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Biomedical subjects

K Rudo

Publications and source records attributed to K Rudo.

16 recordsLinked to original sources
PubMed1992

Generalized elastophagocytic granuloma.

The case of an eighty-three-year-old woman with the sudden onset of a generalized pruritic eruption is reported. The skin lesions resembled disseminated subacute lupus erythematosus on clinical examination, but actinic granuloma or annular elastolytic giant cell granuloma was seen in biopsy specimens of the lesions. Our case was differentiated from generalized granuloma annulare by the distinct zoning of elastolysis and the distribution of giant cells.

Aged↗
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

Transfection of cytochrome P450 cDNAs into mammalian cells used in mutation and transformation assays.

The present work demonstrates that cDNAs coding for cytochrome P450 enzymes can be transfected into mammalian cells and expressed. In the present studies, two different cell systems were used for transfection: 10T1/2 cells which can be used to study initiation and promotion (Diamond, 1984) and AHH-1 cells which can be used to study mutation and clastogenesis (Crespi and Thilly, 1984, Crespi and Penman, 1989). Thus, a diversity of endpoints can be studied in cells which have increased metabolic capability. By increasing the metabolic capability of the target cell, the effects of nongenotoxic as well as genotoxic chemicals, can be examined in the appropriate in vitro systems. For example, the 10T1/2 cells can be treated with a nontransforming dose of an initiator followed by continuous treatment with a second chemical that requires cytochrome P450 specific metabolism to manifest its promoting activity. By this approach, greater insight into the role of chemical metabolism in the promotion process (and presumably other nongenotoxic effects) can be obtained. Additionally, the role of specific cytochrome P450s in the metabolism of different classes of carcinogens/drugs can be elucidated. A major advantage of having the metabolizing enzymes actually present in the target cell is that effects of chemicals can be studied in long-term, low-dose exposure protocols which will eliminate the acute toxic effects which are associated with many current protocols. Thus, more realistic environmental exposure conditions can be achieved by using these in vitro systems containing endogenous metabolism systems.

Animals↗
PubMed1989

Transfection of a rat cytochrome P450b cDNA into C3H10T1/2CL8 mouse embryo fibroblasts.

A cDNA clone of a rat cytochrome P450b gene was used to construct an expression vector driven by an SV40 promoter and containing a G418-resistance selectable marker. This bifunctional plasmid (pJRSL100) was transfected into the C3H 10T1/2CL8 mouse embryo fibroblast cell line. G418-resistant clones were selected and tested for enhanced sensitivity to the carcinogen 2-acetylaminofluorene (2-AAF), a compound that does not normally induce cytotoxicity or morphological transformation in these cells. One subclone, 19P450b-4, exhibited an increased cytotoxic response to 2-AAF compared to the parental C3H10T1/2CL8 cells. DNA analyses of this subclone showed increased number of copies of the cytochrome P450b and the appearance of unique restriction fragment bands relative to parental and control transfected cells. This subclone also exhibited increased levels of mRNA complementary to the P450b cDNA. Metabolism studies of 2-AAF in this subclone demonstrated an increase in the C-hydroxylated metabolites 1-, 3-, 5/9-, and 7-hydroxy-AAF compared with parental C3H 10T1/2CL8 cells. The results indicate that C3H 10T1/2CL8 cells can be transfected with gene/cDNAs to increase their metabolic competency and that such transfection may enhance the usefulness of the C3H 10T1/2CL8 cells in studies on chemically induced cytotoxicity and morphological transformation.

2-Acetylaminofluorene↗
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↗
PubMed1988

Human hepatocyte and kidney cell metabolism of 2-acetylaminofluorene and comparison to the respective rat cells.

The metabolism and mutagenic activation of 2-acetylaminofluorene by human and rat hepatocytes and kidney cells were measured. High performance liquid chromatography was used to separate the 2-acetylaminofluorene metabolites, and a cell-mediated Salmonella typhimurium mutagenesis assay was used to detect mutagenic intermediates. Rat and human differences were observed with cells from both organs and levels of metabolism and mutagenesis were higher in human cells. Within a species, liver and kidney cell differences were also evident, with levels of hepatocyte-mediated metabolism and mutagenesis being greater than kidney cells. Human inter-individual variation was apparent with cells from both organs, but the variation observed was significantly greater in hepatocytes than kidney cells. A knowledge of such differences, including an understanding that they may vary with the chemical being studied, should be useful in the extrapolation of rodent carcinogenesis data to humans.

