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Inhalation exposure to 1,2-dichloropropane: Distribution of blood and tissue concentrations of 1,2dichloropropane in rats during and after exposure a
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Makoto Take , Michiharu Matsumoto , Tetsuya Takeuchi , Mitsuru Haresaku , Hitomi Kondo , a
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Hideki Senoh , Yumi Umeda , Takeji Takamura-Enya & Shoji Fukushima a
Japan Bioassay Research Center, Japan Industrial Safety and Health Association, Hadano, Kanagawa, Japan b
Department of Applied Chemistry, Faculty of Engineering, Kanagawa Institute of Technology, Atsugi, Kanagawa, Japan Published online: 29 Jul 2014.
To cite this article: Makoto Take, Michiharu Matsumoto, Tetsuya Takeuchi, Mitsuru Haresaku, Hitomi Kondo, Hideki Senoh, Yumi Umeda, Takeji Takamura-Enya & Shoji Fukushima (2014) Inhalation exposure to 1,2-dichloropropane: Distribution of blood and tissue concentrations of 1,2-dichloropropane in rats during and after exposure, Journal of Environmental Science and Health, Part A: Toxic/Hazardous Substances and Environmental Engineering, 49:12, 1341-1348, DOI: 10.1080/10934529.2014.928193 To link to this article: http://dx.doi.org/10.1080/10934529.2014.928193
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Journal of Environmental Science and Health, Part A (2014) 49, 1341–1348 Copyright © Taylor & Francis Group, LLC ISSN: 1093-4529 (Print); 1532-4117 (Online) DOI: 10.1080/10934529.2014.928193
Inhalation exposure to 1,2-dichloropropane: Distribution of blood and tissue concentrations of 1,2-dichloropropane in rats during and after exposure
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MAKOTO TAKE1, MICHIHARU MATSUMOTO1, TETSUYA TAKEUCHI1, MITSURU HARESAKU1, HITOMI KONDO1, HIDEKI SENOH1, YUMI UMEDA1, TAKEJI TAKAMURA-ENYA2 and SHOJI FUKUSHIMA1 1 2
Japan Bioassay Research Center, Japan Industrial Safety and Health Association, Hadano, Kanagawa, Japan Department of Applied Chemistry, Faculty of Engineering, Kanagawa Institute of Technology, Atsugi, Kanagawa, Japan
The present investigation was undertaken to determine the distribution and accumulation of 1,2-dichloropropane (DCP) in the blood, lung, liver, kidney, and abdominal fat of rats during and after inhalation exposure. Male rats were exposed to 80 or 500 ppm (v/v) DCP vapor for 360 min and the concentrations of DCP in the blood and tissues during the inhalation exposure period and after the end of the exposure period were measured. DCP accumulation in the abdominal fat was much greater than that in the blood and other tissues. Eighteen hours after the end of inhalation exposure, DCP could still be detected in the abdominal fat in the 80-ppm group, and in the blood, liver, kidney, and abdominal fat in the 500-ppm group. Our results are valuable data pertaining to the pharmacokinetics of DCP and to human health risk assessment of exposure to DCP vapor by inhalation. Keywords: 1,2-Dichloropropane, blood concentration, tissue concentrations, inhalation exposure.
Introduction The chemical 1,2-Dichloropropane (DCP) was used in the past as a soil fumigant, chemical intermediate, and industrial solvent and was found in paint strippers, varnishes, and furniture finish removers.[1] Most uses of DCP have now been discontinued; however, DCP is still detected in the ambient air and public water in Japan.[2] Because DCP is highly volatile,[3] the primary route of exposure of humans to DCP is inhalation of its vapor. The International Agency for Research on Cancer (IARC) evaluated DCP as a Group 3 agent:[4] an agent which is not classifiable as to its carcinogenicity to humans. However, in a recent report, workers in a small printing company in Japan were exposed to the chlorinated organic solvents DCP and/or dichloromethane (DCM) between 1985 and 2006 (DCP) and 1985 to 1997/ 1998 (DCM) and were affected with cholangiocarcinoma.[5] In two-year animal studies, the National Toxicology Program (NTP) reported a dose-related increase of Address correspondence to Makoto Take, Japan Bioassay Research Center, Japan Industrial Safety and Health Association, 2445 Hirasawa, Hadano, Kanagawa 257-0015, Japan E-mail: [email protected] Received February 4, 2014.
liver tumors in mice of both sexes, but not in rats, administered DCP by gavage.[6] However, while DCP administration did not result in induction of liver tumors in rats, a dose-related increase in mammary gland adenocarcinomas in female rats was observed.[6] Our two-year inhalation carcinogenicity study of 200 ppm (mice) or 500 ppm (rats) DCP vapor showed that inhalation exposure to mice and rats of both sexes resulted in significantly increased incidences of tumors in the Harderian gland of male mice, the lung of female mice,[7] and the nasal cavity of male and female rats.[8] To better evaluate the toxicity of volatile chemicals, such as DCP, it is necessary to know the time-course changes of the chemical’s concentration in the blood and tissues consequent to exposure as this is a key factor affecting toxicity. Therefore, understanding the pharmacokinetics of exposure in animal models is one of the fundamental considerations in human risk assessment of exposure to a chemical. To date, only limited information is available about time-course changes of DCP concentration in the blood of animals exposed to DCP by inhalation.[9] The present study was designed to investigate DCP concentrations in the blood and tissues of rats exposed to 80 or 500 ppm DCP vapor, concentrations corresponding to the low and high doses used in the two-year carcinogenicity study by Umeda et al. [8]
1342 Material and methods Chemical DCP of analytical grade (greater than 99.5% pure) was obtained from Wako Pure Chemical Industries, Ltd. (Osaka, Japan).
Take et al. blood samples were collected into 10-mL headspace sampler (HS)-vials, and 0.2 mL of distilled water was added to each sample.[11–13] The lung, liver, kidney, and abdominal fat were removed from each rat, and tissue samples (range from about 0.1 to 0.5 g) were placed into separate 10-mL HS-vials. Then, 5 mL of distilled water was added to the vials, and the vials were immediately sealed with an aluminum crimp cup.[11–13]
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Animals Eighty-four F344/DuCrlCrlj (SPF) male rats were obtained at 6–8 weeks of age from Charles River Laboratories Japan, Inc. (Kanagawa, Japan). All animals were quarantined for one week and were then housed in our laboratory until the age of 18 weeks. The body weights of rats at the start of experiment ranged from 285 to 332 g. Rats were housed in individual cages in a temperature controlled room with a 12-h light/dark cycle (light: 8:00– 20:00). The room temperature and relative humidity were maintained in the ranges of 22 § 2 C and 55 § 15%, respectively. All animals had access to food (CRF-1; Oriental Yeast Co., Ltd., Tokyo, Japan) and tap water ad libitum, except during the inhalation exposure period. Animals were cared for in compliance with the “Regulation for Proper Conduct of Animal Experiments in the Japan Bioassay Research Center,” and the present study was approved by the Ethics Committee of Animal Experiments of the Japan Bioassay Research Center (JBRC). Experiment design The rats were divided into 2 groups, 42 rats in each group, and exposed to 80 or 500 ppm DCP by inhalation for 360 min using whole-body inhalation chambers, which were developed on the JBRC’s own with volume of 1.1 L/ animal.[10] During inhalation exposure period, the rats had no access to food and water. DCP vapor at 80 or 500 ppm was generated by bubbling clean air through liquid DCP. DCP vapor concentrations in the inhalation chambers were measured by gas chromatography (GC) using Agilent Technologies 6890 (Agilent Technologies, Santa Clara, CA, USA) every 15 min during the inhalation exposure period. The target DCP vapor concentrations in the inhalation chambers were 80 and 500 ppm (v/v), and the measured concentrations were 80.2 § 0.9 and 502.1 § 8.6 ppm (mean § S.D.).
Measurement of DCP in the blood and tissues DCP concentrations in the blood and tissue samples were analyzed by HS-GC/mass spectrometer (MS) using Agilent Technologies 7694 HS (oven temperature, 100 C; loop temperature, 130 C; 10 min vial equilibration time for blood samples and 30 min for tissue samples) and Agilent Technologies 5973N GC/MS system (column, J&W DB-1 60 m £ 0.25 mm ID £ 0.25 mm; oven temperature, 100 C; ion source temperature, 230 C; carrier gas, helium at 1 mL/min; ionization, EI (electron ionization); fragment peak, 63 m/z).
Results and discussion Blood concentration of DCP In the 80-ppm group, during the inhalation exposure period, DCP concentration in the blood was increased at 60 min after the start of inhalation exposure and showed a tendency to slightly increase from 60 to 360 min (Fig. 1), background concentration of DCP was not measureable in blood at 0 min. After the end of the exposure period, the DCP concentrations in the blood decreased in a timerelated manner: DCP remained detectable in the blood at 180 min after the end of the exposure period, but it was not detected in the blood at 1080 min (18 h after the end of the inhalation exposure).
