Toxicology Letters, 58 (1991) 59-61 0 1991 Elsevier Science Publishers B.V. 037%4274/91/$3.50 ADONIS 0378427491001115 59 TOXLET 026 11 The effect of extraction solvent on the mutagenicity of airborne particles Huei Lee’, Seok Mooi Law’ and Shaw Tao Lin2 ‘Department of Biochemistry, Chung San Medical and Dental College and ‘Department of Applied Chemistry, Providence University, Taichung, Taiwan (Republic of China) (Received 22 January 1991) (Revision received 17 April 199 1) (Accepted 19 April 1991) Key words: Airborne particles; Extraction methods; Mutagenicity; PAH; Nitro-PAH SUMMARY Two different solvents (acetone and dichloromethane) were compared for their efficacy in extraction of mutagenic compounds from airborne particulate samples. Their mutagenicity was examined with Salmo- nella typhimurium TA98 in presence or absence of S9 mix. The total mutagenic activity of the acetone extract was 1.8-7.0-fold that of the dichloromethane extract. The content of 1 -nitropyrene, 1,6-dinitropy- rene, dibenzo[a,h]anthracene and indo[l,2,3-c,d]pyrene in acetone extracts of airborne particulate samples was 3.8-, 3.6-, 6.6- and 1135-fold that of dichloromethane,extracts, respectively. 1,8_Dinitropyrene, ben- zo[a]pyrene, chrysene, benzo[a]fluoranthene, benzo [a] anthracene, and benzok,h,z]perylene were found in the acetone extract, but were negative in the dichloromethane extract under the same conditions. However, the amount of pyrene in the dichloromethane extract was much higher than in the acetone extract. These results indicate that the extraction efficacy of I-nitropyrene, dinitropyrenes and benzo[a]pyrene is higher with acetone than with dichloromethane. This may be the reason why acetone is the most effective solvent in extraction of mutagens from airborne particulate samples. INTRODUCTION Many researchers have investigated the compounds responsible for mutagenicity in organic extracts of airborne particulate samples. The potent direct-acting muta- genic compounds in the Ames Salmonella test using strain TA98 were found to be dinitropyrenes (DNPs) and 1 -nitropyrene (l-NP) which are frequently extracted us- Address for correspondence: Huei Lee, Department of Biochemistry, Chung San Medical and Dental College, Taichung, Taiwan (Republic of China).
Two different solvents (acetone and dichloromethane) were compared for their efficacy in extraction of mutagenic compounds from airborne particulate samples. Their mutagenicity was examined with Salmo-
nella typhimurium TA98 in presence or absence of S9 mix. The total mutagenic activity of the acetone extract was 1.8-7.0-fold that of the dichloromethane extract. The content of 1 -nitropyrene, 1,6-dinitropy- rene, dibenzo[a,h]anthracene and indo[l,2,3-c,d]pyrene in acetone extracts of airborne particulate samples was 3.8-, 3.6-, 6.6- and 1135-fold that of dichloromethane,extracts, respectively. 1,8_Dinitropyrene, ben- zo[a]pyrene, chrysene, benzo[a]fluoranthene, benzo [a] anthracene, and benzok,h,z]perylene were found in the acetone extract, but were negative in the dichloromethane extract under the same conditions. However, the amount of pyrene in the dichloromethane extract was much higher than in the acetone extract. These results indicate that the extraction efficacy of I-nitropyrene, dinitropyrenes and benzo[a]pyrene is higher with acetone than with dichloromethane. This may be the reason why acetone is the most effective solvent in extraction of mutagens from airborne particulate samples.
INTRODUCTION
Many researchers have investigated the compounds responsible for mutagenicity in organic extracts of airborne particulate samples. The potent direct-acting muta- genic compounds in the Ames Salmonella test using strain TA98 were found to be dinitropyrenes (DNPs) and 1 -nitropyrene (l-NP) which are frequently extracted us-
Address for correspondence: Huei Lee, Department of Biochemistry, Chung San Medical and Dental College, Taichung, Taiwan (Republic of China).
