G.D. DiVincenzo

Characterization of the metabolites of methyl n-butyl ketone, methyl iso-butyl ketone, and methyl ethyl ketone in guinea pig serum and their clearance

Abstract Methyl n -butyl ketone (MnBK) has been reported to produce peripheral neuropathy in experimental animals and in man. The metabolism of MnBK and related ketones was studies as part of a program to elucidate the molecular events in MnBK-induced neuropathy. Guinea pigs were given single 450 mg/kg ip doses of MnBK, methyl iso-butyl ketone (MiBK) or methyl ethyl ketone (MEK). Metabolites in serum were identified and quantitated by gas chromatography and gas chromatography-mass spectrometry. MnBK metabolites were 5-hydroxy-2-hexanone, 2,5-hexanedione, and 2-hexanol. MiBK was converted to 4-hydroxy-2-methyl-2-pentanone and 4-methyl-2-pentanol. MEK produced 2-butanol, 3-hydroxy-2-butanone, and 2,3-butanediol. The fate of several of the above metabolites was also investigated. Our results show that aliphatic ketones are metabolized both by reduction of the carbonyl group to form a secondary alcohol and by oxidation at the ω-1 carbon atom to form an hydroxylated ketone. Guinea pigs and other mammalian species receiving MnBK by various routes of administration showed qualitatively similar metabolic patterns in serum. Serum half-lives and clearance times for MnBK, MiBK, and MEK in the guinea pig were 78 min, 6 hr; 66 min, 6 hr; and 270 min, 12 hr; respectively. Our studies have shown that 2-hexanol, 5-hydroxy-2-hexanone, and 2,5-hexanediol may be neurotoxic by virtue of their conversion to the neurotoxin 2,5-hexanedione. n -Hexane, a reputed neurotoxin, was also investigated and was found to produce 5-hydroxy-2-hexanone and 2,5-hexanedione in guinea pig serum. The neurological effects of MnBK, n -hexane, and 2,5-hexanedione, therefore, may have a common metabolic origin.

The relative neurotoxicity of methyl-n-butyl ketone, n-hexane and their metabolites.

Methyl n-butyl ketone (MnBK) and n-hexane produce a polyneuropathy in experimental animals. Metabolic studies have revealed that both of these compounds are metabolized to similar metabolites; that is 2-hexanol, 5-hydroxy-2-hexanone, 2,5-hexanediol, and 2,5-hexanedione. The latter two metabolites have been shown to be neurotoxins and the former two may be neurotoxic by virtue of the fact that both are metabolized to 2,5-hexanedione. The relative neurotoxicity of MnBK, n-hexane, and their metabolites was compared by administering equimolar doses of each compound by gavage to groups of male rats, 5 days/week over a 90-day period. A control group was given distilled water. The endpoint used to determine relative neurotoxicity was the time (days) required to produce clinical evidence of severe hindlimb weakness or paralysis. All test compounds produced both clinical and histologic evidence of neuropathy. Morphologically, this nerve damage was identical to that previously described following MnBK, n-hexane, and 2,5-hexanedione administration. The relative neurotoxicity of the test compounds in decreasing order of potency was: 2,5-hexanedione, 5-hydroxy-2-hexanone, 2,5-hexanediol, methyl-n-butyl ketone, 2-hexanol, and n-hexane. The neurotoxic potency was directly related to the amount of 2,5-hexanedione produced by each compound as determined by measuring the area under the serum concentration-time curve of 2,5-hexanedione. Additionally, atrophy of testicular germinal epithelium similar to that previously reported for 2,5-hexanedione occurred in the animals receiving 2,5-hexanedione, 2,5-hexanediol, 5-hydroxy-2-hexanone, MnBK, and 2-hexanol. n-Hexane administration had a lesser effect on the germinal epithelium than did the other compounds. Effects on body weight response and feed consumption paralleled the neurotoxic potency of each compound.

