Inhibition of aflatoxin B1 -hepatocarcinogenesis in rats by {beta}-naphthoflavone

Effects of ß-naphthonflavoe (ßNF) on theactivity of hepatic microsomal aflatoxin B₁ (AFB₁)-4-hydroxylase- the cytochrome P-450-dependent enzyme system which catalyzesthe metabolism of AFB₁ to AFM₁ - and on AFB₁ induced in vivohepatocarcinogenesis were investigated in weanling male Fischerrats. A single i.p. injection of ßNF in doses of 20mg/kg and 150 mg/kg induced AFB₁ -4-hydroxylase 3- and 4-fold,respectively, 48 h post injection. Feeding of diet containing0.01% ßNF for a period of 9-weeks induced AFB₁ 2-fold.AFB₁, given by intubatlon in a dose of 25 µg five times/weekfor 8 weeks, produced 42 weeks later a 100% incidence of liverlesions (neoplastic foci, nodules or tumors), but feeding ßNFin diet at a con centration of 0.015% for one week prior toand during the 8 weeks of AFB treatment inhibited AFB₁ hepatocarcinogenesis by -7%. These results are in accord with the suggestionthat AFB₁ induction may be associated with the inhibition ofAFB₁ carcinogenesis, possibly occurring as a consequence ofaccelerated detoxi-fication of AFB₁ via its conversion to AFM₁     

Effects of vitamins A, C, and E on aflatoxin B1-induced mutagenesis in Salmonella typhimurium TA-98 and TA-100

The effects of retinoids (vitamin A analogs) and vitamins C and E on the aflatoxin B1 (AFB1)-induced mutagenesis in Salmonella typhimurium TA-98 and TA-100 were investigated. The bioassay was performed under conditions that permitted the effects of vitamins on carcinogen metabolism to be assessed separately from effects on the expression of the mutated bacterial cell. Both retinoic acid and retinol inhibited (up to 50%) AFB1-induced mutagenesis in S. typhimurium TA-98, but only retinol inhibited (up to 75%) mutagenesis in TA-100. Retinoic acid inhibition of mutagenesis in S. typhimurium TA-98 was pronounced over a wide concentration range (i.e., 2 X 10(-10) to 2 X 10(-8) M) however, at the higher concentrations (i.e., 2 X 10(-8) to 2 X 10(-6) M range) the predominant effect was the inhibition of the metabolism of AFB1 to its mutagenic metabolites. Vitamin E was more potent in inhibiting the expression of AFB1-induced mutagenesis than vitamin C. However, the major inhibitory effects of vitamin E were related to the metabolism of AFB1, whereas vitamin C was inhibitory at both metabolic and the post-metabolic levels of the AFB1 mutagenesis assay. The results of these investigations suggest that vitamins A, C, or E inhibit both AFB1 metabolism to its mutagenic metabolites as well as the expression of AFB1-induced mutated bacterial cells.     

A cyclophosphamide/DNA phosphoester adduct formed in vitro and in vivo

The antitumor activity of cyclophosphamide is thought to be due to the alkylating activity of phosphoramide mustard, a metabolite of cyclophosphamide. Reaction of 2'-deoxyguanosine 3'-monophosphate and phosphoramide mustard resulted in the formation of several adducts that could be detected by high performance liquid chromatography (HPLC). One of these adducts, isolated and purified by HPLC, could be detected by 32P postlabeling. This product was identified by UV, nuclear magnetic resonance, and mass spectrometry and by acid, base, and enzymatic hydrolysis to be 2'-deoxyguanosine 3'-monophosphate 2-(2-hydroxyethyl)aminoethyl ester. A combination of HPLC fractionation of digested DNA and 32P postlabeling was used to detect this adduct in calf thymus DNA incubated in vitro with metabolically activated cyclophosphamide and in DNA from the liver of mice treated with cyclophosphamide. In these DNA samples the adduct occurred at a level of 1/10(5) and 1/3 x 10(7) nucleotides, respectively.     

