TOXICOUHjY AND APPLIED PHARMACOLOGY%,295-302
(1979)
Perinatal Toxicity and Metabolism of n-Hexane in Fischer-344 Rats after Inhalation Exposure during Gestation* JAMES S. Bus, EARL L. WHITE, ROCHELLE W. TYL, AND CRAIG S. BARROW Chemical
Industry
Institute
of Toxicology, Departments of Biochemical Toxicology Research Triangle Park, North Carolina 27709
Received April
and Toxicology,
20, 1979; accepted August 10, 1979
Perinatal Toxicity and Metabolism of n-Hexane in Fischer-344 Rats after Inhalation Exposure during Gestation. Bus, J. S., WHITE, E. L., TYL, R. W., AND BARROW, C. S. (1979). Toxicol. Appl. Pharmacol. 51,295302. Repeated exposure of adult rats to n-hexane produces a central and peripheral neuropathy which may be mediated through metabolic activation to methyl n-butyl ketone (MBK) and 2,5hexanedione (2,5-HD). The perinatal toxicity of n-hexane and its metabolism in the pregnant rat have not been investigated. Pregnant rats were exposed for 6 hr per day to 1000 ppm n-hexane on Days 8-12, 12-16, or 8-16 of gestation. No significant alterations in fetal resorptions, body weights, visible anomalies, and the incidence of soft tissue and skeletal anomalies were noted in any of the treatment groups. The postnatal growth of pups born from dams exposed to 1000 ppm n-hexane 6 hr/day on Days 8-16 of gestation was significantly depressed compared to controls up to 3 weeks after birth (mean treated litter weight 13.9% less than control at 3 weeks). Litter weights of treated pups had returned to control values by 7 weeks after birth. n-Hexane was metabolized to MBK and 2,5-HD in pregnant rats exposed to 1000 ppm n-hexane on Day 20 of gestation. Concentrations of the three compounds in the fetus were approximately equal to those in maternal blood at all times after exposure. The half-life of 2,5-HD in maternal blood was significantly greater than n-hexane and MBK (3.90 hr vs 1.24 and 0.99 hr, respectively). Thus, n-hexane and its metabolites MBK and 2,5-HD may have only a minimal potential to alter perinatal development of rats.
n-Hexane is a widely used industrial solvent which produces a central and peripheral neuropathy in animals (Miyagaki, 1967; Schaumburg and Spencer, 1976; Altenkirch et al., 1978) and in man in the presence of other solvents (Herskowitz et al., 1971; Altenkirch, 1977). The nerve lesion is characterized by multifocal giant axonal swellings associated with accumulations of lo-nm neurofilaments within the axon (Schaumburg and Spencer, 1976). The similarity in the nerve lesion to that induced by methyl n-butyl ketone (MBK) and 2,5hexanedione (2,5-HD), coupled with the I This manuscript presented in part at the Annual Meeting of the Teratology Society, 1978.
observation that 2,5-HD has been detected as a metabolite of both n-hexane and MBK (Abdel-Rahman et al., 1976; DiVincenzo et al., 1976, 1977), suggests that 2,5-HD may be the common neurotoxic metabolite in hexacarbon neuropathy. The biochemical mechanism responsible for hexacarbon neuropathy is unclear, although an interruption of fast axonal transport (Mendell et al., 1977) possibly mediated through an interference of glycolytic energy production (Sabri et af., 1979) has been suggested, The devloping animal undergoing rapid nervous system development, therefore, may be particularly sensitive to n-hexane exposure. The purpose of this study was to in-
295 All
0041-008x/79/140295-08s02.00/0 Copyright 0 1979 by Academic Press, Inc. rights of reproduction in any form reserved. Printed in Great Britain
296
BUS ET AL.