Metabolic fate and disposition of 14C-labeled methyl n-butyl ketone in the rat

Methyl n -butyl ketone (MnBK) has been associated with peripheral neuropathy in laboratory animals. 2,5-Hexanedione, a metabolite of MnBK, has produced peripheral neuropathy in laboratory animals, thus suggesting that neuropathy may be produced by metabolic activation of MnBK. [1- 14 C]MnBK was given to male Charles River CD rats by gavage in corn oil in doses of 20 or 200 mg/kg of body weight. [1- 14 C]MnBK was rapidly absorbed from the gastrointestinal tract after ingestion and radioactivity was eliminated in the breath and urine. Radioactivity in the breath was identified as unchanged MnBK (6% of the dose) and respiratory CO 2 (38%). Urinary radioactivity accounted for 40% of the dose; fecal radioactivity accounted for 1.4%. About 14% of the radioactivity remained in the carcass after 48 hr and 8% remained after 6 days. Carbon-14 was widely distributed throughout the tissues of the rat with highest concentrations in blood and liver. The serum elimination time of MnBK was 6 hr. Metabolites of MnBK in serum and in urine were identified by gas chromatography and by gas chromatography-mass spectroscopy. MnBK metabolites detected in rat serum were 2-hexanol, 5-hydroxy-2-hexanone, and 2,5-hexanedione. Metabolites in urine were 2-hexanol, 5-hydroxy-2-hexanone, 2,5-hexanedione, 2,5-dimethylfuran, γ -valerolactone, norleucine, and urea. Principal metabolic pathways were the reduction of the carbonyl group, oxidation at the α and ω -1 carbon atom, and evidently the decarboxylation of metabolites possessing an α -keto acid moiety. This latter route appears to account for most of the respiratory 14 CO 2 formed rather than utilization by intermediary metabolism and thus may represent a significant route for the metabolism of ketones. A minor metabolic pathway is the transmination of such α -keto acid intermediates to amino acids. Rats were pretreated with MnBK, phenobarbital, or SKF 525A prior to 200-mg/kg doses of [1- 14 C]MnBK. MnBK and phenobarbital pretreatment did not substantially affect the metabolism of [1- 14 C]MnBK. Pretreatment with SKF 525A markedly increased the excretion of 14 CO 2 (49.6 vs 37.6%) and decreased urinary radioactivity (22.5 vs 39%), suggesting that the ω -1 oxidation was mediated by the microsomal mixed function oxidase system. Apparently, α -oxidation of MnBK to CO 2 is the detoxification mechanism and ω -1 oxidation leads to metabolic activation.

Uptake, metabolism, and elimination of methylene chloride vapor by humans

Abstract Methylene chloride (CH2Cl2) is extensively utilized both in industry and in commerce. The finding that CH2Cl2 was metabolized to carbon monoxide (CO) by humans and experimental animals prompted us to measure blood carboxyhemoglobin saturations in workers exposed to CH2Cl2 and to conduct controlled experimental human exposures. Experimental exposures to CH2Cl2 vapor were carried out with sedentary nonsmokers for 7 1 2 hr at 50, 100, 150, or 200 ppm or for 7 1 2 hr each day at 100, 150, or 200 ppm for 5 consecutive days. During controlled exposures as much as 70% of the inhaled vapor was absorbed by the pulmonary route. Between 25 and 34% of the absorbed CH2Cl2 was excreted in the expired air as CO during and after the exposure and less than 5% was eliminated unchanged in the expired air after the exposure. Exposure to 50, 100, 150, or 200 ppm of CH2Cl2 produced peak blood carboxyhemoglobin saturations of 1.9, 3.4, 5.3, and 6.8%, respectively. Peak end tidal air and blood concentrations of CH2Cl2 and CO were essentially unchanged during repeated exposures. The average peak blood carboxyhemoglobin saturation for workers occupationally exposed to CH2Cl2 vapor was 3.9%. The above findings show that end tidal air and blood concentrations of CH2Cl2 and CO are directly proportional to the magnitude of the exposure and that an 8-hr exposure to 100 ppm of CH2Cl2 vapor will produce a blood carboxyhemoglobin saturation of about 3%, less than the increase in blood carboxyhemoglobin saturations produced by an exposure to CO at its recommended TLV of 35 ppm.

Serum Ornithine Carbamyl Transferase as a Liver Response Test for Exposure to Organic Solvents

Ornithine carbamyl transferase (OCT), an enzyme found predominantly in the liver, is released into the bloodstream when liver cells are ruptured. The measurement of serum OCT activity is a convenient, specific, and sensitive assay of liver damage. This test was used to evaluate the effect of several widely used solvents on the livers of guinea pigs. Each solvent was administered intraperitoneally, and 24 hours later serum OCT activity was measured. Many of the solvents tested failed to increase serum OCT activity even at near-lethal doses. Of the thirty-three solvents evaluated, two produced elevations in serum OCT activity at relatively low doses (less than 50 mg/kg), five at moderate doses (50 to 500 mg/kg), and nine at high doses (greater than 500 mg/kg).

