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1.
Infect Prev Pract ; 3(1): 100122, 2021 Mar.
Article in English | MEDLINE | ID: mdl-34368739

ABSTRACT

BACKGROUND: Hand washing is an important targeted hygiene intervention for limiting the spread of infectious agents, including the Ebola virus, which continues to re-emerge. We have assessed the virucidal efficacy of a commercially available liquid hand wash product (LHW) for inactivating Ebola virus. METHODS: The ASTM E1052-11 Standard was used to evaluate the efficacy of an LHW containing the microbicidal active salicylic acid for inactivating Ebola virus - Makona variant suspended in an organic load. Three concentrations (12.5%, 25%, 50%) of three lots of LHW prepared in 440 ppm hard water were evaluated at room temperature for 20, 30, and 60 s contact time. RESULTS: A 25% solution of the LHW caused 4.5 log10 and 4.8 log10 reduction in Ebola virus titer within 20 and 30 s, respectively. The efficacy of a 12.5% LHW solution was lower (1.9 and 2.0 log10 reduction in titer within 20 and 30 s, respectively). The efficacy of the 50% LHW solution could not be measured, due to inability to sufficiently neutralize the LHW at the end of exposure. CONCLUSION: These results suggest the potential utility of an appropriately formulated liquid hand wash agent during Ebola virus disease outbreaks for use within healthcare, community, and home settings. Such an LHW should also be effective against other enveloped viruses, such as the pandemic coronavirus SARS-CoV-2.

3.
Dev Biol (Basel) ; 123: 153-64; discussion 183-97, 2006.
Article in English | MEDLINE | ID: mdl-16566443

ABSTRACT

Adventitious virus assays are performed as part of raw materials testing, cell-line characterization, and lot-release testing of biologicals such as monoclonal antibodies, gene therapy vectors, recombinant proteins, and vaccines. The testing methods follow guidance provided in the 9 CFR (bovine and porcine raw materials testing, and certain vaccine products) or Points to Consider documents (cell line characterization and evaluation of the majority of biologicals). The methodologies used and the types of adventitious viruses detected during testing of the various types of samples are discussed in this paper. The detection of adventitious viruses is quite rare, especially during evaluation of cell banks and biologicals produced in human, mouse, or insect cell substrates. The most common detection scenarios include bovine viral diarrhoea virus in foetal bovine serum samples, porcine parvovirus in porcine substrates, and murine minute virus, REO virus, and Cache Valley virus in Chinese hamster cell-derived bulk harvests. The two last-named viral entities are believed to be introduced via bovine serum used during the manufacturing process (during scale-up or during the entire process). Knowledge of the types of agents being detected is useful in designing viral clearance methodologies for purification processes and in engineering manufacturing processes and facilities.


Subject(s)
Vaccines/standards , Viruses/isolation & purification , Animals , Cell Line , Drug Contamination , Genetic Therapy/standards , Humans , Legislation, Drug , Safety , United States , Virus Diseases/prevention & control , Virus Diseases/transmission
4.
Xenobiotica ; 32(4): 339-47, 2002 Apr.
Article in English | MEDLINE | ID: mdl-12028666

ABSTRACT

1. The single-dose plasma pharmacokinetics of O(2)-vinyl 1-(pyrrolidin-1-yl)diazen-1-ium-1,2-diolate (V-PYRRO/NO) following intravenous (i.v.) and intraperitoneal (i.p.) bolus administration to the male C57BL/6 mouse was studied in an effort to characterize the disposition of the agent and to serve as a basis for the design of in vivo efficacy studies. 2. Plasma V-PYRRO/NO concentrations declined rapidly in a bi-exponential manner after i.v. administration of 5 mg kg(-1) body weight to mouse. The terminal half-life was 9.4 min and the mean residence time was 3.4 min. 3. V-PYRRO/NO was absorbed rapidly following i.p. administration, with peak plasma concentrations being observed 3 min after injection. Levels then declined with a terminal half-life of 11.7 min. The bioavailable fraction from the i.p. compartment was 19%, indicating a high first-pass effect. 4. The results provide additional evidence for a liver-selective metabolism of this nitric oxide-donating prodrug.


