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C Melcion

Publications and source records attributed to C Melcion.

At least 19 recordsLinked to original sources
PubMed1994

Recommendations for the performance of bacterial mutation assays.

At the International Workshop on the Standardisation of Genotoxicity Test Procedures, in Melbourne (27-28 February 1993), the current international guidelines for the correct conduct of bacterial mutation assays were considered, and the major differences between them were examined. An attempt was made to construct a scientifically based, internationally harmonized protocol. The main points of agreement were as follows. The consensus opinion was that there are currently insufficient data to justify a preference for either the preincubation or plate-incorporation methodologies as the initial test. Whichever method is used there was consensus agreement that the bacterial test battery should consist of S. typhimurium TA1537, TA1535, TA98 and TA100. There was also consensus that the 3 strains TA97a, TA97 and TA1537 could be used interchangeably. Although it was not possible to achieve a consensus, the majority of the working group members agreed that strains for the detection of mutagens acting specifically on AT base pairs should be routinely included within the test battery. These strains may be S. typhimurium TA102 or E. coli WP2 strains (WP2 pKM101 and WP2 uvrA or WP2 uvrA pkM101). With regard to study design it was universally agreed that 5 doses of test compound should be used in each experiment, and a majority agreement was obtained for 3 plates per dose. The use of 2 plates per dose is acceptable ONLY if the experiment is repeated. It is recommended that the negative controls may consist of solvent control alone provided that historical data are available to demonstrate lack of effect of the solvent in question. Positive control compounds should be included in all experiments, although the nature of these control compounds need not be specified in the guidelines. There was consensus agreement that for non-toxic freely soluble test agents, an upper limit of 5 mg/plate should be tested (5 microliters per plate for liquids). For insoluble or toxic compounds, the recommendations were the same as those for other in vitro tests (see appropriate paper). A consensus agreement was reached on the need to carry out further tests if equivocal results are obtained in the initial test, although it was generally agreed that the design of the repeat study should be left flexible. As there are little or no data to support the use of an exact repeat assay, a majority of the group recommended that negative results in the first test should be further investigated by either conducting a modified repeat (e.g. S9 titration) or by conducting the alternative methodology.(ABSTRACT TRUNCATED AT 400 WORDS)

Biotransformation↗
PubMed1994

Validation of the in vivo CD1 mouse splenocyte micronucleus test.

In order to validate the in vivo micronucleus test in mouse splenocytes using the cytokinesis block method, 14 compounds with various mechanisms of action were tested: three direct alkylating agents (mitomycin C, ethylnitrosourea, beta-propiolactone), seven indirect alkylating agents (cyclophosphamide, benzo[a]pyrene, diethylnitrosamine, dimethylnitrosamine, 4-aminophenol, 4-aminobiphenyl, 1,1-dimethylhydrazine), two intercalating agents (acridine orange, ethidium bromide) and two spindle poisons (vincristine, colchicine). Male mice were dosed once with the compound, and spleen samples were taken 2 or 14 days after treatment. A significant increase in the binucleated micronucleated splenocyte rate was observed with all the alkylating and intercalating agents at at least one sampling time. In contrast, no increase in the binucleated micronucleated splenocyte rate was observed with the spindle poisons. In conclusion, under these experimental conditions, this in vivo test seems appropriate for the detection of clastogenic compounds including compounds that cannot be detected in the bone marrow micronucleus test. The limit of this test, as expected, is the lack of detection of aneugenic compounds.

Alkylating Agents↗
PubMed1993

Lack of predictivity of bone marrow micronucleus test versus testis micronucleus test: comparison with four carcinogens.

In vivo somatic chromosome mutation tests are usually carried out using the bone marrow micronucleus test in the mouse. This test is also considered predictive for the study of clastogenic effects in germ cells. However, it has been reported that the sensitivity of the bone marrow micronucleus test is insufficient to detect unstable compounds or short-lived metabolites and the use of target cells with metabolic activity (hepatocytes) has been questioned. In order to analyze in vivo micronucleus induction in cells with metabolic enzyme activity, we compared the sensitivity of somatic and germ cells to four carcinogens in the bone marrow and spermatid micronucleus test in the mouse. Three procarcinogens with a complex metabolic pattern (dimethylnitrosamine, diethylnitrosamine and 1,1-dimethylhydrazine) and one direct unstable mutagen (beta-propiolactone) were tested. All four carcinogens were not detected by the bone marrow micronucleus test but were detected in the mouse spermatid micronucleus test in which they induced clear clastogenic effects, as was the case in a previous study in liver micronucleus test. In conclusion, this study demonstrates that the bone marrow micronucleus test is not sufficient for the prediction of a clastogenic hazard in germ cells. In addition to a second in vivo test in an organ with metabolic enzymes, i.e., the liver, the spermatid micronucleus test can be performed when a specific risk to the testis is likely.