Blood and tissue collection and treatment Blood was collected from the tail vein of each rat, and blood collection was immediately followed by necropsy under isoflurane anesthesia. In each group, blood collection and necropsy were performed at 0, 60, 180, and 360 min during the 360-min inhalation exposure period and at 60, 180, and 1080 min after the end of the exposure period. Six rats were used for each collection. 0.2 mL
Fig. 1. The concentration of DCP in the blood of rats exposed to DCP by inhalation (n D 6 for each collection time point).
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Effect of 1,2-dichloropropane exposure in rats During the inhalation exposure period, the DCP concentration in the blood was governed by the blood-to-gas partition coefficient [14] with the DCP concentration in the gas phase of the lung remaining constant. DCP concentration in the blood increased at each blood-collection time point, suggesting that DCP may not have reached steady state during the 6-h exposure period. During the exposure period, DCP was lost from the blood by exhalation and by partitioning into target tissues; however, DCP lost from the blood was replenished from the DCP in the air. In the 500-ppm group, during the inhalation exposure period, DCP concentration in the blood increased from 0 until 360 min in a time-related manner after the start of inhalation exposure (Fig. 1), background concentration of DCP was not measureable in blood at 0 min. After the end of the exposure period, the DCP concentrations in the blood decreased in a time-related manner; however, DCP remained detectable in the blood at 1080 min after the end of the exposure period. The time-related increase of DCP concentration in the blood throughout the exposure period in the 500-ppm group indicates that steady state was not reached between the DCP in the gas phase of the lung and the concentration of DCP in the blood during the 6-h exposure period. In addition, the 10 to 15-fold higher concentration of DCP in the blood of the 500-ppm group compared to the 80-ppm group suggests that the metabolic saturation point was exceeded in rats exposed to 500 ppm DCP, resulting in a proportionally greater build-up of DCP in the bodies of rats exposed to 500 ppm DCP than in rats exposed to 80 ppm DCP. Timchalk et al.[9] reported the concentration of [14C] DCP in the blood of 7-week-old F344 rats exposed to 5, 50, or 100 ppm [14C]DCP for 6 h using a head-only inhalation chamber. In the Timchalk et al.[9] study, the maximum concentrations of [14C]DCP in the blood of rats exposed to 50 ppm was higher than the maximum concentration of DCP in the blood of rats exposed to 80 ppm in our study. This apparent discrepancy is due to such differences as the age of the test animals [15] and use of a headonly rather than a whole-body inhalation chamber. Half-lives (T1/2) of the concentration of DCP in the blood after the end of the inhalation exposure period are shown in Table 1. The T1/2 values were similar in the 80ppm and 500-ppm groups. The area-under-the-curve (AUC) values for the blood from 0 to 1440 min (AUC01440: the 360 min inhalation exposure period and the 1080 min following the exposure period) are shown in Table 1. The AUC0-1440 value for the blood of the 500ppm group was higher than that of the 80-ppm group. The ratio of the AUC0-1440 value for the 500-ppm group to the AUC0-1440 value for the 80-ppm group is 13, and this value is higher than the ratio of DCP concentrations (500/ 80 ppm D 6.25). This is the first report of AUCs for DCP in the blood consequent to exposure to DCP vapor by inhalation and will be valuable in the further development
Table 1. T1/2, AUC, and ratios in the blood and tissues. 80-ppm group
500-ppm group
T1/2 AUC0-1440 Ratio T1/2 AUC0-1440 Ratio Blood 182a Lung 39 Liver 57 Kidney 59 Abdominal fat 154
251b 122c 425 317 9553
1.00 0.49d 1.69 1.26 38.06
168 61 125 127 186
3272 1.00 2352 0.73 7113 2.20 4951 1.53 139711 43.12
Units include amin; bmg/mL £ min; and, cmg/g £ min. d AUC0-1440 value of the lung, liver, kidney or abdominal fat/AUC0-1440 value of the blood.
of DCP physiologically based pharmacokinetic (PBPK) models.
Tissue concentrations of DCP In the 80-ppm group, during the inhalation exposure period, the time-course changes of DCP concentrations in the lung, liver, and kidney showed similar patterns (Figs. 2–4). The concentrations of DCP in these tissues reached a maximum by 60 min and then remained constant from 60 to 360 min, background concentration of DCP was not measureable in these tissues at 0 min. The distribution of DCP into these tissues was governed by the partition coefficients between the blood and the tissues and by metabolism and excretion of DCP in the tissues.[14] DCP concentration in these tissues is expected to increase due to uptake of increasing amounts of DCP from the blood until steady state is reached between the gas phase of the lung and the blood and between the blood and the target tissue. DCP concentration in these tissues is expected to decrease due to metabolism (primarily liver), exhalation (lung), and excretion (primarily kidney). DCP
Fig. 2. The concentration of DCP in the lung of rats exposed to DCP by inhalation (n D 6 for each collection time point).
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Fig. 3. The concentration of DCP in the liver of rats exposed to DCP by inhalation (n D 6 for each collection time point).
Fig. 5. The concentration of DCP in the abdominal fat of rats exposed to DCP by inhalation (n D 6 for each collection time point).
lost from these tissues will be replenished from the DCP in the blood. Steady-state levels of DCP in these tissues was established by 60 min, after which uptake from the blood was balanced by removal through metabolism, exhalation, and excretion. In contrast, the concentrations of DCP in the abdominal fat of the 80-ppm group increased in a time-related manner throughout the exposure period (Fig. 5), and were much higher than in the other tissues at each collection time point: background concentration of DCP was not measureable in the abdominal fat at 0 min. The high concentration of DCP in the abdominal fat was due to the very high lipid solubility of DCP [3] coupled with low metabolism and excretion of DCP in this tissue. As with the other tissues, the DCP concentration in the abdominal fat will increase as it reaches steady state with the blood as governed by the blood-fat partition coefficient [16] and will decrease due to metabolism and excretion. The high lipid solubility of DCP leads to a longer period of time being
required to reach steady state in the abdominal fat than in the other tissues. After the end of the exposure period, the DCP concentrations in all the tissues of the 80-ppm group decreased in a time-related manner. DCP remained detectable in all the tissues at 180 min after the end of the exposure period. DCP was not detected in the lung, liver, or kidney at 1080 min, but a measurable level DCP was still present in the abdominal fat at 1080 min. The concentration of DCP in the tissues was governed primarily by two functions: (1) removal of DCP from the blood by exhalation, excretion, and metabolism;[9,17–19] and (2) redistribution of DCP from the storage tissues into the blood to reestablish steady state with the blood as DCP was removed from the body. However, because the accumulation of DCP in the abdominal fat was much higher than that in the other tissues and because of the low partitioning of DCP into the blood, DCP had not been cleared from the abdominal fat 18 h after the end of the inhalation exposure period. In the 500-ppm group, during the inhalation exposure period, the time-course changes of DCP concentration in the lung, liver, kidney, and abdominal fat showed a pattern similar to the time-course changes of DCP concentration in the blood (Figs. 1–5) with the concentrations of DCP in these tissues increasing in a time-related manner throughout the exposure period. These results are consistent with steady state not being reached between the DCP in the gas phase of the lung and the DCP in the blood during the 6-h exposure period. In addition, the 4- to 23-fold higher concentration of DCP in the tissues of the 500-ppm group compared to the 80-ppm group suggests that it is possible that the metabolic saturation point was exceeded at the 500 ppm concentration, resulting in a greater buildup of DCP in the body in the 500-ppm group than in the 80-ppm group. Similarly to the 80-ppm group, the concentration of DCP in the abdominal fat was much higher than in the other tissues at each collection time point. As discussed for
Fig. 4. The concentration of DCP in the kidney of rats exposed to DCP by inhalation (n D 6 for each collection time point).
