Sister-chromatid exchanges induced by ultraviolet light in Bloom's syndrome fibroblasts

The relationships between the cytotoxic effect of ultraviolet light and the UV-induced sister-chromatid exchanges (SCEs) were compared among fibroblast cell strains from two unrelated Bloom's syndrome (BS) patients, one xeroderma pigmentosum (XP) patient belonging to complementation group A and two unrelated normal controls. The "net" induced SCEs as a function of UV fluence, obtained by subtracting spontaneous SCEs from observed SCEs, were much higher in both BS cells and XP group A cells than in normal cells. The relative efficiency of induced SCE, defined as the "net" induced SCEs as a function of surviving fraction after UV irradiation, was higher in BS cells than in normal and XP cells, and there was essentially no difference between XP and normal cells. These results imply that in addition to the extremely high frequency of spontaneous SCEs, the increased efficiency in UV induction of SCEs may reflect the intrinsic defect(s) in BS cells.     

Pyrimidine dimers are not the principal pre-mutagenic lesions induced in lambda phage DNA by ultraviolet light

Experiments were performed to examine the role of cyclobutyl pyrimidine dimers in the process of mutagenesis by ultraviolet (u.v.) light. Lambda phage DNA was irradiated with u.v. and then incubated with an Escherichia coli photoreactivating enzyme, which monomerizes cyclobutyl pyrimidine dimers upon exposure to visible light. The photoreactivated DNA was packaged into lambda phage particles, which were used to infect E. coli uvr- host cells that had been induced for SOS functions by ultraviolet irradiation. Photoreactivation removed most toxic lesions from irradiated phage, but did not change the frequency of induction of mutations to the clear-plaque phenotype. This implies that cyclobutyl pyrimidine dimers can be lethal, but usually do not serve as sites of mutations in the phage. The DNA sequences of mutants derived from photoreactivated DNA showed that almost two-thirds (16/28) were transitions, the same fraction found for u.v. mutagenesis without photoreactivation. These results show that in this system, the lesion inducing transitions (the major type of u.v.-induced mutation) is not the cyclobutyl pyrimidine dimer; a strong candidate for a mutagenic lesion is the Pyr(6-4)Pyo photoproduct. On the other hand, photoreactivation of SOS-induced host cells before infection with u.v.-irradiated phage reduced mutagenesis substantially. In this case, photoreversal of cyclobutyl dimers serves to reduce expression of the SOS functions that are required in the process of targeted u.v. mutagenesis.     

Mechanism of DNA strand breakage induced by photosensitized tetracycline-Cu(II) complex

Tetracyclines (TCs) in combination with Cu(II) ions exhibited significant DNA damaging potential vis a vis tetracyclines per se. Interaction of tetracyclines with DNA resulted in alkylation at N-7 and N-3 positions of adenine and guanine bases, and caused destabilization of DNA secondary structure. Significant release of acid-soluble nucleotides from tetracycline-modified DNA upon incubation with S(1) nuclease ascertained the formation of single stranded regions in the DNA. Also, the treatment of tetracycline-modified DNA with 0.1 and 0.5M NaOH resulted in 62 and 76% hydrolysis compared to untreated control. Comparative alkaline hydrolysis of DNA modified with tetracycline derivatives showed differential DNA damaging ability in the order as DOTC > DMTC > TC > OTC > CTC. Addition of Cu(II) invariably augmented the extent of tetracycline-induced DNA damage. The alkaline unwinding assay clearly demonstrated the formation of approximately six strand breaks per unit DNA at 1:10 DNA nucleotide/TC molar ratio in the presence of 0.1mM Cu(II) ions. At a similar Cu(II) concentration, a progressive transformation of covalently closed circular (CCC) (form-I) plasmid pBR322 DNA to forms-II and -III was noticed with increasing tetracycline concentrations. The results obtained with the free-radical quenchers viz. mannitol, thiourea, sodium benzoate and superoxide dismutase (SOD) suggested the involvement of reactive oxygen species in the DNA strand breakage. It is concluded that the tetracycline-Cu(II)-induced DNA damage occurs due to (i) significant binding of tetracycline and Cu(II) with DNA, (ii) methyl group transfer from tetracycline to the putative sites on nitrogenous bases, and (iii) metal ion catalyzed free-radical generation in close vicinity of DNA backbone upon tetracycline photosensitization. Albeit, the DNA alkylation and strand cleavage are repairable lesions, but any defect in the critical repair pathway may augment the damage accumulation and mutagenesis.     

