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D E Stevenson

Publications and source records attributed to D E Stevenson.

At least 37 records · Page 2Linked to original sources
PubMed1994

Another flaw in the linearized multistage model upper bounds on human cancer potency.

The statistical methodology used by the EPA and other federal agencies to characterize human cancer potencies based on animal experiments greatly exaggerates the estimates and bounds for human cancer risks in some instances. The current methodology incorporates simplified assumptions and approximations that fail to properly assess the impact of quantitative differences in human and experimental animal background transition rates from stage to stage in the multistage carcinogenic process. Because the majority of tumorigenic responses in rats and mice occur in organs with a high background tumor incidence and, hence, high background stage transition rates, the current simplified methodology often significantly overstates human risk. This newly recognized flaw in conjunction with the several previously recognized flaws in the current characterizations of human cancer potencies argues strongly for a change away from the current characterizations based solely on a default screening methodology to a more comprehensive, biologically based risk assessment methodology.

Animals↗
PubMed1994

Incorporating additional biological phenomena into two-stage cancer models.

Several difficulties arise in the implementation of the Armitage-Doll-type carcinogenesis model. Some of these difficulties have been briefly discussed herein, with particular attention to the recent finding that the magnitude of current animal-based bounds on human cancer potency is influenced by animal background transition rates. Although the ideal method of accounting for the background transition rates necessitates more direct biological information, this paper offers risk assessors some alternatives for improving current quantitative cancer potency assessment. Computer software has been developed to facilitate the use of not only the multistage model but also other dose-response models, including the following: 1) tolerance models (probit, logit, multihit, and Weibull), which assume that a distribution of tolerances exists in the population and that when a tolerance is exceeded, a carcinogenic response occurs; 2) multihit models, which assume that a carcinogenic response occurs when a tissue receives more than a specified number of hits; 3) Armitage-Doll multistage models, which explicitly model the time and/or dose dependence of each transition stage; 4) time-to-response extensions of the multistage model; and 5) extensions of the multistage model including cell proliferation (Lu and Sielken 1991, Holland and Sielken 1993). In any of the dose-response models, the dose scale is not restricted to the administered dose scale. Computer software that facilitates the use of more biologically relevant dose scales (such as delivered and biologically effective dose scales) is now available. Furthermore, the differences between species and routes of exposure in the amount of delivered or biologically effective dose corresponding to a particular administered dose can be incorporated. The dependence of an individual's dose on not only the individual's administered dose but also the individual's background dose and susceptibility can be incorporated as well. Hence, interindividual variability in dose levels and probabilities of a carcinogenic response can be considered (Holland and Sielken 1993). The possibility also exists of incorporating more cell biology into carcinogenesis theory using the MVK two-stage model. In this model, cell proliferation parameters are included, in contrast to models of the Armitage-Doll family. The MVK model offers increased potential for obtaining estimates of low-dose behavior that reflect more of the available biological data, including microdosimetry data. A Monte Carlo simulator for this model can be used in cases in which more complex biological mechanisms are included, information other than the means of population distributions is desired, and information on subpopulations is desired.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1992

Mechanistic and structural studies on Rhodococcus ATCC 39484 nitrilase.

Rhodococcus ATCC 39484 produced a nitrilase when induced with isovaleronitrile. The enzyme was obtainable pure in milligram amounts, had a subunit Mr of 40 kDa, and demonstrated a substrate-induced activation related to aggregation of subunits to form a 560-kDa complex. The enzyme had a broad substrate specificity, had a pH optimum of 7.5, was stable up to 40 degrees C, and had one disulfide bridge and two free cysteine residues, one of which appeared to be catalytically essential. The N-terminal sequence was determined and found to have 78.3% homology, in a 23-residue overlap, with Klebsiella ozaenae nitrilase. The enzyme was inhibited competitively by benzylamine and benzaldehyde and irreversibly by benzyl bromide. However, benzyl bromide was shown to be nonspecific, causing multiple alkylation. Acid quenching of enzyme-substrate mixtures allowed for the detection of covalent enzyme-substrate complexes using mass spectrometry. The covalent intermediate is suggested to be either a thioimidate or an acylenzyme and a reaction mechanism consistent with this observation and also the inhibitor results is proposed. The rate of breakdown of the covalent intermediates was found to be rate limiting even for substrates with undetectable rates of hydrolysis or those with very slow rates of intermediate formation. For phenylacetonitrile, a poor substrate, in addition to acid, approximately 2% of the product was the corresponding amide. This result suggests that a tetrahedral intermediate is formed which, for selected substrates, can break down anomalously to produce amide in place of the normal acid product. Under the conditions used in this study all other substrates tested were converted to acid.

