Received September 2, 1996 and accepted October 29, 1996

Transcription

1 Industrial Health 1997, 35, Mutagenicity of 2-Bromopropane Seung Hee MAENG and II Je YU* Department of Industrial Toxicology, Industrial Health Research Institute, 34-6, Kusan-long, Bupyung-gu Inchon , Korea Korea Industrial Safety Corporation, Received September 2, 1996 and accepted October 29, 1996 Abstract: 2-Bromopropane (2BP, isopropyl bromide), a substitute for freon, has recently been suspected to be the causative chemical for the outbreak of some reproductive dysfunctions such as amenorrhea and oligospermia in workers who has been exposed to this solvent in an electronic factory. Bacterial mutation assays, chromosome aberration analysis in vitro, and micronucleus tests in vivo, were carried out to clarify the mutagenicity of 2BP. 2BP induced mutagenicity in Salmonella typhimurium TA100 with metabolic activation in a dose-dependant manner. 2BP induced mutagenicity in TA1535 as well, with or without metabolic activation. These observations indicated that 2BP induced the base-pair substitution type mutations in salmonella strains. The chromosome aberration analysis showed negative results in Chinese hamster lung cells treated with different concentrations, ranging from to 2.46 mg/ml for 6 h with metabolic activation and for 24 h without metabolic activation. The micronucleus frequencies were recorded by examining polychromatic erythrocytes in the bone marrows of rats which were intraperitoneally injected with 2BP for 28 days. There was no significant increase in the micronucleus frequencies at any of the different doses of 2BP (125 mg/ kg b.wfday, 250 mg/kg b.widay, and 500 mg/kg b.wfday). However, in comparison to controls, there was a significant decrease in the percentage of polychromatic erythrocytes in the total number of erythrocytes. This suggests that there may be bone marrow depression in hematopoiesis at these dose levels of 2BP. Despite the dose levels which showed hematopoietic inhibition in the bone marrow, no micronucleus formation was induced. Key words: 2-Bromopropane, Isopropyl bromide, Mutagenicity, Bacterial mutation assay, Chromosome aberration, Micronucleus, Polychromatic erythrocyte Introduction Recently, the possible health hazards caused by 2BP exposure have become one of major problems in the field of occupational health in Korea'. In 1995, a number of female workers were found to have amenorrhea and male workers were diagnosed with oligospermia while working in an electronic company in Korea. A detailed epidemiologic study revealed that the outbreak of these symptoms followed the substitution of freon 113 for 2BP that had been imported from Japan. This study suggested that 2BP might be the causative agent for the health hazards'. However, data *To whom correspondence should be addressed containing information on the toxicity of 2BP only states LD50 and LC50z, 3), In this study, we examined the mutagenicity of 2BP by using in vitro and in vivo tests in order to provide basic information for further toxicological studies including carcinogenicity and teratogenicity. Materials and Methods Chemicals 2BP (CAS No ) was purchased from Tokyo Kasei Co. Ltd. (Japan). Its purity was 99.0% and no significant contaminants were detected. Liver homogenate (S9) was purchased from Kikkoman Co. (Japan), which was prepared

