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Biomedical subjects

R Langenbach

Publications and source records attributed to R Langenbach.

At least 55 records · Page 3Linked to original sources
PubMed1990

Comparative metabolism and genotoxicity of the structurally similar nitrophenylenediamine dyes, HC Blue 1 and HC Blue 2, in mouse hepatocytes.

Previous studies indicated that HC Blue 1 induced heptocellular carcinomas in B6C3F1 mice whereas the structurally similar nitroaromatic amine HC Blue 2 did not. In an attempt to elucidate the biochemical mechanisms responsible for their different carcinogenic potencies, comparative metabolism and genetic toxicity studies were undertaken. Eighteen-hour urinary recovery of administered radioactivity was equivalent for both compounds following oral gavage (100 mg/kg) in female B6C3F1 mice. By HPLC analysis, HC Blue 1 yielded 3 major polar metabolite peaks, one of which was susceptible to glucuronidase. In vivo metabolism of HC Blue 2 yielded a single major metabolite peak which was not hydrolyzed by glucuronidase. Metabolism by B6C3F1 mouse hepatocytes yielded metabolite profiles which were qualitatively similar to the profiles observed after in vivo metabolism. HC Blue 1 was metabolized by hepatocytes at approximately twice the rate of HC Blue 2. Cytogenetic evaluations of mouse hepatocytes after in vitro treatment indicated HC Blue 1 was more potent than HC Blue 2 in inducing chromosomal aberrations while both chemicals showed weak activity for inducing sister-chromatid exchanges. Furthermore, in the V79 cell metabolic cooperation assay, HC Blue 1, but not HC Blue 2, inhibited cell-to-cell communication suggesting a non-genotoxic activity may be present for HC Blue 1. It is concluded that qualitative and quantitative differences exist in the metabolism of these compounds and that genotoxic as well as nongenotoxic effects may contribute to their different carcinogenic potencies.

Animals↗
PubMed1990

The chemotherapeutic potential of glycol alkyl ethers: structure-activity studies of nine compounds in a Fischer-rat leukemia transplant model.

Structure-activity studies with nine glycol alkyl ethers were conducted with a cellular leukemia transplant model in male Fischer rats. This in vivo assay measures the effects of chemical treatment on neoplastic progression in transplant recipients. Chemicals were given ad libitum in the drinking water simultaneously with the transplants and continued throughout the study. In all, 20 million leukemic cells were injected s.c. into syngeneic rats, which after 60 days resulted in a 10-fold increase in relative spleen weights, a 100-fold increase in white blood cell counts, and a 50% reduction in red blood cell (RBC) indices and platelet counts. At this interval, ethylene glycol monomethyl ether (2-ME) given at a dose of 2.5 mg/ml in the drinking water completely eliminated all clinical, morphological, and histopathological evidence of leukemia, whereas the same dose of ethylene glycol monoethyl ether (2-EE) reduced these responses by about 50%. Seven of the glycol ethers were ineffective as anti-leukemic agents, including ethylene glycol, the monopropyl, monobutyl, and monophenyl ethylene glycol ethers, diethylene glycol, and the monomethyl and monoethyl diethylene glycol ethers. 2-ME more than doubled the latency period of leukemia expression and extended survival for at least 210 days. A minimal effective dose for a 50% reduction in the leukemic responses was 0.25 mg/ml 2-ME in the drinking water (15 mg/kg body weight), whereas a 10-fold higher dose of 2-EE was required for equivalent antileukemic activity. In addition, the in vitro exposure of a leukemic spleen mononuclear cell culture to 2-ME caused a dose- and time-dependent reduction in the number of leukemia cells after a single exposure to 1-100 microM concentrations, whereas the 2-ME metabolite, 2-methoxyacetic acid, was only half as effective. The two glycol alkyl ethers with demonstrable anti-leukemic activity, 2-ME and 2-EE, also exhibited a favorable efficacy-to-toxicity ratio and should be considered for further development as chemotherapeutic agents.

