Raymond W. Nims

Dealkylation of pentoxyresorufin: A rapid and sensitive assay for measuring induction of cytochrome(s) P-450 by phenobarbital and other xenobiotics in the rat

The O-dealkylation of pentoxyresorufin (7-pentoxyphenoxazone) by rat liver microsomes was examined. The reaction appeared highly specific for certain phenobarbital inducible forms of cytochrome P-450 and was increased 95- to 140-fold by animal pretreatment with phenobarbital (75 mg/kg/day, four ip injections) and approximately 50-fold by Aroclor 1254 (500 mg/kg, one ip injection) while animal pretreatment with 3-methylcholanthrene (50 mg/kg/day, three ip injections) resulted in less than a 2-fold increase over the rate detected in control microsomes. It was observed that this activity, in microsomes for Aroclor-pretreated rats, was dependent on O2 and was inhibited by metyrapone and SKF 525-A, indicative of cytochrome(s) P-450 mediation in the reaction. When antibodies directed against purified cytochrome(s) P-450s were employed to inhibit the pentoxyresorufin O-dealkylation reaction, antibodies to P-450PB-B greatly inhibited the reaction (greater than 90%), while antibodies to P-450PB-C or P-450PB/PCN-E had minimal effects. Assay of hepatic microsomes from rats which were pretreated with varying doses of phenobarbital (0.9-75 mg/kg/day, four ip injections) indicated that while aminopyrine-N-demethylase activity was induced only 2-fold at the maximum dose (75 mg/kg/day), pentoxyresorufin O-dealkylase activity was induced approximately 140-fold at this dose and approximately 4-fold by a dose of phenobarbital as low as 0.9 mg/kg.

“NONOates” (1-substituted diazen-1-ium-1,2-diolates) as nitric oxide donors: Convenient nitric oxide dosage forms

Abstract 1-Substituted diazen-1-ium-1,2-diolates have proved useful as tools in enzymology and other pharmacological research applications in which spontaneous generation of nitric oxide according to a reasonably well-defined time course is required. This chapter summarizes relevant physicochemical data, including the NO release rates and product profiles, for a selection of these compounds. Guidelines for quality control and a systematic nomenclature scheme are also presented. It is hoped that, in summarizing this information here, our chapter will help those contemplating new applications of diazeniumdiolate technology as they seek to capitalize on what we believe are the inherent advantages of this compound type in research on the pharmacological properties of nitric oxide.

Chemical biology of nitric oxide: Regulation and protective and toxic mechanisms

Publisher Summary This chapter discusses the important aspects of the solution chemistry of nitrogen oxide (NO) and reactive nitrogen oxide species (RNOS), biochemical targets of NO and intermediates in the autoxidation (NO X ), and the effect of NO in the presence of other toxic molecules, such as reactive oxygen species (ROS). There are two types of nitric-oxide synthase: constitutive (cNOS) and inducible (iNOS). Since cNOS generates low levels of NO, direct effects rather than indirect effects of NO would be particularly relevant. In case of iNOS, considerably higher concentrations of NO are formed for longer periods of time; therefore, both direct and indirect effects could be relevant. This chapter discusses, from a chemical perspective, those processes that are involved in the interactions with key cellular components as well as detoxification and control of NO in vivo . Defining the chemical, biochemical, and cellular pathways of NO quantitatively can provide insights into the role that NO plays in the etiology of various diseases that in turn can provide a basis for the development of new therapeutic agents. The chemical biology of NO will provide the understanding as to how NO can be regulatory, toxic, and protective in biological systems.

Induction of hepatic cytochrome P-450 mediated alkoxyresorufin O-dealkylase activities in different species by prototype P-450 inducers

The induction of cytochrome P-450-mediated alkoxyresorufin O-dealkylase activities by various xenobiotics was examined in liver from a variety of animal species in order to gain insights into the substrate specificities of the induced P-450s. We found that forms of cytochrome P-450 capable of mediating the O-dealkylation of the short-chain phenoxazone ethers methoxy-, ethoxy- and propoxyresorufin were highly induced by 3-methylcholanthrene-type inducers and by Aroclor-1254 in all species tested, although there were species differences in the relative turnover rates for the various substrates. For example, in hamster liver the turnover rates for the short-chain resorufin ethers decreased in the following order: methoxy greater than ethoxy much greater than propoxy, while in the rat liver almost the exact opposite order was observed: ethoxy = propoxy much greater than methoxy. In contrast, the degree of induction by phenobarbital-type inducers of isozymes catalyzing the O-dealkylation of pentoxy- or benzyloxyresorufin was highly species-dependent. Thus, F344/NCr rats, B6C3F1 mice and NZB rabbits showed the greatest (greater than 20-fold) induction of these activities, either by phenobarbital or Aroclor-1254, while Mongolian gerbils showed intermediate levels of induction and Syrian golden hamsters exhibited very low induction. In the Japanese quail, phenobarbital- or DDT-treatment resulted in minimal induction of pentoxy- or benzyloxyresorufin O-dealkylase activity, although significant induction of the latter activity occurred following treatment with 5,6-benzoflavone or with Aroclor-1254. Since substrate specificities of most enzymes can be rationalized based upon differences in the steric requirements at the enzyme active site, we employed molecular modeling techniques to calculate the molecular dimensions of the alkoxyresorufins. Surprisingly, the minimal energy conformations in vacuo of each of the resorufin ethers examined are essentially planar. However, alternative configurations, especially for the pentoxy- and benxyloxy-ethers, having greater three-dimensional bulk are also energetically possible.

