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R W Benson

Publications and source records attributed to R W Benson.

At least 37 records · Page 2Linked to original sources
PubMed1988

Immunochemical quantitation of DNA adducts derived from the human bladder carcinogen 4-aminobiphenyl.

To assess target-tissue exposure to the human urinary bladder carcinogen 4-aminobiphenyl (ABP), we have developed a sensitive immunochemical method for measuring the major arylamine-DNA adduct formed, N-(guan-8-yl)-ABP (Gua-C8-ABP). High-affinity polyclonal antisera from rabbits immunized with N-(guanosin-8-yl)-ABP coupled to keyhole limpet hemocyanin were characterized and shown to have high specificity for antigenic determinants on the purine and biphenyl rings of Gua-C8-ABP and minimal cross-reactivity with ABP, deoxyguanosine, or hydrolyzed DNA. Assay standards containing ABP-modified DNA were prepared by reacting [3H]N-hydroxy-ABP with calf thymus DNA. DNA samples were hydrolyzed with trifluoroacetic acid and dried under vacuum, and the residues were dissolved in dimethyl sulfoxide under argon. Using a streptavidin-biotin amplified enzyme-linked immunosorbent assay, DNA hydrolysates competed at 25 micrograms DNA/microtiter well for a limiting amount of anti-keyhole limpet hemocyanin-(Gua-C8-ABP) in the presence of excess solid-phase bovine serum albumin-(Gua-C8-ABP) coating antigen. The limit of sensitivity for this assay using 25 micrograms DNA was 2 adducts/10(8) nucleotides. Gua-C8-ABP adducts in liver and bladder epithelial DNAs were readily quantified after p.o. administration of 5 mg/kg ABP to dogs. This methodology is capable of detecting adducts at levels of biological significance and should be applicable to human target-tissue dosimetry.

Aminobiphenyl Compounds↗
PubMed1988

Behaviorally conditioned suppression of murine T-cell dependent but not T-cell independent antibody responses.

The aversive and immunosuppressive effects of cyclophosphamide (CY, 250 mg/kg IP), an unconditioned stimulus (UCS), were paired with the presentation of a novel saccharine flavored drinking solution (SAC), a conditioned stimulus (CS), in female Balb/c mice. The objective was to determine the temporal relationship between presentation of the CS (SAC) and immunization with sheep red blood cell (SRBCs), a T-cell dependent antigen, and type III pneumococcal polysaccharide (S3), a T-cell independent antigen, on subsequent antibody responses. Reexposure to the CS or UCS occurred on days -4, -2, 0, +2, or +4 relative to immunization. Primary antibody responses in each group were measured six days following immunization. A strong association between the CS and the UCS developed, producing flavor aversions as evidenced by decreased SAC consumption. CY administration by itself consistently suppressed both types of antibody responses. CS presentation (i.e., SAC) had no significant effect on anti-S3 antibody response. However, the anti-SRBC response was significantly depressed following CS exposure. Exposure to the CS only on days -4 or +2 relative to immunization resulted in statistically significant suppression of antibody response to SRBC's while exposure on days -2, 0, and +4 resulted in anti-SRBC antibody suppression that did not reach significance. These results support the hypothesis that conditioning of antibody responses is relatively specific for T-cell dependent antigens, and that the timing of CS presentation relative to immunization is important in conditioning a suppression of antibody responses.

Animals↗
PubMed1988

Assessment of exposure and susceptibility to aromatic amine carcinogens.

As a consequence of human exposure to carcinogenic aromatic amines, biochemical approaches to risk assessment have emphasized metabolic determinants of individual susceptibility and quantification of arylamine-macromolecular adducts. A known genetic polymorphism in humans, hepatic arylamine acetyltransferase activity, has been associated with differences in individual susceptibility to urinary bladder (slow acetylators) and colorectal (rapid acetylators) cancers. Similarly, the high specificity of an inducible human cytochrome P450 towards the N-oxidation of 4-aminobiphenyl and other aromatic amines is consistent with metabolic differences that can be used to predict relative human risk. Exposure to aromatic amines has also been documented, primarily by quantification of adducts with protein or DNA. Using 32P-postlabelling methods and a competitive avidin/biotin-amplified enzyme-linked immunoassay, we have estimated 4-aminobiphenyl-DNA adduct levels in surgical samples of human peripheral lung and urinary bladder epithelium and report values ranging from 2 to 97 adducts per 10(8) nucleotides.

Acetylation↗
PubMed1987

A sensitive immunochemical assay for acetaminophen-protein adducts.

