Photomutagenicity of anhydroretinol and 5,6-epoxyretinyl palmitate in mouse lymphoma cells

Abstract

Retinyl palmitate (RP) is frequently used as an ingredient in cosmetics and other retail products. We previously reported that, under UVA light irradiation, RP is facilely decomposed into multiple products, including anhydroretinol (AR) and 5,6-epoxyretinyl palmitate (5,6-epoxy-RP). We also determined that combined treatment of mouse lymphoma cells with RP and UVA irradiation produced a photomutagenic effect. In this study, we evaluated the photomutagenicity of AR and 5,6-epoxy-RP, in L5178Y/Tk+/- mouse lymphoma cells. Treatment of cells with AR or 5,6-epoxy-RP alone at 10 and 25 microg/mL for 4 h did not show a positive mutagenic response. However, because these doses did not induce the required amount of cytotoxicity for mouse lymphoma assay, we are unable to determine whether or not these two compounds are mutagenic. Treatment of cells with 1-25 microg/mL AR or 5,6-epoxy-RP under UVA light (315-400 nm) for 30 min (1.38 mW/cm2) produced a synergistic photomutagenic effect. At 10 microg/mL (37.3 microM) AR with UVA exposure, the mutant frequency (MF) was about 3-fold higher than that for UVA exposure alone, whereas the MF for 25microg/mL (46.3microM) of 5,6-epoxy-RP + UVA was approximately 2-fold higher than that for UVA exposure alone. Compared with previous results for RP + UVA treatment, the potency of the induced phototoxicity and photomutagenicity was AR > RP > 5,6-epoxy-RP. To elucidate the underlying photomutagenic mechanism, we examined the loss of heterozygosity (LOH) at four microsatellite loci spanning the entire chromosome 11 for mutants induced by AR or 5,6-epoxy-RP. Most mutants lost the Tk+ allele, and more than 70% of the chromosome damage extended to 38 cM in chromosome length. AR + UVA induced about twice as many mutants that lost all four microsatellite markers from the chromosome 11 carrying the Tk+ allele as RP + UVA or 5,6-epoxy-RP + UVA. These results suggest that two of RP's photodecomposition products are photomutagenic in mouse lymphoma cells, causing events that affect a large segment of the chromosome.

Figure 1.

Structures and abbreviations for retinol, retinyl palmitate, 5,6-epoxyretinyl palmitate, and anhydroretinol.

Figure 2.

Comparison of photomutagenicity of RP (▼), AR (●), and 5,6-epoxy-RP (O) in mouse lymphoma cells. The cells were treated with different concentrations of compounds in conjunction with UVA irradiation of 2.48 J/cm² (1.38 mW/cm² for 30 min). The data points for AR + UVA and 5,6-epoxy-RP + UVA represent the mean of two independent experiments from Table 1. The RP + UVA data were from our previous results (19). UVA alone was the mean of mutant frequencies generated from all experiments with UVA treatment alone. The data for RP, AR, and 5,6-epoxy-RP were adjusted according to the mean of UVA alone as the origin of regression lines.

Figure 3.

(A) Comparison of the percentage of mutational types for all (large and small) colonies produced in mouse lymphoma cells treated with control, UVA, RP + UVA, AR + UVA, and 5,6-epoxy-RP + UVA. The data for control, UVA alone, and RP + UVA are from our previous study (19). (B) Different types of mutations shown by histograms indicating the range of LOH, at the same scale as used in the ideogram of mouse chromosome 11. The loci that were analyzed for LOH (Tk1, D11Mit42, D11Mit29, and D11Mit74) are marked. The ruler in centimorgans indicates the distance from the top of the chromosome. Type 1 mutation, non-LOH; type 2, LOH at Tk locus only; type 3, LOH extending to D11Mit42 (about 6 cM); type 4, LOH extending to D11Mit29 (about 38 cM); and type 5, LOH extending to the top of chromosome 11.

Figure 4.

Proposed activation pathways leading to chromosome mutations induced by photoirradiation of RP and two of its photodecomposition products, AR and 5,6-epoxy-RP.