Document vBRBBbwwjm91aZOdN91qewMxb

5170 BIOCHEMISTRY LAKOWir.z A;,'3 SEVAN Effects of Asbestos, Iron Oxide, Silica, and Carbon Black on the Microsomal Availability of Benzo[a]pyrene+ Joseph R. Lakowicz* and David R. Bcvan PLAINTIFF'S EXHIBIT CAP-1437 abstract; Particulate matter and polynuclear aromatic hy drocarbons are known to be cocarcinogcnic. Using fluores cence spectroscopy, we determined that adsorption of benzo[a]pyrene (BP) to iron oxide, siiica, and asbestos (anihophvllite and Canadian chrysotile) results in a greatly enhanced rate of BP uptake into rat liver microsomes when compared to uptake from aqueous dispersions of BP microcrystals. Simple mixtures of BP microcrystals and particuiates do not display enhanced microsomal uptake rates, an observation which in dicates that adsorption of BP to the surface of the particle is necessary for enhanced microsomal uptake. BP was not re leased into microsomes from carbon black. Most importantly, the data indicate that asbestos particles are more effective than silica and iron oxide in enhancing the microsomal availability of BP. These observations suggest that particles. and especially the flbrous mineral particulates, could be cocarcinogenic as a result of their ability to adsorb polynuclear aromatic hy drocarbons and to transport these carcinogens into cells. Ex cept for chrysotile. the partides did not disrupt microsomal integrity as determined by N'ADPH-dependent lipid per oxidation activity. Binding of the microsomes to the partides did not affect the BP uptake rates. In addition, these BP uptake rates were independent of both the concentrations of microsomes and of panides. These observations are consistent with the mechanism of panicle-enhanced transport br.r.g an increased rate of BP solubilization from the adsorbed scale into the aqueous phase, followed by rapid partitioning of BP into the microsomal membranes. I^lynuclear aromatic hydrocarbons (PAH)1 are known hu man and animal carcinogens, and carcinogenesis by these compounds requires metabolic activation (Heidelberger, 1975; Sims & Grover. 1974). This activation occurs in the micro somal fraction of cells (Sims et al., 1974; Yang et al.. 1977). Inhaled and instilled particulate matter is known to increase the carcinogenic potency of the PAH in lung tissue. For example, intratracheal instillation of benzo[a]pyrene (BP) results in only a low incidence of lung cancer (Saffiotti et al.. 1965) unless particulates are also instilled. The known co careinogenic particulates include hematite or iron oxide (Saf fiotti et al-, 1968). asbestos (Pylev & Shabad. 1973), aluminum and titanium oxide (Stenback et al., 1976). and India ink (Pylev, 1961). In humans, cigarette smoking and asbestos inhalation are known to be highly cocarcinogenic (Sciikoff et al- 1968). These observations stimulated this investigation of the effects of particles on the microsomal availability of benzo [a] pyrene. The mechanisms bywhich panicles enhance PAH carcino genesis have not been elucidated. The major route of PAH entry into the lungs is via retention of inhaled particulates which contain these adsorbed PAH. At present it is not known if particle-adsorbed PAH are eluted directly into lung surfac tant and then transferred to cells or if the PAH are eluted after phagocytosis. However, regardless of the site of elution, par ticles which rapidly release adsorbed PAH could increase the effective dose of carcinogens in the lungs by elution of these compounds prior to clearance of the particles from the lungs. Phagocytosis of such particles with adsorbed PAH could aiso increase the intracellular availability of the PAH for micro *From the Department of Biochemistry and the Gray Freshwater Biological Institute. University of Minnesota. Navarre. Minnesota 55392. Received May ti. 1979. These studies were supported by Grant BC-261 from the American Cancer Society. D.R.B. was supported in part by a Postdoctoral Fellowship tCA-6405) from the Nauonal Cancer Institute. This work was done during ihe tenure of an Established Invesugatorship (to J.R.L.) of the American Heart Association. A preiiminary account of this work was presented at the 23m Annual Meeting of the Biophysical Society. AUania. GA. Feb 1979. somal activation. Although the phagocytidic macrophages may not be transformed themseives. cells are known to release mutagenic BP metabolites (Langeabach et al.. 1978). In addition, an altered BP availability can alter the metabolic profile (Nemoto et al- 1978). and different BP metabolites have different carcinogenic effect (Kapitulnik et aL, 1978). In previous studies we determined that adsorption of chrysene and 1 Jl-benzanthracene to particulates greatly en hanced their rates of uptake into phospholipid vesicles of dipalmitovl-L-a-phosphatidylcholine (DPPC). when compared with uptake from microcrystalline states (Lakowicz et al., 1977. 