The Oxidation Catalytic Converter Reduces the Inhibitory Activity of

The genotoxicity of diesel particles has been widely documented, and their tumor promoting effect has been reported recently using the gap junction in...
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Environ. Sci. Technol. 2000, 34, 1352-1358

The Oxidation Catalytic Converter Reduces the Inhibitory Activity of Soluble Organic Fractions of Diesel Particles on Intercellular Communication ISABELLE VANRULLEN, CATHERINE CHAUMONTET,* PHILIPPE PORNET,† F R EÄ D EÄ R I C V EÄ R A N , A N D P A U L E M A R T E L Laboratoire de Nutrition et Se´curite´ alimentaire, Institut National de la Recherche Agronomique, 78352 Jouy-en-Josas Cedex, France

The genotoxicity of diesel particles has been widely documented, and their tumor promoting effect has been reported recently using the gap junction intercellular communication (GJIC) assay. In our study, the ability of soluble organic fractions (SOF) of diesel particles emitted from cars equipped (WC) or not (WoutC) with the oxidation catalytic converter (OCC) and of particle SOF from an outdoor high polluted place (OHPP) to inhibit GJIC has been evaluated with two cell lines: a rat liver epithelial cell line (REL cells) and a rat pulmonary alveolar type II cell line (3T cells). With both cell lines, our results demonstrate that GJIC is strongly inhibited by WoutC, whereas it is much less reduced by WC ones: for REL cells, the activity of WC particles is 1/4 of the one of WoutC. Also, we show that the inhibition induced by WoutC is associated with a change in the GJ protein localization. Our results clearly show the effectiveness of the OCC technology in reducing both the tumor promoting activity and the genotoxicity of diesel particle SOF.

Introduction Several epidemiological studies have reported associations between daily exposure to particulate matter and increased incidence of respiratory symptoms, hospitalization for respiratory diseases, and premature mortality among the general population (1-5). Particles have different origins, and special attention is given to those emitted from diesel engines. Indeed, these engines constitute important urban sources of fine particles (467

>1000

275

a The cell density is expressed as the percentage of cell number compared to the control.

1/3000th diluted peroxidase-conjugated antimouse antibody (Jackson Immuno Research Laboratory). Immunofluorescence. Cells were cultured in 24-well plates, on 10 mm thin glass plates. Cells were treated as previously described (32). After permeabilization and fixation (triton 0.25% + paraformaldehyde 4%), cells were incubated successively with BSA 2%, avidin, and biotin (blocking kit, Vector), 1/100th diluted anti-Cx43 monoclonal antibody (Transduction Laboratory), 1/200th diluted biotinylated antimouse IgGs (Vector Laboratories), and 1/100th extrAvidin-fluorescein Isothiocyanate (Sigma). Ames Assay. The particle SOF were tested for mutagenicity toward Salmonella typhimurium strains TA98 and TA100 according to the modified Ames assay (33, 34). Bacteria were incubated with or without an exogenous metabolic activation mixture (S9mix) prepared from Wistar rats injected with Aroclor 1254. The studies included positive controls to check mutagenicity with activation (benzo[a]pyrene) and without activation (4-nitroquinoline-N-oxide). Linear model slopes were determined from the dose-response data.

