Inhibitory effect of CDA-II, a urinary preparation, on aflatoxin B(1)-induced oxidative stress and DNA damage in primary cultured rat hepatocytes.

The effects of CDA-II (cell differentiation agent II; a urinary preparation) on both aflatoxin B(1) (AFB(1))-induced cell injury and DNA damage were investigated using cultured rat hepatocytes. CDA-II was able to suppress both the lipid peroxidation and lactate dehydrogenase leakage induced by AFB(1). Glutathione (GSH) depletion by AFB(1) was replenished by CDA-II treatment. Under these experimental conditions, CDA-II enhanced the activity of GSH peroxidase, but not GSH S-transferase. By evaluation of unscheduled DNA synthesis, CDA-II reduced AFB(1)-induced DNA damage in hepatocyte cultures. These findings suggest that CDA-II can inhibit cytotoxicity of AFB(1) through enhancing the activity of GSH peroxidase and preventing GSH depletion.

Effect of CDA-II, urinary preparation, on lipofuscin, lipid peroxidation and antioxidant systems in young and middle-aged rat brain.

The levels of lipofuscin and lipid peroxidation reflect the degree of free radical-induced oxidative damage in the brain. We examined the effects of CDA-II, a preparation of human urine, on lipofuscin and lipid peroxidation in the brain of young (3.5 months) and middle-aged rats (17 months). The rats were given CDA-II orally at dosages of 0.3 or 1.0 g/kg daily for 8 weeks. CDA-II significantly suppressed the contents of lipofuscin and lipid peroxidation in both young and middle-aged rats. CDA-II also elevated the activity of superoxide dismutase, and the amounts of glutathione and ascorbic acid in the middle-aged rats, but not in the young ones. Our results suggest that the protection against oxidative damage by CDA-II in the young rat brain may be due to its scavenging activity against free radicals. In the middle-aged rats, in addition to the scavenging activity, the levels of endogenous antioxidants were also enhanced by the CDA-II treatment.

Pharmacokinetic study of radioactive antineoplaston A10 following oral administration in rats.

Radiolabelled (3H-labelled) Antineoplaston A10 was administered in a single dose of 220 mg to 230 mg/kg to female Sprague Dawley rats. Blood and urine samples for determination of radioactivity were collected one hour prior to, and then at different time intervals after, the administration of the drug. Rats were sacrificed 6 h or 36 h later for the study of radioactivity in the various organs. The concentration of radioactivity in blood reached a maximum after 2 to 3 h after the administration of Antineoplaston A10, whereas the highest concentration of radioactivity in urine was observed in the 3.5-h to 4-h samples. It was observed by quantitative HPLC analysis that in rats sacrificed 6 h after Antineoplaston A10 administration, between 61% to 69% of the drug was absorbed, whereas between 37% to 28% was found in the stomach and between 2% to 3% was present in the intestinal contents and faeces. After 36 h, none could be detected in the stomach, intestinal contents or faeces. Organ distribution studies indicated greater accumulation of radioactivity in ileum, bladder, duodenum, kidneys and jejunum, and relatively low accumulation in the heart, lung, liver and brain. The concentration of radioactivity after 36 h was very low. By quantitative measurement, between 40% to 42% of the drug was excreted in the urine in 6 h and 75% of the radioactive material was in the form of Antineoplaston A10. The identification of the major radioactive material as Antineoplaston A10 was confirmed by TLC and analysis of the products of acid hydrolysis and by determination of melting range.

Chemoprevention by antineoplaston A10 of benzo(a)pyrene-induced pulmonary neoplasia.

The effect of Antineoplaston A10 (AA10), an amino acid derivative isolated from human urine, has been studied on pulmonary adenoma formation resulting from intragastric administration of benzo(a)pyrene(BP) to A/HeJ mice. Two doses of BP, 3 mg each, administered two weeks apart, induced an average of 6.86 tumours within 157 days in the control animals (Tumorigenic Index 437). One per cent of AA10 (w/w) given in mouse food for one week prior to, and then continued after the administration of BP, produced a 70% reduction in the total number of tumours in the test groups.

Quantitative assay of plasma and urinary peptides as an aid for the evaluation of cancer patients undergoing antineoplaston therapy.

