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C L Crespi

Publications and source records attributed to C L Crespi.

At least 73 records · Page 4Linked to original sources
PubMed1990

Human cytochrome P450IIA3: cDNA sequence, role of the enzyme in the metabolic activation of promutagens, comparison to nitrosamine activation by human cytochrome P450IIE1.

We report that, in a human cell line, human cytochrome P450IIA3 is capable of metabolizing aflatoxin B1, benzo[a]-pyrene, N-nitrosodimethylamine (NDMA) and N-nitrosodiethylamine (NDEA) to cytotoxic and mutagenic species. Cytochrome P450IIA3-mediated activation of NDMA and NDEA was compared with human cytochrome P450IIE1-mediated activation in the same cell system. P450IIE1 was more effective at activating NDMA than P450IIA3, while P450IIA3 was more effective at activating NDEA than P450IIE1. Whole cells and microsomal fractions obtained from control cells and from cells expressing the P450IIA3 cDNA were characterized for expression of P450IIA3. Microsomal coumarin 7-hydroxylase activity was some 40 times greater in the transfected cells than in the control cells and was catalyzed by a protein that was immunochemically related to the rat liver cytochrome P450IIA gene family. Immunoblot analysis demonstrated that this protein was readily detectable in transfected cells but barely detectable in control cells. We also report the DNA and deduced amino acid sequence of the P450IIA3 cDNA isolate used in this study. Our isolate encodes a protein 489 amino acids that is five amino acids shorter at the N terminus but otherwise identical to a previously reported human P450IIA3 cDNA sequence.

Aflatoxin B1↗
PubMed1990

Mapping of cytochrome P-450 distribution and function with monoclonal antibodies and cDNA expression.

A large number of cytochrome P-450 forms are responsible for the metabolism and detoxification as well as the mutagenic and carcinogenic activation of numerous classes of xenobiotics. These include drugs and carcinogens and other environmental chemicals. The cytochrome P-450 enzymes are also responsible for the metabolism of endogenous substrates including fatty acids, prostaglandins and all classes of steroids. In order to fully understand the role of each P-450 in a specific metabolic reaction, it is necessary to know both the substrate and product specificity of each P-450 as well as the contribution of the individual P-450 to the total metabolism catalyzed in a tissue containing many P-450 forms. Our laboratory has cloned and expressed, with various vectors, a number of rodent and human P-450s. This technique has enabled us to determine the specificity of individual forms of P-450 for certain substrate utilization and product formation, as well as for the metabolic activation of promutagens to their mutagenic forms. The latter has been measured with the Ames mutagen detection system coupled to mutagen activation by vaccinia expressed single P-450s. Mutagenesis and DNA binding have also been measured with a human cell line stably expressing a single P-450. The former approach determines the specificity of single P-450s in cell lysates and the latter determines P-450 specificity in the living cell. In a complementary approach, we have prepared, characterized and utilized inhibitory monoclonal antibodies to six epitope specific classes of P-450. The inhibitory antibodies block the enzymatic activities of specific P-450s in tissue preparations and thus define the contribution of the single P-450 to the total reaction. This approach can be used to measure many P-450 functions, e.g., substrate utilization, product formation, stereochemical metabolism, mutagen activation, drug toxicity, and DNA binding. The two complementary techniques of cDNA expression and immunoinhibition have been used to examine a number of different classes of compounds with respect to their metabolism and mutagen activation. These approaches may eventually yield an atlas of individual P-450 function and their contribution to the total metabolism of xenobiotics and endobiotics.

Animals↗
PubMed1990

The development of a panel of human cell lines expressing specific human cytochrome P450 cDNAs.

We have isolated a human cytochrome P450IIE1 cDNA. This cDNA was transfected to AHH-1 human lymphoblasts and recombinant cell lines were isolated. Two means of selection for the presence of the vector were used, resistance to histidinol and resistance to hygromycin. Histidinol selection was found to give higher vector copy number than hygromycin B selection. AHH-1 cells expressing the P450IIE1 cDNA were sensitive to the cytotoxic and mutagenic effects of exposure to DMN at concentrations of 10 to 30 ng/ml for 28 hours. This system is more sensitive to DMN than systems using hepatocyte mediated activation.