Subject(s)
Azo Compounds/blood , Azo Compounds/pharmacokinetics , Liver/blood supply , Liver/chemistry , Nitric Oxide/metabolism , Animals , Infusions, Intravenous , Injections, Intravenous , Male , Mice , Mice, Inbred C57BL , Pyrrolidines/administration & dosage
5.
Int J Toxicol ; 20(2): 81-7, 2001.
Article in English | MEDLINE | ID: mdl-11354469

ABSTRACT

The effect of the clinically important anticonvulsant phenytoin (DPH) on hepatocarcinogenesis of male F344/NCr rats initiated with a single i.p. dose of N-nitrosodiethylamine (75 mg/kg b.w.) was studied. Beginning 2 weeks post-initiation, the rats received control diet or diet containing 500 or 1,500 ppm DPH or 500 ppm phenobarbital. At 52 weeks age, the incidences (and multiplicities, in units of tumors per tumor-bearing rat) of hepatocellular adenomas were 0%, 17% (1 +/- 0), 42% (1.8 +/- 0.8), or 67% (2.5 +/- 1.9) in rats exposed to N-nitrosodiethylamine alone, or the carcinogen followed by 500 ppm DPH, 1,500 ppm DPH, or 500 ppm phenobarbital, respectively. Between 53 and 79 weeks of age, 39% of rats receiving N-nitrosodiethylamine alone developed multiple (1.5 +/- 0.8) hepatocellular adenomas. A similar incidence (41%) occurred in the rats administered the carcinogen followed by 500 ppm DPH. The incidence of hepatocellular adenomas (88% and 89%) was significantly greater in rats exposed to N-nitrosodiethylamine followed by 1,500 ppm DPH or 500 ppm phenobarbital, respectively. Multiplicities of hepatocellular adenomas were significantly greater than the control value in rats fed 1,500 ppm DPH or 500 ppm phenobarbital (5.9 +/- 4.8 and 10.1 +/- 6.7, respectively), but not in the rats receiving 500 ppm DPH (2.3 +/- 1.6). No rats exposed to N-nitrosodiethylamine alone or the carcinogen followed by 500 ppm DPH developed hepatocellular carcinomas, while hepatocellular carcinomas occurred in 29% or 67% of the rats given 1,500 ppm DPH or 500 ppm phenobarbital, respectively, following initiation. Increases in hepatic CYP2B-mediated benzyloxyresorufin O-dealkylation activity in rats exposed to 500 and 1,500 ppm DPH for 2 or 23 weeks were approximately 50% and approximately 100%, respectively, of the maximal induction caused by 500 ppm phenobarbital. Thus, in the rat model, DPH enhanced N-nitrosodiethyl-amine-initiated hepatocarcinogenesis when administered at a dose causing maximal CYP2B induction.


Subject(s)
Anticonvulsants/toxicity , Aryl Hydrocarbon Hydroxylases , Carcinogens/toxicity , Cytochrome P-450 Enzyme System/biosynthesis , Diethylnitrosamine/toxicity , Liver Neoplasms, Experimental/chemically induced , Oxidoreductases, N-Demethylating/biosynthesis , Phenytoin/toxicity , Adenoma, Liver Cell/chemically induced , Adenoma, Liver Cell/pathology , Administration, Oral , Animals , Anticonvulsants/administration & dosage , Carcinogens/administration & dosage , Carcinoma, Hepatocellular/chemically induced , Carcinoma, Hepatocellular/pathology , Cytochrome P-450 CYP2B6 , Diet , Diethylnitrosamine/administration & dosage , Dose-Response Relationship, Drug , Enzyme Induction , Injections, Intraperitoneal , Liver Neoplasms, Experimental/pathology , Male , Neoplasms, Experimental , Phenobarbital/toxicity , Phenytoin/administration & dosage , Rats , Rats, Inbred F344
6.
Arch Environ Contam Toxicol ; 35(1): 104-8, 1998 Jul.
Article in English | MEDLINE | ID: mdl-9601927

