Charles Heidelberger

Fluorinated Pyrimidines and Their Nucleosides

Since their introduction (Heidelberger et al., 1957), the fluorinated pyrimidines and their nucleosides have been widely used as biochemical tools for the elucidation of a number of problems encountered in cell biology and molecular biology. Of more practical importance, however, has been their extensive use as drugs for the palliative treatment of patients suffering from disseminated cancer. One of the compounds is now clinically useful in the curative treatment of herpes simplex viral infections of the eye. Although these compounds have generated an enormous literature, this chapter will deal primarily with a review of the major pharmacological and biochemical effects of these compounds; the coverage of the literature, by necessity, will focus only on key references that are germane to the topics being described. Several reviews of this topic have appeared elsewhere (Heidelberger and Ansfield, 1963; Heidelberger, 1965, 1966, 1967, 1969, 1970, 1973; Mandel, 1969; Carter, 1970).

Thymidylate synthetase: Mechanism of inhibition by 5-fluoro-2′-deoxyuridylate☆

Abstract Inhibition of thymidylate synthetase by 5-fluoro-2′-deoxyuridylate requires methylenetetrahydrofolate. Both 5-fluoro-2′-deoxyuridylate and methylenetetrahydrofolate are covalently bound to the enzyme, as shown by the stability of the complex to denaturation by sodium dodecyl sulfate and urea, and to trichloroacetic acid precipitation. By contrast, 5-trifluoromethyl-2′-deoxyuridylate is not covalently bound. We postulate that in the enzyme-inhibitor-cofactor complex, the 6-carbon of 5-fluoro-2′-deoxyuridylate is bound covalently to the enzyme, with the methylene group covalently linking to the 5-carbon of the nucleotide to either the N-5 or N-10 position of tetrahydrofolate.

An epoxide is an intermediate in the microsomal metabolism of the chemical carcinogen, dibenz(a,h)anthracene.

Abstract Washed rat liver microsomes that had been heated for 5 minutes at 50°C were incubated at room temperature for 30 seconds with 3 H-dibenz(a,h)-anthracene, TPNH, MgCl 2 , and unlabeled 5,6-epoxy-dibenz(a,h)anthracene. Analysis of the incubation mixture by TLC revealed the presence of an epoxide. This epoxide was characterized by its behavior on TLC and by its conversion under acidic conditions to the corresponding phenol. The possible role of epoxides in hydrocarbon carcinogenesis is discussed.

Chemical Oncogenesis In Culture

Publisher Summary Although the field of chemical oncogenesis in culture depends upon the fundamentals and advances in cell biology, virology, biochemistry, and other related disciplines, it is clearly impossible to cover these other fields. Although most of the work has been done with cell cultures, but organ cultures are also considered in this chapter. Organ cultures consist of small 3-dimensional pieces of differentiated tissues that are usually maintained on top of the nutrient medium. Cell cultures consist of individual cells submerged in the medium that grow either on a glass or plastic substrate or in suspension. The more important systems, such as 3T3-like systems, other cell systems, and liver cell systems are described in the chapter, followed by some of the major generalizations that can be drawn from the various researches involving these systems. The many advantages of studying biological phenomena in cell cultures, rather than in intact animals, include the ability to control the environment, to do quantitative bookkeeping of the cells, to vary the exposure to chemicals, to synchronize the cells, and to be free of the immunological milieu of the host.

Liver homogenate-mediated mutagenesis in chinese hamster V79 cells by polycyclic aromatic hydrocarbons and aflatoxins.

Several chemical carcinogens that require metabolic activation have been examined for their cytotoxic and mutagenic activity in Chinese hamster V79 cells. Mutagenic activity was measured as the induced frequency of 6-thioguanine-resistant colonies. Metabolic activation was provided by the 9000 g supernatant fraction of rat liver plus cofactors. The cytotoxicity and mutagenicity of aflatoxin B1 and B2, benzo(a)pyrene, 3-methylcholanthrene, 7,12-dimethylbenz(a)anthracene, dibenz(a,h)-anthracene, dibenz(a,c)anthracene, and benz(a)anthracene were examined as functions of concentration. Except for the two isomers of dibenzanthracene, the mutagenic activity in general paralleled the carcinogenic activity. An assay of this type may be useful as a prescreen for environmental chemicals that require metabolic activation.

Studies on fluorinated pyrimidines: VII—the degradative pathway☆☆☆

Abstract A study of the metabolic degradation of 5-fluorouracil has been carried out in mice and human cancer patients. 5-Fluoroorotic acid is not significantly degraded by mice. The first steps of 5-fluorouracil degradation, the conversion to dihydrofluorouracil and α-fluoro-β-ureidopropionic acid, are exactly analogous to those of uracil. In human subjects there is extensive conversion of FU to urea. Most normal tissues, except spleen, degrade FU in vivo . The liver is probably the main site of degradation, and studies with high-speed supernatant fractions have been carried out. Two mouse tumours have been shown to be incapable of degrading the drug. In human beings there is more extensive catabolism when the drug is administered orally than by intravenous injection. There is a higher concentration of radioactivity in a human malignant carcinoma and intestinal mucosa than in the normal tissues and a benign fibro-adenoma. The presence of α-fluoro-β-guanidopropionic acid as a metabolite of 5-fluorouracil has been suggested.

