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Erdtmann, G. ”Neutron Activation Tables”; Verlag Chemie: Weinheim, 1978. Bouten, P.; Hoste, J. Anal. Chlm. Acta 1962, 27, 315-319. Nadkarni, A. R.; Haldar, B. S. Anal. Chim. Acta 1968, 42, 279-284. Byrne, A. R. J. Radloanal. Chem. 1977, 37, 591-597. (18) Maoliang, Li; Filby, R. H. Radiochem. Radloanal. Lett., In press. (19) Certificate of Analysis; National Bureau of Standards, Office of Standard Reference Materials, Washington, DC 20234. (20) Jacobs, F. S.;Fllby, R. H. Anal. Chem. 1989, 55, 74-77.
(21) Gladney, E. S. “Compilation of Elemental Concentratlon Data for NBS Biological and Environmental Standard Reference Materlals”; Los Alamos National Laboratory Informal Report LA-8438MS; Los Aiamos National Laboratory: Los Alamos, NM, 1980.
RECEIVED for review July 18, 1983. Accepted September 1, 1983.
Two-Dimensional Electrophoresis for Determining Toxicity of Environmental Substances Egil Jellurn* and A. K. Thorsrud Institute of Clinical Biochemistry, University of Oslo, Rikshospitalet, Oslo 1, Norway
F. W. Karasek Department of Chemistry, University of Waterloo, Waterloo, Ontario, Canada N2L 3Gl
A short-term test for human cellular response to toxic environmental chemicals using perlpheral blood leukocytes and high-resolution two-dlmenslonai electrophoresis Is demonstrated to be feasible by udng extracts of organic compounds from munlcipai Incinerator fly ash and from particulates of diesel exhaust and air-borne particulates. The effect of the chemlcals on the normal synthesls of about 2000 proteins by the leukocytes is observed. Three types of effects by adding Increasing amounts of chemicals are found proteins whose syntheds is blocked easily; those that are quite resistant; and the appearance of new protelns. The type of effect which predominates appears to be related to composition of the organlc extract. It is anticipated that this test can dlrectiy measure the toxic, genotoxic, and mutagenic behavlor of indlvidual chemlcals or mixtures on human cells.
A number of short-term tests to evaluate the genotoxic, carcinogenic, and mutagenic properties of a chemical or an environmentalmixture have been developed (1). Most of these tests use plants, bacteria, or mammalian cells to look for DNA damage or mutation, followed by longer term animal experimentation for confirmation. The widely accepted Ames test uses mutants of Salmonella typhimurium bacteria to indicate the mutagenic and carcinogenic behavior of a chemical (2). The effects of a given chemical or mixture on man must then be obtained by an uncertain extrapolation of data from animal experimentation and through epidemiological studies. The research described here concerns a short-term test of environmental chemicals and mixtures to directly measure the toxic and genotoxic effects of such chemicals on living, human cells. The test is based on observing the way in which the toxic agents affect the ability of leukocytes from human blood to synthesize proteins. Human leukocytes may easily be isolated from freshly drawn blood using gradient centrifugation. The isolated cells are mainly lymphocytes with some (10-20%) granulocytes and monocytes. They are living, human cells having the ability to synthesize proteins. Protein synthesis involves a highly complex chain of biochemical events, beginning with the synthesis of messenger RNA (mRNA) using the genes (DNA) as templates. The mRNAs contain the code for the amino 0003-2700/83/0355-2340$01.50/0
acid sequence of the protein being constructed. The process of synthesizing proteins within the living leukocytes involves many complex biomolecules, enzymes, and cofactors and is regulated by complex mechanisms. Recently a high-resolutiontwo-dimensional electrophoresis technique for separating proteins has been developed (3-5). It involves separation of the proteins according to isoelectric point in the first dimension, followed by separation according to molecular weight in the second dimension. The technique is able to detect around 2000 proteins in the leukocytes, and it has been claimed that this is close to the total number of proteins (gene products) actually present (6). Prior to separation the isolated living cells are incubated with a mixture of nutrients including amino acids, one of which is [35S]methionine. During a 20-h incubation period the proteins synthesized all contain radioactive 35S.After separation by high-resolution two-dimensional electrophoresis, the proteins are visualized on films by autoradiography (7). If chemical compounds are added to the living cell-nutrient mixture prior to the incubation period and these compounds affect any of the molecules (e.g., DNA, RNA), particles (ribosomes), enzymes, or cofactors involved in protein sythesis, then deviations from a normal protein pattern will be observed after completion of the two-dimensional electrophoresis and autoradiography. Thus, studies of the two-dimensional protein pattern of human leukocytes incubated in the presence and absence of potentially toxic compounds would be a sensitive system for evaluating the short-term toxicity toward human cells. One can then observe how different chemicals may cause specific changes in the pattern of proteins synthesized to reveal the short-term toxic effects and possibly indicate the long-term genotoxic effects. Some chemicals may even induce the synthesis of new proteins. Our initial studies with complex environmental mixtures which contain known toxic compounds indicate that this procedure does produce a two-dimensional electrophoresis pattern of proteins showing such effects caused by the toxic chemicals present. These data are the subject of this paper.
