Sensitivity of drosophila mutants to chemical carcinogens.

7 single-mutant and five double-mutant strains of Drosophila melanogaster were tested for their relative sensitivity to the chemical carcinogens: 1-acetylaminofluorene, benzo(alpha)pyrene, N-methyl-N'-nitro-N-nitrosoguanidine, 4-nitro quinoline-1-oxide and aflatoxin B1. Among the single mutants, mei-9a, mei-41D5 and mus(1)104D1 are hypersensitive to all 5 chemicals, whereas mus(1)107D1 is hypersensitive only to 4-nitroquinoline-1-oxide and is slightly sensitive to benzo(alpha)pyrene. The mei-9a mei-41D5 double-mutant is the most sensitive of 5 tested double-mutants which carry the mei-9a allele. When treated with 0.025 mM benzo(alpha)pyrene this double-mutant produces significantly more sex-linked recessive lethals and dominant lethals than does the control. Analysis of double-mutants reveals that the mei-9+ product functions in a different repair pathway of methyl methanesulfonate-induced damage than do the normal products of the mus(1)103, mus(1)104 and mus(1)107 loci. Our findings suggest that the sensitivity of Drosophila repair-deficient mutants could be exploited in screening for potential mutagens and carcinogens.     

Multi-system approach to study mutagenesis induced by chemical carcinogens.

To understand molecular mechanisms of the mutation fixation process induced by a mutagen and carcinogen, a multi-system approach is suggested to reduce the probability that the results are biased by the assay used. In this light we described our different approaches to answer basic questions on the mutagenesis induced by the chemical carcinogen 4-Nitroquinoline-1-oxide. We determined mutations at the molecular level in three experimental systems: a) in prokaryotes (ss M13mp19 lacZ'/E. coli F'lacZ delta M15); b) in eukaryotes (i) ss and ds pZ189 supF/CV1-P/E.coli lacZam and (ii) HPRT in CHO cells with different repair capacity. We think this type of approach can be used to study the genetic effects of new cancer drugs for which the molecular mechanisms of action at the molecular level are still not well understood. We think to apply the know-how to study mutational spectra in tumor derived tumor suppressor genes.     

Analysis of DNA-protein complexes induced by chemical carcinogens.

DNA-protein complexes induced in intact cells by chromate have been isolated and compared with those formed by other agents such as cis-platinum. Actin has been identified as one of the major proteins that is complexed to the DNA by chromate based upon a number of criteria including, a molecular weight and isoelectric point identical to actin, positive reaction with actin polyclonal antibody, and proteolytic mapping. Chromate and cis-platinum both complex proteins of very similar molecular weight and isoelectric points and these complexes can be disrupted by exposure to chelating or reducing agents. These results suggest that the metal itself is participating in rather than catalyzing the formation of a DNA-protein complex. An antiserum which was raised to chromate-induced DNA-protein complexes reacted primarily with a 97,000 protein that could not be detected by silver staining. Western blots and slot blots were utilized to detect p97 DNA-protein complexes formed by cis-platinum, UV, formaldehyde, and chromate. Other work in this area, involving studying whether DNA-protein complexes are formed in actively transcribed DNA compared with genetically inactive DNA, is discussed. Methods to detect DNA-protein complexes, the stability and repair of these lesions, and characterization of DNA-protein complexes are reviewed. Nuclear matrix proteins have been identified as a major substrate for the formation of DNA-protein complexes and these findings are also reviewed.     

ras-transformation of MDCK cells alters responses to phorbol ester without altering responses to bradykinin

