Activated Toxicity of Diesel Particulate Extract by Ultraviolet A Radiation in Mammalian Cells: Role of Singlet Oxygen

Background Diesel exhaust [diesel exhaust particles (DEPs) and their extracts (DPE)] and ultraviolet A radiation (UVA) are two ubiquitous environmental factors that have been identified as essential risk factors for various benign or malignant human diseases, either alone or in combination with other agents. Objectives We aimed to investigate the synergistic effects of DPE and UVA at low-dose exposures in human–hamster hybrid (AL) cells and their underlying mechanisms. Methods We exposed exponentially growing AL cells to DPE and/or UVA radiation with or without reactive oxygen species (ROS) quenchers and then assayed the cells for survival, mutation induction, apoptosis, and micronucleus generation. In addition, using a singlet oxygen (1O2) trapping probe, 2,2,6,6-tetramethyl-4-piperidone, coupled with electron paramagnetic resonance spectroscopy, we determined the production of 1O2. Results Treatment of AL cells with DPE + UVA induced significant cytotoxic and genotoxic damage. In contrast, we found no significant damage in cells treated with either UVA or DPE alone at the same doses. Mutation spectra of CD59− mutants showed that treatment with DPE + UVA easily induces multilocus deletions. Sodium azide significantly inhibited both cellular and DNA damage induced by DPE + UVA treatment, whereas other ROS inhibitors had little protecting effect. Furthermore, we found a significant increase of 1O2 in the cells that received DPE + UVA treatment. Conclusion These findings suggest that UVA activated the genotoxicity and cytotoxicity of DPE in mammalian cells and that 1O2 played an important role in these processes.


Research
The popularity of diesel engines has been steadily increasing recently because of fuel efficiency, longevity, high torque at highway speeds, safety, and economy concerns (Lloyd and Cackette 2001). Diesel exhaust emitted at ground level is generated during the com bustion process and consists of hundreds of organic and inorganic compounds in either gaseous or particulate phases (Kagawa 2002). The dominant pollutant in ambient air, die sel exhaust particles (DEPs) consist of inert carbonaceous cores with large surface areas. This property is ideal for the adsorption of trace transition metals and various organic sub stances, including polycyclic aromatic hydro carbons (PAHs), nitroaromatic hydrocarbons, quinine, and acids (McClellan 1987). Several national and international agencies have clas sified DEPs as a "potential" or "probable" human carcinogen [International Agency for Research on Cancer (IARC) 1989]. DEP expo sure causes DNA and chromosomal damage, including bulky DNA adducts, oxidized bases, deletions, and chromosomal aberrations, which may lead to a broad spectrum of mutations (DeMarini et al. 2004;Müller et al. 2004;Tsurudome et al. 1999). Recent evidence has demonstrated that DEP and diesel particle extracts (DPEs) are highly mutagenic to TA98 and TA100 Salmonella strains in the Ames test and induce a dosedependent increase in muta tion yield that is suppressed by the S9 mixture (DeMarini et al. 2004). However, results at the hypoxanthineguanine phosphoribosyl trans ferase (Hprt) locus in mammalian cells and the λ/lacI locus in transgenic mice varied in dif ferent studies (Dybdahl et al. 2004;Gu et al. 2005;Risom et al. 2003;Sato et al. 2000).
