Genotoxicity of Air Borne Particulates Assessed by Comet and the Salmonella Mutagenicity Test in Jeddah, Saudi Arabia

Fine airborne respirable particulates less than 10 micrometer (PM10) are considered one of the top environmental public health concerns, since they contain polycyclic aromatic hydrocarbons (PAHs) which are among the major carcinogenic compounds found in urban air. The objective of this study is to assess the genotoxicity of the ambient PM10 collected at 11 urban sites in Jeddah, Saudi Arabia. The PM10 extractable organic matter (EOM) was examined for its genotoxicity by the single cell gel electrophoresis (SCGE) comet assay and the Salmonella mutagenicity (Ames) test. Gas chromatography-mass spectrometry was used to quantify 16 PAH compounds in four sites. Samples from oil refinery and heavy diesel vehicles traffic sites showed significant DNA damage causing comet in 20–44% of the cells with tail moments ranging from 0.5–2.0 compared to samples from petrol driven cars and residential area, with comet in less than 2% of the cells and tail moments of < 0.02.In the Ames test, polluted sites showed indirect mutagenic response and caused 20–56 rev/ m3, mean while residential and reference sites caused 2–15 rev /m3. The genotoxicity of the EOM in both tests directly correlated with the amount of organic particulate and the PAHs concentrations in the air samples. The PAHs concentrations ranged between 0.83 ng/m3 in industrial and heavy diesel vehicles traffic sites to 0.18 ng /m3 in the residential area. Benzo(ghi)pyrene was the major PAH components and at one site it represented 65.4 % of the total PAHs. Samples of the oil refinery site were more genotoxic in the SCGE assay than samples from the heavy diesel vehicles traffic site, despite the fact that both sites contain almost similar amount of PAHs. The opposite was true for the mutagenicity in the Ames test. This could be due to the nature of the EOM in both sites. These findings confirm the genotoxic potency of the PM10 organic extracts to which urban populations are exposed.


Introduction
Over the last decade, there has been an increasing concern about the adverse health effects caused by human exposure to airborne chemical pollutants. These concerns have been reinforced by laboratory studies that confirmed the carcinogenic potential of airborne particulate matter in animal experiments [1,2] and by epidemiological studies that supported a possible correlation between lung cancer and the mutagenic activity of air organic matter in urban areas [3,4,5]. Also, air pollutants are potentially significant reproductive and developmental toxicants [6,7]. Ambient air genotoxicants can originate from emissions of fuel combustion (motor vehicle exhaust, space heating, power generation); waste incineration and industrial processing [8,9]. Exhaust from diesel engines is considered to be a major contributor to the mutagenicity of urban air [2], and its particulates is characterized by a carbonic mixture composed of approximately 18,000 different high-molecular-weight organic compounds [10]. Diesel engines release 10 times the amount of breathable PM compared to unleaded gasoline engines. Diesel exhaust particulate PM acts particularly through polyaromatic hydrocarbons (PAHs), which can easily pass through epithelial cells membranes, bind themselves to cytosolic receptors and then affect cell growth and differentiation [11,12]. PAHs are human mutagens and carcinogens [2] and are potentially significant reproductive and developmental toxicants [6,13]. In a study in New Jersey, USA, the risk of fetal death, premature birth, and low birth weight was significantly higher for those with a high prenatal polycyclic organic matter exposure [14]. Mutagenicity and genotoxicity of organic complex mixture of urban airborne particles have been demonstrated with different short-term tests on prokaryotic and eukaryotic organisms [15][16][17][18]; including mammalian cells cultured in vitro [19,20] and laboratory animals [21]. Of the short term in vitro bioassays for detecting mutagens is the salmonella/mammalian microsome mutagenicity test [22,23]. The salmonella test developed by Ames is about 90% accurate in detecting a wide variety of carcinogens as mutagens. The test is based on the fact that essential properties of most carcinogens are their ability to damage DNA. The test uses bacteria as a sensitive indicator of DNA damage and uses mammalian liver extract for metabolic conversion of chemicals to their active mutagenic forms. The bacterial strains are a set of histidine operon mutants. The standard tester strains contain other mutation that greatly increases their ability to detect mutagens. Another short term bioassay system for detecting DNA damaging agents is the single cell gel electrophoresis (SCGE) test or comet test [24]. This is one of the most sensitive methods for detecting DNA damage; it detects DNA single/double strand breaks and alkali-labile sites, in individual eukaryotic cells. It is reliable and rapid Therefore, this method has been widely used for studying mutagenic environmental chemicals [25][26][27]. The objective of this study is to assess the genotoxicity of the ambient PM10 fraction of outdoor airborne particulates in Jeddah, Saudi Arabia. The acetone extracts of these PM10 particulates were examined for its genotoxicity by the single cell gel electrophoresis (comet assay) and the Salmonella mutagenicity Ames test.

