Supersulfide prevents cigarette smoke extract-induced mitochondria hyperfission and cardiomyocyte early senescence by inhibiting Drp1-filamin complex formation.

Smoking is one of the most serious risk factors for cardiovascular diseases. Although cigarette mainstream and sidestream smoke are significant contributors to increased cardiovascular mortality and morbidity, the underlying mechanism is still unclear. Here, we report that exposure of rat neonatal cardiomyocytes to cigarette smoke extract (CSE) induces mitochondrial hyperfission-mediated myocardial senescence. CSE leads to mitochondrial fission and reactive oxygen species (ROS) production through the complex formation between mitochondrial fission factor Drp1 and actin-binding protein, filamin A. Pharmacological perturbation of interaction between Drp1 and filamin A by cilnidipine and gene knockdown of Drp1 or filamin A inhibited CSE-induced mitochondrial hyperfission and ROS production as well as myocardial senescence. We previously reported that Drp1 activity is controlled by supersulfide-induced Cys644 polysulfidation. The redox-sensitive Cys644 was critical for CSE-mediated interaction with filamin A. The administration of supersulfide donor, Na2S3 also improved mitochondrial hyperfission-mediated myocardial senescence induced by CSE. Our results suggest the important role of Drp1-filamin A complex formation on cigarette smoke-mediated cardiac risk and the contribution of supersulfide to mitochondrial fission-associated myocardial senescence.

Protein Photodamaging Activity and Photocytotoxic Effect of an Axial-Connecting Phosphorus(V)porphyrin Trimer.

An axial-connecting trimer of the porphyrin phosphorus(V) complex was synthesized to evaluate the relaxation process of the photoexcited state and the photosensitizer activity. The photoexcitation energy was localized on the central unit of the phosphorus(V)porphyrin trimer. The photoexcited state of the central unit was relaxed through a process similar to that of the monomer phosphorus(V)porphyrin. The excited state of this axially connected type of phosphorus(V)porphyrin trimer was not deactivated through intramolecular electron transfer. The singlet oxygen generation quantum yield of the trimer was almost the same as that of the monomer. The phosphorus(V)porphyrin, trimer, and monomer bound to human serum albumin and oxidized the tryptophan residue via singlet oxygen generation and electron transfer during visible light irradiation. The photocytotoxicity of these phosphorus(V)porphyrins on two cell lines was examined. The monomer induced photocytotoxicity; however, the trimer did not show cytotoxicity with or without photoirradiation. In summary, the photoexcited state of the trimer was almost the same as that of the monomer, and these phosphorus(V)porphyrins demonstrated a similar protein-photodamaging activity. The difference in association between the photosensitizer molecules and cells is the key factor of phototoxicity by these phosphorus(V)porphyrins.

Glucose starvation impairs NER and γ-H2AX after UVB irradiation.

Glucose is the major source for energy production. As tumor cells have higher glucose requirement, combination of glucose restriction and radio- and chemo-therapy has been explored. In this study, impairment of UVB-induced DNA damage repair response (DDR) by glucose starvation was revealed. Human keratinocytes and skin carcinoma cells were cultured in medium containing 0, 2.5, 5.5 and 25 mM glucose. Glucose restriction suppressed cell proliferation and histone acetylation. UVB exposure formed similar levels of pyrimidine dimers in all glucose conditions, whereas the repair tended to be delayed in low glucose medium. The repair molecules, TFIIH and XPG, were accumulated to DNA damaged sites regardless of glucose supply levels, but the release was delayed in glucose-starved cells. The remaining pyrimidine dimers would induce the collapse of replication forks during S phase, resulting in phosphorylation of histone H2AX (γ-H2AX), but the γ-H2AX in cells cultured in glucose-deleted medium was unexpectedly decreased. This might be due to the suppression of DNA replication by glucose deletion. This was further confirmed by the decrease in the formation of DNA double strand breaks in glucose-starved cells. These results suggested that condition of energy supply might affect UV-induced DDR.

