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682
Articles of general interest-Fd
bated with VC and the microsomal system, labelled 3,N4-ethenocytidine moieties being formed (Laib & Bolt 1978, lot. cit.). The reaction of chloroacetaldehyde with calfthymus DNA at pH 4.5 gave a modified DNA product (Green & Hathway, Chemico-EioL Interactions 1978, 22, 21 I). Enzyme hydrolysis of the DNA produced a mixture of naturally occurring deoxyribonucleosides and chloroacetaldehyde reaction products, predominantly ethenodeoxycytidine and ethenodeoxyadenosine. The same products resulted from hydrolysis of liver DNA prepared from rats that had been exposed to 250ppm VC in drinking-water for 2 yr. According to the studies of Kappus et al. (Toxic. appl. Pharmac. 1976.37, 461). the alkylation of protein and uptake of VC by rat-liver microsomes is dependent on concentration, incubation time, enzymatic activity, NADPH and oxygen, and is almost completely blocked by CO. These authors found that only about 1% of the VC taken up by the microsomes became irreversibly bound, a finding that contrasts with the corresponding figure of around 50% observed in the in uiuo studies of Bolt et al. (lot cit.). Glutathione added to the microsomal incubation mixture decreased the level of irreversible protein binding. When trichloropropene oxide, an inhibitor of epoxide hydrase, was present, the irreversible protein binding in-
BLACK
MARKS
The industrial solvent methyl rt-butyl ketone (MBK) has been implicated in cases of peripheral neuropathy among spray-painters and other workers (Cited in F.C.T. 1977, 15, 159) and has been shown to induce axonal degeneration of the central nervous system and polyneuropathy in experimental animals (ibid 1977, 15. 492). The metabolic fate and disposition of MBK in the rat has been studied by DiVincenzo et al. (Toxic. appl. Pharmac. 1977,41, 547) in male rats dosed by gavage with [I-‘4C]MBK at 20 or 200mg/kg. Absorption was rapid, and activity was eliminated in the breath and urine, mostly within 2 days. Unchanged MBK in the expired air amounted to 6.2% of the dose after 200 mg/kg and to 2.2% after 20 mg/kg. Total activity in the breath represented about 44% of either dose, with unchanged MBK and COZ as the only labelled compounds. Excretion of 14C in the urine amounted to 35% after 20mg/kg and to 40% after 2OOmg/kg; faecal excretion of 14C was less than 1.5% of the dose. About 15% of radioactivity remained in the carcass after 48 hr and 8% after 6 days. It was widely distributed throughout the tissues with highest concentrations in the blood and liver. The elimination time for MBK in the serum was about 6 hr, and the serum metabolites were 2-hexanol, 5-hydroxy-2-hexanone and 2,5-hexanedione. These three metabolites were also detected in the urine, together with 2,5-dimethylfuran, norleucine, y-valerolactone and urea. 2-Hex-
Cosmet. Toxicol.
Vol. 17, No. 6
creased two-fold, even though VC uptake by the microsomes was unaffected. Other in vitro studies have produced evidence of chloroethylene oxide’s participation in the metabolism of VC. The reaction product of chloroacetaldehyde or chloroethylene oxide with adenosine was tentatively characterized by Barbin et al. (Biochem. biophys. Res. Commun. 1975, 67, 596) as 3$-ribofuranosylimidazo-(2,I-i)-purine (I,@-ethenoadenosine); a product with the same RF value and elution characteristics on a Sephadex column was formed when VC was incubated with adenosine in the presence of a microsomal fraction. Barbin er al. (lot. cit.) passed a mixture of VC and air or oxygen into a medium containing both liver microsomes from a phenobarbitone-treated mouse and an NADPH-generating system. The volatile metabolite trapped by reaction with 4-(4nitrobenzyl)pyridine (CNBP) in ethylene glycol had a UV absorption spectrum identical to the product formed from the reaction of chloroethylene oxide (but not 2-chloroacetaldehyde) with 4-NBP. The evidence from these studies strongly supports the view that chloroethylene oxide is the major reactive VC metabolite. While it is clear that metabolites of VC do bind to cellular macromolecules in rat and mouse liver, the extent of nucleic acid binding has not been clearly resolved. [J. Hopkins-BIBRA]