Redundancy of mammalian Y family DNA polymerases in cellular responses to genomic DNA lesions induced by ultraviolet light

Short-wave ultraviolet light induces both mildly helix-distorting cyclobutane pyrimidine dimers (CPDs) and severely distorting (6–4) pyrimidine pyrimidone photoproducts ((6–4)PPs). The only DNA polymerase (Pol) that is known to replicate efficiently across CPDs is Polη, a member of the Y family of translesion synthesis (TLS) DNA polymerases. Phenotypes of Polη deficiency are transient, suggesting redundancy with other DNA damage tolerance pathways. Here we performed a comprehensive analysis of the temporal requirements of Y-family Pols ι and κ as backups for Polη in (i) bypassing genomic CPD and (6–4)PP lesions in vivo, (ii) suppressing DNA damage signaling, (iii) maintaining cell cycle progression and (iv) promoting cell survival, by using mouse embryonic fibroblast lines with single and combined disruptions in these Pols. The contribution of Polι is restricted to TLS at a subset of the photolesions. Polκ plays a dominant role in rescuing stalled replication forks in Polη-deficient mouse embryonic fibroblasts, both at CPDs and (6–4)PPs. This dampens DNA damage signaling and cell cycle arrest, and results in increased survival. The role of relatively error-prone Pols ι and κ as backups for Polη contributes to the understanding of the mutator phenotype of xeroderma pigmentosum variant, a syndrome caused by Polη defects.     

Histone hyperacetylation induced by histone deacetylase inhibitors is not sufficient to cause growth inhibition in human dermal fibroblasts

Use of specific histone deacetylase inhibitors has revealed critical roles for the histone deacetylases (HDAC) in controlling proliferation. Although many studies have correlated the function of HDAC inhibitors with the hyperacetylation of histones, few studies have specifically addressed whether the accumulation of acetylated histones, caused by HDAC inhibitor treatment, is responsible for growth inhibition. In the present study we show that HDAC inhibitors cause growth inhibition in normal and transformed keratinocytes but not in normal dermal fibroblasts. This was despite the observation that the HDAC inhibitor, suberic bishydroxamate (SBHA), caused a kinetically similar accumulation of hyperacetylated histones. This cell type-specific response to SBHA was not due to the inactivation of SBHA by fibroblasts, nor was it due to differences in the expression of specific HDAC family members. Remarkably, overexpression of HDACs 1, 4, and 6 in normal human fibroblasts resulted in cells that could be growth-inhibited by SBHA. These data suggest that, although histone acetylation is a major target for HDAC inhibitors, the accumulation of hyperacetylated histones is not sufficient to cause growth inhibition in all cell types. This suggests that growth inhibition, caused by HDAC inhibitors, may be the culmination of histone hyperacetylation acting in concert with other growth regulatory pathways.     

Isoprene synthase expression and protein levels are reduced under elevated O3 but not under elevated CO2 (FACE) in field-grown aspen trees

Emission of hydrocarbons by trees has a crucial role in the oxidizing potential of the atmosphere. In particular, isoprene oxidation leads to the formation of tropospheric ozone and other secondary pollutants. It is expected that changes in the composition of the atmosphere will influence the emission rate of isoprene, which may in turn feedback on the accumulation of pollutants and greenhouse gases. We investigated the isoprene synthase (ISPS) gene expression and the ISPS protein levels in aspen trees exposed to elevated ozone (O(3)) and/or elevated carbon dioxide (CO(2)) in field-grown trees at the Aspen Free-Air Carbon Dioxide Enrichment (FACE) experimental site. Elevated O(3) reduced ISPS mRNA and the amount of ISPS protein in aspen leaves, whereas elevated CO(2) had no significant effect. Aspen clones with different O(3) sensitivity showed different levels of inhibition under elevated O(3) conditions. The drop in ISPS protein levels induced a drop in the isoprene emission rate under elevated O(3). However, the data indicated that other mechanisms also contributed to the observed strong inhibition of isoprene emission under elevated O(3).     

Human diploid fibroblasts (HDF) can induce DNA synthesis in cycling HDF but not in quiescent HDF or senescent HDF

HeLa cells in S phase induce DNA synthesis in cycling cells, serum-deprived quiescent cells, and non-replicative senescent cells following cell fusion. In contrast normal human diploid fibroblasts (HDF) do not induce DNA synthesis in either quiescent cells or senescent cells. Instead, the replicative HDF nuclei are inhibited from entering S phase in heterokaryons formed with these two types of non-replicative cells. These differences in the inducing capabilities of normal HDF and HeLa cells raise the question whether normal HDF in S phase can induce DNA synthesis in cycling cells. This paper demonstrates that young HDF in S phase can induce DNA synthesis in cycling HDF. Thus, the hypothesis that initiation of DNA synthesis in cycling cells is positively controlled by inducer molecules appears to be valid for normal HDF as well as for transformed cells such as HeLa.     

