A Case of Congenital Pulmonary Vein Stenosis with Secondary Post-Capillary Pulmonary Hypertension and Left Sided Congestive Heart Failure in a Cat.

A five-month-old, 3.8 kg intact male Maine coon cat presented for dyspnea characterized by increased respiratory effort in addition to open-mouth breathing. Thoracic radiographs showed pectus excavatum, enlarged cardiac silhouette, and generalized interstitial patterns. Echocardiography revealed normal left atrial (LA) and left ventricular dimensions. A large tubular structure, suspected to be a distended pulmonary vein (PV), was identified as draining into the LA. Severe eccentric and concentric right ventricular hypertrophy and paradoxical septal motion were noted. Based on Doppler echocardiography, both pulmonary venous and pulmonary artery pressure was severely elevated. Clinical, radiographic, and echocardiographic abnormalities were hypothesized to result from pulmonary vein stenosis (PVS), causing severely elevated pulmonary venous pressures and resulting in clinical signs of left-sided congestive heart failure (L-CHF) and severe post-capillary pulmonary hypertension (Pc-PH). The prognosis for good quality of life was assessed as poor, and the owner elected euthanasia. Necropsy confirmed the presence of PVS with severe dilation of the PVs draining all but the left cranial lung lobe. All lung lobes except the left cranial lobe had increased tissue density and a mottled cut surface. This case report shows that, in rare cases, both L-CHF and Pc-PH may be present without LA enlargement. To the authors' knowledge, this is the first report on PVS in veterinary medicine.

Reduction of hazardous chemicals in Swedish preschool dust through article substitution actions.

Consumer goods and building materials present in the preschool environment can be important sources of hazardous chemicals, such as plasticizers, bisphenols, organophosphorus and brominated flame retardants, poly- and perfluoroalkyl substances, which may pose a health risk to children. Even though exposure occurs via many different pathways, such as food intake, inhalation, dermal exposure, mouthing of toys etc., dust has been identified as a valuable indicator for indoor exposure. In the present study, we evaluate the efficiency of product substitution actions taken in 20 Swedish preschools from the Stockholm area to reduce the presence of hazardous substances in indoor environments. Dust samples were collected from elevated surfaces in rooms where children have their everyday activities, and the concentrations found were compared to the levels from a previous study conducted in 2015 at the same preschools. It was possible to lower levels of hazardous substances in dust, but their continued presence in the everyday environment of children was confirmed since bisphenol A, restricted phthalates and organophosphate esters were still detectable in all preschools. Also, an increase in the levels of some of the substitutes for the nowadays restricted substances was noted; some of the alternative plasticizers to phthalates, such as DEHA and DEHT, were found with increased concentrations. DINP was the dominant plasticizer in preschool dust with a median concentration of 389 µg/g, while its level was significantly (p = 0.012) higher at 716 µg/g in preschools with polyvinyl chloride (PVC) flooring. PBDEs were now less frequently detected in dust and their levels decreased 20% to 30%. This was one of the few times that PFAS were analyzed in preschool dust, where 6:2 diPAP was found to be most abundant with a median concentration of 1140 ng/g, followed by 6:2 PAP 151 ng/g, 8:2 diPAP 36 ng/g, N-Et-FOSAA 18 ng/g, PFOS 12 ng/g, PFOA 7.7 ng/g and PFNA 1.1 ng/g. In addition, fluorotelomer alcohols were detected in 65-90% of the samples. Children's exposure via dust ingestion was evaluated using intermediate and high daily intake rates of the targeted chemicals and established health limit values. In each case, the hazard quotients (HQs) were < 1, and the risk for children to have adverse health effects from the hazardous chemicals analyzed in this study via dust ingestion was even lower after the product substitution actions were taken in preschools.

Homologous recombination mediates cellular resistance and fraction size sensitivity to radiation therapy.

PURPOSE: Cellular sensitivity to radiotherapy total dose and fraction size is strongly influenced by DNA double strand break (DSB) repair. Here, we investigate response to radiotherapy fraction size using CHO cell lines deficient in specific DNA repair pathways in response to radiation induced DNA double strand breaks (DSB). EXPERIMENTAL DESIGN: We irradiated CHO cell lines, AA8 (WT), irs1SF (XRCC3-), V3-3 (DNA-PKcs-) and EM9 (XRCC1-) with 16 Gy in 1 Gy daily fractions over 3 weeks or 16 Gy in 4 Gy daily fractions over 4 days, and studied clonogenic survival, DNA DSB repair kinetics (RAD51 and 53BP1 foci staining) and cell cycle profiles (flow cytometry). RESULTS: In response to fractionated radiotherapy, wild-type and DNA repair defective cells accumulated in late S/G2 phase. In cells proficient in homologous recombination (HR), accumulation in S/G2 resulted in reduced sensitivity to fraction size and increased cellular resistance (clonogenic survival). Sensitivity to fraction size was also lost in NHEJ-defective V3-3 cells, which likely rely on functional HR. By contrast, HR-defective irs1SF cells, with functional NHEJ, remained equally sensitive to fractionation throughout the 3-week treatment. CONCLUSIONS: The high fidelity of HR, which is independent of induced DNA damage level, is postulated to explain the low fractionation sensitivity and cellular resistance of cells in S/G2 phase. In conclusion, our results suggest that HR mediates resistance to fractionated radiotherapy, an observation that may help future efforts to improve radiotherapy outcome.