EXPERIMENTAL SECTION Reagents. Acrylamide, Bisacrylamide ( N - (l-ox0-2propenyl-2-propenamide),urea, and SDS (sodium dodecyl sulfate) were products of Bio-Rad Laboratories, Richmond, CA, and of Serva Feinbiochemica, Heidelberg, F.R.G. Ampholytes for isoe0 1983 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 55. NO. 14. DECEMBER 1983
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1 Flgure 1. Twodimensional protein panern of human peripheral b l d leukocytes. Me,SO. used as solvent in the toxicity experiments. was added to the incubation mixture: (a) control with no Me,SO added: (b) 5 pL 01 Me,SO: (c) 10 pL of Me,SO. In this and all other figures the gels are oriented with the acidic end to the left and higher molecular masses at the top. Two known proteins are marked, (Ac) actin and (C)calmodulin. Spots V2. V3. and 3 (circle)are discussed in the text. IQCtricfocusing were Ampholines tI.KH, Stockholm, Sweden1 or S ~ r v d y t e~ S e r v aFPinbiurhemira,. Firoil-Paque wns produced by Pharmaria Fine Chemicals, Uppsala. Sweden, and RPMl 1620 medium was ohtained from Flow Lahs., Iwine, Scotland. Selectamine kit WRI purchwd lrum GIllCO. Grand lslmd. NY. and ["S]merhionine from the Radiochemical Center, Amersham international Ltd., Amer.iham, Buckinghamshire, England. Dimethyl sulfoxide rMe,SO) and NONII)KI' PNi were products ol Sigma Chemical Co.. SI. I.uuis. MO. A l l other chemicals were commerriaily available analyticai-grade products Apparatus. The equipment for high-resolution two-dimensional electrophoresis was ohlained from EIectroNurlmnic4. Oak Ridge, 1'N. This appnraru.i ix based on the iSO.l).4l.'i' system described by Anderson and Anderson ( 4 . 5 ) . The IS0 apparatus allows isoelectric focusing of 20 samples at a time. Thv serond R - I M IinPar gradient polyacrylamide slab dimensinn gels gelx. and a special apparatus alluws 20 gels to he cast simultaneulwly. Twu DALT tanks were used for electrophoresis in the aecond direction. The power supplies were an LKB RRTI ior imlecrric i m q i n g and a HPM48H (Hrwktr-Pncknrd. Snn I)ieyo. CA) lor the s ~ ~ separation. n d A Model 2161) refrigerating bath (Forma Scientific, Marietta. OH, waz used to keep the temper. ature near 10 "c' during the larter elertrophoreris. After the two-dimensional separation. the gel" were dried in a Hio-lbd Slab Gel Drier, Model 1125. Fur autoradiovaphy Kodak X-Omat h R film (Eastman Kodak, Rochester. S Y i was used. Procrdur?. Experiments were perfnrmd on human peripheral blood leukocytes k?J. Peripheral blood wax collected by venipuncture in heparinized tubes and centrifuged tocollert the buffs coat. lluify coat leukocytes were diluted 3-fold i n RPA11 I M U medium and were isolated by FicoU-Paquemadient centritugation. The isolated fraction consisted mainly o i lymphoryted with a smaller percenurpe uf monwytps and vanulocytes. The cells were washed twice in RPMl I640 medium and incuhared in partly methionine-deficient RP.MI 16-10 1 1 mg oi-wld" methimine 1.) supplemented with fetal Imvine serum t5 ml. 1.1. 2-mercaptuethanol (4 X 10.' moi 1.1. and appruximately 3u gCi of [.'