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S Madle

Publications and source records attributed to S Madle.

At least 37 records · Page 2Linked to original sources
PubMed1991

The quality of genotoxicity testing of drugs. Experiences of a regulatory agency with new and old compounds.

The state of the art of mutagenicity testing of drugs based upon new entities submitted for registration in the Federal Republic of Germany is reviewed for the period between 1986 and 1989. In the initial phase of registration 50 out of 107 new compounds were tested insufficiently, thus making safety assessment for therapeutic use difficult. The main shortcomings applied to both missing as well as insufficiently performed mutagenicity tests. In some cases indications for a genotoxic activity were obtained due to inadequate testing giving rise to a suspicion of a mutagenic potential which had to be clarified. Upon additional testing during the subsequent phases of the registration procedure almost all insufficiencies and unclarified suspicions could be eliminated. Several examples on this point are discussed. When evaluating data for 'old compounds' regulatory authorities are confronted with considerable problems. This publication gives some practical advice for judging the plausibility and relevance of published data. Two examples (quercetin and malathion) for which published data are summarized, illustrate that even extensive literature data may not be sufficient to draw a definitive conclusion, if the relevance of most of the results is questionable.

Animals↗
PubMed1989

Mutagenicity testing of doxylamine succinate, an antinauseant drug.

Doxylamine succinate (DA), a compound which was formerly used as an antinauseant during pregnancy, showed no substantial mutagenicity in mouse embryos following transplacental exposure. A small dose-dependent induction of chromosomal aberrations was found in mouse embryos on day 11 of gestation. No induction of sister chromatid exchanges (SCE) was found in embryos on day 11 of gestation. A micronucleus test with fetal blood on day 17 of gestation was negative. Additionally, DA was negative in Chinese hamster bone marrow in vivo (micronuclei) and in human lymphocyte cultures in vitro (SCE).

Animals↗
PubMed1988

Induction of SCE by indirect mutagens in cultured rat hepatoma cells and in Chinese hamster V79 cells co-cultivated with hepatocyte primary cultures.

The continuous rat hepatoma cell line H4IIEC3/G- and rat hepatocyte primary cultures (hpc) were compared with regard to their capacity to metabolize structurally different promutagens. The sensitivities of both activation systems were evaluated by comparing the induction of SCE in H4IIEC3/G- cells themselves with that ih V79 cells co-cultured with hpc. Of the six chemicals tested, aflatoxin B1 (AFB1), cyclophosphamide, dimethylnitrosamine and nitrosomorpholine (NM) were shown to be inducers of SCE in H4IIEC3/G- cells as well as in V79 cells with hepatocyte activation. 7,12-Dimethylbenzanthracene gave positive responses in hpc/V79 co-cultures but not in H4IIEC3/G- cells whereas benzo[a]pyrene was negative in both systems. These results suggest that H4IIEC3/G- cells retain metabolic activities to convert different indirect mutagens into their active forms and clearly indicate the presence of liver specific cytochrome P-450-dependent mono-oxygenases. However, freshly isolated hepatocytes are more efficient in metabolizing the test compounds. Although hpc provide only external activation, the V79 cells system appears to be more sensitive for the detection of promutagens.

Animals↗
PubMed1987

Cyclophosphamide: interstrain differences in the production of mutagenic metabolites by S9-fractions from liver and kidney in different mutagenicity test systems in vitro and in the teratogenic response in vivo between CBA and C 57 BL mice.

The formation of mutagenic compounds from cyclophosphamide (CPA) by S9-fractions from liver (S9L) or kidney (S9K) of pregnant CBA and C 57 BL mice was investigated, using point mutations in Salmonella typhimurium (TA 1535) and the induction of sister chromatid exchanges (SCE) in human peripheral lymphocytes (HPL) or Chinese hamster ovary (CHO) cells as end points. In addition, the teratological response of CBA and C 57 BL mice to CPA on day 11 of pregnancy was analysed in vivo. The results are as follows: (1) S9L from CBA mice was more effective than S9L from C 57 BL mice in metabolizing CPA to products inducing mutations in Salmonella and SCEs in HPL and CHO cells. (2) S9L was more effective than S9K from both strains of mice. (3) In vivo pretreatment of mice with a single dose of CPA (20 mg/kg) reduced the in vitro metabolizing capacity of S9L and S9K significantly and led to the disappearance of the interstrain difference. (4) The embryolethal and teratogenic effects of CPA were stronger in C 57 BL than in CBA mice; the types of teratological effects were partially different in the two strains.