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ing a moderately polar organic solvent, such as dichloromethane [l-7]. Other organic solvents have also been used for extraction of the mutagenic compounds from airborne particulate matters, such as acetone [8,9], methanol [lO,l 11, benzene/meth- anol [12], and benzene/ethanol [13] etc. Of these solvent systems, Krishna et al. [8] found acetone to be the most effective for extraction of mutagens from airborne particles. However, the extraction efficacy of polycyclic aromatic hydrocarbons (PAHs) and nitro- polycyclic aromatic hydrocarbons (nitro-PAHs) from the different organic solvent extracts has not been studied. In the present paper, two commonly used solvent systems (acetone and dichloromethane) are compared for their efficacy in extraction of compounds from airborne particulate samples which are mutagenic in the Ames test.
dinitropyrene (1,8-DNP), 1,3-dinitropyrene (1,3-DNP), glucose-6-phosphate (G-6-P) and nicotinamide adenine dinucleotide phosphate (NADP) were purchased from the Sigma Chemical Co. (St. Louis, MO., U.S.A.) The PAH mixture was obtained from Supelco Co. (Bellefonte, PA., U.S.A.). Acetone, dichloromethane and other chemi- cals were all provided by E. Merck Co. (Darmstadt, F.R.G.).
Sample collection and preparation Airborne particulate samples were collected from the roof of the Dental Depart-
ment building (elevation -9 m) at Chung Shan Medical and Dental College in Tai- chung, Taiwan, on October 11-12, 1989. The procedures of sample collection and preparation were similar to those reported previously [14-161. The samples were collected for 24 h continuously at a flow rate of approximately 1 m3/min. Two pieces of glass fiber filters were used for each experiment. Each filter was divided into l-cm* pieces with a paper cutter and the pieces were randomly selected for extraction with acetone or dichloromethane in a shaker. The organic extracts were filtered through Whatman No. 2 filter paper. The filtrates were concentrated to approximately 10 ml with a rotatory evaporator at 40°C and then evaporated to dryness under nitrogen stream. The residue was weighed and redissolved in the original solvent and stored at -80°C until use for mutagenicity testing and HPLC analysis.
Sephadex LH-20 column purification The organic solvent extracts of airborne particulate samples were passed through
a Sephadex LH-20 column (15 mm i.d. x 190 mm) at a flow rate of 0.5 ml/min using 2-propanol as eluant. Fractions were collected by a fraction collector every 2 min. An aliquot of 0.1 ml of each fraction was evaporated and the residue was dissolved in 0.1 ml of DMSO for the mutagenicity test. The mutagenic fractions were pooled and used for further determination of PAH and nitro-PAH content by HPLC.
61
HPLC puriJication HPLC was performed with a Waters 600E controlled system and a model U6K
injector (Millipore Co., U.S.A.) coupled to a Soma S-3350 variable wavelength fluo- rescence detector (Soma, Japan). The chromatographic peak area was calculated with a Waters data workstations model 810 (Millipore Co., U.S.A.). The active fractions were partially purified by HPLC with a semipreparative PBondapack column (10 ,um particle, 10 x 250 mm, Millipore Co., U.S.A.) as previously reported [16]. The eluants were collected, evaporated to dryness and dissolved in methanol. These sam- ples were used for the mutagenicity test and for determination of the content of nitro-PAHs.
Determination of nitropyrenes DNPs and l-NP were measured by HPLC as described by Hisamatsu et al. [17].
The active fractions, partially purified by the LH-20 column and semipreparative HPLC as described above, were treated with sodium hydrogen sulfide in order to convert the nitropyrenes to aminopyrenes which have higher fluorescent efficiency. The column for determination of DNPs and l-NP was a 25cm Nucleosil 7Cr8 column and the wavelengths of the fluorescence detector were 3751450 or 3601435 nm. The mobile phase for DNPs and l-NP was methanol/McIlvaine’s buffer in the ratio of 50:50 and 65:35, respectively, at a flow rate 1 mlhnin.
Determination of PAHs The quantitative analysis of pyrene, chrysene, benzo[a]pyrene (B[a]P) and ben-
zo[g,h,z]perylene (B[g,h,i]P), which have higher mutagenic activity in S. typhimurium TA98 with S9 mix, and some other PAHs, benzo[b]fluoranthene (B[b]FA), ben- zo[a]anthracene (B[a]A), dibenzo[a,h]anthracene (DB[a,h]A) and indo[l,2,3-c,d]py- rene (ID[ 1,2,3-c,d]P) were also determined using HPLC. The active fraction from the LH-20 column was injected into a Supelco LC-PAH column at 2 mlhnin with a linear gradient of 50-100% acetonitrile in water for 14 min and then held at 100% acetoni- trile for an additional 20 min. The PAHs were monitored by fluorescence at a wave- length of 365/410 mn.