Amino Acid Sequence↗
PubMed1992

Evaluation of sustained hyperplasia and other short-term tests as predictors of tumorigenic potential in oil products.

Some oil products are known to cause skin tumors following long-term application while others do not. The ability to predict which ones might cause tumors is important. Development of reliable short term tests which can accurately predict tumorigenic potential of oil products is needed to avoid the high cost and long time required for traditional animal bioassays. Several short term tests were evaluated for their ability to predict tumorigenic potential of 10 coded oil samples and results were compared to results of mouse bioassays. Analytical determinations of PAC content (DMSO extraction) and 3-6 ring PAC content were also made for each of the 10 samples for further comparison. Tests which showed good correlation with bioassay results and thus were considered good predictors of tumorigenic potential were: Sustained Epidermal Hyperplasia as measured by epidermal thickness, Nuclear Area of epidermal basal cells, Modified Ames Test and DMSO extraction for PAC content. Tests which did not show good correlation with bioassay results and which were not considered good predictors of tumorigenic potential were: Polymorphonuclear leukocyte (PMN) infiltration into the dermis, Unscheduled DNA Synthesis in epidermal cells and Changes in Nuclear DNA Content of CHO cells.

Animals↗
PubMed1990

Detection of covalent enzyme-substrate complexes of nitrilase by ion-spray mass spectroscopy.

Nitrilase from Rhodococcus ATCC 39484 was found to consist of two species of Mr 40,258 +/- 2 and 40,388 +/- 2 Da. When the enzyme was incubated with nitrile substrates and the reaction quenched with acid, higher Mr species were observed. The mass differences were consistent with addition of a substrate molecule to each species. These results represent the first reported demonstration that this, or any other nitrilase forms a covalent intermediate with its substrates. The observation that the intermediate, suggested to be either a thioimidate or an acylenzyme, can be trapped by acidification indicates that the rate of breakdown of the intermediate is rate-limiting.

Aminohydrolases↗
PubMed1990

L-methionine decarboxylase from Dryopteris filix-mas: purification, characterization, substrate specificity, abortive transamination of the coenzyme, and stereochemical courses of substrate decarboxylation and coenzyme transamination.

L-Methionine decarboxylase from the male fern Dryopteris filix-mas has been purified 256-fold from acetone powder extracts to very near homogeneity. The enzyme is membrane-associated and requires detergent for solubilization during the initial extraction. The enzyme is a homodimer of subunit Mr 57,000 and shows a pH optimum at approximately 5.0 with 20 mM (2S)-methionine as substrate. The specific activity, kcat, for methionine is approximately 50 mol s(-1) (mol of active site)(-1) at pH 4.5 and below. A wide range of straight- and branched-chain (2S)-alkylamino acids are substrates for the enzyme. The values for the rate of decarboxylation, Vmax, and for the apparent Michaelis constant, Km, however, vary with structure and with the chirality at C-3. The pH dependence of V and V/K has been examined for three substrates: (2S)-methionine, valine, and leucine. Pyridoxal 5'-phosphate (PLP) is required for activity, and in the absence of excess PLP, the activity of the enzyme in incubations reduced with respect to time. The addition of PLP fully restores the activity, indicating that an abortive decarboxylation-transamination accompanies the normal decarboxylation reaction. The occurrence of the abortive reaction was confirmed by showing that [35S]methionine is converted to labeled 3-(methylthio)propionaldehyde while [4'-3H]PLP is converted to labeled pyridoxamine 5'-phosphate (PMP). The decarboxylation of (2S)-methionine gave 3-(methylthio)-1-aminopropane. Preparation of the N-camphanamide derivative of the amine allowed the C-1 methylene protons to be distinguished by 1H NMR spectroscopy. Synthetic samples of the camphanamide were prepared in which each of the C-1 methylene protons was replaced by deuterium. When (2S)-methionine and the C-2 deuteriated isotopomer were incubated with the enzyme in deuterium oxide and protium oxide, respectively, and the products were converted to their camphanamide derivatives and analyzed by 1H NMR spectroscopy, it was evident that decarboxylation occurred with retention of configuration at C-2. When the decarboxylation of six other substrates was studied, examination of the N-camphanamide derivatives of the amines indicated that decarboxylation occurred stereospecifically and, by analogy, with retention of configuration at C-2. When tritiated pyridoxal phosphate was incubated with the enzyme, tritiated pyridoxamine phosphate was formed. Analysis of the chirality of the methylene group at C-4' indicated that, during abortive transamination, protonation occurred from the 4'-si face of the coenzyme, the same stereochemical result as that obtained for several bona fide transaminase enzymes.(ABSTRACT TRUNCATED AT 400 WORDS)

Carboxy-Lyases↗
PubMed1990

Fern L-methionine decarboxylase: kinetics and mechanism of decarboxylation and abortive transamination.