.'SI. methionine of high specific acti\ity. Approximately 2 X IO6 cellq r in t l ~ ttinttom. in 401) UL of this medium were cllltured p ~ well multiwell tissue cuhure phtQs. Cultures were incubated for 20 h a t 37 "C in humidified atmosphere containing 5% C 0 2 . The environmental samples were dissolved i n Me,SO and added t~ the cultures prior to mcuhation. The volume oi Me-SO solution added t o the 400.~1.incubation mixture w89 usually hetwecn 2 and 10 01. At the end of the labeling period the cells W P ~ Qharvested hy brief centrifugation in a Rerkmm Mivrufuge I3 tllrrkman Instruments. Palo Alm. CAJ and I he pellets were Iyaed in a buffer containing WPH (9 mol I.).2-mercapmethanol t i 0 mL Li. .4mpholinea 120 mL LI, and a nnninnic detergent. Sonidet P40 (NP-10) (40mL, L). The lyied sample3 were centrifuged fur 1
min in the microfuge to sediment insoluble material, and aliquots (15 pL) of the supernatants were analyzed by high-resolution two-dimensional electrophoresis (8). The first dimension gels (4% polyacrylamide) contained urea (9 mol/L), Ampholines, pH 3.5-10 (50 mL/L), and NP40 (30 mL/L). Internal isoelectric point standards, produced by carhamylation of rahhit muscle creatine kinase, were used in all gels (9). After separation in the second dimension, the gels were stained with Coomassie Blue to visualize the nonradioactive internal standards which do not show on the autoradiograms. The fixed and stained gels were dried and allowed to he in contact with the film for 2 weeks. Samples of fly ash were subjected to the same extraction and concentration procedures as described in an earlier study (IO). The procedures include 16 b of Soxblet extraction of 50 g sample weight with 400 mL of distilled-in-glass benzene (Caledon Laboratories, Georgetown, Ontario) and concentration under aspirator vacuum followed by a gentle stream of nitrogen to a volume of 100 pL. After GC/MS analysis the fly ash samples were evaporated to dryness and reconstituted to 100 ULvolume with MQSO for toxicity tests. The diesel particulate sample was collected from an in-usediesel automobile according to the 1981EPA procedures hy particulate sampling in a dilution tunnel. The 50 X 50 cm filter used to collect particulates was extracted for 24 h with dichloromethane. After solvent reduction by rotary evaporation, sample extracts were transferred to tared vials for evaporation to dryness. The residue was weighed and redissolved in dichloromethane for GC/MS analysis. A portion of this sample equivalent to 830 pg was evaporated to dryness and then was reconstituted to 300 pL with Me,SO solvent for the toxicity tests. The air pollutants were collected by a Sierra Impactor used by the Institute for Air Pollution Research (NILU) in Oslo. It involved the passage of approximately 8000 m3 of air through a glass fiber filter placed before a polyurethane plug. Particulated matter (-3.5 r m size) collected on the filter, and gaseous pollutants were absorbed by the polyurethane plug. The plug was extracted in a Soxhlet apparatus with acetone, the solvent was evaporated off, and the residue was dissolved in 9 mL of Me,SO. The concentraton of organic material was 6400 ng/pL, and in the toxicity test the leukocytes were incubated in the presence of 2-10 p L of extract. Table I summarizes some pertinent analytical data for all the samples used in these experiments.
