Mutagenesis vol. 18 no. 5 pp. 449-455,
September 2003
© 2003 UK Environmental Mutagen Society/Oxford University Press
Potent genotoxic activity of benzo[a]pyrene coated onto hematite measured by unscheduled DNA synthesis in vivo in the rat
1Laboratoire de Toxicologie Génétique and 2Laboratoire Ecologie du Parasitisme, Institut Pasteur de Lille, 1 Rue du Pr. Calmette, BP 245, 59019 Lille Cedex, 3Faculté des Sciences Pharmaceutiques et Biologiques, 3 Rue du Pr. Laguesse, BP 83, 59006 Lille Cedex and 4Laboratoire de Toxicologie, GIP-CERESTE, Equipe d’Accueil 2690, 1 Place de Verdun, 59000 Lille Cedex, France
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Since epidemiological studies have firmly implicated co-exposure to iron oxides and polycyclic aromatic hydrocarbons as a potential etiological factor involved in the excess mortality due to lung cancer in miners, experimental studies have been performed to investigate the role of iron particles in benzo[a]pyrene (B[a]P)-induced lung pathogenesis. In a previous study using the Comet assay in vivo in the rat, we demonstrated that iron particles enhanced B[a]P genotoxicity. To determine whether co-exposure (B[a]P/iron oxides) induces a real genotoxic activity or is only due to inhibition of DNA repair, the unscheduled DNA synthesis (UDS) assay was implemented in vivo in the rat. The UDS assay was used to measure DNA repair in two cell types (lung cells and hepatocytes) of OFA Sprague–Dawley rats, 24 h after endotracheal administration of a single dose of an iron oxide (hematite, Fe2O3) (0.75 mg), of B[a]P (0.75 mg) or of B[a]P (0.75 mg) coated on hematite particles (0.75 mg). No difference in UDS was observed in the two organs investigated in rats treated with iron oxide alone compared with control animals, while a significant increase in UDS was observed in lungs and liver of rats treated with B[a]P alone compared with control animals. The main finding was a significant increase in UDS observed in both lung and liver cells of rats treated with B[a]P coated on hematite when compared with those treated with B[a]P alone. The current study demonstrates (i) that iron particles did not inhibit UDS in lung cells and hepatocytes of OFA Sprague–Dawley treated rats with B[a]P coated on hematite and (ii) a potent genotoxic activity of co-exposure to B[a]P coated on hematite. Therefore, our data may contribute to explaining the excess mortality due to lung cancer in epidemiological studies and overall why exposure to B[a]P coated on Fe2O3 particles resulted in a higher tumor incidence in rodents compared with exposure to B[a]P alone.
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Introduction |
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Polycyclic aromatic hydrocarbons (PAH) constitute a large class of chemicals with widespread occurrence in the environment. Among these PAH, benzo[a]pyrene (B[a]P) is a powerful environmental mutagen and carcinogen referred to by the IARC as a probable human carcinogen (International Agency for Research on Cancer, 1973, 1983, 1987
). B[a]P has been shown to induce gene mutations, chromosomal aberrations and other types of genotoxic effects in both in vitro and in vivo studies (International Agency for Research on Cancer, 1973, 1983, 1987).
The literature implicates concomitant exposure to PAH and iron oxides as the main etiological factor involved in the excessive mortality due to lung cancer in iron miners (Boyd et al., 1970; Mur et al., 1987; Pham et al., 1992; Chau et al., 1993; Pham et al., 1993). To investigate the role of iron oxides in lung pathogenesis, rodent studies were performed using a ubiquitous and carcinogenic PAH, B[a]P, which was coated on hematite (Fe2O3) particles (Saffiotti et al., 1968; Warshawsky et al., 1984; Greife et al., 1993; Warshawsky et al., 1994; Wolterbeek et al., 1995; Cheu et al., 1997). These experimental studies have already demonstrated that repeated exposure to B[a]P coated on hematite results in a decreased latency (Wolterbeek et al., 1995) and an increased incidence of lung tumors (Saffiotti et al., 1968) in rodents when compared with exposure to B[a]P alone.