L-Methionine decarboxylase from Dryopteris filix-mas catalyzes the decarboxylation of L-methionine and a range of straight- and branched-chain L-amino acids to give the corresponding amine products. The deuterium solvent isotope effects for the decarboxylation of (2S)-methionine are D(V/K) = 6.5 and DV = 2.3, for (2S)-valine are D(V/K) = 1.9 and DV = 2.6, and for (2S)-leucine are D(V/K) = 2.5 and DV = 1.0 at pL 5.5. At pL 6.0 and above, where the value of kcat for all of the substrates is low, the solvent isotope effects on Vmax for methionine are 1.1-1.2 whereas the effects on V/K remain unchanged, indicating that the solvent-sensitive transition state occurs before the first irreversible step, carbon dioxide desorption. The enzyme also catalyzes an abortive decarboxylation-transamination reaction in which the coenzyme is converted to pyridoxamine phosphate [Stevenson, D. E., Akhtar, M., & Gani, D. (1990a) Biochemistry (first paper of three in this issue)]. At very high concentration, the product amine can promote transamination of the coenzyme. However, the reaction occurs infrequently and does not influence the partitioning between decarboxylation and substrate-mediated abortive transamination under steady-state turnover conditions. The partition ratio, normal catalytic versus abortive events, can be determined from the amount of substrate consumed by a known amount of enzyme at infinite time, and the rate of inactivation can be determined by measuring the decrease in enzyme activity with respect to time. For methionine, the values of Km as determined from double-reciprocal plots of concentration versus inactivation rate are the same as those calculated from initial catalytic (decarboxylation) rate data, indicating that a single common intermediate partitions between product formation and slow transamination. The partition ratio is sensitive to changes in pH and is also dependent upon the structure of the substrate; methionine causes less frequent inactivation than either valine or leucine. The pH dependence of the partition ratio with methionine as substrate is very similar to that for V/K. Both curves show a sharp increase at approximately pH 6.25, indicating that a catalytic group on the enzyme simultaneously suppresses the abortive reaction and enhances physiological reaction in its unprotonated state. Experiments conducted in deuterium oxide allowed the solvent isotope effects for the partition ratio and the abortive reaction to be determined.(ABSTRACT TRUNCATED AT 400 WORDS)

Amines↗
PubMed1990

Streptomyces L-methionine decarboxylase: purification and properties of the enzyme and stereochemical course of substrate decarboxylation.

L-Methionine decarboxylase from Streptomyces species ATCC 21020 has been purified to near homogeneity, characterized, and compared to the enzyme from the fern Dryopteris filix-mas [Stevenson, D.E., Akhtar, M., & Gani, D. (1990) Biochemistry (first paper of three in this issue)]. The enzyme catalyzes the decarboxylation of a range of alkylamino acid substrates, but the substrate specificity is different from that for the fern enzyme. In accord with the properties of the fern enzyme, the Streptomyces enzyme is also a homodimer of Mr 100,000 +/- 5000 and requires PLP for activity. At low pH where the value of Vmax for both enzymes is maximal and essentially pH independent, kcat for the Streptomyces enzyme with (2S)-methionine as substrate is slightly higher (60 s-1) than the value for the eukaryotic protein (50 s-1). The pH optimum for V/K is much higher than that for the fern enzyme although many features of the pH dependence are similar, including the shape of the curve for the pH dependence of Km. When the decarboxylations of (2S-methionine, (2S)-norleucine, and (2R)-S-ethyl-L-cysteine were conducted on a preparative scale in protium and deuterium oxide, unlabeled and deuteriated amines were formed. 1H NMR spectroscopic analysis of the stereochemistry at C-1 of the camphanamide derivatives of the products [Stevenson, D. E., Akhtar, M., & Gani, D. (1990) Biochemistry (first paper of three in this issue)] indicated that each conversion was stereospecific and occurred with retention of configuration at C-2 of the substrates.(ABSTRACT TRUNCATED AT 250 WORDS)

Carboxy-Lyases↗