The further development of a mammalian DNA alkaline unwinding bioassay with potential application to hazard identification for contaminants from environmental samples.

Recently, we have detailed a DNA alkaline unwinding assay (DAUA) that can be used to rapidly measure chemically induced strand breaks in mammalian cells (Daniel et al., 1985). In this paper we present further development of this assay, including: (1) studies on the relationship between DNA adducts and DNA strand breaks; (2) evaluation of the role of cytotoxicity in DNA strand breaks; and (3) application of the DAUA to cell preparations from the liver of mice dosed with methylating agents. The level of DNA adducts produced in human CCRF-CEM cells by treatment with benzo(a)pyrene diol-epoxide (BPDE), N-acetoxy-2-acetyl aminofluorene (AAAF), and various methylating agents was linear with concentration over several orders of magnitude. Likewise, the level of strand breaks increased with the concentration over the same dose range. The strand breaks/adduct ratio ranged from 0.05 for the methyl adducts to 0.001 for the BPDE adducts. Using these values and the inherent sensitivity of the DAUA (circa 100 to 1000 breaks/cell), (Daniel et al., 1985), the ability of the assay to detect DNA damage induced by various classes of chemical carcinogens can be calculated. The DAUA appears to be useful for assessing the relative potency of various environmental genotoxic effects on mammalian cells. In addition, it can be conducted on cells isolated from target organs of whole animals.

Properties of a local anesthetic receptor in rat brain.

Saturable binding of local anesthetics in rat brain homogenates was demonstrated using (14C)-lidocaine and (3H)-bupivacaine. Saturation analyses revealed a single class of binding sites for lidocaine and bupivacaine. A series of drugs with local anesthetic properties inhibited this binding, while drugs without local anesthetic activity did not affect the specific binding. Specific binding of lidocaine and bupivacaine was maximal from pH 8 to 10; the pH versus binding profile was similar to that reported for local anesthetic blocking of peripheral nerve conduction. These characteristics suggest that binding of local anesthetics to this or similar sites mediates their pharmacological activity.

Differences in pulmonary metabolism of benzo(a)pyrene in inbred mouse strains, using the isolated perfused lung.

The metabolism of benzo(a)pyrene (BaP) was studied in isolated perfused and ventilated lungs from two inbred strains of mice. C57BL/6 and DBA/2 mice were treated with beta-naphthoflavone (BNF) or corn oil alone in order to reveal strain differences in inducibility of the microsomal enzymes responsible for BaP oxidation. BaP metabolism was quantified by the rate of appearance of free and conjugated 14C-metabolites released into the perfusion medium, and the production of nonextractable metabolites covalently bound to lung macromolecules. In both strains, lungs from mice pretreated with BNF released BaP metabolites at a rate somewhat greater than vehicle-treated controls, without a distinct change in the profile of metabolites. However, the amount of BaP covalently bound to lung macromolecules was increased by BNF pretreatment in C57BL/6 mice only. Thus, release of BaP metabolites into the perfusion medium was not a predictor of the rate of production of reactive metabolites.

Benzo(a)pyrene metabolism in the isolated perfused mouse lung.

A method is described for preparing and maintaining an isolated perfused and ventilated mouse lung. The preparation is especially suited for studying xenobiotic metabolism or toxicological interactions, in a species with a broad spectrum of studies in pulmonary toxicology. The preparation is viable with respect to drug metabolism for up to two hours, as judged from studies of aniline oxidation to p-aminophenol. With [14C]-benzo(a)pyrene as substrate for the lungs of male ICR Swiss mice, the major ethyl acetate-extractable metabolites are the 3-hydroxy, 9,10-dihydrodiol, 7,8-dihydrodiol, and 4,5-dihydrodiol derivatives. The rates of individual BaP metabolite production are increased in lungs from mice pretreated with Aroclor 1254 or beta-naphthoflavone, substances known to induce increased synthesis of cytochrome P-450. Small amounts of water-soluble BaP metabolites were hydrolyzed by beta-glucuronidase and aryl sulfatase, suggesting the presence of enzymes required for these conjugations. These results support the existence of significant cytochrome P-450-dependent and conjugative BaP metabolism in the intact mouse lung, similar to that examined in other species, and capable of contributing to the systemic metabolism of this carcinogen.