Base Sequence↗
PubMed1989

Transfection of a human cytochrome P-450 gene into the human lymphoblastoid cell line, AHH-1, and use of the recombinant cell line in gene mutation assays.

We have demonstrated that the human cytochrome P1-450 gene can be transfected into the AHH-1 human lymphoblastoid cell line using the pHEBo vector and hygromycin selection. The transfected gene was expressed when regulatory sequences derived from the herpes simplex virus thymidine kinase gene were incorporated in appropriate orientations. Gene expression was monitored at the enzyme level using assays for 7-ethoxyresorufin deethylase, 7-ethoxycoumarin deethylase and benzo[a]pyrene hydroxylase activities. Bulk transformed cell populations had 2- to 3-fold more of these enzyme activities compared with control populations. Subclones of the bulk population expressing still higher levels of 7-ethoxyresorufin deethylase activity were also obtained. Expression of the transfected cytochrome P1-450 gene was stable for 20-30 days in the presence of hygromycin B. The transformed cell populations were found to be suitable for use in gene locus mutation assays and the mutagenicity of aflatoxin-B1 and 2-acetylaminofluorene (AAF) were examined. Aflatoxin-B1 was found to be 2-3 times more mutagenic to cells bearing the transfected cytochrome P1-450 activity as compared with control cells. In contrast, no difference in AAF mutagenicity was observed. Analysis of the AAF metabolite profile indicated that cells expressing the transfected cytochrome P1-450 gene produced 8-fold more N- and 7-hydroxy-AAF than control cells. The similarity in mutagenic responses between control cells and cells bearing the transfected cytochrome P1-450 gene may be due to the low deacetylase activity of AHH-1 cells. These observations indicate that this vector and expression system are suitable for introducing novel metabolic activities into the AHH-1 cell line.

Cell Line↗
PubMed1989

Development of a human cell line by selection and drug-metabolizing gene transfection with increased capacity to activate promutagens.

We have isolated a human lymphoblastoid cell line with higher levels of native cytochrome P450IA1 activity and by DNA transfection introduced human cDNAs for a putative cytochrome P450IIA2 and epoxide hydrolase (E.C. 3.3.2.3). The resultant cell line, designated MCL-1, was substantially more sensitive to the mutagenicity of dimethylnitrosamine and benzo[a]pyrene than the AHH-1 cell line and was found to have increased metabolism of benzo[a]pyrene to dihydrodiols. The increase in native cytochrome P450IA1 activity was achieved by mutation and selection based on resistance to the phototoxicity of benzo[ghi]perylene. One resistant clone, designated L3, was used for subsequent studies. Two complete cDNAs, one encoding a putative cytochrome P450IIA2 and the other a microsomal epoxide hydrolase, were isolated from a human liver cDNA library. After introduction of the cDNAs into an expression vector and transfection into AHH-1 cells, gene expression was detected at the level of enzyme activity (epoxide hydrolase) or by increased sensitivity to dimethylnitrosamine cytotoxicity/mutagenicity (putative P450IIA2). A vector containing both cDNAs was then constructed and transfected into L3 cells to produce MCL-1 cells. The potential usefulness of drug-metabolizing gene transfection and of the MCL-1 cell line, in particular, for genetic toxicity testing is discussed.

Benzo(a)pyrene↗
PubMed1987

Analysis of human lymphoblast mutation assays by using historical negative control data bases.