Animals↗
PubMed1987

Experience with mutagenicity testing of new drugs: viewpoint of a regulatory agency.

Quality and quantity of mutagenicity testing were analyzed for drugs with new active compounds which were submitted for registration in the Federal Republic of Germany from mid 1982 to mid 1986. A large variety of deficiencies was found, applying to selection and number of mutagenicity tests as well as to test performances. Only 65 out of the 144 drugs submitted for registration were tested sufficiently in the initial phase of registration. From 1982 to 1986 this situation has not been changed markedly. Inadequate test performance still remains the main reason for insufficient testing, leading in some cases to artificially positive results. For in vivo tests the selection of test species was mainly motivated by technical reasons and not by characteristics of the test compound. Most of the insufficiencies were eliminated during the second phase of registration. In some cases insufficient mutagenicity testing led to consequences concerning risk-benefit assessment of the drug and its regulation.

Animals↗
PubMed1987

Hepatocyte-mediated SCE induction by indirect mutagens: importance of hepatocyte density and cell-to-cell contact.

The SCE-inducing effects of the indirectly acting mutagens cyclophosphamide (CP), dimethylnitrosamine (DMN) and aflatoxin B1 (AFB1) were analysed in hepatocyte (hpc)/mammalian cell coculture systems with regard to the importance of the hpc density. V79 cells and human lymphocytes served as target cells. For all 3 compounds steadily increasing genetic effects were observed when the hpc density was increased from 3.2 X 10(4) up to 3.2 X 10(6) viable hpc per culture (25-cm2 flask), i.e. the more hpc available for metabolisation, the more genetic effects induced. The frequency distributions of the CP-induced SCE values were clearly different from those obtained with DMN, especially when high hpc densities were used: distribution patterns obtained for the mutagen with stable metabolites (CP) are characterized by the presence of distinct maxima and the absence of cells with SCE control values, whereas distribution patterns for the mutagen with very short-lived metabolites (DMN) can be described by the absence of maxima and the presence of cells with SCE control values. The frequency distributions of the AFB1-induced SCE values were more similar to the CP type than to the DMN type. From these results it is deduced that close contact between metabolising and target cells is necessary for the detection of the genotoxic effect of DMN. For CP and AFB1 a direct contact seems not to be essential, i.e. reactive intermediates may also be transported via the culture medium to the target cells.

Aflatoxin B1↗
PubMed1987

Hepatocyte activation of indirect mutagens during the first days in primary culture.

Changes in activation capacity of hepatocytes during the first days after hepatocyte isolation were investigated by measuring the cytogenetic effects of the indirect mutagens cyclophosphamide (CP), dimethylnitrosamine (DMN) and aflatoxin B1 (AFB1). On the day of hepatocyte isolation and on the subsequent 4 days, hepatocytes were co-cultured with V79 cells, and SCE- and chromosome aberration-tests were performed. CP- and DMN-induced SCE frequencies declined with increasing hepatocyte age. Such a decline was also found for AFB1-induced aberration frequencies, whereas the SCE-inducing effect of AFB1 remained stable up to 4 days after hepatocyte isolation. This indicates that SCE-inducing and clastogenic AFB1 metabolites are generated by different enzymatic pathways. The clastogenic metabolites of AFB1--as well as the SCE-inducing CP- and DMN-metabolites--are generated by cytochrome P450-dependent monooxygenases whose activity is obviously lost during the first days of primary hepatocyte culture. Activation of AFB1 to SCE-inducing metabolites may be performed by more stable monooxygenases. Our results show that the ability of hepatocytes to activate indirect mutagens decreases during the first few days after hepatocyte isolation. Furthermore, the results with AFB1 indicate that routine culture methods are not suitable to maintain the balance of the different enzymatic functions which are involved in metabolism of indirect mutagens.