Butyrate Enhances γ-H2AX Induced by Benzo[a]pyrene.

Benzo[a]pyrene (BaP) is known to form DNA adduct following metabolic activation, which causes phosphorylation of histone H2AX (γ-H2AX). Recent studies have shown that histone deacetylase (HDAC) inhibitors enhanced BaP-induced CYP1A1 gene expression. In this study, we examined the relationship between the HDAC inhibitor-augmented metabolic activation and BaP-induced γ-H2AX. Sodium butyrate (SB), a typical HDAC inhibitor, enhanced BaP-induced γ-H2AX. The enhanced DNA damage was further confirmed by biased sinusoidal field gel electrophoresis, which detects DNA double-strand breaks. SB remarkably augmented BaP-induced CYP1A1 gene expression, and CYP1A1-overexpressing cells showed elevated generation of γ-H2AX. Furthermore, SB enhanced intracellular oxidation after treatment with BaP. These results suggested that SB-induced CYP1A1 upregulation facilitated BaP metabolism, which might result in excess DNA adducts or oxidative DNA damages, leading to augmentation of γ-H2AX.

Protective role of electrophile-reactive glutathione for DNA damage repair inhibitory effect of dibromoacetonitrile.

Dibromoacetonitrile (DBAN) is a disinfection byproduct (DBP) and linked with cancer in rodents, but the mechanism of its carcinogenicity has not been fully elucidated. We recently reported that DBAN induced inhibition of nucleotide excision repair (NER). In this study, we investigated if glutathione (GSH) is involved in the DBAN-induced inhibition of NER. Human keratinocytes HaCaT were pretreated with L-buthionine-(S,R)-sulfoximine (BSO) to deplete intracellular GSH. BSO treatment markedly potentiated the DBAN-induced NER inhibition as well as intracellular oxidation. The recruitment of NER proteins (transcription factor IIH, and xeroderma pigmentosum complementation group G) to DNA damage sites was inhibited by DBAN, which was further exacerbated by BSO treatment. Our results suggest that intracellular GSH protects cells from DBAN-induced genotoxicity including inhibition of DNA damage repair.

TRPC3-Nox2 Protein Complex Formation Increases the Risk of SARS-CoV-2 Spike Protein-Induced Cardiomyocyte Dysfunction through ACE2 Upregulation.

Myocardial damage caused by the newly emerged coronavirus (SARS-CoV-2) infection is one of the key determinants of COVID-19 severity and mortality. SARS-CoV-2 entry to host cells is initiated by binding with its receptor, angiotensin-converting enzyme (ACE) 2, and the ACE2 abundance is thought to reflect the susceptibility to infection. Here, we report that ibudilast, which we previously identified as a potent inhibitor of protein complex between transient receptor potential canonical (TRPC) 3 and NADPH oxidase (Nox) 2, attenuates the SARS-CoV-2 spike glycoprotein pseudovirus-evoked contractile and metabolic dysfunctions of neonatal rat cardiomyocytes (NRCMs). Epidemiologically reported risk factors of severe COVID-19, including cigarette sidestream smoke (CSS) and anti-cancer drug treatment, commonly upregulate ACE2 expression level, and these were suppressed by inhibiting TRPC3-Nox2 complex formation. Exposure of NRCMs to SARS-CoV-2 pseudovirus, as well as CSS and doxorubicin (Dox), induces ATP release through pannexin-1 hemi-channels, and this ATP release potentiates pseudovirus entry to NRCMs and human iPS cell-derived cardiomyocytes (hiPS-CMs). As the pseudovirus entry followed by production of reactive oxygen species was attenuated by inhibiting TRPC3-Nox2 complex in hiPS-CMs, we suggest that TRPC3-Nox2 complex formation triggered by panexin1-mediated ATP release participates in exacerbation of myocardial damage by amplifying ACE2-dependent SARS-CoV-2 entry.