2-Acetylaminofluorene↗
PubMed1987

Comparison of human and rat hepatocyte metabolism and mutagenic activation of 2-acetylaminofluorene.

A method has been developed to assess the metabolism and mutagenic activation of carcinogens using human and rodent hepatocytes in vitro. A slicing technique which was especially useful for nonperfusable biopsy and resected surgical human liver tissue was used to prepare the hepatocytes. Metabolites of the model carcinogen 2-acetylaminofluorene (AAF) produced by human and rat hepatocytes were similar and consisted primarily of 2-aminofluorene with ring hydroxylated products at the 1-, 3-, 5/9-, 7-, and 8-positions produced in addition to N-hydroxy-AAF. Sulphate and glucuronide conjugates of ring-hydroxylated metabolites and 2-aminofluorene were detected. Metabolism and cell-mediated Salmonella mutagenicity illustrated interindividual variation with human hepatocytes. Levels of metabolism and mutagenesis were generally higher with human hepatocytes compared to rat hepatocyte results. The increased levels of metabolism and mutagenesis of AAF by human hepatocytes compared to rat hepatocytes probably indicates a different sensitivity to hepatocarcinogenic effects of AAF on humans as compared to rats. Understanding differences and similarities between human and rodent carcinogen activation capabilities should be useful in the extrapolation of rodent carcinogenesis data to humans.

2-Acetylaminofluorene↗
PubMed1987

Species variation in bladder cell and liver cell activation of acetylaminofluorene.

The metabolism and mutagenicity of 2-acetylaminofluorene were measured using freshly prepared intact bladder and liver cells from the cow, dog and rat. High pressure liquid chromatography was used to separate 2-acetylaminofluorene metabolites, and Salmonella typhimurium, strain TA98, was used to detect mutagenic intermediates. Species differences as well as animal-to-animal variation within a species were observed. Mutagenic activity with 2-acetylaminofluorene was greater with cow bladder cells than with dog or rat bladder cells. However, dog bladder cells were most active in metabolizing 2-acetylaminofluorene, and rat bladder cells were least active. Liver cells from all three species metabolized 2-acetylaminofluorene to mutagens for Salmonella, with dog and cow cells being more active than rat liver cells. However, cow liver cells were the most active in metabolizing 2-acetylaminofluorene, followed by rat and dog cells. With all cell types studied, except rat bladder cells, aminofluorene was the major metabolite detected. Carbon and N-hydroxylated products were produced by liver and bladder cells of the three species and glucuronide and sulfate conjugates of the metabolites were detected from both cell types. Correlations between mutagenic activity and the level of metabolism or any individual metabolite were not apparent. The data suggest that the relative contribution of bladder cell metabolism in aromatic amine induced bladder cancer may vary with the species.

2-Acetylaminofluorene↗
PubMed1987

Morphological transforming activity and metabolism of cyclopenta-fused isomers of benz[a]anthracene in mammalian cells.

4 isomeric cyclopenta-derivatives of benz[e]anthracene (benz[a]aceanthrylene, benz[j]aceanthrylene, benz[l]aceanthrylene, and benz[k]acephenanthrylene) were examined for their ability to morphologically transform C3H10T1/2CL8 mouse-embryo fibroblasts. All of these polycyclic aromatic hydrocarbons studied except benz[k]acephenanthrylene transformed C3H10T1/2CL8 cells to both type II and type III foci in a concentration-dependent fashion. Benz[j]aceanthrylene was the most active, equivalent in activity to benzo[a]pyrene on a molar basis, in producing dishes of cells with transformed foci (94% at 1.0 microgram/ml). Benz[e]aceanthrylene, and benz[l]aceanthrylene produced 58% and 85% of the dishes with foci respectively at 10 micrograms/ml. Metabolism studies with [3H]benz[j]aceanthrylene in C3H10T1/2CL8 cells in which unconjugated, glucuronic acid conjugated, and sulfate conjugated metabolites were measured indicated that the dihydrodiol precursor to the bay-region diol-epoxide, 9,10-dihydroxy-9,10-dihydrobenz[j]aceanthrylene, was the major dihydrodiol formed (55%). Smaller quantities of the cyclopenta-ring dihydrodiol, 1,2-dihydroxy-1,2-dihydrobenz[j]aceanthrylene (14%), and the k-region dihydrodiol, 11,12-dihydroxy-11,12-dihydrobenz[j]aceanthrylene (5%) were also formed. Similar studies with [14C]benz[l]aceanthrylene indicated that the k-region dihydrodiol, 7,8-dihydroxy-7,8-dihydrobenz[l]aceanthrylene was the major metabolite formed (45%). The cyclopenta-ring dihydrodiol, 1,2-dihydroxy-1,2-dihydrobenz[l]aceanthrylene and 4,5-dihydroxy-4,5-dihydrobenz[l]aceanthrylene were formed in minor amounts (less than 6%). Therefore, metabolism at the cyclopenta-ring of B(j)A and B(l)A is a minor pathway in C3H10T1/2CL8 cells in contrast to previously reported studies with cyclopenta[cd]pyrene in which the cyclopenta-ring dihydrodiol was the major metabolite. These results suggest that routes of metabolic activation other than oxidation at the cyclopenta-ring such as bay region or k-region activation may play an important role with these unique polycyclic aromatic hydrocarbons in C3H10T1/2CL8 cells.