Identification, characterization, and cloning of TIP-B1, a novel protein inhibitor of tumor necrosis factor-induced lysis.

Some cancer cells evade elimination by virtue of their insensitivity to agents that induce apoptosis. Conversely, the side effects of anticancer agents could be diminished if normal cells were more resistant. To further elucidate the factors that contribute to the susceptibility of a cell to apoptosis, these investigations were designed to identify proteins isolated from cells exposed to low concentrations of tumor necrosis factor (TNF) that, when incubated with normally TNF-sensitive cells, protect these cells from TNF-induced cytotoxicity. TIP-B1, a novel protein, has been identified, purified, and characterized from cytosolic extracts of TNF-treated human fibroblasts. The approximately 27 kDa pI-4.5 TIP-B1 protein is unique based on both the sequence of three internal peptides (comprising 51 amino acids) and the nucleotide sequence of the corresponding 783-bp cDNA partial clone. Western blot analyses using polyclonal antisera raised against both the purified native TIP-B1 and the approximately 14 kDa product of the cDNA partial TIP-B1 clone, as well as Northern blot analyses using the cDNA insert as a probe, indicate that TIP-B1 may belong to a family of proteins that are expressed in a number of cell lines from diverse tissues. TNF-sensitive cells, when exposed to 4-10 microg/ml concentrations of TIP-B1 prior to the addition of TNF, are completely protected from TNF-induced lysis. Furthermore, TIP-B1 protects cells from apoptotic lysis induced by TNF. Preincubation of TIP-B1 with TNF does not affect the ability of TNF to induce lysis. Moreover, TIP-B1 does not seem to interfere with the interactions between TNF and the TNF receptors, based on a preliminary flow cytometric analysis of the cellular binding of biotinylated TNF. On the basis of these characteristics, TIP-B1 is not a soluble TNF receptor, an anti-TNF antibody, nor a protease that degrades TNF; yet TIP-B1 functions when added exogenously to cells. These characteristics, its novel sequence, and its function when added exogenously to cells indicate that TIP-B1 is unique and is not one of the other proteins reported previously to be involved in resistance to TNF. The ability of TIP-B1 to function after exogenous incubation with target cells makes TIP-B1 a likely candidate for therapeutic manipulation of TNF-induced effects.     

Combinations of mesna with cyclophosphamide or adriamycin in the treatment of mice with tumors

Following therapeutic administration, cyclophosphamide and Adriamycin are biotransformed to reactive metabolites, some of which are responsible for undesirable systemic toxicities of these chemicals, whereas others are responsible for their chemotherapeutic effectiveness. Microsomal mixed function oxidases activate cyclophosphamide to produce phosphoramide mustard and acrolein, while cytochrome reductase and xanthine oxidase are capable of transforming Adriamycin and forming free radicals. These reactive metabolites produce unwanted toxic side effects; however, their action may be partially ameliorated by the concomitant administration of thiols. In this study we evaluated the therapeutic activity of combinations of mesna (2-mercaptoethanesulfonate) with cyclophosphamide or Adriamycin in mice with a variety of transplantable tumors (L1210 and P-388 leukemia, Lewis lung and colon 26 carcinoma, B16 melanoma, and M5076 sarcoma). In all cases the administration of mesna prior to cyclophosphamide or Adriamycin treatment did not reduce the antitumor effectiveness of these agents and in some instances (C57BL/6 mice with B16 melanoma or M5076 sarcoma) small improvements were observed. Therefore, the addition of thiols, to reduce effectively the buildup of toxic metabolites of cyclophosphamide or Adriamycin may result in the improved therapeutic effectiveness for these agents in the treatment of cancer.     

Effects of inducers and inhibitors on the metabolism of aflatoxin B1 by rat and mouse.

