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Dose-dependent suppression of toluene metabolism by isopropyl alcohol and methyl ethyl ketone after experimental exposure of rats
Toxicology Letters ELSFVIER
Toxicology Letters 81 (1995) 229-234
Dose-dependent suppression of toluene metabolism by isopropyl alcohol and methyl ethyl ketone after experimental exposure of rats Hirohiko Uakia.b, Toshio Kawaic, Kazunori Mizunumac, Chan-Seok Moon b, Zuo-Wen Zhangb, Shunen Inui”, Shiro Takada”, Masayuki Ikeda*b aKyoto Indusirial Health Association, Kyoto 604, Japan bDepartment of Public Health, Kyoto Universiry Faculty of Medicine, Kyoto 606-01, Japan COsaka Occupational Health Service Center, Osaka 550, Japan
Received 14 June 1994;revision received 1I August 1995; accepted 11 August 1995
Abstract In order to examine possible suppression of toluene metabolism due to coexposure to other solvents, female Wistar rats were exposed for 8 h to toluene alone (at 50 or 100 ppm), or in combination with either methyl ethyl ketone (at 50, 100, 200 or 400 ppm) or isopropyl alcohol (at 50, 100, 200, 400, 800 or 1600 ppm). Urine samples were collected for 24 h after initiation of each exposure, and subjected to analysis for two toluene metabolites, hippuric acid and ocresol, both by HPLC. The excretion of hippuric acid, a major metabolite, was not moditied when the concentrations of methyl ethyl ketone or isopropyl alcohol were low, i.e. 100 ppm or below, whereas it was reduced when methyl ethyl ketone or isopropyl alcohol concentrations were twice or more times higher than that of toluene. There were no changes in any cases in excretion of o-cresol, a minor metabolite. The observation after coexposure to methyl ethyl ketone or isopropyl alcohol at low concentration is in line with the negative interaction between toluene and methyl ethyl ketone as well as between toluene and isopropyl alcohol after occupational exposures at low concentrations. Metabolic interaction may take place when the exposure intensity is high, as observed in the present study and also after experimental exposure of volunteers to toluene and m-xylene, or occupational exposure to benzene and toluene. Keyworris:
Combined exposure; Isopropyl alcohol; Metabolism modification;
1. Iatroduetion Possible modification of toxicity of individual chemicals after combined exposure has been a * Corresponding author. Tel.: +81 75 753 4460; fax: +81 75 753 4466.
focus of interest in toxicology of chemicals [l-3], especially solvents [4,5] because solvents in industrial products are generally mixtures except for degreasers [6,7]. During a course of a study to investigate the possible metabolic interaction between solvents as the basis for toxicity modification [8], in a factory survey, our study group has
0378-4274/95/$09.50 0 1995 Elsevier Science Ireland Ltd. All rights reserved SSDI 0378-4274(95)03445-Q
Methyl ethyl ketone; Toluene
230
H. Uuki CI ul : T’ox~colog~~Lerrrrs HI i 1995) 220-234
observed that the metabolism of toluene in solvent workers will not be affected by the coexposure to either isopropyl alcohol or methyl ethyl ketone when the intensities of exposure to these solvents are low [9]. It is highly probable, however, that the extent of metabolism modification is dose-dependent and that metabolic interaction will take place at higher doses, if not detectable at lower doses. A series of animal exposure experiments were conducted to confirm the negative findings on metabolic interaction at lower doses and to investigate possible metabolic interaction at higher doses. 2. Materials and methods 2.1. Exposure of animals and urine sample collection
Female Wistar rats weighing about 200 g were used. For experimental exposure to solvent vapors, two animals were housed together in a metabolic cage (for rats; Natsume Seisakusho. Tokyo, Japan) to obtain enough urine samples for analysis, and four pairs were exposed per exposure condition. Solvent vapor exposure was conducted with a servomechanized dynamic flow-type exposure system [lo], in which the exposure to a mixture of two solvent vapors was possible. The vapor concentrations were automatically measured every 24 min by FID-gas chromatography so that, even when mixed, the two solvent vapors were monitored separately. The coefficient of variation in vapor concentration during each exposure was less than 5% [lo]. Exposure continued for 8 h and urine samples were collected for 24 h after the initiation of exposure, taking the biological half-time of toluene (about 7-8 h in man [l I], and presumably shorter than 8 h in rats) into consideration. At the end of the 24-h urine collection period, the funnel at the bottom of the cage was rinsed with a minimum amount of water, and the wash was pooled with urine to make up the volume to about 50 ml. 2.2. Analysis for urinary metabolites The urine samples were analyzed for two urinary toluene metabolites, hippuric acid and o-