2 88 SH MAENG AND IJ YU from male Sprague-Dawley rats (7-weeks-old with body weight ranging from g) using the method of Ames et al.4~. Phenobarbital and 5, 6-benzoflavone were used as drug-metabolizing inducerss~. NADPH, NADH and glucose- 6-phosphate (G-6-P) were purchased from Sigma Chemical Co. (USA). DMSO, mitomycin C and cyclophosphamide (CYM) were also purchased from Sigma Chemical Co. (USA) and olive oil was purchased Chemical Co. (Japan). Bacterial mutation assays from Yakuri Pure Bacterial mutation assays were carried out by using the pre-incubation method (37 C, 30 min) with and without 59 mix6> using S. typhimurium TA98, TA100, TA1535, TA1537 and E. coli WP2 uvra'~. These bacteria were kindly supplied by Dr. Matsushima, Japan Bioassay Research Center, Kanagawa, Japan. Oxoid nutrient broth was used to preculture tester strains, and Difco Bacto agar was used to make 2 ml of top agar. The top agar layer contained 0.1 µ mole of L-histidine and biotin, or 0.1 u mole of L-tryptophan. The 59 mix contained 4 mm of NADPH, 4 mm of NADH, 5 mm of G-6-P, 8 mm of MgC12, 33 mm of KC1,100 mm of sodium phosphate buffer (ph 7.4) and 10% 59. 2BP was tested at 5 concentrations: 5,000, 1,000, 500, 100 and 50 jig/plate in the preliminary assay, and 5,000, 2,500, 1,250, 625, and 313 µg/plate in the second assay. The concentrations for the second assay were based on the results of the preliminary assay. Duplicated plates were used at each concentration of 2BP, while four plates were used for negative (DMSO) and positive controls. 2BP at each concentration was prepared with DMSO. The revertant colonies per plate were counted with an automatic colony counter (UNITRON, IMAGE TEK SYSTEM, IA 100, USA). Chromosome aberration analysis The CHL (Chinese hamster lung) cells for the chromosome aberration analysis were supplied by Dr. Matsushima, Japan Bioassay Research Center, Kanagawa, Japan. The CHL cells, stored in liquid nitrogen, were cultured with minimum essential medium (MEM, GIBCO BRL, Life Technologies Inc., USA) supplemented with 10% fetal bovine serum (GIBCO BRL, Life Technologies, Inc., USA) in a CO2 incubator at 37 C. The mitotic indices at each concentration were checked for cell growth inhibition. The present chromosome aberration tests were carried out with concentrations of 2BP ranging from to 2.46 mg/ml because 4.92 mg/ml concentration growth inhibition (Table 3). The chromosome of 2BP caused the cell aberration analysis were carried out according to the method of Sofuni8~. The cells (4 x l03 /ml) were seeded in 60 mm plastic petri dishes containing 5 ml of culture medium, and cultured for 3 days in a CO2 incubator at 37 C. They were then treated with 6 different concentrations of 2BP, ranging from to 2.46 mg/ml, for 24 hours. For metabolic activation, the 3 day cultured monolayer cells were treated simultaneously with the 59 mix (5% final concentration) and 2BP for 6 hours. The cells were then washed with phosphate buffered saline and supplied with fresh medium. The chromosome preparations were made after 18 hour additional culture. Duplicated culture plates per each concentration were used. To analyze chromosome aberration, a hundred well-spread and Giemsa stained metaphases per plate were examined under the light microscope (Olympus BH-2, Japan). The Ishidate method9~ was used to determine any chromosome aberrations. Micronucleus test The present micronucleus test was carried out simultaneously with the 28 days repeated dose toxicity test of 2BP. The micronucleus tests were performed as previously described10>, but the treatment condition and dose selection were somewhat changed. Treatments were given once a day, 6 days per week, and the injection period lasted for 28 days. Three doses, 500, 250, and 125 mg/kg b.w./day, were selected as treatments according to rat's body weights. 2BP was dissolved in olive oil and intraperitoneally injected into the rats. Specific pathogen-free Sprague-Dawley rats were purchased from Dae-Han Experimental Animal Center (Korea). Ten-week-old rats were purchased and acclimatized for 2 weeks in a safety clean rack (MJ-721 CS (P), Myungjin, Korea). Temperature and relative humidity were regulated within limits of 23 ± 2 C and 55 ± 7%, respectively. The rats were maintained in a 12 hour light/dark cycle. Purina rodent chow (Korea) and deionized water were offered ad libitum. Each of the test groups consisted of 20 rats (10 males + 10 females), which were allocated randomly according to their body weight (Tables 5, 6). One day after the final injection, bone marrow cells were then collected, smeared and stained. The cells were stained with acridine orange to detect micronuclei according to the method of Hayashi et al.' 1 and with Giemsa solution to observe the polychromatic erythrocytes (PCEs)12~. The number of micronucleated polychromatic erythrocytes (MNPCEs) among 1,000 polychromatic erythrocytes per animal were examined by fluorescence microscopy (OPTIPHOT 2, Nikon, Japan). The number of PCE among Industrial Health 1997, 35, 87-95

