Role of PPARα in inflammatory response of C2C12 myotubes.

Recent studies have shown a role of inflammation in muscle atrophy and sarcopenia. However, no anti-inflammatory pharmacotherapy has been established for the treatment of sarcopenia. Here, we investigate the potential role of PPARα and its ligands on inflammatory response and PGC-1α gene expression in LPS-treated C2C12 myotubes. Knockdown of PPARα, whose expression was upregulated upon differentiation, augmented IL-6 or TNFα gene expression. Conversely, PPARα overexpression or its activation by ligands suppressed 2-h LPS-induced cytokine expression, with pemafibrate attenuating NF-κB or STAT3 phosphorylation. Of note, reduction of PGC-1α gene expression by LPS treatment for 24 hours was partially reversed by fenofibrate. Our data demonstrate a critical inhibitory role of PPARα in inflammatory response of C2C12 myotubes and suggest a future possibility of PPARα ligands as a candidate for anti-inflammatory therapy against sarcopenia.

Semiconducting Transition Metal Dichalcogenide Heteronanotubes with Controlled Outer-Wall Structures.

Transition metal dichalcogenide (TMDC) nanotubes exhibit unique physical properties due to their nanotube structures. The development of techniques for synthesizing TMDC nanotubes with controlled structures is very important for their science and applications. However, structural control efforts have been made only for the homostructures of TMDC nanotubes and not for their heterostructures that provide an important platform for their two-dimensional counterparts. In this study, we synthesized heterostructures of TMDC nanotubes, MoS2/WS2 heteronanotubes, and demonstrated a technique for controlling features of their structures, such as diameters, layer numbers, and crystallinity. The diameter of the heteronanotubes could be tuned with inner nanotube templates and was reduced by using small-diameter WS2 nanotubes. The layer number and crystallinity of the MoS2 outer wall could be controlled by controlling their precursors and synthesis temperatures, resulting in the formation of high-crystallinity TMDC heteronanotubes with specific chirality. This study can expand the research of van der Waals heterostructures.

Development of a yeast reporter gene assay to detect ligands of freshwater cladoceran Daphnia magna ultraspiracle, a homolog of vertebrate retinoid X receptors.

Endocrine-disrupting chemicals (EDCs) often affect homeostatic regulation in living organisms by directly acting on nuclear receptors (NRs). Retinoid X receptors (RXRs), the most highly conserved members of the NR superfamily during evolution, function as partners to form heterodimers with other NRs, such as retinoic acid, thyroid hormone, and vitamin D3 receptors. RXRs also homodimerize and induce the expression of target genes upon binding with their natural ligand, 9-cis-retinoic acid (9cRA), and typical EDCs organotin compounds, such as tributyltin and triphenyltin. In the present study, we established a new yeast reporter gene assay (RGA) to detect the ligands of freshwater cladoceran Daphnia magna ultraspiracle (Dapma-USP), a homolog of vertebrate RXRs. D. magna has been used as a representative crustacean species for aquatic EDC assessments in the Organization for Economic Corporation and Development test guidelines. Dapma-USP was expressed along with the Drosophila melanogaster steroid receptor coactivator Taiman in yeast cells carrying the lacZ reporter plasmid. The RGA for detecting agonist activity of organotins and o-butylphenol was improved by use of mutant yeast strains lacking genes encoding cell wall mannoproteins and/or plasma membrane drug efflux pumps as hosts. We also showed that a number of other human RXR ligands, phenol and bisphenol A derivatives, and terpenoid compounds such as 9c-RA exhibited antagonist activity on Dapma-USP. Our newly established yeast-based RGA system is valuable as the first screening tool to detect ligand substances for Dapma-USP and for evaluating the evolutionary divergence of the ligand responses of RXR homologs between humans and D. magna.

A mouse model of weight gain after nicotine withdrawal.