Cytosol-mediated reduction of resorufin: A method for measuring quinone oxidoreductase

The reduction of resorufin (7-hydroxyphenoxazone) fluorescence was catalyzed by enzymes present in the hepatic cytosol of rats and hamsters. This reaction was mediated by either NADH or NADPH, was completely inhibited by 10 microM dicumarol, and was not affected by anaerobic conditions (purging the reaction cuvette with nitrogen). The enzyme-mediated decrease in resorufin fluorescence was also associated with the loss of the primary absorbance maximum at 570 nm as well as the shoulders at 530 and 600 nm. Similar spectral changes were observed after resorufin was nonenzymatically reduced by sodium dithionite. The enzymatic activity was induced 20- to 40-fold by animal pretreatment with Aroclor-1254 or methylcholanthrene, but only minimally by phenobarbital. A 2.5-fold increase in the rate of the reaction was noted when the pH of the reaction mixture was lowered from pH 7.5 to 6.0. This pH optimum was not a result of slower rates of reoxidation of the reduced resorufin at lower pH, but was due to increased rates of reduction of the compound. Several of the characteristics of the reaction were congruent with the involvement of DT-diaphorase (quinone oxidoreductase, EC 1.6.99.2), and this newly developed fluorimetric assay would appear to be a rapid, sensitive, and direct method for measurement of DT-diaphorase activity.

Comparative pharmacodynamics of CYP2B induction by DDT, DDE, and DDD in male rat liver and cultured rat hepatocytes

In this study the pharmacodynamics were characterized of rat hepatic cytochrome P-450 2B (CYP2B) induction by the pesticide DDT [1,1,1-trichloro-2,2-bis(p-chlorophenyl)ethane] and its metabolites DDE [1,1-dichloro-2,2-bis(p-chlorophenyl)ethylene], which is bioretained, and DDD [1,1-dichloro-2,2-bis(p-chlorophenyl)ethane], which is metabolized further and therefore less prone to bioaccumulate. DDT, DDE, and DDD were each found to be pure phenobarbital-type cytochrome P-450 inducers in the male F344/NCr rat, causing induction of hepatic CYP2B and CYP3A, but not CYP1A. The ED50 values for CYP2B induction (benzyloxyresorufin O-dealkylation) by DDT, DDE, and DDD were, respectively, 103, 88, and > or = 620 ppm in diet (14 d of exposure). The efficacies (Emax values) for induction of benzyloxyresorufin O-dealkylation by DDT, DDE, and DDD were 24-, 22-, and > or = 1-fold, respectively, compared to control values. The potencies of the three congeners for CYP2B induction appeared also to be similar, with EC50 values (based on total serum DDT equivalents) of 1.5, 1.8, and > or = 0.51 microM, respectively. The EC50 values based on DDT equivalents in hepatic tissue were 15, 16, and > or = 5.9 micromol/kg liver tissue, respectively. In primary cultures of adult rat hepatocytes, DDT, DDE, and DDD each displayed ability to induce total cellular RNA coding for CYP2B (ED50 values of 0.98, 0.83, and > or = 2.7 microM, respectively). These results suggest that DDT, DDE, and DDD each possess a high degree of intrinsic CYP2B-inducing ability for rat liver, despite marked differences in bioretention among the congeners.

The induction of alkoxyresorufin metabolism: A potential indicator of environmental contamination

Methods of biochemical monitoring of individual animals for exposure to environmental contaminants are of great potential use. The hepatic metabolism of various alkoxyresorufins, which are highly specific substrates for certain forms of cytochrome(s) P450, is highly induced by a variety of environmental contaminants. Thus, theO-deal-kylation of pentoxy- or benzyloxyresorufin was induced greater than 20-fold in the rat bya-hexachlorocyclohexane, 2,4,5,2′,4′,5′-hexabromobiphenyl, DDT and Aroclor®-1254, while the metabolism of ethoxyresorufin was highly induced by 5,6-benzoflavone, 3,4,5,3′,4′,5′-hexabromobiphenyl and Aroclor®-1254. Additionally, rats exposed to diets containing as little as 12 ppm DDT displayed >five-fold increases in the rate of hepaticO-dealkylation of benzyloxyresorufin. Induction of the hepatic metabolism of these resorufin ethers in 9000xg supernatant fractions taken from rats exposed to potential environmental contaminants may constitute a valuable diagnostic indicator of the presence of a variety of pollutants, including poly cyclic aromatic hydrocarbons, organochlorine pesticides, polyhalogenated biphenyls and 2,3,7,8-tetrachlorodibenzo-p-dioxin. These results suggest the potential applicability of these substrates in detecting chemical contamination in the environment.