RESULTS AND DISCUSSION Figure l a shows the two-dimensional protein pattern of leukocytes incubated in the absence of Me,SO solvent; Figure lb,c shows the protein pattern, with 5 and 10 pL of Me,SO added. The protein spots have been visualized by autoradiography after the electrophoretic separations. Detailed counting with a densitometer and computer has shown that over 2000 spots are present in patterns such as shown in the
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NO. 14, DECEMBER 1983
Table I. Data for Selected Compounds.in Environmental Samples total organic compounds, no. of PAH ng/pL identified French fly ash, A French fly ash, B Canadian fly ash airborne particulate matter, Oslo diesel exhaust particulates ...
2100 4000
TCDD
6400
7 36 26
ND 2 67 ND
2770
29
ND
3100
4
dioxin isomers, ngipL P,CDD H,CDD H,CDD 1 3 80
OCDD
5
14
24
4 100
5 44
ND
ND
ND
7 30 ND
ND
ND
ND
ND
~~.
;rn
2. Twc-jlmenskiml pmtsin paaSm of human k k x y t e s d-ovkg the etfect of increasing amounts of toxic chemicab exbacted from Canadian municipal incinerator fly ash. The extract was dissolved in Me,SO and the foliowing amounts were added to the cell incubation mixture: (a) no addition (control). (b) 2 pk (d) 5 pL; (e) 6 pk (1) 7 pk (e) 8 pk (H) 9 pL; (i) to pk (i) 15 pL.
figure (11). These data show that the MezSO does not alter the two-dimensional protein pattern to any significant extent. Spot 3 is induced hy 10 pL of MeaO. V2 and V:3 are proteins !mown to be affected hy a number of unrelated chemicals and thus are not significant changes (7). In Figure 2 the leukocytes have heen allowed to synthesize proteins in the presence of increasing amounts of the extract of organic chemicals from fly ash generated in a Canadian municipal incinerator burning household garhage. The concentration of total organic material in the MezSO solvent added to the incubation mixture was 3100 ng/pL. It is evident that the organic compounds from the fly ash particles have a profound effect on the proteins synthesized. The typical trend that most proteins in the leukocytes seemed to follow was that of increased suppression of their synthesis leading to fainter and fainter spots. This effect is illustrated by the protein spots marked (F). Above a certain concentration the toxic mixture completely inhibited the synthesis of proteins and killed the cells and no spots could be seen on the autoradiogram. Certain proteins were considerably more resistant toward the chemicals than others, as can be seen for the spots marked A and B in Figure 2. New proteins not present in the controls also appeared to he induced in the presence of the added chemical mixture. Spots marked 1-8 indicate this. Some of these induced proteins are rather persistent once they are formed. Proteins of the spots 1,2, and 8 were clearly seen even when 10 p L of the toxic mixture had heen added to the cella (Figure 2i). The circle marked 8 includes one spot that decreases in intensity,
MQSt O f 2000
< I O 0 Resistant J Roteins
Roteirs
intemity
Pmteins (Inluetim-Mutatim)
+New
0
Quantity of Toxic Compwnd
-
Flgure 3. Summary of some typical effects on different types of proteins synthesized by human leukocytes under the influence of increasing amounts of toxlc chemicais. from data in Figure 2. and one new spot being formed in the presence of 8 p L of the toxic sample (Figure 29). Spots 6 and 7 increase with increasing amount of the chemical mixture added up to a certain point, then decrease, and f m d y disappear. Frame 5 includes three spots, two of which appear to be induced. Formation of these new proteins may be due to mutations, activation of genes, and postsynthetic modifications of the proteins, poasihly including the hinding of an added chemical to proteins. The latter possihlity is uncertain, because it implies that the hinding forces to the protein polypeptide chain must withstand the mixture of mercaptoethanol, urea, and SDS used in the electrophoretic technique. The large amount of data shown in Figure 2 may be simplified and summarized as indicated in Figure 3. The number of proteins that follow these general curves will vary depending
ANALYTICAL CHEMISTRY, VOL. 55, NO. 14, DECEMBER 1983 2343
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. .