Inhibition of nucleotide excision repair and damage response signaling by dibromoacetonitrile: A novel genotoxicity mechanism of a water disinfection byproduct.

Dibromoacetonitrile (DBAN) is a carcinogenic disinfection byproduct (DBP) but how it precipitates cancer is unknown. Nucleotide excision repair (NER) is a versatile repair mechanism for removing bulky DNA lesions to maintain genome stability, and impairment of this process is associated with cancer development. In this study, we found that DBAN inhibited NER and investigated its mechanism with other DNA damage responses. Human keratinocytes HaCaT were treated with DBAN followed by ultraviolet (UV) as a model inducer of DNA damage, pyrimidine dimers, which require NER for the removal. DBAN pretreatment exacerbated UV-cytotoxicity, and inhibited the repair of pyrimidine dimers. DBAN treatment delayed the recruitment of NER proteins, transcription factor IIH (TFIIH) and xeroderma pigmentosum complementation group G (XPG), to DNA damaged sites, and subsequent gap filling process. Moreover, DBAN suppressed the UV-induced double strand breaks (DSBs) formation, as well as phosphorylated histone H2AX (γ-H2AX), a widely used DNA damage marker. Altogether, DBAN could negatively impact the NER process and phosphorylation pathway responding to DNA damage. This study was the first to identify the inhibition of NER and damage response signaling as a genotoxicity mechanism of a class of DBPs and it may serve as a foundation for DBP carcinogenesis.

4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone-Induced Histone Acetylation via α7nAChR-Mediated PI3K/Akt Activation and Its Impact on γ-H2AX Generation.

A typical tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is known as a strong carcinogen. We previously reported that metabolized NNK induced histone H2AX phosphorylation (γ-H2AX), a DNA damage-induced histone modification. In this study, we found that NNK globally acetylated histone H3, which affected γ-H2AX generation. Human lung adenocarcinoma A549 was treated with several doses of NNK. NNK induced dose-dependent global histone H3 acetylation (Ac-H3), at 2 to 12 h after the treatment, independent of the cell cycle. The Ac-H3 pattern was not affected by CYP2A13 overexpression unlike γ-H2AX, indicating no requirement of NNK metabolism to induce Ac-H3. Immunofluorescence staining of Ac-H3 was uniform throughout the nucleus, whereas γ-H2AX was formed as foci and did not coincide with Ac-H3. Nicotinic receptor antagonist methyllycaconitine inhibited Ac-H3 and also γ-H2AX. Phosphoinositide-3-kinase (PI3K)/Akt inhibitors, LY294002, wortmannin, and GSK690693, also suppressed both Ac-H3 and γ-H2AX, whereas KU-55933, an inhibitor of ataxia telangiectasia mutated (ATM) upstream of γ-H2AX, inhibited γ-H2AX but not Ac-H3. These results suggested that binding of NNK to the nicotinic acetylcholine receptor (α7nAChR) activated the PI3K/Akt pathway, resulting in Ac-H3. The activated pathway leading to Ac-H3 enhanced γ-H2AX, suggesting that NNK-induced DNA damage is impacted by the α7nAChR-mediated signal transduction pathway.

Low extracellular pH inhibits nucleotide excision repair.

Nucleotide excision repair (NER) is the main pathway to repair bulky DNA damages including pyrimidine dimers, and the genetic dysregulation of NER associated proteins is well known to cause diseases such as cancer and neurological disorder. Other than the genetic defects, 'external factors' such as oxidative stress and environmental chemicals also affect NER. In this study, we examined the impact of extracellular pH on NER. We prepared the culture media, whose pH values are 8.4 (normal condition), 7.6, 6.6 and 6.2 under atmospheric CO2 conditions. Human keratinocytes, HaCaT, slightly died after 48 h incubation in DMEM at pH 8.4, 7.6 and 6.6, while in pH 6.2 condition, marked cell death was induced. UV-induced pyrimidine dimers, pyrimidine (6-4) pyrimidone photoproducts (6-4PPs) and cyclobutane pyrimidine dimers (CPDs), were effectively repaired at 60 min and 24 h, respectively, which were remarkably inhibited at pH 6.6 and 6.2. The associated repair molecule, TFIIH, was accumulated to the damaged sites 5 min after UVC irradiation in all pH conditions, but the release was delayed as the pH got lower. Furthermore, accumulation of XPG at 5 min was delayed at pH 6.2 and 6.6, and the release at 60 min was completely suppressed. At the low pH, the DNA synthesis at the gaps created by incision of oligonucleotides containing pyrimidine dimers was significantly delayed. In this study, we found that the low extracellular pH inhibited NER pathway. This might partially contribute to carcinogenesis in inflamed tissues, which exhibit acidic pH.

