Inhibition of protein kinase Calpha enhances anticancer agent-induced loss of anchorage-independent growth regardless of protection against apoptosis by Bcl-2.

In the present study, we investigated the effects of several selective protein kinase C (PKC) inhibitors (Gö6976, Gö6983, bisindolylmaleimide I, and rottlerin) in combination with conventional anticancer drugs on apoptosis and long-term anchorage-independent growth of both parental and Bcl-2-overexpressing mammary adenocarcinoma MTLn3 cells. In normal MTLn3 cells, doxorubicin- and etoposide-induced apoptosis was not affected by any of the PKC inhibitors. However, Bcl-2-mediated cytoprotection against apoptosis was slightly counteracted by Gö6976, a selective inhibitor of PKCalpha, as well as by transient overexpression of dominant-negative PKCalpha. Doxorubicin and etoposide both inhibited anchorage-independent growth; for doxorubicin, this occurred at concentrations that did not yet cause apoptosis. Overexpression of Bcl-2 did not overcome these growth-inhibitory effects. The effects of doxorubicin on colony formation were potentiated by Gö6976, Gö6983, and bisindolylmaleimide I but not rottlerin. In contrast, etoposide-induced loss of clonogenicity was primarily enhanced by Gö6976. Gö6976 alone, but not Gö6983, bisindolylmaleimide I, or rottlerin, inhibited colony formation in soft agar. This effect of Gö6976 correlated with inhibition of cell cycle progression. Overall, the data indicate that pharmacological inhibitors of PKCalpha in combination with anticancer drugs, act additively to inhibit long-term anchorage-independent tumor cell growth, independent of apoptosis induction. Importantly, similar additive effects are observed in Bcl-2 overexpressing cells.

Phosphorescent platinum/palladium coproporphyrins for time-resolved luminescence microscopy.

Streptavidin and antibodies were labeled with phosphorescent platinum and palladium coproporphyrin. The optimal conjugates were selected on the basis of spectroscopic analysis (molar extinction coefficient, quantum yield, lifetime) and using ELISA assays to determine the retention of biological activity and immunospecificity. They were subsequently tested for the detection of prostate-specific antigen, glucagon, human androgen receptor, p53, and glutathione transferase in strongly autofluorescent tissues. Furthermore, platinum and palladium coproporphyrin-labeled dUTPs were synthesized for the enzymatic labeling of DNA probes. Porphyrin-labeled DNA probes and porphyrin-labeled streptavidin conjugates were evaluated for DNA in situ hybridization on metaphase spreads, using direct and indirect methods, respectively. The developed in situ detection technology is shown to be applicable not only in mammals but also in plants. A modular- based time-resolved microscope was constructed and used for the evaluation of porphyrin-stained samples. The time-resolved module was found suitable for detection of antigens and DNA targets in an autofluorescent environment. Higher image contrasts were generally obtained in comparison with conventional detection systems (e.g., fourfold improvement in detection of glutathione transferase).

Lack of mammalian mutagenicity of the potent bacterial mutagen tris(2,3-dibromopropyl) phosphate and its metabolite 2-bromoacrolein.

The flame retardant tris(2,3-dibromopropyl)phosphate (Tris-BP) and its metabolite 2-bromoacrolein (2BA) are very potent bacterial mutagens in Salmonella typhimurium (S. typhimurium) TA 100. In this study, we showed that 2BA and Tris-BP are also mutagenic in S. typhimurium TA 104, which detects mutations at AT base pairs, while TA 100 detects mutations at CG basepairs. We also studied the mutagenicity of 2BA in mammalian cells in vitro and in the rat in vivo. Firstly, 2BA was tested in the human lymphoblastoid cell line TK6. The results showed that there was no increase in mutation frequency at the hprt locus, whereas there was a large decrease in cell survival. Secondly, a shuttle vector system was used to study the induction of mutations by 2BA:DNA adducts. The vector was modified by insertion of a single-stranded oligonucleotide containing on average one 2BA:DNA adduct. No increase in mutation frequency above background was detected after replication of this vector in SV40 transformed normal human fibroblasts. Because the liver is a major site for bioactivation of Tris-BP to 2BA in vivo, we tested the initiating capacity of Tris-BP in the rat liver in a modified Solt & Farber initiation and promotion system. Administration of Tris-BP resulted in a small increase in the number of preneoplastic gamma-glutamyl-transpeptidase positive (GGT+) foci in the liver compared to control animals (only significant in the lowest size class). Modification of the experimental protocol by performing partial hepatectomy 24 h after the administration of Tris-BP, did not increase the number of GGT+ or glutathione S-transferase-P (GST-P+) positive foci above the control level. Taken together, these results indicate that, in spite of a high mutagenicity in S. typhimurium, 2BA and Tris-BP have low or negligible mutagenic effects in mammalian systems. The lack of mutagenic activity may explain why Tris-BP is not a carcinogen in the rat liver.

