Marek Banasik

Specific inhibitors of poly(ADP-ribose) synthetase and mono(ADP-ribosyl)transferase.

Two classes of enzymes, poly(ADP-ribose) synthetase and mono(ADP-ribosyl)transferases, catalyze covalent attachment of multiple or single residues, respectively, of the ADP-ribose moiety of NAD+ to various proteins. In order to find good inhibitors of poly(ADP-ribose) synthetase free of side actions and applicable to in vivo studies, we made a large scale survey using an in vitro assay system, and found many potent inhibitors. The four strongest were 4-amino-1,8-naphthalimide, 6(5H)- and 2-nitro-6(5H)-phenanthridinones, and 1,5-dihydroxyisoquinoline. Their 50% inhibitory concentrations, 0.18-0.39 microM, were about two orders of magnitude lower than that of 3-aminobenzamide that is currently most popularly used. A common structural feature among all potent inhibitors, including 1-hydroxyisoquinoline, chlorthenoxazin, 3-hydroxybenzamide, and 4-hydroxyquinazoline, in addition to the four mentioned above, was the presence of a carbonyl group built in a polyaromatic heterocyclic skeleton or a carbamoyl group attached to an aromatic ring. Most of the inhibitors exhibited mixed-type inhibition with respect to NAD+. Comparative studies of the effects on poly(ADP-ribose) synthetase and mono(ADP-ribosyl)transferase from hen heterophils revealed high specificity of most of the potent inhibitors for poly(ADP-ribose) synthetase. On the other hand, unsaturated long-chain fatty acids inhibited both enzymes, and saturated long-chain fatty acids and vitamin K1 acted selectively on mono(ADP-ribosyl)transferase. The finding of many inhibitors of ADP-ribosyltransferases, especially poly(ADP-ribose) synthetase, supports the view that ADP-ribosylation of proteins may be regulated by a variety of metabolites or structural constituents in the cell.

Vascular Factors and Risk of Dementia: Design of the Three-City Study and Baseline Characteristics of the Study Population

Objective: To describe the baseline characteristics of the participants in the Three-City (3C) Study, a study aiming to evaluate the risk of dementia and cognitive impairment attributable to vascular factors. Methods: Between 1999 and 2001, 9,693 persons aged 65 years and over, noninstitutionalized, were recruited from the electoral rolls of three French cities, i.e. Bordeaux, Dijon and Montpellier. Health-related data were collected during face-to-face interviews using standardized questionnaires. The baseline examination included cognitive testing and diagnosis of dementia, and assessment of vascular risk factors, including blood pressure measurements, ultrasound examination of the carotid arteries, and measurement of biological parameters (glycemia, total, high-density lipoprotein and low-density lipoprotein cholesterol, triglycerides, creatinemia); 3,442 magnetic resonance imaging (MRI) examinations were performed in subjects aged 65–79. Measurements of ultrasound, blood, and MRI parameters were centralized. Two follow-up examinations (at 2 and 4 years) were planned. Results: After exclusion of the participants who had subsequently refused the medical interview, the 3C Study sample consisted of 3,649 men (39.3%) and 5,645 women, mean age 74.4 years, with a relatively high level of education and income. Forty-two percent of the participants reported to be followed up for hypertension, about one third for hypercholesterolemia, and 8% for diabetes; 65% had elevated blood pressure measures (systolic blood pressure ≧140 or diastolic blood pressure ≧90). The proportion of Mini-Mental State Examination scores below 24 was 7% and dementia was diagnosed in 2.2% of the participants. Conclusion: Distribution of baseline characteristics of the 3C Study participants suggests that this study will provide a unique opportunity to estimate the risk of dementia attributable to vascular factors.

Inhibitors and activators of ADP-ribosylation reactions

ADP-ribosylation reaction, that is the transfer of the ADP-ribose moiety of NAD+ to acceptor protein, is catalyzed by two classes of ADP-ribosyltransferases,i.e., poly(ADP-ribose) synthetase and mono (ADP-ribosyl)transferases. These two types differ not only in the number of transferring ADP-ribose units but also in the acceptor amino acid(s) and protein. Their in hibitors, particularly those of poly(ADP-ribose) synthetase, have been successfully employed in studies on biological functions of the enzymes and other related fields of research. Recently, we found many potent and specific inhibitors of poly-(ADP-ribose) synthetase, and broadened their chemical as well as biochemical variety. More recently, we found several potent inhibitors of arginine-specific mono(ADP-ribosyl)transferases and activators of poly(ADP-ribose) synthetase.