cresol. Hippuric acid was determined by the HPLC method as previously described [ 111. For ocresol determination, the HPLC system consisted of an automated liquid sampler (Hitachi 655-40), an HPLC pump (Hitachi L-6200), a fluorescence spectrophotometer (Hitachi F-4010), and a data processor (Hitachi D-2000) was used. A column (30 cm in length and 4 mm in diameter, packed with ODS-80A (4 5 pm)) was used for chromatography with a mixture of acetonitrile and water (30:70 by volume) as a mobile phase. The mobile phase was allowed to flow at 0.8 ml/min. The column was kept at 35°C. Fluorescence of the effIuent was monitored with excitation at 278 nm and emission at 302 nm (band pass 5 nm for both). For analysis, 5 ml of each urine sample was taken in a well-capped tube, mixed with 2 ml cont. hydrochloric acid and heated at 100°C for I h for hydrolysis. After cooling down to room temperature, the hydrolyzate was extracted with 2 ml isopropyl ether by vigorous shaking for 1 min. A 5-~1 aliquot of the extract was introduced to the HPLC system per injection. The detection limit for o-cresol was 0.1 mg/l under the standard analytical conditions. The recovery of o-cresol added to urine was 99% of o-cresol added to water. The coefficient of variation was 3.2% when the same sample was analyzed five times. 2.3. Statistical analysis The possible difference in excretion amounts was analyzed by Student’s t-test. 3. Results 3.1. Dose-dependent increase in urinary hippuric acid and o-cresol It is known that hippuric acid is endogenous and is excreted in urine even when there is no exposure to toluene [ 111. Accordingly, the exposureexcretion relationship was examined between toluene vapor concentration and either hippuric acid or o-cresol in urine. The results are summarized in Fig. 1. It is clear from the figure that urinary hippuric acid excretion increased from a physiological level of 36 mg/kg/24 h (as a mean) to higher levels as a linear function of the intensity of exposure to toluene. No o-cresol was detected in
H. Uaki et al. / Toxicology Letters 81 (1995) 229-234
231
the urine of nonexposed animals and the amount of ocresol increased in a linear relation to toluene exposure. 3.2. Suppression of toluene metabolism by coexposure to methyl ethyl ketone
In the first series of experiments, rats were exposed to methyl ethyl ketone in combination with toluene. The results are expressed as the ratios to hippuric acid and ocresol after exposure to toluene alone, to make the changes more readily understandable. When rats were exposed to methyl ethyl ketone at O-200 ppm in addition to 50 ppm toluene (Table l), there was a slight reduction in hippuric acid as an inverse function of an increase in coexposed methyl ethyl ketone concentration. The reduction became statistically signilicant (P < 0.05) when methyl ethyl ketone coexposure was at 200 ppm. In contrast, there was essentially no change in ocresol. When toluene concentration was elevated to 100 ppm, and that of methyl ethyl ketone to 200 or 400 ppm, the reduction in hippuric acid was significant (P < 0.05) at 200 ppm methyl ethyl ketone and even more evident (P c 0.01) at 400 ppm methyl ethyl ketone. No change was observed, however, in o-cresol excretion.
TOLUENE IN AIR (ppm)
Fig. I. Amount of hippuric acid and ocresol excreted in urine during the 34-h period after initiation of 8-h toluene vapor exposure. Solid circles and arrows show mean and S.D. of four determinations each. The lines are regression lines.
3.3. Suppression of toluene metabolism by coexposure to isopropyl alcohol
In the second series of experiments, rats were ex-
Table I Amount of hippuric acid and o-cresol in urine after coexposure to methyl ethyl ketone, relative to the amount after exposure to toluene alone Coexposure (ppm) to
Urinary metabolitea
Toluene
Methyl ethyl ketone
Hippuric acid
o-Cresol
50 50 50 50 50
50 100 200 200 400
98.9 91.4 88.8 79.6 64.5
101.6 f 9.5 105.1 zt 6.8 94.5 f 4.0 103.8 zt 24.0 91.5 f 16.9
zt f f zt f
1.9 8.2 s.o** l3.7* 7.9.’
