Comparison of pulmonary deposition of nebulized 99m technetium-diethylenetriamine-pentaacetic acid through 3 inhalation devices in healthy dogs.

BACKGROUND: Inhalation treatment frequently is used in dogs and cats with chronic respiratory disease. Little is known however about the performance of delivery devices and the distribution of aerosolized drugs in the lower airways. OBJECTIVE: To assess the performance of 3 delivery devices and the impact of variable durations of inhalation on the pulmonary and extrapulmonary deposition of nebulized 99m technetium-diethylenetriamine-pentaacetic acid (99m Tc-DTPA). ANIMALS: Ten university-owned healthy Beagle dogs. METHODS: Prospective crossover study. Dogs inhaled the radiopharmaceutical for 5 minutes either through the Aerodawg spacer with a custom-made nose-muzzle mask, the Aerochamber spacer with the same mask, or the Aerodawg spacer with its original nose mask. In addition, dogs inhaled for 1 and 3 minutes through the second device. Images were obtained by 2-dimensional planar scintigraphy. Radiopharmaceutical uptake was calculated as an absolute value and as a fraction of the registered dose in the whole body. RESULTS: Mean (±SD) lung deposition for the 3 devices was 9.2% (±5.0), 11.4% (±4.9), and 9.3% (±4.6), respectively. Differences were not statistically significant. Uptake in pulmonary and extrapulmonary tissues was significantly lower after 1-minute nebulization, but the mean pulmonary/extrapulmonary deposition ratio (0.38 ± 0.27) was significantly higher than after 5-minute nebulization (0.16 ± 0.1; P = .03). No significant differences were detected after 3- and 5-minute nebulization. CONCLUSION AND CLINICAL IMPORTANCE: The performance of a pediatric spacer with a custom-made mask is comparable to that of a veterinary device. One-minute nebulization provides lower pulmonary uptake but achieves a better pulmonary/extrapulmonary deposition ratio than does 5-minute nebulization.

ASH1L histone methyltransferase regulates the handoff between damage recognition factors in global-genome nucleotide excision repair.

Global-genome nucleotide excision repair (GG-NER) prevents ultraviolet (UV) light-induced skin cancer by removing mutagenic cyclobutane pyrimidine dimers (CPDs). These lesions are formed abundantly on DNA wrapped around histone octamers in nucleosomes, but a specialized damage sensor known as DDB2 ensures that they are accessed by the XPC initiator of GG-NER activity. We report that DDB2 promotes CPD excision by recruiting the histone methyltransferase ASH1L, which methylates lysine 4 of histone H3. In turn, methylated H3 facilitates the docking of the XPC complex to nucleosomal histone octamers. Consequently, DDB2, ASH1L and XPC proteins co-localize transiently on histone H3-methylated nucleosomes of UV-exposed cells. In the absence of ASH1L, the chromatin binding of XPC is impaired and its ability to recruit downstream GG-NER effectors diminished. Also, ASH1L depletion suppresses CPD excision and confers UV hypersensitivity. These findings show that ASH1L configures chromatin for the effective handoff between damage recognition factors during GG-NER activity.

Chromatin remodeler CHD1 promotes XPC-to-TFIIH handover of nucleosomal UV lesions in nucleotide excision repair.

Ultraviolet (UV) light induces mutagenic cyclobutane pyrimidine dimers (CPDs) in nucleosomal DNA that is tightly wrapped around histone octamers. How global-genome nucleotide excision repair (GG-NER) processes CPDs despite that this chromatin arrangement is poorly understood. An increased chromatin association of CHD1 (chromodomain helicase DNA-binding 1) upon UV irradiation indicated possible roles of this chromatin remodeler in the UV damage response. Immunoprecipitation of chromatin fragments revealed that CHD1 co-localizes in part with GG-NER factors. Chromatin fractionation showed that the UV-dependent recruitment of CHD1 occurs to UV lesions in histone-assembled nucleosomal DNA and that this CHD1 relocation requires the lesion sensor XPC (xeroderma pigmentosum group C). In situ immunofluorescence analyses further demonstrate that CHD1 facilitates substrate handover from XPC to the downstream TFIIH (transcription factor IIH). Consequently, CHD1 depletion slows down CPD excision and sensitizes cells to UV-induced cytotoxicity. The finding of a CHD1-driven lesion handover between sequentially acting GG-NER factors on nucleosomal histone octamers suggests that chromatin provides a recognition scaffold enabling the detection of a subset of CPDs.

Global-genome Nucleotide Excision Repair Controlled by Ubiquitin/Sumo Modifiers.