Toxicology and human health assessment of decabromodiphenyl ether

Recent public concern has focused on potential reproductive and developmental effects from exposure to low levels of bisphenol A (BPA, CAS number 80-05-7). Two previous published reviews (Gray et al., 2004a; Goodman et al., 2006) conducted weight-of-evidence evaluations of in vivo reproductive/developmental toxicity from BPA exposure < or = 5 mg/kg-d based on studies published through February 2006. Here, an update of those analyses presents additional relevant studies that were published through July 25, 2008, and a weight-of-evidence analysis of the studies evaluated in all three reviews. As with the earlier literature, positive findings: (1) are countered by null findings in more numerous studies; (2) have not been replicated; (3) do not exhibit coherence and plausibility; (4) do not show consistency across species, doses, and time points; and/or (5) were from studies using non-oral exposure routes. Owing to the lack of first-pass metabolism, results from non-oral studies are of limited relevance to human exposure. Exposure levels in most of the low-dose oral and non-oral animal studies are generally much higher than those experienced by even the most exposed people in the general population. The weight of evidence does not support the hypothesis that low oral doses of BPA adversely affect human reproductive and developmental health.We evaluated the available pharmacokinetic data and human and animal toxicity data for 2,2′,3,3′,4,4′,5,5′,6,6′-decabromodiphenyl ether (BDE-209) (CASRN 1163-19-5) with the objective of deriving a reference dose (RfD) based on the best available science. The available studies for deriving an RfD were first screened using the Klimisch criteria and further evaluated using the United States Environmental Protection Agency’s general assessment factors for data quality and relevance (i.e., soundness, applicability and utility, clarity and completeness, uncertainty and variability, and evaluation and review). The chronic 2-year dietary feeding study conducted by the United States National Toxicology Program (, Technical Report Series No. 309) was selected for RfD derivation. Hepatocellular degeneration in male rats was chosen as the critical endpoint in the development of an RfD. For dose-response characterization, we applied benchmark-dose modeling to animal data and determined a point of departure (the 95% lower confidence limit for a 10% increase in hepatocellular degeneration) of 419 mg/kg-day for oral exposures. Based on the similar pharmacokinetic characteristics of BDE-209 across species, this value was converted to a human equivalence dose of 113 mg/kg-day by applying a dosimetric adjustment factor based on body weight scaling to the ¾ power. An oral RfD of 4 mg/kg-day was calculated by using a composite uncertainty factor of 30, which consisted of 10 for intraspecies uncertainty, 3 for interspecies uncertainty (i.e., 3 for toxicodynamics × 1 for toxicokinetics), and 1 for deficiencies with the database. We consider the RfD to be adequately protective of sensitive subpopulations, including women, their fetuses, children, and people with hepatocellular diseases.

Mitochondrial impairment induced by poly(ADP-ribose) polymerase-1 activation in cortical neurons after oxygen and glucose deprivation

Neuronal cells injured by ischemia and reperfusion to a certain extent are committed to death in necrotic or apoptotic form. Necrosis is induced by gross ATP depletion or ‘energy crisis’ of the cell, whereas apoptosis is induced by a mechanism still to be defined in detail. Here, we investigated this mechanism by focusing on a DNA damage‐sensor, poly(ADP‐ribose) polymerase‐1 (PARP‐1). A 2‐h oxygen and glucose deprivation (OGD) followed by reoxygenation (Reox) induced apoptosis, rather than necrosis, in rat cortical neurons. During the Reox, PARP‐1 was much activated and autopoly(ADP‐ribosyl)ated, consuming the substrate, NAD+. Induction of apoptosis by OGD/Reox was suppressed by overexpression of Bcl‐2, indicating mitochondrial impairment in this induction process. Mitochondrial permeability transition (MPT), or membrane depolarization, and a release of proapoptotic proteins, i.e. cytochrome c, apoptosis‐inducing factor and endonuclease G, from mitochondria were observed during the Reox. These apoptotic changes of mitochondria and the nucleus were attenuated by PARP‐1 inhibitors, 1,5‐dihydroxyisoquinoline and benzamide, and also by small interfering RNA specific for PARP‐1. These results indicated that PARP‐1 plays a principal role in inducing mitochondrial impairment that ultimately leads to apoptosis of neurons after cerebral ischemia.

Airborne asbestos concentration from brake changing does not exceed permissible exposure limit

The use in the past, and to a lesser extent today, of chrysotile asbestos in automobile brake systems causes health concerns among professional mechanics. Therefore, we conducted four separate tests in order to evaluate an auto mechanics exposure to airborne asbestos fibers while performing routine brake maintenance. Four nearly identical automobiles from 1960s having four wheel drum brakes were used. Each automobile was fitted with new replacement asbestos-containing brake shoes and then driven over a predetermined public road course for about 2253 km. Then, each car was separately brought into a repair facility; the brakes removed and replaced with new asbestos-containing shoes. The test conditions, methods, and tools were as commonly used during the 1960s. The mechanic was experienced in brake maintenance, having worked in the automobile repair profession beginning in the 1960s. Effects of three independent variables, e.g., filing, sanding, and arc grinding of the replacement brake shoe elements, were tested. Personal and area air samples were collected and analyzed for the presence of fibers, asbestos fibers, total dust, and respirable dust. The results indicated a presence in the air of only chrysotile asbestos and an absence of other types of asbestos. Airborne chrysotile fiber exposures for each test remained below currently applicable limit of 0.1 fiber/ml (eight-hour time-weighted average).