ABSTRACT

1,1-Dichloro-2,2-bis(p-chlorophenyl)ethane (DDD) and 1,1-dichloro-2, 2-bis(p-chlorophenyl)ethylene (DDE) levels were measured by capillary gas chromatography with electron capture detection in liver and blood serum of male F344/NCr rats exposed for 2 weeks to DDD at dietary concentrations ranging from 8.51 ppm to 2,000 ppm. DDD burdens in serum ranged from <0.006 microM (limit of detection) in control rats to 1.1 microM in the rats fed DDD at 2,000 ppm. The corresponding liver burdens in these animals ranged from <0.006 micromol/kg liver (controls) to 11 micromol/kg liver in rats fed DDD at 2,000 ppm. Levels of DDE in serum or liver were undetectable (<0. 006 microM in serum; <0.006 micromol/kg liver) in rats fed control diet or diet containing 8.51 or 25.5 ppm DDD. The liver and serum burdens of DDE increased with dietary DDD concentration, reaching a maximum of 0.53 microM in serum and 4.7 micromol/kg liver in rats fed 2,000 ppm DDD. As a percentage of total DDD equivalents detected in liver or serum, the DDE burdens increased to a maximum of 36% and 31% in the serum and liver, respectively, of rats fed 689 ppm DDD. The possibility that the DDE might have been generated artifactually in the diet prior to administration to the rats was ruled out by analysis with capillary gas chromatography of the diet containing 2, 000 ppm DDD. The identification of DDE as a metabolite in liver extracts of rats fed 2,000 ppm DDD was confirmed with GC-MS. The results confirmed the presence of DDE as a metabolite of DDD.


Subject(s)
Dichlorodiphenyl Dichloroethylene/metabolism , Dichlorodiphenyldichloroethane/pharmacokinetics , Animal Feed/analysis , Animals , Biotransformation , Dichlorodiphenyl Dichloroethylene/blood , Dichlorodiphenyldichloroethane/blood , Drug Administration Schedule , Gas Chromatography-Mass Spectrometry , Insecticides , Liver/metabolism , Male , Rats , Rats, Inbred F344
7.
In Vitro Cell Dev Biol Anim ; 34(1): 35-9, 1998 Jan.
Article in English | MEDLINE | ID: mdl-9542633

ABSTRACT

The analysis of the gel electrophoresis banding patterns and relative migration distances for the individual isoforms of intracellular enzymes, such as lactate dehydrogenase, purine nucleoside phosphorylase, glucose-6-phosphate dehydrogenase, and malate dehydrogenase, is used routinely in the biopharmaceutical industry for confirmation of cell line species of origin. In the present study, the sensitivity of the technique (AuthentiKit, Innovative Chemistry, Marshfield, MA) for determining interspecies cell line cross-contamination was examined. Extracts were prepared from a CHO-K1 line (AA8, Chinese hamster), MRC-5 (human) cells, and L929 (mouse) cells and from several proportional mixtures of the various binary combinations of cells. The isoenzymes were analyzed according to standard procedures for the technique. Contamination of MRC-5 cells with CHO-K1 or with L929 cells was clearly detectable with each enzyme analyzed. Similarly, the contamination of L929 or CHO-K1 cells with MRC-5 cells was readily apparent with each enzyme. On the other hand, contamination of CHO-K1 cells with L929 cells was only detected with lactate dehydrogenase analysis, and contamination of L929 cells with CHO-K1 cells was not detected with any of the four enzymes examined. For the latter case, the analysis of an additional enzyme (peptidase B) was required. The results indicate that interspecies cross-contamination should be detectable with isoenzyme analysis if the contaminating cells represent at least 10% of the total cell population.


Subject(s)
Cell Culture Techniques/standards , Isoenzymes/analysis , Reagent Kits, Diagnostic , Animals , CHO Cells , Cell Line , Cricetinae , Electrophoresis, Agar Gel/methods , Humans , Mice , Sensitivity and Specificity
8.
J Toxicol Environ Health A ; 53(6): 455-77, 1998 Mar 27.
Article in English | MEDLINE | ID: mdl-9537282

ABSTRACT

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.


Subject(s)
Cytochrome P-450 CYP2B1/biosynthesis , DDT/pharmacology , Dichlorodiphenyl Dichloroethylene/pharmacology , Dichlorodiphenyldichloroethane/pharmacology , Insecticides/pharmacology , Liver/drug effects , Animals , Cells, Cultured , Cytochrome P-450 CYP1A1/biosynthesis , Cytochrome P-450 CYP3A , Cytochrome P-450 Enzyme System/biosynthesis , Enzyme Induction , Liver/anatomy & histology , Liver/enzymology , Male , Mixed Function Oxygenases/biosynthesis , Organ Size/drug effects , Rats , Rats, Inbred F344
9.
Article in English | MEDLINE | ID: mdl-9972450