The hepatotoxicity of acetaminophen may be mediated by the reactive metabolite N-acetyl-p-benzoquinone imine which binds covalently to protein primarily as 3-(cystein-S-yl)acetaminophen. We have developed an avidin biotin-amplified competitive enzyme-linked immunosorbent assay to detect protein-bound acetaminophen. This assay utilizes antisera from rabbits immunized with 3-(N-acetyl-L-cystein-S-yl)acetaminophen coupled via the carboxyl group to primary amino groups on the protein keyhole-limpet hemocyanin. The competitive enzyme-linked immunosorbent assay utilizes metallothionein derivatized with N-acetyl-p-benzoquinone imine (acetaminophen-bound metallothionein) and quantitation was obtained by competition of acetaminophen-derivatives for a limited amount of antibody in the presence of excess solid phase acetaminophen-bound metallothionein. Synthetic 3-(N-acetyl-L-cystein-S-yl)acetaminophen, acetaminophen bound to mouse 9,000 X g supernatant, 100,000 X g supernatant, microsomes, as well as acetaminophen-bound metallothionein were inhibitory. The 50% inhibition for 3-(N-acetyl-L-cystein-S-yl)acetaminophen was 110 fmol/well. In contrast, free acetaminophen was 6200 times less efficient as an inhibitor. The mean 50% inhibition for protein-bound acetaminophen was 2.89 pmol/well. The utility of the method to detect acetaminophen-protein adducts in biological samples was confirmed by detection of NADPH-dependent binding of acetaminophen to microsomal proteins.

Acetaminophen↗
PubMed1986

Development of an avidin-biotin amplified enzyme-linked immunoassay for detection of DNA adducts of the human bladder carcinogen 4-aminobiphenyl.

4-Aminobiphenyl (ABP) is a known human urinary bladder carcinogen which is present in tobacco smoke and may be ubiquitous in the environment. As a biological monitor of carcinogen exposure, we have developed an immunological method for measuring the predominant carcinogen-DNA adduct of ABP, N-(deoxyguanosin-8-yl)-ABP (dG-C8-ABP). Rabbits were immunized with keyhole limpet hemocyanin (KLH) conjugate prepared by a periodate oxidation and coupling of N-(guanosin-8-yl)-ABP (rG-C8-ABP) to the protein. The resulting polyclonal antisera was systematically characterized using dual inhibitor methodology augmented by specialized computer and software support; and a competitive avidin-biotin enzyme-linked immunoassay (A-B ELISA) assay employing polyclonal rabbit anti-KLH-(rG-C8-ABP) was developed. Under the assay conditions described, the detection limit for dG-C8-ABP was 18 fmol/well. The relative lack of reactivity toward ABP, N-acetyl-4-aminobiphenyl, N-(deoxyadenosin-8-yl)-ABP, N-(deoxyguanosin-8-yl)-2-aminofluorene and deoxyguanosine as inhibitors indicated that primary specificity involves epitopes found on the purine and biphenyl rings. Results emphasize the need to define polyclonal anti-adduct sera operationally in the context of the antigen/assay system used to evaluate it. Assay sensitivity was achieved by decreasing the amount of antibody and solid-phase antigen in the competitive portion of the assay and the use of avidin-biotin as well as enzymatic amplification. This methodology is a useful alternative to other ultrasensitive techniques and should be directly applicable to the detection of ABP-DNA adducts in exposed human populations.

Aminobiphenyl Compounds↗
PubMed1982

Evaluation of the immune response to type III pneumococcal polysaccharide as a means to evaluate T-independent immune function in the rat.

Since the rat is frequently the experimental animal of choice for toxicology testing, studies were undertaken to adapt assays routinely used to evaluate immune function in mice so that immune function could likewise be evaluated in collaborative projects employing toxicant-treated rats. Contrary to previous reports in the literature, Type III pneumococcal polysaccharide (S3) was immunogenic in rats. Specific antibody responses to S3 were demonstrated in two strains of rats following immunization by either the subcutaneous (sc) or intraperitoneal (ip) route with purified S3, with S3 contained in polyvalent pneumococcal polysaccharide vaccine (pneumovax), or with heat-killed Type III Streptococcus pneumoniae. Dose-response studies demonstrated that the optimal immunizing dose in Sprague-Dawley or Fischer rats was 25 micrograms S3. Reimmunization with S3 on d 21 did not produce an anamnestic response, and the kinetic data were consistent with S3 being a thymus-independent (T-independent) antigen in the rat. In contrast to our previous studies in the mouse, concurrent sc or ip injections of pertussis vaccine did not modify the response to S3 in rats. Sprague-Dawley rats acquired the capacity to respond immunologically to S3 between 24 and 31 d of age. In mature animals, sex had no effect on the ability to respond to S3. The utility of this model as a means of characterize toxicant-induced immune dysfunction was demonstrated using the prototype immunotoxicant cyclo-phosphamide.