Mutagenicity assay The samples were tested for mutagenicity by the Salmonellalmicrosomal test as
described by Maron and Ames [19]. The liver homogenate supernatant (S9) was prepared from the livers of male Sprague-Dawley rats treated with Aroclor 1254 as described by Maron and Ames [19]. The mutagenicity of acetone and dichlorometh- ane extracts of the unused filters had negative responses (data not shown).
RESULTS
Table I shows the amounts and mutagenic activity of acetone and dichloro- methane extracts from airborne particulate samples. The amount and mutagenic ac-
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TABLE I
COMPARISON OF THE AMOUNT AND MUTAGENICITY OF AIRBORNE PARTICULATE SAMPLES EXTRACTED WITH ACETONE ON DIC~LOROMETHANE
Extraction solvent Amount of organic extract (mg)
Acetone 48.7 DCMb 20.7
Revertants/mga
+s9 -s9
4396 zk 68 1779+22 1415rtll 848+25
Total revertants x 10’
+s9 -s9
2.1 0.7 0.3 0.2
a 1 mg of organic extract used for the mutagenicity test with S. ~yp~~~~r~u~ TA98 in the presence or absence of S9 mix.
b DCM = dichloromethane. Values are means f standard deviation. Spontaneous revertants of TAPS were 27 + 2 (-S9) and 35 + 4 (+s9).
tivity of the acetone extracts were about 2-fold and 1.8-7.0-fold those of the dichlo- romethane extract, respectively.
In order to compare the major mutagenic fractions of two organic extracts of airborne particulate samples, they were partially purified by the LH-20 column and semipreparative HPLC. The direct-acting mutagenic fractions of the acetone and dichloromethane extracts from the LH-20 column were concentrated in the region of fraction M-100, whereas the metabolic-acting mutagenic fractions were in fractions 58-96 and 82-96, respectively (Fig. 1). The active fractions of the acetone and dichlo- romethane extracts from semipreparative HPLC were generally similar. Most muta- genicity with or without the S9 mix was in the retention time range of 28-38 min (Fig. 2). After following our purification procedures, the percentage of recovery of internal standards (1-NP, DNPs and B[u]P) in the extracts of airborne particulate samples is as shown in Table II. From Table II, we find that the extraction efficacy of acetone is 1.4-4.5-fold that of dichloromethane. The percentage of recovery seems to be low due to the multiple steps of purification. The results (Table III) show that the mutage- nicity of the active fraction of both extracts from the LH-20 column and semiprepara- tive HPLC purification decreases in TA98NR like that of the DNPs and l-NP stan- dards. Accordingly, the major contributors to the mutagenicity in acetone and dichlo- romethane extracts might be nitropyrenes. The amount of DNPs, l-NP and PAHs in two organic extracts of air samples was analyzed by HPLC as shown in Table IV. The content of 1-NP, 1,6-DNP, DB[a,h]A and ID[l,2,3-c,d]P in acetone extracts of airbor- ne particulate samples is 3.8-, 3.6, 6.6- and 1135fold that of the dichloromethane extracts, respectively. 1,8-DNP, B[a]P, chrysene, Bfb]FA, B[u]A and B[g,h,flP were found in the acetone extract, but not in the dichloromethane extract by using the same conditions. The amount of pyrene in the dichloromethane extract was much higher than in the acetone extract (about 840-fold), although the polarity of acetone is higher than that of di~hloromethane. Thus, acetone is expected to be a better solvent for the extraction of pyrene. However, the reverse results obtained with pyrene in the dichlo-
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Fig. 1. Comparison of direct (A) and metabolism-dependent (B) mutagenicity of the 1 -min LH-20 column fraction of the acetone extract (----) and the dichloromethane extract (-) of airborne particulate samples
tested in 5’. typhimurium TA98.
ri ,_-1
F
20
‘,-I 8
40 60 so L
Raetim No. Fractbn NO.
Fig. 2. Comparison of the mutagenic activity of the I-min HPLC fraction of the active fraction of the acetone (A) and dichloromethane (B) extract of airborne particulate samples eluted from an LH-20 column
tested in S. typhimurium TA98 with (-) and without (----) S9 mix.