FOR MBK anol was probably eliminated in the form of both the sulphate ester and the glucuronide. These findings identify the principal metabolic pathways as reduction of the ketone group or oxidation at the tl or w-1 carbon, followed apparently by decarboxylation of the metabolites with an a-keto acid component, the latter stage being the probable source of most of the respiratory 14C02. The a-keto acid intermediates may also undergo transamination to amino acids. Pretreatment of rats with unlabelled MBK or with phenobarbital did not materially alter the metabolism of [ I-14C]MBK, but inhibition of the microsomal mixed-function oxidase system by pretreatment with SKF 525A increased 14C02 excretion from 37.6 to 49.6x, after an initial 4-hr decrease, and decreased urinary activity from 39.9 to 22.50/ indicating the involvement of this microsomal-enzyme system in the -1 oxidation of MBK DiVincenzo et al. (ibid 1978, 44, 593) continued their studies with an investigation of the respiratory uptake and percutaneous absorption of MBK in dogs and man. Male beagles exposed to 50 or IOOppm MBK vapour for 6 hr had average breath concentrations of I6 and 35 ppm MBK, respectively, indicating an absorption rate of 65-68%. On cessation of exposure, the concentration of MBK in the breath fell rapidly and was below the level of detection after 3-5 hr. In male volunteers exposed to 10 or 50 ppm MBK for 7.5 hr or to 100 ppm MBK for 4 hr, mean
Articles of general interest--Fd Cosmet. Toxicol. Vol. 17, No. 6 concentrations of MBK in the breath were 1.4 and 9.3 ppm, respectively, for the two lower concentrations, with a rapid fall after cessation of exposure, and 22 ppm for the highest exposure. In no instance was MBK detected in the expired air 3 hr after cessation of exposure. Absorption was calculated to be 75-92x of the dose. Only after inhalation of 100 ppm MBK was the ketone detectable in serum. No MBK metabolites were detected in expired air or in urine, but 2,Shexanedione appeared in the serum after exposure to 50 or 1OOppm MBK, although not during the period of exposure. Skin absorption was determined in pairs of volunteers exposed to [I-“‘CIMBK or to a 9:1 (v/v) mixture of methyl ethyl ketone and [1-‘4C]MBK for 60 min (DiVincenzo et al. lot. cit.). Individual rates of absorption were 4.8 and 8.0pg/min/cm2 for MBK and 4.2 and 5.6 pg/min/cm’ for the mixture. After administration of an oral dose of 0.1 mg MBK/kg to two volunteers, respiratory excretion was 49.9 and 29.0% and urinary excretion was 27.6 and 25,0x, giving &day recoveries of 77.5 and 54.0%. Although most of the respiratory and urinary excretion occurred in the first 24 and 48 hr, respectively, some radioactivity was present in the expired air over the following 3-5 days and in the urine up to day 8. Thus excretion of MBK in man after percutaneous oral exposure seems to be relatively slow, so that repeated daily exposure to high concentrations of the solvent may result in prolonged exposure to its neurotoxic metabolites. The effects of MBK on nervous system function and behaviour in monkeys and rats have been reported by Johnson et al. (Am ind. Hyg. Ass. J. 1977, 38, 567) who exposed both species to 100 or 1000 ppm MBK for 6 hr daily on 5 days/wk. The animals were given neurological tests on a consistent monthly schedule, in which exposure on at least three consecutive days and then a l6-hr time lapse preceded each test period. After 25 wk, animals exposed to 1000 ppm MBK were removed from further exposure, because neurological and clinical observations indicated hind-limb neuropathy. Those exposed to IOOppm MBK continued for longer periods, the monkeys for 41 wk and the rats for 29 wk. After exposure to IOOOppm MBK, motor conduction velocities in ulnar and sciatic-tibia1 nerves were reduced in both animal species, and the amplitude of evoked muscle potentials decreased. The latency of specific components of evoked visual potentials was
683
lengthened in monkeys, while operant behavioural performance in a bar-pressing test was impaired in rats. Both species showed a reduction in body weight. After exposure to 1OOppm MBK there were less marked reductions in nerve conduction velocity and, in rats, also in evoked muscle action potential. At this level of exposure, it took 9 months to induce neuropathy in monkeys. Nerve conduction velocity returned to pre-exposure levels 6 months after cessation of exposure to IOOOppm MBK and 2 months after IOOppm MBK. Anger & Lynch (Enuir. Res. 1977, 14, 204) tested rats daily (in a I-hr test period) on a multiple fixedration fixed-interval 3-min reinforcement schedule. The rats were given MBK in an oral dose of 68, 135, 270 or 406 mg/kg on one day each week, control