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Biomedical subjects

D W Roberts

Publications and source records attributed to D W Roberts.

At least 19 recordsLinked to original sources
PubMed1999

Repeat exposure to incremental doses of acetaminophen provides protection against acetaminophen-induced lethality in mice: an explanation for high acetaminophen dosage in humans without hepatic injury.

In studies designed to simulate a clinical observation in which an individual became tolerant to normally lethal doses of acetaminophen (APAP), mice were pretreated with increasing doses of APAP for 8 days and challenged on day 9 with normally supralethal doses of APAP. These animals developed minimal hepatotoxicity after a challenge dose with a fourfold increase in LD50 to 1,350 mg/kg. The pretreatment regimen resulted in hepatic changes including: centrilobular localization of 3-(cysteine-S-yl)APAP protein adducts, selective down-regulation of cytochrome P4502E1 (CYP2E1) and CYP1A2 that produced the toxic metabolite, N-acetyl-p-benzoquinone imine, higher levels of reduced glutathione (GSH), centrilobular inflammation, and a fourfold increase in hepatocellular proliferation. The protection against the lethal APAP doses afforded by pretreatment is secondary to these changes and to the associated regional shift in the bioactivation of the APAP challenge dose from centrilobular to periportal regions where CYP2E1 is not found, protective GSH is more abundant, and where cell-proliferative responses are better able to sustain repair. This shift in APAP bioactivation results in less-intense covalent binding that is more diffuse and spread uniformly throughout the hepatic lobe, most likely contributing to protection by delaying the early onset of liver injury that has been generally associated with centrilobular localization of the adducts. Intervention of APAP pretreatment-induced cell division in mice with colchicine left them resistant to a 500-mg/kg (normally lethal) dose of APAP, but unable to survive a 1,000-mg/kg APAP challenge dose. The data demonstrate multiple mechanistic components to the protection afforded by APAP pretreatment. Whereas metabolic and physiological changes not dependent on cell proliferation are adequate to protect against 500 mg/kg APAP, these changes plus a potentiated cell-proliferative response are necessary for protection against the supralethal 1,000-mg/kg APAP dose. Furthermore, the data document an uncoupling of the traditional association between covalent binding and toxicity, and suggest that the assessment of toxicity following repeated or chronic APAP exposure must consider altered drug interactions and parameters besides those historically used to assess acute APAP overdose.

Acetaminophen↗
PubMed1999

A computational model for tracking subsurface tissue deformation during stereotactic neurosurgery.

Recent advances in the field of stereotactic neurosurgery have made it possible to coregister preoperative computed tomography (CT) and magnetic resonance (MR) images with instrument locations in the operating field. However, accounting for intraoperative movement of brain tissue remains a challenging problem. While intraoperative CT and MR scanners record concurrent tissue motion, there is motivation to develop methodologies which would be significantly lower in cost and more widely available. The approach we present is a computational model of brain tissue deformation that could be used in conjunction with a limited amount of concurrently obtained operative data to estimate subsurface tissue motion. Specifically, we report on the initial development of a finite element model of brain tissue adapted from consolidation theory. Validations of the computational mathematics in two and three dimensions are shown with errors of 1%-2% for the discretizations used. Experience with the computational strategy for estimating surgically induced brain tissue motion in vivo is also presented. While the predicted tissue displacements differ from measured values by about 15%, they suggest that exploiting a physics-based computational framework for updating preoperative imaging databases during the course of surgery has considerable merit. However, additional model and computational developments are needed before this approach can become a clinical reality.

Animals↗
PubMed1998

Intraoperative brain shift and deformation: a quantitative analysis of cortical displacement in 28 cases.