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Effect of 1,2-dichloropropane exposure in rats the 80-ppm group, the high concentration of DCP in the abdominal fat was due to the very high lipid solubility of DCP [3] coupled with low metabolism and excretion of DCP in this tissue. After the end of the exposure period, the DCP concentrations in the tissues decreased in a time-related manner. DCP remained detectable in the tissues at 180 min after the end of the exposure period. DCP was not detected in the lung at 1080 min, but a measurable level DCP was still present in the liver, kidney, and abdominal fat at 1080 min. The level in the abdominal fat at 1080 min was approximately 100-fold higher than in the liver and kidney. Timchalk et al.[9] reported that the metabolites of DCP following exposure by inhalation are eliminated primarily in the urine and the expired air with a lesser amount excreted in the feces, and that while the majority of DCP was eliminated 24 h after the end of the exposure period, [14C]-volatile organics identified as DCP were still detected in the expired air 42 h after the end of the 6-h inhalation exposure period. Our results are consistent with their results, while the majority of DCP was rapidly eliminated from the body, minor amounts of DCP were detected in the abdominal fat of the 80-ppm group and the blood, liver, kidney, and abdominal fat of the 500-ppm group 18 h post-exposure. The T1/2 values of the concentration of DCP in the tissues after the end of the inhalation exposure period are shown in Table 1. In the two groups, the T1/2 value of the lung was the shortest, and the T1/2 value of the abdominal fat was the longest. The T1/2 values of the lung, liver, and kidney in the 500-ppm group were longer than that in the 80-ppm group. The T1/2 values of the abdominal fat were similar to the T1/2 values of the blood in the two groups. The similar T1/2 values of DCP in the blood and abdominal fat in the two groups coupled with the longer T1/2 values of DCP in the lung, liver, and kidney in the 500-ppm group suggest that DCP was transported from the abdominal fat to other tissues by the blood, thereby lengthening the T1/2 values in these tissues. The tissue AUC0-1440 values and the ratio of the AUC01440 value for each tissue to the AUC0-1440 value for blood are shown in Table 1. As expected, all of the tissue AUC01440 values were higher in rats in the 500-ppm group than in the 80-ppm group. Similarly to the tissue concentrations of DCP at individual time points, the ratio of the tissue AUC0-1440 values for the 500-ppm group to the 80-ppm group–19 (lung), 17 (liver), 16 (kidney), and 15 (abdominal fat)–are higher than the ratio of exposure concentrations (500/80 ppm D 6.25). In addition, the AUC0-1440 value for the abdominal fat and the ratio of the AUC0-1440 value for the abdominal fat to the AUC0-1440 value for the blood are markedly higher than the values for the blood and other tissues. However, the ratios of the AUC0-1440 values for the lung, liver, kidney, and abdominal fat to the AUC0-1440 value for the blood in the 80-ppm group are about the same
as in the 500-ppm group. These results are consistent with abdominal fat being the major reservoir of DCP in the rat body after exposure to DCP vapor. As discussed above in relation to the T1/2 of DCP in the various tissues in the two exposure groups, the large amount of DCP accumulated in the abdominal fat affected the metabolism and elimination of DCP from the body. This is the first report of the distribution of DCP in tissues during and after exposure by inhalation and will be valuable in the further development of DCP PBPK models.
DCP partition coefficients and tissue concentrations Table 2 shows the relationship between the partition coefficients and the ratios of the concentrations of DCP in the blood and tissues. The partition coefficients of the blood/ air, liver/air, and abdominal fat/air are 18.7, 28.4, and 499, respectively,[16] and the ratios of the partition coefficients of the liver and abdominal fat to the blood are 1.52 and 26.68. During the inhalation exposure period, the ratios of the concentrations of DCP in the liver/blood ranged from 2.41 to 3.84 for the 80-ppm group and from 2.76 to 3.26 for the 500-ppm group. The liver/blood ratios in the two groups were similar and about 2-fold higher than the ratio of the partition coefficients of liver/blood (1.52). The ratios of the DCP concentrations in the lung/blood and kidney/blood were also similar for the two groups. The ratios were 15–34% (80ppm group) and 30–36% (500-ppm group) lower in the kidney than in the liver and were 43–63% (80-ppm group) and 50–57% (500-ppm group) lower in the lung than in the kidney. The ratios of the DCP concentrations in the abdominal fat/blood ranged from 24.76 to 49.98 for the 80-ppm group; from 10.45 to 46.26 for the 500-ppm group. These values increased in time-related manner throughout the exposure period. The values at 180 and 360 min for both groups were higher than the ratio of the partition coefficients of abdominal fat/blood (26.68). Taken together, these ratios are indicative of a large distribution in abdominal fat and liver of DCP in the rat body. After the end of the inhalation exposure period, the ratios of the DCP concentrations in the liver/blood, lung/ blood, and kidney/blood for the two groups decreased in time-related manner and were lower than the partition coefficient ratios (Table 2). Similarly, the ratios of the DCP concentrations in the abdominal fat/blood for the 80-ppm group decreased in a time-related manner; however, the values at 60 and 180 min were higher than the ratio of the partition coefficients of abdominal fat/blood. In contrast to all the other tissue/blood ratios, the ratios of the DCP concentrations in the abdominal fat/blood for the 500-ppm group did not decrease in a time-related manner after the end of the exposure period and were markedly higher than the ratio of the partition coefficients of the abdominal fat/blood. Taken together, these ratios are
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Table 2. Ratios of the blood and tissues partition coefficients and ratios of the concentrations of DCP, DCE, and CHCl3 in the blood and tissues. Collection time (min) During exposure period
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Partition coefficient (Ratio) DCP 80-ppm group Blood Liver Abdominal fat Lung Kidney DCP 500-ppm group Blood Liver Abdominal fat Lung Kidney DCE 160 ppmg Blood Liver Abdominal fat CHCl3 100 ppmi Blood Liver Abdominal fat
After end of exposure period
60
180
360
30
60
120
180
1080 (min)
18.7a 28.4 499 —f —
1.00 1.52d 26.68 — —
1.00 3.84e 24.76 1.44 2.52
1.00 2.83 42.17 0.90 2.40
1.00 2.41 49.98 0.66 1.71
—b — — — —
1.00 0.97 35.32 0.39 0.90
—b — — — —
1.00 0.62 33.38 0.05 0.43
—c — — — —
18.7a 28.4 499 —f —
1.00 1.52d 26.68 — —
1.00 2.76e 10.45 0.82 1.92
1.00 3.23 33.61 1.02 2.07
1.00 3.26 46.26 1.06 2.10
—b — — — —
1.00 1.99 52.01 0.82 1.80
—b — — — —
1.00 0.95 47.86 0.32 0.72
1.00 0.86 71.29 —c 0.57
30.4a 35.7 344
1.00 1.17d 11.32
1.00 0.91h 8.26
1.00 1.12 12.14
1.00 1.15 15.37
—b — —
1.00 0.79 9.86
—b — —
1.00 0.30 5.83
—c — —
20.8a 21.1 203
1.00 1.01d 9.76
1.00 0.68h 5.66
1.00 0.60 8.29
1.00 0.68 11.63
1.00 0.40 11.36
—b — —
1.00 0.27 9.20
—b — —
—c — —
a
Gargas et al.[16] Blood not collected. c Compound not detected. d Partition coefficient value of the liver or abdominal fat/ partition coefficient value of the blood e Mean concentration in the liver, abdominal fat, lung, or kidney (n D 6)/mean concentration in the blood (n D 6). f Partition coefficient not determined. g Take et al.[11] h Mean concentration in the liver or abdominal fat (n D 5)/mean concentration in the blood (n D 5). i Take et al.[12] b
consistent with a large accumulation of DCP being maintained in the abdominal fat. In our 13-week inhalation study, male rats were exposed to DCP vapor at 125, 250, 500, 1000, or 2000 ppm.[8] Consequently, centrilobular hypertrophy (centrilobular swelling) of liver was observed at 2000 ppm DCP vapor. However, any histopathological lesions were not observed in the lung, kidney, and abdominal fat at the all concentrations.
Relationship between DCP, DCE, and CHCl3 We previously reported the distribution in the blood and tissues of 1,2-dichloroethane (DCE)[11] and chloroform (CHCl3)[12] consequent to exposure of 18-week-old male F344 rats to these compounds by inhalation. DCE and CHCl3, like DCP, are highly lipid-soluble, volatile organic compounds (VOCs) with chlorinated hydrocarbons, and like DCP, DCE and CHCl3 also accumulated in the abdominal fat.[11,12]
The partition coefficients of the blood/air, liver/air, and abdominal fat/air (Table 2) are 30.4, 35.7, 344 (DCE) and 20.8, 21.1, 203 (CHCl3),[16] respectively, and the ratios of the partition coefficients of the liver and abdominal fat to the blood are 1.17, 11.32 (DCE) and 1.01, 9.76 (CHCl3), respectively. During the exposure period, the ratios of the liver/blood concentrations of DCE and CHCl3 were similar to or lower than the partition coefficient ratios (Table 2), and the ratios of the abdominal fat/blood concentrations of DCE and CHCl3 became slightly higher than the partition coefficient ratios. After the end of the exposure period, the ratios of the liver/blood and abdominal fat/blood of the DCE and CHCl3 decreased in a timerelated manner, and the tissue concentration ratios were generally lower than the partition coefficient ratios. The ratios of the liver/blood and abdominal fat/blood DCP concentrations in both the 80-ppm and 500-ppm groups were higher than those in the DCE and CHCl3 exposed rats. Taken together, these data suggest that the accumulation of DCP in the liver and abdominal fat is greater than for DCE and CHCl3, and that the clearance
Effect of 1,2-dichloropropane exposure in rats of DCP from rat body by metabolism and excretion is slower than for DCE and CHCl3.