saline on three other days, and no treatment at all on the fifth day. Each animal received each concentration on three to six occasions in the course of the 6-month administration period. The performance response rate after MBK was reduced by 20, 30, 40 and 57% respectively, by these doses, but there were considerable individual differences. At the highest dose, MBK induced sluggishness of movement and unsteadiness in some animals, but no permanent weakness or foot drop was observed throughout the study. The effects of MBK (at levels of @I and 0.25%) and two of its metabolites, 2-hexanol (0.1 and 0.5%) and 2.5-hexanedione (0.1%) administered to guinea-pigs in drinking-water for 24 wk were examined by AbdelRahman et al. (Am. ind. Hyg. Ass. J. 1978, 39, 94). A group given 0.5% 2,5-hexanedione all died by wk 8, so this level of exposure was not studied further. All three compounds decreased pupillary response throughout the first 5 wk and greatly impaired the response by wk 24. Body weight increased and locomotor activity decreased. It was demonstrated that 2-hexanol was excreted in guinea-pig urine as MBK, 2-hexanol and 2,5-hexanedione, while the dione was excreted unchanged. The plasma of animals drinking 2-hexanol contained MBK and the dione. Severe polyneuropathy and mortality occurred in the group drinking @5% 2,5-hexanedione for a short period. The effect of 2-hexanol on pupillary response may have been due to its conversion to MBK and 2,5-hexanedione, and it is suggested that the observation of pupillary change may serve as an index in monitoring the degree of exposure of industrial workers-to MBK. [P. Cooper-BIBRA]
Articles of general interest-Fd
bated with VC and the microsomal system, labelled 3,N4-ethenocytidine moieties being formed (Laib & Bolt 1978, lot. cit.). The reaction of chloroacetaldehyde with calfthymus DNA at pH 4.5 gave a modified DNA product (Green & Hathway, Chemico-EioL Interactions 1978, 22, 21 I). Enzyme hydrolysis of the DNA produced a mixture of naturally occurring deoxyribonucleosides and chloroacetaldehyde reaction products, predominantly ethenodeoxycytidine and ethenodeoxyadenosine. The same products resulted from hydrolysis of liver DNA prepared from rats that had been exposed to 250ppm VC in drinking-water for 2 yr. According to the studies of Kappus et al. (Toxic. appl. Pharmac. 1976.37, 461). the alkylation of protein and uptake of VC by rat-liver microsomes is dependent on concentration, incubation time, enzymatic activity, NADPH and oxygen, and is almost completely blocked by CO. These authors found that only about 1% of the VC taken up by the microsomes became irreversibly bound, a finding that contrasts with the corresponding figure of around 50% observed in the in uiuo studies of Bolt et al. (lot cit.). Glutathione added to the microsomal incubation mixture decreased the level of irreversible protein binding. When trichloropropene oxide, an inhibitor of epoxide hydrase, was present, the irreversible protein binding in-
BLACK
MARKS
The industrial solvent methyl rt-butyl ketone (MBK) has been implicated in cases of peripheral neuropathy among spray-painters and other workers (Cited in F.C.T. 1977, 15, 159) and has been shown to induce axonal degeneration of the central nervous system and polyneuropathy in experimental animals (ibid 1977, 15. 492). The metabolic fate and disposition of MBK in the rat has been studied by DiVincenzo et al. (Toxic. appl. Pharmac. 1977,41, 547) in male rats dosed by gavage with [I-‘4C]MBK at 20 or 200mg/kg. Absorption was rapid, and activity was eliminated in the breath and urine, mostly within 2 days. Unchanged MBK in the expired air amounted to 6.2% of the dose after 200 mg/kg and to 2.2% after 20 mg/kg. Total activity in the breath represented about 44% of either dose, with unchanged MBK and COZ as the only labelled compounds. Excretion of 14C in the urine amounted to 35% after 20mg/kg and to 40% after 2OOmg/kg; faecal excretion of 14C was less than 1.5% of the dose. About 15% of radioactivity remained in the carcass after 48 hr and 8% after 6 days. It was widely distributed throughout the tissues with highest concentrations in the blood and liver. The elimination time for MBK in the serum was about 6 hr, and the serum metabolites were 2-hexanol, 5-hydroxy-2-hexanone and 2,5-hexanedione. These three metabolites were also detected in the urine, together with 2,5-dimethylfuran, norleucine, y-valerolactone and urea. 2-Hex-
Cosmet. Toxicol.