Mean & SD. of four determinations each are given. ‘The amount of hippuric acid and otresol are expressed relative (i.e. in percent) to the corresponding amounts after exposure to toluene alone at 50 or 100 ppm, respectively. Asterisks indicate that the difference from the controls (i.e. the amount after exposure to toluene alone at 50 or 100ppm) is statistically significant (**P < 0.01; *P < 0.05).
Table 2 Amount of hippuric alone Coexposure
acid and o-cresol in urine after coexposure
(ppm) to
Toluene
Isopropyl
50 50
50 100
50 100 100 100
200 200 400 800
100
1600
IJrinary alcohol
to isopropyl
alcohol,
relative to the amount
after exposure
to toluene
metabolite”
Hippuric acid _I__.__ 99.7 f 6.5 88.7 f 4.1’ 88.8 f 4.2* X6.3 f Il.1 x7.3 f 9.2 79.3 f 8.0** 64.4 f 1.5” -.
0-C‘resol --_~.91.2 * 949 f liJO.0 f 106.5 f 102.3 f 104.2 f IOX. f -.-
77 16.3 14.3 22.7 4.8 10.2 4.0 -
Notes are as for Table I.
posed to isopropyl alcohol in addition to toluene (Table 2). When rats were exposed to isopropyl alcohol at various concentrations and toluene at 50 ppm, a slight but significant (P < 0.05) reduction in hippuric acid was observed at 100 ppm and 200 ppm isopropyl alcohol. When toluene concentration was elevated to 100 ppm and isopropyl alcohol coexposure was increased up to 1600 ppm, a significant (P < 0.01) reduction was observed in hippuric acid at 800 ppm isopropyl alcohol, and the reduction was more marked (P < 0.01) at 1600 ppm isopropyl alcohol. The amount of o-cresol however stayed unchanged in all cases. 4. Discussion In this study using rats, a linear relationshlp was observed between toluene exposure and urinary excretion of hippuric acid as well as o-cresol as observed in humans [l 11. When the animals were coexposed to methyl ethyl ketone or isopropyl alcohol, there was a reduction in hippuric acid excretion in a manner inversely related to the concentration of the solvent coexposed. Thus, the reduction was insignificant when the concentration of the coexposed solvent was low, e.g. 50 ppm methyl ethyl ketone or isopropyl alcohol in combination with 50 ppm toluene (Tables 1,2) and turned out to be significant only when the coexposure was at high concentrations (e.g. 100 ppm toluene plus 400 ppm methyl ethyl ketone, or 100 ppm toluene plus 800 ppm isopropyl alcohol). It is
worth noting that the current occupational exposure limits for toluene, methyl ethyl ketone and isopropyl alcohol are set at 50 ppm, 200 ppm and 400 ppm, respectively [ 12-141. No changes were observed in the minor metabolite, o-cresol. The reduction in hippuric acid after coexposure to methyl ethyl ketone or isopropyl alcohol at the concentration of twice or four times that of toluene tended to be more evident at 100 ppm toluene than at 50 ppm toluene. This may be due to the fact that hippuric acid is present in the urine from nonexposed rats. The negative observation of hippuric acid excretion after coexposure to methyl ethyl ketone or isopropyl alcohol at low levels is in agreement with the recent observation by this study group in a survey of factory workers 191. In the factories surveyed, the workers were exposed to solvent mixtures in which toluene was a major contaminant in the workroom air (geometric mean vapor concentration; 18 ppm) but methyl ethyl ketone (16 ppm) and isopropyl alcohol (7 ppm) were also present. The present negative findings on possible metabolic interactions between toluene and methyl ethyl ketone or toluene and isopropyl alcohol when the concentrations were low are also in line with the study results by Tardif et al. [5], Kawai et al. [15], Inoue et al. [16] and Huang et al. [17], and offer experimental support to the hypothesis that the chances of metabolic interaction occurring between the solvents coexposed are remote when the exposure intensity is low [9]. It is also worth noting that metabolism of tol-
H. Uaki et al. / Toxicology Letters 81 (1995) 229-234