Long-wavelength UVA enhances UVB-induced cell death in cultured keratinocytes: DSB formation and suppressed survival pathway.

Solar UV radiation consists of both UVA and UVB. The wavelength-specific molecular responses to UV radiation have been studied, but the interaction between UVA and UVB has not been well understood. In this study, we found that long-wavelength UVA, UVA1, augmented UVB-induced cell death, and examined the underlying mechanisms. Human keratinocytes HaCaT were exposed to UVA1, followed by UVB irradiation. Irradiation by UVA1 alone showed no effect on cell survival, whereas the UVA1 pre-irradiation remarkably enhanced UVB-induced cell death. UVA1 delayed the repair of pyrimidine dimers formed by UVB and the accumulation of nucleotide excision repair (NER) proteins to damaged sites. Gap synthesis during NER was also decreased, suggesting that UVA1 delayed NER, and unrepaired pyrimidine dimers and single-strand breaks generated in the process of NER were left behind. Accumulation of this unrepaired DNA damage might have led to the formation of DNA double-strand breaks (DSBs), as was detected using gel electrophoresis analysis and phosphorylated histone H2AX assay. Combined exposure enhanced the ATM-Chk2 signaling pathway, but not the ATR-Chk1 pathway, confirming the enhanced formation of DSBs. Moreover, UVA1 suppressed the UVB-induced phosphorylation of Akt, a survival signal pathway. These results indicated that UVA1 influenced the repair of UVB-induced DNA damage, which resulted in the formation of DSBs and enhanced cell death, suggesting the risk of simultaneous exposure to high doses of UVA1 and UVB.

Cell line-dependent difference in glutathione levels affects the cigarette sidestream smoke-induced inhibition of nucleotide excision repair.

We recently reported that cigarette sidestream smoke (CSS) induced inhibition of nucleotide excision repair (NER) and the cause was NER molecule degradation by aldehydes contained in CSS [Carcinogenesis39, 56-65, 2018; Mutat. Res.834, 42-50, 2018]. In this study, we examined the relationship between intracellular glutathione (GSH) levels and CSS-induced NER inhibition. CSS treatment decreased the intracellular GSH level in human keratinocytes HaCaT, in which the repair of pyrimidine (6-4) pyrimidone photoproducts (6-4PPs) after UVB irradiation was suppressed. We used l-buthionine-(S,R)-sulfoximine (BSO) to artificially deplete intracellular GSH level. BSO treatment remarkably accelerated the CSS-induced NER inhibition. The NER inhibition by CSS was attributed to the delay of accumulation of NER molecules (TFIIH and XPG) to DNA damaged sites, which was further enhanced by BSO treatment. CSS degraded TFIIH, and BSO promoted it as expected. Formaldehyde (FA), a major constituent of CSS, showed similar intracellular GSH reduction and NER inhibition, and BSO promoted its inhibitory effect. Five cultured cell lines showed considerable variability in intrinsic GSH levels, and CSS-induced NER inhibitory effect was significantly correlated with the GSH levels. Chemicals like aldehydes are known to react not only with proteins but also with DNA, causing DNA lesions targeted by NER. Our results suggest that the tissues and cells with low intrinsic GSH levels are susceptible to treatment with CSS and electrophilic compounds like aldehydes through NER inhibition, thus leading to higher genotoxicity and carcinogenicity.