Vol. 17, No. 6
creased two-fold, even though VC uptake by the microsomes was unaffected. Other in vitro studies have produced evidence of chloroethylene oxide’s participation in the metabolism of VC. The reaction product of chloroacetaldehyde or chloroethylene oxide with adenosine was tentatively characterized by Barbin et al. (Biochem. biophys. Res. Commun. 1975, 67, 596) as 3$-ribofuranosylimidazo-(2,I-i)-purine (I,@-ethenoadenosine); a product with the same RF value and elution characteristics on a Sephadex column was formed when VC was incubated with adenosine in the presence of a microsomal fraction. Barbin er al. (lot. cit.) passed a mixture of VC and air or oxygen into a medium containing both liver microsomes from a phenobarbitone-treated mouse and an NADPH-generating system. The volatile metabolite trapped by reaction with 4-(4nitrobenzyl)pyridine (CNBP) in ethylene glycol had a UV absorption spectrum identical to the product formed from the reaction of chloroethylene oxide (but not 2-chloroacetaldehyde) with 4-NBP. The evidence from these studies strongly supports the view that chloroethylene oxide is the major reactive VC metabolite. While it is clear that metabolites of VC do bind to cellular macromolecules in rat and mouse liver, the extent of nucleic acid binding has not been clearly resolved. [J. Hopkins-BIBRA]
FOR MBK anol was probably eliminated in the form of both the sulphate ester and the glucuronide. These findings identify the principal metabolic pathways as reduction of the ketone group or oxidation at the tl or w-1 carbon, followed apparently by decarboxylation of the metabolites with an a-keto acid component, the latter stage being the probable source of most of the respiratory 14C02. The a-keto acid intermediates may also undergo transamination to amino acids. Pretreatment of rats with unlabelled MBK or with phenobarbital did not materially alter the metabolism of [ I-14C]MBK, but inhibition of the microsomal mixed-function oxidase system by pretreatment with SKF 525A increased 14C02 excretion from 37.6 to 49.6x, after an initial 4-hr decrease, and decreased urinary activity from 39.9 to 22.50/ indicating the involvement of this microsomal-enzyme system in the -1 oxidation of MBK DiVincenzo et al. (ibid 1978, 44, 593) continued their studies with an investigation of the respiratory uptake and percutaneous absorption of MBK in dogs and man. Male beagles exposed to 50 or IOOppm MBK vapour for 6 hr had average breath concentrations of I6 and 35 ppm MBK, respectively, indicating an absorption rate of 65-68%. On cessation of exposure, the concentration of MBK in the breath fell rapidly and was below the level of detection after 3-5 hr. In male volunteers exposed to 10 or 50 ppm MBK for 7.5 hr or to 100 ppm MBK for 4 hr, mean
Articles of general interest--Fd Cosmet. Toxicol. Vol. 17, No. 6 concentrations of MBK in the breath were 1.4 and 9.3 ppm, respectively, for the two lower concentrations, with a rapid fall after cessation of exposure, and 22 ppm for the highest exposure. In no instance was MBK detected in the expired air 3 hr after cessation of exposure. Absorption was calculated to be 75-92x of the dose. Only after inhalation of 100 ppm MBK was the ketone detectable in serum. No MBK metabolites were detected in expired air or in urine, but 2,Shexanedione appeared in the serum after exposure to 50 or 1OOppm MBK, although not during the period of exposure. Skin absorption was determined in pairs of volunteers exposed to [I-“‘CIMBK or to a 9:1 (v/v) mixture of methyl ethyl ketone and [1-‘4C]MBK for 60 min (DiVincenzo et al. lot. cit.). Individual rates of absorption were 4.8 and 8.0pg/min/cm2 for MBK and 4.2 and 5.6 pg/min/cm’ for the mixture. After administration of an oral dose of 0.1 mg MBK/kg to two volunteers, respiratory excretion was 49.9 and 29.0% and urinary