Air Sampling Sites
Ambient Air particulates matter (PM10) were collected uniformly using Staplex high volume air sampler at an average flow rate of 1.4 m 3 /min. Sampling lasted 24 hrs and was carried out between Jan 17, 2004 and Feb 9, 2004 at 11 urban sites (Table 1) within a radius of 15 km in Jeddah city which is the second largest city in the Kingdom of Saudi Arabia. It is in the western region of Saudi Arabia, at the coast of the Red Sea with nearly 2.5 of permanent inhabitants. The territory of the city is well ventilated. Air collection sites were selected to be representative of industrial area which is impacted by emissions of chemicals (sites 1, 2, 8); Business area with heavy traffic of petrol and diesel driven motor vehicle (sites 3, 4, 5, 6) in addition to diesel driven trucks (site 7); residential area with petrol driven vehicle (sites 9); highway (site 10 ) and reference site which is the roof of King Fahd Medical Research Center at a quiet neighborhood (site 11). All these sites are assigned on a map for Jeddah area. Air sampler was placed 8 feet above the ground with electric supplies as a source of energy to avoid creating interference when using petrol generator. Following sampling, all the glass fiber filters, 8" x 10" (TFAGF 810) were immediately placed in plastic bag and stored at -20ºC until processed. The amount of air particulate matters was determined gravimetrically by weighing the filters before and after air sampling. Table 1: Sampling sites in Jeddah city and the amount of EOM of particle matter (PM10) collected in 24 hours on glass fiber filters.

Extraction of the Filters
The materials collected onto or absorbed to the glass fiber filters used in the PM10 air sampler were extracted with acetone in Soxhlet apparatus. The glass fiber filter was cut into small strips and placed in a Soxhlet tube and immersed in 300 ml of acetone. The mixture was refluxed 06.0 for 18-24 h. The extract was then concentrated using rotary evaporator. The condensate was weighed and dissolved in appropriate amount of dimethylsulfoxide (DMSO). All processes were carried out without direct exposure to light. This extract was used for the assay of genotoxicity.

Quantification of PAH
Gas chromatography-mass spectrometry (GC-MS) was used to quantify 16 PAH compounds in the PM10 extractable organic matter (EOM). For the GC/MS analysis, the filters were extracted by ultrasonication with tetrahydrofuran. The PAHs were separated on a reversephase column (C18-RP-MZ -PAH) and analyzed with GC-MS using RTX-XLB column 30 m, 0.25 mm i.d., 25 um ft.

Single Cell Gel Electrophoresis (Comet Assay)
DNA strand breaks were determined by way of the alkaline comet assay [24,27], were 1 ml of peripheral blood were exposed to 250 μg of the filter extracts for different periods of time. After treatment, 1x10 5 cells combined with 100 μl of molten low melting agarose (LMA) and immediately pipetted 75 μl onto comet slide (Trevigen, USA), then the slides were immersed in freshly prepared cold lysing solution (2.5M NaC1, 100mM Na2 EDTA, 10mM Tris, pH 10, 1% sodium sarcosinate), 1% Triton X-100 and 10% DMSO was added just before use for a minimum of 1 h at 4°C. Slides then were laid in horizontal electrophoresis apparatus filled with freshly prepared alkaline solution, pH ≥ 13, the slides were immersed in the lysing solution for 20 minutes, then electrophoreses was performed at 1 Volt/cm for 20 minutes. Slides were rinsed for 5 minutes three times and then immersed in 70% ethanol for 5 minutes. After air drying slides were stained with 50µl ethedium bromide (20 μg/ml), covered and placed in a humidified air-tight container to prevent drying of the gel and were analyzed within 3-4 hours.

