Regulation of the binding of 7,12-dimethylbenz[a]-anthracene to DNA of cultured murine epidermal cells at metabolic level: competition of the binding by polycyclic aromatic hydrocarbons and by other precarcinogens.

The binding of labeled carcinogen [3H]DMBA to murine epidermal cells (MEC) DNA in culture has been studied. The influence of unlabeled noncarcinogenic and carcinogenic polycyclic aromatic hydrocarbons (PAH), several PAH metablites, and various directly and indirectly acting non-PAH carcinogens on the binding of [3H]DMBA to MEC DNA has been examined. All the carcinogenic PAH and some of non-carcinogenic PAH effectively inhibit the binding of [3H]DMBA to MEC DNA. The non-PAH chemical carcinogens requiring metabolic activation also reduce the binding of labeled DMBA to MEC DNA; however, a higher concentration of these compounds is required for 50% inhibition of binding than the concentrations of PAH for the same degree of inhibition of binding of [3H]DMBA to MEC DNA. The directly acting carcinogens do not significantly inhibit the binding of [3H]DMBA to DNA. The relationship between structures of PAH and their ability to inhibit the binding of [3H]DMBA to MEC DNA is also discussed. Thus, it appears that the binding of DMBA to cellular DNA is primarily controlled at a level of metabolism and to some extent at the level of binding of reactive metabolites to DNA.     

HMGB1 Promotes the Development of Pulmonary Arterial Hypertension in Rats

Rationale

Pulmonary arterial hypertension (PAH) is characterized by increased pulmonary vascular resistance leading to right ventricular failure and death. Recent studies have suggested that chronic inflammatory processes are involved in the pathogenesis of PAH. However, the molecular and cellular mechanisms driving inflammation have not been fully elucidated.

Objectives

To elucidate the roles of high mobility group box 1 protein (HMGB1), a ubiquitous DNA-binding protein with extracellular pro-inflammatory activity, in a rat model of PAH.

Methods

Male Sprague-Dawley rats were administered monocrotaline (MCT). Concentrations of HMGB1 in bronchoalveolar lavage fluid (BALF) and serum, and localization of HMGB1 in the lung were examined over time. The protective effects of anti-HMGB1 neutralizing antibody against MCT-induced PAH were tested.

Results

HMGB1 levels in BALF were elevated 1 week after MCT injection, and this elevation preceded increases of other pro-inflammatory cytokines, such as TNF-α, and the development of PAH. In contrast, serum HMGB1 levels were elevated 4 weeks after MCT injection, at which time the rats began to die. Immunohistochemical analyses indicated that HMGB1 was translocated to the extranuclear space in periarterial infiltrating cells, alveolar macrophages, and bronchial epithelial cells of MCT-injected rats. Anti-HMGB1 neutralizing antibody protected rats against MCT-induced lung inflammation, thickening of the pulmonary artery wall, and elevation of right ventricular systolic pressure, and significantly improved the survival of the MCT-induced PAH rats.

Conclusions

Our results identify extracellular HMGB1 as a promoting factor for MCT-induced PAH. The blockade of HMGB1 activity improved survival of MCT-induced PAH rats, and thus might be a promising therapy for the treatment of PAH.     

Pyrene fate affected by humic acid amendment in soil slurry systems

Background  

Humic acid (HA) has been found to affect the solubility, mineralization, and bound residue formation of polycyclic aromatic hydrocarbons (PAHs). However, most of the studies on the interaction between HA and PAH concentrated on one or two of the three phases. Few studies have provided a simple protocol to demonstrate the overall effects of HA on PAH distribution in soil systems for all three phases.     

Perilaldehyde activates AMP-activated protein kinase to suppress the growth of gastric cancer via induction of autophagy