vestigate the perinatal toxicity of n-hexane following inhalation exposure. In addition, the metabolism of n-hexane to MBK and 2,5-HD by the pregnant rat and the potential for these metabolites to reach the fetus was investigated. MATERIALS
AND
METHODS
Animals Timed pregnant Fischer-344 rats were obtained from the Charles River Company (Wilmington, Mass.). The day on which a vaginal plug was found was designated Day I of gestation. All rats were housed in plastic cages containing hardwood chip bedding (Betta-Chip, Northeastern Products, Warrensburg, N.Y.) and allowed access to food (Wayne Lab Blox, Allied Mills, Chicago, Ill.) and water ad libitum. Inhalation Exposures Pregnant rats were exposed to 1000 ppm n-hexane (99.0x, Phillips Chemical Co., Bartlesville, Ok.) in a 1lOO-liter glass and stainless-steel chamber operated at an air flow of 300 liters/min for 6 hr/day during various periods of gestation. n-Hexane was introduced into the chamber by volatilizing 1.6 mI/min n-hexane in a heated air stream (7.5 literslmin) which was subsequently merged with the main chamber air intake. Chamber n-hexane concentrations were monitored three times per hour by infrared spectrometry (Wilkes Model 80, Norwalk, Conn.) at a wavelength of 3.33 pm. Concentrations of n-hexane in the chamber did not vary more than 5 % of nominal in any exposure period. All rats were continuously housed in hanging stainless-steel cages in the chamber during the treatment periods, with food and water removed for each 6-hr n-hexane exposure period. In the perinatal nhexane toxicity studies, control rats were housed under identical conditions in a separate inhalation chamber and exposed only to room air. Perinatal Toxicity Pregnant rats were exposed to n-hexane on Days 8-12, 12-16, or 8-16 of gestation. On Day 22 of gestation the females were sacrificed by ether anesthesia, the uterine horns externalized, and the number and position of live, dead, and resorbed fetuses recorded. Fetuses were removed, dried, weighed, and examined for externally visible defects. The litters were equally divided for fixation in Bouin’s solution or 9.5% ethanol for soft tissue or skeletal examination, respectively. Soft-tissue examination of the head
was conducted by hand sectioning as described by Wilson (1965). Soft tissues of the thoracic and abdominal cavities were examined in situ under a dissecting microscope. Heart and kidneys were further studied after transverse sectioning. Fetuses fixed in ethanol were cleared, stained with alizarin red S, and examined for skeletal anomalies (Hurley, 1965). In other experiments, pregnant rats were exposed to 1000 ppm n-hexane on Days 8-16 of gestation. Following delivery of the pups on Day 23 of gestation, litters were culled to six pups per litter and total litter body weights and mortality recorded at weekly intervals up to 7 weeks after birth. All litters were weaned 4 weeks after birth. Disposition Studies Pregnant rats were exposed to 1000 ppm n-hexane on Day 12 or 20 of gestation alone or on Days 15-18 of gestation. At various times directly after the exposure regimens the rats were killed by decapitation, maternal blood collected in tubes containing heparin, and samples of maternal liver, kidney, and brain and whole fetus (three fetuses/litter on Day 20 gestation; entire litter on Day 12 gestation) obtained. All tissues were weighed, frozen in a dry ice bath, and stored at - 20°C prior to analysis. Tissue samples were simultaneously analyzed for the presence of n-hexane, MBK, and 2,5-HD by gas chromatography-mass spectrometry (gems) employing stable isotope-labeled internal standards (White et al., 1979). n-Hexane (Burdick and Jackson, UV grade) was used as received. Acetone (Fisher Chemical Co., Springfield, NJ.) was purified by heating the solvent to reflux with KMnO, followed by distillation. n-[zH,,]Hexane (98 atom% deuterium) was purchased from Merck. 2-[*H,]Hexanone and 2,5[ZH,,]hexanedione were synthesized by base-catalyzed exchange in 2H20. Purity of the synthesized products was established by gcrns. Prior to gems analysis the tissues were thawed, placed in an ice bath, and acetone containing the deuterated standards (3.0 pg/ml for each of the three standards) was added (l/l w/v or l/l v/v). Tissues were homogenized with a Tekmar Tissumizer (Cincinnati, Ohio) at 0°C for 30 set and centrifuged for 15 min at 2000g at 4°C. No samples were allowed to stand at 0°C for more than 30 min before gems analysis. Analyses were conducted using a Finnigan Model 4023 gas chromatograph-mass spectrometer (Sunnyvale, Calif.) equipped with an Incas data system. Gas-liquid chromatography was performed using a 6 ft x 2 mm id glass column packed with SO/l00 mesh Carbopack C/O.1 % SP-1000 (Supelco) and operated isothermally at 180°C. For liver the temperature was programmed from 120 to 170°C at S”C/min. The injector and glass jet separator temperatures were