uene is suppressed by methyl ethyl ketone and isopropyl alcohol when the concentration of the latter solvents are several times higher than that of toluene. Metabolic interactions at higher concentrations are also reported by Inoue et al. [6] and Tardif et al. [5], between benzene and toluene, and toluene and m-xylene, respectively. There is no proposed mechanism to explain the observed suppression of toluene metabolism by methyl ethyl ketone and isopropyl alcohol. However, the suppression may be due to competition for alcohol dehydrogenase which oxidizes benzyl alcohol (an oxidation product of toluene at sidechain methyl moiety) to benzoic acid, as is the case with suppression of methanol oxidation by coadministered ethanol [19]. Methyl ethyl ketone is also known to be converted in vivo to 2-butanol [20]. From a quantitative viewpoint, however, it may be the case that the amount of the 2-butanol formed in vivo is too low to explain the observed suppression. In fact, methyl ethyl ketone appears to be as potent as or even more potent than isopropyl alcohol, when the same absorption rate in the lungs is assumed; methyl ethyl ketone at 400 ppm (Table 1) is equally as suppressive as isopropyl alcohol at 1600 ppm (Table 2) when coexposed to 100 ppm toluene. Thus, it might be necessary to look for a mechanism other than competition for the alcohol derived from methyl ethyl ketone. Acknowledgements This work was supported in part by a grant-inaid for cooperative study (grant No. 05304030: principal investigator, M. Ikeda) from the Ministry of Education, Science and Culture of the Government of Japan to T.K. and M.I. References 111Jonker, D., Woutersen, R.A., van Bladernen, P.J., Til, H.P. and Peron, V.J. (1990) Cweek oral toxicity study of combination of eight chemicals in rats: comparison with the toxicity of the individual compounds. Food Chem. Toxicol. 28, 623-63 I. I21 Jonker, D., Jones, M.A., van Bladernen, P.J., Woutersen, R.A., Til, H.P. and Peron, V.J. (1993) Acute oral toxicity of a combination of four nephrotoxicants in
233
rats compared with the toxicity of the individual compounds. Food Chem. Toxicol. 31, 45-52. 131 Ikeda, M. (1994) Complex exposures: potentials for assessing integrated exposure. Clin. Chem. 40, 1444-1447. I41 Ikeda, M. (1988) Multiple exposure to chemicals. Regul. Toxicol. Pharmacol. 8, 414-421. 151 Tardif, R., Lapare, S., Plaa, G.L. and Brodeur, J. (1991) Effect of simultaneous exposure to toluene and xylene on their respective biological exposure indices in humans. Int. Arch. Occup. Environ. Health 63, 279-284. b51 Inoue, T., Takeuchi, Y., Hisanaga, N., Ono, Y., Iwata, M., Ogata, M., Saito, K., Sakurai, H., Hara, I., Matsushita, T. and Ikeda, M. (1983) A nationwide survey on organic solvent components in various solvent products: Part I. Homogeneous products such as thinners, degreasers and reagents. Ind. Health 2 1, 175- 183. I71 Kumai, M., Koizumi, A., Saito, K., Sakurai, H., Inoue, T., Takeuchi, Y., Hara, I., Ogata, M., Matsushita, T. and Ikeda, M. (I 983) A nationwide survey on organic solvent components in various solvent products: Part II. Heterogeneous products such as paints, inks and adhesives. Ind. Health 21, 185-197. 181 Ikeda, M. (1983) Interaction and health aspects of exposure to mixtures of organic solvents. In: SW. Andnejewski and S. Tarkowski (Eds.), Health Aspects of Combined Exposure to Chemicals in Work and Community Environments. WHO Regional Office for Europe, Copenhagen, Denmark, pp. 77-97. 191 Ukai, H., Tanaka, S., Inui, S., Imai, Y., Kawai, T., Shimbo, S., and Ikeda, M. (1994) Occupational exposure to solvent mixtures; effects on health and metabolism. Occup. Environ. Med. IlO1 Koizumi, A. and Ikeda, M. (1981) A servomechanism for vapor concentration control in experimental exposure chambers. Am. Ind. Hyg. Assoc. J. 42, 417-425. HII Hasegawa, K., Shiojima, S., Koizumi, A. and Ikeda, M. (1983) Hippuric acid and o-cresol in the urine of workers exposed to toluene. Int. Arch. Occup. Environ. Health 52, 197-208. Industrial iI21 American Conference of Governmental Hygienists (I 994) I994- 1995 Threshold Limit Values and Biological Exposure Indices. ACGIH, Cincinnati, USA. 1131 Deutsche Forschungsgemeinschaft (1994) List of MAK and BAT Values 1994. VCH, Weinheim. [I41 Japan Association of Industrial Health (1994) Recommended occupational exposure limits, 1994. Jpn. J. Ind. Health 36, 236-260 (in Japanese). [IS1 Kawai, T., Yasugi, T., Mizunuma, K., Horiguchi, S., Morioka, I., Miyashita, K., Uchida, Y. and Ikeda, M. (1992) Monitoring of workers exposed to a mixture of toluene, styrene and methanol vapours by means of diffusive air sampling, blood analysis and urinalysis. Int. Arch. Occup. Environ. Health 63, 429-435. 1161 Inoue, 0.. Seiji, K., Kawai, T., Watanabe, T., Jin, C., Cai, S.-X., Chen, Z., Qu, Q.-S., Zhang, T. and I keda, M.