To explain the enhancement of B[a]P lung carcinogenic properties in the presence of iron particles, it has been proposed that the higher genotoxicity could be due to an increase in the amount of B[a]P carcinogenic metabolites and reactive oxygen species (ROS) (Sullivan et al., 1985; Cavalieri et al., 1995; Cheu et al., 1997; Kim et al., 1997). Indeed, one of the hypotheses previously suggested in epidemiological studies is that the high granulometry of iron particles increases lung retention and modifies B[a]P uptake during exposure to B[a]P coated on hematite (Henry et al., 1975), thus leading to greater availability and intensive metabolism of B[a]P. The cellular consequence is the production of a greater amount of the ultimate B[a]P carcinogenic metabolite (benzo[a]pyrene 7,8-diol-9,10-epoxide, BPDE) (Greife et al., 1993) and of more ROS (Demple et al., 1994; Cheu et al., 1997), resulting in more damage to the DNA, as already demonstrated in several experimental studies.
If the adducts induced by BPDE or by ROS are present in excessive amount and not correctly repaired by nucleotide excision repair (Fridovich et al., 1986; Celotti et al., 1993) and base excision repair (Krokan et al., 2000), respectively, they could lead to the formation of DNA single-strand breaks and/or induction of mutations (especially G
T and AT transversions) (Eastman et al., 1992; Li et al., 1996). A clear linear relationship between DNA adducts formed by ROS and the formation of DNA single-strand breaks has also been demonstrated, suggesting that these two different lesions might share a common chemical mechanism (Toyokuni et al., 1996). The DNA single-strand breaks and/or mutations can activate protooncogenes and/or inactivate tumor suppressors genes, like p53, that could be encountered in some lung tumors (Eastman et al., 1992).
This hypothesis may explain the higher genotoxicity of co-exposure to B[a]P and iron particles versus B[a]P alone observed in epidemiological (Henry et al., 1975) and experimental (Saffiotti et al., 1968; Greife et al., 1993; Warshawsky et al., 1994; Wolterbeek et al., 1995; Cheu et al., 1997) studies.
So far, however, the exact mechanism underlying lung pathogenesis after co-exposure to B[a]P coated on hematite particles in exposed workers remains unclear (Steinhoff et al., 1991). Furthermore, it seems that DNA repair plays a key role in lung pathogenesis and, until now, DNA repair associated with co-exposure to B[a]P coated on hematite particles has not been studied.
The measurement of DNA repair as unscheduled DNA synthesis (UDS) following chemically induced DNA damage has been shown to be a valuable tool in assessing the genotoxic activity of chemicals in a number of cell lines (Steinmetz et al., 1984). The most widely used UDS assay employs hepatocyte cultures following in vivo treatments (Mirsalis et al., 1980). Using the UDS assay in hepatocyte cultures provides an accurate profile of the genotoxic metabolites formed in the liver, however, they give little information on the effects of chemicals in other key target tissues, such as the lungs (Steinmetz et al., 1984). A method for isolating lung cells was introduced in a previous study (Garry et al., 2003a).
In order to confirm the genotoxicity of B[a]P–hematite found in our previous study using the Comet assay (Garry et al., 2003b), the UDS method was used to measure DNA repair in two cell types (lung cells and hepatocytes) of OFA Sprague–Dawley rats 24 h after endotracheal administration of a single dose of an iron oxide (hematite, Fe2O3) (0.75 mg), of B[a]P (0.75 mg) or of B[a]P (0.75 mg) coated on hematite particles (0.75 mg).
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Chemicals
Sodium chloride, collagenase 1A, HEPES buffer, potassium chloride, sodium phosphate, collagen VII and collagen G were purchased from Sigma-Aldrich Chemie (Steinheim, Germany), pentobarbital from Sanofi Santé Nutrition Animale (Libourne, France), Hank’s balanced salt solution (HBSS), Williams culture medium and fetal calf serum from Gibco (Paisley, UK), fetal calf serum, phosphate-buffered saline and RPMI-1640 medium from Life Technologies (Cergy Pontoise, France) and Histopaque from Amersham Biosciences AB (Uppsala, Sweden). Benzo[a]pyrene (purity >98%) was provided by Sigma-Aldrich (Saint-Quentin Fallavier, France). Fe2O3 (purity 99%, particle size 1 µm) was purchased from Merck (Nogent-sur-Marne, France).
The choice of dose of B[a]P or Fe2O3 (0.75 mg/animal), 
3.75 mg/kg of each component, was made according to the results of a previous study (Garry et al., 2003b). This dose induced a significant effect in the Comet assay and was able to demonstrate the potentiation of B[a]P by Fe2O3.