ABSTRACT

The constitutive and inducible hepatic cytochromes P450 of various feral Cricetid rodents (family Cricetidae, comprising various New World rats and mice, hamsters, gerbils and voles), have been examined in a relatively limited number of field and laboratory investigations. These studies, reviewed herein, have employed substrates and immunochemical reagents that are diagnostic for individual P450 subfamilies of Rattus norvegicus (the common laboratory species derived from the Norway rat, a member of the family Muridae). The results have demonstrated that the feral rodents display hepatic responses to prototypic CYP1A inducers (3-methylcholanthrene, beta-naphthoflavone) similar to those displayed by R. norvegicus and Mus musculus (the common laboratory species derived from the house mouse, another member of the family Muridae). At least one study has demonstrated the induction, by ethanol, of a protein immunochemically similar to CYP2E1 in a Cricetid rodent. In Cricetid rodents, phenobarbital-type inducers cause the induction of a hepatic protein immunologically similar to that primarily induced (CYP2B) in R. norvegicus and M. musculus. The proteins induced in the Cricetid rodents, however, exhibit striking differences in substrate specificity, compared to the proteins induced in R. norvegicus. These results indicate that the previously described differences between the P450 induction responses exhibited by the commonly utilized laboratory species R. norvegicus and M. musculus (family Muridae) and the Syrian hamster and gerbil (family Cricetidae) are observed as a generality for members of the Cricetid family of rodents.


Subject(s)
Arvicolinae/metabolism , Cytochrome P-450 Enzyme System/biosynthesis , Animals , Environmental Monitoring , Enzyme Induction , Rats , Species Specificity
10.
Article in English | MEDLINE | ID: mdl-9972461

ABSTRACT

The induction of a hepatic pleiotropic response, including increase in liver/body weight ratio, induction of hepatic CYP2B and CYP3A protein and catalytic activity, and hepatic microsomal epoxide hydration activity, was investigated in male cotton rats (Sigmodon hispidus) administered graded dietary concentrations (0-1500 ppm) of phenobarbital (PB) for 14 days. A dose-dependent induction of each endpoint was observed, although plateaus in the various dose-response curves were not obtained, and ED50 values (PB concentrations associated with half-maximal responses) for the various endpoints were not able to be calculated. A maximal 1.31-fold increase, compared to the control value, in live/body weight ratio was observed, while microsomal epoxide hydration activity was increased as much as 3.6-fold by PB administration. Pentoxy- and benzyloxyresorufin O-dealkylation and testosterone 16 beta-hydroxylation activities (considered to be relatively selective for CYP2B in the Norway rat (Rattus norvegicus)), were induced maximally less than five-fold. Testosterone 6 beta-hydroxylation (considered to be relatively selective for CYP3A in R. norvegicus) was induced maximally less than two-fold. Maximal induction of 7-ethoxy-4-trifluoromethyl-coumarin O-deethylation was 18-fold, compared to the control rate. Western blotting studies indicated that hepatic microsomal proteins immunoreactive with polyclonal antisera to R. norvegicus CYP2B1 or CYP3A1 were induced, in a dose-responsive manner, by PB in the cotton rats. These results indicate that the cotton rat responds to PB treatment with a coordinate pleiotropic response similar to that displayed by R. norvegicus, although the substrate specificity of the induced proteins appears to differ between the two rodent species.


Subject(s)
Aryl Hydrocarbon Hydroxylases , Cytochrome P-450 Enzyme System/biosynthesis , Phenobarbital/pharmacology , Animals , Body Weight , Catalysis , Cytochrome P-450 CYP3A , Cytochrome P-450 Enzyme System/metabolism , Dose-Response Relationship, Drug , Enzyme Induction , Liver/enzymology , Male , Organ Size , Rats , Rats, Inbred F344 , Sigmodontinae
11.
Carcinogenesis ; 18(7): 1381-7, 1997 Jul.
Article in English | MEDLINE | ID: mdl-9230284

