Detection of the small oligonucleotide products of nucleotide excision repair in UVB-irradiated human skin

Abstract

UVB radiation results in the formation of potentially mutagenic photoproducts in the DNA of epidermal skin cells. In vitro approaches have demonstrated that the nucleotide excision repair (NER) machinery removes UV photoproducts from DNA in the form of small (~30-nt-long), excised, damage-containing DNA oligonucleotides (sedDNAs). Though this process presumably takes place in human skin exposed to UVB radiation, sedDNAs have not previously been detected in human skin. Using surgically discarded human skin, we have optimized the detection of the sedDNA products of NER from small amounts of human epidermal tissue ex vivo within minutes of UVB exposure and after UVB doses that normally lead to minimal erythema. Moreover, sedDNA generation was inhibited by treatment of skin explants with spironolactone, which depletes the epidermis of the essential NER protein XPB to mimic the skin of xeroderma pigmentosum patients. Time course experiments revealed that a partially degraded from of the sedDNAs could be readily detected even 12 hours following UVB exposure, which indicates that these repair products are relatively stable in human skin epidermis. Together, these data suggest that sedDNA detection may be a useful assay for determining how genetic, environmental, and other factors influence NER activity in human skin epidermis and whether abnormal sedDNA processing contributes to photosensitive skin disorders.

Topical Treatment of Human Skin and Cultured Keratinocytes with High-Dose Spironolactone Reduces XPB Expression and Induces Toxicity

“…As we have previously reported ( Choi et al., 2020 ; Kemp et al., 2019 ), the addition of 20 μM SP (10 nmol) twice over 2 days to cell culture medium maintained below small, 8 mm human skin explants reduced epidermal XPB protein expression by 50 to >95%, depending on the individual skin sample ( Figure 1 a). In contrast, the addition of 20 μM SP topically on the skin did not affect XPB protein levels (data not shown).…”

“…Using cultured keratinocytes (KCs) in vitro, we found that both SP and Can induced replication stress and apoptosis but that these responses are not correlated with a loss of XPB protein. Coupled with previous research showing that SP-mediated loss of XPB negatively influences responses to UVR in the skin ( Choi et al., 2020 ; Kemp et al., 2019 ), these results indicate that high concentrations of SP and Can induce toxicity in epidermal skin cells through an XPB-independent mechanism.…”

“…Recent studies have indicated a role for CDK7, the E3 ubiquitin ligase SCF FBXL18 ( Ueda et al., 2019 ), and the ubiquitin-selective segregase VCP/p97 ( Chauhan et al., 2021 ) in this process. SP-mediated loss of XPB has been reported to occur in a variety of cell types in vitro ( Alekseev et al., 2014 ; Elinoff et al., 2018 ; Kemp et al., 2019 ; Lacombe et al., 2016 ; Martella et al., 2020 ; Szalat et al., 2018 ), in human skin explants ex vivo ( Choi et al., 2020 ; Kemp et al., 2019 ), and in rat lungs in vivo ( Elinoff et al., 2018 ). The consequences of SP treatment on XPB protein stability, DNA repair, and transcription have all appeared to be specific to SP and not specific to either its metabolites or the related MR antagonist eplerenone ( Gabbard et al., 2020 ).…”

Spironolactone and XPB: An Old Drug with a New Molecular Target

“…The mechanism by which SP induces the loss of XPB protein was initially investigated by Alekseev et al and was shown to occur rapidly after addition of SP to the culture medium and to be independent of XPB mRNA levels [3]. Several other groups have confirmed these general findings in different cell types in vitro and in tissues ex vivo and in vivo [37,48,62,64,65]. The effect of SP on XPB protein levels was also reversible, as XPB protein levels could be fully restored within a few hours of withdrawing the drug in vitro.…”

“…The observation that SP promotes the degradation of the critical DNA repair protein XPB might indicate that patients taking the drug orally or topically are at an elevated risk of skin cancer development in regions of sun-exposed skin. Though in vitro studies with keratinocytes and ex vivo studies with skin explants showed that SP can deplete these cells and tissues of XPB, inhibit UV photoproduct removal, and increase mutagenesis [37,65], epidemiological studies have found no evidence that SP use is associated with increased cancer risk of any cancer type, including in the skin [78]. There are several possible explanations for these observations, including the fact that orally administered SP is rapidly metabolized (Figure 3) by the liver into compounds [79] such as canrenone and 7α-thiomethylspironolactone (TMS) (Figure 2) that do not affect XPB protein levels [37,64].…”