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Conclusions In the present study, the distribution of DCP in the blood and tissues during and after inhalation exposure was investigated. The accumulation of DCP in the abdominal fat was markedly greater than in the blood and other tissues, and DCP could still be detected in the rat body 18 h after the end of inhalation exposure period. The data reported here greatly extends previously reported time-course changes of DCP concentrations in the blood consequent to inhalation exposure.[9] Our data will improve our understanding of the pharmacokinetics of inhalation exposure to DCP, which is a fundamental consideration for human health risk assessment of exposure to DCP vapor by inhalation.
Acknowledgment We wish to express our thanks to Professor David B. Alexander, Graduate School of Medical Sciences, Nagoya City University and Ms. Yuko Minoshima, Japan Bioassay Research Center, Japan Industrial Safety and Health Association, for proofreading this manuscript. We are also grateful to Mr. Masahiro Yamamoto, Mr. Shigeyuki Hirai, and Ms. Akiko Noda, Japan Bioassay Research Center, Japan Industrial Safety and Health Association, for technical support of this study.
Supplemental material Supplemental data for this article can be accessed on the publisher’s website.
References [1] Agency for Toxic Substances and Disease Registry (ATSDR). 1,2Dichloropropane. Author: Atlanta, GA, 1999. Available at www. atsdr.cdc.gov/toxprofiles/tp134-C2.pdf (accessed Jan 2014). [2] Ministry of the Environment Government of Japan. Pollutant Release and Transfer Register. Environmental Health and Safety Division, Environmental Health Department, Ministry of the Environment Government of Japan: Tokyo, 2011. Available at www.env.go.jp/en/chemi/prtr/substances/index.html (accessed Jan 2014). [3] International Agency for Research on Cancer (IARC). Some halogenated hydrocarbons and pesticide exposures. IARC Monogr. Eval. Carcinog. Risk Chem. Hum. 1986, 41, 131–147.
1347 [4] IARC. Re-evaluation of some organic chemicals, hydrazine and hydrogen peroxide (Part three). IARC Monogr. Eval. Carcinog. Risks Hum. 1999, 71, 1393–1400. [5] Kumagai, S.; Kurumatani, N.; Arimoto, A.; Ichihara, G. Cholangiocarcinoma among offset colour proof-printing workers exposed to 1,2-dichloropropane and/or dichloromethane. Occup. Environ. Med. 2013, 70 (7), 508–510. [6] National Toxicology Program (NTP). Toxicology and carcinogenesis studies of 1,2-dichloropropane (propylene dichloride) in F344/ N rats and B6C3F1 mice (gavage studies). NTP Technical Report Series No. 263. Author: Bethesda, MD, 1986. [7] Matsumoto, M.; Umeda, Y.; Take, M.; Nishizawa, T.; Fukushima, S. Subchronic toxicity and carcinogenicity studies of 1,2dichloropropane inhalation to mice. Inhal. Toxicol. 2013, 25(8), 435–443. [8] Umeda, Y.; Matsumoto, M.; Aiso, S.; Nishizawa, T.; Nagano, K.; Arito, H.; Fukushima, S. Inhalation carcinogenicity and toxicity of 1,2-dichloropropane in rats. Inha. Toxicol. 2010, 22(13), 1116– 1126. [9] Timchalk, C.; Dryzga, M.D.; Smith, F.A.; Bartels, M.J. Disposition and metabolism of [14C]1,2-dichloropropane following oral and inhalation exposure in Fischer 344 rats. Toxicology 1991, 68 (3), 291–306. [10] Take, M.; Ohnishi, M.; Nagano, K.; Yamamoto, S.; Fukushima, S. Design and performance of a system for blood collection of rats under whole-body inhalation exposure. J. Toxicol. Sci. 2009, 34(2), 221–226. [11] Take, M.; Takanobu, K.; Takeuchi, T.; Haresaku, M.; Matsumoto, M.; Nagano, K.; Yamamoto, S.; Fukushima, S. Distribution of blood and tissue concentrations in rats by inhalation exposure to 1,2-dichloroethane. J. Environ. Sci. Health Pt. A 2013, 48(9), 1031–1036. [12] Take, M.; Yamamoto, S.; Ohnishi, M.; Matsumoto, M.; Nagano, K.; Hirota, T.; Fukushima, S. Chloroform distribution and accumulation by combined inhalation plus oral exposure routes in rats. J. Environ. Sci. Health Pt. A 2010, 45(12), 1616–1624. [13] Take, M.; Ohnishi, M.; Yamamoto, S.; Matsumoto, M.; Nagano, K.; Fukushima, S. Distribution of 1,4-dioxane by combined inhalation plus oral exposure routes in rats. Intern. J. Environ. Anal. Chem. 2012, 92(15), 1715–1728. [14] Rozman, K.K.; Klaassen, C.D. Absorption, distribution, and excretion of toxicants. In Casarett & Doull’s Toxicology, 6th Ed.; McGraw-Hill Companies, Inc.: New York, 2001; 91–111. [15] Rodriguez, C.E.; Mahle, D.A.; Gearhart, J.M.; Mattie, D.R.; Lipscomb, J.C.; Cook, R.S.; Barton, H.A. Predicting age-appropriate pharmacokinetics of six volatile organic compounds in the rat utilizing physiologically based pharmacokinetic modeling. Toxicol. Sci. 2007, 98(1), 43–56. [16] Gargas, M.L.; Burgess, R.J.; Voisard, D.E.; Cason, G.H.; Andersen, M.E. Partition coefficients of low-molecular-weight volatile chemicals in various liquids and tissues. Toxicol. Appl. Pharmacol. 1989, 98(1), 87–99. [17] Hutson, D.H.; Moss, J.A.; Pickering, B.A. The excretion and retention of components of the soil fumigant D-D and their metabolites in the rat. Fd. Cosmet. Toxicol. 1971, 9(5), 677– 680. [18] Jones, A.R.; Gibson, J. 1,2-Dichloropropane: metabolism and fate in the rat. Xenobiotica 1980, 10(11), 835–846. [19] Bartels, M.J.; Timchalk, C. 1,2-Dichloropropane: investigation of the mechanism of mercapturic acid formation in the rat. Xenobiotica 1990, 20(10), 1035–1042.
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Appendices Appendix Table A1. DCP concentration in the blood. DCP concentration
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Collection time (min) During the exposure period 0 60 180 360 After the end of the exposure period 60 180 1080 a b
80-ppm group
500-ppm group
N.D.a 0.25 § 0.13b 0.30 § 0.07 0.41 § 0.03
N.D. 2.51 § 0.56 4.60 § 0.89 5.85 § 1.08
0.31 § 0.02 0.21 § 0.03 N.D.
4.35 § 0.59 2.42 § 0.45 0.07 § 0.03
N.D.: not detected. mg/mL (mean § S.D.).
Appendix Table A2. DCP concentration in each tissue. DCP concentration 80 ppm group Collection time (min)
Lung
During exposure period 0 N.D.a 60 0.36 § 0.09b 180 0.27 § 0.08 360 0.27 § 0.10 After end of exposure period 60 0.12 § 0.07 180 0.01 § 0.01 1080 N.D. a
N.D.: not detected. mg/g (mean § S.D.).
b
Liver
Kidney
500 ppm group Abdominal fat
Lung
Liver
Kidney
Abdominal fat
N.D. N.D. 0.96 § 0.20 0.63 § 0.16 0.85 § 0.12 0.72 § 0.20 0.99 § 0.12 0.70 § 0.08
N.D. 6.19 § 1.30 12.65 § 2.29 20.49 § 3.67
N.D. N.D. N.D. N.D. 2.05 § 0.67 6.94 § 1.26 4.82 § 1.04 26.22 § 4.57 4.71 § 2.19 14.85 § 2.87 9.52 § 1.54 154.62 § 68.79 6.23 § 1.47 19.06 § 2.93 12.26 § 1.67 270.65 § 55.83
0.30 § 0.03 0.28 § 0.08 0.13 § 0.02 0.09 § 0.01 N.D. N.D.
10.95 § 1.78 7.01 § 1.12 0.17 § 0.09
3.58 § 0.82 0.77 § 0.33 N.D.