3 MUTAGENICITY OF 2-BROMOPROPANE total erythrocytes was also counted (Olympus BH-2, Japan). Statistical analysis was performed using the method of Kastenbaum and Bowman13~ for percent of micronucleus formed in PCE, and t-test was used for percent of PCE in total erythrocytes and after administration. Results and for changes in body weight before Bacterial mutation assay The results of the preliminary and the second test are shown in Tables 1, 2 and Figure 1. The mutagenicity of 2BP tested positive in Salmonella typhimurium TA100 with 59 mix (Fig. 1, closed circles) and in TA1535 with (Fig. 1, closed squares) and without the S9 mix (Fig. 1, open squares). The number of revertants increased in a dose-dependent manner with the 2BP concentration gradients. The Salmonella typhimurium TA98, TA1537, and E. coli WP2 uvra strains did not display any mutagenic activity in either the presence or absence of the 59 mix. Chromosome aberration analysis The mitotic index data for cell proliferation rates were shown in Table 3. There was a significant inhibition of cell proliferation in the concentration of 4.92 mg/ml. From these results, the three-day cultured CHL cells were treated with 2BP using 6 concentrations ranging from to 2.46 mg/ ml. The results of the chromosome aberration test are shown in Table 4. Chromosomal aberrations did not appear at any concentration either with or without the 59 mix. The frequencies of chromosomal aberrations and the polyploid cells at every concentration were below 5%. Micronucleus test The body weight data of each test group before and after administration of 2BP were shown in Tables 5 and 6. During the experiment period, one female rat had died due to internal bleeding caused by a perforation of blood vessel by an injection needle during administration. Compared with the control, male rats treated with 2BP for 28 days showed significant decreases in body weight at the dose of 250 and Table 1. Results of the preliminary bacterial reverse mutation assay of 2BP

4 90 SH MAENG AND IJ YU Table 2. Results of the second bacterial reverse mutation assay of 2BP 500 mg/kg b.widay. In female rats, 2BP treatment induced significant decrease in body weight at all doses. The MNPCE frequencies are summarized in Tables 7 and 8. Compared with the control group, the mean MNPCE frequencies at any of the 2BP concentrations did not show any significant increase. 2BP did not cause micronuclei formation in the rat bone marrow by repeated intraperitoneal injection for 28 days. The percentage of PCE (Tables 7 and 8), however, did decrease significantly in all treatment groups compared with the control group. The percentages of polychromatic erythrocytes (PCEs) in total erythrocytes in males were ± 8.45% (p<0.05) for 125 mg/kg/b.w./day, ± 3.23% Fig. 1. Number of Revertantslplate in Salmonella typhimurium TA100 and TA1535 with or without metabolic activation A, data from the preliminary assay; B, data from the second assay; Closed circles, TA100 with metabolic activation; Open circles, TA100 without metabolic activation; Closed squares, TA1535 with metabolic activation; Open squares, TA1535 without metabolic activation. Industrial Health 1997, 35, 87-95

5 MUTAGENICITY OF 2-BROMOPROPANE 91 Table 3. Mitotic indices (%) and cell proliferation chromosome preparations rates (%) in the preliminary Table 4. Frequencies (%) of chromosome aberration in CHL cells treated with 2BP

6 92 SH MAENG AND IJ YU Table 5. The body weights of male rats before and after 28 day treatment. Table 6. The body weights of female rats before and after 28 day treatment. Table 7. Results of the bone marrow micronucleus assay by repeated intraperitoneal with 2BP for 28 days in male rats injection AT,.,.C AT,.,.~ nre, a RATnn9!n! +~ Dr IT Table 8. Results of rat bone marrow micronucleus assay by repeated intraperitoneal with 2BP for 28 days in female rats injection