H. Uuki r/ ul. , Tosico/o~~ Lrrrws 81 I IYYS) 229-234
234
(1993) Excretion
of methylhippuric
acids
in urine
of
workers exposed to xylene mixture; comparison among three xylene isomers and toluene. Int. Arch. Occup. Environ. Health 64, 533-539. [17] Huang, M.-Y., Jin, C., Liu, Y.-T., Li, B.-H.. Qu, Q.-S., Uchida, Y., Inoue, O., Nakatsuka, H.. Watanabe, T. and Ikeda, M. (1994) Exposure of workers to a mixture of toluene and xylenes. I. Metabolism. Occup. Environ, Med. 5 I, 42-46. [18] Inoue, O., Seiji, K., Watanabe, T., Kasahara, M., Nakatsuka, H., Yin, S.-N., Li, G.-L., Cai, S.-X., Jin, C. and
Ikeda, M. (1988) Mutual metabolic suppression benzene and toluene in man. Int. Arch. Occup. Health 60, 15-20. [19]
[20]
between Environ.
Proctor, N.H. and Hughes, J.P. (1978) Chemical Hazards of the Workplace. Lippincott, Philadelphia, PA, pp. 329-330. DiVincenzo, G.D., Kaplan. C.J. and Dedinas, J. (1976) Characterization of the metabolites of methyl n-butyl ketone, methyl isobutyl ketone and methyl ethyl ketone in guinea pig serum and their clearance. Toxicol. Appt. Pharmacol. 36, 5 I I-522.
Toxicology Letters 81 (1995) 229-234
Dose-dependent suppression of toluene metabolism by isopropyl alcohol and methyl ethyl ketone after experimental exposure of rats Hirohiko Uakia.b, Toshio Kawaic, Kazunori Mizunumac, Chan-Seok Moon b, Zuo-Wen Zhangb, Shunen Inui”, Shiro Takada”, Masayuki Ikeda*b aKyoto Indusirial Health Association, Kyoto 604, Japan bDepartment of Public Health, Kyoto Universiry Faculty of Medicine, Kyoto 606-01, Japan COsaka Occupational Health Service Center, Osaka 550, Japan
Received 14 June 1994;revision received 1I August 1995; accepted 11 August 1995
Abstract In order to examine possible suppression of toluene metabolism due to coexposure to other solvents, female Wistar rats were exposed for 8 h to toluene alone (at 50 or 100 ppm), or in combination with either methyl ethyl ketone (at 50, 100, 200 or 400 ppm) or isopropyl alcohol (at 50, 100, 200, 400, 800 or 1600 ppm). Urine samples were collected for 24 h after initiation of each exposure, and subjected to analysis for two toluene metabolites, hippuric acid and ocresol, both by HPLC. The excretion of hippuric acid, a major metabolite, was not moditied when the concentrations of methyl ethyl ketone or isopropyl alcohol were low, i.e. 100 ppm or below, whereas it was reduced when methyl ethyl ketone or isopropyl alcohol concentrations were twice or more times higher than that of toluene. There were no changes in any cases in excretion of o-cresol, a minor metabolite. The observation after coexposure to methyl ethyl ketone or isopropyl alcohol at low concentration is in line with the negative interaction between toluene and methyl ethyl ketone as well as between toluene and isopropyl alcohol after occupational exposures at low concentrations. Metabolic interaction may take place when the exposure intensity is high, as observed in the present study and also after experimental exposure of volunteers to toluene and m-xylene, or occupational exposure to benzene and toluene. Keyworris:
Combined exposure; Isopropyl alcohol; Metabolism modification;
1. Iatroduetion Possible modification of toxicity of individual chemicals after combined exposure has been a * Corresponding author. Tel.: +81 75 753 4460; fax: +81 75 753 4466.