OBJECTIVE: A quantitative analysis of intraoperative cortical shift and deformation was performed to gain a better understanding of the nature and extent of this problem and the resultant loss of spatial accuracy in surgical procedures coregistered to preoperative imaging studies. METHODS: Three-dimensional feature tracking and two-dimensional image analysis of the cortical surface were used to quantify the observed motion. Data acquisition was facilitated by a ceiling-mounted robotic platform, which provided a number of precision tracking capabilities. The patient's head position and the size and orientation of the craniotomy were recorded at the start of surgery. Error analysis demonstrated that the surface displacement measuring methodology was accurate to 1 to 2 mm. Statistical tests were performed to examine correlations between the amount of displacement and the type of surgery, the nature of the cranial opening, the region of the brain involved, the duration of surgery, and the degree of invasiveness. RESULTS: The results showed that a displacement of an average of 1 cm occurred, with the dominant directional component being associated with gravity. The mean displacement was determined to be independent of the size and orientation of the cranial opening. CONCLUSION: These data suggest that loss of spatial registration with preoperative images is gravity-dominated and of sufficient extent that attention to errors resulting from misregistration during the course of surgery is warranted.

Adult↗
PubMed1997

Isolation of a cDNA encoding a novel subtilisin-like protease (Pr1B) from the entomopathogenic fungus, Metarhizium anisopliae using differential display-RT-PCR.

Reverse transcription differential display PCR (RT-DD-PCR) was used to identify genes that are specifically expressed by Metarhizium anisopliae when it contacts the host insect cuticle. Using a homology-based subtilisin-like protease primer we identified a hitherto unsuspected differentially expressed subtilisin-like protease (Pr1B) encoding gene. The deduced amino acid sequence shows 54% similarity to the well characterized Pr1A subtilisin of M. anisopliae and karyotype analysis revealed that Pr1A and Pr1B are located on separate chromosomes. Like Pr1A, Pr1B is synthesized as a large precursor (1158 nucleotides; deduced molecular mass = 40031 Da) containing a signal peptide, a propeptide and the mature protease (283 aa; deduced molecular mass = 28714 Da). However, Pr1B possesses several substitutions in the highly conserved sequences comprising the active sites of subtilisins. In particular, the substitution of Thr220 by serine is unique to Pr1B. Substitution of Asn155 by glycine is also very unusual, and we discuss the likely effects these changes will have on the catalytic efficiency of Pr1B.

Amino Acid Sequence↗
PubMed1997

Immunochemical, 32P-postlabeling, and GC/MS detection of 4-aminobiphenyl-DNA adducts in human peripheral lung in relation to metabolic activation pathways involving pulmonary N-oxidation, conjugation, and peroxidation.

4-Aminobiphenyl (ABP) is a recognized human bladder carcinogen, whose presence in cigarette smoke results in DNA adduct formation in the human urothelium. Since preliminary studies indicated that even higher levels of ABP-DNA adducts may be present in human peripheral lung, we utilized a sensitive immunochemical assay, in combination with 32P-postlabeling, to quantify the major 4-aminobiphenyl (ABP)-DNA adduct, N-(guan-8-yl)-ABP, in surgical samples of peripheral lung tissue from smokers and ex-smokers. No differences in adduct levels were detected between smokers and ex-smokers by immunoassay. In contrast, the 32P-postlabeling method showed statistically significant differences between adduct levels in smokers and ex-smokers; however, a relatively high background of smoking-related adducts chromatograph near the major ABP adducts and may compromise estimation of the level of ABP-DNA adducts in smokers. Furthermore, the levels measured by 32P-postlabeling were 20- to 60-fold lower than that measured by immunoassay. Since 32P-postlabeling may underestimate and immunochemical assays may overestimate adduct levels in the lung, selected samples were also evaluated by GC/MS. The immunochemical and GC/MS data were concordant, leading us to conclude that N-(guan-8-yl)-ABP adducts were not related to smoking status. Since ABP-DNA adduct levels in human lung did not correlate with smoking status as measured by immunoassay and GC/MS, the metabolic activation capacity of human lung microsomes and cytosols was examined to determine if another exposure (e.g., 4-nitrobiphenyl) might be responsible for the adduct. The rates of microsomal ABP N-oxidation were below the limit of detection, which was consistent with a lack of detectable cytochrome P4501A2 in human lung. N-Hydroxy-ABP O-acetyltransferase (but not sulfotransferase) activity was detected in cytosols and comparative measurements of N-acetyltransferase (NAT) using p-aminobenzoic acid and sulfamethazine indicated that NAT1 and NAT2 contributed to this activity. 4-Nitrobiphenyl reductase activity was found in lung microsomes and cytosols, with the reaction yielding ABP and N-hydroxy-ABP. Lung microsomes also demonstrated high peroxidative activation of ABP, benzidine, 4,4'-methylene-bis(2-chloroaniline), 2-aminofluorene, and 2-naphthylamine. The preferred co-oxidant was hydrogen peroxide and the reaction was strongly inhibited by sodium azide but not by indomethacin or eicosatetraynoic acid, which suggested the primary involvement of myeloperoxidase rather than prostaglandin H synthase or lipoxygenase. This was confirmed by immunoinhibition and immunoprecipitation studies using solubilized human lung microsomes and antisera specific for myeloperoxidase. These data suggest that ABP-DNA adducts in human lung result from some environmental exposure to 4-nitrobiphenyl. The bioactivation pathways appear to involve: (1) metabolic reduction to N-hydroxy-ABP and subsequent O-acetylation by NAT1 and/or NAT2; and (2) metabolic reduction to ABP and subsequent peroxidation by myeloperoxidase. The myeloperoxidase activity appears to be the highest peroxidase activity measured in mammalian tissue and is consistent with the presence of neutrophils and polymorphonuclear leukocytes surrounding particulate matter derived from cigarette smoking.