The procedure for hematite and B[a]P preparation was established by Keenan et al. (1989a,b). Briefly, B[a]P (0.75 mg) was dissolved in chloroform and hematite (0.75 mg) was suspended in chloroform. The residues were evaporated to dryness under a gentle stream of nitrogen at 37°C to remove the chloroform solvent. Solutions were resuspended in physiological saline solution and, finally, sonicated in an ultrasonic disrupter (Bransonic Ultrasonic Cleaner, Shelton, USA) for 5 min.
The preparation of B[a]P coated on hematite particles was according to the method outlined by Saffiotti et al. (1968) and recapitulated in several publications (Keenan et al., 1989a,b). No verification of the coating of B[a]P on hematite was performed after each experiment, however, the method has been validated in several published papers (Saffiotti et al., 1968; Henry et al., 1975; Keenan et al., 1989b). Briefly, B[a]P (0.75 mg) was dissolved in chloroform and then added to Fe2O3 particles (0.75 mg). After homogenization, the mixture was evaporated under a stream of nitrogen. Residues were resuspended in physiological saline solution and, finally, sonicated in an ultrasonic disrupter for 5 min. The particle size (by number) of B[a]P–Fe2O3 preparation was 92–96% <3 µm, 4–6% between 3 and 15 µm, 1–2% >15 µm. The number of large B[a]P crystals (>15 µm) was very low (1–2%) (Henry et al., 1975). This shows that for the solution of B[a]P coated on hematite particles, the solution is essentially composed of B[a]P–ferric oxide and the percentage (1–2%) of free B[a]P is very low.
Animals
Male OFA Sprague–Dawley rats (6–7 weeks of age, weighing 180–210 g) were obtained from IFFA CREDO (l’Arbresle, France). Animals were housed in cages with solid plastic sides and stainless steel grid tops in a temperature (22 ± 2°C) and humidity (55 ± 15% relative humidity) controlled room on a constant light cycle (12 h on/12 h off). Food and tap water were supplied ad libitum. The feedstuff used was no. A04C10 irradiated feed from U.A.R. (batch 20425). The rats were assigned randomly either to the control or to treatment groups and were housed in labeled cages. All animals were submitted to a minimum of 1 week acclimatization period before the beginning of the experimental procedures.
Endotracheal treatment
A mix of drugs (75 mg/kg ketamine, 1.2 mg/kg diazepam and 0.4 mg/kg atropine) was first administered i.p. to anesthetize male OFA Sprague–Dawley rats. Endotracheal treatment is a recently developed pulmonary exposure rat model (Aliouat et al., 2001; Garry et al., 2003a,b) which allows homogeneous exposure of the whole lung.
Briefly, a cold light, a blunt wire, a mouth-opening device and a catheter (SURFLO® i.v. catheter 16Gx2"; Terumo®, Guyancourt, France) are needed. For the treatment, each anesthetized rat was suspended by its upper incisors on a wire rod at the top of a Teflon slant board (16 x 25 cm, 60° angle). The mouth of the rat was opened and the cold light was placed in front of the neck area. The tongue was stretched by hand or using non-traumatic forceps. The white spot, which could be easily seen through the open glottis, corresponded to the illumination of the trachea. Then the blunt wire was introduced inside the trachea when the glottis was open. The catheter was threaded onto the blunt wire, introduced inside the trachea and the wire was then rapidly removed. In order to check that the catheter was really inside the trachea, soap foam was introduced using a 2 ml syringe to demonstrate rat respiration.
Four groups of three rats were used in this research (12 rats). The first group (three rats) constituted the control group; each animal was treated with 150 µl of sterile physiological saline solution (0.9% NaCl, 0.5% gelatin; Merck, Nogent/Marne, France).
The other three groups of three rats each received a single dose of 0.75 mg Fe2O3 (3.75 mg/kg), 0.75 mg B[a]P (3.75 mg/kg) or 0.75 mg of B[a]P coated on 0.75 mg of Fe2O3 endotracheally. All these compounds were injected as a suspension in 150 µl of physiological saline solution using a 1 ml syringe.
All animals were maintained three per cage under controlled ambient conditions and with free access to food and water. The 12 rats were killed by i.p. injection of 60 mg/kg pentobarbital at 24 h exposure time and organs were then collected.