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TABLE II
RECOVERY (PERCENTAGE) OF INTERNAL STANDARDS (I-NP, DNPs, B[u]P) IN EXTRACTS OF AIRBORNE PARTICULATE SAMPLES
2pg of DNPs and B[u]P were added to 20 mg extracts of airborne particulate samples which were purified by Sephadex LH-20, semipreparative HPLC and then analyzed by analytical HPLC as described in the text.
TABLE III
THE MUTAGENICITY OF ACTIVE FRACTIONS OF ACETONE AND DICHLOROMETHANE EXTRACTS OF AIRBORNE PARTICULATE SAMPLES ELUTED FROM AN LH-20 COLUMN AND SEMIPREPARATIVE HPLC TESTED IN S. TYPHZMURZUM TA98 AND TA98NR WITHOUT S9 MIX
Extraction solvent LH-20
TA98 TA98NR Inhibition”
Semipreparative HPLC
TA98 TA98NR Inhibition
Acetone DCM Control DNPs I-NP
256128 551 f 10 78.5 3239f6 686+5 78.8 2041 f 8 569f 6 12.2 2593 f 5 703+3 12.9
519f4 124f3 78.6 385f8 4323 88.8
a 1 mg of the active fraction of airborne particulate samples was prepared for the mutagenicity test. Values are means f SD. The spontaneous revertants were 23 f 3 in the absence of S9 mix.
bInhibition: % of inhibition = (1 - revertants of TA98NR/revertants of TA98) x 100%. ’ DNPs: 10 @plate; l-NP: 1 .O @plate.
romethane extract suggest that its higher dispersion force and dipolar moment char- acteristics play some important role during extraction.
DISCUSSION
Organic compounds in airborne particulate samples with mutagenic potential in- clude PAHs, heterocyclic PAHs, aminoarenes, nitroarenes, arene ketones and arene quinones [20]. Nitropyrenes are ubiquitous in the environment as a result of various incomplete combustion processes [21,22]. l-NP and DNPs are major contributors to the mutagenicity of airborne particles observed in the S. typhimurium test in the absence of S9 mix and their mutagenicity decreases with the S9 mix [2,22-241. Figures
65
TABLE IV
AMOUNT OF l-NP, DPNs AND SOME PAHs IN THE MUTAGENIC FRACTION OF AIRBORNE PARTICULATE SAMPLES PASSED THROUGH A SEPHADEX LH-20 COLUMN AND SEMI- PREPARATIVE HPLC PURIFICATION”
“Values are ng/mg of extracted organic material of airborne particulate samples. ND = not detected.
1 and 2 show that the direct mutagenicity of partially purified airborne particulate samples was higher than the indirect and that nitropyrenes might be the major mu- tagens in the active fraction from Sephadex LH-20 and semipreparative HPLC. How- ever, the overall mutagenicity decreases in the presence of S9 mix after fractionation by a LH-20 column and semipreparative HPLC. The reason for this remains un- known.
The choice of solvent for use in extraction of airborne particles is usually deter- mined by the desired class of compounds. Numerous solvents have been used by dif- ferent investigators for the extraction of organic compounds from airborne particu- late samples for mutagenicity testing in the Ames test. Krishna et al. [8] compared 7 solvent systems and concluded that the acetone extract gave the highest response. Talcott and Wei [25] reported similar results, namely that acetone extracts contain more mutagens than benzene, chloroform and methanol extracts. More recently, Allheim and Ramdahl[26] used acetone to investigate the mutagenicity of wood and side-stream cigarette smoke. Morin et al. [27] also indicated that acetone was the solvent of choice for removing organic compounds from sidestream cigarette parti- cles. The ability to detect mutagens with TAlOO could be greatly reduced by using methanol and dichloromethane as extraction solvents. Dichloromethane is common- ly chosen as solvent for analysis of PAHs and nitro-PA&Is in air samples [ 1,3-61. Our results show that the extraction efficacy of acetone for l-NP, DNPs and B[u]P is higher than that of the dichloromethane extract. This may be the reason why a num- ber of investigators believe that acetone is the most effective solvent for extraction of mutagens from airborne particulate samples. It is important to note that this study is based on only one air sample site. There will be some variations in the distribution of
66
mutagens in fractions from airborne particulate samples collected from the same locations at different seasons.
ACKNOWLEDGEMENTS
The authors acknowledge with gratitude the financial support of this work by the National Science Council, Taipei, Taiwan, Republic of China (Grant No. NSC 79% 042 l-B040-0 1 Z).
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