Background and Aim: Perillaldehyde (PAH), one of the major oil components in Perilla frutescens, is very critical to health maintenance, for a wide range of human chronic diseases, including cancers. AMP-activated protein kinase (AMPK) has been implicated in the activation of autophagy in distinct tissues. This study was designed to explore whether PAH prevents gastric cancer growth and to investigate the molecular mechanism. Methods and Results: In cultured mouse gastric cancer cell line MFCs and human gastric cancer cell lines GC9811-P, PAH activated AMPK by increasing the Thr172 phosphorylation and activity in a time-/concentration-dependent manner. Furthermore, incubation of MFCs with PAH also increased autophagy as determined by monodansylcadaverine (MDC) staining, which was reversed by AMPK inhibitor compound C. PAH further decreased MFCs cell survival, which was abolished by compound C or autophagy inhibitor 3-Methyladenine (3-MA). In vivo studies indicated that 4-week administration of PAH (100 mg/kg/day) suppressed the growth of gastric cancer and increased the levels of autophagy-related proteins, including beclin-1, LC3-II, cathepsin, caspase-3, p53, and cathepsin in tumors isolated from the xenograft model of gastric cancer in mice. Moreover, these anticancer effects produced by PAH were abolished by coadministration of compound C or 3-MA in vivo. Conclusions: PAH increases AMPK phosphorylation and activity to induce gastric cancer cell autophagy to inhibit the growth of gastric cancer. In perspective, therapy of PAH should be applied to treat patients with gastric cancer.     

Advances in the field of high‐molecular‐weight polycyclic aromatic hydrocarbon biodegradation by bacteria

Interest in understanding prokaryotic biotransformation of high-molecular-weight polycyclic aromatic hydrocarbons (HMW PAHs) has continued to grow and the scientific literature shows that studies in this field are originating from research groups from many different locations throughout the world. In the last 10 years, research in regard to HMW PAH biodegradation by bacteria has been further advanced through the documentation of new isolates that represent diverse bacterial types that have been isolated from different environments and that possess different metabolic capabilities. This has occurred in addition to the continuation of in-depth comprehensive characterizations of previously isolated organisms, such as Mycobacterium vanbaalenii PYR-1. New metabolites derived from prokaryotic biodegradation of four- and five-ring PAHs have been characterized, our knowledge of the enzymes involved in these transformations has been advanced and HMW PAH biodegradation pathways have been further developed, expanded upon and refined. At the same time, investigation of prokaryotic consortia has furthered our understanding of the capabilities of microorganisms functioning as communities during HMW PAH biodegradation.     

Reversal of MicroRNA Dysregulation in an Animal Model of Pulmonary Hypertension

Background

Animals models have played an important role in enhancing our understanding of the pathogenesis of pulmonary arterial hypertension (PAH). Dysregulation of the profile of microRNAs (miRNAs) has been demonstrated in human tissues from PAH patients and in animal models. In this study, we measured miRNA levels in the monocrotaline (MCT) rat model of PAH and examined whether blocking a specific dysregulated miRNA not previously reported in this model, attenuated PAH. We also evaluated changes in miRNA expression in lung specimens from MCT PAH rats overexpressing human prostacyclin synthase, which has been shown to attenuate MCT PAH.

Methods

Expression levels of a panel of miRNAs were measured in MCT-PAH rats as compared to naïve (saline) control rats. Subsequently, MCT PAH rats were injected with a specific inhibitor (antagomiR) for miR-223 (A223) or a nonspecific control oligonucleotide (A-control) 4 days after MCT administration, then weekly. Three weeks later, RV systolic pressure and RV mass were measured. Total RNA, isolated from the lungs, microdissected pulmonary arteries, and right ventricle, was reverse transcribed and real-time quantitative PCR was performed. MiRNA levels were also measured in RNA isolated from paraffin sections of MCT-PAH rats overexpressing prostacyclin synthase.

Results

MiRs 17, 21, and 223 were consistently upregulated, whereas miRs 126, 145, 150, 204, 424, and 503 were downregulated in MCT PAH as compared to vehicle control. A223 significantly reduced levels of miR-223 in PA and lungs of MCT PAH rats as compared to levels measured in A-control or control MCT PAH rats, but A223 did not attenuate MCT PAH. Right ventricular mass and right ventricular systolic pressure in rats treated with A223 were not different from values in A-control or MCT PAH rats. In contrast, analysis of total RNA from lung specimens of MCT PAH rats overexpressing human prostacyclin synthase (hPGIS) demonstrated reversal of MCT-induced upregulation of miRs 17, 21, and 223 and an increase in levels of miR-424 and miR-503. Reduction in bone morphogenetic receptor 2 (BMPR2) messenger (m)RNA expression was not altered by A223, whereas human prostacyclin synthase overexpression restored BMPR2 mRNA to levels in MCT PAH to levels measured in naive controls.