ABSTRACT

A series of promoting and non-promoting barbiturates and hydantoins were examined for their ability to sustain the growth of a phenobarbital (PB)-dependent hepatocyte line in cell culture. The effective liver tumor promoters, pentobarbital, allobarbital and 5-ethyl-5-phenylhydantoin, replaced PB and supported 6/27C1 hepatocyte colony formation in vitro at 52-87% of the level induced by PB. The weak promoters secobarbital and amobarbital supported colony formation at only 11-19% of the PB control. A significant correlation was observed for in vivo and in vitro promotion activities of barbiturates and hydantoins, indicating that clonal expansion by 6/27C1 hepatocytes was promoter-dependent. Cell density also appeared to influence hepatocyte growth in vitro. Hepatocyte colonies acquired the ability to grow in the absence of PB, such that after 10 days incubation with PB, approximately 50% of colonies continued to grow in the absence of promoter. This phenomenon of clone-size-dependent hepatocyte growth suggested the operation of an autocrine growth factor pathway. Addition of the hepatocyte mitogen and autocrine growth factor, transforming growth factor-alpha (TGF-alpha), to culture medium lacking PB induced a dose-dependent increase in 6/27C1 hepatocyte colony formation. At the optimal concentration of 3 ng/ml, TGF-alpha sustained hepatocyte clonal expansion at 84% of the level induced by 2 mM PB. Individual 6/27C1 colonies that grew from single cells in the presence of TGF-alpha were tested for promoter-dependent colony formation. Either PB or TGF-alpha supported colony formation by these cells at similar levels and when combined at optimal concentrations, the response appeared to be saturated. When these factors were tested in combination at suboptimal concentrations, the two compounds were additive for supporting colony formation by the parental 6/27C1 line. The ability of TGF-alpha to replace PB and sustain hepatocyte clonal expansion was confirmed with the tumorigenic 6/15 hepatocyte line. These results suggest that TGF-alpha and PB may promote hepatocarcinogenesis by stimulating a common signal transduction pathway.


Subject(s)
Carcinogens/toxicity , Liver Neoplasms, Experimental/chemically induced , Liver/drug effects , Transforming Growth Factor alpha/pharmacology , Animals , Cell Division/drug effects , Cell Line , Dose-Response Relationship, Drug , Phenobarbital/toxicity , Rats
12.
Drug Metab Dispos ; 25(6): 750-6, 1997 Jun.
Article in English | MEDLINE | ID: mdl-9193878

ABSTRACT

The ability of the benzodiazepines, as a chemical class, to cause the induction and/or inhibition of cytochromes P450 has not been well characterized. In the present study, the induction of the cytochrome P450 2B subfamily (CYP2B) in vivo and the inhibition of CYP2B activity in vitro by selected benzodiazepines was examined in hepatic tissues derived from male F344/NCr rats. Initial studies of the in vivo induction or in vitro inhibition of benzyloxyresorufin O-dealkylation activity revealed that both clonazepam and diazepam were relatively effective in vivo inducers of CYP2B when administered in the diet at 500 ppm for 5 days and also were fairly potent inhibitors of the activity of these hemoproteins in vitro. Oxazepam, in contrast, was ineffective as an inducer or an inhibitor of this activity. Further studies were performed to characterize the subfamily selectivity of the P450 induction and inhibition displayed by clonazepam. Specifically, microsomes from rats treated with clonazepam (1000 or 1800 ppm in the diet for 5 days) were found to be highly induced with respect to catalytic activities mediated by CYP2B, including benzyloxyresorufin and pentoxyresorufin O-dealkylation or testosterone 16 beta-hydroxylation, but other CYP proteins were minimally induced. In addition to inducing the CYP2B subfamily, clonazepam also induced the RNA encoding other drug metabolizing enzymes (e.g., epoxide hydrolase and the glutathione S-transferase alpha-subfamily) that are typically induced by phenobarbital-type inducers. Finally, clonazepam proved to be a potent noncompetitive or "mixed-type" competitive inhibitor of catalytic activities mediated by CYP2B, but not by other CYP proteins (e.g. CYP2A, CYP3A) in microsomes derived from phenobarbital-pretreated rats.


Subject(s)
Anticonvulsants/pharmacology , Clonazepam/pharmacology , Cytochrome P-450 CYP2B1/antagonists & inhibitors , Cytochrome P-450 CYP2B1/biosynthesis , Diazepam/pharmacology , Liver/drug effects , Animals , Enzyme Induction , Liver/enzymology , Male , Rats , Rats, Inbred F344
13.
Biochem Pharmacol ; 52(10): 1561-8, 1996 Nov 22.
Article in English | MEDLINE | ID: mdl-8937471

ABSTRACT

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).