Framework analysis for the carcinogenic mode of action of nitrobenzene.

Nitrobenzene (CASRN: 98-95-3) has been shown to induce cancers in many tissues including kidney, liver, and thyroid, following chronic inhalation in animals. However, with a few exceptions, genotoxicity assays using nitrobenzene have given negative results. Some DNA binding/adduct studies have brought forth questionable results and, considering the available weight of evidence, it does not appear that nitrobenzene causes cancer via a genotoxic mode of action. Nitrobenzene produces a number of free radicals during its reductive metabolism, in the gut as well as at the cellular level, and generates superoxide anion as a by-product during oxidative melabolism. The reactive species generated during nitrobenzene metabolism are considered candidates for carcinogenicity. Furthermore, several lines of evidence suggest that nitrobenzene exerts its carcinogenicity through a non-DNA reactive (epigenetic) fashion, such as a strong temporal relationship between non-, pre-, and neoplastic lesions leading to carcinogenesis. In this report, we first describe the absorption, distribution, metabolism, and excretion of nitrobenzene followed by a summary of the available genotoxicity studies and the only available cancer bioassay. We subsequently refer to the mode of action framework of the U.S. Environmental Protection Agencys 2005 Guidelines for Carcinogen Risk Assessment as a basis for presenting possible modes of action for nitrobenzene-induced cancers of the liver, thyroid, and kidney, as supported by the available experimental data. The rationale(s) regarding human relevance of each mode of action is also presented. Finally, we hypothesize that the carcinogenic mode of action for nitrobenzene is multifactorial in nature and reflective of free radicals, inflammation, and/or altered methylation. †These authors contributed equally to this work.

The application of non-default uncertainty factors in the U.S. EPA's Integrated Risk Information System (IRIS). Part I: UF(L), UF(S), and "other uncertainty factors".

The United States Environmental Protection Agencys Integrated Risk Information System (IRIS) includes hazard identification and dose-response assessment values developed by Agency scientists. Uncertainty factors (UFs) are used in the development of IRIS values to address the lack of information in five main areas. The standard UFs account for interspecies uncertainty (UFA) and intraspecies variability (UFH). The UFA addresses uncertainty related to the extrapolation of data from animals to humans, whereas the UFH addresses variability amongst individuals (i.e., intrahuman). Additional UFs have been employed to account for database incompleteness, extrapolations from a lowest-observed-adverse-effect level in the absence of a no-observed-adverse-effect level (UFL), and subchronic-to-chronic extrapolation (UFS). A sixth UF designated as “other uncertainty factors” (UFO) has also been applied in place of the UFL to account for uncertainty with the adversity of points of departure obtained using benchmark dose modeling. This review will discuss how UFL, UFS, and UFO have been applied in IRIS assessments, along with the rationale used to describe the choice of UF values that deviate from the standard default of 10. †These authors contributed equally to this work.

Dual Inhibitory Effects of Dimethyl Sulfoxide on Poly(ADP-Ribose) Synthetase

Dimethyl sulfoxide (DMSO), a solvent popularly used for dissolving water-insoluble compounds, is a weak inhibitor of poly(ADP-ribose) synthetase, that is a nuclear enzyme producing (ADP-ribose)n from NAD+. The inhibitory mode and potency depend on the concentration of substrate, NAD+, as well as the temperature of the reaction; at micromolar concentrations of NAD+, the inhibition by DMSO is biphasic at 37 degrees C, but is monophasic and apparently competitive with NAD+ at 25 degrees C. DMSO, on the other hand, diminishes dose-dependently and markedly the inhibitory potency of benzamide and other inhibitors. Other organic solvents, ethanol and methanol, also show a biphasic effect on the synthetase activity at different concentrations.

Septin 3 gene polymorphism in Alzheimer's disease.

Septin 3 is a novel member of the septin subfamily of GTPase domain proteins that was recently identified in human neuronal cells. These proteins are involved in vesicle trafficking, neurite outgrowth, and neurofibrillary tangle formation; however, the expression and functional role of septin 3 in normal neuronal tissues and as an etiological agent in neurological disorders is currently unclear. To further characterize these parameters, the present study analyzed the expression of three isoforms of septin 3 (A, B, and C) in fetal and adult human brains and polymorphism of the septin 3 exon 11 microsatellite in control, pure Alzheimers disease (AD), Lewy body variant (LBV) of AD, and Parkinsons disease. Septin 3 mRNAs for isoforms A and B, but not C, were detected in the frontal cortex of fetus and adult human samples, as measured by reverse transcription-coupled polymerase chain reaction. Genotype analyses indicated that polymorphic septin 3 alleles were distributed in two peaks of frequency in both control and disease groups. Categorization of the alleles into short (S) and long (L) types revealed a significant difference between AD patients and controls (p = 0.034 by chi-square test). Furthermore, the S-allele homozygosity was significantly underrepresented in AD compared with control (p = 0.015 by chi-square test). These results suggest that polymorphism in exon 11 of septin 3 may have a determinative role in the pathogenesis of AD.