Acyltransferases↗
PubMed1997

Comparison of covalent binding of acetaminophen and the regioisomer 3'-hydroxyacetanilide to mouse liver protein.

The hepatotoxicity of the analgesic acetaminophen has been previously attributed to metabolic activation by cytochrome P450 to the reactive intermediate N-acetyl-p-benzoquinone imine. At therapeutic doses this species is detoxified by reaction with glutathione; however, following a hepatotoxic dose, liver glutathione levels are depleted and the metabolite covalently binds primarily to cysteine groups on proteins as 3-(cystein-S-yl)acetaminophen adducts. Altered function of critical proteins has been postulated to be the mechanism of hepatotoxicity. Covalent binding has been studied by both radiochemical methods and immunochemical methods. Utilizing Western blot analysis with an antiserum which recognizes acetaminophen we have previously shown that covalent binding occurs on a number of proteins in various hepatic fractions. In an effort to better understand the role of covalent binding in the toxicity, others have studied the non-hepatotoxic isomer 3'-hydroxyacetanilide. Administration of large doses of radiolabeled acetaminophen or 3'-hydroxyacetanilide resulted in similar levels of covalent binding to proteins. To better understand the role of covalent binding in toxicity we have administered mice 3'-hydroxyacetanilide and acetaminophen, and analyzed liver fractions for protein adducts using anti-3-(cystein-S-yl)acetaminophen and anti-arylacetamide antisera in Western blot assays. Analysis of liver fractions from acetaminophen-treated mice, with both antisera showed, as has been previously reported, that acetaminophen covalently binds to a number of hepatic proteins. In liver from 3'-hydroxyacetanilide-treated mice, covalent adducts were detected with an anti-arylacetamide antiserum only. A major 3'-hydroxyacetanilide protein adduct was observed in microsomes at 50 kDa. Minor adducts were observed at 47 kDa in microsomes and 56 kDa in cytosol. 3'-Hydroxyacetanilide protein adducts were not observed in the 10,000 x g pellet. Densitometric analysis of a time course of 3'-hydroxyacetanilide protein adducts indicated that peak levels of the 50 kDa microsomal protein adduct occurred at 1 h and subsequently decreased.

Acetaminophen↗
PubMed1997

Skin sensitization to eugenol and isoeugenol in mice: possible metabolic pathways involving ortho-quinone and quinone methide intermediates.