1978a,b). In addition, we demonstrated that the fibrous mineral asbestos (amosite) is superior to the nonfibrous mineral silica in adsorbing BP in the monomeric state and transporting this carcinogen into DPPC vesicles (Lakowicz & Hvlden. 1978). Thus, it is clear that adsorption of PAH to particles can enhance the transport of PAH into lung suriactanL which is composed of at least 50% DPPC (Tierney, 1974; King <fc Clements, 1972). In this report we investigate the effects of particles on the uptake of BP into microsomes. with particular attention being given to the mechanism of enhanced uptake. Materials and Methods Source and Physical Properties ofParticulates. .Amorphous silica was obtained from Analabs. By nitrogen adsorption, its surface area was 381 nr/8 and the average particle size was 2 um (Lakowicz et al- 1978b). Iron oxide (99.9%. lot 121377) was obtained from Ventron Corp. (8.0 nr/g). and carbon black was from Fisher (31.1 nr/g). Microscopic examination indi cated the iron oxide particles to be of varying size, with most being less than 2 urn. and the carbon particles to be about 25 urn in diameter. Anthophyllite and Canadian chrysotile were standard samples supplied by the International Union Against Cancer. Johannesburg. The reported surface areas are 11.8 1 PAH. polynuclear aromauc hydrocarbons: AHH. aryl hydrocarbon hydroxylase; BP. Denzo[ajpyrene: DPPC. dipalmitoyM.-a-phosphaudyi. choline. ... [Reprinted from oiocnemistrv.-19791 IS.5170.| Cupyneni c 1979 by the American Chemical society and reprinted by permission of the copyright owner. ASARCO ALV 00054-14 PARTICLE-ENHANCED microsomal uptake of and 26.8 m:/g. respectively. The panicle size distributions are heterogeneous, with the average size being about 2 um for both anthophyUite and chrvsotiie. For more detailed informa tion see Timorell (1970). Preparation ofParticulates with Adsorbed BP. Particulates containing adsorbed BP were prepared as described previously (Lakowicz & Hylden. 1978). Briefly, the paniculates were mixed with a benzene solution of BP, followed by evaporation of the benzene under reduced pressure. A total of 0.3 mg of BP was added for each gram of paniculate. These samples were stored in the dark under an argon atmosphere and were used within 1 week of their preparation. The fluorescence emission spectra of BP which was extracted from the particles by using benzene was identical with that of the starting ma terial. Thus, there did not appear to be any degradation of BP on these paniculates which interiered with our measure ments. Aqueous dispersions of BP crystals were prepared by evap oration of a benzene solution of BP to dryness, addition of buffer, and sonicarion for 30 rain at 40 W using a Cole-Parmer Model 8845-2 bath-type sonicator. Microscopic examination of these preparations reveals a heterogeneous size distribution with 90% of the crystais being less than 15 jim and 50% being less than 5 um. We refer to this preparation as being microcrystalline. Preparation of Rat Liver Microsomes. Microsomes were prepared according to Ames et al. (.1975) 5 days after inducing the rats by peritoneal injection of Aroclor 1254. The 105000g pellet was resuspended in buffer at a concentration of 0.6 mg of protein per mL (Lowry et al.. 1951). Fluorescence Spectral Data. Fluorescence spectral data were obtained by using a computerized, photon-counting spectrofluorometer (SLM Instruments. Inc.. Urbana, IL) as described previously (Lakowicz & Hylden. 1978). The back ground fluorescence seen above 395 tun (shorter waveiengths are not transmitted by the emission filters. Coming 0-52 and 2 mm of 1 M NaNO-j was typically less than 10% of the total intensity. Since a similar intensity and spectral distribution for this background were observed for all particulates, we conclude that it results from stray light scattered off the turbid suspensions of particulates." These backgrounds were quanti fied by using panicles without BP and subtracted from the spectra shown. The fluorescence of the microsomes was minor in relation to that resulting from the BP and did not interfere with our observations. In spite of the turbidity of the suspen sions of particles, the scattered light did not interfere with our measurements. In addition. BP emission from microsomebound BP could be observed even in opaque suspensions ofiron oxide and carbon black. Measurement of Microsomal Uptake ofBP. BP uptake into microsomes was quantified by the increase in fluorescence intensity at 405 nm which occurs upon transfer of BP from the surface of the particle into the microsomes. For all the BP uptake kinetics reported here, we used 5 ug of BP and 1 mL of rat liver microsomes. which was equivalent to 0.6 mg of microsomal protein. The buffer used was 0.1 M potassium phosphate. pH 7.7, containing 3 mM MgCl; and 0.1 mM EDTA. An amount of particulate (16.7 mg) containing 5 ug of adsorbed BP was suspended in 10 mL of buffer and dis persed by sonication in a bath-type sonicator for 30 min at room temperature. In addition to dispersing the particles, this procedure facilitated equilibration of BP with the aqueous phase. After measurement of the initial fluorescence spemrum and intensity, microsomes were added in 1 mL of buffer to initiate the reaction. Complete BP transfer to the microsomes VOL. IJ, NO. :3. 