The results of studies to evaluate the hypothesis that the 21 kDa GTP-binding protein derived from the ras oncogene is involved in regulation and coupling of hormone receptors to phospholipase activity have thus far been inconsistent. We therefore examined the effect of H-ras transformation on basal, tumor-promoting phorbol ester (TPA)-stimulated, and bradykinin-mediated phospholipid hydrolysis in Madin Darby canine kidney cells (MDCK) by comparing H-ras-transformed MDCK cells (MDCK-RAS) to two non-transformed strains of MDCK cells (MDCK-D1 and MDCK-ATCC). In unstimulated MDCK-RAS, diacylglycerol (DAG), inositol phosphate accumulation, and choline phosphate release were increased while arachidonic acid and arachidonic acid metabolite (AA) release was not increased, suggesting that ras transformation increased phospholipase C activity. Protein kinase C (PK-C) activity was decreased, and specific binding of [3H]phorbol ester was reduced in MDCK-RAS relative to the non-transformed MDCK cells suggesting that elevated DAG may activate and thereby down-regulate PK-C. Consistent with this finding in MDCK-RAS, TPA-stimulated AA release and subsequent prostaglandin E2 production were decreased, while TPA-stimulated choline phosphate release was increased. Bradykinin receptor-stimulated phospholipid hydrolysis in MDCK-RAS was similar to that of non-transformed cells, suggesting that the ras-derived protein does not directly couple bradykinin receptors to phospholipases in MDCK cells. However, the ability of TPA-treatment to inhibit bradykinin-stimulated phosphoinositide hydrolysis and enhance bradykinin-stimulated AA release was attenuated in MDCK-RAS. Additionally, in MDCK-RAS the conversion of arachidonic acid to prostaglandin E2 was substantially reduced. We conclude that ras transformation of MDCK cells increases DAG levels, thereby activating and, in turn, down-regulating PK-C and certain responses to TPA. Since activation of PK-C may result in a variety of effects on signal transduction pathways, we propose that increased DAG and altered PK-C levels associated with ras transformation may account for the inconsistent effects previously observed in studies evaluating the effect of ras transformation on phospholipases and other signal transduction systems.     

Metabolism and activation of chemical carcinogens

Summary A great deal of effort has gone into research on the mechanisms of action of chemical carcinogens. The action of the host on the carcinogen represents one approach — to determine what metabolic products may be involved. Study of the action of the carcinogen on the host requires a thorough comparison of changes wrought in cellular constituents by the carcinogen. Meshing of these two approaches increases the potential to discover the detailed mechanisms involved. Despite the increase in knowledge, many crucial molecular events and their consequences in the process of carcinogenesis still remain a mystery.     

Immunostimulation exacerbates the biological effects of chemical carcinogens

The reciprocal relation between the high regenerative ability of various animal species and the low incidence of haphazard or experimentally induced malignant tumours in these animal species is well documented. Equally well documented is the repeated observation that the decline in regenerative potential coincides with an increase in the incidence of cancers, a fact which, on an evolutionary scale, parallels with the development of a sophisticated immune system. The combination of the above observations led to the hypothesis that at least parts of an immune reaction might promote tumour development, and indeed, many experiments specifically designed to answer this question support this prediction. However, this “immunostimulation theory of tumour development” is neither explained in a satisfactory fashion nor universally adopted. The aim of the present investigation was to approach this issue by exploiting the dual, spectacular ability of urodele amphibians to regenerate a lot of organs and to make a stand to carcinogenesis. To this end, urodele amphibians of the species Triturus cristatus were immunologically challenged by intra-abdominal injections of sheep serum, they had then both their hind limbs amputated, and crystals of MNNG (N-Methyl-N″-nitro-N-nitrosoguanidine) were implanted into the stumps. The results show that the effects of MNNG on the immunostimulated animals display significant quantitative augmentation with respect to non-immunized controls. This augmentation consists in higher animal mortality, extension of the dedifferentiating stump tissue and concomitant retardation of limb restoration, and increase in the incidence of abnormal regenerates.     

Streptozotocin-induced diabetes modulates the metabolic activation of chemical carcinogens

The effect of chemically-induced diabetes on the hepatic microsomal mixed-function oxidase system and the activation of chemical carcinogens was investigated in animals treated with streptozotocin (STZ). In order to distinguish between the effects of the diabetogenic chemical per se and that of the diabetic state, groups of STZ-treated animals received either nicotinamide simultaneously with STZ to prevent the onset of diabetes, or daily treatment with insulin in order to reverse the effects of diabetes. STZ-treated animals exhibited higher pentoxyresorufin O-dealkylase, ethoxy-resorufin O-deethylase, ethoxycoumarin O-deethylase, aniline p-hydroxylase and NADPH-cytochrome c reductase activities; similarly, increases were seen in cytochrome P-450 and b5 levels. All of these effects were prevented by nicotinamide and, at least partly, antagonised by insulin therapy. Treatment of animals with STZ markedly increased the activation, by liver microsomes in vitro, of Trp-P-1 and Trp-P-2 to mutagens, the effect being totally preventable by nicotinamide and successfully antagonised with insulin therapy. The diabetic animals were similarly more efficient in activating MeIQ but the effect was not preventable by nicotinamide or reversed by insulin. In contrast no changes were seen in the activation of IQ and only a modest increase in the case of MeIQx. It is concluded that diabetes may modulate the metabolic activation of some chemical carcinogens, presumably by changing the ratio of the various cytochrome P-450 isoenzymes.     