Plasma and urinary peptides can be reproducibly assayed by a procedure involving reverse phase chromatography on a column of C18 and HPLC on a column of sulfonated polystyrene. The average plasma and urinary peptide levels of normal persons are 79.4 nmoles/ml and 73.6 nmoles/mg creatinine, respectively. Ninety-one percent of 108 confirmed cancer patients referred to the authors' Clinic for antineoplaston therapy showed various degrees of deficiency of peptides in the plasma, and 98% of the patients had plasma/urine peptide ratios below the normal ranges of 0.82 to 1.15, indicating that the overwhelming majority of cancer patients excreted greater than normal amounts of peptides in the urine. When a patient responded favourably to the antineoplaston therapy, the excessive urinary excretion of peptides was reversed. Plasma levels of peptides and plasma/urine peptide ratios showed a steady increase after one to two months and eventually approached those of normal persons. On the other hand, if a patient was not responding to the antineoplaston therapy, the excessive urinary excretion of peptides remained unaltered or even became greater. Patients in remission following antineoplaston therapy tended to show plasma and urinary peptide levels comparable to those of normal persons. Thus the quantitative assay of plasma and urinary peptides provides a useful parameter for the evaluation and prognosis of cancer patients undergoing antineoplaston therapy. A growing colon tumour was found to have the lowest level of peptides among mice tissues analysed, suggesting that deficiency of inhibitory peptide components may be associated with malignant growth. The results of this study are consistent with the interpretation that a deficiency of antineoplastons may be required for malignant cells to proliferate, and the consequent malignant growth further aggravates this deficiency. Antineoplaston therapy can halt such a "vicious cycle" and induce long-term remission in some cancer patients.

Chemo-surveillance: a novel concept of the natural defence mechanism against cancer.

Synthetic Antineoplaston A10, 3-phenylacetylamino-2,6-piperidinedione, has been shown to produce promising clinical results, similar to those obtained with antineoplastons derived from urine. Antineoplaston A10 is capable of reducing the excessive excretion of peptides often associated with cancer patients, thereby boosting endogenous levels of antineoplaston. Its therapeutic effect is partially attributable to endogenous antineoplastons, the level of which Antineoplaston A10 can help to raise. A patient must have a certain reserve of endogenous antineoplastons in order to benefit from therapy with Antineoplaston A10. The critical plasma peptide level seems to be above 30 nmoles/ml. Patients who responded favourably to the treatment with Antineoplaston A10 invariably showed an increase of plasma peptide levels and a decrease of urinary excretion of peptides. These results suggest that the body, under normal circumstances, is protected by antineoplastons against cancer. This chemical mechanism of protection is hereby termed chemo-surveillance.

Preclinical studies on antineoplaston A10 injections.

Antineoplaston A10 (3-phenylacetylamino-2, 6-piperidinedione) has poor solubility in water. In order to make it more soluble for the preparation of Antineoplaston A10 injections, the compound has to be converted into its sodium salt. It was found that during neutralization A10 undergoes basic hydrolysis with formation of two components, IIa and IIb. However, A10 was found to be fairly resistant to acid hydrolysis at room temperature. At a higher temperature (110 degrees C) the reaction proceeded easily and after 60 min the compound was completely hydrolysed. The ratio of 4:1 of products of basic hydrolysis remained very constant in a number of experiments. They were subsequently identified as phenylacetylglutamine and phenylacetylisoglutamine. A similar ratio of these two compounds was found during partial hydrolysis of A10 in simulated pancreatic juice, indicating a possibility that at least some of the anticancer effects of A10 are attributable to the action of these two degradation products. A decision was therefore made to produce a formulation of Antineoplaston A10 injections, 100 mg/ml as a 4 : 1 mixture of sodium salts of phenylacetylglutamine and phenylacetylisoglutamine. This formulation did not show any significant toxic effects when tested for one year in chronic toxicity studies in a group of 160 HA/1CR Swiss white mice.

Altered methylation complex isozymes as selective targets for cancer chemotherapy.

MC2 is a ternary enzyme complex consisting of MAT, methyltransferase and SAHH. Three isozymes of SAHH have been identified from rat and mouse livers based on different kinetic properties. The Km values are 0.35 +/- 0.05 microM, 1.63 +/- 0.38 microM and 0.37 +/- 0.07 mM for SAHH-L, SAHH-I, and SAHH-H respectively. The corresponding low Km isozymes of MAT and SAHH form MCs-L which include RNA MCs, the intermediate Km isozymes form MC-I, and the high Km isozymes form MC-H which is glycine MC. MCs-L are common to all tissues whereas MC-I and MC-H are organ specific enzyme complexes. Low levels of MC-H in the liver of C3H/HeN mouse are correlated with the slow maturation of hepatocytes and the genetic predisposition to develop spontaneous PHC. Rat Novikoff ascites hepatoma and mouse spontaneous PHC have been shown to contain a SAHH isozyme displaying kinetic properties different from the corresponding normal SAHH-L. The abnormal kinetic properties of tumour SAHH are analogous to those of tumour MAT previously shown by the authors to be uniquely associated with malignant tissues. The tumour isozyme, which is named SAHH-LT, has a Km (AR) value of 2.18 +/- 0.22 microM. The altered tumour MC isozymes appear to play an important role in perpetuating malignant growth, because once the tumour growth was inhibited by poly (I) (C), the abnormal kinetic properties were no longer detectable. Thus abnormal tumour MCs may be exploited as selective targets for cancer chemotherapy. Evidence is presented to indicate that antineoplaston is a potent inhibitory effector of tumour rRNA MCs and an effective antitumor agent.