8.65 § 1.35 2.29 § 0.43 0.06 § 0.02
7.85 § 1.97 1.75 § 0.23 0.04 § 0.01
226.23 § 21.78 115.83 § 21.14 4.99 § 2.19
Journal of Environmental Science and Health, Part A: Toxic/Hazardous Substances and Environmental Engineering Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/lesa20
Inhalation exposure to 1,2-dichloropropane: Distribution of blood and tissue concentrations of 1,2dichloropropane in rats during and after exposure a
a
a
a
a
Makoto Take , Michiharu Matsumoto , Tetsuya Takeuchi , Mitsuru Haresaku , Hitomi Kondo , a
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Hideki Senoh , Yumi Umeda , Takeji Takamura-Enya & Shoji Fukushima a
Japan Bioassay Research Center, Japan Industrial Safety and Health Association, Hadano, Kanagawa, Japan b
Department of Applied Chemistry, Faculty of Engineering, Kanagawa Institute of Technology, Atsugi, Kanagawa, Japan Published online: 29 Jul 2014.
To cite this article: Makoto Take, Michiharu Matsumoto, Tetsuya Takeuchi, Mitsuru Haresaku, Hitomi Kondo, Hideki Senoh, Yumi Umeda, Takeji Takamura-Enya & Shoji Fukushima (2014) Inhalation exposure to 1,2-dichloropropane: Distribution of blood and tissue concentrations of 1,2-dichloropropane in rats during and after exposure, Journal of Environmental Science and Health, Part A: Toxic/Hazardous Substances and Environmental Engineering, 49:12, 1341-1348, DOI: 10.1080/10934529.2014.928193 To link to this article: http://dx.doi.org/10.1080/10934529.2014.928193
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Journal of Environmental Science and Health, Part A (2014) 49, 1341–1348 Copyright © Taylor & Francis Group, LLC ISSN: 1093-4529 (Print); 1532-4117 (Online) DOI: 10.1080/10934529.2014.928193
Inhalation exposure to 1,2-dichloropropane: Distribution of blood and tissue concentrations of 1,2-dichloropropane in rats during and after exposure
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MAKOTO TAKE1, MICHIHARU MATSUMOTO1, TETSUYA TAKEUCHI1, MITSURU HARESAKU1, HITOMI KONDO1, HIDEKI SENOH1, YUMI UMEDA1, TAKEJI TAKAMURA-ENYA2 and SHOJI FUKUSHIMA1 1 2
Japan Bioassay Research Center, Japan Industrial Safety and Health Association, Hadano, Kanagawa, Japan Department of Applied Chemistry, Faculty of Engineering, Kanagawa Institute of Technology, Atsugi, Kanagawa, Japan
The present investigation was undertaken to determine the distribution and accumulation of 1,2-dichloropropane (DCP) in the blood, lung, liver, kidney, and abdominal fat of rats during and after inhalation exposure. Male rats were exposed to 80 or 500 ppm (v/v) DCP vapor for 360 min and the concentrations of DCP in the blood and tissues during the inhalation exposure period and after the end of the exposure period were measured. DCP accumulation in the abdominal fat was much greater than that in the blood and other tissues. Eighteen hours after the end of inhalation exposure, DCP could still be detected in the abdominal fat in the 80-ppm group, and in the blood, liver, kidney, and abdominal fat in the 500-ppm group. Our results are valuable data pertaining to the pharmacokinetics of DCP and to human health risk assessment of exposure to DCP vapor by inhalation. Keywords: 1,2-Dichloropropane, blood concentration, tissue concentrations, inhalation exposure.
Introduction The chemical 1,2-Dichloropropane (DCP) was used in the past as a soil fumigant, chemical intermediate, and industrial solvent and was found in paint strippers, varnishes, and furniture finish removers.[1] Most uses of DCP have now been discontinued; however, DCP is still detected in the ambient air and public water in Japan.[2] Because DCP is highly volatile,[3] the primary route of exposure of humans to DCP is inhalation of its vapor. The International Agency for Research on Cancer (IARC) evaluated DCP as a Group 3 agent:[4] an agent which is not classifiable as to its carcinogenicity to humans. However, in a recent report, workers in a small printing company in Japan were exposed to the chlorinated organic solvents DCP and/or dichloromethane (DCM) between 1985 and 2006 (DCP) and 1985 to 1997/ 1998 (DCM) and were affected with cholangiocarcinoma.[5] In two-year animal studies, the National Toxicology Program (NTP) reported a dose-related increase of Address correspondence to Makoto Take, Japan Bioassay Research Center, Japan Industrial Safety and Health Association, 2445 Hirasawa, Hadano, Kanagawa 257-0015, Japan E-mail: [email protected] Received February 4, 2014.
liver tumors in mice of both sexes, but not in rats, administered DCP by gavage.[6] However, while DCP administration did not result in induction of liver tumors in rats, a dose-related increase in mammary gland adenocarcinomas in female rats was observed.[6] Our two-year inhalation carcinogenicity study of 200 ppm (mice) or 500 ppm (rats) DCP vapor showed that inhalation exposure to mice and rats of both sexes resulted in significantly increased incidences of tumors in the Harderian gland of male mice, the lung of female mice,[7] and the nasal cavity of male and female rats.[8] To better evaluate the toxicity of volatile chemicals, such as DCP, it is necessary to know the time-course changes of the chemical’s concentration in the blood and tissues consequent to exposure as this is a key factor affecting toxicity. Therefore, understanding the pharmacokinetics of exposure in animal models is one of the fundamental considerations in human risk assessment of exposure to a chemical. To date, only limited information is available about time-course changes of DCP concentration in the blood of animals exposed to DCP by inhalation.[9] The present study was designed to investigate DCP concentrations in the blood and tissues of rats exposed to 80 or 500 ppm DCP vapor, concentrations corresponding to the low and high doses used in the two-year carcinogenicity study by Umeda et al. [8]
1342 Material and methods Chemical DCP of analytical grade (greater than 99.5% pure) was obtained from Wako Pure Chemical Industries, Ltd. (Osaka, Japan).
Take et al. blood samples were collected into 10-mL headspace sampler (HS)-vials, and 0.2 mL of distilled water was added to each sample.[11–13] The lung, liver, kidney, and abdominal fat were removed from each rat, and tissue samples (range from about 0.1 to 0.5 g) were placed into separate 10-mL HS-vials. Then, 5 mL of distilled water was added to the vials, and the vials were immediately sealed with an aluminum crimp cup.[11–13]
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Animals Eighty-four F344/DuCrlCrlj (SPF) male rats were obtained at 6–8 weeks of age from Charles River Laboratories Japan, Inc. (Kanagawa, Japan). All animals were quarantined for one week and were then housed in our laboratory until the age of 18 weeks. The body weights of rats at the start of experiment ranged from 285 to 332 g. Rats were housed in individual cages in a temperature controlled room with a 12-h light/dark cycle (light: 8:00– 20:00). The room temperature and relative humidity were maintained in the ranges of 22 § 2 C and 55 § 15%, respectively. All animals had access to food (CRF-1; Oriental Yeast Co., Ltd., Tokyo, Japan) and tap water ad libitum, except during the inhalation exposure period. Animals were cared for in compliance with the “Regulation for Proper Conduct of Animal Experiments in the Japan Bioassay Research Center,” and the present study was approved by the Ethics Committee of Animal Experiments of the Japan Bioassay Research Center (JBRC). Experiment design The rats were divided into 2 groups, 42 rats in each group, and exposed to 80 or 500 ppm DCP by inhalation for 360 min using whole-body inhalation chambers, which were developed on the JBRC’s own with volume of 1.1 L/ animal.[10] During inhalation exposure period, the rats had no access to food and water. DCP vapor at 80 or 500 ppm was generated by bubbling clean air through liquid DCP. DCP vapor concentrations in the inhalation chambers were measured by gas chromatography (GC) using Agilent Technologies 6890 (Agilent Technologies, Santa Clara, CA, USA) every 15 min during the inhalation exposure period. The target DCP vapor concentrations in the inhalation chambers were 80 and 500 ppm (v/v), and the measured concentrations were 80.2 § 0.9 and 502.1 § 8.6 ppm (mean § S.D.).
Measurement of DCP in the blood and tissues DCP concentrations in the blood and tissue samples were analyzed by HS-GC/mass spectrometer (MS) using Agilent Technologies 7694 HS (oven temperature, 100 C; loop temperature, 130 C; 10 min vial equilibration time for blood samples and 30 min for tissue samples) and Agilent Technologies 5973N GC/MS system (column, J&W DB-1 60 m £ 0.25 mm ID £ 0.25 mm; oven temperature, 100 C; ion source temperature, 230 C; carrier gas, helium at 1 mL/min; ionization, EI (electron ionization); fragment peak, 63 m/z).