Differential repair of 1-beta-D-arabinofuranosylcytosine-detectable sites in DNA of human fibroblasts exposed to ultraviolet light and 4-nitroquinoline 1-oxide

The extent of DNA excision repair was determined in dermal fibroblast strains from clinically normal and xeroderma pigmentosum (XP; complementation group A) human donors after single or combined exposures to 254-nm ultraviolet light and 4-nitroquinoline 1-oxide (4NQO). The repair was monitored by incubation of the treated cultures in the presence of 1-beta-D-arabinofuranosylcytosine (araC), a potent inhibitor of long-patch excision repair, followed by quantitation of araC-accumulated DNA single-strand breaks (representing repair events) by velocity sedimentation analysis in alkaline sucrose gradients. The amount of repair in normal fibroblast strains increased as a function of UV fluence and reached a plateau at 15 J/m2; strand breaks were not detected when these same cultures were irradiated with as much as 60 J/m2 UV and incubated in the absence of araC, implying that an initial (incision) step is rate-limiting in the repair of UV damage. In normal fibroblasts (i) the incidence of araC-detectable lesions removed during fixed intervals following exposure to 4NQO (4 microM; 30 min) was approximately 2.5 times greater than that seen following irradiation with repair-saturating fluences (greater than or equal to 15 J/m2) of UV-rays; and (ii) the amount of repair in cultures treated simultaneously with 4NQO (0.5-6 microM; 30 min) and a repair-saturating fluence of UV (20 J/m2) was found to approach the sum of that arising from exposure to each separately. The XP cells (XP12BE) exhibited a deficiency in the removal of araC-detectable DNA lesions following exposure to either of the carcinogens. Since araC is known to inhibit the repair of alkali-stable 4NQO-DNA adducts (i.e., lesions assumed to be removed by the UV-like excision pathway) but not that of alkali-labile sites (i.e., DNA lesions operated on by the X-ray-like repair pathway), our results strongly imply that the multistep excision-repair pathway operative on UV photoproducts in human fibroblasts differs from that responsible for removing alkali-stable (araC-detectable) 4NQO adducts by at least one step, presumably the rate-limiting incision reaction mediated by a lesion-recognizing endonuclease.     

DNA fragmentation induced in human fibroblasts by accelerated (56)fe ions of differing energies

DNA fragmentation was studied in the fragment size range 0.023-5.7 Mbp after irradiation of human fibroblasts with iron-ion beams of four different energies, i.e., 200 MeV/nucleon, 500 MeV/nucleon, 1 GeV/nucleon and 5 GeV/nucleon, with gamma rays used as the reference radiation. The double-strand break (DSB) yield (and thus the RBE for DNA DSB induction) of the four iron-ion beams, which have LETs ranging from 135 to 442 keV/mum, does not vary greatly as a function of LET. As a consequence, the variation of the cross section for DSB induction mainly reflects the variation in LET. However, when the fragmentation spectra were analyzed with a simple theoretical tool that we recently introduced, the results showed that spatially correlated DSBs, which are absent after gamma irradiation, increased markedly with LET for the iron-ion beams. This occurred because iron ions produce DNA fragments smaller than 0.75 Mbp with a higher probability than gamma rays (a probability that increases with LET). These sizes include those expected from fragmentation of the chromatin loops with Mbp dimensions. This result does not exclude a correlation at distances smaller than the lower size analyzed here, i.e. 23 kbp. Moreover, the DSB correlation is dependent on dose, decreasing when dose increases; this can be explained with the argument that at increasing dose there is an increasing fraction of fragments produced by DSBs caused by separate, uncorrelated tracks.     

Irradiations of rabbit myofibrils with an ultraviolet microbeam. II. Phalloidin protects actin in solution but not in myofibrils from depolymerization by ultraviolet light

We tested whether phalloidin protects actin in myofibrils from depolymerization by ultraviolet light (UV). I bands in glycerinated rabbit psoas myofibrils were irradiated with a UV microbeam in the presence and absence of phalloidin. We used the retention of contractility of the irradiated I band as the assay for protection of actin by phalloidin, since previous experiments indicated that UV blocks contraction of an irradiated I band by depolymerizing the thin filaments. The I bands of myofibrils incubated in phalloidin were as sensitive to UV as control I bands, indicating that phalloidin did not protect the thin filaments. However, phalloidin did protect F-actin in solution from depolymerization by UV. This apparent contradiction between F-actin in myofibrils and F-actin in solution was resolved by observing unirradiated myofibrils that were stained with rhodamine-phalloidin. It was found that phalloidin does not bind uniformly to the thin filaments, though as the fluorescence image is observed over time the staining pattern changes until it does appear to bind uniformly. We conclude that phalloidin does not protect F-actin in myofibrils from depolymerization by UV because it does not bind uniformly to the filaments.     