Smoking cessation increases body weight. The underlying mechanisms, however, have not been fully understood. We here report an establishment of a mouse model that exhibits an augmented body weight gain after nicotine withdrawal. High fat diet-fed mice were infused with nicotine for two weeks, and then with vehicle for another two weeks using osmotic minipumps. Body weight increased immediately after nicotine cessation and was significantly higher than that of mice continued on nicotine. Mice switched to vehicle consumed more food than nicotine-continued mice during the first week of cessation, while oxygen consumption was comparable. Elevated expression of orexigenic agouti-related peptide was observed in the hypothalamic appetite center. Pair-feeding experiment revealed that the accelerated weight gain after nicotine withdrawal is explained by enhanced energy intake. As a showcase of an efficacy of pharmacologic intervention, exendin-4 was administered and showed a potent suppression of energy intake and weight gain in mice withdrawn from nicotine. Our current model provides a unique platform for the investigation of the changes of energy regulation after smoking cessation.

A luciferase reporter assay for ecdysone agonists using HEK293T cells.

Ecdysone agonists are a class of insecticides that activate the ecdysone receptor (EcR) heterodimerized with the ultraspiracle (USP). Here, we report a new luciferase reporter assay for ecdysone agonists. The assay employs mammalian HEK293T cells transiently transfected with the EcR and USP genes of Chilo suppressalis, along with the taiman (Tai) gene of Drosophila melanogaster that encodes a steroid receptor coactivator. This assay system gave results consistent with those of radioligand binding assays and showed sensitivity superior to that of the existing in vitro methods. In addition, use of the heterologous host cells precludes perturbation from intrinsic players of the ecdysone signaling, which is a potential drawback of insect cell-based methods. This reporter system is suitable for detailed structure-activity analysis of ecdysone agonists and will serve as a valuable tool for the rational design of novel insect growth regulators.

Sm-Co-based amorphous alloy films for zero-field operation of transverse thermoelectric generation.

Transverse thermoelectric generation using magnetic materials is essential to develop active thermal engineering technologies, for which the improvements of not only the thermoelectric output but also applicability and versatility are required. In this study, using combinatorial material science and lock-in thermography technique, we have systematically investigated the transverse thermoelectric performance of Sm-Co-based alloy films. The high-throughput material investigation revealed the best Sm-Co-based alloys with the large anomalous Nernst effect (ANE) as well as the anomalous Ettingshausen effect (AEE). In addition to ANE/AEE, we discovered unique and superior material properties in these alloys: the amorphous structure, low thermal conductivity, and large in-plane coercivity and remanent magnetization. These properties make it advantageous over conventional materials to realize heat flux sensing applications based on ANE, as our Sm-Co-based films can generate thermoelectric output without an external magnetic field. Importantly, the amorphous nature enables the fabrication of these films on various substrates including flexible sheets, making the large-scale and low-cost manufacturing easier. Our demonstration will provide a pathway to develop flexible transverse thermoelectric devices for smart thermal management.

Pemafibrate, a selective PPARα modulator, and fenofibrate suppress microglial activation through distinct PPARα and SIRT1-dependent pathways.

Pemafibrate, a selective peroxisome proliferator-activated receptor (PPAR) α modulator, is a new drug that specifically modulates PPARα conformation and co-activator recruitment, thereby lowers plasma triglycerides with less off-target effects. Classical PPARα ligands such as fenofibrate suppress inflammatory cells including microglia. However, effects of pemafibrate on microglia have never been addressed. Here we show that pemafibrate, like other PPARα ligands, potently suppressed NF-κB phosphorylation and cytokine expression in microglial cells. PPARα knockdown significantly amplified LPS-induced cytokine expression. Pemafibrate-induced suppression of IL-6 expression was reversed by PPARα knockdown. However, suppression by fenofibrate was not reversed by PPARα knockdown but by Sirtuin 1 (SIRT1) knockdown. In conclusion, pemafibrate and fenofibrate similarly suppresses microglial activation but through distinct PPARα and SIRT1-dependet pathways.

Growth hormone activates X-box binding protein 1 in a sexually dimorphic manner through the extracellular signal-regulated protein kinase and CCAAT/enhancer-binding protein ß pathway in rat liver.