Electrochemical detection and quantification of the acetylated and deacetylated C8-deoxyguanosine DNA adducts induced by 2-acetylaminofluorene.

The genotoxic agent 2-acetylaminofluorene induces, upon metabolic activation, two main types of DNA adducts in animal tissue, i.e., (deoxyguanine-8-yl)-aminofluorene (dG-C8-AF) and N-(deoxyguanine-8-yl)-acetylaminofluorene (dG-C8-AAF). Quantification of the frequency of these adducts usually relies on the use of radioactively labeled 2-acetylaminofluorene. Here, we report the development of a sensitive, non-radioactive method for the quantification of dG-C8-AF and dG-C8-AAF. Essentially, the modified DNA bases are separated by high-performance liquid chromatography (HPLC) and quantified by electrochemical detection. We established that both modified bases guanine-C8-aminofluorene and guanine-C8-acetylaminofluorene are electrochemically active. Subsequently, a procedure was developed to quantify dG-C8-AF and dG-C8-AAF in genomic DNA. Following DNA hydrolysis the adducted bases were extracted by ethyl acetate, separated by HPLC, and detected electrochemically. This procedure has been applied in the analysis of dG-C8-AAF in N-acetoxy-2-acetylaminofluorene-modified calf thymus DNA and in the detection of dG-C8-AAF and dG-C8-AF in liver DNA of mice injected intraperitoneally with 150-450 mg N-hydroxy-2-acetylaminofluorene/kg. The quantification of relatively low dG-C8-AF and dG-C8-AAF adduct levels (i.e., 0.1-1 adduct/10(6) nucleotides) in mouse liver DNA demonstrates the sensitivity of this electrochemical detection procedure. The detection limit of the method is 1 adduct per 10(6) nucleotides for both adducts using 20 micrograms of DNA and 4 adducts per 10(8) nucleotides using 500 micrograms DNA.

The role of sulfation in the metabolic activation of N-hydroxy-4'-fluoro-4-acetylaminobiphenyl.

The role of sulfation in the metabolic activation of the liver carcinogen N-hydroxy-4'-fluoro-4-acetylaminobiphenyl (N-OH-FAABP) in male rat liver was investigated. N-OH-FAABP was a substrate for sulfotransferases in vitro and sulfation was inhibited by the sulfotransferase inhibitors pentachlorophenol (PCP) and 2,6-dichloro-4-nitrophenol (DCNP). The main metabolite of N-OH-FAABP excreted in bile in vivo, and in the isolated perfused liver, was identified as the N-O-glucuronide conjugate. Inhibition of sulfation in vivo by PCP or DCNP, or in vitro by omission of inorganic sulfate, resulted in a large increase in the excretion of the N-O-glucuronide conjugate. It was estimated that at least 21% of the dose was sulfated in control animals. Inhibition of sulfation in vivo by PCP or DCNP prevented the covalent binding of N-OH-FAABP to liver (and kidney) macromolecules by 70% and 20% respectively. HPLC analysis of the fluorobiphenyl DNA and RNA adducts showed that the formation of both N-acetylated and deacetylated (deoxy)-guanosine adducts was prevented. Furthermore, omission of inorganic sulfate in the isolated perfused liver prevented the formation of all fluorobiphenyl DNA adducts by 70-80%. It is concluded that two sulfotransferase-dependent pathways exist for the metabolic activation of N-OH-FAABP in male rat liver: (i) direct sulfation of the hydroxamic acid, resulting, upon decomposition of the FAABP-N-sulfate ester, in the formation of N-acetylated DNA adducts and (ii) deacetylation followed by sulfation of the hydroxylamine to FABP-N-sulfate, leading to the formation of deacetylation DNA adducts.

Inhibition of the oxidative metabolism of theophylline in isolated rat hepatocytes by the quinolone antibiotic enoxacin and its metabolite oxoenoxacin, but not by ofloxacin.