Comet Scoring
A total of 50 cells from each of the duplicated slides were examined randomly under fluorescence microscope (Olympus BX-51, Japan). The extent of DNA damage was measured quantitatively using comet analysis system (Loats, USA) based on extended dynamic range imaging (EDRI) technology, which expressed the comet tail moment automatically. Parallel to each experiment a series of negative controls were done in order to determine any non-specific formation or reduction in the comet.

Salmonella Mutagenicity Test (AmesTest)
Mutagenicity test was performed by the plateincorporation technique using the Salmonella typhimirium strain TA98 as described by Maron and Ames [22] with and without metabolic activation S9 fraction. The liver S9 fraction was prepared as described by Ames [28], were male Sprague-Dawley rat was injected intraperitoneal with sodium Phenobarbital, dissolved in saline at doses of 30 mg/kg for the first 4 days and 60 mg/kg on each of the following 3 days. Prior to scarifying the animal, β-naphthoflavone, suspended in corn oil, was given by the same route at a dose of 80 mg/kg for 2 days. The rat was sacrificed by decapitation. Liver was homogenized in ice-cold 0.15 M KCI solution in a Teflon homogenizer, centrifuged at 9000 x g at 4ºC for 10 min. The supernatant (S9) was stored at -86ºC until used. Sterility of the preparation was determined by plating 0.1 ml of the (S9) preparation on bacto-pepton agar plate. EOM was tested with and without the metabolic activation (S9) fraction. Each PM10 OEM sample was assayed in three doses 25, 50, and 100 μg of condensate/plate using two replica plates per dose. DMSO (100 μ1) was used as a negative control. Revertants colonies were scored after 48 h incubation at 37°C using Image pro-plus software. Standard mutagens were used as positive controls including 4-nitroquinoline-1-oxide, 0.5 pg/plate; sodium azide, 5 pg/plate (without S9); 2aminoanthracene, 2.5 pg/plate (with S9). Positive response was defined by at least a two fold increase in revertants over the negative control. Results were expressed as the number of adjusted revertant colonies per cubic meter of air (revertants/m 3 ). Table 1 shows the amount of acetone extractable organic materials on filters at different sites per cubic meter of air. The EOM is ranging from 40 μg/m 3 in the highly polluted locations to 6 µg/m 3 in the reference site (11). It is clear that 'Breeman' (site 8) has the highest amount of organic materials (40 μg/m 3 ). The major contributors to this pollution at this site are diesel emissions and the municipal incinerator of all Jeddah dumps which is located east of this road. Second highest EOM was found at 'Kubry Almurabah' (site 7) which has 31 μg/m 3 . This site is road intersection in a business area with a lot of petrol driven cars and diesel vehicles which are allowed to move during night time. As for site 2 which has 25.4 μg/m 3 , it is considered to be in an industrial area with sugar factory nearby beside being near to Jeddah main sea port with all the emission coming from the ships and from trucks loading and unloading these ships. Results for the remaining sites are in agreement with their traffic status with the lowest level of organic particulates found near the sea coast 'Khafar Alsawahel' (site 9) 10 μg/m3 and the reference (site 11) with 6.0 μg/m 3 .