Unscheduled DNA synthesis (UDS) assay
The procedure described below is in accordance with the OECD Guidelines (OECD, 1997) and Commission Directive no. EC-2000/32/EC-B39 (European Commission, 2000) concerning the UDS assay with rat liver cells in vivo.
Principle of the measurement of UDS The ex vivo UDS test indicates DNA repair synthesis after damage induced by the test compound after dosing. The end-point of UDS is assessed by determining the incorporation of [3H]thymidine in DNA of cells that are not undergoing scheduled (S phase) DNA synthesis. The uptake of [3H]thymidine is determined by autoradiography.
Animals were killed 24 h after treatment and cells from the target organs were isolated. The resulting cells were exposed to [3H]thymidine, which was incorporated into the DNA if UDS occurs. Normal S phase synthesis is rare in major cell types and can readily be autoradiographically distinguished from UDS. Incorporation was followed by autoradiography of the cells and grain counting.
The technique described here was developed by Mirsalis and Butterworth (Mirsalis et al., 1980), modified by Ashby et al. (1985) and detailed by Kennelly et al. (1993). The advantage of this test is that it is an ex vivo assay, which more accurately represents the pharmacokinetics and pharmacodynamics of the compound than in vitro systems.
Isolation of lung cells
After BALF extraction, collagenase 1A (10 ml, 0.1%) was injected into the lungs via the trachea using a syringe and incubated for 30 min at 37°C. The lungs were carefully dilacerated with a scalpel and the cell suspension was filtered (60 µm) and centrifuged for 3 min at 90 g. The supernatant was removed and 15 ml of HBSS was supplied. An aliquot of 1 ml of this solution was mixed with 1 ml of RMPI-1640 medium and 100 µl of Histopaque solution was layered at the bottom of the tube. This separation by Histopaque eliminated a large number of mononuclear blood cells. The samples were centrifuged at 500 g for 3 min at room temperature. Lung cells at the top and interface of the tube were pipetted out and mixed with 1 ml of RPMI-1640 medium. For all samples, cell viability was >95%. All of the 40 cell types were collected (Boyd et al., 1984) and thus the results observed correspond to the global response of the lung.
Isolation of hepatocytes
Hepatocytes were isolated from OFA Sprague–Dawley rat liver by a two-stage perfusion system. The portal vein was cannulated and clamped. As soon as perfusion started, the superior vena cava was cut. Then the cannula was connected to a pump for perfusion. The liver was first perfused with HEPES buffer kept at 37°C (10 mM HEPES, 125 mM NaCl, 3 mM KCl and 1 mM Na2HPO4) at 40 ml/min for 5 min. The liver was then perfused using a collagenase 1A buffer (HEPES buffer containing 4 mM CaCl2 and 0.025% collagenase 1A) at 20 ml/min for 5 min at 37°C. At the end of perfusion, the liver became spongy. It was cut free into a 150 µm nylon filter funnel placed on a sterile tube. The Glisson’s capsule was carefully cut open and the hepatocytes were broken up by adding 40 ml of Williams’ medium supplemented with 10% FCS. The hepatocyte suspension was centrifuged at 40 g for 1 min. The resulting pellet was resuspended in 40 ml of Williams’ medium. For all samples, cell viability was >96%.
Radiolabeling of hepatocyte and lung cell cultures
The cultures of lung cells or hepatocytes were diluted to provide 1.5 x 105 viable cells/ml. Lung cells were diluted with airway epithelial cell growth medium (PromoCell®; Bioscience Alive, Heidelberg, Germany) and hepatocytes were diluted with Williams’ medium. Aliquots of 3 ml were transferred to each well of 6-well multiplates containing 25 mm round plastic coverslips (Merck, Nogent/Marne, France). The coverslips were first coated with collagen VII for hepatocyte cultures and with collagen G (collagen 1 + collagen 3) for lung cell cultures. Nine replicate wells per animal were prepared. The cultures were incubated at 37°C in an atmosphere of 5% CO2 for 90 ± 10 min to allow attachment of cells to the coverslips. After this attachment period, the supernatant medium was removed and the cells were gently washed with 2 ml of Williams’ E medium–Incomplete (WE-I) for hepatocytes or 2 ml Airway Epithelial Cell Growth Medium for lung cells, which was replaced with 1.5 ml of buffer (WE-I for hepatocytes or Airway Epithelial Cell Growth Medium for lung cells) containing 10 µCi/ml [3H]thymidine. Cultures were incubated for 3–8 h at 37°C in an atmosphere of 5% CO2. At the end of the labeling period, the supernatant was removed and the cells were then incubated overnight with a 250 µM unlabeled thymidine solution to reduce unincorporated radioactivity (cold chase). Then, the cells were rinsed with WE-I medium for hepatocytes or Airway Epithelial Cell Growth Medium for lung cells, fixed three times using Carnoy mix (ethanol/acetic acid) (Merck) and washed with distilled water. After that, the coverslips were dried at room temperature and, finally, mounted on slides with EUKITT (O.Kindler GmbH, Freiburg, Germany).