Timing of ultraviolet light induced mutagenesis in simian virus 40 relative to viral DNA replication in monkey cells

Summary Simian virus 40 (SV40) was used to probe ultraviolet light (UV) — induced mutation in mammalian cells. Viral mutations were scored as reversions of early and late temperature-sensitive (ts) mutants to the wild-type (WT) phenotype. When virus was exposed to moderate or high UV doses, WT revertants were obtained at a frequency related to the square of the dose from two early (tsA) and one late (tsBC) mutant grown at the restrictive temperature. The reversions generated in the progeny of UV-irradiated early mutants presumably arose before the onset of viral DNA replication because, at the non-permissive temperature, tsA mutants are unable to express the functions responsible for the initiation of viral DNA synthesis. Moreover, the early mutant tsA209 underwent similar levels of induced reversion at the permissive and restrictive temperatures, suggesting that the pre-replicative mutational pathway might predominate for moderately and heavily irradiated virus, even under conditions where DNA synthesis can be initiated. The analysis of bursts from revertant plaques produced at the restrictive temperature was consistent with this interpretation. Although the mechanism of pre-replicative mutagenesis is not known, it is likely to be mediated by cellular activities owing to the low genetic complexity of the virus.     

Non-thermal DNA breakage by mobile-phone radiation (1800 MHz) in human fibroblasts and in transformed GFSH-R17 rat granulosa cells in vitro

Cultured human diploid fibroblasts and cultured rat granulosa cells were exposed to intermittent and continuous radiofrequency electromagnetic fields (RF-EMF) used in mobile phones, with different specific absorption rates (SAR) and different mobile-phone modulations. DNA strand breaks were determined by means of the alkaline and neutral comet assay. RF-EMF exposure (1800 MHz; SAR 1.2 or 2 W/kg; different modulations; during 4, 16 and 24h; intermittent 5 min on/10 min off or continuous wave) induced DNA single- and double-strand breaks. Effects occurred after 16 h exposure in both cell types and after different mobile-phone modulations. The intermittent exposure showed a stronger effect in the comet assay than continuous exposure. Therefore we conclude that the induced DNA damage cannot be based on thermal effects.     

Chromatid damage induced by fluorescent light during G2 phase in normal and Gardner syndrome fibroblasts. Interpretation in terms of deficient DNA repair

Skin fibroblasts from Gardner syndrome (GS) compared with those from normal donors showed a significantly higher incidence of chromatid gaps and breaks following exposure to low-intensity, cool-white fluorescent light during G2 phase of the cell cycle. Considerable evidence supports the concept that chromatid gaps and breaks seen directly after exposure to DNA-damaging agents represent unrepaired DNA single- and double-strand breaks respectively. The changes in incidence of chromatid aberrations with time after light exposure are consistent with the sequence of events known to follow DNA damage and repair. Initially, the incidence of light-induced chromatid gaps was equivalent in GS and normal fibroblasts. In the normal cells, the chromatid gaps disappeared by 1 h post-exposure, presumably as a result of efficient repair of DNA single-strand breaks. In contrast, the incidence of gaps increased in GS cells by 0.5 h followed by a decrease at 1 h and concomitant increase in chromatid breaks. It appears from these findings that the increased incidence of chromatid damage in GS fibroblasts results from deficient repair of DNA single-strand breaks which arise from incomplete nucleotide excision of DNA damage during G2 phase.     

Norbixin ingestion did not induce any detectable DNA breakage in liver and kidney but caused a considerable impairment in plasma glucose levels of rats and mice