Demonstration of an altered S-adenosylmethionine synthetase in human malignant tumors xenografted into athymic nude mice.

S-Adenosylmethionine synthetase isoenzymes (EC 2.5.1.6) were studied in human malignant tumors xenografted into athymic nude mice and were studied in normal human and rat tissues. The tumors included 7 melanomas; 4 colon carcinomas; 3 each of mammary, cervical, and ovary carcinomas; 2 each of lung carcinomas and sarcomas; and 1 each of lymphoma and stomach and nasopharyngeal carcinomas. The presence of an altered intermediate Michaelis constant (Km) isoenzyme, previously shown to be a unique characteristic of rat neoplastic tissues, in these tumors was invariably detectable, though the presence of the low-Km isoenzyme characteristic of the enzyme of normal tissues was also evident in many of these tumors. Only tumors demonstrating high rates of growth were devoid of the low-Km isoenzyme. The low-Km isoenzyme was the only enzyme detectable in many normal human and rat tissues. Thus this isoenzyme was probably the common enzyme of all tissues. Some additional organ-specific isoenzymes were found, e.g., in the liver and lactating mammary gland. The low-Km isoenzyme in malignant tumors was altered to assume a considerably higher Km value, and this alteration appeared to be a common aberration of malignant tumors.

Partial purification and characterization of tumor and liver S-adenosylmethionine synthetases.

The S-adenosylmethionine synthetase activities of rat liver and Novikoff ascites tumor have been partially purified and characterized by chromatographic behavior, kinetic analysis, sulfhydryl dependency, and response to inhibitors. We have shown that the tumor contains a single form of the enzyme, with a Km (methionine) of 21 muM, and that the liver contains two isofunctional forms, a minor form with a Km (methionine) of 21 muM, as well as a major form with a Km of 1 mM. The tumor contained more of the low Km form of the enzyme than the liver, although the total enzyme activity of liver (measured at high substrate concentrations) exceeded that of the tumor severalfold. The tumor enzyme also corresponded to the minor form of liver enzyme in elution position from Sephadex G-150 and diethylamino-ethyl cellulose, and both had a Km (adenosine 5'-triphosphate) of 0.14 mM. The tumor enzyme differed from the major form of the liver enzymes in elution position, and the Km (adenosine 5'-triphosphate) for the latter was 1.5 mM. In contrast to the major liver enzyme, the tumor enzyme did not appear to require sulfhydryl agents for the activity to be detected, was inhibited by S-adenosylmethionine, and was inhibited to a greater degree by tripolyphosphate. It is suggested that the two forms of the enzyme are involved in the production of S-adenosylmethionine for different biological functions, and their different properties may allow selective inhibition of tumor growth by chemotherapeutic agents.

Role of ribosomal RNA methylases in the regulation of ribosome production in mammalian cells.

The activity of rRNA methylases was stimulated by high-energy precursors of RNA (ribonucleoside triphosphates) and inhibited by degradation products of RNA (ribonucleotides and oligoribonucleotides). The response of methylases from rat Novikoff ascites tumor and liver to these metabolites was strikingly different. The highly active tumor enzymes responded preferentially to inhibition by catabolic metabolites, whereas the less active liver enzymes responded exclusively to stimulation by anabolic metabolites. When the activity of rRNA methylases was assayed in response to increasing concentration of S-adenosylmethionine, the tumor enzymes responded with a hyperbolic substrate dependence curve and the liver enzymes with a sigmoidal curve. In the presence of an inhibitory dinucleotide, ApA, the tumor enzymes responded with a sigmoidal curve; in the presence of a stimulator, adenosine 5'-triphosphate, the liver enzymes responded with a hyperbolic substrate concentration curve. When normal rats were subject to a series of treatments by thioacetamide, a hepatocarcinogen, the liver nucleolar rRNA methylases became responsive to inhibition by ApA and relatively unresponsive to stimulation by adenosine 5'-triphosphate. When tumor-bearing rats were treated with polyinosinate:polycytidylate, an antitumor agent, the tumor nucleolar rRNA methylases became unresponsive to inhibition by ApA and more responsive to stimulation by adenosine 5'-triphosphate. A correlation was noted between increased methylation efficiency in vivo and increased stability of nucleolar RNA during incubation in vitro, or vice versa. These results are interpreted to indicate that rRNAmethylases are regulated by cellular metabolites during the nucleolar biosynthesis of ribosomes and that rRNA methylases may provide a favorable site for selective action by cancer chemotherapeutic agents.