Ultraviolet (UV) radiation from sunlight, including UVA and UVB, is a major factor for causing skin aging, skin cancer, and cyto genetic damage in lung, bone marrow, and peripheral blood erythrocytes (Balansky et al. 2003;Placzek et al. 2004;Yin et al. 2001). UVB (280-320 nm) is directly absorbed by DNA and induces damage, such as cyclo butane pyrimidine dimers and pyrimidine (64)pyrimidone photoproducts (de Gruijl 2000;Ravanat et al. 2001), which may con tribute to the carcinogenicity of UVB. In contrast, UVA (320-400 nm), the major component of solar UV radiation, is consid ered to be less carcinogenic than UVB because the DNA absorption of UVA is extremely weak. However, recent evidence shows that UVA also induces various forms of DNA damage in the presence of either endogenous or exogenous photosensitizers, such as PAHs like benzo[a]pyrene (Kawanishi and Hiraku 2001;Ravanat et al. 2001). The toxicity of exposure to PAHs plus UV has been observed in laboratory animals (Cavalieri and Calvin 1971;Clark 1964;Stenbäck 1975) and vari ous cells (Kagan et al. 1989;Schirmer et al. 1998;Utesch et al. 1996). More recently, several groups have demonstrated that coex posure to PAHs and UVA significantly aug mented DNA damage, such as singlestrand breaks (Dong et al. 2000), doublestrand breaks (Toyooka et al. 2004, and the formation of 8hydroxy2´deoxyguanosine, (8oxodG; Mauthe et al. 1995), which are closely associated with mutation and carcino genesis. UV or sunlight exposure per se as well as in combination with other agents has been identified as a risk factor for various benign or malignant human diseases. For example, combined exposure to tobacco smoke and sunlight is associated with dysplastic and malignant lip lesions and squamous cell car cinoma of the skin (King et al. 1995;Vander Straten et al. 2001). In aquatic organisms, the toxicity of PAHs increased considerably when combined with UV radiation (Boese et al. 2000;Laycock et al. 2000;Nikkilä et al. 1999). Exposure of organic extracts of air particulates to sunlight was also found to lead to an increase in mutagenicity in a Salmonella assay (alKhodairy and Hannan 1997).
Diesel exhaust and UV radiation are two ubiquitous environmental factors. Individuals such as road maintenance workers and traf fic policemen are easily exposed to these two factors simultaneously in their daily lives. Although the exposure to either diesel exhaust or UVA radiation alone or in combination with other agents has been identified as essential risk factors for various benign or malignant human diseases (King et al. 1995;Morgan et al. 1997;Siegel et al. 2004;Vander Straten et al. 2001), the synergistic effects of DPE and UVA have not been clearly clari fied. In the present study, we focused on the cytotoxicity and genotoxicity of either DPE or UVA exposure alone or simultaneously in a human-hamster hybrid (A L ) cell line. Our results indicated the high risk of coexposure to diesel exhaust and sunlight, which might be mediated by singlet oxygen ( 1 O 2 ).

Materials and Methods
Cell culture. In this study, we used the A L cell line, a human-hamster hybrid formed by fusion of human fibroblasts and the gly2A mutant of Chinese hamster ovary (CHO) cells (Ueno et al. 1996). In addition to a stan dard set of CHOK1 chromosomes, these hybrid cells contain a single copy of human chromosome 11, which encodes several cell surface antigenic markers that render the cells sensitive to killing by specific monoclonal antibodies in the presence of rabbit serum complement (HPR, Inc., Denver, PA, USA). This cell line is sensitive in detecting mutagens that induce mostly large, multilocus deletions such as ionizing radiation, asbestos fibers, and certain heavy metals (Liu et al. 2005;Ueno et al. 1996;Xu et al. 2002). Because only a small segment of the human chromo some (11p15.5) is required for the viability of A L cells, mutations based on marker genes located in the human chromosome ranging in size up to 140 Mbp of DNA can be detected (Wilson et al. 1999).
DPE preparation. In this study, we used DPE [standard reference material (SRM) 1975], defined by the National Institute of Standards and Technology (NIST; Gaithersburg, MD, USA). SRM 1975 is a dichloromethane extract of the diesel particu late matter SRM 2975, which was generated by a forklift truck using an industrial diesel powered engine and collected under specifically designed heavyduty conditions (NIST 2000).
Exposure to DPE and UVA. Exponentially growing A L cells were trypsinized and replated in 30mmdiameter petri dishes at 1 × 10 5 cells/dish for 48 hr, and then treated with either DPE or UVA alone or in combination (DPE + UVA). In the DPE + UVA group, cells were pretreated with DPE in phosphate buffered saline (PBS) for 30 min and then irradiated with UVA. For UVA radiation, three UV lamps (BLEIT151, Spectronics Co., Westbury, New York, USA) with an emission wavelength peak at 365 nm were used to irra diate the cells. The culture plates were placed on a table that was 15 cm away from the UV lamps. During UV exposure, the dose rate was simultaneously measured by a radiometer (Photoelectric Instrument Factory of Beijing Normal University, Beijing, China) with a 365nm detector located the same distance as the culture plates from the UV source.