Results Effects of Particle SOF on GJIC of REL Cells. As the ability of cells to communicate depends on their contacts, the influence of particles on cell density was evaluated first (Table 1). Also, cell density was used to evaluate cell cytotoxicity. Finally, to test the effect of a given compound on GJIC, we consider (on past experience) that it must not reduce cell density by a percentage superior to 25%. When REL cells are incubated for 8 h, none of the particle SOF reduce cell density (not shown). During a 24-h incubation (Table 1), whereas WC1 and WC2 do not modify cell density, the other particle SOF tested reduce it and are cytotoxic at high concentration. The calculated concentrations of WoutC1, WoutC2, and OHPP inducing a 25% reduction of cell density (IC25) after 24 h are respectively 160, 200, and 275 µg/mL. Such high concentrations could be used only for a 1-h incubation. Then, we have studied the kinetics of GJIC inhibition by WoutC1 and WoutC2 using the dye transfer assay (Figure VOL. 34, NO. 7, 2000 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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FIGURE 1. Effect of Particle SOF on REL cell GJIC. (a) Kinetic study; b WoutC1, 142 µg/mL; O WoutC2, 142 µg/mL; [ TPA 100 ng/mL. (b) In experiment 1 WoutC2 inhibits GJIC of REL cells for a 1-h incubation; 100% of GJIC is recovered after 24-h incubation and inhibited again when cells are reincubated for 1 h with fresh WoutC2 medium (F-M). In experiment 2 GJIC of REL cells are not inhibited when cells are incubated with the 24-h conditioned medium (C-M). (c) Dose-response for a 1-h incubation; b WoutC1, 9 WC2, [ OHPP. * Statistically different from the control (P < 0.05) using the Kruskal-Wallis test. 1a). At the concentration of 142 µg/mL, both WoutC1 and WoutC2 inhibit GJIC from 10 min of incubation. Maximum inhibition is reached at 4 h (50% and 60% of inhibition, for WoutC1 and WoutC2, respectively), then GJIC is progressively recovered, and it becomes equivalent to the control at 24 h. The same kinetic is obtained with TPA 100 ng/mL (taken as a reference tumor promoter), but the maximum inhibition is obtained at 1 h and reaches 93%. We have analyzed the recovery of GJIC after 24 h with two approaches (Figure 1b): (i) REL cells incubated for 24 h with WoutC2 and reincubated for 1 h with fresh WoutC2supplemented medium undergo a new inhibition of GJIC and (ii) the conditioned-medium, obtained from REL cells incubated for 24 h with WoutC2 and transferred on new cell cultures for a 1-h incubation, is unable to inhibit GJIC again. Finally, the dose-response of REL cells to WoutC1-2-3, WC1-2, and OHPP has been investigated for a 1-h incubation. 1354

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Figure 1c illustrates the inhibitory effect on GJIC of three representative particle SOF (WoutC1, WC2, OHPP). Reproducible dose-dependent responses were observed for each extract. Table 2 gives for all particle SOF the linear regression equation, the calculated concentration inducing a 50% reduction of GJIC (IC50), and the IC50 ratio (Fm) of WC and OHPP versus WoutC. Moreover IC50 can be related to the equivalent IK50 and to the equivalent ISOF50 by dividing IC50 respectively by the corresponding covered kilometric distance and the corresponding mass of SOF. The IK50 ratio (Fk) and the ISOF50 ratio (Fs) were also calculated for WC versus WoutC. For the two parameters Fm and Fk, on average, the capacity of WC to inhibit GJIC is 1/4 of the one of WoutC, therefore reflecting the effectiveness of the OCC technology on the particle SOF activity. Since the OCC reduces the total amount of SOF extracted for the same quantity of particle (data not shown), the decrease of the activity of the SOF evidenced by

TABLE 2. Comparison of the Activities of Particle SOF as Inhibitors of REL Cell GJIC and as Mutagens WoutC1 linear regression equation r2 IC50a

WC2

OHPP

-0.35x + 103

GJIC Inhibition of REL Cells for a 1-h Incubation -0.32x + 95 -0.30x + 95 -0.12x + 106