Ambient levels of PAHs
The results of chemical analysis of the EOM in 4 places are given in Table 2. A sum of 16 PAHs collected on individual filters was analyzed. The PAHs varied from 0.83 ng/m 3 in the oil refinery (site 1), in the garbage burning, and heavy diesel-powered motor vehicles traffic location (site 8) to 0.18 ng/m 3 in residential areas (site 3) and 0.18 in the reference (site 11). Six of these PAHs compounds are known to be carcinogens and to induce reversions to histidine independence in strain TA98 of Salmonella [29]. At site 8 the benzo(ghi)perylene represent 65.4% (0.53 ng/m 3 ) of the total PAHs, this was followed by the known carcinogens indeno (123-cd) pyrene and Benzo(a)pyrene at concentrations of 0.12 ng/m 3 and 0.03 ng/m 3 respectively. At the oil refinery location (site 1), benzo(ghi)perylene was highest (0.17 ng/m 3 ) followed by Phenanthrene (0.13 ng/m 3 ), then fluoranthene and Benzo(a)pyrene at a concentrations of 0.08 and 0.07 ng/m 3 respectively. The other PAHs compounds occurred as minor components.

Genotoxicity in SCGE (Comet) Assay
In single cell gel electrophoresis assay, cells with increased DNA strands breaks display, increase migration of the DNA from the nucleus toward the anode. Comets formed as broken ends of the negatively charged DNA molecules become free to migrate in the electric field toward the anode. Two main principles are believed to determine the pattern of the comet formation. The ability of DNA to migrate, which is a function of both size of the DNA fragment and the number of broken ends that may be attached to a larger pieces can migrate a short distance from the comet head. Results of the EOM genotoxicity in the comet assay are shown in Table 3 and are expressed as comet tail moment. EOM at 'Breeman' (site 8) with heavy diesel emission in addition to the garbage burning presented the strongest genotoxic responses with 44% of the cells exhibited comet with an average tail moment of 2.0. Next highest genotoxicant sample was from 'Alnujoom Square' (site 1) it caused comet in 20% of the cell with an average tail moment of 0.5. The remaining locations of heavy petrol driven cars of city main road or residential areas showed very weak DNA damaging effects with less than 2% of the cells exhibited comet with an average tail moment of less than 0.02. There was a good correlation between DNA damage in the comet test and the amounts of pollutants and PAHs sequestered by the filters ( Table 1). The genotoxicants in the comet assay does not seem to be the direct toxicants, since the toxicity appeared only in the presence of the metabolic activation system (S9) and there was no comet in absence of (S9).

Mutageicity in the Ames Test
Mutagenicity of the acetone extractable matters in the TA-98 Ames test is shown in Table 4. Out of the 11 sites investigated, 3 exhibited moderate mutagenic activities ranging from 20rev/m 3 to 56rev/m 3 . The highest mutagenic response was found at the garbage burning in addition to heavy diesel-powered motor vehicles traffic location 'Breeman' (8) with 56 rev/ m 3 . This was followed by extracts from heavy traffic intersection 'Kubry  Almurabah' (7) with 34rev/m 3 , then the sugar factory (site 2) with 20 rev/ m 3 . Three sites exhibited weak mutagenic activities ranging from 9 to 15 rev/m 3 corresponding to area of oil burning toursh 'Alnujoom square' (1) and city main roads (5,6). Four open areas or sea shore have 4 to 6 rev/ m 3 . The lowest mutagenic activity corresponding to 2rev/m 3 was measured at the background location 'KFMRC' (11).All filter extracts required the metabolic activation fraction (S9) for their mutagenic response which indicates that ambient PM10 particulates extracts are indirect mutagens.

