Cell-intrinsic melanin fails to protect melanocytes from ultraviolet-mutagenesis in the absence of epidermal melanin.

Melanin is a free-radical scavenger, antioxidant, and broadband absorber of ultraviolet (UV) radiation which protects the skin from environmental carcinogenesis. However, melanin synthesis and UV-induced reactive melanin species are also implicated in melanocyte genotoxicity. Here, we attempted to reconcile these disparate functions of melanin using a UVB-sensitive, NRAS-mutant mouse model, TpN. We crossed TpN mice heterozygous for an inactivating mutation in Tyrosinase to produce albino and black littermates on a C57BL/6J background. These animals were then exposed to a single UVB dose on postnatal day three when keratinocytes in the skin have yet to be melanized. Approximately one-third (35%) of black mice were protected from UVB-accelerated tumor formation. However, melanoma growth rates, tumor mutational burdens, and gene expression profiles were similar in melanomas from black and albino mice. Skin from albino mice contained more cyclobutane pyrimidine dimer (CPD) positive cells than black mice 1-h post-irradiation. However, this trend gradually reversed over time with CPDs becoming more prominent in black than albino melanocytes at 48 h. These results show that in the absence of epidermal pigmentation, melanocytic melanin limits the tumorigenic effects of acute UV exposure but fails to protect melanocytes from UVB-induced mutagenesis.

Aspirin Protects against UVB-Induced DNA Damage through Activation of AMP Kinase.

The anti-inflammatory and chemopreventive activities of aspirin (ASA) may be mediated through its cyclooxygenase inhibitor function. We have previously shown that ASA can protect against UVR-induced skin inflammation and DNA damage; however, the role of inflammation in UV-induced DNA damage and the mechanism underlying ASA protection are poorly characterized. Using immunodeficient NOD scid gamma mice and immunocompetent C57BL/6 mice treated with immune cell‒depleting antibodies, we found that inflammation was not required for UVB-induced 8-oxoguanine and cyclobutane pyrimidine dimers in vivo. Unlike ASA, neither its immediate metabolite salicylate nor the cyclooxygenase inhibitor indomethacin reduced UVB-induced 8-oxoguanine or cyclobutane pyrimidine dimers in melanocyte Melan-a or keratinocyte HaCat cells in vitro. Moreover, addition of prostaglandin E2 did not reverse the protective effect of ASA on UVB-treated cells. Phosphorylation of the 5' AMP protein kinase, observed in ASA-treated cells, could be blocked by the 5' AMP protein kinase inhibitor compound C. Compound C or 5' AMP protein kinase knockdown partially reduced ASA-mediated protection against UVB-induced DNA damage. Finally, injection of compound C partially reversed the protective effect of ASA on UVB-treated mouse skin in vivo. These studies suggest that ASA confers protection against UVB-induced DNA damage through the activation of 5' AMP protein kinase rather than through cyclooxygenase inhibition.

A Randomized Double-blind Placebo-controlled Trial of Oral Aspirin for Protection of Melanocytic Nevi Against UV-induced DNA Damage.

DNA damage plays a role in ultraviolet (UV)-induced melanoma. We previously showed that aspirin (ASA) can suppress prostaglandin-E2 (PGE2) and protect melanocytes from UV-induced DNA damage in mice, and suggested that taking ASA before acute sun exposure may reduce melanoma risk. We conducted a prospective randomized placebo-controlled trial to determine if orally administered ASA could suppress PGE2 in plasma and nevi and protect nevi from UV-induced DNA damage. After obtaining plasma and determining the minimal erythemal dose (MED) in 95 subjects at increased risk for melanoma, they were randomized to receive a daily dose of placebo, 81 mg ASA, or 325 mg ASA, in double-blind fashion for one month. After this intervention, one nevus was irradiated (dose = 1 or 2 MED) using a solar simulator. One day later, MED was re-determined, a second plasma sample was obtained, and the UV-irradiated nevus and an unirradiated nevus were removed. ASA metabolites were detected in the second plasma sample in subjects in the ASA arms. There were no significant differences in the pre- and post-intervention MED between those patients receiving ASA and placebo. Significantly reduced PGE2 levels were detected in plasma (second vs. first samples) and in nevi (both unirradiated and UV-treated) in subjects receiving ASA compared to placebo. Comparing UV-treated nevi from the ASA and placebo cohorts, however, did not reveal significant reductions in CD3-cell infiltration or 8-oxoguanine and cyclobutane pyrimidine dimers. Thus ASA did not effectively protect nevi from solar-simulated UV-induced inflammation and DNA damage under the conditions examined. PREVENTION RELEVANCE: Despite promising rationale, ASA at conventional dosing was not able to protect nevi against UV-induced DNA damage under the conditions examined.See related Spotlight, p. 71.