Animals↗
PubMed1992

Flow cytometric detection of drugs altering the DNA methylation pattern.

We have developed a model system for assessing the demethylating potential of external agents. Disruption in the DNA methylation pattern was evaluated at the translational level of the Escherichia coli beta-galactosidase coding gene (lacZ). We have constructed a clonal cell line (A4/4 cells) derived from the adenovirus-transformed human embryonic kidney 293 strain. The A4/4 cells contain the E. coli lacZ gene under the control of the mouse metallothionein 1 promoter which is down-regulated by a natural DNA methylation pattern. Furthermore, the lacZ transcription is also regulated by the E. coli lac operator/repressor system and by mouse metallothionein 1 metal responsiveness offering a wide range in lacZ expression. In this system, the beta-galactosidase activity was only recovered in the presence of a demethylating agent such as 5-azacytidine. The demethylating potential of 5-azacytidine, 5-aza 2'-deoxycytidine and sodium butyrate was rapidly assessed by a flow cytometric method using fluorescein di-beta-D galactopyranoside as a fluorescent probe. A tremendous induction of lacZ expression was triggered by these drugs. Analysis of cell cycles showed little disruptions with 5-azacytidine and sodium butyrate, but an important blockage in the S-phase following 5-aza 2'-deoxycytidine treatment was observed. This approach allows a rapid identification and study of environmental demethylating agents.

Azacitidine↗
PubMed1992

Detection of micronuclei after exposure to mitomycin C, cyclophosphamide and diethylnitrosamine by the in vivo micronucleus test in mouse splenocytes.

A micronucleus detection test using mouse splenocytes has been adapted from a method previously carried out using human lymphocytes. An ex vivo protocol was chosen: male C57B16 mice were treated with various compounds. Splenocytes were then isolated and placed in culture for 48 h and stimulated with concanavalin A and conditioned medium. The cytokinesis-block method reported by Fenech and Morley was used to detect and score micronuclei in the proliferating lymphocytes (3 micrograms/ml of cytochalasin B for 16 h). Three mutagenic clastogens, mitomycin C (MMC), a direct alkylating agent (0.4, 0.8 and 1.6 mg/kg), cyclophosphamide (CP), an indirect alkylating agent (25, 50 and 100 mg/kg) and diethylnitrosamine (DEN), an indirect alkylating agent with labile metabolites (25, 50 and 100 mg/kg), were tested at four sampling times (2, 4, 8 and 15 days). All three compounds were detected from 48 h after treatment. This method was indeed able to detect clastogenic compounds normally detected by the mouse bone marrow micronucleus test (MMC, CP) as well as a compound with labile metabolites which is not usually detected by this test (DEN). Maximum micronucleus induction was observed after 4 days for MMC, 2 days for CP and 15 days for DEN. This method thus appears to offer a potentially useful toxicological test for assessing in vivo clastogenicity.

Animals↗
PubMed1991

Rat adult hepatocytes in primary pure and mixed monolayer culture. Comparison of the maintenance of mixed function oxidase and conjugation pathways of drug metabolism.