Conclusions

Inhibition of miR-223 did not attenuate MCT PAH, whereas human prostacyclin synthase overexpression restored miRNA levels in MCT PAH to levels detected in naïve rats. These data may establish a paradigm linking attenuation of PAH to restoration of BMPR2 signaling.     

Nucleotide release and associated conformational changes regulate function in the COOH-terminal Src kinase, Csk

The COOH-terminal Src kinase (Csk) regulates a broad array of cellular processes via the specific phosphorylation and downregulation of Src family protein kinases. While Csk has been a topic for steady-state kinetic studies, the individual steps associated with substrate phosphorylation have not been investigated. To understand active-site phenomena, pre-steady-state and transient-state kinetic methods were applied to develop a catalytic pathway for substrate processing. Rapid quench flow techniques show that the phosphorylation of a substrate peptide, generated from a random library, occurs in two kinetic phases: a rapid, exponential "burst" phase followed by a slow, linear phase. The amplitude of the burst phase increases as a function of enzyme concentration, indicating that the biphasic kinetics are not the result of product inhibition. Analysis of the burst rate as a function of substrate concentration indicates that the phosphoryl transfer step is fast (k3 > or = 140 s(-1) and highly favorable (k3/k-3 > or = 6). The apparent dissociation rate constant for ADP (0.6 s(-1), measured using stopped-flow kinetic methods and a fluorescent trapping agent, mant-ATP, is close to kcat. Since the substrate dissociation constant is high, the release of phosphopeptide is not likely to limit turnover. These findings indicate that Csk rapidly delivers the gamma-phosphate of ATP to the substrate and rapidly releases the phosphoproduct. Overall rate limitation in the steady state is then attributed to the slow, net dissociation of ADP. Viscosometric studies suggest that this final event in the catalytic cycle is coupled with slow conformational changes.     

Microbial Transformation of Polycyclic Aromatic Hydrocarbons in Pristine and Petroleum-Contaminated Sediments

To determine rates of microbial transformation of polycyclic aromatic hydrocarbons (PAH) in freshwater sediments, ¹⁴C-labeled PAH were incubated with samples from both pristine and petroleum-contaminated streams. Evolved ¹⁴CO₂ was trapped in KOH, unaltered PAH and polar metabolic intermediate fractions were quantitated after sediment extraction and column chromatography, and bound cellular ¹⁴C was measured in sediment residues. Large fractions of ¹⁴C were incorporated into microbial cellular material; therefore, measurement of rates of ¹⁴CO₂ evolution alone would seriously underestimate transformation rates of [¹⁴C]naphthalene and [¹⁴C]anthracene. PAH compound turnover times in petroleum-contaminated sediment increased from 7.1 h for naphthalene to 400 h for anthracene, 10,000 h for benz(a)anthracene, and more than 30,000 h for benz(a)pyrene. Turnover times in uncontaminated stream sediment were 10 to 400 times greater than in contaminated samples, while absolute rates of PAH transformation (micrograms of PAH per gram of sediment per hour) were 3,000 to 125,000 times greater in contaminated sediment. The data indicate that four- and five-ring PAH compounds, several of which are carcinogenic, may persist even in sediments that have received chronic PAH inputs and that support microbial populations capable of transforming two- and three-ring PAH compounds.     

Correlations between 15 polycyclic aromatic hydrocarbons (PAH) and the mutagenicity of the total PAH fraction in ambient air particles in La Spezia (Italy)

Airborne particulate matter has been monitored 4 times a month for 1 year (1988) in the city of La Spezia (Italy). The polycyclic aromatic hydrocarbon (PAH) fractions were extracted, purified and characterized for the content of 15 individual PAH. In general when concentrations of individual PAH were compared statistical correlation was obtained. Mutagenicity studies were performed by the use of the Ames plate test with the Salmonella strains TA98, TA100, TA98NR and TA98DNP6 with and without metabolic activation (S9 mix). The TA98 strain was by far the most responsive and the S9 mix was absolutely required as expected when PAH are assayed. Besides mutagenicity, toxicity was also considered and it proved to be correlated with mutagenicity in TA98, +S9. The TA98NR and TA98DNP6 strains showed no appreciable differences from the parental strain TA98 indicating the absence of significant amounts of direct-acting nitro derivatives in our PAH samples. Of the 15 PAH considered in this study the amounts of cyclopental[c,d]pyrene (CPP) correlated best with mutagenicity. The role of CPP in contributing to the indirect mutagenicity of urban air PAH samples is discussed.     