Aflatoxin B1↗
PubMed1986

Comparison of S9 mix and hepatocytes as external metabolizing systems in mammalian cell cultures: cytogenetic effects of 7,12-dimethylbenzanthracene and aflatoxin B1.

Two external metabolizing systems, S9 mix from Aroclor-induced rat livers and freshly isolated hepatocytes, were used for activation in cultures of human lymphocytes and V79 cells. 7, 12-dimethylbenzanthracene (DMBA) and aflatoxin B1 (AFB1) were employed as indirectly acting reference mutagens. Mutagenic effects were measured by induction of sister chromatid exchange (SCE). With DMBA, SCE-inducing effects were found to be quite similar after activation by S9 mix and activation by hepatocytes. In human lymphocytes nearly the same dose-effect relationships were found with both metabolizing systems; in V79 cells the hepatocyte-mediated induction of SCE was detectable at slightly lower concentrations than the S9-mediated SCE induction. In contrast with AFB1, S9 activation led to a stronger SCE induction than hepatocyte activation in both target cells. The induction of chromosomal aberrations by AFB1 after activation by the two metabolizing systems was also analysed in V79 cells. This experiment again revealed that AFB1 was more efficiently activated by S9 mix than by hepatocytes, and it appeared that AFB1 is a more potent inducer of chromosomal aberrations than of SCE. The different activation capacities of the two metabolizing systems for AFB1 may be due to the maintenance of inactivation mechanisms in hepatocytes or to the Aroclor induction of the S9 fraction. Our experiments have shown that the suitability of hepatocytes as an activation system is not restricted to microbial or eukaryotic point mutation assays, but that hepatocyte metabolism can also be successfully included in cytogenetic tests with short- and long-term cultures of mammalian target cells.

9,10-Dimethyl-1,2-benzanthracene↗
PubMed1984

Mutagenic activities of cyclophosphamide (NSC-26271) and its main metabolites in Salmonella typhimurium, human peripheral lymphocytes and Chinese hamster ovary cells.

Cyclophosphamide (CPA) and its main metabolites were analyzed with respect to their mutagenic activities in Salmonella, human peripheral lymphocytes (PL), and Chinese hamster ovary (CHO) cells. In Salmonella, the compounds were activated with S9 mix from rat livers, which were unstimulated or stimulated with Aroclor 1254 or phenobarbital. For the enzyme inducers the following order of efficiency was found for all test compounds except carboxyphosphamide: phenobarbital greater than Aroclor 1254 greater than non-induced. The most potent mutagens in all 3 test systems were 4-OH-CPA, PAM and nor-HN2. S9 mix transforms 4-OH-CPA to strong mutagenic compounds in the Salmonella assay. All metabolites tested in the Salmonella assay were activated by S9 mix to higher mutagenic potential.

Animals↗
PubMed1982

Double-blind study on the effect of cigarette smoking on the chromosomes of human peripheral blood lymphocytes in vivo.

A double-blind study with 170 smokers and 124 non-smokers revealed a doubling of the frequencies of exchange-type aberrations in the peripheral blood lymphocytes of smokers as compared with non-smokers. Smokers (N = 24) had 1 SCE more per metaphase than non-smokers (N = 20), an effect that is significant but low when compared with the effect on structural chromosomal aberrations. These results show that structural chromosomal aberrations are better indicators of the genetical effects of low chronic exposures to mutagens in man than SCEs. The frequencies of micronuclei in 3-day lymphocyte cultures showed no differences in smokers (N = 95) and non-smokers (N = 39). Non-smokers (N = 99) had significantly higher frequencies of 2nd metaphases in 48-h cultures in vitro than smokers (N = 141), indicating an effect of smoking on the stimulation of lymphocytes by PHA in vitro.