297
n-HEXANE TOXICITY AND METABOLISM maintained at 200 and 235°C respectively. The column flow rate was 13 ml/min. Aliquots (1 ~1) of the acetone supernatants were injected and the column effluent monitored selectively at the molecular ions of the labeled and unlabeled isotopic variants (m/z 100 and 114 for MBK and 2,5-HD respectively, m/z 105 and 124 for deuterated MBK and 2,5-HD) and fragment ion m/z 57 of n-hexane. Statistics
Data were analyzed by analysis of variance, completely randomized design. The level of significance was chosen as p
(1979)
Perinatal Toxicity and Metabolism of n-Hexane in Fischer-344 Rats after Inhalation Exposure during Gestation* JAMES S. Bus, EARL L. WHITE, ROCHELLE W. TYL, AND CRAIG S. BARROW Chemical
Industry
Institute
of Toxicology, Departments of Biochemical Toxicology Research Triangle Park, North Carolina 27709
Received April
and Toxicology,
20, 1979; accepted August 10, 1979
Perinatal Toxicity and Metabolism of n-Hexane in Fischer-344 Rats after Inhalation Exposure during Gestation. Bus, J. S., WHITE, E. L., TYL, R. W., AND BARROW, C. S. (1979). Toxicol. Appl. Pharmacol. 51,295302. Repeated exposure of adult rats to n-hexane produces a central and peripheral neuropathy which may be mediated through metabolic activation to methyl n-butyl ketone (MBK) and 2,5hexanedione (2,5-HD). The perinatal toxicity of n-hexane and its metabolism in the pregnant rat have not been investigated. Pregnant rats were exposed for 6 hr per day to 1000 ppm n-hexane on Days 8-12, 12-16, or 8-16 of gestation. No significant alterations in fetal resorptions, body weights, visible anomalies, and the incidence of soft tissue and skeletal anomalies were noted in any of the treatment groups. The postnatal growth of pups born from dams exposed to 1000 ppm n-hexane 6 hr/day on Days 8-16 of gestation was significantly depressed compared to controls up to 3 weeks after birth (mean treated litter weight 13.9% less than control at 3 weeks). Litter weights of treated pups had returned to control values by 7 weeks after birth. n-Hexane was metabolized to MBK and 2,5-HD in pregnant rats exposed to 1000 ppm n-hexane on Day 20 of gestation. Concentrations of the three compounds in the fetus were approximately equal to those in maternal blood at all times after exposure. The half-life of 2,5-HD in maternal blood was significantly greater than n-hexane and MBK (3.90 hr vs 1.24 and 0.99 hr, respectively). Thus, n-hexane and its metabolites MBK and 2,5-HD may have only a minimal potential to alter perinatal development of rats.
n-Hexane is a widely used industrial solvent which produces a central and peripheral neuropathy in animals (Miyagaki, 1967; Schaumburg and Spencer, 1976; Altenkirch et al., 1978) and in man in the presence of other solvents (Herskowitz et al., 1971; Altenkirch, 1977). The nerve lesion is characterized by multifocal giant axonal swellings associated with accumulations of lo-nm neurofilaments within the axon (Schaumburg and Spencer, 1976). The similarity in the nerve lesion to that induced by methyl n-butyl ketone (MBK) and 2,5hexanedione (2,5-HD), coupled with the I This manuscript presented in part at the Annual Meeting of the Teratology Society, 1978.
observation that 2,5-HD has been detected as a metabolite of both n-hexane and MBK (Abdel-Rahman et al., 1976; DiVincenzo et al., 1976, 1977), suggests that 2,5-HD may be the common neurotoxic metabolite in hexacarbon neuropathy. The biochemical mechanism responsible for hexacarbon neuropathy is unclear, although an interruption of fast axonal transport (Mendell et al., 1977) possibly mediated through an interference of glycolytic energy production (Sabri et af., 1979) has been suggested, The devloping animal undergoing rapid nervous system development, therefore, may be particularly sensitive to n-hexane exposure. The purpose of this study was to in-
295 All
0041-008x/79/140295-08s02.00/0 Copyright 0 1979 by Academic Press, Inc. rights of reproduction in any form reserved. Printed in Great Britain
296
BUS ET AL.
