Oncogenic purine derivatives: evidence for a possible proximate oncogen.

Two additional urinary metabolites of the chemical oncogen 3-hydroxyxanthine are now identified as 8-chloroxanthine and 8-methylmercaptoxanthine. Such products are thought to be derived from a reactive intermediate which can be tentatively considered to be a proximate oncogen. Since each of these 8-substituted xanthines has also been obtained in vitro by reactions of 3-acetoxyxanthine with chloride ion or methionine, their production in vivo can be explained as resulting through the metabolic formation of an activated ester with a reactivity similar to that of the chemical model.

Oxidizing action of purine N-oxide esters.

A technique involving O-acetylation of purine N-oxide derivatives in buffered aqueous solutions has permitted studies of the reactivity of many compounds for which the O-acetyl derivatives are not otherwise available. The oxidizing properties of a variety of N-acetoxypurines have been measured through their ability to oxidize iodide ion ot iodine, a reaction which is representative of a more general oxidizing ability. Those esters that oxidize iodide ion also catalyze the autoxidation of sulfite, a property characteristic of radicals. The same esters also oxidize cysteine to cysteic acid and tryptophan, tyrosine, and uric acid to yet uncharacterized products. Their oxidizing reactivity was compared with the ability of the same esters to react as electrophiles in another assay that measured the rate of formation of pyridine substitution products. The sulfate ester of 3-hydroxyxanthine has been synthesized. Its reactivity is qualitatively the same as that of 3-acetoxyxanthine but proceeds at a higher rate. Syntheses of S-(8-xanthyl)-N-acetylcysteine, 8-(2-hydroxyethylthio)xanthine, and 1-methyl-8-mehtylmercaptoguanine are also described.

Conversion of the oncogenic 1-methylguanine 3-oxide to 3-hydroxy-1-methylxanthine.

Deamination of the oncogenic 1-methylguanine 3-oxide occurs to a significant extent in rats to yield 3-hydroxy-1-methylxanthine and its metabolites. When 3-hydroxy-1-methylxanthine is administered, 1-methyl-8-methylthioxanthine can be recovered from urine and released from hepatic protein. No 1-methyl-8-methylthioguanine was detected in urine or bound to protein. There is no evidence of significant activation of 1-methylguanine 3-oxide by sulfotransferase, but deamination to the oncogenic 3-hydroxy-1-methylxanthine suffices to explain its oncogenicity.

Oncogenicity of purine 3-oxide and unsubstituted purine in rats.

Injection s.c. of purine 3-oxide into Wistar rats resulted in the appearance of sarcomas and fibromas at the interscapular site of administration, carcinomas in the liver, and a high incidence of s.c. fibromas in the hip at a distance from the site of injection. A small number of liver tumors but not tumors at the injection site appeared in rats to which the parent compound, purine, was administered. Oxidation of purine 3-oxide by xanthine oxidase was found to occur in two steps to yield the potent oncogen 3-hydroxyxanthine. A similar process may occur in vivo since a protein preparation from rat s.c. tissue has similar oxidizing activity.

Mutagenesis originating in site-specific DNA damage.

Covalent monoadducts are the major types of gene damage after exposure to chemical mutagens and carcinogens. These and other damage structures together give rise to the spectrum of mutations that includes base-pair substitutions, insertions and deletions. In this study we introduced the bulky adduct guanine-8-aminofluorene into defined sites on one or both strands of the lactose operator. After insertion into plasmid pBR322 and replication in Escherichia coli COEC40, operator mutants were recognized on 5-bromo,4-chloro,3-indolyl-beta-D-galactoside plates and by hybridization probing. Out of ten randomly selected mutants, nine were single-base deletions and one was a two-base deletion. All mutations were at the site of modification or immediately adjacent to that site. If modifications were placed into both plasmid strands, preventing excision repair, operator mutants comprised close to 100% of operator-containing plasmids.

Carcinogenic purine N-oxide ester modifies covalently all common bases in polynucleotides.

The carcinogen 1-methyl-3-hydroxyxanthine after esterification binds covalently to polynucleotides, RNA and DNA. All four ribopolynucleotides and poly(dT) are targets. Depending on reaction conditions, covalent binding is greatest to poly(A) followed by poly(U), poly(dT), poly(G), poly(C), RNA and DNA. Maximal covalent modification of DNA is one moiety per 360 nucleotides. All modified polynucleotides, RNA and DNA, except poly guanylic acid have been enzymatically digested and the major adducts characterized as nucleosides.

A uridine adduct with an ester of the carcinogen 3-hydroxy-1-methylxanthine.

The structure of the uridine adduct with the acetate ester of the carcinogen 3-hydroxy-1-methylxanthine has been determined. Covalent binding is between C-8 of xanthine and O-2 of uracil. This was determined from studies of the NMR spectrum, mass spectra and solvolysis in liquid hydrogen sulfide. The nucleoside adduct, formed with uridine, is identical with the adduct with polyuridylic acid after enzymatic hydrolysis. Treatment with aqueous ammonia or pH 7 at 100 degrees C leads to the loss of ribose. Thymidine also forms an adduct with 3-acetoxy-1-methylxanthine in a similar yield. Model studies with a space-filling model suggest that the methylxanthine moiety can fit into the major groove of DNA and cause minimal helix distortion if the thymine base is rotated into the unnatural syn conformation.

Realistic risk assessment.

A controversy over cancer risk has undermined trust in the scientific basis of regulation. Drinking water and pesticide programs are in chaos because "potential cancer risk" cannot produce practical standards. The risk controversy involves a dispute over the most fundamental scientific standards, namely what is and what is not a positive result. Here it is shown that the lowest effective dose (LOEL) of well-studied carcinogens is a firm and reproducible quantity that can serve as undisputed basis for safety standards. "Realistic risk assessment" is proposed, based on the immediately available LOEL, as a transitional measure to eliminate the controversy over assumed risks. A traditional regulatory safety margin, over which regulators have explicit authority, would produce traditional safety standards when based on the lowest effective dose. Superfund clean-up targets based on one in one-million potential cancer risk are equivalent to an approximately 10,000-fold safety margin over real risk. Realistic risk opens the way for a reevaluation of regulatory priorities based on the fact that a 5-fold safety margin for the real carcinogen arsenic is now in use and has proven safe.

Arsenic: opportunity for risk assessment.

Arsenic is a human carcinogen that in small amounts is widely distributed in food and water. It has been regulated for almost 100 years worldwide and in the United States, on the judgment of the Royal Commission on Arsenic that a classical threshold of toxicity exists and that a daily intake of 400 micrograms/day is safe. Modern regulatory thinking in the United States has not accepted safe levels for carcinogens and is thus in conflict with the arsenic standard. Recent epidemics of arsenicism have quantitatively confirmed that threshold not only for the non-cancerous arsenical skin lesions but also for arsenical skin and internal cancers. Research shows that arsenic is a general gene inducer. Genes induced are involved in proliferation, recombination, amplification and the activation of viruses. This characterizes arsenic as an indirect carcinogen and provides a molecular basis for risk assessment and the observed threshold dose response. In the United States at present, about 300 cases of occupational arsenical cancer, declining in numbers, are known. Background arsenic below the drinking water standard is not known to have produced disease. The conspicuous nature of arsenical skin disease presents an unusual opportunity for a simplified survey of arsenical skin disease to support regulatory standards for arsenic.