Formaldehyde inhibits UV-induced phosphorylation of histone H2AX.

Formaldehyde (FA) is widely known to cause DNA damage. Recently, our study showed that FA can also inhibit a repair process of DNA damage, nucleotide excision repair (NER). DNA damage response (DDR) involving activation of phosphorylation pathways is important for the accuracy of the repair process, and the inhibition of the accurate repair would raise mutation rate, leading to cancer. We herein investigated whether FA influences phosphorylation of histone H2AX (γ-H2AX), an intermediate player of DDR signaling pathways. Human keratinocytes HaCaT were treated with FA and then exposed to UV known to generate clear γ-H2AX signal. UV-induced γ-H2AX was inhibited by FA in a dose-dependent manner. The repair of pyrimidine dimers was inhibited by FA, while the recruitments of γ-H2AX-related proteins, Mre11 and 53BP1, to damaged sites were also delayed. Mre11, Nbs-1, H2AX and ATM were not degraded after treatment with FA as opposed to NER-related protein, TFIIH. On the other hand, FA inhibited phosphorylation of ATM which acts upstream of γ-H2AX. These results suggest that FA can affect the repair of DNA damage via inhibition of the phosphorylation pathways of H2AX.

Controlled Photodynamic Action of Axial Fluorinated DiethoxyP(V)tetrakis(p-methoxyphenyl)porphyrin through Self-Aggregation.

DiethoxyP(V)tetrakis(p-methoxyphenyl)porphyrin (EtP(V)TMPP) and its fluorinated derivative (FEtP(V)TMPP) were synthesized to examine their photodynamic action. These P(V)porphyrins were aggregated in an aqueous solution, resulting in the suppression of their photodynamic activity. In the presence of human serum albumin (HSA), a water-soluble protein, the aggregation states were resolved and formed a binding complex between P(V)porphyrin and HSA. These P(V)porphyrins photosensitized the oxidation of the tryptophan residue of HSA under the irradiation of long-wavelength visible light (>630 nm). This protein photodamage was explained by the electron transfer from tryptophan to the photoexcited state of P(V)porphyrins and singlet oxygen generation. The axial fluorination reduced the redox potential of the one-electron reduction of P(V)porphyrin and increased the electron transfer rate constant. However, this axial fluorination decreased the binding constant with HSA, and the quantum yield of photosensitized HSA damage through electron transfer was decreased. The photocytotoxicity of these P(V)porphyrins to HaCaT cells was also confirmed, and FEtP(V)TMPP demonstrated stronger phototoxicity than EtP(V)TMPP. In summary, a self-aggregation of porphyrin photosensitizers and resolving by targeting biomacromolecules may be used to target selective photodynamic action. The redox potential and an association with a targeting biomolecule are the important factors of the electron transfer-mediated mechanism, which is advantageous under hypoxic tumor conditions.

Ibudilast attenuates doxorubicin-induced cytotoxicity by suppressing formation of TRPC3 channel and NADPH oxidase 2 protein complexes.

BACKGROUND AND PURPOSE: Doxorubicin is a highly effective anticancer agent but eventually induces cardiotoxicity associated with increased production of ROS. We previously reported that a pathological protein interaction between TRPC3 channels and NADPH oxidase 2 (Nox2) contributed to doxorubicin-induced cardiac atrophy in mice. Here we have investigated the effects of ibudilast, a drug already approved for clinical use and known to block doxorubicin-induced cytotoxicity, on the TRPC3-Nox2 complex. We specifically sought evidence that this drug attenuated doxorubicin-induced systemic tissue wasting in mice. EXPERIMENTAL APPROACH: We used the RAW264.7 macrophage cell line to screen 1,271 clinically approved chemical compounds, evaluating functional interactions between TRPC3 channels and Nox2, by measuring Nox2 protein stability and ROS production, with and without exposure to doxorubicin. In male C57BL/6 mice, samples of cardiac and gastrocnemius muscle were taken and analysed with morphometric, immunohistochemical, RT-PCR and western blot methods. In the passive smoking model, cells were exposed to DMEM containing cigarette sidestream smoke. KEY RESULTS: Ibudilast, an anti-asthmatic drug, attenuated ROS-mediated muscle toxicity induced by doxorubicin treatment or passive smoking, by inhibiting the functional interactions between TRPC3 channels and Nox2, without reducing TRPC3 channel activity. CONCLUSIONS AND IMPLICATIONS: These results indicate a common mechanism underlying induction of systemic tissue wasting by doxorubicin. They also suggest that ibudilast could be repurposed to prevent muscle toxicity caused by anticancer drugs or passive smoking.