Subject(s)
Aryl Hydrocarbon Hydroxylases , Estrogen Antagonists/pharmacology , Liver/drug effects , Liver/enzymology , Tamoxifen/pharmacology , Animals , Cytochrome P-450 CYP3A , Cytochrome P-450 Enzyme System/genetics , Epoxide Hydrolases/genetics , Female , Gene Expression Regulation, Enzymologic/drug effects , Glucuronosyltransferase/genetics , Glutathione Transferase/genetics , Liver/metabolism , Male , RNA, Messenger/genetics , RNA, Messenger/metabolism , Rats , Rats, Inbred F344 , Sulfotransferases/genetics
14.
Anal Biochem ; 238(2): 150-8, 1996 Jul 01.
Article in English | MEDLINE | ID: mdl-8660604

ABSTRACT

S-nitrosothiols have been shown to affect a number of physiological functions. Several techniques have been used to detect these species in biological systems, primarily by methods utilizing chemiluminescence. Since the apparatus required for measurement of chemiluminescence are not readily available in most laboratories, methods employing more conventional techniques such as uv-vis and fluorescence spectroscopy may be of greater use. Herein, we report the development of colorimetric and fluorometric methods for the reliable quantitation of S-nitrosothiols. Solutions containing sulfanilamide/N-(1-naphthyl)- ethylenediamine dihydrochloride or 2,2'-azinobis (3-ethylbenzthiazoline-6-sulfonic acid), when exposed to S-nitrosoglutathione (GSNO), S-nitrosocysteine, or S-nitrosoacteylpenicillamine, resulted in no absorbance changes in the range of 400-800 nm. Exposure to HgCl2 or Cu(acetate)2 resulted in release of nitric oxide (NO) from the S-nitrosothiols. The liberated NO reacted subsequently with oxygen and formed a chemical species which reacted with either analysis solution, resulting in an increase in absorption between 400 and 800 nm. A plot of RSNO versus absorbance was linear for both mercury(II) and copper(II) ions where the slope in the presence of mercury ion was significantly greater than that for copper ion. The sensitivity was as low as 5 microM RSNO using HgCl2. The fluorometric method using 2, 3-diaminonaphthalene as the scavenger of the NOsolidusO2 products gave a sensitivity of 50 nM for GSNO. In addition, S-nitrosylated proteins were quantitated using the fluorometric technique. These methods provide accurate determination of low concentrations of S-nitrosothiols, utilizing conventional spectroscopic techniques available in most laboratories.


Subject(s)
Mercaptoethanol , Nitric Oxide/analysis , Nitroso Compounds/analysis , S-Nitrosothiols , Benzothiazoles , Colorimetry/methods , Copper/chemistry , Ethylenediamines/chemistry , Fluorometry/methods , Mercury/chemistry , Nitroso Compounds/chemistry , Oxygen/chemistry , Serum Albumin, Bovine/chemistry , Sulfanilamide , Sulfanilamides/chemistry , Sulfonic Acids/chemistry , Time Factors
19.
Carcinogenesis ; 17(1): 37-43, 1996 Jan.
Article in English | MEDLINE | ID: mdl-8565134