1979 5171 was obtained by heating the sample to 50 C for 60 min. The frnai fluorescence intensity was measured after reequilibration at the experimental temperature of 25 8C. BP uptake rates from the microcrystalline state, and from BP microcrystals in the presence of paniculates, were obtained in a similar manner, except that the 5 ug of BP microcrystals was sus pended in 10 mL of aqueous buffer. Measurement of Microsomal Integrity in the Presence of Particulates. Microsomal integrity was assayed by lipid per oxidation activity (Ernster & Nordenbrand, 1967: Wills, 1969). NADPH and oxygen are consumed in lipid per oxidation. and we quantified this activity by the loss of the NADPH fluorescence which occurs upon its oxidation. The fluorescence of NADPH was convenient because we found it is possible to quantify consumption of NADPH even in the optically dense paniculate suspensions which are used in the BP uptake measurements. The reported measurements were made in 0.1 M potassium phosphate. pH 7.7, 25 C. but equivalent activities were ob tained in this same buffer when it also contained 3 mM MgCL and 0.1 mM EDTA. The same front face illumination was used as for the BP uptake measurements. The instrumental conditions were as follows: excitation wavelength and filter. 340 nm and 7-54; emission filters. Corning 3-144 and 2 mm of 1 M NaN03; emission wavelength. 464 nm. The 10-mL assay mixture contained 16.7 mg of panicles and 0.6 mg of microsomal protein. To simulate the conditions used in the BP uptake studies, we stirred this mixture for 30 min a: 25 C prior to initiation of lipid peroxidation by addition of 0.5 mL of 5 x 10~* M NADPH. The activity was obtained from the loss of NADPH fluorescence which occurred during the first 2 min. At this time 50 uL of 0.2 M ADP in 5 mM FeClj was added, and the fluorescence intensity was monitored for an additional 2 min. Essentially identical activities were ob tained during both incubation periods, probably as a result of our use of phosphate buffer (Wills. 1969). Upon incubation of particles and NADPH in the absence of microsomes. the fluorescence intensity of NADPH was constant This control indicates the particles themselves did not adsorb the NADPH. they did not catalyze reduction of the NADPH. nor did they cleave the phospbodiester bond. Geavage of this bond results in an approximate fourfold increase in the fluorescence vieid of NADPH. Results Fluorescence Emission Spectra of Benao[a]pyrene. The fluorescence spectral distribution of benzo[a]pyrene is de pendent upon its environment. In Figure 1 we compare the emission spectrum of BP when bound to rat liver microsomes and when present as a microcrystalline dispersion in aqueous buffer. The spectrum of the microsome-bound BP is seen to be highly structured, and it is essentially identical with that observed in dilute benzene solution (Lakowicz & Hvlden, 1978). Microcrystalline aqueous dispersions of BP display markedly different emission spectra compared to microsome-bound BP. These spectra, when normalized with spectra of microsomebound BP. show a greatly increased relative intensity in the region of 490 nm (Figure I). This structureless long-wave length emission, which we will call the exrimer emission, mav resuit from the formation of a charge-transfer complex between a BP molecule in the excited state and an adjacent BP mole cule in the ground state (Birks & Cameron. 1959). Tne apparently large contribution of the excimer emission in these normalized spectra could result from (1) excimer formation between adjacent BP molecules in the microcrystalline aggre- ASARCO ALV 0005415 5172 BIOCHEMISTRY LAKOWICZ AND BE van figure!: Fluorescence emission spectra of benzo[a]pyrene. Microsomes containing BP were obtained by incubation of microsomes (equivalent to 0.6 mg of microsomal protein) with 5 ug of BP mi crocrystals. in a totai volume of 1! mL BP microcrystals (i ug) were suspended in 10 mL of buffer. Antbophyllite and Canadian chrysouie (16.7 mg) were added after formation of the microcrystals. gates. (2) the quenching of the structured monomer emission which results upon excimer formation, or (3) reabsorption of the fluorescence emission from the monomeric state prior to its escape from the crystal In addition. BP is known to form two types of crystal structures (Stevens. 