The microsomal metabolism of aflatoxin B₁ (AFB₁) via various pathways and the induction and inhibition specificities of these pathways were examined in Sprague-Dawley rats, inbred strains of mice, and recombinant inbred lines derived from AKR/J and C57L/J cross. The data suggests that the metabolism of aflatoxin B₁ is catalyzed by at least three different enzymes of the microsomal mixed function oxygenase system: one that mediates conversion to aflatoxin M₁ (AFM₁-hydroxylase) is cytochrome P-448-linked and is associated with the Ah locus at the level of regulatory genetic factors: the other two enzymes which are cytochrome P-450-linked, mediate the metabolism of aflatoxin B₁ to aflatoxin Q₁ (AFQ₁-hydroxylase) and to AFB₁-2,3-oxide (metabolic activation). Although AFQ₁-hydroxylase and metabolic activation, measured in vitro as the formation of DNA-alkylating metabolite(s), are not clearly distinguishable on the basis of induction and inhibition responses, the two activities can be distinguished on the basis of their kinetic parameters and genetic regulation: (a) the apparent K_m of the metabolic activation pathway is almost three orders of magnitude (19.7 × 10^?4 M) higher than that of AFQ₁-hydroxylase (0.7 × 10^?4), [B. D. Roebuck and G. N. Wogan, Cancer Res.37, 1649 (1977)]; and (b) while both activities are induced by phenobarbital (PB) and depressed by 3-methylcholanthrene (3-MC), unlike the metabolic activation, differences in the 3-MC-induced depression of AFQ₁-hydroxylase activity were noted in Ah responsive and nonresponsive strains. Kinetic studies revealed that metabolic activation of AFB₁ is linear with the time of incubation up to 20 min, with up to 2 mg of microsomal protein in the incubation, and has a pH optimum of 7 to 7.4. While PB pretreatment, relative to control, enhanced the apparent V_max 3-fold, 3-MC depressed it about 60 per cent; however, the apparent K_m values of the three microsomal preparations were of the same order of magnitude (1.3 to 2 × 10^?3 M). Under the conditions of the incubation, the minimum number of binding sites in DNA was estimated to correspond to a resultant specific activity of 8–9 nmoles AFB₁-metabolite bound/μmole DNA-P.     

Properties, inducibility, and an improved method of analysis of aryl hydrocarbon hydroxylase in cultured human lymphocytes.

The properties and inducibility of aryl hydrocarbon hydroxylase (AHH) in cultured human lymphocytes were studied, and a sensitive method of analysis has been developed. In agreement with other reports, peripheral blood lymphocytes per se had no activity and required pretreatment in culture with a mitogen for conversion to lymphoblasts to possess AHH activity. This activity had an absolute requirement for reduced nicotinamide adenine dinucleotide phosphate. Under our conditions of incubation, reduced nicotinamide adenine dinucleotide (1.3 mM) or ethylenediaminetetraacetic acid disodium salt (1 mM), when added to reduced nicotinamide adenine dinucleotide phosphate-fortified incubation mixture, caused about 30% increase in activity and MnCl2 caused an appreciable inhibition. The reaction was linear with the number of cells, with up to 16.8 X 10-6 cells contained in the incubation mixture, and had a pH optimum of 8.5. The enzyme was fairly stable at -70 degrees and retained about 90% of the original activity for 15 days. The enzyme activity in the mitogen-stimulated lymphocyte cultures appeared slowly and reached a maximum at 48 to 72 hr of incubation at 37 degrees, after which it considerably decayed with time. Viability of the cells in culture also decayed considerably after 72 hr and reached about 50% level at 120 hr. The enzyme was inducible with 3-methylcholanthrene and dibenz(a,h)anthracene. Dibenz(a,h)anthracene was a much more potent inducer than 3-methylcholanthrene and evoked a response in cells that would be considered noninducible with 3-methylcholanthrene. By using purified lymphocytes and Roswell Park Memorial Institute Medium 1640, providing larger surface area during culture, harvesting the cells at the time of minimum cell death and maximum AHH activity, and assaying AHH at its pH optimum of 8.5 instead of 7.6, it was possible to enhance the sensitivity of the assay an average of 17-fold.