Determination of cytotoxicity. After treatment, cultures were washed with PBS, trypsinized, and replated into 100mmdiameter petri dishes for colony formation. The cultures were incubated for 7 days and then fixed with formaldehyde, stained with Giemsa, and the number of colonies was counted to determine the survival fraction (Xu et al. 2002). We defined the survival fraction as the plating effi ciency of treated group divided by the plating efficiency of the control group.
Quantification of mutations at the CD59 locus and analysis of mutant spectrum. After treatment, we replated cultures in T75 flasks and cultured them for 5-7 days. This expres sion period permits surviving cells to recover from the temporary growth lag from DPE and UVA treatment and to multiply such that the progeny of the mutated cells no longer express lethal amounts of the CD59 surface antigen. To determine mutant fractions, 5 × 10 4 cells were plated into each of six 60mm dishes in a total of 2 mL of growth medium as previously described (Xu et al. 2002). The cultures were incubated for 2 hr to allow for cell attach ment, after which 0.2% CD59 antiserum and 1.5% (vol/vol) freshly thawed complement were added to each dish. The cultures were further incubated for 7-8 days, and then they were fixed, stained, and the number of CD59 − mutants scored. Controls included identical sets of dishes containing antiserum alone, complement alone, or neither agent. We cal culated mutant fractions as the number of sur viving colonies divided by the total number of cells plated after correction for any nonspecific killing due to complement alone.
CD59mutants were isolated by cloning and expanded in culture as previously described (Hei et al. 1998). To ensure that all mutants analyzed were independently generated, we isolated only one and, occasionally, no more than two wellseparated mutants per dish for analysis. We chose five marker genes located on either the short arm (WT, PTH, CAT, RAS) or the long arm (APO-A1) of human chromo some 11 for multiplex polymerase chain reac tion (PCR) because of their mapping positions relative to the CD59 gene and the availability of PCR primers for the coding regions of these genes. PCR amplifications were performed for 30 cycles using a DNA thermal cycle model 480 (PerkinElmer/Cetus, Waltham, MA, USA) in 20 µL reaction mixture containing 0.2 µg of the EcoRIdigested DNA sample in 1× Stoffel fragment buffer, all four deoxyribo nucleotide triphosphates (each at 0.2 mM), 3 mM MgCl 2 , 0.2 mM each primer, and 2 U Stoffel fragment enzyme. Each PCR cycle consisted of denaturation at 94°C for 1 min, annealing at 55°C for 1 min, and extension at 72°C for 1 min. After the last cycle, we incu bated samples at 72°C for an additional 20 min, electrophoresed them on 3% agarose gels, and stained them with ethidium bromide.
Apoptosis assay. For detection of apop tosis, we stained cells with Hoechst stain (Kishikawa et al. 2008). After treatment, cells were cultured for 24 hr and then rinsed with PBS twice, and fixed in a 2% paraformalde hyde solution for 15 min at room tempera ture, and rinsed again three times with PBS. Then the cells were stained with Hoechst 33342 (Sigma, St. Louis, MO, USA) at a final concentration of 5 µg/mL for 20 min at room temperature. We assayed apoptosis under an Olympus 1X71 fluorescence microscope (Olympus, Tokyo, Japan) and considered cells with shrunken, dense morphology and a fragmented nucleus to be apoptotic cells.
Determination of micronucleus formation. We measured the frequency of micronucleus (MN) formation with the cytokinesisblock technique developed by Fenech and Morley (1986). Briefly, 30 min after treatment, cells were trypsinized and replated into 30mm petri dishes at a density of 1 × 10 4 cells. After incubation for 4-6 hr, the growth medium was changed with the medium containing 2.5 µg/mL cytochalasin B (Sigma) and further incubated for 28 hr. Cells were then rinsed with PBS once, and fixed in a 9:1 solution of methanol:acetic acid for 20 min, and rinsed twice with water. The fixed cells were stained with 0.01% (wt/vol) acridine orange for 4 min before observation. MN in the binucleated cells were assayed under an Olympus 1X71 fluorescence microscope and identified mor phologically using the criteria of Fenech (1993). At least 1,000 binucleated cells were scored in each experiment for each data point to measure frequency of MN induction.