Growth hormone (GH) has multiple physiological roles, acting on many organs. In order to investigate its roles in rat liver, we tried to identify novel genes whose transcription was regulated by GH. We identified X-box binding protein 1 (Xbp1) as a candidate gene. XBP1 is a key transcription factor activated in response to endoplasmic reticulum (ER) stress. The purpose of this study was to investigate the mode of action of GH on XBP1, including the relation with ER stress, sex-dependent expression of the mRNA, and the signaling pathway. Intravenous administration of GH rapidly and transiently increased Xbp1 mRNA in hypophysectomized rat livers. Neither phosphorylated inositol-requiring-1α (IRE1α) nor phosphorylated PKR-like ER kinase (PERK) increased, suggesting that Xbp1 expression is induced by an ER stress-independent mechanism. The active form of XBP1(S) protein was increased by GH administration and was followed by an increased ER-associated dnaJ protein 4 (ERdj4) mRNA level. XBP1(S) protein levels were predominantly identified in male rat livers with variations among individuals similar to those of phosphorylated signal transducer and activator of transcription 5B (STAT5B), suggesting that XBP1(S) protein levels are regulated by the sex-dependent secretary pattern of GH. The GH signaling pathway to induce Xbp1 mRNA was examined in rat hepatoma H4IIE cells. GH induced the phosphorylation of CCAAT/enhancer-binding protein ß (C/EBPß) following extracellular signal-regulated protein kinase (ERK) phosphorylation. Taken together, the results indicated that XBP1 is activated by GH in rat liver in a sexually dimorphic manner via ERK and C/EBPß pathway.

Low-protein diet enhances adiponectin secretion in rats.

Previous studies including ours have shown that a low-protein diet up-regulates insulin signaling in the liver and muscle and induces fatty liver in rats. Adiponectin is known as an insulin-sensitizing adipocytokine. We, therefore, examined the effect of a low-protein diet on the adiponectin levels in rats. The low-protein diet significantly increased serum adiponectin level. However, mRNA and protein levels of adiponectin in white adipose tissue (WAT) were not changed by the low-protein diet. Since it is known that oligomerization is important to control serum adiponectin level, we examined the population of adiponectin oligomeric forms in WAT and found that low-protein diet did not change it. Despite these events, the amount of its secretion was significantly increased in the adipocytes isolated from WAT of low-protein diet-fed rats. These results indicate that a low-protein diet enhances adiponectin secretion, which is not due to the increased intracellular amount and oligomerization of adiponectin.

Efficacy and safety of telaprevir with natural human interferon-ß and ribavirin in Japanese chronic hepatitis C patients with depression.

AIM: To assess the efficacy and safety of telaprevir (TVR) when used in combination with natural human interferon-ß (IFN-ß) and ribavirin (RBV) for genotype 1 patients with depression compared to IFN-ß/RBV therapy in Japan. We also examined the efficacy of the TVR/IFN-ß/RBV therapy in treatment failure genotype 2 patients with depression. METHODS: For the genotype 1 patients, 30 patients received TVR (750 mg every 8 h) for 12 weeks combined with IFN-ß and RBV for 24 weeks (Group A), and 30 received IFN-ß and RBV for 48 weeks (Group B). For the genotype 2 patients, 14 patients were dosed only with the TVR-based regimen. RESULTS: The sustained virologic response (SVR) rates for Group A and Group B were 63.3% and 20.0%, respectively (P = 0.001, likelihood ratio test). The SVR rate for genotype 2 patients previously treated with pegylated IFN and/or RBV was 71.4%. No patient dropped out due to exacerbation of depression. The trend of platelet counts after the drugs were given was similar in the TVR/IFN-ß/RBV therapy group and the IFN-ß/RBV therapy group. Common resistance-associated variants of TVR were identified in 4 of the 13 patients who did not achieve SVR. CONCLUSION: This study showed that an addition of TVR to IFN-ß/RBV therapy raised SVR in previously treated and untreated genotype 1 patients and previously treated genotype 2 patients with chronic hepatitis C and depression.

Hepatocyte ß-Klotho regulates lipid homeostasis but not body weight in mice.