Animals↗
PubMed1986

Quantitative analysis of the metabolism of benzo(a)pyrene by transformable C3H10T1/2CL8 mouse embryo fibroblasts.

The metabolism of benzo(a)pyrene [B(a)P] to organic soluble and water soluble metabolites by transformable C3H10T1/2CL8 mouse embryo fibroblasts was studied as a function of time, B(a)P concentration, and cell density. The total formation of organic-soluble and water-soluble metabolites increased with incubation time from 4 to 48 h and with B(a)P concentration from 4 to 40 microM. As cell density increased, the metabolic rate decreased for organic-soluble and water-soluble products between 6,300 and 54,000 cells/cm2 probably due to decreases in B(a)P concentrations to values below saturation. Specific organic-soluble metabolites identified were B(a)P-pre-9,10-diols, B(a)P-9,10-diol, B(a)P-7,8-diol, B(a)P-3,6-quinone, B(a)P-3-phenol, and B(a)P-9-phenol. Water-soluble metabolites were subjected to enzymatic hydrolysis with beta-glucuronidase and aryl sulfatase to identify specific conjugated products. The sulfate conjugated metabolites identified were B(a)P-7,8-diol, B(a)P-pre-9,10-diols, B(a)P-9,10-diol, and B(a)P-3,6-quinone. The beta-glucuronic acid metabolites identified were B(a)P-pre-9,10-diols, B(a)P-3,6-quinone, and B(a)P-3-phenol. Patterns of metabolite formation rates are discussed as to their possible effect on morphological transformation rates in C3H10T1/2 cells with respect to incubation time and cell density.

Animals↗
PubMed1986

Metabolism of benzo(a)pyrene in monolayer cultures of human bronchial epithelial cells from a series of donors.

Benzo(a)pyrene [B(a)P] metabolism was measured in monolayer cultures of human bronchial epithelial cells derived from 18 specimens of explanted tissue. Bronchial epithelial cells converted B(a)P to dihydrodiols, phenols, quinone derivatives, and polyhydroxylated forms. Sulfate and glucuronide conjugates of B(a)P metabolites were also detected. Both total metabolism and distribution of metabolites showed a 10-fold or greater variation in cultures from different specimens. When the data were divided according to smoking status, however, no differences in total metabolism, extent of conjugation, or distribution of metabolites could be demonstrated between the two groups. Wide variation (over 1000-fold) in the cytotoxicity of B(a)P towards cells derived from different specimens was demonstrated but could not be directly correlated to the extent of metabolic activation. The results suggest that human bronchial epithelial cells which are newly grown from explanted tissue of smokers in culture do not demonstrate enzymatic induction. Variation among individuals observed in these studies probably represents basal differences in metabolic capability.

Benzo(a)pyrene↗
PubMed1983

Reanalysis and clarification of the structures of alpha-naphthoflavone dihydrodiols formed by uninduced and induced rat liver microsomes from Charles River CD and Sprague-Dawley rats.

The structures of alpha-naphthoflavone (ANF) dihydrodiols formed by uninduced and induced rat liver microsomes are identified by conversion of the metabolically formed ANF-dihydrodiols to the corresponding phenols. Comparison of these phenols with synthetic standards provides an unambiguous method for structural identification. The results of these studies are that hepatic microsomes from uninduced or phenobarbital, Aroclor-1254, 3-methylcholanthrene, or 5,6-benzoflavone induced Sprague-Dawley or Charles River CD rats each produce a major and a minor ANF-dihydrodiol identified as ANF-7,8-dihydrodiol and ANF-5,6-dihydrodiol, respectively.

Animals↗