vestigate the perinatal toxicity of n-hexane following inhalation exposure. In addition, the metabolism of n-hexane to MBK and 2,5-HD by the pregnant rat and the potential for these metabolites to reach the fetus was investigated. MATERIALS
AND
METHODS
Animals Timed pregnant Fischer-344 rats were obtained from the Charles River Company (Wilmington, Mass.). The day on which a vaginal plug was found was designated Day I of gestation. All rats were housed in plastic cages containing hardwood chip bedding (Betta-Chip, Northeastern Products, Warrensburg, N.Y.) and allowed access to food (Wayne Lab Blox, Allied Mills, Chicago, Ill.) and water ad libitum. Inhalation Exposures Pregnant rats were exposed to 1000 ppm n-hexane (99.0x, Phillips Chemical Co., Bartlesville, Ok.) in a 1lOO-liter glass and stainless-steel chamber operated at an air flow of 300 liters/min for 6 hr/day during various periods of gestation. n-Hexane was introduced into the chamber by volatilizing 1.6 mI/min n-hexane in a heated air stream (7.5 literslmin) which was subsequently merged with the main chamber air intake. Chamber n-hexane concentrations were monitored three times per hour by infrared spectrometry (Wilkes Model 80, Norwalk, Conn.) at a wavelength of 3.33 pm. Concentrations of n-hexane in the chamber did not vary more than 5 % of nominal in any exposure period. All rats were continuously housed in hanging stainless-steel cages in the chamber during the treatment periods, with food and water removed for each 6-hr n-hexane exposure period. In the perinatal nhexane toxicity studies, control rats were housed under identical conditions in a separate inhalation chamber and exposed only to room air. Perinatal Toxicity Pregnant rats were exposed to n-hexane on Days 8-12, 12-16, or 8-16 of gestation. On Day 22 of gestation the females were sacrificed by ether anesthesia, the uterine horns externalized, and the number and position of live, dead, and resorbed fetuses recorded. Fetuses were removed, dried, weighed, and examined for externally visible defects. The litters were equally divided for fixation in Bouin’s solution or 9.5% ethanol for soft tissue or skeletal examination, respectively. Soft-tissue examination of the head
was conducted by hand sectioning as described by Wilson (1965). Soft tissues of the thoracic and abdominal cavities were examined in situ under a dissecting microscope. Heart and kidneys were further studied after transverse sectioning. Fetuses fixed in ethanol were cleared, stained with alizarin red S, and examined for skeletal anomalies (Hurley, 1965). In other experiments, pregnant rats were exposed to 1000 ppm n-hexane on Days 8-16 of gestation. Following delivery of the pups on Day 23 of gestation, litters were culled to six pups per litter and total litter body weights and mortality recorded at weekly intervals up to 7 weeks after birth. All litters were weaned 4 weeks after birth. Disposition Studies Pregnant rats were exposed to 1000 ppm n-hexane on Day 12 or 20 of gestation alone or on Days 15-18 of gestation. At various times directly after the exposure regimens the rats were killed by decapitation, maternal blood collected in tubes containing heparin, and samples of maternal liver, kidney, and brain and whole fetus (three fetuses/litter on Day 20 gestation; entire litter on Day 12 gestation) obtained. All tissues were weighed, frozen in a dry ice bath, and stored at - 20°C prior to analysis. Tissue samples were simultaneously analyzed for the presence of n-hexane, MBK, and 2,5-HD by gas chromatography-mass spectrometry (gems) employing stable isotope-labeled internal standards (White et al., 1979). n-Hexane (Burdick and Jackson, UV grade) was used as received. Acetone (Fisher Chemical Co., Springfield, NJ.) was purified by heating the solvent to reflux with KMnO, followed by distillation. n-[zH,,]Hexane (98 atom% deuterium) was purchased from Merck. 2-[*H,]Hexanone and 2,5[ZH,,]hexanedione were synthesized by base-catalyzed exchange in 2H20. Purity of the synthesized products was established by gcrns. Prior to gems analysis the tissues were thawed, placed in an ice bath, and acetone containing the deuterated standards (3.0 pg/ml for each of the three standards) was added (l/l w/v or l/l v/v). Tissues were homogenized with a Tekmar Tissumizer (Cincinnati, Ohio) at 0°C for 30 set and centrifuged for 15 min at 2000g at 4°C. No samples were allowed to stand at 0°C for more than 30 min before gems analysis. Analyses were conducted using a Finnigan Model 4023 gas chromatograph-mass spectrometer (Sunnyvale, Calif.) equipped with an Incas data system. Gas-liquid chromatography was performed using a 6 ft x 2 mm id glass column packed with SO/l00 mesh Carbopack C/O.1 % SP-1000 (Supelco) and operated isothermally at 180°C. For liver the temperature was programmed from 120 to 170°C at S”C/min. The injector and glass jet separator temperatures were
297
n-HEXANE TOXICITY AND METABOLISM maintained at 200 and 235°C respectively. The column flow rate was 13 ml/min. Aliquots (1 ~1) of the acetone supernatants were injected and the column effluent monitored selectively at the molecular ions of the labeled and unlabeled isotopic variants (m/z 100 and 114 for MBK and 2,5-HD respectively, m/z 105 and 124 for deuterated MBK and 2,5-HD) and fragment ion m/z 57 of n-hexane. Statistics
Data were analyzed by analysis of variance, completely randomized design. The level of significance was chosen as p