The stabilities of several drug oxidation and conjugation pathways in adult rat hepatocytes were investigated in two systems: a primary pure culture lasting 3 days and a primary mixed culture (hepatocytes co-cultured with epithelial cells) lasting 10 days. The cytochrome P450 content in hepatocytes drastically declined within 48 hr in both culture systems. Cytochrome P450-dependent mixed function oxidase was measured by the O-dealkylation of ethoxyresorufin (EROD) and of pentoxyresorufin (PROD). UPD-glucuronosyl transferase (UDP-GT) activity was measured using 1-naphthol and morphine as substrates. In both culture systems, the activities of enzymes belonging to the 3-methylcholanthrene-inducible family, namely EROD and 1-naphthol UDP-GT, were much better maintained than those of PROD and morphine UDP-GT, which belong to the phenobarbitone-inducible family: in pure cultures, EROD and 1-naphthol UDP-GT activities declined to 60% of initial values within 3 days; in mixed cultures, EROD activity was stable throughout the 10 day culture period, whereas that of 1-naphthol UDP-GT was stable until day 4 but had declined to 70% of the initial value by day 8. In contrast, PROD and morphine UDP-GT activities declined to approx. 30% of the initial values within 2 days in both culture systems, and had dropped to approx. 10% of the initial value within 8 days in mixed culture. Reduced glutathione (GSH) levels fluctuated, but remained high throughout culture. GSH conjugation declined to 40% of initial values within 3 days in pure culture, whereas it remained relatively constant in mixed culture. Comparison of these two culture systems therefore showed that although the inclusion of epithelial cells did prolong hepatocyte viability, there was a change in relative enzyme activities in both systems, suggesting a shift towards a more de-differentiated drug metabolism pattern.

Animals↗
PubMed1991

Comparison of the effects of propylthiouracil, amiodarone, diphenylhydantoin, phenobarbital, and 3-methylcholanthrene on hepatic and renal T4 metabolism and thyroid gland function in rats.

We studied the effects of propylthiouracil (PTU), amiodarone (AMIO), diphenylhydantoin (DPH), phenobarbital (PB), and 3-methylcholanthrene (MC) on thyroid histomorphology, on the hepatic and renal enzymes involved in endogenous and exogenous metabolism, and on the plasma levels and pharmacokinetics of thyroid hormones after 7 and 14 days of treatment. PTU and PB, by decreasing both serum tetraiodothyronine (T4) and triiodothyronine (T3), induced a massive increase in serum thyrotropin (TSH) and thus induced thyroid hypertrophy. AMIO and MC, by decreasing respectively serum T3 and T4, also induced an increase of TSH, but to a lesser extent, not sufficient to induce thyroid hypertrophy. Hepatic 5'-deiodinase activity was decreased in all treated rats. Inhibition of this enzyme by PTU was demonstrated in vitro; AMIO also decreased the enzyme activity by a still unelucidated mechanism, which probably requires intact cell plasma membranes, whereas in PB- and MC-treated rats the decrease in enzyme activity certainly resulted from decreased serum concentrations of T4. In PTU-treated rats, and probably in MC-treated rats, decreases in circulating thyroid hormones were primarily due to impairment of synthesis and/or of secretion by the thyroid. In contrast, in PB-treated rats, the decrease in serum thyroid hormone levels seems to be due to increased excretion of these hormones, as T4 serum clearance was significantly increased. PB, a microsomal enzyme inducer, increased the cytochrome b5 and P450 content as well as the cytochrome P450-dependent O-depentylation of pentoxyresorufin. The other type of enzyme inducer, MC, did not affect cytochrome b5 and P450 levels, but did increase the cytochrome P450 dependent O-deethylation of ethoxyresorufin. PB increased the glucuronidation of morphine, whereas MC increased the glucuronidation of 1-naphthol. However, serum T4 clearance, mainly determined by its hepatic conjugation rate, was increased only in PB-treated rats. It appears from this study that the close metabolic relationship between the liver/kidney and the thyroid should be taken into consideration when the findings of chronic toxicology and carcinogenicity studies are interpreted.

Amiodarone↗
PubMed1991

Pig Leydig cell culture: a useful in vitro test for evaluating the testicular toxicity of compounds.

In vivo studies have shown that the testis is a target organ for drugs and chemicals. In order to evaluate the testicular toxicity of compounds and to identify the mechanisms of their toxicity, we have developed a miniaturized primary culture of immature pig Leydig cells. Five well-known drugs with differing mechanisms of toxicity on testicular functions were tested to validate the model. Testosterone and progesterone secretion were measured to evaluate testicular function. Cell viability was assessed quantitatively using a colorimetric assay based on the reduction of a tetrazolium salt (3-(4,5-dimethylthiazol)-2,5-diphenyltetrazolium bromide) which stains viable cells only, thus allowing discrimination between specific inhibitors of Leydig cell function and nonspecific cytotoxic drugs. Ketoconazole and aminoglutethimide inhibited both testosterone and progesterone secretion, but without modifying cell viability. Spironolactone specifically blocked testosterone secretion and increased progesterone concentration without inducing cell mortality. Cycloheximide altered testicular steroid secretion by another mechanism of action. Chlorpromazine, which interferes with the secretion of gonadotropins in vivo, produced a significant inhibition of progesterone and testosterone secretion as a result of the cytotoxic effects of the drug. In conclusion, this in vitro test enables one to discriminate accurately between specific and nonspecific inhibitors of steroidogenesis and could reduce the number of false positives when screening for potential testicular toxins.