Induction of cytochrome P450 and other drug metabolizing enzymes in rat liver following dietary exposure to Aroclor 1254

Selected drug metabolizing activities were measured in female F344/NCr rats exposed to graded dietary concentrations of Aroclor 1254 (1 to 1000 ppm) for 7 days or to lower concentrations of Aroclor (1 to 10 ppm) for up to 28 days. Following the 7-day exposure, the hepatic O-dealkylation of ethoxyresorufin (ETR), mediated primarily by cytochrome P450IA, was increased 60-, 10-, and 4-fold by 33, 10, and 3 ppm Aroclor, respectively. In rats exposed to 10 and 3 ppm Aroclor for 28 days, this activity was increased approximately 30- and 10-fold, respectively. Hepatic ETR O-dealkylase activities correlated with Aroclor concentrations in the livers of exposed rats (r = 0.99, p less than 0.01). Although the O-dealkylation of benzyloxyresorufin was highly increased by 7-days dietary exposure to 1000 ppm Aroclor, the levels of Aroclor necessary for detection of induction were substantially higher than those required for detection of ETR O-dealkylase induction. Examination of the non-P450-mediated drug metabolizing activities, epoxide hydrolase and DT-diaphorase, similarly showed limited (approximately 10-fold) increases. In contrast, aldehyde dehydrogenase (benzaldehyde, NADP+) activity was highly increased (greater than 40-fold) at 1000 ppm, however this activity was increased to only a limited extent at lower Aroclor concentrations (e.g. approximately 3-fold at 33 ppm). These results support the potential use of cytochrome P450 activities as potential biomarkers for environmental exposure to PCBs and related compounds.

Regulation of gene expression of various phase I and phase II drug-metabolizing enzymes by tamoxifen in rat liver.

The objective of the present investigation was to evaluate the effect of tamoxifen (TAM) on the gene expression of different phase I and phase II drug-metabolizing enzymes. Groups of male and female F344/NCr rats were administered either corn oil or TAM (2.8 to 45 mg/kg body wt x 14 days) dissolved in corn oil by gavage. An additional group of rats received a diet supplemented with phenobarbital (PB, 500 ppm). Northern blot analyses of total liver RNA were conducted using [32P]-labeled cDNA or oligonucleotide probes coding for different sulfotransferase (ST); UDP-glucuronosyltransferase (UGT), glutathione S-transferase (GST), epoxide hydrolase (EPH) or cytochrome P450 (CYP) mRNA transcripts. In male rats, TAM increased the levels of STel, STa and STpl mRNAs, whereas PB increased only the STel mRNA. In female rats, there was no expression of STel and STHA mRNA in either control or TAM-treated animals. TAM and PB increased UGTBe/p mRNAs in all rats, whereas UGTml mRNA was elevated only in PB-treated animals. EPH mRNA was elevated markedly in all rats treated with TAM and PB, whereas GSTya/ye mRNA was highly increased by PB, but only marginally increased by TAM. Finally, TAM increased CYP3A1 mRNA, and slightly increased CYP2B1 mRNA, whereas PB highly elevated mRNAs for both of these CYP genes. In conclusion, treatments of rats with TAM increased the mRNA levels of many phase I and phase II drug-metabolizing enzymes, and this pleiotypic response to TAM seems to be different from other prototype inducers such as PB or dioxin (TCDD).

Induction of hepatic CYP1A activity as a biomarker for environmental exposure to Aroclor® 1254 in feral rodents

Specimens of the feral mouse species Reithrodontomys fulvescens trapped from a polychlorinated biphenyl (PCB)-contaminated field location had hepatic ethoxy-resorufin (ETR) O-dealkylase activities and immunoreactive CYP1A protein contents which were two- to threefold higher than those measured in animals of the same species and sex collected from non PCB-contaminated reference sites. Specimens with hepatic ETR O-dealkylase activities differing by as little as 50% could readily be assigned as originating from the PCB or reference sites by the use of a specific chemical inhibitor of cytochrome P450IA (CYP1A). The relative levels of ETR O-dealkylase activity in R. fulvescens significantly correlated with hepatic PCB burdens (r=0.819, P<0.01). When the magnitudes of the induced ETR O-dealkylase activities corresponding to given hepatic PCB burdens were compared between the feral animals, F344/NCr rats (Rattus norvegicus) or B6C3F1 mice (Mus musculus) exposed in the laboratory to dietary Aroclor® 1254, the order of sensitivity to the inducing effects of PCBs were F344/NCr rat>B6C3F1 mouse>R. fulvescens.