CHK1 activity is required for continuous replication fork elongation but not stabilization of post-replicative gaps after UV irradiation.

Ultraviolet (UV)-induced DNA damage causes an efficient block of elongating replication forks. The checkpoint kinase, CHK1 has been shown to stabilize replication forks following hydroxyurea treatment. Therefore, we wanted to test if the increased UV sensitivity caused by the unspecific kinase inhibitor caffeine--inhibiting ATM and ATR amongst other kinases--is explained by inability to activate the CHK1 kinase to stabilize replicative structures. For this, we used cells deficient in polymerase η (Polη), a translesion synthesis polymerase capable of properly bypassing the UV-induced cis-syn TT pyrimidine dimer, which blocks replication. These cells accumulate gaps behind progressing replication forks after UV exposure. We demonstrate that both caffeine and CHK1 inhibition, equally retards continuous replication fork elongation after UV treatment. Interestingly, we found more pronounced UV-sensitization by caffeine than with the CHK1 inhibitor in clonogenic survival experiments. Furthermore, we demonstrate an increased collapse of replicative structures after caffeine treatment, but not after CHK1 inhibition, in UV-irradiated cells. This demonstrates that CHK1 activity is not required for stabilization of gaps induced during replication of UV-damaged DNA. These data suggest that elongation and stabilization of replicative structures at UV-induced DNA damage are distinct mechanisms, and that CHK1 is only involved in replication elongation.

Homologous recombination repairs secondary replication induced DNA double-strand breaks after ionizing radiation.

Ionizing radiation (IR) produces direct two-ended DNA double-strand breaks (DSBs) primarily repaired by non-homologous end joining (NHEJ). It is, however, well established that homologous recombination (HR) is induced and required for repair of a subset of DSBs formed following IR. Here, we find that HR induced by IR is drastically reduced when post-DNA damage replication is inhibited in mammalian cells. Both IR-induced RAD51 foci and HR events in the hprt gene are reduced in the presence of replication polymerase inhibitor aphidicolin (APH). Interestingly, we also detect reduced IR-induced toxicity in HR deficient cells when inhibiting post-DNA damage replication. When studying DSB formation following IR exposure, we find that apart from the direct DSBs the treatment also triggers formation of secondary DSBs peaking at 7-9 h after exposure. These secondary DSBs are restricted to newly replicated DNA and abolished by inhibiting post-DNA damage replication. Further, we find that IR-induced RAD51 foci are decreased by APH only in cells replicating at the time of IR exposure, suggesting distinct differences between IR-induced HR in S- and G2-phases of the cell cycle. Altogether, our data indicate that secondary replication-associated DSBs formed following exposure to IR are major substrates for IR-induced HR repair.

The PCNA pseudogenes in the human genome.

BACKGROUND: The proliferating cell nuclear antigen (PCNA) is a key protein in the eukaryotic DNA replication and cell proliferation. Following the cloning and characterisation of the human PCNA gene, the question of the existence of pseudogenes in the human genome was raised. FINDINGS: In this short communication we summarise the existing information about the PCNA pseudogenes and critically assess their status. CONCLUSIONS: We propose the existence of at least four valid PCNA pseudogenes, PCNAP1, PCNAP2, LOC392454 and LOC390102. We would like to recommend assignment of a name for LOC392454 as "proliferating cell nuclear antigen pseudogene 3" (alias PCNAP3) and a name for LOC390102 as "proliferating cell nuclear antigen pseudogene 4" (alias PCNAP4). We prompt for more critical evaluation of the existence of a PCNA pseudogene, designated as PCNAP.

CK2 phosphorylation of XRCC1 facilitates dissociation from DNA and single-strand break formation during base excision repair.