Aging↗
PubMed1979

Modification of antibody response to type III pneumopolysaccharide by route of injection of pertussis vaccine.

Pertussis vaccine (PV) or diphtheria toxoid-PV-tetanus toxoid (DPT) altered the antibody response of BALB/c female mice to type III pneumococcal polysaccharide antigen (S3). The key factor affecting the magnitude of the response to S3 was the route of injection of PV or DPT, whereas the route of injection of S3 was not crucial. Subcutaneous injections of DPT augmented the antibody response to low, optimal, and tolerogenic (high) doses of S3 injected either subcutaneously or intraperitoneally. This enhancement was persistent and was observed both when S3 and DPT were mixed and injected subcutaneously and when S3 and DPT were injected concurrently at separate subcutaneous sites. When either PV or DPT was injected intraperitoneally, the antibody response to subcutaneously or intraperitoneally injected S3 was significantly decreased. These experiments demonstrate a dichotomy of effect dependent on the route of administration of PV. Most studies in mice utilizing PV employ the intraperitoneal injection route, and it is important to consider whether PV treatment by this route may have unique effects. Our data suggest that intraperitoneal injection of PV suppresses B cells, possibly by influencing regulatory cell function.

Animals↗
PubMed1978

Saccharomyces cerevisiae DNA-dependent RNA polymerase III: a zinc metalloenzyme.

Yeast nuclear RNA polymerase III was purified by batch adsorption to phosphocellulose, followed by ion-exchange chromatography on DEAE-Sephadex and affinity chromatography on DNA-Sepharose. Polyacrylamide gel electrophoresis of the purified enzyme showed a single protein band which contained polymerase activity. The molecular weight estimated by sedimentation velocity centrifugation in a glycerol gradient was 380 000. Enzyme activity was inhibited 50% at 0.1 mM 1,10-phenanthroline and 100% of 1.0 mM, but was restored when 1,10-phenanthroline was removed by dialysis. Enzyme activity was not inhibited by 7,8-benzoquinoline, a nonchelating structural analogue of 1,10-phenanthroline. These results strongly suggest that inhibition of enzyme activity occurs by the formation of a reversible enzyme-zinc-phenanthroline ternary complex. The zinc content, measured by atomic absorption spectroscopy, was 2 g-atoms per mol of enzyme. Zinc was not removed from the enzyme by gel filtration on Sephadex G-25, by passage through Chelex-100 resin, or by dialysis against buffer containing 1,10-phenanthroline. Enzyme-bound zinc was removed by dialysis after denaturation of the enzyme with heat and sodium dodecyl sulfate. Enzyme-bound zinc did not exchange with free zinc. These results establish yeast nuclear RNA polymerase III as a zinc metalloenzyme.

Cell Nucleus↗
PubMed1978

RNA polymerase activities in liver and brain tissue of aging mice.

RNA polymerase activity has been measured in liver and brain of C57BL-6J mice to determine if a change in enzyme activity can be correlated with decreasing survivorship of the animals. The RNA polymerases in tissue homogenates were solubilized by treatment with a buffer of high ionic strength and resolved by DEAE-Sephadex chromatography. Enzyme activity was quantitated by measuring the incorporation of [3H]UMP into RNA using heat denatured calf thymus DNA as the template. Statistically significant differences in polymerase activities were not observed in liver tissue from 18-, 25-, and 29-month-old animals or in brain tissue from 23- to 31-month-old animals. These age groups span the period of most rapid decrease in survivors,ip in our colony of mice (from 93% to 16%). The evidence indicates that changes in liver or brain RNA polymerase activities are not correlated with the rapid decrease in survivorship of these animals.

Aging↗
PubMed1975

Evaluation of prosthetic grafts of different porosity for arterial reconstruction.

Autogenous jugular veins and Dacron grafts of different porosities were implanted as short bypass grafts around ligated femoral arteries in the dog. Patency rates for periods up to eight months were: vein, 79 per cent; DeBakey Dacron (lowest porosity), 39 per cent; Wesolowski Weavenit (imtermediate porosity), 56 per cent; high porosity Dacron, 8 per cent. The failure of sixteen of the twenty-four high porosity grafts was related to perigraft hematomas that occurred two to seven days after operation and later thrombosis. The higher the porosity of the graft, the better the healing pattern as evidenced by fibrous bonding of the inner and outer linings of the grafts through the interstices. In the spectrum of grafts with increasing porosity there is apparently an optimal porosity beyond which late bleeding counterbalances the better healing properties of the higher porosity graft.

Animals↗