- .d
-- 1 Id
Flgura 4. Two-dimensional protein panern of human leukocytes incubated with increasing amounts of extract of airborne particulates collected in iha city of Oslo: (a) no addition. (b) 2 pL, (c) 5 pL, (d) 10 pL. I n Umse gels ampholytes used in me frst dimension were Servalyi 3-10, and Wm secaxl dimendon potyaay!aW gadient was tC-20%.
upon the type of toxic chemicals that are added to the test system. Figures 4 and 5 illustrate the effect of two other types of environmental samples on the human leukocyte protein pattern. In Figure 4 the cells have been exposed to a chemical mixture extracted from air-borne pollutants collected in the central area of the city of Oslo. Figure 5 shows the effect of a mixture extracted from particular matter from the exhaust of a diesel engine. The same trend as observed in Figure 2 is also seen with these samples; the synthesis of most proteins is gradually inhibited by the toxic mixtures (spots F), other proteins are more resistant (spots A & B) and the appearance of new proteins indicate mutations, inductions, or postsynthetic modifications caused by the chemicals (spots 1-5, and
I i
8). The responses shown in Figure 4 for the Oslo sample were seen at one-tenth of the concentration that gave a response in the Ames test. Comparison of Figures 2,4, and 5 illustrates that the three different environmental samples tested have different effects on certain proteins. This might reflect differences in the chemical composition of the samples and differences in toxicity. In these studies complex mixtures rather than single components have been used to establish the data seen in Figures 2-5. For instance, the extract from the municipal incinerator fly ash contains about 300 compounds, many of which are known toxic compounds, such as polycyclic aromatic hydrocarbons (PAH), polychlorinated dibenzofurans (PCDF), and polychlorinated dihenzodioxins (PCDD) (12). This sample has been subjected to an extensive analysis involving preseparation into fractions by HPLC and identification by GC/MS selected ion monitoring technique to give quantitites for each isomeric group from tetra- to octachlorodioxins in the E(t120 ng/pL range in the extract. Although some of these PCDD isomers are considered to be the most toxic in the mixture, other compounds present, e.g., the PAH are also of high toxicity. The diesel extract and the extract of air-borne particulates are also highly complex mixtures containing many of the same compounds as found in the fly ash extract. However, the polychlorinated dioxins are not present in the diesel exhaust and in the air samples. At the present stage we cannot say which chemical compound or groups in the various environmental samples contribute most to the observed toxicity. The test measures the additive or synergetic net toxicity of the complex mixture. Subfractations with HPLC into compound groups are now being carried out and fractions exhibiting positive response in the toxicity test will he further analyzed by GC/MS to identify individual compound types contributing to the toxicity. These data will he the subject of a future paper. Not all environmental mixtures tested gave positive results under comparahle conditions. Figure 6 shows the results from testing two different samples of organic mixtures extracted from fly ash samples obtained from a municipal incinerator in Paris, France. It can he seen that sample A produced little observable effect on the protein pattern up to the 10 pL addition, while the effect produced hy sample B was profound. Although sample B contained twice the concentration of total organic compounds as that of A, there were major differences in composition found when each was analyzed by GC and GC/MS methods. Limited analytical data showed that sample
I
mure 5. Two-dimensional protein panern of human leukocytes incubated wRh increasing amounts of extract of particulate matter from exhaust of a diesel engine: (a) no additions, (b) 2 pL. (c) 10 pL.
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ANALYTICAL CHEMISTRY. VOL. 55, NO. 14, DECEMBER 1983
.
--.
.
.
Fbue 6. Twodimensional protein panem of human leukocytes incubated wkh extracts from two different samples from an incinerator in Paris, A and E contml,no extract added: A,. 5 itL extract from indneratw sample A added: A,,, 10 pL extract from A E,, 2 pL extract from incineratw sample B: E,. 5 pL from same: Elo, 10 pL from same.