CK2 phosphorylates the scaffold protein XRCC1, which is required for efficient DNA single-strand break (SSB) repair. Here, we express an XRCC1 protein (XRCC1(ckm)) that cannot be phosphorylated by CK2 in XRCC1 mutated EM9 cells and show that the role of this post-translational modification gives distinct phenotypes in SSB repair and base excision repair (BER). Interestingly, we find that fewer SSBs are formed during BER after treatment with the alkylating agent dimethyl sulfate (DMS) in EM9 cells expressing XRCC1(ckm) (CKM cells) or following inhibition with the CK2 inhibitor 2-dimethylamino-4,5,6,7-tetrabromo-1H-benzimidazole (DMAT). We also show that XRCC1(ckm) protein has a higher affinity for DNA than wild type XRCC1 protein and resides in an immobile fraction on DNA, in particular after damage. We propose a model whereby the increased affinity for DNA sequesters XRCC1(ckm) and the repair enzymes associated with it, at the repair site, which retards kinetics of BER. In conclusion, our results indicate that phosphorylation of XRCC1 by CK2 facilitates the BER incision step, likely by promoting dissociation from DNA.

DNA repair and replication influence the number of mutations per adduct of polycyclic aromatic hydrocarbons in mammalian cells.

Polycyclic aromatic hydrocarbons (PAH) are an important class of environmental contaminants many of which require metabolic activation to DNA-reactive bay or fjord region diolepoxides (DE) in order to exert their mutagenic and carcinogenic effects. In this study, the mutagenicity of the bay region diolepoxides (+)-anti-7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE) and (±)-anti-1,2-dihydroxy-3,4-epoxy-1,2,3,4-tetrahydrodibenzo[a,h]anthracene (DBADE) and the fjord region diolepoxides (±)-anti-11,12-dihydroxy-13,14-epoxy-11,12,13,14-tetrahydrodibenzo[a,l]-pyrene (DBPDE) and (±)-anti-3,4-dihydroxy-1,2-epoxy-1,2,3,4-tetrahydrobenzo[c]-phenanthrene (BPhDE) was compared in nucleotide excision repair (NER) proficient and deficient hamster cell lines. The (32)P-postlabelling assay was applied to analyze DNA adduct levels and the Hprt gene mutation assay for monitoring mutations. Previously, we found that the mutagenicity per adduct was four times higher for DBPDE compared to BPDE in NER proficient cells. In these same cells, the mutagenicity of DBADE and BPhDE adducts was now found to be significantly lower compared to that of BPDE. In NER deficient cells the highest mutagenicity per adduct was found for BPDE and there was a tenfold and fivefold difference when comparing the BPDE data with the DBADE and BPhDE data, respectively. In order to investigate to what extent the mutagenicity of the different adducts in NER proficient cells was influenced by repair or replication bypass, we measured the overall NER incision rate, the rate of adduct removal, the rate of replication bypass and the frequency of induced recombination in the Hprt gene. Since NER turned out to be an important pathway for the yield of mutations, we further analyzed the role of transcription coupled NER versus global genome NER. However, our data demonstrate that neither of these pathways seems to be the sole factor determining the mutation frequency of the four PAH-DE and that the differences in the repair efficiency of these compounds could not be related to the presence of a bay or fjord region in the parent PAH.

Structural requirements for mutation formation from polycyclic aromatic hydrocarbon dihydrodiol epoxides in their interaction with food chemopreventive compounds.

Chinese hamster V79 cells were used to investigate the protective effect of four known antimutagens present in food, chlorophyllin (CHL), ellagic acid (EA), epigallocathechingallate (EGCG) and benzylisothiocyanate (BITC), against potent mutagenic polycyclic aromatic hydrocarbon diol epoxides (PAH-DE) derived from benzo[a]pyrene (BP), dibenzo[a,h]anthracene (DBA), dibenzo[a,l]pyrene (DBP), and benzo[c]phenanthrene (BPh) known to be deposited on crops from polluted ambient air or formed during food processing. As fjord-region PAH-DE are more toxic and mutagenic than bay-region PAH-DE, we adjusted the concentrations of PAH-DE to induce approximately the same levels of adducts. The studies were performed using an assay indicating toxicity in terms of reduced cell proliferation together with the V79 Hprt assay for monitoring mutant frequencies. CHL significantly increased the survival and showed a protective effect against the mutagenicity of all PAH-DE. A significant protective effect of EA was found towards the mutagenicity of BPDE, DBPDE and BPhDE and with EGCG for BPDE and BPhDE. BITC had a slight positive effect on the mutagenicity of DBADE and BPhDE. Taken together, a novel and unexpected finding was that the antimutagenic activity could differ as much as by a factor of 7 towards four carcinogenic PAH metabolites being relatively similar in structure and genotoxic activity.

Both replication bypass fidelity and repair efficiency influence the yield of mutations per target dose in intact mammalian cells induced by benzo[a]pyrene-diol-epoxide and dibenzo[a,l]pyrene-diol-epoxide.