focus of interest in toxicology of chemicals [l-3], especially solvents [4,5] because solvents in industrial products are generally mixtures except for degreasers [6,7]. During a course of a study to investigate the possible metabolic interaction between solvents as the basis for toxicity modification [8], in a factory survey, our study group has
0378-4274/95/$09.50 0 1995 Elsevier Science Ireland Ltd. All rights reserved SSDI 0378-4274(95)03445-Q
Methyl ethyl ketone; Toluene
230
H. Uuki CI ul : T’ox~colog~~Lerrrrs HI i 1995) 220-234
observed that the metabolism of toluene in solvent workers will not be affected by the coexposure to either isopropyl alcohol or methyl ethyl ketone when the intensities of exposure to these solvents are low [9]. It is highly probable, however, that the extent of metabolism modification is dose-dependent and that metabolic interaction will take place at higher doses, if not detectable at lower doses. A series of animal exposure experiments were conducted to confirm the negative findings on metabolic interaction at lower doses and to investigate possible metabolic interaction at higher doses. 2. Materials and methods 2.1. Exposure of animals and urine sample collection
Female Wistar rats weighing about 200 g were used. For experimental exposure to solvent vapors, two animals were housed together in a metabolic cage (for rats; Natsume Seisakusho. Tokyo, Japan) to obtain enough urine samples for analysis, and four pairs were exposed per exposure condition. Solvent vapor exposure was conducted with a servomechanized dynamic flow-type exposure system [lo], in which the exposure to a mixture of two solvent vapors was possible. The vapor concentrations were automatically measured every 24 min by FID-gas chromatography so that, even when mixed, the two solvent vapors were monitored separately. The coefficient of variation in vapor concentration during each exposure was less than 5% [lo]. Exposure continued for 8 h and urine samples were collected for 24 h after the initiation of exposure, taking the biological half-time of toluene (about 7-8 h in man [l I], and presumably shorter than 8 h in rats) into consideration. At the end of the 24-h urine collection period, the funnel at the bottom of the cage was rinsed with a minimum amount of water, and the wash was pooled with urine to make up the volume to about 50 ml. 2.2. Analysis for urinary metabolites The urine samples were analyzed for two urinary toluene metabolites, hippuric acid and o-
cresol. Hippuric acid was determined by the HPLC method as previously described [ 111. For ocresol determination, the HPLC system consisted of an automated liquid sampler (Hitachi 655-40), an HPLC pump (Hitachi L-6200), a fluorescence spectrophotometer (Hitachi F-4010), and a data processor (Hitachi D-2000) was used. A column (30 cm in length and 4 mm in diameter, packed with ODS-80A (4 5 pm)) was used for chromatography with a mixture of acetonitrile and water (30:70 by volume) as a mobile phase. The mobile phase was allowed to flow at 0.8 ml/min. The column was kept at 35°C. Fluorescence of the effIuent was monitored with excitation at 278 nm and emission at 302 nm (band pass 5 nm for both). For analysis, 5 ml of each urine sample was taken in a well-capped tube, mixed with 2 ml cont. hydrochloric acid and heated at 100°C for I h for hydrolysis. After cooling down to room temperature, the hydrolyzate was extracted with 2 ml isopropyl ether by vigorous shaking for 1 min. A 5-~1 aliquot of the extract was introduced to the HPLC system per injection. The detection limit for o-cresol was 0.1 mg/l under the standard analytical conditions. The recovery of o-cresol added to urine was 99% of o-cresol added to water. The coefficient of variation was 3.2% when the same sample was analyzed five times. 