* Division of Genetic and Reproductive Toxicology, National Center for Toxicological Research, FDA, Jefferson, Arkansas 72079; Division of Biochemical Toxicology, National Center for Toxicological Research, FDA, Jefferson, Arkansas 72079
1To whom correspondence should be addressed at (Tao Chen) HFT-130, 3900 NCTR Road, Jefferson, AR 72079. Fax: 8705437682. E-mail: tchen{at}nctr.fda.gov; or (Peter P. Fu) HFT-110, 3900 NCTR Road, Jefferson, AR 72079. Fax: 8705437136. E-mail: pfu{at}nctr.fda.gov.
Received May 24, 2005; accepted June 27, 2005
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ABSTRACT |
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Key Words: retinyl palmitate; UVA; mouse lymphoma assay; photomutagenicity; loss of heterozygosity; mutant frequency.
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INTRODUCTION |
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The UV radiation that reaches the surface of the earth from the sun is divided into two wavebands, UVA (between 320 and 400 nm) and UVB (between 290 and 320 nm), with visible and infrared light at longer wavelengths (FDA, 1999). Retinyl palmitate shows maximum UV-visible absorption at 326 nm (Tee, 1992
). Therefore, UVA may play an important role in the photobiological activity of RP. UVA itself is a carcinogen and mutagen. UVA can induce cutaneous squamous cell carcinoma in mice (de Gruijl et al., 1993
; Matsui and DeLeo, 1991
) and increase melanoma risk in humans (Swerdlow et al., 1988
; Walter et al., 1990
; Westerdahl et al., 2000
). UVA radiation increases mutant frequency (MF) and micronucleus formation (Phillipson et al., 2002
).
In our previous study on the effects of UVA on RP (Cherng et al., 2005), we determined that photoirradiation of RP and its photodecomposition products generated reactive oxygen species (ROS) and resulted in lipid peroxidation. Generally, ROS and lipid peroxidation produce oxidative damage to DNA and result in mutations. Retinyl palmitate and its photodecomposition products in combination with UVA exposure, however, were not mutagenic in Salmonella typhimurium tester strains TA98, TA100, TA102, and TA104 in the presence or absence of S9 activation enzymes and were not photomutagenic in S. typhimurium TA102 when irradiated with UVA. Oxidative damage frequently results in mutations with large chromosomal alterations (Harrington-Brock et al., 2003
; Rothfuss et al., 2000
; Singh et al., 2005
; Takeuchi et al., 1997
), which cannot be detected by the S. typhimurium gene mutation test system. Therefore, we hypothesized that mutations generated by the UVA irradiation of RP may be due primarily to large-scale chromosome damage.
To test our hypothesis, we have used the L5178Y mouse lymphoma assay (MLA) to evaluate whether RP can interact with UVA to produce a photomutagenic effect. Unlike the microbial assays, the MLA detects a broad spectrum of genetic damage, including both point mutations and chromosomal mutations. This feature makes the MLA particularly useful for detecting mutational events that result from oxidative DNA damage. We also examined the types of mutations induced by UVA and RP + UVA to understand the underlying mechanism of action for the photomutagenicity of RP in combination with UVA exposure.
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MATERIALS AND METHODS |
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Cell treatment with RP alone.
Retinyl palmitate was purchased from the Sigma (St. Louis, MO). The RP working solution (100x) was prepared just prior to use by dissolving it with anhydrous dimethyl sulfoxide (DMSO). The cells were suspended in 100-mm-diameter tissue culture dishes at a concentration of 6 x 106 cells in 10 ml of treatment medium. Because we focused on the photomutagenicity of RP by UVA, we did not test the mutagenic potential of RP more than 100 µg/ml. One hundred µl of the RP stock solutions at concentrations between 25 and 100 µg/ml were added to the medium, and the cells were incubated for 4 h at 37°C. In all cases, including negative controls (DMSO only) and positive controls [0.1 µg/ml 4-nitroquinoline-1-oxide (4-NQO)], the final concentration of DMSO in the medium was 1%. After treatment, the cells were centrifuged and washed once with fresh medium and then resuspended in growth medium at a density of 3 x 105 cells /ml in 25-cm2 cell culture flasks to begin phenotypic expression.
Cell treatment with UVA alone.
The light box, a custom-made 4-lamp unit uses UVA lamps (National Biologics, Twinsburg, OH) (Cherng et al., 2005). The irradiance of light was determined with an Optronics OL754 Spectroradiometer (Optronics Laboratories, Orlando, FL), and the light dose was routinely measured with a Solar Light PMA-2110 UVA detector (Solar Light Inc., Philadelphia, PA). The maximum emission of the UVA was between 340 and 355 nm. The light intensities at wavelengths below 320 nm (UVB light) and above 400 nm (visible light) were about two orders of magnitude lower than the maximum at 340355 nm. The 6 x 106 cells in 10 ml of treatment medium in 100-mm diameter tissue culture dish were exposed to UVA light at a rate of 82.8 mJ/cm2/min for various times from 5 to 45 min.