ß-Klotho (ß-Kl), a transmembrane protein expressed in the liver, pancreas, adipose tissues, and brain, is essential for feedback suppression of hepatic bile acid synthesis. Because bile acid is a key regulator of lipid and energy metabolism, we hypothesized potential and tissue-specific roles of ß-Kl in regulating plasma lipid levels and body weight. By crossing ß-kl(-/-) mice with newly developed hepatocyte-specific ß-kl transgenic (Tg) mice, we generated mice expressing ß-kl solely in hepatocytes (ß-kl(-/-)/Tg). Gene expression, metabolomic, and in vivo flux analyses consistently revealed that plasma level of cholesterol, which is over-excreted into feces as bile acids in ß-kl(-/-), is maintained in ß-kl(-/-) mice by enhanced de novo cholesterogenesis. No compensatory increase in lipogenesis was observed, despite markedly decreased plasma triglyceride. Along with enhanced bile acid synthesis, these lipid dysregulations in ß-kl(-/-) were completely reversed in ß-kl(-/-)/Tg mice. In contrast, reduced body weight and resistance to diet-induced obesity in ß-kl(-/-) mice were not reversed by hepatocyte-specific restoration of ß-Kl expression. We conclude that ß-Kl in hepatocytes is necessary and sufficient for lipid homeostasis, whereas nonhepatic ß-Kl regulates energy metabolism. We further demonstrate that in a condition with excessive cholesterol disposal, a robust compensatory mechanism maintains cholesterol levels but not triglyceride levels in mice.

A Case of Ruptured Vertebrobasilar Junction Aneurysm Associated with Subclavian Steal Phenomenon.

A 77-year-old woman with arteriovenous shunt for hemodialysis in the left forearm suffered from subarachnoid hemorrhage due to the rupture of a saccular aneurysm located on the left lateral wall of vertebrobasilar junction. Her left subclavian artery was severely stenosed and subclavian steal phenomenon was demonstrated on the digital subtraction angiography. Embolization of the parent artery including the aneurysm using detachable coils resulted in the successful obliteration of the aneurysm through the revascularized left subclavian artery. This is the first case in which the vertebrobasilar junction aneurysm would be caused by the hemodynamic stress due to the subclavian steal phenomenon combined with the shunt for hemodialysis in the left forearm.

Acute coronary syndromes occurring while driving: frequency and patient characteristics.

BACKGROUND: Acute coronary syndrome (ACS) may occur during any human activity, including driving. The objectives of this study were to report the frequency of ACS occurring while driving, clarify patient characteristics, and analyze the behavioral patterns of drivers who sustained ACS. METHODS: A single-center, retrospective observational study was conducted using prospectively acquired data. Among 1605 ACS patients admitted between January 2011 and December 2016, 65 (60 men/5 women) patients who sustained ACS while driving were identified. Clinical variables were compared between these 65 patients and 1540 patients who sustained ACS while performing other activities. Furthermore, multivariable regression analysis was performed to identify variables associated with ACS. RESULTS: The frequency of ACS occurring while driving was 4.0% (65/1605). Compared with patients who sustained ACS while performing other activities, those who sustained ACS while driving were significantly younger (66.2 ± 13.0 vs. 57.5 ± 12.2 years, p < 0.001) and more likely to smoke (34.2 vs. 60.0%, p < 0.001). Multivariable regression analysis showed that age (OR 0.961; 95% CI 0.940-0.982) and current smoking (OR 1.978; 95% CI 1.145-3.417) were associated with ACS. While 55 drivers (85%) who remained conscious after ACS could seek medical attention without causing accidents, the other 10 (15%) who sustained cardiac arrest caused accidents. CONCLUSIONS: The association between current smoking and ACS occurring while driving suggests that smoking cessation is advised for smokers who drive from the standpoint of driving safety. We expect that prospective studies be conducted to verify our findings and identify individuals at risk for ACS while driving.

Identification of amino acid residues in the ligand-binding domain of the aryl hydrocarbon receptor causing the species-specific response to omeprazole: possible determinants for binding putative endogenous ligands.