ABSTRACT

To investigate the promoting effects of 1,4-bis[2-(3,5- dichloropyridyloxy)]benzene (TCPOBOP) on liver and thyroid carcinogenesis of rats at doses that cause maximal induction of hepatic CYP2B, 5-week-old male F344 rats were given either a single i.p. dose of 75 mg N-nitrosodiethylamine (NDEA)/kg body wt in saline or saline alone. After 2 weeks the rats were fed control diet or a diet containing 330 or 1000 p.p.m. TCPOBOP or 500 p.p.m. phenobarbital (PB; a positive control group). A total of four sequential sacrifices (9, 30, 52 and 79 weeks of age) was performed. At 30 weeks the mean volume (mm3) of hepatocellular foci in NDEA-initiated rats exposed to either dose of TCPOBOP or to PB was significantly increased as compared with rats exposed to NDEA followed by control diet (P < 0.05). In addition, the volume percentage of liver occupied by foci was significantly greater in NDEA-initiated/1000 p.p.m. TCPOBOP-promoted rats as compared with rats exposed to NDEA alone (P < 0.05, n = 6). At 52 weeks of age the incidences (and multiplicities, in units of tumors per tumor-bearing rat) of hepatocellular adenomas were 0, 83 (2.6 +/- 1.3), 100 (3.4 +/- 2.1) or 67% (2.5 +/- 1.9) in rats exposed to NDEA alone or NDEA followed by 330 or 1000 p.p.m. TCPOBOP or 500 p.p.m. PB respectively (n = 12). Hepatocellular carcinomas were found only in rats given 1000 p.p.m. TCPOBOP (17% incidence) or PB (8% incidence) following NDEA initiation. The incidences of thyroid follicular cell adenomas were 0, 17, 33 or 8% in rats exposed to NDEA alone or NDEA followed by 330 or 1000 p.p.m. TCPOBOP or 500 p.p.m. PB respectively. Between 53 and 79 weeks of age 38% of rats treated with NDEA alone developed multiple (1.5 +/- 0.8) hepatocellular adenomas. This incidence was enhanced to 100% in rats exposed to NDEA followed by either 330 or 1000 p.p.m. TCPOBOP. Multiplicities of hepatocellular adenomas were also increased significantly (10.5 +/- 3.9, 10.4 +/- 7.0 and 10.1 +/- 6.7 respectively) in rats promoted with 330 or 1000 p.p.m. TCPOBOP or 500 p.p.m. PB. None of the rats exposed to NDEA alone developed hepatocellular carcinomas, while multiple hepatocellular carcinomas occurred in 38% of the rats exposed to 330 p.p.m. and 78% of the rats given 1000 p.p.m. TCPOBOP following NDEA initiation. Thyroid follicular cell tumors occurred at 79 weeks in more than 40 and 50% incidences in rats exposed to NDEA followed by 330 or 1000 p.p.m. TCPOBOP respectively. Also, a significant decrease in serum levels of triiodothyronine and thyroxine were observed in non-initiated 79-week-old rats fed 1000 p.p.m. TCPOBOP, compared with age-matched untreated controls (n = 6). Increases in hepatic CYP2B-mediated benzyloxyresorufin O-dealkylase activity detected in rats exposed to 330 and 1000 p.p.m. TCPOBOP for 2 or 23 weeks were similar in magnitude to those caused by 500 p.p.m. PB. Thus TCPOBOP at maximal CYP2B induction doses exhibits a strong promoting activity for both liver and thyroid of rats.


Subject(s)
Carcinogens/toxicity , Cytochrome P-450 Enzyme System/biosynthesis , Liver Neoplasms, Experimental/chemically induced , Pyridines/toxicity , Thyroid Neoplasms/chemically induced , Animals , Cytochrome P-450 Enzyme System/drug effects , Diethylnitrosamine , Enzyme Induction/drug effects , Male , Precancerous Conditions/chemically induced , Rats , Rats, Inbred F344 , Thyroid Hormones/blood , Weight Gain/drug effects
20.
Biochem Pharmacol ; 50(12): 2099-104, 1995 Dec 22.
Article in English | MEDLINE | ID: mdl-8849338

ABSTRACT

Diallyl sulfide (DAS), a known chemopreventive agent, was administered i.g. (200 or 500 mg/kg body wt/day) to male F344/NCr rats for 4 days. Livers were removed, and hepatic levels of a variety of drug-metabolizing enzymes were determined with either catalytic assays or by quantifying levels of total cellular RNA coding for the individual genes of interest. The high dose of DAS induced the cytochrome P450 (CYP) 2B subfamily to near maximal levels [i.e. similar to those induced by phenobarbital (PB)] and induced the CYP3A subfamily, while having minimal effects on the levels of the CYP1A subfamily. In addition, DAS induced the glutathione S-transferase alpha subfamily, the glutathione S-transferase mu subfamily, and epoxide hydrolase. Unlike PB, however, DAS was also able to induce quinone oxidoreductase. In fact, the pleiotropic hepatic response to DAS appeared to be similar to that elicited by PB, with the exception that only DAS induced quinone oxidoreductase. Finally, we determined that DAS induced the levels of a specific nuclear binding protein that appears to be associated with the induction of various genes that are part of the pleiotropic response caused by PB-type inducers.


Subject(s)
Allyl Compounds , Antioxidants/pharmacology , Cytochrome P-450 Enzyme System/biosynthesis , Liver/drug effects , Sulfides/pharmacology , Animals , Base Sequence , Cytochrome P-450 Enzyme Inhibitors , Cytochrome P-450 Enzyme System/genetics , DDT/pharmacology , Enzyme Induction , Glutathione Transferase/biosynthesis , Liver/enzymology , Male , Molecular Sequence Data , Phenobarbital/pharmacology , Rats , Rats, Inbred F344 , Structure-Activity Relationship , Transcription, Genetic
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