In vitro transformation assays for chemical carcinogens

A variety of in vitro mammalian cell assays, designed specifically for the identification of carcinogenic compounds, have been in operation for more than a decade. Although no individual transformation system has won universal acceptance during this time, recent advances have led to the improved reliability and sensitivity of a number of these short-term tests. The underlying problems associated with the most widely used assays are identified and new developments in this rapidly expanding field are noted and discussed.     

Histofluorescence technique for the intracellular localization of chemical carcinogens

Summary Many carcinogens exhibit a fluorescent emission when excited with ultraviolet light. Advantage has been taken of this property to develop a technique that can detect microquantities of these carcinogens on conventional microscopic tissue preparations. This work describes the localization of aflatoxin B₁, N-2-fluorenylacetamide and benzo(a)pyrene both in the cell cytoplasm and nucleus afterin vivo administration of these compounds.     

Hyperketonaemia markedly modulates the metabolic activation of chemical carcinogens

Male Wistar albino rats were rendered hyperketonaemic by oral administration of medium chain triacylglycerols or by a single intraperitoneal injection of the diabetogenic agent streptozotocin. Hepatic post-mitochondrial preparations from these animals were employed as activation systems in the Ames mutagenicity test. Activation systems from both groups of hyperketonaemic rats were more efficient than those of control rats in metabolically converting the precarcinogens Glu-P-1, Trp-P-1, Trp-P-2, N-nitrosopiperidine and N-nitrosopyrrolidine to mutagens. In contrast, when 2-aminofluorene was used as the precarcinogen, the preparations from the hyperketonaemic animals were less efficient than controls in activating this carcinogen. In all cases, the preparations from streptozotocin-treated rats displayed a more pronounced effect than those from triacylglycerol-treated rats, possibly reflecting the greater extent of hyperketonaemia in the former group. It is concluded that hyperketonaemia modulates the bioactivation of chemical carcinogens.     

An overview of bioactivation of chemical carcinogens

Most environmental carcinogens require metabolic activation to reactive intermediates and are mutagenic in appropriate test systems. During the last decade, the cDNAs of numerous xenobiotic-metabolizing enzymes have been cloned. The individually expressed enzymes were used to study their substrate specificities and their inhibition by other compounds. Various enzymes were expressed directly in target cells of in vitro mutagenicity tests. This is illustrated in the present study for rat and human sulphotransferases (SULTs) expressed in Salmonella typhimurium TA1538. Numerous compounds were mutagenic in the new test system. Some of these promutagens were activated by several different SULT forms, whereas many other promutagens were activated with high selectivity by a specific enzyme form, but not by genetically closely related forms from the same species (e.g. allelic variants) or orthologous enzymes from other species. Similar findings have been made using recombinant test systems for specific forms of other classes of enzymes (e.g. cytochromes P450). This high selectivity in activation (and inactivation) may explain some organotropisms as well as species and inter-individual differences in the action of carcinogens. Many carcinogen-metabolizing enzymes are induced or inhibited by other xenobiotics. Such interactions can be exploited for chemo-prevention, which however may be carcinogen- and tissue-dependent.     

Dietary fat modifies the in vivo mutagenicity of some food-borne carcinogens

Female BALB/c mice were fed a low fat diet (1% safflower oil, by weight) or one supplemented with 25% (by weight) of beef fat or olive oil. The abilities of these diets to modify the in vitro and in vivo hepatic conversion of the dietary carcinogens aflatoxin B1, 2-amino-3, 4-dimethylimidazo[4,5-f]quinoline (MeIQ) and 3-amino-1-methyl-5H-pyrido[4,3-b]indole (Trp-P-2) to bacterial mutagens was evaluated. Dietary olive oil appeared to increase the metabolism of both MeIQ and Trp-P-2 to bacterial mutagens in vivo using the intrasanguineous host-mediated assay. Feeding mice either of the high-fat diets increased hepatic conversion of these two compounds to bacterial mutagens in vitro. Dietary fat had no effect on the metabolism of aflatoxin B1. Subsequent experiments suggested that the in vivo effects of dietary olive oil on MeIQ and Trp-P-2 mutagenesis were due to the induction of hepatic enzyme activities rather than to increased rates of uptake of the carcinogen from the gut-lumen.     