Fluorinated pyrimidines. VI. Effects of 5-fluorouridine and 5-fluoro-2'-deoxyuridine on transplanted tumors.

Summary 1. Activity of 5-fluorouridine (FUR) and 5-fluoro-2′-deoxyuridine (FUDR) against several transplanted tumors has been compared with that of 5-fluorouracil (FU). 2. In Adenocarcinoma 755 and L-1210 leukemia FU is more effective than either nucleoside, although the latter have significant tumor-inhibitory activities. 3. In Sarcoma 180, FUDR is much more effective than FU; FUR has no significant activity. 4. In the Ehrlich ascites carcinoma, both nucleosides are more effective in prolonging life than is 5-fluorouracil, with FUR demonstrating activity at a low dose. In a 5-fluorouracil-resistant line of the Ehrlich ascites carcinoma, 5-fluorouridine is as active as in the parent strain. 5. In mice, the toxicity by repeated doses of FUR is much greater than that of FU; FUDR is less toxic than FU. In rats, the toxicity of FUR is much less pronounced than in mice, and the compound was very effective in inhibiting the growth of the Novikoff hepatoma.

The incorporation of 5-fluoro-2′-deoxyuridine into DNA of mammalian tumor cells

Summary Treatment of L1210 mouse leukemia cells with 10 −6 M [ 3 h]5-fluoro-2′-deoxy-uridine resulted in the incorporation of radioactivity into DNA. The DNA hydrolysate was shown to contain [ 3 h]5-fluoro-2′-deoxyuridylic acid by an unequivocal assay involving the formation of a tightly-bound complex with thymidylate synthetase in the presence of 5,10-methylenetetrahydrofolate. The extent of incorporation was estimated to be 1 molecule of 5-fluoro-2′-deoxyuridylic acid per 10 DNA nucleotides.

Binding of K-region epoxides and other derivatives of benz[a]anthracene and dibenz[a,h]anthracene to DNA, RNA and proteins of transformable cells

Abstract The firm binding of benz[a]anthracene and dibenz[a,h]anthracene and their K-region epoxides, cis -dihydrodiols, and phenols to the DNA, RNA, and proteins of exponentially growing cells has been studied. In hamster embryonic cells that undergo malignant transformation, the epoxide of benz[a]anthracene was bound to all macromolecules to a much greater extent than the hydrocarbon and other derivatives. The binding to DNA reached its maximum at 3 h. In the dibenz[a,h]anthracene series, the epoxide was highly bound to RNA and protein, but only to a small extent to DNA. The extent of binding of all compounds to DNA, RNA, and proteins of transformable G23 cells was less than to hamster cels, and the binding to malignant T24 cells was very much lower. The relationship of metabolism to binding and carcinogenesis is discussed.

Endogenous oncornaviruses in chemically induced transformation. I. Transformation independent of virus production.

Abstract The C3H/10T1/2 cell line, established from mouse embryo fibroblasts, is highly sensitive to postconfluence inhibition of division and is susceptible to malignant transformation by chemical carcinogens in culture. Normal, chemically transformed and spontaneously transformed clones of C3H/10T1/2 cells were examined for the production of infectious murine leukemia virus (MuLV) and MuLV and mouse mammary tumor virus (MTV) antigens. Criteria for the expression of oncornaviruses included the production of: (1) MuLV and MTV gs-antigens, (2) MuLV GL and GT cell surface antigens, (3) DNA polymerase-containing particles, and (4) infectious virions. Normal C3H/10T1/2 cells were free of oncornaviruses by all four of these criteria. Transformation of these cells to malignancy by chemical carcinogens did not result in an increased production of MuLV or MTV products; hence, these cells demonstrated the same virus-free phenotype as the parental cell line. Attempts to rescue transforming viral information from transformed C3H cell lines by superinfection with leukemia viruses were unsuccessful. Repressed endogenous MuLV (but not MTV) could be induced from all cell lines by treatment with 5-iododeoxyuridine (IUdR). Induction of MuLV in normal cells occurred in three phases: I, an initial transient production of low-titered virus; II, a 3–4-week interval where virus production fell to a lower level and then gradually increased. This was accompanied by the recruitment of virus-free cells in the culture to produce MuLV gs-antigens and DNA polymerase-containing particles; and III, the constitutive production of high-titered virus. Transformed cells were more sensitive to treatment with IUdR than normal C3H/10T1/2 cells. These cells underwent a more rapid induction of MuLV, contained a larger fraction of cells that expressed MuLV functions, continued the production of MuLV throughout the second phase of induction, and had an earlier onset of the third phase of high-titered MuLV production. Oncornaviruses induced from IUdR-treated cells showed diversity in their host range and in their ability to produce XC plaques; none of these viruses, however, were capable of transforming normal C3H/10T1/2 fibroblasts in culture.