B different from A in distribution and concentrations of compounds such as the PCDD and the PAH compounds. It is evident that if the test were stopped prior to the two-dimensional electrophoresis analysis that simple measurements of net radioactivity incorporated into the leukocyte p r o t e i in the absence and presence of toxic chemicals would reflect total toxicity but would not contain much of the fine details revealed hy the two-dimensional electrophoresis. The potential of the test using two-dimensional electrophoresis data is the possihlity of measuring the effect of toxic agents on every single polypeptide chain h e i synthesized. huming that there is a coded gene behind almost every spot on the two-dimensional autoradiogram, then the test permits, indirectly and in principle, the detection of damage to single genes. Moreover, the test system, in contrast to simple measurements of protein synthesis, is able to pick up changes due to mutations, inductions, or other changes in single polypeptide chains. The experiments described in this paper have used freshly drawn human blood samples. Within each analytical series reported it is alwaya leukocytes from the same individual that have been employed. The two-dimensional protein patterns of leukocytes from different persons are surprisingly constant as long as the subpopulations of lymphocytes, granulocytes, and monocytes are roughly the same. Different healthy persons with approximately the same blood count can therefore in principle be used as sources of leukocytes. In the long run,however, it may be advantageous to adopt a standard cell line rather than to depend on freshly collected blood specimens. Human fibroblasts and human lymphoblastoid cells grown in culture seem to be suitable, and identical cells can be grown in culture for years. Preliminary data show that the toxic chemicals have the same type of effects on the two-dimensional protein pattern of these cells as described
for human leukocytes in this report. Although the major purpose of the data presented here has been to show that the test will reveal toxic effects of complex environmental mixtures, it is evident that a more lengthy evaluation will need to be done using pure samples of individual chemicals known to be toxic to relate effect on specific proteins to specific chemicals. That work is now under way. ACRNO WLEDGMENT We are grateful to T. Sanner, The Norwegian Radiumhospital, Oslo, for supplying the sample of air-borne pollutants. LITERATURE CITED (1) '"ShortTerm Tests tw Carcinogens. Mutagens and Other OBnOtoxlc Agents": EPA Report-62519-79-003; Envlronmental Research Intwmalo" Center. Clnclnnau. OH. (2) Ames, 0 . N.; McCann, J.: Yamsaki. E. Mvtat. Res. 1975. 3 4 , 347. (3) OFmeII. P. H. J . Biol. Chem. 1975, 250. 4007. (4) Anderson. N. L.: Anderson. N. G. Roc. MU. Acad. Scl.. U . S . A . 1977. 74, 5421. (5) Anderson. N. G.: Anderson. N. L. Anal. Eiochem. 1978, 85.331. (6) Duncan. R.: McCankey. E. H. Ctln. U n m . (Whston-Salem, N.C.) 1982. 28. 749. (7) Willard, K. E.; Acd3rmn. N. 0.Clh. Chem. (WlnshM-%fem. N.C.) 1981. 2 7 , 1327. (8) Willard. K. E.: Thorsryd. A. K.: MunW. E.: Jelium. E. &. chem. (Winston-Sa18m. N.C.) 1982. 28. 1067. (9) Anderm. N. L.: Hickman. 8. J. Anal. Biochem. 1979, 93,312. (IO) Eiceman. E. A.: Cbment, R. E.: Karasek, F. W. Anal. Chem. 1979. 51, 2344. (11) Taylor. J.: Anderson, N. L.: Scandara. A. E.; Willard. K.: Anderson. N. 0.Clln. Cham. ~U'hwton-Salem. N.C.) 1982. 28. 861. (12) Tang. H. Y.: Shwe. D. L.: Karasek. F. W.: Helland. P.: Jelium, E. sub mined tor publication In Anal. Chem.
RECEIVED for review June 17,1983. Accepted September 8, 1983. This work has been supported by the Norwegian Cancer Society, hy the National Science and Engineering Research Council of Canada, and hy NATO Research Grant 266.80.