Cell treatment with pre-irradiated RP.
The pre-irradiated RP was obtained by irradiating the stock RP solution with 82.8 mJ/cm2/min UVA for 30 min immediately before adding the solution to the cell culture to make a final concentration of pre-irradiated RP at 25 µg/ml. The cells were treated with the UVA pre-irradiated RP for 4 h at 37°C.
Cell treatment with RP and UVA.
Cells were treated with different concentrations (125 µg/ml) of RP and concomitantly exposed to 82.8 mJ/cm2/min UVA for 30 min. The treated cultures were then incubated with the RP at 37°C for an additional 3.5 h.
The Tk microwell mutation assay.
Mutant selection was performed as described previously (Chen and Moore, 2004). Briefly, the cells were counted and the densities were adjusted using fresh medium at approximately 1 and 2 days after exposure. For mutant enumeration, trifluorothymidine (TFT, 3 µg/ml) was added to the cell culture in cloning medium, and cells were seeded into four 96-well flat-bottom microtiter plates using 200 µl per well and a density of 2000 cells/well. For the determination of plating efficiency, approximately 1.6 cells were aliquoted in 200 µl per well into two 96-well flat-bottom microtiter plates. All plates were incubated at 37°C in a humidified incubator with 5% CO2 in air. After 11 days of incubation, colonies were counted and mutant colonies were categorized as small or large. Small colonies are defined as those smaller than 25% of the diameter of the well. Mutant frequencies were calculated using the Poisson distribution. The induced MF was obtained by subtracting the background MF (negative control MF) from the MF in the treatment group. Cytotoxicity was measured using relative total growth (RTG), which includes a measure of growth during treatment, expression, and cloning (Chen and Moore, 2004
).
Detection of loss of heterozygosity (LOH) at the Thymidine kinase (Tk1) and other three microsatellite loci spanning the entire chromosome 11 for Tk mutants.
Mutant cells were directly taken from TFT-selected plates. Only mutants obtained from the 30-min UVA exposure, the concomitant treatment of 25 µg/ml RP and UVA, and the negative control were isolated and analyzed. The mutants from the RP treatment alone were not analyzed for LOH because RP was not mutagenic under the conditions used (see Results). The mutant cells were washed once with PBS by centrifugation, and cell pellets were quickly frozen and stored at 80°C. Genomic DNA was extracted by digesting the cells in lysis buffer (10 mM Tris-HCl [pH 7.5], 5 mM MgCl2, 1% [v/v] Triton X-100, 1% [v/v] Tween 20) with 200 µg/ml of proteinase K at 60°C for 90 min, followed by inactivation of proteinase K at 95°C for 10 min. The procedure for the polymerase chain reaction (PCR) analysis of LOH at the Tk locus was performed as previously described (Chen et al., 2002a). For PCR analysis of LOH at other loci (D11Mit42, D11Mit29, and D11Mit74 loci; Fig. 1), the amplification reactions were carried out in a total volume of 20 µl using 2x PCR master Mix (Promega, Madison, WI) and pairs of primers described previously (Singh et al., 2005
). The thermal cycling conditions were as follows: initial incubation at 94°C for 3 min, 40 cycles of 94°C denaturation for 30 s, 55°C annealing for 30 s, and 72°C extension for 30 s, and a final extension at 72°C for 7 min. The amplification products were scored for the presence of one band (indicating LOH) or two bands (retention of heterozygosity at the given locus) after 2% agarose gel electrophoresis.
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RESULTS |
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To determine the mutagenicity of the photodecomposition products of RP created by UVA irradiation, we pre-irradiated RP with 2.48 J/cm2 in the absence of cells. The pre-irradiated RP then was used to treat the cells at a concentration of 25 µg/ml. The MF for the pre-irradiated RP treatment was higher than that for 25 µg/ml RP treatment alone, but lower than that for 2.48 J/cm2 UVA treatment alone, although there were no statistically significant differences. The MF for the pre-irradiated RP treatment was significantly less than the concomitant cellular exposure of RP and UVA (Fig. 4).
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DISCUSSION |
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Retinyl palmitate alone, in concentrations of 25100 µg/ml, did not increase the MF over control (Fig. 3), which is consistent with our previous study on RP mutagenicity using the S. typhimurium mutation test system (Cherng et al., 2005). In the present study, the combined treatment of RP in concentrations of 125 µg/ml with a UVA dose of 2.48 J/cm2 resulted in a significant increase of MFs, in a dose-dependent manner. The induced MF (subtracting the MF of the negative control from the MF observed in the test culture) at the 25 µg/ml RP with UVA exposure was about threefold higher than that for UVA-irradiation alone (Table 1). This is different from our previous studies using the microbial mutation assay (Cherng et al., 2005
) in which RP treatment of S. typhimurium TA102 under UVA irradiation was not mutagenic. This inconsistency is most likely related to the fact that combined treatment of RP and UVA causes clastogenicity. Compounds acting primarily by a clastogenic mechanism induce detectable mutagenicity in the MLA but are only weakly mutagenic or nonmutagenic in the microbial assays (Chen et al., 2002b
).