Cells, Cultured↗
PubMed1981

Evaluation of experimental parameters in an S9 /human leukocyte SCE test with cyclophosphamide.

The experimental parameters S9 concentration, cofactor concentration and exposure time were investigated in a microsome test with human leukocyte cultures, To evaluate these parameters, their effects on cyclophosphamide-induced SCE frequencies and changes in cell proliferation were analyzed. By varying S9 concentration and exposure time, dose-effect relationships were found with respect to the induction of SCE. A relatively small effect was found when the concentrations of the cofactors NADP and G-6-P were varied. In general, an increase in SCE frequencies was accompanied by delayed cell proliferation. The importance of varying the experimental parameters to avoid false- negative results in microsome tests is discussed.

Animals↗
PubMed1981

The use of rat liver perfusion to detect indirect mutagens/carcinogens in human leukocytes in vitro.

The rat liver perfusion technique in situ is used to metabolize the indirect mutagen cyclophosphamide (CP). Chromosomal damage (sister chromatid exchanges, chromatid translocations) caused by activated CP is investigated with human lymphocyte cultures in vitro, demonstrating dose- and time-effect relationships. Especially the treatment of human whole blood with CP perfusate of a recirculating perfusion system is recommended as a useful model system for the induction of chromosomal damage in peripheral lymphocytes in vivo in man.

Animals↗
PubMed1980

Methods for analysis of the mutagenicity of indirect mutagens/carcinogens in eukaryotic cells.

The review discusses the variety of methods for activation of indirect mutagens/carcinogens and testing them in cell cultures, especially in mammalian cell cultures. After the necessity for including metabolizing components in mutagenicity tests has been pointed out, the enzymes that transform foreign compounds metabolically, and the factors influencing them, are described. In the main section the various methods of activating indirect mutagens/carcinogens are presented. The methods of including in vivo metabolism in mutagenicity tests are: Analysis of cells from organisms contaminated with a chemical (III.l.a); body fluid-mediated mutagenesis (III.l.b); host-mediated assay (III.l.c). The following activation systems are suitable for including in vitro metabolism of test compounds in mutagenicity tests: Liver and lung perfusion (III.2.a.alpha); organ slices and homogenates (III.2.a.beta); subcellular fractions (III.2.a.gamma); cultivated cells (cell-mediated mutagenesis) (III.2.b); nonenzymatic activation systems (III.2.c). Finally the main factors that influence the metabolism of test substances are summarized. Two factors illustrate the mutagenicity tests with regard to the metabolism of mammalian livers and the methods of performing mutagenicity tests in man.

Animals↗
PubMed1980

Possible mutagenicity of the psychoactive phenothiazine derivative perazine in vivo and in vitro.

Human lymphocyte cultures from 55 schizophrenic subjects and one manic-depressive subject being treated with the phenothiazine derivative perazine and with other drugs were analyzed with respect to chromosomal damage. The frequency of exchange-type aberrations in these subjects was more than double that in clinically normal control subjects. No correlation was detectable between the aberration frequency and sex, age, smoking and drinking habits, and treatment conditions. It is possible that the elevation of the chromosomal aberration frequency is due to perazine. In vitro studies with perazine and two main metabolites (desmethylperazine and perazine sulfoxide) with human lymphocytes and CHO cells with and without metabolic activation by liver microsomes gave negative results with respect to the induction of sister chromatid exchanges. Possible differences in the metabolism of perazine in vivo and in vitro are discussed.

Adolescent↗
PubMed1978

Testing in vitro of an indirect mutagen (cyclophosphamide) with human leukocyte cultures: Activation by liver perfusion and by incubation with crude liver homogenate.

The indirect mutagen cyclophosphamide was tested in human whole blood cultures with respect to chromosome-breaking activities and suppression of mitotic indices after activation by liver perfusion and crude liver homogenates with and without cofactors. Both methods produced nearly the same effects, with the exception of liver homogenate without cofactors which had only weak metabolic activities.

Animals↗