Mutations induced by polycyclic aromatic hydrocarbons (PAH) are expected to be produced when error-prone DNA replication occurs across unrepaired DNA lesions formed by reactive PAH metabolites such as diol epoxides. The mutagenicity of the two PAH-diol epoxides (+)-anti-7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE) and (+/-)-anti-11,12-dihydroxy-13,14-epoxy-11,12,13,14-tetrahydrodibenzo[a,l]pyrene (DBPDE) was compared in nucleotide excision repair (NER) proficient and deficient hamster cell lines. We applied the (32)P-postlabelling assay to analyze adduct levels and the hprt gene mutation assay for monitoring mutations. It was found that the mutagenicity per target dose was 4 times higher for DBPDE compared to BPDE in NER proficient cells while in NER deficient cells, the mutagenicity per target dose was 1.4 times higher for BPDE. In order to investigate to what extent the mutagenicity of the different adducts in NER proficient cells was influenced by repair or replication bypass, we measured the overall NER incision rate, the rate of adduct removal, the rate of replication bypass and the frequency of induced recombination in the hprt gene. The results suggest that NER of BPDE lesions are 5 times more efficient than for DBPDE lesions, in NER proficient cells. However, DBPDE adducts block replication more efficiently and also induce 6 times more recombination events in the hprt gene than adducts of BPDE, suggesting that DBPDE adducts are, to a larger extent, bypassed by homologous recombination. The results obtained here indicate that the mutagenicity of PAH is influenced not only by NER, but also by replication bypass fidelity. This has been postulated earlier based on results using in vitro enzyme assays, but is now also being recognized in terms of forward mutations in intact mammalian cells.

Caffeine delays replication fork progression and enhances UV-induced homologous recombination in Chinese hamster cell lines.

The ability to bypass DNA lesions encountered during replication is important in order to maintain cell viability and avoid genomic instability. Exposure of mammalian cells to UV-irradiation induces the formation of DNA lesions that stall replication forks. In order to restore replication, different bypass mechanisms are operating, previously named post-replication repair. Translesion DNA synthesis is performed by low-fidelity polymerases, which can replicate across damaged sites. The nature of lesions and of polymerases involved influences the resulting frequency of mutations. Homologous recombination represents an alternative pathway for the rescue of stalled replication forks. Caffeine has long been recognized to influence post-replication repair, although the mechanism is not identified. Here, we found that caffeine delays the progress of replication forks in UV-irradiated Chinese hamster cells. The length of this enhanced delay was similar in wild-type cells and in cell deficient in either homologous recombination or nucleotide excision repair. Furthermore, caffeine attenuated the frequency of UV-induced mutations in the hprt gene, whereas the frequency of recombination, monitored in this same gene, was enhanced. These observations indicate that in cells exposed to UV-light, caffeine inhibits the rescue of stalled replication forks by translesion DNA synthesis, thereby causing a switch to bypass via homologous recombination. The biological consequence of the former pathway is mutations, while the latter results in chromosomal aberrations.

A method to monitor replication fork progression in mammalian cells: nucleotide excision repair enhances and homologous recombination delays elongation along damaged DNA.

The capacity to rescue stalled replication forks (RFs) is important for the maintenance of cell viability and genome integrity. Here, we have developed a novel method for monitoring RF progression and the influence of DNA lesions on this process. The method is based on the principle that each RF is expected to be associated with a pair of single-stranded ends, which can be analyzed by employing strand separation in alkali. This method was applied to examine the rate of RF progression in Chinese hamster cell lines deficient in ERCC1, which is involved in nucleotide excision repair (NER), or in XRCC3, which participates in homologous recombination repair, following irradiation with ultraviolet (UV) light or exposure to benzo(a)pyrene-7,8-diol-9,10-epoxide (BPDE). The endpoints observed were cell survival, NER activity, formation of double-strand breaks and the rate of RF progression. Subsequently, we attempted to explain our observation that cells deficient in XRCC3 (irs1SF) exhibit enhanced sensitivity to UV radiation and BPDE. irs1SF cells demonstrated a capacity for NER that was comparable with wild-type AA8 cells, but the rate of RF progression was even higher than that for the wild-type AA8 cells. As expected, cells deficient in ERCC1 (UV4) showed no NER activity and were hypersensitive to both UV radiation and BPDE. The observation that cells deficient in NER displayed a pronounced delay in RF progression indicates that NER plays an important role in maintaining fork progression along damaged DNA. The elevated rate of RF progression in XRCC3-deficient cells indicates that this protein is involved in a time-consuming process which resolves stalled RFs.