-0.08x + 110

-0.17x + 99

0.99 151

0.94 135

0.92 600

0.98 288

mean (IC50) ( SD Fmb Fkc Fsd

WoutC2

WoutC3

0.97 148

WC1

0.96 463

WoutC

WC

OHPP

144 ( 8 100% 100% 100%

531( 96 27% 24% 65%

288 50%

Mutagenicitye

Salmonella typhimurium strain TA98 TA98+S9mix TA100 TA100+S9mix

WoutC1

WC1

1.2 ( 0.5 3.2 ( 0.2 4.3 ( 0.6 5.6 ( 0.1

0.5 ( 0.1 1.4 ( 0.1 0.8 ( 0.2 1.8 ( 0.3

OHPP 1.2 1.7 1.2

a Concentration inducing a 50% reduction of GJIC, expressed as µg/mL. b F ) 100 × [IC (WoutC):IC (SOF)]. c F ) 100 × [IK (WoutC):IK (SOF)] m 50 50 k 50 50 where IK50 is the distance (in km) corresponding to the IC50. d Fs ) 100 × [ISOF50(WoutC):ISOF50(SOF). e Mutagenicity is determined using a modified Ames test. The mean slopes, expressed as histidine revertants/µg of particles (SD, are determined from the dose-response data from one (OHPP) or two (WoutC1, WC1) independent experiments.

Fs (65%) clearly demonstrates a change in the specific activity of the SOF. OHPP shows less inhibition ability than WoutC (Fm ) 50%). Effects of Particle SOF on GJIC of 3T Cells. The effect of particle SOF on cell density and GJIC of rat lung 3T cells has been also investigated. No effect on cell density is observed, even for a 24-h incubation (data not shown) enabling to test higher concentrations than in the case of REL cells. As represented for a 1-h incubation in Figure 2a, WoutC1 inhibits GJIC in a dose-dependent manner, in the same concentration range as for REL cells (0-285 µg/mL). Then (Figure 2b) 3T or REL cells were incubated concurrently with WoutC1-2-3, WC1-2, and OHPP, for a 1-h incubation at the unique concentration of 300 µg/mL. In these conditions, WoutC12-3 inhibit strongly GJIC of both 3T cells and REL cells (at least 80% inhibition), whereas WC1-2 and OHPP inhibit it lesser in REL cells and not significantly in the case of 3T cells. The inhibitory effect of WoutC1 on 3T cells was then analyzed for a 24-h incubation, in comparison with TPA. As shown in Figure 2c, TPA induces a moderate inhibition at 1 h, which is transient. Interestingly, Wout C1 effect at 140 or 280 µg/ mL is stronger, and no recovery of GJIC is observed until 24 h. Mechanisms Involved in the Inhibition of REL Cells. We have attempted to relate the inhibition of GJIC by the particle SOF, at the highest noncytotoxic concentrations, to their effect on the proteins that constitute the GJ channel, namely Cx43. The Cx43 protein amount and its phosphorylation status have been concurrently analyzed by western blot with protein extracts from cells treated or not by WoutC1. As shown in Figure 3, the Cx43 separated into several bands (42, 45, and 47 kDa) on a 10% polyacrylamide gel. The lowest band corresponds to the nonphosphorylated Cx43, whereas the others correspond to phosphorylated forms (33). No variation of any band, at any concentration or time, is detected, attesting that neither the total amount nor the phosphorylation status of Cx43 are affected by WoutC1. Then, we have investigated the cell localization of Cx43 (Figure 4) in REL cells. For control cells, as expected, the fluorescent spots corresponding to Cx43 are located in the plasma membrane. In WoutC2-treated cells, before 1 h of incubation, the spot localization is not affected, and then it

is slightly disorganized. After a 4-h incubation, there is a dramatic decrease of the spots corresponding to membranebound gap junction plaques, and spots are only perinuclear suggesting gap junction internalization (Figure 3b, photography E, f). The signal is restored at 24 h. Mutagenicity of WoutC1, WC1, and OHPP Particle SOF. The mutagenicity of WoutC, WC, and OHPP has been evaluated using a modified Ames test. Two strains of Salmonella typhimurium, TA98 and TA100, are used. The number of revertants per microgram of particles (Table 2) is higher with TA100 than with TA98. When the exogenous activation system (S9mix) is used, to mimic the detoxification/ bioactivation of chemicals due to the mammal cytochrome P450 and phase II enzymatic systems, the mutagenicity of particle SOF is enhanced with both TA98 and TA100 strains. Although this increase is not usually reported (35), it is consistent with the presence of polycyclic aromatic hydrocarbons (PAH) in the SOF (6). Moreover, when bacteria are exposed to particle SOF originated from an OCC-equipped car, the mutagenicity of particle SOF is lower on TA98 and TA100 strains, with and without S9mix. For instance, when this technology is used, a 70% decrease of mutagenicity on TA100 is observed.