From the seeds of Bixa orellana are extracted the carotenoids bixin and norbixin that have been widely used for coloring food. In this study, the toxicity of norbixin, purified or not (annatto extract containing 50% norbixin), was investigated in mice and rats after 21 days of ingestion through drinking water. Mice were exposed to doses of 56 and 351 mg/kg (annatto extract) and 0.8, 7.6, 66 and 274 mg/kg (norbixin). Rats were exposed to doses of 0.8, 7.5 and 68 mg/kg (annatto extract) and 0.8, 8.5 and 74 mg/kg (norbixin). In rats, no toxicity was detected by plasma chemistry. In mice, norbixin induced an increase in plasma alanine aminotransferase activity (ALT) while both norbixin and annatto extract induced a decrease in plasma total protein and globulins (P < 0.05). However, no signs of toxicity were detected in liver by histopathological analysis. No enhancement in DNA breakage was detected in liver or kidney from mice treated with annatto pigments, as evaluated by the comet assay. Nevertheless, there was a remarkable effect of norbixin on the glycemia of both rodent species. In rats, norbixin induced hyperglycemia that ranged from 26.9% (8.5 mg/kg norbixin, to 52.6% (74 mg/kg norbixin, P < 0.01) above control levels. In mice, norbixin induced hypoglycemia that ranged from 14.4% (0.8 mg/kg norbixin, P < 0.05) to 21.5% (66 mg/kg norbixin, P < 0.001) below control levels. Rats and mice treated with annatto pigments showed hyperinsulinemia and hypoinsulinemia, respectively indicating that pancreatic beta-cells were functional. More studies should be performed to fully understand of how species-related differences influences the biological fate of norbixin.     

Lipopolysaccharide (LPS)-induced intra-uterine fetal death (IUFD) in mice is principally due to maternal cause but not fetal sensitivity to LPS

The present study deals with whether lipopolysaccharide (LPS)-induced intra-uterine fetal death (IUFD) is related to LPS-susceptibility of either mother or fetus and how LPS or LPS-induced TNF causes IUFD. LPS-susceptible C3H/HeN or -hypo-susceptible C3H/HeJ pregnant mice and the mice mated reciprocally with these mice were used on days 14 to 16 of gestation for experiments. All of fetuses in pregnant C3H/HeN mice mated with either C3H/HeN males [HeN(HeN)] or C3H/HeJ males [HeN(HeJ)] were killed within 24 hr when injected intravenously (i.v.) with 50 or 100 microg of LPS. On the other hand, the majority of fetuses in C3H/HeJ females mated with either C3H/HeJ males [HeJ(HeJ)] or C3H/HeN males [HeJ(HeN)] survived when injected i.v. with even 400 microg of LPS. These findings indicate that LPS-induced IUFD depends on the maternal LPS-responsiveness. LPS injected into mothers could pass through placenta to fetuses, since an injection with 125I-labeled LPS or IgG into pregnant mice resulted in considerable levels of radioactivity in fetuses as well as placenta. Cultured peritoneal macrophages derived from F1 mice of HeJ(HeN) or HeN(HeJ) mice, produced nitric oxide (NO) and tumor necrosis factor (TNF) in response to LPS, although the levels of NO and TNF were lower in comparison with those of C3H/HeN macrophage cultures, suggesting a possibility that the fetus as well as F1 cells might be responsible to LPS. LPS-induced IUFD was not blocked by treatment with anti-TNF antibody which inhibited LPS-induced TNF production in pregnant females, although an injection of recombinant TNFalpha instead of LPS could induce IUFD, suggesting that the cause of IUFD cannot be attributed to mother-derived TNF alone. The roles of LPS passed through placenta and LPS-induced mediators on IUFD were discussed.     

Xeromorphy increases in shoots of Pseudotsuga menziesii (Mirb.) Franco seedlings with exposure to elevated temperature but not elevated CO2

Seedling structure influences tree structure and function, ultimately determining the potential productivity of trees and their competitiveness for resources. We investigated changes in shoot structure for seedlings of Pseudotsuga menziesii (Douglas-fir) grown under climate change scenarios of ambient or elevated CO₂ (+180 mol mol^–1) plus ambient or elevated temperature (+3.5°C), for 4 years in outdoor, sunlit chambers. Mass allocation and allometry were measured for buds, leaves, branches, and stems, and anatomy was evaluated for leaves and stems. Seedlings became more xeromorphic with elevated temperature: allocation of total mass to branches over stems and leaves increased, sapwood area to height ratio increased, number of growing points relative to seedling size increased, and stem and branch length and mass decreased for sections initiated during the three full CO₂ and temperature seasons. Neither stem nor leaf anatomy was affected by elevated temperature. Elevated CO₂ increased specific mass of leaves, but had few other effects on mass allocation, allometry, or anatomy for any shoot organ. There were no CO₂ × temperature interactions for any important parameter. Thus, under realistic simulated field environmental conditions representative for in at least some P. menziesii forests (i.e., OR, USA, forests with limited soil nitrogen and summer soil moisture), elevated temperature, but not elevated CO₂, may affect seedling shoot structure and, hence, function.