Results and discussion Blood concentration of DCP In the 80-ppm group, during the inhalation exposure period, DCP concentration in the blood was increased at 60 min after the start of inhalation exposure and showed a tendency to slightly increase from 60 to 360 min (Fig. 1), background concentration of DCP was not measureable in blood at 0 min. After the end of the exposure period, the DCP concentrations in the blood decreased in a timerelated manner: DCP remained detectable in the blood at 180 min after the end of the exposure period, but it was not detected in the blood at 1080 min (18 h after the end of the inhalation exposure).
Blood and tissue collection and treatment Blood was collected from the tail vein of each rat, and blood collection was immediately followed by necropsy under isoflurane anesthesia. In each group, blood collection and necropsy were performed at 0, 60, 180, and 360 min during the 360-min inhalation exposure period and at 60, 180, and 1080 min after the end of the exposure period. Six rats were used for each collection. 0.2 mL
Fig. 1. The concentration of DCP in the blood of rats exposed to DCP by inhalation (n D 6 for each collection time point).
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Effect of 1,2-dichloropropane exposure in rats During the inhalation exposure period, the DCP concentration in the blood was governed by the blood-to-gas partition coefficient [14] with the DCP concentration in the gas phase of the lung remaining constant. DCP concentration in the blood increased at each blood-collection time point, suggesting that DCP may not have reached steady state during the 6-h exposure period. During the exposure period, DCP was lost from the blood by exhalation and by partitioning into target tissues; however, DCP lost from the blood was replenished from the DCP in the air. In the 500-ppm group, during the inhalation exposure period, DCP concentration in the blood increased from 0 until 360 min in a time-related manner after the start of inhalation exposure (Fig. 1), background concentration of DCP was not measureable in blood at 0 min. After the end of the exposure period, the DCP concentrations in the blood decreased in a time-related manner; however, DCP remained detectable in the blood at 1080 min after the end of the exposure period. The time-related increase of DCP concentration in the blood throughout the exposure period in the 500-ppm group indicates that steady state was not reached between the DCP in the gas phase of the lung and the concentration of DCP in the blood during the 6-h exposure period. In addition, the 10 to 15-fold higher concentration of DCP in the blood of the 500-ppm group compared to the 80-ppm group suggests that the metabolic saturation point was exceeded in rats exposed to 500 ppm DCP, resulting in a proportionally greater build-up of DCP in the bodies of rats exposed to 500 ppm DCP than in rats exposed to 80 ppm DCP. Timchalk et al.[9] reported the concentration of [14C] DCP in the blood of 7-week-old F344 rats exposed to 5, 50, or 100 ppm [14C]DCP for 6 h using a head-only inhalation chamber. In the Timchalk et al.[9] study, the maximum concentrations of [14C]DCP in the blood of rats exposed to 50 ppm was higher than the maximum concentration of DCP in the blood of rats exposed to 80 ppm in our study. This apparent discrepancy is due to such differences as the age of the test animals [15] and use of a headonly rather than a whole-body inhalation chamber. Half-lives (T1/2) of the concentration of DCP in the blood after the end of the inhalation exposure period are shown in Table 1. The T1/2 values were similar in the 80ppm and 500-ppm groups. The area-under-the-curve (AUC) values for the blood from 0 to 1440 min (AUC01440: the 360 min inhalation exposure period and the 1080 min following the exposure period) are shown in Table 1. The AUC0-1440 value for the blood of the 500ppm group was higher than that of the 80-ppm group. The ratio of the AUC0-1440 value for the 500-ppm group to the AUC0-1440 value for the 80-ppm group is 13, and this value is higher than the ratio of DCP concentrations (500/ 80 ppm D 6.25). This is the first report of AUCs for DCP in the blood consequent to exposure to DCP vapor by inhalation and will be valuable in the further development
Table 1. T1/2, AUC, and ratios in the blood and tissues. 80-ppm group
500-ppm group
T1/2 AUC0-1440 Ratio T1/2 AUC0-1440 Ratio Blood 182a Lung 39 Liver 57 Kidney 59 Abdominal fat 154
251b 122c 425 317 9553
1.00 0.49d 1.69 1.26 38.06
168 61 125 127 186
3272 1.00 2352 0.73 7113 2.20 4951 1.53 139711 43.12
Units include amin; bmg/mL £ min; and, cmg/g £ min. d AUC0-1440 value of the lung, liver, kidney or abdominal fat/AUC0-1440 value of the blood.
of DCP physiologically based pharmacokinetic (PBPK) models.
Tissue concentrations of DCP In the 80-ppm group, during the inhalation exposure period, the time-course changes of DCP concentrations in the lung, liver, and kidney showed similar patterns (Figs. 2–4). The concentrations of DCP in these tissues reached a maximum by 60 min and then remained constant from 60 to 360 min, background concentration of DCP was not measureable in these tissues at 0 min. The distribution of DCP into these tissues was governed by the partition coefficients between the blood and the tissues and by metabolism and excretion of DCP in the tissues.[14] DCP concentration in these tissues is expected to increase due to uptake of increasing amounts of DCP from the blood until steady state is reached between the gas phase of the lung and the blood and between the blood and the target tissue. DCP concentration in these tissues is expected to decrease due to metabolism (primarily liver), exhalation (lung), and excretion (primarily kidney). DCP
Fig. 2. The concentration of DCP in the lung of rats exposed to DCP by inhalation (n D 6 for each collection time point).
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Fig. 3. The concentration of DCP in the liver of rats exposed to DCP by inhalation (n D 6 for each collection time point).
Fig. 5. The concentration of DCP in the abdominal fat of rats exposed to DCP by inhalation (n D 6 for each collection time point).
lost from these tissues will be replenished from the DCP in the blood. Steady-state levels of DCP in these tissues was established by 60 min, after which uptake from the blood was balanced by removal through metabolism, exhalation, and excretion. In contrast, the concentrations of DCP in the abdominal fat of the 80-ppm group increased in a time-related manner throughout the exposure period (Fig. 5), and were much higher than in the other tissues at each collection time point: background concentration of DCP was not measureable in the abdominal fat at 0 min. The high concentration of DCP in the abdominal fat was due to the very high lipid solubility of DCP [3] coupled with low metabolism and excretion of DCP in this tissue. As with the other tissues, the DCP concentration in the abdominal fat will increase as it reaches steady state with the blood as governed by the blood-fat partition coefficient [16] and will decrease due to metabolism and excretion. The high lipid solubility of DCP leads to a longer period of time being
required to reach steady state in the abdominal fat than in the other tissues. After the end of the exposure period, the DCP concentrations in all the tissues of the 80-ppm group decreased in a time-related manner. DCP remained detectable in all the tissues at 180 min after the end of the exposure period. DCP was not detected in the lung, liver, or kidney at 1080 min, but a measurable level DCP was still present in the abdominal fat at 1080 min. The concentration of DCP in the tissues was governed primarily by two functions: (1) removal of DCP from the blood by exhalation, excretion, and metabolism;[9,17–19] and (2) redistribution of DCP from the storage tissues into the blood to reestablish steady state with the blood as DCP was removed from the body. However, because the accumulation of DCP in the abdominal fat was much higher than that in the other tissues and because of the low partitioning of DCP into the blood, DCP had not been cleared from the abdominal fat 18 h after the end of the inhalation exposure period. In the 500-ppm group, during the inhalation exposure period, the time-course changes of DCP concentration in the lung, liver, kidney, and abdominal fat showed a pattern similar to the time-course changes of DCP concentration in the blood (Figs. 1–5) with the concentrations of DCP in these tissues increasing in a time-related manner throughout the exposure period. These results are consistent with steady state not being reached between the DCP in the gas phase of the lung and the DCP in the blood during the 6-h exposure period. In addition, the 4- to 23-fold higher concentration of DCP in the tissues of the 500-ppm group compared to the 80-ppm group suggests that it is possible that the metabolic saturation point was exceeded at the 500 ppm concentration, resulting in a greater buildup of DCP in the body in the 500-ppm group than in the 80-ppm group. Similarly to the 80-ppm group, the concentration of DCP in the abdominal fat was much higher than in the other tissues at each collection time point. As discussed for
Fig. 4. The concentration of DCP in the kidney of rats exposed to DCP by inhalation (n D 6 for each collection time point).