Discussion GJIC Inhibition by Particle SOF. To our knowledge, this paper is the first one analyzing in detail the capacity of the SOF of diesel particles to inhibit GJIC. Until now, only fragmentary data on the inhibitory effects of some types of particles on GJIC were available (14, 36, 37). We demonstrate that all the particle SOF inhibit GJIC on both cell systems used: the liver REL cells and the lung 3T cells. They inhibit GJIC quickly and in a dose-dependent manner (Figures 1 and 2). The active concentrations herein reported are similar to those described for atmospheric and motorcycle particles (36, 37). The SOF from diesel particles constitutes a complex mixture of chemicals and limited information is available on the precise chemicals adsorbed which could inhibit GJIC. The neutral fraction of particle SOF, that contains PAHs, has VOL. 34, NO. 7, 2000 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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FIGURE 2. Effects of Particle SOF on 3T Cell GJIC and Comparison with their Effects on REL Cells. (a) Dose-response to WoutC1 for a 1-h incubation. (b) Comparison of the sensitivity of (9) 3T cells and (0) REL cells to WoutC1-2-3, WC1-2 and OHPP for a 1-h incubation at 300 µg/mL. (c) Kinetic study; O WoutC2, 280 µg/mL; b WoutC2, 140 µg/mL; [ TPA 100 ng/mL. * Statistically different from the control (P < 0.05) using the Kruskal-Wallis test.

FIGURE 3. Effects of WoutC1 on Cx43 Expression. Effect of particle SOF on the amount and the phosphorylation of connexin 43 (Cx43) demonstrated by western blot analysis. For different incubation periods, total proteins were extracted from rat heart (H) or REL cells treated or not (control) with WoutC1 at 71 µg/mL (W71), 142 µg/mL (W142), or 285 µg/mL (W285), which are noncytotoxic concentrations for 24-h incubation. Under 10% SDS-PAGE, Cx43 separated into three bands named NP for the nonphosphorylated form (42kDa), P1 and P2 for the 2 phosphorylated forms (respectively 45kDa and 47kDa). been shown to be the most active one, exhibiting 95% of the total activity (36). Some studies have shown that the GJIC of rat liver epithelial cells exposed to various PAHs exhibiting bay and baylike regions is inhibited (38-40). In one study, 1356

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these compounds failed to inhibit GJIC fibroblasts (41). These compounds which have been already involved in the genotoxicity of diesel particle extracts could also partly explain the inhibitory effect of our particle SOF on GJIC.