We describe here historical negative control data bases for gene locus mutation assays at the thymidine kinase locus and hypoxanthine-guanine phosphoribosyl transferase locus in TK6 and AHH-1 human lymphoblasts, respectively. Protocols have been designed which minimize the variability among independent experiments and thus facilitate the use of historical negative control data bases for assay analysis. The historical negative control data bases for both cell lines can be accurately modeled as gamma or Poisson distributions; confidence limits can be calculated from these distributions. We describe and justify a mutagenicity assay analysis procedure which uses a comparison to the concurrent negative control cultures via a t-test in conjunction with a comparison to the historical negative control data base. The incorporation of a comparison to the historical negative control data base allows the use of a higher confidence level without substantially sacrificing the sensitivity of the mutation assays. The analysis of the mutagenicity of saccharin in TK6 cells is presented as an example; saccharin was found to be nonmutagenic under the conditions tested.

Analysis of Variance↗
PubMed1987

The induction of aneuploidy by nitrogen mustard in a human lymphoblastoid cell line.

We report that the presence of an extra Y chromosome can be used as a marker for the induction of aneuploidy (mitotic non-disjunction) in a human lymphoblastoid cell line. This endpoint is easily visualized in metaphase chromosome preparations after staining with quinacrine mustard. The induction of cells with two Y chromosomes by nitrogen mustard (NM) was examined. Exposure to 150 ng/ml nitrogen mustard induced a 6-fold increase in aneuploid frequency relative to untreated control levels; maximal induction of aneuploidy was observed 2 days after treatment. Lower concentrations of nitrogen mustard (36 and 75 ng/ml) induced smaller increases in aneuploid frequency, with maximal induction observed 1 day after treatment. This system has the potential to be used as an assay for the induction of aneuploidy in cultured human cells.

Aneuploidy↗
PubMed1985

A human cell line sensitive to mutation by particle-borne chemicals.

A human lymphoblastoid cell line with ability to perform oxidative metabolism of various chemicals is mutated by the direct addition of an intact particulate soot. This experiment demonstrates that materials associated with combustion-generated particulates are biologically available and able to cause genetic changes in metabolically competent human cells.

Air Pollutants↗
PubMed1985

Mutagenicity of 1,2-dichloroethane and 1,2-dibromoethane in two human lymphoblastoid cell lines.

1,2-Dichloroethane (DCE) and 1,2-dibromoethane (DBE) were tested for the ability to induce gene mutations in two human lymphoblastoid cell lines, designated AHH-1 and TK6. Both chemicals were 'direct-acting' mutagens in both cell lines. DBE was essentially equally mutagenic in TK6 cells and AHH-1 cells. In contrast, DCE was 25-fold more mutagenic in the AHH-1 cell line than in the TK6 cell line. This differential sensitivity between AHH-1 cells and TK6 cells was related to the levels of glutathione S-transferase activity in these two cell lines.

Cell Line↗
PubMed1985

Xenobiotic metabolism and mutation in a human lymphoblastoid cell line.

Aryl hydrocarbon hydroxylase-1 (AHH-1) cells are a human lymphoblastoid cell line competent in some aspects of xenobiotic metabolism. This cell line contains stable mixed function oxidase activity which is inducible by polycyclic aromatic hydrocarbons (PAHs) but not by phenobarbital or Arochlor 1254. Two substrates for the cellular mixed function oxidase activity, benzo[a]pyrene (B[a]P) and 7-ethoxyresorufin, have been examined. The basal and induced activities have different kinetic parameters toward these two substrates. In contrast, basal and induced activities had similar sensitivities to two cytochrome P-450 suicide substrates. B[a]P metabolism and mutagenicity were studied in this cell line. AHH-1 cells were found to produce predominantly B[a]P phenols and quinones. The major phenol metabolite cochromatographed with authentic 9-hydroxy B[a]P. AHH-1 cells were capable of forming glucuronic acid conjugates of B[a]P phenols; the major product after hydrolysis cochromatographed with 3-hydroxy B[a]P standard. AHH-1 cells did not contain detectable epoxide hydrolase activity using B[a]P-4,5-oxide as substrate. This observation is consistent with the absence of trans-dihydrodiol B[a]P metabolites in the metabolic profile. B[a]P-induced mutagenicity at the hypoxanthine guanine phosphoribosyl transferase (hgprt) locus in AHH-1 cells was found to be linearly related to phenol production during treatment and inhibited by alpha-naphthoflavone (ANF).