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Effect of 1,2-dichloropropane exposure in rats the 80-ppm group, the high concentration of DCP in the abdominal fat was due to the very high lipid solubility of DCP [3] coupled with low metabolism and excretion of DCP in this tissue. After the end of the exposure period, the DCP concentrations in the tissues decreased in a time-related manner. DCP remained detectable in the tissues at 180 min after the end of the exposure period. DCP was not detected in the lung at 1080 min, but a measurable level DCP was still present in the liver, kidney, and abdominal fat at 1080 min. The level in the abdominal fat at 1080 min was approximately 100-fold higher than in the liver and kidney. Timchalk et al.[9] reported that the metabolites of DCP following exposure by inhalation are eliminated primarily in the urine and the expired air with a lesser amount excreted in the feces, and that while the majority of DCP was eliminated 24 h after the end of the exposure period, [14C]-volatile organics identified as DCP were still detected in the expired air 42 h after the end of the 6-h inhalation exposure period. Our results are consistent with their results, while the majority of DCP was rapidly eliminated from the body, minor amounts of DCP were detected in the abdominal fat of the 80-ppm group and the blood, liver, kidney, and abdominal fat of the 500-ppm group 18 h post-exposure. The T1/2 values of the concentration of DCP in the tissues after the end of the inhalation exposure period are shown in Table 1. In the two groups, the T1/2 value of the lung was the shortest, and the T1/2 value of the abdominal fat was the longest. The T1/2 values of the lung, liver, and kidney in the 500-ppm group were longer than that in the 80-ppm group. The T1/2 values of the abdominal fat were similar to the T1/2 values of the blood in the two groups. The similar T1/2 values of DCP in the blood and abdominal fat in the two groups coupled with the longer T1/2 values of DCP in the lung, liver, and kidney in the 500-ppm group suggest that DCP was transported from the abdominal fat to other tissues by the blood, thereby lengthening the T1/2 values in these tissues. The tissue AUC0-1440 values and the ratio of the AUC01440 value for each tissue to the AUC0-1440 value for blood are shown in Table 1. As expected, all of the tissue AUC01440 values were higher in rats in the 500-ppm group than in the 80-ppm group. Similarly to the tissue concentrations of DCP at individual time points, the ratio of the tissue AUC0-1440 values for the 500-ppm group to the 80-ppm group–19 (lung), 17 (liver), 16 (kidney), and 15 (abdominal fat)–are higher than the ratio of exposure concentrations (500/80 ppm D 6.25). In addition, the AUC0-1440 value for the abdominal fat and the ratio of the AUC0-1440 value for the abdominal fat to the AUC0-1440 value for the blood are markedly higher than the values for the blood and other tissues. However, the ratios of the AUC0-1440 values for the lung, liver, kidney, and abdominal fat to the AUC0-1440 value for the blood in the 80-ppm group are about the same
as in the 500-ppm group. These results are consistent with abdominal fat being the major reservoir of DCP in the rat body after exposure to DCP vapor. As discussed above in relation to the T1/2 of DCP in the various tissues in the two exposure groups, the large amount of DCP accumulated in the abdominal fat affected the metabolism and elimination of DCP from the body. This is the first report of the distribution of DCP in tissues during and after exposure by inhalation and will be valuable in the further development of DCP PBPK models.
DCP partition coefficients and tissue concentrations Table 2 shows the relationship between the partition coefficients and the ratios of the concentrations of DCP in the blood and tissues. The partition coefficients of the blood/ air, liver/air, and abdominal fat/air are 18.7, 28.4, and 499, respectively,[16] and the ratios of the partition coefficients of the liver and abdominal fat to the blood are 1.52 and 26.68. During the inhalation exposure period, the ratios of the concentrations of DCP in the liver/blood ranged from 2.41 to 3.84 for the 80-ppm group and from 2.76 to 3.26 for the 500-ppm group. The liver/blood ratios in the two groups were similar and about 2-fold higher than the ratio of the partition coefficients of liver/blood (1.52). The ratios of the DCP concentrations in the lung/blood and kidney/blood were also similar for the two groups. The ratios were 15–34% (80ppm group) and 30–36% (500-ppm group) lower in the kidney than in the liver and were 43–63% (80-ppm group) and 50–57% (500-ppm group) lower in the lung than in the kidney. The ratios of the DCP concentrations in the abdominal fat/blood ranged from 24.76 to 49.98 for the 80-ppm group; from 10.45 to 46.26 for the 500-ppm group. These values increased in time-related manner throughout the exposure period. The values at 180 and 360 min for both groups were higher than the ratio of the partition coefficients of abdominal fat/blood (26.68). Taken together, these ratios are indicative of a large distribution in abdominal fat and liver of DCP in the rat body. After the end of the inhalation exposure period, the ratios of the DCP concentrations in the liver/blood, lung/ blood, and kidney/blood for the two groups decreased in time-related manner and were lower than the partition coefficient ratios (Table 2). Similarly, the ratios of the DCP concentrations in the abdominal fat/blood for the 80-ppm group decreased in a time-related manner; however, the values at 60 and 180 min were higher than the ratio of the partition coefficients of abdominal fat/blood. In contrast to all the other tissue/blood ratios, the ratios of the DCP concentrations in the abdominal fat/blood for the 500-ppm group did not decrease in a time-related manner after the end of the exposure period and were markedly higher than the ratio of the partition coefficients of the abdominal fat/blood. Taken together, these ratios are
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Table 2. Ratios of the blood and tissues partition coefficients and ratios of the concentrations of DCP, DCE, and CHCl3 in the blood and tissues. Collection time (min) During exposure period
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Partition coefficient (Ratio) DCP 80-ppm group Blood Liver Abdominal fat Lung Kidney DCP 500-ppm group Blood Liver Abdominal fat Lung Kidney DCE 160 ppmg Blood Liver Abdominal fat CHCl3 100 ppmi Blood Liver Abdominal fat
After end of exposure period
60
180
360
30
60
120
180
1080 (min)
18.7a 28.4 499 —f —
1.00 1.52d 26.68 — —
1.00 3.84e 24.76 1.44 2.52
1.00 2.83 42.17 0.90 2.40
1.00 2.41 49.98 0.66 1.71
—b — — — —
1.00 0.97 35.32 0.39 0.90
—b — — — —
1.00 0.62 33.38 0.05 0.43
—c — — — —
18.7a 28.4 499 —f —
1.00 1.52d 26.68 — —
1.00 2.76e 10.45 0.82 1.92
1.00 3.23 33.61 1.02 2.07
1.00 3.26 46.26 1.06 2.10
—b — — — —
1.00 1.99 52.01 0.82 1.80
—b — — — —
1.00 0.95 47.86 0.32 0.72
1.00 0.86 71.29 —c 0.57
30.4a 35.7 344
1.00 1.17d 11.32
1.00 0.91h 8.26
1.00 1.12 12.14
1.00 1.15 15.37
—b — —
1.00 0.79 9.86
—b — —
1.00 0.30 5.83
—c — —
20.8a 21.1 203
1.00 1.01d 9.76
1.00 0.68h 5.66
1.00 0.60 8.29
1.00 0.68 11.63
1.00 0.40 11.36
—b — —
1.00 0.27 9.20
—b — —
—c — —
a
Gargas et al.[16] Blood not collected. c Compound not detected. d Partition coefficient value of the liver or abdominal fat/ partition coefficient value of the blood e Mean concentration in the liver, abdominal fat, lung, or kidney (n D 6)/mean concentration in the blood (n D 6). f Partition coefficient not determined. g Take et al.[11] h Mean concentration in the liver or abdominal fat (n D 5)/mean concentration in the blood (n D 5). i Take et al.[12] b
consistent with a large accumulation of DCP being maintained in the abdominal fat. In our 13-week inhalation study, male rats were exposed to DCP vapor at 125, 250, 500, 1000, or 2000 ppm.[8] Consequently, centrilobular hypertrophy (centrilobular swelling) of liver was observed at 2000 ppm DCP vapor. However, any histopathological lesions were not observed in the lung, kidney, and abdominal fat at the all concentrations.
Relationship between DCP, DCE, and CHCl3 We previously reported the distribution in the blood and tissues of 1,2-dichloroethane (DCE)[11] and chloroform (CHCl3)[12] consequent to exposure of 18-week-old male F344 rats to these compounds by inhalation. DCE and CHCl3, like DCP, are highly lipid-soluble, volatile organic compounds (VOCs) with chlorinated hydrocarbons, and like DCP, DCE and CHCl3 also accumulated in the abdominal fat.[11,12]
The partition coefficients of the blood/air, liver/air, and abdominal fat/air (Table 2) are 30.4, 35.7, 344 (DCE) and 20.8, 21.1, 203 (CHCl3),[16] respectively, and the ratios of the partition coefficients of the liver and abdominal fat to the blood are 1.17, 11.32 (DCE) and 1.01, 9.76 (CHCl3), respectively. During the exposure period, the ratios of the liver/blood concentrations of DCE and CHCl3 were similar to or lower than the partition coefficient ratios (Table 2), and the ratios of the abdominal fat/blood concentrations of DCE and CHCl3 became slightly higher than the partition coefficient ratios. After the end of the exposure period, the ratios of the liver/blood and abdominal fat/blood of the DCE and CHCl3 decreased in a timerelated manner, and the tissue concentration ratios were generally lower than the partition coefficient ratios. The ratios of the liver/blood and abdominal fat/blood DCP concentrations in both the 80-ppm and 500-ppm groups were higher than those in the DCE and CHCl3 exposed rats. Taken together, these data suggest that the accumulation of DCP in the liver and abdominal fat is greater than for DCE and CHCl3, and that the clearance
Effect of 1,2-dichloropropane exposure in rats of DCP from rat body by metabolism and excretion is slower than for DCE and CHCl3.