1962). each with different fluorescence spectral properties. Sublimation or rapid removal of solvent, which is the procedure we used to form BP microcrystals. could resuJt in the formation of a metasiaoie crystal form of BP in which excimer formation does not occur. In this crystal state oniy reabsorption processes would be expected to affect the observed emissions. At present we do not know the proportion of each crystal type which we obtain by solvent evaporation. We note that the relative intensities of the monomer (405 nm) and excimer (490 nm) emission from these aqueous dispersions are some what variable, as is indicated by the error bar on Figure 1. This variability could be a result of variations in the crystal size, the proportion of each crystal form, and the amount of BP which is dissolved in the aqueous phase. These parameters are iikely to be strongly dependent upon the rate of solvent evaporation, temperature, and other factors such as the size and surface properties of the container used in preparation of the BP microcrystals. More detailed spectroscopic investiga tions are required to quantitate the relative importance of these excited-state processes, and different forms of BP. as determi nants of the observed emission spectra. Also shown in Figure 1 are the emission spectra of BP microcrystals to which anthophyllite or Canadian cnrysotile was added. These spectra are essentially identical with that of BP microcrystals alone. The smaller excimer contribution of the anthophyllite-containing sample is not significant in comparison with the variability observed in the spectra of BP microcrystals alone. The spectra of microcrystalline dispersions of BP which contain silica or iron oxide are also similar to that of microcrystals aione and are not shown for the sake of clarity. Thus, addition of particulates to preformed BP microcrystals does not result in a shift of the BP emission spectra to that observed for BP which is adsorbed to the surface of these particuiates. Tnese spectra for particle-adsorbed BP will bedescribed oeiow. We conclude that, upon addition of panicles to microcrystalline dispersions of BP. the carcinogen does not readiiy adsorb to the surface of the panicuiates. As a resuit we are abie to compare the microsomal availabilities of particie-adsorbed BP with the availability of BP in the presence FIGURE 2: Fluorescence emission spectra of benzoiajpyrent adsorbed to panicuiates. Emission spectra of adsoroed BP are shown at the same instrumental ampiiucauon (A) and normalized to the same peak intensities (B). Each 10-mL suspension in buffer contained 16.* mg of panicles and 5 ug of BP. i.e.. 0.3 mg of BP per g of pameuiate. of. but not adsorbed to. these same particles. Emission Spectra of Particle-Adsorbed Benzo[a\pyrene. The emission spectra of particle-adsorbed BP are dependent upon the type of particle and the surface density of BP on the particles (Lakowicz et al.. unpublished observation). From the intensity-normalized spectra (Figure 2B). we see that the contribution of the excimer fluorescence is greater for silica than for anthophyllite and chrysotiie. Precise interpretation of these spectra is complicated by the effects of the particles themselves on the fluorescence intensities, the existence of a heterogeneous population of BP molecules on the surface of the panicles, and the contribution of the BP which is soiubilized in the aqueous phase to the total emission. We investi gated the effects of the panicles themselves on the fluorescence intensities by examining the emission spectra of BP microcrystals in the absence and presence of panicles. Relative to BP microcrystals alone (1.0). the intensities of these crystals in the presence of anthophyllite. Canadian chrysotiie. silica, and iron oxide were 1.5,1.4,10. and 0.11 respectively. We interpret the increased intensities as being a resuit of sample turbidity. Light scattering by the turbid sampies may intensify the exciting light at that location in the sample where the fluorescence emission is collected. However, small quantities of BP adsorbed to the surface of the panicles could also ac count for the observed enhancements. On the other hand, hematite, which is red in color, probably attenuates the fluorescence intensity by absorption processes. Oniy minor changes in spectral distribution of the microcrystals occur upon addition of panicles. Thus, the filtering or enhancing eiiens of the panicles appear to be fairly uniform across the emission bands of BP. ASARCO ALV 0005416 particle-enhanced microsomal uptake of bp VOL. IS. NO. :3. 