Aryl Hydrocarbon Hydroxylases↗
PubMed1985

Monoclonal antibody-directed determination of cytochrome P-450 types expressed in a human lymphoblastoid cell line.

Cytochrome P-450-dependent aryl hydrocarbon hydroxylase (AHH) and 7-ethoxycoumarin O-deethylase activities of a cloned line of human lymphoblastoid AHH-1 cells are inhibited by a monoclonal antibody (MAb 1-7-1) prepared to a 3-methylcholanthrene-induced rat liver cytochrome P-450. The monoclonal antibody inhibition determined that a single MAb 1-7-1-sensitive type of cytochrome P-450 is responsible for all of AHH expression in both the basal and benz[a]anthracene-induced cells. Partial inhibition by the MAb 1-7-1, however, indicates that at least two forms of cytochrome P-450 catalyze 7-ethoxycoumarin O-deethylase in both the basal and the induced cells, one form of which is identical to the MAb-sensitive cytochrome P-450 responsible for all of the AHH. Thus, a single cloned cell line is capable of expressing two classes of cytochromes P-450, and the observed multiplicity of cytochrome P-450 in animal tissues does not necessarily depend on cell heterogeneity. A sensitive MAb 1-7-1-based radioimmunoassay also directly demonstrates the presence in these cells of a MAb 1-7-1-specific type of cytochrome P-450 as well as its elevation in the induced cells. These MAb-based methods thus can determine the contribution of specific MAb-defined types of cytochromes P-450 to the cellular metabolism of specific xenobiotics.

7-Alkoxycoumarin O-Dealkylase↗
PubMed1984

Assay for gene mutation in a human lymphoblast line, AHH-1, competent for xenobiotic metabolism.

A novel quantitative gene-locus mutation assay has been developed using a line of human lymphoblast cells, designated AHH-1, competent in oxidative xenobiotic metabolism. AHH-1 cells are sensitive to the mutagenic action of both chemically reactive mutagens and mutagens which require oxidative metabolism to exert their mutagenicity. These cells are readily mutated by direct exposure to ethyl methanesulfonate, ICR-191, 2-acetoaminofluorene, aflatoxin B1, benzo[a]pyrene (BP), cyclopenta[c, d]pyrene, dimethylnitrosamine, lasiocarpine, and 1-methylphenanthrene.

Aryl Hydrocarbon Hydroxylases↗
PubMed1983

Mutation of human lymphoblasts exposed to low concentrations of chemical mutagens for long periods of time.

Methylnitrosourea (MNU), ethyl methanesulfonate (EMS), and 4-nitroquinoline-N-oxide (4NQO) induce mutation to 6-thioguanine resistance and trifluorothymidine resistance in diploid human lymphoblasts (TK6). In single exposure experiments in which greater than 10% of treated cells survive, mutation as a function of concentration is linear for MNU, accelerates for EMS and appears to reach a plateau for 4NQO. In order to probe the bases of these concentration dependencies, human lymphoblasts were exposed for 20 days to each of the three mutagens. Each individual exposure chosen was in itself insufficient to induce statistically significant mutation and each resulted in a cellular survival of greater than 95%. Under this regimen, induced mutation as a function of the number of exposures was linear for all three mutagens. Prior exposure to low concentrations of mutagens was found to have no significant effect on the amount of mutation induced in subsequent exposures. Thus, no biological evidence was found for the induction of repair of misrepair systems.

4-Nitroquinoline-1-oxide↗
PubMed1982

Selection of mitotic Chinese hamster ovary cells from microcarriers. Cell cycle-dependent induction of mutation by 5-bromo-2'-deoxyuridine and ethyl methanesulfonate.