Discussion
Several studies have pointed to the relationship between ambient air pollution and adverse health effect. The biological mechanisms that could be responsible for these effects are revealed by bioassay-directed chemical analysis of the crude organic extracts. Several studies suggested that polycyclic aromatic hydrocarbons might be responsible for most of the mutagenic and genotoxic activity [30]. Several in vitro studies with fractionated crude organic complex mixtures demonstrated that the highest mutagenic and DNA damaging activities is associated with the fractions, which contain most of the PAHs and nitro-PAHs derivatives [18,31,32]. Chemical analyses of PAHs provide specific information about a number of known mutagenic and carcinogenic PAHs [33][34][35][36]. Levels of 16 PAHs measured in this study are ranging from 0.18 ng/m 3 for non polluted and residential locations (n=2) to 0.82 ng/m 3 for the industrial and polluted locations (n=2). These concentrations are much lower compared to those measured in other large cities around the world, including Montreal, Canada with a level of 22.9 ng/m 3 in a polluted and 1.9 ng/m 3 in nonpolluted areas; [37], in Athens, Greece the level of PAHs was 10.87 ng/m 3 measured outdoors [38], in Cairo (Egypt) concentrations of PAHs was 14.79 ng/m 3 during the winter season and 7.53 ng/m 3 in summer [39] and in Kuwait PAHs concentrations measured at 14 sites ranged from 5 to 13 ng/m 3 with phenanthrene representing 35% of the total PAHs. The proportion of five and six-ring PAHs increased around the oil lakes [40]. In Jeddah, the high-molecular-weight PAHs such as benzo(ghi)perylene and B[a]P was the most abundant among the 16 PAHs analyzed, measuring respectively 0.53 ng/m 3 and 0.05 ng/m 3 in polluted areas compared to 0.035 ng/m 3 and 0.02 ng/m 3 in non polluted areas. The lower molecular-weight PAHs were only present as a minor fraction. The highmolecular-weight PAHs are, however, of greater concern because of their significant mutagenic/carcinogenic potency, [35]. B[a]P in the present study is significantly lower than that reported in Birmingham (UK) where the concentrations ranged between 0.81 ± 0.65 ng/m 3 in winter and 0.25 ± 0.20 ng/m 3 in summer [41] and in Heraklion (Greece) the B[a]P concentrations ranged between 0.14 and 3.47 with an average of 1.16 ng/m 3 during the period 2000-2002 (42).
The genotoxicity of the PM10 EOM was also assessed using the SCGE assay. This assay is a rapid and sensitive method to examine single and double-strand DNA breaks [43], and it has been used to evaluate ambient air genotoxicity in several studies [44][45][46]. In the present study, SCGE assay demonstrated good correlations between PAHs concentrations and DNA damageas shown in tables 2 and 3. Extractable organic matters from sites 1 and 8 which are relatively more polluted caused comet formation in 20 and 44 % of the cells with a tail moment of 5 (± 0.2) and 2 (± 0.5). Meanwhile in the less polluted areas, cells with comet were < 2% with a tail moment of 0.01-0.02. Other studies found similar result where the genotoxicity of organic extracts of particles collected from dense traffic area were greater than in limited traffic area according to the same comet assay [47].
Results of the Salmonella mutagenicity assay provides reproducible results that correlate with measured PAH concentrations. Samples taken at sea port in addition to sugar factory (site2), at diesel driven trucks (site 7) and at garbage burning in addition to truck routes (site 8) showed higher mutagenic activities in the Salmonella test with 20, 34, and 56 adjustable rev/m3 respectively, meanwhile samples from the residential or petrol driven motor vehicle sites (sites 3,4,5,9,10,11) showed an average of 6 rev/m 3 . Similar studies using Ames assay, showed that revertants number of TA98 strain was higher in dense traffic sectors than in sectors where traffic was more limited [44,47]. It worth noticing that EOM from site 1 was more genotoxic in comet assay than in the mutagenicity Ames test .This could be due to the nature of the comopounds at the two sites; this emphasizes the need to use more than one test for genotoxicity screening of air pollutants. The EOM were also proven to be indirect mutagens in both tests since it require activation system (S9). This may imply that the organic materials in the air filter extracts could be polyaromatic hydrocarbon which is very well known carcinogen and not nitropolycyclic aromatic compounds which is known to be direct mutagen in the TA-98 Ames [48]. Our results are in accordance with other studies showing greater mutagenic and genotoxic responses of particles' organic extracts from dense diesel traffic areas compare to residential areas. Results of these studies can play a central role in a long term program of cancer prevention aim to identify and minimize human exposure to environmental carcinogens and to aid in the development of preventive program to avoid future human exposure