Autoradiography Six slides from each animal were coated in Kodak NTB-2 liquid emulsion. Each slide was dipped individually into the emulsion, ensuring that no air bubbles were generated. After gelling, the slides were incubated in a light-tight box and left refrigerated for 10–14 days. At the end of this time, the emulsion was developed and fixed. The cell nuclei and cytoplasm were stained with Meyers hemalum. Slides were dehydrated in ethanol, cleared in xylene and mounted with coverslips for microscopic examination.
Grain counting Grain counting was performed using an image analysis system (Visilog, Noesis, France). Nuclear and cytoplasmic grain counts were obtained from 50 cells/slide, 3 slides/animal. Each slide was examined to ensure that the culture was viable. Nuclear (NC) and cytoplasmic (CC) grain counts were recorded and the net nuclear grain count (NNGC) per cell was determined (NNGC = NC – CC).
Criteria for autoradiographic analysis The following criteria were used for analysis of slides: (i) only cells with normal morphology were scored; (ii) isolated nuclei with no surrounding cytoplasm were not scored; (iii) cells with unusual staining artefacts were not scored; (iv) heavily labeled cells in S phase were not scored for the genotoxicity assessment; (v) all other normal cells, 50/slide, were scored.
Expression and interpretation of the results
Expression of results. For each slide, the following were calculated and are presented in tables: the average NNGC with standard deviation (SD); the percent of cells in repair (characterized by a NNGC 
5) with SD; the average NNGC in cells in repair with SD; the percent of cells in S phase.
Criteria for genotoxic activity. A test compound was considered as positive in this assay if: the test compound yields group mean NNGC values >0 for liver (historical data) (Brambilla et al., 1992; Madle et al., 1994) and >1 for lung and 10% or more of cells are in repair (NNGC 5) (Brambilla et al., 1992; Madle et al., 1994) and/or an increase is observed in mean NNGC in cells in repair.
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The results for NNGC are presented in Tables 1 and 2 and illustrated by Figures 1 and 2.
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No difference in UDS was observed in the two organs investigated in rats treated with iron oxide alone when compared with controls. Indeed, values for NNGC for the controls and for the hematite treatment were <1 in the lung cells and <0 in the liver cells. An increase in NNGC was observed in lungs and liver of rats treated with B[a]P alone, with a NNGC of 3.32 in lung cells and of –0.19 in liver cells. A significant increase in NNGC was observed in lungs and liver of rats treated with B[a]P coated on hematite versus B[a]P alone (lung cells, with a NNGC value of 12.63 versus 3.32; liver cells, with a NNGC value of 4.03 versus –0.19).
Analysis of the second parameter, the percentage of cells in repair (NNGC >5), showed (Figure 2) for both organs investigated that no difference was observed between the treatment with hematite and the controls. Indeed, the percentage of cells in repair was 0 for both the control and hematite groups in the lung cells and 0.74 for the controls and 0.63 for the hematite treatment in the liver. An increase in the percentage of cells in repair was observed in lungs and liver of rats treated with B[a]P alone compared with the controls (lung cells, 14.38 versus 0%; liver cells, 26.09 versus 0.74%). A significant increase in the percentage of cells in repair was observed in lungs and liver of rats treated with B[a]P coated on hematite versus B[a]P alone (lung cells, 54.47 versus 14.38%; liver cells, 86.31 versus 26.09%).