Studies on the respiratory uptake and excretion and the skin absorption of methyl n-butyl ketone in humans and dogs

Abstract Methyl n-butyl ketone (MnBK) has produced peripheral neuropathy in experimental animals and is implicated in an occupationally produced neuropathy. Since occupational exposure to MnBK is by inhalation or skin contact, both the absorption and elimination of MnBK vapor and its absorption through skin were investigated. Studies were carried out first with male beagle dogs and subsequently with human volunteers. Humans exposed for 7.5 hours to 10 or 50 ppm or for 4 hr to 100 ppm of MnBK vapor absorbed between 75 and 92% of the inhaled vapor. Unchanged MnBK was not eliminated extensively in the postexposure breath or in urine. 2,5-Hexanedione, a metabolite of MnBK known to be neurotoxic in rats, was found in the serum of humans exposed to either 50 or 100 ppm of MnBK. The absorption and elimination of MnBK in dogs was similar to that observed in humans. The skin absorption of [1-14C]MnBK or a 9 1 ( v v ) mixture of methyl ethyl ketone (MEK) [1- 14 C]MnBK was determined by excretion analysis. Two volunteers exposed by skin contact to [1-14C]MnBK absorbed 4.8 μg min−1 cm−2 and 8.0 μg min−1 cm−2, respectively. Skin exposure to MEK [1- 14 C]MnBK resulted in the respective absorption of 4.2 and 5.6 μg min−1 cm−2 by two individuals. Two volunteers given an oral dose of [1-14C]MnBK (2 μCi; 0.1 mg/kg) excreted 49.9 and 29.0% of the dose, respectively, as respiratory 14CO2 within 3 to 5 days and 27.6 and 25.0% of the dose, respectively, in urine within 8 days. Both [1-14C]MnBK and MEK [1- 14 C]MnBK were absorbed through the skin of dogs. These findings show that MnBK is readily absorbed by the lungs, the gastrointestinal tract, and through the skin, is not eliminated extensively unchanged in breath or urine, and is metabolized to CO2 and 2,5-hexanedione. Radioactivity derived from [1-14C]MnBK was excreted slowly by man, suggesting that repeated daily exposure to high concentrations of MnBK may lead to a prolonged exposure to neurotoxic metabolites.

Fate and disposition of [14C]methylene chloride in the rat

Abstract [ 14 C]Methylene chloride ( 14 CH 2 Cl 2 ) was administered ip to Sprague-Dawley rats at doses ranging from 412 to 930 mg/kg. Animals were sacrificed 2, 8, and 24 hr after dosing. CH 2 Cl 2 was largely eliminated in the breath unchanged during the first 2 hr. A fraction of the original dose was metabolized to carbon monoxide (CO), carbon dioxide (CO 2 ), and an uncharacterized metabolite. At 24 hr 91.5% of the dose was eliminated in the breath unchanged, 2% was eliminated as CO, 3% as CO 2 , 1.5% as an uncharacterized metabolite, 1% was excreted in urine, and 2% remained in the carcass. Although the dissemination of radioactivity was widespread in rat tissue, the overall tissue uptake of 14 C was relatively small. The highest tissue specific activities were found in the liver, kidneys, and adrenal glands. There was no significant accumulation of radioactivity in the fat. In a separate study serum and tissue formaldehyde concentrations (CH 2 O) were measured in rats dosed with CH 2 Cl 2 . A substantial increase in serum CH 2 O (62%) and a decrease in liver CH 2 O (64%) were noted in rats treated with CH 2 Cl 2 . Formaldehyde concentrations in other tissues remained unchanged. 14 CH 2 O was not found in the breath, serum, or tissues of rats treated with 14 CH 2 Cl 2 . Apparently the changes in serum and liver CH 2 O were physiologically induced by CH 2 Cl 2 . There was no evidence from these experiments that CH 2 Cl 2 was metabolized to CH 2 O.