7 MUTAGENICITY OF 2-BROMOPROPANE 93 (p<0.01) for 250 mg/kg b.w./day and ± 3.24% (p<0.01) for 500 mg/kg b.w./day group. The percentages of polychromatic erythrocytes (PCEs) in total erythrocytes in females were ± 5.48% (p<0.01), ± 3.10% (p<0.o l ), and ± 5.52% (p<0.01) for 125 mg, 250 mg, and 500 mg/kg b.widay group, respectively. These suggest that 2BP may have some effects on hematopoiesis bone marrow at these dose levels. Discussion in the In order to assess the potential carcinogenicity of 2BP, we carried out three genetic toxicologic assays using bacterial mutation assays', chromosome aberration tests in vitro9' 14) and micronucleus tests in vivo10. Our results from the reverse mutation assays using Salmonella typhimurium TA100 and TA1535 tested positive, showing a dose-response relationship. 2BP was strongly mutagenic to TA 1535 with and without metabolic activation. However, it showed clear mutagenicity to TA 100 only when the S9 mix was added. This result was similar to that of Warner et al. 15). They also reported the positive mutagenicity of 2BP in vaporization technique with Salmonella typhimurium TA 100 with hamster liver S9. In present study using preincubation method, the level of dose dependency in TA100 without the S9 mix was weak, and 2BP did not produce any mutagenic effects in E. coli WP2 uvra. These results indicated that 2BP induced a base-pair substitutiontype mutagenicity, primarily at one of G-C pairs in the Salmonella typhimurium and that TA1535 is more advantageous for the detection of mutagen that do not preferentially revert TA100, as previously reported16> The series of halogenated propanes and propenes have been used for mutagenic effects in Salmonella typhimurium TA1004,18-21), All of the tihalogenated propane analogues (1, 2, 3-tribromopropane, 1, 2-dibromo-3-chloropropane (DBCP), 1, 3-dibromo-2-chloropropane) showed mutagenicity when the microsome activation system was included17~. The least mutagenic tihalogenated propane was 1, 2, 3- trichloropropane which did not include bromine17~. Trihalogenated propane, DBCP, was reported as mutagenic in the presence of exogenous activation in Salmonella TA 100 and TA1535; DBCP was weakly active in TA1535 in absence of metabolic activation21~. In comparison to DBCP, the dihalogenated propanes were substantially less mutagenic, although 1, 2-dibromopropane, which did not include chlorine, showed a similar mutagenic activity17>. From our present results, when compared with DBCP, 2BP showed a similar mutagenic activity with metabolic activation in Salmonella typhimurium TA100, and showed a high mutagenic activity with and without the addition of an activation system in Salmonella typhimurium TA1535. From these findings, bacterial mutagenic potentials and activity of halogenated propanes may be related to the number and species of the halogen in chemicals. In present chromosome aberration test in vitro, no aberrant cells were induced after 2BP treatments, either with or without metabolic activations. Because the 4.92 mg/ml concentration used in the preliminary assay aroused cell growth inhibition, the highest concentration used was 2.46 mg/ml. However, frequencies of chromosome aberration induced by 2BP treatment at each concentration were below 5% and did not increase compared with controls. The formation of micronuclei was not induced by 2BP at any concentration. Here, the micronuclei were counted by examining the PCEs from the bone marrows of rats which had been repeatedly intraperitoneally injected for 28 days. The highest dose of 2BP was equivalent to 1110 of LD50 (500 mg/kg b.w.) for rodents as previously reported2~. The recommended dose for micronucleus test has been the maximum tolerate dose or that producing some indications of cytotoxicity such as a change in ratio of polychromatic or normochromatic erythrocytes22~. The highest dose of 2BP used in our study might be too low to induce micronulei, compared with conventional administration. However, it could be enough to induce micronucleus formation considering cumulative doses during 28 day experiment, ranging from 3,000 mg/kg b.w. to 12,000 mg/kg b.w., changes in body weights (Tables 5 and 6), and changes in percentages of PCE among total erythrocytes with 2BP administration (Tables 7 and 8). Present results are also different from those that were conducted with DBCP, which show increases in chromosomal aberrations in vivo and in vitro23-25~, and sister chromatid exchanges23, 26). DBCP was known to be metabolized to halogenated epoxides such as epibromohydrin that produced transient infertility in male rats, and a marked preimplantation loss in impregnated female rats27~. Furthermore, epichlorohydrin was capable of increasing the frequency of aberrant cells in occupationally exposed workers28~. These findings suggest that halogenated epoxides may be reactive metabolic intermediates, and might participate in the induction of mutation including cytogenetic effects. From our cytogenetic results, however, 2BP did not induce chromosome aberrations in the CHL with or without