1979 5173 We attempted to compensate for these effects on the fluorescence emissions by normalizing the emission spectra of panicle-bound BP to equivalent intensities at 405 nm. This peak is probably due to both water-solubilized BP and BP which is adsorbed in the monomeric state. The larger con tribution of the excimer emission for silica-adsorbed BP than for BP which is adsorbed to anthophyllite and chrysotile sug gests that the latter fibrous minerals have a superior ability to adsorb BP in the monomeric state. Note from Figure 2B that hematite-adsorbed BP does not display significant excimer emission. This absence appears to be a result of the highly quenched nature of BP which is adsorbed to hematite (Figure 2A). This conclusion is based on the spectra of the dry BP-particle powders. These spectra are essentially identical with those shown in Figure 2B. except that no spectrum was observable for the dry- hematite-BP powder and somewhat less excimer emission was observed from dry BP-silica than from the aqueous suspension. That is, in the dry state the fluorescence of BP adsorbed to hematite is completely quenched. Thus, the observed emission in aqueous suspension is due to BP which is solubilized in the aqueous phase. Quenching of BP fluorescence by energy transfer to the hematite seems a likely quenching mechanism in that hematite adsorbs light strongly in the region of BP emission. This overlap of BP fluorescence with hematite absorption favors energy transfer (Forster. 1948). No fluorescence emission was observed for BP bound to carbon black, but the emission of microsome-bound BP was observed in suspensions of carbon black. For the latter sam ples. the BP fluorescence decreased with time, presumably as a result of BP adsorption by the carbon black. Thus, our inability to observe fluorescence from carbon black bound BP is not a result of the inner Sixer effects in these opaque samples. We conclude that the fluorescence of carbon biack bound BP is also quenched by an energy-transfer mechanism. This is not surprising in light of the broad absorption bands of carbon black. Unlike the emission spectrum observed for suspensions of hematite-bound BP. no water-solubilized spectrum was visible for suspensions of carbon black adsorbed BP. probably as a result of the high affinity of carbon black for polynuclear aromatic hydrocarbons. The relative fluorescence intensities of particle-adsorbed BP are revealed by the unnormalized emission spectra (Figure 2A). The most intense monomer emission was observed for BP adsorbed to the fibrous minerals anthophyllite and chry sotile. The emission of hematite-adsorbed BP is seen to be weak in comparison with that of BP which is adsorbed to the other panicles. As indicated above, only water-solubilized BP appears to contribute significantly to the fluorescence of the BP-hematite suspensions. Since the presence of hematite results in a 10-foid attenuation of the BP fluorescence, it appears that water-solubiiized BP accounts for about 0.2 unit of the "monomer" fluorescence observed in Figure 2A for the other panicles. Hence, these spectra result primarily from BP which is adsorbed to the surface of these panicles. The larger relative intensities of the monomeric emission of BP on the asbestos samples as compared to silica arc probably indi cative of their superior abiiitv to adsorb BP in the monomeric state. This ability is surprising in light of the surface areas of these panicles. By nitrogen adsorption these areas are 381. 26.8. and 11.8 m:-`g for silica, chrysotile. and anthophyllite. respectively (set Materials and Methods). However, we stress that our conclusions must be regarded as tentative until more accurate methods are developed to quantify the fluorescence emissions of compounds which are adsorbed to surfaces. FIGURE 3: Fluorescence intensity of benzofajpyrene during transfer from particulates to rat liver mlcrosomes. Fluorescence Spectral Changes ofBenzo[a]pyrene upon Its Microsomal Uptake. The fluorescence spectral distributions and intensities of microcrystalline and particulate-adsorbed BP remain constant over a period of hours when suspended in aqueous buffer. Upon addition of microsomes a rapid increase in fluorescence intensity occurs at 405 nm (Figure 3), the excimer emission becomes less significant, and the emission spectra become similar to that of the solution form of BP (Figure 1). We attribute these spectral changes to the transfer of BP from the particies to the microsomes. Calculation of the percentage of BP which is transferred to the microsomes requires knowledge of the fluorescence intensity after complete microsomal uptake of BP. These final fluorescence intensities (/.) are obtained by heating the sam ples to 50 "C for 1 h. followed by reequilibration at the ex perimental temperature of 25 C. Other studies (Lakowicz et al,, unpublished observation) indicated that increased tem peratures resulted in increased transfer rates of BP off particies into vesicles of dipalmitoyl-L-o-phosphatidyichoiine (DPPC). However, unlike DPPC vesicles, the microsomes are likely to be unstable at these high temperatures. Inactivation of the microsomes by heating could result in an alteration in the observed value for /.. We tested the validity of this heating procedure for obtaining the final fluorescence intensity in two ways. First, uptake