Large quantities of mitotic cells may be collected by mitotic detachment from a population of Chinese hamster ovary cells growing on positively charged dextran microcarriers in suspension culture. Exponentially growing cells are treated for 2.5 h with colcemid and mitotic cells are detached from the microcarriers by increasing the stirring speed. A yield of 4-6% of the total population is obtained and, of the cells collected, 85-95% are arrested in metaphase. Using this means to synchronize cells we have determined the cell cycle dependence of the toxic and mutagenic effects of 5-bromo-2'-deoxyuridine (BUdR) and ethyl methanesulfonate (EMS). Mutation was measured at two independent loci: resistance to 6-thioguanine and resistance to ouabain. Both mutagens were more toxic during S phase as compared to G1 or G2 or mitosis. BUdR induced significant mutation only during S phase. The maximum induction of 6-thioguanine resistance was observed in cultures treated 10 h after plating of mitotic cells (2 h into S phase), while the maximum induction of ouabain resistance was observed in cultures treated 10-12 h after plating of mitotic cells (2-4 h into S phase). EMS induced significant mutation at all points in the cell cycle. Mutation induction reached a minimum during S phase but the magnitude of difference between any two points in the cell cycle was found to be less than two-fold.

Animals↗
PubMedDate not supplied

Fluorometric screening for metabolism-based drug--drug interactions.

Inhibition of cytochromes P-450 (CYP) is a principal mechanism for metabolism-based drug interactions. In vitro methods for quantitatively measuring the extent of CYP inhibition are well-established. Classical methods use drug molecules as substrates and HPLC-based analysis. However, methodologies, which do not require HPLC separations for data acquisition generally offer higher throughputs and lower costs. Multiwell plate-based, direct, fluorometric assays for the activities of the five principal drug-metabolizing enzymes are available and parameters for the use of these substrates to measure CYP inhibition have been established. This methodology is quantitative, rapid, reproducible, and compatible with common high throughput screening instrumentation. This article describes approaches to establishing this methodology in a drug-discovery support program.

2-Pyridinylmethylsulfinylbenzimidazoles↗
PubMedDate not supplied

A metabolically competent human cell line expressing five cDNAs encoding procarcinogen-activating enzymes: application to mutagenicity testing.

A human B-lymphoblastoid cell line, designated MCL-5, constitutively expressing human cytochrome P-450 CYP1A1 and also expressing five transfected human cDNAs encoding drug-metabolizing enzymes, has been developed. cDNAs encoding CYP1A2, CYP2A6, and microsomal epoxide hydrolase (mEH) were introduced by using a vector conferring hygromycin B resistance, and cDNAs encoding CYP2E1 and CYP3A4 were introduced by using a vector conferring resistance to 1-histidinol. MCL-5 cells stably expressed all five cDNAs and the native CYP1A1 as determined by measurement of form-specific enzyme activity levels. The mutagenicity of seven model procarcinogens to MCL-5 cells was examined at the hypoxanthine guanine phosphoribosyltransferase (hprt) and thymidine kinase (tk) loci. Exposure to benzo[a]pyrene (BP), 3-methylcholanthrene (3MC), N-nitrosodimethylamine (NDMA), N-nitrosodiethylamine (NDEA), aflatoxin B1, (AFB1), 2-(acetylamino)fluorene (AAF), or benzidine (BZD) induced a statistically significant increase in mutant frequency. Linear interpolation of the concentration of procarcinogen necessary to produce a doubling of the mutant fraction at the hprt locus in MCL-5 cells and the parent AHH-1 cell line revealed that, for each of the chemicals examined, except BZD, MCL-5 cells were significantly more sensitive than the parent AHH-1 cells. The increase in sensitivity to mutagenicity ranged from 3-fold for AAF to greater than 40,000-fold for NDMA. MCL-5 cells have great potential as a screening system for the analysis of human procarcinogen/promutagen activation.

Carcinogenicity Tests↗