Aminoglutethimide↗
PubMed1991

Optimum associations of tester strains for maximum detection of mutagenic compounds in the Ames test.

The Ames test is now widely used as a short-term test for the detection of mutagens. Different strains are available with various genetic characteristics, and in the past decade various authors have recommended different associations of strains to give maximum detection potential. However, few studies have been done to compare the sensitivity of individual strains towards a wide range of compounds in a single study. In order to define the best association of strains for screening or regulatory purpose, we have tested 103 direct mutagens (reference genotoxins or in-house compounds) on 7 strains of Salmonella typhimurium: TA1535, TA1537, TA1538, TA97, TA98, TA100 and TA102. 126 different associations of strains have been studied in terms of sensitivity and percentage overlap. Optimum associations of 2, 3, 4 or 5 strains included strains both with and without plasmid pKM101. However, the specificity of detection is greatly diminished by the presence of plasmid pKM101 in the strain, as shown by the high degree of overlap in associations constituted entirely of strains containing the plasmid. The association of strains TA1538 and TA100 detected 86% of the chemicals tested and is therefore recommended for large-scale screening. A rate of detection of 100% was obtained when 6 strains were used. The best associations of 4 and 5 strains, which detected 97 and 99% chemicals respectively, all contained strains TA1537, TA1538 and TA102. Finally, the associations of 4 strains (TA1537, TA1538, TA100, TA102) or 5 strains (TA1535, TA1537, TA1538, TA97, TA102) seemed well adapted to the optimum detection of mutagenic compounds.

Mutagenicity Tests↗
PubMed1989

Limb bud cell culture for in vitro teratogen screening: validation of an improved assessment method using 51 compounds.

Rat embryo limb bud cells multiply and undergo chondrogenesis in micromass culture. Teratogenic agents are identified from their inhibition of chondrogenesis, which is quantified by determination of cartilaginous foci number or proteoglycan production. In other in vitro systems, the detection is based on their ability to affect cell proliferation. So far, these methods have failed to distinguish among true inhibition of differentiation, inhibition of cell proliferation, and nonspecific cytotoxicity. The improved technique involves simultaneous measurement of cartilage synthesis and cell multiplication. Differentiation was evaluated by measurement, using an Artek Counter, of nodule areas after Alcian blue staining and proliferation by spectrophotometric quantification of Crystal-Violet bound to micromass cells. Using this method, retinoic acid was shown to inhibit chondrogenesis without affecting cell multiplication, whereas 6-aminonicotinamide preferentially inhibited cell multiplication without affecting nodule size. Doxylamine (succinate), a known nonteratogen, induced inhibition of chondrogenesis, but with a parallel inhibition of cell multiplication, reflecting a nonspecific toxic effect. This improvement increases the specificity of the micromass culture test. Validation was performed using 51 compounds. Compounds were classified according to their inhibitory activity and their active concentration. The sensitivity of the test was 61%; the specificity, 100%; and the final accuracy, 75%. The method is fully miniaturised, automated, and computerised, allowing numerous compounds to be rapidly tested at very low cost.

Animals↗
PubMed1989

In vivo micronucleus test using mouse hepatocytes.