2.3. Statistical analysis The possible difference in excretion amounts was analyzed by Student’s t-test. 3. Results 3.1. Dose-dependent increase in urinary hippuric acid and o-cresol It is known that hippuric acid is endogenous and is excreted in urine even when there is no exposure to toluene [ 111. Accordingly, the exposureexcretion relationship was examined between toluene vapor concentration and either hippuric acid or o-cresol in urine. The results are summarized in Fig. 1. It is clear from the figure that urinary hippuric acid excretion increased from a physiological level of 36 mg/kg/24 h (as a mean) to higher levels as a linear function of the intensity of exposure to toluene. No o-cresol was detected in
H. Uaki et al. / Toxicology Letters 81 (1995) 229-234
231
the urine of nonexposed animals and the amount of ocresol increased in a linear relation to toluene exposure. 3.2. Suppression of toluene metabolism by coexposure to methyl ethyl ketone
In the first series of experiments, rats were exposed to methyl ethyl ketone in combination with toluene. The results are expressed as the ratios to hippuric acid and ocresol after exposure to toluene alone, to make the changes more readily understandable. When rats were exposed to methyl ethyl ketone at O-200 ppm in addition to 50 ppm toluene (Table l), there was a slight reduction in hippuric acid as an inverse function of an increase in coexposed methyl ethyl ketone concentration. The reduction became statistically signilicant (P < 0.05) when methyl ethyl ketone coexposure was at 200 ppm. In contrast, there was essentially no change in ocresol. When toluene concentration was elevated to 100 ppm, and that of methyl ethyl ketone to 200 or 400 ppm, the reduction in hippuric acid was significant (P < 0.05) at 200 ppm methyl ethyl ketone and even more evident (P c 0.01) at 400 ppm methyl ethyl ketone. No change was observed, however, in o-cresol excretion.
TOLUENE IN AIR (ppm)
Fig. I. Amount of hippuric acid and ocresol excreted in urine during the 34-h period after initiation of 8-h toluene vapor exposure. Solid circles and arrows show mean and S.D. of four determinations each. The lines are regression lines.
3.3. Suppression of toluene metabolism by coexposure to isopropyl alcohol
In the second series of experiments, rats were ex-
Table I Amount of hippuric acid and o-cresol in urine after coexposure to methyl ethyl ketone, relative to the amount after exposure to toluene alone Coexposure (ppm) to
Urinary metabolitea
Toluene
Methyl ethyl ketone
Hippuric acid
o-Cresol
50 50 50 50 50
50 100 200 200 400
98.9 91.4 88.8 79.6 64.5
101.6 f 9.5 105.1 zt 6.8 94.5 f 4.0 103.8 zt 24.0 91.5 f 16.9
zt f f zt f
1.9 8.2 s.o** l3.7* 7.9.’
Mean & SD. of four determinations each are given. ‘The amount of hippuric acid and otresol are expressed relative (i.e. in percent) to the corresponding amounts after exposure to toluene alone at 50 or 100 ppm, respectively. Asterisks indicate that the difference from the controls (i.e. the amount after exposure to toluene alone at 50 or 100ppm) is statistically significant (**P < 0.01; *P < 0.05).
Table 2 Amount of hippuric alone Coexposure
acid and o-cresol in urine after coexposure
(ppm) to
Toluene
Isopropyl
50 50
50 100
50 100 100 100
200 200 400 800
100
1600
IJrinary alcohol
to isopropyl
alcohol,
relative to the amount
after exposure
to toluene
metabolite”
Hippuric acid _I__.__ 99.7 f 6.5 88.7 f 4.1’ 88.8 f 4.2* X6.3 f Il.1 x7.3 f 9.2 79.3 f 8.0** 64.4 f 1.5” -.
0-C‘resol --_~.91.2 * 949 f liJO.0 f 106.5 f 102.3 f 104.2 f IOX. f -.-
77 16.3 14.3 22.7 4.8 10.2 4.0 -
Notes are as for Table I.