Metabolic fate and disposition of [14C]hydroquinone given orally to Sprague-Dawley rats

Hydroquinone (HQ) is used widely in industry and in commerce and is considered to have a low degree of toxicity. Although the metabolism of HQ has been studied elsewhere, a complete materials balance has not been reported. We investigated the metabolism of HQ in naive and HQ pretreated male Sprague-Dawley rats. [14C]HQ was administered by gavage in single doses of 5, 30, or 200 mg/kg to naive rats. HQ was given repeatedly by gavage to male rats at 200 mg/kg for 4 consecutive days followed by a single dose with 200 mg/kg of [14C]HQ. In separate studies rats were fed 5.6% unlabeled HQ in the diet for 2 days or were dosed by gavage with 311 mg/kg [14C]HQ. The excretion patterns of [14C]HQ and its metabolites were similar for rats dosed singly or repeatedly. Rats given a single dose of 200 mg/kg of [14C]HQ excreted 91.9% of the dose in the urine within 2-4 days; 3.8% was excreted in the feces, about 0.4% was excreted in expired air, and 1.2% remained in the carcass. Radioactivity was widely distributed throughout the tissues with higher concentrations in the liver and kidneys. A decrease in 14C tissue concentrations occurred from 48 to 96 h. The only radiolabeled compounds in the urine were HQ (1.1-8.6% of the dose), hydroquinone monosulfate (25-42%), and hydroquinone monoglucuronide (56-66%). Similar findings were observed for rats given HQ in the feed. There were no significant increases from controls for absolute or relative liver weights, liver microsomal protein concentrations, cytochrome b-5, cytochrome P-450 or cytochrome c reductase activity in rats dosed repeatedly with 200 mg/kg HQ. Cytochrome P-450 values were slightly but significantly decreased in rats dosed repeatedly with HQ compared with controls.

Fate of n-butanol in rats after oral administration and its uptake by dogs after inhalation or skin application

Abstract n -[1- 14 C]Butanol was mixed with corn oil and administered by gavage to male Charles River C.D. rats in doses of 4.5, 45, or 450 mg/kg of body wt. Rats dosed with 450 mg/kg excreted 83.3% of the dose as 14 CO 2 at 24 hr; 4.4% was excreted in the urine; less than 1% was eliminated in feces and 12.3% remained in the carcass. n -[1- 14 C]Butanol was excreted in the urine apparently as an O -sulfate and as an O -glucuronide, both of which accounted for 75% of the radioactivity. Urea accounted for the remainder. Rats dosed with 4.5 or 45 mg/kg showed a similar excretion pattern to that of rats dosed with 450 mg/kg. Excretion studies were performed to quantify the percutaneous absorption of n -butanol in male beagle dogs. n -[1- 14 C]Butanol was absorbed through the skin of dogs at a rate of 8.8 μg min −1 cm −2 . Dogs exposed by inhalation to 50 ppm of n -butanol vapor absorbed about 55% of the inhaled vapor. The elimination of n -butanol in the postexposure breath was relatively low compared to the quantity absorbed during the exposure. Dogs dosed with ethanol (200 mg/kg, po) and then exposed to 50 ppm of n -butanol vapor for 6 hr showed no evidence that the exposure to n -butanol vapor inhibited the metabolism of ethanol. These findings suggest that occupational exposure to n -butanol vapor at the current threshold limit value is not likely to affect the metabolism of low doses of ethanol taken concurrently.

Bacterial mutagenicity testing of urine from rats dosed with 2-ethylhexanol derived plasticizers.

Di-(2-ethylhexyl)phthalate (DEHP) produced hepatocellular carcinomas in rodents at high doses in a NTP/NCI bioassay. DEHP has not shown evidence of genotoxic activity in in vitro mutagenicity tests. We extended these studies by examining the mutagenicity of urine from rats dosed with DEHP, 2-ethylhexanol (2-EH), and several other 2-EH derived plasticizers, i.e. di-(2-ethylhexyl)adipate (DEHA), di-(2-ethylhexyl)terephthalate (DEHT) and tri-(2-ethylhexyl)trimellitate (TEHT). A modified Ames Salmonella/microsome assay was used to determine mutagenicity. Urine was pooled from male Sprague--Dawley rats dosed daily for 15 days with 2000 mg/kg of each test substance with the exception of 2-EH which was given at 1000 mg/kg. Direct plating procedures were used to determine the presence of mutagens in urine. Urine from rats dosed with 8-hydroxyquinoline was used as a positive control. There was no evidence that mutagenic substances were excreted in the urine by rats dosed with either DEHP, DEHA, DEHT, TEHT or 2-EH as determined in the presence or absence of rat liver microsomes, and with or without treatment with beta-glucuronidase/aryl sulfatase. Our findings indicate that the above test compounds were not converted to urinary metabolites that were mutagenic. These observations provide no evidence for a genotoxic mechanism for DEHP carcinogenicity in rodents.