Effects of reactive oxygen species quenchers on toxicity of DPE + UVA. We treated exponentially growing A L cells with 20 mM sodium azide (NaN 3 ; Sigma), 500 U/mL superoxide dismutase (SOD; Sigma), 20 mM mannitol (Sigma), 500 U/mL catalase (CAT; Sigma), or 1% dimethyl sulfoxide (DMSO; Sigma) with or without concurrent treatment with DPE for 30 min, and then irradiated them with UVA. Then the survival fraction and MN induction were tested as described above. The dose of reactive oxygen species (ROS) quenchers used in the present study was nontoxic and nonmutagenic.

Electron paramagnetic resonance (EPR) detection of 4-O-TEMPO.
To detect 1 O 2 , we used the trap probe 2,2,6,6tetra methyl4 piperidone hydrochloride (TEMP; purity of 95%). This probe, which has been shown to be volume 117 | number 3 | March 2009 • Environmental Health Perspectives specific for 1 O 2 detection (Zang et al. 1995), reacts with 1 O 2 to yield a stable nitroxide radical 4oxo2,2,6,6tetra methylpiperidine Noxyl (4OTEMPO), having a known threeline EPR spectrum. TEMP (Sigma; 0.05 M) or the stable radical 2,2,6,6tetra methyl piperidineNoxyl (TEMPO; 10 -6 M; Sigma) was added to cells 30 min before UVA radia tion and the culture medium was collected immediately after radiation. Samples in 25µL capillaries inserted into 4mm quartz tubes were used for EPR analysis. EPR spectra were recorded at room temperature on a JEOL JESFA 200 EPR spectrometer (JEOL, Tokyo, Japan). The measurements were repeated at least three times for each sample. We set the microwave source of the EPR at 9.0 GHz and the power at 3.0 mW. Modulation frequency and modu lation amplitude were 100 kHz and 0.1 mT, respectively. The time constant was 0.3 sec, and scan time was 120 sec. The relative signal intensity of 4OTEMPO is represented by dividing the ratio of the 4OTEMPO sig nal intensity of the treated group by that of the control group.
Data analysis. All values were expressed as means ± SD. We tested significant differ ences at the p < 0.01 level using analysis of variance followed by Dunnett ttests or two tailed Student ttests.

Lethality of DPE and UVA in A L cells. The survival fractions of A L cells treated with DPE
(10-20 µg/mL) and/or UVA (0.2-1.0 J/cm 2 ) were determined by colony formation assay. The normal plating efficiency of A L cells used in the present study was about 80%. As shown in Figure 1, single treatment with DPE (20 µg/mL) or UVA (1.0 J/cm 2 ) slightly changed the survival fractions of A L cells. However, with the cotreatment of DPE and UVA, the survival fractions of A L cells showed a dose dependent decrease. For example, at doses of 10 µg/mL DPE + 1.0 J/cm 2 UVA, 20 µg/mL DPE + 0.5 J/cm 2 UVA, and 20 µg/mL DPE + 1.0 J/cm 2 UVA, the survival fractions were sig nificantly decreased to 54.87 ± 16.6%, 44.6 ± 8.97%, and 18.43 ± 1.56%, respectively, com pared with the untreated group (p < 0.01).
Mutation frequencies at CD59 gene and mutant spectra. The average background mutant fraction of A L cells was about 67 ± 27 mutants per 10 5 survivors. As shown in Figure 2, the mutation fractions induced by DPE (20 µg/mL) or UVA (0.5 J/cm 2 ) alone were 73 ± 27 and 73 ± 20 mutants per 10 5 survivors, respectively. However, with the cotreatment of DPE and UVA, the mutation fractions of A L cells showed a dosedependent increase. For example, at doses of 20 µg/mL DPE + 0.5 J/cm 2 UVA and 20 µg/mL DPE + 1.0 J/cm 2 UVA, the mutation yield at the CD59 locus was dramatically increased to 161 ± 41% and 177 ± 27% (p < 0.01).