In this study, most of the mutants from concomitant treatment of RP and UVA were small colony mutants (66%, at a dose of 25 µg/ml, a MF of 287 of a total MF of 437), whereas the percentage of small colony mutants in the negative control cultures was 46% (a MF of 26 of a total MF of 57). Also, LOH at the Tk locus occurred in 85% in large colony mutants and 100% in small colony mutants (a total of 94% overall) from RP + UVA treatment, indicating that the photomutagenicity of RP by UVA irradiation results from a clastogenic mode of action. In the MLA, compounds that induce point mutations result in a high proportion of large colony Tk mutants and little LOH at the Tk locus, whereas clastogens tend to result in a high proportion of small colony mutants and predominantly LOH at the Tk locus (Applegate et al., 1990; Chen et al., 2002a
, 2002b
; Harrington-Brock et al., 2003
). LOH is an important mutational event in tumorigenesis and is frequently observed in a variety of human cancers at loci that are tumor-suppressor genes. LOH can result from any of several mechanisms, including large deletions, mitotic recombination, and whole chromosome loss (Honma et al., 2001
). Generally, depending on the severity of DNA damage of the Tk mutants, LOH will also occur at other loci along chromosome 11 in addition to the Tk gene.
To determine whether RP under UVA irradiation produces mutations through its photodecomposition products or through short-lived products like ROS and lipid peroxides, we irradiated RP for 30 min in the absence of cells, and the resulting pre-irradiated RP reaction mixture then was used to treat cells. The MF for pre-irradiated RP treatment was significantly lower than that for the concomitant exposure to RP and UVA, and it was not significantly different from that for RP treatment or the DMSO control (Fig. 4). This is consistent with our previous results that RP and its identified photodecomposition products did not bind with DNA in the presence of microsomal metabolizing enzymes (Cherng et al., 2005). These results indirectly demonstrate that UVA irradiation of RP produces short-lived species like ROS that damage DNA and result in mutations.
Ultraviolet light causes DNA damage both directly and indirectly. Direct DNA damage is principally by UVB, forming cyclobutane pyrimidine dimers, pyrimidine-pyrimidones, and point mutations. Indirect DNA damage is principally caused by UVA-dependent photoactivation of organic compounds that generates short-lived species (Brendler-Schwaab et al., 2004). Our previous research has shown that irradiation of RP with UVA light can generate ROS and lipid peroxides (Cherng et al., 2005
). Photodynamic action results in the production of free radicals, including ROS. Because of their high reactivity, ROS can attack all cellular constituents, including proteins, nucleic acids, and lipids. Hydroxyl radicals can initiate a chain reaction that produces multiple lipid hydroperoxide molecules from a single initial event (Gutteridge and Halliwell, 1990
). The chain reaction is in effect amplifying the initial oxidative insult, and the resulting active oxygen species can damage DNA and produce mutations.
UVA itself induces genetic damage in cells via an oxidative stress mechanism (Drobetsky et al., 1995; Kamiya, 2003
; Phillipson et al., 2002
). In this study, the mutational spectra induced by UVA and RP + UVA are not significantly different (p = 0.448 in large colonies and p = 0.816 in small colonies; Table 2). This similarity suggests that RP + UVA induces mutations through the same mechanism as UVA, mainly oxidative DNA damage.
Chemicals that absorb UVA and visible light and generate ROS constitute the largest class of photosensitizers. After absorption of UVA light, RP potentially acts as a photosensitizer (Fu et al., 2003). The mutagenicity of photosensitizers probably results from oxidative radicals formed in the photosensitization reaction (Rerko et al., 1992
). Several studies using different oxidative agents provide evidence that oxidative DNA damage generated from these agents can lead to different types of mutations, with a large proportion of chromosome mutations that result mainly from chromosome breakage (Harrington-Brock et al., 2003
; Nakajima et al., 2002
; Rothfuss et al., 2000
; Takeuchi et al., 1997
). In a recent study on the mutagenicity of the lipid peroxidation product 4-hydroxynonenal (4-HNE) in mouse lymphoma cells (Singh et al., 2005
), we found that the major type of mutation was LOH extending to D11Mit42 (see Fig. 1 for the position), the same mutation as currently seen with RP + UVA treatment. The Tk mutants from cells exposed to either of these two compounds are about 60% this type of LOH mutation. A mechanistic pathway initiated by photoirradiation of RP with UVA light leading to induction of chromosome mutations has been suggested and is illustrated in Figure 6.
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NOTES |
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ACKNOWLEDGMENTS |
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The views presented in this article do not necessarily reflect those of the U.S. Food and Drug Administration.
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