Administration, Oral↗
PubMed1990

Cationic liposomes (Lipofectin) mediate retroviral infection in the absence of specific receptors.

We have used cationic liposomes (Lipofectin) to facilitate retrovirus infection of cells lacking the homologous viral receptor. Ecotropic murine leukemia virus and packaged retroviral vectors were shown to infect mink cells, and amphotropic packaged retroviral vectors were shown to infect hamster cells in the presence of Lipofectin but not in the presence of Polybrene. Lipofectin-mediated infection of cells lacking the homologous receptor results in a titer approximately 0.1% of the titer in cells with the homologous receptor, using the standard Polybrene protocol. The use of Lipofectin may provide a simple means to experimentally infect a wide variety of cells with viruses not normally infectious for the species, tissue, or cell type of interest.

Animals↗
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↗
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

Human and rat kidney cell metabolism of 2-acetylaminofluorene and benzo(a)pyrene.

The metabolism and mutagenic activation of the model carcinogens benzo(a)pyrene [B(a)P] and 2-acetylaminofluorene (AAF) by human and rat kidney cells were measured. A slicing technique followed by enzyme digestion was utilized to obtain the kidney cells. Although levels of total metabolism of B(a)P by rat and human kidney cells were similar, analysis of specific metabolites of B(a)P indicated that species differences existed. Human kidney cells produced the organic-soluble metabolites B(a)P-9,10-diol, B(a)P-4,5-diol, B(a)P-7,8-diol, B(a)P-3,6-quinone, and B(a)P-9-phenol. Rat kidney cells produced organic-soluble B(a)P-pre-9,10-diols, B(a)P-9,10-diol, B(a)P-4,5-diol, and B(a)P-6,12-quinone. Both species produced sulfate and glucuronide conjugates of all products. For AAF, kidney cells from some human tissues produced up to four times the level of total metabolites compared to rat kidney cells. Organic-soluble metabolites were qualitatively similar between the species and consisted of 2-aminofluorene (AF), N-hydroxy-AAF and ring-hydroxylated products at the 1, 3, 5/9, 7, and 8 positions. Sulfate and glucuronide conjugates of these metabolites were also detected. Human interindividual variation with kidney cells was about 2.5-fold for total AAF metabolism and up to 6-fold for individual AAF metabolites. For B(a)P metabolism, human interindividual variation in total metabolism was low while for specific metabolites there was up to a 4-fold variation. Levels of AAF and AF cell-mediated Salmonella typhimurium mutagenesis were significantly higher with human cells as compared to rat kidney cells. It appears that the differences between human and rodent kidney cell metabolism of chemical carcinogens vary with the chemical class and understanding these differences will be necessary in the extrapolation of rodent carcinogenesis data to humans.

2-Acetylaminofluorene↗
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

Interlaboratory studies with the Chinese hamster V79 cell metabolic cooperation assay to detect tumor-promoting agents.

Three laboratories participated in an interlaboratory study to evaluate the usefulness of the Chinese hamster V79 cell metabolic cooperation assay to predict the tumor-promoting activity of selected chemicals. Twenty-three chemicals of different chemical structures (phorbol esters, barbiturates, phenols, artificial sweeteners, alkanes, and peroxides) were chosen for testing based on in vivo promotion activities, as reported in the literature. Assay protocols and materials were standardized, and the chemicals were coded to facilitate unbiased evaluation. A chemical was tested only once in each laboratory, with one of the three laboratories testing only 15 out of 23 chemicals. Dunnett's test was used for statistical analysis, and differences between treated- and control-cell responses were analyzed at P less than or equal to .01. Chemicals were scored as positive (at least two concentration levels statistically different than control), equivocal (only one concentration statistically different), or negative. For 15 chemicals tested in all three laboratories, there was complete agreement among the laboratories for nine chemicals. For the 23 chemicals tested in only two laboratories, there was agreement on 16 chemicals. With the exception of the peroxides and alkanes, the metabolic cooperation data were in general agreement with in vivo data. However, an overall evaluation of the V79 cell system for predicting in vivo promotion activity was difficult because of the organ specificity of certain chemicals and/or the limited number of adequately tested nonpromoting chemicals.