With the aim of providing further mechanistic insights into the mode of action of eugenol (4-allyl-2-methoxyphenol) and isoeugenol (4-propenyl-2-methoxyphenol), we have synthesized two series of modified compounds which were tested in the mouse local lymph node assay for their skin sensitizing potential. The replacement of the methoxy group by an isopropoxy group led to a complete loss of sensitization for the eugenol derivative 6a, while no significant effect was observed for the isoeugenol derivative 6b. In the eugenol series, when methyl groups were present in the 3-, 5-, or 6-position a significant reduction in sensitization potential was observed while in the isoeugenol series only methyl substitution in the 3- and 5-position had a discernable effect. Introduction of three methyl groups on the aromatic ring of eugenol (3,5,6-trimethyl-4-allyl-2-methoxyphenol, 7) and of a tert-butyl substituent at the gamma-position of the alkyl chain of isoeugenol (4-[3',3',3'-trimethylpropenyl]-2-methoxyphenol, 8) led to a strong decrease of the sensitizing capacity. Our findings indicate that, at least in the mouse, eugenol could sensitize via a demethylation pathway followed by oxidation to the o-quinone which could act directly as a hapten even if we cannot exclude a reaction via its tautomeric p-quinone methide. Isoeugenol, on the other hand, could act via a mechanism not involving demethylation and for which the evidence is consistent with a direct oxidation to the p-quinone methide.

Animals↗
PubMed1997

Molecular orbital parameters as predictors of skin sensitization potential of halo- and pseudohalobenzenes acting as SNAr electrophiles.

The electrophilic reactivity of a training set of 20 halo- and pseudohalobenzenes, 10 of which are reported skin sensitizers and 10 of which are reported nonsensitizers, has been modeled by MO-calculated indices using the AM1 and PM3 Hamiltonians. The electronic structures of parent molecules and the corresponding Meisenheimer intermediates (sigma-complexes) were evaluated. The NH2 group and the H atom were both studied as model nucleophile-derived substituents in the sigma-complexes. The LUMO energy differences between the parent compounds and their Meisenheimer complexes together with the maximum acceptor superdelocalizabilities determined over the aromatic reaction sites were found to discriminate correctly the sensitizing/reactive from nonsensitizing/unreactive compounds of the training set of 20 compounds. The predictive applicability of these MO indices was confirmed with a test set of seven further compounds for which sensitization data are reported in the literature. A statistically based discriminant analysis provides a model which predicts whether or not an SNAr electrophile will be a sensitizer and estimates the degree of confidence in the prediction.

Aniline Compounds↗
PubMed1997

Further evaluation of the quantitative structure-activity relationship for skin-sensitizing alkyl transfer agents.

The biological activity of skin-sensitizing chemicals can be expressed in terms of physicochemical properties which relate to the propensity of those chemicals to behave as electrophiles and which describe their ability to partition into the epidermis and between compartments within it. For defined series of chemicals, it has proved possible to express such structure-activity relationships quantitatively. Such quantitative relationships can provide valuable insights into the mechanisms of skin sensitization and/or are of use in predictive toxicology. In the present work the quantitative structure-activity relationship (QSAR) previously derived for a series of alkyl transfer agents based on alkanesulfonate leaving groups has been critically examined in the light of skin sensitization data obtained for new members of that series and also for alkyl transfer agents based on different leaving groups. The QSAR predictions were broadly accurate, but demonstrated that further refinement was both necessary and possible. In particular, the physicochemical parameters which relate to the disposition of the chemical in the epidermis, i.e., its penetration through the stratum corneum, cell surface/cytoplasmic distribution and the associated dynamics, will need to be understood more fully in order to enhance the precision of the QSAR and its predictive power.

Alkanesulfonates↗
PubMed1996

Construction of an improved mycoinsecticide overexpressing a toxic protease.

Mycoinsecticides are being used for the control of many insect pests as an environmentally acceptable alternative to chemical insecticides. A key aim of much recent work has been to increase the speed of kill and so improve commercial efficacy of these biocontrol agents. This might he achieved by adding insecticidal genes to the fungus, an approach considered to have enormous potential for the improvement of biological pesticides. We report here the development of a genetically improved entomopathogenic fungus. Additional copies of the gene encoding a regulated cuticle-degrading protease (Pr1) from Metarhizium anisopliae were inserted into the genome of M. anisopliae such that Pr1 was constitutively overproduced in the hemolymph of Manduca sexta, activating the prophenoloxidase system. The combined toxic effects of Pr1 and the reaction products of phenoloxidase caused larvae challenged with the engineered fungus to exhibit a 25% reduction in time of death and reduced food consumption by 40% compared to infections by the wild-type fungus. In addition, infected insects were rapidly melanized, and the resulting cadavers were poor substrates for fungal sporulation. Thus, environmental persistence of the genetically engineered fungus is reduced, thereby providing biological containment.