To compare the type and size of mutations either of spontaneous origin or induced by DPE + UVA treatment, multiplex PCR and primer sequences for five marker genes (WT, PTH, CAT, RAS, and APO-A1) were used as previously described (Hei et al. 1998). As shown in Table 1 and Figure 3, most spon taneous CD59mutants (21 of 30, 70.0%) showed no detectable changes in any of the marker genes examined. In contrast, only 4 of 30 (13.3%) of mutants derived from cells exposed to DPE (20 µg/mL) + UVA (0.5 J/ cm 2 ) retained all the marker genes examined, whereas 26 of 30 (86.7%) of the mutants had lost at least one additional marker, which included 8 of 30 (26.7%) that lost the proxi mal APO-A1 located on the long arm of the chromosome. These results indicated that DPE + UVA easily induced multilocus deletions. Figure 4 shows the percentage of cells with apoptotic morphology after treatments. In control A L cells, 5.9 ± 2.3% were apoptotic. The apopto sis fractions induced by treatment with either DPE (20 µg/mL) or UVA (0.5 J/cm 2 ) alone were 7.0 ± 1.0% and 6.9 ± 1.6%, respec tively. However, when the cells were treated with DPE (20 µg/mL) + UVA (0.5 J/cm 2 ), the fraction of apoptotic cells was remarkably increased, from 5.9 ± 2.3% to 28.0 ± 6.1% (p < 0.01). Figure 5 shows the fractions of binucleated cells with MN. The average fraction of cells with MN in the control group was 1.92 ± 0.25%. The MN fractions induced by DPE (20 µg/mL) or UVA (0.5 J/cm 2 ) alone were 2.01 ± 0.25% and 2.24 ± 0.58%, respectively. A significant increase of MN induction was observed in the DPE (20 µg/mL) + UVA (0.5 J/cm 2 ) treat ment group that was more than twice as high as in the controls (p < 0.01).

Effects of ROS quenchers on survival fraction and MN induction.
We used NaN 3 , CAT, SOD, mannitol, and DMSO to deter mine the role of ROS in the DPE + UVAinduced cytotoxicity and genotoxicity. As shown in Figure 6, CAT, SOD, mannitol, or DMSO had little protective effect on DPE (20 µg/mL) + UVA (0.5 J/cm 2 ) caused cytotoxicity and genotoxicity. NaN 3 , a specific 1 O 2 scaven ger, effectively protected cells from DPE + UVA-induced cell damage and DNA damage. In the presence of NaN 3 , the survival fraction of A L cells treated with DPE + UVA increased from 49.13 ± 8.00% to 91.37 ± 1.81% (p < 0.01; Figure 6A), and MN induction in A L cells significantly decreased from 4.7 ± 0.4% to 2.5 ± 0.4% (p < 0.01; Figure 6B). Figure 7A, 4OTEMPO triplet spectra increased in cells treated with DPE (20 µg/ mL) + UVA (0.5 J/cm 2 ), and NaN 3 (20 mM) significantly reduced this signal. DPE alone and UVA radiation alone did not nota bly change the signal of 4OTEMPO. As shown in Figure 7B, relative signal intensity of 4OTEMPO was significantly increased in cells treated with DPE + UVA (p < 0.01), by about 1.1fold higher than the untreated group. In addition, NaN 3 efficiently decreased the relative signal intensity of 4OTEMPO induced by DPE and UVA to nearly the same as the control group.

Discussion
Both diesel exhaust and UV radiation are ubiq uitous in the environment. Most Ames tests have demonstrated that DEPs and DPEs are mutagenic and are closely related to the gen eration of ROS (DeMarini et al. 2004;Pohjola et al. 2003;Tsurudome et al. 1999). At doses greater than 100 µg/mL, Li and colleagues found that the organic extracts of DEPs were able to generate ROS and induce cell death and apoptosis in macrophages (Hiura et al. 1999;Li et al. 2002). Our previous work also demonstrated the mutagenicity of DEPs in mammalian cells (Bao et al. 2007). However, there is limited evidence on the effects of UVA on the genotoxicity of DPE, especially at lowdose exposures. In the present study in human-hamster hybrid (A L ) cells, we found that coexposure of DPE and UVA radiation severely decreased survival fractions and greatly enhanced cellular apoptosis, mutation fractions, and MN induction even though the treatments of cells with either UVA or DPE alone induced minimal cytotoxicity and genotoxicity. UVA or diesel exhaust exposure alone is most likely to cause singlebase substitutions or less frequent insertions, deletions, and multiplebase sub stitutions/deletions, respectively (Besaratinia et al. 2004;Sato et al. 2000). By analyzing the mutation spectrum on chromosome 11 of A L cells, we found that treatment with DPE + UVA tended to cause multilocus deletions, which is similar to the DNA damage produced by ionizing radiation, suggesting that exposure to DPE + UVA has a risk of cancer similar to that of ionizing