Aldehyde-mediated protein degradation is responsible for the inhibition of nucleotide excision repair by cigarette sidestream smoke.

We recently reported that cigarette sidestream smoke (CSS) can delay nucleotide excision repair (NER), which was due to the inhibition of repair protein accumulation to DNA damage sites. However, the mechanisms how the protein recruitment was inhibited remains unclear. We hypothesized that aldehydes in CSS could be a candidate taking a role for the inhibition, and tested our hypothesis by removing aldehydes from CSS using cigarette-filters. The NER inhibition potency of CSS or filtered CSS (F-CSS) was compared using human keratinocyte cell line, HaCaT. Cigarette-filters were able to reduce total aldehydes in CSS by half. Pretreating cells with CSS and F-CSS enhanced UVB-induced cell death, with the effect of CSS weakened by filtration. CSS strongly inhibited the repair of UVB-induced DNA damage, pyrimidine(6-4)pyrimidone photoproducts (6-4PPs), where the recruitments of repair molecules, TFIIH and XPG, were slowed down. F-CSS showed similar inhibition of NER and accumulation of related proteins, but the effect was weaker than CSS. Semicarbazide (SEM), an aldehyde-trapping agent, alleviated the NER delay induced by both CSS and F-CSS, further confirming that aldehydes in CSS were the main cause for the inhibition of NER and that the different amounts of aldehydes in CSS and F-CSS were responsible for the different inhibition efficiency. Furthermore, TFIIH level was decreased by treatment with CSS and restored in the presence of proteasome inhibitor, indicating that the degradation of NER proteins might be the cause of the inhibition of NER-protein recruitment. These results supported our hypothesis that aldehydes in CSS are the main contributor for the NER inhibition via protein degradation, and reconfirmed that exposure to CSS without filtration could be a severe threat to human health.

Trichloroethylene exposure results in the phosphorylation of histone H2AX in a human hepatic cell line through cytochrome P450 2E1-mediated oxidative stress.

Trichloroethylene (TCE), a chlorinated hydrocarbon, was recently reclassified as a human carcinogen by the International Agency for Research on Cancer. Genotoxic events are known to be crucial steps in the initiation of cancer. The genotoxic properties of TCE have been examined in many studies using a standard battery of genotoxicity tests both in vitro and in vivo. However, consistent results have not been obtained, and studies investigating the mechanism behind the genotoxicity of this compound are lacking. In the present study, we examined the genotoxicity of TCE by assessing phosphorylated histone H2AX (γ-H2AX), a new sensitive and reliable marker of DNA damage, in WRL-68 cells, cultured human hepatocytes and mouse livers. Our results showed that TCE exposure results in the generation of γ-H2AX, both in vitro and in vivo. By investigating the in vitro mechanism, we found that TCE increases the levels of intracellular reactive oxygen species (ROS) and that this increase in ROS levels is attenuated in the presence of disulfiram, a specific cytochrome P450 2E1 (CYP2E1) inhibitor. Furthermore, γ-H2AX induced by TCE was also attenuated by CYP2E1 inhibitors and the antioxidant N-acetylcysteine. These results suggested that ROS, produced via cytochrome P450 2E1-mediated metabolic processing, is a major causal factor for γ-H2AX generation upon exposure to TCE.

