B. L. Van Duuren

CARCINOGENIC EPOXIDES, LACTONES, AND HALO-ETHERS AND THEIR MODE OF ACTION*

Alkylating agents are being introduced into our environment with increasing frequency. Some of them are used in chemotherapy; others are used as industrial intermediates in organic synthesis, as organic solvents for various chemical processes, as cross-linking agents in manufacturing processes, and as antibacterial and fungistatic agents. Coupled with this increase in possible environmental or occupational exposure is the occurrence of alkylating agents as plant natural products, some aspects of which are discussed in other sections of this monograph. The known biological properties of these compounds i.e., their mutagenic, carcinogenic, and cytotoxic properties have, therefore, received increasing attention. Biochemical studies have focused on their possible modes of interaction with tissue constituents: proteins, purines, pyrimidines, nucleic acids, et cetera. In addition, organic chemists have studied the reactivity of these compounds with a variety of nucleophilic agents. Alkylating agents cover a broad range of chemical compounds, and the present report will be concerned mainly with those types that have been investigated in this laboratory; the carcinogenicity, structure-activity relationships, and mode of action of these compounds have been the main concern in our work. The compound types that were explored are epoxides (mono-, di-, and polyfunctional), pand y-lactones, and halo-ether~.~-~ This report will be concerned only with selected aspects of this subject. The compounds of interest, their carcinogenicity or lack thereof, and various routes of administration used in mice and rats are listed in TABLE 1. The structures for these compounds are shown in FIGURES 1,2, and 3. The compounds were usually tested at highest tolerable doses for the lifespans of the animals. Thirty to fifty animals were usually included per group; in the gastric feeding experiments, smaller groups weie used; this is discussed below. The details of these experiments have appeared in our earlier It is clear from TABLE 1 that compounds that were active or inactive when applied on mouse skin showed, with one exception, the same activity or were

Activation of cell membrane enzymes in the stimulation of cell division.

Summary The tumor promoter, phorbol myristate acetate, increases the specific activities of the cell membrane enzymes Na + -K + -ATPase and 5′-nucleotidase in microsomal preparations from stationary cultures of BALB/c-3T3 mouse embryo fibroblasts. Under these conditions of in vitro exposure, the microsomal enzymes NADH diaphorase and glucose-6-phosphatase were not affected.

Cellular interactions of phorbol myristate acetate in tumor promotion.

Abstract The radioactivity from tritium-labeled phorbol myristate acetate is bound to mouse skin with a half-life of approx. 24 h. Autoradiographs revealed that most of the label was associated with the kerato-sebaceous layer over the squamous epithelial layer. The major portion of the radioactivity was recovered in the lipid-containing fractions of a Schmidt-Thannhauser extraction. In cell cultures of 3T3 mouse embryo fibroblasts, maximal binding of [3H]PMA was achieved in 18 h and was stable for up to 4 days in the presence of the labeled tumor-promoting agent. Autoradiographs of formalin-fixed cells showed extensive cytoplasmic labeling with no substantial nuclear activity. Methanol fixation resulted in complete removal of the tritium. Sedimentation analysis in Dextran gradients of subcellular fractions of 3T3 cells exposed to [3H]PMA demonstrated that the bulk of bound label was associated with a Na+-K+ ATPase-rich protein peak which has the properties of cell membranes.

Studies with carcinogens and tumor-promoting agents in cell culture.

Abstract Transformation into permanent cell lines of hamster embryo fibroblasts was induced by benzo(a)pyrene and 7,12-dimethylbenz(a)anthracene. Cells of the transformed lines had altered karyotypes, and the benzo(a)pyrene-induced line was tumorigenic in hamsters. Several lines of mouse fibroblasts of altered morphology were obtained following pyrene and benzo(a)pyrene exposure, but these cells were not tumorigenic when injected in cortisonized mice. 7,12-Dimethylbenz(a)anthracene inhibited the cellular proliferation of an untransformed line (3T3) of mouse fibroblasts to a lesser extent than in a polyoma virus-transformed variant of the same line. At doses that were not toxic to growth of mass cultures, exposure to 7,12-dimethylbenz(a)anthracene caused a reduction in the subsequent cloning efficiency of the 3T3 line. Similar inhibitory effects were obtained with tobacco smoke condensate, a phorbol ester fraction from Croton tiglium L. and benzo(a)pyrene. The toxic effect of bcnzo(a)pyrene was demonstrable 20 generations after exposure. No transformation was observed in the 3T3 cell line with any chemical treatment. The toxic and transforming effects of carcinogens in cell culture systems is discussed.

Epoxides, hydroperoxides and peroxides in air pollution.

Abstract A major source of air pollutants in urban areas is automobile exhaust. Olefins constitute a substantial proportion of the chemicals emitted by this source. Olefins undergo autoxidation and photochemical oxidation in air to hydroperoxides, peroxides, epoxides and other oxygenated aliphatics, frequently of low molecular weight. Long-term carcinogenicity assays of these compounds in mice and rats by various routes of administration have shown that some of these compounds are carcinogenic. Hence, their detection and elimination as air pollutants should be vigorously pursued. This report describes the current status of air pollution research on these compounds, their carcinogenicity, structure-activity relationships and areas which deserve attention in future research. Oxidation products of aromatic hydrocarbon pollutants are also important and will be described.

Cocarcinogens and Tumor Promoters and Their Environmental Importance

In recent years a great deal of effort has been spent on uncovering new carcinogens and studying the mode of action of well-known carcinogens such as nitroso compounds, aromatic hydrocarbons and aromatic amines and their occurrence in air, water, foods and other environmental sources. It has been known for about thirty years that some materials, while non-carcinogenic, can enhance greatly the potency of low-levels of known environmental carcinogens. The first such material to be discovered was the tumor promotor, croton oil, derived from the seeds of a tropical plant, Croton tiglium L, indigenous to India and Sri Lanka. Researchers interested in chemical carcinogenesis have examined its unusual behavior for many years. The active principles of croton oil, the phorbol esters, were finally isolated and chemically characterized. Recently these phorbol esters have become widely available. Consequently, their effects have been examined in a wide range of biochemical and biological studies. The phorbol esters are not environmental factors in cancer causation since they occur in a toxic plant which is not used as a source of animal or human food. Other known tumor promoters are much less active, e.g., anthralin (1,8-dihydroxy-9-anthrone) and phenol. Thus, the phorbol esters, although valuable tools in studies on chemical carcinogenesis, are less relevant to environmental health and disease than another group of compounds known as cocarcinogens. Many cocarcinogens are ubiquitous environmental agents and only recently have these compounds been given more attention. It is expected that as more of these agents become known as environmental agents, new advances in cancer prevention will be made.

Interaction of a hydrophobic fluorescent probe with mouse embryo fibroblasts

Abstract Fluorescence photomicrographs show that the hydrophobic fluorescent probe 1-anilinonaphthalene-8-sulfonate (ANS) binds to hydrophobic components of intact 3T3 cells. Cells exposed to ANS exhibit fluorescence in the cytoplasm, intense nuclear membrane fluorescence, and well-defined fluorescent nucleoli. Fluorescence titrations of 3T3 cells with ANS show a decrease in fluorescence intensity, a blue shift of native cell emission with increasing ANS concentration and the appearance of a new peak due to ANS fluorescence. These fluorescence effects are ascribed to energy transfer processes involving bound ANS and the tryptophan and tyrosine residues of cellular proteins. ANS bound to 3T3 cells appears to quench the long wavelength component of the cellular tryptophan fluorescence, resulting in an unmasking of tryptophan and tyrosine emission at shorter wavelengths.