Omeprazole (OME) induces the expression of genes encoding drug-metabolizing enzymes, such as CYP1A1, via activation of the aryl hydrocarbon receptor (AhR) both in vivo and in vitro. However, the precise mechanism of OME-mediated AhR activation is still under investigation. While elucidating species-specific susceptibility to dioxin, we found that OME-mediated AhR activation was mammalian species specific. Moreover, we previously reported that OME has inhibitory activity toward CYP1A1 enzymes. From these observations, we speculated that OME-mediated AhR target gene transcription is due to AhR activation by increasing amounts of putative AhR ligands in serum by inhibition of CYP1A1 activity. We compared the amino acid sequences of OME-sensitive rabbit AhR and nonsensitive mouse AhR to identify the residues responsible for the species-specific response. Chimeric AhRs were constructed by exchanging domains between mouse and rabbit AhRs to define the region required for the response to OME. OME-mediated transactivation was observed only with the chimeric AhR that included the ligand-binding domain (LBD) of the rabbit AhR. Site-directed mutagenesis revealed three amino acids (M328, T353, and F367) in the rabbit AhR that were responsible for OME-mediated transactivation. Replacing these residues with those of the mouse AhR abolished the response of the rabbit AhR. In contrast, substitutions of these amino acids with those of the rabbit AhR altered nonsensitive mouse AhR to become sensitive to OME. These results suggest that OME-mediated AhR activation requires a specific structure within LBD that is probably essential for binding with enigmatic endogenous ligands.

Microbial metabolites of omeprazole activate murine aryl hydrocarbon receptor in vitro and in vivo.

Omeprazole (OME), a proton pump inhibitor used to treat gastritis, is also an aryl hydrocarbon receptor (AhR) activator. OME activates AhR in human hepatocytes and hepatoma cells, but not in mice in vivo or in vitro. We recently discovered that this species-specific difference results from a difference in a few amino acids in the ligand-binding domain of AhR. However, OME activates both mouse and human AhRs in the yeast reporter assay system. Nevertheless, the cause of this discrepancy in OME responses remains unknown. Here, we report that CYP1A1 mRNA expression in mouse cecum was elevated after OME administration, although the mouse is regarded as an OME-unresponsive animal. Using the yeast reporter assay system with human and murine AhRs, we found AhR agonist-like activity in the cecal extracts of OME-treated mice. We speculated that OME metabolites produced by cecal bacteria might activate murine AhRs in vivo. In high-performance liquid chromatography (HPLC) analysis, AhR agonist-like activity of cecal bacterial culture and cecal extracts were detected at the same retention time. AhR agonist-like activity was also detected in the HPLC fractions of yeast culture media containing OME. This unknown substance could induce reporter gene expression via mouse and human AhRs. The agonist-like activity of the OME metabolite was reduced by concomitant α-naphthoflavone exposure. These results indicate that a yeast-generated OME metabolite elicited the response of mouse AhR to OME in the yeast system, and that bacterial OME metabolites may act as AhR ligands in human and mouse intestines.

DNA Repair and Mutagenesis of ADP-Ribosylated DNA by Pierisin.

Pierisin is a DNA-targeting ADP-ribosyltransferase found in cabbage white butterfly (Pieris rapae). Pierisin transfers an ADP-ribosyl moiety to the 2-amino group of the guanine residue in DNA, yielding N2-(ADP-ribos-1-yl)-2'-deoxyguanosine (N2-ADPR-dG). Generally, such chemically modified DNA is recognized as DNA damage and elicits cellular responses, including DNA repair pathways. In Escherichia coli and human cells, it has been experimentally demonstrated that N2-ADPR-dG is a substrate of the nucleotide excision repair system. Although DNA repair machineries can remove most lesions, some unrepaired damages frequently lead to mutagenesis through DNA replication. Replication past the damaged DNA template is called translesion DNA synthesis (TLS). In vitro primer extension experiments have shown that eukaryotic DNA polymerase κ is involved in TLS across N2-ADPR-dG. In many cases, TLS is error-prone and thus a mutagenic process. Indeed, the induction of G:C to T:A and G:C to C:G mutations by N2-ADPR-dG in the hypoxanthine phosphoribosyltransferase gene mutation assay with Chinese hamster cells and supF shuttle vector plasmids assay using human fibroblasts has been reported. This review provides a detailed overview of DNA repair, TLS and mutagenesis of N2-ADPR-dG induced by cabbage butterfly pierisin-1.