of BP from anthophyllite was followed to completion without heating. Subsequent heating to 50 C did not alter the spectral distribution or intensity of BP. Second, longer incubation periods at 50 "C did not affect the fluorescence intensity. We conclude that the I. values are representative of complete BP transfer to microsomes even if the heating procedure results in loss of microsomal activity. If less than 0.4 mg of microsomes (as protein) is added, the full fluorescence enhancements are not observed. That is. after complete transfer of BP. subsequent addition of more micro somes results in an additional rapid increase in fluorescence intensity. At higher microsome concentrations the final fluorescence intensities are independent of the amount of mi crosomes added, except for iron oxide (see below). This in dependence indicates that the amount of microsomes added is adequate to bind ail the BP. We attribute the lower final intensities at lower microsome concentrations to excimer for mation in the microsomes themselves which results in a de creased relative intensity at 405 nm. For iron oxide the final fluorescence intensity is dependent upon the amount of microsomes added even under conditions where excimer formation within the microsomes does not oc cur. Microsomes bind to all these particuiates isee beiow i and. hence, a portion of the microsome-bound BP remains ir. close proximity to the surface of these particies. We suspect that ASARCO ALV 0005417 5174 BIOCHEMISTRY LAKOWIC2 AND BEVaN figure 4: Benzo[a]pyrcne uptake into rat liver tnicrosomes. (A) The open symbols represent BP uptake into microsomes from 16.7 ag of particles, and the doited symbols represent BP uptake into microsomes in an identical fashion, except that the microtomes were preincusated for 30 min with an additional 16.7 mg of partides which did not contain BP. Similar results were obtained upon addition of microsomes to a suspension containing both 16.7 mg of unlabeled panicles and 16.7 mg of panicles with adsorbed BP. (B) As indicated, the partides were adaeo to preformed BP microcrystals. BP fluorescence can be quenched by energy transfer from BP in the microsomes to the surface of hematite. By adding more microsomes. we can increase the average distance between the BP and this surface, decrease the probability of energy transfer, and. hence, increase the fluorescence yield. Microsomal Uptake ofBenzo[a\pyrcne. We used the timedependent changes in the fluorescence intensity at 405 nm [/(/)] to quantify the amount of BP transferred to the micro somes. In particular, we assumed % benzo[uj pyrene transferred to microsomes * m-h x 100 (1) where f0 and I. are the fluorescence intensities prior to the addition of microsomes and after complete BP transfer, re spectively. We compared the microsomal uptake rates of BP from the microcrystalline and particulate-adsorbed states (Figure 4). Adsorptiofi of BP to ail four mineral particulates results in enhanced microsomal uptake when compared with the microcrystalline state. Perhaps most importantly, chrysotile- and anthophyllite-adsorbed BP show the highest rates of microso mal uptake. The nonfi'orous minerals silica and hematite also enhance BP uptake but are less effective than the asbestos particies. BP was not released into the microsomes from carbon black. As controls we investigated the effects of partides on the microsomal uptake of BP when the particles are added to preformed microcrystals (Figure 4B). We previously indicated that such mixtures displayed the fluorescence spectral prop erties of BP microcrystals and not the properties of partideadsorbed BP. As a result we expected, and found, that all these mixtures displayed BP uptake rates which were identical with that of BP microcrystals to within our experimental errors. These errors are probabiv a result of the variability in the microcrystalline dispersions of BP. We conclude that adsorp tion of BP to the surface of particulates is necessary for partide-enhanced uptake to occur. Tabic I: Binding of Microsomes ;o Parncuiates paniculate unoun: (mg) =r of microsomes boundc amhophyUice aninopnyUite amnopnyllite anthophylliie amhophylliie iron oxide iron oxide iron oxide iron oxide iron oxide silica Canadian chrysotile carbon black 4.2 8.3 "3 16.7 91 :o.o 36 33.4 ?1 4.; 38 8.3 63 16.7 S3 :o.o 87 33.4 E9 16.7 40 16.7 84 16.7 33 a Binding of microsomes to particulates was quanttuied by incu bation of microsomes (equivalent to 0.6 m; of microsomal pro tein) with the amount of parucies shown for 3G mm a: If cC wuh continual suiting. The 480r supernatant was men assayed for pro tein to determine the extent of binding. Mechanism of Particle-Enhanced Microsomal Uptake of Benzo[a]pyrene. Partide-enhanctd uptake of BP could result from various processes. Among these are binding of the mi crosomes to the particulates, disruption of the integrity of the microsomes