Amino Acid Sequence↗
PubMed1996

Fate of biological control introductions: monitoring an Australian fungal pathogen of grasshoppers in North America.

In North America there are two generally recognized pathotypes (pathotypes 1 and 2) of the fungus Entomophaga grylli which show host-preferential infection of grasshopper subfamilies. Pathotype 3, discovered in Australia, has a broader grasshopper host range and was considered to be a good biocontrol agent. Between 1989 and 1991 pathotype 3 was introduced at two field sites in North Dakota. Since resting spores are morphologically indistinguishable among pathotypes, we used pathotype-specific DNA probes to confirm pathotype identification in E. grylli-infected grasshoppers collected at the release sites in 1992, 1993, and 1994. In 1992, up to 23% of E. grylli-infected grasshoppers of the subfamilies Melanoplinae, Oedipodinae, and Gomphocerinae were infected by pathotype 3, with no infections > 1 km from the release sites. In 1993, pathotype 3 infections declined to 1.7%. In 1994 grasshopper populations were low and no pathotype 3 infections were found. The frequency of pathotype 3 infection has declined to levels where its long-term survival in North America is questionable. Analyses of biocontrol releases are critical to evaluating the environmental risks associated with these ecological manipulations, and molecular probes are powerful tools for monitoring biocontrol releases.

Animals↗
PubMed1996

Acetaminophen toxicity in children: diagnostic confirmation using a specific antigenic biomarker.

Chronic acetaminophen (APAP) toxicity poses a difficult diagnostic challenge to the clinician. Signs and symptoms are nonspecific and no currently available laboratory study can confirm APAP as the causative agent of hepatic injury. In this study an antigenic biomarker for APAP toxicity was used to confirm the diagnosis of APAP-induced hepatic failure in two children with chronic APAP toxicity. APAP that has been metabolized to N-acetyl-benzoquinone imine (NAPQI) reacts with cellular proteins to form 3-(cystein-S-yl)-APAP protein adducts (3-Cys-A). Serum from both patients was submitted for quantitation of 3-Cys-A by a competitive inhibition enzyme-linked immunosorbent assay (ELISA). Concentrations of 3-Cys-A in the two patients were 1.97 and 2.77 nmol/mg protein, which are similar to concentrations found in adults with hepatic injury secondary to an overdose of APAP. Individuals with no exposure to APAP have no detectable 3-Cys-A in serum. It was concluded that 3-Cys-A is a useful marker of APAP intoxication after long-term ingestion of APAP when total dose and time course of ingestion are uncertain, and may prove to be a useful clinical and investigative tool in the study of APAP intoxication.

Acetaminophen↗
PubMed1996

Preoperative ketorolac increases bleeding after tonsillectomy in children.

PURPOSE: To compare the incidence of vomiting following codeine or ketorolac for tonsillectomy in children. METHODS: We had planned to enrol 240 patients, aged 2-12 yr undergoing elective tonsillectomy into a randomized, single-blind study in University Children's Hospital. The study was terminated, after 64 patients because interim analysis of the data by a blinded non-study scientist concluded that the patients were at undue risk of excessive perioperative bleeding. After induction of anaesthesia by inhalation with N2O/halothane or with propofol 2.5-3.5 mg.kg-1 i.v., the children were administered 150 micrograms.kg-1 ondansetron and 50 micrograms.kg-1 midazolam. Maintenance of anaesthesia was with N2O and halothane in O2. Subjects were administered either 1.5 mg.kg-1 codeine im or 1 mg.kg-1 ketorolac i.v. before the commencement of surgery. Intraoperative blood loss was measured with a Baxter Medi-Vac Universal Critical Measurement Unit. Postoperative management of vomiting and pain was standardized. Vomiting was recorded for 24 hr after anaesthesia. Data were compared with ANOVA, Chi-Square analysis and Fisher Exact Test. RESULTS: Thirty-five subjects received ketorolac. Demographic data were similar. The incidence of vomiting during the postoperative period was 31% in the codeine-group and 40% in the ketorolac-group. Intraoperative blood losses was 1.3 +/- 0.8 ml.kg-1 after codeine and 2.2 +/- 1.9 ml.kg-1 after ketorolac (mean +/- SD) P < 0.05. Five ketorolac-treated patients had bleeding which led to unscheduled admission to hospital, P < 0.05, Exact Test. CONCLUSION: Preoperative ketorolac increases perioperative bleeding among children undergoing tonsillectomy without beneficial effects.