Concomitant pyogenic spondylodiscitis and empyema following tongue cancer resection and wisdom tooth extraction: A case report and literature review.

RATIONALE: Pyogenic spondylodiscitis is an infectious spinal disease that causes significant motor dysfunctions. Its diagnosis can be challenging owing to its rapid onset and nonspecific symptoms. PATIENT CONCERNS: A 79-year-old Japanese man with a history of type 2 diabetes mellitus and polymyalgia rheumatica presented to our department with tongue pain. Following partial glossectomy and wisdom tooth extraction under general anesthesia, on 10 postoperative day (POD) the patient developed right-sided abdominal pain and difficulty in walking. On 12 POD, the patient was admitted to a municipal hospital due to respiratory distress and paraplegia. DIAGNOSES: The patient was diagnosed with pyogenic spondylodiscitis and empyema. Blood tests revealed elevated C-reactive protein levels (36.5), white blood cell count (19,570), and neutrophil count (17,867). INTERVENTIONS: The patient received meropenem hydrate 3 g/2 days as empiric antibiotic treatment for acute infection. Upon admission to the emergency department on 16 POD, the lung abscess was drained, hemilaminectomy was performed. OUTCOMES: Blood cultures, sputum tests, and cultures from the thoracic and spinal abscesses drained during surgery revealed methicillin-sensitive Staphylococcus aureus. The infection was successfully managed, and the respiratory disturbance and inflammatory response improved. However, the lower half of the patient body remained paralyzed. Subsequently, the patient was transferred to a rehabilitation facility on 45 POD. The patient continued to undergo functional restoration training, gradually regained function, and eventually achieved the ability to walk with grasping gait. LESSONS: This is the first case report of S aureus causing pyogenic spondylodiscitis and empyema due to blood stream infection from a post-oral surgical wound. Pyogenic spondylodiscitis arising from a secondary hematogenous infection is difficult to diagnose and can lead to severe functional impairment. Prompt and appropriate diagnosis and treatment based on detailed patient interviews, additional blood tests, and computed tomography are essential.

Identification of small molecule proliferating cell nuclear antigen (PCNA) inhibitor that disrupts interactions with PIP-box proteins and inhibits DNA replication.

We have discovered that 3,3',5-triiodothyronine (T3) inhibits binding of a PIP-box sequence peptide to proliferating cell nuclear antigen (PCNA) protein by competing for the same binding site, as evidenced by the co-crystal structure of the PCNA-T3 complex at 2.1 Å resolution. Based on this observation, we have designed a novel, non-peptide small molecule PCNA inhibitor, T2 amino alcohol (T2AA), a T3 derivative that lacks thyroid hormone activity. T2AA inhibited interaction of PCNA/PIP-box peptide with an IC(50) of ~1 µm and also PCNA and full-length p21 protein, the tightest PCNA ligand protein known to date. T2AA abolished interaction of PCNA and DNA polymerase δ in cellular chromatin. De novo DNA synthesis was inhibited by T2AA, and the cells were arrested in S-phase. T2AA inhibited growth of cancer cells with induction of early apoptosis. Concurrently, Chk1 and RPA32 in the chromatin are phosphorylated, suggesting that T2AA causes DNA replication stress by stalling DNA replication forks. T2AA significantly inhibited translesion DNA synthesis on a cisplatin-cross-linked template in cells. When cells were treated with a combination of cisplatin and T2AA, a significant increase in phospho(Ser(139))histone H2AX induction and cell growth inhibition was observed.

Dioxin suppresses benzo[a]pyrene-induced mutations and DNA adduct formation through cytochrome P450 1A1 induction and (±)-anti-benzo[a]pyrene-7,8-diol-9,10-epoxide inactivation in human hepatoma cells.