Photosensitized Protein-Damaging Activity, Cytotoxicity, and Antitumor Effects of P(V)porphyrins Using Long-Wavelength Visible Light through Electron Transfer.

Photodynamic therapy (PDT) is a less-invasive treatment for cancer through the administration of less-toxic porphyrins and visible-light irradiation. Photosensitized damage of biomacromolecules through singlet oxygen (1O2) generation induces cancer cell death. However, a large quantity of porphyrin photosensitizer is required, and the treatment effect is restricted under a hypoxic cellular condition. Here we report the phototoxic activity of P(V)porphyrins: dichloroP(V)tetrakis(4-methoxyphenyl)porphyrin (CLP(V)TMPP), dimethoxyP(V)tetrakis(4-methoxyphenyl)porphyrin (MEP(V)TMPP), and diethyleneglycoxyP(V)tetrakis(4-methoxyphenyl)porphyrin (EGP(V)TMPP). These P(V)porphyrins damaged the tryptophan residue of human serum albumin (HSA) under the irradiation of long-wavelength visible light (>630 nm). This protein photodamage was barely inhibited by sodium azide, a quencher of 1O2. Fluorescence lifetimes of P(V)porphyrins with or without HSA and their redox potentials supported the electron-transfer-mediated oxidation of protein. The photocytotoxicity of these P(V)porphyrins to HeLa cells was also demonstrated. CLP(V)TMPP did not exhibit photocytotoxicity to HaCaT, a cultured human skin cell, and MEP(V)TMPP and EGP(V)TMPP did; however, cellular DNA damage was barely observed. In addition, a significant PDT effect of these P(V) porphyrins on a mouse tumor model comparable with the traditional photosensitizer was also demonstrated. These findings suggest the cancer selectivity of these P(V)porphyrins and lower carcinogenic risk to normal cells. Electron-transfer-mediated oxidation of biomacromolecules by P(V)porphyrins using long-wavelength visible light should be advantageous for PDT of hypoxic tumor.

Cigarette sidestream smoke delays nucleotide excision repair: inhibited accumulation of repair proteins at DNA lesions.

Cigarette sidestream smoke (CSS) contains many carcinogens that induce DNA damage. DNA damage plays an important role in the initiation of cancer and several diseases, and repair is the major defense mechanism; however, the relationship between CSS and the repair of DNA damage remains unclear. We herein investigated whether CSS influences nucleotide excision repair (NER) in vivo and in vitro. HR-1 hairless mouse skin treated with CSS was exposed to UVB, as a result of which pyrimidine dimers (cyclobutane pyrimidine dimers (CPDs) and pyrimidine(6-4)pyrimidone photoproducts (6-4PPs)) were formed and repaired via the NER pathway. The immunohistochemical staining of CPDs revealed that their repair was delayed by the CSS treatment. This delay in NER and the underlying mechanisms were examined in the human skin cell lines, HaCaT and HSC-1. Dot-blot assays, enzyme-linked immunosorbent assay and local ultraviolet irradiation assays demonstrated that CSS delayed the repair of CPDs and 6-4PPs. The recruitment of the repair molecules, TFIIH, XPA and XPG to pyrimidine dimers was markedly inhibited by CSS. Semicarbazide, which reacts with aldehydes, recovered the CSS-induced inhibition of NER, and formaldehyde exerted similar inhibitory effects to those of CSS. These results suggest that aldehydes in CSS interfere with the recruitment of NER molecules to damaged sites, leading to a delay in the repair of pyrimidine dimers.

α,ß-Unsaturated Aldehyde-Induced Delays in Nucleotide Excision Repair and the Contribution of Reactive Oxygen Species.