Early effects of chemical carcinogens as compared to induced cell proliferation. II. Automated image analysis

Static automated image analysis was applied to study early variations of chromatin structure in Feulgen-stained liver nuclei from rats injected i.p. with a single dose of dimethylnitrosamine (DMNA), a well known hepatocarcinogen. An increase of nuclear area and a correspondent decrease of average optical density (integrated optical density/area) was observed, as compared with controls, in nuclei from rats treated with 5.4 mg/kg of DMNA. These findings, which were comparable with those induced by partial hepatectomy, indicate the existence in DMNA-treated cells of a chromatin DNA relaxation similar to the G0-G1 transition previously described for human diploid fibroblasts stimulated to proliferate. Because similar results were independently obtained by flow microfluorimetry, it seems reasonable to hypothesize that chromatin decondensation could be a prerequisite for cancer induction.     

斑地锦的化学成分

斑地锦 (ＥｕｐｈｏｒｂｉａｍａｃｕｌａｔａＬ .)系大戟科 (Ｅｕｐｈｏｒｂｉａｃｅａｅ)植物 ,一年生匍匐小草本 ,具有清湿热、止血、通乳的功效 ,常用以治疗痢疾、疳积、外伤出血、痈肿疮毒。药典上将其作为地锦草入药 ,近代药理研究表明斑地锦具有抗菌及抗寄生虫的作用[1 ,2 ] 。文献[3～ 6 ] 报道斑地锦的化学成分主要为黄酮、甾醇、三萜及鞣质类化合物。为进一步寻找斑地锦的抗菌活性物质 ,进行了化学成分分析 ,从中分离得到 9个化合物 ,经理化性质和波谱分析鉴定为 :槲皮素 (Ⅰ )、山柰酚 (Ⅱ )、芹菜素 7 Ｏ 葡萄糖甙 (Ⅲ )、木犀…     

苗族药马蹄金化学成分的研究

从苗族药马蹄金（Dichondra erpen Forst.）全草中分离得到5个化合物,经波谱分析鉴定为委陵莱酸(tormentic acid,I）、尿嘧啶（uracil,2）,茵芋甙（skimmin,3）,甘油（glycerin,4）和N-（-N-苯甲酰基-L-苯丙氨酰基）-O-乙酰基-L-苯丙氨醇（N-（N-benzoyl-L-phenylalanyl-）-O-actyl-L-phenylalanol,5),以上化合物均首次从马蹄金中获得。     

In vivo interactions between ionizing radiation, inflammation and chemical carcinogens identified by increased DNA damage responses

Exposure to ionizing radiation or a variety of chemical agents is known to increase the risk of developing malignancy and many tumors have been linked to inflammatory processes. In most studies, the potentially harmful effects of ionizing radiation or other agents are considered in isolation, mainly due to the large number of experiments required to assess the effects of mixed exposures with different doses and different schedules, and the length of time and expense of studies using disease as the measure of outcome. Here, we have used short-term DNA damage responses to identify interactive effects of mixed exposures. The data demonstrate that exposure to ionizing radiation on two separate occasions ten days apart leads to an increase in the percentage of cells with a sub-G(0) DNA content compared to cells exposed only once, and this is a greater than additive effect. Short-term measurements of p53 stabilization, induction of p21/Cdkn1a and of apoptosis also identify these interactive effects. We also demonstrate similar interactive effects of radiation with the mutagenic chemical methyl-nitrosourea and with a nonspecific pro-inflammatory agent, lipopolysaccharide. The magnitude of the interactive effects is greater in cells taken from mice first exposed as juveniles compared to adults. These data indicate that short-term measurements of DNA damage and response to damage are useful for the identification of interactions between ionizing radiation and other agents.