Animals↗
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↗
PubMed1988

The mutagenic activity of selected compounds at the TK locus: rodent vs. human cells.

The mutagenic (TFT resistance) and toxic responses of mouse lymphoma (MOLY) L5178Y cells and human lymphoblast (HULY) TK6 cells were compared for 13 chemicals. The mutagenic activities of 8 of the 13 chemicals (62%) examined in the HULY and MOLY assays are in agreement - the results being judged positive in both assays. However, a dramatic difference is observed when the two conditions of metabolic activation are considered separately; the overall concordance of 8/13 has been achieved by combining a 13/13 (100%) agreement in the absence of S9 with a 1/6 (17%) agreement in the presence of S9. In the absence of S9, the concentration ranges, lowest significant doses, and shapes of the concentration-response curves for both toxicity and mutagenicity were similar in spite of the differences in exposure times (4 h for MOLY, 20 for HULY) and expression times (2 days for MOLY, 3 days for HULY). The general agreement observed in the absence of S9 contrasted with the differences manifested in its presence. 6 compounds which were negative in the absence of S9 were tested in both the MOLY and HULY assays in the presence of S9. Of the 6 chemicals, only 1 was positive in both MOLY and HULY under the latter condition; 4 others were positive in MOLY and negative in HULY whereas 1 was positive in HULY and negative in MOLY.

Animals↗
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↗
PubMed1986

Mutation and enhanced virus transformation of cultured hamster cells by exposure to gaseous ethylene oxide.

Ethylene oxide is a classical mutagen and a carcinogen based on evidence from studies in experimental animals. It is widely distributed in industrial, research, hospital, and food environments. In an effort to explore the use of newly developed methods for exposing mammalian cells to gaseous or volatile mutagens/carcinogens, Chinese hamster V79 cells were treated for 2 hr with gaseous ethylene oxide, in sealed treatment chambers, and assayed for survival and mutagenic response by analysis of induced resistance to 6-thioguanine or ouabain. Significant numbers of mutants were produced at both genetic markers by 1,250-7,500 ppm ethylene oxide. Similarly, primary Syrian hamster embryo cells were treated for 2 or 20 hr with gaseous ethylene oxide in sealed treatment chambers and subsequently assayed for survival and increased sensitivity to SA7 virus transformation. Treatment concentrations extended from toxic to several nontoxic concentrations. After 2-hr ethylene oxide treatment at 625-2,500 ppm a significant enhancement of virus transformation was observed. At 20 hr after treatment no enhancement was observed. Treatment of hamster cells with ethylene oxide in both bioassay systems yielded concentration-related, quantitative results.

Adenoviruses, Simian↗
PubMed1986

Mutagenic activity and structure-activity relationships of short-chain dialkyl N-nitrosamines in a hamster hepatocyte V79 cell-mediated system.

A series of 19 short chain dialkyl N-nitrosamines was studied for mutagenic activity in an uninduced hamster hepatocyte V79 cell-mediated mutagenesis system. Ouabain was used as the selective agent to quantitatively analyze for chemically induced mutants. None of the nitrosamines was mutagenic in the absence of hamster hepatocyte activation. The relative mutagenic activities of the nitrosamines at an equimolar dose are presented. The results of the study indicated that: increasing alkyl chain length decreased mutagenic activity; oxidation of the 2-carbon position to a carbonyl group increased the mutagenic activity of symmetrical and asymmetrical nitrosamines, whereas oxidation to a hydroxyl group only increased the mutagenic activity of the asymmetrical nitrosamines tested; and the carbon position at which oxidation occurred was important in determining mutagenic activity. The relationships between structure, metabolic activation, and mechanisms of mutagenic activity are discussed.

Animals↗