FIGURE 4. Effects of WoutC2 on Cx43 localization. Immunofluorescence of REL cells treated or not with WoutC2 (A) control, (B) 218 µg/mL for 10 min, (C) 218 µg/mL for 30 min, (D) 218 µg/mL for 1 h, (E) 218 µg/mL for 4 h, (F) 109 µg/mL for 24 h, which are noncytotoxic concentrations for the different incubation times. The arrow indicates the perinuclear localization of Cx43. Mechanisms Involved in GJIC Inhibition by Particle SOF. GJIC can be regulated at several steps of Cx expression, GJ assembly, and gating (42, 43). The absence of variation of the Cx43 amount observed with REL cells throughout the kinetic of GJIC inhibition by WoutC indicates that transcription and translation are not affected by diesel particle SOF. The perinuclear localization of Cx43 observed in the 1-h- and 4-h-treated cells suggests that WoutC could induce either the removal of GJs from the plasma membrane or a disorganization of the cell traffic involved in the addressing of connexons into the plasma membrane. On the other hand, GJIC inhibition occurs after a 10-min incubation, but no disorganization of the GJs is detectable at this time. Two explanations can be proposed: (1) connexons are already dissociated, but this cannot be evidenced by the immunofluorescence technique and (2) only GJ gating is affected. Such variety of mechanisms would be consistent with the complex chemical nature of the SOF of diesel particle. Participation of Diesel Particles in Tumor Promotion. For 20 years, many studies have evidenced the key role of GJIC inhibition in the tumor promotion step of carcinogenesis. This is particularly supported by the GJIC inhibitory effect of numerous tumor promoting agents (15, 16, 18). Since diesel particles inhibit GJIC, they can act as tumor promoters. Furthermore, as shown in Figure 1b for WoutC, although REL cells recover GJIC after a 24-h incubation, GJIC is inhibited again when these cells are reincubated for 1 h, thus demonstrating that REL cells become nonresponsive to particle extracts. As the 24-h-conditioned medium is not able to inhibit GJIC of new cultures, the chemicals initially active on GJIC should have been either metabolized or degraded. Since most lung carcinoma develop from bronchioloalveolar cells, a type II cell line like 3T cells can be considered as a relevant model for in vitro study of tumor promoting effects of airborne particles. 3T cells, which are direct targets of airborne particles, remain sensitive to WoutC even after a 24-h incubation (Figure 2c). Interestingly, 3T cells were more responsive to WoutC than to the positive control TPA. Finally, we can consider that both cases, inhibition of GJIC for a few hours in a repeated manner (REL cells) or steady inhibition of GJIC (3T cells), isolate cells for a time sufficient to let mutated cells escape from the control of surrounding healthy cells. When transposed to the in vivo situation, where cell multiplication is controlled in a very strict manner, this escape would be favorable to the clonal expansion of mutated cells whose proliferation could have been induced by oncogene activation, for instance.

On the whole, these results support the assumption that environmental compounds such as atmospheric particles, which are generally widespread at low doses, are particularly convenient for acting through the tumor promotion process. Comparison of GJIC Inhibition by WoutC1-2-3, WC1-2, and OHPP. The GJIC inhibition assay was useful to compare the potential tumor promoting activity of SOF particle emitted from cars equipped or not with the OCC. If we consider the different parameters characterizing the ability of particle SOF to inhibit GJIC, it appears that the OCC reduces the activity of particles at several levels. The 35% reduction of Fs is directly linked to a reduction of the global toxicity of the compounds of the SOF. It can be related to the 85% reduction of PAHs emission (data not shown) that constitute a small part of the compounds of the SOF. In addition, the OCC reduces the total amount of SOF extracted for the same quantity of particle. These two effects result in the 75% reduction of the global activity of particle SOF illustrated by Fm. Furthermore, we must mention that the OCC has been shown, previously, to reduce the particulate matter quantity (25). In our case, the weighing of the impacted filters showed that the particulate matter exhaust fell off 15% when the car was equipped with the OCC (data not shown). As expected, the atmospheric particles, which were studied as positive control, were active on GJIC. They were less active than WoutC and more active than WC. Since few diesel engines are OCC-equipped, the intermediate activity of atmospheric particle SOF should be due to the presence of less active particle SOF from other sources or to photochemical transformation of the active compounds. Moreover, our study clearly shows that the mutagenicity of diesel particle SOF is reduced, by a factor of 2-5 depending on the Ames test conditions, when the OCC technology is employed. Such a reduction has also been reported in a recent study (44). On the whole, our results clearly show the effectiveness of the OCC technology in reducing both the tumor promoting activity and the genotoxicity of diesel particles, in the same extent.

Acknowledgments The authors are grateful to Mr. J.-Y. Lecoz (PSA Renault) for providing diesel particles and Dr. A. Cle´ment (INSERM 515) for giving 3T cells. They also wish to thank Mrs. M.C. Guyot, C. Lachenal, and M. C. Coue¨ffe´ (Laboratoire d’hygie`ne de la ville de Paris) for their participation to the extraction of VOL. 34, NO. 7, 2000 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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particles and to the Ames mutagenicity assay and Mrs. C. Blondel, Mrs. B. Huet and Mr. P. Flanzy (INRA) for the preparation of the manuscript and the figures.

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Received for review June 7, 1999. Revised manuscript received December 1, 1999. Accepted December 17, 1999. ES9906453