Isolated rat hepatocytes obtained from Aroclor-pretreated rats were incubated with theophylline in the presence or absence of the quinolone antibiotics enoxacin, its metabolite oxoenoxacin, or ofloxacin. The hepatocytes converted theophylline by cytochrome P-450 activity mainly to two metabolites: 1,3-dimethyluric acid and 3-methylxanthine. Enoxacin inhibited the formation of 1,3-dimethyluric acid by 67% at 1.0 mM. Oxoenoxacin or ofloxacin had no inhibitory effect. The oxidation of theophylline to 3-methylxanthine was not inhibited by any of the three compounds. The quinolones had no effect on cell viability. These results show that the inhibition by enoxacin is not due to the formation of its oxoenoxacin metabolite.

The covalent binding of cyclosporin A to rat liver macromolecules in vivo and in vitro: the role of cytochrome P-450.

Incubation of rat liver S9 with [3H]cyclosporin A ([3H]CSA) resulted in covalent binding of CSA to macromolecules. Binding was dependent on the presence of NADPH and could be inhibited by SKF-525A. Incubation of isolated rat liver parenchymal cells with CSA resulted in a concentration-dependent binding which increased with incubation time for at least 2 h. Addition of SKF-525 A (0.5 mM) decreased the binding by 83% while viability of the cells was unchanged. Pretreatment of the cells with diethylmaleate doubled the binding of CSA, indicating that glutathione can prevent binding of CSA. When [3H]CSA was injected i.v. radioactivity was covalently bound to liver and kidney macromolecules. Induction of cytochrome P-450 by phenobarbital resulted in enhanced in vivo covalent binding in liver and kidney. Whether this covalent binding of CSA is related to its cytotoxicity is yet unclear.

Autoradiographic studies on in vivo covalent binding of N-hydroxy-2-acetylaminofluorene in rat liver containing gamma-glutamyltranspeptidase-positive foci. The effect of the sulfation-inhibitor pentachlorophenol.

The role of sulfation in the covalent binding of [ring-3H]-N-hydroxy-2-acetylaminofluorene (N-OH-AAF) in rat liver containing gamma-glutamyltranspeptidase-positive (GGT+) foci was studied in vivo by autoradiography. GGT+ foci were induced by initiation with diethylnitrosamine, followed by a selection protocol consisting of a 2-week exposure to 2-aminofluorene in the drinking water and a single administration of CCl4. Both surrounding ('normal') liver tissue and, to a lesser extent, GGT+ foci covalently bound N-OH-AAF, administered 10 days after selection. The sulfation inhibitor, pentachlorophenol (PCP), reduced the covalent binding of N-OH-AAF in cells surrounding the foci. However, PCP had no effect on binding of radiolabel in GGT+ foci. Thus, reduced sulfotransferase activity may contribute to the resistance of GGT+ preneoplastic lesions to carcinogen cytotoxicity. The results suggest also, that cells in GGT+ foci are able to bind N-OH-AAF covalently by a sulfotransferase-independent pathway.

Prevention by thioethers of the hepatotoxicity and covalent binding to macromolecules of N-hydroxy-2-acetylaminofluorene and its sulfate ester in rat liver in vivo and in vitro.

In order to find potentially effective compounds that could prevent the covalent binding of the carcinogen N-hydroxy-2-acetylaminofluorene (N-OH-AAF) to rat liver macromolecules in vivo, the prevention of the covalent binding to RNA of the sulfate ester of the carcinogen N-OH-AAF by a series of thioethers was investigated in vitro. The most effective thioethers, which inhibited the covalent binding by 70% or more, were studied for their protection against acute hepatotoxicity of N-OH-AAF in the rat in vivo. Three of these thioethers, thiazolidine, methyl 4-(methylthio)benzoate, and 2-(methylthio)benzimidazole significantly decreased the hepatoxicity of N-OH-AAF, by 45, 71 and 83%, respectively. The effects of these thioethers on the covalent binding of N-OH-AAF to cellular macromolecules in vivo were also studied. Methyl 4-(methylthio)benzoate and 2-(methylthio)benzimidazole decreased the adduct formation of N-OH-AAF to DNA by 54 and 44%, respectively, but had no effect on protein adduct formation. Only 2-(methylthio)benzimidazole caused a slight decrease (23%) in the AAF-- protein adduct formation. 2-Acetylaminofluorene (AAF) and methyl 4-(methyl-sulfinyl)benzoate were the main products in the incubation of methyl 4-(methylthio)benzoate with AAF-N-sulfate in vitro. This suggests that the thioether attacks the nitrenium ion which is formed by spontaneous breakdown of AAF-N-sulfate; the formation of a sulfonium--AAF conjugate is postulated which decomposes into AAF and a sulfinyl compound.