excretion was 27.6 and 25,0x, giving &day recoveries of 77.5 and 54.0%. Although most of the respiratory and urinary excretion occurred in the first 24 and 48 hr, respectively, some radioactivity was present in the expired air over the following 3-5 days and in the urine up to day 8. Thus excretion of MBK in man after percutaneous oral exposure seems to be relatively slow, so that repeated daily exposure to high concentrations of the solvent may result in prolonged exposure to its neurotoxic metabolites. The effects of MBK on nervous system function and behaviour in monkeys and rats have been reported by Johnson et al. (Am ind. Hyg. Ass. J. 1977, 38, 567) who exposed both species to 100 or 1000 ppm MBK for 6 hr daily on 5 days/wk. The animals were given neurological tests on a consistent monthly schedule, in which exposure on at least three consecutive days and then a l6-hr time lapse preceded each test period. After 25 wk, animals exposed to 1000 ppm MBK were removed from further exposure, because neurological and clinical observations indicated hind-limb neuropathy. Those exposed to IOOppm MBK continued for longer periods, the monkeys for 41 wk and the rats for 29 wk. After exposure to IOOOppm MBK, motor conduction velocities in ulnar and sciatic-tibia1 nerves were reduced in both animal species, and the amplitude of evoked muscle potentials decreased. The latency of specific components of evoked visual potentials was
683
lengthened in monkeys, while operant behavioural performance in a bar-pressing test was impaired in rats. Both species showed a reduction in body weight. After exposure to 1OOppm MBK there were less marked reductions in nerve conduction velocity and, in rats, also in evoked muscle action potential. At this level of exposure, it took 9 months to induce neuropathy in monkeys. Nerve conduction velocity returned to pre-exposure levels 6 months after cessation of exposure to IOOOppm MBK and 2 months after IOOppm MBK. Anger & Lynch (Enuir. Res. 1977, 14, 204) tested rats daily (in a I-hr test period) on a multiple fixedration fixed-interval 3-min reinforcement schedule. The rats were given MBK in an oral dose of 68, 135, 270 or 406 mg/kg on one day each week, control saline on three other days, and no treatment at all on the fifth day. Each animal received each concentration on three to six occasions in the course of the 6-month administration period. The performance response rate after MBK was reduced by 20, 30, 40 and 57% respectively, by these doses, but there were considerable individual differences. At the highest dose, MBK induced sluggishness of movement and unsteadiness in some animals, but no permanent weakness or foot drop was observed throughout the study. The effects of MBK (at levels of @I and 0.25%) and two of its metabolites, 2-hexanol (0.1 and 0.5%) and 2.5-hexanedione (0.1%) administered to guinea-pigs in drinking-water for 24 wk were examined by AbdelRahman et al. (Am. ind. Hyg. Ass. J. 1978, 39, 94). A group given 0.5% 2,5-hexanedione all died by wk 8, so this level of exposure was not studied further. All three compounds decreased pupillary response throughout the first 5 wk and greatly impaired the response by wk 24. Body weight increased and locomotor activity decreased. It was demonstrated that 2-hexanol was excreted in guinea-pig urine as MBK, 2-hexanol and 2,5-hexanedione, while the dione was excreted unchanged. The plasma of animals drinking 2-hexanol contained MBK and the dione. Severe polyneuropathy and mortality occurred in the group drinking @5% 2,5-hexanedione for a short period. The effect of 2-hexanol on pupillary response may have been due to its conversion to MBK and 2,5-hexanedione, and it is suggested that the observation of pupillary change may serve as an index in monitoring the degree of exposure of industrial workers-to MBK. [P. Cooper-BIBRA]