Aspirin Protects Melanocytes and Keratinocytes against UVB-Induced DNA Damage In Vivo.

UVR promotes skin cancer through multiple mechanisms, including induction of inflammation, oxidative stress, and DNA damage such as 8-oxoguanine and cyclobutane pyrimidine dimers. We investigated whether the anti-inflammatory activities of aspirin (acetylsalicylic acid [ASA]) could protect against UVB-induced DNA damage and skin carcinogenesis. ASA reduced UVB-induced 8-oxoguanine and cyclobutane pyrimidine dimers in Melan-A melanocytes and HaCaT keratinocytes. Skin from UVB-irradiated C57BL/6 mice receiving 0.4 mg ASA daily by gavage exhibited less inflammation, fewer sunburn cells, and reduced 8-oxoguanine lesions than skin from irradiated control animals. ASA similarly reduced UVB-induced sunburn cells, 8-oxoguanine, and cyclobutane pyrimidine dimer lesions in skin of melanoma-prone TN61R mice, and this was associated with decreased prostaglandin E2 in plasma and skin. These effects of ASA, however, did not delay melanoma onset in TN61R mice exposed to a single neonatal dose of UVB. In SKH1-E mice prone to squamous cell carcinoma, ASA reduced plasma and skin prostaglandin E2 levels and indices of UVB-induced DNA damage and delayed squamous cell carcinoma onset induced by chronic UVB. These results indicate that ASA can protect against UVB-induced inflammation in skin and reduce UVB-induced DNA damage in both melanocytes and keratinocytes. These effects translated into greater chemopreventive efficacy for UVB-induced squamous cell carcinoma than melanoma mouse models.

ASA Suppresses PGE2 in Plasma and Melanocytic Nevi of Human Subjects at Increased Risk for Melanoma.

Potential anti-inflammatory and anticarcinogenic effects of aspirin (ASA) may be suitable for melanoma chemoprevention, but defining biomarkers in relevant target tissues is prerequisite to performing randomized controlled chemoprevention trials. We conducted open-label studies with ASA in 53 human subjects with melanocytic nevi at increased risk for melanoma. In a pilot study, 12 subjects received a single dose (325 mg) of ASA; metabolites salicylate, salicylurate, and gentisic acid were detected in plasma after 4-8 h, and prostaglandin E2 (PGE2) was suppressed in both plasma and nevi for up to 24 h. Subsequently, 41 subjects received either 325 or 81 mg ASA (nonrandomized) daily for one week. ASA metabolites were consistently detected in plasma and nevi, and PGE2 levels were significantly reduced in both plasma and nevi. Subchronic ASA dosing did not affect 5" adenosine monophosphate-activated protein kinase (AMPK) activation in nevi or leukocyte subsets in peripheral blood, although metabolomic and cytokine profiling of plasma revealed significant decreases in various (non-ASA-derived) metabolites and inflammatory cytokines. In summary, short courses of daily ASA reduce plasma and nevus PGE2 and some metabolites and cytokines in plasma of human subjects at increased risk for melanoma. PGE2 may be a useful biomarker in blood and nevi for prospective melanoma chemoprevention studies with ASA.

Aspirin Suppresses PGE2 and Activates AMP Kinase to Inhibit Melanoma Cell Motility, Pigmentation, and Selective Tumor Growth In Vivo.