Global-genome nucleotide excision repair (GG-NER) prevents genome instability by excising a wide range of different DNA base adducts and crosslinks induced by chemical carcinogens, ultraviolet (UV) light or intracellular side products of metabolism. As a versatile damage sensor, xeroderma pigmentosum group C (XPC) protein initiates this generic defense reaction by locating the damage and recruiting the subunits of a large lesion demarcation complex that, in turn, triggers the excision of aberrant DNA by endonucleases. In the very special case of a DNA repair response to UV radiation, the function of this XPC initiator is tightly controlled by the dual action of cullin-type CRL4(DDB2) and sumo-targeted RNF111 ubiquitin ligases. This twofold protein ubiquitination system promotes GG-NER reactions by spatially and temporally regulating the interaction of XPC protein with damaged DNA across the nucleosome landscape of chromatin. In the absence of either CRL4(DDB2) or RNF111, the DNA excision repair of UV lesions is inefficient, indicating that these two ubiquitin ligases play a critical role in mitigating the adverse biological effects of UV light in the exposed skin.

Xeroderma pigmentosum group C sensor: unprecedented recognition strategy and tight spatiotemporal regulation.

The cellular defense system known as global-genome nucleotide excision repair (GG-NER) safeguards genome stability by eliminating a plethora of structurally unrelated DNA adducts inflicted by chemical carcinogens, ultraviolet (UV) radiation or endogenous metabolic by-products. Xeroderma pigmentosum group C (XPC) protein provides the promiscuous damage sensor that initiates this versatile NER reaction through the sequential recruitment of DNA helicases and endonucleases, which in turn recognize and excise insulting base adducts. As a DNA damage sensor, XPC protein is very unique in that it (a) displays an extremely wide substrate range, (b) localizes DNA lesions by an entirely indirect readout strategy, (c) recruits not only NER factors but also multiple repair players, (d) interacts avidly with undamaged DNA, (e) also interrogates nucleosome-wrapped DNA irrespective of chromatin compaction and (f) additionally functions beyond repair as a co-activator of RNA polymerase II-mediated transcription. Many recent reports highlighted the complexity of a post-translational circuit that uses polypeptide modifiers to regulate the spatiotemporal activity of this multiuse sensor during the UV damage response in human skin. A newly emerging concept is that stringent regulation of the diverse XPC functions is needed to prioritize DNA repair while avoiding the futile processing of undamaged genes or silent genomic sequences.

Chromatin retention of DNA damage sensors DDB2 and XPC through loss of p97 segregase causes genotoxicity.

DNA damage recognition subunits such as DDB2 and XPC protect the human skin from ultraviolet (UV) light-induced genome instability and cancer, as demonstrated by the devastating inherited syndrome xeroderma pigmentosum. Here we show that the beneficial DNA repair response triggered by these two genome caretakers critically depends on a dynamic spatiotemporal regulation of their homeostasis. The prolonged retention of DDB2 and XPC in chromatin, because of a failure to readily remove both recognition subunits by the ubiquitin-dependent p97/VCP/Cdc48 segregase complex, leads to impaired DNA excision repair of UV lesions. Surprisingly, the ensuing chromosomal aberrations in p97-deficient cells are alleviated by a concomitant downregulation of DDB2 or XPC. Also, genome instability resulting from an excess of DDB2 persisting in UV-irradiated cells is prevented by concurrent p97 overexpression. Our findings demonstrate that DNA damage sensors and repair initiators acquire unexpected genotoxic properties if not controlled by timely extraction from chromatin.

DNA quality control by a lesion sensor pocket of the xeroderma pigmentosum group D helicase subunit of TFIIH.

BACKGROUND: Nucleotide excision repair is a versatile DNA repair reaction that removes bulky adducts generated by environmental mutagens such as the UV spectrum of sunlight or chemical carcinogens. Current multistep models of this excision repair pathway accommodate its broad substrate repertoire but fail to explain the stringent selectivity toward damaged nucleotides among excess native DNA. To understand the mechanism of bulky lesion recognition, we postulated that it is necessary to analyze the function of xeroderma pigmentosum group D (XPD) protein beyond its well-known role in the unwinding of double-stranded DNA. RESULTS: We engineered two new XPD mutants (Y192A and R196E), involving amino acid substitutions near its central protein pore, that confer defective DNA repair despite normal transcription. In situ fluorescence-based protein dynamics studies in living cells demonstrated that both new mutants were unable to recognize DNA damage and failed to form stable associations with lesion sites. However, when their biochemical properties were tested in the framework of an archaeal protein homolog, they both retained ATPase and DNA-unwinding activity. The outstanding difference versus the wild-type control was that their directional 5'-3' translocation along DNA was not stopped by a bulky lesion, and moreover, they were unable to build long-lived demarcation complexes at damaged sites. CONCLUSIONS: By uncoupling for the first time the unwinding and damage sensor activities of XPD, we describe an unprecedented genome quality control process whereby a recognition pocket near the central DNA helicase pore scans individual substrate strands to capture base adducts.