The analysis of the percentage of cells in S phase showed that for both organs no difference was observed between the treatments (lung cells, controls 0.4%, hematite 0.9%, B[a]P 0.4% and B[a]P coated on hematite 0.7%; liver cells, controls 0.1%, hematite 0.1%, B[a]P 0.1% and B[a]P coated on hematite 0%). This observation demonstrated that cells were in G0 stage in the two organs studied and that neither hematite nor B[a]P nor B[a]P coated on hematite induced cell proliferation in liver and lung.
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Discussion |
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PAH are ubiquitous mutagenic and carcinogenic environmental pollutants (Jernström et al., 1996
). B[a]P, a common environmental and occupational PAH (Spurny et al., 1996), which requires metabolic activation and subsequent covalent interaction with DNA to be biologically active, is a well-known mutagenic compound and its involvement in carcinogenesis is widely acknowledged (Hattemer-Frey et al., 1991).
In occupational settings where B[a]P is encountered, numerous chemical and industrial dusts are also present and it has been particularly known for several years that B[a]P can be found in association with particles, especially with iron oxides (Boffetta et al., 1997; Cheu et al., 1997).
Several epidemiological studies have implicated co-exposure to iron oxides and B[a]P as a potential etiological factor involved in excessive mortality due to lung cancer and these studies have suggested that iron oxides might be responsible for a higher B[a]P pulmonary toxicity (Boyd et al., 1970; Mur et al., 1987; Pham et al., 1992, 1993).
Experimental studies have confirmed the epidemiological results, showing that repeated concurrent exposures to B[a]P and hematite resulted in a decreased latency (Wolterbeek et al., 1995) and an increased incidence (Saffiotti et al., 1968) of lung tumors in rodents compared with exposure to B[a]P alone. One of the main hypotheses to explain the enhancement of B[a]P mutagenic properties after co-exposure with iron oxides is an increase in the amount of reactive metabolites when hematite is present (Saffiotti et al., 1968; Warshawsky et al., 1984; Cheu et al., 1997). So far, however, the exact mechanism underlying lung pathogenesis after concurrent exposure to B[a]P and hematite particles remains unclear (Steinhoff et al., 1991; Wolterbeek et al., 1995). If all the experimental studies already conducted have given us information about the influence of iron oxide on the B[a]P metabolite profile, they did not study DNA repair. In a previous study using the Comet assay we have already shown that hematite clearly enhances B[a]P genotoxicity (Garry et al., 2003b). One of our hypotheses to explain the higher toxicity of B[a]P when hematite particles are present is inhibition of DNA repair. Until now, no research has studied both the effects and the consequences of co-exposure to B[a]P coated on hematite particles on DNA repair. It clearly appears though that DNA repair plays a key role in B[a]P lung pathogenesis (Eastman et al., 1992).
OFA Sprague–Dawley rats were chosen in this assay because this rodent strain has the capacity, like humans, to metabolize B[a]P by cytochrome P450 1A1 (Vanden Heuvel et al., 1994; Santostefano et al., 1997; Jana et al., 1998; Roman et al., 1998) and it was in this strain that the genotoxic effect of B[a]P was demonstrated using the in vivo Comet assay (Garry et al., 2003a).
Endotracheal treatment (Aliouat et al., 2001) was chosen for our experiment for the following reasons: it was histologically demonstrated than neither lesions nor inflammation occurred following endotracheal treatment; this mode of exposure is a fast procedure; there is no risk of regurgitation and the distribution of the toxic agents in the lungs is homogeneous; no deaths or clinical signs were observed after endotracheal treatment; the quantity of toxic agents is well controlled for each animal, which is not the case after inhalation exposure.
The choice of exposure time (24 h) and dose of B[a]P (0.75 mg) were made on the basis of the results of a previous study (Garry et al., 2003a). A 1:1 mass ratio between B[a]P and hematite particles was chosen from the literature data (Saffiotti et al., 1972; Henry et al., 1973; Sellakumar et al., 1973; Garry et al., 2003a,b) to investigate the deleterious mechanism of this co-exposure. The rationale for the choice of endotracheal treatment, of a dose level of 0.75 mg and of the 24 h exposure time is detailed in our previous study (Garry et al., 2003b).
The results for the UDS assay are presented in Figures 1 and 2. For liver cells, the NNGC value in controls was equal to –4.09 (negative value) and for the lung cells the NNGC was equal to 0.70 (positive value). The difference in NNGC values for the controls between the two organs investigated is due to the low number of mitochondria in lungs (Albrecht et al., 2001), which leads to a low value of cytoplasmic grain counts (CC) and, thus, a high value of NNGC. Thus, for each treatment, all the NNGC values observed for lung cells are higher than in liver cells.