8 94 SH MAENG AND IJ YU metabolic activation in vitro, nor induce micronucleus in the rat bone marrow cells in vivo. All chlorinated 3-C hydrocarbons, except 1, 2, 3-trichloropropane, have been proven to be potent sister chromatid exchange (SCE) inducers in V79 cells without the S9 mix, whereas 1, 2, 3- trichloropropane had to be transformed to an SCE-inducing metabolite29~. Although there was little information about the cytogenetic effects of 1-bromopropane, 1, 2- dibromopropane, and 1, 2, 3-tribromopropane, it is possible that these bromopropanes may be transformed to bromine metabolites. These bromine metabolites can not induce cytogenetic effects. In addition, the chlorine in halogenated propane may have great part in the induction of cytogenetic effects in mammalian cells. Recently, Park et al.'s reported in their epidemiologic research that a number of 2BP exposed workers showed the signs of reproductive dysfunction such as amenorrhea and oligospermia, and some hemopoietic dysfunctions. Although 2BP showed a potent mutagenicity in Salmonella typhimurium TA100 and TA1535, clastogenicity was not evident in either in vitro or in vivo tests in our study. Araki et a1.30~ reported that several volatile chemicals, which were negative in the preincubation method of bacterial mutation assay, turned out to be positive in the gas exposure method. Although our present result from preincubation method of bacterial mutagenic assay using Salmonella typhimurium TA100 is similar to that of Waners et al.15~, which was obtained from vaporization technique, the negative result of in vitro cytogenetic tests might arise from different exposure condition. Therefore, further investigations using modified methods such as gas exposure are recommended to clarify the clastogenicity of 2BP in detail. A significant decrease in the PCE percentage was observed at every dose during the micronucleus test. These PCE percentage results might indicate hematopoietic damage in bone marrows induced by 2BP. Despite the dose levels of 2BP showing the hematopoietic change in the bone marrow, no micronucleus formation was observed. Acknowledgments We thank for 4 years of Korean-Japanese Cooperative Project on Prevention of Occupational Diseases for Korean Workers. Without this collaborative efforts, this paper would not be completed. We really appreciate the assistance and guidance given by researchers of Japan Bioassay Research Center and Japan National Institute of Industrial Health. References 1) Park JS, Kim YH, Park DW, Park SH, Choi KS (1995) An epidemiological study on workers of LG electronics at Yangsan, Industrial Health Research Institute, Korea Industrial Health Research Institute, Korea Industrial Safety Corporation. 2) Registry of Toxic Effects of Chemical Substances (RTECS), National Institute for Occupational Safety and Health (NIOSH) (1995) RTECS number TX ) Kim HY, Chung YH, Yi KH, Kim JG, Yu IJ (1996) LC50 of 2-Bromopropane. Ind Health 34, ) Ames BN, McCann J, Yamasaki E (1975) Methods for detecting carcinogens and mutagens with the Salmonella/mammalian-microsome mutagenicity test. Mutation Res 31, ) Matsushima T, Sawamura M, Hara K, Sugimura T (1976) A safe substitute for polychlorinated biphenyls as an inducer of metabolic activation system. In: In vitro metabolic activation in mutagenesis testing. eds. by de Serres FJ, Fouts JR, Bend JR, Philpot RM, 85-8, Elsevier, North-Holland, Amsterdam. 6) Yahagi T, Nagao M, Seino Y, Matsushima T, Sugimura T (1977) Mutagenicities of N-nitrosamines on Salmonella. Mutation Res 48, ) Maron DM, Ames BN (1983) Revised methods for the Salmonella mutagenicity test. Mutation Res 113, ) Sofuni T (1991) Chromosome aberration test using cultured cell. In: Mutagenicity and genotoxicity. Lecture series on toxicological tests of chemicals 12. ed. by Ishidate M Jr, 79-87, Chijin Shokan, Tokyo. 9) Ishidate M Jr (1981) Application of chromosomal aberration tests in vitro to the primary screening for chemicals with carcinogenic and/or genetic hazards. In: Proceedings of a symposium held at Montpelier. France (Excerpta Medica), ) The Collaborative Study Group for the Micronucleus Test (1990) Single versus multiple dosing in the micronucleus test: The summary report of the collaborative study by CSGMT/JEMS.MMS. Mutation Res 234, ) Hayashi M, Sofuni T, Ishidate M Jr (1983) Ann application of acridine orange fluorescent staining to the micronucleus test. Mutation Res 105, ) Hayashi M (1991) The micronucleus test. Japanese Society for Bio-pharmaceutical Statistics Monograph Industrial Health 1997, 35, 87-95

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