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Conclusions In the present study, the distribution of DCP in the blood and tissues during and after inhalation exposure was investigated. The accumulation of DCP in the abdominal fat was markedly greater than in the blood and other tissues, and DCP could still be detected in the rat body 18 h after the end of inhalation exposure period. The data reported here greatly extends previously reported time-course changes of DCP concentrations in the blood consequent to inhalation exposure.[9] Our data will improve our understanding of the pharmacokinetics of inhalation exposure to DCP, which is a fundamental consideration for human health risk assessment of exposure to DCP vapor by inhalation.
Acknowledgment We wish to express our thanks to Professor David B. Alexander, Graduate School of Medical Sciences, Nagoya City University and Ms. Yuko Minoshima, Japan Bioassay Research Center, Japan Industrial Safety and Health Association, for proofreading this manuscript. We are also grateful to Mr. Masahiro Yamamoto, Mr. Shigeyuki Hirai, and Ms. Akiko Noda, Japan Bioassay Research Center, Japan Industrial Safety and Health Association, for technical support of this study.
Supplemental material Supplemental data for this article can be accessed on the publisher’s website.
References [1] Agency for Toxic Substances and Disease Registry (ATSDR). 1,2Dichloropropane. Author: Atlanta, GA, 1999. Available at www. atsdr.cdc.gov/toxprofiles/tp134-C2.pdf (accessed Jan 2014). [2] Ministry of the Environment Government of Japan. Pollutant Release and Transfer Register. Environmental Health and Safety Division, Environmental Health Department, Ministry of the Environment Government of Japan: Tokyo, 2011. Available at www.env.go.jp/en/chemi/prtr/substances/index.html (accessed Jan 2014). [3] International Agency for Research on Cancer (IARC). Some halogenated hydrocarbons and pesticide exposures. IARC Monogr. Eval. Carcinog. Risk Chem. Hum. 1986, 41, 131–147.
1347 [4] IARC. Re-evaluation of some organic chemicals, hydrazine and hydrogen peroxide (Part three). IARC Monogr. Eval. Carcinog. Risks Hum. 1999, 71, 1393–1400. [5] Kumagai, S.; Kurumatani, N.; Arimoto, A.; Ichihara, G. Cholangiocarcinoma among offset colour proof-printing workers exposed to 1,2-dichloropropane and/or dichloromethane. Occup. Environ. Med. 2013, 70 (7), 508–510. [6] National Toxicology Program (NTP). Toxicology and carcinogenesis studies of 1,2-dichloropropane (propylene dichloride) in F344/ N rats and B6C3F1 mice (gavage studies). NTP Technical Report Series No. 263. Author: Bethesda, MD, 1986. [7] Matsumoto, M.; Umeda, Y.; Take, M.; Nishizawa, T.; Fukushima, S. Subchronic toxicity and carcinogenicity studies of 1,2dichloropropane inhalation to mice. Inhal. Toxicol. 2013, 25(8), 435–443. [8] Umeda, Y.; Matsumoto, M.; Aiso, S.; Nishizawa, T.; Nagano, K.; Arito, H.; Fukushima, S. Inhalation carcinogenicity and toxicity of 1,2-dichloropropane in rats. Inha. Toxicol. 2010, 22(13), 1116– 1126. [9] Timchalk, C.; Dryzga, M.D.; Smith, F.A.; Bartels, M.J. Disposition and metabolism of [14C]1,2-dichloropropane following oral and inhalation exposure in Fischer 344 rats. Toxicology 1991, 68 (3), 291–306. [10] Take, M.; Ohnishi, M.; Nagano, K.; Yamamoto, S.; Fukushima, S. Design and performance of a system for blood collection of rats under whole-body inhalation exposure. J. Toxicol. Sci. 2009, 34(2), 221–226. [11] Take, M.; Takanobu, K.; Takeuchi, T.; Haresaku, M.; Matsumoto, M.; Nagano, K.; Yamamoto, S.; Fukushima, S. Distribution of blood and tissue concentrations in rats by inhalation exposure to 1,2-dichloroethane. J. Environ. Sci. Health Pt. A 2013, 48(9), 1031–1036. [12] Take, M.; Yamamoto, S.; Ohnishi, M.; Matsumoto, M.; Nagano, K.; Hirota, T.; Fukushima, S. Chloroform distribution and accumulation by combined inhalation plus oral exposure routes in rats. J. Environ. Sci. Health Pt. A 2010, 45(12), 1616–1624. [13] Take, M.; Ohnishi, M.; Yamamoto, S.; Matsumoto, M.; Nagano, K.; Fukushima, S. Distribution of 1,4-dioxane by combined inhalation plus oral exposure routes in rats. Intern. J. Environ. Anal. Chem. 2012, 92(15), 1715–1728. [14] Rozman, K.K.; Klaassen, C.D. Absorption, distribution, and excretion of toxicants. In Casarett & Doull’s Toxicology, 6th Ed.; McGraw-Hill Companies, Inc.: New York, 2001; 91–111. [15] Rodriguez, C.E.; Mahle, D.A.; Gearhart, J.M.; Mattie, D.R.; Lipscomb, J.C.; Cook, R.S.; Barton, H.A. Predicting age-appropriate pharmacokinetics of six volatile organic compounds in the rat utilizing physiologically based pharmacokinetic modeling. Toxicol. Sci. 2007, 98(1), 43–56. [16] Gargas, M.L.; Burgess, R.J.; Voisard, D.E.; Cason, G.H.; Andersen, M.E. Partition coefficients of low-molecular-weight volatile chemicals in various liquids and tissues. Toxicol. Appl. Pharmacol. 1989, 98(1), 87–99. [17] Hutson, D.H.; Moss, J.A.; Pickering, B.A. The excretion and retention of components of the soil fumigant D-D and their metabolites in the rat. Fd. Cosmet. Toxicol. 1971, 9(5), 677– 680. [18] Jones, A.R.; Gibson, J. 1,2-Dichloropropane: metabolism and fate in the rat. Xenobiotica 1980, 10(11), 835–846. [19] Bartels, M.J.; Timchalk, C. 1,2-Dichloropropane: investigation of the mechanism of mercapturic acid formation in the rat. Xenobiotica 1990, 20(10), 1035–1042.
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Appendices Appendix Table A1. DCP concentration in the blood. DCP concentration
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Collection time (min) During the exposure period 0 60 180 360 After the end of the exposure period 60 180 1080 a b
80-ppm group
500-ppm group
N.D.a 0.25 § 0.13b 0.30 § 0.07 0.41 § 0.03
N.D. 2.51 § 0.56 4.60 § 0.89 5.85 § 1.08
0.31 § 0.02 0.21 § 0.03 N.D.
4.35 § 0.59 2.42 § 0.45 0.07 § 0.03
N.D.: not detected. mg/mL (mean § S.D.).
Appendix Table A2. DCP concentration in each tissue. DCP concentration 80 ppm group Collection time (min)
Lung
During exposure period 0 N.D.a 60 0.36 § 0.09b 180 0.27 § 0.08 360 0.27 § 0.10 After end of exposure period 60 0.12 § 0.07 180 0.01 § 0.01 1080 N.D. a
N.D.: not detected. mg/g (mean § S.D.).
b
Liver
Kidney
500 ppm group Abdominal fat
Lung
Liver
Kidney
Abdominal fat
N.D. N.D. 0.96 § 0.20 0.63 § 0.16 0.85 § 0.12 0.72 § 0.20 0.99 § 0.12 0.70 § 0.08
N.D. 6.19 § 1.30 12.65 § 2.29 20.49 § 3.67
N.D. N.D. N.D. N.D. 2.05 § 0.67 6.94 § 1.26 4.82 § 1.04 26.22 § 4.57 4.71 § 2.19 14.85 § 2.87 9.52 § 1.54 154.62 § 68.79 6.23 § 1.47 19.06 § 2.93 12.26 § 1.67 270.65 § 55.83
0.30 § 0.03 0.28 § 0.08 0.13 § 0.02 0.09 § 0.01 N.D. N.D.
10.95 § 1.78 7.01 § 1.12 0.17 § 0.09
3.58 § 0.82 0.77 § 0.33 N.D.
8.65 § 1.35 2.29 § 0.43 0.06 § 0.02
7.85 § 1.97 1.75 § 0.23 0.04 § 0.01
226.23 § 21.78 115.83 § 21.14 4.99 § 2.19
