [14C] MBOCA by either gavage or skin application. Concentrations and amounts of 14C were measured in urine, feces, skin and total carcasses, and parent MBOCA in urine at several intervals after application. The percentages of administered doses excreted and retained in the animals were calculated and comparisons made. Within 72 hr after gavage 16.5% of the administered compound was excreted in urine as 14C but only 0.25% as parent MBOCA . In the same interval after skin application a maximum of 2.54% of administered MBOCA was excreted as 14C but only 0.008% as parent MBOCA . Seventy-two hours after gavage 13.7% of the administered dose was retained in the tissues, and after skin absorption 5-13% was retained. With gavage the rate of excretion of 14C in urine and feces was very high in the first 24 hr (68.3%) but fell off rapidly (2.07%) by the third day. After skin absorption the rates of excretion of 14C were fairly constant over a 3-day period. Less MBOCA was absorbed from the skin if the skin was washed within 8 hr after application, as compared to waiting 24 hr or not washing at all. The amount of parent MBOCA detected in urine is a very small amount of that applied or absorbed. The percentage detected and the rates of excretion depend upon the route of administration, and the interval between exposure and sampling. For these reasons urinary analysis for MBOCA can be used only as very imprecise indicators of extent of recent exposure.

The effect of dietary disulfiram upon the tissue distribution and excretion of 14C-1,2-dibromoethane in the rat.

Dietary disulfiram enhances the toxicity of inhaled 1,2-dibromoethane in rats. This study was undertaken to determine whether the differential toxicity noted was associated with alterations in the levels of the compound and/or its metabolites in the target organs. A comparison of the levels of 14C in selected tissues of male rats, with and without dietary disulfiram, following the oral administration of 14C-1,2-dibromoethane was made. The results indicated that levels of radioactivity in the target organs of animals in the disulfiram group were significantly elevated both at 24 and 48 hours following compound administration. The data indicate a direct correlation between tissue levels and the enhancement of toxicity noted in the disulfiram-treated rats in the inhalation study. A significant elevation in the levels of radioactivity in washed liver nuclei obtained from animals receiving dietary disulfiram was also noted, suggesting a relationship between nuclear uptake and the increased incidence of liver tumors appearing in the disulfiran group in the inhalation study.