Aldehydes are widespread environmental and industrial compounds to which humans are frequently exposed. Despite their significant health risk, the mechanisms underlying aldehyde toxicity are poorly understand. We recently demonstrated that cigarette sidestream smoke (CSS) inhibited nucleotide excision repair (NER), and this was attributed to aldehydes in CSS. In the present study, we examined the influence of saturated and unsaturated aldehydes on NER. The human keratinocyte cell line, HaCaT, was treated with aldehydes and then exposed to UVB. Saturated aldehydes did not show toxicity, whereas α,ß-unsaturated aldehydes caused cell death, which was markedly enhanced by UV exposure. The speed of NER was examined by the detection of pyrimidine (6-4) pyrimidone photoproducts (6-4PPs) using ELISA and local UV irradiation assay. The repair of 6-4PPs was markedly reduced by α,ß-unsaturated aldehydes, but not by saturated aldehydes, and this was attributed to a delay in the recruitment of repair proteins (TFIIH and XPG) to DNA damage sites. Reactive oxygen species (ROS) were produced after a treatment with α,ß-unsaturated aldehydes, and hydrogen peroxide (H2O2) inhibited the repair of 6-4PPs, similar to α,ß-unsaturated aldehydes. H2O2 inhibited the accumulation of XPA and XPG at DNA damage sites, whereas TFIIH showed the same recruitment with or without H2O2. These results suggest that an exposure to α,ß-unsaturated aldehydes, not saturated aldehydes inhibits NER by delaying the recruitment of NER proteins to DNA damage sites, and α,ß-unsaturated aldehyde-induced ROS production may partially play a role in this process.

Effect of physicochemical character differences on the genotoxic potency of kaolin.

BACKGROUND: Kaolin is white clay mineral with the chemical composition Al2Si2O5(OH)4, and many varieties of kaolins having different crystal structures are utilized in industrial, cosmetic and medical fields. To evaluate the effect of physicochemical character differences on the genotoxicity of kaolin, two types of kaolin, kaolin-S with smooth, sphere-shaped crystals, and kaolin-P with clusters of thin pseudohexagonal plates, were used in the study. RESULTS: ICR mice were intratracheally instilled with the kaolins (0.05 and 0.2 mg/mouse), and comet assay was performed on their lungs. Both kaolins showed DNA damage in the lungs of the mice, however the DNA damaging potency was much higher with kaolin-P than that with kaolin-S. In order to clarify the mechanisms for the different genotoxic potency, we examined the incorporation rate and ROS generation of these two types of kaolin in alveolar epithelial A549 and macrophage-like RAW264 cells, using flow cytometric (FCM) analysis. Kaolin-P showed a higher incorporation rate into the mammalian cells and ROS generation than that of kaolin-S. Especially, RAW264 cells aggressively incorporated kaolins, and generated ROS, whereas almost no ROS generation was observed in A549 cells. In addition, inflammatory cytokines were quantified, using the ELISA method, to understand further genotoxic potency differences of kaolins. Concentrations of interleukin-1ß (IL-1ß) and tumor necrosis factor-α (TNF-α) in the media were increased by exposure to both kaolins, but in the case of kaolin-P, these inflammatory cytokines were significantly elevated. Based on these findings, differences of genotoxic potency may contribute to incorporation rates into immune cells. Furthermore, it is likely that immune cells and epithelial cells might closely interact with each other for the appearance of genotoxocity in vivo. In order to clarify the interaction between epithelial and immune cells, A549 and RAW264 were co-cultured and RAW264 cells only were exposed to kaolins, then subsequently A549 was applied to FCM analysis and comet assay. DNA damage observed in the A549 cells markedly increased in the presence of kaolin-exposed RAW264 cells compared to the single culture. CONCLUSION: From these observations, it is suggested that mechanisms of kaolin genotoxicity against epithelial cells are through the activation of macrophage cells. Therefore, it is thought that interactions between epithelial and immune cells would be very important for evaluation of the genotoxicity of fine particulate matter. We also showed here that co-culture models of epithelial and immune cells could be used as suitable models for evaluation of lung genotoxicity of fine particulate matter, including nanomaterials, as in vivo mimicking systems.