Benzo[a]pyrene (BaP) is metabolically activated by cytochrome P450 enzymes, and forms DNA adduct leading to mutations. Cytochrome P450 1A1 plays a central role in this activation step, and this enzyme is strongly induced by chemical agents that bind to the aryl hydrocarbon receptor (AhR), which is also known as a dioxin receptor. 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD), a potent AhR ligand has not been shown to form any DNA adduct, but has a possibility to aggravate the toxicity of precarcinogenic polycyclic hydrocarbons through the induction of metabolic enzymes. We treated human hepatoma cells (HepG2) with TCDD, and subsequently exposed them to BaP to elucidate the synergistic effects on mutations. Surprisingly, mutant frequency induced by BaP at the hypoxanthine-guanine phosphribosyltransferase (HPRT) locus was decreased by pretreatment with TCDD. In correlation with decrease in the mutant frequencies, BaP-DNA adduct formation was also decreased by TCDD pretreatment. This suppressive effect of TCDD was more potent when the cells were exposed to (±)-anti-benzo[a]pyrene-7,8-diol-9,10-epoxide (BPDE), a reactive metabolic intermediate of BaP. Among the enzymes catalyzing BaP oxidation and conjugation, cytochrome P450 1A1, 1A2, 3A4 and UDP-glucuronosyltransferase 1A1 mRNAs were induced by the exposure to TCDD. In cytochrome P450 1A1-deficient murine cells and cytochrome P450 1A1-uninducible human cells, TCDD could not suppress BPDE-DNA adduct formation. Further experiments using "Tet-On" cytochrome P450 1A1-overexpressing cells and a recombinant cytochrome P450 1A1 enzyme demonstrated that this is the key enzyme involved in the biotransformation of BaP, that is, both production and inactivation of BPDE. We conclude that TCDD-induced cytochrome P450 catalyzes the metabolism of BPDE to as yet-unidentified products that are not apparently DNA-reactive, thereby reducing mutations in hepatoma cells.

Translesion DNA synthesis across various DNA adducts produced by 3-nitrobenzanthrone in Escherichia coli.

To analyze translesion DNA synthesis (TLS) across lesions derived from the air pollutant 3-nitrobenzanthrone in Escherichia coli, we constructed site-specifically modified plasmids containing single molecule adducts derived from 3-nitrobenzanthrone. For this experiment, we adopted a modified version of the method developed by Fuchs et al. [29]. Each plasmid contained one of the following lesions in its LacZ' gene: N-(2'-deoxyguanosin-8-yl)-3-aminobenzanthrone (dG-C8-N-ABA); 2-(2'-deoxyguanosin-N(2)-yl)-3-aminobenzanthrone (dG-N(2)-C2-ABA); 2-(2'-deoxyguanosin-8-yl)-3-aminobenzanthrone (dG-C8-C2-ABA); 2-(2'-deoxyadenosin-N(6)-yl)-3-aminobenzanthrone (dA-N(6)-C2-ABA); N-(2'-deoxyguanosin-8-yl)-3-acetylaminobenzanthrone (dG-C8-N-AcABA); or 2-(2'-deoxyguanosin-8-yl)-3-acetylaminobenzanthrone (dG-C8-C2-AcABA). All of the adducts inhibited DNA synthesis by replicative DNA polymerases in E. coli; however, the extent of the inhibition varied among the adducts. All five dG-adducts strongly blocked replication by replicative DNA polymerases; however, the dA-adduct only weakly blocked DNA replication. The induction of the SOS response increased the frequency of TLS, which was higher for the dG-C8-C2-ABA, dG-C8-N-AcABA and dG-C8-C2-AcABA adducts than for the other adducts. In our previous study, dG-C8-N-ABA blocked DNA replication more strongly and induced mutations more frequently than dG-N(2)-C2-ABA in human cells. In contrast, in E. coli the frequency of TLS over dG-N(2)-C2-ABA was markedly reduced, even under the SOS(+) conditions, and dG-N(2)-C2-ABA induced G to T mutations. All of the other adducts were bypassed in a less mutagenic manner. In addition, using E. coli strains that lacked particular DNA polymerases we found that DNA polymerase V was responsible for TLS over dG-C8-N-AcABA and dG-C8-C2-AcABA adducts.