posed to isopropyl alcohol in addition to toluene (Table 2). When rats were exposed to isopropyl alcohol at various concentrations and toluene at 50 ppm, a slight but significant (P < 0.05) reduction in hippuric acid was observed at 100 ppm and 200 ppm isopropyl alcohol. When toluene concentration was elevated to 100 ppm and isopropyl alcohol coexposure was increased up to 1600 ppm, a significant (P < 0.01) reduction was observed in hippuric acid at 800 ppm isopropyl alcohol, and the reduction was more marked (P < 0.01) at 1600 ppm isopropyl alcohol. The amount of o-cresol however stayed unchanged in all cases. 4. Discussion In this study using rats, a linear relationshlp was observed between toluene exposure and urinary excretion of hippuric acid as well as o-cresol as observed in humans [l 11. When the animals were coexposed to methyl ethyl ketone or isopropyl alcohol, there was a reduction in hippuric acid excretion in a manner inversely related to the concentration of the solvent coexposed. Thus, the reduction was insignificant when the concentration of the coexposed solvent was low, e.g. 50 ppm methyl ethyl ketone or isopropyl alcohol in combination with 50 ppm toluene (Tables 1,2) and turned out to be significant only when the coexposure was at high concentrations (e.g. 100 ppm toluene plus 400 ppm methyl ethyl ketone, or 100 ppm toluene plus 800 ppm isopropyl alcohol). It is
worth noting that the current occupational exposure limits for toluene, methyl ethyl ketone and isopropyl alcohol are set at 50 ppm, 200 ppm and 400 ppm, respectively [ 12-141. No changes were observed in the minor metabolite, o-cresol. The reduction in hippuric acid after coexposure to methyl ethyl ketone or isopropyl alcohol at the concentration of twice or four times that of toluene tended to be more evident at 100 ppm toluene than at 50 ppm toluene. This may be due to the fact that hippuric acid is present in the urine from nonexposed rats. The negative observation of hippuric acid excretion after coexposure to methyl ethyl ketone or isopropyl alcohol at low levels is in agreement with the recent observation by this study group in a survey of factory workers 191. In the factories surveyed, the workers were exposed to solvent mixtures in which toluene was a major contaminant in the workroom air (geometric mean vapor concentration; 18 ppm) but methyl ethyl ketone (16 ppm) and isopropyl alcohol (7 ppm) were also present. The present negative findings on possible metabolic interactions between toluene and methyl ethyl ketone or toluene and isopropyl alcohol when the concentrations were low are also in line with the study results by Tardif et al. [5], Kawai et al. [15], Inoue et al. [16] and Huang et al. [17], and offer experimental support to the hypothesis that the chances of metabolic interaction occurring between the solvents coexposed are remote when the exposure intensity is low [9]. It is also worth noting that metabolism of tol-
H. Uaki et al. / Toxicology Letters 81 (1995) 229-234
uene is suppressed by methyl ethyl ketone and isopropyl alcohol when the concentration of the latter solvents are several times higher than that of toluene. Metabolic interactions at higher concentrations are also reported by Inoue et al. [6] and Tardif et al. [5], between benzene and toluene, and toluene and m-xylene, respectively. There is no proposed mechanism to explain the observed suppression of toluene metabolism by methyl ethyl ketone and isopropyl alcohol. However, the suppression may be due to competition for alcohol dehydrogenase which oxidizes benzyl alcohol (an oxidation product of toluene at sidechain methyl moiety) to benzoic acid, as is the case with suppression of methanol oxidation by coadministered ethanol [19]. Methyl ethyl ketone is also known to be converted in vivo to 2-butanol [20]. From a quantitative viewpoint, however, it may be the case that the amount of the 2-butanol formed in vivo is too low to explain the observed suppression. In fact, methyl ethyl ketone appears to be as potent as or even more potent than isopropyl alcohol, when the same absorption rate in the lungs is assumed; methyl ethyl ketone at 400 ppm (Table 1) is equally as suppressive as isopropyl alcohol at 1600 ppm (Table 2) when coexposed to 100 ppm toluene. Thus, it might be necessary to look for a mechanism other than competition for the alcohol derived from methyl ethyl ketone. Acknowledgements This work was supported in part by a grant-inaid for cooperative study (grant No. 05304030: principal investigator, M. Ikeda) from the Ministry of Education, Science and Culture of the Government of Japan to T.K. and M.I. References 111Jonker, D., Woutersen, R.A., van Bladernen, P.J., Til, H.P. and Peron, V.J. (1990) Cweek oral toxicity study of combination of eight chemicals in rats: comparison with the toxicity of the individual compounds. Food Chem. Toxicol. 28, 623-63 I. I21 Jonker, D., Jones, M.A., van Bladernen, P.J., Woutersen, R.A., Til, H.P. and Peron, V.J. (1993) Acute oral toxicity of a combination of four nephrotoxicants in
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