Analgesics, Non-Narcotic↗
PubMed1996

Biochemical characterization and ultrastructural localization of two extracellular trypsins produced by Metarhizium anisopliae in infected insect cuticles.

Proteinase 2 (Pr2) is a fungal (Metarhizium anisopliae) serine proteinase which has a tryptic specificity for basic residues and which may be involved in entomopathogenicity. Analytical and preparative isoelectric focusing methods were used to separate two trypsin components, produced during growth on cockroach cuticle, with isoelectric points of 4.4 (molecular mass, 30 kDa) and 4.9 (27 kDa). The catalytic properties of the proteases were analyzed by their kinetic constants and by a combination of two-dimensional gelatin-sodium dodecyl sulfate-polyacrylamide gel electrophoresis and enzyme overlay membranes. Both Pr2 isoforms preferentially cleave at the carboxyl sides of positively charged amino acids, preferring arginine; the pI 4.4 Pr2 isoform also possessed significant activity against lysine. Compared with the pathogen's subtilisin-like enzyme (Pr1), the pI 4.4 Pr2 isoform shows low activity against insoluble proteins in a host (Manduca sexta) cuticle. However, it degrades most cuticle proteins when they are solubilized, with high-molecular-weight basic proteins being preferentially hydrolyzed. Polyclonal antibodies raised against each Pr2 isoform were isotype specific. This allowed us to use ultrastructural immunocytochemistry to independently visualize each isoform during penetration of the host (M. sexta) cuticle. Both isoforms were secreted by infection structures (appressoria) on the cuticle surface and by the penetrant hyphae within the cuticle. The extracellular sheath, which is commonly observed around fungal cells, often contained Pr2 molecules. Intracellular labelling was sparse.

Amino Acid Sequence↗
PubMed1996

Acetaminophen-induced hepatotoxicity. Analysis of total covalent binding vs. specific binding to cysteine.

Acetaminophen-induced hepatotoxicity is believed to be mediated by covalent binding of the reactive metabolite N-acetyl-p-benzoquinone imine to essential proteins in liver. It has been shown that the primary reaction of this metabolite with hepatic proteins is the formation of 3-(cysteine-S-yl)-acetaminophen adducts. The importance of covalent binding to other amino acids that may be formed by reaction of N-acetyl-p-benzoquinone imine with protein is unclear. Previously, we developed immunochemical assays for the acetaminophen cysteine adducts by immunizing animals with the conjugate 3-(N-acetylcystein-S-yl)acetaminophen-keyhole limpet hemocyanin, wherein the carboxyl group of the N-acetyl-cysteine moiety was coupled to amino groups on the protein. A very sensitive and specific immunochemical assay was developed for acetaminophen specifically bound to cysteine groups on protein [3-(cystein-S-yl)acetaminophen protein adducts]. Analysis of protein adducts indicated that after toxic doses, acetaminophen covalently bound at high levels to cysteine residues on a relatively small number of hepatic proteins. In the present work, a new antiacetaminophen antiserum was prepared by immunizing mice with 4-acetamidobenzoic acid coupled to keyhole limpet hemocyanin. Competitive ELISA data indicate that the resulting antiserum has excellent recognition of acetaminophen and related arylacetamide derivatives. Using this new antiserum, Western blot analyses of liver proteins from acetaminophen-intoxicated mouse livers were performed and compared with similar assays using the anti-3-(cystein-S-yl)acetaminophen antiserum. Visual and densitometric analyses of the Western blots indicate that the two antisera detect the same primary acetaminophen protein adducts; however, minor differences in the intensity of certain bands were observed. These differences may represent either differences in antibody accessibility to 3-(cystein-S-yl)acetaminophen adducts or differences in the proportion of acetaminophen bound to cysteine vs. binding to other amino acids.

Acetaminophen↗