Gene expression of retinoic acid receptors and cellular retinoic acid-binding proteins in rhino and hairless mouse skin.

The rhino mouse comedolytic model and the hairless mouse photoaging model are established animal models for screening the in vivo activity of retinoids. However, the expression of the retinoic acid receptors (RARs) and cellular retinoic acid-binding proteins (CRABPs), known to regulate retinoid activity, is not completely understood in these mouse mutants. For this purpose, mRNA was isolated from rhino and hairless mouse skin and the gene expression of the RARs and CRABPs was measured by Northern blot hybridization. Results showed that RAR gamma was the predominantly expressed RAR in both mouse strains. Two isoforms of RAR gamma, RAR gamma 1 and RAR gamma 2, were detected with RAR gamma 1 being the more strongly expressed. RAR alpha was also detected, but to a lesser degree than RAR gamma. RAR beta expression was not detectable by our methodology. Additionally, topical treatment of these mice with 0.1% all-trans-retinoic acid (tRA) cream resulted in no significant alteration in the expression of the RAR genes. By contrast, CRABP-II was induced 2-4 fold by topical tRA treatment. CRABP-I, expressed to a lesser degree than CRABP-II, was not inducible. The relative expression of the RARs, CRABPs, and inducibility of CRABP-II by tRA in both rhino and hairless mouse skin paralleled that reported for human and mouse skin. These observations suggest that the altered phenotype observed in the rhino mouse most likely does not result from an altered expression level of these genes. The results also support these two animals as models for evaluating the therapeutic potential of retinoids.

Formation of 8-hydroxydeoxyguanosine within DNA of mouse keratinocytes exposed in culture to UVB and H2O2.

Exposure to UV light contributes to the development of skin cancer. The importance of reactive oxygen species in UV-radiation carcinogenesis has been recognized for some time and several associated DNA base modifications have been identified. In particular, 8-hydroxydeoxyguanosine (8-OHdG) has been well studied as an indicator of oxidative damage to calf thymus DNA exposed to a variety of oxygen-generating systems, including UV light. However, to date, few studies of 8-OHdG have been conducted in cell or animal systems and those in vitro investigations that studied UV exposure have used UVC (< 290 nm), not the UVB (290-320 nm) or UVA (320-400 nm) ranges to which organisms are exposed through sunlight. The objective of this study was to measure 8-OHdG formation in the DNA of cultured mouse keratinocytes exposed to UVB. Using HPLC with electrochemical detection, background levels of 8-OHdG were approximately 6 fmol/micrograms DNA in DNA isolated and digested to the nucleoside level. UVB induced 8-OHdG up to 100% above that for mock-treated cells at a dose of 630 mJ/cm2 (dose-response range: 210-630 mJ/cm2). UVB exposure at 630 mJ/cm2 combined with 5 mM H2O2 elevated 8-OHdG formation up to 280% above that in control cells, whereas H2O2 alone had no effect. These results suggest that factors which increase the generation of reactive oxygen species by UV light may be potent cofactors of UV-radiation carcinogenesis.

Elevated pentose cycle and glucuronyltransferase in daunorubicin-resistant P388 cells.

Anthracycline resistance of P388 daunorubicin-resistant cells cannot be accounted for merely by differences in drug uptake and retention; protection against intracellular drug was also indicated. Cytotoxicity of daunorubicin may be partially due to the formation of free radicals and reactive oxygen species (hydrogen peroxide, hydroxyl radical, singlet oxygen, and superoxide anion radical). Protection against free radicals and peroxides is largely dependent upon the availability of reduced glutathione, which in turn requires NADPH for its continual regeneration. Pentose phosphate cycle (also called hexose monophosphate shunt) is known to provide NADPH for maintenance of glutathione. Activities of the two NADPH-producing dehydrogenases of the cycle, glucose-6-phosphate and 6-phosphogluconate dehydrogenase, were 40% higher (P less than 0.05) and activity of the cycle in intact cells was 2-fold higher in the resistant than the sensitive cells. The cycle was as active in these cells as it is known to be in macrophages, indicating a very effective protection against oxidative stress, free radicals, and alkylating electrophiles. Elevated activity of the pentose phosphate pathway in drug-resistant cells can represent a mechanism of resistance against multiple structurally unrelated drugs. Efflux of daunorubicin may be aided by further metabolism to glucuronides. Daunorubicinol, a known active metabolite of daunorubicin, can be metabolized to a glucuronide by the cells and eliminated into the surrounding medium. Glucuronidation of daunorubicinol was evidenced by (a) release of daunorubicinol following glucuronidase hydrolysis of media from cell incubations with 1.8 microM daunorubicin and (b) production of radioactive glucuronide when cell homogenates were incubated with UDP-[14C]glucuronic acid plus daunorubicinol. Glucuronyltransferase activity with a broad substrate specificity was found in the cells. Using model substrates, 1-naphthol and o-aminophenol, it was determined that glucuronyltransferase activity was 4 times higher in daunorubicin-resistant than -sensitive P388 cells. Elevated glucuronyltransferase could contribute to daunorubicin and multidrug resistance.