The optimization of the diagnostic work-up in patients with suspected obstructive lung disease

Background

Liver dysfunction reflects the status of heart failure, with congestion and low perfusion of the liver serving as causative mechanisms. Previous studies demonstrated relationship between the results of liver function test and the prognosis in patients with heart failure. However, few studies have examined this relationship in patients with pulmonary arterial hypertension (PAH).

Methods

The subjects were 37 patients with PAH (8 men and 29 women; 18 with idiopathic PAH and 19 with connective tissue disease-associated PAH). A blood test was performed after a 3-month period free from hospitalization and without changes in functional class, treatment, heart sounds, body weight, or heart rate.

Results

In a mean follow-up period of 635 ± 510 days, 12 patients died due to heart failure, 2 died due to pulmonary hemorrhage, and 23 patients survived. Cox proportional hazard analyses identified functional class (p < 0.001), plasma concentration of brain natriuretic peptide (BNP) (p = 0.001), and hyperbilirubinemia (serum total bilirubin > 1.2 mg/dL; p < 0.001; hazard ratio = 13.31) as predictors of mortality. Patients with hyperbilirubinemia had a worse functional class (P = 0.003), a higher right atrial pressure (p < 0.001), a higher plasma concentration of BNP (p = 0.004), and a larger Doppler right ventricular index of the right ventricle (p = 0.041).

Conclusion

Elevated serum bilirubin is a risk factor for death in patients with PAH.     

Modeling the model organism Dictyostelium discoideum

The cellular slime mold Dictyostelium discoideum is a fascinating organism, not only for biologists, but also for physicists. Since the Belousov-Zhabotinskii reaction pattern, a well-known non-linear phenomenon in chemistry, was observed during aggregation of Dictyostelium amoebae, Dictyostelium has been one of the major subjects of non-linear dynamics studies. Macroscopic theory, such as continuous cell density approximation, has been a common approach to studying pattern formation since the pioneering work of Turing. Recently, promising microscopic approaches, such as the cellular dynamics method, have emerged. They have shown that Dictyostelium is useful as a model system in biology, The synchronization mechanism of oscillatory production of cyclic adenosine 3',5'-monophosphate in Dictyostelium is discussed in detail to show how it is a universal feature that can explain synchronization in other organisms.     

A Perspective on the Toxicity of Petrogenic PAHs to Developing Fish Embryos Related to Environmental Chemistry

Numerous studies demonstrate polynuclear aromatic hydrocarbons (PAHs) dissolved from weathered crude oil adversely affect fish embryos at 0.5 to 23 μg/l. This conclusion has been challenged by studies that claim (1) much lower toxicity of weathered aqueous PAHs; (2) direct contact with dispersed oil droplets plays a significant role or is required for toxicity; (3) that uncontrolled factors (oxygen, ammonia, and sulfides) contribute substantively to toxicity; (4) polar compounds produced by microbial metabolism are the major cause of observed toxicity; and (5) that based on equilibrium models and toxic potential, water contaminated with weathered oil cannot be more toxic per unit mass than effluent contaminated with fresh oil. In contrast, several studies demonstrate high toxicity of weathered oil; shifts in PAH composition were consistent with dissolution (not particle ablation), embryos accumulated dissolved PAHs at low concentrations and were damaged, and assumed confounding factors were inconsequential. Consistent with previous empirical observations of mortality and weathering, temporal shifts in PAH composition (oil weathering) indicate that PAHs dissolved in water should (and do) become more toxic per unit mass with weathering because high molecular weight PAHs are more persistent and toxic than the more abundant low molecular weight PAHs in whole oil.     