There are conflicting epidemiologic data on whether chronic aspirin (ASA) use may reduce melanoma risk in humans. Potential anticancer effects of ASA may be mediated by its ability to suppress prostaglandin E2 (PGE2) production and activate 5'-adenosine monophosphate-activated protein kinase (AMPK). We investigated the inhibitory effects of ASA in a panel of melanoma and transformed melanocyte cell lines, and on tumor growth in a preclinical model. ASA and the COX-2 inhibitor celecoxib did not affect melanoma cell viability, but significantly reduced colony formation, cell motility, and pigmentation (melanin production) in vitro at concentrations of 1 mmol/L and 20 µmol/L, respectively. ASA-mediated inhibition of cell migration and pigmentation was rescued by exogenous PGE2 or Compound C, which inhibits AMPK activation. Levels of tyrosinase, MITF, and p-ERK were unaffected by ASA exposure. Following a single oral dose of 0.4 mg ASA to NOD/SCID mice, salicylate was detected in plasma and skin at 4 hours and PGE2 levels were reduced up to 24 hours. Some human melanoma tumors xenografted into NOD/SCID mice were sensitive to chronic daily ASA administration, exhibiting reduced growth and proliferation. ASA-treated mice bearing sensitive and resistant tumors exhibited both decreased PGE2 in plasma and tumors and increased phosphorylated AMPK in tumors. We conclude that ASA inhibits colony formation, cell motility, and pigmentation through suppression of PGE2 and activation of AMPK and reduces growth of some melanoma tumors in vivo This preclinical model could be used for further tumor and biomarker studies to support future melanoma chemoprevention trials in humans. Cancer Prev Res; 11(10); 629-42. ©2018 AACR.

Transverse Vaginal Septum With Secondary Infertility: A Rare Case.

STUDY OBJECTIVE: To demonstrate the technique of laparoscopic vaginal reconstruction in a rare case of mid-vaginal septum with secondary infertility. DESIGN: A step-by-step explanation of the technique using videos and pictures (Canadian Task Force classification IV). SETTING: Transverse vaginal septum is a rare condition, with an incidence of only 1 in 30,000 women. It is usually a congenital mullerian fusion defect; few cases of acquired septum have been reported. Roughly 40% of cases occur in the mid-vagina. Transverse vaginal septum typically presents with primary amenorrhea and hematocolpos. The goal of surgery is to create a patent vagina with restoration of fertility. The laparoscopic approach has proven superiority over the open technique. INTERVENTIONS: Laproscopic vaginal reconstruction was performed in a 24 year female with transverse vaginal septum. Dissection was done laproscopically up to mid vagina. Incision was given on vagina excising the septum followed by reconstruction. CONCLUSION: The laparoscopic approach to vaginal reconstruction avoids the abdominal incision, with its associated pain and possible adhesion formation. It also provides a better view for dissection. In this patient, a patent vagina was created in a single operation, with no postoperative dyspareunia, and fertility was restored.

Environmental interaction, additive and non-additive genetic variability is involved in the expression of tissue and whole-plant heat tolerance in upland cotton (Gossypium hirsutum. L)

Heat tolerance is measured at tissue level by cellular membrane thermostability (CMT) and at the whole plant level by the heat tolerance index (HTI). Eight upland cotton cultivars and 15 crosses were used to determine the type and extent of genetic variability associated with the expression of these traits between and within environments. Heat stress and non-stress conditions were used as the CMT environments and years for HTI. The wide variation in heterotic expression and combining ability effects observed for CMT and HTI suggest multigenic inheritance of these traits. Significant genetic variability across environments was evident but the traits were not highly heritable because of substantial environmental interaction. The available genetic variability included both additive and non-additive components, but the proportion of additive genetic variability was high for HTI. The parental cultivars CRIS-19 and CIM-448 were good donor parents for high CMT under heat-stressed conditions, and MNH-552 and N-Karishma under non-stressed conditions. Cultivar FH-634 was a good donor parent for HTI. The results show two types of general combining ability (GCA) inheritance among high CMT parents: positive GCA inheritance expressed by CRIS-19 in the presence of heat stress and MNH-552 and N-Karishma in the absence of heat stress; and negative GCA inheritance expressed by FH-900 in the presence of heat stress. It was also evident that genes controlling high CMT in cultivar CRIS-19 were different from those present in the MNH-552, N-Karishma and FH-900 cultivars. Similarly, among high HTI parents, FH-634 showed positive and CIM-443 negative GCA inheritance. No significant relationship due to genetic causes existed between tissue and whole plant heat tolerance, diminishing the likelihood of simultaneous improvement and selection of the two traits.