The bone-marrow micronucleus (BMM) test is highly specific for clastogenic effects but its sensitivity is determined to a great extent by the substances tested, particularly by their metabolism. Some compounds, such as unstable mutagens or those which generate short-lived metabolites, are not detected in this test because the metabolites produced in the liver do not reach the bone marrow. In an attempt to provide qualitative and quantitative assessments of chromosomal mutations produced in vivo by genotoxic agents not detected in the mouse BMM test, a mouse-liver micronucleus test, adapted from Tates model, was developed. The animals were treated twice, with an interval of 24 h between treatments, and then subjected to partial hepatectomy (PH) 24 h after the second treatment in order to induce mitotic stimulation. The incidence of micronucleated hepatocytes was determined 96 h after PH. The test was evaluated with 5 procarcinogens, each with a complex metabolic pattern: dimethylnitrosamine (DMN), diethylnitrosamine (DEN), 1,1-dimethylhydrazine (1,1-DMH), 4-aminophenol (4-APOL), 4-aminobiphenyl (4-ABPYL) and one direct unstable mutagen, beta-propiolactone (BPL). All these compounds are negative in the mouse BMM test but caused a major increase in the incidence of micronuclei in mouse hepatocytes. This test is simple and can be readily compared with the BMM test. Furthermore, it offers a better assessment of the impact of a compound at the chromosomal level in a metabolically competent cell and can therefore be used for the evaluation of the genotoxic activity of compounds with complex metabolic pathways.

Aminobiphenyl Compounds↗
PubMed1986

Characterization of monoclonal antibodies to rabbit renal cortical cells.

The immunological heterogeneity of the rabbit nephron was investigated using monoclonal antibodies. Seventeen antibodies have been produced by fusion of NS1 myeloma cells with spleen cells from BALB/c mice immunized with unfractionated rabbit renal cortical cell preparations. Sixteen antibodies reacted with proximal tubular cells: 11 with the brush border and 5 with basolateral membrane or intracytoplasmic components. Only one of the latter was specific for constituents of the proximal tubule. One antibody reacted with the cortical collecting tubule. Eight of the anti-brush-border antibodies were further characterized by immunoprecipitation of detergent-solubilized radiolabeled brush-border membrane vesicles. Seven proteins with subunits ranging in molecular weight from 90,000 to greater than 340,000 were identified. Systematic survey showed that one of these proteins with a subunit molecular weight of 115,000 exhibited leucine aminopeptidase activity. Selected monoclonal antibodies bound to Sepharose 4B immunoadsorbents were used to deplete solubilized brush-border membrane vesicles of a given antigen and to identify leucine aminopeptidase. Furthermore, the obtention of specific antibodies directed against the proximal tubule allowed us to set up a simple method for renal cell separation: isolated renal cortical cells could be depleted by 80% in proximal cells by passage over columns of Sepharose 6MB covalently linked with three different monoclonal anti-brush-border antibodies, thus leading to cell suspensions considerably enriched in tubule cells originating from the more distal segments of the nephron.

Animals↗
PubMed1984

Ferritin deposition in the glomerular deposits of focal glomerular sclerosis in the rat.

The glomerular lesions of focal sclerosis clinically associated with a steroid-resistant nephrotic syndrome, are of unknown origin. IgM and C3 deposits and electron dense material found in areas of sclerosis are not convincing evidence of an immune pathogenesis. These deposits have been studied in a rat model of focal sclerosis induced by uninephrectomy and repeated aminonucleoside administration. Sclerotic lesions closely resembling human disease developed in the remaining kidney. There was a severe progressive proteinuria. Seventy-eight days after initial aminonucleoside injection 65 per cent of glomeruli were sclerotic with IgM, IgG, C3 and fibrinogen deposits, and electron dense deposits by electron microscopy. To study macromolecule in this model of focal sclerosis, ferritin uptake 4 and 24 h after intravenous ferritin given at 77 days was compared in focal sclerosis rats with control rats without sclerosis (uninephrectomy plus saline-only injections). In focal sclerosis rats sclerotic areas contained massive accumulations of ferritin. In unaffected segments of sclerotic glomeruli, and normal glomeruli of focal sclerosis rats, ferritin concentration was no different from controls. Abnormal ferritin trapping in areas of sclerosis suggests that the presence of IgM and C3 may be due to a similar mechanism, and is not indicative of an immune pathogenesis for focal sclerosis.

Animals↗
PubMed1984

A monoclonal antibody to brush border and passive Heymann nephritis.