In both lung and liver cells, the analysis of NNGC in Figure 1 showed that no difference in UDS was observed in rats treated with iron oxide (Fe2O3) when compared with controls. This result confirmed our previous findings using the Comet assay, where no single-stand breaks were observed in rats treated with hematite (Garry et al., 2003b). It is generally assumed that iron plays a critical role in the Haber–Weiss reaction, which could produce free radicals and ROS (Kehrer et al., 2000), both able to cause DNA adducts and DNA strand breaks and thus induce DNA repair (Okada et al., 1996). The dose of hematite used in our experiment (0.75 mg) seems, however, to be too low to generate enough free radicals and ROS. This fact can explain why (i) no DNA damage was scored in our previous study using the Comet assay (Garry et al., 2003b) and (ii) no difference in UDS was observed in rats treated with iron oxide when compared with the controls in the present study.
In both lung and liver cells, the analysis of NNGC in Figure 1 showed an increase in UDS in rats treated with B[a]P when compared with controls. In our previous study we showed induction of DNA single-strand breaks in rats treated with B[a]P (Garry et al., 2003a,b).
Lung cells, such as non-ciliated Clara cells (which are stem cells of the bronchiolar epithelium and the main carrier of the cytochrome P450 isoenzyme system in lungs) (Boyd et al., 1984; Albrecht et al., 2001) and type II pneumocytes contain a high level of activity of CYP 1A1 and represent a defense system against environmental xenobiotics. These specific cells were directly exposed to B[a]P after endotracheal administration and were thus able to metabolize B[a]P and generate ROS and electrophilic metabolites such as BPDE (Albrecht et al., 2001). These high levels of metabolic capacity might explain the high damage level (DNA single-strand breaks) observed in lung cells in both our previous studies with B[a]P treatment (Garry et al., 2003b) and in the current study.
B[a]P was essentially metabolized by lung cells because administration was made directly into the lungs by endotracheal treatment, and liver exposure was more limited than by the oral route. The high amount of BPDE produced could cross the alveola–capillary barrier, reach the blood (systemic circulation) and damage liver cell DNA (Figure 1). Lung cells and hepatocytes would then be directly exposed to B[a]P and/or its metabolites.
The main observation of this study was a significant increase in DNA repair as measured by UDS induced in lung cells and hepatocytes in rats treated with B[a]P coated on hematite compared with B[a]P alone. In our previous study we demonstrated that B[a]P coated on hematite particles produced more DNA single-strand breaks than exposure to B[a]P alone (Garry et al., 2003b). The current results show that the high genotoxicity of B[a]P coated on hematite particles was not due to inhibition of DNA repair, but that, in contrast, repair was increased. This fact confirmed that co-exposure to B[a]P and hematite particles induced a clear and potent genotoxic activity as revealed by the increase in UDS.
The increase in DNA damage could be due to the high granulometry (specific area and particle size) of iron particles, which increases the lung retention and modifies the uptake of B[a]P. This fact could lead to a greater availability and an intensive exposure to B[a]P, as already proposed by Henry et al. (1975). The consequence would be greater formation of DNA adducts, which could lead to the formation of more DNA single-strand breaks (Eastman et al., 1992; Li et al., 1996), as demonstrated in our previous study (Garry et al., 2003b).
The current study has shown that iron particles do not inhibit DNA repair in lung and liver cells of OFA Sprague–Dawley rats treated with B[a]P coated on hematite.
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Acknowledgements |
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We want to thank Sophie Meintières, Frank Le Curieux, Isabelle Deraugnaucourt, Eric Vercauteren, Doris Lagache and Gonzague Dourdin for their help. This work was supported by GIP-CERESTE.
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5To whom correspondence should be addressed at: Faculté des Sciences Pharmaceutiques et Biologiques, 3 Rue du Pr. Laguesse, BP 83, 59006 Lille Cedex, France. Tel: +33 3 20 87 79 75; Fax: +33 3 20 87 73 10; Email: daniel.marzin{at}pasteur-lille.fr
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Received on March 20, 2003; revised on May 19, 2003; accepted on May 22, 2003.
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