Pharmacokinetics of xamoterol glucuronidation in the rat in vivo and in liver perfusion.

Xamoterol has a phenolic hydroxyl group at which both sulphation and glucuronidation may occur. In the rat in vivo it is almost exclusively glucuronidated. In bile the glucuronide conjugate is the only metabolite of xamoterol. In urine equal amounts of the glucuronide conjugate and unchanged xamoterol are present, together with a small amount of the sulphate conjugate (approx. 1% of the dose). The t1/2 of excretion of the glucuronide in bile was the same as the t1/2 for excretion of unchanged xamoterol in urine. The t1/2 for urinary excretion of the glucuronide conjugate was longer than that for its biliary excretion. In the isolated perfused rat liver glucuronidation was also the almost exclusive metabolic pathway. The extraction ratio of xamoterol by the liver was approx. 0.15. Since xamoterol is a very water-soluble compound, and is almost exclusively glucuronidated in the rat, it is an excellent model substrate for investigations on the pharmacokinetics of glucuronidation in the rat in vivo and in isolated perfused organs.

The binding of reactive metabolites of the carcinogen N-hydroxy-2-acetylaminofluorene to DNA and protein in isolated rat liver nuclei: effects of glutathione and methionine.

The covalent binding of reactive metabolites of the carcinogen N-hydroxy-2-acetylaminofluorene to DNA and protein in isolated, intact rat liver nuclei was studied. The chemically synthesized 2-acetylaminofluorene-N-sulfate became covalently bound to DNA and protein to form adducts, 50% to 60% of which retained the N-acetyl group. Glutathione decreased the covalent binding of acetylated adducts to DNA by 18% and to protein by 50%. Methionine was more effective; it decreased DNA binding by 52% and protein binding by 79%. N-Hydroxy-2-acetylaminofluorene was deacetylated by the nuclear preparation. Almost exclusively, deacetylated 2-aminofluorene adducts to DNA and protein were formed. Glutathione decreased the covalent binding of deacetylated adducts to DNA by only 14%. Protein binding, however, was decreased by 57%. Methionine had no effect on the formation of these adducts to DNA and protein. Formation of 2-aminofluorene-glutathione conjugates was reduced by ascorbic acid by 65%. Covalent binding of deacetylated adducts to DNA and protein, however, was not decreased by ascorbic acid. These data suggest that "harder" nucleophiles like methionine can be used to protect macromolecules in vivo from damage by "hard" electrophiles such as those generated from the reactive 2-acetylaminofluorene-N-sulfate. However, such nucleophiles seem not to be effective with N-hydroxylamines, such as N-hydroxy-2-aminofluorene, formed by deacetylation of N-hydroxy-2-acetylaminofluorene.

Effect of glutathione depletion on the hepatotoxicity and covalent binding rat liver macromolecules of N-hydroxy-2-acetylaminofluorene.

Glutathione plays an important role in the protection of the liver against several hepatotoxins. The hepatocarcinogen N-hydroxy-2-acetylaminofluorene is converted in the rat in vivo to reactive metabolites that bind covalently to cellular macromolecules. These metabolites may also react with glutathione, resulting in the formation of glutathione conjugates and in the detoxification of reactive metabolites. The role of glutathione in detoxification was investigated by depletion of glutathione in the rat in vivo with diethyl maleate. When rats were pretreated with diethyl maleate, 45 min before the administration of N-hydroxy-2-acetylaminofluorene, excretion of 2-acetylaminofluorene:glutathione conjugates in bile was decreased by 60% as compared to controls. However, total covalent binding to rat liver protein was not increased, and total binding to DNA was even decreased (p less than 0.1), apparently at the expense of the acetylated carcinogen-DNA adducts. Formation of deacetylated, 2-aminofluorene adducts to DNA was not affected by diethyl maleate. Pretreatment with diethyl maleate had no major effect on the acute hepatotoxic effects of N-hydroxy-2-acetylaminofluorene. The results indicate that glutathione does not play a vital role in the detoxification of reactive metabolites generated from the carcinogen N-hydroxy-2-acetylaminofluorene, since glutathione is not very effective in competing with macromolecules for trapping of reactive metabolites of N-hydroxy-2-acetylaminofluorene. Thus, 1 mM glutathione did not decrease the covalent binding of 2-acetylaminofluorene-N-sulfate (one of the main reactive metabolites that is formed in vivo) to DNA in vitro, while 10 mM glutathione decreased the covalent binding to RNA by only 20% and to DNA by only 40%.