Preferential inhibition by homopolyribonucleotides of the methylation of ribosomal ribonucleic acid and disruption of the production of ribosomes in a rat tumor.

The literature indicates that some mechanism other than the interferon or host-mediated immune enhancement might also be responsible for an antitumor effect of polyinosinate-polycytidylate [poly(I)-poly(C)]. We have examined the effect of this drug on the synthesis of ribosomes and other macromolecules in a rat tumor, the Novikoff ascites hepatoma. The nucleolus was one of the primary targets affected by the administration of poly(I)-poly(C) in vivo. A progressive decline of the activity of nucleolar ribosomal RNA methylases began within 2 hr, followed by a decline of the nucleolar RNA content. The activity of nucleolar RNA polymerase was inhibited only at later time intervals. Labeling of tumor macromolecules in vivo revealed that the methylation of ribosomal RNA and the production of ribosomes, particularly in the small subunits, were immediately and progressively affected, followed by inhibition of the synthesis of DNA, RNA, and protein at later times. In addition, poly(I)-poly(C) also induced disaggregation of polyribosomes and restricted the movements of nuclear RNA to cytoplasm and of cytoplasmic protein to nucleus. These in vivo effects of poly(I)-poly(C) on tumor cells was observed neither on the host livers nor on livers of normal rats. Studies on isolated nucleoli showed that the in vitro addition of polyinosinate and several other compounds actively inhibited tumor ribosomal RNA methylases but were devoid of inhibitory effect against liver ribosomal RNA methylases; these results augment other studies in the literature in suggesting a selective effect of the polyinosinate moiety on tumor cells. We conclude from this study that initial impairment of the methylation of ribosomal precursor RNA, following exposure of tumor cells to poly(I)-poly(C), is responsible for the destruction of ribosomes, preferentially the small subunits, during the maturation processes. Failure to provide new ribosomes thus triggers the events limiting the growth of tumor cells.

Interrelationships between synthesis and methylation of ribosomal RNA in isolated Novikoff Tumor nucleoli.

Nucleoli isolated from Novikoff hepatoma cells of the rat were previously shown to carry out synthesis of predominantly ribosomal precursor RNA and methylation of this RNA in vitro. In order to develop in vitro systems for further detailed study of these processes and their interrelationships, isolated nucleoli were incubated in a complete RNA-synthesizing medium using (5-3H)cytidine 5'-triphosphate or S-adenoxyl(methyl-3H)methionine to measure the activities of RNA synthesis and methylation, respectively, under the same reaction conditions. Methylation of the ribose of the nascent ribosomal precursor RNA predominated. It occurred in close coordination with the transcriptional step by RNA polymerase as shown by the kinetic data, the analysis of labeled RNA in sucrose gradients, the inhibition by increased ionic strength or actinomycin D, and the release of labeled nucleotides by a 3'-exonuclease, venom phosphodiesterase. Methylation of the RNA bases occurred more slowly, continued longer after transcription ceased, and appeared to follow later in the processing of the RNA. Certain divalent cations (Mg2+, Mn2+, and Ca2+ at higher concentrations, and Zn2+ and Cu2+) inhibited both RNA synthesis and methylation to similar extents. RNase inhibitors (bentonite and dextran sulfate) at low concentration inhibited methylation while stimulating RNA synthesis, and pyrophosphate greatly decreased RNA synthesis with relatively little effect on methylation. These results indicated that RNA polymerase and ribosomal RNA methylases can function independently despite their close relationship. An exogenous substrate for the nucleolar rRNA methylases was found: nuclear RNA prepared from Novikoff hepatoma cells, cultured in the absence of methionine, served as a good substrate for methylation of both ribose and bases. Other exogenous RNAs, including cytoplasmic ribosomal RNA from these methionine-starved cells, nucleolar RNA from normal cells, and wheat germ ribosomal RNA were almost devoid of methyl-acceptor activity. A description of these parameters helps establish isolated nucleoli as a suitable system for further study of interaction of RNA polymerase, methylases, and nucleases in control of synthesis of ribosomal RNA.