by the particulates, and an increased rate of sol ubilization of the BP into the aqueous phase from the surface of the particulates as compared to that of soiubiiization from the microcrystalline state. We investigated the roie of partide-microsome binding on the microsomal uptake rates of BP. We reasoned that, if binding were important for BP transport, then the presence of particles in the reaction mixture which do not contain BP should decrease its microsomal uptake rate. A decreased rate would be observable under conditions where the amount of uniabeled particuiaies is adequate to bind a significant fraction of the microsomes. ASARCO ALV 0005418 PARTICLE-ENHANCED MICROSOMAL UPTAKE OF BP vol. is. no. 19T9 51"5 From Table I we see that 16.7 mg of amhophyllite. chrysotilc. or iron oxide is adequate to bind essentially ail the microsomes under our experimental conditions, and 16.7 mg of silica binds approximately half of the microsomes. We assumed that microsomes would bind randomly to both the labeled and the uniabeied particles. Hence, if binding were important for BP uptake, a decreased uptake rate wouid be observed in the presence of excess uniabeied particles. How ever. identical uptake kinetics were observed in the presence of 16.7 or 33.4 mg of uniabeied particulates. Second, to increase further the potential sensitivity of this experiment to binding, we preincubated the microsomes with uniabeied particles prior to their addition to the BP-parriculate suspensions. In particular, we incubated microsomes with the uniabeied particles (16.7 mg) for 30 min at 25 C. These microsomes were subsequently added to the 10-mL suspension of particles which contained adsorbed BP. If binding were important for BP uptake, we expea a decreased uptake rate under these conditions in which the microsomes are bound to particles not containing BP. The preincubation with uniabeied particles had no significant effect on the BP uptake rate (Figure 4A). We conclude that microsome-particle binding is not a significant determinant of the microsomal uptake rate of BP. We note that a rapid reequilibration of the microsomes between the particles with and without adsorbed BP would invalidate our conclusion. Since the initial rates of BP uptake were identical within our experimental limits, such redistribu tion must be complete within 30 s. Assuming this redistribu tion requires times in excess of 30 s. these data indicate that particie-microsome binding is not a significant faaor in de termining the rates of BP uptake. Because of the known cytotoxic effects of particulates (Harington. 1974; Wade et ai.. 1976). we investigated the possibility that disruption of the microsomes was responsible for particle-enhanced uptake of BP. We assayed microsomal integrity by measuring lipid peroxidation activity. This activity was iost upon addition of deoxvchoiaie and. therefore, provides a measure of the integrity of the microsomal membranes. Lipid peroxidation consumes both N.ADPH and oxygen. We monitored this activity fiuorometricallv by the oxidation of NADPH to NADF', a measurement we could make in the presence of particulates. Anthophyllite. silica, and hematite had no significant effect qp the rate of lipid peroxidation (Table II), but chrysotile was found to have a significant inhibitory effect. These results indicate no correlation between the rates of BP uptake and disruption of the microsomes. That is. anthophyllite and chrysotile show nearly identical enhance ments of uptake, but anthophyllite does not disrupt the mi crosomes and chrysotile does. Likewise, the enhancements of amhophyllite. hematite, and silica all differ, yet none of these panicles significantly affea the integrity of the microsomes. The disruptive effects of chrysotile are easily understood in terms of its unique surface chemistry (Spiel &. Leineweoer. 1969). As opposed to the other paniculates whose surface charges are neutral or slightly negative, the surface of chry sotile is strongly positive (the isoeiectric point is 11.8) and highly basic. In addition, chrysotile has a stronger affinity for polar molecules than the other panicles and is known to be more highly cytotoxic. In control experiments we showed that aeoxycholate and p-'chloromercuri)benzoate inhibited the iipid peroxidase ac tivity. as did boiling of the microsomes (Wills. 1969). In addition. NaDH did not suppor. the lim'd peroxidation aaivuy to the same extent as did NADPH. Tnese experiments dem onstrate that the aaivitv we measured had the propemes of Table 11: Microsomai Lipid Peroxicase Acuvuy in tne Presence of Parucuiaies S3 iCl. (nmol of NADPH oxidized per min per mg of protein! 