An IgG2a monoclonal antibody (MoAb) reacting with the brush border of the renal proximal tubule and glomerular capillary wall was produced by fusion of NS1 myeloma cells with spleen cells from BALB/c mice immunized with renal brush border preparations from rat kidney cortex. This antibody reacts with a 90,000 mol. wt protein which can be isolated by immunoprecipitation of radiolabelled brush border or glomerular preparations and localized on these structures by immunoperoxidase electron microscopy, thus demonstrating the presence of common antigenic determinants. Survey of various organs showed that the MoAb reacted with the brush border of the gut, but also with antigens associated with the distal vascular system. In the liver antigenic determinants were located along the sinusoid walls but mainly on bile canaliculi. Specific glomerular binding could be demonstrated in vivo by immunofluorescence after an intravenous injection of 2 mg of antibody or by paired label methodology using tracer amounts. Kinetics however were dramatically different from those observed in classical passive Heymann nephritis since glomerular binding was transient during the first hours after injection. Binding was also found in tubular structures, as well as in lung, liver, spleen and heart. These results identify a well defined antigen-antibody system responsible for the formation of transient extramembranous glomerular deposits and may be relevant to some human cases of glomerulonephritis. They may also provide new models to study glomerular and tubular transfer of membrane bound antibodies.

Animals↗
PubMed1984

Production and characterization of monoclonal antibodies against rat brush border antigens of the proximal convoluted tubule.

In order to understand further the processes involved in immunological injury of the kidney, we have prepared monoclonal antibodies against brush border (BB) antigens of rat proximal tubule. The 27 antibodies which constitute the basis of this report have been cloned, characterized immunochemically, and classified in three specificity groups on the basis of tissue reactivity. The first group is made up of six antibodies reacting with antigens simultaneously present on BB and glomerulus: three are directed against a high molecular weight (MW) protein which migrates with an apparent MW of 330,000; two react with a 90,000 MW protein that is present diffusely on renal and intestinal BB as well as on endothelial cells; one recognizes an antigen exclusively present on superficial tubules and glomerular epithelial cells, which could not be chemically characterized. The second group is made up of eight antibodies present on renal and intestinal BB: five react with a 120,000 MW antigen, one with a 300,000 MW antigen. The third group comprises 13 antibodies. Two are directed against antigens present within the cytoplasm or the basolateral membranes of renal tubules. Eleven react with intracellular antigens probably related to the cytoskeleton. Since they have been identified through several fusions, some of the monoclonal antibodies described are probably directed against immunodominant proteins of the BB. They open new possibilities for purifying the corresponding antigens by affinity chromatography as well as for obtaining BB preparations selectively depleted of the strongest immunogens thus favouring antibody production to previously unrecognized antigens.

Animals↗
PubMed1984

Renal failure in myeloma: relationship with isoelectric point of immunoglobulin light chains.

Renal failure is a frequent but inconstant complication of myeloma related to light chain excretion. Since it has been suggested that cationic light chains (lc) are most likely to induce renal damage, we have studied the isoelectric point (pI) of light chains produced by 17 patients with myeloma and related the results to the type and severity of renal damage assessed clinically and pathologically. In order to do so, we have applied immunoenzymatic techniques which allow identification of light chain types as well as measurement of pI without prior purification. Ten of fifteen patients with renal failure produced lambda light chains. There was no simple relationship between the isoelectric point and nephrotoxicity. However, light chains with the lowest pI observed in this series were associated with normal renal function in two cases and with acute reversible but severe renal failure requiring dialysis in five cases. By contrast, pI values above 6.0 observed in the remaining patients were associated with moderate renal failure in six patients with recently diagnosed myeloma and with irreversible renal failure, and in two patients in whom myeloma had been evolutive for several years. We thus suggest that further pI measurements may help to identify light chains with different nephrotoxic potentials.

Acute Kidney Injury↗
PubMed1982

[Protective effect of procyanidolic oligomers on the heterologous phase of glomerulonephritis induced by anti-glomerular basement membrane antibodies].

Treatment by procyanidolic oligomers can significantly decrease the proteinuria indiced in the Rat by intravenous injection of anti glomerular basement membranes antibodies. Immunohistological analysis shows that procyanidolic oligomers do not interfere with the mechanisms of immunopathological injury involved in this model (antibody binding to glomerular basement membrane, complement activation, glomerular influx of polymorphonuclear leucocytes). Their protective effect may be due to an increased resistance of the glomerular capillary to the inflammatory mediators released by neutrophils.

Animals↗