radiation (Hei et al. 1997). This result might indicate the different muta genic mechanisms between single treatment by DPE/UVA and their coexposure. DEPs are heterogeneous, consisting of more than 450 different organic compounds, including xenobiotics such as PAHs, halo genated aromatic hydrocarbons, and redox active quinones (Athanasiou et al. 1987;Li et al. 2002;Whong et al. 1981). Some of the individual components are cytotoxic and mutagenic in mammalian cells, but most are promutagens that require activation to elec trophilic metabolites to exert their mutagenic or carcinogenic effects (Xue and Warshawsky 2005). Some studies have demonstrated an increase in directacting mutagens in air sam ples collected in the summer months, which suggests that photochemical reactions might activate air samples (e.g., DPE) to be more mutagenic (Casellas et al. 1995;CluniesRoss et al. 1996). Photoirradiation has been found to enhance both mutagenicity and cytotox icity of chemicals, such as azido analogues of amsacrine and other 9anilinoacridines (Iwamoto et al. 1992), whereas other studies have shown that an exposure to nearUV light converted the promutagens, including PAHs, into directacting mutagens (Barnhart and Cox 1980;de Wiest et al. 1982). These find ings suggest that sunlight, especially UV, as a major modifying factor played an essential role in the adverse health effects induced by chemical pollutions, such as PAH, DPE, and organic extracts of urban particulates. In the present study, treatment with UVA and DPE alone or UVA radiation followed by DPE exposure (data not shown) had slight toxic effects on A L cells, which indicates that DPE might be photosensitized by UVA, leading to an enhancement of cellular and genomic damage in mammalian cells. . Mutational spectra of CD59 mutants either of spontaneous origin or from cells exposed to DPE (20 µg/mL) + UVA (0.5 J/cm 2 ), determined by multiplex PCR. Each line represents the spectrum for a single, independent mutant. Blank spaces indicate missing markers. There are two possible ways to activate the organic compounds of DPE, such as PAHs, to become toxic and carcinogenic. One way is metabolic activation. Metabolic products, such as diol epoxides and diones, are highly carcino genic and induce covalent DNA adducts and oxidative DNA lesions (Ohnishi and Kawanishi 2002;Sims et al. 1974). Metabolic activated xenobiotic in DPE can also exert stimulatory or toxic effects via the generation of ROS (Ichinose et al. 1997;Kumagai et al. 1997;Park et al. 1996;Pinkus et al. 1996). Another way is pho toactivation. It is possible that after absorbance of UVA energy, xenobiotic molecules in DPE, especially PAHs, are elevated from the ground state to an excited state. The excited PAHs can react directly with biological molecules (type I) or can react with tripletstate oxygen to form excited singletstate oxygen (major) or other ROS (minor; type II) . 1 O 2 has been shown to play an impor tant role in cellular and DNA damage induced by coexposure to PAHs and UVA (Ibuki et al. 2002;Liu et al. 1999;). 1 O 2 can produce singlestrand breaks in cellfree DNA and oxidative DNA base modifications (Schulz et al. 2000;Yang et al. 1999). These DNA lesions may inevitably contribute to the mutation induction and MN formation. Using NaN 3 , which is widely used as an efficient 1 O 2 quencher (Li et al. 2001;Sparrow et al. 2002Sparrow et al. , 2003, we found that the decrease in survival fraction and MN generation by DPE + UVA exposure were effectively inhibited, whereas the ROS quenchers CAT, SOD, mannitol, and DMSO had little effect. Furthermore, using a 1 O 2 trapping probe, TEMP, coupled with EPR spectroscopy, we found enhanced production of 1 O 2 in the DPE + UVA exposure group, but not in the groups treated with DPE or UVA alone. These results indicate that photoactive production of 1 O 2 is mainly involved in the process of UVA activated toxicity of DPE in mammalian cells.

Spontaneous mutants DPE + UVA
In summary, our study provides direct evi dence of the augmented cytotoxicity and geno toxicity of DPE activated by UVA through the photoactive production of 1 O 2 . Because increasing amounts of diesel exhaust have been released into the environment and the depletion of ozone layer has led to the more UV exposure for humans, it is important to determine whether diesel exhaust may syner gize with UVA radiation to amplify genetic damage. The underlying mechanisms of these synergistic effects need to be further elucidated both in vitro and in vivo.