5 of act. with no parudes paniculate" none amhophyllite chrysotile silica hematite 25 (100) :i 84 9 36 23 92 34 136 controls deoxycholate (0.351 100 `C. i run p-(chloromercuri)benzoate (1 mM) NADH (no NADPH) S 2 8 9 20 8 32 36 " A total of 16.7 me of each parnculate was used. the NADPH-dependent lipid peroxidation described by Wills (1969) and was not a nonspecific oxidation of NADPH. We conclude that disruption of the microsomes by the panicles cannot account for the panicle-enhanced uptake of BP. Having eliminated binding of microsomes to the particulates and disruption of the microsomes by the paniculates as im portant faaors in the particle-enhanced uptake of 3P. we felt that an increased rate of solubilization of BP in the aqueous phase may be the most important faaor in enhanced uptake. We quantified the rates of BP uptake under conditions where both the particle and microsome concentrations were increased twofold. By increasing both concentrations, we were able to keep the panicie-to-microsome and BP-to-microsome ratios iconstant. If collisional encounters between particle and mi icrosomes were responsible for transfer of BP. then the BP uptake rate should increase fourfold under thee conditions. The observed rate for all particle were identical with those :shown in Ftgure 4. These observations indicate that collisional i encounters between particle and microsome are not of imechanistic significance for the transfer of BP from particle to microsome. Due to the low water solubility of BP in ;aqueous solutions (about 3.8 *ig/L; Davis et al,, 1942) oniv a small fraction of the total BP could be dissolved in the aqueous phase of our 10-mL sample. Thus, it appears unlikeiv that the rate-limiting step for BP uptake into microsome would be its rate of entry into the microsomal membrane. Tne zero-order dependence of the BP uptake rate on both particle and microsome concentrations probably indicate that the idesorption of BP from the surface of the particle is the ratelimiting step for BP uptake into microsome. Similarly, the rate of solubilization of 1,2-benzanthracene into the aqueous phase of silica was found to control the rate of benzanthracene 1uptake by lipid veicie (Lakowicz et al,, 1978b). Discussion We demonstrated that fibrous minerals are superior to nonfibrous minerals in their ability to transport carcinogens into microsome. We sugget that this ability may provide a partial explanation for the cocarcinogenic effects of asbestos inhalation and cigarette smoking in humans and for the cocarrinogenic effects between particle and PAH found in an imal carcinogenicity teting. However, we point out that mechanisms other than particle-enhanced transport may be operative in particie-PAH cocaranogenesis. Tnese mecha- ASARCO ALV 0005419 5176 BIOCHEMISTRY nisms include (1) tissue damage caused by smoking (Auerbach et al.. 1961) or by the inhaled particles. (2) inhibited clearance of smoke particles resulting from the presence of inhaled mineral particulates (Blenkinsopp. 1968: Ferin & Leach. 1976). and (3) altered metabolic profiles of the PAH in the presence of particulates. However, the known cocarcinogenic effects of particles and PAH which are observed in experimental animals correlate well with the microsomal availabilities described in this paper. For example, iron oxide with adsorbed BP is more carcinogenic than simple mixtures of BP and iron oxide (Henry et al.. 1975). Similar results were found with asbestos-adsorbed BP and asbestos-BP mixtures (Pvlev & Shabad. 1973). Our results indicate that BP must be adsorbed to the particle for enhanced transport to occur. Carbon black with adsorbed BP has been found to be less carcinogenic than BP alone (Davis et al., 1975: Steiner, 1956), although, depending on panicle size, cocarci nogenic effects have also been observed (Pylev, 1961). The former results agree with our own, in that carbon black did not release BP into microsomes. In summary, our results seem to be relevant to carcinogen testing in animals, but alternative mechanisms of cocarcino genesis may also be of imponance. We hope our methods, which allow the PAH delivery rates to be quantified, will facilitate the design of experiments which funher elucidate the mechanism of particle-PAH cocarcinogenesis and thereby increase our understanding of the multiple etiology of human cancer. Acknowledgments We express our appreciation to the Freshwater Foundation and especially to its founder. Richard Gray, Sr., without whose support this work would not have been possible. References Ames, B. N_ McCann. J., & Yamasaki. E. (1975) Mutat. Res. 31. 347-364. Auerbach, 0.. Stout. A. P.. Hammond. O. C.. & Garfmkel, L. (1961) N. Engl. J. Med. 265. 253-267. Birks, J. B.. & Cameron, J. W. (1959) Proc. R. Soc. London. Ser. A 249. 297-317. Blenkinsopp. W. K. (1968) J. Pathol. Bactcriol. 96. 297-304. Davis. B. R., Whitehead. J. K.. Gill. M. E.. Lee. P. N., But- terworth. A. D., &. Roe. F. J. R. (1975) Br. J. Cancer 31, 443-452. Davis, W. W,, Krahl/M. E.. & Clowes. G. H. A. (1942) J. Am. Chem. Soc.'64, 108-110. Emster. 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