Functional Characterization of Novel Phenylalanine Hydroxylase p.Gln226Lys Mutation Revealed Its Non-responsiveness to Tetrahydrobiopterin Treatment in Hepatoma Cellular Model

Treatment with tetrahydrobiopterin (BH4) is the latest therapeutic option approved for patients with phenylketonuria (PKU)—one of the most frequent inborn metabolic diseases. PKU or phenylalanine hydroxylase (PAH) deficiency is caused by mutations in the PAH gene. Given that some PAH mutations are responsive to BH4 treatment while others are non-responsive, for every novel mutation that is discovered it is essential to confirm its pathogenic effect and to assess its responsiveness to a BH4 treatment in vitro, before the drug is administered to patients. We found a c.676C>A (p.Gln226Lys) mutation in the PAH gene in two unrelated patients with PKU. The corresponding aberrant protein has never been functionally characterized in vitro and its response to BH4 treatment is unknown. Computational analyses proposed that glutamine at position 226 is an important, evolutionary conserved amino acid while the substitution with lysine probably disturbs tertiary protein structure and impacts posttranslational PAH modifications. Using hepatoma cellular model, we demonstrated that the amount of mutant p.Gln226Lys PAH detected by Western blot was only 1.2% in comparison to wild-type PAH. The addition of sepiapterin, intracellular precursor of BH4, did not increase PAH protein yield thus marking p.Gln226Lys as BH4-non-responsive mutation. Therefore, computational, experimental, and clinical data were all in accordance showing that p.Gln226Lys is a severe pathogenic PAH mutation. Its non-responsiveness to BH4 treatment in hepatoma cellular model should be considered when deciding treatment options for PKU patients carrying this mutation. Consequently, our study will facilitate clinical genetic practice, particularly genotype-based stratification of PKU treatment.     

New Polymorphic Sites in the Human Phenylalanine Hydroxylase Gene

A previously unknown sequence of the human phelylalanine hydroxylase (PAH) gene intron 7 (GeneBank AN AF204239) has been reported. Screening of the group of phenylketonuria patients from Nobosibirsk region for polymorphic sites within intron 7 revealed single nucleotide substitutions at intron positions 332, 451, 574 and 791. Polymorphic site at intron position 791 corresponds to one of the eight restriction sites (MspI) utilized for haplotype construction. Analysis of the MspI allele frequencies in 29 phenylketonuria patients showed that the frequency of the MspI+ allele in this group was 79.4%. Polymorphic sites at nucleotide position +97 from the beginning of intron 10, and at nucleotide position –54 from the end of intron 5, were also described. The polymorphic sites revealed can be used as markers for identification of the PAH alleles in population genetic studies, and also serve for diagnostics of phenylketonuria (PKU). The presence of numerous nucleotide substitutions within the intronic sequences confirms highly polymorphic structure of the PAH gene.     

Detoxication of structurally diverse polycyclic aromatic hydrocarbon (PAH) o-quinones by human recombinant catechol-O-methyltransferase (COMT) via O-methylation of PAH catechols

Polycyclic aromatic hydrocarbons (PAH) are environmental and tobacco carcinogens. Metabolic activation of intermediate PAH trans-dihydrodiols by aldo-keto reductases (AKRs) leads to the formation of electrophilic and redox-active o-quinones. We investigated whether O-methylation by human recombinant soluble catechol-O-methyltransferase (S-COMT) is a feasible detoxication step for a panel of structurally diverse PAH-catechols produced during the redox-cycling process. Classes of PAH non-K-region o-quinones (bay region, methylated bay region, and fjord region o-quinones) produced by AKRs were employed in the studies. PAH o-quinones were reduced to the corresponding catechols by dithiothreitol under anaerobic conditions and then further O-methylated by human S-COMT in the presence of S-[3H]adenosyl-l-methionine as a methyl group donor. The formation of the O-methylated catechols was detected by HPLC-UV coupled with in-line radiometric detection, and unlabeled products were also characterized by LC-MS/MS. Human S-COMT was able to catalyze O-methylation of all of the PAH-catechols and generated two isomeric metabolites in different proportions. LC-MS/MS showed that each isomer was a mono-O-methylated metabolite. 1H NMR was used to assign the predominant positional isomer of benzo[a]pyrene-7,8-catechol as the O-8-monomethylated catechol. The catalytic efficiency (k(cat)/K(m)) varied among different classes of PAH-catechols by 500-fold. The ability of S-COMT to produce two isomeric products from PAH-catechols was rationalized using the crystal structure of the enzyme. We provide evidence that O-8-monomethylated benzo[a]pyrene-7,8-catechol is formed in three different human lung cell lines. It is concluded that human S-COMT may play a critical role in the detoxication of PAH o-quinones generated by AKRs.